Article

Steroidogenesis during in vitro maturation of bovine cumulus oocyte complexes and possible effects of tri-butyltin on granulosa cells

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Abstract

Steroids are known as important factors on the route of oocytes development and cumulus oocyte complexes (COC) as well as follicular granulosa cells (GC) are suggested to be themselves involved in steroidogenesis. The aim of this study was to characterize such a local sex steroidogenic system during in vitro maturation (IVM) of bovine COCs according to the production of estradiol (E), testosterone (T) and progesterone (P). The expression of two steroid-converting key-enzymes was measured in parallel by quantitative RT-PCR. Furthermore, possible effects of the environmental pollutant tri-butyltin (TBT) were elucidated for the first time on bovine COC and GC in vitro concerning that steroidogenic system. During IVM of bovine COCs concentrations of P increased continuously, corresponding with steady-state levels of 3-beta-hydroxy-steroid-dehydrogenase (HSD) transcripts. In contrast, E together with P450 aromatase mRNA (ARO) increased in the first hours of IVM but declining thereafter, whereas T reached almost balanced levels. However, TBT showed only slight effects during IVM of COC. In cultured GC, LH caused highest P- and E-production within 24h and treatment with 50pM TBT induced a significant decrease of E in contrast to 100pM TBT and the control. These results indicate, that (1) COCs were able to modulate their steroidogenic environment in vitro and that (2) TBT may possibly influence or disturb steroidogenesis in the cows reproductive tract shown here for GC.

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... ROS level per cumulus cell diminished through IVM [15], and in oocytes it was minimal at 12 h of IVM [31]. Also, increase of progesterone and decrease of estradiol concentrations in maturation medium after germinal vesicle break down (GVBD) due to their secretion by COCs were observed [28,32,33]. The most significant changes in the kinetics of these processes occurred since 8 -10 h after the beginning of IVM, closely associated with the period of oocyte metaphase-I to metaphase-II transition and the most extensive cumulus expansion (10 -22 h). ...
... The phosphatidylinositol 3-ki- Fig. 1. Kinetics of oocyte maturation in relation to cumulus cells activities as function of oocyte nuclear maturation stages: steroids' secretion, cumulus apoptosis rate, cumulus expansion and ROS concentration as determined by our experiences and/or bibliography analysis: estrogens [32]; progesterone [33]; apoptosis [24]; reactive oxygen species (ROS) in cumulus [15] and in oocyte [31]. Oocyte stages: GV: germinal vesicle; GVBD: germinal vesicle breakdown; Meta-I: metaphase-I; Meta-II: metaphase-II. ...
... Decrease of GSTA1 protein abundance correlated with diminished ROS concentration per cumulus cell during IVM [15]. The reduction of GSTA1 expression in CC at the end of IVM was concomitant with the reduction of estrogens concentration in IVM medium [32]. GSTA1 could be probably secreted to IVM medium for detoxification of the xenobiotics arising from the catabolism of estrogens. ...
... Schoenfelder et al. [9] reported that bovine COCs were able to secrete several steroid hormones during in vitro maturation (IVM) without the support of follicular granulosa or theca cells and that the COCs possessed some important steroidogenic enzymes, such as 3β hydroxysteroid dehydrogenase (3βHSD) and aromatase. Their results indicated that COCs were able to modulate their steroidogenic environment in vitro. ...
... Recently, the COC was suggested to be involved in steroidogenesis [9,10]. Follicular maturation and development are complex processes influenced by both intra- and extra-ovarian events that lead to successful ovulation. ...
... Supporting such unaffected progesterone levels, 3βHSD mRNA expression showed no significant changes during gonadotropin treatment. In contrast, the initial increase in aromatase mRNA that was caused by either FSH or LH led to delayed secretion of estradiol, whereas co-treatment induced just slightly increased estradiol levels [9]. These results may indicate an estrus cycle-dependent reactivity of granulosa cells to different gonadotropin ratios, which exist in distinct but changing ratios in vivo. ...
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Background The purpose of this study was to localize the expression of steroid sulfatase (STS) in cumulus cells and to determine the relationship between STS mRNA expression and the serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol and progesterone. Methods The subject group included 49 women (29 to 44 years old) for whom in vitro fertilization treatment was indicated. All subjects gave informed consent. One hundred fourteen samples of cumulus-oocyte complex (COC) were obtained under microscopic observation. Part of the COC was stained by STS antibody. RNA was extracted by phenol-chloroform method and real-time PCR was performed. Serum of each patient was collected and was measured by ELISA. Results Some of the cumulus samples were stained by STS antibody. The expression of STS mRNA in all samples was confirmed by quantitative RT-PCR. Although there was no significant correlation between the level of STS mRNA and the serum levels of estradiol, progesterone and LH, there was a statistically significant negative correlation between the level of STS mRNA expression and the serum level of FSH (n = 105, p = 0.018, r = -0.22). Conclusion These results have demonstrated for the first time the expression of STS in cumulus cells by immunohistological stainings and real-time RT-PCR. STS expression in cumulus cells may be related to the control of the local steroidal environment in the oocyte. Serum FSH may control STS mRNA expression from the results of RT-PCR, although the correlation was low.
... In the present study the propagated steroidogenesis in bovine COC during artificial maturation under FSH+LH was examined [240]. ...
... Initial experiments using RT-PCR showed remarkable amounts of ARO and HSD. Both key enzymes of the steroidogenic pathway were detectable at all stages of IVM [240]. The initial high levels of ARO declined dramatically to 0.3 % of onset levels after several hours (Fig. 4a). ...
... Additionally, the HSD mRNA levels decreased during IVM too, but did not show this excessive drop like demonstrated for ARO [240]. The outcome of steroid hormone detection mirrored mRNA expression dynamics in part (Fig. 4b/c). ...
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Erscheinungsjahr an der Haupttitelstelle: 2003 München, Techn. Univ., Diss., 2004. Computerdatei im Fernzugriff.
... ROS level per cumulus cell diminished through IVM [15], and in oocytes it was minimal at 12 h of IVM [31]. Also, increase of progesterone and decrease of estradiol concentrations in maturation medium after germinal vesicle break down (GVBD) due to their secretion by COCs were observed [28,32,33]. The most significant changes in the kinetics of these processes occurred since 8 –10 h after the beginning of IVM, closely associated with the period of oocyte metaphase-I to metaphase-II transition and the most extensive cumulus expansion (10 –22 h). ...
... The phosphatidylinositol Fig. 1. Kinetics of oocyte maturation in relation to cumulus cells activities as function of oocyte nuclear maturation stages: steroids' secretion, cumulus apoptosis rate, cumulus expansion and ROS concentration as determined by our experiences and/or bibliography analysis: estrogens [32]; progesterone [33]; apoptosis [24]; reactive oxygen species (ROS) in cumulus [15] and in oocyte [31]. Oocyte stages: GV: germinal vesicle; GVBD: germinal vesicle breakdown; Meta-I: metaphase-I; Meta-II: metaphase-II. ...
... Decrease of GSTA1 protein abundance correlated with diminished ROS concentration per cumulus cell during IVM [15]. The reduction of GSTA1 expression in CC at the end of IVM was concomitant with the reduction of estrogens concentration in IVM medium [32] . GSTA1 could be probably secreted to IVM medium for detoxification of the xenobiotics arising from the catabolism of estrogens. ...
Article
In vitro maturation of oocytes is a crucial step in assisted reproductive technologies in cattle; however, the molecular mechanisms of cumulus contribution to oocyte developmental potential require more investigation. Based on transcriptomic data, we studied by using real-time RT-PCR and western blot in bovine cumulus cells, the kinetics of expression of several candidate genes involved in oxidative stress response, apoptosis, steroid metabolism and signal transmission throughout IVM. Phosphorylations of the components of the main signaling pathways were also analyzed. In addition, IVM was performed in different maturation mediums which influenced the cumulus apoptosis, progesterone secretion and oocyte developmental competence.
... In addition, a few studies report TBT affects the ovary using in vitro models (Saitoh et al., 2001;Schoenfelder et al., 2003) ( Table 2). In a human granulosa-like tumor cell line KGN, TBT exposure for 72 h at 20 ng/ml ($6 nM) suppressed both aromatase activity and aromatase gene expression by 30% compared with control (Saitoh et al., 2001). ...
... Interestingly, the doses of TBT in the study were nearly pharmacologically equivalent to the doses that reportedly induce imposex in female gastropods (Bryan et al., 1986). TBT exposure for 4, 12, and 24 h (50 pM) also increased progesterone levels, reduced testosterone and estrogen levels, reduced expression of aromatase CYP19 and 3 beta-HSD mRNA in bovine cumulus-oocyte complex cultures (Schoenfelder et al., 2003). In addition, 50 pM TBT, but not 100 pM TBT, blunted the LHmediated estrogen production in follicular granulosa cell culture (Schoenfelder et al., 2003), showing the contradictory and complex effect of TBT on ovarian steroidogenesis. ...
... TBT exposure for 4, 12, and 24 h (50 pM) also increased progesterone levels, reduced testosterone and estrogen levels, reduced expression of aromatase CYP19 and 3 beta-HSD mRNA in bovine cumulus-oocyte complex cultures (Schoenfelder et al., 2003). In addition, 50 pM TBT, but not 100 pM TBT, blunted the LHmediated estrogen production in follicular granulosa cell culture (Schoenfelder et al., 2003), showing the contradictory and complex effect of TBT on ovarian steroidogenesis. ...
Article
The hypothalamic-pituitary-gonadal (HPG) axis is the principal modulator of reproductive function. Proper control of this system relies on several hormonal pathways, which make the female reproductive components susceptible to disruption by endocrine-disrupting chemicals such as tributyltin (TBT). Here, we review the relevant research on the associations between TBT exposure and dysfunction of the female HPG axis components. Specifically, TBT reduced hypothalamic gonadotropin-releasing hormone (GnRH) expression and gonadotropin release, and impaired ovarian folliculogenesis, steroidogenesis, and ovulation, at least in part, by causing abnormal sensitivity to steroid feedback mechanisms and deleterious ovarian effects. This review covers studies using environmentally relevant doses of TBT in vitro (1 ng—20 ng/mL) and in vivo (10 ng—20 mg/Kg) in mammals. The review also includes discussion of important gaps in the literature and suggests new avenue of research to evaluate the possible mechanisms underlying TBT-induced toxicity in the HPG axis. Overall, the evidence indicates that TBT exposure is associated with toxicity to the components of the female reproductive axis. Further studies are needed to better elucidate the mechanisms through which TBT impairs the ability of the HPG axis to control reproduction.
... It is known that cultured COCs of various species, in addition to progesterone, also release androgens and estradiol (e.g. Bar-Ami et al., 1989;Wójtowicz and Szołtys, 1998;Schoenfelder et al., 2003). The function of androgens is mainly considered as a metabolic one. ...
... As already described, in vitro androgen release by mature COCs is well established (e.g. Bar-Ami et al., 1989;Wójtowicz and Szołtys, 1998;Schoenfelder et al., 2003), but our study showed, for the first time, the immunolabelling of P450c17 in the post-ovulatory cumulus granulosa cells as well as in the post-ovulatory ampullary epithelial cells. Additionally, the latter cells exhibited cytoplasmic AR immunolabelling suggestive of a non-genomic androgen action. ...
Article
Immunolocalization of 3β-hydroxysteroid dehydrogense (3β-HSD), cytochrome P450c17 (P450c17) and androgen receptor (AR) were investigated in rat cumuli oophori (COCs) of late pre-ovulatory follicles and in post-ovulatory COCs bearing fertilized oocytes. A gradient of intensity of 3β-HSD immunolabelling was observed in the granulosa layer of pre-ovulatory follicles, with almost negative immunolabelling in COCs and with the strongest immunoreaction in the mural granulosa cells. Post-ovulatory COCs showed strong 3β-HSD immunolabelling in the peripheral regions and weak labelling near the oocyte, suggestive of responsiveness of cumulus cells to an anti-luteinizing effect exerted by the fertilized oocyte. In pre-ovulatory follicles, a weak P450c17 immunopositivity was limited to expanded cumulus granulosa cells and the positive labelling persisted in post-ovulatory COCs. P450c17 immunopositivity was also found in ampullary epithelial cells. A strong AR immunopositivity was confined mainly to the COCs in pre-ovulatory follicles and a similar immunoreaction was present in the granulosa cells of ovulated COCs. Simultaneous AR and cytochrome P450c17 immunolabelling in the pre- and post-ovulatory COCs is suggestive of an intra- and paracrine androgen regulation of the cumulus granulosa cell function.
... Gonadal function is responsible for steroidogenesis and gamete production, both of which are affected by TBT exposure (Kishta et al., 2007;Saitoh et al., 2001). Schoenfelder et al. (2003) reported greater progesterone, testosterone, and estrogen levels in bovine ovary cells exposed to 50 pM TBT for 24 h. These ovarian sex hormone abnormalities were associated with TBT's toxic effects on steroidogenic control (Saitoh et al., 2001;Ahn et al., 2007). ...
... Notably, a common feature of TBT's effects on steroidogenesis is the varying degree of irregularities, possibly resulting from the sensitivity and/or control of abnormal enzyme expression in different models that are also dependent on species, sex, age, TBT dose, and the time and type of chemical used (Saitoh et al., 2001;Ohno et al., 2005;Yamazaki et al., 2005;Ahn et al., 2007;Sena et al., 2017). TBT exposure could be act as adverse condition that leads an increase in progesterone levels, as result of altered luteinization (Schoenfelder et al., 2003). Other model of adverse condition, such as a hipoxia exposure was responsible to abnormal luteinization with increase in progesterone levels (Yoshioka et al., 2017). ...
Article
Tributyltin chloride (TBT) is an obesogen associated with various metabolic and reproductive dysfunctions after in utero exposure. However, few studies have evaluated TBT's obesogenic effect on adult ovaries. In this study, we assessed whether TBT's obesogenic effects resulted in adult ovarian adipogenesis and other reproductive abnormalities. TBT was administered to adult female Wistar rats, and their reproductive tract morphophysiology was assessed. We further assessed the ovarian mRNA/protein expression of genes that regulate adipogenesis. Rats exposed to TBT displayed abnormal estrous cyclicity, ovarian sex hormone levels, ovarian follicular development and ovarian steroidogenic enzyme regulation. Rats exposed to TBT also demonstrated abnormal ovarian adipogenesis with increased cholesterol levels, lipid accumulation, and PPARγ, C/EBP-β and Lipin-1 expression. A negative correlation between the ovarian PPARγ expression and aromatase expression was observed in the TBT rats. Furthermore, TBT exposure resulted in reproductive tract atrophy, inflammation, oxidative stress and fibrosis. Ovarian dysfunctions also co-occurred with the uterine irregularities. Abnormal ovarian adipogenic markers occurring after TBT exposure may be associated with uterine irregularities. A positive correlation between the ovarian cholesterol levels and uterine inflammation was observed in the TBT rats. These findings suggest that TBT leads to ovarian obesogenic effects directly by abnormal adipogenesis and/or indirectly through adult reproductive tract irregularities.
... Cumulus cells continue to envelop the oocyte and provide critical support even though gap junction connections breakdown early during maturation (Hyttel et al., 1986a;1986b) and precede/coincident with resumption of meiosis (Thomas et al., 2004;Lodde et al., 2007;Luciano et al., 2011;Campen et al., 2018). Despite having few to no LH receptors initially, cumulus cells begin differentiation and production of progesterone as oocyte maturation progresses (Schoenfelder et al., 2003;Tosca et al., 2007) which has been attributed to a reversion to a granulosa cell state in other species (Chaffin et al., 2012). Under the influence of follicle stimulating hormone and other factors such as IL6 (Liu et al., 2009), cumulus undergo expansion by secreting an extracellular matrix (e.g., hyaluronan, other proteoglycans, and glycoproteins). ...
Article
Cows acutely heat stressed after a pharmacologically induced luteinizing hormone (LH) surge had periovulatory changes in the follicular fluid proteome that may potentiate ovulation and impact oocyte developmental competence. Because the cellular origins of differentially abundant proteins were not known, we have examined the cumulus and granulosa cell transcriptomes from the periovulatory follicle in cows exhibiting varying levels of hyperthermia when occurring after the LH surge. After pharmacological induction of a dominant follicle, lactating dairy cows were administered gonadotropin releasing hormone (GnRH) and maintained in thermoneutral conditions (~67 temperature-humidity index [THI]) or heat stress conditions where THI was steadily increased for ~12 h (71 to 86 THI) and was sufficient to steadily elevate rectal temperatures. Cumulus-oocyte complexes and mural granulosa cells were recovered by transvaginal aspiration of dominant follicle content ~16 h after GnRH. Rectal temperature was used as a continuous, independent variable to identify differentially expressed genes (DEGs) increased or decreased per each 1 °C change in temperature. Cumulus (n = 9 samples) and granulosa (n = 8 samples) cells differentially expressed (false discovery rate [FDR] < 0.05) 25 and 87 genes, respectively. The majority of DEGs were upregulated by hyperthermia. Steady increases in THI are more like the "turning of a dial" than the "flipping of a switch." The moderate but impactful increases in rectal temperature induced modest fold changes in gene expression (<2-fold per 1 °C change in rectal temperature). Identification of cumulus DEGs involved in cell junctions, plasma membrane rafts, and cell-cycle regulation are consistent with marked changes in the interconnectedness and function of cumulus after the LH surge. Depending on the extent to which impacts may be occurring at the junctional level, cumulus changes may have indirect but impactful consequences on the oocyte as it undergoes meiotic maturation. Two granulosa cell DEGs have been reported by others to promote ovulation. Based on what is known, several other DEGs are suggestive of impacts on collagen formation or angiogenesis. Collectively these and other findings provide important insight regarding the extent to which the transcriptomes of the components of the periovulatory follicle (cumulus and mural granulosa cells) are affected by varying degrees of hyperthermia.
... Follicle cells have been implicated in a number of gonadotrophin-regulated processes involved in oocyte growth and maturation. These include steroidogenesis (Redshaw, 1972;Fortune, 1983;Sretarugsa & Wallace, 1997;Hernandez et al., 2003;Schoenfelder et al., All correspondence to: S.S. Sánchez (Dumont, 1978), meiotic arrest (Villecco et al., 1996) and other processes such as vitelline envelope formation (Dumont and Brummett, 1978;Cabada et al., 1996), RNA transfer (Sanchez Riera et al., 1988;Motta et al., 1995) and organelle transfer (Andreuccetti, 1992;Motta et al., 1995). ...
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In this work we carried out ultrastructural, autoradiographic and biochemical analyses of the follicular epithelium during C. cranwelli previtellogenesis. This study revealed that the follicular epithelium in early previtellogenesis is constituted of a single layer of squamous homogeneous cells. During mid-previtellogenesis two types of cells develop: dark cells and clear cells. The follicular dark cells are actively involved in the synthesis of RNA, which is transferred to the oocyte through the interface. In late previtellogenesis the dark cells show apoptotic characteristics such as chromatin condensation, DNA fragmentation and cytoplasm shrinkage. This process forms apoptotic bodies that seem to be engulfed by the oocyte. Our results show evidence that, during mid- and late C. cranwelli previtellogenesis, the follicular epithelium undergoes remodelling processes interacting with the oocyte.
... These changes in intrafollicular steroid concentrations in vivo indicate that a precise balance or sequence of steroids may be necessary for the full maturation of follicle-enclosed oocytes. In addition, mammalian COCs secrete several steroid hormones , including testosterone, during in vitro maturation without support of follicular granulosa or theca cells and possess a selection of important steroidogenic enzymes (bovine, Schoenfelder et al. 2003; porcine, Dode and Graves 2002; Shimada et al. 2002). Furthermore, the addition of exogenous testosterone (100 nM) to oocyte maturation medium, selected on the basis of physiological concentrations reported in preovulatory bovine follicles (∼165 nM, Dieleman et al. 1983) improved the developmental competence of bovine oocytes after IVF (Younis et al. 1989; Silva and Knight 2000). ...
Article
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Recent studies have suggested a relationship between bovine follicular fluid testosterone concentration and the likelihood of the oocyte being fertilised by an X- or Y-bearing spermatozoon; however, this theory has been challenged. To further test this hypothesis, follicles were dissected from the ovaries of slaughtered heifers, measured and carefully ruptured. The cumulus-oocyte complex (COC) was removed and the follicular fluid collected and testosterone concentration determined by radioimmunoassay. COCs were matured, fertilised and cultured in an individually identifiable manner; all cleaved embryos (2- to 4-cell stage, n = 164) had their sex determined by PCR. Testosterone concentrations were positively skewed. There was no significant difference between follicular fluid testosterone concentrations in male and female embryos (mean + or - s.e.m. 51.5 + or - 5.59 and 49.5 + or - 7.42 ng mL(-1), respectively). Linear, quadratic and cubic logistical regression showed that follicular testosterone concentration could not reliably predict the sex of the embryo with odds ratios of 1.001, 1.013 and 1.066, respectively, and coefficient of determination (R(2)) values of 0.0003, 0.0126 and 0.0567, respectively. Follicular size and testosterone concentration were not related (R(2) = 0.087). Finally, follicular size had no influence on embryo sex determination (P = 0.70). In conclusion, under the conditions of the present study, the likelihood of an oocyte being fertilised by an X- or Y-bearing spermatozoon was not affected by the size of the follicle from which it was derived, nor by the testosterone concentration in the follicular fluid.
... PGR (Fig. 2) could play an essential role especially in CCs (Li et al. 2004) through an increased sensitivity to progesterone-induced genes. The PGR is expressed in CCs and associated with the preovulatory stage in many mammals including mice , bovine (Mingoti et al. 2002, Schoenfelder et al. 2003, pig (Lucidi et al. 2003), and human (Chian et al. 1999). The changes in steroid output and sensitivity, especially in progesterone, could be required not only for mammalian oocyte final maturation, but also for ovulation and subsequent embryo survival (Wise et al. 1994, Richards 2005, Christian & Moenter 2010, Lynch et al. 2010. ...
Article
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Cumulus cells (CCs) are essential for oocytes to reach full development competency and become fertilized. Many major functional properties of CCs are triggered by gonadotropins and governed by the oocyte. Consequently, cumulus may reflect oocyte quality and is often used for oocyte selection. The most visible function of CCs is their ability for rapid extracellular matrix expansion after the LH surge. Although unexplained, LH induces the final maturation and improves oocyte quality. To study the LH signaling and gene expression cascade patterns close to the germinal vesicle breakdown, bovine CCs collected at 2 h before and 6 h after the LH surge were hybridized to a custom-made microarray to better understand the LH genomic action and find differentially expressed genes associated with the LH-induced oocyte final maturation. Functional genomic analysis of the 141 overexpressed and 161 underexpressed clones was performed according to their molecular functions, gene networks, and cell compartments. Following real-time PCR validation of our gene lists, some interesting pathways associated with the LH genomic action on CCs and their possible roles in oocyte final maturation, ovulation, and fertilization are discussed. A list of early potential markers of oocyte competency in vivo and in vitro is thereafter suggested. These early biomarkers are a preamble to understand the LH molecular pathways that trigger the final oocyte competence acquisition process in bovine.
... In addition, mammalian COCs also secrete testosterone during in vitro maturation and possess a selection of important steroidogenic enzymes (Schoenfelder et al. 2003). The addition of exogenous testosterone (100 nM) to oocyte maturation medium selected on the basis of physiological concentrations reported in preovulatory bovine follicles ( 165 nM;Dieleman et al. 1983) improved the developmental competence of bovine oocytes after IVF (Younis et al. 1989;Silva and Knight 2000). ...
Article
In vitro production (IVP) of bovine embryos has been improved immensely throughout the last decades. Nevertheless, embryos generated in vitro still differ from their in vivo-produced counterparts. It is possible to achieve blastocyst rates of up to 70% if in vivo-matured oocytes are used. In contrast, if oocytes are matured in vitro, blastocyst rates are only half that of those matured in vivo. This rather limited success may be attributed to the heterogeneous population of oocytes which are normally retrieved from follicles of 3-8 mm rather than from preovulatory follicles. In contrast to the in vivo-ovulated oocyte, these oocytes lack development up to the preovulatory stage and are matured in vitro. Therefore, much effort has been devoted to the establishment of non-invasive and non-perturbing means for selecting the most competent oocytes, for example the extensiveness and compactness of the cumulus-corona investment and the granulation of the ooplasm. In vitro culture (IVC) conditions have been enhanced in the last few years, mainly by adjustment of media formulations, whereas the in vitro maturation (IVM) protocols stay invariable. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting matured oocyte. The scope of this review is to give an overview of the current situation of in vitro maturation of mammalian oocytes with emphasis on the bovine species. Special attention has been paid to the in vivo situation in the follicle and how a better understanding of these intrafollicular factors will aid to improve the in vitro maturation conditions.
... Moreover, in vivo studies in the rat have suggested that the strong steroidogenic activity of the CC mass may be maintained in the postovulatory COC (Schuetz & Dubin 1981, Goldschmit et al. 1989). The ability of maturing bovine COCs to secrete progesterone in culture systems has been reported (Armstrong et al. 1996, Mingoti et al. 2002, Schoenfelder et al. 2003). However, neither the pattern of steroidogenic gene expression nor its relationship with PGE biosynthetic activity within the periconceptional COC has been described in cattle. ...
Article
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Prostaglandin E(2) (PGE(2)) and progesterone appear to be critical mediators of cumulus expansion and the resumption of oocyte meiosis. The aim of this study was to identify the types of prostaglandin E synthase (PTGES) expressed in the bovine cumulus-oocyte complex (COC), to characterize their temporal expression during the periconceptional interval using an in vitro model of maturation (IVM) and fertilization (IVF), and to compare their expression with the level of steroidogenic gene expression. Real-time RT-PCR analysis revealed that enzymes related to the PGE(2) biosynthesis pathway were mainly expressed during IVM. Transcripts encoding PTGES1-3 were detected in bovine COCs. Only the expression of PTGES1 significantly increased during IVM whereas that of PTGES2 and PTGES3 remained unchanged. The induction of PTGES1 expression paralleled the induction of prostaglandin G/H synthase-2 (PTGS2) expression and the amounts of PGE(2) secreted by maturing COCs. Concomitantly, cholesterol side chain cleavage cytochrome P450 expression was significantly upregulated in maturing COCs and the high level of expression persisted in fertilized COCs. The expression of the StAR protein remained constant during IVM and then decreased significantly during IVF. Expression of the progesterone catabolic-related enzyme, 20alpha-hydroxysteroid dehydrogenase significantly decreased throughout the periconceptional interval. This was associated with a rising level of progesterone released by COCs in the culture media. In conclusion, our results suggest that the periconceptional differentiation of the bovine COC includes the transient induction of PGE(2) biosynthetic activity via the PTGS2/PTGES1 pathway during the maturation period and the increasing ability to produce progesterone from the immature to the fertilized stages.
... Grote et al. (29) demonstrated that rats treated with 6 mg/kg of TPTCl had increased serum estrogen levels. By contrast, other studies have shown a reduction in the serum estrogen levels and increased testosterone in TBTCl-treated rats (14,27,31,36). Ma et al. (24) demonstrated that azocyclotin treatment caused an increase in testosterone levels and a decrease in estrogen levels in the ovaries of female zebra fish. ...
Article
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Organotin (OTs) compounds are organometallic compounds that are widely used in industry, such as in the manufacture of plastics, pesticides, paints, and others. OTs are released into the environment by anthropogenic actions, leading to contact with aquatic and terrestrial organisms that occur in animal feeding. Although OTs are degraded environmentally, reports have shown the effects of this contamination over the years because it can affect organisms of different trophic levels. OTs act as endocrine-disrupting chemicals (EDCs), which can lead to several abnormalities in organisms. In male animals, OTs decrease the weights of the testis and epididymis and reduce the spermatid count, among other dysfunctions. In female animals, OTs alter the weights of the ovaries and uteri and induce damage to the ovaries. In addition, OTs prevent fetal implantation and reduce mammalian pregnancy rates. OTs cross the placental barrier and accumulate in the placental and fetal tissues. Exposure to OTs in utero leads to the accumulation of lipid droplets in the Sertoli cells and gonocytes of male offspring in addition to inducing early puberty in females. In both genders, this damage is associated with the imbalance of sex hormones and the modulation of the hypothalamic–pituitary–gonadal axis. Here, we report that OTs act as reproductive disruptors in vertebrate studies; among the compounds are tetrabutyltin, tributyltin chloride, tributyltin acetate, triphenyltin chloride, triphenyltin hydroxide, dibutyltin chloride, dibutyltin dichloride, diphenyltin dichloride, monobutyltin, and azocyclotin.
... However, unlike the studies by Sena et al. (2017) and de Araújo et al. (2018), our studies indicates that TBT exposure does not result in altered progesterone and estrogen levels, or ovarian aromatase protein expression. Particularly, a common feature of the consequences of TBT exposure on steroidogenesis control is the varying degree of irregularities, possibly resulting from the sensitivity and/or control of abnormal enzyme expression in different models that are also dependent on species, sex, age, TBT dose, and the time and type of chemical used (Saitoh et al., 2001;Ahn et al., 2007;Schoenfelder et al., 2003). ...
Article
Tributyltin chloride (TBT) is an endocrine disrupting chemical (EDC) associated with reproductive complications. Studies have shown that TBT targets the reproductive tract, impairing ovarian folliculogenesis and uterine morphophysiology. In this investigation, we assessed whether subchronic and low dose of TBT exposure results in abnormal ovarian follicular reserve and other irregularities in female mice. TBT was administered to female mice (500ng/kg/day for 12 days via gavage), and reproductive tract morphophysiology was assessed. We further assessed reproductive tract inflammation and oxidative stress (OS). Improper functioning of the reproductive tract in TBT mice was observed. Specifically, irregular estrous cyclicity and abnormal ovarian morphology coupled with reduction in primordial and primary follicle numbers was observed, suggesting ovarian reserve depletion. In addition, improper follicular development and a reduction in antral follicles, corpora lutea, and total healthy ovarian follicles together with an increase in cystic follicles was apparent. Evidence of uterine atrophy, reduction in endometrial gland number, and inflammation and OS were seen in TBT miceFurther, strong negative correlations were observed between testosterone levels and primordial, primary, and total healthy ovarian follicles. Thus, these data suggest that the subchronic and low dose of TBT exposure impaired ovarian follicular reserve, uterine gland number, and other reproductive features in female mice.
... However, TBT's effect on ovarian steroidogenic cells has been restricted to granulosa cells. TBT reduces estradiol synthesis in human granulosa-like tumor cells and is associated with aromatase activity inhibition in bovine granulosa cells (Saitoh et al. 2001;Schoenfelder et al. 2003). However, whether TBT can affect theca cells' steroidogenic function remains unknown. ...
Article
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Tributyltin (TBT), an organotin chemical used as a catalyst and biocide, can stimulate cholesterol efflux in non-steroidogenic cells. Since cholesterol is the first limiting step for sex hormone production, we hypothesized that TBT disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. We investigated TBT’s effect on cholesterol trafficking, luteinization, and steroidogenesis in theca cells of five species (human, sheep, cow, pig, and mice). Primary theca cells were exposed to an environmentally relevant dose of TBT (1 or 10 ng/ml) and/or retinoid X receptor (RXR) antagonist. The expression of RXRα in sheep theca cells was knocked down using shRNA. Steroidogenic enzymes, cholesterol transport factors, and nuclear receptors were measured by RT-qPCR and Western blotting, and intracellular cholesterol, progesterone, and testosterone secretion by ELISA. TBT upregulated StAR and ABCA1 in ovine cells, and SREBF1 mRNA in theca cells. TBT also reduced intracellular cholesterol and upregulated ABCA1 protein expression but did not alter testosterone or progesterone production. RXR antagonist and RXRα knockdown demonstrates that TBT’s effect is partially through RXR. TBT’s effect on ABCA1 and StAR expression was recapitulated in all five species. TBT, at an environmentally relevant dose, stimulates theca cell cholesterol extracellular efflux via the RXR pathway, triggers a compensatory upregulation of StAR that regulates cholesterol transfer into the mitochondria and SREBF1 for de novo cholesterol synthesis. Similar results were obtained in all five species evaluated (human, sheep, cow, pig, and mice) and are supportive of TBT’s conserved mechanism of action across mammalian species.
... TBT inhibiting intracellular cholesterol biosynthesis that resulting in abnormal sex steroid production and in turn testicular dysfunction has been reported in male Syrian hamsters (Kanimozhi et al., 2014). TBT was also found disturbing the reproductive endocrine system in cow granulosa cells through increasing progesterone (Schoenfelder et al., 2003). Treatment of bovine adrenal fasciculata-reticularis cells with 100 nM TBT inhibited the production of cortisol and androstenedione, but induced the accumulation of intermediate steroids, indicating the inhibition of the P450s activities by TBT (Yamazaki et al., 2005). ...
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Organotin compounds (OTs) are used in a range of industrial products, such as antifouling paints, agricultural pesticides and stabilizers. Owing to potential endocrine-disrupting effects, human exposure to such compounds is a concern. Nevertheless, little is known about the adverse effect of OTs on adrenocortical function in organisms. In this study, the human adrenocortical carcinoma cell (H295R) model was used to investigate effects of OTs on steroidogenesis and potential causes for such endocrine disruption was examined. H295R cells were exposed to several commonly used OTs, including triphenyltin (TPT), tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT), and the production level of steroid hormones were quantified. TPT and TBT decreased the production levels of 17β-estradiol, aldosterone, and cortisol, but increased that of testosterone. Furthermore, the expression levels of ten major steroidogenic genes (HMGR, StAR, CYP11A1, 3βHSD2, CYP17, CYP19A1, CYP21, CYP11B1, CYP11B2, and 17βHSD) were examined and both up-regulation of CYP11B2 and down-regulation of StAR, 3βHSD2, CYP19A1, CYP21 and CYP11B1 by TPT and TBT were observed. Intracellular levels of ATP and cyclic adenosine monophosphate (cAMP) and the activity of adenylate cyclase (AC) decreased in the H295R cells treated with TPT and TBT. No obvious changes in H295R were found with the treatment of DBT and MBT. These results suggest that OTs may stimulate steroidogenesis in vitro via inhibition of cAMP signaling pathway.
... There is evidence that the cumulus cells of the ovulated cumulus-oocyte complex can release chemical signals, such as progesterone (Schoenfelder et al., 2003;Tosca et al., 2007), which might activate localized sperm release by promoting Ca 2+ influx through CatSper channels (Lishko et al., 2012). Release may also be controlled by components from the oviduct itself, such as disulfide reducants (Talevi et al., 2007;Brussow et al., 2008), glycosidases that cleave oviduct glycans from the epithelium (Carrasco et al., 2008a and2008b), and oviduct smooth muscle contractions (Chang and Suarez, 2012). ...
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Millions or billions of sperm are deposited by artificial insemination or natural mating into the cow reproductive tract but only a few arrive at the site of fertilization and only one fertilizes an oocyte. The remarkable journey that successful sperm take to reach an oocyte is long and tortuous, and includes movement through viscous fluid, avoiding dead ends and hostile immune cells. The privileged collection of sperm that complete this journey must pass selection steps in the vagina, cervix, uterus, utero-tubal junction and oviduct. In many locations in the female reproductive tract, sperm interact with the epithelium and the luminal fluid, which can affect sperm motility and function. Sperm must also be tolerated by the immune system of the female for an adequate time to allow fertilization to occur. This review emphasizes literature about cattle but also includes work in other species that emphasizes critical broad concepts. Although all parts of the female reproductive tract are reviewed, particular attention is given to the sperm destination, the oviduct.
... Bovine COCs secreted E 2 and P 4 during IVM (Mingoti et al., 2002;Schoenfelder et al., 2003;Salhab et al., 2011;Blaschka et al., 2015). During IVM of bovine COCs (30 COCs/300 µL IVM medium) for 24 h, P 4 concentration in the medium significantly increased (3.3 to 10.4 ng mL −1 ), but E 2 concentration did not change (52.8 to 74.7 pg mL −1 ; Blaschka et al., 2015). ...
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The objective of the article is to evaluate the effect of three-step in vitro maturation (IVM) culture system imitating estradiol-17β (E2) and progesterone (P4) concentrations in preovulatory follicles on in vitro bovine embryo production. The cumulus–oocyte complexes (COCs) were collected from follicles (2 to 8 mm in diameter) of bovine ovaries obtained from a local slaughterhouse. For IVM, the COCs were cultured for 22 h in a three-step system: (1) culture in medium 199, containing 700 ng mL⁻¹ E2 and 50 ng mL⁻¹ P4, for 5 h, followed by the medium containing 150 ng mL⁻¹ E2 and 150 ng mL⁻¹ P4 for 11 h, and then the medium containing 20 ng mL⁻¹ E2 and 300 ng mL⁻¹ P4 for 6 h (EP group); (2) culture in the medium containing 700 ng mL⁻¹ E2 for 5 h, followed by the medium containing 150 ng mL⁻¹ E2 for 11 h, and then the medium containing 20 ng mL⁻¹ E2 for 6 h (E group); or (3) culture in the medium containing 50 ng mL⁻¹ P4 for 5 h, followed by the medium containing 150 ng mL⁻¹ P4 for 11 h, and then the medium containing 300 ng mL⁻¹ P4 for 6 h (P group). The COCs were cultured in the medium containing 1000 ng mL⁻¹ E2 for 22 h (control group). After IVM, the COCs were co-incubated with sperm and further cultured. At 48 h after insemination, the cleavage rate of embryos was not different among the groups. At 192 h after insemination, the blastocyst formation rate of EP group was significantly higher than that of the other groups. The total cell number of blastocysts did not differ among the groups. In conclusion, these results demonstrate that the three-step IVM culture system of bovine oocytes imitating temporal changes of E2 and P4 concentrations in preovulatory follicular fluid improves the developmental potential of embryos in vitro.
... Another hypothesis is the paracrine contribution of cumulus cells to the OF content in the Post-ov period. Indeed, the secretion of steroid hormones by bovine cumulus cells after gonadotropin stimulation was reported by several authors [49,50]. ...
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Ovarian steroid hormones are major regulators of the physiology of the oviduct and reproductive events occurring within the oviduct. To establish a whole steroid profiling of the bovine oviductal fluid (OF) during the estrous cycle, contralateral and ipsilateral (to the corpus luteum or preovulatory follicle) oviducts were classified into four stages of the estrous cycle (n = 18-27 cows per stage): postovulatory (Post-ov), mid-luteal (Mid-lut), late luteal (Late-lut), and preovulatory on the basis of the ovarian morphology and intrafollicular steroid concentrations. Steroids were extracted from pools of 150 to 200 mu L OF (three to 10 cows per pool; three to four pools per "stage x side" group), purified, fractioned by high-performance liquid chromatography, and analyzed by gas chromatography coupled with tandem mass spectrometry. The concentrations of progesterone (P4) in ipsilateral OF increased from Post-ov (56.9 +/- 13.4 ng/mL) to Mid-lut (120.3 +/- 34.3 ng/mL), then decreased from Late-lut (76.7 +/- 1.8 ng/mL) to Pre-ov (6.3 +/- 1.7 ng/mL), and were four to 16 times higher than in contralateral OR Most P4 metabolites followed similar patterns of variation. Concentrations of 17beta-estradiol (E2) were significantly higher at Pre-ov (290.5 +/- 63.2 pg/mL) compared with. all other stages (<118.3 pg/mL), with no difference regarding the side of ovulation. Concentrations of androstenedione displayed a pattern similar to that of E2, whereas other androgens, estrone, and corticoids did not vary between stages or sides. In conclusion, a highly concentrated and fluctuating hormonal environment was evidenced in the bovine OF. These results could be useful to improve media for IVF, embryo development, and culture of oviductal cells.
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The effects of butyltin compounds on follicular steroidogenesis in amphibians were examined using ovarian follicles of Rana catesbeiana. Isolated follicles were cultured for 18h in the presence and absence of frog pituitary homogenate (FPH) or various steroid precursors, and the steroid levels in the follicles or culture media were measured by radioimmunoassay (RIA). Among the butyltin compounds, tributyltin (TBT) strongly inhibited the FPH-induced synthesis of pregnenolone (P(5)), progesterone (P(4)) and testosterone (T). It also inhibited the conversion of P(5)-P(4) and T to estradiol-17β(E(2)) and it partially suppressed the conversion of androstenedione (AD) to T, but not P(4) to 17α-hydroxyprogesterone (17α-OHP(4)). A high concentration of dibutyltin (DBT) also inhibited steroidogenesis by the follicles while monobutyltin (MBT) and tetrabutylin (TeBT) had no effect. These results suggest that the initial step of steroidogenesis (P(5) synthesis) and enzymes such as 3β-HSD, 17β-HSD and aromatase are inhibited by TBT or DBT. However, 17α-hydroxylase was not suppressed by TBT or the other butyltin compounds.
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Recently, we have shown that some endocrine disruptors, heavy metals, organotins and azoles suppressed steroidogenic enzymes such as P450 side‐chain cleavage enzyme (P450scc) and aromatase in bullfrog ovarian follicles. In the present study, by using an amphibian ovarian follicle culture system, we examined the effects of these endocrine disruptors on maturation and ovulation of oocytes from Rana dybowskii in vitro. Ovarian fragments or isolated follicles were cultured for 24 h in a medium containing frog pituitary homogenate (FPH) or progesterone (P4) with or without endocrine disruptors, and oocyte maturation (germinal vesicle breakdown, GVBD) and ovulation were examined. Among the organotins, tributyltin (TBT) strongly inhibited both FPH‐ and P4‐induced oocyte maturation (ED50: 0.6 and 0.7 μM, respectively); however, tetrabutyltin (TTBT) and dibutyltin (DBT) showed only partial suppression, while monobutyltin (MBT) showed no inhibitory effect. All of the organotins suppressed P4‐induced oocyte ovulation very effectively at a low concentration, and TBT and DBT exerted an inhibitory effect on FPH‐induced ovulation. Among the heavy metals, mercury (Hg), cadmium (Cd) and cobalt (Co) were very effective in inhibiting FPH‐induced oocyte maturation and ovulation, while lead (Pb), arsenite (As) and zinc (Zn) were less effective. However, all of the heavy metals suppressed FPH‐induced oocyte ovulation at a high dose (100 μM). Among the azoles, itraconazole (ICZ), ketoconazole (KCZ) and clotrimazole (CTZ) effectively inhibited FPH‐induced oocyte maturation and ovulation, while econazole (ECZ), miconazole (MCZ) and fluconazole (FCZ) were considerably less effective. These results demonstrated that the abovementioned endocrine disruptors exhibited differential effects on oocyte maturation and ovulation in amphibian follicles and that the frog ovarian culture system could be used as an effective experimental tool to screen and evaluate the toxicity of various endocrine disruptors in vitro.
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β-Thymosins are small proteins that regulate the actin cytoskeleton and are involved in cell motility, differentiation, the induction of metalloproteinases, in anti-inflammatory processes and tumourigenesis. However, their roles in the ovary have not yet been elucidated. Using transcriptomics and real time reverse transcription-polymerase chain reaction validation, the present study demonstrates that thymosin β-4 (TMSB4) and thymosin β-10 (TMSB10) are upregulated in bovine cumulus cells (CCs) during in vitro maturation of cumulus-oocyte complexes (COCs) in parallel with an increase in mRNA expression of HAS2, COX2 and PGR genes. Using immunocytochemistry, both proteins were found to be localised mainly in granulosa cells, CCs and oocytes, in both the cytoplasm and nucleus, as well as being colocalised with F-actin stress fibres in CCs. Using different maturation mediums, we showed that the expression of TMSB10, but not TMSB4, was positively correlated with COC expansion and progesterone secretion and negatively correlated with apoptosis. Immunofluorescence, coupled with terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL), demonstrated the absence of TMSB4 and/or TMSB10 in apoptotic cells. TMSB10 expression was higher in COCs matured in vivo than in vitro, and differences related to the age of the animal were observed. TMSB4 and/or TMSB10 expression was unchanged, whereas HAS2 overexpressed in CCs from oocytes that developed to the blastocyst stage in vitro compared with those that did not. Thus, TMSB4 and/or TMSB10 ovarian expression patterns suggest that these two thymosins may be involved in cumulus modifications during maturation.
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Today, humans and wildlife are constantly exposed to thousands of chemical residues, through air, food and water. Fungicides have been used widely in order to control fungal diseases and increase crop production. However, the regular use of fungicides can potentially pose a risk to the environment and public health. Fungicides can be classified by chemical group, general mode of action, specific mode of action, or by physical properties once in the plant. These are found to be teratogenic, carcinogenic, mutagenic, reproductive toxicant as well as show harmful effects on ecology including non target plants and animals. This review deals with structure, uses and adverse effects of fungicides on different systems, thus indicates limited use of fungicides to improve the quality of life for human welfare.
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When heat stress effects are detrimental during maturation, cumulus cells are intimately associated with the oocyte. To determine the extent to which heat stress affects these cells, transcriptome profiles of the cumulus that surrounded control and heat-stressed oocytes (41 °C first 12 h only, then shifted back to 38.5 ºC) during in vitro maturation (IVM) were compared using Affymetrix bovine microarrays. Comparison of cumulus-derived profiles revealed a number of transcripts that were increased (n=11) or decreased (n=13) ≥ 2.0 fold after heat stress exposure (P<0.01) sufficient to reduce blastocyst development by 46.4%. In a separate study, qPCR was used to confirm heat-induced differences in the relative abundance of five different genes (caveolin 1, matrix metallopeptidase 9, follicle stimulating hormone receptor, Indian hedgehog homolog and inducible nitric oxide synthase). Heat stress exposure resulted in > 1.7 fold decrease in the protein levels of latent matrix metallopeptidase 9 (proMMP9). Heat-induced reductions in transcript levels were noted at 6 hIVM with reductions in proMMP9 protein levels at 18 hIVM (P=0.0002). Independent of temperature, proMMP9 levels at 24 hIVM were positively correlated with blastocyst development rate (R(2)=0.36; P=0.002). Progesterone production increased during maturation; heat-induced increases were evident by 12 hIVM (P=0.002). Both MMP9 and progesterone are associated with developmental competence of the oocyte; thus it seems plausible for some of the negative consequences of heat stress on the cumulus-oocyte complex to be mediated through heat-induced perturbations occurring in the surrounding cumulus.
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Steroid hormones are regulators in the fine-tuned process of follicular development. During final maturation in vivo a switch from oestradiol (E2) to progesterone (P4) dominance within the follicle is well-described. This change is accompanied by the resumption of meiosis and results in the maturation of the oocyte. It also suggests the important role of these hormones. However, present in vitro maturation (IVM) systems do not completely mimic the in vivo situation, resulting in oocytes of reduced quality. Aim of the study was to determine the temporal pattern of steroid hormone concentrations in the IVM medium of bovine cumulus-oocyte-complexes (COC) at defined time points. The influence of different gonadotropin supplementations during IVM on oocyte maturation, as well as the molecular quality of the oocytes and their corresponding cumulus cells was investigated. COCs were obtained from abattoir-derived ovaries and matured in medium added with different compounds of gonadotropins (eCG/hCG; FSH/LH, each at 0.05 IU or 0.01 IU; only FSH; without gonadotropins) employing a standard protocol without oil overlay. In experiment 1, medium, oocytes and cumulus cells were collected at different time points (0 h [control], 4 h, 8 h, 12 h, 16 h, 20 h, 24 h) after IVM in just eCG/hCG-supplemented medium. In experiment 2, medium, oocytes and cumulus cells were collected at 0 h (control) and after 24 h of IVM with all above-named supplements. The E2 concentration remained similar during IVM whereas P4 concentration increased during experiment 1. No significant changes could be determined after the addition of different gonadotropins (experiment 2). These results suggest that during IVM the temporal pattern of E2 and P4 did not correspond with the pattern during final maturation in vivo. RT-qPCR was used to assess the relative abundance of developmentally important genes in oocytes (BMP15; GDF9; ZAR1; PGR; PGRMC1/2; G6PD; StAR; ESR1/2; SULT1E1; STS; SOAT) and cumulus cells (ESR1/2; FSHR; LHCGR; CYP19A1; HSD3B1; PGR; PGRMC1/2; SULT1E1; STS; SOAT) at all collection points in both experiments. Most transcripts follow a time-regulated mRNA expression pattern during the entire in vitro maturation period. In addition, the expression of the analyzed transcripts was not influenced by the different gonadotropin supplementations during the IVM period. In all, this underlines that present conditions of IVM do not reflect the in vivo situation and require further optimisation.
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In response to the gonadotropin surge, the compact cumulus-oocyte complex (COC) undergoes expansion by synthesis of the mucopolysaccharide hyaluronan (HA) accompanying oocyte maturation. The objective of the present study was to quantify mRNA transcripts of the HA synthase (HAS) 1, HAS2, and HAS3 and the HA-receptors CD44 and RHAMM (receptor for HA-mediated motility). Additionally, we determined the histological localization of HA and its receptor, CD44, in maturing bovine COCs and cultured granulosa cells (GCs). Full-length transcript of bovine HAS2 and a part of the bovine RHAMM sequence has been made available. Real-time reverse transcriptase-polymerase chain reaction was used for individual mRNA expressions of bovine COCs in comparison to follicular GC gonadotropin treatment. Localization of CD44 and HA were done by immunohistochemistry and biotinylated HA-binding protein, respectively. Gonadotropins caused a rapid, 120-fold increase of HAS2 mRNA, whereas a delayed, 2-fold up-regulation of HAS3 mRNA was observed. The HAS1 transcripts were barely detected. Expression of CD44 mRNA greatly increased during in vitro maturation of COCs, indicating an important role when compared to an unchanged, steady-state RHAMM expression. As a consequence, HA was locally enriched after COC expansion, but only limited change was observed in the GCs. In cultured GCs, HAS2 expression was stimulated through FSH application, followed by the effective treatments of FSH+LH and LH. Treatment with LH induced the highest increase of the CD44 receptor, followed by FSH and FSH+LH treatments. These results suggest that HAS2 is mainly responsible for rapid HA synthesis in bovine COCs and GCs. In bovine COCs, the transcriptional up-regulation of both HAS2 and the receptor CD44 appear to be important prerequisites for initiating HA-mediated effects during final oocyte development and sperm-egg interaction.
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The bovine cumulus-oocyte complex (COC) is capable of converting cortisone, an inert glucocorticoid to active cortisol. This mechanism is mediated by 11β-hydroxysteroid oxidoreductase type 1 (HSD11B1), whose expression dramatically increases in the mature COC. In this study, we investigate the time course expression of HSD11B1 and the enzyme activity in the bovine COC undergoing maturation and fertilization in relation to key events taking place in the COC. Bovine COCs were subjected to in vitro maturation (IVM) and fertilization (IVF). The activities of HSD11B1 and HSD11B2, which mediates the opposite reaction, were measured by using a radiometric conversion assay. In parallel studies, cumulus expansion, P4 production, and the expression of genes associated with ovulation were measured. The reductive activity of HSD11B1 increased in the latter half of IVM and remained high during IVF, whereas the oxidative activity of HSD11B2 remained unchanged over both periods. Consequently, the net glucocorticoid metabolism in the bovine COC shifted from inactivation to activation around the time of ovulation and fertilization. The increase in HSD11B1 expression lagged behind that of P4 increase and cumulus expansion but ahead of the expressions of genes responsible for PGE2 synthesis. The reductive activity of HSD11B1 was well correlated with the cumulus expansion rate. This outcome indicates that the ability of the cumulus to activate glucocorticoids is related to its ability to synthesize hyaluronan. These results also indicate that the activation of HSD11B1 is an integral part of the sequential events taking place at the ovulation and fertilization in the bovine COC.
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Tributyltin, an environmental pollutant, affected adrenal steroid hormone biosynthesis by two modes of action. Treatment of bovine adrenal cultured cells with 10-100 nM tributyltin for 48 h suppressed cortisol and androstenedione secretion, but induced the accumulation of 17alpha-hydroxyprogesterone and deoxycortisol, indicating that the P450(C21) and P450(11beta) activities were specifically suppressed. Direct inhibition of the enzymatic activities due to tributyltin was not observed in isolated organelles of untreated cells at concentrations less than 10 microM. Western blotting experiments using specific antibodies against steroidogenic enzymes showed that treatment with 1-100 nM tributyltin caused a decrease in cellular P450(C21) and P450(11beta) protein levels, and real-time PCR experiments showed that the decrease in protein content was attributable to decreases in mRNA of the enzymes. Tributyltin at concentrations higher than 100 nM suppressed all steroid biosynthesis in the adrenal cells. This suppression was closely correlated to the decrease in steroidogenic acute regulatory protein. Since nanomolar concentrations of tributyltin disturbed steroidogenesis in mammalian cells, there is the possibility that steroid hormone synthesis in polluted wild animals is affected by this compound.
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To investigate the localization and expression of steroid sulfatase (STS) in cumulus cells obtained from subjects with and without endometriosis. Case-control study. In vitro fertilization program at the Showa University School of Medicine. Cumulus cells (142 samples) were obtained from 49 patients for whom IVF was indicated. Some of these samples were taken from cases complicated by endometriosis (35 samples), polycystic ovary syndrome (PCOS; 16 samples), or latent hyperprolactinemia (16 samples). Immunohistochemical staining for STS. Measurement of STS mRNA expression by real-time polymerase chain reaction (PCR). Expression of STS mRNA and localization of STS. Steroid sulfatase was localized in the cytoplasm of the cumulus cells, and expression of STS mRNA was observed. The expression level of STS mRNA from patients with endometriosis was significantly higher (11.8-fold) than that of patients without endometriosis. These results suggest a local steroidal regulation mechanism in cumulus cells. Although the physiological role of STS in cumulus cells remains unclear, STS may be involved in the quality of eggs in patients with endometriosis.
Article
Ovarian steroid hormones are major regulators of the physiology of the oviduct and reproductive events occurring within the oviduct. In order to establish a whole steroid profiling of the bovine oviductal fluid (OF) during the estrous cycle, contra- and ipsilateral (to the corpus luteum or pre-ovulatory follicle) oviducts were classified into four stages of the estrous cycle (n=18-25 cows/stage): post-ovulatory (Post-ov), mid luteal (Mid-lut), late luteal (Late-lut) and pre-ovulatory (Pre-ov) based on ovarian morphology and intra-follicular steroid concentrations. Steroids were extracted from pools of 150-200 μl OF (4-10 cows/pool; 3-4 pools per 'stage × side' group), purified, fractioned by high performance liquid chromatography and analyzed by gas chromatography coupled with tandem mass spectrometry. The concentrations of progesterone (P4) in ipsi-lateral OF increased from Post-ov (56.9 ± 13.4 ng/ml) to Mid-lut (120.3 ± 34.3 ng/ml) then decreased from Late-lut (76.7 ± 1.8 ng/ml) to Pre-ov (6.3 ± 1.7 ng/ml) and were 4 to 16 times higher than in contralateral OF. Most P4 metabolites followed similar patterns of variation. Concentrations of 17beta-estradiol (E2) were significantly higher at Pre-ov (290.5 ± 63.2 pg/ml) compared with all other stages (<118.3 pg/ml), with no difference regarding the side of ovulation. Concentrations of androstenedione displayed a pattern similar to that of E2 whereas other androgens, estrone and corticoids did not vary between stages or sides. In conclusion, a highly concentrated and fluctuating hormonal environment was evidenced in the bovine OF. These results could be useful to improve media for in vitro fertilization, embryo development and culture of oviductal cells.
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The toxicity of selected tin compounds is reviewed. Over the years, a variety of uses has been found for organic and inorganic tin compounds, as fungicides, as stablizers in plastics, moluscicides, and miticides; they have also been suggested as insect chemosterilants and for other industrial uses. Many of these products are unpalatable when mixed into diets and have been suggested as rodent repellents. Inhaling tin as dust or fumes may cause a benign pneumoconiosis in exposed workers. The organotin compounds can be divided into alkyltin and aryltin compounds. The trimethyl and triethyltin compounds are well absorbed from the gastrointestinal tract and are the most toxic in this group. Triethyltin particularly produces status spongiosus of the white matter of the central nervous system. Most of the other alkyl and aryl tin compounds are poorly absorbed from the gastrointestinal tract, and are less toxic when given orally than when given parentally. Only one compound, tricyclohexyltin hydroxide, is now registered by the Environmental Protection Agency as a miticide. This product produces skin irritation in rabbits. Studies should be conducted to determine whether it causes contact dermatitis in humans.
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Preovulatory bovine follices (n = 73) were collected at different times after the onset of oestrus until shortly before ovulation, which occurred at 24 +/- 1 X 4 h after the peak concentration of LH in the peripheral blood. Non-atretic antral follicles (n = 9) of 15-19 mm were also collected from cows during the luteal phase of the oestrous cycle. Follicular fluid concentrations of dehydroepiandrosterone, androstenedione and oestrone, and of LH, FSH and prolactin were compared in 2-h periods relative to the LH plasma peak. Before the LH surge the concentrations of the steroids were much higher than in non-atretic luteal-phase follicles of similar size. From 0 to 6 h after the LH peak the steroid concentrations decreased sharply to remain low until ovulation; only that of androstenedione increased again after 14 h to remain constant. The ratio between the concentrations of androstenedione and dehydroepiandrosterone remained constant until 14 h after the LH peak; at 14 h it increased about 4-fold and remained high until ovulation. The ratio between the oestrone and androstenedione concentration increased gradually to a 10-fold higher value until at 14 h an abrupt decrease was observed. These changes indicate that after the LH peak androgen production is directly inhibited and, at a slower rate, the aromatizing activity. Androstenedione appeared to be the major aromatase substrate. Before the plasma LH peak the follicular fluid concentration of FSH was higher than in luteal-phase follicles; the concentrations of LH and prolactin were not different from those in luteal-phase follicles.(ABSTRACT TRUNCATED AT 250 WORDS)
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Secretion of interferon tau (IFN tau) by trophoblastic tissue has been shown to be the first embryonic signal for pregnancy recognition. Therefore we tried to derive biologically active trophoblastic tissue by in vitro techniques. Since conventional in vitro conditions for bovine embryo development were not sufficient for long-term culture, we tested more complex culture conditions, including Ménézo B2 or Buffalo rat liver (BRL) cell-conditioned medium, for their ability to support proliferation and IFN tau secretion by in vitro-derived trophoblastic tissue. IFN tau activity was determined by using a biological assay based on the inhibition of the cytopathic effect of vesicular stomatitis virus on Madin-Darby bovine kidney cells. When cultures of individual hatched blastocysts were started in 60-microliters drops of BRL cell-conditioned medium, mean IFN tau secretion (antiviral units/ml/48 h) corresponded to 1200 on Day 11 and to 5000 on Day 13 (p < 0.01). To characterize trophoblast cell-specific secretions, the inner cell mass was removed from all embryos by microsurgery on Day 13. IFN tau secretion by trophoblastic tissue increased to mean levels of > 10(5) antiviral units/ml/48 h on Day 23m, stayed high for about 1 wk, and then slowly declined to levels below 10(3) antiviral U/ml/48 h. The specificity of the cytoprotective effect of IFN tau was tested by Western blot analysis and by immunoneutralization with use of a polyclonal antiserum specific to IFN tau. Our results demonstrate that viable trophoblastic tissue can be maintained entirely in vitro and secretes high amounts of IFN tau.
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The aim of the present study was to determine whether androgens and progesterone influence the in vitro maturation of bovine oocytes as assessed by cleavage rates and competence to form blastocysts after in vitro fertilization. Bovine cumulus-oocyte complexes were cultured (n = 20 per drop) for 22-24 h at 38.5 degrees C in TCM-199 medium supplemented with 10% oestrous cow serum, eCG (2.5 iu ml(-1)) and a range of treatments that included aromatizable (testosterone; 100 nmol l(-1)) and non-aromatizable (dihydrotestosterone; 100 nmol l(-1)) androgens, an androgen antagonist (flutamide; 36 micromol l(-1)), progesterone (300 nmol l(-1)) and a progesterone antagonist (mifeprisone, RU486; 100 nmol l(-1)). Production of inhibin A, total alpha-subunit, activin A and follistatin by each group of cumulus-oocyte complexes was also measured, since inhibin-related peptides have been implicated as modulators of oocyte maturation and their production may be influenced by steroids and anti-steroids. Both testosterone and dihydrotestosterone increased oocyte cleavage rate (25%; P < 0.01) and dihydrotestosterone also increased (24%; P < 0.05) the proportion of oocytes that reached the >/= eight-cell stage. However, neither androgen affected blastocyst yield, or the proportion of blastocysts that hatched. The stimulatory effect of dihydrotestosterone on cleavage rate was reduced by flutamide but the anti-androgen had no effect when tested alone. Treatment with testosterone, but not dihydrotestosterone, decreased (P < 0.05) endogenous follistatin and increased (P < 0.05) the activin A:follistatin ratio in maturation medium. Concentrations of inhibin A, total alpha-subunit and activin A were not affected significantly by androgen or flutamide. Addition of progesterone or the anti-progestin mifepristone to cumulus-oocyte complexes had no effect on cleavage rate. However, progesterone reduced by approximately 40% (P < 0.05) the proportions of both total oocytes and cleaved oocytes that formed blastocysts. This effect was partially reversed by mifepristone. Neither progesterone nor mifepristone affected inhibin A, activin A or follistatin production. However, total alpha-subunit concentration was significantly greater in the progesterone-treated group than in the controls (50%; P < 0.05), indicating that the negative effect of progesterone on blastocyst yield may be mediated by increased inhibin alpha-subunit expression by cumulus cells.
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This second edition of Laboratory Production of Cattle Embryos reviews advances in embryo production technology, with emphasis on the common ground between cattle and human embryology. The 10 chapters discuss developments in embryo in vitro production (IVP) technology, the oestrous cycle, oocyte recovery, oocyte maturation, sperm capacitation, in vitro fertilization, culture and evaluation of early cattle embryos, embryo and oocyte preservation, establishing pregnancies with IVP embryos and embryos and oocytes in research and commerce. Appendices that present embryo production protocols, preparation of culture media, cryopreservation procedures and various sources of information concerning in vitro production and transfer of cattle embryos are also included, as well as an index and a list of tables and figures. This book will be of interest to those working in areas of animal breeding and reproduction and in animal and veterinary science.
Article
Real-time reverse transcription followed by polymerase chain reaction (RT–PCR) is the most suitable method for the detection and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene. Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the calculation of relative expression ratio allow only for the determination of a single transcription difference between one control and one sample. Therefore a new software tool was established, named REST© (relative expression software tool), which compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance by a randomisation test. Herein, development and application of REST© is explained and the usefulness of relative expression in real-time PCR using REST© is discussed. The latest software version of REST© and examples for the correct use can be downloaded at http://www.wzw.tum.de/gene-quantification/.
Chapter
Over the last 15 years, PCR has become an essential part of most laboratories involved in biomedical research. PCR amplification turns a few attograms of a specific fragment of nucleic acid (far too little to be analyzed directly or used in biochemical reactions) into as much as a microgram of DNA.
Article
1. A definite chronological sequence of events occurs in the eggs and follicles of rabbits after mating or after the injection of ovulation-inducing substances. The follicle secretes secondary liquor folliculi, and there occurs a separation of the corona radiata from strands connecting it to the follicle cells. The ovum goes through nuclear maturation with as climax the production of the first polar body by the 8th hour after copulation. 2. Thyroxin injections cause indirectly the same effects as mating or pituitary injections but no ovulation occurs. The thyroxin effect occurs later than the pituitary effect and is due to an initiation of atresia in the follicles. 3. Explantation of ova results in typical maturation phenomena which are apparently unaffected by the presence of pituitary hormones or of thyroxin in the culture medium. 4. It is concluded that maturation of the ovum can be obtained simply by isolating it from the normal follicular environment. 5. Normal fertilization can be secured with eggs removed from the follicles.
Article
This study was designed to determine whether cumulus oophorus cells secrete progesterone. Immature PMS-primed, hCG-treated rats were used. Their cumuli were isolated from pre-ovulatory (no hCG) and peri-ovulatory (after hCG) follicles, and from post-ovulatory oviducal ampullae. Treatment with hCG increased progesterone secretion by almost two-and-one-half-fold. It was speculated that the cumulus oophorus secretion of progesterone modifies the accumulation of fluid in the ampulla, thus providing the medium in which fertilization takes place.
Article
In a first paper, the structural and functional relationship of granulosa and theca of follicles during early development stages was reported. On the special question of whether and from which moment these two tissue formations are involved in the steroidbiosynthesis, our electronmicroscopic examinations have given a further insight to this. Neither the granulosa nor the theca folliculi of primordial, primary and secondary follicles show definite morphologic submicroscopic criteria of the steroidbiosynthesis. In the resting tertiary follicle, the electronmicroscopy defines the theca cells as steroidbiosynthetic active cells, whereas the granulosa cells of this stages of follicle demonstrate the morphologic characteristics of proteinsynthetic active cells. Our, under physiological conditions, systematically conducted light- and electronmicroscopic studies of preovulatory and freshly ruptured follicles showed remarkable structural changes in the granulosa, as well as in the theca folliculi. For the follicle granulosa cells of the preovulatory follicle, the studies could demonstrate a structural transformation process of proteinsynthetic active to steroidbiosynthetic active cells. This transformation process can be seen in the nucleus. Especially recognizable is the continuous change from rough to smooth endoplasmatic reticulum in the cytoplasm of most of the follicle granulosa cells, which apart from that, often displayed whorle-like formations. Furthermore, we noticed striking changes of the paraplasmatic structures, especially of the fat, present in various stages in the cytoplasm of the follicle granulosa cells. We also noticed large mitochondria, which showed a lot of vesicular cristae. Numerous existing Golgi-fields contained many little homogenous fat-like droplets, which were surrounded by a thin osmiophilic membrane. This recognizable transformation process of the proteinsynthetic active granulosa cells to the steroid cells is quite completed in freshly ruptured follicles, according to our examinations. Although the theca cells have already been defined submicroscopically in the resting tertiary follicle as steroidbiosynthetic active cells, we see in this cellgroup in the preovulatory, as well as in the freshly ruptured follicle a remarkable conspicuous size-increase of the mitochondria. Apart from that, there are wide areas which are solely occupied by smooth endoplasmatic reticulum, whereas other structures, for example fat, are scarcely seen. As our examinations have shown, there is a close relationship between the transformation process in the granulosa and the theca of preovulatory follicles and the concentration movement of the gonadotropins. We are of the opinion that, the increasing concentration of progesteron in serum, which begins prior to the ovulation, can be regarded as a product of the follicle granulosa cells being transformed to steroid cells.
Article
1. A definite chronological sequence of events occurs in the eggs and follicles of rabbits after mating or after the injection of ovulation-inducing substances. The follicle secretes secondary liquor folliculi, and there occurs a separation of the corona radiata from strands connecting it to the follicle cells. The ovum goes through nuclear maturation with as climax the production of the first polar body by the 8th hour after copulation. 2. Thyroxin injections cause indirectly the same effects as mating or pituitary injections but no ovulation occurs. The thyroxin effect occurs later than the pituitary effect and is due to an initiation of atresia in the follicles. 3. Explantation of ova results in typical maturation phenomena which are apparently unaffected by the presence of pituitary hormones or of thyroxin in the culture medium. 4. It is concluded that maturation of the ovum can be obtained simply by isolating it from the normal follicular environment. 5. Normal fertilization can be secured with eggs removed from the follicles.
Article
In a first paper, the structural and functional relationship of granulosa and theca of follicles during early development stages was reported. On the special question of whether and from which moment these two tissue formations are involved in the steroidbiosynthesis, our electronmicroscopic examinations have given a further insight to this. Neither the granulosa nor the theca folliculi of primordial, primary and secondary follicles show definite morphologic submicroscopic criteria of the steriodbiosynthesis. In the resting tertiary follicle, the electronmicroscopy defines the theca cells as steroidbiosynthetic active cells, whereas the granulosa cells of this stages of follicle demonstrate the morphologic characteristics of protein-synthetic active cells. Our, under physiological conditions, systematically conducted light and electronmicroscopic studies of preovulatory and freshly ruptured follicles showed remarkable structural changes in the granulosa, as well as in the theca folliculi. For the follicle granulosa cells of the preovulatory follicle, the studies could demonstrate a structural transformation process of proteinsynthetic active to steroidbiosynthetic active cells. This transformation process can be seen in the nucleus. Especially recognizable is the continuous change from rough to smooth endoplasmatic reticulum in the cytoplasm of most of the follicle granulosa cells, which apart from that, often displayed whorle-like formations. Furthermore, we noticed striking changes of the paraplasmatic structures, especially of the fat, present in various stages in the cytoplasm of the follicle granulosa cells. We also noticed large mitochondria, which showed a lot of vesicular cristae. Numerous existing Golgi-fields contained many little homogenous fat-like droplets, which were surrounded by a thin osmiophilic membrane. This recognizable transformation process of the proteinsynthetic active granulosa cells to the steroid cells is quite completed in freshly ruptured follicles, according to our examinations. Although the theca cells have already been defined submicroscopically in the resting tertiary follicle as steroidbiosynthetic active cells, we see in this cellgroup in the preovulatory, as well as in the freshly ruptured follicle a remarkable conspicuous size-increase of the mitochondria. Apart from that, there are wide areas which are solely occupied by smooth endoplasmatic reticulum, whereas other structures, for example fat, are scarely seen. As our examinations have shown, there is a close relationship between the transformation process in the granulosa and the theca of preovulatory follicles and the theca of preovulatory follicles that, the increasing concentration of progesteron in serum, which begins prior to the ovulation can be regarded as a product of the follicle granulosa cells being transformed to steroid cells.
Article
A sensitive test system has been developed for estimation of estradiol-17 beta (E2) in bovine plasma. Plasma extracts are first purified by a selective immunoaffinity chromatography (IAC) using an antibody raised against estradiol-6-carboxymethyloxime-bovine serum albumin and immobilized to Sepharose. The eluate was analysed by a competitive enzyme immunoassay (EIA) on microtitration plates. For the assay the wells of microtitration plates were coated with affinity purified sheep IgG (antirabbit IgG) that binds the hormone specific antibody raised in rabbits against estradiol-17-hemisuccinate-bovine serum albumin. E2 is estimated by displacement of biocytinyl-E2, that was produced by ligation of estradiol-17 beta, D-glucuronic acid and biocytin. Bound biocytinyl-E2 is detected after binding of streptavidin-peroxidase and colour production by the enzyme. A very high amplification was possible with this technique and the absolute detection limit amounted to approximately 120 fg/well at 94% relative binding. By combination of IAC and EIA the following levels of E2 were found in bovine plasma: male or female calves less than 2.7 pg/ml, cycling cow 0.5-7 pg/ml, cow during the last month of pregnancy 9-310 pg/ml, mature bull 5-30 pg/ml. However, up to 1110 pg E2/ml were found in plasma of a calf after treatment with an illicit hormone preparation used for growth promotion; after 21 days levels declined to 6 pg/ml which is hardly different from controls. In conclusion, the IAC/EIA can be used for sensitive estimation of estradiol-17 beta in plasma from all type of cattle and for control of improper use of E2 after commitment of a threshold level.
Article
A simple direct enzymeimmunoassay (EIA) on microtiter plates for plasma progesterone using the second antibody coating technique and horseradish peroxidase (HRP) as the enzyme label (EIA-HRP) is described and compared with an identical EIA procedure which employed alkaline phosphatase (AP) as the enzyme label (EIA-AP). The assays used antiserum raised against progesterone-7-carboxyethlthioether-BSA in rabbits. Both systems were further compared with the conventional direct progesterone radioimmunoassay (RIA) in regular use. The enzymes HRP and AP were coupled to progesterone-6 beta-hydroxy-hemisuccinate by a mixed anhydride method. While the precision of EIA-HRP was comparable to RIA, the sensitivity in terms of the lowest detection limit obtained in EIA-HRP was about 10 times better than that seen in RIA. Progesterone estimates from plasma samples in EIA-HRP showed good correlation (r = 0.94) with the RIA values and the levels measured in the two systems were identical. Progesterone estimates from plasma samples in EIA-AP were at least three times higher than those obtained by either EIA-HRP or RIA. Thus, only the EIA-HRP but not the EIA-AP was suitable for the reliable direct measurement of progesterone in plasma.
Article
To investigate some biochemical changes during bovine follicle development, ovaries were obtained from cyclic heifers (7 to 11 heifers/d on each day of the 21-d estrous cycle; N = 152). Follicular fluid from the two largest follicles from both ovaries and a pool from small follicles (N = 30/cow) were collected from each animal and analyzed for ionic, enzymatic and endocrine changes in relation to day of the estrous cycle, follicle size, rank and atretic or growing status. Follicular fluid alkaline phosphatase activity and ascorbate concentrations were highest in all follicular sizes during the earlier portion of the estrous cycle (d 1 to 12; P less than .05), then decreased to the lowest levels (d 13 to 21). As follicular size (diameter) increased lactate dehydrogenase (LDH), acid and alkaline phosphatase activity was reduced in follicular fluid (P less than .05). Alkaline phosphatase and LDH activity tended to be increased in atretic follicles (P less than .10), and was correlated with increased progesterone and androgen concentrations of follicular fluid (r = .4, P less than .05). Both albumin and total protein concentrations decreased as follicular diameter increased (P less than .05). Sodium concentrations in follicular fluid were greater in growing-antral than atretic follicles, and increased with follicular enlargement (P less than .05). Follicular potassium concentrations increased as the estrous cycle progressed (P less than .05), and tended to be elevated in atretic follicles (nonsignificant). Both Ca and Mg concentrations increased with follicular enlargement (P less than .05). Dehydroepiandrosterone and testosterone were the predominant androgens in follicular fluid (androstenedione, the lowest concentration); their concentration decreased with follicle development (P less than .05), but were quite variable. Estradiol was increased in growing follicles (P less than .01). Estrone and estradiol concentrations increased as ovulation approached, particularly in small follicles (less than or equal to 4 mm diameter). Changes of biochemical components found in follicular fluid that relate to the growth and atresia process may provide a more sensitive and accurate method to classify follicle status, and thus aid in understanding the complexity of events associated with maturation of the bovine follicle and oocyte.
Article
Follicular maturation and development is a complex process of interrelated intra- and extraovarian events that ultimately lead to ovulation of a mature oocyte and transformation of the ruptured follicle into a corpus luteum. The primordial follicle consists of an immature oocyte arrested in the dictyate stage of meiosis, surrounded by a single layer of relatively undifferentiated granulosa cells. The oocyte remains in the immature state because of many factors, one of which is the oocyte maturation inhibitor (OMI) secreted by granulosa cells. The oocyte subsequently increases in size, and as the antrum forms it becomes surrounded by cumulus cells. The cumulus cells may be intimately involved in the action of O,I to arrest the oocyte in the immature state within the follicle, as well as the resumption of meiosis during the LH surge. The compartments of the follicle that change most dramatically during follicular maturation are the cells lining the follicle--the granulosa and thecal cells. Under the influence of estrogen and FSH, the granulosa cells proliferate and also acquire FSH receptors. At this time, the thecal compartment differentiates and surrounds the granulosa cells, but remains separated from them by a basement membrane. Steroid secretion by the antral follicle involves the interplay of androgens, estrogens, and progestins. Both the granulosa and thecal cell compartments contribute to follicular fluid and serum levels of steroids; the interaction of both cell types may be necessary for estrogen and progesterone secretion in some species. As a consequence of the presence of an elevated number of FSH receptors, the granulosa cells of the small antral follicle are able to respond to FSH in many ways, including increased cyclic AMP accumulation, activation of the aromatase system, and induction of LH receptors, which permits the granulosa cells to later respond to LH. The mechanism by which thecal cells acquire their LH receptors is presently unknown. The granulosa cells of the follicle may indirectly control their own maturation and the number of follicles maturing through the secretion of follicular inhibin, which decreases the pituitary output of FSH. Even though the granulosa cells have acquired large numbers of LH receptors, they are prevented from luteinizing prematurely by factors in follicular fluid, including estrogen and a luteinizing inhibitor (LI). As serum LH levels increase during the preovulatory LH surge, a number of events occur: resumption of oocyte meiosis, transformation of the steroid enzyme complex from estrogen to progesterone secretion, follicular rupture, and formation of the corpus luteum. Granulosa cells form the bulk of the corpus luteum, which secretes elevated amounts of progesterone for a fixed time period depending on the species. Before ovulation the preovulatory follicle must be exposed to and respond to adequate LH and FSH levels in order for the eventual corpus luteum to secrete elevated amounts of progesterone for its normal lifespan...
Article
In order to compare the properties of isolated cumulus and granulosa cells, granulosa cells and cumulus cells surrounding oocytes were harvested from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) porcine antral follicles and the number of LH/hCG receptors was measured by the binding of [125I]hCG. The ability of the cells to secrete progesterone in culture was examined in the presence and absence of hCG and LH. In 3 separate experiments of 1-h incubations at 37 degrees C using cells harvested from medium-sized follicles, granulosa cells bound 10--15-fold more iodinated hCG than an equivalent number of cumulus cells. During a 2-day culture period, cumulus cells secreted less progesterone than granulosa cells from medium- and large-sized antral follicles (p less than 0.01). The potential of both cumulus and granulosa cells to secrete progesterone in culture increased as the follicle progressed from small to large size. Also, the ability of the oocyte to mature in culture increased with antral follicle size. Concurrently the ability of cumulus-oocyte complexes to form monolayers in culture decreased as the follicle matured. Cumulus and granulosa cells harvested from small- and medium-sized follicles responded similarly to LH and hCG with a stimulation in progesterone secretion after 2-6 days in culture.
Article
Preovulatory bovine follicles (n = 58) were collected at different times after the onset of standing oestrus when cows would allow mounting until shortly before ovulation, which occurred 24 +/- 1.4 h after the peak level of LH in the peripheral blood. Non-atretic antral follicles (n = 71) of 3-20 mm were also collected from cows during the luteal phase of the oestrous cycle. The follicular fluid was aspirated for the radioimmunoassay of oestradiol-17 beta, progesterone and testosterone. The follicular wall was examined micromorphologically. Follicular fluid steroid levels were compared in 2-h periods relative to the LH peak. The development of the preovulatory follicles from onset of oestrus to ovulation can be divided into four phases. Phase 0 (after onset of oestrus but before LH surge) was characterized by a high level of oestradiol (6.05 mumol/l); the levels of progesterone and testosterone were lower (0.387 and 0.165 mumol/l respectively) but higher than in non-atretic luteal follicles of similar size. The theca interna (TI) was wider and the membrana granulosa (MG) cells were larger than those of non-atretic antral follicles. During phase 1 (0-6 h after the LH peak) oestradiol remained constant but at a lower level, progesterone increased (0.727 mumol/l) and testosterone was higher from 0 to 2h after the LH peak (0.241 mumol/l). The TI was 40% wider, whereas the size of the MG cells remained the same. In phase 2 (6-20 h after the LH peak) the level of oestradiol dropped rapidly during the period from 6 to 10 h, that of progesterone decreased to the same level as in phase 0 and that of testosterone was low (0.031 mumol/l). The width of the TI decreased to that of preovulatory follicles in phase 0 and the MG cells were slightly larger. In phase 3 (20 h after the LH peak until ovulation) the level of oestradiol decreased further (0.461 mumol/l) and that of testosterone remained low. Progesterone increased to the highest levels observed (1.51 mumol/l), however, and this coincided with a 39% increase in the size of the MG cells, whereas the width of the TI remained the same as in the preceding phases 0 and 2. In phase 3 the basement membrane began to disintegrate and phagocytic cells could be observed. This points to a simultaneous functional and morphological luteinization. It is suggested that these changes in follicular steroid levels and micromorphology are regulated by the preovulatory LH peak.
Article
The resumption of meiosis in mammalian oocytes is associated with sequential changes in follicular steroidogenesis, the principal features of which are an initial stimulation and then suppression of the secretion of oestrogens and androgens, followed by a steady increase in the secretion of progesterone. The addition of steroid enzyme inhibitors to isolated follicles in vitro alters the normal profile of steroids secreted during maturation and induces intracellular changes in the oocytes which are expressed as nuclear abnormalities at fertilization. Different abnormalities are induced by selectively modifying steroid biosynthesis during maturation, the maturing oocyte being more sensitive to an imbalance in the steroid profile rather than the total inhibition of steroid secretion. The cause of the developmental aberrations may either result from or be associated with the abnormal patterns of proteins synthesized by oocytes denied the support of the correct balance or sequence of steroids during maturation.
Article
Oestrone and dihydrotestosterone had no significant action. Other steroids inhibited maturation. The stage of maturation most affected and the median effective concentration (MEC) at this stage varied with different steroids. The predominant effect of pregnenolone (MEC = 6.4 microM), progesterone (MEC = 5.3 microM), androstenedione (MEC = 28.0 microM) and oestradiol-17 beta (MEC = 23.0 microM) was to block maturation after the resumption of meiosis but before completion of the first meiotic division. Testosterone was also effective at this stage (MEC = 23.5 microM) but at higher concentrations it prevented germinal vesicle breakdown (MEC = 40 microM) without causing oocyte degeneration. The inhibitory actions of all steroids were reversible in oocytes exposed for 4 or 18 h.
Article
The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.
Article
We showed previously that the abundance of serum albumin mRNA is decreased in H4-II-E rat hepatoma cells limited for a single essential amino acid (phenylalanine, methionine, leucine, or tryptophan). To define the specificity of this phenomenon, we examined the effect of amino acid limitation on the abundance of mRNAs for 19 genes in the H4-II-E cells. These genes included six genes whose expression is either completely liver-specific or highly enriched in the liver compared with other tissues [albumin, transthyretin (TTR), transferrin, carbamyl phosphate synthetase-I, urate oxidase, class I alcohol dehydrogenase], as well as a number of ubiquitously expressed "housekeeping" genes. The results indicated that the 19 genes could be divided into three classes based on their response to amino acid limitation. Class I genes (the six liver-specific genes and alpha-tubulin) exhibit decreased expression in response to amino acid limitation. The expression of class II genes [beta 2-microglobulin, hypoxanthine-guanine phosphoribosyl transferase (HPRT), H-ferritin, ubiquitin (UbB), insulin-like growth factor binding protein-4, HNF-1 alpha] is not significantly affected by amino acid limitation. Class III genes [gadd153, beta-actin, ubiquitin (UbC), phosphoglycerate kinase-1, C/EBP alpha, C/EBP beta] exhibit increased expression in response to amino acid limitation. Thus, specific inductive as well as repressive effects on gene expression are quite common in amino acid-limited cells. The observation that all six genes whose expression is liver-specific exhibited decreased expression in amino acid-limited cells suggests a common mode of regulation of these genes by amino acid availability. The strong induction by amino acid limitation of the C/EBP inhibitor gadd153 is of interest in this regard, as increased levels of gadd153 could interfere with C/EBP, which is required for high expression of most liver-specific genes. To investigate further the molecular mechanism for the decrease in albumin mRNA abundance, albumin nuclear transcript levels were quantified in control and tryptophan-limited cells. Tryptophan limitation caused a decrease in albumin nuclear transcript abundance, and this decrease preceded the decrease in albumin mRNA, suggesting that the decrease in albumin mRNA was caused at least partly by a decrease in albumin gene transcription. Additional experiments with actinomycin D indicated that albumin mRNA was also destabilized in the tryptophan-limited cells. Thus, the overall results indicate that the decrease in albumin mRNA in the tryptophan-limited cells is caused by a specific decrease in albumin nuclear transcript abundance and destabilization of albumin mRNA.
Article
Tributyltin (TBT) immunotoxicity in rodent species is primarily characterised by T-lymphocyte deficiency resulting from a depletion of cortical thymocytes. In this study, bis(tri-n-butyltin) oxide (TBTO) was administered to male rats as a single oral dose of 30 or 60 mg/kg, and assessments were made of thymic cytopathology and the integrity of cellular DNA. TBTO treatment did not cause severe toxicity or overt clinical signs; however, by 48 h post-dosing relative thymus weights at 30 and 60 mg/kg were reduced to 66 and 43%, respectively, of control values. Increased DNA fragmentation was evident in thymic cell isolates (principally thymocytes) obtained from treated animals during the period of thymic involution. When DNA purified from these cells was visualised by agarose gel electrophoresis a multimeric internucleosomal fragmentation pattern, indicative of supra-physiological levels of apoptosis, was detected. Although unassociated apoptotic or necrotic thymocytes were essentially absent in cell preparations from TBTO-treated rats, significantly increased numbers of mononuclear phagocytic cells were observed. Many of these cells contained either apoptotic thymocytes, with nuclear morphologies exhibiting chromatin condensation, or cell remnants which were characterised as apoptotic bodies. Dibutyltin, which is a major metabolic dealkylation product of tributyltin, failed to significantly stimulate apoptosis when added to isolated thymocytes in vitro. Collectively, these findings suggest that activation of apoptosis contributes to TBT-induced thymocyte depletion in vivo, and indicate that it is unlikely that the metabolite dibutyltin is responsible for this effect.
Article
Tributyltin (TBT) salts are well-known skin irritants in both human and rodents. This study investigated the role of interleukin-1alpha (IL-1alpha) in the process in mice and in murine keratinocytes. The ears of Balb/c mice were painted with different amounts of TBT (67-536 nmol in acetone) or with acetone alone. Two hours later there was dose-related production of IL-1alpha along with ear swelling and accumulation of skin water, all of which were partially prevented by intraperitoneal injection of antibody against murine IL-1alpha. By reverse transcription-polymerase chain reaction we were able to show that the neutralizing antibody also partially prevented TBT-induced in vivo IL-6 expression but no TBT-induced TNF-alpha expression, suggesting a paracrine effect of IL-1alpha on IL-6 production but not TNF-alpha expression and indicating that other inflammatory mediators are involved. TBT induced both intracellular production of IL-1alpha and its release into culture medium in a murine keratinocyte cell line (HEL30). IL-1alpha production was inhibited by addition of a neutralizing antibody against IL-1alpha, which suggests an autocrine effect of IL-1alpha on its own production. The intracellular production of IL-1alpha could he significantly inhibited by prior treatment with antioxidants, which strongly suggests a role for oxidative species in the mechanism of action of TBT in IL-1alpha induction. The complex-1 inhibitor rotenone also significantly inhibits IL-1alpha production. Since TBT causes disturbances in the respiratory chain in mitochondria, the mechanism of its action may be the production of reactive oxygen intermediates at the ubiquinone site, which activate transcription factors and promote IL-1alpha synthesis.
Article
Skin irritant reactions are under the control of a network of cytokines and lipid mediators. This study characterized the production of tumor necrosis factor-alpha (TNF) induced by a skin irritant treatment, tributyltin (TBT), in mice through transcription factor activation and its pharmacologic modulation by anti-inflammatory agents. The ears of BALB/c mice were painted with different amounts of TBT (67-536 nmol in acetone) or with acetone alone. At different times thereafter, TNF production was analyzed both at the mRNA and protein level, by semiquantitative RT-PCR and L929 cytotoxicity assay, respectively. TBT induced rapid (1 h) TNF gene expression and protein synthesis. Maximal TNF production was observed 2 h after treatment. The production of TNF was paralleled by accumulation of skin water; this was partially prevented by intraperitoneal injection of antibody against murine TNF. These data indicate that skin irritation induced by TBT is attributable, in addition to the actions of other inflammatory mediators, to the action of keratinocyte-derived TNF. TNF production was preceded by a rapid (5 min) activation of nuclear factor-kappaB (NF-kappaB), which was also maximal 30 min after treatment. TBT-induced accumulation of skin water and TNF production were significantly reduced by topical treatment with dexamethasone and pentamidine, two anti-inflammatory agents. Interestingly, dexamethasone, but not pentamidine, decreased TBT-induced NF-kappaB activation, confirming in vivo that the glucocorticoid receptor interacts functionally within the nucleus with other transcription factors opposing one another's activity.
Article
Previously published data have suggested that oestradiol exerts direct beneficial effects on human oocytes during in-vitro maturation and that these effects are at least partly due to a non-genomic action of the steroid at the oocyte surface. Here we provide evidence showing that a non-genomic effect of oestradiol is counteracted by androstenedione. In contrast to these results from in-vitro experiments, in which changes in steroid concentrations are abrupt and the non-genomic responses are rapid, the progressively changing follicular steroid concentrations which occur during in-vivo development may rather have permissive or restrictive effects on the events of spontaneous oocyte cytoplasmic maturation. The oocyte is particularly sensitive at the germinal vesicle stage of development to non-genomic steroid actions. Ovarian stimulation protocols should thus be adjusted so as to avoid androgen predominance at the mid-follicular phase. In patients in whom this condition cannot be met, in-vitro maturation of oocytes may be a solution.
Article
To evaluate the output of E2 and progesterone produced by cumulus cells, derived from mature and immature oocytes, in culture medium. Prospective randomized study. McGill Reproductive Center, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada. Twenty-one women, <38 years of age and with normal menstrual cycles, who were undergoing intracytoplasmic sperm injection for assisted reproduction. Culture medium with or without fetal bovine serum (FBS) supplemented with either a physiologic (75 mIU/mL) or a supraphysiologic (7,500 mIU/mL) concentration of gonadotropins. Comparison of steroid levels in culture medium. Estradiol secretion was significantly increased in the culture medium with FBS supplemented with both concentrations of FSH alone compared with control. However, E2 secretion was inhibited by both concentrations of FSH with LH. The level of E2 was undetectable in the medium without FBS even after supplementation with both concentrations of FSH alone, hCG alone, and FSH with LH. Progesterone production was increased in the medium with FBS supplemented with FSH alone, hCG alone, and FSH with LH compared with control. There was no difference in progesterone levels in the culture medium without FBS supplemented with both concentrations of FSH alone and hCG alone compared with control. However, progesterone secretion was increased in the medium without FBS supplemented with a physiologic concentration of FSH with LH. Culture medium with FBS supplemented with a physiologic and a supraphysiologic concentration of FSH stimulates E2 secretion from cumulus cells derived from mature and immature oocytes. This suggests that it may be not necessary to add E2 to the culture medium for maturation in vitro of immature human oocytes retrieved from patients undergoing stimulated cycles.
Article
The widespread environmental contamination, bio-accumulation, and toxic effects of butyltins (BTs) in wildlife is well documented, but the role of BTs in debilitating human immune function mediated through natural killer (NK) lymphocytes (a primary immune defense against tumor and virally infected cells) has not been described. In this study, we assessed the effects of in vitro exposure to a range of concentrations (encompassing environmentally relevant concentrations) of MBT, DBT, and TBT on human natural killer lymphocytes obtained from adult male and female donors. TBT inhibited the tumor-killing capacity of NK cells when the NK cells were pretreated in vitro at 200 nM for as little as 1 h. Inhibition of NK cytotoxic function ranged from 40 to greater than 90%. The toxic potential of butyltins followed the order of TBT > DBT > MBT. Conjugation assays revealed that after a 24-h exposure to TBT, there was about a 50% decrease in NK cell binding to tumor cells, indicating alteration of the NK cell receptors for tumor cells. Analysis of whole-blood samples for BTs revealed the presence of detectable concentrations of MBT, DBT, and TBT in all of the donors, indicating possible exposure of NK cells to BTs in the blood. The results of this study provide evidence that butyltin compounds significantly inhibit NK cell function and possible NK cell-mediated immunotoxic potential in humans.
Article
Many pesticides are able to block or activate the steroid hormone receptors and/or to affect the levels of sex hormones, thereby potentially affecting the development or expression of the male and female reproductive system or both. This emphasizes the relevance of screening pesticides for a wide range of hormone-mimicking effects. Twenty-two pesticides were tested for their ability to affect CYP19 aromatase activity in human placental microsomes using the classical [(3)H](2)O method. Prochloraz, imazalil, propioconazole, fenarimol, triadimenol, triadimefon (all fungicides), and dicofol (an acaricide) gave rise to a statistically significant inhibition of aromatase activity. The IC(50)s of prochloraz, imazalil, propioconazole fenarimol, triadimenol, and triadimefon were calculated from dose-response curves to be 0.04, 0.34, 6.5, 10, 21 and 32 microM, respectively. The IC(50) of dicofol was greater than 50 microM. The positive control 4-hydroxyandrostendione (1 microM) caused an inhibition of aromatase activity by 74%. The compounds, which did not affect the aromatase activity, were bromopropylate, chlorfenvinphos, chlorobenzilate, chlorpyrifos, diuron, heptachlor, iprodion, linuron, pentachlorphenol, procymidon, propyzamide, quintozen, tetrachlorvinphos and tetradifon. With the purpose of comparing the results for fenarimol obtained with the microsomal system with data from an intact cell system, an aromatase assay based on JEG-3 cells was established. 4-Hydroxyandrostendione (1 microM) inhibited the aromatase activity in JEG-3 cells by 94%. The IC(50) for fenarimol in this system was 2 microM, slightly lower than that observed in the microsomal system. For the first time, fenarimol has been demonstrated to inhibit aromatase activity in human tissues and, furthermore, propioconazole, triadimefon, and triadimenol were identified as weak aromatase inhibitors. In conclusion, seven out of 22 tested pesticides turned out to be weak to moderate aromatase inhibitors in vitro, indicating the relevance of elucidating the endocrine effects in vivo of these- compounds.
Article
The circuit of gonadotropins (FSH, LH) and ovaries (theca and granulosa cells) in ovarian estrogen and androgen production is well established. Recent research has revealed an intraovarian network that may ultimately prove relevant to the understanding of ovarian hyperandrogenism. Most of these substances, such as growth factors and cytokines, do not have independent effects on basal androgen production, but exhibit their regulatory potential by modulating hCG- or LH-stimulated steroid production. Precise understanding of the regulatory role of intraovarian factors in ovarian androgen production would shed new light on the pathophysiology and therapy of hyperandrogenemic excess in women.
Article
The organogenesis of the ovary encompasses the formation of a great variety of structures, both germinal and nongerminal. Primordial follicle (PF) formation is of the utmost importance because PFs are obligatory for the reproductive cycle and female fertility. The major events involved in PF formation are described. Areas that could benefit from more investigation are discussed. The working premise is that the number of PFs formed during normal ovary organogenesis varies from one female to the next (ranging from high to low), and that this variability is revealed by the timing of age-related infertility and the menopause. Implicit in this supposition is the concept that anything that alters the sequence of events involved in the process of PF development will have important consequences on female fertility and health.
Article
Natural killer (NK) cells are a subset of lymphocytes that are capable of killing tumor cells, virally infected cells, and antibody-coated cells. Butyltins (BTs) are used in a variety of consumer products and industrial applications. Tributyltin (TBT) is found in dairy products, meat, and fish. Dibutyltin (DBT) is found in plastic products, beverages stored in PVC pipes during manufacturing, and poultry products. BTs appear to increase the risk of cancer and viral infections in exposed individuals. This increased risk may be due in part to the inhibitory effect of these compounds on the cytotoxic function of NK cells. A 24-h exposure of NK cells to 200 nM TBT or 1.5 microM DBT decreased the cytotoxic function of NK cells by greater than 90%. Higher concentrations of TBT and DBT decreased the cytotoxic function of NK cells (by greater than 90%) after only a 1-h exposure. A 24-h exposure to either TBT or DBT decreased intracellular ATP levels by about 30%. However, as much as a 1-h exposure to either 300 nM TBT or 10 microM DBT caused no significant decrease in ATP levels. Thus, a decrease in ATP levels is a longer-term consequence of BT exposure. Intracellular levels of cAMP are decreased by as much as 80% within 5 min of exposure to either TBT or DBT. This rapid decline in cAMP levels in NK cells may be a consequence of BT exposure that is related to the rapid decrease in the cytotoxic function of NK cells.
Article
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
Article
Organotin compounds, including tributyltin (TBT), are a class of the most toxic xenobiotics occurring in aquatic systems. High concentration levels in waters and sediments are mainly due to their extensive use as biocides and high persistence when present in sediments under anaerobic conditions. Toxicity studies have revealed the acute effects of TBT for aquatic organisms at concentrations as low as 1 microgram/L, and the induction of imposex at levels below 0.5 ng/L TBT (as Sn). At 20 ng/L TBT (as Sn) causes sterility and is followed with the disappearance of the most sensitive neogastropods on a given shore. Imposex is the most sensitive response of all known pathological conditions for nontarget organisms following an exposure to tributyltin. In this study results are discussed that were obtained from two monitoring sites with different anthropogenic background using imposex monitoring as an indicator of TBT concentrations, as well as chemical analysis of tissue of Hinia (= Nassarius) reticulata (L.) (Gastropoda).
Article
Organotin compounds are widely used as antifouling agents and bioaccumulate in the food chain. Tributyltin chloride (TBT) has been shown to induce imposex in female gastropods. On the basis of this observation it has been suggested that TBT acts as an endocrine disrupter inhibiting the conversion of androgens to estrogens mediated by the aromatase cytochrome P450 enzyme. However, to date, the molecular basis of TBT-induced imposex and in particular its putative inhibitory effects on human aromatase cytochrome P450 activity have not been investigated. Therefore, we examined the effects of the organotin compounds tetrabutyltin (TTBT), TBT, dibutyltin dichloride (DBT) and monobutyltin trichloride (MBT) on human placental aromatase activity. TBT was found to be a partial competitive inhibitor of aromatase activity with an IC(50) value of 6.2 microM with 0.1 microM androstenedione as substrate. TBT impaired the affinity of the aromatase to androstenedione but did not affect electron transfer from NADPH to aromatase via inhibiting the NADPH reductase. DBT acted as a partial but less potent inhibitor of human aromatase activity (65% residual activity), whereas TTBT and MBT had no effect. The residual activity of TBT-saturated aromatase was 37%. In contrast, human 3beta-HSD type I activity was only moderately inhibited by TBT (80% residual activity). Moreover, neither TTBT or DBT nor MBT inhibited the 3beta-HSD type I activity. Together, these results suggest that the environmental pollutants TBT and DBT, both present in marine organisms, textile and plastic products, may have specific impacts on the metabolism of sex hormones in humans.
Article
The interaction between the human aromatase enzyme and some organotins was investigated. Tributyltin (TBT) at 12 and 59 microM and dibutyltin at 74 microM inhibited aromatase activity in vitro but monobutyltin and tri-, di- and monooctyltins were without effect. In four separate kinetic studies of aromatase, the K(m(app)) for testosterone was 0.24, 0.21, 0.16 and 0.24 microM. TBT inhibited aromatase activity by causing the K(m(app)) to be increased without affecting the V(max), indicative of competitive inhibition. Slope and intercept replots confirmed the effect of aromatase on the K(m(app)). Slope replots from three separate kinetic studies provided Ki values for TBT of 64.5, 40.9 and 37.3 microM. Consequently, TBT is a competitive inhibitor of human aromatase with a Ki approximately 300-fold the K(m(app)) value.
Article
The present in vitro experiments were designed to evaluate the ability of bovine cumulus-oocyte-complexes (COCs) to produce steroids and also to evaluate the modulatory effects of added estradiol, progesterone and testosterone on the steroidogenic activity of COCs. Considerable estradiol accumulation was observed in the control maturation medium for in vitro maturation of bovine COCs during the 24h of maturation (P<0.05). When testosterone was added to the medium at various concentrations, a slight estradiol accumulation occurred, which, however, was lower (P<0.05) than that observed in the control medium. Slight estradiol accumulation was observed in maturation medium containing progesterone at concentrations of 2.5, 5.0 and 10.0 microg/ml, but these increases were less (P<0.05) than those observed in the control medium. However, in the presence of 1.0 microg/ml progesterone, estradiol accumulation was equal to that of the control medium (P>0.05). Progesterone accumulation (P<0.05) was observed in the control medium for in vitro maturation of bovine COCs. When estradiol was added to the maturation medium, progesterone accumulation was observed, but was significant (P<0.05) only when the medium was supplemented with the lesser concentrations of estradiol utilized in the experiment (1.0 microg/ml). The results demonstrated that (1) cumulus cells of bovine COCs are able to secrete estradiol and progesterone in culture systems for in vitro maturation, and this steroidogenesis is modulated by the steroids progesterone, testosterone and estradiol, and (2) the addition of estradiol to the in vitro maturation medium of bovine oocytes should be reviewed, since cumulus cells of COCs have been demonstrated to secrete estradiol in the maturation medium.
Article
A complete VEGF system consisting of the ligand and two of its receptors has been detected for the first time in the bovine cumulus-oocyte-complex (COC). In the course of a 24 hr in vitro maturation procedure (IVM), expression of the smaller VEGF transcripts and their specific receptors flt and flk changed remarkably in a time-dependent manner as observed by RT-PCR. The transcript concentrations of VEGF declined within 24 hr of culture, whereas both receptor mRNAs were found enriched between 6 and 12 hr of IVM. In the follicular fluid of growing ovarian follicles, immunoreactive VEGF, measured by RIA, increased significantly, reaching highest concentrations immediately before ovulation of the oocyte. The immunohistochemical localization of VEGF in bovine COCs revealed strong signals within the cumulus cell complex clearly extending beyond the oocyte cytoplasm at the beginning of in vitro maturation. After 24 hr, IVM immunoreactive VEGF disappeared remarkably from cumulus cells and the oocyte cytoplasm. An exogenous application of VEGF at the beginning of a 24 hr IVM significantly improved cleavage rates of zygotes and their development into bovine embryos showing obvious synergistic effects in combination with FSH, when compared with untreated control embryos. In addition, the number of blastomeres in deriving blastocysts increased after VEGF supplementation. These results indicate a functional VEGF system controlling important events beside the known angiogenetic effect during in vivo and in vitro maturation of the bovine COC, possibly affecting the early embryonic viability.
Article
Real-time reverse transcription followed by polymerase chain reaction (RT-PCR) is the most suitable method for the detection and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene. Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the calculation of relative expression ratio allow only for the determination of a single transcription difference between one control and one sample. Therefore a new software tool was established, named REST (relative expression software tool), which compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance by a randomisation test. Herein, development and application of REST is explained and the usefulness of relative expression in real-time PCR using REST is discussed. The latest software version of REST and examples for the correct use can be downloaded at http://www.wzw.tum.de/gene-quantification/.
Article
Pesticides are synthetic chemicals used not only for improving food and feed production but also for the protection of materials and of human health and well-being. Some of these substances are suspected for adverse effects attributable to an interaction with the endocrine system of vertebrates by mimicking or inhibiting endogenous hormones. One of the biological targets important in this relation is the androgen receptor (AR). To be able to screen environmental samples for the presence of compounds which might interfere with androgen action, we aimed to develop a receptor assay based on recombinant human AR (rhAR). We herein describe an rhAR assay in which the receptor is immobilized in microtiter plates via a specific antibody. The assay can be used for high throughput screening of chemicals spread into the environment. 29 of the most recommended pesticides of the Federal Country Hessen, Germany, were tested for their ability to displace [3H]-DHT bound to the rhAR. This evaluation included the major part of the most common herbicides, insecticides and fungicides and covered three potential groups of endocrine disrupting chemicals. For 28 of the substances evaluated, the relative binding affinity to the rhAR was below 0.1% when compared to dihydrotestosterone (DHT) (100%), only fentinacetate exhibited an affinity of 1.42%. An exchange assay indicated that the binding inhibition was reversible. In consequence, fentinacetate seems to be a hormonally active substance which may be present in vegetables or fish, but also on clothing. We conclude that further investigations on this compound and its metabolites are necessary.
Solvents, fumigants, and related compounds Handbook of Pesticide Toxicology
  • P J Gehring
  • R J Nolan
  • P G Watanabe
  • A Schumann
P.J. Gehring, R.J. Nolan, P.G. Watanabe, A. Schumann, Solvents, fumigants, and related compounds, in: W.J. Hayes Jr., E.R. Laws, (Ed.) Handbook of Pesticide Toxicology, Academic Press, NY, 1991, pp. 10–189.
) Handbook of Pesticide Toxicology
  • P J Gehring
  • R J Nolan
  • P G Watanabe
  • A Schumann