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Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.

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A theory of follicle selection (Lacker, 1981) is tested in the primate by simulating the effects of estradiol administration at different times, strengths, and durations during the follicular phase of the menstrual cycle (Clark et al., 1981; Zeleznik, 1981; Dierschke et al., 1985). The theory can account for the observed atretogenic effects of circulating estradiol on follicle development including full, partial, and delayed atresia of the dominant follicle (Dierschke et al., 1985) and can explain why similar estradiol doses achieve different qualitative effects when given at different times during the cycle. The theory predicts that recovery from early atresia may be possible, and it can also account for the loss of control in the number of maturing follicles that has been observed when estradiol antibodies are given in the midfollicular phase (Zeleznik et al., 1985). These results support the hypothesis that the selection mechanism in the primate is a consequence of feedback involving an essentially equipotent follicle population interacting through circulating estradiol and pituitary gonadotropins. A quantitative test of the theory awaits experimental identification of the maturation surfaces that are predicted by it. An experimental design for this purpose is proposed.

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A theory of follicle selection is developed in which all follicles, ovulatory and atretic, inherit the same developmental plan for responding to circulating concentrations of estradiol and gonadotropins. In the model, this plan is represented by a maturation surface that determines the rate of follicle growth as a function of follicle maturity and circulating hormone concentration. Examples of maturation surfaces are constructed for single and multiple spontaneous ovulators. When model follicles are activated from the reserve pool at random times, spontaneous and coordinated cycles of maturation develop in which the number of ovulatory follicles is controlled within a small and predictable range. For a given maturation surface, this range is nearly independent of the number of follicles in the interacting population, their activation times, and their initial maturities. The theory is therefore consistent with Lipschütz's Law of Follicular Constancy. The theory can account for certain statistical effects that occur with age on the timing of ovulation and the control of ovulation number. Maturation surfaces are also constructed that exhibit spontaneous transitions from cyclic to steady anovulatory behavior. The theory predicts an important regulatory role for atretic follicles in controlling both the timing of maturation and the number of follicles that reach ovulatory maturity.

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cAMP synthesis by the rat oocyte and cumulus-oocyte complex was studied using direct labeling techniques. Cumulus-oocyte complexes synthesized cAMP in response to luteinizing hormone, follicle-stimulating hormone, cholera toxin, and forskolin. However, naked oocytes prepared from cumulus-oocyte complexes by mechanically removing the cumulus cells synthesized cAMP only in response to forskolin and follicle-stimulating hormone; cholera toxin and luteinizing hormone did not stimulate cAMP synthesis. Cholera toxin could augment the response of the oocytes to FSH, indicating an intact, though atypical, adenylate cyclase system. Forskolin was found to inhibit the onset of oocyte maturation in both cumulus-oocyte complexes and naked oocytes. The implications of these findings for the relationship between cAMP synthesis and oocyte maturation in the rat are discussed.

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Cumulus oocyte complexes isolated from PMS gonadotropin-primed immature rats bind [125I]hCG. Unlabeled hCG and ovine LH compete with the labeled hormone for binding sites, while rat FSH and ovine PRL are without effect. Since specific binding of [125I]hCG could not be detected in preparations of oocytes from which the cumuli had been removed, it appears that the binding exhibited by the complex can be attributed to the cumulus cells. The concentration dependence of binding is consistent with the presence of one population of high affinity (apparent Kd, 1.4 X 10(-10) M) binding sites (223 +/- 33 sites/cumulus cell). Rat granulosa cells bind hCG with equivalent apparent affinity and specificity but contain more sites (2060 +/- 180/cell) than cumulus cells.

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Cultured rat ovarian granulosa cells undergo a dramatic morphological change when exposed to follicle-stimulating hormone (FSH). Exposure to FSH causes the flattened epithelioid granulosa cells to assume a nearly spherical shape while retaining cytoplasmic processes which contact the substrate as well as adjacent cells. This effect of FSH is preceded by a dose-dependent increase in intracellular cAMP, is potentiated by cyclic nucleotide phosphodiesterase inhibitors, and is mimicked by dibutyryl cAMP. Prostaglandins E1 or E2 and cholera enterotoxin also cause the cells to change shape. A subpopulation of the cells responds to luteinizing hormone. These morphological changes, which are blocked by 2,4-dinitrophenol, resemble those produced by treating cultures with cytochalasin B. Electron microscopy shows that the unstimulated, flattened cells contain bundles of microfilaments particularly in the cortical and basal regions. After FSH stimulation, microfilament bundles are not found in the rounded granulosa cell bodies but they are present in the thin cytoplasmic processes. These data suggest that the morphological change results from a cAMP-mediated, energy-dependent mechanism that may involve the alteration of microfilaments in these cells.

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The hormone-independent, spontaneous maturation that rat oocytes undergo in vitro can be inhibited by derivatives of cyclic AMP and inhibitors of cyclic nucleotide phosphodiesterase. In this study, we have shown that this inhibition of maturation can be partially relieved by preparations of ovine and rat luteinizing hormone or follicle-stimulating hormone. The ability of gonadotropins to foster the resumption of maturation in cultures of cyclic AMP-inhibited oocytes suggests that this system is suitable for studies of the hormonal control of oocyte development. The dose and time dependency of the response to gonadotropins has been examined in order to study the role of these hormones in oocyte maturation and to compare this effect to other known responses of the cumulus-oocyte complex. These studies show that highly purified preparations of rat gonadotropins are less effective inducers of maturation than the more commonly used, but considerably less purified, preparations of ovine gonadotropins. Almost complete relief of inhibition is observed, however, when the oocytes are exposed to a combination of rat luteinizing hormone and follicle-stimulating hormone. Oocyte maturation was not influenced by the sex steroids progesterone or 17beta-estradiol. Our results suggest that: (i) cyclic AMP is involved in the intrafollicular inhibition of oocyte maturation; (ii) both gonadotropins are required for maximal stimulation of the resumption of oocyte meiosis; and (iii) steroids are not involved in this response to gonadotropins.

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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.

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Cultured rat ovarian granulosa cells respond to follicle-stimulating hormone (FSH) by synthesizing and secreting plasminogen activator. The specificity of the response for FSH prompted us to explore the use of this system as an in vitro bioassay for FSH. The release of FSH by pituitary cell cultures has been examined by this method, as have been preparations of FSH of known biological activity. The results indicate that the granulosa cell system allows accurate, rapid, and convenient determinations of FSH activity. Furthermore, the method obviates metabolic clearance problems associated with whole animal assays and it is extremely sensitive: as little as 10(-15) mol (approximately 100 micronIU) of FSH can be detected.

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Rat ovarian granulosa cells and mouse myocardial cells respond to cell-specific hormones by cyclic AMP-dependent mechanisms. In coculture, these heterologous cells communicate by means of gap junctions. Exposure of the cocultures to a hormone specific for one cell type causes the heterologous cells to respond through a cell contact-dependent mechanism. These studies suggest that this cross-stimulation results from the intercellular communication of a mediator that is common to both cell types. The communicated mediator may be cyclic AMP.

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A quantitative method is described for measuring the amount of plasminogen activator produced by rat ovarian granulosa cells following exposure to hormones in vivo or in vitro. The results confirm the previously reported observation (Beers, W. H., Strickland, S., and Reich, E. (1975) Cell 6, (387-394) that granulosa cells in vivo produce increasing amounts of plasminogen activator as the time of ovulation approaches and that the enzyme is produced only be cells obtained from follicles destined to ovulate. Inactive cells can be stimulated in vitro by gonadotropins to produce plasminogen activator. This response is time- and dose-dependent, and results in an increase of intracellular and extracellular enzyme. Studies of the specificity of this response indicate that preparations of follicle-stimulating hormone are much more effective than corresponding preparations of luteinizing hormone. The effect of other pituitary hormones is also presented. Molecules other than gonadotropins are also capable of stimulating the cells to produce the enzyme. Prostaglandins E1 and E2 and analogues of cAMP effectively stimulated the cells to produce plasminogen activator, cGMP and its analogues and prostaglandins F1a and F2a were without effect as were the six steroids studied. The inactive compounds also did not inhibit the response of the cells to gonadotropins. The granulosa cell plasminogen activator has been analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has an apparent molecular weight of 75,000. By this and other criteria, the granulosa cell enzyme is similar to one of the species of plasminogen activators obtained from cultures of simian virus 40-transformed rat embryo fibroblasts.

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A technique is described for detecting fibrinolytic activity of single cells in culture. This method was applied to the analysis of rat ovarian granulosa cells. Cells obtained from follicules shortly before ovulation show high levels of fibrinolytic activity. This activity is plasminogen-dependent, indicating that it is due to plasminogen activator. The appearance of this activity is correlated with ovulation by temporal and functional criteria, and can be demonstrated both in immature animals primed with hormones and in mature cycling animals. Granulosa cell cultures can be stimulated to release plasminogen activator by exposure in vitro either to luteinizing hormone or to dibutyryl cyclic AMP.

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Plasminogen, plasminogen activator, protease inhibitors, and a proteolytic activity are shown to be present in bovine follicular fluid. Much of the proteolytic activity appears to be due to plasmin. In addition, plasminogen activator activity can be demonstrated in follicle wall homogenates. Evidence that plasmin decreases the tensile strength of follicle wall preparations is also reported. The potential for the involvement of these substances in ovulation is discussed.

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