This study investigated the effects of supplementing bucks semen extenders with Vitamin A (Vit A) and Lupeol (Lup) on post-thawing quality. Pooled semen from six bucks was cryopreserved using extenders containing varying concentrations of Vit A and Lup. Post-thawing assessments included sperm kinematic parameters, plasma membrane and acrosome integrity, antioxidant enzyme activities, and the expression of antioxidant and apoptosis related genes. The results showed that the addition of 0.75 μM Vit A significantly improved post-thaw sperm motility, kinematic velocities (VSL, VCL, VAP), membrane integrity, and acrosome integrity compared to the control. Compared with the control group, this group also exhibited the significantly improved activities of SOD, CAT, and GSH-Px, along with significantly upregulated expression of the antioxidant genes SOD1 and NRF2. Based on these findings, we further evaluated Lup. Compared with the control group, the addition of 2 μM Lup significantly enhanced sperm motility, VSL, WOB, and antioxidant enzyme activity. It also increased the expression of SOD1, NRF2, GPX4, and CAT, while reducing the transcriptional levels of the apoptotic genes Caspase 3 and P53. Furthermore, compared to fresh semen, frozen semen supplemented with 0.75 μM Vit A and 2 μM Lup exhibited good fertilization capacity, although no significant differences were observed in cleavage rate or blastocyst rate. In conclusion, Vit A and Lup act synergistically in bucks semen cryopreservation. They enhance antioxidant capacity, suppress apoptosis-related gene expression, and mitigate oxidative stress during freeze-thawing, thereby improving semen quality.

Objective: This study aimed to investigate the potential of the ASAP1 gene as a genetic biomarker for brucellosis resistance/susceptibility in goats.Methods: This study collected samples from female Shaanbei white cashmere (SBWC) goats to investigate the association between the ASAP1 gene and brucellosis susceptibility. Peripheral blood mononuclear cells (PBMCs) were isolated from goats with various haplo types and Brucella statuses, and the association was evaluated using polymerase chain re action (PCR), quantitative reverse transcription (qRT)-PCR, and lipopolysaccharide (LPS) stimulation assays.Results: The ASAP1 gene was expressed most in th e spleen, significantly more than in the kidney and heart (p<0.05). In the SBWC goats population, three genotypes insertion/insertion (II), insertion/deletion (ID), and deletion/deletion (DD) were identified at the P2, P5, and P7 sites of goat ASAP1 gene. Association analysis showed that P2 and P7 sites variants were associated with host resistance to Brucella infection with the II genotype used as reference (p<0.05; p<0.01) and maintained after multiple testing correction. The ASAP1 gene was observed lower expression in testicular tissues of Brucella-infected adult SBWC goats compared to healthy controls (p<0.01). Haplotype analysis revealed Hap3 and Hap5 were associated with brucellosis-resistant compared to Hap1 (p<0.05). PBMCs were isolated from goats carrying Hap1, Hap3, and Hap5. After LPS stimulation, significantly reduced ASAP1 expression was detected in the susceptible haplotype Hap1 compared to the resistant haplotypes. The highest expression level was exhibited by the most resistant haplotype, Hap5. Furthermore, resistant haplotypes showed more rapid activation of key inflammatory pathways and pro-inflammatory cytokines (NF-κB, IL-6, TNF-α, IFN-γ) compared to susceptible Hap1, with faster resolution of the inflammatory response observed, particularly in the most resistant haplotype Hap5.Conclusion: The present study demonstrates that ASAP1 gene InDel variants influence brucellosis resistance in SBWC goats, providing a theoretical basis for breeding resistant populations.

Objective This study aimed to investigate the potential of the ASAP1 gene as a genetic biomarker for brucellosis resistance/susceptibility in goats. Methods This study collected samples from female Shaanbei white cashmere (SBWC) goats to investigate the association between the ASAP1 gene and brucellosis susceptibility. Peripheral blood mononuclear cells (PBMCs) were isolated from goats with various haplo types and Brucella statuses, and the association was evaluated using polymerase chain re action (PCR), quantitative reverse transcription (qRT)-PCR, and lipopolysaccharide (LPS) stimulation assays. Results The ASAP1 gene was expressed most in the spleen, significantly more than in the kidney and heart (p<0.05). In the SBWC goats population, three genotypes insertion/insertion (II), insertion/deletion (ID), and deletion/deletion (DD) were identified at the P2, P5, and P7 sites of goat ASAP1 gene. Association analysis showed that P2 and P7 sites variants were associated with host resistance to Brucella infection with the II genotype used as reference (p<0.05; p<0.01) and maintained after multiple testing correction. The ASAP1 gene was observed lower expression in testicular tissues of Brucella-infected adult SBWC goats compared to healthy controls (p<0.01). Haplotype analysis revealed Hap3 and Hap5 were associated with brucellosis-resistant compared to Hap1 (p<0.05). PBMCs were isolated from goats carrying Hap1, Hap3, and Hap5. After LPS stimulation, significantly reduced ASAP1 expression was detected in the susceptible haplotype Hap1 compared to the resistant haplotypes. The highest expression level was exhibited by the most resistant haplotype, Hap5. Furthermore, resistant haplotypes showed more rapid activation of key inflammatory pathways and pro-inflammatory cytokines (NF-κB, IL-6, TNF-α, IFN-γ) compared to susceptible Hap1, with faster resolution of the inflammatory response observed, particularly in the most resistant haplotype Hap5. Conclusion The present study demonstrates that ASAP1 gene InDel variants influence brucellosis resistance in SBWC goats, providing a theoretical basis for breeding resistant populations.

This study aimed to evaluate the efficacy of estrus synchronization protocols in goats and to identify the most cost-effective regimen for intensive farming. The protocols assessed used progesterone (P4) sponges or controlled internal drug release (CIDR) devices, with supplemented with pregnant mare serum gonadotropin (PMSG) and prostaglandin (PG), for inducing estrus and pregnancy in goats, and to identify the most cost-effective regimen for intensive farming. During both breeding and non-breeding seasons, does were assigned totreatments: Group I (P4 sponge + PMSG), Group II (P4 sponge + PMSG + PG), and Group III (CIDR + PMSG + PG). We measured estrus response after device removal and pregnancy rates after artificial insemination (AI) were evaluated and analyzed serum hormone dynamics. Additionally, medication costs were calculated for each protocol. For the most effective protocol, we also determined ovulation timing, performed laparoscopic-assisted AI. Estrus onset occurred earlier in Groups II and III than in Group I during both trial seasons. Nevertheless, overall estrus and pregnancy rates did not differ significantly among the groups (P > 0.05), with Group II exhibiting the lowest pregnancy rate. Medication cost per does and per pregnancy were lowest for Group I. Under the Group I protocol, ovulation occurred at 32 h after estrus onset, and laparoscopic-assisted AI yielded a significantly higher pregnancy rate with fresh semen than with chilled semen (P < 0.05). Serum estradiol (E2) concentration at estrus onset was significantly higher in Group I than in Group II (P < 0.05), and luteinizing hormone (LH) levels at estrus onset and ovulation were significantly higher in Groups I and III than in Group II (P < 0.05). Serum P4, LH, and E2 profiles during the estrous cycle followed similar patterns across all groups. In conclusion, there was no significant difference in pregnancy rates between the three estrus synchronization protocols. However, Protocol I demonstrated the lowest per doe and per pregnancy medication costs. Based on cost-effectiveness considerations, the P4 sponge + PMSG protocol is recommended for use in intensive goat production systems.

Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1, also known as BLOS1) is a key gene involved in phagosome-lysosome maturation, transport, and autophagosome fusion, and it plays a crucial role in host resistance to Brucella infection. This study aimed to examine the effects of BLOS1 overexpression (oeBLOS1) on the stress response of goat macrophages and on intestinal microbiota composition. Peripheral blood mononuclear cells (PBMCs) were isolated from oeBLOS1 and wild-type (WT) goats and differentiated into macrophages. These macrophages were then stimulated with Brucella LPS to assess cytokine secretion and autophagy levels. Metagenomic sequencing was also performed to analyze the structural and functional profiles of the rectal fecal microbiota in these goats. After Brucella LPS stimulation, oeBLOS1 goat macrophages rapidly activated the NF-κB and TLR4 signaling pathways, promoting the synthesis and secretion of cytokines such as TNF-α (P < 0.05). Brucella LPS challenge also significantly increased the transcription of autophagy-related genes such as LAMP2 and BECN1, enhancing autophagic activity and bacterial clearance (P < 0.05). Furthermore, oeBLOS1 altered the intestinal microbiota, significantly enriching pathways linked to membrane transport and cell motility, and reducing the abundance of virulence factors and opportunistic pathogens, which may contribute to intestinal immune homeostasis. In summary, oeBLOS1 may help counteract Brucella LPS-induced infection by promoting the immune response, enhancing autophagy. In addition, it is associated with remodeling gut microbial function, suggesting a potential role in disease resistance.

Brucellosis, a zoonotic disease caused by Brucella infection, poses a major threat to both global health and livestock productivity. Although reproductive impairment is well established, the molecular mechanisms driving testicular immunopathology remain poorly understood. In this study, single-cell RNA sequencing was used to delineate transcriptional changes in goat testicular tissues under physiological and Brucella-infected conditions, revealing dynamic immunological remodeling of the testicular microenvironment. Infection induced marked shifts in T cell and macrophage phenotypes, with T cells exhibiting pronounced hyperactivation linked to CD45-mediated signaling cascades. Thioredoxin-interacting protein (TXNIP), a gene strongly up-regulated in response to infection, emerged as a potential immunotherapeutic target. Intercellular communication networks were significantly disrupted in infected testes, with CD39-and JAM-dependent signaling pathways implicated in the erosion of immune privilege. Regulon analysis further identified GATA3, IRF5, SEMA4A, and HCLS1 as transcriptional regulators associated with T cells and macrophages in infected testes. These findings provide novel insights into the molecular mechanisms driving testicular immunopathology during Brucella infection and highlight candidate targets for immunomodulatory intervention in disease control and livestock reproductive health.

Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is considered to have anti-Brucella potential. However, the effect of BLOC1S1 on Brucella autophagy has not yet been studied. This study investigates the interplay between Brucella lipopolysaccharide (LPS) and BLOC1S1 in modulating autophagy within goat spermatogonial stem cells (mGSCs-I-SB). Using LPS from B. melitensis 16M, its capacity is demonstrated to induce AMPK-dependent autophagy, contrasting with Escherichia coli LPS, which shows no significant effect. Mechanistically, B. melitensis 16M LPS activates AMPK signaling, elevates LC3B-II/LC3B-I ratios, and upregulates lysosomal and pro-inflammatory genes. BLOC1S1 overexpression attenuates autophagy, reducing autolysosome formation (TEM) and LC3B-II/I ratio. RNA sequencing and proteomic analyses reveal BLOC1S1-mediated transcriptional reprogramming of lysosomal pathways and mitochondrial metabolism. Co-immunoprecipitation and subcellular localization studies reveal that TDP-43 is a key interacting partner and that BLOC1S1 sequesters TDP-43 in the cytoplasm, inhibiting its nuclear translocation-dependent ATG7 mRNA stability and enhancing autophagy. These findings delineate a dual regulatory mechanism: B. melitensis 16M LPS-driven, AMPK-dependent autophagy induction, and BLOC1S1-mediated autophagic suppression through spatial control of TDP-43. These results advance understanding of host-pathogen interactions in brucellosis and identify BLOC1S1 as a potential therapeutic target for bacterial persistence and TDP-43-related pathologies.

Brucella, an intracellular facultative coccidia, causes brucellosis, which poses a significant threat to livestock farming and public health, and screening for candidate genes associated with resistance to brucellosis is considered an effective strategy for controlling the transmission and infection of this disease. In this context, we detected InDel genetic variants of the tumor necrosis factor (TNF) and protein tyrosine phosphatase receptor T (PTPRT) genes in the Shaanbei White Cashmere (SWBC) goat and analyzed the correlation between their polymorphisms and the risk of brucellosis infection in goats. The results indicated that the TNF rs669191919 and PTPRT rs639317914 loci were polymorphic in the examined goat populations. Both loci exhibited a 13 bp InDel deletion and resulted in three genotypes: insertion/insertion (II), insertion/deletion (ID), and deletion/deletion (DD), with II genotypes and I alleles occurring at higher frequencies. The polymorphism information content (PIC) values suggested that both InDel variant loci were moderately polymorphic (0.25 < PIC <0.50). Furthermore, association analysis revealed that none of the four established genetic models codominant, dominant, recessive, and allele showed an association between the polymorphisms at the rs669191919 and rs639317914 loci and the risk of brucellosis in goats (P > 0.05). Bioinformatics analyses indicated that the rs669191919 and rs639317914 loci specifically bind to the transcription factors upstream transcription factor 1 (USF1) and nescient helix-loop-helix 1 (NHLH1), respectively. In summary, our findings suggest that polymorphisms at the TNF rs669191919 and PTPRT rs639317914 loci do not influence resistance to brucellosis in goats. However, investigations into the specific binding of these polymorphic loci to transcription factors may represent a novel avenue for exploring the mechanisms underlying resistance to brucellosis in livestock.

Spermatogonial stem cells (SSCs) are the key to maintaining production of the sperms and healthy offsprings, and also treating breeding livestock’s reproductive damage and infertility. microRNAs act a decisive role in regulating gene expression in many cells and tissues, including in processes such as proliferation, self-renewal, differentiation, and apoptosis of stem cells. However, the miRNA mechanism in regulation of SSCs is still unclear. Here, high-throughput sequencing was used to identify specific miRNAs. We confirmed that miR-21-5p was concentrated in both goat and mouse SSCs, and enhanced the proliferation and antiapoptotic ability of SSCs. In vivo experiments have shown that miR-21-5p resisted the damage of the chemotherapy drug Busulfan to germ cells, ameliorated Busulfan-induced testicular dysfunction, and maintained spermatogenesis. Further RNA-seq and target gene prediction revealed that SPRY1 and FASLG are targets of miR-21-5p, thereby activating downstream signaling pathways such as MAPK/ERK, PI3K-AKT, and apoptosis. In summary, miR-21-5p is crucial for the self-renewal and maintenance of SSCs. This study provides new avenues for treating breeding livestock’s reproductive damages, infertility, oligospermia, and other conditions.

Prolactin is a single-chain peptide hormone produced by the anterior pituitary gland and plays an important role in the reproductive development of mammals. Therefore, we investigated the expression of the PRLR gene in various goat tissues during distinct developmental stages, as well as the correlation between the 16 bp InDel and growth traits in goats. The study findings demonstrated a consistent expression and distribution of PRLR in both yearling and adult goat tissues, with the highest levels observed in the testis. Association analysis showed that in the yearling IMWC goat population (n = 463), this 16 bp InDel variation was associated with body weight (P = 0.026), body height (P = 0.005), body length (P = 0.008), heart girth (P = 0.001), chest depth (P = 0.000075), and chest width (P = 0.005); In the adult IMWC goat population (n = 211), the 16 bp InDel variation was associated with body height (P = 0.011), height across the hip (P = 0.0003), chest depth (P = 0.001), and cashmere fineness (P = 0.022); In all IMWC goat populations (n = 674), this 16 bp InDel was related to body weight (P = 0.042), body height (P = 0.001), body length (P = 0.013), heart girth (P = 0.012), height across the hip (P = 0.038), chest depth (P = 0.001), and chest width (P = 0.006). The bioinformatics analysis has revealed that the significant impact of a 16 bp InDel on growth traits in goats may be attributed to its specific binding to the transcription factors AP-2α and Sp1. These findings suggest that this genetic variant plays a crucial role in the growth and development of goats, making it a valuable DNA marker for selecting goats with superior growth traits.

Disruptions in testicular homeostasis can lead to impaired spermatogenesis and male infertility. Such disturbances may result from various factors, including viral or bacterial infections, toxic injuries, and genetic mutations or deletions. The maintenance of testicular homeostasis is governed by a complex interplay of various cells, hormones, paracrine factors, genes, and enzymes. UCHL1, a member of the deubiquitinating enzyme family, is recognized for its role in neuronal function. However, its contribution to testicular homeostasis and spermatogenesis remains unclear. This study uncovers a critical role for Uchl1 in maintaining testicular homeostasis, acting as a regulatory switch for spermatogenesis. We demonstrate that Uchl1 knockout (Uchl1_KO) mice exhibit reduced body weight, decreased testicular specific gravity, and impaired spermatogenesis. Single-nucleus RNA sequencing (snRNA-seq) analysis of Uchl1_KO testes reveals a significant decrease in oxidative phosphorylation (OXPHOS) levels and an increase in Sertoli cell abnormalities. Notably, Uchl1_KO/knockdown downregulates metabolism-related adiponectin signaling (ADIPOR1/AMPK) and upregulates the inflammation-related SEMA7A/PLXNC1 pathway. Sertoli cell lines (oeAdipor1/shUchl1) confirm UCHL1's dual regulatory role in these signaling pathways in vitro experiments. Our findings identify UCHL1 as a key regulator of testicular homeostasis and spermatogenesis, and it dynamically controls the balance between metabolic and inflammatory signaling in the testis. This study provides a valuable theoretical foundation for exploring the molecular mechanisms underlying testicular homeostasis balance and for advancing human reproductive health.

TYROBP (also known as DAP12), a transmembrane signaling adaptor protein, activates downstream signaling cascades through its immunoreceptor tyrosine-based activation motif (ITAM) by recruiting tyrosine kinase SYK. TYROBP plays an important role in various immune responses, including inflammation and phagocytosis. Although the role of TYROBP in defense against bacterial infections has been partially investigated, its mechanism in Brucella infection is unclear. Using Tyrobp-knockout RAW264.7 macrophages, we demonstrated that gene deletion impaired macrophage phagocytosis. Following B. abortus lipopolysaccharide (LPS) stimulation, Tyrobp deficiency exacerbated mitochondrial damage and enhanced NF-κB phosphorylation. Quantitative proteomics identified SQSTM1 as a TYROBP-interacting partner, and its expression in knockout cells was reduced under LPS treatment conditions in Western blot results (P = 0.0887). RNAi-mediated SQSTM1 depletion phenocopied TYROBP ablation, recapitulating mitochondrial dysfunction and NF-κB hyperactivation. These results suggest that TYROBP inhibits NFκB phosphorylation through upregulation of SQSTM1, affecting macrophage activation and intracellular bacterial clearance. The study also suggests that the TYROBP-SQSTM1-NFκB signaling pathway may play a key role in Brucella infection, providing a new perspective for understanding the mechanism of Brucella infection.

The cytotoxic T lymphocyte-associated antigen-4 (CTLA4) gene, a member of the immunoglobulin superfamily, is crucial for maintaining immune homeostasis and preventing autoimmune diseases. Studies have shown that polymorphisms in the CTLA4 gene are linked to an increased risk of brucellosis in humans, but its association with brucellosis in goats remains unexplored. In this study, the tissue expression profile of CTLA4 in goats was investigated, and the correlation between InDel polymorphisms in the CTLA4 gene and susceptibility to brucellosis in goats was examined. The findings reveal the widespread expression of CTLA4 in goat tissues, particularly in the spleen and testes. The tested goat populations presented genotypes insertion/insertion (II), insertion/deletion (ID), and deletion/deletion (DD) at both the P1 and P2 loci, and an association analysis revealed significant differences in the distribution of genotypes and allele frequencies at the P1 and P2 loci of the CTLA4 gene between the Brucella goat case and the control groups (p < 0.05). Specifically, compared with the II genotype, the P1 and P2 loci were significantly associated with an elevated risk of brucellosis development in goats under both the codominant (ID/II) and dominant (ID + DD/II) models (P1, p = 0.042, p = 0.016; P2, p = 0.011, p = 0.014). Additionally, haplotype analysis indicated that haplotypes IP1DP2, DP1IP2, and DP1DP2 were significantly associated with an increased risk of brucellosis in goats compared to the reference haplotype IP1IP2 (p = 0.029, p = 0.012, p = 0.034). Importantly, the Lipopolysaccharide (LPS) stimulation of peripheral blood monocytes and/or macrophages from goats with the II, ID, and DD genotypes resulted in increased CTLA4 expression levels in the II genotype, leading to a robust LPS-induced inflammatory response. Through bioinformatic analysis, the observed effect of the InDel locus on Brucella pathogenesis risk in goats could be attributed to the differential binding of the transcription factors nuclear factor kappaB (NF-κB) and CCAAT/enhancer-binding protein α (C/EBPα). These findings offer potential insights for breeding strategies against brucellosis.

The male reproductive system has a standard immune response regulatory mechanism, However, a variety of external stimuli, including viruses, bacteria, heat, and medications can damage the testicles and cause orchitis and epididymitis. It has been shown that various RNA viruses are more likely to infect the testis than DNA viruses, inducing orchitis and impairing testicular function. It was found that local injection of the viral RNA analog poly(I:C) into the testes markedly disrupted the structure of the seminiferous tubules, accompanied by apoptosis and inflammation. Poly(I:C) mainly inhibited the expression of testosterone synthesis-associated proteins, STAR and MGARP, and affected the synthesis and metabolism of amino acids and lipids in the testis. This led to the disruption of the metabolite levels in the testis of mice, thus affecting the normal spermatogenesis process. The present study analyzed the acute inflammatory response of the testis to viral infection using a multi-omics approach. It provides insights into how RNA virus infection impairs testicular function and offers a theoretical basis for future studies on immune homeostasis and responses under stress conditions in male reproduction.

Spermatogonial stem cells (SSCs) are the key to maintaining production of the sperms and healthy offsprings, and also treating breeding livestock's reproductive damage and infertility. MicroRNAs act a decisive role in regulating gene expression in many cells and tissues, including in processes such as proliferation, self-renewal, differentiation, and apoptosis of stem cells. However, the miRNA mechanism in regulation of SSCs is still unclear. Here, high-throughput sequencing was used to identify specific miRNAs. We confirmed that miR-21-5p was concentrated in both goat and mouse SSCs, and enhanced the proliferation and antiapoptotic ability of SSCs. In vivo experiments have shown that miR-21-5p resisted the damage of the chemotherapy drug Busulfan to germ cells, ameliorated Busulfan-induced testicular dysfunction, and maintained spermatogenesis. Further RNA-seq and target gene prediction revealed that SPRY1 and FASLG are targets of miR-21-5p, thereby activating downstream signaling pathways such as MAPK/ERK, PI3K-AKT, and apoptosis. In summary, miR-21-5p is crucial for the self-renewal and maintenance of SSCs. This study provides new avenues for treating breeding livestock's reproductive damages, infertility, oligospermia, and other conditions.

Spermatogonial stem cells (SSCs) play a crucial role in mammalian spermatogenesis and maintain the stable inheritance of the germline in livestock. However, stress and bacterial or viral infections can disrupt immune homeostasis of the testes, thereby leading to spermatogenesis destruction and infertility, which severely affects the health and productivity of mammals. This study aimed to explore the effect of ubiquitin C-terminal hydrolase L1 (UCHL1) knockdown (KD) in goat SSCs and mouse testes and investigate the potential anti-inflammatory function of UCHL1 in a poly(I:C)-induced inflammation model to maintain microenvironmental homeostasis. In vitro, the downregulation of UCHL1 (UCHL1 KD) in goat SSCs increased the expression levels of apoptosis and inflammatory factors and inhibited the self-renewal and proliferation of SSCs. In vivo, the structure of seminiferous tubules and spermatogenic cells was disrupted after UCHL1 KD, and the expression levels of apoptosis- and inflammation-related proteins were significantly upregulated. Furthermore, UCHL1 inhibited the TLR3/TBK1/IRF3 pathway to resist poly(I:C)-induced inflammation in SSCs by antagonizing HSPA8 and thus maintaining SSC autoimmune homeostasis. Most importantly, the results of this study showed that UCHL1 maintained immune homeostasis of SSCs and spermatogenesis. UCHL1 KD not only inhibited the self-renewal and proliferation of goat SSCs and spermatogenesis but was also involved in the inflammatory response of goat SSCs. Additionally, UCHL1 has an antiviral function in SSCs by antagonizing HSPA8, which provides an important basis for exploring the specific mechanisms of UCHL1 in goat spermatogenesis.

With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.

Eif2s3y (eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked, Eif2s3y) is an essential gene for spermatogenesis. Early studies have shown that Eif2s3y can promote the proliferation of spermatogonial stem cells (SSCs) and can replace the Y chromosome together with sex-determining region Y (Sry) to transform SSCs into round spermatozoa. We injected lentiviral particles into the seminiferous tubules of mouse testes by sterile surgery surgically to establish overexpressing Eif2s3y testes. And then the mice were intraperitoneally injected with LPS to established the model of testis inflammation. Through RNA sequencing, qRT-PCR analysis, Western blot, co-culture etc., we found that Eif2s3y alleviated LPS-induced damage in mouse testes and maintained spermatogenesis. In testes with Eif2s3y overexpression, the seminiferous tubules were more regularly organized after exposure to LPS compared with the control. Eif2s3y performs its function by negatively regulating Adamts5 (a disintegrin and metalloproteinase containing a thrombospondin-1 motif), an extracellular matrix-degrading enzyme. ADAMTS5 shows a disruptive effect when the testis is exposed to LPS. Overexpression of Eif2s3y inhibited the TLR4/NFκB signaling pathway in the testis in response to LPS. Generally, our research shows that Eif2s3y protects the testis from LPS and maintains spermatogenesis by negatively regulating Adamts5.

[目的]探究内蒙古绒山羊KAP基因家族角蛋白相关蛋白9.2(KAP9.2)基因插入/缺失(insertion/deletion,InDel)突变及其与生长性状的关系,以期为内蒙古绒山羊分子育种提供理论基础.[方法]选取606只雌性阿拉善型内蒙古绒山羊为研究对象,采集耳组织提取DNA并测定其体重及体高、体长和胸围等体尺数据,利用琼脂糖凝胶电泳方法与直接测序技术检测KAP9.2基因的InDel突变,检测遗传多样性指标,利用SPSS 23.0软件分析突变位点与生长性状的关联性.[结果]在内蒙古绒山羊KAP9.2基因外显子区存在30 bp的InDel突变,产生II、ID和DD 3种基因型.多态信息含量(PIC)显示,该位点在育成羊群体中属于中度多态(0.25＜PIC＜0.50),在成年羊群体中属于低度多态(PIC＜0.25),且均处于哈代-温伯格平衡状态(P＞0.05);在整个群体中,属于低度多态(PIC＜0.25),但偏离哈代-温伯格平衡状态(P＜0.05).关联分析结果表明,在育成羊群体中,此30 bp的InDel突变对体长和十字部高有显著影响(P＜0.05),对体高、胸深和胸宽有极显著影响(P＜0.01);在成年羊群体中,此突变对管围、胸深和胸宽有极显著影响(P＜0.01).进一步的分析发现,在育成羊和成年羊全部群体中,此突变对体长和十字部高有显著影响(P＜0.05),对体高、胸深和胸宽有极显著影响(P＜0.01).[结论]KAP9.2基因InDel突变对阿拉善型内蒙古绒山羊多个生长性状有显著或极显著影响,可作为内蒙古绒山羊优良性状选育的分子标记.