Identification and expression of epidermal growth factor gene in mouse testis

INTRODUCTIONEpidermalgrowthfactor(EGF)wasinitiallyisolatedandpurifiedfromthesubmaxillarygland(SMG)ofmalemouse[1].Itisapolypeptidecomposedof53aminoacidresidues[2].Itinfluencescellproliferationanddifferentiationandmodulatesthegrowthanddevelopmentofmammalianorgans[3--7].AnoteworthyfindingisthatextirpationofmouseSMGresultsinamarkedreductionofserumEGFconcentrationassociatedwithanimpairedspermatogenesis[3].ThisfindingsuggeststhatEGFmayregulatespermproductionanddifferentiation.Inhumantest…     

Murine Asb-17 expression during mouse testis development and spermatogenesis

In this study we isolated a murine mAsb-17 from mouse testis by RT-PCR using primers designed based on the sequences from the GenBank database. The sequence analysis showed that mAsb-17 encodes a 295 amino acid polypeptide with a molecular weight of approximately 34 kDa containing two ankyrin repeats and one SOCS box. The amino acid sequence of mASB-17 showed 87.5%, 98.3% and 92.9% identity with that of human, rat and dog, respectively. Interestingly, northern blot analysis showed that mAsb-17 was expressed only in the testis. The expression analysis by RT-PCR for mAsb-17 in mouse indicates that mAsb-17 is expressed from the fourth week after birth to adult, with the highest expression in round spermatids. Both northern blot and RT-PCR analyses suggest that mASB-17 may play essential roles in testis development and spermatogenesis.     

Identification of Hedgehog signaling outcomes in mouse testis development using a hanging drop-culture system

The Hedgehog (Hh) signaling pathway affects fetal testis growth. Recently, we described the dynamic cellular production of Hh signaling pathway components in juvenile and adult rodent testes. The Hh signaling is understood to regulate cord formation in the fetal testis, but minimal knowledge exists regarding how Hh signaling impacts the postnatal testis. To investigate this, we employed hanging drop cultures, which are used routinely in embryoid body formation. This approach has the advantage of using small media volume, and we examined its suitability for short-term culture of both murine embryonic gonads and adult testis tubules. The effects of cyclopamine, a specific Hh signaling inhibitor, were examined following culture of Embryonic Day 11.5 urogenital ridges (as control) and adult seminiferous tubule fragments for 24-48 h using histological, cell proliferation, and gene expression analyses. Cultured embryonic testes displayed generally normal cord structure, anti-Müllerian hormone (Amh) expression, and cell proliferation; known Hh target gene expression (Gli1, osteopontin, official symbol Spp1, and Amh) was altered in response to cyclopamine. Cultured adult tubules exhibited some loss of seminiferous epithelium organization over 48 h. Spermatogonia continued to proliferate, however, and no significant loss of viability was noted overall. Addition of cyclopamine significantly affected levels of Gli1, Igfbp6, Ccnd2 (cyclin D2), Ccnb1 (cyclin B1), Spp1, Kit, and Amh mRNAs; these genes have been shown previously to be expressed in Sertoli and germ cells. These novel results identify Hh target genes in the testis and demonstrate this signaling pathway likely affects cell survival and differentiation in the context of normal adult testis.     

Proteome profile changes during mouse testis development

Spermatogenesis is a process of terminal differentiation that results in the formation of mature sperm. In the first wave of this differentiation in the mouse testis, different cell types appear in the seminiferous epithelium at specific times. These cytological changes must be accompanied by changes in protein expression patterns. The aim of the present study was the comparative analysis of proteomic profiles of the soluble proteins expressed at different stages of mouse testis development (8, 18 and 45 postnatal days). Conspicuous variations in their accumulation (representing up or downregulation) were detected over the course of development. Using mass spectrometry (MALDI-TOF), 44 proteins or variant forms were identified. Proteins with redox or antioxidant activity were identified in high proportions; others involved in lipid and carbohydrate metabolic pathways, as well as a number of proteins or isoforms not previously characterized in testis were also detected. These results contribute to identify changes in soluble protein associated to the complex process of male germ cell differentiation.     

Cell-surface expression of a new splice variant of the mouse signal peptide peptidase

The signal peptide peptidase (SPP) is an intramembrane-cleaving aspartyl protease that acts on type II transmembrane proteins. SPP substrates include signal peptides after they have been cleaved from a preprotein, hence the name. The known SPP isoform, which we renamed SPPalpha, contains an endoplasmic reticulum retention signal at the carboxy terminus. We found a new splice variant, SPPbeta, with an additional in-frame exon inserted between exons 11 and 12 of SPPalpha. Insertion of the new exon led to a complete change in the amino-acid sequence of the carboxy tail. A stop codon within this new exon resulted in silencing of exon 12 and eliminated the endoplasmic reticulum retention signal. The new SPP isoform predominantly localised to the cell surface in contrast to the more restricted localisation of SPPalpha in the endoplasmic reticulum. Differential expression in mouse tissues and in subcellular compartments suggests new functions for SPP in addition to cleaving signal peptides.     

DNA methylation and demethylation events during meiotic prophase in the mouse testis.

The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis.     

Neuregulins are essential for spermatogonial proliferation and meiotic initiation in neonatal mouse testis

The transition from mitosis to meiosis is unique to germ cells. In murine embryonic ovaries and juvenile testes, retinoic acid (RA) induces meiosis via the stimulated by retinoic acid gene 8 (Stra8), but its molecular pathway requires elucidation. We present genetic evidence in vivo and in vitro that neuregulins (NRGs) are essential for the proliferation of spermatogonia and the initiation of meiosis. Tamoxifen (TAM) was injected into 14-day post-partum (dpp) Sertoli cell-specific conditional Nrg1(Ser-/-) mutant mice. TAM induced testis degeneration, suppressed BrdU incorporation into spermatogonia and pre-leptotene primary spermatocytes, and decreased and increased the number of STRA8-positive and TUNEL-positive cells, respectively. In testicular organ cultures from 5-6 dpp wild-type mice and cultures of their re-aggregated spermatogonia and Sertoli cells, FSH, RA [all-trans-retinoic acid (ATRA), AM580, 9-cis-RA] and NRG1 promoted spermatogonial proliferation and meiotic initiation. However, TAM treatment of testicular organ cultures from the Nrg1(Ser-/-) mutants suppressed spermatogonial proliferation and meiotic initiation that was promoted by FSH or AM580. In re-aggregated cultures of purified spermatogonia, NRG1, NRG3, ATRA and 9-cis-RA promoted their proliferation and meiotic initiation, but neither AM580 nor FSH did. In addition, FSH, RAs and NRG1 promoted Nrg1 and Nrg3 mRNA expression in Sertoli cells. These results indicate that in juvenile testes RA and FSH induced meiosis indirectly through Sertoli cells when NRG1 and NRG3 were upregulated, as NRG1 amplified itself and NRG3. The amplified NRG1 and NRG3 directly induced meiosis in spermatogonia. In addition, ATRA and 9-cis-RA activated spermatogonia directly and promoted their proliferation and eventually meiotic initiation.     

An integrated bioinformatics analysis of mouse testis protein profiles with new understanding

The testis is major male gonad responsible for spermatogenesis and steroidogenesis. Much knowledge is still remained to be learned about the control of these events. In this study, we performed a comprehensive bioinformatics analysis on 1,196 mouse testis proteins screened from public protein database. Integrated function and pathway analysis were performed through Database for Annotation, Visualization and Integrated Discovery (DAVID) and ingenuity Pathway Analysis (IPA), and significant features were clustered. Protein membrane organization and gene density on chromosomes were analyzed and discussed. The enriched bioinformatics analysis could provide clues and basis to the development of diagnostic markers and therapeutic targets for infertility and male contraception.     

Vitamin A deficiency results in meiotic failure and accumulation of undifferentiated spermatogonia in prepubertal mouse testis

Vitamin A (retinol) is required for maintenance of adult mammalian spermatogenesis. In adult rodents, vitamin A withdrawal is followed by a loss of differentiated germ cells within the seminiferous epithelium and disrupted spermatogenesis that can be restored by vitamin A replacement. However, whether vitamin A plays a role in the differentiation and meiotic initiation of germ cells during the first round of mouse spermatogenesis is unknown. In the present study, we found that vitamin A depletion markedly decreased testicular expression of the all-trans retinoic acid-responsive gene, Stra8, and caused meiotic failure in prepubertal male mice lacking lecithin:retinol acyltransferase (Lrat), encoding for the major enzyme in liver responsible for the formation of retinyl esters. Rather than undergoing normal differentiation, germ cells accumulated in the testes of Lrat(-/-) mice maintained on a vitamin A-deficient diet. These results, together with our previous observations that germ cells fail to enter meiosis and remain undifferentiated in embryonic vitamin A-deficient ovaries, support the hypothesis that vitamin A regulates the initiation of meiosis I of both oogenesis and spermatogenesis in mammals.     

Identification and characterization of mouse SSX genes: a multigene family on the X chromosome with restricted cancer/testis expression

Human SSX was first identified as the gene involved in the t(X;18) translocation in synovial sarcoma. SSX is a multigene family, with 9 complete genes on chromosome Xp11. Normally expressed almost exclusively in testis, SSX mRNA is expressed in various human tumors, defining SSX as a cancer/testis antigen. We have now cloned the mouse ortholog of SSX. Mouse SSX genes can be divided into Ssxa and Ssxb subfamilies based on sequence homology. Ssxa has only one member, whereas 12 Ssxb genes, Ssxb1 to Ssxb12, were identified by cDNA cloning from mouse testis and mouse tumors. Both Ssxa and Ssxb are located on chromosome X and show tissue-restricted mRNA expression to testis among normal tissues. All putative human and mouse SSX proteins share conserved KRAB and SSX-RD domains. Mouse tumors were found to express some, but not all, Ssxb genes, similar to the SSX activation in human tumors.     

Interleukin-6 expression during normal maturation of the mouse testis

In this study, we examined the cellular origin and the expression levels of interleukin-6 (IL-6) during normal maturation of mouse testis. The levels of IL-6 (protein and mRNA) were higher in testicular homogenates of sexually immature than mature mice. Immunohistochemical staining of testicular tissues of sexually immature and adult mice show that testicular germ cells, at different stages of differentiation, Leydig cells/interstitial cells and peritubular cells express IL-6. Our results demonstrate, for the first time, overexpression of IL-6 in testicular tissues of immature mice, as compared to mature mice, as well as the expression of IL-6 in germ cells of testicular tissues of adult and sexually immature mice. Thus, our results may indicate the involvement of the endocrine system (gonadotropins and testosterone) in the regulation of IL-6, which is involved in the regulation of testicular development, functions and spermatogenesis.     

Stage and cell-specific expression of calmodulin-dependent phosphodiesterases in mouse testis

Calcium and cyclic nucleotides are second messengers that regulate the development and functional activity of spermatozoa. Calcium/calmodulin-dependent phosphodiesterases (CaM-PDEs) are abundant in testicular cells and in mature spermatozoa and provide one means by which calcium regulates cellular cyclic nucleotide content. We examined the spatial and temporal expression profiles of three knownCaM-PDE genes, PDE1A, PDE1B, and PDE1C, in the testis. In situ hybridization and immunofluorescent staining showed that both PDE1A and PDE1C are highly expressed but at different stages in developing germ cells. However, a very low hybridization signal of PDE1B exists uniformly throughout the seminiferous epithelium and the interstitium. More specifically, PDE1A mRNA is found in round to elongated spermatids, with protein expression in the tails of elongated and maturing spermatids. In contrast, PDE1C mRNA accumulates during early meiotic prophase and throughout meiotic and postmeiotic stages. Immunocytochemistry showed a diffuse, presumably cytosolic distribution of the expressed protein. The distinct spatial and temporal expression patterns of CaM-PDEs suggest important but different physiological roles for these CaM-PDEs in developing and mature spermatozoa.     

Hepatocyte growth factor (HGF) receptor expression and role of HGF during embryonic mouse testis development

The hepatocyte growth factor (HGF) receptor, c-met, transduces the HGF multiple biological activities. During embryonic development the system HGF/c-met regulates the morphogenesis of different organs and tissues. In this study we examined c-met gene expression during mouse testis development and, by means of Northern blot and in situ hybridization, we report the receptor expression pattern. C-met expression is not detectable in male genital ridges isolated from embryos at 11.5 days postcoitum (dpc). In testes isolated from 12.5 and 13.5 dpc, c-met expression is detectable and essentially localized in the developing cords. Male genital ducts do not express c-met at the reported ages, whereas female ducts appear c-met positive. Moreover, we report that HGF is able to induce testicular morphogenesis in vitro. Male genital ridges isolated from embryos at 11.5 dpc are morphologically nonorganized. Culturing 11.5 dpc urogenital ridges in the presence of HGF we obtained testis organization and testicular cord formation. Our data demonstrate that c-met is expressed during the beginning period of testis differentiation and that HGF is able to support testicular differentiation in vitro. All these data indicate that this growth factor, besides its role as mitogenic factor, plays a fundamental role during testicular cord formation probably inducing cell migration and/or cell differentiation.     

Biochemical and immunological characterization of an H2A variant from the mouse testis

A histone H2A variant, protein 'A', has been isolated and purified from the mouse testis. Amino acid composition analysis and electrophoretic properties indicate it to be apparently similar to H2A X X variant present in the mouse L1210 cells. Specific antibodies against protein 'A' have been generated in rabbits and used to study tissue and species distribution in mammals. Protein transfer experiments indicate the presence of antigenically similar proteins in somatic tissues of the mouse. Immunologically similar proteins were also detected in other mammalian testes. The data further indicate that protein A is antigenically distinct from the other members of the H2A family, H2A X 1, H2A X 2 and H2A X Z.     

A new variant of serum leucine aminopeptidase in the mouse: Its development and possible regulation

Serum leucine aminopeptidase (LAP) isozymes were compared in four strains of inbred mice during postnatal development, adult life, and pregnancy. In pregnancy, no changes in the maternal serum LAP pattern were observed, in contrast to human studies. One strain, DD/S, differs from the other three in serum LAP. Polymorphism in serum LAP has not been previously described in the mouse. Neonatal DD/S mice exhibit a single band of serum LAP upon starch gel electrophoresis; however, between 14 and 18 days of age, two distinct bands appear, which persist throughout adult life. In the strains C57BL/6J, BALB/cJ, and DBA/2J there is a single band of activity at all stages. Crosses and backcrosses between DD/S and C57BL/6J show that the double-band variant is inherited as an autosomal recessive. The variant is independent of both the supernatant malic enzyme (Mod-1) and the intestinal LAP (Lap-1) loci, which are known to be linked on chromosome 9. The serum LAP variant is linked to an intestinal alkaline phosphatase variant. The presence of a separate structural gene is suggested by the genetic independence of the serum LAP variant from Lap-1. Also, the two serum LAP bands of DD/S are not interconverted by treatment with neuraminidase, -mercaptoethanol, or heat or by mixing the sera of DD/S and C57BL/6J prior to electrophoresis. The level of serum LAP activity in DD/S is approximately twice that in C57BL/6J. While these observations imply two structurally distinct proteins, the absence of any trace of the second LAP band in the heterozygote strongly suggests that the LAP variant protein is not the result of a separate structural gene. Intestinal LAP in DD/S migrates with the same electrophoretic mobility as the serum LAP variant, implying that the variant might originate in the intestine and its appearance in the serum be modulated by some factor at an unlinked locus.This work was supported by National Institutes of Health Grant RR08117.