Abstract
Chicken somatic cells exhibit cell autonomous sex identity (CASI), where sexual features are determined by sex chromosomes rather than hormones. CASI arises from sex‑biased gene expression due to lack of dosage compensation on sex chromosomes. To identify CASI‑determining genes (CASIDGs), we analyzed transcriptomes of wattles (phenotypically hormone‑sensitive) and pectoral muscles (phenotypically hormone‑unresponsive) from 6‑week‑old roosters and hens, with or without sex hormone treatment. Inter‑sex differentially expressed genes (DEGs) mainly located on the Z chromosome and autosomes 1‑2, while hormone‑affected DEGs predominantly on autosomes 1‑3. After removing hormone‑affected DEGs from the inter‑sex DEGs, 285 DEGs (51.23% on sex chromosome) remained for pectoral muscle, and 255 DEGs (63.93% on sex chromosome) for wattle. Out of the 89 DEGs shared by these two tissues, 88 were located on sex chromosomes and were considered candidate CASIDGs. These candidate CASIDGs were mainly enriched in mitochondrial protein synthesis and isomerase activity-related pathways. Among the CASIDGs, HINT1 and MED18 were validated according to the screening criteria. Overexpressing these two genes in primary muscle cells could affect immune/inflammatory responses, cell proliferation and migration, cytoskeleton, and extracellular matrix interactions. Additionally, 139 autosomal DEGs for pectoral muscle and 92 for wattle were identified as candidate sex marker genes. Among these, IRF9 and CCL4 were co‑regulated by HINT1 and MED18. In conclusion, the sex characteristics of chicken somatic cells reflect the combined effects of CASIDGs and sex hormones. HINT1 and MED18 are confirmed as candidate CASIDGs. These findings lay a solid foundation for elucidating the CASI mechanism in avian somatic cells.
Keywords: Chicken, Cell autonomous sex identity, Sex hormone, Sex determining gene, Sex marker gene
Introduction
Sex is closely related to many biological traits in birds, especially economic traits. The chicken (Gallus gallus) is an important economic animal and model organism. Discovering sex-determining genes and elucidating the relevant mechanisms have important theoretical and practical values for understanding sex evolution in avian species and for sex regulation in poultry. Currently, there are few studies on the determination of sex characteristics in non-gonadal tissues of chickens, such as wattles and muscles. Our recent study indicates that non-gonadal tissues in chickens differ in their sensitivity to sex hormones, for example, wattles are phenotypically sensitive to sex hormone treatment, while muscles are phenotypically unresponsive. This sensitivity is related to the protein abundance of the sex hormone receptor ERα in the tissues (Jin et al., 2026). However, muscles and other phenotypically hormone-unresponsive non-gonadal tissues still show obvious sex differences between roosters and hens. This suggests the presence of other determining factors.
To explain this, previous researchers proposed the concept of cell autonomous sex identity (CASI) (Zhao et al., 2010). The CASI hypothesis was initially based on studies of three naturally occurring gynandromorphic chickens. In these chickens, the levels of sex hormones circulating in the blood were the same on the left and right sides of the body, but the sex characteristics on the two sides were different. In situ hybridisation investigations revealed that the body side exhibiting male traits predominantly had ZZ sex chromosome constitution, while the female phenotype side predominantly had ZW constitution. This discovery indicates that the genes responsible for sex traits in chicken somatic cells reside on the sex chromosomes.
The prevailing consensus holds that sex chromosomes harbor genes responsible for sexual characteristics in chicken somatic cells, i.e., CASI-determining genes (CASIDGs). Genes located on the chicken Z chromosome typically lack dosage compensation (Kuroda et al., 2001; Kuroiwa et al., 2002; Ellegren et al., 2007; Sun et al., 2019). Therefore, the expression level of CASIDGs in males is about twice that in females. This dosage difference causes females to exhibit female characteristics and males to show male characteristics(Clinton et al., 2012). In addition, because males lack the W chromosome, genes on the W chromosome may also be candidates for CASIDGs due to dosage effects.
Previous studies have analyzed transcriptome of chicken blastoderms at Hamburger‑Hamilton stage 4 (HH4), when gonadal sex has not yet differentiated. More than 200 differentially expressed genes (DEGs) on sex chromosomes were identified between males and females (Zhang et al., 2010). At this stage, undifferentiated gonads cannot produce sex hormones. Therefore, the expression differences of these DEGs are not caused by sex hormones but by gene dosage effects. These DEGs on sex chromosomes can be considered candidates for CASIDGs. In addition, other studies have sequenced gonads of 4.5-day-old male and female embryos and brains at different embryonic stages. It was estimated that there are about 300 candidate CASIDGs (Ayers et al., 2013). However, most of the candidate CASIDGs have been investigated in studies on sex differentiation of chicken gonads. For example, DMRT1 on the Z chromosome has been demonstrated to be involved in testis differentiation (Ioannidis et al., 2021). miR‑2954 plays a key role in dosage compensation of avian sex chromosomes (Fallahshahroudi et al., 2025). Except for the gonads, the roles of these genes in sex determination of other non-gonadal tissues have not been reported.
Given that differentiation of different tissues involves epigenetic mechanisms, different tissues may share common CASIDGs and also have distinct ones. Previously identified candidate CASIDGs were based on chicken embryos at very early developmental stages. At those stages, many tissues have not yet begun to differentiate or have not completed differentiation. Therefore, such screening has certain limitations. This study aimed to screen for DEGs between sexes in the wattles and pectoral muscles of 6‑week‑old chickens, and then to remove those genes affected by sex hormones. In this way, putative CASIDGs situated on sex chromosomes could be identified, which will establish a basis for clarifying the mechanisms of sex determination in non-gonadal tissues.
Materials and methods
Construction of sex hormone injection model chickens
Fifty Hy-Line White commercial day-old chicks with similar body weights were selected. Their sex was determined by vent sexing (25 roosters and 25 hens). They were raised under conventional management conditions until 2 weeks of age. Healthy roosters and hens were selected (24 of each sex) for sex hormone injection. Roosters were randomly divided into two groups: a control group (injected with saline) and a hormone treatment group (injected with β‑estradiol). Hens were also randomly assigned to a control group (saline injected) and a hormone treatment group (injected with testosterone). Starting at 2 weeks of age, each chicken received an intramuscular injection into the pectoral muscle every two days. The injection was either saline or the corresponding sex hormone. The dosage was 5 mg per kg body weight (according to preliminary unpublished data).
The injections continued until 6 weeks of age. During this period, all experimental chickens were raised on wire floors in the same room, managed under conventional conditions, and had free access to food and water.
The β‑estradiol (Solarbio, Beijing, China) and testosterone (Solarbio, Beijing, China) solutions were both dissolved in DMSO (Solarbio, Beijing, China). Before injection, they were diluted to the appropriate concentration with saline. Before slaughter, the chickens were fasted for 12 h. After fasting, each chicken was weighed. The comb and wattle were photographed and measured. The chickens were then euthanized by carbon dioxide asphyxiation. The pectoral muscle was dissected and weighed. Tissue samples of the gonads, pectoral muscle, and wattle were collected from the same site of each tissue. These samples were snap-frozen in liquid nitrogen and then transferred to a -80 °C freezer for long‑term storage. In addition, a small piece of pectoral muscle tissue was taken from each chicken. PCR-based sex identification was then performed to confirm the sex determined by vent sexing.
PCR-based sex identification
Sex identification was performed following the manufacturer's instructions. The reagents used were MightAmpTM DNA Polymerase Ver.2 (TaKaRa, Tokyo, Japan) and chicken CHD1 primers. The samples tested were pectoral muscle tissue samples or blood samples from E6 chicken embryos(Ellegren, 1996).
Transcriptome sequencing analysis of muscle and wattle tissues
The general procedure for transcriptome sequencing was as follows. Total RNA was extracted from tissues or cells using TRIzol reagent. The RNA was then treated with DNase I to remove genomic DNA. RNA integrity was assessed using an Agilent 2100 Bioanalyzer. The RNA Integrity Number (RIN) for all samples was ensured to be ≥ 7.0. Next, mRNA was enriched using oligo(dT) magnetic beads. The mRNA was fragmented and used as a template to synthesize cDNA. After constructing the sequencing library, paired-end 150 bp (PE150) sequencing was performed on the Illumina NovaSeq 6000 platform.
Raw sequencing data were subjected to quality control and filtering to obtain clean reads. Clean reads were aligned to the chicken reference genome (GRCg6a) using HISAT2. Gene expression was quantified using featureCounts. DEGs were identified using DESeq2 with different filtering criteria based on the experimental requirements.
To identify the DEGs between untreated males (roosters) and females (hens) separately for pectoral muscle and wattle tissues, the screening criteria were |log₂ (Fold Change) | > 0.585 and P < 0.05.
To identify the DEGs regulated by different sex hormones separately for pectoral muscle and wattle tissues, the transcriptomic data of β-estradiol treated-roosters were compared with the untreated-roosters, or the transcriptomic data of testosterone treated-hens were compared with the untreated-hens. The screening criteria were (|log₂ (Fold Change) | > 0.585 and P < 0.05).
To identify the putative CASIDGs separately for pectoral muscle and wattle tissues, substracting the genes regulated by sex hormones from inter-sex DEGs was performed. The remaining DEGs on the sex chromosomes are putative CASIDGs. It is noteworthy that here the criteria for identification of genes regulated by sex hormones were only |log₂ (Fold Change) | > 0.585 without considering P value. Applying this lenient threshold allows for capturing as many hormone-affected genes as possible (including those with marginal significance), ensuring that the remaining CASIDGs retain high specificity. This approach prioritizes sensitivity in removing hormone-affected genes rather than excluding false positives in the traditional statistical sense. The putative CASIDGs common to the two tissues were also identified.
To identify the pathways enriched by DEGs, Gene Ontology (GO) analysis and KEGG pathway annotation were performed using R 4.5.1. Pathways or biological processes with P < 0.05 considered as statistically significant.
Screening method for sex marker genes in chicken somatic tissues
To identify candidate sex marker genes separately for pectoral muscle and wattle tissues, the gene sets obtained after removal of hormone-affected genes using the lenient criterion described above were used. From these two gene sets, genes located on autosomes were retained. This yields the candidate sex marker gene set for chicken pectoral muscle tissue and the candidate sex marker gene set for chicken wattle tissue, respectively. The obtained candidate sex marker genes were further validated by quantitative PCR (qPCR).
Collection of chicken embryo tissue samples
Thirty fertilized eggs of similar size were selected from Hy‑Line White commercial layer hens. The eggs were incubated using a standard incubation method. On day 6 and day 12 of incubation, the fertilized eggs were taken out and the eggshells were wiped with 75% alcohol for disinfection. The eggs were then opened, and the embryos were removed. The embryos were euthanized by cervical dislocation. Pectoral muscle tissue was carefully dissected and quickly frozen in liquid nitrogen. The samples were then transferred to a -80 °C freezer for storage. For embryos collected on day 6 of incubation, a small amount of blood was taken, and PCR-based sex identification was performed to confirm the genetic sex of each embryo. For embryos collected on day 12 of incubation, the morphology of the gonads was observed to confirm the genetic sex.
For the nucleocytoplasmic separation experiment, one male and one female embryo were collected at 12 days of incubation. Fresh gonad and pectoral muscle tissues were carefully dissected and placed in sterile PBS. These tissues were immediately used for nuclear and cytoplasmic separation.
Nucleocytoplasmic separation of tissues
Nuclear and cytoplasmic separation was performed using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) following the manufacturer's instructions. Fresh gonad and pectoral muscle tissues from 12-day-old chicken embryos were used. The general procedure was as follows. Under hypotonic conditions, a cytoplasmic protein extraction reagent was used to fully swell the cells and disrupt the cell membranes., releasing the cytoplasmic proteins. After centrifugation, the cytoplasmic supernatant and the nuclear pellet were collected. Then, a high-salt nuclear protein extraction reagent was used to extract the nuclear proteins.
RNA extraction and RT-qPCR
Total RNA was extracted from tissues using TRNzol Universal (TIANGEN, Beijing, China) following the manufacturer's instructions. The general procedure was as follows. Tissues were fully homogenized or cells lysed in TRNzol reagent. Chloroform was added to achieve phase separation. After centrifugation, the upper aqueous phase containing RNA was collected. Isopropanol was added to precipitate the RNA. The RNA pellet was washed with 75% ethanol to remove impurities. Finally, the RNA was dried and dissolved in nuclease-free water to obtain total RNA.
A commercial kit (Vazyme, Nanjing, China) was used to reverse transcribe total RNA into cDNA. The general procedure was as follows. Total RNA was mixed with primers and heated for denaturation. Then reverse transcriptase, dNTPs, buffer, and RNase inhibitor were added. The mixture was incubated at 42 °C to synthesize first-strand cDNA. Finally, the reverse transcriptase was inactivated by heating.
All qPCR primers were designed using Primer 5.0 software. They were synthesized by GENEWIZ Biotechnology Co., Ltd. (Suzhou, China). Primer information is listed in Supplementary Table 1. qPCR was performed on a real-time quantitative PCR instrument using a commercial kit (Yeasen, Shanghai, China). The reaction mixture contained SYBR Green master mix, forward and reverse primers, and cDNA template. 28S was used as the internal reference gene. Results were analyzed using the 2‑ΔΔCt method.
Protein extraction and western blot
Total protein was extracted from tissues using RIPA lysis buffer (APPLYGEN, Beijing, China). The general procedure was as follows. Cell pellets or homogenized tissues were mixed with pre-chilled RIPA lysis buffer containing protease and phosphatase inhibitors. The mixture was thoroughly homogenized and lysed on ice for 20 minutes. The lysate was then centrifuged at 4 °C, and the supernatant was collected.
Protein concentration was measured using a BCA Protein Assay Kit (Beyotime, Shanghai, China) following the manufacturer's instructions to ensure equal loading amounts of protein. The general procedure was as follows. BCA working solution was prepared. The test samples and standards were added to a 96-well plate. The working solution was then added and mixed. The plate was incubated at 37 °C for 25 minutes. Absorbance was measured at 562 nm. Protein concentrations were calculated based on a standard curve.
Protein samples with measured concentrations were mixed with protein loading buffer. The mixture was heated in a metal bath at 95 °C for 10 minutes for denaturation. Protein samples were then separated by SDS-PAGE electrophoresis. The separated proteins were transferred to a PVDF membrane using a wet transfer method. The PVDF membrane was blocked with 5% non-fat milk. The membrane was then incubated with specific primary antibodies at 4 °C overnight. After that, the membrane was incubated with the corresponding HRP-labelled secondary antibodies at room temperature for 1 h. Finally, signals were detected using enhanced chemiluminescence (ECL) substrate. The antibodies used in this study were as follows. SMAD2Z antibody (12570-1-AP), HINT1 antibody (10717-1-AP), CDK7 antibody (27027-1-AP), MED18 antibody (15352-1-AP), and GAPDH antibody (60004-1-Ig) were purchased from Proteintech Group (Wuhan Sanying Biotechnology Co., Ltd.). Goat anti rabbit IgG (CW0103S) and Goat anti mouse IgG (CW0102S) were purchased from Jiangsu Coen Biotechnology Co., Ltd.
Isolation and culture of primary muscle cells
Ten fertilized eggs of similar size were selected from Hy‑Line White commercial layer hens. The eggs were incubated using a standard incubation method. After 12 days of incubation, primary muscle cells were isolated. The fertilized eggs were taken out and wiped with 75% alcohol for disinfection. The eggshells were opened, and the embryos were removed. The embryos were euthanized by cervical dislocation. The sex of each embryo was confirmed by observing the morphology of the gonads under a stereomicroscope. The pectoral muscle tissue was carefully dissected and placed in sterile PBS. The tissue was washed twice with sterile PBS. Pre-warmed trypsin (Gibco, NY, USA) was then added. The mixture was digested in a 37 °C-water bath. After complete digestion, DMEM (Gibco, NY, USA) containing 10% FBS (Gibco, NY, USA) was added to stop digestion. The mixture was then centrifuged. The cell pellet was washed once with sterile PBS. Complete medium (DMEM, 10% FBS, and 100× penicillin streptomycin) was added to resuspend the cells. The cell suspension was filtered through a 40μm cell strainer. The cells were then counted and diluted to an appropriate density. The cells were seeded into cell culture plates and cultured in a 37 °C incubator with 5% CO2.
Overexpression of HINT1 or MED18 in chicken primary muscle cells
The coding sequences (CDS) of chicken HINT1 (GenBank: NM_204689.2) and MED18 (GenBank: XM_003642960.6) were amplified by RT‑PCR from chicken ovarian cDNA. EcoRI and HindIII restriction sites were introduced at both ends of each amplicon. Each PCR product and the pcDNA3.1(+) vector were double‑digested with EcoRI and HindIII, then ligated and transformed into DH5α competent cells. Expression of the exogenous sequences in pcDNA3.1(+) is driven by the CMV promoter and terminated by the bGH poly(A) signal. The recombinant plasmids (named pcDNA3.1‑HINT1 and pcDNA3.1‑MED18) were verified by DNA sequencing and then used in subsequent experiments.
Transfection of chicken primary muscle cells with the recombinant plasmids pcDNA3.1‑HINT1 and pcDNA3.1‑MED18 (separately) was performed using Lipofectamine™ 3000 (Invitrogen, CA, USA) following the manufacturer's instructions. The general procedure was as follows. Lipofectamine 3000 reagent and DNA were each diluted with Opti MEM. P3000 reagent was added to the DNA dilution. The two dilutions were then mixed and incubated at room temperature for 15 minutes to form liposome-DNA complexes. Finally, the complexes were added to the cell culture medium. The cells were cultured in a 37 °C incubator with 5% CO2. Cells in 12-well plates were harvested after 24 hours for transcriptome sequencing. After 48 hours, cells in 6-well plates were harvested, and proteins were extracted to assess the efficacy of overexpression.
Screening of DEGs induced by overexpression of HINT1 or MED18 in chicken primary muscle cells
Considering that cellular studies exhibit greater repeatability and less inter-individual variability relative to tissue samples, a more rigorous screening criterion was employed for the identification of DEGs in the cellular experiments. This facilitated the control of the false positive rate and enhanced the dependability of the outcomes.
After measuring gene expression with featureCounts, DESeq2 software was used to detect DEGs in chicken primary muscle cells. The criteria were |log2(Fold Change) | > 1 and padj < 0.05. GO functional enrichment analysis and KEGG signaling pathway annotation were performed on the identified DEGs using R 4.5.1. GO terms and KEGG pathways with padj < 0.05 were considered significant.
Data analysis
All data were analyzed using R 4.5.1. Data are presented as mean ± standard deviation (SD). For comparisons between two groups, an independent samples t-test or Mann‑Whitney U test was used depending on the data distribution. For comparisons among multiple groups, one‑way analysis of variance (ANOVA) followed by Tukey's HSD post hoc test, or the Kruskal‑Wallis test followed by Dunn's post hoc test, was used depending on the data distribution. Statistical significance was defined as P < 0.05. For two‑group comparisons, significant differences are indicated by asterisks (*P < 0.05, **P < 0.01). For multiple group comparisons, significant differences among groups are indicated by letters. Groups sharing the same letter are not statistically different (P > 0.05), while groups with completely different letters are statistically different (P < 0.05).
Results
Sex hormone injection causes tissue-specific changes in sex phenotypes in chickens
Starting at 2 weeks of age, hens were injected with testosterone, and roosters were injected with β‑estradiol. The dose was 5 mg per kg body weight. The injections lasted for 4 weeks. The results showed that compared with control hens, the testosterone-injected hens had significantly larger combs and wattles. Their comb and wattle color was the same as that of control roosters. On the other hand, compared with control roosters, the β‑estradiol-injected roosters had significantly smaller combs and wattles. Their comb and wattle color was the same as that of control hens (Fig. 1A). This indicates that the comb and wattle are phenotypically hormone-sensitive somatic tissues, and their sex phenotypes are easily affected by sex hormones. However, measurement of pectoral muscle weight showed that muscle weight was not significantly affected by testosterone or β‑estradiol injection. This suggests that chicken pectoral muscle is a phenotypically hormone-unresponsive somatic tissue (Fig. 1B).
Fig. 1.
Effects of sex hormone treatment on sexual phenotypes. (A) effects on comb and wattle. (B) effects on pectoral muscle weight. F-control, hen control group; F-T, hen testosterone-treated group; M-control, rooster control group; M-E2, rooster β-estradiol-treated group. Data are presented as mean ± SD, n=6.
Screening and analysis of DEGs between sexes in the pectoral muscle and wattle of control roosters and hens
Through transcriptome sequencing analysis of pectoral muscle tissue from control roosters and hens, 854 DEGs were identified (Supplementary Fig. 1A). Among these, 480 genes were highly expressed in hen pectoral muscle, and 374 genes were highly expressed in rooster pectoral muscle (see Table 1 for the top 10 genes with the highest expression fold change in each group). The chromosomal distribution of these DEGs is shown in Supplementary Fig. 1B. They were mainly concentrated on the Z chromosome (158 DEGs, 18.50%), autosome 1 (111 DEGs, 13.00%), and autosome 2 (62 DEGs, 7.26%).
Table 1.
Top 10 upregulated DEGs in hen and rooster pectoral muscle tissue.
| Ensembl ID | Gene Symbol | log2FoldChange | Chromosome | Description |
|---|---|---|---|---|
| ENSGALG00000035998 | HINTW | 13.00512 | W | histidine triad nucleotide binding protein W |
| ENSGALG00000033705 | - | 12.84100 | W | transitional endoplasmic reticulum ATPase-like |
| ENSGALG00000043758 | - | 12.21445 | W | ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle |
| ENSGALG00000035780 | - | 11.57887 | W | E3 ubiquitin-protein ligase KCMF1-like |
| ENSGALG00000040086 | HNRNPKL | 11.36652 | W | heterogeneous nuclear ribonucleoprotein K-like |
| ENSGALG00000040263 | - | 11.11886 | W | 60S ribosomal protein L17 |
| ENSGALG00000014184 | - | 10.76942 | W | mothers against decapentaplegic homolog 2-like |
| ENSGALG00000041221 | - | 10.50820 | W | - |
| ENSGALG00000048542 | - | 10.16206 | W | ubiquitin-conjugating enzyme E2 R2-like |
| ENSGALG00000040704 | SPIN1W | 9.84024 | W | spindlin 1-W |
| ENSGALG00000043433 | SCX | -4.48921 | 2 | scleraxis bHLH transcription factor |
| ENSGALG00000005831 | DNAI1 | -4.27031 | Z | dynein axonemal intermediate chain 1 |
| ENSGALG00000030687 | LGI3 | -4.24129 | 22 | leucine rich repeat LGI family member 3 |
| ENSGALG00000041042 | GABRG3 | -3.75878 | 1 | gamma-aminobutyric acid type A receptor gamma3 subunit |
| ENSGALG00000029035 | - | -3.75049 | Z | C-type lectin domain family 5, member A |
| ENSGALG00000003025 | - | -3.74979 | 9 | - |
| ENSGALG00000042664 | CBLN1 | -3.52142 | 11 | cerebellin 1 precursor |
| ENSGALG00000005180 | - | -3.32449 | 8 | - |
| ENSGALG00000015599 | SOHO-1 | -3.31834 | 4 | sensory organ homeobox protein SOHo |
| ENSGALG00000037011 | CILP2 | -3.25548 | 28 | cartilage intermediate layer protein 2 |
Note: These data are derived from the same transcriptome sequencing comparison (hen vs. rooster). Therefore, log2FoldChange > 0 indicates genes upregulated in hen pectoral muscle tissue relative to roosters, while log2FoldChange < 0 indicates genes upregulated in rooster pectoral muscle tissue relative to hens.
GO analysis showed that these DEGs were mainly enriched in 125 Biological Process (BP) terms, 42 Molecular Function (MF) terms, and 30 Cellular Component (CC) terms (Supplementary Fig. 1C). The top 10 BP terms included Translation, Cytoplasmic translation, Regulation of cell shape, Regulation of anatomical structure morphogenesis, Oligosaccharide metabolic process, Phosphatidylinositol mediated signaling, Negative regulation of developmental growth, Cellular response to hormone stimulus, Peptidyl threonine phosphorylation, and Response to peptide hormone. The top MF 10 terms included Structural constituent of ribosome, Protein kinase activity, Protein serine/threonine kinase activity, Phosphoric ester hydrolase activity, Calmodulin binding, Phosphatase activity, Ubiquitin protein ligase binding, Cytoskeletal protein binding, Sugar phosphatase activity, and Fatty acid binding. The top 10 CC terms included Cytosolic ribosome, Ribosome, Ribosomal subunit, Cytosolic large ribosomal subunit, Large ribosomal subunit, Ribonucleoprotein complex, Cytosolic small ribosomal subunit, Nuclear speck, Small ribosomal subunit, and Sarcomere. KEGG analysis showed that these DEGs were mainly enriched in 15 pathways (Fig. 2A). These pathways included Ribosome, Focal adhesion, Primary bile acid biosynthesis, ErbB signaling pathway, Apelin signaling pathway, Glycosphingolipid biosynthesis ganglio series, PPAR signaling pathway, TGF beta signaling pathway, Nucleotide excision repair, and Salmonella infection.
Fig. 2.
KEGG pathway enrichment of inter‑sex DEGs. (A) pectoral muscle. (B) wattle. (C) common DEGs shared by pectoral muscle and wattle.
Through transcriptome sequencing analysis of wattle tissue from control roosters and hens, a total of 1156 DEGs were identified (Supplementary Fig. 2A). Among these, 546 genes were highly expressed in hen wattle tissue, and 610 genes were highly expressed in rooster wattle (see Table 2 for the top 10 genes with the highest expression fold change in each group). The chromosomal distribution of these DEGs is shown in Supplementary Fig. 2B. They were mainly concentrated on the Z chromosome (194 DEGs, 16.78%), autosome 1 (148 DEGs, 12.80%), and autosome 2 (87 DEGs, 7.53%).
Table 2.
Top 10 upregulated DEGs in hen and rooster wattle tissue.
| Ensembl ID | Gene Symbol | log2FoldChange | Chromosome | Description |
|---|---|---|---|---|
| ENSGALG00000035998 | HINTW | 14.00979 | W | histidine triad nucleotide binding protein W |
| ENSGALG00000040086 | HNRNPKL | 13.26491 | W | heterogeneous nuclear ribonucleoprotein K-like |
| ENSGALG00000043758 | - | 12.68503 | W | ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle |
| ENSGALG00000033705 | - | 12.48907 | W | transitional endoplasmic reticulum ATPase-like |
| ENSGALG00000040704 | SPIN1W | 12.45166 | W | spindlin 1-W |
| ENSGALG00000030382 | - | 11.56114 | W | Golgi phosphoprotein 3-like |
| ENSGALG00000040263 | - | 11.51514 | W | 60S ribosomal protein L17 |
| ENSGALG00000048542 | - | 11.24611 | W | ubiquitin-conjugating enzyme E2 R2-like |
| ENSGALG00000041221 | - | 11.13468 | W | - |
| ENSGALG00000039023 | - | 11.09506 | W | Nipped-B homolog-like |
| ENSGALG00000042684 | - | -6.29646 | 16 | - |
| ENSGALG00000047927 | - | -5.71604 | 8 | - |
| ENSGALG00000053077 | - | -5.51470 | 33 | - |
| ENSGALG00000023760 | CHIA | -4.99039 | 26 | chitinase, acidic |
| ENSGALG00000019175 | - | -4.54592 | 1 | cystathionine beta-synthase-like |
| ENSGALG00000049364 | - | -4.51028 | Z | uncharacterized LOC112530649 |
| ENSGALG00000005226 | MYOZ1 | -4.45976 | 6 | myozenin 1 |
| ENSGALG00000017016 | FAM124A | -4.45903 | 1 | family with sequence similarity 124 member A |
| ENSGALG00000017168 | SLN | -4.20082 | 1 | sarcolipin |
| ENSGALG00000015753 | - | -4.13608 | Z | natriuretic peptide receptor A/guanylate cyclase A |
Note: These data are derived from the same transcriptome sequencing comparison (hen vs. rooster). Therefore, log2FoldChange > 0 indicates genes upregulated in hen wattle tissue relative to roosters, while log2FoldChange < 0 indicates genes upregulated in rooster wattle tissue relative to hens.
GO analysis showed that these DEGs were mainly enriched in 210 BP terms, 70 MF terms, and 22 CC terms (Supplementary Fig. 2C). The top BP 10 terms included Anatomical structure morphogenesis, Neurogenesis, Chemotaxis, Axon guidance, Taxis, Neuron projection guidance, Positive regulation of cell migration, Negative regulation of cell differentiation, Cell morphogenesis, and Sphingolipid metabolic process. The top 10 MF terms included Extracellular matrix structural constituent, Growth factor binding, Cytokine binding, Chemorepellent activity, Neutral L-amino acid transmembrane transporter activity, Postsynaptic neurotransmitter receptor activity, Transmembrane receptor protein tyrosine kinase activity, Amino acid: sodium symporter activity, L-glutamate transmembrane transporter activity, and Semaphorin receptor binding. The top 10 CC terms included Extracellular matrix, Collagen containing extracellular matrix, External encapsulating structure, Myofibril, Sarcomere, Contractile muscle fiber, Receptor complex, Striated muscle thin filament, Myofilament, and Z disc. KEGG analysis showed that these DEGs were mainly enriched in 14 pathways (Fig. 2B). These pathways included Cytoskeleton in muscle cells, Integrin signaling, ECM receptor interaction, Neuroactive ligand signaling, Focal adhesion, Fatty acid elongation, Mucin type O-glycan biosynthesis, Cytokine-cytokine receptor interaction, Folate biosynthesis, and Histidine metabolism.
Comparing the 854 DEGs in pectoral muscle and the 1156 DEGs in wattle (between roosters and hens), we identified 199 common DEGs shared by both tissues. Their chromosomal distribution is shown in Supplementary Fig. 3A. They were mainly concentrated on the Z chromosome (92 DEGs, 46.23%), the W chromosome (31 DEGs, 15.58%), and autosome 1 (17 DEGs, 8.54%).
GO analysis showed that these common DEGs were mainly enriched in 55 BP terms, 9 MF terms, and 16 CC terms (Supplementary Fig. 3B). The top 10 BP terms included rRNA metabolic process, Carboxylic acid transport, Organic acid transport, Regulation of cell shape, Peptide catabolic process, Regulation of cell morphogenesis, Macroautophagy, rRNA processing, Organic anion transport, and NADP metabolic process. The 9 MF terms included Tubulin binding, Structural constituent of ribosome, Neutral L-amino acid transmembrane transporter activity, Organic cation transmembrane transporter activity, Peptidyl-prolyl cis-trans isomerase activity, ATP hydrolysis activity, L-amino acid transmembrane transporter activity, Cis-trans isomerase activity, and Microtubule binding. The top 10 CC terms included Ribosome, Ribonucleoprotein complex, Organellar large ribosomal subunit, Mitochondrial large ribosomal subunit, Ribosomal subunit, Large ribosomal subunit, RNA polymerase II transcription regulator complex, Mitochondrial protein-containing complex, Organellar ribosome, and Mitochondrial ribosome. KEGG analysis showed that these common DEGs were mainly enriched in the Ribosome, Folate biosynthesis, and Salmonella infection pathways (Fig. 2C).
Screening and analysis of hormone-responsive DEGs in the pectoral muscle and wattle of roosters and hens
Transcriptome sequencing analysis of pectoral muscle from control roosters vs. estrogen-treated roosters identified 1090 DEGs (Supplementary Fig. 4A). Among these, 608 DEGs were upregulated after estrogen treatment, and 482 DEGs were downregulated (see Table 3 for the top 10 genes with the highest fold change in each group). The chromosomal distribution of these DEGs is shown in Supplementary Fig. 4B. They were mainly concentrated on autosome 1 (141 DEGs, 12.94%), autosome 2 (113 DEGs, 10.37%), and autosome 3 (82 DEGs, 7.52%).
Table 3.
Top 10 upregulated and downregulated DEGs in the pectoral muscle of roosters induced by estrogen treatment.
| Ensembl ID | Gene Symbol | log2FoldChange | Chromosome | Description |
|---|---|---|---|---|
| ENSGALG00000025896 | PLPPR4 | 5.88587 | 8 | phospholipid phosphatase related 4 |
| ENSGALG00000054397 | - | 4.90733 | 16 | - |
| ENSGALG00000006926 | - | 4.56813 | 24 | - |
| ENSGALG00000006258 | SPOCK1 | 4.29774 | 13 | SPARC/osteonectin, cwcv and kazal like domains proteoglycan 1 |
| ENSGALG00000004574 | NPPA | 4.25778 | 21 | natriuretic peptide A |
| ENSGALG00000006130 | STMN3 | 4.02806 | 20 | stathmin 3 |
| ENSGALG00000007666 | KCNIP2 | 3.98534 | 6 | potassium voltage-gated channel interacting protein 2 |
| ENSGALG00000054689 | - | 3.92216 | 1 | inositol 1,4,5-trisphosphate receptor-interacting protein-like 1-like |
| ENSGALG00000031018 | RNF165 | 3.73954 | Z | ring finger protein 165 |
| ENSGALG00000010926 | SPP1 | 3.53425 | 4 | secreted phosphoprotein 1 |
| ENSGALG00000033700 | CA3A | -9.16676 | 2 | carbonic anhydrase 3A |
| ENSGALG00000051054 | - | -6.33292 | KZ626819.1 | class I histocompatibility antigen, F10 alpha chain-like |
| ENSGALG00000046715 | - | -6.15102 | Z | - |
| ENSGALG00000012783 | MYBPC1 | -5.34493 | 1 | myosin binding protein C, slow type |
| ENSGALG00000004028 | MATN4 | -5.07642 | 20 | matrilin 4 |
| ENSGALG00000037011 | CILP2 | -4.80678 | 28 | cartilage intermediate layer protein 2 |
| ENSGALG00000015599 | SOHO-1 | -4.72773 | 4 | sensory organ homeobox protein SOHo |
| ENSGALG00000034067 | FMOD | -4.61976 | 26 | fibromodulin |
| ENSGALG00000005180 | - | -4.57358 | 8 | - |
| ENSGALG00000053626 | - | -4.52036 | 31 | natural cytotoxicity triggering receptor 1-like |
Note: These data are derived from the same transcriptome sequencing comparison between estrogen treated roosters and control roosters (treated vs. control). Therefore, log2FoldChange > 0 indicates genes upregulated in the pectoral muscle of treated roosters relative to control roosters, while log2FoldChange < 0 indicates genes upregulated in the pectoral muscle of control roosters relative to treated roosters.
GO analysis showed that these DEGs were mainly enriched in 141 BP terms, 50 MF terms, and 23 CC terms (Supplementary Fig. 4C). The top 10 BP terms i included Circulatory system development, Actin filament-based process, Vasculature development, Tube morphogenesis, Anatomical structure formation involved in morphogenesis, Blood vessel development, Blood vessel morphogenesis, Actin cytoskeleton organization, Angiogenesis, and Tube development. The top 10 MF terms included Protein kinase activity, Protein serine/threonine kinase activity, Actin binding, Cytoskeletal protein binding, Phosphoric ester hydrolase activity, Phosphatase activity, Transmembrane receptor protein kinase activity, Solute: inorganic anion antiporter activity, Antiporter activity, and Inorganic anion transmembrane transporter activity. The top 10 CC terms included Actin cytoskeleton, Basal plasma membrane, Receptor complex, Basal part of cell, Sarcomere, I band, Myofibril, Contractile muscle fiber, Basolateral plasma membrane, and Z disc. KEGG analysis showed that these DEGs were mainly enriched in 18 pathways (Fig. 3A). These pathways included Cytoskeleton in muscle cells, Apelin signaling pathway, Mucin type O-glycan biosynthesis, ErbB signaling pathway, Other types of O-glycan biosynthesis, Efferocytosis, Insulin signaling pathway, Focal adhesion, Salmonella infection, and C-type lectin receptor signaling pathway.
Fig. 3.
KEGG pathway enrichment of DEGs induced by sex hormone treatment in pectoral muscle. (A) estrogen-induced. (B) androgen-induced. (C) common DEGs induced by both estrogen and androgen.
Transcriptome sequencing analysis of pectoral muscle from control hens vs. androgen-treated hens identified 1008 DEGs (Supplementary Fig. 5A). Among these, 394 DEGs were upregulated after androgen treatment, and 614 DEGs were downregulated (see Table 4 for the top 10 genes with the highest fold change in each group). The chromosomal distribution of these DEGs is shown in Supplementary Fig. 5B. They were mainly concentrated on autosome 1 (150 DEGs, 14.88%), autosome 2 (102 DEGs, 10.12%), and autosome 3 (95 DEGs, 9.42%).
Table 4.
Top 10 upregulated and downregulated DEGs in the pectoral muscle of hens induced by androgen treatment.
| Ensembl ID | Gene Symbol | log2FoldChange | Chromosome | Description |
|---|---|---|---|---|
| ENSGALG00000049589 | - | 5.47245 | 26 | polymeric immunoglobulin receptor-like |
| ENSGALG00000009603 | - | 5.14432 | 1 | prolactin-like protein |
| ENSGALG00000016286 | TASL | 5.06620 | 1 | chromosome X open reading frame 21 |
| ENSGALG00000050736 | - | 4.91552 | 8 | - |
| ENSGALG00000037467 | NEU2 | 4.82985 | 9 | neuraminidase 2 |
| ENSGALG00000045607 | CXCL13 | 4.81646 | 4 | C-X-C motif chemokine ligand 13 |
| ENSGALG00000044569 | - | 4.64488 | 16 | uncharacterized LOC101747901 |
| ENSGALG00000029235 | CPNE4 | 4.63040 | 2 | copine 4 |
| ENSGALG00000017485 | TLR1A | 4.11453 | 4 | toll-like receptor 1 family member A |
| ENSGALG00000051119 | PCP4L1 | 4.09361 | 25 | Purkinje cell protein 4 like 1 |
| ENSGALG00000009724 | GREM1 | -5.57378 | 5 | gremlin 1, DAN family BMP antagonist |
| ENSGALG00000011287 | SULT | -5.16001 | 3 | sulfotransferase |
| ENSGALG00000016985 | CPB2 | -5.16001 | 1 | carboxypeptidase B2 |
| ENSGALG00000005323 | DIRAS2 | -5.1162 | Z | DIRAS family GTPase 2 |
| ENSGALG00000016805 | SULT1C3 | -5.07738 | 1 | sulfotransferase family 1C member 3 |
| ENSGALG00000043480 | GPM6A | -4.924 | 4 | glycoprotein M6A |
| ENSGALG00000019277 | SLCO1B3 | -4.89645 | 1 | solute carrier organic anion transporter family member 1B3 |
| ENSGALG00000007596 | - | -4.81784 | 12 | monocarboxylate transporter 2-like |
| ENSGALG00000011117 | AGT | -4.75966 | 3 | angiotensinogen |
| ENSGALG00000010853 | C8B | -4.18531 | 8 | complement C8 beta chain |
Note: These data are derived from the same transcriptome sequencing comparison between androgen treatment hens and control hens (treated vs. control). Therefore, log2FoldChange > 0 indicates genes upregulated in the pectoral muscle of treated hens relative to control hens, while log2FoldChange < 0 indicates genes upregulated in the pectoral muscle of control hens relative to treated hens.
GO analysis showed that these DEGs were mainly enriched in 167 BP terms, 38 MF terms, and 84 CC terms (Supplementary Fig. 5C). The top 10 BP terms included Translation, Cytoplasmic translation, Regulation of response to external stimulus, Purine nucleoside triphosphate biosynthetic process, Purine ribonucleoside triphosphate biosynthetic process, Purine ribonucleotide biosynthetic process, Membraneless organelle assembly, Ribonucleoside triphosphate biosynthetic process, Axon extension, and Negative regulation of developmental growth. The top 10 MF terms included Structural constituent of ribosome, Protein serine/threonine kinase activity, rRNA binding, Protein kinase activity, Cytoskeletal protein binding, Actin binding, 5′-nucleotidase activity, Semaphorin receptor binding, Ubiquitin protein ligase binding, and Protein N-acetyltransferase activity. The top 10 CC terms included Cytosolic ribosome, Ribosome, Ribosomal subunit, Cytosolic large ribosomal subunit, Cytosolic small ribosomal subunit, Small ribosomal subunit, Large ribosomal subunit, Ribonucleoprotein complex, Sarcomere, and Myofibril. KEGG analysis showed that these DEGs were mainly enriched in 12 pathways (Fig. 3B). These pathways included Ribosome, mTOR signaling pathway, Nicotinate and nicotinamide metabolism, Focal adhesion, Salmonella infection, Regulation of actin cytoskeleton, p53 signaling pathway, Integrin signaling, Efferocytosis, and Inositol phosphate metabolism.
By comparing the DEGs screened from the above transcriptome analyses of rooster and hen pectoral muscles (estrogen‑treated vs. control, and androgen‑treated vs. control), we identified 181 common DEGs. Their chromosomal distribution is shown in Supplementary Fig. 6A. They were mainly concentrated on autosome 1 (34 DEGs, 18.78%), autosome 2 (25 DEGs, 13.81%), and autosome 3 (19 DEGs, 10.50%).
GO analysis showed that these common DEGs were mainly enriched in 73 BP terms, 33 MF terms, and 22 CC terms (Supplementary Fig. 6B). The top 10 BP terms included Phospholipid transport, Translation, Phosphatidylinositol-mediated signaling, Peptide metabolic process, Phospholipid translocation, Lipid translocation, Lipid transport, Organophosphate ester transport, Regulation of membrane lipid distribution, and Lipid localization. The top 10 MF terms included mRNA 3′-UTR binding, 5′-nucleotidase activity, Phosphoric ester hydrolase activity, Nucleotidase activity, Protein serine/threonine kinase activity, Ubiquitin protein ligase binding, Phosphatidylinositol kinase activity, Ubiquitin-like protein ligase binding, Protein tyrosine phosphatase activity, and Phosphatase activity. The top 10 CC terms included Cytosolic ribosome, Phosphatidylinositol 3-kinase complex, Ribosome, Transferase complex, Ribosomal subunit, Cytosolic large ribosomal subunit, Cul4-RING E3 ubiquitin ligase complex, Nucleoplasm, Sarcomere, and Protein acetyltransferase complex. KEGG analysis showed that these common DEGs were mainly enriched in 8 pathways (Fig. 3C). These pathways included Salmonella infection, Inositol phosphate metabolism, Apelin signaling pathway, Progesterone-mediated oocyte maturation, Toll-like receptor signaling pathway, Focal adhesion, Viral life cycle - HIV-1, and Ribosome.
Transcriptome sequencing analysis of wattle from control roosters vs. estrogen-treated roosters identified 1707 DEGs (Supplementary Fig. 7A). Among these, 833 DEGs were upregulated after estrogen treatment, and 874 DEGs were downregulated (see Table 5 for the top 10 genes with the highest fold change in each group). The chromosomal distribution of these DEGs is shown in Supplementary Fig. 7B. They were mainly concentrated on autosome 1 (236 DEGs, 13.83%), autosome 2 (164 DEGs, 9.61%), and autosome 3 (129 DEGs, 7.56%).
Table 5.
Top 10 upregulated and downregulated DEGs in the wattle of roosters induced by estrogen treatment.
| Ensembl ID | Gene Symbol | log2FoldChange | Chromosome | Description |
|---|---|---|---|---|
| ENSGALG00000027053 | AKAIN1 | 5.45482 | 2 | A-kinase anchor inhibitor 1 |
| ENSGALG00000021235 | NPY6R | 5.42996 | 13 | neuropeptide Y receptor Y6 (pseudogene) |
| ENSGALG00000017052 | CCNA1 | 5.41910 | 1 | cyclin A1 |
| ENSGALG00000046106 | - | 5.26776 | 31 | - |
| ENSGALG00000031564 | - | 5.03407 | 3 | SOGA family member 3 |
| ENSGALG00000004966 | NYAP2 | 4.92968 | 9 | neuronal tyrosine-phosphorylated phosphoinositide- 3-kinase adaptor 2 |
| ENSGALG00000047053 | - | 4.79017 | Z | - |
| ENSGALG00000014913 | ROS1 | 4.67895 | 3 | ROS proto-oncogene 1, receptor tyrosine kinase |
| ENSGALG00000002266 | OGDHL | 4.64705 | 6 | oxoglutarate dehydrogenase like |
| ENSGALG00000007814 | - | 4.56397 | 9 | alkaline phosphatase, placental |
| ENSGALG00000014994 | CRHBP | -8.40017 | Z | corticotropin releasing hormone binding protein |
| ENSGALG00000027166 | ART7B | -7.84842 | 1 | GPI-anchored ADP-ribosyltransferase |
| ENSGALG00000053626 | - | -6.52045 | 31 | natural cytotoxicity triggering receptor 1-like |
| ENSGALG00000000911 | IL19 | -5.71928 | 26 | interleukin 20 |
| ENSGALG00000049086 | - | -5.62432 | 27 | feather keratin Cos1-1/Cos1-3/Cos2-1-like |
| ENSGALG00000047927 | - | -5.57275 | 8 | - |
| ENSGALG00000053077 | - | -5.36454 | 33 | - |
| ENSGALG00000007647 | MYO3A | -5.31555 | 2 | myosin IIIA |
| ENSGALG00000008732 | CTAGE1 | -5.14949 | 3 | cutaneous T-cell lymphoma-associated antigen 1 |
| ENSGALG00000046715 | - | -5.10911 | Z | - |
Note: These data are derived from the same transcriptome sequencing comparison between estrogen treated roosters and control roosters (treated vs. control). Therefore, log2FoldChange > 0 indicates genes upregulated in the wattle of treated roosters relative to control roosters, while log2FoldChange < 0 indicates genes upregulated in the wattle of control roosters relative to treated roosters.
GO analysis showed that these DEGs were mainly enriched in 340 BP terms, 103 MF terms, and 81 CC terms (Supplementary Fig. 7C). The top 10 BP terms included Cell adhesion, Anatomical structure morphogenesis, Cell migration, Neurogenesis, Generation of neurons, Neuron differentiation, Cell motility, Cell-cell adhesion, Cell development, and Enzyme-linked receptor protein signaling pathway. The top 10 MF terms included Cell adhesion molecule binding, Integrin binding, Growth factor activity, Cytoskeletal protein binding, Purinergic nucleotide receptor activity, Nucleotide receptor activity, Extracellular matrix structural constituent, Receptor ligand activity, Organic anion transmembrane transporter activity, and Signaling receptor activator activity. The top 10 CC terms included Plasma membrane protein complex, Extracellular matrix, External encapsulating structure, Cell surface, Collagen-containing extracellular matrix, Integrin complex, Anchoring junction, Protein complex involved in cell adhesion, Receptor complex, and Plasma membrane signaling receptor complex. KEGG analysis showed that these DEGs were mainly enriched in 20 pathways (Fig. 4A). These pathways included Integrin signaling, ECM-receptor interaction, Focal adhesion, Cytoskeleton in muscle cells, Calcium signaling pathway, IgSF CAM signaling, Cell adhesion molecule (CAM) interaction, Neuroactive ligand signaling, MAPK signaling pathway, and Regulation of actin cytoskeleton.
Fig. 4.
KEGG pathway enrichment of DEGs induced by sex hormone treatment in wattle. (A) estrogen-induced. (B) androgen-induced. (C) common DEGs induced by both estrogen and androgen.
Transcriptome sequencing analysis of wattle from control hens vs, androgen-treated hens identified 1955 DEGs (Supplementary Fig. 8A). Among these, 919 DEGs were upregulated after androgen treatment, and 1036 DEGs were downregulated (see Table 6 for the top 10 genes with the highest fold change in each group). The chromosomal distribution of these DEGs is shown in Supplementary Fig. 8B. They were mainly concentrated on autosome 1 (262 DEGs, 13.40%), autosome 2 (188 DEGs, 9.62%), and autosome 3 (168 DEGs, 8.59%).
Table 6.
Top 10 upregulated and downregulated DEGs in the wattle of hens induced by androgen treatment.
| Ensembl ID | Gene Symbol | log2FoldChange | Chromosome | Description |
|---|---|---|---|---|
| ENSGALG00000035803 | THRSP | 6.59414 | 1 | thyroid hormone responsive |
| ENSGALG00000026945 | CIDEC | 6.29340 | 11 | cell death inducing DFFA like effector c |
| ENSGALG00000049358 | - | 4.73972 | 22 | - |
| ENSGALG00000004230 | LIPC | 4.70336 | 10 | lipase C, hepatic type |
| ENSGALG00000010822 | SPATA4 | 4.68632 | 4 | spermatogenesis associated 4 |
| ENSGALG00000029024 | TCF21 | 4.68358 | 3 | transcription factor 21 |
| ENSGALG00000006653 | CSF2 | 4.64072 | 13 | colony stimulating factor 2 |
| ENSGALG00000043252 | OVALX | 4.62005 | 2 | ovalbumin-related protein X (SERPINB14C) |
| ENSGALG00000017168 | SLN | 4.59003 | 1 | sarcolipin |
| ENSGALG00000034907 | HOXB5 | 4.57644 | 27 | homeobox B5 |
| ENSGALG00000008273 | CALHM3 | -4.70872 | 6 | calcium homeostasis modulator 3 |
| ENSGALG00000001132 | APBB3 | -4.29150 | 13 | amyloid beta (A4) precursor protein-binding, family B, member 3 |
| ENSGALG00000034567 | POU4F3 | -4.28018 | 4 | POU class 4 homeobox 3 |
| ENSGALG00000042443 | - | -4.15863 | 2 | - |
| ENSGALG00000006710 | RAB36 | -4.02243 | 15 | RAB36, member RAS oncogene family |
| ENSGALG00000021303 | NPY7R | -3.96550 | 13 | neuropeptide Y7 receptor |
| ENSGALG00000010781 | GLRA3 | -3.84636 | 4 | glycine receptor alpha 3 |
| ENSGALG00000036097 | CDH18 | -3.84142 | 2 | cadherin 18 |
| ENSGALG00000012094 | SLC35F4 | -3.65663 | 5 | solute carrier family 35 member F4 |
| ENSGALG00000048560 | - | -3.65554 | 1 | - |
Note: These data are derived from the same transcriptome sequencing comparison between androgen treatment hens and control hens (treated vs. control). Therefore, log2FoldChange > 0 indicates genes upregulated in the wattle of treated hens relative to control hens, while log2FoldChange < 0 indicates genes upregulated in the wattle of control hens relative to treated hens.
GO analysis revealed that these DEGs were mainly enriched in 319 BP terms, 86 MF terms, and 35 CC terms (Supplementary Fig. 8C). The top 10 BP terms included Anatomical structure morphogenesis, Circulatory system development, Vasculature development, Tube development, Positive regulation of cell migration, Animal organ development, Blood vessel development, Blood vessel morphogenesis, Anatomical structure formation involved in morphogenesis, and Cell adhesion. The top 10 MF terms included Growth factor binding, Transmembrane receptor protein kinase activity, Transmembrane receptor protein tyrosine kinase activity, Cell adhesion molecule binding, Receptor ligand activity, Protein tyrosine kinase activity, Signaling receptor activator activity, Growth factor activity, Fatty acid binding, and Glycosaminoglycan binding. The top 10 CC terms included Anchoring junction, Cell-cell junction, Receptor complex, Extracellular matrix, External encapsulating structure, Adherens junction, Collagen-containing extracellular matrix, Chromosomal region, Cell-substrate junction, and Basement membrane. KEGG analysis showed that these DEGs were mainly enriched in 27 pathways (Fig. 4B). These pathways included Integrin signaling, PPAR signaling pathway, ECM-receptor interaction, Cornified envelope formation, Fatty acid metabolism, Calcium signaling pathway, Biosynthesis of unsaturated fatty acids, Cytoskeleton in muscle cells, Cytokine-cytokine receptor interaction, and MAPK signaling pathway.
By comparing the DEGs screened from the above transcriptome sequencing analyses of rooster and hen wattles (estrogen‑treated vs. control, and androgen‑treated vs. control), we identified 543 common DEGs. Their chromosomal distribution is shown in Supplementary Fig. 9A. They were mainly concentrated on autosome 1 (80 DEGs, 14.73%), autosome 2 (53 DEGs, 9.76%), and autosome 3 (45 DEGs, 8.29%).
GO analysis showed that these common DEGs were mainly enriched in 199 BP terms, 67 MF terms, and 32 CC terms (Supplementary Fig. 9B). The top 10 BP terms included Anatomical structure morphogenesis, Cell adhesion, Cell-cell adhesion, Axon development, Positive regulation of cell migration, Tube development, Response to wounding, Cell migration, Positive regulation of cell motility, and Positive regulation of locomotion. The top 10 MF terms included Cell adhesion molecule binding, Oxidoreductase activity acting on paired donors with incorporation or reduction of molecular oxygen, Integrin binding, Cell-cell adhesion mediator activity, Cytokine binding, Cell adhesion mediator activity, Extracellular matrix structural constituent, Purinergic nucleotide receptor activity, Nucleotide receptor activity, and Growth factor activity. The top 10 CC terms included Extracellular matrix, External encapsulating structure, Collagen-containing extracellular matrix, Anchoring junction, Condensed chromosome centromeric region, Receptor complex, Plasma membrane protein complex, Cell-cell junction, Chromosome centromeric region, and Catenin complex. KEGG analysis showed that these common DEGs were mainly enriched in 18 pathways (Fig. 4C). These pathways included ECM-receptor interaction, Integrin signaling, Focal adhesion, Cytoskeleton in muscle cells, Retinol metabolism, Cornified envelope formation, PPAR signaling pathway, Fatty acid metabolism, Calcium signaling pathway, and IgSF CAM signaling.
By comparing the common DEGs shared between rooster and hen pectoral muscles (181 genes) and those shared between rooster and hen wattles (543 genes), we found that only 8 DEGs were common to both tissues. They were SLK, PTGR2, FRMD3, MX1, CD320, CCN3, SECISBP2L, and ASPH.
Screening and analysis of chicken CASIDGs
To screen for candidate CASIDGs, we removed DEGs regulated by sex hormones (using the lenient criterion described in Methods) from the DEGs present between normal roosters and hens. The results showed that 285 DEGs were obtained in pectoral muscle. Among these, 95 DEGs were highly expressed in hen pectoral muscle, and 190 DEGs were highly expressed in rooster pectoral muscle (see Table 7 for the top 10 DEGs with the highest expression fold change in each group). The chromosomal distribution of these DEGs is shown in Supplementary Fig. 10A. Among them, 123 DEGs (43.16%) were located on the Z chromosome, and 23 DEGs (8.07%) were located on the W chromosome. The list of genes located on the sex chromosomes is provided in Supplementary Table 2.
Table 7.
Top 10 DEGs highly expressed in hen and rooster pectoral muscle after screening.
| Ensembl ID | Gene Symbol | log2FoldChange | Chromosome | Description |
|---|---|---|---|---|
| Hen | ||||
| ENSGALG00000035998 | HINTW | 13.0051 | W | histidine triad nucleotide binding protein W |
| ENSGALG00000043758 | - | 12.21445 | W | ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle |
| ENSGALG00000035780 | - | 11.57887 | W | E3 ubiquitin-protein ligase KCMF1-like |
| ENSGALG00000040086 | HNRNPKL | 11.36652 | W | heterogeneous nuclear ribonucleoprotein K-like |
| ENSGALG00000040263 | - | 11.11886 | W | 60S ribosomal protein L17 |
| ENSGALG00000014184 | - | 10.76942 | W | mothers against decapentaplegic homolog 2-like |
| ENSGALG00000041221 | - | 10.50820 | W | - |
| ENSGALG00000048542 | - | 10.16206 | W | ubiquitin-conjugating enzyme E2 R2-like |
| ENSGALG00000040704 | SPIN1W | 9.84024 | W | spindlin 1-W |
| ENSGALG00000030382 | - | 9.32653 | W | Golgi phosphoprotein 3-like |
| Rooster | ||||
| ENSGALG00000028005 | GADD45G | 1.87433 | Z | growth arrest and DNA damage inducible gamma |
| ENSGALG00000028142 | HACD4 | 1.49616 | Z | 3-hydroxyacyl-CoA dehydratase 4 |
| ENSGALG00000026300 | TOMM5 | 1.49136 | Z | translocase of outer mitochondrial membrane 5 |
| ENSGALG00000015543 | MRPL50 | 1.45988 | Z | mitochondrial ribosomal protein L50 |
| ENSGALG00000015677 | SUSD1 | 1.43023 | Z | sushi domain containing 1 |
| ENSGALG00000002605 | MRPL17 | 1.41105 | Z | mitochondrial ribosomal protein L17 |
| ENSGALG00000020528 | INIP | 1.40338 | Z | INTS3 and NABP interacting protein |
| ENSGALG00000027963 | COX7C | 1.40329 | Z | cytochrome c oxidase subunit 7C |
| ENSGALG00000036774 | RPL17 | 1.35059 | Z | ribosomal protein L17 |
| ENSGALG00000012624 | PRXL2C | 1.27643 | Z | AhpC/TSA antioxidant enzyme domain containing 1 |
GO analysis showed that these DEGs were mainly enriched in 89 BP terms, 24 MF terms, and 28 CC terms (Fig. 5A; see Supplementary Table 3 for the complete list). The top 10 BP terms included rRNA metabolic process, Ribosome biogenesis, Ribosomal large subunit assembly, Ribosome assembly, NAD metabolic process, Organophosphate biosynthetic process, Ribosomal large subunit biogenesis, Ribonucleoprotein complex biogenesis, Regulation of cell cycle, and Organophosphate metabolic process. The top 10 MF terms included Oxidoreductase activity acting on aldehyde or oxo donors with NAD or NADP as acceptor, Oxidoreductase activity acting on aldehyde or oxo donors, Aldehyde dehydrogenase (NAD+) activity, Aldehyde dehydrogenase [NAD(P)+] activity, Structural constituent of ribosome, Calcium/calmodulin-dependent protein kinase activity, Calmodulin binding, Oxidoreductase activity acting on paired donors with incorporation or reduction of molecular oxygen (O2 to H2O), Sugar-phosphatase activity, and 5′-3′ exonuclease activity. The top 10 CC terms included Ribosome, Large ribosomal subunit, Mitochondrial inner membrane, Ribonucleoprotein complex, Ribosomal subunit, Organelle inner membrane, Mitochondrial membrane, Mitochondrial envelope, Organellar ribosome, and Mitochondrial ribosome. KEGG analysis showed that these DEGs were mainly enriched in 9 pathways (Fig. 5B). These pathways included Biosynthesis of unsaturated fatty acids, Fatty acid metabolism, Nicotinate and nicotinamide metabolism, Ribosome, Sulfur relay system, Nucleotide excision repair, Fatty acid elongation, PPAR signaling pathway, and Primary bile acid biosynthesis.
Fig. 5.
Transcriptome analysis of DEGs in pectoral muscle after screening. (A) GO enrichment analysis. (B) KEGG enrichment analysis.
Using a similar screening method, we obtained 255 DEGs in wattle. Among these, 85 DEGs were highly expressed in hen wattle, and 170 DEGs were highly expressed in rooster wattle (see Table 8 for the top 10 DEGs with the highest expression fold change in each group). The chromosomal distribution of these DEGs is shown in Supplementary Fig. 10B. Among them, 137 DEGs (53.73%) were located on the Z chromosome, and 26 DEGs (10.20%) were located on the W chromosome. The list of genes located on the sex chromosomes is provided in Supplementary Table 4.
Table 8.
Top 10 DEGs highly expressed in hen and rooster wattle after screening.
| Ensembl ID | Gene Symbol | log2FoldChange | Chromosome | Description |
|---|---|---|---|---|
| Hen | ||||
| ENSGALG00000040086 | HNRNPKL | 13.26491 | W | heterogeneous nuclear ribonucleoprotein K-like |
| ENSGALG00000033705 | - | 12.48907 | W | transitional endoplasmic reticulum ATPase-like |
| ENSGALG00000040704 | SPIN1W | 12.45166 | W | spindlin 1-W |
| ENSGALG00000030382 | - | 11.56114 | W | Golgi phosphoprotein 3-like |
| ENSGALG00000048542 | - | 11.24611 | W | ubiquitin-conjugating enzyme E2 R2-like |
| ENSGALG00000041221 | - | 11.13468 | W | - |
| ENSGALG00000039023 | - | 11.09506 | W | Nipped-B homolog-like |
| ENSGALG00000035780 | - | 10.84624 | W | E3 ubiquitin-protein ligase KCMF1-like |
| ENSGALG00000031327 | - | 10.59848 | W | chromodomain helicase DNA binding protein 1 |
| ENSGALG00000048307 | - | 10.32157 | W | ras GTPase-activating protein 1-like |
| Rooster | ||||
| ENSGALG00000054033 | - | -2.69037 | 5 | - |
| ENSGALG00000006591 | TNNI2 | -2.18377 | 5 | troponin I2, fast skeletal type |
| ENSGALG00000045092 | CCDC112 | -1.74073 | Z | coiled-coil domain containing 112 |
| ENSGALG00000027345 | SLC46A2 | -1.67630 | Z | solute carrier family 46 member 2 |
| ENSGALG00000005423 | GRHPR | -1.49644 | Z | glyoxylate and hydroxypyruvate reductase |
| ENSGALG00000015382 | CHRNA6 | -1.39085 | Z | cholinergic receptor nicotinic alpha 6 subunit |
| ENSGALG00000015628 | MSANTD1 | -1.29980 | 4 | Myb/SANT DNA binding domain containing 1 |
| ENSGALG00000028998 | XPA | -1.29280 | Z | XPA, DNA damage recognition and repair factor |
| ENSGALG00000050155 | CLCA2 | -1.27319 | 8 | chloride channel accessory 2 |
| ENSGALG00000048320 | HINT1 | -1.25915 | Z | histidine triad nucleotide binding protein 1 |
GO analysis showed that these DEGs were mainly enriched in 63 BP terms, 9 MF terms, and 18 CC terms (Fig. 6A; see Supplementary Table 5 for the complete list). The top 10 BP terms included rRNA metabolic process, Response to radiation, rRNA processing, DNA repair, DNA damage response, Ribosome biogenesis, Response to light stimulus, Negative regulation of transport, DNA metabolic process, and Cellular response to stress. The 9 MF terms included Oxidoreductase activity acting on metal ions, RNA polymerase II CTD heptapeptide repeat modifying activity, Peptidyl-prolyl cis-trans isomerase activity, Cis-trans isomerase activity, Sulfuric ester hydrolase activity, Ligand-gated monoatomic anion channel activity, Structural constituent of ribosome, SMAD binding, and Molecular function inhibitor activity. The top 10 CC terms included Mitochondrial matrix, Organellar large ribosomal subunit, Mitochondrial large ribosomal subunit, Guanyl-nucleotide exchange factor complex, Ribosome, Striated muscle thin filament, Myofilament, Bounding membrane of organelle, Large ribosomal subunit, and Ribosomal subunit. KEGG analysis showed that these DEGs were mainly enriched in the p53 signaling pathway, Nucleotide excision repair, and Ribosome pathways (Fig. 6B).
Fig. 6.
Transcriptome analysis of DEGs in wattle after screening. (A) GO enrichment analysis. (B) KEGG enrichment analysis.
By comparing the DEGs screened from pectoral muscle and wattle (after removal of hormone‑affected genes using a lenient criterion), we found 89 DEGs shared by both tissues. Their chromosomal distribution is shown in Supplementary Fig. 10C. Among them, 70 DEGs (78.65%) were located on the Z chromosome, and 18 DEGs (20.22%) were located on the W chromosome. Thus, the total proportion of DEGs on sex chromosomes was 98.88%. The list of genes located on the sex chromosomes is provided in Supplementary Table 6.
These shared DEGs were mainly enriched in 55 BP terms, 14 MF terms, and 24 CC terms (Fig. 7A). The top 10 BP terms included rRNA metabolic process, Transcription by RNA polymerase I, rRNA processing, Protein peptidyl-prolyl isomerization, Macroautophagy, Ribosome biogenesis, Peptidyl-proline modification, Response to radiation, Cellular response to stress, and Autophagy. The top 10 MF terms included Peptidyl-prolyl cis-trans isomerase activity, Cis-trans isomerase activity, Structural constituent of ribosome, Tropomyosin binding, Sulfurtransferase activity, Nucleoside monophosphate kinase activity, RNA polymerase II CTD heptapeptide repeat modifying activity, Proton channel activity, I-SMAD binding, and C-acyltransferase activity. The top 10 CC terms included Large ribosomal subunit, Organellar large ribosomal subunit, Mitochondrial large ribosomal subunit, Mitochondrial protein-containing complex, Ribonucleoprotein complex, Ribosomal subunit, Organellar ribosome, Mitochondrial ribosome, Ribosome, and RNA polymerase II transcription regulator complex. KEGG analysis showed that these shared DEGs were mainly enriched in three pathways (Fig. 7B): Ribosome, Nucleotide excision repair, and Sulfur relay system. Since CASIDGs should be located on sex chromosomes, only those DEGs located on the Z or W chromosomes obtained from the above screening were considered candidates for CASIDGs.
Fig. 7.
Transcriptome analysis of common DEGs shared by pectoral muscle and wattle after screening. (A) GO enrichment analysis. (B) KEGG enrichment analysis.
Validation of candidate CASIDGs
From the 88 shared candidate CASIDGs, we prioritized four genes (HINT1, MED18, CDK7, and SMAD2Z) for Western blot validation. The selection criteria were as follows: (1) these genes are located on the Z chromosome and showed consistent differential expression between males and females in both pectoral muscle and wattle; and (2) they exhibited robust and reproducible expression signals in our transcriptome data, making them suitable for further validation.
Mouse liver protein samples were used as positive controls. We tested the feasibility of antibodies against the proteins encoded by these genes in chicken pectoral muscle and wattle protein samples by Western blotting (WB). As predicted, chicken HINT1 showed a band near 15 kDa; chicken MED18 showed a band near 25 kDa; chicken CDK7 showed a band near the predicted size of 40 kDa; chicken SMAD2Z showed a band between 55 and 70 kDa (Supplementary Fig. 11).
Using these antibodies, we measured the protein expression levels of CDK7, SMAD2Z, HINT1, and MED18 by WB in the wattle and pectoral muscle tissues of control hens, androgen-treated hens, control roosters, and estrogen-treated roosters. The results showed that the protein abundance of CDK7 in wattle tissue was not significantly different among the groups. In pectoral muscle, CDK7 protein abundance was extremely low and could not be detected by WB (n = 6, Fig. 8, Fig. 9). The protein abundance of SMAD2Z in both wattle and pectoral muscle showed no significant differences among the groups either (n = 6, Fig. 8, Fig. 9). For HINT1 and MED18, their protein abundances in both wattle and pectoral muscle showed significant differences or a trend of difference between roosters and hens, and sex hormone injection did not significantly change their protein abundances (n = 6, Fig. 8, Fig. 9). Similar results were then obtained for HINT1 and MED18 protein abundances in gonadal tissues (n = 6, Supplementary Fig. 12): both genes showed significant expression differences between roosters and hens and were unaffected by sex hormone treatment. Based on these Western blot results showing that HINT1 and MED18, but not CDK7 or SMAD2Z, exhibited significant sex-differential protein abundance that was unaffected by hormone treatment, we selected HINT1 and MED18 for subsequent overexpression experiments.
Fig. 8.
Protein expression levels of CDK7, SMAD2Z, HINT1, and MED18 in wattle of 6‑week‑old chickens from different treatment groups. (A) protein expression levels of CDK7 and SMAD2Z. (B) protein expression levels of HINT1 and MED18. (C) quantitative analysis of relative protein expression of CDK7 and SMAD2Z. (D) quantitative analysis of relative protein expression of HINT1 and MED18. F, hen; M, rooster; C, control group; T, sex hormone‑treated group. Data are presented as mean ± SD. Different letters indicate statistically significant differences between groups (P < 0.05), while the same letter indicates no statistically significant difference (P > 0.05).
Fig. 9.
Protein expression levels of CDK7, SMAD2Z, HINT1, and MED18 in pectoral muscle of 6‑week‑old chickens from different treatment groups. (A) protein expression levels of CDK7 and SMAD2Z. (B) protein expression levels of HINT1 and MED18. (C) quantitative analysis of relative protein expression of SMAD2Z. (D) quantitative analysis of relative protein expression of HINT1 and MED18. F, hen; M, rooster; C, control group; T, sex hormone‑treated group. Data are presented as mean ± SD. Different letters indicate statistically significant differences between groups (P < 0.05), while the same letter indicates no statistically significant difference (P > 0.05).
In addition, we measured the protein abundances of HINT1 and MED18 in the pectoral muscle of chicken embryos before gonadal differentiation (embryonic day 6, E6) and after gonadal differentiation (embryonic day 12, E12). The results showed that, regardless of whether before or after gonadal differentiation, the protein abundances of both HINT1 and MED18 in pectoral muscle were significantly different between rooster and hen embryos, with roosters having higher levels than hens (n = 6, Supplementary Fig. 13 and Supplementary Fig. 14). This suggests that sex differences in HINT1 and MED18 protein abundances exist before gonadal sex differentiation.
Subcellular distribution of HINT1 and MED18 proteins
After nucleocytoplasmic separation of gonadal and pectoral muscle tissues from E12 chicken embryos, we quantified the protein abundances of HINT1 and MED18 in whole cell fractions, nuclear fractions, and cytoplasmic fractions by WB. The results showed that HINT1 protein was mainly distributed in the cytoplasm, while MED18 protein was expressed in both the nucleus and the cytoplasm (Supplementary Fig. 15).
Effects of HINT1 and MED18 overexpression on chicken primary muscle cells
HINT1 was overexpressed in primary muscle cells derived from E12 hen embryos. WB analysis validated the effective overexpression of HINT1 in these cells (Supplementary Fig. 16A). Thereafter, transcriptome sequencing was conducted on control cells (empty vector group) and cells overexpressing HINT1. The results showed that HINT1 overexpression caused significant expression changes in 1262 genes. Among these, 495 genes were upregulated and 767 genes were downregulated relative to the control group (Supplementary Fig. 17A).
GO analysis showed that these DEGs were mainly enriched in 178 BP terms, 37 MF terms, and 10 CC terms (Supplementary Fig. 17B). The top 10 BP terms included Cell adhesion, Locomotion, Anatomical structure morphogenesis, Cell migration, Axon guidance, Neuron projection guidance, Response to external stimulus, Cell motility, Cell morphogenesis, and Regulation of cell migration. The top 10 MF terms included Extracellular matrix structural constituent, Signaling receptor regulator activity, Receptor ligand activity, Signaling receptor activator activity, Cell adhesion molecule binding, Cytokine activity, Chemokine activity, Integrin binding, Glycosaminoglycan binding, and Extracellular matrix binding. The top 10 CC terms included Extracellular matrix, External encapsulating structure, Collagen-containing extracellular matrix, Basement membrane, Axon, Receptor complex, Cell surface, Plasma membrane protein complex, Neuron projection, and Plasma membrane signaling receptor complex. KEGG analysis showed that the DEGs induced by HINT1 overexpression were mainly enriched in the ECM-receptor interaction, Cytoskeleton in muscle cells, Focal adhesion, Cytokine-cytokine receptor interaction, and IgSF CAM signaling pathways (Fig. 10A).
Fig. 10.
KEGG enrichment analysis of HINT1 overexpression in chicken embryonic primary muscle cells. (A) hen primary muscle cells. (B) rooster primary muscle cells.
After successful overexpression of HINT1 in primary muscle cells from E12 rooster embryos (Supplementary Fig. 16B), transcriptome sequencing analysis showed that HINT1 overexpression caused significant expression changes in 995 genes. Among these, 442 genes were upregulated and 553 genes were downregulated relative to the control group (Supplementary Fig. 18A).
GO analysis showed that these DEGs were mainly enriched in 235 BP terms, 22 MF terms, and 11 CC terms (Supplementary Fig. 18B). The top 10 BP terms included Response to external stimulus, Immune system process, Immune response, Cell adhesion, Biological process involved in interspecies interaction between organisms, Defense response, Response to external biotic stimulus, Response to other organism, Response to biotic stimulus, and Defense response to other organism. The top 10 MF terms included Extracellular matrix structural constituent, Signaling receptor regulator activity, Receptor ligand activity, Signaling receptor activator activity, Cytokine activity, Integrin binding, Cell adhesion molecule binding, Chemokine activity, Cytokine receptor binding, and Cytokine binding. The top 10 CC terms included Extracellular matrix, External encapsulating structure, Collagen-containing extracellular matrix, Basement membrane, Receptor complex, Cell surface, Plasma membrane protein complex, Plasma membrane signaling receptor complex, Integrin complex, and Protein complex involved in cell adhesion. KEGG analysis showed that the DEGs induced by HINT1 overexpression were mainly enriched in the ECM-receptor interaction, Cytokine-cytokine receptor interaction, Cytoskeleton in muscle cells, Focal adhesion, Toll-like receptor signaling pathway, Cell adhesion molecule (CAM) interaction, ABC transporters, and MAPK signaling pathway pathways (Fig. 10B).
By comparing the DEGs induced by HINT1 overexpression in primary muscle cells from roosters and hens, we found 585 common DEGs (224 upregulated and 361 downregulated relative to the control group). Among these, 584 showed the same expression trend in both sexes. GO analysis showed that these common DEGs were enriched in 138 BP terms, 22 MF terms, and 6 CC terms. KEGG analysis showed that these common DEGs were mainly enriched in the Cytoskeleton in muscle cells, ECM-receptor interaction, Integrin signaling, Cytokine-cytokine receptor interaction, and Focal adhesion pathways.
In primary muscle cells from E12 hen embryos, MED18 was overexpressed. WB analysis confirmed successful overexpression of MED18 in the cells (Supplementary Fig. 16A). Subsequently, transcriptome sequencing analysis was performed on control cells (empty vector group) and MED18-overexpressing cells. The results showed that MED18 overexpression caused significant expression changes in 490 genes. Among these, 367 genes were upregulated and 123 genes were downregulated relative to the control group (Supplementary Fig. 19A).
GO analysis showed that these DEGs were mainly enriched in 102 BP terms, 11 MF terms, and 1 CC term (Supplementary Fig. 19B). The top 10 BP terms included Leukocyte migration, Cell migration, Mononuclear cell migration, Locomotion, Lymphocyte migration, Cell motility, Response to external stimulus, Leukocyte chemotaxis, Positive regulation of ERK1 and ERK2 cascade, and Chemotaxis. The top 10 MF terms included Cytokine activity, Receptor ligand activity, Signaling receptor regulator activity, Signaling receptor activator activity, Cytokine receptor binding, Growth factor activity, Chemokine activity, CCR chemokine receptor binding, Phosphatidylserine binding, and Heme binding. The single CC term was Cell surface. KEGG analysis showed that the DEGs induced by MED18 overexpression were mainly enriched in the Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, Cytosolic DNA-sensing pathway, Neuroactive ligand-receptor interaction, and Neuroactive ligand signaling pathways (Fig. 11A).
Fig. 11.
KEGG enrichment analysis of MED18 overexpression in chicken embryonic primary muscle cells. (A) hen primary muscle cells. (B) rooster primary muscle cells.
After successful overexpression of MED18 in primary muscle cells from E12 rooster embryos (Supplementary Fig. 16B), transcriptome sequencing analysis showed that MED18 overexpression caused significant expression changes in 676 genes. Among these, 332 genes were upregulated and 344 genes were downregulated relative to the control group (Supplementary Fig. 20A).
GO analysis showed that these DEGs were mainly enriched in 163 BP terms, 16 MF terms, and 4 CC terms (Supplementary Fig. 20B). The top 10 BP terms included Response to external stimulus, Immune response, Immune system process, Cell adhesion, Defense response, Inflammatory response, Regulation of multicellular organismal process, Biological process involved in interspecies interaction between organisms, Locomotion, and Response to external biotic stimulus. The top 10 MF terms included Signaling receptor regulator activity, Receptor ligand activity, Signaling receptor activator activity, Cytokine activity, Extracellular matrix structural constituent, Cytokine receptor binding, Cytokine binding, Chemokine activity, Cell adhesion molecule binding, and Chemokine receptor binding. The CC terms included Extracellular matrix, External encapsulating structure, Receptor complex, and Collagen-containing extracellular matrix. KEGG analysis showed that the DEGs induced by MED18 overexpression were mainly enriched in the Cytokine-cytokine receptor interaction, Cytoskeleton in muscle cells, ECM-receptor interaction, Toll-like receptor signaling pathway, Focal adhesion, MAPK signaling pathway, Cell adhesion molecule (CAM) interaction, Arginine and proline metabolism, and Calcium signaling pathway pathways (Fig. 11B).
By comparing the DEGs induced by MED18 overexpression in primary muscle cells from roosters and hens, we found 110 common DEGs (83 genes upregulated and 27 genes downregulated relative to the control group). Among these, 91 DEGs showed the same expression trend in both sexes. GO analysis showed that these DEGs were enriched in 105 BP terms, 13 MF terms, and 0 CC terms. KEGG analysis showed that these common DEGs were mainly enriched in the Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway, Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, and Herpes simplex virus 1 infection pathways.
Screening of sex marker genes in somatic tissues
Since there is currently a lack of sex marker genes for somatic tissues, this study screened for sex marker genes in somatic tissues. In this study, sex marker genes were defined as autosomal genes that show significant expression differences between roosters and hens and are not affected by sex hormones. These sex marker genes serve as downstream sex-biased biomarkers rather than sex-determining factors per se, as their sex-biased expression is driven by upstream sex-linked CASIDGs. Using transcriptome sequencing data from the pectoral muscles of untreated control roosters and hens, we identified inter-sex DEGs. From these, we removed DEGs affected by estrogen or androgen treatment in the pectoral muscle. This yielded 139 DEGs located on autosomes (see Supplementary Table 7 for the complete gene list). Their distribution is shown in Supplementary Fig. 21A. They were mainly concentrated on autosome 1 (18 DEGs, 12.95%), autosome 2 (11 DEGs, 7.91%), and autosome 4 (10 DEGs, 7.19%). These DEGs were considered sex marker genes for pectoral muscle tissue. Eight genes were randomly selected from these genes for qPCR validation. The results showed that the expression changes of all eight genes were consistent with the transcriptome sequencing data (Supplementary Fig. 21B), indicating that the screening results for sex marker genes were reliable.
Similarly, for wattle tissue, we removed DEGs affected by estrogen or androgen treatment in wattle from the inter-sex DEGs. This yielded 92 DEGs located on autosomes (see Supplementary Table 8 for the complete gene list). Their distribution is shown in Supplementary Fig. 22A. They were mainly concentrated on autosome 1 (16 DEGs, 17.39%), autosome 4 (12 DEGs, 13.04%), and autosome 2 (11 DEGs, 11.96%). These DEGs were considered sex marker genes for wattle tissue. Similarly, eight genes were randomly selected from these genes for qPCR validation. The results showed that among the eight genes, seven genes (TREMB2, STEAP3, CFH, CST3, ADA, PDZK1IP1, and S100A10) showed expression changes consistent with the transcriptome sequencing data (Supplementary Fig. 22B).
By comparing the sex marker genes screened from pectoral muscle and wattle tissues, we found that the two tissues shared almost no common sex marker genes. This indicates that sex marker genes are tissue-specific.
Effects of HINT1 and MED18 overexpression on the expression of sex marker genes in pectoral muscle tissue
By comparing the sex marker genes of pectoral muscle with the DEGs induced by HINT1 overexpression, we identified five sex marker genes affected by HINT1 overexpression. qPCR validation showed that among these, the expression changes of APOA1, IRF9, and CCL4 were consistent with the transcriptome sequencing data in both roosters and hens (Fig. 12A-B). Similarly, we identified two sex marker genes affected by MED18 overexpression, namely IRF9 and CCL4. qPCR validation confirmed that the expression changes of these two genes were consistent with the transcriptome sequencing data in both roosters and hens (Fig. 12C-D). Combining the results of HINT1 and MED18 overexpression, IRF9 and CCL4 are sex marker genes in pectoral muscle tissue that are regulated by both HINT1 and MED18.
Fig. 12.
Effects of HINT1 or MED18 overexpression on candidate sex marker genes. (A) HINT1 overexpression in hen primary muscle cells. (B) HINT1 overexpression in rooster primary muscle cells. (C) MED18 overexpression in hen primary muscle cells. (D) MED18 overexpression in rooster primary muscle cells.
Discussion
Although previous studies have observed the phenomenon of cell autonomous sex identity, the underlying mechanisms remain unclear. Identifying CASIDGs will help elucidate these mechanisms. Current evidence suggests that CASIDGs are located on sex chromosomes and exhibit dosage effects (Clinton et al., 2012). Previous studies have performed transcriptome sequencing analysis using chicken embryos before gonadal sex differentiation and identified some DEGs located on sex chromosomes. These DEGs can be considered candidates for CASIDGs (Melamed and Arnold, 2007; Zhang et al., 2010; Ayers et al., 2013). Given that tissues are less differentiated at early developmental stage and that tissue differentiation is substantially influenced by epigenetic mechanisms, the present study identified CASIDGs after gonadal sex differentiation using 6-week-old sex hormone-treated and untreated roosters and hens.
The screening strategy was as follows: from the inter-sex DEGs, we removed DEGs affected by sex hormones (using a lenient criterion without P-value threshold to prioritize sensitivity, ensuring that the remaining CASIDGs have high specificity). The remaining DEGs located on sex chromosomes were considered candidates for CASIDGs.
In this study, wattle and pectoral muscle were selected for screening CASIDGs. Our previous results showed that the sensitivity of different tissues to sex hormones is related to the protein abundance of the estrogen receptor ERα(Jin et al., 2026). The protein abundance of ERα in wattle is high, making it sensitive to sex hormones. In contrast, ERαabundance in pectoral muscle is low, making it insensitive. Indeed, androgen injection caused hen wattles and combs to become larger and redder, while estrogen injection caused rooster wattles and combs to become smaller and yellower. This indicates that the sex hormone injection experiments were successful, and that wattle and comb can represent phenotypically hormone-sensitive somatic tissues. In contrast, for both roosters and hens, injection of testosterone or β‑estradiol did not cause significant changes in pectoral muscle weight. This indicates that pectoral muscle can represent a phenotypically hormone-unresponsive somatic tissue. Therefore, using these two tissues allowed us to screen for tissue-specific CASIDGs.
In 6-week-old control roosters and hens, the inter-sex DEGs in somatic tissues reflect the influence of CASIDGs as well as the regulatory impact of sex hormones. We identified a total of 854 DEGs in the pectoral muscle. These genes are mainly located on the Z chromosome (18.50%), autosome 1 (13.00%), and autosome 2 (7.26%). In wattle, we identified a total of 1156 DEGs. These genes are mainly located on the Z chromosome (16.78%), autosome 1 (12.80%), and autosome 2 (7.53%). Upon comparison of DEGs between the pectoral muscle and wattle, we identified 199 shared DEGs. These genes are mainly located on the Z chromosome (46.23%), the W chromosome (15.58%), and autosome 1 (8.54%). Consequently, whether in specific tissues or in the collective DEGs, the DEGs were predominantly localized on the sex chromosomes. The differential expression of these DEGs on sex chromosomes primarily pertains to gene dosage effects; however, some may also result from sex hormone influences.
In contrast, the expression differences of DEGs on autosomes between sexes can be attributed to regulation by sex‑linked DEGs and to sex hormone effects. Although the sex‑linked DEGs had the highest numbers among the inter‑sex DEGs, the majority of DEGs were still distributed on autosomes. DEGs on autosomes 1 and 2 accounted for the highest proportions, which is related to their lengths and gene numbers (Elferink et al., 2010). It is worth noting that the number of DEGs in wattle was 35.36% higher than that in pectoral muscle. This is likely because wattle is more sensitive to sex hormones than pectoral muscle. Furthermore, pathway enrichment analysis showed that the DEGs in pectoral muscle were enriched in pathways involved in protein synthesis, response to hormone stimulus, activation of signaling pathways, regulation of development and growth, muscle structure and contraction, lipid metabolism and transport, cell adhesion, and folate synthesis. These pathways may participate in the formation of sex characteristics in pectoral muscle, such as roosters having heavier pectoral muscle than hens but lower fat content. On the other hand, the DEGs in wattle were enriched in pathways involved in anatomical structure morphogenesis, response to growth factor and hormone signals, regulation of cell proliferation and differentiation, cell migration, extracellular matrix composition and cell adhesion, and folate synthesis. These pathways may participate in the formation of sex characteristics in wattle, such as roosters having larger wattles than hens, and differences in wattle morphology and texture between the sexes. Because there were few common DEGs between the pectoral muscle and wattle, the number of pathways obtained from enrichment analysis was also small. The common pathways involved protein synthesis, folate biosynthesis, and cell morphology regulation. The small number of common DEGs between pectoral muscle and wattle is likely related to epigenetic regulation of somatic tissue differentiation (Yu et al., 2008; Wang et al., 2025). This also suggests that different tissues may share some common CASIDGs while also having distinct ones.
To remove DEGs affected by sex hormones and obtain candidate CASIDGs, we treated young chicks with different sex hormones. In 6‑week‑old roosters, we compared estrogen-treated and untreated control pectoral muscles and screened 1090 DEGs. They were mainly concentrated on autosome 1 (12.94%), autosome 2 (10.37%), and autosome 3 (7.52%). In 6‑wk‑old hens, we compared androgen-treated and untreated control pectoral muscles and screened 1008 DEGs. They were mainly concentrated on autosome 1 (14.88%), autosome 2 (10.12%), and autosome 3 (9.42%). In comparison to the number of DEGs seen between normal roosters and hens in pectoral muscle, both sex hormones stimulated a greater number of DEGs, as the hormonal influence was more significant in the treated groups.
By comparing DEGs induced by estrogen treatment with those induced by androgen treatment in pectoral muscle, we found 181 common DEGs. They were mainly concentrated on autosome 1 (18.78%), autosome 2 (13.81%), and autosome 3 (10.50%). Notably, these DEGs were not concentrated on sex chromosomes. This is because the expression differences of CASIDGs on sex chromosomes and the differences they induce in other sex‑linked genes were smaller than the expression differences induced by sex hormone treatment. Similarly, in 6‑week‑old roosters, we compared estrogen-treated and untreated control wattles and screened 1707 DEGs. They were mainly concentrated on autosome 1 (13.83%), autosome 2 (9.61%), and autosome 3 (7.56%). In 6‑week‑old hens, we compared androgen-treated and untreated control wattles and screened 1955 DEGs. They were mainly concentrated on autosome 1 (13.40%), autosome 2 (9.62%), and autosome 3 (8.59%). Again, these DEGs were not concentrated on sex chromosomes, and their numbers were clearly higher than the number of DEGs between normal roosters and hens in wattle. By comparing DEGs induced by estrogen treatment with those induced by androgen treatment in wattle, we found 543 common DEGs. They were mainly concentrated on autosome 1 (14.73%), autosome 2 (9.76%), and autosome 3 (8.29%). Compared with the pectoral muscle, the number of DEGs screened in wattle after hormone treatment was higher in roosters, in hens, and among the common DEGs. This is consistent with wattle having higher sensitivity to sex hormones than pectoral muscle. Previous studies have shown that tissue specificity is closely related to epigenetic regulation (Yu et al., 2008; Wang et al., 2025). These DEGs, especially those common to both tissues, reflect the molecular mechanisms of sex hormone action to some extent. Pathway enrichment analysis showed that the effects of estrogen in rooster pectoral muscle were mainly reflected in pathways related to blood vessel formation, cell adhesion, anatomical structure formation, muscle cytoskeleton, actin, muscle contraction, and immunity. The effects of androgen in hen pectoral muscle were mainly reflected in pathways related to cell adhesion, protein synthesis, muscle cytoskeleton, anatomical structure morphogenesis, and response to external stimuli. The effects of estrogen and androgen in muscle exhibits some common features (such as effects on cell adhesion, muscle cytoskeleton, and anatomical structure formation) but also had some distinctions, such as estrogen in roosters being related to muscle movement, whereas androgen in hens was related to protein synthesis. For wattle, the effects of estrogen in roosters were mainly reflected in pathways related to cell adhesion, cell movement and migration, anatomical structure morphogenesis, muscle cytoskeleton, actin, neuroactive ligand signaling, calcium signaling, and response to external stimuli. The effects of androgen in hens were mainly reflected in pathways related to blood vessel formation, cell adhesion, muscle cytoskeleton, anatomical structure formation, fatty acid metabolism, calcium signaling, and response to external stimuli. The effects of estrogen and androgen in wattle also shared some common features (such as cell adhesion, muscle cytoskeleton, anatomical structure morphogenesis, calcium signaling, and fat metabolism) and differences (such as neuroactive ligand signaling for estrogen and blood vessel formation for androgen). These sex hormone effects may lead to phenotypic changes in chicken pectoral muscle and wattle tissues. In addition, by comparison, only 8 DEGs were commonly responsive to sex hormone treatment in both pectoral muscle and wattle. They were SLK, PTGR2, FRMD3, MX1, CD320, CCN3, SECISBP2L, and ASPH. Except for FRMD3, which is located on the Z chromosome, the other 7 genes are located on autosomes. Although no previous studies have directly linked these genes to sex hormone responses, one study showed that CCN3 may participate in the formation of certain female‑specific phenotypes in mammals. It was identified as a brain‑derived maternal hormone highly expressed in lactating women and is essential for maintaining bone health during lactation (Babey et al., 2024).
During the screening process for candidate CASIDGs, we removed DEGs regulated by sex hormones from the inter‑sex DEGs present between normal roosters and hens. After this removal, we obtained 285 DEGs in pectoral muscle. Among these, 43.16% were located on the Z chromosome, and 8.07% on the W chromosome. In wattle, we obtained 255 DEGs. Among these, 53.73% were located on the Z chromosome, and 10.20% on the W chromosome. By comparing the DEGs obtained from pectoral muscle and wattle, we found 89 DEGs common to both tissues. Among these common DEGs, 78.65% were located on the Z chromosome, and 20.22% on the W chromosome. Thus, after removing hormone‑affected DEGs, the vast majority of DEGs (98.88%) were located on the sex chromosomes. This is consistent with the hypothesis that CASIDGs reside on the sex chromosomes. Pathway enrichment analysis showed that the candidate CASIDGs were mainly enriched in pathways related to protein synthesis, especially mitochondrial protein synthesis, and isomerase activity. This suggests that candidate CASIDGs may influence the sex characteristics of chicken somatic cells through mechanisms such as regulating mitochondrial protein synthesis.
Previous studies performed transcriptome sequencing analysis on blastoderms of chicken embryos at HH4 (Zhang et al., 2010) and on gonads of 4.5-day-old chicken embryos of different sexes (Ayers et al., 2013). Based on their results, they estimated that the number of CASIDGs could reach 200 to 300. By comparison, among the candidate CASIDGs identified in this study, 40 DEGs were the same as those found in the HH4 blastoderm analysis. All 40 of these DEGs were located on the Z chromosome. In comparison to the sequencing results from 4.5-day embryonic gonads, 30 DEGs were identical, all of which were situated on the Z chromosome.
However, it is worth noting that there are several differences between this study and previous studies. First, the samples used were different. This study used pectoral muscle and wattle tissues, which are already well differentiated. Previous studies used whole blastoderms (containing all tissues that were not yet fully differentiated) or gonads that had not yet undergone sex differentiation. Second, in this study, the gonads had already differentiated and were capable of producing different sex hormones at the time of sampling. In previous studies, the gonads had not yet differentiated and did not produce different sex hormones. Third, the sequencing technology used in this study was considerably more advanced than that used in previous studies, providing higher sequencing accuracy. Fourth, the reference genome used in this study has been updated many times compared with that used in previous studies, and the gene annotation is more complete. These differences may explain why only some of the candidate CASIDGs overlap between this study and previous studies. For example, DMRT1, a sex determining gene for male gonads identified previously, was not present in the list of candidate CASIDGs identified in this study. This may reflect that different tissues in chickens have different CASIDGs.
In this study, we validated four candidate CASIDGs: HINT1, MED18, CDK7, and SMAD2Z. The results showed that HINT1 and MED18 met the selection criteria for CASIDGs at the protein level (protein abundance showed significant differences between roosters and hens and was not affected by sex hormone treatment). Moreover, sex differences in their protein abundances existed before gonadal sex differentiation. The HINT1 gene located on the chicken Z chromosome (also known as chPKCI, HINTZ) belongs to the histidine triad superfamily. It contains a His-X-His-X-His-X motif (where X is a hydrophobic amino acid). HINT1 plays important roles in various neuropsychiatric diseases (Zimoń et al., 2012; Liu et al., 2017) and is involved in biological processes such as apoptosis regulation, gene transcription, and cell cycle control (Razin et al., 1999; Weiske and Huber, 2006; Cen et al., 2009). In endothelial cells, HINT1 maintains mitochondrial homeostasis and energy metabolism(Gao et al., 2023). Although research on HINT1 in chickens is relatively limited, its family member HINTW located on the W chromosome was previously studied in depth as a candidate gene for ovary determination (Smith et al., 2009; C. Sun et al., 2023; C. Sun et al., 2025). HINT1 is mainly distributed in the cytoplasm, suggesting that it may function through interactions with cytoplasmic proteins. Pathway enrichment analysis after HINT1 overexpression in pectoral muscle cells showed that the DEGs were mainly enriched in pathways related to immune/inflammatory responses, cell proliferation and development regulation, key growth and survival signaling pathways (such as Wnt and MAPK/ERK), extracellular matrix-cytoskeleton signaling, and intercellular communication. This suggests that HINT1 may influence sex characteristics of somatic tissues through these pathways.
The MED18 gene is located on the chicken Z chromosome. The protein it encodes is an important component of the mediator complex during transcription and plays a key regulatory role in transcription initiation and elongation (Kumafuji et al., 2014). Studies have shown that MED18 can negatively regulate transcription through the CDK/cyclin module (Kumafuji et al., 2014). However, no studies have been reported on the involvement of MED18 in chicken sex determination. Transcriptome sequencing analysis after MED18 overexpression in pectoral muscle cells showed that MED18 may influence sex characteristics of somatic tissues by participating in biological processes such as immune/inflammatory responses, cell chemotaxis and migration, cell proliferation, and regulation of development and growth.
It should be noted that in this study, both HINT1 and MED18 were overexpressed in primary muscle cells of rooster and hen embryos. This was mainly because these two genes are Z‑linked and may have a dosage‑dependent synergistic effect. Roosters have two Z chromosomes, while hens have only one. Furthermore, the dosage compensation system in chickens is imperfect, resulting in nearly all genes on the Z chromosome exhibiting differential expression levels between the sexes. Therefore, when HINT1 and MED18 function in roosters, they may rely on the synergistic effect of genes on the Z chromosome at twice the dose present in hens. Overexpressing them only in hens may not fully reveal this synergistic effect due to the single‑dose background. Overexpressing them in roosters as well can, on one hand, avoid the potential endogenous dosage limitation in hens, and on the other hand, help to more comprehensively analyze the functions of these two genes, making the conclusions more reliable.
In addition to the Z‑linked candidates discussed above, some W‑linked candidate CASIDGs were also identified, such as HINTW and SPIN1W (a spindlin family member implicated in chromatin remodeling and transcriptional regulation). Although these W‑linked genes are exclusively present in females (ZW) and absent in males (ZZ), their potential roles in somatic sex determination have not been experimentally explored. Based on their known or predicted molecular functions, we speculate that they may contribute to the establishment of female somatic sex characteristics through mechanisms such as dosage‑dependent modulation of chromatin structure, RNA processing, and protein ubiquitination. However, we acknowledge that these hypotheses require direct experimental validation in future studies, and the precise functions of W‑linked CASIDGs in somatic tissues remain to be elucidated.
Sex marker genes are important tools in sex research. Sex marker genes for chicken gonads are already well defined, such as DMRT1, SOX9, FOXL2, and CYP19A1(Luo et al., 2024). However, sex marker genes for gonads do not apply to other somatic tissues. This is because the gonads of roosters and hens have obvious morphological differences. The hen's gonad is mainly the ovary, which develops from the cortex, while the rooster's testis develops mainly from the medulla (Guioli et al., 2020; Mizia et al., 2023). Currently known sex marker genes function mainly due to their specific expression in the cortex or medulla. For other somatic tissues, there are no obvious anatomical differences between roosters and hens like those in gonadal tissues. Therefore, sex marker genes for gonads cannot be applied to other somatic tissues. In this study, we screened for sex marker genes in pectoral muscle and wattle according to our defined criteria. A sex marker gene was defined as a gene located on an autosome that shows significant expression differences between the sexes and is not affected by sex hormones, and these genes are downstream sex-biased biomarkers rather than sex-determining factors per se. The results showed 139 candidate sex marker genes for pectoral muscle and 92 for wattle. However, the two tissues shared almost no common sex marker genes, indicating that sex marker genes are tissue specific. Validation showed that 15 out of 16 randomly selected genes had expression patterns consistent with the transcriptome sequencing results. This confirms that the screening results are reliable, and the genes identified in this study can serve as sex marker genes. Consistently, the candidate CASIDGs HINT1 and MED18 both significantly affected the expression of sex marker genes in pectoral muscle. This included their common regulation of the sex marker genes IRF9 and CCL4. These findings further support the view that HINT1 and MED18 are CASIDGs and also support the application value of the identified sex marker genes.
Currently, there are no knockout or gene‑edited chickens available for HINT1, MED18, or the sex marker genes. Therefore, substantial validation of these genes remains to be completed in the future. In addition, the candidate CASIDGs and the somatic sex marker genes identified in this study require further characterization.
Conclusion
This study reveals that the sex characteristics of chicken somatic cells are determined by both the dosage effects of CASIDGs on sex chromosomes and the effects of sex hormones. Genes associated with the effects of sex hormones are predominantly situated on autosomes 1 to 3. Wattle exhibits higher sensitivity to sex hormones than pectoral muscle. The effects of sex hormones in pectoral muscle and wattle involve both common pathways (such as hormone response and cell adhesion) and distinct pathways. For example, the pectoral muscle focuses on muscle contraction and protein synthesis, whereas the wattle focuses on morphogenesis and cell migration.
By removing sex hormone‑affected genes, this study screened out 88 candidate CASIDGs mainly located on sex chromosomes. Among them, 40 overlapped with previous findings in early chicken blastoderms. Both tissues shared common CASIDGs, and each tissue also had its own tissue‑specific CASIDGs. The CASIDGs were mainly enriched in pathways related to mitochondrial protein synthesis and isomerase activity. HINT1 (cytoplasmic) and MED18 (nuclear) were validated as candidate CASIDGs that met the screening criteria. HINT1 may participate in sex determination by regulating cell proliferation, survival, and cytoskeletal signaling. MED18 may mediate immune responses, cell migration, and developmental regulation. This study also found sex marker genes specific to various tissues. These findings clarify the molecular mechanisms of sex hormone action and identify HINT1 and MED18 as significant candidate genes for CASI determination. This work establishes a robust basis for a deeper understanding of the mechanisms underlying cell autonomous sex identity in somatic cells.
Ethics approval
All animal experimental procedures in this study complied with relevant guidelines for animal welfare and use, and were approved by the Animal Welfare and Use Committee of Yangzhou University (SYXK(SU)2021-0020).
Disclosures
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funding statement
The work was funded by the ‘JBGS’ Project of Seed Industry Revitalization in Jiangsu Province [JBGS-2021(105), JBGS-2021(027)], the Enterprise-Academy Cooperation Project of the First Affiliated Hospital of Xi'an Jiaotong University [204022556], the Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
Data availability statement
The datasets supporting the conclusions of this article are available in the NCBI Sequence Read Archive (SRA) repository, BioProjects: PRJNA1458637 and PRJNA1458752 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1458637 and https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1458752).
Author Contribution
Zidi Jin designed the experiment for CASIDGs identification, performed the formal analysis of transcriptomic data, carried out Western blot and qPCR validation experiments, interpreted the data, and drafted the manuscript. Xiaolin Wu designed and conducted the experiment for identification of candidate sex marker genes, interpreted the data, took charge of data curation and organization, drafted the manuscript. Aneeqa Imtiaz designed and conducted the animal experiment, interpreted the data, and drafted the manuscript. Jing Ge provided critical suggestions on the design of the experiment for CASIDGs identification and the data interpretation, managed project administration, and critically reviewed the manuscript. Haizhou Gong provided critical suggestions on the design of the experiment for identification of candidate sex marker genes and the data interpretation, performed software-based processing of RNA-seq raw data and data visualization, and critically reviewed the manuscript. Minmeng Zhao provided critical suggestions on the design of the animal experiment and the data interpretation, and critically reviewed the manuscript. Daoqing Gong contributed to the conception of this study and fund acquisition, and critically reviewed the manuscript. Long Liu conceived this study, contributed to fund acquisition, and critically reviewed the manuscript. Tuoyu Geng conceived and supervised this study, contributed to fund acquisition, critically reviewed and finalized the manuscript. All authors read and approve the final version of the manuscript, are personally and publicly responsible for all aspects of the work, and ensure that any questions regarding the accuracy or integrity of the research are thoroughly investigated and resolved.
Acknowledgments
None.
Declaration of generative AI in scientific writing
During the preparation of this work, the authors used DeepSeek-R1 for the purpose of language polishing and grammar correction. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the final content of the publication.
Footnotes
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2026.107733.
Contributor Information
Long Liu, Email: liulong@yzu.edu.cn.
Tuoyu Geng, Email: tygeng@yzu.edu.cn.
Appendix. Supplementary materials
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets supporting the conclusions of this article are available in the NCBI Sequence Read Archive (SRA) repository, BioProjects: PRJNA1458637 and PRJNA1458752 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1458637 and https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1458752).