Effects Of Melatonin On Follicular Development And Atresia In The Porcine

The follicle is the structural and functional unit of the mammalian ovary.Its life cycle is defined by rapid growth that fosters oocyte maturation or by involution that results in follicular atresia,the latter occurring at any stage of development in response to endocrine,paracrine,and gene expression signaling within the follicle.Studies on the regulation mechanisms of follicular development and atresia are of important theoretical value and practical significance to better understand the complex reproductive processes in the porcine.Melatonin(N-acetyl-5-methoxytryptamine),an indole amine produced in many organs including the pineal gland,is involved in many biological rhythm related processes,such as cell growth,dormancy cycle regulation,energy metabolism,angiogenesis,neuroprotection,cell senescence.Evidence from cell culture and animal studies revealed the beneficial effects of melatonin on oocyte maturation,development of growing follicles,and embryonic development in vitro and in vivo.However,there is little systematic report on the effect of melatonin on follicular development and atresia in the porcine.Herein,the expression profile of melatonin receptor genes in different tissues of porcine,the biological effect of melatonin on porcine follicular development and atresia,associated with the expression of mRNA and protein of related genes,as well as the potential regulation mechanism of melatonin on granulosa cell apoptosis were explored.The following results were obtained here:1.The expressions of melatonin receptors MT1 and MT2 were detected in brain,liver,pancreas,ovary,fat,kidney,heart and spleen tissue in the female porcine,but with significant difference among different tissues.MT and MT2 were highly expressed in the ovary and brain,compared with the other tissues.2.A total of 9 groups of hierarchical follicles was stripped followed by ELISA analysis of melatonin concentration in follicular fluid.Results showed that endogenous,intrafollicular melatonin concentration decreased as follicular atresia progressed.Melatonin concentrations in healthy follicles were higher(range of 86.4±2.4 to 126.6±6.2 ng/L)than in serum(29.3±2.2 ng/L;P<0.05).The percentages of apoptotic cells in granulosa cells increased as follicular atresia advanced: atretic follicles > early atretic follicles > healthy follicles.The Pearson correlation coefficient of melatonin level and the apoptotic rate of granulosa cells in follicles was-0.847.Reletive mRNA levels of MT1 and MT2 in granulosa cells were: healthy follicles > early atretic follicles > progressed atretic follicles.3.Healthy antral follicle with diameter of 3~5 mm was treated by different concentrations of melatonin supplementation(0,0.05,0.2,0.8 ng/mL)in vitro during a 6 d incubation.The results demonstrated that the culumus layers was stimulated by melatonin treatments after 48 h incubation(P<0.05),the rate of A level COCs was highest(17/48)in 0.8 ng/mL melatonin treatment,as compared with other groups(P<0.05).The survival rate and membrane integrity of antral follicles was remarkedly improved by 0.05,0.2 and 0.8 ng/mL melatonin after 6 d incubation(P<0.05),among which,the survival rate of antral follicles was as high as 95.83%,and the membrane integrity rate of antral follicles was reached to 72.22% in 0.2 ng/mL melatonin group,when compared with the other treatments(P<0.05).After 3 d incubation,the diffusion rate of culumus derived from in vitro cultured antral follicles was reached to 88.89% in 0.05% ng/mL melatonin group(P<0.05),the release of polar body of oocyte(56.94%)was significantly higher than the control(P<0.05).0.05,0.2,0.8 ng/mL melatonin supplementation significantly decresed percentages of progressed apoptotic granulosa cells(P<0.05).4.Porcine granulosa cells from medium-sized(3–5 mm),healthy follicles were cultivated with variable concentrations of melatonin(0,0.001,0.01,0.1,1.0,and 10 ng/mL)for 48 h,in combination with its receptor antagonist,luzindole and 4P-PDOT,followed by evaluation of apoptotic markers and steroidogenesis activities in the treated cells.Results revealed that 0.01 ng/mL melatonin was the optimal dosage for melatonin supplementation in the current condition,since the cell viability and colony-forming efficiency of porcine granulosa cells was significantly stimulated,accomplished with reduced percentages of apoptotic granulosa cells by 0.01 ng/mL melatonin for the 48-h incubation period(P<0.05).Moreover,estradiol(E2)biosynthesis was significantly stimulated by melatonin supplementation and suppressed for the progesterone(P4)secretion;the minimum ratio of progesterone to estradiol was 1.82 in 0.01 ng/mL melatonin treatment after 48 h of cultivation.The expression of CYP17A1 and CYP19A1 were up-regulated by 0.01 ng/mL melatonin.In addition,the mRNA levels of melatonin receptor MT2,as well as the apoptotic inhibition factor Bcl-2 and antioxdative enmyze genes GPX4,SOD1 were significantly improved by 0.01 ng/mL melatonin(P<0.05),whereas that of the apoptotic inducing factors Bax,P53 and Caspase-3 was remarkedly down-regulated(P<0.05),however,the stimulatory effects of melatonin on granulosa cell preferation and the inhibition effects on apoptosis was significantly blocked by the addition of melatonin receptor antagonist luzindole and the selective MT2 antagonist 4P-PDOT(P<0.05).5.Porcine granulosa cells were treated by ER stress inducer or serum withdraw for the first 24 h period,and continue to be cultivated with variable concentrations of melatonin(0,0.001,0.01,0.1,1.0,and 10 ng/mL)for another 24 h,followed by evaluation of apoptotic markers in the treated cells.The results revealed that the spontaneous apoptosis of granulosa cells,triggered by serum deprivation in vitro,was remarkably alleviated by melatonin in dose-dependent manner(1.0 ng/mL melatonin,32.7±0.5%,vs.control,47.0±1.0%;P<0.05).Furthermore,1.0 ng/mL melatonin significantly decreased the total percetages of apoptotic granulosa cells in vitro,as compared to the treatments of the positive control Tunicamycin or serum deprivation group(P<0.05).Treatment with 1.0 ng/mL of melatonin also significantly elevated MT2,SOD1,and GPX4 while lowering FAS L,CHOP,and GRP78 mRNA abundance in serum-free cultured granulosa cells,as compared to the control(P<0.05).The anti-apoptotic effect and some changes of apoptotic-relevant genes in granulosa cells invoked by melatonin supplementation were markedly blocked by luzindole(P<0.05),suggesting that melatonin could prevent the apoptosis of porcine granulosa cells during follicular atresia via its membrane receptors and its radical scavenging activity.In summary,this study revealed the higher expression of MT1 and MT2 in ovary,and that melatonin levels was negatively correlated with follicular atresia.Furthermore,melatonin played promotion effctes on survival,membrane integrity,diameter growth,and culumus layers proliferation,accomplished with stimulation effects on clumus diffusion and polar body release of oocyes derived from in vitro cultured anral follicles.On the other hand,melatonin exhibited apoptosis-inhibition effects on ex-vitro and in vitro cultured granulosa cells and regulated the synthesis of steroid hormone.Melatonin and its receptors played important roles in follicular development and functions of granulosa cells.The study may provide a new theoretical basis for further understanding of the regulatory mechanism of melatonin in follicular atresia-related functions in the porcine.