Comparison of Sertoli cells in duck testes pre- and post-sexual maturation

Abstract

To investigate the role of Sertoli cells in duck testicular development and maturation, we conducted single-cell RNA sequencing on testes from immature (IMT) and mature (MT) ducks. Sertoli cells accounted for 27.75 % and 33.42 % of testicular cells in IMT and MT, respectively. We identified 2,630 differentially expressed genes (DEGs) in mature Sertoli cells, with 1,540 upregulated and 1,090 downregulated. Subtype analysis revealed 2,498 DEGs in Sertoli cell type 1 (SC1) (898 up, 1,600 down) and 2,515 DEGs in SC2 (1,291 up, 1,224 down), including 1,090 SC1-specific, 1,107 SC2-specific, and 1,408 shared genes. Gene Ontology analysis showed DEGs were mainly linked to cellular components: SC1 DEGs were enriched in intracellular structures (e.g., nucleus), whereas SC2 DEGs were enriched in membrane- and extracellular-related components (e.g., cell periphery). KEGG pathway analysis highlighted significant enrichment of "Spliceosome," "Neurotrophin signaling," and "MAPK signaling" pathways in MT and IMT, with Neurotrophin signaling predominant in SC1 and MAPK signaling in SC2. Furthermore, the Notch signaling pathway was significantly enriched for SC1-specific genes, whereas the MAPK signaling pathway was significantly enriched for SC2-specific genes. Protein–protein interaction analysis of these pathways identified core genes, including SF3A2, SNRPF, SNRPB2, SNRPG, SF3B4, MAP3K1, NRAS, MET, and EGF. This study delineates developmental dynamics of duck Sertoli cells and offers preliminary insights into regulatory mechanisms underlying testicular maturation, providing a key theoretical and data-driven foundation for understanding Sertoli cell functions in avian testis development.

Introduction

Spermatogenesis, the process of sperm production, occurs in the seminiferous tubules of the testes. This process begins with spermatogonial stem cells and progresses through multiple stages of cell differentiation, ultimately producing mature sperm (Higuchi et al., 2021). During development, germ cells maintain close contact with Sertoli cells, which support them and are vital for their maturation (O'Donnell et al., 2022). The number and functional capacity of Sertoli cells directly influence testis size and the ability to sustain germ cell development (Ruthig et al., 2022; Johnsonet al., 2008). Additionally, Sertoli cells supply essential nutritional support by secreting key metabolites, such as amino acids, sugars, lipids, vitamins, and metal ions, to meet the metabolic demands of developing germ cells (Erkkilä et al., 2002).

Single-cell RNA sequencing has revealed numerous Sertoli cell regulatory genes essential for spermatogenesis, offering improved mechanistic insights. For instance, BMP6 promotes Sertoli cell proliferation and suppresses apoptosis by activating DACH1, TFAP2A, and the Smad2/3 pathway (Wang et al., 2017). Similarly, BMP4 stimulates proliferation through the Smad1/5 pathway and upregulation of ID2/3 (Hai et al., 2015). Conversely, GLI3 negatively regulates human Sertoli cell growth (Yao et al., 2016). Genetic evidence further confirms this. Mdm2 knockout in rodent Sertoli cells causes severe apoptosis and infertility (Fouchécourt et al., 2016), and dual deletion of Insr and Igf1r in mice reduces testis weight and sperm output by 75 % (Pitetti et al., 2013). Collectively, these data establish that Sertoli cell-derived factors are vital for normal spermatogenesis across species, and their dysfunction leads directly to male infertility (Du et al., 2021).

Similar to their mammalian counterparts, avian Sertoli cells form intimate associations with germ cells during post-embryonic development, creating the specialized microenvironment required for spermatogenesis in poultry (Kirby, 1999). In chickens, mature Sertoli and germ cells can be isolated and cultured for functional studies (Guibert et al., 2011), and several Sertoli cell-derived genetic regulators of testis development have been identified. For instance, the Sertoli cell marker FOXD1 is a pivotal regulator that orchestrates both embryonic testicular differentiation and subsequent functional maturation (Yu et al., 2019). Conversely, FOXL2 overexpression in male chicken gonads antagonizes testis development by transcriptionally repressing male pathway genes (e.g., SOX9, DMRT1, and AMH) and disrupting Sertoli cell ontogeny (Major et al., 2019).

However, the role of Sertoli cells in duck testicular development remains poorly characterized. Our preliminary studies on Jinding ducks identified distinct developmental stages: testes were immature with no observable spermatogenesis at 10 weeks post-hatching, but reached full maturity with active sperm production by 23 weeks. Using single-cell RNA sequencing, we profiled the cellular heterogeneity of duck testes at these two stages and identified Sertoli cells based on established marker genes (Tao et al., 2025). This study focuses on comparing the Sertoli cells between the two stages to establish their regulatory roles in testicular maturation and the initiation of spermatogenesis.

Materials and methods

Experimental animals and sample collection

All Jinding ducks were raised under identical rearing conditions with standardized management practices. The feed used was commercially available duck feed, including starter feed and grower feeds, purchased from New Hope Feed Group Co., Ltd. in Jiangsu, China. For ducklings, 22 hours of light is provided within the first week after hatching, and then the light duration is gradually reduced and transitioned to natural light. Testes were collected from three biologically distinct males (n = 3 per group, serving as biological replicates) at two developmental stage:10 weeks (IMT group) and 23 weeks (MT group). Body weight and total testis weight (combined weight of both testes) were reported by our research team (Tao et al., 2025), with body weights of 1207.222±38.864 and 1451.778±106.389, and total testis weight of 1.471±1.427 and 34.60±32.26, respectively. The One testis from each animal was used for single-cell RNA sequencing.

Preparation of testicular tissue single-cell samples

Testes from six ducks (three per group: IMT and MT) were processed for single-cell suspension. Briefly, the testes were rinsed with pre-cooled DMEM (Dulbecco’s Modified Eagle Medium), trimmed of excess tissue, and minced with ophthalmology scissors. The tissue fragments were collected in a 50 mL tube, centrifuged at 300 × g for 1 min, and the supernatant was discarded. The pellet was then subjected to enzymatic digestion in 10 mL of DMEM containing 1 % collagenase, 0.6 % DNase, and 0.5 % hyaluronidase for 15 min in a 37°C water bath. The digestion was terminated by adding pre-cooled DMEM and incubating for 2 min. The cell suspension was filtered through a 70 µm sieve and centrifuged at 500 × g for 5 min at 4°C. After supernatant removal, the pellet was resuspended in DMEM and treated with red blood cell lysis buffer. Following another centrifugation (400 × g, 5 min), the pellet was resuspended in 100–500 µL of pre-cooled DMEM. Cell viability was confirmed at ≥90 % by 0.4 % trypan blue exclusion using a Countess® II Automated Cell Counter (Thermo Scientific^TM, USA). Finally, the live cell concentration was adjusted to 1000–2000 cells/µL for subsequent sequencing.

10 × Genomics single-cell library preparation

Testicular cells from the IMT and MT groups were processed on the 10 × Genomics Chromium NextGEM Single Cell 3′ HT Kit v3.1) (10X GENOMICS, PN- 1000348) to generate barcoded libraries. Single-cell GEMs (Gel Bead in Emulsion) were formed on a Chromium Chip, where cell lysates were mixed with Master Mix and Gel Beads carrying oligonucleotides for barcoding and cDNA synthesis. The released primers, containing Illumina R1, a 16 nt barcode, a 10 nt UMI (Unique Molecular Identifier) , and a poly-dT tail, enabled reverse transcription of mRNA into barcoded, full-length cDNA. Following reagent cleanup with silane magnetic beads and cDNA amplification, final libraries were prepared with the Chromium Next GEM Single Cell 3′ Kit v3.1 and sequenced on an Illumina NovaSeq 6000 (150 bp paired-end) at Gene Denovo Biotechnology (Guangzhou, China).

Analysis of raw single-cell RNA-seq data

The Cell Ranger pipeline (v3.1.0) was used for primary data analysis. After filtering low-quality barcodes and UMIs, reads were aligned to the reference genome. Only exon-mapping, uniquely aligned reads were used for UMI counting, which included error correction by Cell Ranger. This process generated a cell-by-gene matrix for each sample, which was then imported into Seurat (v3.1.1) for subsequent analysis.

Differentially expressed gene analysis

Differential gene expression between the MT and IMT groups was assessed using the Wilcoxon rank-sum test (Camp et al., 2017). Genes with a ≥ 2-fold increase in expression in the MT group and an adjusted p-value < 0.01 were defined as significantly upregulated.

Gene Ontology and KEGG enrichment analyses

Gene Ontology (GO) enrichment analysis was performed on DEGs. Significantly enriched GO terms were identified using a false discovery rate (FDR) correction. An FDR≤0.01 was set as the significance threshold.

Pathway enrichment analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.kegg.jp/) (Griswold et al., 1998). An FDR-corrected p-value ≤ 0.01 was used to determine significantly enriched KEGG pathways for the DEGs.

Protein–protein interaction network analysis

Protein–protein interaction (PPI) networks were constructed using the STRING online database (https://cn.string-db.org/) and visualized with Cytoscape software.

Relative expression levels of core genes by RT-PCRRT-PCR

qRT-PCR (quantitative reverse transcription polymerase chain reaction) was performed on nine core genes in testis to validate the PPI result with β-actin used as a reference gene. All primer sequences were designed using Premer Premier 5 software (Premier Biosoft, Palo Alto, CA) and are listed in Supplementary Table S1. The reactions were conducted in triplicate using SuperReal qPCR Premix (SYBR Green) master mix on an Agilent Stratagene Mx3000p qPCR system under the following amplification conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 25 s, 60 °C for 25 s, 75°C for 25 s, and 72 °C for 10 min.

Results

RNA sequences in duck testes

More than 30 M reads were obtained in each duck testis sample, with high-quality metrics supporting robust downstream analysis (Table 1). Specifically, valid barcodes accounted for >93 % of the data, and Q30 scores were consistently high (>91 % for barcodes, RNA reads, and UMIs). Over 83 % of reads were uniquely mapped to the reference genome, leading to the identification of approximately 21,063–21,483 genes per sample. Following standard cell filtering, we retained 6,567 to 12,446 high-quality cells per sample, with a median of over 1,000 genes detected per cell.

Table 1.

Overview of duck testicular RNA sequences.

Index	IMT-1	IMT-2	IMT-3	MT-1	MT-2	MT-3
Number of Reads	332505029	340882637	336445006	327578469	343434190	300961645
Valid Barcodes (%)	94.2	93.8	94	97.2	97.3	96.9
Reads mapped confidently to genome (%)	83.7	84.6	83.4	83	83.9	84.4
Before filter Number of Cells	7125	14059	9518	11258	9864	8471
After filter Number of Cells	6567	12446	8743	10109	9010	7827
total genes detected	21063	21483	21282	21399	21475	21308
Before filter median UMI counts per cell	1543	2992	3120	6913	8360	8473
After filter median UMI counts per cell	1434	2155	2567	6750	7992	8224
Before filter median genes per cell	1081	1737	1888	2735	3034	2892
After filter median genes per cell	1026	1379	1647	2660	2957	2797

Variation in duck Sertoli cell counts at different developmental stages

Quality filtering yielded a final dataset of 54,702 testicular single cells, with 27,756 from IMT and 26,946 from MT ducks. Subsequent clustering analysis identified a total of 16,754 Sertoli cells, of which 9,277 were from the IMT group and 7,477 from the MT group. This corresponded to a significantly larger Sertoli cell fraction in the IMT group (33.21±0.65) than in the MT group (27.86±1.73), P value is 0.045 (Table 2).

Table 2.

The difference in the ratio of Sertoli cell numbers between IMT and MT.

Group	sample	Testis cells	Sertoli cells	Ratio of Sertoli cells (%)	The mean of the ratio of Sertoli cells (%)	P value
IMT	IMT1	6567	2099	31.96	33.21±0.65	0.045
	IMT2	12446	4253	34.17
	IMT3	8734	2925	33.49
MT	MT1	10109	2517	24.90	27.86±1.73
	MT2	9010	2784	30.90
	MT3	7827	2176	27.80

Analysis of DEGs in duck Sertoli cells at different developmental stages

A comparative analysis revealed 2,630 DEGs between the MT and IMT groups, comprising 1,090 upregulated and 1,540 downregulated genes (Fig. 1A, Supplemental Table 2). A heatmap displays the expression patterns of the top 20 most significant DEGs. Among these, 19 genes, including SULT6B1, TSSK6, FAM166C, Armc12, SPATC1, Tex33, ODF1, ncbi_113845535, CBY2, ncbi_106019812, SMRP1, ENTR1, KIF2A, C19orf71, ncbi_119715751, TENT5C, C1orf158, and Klhl10, ncbi_101794437 were upregulated. Only one gene, LAMA1, was downregulated (Fig. 1B). The details of these 20 genes are provided in Table 3.

Fig. 1.

Analysis of DEGs in testes of immature (IMT) and mature (MT) ducks.

A. Volcano plot illustrating DEGs between the IMT and MT groups. A total of 1,090 genes were downregulated and 1,540 were upregulated.

B. Heatmap displaying the top 20 DEGs between the IMT and MT groups. Among these, 19 genes were upregulated: LAMA1, SULT6B1, TSSK6, FAM166C, Armc12, SPATC1, Tex33, ODF1, ncbi_113845535, CBY2, ncbi_106019812, SMRP1, ENTR1, KIF2A, C19orf71, ncbi_119715751, TENT5C, C1orf158, and Klhl10. Only one gene, LAMA1, was downregulated.

C. GO analysis of DEGs in duck testes. The bar chart displays the top 20 significantly enriched GO terms for the MT group compared to the IMT group, ranked by false discovery rate (FDR)-corrected p-values (FDR < 0.01).

D. KEGG analysis of DEGs in duck testes. The plot displays the top 10 significantly enriched KEGG pathways in the MT group compared to the IMT group, ranked by FDR-corrected p-values (FDR < 0.01).

Table 3.

List of the 20 most significant DEGs in duck testes.

Gene ID	Gene Name	log2FC	Description	function
ncbi_101791570	LAMA1	−10.81	laminin subunit alpha-1	Binding to cells via a high affinity receptor, laminin is thought to mediate the attachment, migration and organization of cells into tissues during embryonic development by interacting with other extracellular matrix components.
ncbi_101790520	SULT6B1	10.70	Sulfotransferase 6B1, partial	Predicted to enable sulfotransferase activity. Predicted to be involved in sulfation. Predicted to be located in cytosol. Predicted to be active in cytoplasm.
ncbi_113844195	TSSK6	10.63	testis-specific serine/threonine-protein kinase 6	Required for sperm production and function.
ncbi_101789599	FAM166C	10.51	UPF0573 protein C2orf70 homolog	Microtubule inner protein (MIP) part of the dynein-decorated doublet microtubules (DMTs) in cilia axoneme, which is required for motile cilia beating.
ncbi_113839899	Armc12	10.38	armadillo repeat-containing protein 12 isoform X2	Essential for male fertility and sperm mitochondrial sheath formation.
ncbi_119716153	SPATC1	10.35	Spermatogenesis and centriole associated 1	Predicted to enable gamma-tubulin binding activity.
ncbi_101799968	Tex33	10.28	testis-expressed protein 33	Seems to be associated with spermiogenesis but is not essential for sperm development and male fertility.
ncbi_101799903	ODF1	10.22	outer dense fiber protein 1	maintaining the elastic structure and recoil of the sperm tail as well as in protecting the tail from shear forces during epididymal transport and ejaculation.
ncbi_113845535	ncbi_113845535	10.15	-	-
ncbi_101800199	CBY2	10.10	spermatid-associated protein	Enables identical protein binding activity. Predicted to be located in cytoplasmic vesicle.
ncbi_106019812	ncbi_106019812	9.99	-	-
ncbi_106017369	SMRP1	9.75	spermatid-specific manchette-related protein 1	may play a role in ciliogenesis.
ncbi_101797228	ENTR1	9.57	endosome-associated-trafficking regulator 1	Involved in several processes, including endocytic recycling; positive regulation of cilium assembly.
ncbi_101789767	KIF2A	9.52	kinesin-like protein KIF2B	The protein encoded by this gene is a plus end-directed motor required for normal mitotic progression. The encoded protein is required for normal spindle activity during mitosis and is necessary for normal brain development.
ncbi_113845591	C19orf71	9.46	uncharacterized protein C19orf71-like	Microtubule inner protein (MIP) part of the dynein-decorated doublet microtubules (DMTs) in cilia axoneme, which is required for motile cilia beating. Located at the center of the tektin bundle where may function to recruit tektins or stabilize the bundle.
ncbi_119715751	ncbi_119715751	9.35	_
ncbi_101799906	TENT5C	9.29	terminal nucleotidyltransferase 5C	Enables RNA adenylyltransferase activity. Involved in mRNA stabilization.
ncbi_101795344	C1orf158	9.21	uncharacterized protein C1orf158 homolog	Predicted to be involved in flagellated sperm motility.
ncbi_101792980	Klhl10	9.14	kelch-like protein 10 isoform X2	Mutations in this gene have been associated with oligozoospermia in some infertile males.
ncbi_101794437	ncbi_101794437	9.09	_	_

GO analysis showed that the top 20 GO terms in Fig. 2C were dominated by cellular component (16 terms), with biological process and molecular function comprising the remaining 3 and 1 term(s), respectively. The top three terms were axoneme, ciliary plasm, and cell part (Fig. 1C).

Fig. 2.

Differential analysis of Sertoli cell subtypes in the testes of immature (IMT) and mature (MT) ducks.

A. Bar plot of DEGs among groups SC1, SC2, and SC3 between IMT and MT. In SC1 cells, 898 genes were upregulated and 1,600 genes were downregulated, while in SC2 cells, 1,291 genes were upregulated and 1,224 genes were downregulated between IMT and MT. No DEGs were detected in SC3 between IMT and MT (There was only one cell in the SC3 group of the IMT).

B. Venn Diagram of DEGs between Groups SC1 and SC2 of IMT and MT duck testes. A total of 1,090 genes were specifically expressed in SC1, 1,107 genes in SC2, and 1,408 genes were co-expressed in both groups.

C. Bubble plot of the top 20 enriched GO terms for the SC1 and SC2 cells of IMT and MT groups.

D. Bubble plot of the top 20 enriched KEGG for the SC1 and SC2 cells of IMT and MT groups.

E. Bubble plot of the top 10 significantly enriched GO terms for SC1-specific, SC2-specific, and shared DEGs.

F. Bubble plot of the top 10 significant KEGG pathways for SC1-specific, SC2-specific, and shared DEGs.

KEGG pathway enrichment analysis identified the "Spliceosome" as the most significantly altered pathway, with the Neurotrophin and mitogen-activated protein kinase (MAPK) signaling pathways also among the most significant (Fig. 1D).

Differential analysis of Sertoli cell subtypes at different developmental stages

Supporting cells are divided into three subtypes, among which SC1 cells are the most abundant, SC2 cells are less abundant, and SC3 cells are the least (Table 4). Analysis of DEGs between IMT and MT duck testes revealed distinct expression patterns. In Sertoli cell type1 (SC1), 898 genes were upregulated and 1,600 were downregulated, whereas in SC2 cells, 1,291 genes were upregulated and 1,224 were downregulated (Fig. 2A, Supplemental Tables 3 and 4). No DEGs were detected in SC3, attributable to the presence of only one cell in the IMT group. Comparative analysis identified 1,090 SC1-specific genes, 1,107 SC2-specific genes, and 1,408 genes co-expressed in SC1 and SC2 groups (Fig. 2B, Supplement Tables 5–7).

Table 4.

The number of the three Sertoli cell subtypes between IMT and MT.

Cluster	IMT	MT	total
SC1	4477 (48.26 %)	5647 (75.52 %)	10124
SC2	4799 (51.73 %)	1513 (20.24 %)	6312
SC3	1 (0.01 %)	317 (4.24 %)	318
total	9277	7477	16754

The top 20 enriched GO terms for SC1 and SC2 are presented in Fig. 2C. In SC1, 17 of these terms were classified as cellular components (e.g., intracellular, nuclear part, and organelle) and three were associated with biological processes (RNA metabolic process, nucleic acid metabolic process, and gene expression) (Fig. 2C1). In SC2, among the top 20 terms, 14 were cellular components (e.g., motile cilium, cell projection, and cell periphery), four were biological processes (e.g., cell projection organization, and movement of cell or subcellular component), and one term (protein binding) was categorized under molecular function (Fig. 2C2). KEGG pathway enrichment analysis revealed distinct profiles for SC1 and SC2 (Fig. 2D). In SC1, the top three significantly enriched pathways were the spliceosome, ribosome biogenesis in eukaryotes, and the Notch signaling pathway. The Neurotrophin signaling pathway was also detected in SC1 (Fig. 2D1). In SC2, the top 20 enriched terms included several notable pathways, such as axon guidance, gap junction, and the Relaxin signaling pathway. The MAPK signaling pathway was also detected in SC2 (Fig. 2D2).

GO and KEGG enrichment analyses were conducted on the SC1-specific, SC2-specific, and common (co-expressed) genes (Fig. 2E and F). GO analysis revealed that the most significantly enriched cellular component terms were the nucleus in SC1-specific genes (Fig. 2E1), the cell periphery in SC2-specific genes (Fig. 2E2), and the motile cilium in common genes (Fig. 2E3). Correspondingly, KEGG pathway analysis identified the Notch signaling pathway (Fig. 2F1), the MAPK signaling pathway (Fig. 2F2), and the spliceosome (Fig. 2F3) as significantly enriched in the SC1-specific, SC2-specific, and common gene sets, respectively.

Protein–protein interaction analysis of significantly enriched pathways

PPI analysis was performed on the top three KEGG pathways: Spliceosome, Neurotrophin signaling pathway, and MAPK signaling pathway. The analysis identified SF3A2, SNRPF, SNRB2, SNRPG, and SF3B4 as core genes within the Spliceosome network (Fig. 3A). Similarly, MAP3K1 and NRAS were core genes in the Neurotrophin signaling pathway (Fig. 3B), while MET and EGF were core genes in the MAPK signaling pathway (Fig. 3C). Comparative analysis of core genes in SC cells between MT and IMT groups revealed downregulated of SF3A2, SNRPF, SNRPB2, SNRPG, SF3B4, MAP3K1, and NRAS in MT, while MET and EGF showed upregulation compared to the IMT group (Fig. 3D), as detected by single-cell RNA sequencing. Subsequent RT-PCR validation in duck testicular tissues revealed distinct fold changes across target genes: SF3A2 (0.45), SNRPF (0.54), SNRPB2 (0.68), SNRPG (0.35), SF3B4 (0.68), MAPK3K1 (0.46), NRAS (0.59), MET (3.49) and EGF (2.34).

Fig. 3.

Protein–protein interaction (PPI) networks of the top three enriched KEGG pathways in Sertoli cells between immature (IMT) and mature (MT) testes.

(A) The core hub genes in the Spliceosome pathway are SF3A2, SNRPG, SF3B4, SNRPB2, and SNRPF.

(B) In the Neurotrophin signaling pathway, MAP3K1 and NRAS are the core hub genes.

(C) In the MAPK signaling pathway, MET and EGF are the core hub genes.

(D) The expression of core genes in SC cells of MT and IMT group.

(E) Relative expression level of core genes in testis of MT/IMT by RT-PCR.

Discussion

Here, we investigated the differences in cell populations and evolutionary relationships between the IMT and MT groups using single-cell RNA-seq technology (10 × Genomics). A total of 54,702 single cells were obtained from six duck testes (three from each of the IMT and MT groups). The high-quality transcriptomes, collectively expressing >21,000 genes per sample, provided strong statistical power for unbiased analyses of these cell populations.

Using a conservative statistical threshold, we identified DEGs between testicular cell populations, enabling functional categorization into distinct clusters. The proportion of Sertoli cells was lower in the MT group (27.75 %) than in the IMT group (33.42 %). This difference may be attributed to the cessation of Sertoli cell mitosis and the increase in germ cells undergoing meiosis and differentiation during puberty (Griswold, 1998).

We identified 2,630 DEGs, indicating significant differences in levels of gene expression between the groups. Among the top 20 DEGs, several genes are known to play important roles in spermiogenesis or sperm function. For example, LAMA1 was upregulated from pubertal to adult stages in Bactrian camel testes, suggesting a potential role in spermatogenesis (Hasi et al., 2023). TSSK6 is essential for regulating male fertility (Salicioni et al., 2020). ARMC12 deficiency causes abnormal mitochondrial coiling along the flagellum, leading to reduced sperm motility and male sterility (Shimada et al., 2021). SPATC1 is involved in sex determination and spermatogenesis (An et al., 2022). SPATC1L, a paralog of SPATC1, maintains the integrity of the sperm head-tail junction (Kim et al., 2018). TEX33 is associated with spermiogenesis but is not essential for sperm development or male fertility (Xia et al., 2021). ODF1, besides being involved in the correct arrangement of the mitochondrial sheath and outer dense fibers, is essential for the rigid junction between the sperm head and tail (Yang et al., 2012). CBY2 may be related to spermatogenesis, sperm flagellar structure, and energy metabolism (Xue et al., 2025). SMRP1 expression was specific to the testis and localized to the cytoplasm of elongated spermatids (Matsuoka et al., 2008). KIF2A upregulates the PI3K/AKT signaling pathway through PLK1 during spermatogenesis, thereby affecting the proliferation and apoptosis levels of spermatogenic cells (Zhao et al., 2024). The poly(A) polymerase activity of TENT5C may regulate the spatial translation of Odf1 mRNAs during spermiogenesis, a process critical for sperm morphogenesis and fertility (Baptissart et al., 2025). Klhl10 was associated with asthenozoospermic spermatozoa in humans (Liu et al., 2018). However, the functions of the following genes in sperm have not been reported: SULT6B1, FAM166C, ncbi_113845535, ncbi_106019812, ENTR1, C19orf71, ncbi_119715751, C1orf158, ncbi_101794437, and ZC2HC1B.

We identified a substantial number of DEGs in SC1 (2,498) and SC2 (2,515), including 1,090 SC1-specific, 1,107 SC2-specific, and 1,408 shared genes. This DEG profile offers a data foundation for comparing these two subtypes. Notably, DEG analysis was not feasible for SC3, as only one cell was found in the IMT group compared to 317 in the MT group. The general rarity of SC3 cells and their primary existence in mature testes further indicate that SC3 likely corresponds to the mature Sertoli cell type.

GO analysis revealed that cellular components underwent the most significant changes among the three major functional categories, indicating substantial alterations in the subcellular localization or structural composition of these DEGs between IMT and MT. GO analysis also revealed substantial differences between the two types of Sertoli cells. The DEGs in SC1 were primarily enriched in intracellular-related cellular components (e.g., intracellular cell, nucleus, etc.), whereas those in SC2 were mainly enriched in cell membrane and extracellular-related cellular components (e.g., motile cilium, cell projection, etc.). The nucleus is the most significant GO term of SC1-specific genes, whereas the cell periphery is the most significant GO term of SC2-specific genes.

KEGG analysis showed that the Spliceosome, Neurotrophin signaling pathway, and MAPK signaling pathway were the top three differentially expressed pathways, suggesting that these signaling pathways play important roles in promoting duck testicular development. Furthermore, the Notch signaling pathway was a significantly enriched pathway for SC1-specific genes, while the MAPK signaling pathway was a significantly enriched pathway for SC2-specific genes. The spliceosome pathway was identified as a significant pathway common to both cell types. A study in mice has shown that during early testis development, Notch signaling exhibits high activity and tends to maintain Sertoli cells in an immature, proliferative state. As development progresses, the timely downregulation of Notch signaling allows Sertoli cells to exit the cell cycle and enter functional maturation (Garcia and Hofmann, 2013; Garcia et al., 2013). Thus, Notch signaling may have an important role in pre-pubertal spermatogonia quiescence, onset of spermatogenesis, and regulation of the spermatogenic cycle (Murta et al., 2013). MAPK signaling critically regulates testis development by controlling spermatogenesis, along with the fate determination of pre-Sertoli and primordial germ cells during embryogenesis, postnatal testicular cell proliferation, and the functions of mature Sertoli and Leydig cells (Luo et al., 2022). The analysis revealed that the Notch signaling pathway was significantly enriched in duck SC1 genes, whereas the MAPK signaling pathway was significantly enriched in duck SC2 genes. This suggests that the Notch pathway may function to maintain SC1 in an immature, proliferative state, whereas the MAPK pathway may be involved in the functions of mature Sertoli cells (SC2). In short, the balance and crosstalk between these two pathways are crucial for maintaining the normal physiological state of Sertoli cells.

PPI analysis identified several hub genes: SF3A2, SNRPF, SNRB2, SNRPG, and SF3B4 in the spliceosome; MAP3K1 and NRAS in the Neurotrophin signaling pathway; and MET and EGF in the MAPK signaling pathway. These findings indicate that these genes play crucial roles in regulating testicular development in Sertoli cells.

Splicing factor 3a subunit 2 (SF3A2) is a component of the splicing factor SF3A complex that contributes to the assembly of the 17S U2 snRNP. SF3A2 was reported to play a direct role in mitotic chromosome segregation, functioning as a microtubule-binding and bundling protein (Pellacani et al., 2018; Takenaka et al., 2004). SNRPF, SNRPG, and SNRPB are members of the Sm protein ring, which forms part of the spliceosome (Ge et al., 2017). Splicing factor SF3b4, as a core subunit of the U2 spliceosome, not only plays a crucial role in the splicing process but also functions in transcription, translation, and cell signal transduction, participating in the regulation of cell cycle progression, cell differentiation, and immune responses (Yan et al., 2021). The identification of these genes in our study suggests that their interactions play an important role in regulating testicular development through the spliceosome pathway.

MAP3K1 is a key component of the MAPK signal transduction pathway (Warr et al., 2011) and has been associated with human testis determination (Pearlman et al., 2010). In male sheep, MAP3K1 serves as a marker of spermatogenesis and apoptosis (Guan et al., 2017). In duck Sertoli cells during development from 10 to 23 weeks, MAP3K1 functions as a hub gene in both the Neurotrophin signaling pathway and the MAPK signaling pathway. These findings suggest that MAP3K1 probably plays a significant role in duck testicular development and spermatogenesis through these two interconnected signaling pathways.

NRAS is another hub gene of the Neurotrophin signaling pathway. As an N-ras oncogene, NRAS encodes a membrane protein that shuttles between the Golgi apparatus and plasma membrane and has been associated with melanoma (Muñoz-Couselo et al., 2017). However, the function of NRAS in testis development remains unreported. In this study, we identified NRAS as a hub gene in the Neurotrophin signaling pathway, suggesting it may interact with MAP3K1 to regulate testicular development and spermatogenesis through this pathway.

EGF has long been recognized for its role in testis development and spermatogenesis (Yan et al., 1998). The MAPK signaling pathway plays crucial roles in testicular development by regulating spermatogenesis, the fate determination of pre-Sertoli and primordial germ cells during embryogenesis, the proliferation of testicular cells in the postnatal period, and the functions of mature Sertoli and Leydig cells (Luo et al., 2022). Poultry studies also have confirmed that the MAPK signaling pathway has a key role in regulating testicular development and sperm motility (Song et al., 2025; Guo et al., 2023). In our study, EGF emerged as a hub gene in the MAPK signaling pathway in duck testis, suggesting its important regulatory role in testis development through this pathway in duck Sertoli cells, consistent with previous findings.

MET (MET proto-oncogene, receptor tyrosine kinase) is another hub gene in the MAPK signaling pathway. It is expressed in testes and spermatozoa and contributes to testis development and sperm maturation (Catizone et al., 2002). Although the function of MET in the testes of poultry remains uncharacterized, our results indicate that it probably regulates duck testis development and spermatogenesis through the MAPK signaling pathway.

Comparative analysis of SC cells identified core genes expression profiles;subsequent RT-PCR validation of these genes in duck testicular tissues conformed concordance with single-cell sequencing data, suggesting results are reliable. The results showed that SF3A2, SNRPF, SNRPB2, SNRPG, SF3B4, MAP3K1, and NRAS were downregulated, suggesting that these genes play a negative regulatory role; however, MET and EGF were upregulated, suggesting that these two play a positive regulatory role.

Conclusion

In summary, we used single-cell RNA sequencing to reveal the dynamic changes in Sertoli cells during duck testicular development. The analysis demonstrated a significantly lower proportion of Sertoli cells in mature (MT, 27.75 %) compared to immature (IMT, 33.42 %) testes, with 2,630 DEGs identified. Subtype analysis revealed 2,498 DEGs in SC1 (898 upregulated and 1,600 downregulated) and 2,515 DEGs in SC2 (1,291 upregulated and 1,224 downregulated). Among these, 1,090 DEGs were specific to SC1, 1,107 to SC2, and 1,408 were shared between the two subtypes. Functional analysis indicated that these genes were primarily involved in cellular component organization and significantly enriched in Spliceosome, Neurotrophin signaling, and MAPK signaling pathways. Neurotrophin signaling was enriched in SC1, whereas the MAPK signaling pathway was enriched in SC2. PPI networks identified key regulatory genes, including SF3A2, MAP3K1, NRAS, EGF, and MET, with MAP3K1 serving as a dual hub gene participating in two signaling pathways. These findings not only confirm the conserved functions of EGF and MET in the MAPK pathway but also reveal the potential role of NRAS in avian testicular development, providing novel molecular targets for avian reproductive biology research.

Although this single-cell transcriptomic analysis of duck Sertoli cells provides novel insights, several limitations must be acknowledged. First, the experimental design captured only static snapshots of two developmental time points (10 weeks, immature; 23 weeks, mature), which precludes inference of the continuous dynamic trajectory of Sertoli cell differentiation or the precise causal relationships underlying the initiation of spermatogenesis. Second, while key hub genes (e.g., SF3A2, MAP3K1, NRAS) and signaling pathways (Notch, MAPK, Neurotrophin) were identified, functional experiments (e.g., gene knockdown/overexpression or in vivo knockout models) are lacking to establish their causal regulatory roles. To address these limitations, future research should focus on functional validation of core genes using approaches such as CRISPR-Cas9, time-series single-cell sequencing to reconstruct Sertoli cell differentiation trajectories, and exploration of ligand-receptor interaction networks between Sertoli cells and other testicular cell types using methods such as CellChat.

Funding

This research was funded by the Frontier Technology Research and Development Plan of Jiangsu Frontier Technology R&D Program (Modern Agriculture) (BF2025306), 2025 Yangzhou Municipal Plan-Natural Science Foundation Project (YZ2025114), and the JBGS Project of Seed Industry Revitalization in Jiangsu Province, China (JBGS (2021) 030, JBGS (2021)111). The authors thank The National Germplasm Center of Domestic Animal Resources for providing experimental ducks, and Guangzhou Jidiao Biotechnology Co., Ltd., for their technical support in this experiment.

Lead contact

Further information and requests for resources and reagents should be directed to the lead contacts, Chunhong Zhu (zhuch_1304428@126.com) and Huifang Li (lhfxf_002@aliyun.com.cn).

Data and code availability

The data in this study have been deposited at the GSA database of the China National Center for Bioinformation (CRA020737). Raw data can be obtained through the Shared URL: https://share.cncb.ac.cn/i2o5A6aG.

CRediT authorship contribution statement

Zhiyun Tao: Writing – original draft. Wenjuan Xu: Project administration. Weitao Song: Resources. Lizhi Lu: Funding acquisition. Shuangjie Zhang: Resources. Hongxiang Liu: Data curation. Zhicheng Wang: Writing – review & editing. Haotian Gu: Resources. Chunhong Zhu: Writing – review & editing. Huifang Li: Funding acquisition.

Disclosures

The authors declare no competing interests. The funders had no role in the design of the study, the collection, analyses, or interpretation of data, the writing of the manuscript, or the decision to publish the results.

Appendix. Supplementary materials

Supplementary Table 1: The primer list of nine core genes.

Supplementary Table 2: DEGs in Sertoli cells between MT and IMT.

Supplementary Table 3: DEGs in Sertoli cells 1 between MT and IMT.

Supplementary Table 4: DEGs in Sertoli cells 2 between MT and IMT.

Supplementary Table 5: SC1-specific DEGs between MT and IMT.

Supplementary Table 6: SC2-specific DEGs between MT and IMT.

Supplementary Table 7: Common DEGs in SC1 and SC2 between MT and IMT.