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Impact of genistein on maturation of mouse oocytes, fertilization, and fetal development

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Abstract

Genistein (GNT), a natural isoflavone compound found in soy products, affects diverse cell functions, including proliferation, differentiation and cell death. An earlier study by our group showed that GNT has cytotoxic effects on mouse blastocysts and is associated with defects in their subsequent development in vitro. Here, we further investigate the effects of GNT on oocyte maturation, and subsequent pre- and postimplantation development, both in vitro and in vivo. GNT induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryo development. Treatment of oocytes with GNT during in vitro maturation (IVM) led to increased resorption of postimplantation embryos, and decreased placental and fetal weights. With the aid of an in vivo mouse model, we showed that consumption of drinking water containing GNT led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Moreover, our findings support a degree of selective inhibition of retinoic acid receptors in blastocysts treated with GNT during oocyte maturation. To our knowledge, this is the first study investigating the impact of GNT on maturation of mouse oocytes, fertilization, and sequential embryonic development.

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... The time of vaginal opening was advanced in female rats after neonatal exposure to genistein [10]. Additionally, fertility in adult rodents was reduced after neonatal genistein exposure, which was closely related to the disruption of estrous cyclicity [11], ovulation failure [12,13], poor oocyte quality [14], and microenvironmental alterations of the reproductive tract [15,16]. However, these studies showed 4 limited data that described the state of the corresponding cells for genistein-caused pathologies. ...
... Furthermore, these MOFs developed continuously because MOFs at different stages persisted in the ovaries of mice at PND 21. In rodents and wildlife, MOFs are correlated with reduced fertility and decreased embryonic survival rates [14,30]. Previous studies utilizing neonatal genistein injections and oral genistein administration have shown increased MOFs associated with decreased fertility as well as premature reproductive senescence [13,31,32]. ...
... These phenomena indicate that adult fertility was impaired or abolished following developmental exposure to high doses of genistein, which has also been reported in previous studies for mice (50 mg/kg) [31] and rats (100 mg/kg) [38]. Our microscopic examination further showed that most of the ovaries from genistein-treated mice manifested 14 cystic follicles, with differing degrees of hypertrophy in thecal, cortical, and medullary cells. The altered expression levels of HSD17B and HSD3B (the two enzymes that are responsible for estradiol and progesterone synthesis, respectively), indirectly reflected the disorder of intercellular steroidogenesis in the various functional cells of the ovary. ...
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Soy-based formula contains high concentrations of the isoflavone genistein. Genistein possesses estrogenic and tyrosine kinase inhibitory activity and interferes with cellular proliferation and development. To date, the acute and chronic effects of genistein on ovarian and uterine development have not been fully elucidated. In this study, mice at postnatal day 1 were subcutaneously injected with 100 mg/kg genistein for 10 consecutive days, and then their ovaries and uteri were collected on days 10, 21, and 90. Histological evaluation was performed after hematoxylin and eosin staining. The proliferating activity was indicated by the proliferating indicator protein Ki67. Results showed that the subcutaneous injection of genistein to neonatal mice induced the formation of multi-oocyte follicles and delayed the primordial follicle assembly in the ovaries. Genistein significantly enlarged the cross-sectional area of the uterine cavity and wall and disrupted the regularity between the uterine stroma and myometrium. Genistein exposure inhibited proliferative activity because fewer Ki67-positive nuclei were detected in ovarian and uterine cell populations than in the control. Furthermore, most ovaries from adult mice given neonatal genistein were without corpora lutea, and there appeared to be cystic follicles and hypertrophy of the theca, and cortical and medullary layers. Considering the high concentration of isoflavone in soy-based infant formulas and livestock feed, we suggest that the use of isoflavone-rich diets in humans and livestock receive closer examination. Graphical Abstract Fullsize Image Graphical Abstract
... In experiments using mice oocyte, genistein is known to inhibit maturity, but in yet another research also using mice oocyte, it is known that genistein increase post-cryopreservation survivability. 8,9 These conflicting results demands further research on the supplementation of both nutrients, which are both found in soybeans (Glycine max). ...
... However, the results of other studies show that the amount of genistein circulating in the circulation is very small when given in the form of soy. 9,31 The effect of soybean supplementation on embryo development Evaluation of embryo development after ICSI was performed repeatedly using an inverted microscope. Evaluation includes observations of cytoplasm, cell division, and zona pellucida. ...
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Introduction: One of the important factors in Assisted Reproductive Technology (ART) is embryo quality that depends on oocyte quality. Maternal nutrition in form of soybean supplementation is thought to have benefits in oocyte quality. To determine the effect soybean supplementation to the embryo quality from oocyte side. Methods: This experimental study involved female mice from Swiss strain aged 6 weeks that were divided into two groups, group with soybean (soybean group/SG) and group without soybeans (pellet group/PG). Follicles were collected and denuded to get oocytes. The oocytes were stained with MitoTracker for assessing the mitochondrial membrane potential and TUNEL for assessing the apoptotic level. Colour intensity was assessed using a confocal microscope and determined using ImageJ software. Sperms were extracted surgically from the epididymis-vas deferens and performed preparation prior to intra cytoplasmic sperm injection (ICSI) procedure. Then, the embryos were cultured and observed for the quality. Result: In TUNEL test, the average colour intensity of the SG was lower compared to the PG, significantly (p=0.03). While in the Mito Tracker test, the average colour intensity for the SG was higher compared to PG, not significantly (p=1.08). In addition, the embryo development on Day-1 and Day-3 showed more good embryo quality of SG compared to PG, significantly (p=0.03). Conclusion: Soybean supplementation improved embryo quality at the cleavage stage by decreasing apoptosis of the oocytes rather than increasing the viability the oocytes.
... New pieces of evidence are emerging regarding the mechanisms by which genistein advances its deleterious effects on pregnancy and foetal development. In agreement with previous studies, the present study showed that oral exposure of pregnant rats to genistein significantly reduced their body, placental, and foetal weights (Chan 2009;Awobajo et al., 2013;Amir et al., 2018;Awobajo et al., 2018;2020a;2020b). Adequate and proper growth of the placenta is key to normal foetal development, and the significant decrease in placental weight, especially at GD-16 in the 2 mg rats is linked to the reduction in foetal weight throughout the GD-20. ...
... This study also revealed that circulating PIGF levels in rats can reach a concentration of 300-400 pg/ml during late gestational period; this indicates important role played by this protein in the sustenance of pregnancy (Chan 2009;Khaliq et al., 1996). PlGF is encoded by the PlGF gene and is a member of the vascular endothelial growth factor (VEGF) subfamily (Smith et al., 2007). ...
Article
The mechanisms by which genistein, a phytoestrogen, affects fetoplacental development adversely are still poorly understood. It is reported that genistein ingestion modulates thyroid functions, leptin hormone, C-reactive protein, and thyroxin kinase activities. In this study, we evaluated changes in serum and placental insulin-like growth factor-I (IGF-1), placental growth factor (PIGF), and soluble fms-like tyrosine kinase-1 (sFLT-1) in pregnant rats exposed to genistein using ELISA. According to the treatments, Rats were divided into control, 2 mg genistein, and 4 mg genistein groups. Genistein groups were administered with the doses orally from gestational day (GD) one onwards until sacrifice, while the control group received an equal volume of distilled water the vehicle. At GD-12, GD-16, and GD-20, serum samples and placenta homogenates were prepared from maternal blood samples and the placenta and were analysed to determine the concentration of IGF-1, sFLT-1, and PIGF. Serum IGF-1 and PIGF were both increased in all genistein groups at GD-12 and GD-16, and at GD-20 in the 4 mg group. However, serum IGF-1and PIGF levels were decreased in the placenta from all genistein groups at GD-20. Placenta sFLT-1 levels increased at both GD-16 and GD-20 in genistein-treated rat serum. An initial decrease in placental sFLT-1 at GD-12 was followed by an increase at GD-16 and finally a decrease at GD-20 in all genistein treated rats. The sFL-1/PlGF ratio in placenta samples of genistein-exposed rats was decreased at GD-16 and increased at GD-20, while the reverse was recorded in the serum sample at the same gestational periods. The fetoplacental growth disruption mechanism of genistein can be partly explained by its interference with placental growth factor signaling. https://authors.elsevier.com/a/1fiDE3oGhS0T0
... Oocyte fertilization and fetal development are complex and conditioned by the microenvironment [20]. For this reas pair these processes leading to developmental problems [50]. eficial effects of curcumin have been highlighted in many in v that it has been proven safe in humans, there are still conflic fect on oocyte maturation, fertilization and development of t leagues examined the cytotoxic effects of curcumin by trea different concentrations of curcumin. ...
... Oocyte fertilization and fetal development are complex processes tightly regulated and conditioned by the microenvironment [20]. For this reason, chemical injury may impair these processes leading to developmental problems [50]. Despite the fact that the beneficial effects of curcumin have been highlighted in many in vitro and in vivo studies and that it has been proven safe in humans, there are still conflicting studies regarding its effect on oocyte maturation, fertilization and development of the blastocyst. ...
Article
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Curcumin, also known as diferuloylmethane, is the main polyphenolic substance present in the rhizomes of Curcuma longa L. This plant showed many beneficial effects and has been used since ancient times for both food and pharmaceutical purposes. Due to its pleiotropic functions, curcumin consumption in the human diet has become very common thanks also to the fact that this natural compound is considered quite safe as it does not have serious side effects. Its functions as an anti-inflammatory, anti-oxidant, neuroprotective, immunomodulatory, anti-toxicant, anti-apoptotic, and anti-diabetic compound are already known and widely demonstrated. There are numerous studies concerning its effects on various human pathologies including cancer, diabetes and arthritis while the studies on curcumin during pregnancy have been performed only in animal models. Data concerning the role of curcumin as anti-inflammatory compound suggest a possible use of curcumin in managing pregnancy complications such as Preeclampsia (PE), Gestational Diabetes Mellitus (GDM), Fetal Growth Restriction (FGR), PreTerm Birth (PTB), and exposure to toxic agents and pathogens. The aim of this review is to present data to support the possible use of curcumin in clinical trials on human gestation complications.
... Fetal weight of 600 mg was taken as an important indicator of successful embryonic and fetal development, in keeping with our previous findings. 22,24,25,[40][41][42][43] Investigations by our group have repeatedly demonstrated that 35%-40% fetuses weigh >600 mg and the average weight of total surviving fetuses is~600 AE 12 mg in the untreated control group on day 18 of pregnancy in a mouse embryo transfer assay. 22,24,25,[40][41][42][43][44] Fetal weight is considered an important marker of developmental status. ...
... 22,24,25,[40][41][42][43] Investigations by our group have repeatedly demonstrated that 35%-40% fetuses weigh >600 mg and the average weight of total surviving fetuses is~600 AE 12 mg in the untreated control group on day 18 of pregnancy in a mouse embryo transfer assay. 22,24,25,[40][41][42][43][44] Fetal weight is considered an important marker of developmental status. We therefore used average fetal weight of the untreated control group as the key indicator of post-implantation development of ENN B1-exposed blastocysts. ...
Article
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Enniatins are mycotoxins of Fusarium fungi that naturally exist as mixtures of cyclic depsipeptides. Previous reports have documented hazardous effects of enniatins on cells, such as apoptosis. However, their effects on pre‐ and post‐implantation embryonic development require further clarification. Here, we showed for the first time that enniatin B1 (ENN B1) exerts cytotoxic effects on mouse blastocyst‐stage embryos and induces intracellular oxidative stress and immunotoxicity in mouse fetuses. Co‐incubation of blastocysts with ENN B1 triggered significant apoptosis and led to a decrease in total cell number predominantly through loss of inner cell mass. In addition, ENN B1 appeared to exert hazardous effects on pre and postimplantation embryo development potential in an in vitro development assay. Treatment of blastocysts with 1‐10 μM ENN B1 led to increased resorption of post‐implantation embryos and decreased fetal weight in the embryo transfer assay in a dose‐dependent manner. Importantly, in an in vivo model, intravenous injection with ENN B1 (1, 3, and 5 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst‐stage embryos and impairment of embryonic development from the zygote to blastocyst stage, subsequent degradation of embryos, and further decrease in fetal weight. Intravenous injection with 5 mg/kg body weight/d ENN B1 additionally induced a significant increase in total reactive oxygen species (ROS) content and transcription levels of genes encoding antioxidant proteins in mouse fetal liver. Moreover, ENN B1 triggered apoptosis through ROS generation and strategies to prevent apoptotic processes effectively rescued ENN B1‐mediated hazardous effects on embryonic development. Transcription levels of CXCL1, IL‐1β, and IL‐8 related to innate immunity were downregulated after intravenous injection of ENN B1. These results collectively highlight the potential of ENN B1 to exert cytotoxic effects on embryos as well as oxidative stress and immunotoxicity during mouse embryo development.
... Any environmental chemicals affecting these components are likely to impact the oocyte maturation. For example, natural isoflavone genistein has been shown to mimic the actions of estrogen in mouse by inhibiting oocyte maturation, in vivo and in vitro, and fertilization and embryonic development (Chan, 2009;Jung et al., 1993). This effect of genistein appears to be mediated through the estrogen receptor pathways (Wang et al., 1996). ...
... The inhibitory effect of the phytoestrogen genistein observed in this study in zebrafish is consistent with similar observations in rodents (Chan, 2009). Genistein not only impairs oocyte maturation but also disrupts fertilization, induces apoptosis, and disrupts embryogenesis in mice. ...
Article
Oocyte maturation can be a target of endocrine disruption by environmental chemicals capable of acting as hormone mimics, receptor blockers, and/or enzyme inhibitors. Six environmental chemicals (genistein, endosulfan, malathion, iprodione, carbaryl, and glyphosate) were selected to determine their ability to interfere with oocyte maturation in zebrafish. The translucent oocytes undergoing germinal vesicle (nucleus) breakdown (GVBD) were counted and expressed as a ratio of oocytes undergoing GVBD and total oocytes exposed. The GVBD increased significantly in oocytes exposed to 10IU/ml to 100IU/ml human chorionic gonadotropin (hCG). The lowest effective concentration of genistein that inhibited hCG-induced GVBD was 30μM, while endosulfan inhibited it at 0.03μM concentration. In addition, malathion inhibited hCG-induced GVBD at the lowest concentration of 60μM. These inhibitory effects were likely due to the chemicals acting as estrogen mimics, induction of estrogen receptors, or increase in aromatase activity resulting in enhanced estrogen action. Fungicide iprodione, possibly acting as a progestin mimic, promoted hCG-induced GVBD at the lowest concentration of 20μM, while the weed killer glyphosate inhibited hCG-induced GVBD starting at the 50μM concentration. These results demonstrate the feasibility of using fully grown zebrafish oocytes arrested at the prophase I stage in an in vitro incubation system to evaluate the effects of a variety of environmental chemicals on oocyte maturation.
... During normal embryogenesis, apoptosis (a unique morphological pattern of cell death) functions to clear abnormal or redundant cells in preimplantation embryos [15,16]. Apoptotic processes do not occur prior to the blastocyst stage during normal mouse embryonic development [17], and induction of apoptosis during the early stages of embryogenesis (i.e., following exposure to a teratogen) causes embryonic developmental injury [12,13,[18][19][20]. Apoptosis plays an important role in development and disease [21]. ...
... Moreover, placental and fetal weights were lower in the dillapiole-treated group ( Figure 4B,C). Previous studies, including a recent investigation by our laboratory, showed that 35%-40% of fetuses weigh more than 600 mg, and the average weight of total surviving fetuses is ~600 ± 12 mg in the untreated control group at day 18 of pregnancy in [13,19,[28][29][30]. Fetal weight is an important indicator of developmental status. ...
Article
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We examined the cytotoxic effects of dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal, and acaricidal activities, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Blastocysts treated with 2.5-10 μM dillapiole exhibited a significant increase in apoptosis and corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with dillapiole were lower than those of their control counterparts. Moreover, in vitro treatment with 2.5-10 μM dillapiole was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that dillapiole induces apoptosis and retards early post-implantation development, both in vitro and in vivo. However, the extent to which this organic compound exerts teratogenic effects on early human development is not known at present. Further studies are required to establish effective protection strategies against the cytotoxic effects of dillapiole.
... Apoptotic processes do not occur prior to the blastocyst stage during normal mouse embryonic development [14]. Induction of apoptosis during early stages of embryogenesis (i.e., following exposure to a teratogen) compromises embryonic development [15][16][17][18][19]. Additionally, several chemical and physical triggers of apoptosis create oxidative stress via ROS generation [15,20]. ...
... Interestingly, no difference in placental weight was evident between the OTA-treated and untreated groups ( Figure 4B), but fetal weight was lower in the OTA-treated group. Moreover, both earlier and current experiments by our group have shown that 35%-40% of normal mouse fetuses weigh more than 600 mg at day 18 of pregnancy after mouse embryo transfer, and the average weight of all surviving fetuses was ~600 ± 12 mg in the untreated control group of the present experiment [16,17,35]. Fetal weight is an important indicator of developmental status, and the average fetal weight of untreated controls thus serves as a key indicator of the developmental status of OTA-treated blastocysts. ...
Article
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Ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, both in vitro and in vivo. In the present study, we explored the cytotoxic effects exerted by OTA on the blastocyst stage of mouse embryos, on subsequent embryonic attachment, on outgrowth in vitro, and following in vivo implantation via embryo transfer. Mouse blastocysts were incubated with or without OTA (1, 5, or 10 μM) for 24 h. Cell proliferation and growth were investigated using dual differential staining; apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay; and embryo implantation and post-implantation development were assessed by examination of in vitro growth and the outcome of in vivo embryo transfer, respectively. Blastocysts treated with 10 μM OTA displayed a significantly increased level of apoptosis and a reduction in total cell number. Interestingly, we observed no marked difference in implantation success rate between OTA-pretreated and control blastocysts either during in vitro embryonic development (following implantation in a fibronectin-coated culture dish) or after in vivo embryo transfer. However, in vitro treatment with 10 μM OTA was associated with increased resorption of post-implantation embryos by the mouse uterus, and decreased fetal weight upon embryo transfer. Our results collectively indicate that in vitro exposure to OTA triggers apoptosis and retards early post-implantation development after transfer of embryos to host mice. In addition, OTA induces apoptosis-mediated injury of mouse blastocysts, via reactive oxygen species (ROS) generation, and promotes mitochondrion-dependent apoptotic signaling processes that impair subsequent embryonic development.
... However, fetal weight was lower in the 100 μM DHLA-treated group compared to the control group (479 ± 61 mg versus 611 ± 68 mg, respectively). Previous studies, including a recent report by our group, showed that 35–40% of fetuses weigh more than 600 mg, and the average weight of total surviving fetuses is about 600 ± 12 mg in the untreated control group at day 18 of pregnancy in a mouse embryo transfer assay [23,27282930. Fetal weight is an important indicator of developmental status, and the average fetal weight of the untreated control group is used as a key marker of development of blastocysts treated with 100 μM DHLA. Interestingly, only 5.8% of the fetuses in the 100 μM DHLA-pretreated group weighed more than 600 mg (indicative of successful embryonic and fetal development), whereas 43% of control fetuses exceeded this threshold (Figure 4C). ...
... During normal embryogenesis, apoptosis (a unique morphological pattern of cell death) functions to clear abnormal or redundant cells in preimplantation embryos [31,32]. Apoptotic processes do not occur prior to the blastocyst stage during normal mouse embryonic development [33], and induction of apoptosis during the early stages of embryogenesis (i.e., following exposure to a teratogen) causes embryonic developmental injury [22,23,28,34,35]. In the present study, we investigated whether DHLA adversely affects the blastocyst stage of mouse embryos and subsequent early pre-and post-implantation embryonic development. ...
Article
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α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet, taken up by cells and tissues, and subsequently reduced to dihydrolipoic acid (DHLA). In view of the recent application of DHLA as a hydrophilic nanomaterial preparation, determination of its biosafety profile is essential. In the current study, we examined the cytotoxic effects of DHLA on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, in vivo implantation by embryo transfer, and early embryonic development in an animal model. Blastocysts treated with 50 μM DHLA exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with DHLA were lower than that of their control counterparts. Moreover, in vitro treatment with 50 μM DHLA was associated with increased resorption of post-implantation embryos and decreased fetal weight. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 100 μM DHLA led to decreased early embryo development, specifically, inhibition of development to the blastocyst stage. However, it appears that concentrations of DHLA lower than 50 μM do not exert a hazardous effect on embryonic development. Our results collectively indicate that in vitro and in vivo exposure to concentrations of DHLA higher than 50 μM DHLA induces apoptosis and retards early pre- and post-implantation development, and support the potential of DHLA to induce embryonic cytotoxicity.
... Research in rodent models indicates that administering genistein, bisphenol, and other environmental estrogenic compounds in females disrupted embryo implantation in adult female offspring, potentially due to oviduct-uterine abnormalities [45,46]. Regarding the impact of soy isoflavones on the blastocyst, isoflavone-induced inhibition of glucose uptake could inhibit early embryonic development, as glucose serves as the primary source of exogenous energy during the initial stages of pre-implantation embryo development [47]. Furthermore, high concentrations of genistein have been shown to inhibit phosphorylation of tyrosine in cadherin-catenin complexes, which play crucial roles in compaction, adhesive functions, and embryonic cleavage in mouse embryos [48]. ...
Article
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Soybean meal (SBM) is a prevailing plant protein supplement in animal diets because of its nutritional value and availability. This review paper explores the significance of SBM and processed soy products, emphasizing their nutritional and bioactive components, such as isoflavones and soyasaponins. These compounds are known for their antioxidant and anti-inflammatory properties and are associated with a reduced prevalence of chronic diseases. However, the presence of antinutritional compounds in SBM presents a significant challenge. The paper evaluates various processing methods, including ethanol/acid wash, enzyme treatment, and fermentation, which are aimed at enhancing the nutritional value of soy products. It highlights the significance to maintain a balance between nutritional enhancement and the preservation of beneficial bioactive compounds, emphasizing the importance of different processing techniques to fully exploit the health benefits of soy-based products. Therefore, this review illuminates the complex balance between nutritional improvement, bioactive compound preservation, and the overall health implications of soy products.
... Assisted reproductive technologies which involve the collection of immature oocytes followed by in vitro maturation, disrupt this process resulting in a reduction in oocyte quality (Carnevale, 2016). Oocyte ability indicators to be fertilized are represented by the presence of layers of compact cumulus cells around the oocyte and homogeneous cytoplasm (Chan et al., 2009). Oocytes evaluation needs to identify the correct morphological changes, the best procedure for oocytes classification consists of isolation and removing the debris by repeated washings (3times) and separating into groups of quality (Hinrichs, 2010b). ...
Article
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In the current study, 930 equine oocytes were collected, 765 (82.25%) of which were considered viable for in vitro maturation. All selected oocytes were matured for 27 h at 38.50 0 C in an atmosphere of 5% CO2 in humidified air. 25 mm hepes-buffered TCM-199 supplemented with 2 mm sodium pyruvate, 1mm l-glutamine, penicillin (75 mg/ml), streptomycin (50 mg/ml) and 10% fetal calf serum was used for in vitro maturation. Following maturation, matured COCs were examined to investigate for first polar body formation. The morphocytometric assessment was performed using the motic image plus (MIP) software with an inverted microscope. Morphocytometric examination results showed highly significant differences (p<0.001) among groups of matured oocytes (zona pellucida thickness; <13 µm, cumulus oophorus thickness; < 10 µm and oocytes diameter; < 100 µm), where the rates were 63.39%, 33.59% and 30.58%, respectively. The results of morphocytometric evaluation based on zona pellucida thickness, cumulus oophorus thickness and oocyte diameter of the total 765 cultured oocytes showed that 197 oocytes (25.75%) were classified as excellent mature, 108 oocytes (14.11%) as mature good, 203 oocytes (26.53%) as immature and 257 oocytes (33.59%) were considered as degenerated. It is concluded thatthe oocytes rates differed according to the parameters (cumulus oophorus, oocytes diameter, zona pellucida).
... This hypothesis has been supported by numerous results from in vitro and animal models. For instance, genistein treatment of murine oocytes significantly reduced the rate of oocyte maturation, in vitro fertilization, and embryonic development [155]. Similarly, treatment of murine oocytes with high-dose curcumin during in vitro maturation (IVM) impaired blastocyst development from the morula, promoted early-stage death of mouse blastocysts, increased resorption of postimplantation embryos, and decreased fetal weight [156]. ...
Article
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Polyphenols are a group of phytochemicals with extensive biological functions and health-promoting potential. These compounds are present in most foods of plant origin and their increased widespread availability through the intake of nutritional supplements, fortified foods, and beverages, has also led to increased exposure throughout gestation. In this narrative review, we focus on the role of polyphenols in both healthy and pathological pregnancy. General information related to their classification and function is followed by an overview of their known effects in early-pregnancy events, including the current insights into molecular mechanisms involved. Further, we provide an overview of their involvement in some of the most common pregnancy-associated pathological conditions, such as preeclampsia and gestational diabetes mellitus. Additionally, we also discuss the estimated possible risk of polyphenol consumption on pregnancy outcomes. The consumption of dietary polyphenols during pregnancy needs particular attention considering the possible effects of polyphenols on the mechanisms involved in maternal adaptation and fetal development. Further studies are strongly needed to unravel the in vivo effects of polyphenol metabolites during pregnancy, as well as their role on advanced maternal age, prenatal nutrition, and metabolic risk of the offspring.
... The naturally occurring genistein in soy is what has long been studied in relation to its potential effects on human and nonhuman hormonal functions, and specifically as a potential endocrine Thirty years more accurately reflects the time elapsed to date in the literature discussed. 6 disruptor with a range of studied effects on sexual dimorphism of the brain (Rosenfeld 2019;Sirotkin and Harrath 2014;Sumien et al. 2013;Kouki et al. 2003), in debates over male mammary gland development, or in possible disruptions to in vitro fertilization and embryonic development in mouse models (Messina 2010;Hamilton-Reeves et al. 2010;Chan 2009). Yet, as Chan (2009, 56) suggests, "the estrogenic and/or anti-estrogenic activities of [genistein] may reduce or enhance estrogen-dependent tumor growth, depending on the dose and timing of exposure." ...
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This article critically analyzes cultural anxieties around the plant-based phytoestrogens in soy. Drawing from biomedical data on the physiological effects of soy ingestion, I show the ways in which gendered food fear is co-constituted by science and its diffusion within popular media, including through privileging of the perspectives of heterosexual cisgender white men, from conspiracy radio host Alex Jones to Men’s Health or Good Housekeeping magazines. Gendered tropes of determinism, sexual mutation, and panic, such as hyperfocus on semen, sperm counts, or genitalia as determinant of masculinity in rat models, draw from critical biomedical research of the legume, but also center heteronormative, transphobic, and white supremacist understandings of food as a hormonal reproductive toxin. Drawing from a feminist and queer ecologies approach, this article connects the social and life sciences by showcasing the ways popular media propels gendered ecological fear in a historical moment of increased concern over the politics and power of food, health, and toxic exposures.
... We observed that litter size was significantly reduced by genistein, and at 50 mg/kg dose, there was no embryo, suggesting impairment in implantation. This effect could be explained by genistein's inhibitory role on early postimplantation development, leading to an early death of blastocysts (Chan, 2009;Chan et al., 2007). Exposure to genistein has been reported to modulate oviduct morphogenesis during development as a result of changes in Hoxa, wnt and hedgehog signalling (Jefferson et al., 2011). ...
Article
Genistein, an isoflavonoid, is found in a plethora of plant‐based foods, and has been approved for use in various therapies. A couple of studies in adult men observed a negative correlation between genistein exposure and reproductive parameters. To assess the effects of genistein exposure on reproduction and fertility the in males and females, we performed quantitative meta‐analyses by pooling data from published studies on animals that assessed various reproductive parameters. Pooled analysis showed significant decreases in sperm count in males exposed to genistein during adulthood (Hedges's g = −2.51, p = 0.013) and in utero (Hedges's g = −0.861, p = 0.016) compared with controls. In males exposed to genistein in utero, serum testosterone levels decreased (Hedges's g = −6.301, p = 0.000) and luteinizing hormone (LH) (Hedges's g = 7.127, p = 0.000) and FSH (Hedges's g = 6.19, p = 0.000) levels increased in comparison with controls. In females, the number of corpora lutea (Hedges's g = −2.103, p = 0.019) and the litter size (Hedges's g = −1.773, p‐value = 0.000) decreased; however, female reproductive hormones remained unaffected. These meta‐analyses show that genistein has detrimental effects on male reproductive system and on the progression and sustenance of pregnancy, with more pronounced adverse impact in males, particularly when exposed in utero.
... The measurement of placental weights revealed that this parameter was significantly lower in the group treated with 2 μM AOH plus 8 μM OTA compared with those treated with either toxin alone or with 0-1 μM AOH plus 8 μM OTA (Fig. 3B). Our group and others previously reported that about 35-40% of fetuses weigh >600 mg at Day 18 of pregnancy in mouse embryo transfer assays, and the average weight of a Day-18 fetus is ∼600 ± 12 mg [33,[42][43][44][45][46]. Fetal weight is accepted as an important indicator and parameter for assessing fetal developmental status in various pregnancy periods. ...
Article
Alternariol (AOH) and ochratoxin A (OTA), two mycotoxins found in many foods worldwide, exhibit cytotoxicity and embryotoxicity, triggering apoptosis and cell cycle arrest in several mammalian cells and mouse embryos. The absorption rate of AOH from dietary foodstuff is low, meaning that the amount of AOH obtained from the diet rarely approaches the cytotoxic threshold. Thus, the potential harm of dietary consumption of AOH is generally neglected. However, previous findings from our group and others led us to question whether a low dosage of AOH could aggravate the cytotoxicity of other mycotoxins. In the present study, we examined how low dosages of AOH affected OTA-triggered apoptosis and embryotoxicity and investigated the underlying regulatory mechanism in mouse blastocysts. Our results revealed that non-cytotoxic concentrations of AOH (1 and 2 μM) could enhance OTA (8 μM)-triggered apoptotic processes and embryotoxicity in mouse blastocysts. We also found that AOH can enhance OTA-evoked intracellular reactive oxygen species (ROS) generation and that this could be prevented by pretreatment with the potent ROS scavenger, N-acetylcysteine. Finally, we observed that this ROS generation acts as a key inducer of caspase-dependent apoptotic processes and subsequent impairments of embryo implantation and pre- and post-implantation embryonic development. In sum, our results show that non-cytotoxic dosages of AOH can aggravate OTA-triggered apoptosis and embryotoxicity through ROS- and caspase-dependent signaling pathways.
... In the past few years, OM has proved to be a target for endocrine disruption by environmental chemicals capable of acting as hormone mimics, receptor blockers, and/or enzyme inhibitors. The inhibitory nature of organochlorine pesticides (methoxychlor, lindane, and dieldrin) and natural isoflavones (genistein) on OM is well documented in mammals (Picard et al. 2003;Chan 2009). Not limited to mammals, the impact of diverse endocrine disruptors on aquatic species, specifically the fish, has also become a global concern. ...
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Reproduction process is important for fish population productivity and in its resilience to fishing and environmental changes. Reproductive potential is the ability to generate feasible amount of quality eggs in relation to the energy available and parental life expectancy. The important parameters for estimating the reproductive potential of the fish are maturity, fecundity, sex ratio, and fish condition which directly affect fish population productivity. Euthynnus affinis in the Veraval region of Gujarat is caught by using drift gillnets throughout the year with peak during December to March. The overall sex ratio is 0.82. The spawning occurs round the year with peak during December to March. The size at first maturity ranges from 55 to 60 cm, whereas absolute fecundity ranged from 171,550.4 to 827,734.35/kg body weight. The size of ova varied from 0.34 to 0.61 mm. Gonadosomatic index is highest during the month of December. Opportunistic predatory nature is in adults, and the most frequently encountered food was fishes, shrimps, Decapterus, squids, and digested fish.
... During physiologic embryogenesis, albeit after the blastocyst stage, the apoptosis removes the abnormal and redundant cells, leading to embryo development arrest because of DNA fragmentation and/or mutation (7,25). On the other hand, the early apoptosis (mainly through mitochondria-dependent pathway) of pre-implantation embryos (mainly at stages of 2-cells and 8-cells) has been shown to positively correlate with oxidative stress (7,(26)(27)(28). Minding these issues, it could be suggested that the NMC (at dose levels of 15 and 30 mg kg -1 ) was able to potentially induce oxidative stress, and via this mechanism, it could result in embryo development arrest. ...
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The present study was done to uncover the possible beneficial and/or detrimental effect(s) of nano-micelle curcumin (NMC) on oocyte in-vitro maturation and pre-implantation embryo development. Forty-eight mature female Wistar rats were assigned to control, 7.5, 15, and 30 mg/kg-1 NMC-receiving (orally, for 48 days) groups. To assess the cumulus-oocyte complexes (COCs), the ovaries were stimulated by administrating (i.p.) a 25 IU of the pregnant mare's serum gonadotropin (PMSG) hormone. Following 48-h, 15 IU of hCG was injected (i.p.), and the COCs were taken after 16-18-h. To analyze the pre-implantation embryo development ratio, the sperms were collected from clinically healthy male Wistar rats, and 3.0-3.6 × 106 per mL was added into the fertilization drop. The animals in 7.5 mg/kg-1 NMC-receiving group exhibited a higher oocyte number versus control and other NMC-receiving groups. The NMC, in a dose-dependent manner, decreased the Zygote, 2-cell, blastocyst percentages, as well as hatched embryos, compared to the control group (P < 0.05). The 15 and 30 mg/kg-1 NMC-receiving groups represented a remarkable enhancement in type I arrest. Meanwhile, a significant (P < 0.05) reduction was revealed in type III embryo arrest in the same groups. The NMC, at 7.5 mg/kg-1 potentially enhances the oocyte number, while it fairly reduces the pre-implantation embryo development, even when it is administrated in dose levels of 7.5 mg/kg-1 and/or higher. Although more studies are needed, the NMC could be considered as a suppressor of fertility potential, when consumed chronically even in low doses.
... Oocyte growth, maturation and viability are very sensitive to the environment and can be affected by certain microenvironment factors or status, including oxygen concentration, temperature, and glucose content [25][26][27]. Multiple studies by other research groups and our laboratory have reported that chemical or physical teratogens trigger cell death or induce cell damage during oocyte maturation or in preimplantation stage embryos, with harmful consequences for embryonic development [26,[28][29][30][31][32][33][34]. ...
Article
Previous studies have shown that berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines, suppresses growth and induces apoptosis in some tumor cell lines. It has also been shown that berberine possesses anti-atherosclerosis and antioxidant activities in hyperlipidemic model rats. Our previous study in mice found that berberine causes harmful effects on preimplantation and postimplantation embryonic development, both in vitro and in vivo, by triggering reactive oxygen species (ROS)-mediated apoptotic cascades in mouse blastocysts. In the current investigation, we further showed that berberine treatment has distinct dose-dependent effects on oocyte maturation and subsequent development. Preincubation of oocytes with 2.5 μM berberine significantly enhanced maturation and in vitro fertilization (IVF) rates, with subsequent beneficial effects on embryonic development. In contrast, preincubation with 10 μM berberine negatively impacted mouse oocyte maturation, decreased IVF rates and impaired subsequent embryonic development. Similar dose-dependent effects were also demonstrated in vivo. Specifically, intravenous injection of berberine significantly enhanced mouse oocyte maturation, IVF rate and early-stage embryo development after fertilization at a dose of 1 mg/kg body weight but significantly impaired oocyte maturation and IVF rates and caused harmful effects on early embryonic development at a dose of 5 mg/kg. Mechanistically, we found that berberine enhanced intracellular ROS production and apoptosis of oocytes at a concentration of 10 μM but actually significantly decreased total intracellular ROS content and had no apoptotic effect at a concentration of 2.5 μM. Moreover, pretreatment of oocytes with Ac-DEVD-cho, a caspase-3-specific inhibitor, effectively blocked berberine-induced negative impacts on oocyte maturation, fertilization and subsequent development. Collectively, these findings establish the dose-dependent beneficial versus deleterious effects of berberine and suggest that the mechanism underlying the deleterious effects of berberine involves a caspase-3-dependent apoptotic process acting downstream of an increase in intracellular ROS levels.
... Many of these phytoestrogens like resveratrol and trans-resveratrol demonstrate a broad spectrum of pharmacological and therapeutic health benefits (42,43). The uterotrophic property of coumestrol and genistein and many other phytoestrogens' effects on estrous cycle, oocyte maturation, fertilization and sequential embryonic development have been reported earlier (44,45). ...
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Background: Scoparia dulcis Linn. is reported to be used by women of Assam and Arunachal Pradesh in northeast India for treating menstrual disorders. Scoparia dul- cis contains compounds that bind with estrogen receptors (ERα and ERβ) evidenced by increased PCNA in endometrial epithelium. Methods: Crude extract was orally administered at the dose of 500 mg/kg body weight/day to the female mice (60–70 days old) in five different groups. Each group containing six females included: (I) cyclic control, (II) cyclic extract treated, (III) Ovariectomized (OVX)-vehicle treated (Control), (IV) OVX-E2 treated (V) OVX- extract treated. Extract was administered for eight days to the cyclic groups and three days to the OVX groups. PCNA was detected immunohistochemically in uterine tiss ues and signals were analyzed by Image J software (NIH, USA). Compounds were separated by GC-MS and identified using NIST. In silico molecular docking studies was performed with human estrogen receptors (ERα and ERβ). Molecular dynamics (MD) simulations of the best interacting compound was done using gromacs. Results: The results showed cell proliferation in the uterine endometrium evidenced by PCNA. Two phytocompounds, Octadecanoic acid and methyl stearate showed binding affinity with ERα and ERβ. Conclusion: Scoparia dulcis contains compounds having binding affinity with ERα and ERβ. The present study is the first report on compounds from Scoparia dulcis showing binding affinity with human estrogen receptors which may have biological effect on female reproduction.
... During normal embryogenesis, the process of apoptosis removes abnormal or redundant cells from pre-implantation embryos [26]. Induction of apoptosis during early stages of embryogenesis (i.e., following exposure to a teratogen) compromises embryonic development [27,28]. The main methods to study teratogens are either through epidemiological studies in human populations or by controlled exposure in animal models. ...
Article
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Ochratoxin A (OTA) is a mycotoxin produced by different Aspergillus and Penicillium species, and it is considered a common contaminant in food and animal feed worldwide. On the other hand, human embryonic stem cells (hESCs) have been suggested as a valuable model for evaluating drug embryotoxicity. In this study, we have evaluated potentially toxic effects of OTA in hESCs. By using in vitro culture techniques, specific cellular markers, and molecular biology procedures, we found that OTA produces mild cytotoxic effects in hESCs by inhibiting cell attachment, survival, and proliferation in a dose-dependent manner. Thus, we suggest that hESCs provide a valuable human and cellular model for toxicological studies regarding preimplantation stage of human fetal development.
... Specifically, genistein inhibits steroidogenesis or steroidogenic enzymes in cultured rat pre-antral follicles [22], rat granulosa-luteal cells [23], porcine granulosa, theca, or luteal cells [24][25][26][27], and mouse antral follicles [28]. Additionally, studies have shown that genistein exposure alters follicle growth [17], induces follicular atresia [29], inhibits oocyte maturation [30], and inhibits antral follicle growth [28] in rodents. Collectively, these studies show that genistein exposure can affect hormone levels and follicle growth, both of which are necessary for normal female fertility [31]. ...
Article
Genistein is a phytoestrogen found in soy and soy-based products. Previously, we found that genistein adversely affected estradiol levels and follicle growth in vitro. Proper hormone production and follicle growth are key regulators of normal fertility. Therefore, we hypothesized that genistein adversely affects female fertility and pregnancy outcomes. To test this hypothesis, we dosed sexually mature female CD-1 mice (35days) with 0, 300, 500, or 1000ppm genistein for 30, 60, 150, and 240days. At the end of the dosing periods, we measured mating rate, pregnancy rate, fertility rate, gestation time, parturition time, pup mortality, litter size, average pup weight, and estradiol and progesterone levels. We found that chronic, preconception exposure to genistein affects gestation time, parturition time, litter size, pup weight, and pup mortality. Additionally, genistein exposure for 240days appears to have a protective effect on fertility rate, but does not affect hormone levels in vivo.
... Interestingly, the placental weights derived from rhein-pretreated embryos were not significantly different from those of the untreated control group ( Figure 3B). However, the average fetal weight, which is considered an important indicator of developmental status [20,[31][32][33][34], was significantly lower in the 10 and 20 μM rhein-treated groups compared to the control group ( Figure 3C). The embryo transfer assay showed that 46.7% fetal weight of the control group was >600 mg and 42.9% fetal weight was 400-600 mg. ...
Article
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Rhein, a glucoside chemical compound found in a traditional Chinese medicine derived from the roots of rhubarb, induces cell apoptosis and is considered to have high potential as an antitumor drug. Several previous studies showed that rhein can inhibit cell proliferation and trigger mitochondria-related or endoplasmic reticulum (ER) stress-dependent apoptotic processes. However, the side effects of rhein on pre- and post-implantation embryonic development remain unclear. Here, we show that rhein has cytotoxic effects on blastocyst-stage mouse embryos and induces oxidative stress and immunotoxicity in mouse fetuses. Blastocysts incubated with 5–20 μM rhein showed significant cell apoptosis, as well as decreases in their inner cell mass cell numbers and total cell numbers. An in vitro development assay showed that rhein affected the developmental potentials of both pre- and post-implantation embryos. Incubation of blastocysts with 5–20 μM rhein was associated with increased resorption of post-implantation embryos and decreased fetal weight in an embryo transfer assay. Importantly, in an in vivo model, intravenous injection of dams with rhein (1, 3, and 5 mg/kg body weight/day) for four days resulted in apoptosis of blastocyst-stage embryos, early embryonic developmental injury, and decreased fetal weight. Intravenous injection of dams with 5 mg/kg body weight/day rhein significantly increased the total reactive oxygen species (ROS) content of fetuses and the transcription levels of antioxidant proteins in fetal livers. Additional work showed that rhein induced apoptosis through ROS generation, and that prevention of apoptotic processes effectively rescued the rhein-induced injury effects on embryonic development. Finally, the transcription levels of the innate-immunity related genes, CXCL1, IL-1 β and IL-8, were down-regulated in the fetuses of dams that received intravenous injections of rhein. These results collectively show that rhein has the potential to induce embryonic cytotoxicity and induce oxidative stress and immunotoxicity during the development of mouse embryos.
... But what might be positive attributes in the setting of adult cancer may have different consequences in pre-and postnatal life where epigenetic landscapes are rapidly shifting (Gluckman et al. 2008). Phytoestrogens affect epigenetic programs in differentiating embryonic stem cells (Sato et al. 2011), developing embryos (Chan 2009;Dolinoy 2008), and, as we observed here, infants who consume soy formula. ...
Article
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Background: Early life exposure to estrogenic compounds affects the development of the reproductive system in rodent models and humans. Soy products, which contain phytoestrogens such as genistein, are one source of exposure in infants fed soy formula and result in high serum concentrations. Objectives: To determine if soy exposure is associated with differential DNA methylation in vaginal cells from soy-fed infant girls. Methods: Using the Illumina HumanMethylation450 BeadChip we evaluated epigenome-wide DNA methylation in vaginal cells from four soy-formula-fed and six cow-formula-fed girls from the Infant Feeding and Early Development (IFED) study. Using pyrosequencing we followed up the two most differentially methylated sites in 214 vaginal cell samples serially collected between birth and nine months of age from 50 girls (28 soy-formula-fed and 22 cow-formula-fed). With a mouse model, we examined the effect of neonatal exposure to genistein on gene specific mRNA levels in vaginal tissue. Results: The epigenome-wide scan suggested differences in methylation between soy-formula-fed and cow-formula-fed infants at three CpGs in the gene proline rich 5 like (PRR5L) (p<10(4)). Pyrosequencing of the two feeding groups found that methylation levels progressively diverged with age, with pointwise differences becoming statistically significant after 126 days. Genistein exposed mice showed a 50% decrease in vaginal Prr5l mRNA levels compared to controls. Conclusions: Girls fed soy formula have altered DNA methylation in vaginal cell DNA which may be associated with decreased expression of an estrogen-responsive gene.
... Genistein inhibits steroidogenesis or steroidogenic enzymes in cultured rat pre-antral follicles (Myllymaki et al., 2005), rat granulosa-luteal cells (Whitehead and Lacey, 2000), and porcine granulosa, theca, or luteal cells (Gregoraszczuk et al., 1999;Nynca and Ciereszko, 2006;Tiemann et al., 2007;Basini et al., 2010). Exposure to genistein also alters follicle growth (Zhuang et al., 2010), induces follicular atresia (Zin et al., 2013), and inhibits oocyte maturation (Chan, 2009). However, these studies have not examined the effects of genistein on the intact, adult antral follicle, the functional unit of the ovary. ...
... In this study in vitro maturated oocytes to MII stage in oocytes treated with 200 g/ml P. rhoeas extract was significantly lower than 50 g/ml extract; however, no significant difference was observed when compared to other concentrations of extract. Reduction in in vitro maturated oocytes to MII stage might be due to deleterious effects of excessive concentrations of the extract, because some flavonoids have toxic effects (Chan, 2005;Wu et al., 2005;Chan, 2009). Excessive amount of chelators such as ethylene diamine tetra-acetic acid (EDTA) in developmental medium may be harmful (Olson and Seidel, 2000) and high concentrations of anthocyanins reduce the proliferation ability and viability of human cells (Glei et al., 2003). ...
Article
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This study was conducted to determine the effect of Papaver rhoeas L. extract on in vitromaturation (IVM) of sheep oocytes. Sheep ovaries collected from a local abattoir were trans-ported to the laboratory within 2 h after slaughter. The cumulus–oocyte complexes (COCs)were aspirated from follicles (2–6 mm in diameter). Good quality COCs were selected andcultured in TCM 199 supplemented with 0 (control), 25, 50, 100 and 200 �g/ml of P. rhoeasextract. The COCs were incubated at 38.5◦C in humidified atmosphere of 5% CO2in airfor 24 h. After the incubation period, expansion of cumulus cells and the nuclear status ofthe oocytes were assessed by phase contrast microscopy and Hoechst 33258, respectively.There were no significant differences between treatments in the percentage of cumulusexpansion (P > 0.05). The percentage of arrested oocytes at germinal vesicle (GV) stage inthe control group was significantly (P < 0.05) higher than other treatments. There were sig-nificant (P < 0.05) differences between all concentrations of extract and the control group inthe percentage of in vitro maturated oocytes to metaphase II (MII) stage. The oocytes treatedwith 50 �g/ml extract achieved the highest percentage of MII stage compared to the con-trol (P < 0.05); however, no significant difference observed between 25, 50 and 100 �g/mlextract. In conclusion, the results of this study show that supplementation of appropriateconcentrations of P. rhoeas extract (50 �g/ml) in maturation medium improve the sheepoocyte maturation rate. Moreover, effects of P. rhoeas extract on oocyte maturation weredependent on the extract concentration in the maturation medium.
... Additionally, studies have been conducted for evaluating the effects of various compounds on embryonic development: 3-hydroxyflavone [6] has been shown to have a beneficial effect on embryonic development, whereas studies suggest that flavonoids such as genistein [7], ginkgolides [8], and daidzein [9] may be toxic. ...
Article
Treatment with resveratrol at concentrations greater than 0.5 μmol/L resulted in the arrest of mouse embryo development at the two-cell stage. Resveratrol-induced cytotoxicity was investigated in embryos by evaluating morphologic features by using the bromodeoxyuridine assay and acridine orange and ethidium bromide double staining. Resveratrol was found to significantly increase the expressions of p53, p21, Atf3, smac/Diablo, Bax, Bak1, Bok, and Noxa mRNA in the embryos, whereas Cullin 3 and Cdk1 expressions were decreased. Furthermore, active p53 positive signal in embryos arrested at the two-cell stage was localized in the nucleus, whereas no active p53 signal was observed in control embryos. Pretreatment with pifithrin-α, a p53 inhibitor, downregulated active p53 in two-cell embryo nuclei and ameliorated approximately 50% of the embryonic developmental defect caused by resveratrol. The findings of the present study, therefore, suggest that pifithrin-α could be used as an effective cytoprotective agent against a reproductive toxin such as resveratrol. Copyright © 2015 Elsevier Inc. All rights reserved.
... There was also a significant decrease in the foetoplacenta weight at twelve day of pregnancy in pregnant rats pre-exposed to genistein and this observed effect was reversed in the corresponding seven days reversal groups 25 ( Table 2). Similar effects have been reported in mouse . ...
Article
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Background: The occurrence of phytoestrogens in food gaining increasing attention due to the acclaimed health benefits. Objective: This study examined the effect of genistein on estrous cyclicity, fertilization of ovulated oocytes, foetal weight, female hormones and possibilities of reversal of any detrimental effects in rats. Methods: Estrous cycle was monitored before and during genistein administration, blood samples were collected for hormonal analysis at terminal days. Some rats were sacrificed on second day of pregnancy, the oviducts were removed and flushed for collection of fertilized and unfertilized oocyte. Results: Genistein adversely altered the estrous cycle with significant reduction in bodyweight gained, increase in uterine weight, reduction in the percentage of the ovulated oocyte that were fertilized despite no significant change in the number of ovulated oocyte. Pregnancy was characterized with reduction in circulating progesterone level along with significant reduction in number of implants, corpora lutea count, pituitary weight and foeto-placenta weight. The increased resorption of implants was attributed to the reduction in progesterone level and a reduction in the surviving corpora lutea. These adverse effects were not reversed within the seven days recovery allowed of administration. Conclusion: Results also showed that oral exposure to genistein not only alters oestrous cyclicity and fertilization of ovulated oocytes, but exposure during pregnancy in rats, precipitates adverse effects on pregnancy due to its inhibitory effects on corporal luteum survival, and progesterone production.
... Overall, fetal weight was lower in the 25-50 mM EGCG-treated group, compared to the control group ( Figure 4C). Previous investigations, including a recent report by our group, showed that 35-40% of fetuses weigh more than 600 mg, with the average weight of total surviving fetuses being approximately 600 AE 12 mg in the untreated control group at day 18 of pregnancy in a mouse embryo transfer assay (Chan, 2006(Chan, , 2009, 2008Huang et al., 2006). Fetal weight is an important indicator of developmental status. ...
Article
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Abstract Catechins, a family of polyphenols found in tea, evoke various responses, including cell death. However, the side effects of these compounds, particularly those on embryonic development, have not been characterized in detail. A previous study by our group showed that (-)-epigallocatechin-3-gallate (EGCG), a catechin highly abundant in green tea, induces different cell-death modes in MCF-7 cells, depending on the treatment dosage. In the current study, we examined the effects of EGCG on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro and in vivo implantation by embryo transfer. Blastocysts treated with 25-50 μM of EGCG exhibited a significant increase in apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with EGCG was lower than that of their control counterparts. Moreover, in vitro treatment with 25-50 μM of EGCG led to increased resorption of postimplantation embryos and decreased fetal weight. EGCG appeared to induce injury in mouse blastocysts through intrinsic apoptotic signaling processes to impair sequent embryonic development. These results collectively highlight the potential of EGCG to induce embryonic cytotoxicity.
... In this study in vitro maturated oocytes to MII stage in oocytes treated with 200 g/ml P. rhoeas extract was significantly lower than 50 g/ml extract; however, no significant difference was observed when compared to other concentrations of extract. Reduction in in vitro maturated oocytes to MII stage might be due to deleterious effects of excessive concentrations of the extract, because some flavonoids have toxic effects (Chan, 2005;Wu et al., 2005;Chan, 2009). Excessive amount of chelators such as ethylene diamine tetra-acetic acid (EDTA) in developmental medium may be harmful (Olson and Seidel, 2000) and high concentrations of anthocyanins reduce the proliferation ability and viability of human cells (Glei et al., 2003). ...
... Cattle are potentially at risk for reproductive effects of both phytoestrogens and environmental estrogens. Adverse effects of phytoestrogens on fetal development have been reported for rodents (Chan, 2009). Brief exposures to environmental estrogens, such as bisphenol A (Bpa), during fetal development may adversely affect oocyte development in mice (Susiarjo et al., 2007) and in women, exposure to BPA is correlated with recurrent miscarriage (Sugiura-Ogasawara et al., 2005). ...
Article
The ovarian follicular reserve has been linked to fertility in cattle. Young adult cattle with low vs. high numbers of antral follicles >3 mm in diameter in follicular waves also have lower numbers of preantral follicles and decreased fertility. This underscores the importance of understanding the factors that regulate early follicular development and establish the ovarian follicular reserve, but little is known about how the follicular reserve is first established. In ruminants and humans, follicles form during fetal life, but there is a gap (about 50 d in cattle) between the appearance of the first primordial follicles and the first growing, primary follicles. In this review we present evidence that in cattle, fetal ovarian steroids (i.e., estradiol and progesterone) are negative regulators of both follicle formation and of the acquisition by newly formed follicles of the capacity to activate (i.e., initiate growth). The results indicate that capacity to activate is linked to the completion of meiotic prophase I by the oocyte. The inhibitory effects of estradiol on follicle activation were found to be reversible and correlated with inhibition of the progression of meiotic prophase I. Fetal bovine ovaries produce steroid hormones and production varies considerably during gestation and in a pattern consistent with the hypothesis that they inhibit follicle formation and capacity of newly formed follicles to activate in vivo. However, little was known about how steroid production is regulated. In our studies, both LH and FSH stimulated progesterone and estradiol production by ovarian pieces in vitro. The addition of testosterone to the culture medium enhanced estradiol production, especially when FSH was also present, but inhibited progesterone production, even in the presence of gonadotropins. Evidence is also presented for effects of maternal nutrition and health and for potential effects of estrogenic endocrine-disrupting chemicals on the size of the ovarian follicular reserve established during fetal life. In summary, fetal ovarian steroids may be important regulators of the early stages of follicular development in cattle. Therefore, external factors that alter steroid production or action may affect the size of the ovarian follicular reserve.
... We suggest that the reduction of oocyte maturation and blastocyst formation rates with this concentration of quercetin may result from unresponsive signaling to oocytes and embryos or direct embryo toxicity owing to excessive levels of flavonoids. Some groups have reported toxic effects of other flavonoids on embryos from different species [4][5][6]27]. In contrast, several investigations have shown that supplementation of porcine IVM medium with antioxidants such as selenium, vitamin E, and ascorbic acid decreases ROS levels while enhancing the developmental competence of IVF embryos and parthenotes [21,29,30]. ...
Article
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Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels during oocyte maturation. Immature oocytes were untreated or treated with 1, 10, and 50 μg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 μg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 μg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of queretin (50 μg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.
... During normal embryogenesis, apoptosis (a unique morphological pattern of cell death) functions to remove abnormal or redundant cells in preimplantation embryos [16,17]. However, apoptotic processes do not occur prior to the blastocyst stage during normal mouse embryonic development [18], and induction of cell death during oocyte maturation and early stages of embryogenesis (i.e., via exposure to a teratogen) leads to developmental injury [14,[19][20][21][22]. While we have established that emodin promotes cell apoptosis and developmental injury in blastocyst-stage embryos [12], the influence of this compound on early-stage embryogenesis processes, such as oocyte maturation, fertilization, and sequential embryo development from zygotes, is currently unclear. ...
Article
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Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20-40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process.
... We suggest that the reduction of oocyte maturation and blastocyst formation rates with this concentration of quercetin may result from unresponsive signaling to oocytes and embryos or direct embryo toxicity owing to excessive levels of flavonoids. Some groups have reported toxic effects of other flavonoids on embryos from different species [4][5][6]27]. In contrast, several investigations have shown that supplementation of porcine IVM medium with antioxidants such as selenium, vitamin E, and ascorbic acid decreases ROS levels while enhancing the developmental competence of IVF embryos and parthenotes [21,29,30]. ...
Article
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Quercetin is a plant-derived flavonoid found in fruits, vegetables, leaves and grains that has antioxidant properties and act as a free radical scavenger. Although much interest about quercetin was increased, there is limited information available on the effect of quercetin on oocytes and embryos in pigs. Therefore, we investigated the effects of quercetin on nuclear maturation of porcine oocytes and embryonic development after parthenogenetic activation. Then we evaluated the antioxidant effects of quercetin by measuring reactive oxygen species (ROS) levels during oocytes maturation. Immature oocytes were untreated or treated with 1, 10 and 50 µg/ml quercetin during in vitro maturation (IVM). Quercetin treatment did not improve the nuclear maturation of oocytes, but a significantly greater proportion (15.81%, P<0.05) of parthenogenetically activated oocytes developed into blastocysts when the IVM medium was supplemented with adequate quercetin (1 µg/ml) compared to control (9.82%), 10 μg/ml (8.46%) or 50 μg/ml (3.46%) groups; however, cleavage rate and blastocyst cell number were not affected. Oocyte treated with 1 or 10 µg/ml quercetin had significantly lower levels of ROS than the control (0 µg/ml) and highest concentration (50 µg/ml) groups. Moreover, quercetin at the highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. We conclude that exogenous quercetin is beneficial for nuclear maturation during porcine IVM and subsequent embryo development by reducing of ROS levels. This study was supported by MKE (#10033805-2010-12/ #10033839-2010-12), NRF (#M10625030005-508-10N25), BK21 for Veterinary Science, and IPET (#109023-05-1-CG000). (poster)
... During normal embryogenesis, apoptosis (a unique morphological pattern of cell death) functions to remove abnormal or redundant cells in preimplantation embryos [26,27]. However, apoptotic processes do not occur prior to the blastocyst stage during normal mouse embryonic development [28], and induction of cell death during oocyte maturation and early stages of embryogenesis (i.e., via exposure to a teratogen) leads to embryonic developmental injury [21,24,293031. Previous studies by our group have demonstrated that curcumin promotes cell apoptosis and developmental injury in blastocyst stage embryos [22]. ...
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Curcumin, a common dietary pigment and spice, is a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa. Previously, we reported a cytotoxic effect of curcumin on mouse embryonic stem cells and blastocysts and its association with defects in subsequent development. In the present study, we further investigated the effects of curcumin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, curcumin induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with curcumin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments with an in vivo mouse model disclosed that consumption of drinking water containing 40 μM curcumin led to decreased oocyte maturation and in vitro fertilization as well as early embryonic developmental injury. Finally, pretreatment with a caspase-3-specific inhibitor effectively prevented curcumin-triggered injury effects, suggesting that embryo impairment by curcumin occurs mainly via a caspase-dependent apoptotic process.
Article
The reproductive toxicity of endocrine-disrupting chemicals has become a matter of great concern. However, the potential toxicological mechanism of typical environmental estrogens, bisphenol A (BPA) and genistein (GEN), on adult ovary remains ambiguous. In this study, we used laying hens as the experimental model and aimed to clarify the effect of long-term exposure to safe reference doses of BPA and GEN on adult ovary. Results demonstrated that 1/10 no-observable-adverse effect-level dose (1/10 NOAEL, 500 μg/kg body weight [bw]/day) of BPA significantly reduced the production performance and caused the degeneration of follicles and stromal cells and the increase of atretic follicles. Moreover, 1/10 NOAEL dose of BPA undermined the redox homeostasis of the ovary through activating Keap1 and suppressing the Nrf2-signaling pathway (Nrf2, NQO1, and HO-1). On the contrary, GEN (20, 40 mg/kg bw/day) dramatically improved the antioxidant capacity of the ovary by regulating the Nrf2-Keap1 pathway, enhancing the activities of antioxidant-related enzymes (CAT, GSH-Px, and T-SOD), and inhibiting the excessive accumulation of lipid peroxidation products (MDA). Parallel in vitro studies confirmed that the differential role of BPA and GEN on ovarian redox balance was directly mediated by Nrf2-Keap1 antioxidant system. And GEN could ameliorate BPA-induced oxidative stress. Importantly, our research found that exposure to BPA and GEN altered estrogen receptor alpha (ERα) expression in the ovary. And the use of specific ERα agonist/antagonist confirmed that BPA and GEN have opposite regulatory effects on the Nrf2-Keap1 pathway by targeting ERα.
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Oogenesis, an amalgamation of the balanced network of neuroendocrine, endocrine, and autocrine/paracrine factors, is inevitable for the production of fertilizable female gamete and sustenance of a progeny on earth. In today’s up-to-the-minute world, aquatic organisms are exposed to a myriad of environmental anthropogenic contaminants that share structural similarity with natural hormones, putting fish fertility and aquaculture industries at stake. A subset of such endocrine disruptors is the “xenoestrogens” that carry the ability to mimic 17β-estradiol, a natural female hormone, leading to adverse outcomes such as early puberty, premature ovarian failure, and impaired fertility. The present review seeks to elucidate the voyage of a fish oocyte undertaking the endocrine as well as autocrine/paracrine inputs. The effects of EDCs on various ovarian processes have been summarized along with the diverse signaling cascades that might participate to induce significant alterations at the receptor level, steroidogenic potential, maturational competence, ovulatory response, or even epigenetics of the ovary. Since reproduction heavily relies on the metabolic state of an organism, the potential influence of endocrine disruptors on oxidative stress and energy homeostasis has also been taken into consideration.
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Ginsenoside Rb1 (GRb1), the major saponin component of ginseng root, has a wide range of therapeutic applications for various diseases. Previously, our group showed that GRb1 triggers ROS‐mediated apoptotic cascades in mouse blastocysts, leading to decreased cell viability and impairment of pre‐ and postimplantation embryonic development, both in vitro and in vivo. In this study, we further found that GRb1 exerted dose‐dependent effects on oocyte maturation and sequent development in vitro. Oocytes preincubated with 25 μg/mL GRB1 displayed significantly enhanced maturation and in vitro fertilization (IVF) rates, along with progression of subsequent embryonic development. In contrast, treatment with 50 and 100 μg/mL GRB1 led to impairment of mouse oocyte maturation, decreased IVF rates, and injurious effects on subsequent embryonic development. In vivo, intravenous injection of 1 mg/kg body weight GRb1 significantly promoted mouse oocyte maturation, IVF, and early‐stage embryo development after fertilization while administration of 5 mg/kg body weight GRb1 led to a marked decrease in oocyte maturation and IVF rates concomitant with impairment of early embryonic development in our animal model. In terms of the mechanisms underlying the regulatory effects of GRb1 demonstrated increased intracellular reactive oxygen species (ROS) production and apoptosis in the 100 μg/mL GRb1 treatment group. However, we observed a significant decrease in total intracellular ROS content and inhibition of apoptosis events in the 25 μg/mL GRb1 treatment group, signifying that the intracellular ROS content serves as a key upstream regulator of GRb1 that influences its dose‐dependent beneficial or deleterious effects on oocyte maturation and sequent embryonic development. For further clarification of the mechanisms underlying GRb1‐triggered injurious effects, oocytes were pretreated with Ac‐DEVD‐CHO, a caspase‐3‐specific inhibitor, which effectively blocked injury to oocyte maturation, fertilization, and sequent development. In sum, study findings highlight the potential involvement of p53‐, p21‐, and caspase‐3‐dependent regulatory signaling cascades in GRb1‐mediated apoptotic processes.
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Silver nanoparticles (AgNPs) are antibacterial materials widely used in numerous products and medical supplies. Previously, we showed that AgNPs trigger apoptotic processes in mouse blastocysts, leading to a decrease in cell viability and impairment of preimplantation and postimplantation embryonic development in vitro and in vivo. In the present study, we further investigated the hazardous effects of AgNPs on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent preimplantation and postimplantation development in vitro and in vivo. Data from in vitro experiments revealed that AgNPs impair mouse oocyte maturation, decrease IVF rates, and induce injury effects on subsequent embryonic development to a significant extent. In an animal model, intravenous injection of AgNPs (5 mg/kg body weight) led to a significant decrease in mouse oocyte maturation and IVF concomitant with impairment of early embryonic development in vivo. Importantly, pretreatment with N‐acetylcysteine effectively prevented AgNP‐triggered reactive oxygen species (ROS) production and apoptosis, clearly suggesting a critical role of ROS as an upstream initiator or key regulator of AgNP‐induced hazardous effects on oocyte maturation and sequent embryonic development. Furthermore, preincubation of oocytes with Ac‐DEVD‐cho, a caspase‐3‐specific inhibitor, effectively prevented hazardous effects, highlighting the potential involvement of caspase‐dependent apoptotic signaling cascades in AgNP‐mediated events. Expression levels of p53 and p21 of blastocysts were upregulated upon preincubation of mouse oocytes with AgNPs. Our collective results imply that cell apoptosis in mouse blastocysts derived from the AgNP‐pretreated oocytes via intracellular ROS generation, which is further mediated through p53‐, p21‐, and caspase‐3‐dependent regulatory mechanisms.
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Phytoestrogens are increasingly used as dietary supplements due to their suggested health promoting properties, but also by women for breast enhancement and relief of menopausal symptoms. Generally, phytoestrogens are considered to exert estrogenic activity via estrogen receptors (ERs), but they may also affect estrogen synthesis and metabolism locally in breast, endometrial and ovarian tissues. Considering that accurate regulation of local hormone levels is crucial for normal physiology, it is not surprising that interference with hormonal synthesis and metabolism is associated with a wide variety of women’s health problems, varying from altered menstrual cycle to hormone-dependent cancers. Yet, studies on phytoestrogens have mainly focused on ER-mediated effects of soy-derived phytoestrogens, with less attention for steroid synthesis and metabolism or other phytoestrogens. This review aims to evaluate the potential of phytoestrogens to modulate local estrogen levels and the implications for women’s health. For that, an overview is provided of the effects of commonly used phytoestrogens, i.c. 8-prenylnaringenin, biochanin A, daidzein, genistein, naringenin, resveratrol and quercetin, on estrogen synthesizing and metabolizing enzymes in vitro. The potential implications for women’s health are assessed by comparing the in vitro effect concentrations with blood concentrations that can be found after intake of these phytoestrogens. Based on this evaluation, it can be concluded that high-dose supplements with phytoestrogens might affect breast and endometrial health or fertility in women via modulation of steroid hormone levels. However, more data regarding tissue levels of phytoestrogens and effect data from dedicated, tissue-specific assays is needed for a better understanding of potential risks. At least until more certainty regarding the safety has been established, especially young women would better avoid using supplements containing high doses of phytoestrogens.
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Genistein (GEN), an isoflavonoid phytoestrogen, is one of the potent estrogenic compounds derived from plants that can cause disrupting effects on sex organ development in non-mammalian and mammalian species. The present study revealed effect of genistein on germ cell number in the genital ridges during gonadogenesis. Genistein (16 and 24 mu g/g egg) was injected into the egg yolk prior to incubation. Effect of genistein on quail-primordial germ cells (PGCs) number was examined by counting the number of Wisteria floribunda (WFA)-positive cells localized in both left and right genital ridges compared with the control group. Both concentrations of genistein resulted in significant decrease of PGC number compared with the control group. Percentages of the sterility rate of the embryo treated with 16 and 24 pig of genistein/g egg were 19% and 23%, respectively. These results provide evidence that genistein may be a germ cell toxicant causing sterility later in life of adult birds. This is the first report on the effect of genistein on PGC number in the genital ridges of the avian embryo.
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Previously we identified puerarin, an isoflavone compound, as a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to retardation of embryonic development and cell viability. In the current study, we investigated whether puerarin exerts deleterious effects on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, puerarin caused significant impairment of these processes in vitro. Pre-incubation of oocytes with puerarin during in vitro maturation led to increased post-implantation embryo resorption and decreased mouse fetal weight. In an in vivo animal model, intravenous injection with or without puerarin (1, 3 and 5 mg/kg body weight/day) for 4 days caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked puerarin-triggered deleterious effects, clearly implying that embryonic injury induced by puerarin is mediated by a caspase-dependent apoptotic mechanism. These results clearly demonstrate that puerarin has deleterious effects on mouse oocyte maturation, fertilization and subsequent embryonic development in vitro and in vivo.
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Male and female reproductive health is under the influence of uncountable endogenous and exogenous factors. A vast amount of literature is devoted to understanding how such factors operate to disrupt organs and cells of the reproductive tract and, at the same time, which kind of preventive or curative actions might be implemented. Polyphenols, mainly by their antioxidant properties, are promising substances to mitigate reproductive disruptions, most of which are known to involve oxidative stress. Largely found in a vast number of foods, beverages, plants and herbs, polyphenols may also act as phytoestrogens and trigger numerous responses mainly in the female reproductive system. Acting in a dose-dependent way, polyphenols can also exert toxic effects to both males and females, and their safety limits are also largely under investigation. This chapter briefly reviews the literature concerning polyphenol action in the healthy and disrupted male and female reproductive apparatus, aiming to put the reader in contact with the most recent research in such themes.
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Mutant mouse lines are unique models with an enormous scientific potential. Cryopreservation of preimplantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if sufficient specimens have been cryopreserved. Prerequisites to economically cryopreserve embryos are high yields of embryos prepared from donors and a high recovery rate after revitalization. Diets for laboratory animals are often produced from phytoestrogen-containing soy; the present study shows that feeding the donor animals with a phytoestrogen-poor diet is more efficient compared to a phytoestrogen-containing, soy-based diet. Additionally, a uterotrophic bioassay indicating the estrogenic role of compounds showed a significant increase of the relative uterus size of females fed with a phytoestrogen-rich diet. The role of the housing-temperature was investigated, too, showing that a housing-temperature of 24 °C results in the best embryo yields. The production of two-cell embryos is more economic than the production of eight-cell embryos. Investigating the recovery rate of frozen/thawed embryos, a very high recovery rate was determined when both, two- and eight-cell embryos were thawed. However, the capacity to develop to the next embryonic stage in vitro was dramatically reduced when two-cell embryos were compared to eight-cell embryos. After embryo transfer, the sex ratio became uneven and more males were delivered. This effect might be due to the procedures to which animals and embryos were subjected. These data show that many parameters can influence the production of animals when using (frozen/thawed) embryos. These parameters need continuous surveillance. Copyright © 2015 Elsevier Inc. All rights reserved.
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Objective: To investigate the effects of pregestational and gestational exposure to an isoflavone of sexually mature female mice on their reproductive health and on the postnatal development of their offspring. Methods: Sexually-mature ICR female mice were randomly segregated into Control group 1 that was given the normal diet consisting of food pellets, Control group 2 that was given food pellets with additional corn oil of 2 mL/kg bodyweight. The three (3) isoflavone treatment groups were given 50 mg/kg body weight, 100 mg/kg body weight and 150 mg/kg body weight for low dose (LD), medium dose (MD) and high dose (HD) respectively. Treatments were administered 2 weeks prior to mating and gestation and thereafter until parturition. The delivered pups were weaned up until 21 days. On the 21st day, postnatal development were determined, excluding the birth weight of pups which was measured one day post-parturition. The dams were sacrificed as well and the markers of maternal health were determined. Results: There were no significant differences found between the control groups and the treatment groups in terms of the markers of maternal reproductive health. For postnatal development, only HD group displayed a significantly higher mean AGD from the other groups. Conclusions: The data implies that exposure to the isoflavone genestein, with the given dosages, does not impact the maternal reproductive health while the high dose brings about masculinization of the pups which implies that isoflavone exerts its action as an endocrine disruptor affecting postnatal development. This could be attributed to the decrease in estrogen due to the inhibition of aromatase, an enzyme involved in estrogen synthesis.
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Abstract Previously, we reported that dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal and acaricidal activities, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to impaired embryonic development and cell viability. In the current study, we investigated the deleterious effects of dillapiole on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, dillapiole induced significant impairment of mouse oocyte maturation, decrease in the IVF rate and inhibition of subsequent embryonic development in vitro. Pre-incubation of oocytes with dillapiole during in vitro maturation led to an increase in post-implantation embryo resorption and decrease in mouse fetal weight. In an in vivo animal model, 2.5, 5 or 10 μM dillapiole provided in drinking water caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked dillapiole-triggered deleterious effects, clearly implying that embryonic injury induced by dillapiole is mediated via a caspase-dependent apoptotic mechanism. To the best of our knowledge, this is the first study to establish the impact of dillapiole on maturation of mouse oocytes, fertilization and sequential embryonic development.
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We previously reported that ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, and is a risk factor for abnormal embryonic development. More specifically, OTA triggers apoptotic processes in the inner cell mass of mouse blastocysts, decreasing cell viability and embryonic development. In the current study, we investigated the deleterious effects of OTA on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre- and postimplantation development both in vitro and in vivo. Notably, OTA significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with OTA during in vitro maturation increased postimplantation embryonic resorption and decreased mouse fetal weight. In an in vivo animal model, provision of 1–10 μM OTA in the drinking water or intravenous injection of 1 or 2 mg/kg body weight of OTA decreased oocyte maturation and IVF, and had deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase-3-specific inhibitor effectively blocked these OTA-triggered deleterious effects, suggesting that the embryonic injury induced by OTA is mediated via a caspase-dependent apoptotic mechanism. Furthermore, OTA upregulated the levels of p53 and p21 in blastocyst cells derived from OTA-pretreated oocytes, indicating that such cells undergo apoptosis via p53-, p21-, and caspase-3-dependent regulatory mechanisms. This could have deleterious effects on embryonic implantation and fetal survival rates, as seen in our animal models. © 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014.
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Previously, we reported that sanguinarine, a phytoalexin with antimicrobial, anti-oxidant, anti-inflammatory and pro-apoptotic effects, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, causing decreased embryonic development and cell viability. In the current study, we investigated the deleterious effects of sanguinarine on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre- and postimplantation development both in vitro and in vivo. Notably, sanguinarine significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with sanguinarine during in vitro maturation induced an increase in postimplantation embryo resorption and a decrease in mouse fetal weight. In an in vivo animal model, 1 to 5 μM sanguinarine, provided in drinking water, caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase-3-specific inhibitor effectively blocked sanguinarine-triggered deleterious effects, clearly implying that embryonic injury induced by sanguinarine is mediated by a caspase-dependent apoptotic mechanism. © 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014.
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Smoking is still considered to be mainly a male problem. However, it is estimated that there are approximately 250 million women worldwide who smoke cigarettes and millions more women who use smokeless tobacco products. This article addresses the many facets of tobacco use among women. The aim of the paper is to increase recognition among clinicians and researchers of the specific characteristics of female tobacco use. Together with providing epidemiological data on the distribution of tobacco use among women and data from population-based analyses on sociocultural factors that influence it, the article presents tobacco use during pregnancy as a particularly important public health problem. Further, the article points out sex-related differences (ie, physiological, psychological, or behavioral) between male and female tobacco use. A special focus is on the important role of ovarian hormones. Adverse effects of tobacco use to women and their children as well as tobacco-related morbidities and comorbidities are presented, and women's greater susceptibility to tobacco constituents as compared to men is stressed. Awareness of these differences can contribute to improvement of the effectiveness of smoking cessation programs addressed both to the specific female population and to an individual smoking woman.
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The mechanisms of prolactin signal transduction in generative and somatic cells of mammalian ovarian follicles are poorly understood. In this work, participation of tyrosine kinases and protein kinase C in mediation of the previously revealed modulating effects of prolactin on the nuclear maturation of bovine oocytes and the morphologic and functional state of surrounding cumulus cells in vitro has been investigated. It was found that a tyrosine kinase inhibitor genistein suppresses the stimulating action of prolactin on the completion of oocyte nuclear maturation and cumulus expansion, whereas a protein kinase C inhibitor calpostin C does not affect the hormonal effect. Furthermore, both genistein and calpostin C inhibited the inducing influence of prolactin on the proliferative activity of cumulus cells. At the same time, the retarding action of prolactin on destructive processes in cumulus cells was blocked only in the presence of calpostin C. These results show that the stimulating influence of prolactin on oocyte nuclear maturation accompanied by cumulus expansion is achieved with participation of tyrosine kinases, whereas the modulating action of the hormone on the functional state of cumulus cells depends on activation both of tyrosine kinases and protein kinase C.
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Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin inhibits cell proliferation and induces caspase 3-dependent apoptosis. However, its side-effects, particularly those on embryonic development, have not been well characterized as yet. In the current study, we examined the cytotoxic effects of emodin on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation by embryo transfer. Blastocysts treated with 25-75 μM emodin exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with emodin was lower than that of their control counterparts. Moreover, in vitro treatment with 25-75 μM emodin was associated with increased resorption of post-implantation embryos and decreased fetal weight. With the aid of an in vivo mouse model, we showed that consumption of drinking water containing emodin led to apoptosis and decreased cell proliferation, and inhibited early embryonic development to the blastocyst stage. Our findings support a degree of selective inhibition of retinoic acid receptors in blastocysts treated with emodin. In addition, emodin appears to induce injury in mouse blastocysts through intrinsic apoptotic signaling processes to impair sequent embryonic development. These results collectively indicate that emodin has the potential to induce embryonic cytotoxicity.
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The estrogenic soy isoflavone, genistein, stimulates growth of estrogen-dependent human breast cancer (MCF-7) cells in vivo. Genistin is the glycoside form of genistein and the predominant form found in plants. It is generally believed that genistin is metabolized to the aglycone genistein in the lower gut. However, it is unclear if the rate of metabolism of genistin to genistein is sufficient to produce a level of genistein capable of stimulating estrogen-dependent breast cancer cell growth. Our hypothesis was that dietary genistin would stimulate tumor growth similar to that observed with genistein in athymic mice. To test this hypothesis, genistin or genistein was fed to athymic mice containing xenografted estrogen-dependent breast tumors (MCF-7). Mice were fed either genistein at 750 p.p.m. (parts per milllion) or genistin at 1200 p.p.m., which provides equal molar concentrations of aglycone equivalents in both diets. Tumor size was measured weekly for 11 weeks. At completion of the study, half of the animals per treatment group were killed and tumors collected for evaluation of cellular proliferation and estrogen-responsive pS2 gene expression. Incorporation of bromo-deoxyuridine into cellular DNA was utilized as an indicator of cellular proliferation. Dietary genistin resulted in increased tumor growth, pS2 expression and cellular proliferation similar to that observed with genistein. The remaining mice were switched to diets free of genistin and genistein. When mice were placed on isoflavone free diets, tumors regressed over a span of 9 weeks. Next, we examined how effectively and where metabolism of genistin to genistein occurred in the digestive tract. We present evidence that demonstrates conversion of genistin to its aglycone form genistein begins in the mouth and then continues in the small intestine. Both human saliva and the intestinal cell-free extract from mice converted genistin to genistein. In summary, the glycoside genistin, like the aglycone genistein, can stimulate estrogendependent breast cancer cell growth in vivo. Removal of genistin or genistein from the diet caused tumors to regress.
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Genistein, a component of soy, was administered to prepubertal female Sprague-Dawley Co rats and investigated for chemoprevention against mammary cancer, Genistein, at 500 mu g/g body wt or an equivalent volume of the vehicle, dimethylsulfoxide (DMSO), was injected (s.c.) on days 16, 18 and 20 post-partum, At day 50 post-partum all animals were exposed to 80 mu g dimethylbenz[a]anthracene (DMBA) per g body wt, Animals treated prepubertally with genistein as compared to DMSO had reduced incidence and significantly fewer adenocarcinomas per animal. Mammary whole mount analysis showed that prepubertal genistein treatment resulted in mammary glands of 50-day-old rats developing fewer terminal end buds and more lobules II, Cell proliferation studies with bromodeoxyuridine (BrdU) showed that terminal end buds from mammary glands of 50-day-old females treated prepubertally with genistein had significantly fewer cells in S-phase of the cell cycle, Serum genistein concentrations in 21- and 50-day-old females following prepubertal genistein treatment were 4.2 +/- 0.6 mu M and 102 +/- 30 nM, respectively, Animals treated prepubertally with genistein as compared to vehicle spent more time in the estrus phase of the estrus cycle, although all animals did cycle, In 50-day-old females, circulating estradiol-17 beta and progesterone concentrations were not significantly altered by the prepubertal genistein treatment, Oocyte/follicle counts and numbers of atretic follicles and corpora lutea were not significantly different between the genistein- and vehicle-treated animals, We conclude that genistein treatment during the prepubertal period can suppress the development of chemically-induced mammary cancer without significant toxicity to the endocrine/reproductive system.
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Very little is known regarding the effects of ionizing radiation on cytoplasmic signal transduction pathways. Here, we show that ionizing radiation induces enhanced tyrosine phosphorylation of multiple substrates in human B-lymphocyte precursors. This response to ionizing radiation was also observed in cells pretreated with vanadate, a potent protein-tyrosine-phosphatase (PTPase) inhibitor, and phosphotyrosyl [Val5]angiotensin II phosphatase assays showed no decreased PTPase activity in irradiated cells. Thus, enhanced tyrosine phosphorylation in irradiated B-lymphocyte precursors is not triggered by inhibition of total cellular PTPase activity. Immune-complex kinase assays using anti-phosphotyrosine antibodies demonstrated enhanced protein-tyrosine kinase (PTK) activity in the immunoprecipitates from irradiated cells, and the PTK inhibitors genistein and herbimycin effectively prevented radiation-induced tyrosine phosphorylation. Immune-complex kinase assays on irradiated and unirradiated B-lymphocyte precursors using antibodies prepared against unique amino acid sequences of p59fyn, p56/p53lyn, p55blk, and p56lck demonstrated that these Src-family tyrosine kinases were not the primary PTKs responsible for enhanced tyrosine kinase activity in the anti-phosphotyrosine antibody immunoprecipitates or for enhanced tyrosine phosphorylation of multiple substrates. Thus, our findings favor the hypothesis that ionizing radiation induces enhanced tyrosine phosphorylation in B-lymphocyte precursors by stimulation of as yet unidentified PTKs. Tyrosine phosphorylation appears to be an important proximal step in radiation-induced apoptosis and clonogenic cell death because inhibition of PTK prevents DNA fragmentation and loss of clonogenicity of irradiated B-lymphocyte precursors. Since PTKs play myriad roles in the regulation of cell function and proliferation, the activation of a PTK cascade, as detailed in this report, may explain some of the pleiotropic effects of ionizing radiation on cellular functions of B-lymphocytes and their precursors.
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Genistein, an in vitro inhibitor of topoisomerase II and tyrosine kinases, suppressed growth and induced differentiation in HL-205 cells, a clonal population of the human promyelocytic HL-60 leukemia cells, and in K-562-J cells, a clonal population of the human erythroid K-562 leukemia cells. Maturing HL-205 cells acquired either granulocytic or monocytic markers, namely, reactivity with the murine OKM1 monoclonal antibody, expression of nitroblue tetrazolium dye reduction, and staining for nonspecific esterase. The maturing K-562-J cells stained with benzidine, which indicates the presence of hemoglobin, an erythroid maturation marker. Although the acquisition of the maturation markers in both HL-205 and K-562-J cells was time dependent up to 6 days, the kinetics of this induction differed between the two cell types. Despite the in vitro inhibitory effect of genistein, treatment of either HL-205 or K-562-J cells with 150 micrograms/ml genistein for up to 16 h did not alter topoisomerase II activity (as determined by the unknotting assay) in their nuclear extracts. Analysis with the anti-phosphotyrosine PY-20 murine monoclonal antibody indicated that treatment of K-562-J cells with genistein decreased the reactivity of the antibody with two of the cellular proteins. However, no reactivity with the PY-20 antibody was detected in untreated or genistein-treated HL-205 cells. An early event in the HL-205 and K-562-J cells, occurring after only 1 h of treatment with 30-200 micrograms/ml genistein, was the induction of DNA damage as measured by an alkaline elution assay. This damage may be a contributing factor in the genistein-induced cell differentiation in the HL-205 and K-562-J cells.
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Retinoic acid (RA) is a vitamin A derivative that exhibits major effects on biological processes such as cell differentiation and embryo pattern formation. Two human retinoic acid receptors (RAR alpha and beta) have been recently characterized. These receptors are encoded by two genes and their affinities for RA differ, suggesting that these two nuclear receptors may have distinct roles in mediating the varied biological effects of RA. Here we show that RAR alpha and beta differ in the regulation of expression of their mRNAs. Different levels of RAR alpha and beta transcripts were found in the various human tissues analysed. In addition, treatment of human hepatoma cells with RA leads to a rapid 10- to 50-fold increase in RAR beta mRNA levels, whereas RAR alpha mRNA expression is not affected. The induction of RAR beta transcription does not require de novo protein synthesis but is completely abolished by inhibitors of RNA synthesis. Nuclear transcript elongation assays indicate that the mechanism of RAR beta mRNA induction lies at the transcriptional level. These data demonstrate that the RAR beta gene is a primary target for RA. The differences in regulation of RAR gene expression might be a fundamental aspect of retinoid physiology and may prove especially important in the analysis of the morphogenic properties of RA.
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Tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor, pp60v-src and pp110gag-fes was inhibited in vitro by an isoflavone genistein. The inhibition was competitive with respect to ATP and noncompetitive to a phosphate acceptor, histone H2B. By contrast, genistein scarcely inhibited the enzyme activities of serine- and threonine-specific protein kinases such as cAMP-dependent protein kinase, phosphorylase kinase, and the Ca2+/phospholipid-dependent enzyme protein kinase C. When the effect of genistein on the phosphorylation of the EGF receptor was examined in cultured A431 cells, EGF-stimulated serine, threonine, and tyrosine phosphorylation was decreased. Phosphoamino acid analysis of total cell proteins revealed that genistein inhibited the EGF-stimulated increase in phosphotyrosine level in A431 cells.
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The mammalian embryo cannot develop without the placenta. Its specialized cells (trophoblast, endoderm, and extraembryonic mesoderm) form early in development. They attach the embryo to the uterus (implantation) and form vascular connections necessary for nutrient transport. In addition, the placenta redirects maternal endocrine, immune, and metabolic functions to the embryo's advantage. These complex activities are sensitive to disruption, as shown by the high incidence of early embryonic mortality and pregnancy diseases in humans, as well as the numerous peri-implantation lethal mutations in mice. Integration of molecular and developmental approaches has recently produced insights into the molecules that control these processes.
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We have investigated the effect of the soybean isoflavone genistein on the growth and differentiation of human melanoma cells. Four human melanoma cell lines, either completely lacking or containing different levels of wild-type p53, were treated with genistein in vitro in culture. It has been found that genistein significantly inhibited cell growth and that the chemosensitivity might depend on cellular p53 content. Specifically, the data suggest that high levels of wild-type p53 expression make cells resistant to genistein's growth-inhibitory action. Further support for this observation came from the stable transfection studies in which p53 transfectants expressing high levels of wild-type p53 became resistant to genistein. With respect to cell differentiation, our study showed that genistein increased melanin content and tyrosinase activity and caused the cells to form dendrite-like structures. Cells lacking p53 responded more than cells with p53 to dendrite-like structure formation. We also observed that genistein-induced differentiation involved an increase in tyrosinase mRNA level; the mechanisms by which genistein increases tyrosinase transcripts remain to be elucidated. Genistein treatment of the melanoma cell lines resulted in cell cycle arrest at G2/M check point and no significant apoptosis was observed. Images Figure 1 Figure 3 Figure 4 Figure 5
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Genistein is a specific inhibitor of protein tyrosine kinase (PTK) and is considered as a therapeutic candidate for various cancers. In this paper we investigate the effects of genistein on cell proliferation and differentiation in neuroblastoma (NB) cell lines and its possible mechanism of action. Genistein substantially inhibited the growth of five (N2A, JC, SKNSH, MSN and Lan5) of the six tumor cell lines examined in a dose-dependent manner with an IC50 value of approximately 5 microg/ml. The exception was GC cells. N2A cells were treated with genistein for 6 days and exhibited morphological features of differentiation, as evidenced by the development of dendritic extensions. Terminal deoxynucleotidyl transferase (TDT) histochemical staining showed a significant elevation in darkly stained nuclei in genistein-treated N2A cells compared with controls, indicating the occurrence of apoptosis. Fluorescent quantitation of DNA fragments confirmed apoptosis in genistein-treated N2A cells. To further elucidate the possible mechanisms by which genistein modulates NB cell growth and differentiation we investigated the effect of genistein on the activities of PTK and mitogen-activated protein (MAP) kinase and N-myc proto-oncogene expression in N2A cells. The results showed that genistein down-regulated intrinsic PTK activity by approximately 33% and inhibited insulin-like growth factor (IGF)-stimulated PTK activity by 75%. The effect of genistein on the intrinsic activity of MAP kinase was insignificant. In addition, genistein significantly reduced N-myc expression in a dose-dependent fashion. Our study suggests that genistein arrests cell growth and induces NB cell differentiation by mediating apoptosis and modulating PTK activity and N-myc proto-oncogene expression.
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The estrogenic soy isoflavone, genistein, stimulates growth of estrogen-dependent human breast cancer (MCF-7) cells in vivo. Genistin is the glycoside form of genistein and the predominant form found in plants. It is generally believed that genistin is metabolized to the aglycone genistein in the lower gut. However, it is unclear if the rate of metabolism of genistin to genistein is sufficient to produce a level of genistein capable of stimulating estrogen-dependent breast cancer cell growth. Our hypothesis was that dietary genistin would stimulate tumor growth similar to that observed with genistein in athymic mice. To test this hypothesis, genistin or genistein was fed to athymic mice containing xenografted estrogen-dependent breast tumors (MCF-7). Mice were fed either genistein at 750 p.p.m. (parts per milllion) or genistin at 1200 p.p.m., which provides equal molar concentrations of aglycone equivalents in both diets. Tumor size was measured weekly for 11 weeks. At completion of the study, half of the animals per treatment group were killed and tumors collected for evaluation of cellular proliferation and estrogen-responsive pS2 gene expression. Incorporation of bromo-deoxyuridine into cellular DNA was utilized as an indicator of cellular proliferation. Dietary genistin resulted in increased tumor growth, pS2 expression and cellular proliferation similar to that observed with genistein. The remaining mice were switched to diets free of genistin and genistein. When mice were placed on isoflavone free diets, tumors regressed over a span of 9 weeks. Next, we examined how effectively and where metabolism of genistin to genistein occurred in the digestive tract. We present evidence that demonstrates conversion of genistin to its aglycone form genistein begins in the mouth and then continues in the small intestine. Both human saliva and the intestinal cell-free extract from mice converted genistin to genistein. In summary, the glycoside genistin, like the aglycone genistein, can stimulate estrogen-dependent breast cancer cell growth in vivo. Removal of genistin or genistein from the diet caused tumors to regress.
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A diet high in soy is associated with many health benefits, including reduced incidence of breast cancer. The soy phytoestrogen, genistein, is hypothesized to contribute to mammary chemoprevention via interaction with estrogen receptors (ERs) alpha and/or beta. These steroid signaling pathways are believed to exert control over proliferation and differentiation of the mammary gland by a complex bidirectional interaction with the epidermal growth factor (EGF) signaling pathway. The current work was designed to study the role of these two pathways in prepubertal mammary gland growth. Female Sprague-Dawley CD rats were injected with genistein (500 micro g/g body wt) or estradiol benzoate (EB) (500 ng/g body wt) on days 16, 18 and 20. Whole mount analysis of mammary glands from 21-day-old rats showed that both treatments resulted in significantly increased terminal end buds (TEBs), and increased ductal branching, compared with animals given the vehicle, dimethylsulfoxide (DMSO). Both effects were inhibited by blockage of ER function by pre-treating with 2 mg ICI 182,780/kg body wt, a steroidal anti-estrogen. Immunoblotting analyis of mammary gland extracts demonstrated increased epidermal growth factor receptor (EGFR) and progesterone receptor (PR) expression following treatment with EB or genistein. Tyrosine-phosphorylated EGFR, as measured by immunoprecipitation/immunoblotting was also increased, but when normalized to total receptors, there was no net effect. The expression and phosphorylation of downstream targets of the EGFR, mitogen activating kinase kinase (MEK 1 and 2) and extracellular signal regulated kinases 1 and 2 (ERK 1 and 2) were not significantly affected. Anti-estrogen pre-treatment prevented the increase in EGFR, phospho-EGFR and PR. The data indicate an ER-based mechanism of action for genistein in mammary gland proliferation and differentiation, which can lead to protection against mammary cancer.
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New cancer therapeutic strategies must be investigated that enhance prostate cancer treatment while minimizing associated toxicities. We have previously shown that genistein, the major isoflavone found in soy, enhanced prostate cancer radiotherapy in vitro and in vivo. In this study, we investigated the cellular and molecular interaction between genistein and radiation using PC-3 human prostate cancer cells. Tumor cell survival and progression was determined by clonogenic analysis, flow cytometry, EMSA analysis of NF-kappaB, and western blot analysis of cyclin B1, p21WAF1/Cip1, and cleaved PARP protein. Genistein combined with radiation caused greater inhibition in PC-3 colony formation compared to genistein or radiation alone. Treatment sequence of genistein followed by radiation and continuous exposure to genistein showed optimal effect. Cell cycle analysis demonstrated a significant dose- and time-dependent G2/M arrest induced by genistein and radiation that correlated with increased p21WAF1/Cip1 and decreased cyclin B1 expression. NF-kappaB activity was significantly decreased by genistein, yet increased by radiation. Radiation-induced activation of NF-kappaB activity was strongly inhibited by genistein pre-treatment. A significant and striking increase in cleaved PARP protein was measured following combined genistein and radiation treatment, indicating increased apoptosis. A mechanism of increased cell death by genistein and radiation is proposed to occur via inhibition of NF-kappaB, leading to altered expression of regulatory cell cycle proteins such as cyclin B and/or p21WAF1/Cip1, thus promoting G2/M arrest and increased radiosensitivity. These findings support the important and novel strategy of combining genistein with radiation for the treatment of prostate cancer.
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The regulatory effects of retinoic acid (RA) on retinoic acid receptor (RAR) alpha, beta and gamma mRNA were examined in the F9 mouse teratocarcinoma cells. Northern blot hybridization showed that RA regulated all three types of RAR gene expression. The different transcripts for each RAR alpha and beta were differentially regulated by RA. The maximum induction of the 2.8 kb RAR alpha and 3.3 kb RAR beta transcript levels by RA took longer than the induction of the 3.8 kb RAR alpha and 3.5 kb RAR beta transcript levels. The data suggest that these transcripts originated from different promoters. Short term treatment (< or = 24 hours) of RA induced both 3.1 and 3.3 kb RAR gamma transcripts. Long term treatment (> 24 hours) of RA resulted in the inhibition of 3.1 kb mRNA, whereas the 3.3 kb mRNA remained elevated. In addition, a new RAR gamma transcript of 2.9 kb was induced. In contrast to RAR alpha and beta, the effect on RAR gamma gene expression was irreversible. Cycloheximide did not prevent the effect of RA on RAR gene expression, whereas actinomycin D totally abolished the RA effect on the expression of all three receptor genes. The data suggest that the biological effects of RA may be constrained or augmented by differential regulation of its own receptor gene expression.
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Introduction Retinoids control cell differentiation and proliferation by regulating gene expression. The important questions are. which genes and how? During the 7 yr since we last reviewed retinoid action (1) there have been great advances. This article reviews new information about mechanistic relationships between retinoids and transforming growth factor-β (TGF-β), including their respective receptors. We consider these molecules, together with oncogenes and suppressor genes, to be components of a central, unifying regulatory system, flexibly connected, and used in many ways to integrate information relating to the state of differentiation and proliferation of the cell. Here we consider the following topics: 1) a summary of the role of retinoids in cellular defferentiation and proliferation, especially as related to their role in carcinogenesis: 2) a brief review of the current knowledge of the role of newly discovered retinoic acid receptors in modulationg the above processes; 3) a brief summary of the structure of the genes and peptides comprising the immediate TGF-β family and their role in modulating cellular differentiation and proliferation; and 4) the interface between retinoids and TGF-β, and the role of the products of oncogenes and suppressor genes in this interface.
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Three retinoic acid receptors (RAR), structurally homologous to steroid and thyroid hormone nuclear receptors, have recently been cloned. Analysis of the tissue distribution of RARs mRNAs have demonstrated a distinct expression pattern of the receptors' transcripts. Moreover, the RAR beta gene specifically, is autoregulated by RA. These findings suggest that the retinoic acid receptors differentially contribute retinoic acid physiology.
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Late morulae and blastocysts were recovered from streptozocin-induced diabetic pregnant rats and individually examined for numbers of inner cell mass (ICM) cells and trophectoderm (TE) cells. Compared with embryos collected from control rats, exposure to maternal diabetes significantly decreased mean ICM cell number of blastocysts recovered on day 5 of gestation, but the TE population of these embryos remained unaffected. The mean ICM proportion was therefore significantly lower than that of control embryos. These differences were not observed between the two groups of morulae collected on day 5, suggesting that the distinctive susceptibility of the ICM was expressed after blastocyst formation. On day 6, a significant inhibitory effect of diabetes was observed on the growth of both ICM and TE cells, but because the reduction was more severe in the ICM than in the TE, the mean ICM proportion of these blastocysts was again significantly lower than in control embryos. A linear quadratic relationship was obtained between the numbers of ICM cells of individual blastocysts and their respective numbers of TE cells in each of the two experimental groups. However, the slope of the curve was slower in the diabetic group than the control group. The disturbed ICM cell growth in the blastocysts from diabetic rats was found to be associated with a significantly increased incidence of cell death predominantly located in the ICM. Because it is known that excessive reduction of the ICM is incompatible with normal embryogenesis after implantation, our results suggest that the differential sensitivity of ICM and TE cells in preimplantation blastocysts may contribute to the pattern of postimplantation defects described in diabetic pregnancies.
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It has long been suggested that pattern formation depends in part on signalling molecules known as 'morphogens', diffusible substances that determine cell fate in a concentration-dependent way. Retinoic acid, a small hydrophobic molecule that binds to nuclear receptors, is a candidate morphogen for specifying the anteroposterior pattern of vertebrate limbs.
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A cDNA encoding a protein that binds retinoic acid with high affinity has been cloned. The protein is homologous to the receptors for steroid hormones, thyroid hormones and vitamin D3, and appears to be a retinoic acid-inducible trans-acting enhancer factor, suggesting that the molecular mechanisms of the effect of retinoids (vitamin A) on embryonic development, differentiation and tumour cell growth are similar to those described for other members of this nuclear receptor family.
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Studies of steroid receptors have led to the identification of a superfamily of ligand-inducible regulatory proteins that includes receptors for thyroid hormones and retinoic acid. This family of receptors regulates gene expression through binding to short cis-acting sequences referred to as hormone-response elements. Identification of a functional retinoic acid responsive element is crucial to our understanding of the mechanisms by which retinoic acid receptors activate gene expression and regulate cell differentiation. One impediment to such a study is the absence of any identified gene whose transcription is directly dependent on the receptor-hormone complex. Because the DNA-binding domains of the retinoic acid and thyroid hormone receptors are highly related (62% identical in their amino acid sequences), we have investigated the possibility that the retinoic acid receptor could activate gene expression through a thyroid hormone response element. We now report that a human retinoic acid receptor expressed from cloned complementary DNA or the endogenous retinoic acid receptor present in F9 teratocarcinoma cells can activate gene expression from promoters fused to a natural or synthetic thyroid hormone response element. The product translated in vitro from the human retinoic acid receptor cDNA can bind to a thyroid hormone response element with high affinity. The unexpected implication of these findings is that retinoic acid and thyroid hormones, acting through their respective receptors, could control overlapping gene networks involved in the regulation of vertebrate morphogenesis and homeostasis.
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Retinoic acid (RA) binds to a cytosolic protein distinguishable from the cellular retinol (R) binding protein. Recent studies, showing an influence by R and RA on genomic expression, suggest an interaction with the cell nucleus mediated by the specific binding proteins in a manner resembling that of steroid hormones. RA can irreversibly stimulate in vitro differentiation of teratocarcinoma cells and support early embryonic development in vitamin A depleted animals. This study demonstrates a saturable, highly specific and regional accumulation of RA in the neuroepithelium and developing CNS that occurs in early but not in late fetal development in the mouse. The results suggest that a binding protein, or some other cellular mechanism for accumulation of RA is expressed in the neural cells only during restricted periods of development. High levels are recorded also in regions where cranial neural crest cells are known to migrate, and later in the visceral arches and maxillary areas, the mesenchyme of which is known to be partly derived from migrating cranial neural crest cells. The specific accumulation of RA in embryonic neural and cranial neural crest cells is in line with animal experiments and human clinical data, showing that retinoids specifically impair CNS, eye, ear, and facial development.
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Vitamin A and its derivatives, the retinoids, cause specific alterations in the proximo-distal pattern of regenerating axolotl limbs. In some cases complete limbs grow from carpal level amputations. If retinoids are responsible for changing the positional information of limb cells, these compounds may represent valuable probes for investigating the molecular basis of pattern formation.
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The thymus gland is crucial for the formation of thymocytes of diverse TCR specificity. Recent studies have demonstrated that deletion (negative selection) of autoreactive thymocytes occurs through the process of apoptosis in which TCR activates cell death by DNA fragmentation. In addition, in vitro stimulation of thymocytes with anti-CD3 mAb, calcium ionophore, or glucocorticoids results in DNA fragmentation followed by cell death. The availability of various substances capable of inhibiting activation-induced programmed cell death of thymocytes may be used as a tool to help identify several important events occurring during the process of apoptosis. We investigated the effect of protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, on thymocyte apoptosis induced by stimulation of anti-CD3 mAb or glucocorticoid. Anti-CD3 mAb stimulation resulted in removal of CD4+CD8+ thymocytes by DNA fragmentation. However, in PTK inhibitor-pretreated thymocytes, there was a minimal deletion of double positive thymocytes. In contrast, PTK inhibitors did not prevent glucocorticoid-induced thymic apoptosis. Our results suggest that anti-CD3 mAb-induced thymic apoptosis depends on PTK activation via TCR, and that glucocorticoid-induced thymic apoptosis is PTK-independent.
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One-cell mouse embryos of the Balb/c strain normally divide at 18.5 h p.c. (post conception), but they suffer an extremely long G2 arrest when irradiated with 2 Gy X-rays 8 h p.c. at the early pronuclear stage. This could be an indirect effect of radiation on tyrosine dephosphorylation of the p34cdc2 subunit of a maturation or mitosis promoting factor (MPF), which normally occurs at the end of G2. This, in turn, would maintain MPF in an inactivated form and block entry into mitosis. Preliminary studies were undertaken at the morphological level to assess indirectly the validity of this hypothesis. For this purpose, irradiated and control embryos were exposed to different compounds, which are known to interfere, directly or indirectly, with the state of phosphorylation/dephosphorylation of p34cdc2. Caffeine (CAF; 2 mM) did not affect the time of first division of control embryos, but it completely suppressed the radiation-induced G2 arrest of embryos exposed to this compound from 17 h p.c., i.e. 1.5 h before the normal time of first cleavage. Under the same conditions, okadaic acid (OA; 3 microM), a specific inhibitor of phosphatases I and IIA, induced a rapid pronuclear membrane breakdown and a block of all control and irradiated embryos at metaphase. Genistein (GEN; 92 or 185 microM). A potent inhibitor of tyrosine kinases, increased the radiation-induced G2 arrest and even induced a dose-dependent G2 arrest in the control embryos. Embryos were exposed at different times following irradiation to a mixture of either CAF (2 or 5 mM) or OA (3 or 10 microM), and cycloheximide (CH; 5 micrograms/ml), a potent protein synthesis inhibitor. Reversion of G2-arrest by CAF was still seen in embryos exposed to CAF+CH from 17 h p.c. However, the proportion of irradiated embryos eventually able to cleave was lower than that obtained under the conditions of exposure to CAF alone. Embryos exposed to CAF+CH before 17 h p.c. were not able to cleave, regardless of the concentration of CAF used. Nuclear envelope breakdown still occurred in 100% control and irradiated embryos, following exposure to 3 microM OA+CH from 10 h p.c., or to 10 microM OA+CH from 8.5 p.c.(ABSTRACT TRUNCATED AT 400 WORDS)
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Taxol is an antineoplastic agent with significant activity against ovarian as well as breast cancer. To investigate mechanisms by which taxol exerts its cytotoxic action, taxol-induced apoptosis, characterized by morphologic changes and internucleosomal DNA fragmentation, was examined in a human ovarian tumor cell line. Time-dependent morphologic changes, characteristic of apoptosis, were observed over the same time as the appearance of internucleosomal DNA fragmentation. The specific protein tyrosine kinase inhibitors genistein and herbimycin A, and the ATP depletion agent sodium azide, interfered with taxol-induced DNA fragmentation and clonal cell death. Based on a quantitative reverse transcription-polymerase chain reaction technique, bcl-2 alpha oncogene expression was decreased in conjunction with taxol-induced DNA fragmentation, and this decrease could be blocked by genistein. These results strongly implicate protein tyrosine phosphorylation as an event that mediates apoptosis and, thus, the antitumor activity of taxol in ovarian cancer.
Article
Incubation of mouse thymocytes with the protein tyrosine kinase inhibitors herbimycin A and methyl-2,5-dihydroxycinnamate induced a decreased and altered profile of nuclear phosphotyrosine proteins in parallel with an increase in internucleosomal DNA fragmentation and cell death dose-dependently. No change in the profile of cytoplasmic phosphotyrosine proteins was observed. DNA fragmentation was dependent on the synthesis of RNA and protein, suggesting that the inhibition of tyrosine phosphorylation of the nuclear proteins induces apoptosis. DNA fragmentation was enhanced by simultaneous incubation with phorbol esters capable of activating protein kinase C. Genistein, another inhibitor of protein tyrosine kinase, induced DNA fragmentation more rapidly than herbimycin A, but there was no predominant alteration of phosphotyrosine proteins in early incubation, suggesting that genistein may induce apoptosis by a mechanism other than direct inhibition of protein tyrosinekinase activity.
Article
Experimental evidence suggests that hematopoietic growth factors promote cell survival by suppressing apoptosis or programmed cell death. Since interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) induce tyrosine phosphorylation of a common set of proteins in the factor-dependent cell line M07e, we have investigated whether growth-factor-induced tyrosine phosphorylation is involved in the promotion of cell survival and suppression of apoptosis. Experiments were carried out with the leukemic cell lines HL-60 and M07e and the tyrosine kinase inhibitors genistein and tyrphostin AG82. Both the tyrosine kinase inhibitors induced apoptosis of HL-60 and M07e cells. This was indicated by the appearance of DNA degradation and morphologic evidence of nuclear condensation and fragmentation. It was also confirmed by flow cytometry of DNA, which showed apoptotic cells as a fraction of cells characterized by a diminished DNA stainability, represented on the DNA frequency histograms as a distinct peak below the G0/G1 population. Kinase inhibitors also reduced the fraction of cells in the S phase of the cell cycle. That tyrphostin specifically inhibited tyrosine kinases was further suggested by the prevention of its effects by the tyrosine phosphatase inhibitor sodium orthovanadate (vanadate), at least during the first 18-24 h of treatment. The incomplete prevention of genistein effects by vanadate suggests that genistein is a less specific inhibitor of tyrosine kinases than tyrphostin, and may also act as an inhibitor of topoisomerase II. Vanadate also prevented apoptosis an