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.     

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.     

TEX101 is shed from the surface of sperm located in the caput epididymidis of the mouse

It is generally believed that cell-to-cell cross-talk and signal transduction are mediated by cell surface molecules that play diverse and important regulatory roles in spermatogenesis and fertilization. Recently, we identified a novel plasma membrane-associated protein, TES101-reactive protein (TES101RP, or TEX101), on mouse testicular germ cells. In this study, we investigate Tex101 mRNA expression in the adult mouse testis using in situ hybridization, and we examine the fate of TEX101 during sperm transport by immunohistochemical and Western blot analyses. Tex101 mRNA was expressed in a stage-specific manner in spermatocytes and in step 1-9 spermatids of the testis, but not in spermatogonia. Although the TEX101 protein remained on the cell surfaces of step 10-16 spermatids and testicular sperm, it was shed from epididymal sperm located in the caput epididymidis. The results of this study provide additional information on germ cell-specific TEX101 expression during spermatogenesis and post-testicular sperm maturation.     

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.     

Molecular cloning and characterization of phosphatidylcholine transfer protein-like protein gene expressed in murine haploid germ cells

We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.     

Cloning,Characterization and Primary Function Study of a Novel Gene,Cymg1,Related to Family 2 Cystatins

Cystatins are cysteine proteinase inhibitors,We found two expression sequence tags (ESTs),CA463109 and AV042522,from a mouse testis library using Digital differential display (DDD).By electricalhybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb,and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is locatedin the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristicsare seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically inthe male reproductive tract.CYMG1 has a 44%(48/108)identity with mouse CRES and 30%(42/140)identity with mouse cystatin C.Northern blot analysis showed that the Cymgl is specifically expressed inadult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in thecytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mousetestes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCRresults also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expressionlevel varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weekspostpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main-tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop-mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicatethat Cymgl might play an important role in mouse spermatogenesis and sexual maturation. Cystatins are cysteine proteinase inhibitors,We found two expression sequence tags(ESTs),CA463109 and AV042522,from a mouse testis library using Digital differential display (DDD).By electricalhybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb,and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is locatedin the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristicsare seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically inthe male reproductive tract.CYMG1 has a 44%(48/108)identity with mouse CRES and 30%(42/140)identity with mouse cystatin C.Northern blot analysis showed that the Cymgl is specifically expressed inadult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in thecytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mousetestes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCRresults also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expressionlevel varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weekspostpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main-tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop-mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicatethat Cymgl might play an important role in mouse spermatogenesis and sexual maturation.     

A heat-shock protein 40, DNAJB13, is an axoneme-associated component in mouse spermatozoa

DNAJB13 is a type II HSP40/DnaJ protein. Using a specific antibody raised against the recombinant DNAJB13 protein, we characterized DNAJB13 in mouse testes and epididymal spermatozoa. The expression of DNAJB13 protein in testis was undetectable until postnatal Week 4 revealed by Western blot analysis, whereas Dnajb13 mRNA was detectable as early as postnatal Week 1 by RT-PCR. Immunohistochemistry analyses showed that DNAJB13 was localized in the cytoplasm of spermatids from step 2 to 3 onward with the strongest expression in step 9-10, and in the spermatid flagella. In mature spermatozoa, DNAJB13 was present along the entire length of the sperm flagellum, but not in the SDS-resistant tail structures lacking the flagellar axoneme, strongly suggesting that DNAJB13 is an axoneme-associated component. In addition, we showed that the expression of Dnajb13 mRNA and DNAJB13 protein was unaltered after heat shock treatment, indicating that DNAJB13 was constitutively expressed in mouse testis. Taken together, the present study suggested that DNAJB13 might be involved in assembly and stability of axoneme during sperm flagellum development.     

Ca(2+)/calmodulin-dependent protein kinase IV is expressed in spermatids and targeted to chromatin and the nuclear matrix

Ca(2+)/calmodulin-dependent protein kinase IV and calspermin are two proteins encoded by the Camk4 gene. Both are highly expressed in the testis, where in situ hybridization studies in rat testes have demonstrated that CaMKIV mRNA is localized to pachytene spermatocytes, while calspermin mRNA is restricted to spermatids. We have examined the expression patterns of both CaMKIV and calspermin in mouse testis and unexpectedly find that CaMKIV is expressed in spermatogonia and spermatids but excluded from spermatocytes, while calspermin is found only in spermatids. CaMKIV and calspermin expression in the testis are stage-dependent and appear to be coordinately regulated. In germ cells, we find that CaMKIV is associated with the chromatin. We further demonstrate that a fraction of CaMKIV in spermatids is hyperphosphorylated and specifically localized to the nuclear matrix. These novel findings may implicate CaMKIV in chromatin remodeling during nuclear condensation of spermatids.     

Transient appearance of CRES protein during spermatogenesis and caput epididymal sperm maturation

In previous studies we identified an epididymal gene that exhibits homology to the cystatin family of cysteine protease inhibitors. The expression of this gene, termed CRES (cystatin-related epididymal and spermatogenic), was shown to be highly restricted to the proximal caput epididymal epithelium with less expression in the testis and no expression in the 24 other tissues examined. In this report, studies were carried out to examine CRES gene expression in the testis as well as to characterize the CRES protein in the testis and epididymis. In situ hybridization experiments revealed that within the testis CRES gene expression is stage-specific during spermatogenesis and is exclusively expressed by the round spermatids of Stages VII-VIII and the early elongating spermatids of Stages IX and X. Immunohistochemical studies demonstrated that CRES protein was transiently expressed in both the testis and epididymis. Within the testis the protein was localized to the elongating spermatids, whereas within the epididymis CRES protein was exclusively synthesized by the proximal caput epithelium and then secreted into the lumen. Surprisingly, the secreted CRES protein had completely disappeared from the epididymal lumen by the distal caput epididymidis. Western blot analysis of testicular and epididymal proteins showed that the CRES antibody specifically recognized a predominant 19 kDa CRES protein and a less abundant 14 kDa form. These observations suggest that the CRES protein performs a specialized role during sperm development and maturation. © 1995 Wiley-Liss, Inc.     

MARCH7 E3 ubiquitin ligase is highly expressed in developing spermatids of rats and its possible involvement in head and tail formation

Spermatogenesis is a highly complicated metamorphosis process of male germ cells. Recent studies have provided evidence that the ubiquitin–proteasome system plays an important role in sperm head shaping, but the underlying mechanism is less understood. In this study, we localized membrane-associated RING-CH (MARCH)7, an E3 ubiquitin ligase, in rat testis. Northern blot analysis showed that March7 mRNA is expressed ubiquitously but highly in the testis and ovary. In situ hybridization of rat testis demonstrated that March7 mRNA is expressed weakly in spermatogonia and its level is gradually increased as they develop. Immunohistochemical analysis detected MARCH7 protein expression in spermiogenic cells from late round spermatids to elongated spermatids and in epididymal spermatozoa. Moreover, MARCH7 was found to be localized to the caudal end of the developing acrosome of late round and elongating spermatids, colocalizing with β-actin, a component of the acroplaxome. In addition, MARCH7 was also detected in the developing flagella and its expression levels were prominent in elongated spermatids. We also showed that MARCH7 catalyzes lysine 48 (K48)-linked ubiquitination. Immunolocalization studies revealed that K48-linked ubiquitin chains were detected in the heads of elongating spermatids and in the acrosome/acroplaxome, neck, midpiece and cytoplasmic lobes of elongated spermatids. These results suggest that MARCH7 is involved in spermiogenesis by regulating the structural and functional integrity of the head and tail of developing spermatids.     

Molecular Cloning and Putative Functions of KIFC1 for Acrosome Formation and Nuclear Reshaping during Spermiogenesis in Schlegel's Japanese Gecko Gekko japonicus

Spermiogenesis, occurring in the male testis, is a complicated and highly-ordered developmental process resulting in the production of fertile mature sperm. In Gekko japonicus, this process occurs in 7 steps during which the spermatids undergo dramatic changes in the cytoskeleton and nucleus. Here, we cloned and sequenced the cDNA of the mammalian KIFC1 homologue in the testis of G. japonicus. The 2 344 bp full-length cDNA sequence contained a 191 bp 5'-untranslated region, a 134 bp 3'-untranslated region and a 2 019 bp open reading frame encoding a protein of 672 amino acids. Tissue expression analysis revealed the highest expression of kifc1 mRNA was in the testis. Fluorescence in situ hybridization revealed that the kifc1 mRNA signal was hardly detected in step 1 spermatids but became concentrated at the acrosome of step 2 spermatids and abundant in the nucleus of step 5 spermatids where the nucleus then undergoes dramatic elongation and compression. The kifc1 mRNA signal then gradually disappears in mature sperm. This expression of KIFC1 at specific stages of spermiogenesis in G. japonicus implies its important role in the major cytological transformations such as acrosome biogenesis and nucleus morphogenesis.     

Identification of a novel gene SRG4 expressed at specific stages of mouse spermatogenesis

Spermatogenesis is a complex process. Duringspermatogenesis, the production of sperm occurs withinthe testicular seminiferous tubules through three separatedphases. First of all, diploid germ cells, primitivespermatogonia, will self renew to amplify and producetypes A and B spermatogonia. Type B spermatogonia willdifferentiate into primary spermatocytes. Then, meioticdivisions of spermatocytes will produce round spermatids.Finally, after a series of biochemical and morphologicalchanges, sper…     

Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse

The mouse testis contains two isotypes of cytochrome c, which differ in 14 of 104 amino acids: cytochrome cs is present in all somatic tissues and cytochrome cT is testis specific. The regulation of cytochrome cS and cytochrome cT gene expression during spermatogenesis was examined by Northern blot analysis using specific cDNA probes. Total RNA was isolated from adult tissues, enriched germinal cell populations and polysomal gradients of total testis and isolated germinal cells. Three cytochrome cS mRNAs were detected averaging 1.3 kb, 1.1 kb and 0.7 kb in all tissues examined; an additional 1.7 kb mRNA was observed in testis. Isolated germinal cells through prepuberal pachytene spermatocytes contained only the three smaller mRNAs; the 1.7 kb mRNA was enriched in round spermatids. All three smaller cytochrome cS mRNAs were present on polysomes; the 1.7 kb mRNA was non-polysomal. Cytochrome cT mRNA of 0.6-0.9 kb was detected in testis; mRNA levels were low in early spermatogonia and peaked in prepuberal pachytene spermatocytes. In adult pachytene spermatocytes, a subset of the cytochrome cT mRNAs, 0.7-0.9 kb, was present on polysomes; a shortened size class, 0.6-0.75 kb, was non-polysomal. A distinct, primarily non-polysomal, cytochrome cT 0.7 kb mRNA was present in round spermatids. These results indicate that (1) both cytochrome cS and cytochrome cT mRNAs are present in early meiotic cells, (2) a 1.7 kb cytochrome cS mRNA is post-meiotically expressed and non-polysomal and (3) cytochrome cS and cytochrome cT mRNAs are each developmentally and translationally regulated during spermatogenesis.     

Cloning, characterization and primary function study of a novel gene, Cymg1, related to family 2 cystatins

Cystatins are cysteine proteinase inhibitors. We found two expression sequence tags (ESTs), CA463109 and AV042522, from a mouse testis library using Digital differential display (DDD). By electrical hybridization, a novel gene, Cymg1 (GenBank accession No. AY600990), which has a full length of 0.78kb, and contains four exons and three introns, was cloned from a mouse testis cDNA library. The gene is located in the 2G3 area of chromosome 2. The full cDNA encompasses the entire open reading frame, encoding 141 amino acid residues. The protein has a cysteine protease inhibitor domain that is related to the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the CRES subfamily, which are related to the family 2 cystatins and are expressed specifically in the male reproductive tract. CYMG1 has a 44% (48/108) identity with mouse CRES and 30% (42/140) identity with mouse cystatin C. Northern blot analysis showed that the Cymg1 is specifically expressed in adult mouse testes. Cell location studies showed that the GFP-tagged CYMG1 protein was localized in the cytoplasm of HeLa cells. Immunohistochemistry revealed that the CYMG1 protein was expressed in mouse testes spermatogonium, spermatocytes, round spermatids, elongating spermatids and spermatozoa. RT-PCR results also showed that Cymg1 was expressed in mouse testes and spermatogonium. The Cymg1 expression level varied in different developmental stages: it was low 1 week postpartum, steadily increased 2 to 5 weeks postpartum, and was highest 7 weeks postpartum. The expression level at 5 weeks postpartum was maintained during 13 to 57 weeks postpartum. The Cymg1 expression level in the testes over different developmental stages correlates with the mouse spermatogenesis and sexual maturation process. All these indicate that Cymg1 might play an important role in mouse spermatogenesis and sexual maturation.     

Identification of a heat-shock protein Hsp40, DjB1, as an acrosome- and a tail-associated component in rodent spermatozoa

Iba1 is a 17-kDa EF-hand protein highly expressed in the cytoplasm of elongating spermatids in testis. Using Iba1 as a bait, we performed yeast Two-hybrid screening and isolated a heat-shock protein Hsp40, DjB1, from cDNA library of mouse testis. To characterize DjB1 that is encoded by Dnajb1 gene, we carried out immunoblot analyses, in situ hybridization, and immunohistochemistry. Immunoblot analyses showed that DjB1was constitutively expressed in mouse testis and that its expression level was not changed by heat shock. Dnajb1 mRNA was exclusively expressed in spermatocytes and round spermatids in mouse testis, and Dnajb1 protein DjB1 was predominantly expressed in the cytoplasm of spermatocytes, round spermatids, and elongating spermatids. In mature mouse spermatozoa, DjB1 was localized in the middle and the end pieces of flagella as well as in association with the head (acrosomal region). Association of DjB1 with the acrosomal region in sperm head was also observed in rat spermatozoa. These data suggested that DjB1, which was constitutively expressed in postmeiotic spermatogenic cells in testis, was integrated into spermatozoa as at least two components, that is, sperm head and tail of rodent spermatozoa.     

Murine interleukin-11 (IL-11) is expressed at high levels in the hippocampus and expression is developmentally regulated in the testis

IL-11, derived from a bone marrow stromal cell line, has pleiotropic effects on both hematopoietic cells and nonhematopoietic cells. However, no previous studies have systematically addressed expression of IL-11 in primary tissues in vivo and the relationship of IL-11 tissue specific gene expression and function of IL-11 is not clear. In the present study, we examined constitutive IL-11 expression in various murine adult tissues in vivo. IL-11 mRNA is expressed in a wide range of normal tissues (including hematopoietic organs) at levels only detected by RT-PCR. IL-11 protein was detected in brain and testis by Western blot analysis. The in vivo cellular distribution of IL-11 expression was examined by in situ hybridization. In brain, IL-11 message is distributed in granular layer dentate gyrus and pyramidal cell layers of hippocampus. IL-11 is also expressed in anterior horn cells and lateral column neuronal cells of the spinal cord. In testis, Il-11 mRNA is expressed in round spermatids at stage VI-IX seminiferous tubules. IL-11 expression in testis is restricted to developing spermatogonia and is developmentally regulated, since no expression is seen in mice genetically deficient in germ cells and in mice prior to sexual maturation. These expression data correlate with functional data demonstrating that IL-11 stimulates proliferation in vitro of a hippocampus neuronal progenitor cell line and administration of IL-11 in vivo accelerates recovery of spermatogenesis after cytotoxic therapy. These studies suggest that IL-11 may be an important regulator in neural and testicular function. © 1996 Wiley-Liss, Inc.     

Immunochemical and functional characterization of a polyclonal antibody to human sperm antigen

A polyclonal antibody was raised against a 16 kDa human sperm protein identified by a monoclonal antibody to human sperm. The antibody showed significant reactivity with mouse spermatozoa as seen by ELISA. Immunohistochemical analysis showed that the antibody reacted with antigens from mouse testis, prostate as well as seminal vesicle. In both mouse and human testis the antibody localized antigens in round as well as elongated spermatids and mature spermatozoa. By SDS-PAGE and Western blot analysis the antibody reacted with a 16 kDa protein in the testis and seminal vesicle, whereas in the prostate it identified two proteins, one at 20 kDa and another at 25 kDa. Immunofluorescent localization by the antibody showed reactivity with acrosomal and/equatorial and midpiece region of human spermatozoa. The antibody showed extensive agglutination both in mouse and human spermatozoa. The results indicate that the antigen may be a conserved antigen. Cross reactivity of the antibody with mouse spermatozoa enabled us to carry out antifertility trials. Passive immunization of female mice with this antibody caused 67% reduction in fertility. It is likely that the antifertility effect could be partly due to agglutinating nature of the antibody which may have caused inhibition of all processes that depend on forward motility such as cervical mucus penetration and possibly preventing sperm egg interaction. Such well characterized and functionally relevant antibodies will enable to identify sperm antigens relevant for fertility. Identification of such antigens may also help in diagnosis of immuno infertility.     

Molecular cloning and characterization of oppo 1: a haploid germ cell-specific complementary DNA encoding sperm tail protein

We isolated a cDNA clone specifically expressed during spermatogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1085 nucleotides and had an open reading frame of 870 nucleotides encoding a putative protein of 290 amino acid residues. Northern blot analysis revealed a 1.2-kilobase mRNA exclusively expressed in the testis in adult mice; the mRNA was first detected late pachytene stage, and expression increased as the animals matured. The protein encoded by the mRNA had a molecular weight of approximately 33 kDa by Western blot analysis, and was localized to occupy the flagella from the connecting piece through the principal piece. We named this newly isolated gene oppo 1, and we suggest that it plays an important role in sperm tail structure and/or sperm movement.