Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro

Cumulus expansion and cumulus cell-oocyte coupling during in-vivo and in-vitro maturation of pig oocytes were studied by measuring [3H]uridine uptake. In vivo, cumulus expansion started before germinal vesicle breakdown (GVBD) (16 h versus 20 h after hCG) but no significant change occurred in the coupling index until 32 h after hCG. Intercellular coupling was decreasing at 32 h after hCG in oocytes at anaphase I and telophase I. Complete uncoupling was closely correlated with corona radiata expansion. In vitro, partial uncoupling was observed in oocyte-cumulus cell complexes from prepubertal and PMSG-stimulated gilts cultured for 16 and 32 h, respectively. The addition of FSH caused cumulus expansion, and the functional coupling between the cumulus cells and the oocyte was maintained up to at least 16 h of culture in complexes from prepubertal gilts. We conclude that, under our conditions, neither hormone-free nor FSH-supplemented medium ensured the same [3H]uridine uptake and uncoupling kinetics as during in-vivo maturation.     

Metabolic, fluorescent dye and electrical coupling between hamster oocytes and cumulus cells during meiotic maturation in vivo and in vitro

Heterologous intercellular communication was determined qualitatively by lucifer yellow dye transfer and quantitatively by transfer of radiolabeled uridine metabolites and electrical current in hamster oocyte-cumulus complexes during meiotic maturation in vitro and in vivo. In addition, changes in cell resting potentials during maturation were recorded. Significantly less time was required for germinal vesicle breakdown (GVBD) in oocytes matured in vitro than in oocytes stimulated in vivo (1.81 +/- 0.06 hr, N = 13 vs 2.46 +/- 0.07 hr, N = 18, respectively, P less than 0.001). Resting potentials of the oocyte (RP-o) and cumulus cells (RP-c) significantly increased contemporaneously with GVBD in vitro (RP-o: from -18.9 +/- 3.2 mV to -33.2 +/- 2.9 mV, P less than 0.001; RP-c: from -16.3 +/- 1.9 mV to -27.5 +/- 2.6 mV, P less than 0.001) and in vivo after hCG injection (RP-o: from -16.8 +/- 5.9 mV to -30.1 +/- 3.9 mV, P less than 0.001; RP-c: from -15.5 +/- 3.8 mV to -26.3 +/- 3.2 mV, P less than 0.001). RP-o and RP-c progressively increased with time of culture up to 7 hr (maximum time examined) while the values reached maxima in in vivo matured oocytes 4.5 hr post-hCG and subsequently declined concomitant with the onset of cumulus expansion. Cumulus to oocyte coupling decreased progressively with time after release from meiotic arrest both in vitro and in vivo, as assessed by a progressive reduction in transfer of either uridine marker or lucifer yellow from the cumulus cell to the oocyte. By 4.5 hr after hCG injection, cumulus expansion had begun in 100% of complexes examined. Expansion was extensive by 7 hr post-hCG and spread of lucifer yellow from a cumulus cell was limited to very few adjacent cumulus cells. Oocyte to cumulus cell metabolic coupling also decreased progressively with time in both treatment groups. Examination of the extent of heterologous ionic coupling revealed that ionic coupling exhibited biphasic and, bidirectionally parallel, increases during meiotic maturation. While these temporal changes were observed in both groups, the coupling ratios were much greater in those complexes matured in vitro than in vivo. These results show that dye, metabolic, and electrical coupling exist between the immature hamster oocyte and its surrounding cumulus cells but that during the early stages of meiosis, metabolic and dye coupling decrease, while electrical coupling increases biphasically.     

The relationship between parthenogenetic embryonic development and cumulus cell-oocyte intercellular coupling during oocyte meiotic maturation

Intercellular coupling between cumulus cells and oocytes persists after oocyte meiotic maturation has been initiated. The experiments described here focus on the relationship between oocyte-cumulus cell intercellular coupling during maturation and the subsequent embryonic development of spontaneous mouse parthenotes. Several lines of evidence suggest that this coupling during oocyte maturation is required for the acquisition of the capacity for spontaneous mouse parthenotes to develop embryologically. First, the period of time that LT/Sv oocytes remained coupled to cumulus cells during oocyte maturation in vivo corresponded to that required for the acquisition of the capacity for parthenogenetic embryonic development. Second, the longer that cumulus cells were present during Fpontaneous oocyte maturation in vitro, the higher was the percentageofova undergoing subsequent parthenogenetic development. Third, cumulus cell-free oocytes cocultured with cumulus cell-enclosed oocytes during the maturation period in vitro did not develop embryologically. Fourth, intercellular coupling between cumulus cells and oocytes persisted throughout the oocyte maturation period in vitro. Fifth, incubation of oocyte-cumulus cell complexes in medium containing follicle-stimulating hormone (FSH) promoted uncoupling and decreased the percentage of ova undergoing parthenogenetic development. Thus, cell-to-cell communication, mediated via the intercellular coupling pathway between cumulus cells and oocytes, plays an important role during oocyte maturation and relates to subsequent preimplantation development.     

The mechanism of cumulus cell-oocyte uncoupling: Evidence for the participation of both cumulus cells and oocytes

Cumulus cells are metabolically coupled to oocytes via heterologous gap junctions. This coupling terminates near the time of ovulation, and the termination appears to be correlated with the mucification of the cumulus cells lying immediately adjacent to the oocytes. The first objective of this project was to determine whether follicle stimulating hormone (FSH) induction of cumulus cell-oocyte uncoupling could occur independently of FSH-stimulated cumulus mucification (expansion). Intercellular coupling was measured as a percentage of radiolabeled choline (or its metabolites) that was incorporated into the oocyte relative to the total amount of radiolabel incorporated into the entire cumulus cell-oocyte complex. It was found that the complete suppression of FSH-stimulated cumulus expansion with chondroitin sulfate B had no suppressive effect on FSH-stimulated cumulus cell-oocyte uncoupling. This finding showed that FSH-stimulated cumulus expansion was not required for cumulus cell-oocyte uncoupling. Since 17β-estradiol, testosterone, or progesterone could not induce maximal cumulus cell uncoupling, it was concluded that the uncoupling-promoting action of FSH was probably not mediated by steroid hormones. A partial uncoupling of cumulus cells and oocytes was found when spontaneous oocyte maturation had occurred in the absence of FSH. This partial uncoupling was prevented by incubation of cumulus cell-oocyte complexes in concentrations of dibutyryl cyclic adenosine monophosphate (dbcAMP) or 3-isobutyl-1-methyl xanthine (IBMX) (0.25 and 0.10 mM respectively) that suppressed spontaneous oocyte maturation without inducing cumulus expansion. These inhibitors also prevented the maximal induction of uncoupling that would have been provoked by biological grade preparations of either FSH or luteinizing hormone (LH). It was concluded that two factors were required to bring about maximal cumulus cell-oocyte uncoupling: one factor was dependent upon the action of gonadotropins on cumulus cell function, the other factor appeared to be a function of the oocytes, since maximal uncoupling could occur only after the germinal vesicles had broken down.     

Bovine cumulus cell-oocyte gap junctional communication during in vitro maturation in response to manipulation of cell-specific cyclic adenosine 3',5'-monophosophate levels

In the growing follicle, communication between the oocyte and its surrounding follicular cells is essential for normal oocyte and follicular development. Maturation of the fully grown oocyte in vivo is associated with the loss of cumulus cell-oocyte gap junctional communication, preventing entry of meiotic-modulating factors such as cAMP into the oocyte. We have previously shown that oocyte and cumulus cell cAMP levels can be independently regulated using inhibitors of cell-specific phosphodiesterase (PDE) isoenzymes. The objectives of this study were to examine the effects of cell type-specific PDE inhibitors on the maintenance of cumulus cell-oocyte gap junction communication (GJC) and oocyte meiotic progression. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles of abattoir-derived ovaries. Cumulus cell-oocyte GJC during oocyte maturation was quantified using the fluorescent dye, calcein-AM. COCs were cultured in the presence of specific PDE inhibitors, milrinone (an oocyte PDE3 inhibitor) or rolipram (a cumulus cell PDE4 inhibitor), and were pulsed with calcein-AM to allow dye transfer between the two cell types. Following cumulus cell removal, fluorescence in denuded oocytes was measured by microphotometry, and meiotic progression was assessed. In control COCs, dye transfer from cumulus cells to the oocyte fell progressively from 0 to 9 h, after which oocyte-cumulus cell GJC was completely lost. Loss of GJC was significantly attenuated (P < 0.05) during this time in response to treatment with milrinone and rolipram. Forskolin maintained GJC at the initial 0 h level until 3-4 h of culture, whereas treatment with milrinone and forskolin together actually increased the level of dye transfer above that in COCs treated with forskolin alone. Importantly, all treatments that prolonged GJC also delayed meiotic resumption, with meiosis generally resuming when fluorescence had fallen to approximately 40% of initial levels. These results, together with our previous studies, demonstrate that treatments that maintain or elevate cAMP levels in cumulus cells, oocytes, or both result in prolonged oocyte-cumulus cell communication and delayed meiotic resumption.     

Gonadotropin-induced murine oocyte maturation in vivo is not associated with decreased cyclic adenosine monophosphate in the oocyte-cumulus cell complex

The cyclic adenosine monophosphate (cAMP) content of intact oocyte-cumulus cell complexes at various times after the induction of oocyte maturation in mice in vivo was correlated with the time of commitment by the oocytes to undergo germinal vesicle breakdown (GVB) and metabolic coupling between the oocyte and cumulus cells. Seventy-nine percent of the oocytes either underwent GVB or were committed to do so by 2 h after injection of human chorionic gonadotropin (hCG). This occurred without a decrease in the coupling between cumulus cells and the oocyte and with increasing cAMP levels in the oocyte-cumulus cell complex. Maintenance of threshold levels of cAMP within mammalian oocytes appears essential for the maintenance of meiotic arrest, but data presented here suggest that oocyte maturation in mice is induced by gonadotropins in nonatretic follicles in vivo by some mechanism other than one which decreases the cAMP content of the intact oocyte-cumulus cell complex.     

Progression of oocyte maturation from metaphase I to metaphase II is disturbed by previous immunological interference with cumulus cell function.

The effects of an antibody preparation reacting with preovulatory mouse cumuli oophori (anticumulus Ig) on oocyte maturation in vivo and in vitro were studied. Continuous presence of anticumulus Ig in culture medium did not impair oocyte maturation in vitro. Similarly, no effect on oocyte maturation in vivo was observed when anticumulus Ig was given to females superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) at the time of hCG treatment. However, when administered earlier, anticumulus Ig brought about serious disturbances of oocyte meiotic competence, since only immature oocytes were ovulated after anticumulus Ig injection at the time of PMSG treatment and as much as 70% of the ovulated oocytes were immature when the antibody was applied 24 hr later. Previous absorption of anticumulus Ig with isolated cumulus cells removed the inhibitory effect of this preparation on oocyte meiotic competence to the same extent as absorption with whole cumuli oophori, despite the persistence of a strong reactivity of the cumulus cell-absorbed antibody preparations with the cumulus intercellular matrix. The ability of oocytes obtained from antibody-injected animals to mature in vitro was also considerably impaired when the injection was made at the time of PMSG treatment. In all cases the maturation defect concerned the progression of meiosis from metaphase I to metaphase II, while the ability of oocytes to undergo germinal vesicle breakdown (GVBD) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreases in heterologous metabolic and dye coupling, but not in electrical coupling, accompany meiotic resumption in hamster oocyte-cumulus complexes

The temporal relationship between resumption of meiosis and reduction in either heterologous intercellular coupling, or magnitude of oocyte or cumulus cell resting potential in hamster oocyte-cumulus complexes was investigated. Coupling was assessed qualitatively by lucifer yellow dye transfer and quantitatively by transfer of radiolabeled uridine metabolites or electrical current after culture of complexes in various systems previously characterized either to maintain meiotic arrest or to permit meiotic resumption. In each of the three systems which permitted meiotic resumption, cumulus to oocyte metabolic and dye coupling and oocyte to cumulus dye coupling decreased progressively with time after release from meiotic arrest. In contrast, no similar temporal changes in metabolic or dye coupling were observed in any complex after culture in either of the two systems which maintained meiotic arrest. Analysis of the extent of heterologous ionic coupling revealed that in neither direction was a decrease in ionic uncoupling consistently associated with reinitiation of meiosis. Furthermore, while the resting potential of both the oocyte and cumulus cell underwent changes characteristic of each system employed, the level of neither cell membrane potential was specific to meiotic status. These results support the hypothesis that meiotic maturation in hamster oocytes is accompanied by disruption of the integrity of intercellular, non-ionic coupling between the oocyte and its adherent cumulus cells. The data show, however, that no specific alteration either in the extent of ionic coupling or in the oocyte or cumulus cell resting potential is prerequisite for meiotic resumption in this species.     

体外培养条件下促性腺激素对昆明白小鼠卵母细胞成熟及卵丘扩展的影响

研究促卵泡激素（FSH）,人绒毛膜促性腺激素（hCG)对昆明白小鼠卵母细胞成熟和卵丘扩展的影响,以及体外培养时卵丘扩展与卵母细胞成熟之间的关系,FSH可以明显促进次黄嘌吟（HX）抑制条件下的卵丘－卵母细胞复合体CEO卵母细胞成熟及卵丘扩展,其最佳作用剂量为100IU／L,且FSH作用30分钟即可以使CEO获得恢复减数分裂的信息,在HX存在的条件下,FSH处理后10hr,CEO卵丘明显扩展,而生发泡破裂（GVBD）则在16－20hr明显增加,所有卵丘未扩展的CEO中卵母细胞均未发生GVBD,低剂量hCG单独或与FSH共同存在,对CEO卵母细胞成熟及卵丘扩展均无明显影响;高剂量hCG可以部分抑制FSH对卵母细胞成熟的促进作用,结果表明：FSH明显促进CEO卵母细胞成熟及卵丘扩展,而hCG却不具有此作用,体外培养条件下（含次黄嘌呤）,卵丘扩展是卵母细胞成熟的前提条件,但卵母细胞成熟并不需要卵丘完全扩展。     

Maturation of the mouse oocyte-cumulus cell complex: stimulation by lectins.

We have used carbohydrate-binding proteins, or lectins, as tools to investigate the physiological phenomena associated with the preovulatory maturation of the oocyte-cumulus cell complex. Certain lectins are mitogens, and since other mitogenic agents such as growth factors are known to stimulate meiotic maturation and cumulus expansion, we tested the ability of lectins to provoke these physiological responses. Cumulus cell-enclosed oocytes (CEO) from primed mice were maintained in meiotic arrest in vitro with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) and treated with one of eleven different lectins. With the exception of pokeweed mitogen (PWM), all of the mitogenic lectins tested were able to induce germinal vesicle breakdown (GVB) in meiotically arrested oocytes, and this action required the presence of the somatic cumulus cells; in fact, either there was no effect or maturation was suppressed when cumulus cell-free oocytes (denuded oocytes; DO) were treated with lectins. None of the nonmitogenic lectins stimulated meiotic maturation in either CEO or DO. The mitogenic lectin concanavalin A (Con A) also induced maturation in CEO when meiotic arrest was maintained with hypoxanthine, guanosine, or 3-isobutyl-1-methylxanthine. The kinetics of spontaneous oocyte maturation in inhibitor-free medium were not altered by Con A. Only the mitogenic lectins that induced meiotic maturation stimulated cumulus expansion, with Con A the most active lectin. The actions of Con A on the maturation of the oocyte-cumulus cell complex were inhibited by methyl-alpha-D-mannopyranoside as predicted by its sugar-binding specificity. These results demonstrate that (1) lectins can stimulate maturation of the mouse oocyte-cumulus cell complex; (2) mitogenicity is associated with the positive activity of the lectins; and (3) cumulus cells mediate the stimulatory action of lectins on oocyte maturation, while inhibition of GVB occurs at the oocyte level. These data support the idea that common signals mediate the mitogenic and maturation-inducing actions of lectins.     

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.     

Metabolic coupling, cumulus expansion and meiotic resumption in mouse cumuli oophori cultured in vitro in the presence of FSH or dcAMP, or stimulated in vivo by hCG

The temporal changes of metabolic coupling between the mouse oocyte and the cumulus cells which follow hCG injection in vivo and FSH treatment in vitro were studied by measuring what fraction of [3H]uridine taken up by cumulus cells was transferred to the oocyte. Meiotic resumption and a partial coupling loss (to 35% of the initial value) spontaneously occurred in cumuli cultured in control medium. The addition of 1 microgram FSH/ml in vitro, or the injection of hCG in vivo caused a delay of about 3 h in both phenomena and a near total uncoupling, together with cumulus expansion. FSH caused uncoupling even if cumulus expansion was prevented by the addition of heparin. The presence of 2 mM-dcAMP prevented meiotic resumption in cumulus-enclosed oocytes and maintained a high level of co-operation for at least 6 h. The slow uncoupling observed at later times was due to cumulus expansion, because it was totally prevented by heparin. We suggest that metabolic co-operation with the cumulus oophorus and meiotic resumption are both regulated by FSH through variations of intracellular levels of cAMP.     

Preendocytotic alterations in cumulus cell gap junctions precede meiotic resumption in the rat cumulus-oocyte complex

Cumulus cells in the mammalian ovary are normally connected to each other and to their enclosed oocyte by an extensive network of gap junctions (GJs). We have shown that the loss of cumulus cell GJs is correlated temporally with meiotic resumption in the intact preovulatory rat follicle (Larsen et al., 1986). Here we describe morphological changes in GJ particle packing patterns (PPPs) that occur prior to GJ loss and meiotic resumption in hormonally stimulated rat cumulus-oocyte complexes (COCs). In the PMSG-primed rat, 89% of the cumulus cell GJ area detected by freeze-fracture electron microscopy consists of tightly packed junctional particles: 4% exhibit loose PPPs of randomly dispersed particles; and 7% contain a mixture of both tight and loose PPPs. One to 2 hr after stimulation with hCG, the area of GJs containing tight PPPs drops by 50%-60%, while junctions exhibiting loosely organized and mixed patterns increase concomitantly. These shifts in PPPs are accompanied by the appearance of unusual particle-free areas of puckered or ruffled nonjunctional membrane at the GJ periphery. Cumulus cell GJs from isolated COCs incubated in FSH-containing medium demonstrate a similar shift in PPPs prior to meiotic resumption. The appearance of fusing areas of particle-free nonjunctional membrane at the GJ periphery in vitro is correlated with GJ loss and is not seen in COCs treated with dihydrocytochalasin B to inhibit endocytotic removal of cumulus GJs. The structural and temporal nature of these morphological observations supports the hypothesis that interruption of junctional communication plays a role in meiotic maturation of the preovulatory oocyte.     

卵丘在卵母细胞成熟中的作用

卵丘是指在卵母细胞外周并与之进行代谢联系的颗粒细胞群;卵丘对于卵母细胞成熟有极其重要的作用。主要表现在卵丘参与维持卵母细胞减数分裂阻滞,诱导卵母细胞减数分裂恢复、支持卵母细胞细胞质的成熟。卵丘形态和卵丘扩展影响卵母细胞成熟。了解卵丘在卵母细胞成熟中的作用有助于帮助人们进一步揭示哺乳动物卵母细胞成熟的机制。     

Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice

Mucification of the cumulus layer around the oocyte is an obligatory process for female fertility. Tumor necrosis factor-induced protein-6 (TNFIP6 or TSG6) has been shown to be specifically expressed during this process. We have generated TNFIP6-deficient mice and tested the ability of their cumulus cells to undergo mucification. Cumulus cell-oocyte complexes fail to expand in TNFIP6-deficient female mice because of the inability of the cumulus cells to assemble their hyaluronan-rich extracellular matrix. The impaired cumulus matrix formation is due to the lack of covalent complexes between hyaluronan and the heavy chains of the inter-alpha-trypsin inhibitor family. As a consequence, TNFIP6-deficient females are sterile. Cultured TNFIP6-deficient cumulus cell-oocyte complexes also fail to expand when stimulated with dibutyryl cyclic AMP or epidermal growth factor. Recombinant TNFIP6 is able to catalyze the covalent transfer of heavy chains to hyaluronan in a cell-free system, restore the expansion of Tnfip6-null cumulus cell-oocyte complexes in vitro, and rescue the fertility in Tnfip6-null females. These results provide clear evidence that TNFIP6 is a key catalyst in the formation of the cumulus extracellular matrix and indispensable for female fertility.     

Cumulus cells of oocyte-cumulus complexes secrete a meiosis-activating substance when stimulated with FSH

The effect of the different follicular cell types on resumption of meiosis was studied during stimulation with FSH. Cumulus enclosed oocytes (CEO), denuded oocytes (DO), and cumulus and mural granulosa cells were used. The resumption of meiosis and oocyte maturation were assessed by the determination of the germinal vesicle breakdown (GVBD) and polar body formation (PB) at the end of a 24 hr culture period in the presence of 4 mM hypoxanthine (HX). The effects of recombinant LH (r-LH) and hCG were also evaluated. Oocyte exposure to the gonadotrophins varied from 5 min to 24 hr (i.e., priming time). Oocytes were obtained from immature gonadotrophin-stimulated and -unstimulated mice. 1. FSH (1 IU/L-75 IU/L) provoked a dose-dependent increase in GVBD and PB in CEO, but not in DO, in stimulated and unstimulated mice. Eight IU/L was sufficient for inducing resumption of meiosis. In contrast, LH and hCG (both 1 IU/L-1500 IU/L) were without effect on GVBD and PB in CEO and DO of oocytes from stimulated and unstimulated mice. A combination of 8IU/L FSH and 4–8 IU/L hCG produced an additive effect, whereas combinations with LH and higher concentrations of hCG had no such effect. 2. A 2 hr priming with FSH (8 IU/L-75 IU/L) induced a dose-dependent oocyte maturation in CEO. Thirty minutes of priming with FSH (75 IU/L) was sufficient for induction of meiotic resumption in CEO. 3. Priming CEO with FSH for 2 hr followed by the separation and repooling of oocytes and cumulus cells induced oocyte maturation. GVBD of new, unprimed DO added to cumulus cells of primed CEO increased slightly but was significant, whereas GVBD in DO isolated from the primed CEO only increased marginally. DO cocultured with FSH-primed cumulus masses seem to be prevented from resuming meiosis. 4. Priming a coculture of granulosa cells and DO with FSH for 2 hr caused a significant increase in GVBD compared to the control, evaluated after 24 hr. In contrast, a 24 hr FSH-priming of a coculture of granulosa cells and DO was without effect on GVBD. 5. A spent medium in which unstimulated cumulus cells or mural granulosa cells had grown was without effect on GVBD in DO. However, a small fraction of the DO resumed meiosis after culture in a spent medium derived from a 2 hr priming of CEO and spent media from 24 hr priming of CEO induced a 2–3 times higher GVBD frequency in the DO compared to the controls. Heat treatment of spent media (70°C, 30 min) from a 24 hr FSH-priming of CEO still induced GVBD in naive DO. The results showed that FSH, in a concentration of as little as 8 IU/L, but not r-LH and hCG, induced within 30 minutes the cumulus cells to produce and after 2 hr to secrete a diffusible heat stable meiosis activating substance. This substance overcame, in a paracrine fashion, the inhibiting effect of HX and induced oocyte maturation directly in DO. The production of this substance, however, was dependent on the initial connection between the cumulus cells and the oocyte, indicating an important 2-way communication between these 2 cell types. The mural granulosa cells did not produce a meiosis inducing activity by stimulation with FSH, but significantly, more DO matured after coculture with the nonstimulated granulosa cells for 24 hr than for 2 hr. It is proposed that the heat stable meiosis activating component of the spent media from the FSH-stimulated CEO belongs to the meiosis activating sterols, MAS, previously isolated from human follicular fluid and from adult bull testes. Mol. Reprod. Dev. 46:296–305, 1997. © 1997 Wiley-Liss, Inc.     

Metabolic coupling and ligand-stimulated meiotic maturation in the mouse oocyte-cumulus cell complex.

Cumulus cells are metabolically coupled to the mammalian oocyte via heterologous gap junctions. One function attributed to the gap junctional communications is the transfer of regulatory signals that direct the meiotic state of the oocyte. However, the precise role of these junctions in meiotic maturation is still unclear. The aim of this study was to test the hypothesis that meiotic resumption is induced by the transfer of a stimulatory signal(s) from the cumulus cells to the oocyte through the gap junctional coupling pathway. We have previously shown that the mitogenic lectin concanavalin A (Con A) induces oocyte maturation in isolated cumulus cell-enclosed oocytes (CEO) when meiotic arrest is maintained with a number of different inhibitory agents [Biol Reprod 1990; 42:413-423]. In the present study, Con A stimulated maturation in dibutyryl cAMP (dbcAMP)-arrested CEO but not in denuded oocytes cocultured with cumulus cells. Heptanol, a known gap junction uncoupler, effectively prevented Con A- and FSH-induced maturation of intact CEO and dramatically reduced metabolic coupling between cumulus cells and the oocyte. However, this alcohol had no effect on denuded oocytes (DO) or on dbcAMP-arrested CEO in the absence of stimulating ligand. Con A and FSH produced only a minimal loss of coupling. When the effects of heptanol were compared with those of the n-alkanols hexanol and decanol, the efficacies of these agents as suppressors of Con A-stimulated oocyte maturation was directly related to their relative abilities to suppress metabolic coupling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of nitric oxide synthase inhibitors on porcine oocyte meiotic maturation

As an important biological messenger, nitric oxide (NO) exhibits a wide range of effects during physiological and pathophysiological processes, including mammalian oocyte meiotic maturation. The present study investigated whether NO derived from two nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS) or endothelial NOS (eNOS), is involved in the meiotic maturation of porcine oocytes. Meanwhile, the cumulus cells' function in meiotic maturation and their interaction with oocyte development and degeneration were also investigated using cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs). Different inhibitors for NOS were supplemented to the medium. Cumulus expansion, cumulus cell DNA fragmentation and oocyte meiotic resumption were evaluated 48 h after incubation. Aminoguanidine (AG), a selective inhibitor for iNOS, suppressed cumulus expansion and inhibited CEOs to resume meiosis (p < 0.05), but did not inhibit cumulus cell DNA fragmentation. Both Nomega-nitro-L-arginine (L-NNA) and Nomega-nitro-L-arginine methyl ester (L-NAME), inhibitors for both iNOS and eNOS, delayed cumulus expansion, inhibited cumulus cell DNA fragmentation and inhibited CEOs to resume meiosis. Such effects were not seen in DOs. These results indicate that iNOS-derived NO is necessary for cumulus expansion and meiotic maturation by mediating the function of the surrounding cumulus cells, and eNOS-derived NO is also involved in porcine meiotic maturation.     

Influence of hyaluronan accumulation during cumulus expansion on in vitro porcine oocyte maturation

During oocyte maturation, the cumulus-oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process.