Spermatogenesis carries the task of precise intergenerational transmission of genetic information from the paternal genome and involves complex developmental processes regulated by the testicular microenvironment. Studies performed mainly in mouse models have established the theoretical basis for spermatogenesis, yet the wide interspecies differences preclude direct translation of the findings, and farm animal studies are progressing slowly. More than 32,000 cells from prepubertal (3-month-old) and pubertal (24-month-old) buffalo testes were analyzed by using single-cell RNA sequencing (scRNA-seq), and dynamic gene expression roadmaps of germ and somatic cell development were generated. In addition to identifying the dynamic processes of sequential cell fate transitions, the global cell-cell communication essential to maintain regular spermatogenesis in the buffalo testicular microenvironment was uncovered. The findings provide the theoretical basis for establishing buffalo germline stem cells in vitro or culturing organoids and facilitating the expansion of superior livestock breeding.

Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, 8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.

Background: Exosomes are nanosized membranous vesicles secreted by various types of cells, which facilitate intercellular communication by transporting bioactive compounds. Exosomes are abundant in biological fluids including semen, and their protein composition and the potential of seminal plasma exosomes (SPEs) as fertility biomarkers were elucidated in humans, however, little information is available regarding buffalo (Bubalus bubalis). Here, we examined protein correlation between spermatozoa, seminal plasma (SP), and SPEs, and we compared and analyzed protein differences between high-motility (H-motility) and low-motility (L-motility) SPEs in buffalo.Results: SPEs were concentrated and purified by ultracentrifugation combined with sucrose density gradient centrifugation, followed by verification using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. Protein composition in spermatozoa, SP and SPEs, and protein difference in H- and L-motility SPEs were identified by LC-MS/MS proteomic analysis and were functionally analyzed through comprehensive bioinformatics. Many SPEs proteins originated from spermatozoa and SP, and nearly one third were also present in spermatozoa and SP. A series of proteins associated with reproductive processes including sperm capacitation, spermatid differentiation, fertilization, sperm-egg recognition, membrane fusion, and acrosome reaction were integrated in a functional network. Comparative proteomic analyses showed 119 down-regulated and 41 up-regulated proteins in L-motility SPEs, compared with H-motility SPEs. Gene Ontology (GO) enrichment of differentially expressed proteins (DEPs) showed that most differential proteins were located in sperm and vesicles, with activities of hydrolase and metalloproteinase, and were involved in sperm-egg recognition, fertilization, single fertilization, and sperm-zona pellucida binding processes, etc. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differential proteins were mainly involved in the PPRP signaling pathway, calcium signaling pathway, and cAMP signaling pathway, among others. Furthermore, 6 proteins associated with reproduction were validated by parallel reaction monitoring analysis.Conclusion: This study provides a comprehensive description of the seminal plasma exosome proteome and may be of use for further screening of biomarkers associated with male infertility.

Endoplasmic reticulum (ER) stress plays a crucial role in granulosa cell (GCs) apoptosis, which is the main cause of follicular atresia. Quercetin (QC), a plant-derived flavonoid, has antioxidant, anti-inflammatory, and other biological properties. However, whether QC can alleviate the effects of ER stress on buffalo GCs remains unknown. In this study, we constructed an ER stress model in buffalo GCs by using tunicamycin (TM) and pre-treated with QC to explore the effect of QC on cells under ER stress. Apoptosis was detected by Annexin fluorescein 5 isothiocyanate (V-FITC), and the expressions of mRNA and related proteins involved in ER stress and apoptosis were detected via real-time polymerase chain reaction and Western blot. The results revealed that ER stress can cause apoptosis in GCs, whereas QC pre-treatment can prevent apoptosis caused by ER stress. After pre-treatment with QC, the expression levels of ER stress-related genes and proteins significantly decreased, pro-apoptotic genes were significantly down-regulated, and anti-apoptotic genes were significantly up-regulated. Furthermore, the results of Chop gene overexpression suggested that QC alleviated ER stress via the PERK/CHOP signaling pathway. In this study, we preliminarily elucidated that QC alleviates ER stress-induced apoptosis in buffalo GCs, and the results suggest a novel strategy for delaying follicular atresia by inhibiting GCs apoptosis.

To illuminate the mechanisms underlying the high-altitude tolerance of Tibetan pig spermatozoa, proteomes of spermatozoa from Tibetan pigs raised in high and low altitudes were compared using a tandem mass tag (TMT)-labeled quantitative proteomics approach. A total of 77 differentially expressed proteins (DEPs) were identified. Gene Ontology (GO) analysis revealed DEPs that were predominantly associated with the actin cytoskeleton, the tricarboxylic acid (TCA) cycle, and adenosine triphosphate (ATP) metabolism, and were from 12 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Three subnetworks were significantly enriched and 10 centric proteins were identified by protein-protein interaction (PPI) network analysis. Relative expression levels of the proteins (ATP5H, CYCS, MYH9 and FN1) were confirmed using Western blotting. Our study is the first to use a tandem mass tag (TMT) approach to analyze Tibetan pig spermatozoa, and provides a foundation to understand the mechanisms underlying the reproductive adaptations of Tibetan pigs to high-altitude environments.