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Based on our previous study in follicles, the first aim of this work was to evaluate the effect of melatonin in the swine corpus luteum (CL). Luteal cells were exposed to 10 and 20pg mL-1 melatonin. We evaluated the effect on proliferation (bromo-deoxy-uridine uptake), steroidogenesis (progesterone) and redox status by means of Griess test (nitric oxide production), WST-1 test (superoxide anion generation) and FRAP test (non-enzymatic antioxidant power). The results showed a significant increase in antioxidant power, as well as a reduction in the other parameters analysed. These data and the expression of MT2 observed in luteal cells allow us to hypothesise a physiological role of melatonin in the regulation of CL functionality. The reproductive function is dependent on energy reserves stored in adipose tissue. Therefore, we sought to verify the effect of melatonin on adipose stromal cells (ASCs). MT2 receptor expression was detected in ASCs and the presence of gene markers (PPARγ and leptin) before and after adipogenic differentiation was verified. The differentiation was significantly inhibited by melatonin, as well as cell viability. In conclusion, present results suggest that melatonin exerts a potential inhibitory action on luteal function and adipogenesis, possibly mediated by MT2.

Melatonin exerts well-known reproductive effects, mainly acting on hypothalamic gonadotrophin-releasing hormone release. More recent data suggest that melatonin acts directly at the ovarian level, even if, at present, these aspects have been only partly investigated. Swine follicular fluid contains melatonin and its concentration is significantly reduced during follicular growth. Therefore, the present study was undertaken to examine the effects of melatonin, used at physiological concentrations, on cultured swine granulosa cells collected from small (5 mm) follicles on the main parameters of granulosa cell function such as proliferation and steroidogenesis, namely oestradiol 17β and progesterone (P4) production. Moreover, the effects of melatonin on superoxide anion and nitric oxide (NO) generation by swine granulosa cells were also investigated. Finally, since angiogenesis is crucial for follicle growth, the effects of melatonin on new vessel growth were studied. Collected data indicate that melatonin interferes with cultured granulosa cell proliferation and steroidogenesis, specifically in terms of P4 production and NO output. In addition, the events of physiological follicular angiogenesis were stimulated by melatonin as evidenced by angiogenesis bioassay. Therefore, we suggest that physiological melatonin concentrations could potentially be involved in local modulation of swine ovarian follicle function.

This study was designed to investigate the presence of bioactive tumour necrosis factor-alpha (TNF-alpha) in bovine fluid collected from small (5 mm) and large (8 mm) follicles, as well as the production of the cytokine by the granulosa cells collected from the same type of follicles. Moreover, the effectiveness of 10, 1 and 0.1 ng mL(-1) of human TNF-alpha (hTNF-alpha) in affecting the main parameters of granulosa cell function, progesterone (P4) and oestradiol-17beta (E2) production, cell proliferation and apoptosis, was tested. In addition, the study aimed to determine whether the signalling mechanisms of TNF-alpha in these cells involve cAMP, nitric oxide or prostaglandin E2 (PGE2) and F2alpha (PGF2alpha). It emerged that bioactive TNF-alpha is present in follicular fluid from both types of follicles and can be measured in media conditioned by granulosa cells from large follicles. As for the effects of hTNF-alpha, it inhibits P4 production in cells from both types of follicles and stimulates E2 output in those from small follicles; it does not affect proliferation, but it stimulates granulosa cell apoptosis. Finally, the effects of hTNF-alpha on bovine granulosa cells are not mediated by nitric oxide or cAMP, as neither of these substances were affected by treatment with the cytokine; however, in some way, they could be mediated through PGE2 and PGF2alpha, the production of which was inhibited by TNF-alpha in cells from small follicles.

The present study was performed to explore two of the possible signalling mechanisms through which nitric oxide (NO) inhibits steroidogenesis in bovine granulosa cells. Because cGMP is generally known to play a pivotal role in NO signal transduction, the first aim of the present study was to verify the presence of a functional NO–cGMP signalling pathway. Because non-cGMP-dependent pathways could be involved in the inhibition of steroidogenesis by NO, we examined the formation of lipid hydroperoxides (LPOs), possibly induced by NO. Using bovine granulosa cells collected from small (< 5 mm) and large (> 8 mm) follicles, the effectiveness of the NO donor s-nitroso-N-acetylpenicillamine (SNAP; 10–3, 10–4 and 10–5 M) in stimulating cGMP production and the formation of LPOs was examined. The second aim of the present study was to determine whether the effects of NO on steroidogenesis could be mimicked by treatment of cells with a cGMP analogue (8-bromo-cGMP (8-Br-cGMP); 10–3, 10–4 and 10–5 M) and whether these effects could be reversed by [1H]-[1,2,3]oxadiaziolo[4,3a]quinoxaline-1-one (ODQ; 10–5 and 10–4 M) an inhibitor of NO-sensitive soluble guanylate cyclase. The highest dose of SNAP used induced a significant (P