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. 2025 Sep 11;104(11):105837. doi: 10.1016/j.psj.2025.105837

DHCR7 expression, function and estrogen-induced promoter histone modification changes in chicken granulosa cells of pre-hierarchical follicles

Dandan Li a, Qingqing Wei a,b, Li Kang a,b, Yi Sun a,b, Yunliang Jiang a,b,⁎
PMCID: PMC12466222  PMID: 40957294

Abstract

Follicle selection is characterized by granulosa cell differentiation and progesterone production using cholesterol. 7-dehydrocholesterol reductase (DHCR7) is the rate-limiting enzyme in cholesterol synthesis. After follicle selection, the expression of DHCR7 transcripts variants (T1, T3, and T4) significantly increase, and estrogen promotes their expression in a dose-dependent manner in chicken granulosa cells of pre-hierarchical follicles (Pre-GCs). This study investigates DHCR7′s role in follicular development and selection, focusing on its mRNA and protein expression and impact on Pre-GC function, as well as histone modifications in the DHCR7 promoter region under estrogen treatment. RT-qPCR analysis revealed that the mRNA expression of total DHCR7 and its transcripts T1, T3, and T4 gradually increased as follicles developed. After follicle selection, both mRNA and protein levels of total DHCR7, along with mRNA levels of T1, T3, and T4, were upregulated. Further RT-qPCR analysis revealed that DHCR7 suppressed the expression of genes related to proliferation and apoptosis while promoting those involved in progesterone synthesis. Additionally, DHCR7 inhibited Pre-GC proliferation and apoptosis, while enhancing progesterone secretion. CUT&RUN-qPCR analysis demonstrated that estrogen significantly enriched H3K4me1, H3K4me2, H3K27ac, and H4K16ac in the estrogen response region of the DHCR7 promoter, while no significant changes were observed in H3K4me3 and H3K9ac. This study highlights the critical role of DHCR7 in follicular development and selection, demonstrating its regulation of granulosa cell differentiation and progesterone synthesis. The findings also suggest that estrogen-induced histone modifications at the DHCR7 promoter contribute to its transcriptional activation, offering new insights into the molecular mechanisms underlying ovarian function and reproductive performance in poultry.

Keywords: Chicken, DHCR7, Estrogen, Granulosa cells, Histone modification

Abbreviations

CCND1

cyclin D1

CCND2

cyclin D2

CDK1

cyclin-dependent kinase 1

CDK2

cyclin-dependent kinase 2

CCK-8

Cell Counting Kit-8

CASPASE3

cysteine-aspartic acid proteases 3

CASPASE8

cysteine-aspartic acid proteases 8

CASPASE9

cysteine-aspartic acid proteases 9

CYP11A1

cytochrome P450 family 11 subfamily A member 1

DHCR7

7-dehydrocholesterol reductase

EdU

5-Ethynyl-2′-deoxyuridine

ELISA

enzyme-linked immunosorbent assay

E2

estradiol

ER

estrogen receptor

ERE

estrogen response element

FBS

fetal bovine serum

HSD3B

3β-hydroxysteroid dehydrogenase

LWF

large white follicle

Pre-GCs

granulosa cells of pre-hierarchical follicles

Pre-TCs

theca cells of pre-hierarchical follicles

Post-GCs

granulosa cells of hierarchical follicles

Post-TCs

theca cells of hierarchical follicles

PVDF

polyvinylidene fluoride

PBS

phosphate-buffered saline

RT-qPCR

real-time quantitative PCR

SWF

small white follicle

SYF

small yellow follicle

SLOS

Smith-Lemli-Opitz syndrome

SREBP2

sterol-regulatory element binding protein 2

SEM

standard error of the mean

StAR

steroidogenic acute regulatory protein

siRNA

small interfering RNA.

Introduction

The development of chicken follicles is a highly coordinated physiological process that includes the activation of primary follicles, follicular growth, selection of dominant follicles, and ovulation (Johnson and Woods, 2009). According to the size of the follicle, chicken follicles can be divided into two major categories: pre-hierarchical follicles, which include small white follicles (SWFs), large white follicles (LWFs), and small yellow follicles (SYFs); and hierarchical follicles, classified by size from F6 to F1, with F1 being the largest dominant follicle about to ovulate (Onagbesan et al., 2009; Hrabia et al., 2018; Lin et al., 2018; Jiang et al., 2022). Approximately once a day, the hen selects one follicle from a cohort of SWFs to enter the hierarchical stage, where it undergoes rapid growth and development until ovulation. (Onagbesan et al., 2009; Johnson, 2012). The unselected follicles either undergo atresia or remain available for selection in subsequent laying cycles. This process is regulated by reproductive hormones, various secreted factors, and intercellular signaling, directly impacting the egg-laying efficiency of poultry. Among them, estrogen, as a key reproductive hormone, stimulates the liver to produce yolk precursor proteins and lipoproteins, which are transported into the ovary for yolk deposition (Li et al., 2014). Estrogen can promote cell proliferation, differentiation, and steroid hormone synthesis, thereby regulating follicle development (Guo et al., 2018; Liu et al., 2021). Studies have shown that exogenous estrogen can significantly increase the number of follicles, their diameter, and oocyte size in chicks (Zhao et al., 2017).

Cholesterol is an essential lipid molecule that serves not only as a key component of cell membranes but also as a precursor for the synthesis of steroid hormones such as estrogen and progesterone, bile acids (Luo et al., 2020), and vitamin D (Prabhu et al., 2016b). The biosynthesis of cholesterol involves a series of complex enzymatic reactions, among which 7-dehydrocholesterol reductase (DHCR7) is a key rate-limiting enzyme responsible for catalyzing the conversion of 7-dehydrocholesterol to cholesterol (Sharpe and Brown, 2013). As a crucial enzyme in cholesterol metabolism, DHCR7 is involved in lipid metabolic regulation (Li et al., 2009); its dysfunction is closely associated not only with the development of diabetes and insulin resistance (Li et al., 2009) but also with Smith-Lemli-Opitz syndrome (SLOS) (Jira et al., 2003; DeBarber et al., 2011). Additionally, DHCR7 cooperates with emopamil binding protein to promote cell proliferation and differentiation (Prabhu et al., 2016a). Studies have shown that DHCR7 expression is finely regulated at multiple levels, including by the transcription factor sterol-regulatory element binding protein 2 (SREBP2) (Horton et al., 2003), m6A modifications (Zeng et al., 2024; Li et al., 2025), phosphorylation modifications (Prabhu et al., 2017) as well as feedback regulation by cholesterol and vitamin D (Prabhu et al., 2016b). Notably, estrogen can regulate the expression of DHCR7 transcripts variants (T1, T3, and T4) in chicken granulosa cells of pre-hierarchical follicles (Pre-GCs) in a dose-dependent manner (Li et al., 2022). Although the function of DHCR7 has been extensively studied in mammals, its mechanistic role in avian species, particularly in ovarian function and follicle development, remains to be elucidated.

The regulation of gene expression is a complex process involving the synergistic actions of transcription factors, hormonal signals, and epigenetic mechanisms. The expression of genes is influenced by histone modifications; for example, in mouse ovarian granulosa cells, butyrate promotes steroid hormone synthesis by regulating PPARγ and PGC1α pathway genes via H3K9ac (Ye et al., 2021). Meanwhile, methylation of lysine residues on histones H3 and H4 plays key roles in follicle development and oocyte maturation (Shen et al., 2017). Notably, estrogen, as an important reproductive hormone, can regulate target gene expression by altering histone modification patterns, thereby participating in the regulation of reproductive physiological processes (Malik et al., 2010; Frick, 2013). Our previous research has elucidated the molecular mechanisms by which estrogen regulates chicken DHCR7 expression at transcriptional and translational levels (Li et al., 2025); however, systematic studies on the epigenetic regulation of DHCR7 induced by estrogen are still lacking in chickens.

This study systematically analyzes the expression profiles of total DHCR7, and its transcripts variants (T1, T3, and T4) across different follicular tissues and cells. We then investigate the biological functions of DHCR7 in Pre-GCs. Finally, we examine the dynamic changes of histone modifications in the promoter region of the chicken DHCR7 gene under estrogen regulation. These findings not only deepen our understanding of estrogen's role in ovarian physiology but also highlight the critical role of DHCR7 in follicular development, offering potential targets for improving reproductive performance in poultry through the regulation of DHCR7 expression.

Materials and methods

Animals and sample collection

Thirty-five to forty weeks old Hy-Line Brown laying hens with a weekly egg-laying percentage of 90 %−95 % were randomly chosen from the affiliated farm of Shandong Agricultural University. All chickens were raised under the same conditions, which were consistent with standard breeding practices on the farm, with ad libitum access to food and water, and a light cycle of 16 h of light and 8 h of darkness. Each chicken was euthanized by cervical dislocation, and SWFs, LWFs, SYFs, and F6 to F1 follicles were then collected from the ovaries and immediately placed in cold phosphate-buffered saline (PBS). These follicles were collected for cells culture or stored in liquid nitrogen for RNA extraction as required. All animal experiments were approved by the Institutional Animal Care and Use Committee of Shandong Agricultural University (No: SDAUA-2024-088) and conducted in strict accordance with the "Guidelines for Experimental Animals " issued by the Ministry of Science and Technology of China.

Cell culture and treatment

Cells were isolated and cultured using a previously described procedure (Hu et al., 2024). Briefly, granulosa cells from pre-hierarchical follicles including SWFs, LWFs, and SYFs were prepared by crushing the follicles after yolk extrusion, and named as Pre-GCs. The follicles were then digested with 1 mg/mL collagenase II (Coolaber, Beijing, China) in a 37°C water bath for 5 min, followed by filtration through a 200-mesh sieve to isolate Pre-GCs. Granulosa cells of hierarchical follicles (including F6-F1), referred to as Post-GCs, were carefully separated by gently squeezing out the yolk using forceps, and the granulosa cell layer was digested with 0.25 % trypsin-EDTA (Gibco, Grand Island, NY, USA) in a 37°C water bath for 8 min, followed by filtration through a 200-mesh sieve to obtain Post-GCs. Theca cells of pre-hierarchical follicles (Pre-TCs) and theca cells of hierarchical follicles (Post-TCs) were digested with 1 mg/mL collagenase II in a 37°C water bath for 30 min and then filtered through a 200-mesh sieve. After filtration, all cells were centrifuged at 2000 rpm/min for 5 min and resuspended in M199 medium (Gibco, Grand Island, NY, USA) containing fetal bovine serum (FBS; Vazyme, Nanjing, China) and 1 % antibiotic mixture (Solarbio, Beijing, China). Pre-GCs were cultured in medium containing 1 % FBS, while Post-GCs, Pre-TCs, and Post-TCs were cultured in medium containing 5 % FBS. Finally, the cells were seeded in 6-well plates at a density of 1 × 106 cells per well and cultured in a 39°C incubator with 5 % CO2.When the cell density reaches 80 %, they were transiently transfected or treated with 50 nmol/L estradiol (E2; Sigma-Aldrich, Missouri, USA).

Plasmids construction and transfection

The full-length coding sequence of chicken DHCR7 was amplified with TransStart FastPfu DNA polymerase (TransGen, Beijing, China) using cDNA synthesized from total RNA extracted from granulosa cells as the template for construction overexpression vectors. The DHCR7 gene was inserted into the Nhe I and Xho I restriction sites of the pcDNA3.1(+) vector. Plasmid constructs were verified through DNA sequencing and restriction enzyme digestion. The PCR primers used for amplification are provided in Table S1.

When the cell reached about 80 % confluence on 6-well plates, they were transiently transfected with 3 μg DHCR7 overexpression plasmids using Lipofectamine LTX and Plus Reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's guidelines. To knock down DHCR7 expression, small interfering RNA (siRNAs) targeting chicken DHCR7, synthesized by Guangzhou RiboBio Co., Ltd., were transiently transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 75 nM. After 6 h, the culture medium was replaced with fresh medium, and the cells were incubated for an additional 24 h, 36 h or 48 h.

RNA extraction and real-time quantitative PCR (RT-qPCR)

Total RNA was extracted using the RNA simple Total RNA Kit (TIANGEN, Beijing, China). For each sample, 1 µg of RNA was subjected to reverse transcription following a two-step method: genomic DNA was first removed, then reverse transcription was performed, with all procedures carried out according to the instructions of the Evo M-MLV RT Mix Kit with gDNA Clean (Accurate Biotechnology, Hunan, China). qPCR was conducted using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan, China). The reaction system consisted of 10 µL of 2× SYBR Green Pro Taq HS Premix, 7.2 µL of RNase-free water, 0.4 µL of forward primer (10 µmol/L), 0.4 µL of reverse primer (10 µmol/L), and 2 µL of cDNA. The thermal cycling program was set as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s, using Archimed X4 instrument (ROCGENE, Beijing, China) and LightCycler 96 (Roche, Basel, Switzerland) for amplification. Relative gene expression levels were calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001), with GAPDH as the internal reference gene. All RT-qPCR primer sequences were listed in supplementary Table 1.

Protein extraction and Western blotting

Total protein was extracted from follicular cells using RIPA buffer (Beyotime, Shanghai, China) supplemented with a mixture of protease and phosphatase inhibitors (NCM, Suzhou, China). Protein concentration was measured using the BCA Protein Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Protein samples were mixed with 5× SDS-PAGE loading buffer (NCM, Suzhou, China) and denatured at 70°C for 10 min.

Equal amounts of protein were separated by SDS-PAGE on 10 % polyacrylamide gels (Beyotime, Shanghai, China) and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5 % skimmed milk at room temperature for 1 h. After blocking the PVDF membranes, they were incubated with rabbit anti-DHCR7 antibody (ABclonal, Wuhan, China; 1:1,000dilution) and mouse anti-GAPDH monoclonal antibody (Proteintech, Wuhan, China; 1:50,000 dilution) overnight at 4°C. Following five washes with Tris-buffered saline with Tween 20 (5 min each), membranes were incubated with secondary antibodies (Beyotime, Shanghai, China) at a 1:4,000 dilution for 1.5 h at room temperature. Protein bands were detected using BeyoECL Plus (Beyotime, Shanghai, China) and visualized with the C300 imaging system (Azure Biosystems, California, USA). Band intensities were quantified by ImageJ 1.46r software, normalizing target protein signals to the internal reference GAPDH.

CUT&RUN-qPCR

CUT&RUN-qPCR (Vazyme, Nanjing, China) was performed as follows: ConA Beads Pro were prepared by washing and resuspending in Binding Buffer. Cells were collected, washed, and resuspended in Wash Buffer. The cells were then combined with ConA beads pro and incubated for 10 min at room temperature. Subsequently, cells were incubated with rabbit anti-H3K4me1 mAb (ABclonal, Wuhan, China), rabbit anti-H3K4me2 mAb (ABclonal, Wuhan, China), rabbit anti-H3K4me3 mAb (ABclonal, Wuhan, China), rabbit anti-H3K9ac mAb (ABclonal, Wuhan, China), rabbit anti-H4K16ac mAb (Engibody, shanghai, China), rabbit anti-H3K27ac mAb (Engibody, shanghai) and rabbit IgG antibody (CST, Danvers, MA, USA) overnight at 4°C. After washing, pG-MNase enzyme was added and incubated at 4°C for 1 h. Chromatin digestion was initiated by adding CaCl2 and incubated on ice for 1 h. Digestion was stopped with Stop Buffer, and DNA fragments were released by incubation at 37°C. DNA was purified using FastPure gDNA Mini Columns and eluted in ddH2O. Finally, qPCR was performed to quantify target DNA fragments.

Cell counting kit-8 (CCK-8) assay

Proliferation of Pre-GCs was detected using the Cell Counting Kit-8 (Vazyme, Nanjing, China). At 0, 12, 24, 36 and 48 h after transfection, 150 μL of medium with 15 μL of CCK8 solution was added to each well, and further incubated for 2 h at 39°C. The absorbance was measured at 450 nm wave length using an ELx808 Absorbance Reader (BioTek, Vermont, USA).

5-ethynyl-2′-deoxyuridine (EdU) assay

The EdU assay was performed according to the instructions of BeyoClick™ EdU-555 kit manual (Beyotime, Shanghai, China). 24 h after transfection, Pre-GCs were labeled with EdU (50 mmol/L) for 2 h and cell nuclei were stained with Hoechst 33342. Fluorescence detection was then carried out using an OLYMPUS IX73 (Olympus, Tokyo, Japan). The number of positive cells and the total number of cells were counted using ImageJ 1.46r software.

Enzyme-linked immunosorbent assay (ELISA)

The culture medium of Pre-GCs was collected 36 h after transfection. Progesterone concentration in the culture medium was detected using the chicken progesterone ELISA kit (Enzyme-linked Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The absorbance was measured at 450 nm wave length using a RT-6100 (Rayto, Shenzhen, China).

Flow cytometry

Apoptosis of Pre-GCs was detected using the Annexin V-FITC/PI Apoptosis Detection kit (Vazyme, Nanjing, China). 24 h after transfection, cells were resuspended in 1 × Binding Buffer and then incubated with 5 μL of Annexin V-FITC and 5 μL of PI Staining Solution at room temperature for 10 min in the dark. Detection was performed using the Attune® NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). Data were analyzed using AttuneTM Cytometric Software v5.3.0.

Statistical analysis

Each experiment consisted of at least three biological replicates and was repeated at least three times. The results are expressed as the mean ± standard error of the mean (SEM). For two-group comparisons, Student’s t-test was used. For comparisons involving more than two groups, One-Way ANOVA followed by Tukey’s multiple comparison was applied using SPSS 17.0 (SPSS Inc., Chicago, IL, USA), with statistical significance set at p < 0.05. Graphs were generated using GraphPad Prism software (version 8.0; San Diego, CA, USA).

Results

Expression characteristics of total DHCR7 and its transcripts T1, T3, and T4 in chicken ovarian follicles, theca and granulosa cells

In our previous study, through third-generation transcriptome sequencing of chicken follicular granulosa cells before and after follicle selection, we found that DHCR7 produced three differentially expressed transcript variants (T1, T3, and T4) through 5′ alternative splicing (Li et al., 2022). Although these three transcripts encode the same protein, they may differ in aspects such as expression regulation, spatiotemporal distribution, and transcript stability. In view of this, we systematically examined the expression levels of total DHCR7 and its transcripts T1, T3, and T4 in chicken follicle tissues at different developmental stages as well as in follicular granulosa and theca cells.

The results showed that as the follicles developed, the expression of total DHCR7 and its transcripts T1, T3, and T4 in follicle tissues exhibited a gradually increasing trend (Fig. 1A-D). Notably, the expression pattern of T1 differed slightly from that of T3 and T4, with T1 having the highest expression level in F2, exceeding that in F1; meanwhile, T1 expression in F4 was significantly higher than in F3 (Fig. 1B).

Fig. 1.

Fig 1

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Expression pattern of total DHCR7, and each transcript of T1, T3, and T4 in chicken follicles at different developmental stage. mRNA expression level of DHCR7 (A), T1 (B), T3 (C), and T4 (D) in chicken follicles (n = 3). SW, small white; LW, large white; SY, small yellow; F6–F1 represent hierarchical follicles, which are sorted from smallest to largest in diameter. Results are shown as mean±SEM. abc, p < 0.05.

After follicle selection, the mRNA expression of total DHCR7, and each transcript of T1, T3 and, T4 was significantly upregulated in Post-GCs and Post-TCs (Fig. 2A-D). At the protein level, total DHCR7 expression in Post-GCs was significantly higher than in Pre-GCs; however, in theca cells, no obvious changes in total DHCR7 expression were observed before and after follicle selection (Fig. 2E-F). In Post-GCs, total DHCR7 mRNA and protein levels, as well as T1, T3, and T4 transcript abundances, were all markedly higher than in the other three follicular cell types (Fig. 2A-F).

Fig. 2.

Fig 2

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Expression characteristics of total DHCR7, and each transcript of T1, T3, and T4 in chicken follicular granulosa and theca cells. (A-D) DHCR7, T1, T3, and T4 mRNA expression level in follicular granulosa and theca cells detected by RT-qPCR (n = 3). (E-F) DHCR7 protein expression level in follicular granulosa and theca cells detected by Western blotting (n = 3). Results are shown as mean±SEM. abc, p < 0.05, ABC, p < 0.01.

Effects of DHCR7 on the proliferation, differentiation and apoptosis of chicken Pre-GCs

The mRNA and protein levels of total DHCR7 were significantly upregulated in Post-GCs, whereas in theca cells, only a significant increase in mRNA expression was observed without notable changes at the protein level. This suggests that DHCR7 plays a more direct and critical regulatory role in granulosa cells. Previous studies have shown that estrogen can markedly upregulate the mRNA expression of DHCR7 transcript variants (T1, T3, and T4) in chicken Pre-GCs (Li et al., 2022), indicating that this cell type is the primary target for estrogen-mediated regulation of DHCR7 function. Therefore, we focused on investigating the effects of DHCR7 on proliferation, differentiation, and apoptosis of chicken Pre-GCs.

To this end, we systematically studied the function of DHCR7 in chicken Pre-GCs using overexpression (Fig. 3A-C) and siRNA-mediated knockdown (Fig. 3D-F) approaches. DHCR7 overexpression inhibited the mRNA expression of cyclin D1 (CCND1), cyclin D2 (CCND2), cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) which are associated with cell proliferation (Fig. 4A), and significantly inhibited cell proliferation at 24, 36 and 48 h (Fig. 4C), consistent with the EdU assay results (Fig. 4D-E). Conversely, knockdown of DHCR7 by siRNA promoted the expression of proliferation-related genes and cell proliferation (Fig.4B, F-H). Taken together, DHCR7 inhibits the proliferation of chicken Pre-GCs.

Fig. 3.

Fig 3

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DHCR7 overexpression and knock down in chicken Pre-GCs. RT-qPCR and Western blot analyses were conducted to verify DHCR7 overexpression (A-C) and knockdown (D-F) effects (n = 3). Results are shown as mean±SEM.**p < 0.01, and ***p < 0.001.

Fig. 4.

Fig 4

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DHCR7 inhibits the proliferation of chicken Pre-GCs. (A-B) The effect of DHCR7 on the expression of genes related to cell proliferation (n = 3). Cell counting kit-8 (CCK-8) (C, F; n = 5) and 5-ethynyl-2′-deoxyuridine (EdU) (D-E, J-H; n = 6) analyses showing the effects of DHCR7 on cell proliferation. Results are shown as mean±SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

To determine the effect of DHCR7 on progesterone production, we detected the mRNA levels of genes related with progesterone secretion and the progesterone content in chicken Pre-GCs. Results showed that overexpression of DHCR7 promoted the mRNA expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B) (Fig. 5A) and progesterone secretion (Fig. 5B). Conversely, knockdown of DHCR7 inhibited the mRNA expression of StAR, CYP11A1, and HSD3B (Fig. 5C), as well as progesterone secretion (Fig. 5D). These results show that DHCR7 promotes progesterone secretion in chicken Pre-GCs, indirectly supporting its role in differentiation, as follicle selection is marked by granulosa cell differentiation and progesterone production.

Fig. 5.

Fig 5

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DHCR7 promotes the differentiation of chicken Pre-GCs. (A, C) The effect of DHCR7 on the expression of genes related to cell differentiation (n = 3). (B, D) Elisa analyses showing the effect of DHCR7 on cell differentiation (n = 3). P4, progesterone. Results are shown as mean±SEM. *p < 0.05, and **p < 0.01.

Apoptosis is the main inducer of follicle atresia in chickens. To investigate whether DHCR7 influences the apoptosis of chicken Pre-GCs, RT-qPCR and flow cytometric analyses were performed. RT-qPCR results showed that overexpression of DHCR7 suppressed the mRNA expression of cysteine-aspartic acid proteases 3, 8, and 9 (caspase 3, caspase 8, and caspase 9) which are related to cell apoptosis (Fig. 6A). Flow cytometry analysis of cell apoptosis showed that the percentages of cells at early and late apoptosis stages were significantly reduced after DHCR7 was overexpressed (Fig. 6B-D). Conversely, knockdown of DHCR7 increased the mRNA expression of apoptosis marker genes and significantly increased the number of apoptotic cells (Fig. 6E-H). These results demonstrate that DHCR7 inhibits apoptosis in chicken Pre-GCs.

Fig. 6.

Fig 6

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DHCR7 inhibits apoptosis of chicken Pre-GCs. (A, E) The effect of DHCR7 on the expression of genes related to cell apoptosis (n = 3). (B-D, F-H) Flow cytometry analyses showing the effect of DHCR7 on cell apoptosis (n = 5). Results are shown as mean±SEM. *p < 0.05, and **p < 0.01.

Dynamic changes of histone methylation and acetylation modifications at the estrogen response region of the DHCR7 promoter induced by estrogen in chicken Pre-GCs

Given that estrogen was known to promote the expression of DHCR7 transcript variants (T1, T3, and T4) in chicken Pre-GCs in a dose-dependent manner (Li et al., 2022), and that we previously systematically elucidated the molecular mechanisms underlying estrogen-mediated upregulation of DHCR7 at both transcriptional and translational levels (Li et al., 2025), it is of great importance to further investigate the upstream epigenetic regulatory mechanisms. Histone methylation and acetylation modifications at promoter regions serve as key regulatory elements in gene transcription and play critical roles in hormone responses. Therefore, this study focused on analyzing the dynamic changes of histone methylation and acetylation modifications at chicken DHCR7 promoter under estrogen treatment.

Our previous research identified an estrogen responsive region in the promoter region of chicken DHCR7. Considering that CUT&RUN experiments require primers to be designed within 100–200 bp for optimal performance, this estrogen response region was subdivided into three subregions (region 1 to region 3; supplementary figure 1). Subsequently, under E2 stimulation, we assessed the dynamic changes of histone marks, including H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K27ac, and H4K16ac across these three subregions in chicken Pre-GCs.

The CUT&RUN-qPCR results showed that E2 treatment significantly enhanced the binding of H3K4me1, H3K4me2, H3K27ac, and H4K16ac across all three regions (Fig. 7A-B, E-F). However, there was no significant change in the binding of H3K4me3 and H3K9ac (Fig. 7C-D). Furthermore, at the basal level, the binding of H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K27ac, and H4K16ac exhibited a progressively increasing trend from region 1 to region 3 (Fig. 7A-F). These findings indicate that estrogen dynamically regulates chicken DHCR7 gene expression by modulating histone methylation and acetylation modifications at its promoter.

Fig. 7.

Fig 7

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Dynamic changes in histone modifications within the estrogen responsive region of the DHCR7 promoter in chicken Pre-GCs after estrogen treatment. CUT&RUN-qPCR analysis of enrichment changes in (A-C) histone methylation modifications (H3K4me1, H3K4me2, and H3K4me3) and (D-F) histone acetylation modification (H3K9ac, H3K27ac, and H4K16ac) at the DHCR7 estrogen response region after estrogen treatment (n = 3). Results are shown as mean±SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Discussion

The ovary of a sexually mature hen contains approximately 12,000 oocytes, yet fewer than 5 % of follicles ultimately develop to maturity and ovulate (Onagbesan et al., 2009), with the majority undergoing atresia during follicular development (Tilly et al., 1991; Johnson et al., 1996). Differences in egg production performance among hens are closely related to the efficiency of follicle selection. Investigating the factors influencing follicle selection and their underlying genetic mechanisms is therefore important for improving laying performance. DHCR7, as a key enzyme in cholesterol synthesis, determines cholesterol supply in the ovary and other steroidogenic tissues, and cholesterol levels directly affect steroid hormone synthesis and reproductive function (Jiang et al., 2020; Schade et al., 2020; Gao et al., 2023; Li et al., 2023).

In this study, we systematically analyzed the expression dynamics of total DHCR7, and each transcript of T1, T3 and, T4 in chicken follicular tissues and cells. The results showed that total DHCR7 and its transcripts (T1, T3 and, T4) gradually increased during chicken follicle development, suggesting their involvement in follicular regulation. Notably, T1 exhibited an expression pattern distinct from T3 and T4, which may confer differential functions or regulatory mechanisms (Chu et al., 2020; Zhang et al., 2023; Li et al., 2025). Although T1, T3 and, T4 encode the same protein, they differ in expression regulation and spatiotemporal distribution.

During follicle selection, total DHCR7, and each transcript of T1, T3 and, T4 were significantly upregulated at the mRNA level in chicken Post-GCs and Post-TCs. However, only chicken granulosa cells showed a marked increase in total DHCR7 protein, while no significant change was observed in theca cell, indicating a more critical role of DHCR7 in granulosa cells. DHCR7 expression in granulosa cells has also been reported in humans (Kranc et al., 2017) and mice (Nakanishi et al., 2021). The inconsistency between mRNA and protein expression in chicken theca cells before and after follicle selection suggests the presence of post-transcriptional regulation or protein stability control. This phenomenon is consistent with multiple studies indicating complex regulatory layers between mRNA abundance and protein expression (Ma et al., 2008). This study reveals for the first time the dynamic expression characteristics of the DHCR7 gene and its transcripts (T1, T3 and, T4) in chicken follicular tissues and cells, providing an important foundation for further investigation of their functions during follicle development.

A reduction in the number of pre-hierarchical follicles and increased follicular atresia result in fewer pre-ovulatory follicles and subsequently decreased laying rates in hens (Johnson, 1993). Granulosa cell apoptosis plays a central role in follicular atresia (Zhou et al., 2019). Our study showed that chicken DHCR7 mRNA and protein expression in granulosa cells significantly increased after follicle selection, and estrogen dose-dependently promoted the expression of T1, T3, and T4 in Pre-GCs (Li et al., 2022), highlighting Pre-GCs as key targets for DHCR7 functional studies. The data demonstrated that DHCR7 inhibits proliferation and apoptosis of chicken Pre-GCs while promoting their differentiation. This aligns with previous findings linking chicken DHCR7 to cell differentiation (Li et al., 2022). It is well known that chicken Pre-GCs are undifferentiated and produce limited progesterone (Tilly et al., 1991; Johnson and Woods, 2009), whereas after follicle selection, granulosa cells differentiate and synthesize large amounts of progesterone (Grieshaber et al., 2000). Therefore, DHCR7 promotion of chicken Pre-GCs differentiation may accelerate their maturation into Post-GCs and follicle selection. Furthermore, DHCR7 exhibits diverse functions across species and pathological conditions: in pigs, DHCR7 promotes progesterone production in cumulus cells (Yamashita et al., 2005); in goats, it inhibits subcutaneous adipocyte differentiation while promoting intramuscular adipocyte differentiation (Li et al., 2024); in human breast cancer cell lines, DHCR7 is upregulated, and its knockdown inhibits proliferation and induces apoptosis (Wang et al., 2024); in ovarian cancer tissue, DHCR7 expression is downregulated (Pampalakis et al., 2015); while in gastric cancer cells, DHCR7 knockdown suppresses apoptosis and promotes proliferation (Chen et al., 2023). These findings suggest that DHCR7 has complex and context-dependent regulatory roles in different cell types and pathological states.

In poultry reproductive research, epigenetic regulation plays an important role in follicle development and selection by activating or repressing key genes, thereby affecting laying performance (Chamani and Keefe, 2019). For example, decreased levels of H3K9ac and H3K27ac in the promoter of the CYP19A1 gene suppress estradiol synthesis, impairing ovarian function (Fan et al., 2019). Histone methylation is crucial in oocyte maturation and follicle development (He et al., 2021; Bilmez et al., 2022). These observations highlight the important regulatory roles of histone modifications in follicular development. In our previous studies, we found that estrogen activates chicken DHCR7 transcription by binding of estrogen receptors (ERs) to an estrogen response element (ERE) in the DHCR7 promoter (Li et al., 2025). Usually, hormone-regulated gene expression depends not only on transcription factor binding but also on chromatin accessibility and histone modifications. For example, after E2 treatment, estrogen receptor α and H3K27ac enrichment increased in the upstream regulatory region of TET2 (Zhu et al., 2022). Conversely, when ER binding to ERE-associated genes leads to transcriptional repression, there is a marked reduction in H3K4me1 and H3K27ac enrichment (Lyu et al., 2022). This may be related to chromatin remodeling mechanisms, where ER recruits coregulators to induce specific histone modifications, thus modulating gene expression (Green and Carroll, 2007). Accordingly, this study focused on the effects of E2 treatment on histone methylation and acetylation modifications in chicken DHCR7 promoter. The results showed that E2 significantly enhanced the binding of H3K4me1, H3K4me2, H3K27ac, and H4K16ac to the estrogen responsive region of the chicken DHCR7 promoter, while the binding of H3K4me3 and H3K9ac remained unchanged. These modifications are typically associated with gene activation, suggesting that estrogen promotes chromatin opening at the chicken DHCR7 promoter, thereby activating its transcription. Similarly, E2 significantly increases H3K4me1/3, H3K9ac, and H3K27ac levels at the GREB1 promoter and enhancer regions (Tong et al., 2019). Notably, E2 treatment reduces H3K9ac enrichment at the cTnI promoter (Peng et al., 2016) and H3K4me1/2 at the RPRM promoter (Malik et al., 2010), suppressing gene expression. We also found that under basal conditions, multiple activation-related histone modifications (H3K4me1/2/3, H3K9ac, H3K27ac, H4K16ac) exhibit increasing trends across different subregions, indicating that the chicken DHCR7 promoter already possesses a degree of activation potential, and its fine regulation may facilitate rapid hormonal responses. Reports have also indicated that DHCR7 expression is influenced by other epigenetic modification, such as DNA methylation (Kim et al., 2005; Chen et al., 2022), m6A modifications (Zeng et al., 2024; Li et al., 2025), and miRNAs (Monchusi and Kaur, 2022). Future research should further explore DHCR7-related epigenetic regulatory networks and their interactions with hormonal signaling pathways to provide theoretical support and practical strategies for optimizing reproductive performance and production efficiency in livestock and poultry.

In summary, this study reveals the critical role of DHCR7 in chicken follicle development and selection, especially its regulated expression in Pre-GCs and its impact on cell proliferation, apoptosis, and differentiation. Furthermore, estrogen promotes transcriptional activation of chicken DHCR7 by modulating histone methylation and acetylation modifications at its promoter, thereby advancing follicle maturation. This provides an epigenetic basis for understanding the mechanism behind estrogen-mediated transcriptional activation of DHCR7. These findings enrich our understanding of the molecular mechanisms underlying follicle development and provide important clues for elucidating the genetic and epigenetic bases that regulate laying performance in hens.

CRediT authorship contribution statement

Dandan Li: Writing – review & editing, Writing – original draft, Methodology, Data curation. Qingqing Wei: Supervision. Li Kang: Supervision. Yi Sun: Methodology. Yunliang Jiang: Writing – review & editing, Supervision, Funding acquisition.

Disclosures

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be misconstrued as a potential conflict of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant number 32272869).

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2025.105837.

Appendix. Supplementary materials

mmc1.docx (127.1KB, docx)

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