Tian-Yang Wang

Antioxidant supplementation overcomes the deleterious effects of maternal restraint stress-induced oxidative stress on mouse oocytes

In this study, using a mouse model, we tested the hypothesis that restraint stress would impair the developmental potential of oocytes by causing oxidative stress and that antioxidant supplementation could overcome the adverse effect of stress-induced oxidative stress. Female mice were subjected to restraint stress for 24 h starting 24 h after equine chorionic gonadotropin injection. At the end of stress exposure, mice were either killed to recover oocytes for in vitro maturation (IVM) or injected with human chorionic gonadotropin and caged with male mice to observe in vivo development. The effect of antioxidants was tested in vitro by adding them to IVM medium or in vivo by maternal injection immediately before restraint stress exposure. Assays carried out to determine total oxidant and antioxidant status, oxidative stress index, and reactive oxygen species (ROS) and glutathione levels indicated that restraint stress increased oxidative stress in mouse serum, ovaries, and oocytes. Whereas the percentage of blastocysts and number of cells per blastocyst decreased significantly in oocytes from restraint-stressed mice, addition of antioxidants to IVM medium significantly improved their blastocyst development. Supplementation of cystine and cysteamine to IVM medium reduced ROS levels and aneuploidy while increasing glutathione synthesis and improving pre- and postimplantation development of oocytes from restraint-stressed mice. Furthermore, injection of the antioxidant epigallocatechin gallate into restraint-stressed mice significantly improved the blastocyst formation and postimplantation development of their oocytes. In conclusion, restraint stress at the oocyte prematuration stage impaired the developmental potential of oocytes by increasing oxidative stress and addition of antioxidants to IVM medium or maternal antioxidant injection overcame the detrimental effect of stress-induced oxidative stress. The data reported herein are helpful when making attempts to increase the chances of a successful outcome in human IVF, because restraint was applied at a stage similar to the FSH stimulation period in a human IVF program.

Control of Spontaneous Activation of Rat Oocytes by Regulating Plasma Membrane Na+/Ca2+ Exchanger Activities

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+/Ca2+ exchanger (NCX) would decrease with oocyte aging and that SA of rat oocytes could be inhibited if the intraoocyte Ca2+ rises were prevented by activating the 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 maturation-promoting factor and mitogen-activated protein kinase activities. Experiments using the NCX inhibitor bepridil, the Na+/K+-ATPase inhibitor ouabain, and an assay for intraoocyte Ca2+ concentrations showed that extracellular Na+ inhibited rat oocyte SA by enhancing NCX activity and preventing intracellular Ca2+ rises. Immunohistochemical quantification indicated that the density of NCX1 decreased significantly in aged oocytes that were prone to SA compared with 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 that 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 the NCX is active in rat oocytes and its activity decreases with oocyte aging and that activating the NCX by increasing extracellular Na+ inhibits SA of rat oocytes and improves the development of rat somatic cell NT embryos. These data are also important for understanding the mechanisms of oocyte aging.

Cumulus cells accelerate oocyte aging by releasing soluble Fas Ligand in mice

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.

Combined Inhibitory Effects of Pyruvate and Low Temperature on Postovulatory Aging of Mouse Oocytes

ABSTRACT This study tested the hypothesis that oocyte aging could be prevented for a longer time by reducing the culture temperature while supplementing the culture medium with more pyruvate. Newly ovulated mouse oocytes were cultured at various temperatures for various times in HCZB medium (Kimura and Yanagimachi, Biol Reprod 1995; 52:709–720) containing various concentrations of pyruvate before examining 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 6 h, 25°C for 24 h, 15°C for 96 h, and 5°C for 48 h. 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 6 h, 25°C for 24 h, 15°C for 36 h, and 5°C for 24 h. Transfer of two-cell embryos or blastocysts showed no difference between newly ovulated control oocytes and oocytes cultured at 15°C for 36 h 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 96 h and at 5°C for 48 h showed unrecoverable decreases in the content of glutathione, the glutathione/oxidized glutathione ratio, the BCL2 content, and in the numbers of oocytes with normal spindles and cortical granule distribution, suggesting induction of oxidative stress, which caused oocyte apoptosis and cytoskeleton alterations by downregulating BCL2. Because oocytes cultured at 15°C for 36 h were activated or fertilized after a 6-h recovery culture, aging of ovulated mouse oocytes has been successfully prevented for 42 h without impairing their developmental potential.

Non-frozen preservation protocols for mature mouse oocytes dramatically extend their developmental competence by reducing oxidative stress

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.

Restraint Stress on Female Mice Diminishes the Developmental Potential of Oocytes: Roles of Chromatin Configuration and Histone Modification in Germinal Vesicle-Stage Oocytes

ABSTRACT The mechanisms by which restraint stress impairs oocyte developmental potential are unclear. Factors causing differences between the developmental potential of oocytes with surrounded nucleolus (SN) and that of oocytes with nonsurrounded nucleolus (NSN) are not fully characterized. Furthermore, the relationship between increased histone acetylation and methylation and the increased developmental competence in SN oocytes is particularly worth exploring 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 or 48 h or for 23 days before being examined for oocyte chromatin configuration, histone modification, and development in vitro and in vivo. Results showed that restraint for 48 h or 23 days impaired NSN-to-SN transition, histone acetylation and methylation in SN oocytes, and oocyte developmental potential. However, whereas the percentage of stressed SN oocytes returned to normal after a 48-h postrestraint recovery, neither histone acetylation/methylation in SN oocytes nor developmental competence recovered following postrestraint recovery with equine chorionic gonadotropin (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 1) restraint stress impaired oocyte potential with disturbed histone modifications; 2) SN configuration was uncoupled from increased histone acetylation/methylation in the restraint-stressed oocytes; and 3) the developmental potential of SN oocytes is more closely correlated with epigenetic histone modification than with chromatin configuration.

Role of Na+/Ca2+ exchanger (NCX) in modulating postovulatory aging of mouse and rat oocytes.

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.

Role of Cytoskeleton in Regulating Fusion of Nucleoli: A Study Using the Activated Mouse Oocyte Model

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 (Ca2+-free CZB medium 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 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF.

Mechanisms by which a Lack of Germinal Vesicle (GV) Material Causes Oocyte Meiotic Defects: A Study Using Oocytes Manipulated to Replace GV with Primary Spermatocyte Nuclei

ABSTRACT Oocytes with germinal vesicles (GVs) 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 of these mechanisms will help us to understand meiotic control, nuclear-cytoplasmic interactions, and cellular reprogramming. This study showed that although oocytes with prometaphase I chromosomes replaced with primary spermatocyte nuclei (PSN) did not, oocytes with GV replaced with PSN (PSG oocytes) did display meiotic defects. Among the defects, insufficient chromosome condensation with chromosome bridges was associated with spindle abnormalities. Abnormal spindle migration, cortical nonpolarization, and the aberrant spindle caused randomly positioning of cleavage furrows, leading to large first polar bodies (PB1) and unequal allocation of chromosomes and mitogen-activated protein kinases (MAPK) between oocyte and PB1. Spindle assembly checkpoint was activated but did not stop the incorrect 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, whereas 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 and 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.

MicroRNA-21 plays a pivotal role in the oocyte-secreted factor-induced suppression of cumulus cell apoptosis†

Abstract It is known that oocytes and cumulus cells (CCs) are more resistant to apoptosis than other compartments of the antral follicle. However, although oocyte-secreted factors (OSFs) have been found to be involved in suppressing bovine CC apoptosis, little is known about the intracellular mechanisms by which OSFs render CCs resistant to apoptosis. Here, we show that coculture with mouse or pig cumulus-denuded oocytes, culture with recombinant mouse growth differentiation factor-9 (GDF-9), or culture in pig oocyte-conditioned medium (POCM) significantly inhibited CC apoptosis of mouse oocytectomized cumulus oophorus complexes (OOXs). The POCM contained both GDF-9 and bone morphogenetic protein-15, and their levels remained constant during culture of OOXs. The level of microRNA-21 (miR-21) was significantly lower in OOXs than in COCs after culture in a simplified α-MEM medium, but increased significantly when OOXs were cultured with GDF-9 or in POCM. The level of miR-21 in OSF-treated CCs was correlated with that of Dicer1 but not that of Drosha mRNA. Inhibiting activin receptor-like kinase 5 or SMAD3 completely abolished the beneficial effects of GDF-9 or POCM on CC apoptosis and miR-21 levels. Up- and downregulating miR-21 expression significantly reduced and increased CC apoptosis, respectively. The OSF-upregulated miR-21 expression suppressed CC apoptosis with activation of the PI3K/Akt signaling. In conclusion, miR-21 plays a pivotal role in the OSF suppression of CC apoptosis. OSFs upregulated miR-21 expression through the TGF-β superfamily signaling, which worked through DICER. MicroRNA-21 prevented apoptosis via the PI3K/Akt signaling. Summary Sentence MicroRNA-21 plays a pivotal role in the oocyte-secreted factor suppression of cumulus cell apoptosis to explore the intracellular mechanisms by which oocyte-secreted factors suppress cumulus cell apoptosis.