Abstract
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.
Introduction
A considerable number of couples who want to have a child fail to achieve pregnancy. A past survey conducted by the World Health Organization attributed infertility to ‘the male only’ in 24% of cases and ‘both sexes’ in another 24% (1). Whereas the cause of infertility in humans often remains unclarified, studies with gene-modified model animals have increased our knowledge on the physiology of reproduction and the pathology of infertility. The acceleration of such genetic studies by the recent genome editing technology has been unveiling not only the spermatogenesis, which has been one of central themes in reproductive biology, but also the functional maturation of spermatozoa. One of such recent findings on sperm maturation is the lumicrine, a testis–epididymis transluminal signaling by which sperm maturation in the epididymis is governed. The present review describes the mechanism of mammalian sperm maturation focusing on the lumicrine regulation system.
The Testis and Spermatogenesis
The mammalian spermatozoon can be morphologically divided into a head and a tail. The head comprises a nucleus containing highly condensed chromatin, an acrosome (a cytoplasmic vesicle containing hydrolases and molecules required for the interaction with oocytes) and a small amount of cytoplasm, whereas the tail is a eukaryotic flagellum having an axoneme with a 9 + 2 arrangement of microtubules at its center. Spermatozoa are generated from spermatogonial stem cells located inside the seminiferous tubules of the testis (Fig. 1A). In the mouse, spermatogenesis begins a few days after birth and takes 35 days to complete (2).
The epididymis is the site where sperm maturation occurs. (A) Male reproductive organs. Spermatozoa are generated inside seminiferous tubule of testis, whereas they become mature in the epididymis. The anatomical parts of caput, corpus and cauda of epididymis are also indicated. ST, seminiferous tubule; ED, efferent duct; IS, initial segment; VD, vas deferens. (B) A schematic representation of spermatogenesis in the seminiferous tubule. The basally located spermatogonial stem cells differentiate into spermatozoa, which will be released into the lumen of seminiferous tubule. (C) Spermatozoa acquire motility, migratory ability into oviduct and egg binding along their maturation in the epididymis. (D) Histology of untreated (upper) and efferent duct-ligated (lower) epididymal initial segments. Epididymal cell functions are regulated by factors coming from testis. Bars, 50 μm.
After completion of morphogenesis, spermatozoa are released into the lumens of seminiferous tubules (Fig. 1B). At that time, each spermatozoon morphologically appears as a complete gamete equipped with a compact head and a long flagellum. However, the spermatozoa are immature and still lack egg-fertilizing ability; they cannot swim by tail waving or fuse with the egg (3). To become fully fertile, spermatozoa need further functional maturation.
The Epididymis and Sperm Maturation
The spermatozoa released from the seminiferous tubule epithelium are transported to the epididymis via the efferent duct. The epididymis is a highly coiled epithelial duct (Fig. 1A) originating from the mesonephric duct in the intermediate mesoderm. Anatomically, the epididymis consists of three parts: caput (head), corpus (body) and cauda (tail) as first described by Benoit (4). The whole epididymis constitutes a route for sperm transport sperm coming from testis by connecting with testis via efferent duct at the caput and with vas deferens at cauda epididymis. The total length of the epididymal lumen reaches ~1 m in mice and 4–5 m in humans, with some units of the coiled structure divided by connective tissue. The epididymis is found only in land vertebrates that propagate by internal fertilization.
‘Immature’ testicular spermatozoa pass slowly through the lumen from caput to cauda epididymis over a period of 10 days to 2 weeks, during which the spermatozoa become ‘mature.’ The epididymis therefore not only serves as a sperm transport route but also provides an environment for functional sperm maturation. During their maturation, spermatozoa acquire such abilities to migrate and ascend the female genital tract to reach eggs, to exert acrosome reaction for the release of its contents necessary for successful fertilization, and to bind eggs (Fig. 1C). In the head of spermatozoa, the nuclear chromatin becomes highly condensed during the epididymal transit along with the inter and intra-molecular disulfide bond formation of protamine, a small basic protein abundant in the sperm nucleus (5, 6) Since the transcription and translation are silent at the completion of spermatogenesis, sperm maturation is considered to be induced by posttranslational modifications.
Spermatozoa thus acquire the abilities needed for successful reproduction as they mature. However, little has so far been clarified on the molecular mechanism of sperm maturation in the epididymis, mainly because of the lack of development of the appropriate experimental systems and animal models suitable for clarifying the molecular mechanisms of controlling sperm ‘quality’ such as the degree of maturation. The molecular mechanisms of sperm maturation in epididymis have therefore remained poorly characterized.
Activation of Sperm Maturation Mechanism by Epididymis
When considered from the viewpoint of the epididymis itself, an interesting experimental fact was reported >40 years ago in 1978. The initial segment of the rodent epididymis is characterized by a highly differentiated and tall pseudostratified monolayer of epithelial cells. Interestingly, this luminal epithelium of initial segment becomes degenerated and therefore thin when the efferent duct was ligated to interfere with luminal connection between testis and epididymis (7, 8) (Fig. 1D). This phenomenon can be explained by the working hypothesis that secretory factors coming from testis through luminal space reach the epididymis and promote the differentiation of initial segment epithelial cells. This secretory mode was named ‘lumicrine’ (lumi + crine) as secreted factors migrate via the luminal space of male reproductive tract (9). Although some secreted proteins expressed in the testis were deemed lumicrine factor candidates, their molecular roles had remained unclear until gene-modified animals were available.
The receptor tyrosine kinase Ros1 (c-ros) is well known as a proto-oncogene because its fusion product often causes non-small-cell lung cancer (10). In mice, Ros1 gene is expressed in the initial segment of the epididymis. When Ros1 is knocked out, the resulting Ros1−/− males were complete infertile because the spermatozoa ejaculated into the female reproductive tract were unable to ascend the oviduct, thereby causing male infertility (11, 12). Interestingly, the Ros1−/− males exhibited initial segment differentiation failures, similarly to the above-described results of testicular efferent duct ligation. These facts suggest a possibility that ROS1 serves as a receptor of testicular lumicrine factors.
ROS1 is known to be activated constitutively by fusing with various proteins as a consequence of gene rearrangements in human non-small cell lung cancer (10). As the endogenous ligand for ROS1 had not been identified yet, ROS1-binding proteins were screened based on the hypothesis that the endogenous ligand for ROS1 is a lumicrine factor. The NEL-like protein 2 (NELL2) was thus identified as a secretory protein with estimated molecular weight of ~90,000 having a domain structure of extracellular matrix molecules, such as the laminin G domain and EGF-like domain (Fig. 2A). Nell2 is exclusively expressed in brain and testis but not in epididymis (13) (Fig. 2B). While NELL2 was reported to mediate axonal guidance as a ligand for Robo3 in brain (14), its physiological roles in testis remained unclear. When recombinant NELL2 protein was expressed and purified from mammalian cells, it bounds specifically to ROS1 in vitro (Fig. 2C). Ablation of Nell2 by genome editing technology resulted in male infertility despite the absence of abnormalities in spermatogenesis in the testis or mating behavior. In the initial segment of the Nell2−/− epididymis, the differentiation of luminal epithelium was defective (Fig. 2D) as observed in those of efferent duct-ligated or Ros1−/− animals. The phosphorylation of ERK1/2 and the expression of Etv family transcription factors, both are located downstream of ROS1-mediated intracellular signaling, were downregulated. On the other hand, when Nell2 ablation was rescued with testis-specific expression of Nell2 transgene, the impaired initial segment differentiation and male infertility were completely restored. These results led to the conclusion that NELL2 is an endogenous ligand for ROS1 and the testis-derived lumicrine factor (15).
Lumicrine Regulation of Sperm Maturation
The localization of sperm ejaculated into the female reproductive tract can be tracked by illuminating them with fluorescent protein expression (16). This technology elucidated how the epididymis regulates sperm maturation and fertility under the control of lumicrine signaling. Spermatozoa ejaculated by wild-type males ascends from uterus into the oviduct, whereas those from Nell2−/− males remained in the uterus and did not migrate into the oviduct (15) (Fig. 2E and F). Such a defective sperm migration in the female reproductive tract often coincides with the disappearance of a membranous protein present on sperm surfaces, i.e. ‘a disintegrin and metalloprotease domain 3’ (ADAM3) in many distinct mutant mouse lines (17). ADAM3 is a member of what is called the ADAM metalloproteinase family but lacks its protease activity due to amino acid substitution. During spermatogenesis in the testis, ADAM3 starts to express as a precursor with a molecular weight of ~100,000 and processed into a mature form with a molecular weight of ~30,000 by an unknown mechanism during the sperm transit through the epididymis (18) (Fig. 3A). In the sperm isolated from the cauda epididymis of Nell2−/− or Ros1−/− mice, ADAM3 was not processed appropriately into the mature form, and lost eventually (Fig. 3B). These findings suggest that the ADAM3 processing enzymes are not expressed or dysfunctional because of an initial segment differentiation failure.
NELL2 is a lumicrine mediator. (A) A schematic representation of NELL2. (B) Nell2 expressions in urogenital organs. (C) NELL2-conjugated beads specifically pull-down ROS1. (D) Histology of wild-type and Nell2−/− initial segment epididymis. Note the height of luminal epithelium. Bars, 100 μm. (E) A schematic representation of female reproductive tract. Spermatozoa migrate from uterus into oviduct toward oocytes inside ampulla. The boxed area corresponds to the view field in F. (F) Red fluorescence-illuminated wild-type (left) and Nell2−/− (right) spermatozoa ejaculated into female reproductive tract. Nell2−/− spermatozoa do not migrate into oviduct. Bars, 500 μm.
Lumicrine regulation of sperm maturation. (A) A schematic representation of sperm ADAM3 and its maturation by processing in epididymis. (B) Impaired ADAM3 processing in Nell2−/− spermatozoa. (C) A schematic representation of secreted protease OVCH2. (D) OVCH2 is absent in Nell2−/− epididymis. (E) ADAM3 maturation is also aberrant in Ovch2−/− spermatozoa. (F) A working model of sperm maturation by NELL2–ROS1–OVCH2–ADAM3 axis. Top, NELL2 protein coming from testis through luminal space binds ROS1 on the apical surface of epididymal epithelium to trigger its differentiation. Bottom, the differentiated epididymal epithelium secretes OVCH2 that directly or indirectly process ADAM3 on sperm surface.
A comparative analysis of epididymal transcriptomes found that the chymotrypsin-like serine protease ovochymase 2 (OVCH2) and metalloproteinase ADAM28 were abundantly expressed in the epididymis of wild-type mice but significantly decreased in that of Nell2−/− mice (Fig. 3D). When Ovch2 and Adam28 were ablated in mice by genome editing, Adam28−/− males were fertile, whereas male Ovch2−/− males were completely infertile because of the defective sperm migration into the oviduct (15). The initial segment of Ovch2−/− epididymis differentiated normally, but sperm ADAM3 processing into the mature form was found to be abnormal (Fig. 3E). These results demonstrated a cascade of events in sperm maturation regulated by lumicrine signaling. Testicular NELL2 first reaches the epididymis through the luminal space of reproductive tract and activates ROS1 to trigger luminal epithelial differentiation. Then OVCH2 is secreted from the differentiated luminal epithelium into the lumen, where ADAM3 on the surface of spermatozoa is directly or indirectly processed to develop the ability of spermatozoa to migrate into the oviduct (Fig. 3F).
Other Recent Findings About Sperm Maturation
In addition to Ovch2, many genes encoding extracellular or cell surface proteases and secreted protease inhibitors abundantly expressed in the male reproductive tract are shown to be necessary for sperm abilities to migrate into oviduct and bind eggs, suggesting that proteolysis in the lumen plays critical roles in the control of sperm maturation (19). In addition to proteases, many secreted factors expressed in the epididymis are also essential to sperm maturation. Ablation of RNase10, a ribonuclease-A-like secretory protein lacking enzyme activity, also causes sperm failures of oviduct migration and egg zona pellucida binding (20). β-defensins are a group of antibacterial peptide-like molecules of around 70 residues, encoded on mouse chromosome 8 as a cluster of 98 genes including 22 pseudogenes (21). Deletion of nine β-defensin genes highly expressed in the epididymis caused male infertility with defects including reduced sperm motility and zona pellucida binding (22). Cystatin and ‘prostate and testis expressed’ (PATE) are secretory proteins of ~100–150 residues expressed in the male reproductive tract including epididymis. Mice lacking a cystatin or PATE gene cluster were also male infertile because of the defective sperm migration into oviducts and binding to the egg zona pellucida (23). These findings also support the importance of the luminal environment of male reproductive tract in sperm maturation. The precise molecular mechanism explaining how this luminal environment regulates sperm maturation remains to be clarified further.
The molecular mechanisms of sperm maturation other than that including ADAM3 processing have been increasingly evident. Calcineurin is a calcium-dependent protein phosphatase well known to activate the transcription factor NF-AT by dephosphorylation in immunocytes (24). One of its isoforms, i.e. the catalytic subunit PPP3CC, and the regulatory subunit PPP3R2 are expressed in a testis-specific manner and bind to the outer membrane of sperm mitochondria via an adapter protein SPATA33. Ablation of PPP3CC, PPP3R2 or SPATA33 result in the decreased flexibility of sperm flagellar midpiece, which causes the impairment of sperm penetration through egg zona pellucida and male infertility (25, 26). Interestingly, while in vitro supplementation of culture medium with the calcineurin inhibitor FK506 or cyclosporin does not alter sperm flagellar flexibility, the in vivo treatment with these inhibitors makes animals male infertile and the sperm flagellum of treated animals becomes rigid (25). Thus, it is apparent that spermatozoa acquire flagellar flexibility in a calcineurin-dependent manner during when they pass through the epididymis, although further investigations will be necessary to understand its precise molecular mechanism.
Future Prospects
As discussed above, increasing evidence have unveiled the two-step control mechanism of sperm maturation; in the first step, the differentiation of epididymis that governs sperm maturation is regulated by lumicrine signaling; in the second step, secreted factors coming from differentiated epididymal luminal epithelium act on spermatozoa to make them functionally mature. In this respect, it is an epoch-making that NELL2-ROS1 signaling pathway was identified as the molecular entity of lumicrine regulation. However, even the NELL2 binding mechanism by its receptor ROS1 is still poorly understood; further characterization of the precise molecular mechanisms of lumicrine signaling and sperm maturation would still be essential. In addition to ROS1, an adhesion G protein-coupled receptor ADGRG2 (GPR64) is abundantly expressed in the epididymis and found to be essential for male fertility (27). It would be interesting to investigate whether ADGRG2 functions independently or synergistically with ROS1.
As the lumen is one of basic epithelial tissue structures, lumicrine mechanisms are not restricted to the reproductive tract and could be functional elsewhere in the body. Recently, transluminal signaling was found to be functional in the mouse oviduct (28). Ablation of an orphan G protein-coupled receptor ADGRD1 (GPR133), which is expressed in oviductal epithelial cells, prevented fertilized egg migration from the oviductal ampulla to the uterus, causing female infertility. PLXDC2, a single-pass transmembrane protein expressed on the cumulus cell surface, was identified as the ligand for ADGRD1 ligand. Upon the decay of cumulus layer following fertilization, cumulus cells released from cumulus-oocyte complex reach oviductal epithelial cells and bind to ADGRD1. Inside the oviduct, the migration of fertilized eggs is regulated by the luminal fluid flow from the uterus to the ovary. Upon ADGRD1 activation, the lumen fluid flow toward the ovary decreases because of the reduction of luminal fluid production. Fertilized eggs are thus allowed to be transported toward the uterus. Therefore, the reproductive tracts in both sexes play not merely the transport route of gametes or zygotes; signal transduction through the lumen of reproductive tract plays critical roles for successful reproduction.
With regard to sperm maturation, it should be focused further how the modifications of molecules occurred in spermatozoa regulate the cellular functions acquired in the course of sperm maturation. To this end, the combination of a biochemical approach and use of gene-modified animals would be effective. In humans, their reproductive properties are slightly different from those of rodents; the prominently thickened initial segment epithelium in the rodent epididymis is not so evident in the human epididymis, and Adam3, which is considered to play a central role in sperm maturation in mice, occurs as a pseudogene in humans. Further molecular investigation with model animals will clarify whether these interspecies differences reflect underlying differences in physiological mechanism of reproduction.
In humans, not only sperm maturation but also social factors such as increasing lifespan, late marriage and stress are considered to largely influence the risk of infertility; the underlying mechanism of fertilization is of great significance in properly evaluating their impacts. Further investigations focusing on lumicrine is expected to clarify the molecular mechanisms of sperm maturation essential for successful reproduction.
Acknowledgments
This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science KAKENHI grants (JP21H00231 and JP21H02487) and the Japan Science and Technology Agency (JPMJPR2143).
