Iris S. Gademan

The role of the tumor suppressor p53 in spermatogenesis.

The p53 protein appeared to be involved in both spermatogonial cell proliferation and radiation response. During normal spermatogenesis in the mouse, spermatogonia do not express p53, as analyzed by immunohistochemistry. However, after a dose of 4 Gy of X-rays, a distinct p53 staining was present in spermatogonia, suggesting that, in contrast to other reports, p53 does have a role in spermatogonia. To determine the possible role of p53 in spermatogonia, histological analysis was performed in testes of both p53 knock out C57BL/6 and FvB mice. The results indicate that p53 is an important factor in normal spermatogonial cell production as well as in the regulation of apoptosis after DNA damage. First, p53 knock out mouse testes contained about 50% higher numbers of A1 spermatogonia, indicating that the production of differentiating type spermatogonia by the undifferentiated spermatogonia is enhanced in these mice. Second, 10 days after a dose of 5 Gy of X-rays, in the p53 knock out testes, increased numbers of giant sized spermatogonial stem cells were found, indicating disturbance of the apoptotic process in these cells. Third, in the p53 knock out testis, the differentiating A2-B spermatogonia are more radioresistant compared to their wild-type controls, indicating that p53 is partly indispensable in the removal of lethally irradiated differentiating type spermatogonia. In accordance with our immunohistochemical data, Western analysis showed that levels of p53 are increased in total adult testis lysates after irradiation. These data show that p53 is important in the regulation of cell production during normal spermatogenesis either by regulation of cell proliferation or, more likely, by regulating the apoptotic process in spermatogonia. Furthermore, after irradiation, p53 is important in the removal of lethally damaged spermatogonia.

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

Abstract Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms γ-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 γ-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, γ-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit γ-H2AX foci but show homogeneous nuclear γ-H2AX staining, whereas in pachytene spermatocytes γ-H2AX is only present in the sex vesicle. In response to ionizing radiation, γ-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, γ-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear γ-H2AX staining in leptotene spermatocytes demonstrates a function for γ-H2AX during meiosis. γ-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 γ-H2AX foci at DNA double-strand breaks.

Involvement of the D-Type Cyclins in Germ Cell Proliferation and Differentiation in the Mouse

Abstract Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D1 is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D1 in spermatogonial proliferation, in particular during the G1/S phase transition. Cyclin D2 is first expressed at the start of spermatogenesis when gonocytes produce A1 spermatogonia. In the adult testis, cyclin D2 is expressed in spermatogonia around stage VIII of the seminiferous epithelium when Aal spermatogonia differentiate into A1 spermatogonia and also in spermatocytes and spermatids. To further elucidate the role of cyclin D2 during spermatogenesis, cyclin D2 expression was studied in vitamin A-deficient testis. Cyclin D2 was not expressed in the undifferentiated A spermatogonia in vitamin A-deficient testis but was strongly induced in these cells after the induction of differentiation of most of these cells into A1 spermatogonia by administration of retinoic acid. Overall, cyclin D2 seems to play a role at the crucial differentiation step of undifferentiated spermatogonia into A1 spermatogonia. Cyclin D3 is expressed in both proliferating and quiescent gonocytes during testis development. Cyclin D3 expression was found in terminally differentiated Sertoli cells, in Leydig cells, and in spermatogonia in adult testis. Hence, although cyclin D3 may control G1/S transition in spermatogonia, it probably has a different role in Sertoli and Leydig cells. In conclusion, the three D-type cyclins are differentially expressed during spermatogenesis. In spermatogonia, cyclins D1 and D3 seem to be involved in cell cycle regulation, whereas cyclin D2 likely has a role in spermatogonial differentiation.

Apoptosis regulation in the testis: involvement of Bcl-2 family members.

Using immunohistochemical techniques and Western blot analysis, the possible role of Bcl‐2 family members Bax, Bcl‐2, Bcl‐xs, and Bcl‐xl in male germ cell density‐related apoptosis and DNA damage induced apoptosis was studied. The apoptosis inducer Bax was localized in all mouse and human testicular cell types, but despite the fact that irradiation induces its transcriptional activator, p53 in the human, Bax expression did not change after irradiation. The apoptosis inhibitor Bcl‐2 appeared to be present in late spermatocytes and spermatids and was up‐regulated in these cells after a dose of 4 Gy of X‐rays. Finally, Bcl‐x was expressed in both the mouse and human testis. The apoptosis inhibiting long transcripts of Bcl‐x, Bcl‐xl, were expressed in spermatogonia and spermatocytes and were up‐regulated after X‐irradiation. The apoptosis inducing shorter form of Bcl‐x, Bcl‐xs, was found to be expressed only in somatic cells, like peritubular and Leydig cells. While Bax is important in germ cell density regulation, Bax expression did not change after DNA damage inflicted by X‐radiation. Hence, spermatogonial apoptosis after X‐irradiation may not be induced via the apoptosis inducer Bax. Furthermore, as Bcl‐xl, but not Bcl‐2, is present in spermatogonia and spermatocytes, Bcl‐xl may regulate germ cell density, possibly in cooperation with Bax. As Bcl‐xl expression is enhanced after irradiation, this protein may also have a role in the response of spermatogonia and spermatocytes to irradiation. Mol. Reprod. Dev. 56:353–359, 2000.

P21(Cip1/WAF1) expression in the mouse testis before and after X irradiation.

During spermatogenesis, the radiosensitivity of testicular cells changes considerably. To investigate the molecular mechanism underlying these radiosensitivity differences, p21(Cip1/WAF1) expression was studied before and after irradiation in the adult mouse testis. P21(Cip1/WAF1) is a cyclin‐dependent kinase inhibitor (CDI) and has a role in the G1/S checkpoint and differentiation.

Role for c-Abl and p73 in the radiation response of male germ cells

p53 plays a central role in the induction of apoptosis of spermatogonia in response to ionizing radiation. In p53−/− testes, however, spermatogonial apoptosis still can be induced by ionizing radiation, so p53 independent apoptotic pathways must exist in spermatogonia. Here we show that the p53 homologues p63 and p73 are present in the testis and that p73, but not p63, is localized in the cytoplasm of spermatogonia. Unlike p53, neither p63 nor p73 protein levels were found to increase after a dose of 4 Gy of X-rays. Although p73 protein levels did not increase, its interaction with the non-receptor tyrosine kinase c-Abl and its phosphorylation on tyrosine residues did. c-Abl and p73 co-localize in the cytoplasm of spermatogonia and spermatocytes and in the residual bodies. Furthermore, c-Abl protein levels increase after irradiation. p63 was not found to co-localize or interact with c-Abl neither before nor after irradiation. In conclusion, in the testis ionizing radiation elevates cytoplasmic c-Abl that in turn interacts with p73. This may represent an additional, cytoplasmic, apoptotic pathway. Although less efficient than the p53 route, this pathway may cause spermatogonial apoptosis as observed in p53 deficient mice.

Function of DNA-Protein Kinase Catalytic Subunit During the Early Meiotic Prophase Without Ku70 and Ku86

Abstract All components of the double-stranded DNA break (DSB) repair complex DNA-dependent protein kinase (DNA-PK), including Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs), were found in the radiosensitive spermatogonia. Although p53 induction was unaffected, spermatogonial apoptosis occurred faster in the irradiated DNA-PKcs-deficient scid testis. This finding suggests that spermatogonial DNA-PK functions in DNA damage repair rather than p53 induction. Despite the fact that early spermatocytes lack the Ku proteins, spontaneous apoptosis of these cells occurred in the scid testis. The majority of these apoptotic spermatocytes were found at stage IV of the cycle of the seminiferous epithelium where a meiotic checkpoint has been suggested to exist. Meiotic synapsis and recombination during the early meiotic prophase induce DSBs, which are apparently less accurately repaired in scid spermatocytes that then fail to pass the meiotic checkpoint. The role for DNA-PKcs during the meiotic prophase differs from that in mitotic cells; it is not influenced by ionizing radiation and is independent of the Ku heterodimer.

Response to motexafin gadolinium and ionizing radiation of experimental rat prostate and lung tumors

PURPOSE To investigate the responses of two experimental rat tumors to single and fractionated X-ray doses whether or not combined with Motexafin gadolinium (MGd), and the distribution of MGd in R3327-MATLyLu (MLL) tumors using MRI. METHODS L44 lung tumor in BN rats and MLL prostate tumor in Copenhagen rats were grown subcutaneously. MGd at concentrations of 8.7 to 25.1 micro mol/kg was administered 2 h before or just before treatments with single and fractionated X-ray doses. Tumor volume growth delay was the endpoint used. The two-dimensional distribution of the MGd concentration in time was analyzed simultaneously in slices through the center of MLL tumors using MRI. Directly after the MRI experiments, tumor sections were stained for cytoplasm, nuclei, and microvessel endothelium. RESULTS MGd at different concentrations administered a few minutes or 2 h before X-ray doses produced no radiation enhancement in the two tumor models. The MGd concentration as determined by MRI was maximal 5 min after injection and decreased slowly thereafter. In a representative section at the center of the MLL tumor, the microvessel density is nearly homogeneous and correlates with a nearly homogeneous MGd distribution. Hardly any MGd is taken up in underlying muscle tissue. CONCLUSION No radiosensitization was observed for the different irradiation regimens. The distribution of MGd is nearly homogeneous in the MLL tumor and hardly any MGd is taken up in underlying muscles. Our negative results on radiosensitivity in our two tumor models raise questions about the efficacy of MGd as a general radiosensitizing agent.