Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment. VI. Alteration in nuclear factors for interaction with histone gene promoter.

This article reports the effects of gossypol at the genomic level in rat spermatogenic cells. After gossypol treatment for various times (8, 12, and 19 weeks), the spermatogonial cells were allowed to rest for 2 to 4 weeks. The function of histone H4 gene promoter (H4GP) in the repopulating pachytene spermatocytes (RPS) was investigated. The sequences of the oligonucleotides for the H4GP binding sites 1 and 2 were synthesized by an ABI-392 DNA synthesizer. RPS and the control pachytene spermatocytes (CPS) were obtained by centrifugal elutriation and subsequently they were used for the preparation of nuclear protein extracts (NPE). The NPE interaction with the DNA fragment of site 1 or 2 was studied by an electrophoresis mobility shift assay (EMSA). EMSA with NPE-CPS revealed ten major gel shift bands for site 1 and 2. The presence of extra unlabelled DNA fragments competed with 6 of the bands. After 2 to 4 weeks recovery from 8, 12, and 19 weeks of gossypol treatment, NPE-RPS failed to shift four bands (b through e) in site 1. These results suggested that gossypol treatment affected the transcription factors for interaction with site 1. On the contrary, no effect was demonstrated in NPE that interacted with site 2. Furthermore, gossypol treatment did not change the nucleotide sequence in the H4GP site 1 and 2.