Chronic cyclophosphamide treatment alters the expression of stress response genes in rat male germ cells

Increases in the survival rate of men treated with chemotherapeutic drugs and their desire to have children precipitate concerns about the effects of these drugs on germ cells. Azoospermia, oligospermia, and infertility are common outcomes resulting from treatment with cyclophosphamide, an alkylating agent. Exposure of male rats to cyclophosphamide results in dose-dependent and time-specific adverse effects on progeny outcome. Elucidation of the effects of chronic low-dose cyclophosphamide treatment on the expression of stress response genes in male germ cells may provide insight into the mechanisms underlying such adverse effects. Male rats were gavaged with saline or cyclophosphamide (6 mg/kg) for 4-5 wk; pachytene spermatocytes, round spermatids, and elongating spermatids were isolated; RNA was extracted and probed on cDNA arrays containing 216 cDNAs. After saline treatment, 125 stress response genes were expressed in pachytene spermatocytes (57% of genes studied), 122 in round spermatids (56%), and 83 in elongating spermatids (38%). Cyclophosphamide treatment reduced the number of genes detected in all germ cell types. The predominant effect of chronic cyclophosphamide exposure was to decrease the expression level of genes in pachytene spermatocytes (34% of genes studied), round spermatids (29%), and elongating spermatids (4%). In elongating spermatids only, drug treatment increased the expression of 8% of the genes studied. The expression profiles of genes involved in DNA repair, posttranslational modification, and antioxidant defense in male germ cells were altered by chronic cyclophosphamide treatment. We hypothesize that the effects of cyclophosphamide exposure on germ cell gene expression during spermatogenesis may have adverse consequences on male fertility and progeny outcome.     

Impact of the chemotherapy cocktail used to treat testicular cancer on the gene expression profile of germ cells from male Brown-Norway rats

Advances in treatment for testicular cancer that include the coadministration of bleomycin, etoposide, and cisplatin (BEP) have brought the cure rate to higher than 90%%. The goal of this study was to elucidate the impact of BEP treatment on gene expression in male germ cells. Brown-Norway rats were treated for 9 wk with vehicle (0x) or BEP at doses equivalent to 0.3x and 0.6x the human dose. At the end of treatment, spermatogenesis was affected, showing altered histology and a decreased sperm count; spermatozoa had a higher number of DNA breaks. After 9 wk of treatment, round spermatids were isolated, and RNA was extracted and probed on Rat230-2.0 Affymetrix arrays. Of the 31 099 probe sets present on the array, 59%% were expressed in control round spermatids. BEP treatment significantly altered the expression of 221 probe sets, with at least a 1.5-fold change compared with controls; 80% were upregulated. We observed a dose-dependent increase in the expression of oxidative stress response genes and no change in the expression of genes involved in DNA repair. BEP upregulated genes were implicated in pathways related to Jun and Junb protooncogenes. Increased mRNA levels of Jun and Junb were confirmed by quantitative RT-PCR; furthermore, JUN protein was increased in elongating spermatids. Thus, BEP exposure triggers an oxidative stress response in round spermatids and induces many pathways that may lead to the survival of damaged cells and production of abnormal sperm.     

Activité aromatase dans les cellules germinales mâles: quelle signification fonctionnelle?

The ability of the male gonad to convert androgens into estrogens is well known. According to age, aromatase activity has been already measured in immature and mature rat Leydig cells as well as in Sertoli cells. Recently, in different studies, a cytochrome P450_arom has even been immunolocalized not only in Leydig cells but also in germ cells of mouse, brown bear and rooster whereas in pig, ram and human the aromatase is mainly present in Leydig cells. Our purpose was to investigate the testicular cell distribution of cytochrome P450_arom mRNA in adult rat using RT-PCR. With 2 highly specific primers located on exons 8 and 9, we have been able to amplify a 289 bp aromatase fragment not only in Leydig cells and Sertoli cells but more importantly in highlyenriched preparations of pachytene spermatocytes, round spermatids and testicular spermatozoa. These amplified products showed 100% homology with the corresponding fragment of the rat ovary cDNA. In parallel, using an anti-human cytochrome P450_arom antibody we have demonstrated the presence of a 55 kDa protein in seminiferous tubules and crude germ cell (pachytene spermatocytes and round spermatids) preparation of the mature rat. After incubation with tritiated androstenedione, the aromatase activities in the microsomal fractions were 3.12±0.19 pmoles/mg/h in the testis, 1.25±0.13 in the seminiferous tubules and 1.53±0.15 in the crude germ cells. In purified testicular spermatozoa the aromatase activity was 2.96±0.69 pmoles/mg/h and found to be 5-fold higher when compared to that of either purified pachytene spermatocytes or round spermatids. Using a quantitative RT-PCR method with a standard cDNA 29 bp shorter, we have compared the amount of cytochrome P450_arom mRNA in mature rat Leydig cells and Sertoli cells. In purified Leydig cells from 90 day-old rats the P450_arom mRNA level was: 36.2±3.4×10^?3 amoles/μg RNA whereas in Sertoli cells the mRNA level was 10 fold lower. In pachytene spermatocytes, round spermatids and testicular spermatozoa the P450_arom mRNA levels were re pectively 367.2±76.6, 117.6±22.0 and <1×10^?3 amole/μg RNA. In conclusion we have demonstrated that the P450 aromatase is present not only in Sertoli cells and Leydig cells from mature rat testis but a biologically active aromatase exists also in germ cells (pachytene spermatocytes, round spermatids and spermatozoa). The existence of an additional source of estrogens within the genital tract of the male is now well documented and that suggests a putative role for these hormones during the male germ cell development.     

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.     

Dehydrodolichyl diphosphate synthase in Sertoli and spermatogenic cells of prepuberal rats

The specific activity of 2,3-dehydrodolichyl diphosphate synthase in homogenates of protease-treated seminiferous tubules, enriched spermatogenic cells, and Sertoli cells changed as a function of the age of prepuberal rats. The highest enzymatic activity occurred in each case in 23-day-old rats. Homogenates of pachytene spermatocytes, spermatids, or Sertoli cells had higher synthase activity than a whole testicular homogenate prepared by protease treatment of tubules. Enzymatic activity in pachytene spermatocytes expressed per mg of protein was about 1.7-fold higher than in spermatids, 5.3-fold higher than in spermatogonia, and about 8.3-fold higher than in spermatozoa. Therefore, the increase in spermatogenic cell synthase before day 23 can be accounted for by the appearance of the pachytene spermatocytes. Enzymatic activity decreased remarkably after the differentiation of spermatids into spermatozoa. Synthase activity in enriched Sertoli cell preparations was 1.5-2.3-fold higher than in spermatogenic cell preparations between days 15 and 30. Therefore, both spermatogenic cells and Sertoli cells contribute to changes in the enzymatic activity in seminiferous tubules during development. These changes may be important in regulating the availability of dolichyl phosphate for glycoprotein synthesis during early stages of differentiation.     

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.     

The effect of oxidative stress on nucleotide-excision repair in colon tissue of newborn piglets

Nucleotide-excision repair (NER) is important for the maintenance of genomic integrity and to prevent the onset of carcinogenesis. Oxidative stress was previously found to inhibit NER in vitro, and dietary antioxidants could thus protect DNA not only by reducing levels of oxidative DNA damage, but also by protecting NER against oxidative stress-induced inhibition. To obtain further insight in the relation between oxidative stress and NER activity in vivo, oxidative stress was induced in newborn piglets by means of intra-muscular injection of iron (200 mg) at day 3 after birth. Indeed, injection of iron significantly increased several markers of oxidative stress, such as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) levels in colon DNA and urinary excretion of 8-oxo-7,8-dihydroguanine (8-oxoGua). In parallel, the influence of maternal supplementation with an antioxidant-enriched diet was investigated in their offspring. Supplementation resulted in reduced iron concentrations in the colon (P = 0.004) at day 7 and a 40% reduction of 8-oxodG in colon DNA (P = 0.044) at day 14 after birth. NER capacity in animals that did not receive antioxidants was significantly reduced to 32% at day 7 compared with the initial NER capacity on day 1 after birth. This reduction in NER capacity was less pronounced in antioxidant-supplemented piglets (69%). Overall, these data indicate that NER can be reduced by oxidative stress in vivo, which can be compensated for by antioxidant supplementation.     

Characterization and immunolocalization of beta-D-glucuronidase in mouse testicular germ cells and spermatozoa

Spermatozoa released from the seminiferous tubules are terminally differentiated cells with no known synthetic activity. Their components are synthesized in the spermatogenic cells during spermatogenesis. In this study, we report the characterization and immunolocalization of beta-glucuronidase in mouse testicular germ cells and spermatozoa. The enzyme is an exoglycohydrolase with dual localization, being present in lysosomes and endoplasmic reticulum of several mouse and rat tissues. The purified germ cell preparations (spermatocytes, round spermatids, and condensed/elongated spermatids) when assayed for beta-glucuronidase activity showed that the spermatocytes contained five times more enzyme activity per cell than the spermatids. Polyacrylamide gel electrophoresis, carried out under native and denaturing conditions, demonstrated that the germ cells express only the lysosomal form of the enzyme (pI 5.5-6.0) with a subunit molecular mass of 74 kDa. Immunocytochemical studies revealed a positive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosomes of late spermatocytes (pachytene spermatocytes) and a stage-specific localization during spermiogenesis. The forming or formed acrosome of the elongated spermatids (stages 9-16) and epididymal spermatozoa was highly immunopositive. Comparison of immunoprecipitation curves and kinetic properties of the enzyme present in spermatocytes and spermatozoa revealed no major differences. Taken together, our results demonstrate that beta-glucuronidase activities present in the lysosomes of spermatocytes and the sperm acrosome are kinetically and immunologically similar.     

Comparison of Oxidative Stress/DNA Damage in Semen and Blood of Fertile and Infertile Men

Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods.The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/10⁶ dG were respectively 7.85 and 5.87 (p = 0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.     

Telomere length in male germ cells is inversely correlated with telomerase activity

Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity.     

Immunohistochemical study of calmodulin in developing mouse testis

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.     

Pseudomonas aeruginosa deficient in 8-oxodeoxyguanine repair system shows a high frequency of resistance to ciprofloxacin

The 8-oxodeoxyguanine (8-oxodG) repair system participates in the prevention and correction of mutations generated by oxidative DNA damage in prokaryotes and eukaryotes. In this study, we report that Pseudomonas aeruginosa strains deficient in this repair mechanism by inactivation of the mutT, mutM and mutY genes generate a high frequency of cells resistant to the antibiotic ciprofloxacin. In the mutT strain, the increase in ciprofloxacin resistance achieved at threefold minimal inhibitory concentration was about 1600-fold over the wild-type (WT) level, similar to the frequency achieved by the mismatch repair-deficient mutS strain. Molecular analysis of WT, mutT and mutY clones resistant to ciprofloxacin indicated that the nfxB gene was mutated in the majority of the cases, while mutS- derived resistant clones were mainly mutated in gyrA and parC genes. Cell viability analysis after treatment with paraquat or hydrogen peroxide indicated that 8-oxodG repair-deficient strains were considerably more susceptible to oxidative stress than the parental strain. Finally, it is shown that the ciprofloxacin resistance frequency of WT and repair-deficient strains increased significantly after cell exposure to paraquat. Thus, oxidative stress is strongly implicated in the emergence of ciprofloxacin-resistant mutants in P. aeruginosa , and the 8-oxodG repair pathway plays an important role in the prevention of these mutations.     

Stimulation of rat Sertoli cell secretory activity in vitro by germ cells and residual bodies

The direct influence of germ cells and residual bodies on Sertoli cell basal and FSH-stimulated secretion of androgen-binding protein (ABP) was studied using Sertoli cells, recovered from 20-day-old rats, cultured alone or cocultured with a crude germ cell preparation from adult rats or with pachytene spermatocytes, round spermatids or populations of residual bodies enriched by centrifugal elutriation. The effect of a rat liver epithelial cell line (LEC) on Sertoli cell function was also tested. Addition of a crude germ cell preparation increased basal and FSH-stimulated ABP secretion. Pachytene spermatocytes and residual bodies adhered to the Sertoli cell monolayer to a much greater extent than did round spermatids. Addition of pachytene spermatocytes markedly enhanced basal and FSH-stimulated ABP secretion over 12 days of culture. Round spermatids and residual bodies stimulated ABP secretion although to a lesser extent than did spermatocytes. Furthermore, the increase of FSH-stimulated ABP levels was not maintained after 4 or 8 days of culture. LEC also enhanced basal and FSH-induced ABP levels but the increase of FSH-induced ABP production was only observed until Day 8 of culture. The influence of LEC on Sertoli cell secretion could be mediated through the production of an extracellular matrix. It is concluded that germ cells, particularly pachytene spermatocytes, can directly stimulate Sertoli cell secretory activity in vitro.