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 Erβnull 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 Erβnull 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 Erβnull GCs, a subset of PMSG- or hCG-induced genes showed a differential expression in Erβnull 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 Erβnull 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.
1. INTRODUCTION
Ovarian granulosa (GCs) and theca cells (TCs) play essential regulatory roles in follicle development and oocyte maturation (Eppig, 2001; Jones and Shikanov, 2019; Orisaka et al., 2009). While ovarian follicle activation is gonadotropin-independent, later stages of follicle development require follicle stimulating hormone (FSH) and luteinizing hormone (LH) to induce maturation and ovulation (Goldman and Mahesh, 1968; Goldman and Mahesh, 1969; Grimek et al., 1976; Schwartz, 1974). Gonadotropin signaling initiates steroidogenesis in TCs, which produce androgens (Fortune and Armstrong, 1977) that are converted into estrogens by the neighboring GCs (Erickson and Hsueh, 1978; Liu and Hsueh, 1986). Follicular estrogen in turn regulates the gonadotropin secretion from the H-P axis (Yasin et al., 1995). Gonadotropins also induce differentiation of GCs, which is essential for the expression of factors that regulate the final stages of follicle development, oocyte maturation, and ovulation (Greep et al., 1942; LOSTROH and JOHNSON, 1966). FSH acts through binding the FSH receptor (FSHR) expressed exclusively on GCs, whereas LH acts through binding the LH receptor (LHCGR) expressed on both TCs and GCs (Gromoll et al., 1996; Moudgal, 2012; RAJANIEMI et al., 1977; Simoni et al., 1997). The expression of FSHR on GCs is follicular-stage-specific (Oktay et al., 1997; Peters, 1979), however, the expression of LHCGR on GCs is induced by FSH signaling (Dierich et al., 1998; Zeleznik et al., 1974).
Estrogen signaling plays a dynamic role in ovarian folliculogenesis and steroidogenesis, as well as in the hypothalamic-pituitary-ovarian (H-P-O) axis’ regulation of ovarian functions (Richards, 1980; Shupnik, 2002; Wójtowicz et al., 2007). While TCs predominantly express estrogen receptor alpha (ERα), GCs primarily express estrogen receptor beta (ERβ). Mutant mouse and rat models homozygous for the Erβ null mutation suffer from defective follicle maturation and failure of ovulation (Couse et al., 2005a; Rumi et al., 2017). Although Erβ mutants exhibit an attenuated gonadotropin surge (Jayes et al., 2014; Rumi et al., 2017), administration of exogenous gonadotropins fails to compensate for the defective follicle maturation and induction of ovulation, which suggests a primary intraovarian defect(s) caused by the loss of ERβ (Rumi et al., 2017). Due to the predominant expression of ERβ in GCs, we suspect that dysregulation of ERβ-regulated genes in GCs is responsible for the failure of gonadotropin-induced follicle maturation.
Gonadotropin stimulation induces the differentiation of somatic cells in the ovary and regulates the expression of genes that impact follicle development and oocyte maturation (Dekel and Beers, 1978; Gómez et al., 1993). We observed that Erβnull ovarian follicles fail to develop beyond the antral stage. Previous studies have focused on the attenuated gonadotropin surge and defective expression of GC genes involved in the preovulatory process (Khristi et al., 2018; Rumi et al., 2017). However, ERβ is the predominant estrogen receptor expressed in the ovary since the embryonic stage (Byers et al., 1997b; Couse et al., 2004a; Pelletier and El-Alfy, 2000a; Slomczynska et al., 2001). Thus, in the absence of ERβ, ovarian follicle defects may develop due to alerted gene expression prior to gonadotropin stimulation. Therefore, in this study, we have analyzed the GC transcriptome in Erβnull rat ovaries before and after gonadotropin stimulation and compared them with those of wildtype rats. This systematic approach generated an invaluable source of data understanding the molecular mechanisms involved in ERβ-regulated follicle maturation beyond the antral stage.
2. MATERIALS AND METHODS
2.1. Animal models
4-wk-old Erβnull and age-matched wildtype female Holtzman Sprague-Dawley (HSD) rats were used in this study. Erβnull mutant rats were generated by targeted deletion of exon 3 of the rat Erβ gene (Rumi et al., 2017). Experimental animals were generated by breeding heterozygous mutant male and female rats carrying the exon 3 deletion within Erβ gene. Presence of the Erβ gene mutation was screened for using PCR-based genotyping (Rumi et al., 2017). All animal experiments were performed in accordance with the protocols approved by the University of Kansas Medical Center (KUMC) Animal Care and Use Committee.
2.2. Induction of follicle maturation by gonadotropin administration
Synchronized follicular growth was initiated by administration of exogenous gonadotropins to 4-wk-old Erβnull and wildtype female rats as described previously by our laboratory (Chakravarthi et al., 2020a; Chakravarthi et al., 2018; Khristi et al., 2018; Rumi et al., 2017). Briefly, 30IU of pregnant mare’s serum gonadotropin (PMSG, BioVendor, MO) was injected i.p. into immature female rats, and 48h later some of PMSG treated females received an i.p. injection of 30IU of human chorionic gonadotropin (hCG, BioVendor, MO; Fig. 2A).
2.3. Collection of GCs and RNA-sequencing
Ovarian tissues were collected prior to gonadotropin administration (Basal group), 48h after PMSG injection (PMSG group), and in PMSG-treated rats 4h following hCG injection and 10h post-hCG (hCG 4h and hCG 10h groups). GCs were released from the ovaries by needle puncture under microscopic examination, followed by filtration with a 40μM cell strainer (Chakravarthi et al., 2018; Khristi et al., 2018). Total RNA was extracted from the GCs using TRI Reagent (Sigma-Aldrich, St. Louis, MO) following the manufacturer’s instructions. RNA quality was assessed on an Agilent Bioanalyzer, and samples with RIN values over 9 were selected for mRNA-seq library preparation. Each library was prepared using RNA samples from 3 individual rats and 3 independent libraries were made for each experimental time point. 500 ng of total RNA was used for each RNA-seq library preparation using a TruSeq Standard mRNA kit (Illumina, San Diego, CA) as described in our previous publications (Khristi et al., 2018; Khristi et al., 2019). The cDNA libraries were first evaluated for quality at the KUMC Genomics Core and then sequenced on an Illumina HiSeq X sequencer (Novogene Corporation, Sacramento, CA). All RNA-seq data have been submitted to the Sequencing Read Archive as shown in Supplementary Table 1. Additionally, our previously published RNA-seq data of wildtype and Erβnull GCs collected 10h post-hCG can be used for comparisons (Khristi et al., 2018).
2.4. Analyses of RNA-sequencing data
RNA-sequencing data were demultiplexed, trimmed, aligned, and analyzed using CLC Genomics Workbench 20 (Qiagen Bioinformatics, Germantown, MD) as described previously (Chakravarthi et al., 2020a; Chakravarthi et al., 2019; Khristi et al., 2019). Trimming was performed to remove low quality reads, and good quality reads were aligned with the Rattus norvegicus genome (Rnor_6.0) using the default parameters: (a) maximum number of allowable mismatches was 2; (b) minimum length and similarity fraction was set at 0.8; and (c) minimum number of hits per read was 10. Expression values were measured in TPM (transcripts per million). The threshold p-value was determined according to the false discovery rate (FDR). Differentially expressed genes (DEGs) were selected if the absolute fold change in expression was ≥ 2 with an FDR p-value of ≤0.05 as described previously (Chakravarthi et al., 2020a; Chakravarthi et al., 2019; Khristi et al., 2019).
2.5. Pathway analyses
In the first approach, DEGs were identified between the ‘Basal to PMSG’ and ‘PMSG to hCG 4h’ groups for either wildtype or Erβnull GCs. In the second approach, DEGs were identified in Basal, PMSG, or hCG 4h groups comparing the wildtype and Erβnull data. These DEGs were sub-grouped into upregulated and downregulated genes. Using the Meta-Chart (https://www.meta-chart.com/) software, Venn diagrams were generated by overlapping the upregulated or downregulated DEGs in wildtype and Erβnull data as identified in the first approach. For further analyses, these Venn diagrams were overlapped with genes upregulated or downregulated in Erβnull GCs as identified in the second approach. PMSG- or hCG-regulated genes in wildtype GCs that showed a differential expression in Erβnull GCs were selected as ERβ-regulated genes and subjected to Ingenuity Pathway Analysis (IPA; Qiagen Bioinformatics) to determine their involvement in folliculogenesis.
2.6. Validation of RNA-sequencing by RT-qPCR
For validation of RNA-seq data, selected DEGs were analyzed using RT-qPCR. cDNA was prepared from 1 μg of total RNA purified from the GCs collected at the indicated time points (Basal, PMSG, hCG 4h and hCG 10h) (Fig. 2A) using the High Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific). qPCR was performed using Power SYBR Green Master Mix (ThermoFisher Scientific). A list of qPCR primer sequences used in this study is shown in Supplementary Table 2. The results of RT-qPCR were normalized to Rn18s expression and calculated by the comparative ΔΔCT method (Chakravarthi et al., 2020b; Khristi et al., 2018; Khristi et al., 2019).
2.7. Statistical analyses
Each experimental group consisted of a minimum of 6 rats, and all procedures were repeated for reproducibility (except for RNA-sequencing). The experimental results are presented as the mean ± standard error (SE). The results were analyzed for one-way ANOVA, and the significance of mean differences was determined by Duncan’s post hoc test, with p< 0.05. All the statistical calculations were done using SPSS 22 (IBM, Armonk, NY). Each RNA-seq library was prepared using pooled RNA samples from 3 individual wildtype or Erβnull rats. Each group of RNA-sequencing data consisted of three different libraries.
3. RESULTS
3.1. Defective follicle development in Erβnull rats
Wildtype rat ovaries responded to exogenous gonadotropins and follicles were observed to have varying stages of development including preantral, antral, and large Graafian follicles (Fig. 1A-C). In contrast, gonadotropin-stimulated Erβnull rat ovaries exhibited an abundance of antral follicles of similar size (Fig. 1D-F). While the follicles in wildtype ovaries showed the signs of selection and dominance in response to gonadotropin stimulation (Fig. 1G), the antral follicles in Erβnull ovaries failed to undergo such preovulatory development to Graafian follicles (Fig. 1H).
3.2. GC-transcriptome during gonadotropin-induced follicle development
GC mRNA-seq data for Basal, PMSG, and 4h post-hCG groups were analyzed in two different approaches. In the first approach, DEGs were identified across the treatment groups from Basal to PMSG, and from PMSG to 4h post-hCG within the same genotype (i.e., wildtype or Erβnull; Fig. 2B and 2C). PMSG administration resulted in differential expression of 1720 genes in wildtype and 1085 genes in Erβnull GCs. At 4h post-hCG, 3061 genes were differentially expressed in wildtype and 1055 genes in Erβnull GCs. The PMSG and 4h post-hCG DEGs in wildtype and Erβnull GCs are presented in Supplementary Tables 3-6. In the second approach, DEGs were identified within the Basal, PMSG, or 4h post-hCG groups across the wildtype and Erβnull RNA-seq data (Fig. 2D-F). In in Erβnull GCs, 970 genes were differentially expressed in Basal stage, 1162 genes after PMSG stimulation and 1526 genes at 4h post-hCG. A list of the DEGs in Erβnull GCs compared to wildtype at Basal, PMSG, and hCG 4h time points are presented in Supplementary Tables 7-9.
3.3. ERβ-regulated genes in GCs 48h post-PMSG
Comparison of the PMSG group to the unstimulated (Basal) group within the same genotype exhibited differential expression of 1720 (591 + 1129) genes in wildtype GCs, and 1085 (447 + 638) genes in Erβnull GCs. Direct comparison of PMSG-treated Erβnull GCs to PMSG-treated wildtype GCs identified a total of 1162 DEGs with 453 genes showing downregulation and 709 showing upregulation. These results show a wide divergence in the gene expression profiles in GCs from wildtype and Erβnull rat ovaries. In the Fig. 3A Venn diagram, we compared the PMSG-upregulated (from Basal) genes in wildtype GCs (total 591) and Erβnull GCs (total 447) to those DEGs (total 453) that were downregulated in PMSG-treated Erβnull GCs compared to wildtype GCs. In the side by side comparison of Erβnull and wildtype GCs, only 201 (161+40) genes showed a similar pattern in response to PMSG in both, while 431 (296+135) genes were upregulated in wildtype GCs only and 246 (238+8) genes in Erβnull GCs only. Remarkably, 175 (135+40) genes which were upregulated in wildtype and Erβnull GCs after PMSG treatment were downregulated in Erβnull GCs when compared to wildtype PMSG-treated GCs (Fig. 3A).
In the Figure 3B Venn diagram, we compare the genes downregulated (from Basal) after PMSG treatment, total 1129 in wildtype and 638 in Erβnull GCs, to 709 DEGs that were upregulated in PMSG-treated Erβnull GCs compared to wildtype GCs. Again, the side by side comparison of Erβnull and wildtype GCs indicated that only a small proportion of 423 (59+364) genes showed a similar pattern in response to PMSG in both, while 706 (523+183) genes were upregulated in wildtype GCs only and 215 (209+6) genes in Erβnull GCs only (Fig. 3B). Remarkably, 242 (183+59) genes which were downregulated in wildtype and Erβnull GCs were upregulated in Erβnull GCs when compared to wildtype PMSG-treated GCs (Fig. 3B).
3.4. ERβ-regulated genes in GCs 4h post-hCG
Comparative analyses were performed with the 4h post-hCG GCs (Fig 3C and D) and their respective PMSG controls as described above for the post-PMSG group. While hCG treatment altered the expression of 3061 genes (1547 upregulated and 1514 downregulated) in wildtype GCs, expression of only 1108 genes (447 upregulated and 661 downregulated) were significantly modulated in Erβnull GCs. Direct comparison of hCG-treated Erβnull GCs to hCG-treated wildtype GCs indicated a total of 1527 DEGs, with 662 genes showing downregulation and 865 showing upregulation in the Erβnull GCs. Of the total 1547 upregulated genes in wildtype hCG 4h GCs, 789 (577 +212) genes showed similar expression patterns in Erβnull hCG 4h GCs (Fig. 3C). 758 (466 +292) upregulated genes were unique to wildtype GCs and 212 (196 + 6) to Erβnull GCs. Remarkably, 504 (292+212) genes that were upregulated in wildtype hCG 4h GCs were downregulated in Erβnull hCG 4h GCs (Fig. 3C).
Of the total 1514 genes downregulated in wildtype hCG 4h GCs, 393 (280+113) genes were also downregulated in Erβnull hCG 4h GCs (Fig. 3D). 1121 (811+310) downregulated genes were unique to wildtype GCs and 268 (238 +30) to Erβnull GCs (Fig. 3D). Furthermore, we identified 423 (310 +113) genes that were downregulated in wildtype hCG 4h GCs but were upregulated in ERβnull hCG 4h GCs (Fig. 3D).
3.5. ERβ-regulated FSH/FSHR target genes
As PMSG predominantly acts on FSH receptors (FSHRs), we examined the PMSG-regulated genes using Ingenuity Pathway Analysis (IPA) specifically for FSH/FSHR signaling. IPA demonstrated that a large number of the 175 genes (Fig. 3A) that were upregulated by PMSG treatment (from Basal) in wildtype or Erβnull GCs but downregulated in PMSG-treated Erβnull GCs (compared to PMSG-treated wildtype) have roles in folliculogenesis (Fig. 4A). Further analyses determined that these genes downregulated in Erβnull GCs are specifically involved in steroid metabolism, follicle development, molecular transport, cell survival, ovulation, and fertility (Fig. 4A).
We observed that the expression of Fshr was not decreased in PMSG-treated Erβnull GCs (Fig. 4B), but several other genes known to be upregulated by FSH/FSHR signaling failed to respond appropriately (Fig. 4C-G). Importantly, PMSG-induced upregulation of Lhcgr was absent in Erβnull GCs (Fig. 4C). PMSG-induced upregulation of steroidogenesis-related genes including Cyp11a1 and Cyp19a1 was also absent in Erβnull GCs (Fig. 4D and E). Expression of the transcriptional regulator, Gata4, and Npr2, failed to be induced by PMSG in Erβnull GCs (Fig. 4F and G). Interestingly, expression levels of these genes were quite low in both wildtype and Erβnull GCs at the Basal stage before gonadotropin administration, reaching peak levels 48h after PMSG injection but decreasing to Basal levels 4h after hCG injection (Fig. 4C-G).
3.6. ERβ-regulated LH/LHCGR target genes
hCG is known to act on the LH receptors (LHCGRs) expressed on GCs, and we have examined the hCG-regulated genes in IPA specifically for LH/LHCGR signaling. IPA identified that a large number of the 504 genes (Fig. 3C) that were significantly upregulated (from PMSG) in hCG-stimulated wildtype or Erβnull GCs but downregulated in hCG-stimulated Erβnull GCs (compared to hCG-stimulated wildtype). These genes are involved in preovulatory cumulus cell expansion, oocyte maturation, and the induction of ovulation (Fig. 5A).
In wildtype GCs, expression levels of transcriptional regulators Pgr and Runx2 were low after PMSG treatment but increased rapidly after hCG treatment as expected (Fig. 5B and C). Administration of hCG failed to upregulate the expression of Pgr and Runx2 in Erβnull GCs, but not to the extent that was observed in wildtype GCs (Fig. 5B and C). Among the other key genes involved in preovulatory follicle development and ovulation, Egfr (Fig. 5D) and Ptgs2 (Fig. 5F) showed expression patterns in Erβnull GCs similar to that of Pgr in wildtype GCs (Fig. 5B). In contrast, Kiss1 (Fig. 5E) and Adamts1 (Fig. 5G) showed elevated expression in Erβnull GCs similar to that of Runx2 in wildtype GCs (Fig. 5C). hCG-induced upregulation of these genes (Egfr, Ptgs2, Kiss1, and Adamts1) was either blunted (Fig. 5B, C, F and G) or absent (Fig. 5D and E) in Erβnull GCs.
3.7. ERβ-regulated novel genes involved in follicle development
We also identified novel gonadotropin-stimulated genes that were differentially upregulated in wildtype GCs but failed to respond in Erβnull GCs. We speculate that these genes may contribute to gonadotropin-stimulated ovarian follicle development. However, IPA analyses did not link these genes to either gonadotropin or ERβ signaling. We have selected and validated the expression of theses novel genes based on their higher TPM values and relative fold changes.
Among these genes, Jaml (Fig. 6A), Galnt6 (Fig. 6B), Znf750 (Fig. 6C), and Dusp9 (Fig. 6D) were upregulated by PMSG treatment and downregulated after hCG administration in wildtype GCs. Remarkably, the expression of Jaml (Fig. 6A), which is a cell adhesion molecule, was significantly higher at Basal stage in wildtype GCs. Two other novel genes, Wnt16 (Fig. 6E) and Mageb16 (Fig. 6F), were upregulated in wildtype GCs only after hCG treatment. While the expression of Wnt16 (Fig. 6E) came down to Basal level 10h after hCG treatment, Mageb16 (Fig. 6F) level remained elevated. These genes exhibited minimal changes in Erβnull GCs after gonadotropin treatment.
4. DISCUSSION
Erβnull female rodents are infertile due to defective follicle development and failure of ovulation (Couse and Korach, 1999; Couse et al., 2003; Rumi et al., 2017). In order to elucidate the specific defects in follicle development, we studied exogenous-gonadotropin-induced synchronized follicle development using 4-wk-old female rats. We observed that gonadotropin stimulation fails to initiate the selection and dominance of antral follicles in Erβnull rats. The presence of many antral follicles of similar size and lack of preovulatory Graafian follicles suggest that ERβ is essential for follicle development beyond the antral stage.
ERβ is the major estrogen receptor in the ovary (Byers et al., 1997a; Couse et al., 2004b; Pelletier and El-Alfy, 2000b; Słomczyńska et al., 2001), and GCs are the major cell-type in ovarian follicles that express ERβ (Cheng et al., 2002; Paterni et al., 2014). ERβ plays an important role in the transcriptional regulation of GC genes that are involved in the GC-oocyte interactions required for the development of ovarian follicles (Couse et al., 2005b; Khristi et al., 2018; Rumi et al., 2017). In this study, we analyzed the GC transcriptome before and after gonadotropin administration to identify the ERβ-regulated genes (direct or indirect) that are involved in follicle maturation beyond the antral stage. Using a systematic approach, we identified the DEGs that are responsible for PMSG- and hCG-induced development of wildtype ovarian follicles. Next, among these genes, we identified DEGs that showed a similar or opposite expression pattern in Erβnull GCs. This unique approach of differential transcriptome analysis enabled us to identify the ERβ-regulated genes that are dysregulated after PMSG or hCG administration in Erβnull rats. These ERβ-regulated genes were further subjected to IPA to identify key genes involved in steroidogenesis, follicle development, cumulus expansion, oocyte maturation, and ovulation.
It is noteworthy that, for the transcriptome and subsequent IPA analyses, we have considered PMSG treatment to be representative of FSH/FSHR activation and hCG treatment of LH/LHCGR activation. Despite a normal level of Fshr expression in Erβnull GCs, PMSG treatment failed to induce many of the FSH/FSHR target genes including Lhcgr, Cyp11a1, Cyp19a1, Gata4, and Npr2; however, expression of some other known targets like Star remained unaffected. This suggests that upregulation of a subset of PMSG-induced genes also requires the presence of ERβ in GCs. In wildtype GCs, a low level of expression before gonadotropin treatment (Basal stage) and marked upregulation after PMSG administration suggests that a high level of these proteins is necessary during this stage of follicle development or to execute the responses of LH/LHCGR signaling during the subsequent stage. Moreover, a sharp decrease of selective transcript levels after hCG administration suggests that either LH/LHCGR signaling leads to abrupt downregulation of transcription or degradation of those mRNAs. Such spatiotemporal patterns of expression in response to gonadotropins are characteristic of many of the GC genes involved in follicle maturation (Bédard et al., 2003; Chakravarthi et al., 2015; Chakravarthi et al., 2016; Chen et al., 2009; Lakshminarayana et al., 2014; Ronen-Fuhrmann et al., 1998). However, those reports were based on studying one or a few genes, but our study demonstrates the spatiotemporal pattern of gene expression among the whole transcriptome in wildtype and Erβnull GCs.
PMSG stimulation failed to upregulate the expression of Lhcgr in Erβnull GCs, which is essential for follicle development beyond the antral stage (LaPOLT et al., 1990; Lu et al., 1993; Richards and Ascoli, 2018). Lhcgr knockout mice exhibited an ovarian phenotype with normal development of primary, secondary, and antral follicles but a lack of preovulatory Graafian follicles (Lei et al., 2001), which emphasizes the importance of ERβ-regulated Lhcgr expression. Cyp19a1 knockout mice also developed antral follicles that failed to ovulate (Fisher et al., 1998). It has been reported that decreased levels of Cyp11a1 or Cyp19a1 disrupt the development of antral and Graafian follicles (Gershon and Dekel, 2020). The reduced level of Gata4 expression in Erβnull GCs may contribute to reduced GC proliferation and theca cell recruitment, which would impair the maturation of follicles to the preovulatory stage (Bennett et al., 2013; Efimenko et al., 2013; LaVoie, 2014). Additionally, NPR2 plays an important role in GCs by inducing cGMP levels, which inhibits phosphodiesterase 3A, prevents cAMP hydrolysis, and retrieves the oocytes from meiotic arrest (Arroyo et al., 2020; Potter et al., 2006; Zhang et al., 2010).
We identified that LH/LHCGR target genes, particularly the expression of genes involved in cumulus cell expansion, steroidogenesis, oocyte maturation, and ovulation, were affected in Erβnull GCs. This effect was in part due to the failure of PMSG-induced Lhcgr upregulation in Erβnull GCs. We detected that hCG-induced upregulation of Pgr in GCs is also dependent on ERβ. Progesterone signaling is essential for follicle maturation and induction of ovulation (Jo et al., 2002; Kubota et al., 2016; Robker et al., 2009). Progesterone signaling regulates the expression of Egfr, Ptgs2, and Adamts1 (Robker et al., 2000), which was affected in Erβnull GCs. Studies have shown that EGFR signaling can regulate the expression of Runx2 and Plat, induce production of steroids, and promote oocyte maturation (Jamnongjit et al., 2005). EGFR signaling also plays an essential role in the transition of antral follicles to the preovulatory stage (Panigone et al., 2008; Wu et al., 2019). RUNX2 regulates the expression of Ptgs2 and Runx1, impacting the ovulatory process (Lee-Thacker et al., 2018; Liu et al., 2009). We observed an absence of Ptgs2 upregulation in Erβnull GCs, which might be due to the failure of Runx2 induction in response to hCG stimulation. PTGS2 is involved in prostaglandin synthesis, preovulatory follicle maturation, and ovulation (Laurincik et al., 1993; Siddappa et al., 2015). We previously reported that the expression of Kiss1 in GCs is induced by gonadotropins, which is dependent on ERβ signaling (Chakravarthi et al., 2018). Intraovarian administration of kisspeptin increased the number of preovulatory follicles and decreased the number of antral follicles, whereas treatment with kisspeptin antagonist KP234 had the opposite effect, suggesting a requirement for kisspeptin during preovulatory follicle maturation (Fernandois et al., 2016). Recently, we have also shown that GC-derived KISS1 plays a role in follicle development and oocyte maturation (Chakravarthi et al, 2020). In addition to Ptgs2, hCG-induced upregulation of Adamts1 was not detected in Erβnull GCs. It has been suggested that LH/LHCGR-induced ERK activation is regulated by ADAMTS1, a metalloprotease, which is also involved in cumulus expansion and induction of ovulation (Arroyo et al., 2020; Madan et al., 2003; Shozu et al., 2005; Yung et al., 2010).
We also identified a subset of gonadotropin-induced genes in GCs, which were dependent on ERβ, but they were not known to be regulated by gonadotropin or ERβ signaling or to be involved in ovarian follicle development. Among these novel genes, JAML is a cell-cell adhesion protein and engaged in homophilic interactions (Moog-Lutz et al., 2003). Expression of Jaml was high in the Basal and PMSG groups and decreased after hCG stimulation in wildtype, but, in Erβnull, its expression was very low in all stages. The high level of expression in the Basal and PMSG stages suggests that JAML might have a role in preantral follicles, especially in GC-GC or GC-oocyte interactions. GALNT6 has been shown to be essential for the activation of EGFR by phosphorylation and by modification of O-glycosylation (Lin et al., 2017). It is well known that LH-induced EGFR signaling plays a critical role in preovulatory follicle development by activating MAPK pathways and inducing the steroidogenic enzymes (Jamnongjit et al., 2005; Panigone et al., 2008; Wu et al., 2019). ZNF750 is a cell cycle regulator that acts by inducing Klf4 gene expression (Boxer et al., 2014; Liu et al., 2019; Sen et al., 2012). KLF4 increases the susceptibility of GCs to apoptosis by downregulating Bcl2 expression and promotes LH induced luteal transition of GCs (Choi and Roh, 2018). Dual-specific phosphatases (DUSPs) dephosphorylate MAPKs and thereby inhibiting their activities (Chen et al., 2019). We speculate that PMSG-induced Dusp9 suppresses ERK activation to allow proper development of preantral follicles in wildtype ovaries. In contrast, in Erβnull ovaries, the lack of DUSP9 results in aberrant activation of ERK and leads to premature formation of many small antral follicles. In this study, we also observed a failure of PMSG- or hCG-stimulated induction of either Wnt16 or Mageb16. WNT16 is a positive regulator of the canonical WNT pathway and it plays a potential role in the dominant follicle selection (Gupta et al., 2014). Mage proteins interact with nucleic acids or target proteins and regulate different cell processes (Rajagopalan et al., 2011). Mageb16 was reported to be a testis-specific gene (Liu et al., 2014), and the expression of Mageb16 was found to be low in the ovary (Liu et al., 2014). Interestingly, we observed a drastic increase in the expression of Mageb16 in wildtype GCs after gonadotropin injection, which proves that gonadotropin stimulation is necessary for the expression of Mageb16 in ovaries. Based on the expression and its general function, Mageb16 may have a role in the later stages of follicle development and further studies are needed to confirm its role.
Ours results demonstrate the spatiotemporal pattern of gonadotropin-induced gene regulation in GCs that is essential for ovarian follicle maturation. These datasets may serve as the reference for understanding gonadotropin regulation of GC genes. Although a group of gonadotropin-regulated genes showed similar expression patterns in both wildtype and Erβnull GCs, others were unique to each genotype. We were particularly interested in a subset of gonadotropin-regulated genes characterized by a remarkably different expression pattern in Erβnull GCs. These included FSH signaling regulated genes like Lhcgr, Cyp11a1, Cyp19a1, Gata4, Npr2, Jaml, Galnt6, Znf750, and Dusp6 and LH regulated genes Pgr, Egfr, Adamts1, Ptgs2, Kiss1, Runx2, Wnt16, and Mageb16 (Fig.7). We also identified several novel genes including Jaml, Galnt6, Znf750, Dusp6, Wnt16, and Mageb16, which are gonadotropin-induced but dependent on ERβ, however, further studies are required to confirm their involvement in ovarian follicle maturation.
Disclosure
The authors do not have any conflicts of interest.
Acknowledgments
Generation of these datasets was supported by funding from the KUMC SOM, COBRE (P30 GM122731), and K-INBRE.
Footnotes
Grant support: This study was supported in part by the internal grant supports from KUMC SOM, COBRE, and K-INBRE