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. 2024 Mar 6;103(5):103620. doi: 10.1016/j.psj.2024.103620

NR5A1 and NR5A2 regulate follicle development in chicken (Gallus gallus) by altering proliferation, apoptosis, and steroid hormone synthesis of granulosa cells

Ruixue Nie *,1, Haoyu Tian *,1, Wenhui Zhang *, Fuwei Li †, Bo Zhang *, Hao Zhang *,2
PMCID: PMC10959722  PMID: 38492249

Abstract

Chicken ovarian follicle development is regulated by complex and dynamic gene expression. Nuclear receptor 5A1 and 5A2 (NR5A1 and NR5A2, respectively) are key genes that regulate steroid hormone production and gonadal development in mammals; however, studies on follicular development in the chicken ovary are scarce. In this study, we investigated the functions of NR5A1 and NR5A2 on follicle development in chickens. The results showed that the expression of NR5A1 and NR5A2 was significantly higher in small yellow follicles and F5. Furthermore, the expression of NR5A1 and NR5A2 was significantly higher in follicular tissues of peak-laying hens (30 wk) than in follicular tissues of late-laying hens (60 wk), with high expression abundance in granulosa cells (GC). The overexpression of NR5A1 and NR5A2 significantly promoted proliferation and inhibited apoptosis of cultured GC; upregulated STAR, CYP11A1, and CYP19A1 expression and estradiol (E2) and progesterone (P4) synthesis in GC from preovulatory follicles (po-GC); and increased STAR, CYP11A1, and CYP19A1 promoter activities. In addition, follicle-stimulating hormone treatment significantly upregulated NR5A1 and NR5A2 expression in po-GC and significantly promoted FSHR, CYP11A1, and HSD3B1 expression in GC from pre-hierarchical follicles and po-GC. The core promoter region of NR5A1 was identified at the −1,095- to −483-bp and −2,054- to −1,536-bp regions from the translation start site (+1), and the core promoter region of NR5A2 was at −998 to −489 bp. Two single nucleotide polymorphisms (SNP) were identified in the core promoter region of the NR5A1 gene, which differed between high- and low-yielding chicken groups. Our study suggested that NR5A1 and NR5A2 promoted chicken follicle development by promoting GC proliferation and E2 and P4 hormone synthesis and inhibiting apoptosis. Moreover, we identified the promoter core region or functional site that regulates NR5A1 and NR5A2 expression.

Key words: chicken, NR5A1, NR5A2, granulosa cell, follicle development

INTRODUCTION

The number and quality of follicles on the ovaries directly determine the egg production of hens; thus, efficient follicle development is an indispensable prerequisite for maintaining a high laying rate and achieving great economic output in egg production. High-yielding Hy-Line hens have more pre-ovulatory follicles and larger ovarian weights than a Chinese native chicken breed (Leghari et al., 2015).

Follicle development involves the growth of oocytes, granulosa cells (GC) and theca cells (TC). The proliferation, differentiation, and apoptosis of GC are involved in regulating follicular recruitment, selection, atresia, maturation and ovulation processes. Moreover, steroid hormones, cytokines, and growth factors secreted from GC are essential for the development of the whole follicle (Tilly et al., 1991b; Li et al., 2024; Shen et al., 2024). Many genes have been found to be involved in regulating the proliferation and differentiation of GC, such as ras-related C3 botulinum toxin substrate 1 (RAC1), inhibitor of DNA binding 2 (ID2), bone morphogenetic protein 6 (BMP6), and bone morphogenetic protein 15 (BMP15) genes, which promote the expression of follicle-stimulating hormone receptor (FSHR), steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cyclin D2 (CCND2), and proliferating cell nuclear antigen (PCNA) (Ocón-Grove et al., 2012; Stephens and Johnson, 2016; Tyasi et al., 2020; Huo et al., 2023) as well as bone morphogenetic protein 2 (BMP2), inhibitor of DNA binding 1 (ID1), inhibitor of DNA binding 3 (ID3), and inhibitor of DNA binding 4 (ID4), which inhibit the expression of FSHR (Johnson and Woods, 2009; Haugen and Johnson, 2010).

A previous study showed that anti-Müllerian hormone (AMH) could regulate follicle selection in mouse and identify the stage of follicular development (Durlinger et al., 2001; Huang et al., 2021). Meanwhile, the follicle-stimulating hormone (FSH) is involved in follicle development by regulating the proliferation and differentiation of GC (Woods and Johnson, 2005). However, the key genes and functional approach to GC proliferation, differentiation, apoptosis, and hormone synthesis should be identified to clarify their roles in follicular development in chickens.

Nuclear receptor 5A1 (NR5A1, also known as SF-1) and nuclear receptor 5A2 (NR5A2, also known as LRH-1) are members of the NR5A subfamily of orphan nuclear receptors (Guzmán et al., 2021; Kouri et al., 2024). As transcription factors, NR5A1 and NR5A2 often bind directly to the same or highly similar response elements on DNA to regulate gene transcription (Corzo et al., 2015). In mammals, NR5A1 and NR5A2 are usually expressed in the hypothalamus, pituitary, ovary, and uterus (Hinshelwood et al., 2003) and are involved in a wide range of reproductive processes, especially the regulation of steroid hormone production and gonadal development (Meinsohn et al., 2019). The deficiency of NR5A1 leads to ovarian hypoplasia, reduced oocyte numbers, and complete absence of corpus luteum formation in mice (Luo et al., 1994; Pelusi et al., 2008; Rotgers et al., 2021), while that the deficiency of NR5A2 impairs GC proliferation and ovulation failure in mice (Duggavathi et al., 2008; Meinsohn et al., 2018). Recent studies have identified NR5A1 mutation as a possible single-gene cause of polycystic ovarian syndrome (PCOS) (Crespo et al., 2022) and has potential to treat PCOS (Wen et al., 2024). Other studies have also found that NR5A2 is highly expressed in ovarian GC of patients with PCOS (Lizneva et al., 2016), which further suggests that NR5A1 and NR5A2 play important roles in follicular development and ovulation. Dysregulated expression of NR5A1 and NR5A2 has also been found in ovarian cancer (Chand et al., 2013; Miller et al., 2013). The study of NR5A1 and NR5A2 on mammalian follicular development increasingly comprehensive. In our previous transcriptomic profiles, NR5A1 and NR5A2 were differentially expressed in follicles before and after follicle selection, implicating their role in follicular selection and development (Nie et al., 2022). In the research of regulatory sequences, the core region of the promoter plays a crucial role in regulating gene expression levels (Lubliner et al., 2015). Therefore, accurately identifying the core promoter region and exploring genetic variation is of great significance for understanding the regulatory mechanisms of gene expression (Li et al., 2018).

However, few studies of these 2 genes have been conducted in poultry follicle development. Therefore, our study aimed to clarify the expression pattern of NR5A1 and NR5A2 during follicle development, investigate their regulatory roles in GC, and screen the core promoter region for functional mutations to provide a basis for the chicken reproductive process.

MATERIALS AND METHODS

Animal and Sample Collection

All animal experimental protocols were approved by the Animal Care and Use Committee of China Agricultural University and performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (AW80203202-1-1). The yellow-bearded chickens used in this study were raised in individual cages at the Experimental Chicken Farm of China Agricultural University (Beijing, China), where they were housed in a temperature-controlled room (maintained at 20°C–22°C) with a 14L:10D photoperiod throughout the experiment. The peak- (30 wk, n = 6) and late-laying hens (60 wk, n = 6) were euthanized and dissected to collect the follicles, which were immediately stored at −80°C for total RNA extraction. The blood samples of 2 high-yielding layer breeds (White Leghorn and Rock Island Red chickens) and 3 low-yielding breeds (Huiyang bearded, Tibetan, and Beijing You chickens) were stored in a −20°C refrigerator for genomic DNA extraction.

Plasmid Construction

The coding sequence of NR5A1 and NR5A2 were cloned into a pcDNA3.1-EGFP vector (restriction enzymes HindIII and EcoRI) to obtain 2 overexpression vectors pcDNA3.1-NR5A1-EGFP and pcDNA3.1-NR5A2-EGFP. The promoter fragments of NR5A1, NR5A2, STAR, CYP11A1, and CYP19A1 were cloned into a pGL3-Basic vector (restriction enzymes KpnⅠ and HindIII) to obtain the luciferase vector. The primer information is shown in Supplementary Table 1. All plasmid insertion sequences were verified by sequencing.

Cell Culture and Transfection

The isolation and cultivation of GC and TC were carried out according to previous reports (Gilbert et al., 1977; Qin et al., 2020). Briefly, GC and 293T cell lines were maintained in a basal medium consisting of M199 (Gibco, Gaithersburg, MD) and Dulbecco's modified Eagle medium, respectively, supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) (Li et al., 2022). The recombinant FSH protein (Catalog # 5925-FS-010) was reconstituted in phosphate buffered saline containing at least 0.1% bovine serum albumin and then diluted with complete medium and added to GC at 12 h after post-seeding. The transfection was performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA), according to manufacturer instructions.

Cell Counting Kit 8 and 5-Ethynyl-2-Deoxyuridine Assays

The GC were cultured in a 96-well plate and transfected with an overexpression vector, and the Cell Counting Kit 8 (CCK8) (Beyotime Biotechnology, Shanghai, China) assay was performed at 6, 12, and 24 h after transfection, according to the manufacturer's instructions. In 24-well cell culture plates, the GC were cultured and transfected with pcDNA3.1-NR5A1-EGFP or pcDNA3.1-NR5A2-EGFP. The 5-ethynyl-2-deoxyuridine (EdU) assay was performed at 36 h after transfection, according to manufacturer instructions (Beyotime Biotechnology).

RNA Extraction, cDNA Synthesis, and Quantitative Real-Time Polymerase Chain Reaction

The GC were cultured in 12-well cell culture plates, transfected with an overexpression vector, and collected 36 h after transfection. Total RNA was extracted from cultured cells or tissues using TRIzol (Invitrogen). The cDNA was synthesized using a Fast Quant RT Kit with gDNase Eraser (Tiangen, Beijing, China), and ABclonal 2 × Universal SYBR Green Fast qPCR Mix (ABclonal, Wuhan, China) was used for quantitative real-time polymerase chain reaction (qRT-PCR). The primer information is shown in Supplementary Table 1.

ELISA

The GC from pre-hierarchical follicles (ph-GC) and pre-ovulatory follicles (po-GC) were cultured in 24-well plates and transfected with an overexpression vector for 36 h. The cell culture medium was assayed for progesterone (P4) and estradiol (E2) concentrations using the chicken progesterone/estradiol ELISA kit (Beijing SINO-UK Institute of Biological Technology, Beijing, China), according to kit instructions.

Dual-Luciferase Reporter Assay

The po-GC or 293T cells were cultured in 24-well plates and transfected with luciferase plasmids and Renilla luciferase reporter vector (pRL-TK) at a ratio of 25:1 for 36 h. Luciferase activity was measured using the Dual-Luciferase Report Assay Kit (Promega, Madison, WI) on a SpectraMax i3 × Multi-Mode Microplate Reader (Molecular Devices Corporation, Sunnyvale, CA). The Renilla luciferase signal was normalized to the firefly luciferase signal. All reactions were performed in triplicate.

Single Nucleotide Polymorphism Screening and Analysis

Blood genomic DNA was extracted by referring to the instructions of Tiangen Biochemical Blood Genomic DNA Extraction Kit (Tiangen). The DNA was used as template to amplify the promoter region of the NR5A1 and NR5A2 genes in each breed of chickens. The PCR products with successful amplification of target fragments were verified by Sanger sequencing, and the sequencing results were analyzed through sequence comparison using Chromas Pro and SnapGene software to screen differential single nucleotide polymorphism (SNP) loci in high- and low-yielding laying hens. The χ2 test was used to test for Hardy–Weinberg equilibrium. Finally, genetic parameters, including gene frequency, genotype frequency, homozygosity (Ho), heterozygosity (He), effective number of alleles (Ne), and polymorphism information content (PIC), were calculated.

Statistical Analysis

All data were initially processed in Microsoft Excel 2019. One-way ANOVA and t-test were performed using IBM SPSS Statistics version 25 (IBM Corp., Armonk, NY). All plots were drawn using GraphPad Prism version 7 (GraphPad Software, San Diego, CA). Different lowercase and uppercase letters in figures are used to indicate significant differences (P < 0.05) and highly significant differences (P < 0.01), respectively. The same letters indicate non-significant differences (P > 0.05). * P < 0.05, ** P < 0.01, and *** P < 0.001.

RESULTS

Expression Patterns of NR5A1 and NR5A2 during Follicle Development

The mRNA expression patterns of NR5A1 and NR5A2 gradually increased before follicle selection, which then decreased after the selection. The expression of NR5A1 was highest in small yellow follicle (SYF), the stage at which follicular selection occurs (Figure 1A), while the expression of NR5A2 was highest in F5, which is the first hierarchical follicle after the selection (Figure 1D). The expression of NR5A1 in SYF and F5 of peak-laying hens was significantly higher than that in late-laying hens (P < 0.01) (Figure 1B); however, the expression of NR5A2 was significantly higher in peak-laying hens than in late-laying hens only in SYF (P < 0.05) (Figure 1E). We found that the expression of NR5A1 and NR5A2 was significantly higher in GC than in TC in both SYF and F5 (P < 0.05) (Figures 1C and 1F). These results indicate that NR5A1 and NR5A2 may be involved in regulating follicle selection and development through GC.

Figure 1.

Figure 1

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Expression patterns of NR5A1 and NR5A2 in follicle tissues. Expression profiles of NR5A1 (A) and NR5A2 (D) during the whole follicle development stage of peak-laying hens. Expression profiles of NR5A1 (B) and NR5A2 (E) in peak-laying hens (30 wk) and late-laying hens (60 wk). (C and F) The NR5A1 and NR5A2 expression levels in granulosa cells (GC) and theca cells (TC) of peak-laying hens, respectively. Data are expressed as mean ± SEM. Asterisks indicate significant differences: *P < 0.05 and **P < 0.01. Lowercase letters are used to indicate the level of significance of differences, with the same lowercase or uppercase letters indicating non-significant differences. OS, ovarian stromal tissue; SWF, small white follicle; LWF, large white follicle; SYF, small yellow follicle; F5–F1, hierarchal follicles, sorted from smallest to largest in diameter.

NR5A1 and NR5A2 Promote Proliferation and Inhibit Apoptosis of GC

In cultured primary GC, we transfected vectors (pcDNA3.1-NR5A1-EGFP and pcDNA3.1-NR5A2-EGFP) to overexpress NR5A1 and NR5A2, respectively (Figures 2A and 2B). Both CCK8 and EdU assays demonstrated that overexpressed NR5A1 or NR5A2 could promote GC proliferation (Figures 2C–2F). Moreover, overexpression of NR5A1 significantly promoted the expression of pro-proliferative gene cyclin-dependent kinase 2 (CDK2) and anti-apoptotic gene b-cell lymphoma 6 protein (BCL-6) (P < 0.05) and inhibited the expression of apoptosis-mediated genes, fas cell surface death receptor (Fas), Caspase8, and Caspase9 (P < 0.05) (Figure 2G). Meanwhile, overexpression of NR5A2 significantly promoted the expression of cyclin D1 (CCND1), cyclin D2 (CCND2), and BCL-6 (P < 0.05) and inhibited the expression of Caspase3, Caspase8, and Caspase9 (P < 0.05) (Figure 2H).

Figure 2.

Figure 2

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NR5A1 and NR5A2 promote proliferation and inhibit apoptosis of granulosa cells (GC). (A and B) mRNA expression levels of NR5A1 and NR5A2 after transfection with pcDNA3.1-NR5A1-EGFP or pcDNA3.1-NR5A2-EGFP in GC, respectively. The proliferation rates of GC with NR5A1 and NR5A2 overexpression were assessed by CCK8 assay (C and D) or EdU (E and F). The mRNA expression levels of proliferative and apoptotic-related genes in GC with NR5A1 and NR5A2 overexpression. Asterisks indicate significant differences: *P < 0.05 and **P < 0.01.

NR5A1 and NR5A2 Promote E2 and P4 Hormone Synthesis in po-GC

In cultured ph-GC, neither NR5A1 nor NR5A2 overexpression had a significant effect on the expression of steroid hormone production-related genes (Figures 3A and 3G) or on the concentrations of E2 and P4 (P > 0.05) (Figures 3B–3C and 3H–3I). In po-GC, NR5A1 highly significantly upregulated the expression of CYP11A1, cytochrome P450 family 19 subfamily A member 1 (CYP19A1), and the concentration of E2 and P4 (P < 0.01), but it had no significant effect on the expression of FSHR, STAR, and 3 beta- and steroid delta-isomerase 1 (HSD3B1) (P > 0.05) (Figures 3D–3F). Overexpression of NR5A2 significantly upregulated the expression of STAR, CYP11A1, and CYP19A1 and the concentration of E2 and P4 (P < 0.05) but had no significant effect on the expression of FSHR or HSD3B1 (P > 0.05) (Figures 3J–3L).

Figure 3.

Figure 3

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Effects of NR5A1 and NR5A2 overexpression on estradiol (E2) and progesterone (P4) hormone synthesis in chicken granulosa cells (GC). (A) Expression levels of FSHR, STAR, CYP11A1, CYP19A1, and HSD3B1 in GC from pre-hierarchical follicles (ph-GC) with NR5A1 overexpression. (B and C) Concentrations of P4 and E2 in ph-GC with NR5A1 overexpression assessed by ELISA. (D) Expression levels of FSHR, STAR, CYP11A1, CYP19A1, and HSD3B1 in GC from pre-ovulatory follicles (po-GC) with NR5A1 overexpression. (E and F) Concentrations of P4 and E2 in po-GC with NR5A1 overexpression assessed by ELISA. (G) Expression levels of genes in GC from ph-GC with NR5A2 overexpression. (H and I) Concentrations of P4 and E2 in ph-GC with NR5A2 overexpression. (J) Expression levels of genes in GC from po-GC with NR5A2 overexpression. (K and L) Concentrations of P4 and E2 in po-GC with NR5A2 overexpression. *P < 0.05, **P < 0.01.

NR5A1 and NR5A2 Promote Transcription of STAR, CYP11A1, and CYP19A1 Genes in po-GC

The binding sites of NR5A1 and NR5A2 to STAR, CYP11A1, and CYP19A1 were predicted using JASPAR (Castro-Mondragon et al., 2022). The binding sites of NR5A1 and NR5A2 were found in the promoter regions of STAR, CYP11A1, and CYP19A1 genes, and the binding sequences and positions were identical or similar (Table 1). The results of the dual-luciferase reporter assay showed that NR5A1 and NR5A2 significantly increased promoter activities of STAR (Figures 4A and 4D), CYP11A1 (Figures 4B and 4E), and CYP19A1 (Figures 4C and 4F) in po-GC (P < 0.05).

Table 1.

Binding site prediction information for NR5A1 and NR5A2 regulating STAR, CYP11A1, and CYP19A1 transcription.

Transcription factors Regulated genes Predicted binding site sequence information and location (5′-3′)
NR5A1 STAR AGCAAGGTCAG (−644 to −633)
CYP11A1 AGCAAGGCCAC (−162 to −151)
CYP19A1 ATCAAGGTCAT (−280 to −271)
NR5A2 STAR AGGAGCAAGGTCAGC (−647 to −632)
CYP11A1 CAAAGCAAGGCCACC (−165 to −150)
CYP19A1 CATATCAAGGTCATA (−283 to −270)
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Figure 4.

Figure 4

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NR5A1 and NR5A2 promote the transcription of STAR, CYP11A1, and CYP19A1 in chicken pre-ovulatory granulosa cells (po-GC). Results of the dual-luciferase reporter assay of NR5A1 (A–C) and NR5A2 (D–F). Lowercase letters are used to indicate the level of significance of differences.

NR5A1 and NR5A2 Inhibit AMH Gene Expression in ph-GC

The expression of AMH was higher in ovary stroma (OS), small white follicle (SWF), and large white follicle (LWF) than in SYF (P < 0.05). AMH expression rapidly decreased in F5 and maintained very low levels in other pre-ovulatory follicles (Figure 5A). Overexpression of NR5A1 or NR5A2 significantly inhibited AMH expression in ph-GC (P < 0.05) (Figures 5B and 5C).

Figure 5.

Figure 5

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NR5A1 and NR5A2 inhibit AMH mRNA expression in granulosa cells (GC) from pre-hierarchical follicles (ph-GC). (A) Expression of AMH in follicular tissues at all development stages. (B) Effect of NR5A1 overexpression on AMH in chicken ph-GC. (C) Effect of NR5A2 overexpression on AMH in chicken ph-GC. Asterisks indicate significant differences: *P < 0.05 and **P < 0.01. Lowercase letters are used to indicate the level of significance of differences, and the same lowercase or capital letters indicate non-significant differences.

FSH Regulates the Expression of NR5A1, NR5A2, and Steroid Hormone Biosynthesis Genes

To preliminarily investigate whether the mechanism of the effect of FSH on follicle development occurred through NR5A1 and NR5A2, the ph-GC and po-GC were treated with 50 ng/mL FSH for 24 h. The supplementation of FSH had no significant effect on the expression of NR5A1 or NR5A2 in ph-GC (P > 0.05) (Figure 6A), but it significantly upregulated the expression of NR5A1 and NR5A2 in po-GC (P < 0.01) (Figure 6B). The supplementation of FSH also significantly upregulated the expression of FSHR, CYP11A1, and HSD3B1 both in ph-GC and po-GC (P < 0.05) (Figure 6C and 6D).

Figure 6.

Figure 6

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Effects of follicle-stimulating hormone on the expression levels of NR5A1, NR5A2, and steroid hormone biosynthesis-related genes of estradiol (E2) and progesterone (P4) in chicken granulosa cells (GC). (A and B) Expression of NR5A1 and NR5A2 in GC from the pre-hierarchical follicles (ph-GC) and pre-ovulatory follicles (po-GC), respectively. (C and D) Expression of FSHR, STAR, CYP11A1, CYP19A1, and HSD3B1 in ph-GC and po-GC. *P < 0.05, **P < 0.01.

Identification of NR5A1 and NR5A2 Gene Promoter Core Regions and SNP Sites in High- and Low-Yielding Layer Populations

To identify the promoter regions and SNP that may affect gene transcription expression, we transfected 293T cells with different-length fragment vectors of NR5A1 and NR5A2 promoter regions for 36 h and found that the −1,095- to −126-bp and −2,054- to −126-bp regions from the translation start site (+1) of the promoter regions had the strongest relative luciferase activity (P < 0.05), indicating that these regions are the core promoters of NR5A1. Meanwhile, the −998- to −489-bp region was the core promoter region of NR5A2 (Figures 7A and 7B).

Figure 7.

Figure 7

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Identification of promoter core regions of NR5A1 and NR5A2 and SNP screen. Results of the dual-luciferase reporter assay of NR5A1 (A) and NR5A2 (B) in core promoter region identification. (C and D) Sequencing chromatogram at the SNP sites of the NR5A1 gene. (E) Dual-luciferase analysis for promoter activity. Lowercase letters are used to indicate the level of significance of differences.

Two SNP loci, g. −1704 G > A and g. −1784 G > T, were identified in the core promoter region of NR5A1 (Tables 2 and 3 and Figures 7C and 7D). In high-yielding breed populations (White Leghorn and Rocky Island Red chickens), the locus of g. −1,704 G > A presented only GG genotype, whereas the site in low-yielding populations (Huiyang bearded, Tibetan, and Beijing You chickens) exhibited 3 genotypes (GG, GA, and AA). Their distribution met the Hardy–Weinberg equilibrium. In the high-yielding populations, the locus of g. −1,784 G > T showed polymorphism, and the genotype frequency displayed GT > GG > TT, whereas the locus in the low-yielding populations had a much dominant GG genotype. The genotype frequencies of the 2 loci were significantly different between the high- and low-yielding groups. However, no such locus was screened in NR5A2 promoter regions among the populations.

Table 2.

Comparisons of genetic properties of the NR5A1 gene g. −1,704 G > A locus in high- and low-yielding laying hens.

Breed Genotype frequency
 Gene frequency
 Genetic parameters
 χ2 P-value
GG GA AA G A PIC Ho He Ne
White Leghorn 1 0 0 1 0 0 1 0 1 0 1
Rock Island Red 1 0 0 1 0 0 1 0 1 0 1
Huiyang bearded 0.30 0.40 0.30 0.50 0.50 0.38 0.50 0.50 2.00 0.404 0.817
Tibetan 0.37 0.50 0.13 0.62 0.38 0.36 0.53 0.47 1.89 0.018 0.991
Beijing You 0.40 0.42 0.19 0.60 0.40 0.36 0.52 0.48 1.92 0.446 0.800
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Table 3.

Comparisons of genetic properties of the NR5A1 gene g. −1,784 G > T loci in high- and low-yielding laying hens.

Breed Genotype frequency
 Gene frequency
 Genetic parameters
 χ2 P- value
GG GT TT G T PIC Ho He Ne
White Leghorn 0.35 0.43 0.22 0.57 0.43 0.37 0.51 0.49 1.96 0.203 0.903
Rock Island Red 0.30 0.42 0.28 0.51 0.49 0.37 0.49 0.50 2.06 0.992 0.609
Huiyang bearded 1 0 0 1 0 0 1 0 1 0 1
Tibetan 0.83 0.10 0.07 0.88 0.12 0.19 0.79 0.21 1.27 3.067 0.216
Beijing You 1 0 0 1 0 0 1 0 1 0 1
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DISCUSSION

Chicken follicle development follows a strict order with different morphologies characteristics at each stage. Different cellular components of follicular tissue perform various functions, which are regulated by complex gene expression patterns. In addition, the egg-producing period of hens can be divided into 3 stages, pre-laying, peak laying, and late laying periods, and hens in different laying stages have different physiological states and different functional gene expression levels in their bodies. In recent years, many differentially expressed genes have been found in the follicular tissues of hens at different laying stages or high- and low-yielding layer populations using transcriptome sequencing technology. Some genes, such as NR5A1 and NR5A2 have been identified as candidates for regulating follicle development and egg production (Chen et al., 2021; Sun et al., 2021; Xiang et al., 2022). In the present study, we found that NR5A1 and NR5A2 were significantly more highly expressed in SYF and F5, and their expression was significantly higher in SYF in peak-laying hens (30 wk) than in late-laying hens and significantly higher in GC than in TC. Thus, we inferred that NR5A1 and NR5A2 affect egg production mainly through the regulation of follicle selection and development by GC.

Follicular GC proliferation and apoptosis are 2 important cellular processes affecting follicular development that are regulated by related genes (Tilly et al., 1991b; Zhu et al., 2021a; Zhu et al., 2021b; Yang et al., 2022). For example, IGF1 and VGLL1 stimulate proliferation and inhibit apoptosis in chicken follicular GC, whereas VGLL4 inhibits GC proliferation and stimulates apoptosis in GC (Sun et al., 2022; Zhu et al., 2022). The deletion of NR5A2 resulted in a significant reduction in the transcription products of key cell cycle genes (CCND1, CCND2, CCNE1, and CCNE2) that block GC proliferation in mice (Meinsohn et al., 2018). In our study, we also found that the expression of some genes related to cell proliferation (CCND1, CCND2, CDK2, and BCL-6) was significantly upregulated, whereas that of genes related to apoptosis (Fas, Caspase3, Caspase8, and Caspase9) were significantly downregulated in chicken. The overexpression of NR5A1 and NR5A2 in chicken follicular GC, we initially demonstrated that NR5A1 and NR5A2 regulated chicken follicle development by promoting GC proliferation and inhibiting apoptosis. However, further validation, such as assays at the protein level or through the use of flow cytometry, is required.

Steroid hormones, such as E2 and P4, are critical hormones secreted by the ovary that regulate not only reproductive function but also GC proliferation and differentiation (Vitale et al., 2002; Ding et al., 2012; Will et al., 2017; Yuan et al., 2019). Several key genes, STAR, CYP11A1, CYP19A1, and HSD3B1, are involved in the synthesis of E2 and P4 (Stocco, 1999; Miller, 2008; Miller, 2013; Stocco et al., 2017). In mammalian follicular GC, NR5A1 and NR5A2 regulate the expression of genes, such as STAR, CYP11A1, and CYP19A1, and affect steroid hormone production (Duggavathi et al., 2008; Jin et al., 2016). In this study, we found that NR5A1 and NR5A2 significantly increased the expression of the STAR, CYP11A1, and CYP19A1 genes and the synthesis of E2 and P4 hormones in cultured po-GC, which confirms the regulatory role of NR5A1 and NR5A2 on follicle development in chickens. Interestingly, NR5A1 and NR5A2 had no effect on ph-GC, which suggests that these 2 genes cannot initiate steroid hormone synthesis in undifferentiated GC independently. A previous study demonstrated that AMH secreted by GC may inhibit follicle selection (Johnson et al., 2009). Our study also concluded that NR5A1 and NR5A2 significantly inhibited AMH expression in ph-GC, which may be the pathway through which NR5A1 and NR5A2 regulate follicle development in ph-GC. Furthermore, we revealed that NR5A1 and NR5A2 promote STAR, CYP11A1, and CYP19A1 gene expression and E2 and P4 hormone synthesis in po-GC, inhibit AMH expression in ph-GC, and promote follicle development in chickens.

As we know, FSH is an important hormone that regulates follicular development. The receptor of FSH, FSHR, is a G protein-coupled receptor that specifically binds FSH and mediates cyclic adenosine monophosphate (cAMP) to exert selection of pre-hierarchical follicles and development. Thus, FSHR has been identified as a marker of follicular GC differentiation status and follicular selection (Woods and Johnson, 2005). The enhancement of cAMP signaling mediates the expression of hormone-producing genes and hormone synthesis and is considered to be an important process in follicular selection and development. The FSHR has a selective response to FSH signals (Tilly et al., 1991a; Kim et al., 2013; Kim, 2014; Johnson, 2015), and short treatment (1 or 3 h) of po-GC with 10 ng/mL FSH can increase cAMP production; after which, cAMP activates protein kinase A and induces more steroid hormone-related genes STAR, CYP11A1, and hormone synthesis, but the same treatment does not affect ph-GC.

Several recent studies have found that mitogen-activated protein kinase, protein kinase C, and β-arrestin signaling are partly responsible for preventing ph-GC from responding to FSH treatment and remaining undifferentiated. In contrast, prolonged (18 h) FSH treatment was found to increase STAR and CYP11A1 expression and P4 synthesis in ph-GC (1–8 mm), with the strongest response from the soon-to-be-selected SYF (Johnson and Lee, 2016; Kim and Johnson, 2018). In an in vivo study, increasing the dose of exogenous FSH initiated the selection of multiple follicles to the pre-ovulatory level in a dose-dependent manner (Ghanem and Johnson, 2019). In the present study, we treated chicken follicular GC with 50 ng/mL FSH for 24 h and found that FSH did not promote NR5A1 and NR5A2 expression in ph-GC, although FSH significantly upregulated NR5A1 and NR5A2 expression in po-GC. Combined with the results that the upper part of NR5A1 and NR5A2 had no significant effect on ph-GC but promoted hormone synthesis in po-GC, we believe that the regulatory effect of FSH on pre-hierarchical follicle development was not exerted through NR5A1 and NR5A2 and that the regulatory effect on pre-ovulatory follicle development may be partially achieved by promoting NR5A1 and NR5A2 expression or that FSH has a facilitative effect on the functional performance of NR5A1 and NR5A2. This experiment also indicated that 24-h treatment with 50 ng/mL FSH significantly upregulated the expression of steroid hormone production-related genes FSHR, CYP11A1, and HSD3B1 in follicular GC (ph-GC and po-GC), which is consistent with previous findings (Wang et al., 2017; Li et al., 2019), thereby confirming the important regulatory role of FSH on follicular development.

In mice, NR5A1 binds to the −105 to −95-bp region of the STAR promoter, ensuring maximum basal activity of the STAR transcription (Wooton-Kee, 1999). In human ovarian GC, NR5A2 promotes CYP11A1 expression, and 2 binding sites, −1,580 and −40 bp, have been identified in the promoter region (Kim et al., 2005). NR5A1 and AP-1 synergistically activate CYP11A1 transcription in vitro and in vivo (Guo et al., 2007). Moreover, the expression of CYP19A1 is activated by NR5A1 through binding at the −280- to −271-bp (5′-TCAAGGTCA-3′) site of the promoter region (Wang and Gong, 2017). Previous results suggest that NR5A1 and NR5A2 tend to act as transcription factors to regulate steroid hormone production-related gene expression. In the present study, we confirmed that NR5A1 and NR5A2 promote STAR, CYP11A1, and CYP19A1 gene transcription in chicken po-GC by enhancing promoter activity using a dual-luciferase assay, which appears to be the first time that this regulatory function has been confirmed using chicken follicular GC.

The core promoter is a necessary DNA sequence to ensure transcriptional initiation (Xiang et al., 2014). Using prediction analysis and experimental validation in this study, we found that the core promoter of the NR5A1 gene was in the −1,095- to −483-bp and −2,054- to −1,536-bp regions, whereas that of the NR5A2 gene was in the −998- to −489-bp region.

Gene expression is usually regulated by SNP in promoter regions that can function as molecular markers in breeding. In this study, we found two SNP loci located within the identified core promoter region of NR5A1. The genotype distributions of the SNP were different between the high- and low-yielding chicken groups. Thus, we considered that the two SNP sites might be associated with NR5A1 expression and egg production in chickens. Meanwhile, we selected a 20-bp region before and after each of the two mutated sites and used JASPAR online software to predict the changes in the type and number of transcription factors bound to this region. The results suggest that the g. −1,704 G and g. −1,784 T alleles of high-yield laying hens upregulate gene expression.

Furthermore, we did not find simultaneous mutations in the −1,704 nor −1,784 loci in any of the chickens in this study. Future research should verify the functions of these two mutations in high- and low-yielding populations of the same egg breed, correlate them with egg production performance, identify beneficial mutations in high-yielding hens and deleterious mutation loci in low-yielding hens, and develop them as molecular genetic markers to distinguish high- and low-yielding hens to provide molecular theoretical support for egg breeding improvement.

In conclusion, NR5A1 and NR5A2 are highly expressed in chicken ovarian SYF and F5 during the peak laying period, which can promote GC proliferation, inhibit apoptosis, inhibit AMH gene expression in ph-GC, promote STAR, CYP11A1, and CYP19A1 gene expression and E2 and P4 hormone synthesis, and have important regulatory effects on chicken ovarian follicle development. Two mutant sites, g. −1704 G > A and g. −1784 G > T, in the core promoter region of NR5A1 gene may be responsible for its differential expression in laying hens.

Acknowledgments

ACKNOWLEDGMENTS

This study was supported by the National Key Research and Development Program of China (2022YFD1600902), Key Research and Development Program of Shandong (2022LZGC013), and China Agriculture Research System (CARS-40).

DISCLOSURES

The authors declare no conflict of interest.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.103620.

Appendix. Supplementary materials

mmc1.docx (22.7KB, docx)

REFERENCES

  1. Castro-Mondragon J.A., Riudavets-Puig R., Rauluseviciute I., Lemma R.B., Turchi L., Blanc-Mathieu R., Lucas J., Boddie P., Khan A., Pérez N.M., Fornes O., Leung T.Y., Aguirre A., Hammal F., Schmelter D., Baranasic D., Ballester B., Sandelin A., Lenhard B., Vandepoele K., Wasserman W.W., Parcy F., Mathelier A. JASPAR 2022: the 9th release of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 2022;50:D165–D173. doi: 10.1093/nar/gkab1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chand A.L., Pathirage N., Lazarus K., Chu S., Drummond A.E., Fuller P.J., Clyne C.D. Liver receptor homologue-1 expression in ovarian epithelial and granulosa cell tumours. Steroids. 2013;78:700–706. doi: 10.1016/j.steroids.2013.03.001. [DOI] [PubMed] [Google Scholar]
  3. Chen X.X., Sun X., Chimbaka I.M., Qin N., Xu X.X., Liswaniso S., Xu R.F., Gonzalez J.M. Transcriptome analysis of ovarian follicles reveals potential pivotal genes associated with increased and decreased rates of chicken egg production. Front. Genet. 2021;12 doi: 10.3389/fgene.2021.622751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Corzo C.A., Mari Y., Chang M.R., Khan T., Kuruvilla D., Nuhant P., Kumar N., West G.M., Duckett D.R., Roush W.R., Griffin P.R. Antiproliferation activity of a small molecule repressor of liver receptor homolog 1. Mol. Pharmacol. 2015;87:296–304. doi: 10.1124/mol.114.095554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crespo R.P., Rocha T.P., Montenegro L.R., Nishi M.Y., Jorge A.A.L., Maciel G.A.R., Baracat E., Latronico A.C., Mendonca B.B., Gomes L.G. High-throughput sequencing to identify monogenic etiologies in a preselected polycystic ovary syndrome cohort. J. Endocr. Soc. 2022;6:1–10. doi: 10.1210/jendso/bvac106. bvac106-bvac106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ding T., Luo A., Yang S., Lai Z., Wang Y., Shen W., Jiang J., Lu Y., Ma D., Wang S. Effects of basal media and supplements on diethylstilbestrol-treated immature mouse primary granulosa cell growth and regulation of steroidogenesis in vitro. Reprod. Domest. Anim. 2012;47:355–364. doi: 10.1111/j.1439-0531.2011.01879.x. [DOI] [PubMed] [Google Scholar]
  7. Duggavathi R., Volle D.H., Mataki C., Antal M.C., Messaddeq N., Auwerx J., Murphy B.D., Schoonjans K. Liver receptor homolog 1 is essential for ovulation. Genes & Dev. 2008;22:1871–1876. doi: 10.1101/gad.472008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Durlinger A.L.L., Gruijters M.J.G., Kramer P., Karels B., Kumar T.R., Matzuk M.M., Rose U.M., de Jong F.H., Uilenbroek J.T.J., Grootegoed J.A., Themmen A.P.N. Anti-Mullerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology. 2001;142:4891–4899. doi: 10.1210/endo.142.11.8486. [DOI] [PubMed] [Google Scholar]
  9. Ghanem K., Johnson A.L. Response of hen pre-recruitment ovarian follicles to follicle stimulating hormone, in vivo. Gen. Comp. Endocrinol. 2019;270:41–47. doi: 10.1016/j.ygcen.2018.10.004. [DOI] [PubMed] [Google Scholar]
  10. Gilbert A.B., Evans A.J., Perry M.M., Davidson M.H. A method for separating granulosa-cells, basal lamina and theca of preovulatory ovarian follicle of domestic-fowl (gallus-domesticus) J. of Reprod. Fertil. 1977;50:179–181. doi: 10.1530/jrf.0.0500179. [DOI] [PubMed] [Google Scholar]
  11. Guo I.C., Huang C.Y., Wang C.K.L., Chung B.C. Activating protein-1 cooperates with steroidogenic factor-1 to regulate 3′,5′-cyclic adenosine 5′-monophosphate-dependent human CYP11A1 transcription in vitro and in vivo. Endocrinology. 2007;148:1804–1812. doi: 10.1210/en.2006-0938. [DOI] [PubMed] [Google Scholar]
  12. Guzmán A., Hughes C.H.K., Murphy B.D. Orphan nuclear receptors in angiogenesis and follicular development. Reproduction. 2021;162:R35–R54. doi: 10.1530/REP-21-0118. [DOI] [PubMed] [Google Scholar]
  13. Haugen M.J., Johnson A.L. Bone morphogenetic protein 2 inhibits FSH responsiveness in hen granulosa cells. Reproduction. 2010;140:551–558. doi: 10.1530/REP-10-0211. [DOI] [PubMed] [Google Scholar]
  14. Hinshelwood M.M., Repa J.J., Shelton J.M., Richardson J.A., Mangelsdorf D.J., Mendelson C.R. Expression of LRH-1 and SF-1 in the mouse ovary: localization in different cell types correlates with differing function. Mol. Cell. Endocrinol. 2003;207:39–45. doi: 10.1016/s0303-7207(03)00257-0. [DOI] [PubMed] [Google Scholar]
  15. Huang S.J., Purevsuren L., Jin F., Zhang Y.P., Liang C.Y., Zhu M.Q., Wang F., Jia C.L., Wei Z.H. Effect of anti-mueurollerian hormone on the development and selection of ovarian follicle in hens. Poult. Sci. 2021;100 doi: 10.1016/j.psj.2020.12.056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huo Y.A., Li Q.Q., Yang L., Li X.X., Sun C., Liu Y., Liu H.L., Pan Z.X., Li Q.F., Du X. SDNOR, a novel antioxidative lncRNA, is essential for maintaining the normal state and function of porcine follicular granulosa cells. Antioxidants (Basel) 2023;12:21. doi: 10.3390/antiox12040799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jin H., Won M., Park S.E., Lee S., Park M., Bae J. FOXL2 is an essential activator of SF-1-induced transcriptional regulation of anti-mullerian hormone in human granulosa cells. PLOS ONE. 2016;11 doi: 10.1371/journal.pone.0159112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johnson A.L., Woods D.C. Dynamics of avian ovarian follicle development: cellular mechanisms of granulosa cell differentiation. Gen. Comp. Endocrinol. 2009;163:12–17. doi: 10.1016/j.ygcen.2008.11.012. [DOI] [PubMed] [Google Scholar]
  19. Johnson A.L. Ovarian follicle selection and granulosa cell differentiation. Poult. Sci. 2015;94:781–785. doi: 10.3382/ps/peu008. [DOI] [PubMed] [Google Scholar]
  20. Johnson A.L., Lee J. Granulosa cell responsiveness to follicle stimulating hormone during early growth of hen ovarian follicles. Poult. Sci. 2016;95:108–114. doi: 10.3382/ps/pev318. [DOI] [PubMed] [Google Scholar]
  21. Kim D., Ocon-Grove O., Johnson A.L. Bone morphogenetic protein 4 supports the initial differentiation of hen (gallus gallus) granulosa cells. Biol. Reprod. 2013;88:161. doi: 10.1095/biolreprod.113.109694. [DOI] [PubMed] [Google Scholar]
  22. Kim D. 2014. Regulatory mechanisms of G protein-coupled receptor (GPCR) signaling at follicle selection in the hen ovary. [Google Scholar]
  23. Kim D., Johnson A.L. Differentiation of the granulosa layer from hen prehierarchal follicles associated with follicle-stimulating hormone receptor signaling. Mol. Reprod. Dev. 2018;85:729–737. doi: 10.1002/mrd.23042. [DOI] [PubMed] [Google Scholar]
  24. Kim J.W., Havelock J.C., Carr B.R., Attia G.R. The orphan nuclear receptor, liver receptor homolog-1, regulates cholesterol side-chain cleavage cytochrome p450 enzyme in human granulosa cells. J. Clin. Endocrinol. Metab. 2005;90:1678–1685. doi: 10.1210/jc.2004-0374. [DOI] [PubMed] [Google Scholar]
  25. Kouri C., Sommer G., Lapiscina I.M.D., Elzenaty R.N., Tack L.J.W., Cools M., Ahmed S.F., Flück C.E., S. F. n. s. group Clinical and genetic characteristics of a large international cohort of individuals with rare NR5A1/SF-1 variants of sex development. EBioMedicine. 2024;99:16. doi: 10.1016/j.ebiom.2023.104941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Leghari I.H., Li J., Zeng W., Mi Y., Zhang C. Comparative study of reproductive organs and egg quality characteristics in hyline and domestic layer breeds. Pak. Vet. J. 2015;35:315–320. [Google Scholar]
  27. Li L.Y., Hua R., Hu K.Q., Chen H.L., Yin Y.M., Shi X.J., Peng K.Z., Huang Q., Qiu Y., Li X., Liu Q.F., Liu S.F., Wang Z. SIRT6 deficiency causes ovarian hypoplasia by affecting Plod1-related collagen formation. Aging Cell. 2024;23:e14031. doi: 10.1111/acel.14031. 1-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Li D.M., Li X.Y., He H.R., Zhang Y., He H., Sun C.J., Zhang X.Y., Wang X.Z., Kan Z.Y., Su Y., Han S.S., Xia L., Tan B., Ma M.G., Zhu Q., Yin H.D., Cui C. miR-10a-5p inhibits chicken granulosa cells proliferation and progesterone(P4) synthesis by targeting MAPRE1 to suppress CDK2. Theriogenology. 2022;192:97–108. doi: 10.1016/j.theriogenology.2022.08.019. [DOI] [PubMed] [Google Scholar]
  29. Li J., Luo W., Huang T., Gong Y. Growth differentiation factor 9 promotes follicle-stimulating hormone-induced progesterone production in chicken follicular granulosa cells. Gen. Comp. Endocrinol. 2019;276:69–76. doi: 10.1016/j.ygcen.2019.03.005. [DOI] [PubMed] [Google Scholar]
  30. Li Y.X., Zhang J., Qian Y., Meng C.H., Wang H.L., Cao S.X. Mutation-388 C &gt; G of NR5A1 gene affects litter size and promoter activity in sheep. Anim. Reprod. Sci. 2018;196:19–27. doi: 10.1016/j.anireprosci.2018.05.001. [DOI] [PubMed] [Google Scholar]
  31. Lizneva D., Suturina L., Walker W., Brakta S., Gavrilova-Jordan L., Azziz R. Criteria, prevalence, and phenotypes of polycystic ovary syndrome. Fertil. Steril. 2016;106:6–15. doi: 10.1016/j.fertnstert.2016.05.003. [DOI] [PubMed] [Google Scholar]
  32. Lubliner S., Regev I., Lotan-Pompan M., Edelheit S., Weinberger A., Segal E. Core promoter sequence in yeast is a major determinant of expression level. Genome Res. 2015;25:1008–1017. doi: 10.1101/gr.188193.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Luo X.R., Ikeda Y.Y., Parker K.L. A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual-differentiation. Cell. 1994;77:481–490. doi: 10.1016/0092-8674(94)90211-9. [DOI] [PubMed] [Google Scholar]
  34. Meinsohn M.-C., Morin F., Bertolin K., Duggavathi R., Schoonjans K., Murphy B.D. The orphan nuclear receptor liver homolog receptor-1 (Nr5a2) regulates ovarian granulosa cell proliferation. J. Endocr. Soc. 2018;2:24–41. doi: 10.1210/js.2017-00329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Meinsohn M.C., Smith O.E., Bertolin K., Murphy B.D. The orphan nuclear receptors steroidogenic factor-1 and liver receptor homolog-1: structure, regulation, and essential roles in mammalian reproduction. Physiol. Rev. 2019;99:1249–1279. doi: 10.1152/physrev.00019.2018. [DOI] [PubMed] [Google Scholar]
  36. Miller S., Bhasin N., Urrego H., Moroz K., Rowan B.G., Ramayya M.S., Makridakis N.M. Genetic and epigenetic alterations of steroidogenic factor-1 in ovarian tumors. Int. J. Oncol. 2013;42:627–634. doi: 10.3892/ijo.2012.1758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Miller W.L. Steroidogenic enzymes. Endocr. Dev. 2008;13:1–18. doi: 10.1159/000134751. [DOI] [PubMed] [Google Scholar]
  38. Miller W.L. Steroid hormone synthesis in mitochondria. Mol. Cell. Endocrinol. 2013;379:62–73. doi: 10.1016/j.mce.2013.04.014. [DOI] [PubMed] [Google Scholar]
  39. Nie R.X., Zheng X.T., Zhang W.H., Zhang B., Ling Y., Zhang H., Wu C.X. Morphological characteristics and transcriptome landscapes of chicken follicles during selective development. Animals (Basel) 2022;12:713. doi: 10.3390/ani12060713. 1-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ocón-Grove O.M., Poole D.H., Johnson A.L. Bone morphogenetic protein 6 promotes FSH receptor and anti-Mullerian hormone mRNA expression in granulosa cells from hen prehierarchal follicles. Reproduction. 2012;143:825–833. doi: 10.1530/REP-11-0271. [DOI] [PubMed] [Google Scholar]
  41. Pelusi C., Ikeda Y., Zubair M., Parker K.L. Impaired follicle development and infertility in female mice lacking steroidogenic factor 1 in ovarian granulosa cells. Biol. Reprod. 2008;79:1074–1083. doi: 10.1095/biolreprod.108.069435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Qin N., Tyasi T.L., Sun X., Chen X., Zhu H., Zhao J., Xu R. Determination of the roles of GREM1 gene in granulosa cell proliferation and steroidogenesis of hen ovarian prehierarchical follicles. Theriogenology. 2020;151:28–40. doi: 10.1016/j.theriogenology.2020.03.030. [DOI] [PubMed] [Google Scholar]
  43. Rotgers E., Nicol B., Rodriguez K., Rattan S., Flaws J.A., Yao H.H.C. Constitutive expression of steroidogenic factor-1 (NR5A1) disrupts ovarian functions, fertility, and metabolic homeostasis in female mice. FASEB J. 2021;35:e21770. doi: 10.1096/fj.202100304R. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shen X.X., Zhao X.Y., He H.R., Zhang Y., Zhu Q., Yin H.D. Transcriptome profiling reveals SLC5A5 regulates chicken ovarian follicle granulosa cell proliferation, apoptosis, and steroid hormone synthesis. Poult. Sci. 2024;103 doi: 10.1016/j.psj.2023.103241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stephens C.S., Johnson P.A. Bone morphogenetic protein 15 may promote follicle selection in the hen. Gen. Comp. Endocrinol. 2016;235:170–176. doi: 10.1016/j.ygcen.2016.06.027. [DOI] [PubMed] [Google Scholar]
  46. Stocco D.M. Steroidogenic acute regulatory (StAR) protein: what's new? BioEssays. 1999;21:768–775. doi: 10.1002/(SICI)1521-1878(199909)21:9<768::AID-BIES8>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  47. Stocco D.M., Zhao A.H., Tu L.N., Morohaku K., Selvaraj V. A brief history of the search for the protein(s) involved in the acute regulation of steroidogenesis. Mol. Cell. Endocrinol. 2017;441:7–16. doi: 10.1016/j.mce.2016.07.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sun X., Chen X.X., Zhao J.H., Ma C., Yan C.C., Liswaniso S., Xu R.F., Qin N. Transcriptome comparative analysis of ovarian follicles reveals the key genes and signaling pathways implicated in hen egg production. Bmc Genomics. 2021;22:899. doi: 10.1186/s12864-021-08213-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sun X., Liswaniso S., Shan X.S., Zhao J.H., Chimbaka I.M., Xu R.F., Qin N. The opposite effects of VGLL1 and VGLL4 genes on granulosa cell proliferation and apoptosis of hen ovarian prehierarchical follicles. Theriogenology. 2022;181:95–104. doi: 10.1016/j.theriogenology.2022.01.017. [DOI] [PubMed] [Google Scholar]
  50. Tilly J.L., Kowalski K.I., Johnson A.L. Stage of ovarian follicular development associated with the initiation of steroidogenic competence in avian granulosa-cells. Biol. Reprod. 1991;44:305–314. doi: 10.1095/biolreprod44.2.305. [DOI] [PubMed] [Google Scholar]
  51. Tilly J.L., Kowalski K.I., Johnson A.L., Hsueh A.J.W. Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology. 1991;129:2799–2801. doi: 10.1210/endo-129-5-2799. [DOI] [PubMed] [Google Scholar]
  52. Tyasi T.L., Sun X., Shan X.S., Liswaniso S., Chimbaka I.M., Qin N., Xu R.F. Effects of RAC1 on proliferation of hen ovarian prehierarchical follicle granulosa cells. Animals (Basel) 2020;10:1589. doi: 10.3390/ani10091589. 1-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Vitale A.M., Gonzalez O.A., Parborell F., Irusta G., Campo S., Tesone M. Inhibin A increases apoptosis in early ovarian antral follicles of diethylstilbestrol-treated rats. Biol. Reprod. 2002;67:1989–1995. doi: 10.1095/biolreprod.102.003673. [DOI] [PubMed] [Google Scholar]
  54. Wang J., Gong Y.Z. Transcription of CYP19A1 is directly regulated by SF-1 in the theca cells of ovary follicles in chicken. Gen. Comp. Endocrinol. 2017;247:1–7. doi: 10.1016/j.ygcen.2017.03.013. [DOI] [PubMed] [Google Scholar]
  55. Wang Y., Chen Q., Liu Z., Guo X., Du Y., Yuan Z., Guo M., Kang L., Sun Y., Jiang Y. Transcriptome analysis on single small yellow follicles reveals that Wnt4 is involved in chicken follicle selection. Front. Endocrinol. 2017;8:317. doi: 10.3389/fendo.2017.00317. 1-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wen F., Ding Y.X., Wang M.M., Du J., Zhang S., Kee K. FOXL2 and NR5A1 induce human fibroblasts into steroidogenic ovarian granulosa-like cells. Cell Prolif. 2024;16:e13589. doi: 10.1111/cpr.13589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Will E.A., Liu X., Peluso J.J. AG 205, a progesterone receptor membrane component 1 antagonist, ablates progesterone's ability to block oxidative stress-induced apoptosis of human granulosa/luteal cells. Biol. Reprod. 2017;96:843–854. doi: 10.1093/biolre/iox013. [DOI] [PubMed] [Google Scholar]
  58. Woods D.C., Johnson A.L. Regulation of follicle-stimulating hormone-receptor messenger RNA in hen granulosa cells relative to follicle selection. Biol. Reprod. 2005;72:643–650. doi: 10.1095/biolreprod.104.033902. [DOI] [PubMed] [Google Scholar]
  59. Wooton-Kee C.R. Steroidogenic factor-1 influences protein-DNA interactions within the cAMP-responsive regions of the murine StAR gene. Endocrinology. 1999;141:1345–1355. doi: 10.1210/endo.141.4.7412. [DOI] [PubMed] [Google Scholar]
  60. Xiang X., Huang X., Wang J.F., Zhang H.Y., Zhou W., Xu C.H., Huang Y.Y., Tan Y.T., Yin Z.Z. Transcriptome analysis of the ovaries of Taihe black-bone silky fowls at different egg-laying stages. Genes. 2022;13:2066. doi: 10.3390/genes13112066. 1-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Xiang Y., Meng S.S., Wang J.F., Li S.Y., Liu J.R., Li H.M., Li T.Y., Song W.H., Zhou W.H. Two novel DNA motifs are essential for BACE1 gene transcription. Sci. Rep. 2014;4:6864. doi: 10.1038/srep06864. 1-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yang S., Chen J., Ma B.C., Wang J.L., Chen J.X. Role of autophagy in lysophosphatidylcholine-induced apoptosis of mouse ovarian granulosa cells. Int. J. Mol. Sci. 2022;23:1479. doi: 10.3390/ijms23031479. 1-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Yuan X.-H., Yang C.-R., Wang X.-N., Zhang L.-L., Gao X.-R., Shi Z.-Y. Progesterone maintains the status of granulosa cells and slows follicle development partly through PGRMC1. J. Cell. Physiol. 2019;234:709–720. doi: 10.1002/jcp.26869. [DOI] [PubMed] [Google Scholar]
  64. Zhu M.Q., Wang D., Zou K.X., Wang F., Zhang Z.C., Song X.R., Jia C.L., Wei Z.H. Insulin-like growth factor-1 regulates follicle selection of hens by promoting proliferation and inhibiting apoptosis of granulosa cells in prehierarchical follicles in vitro. Anim. Reprod. Sci. 2022;247:107091. doi: 10.1016/j.anireprosci.2022.107091. 1-10. [DOI] [PubMed] [Google Scholar]
  65. Zhu M.K., Miao S.S., Zhou W.T., Elnesr S.S., Dong X.Y., Zou X.T. MAPK, AKT/FoxO3a and mTOR pathways are involved in cadmium regulating the cell cycle, proliferation and apoptosis of chicken follicular granulosa cells. Ecotoxicol. Environ. Saf. 2021:214. doi: 10.1016/j.ecoenv.2021.112091. [DOI] [PubMed] [Google Scholar]
  66. Zhu Y.F., Wang H., Wang J.P., Han S.S., Zhang Y., Ma M.G., Zhu Q., Zhang K.Y., Yin H.D. Zearalenone induces apoptosis and cytoprotective autophagy in chicken granulosa cells by PI3K-AKT-mTOR and MAPK signaling pathways. Toxins. 2021;13 doi: 10.3390/toxins13030199. [DOI] [PMC free article] [PubMed] [Google Scholar]

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