Melatonin influences the sonic hedgehog signaling pathway in porcine cumulus oocyte complexes

Abstract

Melatonin, which is synthesized in the pineal gland and peripheral reproductive organs, has antioxidant properties and regulates physiological processes. It is well known that melatonin affects in vitro maturation (IVM) of oocytes and embryonic development in many species. However, beneficial effects of melatonin on IVM have been explained mainly by indirect antioxidant effects and little information is available on the underlying mechanism by which melatonin directly acts on porcine cumulus oocyte complexes (COCs). Sonic hedgehog (Shh) signaling is important for follicle development, oocyte maturation, and embryo development, and there may be a relationship between melatonin and Shh signaling. To examine this, we designed three groups: (i) control, (ii) melatonin (10 mol/L), and (iii) melatonin with cyclopamine (2 μmol/L; Shh signaling inhibitor). The aim of this study was to investigate the effects of these agents on cumulus expansion, oocyte maturation, embryo development after parthenogenetic activation (PA), gene expression in cumulus cells, oocytes and blastocysts, and protein expression in COCs. Melatonin significantly increased the proportion of COCs exhibiting complete cumulus expansion (degree 4), PA blastocyst formation rates, and total cell numbers, which were inhibited by addition of cyclopamine. Simultaneously, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, and Has2) and Shh signaling-related genes (Shh, Pthc1, Smo, and Gli1) and proteins (Ptch1, Smo, and Gli1) in cumulus cells was upregulated in the melatonin-treated group, and these effects were also inhibited by cyclopamine. In conclusion, our results suggest that Shh signaling mediates effects of melatonin to improve porcine cumulus expansion and subsequent embryo development.

Stimulatory Effects of Melatonin on Porcine In Vitro Maturation Are Mediated by MT2 Receptor

“…Regarding the effects of melatonin with or without selective MT2 antagonists (luzindole or 4-P-PDOT) on nuclear maturation of porcine oocytes, no significant difference was observed in immature, degenerate, and MII oocyte rates between any treatment group and the control. The absence of any effects on nuclear maturation by melatonin is consistent with a previous study [ 24 ]. Also, neither of the MT2 antagonists, luzindole or 4-P-PDOT, affected nuclear maturation of porcine oocytes.…”

“…Expansion of the granulosa and cumulus cell layers plays an important role in oocyte maturation [ 26 ], and facilitates ovulation in the ovulatory follicle [ 20 ]. Recently, we demonstrated that the mRNA of melatonin receptors MT1 and MT2 are expressed in porcine cumulus cells [ 24 ]. The presence of melatonin membrane receptors in cumulus cells and the evidence of MT2 receptor-mediated modulation in porcine granulosa cell functions [ 27 ] infers that the MT2 receptor could mediate the effects of melatonin on cumulus expansion.…”

“…We investigated whether the effects of melatonin on porcine IVM and subsequent embryo development were mediated by the MT2 receptor. Agents were added to the IVM medium during the entire maturation period of 44 h. The concentrations of melatonin (10 −9 M) [ 24 ] and selective MT2 antagonists (luzindole and 4-P-PDOT) (10 −9 M) [ 41 ] were set according to previous studies, and we designated six groups: (1) control; (2) melatonin; (3) luzindole; (4) 4-P-PDOT; (5) melatonin plus luzindole, or (6) melatonin plus 4-P-PDOT. In experiment 1, we investigated the presence of MT2 receptor protein in porcine COCs.…”

“…Recently, we reported that melatonin increases porcine cumulus expansion in vitro and subsequent embryo development through the activation of sonic hedgehog signaling [ 24 ]. In addition, the presence of mRNAs of melatonin membrane receptors MT1 and MT2 in cumulus cells of pigs was demonstrated [ 24 ]. The presence of melatonin membrane receptors in cumulus cells infers that the effects of melatonin on cumulus expansion might be mediated by these receptors.…”

Importance of Melatonin in Assisted Reproductive Technology and Ovarian Aging

“…Because melatonin reduces ROS and oxidative stress via its direct antioxidative functions, it improves cellular integrity ( Figure 3). However, indirect effects of melatonin via membrane receptors (MT1, MT2) and/or nuclear receptors (RORα) are also important to understand the processes by which melatonin alters the level of oxidative stress [56,57]. Figure 3 summarizes the mechanism of melatonin that are assumed to improve oocyte maturation and quality based on reports to date.…”