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Summary Although ethanol treatment is widely used to activate oocytes, the underlying mechanisms are largely unclear. Roles of intracellular calcium stores and extracellular calcium in ethanol-induced activation (EIA) of oocytes remain to be verified, and whether calcium-sensing receptor (CaSR) is involved in EIA is unknown. This study showed that calcium-free ageing (CFA) in vitro significantly decreased intracellular stored calcium (sCa) and CaSR expression, and impaired EIA, spindle/chromosome morphology and developmental potential of mouse oocytes. Although EIA in oocytes with full sCa after ageing with calcium does not require calcium influx, calcium influx is essential for EIA of oocytes with reduced sCa after CFA. Furthermore, the extremely low EIA rate in oocytes with CFA-downregulated CaSR expression and the fact that inhibiting CaSR significantly decreased the EIA of oocytes with a full complement of CaSR suggest that CaSR played a significant role in the EIA of ageing oocytes. In conclusion, CFA impaired EIA and the developmental potential of mouse oocytes by decreasing sCa and downregulating CaSR expression. Because mouse oocytes routinely treated for activation (18 h post hCG) are equipped with a full sCa complement and CaSR, the present results suggest that, while calcium influx is not essential, CaSR is required for the EIA of oocytes.

In this study, diamond crystals were synthesized via the temperature gradient method at 5.6 GPa and 1230–1245 °C by adding a Mn3P2 dopant and FeNi catalyst. Experimental results showed that Mn3P2 shifted the V-shaped growth region to the upper right by influencing the catalytic properties. The scanning electron microscopy (SEM) photographs of the diamonds synthesized at 1245 °C revealed that there were many dendritic structures in the (100) surfaces. The diamond crystal quality could be improved when the added Mn3P2 was less than 6 wt% at 1245 °C, but it would be seriously damaged when the diamond was heavily doped with Mn3P2. Pits and uneven layered structures appeared on the diamond surfaces when the additive was included at 12 wt% at 1245 °C. The Fourier transform infrared spectroscopy (FTIR) results revealed that the Mn3P2 additive increases the N content of the crystal and that N is present in the diamond crystal in the form of a “C” center. It is worth noting that phosphorus could be doped into diamond crystals by using the Mn3P2 additive and that the other impurities in diamonds were mostly C–N and C–O forms, as confirmed by X-ray photoelectron spectroscopy (XPS). The phosphorus in the sample crystals mainly formed C–P bonds with carbon, while a smaller amount of phosphorus formed P–O bonds with oxygen. The test results of the electrical properties as assessed by the van der Pauw method for the diamond crystals with Mn3P2 doped at 1245 °C revealed a resistivity of 0.516 × 106–9.729 × 106 Ω cm and a negative Hall coefficient, indicative of an n-type semiconductor.

High-quality type IIa large diamond crystals are synthesized with Ti/Cu as nitrogen getter doped in an FeNi–C system at temperature ranging from 1230 °C to 1380 °C and at pressure 5.3–5.9 GPa by temperature gradient method. Different ratios of Ti/Cu are added to the FeNi–C system to investigate the best ratio for high-quality type IIa diamond. Then, the different content of nitrogen getter Ti/Cu (Ti : Cu = 4 : 3) is added to this synthesis system to explore the effect on diamond growth. The macro and micro morphologies of synthesized diamonds with Ti/Cu added, whose nitrogen concentration is determined by Fourier transform infrared (FTIR), are analyzed by optical microscopy (OM) and scanning electron microscopy (SEM), respectively. It is found that the inclusions in the obtained crystals are minimal when the Ti/Cu ratio is 4:3. Furthermore, the temperature interval for diamond growth becomes narrower when using Ti as the nitrogen getter. Moreover, the lower edge of the synthesis temperature of type IIa diamond is 25 °C higher than that of type Ib diamond. With the increase of the content of Ti/Cu (Ti : Cu = 4 : 3), the color of the synthesized crystals changes from yellow and light yellow to colorless. When the Ti/Cu content is 1.7 wt%, the nitrogen concentration of the crystal is less than 1 ppm. The SEM results show that the synthesized crystals are mainly composed by (111) and (100) surfaces, including (311) surface, when the nitrogen getter is added into the synthesis system. At the same time, there are triangular pits and dendritic growth stripes on the crystal surface. This work will contribute to the further research and development of high-quality type IIa diamond.

While effects of gestational, neonatal or adolescent stress on psychological alterations in progeny have been extensively studied, much less is known regarding the effects of adult pre-gestational life events on offspring behavior. Although full siblings often display behavioral differences, whether the different parental life events prior to different pregnancies contribute to these behavioral differences among siblings is worth studying. In this study, male and female adult mice were restrained for 60 days before mating with unstressed or stressed partners. F1 offspring were examined for anxiety or mated to generate F2. Both F1 females and males from restrained mothers and/or fathers showed significantly reduced anxiety and serum cortisol and increased mRNA levels of glucocorticoid receptor and brain-derived neurotrophic factor compared to control offspring from unstressed parents. Similar behavioral and molecular changes were also observed in F2 females and males. Although restraint of adolescent mice reduced anxiety in F1 of both sexes, social instability of them increased anxiety predominantly in F1 females. Thus, adult pre-gestational restraint reduced offspring’s anxiety across generations; different stressors on parents may cause different phenotypes in offspring; individual behaviors can depend on adult life experiences of parents.

Objective To study the effect of cumulus denudation on in vitro maturation of rabbit oocytes. Design Experimental animal study. Setting Academic institution. Animal(s) Rabbits and mice. Intervention(s) Rabbit oocytes were observed compared with mouse oocytes. Main Outcome Measure(s) Developmental competence, membrane integrity, and apoptotic status of oocytes after cumulus denudation. Result(s) Although in vitro maturation of mouse cumulus-denuded oocytes was unaffected, rabbit cumulus-denuded oocytes could not mature. However, 50% of rabbit cumulus-intact oocytes matured normally when their gap junctions were sealed with 1-heptanol. Coculture with cumulus cells did not improve maturation of rabbit cumulus-denuded oocytes unless with an intact corona radiata. Staining with Hoechst 33258, Bcl-2 antibodies, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling showed membrane breaches or apoptosis of rabbit cumulus-denuded oocytes, contrary to the mouse cumulus-denuded oocytes. Ultrastructurally, rabbit oocytes showed no perivitelline space but numerous long cell junctions projecting into the egg cortex, contrary to the mouse oocytes. However, the damaging effect of cumulus denudation was much relieved after preincubation of rabbit cumulus-intact oocytes with phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, and some cumulus-denuded oocytes prepared after preincubation matured and developed into blastocysts. Conclusion(s) [1] Cumulus denudation severely damaged rabbit oocytes leading to their apoptosis or degeneration, possibly because of the deep-set junctional complexes anchoring the oocyte and corona cells; and [2] preincubation with phosphodiesterase inhibitor may provide a method to avoid the damaging effect of cumulus denudation on rabbit oocytes.

Although successful embryo development is dependent upon genetic and epigenetic contributions from both the male and female, the male potential to adversely affect embryo development has been scarcely studied. It is unclear whether the sperm variation among different males would affect the outcome of oocyte evaluation by embryo development following fertilization. In the present study, variation in the developmental potential of mouse embryos was first compared between in vitro fertilization with epididymal spermatozoa from different males and Sr2+ parthenogenetic activation using oocytes of different qualities, and then the effect of male on fertilization and embryo development was examined using randomly chosen oocytes and spermatozoa from cauda epididymidis, vas deferens or electro-ejaculates. Rates of fertilization and blastocyst formation were significantly higher with spermatozoa from cauda epididymidis or vas deferens than with ejaculated spermatozoa. Rates of embryonic development differed significantly between different males, but not between different ejaculates of the same male. Analysis of standard errors of means and coefficients of variance indicated that as long as multiple males were involved, the variation in oocyte fertilization/activation and blastocyst formation was always higher after fertilization than after Sr2+ parthenogenetic activation whether spermatozoa were collected from epididymidis, vas deferens or ejaculates and regardless of oocyte qualities. It is concluded that (1) epididymal mouse spermatozoa fertilize more oocytes than ejaculated spermatozoa under identical experimental conditions; (2) like farm animals, the mice also show a remarkable male effect on the developmental potential of in vitro produced embryos although they are supposed to be less genetically diverse; (3) parthenogenetic activation is recommended for assessment of oocyte quality to exclude the effect of male. Mol. Reprod. Dev. 76: 239–245, 2009. © 2008 Wiley-Liss, Inc.

The objective of this article was to study the developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles. M-199 medium was conditioned for 24 hr with cumulus-denuded oocytes (DOs), oocytectomized complexes (OOXs), or mural granulosa cells (MGCs) from goat follicles of different sizes. Mouse OOXs and eCG were added to culture drops of the conditioned medium and cumulus expansion was scored at 18 hr of culture to assess CEEF production. While mouse OOXs did not expand, goat OOXs underwent full cumulus expansion when cultured in nonconditioned eCG-supplemented M-199 medium. When cultured in nonconditioned medium containing 10% follicular fluid (FF) from goat medium (2–4 mm) and small (0.8–1.5 mm) follicles, 71–83% mouse OOXs expanded; but expansion rates decreased (P

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 α-hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.

Using a well-in-drop (WID) oocyte/embryo culture system that allows identification of follicular origin, we have investigated the effects of granulosa cells (GCs) apoptosis, follicle size, cumulus–oocyte complexes (COCs) morphology, and cumulus expansion on the developmental competence of goat oocytes matured and cultured individually following parthenogenetic activation. The WID system supported oocyte maturation and embryo development to a level similar to the conventional group system. The majority of goat oocytes acquired competence for development up to the 8–16 cell stage in follicles larger than 2 mm, but did not gain the ability to form morula/blastocyst (M/Bs) until follicles larger than 3 mm in diameter. The extent of atresia affected M/Bs formation. This effect varied according to the follicle size. Cumulus expansion increased with follicle size and decreased with increasing incidence of GCs apoptosis. Oocyte developmental potential was also correlated with cumulus expansion. Regardless of the degree of follicle atresia, 73–84% of the floating cells in the follicular fluid (FF) underwent apoptosis. Correlation between floating cell density in FF and oocyte developmental potency suggests the possibility to use the floating cell density as a simple and non-invasive marker for oocyte quality. It is concluded that the developmental potential of an oocyte is determined by multifactor interactions, and multiple factors must be considered together to accurately predict the quality of an oocyte.

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.

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.

Studies suggest that oocyte cumulus expansion is regulated by both cumulus expansion-enabling factor (CEEF) and cumulus expansion-inhibiting factors (CEIF). Many reports on CEEF have appeared, but CEIF has rarely been studied. By cumulus expansion assays using mouse cumulus-oocyte complexes (COCs) and oocytectomized complexes, the present study demonstrated that whereas follicular fluid (FF) from medium (diameter, 2–4 mm) goat follicles contained both CEEF and CEIF activities, FF from large (diameter, 5–6 mm) abattoir or large (diameter, 5–7 mm) follicle-stimulating hormone (FSH)-stimulated follicles contained neither. FF from (diameter, 5–7 mm) human chorionic gonadotropin-stimulated follicles showed CEEF but not CEIF activity. Whereas medium conditioned with cumulus or mural granulosa cells from medium goat follicles contained only CEEF activity, theca cell-conditioned medium (CM) showed both CEEF and CEIF activities. Whereas 0.01 mg/ ml of heparin efficiently inhibited cumulus expansion of mouse COCs in vitro, FF from large follicles that showed no CEIF activity contained much higher concentrations (0.23–0.25 mg/ ml) of heparin. None of the glycosaminoglycans (GAGs) tested inhibited cumulus expansion of goat COCs. Among the follicles observed, only FF from medium goat follicles contained a linoleic acid (LA) level sufficient to inhibit cumulus expansion of both mouse and goat COCs in vitro. CM contained some amount of GAGs but no LA. Taken together, the results suggest that 1) the FSH and luteinizing hormone (LH) surges before ovulation promote cumulus expansion by down-regulating CEIF and upregulating CEEF activity, respectively; 2) GAGs are not the CEIF in goat follicles; and 3) LA has CEIF activity but additional factors must be involved, because CM that showed high CEIF activity contained no LA. cumulus cells, cumulus expansion-enabling factor, cumulus expansion-inhibiting factor, follicular development, gamete biology, glycosaminoglycans, linoleic acid, oocyte cumulus expansion, oocyte maturation

Studies suggest that oocyte cumulus expansion is regulated by both cumulus expansion-enabling factor (CEEF) and cumulus expansion-inhibiting factors (CEIF). Many reports on CEEF have appeared, but CEIF has rarely been studied. By cumulus expansion assays using mouse cumulus-oocyte complexes (COCs) and oocytectomized complexes, the present study demonstrated that whereas follicular fluid (FF) from medium (diameter, 2-4 mm) goat follicles contained both CEEF and CEIF activities, FF from large (diameter, 5-6 mm) abattoir or large (diameter, 5-7 mm) follicle-stimulating hormone (FSH)-stimulated follicles contained neither. FF from (diameter, 5-7 mm) human chorionic gonadotropin-stimulated follicles showed CEEF but not CEIF activity. Whereas medium conditioned with cumulus or mural granulosa cells from medium goat follicles contained only CEEF activity, theca cell-conditioned medium (CM) showed both CEEF and CEIF activities. Whereas 0.01 mg/ml of heparin efficiently inhibited cumulus expansion of mouse COCs in vitro, FF from large follicles that showed no CEIF activity contained much higher concentrations (0.23-0.25 mg/ml) of heparin. None of the glycosaminoglycans (GAGs) tested inhibited cumulus expansion of goat COCs. Among the follicles observed, only FF from medium goat follicles contained a linoleic acid (LA) level sufficient to inhibit cumulus expansion of both mouse and goat COCs in vitro. CM contained some amount of GAGs but no LA. Taken together, the results suggest that 1) the FSH and luteinizing hormone (LH) surges before ovulation promote cumulus expansion by down-regulating CEIF and up-regulating CEEF activity, respectively; 2) GAGs are not the CEIF in goat follicles; and 3) LA has CEIF activity but additional factors must be involved, because CM that showed high CEIF activity contained no LA.

Although studies of both humans and animals suggest detrimental effects of psychological (restraint) stress on reproduction, reports concerning the direct effect of psychological (restraint) stress on the oocyte are few and conflicting. In the present study, a restraint system that allows mice free intake of feed and water while restraining their movement was established, and effects of maternal restraint on oocyte competence were examined by observing embryo development in vitro and in vivo. The results indicated that restraint stress applied to both gonadotropin-stimulated and unstimulated females during oocyte growth and maturation increased their plasma cortisol level but impaired ovulation and oocyte developmental potential. Injection of cortisol also decreased oocyte developmental potential in both stimulated and unstimulated mice. However, whereas restraint stress reduced the plasma follicle-stimulating hormone (FSH) level of unstimulated mice, injection of cortisol did not. Because the stimulated mice had received very high doses of FSH and luteinizing hormone from injection with equine chorionic gonadotropin injection, the results suggested that whereas cortisol acts directly on the ovary to damage the oocyte, restraint stress impairs oocyte competence by actions on both the hypothalamic-pituitary-gonadal and the hypothalamic-pituitary-adrenal axes. However, exposing the cumulus-oocyte complexes (COCs) to physiological levels of cortisol did not affect oocyte nuclear and cytoplasmic maturation in vitro. Thus, cortisol might have impaired ovulation and oocyte potential by an indirect effect on ovarian tissues other than the COCs.

Inhibiting oocyte aging is important not only for healthy reproduction but also for the success of assisted reproduction techniques. Although our previous studies showed that cumulus cells accelerated aging of mouse oocytes, the underlying mechanism is unknown. The objective of this paper was to study the effects of pyruvate and cumulus cells on mouse oocyte aging. Freshly ovulated mouse cumulus–oocyte complexes (COCs) or cumulus-denuded oocytes (DOs) were cultured in Chatot-Ziomek-Bavister (CZB) medium or COC-conditioned CZB medium supplemented with different concentrations of pyruvate before being examined for aging signs and developmental potential. Pyruvate supplementation to CZB medium decreased rates of ethanol-induced activation in both COCs and DOs by maintaining their maturation-promoting factor activities, but more pyruvate was needed for COCs than for DOs. Addition of pyruvate to the COC-conditioned CZB also alleviated aging of DOs. Observations on cortical granules, level of BCL2 proteins, histone acetylation, intracellular concentration of glutathione, and embryo development all confirmed that pyruvate supplementation inhibited aging of mouse oocytes. It is concluded that the aging of mouse oocytes, facilitated by culture in COCs, can be partially prevented by the addition of pyruvate to the culture medium.

The objectives of this study were to investigate the effect of heat stress duringin vitromaturation on the developmental potential of mouse oocytes and to determine whether the deleterious effect was on the nuclear or cytoplasmic component. While rates of oocyte nuclear maturation (development to the metaphase II stage) did not differ from 37 to 40 °C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 °C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles maturedin vivoorin vitroat 37, 40 or 40.7 °C were transplanted intoin vivomatured cytoplasts, no blastocyst formation was observed whenin vivospindles were transferred into the 40 °C cytoplasts. While oocytes reconstructed between 37 °C ooplasts and 37 or 40 °C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 °C ooplasts and 37 °C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 °C compared with oocytes matured at 37 °C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 °C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 °C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 °C. It is concluded that 1) oocyte cytoplasmic maturation is more susceptible to heat stress than nuclear maturation, and 2) cytoplasmic rather than nuclear components determine the pre-implantation developmental capacity of an oocyte.

Although demecolcine-assisted enucleation has been performed successfully in porcine and cattle, the mechanism and protocol optimization of chemically assisted enucleation need further investigation. The present study optimized the protocol for goat oocyte enucleation and demonstrated that a 30-min treatment with 0.8 ng/mL demecolcine-induced cytoplasmic protrusions in over 90% of the oocytes. Rates of enucleation, cell fusion, and blastocyst formation were significantly higher after demecolcine-assisted than after blind aspiration enucleation, although differences in rates of live births remain to be unequivocally determined between the two treatments. The ability to form protrusions decreased significantly as spindles became less organized in aged oocytes and the oocytes with a poor cumulus expansion. More than 93% of the demecolcine-induced protrusions persisted for 2 h in the absence of cytochalasin B (CB) but most disappeared within 30 min of CB treatment. The spindle disintegrated, an actin-rich ring formed around the chromosome mass and the MAP kinase activity increased significantly after demecolcine treatment. When oocytes with induced protrusions were treated with CB, however, the contractile ring disappeared, the spindle reintegrated, and both MPF and MAP kinase activities decreased significantly. It is concluded that (1) cytoplasmic protrusions can be induced in goat oocytes with a very low concentration of demecolcine; (2) oocyte selection and enucleation can be achieved simultaneously with demecolcine treatment; and (3) an interactive effect between the MAP kinase, MPF, microfilaments and microtubules might be implicated in the control of cytoplasmic protrusion formation after demecolcine treatment.

Objective To study the mechanisms by which cumulus cells (CCs) promote oocyte maturation by observing the effect of removing the cumulus oophorus on nuclear and cytoplasmic events during in vitro maturation. Design Experimental animal study. Setting Academic institution. Animal(s) Mice of the Kun-ming breed. Intervention(s) Cumulus-free oocytes were cultured alone (DOs) or with a CC monolayer (coDOs), and the nuclear and cytoplasmic events were compared with those of oocytes matured in vivo or in vitro with the cumulus intact (COCs). Main Outcome Measure(s) Nuclear progression, spindle assembly, behavior of cortical granules (CGs) and mitochondria, levels of glutathione (GSH), and dynamics of maturation-promoting factor (MPF) activity during oocyte maturation under different conditions. Result(s) Cumulus removal increased MPF activity and accelerated the transition from the G2 to the M phase and the redistribution of CGs. Spindle assembly and mitochondrial congregation were impaired. In addition, removal of the cumulus caused a precocious exocytosis of CGs, leading to zona hardening and reduced penetrability of oocytes by sperm. After DOs were matured on the CC monolayer, however, these parameters were much improved, and the DOs acquired characteristics closer to those of cumulus-invested oocytes matured in vivo or in vitro. The level of intracellular GSH of DOs, on the other hand, did not differ from that of oocytes matured as COCs, suggesting that the mouse DOs can synthesize GSH on their own. Conclusion(s) While denuding mouse oocytes of CCs impaired in vitro cytoplasmic maturation, coculture with CCs promoted maturation, possibly through the regulation of MPF activity and meiotic progression.