Autophagy, an evolutionarily conserved cell reprogramming mechanism, exists in all eukaryotic organisms. It is a fundamental and vital degradation/recycling pathway that removes undesirable components, such as cytoplasmic organelles, misfolded proteins, viruses, and intracellular bacteria, to provide energy and essential materials for organisms. The success of male reproduction depends on healthy testes, which are mainly composed of seminiferous tubules and mesenchyme. Seminiferous tubules are composed of Sertoli cells (SCs) and various germ cells, and the main functional part of mesenchyme are Leydig cells (LCs). In recent years, a large amount of evidence has confirmed that autophagy is active in many cellular events associated with the testes. Autophagy is not only important for testicular spermatogenesis, but is also an essential regulatory mechanism for the ectoplasmic specialization (ES) integrity of SCs, as well as for the normal function of the blood–testes barrier (BTB). At the same time, it is active in LCs and is crucial for steroid production and for maintaining testosterone levels. In this review, we expanded upon the narration regarding the composition of the testes; summarized the regulation and molecular mechanism of autophagy in SCs, germ cells, and LCs; and concluded the roles of autophagy in the process of spermatogenesis and testicular endocrinology. Through integrating the latest summaries and advances, we discuss how the role of autophagy is a double-edged sword in the testes and may provide insight for future studies and explorations on autophagy in male reproduction.

Infertility is a widespread clinical problem that affects human reproduction and species persistence worldwide. Around 40–70% of cases are due to male reproductive defects. Functional spermatogenesis (sperm production through several coordinated events) is at the heart of male fertility. Non-coding RNAs (ncRNAs) are the primary regulators of gene expression, controlling extensive critical cellular processes, for example proliferation, differentiation, apoptosis, and reproduction. Due to advancements in high-throughput sequencing tools, many studies have revealed that ncRNAs are widely expressed in germ cells, meiosis, spermatogenesis, sperm fertility, early post-fertilization development, and male infertility. The present review examines the biology and function of ncRNAs, including microRNAs, circular RNAs, and long ncRNAs, in spermatogenesis, their correlation with infertility, and their potential as biomarkers for sperm quality and fertility. The function of ncRNA in Sertoli cells (SCs) and Leydig cells (LCs) is also outlined throughout this study, because spermatogenesis requires testicular somatic cells to be involved in testicular development and male fertility. Meanwhile, the future development of ncRNAs for the clinical treatment of male infertility is also anticipated and discussed.

Yaks, a valuable livestock species endemic to China’s Tibetan plateau, have a low reproductive rate. Cryptorchidism is believed to be one of the leading causes of infertility in male yaks. In this study, we compared the morphology of the normal testis of the yak with that of the cryptorchidism, and found dysplasia of the seminiferous tubules, impaired tightness of the Sertoli cells, and a disruption of the integrity of the blood–testis barrier (BTB) in the cryptorchidism. Previous studies have shown that CAV1 significantly contributes to the regulation of cell tight junctions and spermatogenesis. Therefore, we hypothesize that CAV1 may play a regulatory role in tight junctions and BTB in Yaks Sertoli cells, thereby influencing the development of cryptorchidism. Additional analysis using immunofluorescence, qRT-PCR, and Western blotting confirmed that CAV1 expression is up-regulated in yak cryptorchidism. CAV1 over-expression plasmids and small RNA interference sequences were then transfected in vitro into yak Sertoli cells. It was furthermore found that CAV1 has a positive regulatory effect on tight junctions and BTB integrity, and that this regulatory effect is achieved through the FAK/ERK signaling pathway. Taken together, our findings, the first application of CAV1 to yak cryptorchidism, provide new insights into the molecular mechanisms of cell tight junctions and BTB. This paper suggests that CAV1 could be used as a potential therapeutic target for yak cryptorchidism and may provide insight for future investigations into the occurrence of cryptorchidism, the maintenance of a normal physiological environment for spermatogenesis and male reproductive physiology in the yak.

Long noncoding RNAs (LncRNAs) play essential roles in numerous biological processes in mammals, such as reproductive physiology and endocrinology. Cryptorchidism is a common male reproductive disease. Circadian rhythms are actively expressed in the reproductive system. In this study, a total of 191 LncRNAs were obtained from yak testes and cryptorchids. Then, we identified NTRK2’s relationship to circadian rhythm and behavioral processes. Meanwhile, the ceRNA (LncRNA-MSTRG.19083.1/miR-429-y/NTRK2) network was constructed, and its influence on circadian rhythm was revealed. The results showed that NTRK2 and LncRNA-MSTRG.19083.1 were significantly upregulated, and miR-429-y was obviously decreased in cryptorchid tissue; NTRK2 protein was mainly distributed in the Leydig cells of the testis. In addition, the upregulation of the expression level of miR-429-y resulted in the significant downregulation of LncRNA and NTRK2 levels, while the mRNA and protein levels of CREB, CLOCK, and BMAL1 were significantly upregulated; the knockdown of miR-429-y resulted in the opposite changes. Our findings suggested that LncRNA-MSTRG.19083.1 competitively binds to miR-429-y to target NTRK2 to regulate circadian rhythm through the cAMP pathway. Taken together, the results of our study provide a comprehensive understanding of how the LncRNA-miRNA-mRNA networks operate when yak cryptorchidism occurs. Knowledge of circadian-rhythm-associated mRNAs and LncRNAs could be useful for better understanding the relationship between circadian rhythm and reproduction.

The yak (Bos grunniens) is a valuable livestock animal endemic to the Qinghai–Tibet Plateau in China with low reproductive rates. Cryptorchidism is one of the primary causes of infertility in male yaks. Compared with normal testes, the tight junctions (TJs) of Sertoli cells (SCs) and the integrity of the blood–testis barrier (BTB) in cryptorchidism are both disrupted. MicroRNAs are hairpin-derived RNAs of about 19–25 nucleotides in length and are involved in a variety of biological processes. Numerous studies have shown the involvement of microRNAs in the reproductive physiology of yak. In this study, we executed RNA sequencing (RNA-seq) to describe the expression profiles of mRNAs and microRNAs in yaks with normal testes and cryptorchidism to identify differentially expressed genes. GO and KEGG analyses were used to identify the biological processes and signaling pathways which the target genes of the differentially expressed microRNAs primarily engaged. It was found that novel-m0230-3p is an important miRNA that significantly differentiates between cryptorchidism and normal testes, and it is down-regulated in cryptorchidism with p < 0.05. Novel-m0230-3p and its target gene CSF1 both significantly contribute to the regulation of cell adhesion and tight junctions. The binding sites of novel-m0230-3p with CSF1 were validated by a dual luciferase reporter system. Then, mimics and inhibitors of novel-m0230-3p were transfected in vitro into SCs, respectively. A further analysis using qRT-PCR, immunofluorescence (IF), and Western blotting confirmed that the expression of cell adhesion and tight-junction-related proteins Occludin and ZO-1 both showed changes. Specifically, both the mRNA and protein expression levels of Occludin and ZO-1 in SCs decreased after transfection with the novel-m0230-3p mimics, while they increased after transfection with the inhibitors, with p < 0.05. These were achieved via the CSF1/CSF1R/Ras signaling pathway. In summary, our findings indicate a negative miRNA-mRNA regulatory network involving the CSF1/CSF1R/Ras signaling pathway in yak SCs. These results provide new insights into the molecular mechanisms of CSF1 and suggest that novel-m0230-3p and its target protein CSF1 could be used as potential therapeutic targets for yak cryptorchidism.

Hormone-sensitive lipase (HSL) is a key enzyme in animal fat metabolism and is involved in the rate-limiting step of catalyzing the decomposition of fat and cholesterol. It also plays an important regulatory role in maintaining seminiferous epithelial structure, androgen synthesis and primordial germ cell differentiation. We previously reported that HSL is involved the synthesis of steroids in Bactrian camels, although it is unclear what role it plays in testicular development. The present study was conducted to characterize the biological function and expression pattern of the HSL gene in the hypothalamic pituitary gonadal (HPG) axis and the development of testis in Bactrian camels. We analyzed cloning of the cDNA sequence of the HSL gene of Bactrian camels by RT-PCR, as well as the structural features of HSL proteins, using bioinformatics software, such as ProtParam, TMHMM, Signal P 4.1, SOPMA and MEGA 7.0. We used qRT-PCR, Western blotting and immunofluorescence staining to clarify the expression pattern of HSL in the HPG axis and testis of two-week-old (2W), two-year-old (2Y), four-year-old (4Y) and six-year-old (6Y) Bactrian camels. According to sequence analysis, the coding sequence (CDS) region of the HSL gene is 648 bp in length and encodes 204 amino acids. According to bioinformatics analysis, the nucleotide and amino acid sequence of Bactrian camel HSL are most similar to those of Camelus pacos and Camelusdromedarius, with the lowest sequence similarity with Mus musculus. In adult Bactrian camel HPG axis tissues, both HSL mRNA and protein expression were significantly higher in the testis than in other tissues (hypothalamus, pituitary and pineal tissues) (p < 0.05). The expression of mRNA in the testis increased with age and was the highest in six-year-old testis (p < 0.01). The protein expression levels of HSL in 2Y and 6Y testis were clearly higher than in 2W and 4Y testis tissues (p < 0.01). Immunofluorescence results indicate that the HSL protein was mainly localized in the germ cells, Sertoli cells and Leydig cells from Bactrian camel testis, and strong positive signals were detected in epididymal epithelial cells, basal cells, spermatocytes and smooth muscle cells, with partially expression in hypothalamic glial cells, pituitary suspensory cells and pineal cells. According to the results of gene ontology (GO) analysis enrichment, HSL indirectly regulates the anabolism of steroid hormones through interactions with various targets. Therefore, we conclude that the HSL gene may be associated with the development and reproduction of Bactrian camels in different stages of maturity, and these results will contribute to further understanding of the regulatory mechanisms of HSL in Bactrian camel reproduction.