Reproduction Abstracts

Introduction: ALF is a germ cell-specific counterpart of the large (α/β) subunit of general transcription factor TFIIA and has a vital role in the meiotic and post-meiotic development of male germ cells. We have found in vitro a variety of effects of hydrogen peroxide (H₂O₂) on different types of germ cells, including the induction of DNA strand breaks and apoptosis. We therefore examined its effect on the expression of genes such as ALF in order to investigate the mechanisms responsible.

Methods: Following STAPUT separation, the fractions containing only spermatocytes or spermatids were cultured for 16 h before the addition of H₂O₂ at final concentrations of 0, 10, 50, or 100 μM. After 2 h the cells were harvested and the total protein or RNA was extracted. Western blotting and reverse-transcripted quantitative PCR were performed to determine the effects on ALF expression levels.

Results: QRT-PCR and western blotting showed that both ALF mRNA and protein levels were reduced at the higher levels (50 and 100 μM) of H₂O₂ exposure but not at a lower dose (10 μM). The effect was relatively more pronounced in spermatocytes than spermatids.

Discussion: H₂O₂ exposure results in the specific down-regulation of a transcription factor in developing male germ cells. Whether this is a cause or consequence of the known effects of H₂O₂ on such cells remains to be shown. The relatively greater effect on spermatocytes than spermatids correlates with their greater sensitivity to the induction of DNA damage and apoptosis we have found recently.