DIA Proteomics Reveals the Mechanism of cAMP Signaling Pathway-Mediated HPT Axis in Regulating Spermatogenesis of Hu Sheep

Simple Summary

Reproductive efficiency is a cornerstone of sustainable livestock production. This study investigated the molecular basis for the superior fertility of Hu sheep, a prolific indigenous Chinese breed, by performing a comprehensive proteomic comparison of the key reproductive tissues—hypothalamus, pituitary, and testes—between Hu sheep and three conventional meat breeds. Our analysis revealed that Hu sheep exhibit accelerated testicular development and earlier initiation of spermatogenesis. A central finding was the consistent and heightened activity of the cAMP signaling pathway across all three tissues in Hu sheep, indicating enhanced communication within their reproductive axis. Furthermore, we identified several tissue-specific proteins with altered abundance, implicating improved neuroendocrine regulation, antioxidant defense, and blood–testis barrier integrity. These results suggest a potential mechanistic framework for the high fecundity of Hu sheep and identify candidate molecular targets for future functional validation and offer potential molecular targets for enhancing reproductive performance in sheep through breeding or management strategies.

Abstract

Objective: Although Hu sheep are renowned for their high fecundity, the multi-tissue regulatory networks governing spermatogenesis, particularly within the hypothalamic–pituitary–testicular (HPT) axis, remain poorly understood. This study aimed to elucidate these mechanisms by performing a comparative proteomic analysis of the HPT axis in Hu sheep and three other breeds. Methods: We utilized data-independent acquisition (DIA) proteomics to analyze hypothalamic, pituitary, and testis tissues from 36 samples across four breeds. The experimental workflow included protein extraction, enzymatic digestion, LC-MS/MS, and subsequent bioinformatic analyses, complemented by histological examination. Results: Hu sheep exhibited accelerated testicular development and an earlier onset of spermatogenesis. Comprehensive proteomic profiling identified a total of 10,528 proteins, with 771 differentially expressed proteins (DEPs) detected in the testis. These testicular DEPs were significantly enriched in pathways related to spermatogenesis, the blood–testis barrier, and steroid hormone biosynthesis. Notably, the cAMP signaling pathway was consistently enriched across all three tissues, underscoring its pivotal role in regulating spermatogenesis. Protein–protein interaction (PPI) network analysis further highlighted hub proteins, such as MET, suggesting their potential involvement in somatic cell functions and the spermatogenic microenvironment. Key findings were validated by Western blot analysis. Conclusion: This study is the first multi-tissue proteomic investigation proposing a model in which the high reproductive performance of Hu sheep is potentially linked to the efficient, coordinated regulation of spermatogenesis-related proteins and signaling pathways—particularly in the testis. These findings offer novel insights into the molecular mechanisms of male reproduction in sheep and identify potential targets for future research and breeding applications.

1. Introduction

Hu sheep (H), a quintessential indigenous Chinese breed, are globally renowned for their exceptional prolificacy. As China’s only polyparous sheep breed, they display “year-round estrus, high fecundity, tolerance to coarse feed, and adaptability to intensive housing” [1]. Southdown (S) sheep are commonly used as sire or dam lines in crossbreeding due to their superior growth performance and carcass quality [2]. Polled Dorset (PD), a premium meat breed valued for early maturity and rapid growth, is frequently employed as a sire in crossbreeding programs—including with Hu sheep—for genetic improvement [3]. Suffolk (SF), known for its heavily muscled conformation, fast growth, and excellent slaughter traits, is widely used as a terminal sire to enhance offspring growth and meat quality [4].

Reproductive efficiency is a key determinant of economic performance in animal husbandry, and sheep fertility is of major importance to the livestock industry. However, most sheep breeds are uniparous and exhibit seasonal reproductive patterns. While assisted reproductive technologies are commonly used in ewes to improve estrus synchronization and ovulation rates [5]. research on enhancing reproductive efficiency in rams remains limited. Therefore, this study aims to identify key proteins and pathways regulating testicular development and spermatogenesis in sheep, with the goal of promoting sexual maturation and improving male reproductive efficiency.

The testis is a key male reproductive organ responsible for sperm production and androgen secretion [6]. Spermatogenesis occurs within a complex testicular microenvironment and is regulated by the hypothalamic–pituitary–testicular (HPT) axis, involving intricate interactions among Leydig cells, Sertoli cells, and spermatogonial stem cells [7]. The HPT axis tightly controls gonadal differentiation and maturation to maintain normal male reproductive function [8]. The hypothalamus releases gonadotropin-releasing hormone (GnRH), which stimulates the anterior pituitary to secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH) [9]. FSH drives spermatogenesis, while LH regulates testosterone (T) production by Leydig cells [10]. Testosterone then binds to androgen receptors on Sertoli cells, enabling them to indirectly support spermatogenesis [11]. Sertoli cells form the blood–testis barrier (BTB), which partitions the seminiferous tubules into basal and adluminal compartments, providing a stable microenvironment and essential nutrients for germ cell development [12]. In response to signals from Sertoli cells, spermatogonial stem cells either self-renew to sustain sperm production or commit to differentiation, ultimately leading to mature sperm formation [13].

Proteomics is a powerful tool for in-depth characterization of the proteins expressed in an organism. Unlike genomic or transcriptomic analyses, it enables precise identification of candidate biomarkers across multiple biological systems [14]. Mustafa Hitit et al. used proteomics to identify 190 differentially expressed proteins in ram sperm, revealing distinct abundance profiles between high-fertility (HF) and low-fertility (LF) rams and highlighting the functional relevance of the sperm proteome to male fertility [15]. Taylor Pini et al. employed LC-MS/MS to characterize the proteome of ejaculated ram sperm and quantitatively compared it with epididymal sperm, identifying 685 proteins in ejaculated sperm—most enriched in metabolic pathways [16].

In this study, data-independent acquisition (DIA) technology was used to analyze 36 tissue samples from the hypothalamus (H), pituitary gland (P), and testis (T) of Hu sheep (H), Southdown (S), Polled Dorset (PD), and Suffolk (SF) breeds. The experimental design comprised 12 sample groups and 9 comparative groups to assess protein expression differences, with molecular-level validation of protein functions.

The hypothalamic–pituitary–testicular (HPT) axis is the central regulator of male reproduction in mammals. In seasonally breeding sheep breeds, the activity of the HPT axis is precisely modulated by photoperiod via melatonin secretion from the pineal gland, resulting in distinct seasonal fluctuations in the pulsatile secretion of gonadotropin-releasing hormone (GnRH). This, in turn, affects the release of pituitary gonadotropins (FSH and LH) and testicular function, defining breeding and non-breeding seasons. In contrast, Hu sheep, as a prolific breed with year-round estrus, may possess a unique neuroendocrine set-point or signal transduction efficiency within their HPT axis, allowing them to overcome strict seasonal constraints and maintain continuously active reproductive status. These differences are likely manifested not only at the hormonal level but also originate from disparities in the expression and function of key regulatory proteins in hypothalamic neurons, pituitary gonadotropes, and testicular Sertoli and Leydig cells. Consequently, a systematic multi-tissue proteomic comparison of the HPT axis between Hu sheep and seasonal meat breeds is crucial for elucidating the intrinsic molecular mechanisms underlying their high fecundity.

2. Materials and Methods

2.1. Animal Handling and Sample Collection

At Gansu Qinghuan Meat Sheep Breeding Co., Ltd., four sheep breeds—Hu sheep (H), Southdown (S), Polled Dorset (PD), and Suffolk (SF)—were raised. The feeding trial employed a randomized experimental design. All experimental lambs were individually housed in single pens for a total period of 180 days (including a 15-day pre-trial acclimatization phase) under identical nutritional and husbandry management conditions. The sheep barn was well-ventilated, with the ambient temperature maintained at [10–25 °C] and relative humidity between [40% and 60%]. Pens were cleaned and disinfected daily to ensure hygiene. Throughout the trial, animals were fed a complete mixed pelleted diet with ad libitum access to feed and water. The diet was formulated according to the feeding objectives, and the nutrient levels on a dry matter basis calculated based on the China Feed Composition and Nutrient Value Table [17]. The feed formula is presented in (Table S1).

The experiment concluded in November (late autumn), when the local natural photoperiod was approximately 11–12 h per day. Three healthy six-month-old rams from each breed were selected, yielding a total of 36 tissue samples. The experimental design comprised 12 sample groups and 9 comparative groups. At the end of the experiment, the body weights of the experimental sheep (expressed as mean ± standard deviation) and their corresponding percentages of the standard adult ram body weight for each breed were as follows: Hu sheep, 51.13 ± 1.10 kg (78.66%); Southdown, 57.17 ± 1.04 kg (68.04%); Polled Dorset, 65.10 ± 1.55 kg (65.10%); Suffolk, 74.53 ± 1.00 kg (59.62%) (

Table 1. Comparison of different experimental sheep breeds with adult rams based on the midpoint value of the adult ram body weight range.

Breed	Mean Body Weight of Experimental Sheep (kg)	Adult Ram Body Weight (kg)	Percentage (%)
H	51.13 ± 1.10	60 [19]	85.22
PD	65.1 ± 1.55	130 (Span: 110–150) [20]	50.08
SF	74.53 ± 1.00	153.22 (Span: 124.84–181.6) [21]	48.64
S	57.16 ± 1.04	93.07 (Span: 81.72–104.42) [21]	61.42

Note: Adult ram body weight data were sourced from. For the PD, SF, and S breeds, the values represent the midpoint of the commonly reported range in the literature and were used to calculate the percentages. For the HH breed, a fixed standard value was used. All results are presented as mean ± standard error of the mean (SEM).

). At sampling, anesthesia was induced via intramuscular injection of diazepam (410 mg; Jining Ankang Pharmaceutical Co., Ltd.) and scopolamine (90.3 mg; Shanghai ChemGen Co., Shanghai, China), followed by intravenous administration of sodium thiopental (10–20 mg/kg; Shanghai Sine Pharma Corporation, Shanghai, China) [18]. Hypothalamus (H), pituitary (P), and testis (T) tissues were collected from each animal. Animal age was verified using official breeding records. Immediately after collection, tissue samples were placed in cryovials, rapidly frozen in liquid nitrogen, and stored at –80 °C for subsequent proteomic analysis.

2.2. Sample Preparation

Protein extraction was performed as follows: (1) Samples were cryogenically ground in liquid nitrogen. (2) Homogenates were suspended in 200 µL SDT lysis buffer (4% SDS, 150 mM Tris-HCl, pH 8.0, 100 mM DL-dithiothreitol [Sigma, Beijing, China]) and incubated at 95 °C for 3 min. (3) Mixtures were sonicated for 2 min and centrifuged at 16,000× g for 20 min at 4 °C. (4) The supernatant was collected, and protein concentration was determined using a BCA assay kit (Bio-Rad, Hercules, CA, USA).

2.3. Protein Digestion

Protein digestion was carried out using the filter-aided sample preparation (FASP) method.with 200 µg protein per sample. Briefly, proteins were reduced with 100 mM DTT at 95 °C for 5 min and cooled to room temperature. Samples were mixed with 200 µL UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0) and loaded onto a 10 kDa ultrafiltration device, followed by centrifugation at 12,000× g for 15 min; this wash was repeated once. After alkylation with 100 µL of 50 mM iodoacetamide in the dark, filters were washed twice with UA buffer and twice with 50 mM NH₄HCO₃. Trypsin solution (6 µg trypsin in 40 µL NH₄HCO₃, total 60 µL) was added, and digestion proceeded at 37 °C for 16–18 h. Peptides were collected by centrifugation, acidified with 0.1% TFA, desalted using a Thermo spin column, and quantified prior to downstream analysis.

2.4. High-Resolution LC-MS/MS Mass Spectrometry Detection

Peptides were redissolved in 30 µL of solvent A (0.1% formic acid in water) and analyzed by online nanospray LC-MS/MS using an Orbitrap Fusion Lumos mass spectrometer coupled with an EASY-nLC 1200 system (Thermo Fisher Scientific, Waltham, MA, USA)). A 3 µL aliquot was loaded onto an Acclaim PepMap C18 analytical column (75 μm × 25 cm) and separated over a 120 min linear gradient from 5% to 35% mobile phase B (0.1% formic acid in acetonitrile) at a flow rate of 200 nL/min and column temperature of 40 °C. Electrospray ionization was performed at 2.0 kV. The instrument operated in data-dependent acquisition mode: full MS scans (m/z 350–1200) were acquired at 120,000 resolution, followed by HCD-MS/MS scans at 15,000 resolution with 32% collision energy.

2.5. Database Searching

Raw data from data-dependent acquisition (DDA) were processed using Spectronaut X (Biognosys AG, Zürich, Switzerland) with default settings to generate an initial target list. Database searching was performed against a sheep-specific protein database (including common contaminants), with trypsin specified as the protease. Carbamidomethylation of cysteine was set as a fixed modification, and methionine oxidation as a variable modification. A false discovery rate (FDR) threshold of 1% was applied for both peptide precursor and protein identifications.

2.6. Spectral Library Construction

The spectral library used for DIA data analysis was constructed from a pooled sample. Specifically, peptide digests from all 36 individual samples—spanning four sheep breeds and three tissue types (hypothalamus, pituitary, and testis)—were combined in equal amounts to generate a comprehensive pooled sample. This pooled sample was then fractionated using high-pH reversed-phase liquid chromatography to reduce sample complexity. The resulting fractions were analyzed on an Orbitrap Fusion Lumos mass spectrometer in data-dependent acquisition (DDA) mode, following the procedures described in Section 2.4. The raw DDA files were processed and searched against the reference database as detailed in Section 2.5. The resulting collection of high-confidence identified peptides and their corresponding fragment ion spectra formed a project-specific spectral library, comprising 10,528 proteins and 89,898 peptides. This spectral library was subsequently used to deconvolute all single-injection DIA data files.

2.7. DIA: Nano-HPLC-MS/MS Analysis

Peptides were reconstituted in 30 µL of solvent A (0.1% aqueous formic acid) and analyzed by online nanospray LC-MS/MS using an Orbitrap Fusion Lumos mass spectrometer coupled to an EASY-nLC 1200 system (Thermo Fisher Scientific, Waltham, MA, USA). A 3 µL injection was loaded onto an Acclaim PepMap C18 analytical column (75 μm × 25 cm) and separated over a 120 min linear gradient from 5% to 35% mobile phase B (0.1% formic acid in acetonitrile) at a constant flow rate of 200 nL/min and column temperature of 40 °C. Electrospray ionization was performed at 2.0 kV.

Mass spectrometry was conducted in data-independent acquisition (DIA) mode with automatic MS/MS switching. Key parameters included: full MS scans (m/z 350–1200) at 120,000 resolution; HCD-MS/MS at 30,000 resolution with 32% collision energy and 5% stepping; and a variable-window DIA scheme consisting of 60 overlapping (1 m/z) isolation windows.

2.8. DIA Data Acquisition and Analysis

The data-dependent acquisition (DDA) library contained 10,528 protein groups and 89,898 peptides. Raw data-independent acquisition (DIA) files were analyzed in Spectronaut X (Biognos: AG, Zürich, Switzerland) using default settings. Retention time alignment was performed via the dynamic iRT algorithm, and data extraction was guided by extensive mass calibration. Spectronaut Pulsar X automatically determined optimal extraction windows based on iRT calibration and gradient stability. A 1% false discovery rate (FDR) threshold was applied at both precursor and protein levels.

Prior to statistical testing, protein intensity matrices were preprocessed. Missing values were imputed using a minimum value approach (drawing from a normal distribution with a width of 0.3 and a down-shift of 1.8 standard deviations relative to the mean of each sample’s detected values) to simulate the background signal of low-abundance proteins. Potential batch effects were assessed by principal component analysis (PCA) and were found to be negligible; therefore, no batch effect correction was applied. For all pairwise comparisons between experimental groups, differential expression was assessed by Student’s t-test. The resulting p-values were corrected for multiple hypothesis testing using the Benjamini–Hochberg method to control the false discovery rate (FDR) and obtain Q-values. Differentially expressed proteins (DEPs) were defined as those with a Q-value < 0.05 and an absolute average log₂ ratio (|log₂FC|) > 0.58. Given the high-dimensional nature of the DIA data and the sample size (n = 3 per breed), the power to detect subtle expression differences is limited; thus, these pathway enrichment results should be primarily regarded as hypothesis-generating.

2.9. Protein Quantitative Normalization Treatment

For qualitative protein analysis, a 1.0% false discovery rate (FDR) threshold was applied to both peptide precursors and proteins. Peak intensities across all samples were normalized using the local normalization method in Pulsar, yielding highly comparable signal intensity distributions among most samples. Protein quantification was based on the average peak areas of the top three MS1 peptides meeting the 1.0% FDR criterion.

2.10. Bioinformatics Analyses

Data analysis was performed using Perseus software and the R statistical environment. Differentially expressed proteins (DEPs) were defined as those meeting the criteria stated in Section 2.7 (Q-value < 0.05 and |log₂FC| > 0.58, corresponding to a fold change > 1.5 or <0.67). DEPs were functionally annotated against the Kyoto Encyclopedia of Genes and Genomes (KEGG, release 109.0) and Gene Ontology (GO, release 42.442) databases. GO and KEGG enrichment analyses were conducted using Fisher’s exact test with false discovery rate (FDR) correction.

Visualization and advanced analyses included: clustering heatmaps generated via OECloud Tools; gene set enrichment analysis (GSEA v4.1.0) to identify enriched signaling pathways; K-means clustering of DEPs using an online bioinformatics platform; and protein–protein interaction (PPI) network construction using the STRING database (v11.5), visualized in Cytoscape (v3.9.1). Networks with fewer than three nodes were excluded from further analysis [22].

2.11. Western Blotting

To validate the proteomic results, six proteins were randomly selected for Western blot (WB) analysis. Tissue samples were rinsed 2–3 times with ice-cold PBS, minced, and transferred to homogenization tubes. A tenfold volume of lysis buffer(G2002, Servicebio, Wuhan, China) supplemented with PMSF protease inhibitor (G2008, Servicebio, Wuhan, China) was added, followed by thorough homogenization. Homogenates were incubated on ice for 30 min with gentle shaking every 5 min and then centrifuged at 12,000× g and 4 °C for 10 min. The supernatant was collected as total protein extract.

Proteins were separated by SDS-PAGE and transferred onto a PVDF membrane (0.45 μm, WGPVDF45, Servicebio, Wuhan, China). The membrane was probed with the following primary antibodies: MUDENG (26567-1-AP, Wuhan Sanying, Wuhan, China; 1:500), (12353-1-AP, Wuhan Sanying, Wuhan, China; 1:500), PDE8B (20364-1-AP, Wuhan Sanying, Wuhan, China; 1:500), ITGA8 (30714-1-AP, Wuhan Sanying, Wuhan, China; 1:1000), RCAN1 (14869-1-AP, Wuhan Sanying, Wuhan, China; 1:500), TRIM65 (bs-17120r, BIOSS, Beijing, China; 1:500) and ACTIN (GB15003, Servicebio, Wuhan, China; 1:5000).

All images were analyzed using AIWBwell™ software, version v1.0, provided by Servicebio (Wuhan Sino-Bio Co., Ltd., Wuhan, China), and the relative abundance of the target protein in each sample was determined by the ratio of its gray intensity to that of the internal reference protein. All results are presented as mean ± standard error of the mean (SEM).

3. Results

3.1. Histological Observation of Testis

Compared with the PD, SF, and S breeds, H sheep exhibited larger seminiferous tubule diameters, clearer luminal structures, the presence of sperm within the lumen, and well-organized layers of germ cells and Sertoli cells, indicating more advanced testicular development and superior spermatogenic function (Figure 1). These histological observations were further corroborated by quantitative data: as shown in

Table 2. Quantitative histological data: testis weight, seminiferous tubule diameter, and presence of sperm.

Breed	Testis Weight (kg)	Diameter of Seminiferous Tubules (um)	Sperm
H	0.483 ± 0.008	166.7 ± 0.03	Yes
PD	0.343 ± 0.022	58.45 ± 0.01	no
SF	0.453 ± 0.009	75.2 ± 0.001	no
S	0.407 ± 0.007	60.6 ± 0.03	no

All results are presented as mean ± standard error of the mean (SEM).

, H had the heaviest testes (0.483 ± 0.008 kg), the largest seminiferous tubule diameter (166.7 ± 0.03), and detectable sperm in the lumen, whereas the other breeds (PD, SF, S) showed lighter testes, smaller seminiferous tubule diameters—even as low as 58.45 ± 0.01 μm—and no observable sperm. The consistency between quantitative measurements and histological findings collectively demonstrates that Hu sheep possess more mature seminiferous tubule architecture, clearer lumens, more active spermatogenesis, and a higher degree of testicular development.

3.2. Qualitative and Quantitative Proteomic Analysis and Sample Correlation Assessment

By matching DIA data against a DDA reference database with a false discovery rate (FDR) ≤ 0.01, 10,528 protein groups and 89,898 peptides were identified (Figure S1A,B), establishing a robust foundation for exploring key regulators of sheep reproductive performance. Principal component analysis (PCA) and correlation results showed clear sample clustering, indicating high data reliability (Figure 2B, Table S2). Pearson correlation coefficients between all sample pairs revealed intra- and inter-group similarities >0.8 across the nine groups, reflecting high sample consistency (Figure 2C, Table S3). Relative standard deviation (RSD) analysis showed RSD values concentrated in the 0–0.5 range, with low medians and narrow interquartile ranges, confirming low within-group variability and good reproducibility (Figure 2D).

3.3. Identification and Analysis of DAPs

Venn diagrams were used to illustrate the distribution of differentially abundant proteins (DAPs) across comparison groups (Figure 3A). Testicular tissue samples showed the highest proteomic similarity, with only 40 DAPs identified between the two testis comparison groups; hypothalamic comparisons shared 86 overlapping proteins, and pituitary comparisons shared 59. DAPs were defined by an absolute fold change (FC) > 1.5 and an adjusted p-value (Q-value) < 0.05. Comprehensive analysis revealed distinct protein regulation patterns: H-H-vs-PD-H had 239 up- and 143 down-regulated proteins; H-H-vs-SF-H had 181 up- and 152 down-regulated; H-H-vs-S-H had 341 up- and 134 down-regulated; H-P-vs-PD-P had 218 up- and 110 down-regulated; H-P-vs-SF-P had 220 up- and 115 down-regulated; H-P-vs-S-P had 180 up- and 135 down-regulated; H-T-vs-PD-T had 102 up- and 111 down-regulated; H-T-vs-SF-T had 197 up- and 85 down-regulated; and H-T-vs-S-T had 133 up- and 143 down-regulated proteins (Figure 3B, Table S4). Notably, the hypothalamus exhibited the greatest number of DAPs, suggesting a central regulatory role in coordinating reproductive physiological differences among sheep breeds, whereas testicular DAPs appeared to reflect more specialized functions in reproductive regulation.

3.4. GO and KEGG Enrichment Analysis

GO enrichment analysis (Figure 4, Table S5) revealed that in hypothalamic tissue, DAPs from the H-H-vs-PD-H group were significantly enriched (p < 0.05) in 167 GO terms, including DNA replication initiation, gonadotropin hormone-releasing hormone activity, and chromatoid body; the H-H-vs-SF-H group in 166 terms, such as steroid biosynthetic process and gonadotropin hormone-releasing hormone activity; and the H-H-vs-S-H group in 211 terms, including catecholamine biosynthetic process, gonadotropin hormone-releasing hormone activity, and SCF ubiquitin ligase complex.

In pituitary tissue, DAPs from H-P-vs-PD-P were enriched in 139 GO terms (p < 0.05), including signal transduction, calcium:sodium antiporter activity, and acrosomal vesicle; H-P-vs-SF-P in 141 terms, such as signal transduction, spermatid development, and sperm flagellum; and H-P-vs-S-P in 132 terms, including spermatid development, hormone biosynthetic process, dynein intermediate chain binding, and cell junction.

In testicular tissue, DAPs from H-T-vs-PD-T were enriched in 217 GO terms (p < 0.05), covering fructose metabolic process, calcium-dependent phospholipid binding, and bicellular tight junction; H-T-vs-SF-T in 242 terms, including fructose metabolic process, signal transduction, and bicellular tight junction; and H-T-vs-S-T in 200 terms, such as cellular response to cAMP, ATP binding, and ATP-dependent diacylglycerol kinase activity.

Notably, the cAMP signaling pathway was significantly enriched across multiple comparison groups and tissues, suggesting its potential role as a central mediator of the high fecundity traits in Hu sheep.

KEGG enrichment analysis (Figure 5, Table S6) showed that in hypothalamic tissue, the H-H-vs-PD-H group was significantly enriched in Nicotine addiction and the cAMP signaling pathway; H-H-vs-SF-H in the cAMP signaling pathway and Cell cycle; and H-H-vs-S-H in the cAMP signaling pathway and Circadian entrainment.

In pituitary tissue, H-P-vs-PD-P was enriched in Signal transduction and Immune system pathways; H-P-vs-SF-P in FoxO signaling pathway and Cell adhesion molecules; and H-P-vs-S-P in Arginine biosynthesis and Renin–angiotensin system.

In testicular tissue, H-T-vs-PD-T was significantly enriched in Hedgehog signaling pathway and Th1 and Th2 cell differentiation; H-T-vs-SF-T in HIF-1 signaling pathway and AMPK signaling pathway; and H-T-vs-S-T in Efferocytosis and Calcium signaling pathway.

3.5. GSEA-Based Proteomic Enrichment Analysis

To comprehensively assess signaling pathway alterations across the global proteome, Gene Set Enrichment Analysis (GSEA) was performed. The results corroborated KEGG enrichment findings and further revealed that, in comparisons between Hu sheep and other breeds, multiple key reproductive regulatory pathways were coordinately up-regulated in hypothalamic, pituitary, and testicular tissues of Hu sheep (Figure 6).

Notably, both the GnRH signaling pathway (oas04912) and the cAMP signaling pathway (oas04024) were significantly enriched (NES > 0) across multiple tissue comparisons, suggesting enhanced signaling efficiency of the hypothalamic–pituitary–testicular (HPT) axis in Hu sheep—from upstream neuroendocrine regulation in the hypothalamus to downstream gonadal function in the testes. Upstream activation of the GnRH pathway may drive more efficient pituitary secretion of gonadotropins (LH and FSH), which then promote testosterone synthesis and spermatogenesis in Leydig and Sertoli cells via activation of the cAMP pathway, a critical second messenger system.

Furthermore, activation of the Hedgehog signaling pathway (oas04340) and the FoxO signaling pathway (oas04068) provides new insights into the reproductive traits of Hu sheep. The Hedgehog pathway, critical for testicular development during embryogenesis and adult stem cell fate, may reflect superior Sertoli or Leydig cell function in Hu sheep. Meanwhile, activation of the FoxO pathway—a key sensor of cellular stress and metabolism—suggests enhanced antioxidant capacity in Sertoli and other supporting cells, fostering a more stable microenvironment for sustained and efficient spermatogenesis.

3.6. Analysis of the Protein–Protein Interaction Network for Differentially Abundant Proteins

To further explore interactions among differentially abundant proteins and identify key regulatory nodes, a protein–protein interaction (PPI) network was constructed (Figure 7). Connectivity analysis revealed a set of highly connected hub proteins that may play central roles in mediating reproductive trait differences among breeds.

In the hypothalamic PPI network, cyclin-dependent kinase 2 (CDK2) and Fizzy-related protein 1 (FZR1) showed high connectivity with multiple cell cycle–related proteins, suggesting that reproductive differences between Hu sheep and other breeds may partly stem from altered neuroendocrine cell proliferation and differentiation. As a key regulator of the G1/S transition, CDK2 may affect GnRH neuron homeostasis or activity, thereby modulating HPT axis output.

In the testicular PPI network, hepatocyte growth factor receptor (MET) emerged as a critical hub protein. As a core receptor in pathways such as MAPK and PI3K-Akt, MET regulates cell proliferation, migration, and survival; its central role implies potential effects on the spermatogenic microenvironment via Sertoli cell function or blood–testis barrier integrity—consistent with KEGG enrichments like “MAPK signaling pathway” and “Focal adhesion”, highlighting the importance of cell junctions and signal transduction in testicular function.

PPI networks for all nine comparison groups were constructed using a confidence threshold of p < 0.05 to reduce complexity. Key functional associations included CDK2 and FZR1 with cell cycle regulation in the hypothalamus, REM1 and AXL with GTP binding in the pituitary, and MET with the MAPK signaling pathway in the testes.

The left panel shows the protein–protein interaction network of the top 25 connectivity proteins. Circles represent differentially expressed proteins/genes, with red indicating upregulation and blue indicating downregulation. The size of the circles reflects the degree of connectivity, with larger circles representing higher connectivity. The right panel displays a bar chart of the expression levels of the top 25 connectivity proteins. The middle section provides a shared legend for both parts of the figure.

3.7. Validation by Western Blot Analysis

To validate the accuracy of the proteomic sequencing results, six randomly selected differentially expressed proteins (MUDENG, TAF12, PDE8B, ITGA8, RCAN1, and TRIM65) were analyzed by Western blot. Although variations in expression levels were observed, their expression patterns were consistent, confirming the reliability and accuracy of the proteomic data. One-way ANOVA followed by Tukey’s test revealed significant differences among all tested breeds (Figure 8).

4. Discussion

This study establishes a complete workflow encompassing multi-tissue sample collection, DIA mass spectrometry detection, and multi-level bioinformatic analysis (differential analysis, enrichment analysis, GSEA, PPI network). This workflow fully leverages the advantages of high precision and reproducibility offered by DIA technology, successfully identifying over 10,000 proteins and enabling reliable quantitative comparisons despite a relatively modest sample size. Notably, the body weights of the experimental sheep used in this study were carefully selected, ranging from 48.6% to 85.2% of the standard adult ram body weight for their respective breeds (HH: 85.2%; PD: 50.1%; SF: 48.6%; S: 61.4%). Accurate assessment of reproductive maturity during animal development is crucial for understanding life history traits, formulating breeding strategies, and implementing conservation measures [23]. provides a replicable and efficient technical paradigm for future similar multi-tissue, large-cohort comparative proteomic studies in livestock animals. Notably, the application of GSEA to tissue comparisons in non-model animals facilitates the understanding of phenotypic differences from the perspective of overall pathway activity, surpassing the limitations of traditional single differential protein analysis.

4.1. Key Proteins Associated with Reproductive Performance

This study employed DIA proteomics to systematically compare the hypothalamic–pituitary–testicular (HPT) axis between highly prolific Hu sheep and three introduced meat breeds. Our analysis not only identified numerous differentially abundant proteins but also highlighted the central regulatory role of the hypothalamus, revealed the coordinated activation of key signaling pathways—including cAMP—and pinpointed hub proteins associated with reproductive function. These findings offer novel insights into the molecular mechanisms underlying the high fecundity of Hu sheep from a systems biology perspective.

One of the most notable findings is that the hypothalamus harbored substantially more differentially abundant proteins (DAPs; 1190) than the pituitary (978) or testis (771), underscoring its central regulatory role in the HPT axis. The hypothalamus governs reproductive function through pulsatile GnRH secretion, and its distinct proteomic profile in Hu sheep may enhance GnRH pulse frequency or amplitude, thereby driving stronger FSH and LH release from the pituitary. This likely underlies Hu sheep’s year-round estrus and high prolificacy. In contrast, testicular DAPs primarily reflect downstream adaptations—such as in spermatogenesis, blood–testis barrier integrity, and steroidogenesis—in response to upstream hormonal cues. However, the high fecundity of Hu sheep is not solely driven by hormone levels but by the efficiency of signal transduction. Our analysis reveals that the cAMP signaling pathway is consistent with acting as a core mechanistic bridge. In the hypothalamus, cAMP likely modulates GnRH neuronal excitability. Crucially, we hypothesize that the upregulation of GnRH1 may drive a robust cAMP-PKA signaling cascade in the testes. The accumulation of cAMP triggers the PKA-dependent phosphorylation of CREB, which directly activates key meiotic genes (such as Stra8). Thus, the cAMP pathway is proposed to serve as the functional transducer that converts superior neuroendocrine signaling into accelerated meiotic initiation.

4.2. Key Functional Pathways Associated with Reproductive Performance

Both GSEA and KEGG analyses consistently revealed significant enrichment of the cAMP signaling pathway across multiple tissues. As the primary second messenger cascade downstream of FSH and LH signaling, its widespread activation suggests enhanced signal transduction efficiency and cellular responsiveness throughout the HPT axis in Hu sheep. In the hypothalamus, cAMP likely modulates GnRH neuronal excitability and pulsatile secretion; in the pituitary, it mediates GnRH-induced gonadotropin synthesis; and in the testes, the cAMP/PKA pathway coordinately regulates Sertoli cell function and Leydig cell testosterone production. By faithfully transmitting and amplifying hypothalamic signals into robust testicular responses, the cAMP pathway serves as a core mechanistic driver of Hu sheep’s high fecundity.

Multiple signaling pathways have been identified in ovine Sertoli cells, including the androgen signaling pathway [24], the AMP-activated protein kinase (AMPK) signaling pathway [25], and the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway [26]. The cAMP signaling pathway is known to regulate steroid hormone synthesis and sperm development in the testes [27]. In our study, cAMP was significantly up-regulated in testicular tissue. Virtually all stages of spermatogenesis critically depend on dynamic changes in cAMP levels. The cAMP/PKA pathway, an intracellular signaling cascade, mediates non-genomic effects involved in steroid hormone regulation and mammalian sperm capacitation [28]. In the testes, regulators of G-protein signaling (RGS) prevent premature acrosome reaction by inhibiting cAMP accumulation in the sperm head, while simultaneously enhancing the PI3K–Akt signaling pathway in the sperm tail to maintain motility [29]. Accumulating evidence indicates that spermatogenesis is predominantly regulated by the MAPK, AMPK, and TGF-β/Smad signaling pathways. For example, the MAPK pathway modulates tight and adherent junctions, germ cell proliferation and meiosis, as well as Sertoli cell proliferation and lactate production [30].

The enrichment of the Hedgehog signaling pathway in the testes is also noteworthy. Its activation in Hu sheep suggests functional advantages in Sertoli cell maturation, Leydig cell differentiation, or germ stem cell homeostasis, thereby fostering a superior microenvironment for sustained and efficient spermatogenesis. Moreover, the Hedgehog pathway is involved in testicular development [31], is closely associated with spermatogenic processes [32]. Instead of acting in isolation, we suggest a synergistic interaction between the MET and Hedgehog pathways to optimize the germ cell pool. While MET—identified as a central node in our PPI network—likely promotes the expansion of spermatogonial stem cells via the PI3K/AKT/mTOR axis (providing the “quantity” of germ cells), the Hedgehog pathway provides the spatial cues to cease self-renewal and initiate differentiation (controlling the “timing”). This coordinate regulation ensures a sustained high yield of sperm by decoupling proliferation capacity from differentiation pacing, thereby fostering a superior microenvironment for continuous spermatogenesis.

This study successfully identified a series of differentially abundant proteins potentially linked to reproductive performance and preliminarily inferred their regulatory roles through PPI network analysis. Although these proteins showed significant expression differences between Hu sheep and other breeds, their specific functions in reproductive regulation require further experimental validation.

In hypothalamic comparisons between Hu sheep and other breeds, gonadotropin-releasing hormone 1 (GnRH1) exhibited significantly higher expression in Hu sheep than in Southdown (S-H), Polled Dorset (PD-H), and Suffolk (SF-H) sheep—for example, in the H-H-vs-S-H group, fold change >1.5 and Q value <0.05. This is particularly significant, as GnRH1, the most upstream neuroendocrine signal in the hypothalamic–pituitary–testicular (HPT) axis, directly governs pituitary gonadotropin synthesis and release through both its expression level and pulsatile secretion pattern.

The differential expression of gonadotropin-releasing hormone 1 (GnRH1) in hypothalamic tissue merits particular attention. As the most upstream initiator of the hypothalamic–pituitary–testicular (HPT) axis, variations in its expression may indirectly modulate the entire reproductive endocrine axis, offering key insights into breed-level reproductive differences. GnRH1 is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) synthesized by hypothalamic neurons and secreted in a pulsatile manner into pituitary portal capillaries. It binds to gonadotropin-releasing hormone receptor 1 (GnRHR1) on pituitary gonadotropic cells, stimulating synthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which enter systemic circulation to act on gonadal targets [33]. in testes, FSH regulates spermatogenesis and LH stimulates testosterone secretion [34]. Absence of GnRH1 leads to gonadal dysfunction and reproductive arrest [35]. Similarly, GnRH1 immunization impairs fertility in Suffolk rams, reducing epididymal sperm counts, serum testosterone levels, and spermatogenesis [36].

Our findings suggest that upregulated GnRH1 in Hu sheep hypothalamus enhances the amplitude or frequency of GnRH pulses, promoting more robust FSH and LH release from the pituitary. These gonadotropins then act on testes via circulation: LH boosts testosterone production in Leydig cells through the cAMP/PKA pathway—consistently enriched across tissues in this study—while FSH binds Sertoli cell receptors to regulate blood–testis barrier integrity and the spermatogenic microenvironment. This coordinated cascade—from hypothalamic neuroendocrine signaling, through pituitary hormonal amplification, to testicular execution—underlies the reproductive advantages of Hu sheep, including earlier sexual maturity and superior spermatogenic capacity, compared to meat breeds.

In comparative pituitary analyses, we detected significant expression differences in key proteins—ABHD2, NME5, and CEP57—among sheep breeds. Compared with Southdown (S-P), Polled Dorset (PD-P), and Suffolk (SF-P) sheep, Hu sheep exhibited downregulated ABHD2 and significantly upregulated NME5 and CEP57 in pituitary tissue (e.g., in the H-P-vs-S-P group, fold changes > 1.5, Q value < 0.05).

Existing literature links these proteins to sperm capacitation, antioxidant stress response, and centrosomal function, respectively, suggesting previously underappreciated pituitary expression patterns potentially involved in gonadal regulation and offering new avenues for studying pituitary–gonadal signaling mechanisms.

ABHD2 (abhydrolase domain-containing protein 2) functions as a membrane progesterone (P4) receptor in sperm, mediating chemotaxis and the acrosome reaction [37]. It is essential for sperm activation and is enriched in sperm flagella and motile cilia, as reported by Mustafa Hitit [38]. NME5 (NME/NM23 family member 5), an oxidative stress-related gene, is specifically expressed in mouse testes and participates in spermatogenesis [39]. Under physiological conditions, it protects elongating and round spermatids from oxidative damage by modulating antioxidant enzymes such as Gpx5 [40]. Asrat Tera Dolebo [41] further reported that NME5 is involved in ovine spermatogenesis and linked to male fertility and reproductive performance in other species, though its role in highly prolific sheep remains unexplored. CEP57 (Centrosomal Protein 57), also known as translokin, is a centrosome-associated protein of the Cep57 family [3]. Studies by Julia Lisboa Rodrigues et al. show that CEP57 is associated with reproductive traits in rams, is highly expressed in Merino testes, and contributes to sperm motility [42].

The differential expression of these proteins in Hu sheep pituitary tissue suggests the presence of previously undercharacterized regulatory mechanisms governing gonadal function at the pituitary level. ABHD2 downregulation may alter pituitary sensitivity to gonadal steroid hormone feedback, while NME5 upregulation could enhance antioxidant defense in pituitary cells. Changes in CEP57 expression may affect cell division and protein trafficking efficiency in hormone-secreting pituitary cells. Together, these alterations may modulate gonadotropin synthesis and secretion, thereby influencing downstream testicular processes—including sperm capacitation, antioxidant stress response, and sperm motility.

In testicular tissue comparisons, we observed significant differences in the expression of the tight junction protein TJP3/ZO-3 among sheep breeds. Compared with Southdown (S-T), Polled Dorset (PD-T), and Suffolk (SF-T) sheep, Hu sheep showed markedly upregulated TJP3/ZO-3 expression (e.g., in the H-T-vs-S-T group, fold change > 1.5, Q value < 0.05). This is highly significant, as TJP3/ZO-3 is a key component of the blood–testis barrier (BTB), and its altered expression may directly affect BTB structural integrity and functional properties. The upregulation of TJP3/ZO-3 is not merely structural but serves as a prerequisite safeguard for high-efficiency spermatogenesis. Given that the upstream GnRH1-cAMP axis drives rapid germ cell generation, a reinforced BTB mediated by TJP3 is essential to preserve an immune-privileged microenvironment. This prevents autoimmune attacks against the rapidly generated haploid germ cells, ensuring that the high quantity of sperm produced reaches functional maturity.

In testicular tissue, differential expression of the tight junction protein TJP3/ZO-3 suggests structural or functional variations in the BTB among breeds. As the BTB is essential for maintaining the spermatogenic microenvironment, altered TJP3/ZO-3 expression offers new insights into breed differences in spermatogenic efficiency. Meanwhile, protein–protein interaction (PPI) network analysis identified MET as a relatively central node. Previous studies have reported its role in regulating cell proliferation and survival, indicating that MET may indirectly influence spermatogenesis by modulating testicular somatic cell function.

Within the testes, the BTB is a critical structure that maintains the spermatogenic microenvironment. It is located near the base of the seminiferous tubules and forms between adjacent Sertoli cells (SCs) [43]. Tight junctions (TJs)—composed of connexins between SCs and germ cells—are the primary structural component of the BTB and a key type of inter-Sertoli cell connection [44]. MET modulates the expression of molecules such as kininogen 2 (PK2), PKR2, p-Akt, and p-GSK3, which are crucial for spermatogenesis and testicular function. Through its effects on the BTB, MET is implicated in testicular injury, dysfunction, apoptosis, autophagy, and PK2-related signaling mechanisms [45].

These findings suggest that the distinct expression patterns of key proteins such as TJP3/ZO-3 and MET in Hu sheep testes may jointly enhance spermatogenic efficiency by strengthening BTB function and optimizing the testicular microenvironment. This mechanism is consistent with the protein expression changes observed in hypothalamic and pituitary tissues, collectively underpinning the reproductive advantage of the HPT axis in Hu sheep.

However, it should be noted that the specific molecular roles of these proteins in ovine reproductive regulation have not yet been experimentally validated, representing a critical direction for future functional studies. While our proteomic screening and bioinformatic analyses provide valuable protein-level insights and preliminary hypotheses regarding breed differences in reproductive performance, experimental confirmation of their functions and mechanisms remains essential.

4.3. Limitations and Future Perspectives

It should be acknowledged that this study has certain limitations. First, the sample size for proteomic analysis was relatively small (n = 3 per breed). Although a rigorous experimental design and appropriate statistical corrections were applied, the limited sample size may reduce statistical power and hinder a comprehensive assessment of inter-individual variability—particularly for proteins exhibiting subtle expression changes. Second, our findings are primarily correlative; the precise functional roles of the identified key differentially expressed proteins and their associated pathways in spermatogenesis have not been experimentally validated. It should also be noted that since all animals were sampled at six months of age during late autumn, these findings are consistent with, but do not directly demonstrate, the neuroendocrine mechanisms underlying year-round estrus or the overcoming of seasonal reproductive constraints.

To address these limitations, future research could be expanded and deepened in the following directions: Subsequent studies should include a larger number of biological replicates per group and consider incorporating additional sheep breeds or crossbred combinations with diverse reproductive phenotypes to enhance the generalizability and statistical robustness of the findings. Utilize cellular models (e.g., Sertoli cells or germ cell lines) or animal models to perform gain-of-function and loss-of-function experiments—through techniques such as gene overexpression or knockdown (e.g., RNAi)—to directly test the roles of key candidate proteins in spermatogenesis. Collect samples at multiple developmental time points (e.g., pre-pubertal and sexually mature stages) and integrate transcriptomic, proteomic, and phosphoproteomic data to dynamically reconstruct the molecular networks underlying the high prolificacy of Hu sheep. Furthermore, correlate these omics discoveries with detailed phenotypic data—including semen quality parameters and longitudinal hormone profiles—to establish a more complete and causal link from molecular mechanisms to reproductive phenotypes.

5. Conclusions

In summary, this multi-tissue proteomic study proposes a multi-layered regulatory model to explain the high fecundity of Hu sheep (Figure 9). The hypothalamus—showing the highest number of DAPs—likely acts as the initiating center and key regulatory hub of the HPT axis, driving reproductive differences between Hu sheep and other breeds. Its unique proteomic profile may shape gonadotropin-releasing hormone 1 (GnRH1) secretion patterns, thereby governing the entire reproductive endocrine axis. Consistent enrichment and activation of the cyclic adenosine monophosphate (cAMP) signaling pathway across tissues suggest that this core hormonal cascade, potentially operating with higher efficiency, is suggested to support the enhanced HPT axis functionality in Hu sheep, pending further experimental confirmation. Moreover, we identified several candidate proteins closely linked to reproductive function: GnRH1 in the hypothalamus; ABHD2, NME5, and CEP57 in the pituitary; and TJP3/ZO-3 and MET in the testes. Together, these proteins may modulate ovine reproductive performance by regulating neuroendocrine signaling, sperm capacitation, antioxidant stress response, blood–testis barrier (BTB) integrity, and intracellular signal transduction within their respective tissues.