Heat shock protein 2 promoter drives cre expression in spermatocytes of transgenic mice

We generated transgenic mouse line C57BL/6‐Tg(Hspa2‐cre)1Eddy/J (Hspa2‐cre), which expresses cre‐recombinase under the control of a 907‐bp fragment of the heat shock protein 2 (Hspa2) gene promoter. Transgene expression was determined using Gt(ROSA)26Sor^tm1Sor/J (ROSA26) and Tg(CAG‐Bgeo/GFP)21Lbe/J (Z/EG) reporter strains and RT‐PCR and immunohistochemistry assays. Hspa2‐cre expression mimicked the spermatogenic cell‐specific expression of endogenous HSPA2 within the testis, being first observed in leptotene/zygotene spermatocytes. Expression of the transgene also was detected at restricted sites in the brain, as occurs for endogenous HSPA2. Although the results of mating the Hspa2‐cre mice to mice with a floxed Cdc2a allele indicated that some expression of the transgene occurs during embryogenesis, the Hspa2‐cre mice provide a valuable new tool for assessing the roles of genes during and after meiotic prophase in pachytene spermatocytes. genesis 48:114–120, 2010. Published 2009 Wiley‐Liss, Inc.     

A rat H1t‐GFP transgene recapitulates endogenous H1t expression pattern in mouse

H1 histones bind to linker DNA. H1t (H1f6), a testis‐specific linker histone variant, is present in pachytene spermatocytes and spermatids. The expression of H1t histone coincides with the acquisition of metaphase I competence in pachytene spermatocytes. Here we report the generation of H1t‐GFP transgenic mice. The H1t‐GFP (H1 histone testis‐green fluorescence protein) fusion protein expression recapitulates the endogenous H1t expression pattern. This protein appears first in mid pachytene spermatocytes in stage V seminiferous tubules, persists in round spermatids and elongating spermatids, but is absent in elongated spermatids. The strong green fluorescence signal, due to the high abundance of H1t‐GFP, is maintained in spermatocytes after induction towards metaphase I through treatment with okadaic acid. Therefore, H1t‐GFP can be used as a visual marker for monitoring the progression of meiosis in vitro and in vivo, as well as fluorescence‐activated cell sorting (FACS) sorting of germ cells.     

Transgenic Stra8-EYFP pigs: a model for developing male germ cell technologies

The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state. Towards this end, we have produced transgenic pigs expressing mitochondrial localized enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell specific Stimulated by Retinoic Acid 8 (Stra8) promoter. Stra8 has been shown to be expressed in pre-meiotic germ cells of mice. Twelve clones harboring the Stra8-EYFP-mito transgene were produced. Analysis by Western blot indicated that expression of the transgene was limited to testicular tissue in the transgenic pigs. Single cells and seminiferous tubules were cultured in vitro and subsequently examined with epifluorescent microscopy. Expression of EYFP was noted in cells cultured for up to 5 days. Both EYFP-mito and STRA8 antibodies were shown to bind and co-localize in seminiferous tubule cells in whole mounts and in histological sections. EYFP-mito in the transgenic pigs co-localized with the endogenous stem cell marker, NANOG. Expression of the Stra8-EYFP transgene in spermatogenic cells indicates that these pigs will be useful by providing labelled cells for use in such technologies such as germ cell transplantation and in vitro spermatogenic studies.     

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.     

Ablation of Ggnbp2 impairs meiotic DNA double‐strand break repair during spermatogenesis in mice

Gametogenetin (GGN) binding protein 2 (GGNBP2) is a zinc finger protein expressed abundantly in spermatocytes and spermatids. We previously discovered that Ggnbp2 resection caused metamorphotic defects during spermatid differentiation and resulted in an absence of mature spermatozoa in mice. However, whether GGNBP2 affects meiotic progression of spermatocytes remains to be established. In this study, flow cytometric analyses showed a decrease in haploid, while an increase in tetraploid spermatogenic cells in both 30‐ and 60‐day‐old Ggnbp2 knockout testes. In spread spermatocyte nuclei, Ggnbp2 loss increased DNA double‐strand breaks (DSB), compromised DSB repair and reduced crossovers. Further investigations demonstrated that GGNBP2 co‐immunoprecipitated with a testis‐enriched protein GGN1. Immunofluorescent staining revealed that both GGNBP2 and GGN1 had the same subcellular localizations in spermatocyte, spermatid and spermatozoa. Ggnbp2 loss suppressed Ggn expression and nuclear accumulation. Furthermore, deletion of either Ggnbp2 or Ggn in GC‐2spd cells inhibited their differentiation into haploid cells in vitro. Overexpression of Ggnbp2 in Ggnbp2 null but not in Ggn null GC‐2spd cells partially rescued the defect coinciding with a restoration of Ggn expression. Together, these data suggest that GGNBP2, likely mediated by its interaction with GGN1, plays a role in DSB repair during meiotic progression of spermatocytes.     

Developmental changes in cyclin B1 and cyclin-dependent kinase 1 (CDK1) levels in the different populations of spermatogenic cells of the post-natal rat testis

Spermatogenesis is a highly ordered process which requires mitotic and meiotic divisions. In this work, we studied the relative changes in the levels of the two components of the M-phase promoting factor (MPF): the regulatory subunit cyclin B1 (CycB1) and its catalytic subunit cdk1, in spermatogenic cells of rats between 16 and 90 days of life. A multivariate flow cytometry analysis of forward scatter (FSC), side scatter (SSC) and DNA content was used to identify six populations of rat germ cells: spermatogonia with preleptotene spermatocytes, young pachytene spermatocytes, middle to late pachytene spermatocytes, secondary spermatocytes with doublets of round spermatids, round spermatids, and elongated spermatids. For any population studied no significant difference in the relative cellular content of CycB1 or cdk1 proteins between animals of different ages was observed. By contrast, CycB1 and cdk1 levels were different between the different populations of germ cells. CycB1 and cdk1 were rather high in young pachytene spermatocytes and culminated in late spermatocytes, i.e. just before the first meiotic division. The relative levels of the two proteins remained high in secondary spermatocytes then decreased in round spermatids at the exit of meiosis. Similar results were obtained by Western-blot analysis of total proteins obtained from lysates of elutriated fractions of spermatocytes and spermatids. MPF activity was assessed in lysates of germ cells from 32-day-old rats or adult animals using p13suc1 agarose and histone H1 as an exogenous substrate. H1 kinase activity was higher in pachytene spermatocytes than in round spermatid fractions from both adult and young rats. These results indicate that the meiotic G2/M transition is associated to high levels of CycB1 and cdk1 leading to high MPF activity irrespective of the age of the animals.     

兔精子发生过程中cyclin B1基因表达和蛋白定位的发育阶段依赖性

利用原位杂交和免疫组化等方法,研究兔精子发生过程中生精细胞cyclin B1 mRNA的表达和蛋白定位特点,结果显示,兔生精上皮中Cyclin B1 mRNA的主要分布在初级精母细胞中,直至圆形精子细胞仍然存在,于精子细胞的变态过程中逐渐消失,在伸长的精子细胞和精子中未检测出cyclin B1 mRNA,Cyclin B1蛋白在进入分裂期的精原细胞和精母细胞中表达,在圆形精子细胞和伸长的精子细胞中呈现大量的cyclin B1蛋白,上述结果表明,在兔精子发生过程中,cyclin B1 mRNA表达和蛋白定位具有发育阶段依赖性的特征。     

Postnatal male germ‐cell expression of cre recombinase in Tex101‐iCre transgenic mice

We have generated a transgenic mouse line that expresses improved Cre recombinase (iCre) under the control of the testis‐expressed gene 101 (Tex101) promoter. This transgenic mouse line was named Tex101‐iCre. Using the floxed ROSA reporter mice, we found that robust Cre recombinase activity was detected in postnatal testes with weak or no activity in other tissues. Within the testis, Cre recombinase was active in spermatogenic cells as early as the prospermatogonia stage at day 1 after birth. In 30‐ and 60‐day‐old mice, positive Cre recombinase activity was detected not only in prospermatogonia but also in spermatogenic cells at later stages of spermatogenesis. There was little or no Cre activity in interstitial cells. Breeding wild‐type females with homozygous floxed fibroblast growth factor receptor 2 (Fgfr2) males carrying the Tex101‐iCre transgene did not produce any progeny with the floxed Fgfr2 allele. All the progeny inherited a recombined Fgfr2 allele, indicating that complete deletion of the floxed Fgfr2 allele by Tex101‐iCre can be achieved in the male germline. Furthermore, FGFR2 protein was not detected in spermatocytes and spermatids of adult Fgfr2^fl/fl;Tex101‐iCre mice. Taken together, our results suggest that the Tex101‐iCre mouse line allows the inactivation of a floxed gene in spermatogenic cells in adult mice, which will facilitate the functional characterization of genes in normal spermatogenesis and male fertility. genesis 48:717–722, 2010. © 2010 Wiley‐Liss, Inc.     

Differences in DNA double strand breaks repair in male germ cell types: lessons learned from a differential expression of Mdc1 and 53BP1

In male germ cells the repair of DNA double strand breaks (DSBs) differs from that described for somatic cell lines. Irradiation induced immunofluorescent foci (IRIF's) signifying a double strand DNA breaks, were followed in spermatogenic cells up to 16 h after the insult. Foci were characterised for Mdc1, 53BP1 and Rad51 that always were expressed in conjecture with gamma-H2AX. Subsequent spermatogenic cell types were found to have different repair proteins. In early germ cells up to the start of meiotic prophase, i.e. in spermatogonia and preleptotene spermatocytes, 53BP1 and Rad51 are available but no Mdc1 is expressed in these cells before and after irradiation. The latter might explain the radiosensitivity of spermatogonia. Spermatocytes from shortly after premeiotic S-phase till pachytene in epithelial stage IV/V express Mdc1 and Rad51 but no 53BP1 which has no role in recombination involved repair during the early meiotic prophase. Mdc1 is required during this period as in Mdc1 deficient mice all spermatocytes enter apoptosis in epithelial stage IV when they should start mid-pachytene phase of the meiotic prophase. From stage IV mid pachytene spermatocytes to round spermatids, Mdc1 and 53BP1 are expressed while Rad51 is no longer expressed in the haploid round spermatids. Quantifying foci numbers of gamma-H2AX, Mdc1 and 53BP1 at various time points after irradiation revealed a 70% reduction after 16 h in pachytene and diplotene spermatocytes and round spermatids. Although the DSB repair efficiency is higher then in spermatogonia where only a 40% reduction was found, it still does not compare to somatic cell lines where a 70% reduction occurs in 2 h. Taken together, DNA DSBs repair proteins differ for the various types of spermatogenic cells, no germ cell type possessing the complete set. This likely leads to a compromised efficiency relative to somatic cell lines. From the evolutionary point of view it may be an advantage when germ cells die from DNA damage rather than risk the acquisition of transmittable errors made during the repair process.     

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.     

Murine polo like kinase 1 gene is expressed in meiotic testicular germ cells and oocytes

To identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding region shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythroleukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the PlK1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage. © 1995 Wiley-Liss, Inc.     

DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells.

Genomic methylation patterns are established during maturation of primordial germ cells and during gametogenesis. While methylation is linked to DNA replication in somatic cells, active de novo methylation and demethylation occur in post-replicative spermatocytes during meiotic prophase (1). We have examined differentiating male germ cells for alternative forms of DNA (cytosine-5)-methyltransferase (DNA MTase) and have found a 6.2 kb DNA MTase mRNA that is present in appreciable quantities only in testis; in post-replicative pachytene spermatocytes it is the predominant form of DNA MTase mRNA. The 5.2 kb DNA MTase mRNA, characteristic of all somatic cells, was detected in isolated type A and B spermatogonia and haploid round spermatids. Immunobolt analysis detected a protein in spermatogenic cells with a relative mass of 180,000-200,000, which is close to the known size of the somatic form of mammalian DNA MTase. The demonstration of the differential developmental expression of DNA MTase in male germ cells argues for a role for testicular DNA methylation events, not only during replication in premeiotic cells, but also during meiotic prophase and postmeiotic development.