Nongenomic steroid-triggered oocyte maturation: of mice and frogs

Abstract

Luteinizing hormone (LH) mediates many important processes in ovarian follicles, including cumulus cell expansion, changes in gap junction expression and activity, sterol and steroid production, and the release of paracrine signaling molecules. All of these functions work together to trigger oocyte maturation (meiotic progression) and subsequent ovulation. Many laboratories are interested in better understanding both the extra-oocyte follicular processes that trigger oocyte maturation, as well as the intra-oocyte molecules and signals that regulate meiosis. Multiple model systems have been used to study LH-effects in the ovary, including fish, frogs, mice, rats, pigs, and primates. Here we provide a brief summary of oocyte maturation, focusing primarily on steroid-triggered meiotic progression in frogs and mice. Furthermore, we present new studies that implicate classical steroid receptors rather than alternative non-classical membrane steroid receptors as the primary regulators of steroid-mediated oocyte maturation in both of these model systems.

Figure 1

Model for gonadotropin-mediated oocyte maturation in Xenopus Laevis. Prior to ovulation, oocytes are held in meiotic arrest in prophase I by constitutive Gα_s and Gβγ signaling that stimulates adenylyl cyclase to elevate intracellular cAMP. Part of this inhibitory G protein signaling comes from GPR3; however, alternative mechanisms of G protein activation are likely present as well [25]. MNAR also contributes to the constitutive Gβγ signaling via direct interactions with Gβ [35]. Upon gonadotropin stimulation, proteinases might degrade and inactivate GPR3, thus enhancing but not initiating oocyte maturation. The most important signal that triggers oocyte maturation is gonadotropin-induced testosterone production, which acts via classical ARs and possibly MNAR to attenuate Gβγ signaling. These events lead to decreased intracellular cAMP, resulting in increased MOS protein translation. MOS then activates the MAPK cascade as well as CKD1, which in turn promote increased MOS production in a powerful feedback loop that is regulated at least in part by Paxillin. This all-or-none response ultimately leads to meiotic progression.

Figure 2

Xenopus mPR regulates oocyte maturation independent of steroid binding A) COS cells were transfected with pcDNA3.1, an expression vector encoding Myc-tagged Xenopus mPRβ (a generous gift from James Maller) [21], or a cDNA encoding Myc-tagged classical Xenopus AR. Binding assays with increasing amounts of radiolabeled testosterone were performed as described [20]. Note that, while significant testosterone binding to the classical AR was observed, testosterone binding to cells expressing mPRβ was no higher than binding to mock-transfected cells. Expression levels were confirmed by Western blot using an anti-Myc antibody. B) Oocytes were injected with 50.6 nl of either 10 mM Hepes, nonspecific rabbit IgG (400 ng/ul), or anti-mPR immunoglobulin (1000 ng/ul). Oocytes were then treated with 300 nM testosterone or 300 nM progesterone. Oocyte maturation was quantified by percent GVBD (white spot on the animal pole) after 12 hours. Note that injection of the anti-mPR antibody significantly lowered both progesterone and testosterone-induced oocyte maturation, despite the lack of testosterone binding (A). Injection of the anti-mPR antibody also reduced MAPK and CDK1 activity (data not shown). Twenty oocytes were used for each point, and similar experiments were performed three times with nearly identical results.

Figure 2

Xenopus mPR regulates oocyte maturation independent of steroid binding A) COS cells were transfected with pcDNA3.1, an expression vector encoding Myc-tagged Xenopus mPRβ (a generous gift from James Maller) [21], or a cDNA encoding Myc-tagged classical Xenopus AR. Binding assays with increasing amounts of radiolabeled testosterone were performed as described [20]. Note that, while significant testosterone binding to the classical AR was observed, testosterone binding to cells expressing mPRβ was no higher than binding to mock-transfected cells. Expression levels were confirmed by Western blot using an anti-Myc antibody. B) Oocytes were injected with 50.6 nl of either 10 mM Hepes, nonspecific rabbit IgG (400 ng/ul), or anti-mPR immunoglobulin (1000 ng/ul). Oocytes were then treated with 300 nM testosterone or 300 nM progesterone. Oocyte maturation was quantified by percent GVBD (white spot on the animal pole) after 12 hours. Note that injection of the anti-mPR antibody significantly lowered both progesterone and testosterone-induced oocyte maturation, despite the lack of testosterone binding (A). Injection of the anti-mPR antibody also reduced MAPK and CDK1 activity (data not shown). Twenty oocytes were used for each point, and similar experiments were performed three times with nearly identical results.

Figure 3

Oocytes from PR null mice do not mature in response to progestin. Follicles were isolated as described [56] from the ovaries of 4-week old PR null mice or heterozygotic littermates (generous gifts from O. Conneely, Baylor College of Medicine). 10-12 follicles were used for each condition, and were treated with 0.1% ethanol (ETOH), the progestin R5020 (250 nM), or estradiol (250 nM). After 8 hours, follicles were quickly dissected using 30 gauge needles, and oocytes scored for percent GVBD (visual loss of nuclear membrane). Note that oocytes from the heterozygotic mice matured in response to both estradiol and R5020, while oocytes from the PR null mice only responded to estradiol. This suggests that classical PRs are regulating progestin-induced oocyte maturation in mice. The graph represents the average of two different experiments performed by two independent investigators. Each bar represents the average +/- SD (p=0.03 when comparing responses to R5020 by students t-test).