MSJ-1, a mouse testis-specific DnaJ protein, is highly expressed in haploid male germ cells and interacts with the testis-specific heat shock protein Hsp70-2

The MSJ-1 gene encodes a murine DnaJ homologue that is expressed specifically in adult testis. DnaJ proteins act as cochaperones of Hsp70 proteins in promoting diverse cellular functions. In this study we used recombinant MSJ-1 proteins to produce MSJ-1 antiserum and to carry out in vitro binding assays. In a wide immunoscreening of mouse tissues, affinity-purified MSJ-1 antibodies recognize a unique protein of 30 kDa in male germ cells only. MSJ-1 is able to interact with the testis-specific Hsp70-2 protein and can be coimmunoprecipitated with Hsp70-2 from spermatogenic cells; binding of these two chaperones is consistent with the presence of a third component, which is so far unknown. MSJ-1 is weakly detected in early round spermatids, and its protein content increases in cytodifferentiating spermatids where it colocalizes with the developing acrosome and their postnuclear region. Hsp70-2, which is known to be highly expressed in meiotic cells, shows a subcellular localization in late differentiating spermatids that overlaps that of MSJ-1. MSJ-1 is also maintained in testicular and epididymal spermatozoa, where it sharply demarcates into two distinct cell areas; the outer surface of the acrosomal vesicle, and the centrosomal area. On the whole, our findings are consistent with a role for MSJ-1 in acrosome formation and centrosome adjustment during spermatid development, whereas its presence in mature spermatozoa suggests a special function during fertilization, shortly afterward, or both.     

The deubiquitinating enzyme mUBPy interacts with the sperm-specific molecular chaperone MSJ-1: the relation with the proteasome, acrosome, and centrosome in mouse male germ cells

The mouse USP8/mUBPy gene codifies a deubiquitinating enzyme expressed preferentially in testis and brain. While the ubiquitin-specific processing proteases (UBPs) are known to be important for the early development in invertebrate organisms, their specific functions remain still unclear in mammals. Using specific antibodies, raised against a recombinant mUBPy protein, we studied mUBPy in mouse testis. The mUBPy is expressed exclusively by the germ cell component and is maintained in epididymal spermatozoa. The enzyme is functionally active, being able to detach ubiquitin moieties from endogenous protein substrates. Protein interaction assays showed that sperm UBPy interacts with MSJ-1, the sperm-specific DnaJ protein evolutionarily conserved for spermiogenesis. Immunocytochemistry revealed that mUBPy shares with MSJ-1 the intracellular localization during spermatid cell differentiation; intriguingly, we show here that the proteasomes also locate in mUBPy/MSJ-1-positive sites, such as the cytoplasmic surface of the developing acrosome and the centrosomal region. These colocalization sites are maintained in epididymal spermatozoa. The demonstration of a protein interaction between a deubiquitinating enzyme and a molecular chaperone and the documentation on the proteasomes in both differentiating and mature mouse male germ cells suggest that members of the chaperone and ubiquitin/proteasome systems could cooperate in the fine control of protein quality to yield functional spermatozoa.     

Defect of sperm assembly in a neurological mutant of the mouse, wobbler (WR)

In the wobbler (WR) mouse, a neuromuscular mutant characterized by a motoneuron degeneration and male infertility, the cellular basis of the defect in spermiogenesis was studied by light and electron microscopy as well as by lectin binding. Spermatozoa of the wobbler mutant had rounded heads, and their motility was reduced. In histological sections of WR testes, spermatogenesis appeared normal up to the stage of round spermatids, but the elongation and flattening of the nucleus during late spermiogenesis did not occur. Numbers of spermatid nuclei in WR testes were reduced to 70%-80% of controls. The acrosomal marker glycoprotein, peanut agglutinin receptor, was synthesized, but the acrosomal membrane did not attach to the nucleus. The disturbance in spermiogenesis of the wobbler mouse is not due to impaired descent of the testis, nor to a lack of testosterone, and is distinct from that observed in other mouse mutants (quaking, QK; Purkinje cell degeneration, PCD) with combined neurological and spermiogenesis defects.     

MSJ-1, a New Member of the DNAJ Family of Proteins, Is a Male Germ Cell-Specific Gene Product

A cDNA encoding for a new member of the DnaJ protein family has been isolated by screening a mouse spermatogenic cell expression library. The full-length cDNA obtained by extension of the original clone with RT-PCR has been characterized with respect to its DNA sequence organization and expression. The predicted open reading frame encodes a protein of 242 amino acid residues whose sequence is similar to that of bacterial DnaJ proteins in the amino-terminal portion since it contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ protein–protein interactions. In contrast, the middle and carboxyl-terminal regions of the protein are not similar to any other DnaJ proteins, with the exception of the human neuronal HSJ-1 with which displays a 48% identity in a 175-amino-acid overlap. Analysis of RNAs from a wide spectrum of mouse somatic tissues, including the brain, and from ovary and testis reveals that the gene is specifically expressed in testis only. Developmental Northern blot analysis of testis RNA from mice of different ages andin situhybridization on juvenile and adult testis sections demonstrate that the mRNA is first transcribed in spermatids. A similar pattern of expression is exhibited also in rat testis. Based upon all these observations, we have named this novel mouse gene, MSJ-1, for mouse sperm cell-specific DNAJ first homolog.     

The Enzymatic Activity of Cu/Zn Superoxide Dismutase Does Not Fluctuate in Mouse Spermatogenic Cells Despite mRNA Changes

In the mammalian testis, multiple mRNAs encoding the copper zinc superoxide dismutase (SOD-1) are expressed in postmeiotic male germ cells. Here we relate SOD-1 mRNA levels to SOD-1 protein and enzyme activity levels in mouse spermatogenic cells. Although the sizes and relative amounts of the multiple SOD-1 mRNAs vary as male germ cells enter meiosis and proceed into the postmeiotic stages of spermatogenesis, the amount of SOD-1 protein and enzyme activity does not fluctuate significantly, suggesting a precise control of SOD-1 activity in male germ cells.     

Nuclear envelope remodeling during mouse spermiogenesis: postmeiotic expression and redistribution of germline lamin B3

Lamins are members of a multigene family of structural nuclear envelope (NE) proteins. Differentiated mammalian somatic cells express lamins A, C, B1, and B2. The composition and organization of the nuclear lamina of mammalian spermatogenic cells differ significantly from that of somatic cells as they express lamin B1 as well as two short germ line-specific isoforms, namely lamins B3 and C2. Here we describe in detail the expression pattern and localization of lamin B3 during mouse spermatogenesis. By combining RT-PCR, immunoblotting, and immunofluorescence microscopy, we show that lamin B3 is selectively expressed during spermiogenesis (i.e., postmeiotic stages of spermatogenesis). In round spermatids, lamin B3 is distributed in the nuclear periphery and, notably, also in the nucleoplasm. In the course of spermiogenesis, lamin B3 becomes redistributed as it concentrates progressively to the posterior pole of spermatid nuclei. Our results show that during mammalian spermiogenesis the nuclear lamina is composed of B-type isoforms only, namely the ubiquitous lamin B1 and the germline-specific lamin B3. Lamin B3 is the first example of a mammalian lamin that is selectively expressed during postmeiotic stages of spermatogenesis.     

Impaired fertility and spermiogenetic disorders with loss of cell adhesion in male mice expressing an interfering Rap1 mutant

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.     

Expression of a sperm protein gene during spermatogenesis in mammalian testis: an in situ hybridization study.

In previous work a specific membrane protein with an estimated Mr of 20.1 kDa was purified from rabbit sperm tails and designated as rSMP-B protein. Antibodies were raised against rSMP-B protein and used to isolate and identify the cDNA coding the rSMP-B protein from a rat testis lambda gt11 expression library. The nucleotide sequence of the cDNA was determined in a previous study. Single-stranded 35S-labeled RNA probes were prepared. With the techniques of in situ hybridization, rSMP-B mRNA was detected in spermatids of rat and rabbit testis. The present results support our previous observation that immunization of male rabbits with the rSMP-B protein results in the arrest of spermatogenesis at the spermatid stage. Overall, rSMP-B protein appears to be involved in spermiogenesis, and the synthesis of the mRNA encoding the protein occurs in germ cells during the postmeiotic haploid phase of spermatogenesis.     

Poly (A) binding protein is bound to both stored and polysomal mRNAs in the mammalian testis

RNA-binding proteins that bind to the 3′ untranslated region of mRNAs play important roles in regulating gene expression. Here we examine the association between the 70 kDa poly (A) binding protein (PABP) and stored (RNP) and polysomal mRNAs during mammalian male germ cell development. PABP mRNA levels increase as germ cells enter meiosis, reaching a maximum in the early postmeiotic stages, and decreasing to a nearly nondetectable level towards the end of spermatogenesis. Most of the PABP mRNA is found in the nonpolysomal fractions of postmitochondrial extracts, suggesting that PABP mRNA is either inefficiently translated or stored as RNPs during spermatogenesis. Virtually all of the testicular PABP is bound to either polysomal or nonpolysomal mRNAs, with little, if any, free PABP detectable. Analysis of several specific mRNAs reveals PABP is bound to both stored (RNP) and translated forms of the mRNAs. Western blot analysis and immunocytochemistry indicate PABP is widespread in the mammalian testis, with maximal amounts detected in postmeiotic round spermatids. The presence of PABP in elongating spermatids, a cell type in which PABP mRNA is nearly absent, suggests that PABP is a stable protein in the later stages of male germ cell development. The high level of testicular PABP in round spermatids and in mRNPs suggests a role for PABP in the storage as well as in the subsequent translation of developmentally regulated mRNAs in the mammalian testis. © 1995 Wiley-Liss, Inc.