ROCK1 is a multifunctional factor maintaining the primordial follicle reserve and follicular development in mice

Abstract

The follicle is the basic structural and functional unit of the ovary in female mammals. The excessive depletion of follicles will lead to diminished ovarian reserve or even premature ovarian failure, resulting in diminished ovarian oogenesis and endocrine function. Excessive follicular depletion is mainly due to loss of primordial follicles. Our analysis of published human ovarian single-cell sequencing results by others revealed a significant increase in rho-associated protein kinase 1 (ROCK1) expression during primordial follicle development. However, the role of ROCK1 in primordial follicle development and maintenance is not clear. This study revealed a gradual increase in ROCK1 expression during primordial follicle activation. Inhibition of ROCK1 resulted in reduced primordial follicle activation, decreased follicular reserve, and delayed development of growing follicles. This effect may be achieved through the HIPPO pathway. The present study indicates that ROCK1 is a key molecule for primordial follicular reserve and follicular development.NEW & NOTEWORTHY ROCK1, one of the Rho GTPases, plays an important role in primordial follicle reserve and follicular development. ROCK1 was primarily expressed in the cytoplasm of oocytes and granulosa cell in mice. Inhibition of ROCK1 significantly reduced the primordial follicle reserve and delayed growing follicle development. ROCK1 regulates primordial follicular reserve and follicle development through the HIPPO signaling pathway. These findings shed new lights on the physiology of sustaining female reproduction.

Keywords: HIPPO; ROCK1; follicular development; ovary; primordial follicle reserve.

Graphical abstract

Figure 1.

ROCK1 expression was increased during primordial follicle activation. A: single-cell sequencing results (GSE107746) were analyzed for changes in Rock1 and Rock2 expression in granulosa cells and oocytes of human primordial and primary follicles. B and C: the total mRNA levels of Rock1 and Rock2 in female mouse ovaries from 1 to 7 dpp (n = 3). D and E: the total protein levels of ROCK1 and ROCK2 in female mouse ovaries from 1 to 7 dpp. Every group included 10 ovaries, and the experiment was repeated at least three times. F: immunofluorescent staining of ROCK1 in newborn mice ovaries. ROCK1 signals (green) were costained with DDX4 (red). The nuclei were stained with DAPI (blue). White arrows indicate oocytes, yellow arrows indicate granulosa cells, and purple arrows indicate interstitial cells. Scale bar: 15 μm. G: immunofluorescent staining of ROCK1 in newborn mice ovaries. ROCK1 signals (green) were costained with DDX4 (red). The nucleus was stained by DAPI (blue). Scale bar: 15 μm. H: immunofluorescent staining of ROCK1 in newborn mice ovaries. ROCK1 signals (green) were costained with FOXL2 (red). The nuclei were stained with DAPI (blue). Scale bar: 15 μm. Statistical significance was determined using two-tailed unpaired Student’s t test (A) or one-way ANOVA followed by Tukey’s post hoc test (B and C), and values are the means ± SD. Statistically significant values of P < 0.05. PF, primary follicle; PmF, primordial follicle; ROCK1, rho-associated protein kinase 1; 2 dpp, 2 days postpartum.

Figure 2.

The efficacy of Y-27632 inhibitor. A: the protein expression levels of FilaminA, ROCK1, and ROCK2. The 2 dpp ovaries were cultured with or without Y-27632 for 2 days. Each group was 10 ovaries, repeat for at least three times. GAPDH and β-actin served as an internal control. B: immunofluorescence of FilaminA within Y-27632-treated ovaries. The 2 dpp ovaries were cultured with or without Y-27632 for 2 days. ROCK1 signals (green) were costained with DDX4 (red). The nucleus was stained by DAPI (blue). Scale bar: 20 μm. C: immunofluorescence of ROCK1 within Y-27632-treated ovaries. The 2 dpp ovaries were cultured with or without Y-27632 for 2 days. ROCK1 signals (green) were costained with DDX4 (red). The nucleus was stained by DAPI (blue). Scale bar: 20 μm. ROCK1, rho-associated protein kinase 1; 2 dpp, 2 days postpartum.

Figure 3.

ROCK1 regulates follicle activation, maintenance, and development. A: experimental design for culture of the newborn female mouse ovaries from 2 dpp mice with the ROCK1 inhibitor Y-27632 at 10 μM (n = 9). B and C: hematoxylin staining and whole ovary follicle counting of ovarian sections cultured for 3 days with Y-27632. Scale bar: 20 μm (n = 9). D: Western blot to detect changes in DDX4. The 2 dpp female ovaries were cultured for 2 days. Each group included 10 ovaries, and the experiment was repeated at least three times. E: results of the statistical analysis of D (n = 4). F: experimental design for kidney capsule transplantation of Y-27632-treated ovaries. G: hematoxylin staining of ovarian sections treated with Y-27632. Female mouse ovaries at 3 dpp were cultured with Y-27632 for 3 days and transplanted into immunodeficient mice to continue development under the kidney capsule for 14 days. Scale bar: 20 μm. H: primordial follicle (PmF) counting of ovarian sections from kidney capsule transplantation (n = 14). I: Hematoxylin eosin (HE) staining of ovarian sections from kidney capsule transplantation. Scale bar: 20 μm. J: quantitation of granulosa cell thickness from oocytes diameters of 35–40 μm. n = 8. K: quantitation of granulosa cell thickness from oocytes diameters of 40–45 μm (n = 46). L: quantitation of granulosa cell thickness from oocytes diameters of 45–50 μm (n = 42). M: quantitation of granulosa cell thickness from oocytes diameters of 50–55 μm (n = 32). N: quantitation of granulosa cell thickness from oocytes diameters of 55–60 μm (n = 9). Statistical significance was determined using two-tailed unpaired Student’s t test, and values are the means ± SD. Statistically significant values are indicated by P < 0.05. ROCK1, rho-associated protein kinase 1; 2 dpp, 2 days postpartum.

Figure 4.

ROCK1 inhibition leads to reduced primordial follicles and antral follicles in ovarian topical administration model. A: experimental design for ovarian topical administration in vivo. B: photograph of the ovary at 0 h after matrigel injection. The same female mice were injected with matrigel containing Y-27632 on one side of the ovary and blank matrigel on the other side. C: photograph of the ovary at 2 wk after matrigel injection. D and E: hematoxylin staining and whole ovary follicle counting of ovarian sections after ovarian topical administration for 14 days with Y-27632 (n = 3). The results for two experimental groups were compared by two-tailed unpaired Student’s t tests. Statistical significance was determined using two-tailed unpaired Student’s t test, and values are the means ± SD. Statistically significant values are indicated by P < 0.05. ROCK1, rho-associated protein kinase 1.

Figure 5.

Y-27632-treated ovaries showed reduced proliferation and increased apoptosis. A: immunofluorescence of K_i-67 in Y-27632-treated ovaries. ROCK1 signals (green) were costained with DAPI (blue). The 2 dpp ovaries were cultured with or without Y-27632 for 2 days. Scale bar: 25 μm. B: quantitation of K_i-67-positive cells from interstitial cells (n = 4). C: quantitation of K_i-67-positive cell from oocytes of primordial follicle (oocytes-PmF) (n = 4). D: quantitation of K_i-67-positive cells from oocytes of primary follicle (oocytes-PF) (n = 4). E: quantitation of K_i-67-positive cells from pregranulosa cell of primordial follicle (GC-PmF) (n = 4). F: quantitation of K_i-67-positive cells from granulosa cell of primordial follicle (GC-PF) (n = 4). G: immunofluorescence of TUNEL with Y-27632-treated ovary. Scale bar: 25 μm. H: quantitation of TUNEL-positive cell from whole ovary (n = 10). Statistical significance was determined using two-tailed unpaired Student’s t test, and values are the means ± SD. Statistically significant values of P < 0.05. 2 dpp, 2 days postpartum.

Figure 6.

Transcriptome sequencing results indicate that ROCK1 may regulate follicular reserve through the HIPPO pathway. A: volcano plots show the changes in mRNA abundance in RNA-seq of Y-27632-treated ovary. DEGs were identified from Y-27632 vs. the control (n = 3). B: heatmap of significantly altered genes. C–E: representative enriched GO terms, KEGG terms, and GSEA term. DEGs, differentially expressed genes; GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes.

Figure 7.

Y-27632-treated ovaries showed enhancement of HIPPO signaling pathway. A–C: the mRNA expression levels of Ccn2, Tead4, and Yap1. The 2 dpp ovaries were cultured with or without Y-27632 for 2 days (n > 3/group). D: the protein expression levels of LAST1, YAP, p-YAP, CCN2, and TEAD4. The 2 dpp ovaries were cultured with or without Y-27632 for 2 days. Each group included 10 ovaries, and the experiment was repeated at least three times. E: the protein expression levels of YAP and TEAD4. The 2 dpp ovaries were cultured with or without Y-27632 for 3 days. Each group included 10 ovaries, and the experiment was repeated at least three times. F–J: results of the statistical analysis of D. The results are given as the means ± SD. The results for two experimental groups were compared by two-tailed unpaired Student’s t tests. Statistical significance was determined using two-tailed unpaired Student’s t test, and values are the means ± SD. Statistically significant values are indicated by P < 0.05. 2 dpp, 2 days postpartum.

Figure 8.

ROCK1 regulate follicle reserve through HIPPO signaling pathway. A: hematoxylin staining of ovarian sections from Y-27632 and YAP-TEAD4 inhibitor-treated female ovaries. The 2 dpp ovaries were cultured for 3 days. B–D: whole ovary follicle counting. E: the ratio of PmF/PF (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test, and values are the means ± SD. The results are given as the means ± SE. The results for two experimental groups were compared by two-tailed unpaired Student’s t tests. Statistical significance was determined using two-tailed unpaired Student’s t test, and values are the means ± SD. Statistically significant values are indicated by P < 0.05. PF, primary follicle; PmF, primordial follicle; total, total follicle; 2 dpp, 2 days postpartum.