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a Ottawa Regional Cancer Centre, and Departments of Medicine, Cellular and Molecular Medicine, and Obstetrics&Gynecology, University of Ottawa, Ottawa, Ontario, Canada K1H 8L6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability of granulosa cells to undergo spontaneous luteinization (i.e., in the absence of LH) suggests that either the hormonal/growth factor signals required for luteinization are not present in the follicle before the LH surge, but are provided in the culture system, or that substances are present in the intrafollicular environment that inhibit the process of luteinization. Previous studies showing that follicular fluid obtained from small- and medium-sized horse [7] and pig [8] follicles contain a luteinization inhibitor support the latter hypothesis. The origin of the luteinization inhibitor in follicular fluid is unknown, although numerous studies suggest that oocytes secrete a factor that has similar activity [9–16].
A potential role for the oocyte in the regulation of follicular steroidogenesis and luteinization was first suggested by El-Fouly et al. [9], who demonstrated that removal of the oocyte from rabbit graafian follicles in situ resulted in spontaneous luteinization and increased progesterone production. Further studies in rats found that the addition of oocytes to granulosa cell cultures prevented morphological luteinization [10], and that luteinization of rat follicles in vitro [11] or in vivo [12] coincided with degeneration of the oocyte. Using the microsurgical procedure of oocytectomy, we and others have also shown the inhibitory influence of mouse [13] and porcine [14] oocytes and human embryos [15] on progesterone production by both cumulus and mural granulosa cells.
In the current study, the paracrine signals from oocytes that modulate granulosa cell steroidogenesis were examined for possible stage-specific secretion during follicular development and luteinization. In addition, developmental changes in the responsiveness of granulosa cells to these paracrine factors were investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For most experiments, mouse ovaries were obtained from 24- to 26-day-old (C57BL/6 x CBA/J) F1 mice injected 44 h previously with 5 IU eCG. Oocyte-cumulus cell complexes (OCC) were isolated by puncturing antral follicles with 25-gauge needles in Waymouth MB 752/1 culture medium (WAY) containing 75 mg/L penicillin-G, 50 mg/L streptomycin, and 25 mg/L sodium pyruvate (all from Sigma Chemical Co., St. Louis, MO), and 5% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT). After isolation, the complexes were washed three times in fresh WAY/FBS.
For some experiments, oocyte-granulosa cell complexes were isolated from follicles at various stages of development (see Table 1). Meiotically incompetent oocyte-granulosa cell complexes having 2–3 layers of granulosa cells around the oocyte were isolated from the preantral follicles of 12-day-old mice using enzymatic and mechanical dispersion as described previously [16]. Complexes were also isolated from the early antral follicles of 16-day-old mice or from the oviducts of eCG-primed mice induced to ovulate by administration of 5 IU hCG. All complexes were washed three times in medium before transfer to the final culture medium.
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Animals were maintained and handled according to the Guidelines for the Care and Use of Animals established by the Canadian Council on Animal Care.
Removal of the Oocyte from Oocyte-Granulosa Cell Complexes
The microsurgical removal of the oocyte from OCC was performed using a micromanipulation procedure as described previously [17]. These oocytectomized (OOX) complexes, consisting of the zona pellucida surrounded by layers of cumulus cells, but without the oocyte, were washed twice before culture. Oocyte-granulosa cell complexes from the preantral follicles of 12-day-old animals were too sticky to oocytectomize immediately after isolation, and they were therefore first cultured individually in agarose-coated wells as described previously [16]. Ovulated complexes cannot be oocytectomized as the cumulus cells are embedded in a matrix that is far too sticky to be handled by micromanipulation.
Culture of Intact and OOX Complexes
For all experiments, groups of 25 intact or 25 OOX complexes were transferred to 500 µl WAY/FBS in each well of a 24-well plate (Corning Glass Works, Corning, NY). Complexes were treated with FSH (NIADDK-oFSH-18; 150 ng/ml) and with testosterone (Sigma; 500 nM) or pregnenolone (Sigma; 500 nM), and they were cultured for up to 48 h at 37°C in 5% CO2:5% O2:90% N2. The media were then collected and stored at -20°C until assayed for progesterone and estradiol-17ß using RIAs that have been described and validated for direct measurements [18, 19]. Cross-reactivity of the progesterone antibody with pregnenolone is 0.13%.
Coculture of OOX Complexes with Denuded Oocytes
Fully grown oocytes were isolated from the antral follicles of eCG-primed 24- to 26-day-old mice and denuded by repeated pipetting of OCC with a Pasteur pipette. Oocytes collected from the early antral follicles of 16-day-old unprimed mice were similarly denuded. They were cultured in WAY/FBS for 6 h and divided on the basis of the presence (meiotically incompetent) or absence (meiotically competent) of the germinal vesicle. Growing oocytes were obtained by placing the ovaries of 12-day-old mice in 3 ml of Ca2+-free PBS + 0.5% collagenase + 0.01% bovine pancreas DNase I (Sigma), and pipetting the follicles at 4-min intervals. Ovulated oocytes were obtained from oviducts 16 h after hCG administration to eCG-primed mice. Zygotes were collected from the oviducts of mice induced to ovulate with hCG and then paired overnight with fertile males. Groups of 25 OOX complexes were cultured with or without denuded oocytes at a concentration of 1 oocyte/2 µl, with the exception of oocytes from preantral follicles, for which 2.5 oocytes/2 µl were used to approximate similar total cellular volume. Cells were cultured in the presence of FSH and testosterone for 48 h in 500 µl of WAY/FBS in each well of a 24-well plate. Media were stored at -20°C until assayed for progesterone content.
Statistical Analyses
Statistical comparisons between two groups (e.g., to compare intact and OOX groups) were made by unpaired, two-tailed t-tests. For most experiments involving multiple groups, comparisons were made by analysis of variance. When significant effects were observed, Fisher's protected least-significant differences test was used for multiple comparisons. Data are the mean ± SEM of results from 3–7 replicate experiments. Statistical significance was inferred at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocyte-granulosa cell complexes showed stage-specific changes in their ability to produce steroid hormones in culture. Accumulation of estradiol in cultures of FSH + testosterone-stimulated complexes from antral follicles was more than 6- and 13-fold that from preantral follicles or after ovulation, respectively (Fig. 1). Conversely, progesterone production by complexes from both preantral and antral follicles was significantly lower than by ovulated complexes.
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The role of the oocyte in regulating granulosa cell progesterone production during follicular development was investigated by comparing progesterone accumulation in cultures of intact and OOX complexes from preantral, early antral, and large antral follicles. In all cases, levels of progesterone in FSH + testosterone-stimulated cultures of OOX complexes was greater than in cultures of intact complexes (Fig. 2). The accumulation of progesterone by these OOX complexes increased in a stage-specific manner, with OOX antral complexes capable of generating progesterone levels 23-fold higher than intact complexes at the same stage.
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To determine the developmental stages at which oocytes secrete the progesterone-inhibitory factor, denuded oocytes at various stages of development were cocultured with OOX complexes from antral follicles, and progesterone accumulation was determined. To equalize oocyte mass in the cocultures, the number of oocytes from preantral follicles was 2.5-fold that of oocytes from antral follicles. FSH + testosterone-stimulated progesterone production by OOX complexes was reduced when the complexes were cultured with oocytes, regardless of the stage of oocyte used (Fig. 3). Coculture with growing oocytes isolated from preantral follicles of 12-day-old mice reduced progesterone levels to 50% of the levels produced by OOX complexes cultured alone (control). Both meiotically incompetent and competent oocytes from early antral follicles were similarly inhibitory. Fully grown oocytes from antral follicles were most effective in their ability to inhibit progesterone accumulation by the OOX complexes, reducing levels to 20% of control values. However, ovulated oocytes and zygotes retained considerable ability to inhibit progesterone production (to 33% and 41% of control, respectively).
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The effect of meiotic status of meiotically competent oocytes on their ability to regulate progesterone production was determined by culturing intact and OOX complexes in the presence or absence of isobutylmethylzanthine (IBMX), a phosphodiesterase inhibitor that prevents the spontaneous resumption of meiosis of oocytes placed into culture. Appropriate meiotic status of the oocytes was confirmed by denuding the complexes at the end of the culture period. Regardless of the presence or absence of IBMX, FSH + testosterone-stimulated progesterone accumulation by intact complexes was only 4% that of OOX complexes (Table 2), suggesting that the ability of oocytes to produce the progesterone-inhibitory factor is not affected by meiotic activity in meiotically competent oocytes.
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Changing Responsiveness of Granulosa Cells to Oocyte-Secreted Factors
The results presented in Figure 1 indicate that ovulated OCC can produce significant amounts of progesterone, which would suggest that ovulated oocytes are no longer capable of secreting the progesterone-inhibitory factor. However, when ovulated oocytes were cocultured with OOX complexes, progesterone accumulation was reduced, indicating that ovulated oocytes are capable of secreting this inhibitory factor (Fig. 3). An alternative explanation is that ovulated cumulus cells are no longer responsive to the oocyte-secreted factor. To test this hypothesis, denuded fully grown (antral-stage) oocytes were tested for their ability to inhibit progesterone production by ovulated OCC. With OOX complexes from antral follicles used for comparison, denuded oocytes were able to inhibit progesterone production by OOX complexes but had no effect on progesterone accumulation by ovulated complexes (Fig. 4).
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The desensitization of cumulus cells appears to occur during the final stages of follicular development and is directly or indirectly a consequence of the LH surge. To determine whether a similar loss of responsiveness to the progesterone-inhibitory factor occurs during the spontaneous luteinization of OOX antral complexes, OCC and OOX were cultured for 24 h in the presence of FSH and pregnenolone, during which time the accumulation of progesterone by OOX complexes became significantly greater than that by OCC (8526 ± 284 pg/ml, n = 8, vs. 4222 ± 613 pg/ml, n = 3, respectively). The media were changed to fresh media or denuded oocyte-conditioned media, and the complexes were cultured with the same hormonal treatment for a second 24 h. Progesterone accumulation by OCC (3265 ± 239 pg/ml, n = 4) remained low relative to that of the OOX complexes (10 431 ± 864 pg/ml, n = 4). In addition, oocyte-conditioned media were able to inhibit progesterone production by OOX complexes by 25% (7803 ± 762 pg/ml, n = 4), indicating that these cumulus cells could still respond to the actions of the oocyte-secreted progesterone-inhibitory factor.
Oocyte Effects on Progesterone Accumulation in the Presence of Pregnenolone
We hypothesized that oocytes might inhibit progesterone accumulation by granulosa cells by regulating the activity of the steroidogenic enzymes required for its synthesis, thereby limiting the amount of substrate available. To investigate this hypothesis, complexes were isolated from antral follicles, after which intact and OOX complexes, and OOX complexes cocultured with denuded oocytes were cultured in the presence of FSH with or without pregnenolone for 48 h. Overall levels of progesterone accumulation by all complexes were greater when pregnenolone was provided (p < 0.05; Fig. 5). However, in both the absence and the presence of substrate, OOX complexes produced significantly greater amounts of progesterone than did either intact complexes or OOX complexes cocultured with denuded oocytes, suggesting that the presence of the oocyte influences granulosa cells in a manner that reduces the ability of these complexes to produce and/or accumulate progesterone, even in the presence of abundant substrate.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The developmentally specific abundance and/or potency of the progesterone inhibitory activity, where fully grown oocytes most effectively inhibit cumulus cell progesterone production, correlates well with the previously demonstrated influence of the oocyte on LH receptor mRNA expression by granulosa cells [20]. In that study, fully grown oocytes were most effective in suppressing LH receptor expression compared to oocytes from preantral follicles and mature (metaphase II-arrested) oocytes. Although FSH was used to stimulate steroidogenesis in the present study, the ability of oocytes to alter gonadotropin receptor expression may contribute to the reduced steroidogenic capacity. In both cases, the action of the oocyte-derived factor is downstream of FSH-induced generation of cAMP [21, 22], and it is therefore reasonable to speculate that the oocyte-derived factor(s) that suppresses progesterone production is the same factor(s) that suppresses LH receptor expression. This, however, may not fully explain the inhibitory influence of oocytes on progesterone production since metaphase II-stage oocytes were substantially less able to suppress LH receptor expression than meiotically arrested (germinal vesicle-intact) oocytes [20], whereas these two stages of oocytes were equally capable of inhibiting progesterone accumulation by OOX complexes.
The ability of zygotes to suppress progesterone production is in agreement with a previous study demonstrating the inhibitory effects of human embryos on granulosa cell progesterone and estradiol accumulation, suggesting the prolonged production of steroid-regulatory factors after ovulation [15]. However, the inability of two-cell-stage mouse embryos to suppress LH receptor expression in granulosa cells suggests that the embryonic regulation of steroidogenesis may not be mediated by alteration in LH receptor expression [20].
Meiotic incompetence of the oocyte has previously been shown to be accompanied by functional incompetence of its embracing cumulus cells, i.e., their inability to produce steroid hormones [23]. The observation that meiotically incompetent and competent oocytes were equally inhibitory to progesterone production would suggest that the lack of steroidogenic capacity described previously could be accounted for, at least in part, by the inhibitory influence of the oocyte. That being said, the ability of oocyte-deprived granulosa cells to generate progesterone significantly increased at the early antral stage, the stage of development during which oocytes acquire meiotic competence, suggesting that inherent steroidogenic capacity is indeed increasing at the time.
Ovulated oocytes produced sufficient progesterone-inhibitory activity to significantly reduce progesterone production by OOX complexes. The apparently conflicting observation that ovulated OCC produced abundant progesterone could potentially be explained by the fact that ovulated cumulus cells had lost their sensitivity to the steroid-regulating factor. Another possible contributing factor may be that cell-cell contact is required for oocytes to have inhibitory effects on progesterone production, and contact would naturally be lost during cumulus expansion. Certainly, additional factors must play a role in the process of granulosa cell desensitization to the oocyte factors, since spontaneous luteinization of OOX complexes did not result in the same desensitization that was evident in ovulated complexes.
The observation that estradiol [22] and progesterone production (this study) are regulated by oocyte-secreted factors suggests that these factors may act on granulosa cells in a manner that alters the abundance or activity of several enzymes in the steroidogenic pathway, including aromatase to enhance estradiol production and P-450 side-chain-cleavage enzyme (P450scc) and/or 3-beta-hydroxysteroid dehydrogenase (3ß-HSD) to inhibit progesterone production. Goldschmit et al. [23] demonstrated that P450scc is not expressed in cumulus cells earlier than 2–3 h before ovulation, and they proposed the presence of a putative intraovarian suppressive factor(s) that disappears before ovulation and thus renders the cumulus cells permissive for P450scc responsiveness to gonadotropins [23]. Whereas the oocyte appears to secrete such a factor, it is clear that there must be additional factors that facilitate the decreasing responsiveness of the granulosa cell to the oocyte-secreted product after the LH surge. The demonstration in this study that oocyte-secreted factors alter the ability of granulosa cells to accumulate progesterone in the presence of abundant pregnenolone suggests that the abilities of granulosa cells to convert pregnenolone to progesterone and/or to metabolize progesterone are regulated, directly or indirectly, by factors secreted by the oocyte.
The factor(s) released by mouse oocytes modulates gonadotropin-stimulated steroid hormone production by both cumulus and mural granulosa cells [22], although steroidogenesis is more predominantly a function of mural granulosa cells rather than cumulus cells. Despite this fact, the ability of cumulus cells to produce progesterone in a gonadotropin-responsive manner has been demonstrated in many species, including rats [24], pigs [25], and humans [26]. In addition, the ability of oocytes to regulate granulosa cell progesterone production does not appear to be an activity restricted to mammals. Sretarugsa and Wallace [27] have performed experiments indicating that Xenopus oocytes modulate follicular steroidogenesis, and ablation of the germinal disc region in the chicken follicle (the equivalent of the mammalian oocyte) has been shown to prevent the preovulatory rise in progesterone [28].
The secretion of the progesterone-inhibitory factor by oocytes at all stages of development, albeit at variable amounts or potencies, distinguishes it from the cumulus expansion-enabling factor, which seems to be produced only by specific stages of oocyte development, notably after the oocyte has acquired meiotic competence [16]. Both oocyte-secreted factors share a common feature in that continuous exposure of granulosa cells to the oocyte-secreted factors is necessary to maintain the responsiveness of the cells to those factors. Loss of contact with oocytes for as little as 8 h reduces the ability of cumulus cells to expand [29] and to synthesize hyaluronic acid and dermatan sulphate [30], and normal steroid hormone production is altered within 24 h after oocyte loss ([22], this study). However, it is clear that both oocyte-secreted factors operate in conjunction with other follicular factors to regulate granulosa cell activity, since the presence of either factor does not necessarily ensure that the granulosa cells are responsive to it. Meiotically competent oocytes secrete the expansion-enabling factor, but the cumulus cells do not expand until after the LH surge. In contrast, maturing and ovulated oocytes continue to secrete the steroid-regulating factor, whereas the ability of cumulus cells to respond to this factor is lost after ovulation.
Evidence continues to accumulate to support the notion that follicular growth and function is dependent upon the oocyte. In vivo models of germ cell deficiency such as Kit receptor mutants [31], germ cell-deficient mice [32], growth differentiation factor-9-deficient mice [33], and busulphan-treated rats [34] all display impaired follicular development and altered gonadotropic or steroidogenic hormone production. To date, every granulosa cell activity associated with follicle development that has been examined has been found to be regulated, at least in part, by the presence of the oocyte, and it is clear that some of the normal functions of cumulus cells are compromised when these cells are removed from the presence of the oocyte. The data presented here build on the concept that oocytes control many aspects of granulosa cell function and, while the mechanisms by which the germ cells influence granulosa cell activity are unclear, there is no doubt that investigation of these mechanisms will yield results that are essential for our understanding of normal follicular development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted: July 10, 1998.
Received: February 24, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5-3ß-hydroxysteroid dehydrogenase activity in the membrana granulosa of the ovulable type follicle of the rat. Histochemistry 1979; 62:125–135.[CrossRef][Medline]This article has been cited by other articles:
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