Expression of atrial natriuretic peptide, progesterone, apoptosis-related proteins and caspase-3 in in vitro luteinized and leptin-treated porcine granulosa cells.

OBJECTIVE

Our aim was to examine the expression patterns of ANP, the rate of apoptosis bcl-2 and p53 expression and caspase-3 activity and progesterone (P) production in porcine granulosa cells (pGCs) stimulated in vitro for luteinization and after treatment with leptin.

METHODS

Freshly isolated prepubertal pGCs were cultured as monolayers for 24 h, subsequently FSH was supplemented for 24 h, and finally LH was added to a part of the cells for 24 h to induce luteinization. The effect of leptin on in vitro luteinized pGCs was tested by the addition of 10 ng/ml human recombinant leptin (hrL) 24 h after LH administration. Indirect immunofluorescence for ANP, bcl-2 and p53 expression was used, P production was assayed by direct enzyme immunoassay (EIA) and colorimetric AcDEVD-Pna assay for caspase-3 activity was applied.

RESULTS

Stimulation of prepubertal pGCs with FSH resulted in a moderate expression of ANP and elevation in P production. When FSH treatment was followed by LH, the pronounced expression of ANP was observed in all cells. Suppressive effect of FSH and LH on p53 expression and caspase-3 activity with parallel increase in bcl-2 expression and increased P production was observed. The treatment of in vitro luteinized (FSH/LH-stimulated) pGCs with leptin did not influence the expression of ANP. However, in FSH/LH plus leptin treated cells the concomitant increase in bcl-2 expression and parallel inhibitory effect on p53 expression and caspase-3 activity was noted, compared to control cells without any significant increase in P production.

CONCLUSION

The present data demonstrated the positive relationship between ANP expression and P production in pGCs stimulated for luteinization in vitro by FSH and LH, as well as their antiapoptotic role mediated presumably by cGMP accumulation in the luteinized pGCs. A direct anti-apoptotic effect of leptin on in vitro luteinized pGCs, without any significant modulation of P production, was documented.