Apoptosis is a tightly controlled process by which tissues eliminate unwanted cells. Spontaneous germ cell apoptosis in testis has been broadly investigated in mammals that have an associated spermatogenesis pattern. However, the mechanism of germ cell apoptosis in seasonally breeding reptiles following a dissociated spermatogenesis has remained enigmatic. In the present study, morphological evidence has clearly confirmed the dissociated spermatogenesis pattern in Pelodiscus sinensis. TUNEL and TEM analyses presented dynamic changes and ultrastructural characteristics of apoptotic germ cells during seasonal spermatogenesis, implying that apoptosis might be one of the key mechanisms to clear degraded germ cells. Furthermore, using RNA-Seq and digital gene expression (DGE) profiling, a large number of apoptosis-related differentially expressed genes (DEGs) at different phases of spermatogenesis were identified and characterized in the testis. DGE and RT-qPCR analysis revealed that the critical anti-apoptosis genes, such as Bcl-2, BAG1, and BAG5, showed up-regulated patterns during intermediate and late spermatogenesis. Moreover, the increases in mitochondrial transmembrane potential in July and October were detected by JC-1 staining. Notably, the low protein levels of pro-apoptotic cleaved caspase-3 and CytC in cytoplasm were detected by immunohistochemistry and western blot analyses, indicating that the CytC-Caspase model might be responsible for the effects of germ cell apoptosis on seasonal spermatogenesis. These results facilitate understanding the regulatory mechanisms of apoptosis during spermatogenesis and uncovering the biological process of the dissociated spermatogenesis system in reptiles.

Previously, we have shown that human sperm Prohibitin (PHB) expression is significantly negatively correlated with mitochondrial ROS levels but positively correlated with mitochondrial membrane potential and motility. However, the possible role of PHB in mammalian spermatogenesis has not been investigated. Here we document the presence of PHB in spermatocytes and its functional roles in meiosis by generating the first male germ cell-specific Phb-cKO mouse. Loss of PHB in spermatocytes resulted in complete male infertility, associated with not only meiotic pachytene arrest with accompanying apoptosis, but also apoptosis resulting from mitochondrial morphology and function impairment. Our mechanistic studies show that PHB in spermatocytes regulates the expression of STAG3, a key component of the meiotic cohesin complex, via a non-canonical JAK/STAT pathway, and consequently promotes meiotic DSB repair and homologous recombination. Furthermore, the PHB/JAK2 axis was found as a novel mechanism in the maintenance of stabilization of meiotic STAG3 cohesin complex and the modulation of heterochromatin formation in spermatocytes during meiosis. The observed JAK2-mediated epigenetic changes in histone modifications, reflected in a reduction of histone 3 tyrosine 41 phosphorylation (H3Y41ph) and a retention of H3K9me3 at the Stag3 locus, could be responsible for Stag3 dysregulation in spermatocytes with the loss of PHB.

The Cullin-RING ubiquitin-ligase CRL4 controls cell cycle and DNA damage checkpoint response and ensures genomic integrity. Inactivation of the Cul4 component of the CRL4 E3 ligase complex in Caenorhabditis elegans by RNA interference results in massive mitotic DNA re-replication in the blast cells, largely due to failed degradation of the DNA licensing protein, CDT-1, and premature spermatogenesis. Here we show that inactivation of Cul4a by gene-targeting in mice only affected male but not female fertility. This male infertility phenotype resulted from a combination of decreased spermatozoa number, reduced sperm motility and defective acrosome formation. Agenesis of the mutant germ cells was accompanied by increased cell death in pachytene/diplotene cells with markedly elevated levels of phospho-p53 and CDT-1. Despite apparent normal assembly of synaptonemal complexes and DNA double strand break repair, dissociation of MLH1, a component of the late recombination nodule, was delayed in Cul4a -/- diplotene spermatocytes, which potentially led to subsequent disruptions in meiosis II and spermiogenesis. Together, our study revealed an indispensable role for Cul4a during male germ cell meiosis.

Testis development and spermatogenesis are strictly regulated by numbers of genes and non-coding genes. However, long non-coding RNAs (lncRNAs) as key regulators in multitudinous biological processes have not been systematically identified in bovine testes during sexual maturation. In this study, we comprehensively analyzed lncRNA and mRNA expression profiling of six bovine testes at 3 days after birth and 13 months by RNA sequencing. 23,735 lncRNAs and 22,118 mRNAs were identified, in which 540 lncRNAs (P-value < 0.05) and 3,525 mRNAs (P-adjust < 0.05) were significantly differentially expressed (DE) between two stages. Correspondingly, the results of RT-qPCR analysis showed well correlation with the transcriptome data. Moreover, GO and KEGG enrichment analyses showed that DE genes and target genes of DE lncRNAs were enriched in spermatogenesis. Furthermore, we constructed lncRNA-gene interaction networks; consequently, 15 DE lncRNAs and 12 cis-target genes were involved. The target genes (SPATA16, TCF21, ZPBP, PACRG, ATP8B3, COMP, ACE, and OSBP2) were found associated with bovine sexual maturation. In addition, the expression of lncRNAs and cis-target genes was detected in bovine Leydig cells, Sertoli cells, and spermatogonia. Our study identified and analyzed lncRNAs and mRNAs in testis tissues, suggesting that lncRNAs may regulate testis development and spermatogenesis. Our findings provided new insights for further investigation of biological function in bovine lncRNA.

Hereditary primary hypogonadism (HPH), caused by gene mutation related to testosterone synthesis in Leydig cells, usually impairs male sexual development and spermatogenesis. Genetically corrected stem Leydig cells (SLCs) transplantation may provide a new approach for treating HPH. Here, a novel nonsense-point-mutation mouse model (LhcgrW495X ) is first generated based on a gene mutation relative to HPH patients. To verify the efficacy and feasibility of SLCs transplantation in treating HPH, wild-type SLCs are transplanted into LhcgrW495X mice, in which SLCs obviously rescue HPH phenotypes. Through comparing several editing strategies, optimized PE2 protein (PEmax) system is identified as an efficient and precise approach to correct the pathogenic point mutation in Lhcgr. Furthermore, delivering intein-split PEmax system via lentivirus successfully corrects the mutation in SLCs from LhcgrW495X mice ex vivo. Gene-corrected SLCs from LhcgrW495X mice exert ability to differentiate into functional Leydig cells in vitro. Notably, the transplantation of gene-corrected SLCs effectively regenerates Leydig cells, recovers testosterone production, restarts sexual development, rescues spermatogenesis, and produces fertile offspring in LhcgrW495X mice. Altogether, these results suggest that PE-based gene editing in SLCs ex vivo is a promising strategy for HPH therapy and is potentially leveraged to address more hereditary diseases in reproductive system.

Circular RNA (circRNA) is a new class of non-coding RNA that has recently attracted researchers' interest. Studies have demonstrated that circRNA can function as microRNA sponges or competing endogenous RNAs. Although circRNA has been explored in some species and tissues, the genetic basis of testis development and spermatogenesis in cattle remains unknown. We performed ribo-depleted total RNA-Seq to detect circRNA expression profiles of neonatal (one week old) and adult (4 years old) Qinchuan cattle testes. We obtained 91 112 596 and 80 485 868 clean reads and detected 21 753 circRNAs. A total of 4248 circRNAs were significantly differentially expressed between neonatal and adult cattle testes. Among these circRNAs, 2225 were upregulated, and 2023 were downregulated in adult cattle testis. Genomic feature, length distribution and other characteristics of the circRNAs in cattle testis were studied. Moreover, Gene Ontology and KEGG pathway analyses were performed for source genes of circRNAs. These source genes were mainly involved in tight junction, adherens junction, TGFβ signalling pathway and reproduction, such as PIWIL1, DPY19L2, SLC26A8, IFT81, SMC1B, IQCG and TTLL5. CircRNA expression patterns were validated by RT-qPCR. Our discoveries provide a solid foundation for the identification and characterization of key circRNAs involved in testis development or spermatogenesis.

Spermatogenesis is a complex biological process regulated by well-coordinated gene regulation, including MicroRNAs (miRNAs). miRNAs are endogenous non-coding ribonucleic acids (ncRNAs) that mainly regulate the gene expression at post-transcriptional levels. Several studies have reported miRNAs expression in bull sperm and the process of spermatogenic arrest in cattle and yak. However, studies for the identification of differential miRNA expression and its mechanisms during the developmental stages of testis still remain uncertain. In the current study, we comprehensively analyzed the expression of miRNA in bovine testes at neonatal (3 days after birth, n = 3) and mature (13 months, n = 3) stages by RNA-seq. Moreover, the role of bta-miR-146b was also investigated in regulating the proliferation and apoptosis of bovine male germline stem cells (mGSCs) followed by a series of experiments. A total of 652 miRNAs (566 known and 86 novel miRNAs) were identified, whereas 223 miRNAs were differentially expressed between the two stages. Moreover, an elevated expression level of bta-miR-146b was found in bovine testis among nine tissues, and the functional studies indicated that the overexpression of bta-miR-146b inhibited the proliferation of bovine mGSCs and promoted apoptosis. Conversely, regulation of bta-miR-146b inhibitor promoted bovine mGSCs proliferation. This study provides a basis for understanding the regulation roles of miRNAs in bovine testis development and spermatogenesis.

Spermatogenesis defects are important for male infertility; however, the etiology and pathogenesis are still unknown. Herein, we identified two loss-of-function mutations of STK33 in seven individuals with non-obstructive azoospermia. Further functional studies of these frameshift and nonsense mutations revealed that Stk33-/KI male mice were sterile, and Stk33-/KI sperm were abnormal with defects in the mitochondrial sheath, fibrous sheath, outer dense fiber, and axoneme. Stk33KI/KI male mice were subfertile and had oligoasthenozoospermia. Differential phosphoproteomic analysis and in vitro kinase assay identified novel phosphorylation substrates of STK33, fibrous sheath components A-kinase anchoring protein 3 and A-kinase anchoring protein 4, whose expression levels decreased in testis after deletion of Stk33. STK33 regulated the phosphorylation of A-kinase anchoring protein 3/4, affected the assembly of fibrous sheath in the sperm, and played an essential role in spermiogenesis and male infertility.

Leydig cell failure (LCF) caused by gene mutation results in testosterone deficiency and infertility. Serum testosterone levels can be recovered via testosterone replacement; however, established therapies have shown limited success in restoring fertility. Here, we use a luteinizing hormone/choriogonadotrophin receptor (Lhcgr)-deficient mouse model of LCF to investigate the feasibility of gene therapy for restoring testosterone production and fertility. We screen several adeno-associated virus (AAV) serotypes and identify AAV8 as an efficient vector to drive exogenous Lhcgr expression in progenitor Leydig cells through interstitial injection. We observe considerable testosterone recovery and Leydig cell maturation after AAV8-Lhcgr treatment in pubertal Lhcgr-/- mice. Of note, this gene therapy partially recovers sexual development, substantially restores spermatogenesis, and effectively produces fertile offspring. Furthermore, these favorable effects can be reproduced in adult Lhcgr-/- mice. Our proof-of-concept experiments in the mouse model demonstrate that AAV-mediated gene therapy may represent a promising therapeutic approach for patients with LCF.

Spermiogenesis is a highly organized process of the metamorphosis of round spermatids into spermatozoa in the testes. Autophagy is involved in the physiological process of spermiogenesis and its crucial role in germ-plasm clearance conserved across kingdoms. However, the fate of by-products generated through autophagy during spermiogenesis is still largely unknown. In the present study, we showed that the autophagy enhanced lipid droplets (LDs) formation during spermiogenesis in Chinese soft-shelled turtle, Pelodiscus sinensis. TEM and Oil Red O staining results found that the number and size of LDs within spermatid increased considerably during the process of spermiogenesis. RNA-Seq analysis revealed that autophagy was highly activated via the PI3K pathway during spermatogenesis. Inhibiting autophagy with 3-methyladenine (3-MA) significantly decreased testicular triglycerides (TGs) and fatty acid (FAs) content. In comparison with the control group, the number and size of LD within elongating spermatids was reduced significantly in the 3-MA group. Moreover, DGAT1, a diacylglycerol acyltransferase, which normally localize to the endoplasmic reticulum, was found to co-localize with LDs. Taken together, our results showed that FAs released through the autophagic degradation of germ-plasm was replenished LDs of spermatid, increasing LD number and size, during the process of spermiogenesis. These LDs facilitate long-term sperm storage in the epididymis of Chinese soft-shelled turtle.

Rationale: Stem Leydig cells (SLCs) transplantation can restore testosterone production in rodent models and is thus a potential solution for treating testosterone deficiency (TD). However, it remains unknown whether these favorable effects will be reproduced in more clinically relevant large-animal models. Therefore, we assessed the feasibility, safety and efficacy of autologous SLCs transplantation in a testosterone-deficient non-human primate (NHP) model. Methods: Cynomolgus monkey SLCs (CM-SLCs) were isolated from testis biopsies of elderly (> 19 years) cynomolgus monkeys by flow cytometry. Autologous CM-SLCs were injected into the testicular interstitium of 7 monkeys. Another 4 monkeys were injected the same way with cynomolgus monkey dermal fibroblasts (CM-DFs) as controls. The animals were then examined for sex hormones, semen, body composition, grip strength, and exercise activity. Results: We first isolated CD271+ CM-SLCs which were confirmed to expand continuously and show potential to differentiate into testosterone-producing Leydig cells (LCs) in vitro. Compared with CM-DFs transplantation, engraftment of autologous CM-SLCs into elderly monkeys could significantly increase the serum testosterone level in a physiological pattern for 8 weeks, without any need for immunosuppression. Importantly, CM-SLCs transplantation recovered spermatogenesis and ameliorated TD-related symptoms, such as those related to body fat mass, lean mass, bone mineral density, strength and exercise capacity. Conclusion: For the first time, our short-term observations demonstrated that autologous SLCs can increase testosterone levels and ameliorate relevant TD symptoms in primate models. A larger cohort with long-term follow-up will be required to assess the translational potential of autologous SLCs for TD therapy.

As a gene contributing to spermatogenesis, the Boule gene (also called Boll), whose mutations result in azoospermia and sterility of flies and mice, was conserved in reductional maturation divisions. However, in goats, the polymorphisms of Boule, especially with regard to their fundamental roles in female reproduction traits, are still unknown. Therefore, the aims of this study were to detect a potential mutation (rs661484476: g.7254T>C) located in intron 2 of the Boule gene by tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) and to explore its potential association with the litter size of Shaanbei White-Cashmere goats (SBWGs). In this study, g.7254T>C was firstly detected. The TT genotype was the dominant genotype in the single-lamb group, and T was also the dominant allele in all tested groups. Additionally, the detected locus displayed moderate polymorphism with polymorphism information content (PIC) values among all studied goats ranging from 0.303 to 0.344. Notably, according to the χ2 test, the distribution differences for the genotypic frequencies between the single- and multi-lamb groups was significant (p = 0.014). Furthermore, the polymorphisms of the goat Boule gene were significantly associated with the goat litter size in SBWGs (p < 0.05), which indicated that g.7254T>C could be a potential marker in the marker-assisted selection process for potential litter size in goats. These results also indicated that the Boule gene might exercise important functions in female goat reproduction, which provided new insight for female goat breeding and could accelerate the process of goat breeding.

A conserved gene in meiosis, the Boule gene is involved in meiosis and spermatogenesis. The deletion of this gene in males blocks meiosis and results in infertility. Alternative splicing variants of the Boule gene have been identified in humans, bovines, and bats, but in dairy goats remains unknown. This study was therefore to detect splicing variants of the goat Boule gene and explore their potential roles in meiosis. Three isoforms, denoted as Boule-a, Boule-b, and Boule-c, were identified in the testes of goats using real-time PCR (RT-PCR) and cloning sequencing. Compared to the normal Boule gene, Boule-a was found to lack exons 7 and 8, which corresponds to a predicted variant, X4, on the NCBI database. Boule-b lacked exon 8, and Boule-c only retained exons 1 and 2. Of these three variants, two were novel isoforms of the Boule gene. Quantitative RT-PCR (qRT-PCR) showed that the Boule-a and Boule-b expression patterns were significantly different between the adult goat testes and the postnatal testes of 42 and 56 days. Overexpression of Boule-a and Boule-c in GC-1 spg cells of model mice significantly repressed CDC2 expression. Bisulfite sequencing PCR (BSP) results showed that the promoter region of the Boule gene was hypermethylated in goat testes. A negative correlation between the methylation levels of the Boule gene promoter and total mRNA expression of its transcripts was found. Our data showed alternative splicing and promoter methylation in the goat Boule gene, suggesting that this gene may play an important role in the regulation of Boule expression and in meiosis processing.

Testosterone is an important steroid hormone that is indispensable for male sexual development and the reproductive system. Leydig cells (LCs), where autophagy is extremely active, reside in the testicular interstitium and are the major sites of testosterone production. However, the ultrastructural characteristics and the functional role of autophagy in LCs of livestock remain unknown. In this study, the LCs of the dairy goats were investigated to identify the steroidogenic activity and autophagy levels at different ages of development by light microscopy, immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy (TEM). Morphological results showed that the steroidogenic activity (3β-HSD staining) and ultrastructural characteristics of the LCs were changed with increasing age. TEM results demonstrated that the organelles involved in testosterone synthesis, e.g., smooth endoplasmic reticulum, mitochondria, and lipid droplets, were abundantly distributed within the cytoplasm of LCs in pubertal and adult testes. Moreover, autophagy activity was enhanced in the testes at pubertal and adult stages compared with that at the juvenile stage. Several different autophagic vacuoles, including pre-autophagosomes, autophagosomes, and autolysosomes, were observed within the cytoplasm of LCs from pubertal and adult testes. However, immunofluorescent staining and TEM results showed that no typical lipophagic or mitophagic vacuoles were observed in the cytoplasm of LCs. Furthermore, primary LCs from dairy goats were used to study the effect of autophagy on testosterone production. After treatment with 3-methyladenine (3-MA, an autophagy inhibitor), the primary LCs decreased testosterone production. In contrast, treatment with rapamycin (an autophagy activator), enhanced steroidogenesis in LCs. Collectively, these in vivo and in vitro results suggested that autophagy activity is related to steroidogenesis in LCs of dairy goats, which may ultimately influence the spermatogenesis and fertility of these animals.

CKLF-like MARVEL transmembrane domain-containing 2 (CMTM2) plays an important role in animal reproduction, and abnormal CMTM2 expression leads to spermatogenesis disorders. This study was conducted to assess the role of CMTM2 in goat reproduction by investigating an insertion/deletion (indel) variation and its effect on litter size, and to reveal its functional mechanism. A 14-base pair (bp) indel was found at position -861 to -848 nt of the CMTM2 promoter in 1131 female Shaanbei white cashmere goats. Association analyses revealed that the 14-bp indel in dams was significantly correlated with the size of the first litter (P = 0.013), with the 14-bp deletion significantly decreasing litter size. Moreover, litter size at first kidding was significantly correlated with genotype frequencies (P = 0.019). Expression of the goat CMTM2 gene was detected in testes and ovaries. In male, CMTM2 was increased after the initiation of meiosis in the developing testis. In female, CMTM2 expression was higher in ovary from multiple prolific goats at first kidding compared to non-prolific goats. Moreover, both in testis after initiation of meiosis and in ovary, the homozygous 14-bp inserted genotype II was associated with increased CMTM2 expression compared to the heterozygous genotype ID (P < 0.05). To further explore whether the 14-bp indel was located in the core promoter activity region of the CMTM2 promoter, the five recombinant plasmids pGL3-pro1 (-1756 to +83), pGL3-pro2 (-1200 to +83), pGL3-pro3 (-763 to +83), pGL3-pro4 (-496 to +83), and pGL3-pro5 (-205 to +83) were co-transfected with pRL-TK into HEK293T and GC-1 cells. A luciferase reporter assay showed that the pGL3-pro2 and pGL3-pro5 vectors produced significantly stronger luminescence than the other vectors. Interestingly, the 14-bp indel locus was included in the pGL3-pro2 plasmid, suggesting that the indel was functional. Subsequently, the two recombinant plasmids pGL3-pro2-inserted-allele and pGL3-pro2-deleted-allele were co-transfected with pRL-TK into HEK293T and GC-1 cells, respectively. The luciferase reporter assay demonstrated that the deleted allele of CMTM2 showed significantly lower luminescence activity than the inserted allele (P < 0.05), which corresponded to a decrease in litter size in the deletion-deletion genotype. Therefore, our results suggest that a 14-bp deletion in the promoter region of CMTM2 is significantly correlated with litter size in goats; this marker seems promising for breeding selection for improving economically important traits in goats.