In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm.

During epididymal transit, spermatozoa go through several functional maturation steps, resulting from interactions with epididymal secretomes specific to each region. In particular, the sperm membrane is under constant remodeling, with sequential attachment and shedding of various molecules provided by the epididymal lumen fluid and epididymosomes, which also deliver sncRNA cargo to sperm. As a result, the payload of sperm sncRNAs changes during the transit from the epididymis caput to the cauda. This work was designed to study the dynamics of cattle sperm sncRNAs from spermatogenesis to final maturation.

The canonical Wnt signaling pathway is of paramount importance in development and disease. An emergent question is whether the upstream cascade of the canonical Wnt pathway has physiologically relevant roles beyond β-catenin-mediated transcription, which is difficult to study due to the pervasive role of this protein. Here, we show that transcriptionally silent spermatozoa respond to Wnt signals released from the epididymis and that mice mutant for the Wnt regulator Cyclin Y-like 1 are male sterile due to immotile and malformed spermatozoa. Post-transcriptional Wnt signaling impacts spermatozoa through GSK3 by (1) reducing global protein poly-ubiquitination to maintain protein homeostasis; (2) inhibiting septin 4 phosphorylation to establish a membrane diffusion barrier in the sperm tail; and (3) inhibiting protein phosphatase 1 to initiate sperm motility. The results indicate that Wnt signaling orchestrates a rich post-transcriptional sperm maturation program and invite revisiting transcription-independent Wnt signaling in somatic cells as well.

Lipocalin family members, LCN8 and LCN9, are specifically expressed in the initial segment of mouse caput epididymis. However, the biological functions of the molecules in vivo are yet to be clarified. In this study, CRISPR/Cas9 technology was used to generate Lcn8 and Lcn9 knockout mice, respectively. Lcn8-/- and Lcn9-/- male mice showed normal spermatogenesis and fertility. In the cauda epididymis of Lcn8-/- male mice, morphologically abnormal sperm was increased significantly, the proportion of progressive motility sperm was decreased, the proportion of immobilized sperm was elevated, and the sperm spontaneous acrosome reaction (AR) frequency was increased. Conversely, the knockout of Lcn9 did not have any effect on the ratio of morphologically abnormal sperm, sperm motility, and sperm spontaneous AR frequencies. These results demonstrated the role of LCN8 in maintaining the sperm quality in the epididymis, and suggested that the deficiency of LCN8 leads to epididymal sperm maturation defects.

After leaving the testis, sperm undergo two sequential maturational processes before acquiring fertilizing capacity: sperm maturation in the male epididymis, and sperm capacitation in the female reproductive tract. During their transit through the epididymis, sperm experience several maturational changes; the acquisition of motility is one of them. The molecular basis of the regulation of this process is still not fully understood. Sperm are both transcriptionally and translationally silent, therefore post-translational modifications are essential to regulate their function. The post-translational modification by the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) can act as a counterpart of phosphorylation in different cellular processes. Therefore, our work was aimed to characterize the O-GlcNAcylation system in the male reproductive tract and the occurrence of this phenomenon during sperm maturation. Our results indicate that O-GlcNAc transferase (OGT), the enzyme responsible for O-GlcNAcylation, is present in the testis, epididymis and immature caput sperm. Its presence is significantly reduced in mature cauda sperm. Consistently, caput sperm display high levels of O-GlcNAcylation when compared to mature cauda sperm, where it is mostly absent. Our results indicate that the modulation of O-GlcNAcylation takes place during sperm maturation and suggest a role for this post-translational modification in this process.

Extracellular vesicles (EVs) are important players in cell to cell communication in reproductive systems. Notably, EVs have been found and characterized in the male reproductive tract, however, direct functional evidence for their importance in mediating sperm function is lacking. We have previously demonstrated that Arrdc4, a member of the α-arrestin protein family, is involved in extracellular vesicle biogenesis and release. Here we show that Arrdc4-mediated extracellular vesicle biogenesis is required for proper sperm function. Sperm from Arrdc4-/- mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as observed by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and two-cell embryo production. We found a significant reduction in extracellular vesicle production by Arrdc4-/- epididymal epithelial cells, and further, supplementation of Arrdc4-/- sperm with additional vesicles dampened the acrosome reaction defect and restored zona pellucida binding. These results indicate that Arrdc4 is important for proper sperm maturation through the control of extracellular vesicle biogenesis.

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.

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.

Cdc14a is an evolutionarily conserved dual-specific protein phosphatase, and it plays different roles in different organisms. Cdc14a mutations in human have been reported to cause male infertility, while the specific role of Cdc14a in regulation of the male reproductive system remains elusive. In the present study, we established a knockout mouse model to study the function of Cdc14a in male reproductive system. Cdc14a-/- male mice were subfertile and they could only produce very few offspring. The number of sperm was decreased, the sperm motility was impaired, and the proportion of sperm with abnormal morphology was elevated in Cdc14a-/- mice. When we mated Cdc14a-/- male mice with wild-type (WT) female mice, fertilized eggs could be found in female fallopian tubes, however, the majority of these embryos died during development. Some empty spaces were observed in seminiferous tubule of Cdc14a-/- testes. Compared with WT male mice, the proportions of pachytene spermatocytes were increased and germ cells stained with γH2ax were decreased in Cdc14a-/- male mice, indicating that knockout of Cdc14a inhibited meiotic initiation. Subsequently, we analyzed the expression levels of some substrate proteins of Cdc14a, including Cdc25a, Wee1, and PR-Set7, and compared those with WT testes, in which the expression levels of these proteins were significantly increased in Cdc14a-/- testes. Our results revealed that Cdc14a-/- male mice are highly subfertile, and Cdc14a is essential for normal spermatogenesis and sperm function.

Germ cells manifest a unique gene expression program and regain totipotency in the zygote. Here, we perform Hi-C analysis to examine 3D chromatin organization in male germ cells during spermatogenesis. We show that the highly compartmentalized 3D chromatin organization characteristic of interphase nuclei is attenuated in meiotic prophase. Meiotic prophase is predominated by short-range intrachromosomal interactions that represent a condensed form akin to that of mitotic chromosomes. However, unlike mitotic chromosomes, meiotic chromosomes display weak genomic compartmentalization, weak topologically associating domains, and localized point interactions in prophase. In postmeiotic round spermatids, genomic compartmentalization increases and gives rise to the strong compartmentalization seen in mature sperm. The X chromosome lacks domain organization during meiotic sex-chromosome inactivation. We propose that male meiosis occurs amid global reprogramming of 3D chromatin organization and that strengthening of chromatin compartmentalization takes place in spermiogenesis to prepare the next generation of life.

During epididymal sperm maturation, spermatozoa acquire progressive motility through dynamic protein modifications. However, the relationship between sequential protein modifications during epididymal sperm maturation and sperm motility and fertility has not yet been investigated. This study investigated whether sequential changes in fertility-related protein expression including that of enolase 1 (ENO1), ubiquinol-cytochrome c reductase core protein 1 and 2 (UQCRC1 and UQCRC2), and voltage-dependent anion channel 2 (VDAC2) in spermatozoa during epididymal maturation are related to bovine sperm motility. Moreover, we found that mitochondrial metabolism is closely related to fertility-related proteins. Therefore, we investigated how the sequential modification of mitochondrial proteins during epididymal maturation regulates sperm motility.

Sperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29), which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process.

GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant differences observed in 2 out of 3 phosphoforms in asthenozoosperm suggest that GRP78 phosphorylation may have functional relevance in sperm with consequent clinical implications.

Alongside the well-known central modulatory role, the Kisspeptin system, comprising Kiss1, its cleavage products (Kisspeptins), and Kisspeptin receptor (Kiss1R), was found to regulate gonadal functions in vertebrates; however, its functional role in the male gamete and its localization during maturation have been poorly understood. The present study analyzed Kisspeptin system in dog testis and spermatozoa recovered from different segments of the epididymis, with focus on Kiss1R on sperm surface alongside the maturation during epididymal transit, demonstrated by modification in sperm kinetic, morphology, and protamination. The proteins Kiss1 and Kiss1R were detected in dog testis. The receptor Kiss1R only was detected in total protein extracts from epididymis spermatozoa, whereas dot blot revealed Kiss1 immunoreactivity in the epidydimal fluid. An increase of the Kiss1R protein on sperm surface along the length of the epididymis, with spermatozoa in the tail showing plasma membrane integrity and Kiss1R protein (p < 0.05 vs. epididymis head and body) was observed by flow cytometry and further confirmed by epifluorescence microscopy and Western blot carried on sperm membrane preparations. In parallel, during the transit in the epididymis spermatozoa significantly modified their ability to move and the pattern of motility; a progressive increase in protaminization also occurred. In conclusion, Kisspeptin system was detected in dog testis and spermatozoa. Kiss1R trafficking toward plasma membrane along the length of the epididymis and Kiss1 in epididymal fluid suggested a new functional role of the Kisspeptin system in sperm maturation and storage.

Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotypes. Recent studies show that the small RNA repertoire of sperm is remodeled during post-testicular maturation in the epididymis. Epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in shaping the sperm epigenome. Here, we characterize the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. We find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is stable throughout post-testicular maturation. Although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, and was present only in sperm samples obtained from the caput epididymis and vas deferens of virgin males. Curiously, contaminating extracellular DNA was associated with citrullinated histone H3, potentially resulting from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA.

Physiology and metabolism are often sexually dimorphic, but the underlying mechanisms remain incompletely understood. Here, we use the intestine of Drosophila melanogaster to investigate how gut-derived signals contribute to sex differences in whole-body physiology. We find that carbohydrate handling is male-biased in a specific portion of the intestine. In contrast to known sexual dimorphisms in invertebrates, the sex differences in intestinal carbohydrate metabolism are extrinsically controlled by the adjacent male gonad, which activates JAK-STAT signaling in enterocytes within this intestinal portion. Sex reversal experiments establish roles for this male-biased intestinal metabolic state in controlling food intake and sperm production through gut-derived citrate. Our work uncovers a male gonad-gut axis coupling diet and sperm production, revealing that metabolic communication across organs is physiologically important. The instructive role of citrate in inter-organ communication might be significant in more biological contexts than previously recognized.

Pesticides, especially organophosphorus pesticides such as chlorpyrifos (CPF), play an important role in modern agriculture. Studies have shown that pesticide residues are an important cause of male reproductive injury in mammal.

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.

Eutherian spermatozoa are dependent on the environment of the proximal epididymis to complete their maturation; however, no specific epididymal factors that mediate this process have so far been identified. Here, we show that targeted disruption of the novel gene Rnase10 encoding a secreted proximal epididymal protein in the mouse results in a binding defect in spermatozoa and their inability to pass through the uterotubal junction in the female. The failure to gain the site of fertilization in the knockout spermatozoa is associated with a gradual loss of ADAM3 and ADAM6 proteins during epididymal transit. In the distal epididymis, these spermatozoa appear to lack calcium-dependent associations with the immobilizing glutinous extracellular material and are released as single, vigorously motile cells that display no tendency for head-to-head agglutination and lack affinity to the oviductal epithelium. In sperm-egg binding assay, they are unable to establish a tenacious association with the zona pellucida, yet they are capable of fertilization. Furthermore, these sperm show accelerated capacitation resulting in an overall in vitro fertilizing ability superior to that of wild-type sperm. We conclude that the physiological role of sperm adhesiveness is in the mechanism of restricted sperm entry into the oviduct rather than in sperm-egg interaction.

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.