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International Journal of Molecular Sciences logoLink to International Journal of Molecular Sciences
. 2024 Mar 11;25(6):3202. doi: 10.3390/ijms25063202

Loss of ERβ Disrupts Gene Regulation in Primordial and Primary Follicles

Eun Bee Lee 1,†, V Praveen Chakravarthi 1,†, Ryan Mohamadi 1, Vinesh Dahiya 1, Kevin Vo 1, Anamika Ratri 1, Patrick E Fields 1, Courtney A Marsh 2, M A Karim Rumi 1,*
Editor: Mikhail P Ponomarenko
PMCID: PMC10970686  PMID: 38542176

Abstract

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.

Keywords: primordial follicle growth activation, estrogen receptor β, primordial follicles, primary follicles, transcriptome analysis

1. 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 second-wave 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 gonadotropin-independent 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 (Erβnull) 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 Erβnull 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 Erβnull 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.

2. Results

2.1. 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 (Figure 1 and Figure 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.

Figure 1.

Figure 1

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Erβ expression in primordial follicle (PdFs) and primary follicles (PrFs). PdFs and PrFs were isolated from 3-week-old Erβfl/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

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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). Erβnull follicles were negative for ERβ detection (I,J).

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). Erβnull follicles were negative for the IF staining of total ERβ (Figure 2I,J), so we did not examine the localization of pERβ in Erβnull follicles.

2.2. Differential Expression of Follicular Genes in Erβnull Primordial Follicles

We compared the transcriptomes of Erβnull PdFs with those of Erβfl/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 Erβnull 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 enhance or repress gene expression in PdFs (Figure 3A,B). The top 10 upregulated genes in the Erβnull PdFs include Av320801, H2ac19, Or11a4, Gm14147, Gm5795, Gm8947, Gm21103, Gm12184, Pramel28 and Or8b41, whereas the top 10 downregulated genes are Gm49388, Nutf2, Fam151a, Vsx2, Gm49378, Pabpn1l, H3c2, Tead3, Dnmt1, and Gdpd2 (Supplementary Table S1).

Figure 3.

Figure 3

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Differential expression of genes in Erβnull primordial follicle (PdFs). PdFs were isolated from 3-week-old Erβnull and age-matched Erβfl/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 Erβnull PdFs (B). In the absence of ERβ, there was an increased number of downregulated genes in Erβnull 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.

2.3. Differential Expression of Follicular Genes in Erβnull Primary Follicles

We also analyzed the transcriptome profile in Erβnull PrFs and compared it with the genes expressed in the Erβfl/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 Erβnull PrFs (≥2-fold change; FDR p-value ≤ 0.05). In Erβnull 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 upregulating the inactivated PrFs. Thus, ERβ is not only required for gene regulation in ovarian follicles before PFGA (i.e., PdFs) but also in ovarian follicles after PFGA (i.e., PrFs) (Figure 3 and Figure 4). The top 10 upregulated genes in Erβnull PrFs include Gm5128, Rhox4a2, Gn11757, Or4x18, H2bc23, Gm5798, Gm45799, Mageb1, Gm20605 and Vmn1r242, whereas the top 10 downregulated genes are Fam177a, Gm7903, H4c18, Zfp968, Gm14288, Ott, Map11c3a, Thoc7, Pigy1, and Derpc (Supplementary Table S2).

Figure 4.

Figure 4

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Differential expression genes in Erβnull primary follicles (PrFs). PrFs were isolated from 3-week-old Erβnull and age-matched Erβfl/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 Erβnull PrFs. Both heatmaps and volcano plots show that a larger number of genes are downregulated in Erβnull PrFs compared with those of Erβfl/fl PrFs.

2.4. Differential Expression of Follicular Genes during PFGA

We also identified the DEGs during PFGA of Erβfl/fl PdFs (Figure 5A,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 change; FDR p-value < 0.05) in Erβfl/fl PrFs compared with Erβfl/fl PdFs. Additionally, 56% (2765 out of 4909) of the DEGs were upregulated, and 44% of DEGs were downregulated, indicating that, in the ERβ, both gene induction as well as gene repression occur during the normal PFGA process.

Figure 5.

Figure 5

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Differential expression of genes in Erβfl/fl follicles during PFGA. PdFs and PrFs were isolated from 3-week-old Erβfl/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 Erβfl/fl PrFs compared with Erβfl/fl PdFs. Both heatmaps and volcano plots show that a large number of genes are significantly upregulated during PFGA of Erβfl/fl PdFs (A,B).

In contrast, during the PFGA of Erβnull PdFs (Figure 6 A,B), we detected that a total of only 824 out of 20,268 genes with TPM value ≥ 5 were differentially expressed in Erβnull PrFs compared with Erβnull PdFs (≥2-fold change; FDR p-value ≤ 0.05). Of the DEGs, most of the genes (about 93%, 770 out of 824 genes) were downregulated, indicating the importance of proper gene enhancing role of ERβ during PFGA.

Figure 6.

Figure 6

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Differentially expressed genes in Erβnull follicles during PFGA. PdFs and PrFs were isolated from 3-week-old Erβnull 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 Erβnull PRs compared with Erβnull PdFs.

When the DEGs between the two groups (PFGA in Erβfl/fl and PFGA in Erβnull groups) were compared, we detected that only 546 genes were common and the rest of the DEGs were group-specific (Figure 7A). We observed that 4363 genes that were differentially expressed in Erβfl/fl follicles during PFGA were missing in Erβnull follicles during PFGA. Instead, 278 ERβ-independent genes were differentially expressed in Erβnull follicles during their PFGA (Figure 7A).

Figure 7.

Figure 7

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Venn diagram analysis of differential gene expression. (A) Venn diagram representing the differentially expressed genes (DEGs) observed during PFGA of Erβnull follicles (between Erβnull primary follicles (PrFs) and Erβnull primordial follicles (PdFs)) compared with DEGs during PFGA of Erβfl/fl follicles (between Erβfl/fl PrFs and Erβfl/fl PdFs). (B) Venn diagram representing the DEGs between Erβnull and Erβfl/fl PrFs compared with DEGs between Erβnull PrFs and Erβfl/fl PrFs.

To identify the ERβ-regulated genes that play a role in PFGA, we also compared the DEGs between Erβnull PdFs and Erβfl/fl PdFs (866 genes; Figure 3) with the DEGs between Erβnull PrFs and Erβfl/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 Erβnull PrFs during PFGA (Figure 7B). These findings suggest that, while Erβnull follicles lack the genes that are expressed during the PFGA of Erβfl/fl follicles, they nevertheless expressed a large number of aberrant genes, which may be responsible for the abnormal phenotypes of activated Erβnull follicles.

2.5. ERβ Regulation of Epigenetics and Transcription Factors in Primordial Follicles

When we compared the transcriptomes in Erβnull PdFs to Erβfl/fl PdFs, Erβnull PrFs to Erβfl/fl PrFs, and Erβnull PrFs to Erβnull 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 Erβnull PdFs for transcriptional and epigenetic regulators.

Among the 866 DEGs in Erβnull 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 Erβnull PdFs, including Tet3, Npm2, Mbd3, Ezh2, Dnmt1, Chd3 Chd4 and Chd7 (Table 1).

Table 1.

Differentially expressed epigenetic regulators in Erβnull mouse primordial follicles.

Name Chrom ENSEMBL Region Max TPM Fold Change FDR p-Value
Nek6 2 ENSMUSG00000026749 38401655..38484618 9.22 2.38 0.04
Chd4 6 ENSMUSG00000063870 125072944..125107554 102.71 −2.03 0.04
Kat6b 14 ENSMUSG00000021767 21531502..21722546 34.22 −2.03 0.02
Baz1b 5 ENSMUSG00000002748 135216118..135274983 52.87 −2.04 0.02
Ppm1g 5 ENSMUSG00000029147 Comp (31360008..31378031) 50.81 −2.07 0.04
Top2a 11 ENSMUSG00000020914 Comp (98883769..98915015) 41.65 −2.17 0.02
Chd7 4 ENSMUSG00000041235 8690406..8867659 16.81 –2.18 0.04
Scmh1 4 ENSMUSG00000000085 120262478..120387383 28.73 −2.18 0.04
Cul4a 8 ENSMUSG00000031446 13155621..13197940 40.18 −2.20 0.01
Chd3 11 ENSMUSG00000018474 Comp (69234099..69260232) 33.08 −2.25 0.01
Srcap 7 ENSMUSG00000053877 127111155..127160391 38.99 −2.26 0.04
Paf1 7 ENSMUSG00000003437 28092376..28098813 43.45 −2.29 0.03
Safb 17 ENSMUSG00000071054 56891825..56913294 50.16 −2.46 0.03
Idh2 7 ENSMUSG00000030541 Comp (79744594..79765140) 45.59 −2.47 0.02
Phf1 17 ENSMUSG00000024193 27152026..27156882 55.85 −2.49 0.03
Cit 5 ENSMUSG00000029516 115983337..116147006 28.47 −2.50 0.02
Chaf1a 17 ENSMUSG00000002835 56347439..56379289 39.39 −2.65 0.01
Mbd3 10 ENSMUSG00000035478 Comp (80228373..80235384) 49.18 −2.65 0.01
Ezh2 6 ENSMUSG00000029687 Comp (47507073..47572275) 45.82 −2.75 0.03
Ruvbl1 6 ENSMUSG00000030079 88442391..88474554 28.33 −2.87 0.03
Apex1 14 ENSMUSG00000035960 51162425..51164596 42.76 −2.92 0.03
Gse1 8 ENSMUSG00000031822 120955195..121308129 19.72 −2.95 0.03
Phf12 11 ENSMUSG00000037791 77873580..77921365 34.51 −2.97 0.01
Tet3 6 ENSMUSG00000034832 Comp (83339355..83436066) 102.33 −3.49 0.02
Npm2 14 ENSMUSG00000047911 Comp (70884742..70896684) 274.25 −4.45 0.00
Dnmt1 9 ENSMUSG00000004099 Comp (20818505..20871184) 337.80 −8.16 0.00
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We also identified 50 of the DEGs in Erβnull PdFs that were transcription factors, with 21 upregulated and 29 downregulated (Table 2). The upregulated transcription factors include Zfp985, Zfp429, Hmx2, Tbx20, Lin28b, Pax5 and Klf6, whereas the downregulated transcription factors are Foxl2, Tet3, Tead3, Pax1, Dnmt1, E2f1, Kmt2b, Mbd3, Fou5f1, Lin28a, Vax2, and E2f4 (Table 2).

Table 2.

Differentially expressed transcription factors in Erβnull mouse primordial follicles.

Name Chrom ENSEMBL Region Max TPM Fold Change FDR p-Value
Gm9048 10 ENSMUSG00000112495 Comp (118182176..118184584) 45 7.54 0.00
Zfp985 4 ENSMUSG00000065999 147637734..147669655 24.54 6.53 0.00
Zfp429 13 ENSMUSG00000078994 Comp (67536024..67547938) 12.35 5.22 0.03
Hmx2 7 ENSMUSG00000050100 131150502..131159743 10.49 5.18 0.01
Zfp988 4 ENSMUSG00000078498 147390131..147418191 38.74 4.86 0.00
Tigd5 15 ENSMUSG00000103906 75781584..75786384 8.47 4.26 0.02
Zfp595 13 ENSMUSG00000057842 Comp (67461062..67480634) 12.72 4.19 0.03
Bsx 9 ENSMUSG00000054360 40785423..40791353 8.04 4.03 0.04
Tbx20 9 ENSMUSG00000031965 Comp (24629434..24685599) 10.79 3.79 0.00
Zfp488 14 ENSMUSG00000044519 Comp (33689027..33700721) 20.24 3.72 0.01
Zfp994 17 ENSMUSG00000096433 Comp (22416246..22444597) 12.46 3.49 0.02
Zfp831 2 ENSMUSG00000050600 174485327..174552625 6.34 3.10 0.03
Lin28b 10 ENSMUSG00000063804 Comp (45252716..45362491) 12.71 2.98 0.04
Pax5 4 ENSMUSG00000014030 Comp (44524757..44710487) 8.18 2.88 0.03
Zfp850 7 ENSMUSG00000096916 Comp (27684279..27713540) 15.1 2.67 0.02
Hdx X ENSMUSG00000034551 Comp (110479628..110606776) 10.72 2.45 0.02
Zfp992 4 ENSMUSG00000070605 146533480..146554749 67.95 2.44 0.03
Klf6 13 ENSMUSG00000000078 5911481..5920393 99.37 2.34 0.02
Dmrta1 4 ENSMUSG00000043753 89567673..89583009 71.39 2.29 0.01
Csrnp3 2 ENSMUSG00000044647 65676111..65861890 13.47 2.07 0.05
Ikzf2 1 ENSMUSG00000025997 Comp (69570373..69726404) 15.84 2.03 0.04
Foxl2 9 ENSMUSG00000050397 98837341..98840596 95.15 −2.10 0.03
Cenpb 2 ENSMUSG00000068267 Comp (131017102..131021987) 34.64 −2.11 0.02
Scmh1 4 ENSMUSG00000000085 120262478..120387383 28.73 −2.18 0.04
Lin28a 4 ENSMUSG00000050966 Comp (133730641..133746152) 28.17 −2.25 0.03
Srcap 7 ENSMUSG00000053877 127111155..127160391 38.99 −2.26 0.04
Zfp651 9 ENSMUSG00000013419 121588396..121600808 17.33 −2.31 0.01
Ahdc1 4 ENSMUSG00000037692 132738571..132805421 28.43 −2.36 0.03
Cic 7 ENSMUSG00000005442 24967129..24993584 41.22 −2.36 0.01
Hsf1 15 ENSMUSG00000022556 76361622..76386113 18.46 −2.36 0.04
Safb2 17 ENSMUSG00000042625 Comp (56867965..56891585) 35.58 −2.41 0.02
Safb 17 ENSMUSG00000071054 56891825..56913294 50.16 −2.46 0.03
Zfp212 6 ENSMUSG00000052763 47897410..47909573 28.34 −2.48 0.03
E2f4 8 ENSMUSG00000014859 106024295..106032002 47.41 −2.48 0.03
Phf1 17 ENSMUSG00000024193 27152026..27156882 55.85 −2.49 0.03
Drap1 19 ENSMUSG00000024914 Comp (5472833..5475007) 99.42 −2.52 0.03
Pou5f1 17 ENSMUSG00000024406 35816915..35821669 43.05 −2.61 0.03
Mbd3 10 ENSMUSG00000035478 Comp (80228373..80235384) 49.18 −2.65 0.01
Kmt2b 7 ENSMUSG00000006307 Comp (30268283..30288151) 35.67 −2.66 0.00
Tcf7l1 6 ENSMUSG00000055799 Comp (72603361..72766237) 17.64 −2.84 0.04
Aebp1 11 ENSMUSG00000020473 5811947..5822088 26.23 −3.20 0.00
Zfp598 17 ENSMUSG00000041130 24888661..24900990 34.89 −3.25 0.00
Tet3 6 ENSMUSG00000034832 Comp (83339355..83436066) 102.33 −3.49 0.02
Sp110 1 ENSMUSG00000070034 Comp (85504620..85526538) 134.34 −3.62 0.01
Zfp821 8 ENSMUSG00000031728 110432178..110451564 18.46 −3.64 0.02
E2f1 2 ENSMUSG00000027490 Comp (154401327..154411812) 455.5 −4.23 0.01
Zfp414 17 ENSMUSG00000073423 33848064..33850753 22.73 −5.25 0.04
Dnmt1 9 ENSMUSG00000004099 Comp (20818505..20871184) 337.8 −8.16 0.00
Tead3 17 ENSMUSG00000002249 Comp (28550645..28569791) 94.04 −9.96 0.00
Vsx2 12 ENSMUSG00000021239 84616536..84642231 159.32 −16.35 0.00
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2.6. ERβ Regulation of Epigenetics and Transcription Factors in Primary Follicles

We further analyzed the DEGs identified in the Erβnull PrFs. Among the 1786 DEGs in Erβnull PrFs (≥2-fold change; FDR p-value < 0.05, TPM ≥ 5), we identified the differential expression of 97 epigenetic regulators, with 95 downregulated and 2 upregulated (Table 3). The downregulated epigenetic regulators include Tet3, Pcna, Chd4, Sin3a, Sin3b, Ezh2, Kdm1a, Kdm1b, Gatad2a, Smarca2, Npm2, Prmt1, Setd1a, Dppa3, and Dnmt1 (Table 3).

Table 3.

Differentially expressed epigenetic regulators in Erβnull mouse primary follicles.

Name Chrom ENSEMBL Region Max TPM Fold Change FDR p-Value
Usp44 10 ENSMUSG00000020020 93667417..93693950 44.84 3.16 0.01
Taf9 13 ENSMUSG00000052293 100788087..100792568 87.70 2.37 0.03
Anp32a 9 ENSMUSG00000032249 62248575..62286094 97.88 −2.01 0.03
Kmt2d 15 ENSMUSG00000048154 Comp (98729550..98769085) 46.90 −2.02 0.00
Sin3b 8 ENSMUSG00000031622 73449913..73484829 33.06 −2.06 0.02
Sf3b1 1 ENSMUSG00000025982 Comp (55024328..55066640) 73.73 −2.08 0.00
Ywhab 2 ENSMUSG00000018326 163836880..163860508 100.30 −2.08 0.00
Trrap 5 ENSMUSG00000045482 144704542..144796588 34.95 −2.08 0.00
Suz12 11 ENSMUSG00000017548 79883932..79924949 97.49 −2.10 0.00
Parp1 1 ENSMUSG00000026496 180396489..180428819 42.15 −2.11 0.01
Huwe1 X ENSMUSG00000025261 150583803..150718413 62.48 −2.11 0.00
Sf3b3 8 ENSMUSG00000033732 Comp (111536871..111573419) 45.78 −2.12 0.00
Bap1 14 ENSMUSG00000021901 30973407..30981901 34.76 −2.12 0.02
Ogt X ENSMUSG00000034160 100683666..100727957 85.85 −2.13 0.00
Tle4 19 ENSMUSG00000024642 Comp (14425514..14575415) 50.47 −2.14 0.00
Crebbp 16 ENSMUSG00000022521 Comp (3899192..4031861) 65.72 −2.17 0.00
Noc2l 4 ENSMUSG00000095567 156320376..156332073 27.12 −2.18 0.05
Ncl 1 ENSMUSG00000026234 Comp (86272441..86287122) 140.95 −2.19 0.00
Wdr5 2 ENSMUSG00000026917 27405169..27426547 51.40 −2.21 0.01
Psip1 4 ENSMUSG00000028484 Comp (83373917..83404696) 178.10 −2.23 0.00
Mllt6 11 ENSMUSG00000038437 97554240..97576289 25.17 −2.25 0.00
Brd2 17 ENSMUSG00000024335 Comp (34330997..34341608) 42.14 −2.25 0.00
Mphosph8 14 ENSMUSG00000079184 56905705..56934887 82.52 −2.26 0.00
Phf1 17 ENSMUSG00000024193 27152026..27156882 44.37 −2.26 0.01
Max 12 ENSMUSG00000059436 Comp (76984043..77008975) 63.02 −2.27 0.01
Ezh2 6 ENSMUSG00000029687 Comp (47507073..47572275) 82.70 −2.28 0.00
Babam1 8 ENSMUSG00000031820 71849505..71857263 41.46 −2.28 0.04
Kdm1a 4 ENSMUSG00000036940 Comp (136277851..136330034) 39.36 −2.30 0.01
Ruvbl1 6 ENSMUSG00000030079 88442391..88474554 43.20 −2.30 0.03
Ubr5 15 ENSMUSG00000037487 Comp (37967572..38079098) 38.86 −2.31 0.00
Ube2b 11 ENSMUSG00000020390 Comp (51876324..51891589) 97.87 −2.32 0.00
Cul4b X ENSMUSG00000031095 Comp (37622151..37665073) 77.46 −2.32 0.00
Gatad2a 8 ENSMUSG00000036180 Comp (70359726..70449034) 64.10 −2.33 0.00
Nap1l1 10 ENSMUSG00000058799 111309084..111334011 147.30 −2.33 0.00
Atn1 6 ENSMUSG00000004263 Comp (124719507..124733487) 30.78 −2.34 0.04
Smarcc1 9 ENSMUSG00000032481 109946776..110069246 52.40 −2.34 0.00
Rbbp4 4 ENSMUSG00000057236 Comp (129200893..129229163) 91.38 −2.35 0.00
Smarca2 19 ENSMUSG00000024921 26582450..26755722 55.11 −2.37 0.00
Kdm1b 13 ENSMUSG00000038080 47196975..47238755 120.51 −2.39 0.00
Phf13 4 ENSMUSG00000047777 Comp (152074090..152080715) 77.03 −2.40 0.00
Ppm1g 5 ENSMUSG00000029147 Comp (31360008..31378031) 42.81 −2.41 0.02
Cul4a 8 ENSMUSG00000031446 13155621..13197940 41.87 −2.42 0.00
Smarce1 11 ENSMUSG00000037935 Comp (99099873..99121843) 78.03 −2.46 0.00
Pcgf6 19 ENSMUSG00000025050 Comp (47022056..47039345) 83.94 −2.47 0.00
Phf12 11 ENSMUSG00000037791 77873580..77921365 48.87 −2.49 0.00
Setd1a 7 ENSMUSG00000042308 127375842..127399294 34.92 −2.50 0.00
Cxxc1 18 ENSMUSG00000024560 74349195..74354567 25.91 −2.50 0.02
Morf4l2 X ENSMUSG00000031422 Comp (135633691..135644439) 100.27 −2.50 0.00
Senp3 11 ENSMUSG00000005204 Comp (69563941..69572910) 37.78 −2.51 0.03
Ube2d1 10 ENSMUSG00000019927 Comp (71090810..71121092) 47.40 −2.52 0.04
Ddb1 19 ENSMUSG00000024740 10582691..10607183 95.54 −2.52 0.00
Ywhaz 15 ENSMUSG00000022285 Comp (36771014..36797173) 255.41 −2.53 0.00
Hdgf 3 ENSMUSG00000004897 87813628..87823439 56.02 −2.57 0.00
Ruvbl2 7 ENSMUSG00000003868 Comp (45071184..45087520) 26.17 −2.59 0.03
Tet3 6 ENSMUSG00000034832 Comp (83339355..83436066) 110.74 −2.60 0.00
Pcna 2 ENSMUSG00000027342 Comp (132091082..132095234) 161.41 −2.62 0.00
Chd4 6 ENSMUSG00000063870 125072944..125107554 73.48 −2.64 0.00
Hmgn1 16 ENSMUSG00000040681 Comp (95921818..95928929) 173.80 −2.64 0.00
Uhrf1 17 ENSMUSG00000001228 56610321..56630486 143.72 −2.68 0.00
Elp5 11 ENSMUSG00000018565 Comp (69859048..69873343) 48.53 −2.69 0.03
Hmgn2 4 ENSMUSG00000003038 Comp (133692049..133695961) 206.22 −2.70 0.00
Dek 13 ENSMUSG00000021377 Comp (47238251..47259677) 100.04 −2.70 0.00
Ube2e1 14 ENSMUSG00000021774 4137837..4186974 84.58 −2.71 0.02
Sfpq 4 ENSMUSG00000028820 126915117..126930806 135.73 −2.73 0.00
Npm2 14 ENSMUSG00000047911 Comp (70884742..70896684) 552.61 −2.73 0.00
Prmt1 7 ENSMUSG00000109324 Comp (44625413..44635992) 50.43 −2.74 0.01
Mybbp1a 11 ENSMUSG00000040463 72332181..72342594 44.28 −2.74 0.00
Smarcd1 15 ENSMUSG00000023018 99600010..99611872 30.23 −2.78 0.00
Sin3a 9 ENSMUSG00000042557 56979324..57035650 110.49 −2.79 0.00
Ube2t 1 ENSMUSG00000026429 134890303..134901900 84.21 −2.83 0.01
Mbip 12 ENSMUSG00000021028 Comp (56375088..56392679) 49.04 −2.83 0.02
Trim28 7 ENSMUSG00000005566 12733041..12764962 96.42 −2.87 0.00
Dnmt1 9 ENSMUSG00000004099 Comp (20818505..20871184) 484.27 −2.90 0.00
Ube2d3 3 ENSMUSG00000078578 135143910..135173959 339.10 −2.94 0.00
Rnf2 1 ENSMUSG00000026484 Comp (151333755..151376706) 81.36 −2.95 0.00
Maz 7 ENSMUSG00000030678 Comp (126621302..126626209) 70.90 −2.95 0.00
Nbn 4 ENSMUSG00000028224 15957925..15992589 28.10 −2.96 0.01
Ssrp1 2 ENSMUSG00000027067 84867578..84877453 44.65 −2.97 0.00
Rbbp7 X ENSMUSG00000031353 161543398..161562088 249.01 −2.97 0.00
Pkm 9 ENSMUSG00000032294 59563651..59586658 92.08 −2.98 0.00
Exosc9 3 ENSMUSG00000027714 36606755..36619876 35.60 −3.01 0.04
Sap30 8 ENSMUSG00000031609 Comp (57935741..57940894) 91.03 −3.08 0.00
Npm1 11 ENSMUSG00000057113 Comp (33102287..33113206) 596.28 −3.08 0.00
Ywhae 11 ENSMUSG00000020849 75623695..75656671 309.89 −3.09 0.00
Clns1a 7 ENSMUSG00000025439 97345841..97370003 63.72 −3.18 0.00
Mta2 19 ENSMUSG00000071646 8919239..8929667 36.75 −3.18 0.00
Anp32b 4 ENSMUSG00000028333 46450902..46472657 385.30 −3.23 0.00
Skp1 11 ENSMUSG00000036309 52122822..52137685 588.59 −3.49 0.00
Mbd3 10 ENSMUSG00000035478 Comp (80228373..80235384) 51.39 −3.71 0.00
Smyd2 1 ENSMUSG00000026603 Comp (189612689..189654560) 37.39 −3.72 0.01
Dpy30 17 ENSMUSG00000024067 Comp (74606469..74630939) 116.71 −3.80 0.00
Mbd6 10 ENSMUSG00000025409 Comp (127117825..127124887) 14.95 −3.96 0.02
Dppa3 6 ENSMUSG00000046323 122603369..122607231 600.49 −4.10 0.00
Setd4 16 ENSMUSG00000022948 Comp (93380345..93400951) 29.71 −4.18 0.01
Sgf29 7 ENSMUSG00000030714 126248481..126272097 44.93 −4.28 0.00
Smarcb1 10 ENSMUSG00000000902 Comp (75732603..75757451) 61.57 −4.78 0.00
Actb 5 ENSMUSG00000029580 Comp (142888870..142892509) 659.90 −4.87 0.00
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We also detected 79 transcription factors among the DEGs, with 17 upregulated and 62 downregulated (Table 4). Important upregulated transcription factors include Nkx6, Hoxb5, Vsx1, Dbx2, and Pou2af1. The downregulated transcription factors include Epas1, Nr5a2, Lhx8, Nobox, Foxl2, Dnmt1, Wt1, Tet3, Myc, Sox4, Gata4, Hif1a, Ybx2, Ybx3, E2f1, E2f5, Mbd3, Jund, Jun, JunB, and Fos (Table 4). Among the downregulated transcription factors, the crucial roles of Foxl2, Lhx8, Nobox, Nr5a2 and Gata4 in regulating PFGA are already known [23,24,25,26,27,28].

Table 4.

Differentially expressed transcription factors in Erβnull mouse primary follicles.

Name Chrom ENSEMBL Region Max TPM Fold Change FDR p-Value
Gm28230 2 ENSMUSG00000100642 74557072..74578262 14.34 11.27 0.04
Batf3 1 ENSMUSG00000026630 190830044..190841142 48.12 7.92 0.03
Zbtb9 17 ENSMUSG00000079605 27192141..27227350 35.39 5.54 0.02
Barhl1 2 ENSMUSG00000026805 Comp (28797691..28806680) 31.09 3.22 0.01
Zfp786 6 ENSMUSG00000051499 Comp (47796200..47807801) 44.70 3.08 0.01
Nkx6-2 7 ENSMUSG00000041309 Comp (139159292..139162713) 35.37 2.94 0.04
Nkx6-3 8 ENSMUSG00000063672 23643285..23648964 29.95 2.94 0.04
Hoxb5 11 ENSMUSG00000038700 96194162..96196947 47.52 2.70 0.03
Nkx6-1 5 ENSMUSG00000035187 Comp (101806005..101812862) 35.86 2.63 0.02
Rax 18 ENSMUSG00000024518 Comp (66061348..66072858) 41.42 2.60 0.01
Vsx1 2 ENSMUSG00000033080 Comp (150522622..150531280) 43.97 2.40 0.01
Zscan20 4 ENSMUSG00000061894 Comp (128477332..128503891) 49.80 2.29 0.00
Msantd1 5 ENSMUSG00000051246 35065356..35081183 42.68 2.26 0.04
Dbx2 15 ENSMUSG00000045608 Comp (95521444..95553841) 47.73 2.26 0.02
Pou2af1 9 ENSMUSG00000032053 51125008..51151380 75.81 2.16 0.02
Zfp474 18 ENSMUSG00000046886 52748987..52772902 57.25 2.15 0.05
Zfp853 5 ENSMUSG00000093910 Comp (143272793..143279378) 61.54 2.02 0.02
Atf4 15 ENSMUSG00000042406 80139385..80141742 104.74 −2.02 0.01
Foxm1 6 ENSMUSG00000001517 128339930..128353109 37.06 −2.04 0.01
Esr2 12 ENSMUSG00000021055 Comp (76167193..76224033) 49.73 −2.04 0.01
Lhx8 3 ENSMUSG00000096225 Comp (154011931..154036296) 50.13 −2.05 0.04
Klf11 12 ENSMUSG00000020653 24701273..24712788 55.50 −2.06 0.00
Kmt2b 7 ENSMUSG00000006307 Comp (30268283..30288151) 27.28 −2.10 0.01
Tsc22d1 14 ENSMUSG00000022010 76652401..76745205 92.41 −2.16 0.00
Epas1 17 ENSMUSG00000024140 87061128..87140838 114.54 −2.16 0.01
Nr5a2 1 ENSMUSG00000026398 Comp (136770309..136888186) 93.18 −2.18 0.00
Thra 11 ENSMUSG00000058756 98631464..98659832 48.00 −2.18 0.03
Zfp277 12 ENSMUSG00000055917 Comp (40365045..40495901) 44.83 −2.18 0.01
Plagl1 10 ENSMUSG00000019817 12936248..13007438 103.20 −2.20 0.00
Hif1a 12 ENSMUSG00000021109 73948149..73994304 105.08 −2.21 0.00
Nr4a1 15 ENSMUSG00000023034 101152150..101172676 64.97 −2.22 0.04
Nfyc 4 ENSMUSG00000032897 Comp (120614635..120688769) 60.97 −2.23 0.03
Sp110 1 ENSMUSG00000070034 Comp (85504620..85526538) 477.64 −2.24 0.01
Nfic 10 ENSMUSG00000055053 Comp (81232020..81291469) 26.92 −2.24 0.01
Phf1 17 ENSMUSG00000024193 27152026..27156882 44.37 −2.26 0.01
Gtf2i 5 ENSMUSG00000060261 Comp (134266688..134343614) 37.57 −2.27 0.00
Max 12 ENSMUSG00000059436 Comp (76984043..77008975) 63.02 −2.27 0.01
Noto 6 ENSMUSG00000068302 85400868..85405859 63.77 −2.29 0.04
Gatad2a 8 ENSMUSG00000036180 Comp (70359726..70449034) 64.10 −2.33 0.00
Foxp4 17 ENSMUSG00000023991 Comp (48178058..48235570) 31.10 −2.33 0.04
Ski 4 ENSMUSG00000029050 Comp (155238532..155307049) 116.77 −2.37 0.00
Fbxl19 7 ENSMUSG00000030811 127343715..127368655 31.37 −2.38 0.00
Sox4 13 ENSMUSG00000076431 Comp (29132902..29137696) 105.03 −2.40 0.00
Myc 15 ENSMUSG00000022346 61857240..61862223 61.30 −2.42 0.00
Fosb 7 ENSMUSG00000003545 Comp (19036621..19043976) 129.20 −2.45 0.02
Zfp57 17 ENSMUSG00000036036 37312055..37321527 167.57 −2.47 0.00
Tgif1 17 ENSMUSG00000047407 Comp (71151200..71160541) 69.51 −2.47 0.00
Pcgf6 19 ENSMUSG00000025050 Comp (47022056..47039345) 83.94 −2.47 0.00
Cxxc1 18 ENSMUSG00000024560 74349195..74354567 25.91 −2.50 0.02
Srf 17 ENSMUSG00000015605 Comp (46859255..46867101) 40.70 −2.51 0.03
Gata4 14 ENSMUSG00000021944 Comp (63436371..63509141) 72.01 −2.52 0.00
Tcf3 10 ENSMUSG00000020167 Comp (80245348..80269481) 48.30 −2.56 0.00
Tet3 6 ENSMUSG00000034832 Comp (83339355..83436066) 110.74 −2.60 0.00
Tcf7 11 ENSMUSG00000000782 Comp (52143198..52174158) 41.81 −2.60 0.01
Akap8l 17 ENSMUSG00000002625 Comp (32540398..32569581) 23.89 −2.61 0.04
Nacc2 2 ENSMUSG00000026932 Comp (25945547..26013232) 206.00 −2.71 0.01
Ybx3 6 ENSMUSG00000030189 Comp (131341818..131365439) 166.05 −2.80 0.00
Foxl2 9 ENSMUSG00000050397 98837341..98840596 99.70 −2.87 0.00
Dnmt1 9 ENSMUSG00000004099 Comp (20818505..20871184) 484.27 −2.90 0.00
Wt1 2 ENSMUSG00000016458 104956874..105003961 99.07 −2.94 0.00
Gpbp1 13 ENSMUSG00000032745 Comp (111562214..111626645) 155.66 −2.94 0.00
Maz 7 ENSMUSG00000030678 Comp (126621302..126626209) 70.90 −2.95 0.00
Nobox 6 ENSMUSG00000029736 Comp (43280608..43286488) 111.11 −2.96 0.00
Cpeb1 7 ENSMUSG00000025586 Comp (80996774..81105213) 146.89 −2.99 0.00
E2f1 2 ENSMUSG00000027490 Comp (154401327..154411812) 777.45 −3.02 0.01
Klf2 8 ENSMUSG00000055148 73072877..73075500 40.10 −3.18 0.02
E2f5 3 ENSMUSG00000027552 14643701..14671369 215.06 −3.21 0.00
Zbed3 13 ENSMUSG00000041995 95460120..95474349 2120.46 −3.68 0.00
Mbd3 10 ENSMUSG00000035478 Comp (80228373..80235384) 51.39 −3.71 0.00
Jund 8 ENSMUSG00000071076 71151599..71153265 359.34 −3.79 0.00
Zfp213 17 ENSMUSG00000071256 Comp (23775741..23783212) 23.71 −3.82 0.01
Egr1 18 ENSMUSG00000038418 34992876..34998037 398.93 −3.91 0.00
Mbd6 10 ENSMUSG00000025409 Comp (127117825..127124887) 14.95 −3.96 0.02
Nme2 11 ENSMUSG00000020857 Comp (93840640..93847085) 142.47 −4.08 0.00
Jun 4 ENSMUSG00000052684 Comp (94937271..94940459) 285.22 −4.14 0.00
Junb 8 ENSMUSG00000052837 Comp (85701113..85705347) 176.68 −4.42 0.00
Fos 12 ENSMUSG00000021250 85520664..85524047 672.23 −4.51 0.00
Bmyc 2 ENSMUSG00000049086 25596751..25597733 91.04 −5.89 0.00
Ybx2 11 ENSMUSG00000018554 69826622..69832431 205.82 −6.59 0.00
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3. 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 Erβnull 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 Erβfl/fl and Erβnull 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 (Table 1, Table 2, Table 3 and Table 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 Erβnull 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 Erβnull PdFs, including epigenetic and transcriptional regulators (Table 1 and Table 2). Our results suggest that such deregulation may lead to the increased susceptibility of PdFs to undergo PFGA. Moreover, following the PFGA, Erβnull PrFs also suffers from the defective expression of many genes, including many epigenetic and transcriptional regulators (Table 3 and Table 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.

4. Materials and Methods

4.1. Animal Models

An Erβ mutant mouse model carrying a floxed exon 3 allele (Erβfl/fl) [45] was included in this study. A mouse line carrying CMV-Cre [46] (006054, Jax Mice) was mated with the Erβfl/fl mice for deletion of the floxed exon three and established heterozygous mouse lines. Erβfl/null male and female mice were mated to generate the Erβnull mutant females. The mouse lines were maintained in C57BL/6J (000664, Jax Mice) genetic background. In all experiments, Erβfl/fl mice were used as normal control. Three-week-old Erβnull and age-matched Erβfl/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.

4.2. 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.

4.3. 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.

4.4. 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).

4.5. 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).

4.6. 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.

4.7. Statistical Analysis

Each RNA-Seq library was prepared using the pooled follicles of three to five individual Erβfl/fl or Erβnull 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 Erβfl/fl and Erβnull 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).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25063202/s1.

ijms-25-03202-s001.zip (127.5KB, zip)

Author Contributions

M.A.K.R. planned the studies, wrote the manuscript, and edited the figures. V.P.C., R.M., V.D., K.V. and A.R. performed all the experiments, analyzed the data, and prepared the figures. E.B.L. initially assisted with some data analyses only. P.E.F. and C.A.M. critically read and edited the final manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

All procedures were performed in accordance with the protocols approved by the University of Kansas Medical Center Animal Care and Use Committee.

Informed Consent Statement

Not applicable.

Data Availability Statement

All RNA-Seq data have been submitted to the SRA (SUB14127580).

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was partly supported by NIH R21 HD105095 grant funding.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ijms-25-03202-s001.zip (127.5KB, zip)

Data Availability Statement

All RNA-Seq data have been submitted to the SRA (SUB14127580).

Articles from International Journal of Molecular Sciences are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

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