Nitric oxide stimulates the production of atrial natriuretic peptide and progesterone by human granulosa luteinized cells with an antiapoptotic effect.

OBJECTIVE: To study the effect of nitric oxide (NO) on atrial natriuretic peptide (ANP) and progesterone (P) production by human granulose luteinized cells (GLC) in vitro and to elucidate their role on the survival of cultured cells. METHODS: Human GLCs were cultured in HAM's F10/10% FCS as monolayers for 24 h. Subsequently GLCs were treated for 24 h with 0.5 mM Sodium Nitroprusside (SNP, NO donor) and 0.5 mM Aminoglutethimide (AG , P450scc inhibitor). The levels of ANP and P were measured in supernatants of cultured cells by proANP(1-98) kit and RIA, respectively. Caspase-3 activity was determined by Ac-DEVD-pNA as substrate. RESULTS: The production of ANP and P was increased by NO as compared to control cells (p<0.05). AG diminished the production of P compared to SNP (p<0.05). The caspase-3 activity was significantly lower in SNP treated cells (p<0.05) and increased significantly after AG treatment compared to control cells (p<0.05). CONCLUSION: NO generated by SNP in human GLCs culture stimulated the production of ANP and P. The higher levels of ANP and P were closely related to significantly lower caspase-3 activity thus showing the role of ANP, P and NO on the survival of preovulatory human follicle.

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.

Localization of atrial natriuretic peptide in pig granulosa cells isolated from ovarian follicles of various size.

The aim of the current study was to present the spatial distribution of atrial natriuretic peptide (ANP) in short-term cultures of pig granulosa cells obtained from small, medium, and large ovarian follicles. The specific immunoreactivity was detected by three monoclonal antibodies recognizing different epitopes of the ANP molecule (Mab 6C3, Mab 6F11, Mab5D3). The specific ANP immunoreactivity detected by Mab 6C3 and Mab 6F11 showed dense staining of cytoplasm and was similar in granulosa cells from small and medium follicles. The strongest ANP immunostaining was observed in GC obtained from large follicles. The ANP immunostaining detected by Mab 5D3 had granular appearance moderately expressed in the submembrane region of granulosa cells of all types of follicles. Since ANP and ANP receptors are present in reproductive organs, the three anti-ANP antibodies may be an useful tool in further studies concerning the role of ANP in granulosa cell differentiation and function.