Melatonin modulates the functions of porcine granulosa cells via its membrane receptor MT2 in vitro.

Melatonin (N-acetyl-5-methoxytryptamine) is documented as a hormone involved in the circadian regulation of physiological and neuroendocrine function in mammals. Herein, the effects of melatonin on the functions of porcine granulosa cells in vitro were investigated. Porcine granulosa cells were cultivated with variable concentrations of melatonin (0, 0.001, 0.01, 0.1, 1.0, and 10ng/mL) for 48h. Melatonin receptor agonist (IIK7) and antagonist (Luzindole, 4P-PDOT) were used to further examine the action of melatonin. The results showed optimum cell viability and colony-forming efficiency of porcine granulosa cells at 0.01ng/mL melatonin for 48-h incubation period. The percentage of apoptotic granulosa cells was significantly reduced by 0.01 and 0.1ng/mL melatonin within the 48-h incubation period as compared with the rest of the treatments. Estradiol biosynthesis was significantly stimulated by melatonin supplementation and suppressed for the progesterone secretion; the minimum ratio of progesterone to estradiol was 1.82 in 0.01ng/mL melatonin treatment after 48h of cultivation. Moreover, the expression of BCL-2, CYP17A1, CYP19A1, SOD1, and GPX4 were up-regulated by 0.01ng/mL melatonin or combined with IIK7, but decreased for the mRNA levels of BAX, P53, and CASPASE-3, as compared with control or groups treated with Luzindole or 4P-PDOT in the presence of melatonin. In conclusion, the study demonstrated that melatonin mediated proliferation, apoptosis, and steroidogenesis in porcine granulosa cells predominantly through the activation of melatonin receptor MT2 in vitro, which provided evidence of the beneficial role of melatonin as well as its functional mechanism in porcine granulosa cells in vitro.

Effects of melatonin on follicular atresia and granulosa cell apoptosis in the porcine.

The accumulation of reactive oxygen species is detrimental to the health of the ovarian follicle. The protective, antioxidant properties of melatonin, an endogenous component of porcine follicular fluid, on apoptosis of granulosa cells were evaluated in this study. Porcine granulosa cells from medium-sized (3-5 mm), healthy follicles were cultured in serum-free conditions with melatonin (0, 0.01, 0.1, 1.0, 10, and 100 ng/mL) with or without its receptor antagonist, luzindole, followed by evaluation of apoptotic markers in the treated cells. Results revealed that endogenous, intrafollicular melatonin concentration decreased as follicular atresia progressed, whereas the percentage of apoptotic granulosa cells increased. Spontaneous apoptosis of granulosa cells, triggered by serum deprivation in vitro, was remarkably blocked by melatonin (1.0 ng/mL melatonin, 32.7 ± 0.5%, vs. control, 47.0 ± 1.0%; P < 0.05). Treatment with 1.0 ng/mL of melatonin also significantly elevated MT2, SOD1, and GPX4 while lowering FASL, CHOP, and GRP78 mRNA abundance compared to the untreated control. The anti-apoptotic effect and some changes of apoptotic-relevant genes in granulosa cells invoked by melatonin supplementation were markedly blocked by luzindole, suggesting that melatonin could prevent the apoptosis of porcine granulosa cells during follicular atresia via its membrane receptors and its free-radical-scavenging activity. These findings provide new insights into the regulatory mechanism of melatonin in follicular atresia-related functions. Mol. Reprod. Dev. 83: 692-700, 2016 © 2016 Wiley Periodicals, Inc.

Testing Rhodiola sachalinensis saccharide as cryoprotectant for bovine spermatozoa.

Rhodiola sachalinensis saccharide (RSS) was extracted from the rhizome of Herba Rhodiolae and was expected as a novel cryoprotectant. The aim of this study was to test the effects of RSS on motility of bull sperm and the activities of superoxide dismutase (SOD), lactate dehydrogenase (LDH), and glutamic oxaloacetic transaminase (GOT) in bull sperm during cryopreservation. Rhodiola sachalinensis saccharide was added at the concentrations of 0.02, 0.04, 0.06, 0.08, and 0.10 mg/mL to the extenders, which were used to store bovine semen. It was found that the RSS-added extends resulted in a higher percentage of cryopreserved sperm motility, mitochondrial activity, and membrane and acrosome integrity than those of RSS-free extenders. The SOD, LDH, and GOT activities were all decreased during the process of freezing and thawing. The extenders supplemented with RSS improved the SOD, LDH, and GOT activities after cryopreservation compared with the RSS-free groups. In conclusion, RSS conferred great cryoprotective capacity to the basic extender for bull spermatozoa during the process of freezing-thawing, and the optimal concentration of RSS for the extender was 0.06 mg/mL.