αV Integrin Expression and Localization in Male Germ Cells

Integrins are transmembrane receptors that facilitate cell adhesion and cell–extracellular matrix communication. They are involved in the sperm maturation including capacitation and gamete interaction, resulting in successful fertilization. αV integrin belongs to the integrin glycoprotein superfamily, and it is indispensable for physiological spermiogenesis and testosterone production. We targeted the gene and protein expression of the αV integrin subunit and described its membrane localization in sperm. Firstly, in mouse, we traced αV integrin gene expression during spermatogenesis in testicular fraction separated by elutriation, and we detected gene activity in spermatogonia, spermatocytes, and round spermatids. Secondly, we specified αV integrin membrane localization in acrosome-intact and acrosome-reacted sperm and compared its pattern between mouse, pig, and human. Using immunodetection and structured illumination microscopy (SIM), the αV integrin localization was confined to the plasma membrane covering the acrosomal cap area and also to the inner acrosomal membrane of acrosome-intact sperm of all selected species. During the acrosome reaction, which was induced on capacitated sperm, the αV integrin relocated and was detected over the whole sperm head. Knowledge of the integrin pattern in mature sperm prepares the ground for further investigation into the pathologies and related fertility issues in human medicine and veterinary science.

Prothymosin alpha expression and localization during the spermatogenesis ofDanio rerio

SummaryProthymosin α (PTMA) is a highly acidic, intrinsically disordered protein, which is widely expressed and conserved throughout evolution; its uncommon features are reflected by its involvement in a variety of processes, including chromatin remodelling, transcriptional regulation, cell proliferation and death, immunity. PTMA has also been implicated in spermatogenesis: during vertebrate germ cell progression in the testis the protein is expressed in meiotic and post-meiotic stages, and it is associated with the acrosome system of the differentiating spermatids in mammals. Then, it finally localizes on the inner acrosomal membrane of the mature spermatozoa, suggesting its possible role in both the maturation and function of the gametes. In the present work we studied PTMA expression during the spermatogenesis of the adult zebrafish, a species in which two paralogs have been described. Our data show thatptmatranscripts are expressed in the testis, and localize in meiotic and post-meiotic germ cells, namely spermatocytes and spermatids. Consistently, the protein is expressed in spermatocytes, spermatids, and spermatozoa: its initial perinuclear distribution is extended to the chromatin region during cell division and, in haploid phases, to the cytoplasm of the developing and final gametes. The nuclear localization in the acrosome-lacking spermatozoa suggests a role for PTMA in chromatin remodelling during gamete differentiation. These data further provide a compelling starting point for the study of PTMA functions during vertebrate fertilization.

Sperm arylsulfatase A binds to mZP2 and mZP3 glycoproteins in a nonenzymatic manner

We have shown previously that sperm surface arylsulfatase A (ASA) of mouse, pig, and human is involved in sperm–egg zona pellucida (ZP) binding. By treating capacitated mouse sperm with A23187 to induce the acrosome reaction, we demonstrated by immunoblotting that ASA also existed in the acrosomal content and on the inner acrosomal membrane. Since mZP2 and mZP3 are known as sperm receptors, whereas mZP1 as a cross-linker of mZP2/mZP3, we determined whether purified ASA bound to mZP2 and mZP3 selectively. The three mZP glycoproteins were purified from solubilized ovarian ZP by size exclusion column chromatography. Immuno-dot blot analyses revealed that purified sperm ASA bound to mZP2 at the highest level followed by mZP3, whereas the binding of ASA to mZP1 was minimal. The results confirmed the physiological significance of sperm ASA in the ZP binding process. The binding of ASA to mZP2 and mZP3 was, however, not dependent on the active site pocket amino acids, Cys69, Lys123, and Lys302, which are pertinent to the capturing of an arylsulfate substrate, since ASA mutant with Ala substitution at these three residues still bound to mZP2 and mZP3. The availability of the active site pocket of ASA bound to the ZP suggested that ASA would still retain enzymatic activity, which might be important for subsequent sperm penetration through the ZP.

Disruption of Mouse CD46 Causes an Accelerated Spontaneous Acrosome Reaction in Sperm

ABSTRACT Human membrane cofactor protein (MCP, CD46) is a ubiquitously expressed protein known to protect cells from complement attack. Interestingly, when we examined the expression of mouse CD46, which we recently cloned, the message was found only in testis and the protein was found on the inner acrosomal membrane of sperm. In order to elucidate the function of CD46, we produced mice carrying a null mutation in the CD46 gene by using homologous recombination. Despite the absence of CD46, the mice were healthy and both sexes were fertile. However, to our surprise, the fertilizing ability of males appeared to be facilitated by disruption of the CD46 gene, as the average number of pups born from CD46−/− males was significantly greater than that of wild-type males. It was also revealed that the incidence of the spontaneous acrosome reaction doubled in CD46−/− sperm compared to that in wild-type sperm. It was assumed that this increase caused the heightened fertilizing ability found in CD46−/− sperm. These data suggest that CD46 may have some role in regulating sperm acrosome reaction.

Tracing sperm acrosome differentiation in the testis and maturation in the epididymis of the tammar wallaby (Macropus eugenii) with a 45-kDa acrosome-membrane-associated protein

A 45-kDa protein was originally extracted from a depression, where the acrosome is lodged, on the anterior end of the sperm nucleus of ejaculated wallaby spermatozoa. Using immunofluorescent and confocal microscopes, this study demonstrates that the 45-kDa protein is persistently localized to the sperm acrosome throughout the periods of spermiogenesis, spermiation, epididymal maturation and ejaculation in the tammar wallaby. The distribution of the 45-kDa protein is always on the perimeter of the acrosome and associated with the acrosomal membrane, so that changes in the shape of the 45-kDa immunofluorescent labelling mirror changes in the shape of the acrosome during its differentiation in the testis and epididymis. Thus, the 45-kDa protein may be used as a molecular marker to study the marsupial acrosome differentiation and to chart the events of testicular and epididymal maturation of the spermatozoa. Furthermore, the behaviour of the 45-kDa protein during the immunostaining process suggests that this protein is a largely insoluble and detergent-resistant protein and may play an important role in attachment of the acrosome to the nucleus during sperm formation, similar to those inner acrosomal-membrane-associated proteins that have been reported in eutherian spermatozoa.