| dc.description | Primary cultures of Leydig cells are an important tool in the study of steroidogenesis regulation. Unfortunately, Leydig cells, especially of rat origin, are fragile and short-lived once isolated from their intratesticular environment. The present studies showed that more than 99% of the freshly isolated cells were viable and had no signs of apoptotic DNA fragmentation. However, once in culture, rat Leydig cells soon presented biochemical evidence of internucleosomal DNA fragmentation. The number of apoptotic cells increased with the duration of the culture (3--72 h). The higher rate of apoptotic cell death, observed between 0--3 h of incubation, is believed to be induced by the lack of cell-cell contact between Leydig cells following cell plating. The addition of physiological doses of LH (0.1 ng/ml) to the culture media significantly increased androgen production from 0--72 h, but, had no significant effect on the onset of apoptosis from 0 to 24 h. Thereafter, LH at 0.1 ng/ml significantly inhibited the onset of apoptosis. On the contrary, the addition of supraphysiological doses of LH (10.0 ng/ml) promoted the onset of apoptosis in Leydig cells during the initial 24 h of incubation. Apoptotic cell death could also be induced in vivo in Leydig cells by injecting animals with large doses of hCG (100 IU/rat) 12 to 24 h prior to cell preparation. These results implicated the importance of reactive oxygen intermediates, generated during steroidogenesis, in the induction of apoptosis both in vivo and in vitro. Indeed, the addition of exogenous antioxidants, more specifically superoxide dismutase and catalase, was shown to inhibit the onset of apoptosis induced by supraphysiological stimulation of steroidogenesis. Finally, Western analysis of pro and anti-apoptotic proteins, Bcl-2 and Bax, respectively, showed no evidence of their involvement in the regulation of physiological cell death in Leydig cells. However, these results do not rule out the involvement of other apoptosis related proteins. In conclusion, although apoptosis in Leydig cells is complex, these results give a better understanding of the process, help to improve culture conditions and provide better insights on data obtained from in vitro studies. | en_US |
