Downregulation of nuclear progestin receptor (Pgr) and subfertility in double knockouts of progestin receptor membrane component 1 (pgrmc1) and pgrmc2 in zebrafish

Abstract

The progestin receptor membrane components (Pgrmcs) contain two paralogs, Pgrmc1 and Pgrmc2. Our previous research into single knockout of Pgrmc1 or Pgrmc2 suggests that Pgrmc1 and Pgrmc2 regulate membrane progestin receptor or steroid synthesis and therefore female fertility in zebrafish. Additional roles of Pgrmcs may not be determined in using single Pgrmc knockouts due to compensatory roles between Pgrmc1 and Pgrmc2. To address this question, we crossed single knockout pgrmc1 (pgrmc1^-/-) with pgrmc2 (pgrmc2^-/-), and generated double knockouts for both pgrmc1 and pgrmc2 (pgrmc1/2^-/-) in a vertebrate model, zebrafish. In addition to the delayed oocyte maturation and reduced female fertility, significant reduced ovulation was found in double knockout (pgrmc1/2^-/-) in vivo, though not detected in either single knockout of Pgrmc (pgrmc1^-/- or pgrmc2^-/-). We also found significant down regulation of nuclear progestin receptor (Pgr) protein expression only in pgrmc1/2^-/-, which was most likely the cause of reduced ovulation. Lower protein expression of Pgr also resulted in reduced expression of metalloproteinase in pgrmc1/2^-/-. With this study, we have provided new evidence for the physiological functions of Pgrmcs in the regulation of female fertility by regulation of ovulation, likely via regulation of Pgr, which affects regulation of metalloproteinase expression and oocyte ovulation.

Keywords: Metalloproteinase; Ovulation; Pgr; Pgrmc1; Pgrmc2; Progestins.

Fig 1.. Reduced fertility in pgrmc1/2^−/− homozygous female zebrafish.

(A) Mutant female zebrafish produced fewer embryos over a two-week mating period than wt females. (B) Mutant females spawned with less frequency than wildtype. (C) pgrmc1/2^−/− females spawned less embryos per day. (D) pgrmc1/2^−/− females spawned less embryos each time they spawned. (E) Wildtype females produced more embryos daily than pgrmc1/2^−/− females. **, p<0.01; ****, p<0.0001.

Fig 2.. Quantification of different stages of oocytes in wt and pgrmc1/2^−/− female ovaries.

Significant numbers of fully-grown immature Stage IVa follicles and matured but not ovulated Stage IVb oocytes were observed in pgrmc1/2^−/− females 30 minutes after room lights were switched. At the same time point, no Stage IVa and Stage IVb follicles could be observed in wildtype females. Further, pgrmc1/2^−/− females had less Stage V oocytes.

Fig 3.. Reduced Pgr expression in the most advanced follicles of pgrmc1/2^−/−.

The night prior to spawning at 21:00, the most advanced stage of oocytes sampled at 21:00 are Stage III oocytes. These oocytes will grow further and develop into Stage IVa oocytes (fully grown but immature) at 06:00. Then, these advanced oocytes will undergo oocyte maturation to become Stage IVb oocytes around 08:00 about 1 hour prior to lights on and ovulation. (A) Expression of pgr transcripts in most advanced stage oocytes from wt or Pgrmc mutants over different developmental time points (21:00, Stage III; 06:00, Stage IVa; 08:00, Stage IVb). (B) Reduced expression of Pgr protein in Stage IVa follicles (06:00) only in pgrmc1/2^−/−. *, p<0.05; **, p<0.01; ***, p<0.001.

Fig 4.. Expression of metallopeptidase in Pgrmc knockouts in comparison to wt in most advanced follicles

(21:00, Stage III; 06:00, Stage IVa; 08:00, Stage IVb.). Low adamts9 expression can be observed in Stage IVb oocytes of all genotypes (pgrmc1^−/−, pgrmc2^−/−, and pgrmc1/2^−/−). In addition, expression of adamts1, adamts8a, and mmp2 was significantly reduced in Stage IVb follicles in pgrmc1/2^−/−. Asterisks indicate a significant difference of transcripts compared to wt at the same time point. adam8b, a distintegrin and metalloproteinase domain 8b; adamts1, a disintegrin and metalloproteinase with thrombospondin type 1 motif 1; adamts8a; adamts9; mmp2, matrix metalloproteinase 2; and mmp9, matrix metalloproteinase 9. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.