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PubMed2003

Identification of developmentally regulated genes in the somatic cells of the mouse testis using serial analysis of gene expression.

To identify genes developmentally regulated in the somatic cells of the testis, serial analysis of gene expression (SAGE) has been used to generate gene expression profiles from these cells in the fetal and adult mouse. To avoid germ cell transcripts, a fetal SAGE library was generated from germ cell-free fetal Wv/Wv mice, and an adult SAGE library was generated from adult testes depleted of germ cells with busulfan. The combined SAGE libraries contained 147570 tags identifying 12976 unique transcripts. Of these transcripts, 3607 were present in only the fetal library and 3941 were present in only the adult library. Most of the abundant differentially expressed tags in the adult testis library were from characterized genes, whereas 3' rapid amplification of complementary ends was required to identify most differentially expressed tags in the fetal library. These fetal tags were mostly associated with uncharacterized UniGene clusters. These data provide a comprehensive and quantitative analysis of gene expression in the somatic cells of the fetal and adult testis (including unknown transcripts) and identify genes differentially expressed in these cells during testis development. These differentially regulated genes are likely to provide insight into mechanisms regulating testis function both during development and in the adult animal.

Animals↗
PubMed2003

Evidence for similar expression of protein C inhibitor and the urokinase-type plasminogen activator system during mouse testis development.

Plasminogen activators (PAs) and their inhibitors (PAIs) are predicted to be involved in the restructuring events that characterize the testis throughout development. We here demonstrate that PAI-3 or protein C (PC) inhibitor (PCI) was expressed in a sexually dimorphic fashion during mouse gonad genesis, whereas PAI-1 and -2 exhibited no sex differences. PCI transcripts accumulated rapidly in the male gonad, from 12.5 d postcoitum onward. Western blot and immunohistochemistry analyses confirmed that male, but not female, fetal gonads produced PCI, and that Leydig cells are the site of PCI synthesis. The occurrence of testicular target proteases for PCI, i.e. PC and urokinase- and tissue-type PA, was further tracked using RT-PCR, plasminogen zymography, and/or immunohistochemistry. PC and tissue-type PA showed no variation between sexes. By contrast, urokinase-type PA and its receptor (uPAR; which dictates the site and extent of proteolysis) exhibited sex differences from 13.5-14.5 d postcoitum. At that time, uPAR expression was restricted to Leydig cells. At earlier ages, uPAR was uniformly and widely distributed in the gonads of both sexes. In adult testes, PCI and uPAR immunoreactivities were also present in Leydig cells. In addition, PCI, PC, and uPAR had a germinal origin. Collectively, these results support the hypothesis that PCI may contribute to proteolysis equilibrium within the testis by acting in tandem with urokinase in Leydig cells and with PC and/or urokinase in spermatogenic cells. It will be important to determine how this role is linked to the phenotype of sterility reported elsewhere in male mice with pci deleted.

Animals↗
PubMed1990

Expression of a candidate sex-determining gene during mouse testis differentiation.

The development of a eutherian mammal as a male is a consequence of testis formation in the embryo, which is thought to be initiated by a gene on the Y chromosome. In the absence of this gene, ovaries are formed and female characteristics develop. Sex determination therefore hinges on the action of this testis-determining gene, known as Tdy in mice and TDF in humans. In the past, several genes proposed as candidates for Tdy/TDF have subsequently been dismissed on the grounds of inappropriate location or expression. We have recently described a candidate for Tdy, which maps to the minimum sex-determining region of the mouse Y chromosome. To examine further the involvement of this gene, Sry, in testis development, we have studied its expression in detail. Fetal expression of Sry is limited to the period in which testes begin to form. This expression is confined to gonadal tissue and does not require the presence of germ cells. Our observations strongly support a primary role for Sry in mouse sex determination.

Animals↗
PubMed2003

Expression of Tom34 splicing isoforms in mouse testis and knockout of Tom34 in mice.

The 34-kDa translocase of the outer mitochondrial membrane (Tom34) is a putative mammalian-specific factor involved in protein import into mitochondria. We analyzed the genomic sequence of the mouse Tom34 gene and found it has two alternative initial exons. Using reverse transcription and the polymerase chain reaction (RT-PCR), we found that these two mRNAs differs only in the 5'-proximal sequences corresponding to the two initial exons (exon 1a and 1b). Tom34 mRNA with exon 1a (Tom34a) is expressed ubiquitously, while that with exon 1b (Tom34b) is expressed only in mature testicular germ cells. To explore the in vivo function of Tom34 proteins, we generated Tom34-deficient mice by targeted disruption. The Tom34(-/-) mice were viable and grew normally and had a normal Mendelian inheritance pattern. Male as well as female Tom34(-/-) mice were fertile. In vitro-preprotein import into isolated mitochondria showed no apparent difference between Tom34(-/-) and wild-type mice. These results indicate that Tom34 is dispensable for mouse growth and development under optimal conditions.

Alternative Splicing↗
PubMed1991

Effects of dietary zinc deficiency on protein secretory functions of the mouse testis.

The effects of dietary zinc deficiency on testicular protein secretion, mainly that by Sertoli cells, were examined by electron microscopy and two-dimensional polyacrylamide gel electrophoresis of [35S] methionine-labeled secretory proteins from mouse testes. Zinc deficiency caused a significant decrease in the gonadosomatic index and a distinct increase in deoxyribonucleic acid concentration. Sertoli cells maintained normal fine-structural features; junctional complexes among Sertoli cells continued to divide seminiferous tubules into basal and adluminal compartments in the zinc-deficient mouse testes. Severe atrophic changes were observed in spermatogenic cells after meiotic division in the adluminal compartment, but not in spermatogonia located in the basal compartment. Zinc replacement treatment caused spermatogenesis to recover normally. Although total protein secretion was not affected by zinc deficiency, one polypeptide spot appeared due mainly to the loss of its target spermatogenic cells. The present study indicates that zinc is indispensable for spermatogenic cells after meiosis and that testicular protein secretory functions can be preserved in the absence of zinc.

Animals↗
PubMed1985

Separation of mouse testis cells on a Celsep (TM) apparatus and their usefulness as a source of high molecular weight DNA or RNA.

The use of a self-contained unit-gravity cell separation apparatus for separation of populations of mouse testicular cells is described. The apparatus, a Celsep (TM), maximizes the unit area over which sedimentation occurs, reduces the amount of separation medium employed, and is quite reproducible. Cells thus isolated have been good sources for isolation of DNA, and notably, high molecular weight RNA.

Animals↗
PubMed2003

'Side Population' cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells.

Stem cells in various somatic tissues (bone marrow, skeletal muscle) can be identified by the 'Side Population' marker based on Hoechst 33342 efflux. We show that mouse testicular cells also display a 'Side Population' that express Bcrp1 mRNA, the ABC transporter responsible for Hoechst efflux in hematopoietic cells. Inhibition of Hoechst efflux by specific BCRP1 inhibitor Ko143 show that germinal 'Side Population' phenotype is dependent on BCRP1 activity. Analysis of two well-defined models of altered spermatogenesis (W/Wv mutants and cryptorchid male mice) and RNA expression studies of differentiation markers demonstrate that germinal 'Side Population' contains spermatogonial cells. In addition, alpha 6-integrin and Stra8 germinal stem cell markers, are expressed in the 'Side Population'. In vivo repopulation assay clearly establishes that testis 'Side Population' in adult mice is highly enriched in male germ stem cells.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2003

Differential expression of succinyl CoA transferase (SCOT) genes in somatic and germline cells of the mouse testis.

Succinyl CoA:3-oxo acid CoA transferase (SCOT/OXCT; EC 2.8.3.5) is a key mitochondrial enzyme in the metabolism of ketone bodies in various organs (but not in the liver). We identified a cDNA clone of the testicular germ cell-specific succinyl CoA transferase isozyme (SCOT-t). We then isolated a mouse orthologue of the SCOT/OXCT cDNA (SCOT-s) and determined the expression of the two types of SCOT in the testis. The mRNAs of scot-s and scot-t were expressed exclusively in testicular somatic cells (i.e. Leydig and Sertoli cells) and germ cells, respectively. SCOT enzymatic activities were assayed in Leydig cell (SCOT-s) and sperm (SCOT-t) fractions. The SCOT activity in sperm was 2.5-fold higher than that in Leydig cells. We conclude that germ cells and somatic cells differentially express the SCOT enzymes and that the SCOT activity of sperm caused exclusively by SCOT-t should play an important role in sperm activity.

Animals↗
PubMed1982

Further observations on the Sertoli cell junctions of the mouse testis after metal contract freeze-fracture, and comparisons with cellular junctions of other epithelial cells.

Using a metal contact freezing methods, the junctional complexes of Sertoli cells and other epithelial cells (intestine, liver, and epididymis) in the mouse were studied by freeze-fracture in unfixed and in aldehyde-fixed specimens. The tight junctions of the Sertoli cells without fixation consists of discrete rows of particles located preferentially on the E face. Continuous furrows with or without tight-junctional particles are found on both fractured faces. The majority of the tight-junctional particles of th Sertoli cells are situated at one side of the furrow rather than in the center. The "aisle" configuration of the gap junction is seen in developing Sertoli cells prepared without fixation. Tight junctions of epithelial cells other than Sertoli cells consists of discrete rows of particles on the E face and furrows on the P face when prepared without fixation. The tight and gap junctions of the Sertoli cells are not affected by fixation, but the tight junctions of other types of epithelial cells are effected as previously suggested. A distribution pattern of the gap-junctional particles of the Sertoli cells and of other cells shows a pleomorphism. Changes in gap junctions produced by fixation could not be detected clearly. Characteristics of the Sertoli-cell junctions are discussed. Desmosomes between the intestinal epithelial cells without fixation are presented.

Animals↗
PubMed1972

Inorganic cations in the cell nucleus. Selective accumulation during meiotic prophase in mouse testis.

Earlier reports indicated the presence of significant amounts of inorganic salts in the nucleus. In the present study the possibility that this might be related to the transcription process was tested on seminiferous epithelium of the adult mouse, using potassium pyroantimonate as a fixative. The results indicated that a correlation exists between the inorganic cations comprising the pyroantimonate-precipitable fraction and the RNA synthetic activity. During meiotic prophase an accumulation of cation-antimonate precipitates occurs dispersed through the middle pachytene nuclei, the stage in which RNA synthesis reaches a maximum. At other stages (zygotene to diplotene), where RNA synthesis falls to a low level, that pattern is not seen; cation-antimonate deposits are restricted to a few masses in areas apparently free of chromatin. The condensed sex chromosomes, the heterochromatin of the "basal knobs," the axial elements, and the synaptonemal complexes are devoid of antimonate deposits during the meiotic prophase. The Sertoli cells, active in RNA synthesis in both nucleoplasm and nucleolus, show cation-antimonate deposits at these sites. In the nucleoplasm some "patches" of precipitates appear coincident with clusters of interchromatin granules; in the nucleolus the inorganic cations are mainly located in the fibrillar and/or amorphous areas, whereas relatively few are shown by the granular component. The condensed chromatin bodies associated with the nucleolus were always free of antimonate precipitates. It is suggested that the observed sites of inorganic cation accumulation within the nucleus may at least partially indicate the presence of RNA polymerases, the activity of which is dependent on divalent cations.

Animals↗
PubMed2005

Zinc-regulating proteins, ZnT-1, and metallothionein I/II are present in different cell populations in the mouse testis.

Zinc ions play an important role in testis development and spermatogenesis. Thus, nutritional zinc deficiency leads to aberrant testicular development, reduced spermatogenesis, and male sterility. The precise actions of zinc in mediating these functions and the mechanisms by which zinc is itself regulated in the testis, however, have not been adequately elucidated. We have assessed the distribution of the zinc-regulating proteins ZnT-1 and metallothionein I/II (MT I/II) in the mouse seminiferous tubule. Co-labeling for ZnT-1 and MT I/II demonstrated unique patterns of distribution for these proteins, with ZnT-1 present in Sertoli cells in addition to luminal spermatozoa and MT I/II restricted to spermatocytes. These findings were confirmed by dual-label immunofluorescence for ZnT-1 and the Sertoli cell marker, vimentin, and by immunoelectron microscopy. The differential expression patterns of ZnT-1 and MTs support the hypothesis that ZnT-1 and MTs play different roles in the regulation of intracellular zinc in this organ. The specific expression of ZnT-1 in the Sertoli cells, moreover, is consistent with their role in maintaining a nurturing, closely regulated environment for spermatogenesis.

Animals↗
PubMed1997

Spermatogenic cell apoptosis induced by mitomycin C in the mouse testis.

Spermatogenic cell degeneration in the mature mammalian testis occurs both spontaneously during normal spermatogenesis and in response to cytotoxic agents. Mitomycin C (MC) is an antibiotic that affects DNA synthesis. In the present study, we examined the induction of mouse spermatogenic cell apoptosis by MC, using TdT-mediated dUTP-biotin nick end labeling (TUNEL) to detect high levels of DNA fragmentation in situ, transmission electron microscopy (TEM) to observe nuclear chromatin condensation, and molecular methods to detect DNA ladders. This study shows that in the testis of MC-treated mice: (i) apoptotic cell death with fragmentation of nuclear DNA is induced by MC dose-dependently, (ii) apoptotic cell death is most commonly found in the spermatogonia and less frequently in spermatocytes, and (iii) apoptotic cell death induced by MC is not specific for the seminiferous stage of the tubules. The present study suggests that the spermatogenic cell apoptosis induced by MC might be involved in its testicular toxicity.

Animals↗
PubMed1992

Germ cell-specific expression of a proacrosin-CAT fusion gene in transgenic mouse testis.

Acrosin is a serine proteinase located in a zymogen form, proacrosin in the acrosome of the sperm. It is released as a consequence of the acrosome reaction and is believed to be the most important enzyme in the fertilization process. In the mouse, the proacrosin gene is transcribed premeiotically in spermatocytes, but protein biosynthesis starts in haploid spermatids and is restricted to the emerging acrosome. Four lines of transgenic mice harboring 2.3 kb of 5' untranslated region of the rat proacrosin gene fused to the CAT-reporter gene were generated by microinjection of fertilized eggs. The chimeric gene was found to be present in 10-100 copies per genome in the different strains. The 5' untranslated region of rat proacrosin gene could properly direct CAT-gene expression to spermatocytes and CAT-mRNA translation to round spermatids as it is known for mouse proacrosin gene. However, CAT protein is not restricted to the acrosome; rather, it is distributed in the spermatid cytoplasm. This could be due to the lack of DNA sequences for a hydrophobic leader peptide that have been found in all mammalian proacrosins studied until now but that was not present in transgene. It can be concluded from our results that cis-acting sequences required for tissue specific proacrosin expression reside on a 2.3-kb restriction fragment and are conserved in the proacrosin genes of mouse and rat.

Acrosin↗
PubMed1997

Radiation-induced cell death in the mouse testis: relationship to apoptosis.

The killing of male germ cells by radiation and other toxicants has recently been attributed to apoptosis, but a critical evaluation of the presence of the different features of apoptosis has not been performed. In this study, mouse testes exposed to radiation were examined by light microscopy, electron microscopy and terminal transferase-mediated end labeling (TUNEL) to determine whether the cells were apoptotic according to several criteria. Testes were irradiated with single doses of gamma rays of up to 5 Gy. Although the maximum response was produced by 5 Gy, even 0.5 Gy induced marked changes. The numbers of abnormal spermatogonia reached a peak 12 h after irradiation and then declined, and the total number of spermatogonia began to decline at 12 h. These changes were most prominent among the B spermatogonia and early preleptotene spermatocytes. When examined by both light and electron microscopy, the majority of the abnormal spermatogonia showed condensation of nuclear chromatin and some showed features similar to necrosis, but the typical morphological characteristics of apoptosis, margination of chromatin and nuclear fragmentation, were rare. Many of the abnormal spermatogonia were TUNEL-positive, with the maximum number occurring at 12 h after irradiation. Although the morphological features of radiation-induced spermatogonial degeneration were not typical of apoptosis, the TUNEL staining, the rapid onset of degeneration and the sensitivity to low doses suggest that the mechanism of radiation-induced spermatogonial degeneration is closely related to apoptosis.

Animals↗
PubMedDate not supplied

Chemical dosimetry of ethyl nitrosourea in the mouse testis.

[3H-Et]Nitrosourea was administered to male (101 X C3H) mice by i.p. injection at exposure levels of 10 mg/kg or 100 mg/kg. At intervals from 1 h to 6 days following treatment, the ratio of O6-ethylguanine to N7-ethylguanine in testis DNA averaged 1.13 following the 100 mg/kg exposure and 0.72 following the 10 mg/kg exposure. The amount of O6-ethylguanine recovered after the 100 mg/kg exposure was 40% greater than predicted from a linear extrapolation of the amount of O6-ethylguanine recovered after the 10 mg/kg exposure. We suggest that the high (100 mg/kg) exposure to ethyl nitrosourea results in depletion of the O6-alkylguanine acceptor protein within the testis and permits O6-ethylguanine to persist at higher levels than would be predicted from lower exposure data. W.L. Russell et al. (1982), W.L. Russell (1984) have found that specific-locus mutation frequencies induced in mouse spermatogonial stem cells are 5.8-fold greater after a single 100 mg/kg exposure to ethyl nitrosourea than after 10 weekly exposures to 10 mg/kg. The finding that the corresponding ratio for O6-ethylguanine formed in the testis is only 1.4 may be interpreted in a number of possible ways. If O6-ethylguanine is an important lesion for producing specific-locus mutations, then its formation in the stem cells must be at least 4-fold greater than that for the whole testis as the ENU exposure goes from 10 to 100 mg/kg: alternatively, the rate of repair of this lesion by the stem cells must decrease at least 4-fold relative to the average testicular cell. Other explanations for the difference in mutation response of the stem cells to acute vs. chronic ethyl nitrosourea-exposures include the possibility that other DNA lesions may be responsible for many of the mutations or that two hits on the DNA may be required to produce an effect.

Alkylating Agents↗
PubMed2003

Sox15 is up regulated in the embryonic mouse testis.

The mammalian sex determining region on the Y chromosome, SRY, is the founding member of the SOX gene family. SOX genes share a common DNA-binding motif termed the HMG box and have diverse roles in vertebrate embryonic development and tissue differentiation. Sox15 expression was analysed during mouse embryogenesis by whole-mount in situ hybridisation and Real Time RT-PCR. Sox15 was found to be expressed in developing mouse gonads from 11.5 dpc to 13.5 dpc with a peak of expression at 12.5 dpc. Expression was approximately twice as high in the male gonad as in the female gonad.

Animals↗
PubMed2003

Effects of 3,3',4,4',5-pentachlorobiphenyl, a coplanar polychlorinated biphenyl congener, on cultured neonatal mouse testis.

3,3',4,4',5-Pentachlorobiphenyl (PCB126), a congener with a planar configuration, has been established to have relatively strong toxicities similar to those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via aryl hydrocarbon receptors. We investigated the effects of this coplanar PCB on mammalian early spermatogenesis and steroidogenesis in a mouse neonatal testicular organ culture system. Testes collected from newborn mice were subjected to organ culture in medium containing 0, 10, 100 or 1000 nM PCB126. Histochemical analysis revealed that the BrdU-labeling indices of both spermatogenic cells and Sertoli cells were unchanged in all testis specimens exposed to the coplanar PCB. CYP1A1 and steroidogenic enzymes (P450scc, P450c17, 3beta-HSD and 17beta-HSD) mRNA levels were determined by semiquantitative RT-PCR. The CYP1A1 mRNA level in cultured testis was significantly increased by PCB126 in a dose-dependent manner. Although mRNA levels of 3beta-HSD and 17beta-HSD were unchanged, the P450scc mRNA level was significantly down-regulated by PCB126 in a dose-dependent manner. In contrast, the P450c17 mRNA level was significantly higher in 1000 nM PCB126-exposed testis than in control testis. These results suggest that the coplanar PCB does not alter the proliferative activity of spermatogenic cells and Sertoli cells in neonatal testis, but that it directly affects the expression of steroidogenic enzyme genes.

Animals↗
PubMed1996

Reinitiation of spermatogonial mitotic differentiation in inactive old BDF1 mouse seminiferous tubules transplanted to W/Wv mouse testis.

The seminiferous epithelia of old mice (33 mo of age) are composed of spermatogonia and Sertoli cells. Histochemical examination using the anti-c-kit monoclonal antibody demonstrated that the number of differentiating type A spermatogonia decreases with age. To elucidate the differential activity of old mouse spermatogonia, we transplanted extremely thin seminiferous epithelia of old BDF, mice into W/Wv mouse testes and examined whether or not they could reinitiate differentiation. Artificially cryptorchid mice were used as the control. At 2 wk after transplantation, spermatocytes and round spermatids were detected in transplanted seminiferous tubules of the control, whereas the most advanced spermatogenic cells in those of old mice were spermatocytes. At 4 wk after transplantation, although elongated spermatids were detected in transplanted tubules of the control, haploid cells (spermatids) were still undetectable in those derived from old mice. Thus, meiosis was never restored, although spermatogonia of old mice can reinitiate differentiation into spermatocytes under suitable testicular conditions. Since it has been reported in several mammalian species that age-related changes in the testicular microenvironment lead to the gerontal cessation of spermatogenesis, the present results suggest that both a defective extratubular environment and a defective intratubular environment may cause the cessation of spermatogenesis in old BDF, mice.

Aging↗