Quantitative Phosphoproteomic Profiling of Mouse Sperm Maturation in Epididymis Revealed Kinases Important for Sperm Motility.

Transcriptionally and translationally silent sperm undergo functional maturation during epididymis traverse, which provides sperm ability to move and is crucial for successful fertilization. However, the molecular mechanisms governing sperm maturation remain poorly understood, especially at the protein post-translational modification level. In this study, we conducted a comprehensive quantitative phosphoproteomic analysis of mouse epididymal sperm from different regions (caput, corpus, and cauda) to unveil the dynamics of protein phosphorylation during sperm maturation. We identified 6447 phosphorylation sites in 1407 phosphoproteins, and 345 phosphoproteins were differentially phosphorylated between caput and cauda sperm. Gene ontology and KEGG pathway analyses showed enrichment of differentially phosphorylated proteins in energy metabolism, sperm motility, and fertilization. Kinase substrate network analysis followed by inhibition assay and quantitative phosphoproteomics analysis showed that TSSK2 kinase is important for sperm motility and progressive motility. This study systemically characterized the intricate phosphorylation regulation during sperm maturation in the mouse epididymis, which can be a basis to elucidate sperm motility acquisition, and to offer potential targets for male contraception and the treatment of male infertility.

Complexity and modification of the bull sperm glycocalyx during epididymal maturation.

Mammalian spermatozoa have a surface covered with glycocalyx, consisting of heterogeneous glycoproteins and glycolipids. This complexity arises from diverse monosaccharides, distinct linkages, various isomeric glycans, branching levels, and saccharide sequences. The glycocalyx is synthesized by spermatozoa developing in the testis, and its subsequent alterations during their transit through the epididymis are a critical process for the sperm acquisition of fertilizing ability. In this study, we performed detailed analysis of the glycocalyx on the sperm surface of bull spermatozoa in relation to individual parts of the epididymis using a wide range (24) of lectins with specific carbohydrate binding preferences. Fluorescence analysis of intact sperm isolated from the bull epididymides was complemented by Western blot detection of protein extracts from the sperm plasma membrane fractions. Our experimental results revealed predominant sequential modification of bull sperm glycans with N-acetyllactosamine (LacNAc), followed by subsequent sialylation and fucosylation in a highly specific manner. Additionally, variations in the lectin detection on the sperm surface may indicate the acquisition or release of glycans or glycoproteins. Our study is the first to provide a complex analysis of the bull sperm glycocalyx modification during epididymal maturation.

RUNX transcription factors are essential in maintaining epididymal epithelial differentiation.

Apart from the androgen receptor, transcription factors (TFs) that are required for the development and formation of the different segments of the epididymis have remained unknown. We identified TF families expressed in the developing epididymides, of which many showed segment specificity. From these TFs, down-regulation of runt related transcription factors (RUNXs) 1 and 2 expression coincides with epithelial regression in Dicer1 cKO mice. Concomitant deletion of both Runx1 and Runx2 in a mouse epididymal epithelial cell line affected cell morphology, adhesion and mobility in vitro. Furthermore, lack of functional RUNXs severely disturbed the formation of 3D epididymal organoid-like structures. Transcriptomic analysis of the epididymal cell organoid-like structures indicated that RUNX1 and RUNX2 are involved in the regulation of MAPK signaling, NOTCH pathway activity, and EMT-related gene expression. This suggests that RUNXs are master regulators of several essential signaling pathways, and necessary for the maintenance of proper differentiation of the epididymal epithelium.

The expression spectrum of yak epididymal epithelial cells reveals the functional diversity of caput, corpus and cauda regions.

Sperm undergo a series of changes in the epididymis region before acquiring the ability to move and fertilize, and the identification of genes expressed in a region-specific manner in the epididymis provides a valuable insight into functional differences between regions. We collected epididymal tissue from three yaks and cultured epithelial cells from the caput, corpus and cauda regions of the yak epididymis using the tissue block method. RNA sequencing analysis (RNA-seq) technology was used to detect gene expression in yak epididymal caput, corpus and cauda epithelial cells. The results showed that the DEGs were highest in the caput vs. corpus comparison, and lowest in the corpus vs. cauda comparison. Six DEGs were verified by real-time fluorescence quantitative PCR (qRT-PCR), consistent with transcriptome sequencing results. The significantly enriched DNA replication pathway in the caput vs. corpus was coordinated with cell proliferation, while upregulated DEGs such as POLD1 and MCM4 were found in the DNA replication pathway. The AMPK signaling pathway was found significantly enriched in the caput vs cauda, suggesting its involvement in sperm maturation and capacitation. The TGF beta signaling pathway was screened in the corpus vs cauda and is crucial for mammalian reproductive regulation. Upregulated DEGs (TGFB3, INHBA, INHBB) are involved in the TGF beta signaling pathway. This study provides a reference for culturing yak epididymal epithelial cells in vitro, and elucidates the transcriptional profiles of epithelial cells in different segments of the epididymis, revealing the regulatory and functional differences between different segments, providing basic data for exploring the molecular mechanism of yak sperm maturation and improving the reproductive capacity of high-altitude mammals.

Immortalized mouse caput epididymal epithelial (mECap18) cell line recapitulates the in-vivo environment.

Residing between the testes and the vas deferens, the epididymis is a highly convoluted tubule whose unique luminal microenvironment is crucial for the functional maturation of spermatozoa. This microenvironment is created by the combined secretory and resorptive activity of the lining epididymal epithelium, including the release of extracellular vesicles (epididymosomes), which encapsulate fertility modulating proteins and a myriad of small non-coding RNAs (sncRNAs) that are destined for delivery to recipient sperm cells. To enable investigation of this intercellular communication nexus, we have previously developed an immortalized mouse caput epididymal epithelial cell line (mECap18). Here, we describe the application of label-free mass spectrometry to characterize the mECap18 cell proteome and compare this to the proteome of native mouse caput epididymal epithelial cells. We report the identification of 5,313 mECap18 proteins, as many as 75.8% of which were also identified in caput epithelial cells wherein they mapped to broadly similar protein classification groupings. Furthermore, key pathways associated with protein synthesis (e.g., EIF2 signaling) and cellular protection in the male reproductive tract (e.g., sirtuin signaling) were enriched in both proteomes. This comparison supports the utility of the mECap18 cell line as a tractable in-vitro model for studying caput epididymal epithelial cell function.

Commonality and heterogeneity of pacemaker mechanisms in the male reproductive organs.

During emission, the first phase of ejaculation, smooth muscle in organs of the male reproductive tract (MRT) vigorously contract upon sympathetic nerve excitation to expel semen consisting of sperm and seminal plasma. During inter-ejaculation phases, the epididymis, seminal vesicles and prostate undergo spontaneous phasic contractions (SPCs), this transporting and maintaining the quality of sperm and seminal plasma. Recent studies have revealed platelet-derived growth factor receptor α-expressing (PDGFRα+) subepithelial interstitial cells in seminal vesicles subserve the role of pacemaker cells that electrically drive SPCs in this organ. PDGFRα+ smooth muscle cells in the epididymis also appear to function as pacemaker cells implicating PDGFRα as a potential signature molecule in MRT pacemaking. The dominant mechanism driving pacemaking in these organs is the cytosolic Ca2+ oscillator. This operates through entrainment of the release-refill cycle of Ca2+ stores, the released Ca2+ ions opening Ca2+-activated chloride channels, including in some cases ANO1 (TMEM16A), with the resultant pacemaker potential activating L-type voltage-dependent Ca2+ channels in the smooth muscle causing contraction (viz. SPCs). A second pacemaker mechanism, namely the membrane oscillator also has a role in specific cases. Further investigations into the commonality and heterogeneity of MRT pacemakers will open an avenue for understanding the pathogenesis of male infertility associated with deterioration of seminal plasma.

The GgcxK325Q Mutation Does Not Affect the Calcium Homeostasis of the Epididymis and Male Fertility in Mice.

A low-calcium microenvironment is imperative for spermatozoa maturation within the epididymis. Our previous work has shown that γ-glutamyl carboxylase (GGCX), the carboxylation enzyme of the matrix Gla protein (MGP), plays an essential role in epididymal calcium homeostasis and sperm maturation in rats and that the GGCX SNP mutation rs699664 was associated with asthenozoospermia (AZS) in humans. Here, we investigated the expression patterns of GGCX and MGP in the mouse epididymis and generated GgcxK325Q knock-in (KI) mice. We also tested the effects of this mutation on epididymal calcium homeostasis, sperm function, and male fertility in GgcxK325Q-/- mice. The results showed that both GGCX and MGP were enriched in all regions of the mouse epididymis, especially in the initial segment of the epididymis. Double immunofluorescence staining revealed that GGCX colocalized with MGP in the epithelial cells of the initial segment and caput regions as well as in the lumen of the corpus and cauda regions of the mouse epididymis. However, the GgcxK325Q-/- mice were fertile with normal epididymal morphology, sperm functions, and epididymal calcium concentration. Overall, our findings revealed that the GgcxK325Q mutation does not exert any discernible effect on male fertility in mice.

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.

In vivo dynamic volumetric imaging of mouse testis and epididymis with optical coherence tomography†.

The implementation of live imaging in reproductive research is crucial for studying the physiological dynamics. Sperm transport is a highly dynamic process regulated by tubular contractions and luminal flows within the male reproductive tract. However, due to the lack of imaging techniques to capture these dynamics in vivo, there is little information on the physiological and biomechanical regulation of sperm transport through the male reproductive tract. Here, we present a functional in vivo imaging approach using optical coherence tomography, enabling live, label-free, depth-resolved, three-dimensional, high-resolution visualization of the mouse testis and epididymis. With this approach, we spatiotemporally captured tubular contractility in mouse testis and epididymis, as well as microstructures of these reproductive organs. Our findings demonstrated that the contraction frequency varies significantly depending on the epididymal regions, suggesting the spatial regulation of epididymal contractility. Furthermore, we implemented quantitative measurements of the contraction wave and luminal transport through the epididymal duct, revealing the physiological dynamics within the male reproductive tract. The results show that the contraction wave propagates along the epididymal duct and the wave propagation velocity was estimated in vivo. In conclusion, this is the first study to develop in vivo dynamic volumetric imaging of the male reproductive tract, which allows for quantitative analysis of the dynamics associated with sperm transport. This study sets a platform for various studies investigating normal and abnormal male reproductive physiology as well as the pharmacological and environmental effects on reproductive functions in mouse models, ultimately contributing to a comprehensive understanding of male reproductive disorders.

Extracellular vesicles are involved in the paracrine communication between epithelial cells in different regions of the domestic cat epididymis.

Sperm maturation depends on exposure to specific microenvironments within the different segments of the epididymis, but mechanisms underlying how these microenvironments are produced or maintained are not well understood. We hypothesized that epididymal extracellular vesicles (EVs) could play a role in the process of maintaining microenvironments in different regions of the epididymis. Specifically, we tested whether the EVs from different regions of the epididymis can serve as a form of paracrine communication between cells in different segments. Domestic cat tissues were used to develop a reproducible in vitro culture system for corpus epididymis explants that were then exposed to EVs collected from upstream (i.e. caput) segments. The impacts of different culture or exposure conditions were compared by analyzing the morphology, apoptosis, transcriptional activity, and gene expression in the explants. Here, we report the development of the first in vitro culture system for epididymal tissue explants in the domestic cat model. Using this system, we found that EVs from the caput segment have a significant effect on the transcriptional profile of tissue from the corpus segment (1233 differentially expressed genes due to EV supplementation). Of note, expression of genes associated with regulation of epithelial cell differentiation and cytokine signaling in the epididymis were regulated by the presence of EVs. Together, our findings comprise the first report of paracrine control of segmental gene regulation by epididymal EVs in any species. These results contribute to a better understanding of epididymis biology and could lead to techniques to enhance or suppress male fertility.

Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis.

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl- channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential -60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl- concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.

Epididymosomes: Composition and Functions for Sperm Maturation.

This article provides an overview of literature pertaining to epididymosome origin, composition and their functional significance. Broadly, epididymosomes are defined as extracellular vesicles that are secreted by the epididymal epithelium and thereafter facilitate intercellular communication within the male reproductive tract. Epididymosomes fulfil this communication role via their encapsulation and delivery of a diverse macromolecular payload to recipient cells. This complex cargo includes proteins, lipids, and nucleic acids, which are delivered to maturing spermatozoa, thereby influencing their viability and function. Additionally, epididymosomes have been implicated in the post-translational modification of intrinsic sperm proteins, protection of sperm from oxidative stress and immune surveillance, and in the transmission of epigenetic information capable of mediating intergenerational effects. Hence, continued research into the biogenesis, cargo composition, and functional significance of epididymosomes holds promise for advancing male reproductive health and fertility treatments.

Distinct actions of testicular endocrine and lumicrine signaling on the proximal epididymal transcriptome.

The epididymal function and gene expression in mammals are under the control of the testis. Sex steroids are secreted from the testis and act on the epididymis in an endocrine manner. There is another, non-sex steroidal secreted signaling, named lumicrine signaling, in which testis-derived secreted proteins go through the male reproductive tract and act on the epididymis. The effects of such multiple regulations on the epididymis by the testis have been investigated for many genes. The recent development of high-throughput next-generation sequencing now enables us a further comparative survey of endocrine and lumicrine action-dependent gene expression. In the present study, testis-derived endocrine and lumicrine actions on epididymal gene expression were comparatively investigated by RNA-seq transcriptomic analyses. This investigation utilized experimental animal models in which testis-derived endocrine and/or lumicrine actions were interfered with, such as unilateral or bilateral orchidectomy. By bilateral orchidectomy, which interferes with both endocrine and lumicrine actions, 431 genes were downregulated. By unilateral orchidectomy, which also interferes with endocrine and lumicrine actions by the unilateral testis, but the endocrine action was compensated by the contralateral testis, 283 genes were downregulated. The content of such genes downregulated by unilateral orchidectomy was like those of lumicrine action-interfered efferent duct-ligation, W/Wv, and Nell2-/- mice. When genes affected by unilateral and bilateral orchidectomy were compared, 154 genes were commonly downregulated, whereas 217 genes were specifically downregulated only by bilateral orchidectomy, indicating the distinction between endocrine and lumicrine actions on the proximal epididymal transcriptome. Comparative transcriptome analyses also showed that the expressions of genes emerging since Amniota were notably impacted by bilateral orchidectomy, unilateral orchidectomy, and lumicrine action-interfering treatments; the degree of influence from these treatments varied based on the evolutionary stage beyond Amniota. These findings unveil an evolutional transition of regulated gene expression in the proximal epididymis by two different testis-derived signaling mechanisms.

Expression of NELL2/NICOL-ROS1 lumicrine signaling-related molecules in the human male reproductive tract.

The maturation of spermatozoa is a regulated process, influenced by genes expressing essential secreted proteins in the proximal epididymis. Recent genetic studies in rodents have identified the non-sex steroidal molecular signals that regulate gene expression in the proximal epididymis. Germ cells in the testis secrete ligand proteins into the seminiferous tubule lumen The ligand proteins travel through the male reproductive tract lumen to the epididymis, where they bind to receptors, triggering the differentiation of the luminal epithelium for sperm maturation. It is, however, not fully unveiled if such a testis-epididymis trans-luminal signaling mechanism exists in other species, especially humans. In the present study, the rodent-type testis-epididymis trans-luminal signaling in the human male reproductive tract was evaluated in a step-by-step manner by analyzing testis and epididymis gene expression and signaling mediator protein function. There was a significant correlation between the epididymal expressions of mouse genes upregulated by the trans-luminal signaling and those of their human orthologs, as evaluated by the correlation coefficient of 0.604. The transcript expression of NELL2 and NICOL encoding putative ligand proteins was also observed in human testicular cells. In vitro experiments demonstrated that purified recombinant human NELL2 and NICOL formed a molecular complex with similar properties to rodent proteins, which was evaluated by a dissociation equilibrium constant of 110 nM. Recombinant human NELL2 also specifically bound to its putative receptor human ROS1 in vitro. Collectively, these findings suggest that the rodent-type testis-epididymis secreted signaling mechanism is also possible in the human male reproductive tract.

Glucocorticoid exposure modifies the miRNA profile of sperm in the guinea pig: Implications for intergenerational transmission.

Approximately 1%-3% of the adult population are treated with synthetic glucocorticoids (sGCs) for a variety of conditions. Studies have demonstrated that adversities experienced by males prior to conception may lead to abnormal neuroendocrine function and behaviors in offspring and that epigenetic factors including microRNA (miRNA) within sperm may be responsible for driving these effects. However, it remains unclear where in the epididymis sperm miRNA changes are occurring. Here, we hypothesized that sGC exposure will alter the miRNA profile of sperm in the epididymis in a region-specific manner. Adult male guinea pigs were exposed to regular drinking water (Ctrl) or water with the sGC dexamethasone (Dex; 3mg/kg) (n = 6/group) every other day for 48 days. Sperms were collected from epididymal seminal fluid in the caput and cauda regions of the epididymis and total RNA was extracted. miRNAs were assessed by miRNA 4.0 microarray; data were processed by TAC 4.0.1 and R. miRNA analysis revealed one miRNA in the caput that was significantly decreased by Dex in sperm. In the cauda, 31 miRNAs were reduced in sperm following Dex-exposure. The findings of this study demonstrate that Dex-exposure influences miRNA profile of sperm in the cauda but not the caput of the epididymis. This suggests that glucocorticoids target the epididymis to modify sperm miRNA and do not modify the miRNA content during spermiation in the testes.

Single-cell and spatial transcriptomic investigation reveals the spatiotemporal specificity of the beta-defensin gene family during mouse sperm maturation.

Low sperm motility is a significant contributor to male infertility. beta-defensins have been implicated in host defence and the acquisition of sperm motility; however, the regulatory mechanisms governing their gene expression patterns and functions remain poorly understood. In this study, we performed single-cell RNA and spatial transcriptome sequencing to investigate the cellular composition of testicular and epididymal tissues and examined their gene expression characteristics. In the epididymis, we found that epididymal epithelial cells display a region specificity of gene expression in different epididymal segments, including the beta-defensin family genes. In particular, Defb15, Defb18, Defb20, Defb25 and Defb48 are specific to the caput; Defb22, Defb23 and Defb26 to the corpus; Defb2 and Defb9 to the cauda of the epididymis. To confirm this, we performed mRNA fluorescence in situ hybridisation (FISH) targeting certain exon region of beta-defensin genes, and found some of their expression matched the sequencing results and displayed a close connection with epididimosome marker gene Cd63. In addition, we paid attention to the Sertoli cells and Leydig cells in the testis, along with fibroblasts and smooth muscle cells in the epididymis, by demonstrating their gene expression profile and spatial information. Our study provides a single-cell and spatial landscape for analysing the gene expression characteristics of testicular and epididymal environments and has important implications for the study of spermatogenesis and sperm maturation.

The role of the radiologist in the evaluation of male infertility: recommendations of the European Society of Urogenital Radiology-Scrotal and Penile Imaging Working Group (ESUR-SPIWG) for scrotal imaging.

OBJECTIVES: The Scrotal and Penile Imaging Working Group (SPIWG) of the European Society of Urogenital Radiology (ESUR) aimed to produce recommendations on the role of the radiologist in the evaluation of male infertility focused on scrotal imaging. METHODS: The authors independently performed an extensive literature Medline search and a review of the clinical practice and consensus opinion of experts in the field. RESULTS: Scrotal ultrasound (US) is useful in investigating male infertility. US abnormalities related to abnormal sperm parameters (sperm concentration, total count, motility, and morphology) are low testicular volume (TV), testicular inhomogeneity (TI), cryptorchidism, testicular microlithiasis (TML), high-grade varicocele, bilateral absence of vas deferens, bilateral dilation and echotexture abnormalities of the epididymis. The proposed ESUR-SPIWG recommendations for imaging in the evaluation of male infertility are therefore: to measure TV; investigate TI; perform annual (US) follow-ups up to age 55 in men with a history of cryptorchidism/orchidopexy and/or in men with TML plus "additional risk factors" or with "starry sky" TML; perform scrotal/inguinal US in men with nonpalpable testis; perform scrotal US in men with abnormal sperm parameters to investigate lesions suggestive of tumors; evaluate varicocele in a standardized way; evaluate the presence or absence of vas deferens; investigate the epididymis to detect indirect signs suggesting obstruction and/or inflammation. CONCLUSIONS: The ESUR-SPIWG recommends investigating infertile men with scrotal US focusing on TV, inhomogeneity, localization, varicocele, vas deferens, and epididymal abnormalities. Cryptorchidism, TML, and lesions should be detected in relation to the risk of testicular tumors. CLINICAL RELEVANCE STATEMENT: The ESUR-SPIWG recommendations on scrotal imaging in the assessment of male infertility are useful to standardize the US examination, focus on US abnormalities most associated with abnormal semen parameters in an evidence-based manner, and provide a standardized report to patients. KEY POINTS: So far, ESUR-SPIWG recommendations on scrotal imaging in the assessment of male infertility were not available. The ESUR-SPIWG recommends investigating infertile men with scrotal US focusing on testicular volume, inhomogeneity, localization, varicocele, vas deferens and epididymal abnormalities, and assessing cryptorchidism, testicular microlithiasis and lesions in relation to the risk of testicular tumors. The ESUR-SPIWG recommendations on scrotal imaging in the assessment of male infertility are useful to standardize the US examination, focus on US abnormalities most associated with abnormal sperm parameters in an evidence-based manner, and provide a standardized report to patients.

Effect of Spag11a gene knockout on the epididymis in mice: A histopathological and molecular analyses.

The sperm-associated antigen 11a (Spag11a) gene is exclusively expressed in the caput epididymis. Our previous studies demonstrated that small interfering RNA (siRNA)-mediated ablation of this gene resulted in increased proliferation of epididymal epithelial cells. Further, active immunization-mediated ablation of SPAG11A protein increased the susceptibility of male reproductive tract tissues to diethylnitrosamine (DEN)-induced tumorigenesis. In this study, we report that the caput epididymis of Spag11a knockout mice displayed hyperplasia and inflammation, while the caput epididymis of wild-type mice exhibited normal anatomical structure. Global transcriptome analyses in the caput epididymis of knockout mice indicated differential expression of genes involved in a variety of cellular processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested that the absence of Spag11a may activate microRNAs associated with cancer, chemical carcinogenesis-receptor activation, and chemical carcinogenesis-DNA adducts pathways; which may contribute to the promotion of tumorigenesis in the epididymis. The susceptibility of caput epididymis to chemically induced carcinogenesis in Spag11a knockout mice was analyzed. Histological analyses indicated that while the epididymis of wild-type mice did not show any signs of tumorigenesis, knockout mice displayed hyperplasia, anaplasia, dysplasia, neoplasia, and inflammation in the caput epididymis. Our results provide concrete evidence that deletion of Spag11a induces histopathological and molecular changes that contribute to tumorigenesis. It is possible that the expression of Spag11a gene could be one of the reasons for the rarity of epididymal cancers. The involvement of an epididymal gene in tumorigenesis is being demonstrated for the first time.

Characterization of gonadotropins and their receptors in a chondrichthyan, Scyliorhinus canicula, fills a gap in the understanding of their coevolution.

In Gnathostomes, reproduction is mainly controlled by the hypothalamic-pituitary-gonadal (HPG) axis, with the involvement of the pituitary gonadotropic hormones (GTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which activate their cognate receptors, FSHR and LHR, expressed in gonads. Each GTH consists of a common α subunit and of a specific FSHß or LHß subunit. Chondrichthyes (holocephalans and elasmobranchs) is a sister group of bony vertebrates. This position is highly favorable for the understanding of the evolution of endocrine regulations of reproduction among gnathostomes. Surprisingly, the characterization of gonadotropins and their receptors is still limited in chondrichthyes. In the present study, GTH and GTHR sequences have been identified from several chondrichthyan genomes, and their primary structures were analyzed relative to human orthologs. 3D models of GTH/GTHR interaction were built, highlighting the importance of the receptor hinge region for ligand recognition. Functional hormone-receptor interactions have been studied in HEK cells using the small-spotted catshark (Scyliorhinus canicula) recombinant proteins and showed that LHR was specifically activated by LH whereas FSHR was activated by both FSH and LH. Expression profiles of GTHs and their receptors were explored by real-time PCR, in situ hybridization and immunohistochemistry during spermatogenesis, along the male genital tract and other tissues, as well as in some female tissues for comparison. Tissue-expression analyses showed that the highest levels were observed for fshr transcripts in testis and ovary and for lhr in specific extragonadal tissues. The two receptors were expressed at all stages of spermatogenesis by both germ cells and somatic cells, including undifferentiated spermatogonia, spermatocytes, spermatids, somatic precursors and Sertoli cells; differentiated Leydig cells being absent in the testis of S. canicula. Receptors were also expressed by the lymphomyeloid epigonal tissue and the testicular tubules. These results, suggest a wide range of gonadotropin-regulated functions in Elasmobranchs, as well as functional redundancy during spermatogenesis. These extended functions are discussed in an evolutionary context in which the specificity of gonadotropin signaling must have contributed to the evolution of gonadal cells' morphology and function.

Busulfan administration replicated the characteristics of the epididymal initial segment observed in mice lacking testis-epididymis lumicrine signaling.

The physiological functions of the mammalian epididymis are typically regulated by the testes. In addition to sex steroids secreted by testicular Leydig cells, which act on the epididymis in an endocrine manner, there is a non-sex-steroidal signaling pathway known as the lumicrine pathway. This lumicrine signaling pathway involves ligand proteins secreted from germ cells within the testicular seminiferous tubules traversing the male reproductive tract, which induce epithelial differentiation in the epididymis. These findings prompted an inquiry into whether treatments influencing testis physiology can disrupt epididymal function by interfering with testis-epididymis communication. Busulfan, an alkylating agent commonly used to deplete testicular germ cells in reproductive biology, has not been sufficiently explored because of its effects on the epididymis. This study investigated the effects of busulfan administration on the proximal epididymis using histological and transcriptomic analyses. Notably, busulfan, as opposed to the vehicle dimethyl sulfoxide (DMSO), altered the morphology of the initial segment of the epididymis, leading to a reduction in the cell height of the luminal epithelium. RNA sequencing identified 185 significantly downregulated genes in the proximal epididymis of busulfan-administered mice compared to DMSO-administered mice. Comparative transcriptome analyses revealed similarities between the epididymal transcriptome of busulfan-administered mice and lumicrine-deficient mice, such as efferent-duct-ligated W/Wv and Nell2-/- mice. However, this differed from that of bilaterally orchidectomized mice, in which both the endocrine and lumicrine signaling pathways were simultaneously ablated. Collectively, these results suggested that the harmful effects of busulfan on the proximal epididymis are secondary consequences of the ablation of testis-epididymis lumicrine signaling.