Melatonin Improves The Developmental Competence of Goat Oocytes

Abstract

Background: DNA methylation is one the epigenetic mechanisms, which is critically involved in gene expression. This phenomenon is mediated by DNA methyl-transferases and is affected by environmental stress, including in vitro maturation (IVM) of oocytes. Melatonin, as an antioxidant, may theoretically be involved in epigenetic regulation via reductions of reactive oxygen species. This study was performed to investigate DNA methylation and the possibility of goat oocyte development after treatment with different concentrations of melatonin.

Materials and methods: This experimental study was performed to investigate DNA methylation and the possibility of goat oocyte development after treatment with different concentrations of melatonin. For this purpose, oocytes with granulated cytoplasm were selected and co-cultured with at least two layers of cumulus cells in maturation medium with 10^-6 M, 10^-9 M, 10^-12 M and 0-M (as control group) of melatonin. Nucleus status, glutathione content and developmental competence of the oocytes in each experimental group were assessed. Also, expression of genes associated with DNA methylation, including DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3b (DNMT3b) and DNA methyltransferase 3a (DNMT3a) was evaluated by quantitative real time-polymerase chain reaction (RT-PCR).

Results: According to our findings, the percentage of oocytes that reached the M-II stage significantly increased in the 10-12 M group (P<0.05). Also, a significant elevation of glutathione content was observed in melatonin-treated oocytes (P<0.05). Analysis of blastocyst formation revealed that developmental competence of the oocytes was higher than the control group (P<0.05). It was observed that melatonin treatment decreased expression levels of DNA methyltransferases (DNMTs) and global DNA methylation (P<0.05). In addition, the expression of melatonin receptor1A (MTNR1A) was detected in both cumulus and oocyte by RT-PCR.

Conclusion: The results suggested that in goat model melatonin affects DNA methylation pattern, leading to an improvement in the developmental competence of the oocytes.

Keywords: Glutathione; Melatonin; Methylation.

Fig.1

Glutathione (GSH) and reactive oxygen species (ROS) levels. The effect of melatonin on intracellular A. GSH and B. ROS levels in goat oocytes after in vitro maturation. Different letters (a, b) indicate a significant difference (P<0.05).

Fig.2

Immunocytochemical staining of oocyte. Oocytes stained with A, B. Hoechst followed by C, D. Methyl cytosin labeling in goat oocytes. *; Indicated polar body and arrow indicated M-II plate (scale bar: 20 µm), and E. Changes in methylation levels in goat oocytes from the experimental and control groups, as estimated by immunostaining. Different letters (a, b) indicate a significant difference (P<0.05).

Fig.3

Gene expression following oocyte in vitro maturation. A. The expression of DNMTs genes in goat matured oocytes treated and un-treated with melatonin. Different letters in each gene group indicate significant difference in gene expression, B. The expression of melatonin receptor (MTNR1A) in mature goat oocytes treated and un-treated with melatonin (lanes 2 and 3) and cumulus cells from matured oocyte treated and un- treated (lanes 4 and 5). Lane 1 shows the DNA molecular weight marker (100 bp ladder). Lane 7 shows the polymerase chain reaction (PCR) reaction without cDNA substrate as the negative control, and C. The expression of YWHAZ in goat matured oocytes treated and un-treated with melatonin (lanes 10 and 11) and cumulus cells from matured oocyte treated and un-treated (lanes 12 and 13). Lane 8 shows the DNA molecular weight marker (100 bp ladder). Lane 14 shows the PCR reaction without cDNA substrate. Lane 6 and 9 in both pictures show the expression of MTNR1A and YWHAZ in ovary tissue as a positive control.