Roles of progesterone receptor membrane component 1 and membrane progestin receptor alpha in regulation of zebrafish oocyte maturation

Abstract

Although previous studies suggest membrane progesterone receptor alpha (mPRα/Paqr7) mediates 17, 20β-dihydroxy-4-pregnen-3-one (DHP) induction of oocyte maturation (OM) in zebrafish, critical information needed to establish mPRα as the receptor mediating OM is lacking. The relative potencies of progestins and specific mPRα agonists in inducing OM matched their relative binding affinities for zebrafish mPRα, supporting its role in OM. Microinjection of pertussis toxin blocked DHP induction of OM and the progestin-induced decrease in cyclic AMP levels, suggesting mPRα activates an inhibitory G protein (Gi). Microinjection of morpholino antisense oligonucleotides to zebrafish pgrmc1 blocked induction of OM by DHP which was accompanied by decreased levels of Pgrmc1 and mPRα on the oocyte plasma membranes. Similarly, treatment of denuded oocytes with a PGRMC1 inhibitor, AG205, blocked the gonadotropin-induced increase in plasma membrane mPRα levels and attenuated DHP induction of OM. Co-incubation with two inhibitors of epidermal growth factor Erbb2, ErbB2 inhibitor II and AG 879, prevented induction of OM by DHP, indicating the likely involvement of Erbb2 in mPRα-mediated signaling. Treatment with AG205 reversed the inhibitory effects of the Erbb2 inhibitors on OM and also inhibited insulin-like growth factor-1 induction of OM. Close associations between Pgrmc1 and mPRα, and between Pgrmc1 and Erbb2 were detected in zebrafish oocytes with in situ proximity ligation assays. The results suggest progestin induction of OM in zebrafish is mediated through an mPRα/Gi/Erbb2 signaling pathway that requires Pgrmc1 for expression of mPRα on oocyte membranes and that Pgrmc1 also is required for induction of OM through Erbb2.

Keywords: AG205; Adaptor protein; Erbb2; IGF-1; Oocyte maturation; Pgrmc1; Zebrafish; mPRα.

Fig. 1.

Comparison of the activities of various progestins in the OM bioassay and their binding affinities to zebrafish mPRα. (A) Dose-response relationships of progestins and testosterone for induction of maturation of follicle-enclosed oocytes in the zebrafish OM in vitro bioassay. OM was assessed by GVBD (disappearance of germinal vesicle). DHP, Org OD-02–0 (02–0, mPR agonist), Org OD-13–0 (13–0, mPR agonist), R5020 (nPR agonist), and testosterone, were tested over the concentration range of 10^–10–10^–6 M. Each treatment group in experiments A and C consisted of three replicate wells of 20 oocytes per well. The entire experiments were repeated three times and similar results were obtained on each occasion. The data represent means ± S.E.M. (B) Competitive binding of DHP, Org OD 02–0, R5020 and Org OD 13–0 over the concentration range of 10^–10–10^–5 M to plasma membrane preparations of MDA-MB-231 cells stably transfected with zebrafish mPRα, expressed as a percentage of maximum specific DHP binding. Each value is the mean of triplicate estimations (see Supplementary Fig. 2 for statistical comparisons between treatment groups). (C) Effects of co-incubation of 5 nM DHP with RU486 at three different concentrations (1 μM, 0.1 μM, and 0.05 μM) on the percent GVBD of denuded oocytes in the OM bioassay. Results were analyzed by oneway ANOVA followed by the Bonferroni test. Different letters denote significant differences between the treatments groups (p < 0.05) in the post hoc test.

Fig. 2.

Effects of pertussis toxin treatments on DHP induction of OM (A) and DHP downregulation of cAMP production (B). (A) Effects of microinjection with activated pertussis toxin (aPTX 0.5 μg/μl) or heat-inactivated PTX (iPTX) on the maturation response of zebrafish follicle-enclosed oocytes to 5 nM DHP in the in vitro OM bioassay. OM was assessed by GVBD (disappearance of germinal vesicle). Mature vitellogenic oocytes of diameter ~ 550 μm were injected with 1 nl PTX or saline (Sal) control (< 1% of oocyte volume). An entire experiment was conducted on oocytes from a single donor. The experiment was repeated seven times and the results pooled. The total number of oocytes in the vehicle-, DHP- and aPTX-treated groups was over 100, whereas the number of oocytes in the other treatment groups ranged from 31 to 39. (B). Effects of pre-treatment of oocyte membranes with aPTX or iPTX for 3 h on cAMP production in response to treatment with 10 nM Org OD 02–0. Production of cAMP in response to 5 nM DHP alone was included as a positive control. All data represent means ± S.E.M. Results were analyzed by two-way ANOVA followed by the Bonferroni test. Significant interactive effects of aPTX in the presence of DHP on GVBD and cAMP production were detected in two-way ANOVAs, which was predicted because aPTX blocked OM and the decrease in cAMP levels in response to DHP. Different letters denote significant differences between the treatment groups (p < 0.05) in the post hoc test.

Fig. 3.

Involvement of Pgrmcl in DHP induction of OM. (A) Effects 3hr preincubation with AG205 on DHP- and Org OD 02–0-induced OM of denuded full-grown oocytes in the in vitro bioassay. (B) Effects of a 3 h co-treatment with DHP and AG205 (preinc 3 h) and subsequent treatment with DHP or vehicle alone for another 3 h (3 h rescue) on OM of denuded full-grown oocytes in the in vitro bioassay. (C) Effects of DHP and Org OD 02–0 on OM of denuded full- grown oocytes co-treated with IgG or the zebrafish Pgrmcl antibody (Pgrmcl ab, 1:300) in the in vitro bioassay. OM was assessed by GVBD (disappearance of germinal vesicle). All data represent means ± S.E.M (n = 9). All the experiments were repeated three times and similar results were obtained on each occasion. (A) Results were analyzed by two-way ANOVA followed by the Bonferroni test. Significant interactive effects of AG205 in the presence of DHP and Org 02 on GVBD were detected in a two-way ANOVA, which was predicted because AG205 decreased the OM response to the progestins. (B and C) Results were analyzed by one-way ANOVA followed by the Bonferroni test. Different letters denote significant differences between the treatments groups (p < 0.05), in the post hoc test.

Fig. 4.

Effects of co-incubation with Pgrmcl inhibitor, AG205, on hCG upregulation of plasma membrane expression of mPRα. (A) Representative Western blot of mPRα expression on ovarian membranes of zebrafish oocytes after incubation for 6h with 10.I.U. hCG/ml (H), and co-incubation with 10 I.U. hCG/ ml and 50 μM AG205 (HA), or no treatment controls (V), (N = 3). (B) The bar graph shows the relative densitometry of the mPRα bands normalized to the actin loading controls. Results were analyzed by one-way ANOVA followed by the Bonferroni test. Significant differences between the paired groups of hCG alone versus hCG plus AG205 were tested by Student’s t-test. Different letters denote significant differences between the treatments groups (p < 0.05) in the post hoc Bonferroni test.

Fig. 5.

Effects of microinjection with zebrafish pgrmc1 antisense morpholino oligonucleotides on plasma membrane expression of mPRa (A) and DHP induction of OM (B). (A) Western blot analysis of Pgrmc1, mPRα and actin expression on ovarian membranes of zebrafish oocytes after microinjection of zebrafish pgrmc1 antisense morpholino oligonucleotides (PG-As), and non-targeting control morpholino oligonucleotides (NC). (B) Vitellogenic follicle-enclosed oocytes with diameters of 450–500 μm were injected with 1 nl of the oligonucleotides (< 1% of oocyte volume) and incubated with 10nM DHP in the in vitro OM bioassay. OM was assessed by GVBD (disappearance of germinal vesicle). The experiment was repeated two times and the results pooled. The total number of oocytes in each treatment group were: vehicle −30, DHP −14, DHP + PG-As −29, and DHP + NC −16. All data represent means ± S.E.M. Results were analyzed by one-way ANOVA followed by the Bonferroni test. Significant differences between the paired groups of microinjection with Pgrmc1 antisense (PG-As) versus Pgrmc1 non-targeting controls (NC) were tested by Student’s t-test. Different letters denote significant differences between the treatments groups (p < 0.05) in the post hoc Bonferroni test.

Fig. 6.

Interactions between Pgrmc1 and mPRα (A, B), and between Pgrmc1 and Erbb2 (C and D) in zebrafish oocytes detected in the in situ proximity ligation assay. Close associations of Pgrmc1 with mPRα are shown as red dots in the image (A). The mPRα antibody was replaced with IgG in the assay as a negative control (B). Close associations of Pgrmc1 with Erbb2 are shown as red dots in the image (C). The Pgrmc1 antibody was replaced with IgG in the assay as a negative control (D). Nuclei of the follicle cells surrounding the oocytes are stained with DAPI (4’, 6-diamidino-2-phenylindole). Scale bars in the images represent 100 μm (μm). The assay was repeated 3 or more times and similar results were obtained on each occasion. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7.

Effects of two different Erbb2 inhibitors, ErbB2 inhibitor II and AG879, on zebrafish OM. Western blot of Erbb2 expression on zebrafish defolliculated (DO) membranes (A). Effects of co-treatment with DHP or AG205 together with Erbb2 inhibitors on OM of denuded oocytes in the in vitro bioassay (B and C). OM was assessed by GVBD (disappearance of germinal vesicle). All data represent means ± S.E.M (n = 3). The entire experiment was repeated three times and similar results were obtained on each occasion. Results were analyzed by one-way ANOVA followed by the Bonferroni test. Different letters denote significant differences between the treatments groups (p < 0.05) in the post hoc test.

Fig. 8.

Effects of IGF-1 and the IGF-1 receptor inhibitor (NPV) on OM (A), and the effects of AG205 on OM induced by IGF-1 (B) of denuded oocytes in the in vitro bioassay. OM was assessed by GVBD (disappearance of germinal vesicle). Each treatment was replicated three times in each experiment (20 oocytes/ well). The experiments were repeated three or more times and the results were pooled. All data represent means ± S.E.M. (n = 9). Results were analyzed by one-way ANOVA followed by the Bonferroni test. Different letters denote significant differences between the treatment groups (p < 0.05) in the post hoc test.