The Effect of Different Doses of Melatonin on in Vitro Maturation of Human Follicular Fluid-Derived Oocyte-Like Cells

Abstract

Objective: Human follicular fluid (FF) contains different cell populations including mesenchymal stem cells. Studies tried to improve their differentiation to oocyte and use them in infertility treatments. Using an antioxidant may improve the quality of these cells. The present study investigated the effects of different doses of melatonin on FF-derived cells grown to oocyte-like cells (OLC).

Methods: Cell viability (MTT assay), flow cytometry, and ICC staining were utilized to evaluate CD105 and CD34 expression; colony forming unit assay (CFU-F) capability, qRT-PCR were used to investigate ZP1, ZP2, ZP3, GDF9, and SCP3 expression. AMH, Estradiol and Progesterone levels in the supernatant were measured. Morphological characteristics of fibroblast-like cells changing to a round shape were seen specifically in the group treated with melatonin 10-7M after 2 weeks.

Results: There was no difference between control and treatment groups for MTT and CFU assays. ICC staining was positive for CD105 marker and negative for CD34 hematopoietic stem cell marker. qRT-PCR results indicated that ZP1, ZP2, GDF9, and SCP3 expression increased in the group treated with melatonin 10-7M in Week 2, while ZP3 decreased in this group. Progesterone and AMH were detected in differentiation medium.

Conclusions: Melatonin may improve in vitro formation of OLCs.

Keywords: Follicular fluid; infertility; melatonin; oocyte like cell; stem cell.

Figure 1

Morphological changes of follicular fluid cells in minimal culture condition. Control groups without melatonin treatment on day 0(a), week 1(b), week 2(c); groups treated with melatonin 10^-7M on week 1(d) and week 2 (e); groups treated with melatonin 10^-9M on week 1(f) and week 2(g); groups treated with melatonin 10^-11M on week 1(h) and week 2(i); blue arrows: OLCs; yellow arrows: epithelial cells; 20 X.

Figure 2

Viability of FF cells in control and groups treated with melatonin 10^-7M, 10^-9M and 10^-11M for 1 week. Cell viability did not change significantly in the four groups.

Figure 3

Flow cytometry analysis of CD105 antigen expression in the control group compared with melatonin treated groups after 2 weeks. High significant expression. observed in control group confirming the most stemness characteristic of the cells without melatonin treatment. **<0.01; ***<0.001.

Figure 4

Immunostaining of human FF cells for specific cell surface markers after 48h. (a) Red arrows show cells low positive expression of CD105, (c) negative expression of CD34. Cell nuclei were counterstained by DAPI (b, d); 20X.

Figure 5

Adipogenic and osteogenic differentiation of human follicular fluid cells. (a) Adipogenic differentiated cells of human FF showed by Oil Red O staining after 2 weeks. (b) Cells differentiated with osteogenic medium showed brownish red after staining with Alizarin Red; 20X.

Figure 6

Colony forming unit fibroblast assay analysis of human FF cells after 2 weeks. Colony forming did not show significant differences between control and treated groups.

Figure 7

qRT-PCR analysis of ZP1(a, b), ZP2(c, d), ZP3(e, f), GDF9(g, h) and SCP3(I, j). mRNA expression. of human FF-derived OLCs on day 0, week 1(w1) and week 2(w2) in melatonin treatment groups (10^-7, 10^-9,10-¹¹M) compared with control groups; gene expression at a specific concentration in week 1 compared to week 2 (a, c, e, g, i); gene expression of melatonin treatment groups compared with control groups each week (b, d, f, h, j); *: p<0.05.

Figure 8

AMH (a), Estradiol (b), and Progesterone(c) production during FF cell culture in vitro. Hormone levels compared between control and melatonin treated groups (10^-7, 10^-9, 10^-11) in 2 weeks; *: p<0.05.