Type II DNA topoisomerase (topo II) is required for diverse biological functions including DNA replication, maintenance of genome stability, chromosome segregation and chromosome condensation. While the identity of topo II in rodent testis has been established, the regulation of topo II expression during the development of the postnatal testis and gametogenesis is unclear. Here, we report that rat testis topo II is developmentally and hormonally regulated. Topo IIalpha mRNA levels peaked prior to the onset of puberty, declined sharply thereafter and stabilized in adult testis. In contrast, the topo II enzyme content was lower in prepubertal testis but increased after the onset of puberty. Topo II was expressed in a cell-specific manner within germ cells, being detected only in pachytene spermatocytes. While testosterone markedly increased topo IIalpha mRNA levels in prepubertal testis, continued treatment failed to enhance topo IIalpha mRNA above postpubertal control levels. The extent of topo II activity remained steady regardless of the testosterone-induced increase in topo IIalpha mRNA levels. Inhibition of testosterone function in postpubertal animals by ethanedimethane sulphonate (EDS) and flutamide resulted in a significant decrease in topo IIalpha gene expression and topo II activity. The administration of exogenous testosterone (T) to EDS- and flutamide-treated rats restored topo IIalpha mRNA levels and topo II activity similar to the levels seen in the testis of age-matched control animals. Histochemical analyses of testes indicated that the effect of T on spermatogenesis was separable from its effect on topo IIalpha expression. Our results reveal that testosterone acts as a positive regulator of topo IIalpha gene expression and is required for the maintenance of topo IIalpha expression during the development of the postnatal testis and spermatogenesis.
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RP Bakshi, S Galande, P Bali, R Dighe, and K Muniyappa
R A Gadkari, S Roy, N Rekha, N Srinivasan, and R R Dighe
Human chorionic gonadotrophin (hCG) is secreted during early pregnancy and is required for implantation and maintenance of the pregnancy. Active or passive immunoneutralization of hCG results in termination of pregnancy and this forms the basis of the hCG-based female contraceptive vaccine. However, the β subunit of hCG possesses 85% sequence homology with the first 114 amino acids of the β subunit of pituitary human LH (hLH), which is required for ovulation and maintenance of the corpus luteum function during the menstrual cycle. Immunization against hCG or its β subunit leads to generation of antibodies that can neutralize hLH due to many shared epitopes and hence may cause abnormal menstrual cycles. Therefore, it is essential to identify epitopes that are different in the two hormones. In the present study, we report a monoclonal antibody (MAb) specific for hCG that shows no binding to the isolated subunits. Interestingly, the MAb also does not bind hLH at all. The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCGβ that is absent in hLHβ. The MAb, either as IgG or its recombinant single-chain variable region fragment, inhibited the response to hCG, but not to hLH. Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception.