Relationship between chromatin configuration and in vitro maturation ability in guinea pig oocytes.

BACKGROUND: Germinal vesicle (GV) chromatin configurations of oocytes are proposed to be related to oocyte competence and may reflect the quality of oocyte. Currently, a limited number of published studies investigated the GV chromatin configurations of guinea pig oocytes. OBJECTIVE: In this study on the in vitro maturation (IVM) of guinea pig oocytes, we examined the changes in their GV chromatin configurations during meiotic progression. METHODS: Based on the degree of chromatin compaction, the GV chromatin configurations of guinea pig oocytes could be divided into three categories depending on whether the nucleolus-like body (NLB) was surrounded or partly surrounded by compacted chromatin, namely the uncondensed (NSN), the intermediate type (SN-1) and the compacted type (SN-2). RESULTS: The percentage of cells displaying the SN-2 configuration increased with the growth of guinea pig oocytes, suggesting that this configuration presents the potential for maturation in oocytes. Oocytes derived from larger follicle exhibited increased meiotic potential. Serum starvation affected the GV chromatin configurations of guinea pig oocytes. CONCLUSIONS: Collectively, these results suggest that the SN-2 type might be a more mature form of configuration in guinea pig oocyte, whose proportion was associated with the follicle size and susceptible to the environment (e.g. serum concentration).

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.

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.

Characterization of cumulus expansion-inhibiting factor (CEIF) in goat follicles.

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.

Regulation of fusion of the nucleolar precursor bodies following activation of mouse oocytes: roles of the maturation-promoting factors and mitogen-activated protein kinases.

Fusion of nucleoli or nucleolus precursor bodies (NPBs) has been observed during somatic cell interphase and pronuclear development of human zygotes; however, the underlying mechanism is unknown. NPB fusion and its regulation by mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) were studied in activated mouse oocytes. Small NPBs appeared about 4 h after ethanol activation, and took about 1.5 h to fuse into a large NPB, which persisted for about 10 h before disappearance. Analysis of the temporal windows for kinase action indicated that a high MAPK activity during the first 2 h and a low MPF activity during the first 3-4 h after activation were essential for subsequent NPB fusion. A preactivation decline in MAPK activity was associated with decreased NPB fusion following activation of aged oocytes. While MAPK inactivation by regulator U0126 prevented NPB fusion in oocytes activated by ethanol or 5 min Sr2+ treatments, it had no effect on oocytes fertilized or activated by 6 h Sr2+ treatment. In most cases, while rates of pronuclear formation did not differ, rates of NPB fusion differed significantly between different treatments. Our results suggest that: (i) the MAPK and MPF activities at the initial stage of activation regulate NPB fusion after pronuclear formation; (ii) pronuclear assembly and NPB fusion are two separable events that might be controlled by different mechanisms; and (iii) high MAPK activity and low MPF activity at the initial stage of activation is essential for NPB fusion when only one calcium rise is induced by ethanol, while inhibition of MAPK activity does not affect NPB fusion when the repetitive intracellular Ca2+ rises are induced after fertilization.

Maternal restraint stress diminishes the developmental potential of oocytes.

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.

Damaging effect of cumulus denudation on rabbit oocytes.

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.

Pyruvate prevents aging of mouse oocytes.

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.

Dynamic changes of germinal vesicle chromatin configuration and transcriptional activity during maturation of rabbit follicles.

Dynamic changes in configuration of germinal vesicle (GV) chromatin and their effects on oocyte transcriptional activity and developmental competence were studied in rabbit oocytes. The results indicated that global condensation of GV chromatin, rather than the formation of a perinucleolar ring, would better reflect the global transcriptional repression and developmental competence of oocytes. Gonadotropins, by promoting large-scale chromatin remodeling, regulate oocyte transcriptional activity and developmental competence.

Chromatin configurations in the germinal vesicle of mammalian oocytes.

In all the studied mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configuration). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configuration) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. Oocytes may also take on a SN/corresponding configuration during early atresia, but they undergo GVBD at the advanced stage of atresia. As not all the species show the SN configuration while in all the species, gene transcription always stops at the late stage of oocyte growth, it is suggested that not the formation of perinucleolar rings but a thorough condensation of GV chromatin is essential for transcriptional repression. The GV chromatin configuration is highly correlated with oocyte competence; oocytes must end the NSN configuration before they gain the full meiotic competence, and they must take on the SN/corresponding configurations and stop gene transcription before they acquire the competence for early embryonic development. While factors inhibiting follicle atresia tend to synchronize oocytes in a chromatin configuration toward maturation, factors inducing follicle atresia tend to synchronize oocytes in a chromatin configuration reminiscent of early atresia. Furthermore, although condensation of GV chromatin is associated with transcriptional repression, both processes may not be associated with histone deacetylation during oocyte growth.

Effects of heat stress during in vitro maturation on cytoplasmic versus nuclear components of mouse oocytes.

The objectives of this study were to investigate the effect of heat stress during in vitro maturation 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 degrees C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 degrees C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles matured in vivo or in vitro at 37, 40 or 40.7 degrees C were transplanted into in vivo matured cytoplasts, no blastocyst formation was observed when in vivo spindles were transferred into the 40 degrees C cytoplasts. While oocytes reconstructed between 37 degrees C ooplasts and 37 or 40 degrees C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 degrees C ooplasts and 37 degrees C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 degrees C compared with oocytes matured at 37 degrees C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 degrees C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 degrees C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 degrees 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.

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats.

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 alpha-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.

Developmental and hormonal regulation of cumulus expansion and secretion of cumulus expansion-enabling factor (CEEF) in goat follicles.

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 < 0.05) at either lower or higher FF concentrations. FF from large (5-6 mm) follicles did not support mouse OOX expansion at any concentrations. While medium conditioned with DOs from follicles of all the three sizes supported expansion of 80-90% mouse OOXs, medium conditioned with mature DOs had no effect. While cumulus cells from follicles of all the three sizes secreted CEEF in the absence of gonadotropins, MGCs from large follicles became gonadotropin dependent for CEEF production. Both FSH and LH stimulated CEEF production by large follicle MGCs, but FSH had a shorter half-life than LH to expand mouse OOXs. Few meiosis-incompetent goat oocytes from small follicles underwent cumulus expansion when cultured in medium conditioned with goat DOs or cocultured with goat COCs from medium follicles. It is concluded that (1) goat cumulus expansion is independent of the oocyte; (2) the limited CEEF activity in FF from large follicles was due mainly to the inability of MGCs in these follicles to secret the factor in absence or short supply of gonadotropins; (3) the cumulus expansion inability of the meiosis incompetent goat oocytes was due to the inability of their cumulus cells to respond to rather than to produce CEEF.

Coculture with cumulus cells improves maturation of mouse oocytes denuded of the cumulus oophorus: observations of nuclear and cytoplasmic events.

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.

Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytes.

The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system.

Effects of delayed excision of oviducts/ovaries on mouse oocytes and embryos.

To achieve the best and reproducible results of experiments, effects of delayed excision of oviducts/ovaries on mouse ovarian/ovulated oocytes and embryos have been studied. Oviducts/ovaries were excised at different times after death of mice and effects of the postmortem interval on ovarian/ovulated oocytes and embryos were analyzed. When oviduct excision was delayed 10 min, many ovulated oocytes lysed or underwent in vitro spontaneous activation, and this postmortem effect aggravated with the extension of postmortem interval and oocyte aging. Oocytes from different mouse strains responded differently to delayed oviduct removal. Delayed oviduct excision did not cause lysis of zygotes or embryos but compromised their developmental potential. When ovaries were excised at 30 min after death, percentages of atretic follicles increased while blastocyst cell number declined significantly after oocyte maturation in vitro. Preservation of oviducts in vitro, in intact or opened abdomen at different temperatures and histological analysis of oviducts from different treatments suggested that toxic substance(s) were secreted from the dying oviducts which induced oocyte lysis and spontaneous activation and both this effect itself and the sensitivity of oocytes to this effect was temperature dependent. It is concluded that a short delay of oviduct/ovary removal had marked detrimental effects on oocytes and embryos. This must be taken into account in experiments using oocytes or embryos from slaughtered animals. The data may also be important for estimation of the time of death in forensic medicine and for rescue of oocytes from deceased valuable or endangered mammals.

Germinal vesicle chromatin configurations of bovine oocytes.

A common feature in the configuration of germinal vesicle (GV) chromatin in most species is that diffuse chromatin condenses into a perinucleolar ring during follicular growth; however, no such ring was observed in goat oocytes. Reports on whether bovine GV chromatin condenses into a perinucleolar ring are controversial. Besides, it is not known whether the perinucleolar ring in an oocyte represents a step toward final maturation or atresia. Changes in GV chromatin configurations during growth and maturation of bovine oocytes were studied using a new method that allows a clearer visualization of both the nucleolus and the chromatin after Hoechst and chromomycin A(3) staining. On the basis of the degree of condensation and distribution, the GV chromatin of bovine oocytes were classified into five configurations: NSN with diffuse chromatin in the whole nuclear area, N with condensed netlike chromatin, C with clumped chromatin, SN with clumped chromatin surrounding the nucleoli, and F with floccular chromatin near the nucleoli and near the nuclear envelope. Most of the oocytes were at the NSN stage in the <1.4-mm follicles, but the NSN pattern disappeared completely in follicles larger than 1.5mm. The SN pattern began to emerge in 1.5-mm follicles, and the number of SN oocytes increased while the number of oocytes with N and C configurations decreased with follicular growth. During maturation in vivo, while the number of N, C, and SN oocytes decreased, that of the F oocytes increased and reached maximum at 51h post prostaglandin injection. After that, the number of F oocytes decreased significantly because of germinal vesicle breakdown (GVBD). During maturation in vitro, GV chromatin configurations changed in a similar manner as during maturation in vivo. Fewer oocytes were at N, C, and SN stages, but more were at F and GVBD stages in the atretic than in the healthy follicles. Serum starvation slowed the F-GVBD transition of the in vitro maturing oocytes. More oocytes were of the SN or C configuration when ovaries were transported at 45-40 degrees C than at 35-30 degrees C. Most of the heated oocytes were blocked at the SN stage during in vitro maturation. It is concluded that (i) bovine GV chromatin condenses into a perinucleolar ring during follicular growth; (ii) bovine oocytes were synchronized at the F stage before GVBD; (iii) oocyte GV chromatin configurations were affected by serum starvation, high temperature, and follicular atresia.

Configurations of germinal vesicle (GV) chromatin in the goat differ from those of other species.

Configuration of germinal vesicle (GV) chromatin has been studied and found correlated with the developmental competence of oocytes in several mammalian species. A common feature in the configuration of GV chromatin in the species studied so far is that the diffuse chromatin (the so called "NSN" pattern) condenses into a perinucleolar ring (the so called "SN" configuration) with follicular growth. However, no study has been published on the configuration of GV chromatin in the goat. Nor is it known whether the perinucleolar ring of condensed chromatin (CC) in an oocyte represents a step toward final maturation or atresia. Changes in configurations of GV chromatin and RNA synthesis during goat oocyte growth, atresia and maturation in vivo and in vitro were investigated in this study. Based on both the size of nucleoli and the degree of chromatin condensation, the GV chromatin of goat oocytes was classified into GV1 characterized by large nucleoli and diffuse chromatin, GV2 with medium-sized nucleoli and condensed net-like (GV2n) or clumped (GV2c) chromatin, GV3 with small nucleoli and net-like (GV3n) or clumped (GV3c) chromatin, and GV4 with no nucleolus but clumped chromatin. The results showed that (i) the configurations of GV chromatin in the goat differ from those of other species in that the chromatin did not condense into a perinucleolar ring; (ii) most of the goat oocytes are synchronized at the GV3n configuration before GVBD; (iii) the GVn pattern might represent a healthy state, but the GVc an atretic state; (iv) in both goats and mice, the GC-specific (Chromomycin A3, CMA3) and the AT-specific (Hoechst 33342) fluorochromes followed the same pattern of distribution in GV chromatin; (v) the nucleolar size decreased significantly with oocyte growth and maturation in vivo and in vitro; and (vi) goat oocytes began GVBD at 8 hr and had completed it by 20 hr after onset of estrus. The peculiar configuration of GV chromatin of goat oocytes can be a useful model for studies of morphological and functional changes of different nuclear compartments during the cell cycle and cell differentiation, and the functional differentiation between GV3n and GV3c might be used for reference to the question whether the "SN" configuration in other species inclines toward ovulation or atresia.

Apoptosis in granulosa cells during follicular atresia: relationship with steroids and insulin-like growth factors.

It is well known that during mammalian ovarian follicular development, the majority of follicles undergo atresia at various stages of their development. However, the mechanisms controlling this selection process remain unknown. In this study, we investigated apoptosis in granulosa cells during goat follicular atresia by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The changes in the levels of steroids, insulin-like growth factors (IGFs) and IGF receptors were studied by radioimmunoassay (RIA) and semi-quantitative reverse transcription-PCR. We found that the percentage of apoptotic granulosa cells in the atretic (A) follicles was significantly higher than that in the slightly atretic (SA) and healthy (H) follicles. The level of estradiol and the ratio of estradiol to progesterone in H follicles were significantly higher than those in A follicles. On the other hand, the level of progesterone was not significantly different among these follicle types. We also found that the level of IGF-I in H follicles was higher than in SA and A follicles, whereas the amount of IGF-II did not vary significantly. The expression of IGF receptor also decreased in A follicles as compared to that in H and SA follicles. These results suggested that estradiol and IGF-I might be involved in controlling apoptosis in granulosa cells during follicular atresia.