Developmental expression and characterization of FS39, a testis complementary DNA encoding an intermediate filament-related protein of the sperm fibrous sheath.

Proteins immunologically related to intermediate filaments have been identified in the sperm fibrous sheath but remain uncharacterized. We isolated and characterized a novel intermediate filament-related protein (FS39) localized to the fibrous sheath of the sperm tail. We used Northern blot analysis to establish that FS39 is transcribed predominantly in the testis of mice >18-20 days old. At this age, spermatogenesis has proceeded to the development of the first round haploid spermatids. In situ hybridization revealed that FS39 mRNA is first detectable in late step 3 spermatids, is at its highest level during steps 9 and 10, and diminishes in steps 13 and 14. Western blot analysis identified a single protein of 39 kDa in mouse and rat testis and epididymis, suggesting the protein is conserved in rodents. Indirect immunofluorescence localized FS39 to the fibrous sheath of the sperm tail, and in testis sections expression was detected from step 13 and step 14 spermatids onward, indicating FS39 is under translational control. Southern blot analysis showed FS39 to be a single copy gene, and hybridization to human genomic DNA suggested that a human equivalent gene is present. These results demonstrate that FS39 is transcribed in testis tissue during the haploid phase of spermatogenesis, is present in mature sperm, and codes for a novel 39-kDa intermediate filament-related protein of the fibrous sheath.     

NYD-SP15: a novel gene potentially involved in regulating testicular development and spermatogenesis

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, we identified a novel human testis gene, NYD-SP15. NYD-SP15 expression was 3.26-fold higher in adult than in fetal testis; however, there was almost no NYD-SP15 expression in the sperm. NYD-SP15 comprises 3364 base pairs, including a 1545 bp open reading frame encoding a 514 amino acid protein possessing 89% sequence identity with the mouse testis homologous protein. NYD-SP15 is located on human chromosome 13q14.2. The deduced structure of the protein contains two dCMP_cyt_deam domains, indicating a potential functional role for zinc ion binding. The gene is expressed variably in a wide range of tissues, with high expression levels in the testis. Sequence analysis revealed that NYD-SP15 is not a highly conserved protein, with its distribution in high-level species such as vertebrates including Homo, Mus, Rattus, and Canis. The results of semiquantitative polymerase chain reaction in mouse testis representing different developmental stages indicate that NYD-SP15 expression was developmentally regulated. These results suggest the putative NYD-SP15 protein may play an important role in testicular development and spermatogenesis and may be an important factor governing male infertility.     

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.     

Molecular cloning of an acrosomal sperm antigen gene and the production of its recombinant protein for immunocontraceptive vaccine

A monoclonal antibody, HS-63, which reacts specifically with a highly conserved sperm acrosome antigen, was shown to inhibit in vitro fertilization of mouse and human. The corresponding sperm antigen designated as MSA-63 was purified to homogeneity from mouse testes and used as an immunogen to generate polyclonal antisera in rabbits. The cDNA fragments of MSA-63 gene were cloned from mouse testis cDNA library by an immunoscreening method using polyclonal antisera specific for MSA-63. Using the established cDNA clone as a probe, the gene encoding for MSA-63 protein was found to be conserved among different mammalian species. Only one specific mRNA 1.5 kb in size was identified from the adult mouse testis among different mouse tissues. The recombinant fusion protein containing MSA-63 protein fragment was produced in Escherichia coli and used to immunize female mice. Similar to the original HS-63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition to the in vitro fertilization of mouse oocytes. The results of this preliminary study suggest that it is feasible to mass produce sperm-specific antigens or their antigenic fragments by recombinant DNA technology for the development of sperm antigen-based immunocontraceptive vaccines.     

Identification of a novel testis-specific gene mtLR1, which is expressed at specific stages of mouse spermatogenesis

A novel testis-specific gene termed mtLR1 was identified by digital differential display. Sequence analyses revealed that mtLR1 protein contains an amino terminus leucine-rich repeat domain and shows 33% similarities to PIDD which functions in p53-mediated apoptosis. Northern blot analysis showed that mtLR1 mRNA was specifically expressed in adult mouse testis, and RT-PCR results also showed that mtLR1 was exclusively expressed in adult testis and not in spermatogonial cells. The expression of mtLR1 mRNA was developmentally upregulated in the testes during sexual maturation and was, conversely, downregulated by experimental cryptorchidism in vivo. We also showed that the expression of mtLR1 mRNA was relatively highly sensitive to heat stress in vitro. The green fluorescent protein produced by pEGFP-C3/mtLR1 was only detected in the cytoplasm of spermatogonia cell line GC-1 after 24 h posttransfection. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of spermatocytes and round spermatids within seminiferous tubules of the adult testis. The time-dependent expression pattern of mtLR1 in postnatal mouse testes suggested that mtLR1 gene might be involved in the regulation of spermatogenesis and sperm maturation.     

In vivo gene transfer by electroporation allows expression of a fluorescent transgene in hamster testis and epididymal sperm and has no adverse effects upon testicular integrity or sperm quality

The study of gene function in testis and sperm has been greatly assisted by transgenic mouse models. Recently, an alternative way of expressing transgenes in mouse testis has been developed that uses electroporation to introduce transgenes into the male germ cells. This approach has been successfully used to transiently express reporter genes driven by constitutive and testis-specific promoters. It has been proposed as an alternative method for studying gene function in testis and sperm, and as a novel way to create transgenic animals. However, the low levels and transient nature of transgene expression that can be achieved using this technique have raised concerns about its practical usefulness. It has also not been demonstrated in mammals other than mice. In this study, we show for the first time that in vivo gene transfer using electroporation can be used to express a fluorescent transgene in the testis of a mammal other than mice, the Syrian golden hamster. Significantly, for the first time we demonstrate expression of a transgene in epididymal sperm using this approach. We show that expression of the transgene can be detected in sperm for as long as 60 days following gene transfer. Finally, we provide the first systematic demonstration that this technique does not lead to any significant long-term adverse effects on testicular integrity and sperm quality. This technique therefore offers a novel way to study gene function during fertilization in hamsters and may also have potential as a way of creating transgenic versions of this important model species.     

Cloning of a sea urchin sarco/endoplasmic reticulum Ca2+ ATPase

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), a vesicular integral membrane protein, is the best-characterized member of the P-type ion translocating ATPase superfamily. Here we describe the cloning and structural analysis of a sea urchin SERCA (suSERCA) cloned from testis cDNA. The approximately 112 kDa suSERCA is 1022 amino acids with approximately 70% identity and 80% similarity to all known mammalian SERCA isoforms. suSERCA shares all the structural features of mammalian SERCAs, including domains: A, actuator; N, nucleotide-binding; and P, phosphorylation, and also 10 transmembrane helices. Like human SERCA2, the suSERCA has a possible 11th transmembrane segment in its extreme C-terminus. The alignment of three sequences (suSERCA, human SERCA2, and rabbit SERCA1a) shows that the Ca2+ binding residues and kinks (required to form the ion-binding pocket) are 100% conserved. The annotated suSERCA gene consists of 24 exons separated by 23 introns and is approximately 30 kb. Western blots show that suSERCA is present in sea urchin eggs and testis, but not in mature spermatozoa. Treatment of live sperm with SERCA inhibitors has no effect on intracellular calcium, suggesting the absence of SERCA in sea urchin spermatozoa.     

NYD-SP15: A Novel Gene Potentially Involved in Regulating Testicular Development and Spermatogenesis

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, we identified a novel human testis gene, NYD-SP15. NYD-SP15 expression was 3.26-fold higher in adult than in fetal testis; however, there was almost no NYD-SP15 expression in the sperm. NYD-SP15 comprises 3364 base pairs, including a 1545 bp open reading frame encoding a 514 amino acid protein possessing 89% sequence identity with the mouse testis homologous protein. NYD-SP15 is located on human chromosome 13q14.2. The deduced structure of the protein contains two dCMP_cyt_deam domains, indicating a potential functional role for zinc ion binding. The gene is expressed variably in a wide range of tissues, with high expression levels in the testis. Sequence analysis revealed that NYD-SP15 is not a highly conserved protein, with its distribution in high-level species such as vertebrates including Homo, Mus, Rattus, and Canis. The results of semiquantitative polymerase chain reaction in mouse testis representing different developmental stages indicate that NYD-SP15 expression was developmentally regulated. These results suggest the putative NYD-SP15 protein may play an important role in testicular development and spermatogenesis and may be an important factor governing male infertility. These authors contributed equally to this work     

The murine seminiferous epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis

The seminiferous epithelial cycle and spermatogenic wave are conserved features of vertebrate spermatogenic organisation that reflect the need for the rigorous maintenance of sperm production. Although the cycle and the wave of the adult seminiferous epithelium have been well characterised, particularly in rodent species, their developmental origins are unknown. We show that the Sertoli cells of the pre-pubertal mouse, including those of the germ cell-deficient XXSxra mutant, exhibit coordinated, cyclical patterns of gene expression, presaging the situation in the adult testis, where Sertoli cell function is coupled to the spermatogenic cycle. In the case of the galectin 1 gene (Lgals1), localised differential expression in the Sertoli cells can be traced back to neonatal and embryonic stages, making this the earliest known molecular marker of functional heterogeneity in mammalian testis cords. In addition, the timing of germ cell apoptosis in normal pre-pubertal testes is linked to the temporal cycle of the Sertoli cells. These data show that the cycle and wave of the murine seminiferous epithelium originate at a much earlier stage in development than was previously known, and that their maintenance in the early postnatal cords depends exclusively on the somatic cell lineages.     

Marlin-1 is expressed in testis and associates to the cytoskeleton and GABAB receptors

Marlin-1 is a GABA(B) receptor and Jak tyrosine kinase-binding protein that also associates with RNA and microtubules. In humans and rodents, expression of Marlin-1 is predominantly restricted to the brain, but expression in lymphoid cells has also been reported. Here, we have studied the distribution of Marlin-1 in testis and spermatozoa. Our results indicate that Marlin-1 is highly expressed in testis. The protein is abundant in spermatogonia, spermatocytes, spermatozoa, and Sertoli cells. We also have studied the subcellular distribution in spermatozoa. Marlin-1 is present in the tail and to a lesser degree in the head of the sperm cell. Finally, we have explored two protein interactions. Our findings demonstrate that Marlin-1 associates with a microtubule fraction and with GABA(B) receptors in testis suggesting that the set of protein interactions of Marlin-1 are conserved in different tissues.     

A novel human gene, encoding a potential membrane protein conserved from yeast to man, is strongly expressed in testis and cancer cell lines.

We have characterized a novel human gene (C14orf1) which codes for a polypeptide homologous to the yeast protein Yer044c. Both the human and yeast proteins are predicted to be highly basic and to present several potential, evolutionarily conserved, transmembrane domains. C14orf1 mRNA was found to be particularly abundant in the adult testis and in several cancer cell lines. The gene maps to chromosome band 14q24. Further investigations should be performed to understand the role of C14orf1 in the testis and the significance of its strong expression in the cell lines studied here.