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Spermatids are haploid differentiating cells that, in the meantime they differentiate, translocate along the seminiferous epithelium towards the tubule lumen to be just released as spermatozoa. The success of such a migration depends on dynamic of spermatid-Sertoli cell contacts, the molecular nature of which has not been well defined yet. It was demonstrated that the vascular endothelial cadherin (VEC) is expressed transitorily in the mouse seminiferous epithelium. Here, we evaluated the pattern of VEC expression by immunohistochemistry first in seminiferous tubules at different stages of the epithelial cycle when only unique types of germ cell associations are present. Changes in the pattern of VEC localization according to the step of spermatid differentiation were analysed in detail using testis fragments and spontaneously released germ cells. Utilizing the first wave of spermatogenesis as an in vivo model to have at disposal spermatids at progressive steps of differentiation, we checked for level of looser VEC association with the membrane by performing protein solubilisation under mild detergent conditions and assays through VEC-immunoblotting. Being changes in VEC solubilisation paralleled in changes in phosphotyrosine (pY) content, we evaluated if spermatid VEC undergoes Y658 phosphorylation and if this correlates with VEC solubilisation and spermatid progression in differentiation. Altogether, our study shows a temporally restricted pattern of VEC expression that culminates with the presence of round spermatids to progressively decrease starting from spermatid elongation. Conversely, pY658-VEC signs elongating spermatids; its intracellular polarized compartmentalization suggests a possible involvement of pY658-VEC in the acquisition of spermatid cell polarity.

The acrosome is a peculiar vacuole that at fertilization undergoes the acrosome reaction (AR), an event unique in the sperm life. Contents released promote sperm penetration through oocyte's investments; membranous components are involved in sperm-egg interaction/fusion. Therefore, both constituents play a role in fertilization. The biogenesis of this vacuole, however, has not been clarified yet; recently, it has been proposed as a novel lysosome-related organelle (LRO). Our research focuses on the involvement of the endosomal pathway in acrosomogenesis starting from the early phases. The trafficking sorted by USP8/UBPy, an endosomal regulator recently described as a compelling candidate for male fertility gene, was investigated in comparison to that of SP56, a marker of the biosynthetic pathway. Mouse spermatids were double/triple immunolabeled and examined by confocal microscopy. The contribution of the vesicular traffic assisted by the cortical microtubule array was also evaluated in nocodazole-treated spermatids. USP8/UBPy-sorted cargo contributes early to acrosomogenesis and its trafficking is microtubule mediated. It was identified, through co-immunoprecipitation/co-immunolocalization assays, that the membrane receptor MET, described herein for the first time in spermatids, as an USP8/UBPy-target substrate is delivered to the acrosome. MET and USP8/UBPy still colocalize in epididymal spermatozoa. Following the AR, MET and USP8/UBPy show a distinct fate. MET, in particular, translocates at the PAS, the post acrosomal segment known to harbor sperm-borne factors involved in oocyte activation. Overall, our results support the concept of the acrosome as a LRO and provide evidence for the identification of MET as a tyrosine kinase receptor that may play a role in fertilization., (© 2015 Society for Reproduction and Fertility.)

Despite advances in assisted reproductive technologies, infertility remains a consistent health problem worldwide. Spermiation is the process through which mature spermatids detach from the supporting Sertoli cells and are released into the tubule lumen. Spermiation failure leads to lack of mature spermatozoa and, if not occasional, could result into azoospermia, major cause of male infertility in human population. Spermatids are led through their differentiation into spermatozoa by the apical ectoplasmic specialization (aES), a testis-specific, actin-based anchoring junction restricted to the Sertoli-spermatid interface. The aES helps spermatid movement across the seminiferous epithelium, promotes spermatid positioning, and prevents the release of immature spermatozoa. To accomplish its functions, aES needs to undergo tightly and timely regulated restructuring. Even if components of aES are partly known, the mechanism/s through which aES is regulated remains still elusive. In this review, we propose a model by which the small GTPase Rap1 could regulate aES assembly/remodelling. The characterization of key players in the dynamic of aES, such as Rap1, could open new possibility to develop prognostic, diagnostic, and therapeutic approaches for male patients under treatment for infertility as well as it could lead to the identification of new target for male contraception.

Ubiquitin-specific peptidase 8 (USP8) is a deubiquitinating enzyme that works as a regulator of endosomal sorting and vesicle morphology in cultured cells. Its function in vivo is, however, unknown as USP8 gene deletion leads to embryonic lethality. Previously, we have shown that USP8 is highly expressed in male germ cells. These cells develop a peculiar acidic vesicle that is indispensable for fertilization, the acrosome; USP8 might be involved in vivo in acrosomogenesis. The objective of this study was to test this hypothesis by determining if selective components of the early endosomal machinery interact functionally with USP8 during acrosomogenesis using protein-protein interaction assays and double/triple immunolabeling. Moreover, by exploiting the characteristic of USP8 that exhibits a microtubule interacting and trafficking/transport (MIT) domain, we verified whether USP8 effectively associates with spermatid microtubules by microtubule cosedimentation and binding assays. USP8 was able to interact with spermatid ESCRT-0 (endosomal-sorting complex required for transport-0) and microtubule structures; USP8/ESCRT-0-labeled vesicles, monitored by fluorescence microscopy, were found to contribute to acrosome formation while USP8 can directly link, via its MIT domain, the labeled vesicles/developing acrosome to microtubules, which could favor both acrosome assembly and shaping. VPS54, the vacuolar-sorting protein responsible for early endocytic retrograde transport, was here detected for the first time in male germ cells; VPS54 followed the intracellular route of USP8/ESCRT-0-labeled vesicles during acrosomogenesis. We concluded that in vivo USP8 has a role strongly associated with acrosome biogenesis and that the early endosome pathway is significantly involved in the process, which suggests that the acrosome could be a novel lysosome-related organelle.

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell-cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli-germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin-positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ-Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.