DNA Double-Strand Breaks and γ-H2AX Signaling in the Testis1

Hamer, Geert; Roepers-Gajadien, Hermien L.; Duyn-Goedhart, A. Van; Gademan, Iris S.; Kal, Henk B.; Buul, Paul P.W. van; Rooij, Dirk G. de

doi:10.1095/biolreprod.102.008672

Cited by 189 publications

(170 citation statements)

References 45 publications

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“…Phoshorylated γH2AX‐histone protein is associated with both DNA double strand breaks and sex vesicle formation in the testis (Hamer et al ., 2003). It is expressed in a subset of the diploid spermatogonia, step 11–14 haploid spermatids and tetraploid spermatocytes (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…It is expressed in a subset of the diploid spermatogonia, step 11–14 haploid spermatids and tetraploid spermatocytes (Fig. 5A) (Hamer et al ., 2003; Meyer‐Ficca et al ., 2005). FxCycle DNA stain was used to detect the DNA content of the cells in the γH2AX‐S139 assay.…”

Section: Resultsmentioning

confidence: 99%

“…A γH2AX‐Ser139 signal can be detected in intermediate and B spermatogonia, zygotene and pachytene spermatocytes and step 11–14 elongated spermatids (Hamer et al ., 2003; Meyer‐Ficca et al ., 2005). In the adult rat testis, the γH2AX‐positive cell clustered as expected based on the immunohistochemical staining and literature.…”

Section: Discussionmentioning

confidence: 99%

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A detailed protocol for a rapid analysis of testicular cell populations using flow cytometry

et al. 2015

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SummaryAccurate analysis and quantification of different testicular cell populations are of central importance in studies of male reproductive biology. The traditional histomorphometric and immunohistochemical methods remain the gold standard in studying the complex dynamics of the testicular tissue. Through past years advances have been made in the application of flow cytometry for the rapid analysis of testicular cell populations. Detection of DNA content and of surface antigens and fluorescent reporters have been widely used to analyze and sort cells. Detection of intracellular antigens can broaden the possibilities of applying flow cytometry in studies of male reproduction. Here, we report a detailed protocol for the preparation of rat testicular tissue for detection of intracellular antigens by flow cytometry, and a pipeline for subsequent data analysis and troubleshooting. Rat testicular ontogenesis was chosen as the experimental model to validate the performance of the assay using vimentin and γH2AX as intracellular markers for the somatic and spermatogenic cells, respectively. The results show that the assay is reproducible and recapitulates the rat testis ontogenesis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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A detailed protocol for a rapid analysis of testicular cell populations using flow cytometry

et al. 2015

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“…Here, we chose S139‐phophorylated γH2AX (pH2AX) as a proxy for DNA damage and meiotic DSB repair. When spermatocytes accumulate DNA damage due to ionizing radiation or chemotherapeutics, distinct foci of positive pH2AX signal can be observed in the nuclei outside the sex body (Hamer et al ., 2003) and the levels of pH2AX can be used to assess the faithfulness of the meiotic recombination. There were no alterations in the pH2AX staining pattern in the E2F1 −/− ‐testis at P40 compared with E2F1 +/+ , which indicated that increased DNA damage was not the source of the spermatocyte apoptosis.…”

Section: Discussionmentioning

confidence: 99%

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis

et al. 2015

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SummaryCell cycle control during spermatogenesis is a highly complex process owing to the control of the mitotic expansion of the spermatogonial cell population and following meiosis, induction of DNA breaks during meiosis and the high levels of physiological germ‐cell apoptosis. We set out to study how E2F1, a key controller of cell cycle, apoptosis, and DNA damage responses, functions in the developing and adult testis. We first analyzed the expression pattern of E2f1 during post‐natal testis development using RNA in situ hybridization, which showed a differential expression pattern of E2f1 in the adult and juvenile mouse testes. To study the function of E2f1, we took advantage of the E2F1−/− mouse line, which was back‐crossed to C57Bl/6J genetic background. E2f1 loss led to a severe progressive testicular atrophy beginning at the age of 20 days. Spermatogonial apoptosis during the first wave of spermatogenesis was decreased. However, already in the first wave of spermatogenesis an extensive apoptosis of spermatocytes was observed. In the adult E2F1−/− testes, the atrophy due to loss of spermatocytes was further exacerbated by loss of spermatogonial stem cells. Surprisingly, only subtle changes in global gene expression array profiling were observed in E2F1−/− testis at PND20. To dissect the changes in each testicular cell type, an additional comparative analysis of the array data was performed making use of previously published data on transcriptomes of the individual testicular cell types. Taken together, our data indicate that E2F1 has a differential role during first wave of spermatogenesis and in the adult testis, which emphasizes the complex nature of cell cycle control in the developing testis.

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“…Phosphorylated H2AX (g-H2AX) foci are markers of DNA double strand breaks [Rogakou et al, 1998]. g-H2AX content in testicular cells is heterogeneous, higher than in cells from other organs and increases after irradiation [Hamer et al, 2003;Ahmed et al, 2007Ahmed et al, , 2010aBlanco-Rodriguez, 2009;Paris et al, 2011]. The presence of H2AX has been documented in human [Gatewood et al, 1990] and in mouse [Meyer-Ficca et al, 2009] sperm chromatin.…”

Section: Discussionmentioning

confidence: 99%

Direct and delayed X‐ray‐induced DNA damage in male mouse germ cells

Cordelli¹,

Eleuteri²,

Grollino³

et al. 2012

Environ and Mol Mutagen

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Sperm DNA integrity is essential for the accurate transmission of paternal genetic information. Various stages of spermatogenesis are characterized by large differences in radiosensitivity. Differentiating spermatogonia are susceptible to radiationinduced cell killing, but some of them can repair DNA damage and progress through differentiation. In this study, we applied the neutral comet assay, immunodetection of phosphorylated H2AX (g-H2AX) and the Sperm Chromatin Structure Assay (SCSA) to detect DNA strand breaks in testicular cells and spermatozoa at different times following in vivo X-ray irradiation. Radiation produced DNA strand breaks in testicular cells that were repaired within the first few hours after exposure. Spermatozoa were resistant to the induction of DNA damage, but non-targeted DNA lesions were detected in spermatozoa derived from surviving irradiated spermatogonia. These lesions formed while round spermatids started to elongate within the testicular seminiferous tubules. The transcription of pro-apoptotic genes at this time was also enhanced, suggesting that an apoptotic-like process was involved in DNA break production. Our results suggest that proliferating spermatogonia retain a memory of the radiation insult that is recognized at a later developmental stage and activates a process leading to DNA fragmentation. Environ. Mol. Mutagen. 53:429-439, 2012. V V C 2012 Wiley Periodicals, Inc.

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DNA Double-Strand Breaks and γ-H2AX Signaling in the Testis1

Cited by 189 publications

References 45 publications

A detailed protocol for a rapid analysis of testicular cell populations using flow cytometry

A detailed protocol for a rapid analysis of testicular cell populations using flow cytometry

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis

Direct and delayed X‐ray‐induced DNA damage in male mouse germ cells

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