SP1 governs primordial folliculogenesis by regulating pregranulosa cell development in mice

Abstract

Establishment of the primordial follicle (PF) pool is pivotal for the female reproductive lifespan; however, the mechanism of primordial folliculogenesis is poorly understood. Here, the transcription factor SP1 was shown to be essential for PF formation in mice. Our results showed that SP1 is present in both oocytes and somatic cells during PF formation in the ovary. Knockdown of Sp1 expression, especially in pregranulosa cells, significantly suppressed nest breakdown, oocyte apoptosis, and PF formation, suggesting that SP1 expressed by somatic cells functions in the process of primordial folliculogenesis. We further demonstrated that SP1 governs the recruitment and maintenance of Forkhead box L2-positive (FOXL2+) pregranulosa cells using an Lgr5-EGFP-IRES-CreERT2 (Lgr5-KI) reporter mouse model and a FOXL2+ cell-specific knockdown model. At the molecular level, SP1 functioned mainly through manipulation of NOTCH2 expression by binding directly to the promoter of the Notch2 gene. Finally, consistent with the critical role of granulosa cells in follicle survival in vitro, massive loss of oocytes in Sp1 knockdown ovaries was evidenced before puberty after the ovaries were transplanted under the renal capsules. Conclusively, our results reveal that SP1 controls the establishment of the ovarian reserve by regulating pregranulosa cell development in the mammalian ovary.

Keywords: NOTCH2; SP1; pregranulosa cells; premature ovarian insufficiency; primordial follicle formation.

Figure 1

SP1 plays a regulatory role in the formation of PFs. (A) SP1 was expressed in both oocytes and somatic cells from 16.5 dpc to 3 dpp. SP1 in green; oocyte in red (DDX4); nuclear DNA in blue (Hoechst). Scale bar, 40 μm. (B) The protein levels of SP1 from 16.5 dpc to 3 dpp. (C--E) Ovaries phenotype analysis after RNAi. (C) The cell phenotypes and lentivirus infection efficiency were examined using an SP1 (green) immunostaining assay after 5 and 8 days of culture. Oocytes were stained for DDX4 (red), and nuclei were dyed with Hoechst (blue). PF: arrows; nest: arrowheads; scale bar, 40 μm. (D and E) Quantification of the PFs, total oocytes, and oocytes in nests per ovary after 5 (D) and 8 days (E) of culture. The data are presented as mean ± SEM. **P < 0.01 and ***P < 0.001 (t-test).

Figure 2

Sp1 knockdown decreases the number of FOXL2⁺ cells in perinatal ovaries. (A and B) SP1 functioned in OSCs for PF formation. (A) Ovaries at 16.5 dpc were cultured for 4 days, and the ovarian cells were dispersed and reconstituted into follicle-like structures (group 1). Before reconstitution, oocytes and OSCs derived from ovaries pretreated with or without MIT (groups 2 and 3) were exchanged. (B) The tissue structures of the reconstructed cell masses. DDX4: green; propidium iodide: red. Arrows: follicle-like structures, arrowheads: OSCs treated with MIT failed to enclose oocytes. Scale bar, 20 μm. (C) FOXL2 immunofluorescence (red) was assessed after Sp1 knockdown for 5 and 8 days. Nuclei: Hoechst (blue). Scale bar, 40 μm. (D) Quantification of FOXL2⁺ cells after 5 and 8 days of culture. (E and F) The expression of Foxl2 mRNA (E) and protein (F) in Sp1 knockdown ovaries after 5 days of culture. The data are presented as mean ± SEM. **P < 0.01 and ***P < 0.001 (t-test).

Figure 3

SP1 governs the recruitment and maintenance of FOXL2⁺ cells. (A and B) Immunostaining (A) and quantitative analysis (B) show that Sp1 knockdown significantly decreased the number of LGR5⁺BrdU⁺ cells and inhibited the proliferation of LGR5⁺ progenitor cells. EGFP (representing LGR5): green; BrdU: blue; FOXL2: red. Arrowheads: LGR5⁺BrdU⁺ cells; scale bar, 40 μm. (C) Immunostaining of SP1 (red) and FOXL2 (green) in F-Sp1-sh-treated ovaries after 5 and 8 days of culture. Nuclei: Hoechst (blue). Scale bar, 40 μm. (D) SP1 protein levels were clearly lower in F-Sp1-sh OSCs than in controls after 5 days of culture. (E) Quantification of FOXL2⁺ cells in F-SCR-sh and F-Sp1-sh-treated ovaries. The data are presented as mean ± SEM. ***P < 0.001 (t-test).

Figure 4

Pregranulosa cell-specific knockdown of Sp1 suppresses nest breakdown and oocyte apoptosis. (A) The cell phenotypes and lentivirus infection efficiency in the ovaries were examined by SP1 (red) immunostaining after 5 and 8 days of culture. Oocytes were stained for DDX4 (green), and nuclei were dyed with Hoechst (blue). PF: arrows; nest: arrowheads; scale bar, 40 μm. (B and C) Quantification of the PFs, total oocytes, and oocytes in nests per ovary after 5 (B) and 8 days (C) of culture. (D) Representative micrographs showing TUNEL staining (green) in control and Sp1 pregranulosa cell-specific knockdown ovaries. Oocytes in red (DDX4-positive); nuclear in blue (Hoechst). Arrowheads: TUNEL⁺ oocytes; scale bar, 40 μm. (E) Quantification of TUNEL⁺ oocytes. The data are presented as me3 ± SEM. **P < 0.01 and ***P < 0.001 (t-test).

Figure 5

SP1 controls primordial folliculogenesis mainly through NOTCH2 signaling. (A) The NOTCH2 protein levels were assessed via western blot analysis. (B) The Notch2 and Hey2 mRNA levels were analyzed by quantitative real-time PCR. (C) Ovaries were immunolabeled for NOTCH2 (green) and DDX4 (red) and stained with Hoechst (blue). Scale bar, 40 μm. (D and E) The levels of SP1 and NOTCH2 were assayed by western blot analysis (D) and quantitative real-time PCR (E). (F) Western blot analysis of NICD2 domain expression. (G) The mRNA levels of Hey2 in NICD2-overexpressing ovaries. (H) NICD2-overexpressing Sp1 knockdown ovaries restored the protein levels of NOTCH2. (I and J) Immunostaining for DDX4 (green; I) and follicle number counting (J) after overexpression of NICD2 in Sp1 knockdown ovaries. Nuclei were dyed with propidium iodide (red). Scale bar, 40 μm. The data are presented as mean ± SEM. **P < 0.01 and ***P < 0.001 (t-test); different letters (a–c) indicate significant differences between groups (ANOVA and Holm–Šidák test).

Figure 6

SP1 binds directly to the promoter of the Notch2 gene. (A) A ChIP assay reveals that SP1 bound directly to the Notch2 promoter. (B) Luciferase assay results showing that SP1 activated Notch2 expression. Notch2-p, promoter of Notch2 gene (the region from −340 to −156); PCMV-SP1, SP1-overexpressing vector; PCMV, empty vector. (C) The potential binding sites of SP1 on the Notch2 promoter. (D) The ability of SP1 to bind to six GC-rich sequences on Notch2-p. (E) S4–S6, rather than S1–S3, comprised the SP1 binding site. The data are presented as mean ± SEM. Different letters (a–c) indicate significant differences between groups (ANOVA and Holm–Šidák test).

Figure 7

FOXL2⁺ cell deficiency leads to oocyte loss at prepuberty in Sp1 knockdown ovaries. (A) A large loss of oocytes and follicles in Sp1 knockdown ovaries after 12 days of culture. PF: arrows; PrF (primary follicle): arrowheads; degenerating oocytes: red frames; scale bar, 40 μm. (B and C) FOXL2 immunofluorescence (red; B) and FOXL2⁺ cell counting (C). Nuclei were dyed with Hoechst (blue). The data are presented as mean ± SEM. ***P < 0.001 (t-test). (D and E) Hematoxylin staining (D) and SP1 immunofluorescence (green; E) show that Sp1 knockdown resulted in a severe loss of oocytes within nests in prepubescent ovaries. Oocytes in red (DDX4); nuclear DNA in blue (Hoechst). Scale bar, 140 μm. (F) Silencing of Sp1 caused functional granulosa cell deficiency at prepuberty. FOXL2 (red) and DDX4 (green) (left); NOTCH2 (green) and DDX4 (red) (right). Nuclei in blue (Hoechst). Scale bar, 140 μm. (G) Foxl2-driven Sp1 knockdown resulted in largely loss of oocytes in prepubescent ovaries. Scale bar, 140 μm.

Figure 8

Schematic representation showing the proposed role of SP1 in PF formation. (A) Physiologically, the recruitment and maintenance of FOXL2⁺ pregranulosa cells are critical for PF formation. FOXL2⁺ cells are recruited from proliferating LGR5⁺ cells in the ovarian surface epithelium from 15.5 dpc to 1 dpp. Then, from 1 dpp to 3 dpp, the maintained FOXL2⁺ cells start to invade the nest germ cells and surround individual oocytes with B-bodies to form PF structures, while the rest of the oocytes undergo programmed cell death (PCD). From 3 dpp to 7 dpp, some PFs in the ovarian medulla are activated for further development. SP1 in LGR5⁺ cells and FOXL2⁺ cells plays an indispensable role in the recruitment and maintenance of pregranulosa cells by directly regulating Notch2 transcription during PF formation. (B) In Sp1-silenced ovaries, the proliferation of LGR5⁺ cells and the maintenance of FOXL2⁺ cells are inhibited, which consequently decreases the number of FOXL2⁺ cells during PF formation and development. The deficiency in FOXL2⁺ cell development suppresses PF formation and causes large number of oocytes to remain in nests perinatally. However, due to the absence of a sufficient amount of functional granulosa cells to support oocyte development, all of the oocytes in nests undergo cell death following ovarian development (after PF formation at 3 dpp).