Comparison of radiation-and chemically-induced dominant lethal mutations in male mice

Ionizing radiation-induced dominant lethal mutations in all spermatogenic stages. After irradiation of male mice with 200 R the yield of induced mutations in early spermatids was twice the yield in spermatozoa, late spermatids, and spermatocytes. After irradiation with 400 R or 800 R the spermatocytes were the most sensitive stage for the induction of dominant lethal mutations. The frequency of radiation-induced dominant lethal mutations in postspermatogonial stages was dose-dependent. The yield of dominant lethal mutations in spermatogonia was independent of the dose.     

Reaction of the chromatoid body with a monoclonal antibody to a rat histocompatibility antigen

The monoclonal antibody OX3 against a polymorphic class II antigen encoded by the major histocompatibility locus of the rat has been shown to cross-react with the chromatoid body during spermatogenesis. Using an indirect immunofluorescence assay on frozen, fixed testis sections, the antibody revealed a pattern of fluorescent speckling that correlated with specific stages of spermatogenesis. The positive material first appeared in late pachytene spermatocytes as multiple small spots. Larger dots appeared in all regions containing round spermatids, but, as the spermatids matured, only fine dots were seen. Mature spermatids were negative, as were all early cells (spermatogonia to early pachytene spermatocytes). When suspension of fixed testicular cells were tested, the activity was clearly associated with the chromatoid body adjacent to the nucleus in round spermatids and with multiple smaller structures encircling the nucleus in primary spermatocytes. These associations were confirmed in observations on immature testes at various ages. No reactivity was seen in testes of animals whose testes had previously been irradiated to render them aspermatogenic, nor in grc/grc rats in which spermatogenesis is arrested at the primary spermatocyte stage. Because the expression of this reactivity was seen even in rats that do not express the OX3 antigen on their somatic cells, this antibody should prove useful in determining the structure of this body, its origin and fate, and any possible role it may have in spermiogenesis.     

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.     

The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.     

Testicular phenotype in luteinizing hormone receptor knockout animals and the effect of testosterone replacement therapy

The LH receptor knockout model, developed in our laboratory, was used in determining what FSH alone can do in the absence of LH signaling and whether any of the testicular LH actions are not mediated by androgens. The results revealed that null animals contained smaller seminiferous tubules, which contained the same number of Sertoli cells, spermatogonia, and early spermatocytes as wild-type siblings. The number of late spermatocytes, on the other hand, was moderately decreased, the number of round spermatids was dramatically decreased, and elongated spermatids were completely absent. These changes appear to be due to an increase in apoptosis in spermatocytes. While the number of Leydig cells progressively increased from birth to 60 days of age in wild-type animals, they remained unchanged in null animals. Consequently, 60-day-old null animals contained only a few Leydig cells of fetal type. The age-dependent increase in testicular macrophages lagged behind in null animals compared with wild-type siblings. Orchidopexy indicated that -/- testicular phenotype was not due to abdominal location. Rather, it was mostly due to androgen deficiency, as 21-day testosterone replacement therapy stimulated the growth of seminiferous tubules, decreased apoptosis, and increased the number of late spermatocytes and round spermatids and their subsequent differentiation into mature sperm. The therapy, however, failed to restore adult-type Leydig cells and testicular macrophage numbers to the wild-type levels. In summary, our data support the concept that FSH signaling alone can maintain the proliferation and development of Sertoli cells, spermatogonia, and early spermatocytes. LH actions mediated by testosterone are required for completion of spermatogenesis, and finally, androgen-independent actions of LH are required for the formation of adult-type Leydig cells and recruitment of macrophages into the testes.     

Biosynthesis and localization of lactate dehydrogenase X in pachytene spermatocytes and spermatids of mouse testes

The presence and biosynthesis of the testis-specific isozyme of lactate dehydrogenase (LDH-X) in cells at various stages of spermatogenesis have been examined. Enrichment of testicular cells in various stages of spermatogenesis has been achieved by two methods: (1) cell separation by velocity sedimentation in the Elutriator rotor and (2) γ irradiation of testes to eliminate specific classes of testicular cells. Separation of cells from immature mice indicated that cells prior to the midpachytene stage contain no LDH-X. Measurement of LDH-X levels in cells separated from adult mice and in testicular homogenates prepared at various times after irradiation indicated that the highest level of LDH-X per cell (normalized for DNA content) was in spermatids. Synthesis of LDH-X was determined, after in vivo injection of [³H]valine, by measurement of the radioactivity in LDH-X precipitated with specific antiserum. After irradiation, the rate of LDH-X synthesis remained constant, despite the loss of early primary spermatocytes. In separated cells, the rate of LDH-X synthesis was highest in late pachytene spermatocytes, lower in round spermatids, and even lower, but still significant, in elongated spermatids. Therefore, the synthesis of LDH-X begins at a specific point during spermatogenesis, the midpachytene stage of spermatocyte development, and continues throughout spermatid differentiation.     

Ionizing radiation-induced mutant frequencies increase transiently in male germ cells of older mice

Spontaneous mutant frequency in the male germline increases with age, thereby increasing the risk of siring offspring with genetic disorders. In the present study we investigated the effect of age on ionizing radiation-induced male germline mutagenesis. lacI transgenic mice were treated with ionizing radiation at 4-, 15- and 26-month-old, and mutant frequencies were determined for pachytene spermatocytes and round spermatids at 15 days or 49 days after ionizing radiation treatment. Cells collected 15 days after treatment were derivatives of irradiated differentiating spermatogenic cells while cells collected 49 days later were derivatives of spermatogonial stem cells. The results showed that (1) spontaneous mutant frequency increased in spermatogenic cells recovered from nonirradiated old mice (26-months-old), particularly in the round spermatids; (2) mutant frequencies were significantly increased in round spermatids obtained from middle-aged mice (15-months-old) and old age mice (26-months-old) at 15 and 49 days after irradiation compared to the sham-treated old mice; and (3) pachytene spermatocytes obtained from 15- or 26-month-old mice displayed a significantly increased mutant frequency at 15 days post irradiation. This study indicates that age modulates the mutagenic response to ionizing radiation in the male germline.     

DNA double-strand breaks and gamma-H2AX signaling in the testis

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms gamma-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These gamma-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, gamma-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit gamma-H2AX foci but show homogeneous nuclear gamma-H2AX staining, whereas in pachytene spermatocytes gamma-H2AX is only present in the sex vesicle. In response to ionizing radiation, gamma-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, gamma-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear gamma-H2AX staining in leptotene spermatocytes demonstrates a function for gamma-H2AX during meiosis. gamma-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA double-strand breaks.     

Rat germinal cells require PARP for repair of DNA damage induced by gamma-irradiation and H2O2 treatment

The ability of rat germinal cells to recover from genotoxic stress has been investigated using isolated populations of primary spermatocytes and round spermatids. Using a comet assay at pH 10.0 to assess single strand breakage (SSB) in DNA, it was found that a high level of damage was induced by 5 Gy gamma-irradiation and acute exposure to 50 microM H2O2. This damage was effectively repaired during a subsequent recovery period of 1-3 hours culture in vitro but repair was significantly delayed in the presence of the poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide (3-ABA). Immunofluorescence detection of PARP with specific antibodies localised the protein to discrete foci within the nucleus of both spermatocytes and spermatids. Poly(ADP-ribose) (pADPR) could also be detected in spermatid nuclei following gamma-irradiation or H2O2 treatment. Moreover, PARP activation occurs both in spermatocytes and spermatids left to recover after both genotoxic stresses. The NO donors, 3-morpholino-sydnonimine (SIN-1) and S-nitrosoglutathione (SNOG), caused significant SSBs in both spermatocytes and spermatids. The effects of SIN-1 could be prevented by exogenous catalase (CAT), but not superoxide dismutase (SOD), in the cell suspensions. SNOG-induced SSBs were insensitive to both CAT and SOD. It is concluded that DNA in spermatocytes and spermatids is sensitive to damage by gamma-irradiation and H2O2 and that efficient repair of SSBs requires PARP activity.     

Immunohistochemical localization of the calcium/calmodulin-dependent protein phosphatase,calcineurin, in the mouse testis: its unique accumulation in spermatid nuclei

Immunohistochemical localization of a calmodulin-dependent protein phosphatase, calcineurin, was studied in the mouse testis in relation to previous observations showing that calmodulin is unusually rich in spermatogenic stages from mid-pachytene spermatocytes to elongating spermatids. The antibodies raised against calcineurin from scallop testis reacted with subunit B, but not subunit A, of calcineurin isoforms from mouse brain and testis. Indirect immunofluorescence using these antibodies on the mouse testis revealed positive reactions only in the nuclei of round or elongating spermatids: calcineurin started to accumulate in nuclei from the acrosomal cap phase, peaked at the initial stage of nuclear elongation, and decreased thereafter. There was almost no signal in the cytoplasm; spermatogenic cells at other stages, including spermatogonia, spermatocytes, mature sperm, and other somatic cells in the seminiferous tubules were totally negative. Immuno-electron microscopy gave the same result, on the basis of measuring the density of immunogold particles. These results suggest a role for calcineurin in remodeling of the nuclear chromatin in metamorphosing spermatids.     

Rat spermatogenic cell beta-D-galactosidase: characterization, biosynthesis, and immunolocalization

In this study we have demonstrated that the rat sperm acrosomal beta-d-galactosidase is expressed in late spermatocytes and spermatids (round, elongated/condensed) during spermatogenesis. The enzyme is an exoglycohydrolase which, along with other exoglycohydrolases and proteases, is thought to aid in penetration of the zona pellucida, the extracellular glycocalyx that surrounds the mammalian egg. The presence of the enzyme in spermatocytes was confirmed by multiple approaches using biochemical, biosynthetic, and immunohistochemical protocols. The germ cells (spermatocytes, round spermatids, and elongated/condensed spermatids), purified from rat testis, were found to contain beta-galactosidase and four other glycohydrolases (beta-d-glucuronidase, alpha-d-mannosidase, alpha-l-fucosidase, and beta-N-acetylglucosaminidase). With the exception of alpha-l-fucosidase, the other enzymes assayed demonstrated a two- to threefold higher activity per cell in spermatocytes than in round spermatids. Immunoblotting approaches of affinity-purified germ cell extracts demonstrated several molecular forms of beta-galactosidase in spermatocytes and round spermatids; one of these forms (62 kDa) was seen only in round spermatids. The biosynthetic approach demonstrated that the enzyme is synthesized in spermatocytes and round spermatids in culture in high-molecular-weight precursor forms (90/88 kDa) which undergo processing to lower molecular weight mature forms in a cell-specific manner. The net result is the formation of predominantly 64- and 62-kDa forms in spermatocytes and round spermatids, respectively. The conversion of precursor forms to mature forms in the diploid and haploid cells in culture is rapid with t(1/2) of 6.5 and 9.0 h, respectively. Immunohistochemical approaches revealed an immunopositive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosome-like structures in the late spermatocytes and early round spermatids. The forming/formed acrosome in round and elongated spermatids was also immunoreactive.     

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.     

Effects of X-irradiation on the kinetics of abnormal sperm production and sperm loss in the mouse

Exposure of male mice to 6 Gy of X-rays resulted in a very rapid and extensive sloughing of the germinal epithelium as shown by the accumulation of non-sperm cells within the lumen of the epididymis. These cells were identified as stage 1 and 2 round spermatids. After accumulating in the caput, they progressed through the epididymis over the weeks of sampling and, by Week 9 after irradiation, they had completely disappeared from the organ. It is suggested that the precocious loss of round spermatids is responsible for the induction of oligospermy within the testis and the caput epididymidis. Similar sperm losses from the cauda epididymidis were not observed. Radiation also enhanced the frequency of misshapen spermatozoa normally found in this strain. From kinetic considerations, it is suggested that the generation of abnormal spermatozoa may be biphasic with an early component comprising maturing spermatids and a late contingent composed of affected spermatocytes. Return to the pre-irradiation level of abnormal frequency was not observed within the time frame of this study (10 weeks), perhaps indicating residual damage. The synchrony that existed among the various organs in terms of both sperm loss and the generation of abnormal spermatozoa may be the result of a rapid dispersion of gametes from the testis and not due to local responses as would be expected if sperm flow were affected by the irradiation. The distribution of abnormal sperm types was different in the testis from that in the epididymis, presumably because of a testicular spermatophagic mechanism specific for the removal of certain deformities. It is concluded that the kinetics of spermatogenesis, of spermiogenesis, and of sperm transport in the mouse is not affected by exposure to 6 Gy of X-rays.     

beta-Nerve growth factor participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes

NGF appears to be involved in spermatogenesis. However, mice lacking NGF or TrkA genes do not survive more than a few days whereas p75(NTR) knockout mice are viable and fertile. Therefore, we addressed the effect of betaNGF on spermatogenesis by using the systems of rat germ cell culture we established previously. betaNGF did not modify the number of Sertoli cells, pachytene spermatocytes, secondary spermatocytes nor the half-life of round spermatids, but increased the number of secondary meiotic metaphases and decreased the number of round spermatids formed in vitro. These effects of betaNGF were reversible and maximal at about 4 x 10(-11) M. Conversely, K252a, a Trk-specific kinase inhibitor, enhanced the number of round spermatids above that of control cultures. The presence of betaNGF and its receptors TrkA and p75(NTR) was investigated in testis sections, in Sertoli cell and germ cell fractions, and in germ cell and Sertoli cell co-cultures. betaNGF was detected only in germ cells from pachytene spermatocytes of stages VII up to spermatids of stages IX-X. TrkA and p75(NTR) were detected in Sertoli cells and in these germ cells. Taken together, these results indicate that betaNGF should participate in an auto/paracrine pathway of regulation of the second meiotic division of rat spermatocytes in vivo.     

Micronuclei and chromosome aberrations in Xenopus laevis spermatocytes and spermatids exposed to adriamycin and colcemid

Cultured testes and spermatocytes from the frog Xenopus laevis have been incubated (40-42 h) with adriamycin or colcemid followed by quantitation of chromosome aberrations in secondary spermatocytes and quantitation of micronuclei in secondary spermatocytes, early round spermatids, and round spermatids with acrosomal vacuoles (AV) at 18-162 h of culture. Micronucleus frequencies were consistently higher in secondary spermatocytes relative to round spermatids after exposure to either adriamycin or colcemid due to a higher rate of micronucleus formation during meiosis I compared to meiosis II. Also, some of the micronuclei formed during meiosis I did not survive meiosis II to form micronucleated spermatids. Micronucleus formation occurred in 3-7% of secondary spermatocytes with detectable chromosome aberrations, depending upon drug treatment. Thus, the ratio of micronuclei to total chromosome aberrations in secondary spermatocytes was always higher in colcemid-treated cells compared to adriamycin-treated cells following 18- and 42-h treatment periods. Adriamycin induced significant increases in micronuclei in both secondary spermatocytes and spermatids after 162 h of culture, the time for initial pachytene stages to develop into secondary spermatocytes and spermatids. The data show that cultured testes and spermatocytes from Xenopus may be used to quantify specific meiotic chromosome aberrations induced by both clastogens and spindle poisons using either a rapid secondary spermatocyte micronucleus assay or meiotic chromosome analysis.     

Stage specific effect during one seminiferous epithelial cycle following ethylene glycol monomethyl ether exposure in rats.

Stage specific effect of single oral dose (500 mg/kg body wt) of ethylene glycol monomethyl ether (EGME) was characterised during one cycle of seminiferous epithelium in rats. Maximum peritubular membrane damage and germinal epithelial distortion were observed at stages IX-XII. Cell death occurred during conversion of zygotene to pachytene spermatocytes (stage XIII) and between dividing spermatocytes and step I spermatids (stage late XIII-XIV). Profound effect was noted during first meiotic division than during second meiotic division. Presence of multinucleated secondary spermatocytes indicated cytokinesis arrest. The spermatogenesis was delayed and consequently frequency of tubules at stages I-VIII was reduced by day 10. Many of the tubules were devoid of round spermatids on day 12. Possibly, EGME (or it's metabolite) distorted the barrier system at stages IX-XIV and damaged the cells mostly at stages XII-early XIV.     

Rat spermatogenesis in vitro traced by quantitative flow cytometry

In vitro differentiation of germ cells in rat seminiferous tubule segments at stages II-III of the epithelial cycle was studied. DNA flow cytometry was used for quantitation of absolute cell numbers from the cultured tubule segments that were compared to freshly isolated stages of the cycle, as identified by transillumination stereomicroscopy of the seminiferous tubules and phase-contrast microscopy of live cell squashes. Spermatogonia and spermatocytes from stages II-III showed normal morphological differentiation during 7 days in vitro. Round spermatids differentiated to Step 7 of spermiogenesis but Step 16 spermatids failed to develop. Acid phosphatase activity in the spermatogenic cells changed normally during the culture. As compared with freshly isolated control tubule segments, 35% of round spermatids and 42% of pachytene spermatocytes were present in culture after 7 days. The cell numbers recovered from defined stages by DNA flow cytometry were close to those found in morphometric studies. Flow cytometry is an efficient quantitation method for cells liberated from seminiferous epithelium. Spermatogonia, spermatocytes, and early spermatids are able to differentiate in vitro, but spermatids approaching the elongation (acrosome) phase, and particularly the maturation phase, fail to differentiate under present culture conditions.     

Parp1–XRCC1 and the repair of DNA double strand breaks in mouse round spermatids

The repair of DNA double strand breaks (DSBs) in male germ cells is slower and differently regulated compared to that in somatic cells. Round spermatids show DSB repair and are radioresistant to apoptosis induction. Mutation induction studies using ionizing irradiation, indicated a high frequency of chromosome aberrations (CA) in the next generation. Since they are in a G1 comparable stage of the cell cycle, haploid spermatids are expected to repair DSBs by the non-homologous end-joining pathway (NHEJ). However, immunohistochemical evidence indicates that not all components of the classical NHEJ pathway are available since the presence of DNA-PKcs cannot be shown. Here, we demonstrate that round spermatids, as well as most other types of male germ cells express both Parp1 and XRCC1. Therefore, we have determined whether the alternative Parp1/XRCC1 dependent NHEJ pathway is active in these nuclei and also have tested for classical NHEJ activity by a genetic method. To evaluate DSB repair in SCID mice, deficient for DNA-PKcs, and to study the involvement of the Parp1/XRCC1 dependent NHEJ pathway in round spermatids, the loss of γ-H2AX foci after irradiation has been determined in nucleus spreads of round spermatids of SCID mice and in nucleus spreads and histological sections of Parp1-inhibited mice and their respective controls. Results show that around half of the breaks in randomly selected round spermatids are repaired between 1 and 8 h after irradiation. The repair of 16% of the induced DSBs requires DNA-PKcs and 21% Parp1. Foci numbers in the Parp1-inhibited testes tend to be higher in spermatids of all epithelial stages reaching significance in stages I–III which indicates an active Parp1/XRCC1 pathway in round spermatids and a decreased repair capacity in later round spermatid stages. In Parp1-inhibited SCID mice only 14.5% of the breaks were repaired 8 h after irradiation indicating additivity of the two NHEJ pathways in round spermatids.