Lactylation of CREB is required for FSH-induced proliferation and differentiation of ovarian granulosa cells

Abstract

Follicle-stimulating hormone (FSH) promotes follicular development by inducing the proliferation and differentiation of granulosa cells (GCs). This process is primarily attributed to the activation of the canonical G protein-coupled receptor (GPCR)/adenylyl cyclase/cAMP/PKA/CREB signaling pathway. Here, we revealed a novel mechanism wherein FSH promotes GCs proliferation and differentiation by stimulating cAMP response element-binding protein (CREB) lactylation. Specifically, FSH induced CREB lactylation at lysine 136 (K136la), leading to CREB phosphorylation at serine 133, which facilitated CREB/CBP/P300 complex formation for transcription activation. Moreover, K136la alone directly recruited CBP/P300, triggering transcriptional surges of proliferation and differentiation genes by binding with the cAMP response element (CRE), thereby stimulating GCs proliferation and differentiation. By contrast, a CREB mutation at K136 eliminated these effects. Blocking CREB lactylation using oxamate or C646 in vivo suppressed GCs proliferation, differentiation, and follicular development in mouse ovaries. These findings highlight the important role of lactylation between metabolic regulation and folliculogenesis, and its importance in mediating GPCR signaling, providing a theoretical basis for treating female infertility associated with defective follicular development.

Graphical Abstract

Figure 1.

FSH-induced GCs proliferation is associated with enhanced levels of lactate and lactylation. (A) For in vivo EdU assay, mice were intraperitoneally injected with 10, 5, 2, and 2 IU of FSH (dissolved in 0.9% saline) every 12 h, and EdU reagent (5 mg/kg of body weight) was injected intraperitoneally 12 h before collection of ovaries. The ovaries were then processed and sectioned into 5 μm paraffin slices. Then, the cell climbing sheets or ovarian sections were incubated with EdU dye reaction solution for 30 min at room temperature, and the nuclei were counterstained with DAPI for 10 min. The proliferation of follicular cells was measured by counting the proportion of red fluorescent nuclei. Scale bar: 100 μm. (B) Quantitative analysis of GCs proliferation activity in (A). (C) Mice received intraperitoneal injections of FSH (10, 5, 2, and 2 IU dissolved in 0.9% saline) at 12 h intervals, followed by immunohistochemical detection of Ki67 expression and cellular distribution. Scale bar: 100 μm. (D) Quantitative analysis of the Ki67-positive cell ratio among different cell types in (C). (E) Western blot analysis of proliferation-related protein levels following intraperitoneal injections of FSH (10, 5, 2, and 2 IU dissolved in 0.9% saline) at 12 h intervals. (F) l-Lactate levels in follicular fluid were measured after intraperitoneal FSH administration (10, 5, 2, and 2 IU in 0.9% saline) at 12 h intervals. (G) GLUT1 protein expression was analyzed by western blot following intraperitoneal FSH injection (10, 5, 2, and 2 IU in 0.9% saline) at 12 h intervals. (H) Mice were administered FSH intraperitoneally (10, 5, 2, and 2 IU in 0.9% saline) at 12 h intervals, with subsequent immunohistochemical analysis of Pan-Kla expression and cellular localization. Scale bar: 100 μm. (I) Quantitative analysis of Pan-Kla protein levels across different cell types as shown in (H). (J) Western blot analysis was performed to evaluate Pan-Kla expression after intraperitoneal FSH administration (10, 5, 2, and 2 IU in 0.9% saline) at 12 h intervals. The data are presented as mean ± SD. Differences between groups were assessed using t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS indicates no difference.

Figure 2.

Suppression of lactate production/lactylation inhibits FSH-induced GCs proliferation. (A) l-Lactate levels were measured in mGCs and KGN cells after treatment with 15 mM 2-DG or 15 mM oxamate for 2 h followed by 5 IU of FSH for 12 h. (B) KGN cells were treated with either 15 mM 2-DG or 15 mM oxamate for 2 h followed by 5 IU of FSH for 12 h, after which lactyl-CoA levels were measured. (C) Western blot assessing Pan-Kla protein expression after mGCs and KGN treatment with 15 mM 2-DG and 15 mM oxamate for 2 h followed by treatment with 5 IU of FSH for 12 h. (D) Western blot assessing Pan-Kla protein expression after KGN treatment with 3 mM α-CHCA for 2 h followed by 15 mM sodium lactate treatment for 12 h. (E) Cell viability was assessed using a CCK-8 assay following sequential exposure to either 15 mM 2-DG or 15 mM oxamate for 2 h and subsequent treatment with 5 IU of FSH for 12 h. (F) Proliferation-related protein expression was analyzed via western blot in cells sequentially treated with 15 mM 2-DG or 15 mM oxamate for 2 h prior to stimulation with 5 IU of FSH for 12 h. (G) l-Lactate levels were measured in KGN cells following 12 h transfection with LDHA and/or LDHB siRNA and subsequent treatment with 5 IU of FSH for 12 h. (H) Lactyl-CoA production was assessed in KGN cells subjected to: (i) 12 h of LDHA and/or LDHB siRNA transfection and (ii) 12 h of exposure to 5 IU of FSH. (I) Western blot analysis of Pan-Kla protein expression level in KGN cells transfected with siRNAs against LDHA and/or LDHB for 12 h, followed by FSH treatment for 12 h. (J) Cell viability determination using a CCK-8 assay following treatment as indicated in KGN cells transfected with siRNAs against LDHA and/or LDHB 12 h, followed by FSH treatment for 12 h. (K) KGN cells were transfected with LDHA and/or LDHB siRNAs for 12 h followed by a 12 h FSH exposure, then subjected to western blot analysis of proliferation-related proteins. (L) KGN cells transfected with LDHA/LDHB siRNAs for 12 h were subsequently cultured for 12 h with 5 IU of FSH in the presence or absence of 15 mM sodium lactate. Western blot assessing Pan-Kla protein expression. (M) KGN cells underwent LDHA/LDHB siRNA transfection for 12 h followed by 12 h culture with 5 IU of FSH, supplemented or not with 15 mM sodium lactate. Cell viability was assessed using the CCK-8 assay. (N) KGN cells underwent LDHA/LDHB siRNA transfection for 12 h followed by 12 h culture with 5 IU of FSH, supplemented or not with 15 mM sodium lactate. Western blot assessing PCNA and Cyclin D2 proteins expression. (O) Measurement of lactyl-CoA levels in KGN cells transfected with siRNAs against ACSS2 and/or SUCLG1 for 12 h, followed by treatment with 5 IU of FSH for 12 h. (P) Western blot analysis of expression levels of Pan-Kla and PCNA proteins in KGN cells transfected with siRNAs against ACSS2 and/or SUCLG1 for 12 h, followed by treatment with 5 IU of FSH for 12 h. (Q) Cell viability was determined by CCK-8 assay after transfection with siRNAs against ACSS2 and/or SUCLG1 for 12 h, followed by 5 IU of FSH for 12 h. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS indicates no difference.

Figure 3.

FSH promotes CREB lactylation in GCs. (A) Each mouse was intraperitoneally injected with 10, 5, 2, and 2 IU FSH (dissolved in 0.9% saline) every 12 h and control mice were concomitantly injected with the same volume of 0.9% saline. After 48 h, mice in each group were sacrificed for the collection of GCs to identify lactylation by LC-MS/MS. (B) Enumeration of proteins with increased lactylation. (C) Heatmap showing profiles of all quantifiable Kla sites before or after normalization according to quantitative proteomics analysis. Before normalization, lactylation levels were quantified without proteomic normalization; after normalization, lactylation levels were quantified with proteomic normalization. (D) Gene Ontology analysis of the proteins showing increased lactylation. (E) Subcellular classification of the proteins exhibiting increased lactylation. (F) Validation of the conservation of the CREB S133 phosphorylation site and S136 lactylation site across different species. (G) Co-immunoprecipitation assays confirmed the interaction between CREB and Pan-Kla in KGN cells following treatment with 5 IU of FSH for 12 h. (H) The ratio of lactylated CREB to total CREB was quantified in (G) using densitometric analysis. (I) Co-immunoprecipitation revealed CREB–Pan-Kla interaction in KGN cells treated with 15 mM oxamate for 2 h followed by stimulation with 5 IU of FSH for 12 h. (J) The ratio of lactylated CREB to total CREB was quantified in (I) using densitometric analysis. (K) Co-immunoprecipitation detection of CREB interaction with Pan-Kla after simultaneous knockdown of LDHA and LDHB for 12 h and then treatment with 5 IU of FSH in KGN cells. (L) The ratio of lactylated CREB to total CREB was quantified in (K) using densitometric analysis. (M) Co-immunoprecipitation detection of CREB interaction with Pan-Kla after transfection with ACSS2 and SUCLG1 siRNAs for 12 h and then treatment with 5 IU of FSH in KGN cells. (N) The ratio of lactylated CREB to total CREB was quantified in (M) using densitometric analysis. (O) The 3D structure of the K136R mutant CREB. (P) Co-immunoprecipitation detection of CREB interaction with Pan-Kla after overexpression of Flag-tagged WT or K136R CREB in KGN cells. (Q) The ratio of lactylated CREB to total CREB was quantified in (P) using densitometric analysis. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS indicates no difference.

Figure 4.

Suppression of P300 activity inhibits FSH-induced GCs proliferation. (A) Co-immunoprecipitation analysis demonstrated a marked attenuation of CREB binding to Pan-Kla after knockdown of lactyltransferases for 24 h in KGN cells. (B) Western blot analysis was performed to evaluate Pan-Kla levels in mGCs and KGN cells pre-treated with 10 μM C646 for 2 h prior to stimulation with 5 IU of FSH for 12 h. (C) Co-immunoprecipitation analysis demonstrated a marked attenuation of CREB binding to Pan-Kla pre-treated with 10 μM C646 for 2 h prior to stimulation with 5 IU of FSH for 12 h in KGN. (D) Quantitative analysis of the lactylated CREB proportion relative to total CREB was performed in (C). (E) Western blot analysis was performed to evaluate Pan-Kla levels in KGN cells pre-treated with 10 μM C646 for 2 h prior to 15 mM sodium lactate stimulation for 12 h. (F) Co-immunoprecipitation analysis demonstrated a marked attenuation of CREB binding to Pan-Kla pre-treated with 10 μM C646 for 2 h prior to 15 mM sodium lactate stimulation for 12 h in KGN. (G) Quantitative analysis of the lactylated CREB proportion relative to total CREB was performed in (F). (H) Western blot assessing acetylation protein expression in KGN cells treated with 5 IU of FSH. (I) Western blot assessing acetylation protein expression after KGN treatment with 10 μM C646 for 2 h followed by treatment with 5 IU of FSH for 12 h. (J) Western blot assessing acetylation protein expression in different cells after treatment with C646 for 12h. (K) Cell viability was assessed using the CCK-8 assay following treatment with 10 μM C646 followed by 5 IU of FSH in mGCs and KGN cells. (L) Western blot assessing levels of proliferation-related proteins in mGCs and KGN cells treated with 5 IU of FSH and/or 10 μM C646. (M) Cell viability in mGCs and KGN cells was quantitatively evaluated using the CCK-8 assay following sequential transfection with P300 siRNA for 12 h and subsequent stimulation with 5 IU of FSHfor 12 h. (N) Western blot analysis was performed to evaluate expression of proliferation-related proteins in mGCs and KGN cells following sequential treatments: transfected with P300 siRNA for 12 h, followed by FSH stimulation for 12 h. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS indicates no difference.

Figure 5.

FSH-induced CREB lactylation facilitates CREB phosphorylation. (A) Schematic representation of the three-dimensional structure of CREB. (B) Western blot detecting P-CREB (S133) protein levels in KGN cells treated with 15 mM 2-DG for 2 h followed by treatment with 5 IU of FSH for 12 h. (C) Western blot detecting P-CREB (S133) protein levels in KGN cells treated with 15 mM oxamate for 2 h followed by treatment with 5 IU of FSH for 12 h. (D) Western blot detecting P-CREB (S133) protein levels after transfection with LDHA and LDHB siRNAs for 12 h and subsequent stimulation with 5 IU of FSH for 12 h. (E) Western blot detecting P-CREB (S133) protein levels in KGN cells treated with 3 mM α-CHCA for 2 h followed by 15 mM sodium lactate treatment for 12 h. (F) Western blot detecting P-CREB (S133) protein levels in KGN cells treated with 10 μM C646 for 2 h followed by treatment with 5 IU of FSH for 12 h. (G) Western blot detecting P-CREB (S133) protein levels in KGN cells treated with 10 μM C646 for 2 h followed by 15 mM sodium lactate treatment for 12 h. (H) Western blot detecting P-CREB (S133) protein levels after overexpression of WT-Flag-CREB or MUT-Flag-CREB_K136R in CREB knockdown KGN cells with FSH treatment. (I) CREB expression levels were compared between WT KGN cells and CREB knockout (KO) KGN cells. (J) Western blot detecting P-CREB (S133) protein levels after overexpression of WT-Flag-CREB or MUT-Flag-CREB_K136R with treatment with 10 IU of FSH in CREB KO KGN cells. (K) A schematic diagram illustrating the lactylated CREB–PKA kinase reaction system. (L) Detection of the phosphorylation or lactylation level in the Flag-tagged CREB–bead complexes. (M) The levels of phosphorylated CREB and lactylated CREB in (L) were quantified using densitometric analysis. (N) Molecular docking analysis of WT CREB and the K136R mutant CREB with the PRKACA protein. Binding affinity was assessed based on the PIPER pose energy docking score. (O) Co-immunoprecipitation detection of CREB interaction with PRKACA after overexpression of WT-Flag-CREB or MUT-Flag-CREB_K136R with treatment with 5 IU of FSH in KGN cells. (P) Co-immunoprecipitation assays were performed to assess the interaction between CREB and Pan-Kla in KGN cells overexpressing Flag-tagged WT CREB or its phospho-deficient S133A mutant in KGN cells treated with 5 IU of FSH. (Q) Co-immunoprecipitation assays were conducted to compare the physical interaction between CREB and P300 in KGN cells following transient transfection with Flag-tagged WT CREB or its phospho-deficient S133A mutant in KGN cells treated with 5 IU of FSH. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS indicates no difference.

Figure 6.

CREB lactylation promotes GCs proliferation by activating CREB-dependent transcription. (A) qRT–PCR was performed to measure the expression levels of Cyclin D2 and c-FOS mRNAs in CREB KO KGN cells after overexpression of WT-CREB or MUT-Flag-CREB_K136R for 12 h followed by treatment with 5 IU of FSH for 12 h. (B) ChIP assays were performed to evaluate the recruitment of Flag-tagged CREB to Cyclin D2 and c-FOS promoter regions in CREB KO KGN cells after overexpression of WT-CREB or MUT-CREB_K136R for 12 h followed by treatment with 5 IU of FSH for 12 h. (C) Cell viability was assessed in FSH (5 IU)-treated CREB KO KGN cells following introduction of the WT-CREB or lysine-deficient K136R mutation (MUT-CREB_K136R). (D) Western blot detecting proliferation-related protein levels after transfection with WT-Flag-CREB or WT-CREB_K136R for 12 h followed by treatment with 5 IU of FSH for 12 h in CREB KO KGN cells. (E) CREB KO KGN cells underwent WT-CREB transfection for 12 h followed by 2 h culture with 15 mM oxamate prior to treatment with 5 IU of FSH, either supplemented or not with 15 mM sodium lactate. Cell viability was assessed using the CCK-8 assay. (F) CREB KO KGN cells underwent WT-CREB transfection for 12 h followed by 2 h culture with 15 mM oxamate prior to treatment with 5 IU of FSH, either supplemented or not with 15 mM sodium lactate. The proliferation-related protein levels were assessed by western blot. (G) CREB KO KGN cells underwent MUT-CREB_S133D transfection for 12 h followed by 2 h culture with 15 mM oxamate prior to treatment with 5 IU of FSH, either supplemented or not with 15 mM sodium lactate. Cell viability was assessed using the CCK-8 assay. (H) CREB KO KGN cells underwent MUT-CREB_S133D transfection for 12 h followed by 2 h culture with 15 mM oxamate prior to treatment with 5 IU of FSH, either supplemented or not with 15 mM sodium lactate. The proliferation-related protein levels were assessed by western blot. (I) CREB KO KGN cells underwent WT-CREB/MUT-CREB_S133A transfection for 12 h or treatement with 50 mM H-89 for 2 h prior to treatment with 5 IU of FSH for 12 h. Cell viability was assessed using the CCK-8 assay. (J) CREB KO KGN cells underwent WT-CREB/MUT-CREB_S133A transfection for 12 h or treatment with 50 mM H-89 for 2 h prior to treatment with 5 IU of FSH for 12 h. The proliferation-related protein levels were assessed by western blot. (K) CREB KO KGN cells underwent S133WT-S136WT, S133D-K136WT, and S133D-K136R transfection for 12 h prior to treatment with 5 IU of FSH for 12 h. Cell viability was assessed using the CCK-8 assay. (L) CREB KO KGN cells underwent S133WT-S136WT, S133D-K136WT, and S133D-K136R transfection for 12 h prior to treatment with 5 IU of FSH for 12 h. Western blot analysis of proliferation-related protein levels after the indicated treatments. (M) Cyclin D2 promoter activity was examined by a dual-luciferase reporter gene assay after overexpression of WT-CREB or MUT-Flag-CREB_K136R for 12 h followed by treatment with 5 IU of FSH for 12 h in CREB KO cells. (N) A dual-luciferase reporter gene assay of Cyclin D2 promoter activity was assessed in FSH (5 IU)-treated CREB KO KGN cells following introduction of the WT-CREB. (O) Detection of Cyclin D2 promoter activity using dual-luciferase assay after CREB KO KGN cells underwent MUT-CREB_S133D transfection for 12 h followed by 2 h culture with 15 mM oxamate prior to treatment with 5 IU of FSH, either supplemented or not with 15 mM sodium lactate. (P) The dual-luciferase reporter assay system was employed to quantitatively assess transcriptional activity of the Cyclin D2 promoter after KGN cells underwent WT-CREB/MUT-CREB_S133A transfection for 12 h or treatment with 50 mM H-89 for 2 h prior to treatment with 5 IU of FSH for 12 h in CREB KO KGN cells. (Q) The transcriptional activity of the Cyclin D2 promoter was quantitatively evaluated using the dual-luciferase reporter assay system after KGN cells underwent S133WT-S136WT, S133D-K136WT, and S133D-K136R transfection for 12 h prior to treatment with 5 IU of FSH for 12 h in CREB KO KGN cells. (R) Comparative co-immunoprecipitation analysis was performed to characterize the interaction between CBP/P300 and Flag-tagged CREB variants in FSH-treated KGN cells. Specifically, cells were transiently transfected with either WT Flag-CREB or the lactylation-defective Flag-CREB_K136R mutant prior to 12 h FSH stimulation. (S) Co-immunoprecipitation detection of CREB interaction with CBP/P300 after transfection with Flag-CREB/Flag-CREB_S133A for 12 h, or treatment with 50 nM H-89 prior to treatment with 5 IU of FSH for 12 h in CREB KO KGN cells. (T) Co-immunoprecipitation detection of CREB interaction with CBP/P300 after transfection with S133WT-S136WT, S133D-K136WT, and S133D-K136R for 12 h prior to treatment with 5 IU of FSH for 12 h in CREB KO KGN cells. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS indicates no difference.

Figure 7.

CREB lactylation promotes GCs differentiation by activating CREB-dependent transcription. (A) Treatment of mGCs with 15 mM 2-DG or 15 mM oxamate for 2 h followed by 5 IU of FSH for 12 h. The levels of estradiol (E2) in the culture medium were examined by radioimmunoassay. (B) mGC treatment with 15 mM 2-DG or 15 mM oxamate for 2 h followed by 5 IU of FSH for 12 h. The levels of progesterone (P4) in the culture medium were examined by radioimmunoassay. (C) Western blot determining protein levels of 3β-HSD, CYP19A1, and CYP11A1 in ovarian GCs treated with 15 mM 2-DG for 2 h followed by 5 IU of FSH for 12 h. (D) Western blot analysis of 3β-HSD, CYP19A1, and CYP11A1 protein expression levels in ovarian GCs treated with 15 mM oxamate for 2 h followed by stimulation with 5 IU FSH for 12 h. (E) mGCs transfected with LDHA and LDHB siRNAs for 12 h followed or not with 5 IU of FSH for 12 h. The culture medium was collected to examine the estradiol level using radioimmunoassay. (F) The levels of progesterone in the culture medium were examined by radioimmunoassay after mGCs were transfected with LDHA and LDHB siRNAs for 12 h followed or not with 5 IU of FSH for 12 h. (G) Western blot determining protein levels of 3β-HSD, CYP19A1, and CYP11A1 in ovarian GCs after transfection with LDHA and LDHB siRNAs for 12 h followed or not with 5 IU of FSH for 12 h. (H) mGCs treated with 10 μM C646 for 2 h prior to 5 IU of FSH for 12 h. The culture medium was collected for to examine the estradiol level using radioimmunoassay. (I) mGCs treated with 10 μM C646 for 2 h prior to 5 IU of FSH for 12 h. The culture medium was collected to examine the progesterone level using radioimmunoassay. (J) Western blot determining protein levels of 3β-HSD, CYP19A1, and CYP11A1 in ovarian GCs after treatment with 10 μM C646 for 2 h prior to 5 IU of FSH for 12 h. (K) The detection of estradiol levels in culture medium by radioimmunoassay after transfection with WT-CREB or MUT-CREB_K136R followed by treatment with 5 IU of FSH in CREB KO KGN cells. (L) The detection of progesterone levels in culture medium by radioimmunoassay after transfection with WT-CREB or MUT-CREB_K136R followed by treatment with 5 IU of FSH in CREB KO KGN cells. (M) Western blot determining protein levels of 3β-HSD, CYP19A1, and CYP11A1 in CREB KO KGN cells after transfection with WT-CREB or MUT-CREB_K136R followed by treatment with 5 IU of FSH in CREB KO KGN cells. (N) qRT–PCR detecting the mRNA levels of Cyp19A1 and Cyp11A1 after transfection with WT-CREB or MUT-CREB_K136R followed by treatment with 5 IU of FSH in CREB KO KGN cells. (O) The binding of Flag-tagged CREB to the Cyp19A1 and Cyp11A1 promoters in CREB KO KGN cells after transfection with WT-CREB or MUT-CREB_K136R followed by treatment with 5 IU of FSH in CREB KO KGN cells. The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS indicates no difference.

Figure 8.

In vivo validation of the mechanistic model through intraperitoneal injection of C646. (A) A schematic diagram depicts the intraperitoneal injection protocol for C646 and FSH. Briefly, each mouse received intraperitoneal injections of 10, 5, 2, or 2 IU FSH (dissolved in 0.9% saline) along with 15 mg/kg C646 (dissolved in 0.9% saline) every 12 h. Control mice were injected with an equal volume of 0.9% saline. (B) Immunohistochemical analysis of Pan-Kla levels was performed following the protocol described in (A). Scale bar: 100 μm. (C) Quantitative analysis of Pan-Kla levels in (B). (D) Western blot analysis was performed to detect Pan-Kla levels according to the protocol in (A). (E) Co-immunoprecipitation detection of CREB interaction with Pan-Kla according to the protocol in (A). (F) Western blot analysis of P-CREB (S133) protein levels according to the protocol in (A). (G) The interaction of CREB with PRKACA or CBP/P300 was analyzed by co-immunoprecipitation following the method in (A). (H) EdU incorporation assay detects the proliferation activity of mouse ovarian GCs following the method in (A). Scale bar: 100 μm. (I) Western blot analysis of proliferation-related protein levels according to the protocol in (A). (J) The levels of E2 in serum were examined by radioimmunoassay according to the protocol in (A). (K) The levels of progesterone in serum were examined by radioimmunoassay following the method in (A). (L) Western blot analysis of differentiation-related protein levels following the method in (A). (M) Measurement of ovarian size following the method in (A). (N) Measurement of ovarian weight following the method in (A). (O) Measurement of follicle diameter according to the protocol in (A). (P) Quantitative histomorphological analysis of ovarian follicles at various developmental stages was performed using H&E staining in mice according to the protocol in (A). PF, primary follicle; SF, secondary follicle; AF, antral follicle. Scale bar: 100 μm. (Q) Histomorphometric quantification of ovarian follicles at distinct developmental stages was performed as in (P). The data were presented as mean ± SD. Differences between groups were assessed using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS indicates no difference.