Loss of ERβ Disrupts Gene Regulation in Primordial and Primary Follicles

Loss of ERβ increases primordial follicle growth activation (PFGA), leading to premature ovarian follicle reserve depletion. We determined the expression and gene regulatory functions of ERβ in dormant primordial follicles (PdFs) and activated primary follicles (PrFs) using mouse models. PdFs and PrFs were isolated from 3-week-old Erβ knockout (Erβnull) mouse ovaries, and their transcriptomes were compared with those of control Erβfl/fl mice. We observed a significant (≥2-fold change; FDR p-value ≤ 0.05) deregulation of approximately 5% of genes (866 out of 16,940 genes, TPM ≥ 5) in Erβnull PdFs; ~60% (521 out of 866) of the differentially expressed genes (DEGs) were upregulated, and 40% were downregulated, indicating that ERβ has both transcriptional enhancing as well as repressing roles in dormant PdFs. Such deregulation of genes may make the Erβnull PdFs more susceptible to increased PFGA. When the PdFs undergo PFGA and form PrFs, many new genes are activated. During PFGA of Erβfl/fl follicles, we detected a differential expression of ~24% genes (4909 out of 20,743; ≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5); 56% upregulated and 44% downregulated, indicating the gene enhancing and repressing roles of Erβ-activated PrFs. In contrast, we detected a differential expression of only 824 genes in Erβnull follicles during PFGA (≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5). Moreover, most (~93%; 770 out of 824) of these DEGs in activated Erβnull PrFs were downregulated. Such deregulation of genes in Erβnull activated follicles may impair their inhibitory role on PFGA. Notably, in both Erβnull PdFs and PrFs, we detected a significant number of epigenetic regulators and transcription factors to be differentially expressed, which suggests that lack of ERβ either directly or indirectly deregulates the gene expression in PdFs and PrFs, leading to increased PFGA.


Introduction
The earliest step in ovarian folliculogenesis is the formation of primordial follicles (PdFs) with the breakdown of germ cell nests [1].Two classes of PdFs are formed in mammalian ovaries, each exhibiting a distinct developmental dynamic [2,3].While the first wave of PdFs is activated rapidly into primary follicles (PrFs) as they are formed, the second wave of PdFs mostly remains dormant and serves as an ovarian reserve throughout adult life in females [2,3].The second wave of PdFs is selectively activated through a strictly regulated mechanism known as primordial follicle growth activation (PFGA).In the mouse, the first wave of follicles wane in the first 12 weeks of life, and then all activated follicles derive from the second wave of PdFs [2,3].Thus, the initial quantity of second-wave PdFs, the rate of PFGA, and the loss of follicle reserve are the key determinants of female reproductive longevity.
The mammalian ovarian reserve is represented by a fixed number of PdFs of secondwave origin that remain quiescent until recruited into the growing pool [1].An increased rate of PFGA can lead to early depletion of the ovarian reserve, resulting in ovulatory dysfunction, including premature ovarian insufficiency (POI) [4][5][6][7].Thus, understanding the precise molecular mechanisms that maintain PdFs in a dormant state and allow for the gradual activation of PrFs is critical and clinically important [8].PFGA is gonadotropinindependent and involves intraovarian mechanisms [9][10][11][12].It has been shown that secreted factors like AMH from activated ovarian follicles may act on PdFs and exert the inhibitory effect of PFGA [13,14].Previous studies have suggested that PFGA is inhibited by gatekeepers upstream or within the PI3-kinase, mTOR, Hippo, and TGFβ signaling pathways [7,15,16].Several transcription factors, including FOXO3A, and FOXL2, play important roles in controlling PFGA [7,15].However, the role of estrogen signaling in PFGA was not known before our observation that estrogen receptor β (ERβ) is essential for regulating PFGA [17].
There have been contradictory reports on the role of estrogen signaling during oocyte nest breakdown and the formation of PdFs [18][19][20][21].Aromatase knockout (ArKO) mice lacking estrogen synthesis had increased numbers of PrFs at 12 weeks of age and reduced total follicles at one year [22].Despite these findings, it was not suspected that estrogen signaling regulates PFGA [22].We observed that loss of ERβ did not affect the total number of ovarian follicles but markedly increased PFGA [17].Disruption of ERβ signaling, but not ERα, resulted in excessive PFGA, leading to premature depletion of ovarian follicles [17].Thus, ERβ plays a gatekeeping role in maintaining the ovarian reserve [17].Targeted deletion of the ERβ DNA binding domain (DBD) increased PFGA like that of Erβ knockout (Erβ null ) ovaries, indicating that the canonical transcriptional function of ERβ is essential for this regulation [17].
ERβ is a ligand-activated transcription factor that regulates cellular gene expression at the transcription level.Therefore, it is very likely that ERβ either downregulates the expression of genes that activate PFGA or upregulates the genes that inhibit this process.As the core components of PFGA are PdFs and PrFs, we primarily focused on these ovarian follicles.We investigated the transcriptome changes before, during, and after PFGA of PdFs in the absence or presence of ERβ.We isolated the PdFs and PrFs from 3-week-old Erβ null and age-matched wildtype mouse ovaries, examined the expression of ERβ mRNA and protein in isolated PdFs and PrFs, and performed RNA-sequencing analyses.Previous studies on Erβ null mice ovaries have identified genes related to steroidogenesis, preovulatory follicle maturation, and ovulation induction.In this study, we have emphasized the question of whether the loss of ERβ impacts epigenetic and transcriptional regulators in ovarian follicles.Our results indicate that ERβ is essential in upregulating the gene expression in dormant PdFs and activated PrFs.

Both Primordial and Primary Follicles Express ERβ mRNA and Protein
To identify the transcriptional regulatory role of ERβ in PFGA, first, we examined the expression of ERβ in mouse PdFs and PrFs at mRNA and protein levels (Figures 1 and 2).We detected that Erβ mRNA is expressed in both PdFs and PrFs isolated from mouse ovaries (Figure 1A-C).Although the mRNA level was slightly higher in PrFs, it was not statistically significant.
To verify further, we examined the expression of ERβ protein in isolated mouse PdFs and PrFs using immunofluorescence (IF) staining (Figure 2).Isolated PdFs and PrFs were used to prepare cytospin slides, and the follicles were stained with antibodies against total ERβ and phosphorylated ERβ (pERβ, S105).We observed that total ERβ protein is localized within the cytoplasm and nucleus of granulosa cells (GCs) as well as oocytes in both PdFs and PrFs (Figure 2A,B).In contrast, pERβ was detected only within the nuclei To verify further, we examined the expression of ERβ protein in isolated mouse PdFs and PrFs using immunofluorescence (IF) staining (Figure 2).Isolated PdFs and PrFs were used to prepare cytospin slides, and the follicles were stained with antibodies against total ERβ and phosphorylated ERβ (pERβ, S105).We observed that total ERβ protein is localized within the cytoplasm and nucleus of granulosa cells (GCs) as well as oocytes in both PdFs and PrFs (Figure 2A,B).In contrast, pERβ was detected only within the nuclei of GCs and oocytes (Figure 2E,F).Erβ null follicles were negative for the IF staining of total ERβ (Figure 2I,J), so we did not examine the localization of pERβ in Erβ null follicles.

Differential Expression of Follicular Genes in Erβ null Primordial Follicles
We compared the transcriptomes of Erβ null PdFs with those of Erβ fl/fl PdFs.Of 43,230 mouse genes in the reference genome GRCm39, RNA-Seq analyses detected 21,122 genes with a TPM value ≥ 1.0 and 16,940 genes with a TPM value ≥ 5.0 in the PdFs.We observed that approximately 5% of the genes (866 out of 16,940 genes, TPM value ≥ 5) were differentially expressed in Erβ null PdFs (≥2-fold change; FDR p-value ≤ 0.05).Notably, about 60% (521 out of 866) of the differentially expressed genes (DEGs) were markedly upregulated, and the remaining 40% of the DEGs were downregulated, indicating that ERβ can either  To verify further, we examined the expression of ERβ protein in isolated mouse PdFs and PrFs using immunofluorescence (IF) staining (Figure 2).Isolated PdFs and PrFs were used to prepare cytospin slides, and the follicles were stained with antibodies against total ERβ and phosphorylated ERβ (pERβ, S105).We observed that total ERβ protein is localized within the cytoplasm and nucleus of granulosa cells (GCs) as well as oocytes in both PdFs and PrFs (Figure 2A,B).In contrast, pERβ was detected only within the nuclei of GCs and oocytes (Figure 2E,F).Erβ null follicles were negative for the IF staining of total ERβ (Figure 2I,J), so we did not examine the localization of pERβ in Erβ null follicles.

Differential Expression of Follicular Genes in Erβ null Primordial Follicles
We compared the transcriptomes of Erβ null PdFs with those of Erβ fl/fl PdFs.Of 43,230 mouse genes in the reference genome GRCm39, RNA-Seq analyses detected 21,122 genes with a TPM value ≥ 1.0 and 16,940 genes with a TPM value ≥ 5.0 in the PdFs.We observed that approximately 5% of the genes (866 out of 16,940 genes, TPM value ≥ 5) were differentially expressed in Erβ null PdFs (≥2-fold change; FDR p-value ≤ 0.05).Notably, about 60% (521 out of 866) of the differentially expressed genes (DEGs) were markedly upregulated, and the remaining 40% of the DEGs were downregulated, indicating that ERβ can either  C,D,G,H,K,L).While total ERβ was detected in the nucleus and the cytoplasm of oocytes and GCs in PdF and PrF (A,B), pERβ (S105) was localized within the nuclei (E,F).Erβ null follicles were negative for ERβ detection (I,J).
Figure 3. Differential expression of genes in Erβ null primordial follicle (PdFs).PdFs were isolated from 3-week-old Erβ null and age-matched Erβ fl/fl mouse ovaries.Isolated PdFs were subjected to RNA-Seq analyses.Heatmaps (all genes) (A) as well as volcano plots of the differentially expressed genes (DEGs) in Erβ null PdFs (B).In the absence of ERβ, there was an increased number of downregulated genes in Erβ null PdFs.These results also suggest that, despite the PdFs being in a dormant state, ERβ plays an important role in regulating active gene expression within them.

Differential Expression of Follicular Genes in Erβ null Primary Follicles
We also analyzed the transcriptome profile in Erβ null PrFs and compared it with the genes expressed in the Erβ fl/fl PrFs (Figure 4A,B).Out of 43,230 genes in GRCm39, RNA-Seq analyses detected 21,356 genes with a TPM value ≥ 1.0 and 21,221 genes with a TPM value ≥ 5.0.We observed that approximately 8% of the genes (1786 out of 21,221 genes, TPM ≥ 5) were differentially expressed in the Erβ null PrFs (≥2-fold change; FDR p-value ≤ 0.05).In Erβ null PrFs, 83% of the DEGs (1479 out of 1786) were downregulated, whereas only 17% were upregulated, indicating that the presence of ERβ is required for Heatmaps (all genes) (A) as well as volcano plots of the differentially expressed genes (DEGs) in Erβ null PdFs (B).In the absence of ERβ, there was an increased number of downregulated genes in Erβ null PdFs.These results also suggest that, despite the PdFs being in a dormant state, ERβ plays an important role in regulating active gene expression within them.

ERβ Regulation of Epigenetics and Transcription Factors in Primordial Follicles
When we compared the transcriptomes in Erβ null PdFs to Erβ fl/fl PdFs, Erβ null PrFs to Erβ fl/fl PrFs, and Erβ null PrFs to Erβ null PdFs, we observed a consistent deregulation of genes, which suggests that ERβ plays a crucial role in transcriptionally regulating the genes in ovarian follicles before and during PFGA.Accordingly, we further analyzed the DEGs that were identified in Erβ null PdFs for transcriptional and epigenetic regulators.
Among the 866 DEGs in Erβ null PdFs (≥2-fold change; FDR p-value < 0.05, TPM value ≥ 5), we identified a differential expression of 26 epigenetic regulators and chromatin remodelers (Table 1).Remarkably 25 of the 26 differentially expressed epigenetic regulators were significantly downregulated in Erβ null PdFs, including Tet3, Npm2, Mbd3, Ezh2, Dnmt1, Chd3 Chd4 and Chd7 (Table1).To identify the ERβ-regulated genes that play a role in PFGA, we also compared the DEGs between Erβ null PdFs and Erβ fl/fl PdFs (866 genes; Figure 3) with the DEGs between Erβ null PrFs and Erβ fl/fl PrFs (1786 genes; Figure 4).We observed that only 168 genes were common to these two groups suggesting that 1618 genes were differentially expressed in Erβ null PrFs during PFGA (Figure 7B).These findings suggest that, while Erβ null follicles lack the genes that are expressed during the PFGA of Erβ fl/fl follicles, they nevertheless expressed a large number of aberrant genes, which may be responsible for the abnormal phenotypes of activated Erβ null follicles.

ERβ Regulation of Epigenetics and Transcription Factors in Primordial Follicles
When we compared the transcriptomes in Erβ null PdFs to Erβ fl/fl PdFs, Erβ null PrFs to Erβ fl/fl PrFs, and Erβ null PrFs to Erβ null PdFs, we observed a consistent deregulation of genes, which suggests that ERβ plays a crucial role in transcriptionally regulating the genes in ovarian follicles before and during PFGA.Accordingly, we further analyzed the DEGs that were identified in Erβ null PdFs for transcriptional and epigenetic regulators.

Discussion
Expression of ERβ has been detected in the developing oocytes, GCs, and stromal cells surrounding the follicles, and the level of expression changes as the follicles develop [29][30][31][32][33][34][35].While several studies have shown prominent expression of ERβ in PdFs [29,32,33], others have failed to detect expression [36].A lack of antibody specificity has contributed to these challenges in ERβ research [34].We observed that Erβ mRNA and protein are abundantly expressed in PdFs and PrFs isolated from 3-week-old mouse ovaries.Nuclear localization of phospho-ERβ indicates the presence of transcriptionally active ERβ both in the oocytes and GCs of the PdFs and PrFs.Therefore, it is expected that one should observe deregulation of gene expression following the loss of ERβ in ovarian follicles.Despite the apparent dormant state of PdFs, we observed deregulation of many abundantly expressed genes in Erβ null follicles.
Studies have shown that somatic cells initiate PFGA by awakening the dormant oocytes [37], while signaling molecules in oocytes play a crucial role in regulating PFGA [15,38,39].It has been suggested that signaling from activated follicles inhibits the activation of PdFs [40][41][42].However, signaling from PdFs also inhibits the activation of neighboring PdFs [43].These findings highlight the complexity surrounding the events leading to PFGA and the current knowledge gaps.As ERβ is expressed in both GCs and oocytes of PdFs and PrFs, disruption of ERβ signaling may impact ovarian biology, reproduction functions, and women's health.
We observed that loss of ERβ predominantly downregulated the expression of genes both in PdFs and PrFs.This observation indicates that ERβ plays a crucial role in regulating gene expression in dormant and activated ovarian follicles.This was more clearly evident during PFGA of Erβ fl/fl and Erβ null ovarian follicles.While there was no difference in the total number of genes detected by RNA-Seq (20,743 vs. 21,221, TPM ≥ 5), there was a vast difference in gene upregulation among them (2765 vs. 307; FDR p value ≤ 0.05).
ERβ is the major nuclear receptor that mediates estrogen signaling in the mammalian ovaries.Loss of ERβ can directly impair gene regulation.We observed that many epigenetic and transcription regulators are also differentially expressed following the loss of ERβ (Tables 1-4).Expression of those epigenetic and transcriptional regulators in ovarian follicles may be regulated by the transcription function of ERβ.Thus, in addition to the direct impact of ERβ, the differentially expressed transcriptional regulators may also deregulate gene expression in Erβ null PdFs or PrFs.We observed that loss of ERβ increases PFGA and thus leads to premature depletion of PdF reserve [17].As ERβ is a transcription factor, it is expected that this transcriptional regulator either increases the expression of genes that inhibit PFGA or decreases the expression of genes that induce PFGA.
In this study, we made a novel observation that loss of ERβ deregulates genes in Erβ null PdFs, including epigenetic and transcriptional regulators (Tables 1 and 2).Our results suggest that such deregulation may lead to the increased susceptibility of PdFs to undergo PFGA.Moreover, following the PFGA, Erβ null PrFs also suffers from the defective expression of many genes, including many epigenetic and transcriptional regulators (Tables 3 and 4).Such a deregulation of genes in the activated follicles ultimately leads to increased atresia, lack of follicle maturation beyond the antral stage and failure of ovulation [17,44].Future studies are required to elucidate the underlying molecular mechanisms.

Animal Models
An Erβ mutant mouse model carrying a floxed exon 3 allele (Erβ fl/fl ) [45] was included in this study.A mouse line carrying CMV-Cre [46] (006054, Jax Mice) was mated with the Erβ fl/fl mice for deletion of the floxed exon three and established heterozygous mouse lines.Erβ fl/null male and female mice were mated to generate the Erβ null mutant females.The mouse lines were maintained in C57BL/6J (000664, Jax Mice) genetic background.In all experiments, Erβ fl/fl mice were used as normal control.Three-week-old Erβ null and age-matched Erβ fl/fl female mice were euthanized to collect their ovaries and isolate the ovarian follicles.All procedures were performed following the protocols (KUMC ACUP# 2021-2601, 1/19/2022) and approved by the University of Kansas Medical Center Animal Care and Use Committee.

Isolation of Ovarian Follicles
Following our previously published procedure, ovarian follicles were isolated from 3-week-old mouse ovaries [17].Approximately 100 mg of minced ovary tissue was digested in 1 mL of digestion medium (199 media containing 0.08 mg/mL of liberase with medium concentration of thermolysin (Roche Diagnostics GmbH, Mannheim, Germany) supplemented with 5 U/mL of DNase I and 1% bovine serum albumin (Thermo Fisher Scientific, Waltham, MA, USA)).The digestion mix was agitated on an orbital shaker (Disruptor Genie, Scientific Industries, Bohemia, NY, USA) at 1500 rpm for 15 min at room temperature.The enzymatic reaction was stopped by the addition of 10% fetal bovine serum.Digested ovary tissues were passed through a 70 µm cell strainer (Thermo Fisher Scientific) to remove the secondary, and large follicles and tissue aggregates.The filtrate containing the small follicles and cellular components was filtered again through a 35 µm cell strainer (BD Falcon, Franklin Lakes, NJ, USA).The 35 µm strainer was reverse eluted with medium 199 to isolate the PrFs, and the filtrate was subjected to sieving through a 10 µm cell strainer (PluriSelect USA, Gillespie Way, CA, USA) to separate the PdFs from other cellular components.Finally, the 10 µm cell strainer was reverse eluted to isolate the PdFs.Unwanted cellular components were removed from the desired follicles under microscopic examination before proceeding to RNA isolation.

Gene Expression Analyses in Primordial and Primary Follicles
We used 200 to 250 PdFs and 100 to 150 PrFs for cDNA synthesis using the Message Booster cDNA synthesis kit (Lucigen, Palo Alto, CA, USA).Direct cDNA and subsequent cRNA syntheses were performed by following the manufacturer's instructions.In vitro synthesized cRNA was purified by using Monarch RNA cleanup kit (New England Biolabs, Ipswich, MA, USA) and subjected to first-strand and subsequent second-strand cDNA synthesis using the reagents provided in the Message Booster cDNA synthesis kit.The cDNA was diluted 1:10 in 10 mM Tris-HCl (pH 7.4), and 2.5 µL of the diluted cDNA was used in a 10-µL qPCR reaction as described above.The relative quantification of target mRNA expression was calculated by normalizing the data with Actb expression.

Immunofluorescence Staining of Isolated Ovarian Follicles
Isolated PdFs and PrFs were used to prepare the cytospin slides.Approximately 100 PdFs and 100 PrFs were suspended in 150 µL M199 media and loaded into a cytospin funnel, and a coated cytospin slide was placed.Then, cytospin slides were centrifuged at 700× g for 5 min, air-dried, and fixed in cold acetone-methanol for 10 min.Then, the slides were washed with PBST three times and blocked with 5% goat serum (Thermo Fisher Scientific) for 1 h at room temperature.The blocked slides were incubated with a rabbit monoclonal antibody against ERβ (1:250, in 5% goat serum) (Clone 68-4, Millipore Sigma, Burlington, MA, USA) or an antibody against phospho-ERβ (Ser 105) overnight at 2-8 • C. The first antibody-exposed slides were washed three times in PBST and incubated with anti-rabbit AleXa flour 594 conjugated second antibody (1:500, in 5% goat serum) at room temperature for 1 h.Slides were washed three times with PBST and covered with fluor mount with DAPI (Invitrogen), and images were captured using a Nikon-83 fluorescence microscope (Nikon Instruments, Melville, NY, USA).

RNA-Seq Analyses of Primordial and Primary Follicles
Gene expression at the mRNA level was evaluated by RNA sequencing (RNA-Seq).RNA-Seq libraries were prepared using the Ovation Solo RNA-Seq system (Tecan USA, Morgan Hill, CA, USA), optimized for ultra-low input RNA (10 pg to 10 ng of total RNA).Amounts of 300 to 400 PdFs and 150 to 200 PrFs were used to prepare each RNA-Seq library.Follicle lysates were used for the RNA-Seq library preparation and following the manufacturer's instructions.The RNA-Seq libraries were evaluated for quality at the KUMC Genomics Core and then sequenced on an Illumina HiSeq X sequencer using the R1 primer provided with the kit (Psomagen, Rockville, MD, USA).

Detection of Differentially Expressed Genes
All RNA-Seq data have been submitted to the Sequencing Read Archive.RNA-Seq data were analyzed using CLC Genomics Workbench (Qiagen Bioinformatics, Redwood City, CA, USA) as described in our previous publications [44,47,48].Selected RNA-Seq data were validated using the RT-qPCR analyses described above in Section 4.3.

Statistical Analysis
Each RNA-Seq library was prepared using the pooled follicles of three to five individual Erβ fl/fl or Erβ null mice.Each group of RNA sequencing data consisted of three different libraries.For the RT-PCR experiments, each cDNA was prepared from pooled RNA from follicles from three mice ovaries of the same genotype.Both the Erβ fl/fl and Erβ null groups consisted of >3 cDNAs.All of the laboratory investigations were repeated to insure reproducibility.The data are presented as the mean ± standard error (SE).The results were analyzed using one-way ANOVA, and the significance of the mean differences was determined by Duncan's post hoc test, with p ≤ 0.05.The statistical calculations were undertaken using SPSS 22 (IBM, Armonk, NY, USA).

Figure 1 .
Figure 1.Erβ expression in primordial follicle (PdFs) and primary follicles (PrFs).PdFs and PrFs were isolated from 3-week-old Erβ fl/fl mouse ovaries by enzymatic digestion and size fractionation (A,B).cDNAs were prepared from the isolated PdFs and PrFs using direct 'Cell to cDNA' kit reagents and subjected to RT-qPCR analysis.RT-qPCR analysis shows that both PdFs and PrFs expressed Erβ mRNA in a comparable amount (C).RT-qPCR data are shown as mean ± SE, n ≥ 3. Rel., relative, p > 0.05.

Figure 2 .
Figure 2. Detection of ERβ in cytospin preparations of primordial follicles (PdFs) and primary follicles (PrFs).PdFs and PrFs were isolated from 3-week-old mouse ovaries and used for the preparation of the cytospin slides.Immunofluorescence (IF) staining of the cytospin slides identified the expression of ERβ protein in both PdFs and PrFs (A-L).The upper panels show IF staining of ERβ (A,B,E,F,I,J), and the lower panels show DAPI staining (C,D,G,H,K,L).While total ERβ was detected in the nucleus and the cytoplasm of oocytes and GCs in PdF and PrF (A,B), pERβ (S105) was localized within the nuclei (E,F).Erβ null follicles were negative for ERβ detection (I,J).

Figure 1 .
Figure 1.Erβ expression in primordial follicle (PdFs) and primary follicles (PrFs).PdFs and PrFs were isolated from 3-week-old Erβ fl/fl mouse ovaries by enzymatic digestion and size fractionation (A,B).cDNAs were prepared from the isolated PdFs and PrFs using direct 'Cell to cDNA' kit reagents and subjected to RT-qPCR analysis.RT-qPCR analysis shows that both PdFs and PrFs expressed Erβ mRNA in a comparable amount (C).RT-qPCR data are shown as mean ± SE, n ≥ 3. Rel., relative, p > 0.05.

Figure 1 .
Figure 1.Erβ expression in primordial follicle (PdFs) and primary follicles (PrFs).PdFs and PrFs were isolated from 3-week-old Erβ fl/fl mouse ovaries by enzymatic digestion and size fractionation (A,B).cDNAs were prepared from the isolated PdFs and PrFs using direct 'Cell to cDNA' kit reagents and subjected to RT-qPCR analysis.RT-qPCR analysis shows that both PdFs and PrFs expressed Erβ mRNA in a comparable amount (C).RT-qPCR data are shown as mean ± SE, n ≥ 3. Rel., relative, p > 0.05.

Figure 2 .
Figure 2. Detection of ERβ in cytospin preparations of primordial follicles (PdFs) and primary follicles (PrFs).PdFs and PrFs were isolated from 3-week-old mouse ovaries and used for the preparation of the cytospin slides.Immunofluorescence (IF) staining of the cytospin slides identified the expression of ERβ protein in both PdFs and PrFs (A-L).The upper panels show IF staining of ERβ (A,B,E,F,I,J), and the lower panels show DAPI staining (C,D,G,H,K,L).While total ERβ was detected in the nucleus and the cytoplasm of oocytes and GCs in PdF and PrF (A,B), pERβ (S105) was localized within the nuclei (E,F).Erβ null follicles were negative for ERβ detection (I,J).

Figure 2 .
Figure 2. Detection of ERβ in cytospin preparations of primordial follicles (PdFs) and primary follicles (PrFs).PdFs and PrFs were isolated from 3-week-old mouse ovaries and used for the preparation of the cytospin slides.Immunofluorescence (IF) staining of the cytospin slides identified the expression of ERβ protein in both PdFs and PrFs (A-L).The upper panels show IF staining of ERβ (A,B,E,F,I,J), and the lower panels show DAPI staining (C,D,G,H,K,L).While total ERβ was detected in the nucleus and the cytoplasm of oocytes and GCs in PdF and PrF (A,B), pERβ (S105) was localized within the nuclei (E,F).Erβ null follicles were negative for ERβ detection (I,J).

Figure 3 .
Figure 3. Differential expression of genes in Erβ null primordial follicle (PdFs).PdFs were isolated from 3-week-old Erβ null and age-matched Erβ fl/fl mouse ovaries.Isolated PdFs were subjected to RNA-Seq analyses.Heatmaps (all genes) (A) as well as volcano plots of the differentially expressed genes (DEGs) in Erβ null PdFs (B).In the absence of ERβ, there was an increased number of downregulated genes in Erβ null PdFs.These results also suggest that, despite the PdFs being in a dormant state, ERβ plays an important role in regulating active gene expression within them.

Figure 4 .
Figure 4. Differential expression genes in Erβ null primary follicles (PrFs).PrFs were isolated from 3week-old Erβ null and age-matched Erβ fl/fl mouse ovaries.Isolated PrFs were subjected to RNA-Seq analyses.Heatmaps (all genes) (A) and volcano plots (B) show the differential expression of genes in Erβ null PrFs.Both heatmaps and volcano plots show that a larger number of genes are downregulated in Erβ null PrFs compared with those of Erβ fl/fl PrFs.

Figure 4 . 19 2. 4 .
Figure 4. Differential expression genes in Erβ null primary follicles (PrFs).PrFs were isolated from 3-week-old Erβ null and age-matched Erβ fl/fl mouse ovaries.Isolated PrFs were subjected to RNA-Seq analyses.Heatmaps (all genes) (A) and volcano plots (B) show the differential expression of genes in Erβ null PrFs.Both heatmaps and volcano plots show that a larger number of genes are downregulated in Erβ null PrFs compared with those of Erβ fl/fl PrFs.2.4.Differential Expression of Follicular Genes during PFGAWe also identified the DEGs during PFGA of Erβ fl/fl PdFs (Figure5A,B).A large number of new genes are activated during the PFGA, and we observed that about 24% (4909 out of 20,743) of genes with TPM value ≥ 5 were differentially expressed (≥2-fold

Figure 5 .
Figure 5. Differential expression of genes in Erβ fl/fl follicles during PFGA.PdFs and PrFs were isolated from 3-week-old Erβ fl/fl mouse ovaries.Isolated PdFs and PrFs were subjected to RNA-Seq analyses.Heatmaps (all genes) (A) and volcano plots (B) indicate the differential expression of genes in Erβ fl/fl PrFs compared with Erβ fl/fl PdFs.Both heatmaps and volcano plots show that a large number of genes are significantly upregulated during PFGA of Erβ fl/fl PdFs (A,B).

Figure 5 .
Figure 5. Differential expression of genes in Erβ fl/fl follicles during PFGA.PdFs and PrFs were isolated from 3-week-old Erβ fl/fl mouse ovaries.Isolated PdFs and PrFs were subjected to RNA-Seq analyses.Heatmaps (all genes) (A) and volcano plots (B) indicate the differential expression of genes in Erβ fl/fl PrFs compared with Erβ fl/fl PdFs.Both heatmaps and volcano plots show that a large number of genes are significantly upregulated during PFGA of Erβ fl/fl PdFs (A,B).

Figure 6 .
Figure 6.Differentially expressed genes in Erβ null follicles during PFGA.PdFs and PrFs were isolated from 3-week-old Erβ null mouse ovaries.Isolated PdFs and PrFs were subjected to RNA-Seq analyses.Heatmaps (all genes) (A) and volcano plots (B) indicate the differential expression of genes in Erβ null PRs compared with Erβ null PdFs.

Figure 6 .
Figure 6.Differentially expressed genes in Erβ null follicles during PFGA.PdFs and PrFs were isolated from 3-week-old Erβ null mouse ovaries.Isolated PdFs and PrFs were subjected to RNA-Seq analyses.Heatmaps (all genes) (A) and volcano plots (B) indicate the differential expression of genes in Erβ null PRs compared with Erβ null PdFs.

Table 2 .
Differentially expressed transcription factors in Erβ null mouse primordial follicles.

Table 3 .
Differentially expressed epigenetic regulators in Erβ null mouse primary follicles.

Table 4 .
Differentially expressed transcription factors in Erβ null mouse primary follicles.