Granulosa cell genes that regulate ovarian follicle development beyond the antral stage: The role of estrogen receptor β

Abstract

Follicle development beyond the preantral stage is dependent on gonadotropins. FSH signaling is crucial for the advancement of preantral follicles to the antral stage, and LH signaling is essential for further maturation of preovulatory follicles. Estrogen is intricately tied to gonadotropin signaling during the advanced stages of folliculogenesis. We observed that Erβ^null ovarian follicles fail to develop beyond the antral stage, even after exogenous gonadotropin stimulation. As ERβ is primarily expressed in the granulosa cells (GCs), we explored the gonadotropin-regulated GC genes that induce maturation of antral follicles. Synchronized follicle development was induced by administration of exogenous gonadotropins to wildtype 4-wk-old female rats. The GC transcriptome was analyzed via RNA-sequencing before and after gonadotropin stimulation. An Erβ^null mutant model that fails to show follicle maturation was also included in order to identify the ERβ-regulated genes involved at this step. We observed that specific groups of genes were differentially expressed in response to PMSG or hCG administration in wildtype rats. While some of the PMSG or hCG-induced genes showed a similar expression pattern in Erβ^null GCs, a subset of PMSG- or hCG-induced genes showed a differential expression pattern in Erβ^null GCs. These latter ERβ-regulated genes included previously known FSH or LH target genes including Lhcgr, Cyp11a1, Cyp19a1, Pgr, Runx2, Egfr, Kiss1, and Ptgs2, which are involved in follicle development, oocyte maturation, and ovulation. We also identified novel ERβ-regulated genes including Jaml, Galnt6, Znf750, Dusp9, Wnt16, and Mageb16 that failed to respond to gonadotropin stimulation in Erβ^null GCs. Our findings indicate that the gonadotropin-induced spatiotemporal pattern of gene expression is essential for ovarian follicle maturation beyond the antral stage. However, expression of a subset of those gonadotropin-induced genes is dependent on transcriptional regulation by ERβ.

Keywords: ERβ; Follicle maturation; Gonadotropins; Granulosa cells; Transcriptome analyses.

Figure 1.. Defective follicle maturation in Erβ^null ovaries.

Hematoxylin and Eosin stained sections of ovaries collected from gonadotropin-treated 4-wk-old wildtype rats show different stages of follicles including antral, and preovulatory or Graafian follicles (A-B). In contrast, the sections of ovaries collected from Erβ^null rats show numerous antral follicles of similar size (C-D). Follicle counts were conducted to determine the numbers of antral and preovulatory follicle (Graafian follicle) in wildtype and in Erβ^null ovaries (E and G). Ovaries from wildtype rats contain antral and graafian follicles, but Erβ^null ovaries are devoid of preovulatory (Graafian) follicles (>600mm) and showing only numerous antral follicles (A-D, E and G). Erβ^null follicles do not undergo selection and dominance in response to gonadotropin and lack preovulatory maturation, which is observed in wildtype ovaries following stimulation with PMSG and hCG (F and H). hCG 4h, 4h after hCG administration to PMSG treated rats. Follicle count data represented as mean ± SEM. n ≥ 3. *P ≤ 0.05.

Figure 2.. RNA-sequencing of granulosa cells collected from gonadotropin-treated wildtype and Erβ^null rat ovaries.

4-wk-old wildtype and Erβ^null rats were treated with gonadotropins, and ovaries were collected before and after treatment for isolation of granulosa cells (GCs): before treatment (Basal), 48 hours after PMSG injection (PMSG), and 4h after hCG injection (hCG 4h) (A). Sequencing was performed on total RNA extracted from GCs, and data were analyzed by using CLC Genomics Workbench. Hierarchical clustering was performed on the differentially expressed genes (DEGs) among replicates at different stages of gonadotropin treatment in wildtype (B-D) or Erβ^null (E-G) GCs. In addition, clustering was performed on the DEGs between wildtype and Erβ^null rat GCs in Basal (H), PMSG (I), and hCG 4h (J) groups.

Figure 3.. Identification of PMSG- and hCG-regulated genes that are dependent on ERβ signaling.

Upregulated (up reg) genes from the Basal to PMSG treated group in wildtype (WP vs WB) granulosa cells (GCs) were compared with that of Erβ^null GCs (MP vs MB). These two sets of DEGs were then compared with the genes that were differentially downregulated (down reg) in PMSG-treated Erβ^null GCs compared to that of wildtype GCs (MP vs WP) to identify the potential PMSG-induced genes that are dependent on ERβ signaling (A). Downregulated genes from the Basal to PMSG group in wildtype GCs (WP vs WB) were compared with that of Erβ^null GCs (MP vs MB). These two sets of DEGs were then compared with the genes that were upregulated in PMSG-treated Erβ^null GCs (MP vs WP) to identify the PMSG-inhibited genes that are dependent on ERβ (B). Similarly, upregulated genes in PMSG to hCG 4h wildtype GCs (W4h H vs WP) were compared to that of Erβ^null GCs (M4h H vs MP), and subsequently compared with the genes downregulated in Erβ^null GCs 4h after hCG treatment (M4hH vs W4h H), to identify the hCG-induced genes that are dependent on ERβ (C). Finally, downregulated genes from PMSG to hCG 4h wildtype GCs (W4h H vs WP) were compared to that of Erβ^null GCs (M4h H vs MP), and then with the differentially upregulated genes in Erβ^null hCG 4h GCs (M4h H vs W4h H), to detect the hCG inhibited genes that are dependent on ERβ (D). WB, Wildtype Basal; WP, Wildtype PMSG; W4h H, Wildtype 4h post-hCG; MB, Erβ^null Basal; MP, Erβ^null PMSG; M4h H, Erβ^null 4h post-hCG.

Figure 4.. PMSG-regulated genes that are ERβ dependent and involved in folliculogenesis.

PMSG and ERβ dependent target genes that were identified in the Fig. 3A were subjected to Ingenuity Pathway Analysis (IPA). IPA revealed a group of downregulated genes that have FSH/FSHR as an upstream regulator (A) and are involved in steroid metabolism, folliculogenesis, ovulation, molecular transport, cell death, and cell survival (sur). The expression of these genes was further validated by RT-qPCR analysis (B-G). We did not detect any significant difference in the expression levels or pattern of Fshr between wildtype and Erβ^null GCs (B). In wildtype GCs, Lhcgr, Cyp11a1 Cyp19a1, Gata4 and Npr2 expression significantly increased from Basal to PMSG and then returned to basal levels at 4h after hCG and remained unchanged at 10h after hCG (C-G). In Erβ^null GCs the pattern of expression of Lhcgr was similar to that of wildtype GCs, but following PMSG Lhcgr expression was significantly lower in Erβ^null GCs compared to wildtype GCs (C). Cyp11a1 and Cyp19a1 expression showed an increasing pattern from Basal to hCG4h and remained unchanged at hCG10h, however, following PMSG Cyp11a1 and Cyp19a1 expression was significantly lower in Erβ^null GCs compared to wildtype GCs (D, E). There was no significant change in the expression of Gata4 and Npr2 from Basal to 10h hCG. But following PMSG treatment, Gata4 and Npr2 expression was significantly lower in Erβ^null GCs compared to wildtype GCs (F, G). RT-qPCR data are represented as mean ± SEM. n ≥ 6. *P ≤ 0.05.

Figure 5.. hCG-regulated genes that are dependent on ERβ and involved in follicle maturation.

hCG-regulated genes that were identified in Fig. 3C were subjected to Ingenuity Pathway Analysis (IPA). IPA revealed a subset of LH/LHCGR-regulated genes involved in steroidogenesis, cumulus cell expansion, oocyte maturation, and ovulation (A). The expression of some of the key genes involved in follicle maturation and ovulation were further confirmed by RT-qPCR (B-G). The expression of Pgr, Egfr and Ptgs2 remained unchanged from Basal to PMSG but increased significantly at hCG 4h and then came to basal levels at 10h following hCG in wildtype GCs (B, D, F). The expression levels and pattern of Pgr, Egfr and Ptgs2 in Erβ^null GCs were similar to that of wildtype GCs except at hCG4h, where the expression was significantly lower in Erβ^null GCs compared to wildtype GCs (B, D, F). The expression of Runx2, Kiss1 and Adamts1 increased slightly from Basal to PMSG but increased rapidly at hCG 4h and then remained unchanged at hCG 10h in Erβ^null GCs (C, E, G). The expression pattern of Runx2 and Adamts1 in Erβ^null GCs was similar to that of wildtype GCs, but thereafter at hCG4h and hCG 10h expression was significantly lower in Erβ^null GCs compared to wildtype GCs (C, G). The expression of Kiss1 remained unchanged from Basal to hCG 10h in Erβ^null GCs. In contrast, Kiss1 expression level at PMSG, hCG4h and hCG 10h was significantly higher in wildtype GCs as compared to Erβ^null GCs (E). RT-qPCR data are represented as mean ± SEM. n ≥ 6. *P ≤ 0.05.

Figure 6.. Gonadotropin-induced and ERβ-regulated novel genes with potential roles in follicle development.

We identified several PMSG (A-D) and hCG (E, F) regulated genes, which were not previously shown to be regulated by ERβ or gonadotropins. We identified these genes based on their relative abundance (TPM values) and fold change in Erβ^null GCs. PMSG induced genes included Jaml, Galnt6, Znf750, and Dusp9 (A-D). The expression of Jaml was higher basally and following PMSG in wildtype GCs as compared to Erβ^null GCs and declined following stimulation with hCG (A) whereas Erβ^null GCs the expression remained unchanged following gonadotropin stimulation (A). The expression of Galnt6, Znf750, and Dusp9 increased significantly from Basal to PMSG and declined after hCG stimulation in wildtype GCs (B-D). In Erβ^null GCs, expression of Galnt6 remained unchanged from Basal to PMSG, increased slightly 4h after hCG, 4h and then decreased at hCG10h. The PMSG-induced increase in Galnt6 observed in wildtype GCs was completely absent in Erβ^null GCs (B). The expression of Znf750 and Dusp9 in Erβ^null GCs remained unchanged from Basal to hCG10h, which is in contrast to PMSG induction of expression in wildtype GCs (C, D). The expression of Wnt16 remained unchanged from Basal to PMSG, increased significantly from PMSG to hCG 4h and then declined to basal levels at hCG10h in wildtype GCs (E). The expression of Wnt16 remains unchanged from Basal to hCG10h in Erβ^null GCs (E). Expression of Mageb16 was increased at 4 and 10h following hCG stimulation in wildtype GCs (F). Expression of Mageb16 was also increased by hCG in Erβ^null GCs, but this induction was markedly lower than that achieved in wildtype GCs at hCG4h and hCG10h (F). RT-qPCR data are represented as mean ± SEM. n ≥ 6. *P ≤ 0.05.

Figure 7.. ERβ-regulated granulosa cell genes that are crucial for gonadotropin induced follicle maturation.

Expression of FSH/FSHR-induced genes including Cyp11a1, Cyp19a1, Lhcgr, Gata4, Npr2, Jaml, Galnt6, Znf750, and Dusp9 as well as expression of LH/LHCGR-induced genes including Pgr, Runx2, Egfr, Kiss1, Ptgs2, Adamts1, Wnt16, and Mageb16 are dependent on ERβ-signaling. These genes play a crucial role in the preovulatory maturation of ovarian follicles.