Jing-He Tan

Northeast Agricultural University, Charbin, Heilongjiang Sheng, China

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Publications (67)181.78 Total impact

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    ABSTRACT: The developmental capacity of in vitro-matured (IVM) oocytes is markedly lower than that of their in vivo-matured (IVO) counterparts, suggesting the need for optimization of IVM protocols in different species. There are few studies on IVM of rat oocytes, and there are even fewer attempts to improve ooplasmic maturation compared to those reported in other species. Furthermore, rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct; however, whether IVM rat oocytes have lower SA rates than IVO oocytes and can potentially be used for nuclear transfer is unknown. In this study, we investigated the effects of maturation protocols on cytoplasmic maturation of IVM rat oocytes and observed the possibility to reduce SA by using IVM rat oocytes. Ooplasmic maturation was assessed using multiple markers, including pre- and postimplantation development, meiotic progression, CG redistribution, redox state, and the expression of developmental potential- and apoptosis-related genes. The results showed that the best protocol consisting of modified Tissue Culture Medium-199 (TCM-199) supplemented with cysteamine/cystine and the cumulus cell monolayer dramatically improved the developmental competence of rat oocytes and supported both pre- and postimplantation development and other ooplasmic maturation makers to levels similar to that observed in ovulated oocytes. Rates of SA were significantly lower in IVM oocytes than in IVO oocytes when observed at the same intervals after nuclear maturation. In conclusion, we have optimized protocols for IVM of rat oocytes that sustain ooplasmic maturation to a level similar to ovulated oocytes. The results suggest that IVM rat oocytes might be used to reduce SA for rat cloning.
    No preview · Article · Dec 2015
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    ABSTRACT: A specific problem in goat semen preservation is the detrimental effect of seminal plasma on sperm viability in extenders containing yolk or milk. Thus, the use of chemically defined extenders will have obvious advantages. Although previous studies indicate that the initial pH of an extender is crucial to sustain high sperm motility, changes in extender pH during long-term semen storage have not been observed. Monitoring extender pH at different times of semen storage and modeling its variation according to nonlinear models is thus important for protocol optimization for long-term liquid semen preservation. The present results showed that during long-term liquid storage of goat semen, both sperm motility and semen pH decreased gradually, and a strong correlation was observed between the two. Whereas increasing the initial extender pH from 6.04 to 6.25 or storage with stabilized pH improved, storage with artificially lowered pH impaired sperm motility. Extender renewal improved sperm motility by maintaining a stable pH. Sperm coating with chicken (Gallus gallus) egg yolk improved motility by increasing tolerance to pH decline. A new extender (n-mZAP) with a higher buffering capacity was formulated, and n-mZAP maintained higher sperm motility, membrane integrity and acrosome intactness than the currently used mZAP extender did. Goat semen liquid-stored for 12 d in n-mZAP produced pregnancy and kidding rates similar to those obtained with freshly collected semen following artificial insemination. In conclusion, maintenance of a stable pH during liquid semen storage dramatically improved sperm viability and fertilizing potential.
    No preview · Article · Nov 2015 · Animal reproduction science
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    ABSTRACT: Female mice and rats exposed to psychological restraint stress during pregnancy show impaired embryo development and reduced pregnancy rates and litter sizes. Restraint stress in female mice causes chromosome abnormalities and impairs the developmental potential of oocytes. The mechanisms underlying these effects appear to be related to changes in chromatin configuration during maturation and/or epigenetic histone modifications. Two classes of chromatin configuration occur in fully grown germinal vesicle stage oocytes: the nonsurrounded nucleolus (NSN) and the surrounded nucleolus (SN) oocytes. Histone acetylation leads to relaxation of chromatin structure and correlates with gene activation, whereas histone deacetylation leads to condensation of chromatin structure and correlates with gene repression. The relationship between oocyte growth and increased histone acetylation and methylation or that between oocyte growth and chromatin configuration and competence is unclear and can only be determined using a system where the SN configuration can be dissociated from histone modification. The aim of this study was to examine the effects of restraint stress on early stages of oocyte development and to specifically investigate the role of the NSN-to-SN transition and the acquisition of certain histone modifications on the developmental potential of oocytes. Female mice were subjected to restraint stress for 24 or 48 hours or for 23 days before being examined for oocyte chromatin configuration, histone modification, and development both in vitro and in vivo. Restraint stress for 48 hours or 23 days impaired NSN-to-SN transition, histone acetylation, and methylation in SN oocytes and was associated with impaired oocyte developmental potential. Although the percentage of stressed SN oocytes returned to normal following a 48-hour postrestraint recovery, neither histone acetylation/methylation in SN oocytes nor oocyte competence to sustain early embryonic development recovered following postrestraint recovery with equine chorionic gonadotropin injection. Oocyte histone modification was expedited to early completion after priming unstressed mice with equine chorionic gonadotropin. Unlike levels of acetylated and methylated histones, which decreased in the stressed SN oocytes, the level of phosphorylated H3S10 increased significantly following exposure of the oocytes to stress. These data indicate that restraint stress in female mice impaired oocyte development potential through disturbance of histone modifications and suggest that SN configuration was uncoupled from increased histone acetylation/methylation in the stressed oocytes. Epigenetic histone modifications in the SN oocytes are more closely correlated with increased oocyte developmental potential than are changes in chromatin configuration.
    No preview · Article · Jun 2015 · Obstetrical and Gynecological Survey
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    ABSTRACT: Although previous studies have suggested that cumulus cells (CCs) accelerate oocyte aging by secreting soluble and heat-sensitive paracrine factors, the factors involved are not well characterized. Because Fas-mediated apoptosis represents a major pathway in induction of apoptosis in various cells, we proposed that CCs facilitate oocyte aging by releasing soluble Fas ligand (sFasL). In this study, we reported that when the aging of freshly ovulated mouse oocytes were studied in vitro, both the apoptotic rates of CCs and the amount of CCs produced sFasL increased significantly with the culture time. We found that oocytes expressed stable levels of Fas receptors up to 24 h of in vitro aging. Moreover, culture of cumulus-denuded oocytes in CCs-conditioned CZB medium (CM), in CZB supplemented with recombinant sFasL, or in CM containing sFasL neutralizing antibodies all showed that sFasL impaired the developmental potential of the oocytes whereas facilitating activation and fragmentation of aging oocytes. Furthermore, CCs from the FasL-defective gld mice did not accelerate oocyte aging due to the lack of functional FasL. In conclusion, we propose that CCs surrounding aging oocytes released sFasL in an apoptosis-related manner, and the released sFasL accelerated oocyte aging by binding to Fas receptors.
    Preview · Article · Mar 2015 · Scientific Reports
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    ABSTRACT: Although plasma corticosterone is considered the main glucocorticoid involved in regulation of stress responses in rodents, the presence of plasma cortisol and whether its level can be used as an indicator for rodent activation of stress remain to be determined. In this study, effects of estrous cycle stage, circadian rhythm, and acute and chronic (repeated or unpredictable) stressors of various severities on dynamics and correlation of serum cortisol and corticosterone were examined in mice. A strong (r = 0.6-0.85) correlation between serum cortisol and corticosterone was observed throughout the estrous cycle, all day long, and during acute or repeated restraints, chronic unpredictable stress and acute forced swimming or heat stress. Both hormones increased to the highest level on day 1 of repeated-restraint or unpredictable stresses, but after that, whereas the concentration of cortisol did not change, that of corticosterone showed different dynamics. Thus, whereas corticosterone declined dramatically during repeated restraints, it remained at the high level during unpredictable stress. During forced swimming or heat stress, whereas cortisol increased to the highest level within 3 min., corticosterone did not reach maximum until 40 min. of stress. Analysis with HPLC and HPLC-MS further confirmed the presence of cortisol in mouse serum. Taken together, results (i) confirmed the presence of cortisol in mouse serum and (ii) suggested that mouse serum cortisol and corticosterone are closely correlated in dynamics under different physiological or stressful conditions, but, whereas corticosterone was a more adaptation-related biomarker than cortisol during chronic stress, cortisol was a quicker responder than corticosterone during severe acute stress.
    Preview · Article · Feb 2015 · PLoS ONE
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    ABSTRACT: The mechanisms by which restraint-stress impairs oocyte developmental potential are unclear. Factors causing the difference between oocytes with surrounded nucleolus (SN) and oocytes with non-surrounded nucleolus (NSN) in developmental potential are not fully characterized. Furthermore, the relationship between the increased histone acetylation/methylation and the increased developmental competence in SN oocytes is particularly worth exploring by using a system where the SN configuration can be uncoupled (dissociated) from increased histone modifications. In this study, female mice were subjected to restraint for 24 h, 48 h or 23 days before examination for oocyte chromatin configuration, histone modification and development in vitro and in vivo. Results showed that restraints for 48 h or 23 days impaired NSN-to-SN transition, histone acetylation/methylation in SN oocytes and oocyte developmental potential. However, whereas the SN percentage of stressed oocytes returned to normal after a 48-h post-restraint recovery, neither histone acetylation/methylation in SN oocytes nor developmental competence recovered following post-restraint recovery with eCG injection. Priming unstressed mice with eCG expedited oocyte histone modification to an early completion. Contrary to the levels of acetylated and methylated histones, the level of phosphorylated H3S10 increased significantly in the stressed SN oocytes. Together, the results suggest that (i) restraint-stress impaired oocyte potential with disturbed histone modifications; (ii) SN configuration was uncoupled from increased histone acetylation/methylation in the restraint-stressed oocytes; and (iii) the developmental potential of SN oocytes is more closely correlated with epigenetic histone modification than with chromatin configuration. Copyright 2014 by The Society for the Study of Reproduction.
    No preview · Article · Nov 2014 · Biology of Reproduction
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    Full-text · Dataset · Nov 2014
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    ABSTRACT: Efforts to improve the quality of in vitro matured oocytes by blocking germinal vesicle breakdown (GVBD) and allowing more time for ooplasmic maturation have achieved little due to a lack of knowledge on the molecular events during GVBD blocking. Such knowledge is also important for studies aimed at regulating gene expression in maturing oocytes prior to GVBD. We studied species difference and signaling pathways leading to the carrying-over effect of GVBD blocking on post-blocking meiotic progression (PBMP). Overall, GVBD-blocking with roscovitine decelerated PBMP of mouse oocytes but accelerated that of pig oocytes. During blocking culture, whereas cyclin B of pig oocytes increased continuously, that of mouse oocytes declined first and then increased slowly. In both species, (a) whereas active CDC2A showed a dynamics similar to cyclin B, inactive CDC2A decreased continuously; (b) when oocytes were blocked in blocking medium containing cycloheximide, PBMP was decelerated significantly while cyclin B and active CDC2A decreasing to the lowest level; (c) whereas sodium vanadate in blocking medium reduced PBMP, epidermal growth factor (EGF) in blocking medium accelerated PBMP significantly with no effect on cyclin B levels. In conclusion, the EGF signaling cascade accelerated PBMP by promoting the pre-MPF (M-phase-promoting factor) to MPF conversion during GVBD blocking with roscovitine. The significant difference in PBMP observed between mouse and pig oocytes was caused by species difference in cyclin B dynamics during blocking culture as no species difference was observed in either pre-MPF to MPF conversion or the EGF signaling activity.
    Preview · Article · Jul 2014 · PLoS ONE
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    ABSTRACT: Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca(2+)-free CZB containing 10-mM SrCl2) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that (a) microfilaments and intermediate filaments but not microtubules support nucleolar fusion; (b) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized; and (c) microtubule disruption prevents pronuclear formation by activating MPF.
    Preview · Article · Jul 2014 · Biology of Reproduction
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    ABSTRACT: We studied the role of the Na+/Ca2+ exchanger (NCX) in modulating oocyte postovulatory aging by observing changes in NCX contents and activities in aging mouse and rat oocytes. Whereas the NCX activity was measured by observing oocyte activation following culture with NCX inhibitor or activator, the NCX contents were determined by immunohistochemical quantification. Although NCX was active in freshly-ovulated rat oocytes recovered 13 h post hCG injection and in aged oocytes recovered 19 h post hCG in both species, it was not active in freshly-ovulated mouse oocytes. However, NCX became active when the freshly-ovulated mouse oocytes were activated with ethanol before culture. Measurement of cytoplasmic Ca2+ revealed Ca2+ increases always before NCX activation. Whereas levels of the reactive oxygen species (ROS) and the activation susceptibility increased, the density of NCX member 1 (NCX1) decreased significantly with oocyte aging in both species. While culture with H2O2 decreased the density of NCX1 significantly, culture with NaCl supplementation sustained the NCX1 density in mouse oocytes. It was concluded that (a) the NCX activity was involved in the modulation of oocyte aging and spontaneous activation; (b) ROS and Na+ regulated the NCX activity in aging oocytes by altering its density as well as functioning; and (c) cytoplasmic Ca2+ elevation was essential for NCX activation in the oocyte.
    Preview · Article · Apr 2014 · PLoS ONE
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    ABSTRACT: The objective of this study was to test whether aging induces oxidative stress (OS) during oocyte preservation at different temperatures and whether the oocyte competence can be extended by antioxidant supplementation. The increase in activation susceptibility was efficiently prevented when oocytes were preserved at 37°C for 9 h in HCZB medium with 10.27 mM pyruvate and 10 µM α-tocopherol, at 25°C for 30 h with 20.27 mM pyruvate, and at 15°C for 96 h and at 5°C for 48 h with 10.27 mM pyruvate. Satisfactory blastocyst development was achieved after oocyte preservation at 37°C for 9 h, at 25°C for 30 h, at 15°C for 48 h and at 5°C for 24 h using the above protocols but with cysteamine/cystine supplementation. Transfer of blastocysts obtained from the above protocols showed no difference in pregnancy outcome between newly ovulated and preserved oocytes. Because oocytes preserved at 15°C for 48 h were fertilized after a 6-h recovery culture, aging of ovulated mouse oocytes has been successfully prevented for 54 h. Assays for ROS and glutathione indicated that in vitro preservation caused marked OS in oocytes. In conclusion, marked OS was observed following in vitro preservation of mature oocytes at different temperatures. Whereas any protocol that reduced OS could inhibit activation susceptibility, only those protocols that decreased OS while increasing glutathione synthesis could sustain oocyte competence.
    No preview · Article · Nov 2013 · Molecular Human Reproduction
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    ABSTRACT: It is known that psychological stress affects reproduction in women, but it is unknown whether the effect is by impairing implantation. Although studies suggest that long periods of auditory or restraint stress may inhibit implantation in rats and mice, the exact stage of pregnancy at which stress impairs implantation is unclear. Furthermore, whether stress impairs implantation by decreasing the heparin-binding epidermal growth factor-like growth factor (HB-EGF), estrogen and/or progesterone and whether by acting on embryos or on the uterus need further investigations. In this study, a 24-h restraint stress was initiated at 15:30 of day 3 (regimen 1) or at 07:30 (regimen 2) or 15:30 of day 4 (regimen 3) of pregnancy (vaginal plug = day 1) to observe effects of restraint stress applied at different peri-implantation stages on implantation. Among the three regimens, whereas regimens 1 and 3 affected neither term pregnancy nor litter size, regimen 2 reduced both. Further observations indicated that regimen 2 of restraint stress also delayed blastocyst hatching and the attachment reaction, decreased serum concentrations of progesterone and estradiol, and down regulated the expression of HB-EGF in both the endometrium and blastocysts. Taken together, the results suggested that restraint stress inhibited mouse implantation in a temporal window-dependent manner and by impairing blastocyst activation and hatching and uterine receptivity via down-regulating HB-EGF, estrogen and progesterone. Thus, the stress applied within the implantation window impaired implantation by acting on both embryos and the uterus.
    Full-text · Article · Nov 2013 · PLoS ONE
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    ABSTRACT: Oocytes with germinal vesicles (GV) replaced with somatic nuclei exhibit meiotic abnormalities. Although this suggests an exclusive role for GV material in meiosis, mechanisms by which a lack of GV material causes meiotic defects are unknown. Knowledge on these mechanisms will help us to understand meiotic control, nuclear-cytoplasmic interactions and cellular reprogramming. This study showed that whereas oocytes with pro-metaphase I chromosomes replaced with primary spermatocyte nuclei (PSN) did not, oocytes with GV replaced with PSN (PSG oocytes) displayed meiotic defects. Among the defects, insufficient chromosome condensation with chromosome bridges was associated with spindle abnormalities. Abnormal spindle migration, cortical non-polarization and the aberrant spindle caused randomly positioning of cleavage furrows, leading to large first polar bodies (PB1) and unequal allocation of chromosomes and MAPK between oocyte and PB1. Spindle assembly checkpoint (SAC) was activated but did not stop the mal-division. The unequal MAPK allocation resulted in differences in pronuclear formation and PB1 degeneration; oocytes receiving more MAPK were more capable of forming pronuclear rudiments while PB1 receiving more MAPK degenerated sooner than those that received less. Because none of the PSG oocytes or the enucleated GV oocytes injected with sperm heads showed cortical polarization in spite of chromosome localization close to the oolemma and because the PSG oocytes receiving more MAPK could form only pronuclear rudiments but not normal pronuclei, we suggest that the GV material plays essential roles in polarization and pronuclear formation on top of those played by chromosomes or MAPK. In conclusion, using PSG oocytes as models, this study has revealed the primary pathways by which a lack of GV material cause meiotic defects, laying a foundation for future research on the role of GV material in oocyte meiotic control.
    Preview · Article · Aug 2013 · Biology of Reproduction
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    ABSTRACT: This study examined the role of CRH-induced ovarian cell apoptosis in the restraint stress (RS)-induced impairment of oocyte competence. Oocyte percentages of apoptotic cumulus cells (CCs) did not differ between stressed and control mice before in vitro maturation (IVM) but became significantly higher in stressed mice after IVM without serum, growth factor and hormone. The level of Bcl2 mRNA decreased significantly in mural granulosa cells (MGCs) and ovarian homogenates after RS. Whereas ovarian estradiol, testosterone and IGF1 decreased, cortisol and progesterone increased significantly following RS. RS increased the level of CRH in serum, ovary and oocyte while enhancing the expression of CRHR1 in CCs, MGCs and thecal cells. RS down-regulated ovarian expression of glucocorticoid receptor (GR) and brain-derived neurotrophic factor. Furthermore, CRH supplementation to IVM medium impaired oocyte developmental potential while increasing apoptotic CCs, an effect that was completely overcome by addition of CRHR1 antagonist antalarmin. Results suggested that RS impaired oocyte competence by increasing CRH but not glucocorticoids. Increased CRH initiated a latent apoptotic program in CCs and oocytes during their intra-ovarian development, which was executed later during IVM to impair oocyte competence. Thus, elevated CRH interacted with increased CRHR1 on thecal cells and MGCs reducing the production of testosterone, estrogen and IGF1 while increasing the level of progesterone. The imbalance between estrogen and progesterone and the decreased availability of growth factors triggered apoptosis of MGCs and facilitated CCs expression of CRHR1, which interact with the oocyte-derived CRH later during IVM to induce CCs apoptosis and reduce oocyte competence.
    Preview · Article · Jul 2013 · Biology of Reproduction
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    ABSTRACT: Inhibiting oocyte spontaneous activation (SA) is essential for successful rat cloning by nuclear transfer (NT). This study tested the hypothesis that activities of the Na(+)/Ca(2+) exchanger (NCX) would decrease with oocyte aging and that SA of rat oocytes could be inhibited if the intra-oocyte Ca(2+) rises were prevented by activating NCX through increasing Na(+) concentrations in the culture medium. Elevating Na(+) levels in culture medium by supplementing NaCl inhibited SA of rat oocytes while maintaining a constant level of MPF and MAPK activities. Experiments using NCX inhibitor bepridil, Na(+)/K(+)-ATPase inhibitor ouabain and assay for intra-oocyte Ca(2+) concentrations showed that extracellular Na(+) inhibited rat oocyte SA by enhancing NCX activity and preventing intracellular Ca(2+) rises. Immunohistochemical quantification indicated that the density of NCX1 decreased significantly in aged oocytes that were prone to SA compared to that in freshly ovulated oocytes whose SA rates were low during in vitro culture. Cumulus cell NT showed that sham enucleation caused marked SA in freshly ovulated rat oocytes and Na+ supplementation prevented the manipulation-induced SA and improved the in vitro and in vivo development of rat somatic cell NT embryos. Taken together, the results have confirmed our hypothesis that NCX is active in rat oocytes and its activity decreased with oocyte aging and that activating NCX by increasing extracellular Na+ inhibits SA of rat oocytes and improves the development of rat somatic cell NT embryos. Data are also important for understanding mechanisms of oocyte aging.
    No preview · Article · May 2013 · Biology of Reproduction
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    ABSTRACT: Although oocytes from prepubertal animals are found less competent than oocytes from adults, the underlying mechanisms are poorly understood. Using the mouse oocyte model, this paper has tested the hypothesis that the developmental potential of prepubertal oocytes is compromised due mainly to their impaired potential for glutathione synthesis. Oocytes from prepubertal and adult mice, primed with or without eCG, were matured in vitro and assessed for glutathione synthesis potential, oxidative stress, Ca reserves, fertilization and in vitro development potential. In unprimed mice, abilities for glutathione synthesis, activation, male pronuclear formation, blastocyst formation, cortical granule migration and polyspermic block were all compromised significantly in prepubertal compared to adult oocytes. Cysteamine and cystine supplementation to maturation medium significantly promoted oocyte glutathione synthesis and blastocyst development but difference due to maternal age remained. Whereas reactive oxygen species (ROS) levels increased, Ca storage decreased significantly in prepubertal oocytes. Levels of both catalytic and modifier subunits of the γ-glutamylcysteine ligase were significantly lower in prepubertal than in adult oocytes. Maternal eCG priming improved all the parameters and eliminated the age difference. Together, the results have confirmed our hypothesis by showing that prepubertal oocytes have a decreased ability to synthesize glutathione leading to an impaired potential to reduce ROS and to form male pronuclei and blastocysts. The resulting oxidative stress decreases the intracellular Ca store resulting in impaired activation at fertilization, and damages the microfilament network, which affects cortical granule redistribution leading to polyspermy.
    Preview · Article · Mar 2013 · PLoS ONE
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    ABSTRACT: Although the predatory stress experimental protocol is considered more psychological than the restraint protocol, it has rarely been used to study the effect of psychological stress on reproduction. Few studies exist on the direct effect of psychological stress to a female on developmental competence of her oocytes, and the direct effect of predatory maternal stress on oocytes has not been reported. In this study, a predatory stress system was first established for mice with cats as predators. Beginning 24 h after injection of equine chorionic gonadotropin, female mice were subjected to predatory stress for 24 h. Evaluation of mouse responses showed that the predatory stress system that we established increased anxiety-like behaviors and plasma cortisol concentrations significantly and continuously while not affecting food and water intake of the mice. In vitro experiments showed that whereas oocyte maturation and Sr(2+) activation or fertilization were unaffected by maternal predatory stress, rate of blastocyst formation and number of cells per blastocyst decreased significantly in stressed mice compared to non-stressed controls. In vivo embryo development indicated that both the number of blastocysts recovered per donor mouse and the average number of young per recipient after embryo transfer of blastocysts with similar cell counts were significantly lower in stressed than in unstressed donor mice. It is concluded that the predatory stress system we established was both effective and durative to induce mouse stress responses. Furthermore, predatory stress applied during the oocyte pre-maturation stage significantly impaired oocyte developmental potential while exerting no measurable impact on nuclear maturation, suggesting that cytoplasmic maturation of mouse oocytes was more vulnerable to maternal stress than nuclear maturation.
    Full-text · Article · Oct 2012 · PLoS ONE
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    ABSTRACT: This study tested our hypothesis that oocyte aging should be prevented for longer time by reducing culture temperature while supplementing more pyruvate to culture medium. Newly ovulated mouse oocytes were cultured at various temperatures for various times in HCZB medium containing various concentrations of pyruvate before examination for aging parameters and developmental potential. The increase in susceptibility to activating stimuli was efficiently prevented when oocytes were cultured in HCZB with 10.27-mM pyruvate at 37°C for 6h, 25°C for 24h, 15°C for 96h and 5°C for 48h. Satisfactory blastocyst development of both parthenotes and fertilized zygotes was achieved after oocyte culture in HCZB containing 10.27-mM pyruvate at 37°C for 6h, 25°C for 24h, 15°C for 36h and 5°C for 24h. Transfer of two-cell embryos or blastocysts showed no difference between newly ovulated control oocytes and oocytes cultured at 15°C for 36h in either term pregnancy, live young per pregnant recipient, live young/transferred embryos or birth weight of young. Oocytes with impaired developmental potential after culture at 15°C for 96h and at 5°C for 48h showed unrecoverable decreases in GSH contents, GSH/GSSG ratio and BCL2 contents, and in numbers of oocytes with normal spindles and cortical granule distribution, suggesting induction of oxidative stress, which caused oocyte apoptosis and cytoskeleton alterations by down regulating BCL2. Because oocytes cultured at 15°C for 36h were activated or fertilized after a 6-h recovery culture, aging of ovulated mouse oocytes has been successfully prevented for 42h without impairing developmental potential.
    Preview · Article · Sep 2012 · Biology of Reproduction
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    ABSTRACT: Abstract A systematic study was conducted on round spermatids (ROS) injection (ROSI) using the goat model. After ROSI, the oocytes were treated or not with ionomycin (ROSI+I and ROSI-I, respectively) and compared with intracytoplasmic sperm injection (ICSI). After ROSI-I, most oocytes were arrested with premature chromatin condensation and few oocytes formed pronuclei. In contrast, most oocytes formed pronuclei after ROSI+I. Some ROS were observed to form asters that organized a dense microtubule network after ROSI+I, but after ROSI-I, no ROS asters were observed. Whereas most of the oocytes showed Ca(2+) rises and a significant decline in maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities after ROSI+I, no such changes were observed after ROSI-I. Due to the lack of Ca(2+) oscillations after ROSI-I, oocytes were injected with more ROS. Interestingly, different from the results observed in a single ROS injection, injection with four ROS effectively activated oocytes by inducing typical Ca(2+) oscillations. Whereas ROSI+I oocytes and ICSI oocytes both showed extensive microtubule networks, no such a network was observed in parthenogenetic oocytes. Together, the results suggest that goat ROS is not able to trigger intracellular Ca(2+) rises and thus to inhibit MPF and MAPK activities, but artificial activation improved fertilization and development of ROSI goat oocytes. Goat ROS can organize functional microtubular asters in activated oocytes. A ROS-derived factor(s) may be essential for organization of a functional microtubule network to unite pronuclei. Goat centrosome is of paternal origin because both ROS and sperm asters organized an extensive microtubule network after intra-oocyte injection.
    No preview · Article · Aug 2012
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    ABSTRACT: Cyclin B1 turnover and the insensitivity of fully-grown mouse oocytes to cycloheximide (CHX) inhibition of germinal vesicle breakdown (GVBD) were examined by assaying GVBD and cyclin B1 levels after treatment of oocytes with various combinations of eCG and CHX. Whereas over 95% of oocytes underwent GVBD after culture for 24 h with CHX alone, only 10% did so after culture with CHX + eCG (P < 0.05). In addition, preculture with eCG alone had no effect, but preculture with eCG + CHX prevented GVBD during a second culture with CHX alone. Therefore, we inferred that eCG delayed GVBD long enough for CHX inhibition of protein synthesis to allow cyclin B1 to decrease below a threshold where GVBD became dependent upon its de novo synthesis. However, western blot revealed no cyclin B1 synthesis, but cyclin B1 degradation, as long as GVs were maintained intact with eCG. Regarding the function of CHX in preculture without protein synthesis to block subsequent GVBD, whereas eCG delayed GVBD for only 3 h, CHX had an ongoing effect that further postponed GVBD, thus allowing cyclin B1 to decrease below the threshold. When oocytes precultured with eCG + CHX were further cultured without eCG and CHX, cyclin B1 first decreased but then, because of the ongoing effects of CHX, increased to a level sufficient to induce GVBD. The content of P34Cdc2 was not altered under any of the culture conditions (P > 0.05). We concluded that insensitivity of mouse germinal vesicle (GV) oocytes to CHX was due to the presence of sufficient cyclin B1, and that cyclin B1 level in such oocytes was maintained by an equilibrium between synthesis and degradation.
    No preview · Article · Mar 2012 · Theriogenology

Publication Stats

859 Citations
181.78 Total Impact Points

Institutions

  • 2015
    • Northeast Agricultural University
      Charbin, Heilongjiang Sheng, China
  • 2003-2015
    • Shandong Agricultural University
      • College of Animal Science and Veterinary Medicine
      T’ai-an-shih, Shandong Sheng, China