Research Paper Volume 13, Issue 1 pp 1096—1119

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

cGMP/PKG participated in PF activation. (A) The relative mRNA levels of GUCY1b1 and GUCY1a1 in ovaries from 1-7 dpp. (B) Radioimmunoassay of cGMP levels in ovaries from 1-7 dpp (n=20). (C) The relative mRNA levels of PKG in ovaries from 1-7 dpp. (D–G) The relative mRNA levels of (D) the germline marker DEAD box helicase 4 (MVH), (E) the granulosa cell marker forkhead box L2 (FOXL2), (F) GUCY1b1 and (G) PKG in oocytes and somatic cells isolated from ovaries at 3 dpp. (H) Immunofluorescence staining of PKG and MSY2 in ovaries at 3 dpp and 7 dpp. Arrows indicate PFs and triangles indicate GFs. Scale bar, 40 μm. (I, L) Ovaries at 1 dpp were treated with the vehicle, the sGC agonist YC (10 μM), the sGC inhibitor ODQ (1 μM), the cGMP analog 8Br (10 μM) or the PKG inhibitor KT (1 μM) for six days (n=6). The ovarian morphology was analyzed after hematoxylin staining. Triangles indicate GFs. Scale bar, 40 μm. (J, M) The numbers of PFs and GFs/the total number of follicles were analyzed. (K, N) The numbers of oocytes with diameters larger than 20 μm were counted. *, ** and *** denote statistical significance at p p p p

Figure 2. cGMP/PKG participated in PF activation. (A) The relative mRNA levels of GUCY1b1 and GUCY1a1 in ovaries from 1-7 dpp. (B) Radioimmunoassay of cGMP levels in ovaries from 1-7 dpp (n=20). (C) The relative mRNA levels of PKG in ovaries from 1-7 dpp. (DG) The relative mRNA levels of (D) the germline marker DEAD box helicase 4 (MVH), (E) the granulosa cell marker forkhead box L2 (FOXL2), (F) GUCY1b1 and (G) PKG in oocytes and somatic cells isolated from ovaries at 3 dpp. (H) Immunofluorescence staining of PKG and MSY2 in ovaries at 3 dpp and 7 dpp. Arrows indicate PFs and triangles indicate GFs. Scale bar, 40 μm. (I, L) Ovaries at 1 dpp were treated with the vehicle, the sGC agonist YC (10 μM), the sGC inhibitor ODQ (1 μM), the cGMP analog 8Br (10 μM) or the PKG inhibitor KT (1 μM) for six days (n=6). The ovarian morphology was analyzed after hematoxylin staining. Triangles indicate GFs. Scale bar, 40 μm. (J, M) The numbers of PFs and GFs/the total number of follicles were analyzed. (K, N) The numbers of oocytes with diameters larger than 20 μm were counted. *, ** and *** denote statistical significance at p < 0.05, p < 0.01 and p < 0.001, respectively. Different letters with the same color denote statistical significance at p < 0.01 (Red letters represent the proportions of PFs, while black letters represent the proportions of GFs).