Interactions between somatic cells and germ cells throughout mammalian oogenesis

Oocytes and their companion somatic cells maintain a close association throughout oogenesis and this association is essential for normal oocyte and follicular development. This review summarizes current concepts of the role of the somatic cells in the regulation of mammalian oocyte growth, the maintenance of meiotic arrest, the induction of oocyte maturation, and the acquisition of full embryonic developmental competence during oocyte maturation in vitro. Gap junctions appear to mediate these regulatory processes. The regulatory interaction of oocytes and somatic cells, however, is not unidirectional; the oocyte participates in the proliferation, development, and function of the follicular somatic cells. The oocyte secretes factors that enable the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in response to hormonal stimulation. In addition, the oocyte produces factors that promote the proliferation of granulosa cells. These interactions in vitro do not appear to require the mediation of gap junctions. The oocyte also promotes the differentiation of granulosa cells into functional cumulus cells, but this function of the oocyte appears to require the continued presence and close association of the oocyte and granulosa cells. Therefore, oocytes and follicular somatic cells are interdependent for development and function.     

Chemical signals that regulate mammalian oocyte maturation

The nature and functions of the chemical signals involved in the acquisition of competence to resume meiosis, and the maintenance of meiotic arrest in antral follicles are the subjects of this paper. Evidence indicating that gonadotropins are not required for the development of competence to undergo spontaneous maturation is discussed. However, gonadotropins may promote optimal conditions for oocyte development via an estrogen-dependent action on follicular cells. Evidence for the participation of cyclic AMP (cAMP), steroids and a putative maturation-inhibiting factor in the maintenance of meiotic arrest in mammalian oocytes is discussed. Cyclic AMP seems to play a critical role in the maintenance of meiotic arrest by actions in both granulosa/cumulus cells and oocytes. However, cAMP does not appear to equilibrate between cumulus cells and oocytes. In the granulosa/cumulus cells, cAMP may promote the generation/activation of a maturation-inhibiting factor which is transferred to the oocyte. Oocyte cAMP appears to be produced in the oocyte itself. The putative maturation-inhibiting factor may be maintained in an active form by a cAMP-dependent process in the oocyte. Alternatively, the putative maturation-inhibiting factor may play a role in maintaining oocyte cAMP levels. Some steroid hormones act synergistically with a cAMP-dependent process in the oocyte to maintain meiotic arrest. However, the physiological significance of this observation remains in question.     

Meiotic competence of equine oocytes and pronucleus formation after intracytoplasmic sperm injection (ICSI) as related to granulosa cell apoptosis

Follicle atresia and granulosa cell apoptosis may be related to oocyte meiotic and developmental competence. We analyzed the relationships among granulosa cell apoptosis, initial cumulus morphology, oocyte nuclear maturation in vitro, and pronucleus formation after intracytoplasmic sperm injection (ICSI) in the horse. For each follicle, the size was measured and granulosa cells were used for DNA laddering analysis. Oocytes were evaluated for cumulus morphology, cultured for in vitro maturation, and submitted to ICSI. Apoptosis was categorized as absent, intermediate, or advanced according to the relative concentrations of two DNA fragments at 900 and 360 base pairs (bp). In 98 oocyte-follicle pairs, 52 oocytes were classified as expanded (Exp), 39 as compact (Cp), and 7 as having a partial (P) cumulus. Advanced apoptosis was detected in 55% (54/98) of follicles; 37% (36/98) of follicles showed an intermediate level of apoptosis; and 8 follicles (8%) were nonapoptotic. Follicle size was not significantly correlated with granulosa cell apoptosis (P > 0.05). Significantly more Exp than Cp oocytes originated from follicles with advanced apoptosis (P < 0.001). The proportion of oocytes maturing in vitro was significantly higher in oocytes issuing from apoptotic follicles than in oocytes issuing from healthy follicles (P < 0.05). The proportion of normally (two pronuclei) or abnormally fertilized oocytes (one or greater than two pronuclei, or partially decondensed sperm) did not differ in relation to granulosa cell apoptosis. We conclude that, in the mare, granulosa cell apoptosis is related to cumulus expansion and an increase in oocyte meiotic competence but has no effect on the proportion of meiotically competent oocytes that activate after ICSI. These results provide selection criteria for horse oocytes used in assisted reproductive techniques so that embryo production may be maximized.     

Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence

Homologous gap junctions are generally recognized as a means of coordinating cellular behavior under developmental and homeostatic conditions. In the mammalian ovary, heterologous gap junctions between the oocyte and the granulosa cells have been widely implicated in the regulation of meiotic maturation late in oogenesis. However, the role of oocyte-granulosa cell gap junctions at earlier stages of oogenesis is poorly understood. Stage-specific defects in both oocyte and follicle development have been identified in juvenile mice deficient in heterologous oocyte-granulosa cell gap junctions due to targeted deletion of Gja4, the gene encoding connexin-37. Follicle development arrests at the type 4 preantral stage and although oocytes commence growth, oocyte growth ceases at a diameter of 52 microm (74.3% of control size). Analysis of cell cycle and cytoskeletal markers indicates that oocytes arrest in a G(2) state based on uniform decondensed GV chromatin, interphase microtubule arrays, and nonphosphorylated cytoplasmic centrosomes. Functional assays of meiotic competence confirm that oocytes from connexin-37-deficient mice are unable to enter M phase (initiate meiotic maturation) unless treated with the phosphatase inhibitor okadaic acid (OA). Unlike growing oocytes from heterozygous control animals, OA-treated oocytes from connexin-37-deficient mice respond acutely and progress rapidly to the circular bivalent stage of meiosis I and upon removal from OA rapidly revert to an interphase state. In contrast, OA-treated control incompetent oocytes are slow to respond, exhibit a lower proportion of chromosomal bivalent stage oocytes, but remain in and progress into meiotic M phase upon removal from OA. This study demonstrates that heterologous gap-junctional communication is required for the completion of oocyte growth and the acquisition of cytoplasmic meiotic competence.     

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.     

Meiotic state of bovine oocytes is regulated by interactions between cAMP,cumulus, and granulosa

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells.     

All‐trans retinoic acid exposure increases connexin 43 expression in cumulus cells and improves embryo development in bovine oocytes

In developing follicles, cellular coupling within cumulus–oocyte complexes (COCs) creates a functional syncytium allowing for the passage of small molecules. In many species, intercellular coupling between granulosa cells results from the expression of connexin 43 (CX43 or Gja1) and the formation of gap junctional plaques. Previously, our lab has shown that oocytes with a higher developmental potential had higher CX43 expression in their cumulus cells compared with developmentally incompetent oocytes. All‐trans retinoic acid (ATRA) has been shown to increase CX43 expression in several different cell types. In this study we investigated the effect of ATRA treatment, during maturation, on CX43 expression and localization in cumulus cells and the developmental competence of bovine oocytes. COCs and granulosa cells exposed to ATRA during maturation had significantly higher CX43 expression and increased gap junctional coupling, respectively. In addition, there was a significant increase in the maturation, cleavage, and blastocyst rates in ATRA treated COCs. Data from these studies suggest that not only can CX43 be used as a biomarker for oocyte health, it can also potentially be manipulated using ATRA to increase the number of oocytes achieving developmental competence.     

Investigations of oocyte in vitro maturation within a mouse model

This study attempted to develop a 'less meiotically competent' murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze-thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage.     

Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes

To determine the role of cumulus cells in oocyte maturation, we carried out an investigation on the effects of addition of cumulus cells to the maturation medium on the developmental competence of corona-enclosed oocytes and oocytes denuded from their somatic cells. The addition of cumulus cell (1.6 x 10(6) cells/mL) improved the development of bovine corona-enclosed oocytes, however, addition of a similar number of cumulus cells as cumulus-oocyte-complexes (COCs, cumulus cell density: 4.2 x 10(6) cells/mL) had no effect on the development of oocytes denuded from their somatic cells. To determine if corona-enclosed oocytes can obtain developmental competence without the addition of extra cumulus cells, the effects of cell density during in vitro maturation on the developmental competence were studied. A density of 1.6 to 3.2 x 10(6) cumulus cells/mL was the most effective for in vitro maturation of oocytes with intact gap junctions. The effects of the medium conditioned by COCs on the developmental competence of oocytes was also examined. It was demonstrated that COC-conditioned medium improved the development of bovine oocytes to the blastocyst stage. These data suggest that the developmental competence of bovine oocytes surrounded with corona cells is supported in a cell density-dependent manner in the maturation medium. In addition, the data indicate that cumulus cells benefit bovine oocyte development either by secreting soluble factors which induce developmental competence or by removing an embryo development-suppressive component from the medium.     

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: possible role of the hexosamine pathway as a regulator of developmental competence

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.     

Meiotic competence of prepubertal goat oocytes

The object of this work was to evaluate in vitro maturation of follicular oocytes from the ovaries of prepubertal goats obtained from the slaughterhouse. To obtain the oocytes, follicles were dissected and classified according to their diameters. In the first experiment, oocytes were matured in vitro with granulosa cells. No significant differences were detected in the percentages of maturation between adult and prepubertal goat oocytes recovered from follicles of 2.5 to 6.0 mm in diameter (81.82 vs 72.47%, respectively). The percentage of maturation increased to 88.0% in prepubertal goat oocytes from 3.0 to 6.0-mm follicles. In the second experiment, the percentage of maturation of prepubertal goat oocytes was greater after 27 than after 24 h. In the third experiment, the maturational capacity of prepubertal goat oocytes according to follicular diameter was evaluated. The percentages of maturation after 27 h of culture with no granulosa cells were 24.14, 56.60 and 74.78%, respectively, for follicles 1.0 to 1.9 mm, 2.0 to 2.9 mm, and 3.0 to 6.0 mm in diameter. As the follicular diameter increased, growth of the oocyte as well as a greater number of oocytes with more cumulus cell layers were observed. A correlation between the diamter of the oocyte and its competence to complete in vitro maturation was also observed. Oocytes with more cumulus cell layers showed only a slight superiority in their capacity for maturation in large-size follicles (3.0 to 6.0 mm), but the difference was not significant. In conclusion, oocytes from prepubertal goats complete their growth and reach meiotic competence in follicles larger than 3.0 mm. With these oocytes it is possible to obtain in vitro maturation results similar to those from adult goats.     

The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotropin-induced oocyte maturation

The concentration of hypoxanthine in mouse follicular fluid has been estimated to be 2-4 mM, and although this concentration maintains meiotic arrest in fully grown mouse oocytes in vitro, oocyte maturation in vivo is not induced by a decrease in the concentration of this purine in follicular fluid (J. J. Eppig, P. F. Ward-Bailey, and D. L. Coleman, Biol. Reprod. 33, 1041-1049, 1985). In the present study, the effect of 2 mM hypoxanthine on oocyte growth and development in vitro was assessed and the ability of gonadotropins to stimulate oocyte maturation in the continued presence of hypoxanthine was determined. Oocyte-granulosa cell complexes were isolated from 10- to 11-day-old mice and cultured in the presence or absence of 2 mM hypoxanthine. Oocytes from 10- to 11-day-old mice are in mid-growth phase and, without further development, are incompetent of undergoing meiotic maturation. During a 12-day culture period the granulosa cell-enclosed oocytes approximately doubled in size and, regardless of the presence or absence of hypoxanthine, 50-70% developed competence to undergo germinal vesicle breakdown (GVBD). Hypoxanthine promoted the continued association of oocytes with their companion granulosa cells during the 12-day culture period, and therefore had a beneficial effect on oocyte development. Most of the oocytes that acquired GVBD competence in the absence of hypoxanthine underwent spontaneous GVBD. In contrast, 95% of the GVBD-competent oocytes were maintained in meiotic arrest by hypoxanthine. Following withdrawal of the hypoxanthine after the 12-day culture, 75% of the GVBD-competent oocytes underwent GVBD. These results show that hypoxanthine, and/or its metabolites, maintains meiotic arrest in oocytes that grow and acquire GVBD competence in vitro. Follicle-stimulating hormone (FSH), but not luteinizing hormone or human chorionic gonadotropin, induced oocyte GVBD in the continued presence of hypoxanthine. FSH stimulated oocyte maturation at a significantly (P less than 0.01) higher frequency than coculture of the granulosa cell-denuded oocytes with granulosa cells in the continued presence of hypoxanthine. FSH did not induce the maturation of denuded oocytes cocultured with granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro

Current methods for detecting complete oocyte maturation and developmental competence are inadequate. The objectives of this study were to (1) examine the relationship between cat oocyte energy metabolism and development in vitro after fertilization and (2) determine if cumulus cell metabolism could be used to predict development of individual oocytes after fertilization in vitro. The hanging drop method was used to assess metabolism of three different types of cat oocytes: immature (IMO), in vitro matured (IVM), and in vivo matured (IVOM). Stage of oocyte nuclear maturation or developmental competence was assessed after metabolic analysis. Glycolysis and oxidation of glucose, glutamine, palmitate, and lactate increased with the resumption of oocyte meiotic maturation (P<0.05). Pyruvate was the preferred substrate, but uptake was not linked to maturation. IVM oocytes had impaired glucose and palmitate metabolism compared to IVOM oocytes (P<0.05). Oocyte glycolytic activity and oocyte glucose oxidation correlated well with embryo development after insemination in vitro (P<0.05). Furthermore, oocytes that had similar glucose metabolism and that were grouped together for culture on this basis had higher (P<0.05) overall rates of development than oocytes grouped randomly. There was no correlation (P>0.05) between cumulus cell metabolism and individual oocyte development after in vitro fertilization. The data reveal that energy metabolism is linked to oocyte maturation in the cat and that glucose metabolic activity can indicate those oocytes most likely to fertilize and develop in vitro. Measuring cumulus cell metabolism does not accurately predict individual oocyte development after insemination in vitro.     

Inhibition of oocyte maturation in the mouse: Participation of cAMP,steroid hormones,and a putative maturation-inhibitory factor

The hypothesis that cumulus cells inhibit oocyte maturation by a cAMP-dependent process was tested (R. M. Schultz, R. Montgomery, P. F. Ward-Bailey, and J. J. Eppig (1983). Dev. Biol.95, 294–304.). Treatment of isolated cumulus cell-oocyte complexes with follicle-stimulating hormone (FSH) resulted in a dose-dependent increase in both cumulus cell cAMP levels and in the extent of inhibition of germinal vesicle breakdown (GVBD), the first morphological manifestation of oocyte maturation. Furthermore, it was found that concentrations of a membrane-permeable analog of cAMP, dibutyryl cAMP (dbcAMP), that were below those required for complete meiotic inhibition had a greater inhibitory effect on cumulus cell-enclosed oocytes than on denuded oocytes. Cumulus cell-enclosed and denuded oocytes matured at the same time in the absence of dbcAMP. Ablation of the gap junctions that couple cumulus cells to the oocyte abolished the maturation-inhibitory action of cumulus cells that was promoted either by FSH or low concentrations of dbcAMP. These results are consistent with the hypothesis that inhibition of oocyte maturation is mediated by a factor of granulosa/cumulus cell origin, other than cAMP, which requires cAMP for its activity and/or generation, and an intact intercellular coupling pathway between cumulus cells and the oocyte. A variety of steroid hormones potentiated the FSH-induced inhibition of maturation in cumulus cell-enclosed oocytes. In addition, steroid hormones inhibited maturation in denuded oocytes, but only when oocyte cAMP levels were elevated by cAMP analogs or forskolin. Steroids alone did not inhibit maturation of either cumulus cell-enclosed or denuded oocytes. Moreover, the steroids alone or in combination with FSH did not affect metabolic coupling between the cumulus cells and oocytes, nor did testosterone affect the forskolin-induced level of cAMP in denuded oocytes. Therefore, it is proposed that the oocyte is a site for the synergistic activity of steroid hormones with a cAMP-dependent process in inhibiting maturation. Results of these studies are discussed in terms of the roles of intercellular communication, cAMP, a putative maturation-inhibiting factor, and steroid hormones in the inhibition of maturation of mouse oocytes.     

Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity

Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 microM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P < 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P < 0.05) increased embryo development to the blastocyst stage by 33%-39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P < 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential.     

Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Large-scale chromatin remodeling in germinal vesicle bovine oocytes: interplay with gap junction functionality and developmental competence

In mammals, oocyte acquires a series of competencies sequentially during folliculogenesis that play critical roles at fertilization and early stages of embryonic development. In mouse, chromatin in germinal vesicle (GV) undergoes dynamic changes during oocyte growth and its progressive condensation has been related to the achievement of developmental potential. Cumulus cells are essential for the acquisition of meiotic competence and play a role in chromatin remodeling during oocyte growth. This study is aimed to characterize the chromatin configuration of growing and fully grown bovine oocytes, the status of communications between oocyte and cumulus cells and oocyte developmental potential. Following nuclear staining, we identified four discrete stages of GV, characterized by an increase of chromatin condensation. GV0 stage represented 82% of growing oocytes and it was absent in fully grown oocytes. GV1, GV2, and GV3 represented, respectively, 24, 31, and 45% of fully grown oocytes. Our data indicated a moderate but significant increase in oocyte diameter between GV0 and GV3 stage. By dye coupling assay the 98% of GV0 oocytes showed fully open communications while the number of oocytes with functionally closed communications with cumulus cells was significantly higher in GV3 group than GV1 and GV2. However, GV0 oocytes were unable to progress through metaphase II while GV2 and GV3 showed the highest developmental capability. We conclude that in bovine, the progressive chromatin condensation is related to the sequential achievement of meiotic and embryonic developmental competencies during oocyte growth and differentiation. Moreover, gap-junction-mediated communications between oocyte and cumulus cells could be implicated in modulating the chromatin remodeling process.