TMEM225 Is Essential for Sperm Maturation and Male Fertility by Modifying Protein Distribution of Sperm in Mice.

Nonobstructive azoospermia is the leading cause of male infertility. Abnormal levels of transmembrane protein 225 (TMEM225), a testis-specific protein, have been found in patients with nonobstructive azoospermia, suggesting that TMEM225 plays an essential role in male fertility. Here, we generated a Tmem225 KO mouse model to explore the function and mechanism of TMEM225 in male reproduction. Male Tmem225 KO mice were infertile. Surprisingly, Tmem225 deletion did not affect spermatogenesis, but TMEM225-null sperm exhibited abnormalities during epididymal maturation, resulting in reduced sperm motility and an abnormal hairpin-loop configuration. Furthermore, proteomics analyses of cauda sperm revealed that signaling pathways related to mitochondrial function, the glycolytic pathway, and sperm flagellar morphology were abnormal in Tmem225 KO sperm, and spermatozoa lacking TMEM225 exhibited high reactive oxygen species levels, reduced motility, and flagellar folding, leading to typical asthenospermia. These findings suggest that testicular TMEM225 may control the sperm maturation process by regulating the expression of proteins related to mitochondrial function, glycolysis, and sperm flagellar morphology in epididymal spermatozoa.

SperMD: the expression atlas of sperm maturation.

The impairment of sperm maturation is one of the major pathogenic factors in male subfertility, a serious medical and social problem affecting millions of global couples. Regrettably, the existing research on sperm maturation is slow, limited, and fragmented, largely attributable to the lack of a global molecular view. To fill the data gap, we newly established a database, namely the Sperm Maturation Database (SperMD, http://bio-add.org/SperMD ). SperMD integrates heterogeneous multi-omics data (170 transcriptomes, 91 proteomes, and five human metabolomes) to illustrate the transcriptional, translational, and metabolic manifestations during the entire lifespan of sperm maturation. These data involve almost all crucial scenarios related to sperm maturation, including the tissue components of the epididymal microenvironment, cell constituents of tissues, different pathological states, and so on. To the best of our knowledge, SperMD could be one of the limited repositories that provide focused and comprehensive information on sperm maturation. Easy-to-use web services are also implemented to enhance the experience of data retrieval and molecular comparison between humans and mice. Furthermore, the manuscript illustrates an example application demonstrated to systematically characterize novel gene functions in sperm maturation. Nevertheless, SperMD undertakes the endeavor to integrate the islanding omics data, offering a panoramic molecular view of how the spermatozoa gain full reproductive abilities. It will serve as a valuable resource for the systematic exploration of sperm maturation and for prioritizing the biomarkers and targets for precise diagnosis and therapy of male subfertility.

CUB domains are not required for OVCH2 function in sperm maturation in the mouse epididymis.

BACKGROUND: Ovochymase 2 (Ovch2) is an epididymis-specific gene that is required for male fertility. While a multitude of reproductive tract-specific genes required for male fertility have been identified, OVCH2 is thus far the first protein required for male fertility that contains Complement C1r/C1s, Uegf, Bmp1 (CUB) domains located in tandem in the C-terminus of the protein. Identifying the functional significance of this unique domain has implications in better understanding fertility and infertility and as a potential contraceptive target. OBJECTIVE: The goals of these studies were to understand the influence and requirement of OVCH2 CUB domains in the localization and functional requirement of OVCH2 in sperm maturation and function. MATERIALS AND METHODS: To this end, we performed in vivo localization analysis of OVCH2 and reproductive phenotype analysis of mice containing C-terminal FLAG tag on OVCH2, with either the entire protein intact, or CUB2 or both CUB1 and CUB2 genetically ablated. All mice were generated through the CRISPR/Cas9 gene editing approach. RESULTS: We found that OVCH2 is specifically expressed in the proximal caput epididymidis, and the absence of CUB2 did not affect this localization pattern. Although the absence of both CUB domains significantly reduced sperm motility and progressive motility, this effect was not manifested in a reduction in fertility over a 6-month period mating trial, which showed no significant differences between control and CUB deletant mice. Further, the absence of one or both CUB domains did not affect reproductive organ structure or sperm morphology. CONCLUSIONS: Our studies demonstrate that the CUB domains are not required for fertility in male mice, at least under the normal animal housing conditions our mice were tested in, and suggest that the enzymatic activity of the OVCH2 protease, in the absence of its CUB domains, is sufficient for normal sperm processing in the epididymis. Although our findings do not preclude the possibility that OVCH2 CUB domains are required under a yet-identified stress condition, our findings demonstrate that the most likely region for deleterious mutations in men with idiopathic infertility and the most vulnerable site for inhibition of OVCH2 protein function is in its protease domain, and not its CUB domains. Our findings have implications in the genetic screening of infertile men and the development of a novel non-hormonal male contraceptive by honing in on the more critical region of a functionally required protein.

Post-testicular sperm maturation in ancient holostean species.

Fish speciation was accompanied by changes in the urogenital system anatomy. In evolutionarily modern Teleostei, male reproductive tracts are fully separated from the excretory system, while in evolutionarily ancient Chondrostei and Holostei, the excretory and reproductive tracts are not separated. Sturgeon post-testicular sperm maturation (PTSM) occurring as a result of sperm/urine mixing is phenomenologically well described, while, in holosteans, functional intimacy of seminal ducts with kidney ducts and the existence of PTSM still need to be addressed. In Lepisosteus platostomus (Holostei), sperm samples were collected from testes (TS), efferent ducts (EDS), and Wolffian ducts (WDS). While WDS was motile, no motility was found in TS and EDS. The existence of PTSM was checked by in vitro PTSM procedure. After TS and EDS incubation in seminal fluid from WDS, no more than 5% motile spermatozoa were observed in TS, whereas in EDS the motility percentage was up to 75%. Experimental dyeing of urogenital ducts in gars and sturgeons revealed some differences in the interconnection between sperm ducts and kidneys. It is concluded that post-testicular sperm maturation occurs in gars and suggests that infraclass Holostei occupies an intermediate evolutionary position between Teleostei and Chondrostei in the anatomical arrangement of the urogenital system.

[Delivery of epididymis-specific mRNAs from mouse epididymosomes into N2a and TM4 cells].

OBJECTIVE: To investigate whether mouse epididymis-specific mRNAs Adam7 and Crisp1 can be delivered into N2a and TM4 cells, and to provide an experimental basis for exploring the function of epididymal mRNAs. METHODS: Using RT-PCR, we detected the presence of epididymis-specific genes (Adam7, Crisp1, Defb22, Wfdc2, and Wfdc9) in the testis, epididymis, epididymosome and sperm of adult male BALB/c mice as well as in the human testis, seminal vesicles and sperm. We isolated epididymosomes of BALB/c mice by low-speed centrifugation, filtration and ultracentrifugation, fluorescently labeled them by PKH26, co-incubated them for 1 hour with the N2a and TM4 cells after 24 hours of starvation culture, and observed whether they were fused with the N2a and TM4 cells and ingested using the epididymosomes without PKH26 labeling, PKH26 dye without epididymosomes, and non- epididymosome or -PKH26 dye as controls. Then we detected the epididymis-specific genes in the N2a and TM4 cells after 1-hour co-incubation by RT-PCR. RESULTS: Adam7 and Crisp1 were present in the mouse epididymis, epididymosomes and sperm, and in the human seminal vesicles and sperm as well, but not in the testes of either the mice or men. PKH26 and Hoechst33258 fluorescence double-labeling showed that the mouse epididymosomes were fused with the N2a and TM4 cells and ingested; RT-PCR revealed the mRNAs of Adam7 and Crisp1 in the N2a and TM4 cells after 1-hour co-incubation; and Western blot exhibited the CRISP1 protein in the N2a and TM4 cells incubated with epididymosomes. CONCLUSION: Epididymosomes can deliver epididymis-specific mRNAs Adam7 and Crisp1 into N2a and TM4 cells, where Crisp1 may be translated into proteins, though their function and significance need to be further studied.

Effect of Nano-selenium on exosomes secretion associated with sperm maturation within the epididymis.

Selenium is commonly used as a supplement in the poultry diet and plays an important role in male fertility. However, the effect of selenium nanoparticles (Se-NPs) on exosome secretion associated with spermatozoa in the epididymis is largely unknown. H&E staining, Immunohistochemistry, Immunofluorescence and Western blot were performed to study the effect of Se-NPs on exosomes secretion associated with sperm maturation in epididymis. The results indicated that the Se-NPs showed a significant contribution to sperm concentration by light microscopy. It was observed that there was an increase in the spermatozoa concentration in the epididymis of the treated group as compared to the control group. Furthermore, exosome secretion, the expression of tumor susceptibility gene-101 (TSG-101) and cluster of differentiation (CD-63) proteins was identified by immunochemistry, immunofluorescence assay, and western blotting. After nano-selenium treatment, the exosome markers TSG-101 and CD-63 were strong positive immunoreactivity and immunosignaling in the lumen followed by epithelial lining of the epididymis. However weak positive immunoreactivity and immunosignaling were seen of TSG-101 and CD63 in the control group. In addition, highly significant protein expression of TSG-101 and CD63 in the treated group as compared to the control group was confirmed by western blotting. In conclusion, the above findings provide rich evidence about the Se-NPs play a dynamic role in exosome secretion that might be essential for sperm motility and maturation within epididymis.

Progesterone receptors in extratesticular ducts of the Amazonian stingray Potamotrygon wallacei: A potential role in sperm maturation and aggregate formation.

In cururu stingray (Potamotrygon wallacei Carvalho, Rosa and Araújo 2016) males, plasma progesterone (P4) levels appear to be associated with spermiation events. However, the specific contribution of P4 in sperm maturation via extratesticular ducts in this stingray species is unknown. With the aim of filling this knowledge gap, this study examined the morphology and the presence of progesterone receptors (PR) in the ducts, and analyzed the relationship of progesterone (P4) with sperm maturation and formation of aggregates. Morphological analysis showed that a columnar pseudostratified epithelium with stereocilia lined all the attached ducts. In active males, the secretory cells of the epididymis and the Leydig glands presented PR; however, these receptors were not found in the distal region of the epididymis (essential for nurturing and capacitation events) of regressing males. In the seminal vesicles of active males, the spermatozoa are parallelly aligned and embedded in a matrix to form the spermatozeugmata. The matrixes are formed by proteins secreted by the ducts and Sertoli cell cytoplasts. These structures presented PR, which suggests that P4 engages in sperm metabolism during storage. Our findings allude to the potential role of P4 in regulating the development and function of the attached ducts in different reproductive phases. Furthermore, P4 seems to be an essential component for regulating sperm progress, protein secretion, aggregate formation, and maintenance of sperm during storage in this freshwater stingray.

Lumicrine signaling: Extracellular regulation of sperm maturation in the male reproductive tract lumen.

The behaviors of cells, tissues, and organs are controlled by the extracellular environment in addition to their autonomous regulatory system. Dysfunction of extracellular regulatory mechanisms affects not only the development and survival of organisms but also successful reproduction. In this review article, a novel extracellular regulatory mechanism regulating the mammalian male reproductive ability will be briefly summarized. In terrestrial vertebrates, spermatozoa generated in the testis are transported through the lumen of the male reproductive tract and become functionally mature during the transport. Recent studies with gene-modified animals are unveiling the luminal extracellular environment of the reproductive tract to function not only as the pathway of sperm transport and the site of sperm maturation but also as the channel for cellular communication to regulate sperm maturation. Of special interest is the molecular mechanism of lumicrine signaling, a transluminal secreted signal transduction in the male reproductive tract lumen as a master regulator of sperm maturation and male reproductive ability. The general significance of such transluminal signaling in the context of cell biology will also be discussed.

Melatonin alleviated fluoride-induced impairment of spermatogenesis and sperm maturation process via Interleukin-17A.

Fluoride-induced male reproductive failure is a major environmental and human health concern, but interventions are still lacking. Melatonin (MLT) has potential functions in regulating testicular damage and interleukin-17 (IL-17) production. This study aims to explore whether MLT can mitigate fluoride-induced male reproductive toxicity through IL-17A, and screen the potential targets. So the wild type and IL-17A knockout mice were employed and treated with sodium fluoride (100 mg/L) by drinking water and MLT (10 mg/kg.BW, intraperitoneal injection per two days starting from week 16) for 18 weeks. Bone F- concentrations, grade of dental damage, sperm quality, spermatogenic cells counts, histological morphology of testis and epididymis, and the mRNA expression of spermatogenesis and maturation, classical pyroptosis related and immune factor genes were detected respectively. The results revealed that MLT supplementations alleviated fluoride-induced impairment of spermatogenesis and maturation process, protecting the morphology of testis and epididymis through IL-17A pathway, and Tesk1 and Pten were identified as candidate targets from 29 regulation genes. Taken together, this study demonstrated a new physiological role for MLT in the protection against fluoride-induced reproductive injury and possible regulation mechanisms, which providing a useful therapeutic strategy for male reproductive function failure caused by fluoride or other environmental pollutants.

Sialylation: fate decision of mammalian sperm development, fertilization, and male fertility†.

Sperm development, maturation, and successful fertilization within the female reproductive tract are intricate and orderly processes that involve protein translation and post-translational modifications. Among these modifications, sialylation plays a crucial role. Any disruptions occurring throughout the sperm's life cycle can result in male infertility, yet our current understanding of this process remains limited. Conventional semen analysis often fails to diagnose some infertility cases associated with sperm sialylation, emphasizing the need to comprehend and investigate the characteristics of sperm sialylation. This review reanalyzes the significance of sialylation in sperm development and fertilization and evaluates the impact of sialylation damage on male fertility under pathological conditions. Sialylation serves a vital role in the life journey of sperm, providing a negatively charged glycocalyx and enriching the molecular structure of the sperm surface, which is beneficial to sperm reversible recognition and immune interaction. These characteristics are particularly crucial during sperm maturation and fertilization within the female reproductive tract. Moreover, enhancing the understanding of the mechanism underlying sperm sialylation can promote the development of relevant clinical indicators for infertility detection and treatment.

Epididymal DIS3 exosome ribonuclease is not necessary for mouse sperm maturation or fertility.

DIS3 is an RNA exosome associated ribonuclease that degrades a wide range of transcripts that can be essential for cell survival and development. The proximal region of the mouse epididymis (initial segment and caput) plays a pivotal role in sperm transport and maturation required for male fertility. However, whether DIS3 ribonuclease mediates RNA decay in proximal epididymides remains unclear. Herein, we established a conditional knockout mouse line by crossing a floxed Dis3 allele with Lcn9-cre mice in which the recombinase is expressed in the principal cells of initial segment as early as post-natal day 17. Morphological and histological analyses, immunofluorescence, computer-aided sperm analysis and fertility were used for functional analyses. We document that DIS3 deficiency in the initial segment had no effect on male fertility. Dis3 cKO males had normal spermatogenesis and initial segment development. In cauda epididymides of Dis3 cKO mice, sperm abundance, morphology, motility, and the frequency of acrosome exocytosis were comparable to controls. Collectively, our genetic model demonstrates that loss of DIS3 in the initial segment of the epididymis is not essential for sperm maturation, motility, or male fertility.

CpG DNA methylation changes during epididymal sperm maturation in bulls.

BACKGROUND: During epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions. RESULTS: Caput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda. CONCLUSIONS: Specific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

Spectacular role of epididymis and bio-active cargo of nano-scale exosome in sperm maturation: A review.

The epididymis is responsible for post-testicular sperm maturation as it provides a favorable environment for spermatozoa to gain the ability for movement and fertilization. The recent evidence has shown that, the spermatozoa are vulnerable to dynamic variations driven by various cellular exposure mechanisms mediated by epididymosomes. Exosomes provide new insight into a mechanism of intercellular communication because they provide direct evidence for the transfer of several important bio-active cargo elements (proteins, lipid, DNA, mRNA, microRNA, circular RNA, long noncoding RNA) between epididymis and spermatozoa. In broad sense, proteomic analysis of exosomes from epididymis indicates number of proteins that are involved in sperm motility, acrosomal reaction, prevent pre-mature sperm capacitation and male infertility. Pinpointing, how reproductive disorders are associated with bio-active cargo elements of nano-scale exosome in the male reproductive tract. Therefore, the current review presents evidence regarding the distinctive characteristics and functions of nano-scale exosome in the male reproductive tract in both pathological and physiological developments, and argue that these vesicles serve as an important regulator of male reproduction, fertility, and disease susceptibility.

Small RNA shuffling between murine sperm and their cytoplasmic droplets during epididymal maturation.

Reports that mouse sperm gain small RNAs from the epididymosomes secreted by epididymal epithelial cells and that these "foreign" small RNAs act as an epigenetic information carrier mediating the transmission of acquired paternal traits have drawn great attention because the findings suggest that heritable information can flow from soma to germ line, thus invalidating the long-standing Weismann's barrier theory on heritable information flow. Using small RNA sequencing (sRNA-seq), northern blots, sRNA in situ hybridization, and immunofluorescence, we detected substantial changes in the small RNA profile in murine caput epididymal sperm (sperm in the head of the epididymis), and we further determined that the changes resulted from sperm exchanging small RNAs, mainly tsRNAs and rsRNAs, with cytoplasmic droplets rather than the epididymosomes. Moreover, the murine sperm-borne small RNAs were mainly derived from the nuclear small RNAs in late spermatids. Thus, caution is needed regarding sperm gaining foreign small RNAs as an underlying mechanism of epigenetic inheritance.

A small secreted protein NICOL regulates lumicrine-mediated sperm maturation and male fertility.

The mammalian spermatozoa produced in the testis require functional maturation in the epididymis for their full competence. Epididymal sperm maturation is regulated by lumicrine signalling pathways in which testis-derived secreted signals relocate to the epididymis lumen and promote functional differentiation. However, the detailed mechanisms of lumicrine regulation are unclear. Herein, we demonstrate that a small secreted protein, NELL2-interacting cofactor for lumicrine signalling (NICOL), plays a crucial role in lumicrine signalling in mice. NICOL is expressed in male reproductive organs, including the testis, and forms a complex with the testis-secreted protein NELL2, which is transported transluminally from the testis to the epididymis. Males lacking Nicol are sterile due to impaired NELL2-mediated lumicrine signalling, leading to defective epididymal differentiation and deficient sperm maturation but can be restored by NICOL expression in testicular germ cells. Our results demonstrate how lumicrine signalling regulates epididymal function for successful sperm maturation and male fertility.

Trade-off between thermal preference and sperm maturation in a montane lizard.

Temperature is a key abiotic factor that influences performance of several physiological traits in ectotherms. Organisms regulate their body temperature within a range of temperatures to enhance physiological function. The capacity of ectotherms, such as lizards, to maintain their body temperature within their preferred range influences physiological traits such as speed, various reproductive patterns, and critical fitness components, such as growth rates or survival. Here, we evaluate the influence of temperature on locomotor performance, sperm morphology and viability in a high elevation lizard species (Sceloporus aeneus). Whereas maximal values for sprint speed coincides with field active and preferred body temperature, short-term exposure at the same range of temperatures produces abnormalities in sperm morphology, lower sperm concentration and diminishes sperm motility and viability. In conclusion, we confirmed that although locomotor performance is maximized at preferred temperatures, there is a trade-off with male reproductive attributes, which may cause infertility. As a consequence, prolonged exposure to preferred temperatures could threaten the persistence of the species through reduced fertility. Persistence of the species is favored in environments with access to cooler, thermal microhabitats that enhance reproductive parameters.

Fetal Exposure to Endocrine Disrupting-Bisphenol A (BPA) Alters Testicular Fatty Acid Metabolism in the Adult Offspring: Relevance to Sperm Maturation and Quality.

Daily exposure to bisphenols can affect reproductive functions due to their pseudo-estrogenic and/or anti-androgenic effects. Testicular lipids contain high levels of polyunsaturated fatty acids necessary for sperm maturity, motility, and spermatogenesis. Whether prenatal exposure to bisphenols alters testicular fatty acid metabolism in adult offspring is unknown. Pregnant Wistar rats were gavaged from gestational day 4 to 21 with BPA and BPS (0.0, 0.4, 4.0, 40.0 µg/kg bw/day). Despite increased body and testis weight, the total testicular cholesterol, triglyceride, and plasma fatty acids were unaffected in the offspring. Lipogenesis was upregulated by increased SCD-1, SCD-2, and expression of lipid storage (ADRP) and trafficking protein (FABP4). The arachidonic acid, 20:4 n-6 (ARA) and docosapentaenoic acid, 22:5 n-6 (DPA) levels were decreased in the BPA-exposed testis, while BPS exposure had no effects. The expression of PPARα, PPARγ proteins, and CATSPER2 mRNA were decreased, which are important for energy dissipation and the motility of the sperm in the testis. The endogenous conversion of linoleic acid,18:2 n-6 (LA), to ARA was impaired by a reduced ARA/LA ratio and decreased FADS1 expression in BPA-exposed testis. Collectively, fetal BPA exposure affected endogenous long-chain fatty acid metabolism and steroidogenesis in the adult testis, which might dysregulate sperm maturation and quality.

Specific expression of alternatively spliced genes in the turkey (Meleagris gallopavo) reproductive tract revealed their function in spermatogenesis and post-testicular sperm maturation.

The tissue-specific profile of alternatively spliced genes (ASGs) and their involvement in reproduction processes characteristic of turkey testis, epididymis, and ductus deferens were investigated for the first time in birds. Deep sequencing of male turkey reproductive tissue RNA samples (n = 6) was performed using Illumina RNA-Seq with 2 independent methods, rMATs and SUPPA2, for differential alternative splicing (DAS) event prediction. The expression of selected ASGs was validated using quantitative real-time reverse transcriptase-polymerase chain reaction. The testis was found to be the site of the highest number of posttranscriptional splicing events within the reproductive tract, and skipping exons were the most frequently occurring class of alternative splicing (AS) among the reproductive tract. Statistical analysis revealed 86, 229, and 6 DAS events in the testis/epididymis, testis/ductus deferens, and epididymis/ductus deferens comparison, respectively. Alternative splicing was found to be a mechanism of gene expression regulation within the turkey reproduction tract. In testis, modification was observed for spermatogenesis specific genes; the changes in 5' UTR could act as regulator of MEIG1 expression (a player during spermatocytes meiosis), and modification of 3' UTR led to diversification of CREM mRNA (modulator of gene expression related to the structuring of mature spermatozoa). Sperm tail formation can be regulated by changes in the 5' UTR of testicular SLC9A3R1 and gene silencing by producing dysfunctional variants of ODF2 in the testis and ATP1B3 in the epididymis. Predicted differentially ASGs in the turkey reproductive tract seem to be involved in the regulation of spermatogenesis, including acrosome formation and sperm tail formation and binding of sperm to the zona pellucida. Several ASGs were classified as cilia by actin and microtubule cytoskeleton organization. Such genes may play a role in the organization of sperm flagellum and post-testicular motility development. To our knowledge, this is the first functional investigation of alternatively spliced genes associated with tissue-specific processes in the turkey reproductive tract.

In silico docking analysis of beta-defensin 20 against cation channel sperm-associated protein 1-4 to predict its role in the sperm maturation.

Beta-defensin 20 (DEFB20) is widely expressed in the epididymis with gene features involved in epididymal sperm maturation. However, the action mechanism and function of DEFB20 in sperm maturation are still unclear. One of the important roles of beta-defensin is the ion channel activity. The cation channel sperm-associated protein (CatSper) alpha is an ion channel protein found on the sperm surface. This study aimed to investigate the interaction between DEFB20 and CatSper1-4 protein in relation to the sperm maturation process. Protein sequences were obtained from the National Center for Biotechnology Information (NCBI). Protein modeling and validation were carried out by using the Robetta modeling server and the Ramachandran plot method. Rosetta web server was used for the docking analysis. The results revealed a natural interaction between DEFB20 and CatSper1-4. The interaction occurred at the cation channel (close to the casein kinase II), ion transport protein, and kinase c phosphorylation of the CatSper1-4 active site. The DEFB20 region interacting with CatSper2-4 was the beta-defensin domain, while with CatSper1 was the non-beta-defensin domain. Based on the analysis, DEFB20 may interact with CatSper α subunits, particularly CatsSper1, to affect ion channel activity during sperm maturation.

Cathepsin B plays a role in spermatogenesis and sperm maturation through regulating autophagy and apoptosis in mice.

Spermatogenesis and sperm maturation are complex and highly ordered biological processes. Any failure or disorder in these processes can cause defects in sperm morphology, motility, and fertilization ability. Cathepsin B (CTSB) is involved in the regulation of a variety of pathological processes. In the present study, we found that CTSB was abundantly expressed in the male reproductive system, however, the specific role of CTSB in regulating spermatogenesis and sperm maturation remained elusive. Hence, we generated Ctsb -/- mice using CRISPR/Cas9 technology. In Ctsb -/- mice, sperm count was significantly decreased while the level of morphologically abnormal sperm was markedly increased. Additionally, these mice had significantly lower levels of progressive motility sperm and elevated levels of immobilized sperm. Histological analysis showed slight vacuolization in the testis epithelium, as well as the loss of epididymal epithelium cells. Further investigation showed that autophagic activity was inhibited and apoptotic activity was increased in both the testis and epididymis of Ctsb -/- mice. Together, our findings demonstrate that CTSB plays an important role in spermatogenesis and sperm maturation in mice.