Research Paper Volume 13, Issue 1 pp 1096—1119

The endothelial nitric oxide synthase/cyclic guanosine monophosphate/protein kinase G pathway activates primordial follicles

eNOS was involved in PF activation. (A) Immunohistochemical staining of eNOS, iNOS and nNOS in ovaries at 3 dpp and 7 dpp. Scale bar, 40 μm. (B) The relative mRNA levels of eNOS, iNOS and nNOS were assessed in ovaries from 1-7 dpp using qRT-PCR. (C) Immunofluorescence staining of eNOS in ovaries at 3 dpp and 7 dpp. Sections were labeled for eNOS (red), the oocyte marker Y box protein 2 (MSY2, green) and the nuclear marker Hoechst (blue). Arrows indicate PFs and triangles indicate GFs. Scale bar, 40 μm. (D) Ovaries at 1 dpp were treated with the vehicle, the NO donor SN (100 μM) or the NO inhibitor NM (1 mM) for six days (n=6). The ovarian morphology was analyzed after hematoxylin staining. Triangles indicate GFs. Scale bar, 40 μm. (E) The numbers of PFs and GFs/the total number of follicles were analyzed. (F) The numbers of oocytes with diameters larger than 20 μm were counted. **, *** and **** denote statistical significance at p p p p

Figure 1. eNOS was involved in PF activation. (A) Immunohistochemical staining of eNOS, iNOS and nNOS in ovaries at 3 dpp and 7 dpp. Scale bar, 40 μm. (B) The relative mRNA levels of eNOS, iNOS and nNOS were assessed in ovaries from 1-7 dpp using qRT-PCR. (C) Immunofluorescence staining of eNOS in ovaries at 3 dpp and 7 dpp. Sections were labeled for eNOS (red), the oocyte marker Y box protein 2 (MSY2, green) and the nuclear marker Hoechst (blue). Arrows indicate PFs and triangles indicate GFs. Scale bar, 40 μm. (D) Ovaries at 1 dpp were treated with the vehicle, the NO donor SN (100 μM) or the NO inhibitor NM (1 mM) for six days (n=6). The ovarian morphology was analyzed after hematoxylin staining. Triangles indicate GFs. Scale bar, 40 μm. (E) The numbers of PFs and GFs/the total number of follicles were analyzed. (F) The numbers of oocytes with diameters larger than 20 μm were counted. **, *** and **** denote statistical significance at p < 0.01, p < 0.001 and p < 0.0001 respectively. Different letters with the same color denote statistical significance at p < 0.001 (Red letters represent the proportions of PFs, while black letters represent the proportions of GFs).