Developmental program of PGK-1 and PGK-2 isozymes in spermatogenic cells of the mouse: Specific activities and rates of synthesis

The specific activities and synthesis of the ubiquitous isozyme, PGK-1, and the testis-specific isozyme, PGK-2, have been quantitated and localized in spermatogenic cells of the mouse. There is a fivefold increase in total PGK specific activity between immature and adult testes which begins at approximately 30 days of age, coincident with the appearance of late-middle stage spermatids. The increase in total PGK is entirely due to the appearance and increase of the PGK-2 isozyme. Rates of PGK synthesis were measured by labeling testicular cells in vitro with [³H]leucine and purifying the PGK isozymes. When total testicular cells were examined, PGK-2 synthesis was detectable after 22 days of age at very low levels and increased in older testes to a level of 0.5% of total protein synthesis. PGK-1 synthesis remained relatively constant at all ages at a level 100-fold lower (0.005%). Testicular cells were separated into highly enriched fractions of particular spermatogenic stages by centrifugal elutriation. The PGK-1 synthesis rates were, again, very low and not significantly different between the various spermatogenic stages. PGK-2 synthesis was low to nondetectable in pachytene spermatocytes, increased to 0.07% in early spermatids and represented 0.7% of total protein synthesis in late spermatids. This increased rate of PGK-2 synthesis appears to require an increase in the amount of PGK-2 mRNA in late spermatids, cells in which no active RNA synthesis is detectable.     

Haploid accumulation and translational control of phosphoglycerate kinase-2 messenger RNA during mouse spermatogenesis

The intracellular location of the mRNA for the testis-specific isozyme of phosphoglycerate kinase-2 (PGK-2) has been determined for two spermatogenic cell types. The mRNA activity for PGK-2 from the polysomal and nonpolysomal fractions of pachytene primary spermatocytes or round spermatids has been assayed by cell-free translation with the polypeptide products monitored by immunoprecipitation, followed by one-dimensional or two-dimensional electrophoresis and fluorography. The results reveal that the majority of PGK-2 mRNA activity of round spermatids was present in the polysomal fraction while the relatively less abundant PGK-2 mRNA of pachytene primary spermatocytes was present in the nonpolysomal fraction. No PGK-2 mRNA activity was observed in the cytoplasmic RNA from primitive type A spermatogonia or prepubertal Sertoli cells. These data indicate that mature PGK-2 mRNA first appears in the cytoplasm of spermatogenic cells during the prophase of meiosis and increases in amount after meiosis. Although mature PGK-2 mRNA is present in meiotic cells it is not actively translated until after meiosis has been completed. Thus, mRNA accumulation and translational mechanisms are involved in the control of phosphoglycerate kinase-2 synthesis during spermatogenesis.     

Immunofluorescent localization of PGK-1 and PGK-2 isozymes within specific cells of the mouse testis

The ubiquitous isozyme of phosphoglycerate kinase, PGK-1, and the testis-specific isozyme, PGK-2, have been localized to specific cells of the testis by indirect immunofluorescence on Bouin's fixed testis sections. The earliest cell of the spermatogenic series in which PGK-2 is detectable by immunofluorescence is the Stage 12 spermatid. The intensity of fluorescence increases as the spermatids progress to later stages and is strong in both released spermatids and their residual bodies. PGK-2 is not detectable in premeiotic germinal cells or somatic cells of the testis. Specific fluorescence for PGK-1 is localized to the somatic cells of the testis: the interstitial and Sertoli cells.     

A single nucleotide substitution in the phosphoglycerate kinase (PGK)-1 gene occurred after the separation of PGK-1 and PGK-2

Summary The mammalian genome contains two functional loci for the production of phosphoglycerate kinase, PGK-1, an X-linked gene expressed in all somatic cells, and PGK-2, an autosomal intron-less gene expressed exclusively in late spermatogenesis. A nucleotide substitution from guanine to thymine was recently found at position 473 of PGK-1 mRNA in PGK Shizuoka. The mutation was not found in the PGK-2 gene and might have occurred after separation of PGK-1 and PGK-2.     

Immunohistochemical localization of mouse testis-specific phosphoglycerate kinase (PGK-2) by monoclonal antibodies.

Monoclonal antibodies against mouse testis-specific phosphoglycerate kinase (PGK-2) were produced in order to determine immunohistochemically the onset of PGK-2 synthesis in the germinal epithelium of the mouse. PGK-2 was detected in testis sections in spermatids as early as stage 12 and in spermatozoa, but not in earlier stages of spermatogenesis nor in any somatic cells of the testis. During ontogeny, PGK-2 appears within the testis at day 30 post-partum, concomitant with spermatids entering the maturation phase. All three allelic isozymes PGK-2A, -2B, and -2C were detected equally by the monoclonal antibody in testis sections of several inbred mouse strains, each of which expresses a specific PGK-2 variant. Moreover, the monoclonal antibody against mouse PGK-2 reacted with heterologous sperm-specific PGK from rat, rabbit, and bull and, therefore, may serve as a useful immunochemical marker for mammalian spermatogenesis.     

Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.     

Polycomb基因家族在大鼠精子发生过程中的表达

目的检测大鼠精子发生不同阶段细胞中Polycomb-group（Pc-G）家族在mRNA水平上表达是否有差异。方法提纯大鼠精子发生过程中的精原细胞、精母细胞、圆形精子细胞以及支持细胞,用荧光定量PCR方法检测Pc-G家族基因mRNA表达量。结果Pc-G基因家族中Ezh2、Eed、Bmi-1在精子发生中后期高表达;在各生精细胞中,YY1基因表达量低于支持细胞。结论Pc-G基因家族在精子发生各阶段细胞中特征性表达,与精子发生具有相关性,可能对精子发生分化和维持遗传稳定性都有重要的作用。     

Evolution of Ca2+- and cAMP-dependent regulatory mechanisms during ram spermatogenesis

Calmodulin level and cAMP-dependent protein kinase activity of ram germ cells at different stages of spermatogenesis have been determined. Calmodulin levels decrease during maturation. Simultaneously, calmodulin localization changes during cell differentiation. In round, elongating, and elongated spermatids, calmodulin is closely associated with the developing acrosome; in spermatozoa, it becomes present in the postacrosome, the neck region and the tail. Protein kinase activity is relatively low in testicular cells but increases dramatically during epididymal maturation of spermatozoa. A concerted regulation by cAMP and Ca2+ of biochemical events in spermatogenic cells and spermatozoa is suggested.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.     

Expression and localization of DNA topoisomerase II during rat spermatogenesis

The potential role(s) of DNA topoiosmerase II (topo II) during chromatin changes that characterize different stages of spermatogenesis was investigated in the rat by an analysis of the expression and localization of topo II mRNA and protein in individual spermatogenic cells. Expression of topo II was restricted to spermatogonia, spermatocytes, and round and early-elongating spermatids. Two protein bands of 177 and 170 kDa were detected in immunoblots of spermatocytes and round spermatids, while bands of 148 and 142 kDa were prominent in preparations of elongating spermatids. Topo II levels and distribution patterns, as observed by immunofluorescent microscopy, exhibited cell type-specific variations. Differences in topo II staining patterns were also apparent when nuclear matrices of spermatogenic cells were prepared with different extraction conditions. In addition to its possible function as a structural component, topo II, associated with nuclear matrix preparations from spermatogenic cells, possessed catalytic activity. These observations indicate that both the 177 and 170 kDa and the 148 and 142 kDa forms of topo II share similar structural and functional properties. Topo IIβ mRNA was transcribed in rat spermatogenic cells at 6.2 kb. Relative levels of topo IIβ mRNA were high in spermatogonia and spermatocytes, and decreased in both round and early-elongating spermatids. Changes in topo II expression levels and localization patterns represent distinct stage-specific markers for the maturation of spermatogenic cells, and are consistent with the involvement of topo II in mediating DNA modifications and chromatin changes during spermatogenesis. © 1996 Wiley-Liss, Inc.     

Gamma-ray exposure accelerates spermatogenesis of medaka fish,Oryzias latipes

To examine the spermatogenesis (and spermiogenesis) cell population kinetics after gamma-irradiation, the frequency and fate of BrdU-labeled pre-meiotic spermatogenic cells (spermatogonia and pre-leptotene spermatocytes) and spermatogonial stem cells (SSCs) of the medaka fish (Oryzias latipes) were examined immunohistochemically and by BrdU-labeling. After 4.75 Gy of gamma-irradiation, a statistically significant decrease in the frequency of BrdU-labeled cells was detected in the SSCs, but not in pre-meiotic spermatogenic cells. The time necessary for differentiation of surviving pre-meiotic spermatogenic cells without delay of germ cell development was shortened. More than 90% of surviving pre-meiotic spermatogenic cells differentiated into haploid cells within 5 days after irradiation, followed by a temporal spermatozoa exhaust in the testis. Next, spermatogenesis began in the surviving SSCs. However, the outcome was abnormal spermatozoa, indicating that accelerated maturation process led to morphological abnormalities. Moreover, 35% of the morphologically normal spermatozoa were dead at day 6. Based on these results, we suggest a reset system; after irradiation most surviving spermatogenic cells, except for the SSCs, are prematurely eliminated from the testis by spermatogenesis (and spermiogenesis) acceleration, and subsequent spermatogenesis begins with the surviving SSCs, a possible safeguard against male germ cell mutagenesis.     

Loss of TSLC1 causes male infertility due to a defect at the spermatid stage of spermatogenesis

Tumor suppressor of lung cancer 1 (TSLC1), also known as SgIGSF, IGSF4, and SynCAM, is strongly expressed in spermatogenic cells undergoing the early and late phases of spermatogenesis (spermatogonia to zygotene spermatocytes and elongating spermatids to spermiation). Using embryonic stem cell technology to generate a null mutation of Tslc1 in mice, we found that Tslc1 null male mice were infertile. Tslc1 null adult testes showed that spermatogenesis had arrested at the spermatid stage, with degenerating and apoptotic spermatids sloughing off into the lumen. In adult mice, Tslc1 null round spermatids showed evidence of normal differentiation (an acrosomal cap and F-actin polarization indistinguishable from that of wild-type spermatids); however, the surviving spermatozoa were immature, malformed, found at very low levels in the epididymis, and rarely motile. Analysis of the first wave of spermatogenesis in Tslc1 null mice showed a delay in maturation by day 22 and degeneration of round spermatids by day 28. Expression profiling of the testes revealed that Tslc1 null mice showed increases in the expression levels of genes involved in apoptosis, adhesion, and the cytoskeleton. Taken together, these data show that Tslc1 is essential for normal spermatogenesis in mice.