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Endocrinology Vol. 148, No. 9 4458-4465
Copyright © 2007 by The Endocrine Society

Are Steroids Obligatory Mediators of Luteinizing Hormone/Human Chorionic Gonadotropin-Triggered Resumption of Meiosis in Mammals?

Shmulik Motola, Malka Popliker and Alex Tsafriri

Department of Biological Regulation, The Bernhard Zondek Hormone Research Laboratory, The Weizmann Institute of Science, Rehovot 76100, Israel

Address all correspondence and requests for reprints to: Alex Tsafriri, Department of Biological Regulation, The Bernhard Zondek Hormone Research Laboratory, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: alex.tsafriri{at}weizmann.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroids mediate the gonadotropic stimulus of oocyte maturation in fish and amphibians. Such a role of steroids in mammals has not been confirmed until recently. A series of studies presented data suggesting that steroids might be involved in meiosis of mouse oocytes. Here we examined this suggestion using in vitro cultures of rat and mouse follicle-enclosed oocytes (FEOs) and cumulus-enclosed oocytes (CEOs). In FEOs that mature only in response to gonadotropins or other stimuli, we tested the ability of steroids to trigger meiosis and whether addition of steroid receptor antagonists blocks LH/human chorionic gonadotropin stimulation of meiosis. In CEOs that mature spontaneously, we tested whether steroid antagonists block maturation and whether steroids overcome the inhibition of maturation by hypoxanthine (Hx), a mild inhibitor of meiotic resumption. The progesterone antagonists mifepristone (RU 486) and Organon 31710 as well as the estrogen antagonist faslodex did not prevent LH-triggered maturation of rat or mouse FEOs or the spontaneous maturation of CEOs. In accordance, the progesterone agonist promegestone (R5020) and estradiol did not stimulate the resumption of meiosis in rat and mouse FEOs, and both did not overcome the Hx inhibition of meiosis in rat and mouse CEOs. Flutamide, an androgen antagonist, did block meiosis in rat FEOs, but this action could not be affected by adding dihydrotestosterone, suggesting that it was not androgen receptor mediated. Flutamide did not affect spontaneous maturation of rat CEOs, and dihydrotestosterone could not stimulate meiosis inhibited by Hx. Thus, in contrast to lower vertebrates, in mammals, steroids do not seem to serve as an obligatory signal by which the somatic cells of the follicle transfer the gonadotropic stimulation of meiosis to the oocyte.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ESSENTIAL ROLE of follicle-cell steroids in the mediation of gonadotropic stimulation of oocyte maturation in fish and amphibian oocytes is well established (1, 2, 3, 4). Studies with isolated mammalian oocytes [cumulus-enclosed oocytes (CEOs) or oocytes stripped of their cumulus cells, denuded] maturing spontaneously in vitro led to inconsistent findings. In some cases, steroids like testosterone attenuated the spontaneous meiotic resumption, and in other studies, estradiol, testosterone, or progesterone potentiated the inhibitory action of dibutyryl cAMP or forskolin. Yet in other studies, steroids did not affect meiosis in culture (5, 6, 7). Nevertheless, such studies led to the conclusion that steroids may modulate meiosis under some experimental conditions (reviewed in Refs. 8 and 9).

The preovulatory surge of gonadotropins induces a prompt rise in follicular steroidogenesis, especially progesterone, which could mediate LH stimulation of meiotic resumption. Explantation of rat preovulatory follicles before the LH surge allowed examination of follicular responses, including resumption of oocyte maturation and steroidogenesis, after LH/human chorionic gonadotropin (hCG) stimulation in vitro (10, 11). Addition of steroids to the medium could not stimulate the resumption of meiosis in rat preovulatory follicle-enclosed oocyte (FEOs) (10). Similar results were obtained in bovine, porcine, and ovine follicles in culture (12, 13). Furthermore, suppression of follicular steroidogenesis by inhibitors did not impair the resumption of meiosis triggered by LH (14, 15).

Recently, a series of publications presented evidence that steroids mediate LH action on the resumption of meiosis in the mouse (16, 17). In these studies, it was claimed that progesterone, estrogen, or testosterone can stimulate resumption of meiosis and that specific antagonists of each of these steroid receptors can block its action. Here we examine the suggested role of progesterone, estrogen, and androgen in the mediation of LH-stimulated resumption of meiosis in the rat and the mouse.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Rats and mice.
Animals were provided with water and chow without restriction and housed in air-conditioned rooms illuminated 14 h/d. Immature rats or mice were injected with equine chorionic gonadotropin (eCG) (5 IU/mouse; 12 IU/rat; Horizon, Ltd., Sydney, Australia) between 0900 and 0930 h on d 23–24 of age to enhance multiple follicular development. The animals were killed 48–50 h after eCG treatment by cervical dislocation and preovulatory follicles (FEOs) and CEOs were explanted for culture.

Culture of FEOs and cumulus-oocyte complexes (COCs).
The preovulatory follicles were isolated 48 h after pregnant mare serum gonadotropin injection as previously described (10). FEOs (10–15 per dish) were cultured in Leibovitz’s L-15 medium (Life Technologies, Inc., Orchard Park, NY) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml) (Life Technologies), and 5% fetal calf serum (Sera-Lab, Crawley Down, UK), hereafter referred to as L-15 plain medium. FEOs were cultured at 37 C in a controlled atmosphere of 50% O2, 1.3% CO2, and 48.7% N2. After 60 min of preequilibration, ovine LH (1 µg/ml) (generously provided by Dr. A. F. Parlow and the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health), progesterone (Sigma-Aldrich, St. Louis, MO), promegestone (R5020; PerkinElmer Life and Analytical Sciences, Boston, MA), estradiol (Sigma-Aldrich), or dihydrotestosterone (DHT; Sigma-Aldrich) was added. Nonaromatazable DHT was used, rather than testosterone, because of its reported efficiency to induce meiosis in the mouse (17). The steroid receptor antagonists mifepristone (RU-486; Sigma-Aldrich) and Organon 31710 (Org 31710) (generously provided by N.V. Organon, Oss, The Netherlands), both antagonists of progesterone; faslodex (ICI 182,780; Tocris Cookson, Ellisville, MO), an antagonist of estrogen; or flutamide (Sigma-Aldrich), a testosterone antagonist were added to the culture 30 min before LH or steroid stimulation unless indicated otherwise. Org 31710 was dissolved in dimethylsulfoxide (Sigma-Aldrich) and all other steroids and their antagonists in ethanol, and the final concentration of the solvents in culture medium was 0.1%. The same solvent concentration was included in control culture media. At the end of the culture period, the follicles were punctured to release and collect the COCs under a dissecting microscope. Oocyte maturation was assessed by scoring groups of 10–15 oocytes using Nomarski interference microscopy. The mean ± SEM values for each of the treatment groups are given. At least three replicate cultures were used for each treatment group, on two separate experiments.

The COCs were collected by puncturing the largest ovarian follicles and exerting gentle pressure. Plain L-15 medium supplemented with 4 mM hypoxanthine (Hx) (Sigma-Aldrich), hereafter referred to as L-15 Hx medium, previously shown as a mild inhibitor of meiotic resumption (18), was used for oocyte collection to maintain them at the germinal vesicle stage. The oocytes for the control group were washed and subsequently cultured in L-15 plain medium. At the end of the culture period, the COCs were collected and examined by Nomarski interference microscopy, as detailed above for FEOs.

Xenopus laevis oocytes.
Xenopus ovarian fragments were generously provided by Prof. N. Daskal (Tel-Aviv University). Ovarian tissue and oocytes were collected as previously described (19). Briefly, ovarian tissue was incubated for 1 h at 4 C with 2 mg/ml Sigma type II collagenase in modified Bart’s solution (MSBH) without Ca2+ and then washed extensively using MSBH and manually separated in MSBH. Fully grown stage VI oocytes were selected and cultured for 18–19 h at 20 C without or with progesterone or promegestone. Promegestone and progesterone were dissolved in ethanol and reached a final concentration of 0.15 and 0.3% ethanol in the culture medium, respectively. The same solvent concentration was present in control cultures. After the culture, germinal vesicle breakdown (GVBD) in the oocytes was observed by the appearance of a white spot on the animal pole.

All the experiments were carried out in accordance with the principles and guidelines for the use of laboratory animals and were approved by the Weizmann Institute of Science Institutional Animal Care and Use Committee.

Statistics
Data are expressed as mean ± SEM of pooled results obtained from at least three replicate cultures and two separate experiments. Statistical analysis was performed by one-way ANOVA test followed by Fisher’s protected least significant difference for multiple comparisons using StatView Program (Abacus Concepts, Berkeley, CA). Values of P < 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Is progesterone mediating LH/hCG action on resumption of meiosis in murine FEOs?
The progesterone antagonists mifepristone (RU 486; 25–50 µM) and Org 31710 (1–10 µM), did not prevent LH-triggered maturation of rat FEOs. In accordance, addition of the progesterone agonist promegestone (R5020; 0.25–1 µM) failed to simulate the action of LH and trigger the resumption of meiosis in rat FEOs (Fig. 1Go). By contrast, progesterone (10 µM) and promegestone (31 µM) were effective in stimulating the maturation of Xenopus oocytes (93 and 85% GVBD, evidenced by a white spot in the animal hemisphere, respectively, vs. 6% in the control oocytes; n = 198–223). Furthermore, mifepristone (2.5–10 µM) was unable to prevent the spontaneous resumption of meiosis, and promegestone (0.1–0.25 µM) did not overcome the mild Hx (4 mM) inhibition of spontaneous meiosis in rat CEOs (Fig. 2Go).


Figure 1
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  		FIG. 1. The effect of progesterone agonist or receptor antagonists on resumption of meiosis in rat FEOs. Preovulatory follicles were cultured for 6 h in medium alone (control) or with LH (1 µg/ml) or promegestone (0.25–1 µM). The PR antagonist mifepristone (25–50 µM) or Org 31710 (1–10 µM) was added before LH. Different superscripts indicate significant differences (P < 0.05). The number of FEOs examined is indicated on the columns.

 

Figure 2
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  		FIG. 2. The effect of progesterone agonist and PR antagonist on the maturation of rat COCs. CEOs were cultured for 6 h in medium alone (control), with Hx (4 mM) alone, or in combination with promegestone (0.1–0.25 µM) or with mifepristone (2.5–10 µM) alone as indicated. Different superscripts indicate significant difference (P < 0.05). The number of CEOs examined is indicated on the columns.

 
Similar results were obtained using mouse follicles and CEOs. The progesterone antagonist Org 31710 did not affect LH-stimulated resumption of meiosis in FEOs, and promegestone did not induce meiosis in them (Fig. 3Go). Likewise, in mouse CEOs in culture, the spontaneous maturation went undisturbed by the addition of mifepristone and was not stimulated by promegestone upon meiotic suppression by Hx (Fig. 4Go).


Figure 3
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  		FIG. 3. The effect of PR antagonist or agonist on resumption of meiosis in mouse FEOs. Preovulatory follicles were cultured for 6 h in medium alone (control) or with LH (1 µg/ml) or promegestone (0.25–1 µM). The PR antagonist Org 31710 (1–10 µM) was added to the cultures before LH. Other details are as in Fig. 1Go.

 

Figure 4
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  		FIG. 4. The effect of progesterone agonist and PR antagonist on the maturation of mouse COCs. CEOs were cultured for 6 h in medium alone (control), with Hx (4 mM) alone, in combination with promegestone (0.1–0.25 µM) or with mifepristone (2.5–10 µM) alone as indicated. Other details are as in Fig. 1Go.

 
Estrogen as a mediator of meiosis induction in murines
Using the estrogen antagonist faslodex (25–50 µM), we could not block LH-induced resumption of meiosis in rat and mouse FEOs in culture. Addition of estradiol (0.25–1 µM) failed to induce meiosis in FEOs cultured without LH (Figs 5Go).


Figure 5
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  		FIG. 5. The effect of ER antagonist on LH-stimulated resumption of meiosis in rat and mouse FEOs. Rat (A and C) or mouse (B and D) preovulatory follicles were cultured for 6 h in medium alone (control) or with LH (1 µg/ml). The ER antagonist faslodex (25–50 µM) (A and B) or estradiol (0.1–0.25 µM) (C and D) were added to the cultures as indicated. Other details are as in Fig. 1Go.

 
Using CEOs further supported the results obtained in FEOs. Faslodex failed to prevent the spontaneous maturation of rat and mouse CEOs. In concordance, estradiol did not overcome the inhibition of meiosis by Hx in CEOs of the rat and the mouse (Fig. 6Go).


Figure 6
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  		FIG. 6. The effect of estrogen and ER antagonist on the maturation of rat and mouse CEOs. Rat (A) or mouse (B) CEOs were cultured for 6 h in medium alone (control), with Hx (4 mM) alone, in combination with estradiol (0.1–0.25 µM) or with faslodex (2.5–10 µM) alone as indicated. Other details are as in Fig. 1Go.

 
Androgen and meiotic maturation in rat oocytes
Addition of the potent androgen DHT (0.25–1 µM) to rat FEOs in culture did not induce resumption of meiosis. Nevertheless, the androgen receptor (AR) antagonist flutamide exerted a dose-dependent inhibition of LH-induced resumption of meiosis (10–50 µM). But most importantly, preincubation of the follicles with DHT (0.25–1 µM) for 0.5 h before adding flutamide and LH did not affect resumption of meiosis (Fig. 7Go), suggesting that this action of the inhibitor was unrelated to its AR antagonistic activity.


Figure 7
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  		FIG. 7. The effect of testosterone receptor antagonist on LH-stimulated resumption of meiosis in rat FEOs. Preovulatory follicles were cultured for 6 h in medium alone (control) or with LH (1 µg/ml). The testosterone receptor antagonist flutamide (0.25–50 µM) was added to the cultures as indicated. In some of the experiments, DHT (0.25–1 µM) and flutamide (25 µM) were added at 30-min intervals before LH (1 µg/ml). Other details are as in Fig. 1Go.

 
In rat CEO cultures, flutamide (2.5–10 µM) did not affect their spontaneous maturation. Likewise, DHT (0.1–0.25 µM) could not overcome the inhibition of maturation by Hx. Addition of both flutamide and DHT did not affect Hx inhibition of meiosis (Fig. 8Go).


Figure 8
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  		FIG. 8. The effect of testosterone receptor antagonist on the spontaneous maturation of rat COCs. Rat COCs were cultured for 6 h in medium alone (control), with flutamide (2.5 or 10 µM), with Hx (4 mM) alone, or in combination with DHT (0.1–0.25 µM). In some of the experiments, flutamide (2.5 µM) was added to Hx (4 mM) 30 min before DHT (0.25 µM). Other details are as in Fig. 1Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was aimed to examine specifically the putative involvement of steroids in the first step of meiotic resumption, GVBD. In previous studies, meiotic resumption in mammals has been examined with two aims in mind: whether any of the steroids serve as follicular inhibitors of meiosis and whether they mediate the gonadotropin action in the stimulation of meiosis like in amphibians and fishes. Studies with isolated mammalian oocytes maturing spontaneously led to conflicting results. In some studies, testosterone attenuated the spontaneous meiotic resumption, and in others, estradiol, testosterone, or progesterone potentiated the inhibitory action of dibutyryl cAMP or forskolin. But in other studies, steroids did not affect spontaneous maturation consistently (5, 6, 7, 20). The search for meiotic inhibitors among steroids did not lead to the identification of a steroid as such, yet it was clear that steroids can modulate meiosis under some experimental conditions (reviewed in Refs. 8 and 9). Therefore, it was doubted whether steroids serve a physiological role as inhibitors of meiotic resumption.

The increase in follicular steroidogenesis after gonadotropic stimulation allows the participation of steroids, and especially of progesterone, in the transmission of the gonadotropic signal to the oocyte. Addition of steroids to the medium could not stimulate the maturation of bovine, porcine, or rat FEOs (10, 12). Furthermore, inhibition of follicular steroidogenesis, which reduced progesterone, estrogen, and androgen, did not impair the ability of LH to induce resumption of meiosis (14, 15). Similar results were obtained by Moor (13) in the sheep.

Recently, using denuded oocytes isolated from mice that were not primed with eCG and cultured in the presence of 3-isobutyl-1-methylxanthine, Gill et al. (16) have reported stimulation of the resumption of meiosis by testosterone. They have concluded that androgen produced by preovulatory follicles induces oocyte maturation. In a subsequent study, Jamnongjit et al. (17), using several oocyte culture models, including preovulatory follicles primed with eCG, found that each one of the three steroid groups, progesterone, estrogen, and testosterone, was able to induce the resumption of meiosis acting through its own receptor, because their effect could be blocked by its receptor antagonist. In our study, using cultures of rat and mouse preovulatory follicles and COCs (in which spontaneous maturation was prevented by Hx), we did not find stimulation of oocyte maturation by the progesterone agonist promegestone or estradiol and DHT. Furthermore, the progesterone receptor (PR) antagonists mifepristone or Org 31710 and the estrogen receptor (ER) antagonist faslodex did not alter LH-triggered resumption of meiosis in FEOs or spontaneous maturation of COCs in the rat or the mouse. Only the addition to rat follicles (not tested in the mouse) of the AR antagonist flutamide caused dose-dependent inhibition of LH-induced GVBD. Preincubation with DHT did not alter this action of flutamide, suggesting that its inhibitory effect was not mediated through AR. Flutamide did not affect the spontaneous maturation of rat COCs. The steroidogenic response to LH/hCG stimulation of ovulation in the rat (and most likely also in the mouse) is suppression of androgen production (21) and CYP17A activity (22). Finally, the fertility of androgen-resistant (Tfm/Tfm) and AR-deficient mice, albeit reduced and leading to premature ovarian failure, argues against a critical role of androgen in resumption of meiosis, despite its regulatory role in several genes regulating follicle development (23, 24). All these findings make follicular androgen a most unlikely mediator of LH action on oocyte maturation.

In rats and mice, both progesterone and estrogen are transiently elevated at a critical time before meiosis after LH/hCG stimulation (25, 26, 27, 28). Our present studies with rat and mouse FEOs and CEOs did not provide any support for the suggested participation of steroids in mediating the gonadotropin effect on meiotic resumption in mammals (4, 29, 30). The divergent results reported (16, 17) may be related to the experimental procedures used. Notable is the use of animals or oocytes that were not primed previously with gonadotropins. In this case, the effect of the steroid may be associated with promotion of meiotic competence (see below). Clearly, normal follicle growth and response of preovulatory follicles to LH/hCG, including resumption of meiosis, are contingent on previous exposure to FSH (31, 32). Therefore, oocytes from follicles that were not primed with gonadotropin can hardly represent an appropriate model to investigate the physiological regulation of meiotic resumption. Nevertheless, the same laboratory obtained similar results by adding progesterone or testosterone to eCG-primed preovulatory follicles (17), suggesting that eCG priming cannot explain all the differences obtained.

Additional experiments do not support the suggested role of steroids in resumption of meiosis. Administration of inhibitors of steroid synthesis before the ovulatory stimulus to macaque monkeys (33) did not affect resumption of meiosis, despite impairing follicle rupture and subsequent development. Furthermore, mice lacking both isoforms of PR (PR-A and PR-B), PR knockout (PRKO) mice, show normal follicular growth and luteinization and fail to ovulate, but the oocytes mature nevertheless (34). Aromatase knockout (ArKO) mice do not respond to the superovulatory protocol by follicle rupture, but oocytes isolated from eCG-treated mice matured in vitro without estrogen supplementation and showed developmental competence similar to wild-type animals (35). Likewise, ERß knockout (ßERKO) mice showing reduced fertility have large numbers of trapped mature oocytes in the ovary (36), as are those of {alpha}- and ßERKO follicles grown and ovulated in vitro (37).

Cumulatively, these studies in rodents argue against a mediatory role of steroids in the stimulation of oocyte maturation. Yet, in several other mammalian species, resumption of meiosis and its progression to metaphase II seem to be steroid dependent. Thus, in ovine follicles, inhibition of steroidogenesis impaired the progression to metaphase II, although it did not block GVBD (38). But inhibition of steroidogenesis in porcine (39, 40) and bovine (41) CEOs in culture prevented GVBD, which could be restored by the addition of progesterone in porcine, but not bovine, oocytes. Conversely, negative effects of steroids on meiosis were reported in the pig (42). The involvement of steroids in the regulation of meiosis in porcine and bovine follicles awaits further detailed examination. It is possible that the difference in the need for steroid support for maturation between rodents and the ovine, bovine, and porcine species is related to the length of the period between LH/hCG stimulation and GVBD in these two groups of mammals. In rodents GVBD is completed within 3–4 h, whereas it takes approximately 10–11 h in the sheep, 10–12 h in the cow, and 17–18 h in the pig (43). Such long latent periods may require steroid support for maintaining oocyte wellbeing and ability to undergo meiotic maturation.

Steroids play an important role in follicular development and ovarian function (31, 44). Studies with transgenic mouse models extended these studies and confirmed the requirement of steroids in normal ovarian activity (45, 46, 47, 48, 49). Nevertheless, as already indicated, oocytes obtained from ArKO (35), PRKO (34), and ERKO (36, 37) mice are able to mature in vitro.

Steroids were implicated, in addition to resumption of meiosis, also in later stages of oocyte growth in the development of meiotic competence and in the acquisition of fertilizability and developmental competence, the latter often referred to as cytoplasmic maturation.

Meiotic competence, defined as the ability of oocytes to resume maturation (undergo GVBD) spontaneously in vitro, is reached between 15–20 d of age in mice (50, 51) and 20–25 d in rats (52), coinciding with antrum formation and the final stages of oocyte growth. In mammals with larger follicles, meiotic competence is reached only in antral follicles and correlating with follicle size (53). In mouse oocytes, acquisition of meiotic competence was associated with the phosphatase cdc25b, required for the activation of cyclin-dependent kinase-1 (CDK1, p34cdc2) (54), a component of maturation-promoting factor (MPF) (55). Hypophysectomy of 15-d-old rats, before the acquisition of meiotic competence, resulted in a marked reduction in the number and ratio of competent oocytes. This effect could be reversed by administration of eCG and FSH but not LH. This effect of FSH was partially mediated by estrogen (52, 56). Large oocytes from immature hypogonadal mice (hpg) that are deficient in GnRH spontaneously resumed meiosis in vitro. Yet, the number of competent oocytes in hpg mice was reduced markedly, and exogenous gonadotropin increased the number of competent oocytes (57). Accordingly, FSH increased follicular growth in hpg mice (58). Studies with ArKO mice or preantral follicles cultured with an aromatase inhibitor resulted in normal follicular growth, and the oocytes matured in vitro (35, 59, 60). This may be related to the treatment with gonadotropin in these studies. Whether estrogen is required for meiotic competence and whether the oocytes obtained in estrogen-depleted follicles are capable of normal development to live offspring remains to be examined.

Developmental competence from small antral follicles is limited compared with oocytes from large follicles (61, 62, 63, 64, 65). Studies with porcine, ovine, bovine, and primate oocytes (38, 66, 67, 68, 69, 70, 71) implicated steroids in developmental competence, suggesting granulosa cell-oocyte interactions through steroids, among other factors, in this process (for review see Ref. 72).

In conclusion, the suppression of follicular steroid production or action does not abolish the stimulation of meiosis by gonadotropins, and steroids are unable to fully substitute LH in stimulating meiosis, at least in rodents. Thus, in stark contrast to lower vertebrates, in mammals, steroids do not seem to serve as an obligatory signal by which the somatic cells of the follicle transmit the gonadotropic stimulation of meiosis to the oocyte. We suggest that this may be associated with the evolution of hierarchical follicle growth in mammals and ovulation of one or a few oocytes each reproductive cycle. The coexistence of follicles and oocytes at different stages of development and their cyclic exposure to high levels of steroids presumably precludes their use in mammals as a signal for the resumption of meiosis. This may have contributed to the evolution of more elaborate paracrine mechanisms for regulating the resumption of meiosis, such as the recently described local epidermal growth factor-like factors (73, 74). That steroids do not serve as mediators of gonadotropins in meiosis should not invalidate their role in the acquisition of meiotic and developmental competence by the oocyte, cumulus expansion, and rupture of the follicle at ovulation.


   Acknowledgments
 
We thank Dr. N. Dekel and Dr. Y. Orly for helpful discussions; Dr. H. Garty, Dr. C. Asher, and Dr. N. Daskal for their advice and supply of Xenopus ovaries; Dr. R. G. J. M. Hanssen of Organon for Org 31710; Dr. A. F. Parlow and the National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Pituitary Program, National Institute of Child Health and Human Development, for the gonadotropins.


   Footnotes
 
This work was supported by the Maria and Bernhard Zondek Hormone Research Fund and The Israel Sciences Foundation (Grant 436/05).

The authors have nothing to disclose.

First Published Online May 24, 2007

Abbreviations: ArKO, Aromatase knockout; CEO, cumulus-enclosed oocyte; COC, cumulus-oocyte complex; DHT, dihydrotestosterone; eCG, equine chorionic gonadotropin; FEO, follicle-enclosed oocyte; GVBD, germinal vesicle breakdown; Hx, hypoxanthine; MSBH, modified Bart’s solution; Org 31710, Organon 31710; PR, progesterone receptor.

Received April 5, 2007.

Accepted for publication May 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nagahama Y, Yoshikuni M, Yamashita M, Tokumoto T, Katsu Y 1995 Regulation of oocyte growth and maturation in fish. Curr Top Dev Biol 30:103–145[Medline]
  2. Maller JL 1998 Recurring themes in oocyte maturation. Biol Cell 90:453–460[CrossRef][Medline]
  3. Maller JL, Schwab MS, Gross SD, Taieb FE, Roberts BT, Tunquist BJ 2002 The mechanism of CSF arrest in vertebrate oocytes. Mol Cell Endocrinol 187:173–178[CrossRef][Medline]
  4. Hammes SR 2004 Steroids and oocyte maturation: a new look at an old story. Mol Endocrinol 18:769–775[Abstract/Free Full Text]
  5. Rice C, McGaughey RW 1981 Effect of testosterone and dibutyryl cAMP on the spontaneous maturation of pig oocytes. J Reprod Fertil 62:245–256[Abstract/Free Full Text]
  6. Eppig JJ, Freter RR, Ward-Bailey PF, Schultz RM 1983 Inhibition of oocyte maturation in the mouse: participation of cAMP, steroid hormones, and a putative maturation-inhibitory factor. Dev Biol 100:39–49[CrossRef][Medline]
  7. Batten BE, Roh SI, Kim MH 1989 Effects of progesterone and a progesterone antagonist (RU486) on germinal vesicle breakdown in the mouse. Anat Rec 223:387–392[CrossRef][Medline]
  8. Eppig JJ, Downs SM 1984 Chemical signals that regulate mammalian oocyte maturation. Biol Reprod 30:1–11[Abstract]
  9. Tsafriri A 1985 The control of meiotic maturation in mammals. In: Biology of fertilization. New York: Academic Press; 221–252
  10. Tsafriri A, Lindner HR, Zor U, Lamprecht SA 1972 In-vitro induction of meiotic division in follicle-enclosed rat oocytes by LH, cyclic AMP and prostaglandin E2. J Reprod Fertil 31:39–50[Abstract/Free Full Text]
  11. Lindner HR, Tsafriri A, Lieberman ME, Zor U, Koch Y, Bauminger S, Barnea A 1974 Gonadotropin action on cultured Graafian follicles: induction of maturation division of the mammalian oocyte and differentiation of the luteal cell. Recent Prog Horm Res 30:79–138[Medline]
  12. Foote WD, Thibault C 1969 Researches experimentales sur la maturation in vitro des ovocytes de truie et the veau. Annls Biol Anim Biochim Biophys 9:329–349
  13. Moor RM 1978 Role of steroids in the maturation of ovine oocytes. Annls Biol amin Biochim Biophys 18:477–482
  14. Lieberman ME, Tsafriri A, Bauminger S, Collins WP, Ahre’n K, Lindner HR 1976 Oocyte meiosis in cultured rat follicles during inhibition of steroidogenesis. Acta Endocrinol 83:151–157[Medline]
  15. Billig H, Hillensjo T, Tsafriri A, Magnusson C, Brodie A 1983 Nuclear maturation of follicle-enclosed rat oocytes during inhibition of steroidogenesis. Gamete Res 8:79–86[CrossRef]
  16. Gill A, Jamnongjit M, Hammes SR 2004 Androgens promote maturation and signaling in mouse oocytes independent of transcription: a release of inhibition model for mammalian oocyte meiosis. Mol Endocrinol 18:97–104[Abstract/Free Full Text]
  17. Jamnongjit M, Gill A, Hammes SR 2005 Epidermal growth factor receptor signaling is required for normal ovarian steroidogenesis and oocyte maturation. Proc Natl Acad Sci USA 102:16257–16262[Abstract/Free Full Text]
  18. Downs SM 1993 Purine control of mouse oocyte maturation: evidence that nonmetabolized hypoxanthine maintains meiotic arrest. Mol Reprod Dev 35:82–94[CrossRef][Medline]
  19. Williams JA, McChesney DJ, Calayag MC, Lingappa VR, Logsdon CD 1988 Expression of receptors for cholecystokinin and other Ca2+-mobilizing hormones in Xenopus oocytes. Proc Natl Acad Sci USA 85:4939–4943[Abstract/Free Full Text]
  20. Schultz RM, Montgomery RR, Ward-Bailey PF, Eppig JJ 1983 Regulation of oocyte maturation in the mouse: possible roles of intercellular communication, cAMP, and testosterone. Dev Biol 95:294–304[CrossRef][Medline]
  21. Suzuki K, Tamaoki B 1983 Acute decrease by human chorionic gonadotropin of the activity of preovulatory ovarian 17{alpha}-hydroxylase and C-17-C-20 lyase is due to decrease of microsomal cytochrome P-450 through de novo synthesis of ribonucleic acid and protein. Endocrinology 113:1985–1991[Abstract/Free Full Text]
  22. Tsafriri A, Eckstein B 1986 Changes in follicular steroidogenic enzymes following the preovulatory surge of gonadotropins and experimentally-induced atresia. Biol Reprod 34:783–787[Abstract]
  23. Lyon MF, Glenister PH 1980 Reduced reproductive performance in androgen-resistant Tfm/Tfm female mice. Proc R Soc Lond B Biol Sci 208:1–12[Medline]
  24. Shiina H, Matsumoto T, Sato T, Igarashi K, Miyamoto J, Takemasa S, Sakari M, Takada I, Nakamura T, Metzger D, Chambon P, Kanno J, Yoshikawa H, Kato S 2006 Premature ovarian failure in androgen receptor-deficient mice. Proc Natl Acad Sci USA 103:224–229[Abstract/Free Full Text]
  25. Lieberman ME, Barnea A, Bauminger S, Tsafriri A, Collins WP, Lindner HR 1975 LH effect on the pattern of steroidogenesis in cultured Graafian follicles of the rat: dependence on macromolecular synthesis. Endocrinology 96:1533–1542[Abstract/Free Full Text]
  26. Hillensjo T, Dekel N, Ahren K 1976 Effects of gonadotrophins on the cumulus oophorus of isolated rat Graafian follicles. Acta Physiol Scand 96:558–568[Medline]
  27. Uilenbroek JT 1985 Effect of LH on progesterone and oestradiol production in vivo and in vitro by preovulatory rat follicles. J Reprod Fertil 74:303–310[Abstract/Free Full Text]
  28. Su YQ, Nyegaard M, Overgaard MT, Qiao J, Giudice LC 2006 Participation of mitogen-activated protein kinase in luteinizing hormone-induced differential regulation of steroidogenesis and steroidogenic gene expression in mural and cumulus granulosa cells of mouse preovulatory follicles. Biol Reprod 75:859–867[Abstract/Free Full Text]
  29. Jamnongjit M, Hammes SR 2005 Oocyte maturation: the coming of age of a germ cell. Semin Reprod Med 23:234–241[CrossRef][Medline]
  30. Jamnongjit M, Hammes SR 2006 Ovarian steroids: the good, the bad, and the signals that raise them. Cell Cycle 5:1178–1183[Medline]
  31. Richards JS 1994 Hormonal control of gene expression in the ovary. Endocr Rev 15:725–751[Abstract/Free Full Text]
  32. Richards JS, Russell DL, Ochsner S, Espey LL 2002 Ovulation: new dimensions and new regulators of the inflammatory-like response. Annu Rev Physiol 64:69–92[CrossRef][Medline]
  33. Hibbert ML, Stouffer RL, Wolf DP, Zelinski-Wooten MB 1996 Midcycle administration of a progesterone synthesis inhibitor prevents ovulation in primates. Proc Natl Acad Sci USA 93:1897–1901[Abstract/Free Full Text]
  34. Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery Jr CA, Shyamala G, Conneely OM, O’Malley BW 1995 Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 9:2266–2278[Abstract/Free Full Text]
  35. Huynh K, Jones G, Thouas G, Britt KL, Simpson ER, Jones ME 2004 Estrogen is not directly required for oocyte developmental competence. Biol Reprod 70:1263–1269[Abstract/Free Full Text]
  36. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor ß. Proc Natl Acad Sci USA 95:15677–15682[Abstract/Free Full Text]
  37. Emmen JM, Couse JF, Elmore SA, Yates MM, Kissling GE, Korach KS 2005 In vitro growth and ovulation of follicles from ovaries of estrogen receptor (ER){alpha} and ERß null mice indicate a role for ERß in follicular maturation. Endocrinology 146:2817–2826[CrossRef][Medline]
  38. Moor RM, Polge C, Willadsen SM 1980 Effect of follicular steroids on the maturation and fertilization of mammalian oocytes. J Embryol Exp Morphol 56:319–335[Medline]
  39. Yamashita Y, Shimada M, Okazaki T, Maeda T, Terada T 2003 Production of progesterone from de novo-synthesized cholesterol in cumulus cells and its physiological role during meiotic resumption of porcine oocytes. Biol Reprod 68:1193–1198[Abstract/Free Full Text]
  40. Shimada M, Yamashita Y, Nishibori M 2005 Transcripts encoding the enzymes that convert acetyl-CoA to cholesterol are induced in cumulus cells and are essential for progesterone biosynthesis and meiotic resumption of porcine oocytes. Jpn J Reprod Endocrinol 10:15–20
  41. Wang HF, Isobe N, Kumamoto K, Yamashiro H, Yamashita Y, Terada T 2006 Studies of the role of steroid hormone in the regulation of oocyte maturation in cattle. Reprod Biol Endocrinol 4:4–13[CrossRef][Medline]
  42. Dode MA, Graves C 2002 Involvement of steroid hormones on in vitro maturation of pig oocytes. Theriogenology 57:811–821[CrossRef][Medline]
  43. Hunter RHF 2003 Physiology of the Graafian follicle and ovulation. Cambridge, UK: Cambridge University Press; 232
  44. Hirshfield AN 1991 Development of follicles in the mammalian ovary. Int Rev Cytol 124:43–101[Medline]
  45. Hasegawa T, Zhao L, Caron KM, Majdic G, Suzuki T, Shizawa S, Sasano H, Parker KL 2000 Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice. Mol Endocrinol 14:1462–1471[Abstract/Free Full Text]
  46. Fisher CR, Graves KH, Parlow AF, Simpson ER 1998 Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proc Natl Acad Sci USA 95:6965–6970[Abstract/Free Full Text]
  47. Britt KL, Drummond AE, Cox VA, Dyson M, Wreford NG, Jones ME, Simpson ER, Findlay JK 2000 An age-related ovarian phenotype in mice with targeted disruption of the Cyp 19 (aromatase) gene. Endocrinology 141:2614–2623[Abstract/Free Full Text]
  48. Couse JF, Hewitt SC, Korach K 2006 Steroid receptors in the ovary and uterus In: Knobil E, Neill JD, eds. Physiology of reproduction. 3rd ed. New York: Raven Press; 593–678
  49. Conneely OM, Mulac-Jericevic B, Lydon JP, De Mayo FJ 2001 Reproductive functions of the progesterone receptor isoforms: lessons from knock-out mice. Mol Cell Endocrinol 179:97–103[CrossRef][Medline]
  50. Szybek K 1972 In-vitro maturation of oocytes from sexually immature mice. J Endocrinol 54:527–528[Abstract/Free Full Text]
  51. Sorensen RA, Wassarman PM 1976 Relationship between growth and meiotic maturation of the mouse oocyte. Dev Biol 50:531–536[CrossRef][Medline]
  52. Bar-Ami S, Tsafriri A 1981 Acquisition of meiotic competence in the rat: role of gonadotrophin and estrogen. Gamete Res 4:463–472[CrossRef]
  53. Motlik J, Crozet N, Fulka J 1984 Meiotic competence in vitro of pig oocytes isolated from early antral follicles. J Reprod Fertil 72:323–328[Abstract/Free Full Text]
  54. Lincoln AJ, Wickramasinghe D, Stein P, Schultz RM, Palko ME, De Miguel MP, Tessarollo L, Donovan PJ 2002 Cdc25b phosphatase is required for resumption of meiosis during oocyte maturation. Nat Genet 30:446–449[CrossRef][Medline]
  55. Gautier J, Norbury C, Lohka M, Nurse P, Maller J 1988 Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+. Cell 54:433–439[CrossRef][Medline]
  56. Bar-Ami S, Nimrod A, Brodie AM, Tsafriri A 1983 Role of FSH and oestradiol-17ß in the development of meiotic competence in rat oocytes. J Steroid Biochem 19:965–971[CrossRef][Medline]
  57. Schroeder AC, Eppig JJ 1989 Developmental capacity of mouse oocytes that undergo maturation in vitro: effect of the hormonal state of the oocyte donor. Gamete Res 24:81–92[CrossRef][Medline]
  58. Allan CM, Wang Y, Jimenez M, Marshan B, Spaliviero J, Illingworth P, Handelsman DJ 2006 Follicle-stimulating hormone increases primordial follicle reserve in mature female hypogonadal mice. J Endocrinol 188:549–557[Abstract/Free Full Text]
  59. Hu Y, Cortvrindt R, Smitz J 2002 Effects of aromatase inhibition on in vitro follicle and oocyte development analyzed by early preantral mouse follicle culture. Mol Reprod Dev 61:549–559[CrossRef][Medline]
  60. Fatum M, Gyo Y, Diana P, Laufer N, Simon A 2006 Is estradiol mandatory for an adequate follicular and embryo development? A mouse model using aromatase inhibitor (anastrozole). J Assist Reprod Genet 23:407–412[CrossRef][Medline]
  61. Eppig JJ, Schroeder AC, O’Brien MJ 1992 Developmental capacity of mouse oocytes matured in vitro: effects of gonadotrophic stimulation, follicular origin and oocyte size. J Reprod Fertil 95:119–127[Abstract/Free Full Text]
  62. Pavlok A, Kopecny V, Lucas-Hahn A, Niemann H 1993 Transcriptional activity and nuclear ultrastructure of 8-cell bovine embryos developed by in vitro maturation and fertilization of oocytes from different growth categories of antral follicles. Mol Reprod Dev 35:233–243[Medline]
  63. Lonergan P, Monaghan P, Rizos D, Boland MP, Gordon I 1994 Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro. Mol Reprod Dev 37:48–53[CrossRef][Medline]
  64. Crozet N, Ahmed-Ali M, Dubos MP 1995 Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro. J Reprod Fertil 103:293–298[Abstract/Free Full Text]
  65. Cognie Y, Benoit F, Poulin N, Khatir H, Driancourt MA 1998 Effect of follicle size and of the FecB Booroola gene on oocyte function in sheep. J Reprod Fertil 112:379–386[Abstract/Free Full Text]
  66. Osborn JC, Moor RM 1983 The role of steroid signals in the maturation of mammalian oocytes. J Steroid Biochem 19:133–137[Medline]
  67. Mattioli M, Galeati G, Bacci ML, Seren E 1988 Follicular factors influence oocyte fertilizability by modulating the intercellular cooperation between cumulus cells and oocyte. Gamete Res 21:223–232[CrossRef][Medline]
  68. Zelinski-Wooten MB, Hess DL, Baughman WL, Molskness TA, Wolf DP, Stouffer RL 1993 Administration of an aromatase inhibitor during the late follicular phase of gonadotropin-treated cycles in rhesus monkeys: effects on follicle development, oocyte maturation, and subsequent luteal function. J Clin Endocrinol Metab 76:988–995[Abstract]
  69. Zelinski-Wooten MB, Hess DL, Wolf DP, Stouffer RL 1994 Steroid reduction during ovarian stimulation impairs oocyte fertilization, but not folliculogenesis, in rhesus monkeys. Fertil Steril 61:1147–1155[Medline]
  70. Borman SM, Chwalisz K, Stouffer RL, Zelinski-Wooten MB 2003 Chronic low-dose antiprogestin impairs preimplantation embryogenesis, but not oocyte nuclear maturation or fertilization in rhesus monkeys. Steroids 68:1041–1051[CrossRef][Medline]
  71. Zheng P, Si W, Bavister BD, Yang J, Ding C, Ji W 2003 17ß-Estradiol and progesterone improve in-vitro cytoplasmic maturation of oocytes from unstimulated prepubertal and adult rhesus monkeys. Hum Reprod 18:2137–2144[Abstract/Free Full Text]
  72. Moor RM, Dai Y, Lee C, Fulka Jr J 1998 Oocyte maturation and embryonic failure. Hum Reprod Update 4:223–236[Abstract/Free Full Text]
  73. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M 2004 EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 303:682–684[Abstract/Free Full Text]
  74. Ashkenazi H, Cao X, Motola S, Popliker M, Conti M, Tsafriri A 2005 Epidermal growth factor family members: endogenous mediators of the ovulatory response. Endocrinology 146:77–84[Abstract/Free Full Text]



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