Journal of Animal Science and Biotechnology
Research

Unique Sertoli cell adaptations support enhanced spermatogenesis in chickens

1,2 ,1,3 ,1,3 ,1,3 ,1,3 ,1,3 ,1,3 ,1,3 ,1,3 ,1,3 ,1,3

Abstract

Background

The cellular basis of testicular development and spermatogenesis for the extreme sperm density in chickens (100-fold higher than mammals) remains poorly defined. A comprehensive understanding of the molecular characteristics driving poultry testicular development is crucial for explaining this enhanced spermatogenic capacity.

Results

Here, we first established a single-cell transcriptome profile of chicken testes from hatching to maturity, identifying the dynamic transcriptional characteristics of germ cell fate transition and exploring the developmental characteristics of Sertoli cells and Leydig cells. Multi-species comparisons revealed a higher proportion of germ cells and the unique adaptations of Sertoli cells in chicken testes. Most importantly, our results demonstrated that Sertoli cells dominated in somatic composition of mature chicken testes, and proliferating Sertoli cells persisted in chicken testes even after sexual maturity, while no proliferating Sertoli cells in mammals. We also found a richer interaction network between chicken testicular cells, especially the specific activation of Sertoli cell interaction signals, such as TGF-β, BMP, EGF, and activin. These adaptations of Sertoli cells may support the spermatogenic superiority in chickens. Additionally, our results indicated that cAMP responsive element binding protein 5 (CREB5) played a crucial role in maintaining the maturation and function of chicken Sertoli cells, and circadian rhythm promoted testosterone secretion and the development of Leydig cells.

Conclusion

Our study revealed that the sustained proliferative capacity of Sertoli cells, their enriched signaling network, and the regulatory roles of CREB5 and circadian rhythms collectively represented unique testicular adaptations in chickens. These findings may hold extraordinary significance in understanding the molecular characteristics of poultry testicular development, and provide a plausible framework for explaining enhanced spermatogenesis in poultry.

Introduction

The testis is a crucial reproductive organ in male animals, serving as the site for spermatogenesis, which generates sperm to transmit genetic information [1]. Spermatogenesis is a precise and dynamic developmental process that involves the differentiation of spermatogonial stem cells (SSCs), mitosis of spermatogonia, meiosis of spermatocytes, morphological changes in spermatids and the formation of sperm [2]. However, the higher sperm density were demonstrated in poultry (3 × 109 mL−1 in chicken) than that in mammals, such as humans (2 × 107 mL−1) and mice (1 × 107 mL−1) [3,4,5]. Additionally, chickens, as light sensitive animals, have highly complex and diverse circadian rhythm systems, and heavily rely on their circadian rhythms to regulate reproductive processes [6, 7]. Prolonging the photoperiod has become an important means to promote testicular spermatogenesis during chicken production [8, 9]. These characteristics indicate the existence of unique adaptations during chicken spermatogenesis. However, the molecular basis of testicular development and spermatogenic superiority in poultry remains unclear.

During spermatogenesis, Sertoli cells and Leydig cells, and their secretions, especially testosterone secreted by Leydig cells, are important components of the testicular microenvironment that support germ cell development and sperm formation [10]. Sertoli cells, as the only somatic cells that directly interact with developing germ cells, can form the blood-testis barrier (BTB) and have been described as key drivers of spermatogenesis [11]. The number of Sertoli cells directly determines the testicular size and sperm production [12]. Immature Sertoli cells have strong phagocytic ability to promptly engulf underdeveloped or defective germ cells, and mature Sertoli cells provide protection and nutrition to germ cells [13]. In mammals, Sertoli cells exist in an immature state before puberty and transform into mature Sertoli cells that no longer differentiate during puberty [14]. However, there is limited understanding of the developmental characteristics of germ cells, Sertoli cells, and Leydig cells in chicken testes.

Here, we investigated chicken testes at five stages: the hatching stage (chicken_1d), juvenile stage (chicken_3w), developmental stage (chicken_12w), rapid development stage (chicken_18w), and mature stage (chicken_24w) using single-cell transcriptomics (10 × Genomics) to explore the molecular characteristics of spermatogenesis and spermatogenic superiority in chicken testes. We identified and characterized the immature and mature subtypes of Sertoli cells in chicken testes. Most importantly, we verified the unique persistence of proliferating immature Sertoli cells in chicken testes after sexual maturity through immunofluorescence, cell cycle detection and EDU staining. We preliminarily elucidated the effects of cAMP responsive element binding protein 5 (CREB5) on maintaining the maturation and function of chicken Sertoli cells. We also identified precursor cells of Leydig cells and detected the effects of circadian rhythm on testosterone secretion and the development of Leydig cells through retinoic acid receptor-related orphan receptor alpha (RORα) and neuronal PAS domain protein 2 (NPAS2) interference. Our findings not only provide substantial data on the molecular features of testicular development and spermatogenesis in poultry, but also lay the foundation for the developmental mechanisms of Sertoli cells and Leydig cells.

Method details

Testis sample preparation

Arbor Acres male broiler chickens (Gallus gallus domesticus) were randomly selected from a breeding farm with a standardized manner in Jilin Province, China. Chickens were housed in dry, well-ventilated coops with an activity space of 0.2 square meters per bird. A daily photoperiod of 12 h (combining natural and artificial light) is provided to stimulate normal physiological rhythms. The rearing environment is kept clean, quiet, and free from excessive stress. The testicular tissue samples were collected from the bilateral testes under sterile conditions. Specifically, the samples were obtained from the mid-region of the testes. Testis samples for scRNA-seq and immunostaining were obtained from 13 male Arbor Acres broilers of five different ages, namely, 1 d (n = 2), 3 weeks (n = 2), 12 weeks (n = 3), 18 weeks (n = 3), and 24 weeks (n = 3). Fresh testis samples were taken from the broilers and placed in phosphate-buffered saline (PBS). The left testes were selected and divided into two parts for single-cell sequencing and immunofluorescence. The experiments were approved by the Experimental Animal Welfare and Ethics Committee of Jilin Agricultural University (Approval No. 20210422 002) and performed in accordance with the protocol outlined in the “Guide for Care and Use of Laboratory Animals” (Jilin Agricultural University).

Single-cell suspension preparation

Tissues were washed twice, decapsulated and minced into small pieces for adequate digestion. Suitable tissue pieces were then transferred to a 15-mL tube and digested through a standard two-step procedure. Briefly, tissues were sequentially digested with collagenase type IV (Cat# C5138-500MG, Sigma-Aldrich, St. Louis, MO, USA) and 0.25% trypsin/EDTA (Cat# 25300054, Invitrogen, Carlsbad, CA, USA). Subsequently, the digested cells were collected and subjected to a red blood cell (RBC) removal process using red blood cell (RBC) lysis buffer (CST, Cat# 46232). Single cells were obtained by filtering with 70 µm strainers (Thermo Fisher Scientific, Cat# 08-771-2) and collected into 1.5-mL tubes by centrifugation at 400 × g for 5 min. The cells were then detected by an automatic cell fluorescence analyzer (Count Star, Shanghai, China) and resuspended at approximately 400 cells/μL in 1 × PBS containing 0.4% BSA (Thermo Fisher Scientific, Cat# AM2616).

scRNA-seq library preparation and sequencing

scRNA-seq was performed by CapitalBio Technology (Beijing, China) using the 10 × Genomics system. Briefly, after the cells were diluted, the cell suspension was loaded onto a 10 × Chromium Controller to generate single-cell gel beads in the emulsion using a Chromium Single Cell G Chip Kit (1000120, 10 × Genomics, Pleasanton, CA, USA) and a Gel Bead Kit V3.1 (1000121, 10 × Genomics, Pleasanton, CA, USA). In each experiment, approximately 10,000 single cells were captured from each testis sample. Captured cells were lysed to release RNA, and cDNA was generated by reverse transcription and amplified using an S1000 Thermal Cycler (Bio-Rad, USA). The cDNA quality was subsequently assessed on an Agilent 4200 TapeStation. Subsequently, scRNA-seq libraries were prepared using the Single Cell 3′ Library and Gel Bead Kit V3.1 following the manufacturer’s instructions. scRNA-seq libraries were sequenced at 150 bp paired ends (PE150) using an Illumina NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA).

Quality control and analysis of scRNA-seq data

We used Gallus_gallus GRCg6a.101 as the reference genome and then quantified the cell barcodes with CellRanger (https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest). We imported the matrices into Seurat v4.0.4 (https://github.com/satijalab/seurat) and filtered low-quality cells as follows: cells expressing genes in fewer than three cells, cells with fewer than 800 or more than 7,000 genes, and cells with a percentage of mitochondrial genes greater than 5% were removed. Then, we used the “Merge” function to merge the scRNA-seq data of broiler testes from the same period. The merged data were normalized by “LogNormalize” and calculated for 2,000 highly variable genes by “FindVariableFeatures”. Next, we used “FindIntegrationAnchors” and “IntegrateData” to integrate the scRNA-seq data from the five periods, and the integrated data were normalized to “ScaleData”. We then performed principal component analysis (PCA) via “RunPCA” and selected 15 principal components (PCs). After that, we used the “FindNeighbors” and “FindClusters” functions to cluster the cells and visualized the data with the “RunUMAP” function.

Integration analysis of scRNA-seq data

We used the default Wilcoxon rank-sum test in the “FindAllMarkers” function to determine the differentially expressed genes (DEGs) of each cell cluster with a log2 foldchange > 0.25. According to the DEGs of each cell cluster, we identified the cell types of each cluster by using well-known marker genes and integrated the same cell type. Subsequently, we extracted testicular germ cells through the "subset" function to perform germ cell clustering analysis again and identified the cell types of the cell clusters. Additionally, DEGs between different cell types or different periods of the same cell type were obtained through the "FindMarker" function with the parameters “logfc. threshold = 0.25”, “min. pct = 0.25”, and "p_val < 0.05". Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEGs were performed using DAVID (https://david.ncifcrf.gov/) with P < 0.05 as the cutoff for significantly enriched terms and then visualized by the ggplot2 package (v3.3.5, https://r-graph-gallery.com/ggplot2-package.html).

Cell trajectory analysis

The Monocle 2 package (v2.18.0, http://cole-trapnell-lab.github.io/monocle-release/docs/) was installed in R to reconstruct the cell differentiation trajectory. We identified DEGs that varied in pseudotime order and set “qval < 0.01” and “num_cells_expressed > 10” for pseudotime analysis. Next, we applied the "DDRTree" method of the "reduceDimension" function for dimensionality reduction and used the "orderCells" function to sort cells and construct trajectories. Cell visual trajectories of clusters and groups were generated in pseudotime order using the "plot_cell_trajectory" function and colored by pseudotime values, cell states, and original ident. Pseudotime heatmaps were generated using the "plot_pseudotime_heatmap" function, and pseudotime genes were visualized using the “plot_genes.in_pseudotime” function.

Additionally, RNA Velocity analysis was used to detect the development of Leydig cells. The velocyto package (https://velocyto.org/) and scVelo (https://scvelo.readthedocs.io/en/stable/) package was installed in R to draw the cell differentiation trajectory under the uniform manifold approximation and projection (UMAP) clustering results.

Cell‒cell communication analysis

The CellChat package (1.5.0) was installed in R to study the interactions between different cell types in testes [15]. Subsequently, we used the "computeCommunProb" and "aggregateNet" functions to compute communication probabilities and infer cellular communication networks and used the "compareInteractions" function to compare the number and strength of interactions. Subsequently, we visualized the overall signaling patterns between humans and chickens by using the “netAnalysis_signalingRole_heatmap” function.

Cross-species correlation analysis

To compare and observe the differences in cell composition and gene expression in testicles of different species, we reintegrated single-cell transcriptome data from 24-week-old broilers (n = 3) and collected data from adult humans (n = 5) and adult mice (n = 2) to perform cross-species correlation analysis. We eliminated batch and technical differences through a unified integration approach. The single-cell transcriptome data of both adults and mice were obtained from ArrayExpress with the accession codes E-MTAB-11063 (human) and E-MTAB-11071 (mouse) [16].

Histological analysis

The collected fresh testis tissues were soaked in a 4% paraformaldehyde (PFA) solution and dehydrated using gradient ethanol (30%, 50%, 70%, 95%, and 100%, 2 h of each gradient). The dehydrated tissues were placed in xylene for 20 min and embedded in paraffin. Next, the embedded testis tissues were cut into 5 μm sections by a microtome (Leica RM2235, Wetzlar, Germany) and then successively placed in xylene for deparaffinization and gradient alcohol (100%, 95%, 75%, 50%, and 30%) for 5 min each for rehydration. After these steps, hematoxylin and eosin (H&E) was used to stain the sections for a few seconds. Subsequently, the slices were rinsed with water to remove excess dye, sealed with neutral balsam (Biotopped, Beijing, China), and finally analyzed using a Leica microscope (Wetzlar, Germany).

Immunofluorescence of testes

Testis tissues of chicken from different groups and adult mouse were soaked in a 4% PFA solution, dehydrated, and then embedded in paraffin. Next, the embedded testis tissues were cut into 5 μm sections. The sections were successively placed in xylene and gradient alcohol for 5 min each and treated with an 80 °C sodium citrate solution for 20 min. After being washed with PBS, the sections were blocked with 10% sheep serum for 30 min, followed by incubation with a primary antibody overnight at 4 °C, including ACTA2 (Invitrogen, Cat# MA5-41117), CLEC3B (Invitrogen, Cat# MA5-34760), VWF (Proteintech, Cat# 66682-1-lg), DDX4 (Invitrogen, Cat# MA5-15565), TOP2A (Invitrogen, Cat# MA5-32096), TPPP2 (Invitrogen, Cat# PA5-112776), SOX9 (Millipore, Cat# AB5535), and PCNA (Proteintech, Cat# 10205-2-AP). After that, the sections were incubated with secondary antibody for 1 h at room temperature, followed by incubation with DAPI as a nuclear stain. Finally, the sections were detected, and images were collected using a fluorescence microscope.

Extraction of Leydig cells and Sertoli cells

Leydig cells and Sertoli cells were isolated from the testicular tissues of 24-week-old broiler chickens via previously described methods [6, 17, 18]. The cells were cultured in DMEM/F12 (Gibco, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum (Sangon Biotech, Shanghai, China) and placed in a 37 °C incubator with 5% CO2. When the cell purity exceeded 95%, the cells were used for subsequent experiments.

Cell proliferation detection

Sertoli cell proliferation was detected using a BeyoClick™ EdU-594 cell proliferation kit (Beyotime Institute of Biotechnology, Shanghai, China). Sertoli cells were cultured in triplicate in 24-well plates at a density of 5 × 104 cells/well and treated following the manufacturer’s instructions. Finally, the EdU-positive cells were detected by a cell imaging detector (BioTek, Winooski, VT, USA).

Cell cycle detection

The purified chicken Sertoli cells were collected by centrifugation at 500 × g for 5 min. The cells were then washed once with PBS, centrifuged again, and the supernatant was discarded. The 500 μL PBS containing 50 μg/mL PI staining solution (Beyotime Institute of Biotechnology, Shanghai, China) and 100 μg/mL RNase A were add in cells. After gentle mixing, the cells were incubated in the dark for 30 min at 37 °C. Data were collected using a flow cytometer (ACEA Biosciences, CA, USA), with appropriate gating conditions set to exclude debris and doublets. The proportions of cells in the G1, S, and G2/M phases of the cell cycle were analyzed based on PI fluorescence intensity.

Gene interference

First, we designed and synthesized small interfering RNAs (siRNAs), including siCREB5, siRORα, siNPAS2 and NC (GenePharma, Suzhou, China). The sequences of the siRNAs used are shown in Table S1. Second, Leydig cells were seeded onto six-well plates at a density of 1 × 106 cells/well and cultured overnight. When the density of adherent cells reached 70%, we transfected siCREB5 (50 nmol/L) into chicken Sertoli cells, and siRORα or siNPAS2 (50 nmol/L) into Leydig cells by using Lipofectamine 2000 following the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). After 6 h, 10% fetal bovine serum was added to all the wells, and the cells were cultured for 24 h.

RT-qPCR analysis

To obtain total RNA, broiler Leydig cells were lysed by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Next, 1 μg of RNA was reverse transcribed to cDNA using a reverse transcription kit (Takara, Tokyo, Japan). Subsequently, gene expression was detected by RT-qPCR using SYBR Premix reagent (Takara, Tokyo, Japan) and analyzed by the 2−∆∆Ct method. β-Actin was used as a reference gene to calculate relative mRNA expression levels. The primer sequences of the detected genes and their annealing temperatures (Tm) are shown in Table S2.

Testosterone measurement

After NPAS2 interference, testosterone levels in Leydig cells were measured using an ELISA kit following the manufacturer’s instructions (J&L Biological, Shanghai, China). Ten microliters of supernatant, 40 µL of sample diluent and HRP-conjugate reagent were added sequentially to each well. After incubation at 37 °C for 1 h, testosterone levels were measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).

Western blot

Total protein was extracted using RIPA lysis buffer and measured using a protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein (20 µg) were separated by 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. After blocked with 5% non-fat milk for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies, including CREB5 (Proteintech, Cat# 14196-1-AP), NPAS2 (Santa cruz, Cat# sc-134404), RORα (Proteintech, Cat# 10616-1-AP), ZO-1 (Proteintech, Cat# 21773-1-AP), occludin (Proteintech, Cat# 27260-1-AP), STAR (OmnimAbs, Cat# OM153513), HSD3B1 (abcam, Cat# ab65156), CYP11A1 (CellSignalingTechnology, Cat# 14217). Then, the membranes were probed with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, including goat anti-mouse IgG (Proteintech, Cat# SA00001-1) and goat anti-rabbit IgG (Proteintech, Cat# SA00001-2). Protein bands were visualized using an enhanced chemiluminescence (ECL) detection reagent and captured by a chemiluminescence imaging system. β-Actin (Proteintech, Cat# 60008-1-lg) was used as a loading control to ensure equal protein loading.

Statistical analysis

Each experiment was conducted with 3 biological replicates, and all the data were statistically analyzed with GraphPad Prism version 5.0 (GraphPad Prism Software, San Diego, CA, USA). Differences between groups were determined by one-way analysis of variance (ANOVA) and Student’s t-test. A P-value < 0.05 indicated a statistically significant difference.

Results

Single-cell transcriptome of chicken testes from hatching to maturity

To investigate chicken testis development, we collected 13 pairs of whole chicken testes from five different ages: 1 d, 3 weeks, 12 weeks, 18 weeks, and 24 weeks, and observed a significant increase in the size and weight of chicken testicles during the five stages from hatching to maturity (Fig. 1A and Fig. S1B). Histological results showed the formation of seminiferous tubules from 1 d to 3 weeks, spermatogonia proliferation from 3 to 12 weeks, and complete spermatogenesis at 18 weeks and 24 weeks (Fig. 1B). These results suggested the comprehensive histological characterization and foundational insights of chicken testicular development phases.

Fig. 1
figure 1

Single-cell transcriptome profile of chicken testes from hatching to sexual maturity. A Schematic of the experimental workflow. B Hematoxylin-eosin (HE) staining of section of chicken testes from five different ages (1 d, 3 weeks, 12 weeks, 18 weeks, and 24 weeks), demonstrating morphological changes of chicken testes during the development process from hatching to sexual maturity. C A UMAP plot showing the annotated testicular cell types. Each dot represents a single testicular cell and is colored based on its cell type. D Five UMAP plots showing the annotated testicular cell types from 1 d (n = 2), 3 weeks (n = 2), 12 weeks (n = 3), 18 weeks (n = 3), and 24 weeks (n = 3) chickens. E Heatmap showing the markers of each cell cluster. The number on the left shows the number of markers, and is colored based on its cell type (Table S1). The gene expression levels are colored based on Z score. The figure on the right shows 5 representative GO terms enriched in the marker genes of each cluster with −log 10 (P-value) of each GO term colored based on the right color key

We then performed scRNA-seq on all 13 testicular samples (n = 2 or 3) from broiler chickens using the 10 × Genomics Chromium platform. A total of 105,486 cells from the 155,664 collected cells were used for the further analysis after quality control (Fig. S1A and Fig. S1B). To characterize the cell types of chicken testes, the UMAP analysis was performed on scRNA-seq data (Fig. 1C and D; Fig. S1C and S1D). We identified 11 major types of testicular cells using known specific marker genes of each cell type in testes [16, 19]. The somatic compartment comprised Sertoli cells (marked by ALDH1A1+, GSTA2+, SOX9+), Leydig cells (CYP17A1+, CYP11A1+, STAR+), testicular peritubular cells (TPCs; ACTA2+, DCN+, COL1A1+), smooth muscle cells (RGS4+, RGS5+, HTRA1+), endothelial cells (VWF+, CDH5+, SOX18+), nerve cells (CDH19+, NRN1+, ADGRB3+), and immune cells, including macrophages (AVD+, CD83+, C1QA+), T cells (CD3E+, ICOS+, CD28+). The germinal lineage was categorized into spermatogonia (FGFR3+, HMGB3+, DAZL+), spermatocytes (TOP2A+, SMC2+, NUSAP1+), and spermatids (TPPP2+, SPATA4+, TUBA8B+) (Fig. 1C and Fig. S2A). We also identified markers for major cell types through immunofluorescence staining, including ACTA2, VWF, CLEC3B, SOX9, DDX4, TOP2A, and TPPP2 (Fig. S1E). Additionally, a total of 18,294 genes were explored for each cell type, which could serve as potential markers for chicken testicular cells (Table S3). The definition and the physiological functional characteristics of each cell type was further confirmed by Gene Ontology (GO) analysis (Fig. 1E).

Subsequently, we calculated the percentage composition of various testicular cell types at the five stages. The proportion of TPCs in testicular cells was highest at 1 d (81.10%) and 3 weeks (76.60%), while the proportion of Sertoli cells in testicular cells was highest at 12 weeks (40.29%), and Sertoli cells still had the highest proportion in somatic cells at 18 weeks and 24 weeks. Notably, the proportion of germ cells rapidly increased from 12 weeks (11.08%) to 18 weeks (78.54%), and reached as high as 91.19% in the 24 weeks (Fig. S2B). These results suggested that the development of germ cells and Sertoli cells may be related to the spermatogenic superiority in chickens.

Dynamic transcriptional characteristics of germ cells during chicken spermatogenesis

Elucidating the different stages and characteristics of germ cell development is crucial for understanding the process of spermatogenesis [19]. Therefore, we integrated and analyzed 42,561 chicken germ cells and identified 15 main cell clusters. We defined 15 cell clusters as undifferentiated spermatogonia (Undiff_ed S’gonia), Diff_ing S’gonia, and Diff_ed S’gonia in spermatogonia; defined spermatocytes as the preleptotene stage (pre_Lep), leptotene stage (Lep), zygotene stage (Zyg), pachytene stage (Pach), diplotene stage (Dip), diakinesis (Dia), and secondary spermatocytes (Sec_S’cyles) based on the stage of meiosis; and defined spermatids as round spermatids (RS) and elongated spermatids (ES) (Fig. 2A). We detected specific expression of FGFR3 in SSCs; high expression of DMRT1 in Diff_ing S’gonia; high expression of PCNA in pre_Lep; specific expression of DMC1, PIWIL1, TEX12, ASPM, and NME8 in meiosis; high expression of CCNB1 in Sec_S’cyles; high expression of SPAG6 and SPATA2 in RS; and high expression of PLCZ1 in ES (Fig. 2B). According to the statistical analysis of the number and proportion of germ cells, only a small number of germ cells were present in chicken_1d and chicken_3w, and complete spermatogenesis occurred in chicken_18w and chicken_24w. Notably, the proportion of spermatids reached as high as 71.6% in chicken_24w (Fig. S3A).

Fig. 2
figure 2

Dynamic transcriptome of germ cell in chicken testes at the single-cell resolution. A A UMAP plot showing the annotated cell types of testicular germ cells. Each dot represents a single germ cell and is colored based on its cell type. B Marker expression patterns of each cluster of testicular germ cells on UMAP plots. A gradient of blue and gray represents high or low marker expression levels. C UMAP plots showing the annotated cell types of spermatogonia. Dashed lines and arrows represent their developmental trajectory. D Heatmaps showing the representative markers of each cell cluster of spermatogonia. A gradient of red and blue represents high or low marker expression levels. E Pseudotime trajectory of testicular germ cells analyzed by Monocle analysis. Each dot represents a single germ cell and is colored based on the predicted pseudotime. F Expression patterns of representative dynamic genes as predicted by pseudotime during spermatogenesis. Germ cells are colored based on the cluster colors and ordered according to the pseudotime trajectory. Pseudotime (SSCs to ES2) is indicated below each gene plot column. G Heatmap showing the clusters of top 1,000 genes (Table S8) in pseudotime from testicular germ cells of broiler chickens. A gradient of red and blue represents high or low gene expression levels based on the expression color code. The number on the left shows the number of differentially expressed genes, and the right side shows the representative genes and GO terms

Our results revealed 35,798 potential marker genes, most of which were dynamically transcribed and expressed during spermatogenesis (Fig. S3B; Table S4). We analyzed the expression patterns of key markers in each cluster during spermatogenesis. Notably, we identify two types of SSCs in Undiff_ed S’gonia by a detailed analysis of the subtypes, named SSC1 and SSC2, while SSC1 only exists in chicken_1d and chicken_3w. ACTC1, ID3, ID2, CYP2R1, and PROK2 were highly expressed in SSC1, while KIT, DGKB, UCHL1, DMRT1 were highly expressed in SSC2 (Fig. 2C–D and Fig. S4A). Some key markers were also analyzed in each cluster of spermatocytes, and spermatids (Fig. S3C and D). Additionally, the total of 4,217, 13,498, and 4,347 potential markers and the molecular characteristics were explored for each cluster of spermatogonia spermatocytes, and spermatids (Fig. S4; Tables S5–7).

To identify key genes involved in spermatogenesis, we conducted a pseudotime analysis of germ cells via Monocle 2 (Fig. 2E; Table S8). The expression patterns of DDX4, KIT, GOLGB1, FOXP2, HMGA2, DHRS4, and NRF1 suggested their important roles in the proliferation and differentiation of SSCs and Diff_ing S’gonia. Meiosis-related genes, including RNF212, SPO11, HORMAD1, and SPATA22, were highly expressed at the early stage of meiosis, and their expression decreased as meiosis progressed. PGK2 has been reported to be essential for sperm function and male fertility [20]. MYH9 and CA2 were highly expressed in Pach and Dip, respectively. SYCE3, DNAJC2, NME6, SYPL1, LDHA and MCM10 might play important roles in the development of RS. CEP41 and LDHB were more highly expressed in the ES group, which could suggest their involvement in spermatogenesis (Fig. 2F; Fig. S3E). In pseudotime, we divided the spermatogenesis process into five stages based on the first 1,000 DEGs to explore the dynamic molecular characteristics of spermatogenesis. The expression of ORC5, HMGA2, LAMB1, ASZ1, MAEL and DHRS4 increased, and genes related to stem cell population maintenance and differentiation increased in the first stage. KMD5A, MYH9, YBX3, SYCE3, LCA5L and DNAJC2 were observed, and RNA splicing and chromosome organization were enriched in the second and third stages. NME6, RPSH1, SPAG17, TTC26, RRP7A and TTLL1 were observed, and spermatid development was enriched in the fourth stage. FRS2, IFT27, PLCZ1, CACNA2D1, CEP41 and FOXM1 were observed, and cell differentiation occurred in the fifth stage (Fig. 2G). We also conducted pseudotime analysis of spermatogonia, spermatocytes, and spermatids to observe a large number of key pseudotime genes (Fig. S5; Tables S9–11).

To sum up, we conducted a systematic identification of chicken germ cells and studied the dynamic transcriptional characteristics of germ cell development, providing a wealth of data and molecular basis for in-depth research on the regulatory mechanism of spermatogenesis.

The persistence of immature Sertoli cells in chicken testes after sexual maturity

Sertoli cells are indispensable in the process of spermatogenesis, and the number of Sertoli cells directly affects the production of sperm [12, 21]. Thus, we integrated the Sertoli cell cluster and found two main cell clusters as mature and immature Sertoli cells in chicken testes (Fig. 3A–C). Additionally, we identified 1,208 markers in immature Sertoli cells and 2,295 markers in mature Sertoli cells (Fig. S6A; Table S12). GO analysis revealed that immature Sertoli cells were enriched in “translation”, “oxidative phosphorylation”, “ribosome biogenesis”, “mitochondrion organization” and “metabolic process”, while mature Sertoli cells were enriched in “development process”, “protein modification process”, “intracellular signal transduction”, “regulation of response to stimulus” and “RNA biosynthetic process” (Fig. S6B).

Fig. 3
figure 3

The persistence of immature Sertoli cells in chicken testes after sexual maturity. A A UMAP plot of testicular Sertoli cells of broiler chickens from five different groups (1 d, 3 weeks, 12 weeks, 18 weeks, and 24 weeks). Each dot represents a single Sertoli cell and is colored based on its cell subtype. B Identification of mature and immature Sertoli cells by analyzing nFeature_RNA, nCount_RNA, percent.mt and the expression patterns of classic marker genes. C Five UMAP plots showing the annotated types of Sertoli cells from five groups (1 d, 3 weeks, 12 weeks, 18 weeks, and 24 weeks), respectively. Each dot represents a single testicular cell and is colored based on its cell type. D UMAP plot of testicular Sertoli cells from chicken_24w, adult humans and adult mice. Each dot represents a single Sertoli cell and is colored based on its cell subtype. E Marker expression patterns of mature or immature Sertoli cells on UMAP plots. A gradient of blue and gray represents high or low marker expression levels. F Immunofluorescence staining for PCNA (green), SOX9 (red, a marker of Sertoli cells) and DAPI (blue) in testes of adult mice and chicken_24w. Scale bars = 100 μm. G Schematic of Sertoli cell extraction and experimental process. H Detection of primary Sertoli cell cycle in broiler chicken testis by flow cytometry. I Proliferation of testicular Sertoli cells of broiler chickens was measured using the EDU incorporation assay. EDU detection was performed at 0 h, 48 h, and 96 h of Sertoli cell adhesion, respectively. Red fluorescence represents EDU-labeled Sertoli cells. Nuclei were stained with Hoechst 33,342 (blue). Scale bars = 200 μm

Interestingly, we found that immature Sertoli cells persisted in the testes, even after sexual maturity, when analyzing Sertoli cells in chicken testes at five different periods (Fig. 3B). However, it is well known that immature Sertoli cells develop into mature Sertoli cells to provide energy for germ cells, and testicular Sertoli cells exist in a mature form and no longer proliferate after sexual maturity or adolescence in mammals [14]. To investigate the differences in the Sertoli cells of chicken and mammals, we integrated testicular Sertoli cells from chicken_24 weeks, adult human and adult mouse and identified two cell subtypes, namely, immature (TOMM7+, RPL22L1+) and mature (AR+, FSHR+, CREB5+, SHANK2+) Sertoli cells (Fig. 3D and E). We also found that both immature and mature Sertoli cells were present in chicken testes after sexual maturity, while only mature Sertoli cells were present in human and mouse testes.

Next, we detected the proliferation of testicular Sertoli cells in 28-week-old chickens and adult mice to verify the presence of immature Sertoli cells. Immunofluorescence co-staining of the proliferation marker PCNA and the Sertoli cell marker SOX9 revealed that there were proliferating Sertoli cells in the testes of the chickens at 24 weeks, but no co-stained cells were detected in the testes of the adult mice (Fig. 3F). Subsequently, we extracted and purified testicular Sertoli cells from chicken_24w to verify the presence of immature Sertoli cells in vitro (Fig. 3G). We detected the cell cycle distribution of the purified Sertoli cells by flow cytometry and found that 15.15% of the Sertoli cells were in the S phase and 34.93% were in the G2/M phase (Fig. 3H). Additionally, we cultured the extracted and purified Sertoli cells and tested their proliferation status by EdU staining. We observed EDU-positive Sertoli cells, and the number of these cells significantly increased with cultivation time (Fig. 3I). These active proliferation characteristics of immature Sertoli cells were consistent with previous studies [22]. Taken together, these results suggested that proliferating immature Sertoli cells persist in chicken testes after sexual maturity, which demonstrated the uniqueness of Sertoli cell development in chicken testes.

CREB5 played a crucial role in the maturation of chicken Sertoli cells

To explore the maturation mechanism of Sertoli cells, we analyzed single-cell data from immature and mature Sertoli cells. Compared with those in immature Sertoli cells, we discovered 715 DEGs in mature Sertoli cells, including 605 upregulated genes and 110 downregulated genes (Fig. 4A; Table S13). Mature Sertoli cells were enriched in “Sertoli cell development”, “male gonad development”, “BMP signaling pathway”, “SMAD protein signal transduction”, “cellular response to corticotropin-releasing hormone stimulus” and some GO terms related to development and proliferation (Fig. 4B). Subsequently, we characterized some representative genes in these GO terms, such as SOX8, TGFB2, EGFR, ROR2, NR4A2, NR4A3, NR3C1 and NR3C2. Notably, mature Sertoli cells also exhibited “transcription factor activity, sequence-specific DNA binding”, and the expression levels of CREB5, CREM, WT1, and SMAD3 were significantly increased in mature Sertoli cells (Fig. 4C). KEGG analysis revealed that Sertoli cell maturation was associated with the “GNRH signaling pathway”, “MAPK signaling pathway”, “TGF-β signaling pathway”, “Apelin signaling pathway” and “Calcium signaling pathway”, and several related genes were characterized, including RYR2, ADCY6, BMPR2, JUN, CACNA1H, MET, MAPK8, MAP3K1, and EGR1 (Fig. 4D and E). MAPK signaling pathway, TGF-β signaling pathway, and GnRH signaling pathway were crucial for Sertoli cell maturation [23,24,25].

Fig. 4
figure 4

CREB5 played a crucial role in the maturation of chicken Sertoli cells. A Statistical analysis of the number of differentially expressed genes between mature and immature Sertoli cells. Red represents the number of upregulated genes, and blue represents the number of downregulated genes. B A dot bubble chart showing representative GO terms enriched in differentially expressed genes. C Expression patterns of representative genes of mature and immature Sertoli cells. D A dot bubble chart showing representative KEGG pathways enriched in differentially expressed genes. E A chord plot showing the subordinate relationship between representative genes (Left) and KEGG pathways (Right). The color of the boxes in front of the gene names from blue to red represents the fold change in gene expression. F Schematic of Sertoli cell extraction and CREB5 interference. G and H The mRNA and protein expression of CREB5. I and J The mRNA and protein expression of ZO-1, and occludin in chicken Sertoli cells (n = 3). K The mRNA expression of AR, LAMA5, NOTCH2 in chicken Sertoli cells (n = 3). *P < 0.05, **P < 0.01

Combining CREB5 as the gene with the greatest differential expression, we hypothesized that CREB5 might be involved in Sertoli cell maturation. We extracted and purified Sertoli cells from chicken testes, and conducted cell transfection using siCREB5 to detect the effect of CREB5 on Sertoli cell function (Fig. 4F). The results showed that siCREB5 treatment decreased the mRNA and protein expression of CREB5 and siCREB5-1984 was used for future research (Fig. 4G and H). We found that siCREB5 treatment also decreased the mRNA and protein expression of blood-testis barrier (BTB) related genes ZO-1 and occludin (Fig. 4I and J). Additionally, siCREB5 decreased the mRNA expression of mature related markers AR, LAMA5, and NOTCH2 (Fig. 4K). Our results suggested that CREB5 might play a crucial role in maintaining the maturation of chicken Sertoli cells, laying the foundation for studying the mechanisms Sertoli cell maturation.

Circadian rhythms participated in the development of Leydig cells in chicken testes

Leydig cells can synthesize and secrete testosterone, which is particularly important for testicular development and spermatogenesis [6]. Thus, we integrated and analyzed the Leydig cell cluster and identified two main cell clusters (precursor and Leydig cells) by the expression patterns of classic marker genes (Fig. 5A and B). Specifically, the proportion of mitochondrial genes significantly increased, as mitochondria are the main site for testosterone secretion, and of course, genes related to testosterone secretion, including STAR, CYP17A1, and CYP11A1, were upregulated (Fig. 5B). Next, we calculated the markers of each cell cluster and identified 781 markers in precursor cells (C7, EDNRA, APOA1, BTG2, and COL3A1) and 420 markers in Leydig cells (CYP17A1, CYP11A1, TPM2, LHCGR, and ND3) (Fig. S6C; Table S14). GO analysis of the markers revealed that precursor cells were enriched in “cellular developmental process”, “cell population proliferation”, “RNA biosynthetic process”, “mesenchyme development” and “apoptosis process”, while Leydig cells were enriched in “cellular respiration”, “steroid biosynthetic process”, “generation of precursor metabolites and energy”, “lipid biosynthetic process” and “cholesterol metabolic process”, which are known functions of Leydig cells (Fig. S6D).

Fig. 5
figure 5

Circadian rhythm participated in the development of Leydig cells in chicken testes. A A UMAP plot of testicular Leydig cells of broiler chickens from five different groups (1 d, 3 weeks, 12 weeks, 18 weeks, and 24 weeks). Each dot represents a single Leydig cell and is colored based on its cell type. B Identification of precursor and Leydig cells by analyzing percent.mt and the expression patterns of classic marker genes. C RNA velocity analysis showing the dynamic change trend of precursor (blue) and Leydig (yellow) cells. D Statistical analysis of the number of differentially expressed genes between precursor and Leydig cells. Red represents the number of upregulated genes, and blue represents the number of downregulated genes. E A dot bubble chart showing representative GO terms enriched in differentially expressed genes. F Expression patterns of enriched genes in “circadian regulation of gene expression”. G Schematic of Leydig cell extraction and experimental process. H–K Relative mRNA and protein expression of RORα and NPAS2 in Leydig cells (n = 3). L and M The mRNA and protein expression of key genes related to testosterone secretion, including CYP11A1, HSD3B1, and STAR in Leydig cells (n = 3). N Measurement of testosterone level in Leydig cells using ELISA (n = 3). O Relative mRNA expression of key genes related to circadian rhythm, including NPAS2BMAL1, RORα, CRY-1, CRY-2, PER-2, PER-3 (n = 3). P Schematic diagram of circadian rhythm promoting testosterone secretion in chicken Leydig cells. *P < 0.05, **P < 0.01

Importantly, the trend of cell differentiation from cell velocity analysis confirmed the developmental relationship of precursor and Leydig cells (Fig. 5C). Additionally, we found that some marker genes of precursor cells, such as TPM1, APOA1, COL1A2, and COL3A1, were highly expressed in TPCs. The results of UMAP and RNA velocity analysis also indicated that precursor cells could develop from TPCs. These results suggested that TPCs and Leydig cells might have the same source, as it has been reported in human testes that there is a common progenitor for peritubular myoid cells and Leydig cells [26].

To investigate the development mechanisms of Leydig cells, we compared precursor cells and identified 531 DEGs in Leydig cells, including 180 upregulated genes and 351 downregulated genes (Fig. 5D; Table S15). GO analysis of the markers indicated that Leydig cells were enriched in “gonad development”, “cellular response to corticotropin-releasing hormone stimulus”, “aerobic respiration”, “positive regulation of DNA repair”, and “cholesterol biosynthetic process” (Fig. 5E). The molecular characteristics of Leydig cell maturation are similar to those of human and mouse studies, both of which are related to steroid biological processes as well as energy metabolism [10, 27].

Interestingly, “circadian regulation of gene expression” was significantly enriched, and the expression levels of the neuronal PAS domain protein 2 (NPAS2) and the retinoic acid receptor-related orphan receptor-alpha (RORα) increased in Leydig cells, which might be closely related to testosterone secretion and the development of Leydig cells (Fig. 5F). Therefore, we demonstrated this in vitro by extracting testicular Leydig cells and treating them with siRORα and siNPAS2 (Fig. 5G). siRORα and siNPAS2 treatment significantly inhibited the relative mRNA and protein expression of RORα and NPAS2 (Fig. 5H–K). Notably, siRORα and siNPAS2 treatment significantly downregulated the relative mRNA and protein expression of CYP11A1, HSD3B1, and STAR, and decreased testosterone secretion in chicken Leydig cells (P < 0.01) (Fig. 5L–N). Additionally, we found that the relative mRNA expression of RORα, NPAS2, BMAL1, CRY-1, CRY-2, PER-2, and PER-3 decreased significantly after RORα and NPAS2 interference (P < 0.05) (Fig. 5O). The above results suggested that circadian rhythm could participated in testosterone secretion and the development of Leydig cells in chicken testes, which provided a new perspective for studying the development of Leydig cells (Fig. 5P).

The dominant proportion of chicken Sertoli cells revealed by cross-species analysis

The sperm density of chickens is much greater than that of adult humans and mice, which suggests a unique system during spermatogenesis in chickens. Multi species analysis has been applied to explore the conservation and differences among species [28]. To characterize the spermatogenesis of chickens and mammals, we downloaded single-cell transcriptome data of adult human testes (n = 5; 16,015 cells) and adult mouse testes (n = 2; 5,264 cells) for cross-species analysis of chicken_24 weeks testes (n = 3; 23,893 cells). We used UMAP to integrate and analyze testicular cells from chickens, humans and mice.

According to known specific marker genes of each cell type, we identified 7 major types of germ cells and somatic cells, including spermatogonia, spermatocytes, spermatids, Sertoli cells, endothelial cells, immune cells, and myoid & Leydig cells (Fig. 6A). To characterize the cellular composition of the three types of testicles, we calculated the proportions of each cell type. Our results revealed that the proportion of testicular germ cells in chicken, which accounted for 91.30%, was much greater than that in human (85.97%) and mice (67.85%) (Fig. 6B). We observed particularly efficient spermatogenesis, with spermatids accounting for up to 67.01% of the total number of testicular germ cells in chickens (42.94% in humans and 52.66% in mice) (Fig. 6C).

Fig. 6
figure 6

The dominant proportion of chicken Sertoli cells revealed by cross-species analysis. A UMAP plots of the annotated testicular cell types from chicken_24w (n = 3), adult human (n = 5), and adult mouse (n = 2). Each dot represents a single testicular cell and is colored based on its cell type. The single-cell transcriptome data of adults and mice are both from ArrayExpress with the accession codes E-MTAB-11063 (human) and E-MTAB-11071 (mouse). B A bar chart showing ratio of testicular germ cells and somatic cells in chicken_24w, adult human, and adult mouse. C A column chart showing ratio of spermatogonia, spermatocytes and spermatids in chicken_24w, adult human, and adult mouse. D A column chart showing ratio of endothelial cells, immune cells, myoid & Leydig cells, and Sertoli cells testicular somatic cells of chicken_24w, adult human, and adult mouse. E Immunofluorescence staining for SOX9 (red, a marker of Sertoli cells) and DAPI (blue) in testes of five-stage chicken (1 d, 3 weeks, 12 weeks, 18 weeks, and 24 weeks), and adult mouse. Scale bars = 100 μm. F Bar charts showing the number of inferred interactions and interaction strength of chicken_24w and adult human. G Circle plots (by CellChat analysis) showing the differential cell–cell communication network between chicken_24w and adult human, including the number of inferred interactions (left) and interaction strength (right). Circle plots in dashed boxes showing the number of inferred interactions between Sertoli cells and other cells. H Heatmaps showing the overall signaling patterns of each cell type in chicken_24w and adult human. The color of the small grid from light to dark indicates its relative strength

Additionally, we found that the composition proportion of somatic cells in the microenvironment of chicken testes was significantly different from that of humans and mice. The proportion of chicken Sertoli cells in testicular somatic cells was as high as 86.48%, which is much higher than that in humans (36.44%) and mice (27.42%), while myoid and Leydig cells made up the greatest proportion of testicular somatic cells in humans and mice (Fig. 6D). Our results reflected the specificity and differences in the composition of chicken testicular cells. Simultaneously, the fluorescence staining results of SOX9 revealed the distribution of Sertoli cells in chicken testes, and compared with that in mouse testes, more chicken testicular Sertoli cells were distributed in the seminiferous tubules (Fig. 6E).

We then investigated the interactions between somatic cells and germ cells in chicken and human testes via CellChat analysis. Interestingly, the number of inferred interactions in chickens was three times that in humans, although the intensity was lower than that in humans (Fig. 6F). In particular, there were significant differences in the number of interactions and interaction strength between chicken and human testicular Sertoli cells (Fig. 6G). Next, we analyzed the overall signaling patterns in the cell‒cell communication network between chicken and human testes. Overall, there were more signaling patterns in chicken testes than in human testes, such as TGF-β, SEMA3, ANGPTL, BMP, ncWNT, GAS, EGF, MK, periostin, GDF, ANGPT, activin, and CCK signaling in Sertoli cells (Fig. 6H). Taken together, these results demonstrated the uniqueness of Sertoli cell development in chicken testes.

Discussion

It is important to investigate the molecular features of spermatogenesis for understanding the differences and conservatism of testicular development across different species [1, 16, 28]. In this study, through establishing a single-cell transcriptome profile of chicken testes from hatching to maturity, we identified the dynamic transcriptional characteristics of germ cell fate transition and explored the unique adaptations of Sertoli cells in chicken testes. Most importantly, we demonstrated that immature Sertoli cells persist in chicken testes after sexual maturity, contrasting with the known development of Sertoli cells in mammals. We also elucidated the developmental characteristics of Sertoli cells and Leydig cells. These results may supplement the theory of testicular Sertoli cell development and hold extraordinary significance in understanding the unique adaptations in poultry.

Cell recognition

Cell recognition is a fundamental and important step in single-cell data analysis [29]. We identified 11 main cell types, including 8 types of somatic cells and 3 types of germ cells. In addition to nerve cells, other types of testicular cells and their markers have been proven by many single-cell testicular articles [26, 30,31,32]. Notably, we observed nerve cells in chicken testes, which was confirmed by marker identification and GO analysis. Previous studies have shown that the nerves distributed in the testes, originating from the spermatic plexus, are composed of many small nerve fibers from the renal plexus and abdominal aortic plexus, and these nerve fibers are unmyelinated and descend along the testicular artery, known as the superior spermatic nerves [33, 34]. Testicular nerves reportedly play an important role in the regulation of testosterone secretion [35].

Additionally, we identified SSC1 and SSC2 in Undiff_ ed S'gonia, which have different gene expression patterns. ACTC1 served as a marker of early first trimester human trophoblast in human embryonic stem cell model [36]. ID3, ID2, CYP2R1, and PROK2 have also reported to participate in testicular development [37,38,39]. UCHL1 promoted SSCs self-renewal, while KIT and DMRT1 are considered as spermatogonial proliferation signals [40, 41]. These results suggested that SSC1 might be closer to primitive stem cells, and SSC2 could self-renew and differentiate into Undiff_ing S’gonia according to the developmental trajectory. Differently, the single-cell transcriptome study of human testes identified five discrete transcriptional/developmental spermatogonial states, including a new early SSC state (state 0), and identified significant similarities between adult state 0 and infant SSC [19].

Proliferating Sertoli cells persist in chicken testes after sexual maturity

Sertoli cells play a central role in testicular development and spermatogenesis, which has also been proven in single-cell transcriptome studies of human azoospermia [13, 21, 42]. Numerous pieces of evidence indicate that Sertoli cell number directly determines the testamentary size and sperm production [12]. In our study, we revealed the unique adaptations in chicken Sertoli cells. The most important was the persistent presence of proliferating Sertoli cells in chicken testes after sexual maturity, which were confirmed by the immunofluorescence results of co-staining with PCNA and SOX9, as well as cell cycle distribution and EDU positive labeling of chicken Sertoli cells. Conversely, immature Sertoli cells mature during puberty and are no longer proliferating after puberty in mammalian testes [43]. The sustained existence of immature Sertoli cells can develop into mature Sertoli cells to continuously provide nutrition and exert phagocytic function during spermatogenesis [44]. A previous report provided strong evidence that transplanting mouse fetal testicular cells into a Sertoli-depleted adult testis can promote the colonization of mouse fetal Sertoli cells in adult seminiferous tubules and support host testicular spermatogenesis, which may suggest that immature fetal Sertoli cells can become sufficient maturation and functional in the adult testis. [45]. These results reflected the sustained presence of proliferating Sertoli cells in chicken testes after sexual maturity, which might be one of the reasons for the high sperm density.

CREB5 participates in maintaining the maturation and function of chicken Sertoli cells

The maturation of Sertoli cells is accompanied by significant changes in gene expression [44]. We found that the expression levels of AR, FSHR, and CREB5 increased in mature Sertoli cells. It has been reported that AR is a marker of Sertoli cell maturation, and its expression changes during the spermatogenic cycle [44, 46]. FSHR, a marker of Sertoli cell maturation regulation, binds to FSH and stimulates cAMP to control the proliferation, development, and function of Sertoli cells [47, 48]. CREB5, as a binding protein of cAMP, has also been reported to be associated with resistance to AR-targeted therapy [49], which may become a new marker of chicken testicular Sertoli cells. Importantly, our results indicated that CREB5 interference reversed the maturation characteristics of Sertoli cells and disrupted the structure of BTB. A previous study has shown that CREB5 promotes joint formation and the subsequent development of articular chondrocytes, which suggesting the importance of CREB5 for organ and cell development [50]. Taken together, our results indicated that CREB5 participates in maintaining the maturation and function of chicken Sertoli cells.

Circadian rhythm promotes testosterone secretion in Leydig cells

Leydig cells are the main site for testosterone synthesis and secretion, and testosterone is indispensable during spermatogenesis [51]. Our results indicated that the development of Leydig cells was closely related to the circadian rhythm in chicken testes, accompanied by the upregulation of NPAS2 and RORα expression. It is widely known that the photoperiod is particularly important for regulating chicken production and reproduction [52]. There is limited direct evidence that circadian rhythms are involved in regulating testosterone secretion and the development of Leydig cells [53,54,55]. RORα, as an important circadian clock gene has been reported to regulate testosterone secretion of Leydig cells [56]. NPAS2 is an important regulatory factor that generates and maintains periodic rhythms, regulating various physiological functions such as sleep, arousal, endocrine, metabolism, cell proliferation and apoptosis, and immunity [57]. It has been reported that NPAS2 forms a heterodimer with BMAL1, which binds to E-box enhancer elements, thereby promoting the transcriptional activation of circadian rhythm-related genes, such as RORα, CRY1/2 and PER2/3 [58]. However, there is no direct evidence to indicate the role of NPAS2 in regulating testosterone secretion and the development of Leydig cells. In our study, RORα and NPAS2 interference decreased the expression of circadian rhythm related genes BMAL1, CRY-1, CRY-2, PER-2, and PER-3 and inhibited testosterone secretion, accompanied by a decrease in the expression of STAR, CYP17A1, and CYP11A1. Taken together, these results suggested that circadian rhythm participated in testosterone secretion and the development of Leydig cells in chicken testes.

Multispecies analysis of key signaling between chicken testicular cells

Quoting single-cell testicular data from other species from databases has been widely applied for cross species analysis [59]. Compared to adult humans and mice, cross-species analysis revealed a greater proportion of germ cells in mature chickens, especially spermatids. In a previous study on single-cell testes involving species evolution, the proportion of testicular cells in various species was statistically analyzed, indicating that the proportion of germ cells in red jungle fowl was higher than that in mammals, including humans, mice, monkeys, and platypus [16]. These results of high proportion of germ cells were an important foundation for the formation of high sperm density in chickens.

The cellular interactions in chicken testes were three times greater than those in human testes, mainly involving TGF-β signaling, VEGF signaling, BMP signaling, FGF signaling and activin signaling. We also found the specific activation of Sertoli cell interaction signals, such as TGF-β, BMP, EGF, and activin. Studies have reported that TGF-β signaling, VEGF signaling, BMP signaling, FGF signaling and their interactions participate in cell growth and organ development [60, 61]. FGF signaling acted upstream of RA signaling to activate the PI3K/Akt pathway in germ cells and played an important role in spermatogenesis [62, 63]. Activin signaling was involved in Sertoli cell differentiation and function, increased activin signaling induces the proliferation of primary Sertoli cells, and activin A reprograms Sertoli cells to immature and dedifferentiated states [64]. The above results reflected the key signaling of chicken spermatogenesis, which was particularly important for the formation of unique adaptations in chickens.

Conclusion

In summary, we established a complete single-cell transcriptome profile of chicken testes from hatching to maturity, revealing the unique adaptations in chicken Sertoli cells. Most importantly, proliferating Sertoli cells persisted in chicken testes even after sexual maturity. We also observed the dominance of Sertoli cells in somatic composition of mature chicken testes, and the specific activation of Sertoli cell interaction signals. Moreover, our results indicated that CREB5 played a crucial role in maintaining the maturation and function of chicken Sertoli cells, and circadian rhythm promoted testosterone secretion and the development of Leydig cells in chicken testes. These findings might lay a molecular foundation for elucidating the poultry spermatogenic superiority and provided new insights into the formation of high sperm density in poultry. The sample sizes of 1 d and 3 weeks (n = 2) may affect the reliability and statistical ability of the results, as well as the lack of more in-depth mechanism research, which may be a limitation of this study. In the future, we will delve into the key targets and molecular mechanisms underlying the formation of high sperm density.

Data Availability

Raw and processed single-cell transcriptome data of 13 chicken testes at 1 d, 3 weeks, 12 weeks, 18 weeks, and 24 weeks have been deposited in NCBI with the BioProject codes PRJNA1119270. The analysis and experimental data in this study are also available from the corresponding author according to reasonable requirements.

Abbreviations

  • ALDH1A1 :: Aldehyde dehydrogenase 1 family member A1
  • ANOVA:: Analysis of variance
  • AR :: Androgen receptor
  • AVD :: Avidin
  • BMAL1 :: Brain and muscle arnt-like 1
  • BMP :: Bone morphogenetic proteins
  • BTB:: Blood-testis barrier
  • CA2 :: Carbonic anhydrase 2
  • CDH5 :: Cadherin 5
  • CEP41 :: Centrosomal protein 41
  • CREB5 :: cAMP responsive element binding protein 5
  • CRY-1 :: Cryptochrome 1
  • CRY-2 :: Cryptochrome 2
  • CYP11A1 :: Cytochrome P450 family 11 subfamily A member 1
  • DDX4 :: DEAD-box helicase 4
  • DEGs:: Differential expressed genes
  • DHRS4 :: Dehydrogenase/reductase 4
  • DNAJC2 :: DnaJ heat shock protein family (Hsp40) member C2
  • EGF :: Epidermal growth factor
  • ELISA:: Enzyme-linked immunosorbent assay
  • FOXM1 :: Forkhead box M1
  • FOXP2 :: Forkhead box protein P2
  • GO:: Gene Ontology
  • GOLGB1 :: Golgin B1
  • GSTA2 :: Glutathione S-transferase alpha 2
  • H&E:: Hematoxylin and eosin
  • HMGA2 :: High mobility group AT-hook 2
  • HORMAD1 :: HORMA domain-containing 1
  • HSD3B1 :: Hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-Isomerase 1
  • IFT27 :: Phospholipase C zeta 1
  • KEGG:: Kyoto Encyclopedia of Genes and Genomes
  • KIT :: KIT proto-oncogene, receptor tyrosine kinase
  • KMD5A :: Lysine demethylase 5A
  • LAMB1 :: Laminin subunit beta 1
  • LCA5L :: Lebercilin-like protein
  • LDHA :: Lactate dehydrogenase A
  • LDHB :: Lactate dehydrogenase B
  • MAEL :: Maelstrom spermatogenic transposon silencer
  • MCM10 :: Minichromosome maintenance 10 replication initiation factor
  • MYH9 :: Myosin heavy chain 9
  • NME6 :: NME/NM23 nucleoside diphosphate kinase 6
  • NOTCH2 :: Notch receptor 2
  • NPAS2 :: Neuronal PAS domain protein 2
  • NRF1 :: Nuclear respiratory factor 1
  • ORC5 :: Origin recognition complex subunit 5
  • PBS:: Phosphate-buffered saline
  • PCA:: Principal component analysis
  • PCNA :: Proliferating cell nuclear antigen
  • PER-2 :: Period circadian regulator 2
  • PER-3 :: Period circadian regulator 3
  • PFA:: Paraformaldehyde
  • PGK2 :: Phosphoglycerate kinase 2
  • PLCZ1 :: Intraflagellar transport 27
  • RNF212 :: Ring finger protein 212
  • RORα :: Retinoic acid receptor-related orphan receptor alpha
  • RT-qPCR:: Quantitative real-time PCR
  • S’gonia:: Spermatogonia
  • scRNA-seq:: Single-cell RNA sequencing
  • S'cyles:: Spermatocytes
  • SOX18 :: SRY-box transcription factor 18
  • SOX9 :: SRY-box transcription factor 9
  • SPAG17 :: Sperm-associated antigen 17
  • SPATA22 :: Spermatogenesis-associated 22
  • SPATA4 :: Spermatogenesis associated 4
  • SPO11 :: SPO11 initiator of meiotic double-strand breaks
  • SSCs:: Spermatogonial stem cells
  • STAR :: Steroidogenic acute regulatory protein
  • SYCE3 :: Synaptonemal complex central element 3
  • SYPL1 :: Synaptophysin-like 1
  • TGF-β :: Transforming growth factor-β
  • TOP2A :: Topoisomerase II alpha
  • TPCs:: Testicular peritubular cells
  • TTC26 :: Tetratricopeptide repeat domain 26
  • TTLL1 :: Tubulin tyrosine ligase-like 1
  • TUBA8B :: Tubulin alpha 8b
  • UMAP :: Uniform manifold approximation and projection
  • VWF :: Von willebrand factor
  • YBX3 :: Y-box binding protein 3
  • ZO-1 :: Zonula occludens-1

References

  1. 1.Trost N, Mbengue N, Kaessmann H. The molecular evolution of mammalian spermatogenesis. Cells Dev. 2023;175.(2023)203865. https://doi. org/10. 1016/j.cdev.: 203865.
    10.1016/j.cdev.2023.203865
    Article|CAS|PubMed|Google Scholar
  2. 2.Fayomi AP, Orwig KE. Spermatogonial stem cells and spermatogenesis in mice, monkeys and men. Stem Cell Res. 2018;29.(2018)207–14. https://doi. org/10. 1016/j.scr.: 207.
    10.1016/j.scr.2018.04.009
    Article|CAS|PubMed|Google Scholar
  3. 3.Tapeh RS, Zhandi M, Zaghari MA. Effects of guanidinoacetic acid diet supplementation on semen quality and fertility of broiler breeder roosters. Theriogenology. 2017;89.(2017)11.012.: 178.
    10.1016/j.theriogenology.2016.11.012
    Article|CAS|PubMed|Google Scholar
  4. 4.Levitas E, Lunenfeld E, Weisz N, Friger M, Potashnik G. Relationship between age and semen parameters in men with normal sperm concentration.(2007)analysis of 6022 semen samples.Andrologia.: 45.
    10.1111/j.1439-0272.2007.00761.x
    Article|CAS|PubMed|Google Scholar
  5. 5.Albert MRC. Changes from puberty to adulthood in the concentration, motility and morphology of mouse epididymal spermatozoa. Int J Androl. 1983;6(5).(1983)446–60. https://doi. org/10. 1111/j.1365-2605.: 446.
    10.1111/j.1365-2605.1983.tb00559.x
    Article|CAS|PubMed|Google Scholar
  6. 6.Xu G, Yuan Z, Hou J, Zhao J, Liu H, Lu W, et al. Prolonging photoperiod promotes testosterone synthesis of Leydig cells by directly targeting local melatonin system in rooster testes. Biol Reprod. 2021;105(5).(2021)org/10.1093/biolre/ioab155.: 1317.
    10.1093/biolre/ioab155
    Article|CAS|PubMed|Google Scholar
  7. 7.Ding H, Zhao J, Liu H, Wang J, LuW. BMAL1 knockdown promoted apoptosis and reduced testosterone secretion in TM3 Leydig cell line. Gene. 2020;747.(2020)144672. https://doi. org/10. 1016/j.gene.: 144672.
    10.1016/j.gene.2020.144672
    Article|CAS|PubMed|Google Scholar
  8. 8.Sharp PJ. Photoperiodic control of reproduction in the domestic hen. Poult Sci. 1993;72(5).(1993)3382/ps.0720897.: 897.
    10.3382/ps.0720897
    Article|CAS|PubMed|Google Scholar
  9. 9.Bachman SE, Bachman JM, Mashaly MM. Effect of photoperiod on the diurnal rhythm of plasma testosterone, dihydrotestosterone and androstenedione in mature male chickens. Comp Biochem Physiol A Comp Physiol. 1987;87(3).(1987)org/10.1016/0300-9629(87)90399-9.: 775.
    10.1016/0300-9629(87)90399-9
    Article|CAS|PubMed|Google Scholar
  10. 10.Guo J, Sosa E, Chitiashvili T, Nie X, Rojas EJ, Oliver E, et al. Single-cell analysis of the developing human testis reveals somatic niche cell specification and fetal germline stem cell establishment. Cell Stem Cell. 2021;28(4).(2021)12.004.: 764-778.
    10.1016/j.stem.2020.12.004
    Article|CAS|PubMed|Google Scholar
  11. 11.De Reviers M, Hochereau-De Reviers MT, Blanc MR, Brillard JP, Courot MP. Control of Sertoli and germ cell populations in the cock and sheep testes. Reprod Nutr Dev. 1980;20(1b).(1980)241–9. https://doi. org/10.1051/rnd:.: 241.
    10.1051/rnd:19800206
    Article|CAS|PubMed|Google Scholar
  12. 12.Yan RG, Li BY, Yang QE. Function and transcriptomic dynamics of Sertoli cells during prospermatogonia development in mouse testis. Reprod Biol. 2020;20(4).(2020)525–35. https://doi. org/10. 1016/j.repbio.: 525.
    10.1016/j.repbio.2020.09.002
    Article|CAS|PubMed|Google Scholar
  13. 13.Griswold MD. The central role of Sertoli cells in spermatogenesis. Semin Cell Dev Biol. 1998;9(4).(1998)411–6. https://doi. org/10.1006/scdb.: 411.
    10.1006/scdb.1998.0203
    Article|CAS|PubMed|Google Scholar
  14. 14.Archambeault DR, Yao HH. Activin A, a product of fetal Leydig cells, is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion. Proc Natl Acad Sci U S A. 2010;107(23).(2010)1073/pnas.1000318107.: 10526.
    10.1073/pnas.1000318107
    Article|CAS|PubMed|Google Scholar
  15. 15.Jin S, Guerrero-Juarez CF, Zhang L, Chang I, Ramos R, Kuan CH, et al. Inference and analysis of cell-cell communication using cellchat. Nat Commun. 2021;12.(2021)org/10.1038/s41467-021-21246-9.: 1088.
    10.1038/s41467-021-21246-9
    Article|CAS|PubMed|Google Scholar
  16. 16.Murat F, Mbengue N, Winge SB, Trefzer T, Leushkin E, Sepp M, et al. The molecular evolution of spermatogenesis across mammals. Nature. 2023;613(7943).(2023)org/10.1038/s41586-022-05547-7.: 308.
    10.1038/s41586-022-05547-7
    Article|CAS|PubMed|Google Scholar
  17. 17.Dong Y, Zhao J, Zhu Q, Liu H, Wang J, Lu WF. Melatonin inhibits the apoptosis of rooster Leydig cells by suppressing oxidative stress via AKT-Nrf2 pathway activation. Free Radic Biol Med. 2020;160.(2020)1–12. https://doi. org/10. 1016/j.freeradbiomed.: 1.
    10.1016/j.freeradbiomed.2020.06.024
    Article|CAS|PubMed|Google Scholar
  18. 18.Xu K, Wang J, Liu H, Zhao J, Lu W. Melatonin promotes the proliferation of chicken Sertoli cells by activating the erk/inhibin alpha subunit signaling pathway. Molecules. 2020;25(5).(2020)org/10.3390/molecules25051230.: 1230.
    Article|CAS|PubMed|Google Scholar
  19. 19.Guo J, Grow EJ, Mlcochova H, Maher GJ, Lindskog C, Nie X, et al. The adult human testis transcriptional cell atlas. Cell Res. 2018;28(12).(2018)org/10.1038/s41422-018-0099-2.: 1141.
    10.1038/s41422-018-0099-2
    Article|CAS|PubMed|Google Scholar
  20. 20.Danshina PV, Geyer CB, Dai Q, Goulding EH, Willis WD, Kitto GB, et al. Phosphoglycerate kinase 2 (PGK2) is essential for sperm function and male fertility in mice. Biol Reprod. 2010;82(1).(2010)109.079699.: 136.
    10.1095/biolreprod.109.079699
    Article|CAS|PubMed|Google Scholar
  21. 21.Zhao L, Yao C, Xing X, Jing T, Li P, Zhu Z, et al. Single-cell analysis of developing and azoospermia human testicles reveals central role of Sertoli cells. Nat Commun. 2020;11.(2020)org/10.1038/s41467-020-19414-4.: 5683.
    10.1038/s41467-020-19414-4
    Article|CAS|PubMed|Google Scholar
  22. 22.Guibert E, Brière S, Pelletier R, Brillard JP, Froment P. Characterization of chicken Sertoli cells in vitro. Poult Sci. 2011;90(6).(2011)3382/ps.2010-01081.: 1276.
    10.3382/ps.2010-01081
    Article|CAS|PubMed|Google Scholar
  23. 23.Kumar L, Solanki S, Jain A, Botts M, Gupta R, Rajput S, et al. MAPKs signaling is obligatory for male reproductive function in a development-specific manner. Front Reprod Health. 2024;6.(2024)1330161. https://doi. org/10.3389/frph.: 1330161.
    10.3389/frph.2024.1330161
    Article|CAS|PubMed|Google Scholar
  24. 24.Voigt AL, Dardari R, Lara NLM, He T, Steele H, Dufour A, et al. Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche. Mol Hum Reprod. 2023;29(3):gaad004.2023.https://doi.org/10.1093/molehr/gaad004.
    Article|CAS|PubMed|Google Scholar
  25. 25.Iliadou PK, Tsametis C, Kaprara A, Papadimas I, Goulis DG. The Sertoli cell.(2015)novel clinical potentiality.Hormones (Athens).: 504.
    10.14310/horm.2002.1648
    Article|CAS|PubMed|Google Scholar
  26. 26.Guo J, Nie X, Giebler M, Mlcochova H, Wang Y, Grow EJ, et al. The dynamic transcriptional cell atlas of testis development during human puberty. Cell Stem Cell. 2020;26(2).(2020)12.005.: 262-276.
    10.1016/j.stem.2019.12.005
    Article|CAS|PubMed|Google Scholar
  27. 27.Wu J, Xu W, Zhang D, Dai J, Cao Y, Xie Y, et al. Nicotine inhibits murine Leydig cell differentiation and maturation via regulating Hedgehog signal pathway. Biochem Biophys Res Commun. 2019;510(1).(2019)11.107.: 1.
    10.1016/j.bbrc.2018.11.107
    Article|CAS|PubMed|Google Scholar
  28. 28.Shami AN, Zheng X, Munyoki SK, Ma Q, Manske GL, Green CD, et al. Single-cell RNA sequencing of human, macaque, and mouse testes uncovers conserved and divergent features of mammalian spermatogenesis. Dev Cell. 2020;54(4).(2020)529-547. e12. https://doi. org/10. 1016/j.devcel.: 529-547.
    10.1016/j.devcel.2020.05.010
    Article|CAS|PubMed|Google Scholar
  29. 29.Balzer MS, Ma Z, Zhou J, Abedini ASusztak. How to get started with single cell RNA sequencing data analysis. J Am Soc Nephrol. 2021;32(6).(2021)1681/asn.2020121742.: 1279.
    10.1681/asn.2020121742
    Article|CAS|PubMed|Google Scholar
  30. 30.Guo H, Liu J, Xu M, Zhu T, Liu S, Liu P, et al. Single-cell rna sequencing uncovers dynamic roadmap during chicken spermatogenesis. BMC Genomics. 2025;26.(2025)org/10.1186/s12864-025-11944-9.: 746.
    10.1186/s12864-025-11944-9
    Article|CAS|PubMed|Google Scholar
  31. 31.Estermann MA, Williams S, Hirst CE, Roly ZY, Serralbo O, Adhikari D, et al. Insights into gonadal sex differentiation provided by single-cell transcriptomics in the chicken embryo. Cell Rep. 2020;31(1).(2020)107491. https://doi. org/10. 1016/j.celrep.: 107491.
    10.1016/j.celrep.2020.03.055
    Article|CAS|PubMed|Google Scholar
  32. 32.Wang X, Wang Y, Wang Y, Guo Y, Zong R, Hu S, et al. Single-cell transcriptomic and cross-species comparison analyses reveal distinct molecular changes of porcine testes during puberty. Commun Biol. 2024;7(1).(2024)org/10.1038/s42003-024-07163-9.: 1478.
    10.1038/s42003-024-07163-9
    Article|CAS|PubMed|Google Scholar
  33. 33.Zhu BC, Chiocchio SR, Suburo AM, Tramezzani JH. Monoaminergic and peptidergic contributions of the superior and the inferior spermatic nerves to the innervation of the testis in the rat. J Androl. 1995;16(3).(1995)248–58.: 248.
    10.1002/j.1939-4640.1995.tb00521.x
    Article|CAS|PubMed|Google Scholar
  34. 34.Zhu C, Palmada MN, Aguado LI, Cavicchia JC. Administration of acetylcholine to the spermatic nerve plexus inhibits testosterone secretion in an in vitro isolated rat testis-nerve plexus system. Int J Androl. 2002;25(3).(2002)134–8. https://doi. org/10. 1046/j.1365-2605.: 134.
    10.1046/j.1365-2605.2002.00337.x
    Article|CAS|PubMed|Google Scholar
  35. 35.Chiocchio SR, Suburo AM, Vladucic E, Zhu BC, Charreau E, Décima EE, et al. Differential effects of superior and inferior spermatic nerves on testosterone secretion and spermatic blood flow in cats. Endocrinology. 1999;140(3).(1999)3.6569.: 1036.
    10.1210/endo.140.3.6569
    Article|CAS|PubMed|Google Scholar
  36. 36.Karvas RM, Mcinturf S, Zhou J, Ezashi T, Schust DJ, Roberts RM, et al. Use of a human embryonic stem cell model to discover GABRP, WFDC2, VTCN1 and ACTC1 as markers of early first trimester human trophoblast. Mol Hum Reprod. 2020;26(6).(2020)org/10.1093/molehr/gaaa029.: 425.
    10.1093/molehr/gaaa029
    Article|CAS|PubMed|Google Scholar
  37. 37.Yilmaz N, Yildiz A. Intracerebroventricular PROK2 infusion could increase the secretion of male reproductive hormones by stimulating the HPG axis. Mol Biol Rep. 2024;51.(2024)org/10.1007/s11033-024-09604-4.: 656.
    10.1007/s11033-024-09604-4
    Article|CAS|PubMed|Google Scholar
  38. 38.Morais R, Crespo D, Nóbrega RH, Lemos MS, Van De Kant HJG, De França LR, et al. Antagonistic regulation of spermatogonial differentiation in zebrafish (Danio rerio) by Igf3 and Amh. Mol Cell Endocrinol. 2017;454.(2017)112–24. https://doi. org/10. 1016/j.mce.: 112.
    10.1016/j.mce.2017.06.017
    Article|CAS|PubMed|Google Scholar
  39. 39.Blomberg Jensen M, Nielsen JE, Jørgensen A, Rajpert-De Meyts E, Kristensen DM, Jørgensen N, et al. Vitamin D receptor and vitamin D metabolizing enzymes are expressed in the human male reproductive tract. Hum Reprod. 2010;25(5).(2010)org/10.1093/humrep/deq024.: 1303.
    10.1093/humrep/deq024
    Article|CAS|PubMed|Google Scholar
  40. 40.Chen W, Zhang Z, Chang C, Yang Z, Wang P, Fu H, et al. A bioenergetic shift is required for spermatogonial differentiation. Cell Discov. 2020;6.(2020)org/10.1038/s41421-020-0183-x.: 56.
    10.1038/s41421-020-0183-x
    Article|CAS|PubMed|Google Scholar
  41. 41.Yang D, Zhang M, Chen W, Lu Q, Wan S, Du X, et al. Uchl1 maintains microenvironmental homeostasis in goat germline stem cells. FASEB J. 2023;37(12).(2023)e23306. https://doi. org/10.1096/fj.
    10.1096/fj.202301674RR
    Article|CAS|PubMed|Google Scholar
  42. 42.Hofmann MC, McBeath E. Sertoli cell-germ cell interactions within the niche.(2022)paracrine and juxtacrine molecular communications.Front Endocrinol (Lausanne).: 897062.
    10.3389/fendo.2022.897062
    Article|CAS|PubMed|Google Scholar
  43. 43.Sharpe RM, Mckinnell C, Kivlin C, Fisher JS. Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. Reproduction. 2003;125(6).(2003)0.1250769.: 769.
    10.1530/rep.0.1250769
    Article|CAS|PubMed|Google Scholar
  44. 44.You X, Chen Q, Yuan D, Zhang C, Zhao H. Common markers of testicular Sertoli cells. Expert Rev Mol Diagn. 2021;21(6).(2021)613–26. https://doi. org/10.1080/14737159.: 613.
    10.1080/14737159.2021.1924060
    Article|CAS|PubMed|Google Scholar
  45. 45.Yokonishi T, Capel B. Differentiation of fetal Sertoli cells in the adult testis. Reproduction. 2021;162(2).(2021)org/10.1530/rep-21-0106.: 141.
    10.1530/rep-21-0106
    Article|CAS|PubMed|Google Scholar
  46. 46.Beau C, Vivian N, Münsterberg A, Dresser DW, Lovell-Badge RGD. In vivo analysis of the regulation of the anti-müllerian hormone, as a marker of Sertoli cell differentiation during testicular development, reveals a multi-step process. Mol Reprod Dev. 2001;59(3).(2001)1002/mrd.1030.: 256.
    10.1002/mrd.1030
    Article|CAS|PubMed|Google Scholar
  47. 47.Migrenne S, Moreau E, Pakarinen P, Dierich A, Merlet J, Habert R, et al. Mouse testis development and function are differently regulated by follicle-stimulating hormone receptors signaling during fetal and prepubertal life. PLoS ONE. 2012;7(12).(2012)pone.0053257.
    10.1371/journal.pone.0053257
    Article|CAS|PubMed|Google Scholar
  48. 48.Rebourcet D, O’shaughnessy PJ, Monteiro A, Milne L, Cruickshanks L, Jeffrey N, et al. Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis. PLoS ONE. 2014;9(8).(2014)pone.0105687.
    10.1371/journal.pone.0105687
    Article|CAS|PubMed|Google Scholar
  49. 49.Hwang JH, Arafeh R, Seo JH, Baca SC, Ludwig M, Arnoff TE, et al. CREB5 reprograms FOXA1 nuclear interactions to promote resistance to androgen receptor-targeting therapies. Elife. 2022;11.(2022)7554/eLife.73223.
    10.7554/eLife.73223
    Article|CAS|PubMed|Google Scholar
  50. 50.Zhang CH, Gao Y, Hung HH, Zhuo Z, Grodzinsky AJ, Lassar AB. CREB5 coordinates synovial joint formation with the genesis of articular cartilage. Nat Commun. 2022;13.(2022)org/10.1038/s41467-022-35010-0.: 7295.
    10.1038/s41467-022-35010-0
    Article|CAS|PubMed|Google Scholar
  51. 51.Zirkin BR, Papadopoulos V. Leydig cells.(2018)formation, function, and regulation.Biol Reprod.: 101.
    10.1093/biolre/ioy059
    Article|CAS|PubMed|Google Scholar
  52. 52.Geng AL, Zhang Y, Zhang J, Wang HH, Chu Q, Yan ZX, et al. Effects of light regime on circadian rhythmic behavior and reproductive parameters in native laying hens. Poult Sci. 2022;101(5).(2022)101808. https://doi. org/10. 1016/j.psj.: 101808.
    10.1016/j.psj.2022.101808
    Article|CAS|PubMed|Google Scholar
  53. 53.Guo Y, Shen O, Han J, Duan H, Yang S, Zhu Z, et al. Circadian rhythm genes mediate fenvalerate-induced inhibition of testosterone synthesis in mouse Leydig cells. J Toxicol Environ Health A. 2017;80(23–24).(2017)1314–20. https://doi. org/10.1080/15287394.: 1314.
    10.1080/15287394.2017.1384148
    Article|CAS|PubMed|Google Scholar
  54. 54.Lateef OM, Akintubosun MO. Sleep and reproductive health. J Circadian Rhythms. 2020;18.(2020)5334/jcr.190.: 1.
    10.5334/jcr.190
    Article|CAS|PubMed|Google Scholar
  55. 55.Qin F, Cao H, Yuan H, Guo W, Pei H, Cao Y, et al. 1800 MHz radiofrequency fields inhibits testosterone production via CaMKI /RORα pathway. Reprod Toxicol. 2018;81.(2018)229–36. https://doi. org/10. 1016/j.reprotox.: 229.
    10.1016/j.reprotox.2018.08.014
    Article|CAS|PubMed|Google Scholar
  56. 56.Yang M, Guan S, Tao J, Zhu K, Lv D, Wang J, et al. Melatonin promotes male reproductive performance and increases testosterone synthesis in mammalian Leydig cells. Biol Reprod. 2021;104(6).(2021)org/10.1093/biolre/ioab046.: 1322.
    10.1093/biolre/ioab046
    Article|CAS|PubMed|Google Scholar
  57. 57.Peng LU, Bai GY. Roles of NPAS2 in circadian rhythm and disease. Acta Biochim Biophys Sin. 2021;53(10).(2021)org/10.1093/abbs/gmab105.: 1257.
    10.1093/abbs/gmab105
    Article|CAS|PubMed|Google Scholar
  58. 58.Yuan P, Li J, Zhou F, Huang Q, Zhang J, Guo X, et al. NPAS2 promotes cell survival of hepatocellular carcinoma by transactivating CDC25A. Cell Death Dis. 2017;8(3).(2017)e2704. https://doi. org/10.1038/cddis.
    10.1038/cddis.2017.131
    Article|CAS|PubMed|Google Scholar
  59. 59.Li J, Zhang X, Wang X, Wang Z, Li X, Zheng J, et al. Single-nucleus transcriptional and chromatin accessible profiles reveal critical cell types and molecular architecture underlying chicken sex determination. J Adv Res. 2024;70.(2024)29–43. https://doi. org/10. 1016/j.jare.: 29.
    Article|CAS|PubMed|Google Scholar
  60. 60.Cai C, Wan P, Wang H, Cai X, Wang J, Chai Z, et al. Transcriptional and open chromatin analysis of bovine skeletal muscle development by single-cell sequencing. Cell Prolif. 2023;56(9).(2023)1111/cpr.13430.
    10.1111/cpr.13430
    Article|CAS|PubMed|Google Scholar
  61. 61.Valiulyte I, Steponaitis G, Kardonaite D, Tamasauskas A, Kazlauskas A. A SEMA3 signaling pathway-based multi-biomarker for prediction of glioma patient survival. Int J Mol Sci. 2020.https.(2020)org/10.3390/ijms21197396.
    10.3390/ijms21197396
    Article|CAS|PubMed|Google Scholar
  62. 62.Pui HP, Saga Y. Gonocytes-to-spermatogonia transition initiates prior to birth in murine testes and it requires FGF signaling. Mech Dev. 2017;144(Pt B).(2017)125–39. https://doi. org/10. 1016/j.mod.: 125.
    10.1016/j.mod.2017.03.002
    Article|CAS|PubMed|Google Scholar
  63. 63.Xu YR, Dong HS, Yang WX. Regulators in the apoptotic pathway during spermatogenesis: killers or guards? Gene. 2016;582(2):97–111.https://doi.org/10.1016/j.gene.2016.02.007.
    Article|CAS|PubMed|Google Scholar
  64. 64.Nicholls PK, Stanton PG, Chen JL, Olcorn JS, Haverfield JT, Qian H, et al. Activin signaling regulates Sertoli cell differentiation and function. Endocrinology. 2012;153(12).(2012)6065–77. https://doi. org/10.1210/en.: 6065.
    10.1210/en.2012-1821
    Article|CAS|PubMed|Google Scholar

Acknowledgements

We gratefully acknowledge Professor Qien Yang from the Northwest Institute of Plateau Biology, Chinese Academy of Sciences and Professor Qiang Qiu from Northwestern Polytechnical University for their assistance.

Funding

This work was supported by “National Key Research and Development Program of China” (Grant No. 2021YFF1000701) and “National Natural Science Foundation of China” (Grant No. U22A20509).

Ethics Declaration

Ethics approval and consent to participate

The experiments were approved by the experimental animal welfare and ethics committee of Jilin Agricultural University (approval No. 20210422 002) and performed in accordance with the protocol outlined in the “Guide for Care and Use of Laboratory Animals” (Jilin Agricultural University).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Rights and Permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ . The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Reprints and permissions