小鼠排卵前后卵巢纤蛋白溶酶原激活因子活性的变化

给幼龄小鼠注射PMSG刺激滤泡生长,随后注射hCG以诱发排卵。在激素处理的不同时间取出卵巢,制备卵巢匀浆液或从卵巢中分离颗粒细胞和卵丘-卵母细胞复合体,并做离体培养。样品中组织型(tPA)和尿激酶型(uPA)纤蛋白溶酶原激活因子经SDS-凝胶电泳分离,用纤蛋白铺盖技术测定。实验结果表明,注射hCG 8h后15％的受试动物排卵,而卵巢匀浆液和颗粒细胞中tPA和uPA活性分别也在hCG注射后4和8h达到高峰。排卵后酶活性下降。卵丘-卵母细胞复合体主要含tPA,注射hCG 12—24h达到高峰。上述资料证明,tPA和uPA都参入小鼠排卵过程。因为排出的卵子中仍含有大量tPA,卵细胞的tPA除参与排卵外,可能对排卵后的一些生理过程也起重要作用。     

Vasoactive intestinal peptide stimulates plasminogen activator activity by cultured rat granulosa cells and cumulus-oocyte complexes

Vasoactive Intestinal Peptide (VIP), originally considered to be a gut hormone, has recently been found to increase estrogen and progesterone production by ovarian granulosa and luteal cells. Because several studies indicate that granulosa cells and oocytes are capable of producing plasminogen activators, we have studied the effects of VIP on plasminogen activator activity in cultured granulosa cells and cumulus-oocyte complexes collected from the ovaries of hypophysectomized, estrogen-treated immature rats. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a fibrin overlay technique to assess plasminogen activator activity, we observed that treatment with VIP stimulated the secretion of tissue-type plasminogen activator (tPA), but not urinary-type plasminogen activator (uPA), in a dose-dependent manner by cultured granulosa cells as well as by cumulus-oocyte complexes, but not by denuded oocytes. However, preparation of cumulus-free oocytes from cumulus-oocyte complexes which had previously been treated with VIP indicated substantial increases in tPA activity within the oocyte. The actions of VIP on tPA activity in granulosa cells were specific, because other closely related peptides (PHM-27 and glucagon) were ineffective. These effects of VIP, in addition to the previously observed effects on steroidogenesis, suggest that VIP may be an important regulator of ovarian function.     

Plasminogen activator activity in cumulus-oocyte complexes of gonadotropin-treated rats during the periovulatory period

Two types of plasminogen activator (tissue-type, tPA; urokinase-type, uPA) have been demonstrated in ovarian granulosa cells, but only tPA activity was found in denuded oocytes. Immature rats were treated subcutaneously with 20 IU pregnant mare's serum gonadotropin (PMSG) to stimulate follicle maturation, followed 2 days later by an injection of 10 IU human chorionic gonadotropin (hCG) to induce ovulation. Cellular plasminogen activator activities were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by a fibrin-overlay technique. Cumulus-oocyte complexes from rats before and after PMSG treatment contained low amounts of tPA, but not uPA, activity. After hCG treatment, tPA activity showed a time-dependent increase, reaching a maximum at 24 h after injection. At 12 and 24 h after hCG treatment, uPA activity was also detected. The appearance of high molecular weight lysis zones further suggested the formation of plasminogen activator-inhibitor complexes. Morphological analysis indicated that the increases in oocyte tPA activity were correlated with the extent of cumulus cell expansion and dispersion. In denuded oocytes, tPA activity also progressively increased during the periovulatory period to a maximum at 24 h after hCG treatment. In contrast, neither uPA activity nor activator-inhibitor complex was detected. Secretion of the proteases was measured in the conditioned media of cumulus-oocyte complexes cultured for 24 h in vitro. Substantial increases in tPA release were found in complexes obtained at 8 and 12 h after hCG injection, with lower secretion from complexes obtained at 24 h after hCG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte

Although it has been shown that granulosa cells regulate the growth and meiotic maturation of mammalian oocytes, there is little evidence of a role for the oocyte in the differentiation or function of granulosa cells. To test the hypothesis that the oocyte participates in the regulation of granulosa cell function, oocytes were removed from isolated oocyte-cumulus cell complexes by a microsurgical procedure and oocytectomized complexes were tested for their ability to undergo expansion in response to follicle-stimulating hormone (FSH). FSH increased the levels of intracellular cAMP, the activity of the hyaluronic acid-synthesizing enzyme system, and induced cumulus expansion in intact complexes. In contrast, FSH did not induce increased hyaluronic acid-synthesizing enzyme activity or cumulus expansion in oocytectomized complexes. Therefore, the participation of the oocyte is necessary for the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in vitro in response to stimulation with FSH. FSH induced the elevation of intracellular cAMP to the same extent in both intact and oocytectomized complexes and the cAMP analog 8-bromo cyclic adenosine monophosphate (8Br-cAMP) did not stimulate expansion in oocytectomized complexes. Therefore, the influence of the oocyte on cumulus expansion occurs downstream from the elevation of cAMP levels in the cumulus cells. Epidermal growth factor (EGF), a potent stimulator of cumulus expansion in intact complexes, which probably acts by a mechanism at least initially different from FSH, failed to stimulate cumulus expansion after oocytectomy. Next, oocytectomized complexes were either cocultured with germinal vesicle stage denuded oocytes or cultured in medium conditioned by denuded oocytes. In both cases, FSH or EGF stimulated expansion by oocytectomized complexes. The degree of expansion was directly correlated to the number of oocytes used to condition the medium. Contact between the oocyte and the cumulus cells is not necessary for cumulus expansion. Rather, a factor(s) secreted by the oocyte is necessary for the cumulus cells to undergo expansion in response to either FSH or EGF. FSH did not induce expansion of oocytectomized complexes in media conditioned by various somatic cells such as granulosa cells, fibroblasts, and Sertoli cells; by a mixed population of male germ cells; or by spermatozoa. This suggests that the expansion enabling activity is specific to the oocyte. These results demonstrate that the oocyte participates in the regulation of cumulus cell function.     

Cell-to-cell communication and ovulation. A study of the cumulus-oocyte complex

Cell-to-cell communication was characterized in cumulus-oocyte complexes from rat ovarian follicles before and after ovulation. Numerous, small gap junctional contacts were present between cumulus cells and oocytes before ovulation. The gap junction are formed on the oocyte surface by cumulus cell processes that transverse the zona pellucida and contact the oolemma. The entire cumulus mass was also connected by gap junctions via cumulus-cumulus interactions. In the hours preceding ovulation, the frequency of gap junctional contacts between cumulus cells and the oocyte was reduced, and the cumulus was disorganized. Electrophysiological measurements indicated that bidirectional ionic coupling was present between the cumulus and oocyte before ovulation. In addition, iontophoretically injected fluorescein dye was tranferred between the oocyte and cumulus cells. Examination of the extent of ionic coupling in cumulus-oocyte specimens before and after ovulation revealed that ionic coupling between the cumulus and oocyte progressively decreased as the time of ovulation approached. In postovulatory specimens, no coupling was detected. Although some proteolytic mechanism may be involved in the disintegration of the cumulus-oocyte complex, neither the cumulus cells nor the oocyte produced detectable levels of plasminogen activator, a protease which is synthesized by membrana granulosa cells. In summary, cell communication is a characterisitc feature of the cumulus-oocyte complex, and this communication is terminated near the time of ovulation. This temporal pattern of the termination of communication between the cumulus and the oocyte may indicate that communication provides a mechanism for regulating the maturation of the oocyte during follicular development before ovulation.     

Hormonal regulation of tissue-type plasminogen activator messenger ribonucleic acid levels in rat granulosa cells: mechanisms of induction by follicle-stimulating hormone and gonadotropin releasing hormone

FSH and GnRH both stimulate rat granulosa cells to produce tissue-type plasminogen activator (tPA). We have studied the molecular mechanisms involved in the action of these hormones by measuring tPA mRNA levels in primary cultures of rat granulosa cells. When granulosa cells were cultured in the presence of FSH or GnRH the level of tPA mRNA was increased 20- and 12-fold, respectively. The induction of tPA mRNA by FSH and GnRH was additive and the kinetics of induction differed. The effect of FSH could be mimicked by bromo-cAMP or forskolin, and was drastically enhanced by cotreatment with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine. These findings are consistent with the notion that FSH mediates its effect through the protein kinase A pathway. GnRH is believed to augment phospholipid turnover in granulosa cells, leading to the activation of the protein kinase C pathway. Like GnRH, the protein kinase C activator phorbol myristate acetate also induced tPA mRNA in granulosa cells. In the presence of the protein synthesis inhibitor, cycloheximide, FSH-stimulated tPA message levels were enhanced by 30-fold, revealing superinduction of tPA mRNA levels by this pathway. In contrast the induction of tPA mRNA by GnRH was inhibited by cycloheximide indicating that the synthesis of an intermediate protein is required for the GnRH effect. Our data suggest that FSH and GnRH increase the tPA mRNA levels by two distinct pathways in cultured granulosa cells, providing a model system for studying the hormonal regulation of tPA gene expression.     

大鼠卵巢细胞分泌纤溶酶原激活因子抑制因子（PAI—1）的...

Rat ovarian cells produce not only plasminogen activator (tPA) but also plasminogen activator inhibitor type 1 (PAI-1), and their coordinated geneexpression induced by gonadotropins are thought to be responsible for follicular rupture. In this study, it was demonstrated that (1) theca-interstitial compartment synthesizes the majority of PAI-1 activity in the ovary before ovulation, the follicular wall may therefore serve as a specific barrier to prevent the secretion of PA into the extrafollicular compartment; (2) Granulosa cells contribute only small amount of ovarian PAI-1 activity, but synthesize most of tissue-type plasminogen activator activity involved in the process leading to ovulation: (3) Since only matured cumulus-oocyte complexes secrete high level of tPA and PAI-1, both tPA and PAI-1 activity in the conditioned medium may be used as reliable markers for evaluating oocyte quality for in vitro fertilization.     

Oocyte-secreted factor(s) determine functional differences between bovine mural granulosa cells and cumulus cells

Cumulus cells and mural granulosa cells (MGC) are phenotypically different and there is now evidence suggesting that the oocyte plays an active role in determining the fate of follicular somatic cells. This study investigates the role of oocyte-secreted factor(s) in the regulation of the growth and differentiation of cumulus and MGC. Bovine cumulus-oocyte complexes (COC) and MGC were cultured with various hormones for 18 h followed by a further 6-h pulse of [(3)H]thymidine as an indicator of follicular cell DNA synthesis. The COC incorporated 11 to 14 times more [(3)H]thymidine than MGC in either the absence or presence of 50 ng/ml insulin-like growth factor (IGF)-I. Purified porcine FSH (450 ng/ml) added together with IGF-I marginally increased (3)H incorporation in MGC relative to IGF-I alone but dramatically decreased incorporation in COC sixfold. Conversely, mean progesterone production in the presence of IGF-I + FSH was 13-fold higher from MGC than from COC, confirming a distinctive phenotype of cumulus cells. However, this phenotype was found to be dependent on the presence of the oocyte, as microsurgical removal of the oocyte (oocytectomy) resulted in an 11-fold decrease in [(3)H]thymidine incorporation in cumulus cells treated with IGF-I, elimination of the inhibitory effect of FSH on IGF-I-stimulated DNA synthesis, and led to a 2-fold increase in progesterone production in medium with IGF-I and FSH. All of these markers were completely restored to COC levels when oocytectomized complexes were cocultured with denuded oocytes (DO) at a concentration of 0.5 oocytes/microl, demonstrating that oocytes secrete a soluble factor(s) that promotes growth and attenuates cumulus cell progesterone secretion. In the presence of IGF-I, [(3)H]thymidine incorporation in MGC increased ninefold above control levels with the addition of DO. The addition of FSH to IGF-I-increased (3)H counts in MGC, however, led to a decrease in counts in MGC + DO as is also observed in COC. Furthermore, progesterone production was halved when DO were added to MGC cultures, most notably in the presence of IGF-I and/or FSH. These results provide further evidence that MGC and cumulus cells have distinctive phenotypes and that the oocyte is responsible for some of the characteristic features of cumulus cells. Bovine oocytes secrete a soluble factor(s) that simultaneously promotes growth and attenuates steroidogenesis in follicular somatic cells.     

Effect of granulocyte-macrophage colony-stimulating factor deficiency on ovarian follicular cell function

Granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine secreted by lymphohaemopoietic and other cell lineages, is known to influence ovarian cyclicity and embryo development. The aim of this study was to examine the effect of GM-CSF on ovarian follicular cell function using GM-CSF-deficient (GM -/-) mice. Immature GM -/- and GM +/+ mice were stimulated with eCG, and cumulus-oocyte complexes and mural granulosa cells were collected 48 h later. Expression of GM-CSF receptor (GM-CSFR) alpha and beta mRNA subunits by cumulus-oocyte complexes and mural granulosa cells was examined using RT-PCR. Cumulus-oocyte complexes from both genotypes were found to express mRNA for the GM-CSFRalpha-subunit only, while the mural granulosa cells expressed both the alpha and beta receptor subunits. Cumulus-oocyte complexes recovered from GM -/- mice had approximately twice the number of cumulus cells per cumulus-oocyte complex than did those of GM +/+ mice (P < 0.05), even though the growth-promoting activity of denuded GM -/- oocytes was found to be equivalent to that of wild-type oocytes. GM-CSF deficiency was associated with marginally increased DNA synthesis in cumulus cells and significantly (P < 0.05) lower progesterone production by mural granulosa cells recovered from GM -/- compared with those recovered from GM +/+ mice. The addition of rec-mGM-CSF in vitro did not affect DNA synthesis in either cell type or progesterone production by mural granulosa cells, irrespective of GM-CSF status. There was no effect of GM-CSF deficiency on the capacity of FSH and insulin-like growth factor I to stimulate DNA synthesis in cumulus-oocyte complexes (approximately 15- and threefold, respectively) and in mural granulosa cells (approximately two- and threefold, respectively). Taken together, these data show that GM-CSF influences events associated with follicular maturation in mice. The effects of GM-CSF are not exerted directly in granulosa or cumulus cells, but appear to be mediated indirectly, perhaps through the agency of steroidogenesis-regulating secretions of local macrophage populations residing in the theca.     

Regulation of serine protease inhibitor-E2 and plasminogen activator expression and secretion by follicle stimulating hormone and growth factors in non-luteinizing bovine granulosa cells in vitro.

During ovarian follicle growth, there is expansion of the basal lamina and changes in the follicular extracellular matrix (ECM) that are mediated in part by proteolytic enzyme cascades regulated by tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). One PA inhibitor, serine protease inhibitor-E2 (SERPINE2) is expressed in granulosa but not theca cells, and expression changes with follicle development. In this study, we hypothesized that PA and SERPINE2 expression/secretion by granulosa cells are regulated by FSH and growth factors. SERPINE2 mRNA and protein levels, tPA gene expression and uPA secretion were stimulated by FSH. Insulin-like growth factor-I stimulated SERPINE2 secretion and uPA activity, and decreased secreted tPA activity and gene expression. Bone morphogenetic protein-7 increased SERPINE2 secretion and expression and tPA secretion. In contrast, fibroblast growth factor-2 inhibited tPA secretion and SERPINE2 secretion and expression. Epidermal growth factor inhibited SERPINE2 secretion and expression, but increased secreted tPA activity. Estradiol and SERPINE2 secretion were highly positively correlated, but estradiol did not alter SERPINE2 expression. These data demonstrate that SERPINE2 expression and protein secretion are regulated by FSH and growth factors in non-luteinizing bovine granulosa cells. As estradiol is a known marker of follicle health, and SERPINE2 is an anti-apoptotic factor, we propose that SERPINE2 is involved in the regulation of atresia in bovine follicles.     

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.     

Synaptosomal-associated protein 25 gene expression is hormonally regulated during ovulation and is involved in cytokine/chemokine exocytosis from granulosa cells

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.     

Energy substrate metabolism of mouse cumulus-oocyte complexes: response to follicle-stimulating hormone is mediated by the phosphatidylinositol 3-kinase pathway and is associated with oocyte maturation

Successful in vitro maturation (IVM) of oocytes obtained from medium-sized antral follicles could avoid the need for superovulation for in vitro fertilization. The wide range of doses of FSH used in IVM prompted us to study the effect of varying concentrations of FSH on the dynamics of nutrient uptake and production by individual maturing mouse cumulus-oocyte complexes (COCs). COCs isolated from the antral follicles of unprimed, prepubertal B6CBF(1) mice were cultured individually in increasing concentrations of FSH (0-2000 ng/ml). Following culture, pyruvate, glucose, and lactate uptake or production by individual complexes were noninvasively assessed and compared with the stage of nuclear maturation of the enclosed oocyte. FSH significantly increased oocyte maturation and produced a two- to threefold increase in glucose uptake and lactate production by COCs in which the enclosed oocyte completed maturation. In these COCs, pyruvate was taken up under control conditions but was produced in progressively higher quantities in increasing concentrations of FSH. In COCs where the oocyte failed to complete maturation, pyruvate was taken up (rather than produced) and glucose uptake and lactate production were lower and unaffected by the presence or absence of FSH. This suggests that there is dialogue between cumulus cells and the maturing oocyte that influences FSH responsiveness and substrate metabolism of the whole COC. Finally, inhibition of FSH-stimulated glucose uptake by the PI3-kinase inhibitor LY294002 and the finding of GLUT4 protein in granulosa cells suggest that FSH increases glucose uptake by PI3-kinase-mediated translocation of GLUT4 to the granulosa cell membrane.     

Regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured rat Sertoli and Leydig cells

New data are provided to show that (i) rat Sertoli cells produce two types of plasminogen activators, tissue type (tPA) and urokinase type (uPA), and a plasminogen activator inhibitor type-1 (PAI-1); (ii) both tPA (but not uPA) and PAI-1 secretion in the culture are modified by FSH, forskolin, dbcAMP, GnRH, PMA and growth factors (EGF and FGF), but not by hCG and androstenedione (△4); (iii) in vitro secretion of tPA and PA-PAI-1 complexes of Sertoli cells are greatly enhanced by presence of Leydig cells which produce negligible tPA but measurable PAI-1 activity;(iv) combination culture of Sertoli and Leydig cells remarkably increases FSH-induced PAI-1 activity and decreases hCG- and forskolin-induced inhibitor activity as compared with that of two cell types cultured alone. These data suggest that rat Sertoli cells, similar to ovarian granulosa cells, are capable of secreting both tPA and uPA, as well as PAI-1. The interaction of Sertoli cells and Leydig cells is essential for the cells to response to     

Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles

Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps.     

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.