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.

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.

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.

Adhesion G protein-coupled receptor G2 is dispensable for lumicrine signaling regulating epididymal initial segment differentiation and gene expression†.

The mammalian epididymis is the organ for functional sperm maturation. In rodents, the initial segment, the most proximal region of the epididymis, plays a critical role in sperm maturation. The luminal epithelial differentiation and the following gene expression of the initial segment are regulated by the lumicrine signaling, a testis-epididymis transluminal secreted signaling. Adhesion G protein-coupled receptor G2 (ADGRG2) is expressed in the efferent duct and the initial segment epididymis. In the preceding study, Adgrg2 ablation decreased the expression of several genes expressed in the initial segment. Such downregulated genes include those known to be regulated by lumicrine signaling, suggesting the involvement of ADGRG2 in lumicrine signaling. The present study examined whether ADGRG2 is associated with the lumicrine signaling regulating epididymal initial segment differentiation and gene expression. Adgrg2-null mice were generated by CRISPR/CAS9-mediated genome editing. The postnatal differentiation of the Adgrg2-null male epididymal initial segment was histologically comparable with that of control wild-type animals. The RNA-seq of Adgrg2-null mice was performed together with those of efferent duct-ligated and W/Wv mice in both of which lumicrine signaling is defective. The comparative transcriptome analyses clarified that the expressions of genes expressed in the initial segment and regulated by lumicrine signaling were decreased by Adgrg2 nullification. However, the extent of such downregulations observed in Adgrg2-null epididymis was not so prominent compared with those of lumicrine signaling deficient Nell2-/-, efferent duct-ligated, or W/Wv mice. Collectively, these findings indicate that ADGRG2 is dispensable for the lumicrine regulation of epididymal initial segment differentiation.

Segmental differentiation of the murine epididymis: identification of segment-specific, GM1-enriched vesicles and regulation by luminal fluid factors†.

The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.

Regulation of apical blebbing in the porcine epididymis.

Apical blebbing, a non-classical secretion mechanism, occurs in the mature porcine epididymis as part of its normal function. Proteins secreted by this mechanism contribute to the modification of the sperm plasma membrane during epididymal transit and are thought to contribute to acquisition of fertilizing ability. However, little is known about the regulation of this secretion mechanism in an in vivo model. Previous work demonstrated apical blebbing in the epididymis developed pubertally, suggesting androgens, sperm or other luminal factors regulated this process. Hence, the objective was to evaluate the hypothesized regulation of apical blebbing in the epididymis of pubertal boars by androgens and luminal factors. Androgen receptor blockade (flutamide) and surgical interventions (efferent duct ligation, orchidectomy or transection of the caput epididymis) were used to alter signaling, and the subsequent effects on apical blebbing were evaluated histologically. Apical blebbing was not altered by androgen receptor blockade with flutamide, but was significantly reduced 24 h after efferent duct ligation and after orchidectomy, treatments that eliminated luminal flow from the testis (P < 0.05). Like efferent duct ligation, epididymal transection altered luminal flow without removing the androgen source and significantly reduced the appearance of apical blebbing (P < 0.05). In conclusion, apical blebbing in the porcine epididymis appears to be regulated by luminal factors.

Initial Segment Differentiation Begins During a Critical Window and Is Dependent upon Lumicrine Factors and SRC Proto-Oncogene (SRC) in the Mouse.

Without a fully developed and functioning initial segment, the most proximal region of the epididymis, male infertility results. Therefore, it is important to understand the development of the initial segment. During postnatal development of the epididymis, many cellular processes of the initial segment are regulated by lumicrine factors, which are produced by the testis and enter the epididymis with testicular luminal fluid. In this report, we showed that prior to Postnatal Day 15 (P15), the initial segment was lumicrine factor independent in the mouse. However, from P19 onward, lumicrine factors were essential for the proliferation and survival of initial segment epithelial cells. Therefore, P15 to P19 was a critical window that established the dependency of lumicrine factors in the initial segment epithelium. The initial segment-specific kinase activity profile, a marker of initial segment differentiation, was also established during this window. The SFK (SRC proto-oncogene family kinases), ERK pathway (known as the RAF/MEK/ERK pathway) components, and AMPK (AMP-activated protein kinases) pathway components had increased activities from P15 to P19, suggesting that lumicrine factors regulated SFK/ERK/AMPK signaling to initiate differentiation of the initial segment from P15 to P19. Compared with litter mate controls, juvenile Src null mice displayed lower levels of MAPK3/1 (mitogen-activated protein kinase 3/1) activity and a reduced level of differentiation in the initial segment epithelium, a similar phenotype resulting from inhibition of SRC activity within the window of P15 to P19. Therefore, lumicrine factor-dependent SRC activity signaling through MAPK3/1 is important for the initiation of initial segment differentiation during a critical window of development.

The molecular mechanisms of mammalian sperm maturation regulated by NELL2-ROS1 lumicrine signaling.

In terrestrial vertebrates, spermatozoa generated in the testis are transported through the reproductive tract toward outside the body. In addition to as the pathway of sperm transport, the male reproductive tract also functions as the site of post-testicular sperm maturation and the epididymis, which constitutes the majority of male reproductive tract, and plays central roles in such a sperm maturation. Recent studies with gene-modified animals have been unveiling not only the molecular mechanisms of sperm maturation in the epididymis but also the regulatory system by which the epididymis acquires and executes sperm-maturing functions. In this review, the mechanisms of mammalian sperm maturation will be summarized, based on recent findings, including the lumicrine regulation of sperm maturation.

Examination of testicular lumicrine regulation of activins and immunoregulatory genes in the epididymal caput.

BACKGROUND: Immunoregulatory genes encoding activin A (Inhba) and B (Inhbb), and indolamine 2,3-dioxygenase-1 (Ido1) are highly expressed in the murine caput epididymidis, which also has a network of intraepithelial mononuclear phagocytes. This environment is postulated to promote immunological tolerance to epididymal sperm. The factors regulating the immunoregulatory agents in the epididymal caput are poorly understood. OBJECTIVES: This study aimed to investigate the potential role of testicular lumicrine factors in regulating activin and other immune-related genes in the caput epididymidis. MATERIALS AND METHODS: The efferent ducts in adult C57/Bl6 mice were exposed and ligated bilaterally. Serum and tissues were collected seven days later. Animals with bilateral sham ligation and animals with no ligations (collectively referred to as the "intact" group) were used as controls. RESULTS: Pressure-induced seminiferous epithelial damage due to intratubular fluid accumulation was observed in all ligated testes. Testicular inhibin was significantly increased and testosterone was elevated in some animals following bilateral ligation, but serum testosterone, serum LH, and serum inhibin were normal. Ligation caused epithelial regression in the initial segment, with similar but less severe effects in other caput segments. Activin A staining by immunohistochemistry in the epithelium was reduced in bilateral ligation, particularly in the initial segment, with moderately reduced staining intensity in the rest of the caput. Inhba expression within the caput was not significantly affected by bilateral ligation, but Inhbb was reduced by more than 60%. Transcripts encoding the macrophage-specific receptor Cx3cr1 were significantly reduced following bilateral ligation, but other immune cell markers, Ido1, and inflammatory genes were unaffected. CONCLUSION: These data indicate that testicular lumicrine secretion regulates several genes that are preferentially expressed in the initial segment, but has marginal effects on genes such as those encoding activin A and IDO1, which are expressed more widely in the caput.

Mouse CD52 is Predominantly Expressed in the Cauda Epididymis, Regulated by Androgen and Lumicrine Factors.

BACKGROUND: Sperm maturation takes place through contact between sperm and proteins produced in the epididymal lumen. CD52 had been characterised in the sperm; however, the expression and its regulation in the epididymis are mostly unknown. AIM: This study aimed to analyse the expression and regulation of CD52 in the mouse epididymis. SETTING AND DESIGN: Experimental design was used in this study. MATERIALS AND METHODS: Epididymis tissues from mice strain Deutch Democratic Yokohama were used as sources of total RNA. Bioinformatic tool was used to predict signal peptides. Quantitative real-time reverse transcription-polymerase chain reaction was used to analyse tissue distribution, androgen, testicular factors dependency and postnatal development. STATISTICAL ANALYSIS: One-way analysis of variance was used to analyse differences between treatment and control untreated group. P < 0.05 was determined as a significant difference. RESULTS: CD52 amino acid sequence contains a signal peptide, indicating it is a secretory protein. CD52 exhibited region-specific expression in the epididymis, with the highest level being in the cauda. CD52 expression was regulated by androgen indicated by a significant downregulation at day 1 and day 3 following a castration (P < 0.05). Dependency on androgen was confirmed by injection of exogenous testosterone which prevented downregulation by 50%. Moreover, lumicrine factors also influenced CD52 expression indicated by ligation of efferent duct which also reduced expression at day 1 to day 5 following the ligation (P < 0.05). CD52 expression was developmentally regulated. This was shown by increase in the level of expression starting at day 15 postnatally. CONCLUSION: CD52 shows characteristics of genes involved in sperm maturation in the epididymis.