Telomerase activity in pig granulosa cells proliferating and differentiating in vitro

Abstract

The aim of the work was to analyze the telomerase activity (TA) in two different populations of pig granulosa cells (GC) proliferating and differentiating in vitro: (a) in relatively undifferentiated granulosa cells isolated from small (1–2 mm) antral follicles and (b) in functionally advanced, differentiated cells obtained from large (5–7 mm) antral follicles. The proliferative potential in vitro of small follicle granulosa cells (SF-GC) was higher than that of large follicle granulosa cells (LF-GC). EGF stimulated significantly ( p < 0.01) proliferation in SF-GC as well as LF-GC. FSH did not have a stimulating effect on proliferation in both of the GC populations. Steroidogenesis was induced in both SF- and LF-GC in vitro. Significantly higher ( p < 0.01) levels of estradiol were measured in LF-GC cultures. In SF-GC, no significantly different effects of EGF and FSH on estradiol production were found. The production of progesterone in vitro was higher in LF-GC than in SF-GC and its production was specifically promoted by FSH in contrast to estradiol the synthesis of which in vitro was less dependent on culture conditions. Using the TRAP assay telomerase activity was detected in freshly isolated and in vitro cultured pig SF- and LF-GC. In EGF, but not FSH stimulated SF-GC, significantly enhanced ( p < 0.05) TA in comparison with the control was observed at an interval of 24 h of culture. After the 48 h in vitro, levels of TA in both EGF and FSH treated cells were comparable with control. In LF-GC, both EGF and FSH stimulated significantly ( p < 0.05) TA after the 24 h of in vitro culture. At an interval of 48 h, no significant differences in the level of TA were observed between control, EGF and FSH stimulated LF-GC. Comparing the levels of TA in SF- and LF-GC, significantly higher levels of TA were found in control ( p < 0.05) and EGF ( p < 0.01) treated SF-GC after 24 h in vitro. On the other hand, absolutely, but not significantly, higher levels of TA were found in LF-GC versus SF-GC in all culture conditions after 48 h in vitro.