Mesenchymal Stem Cell-Conditioned Medium Modulates Apoptotic and Stress-Related Gene Expression, Ameliorates Maturation and Allows for the Development of Immature Human Oocytes after Artificial Activation

Abstract

The aim of the present study was to determine whether mesenchymal stem cell-conditioned medium (MSC-CM) modulates apoptotic and stress-related gene expression, and ameliorates maturation and developmental potential of immature human oocytes after artificial activation. A total of 247 surplus immature germinal vesicle (GV) oocytes obtained from infertile women were allocated into two in vitro maturation (IVM) groups: 1: GV oocytes (n = 116) matured in vitro (fIVM), and 2: GV oocytes (n = 131) that were vitrified, then in vitro matured (vIVM). Also, two maturation media were used: Alpha-minimum essential medium (α-MEM) and human umbilical cord-derived MSCs (hUCM). After 36 h of incubation, the IVM oocytes were examined for nuclear maturation. In IVM-matured oocytes, cytoplasmic maturation was evaluated after artificial activation through Ionomycin. Moreover, the quantitative expressions of B-cell CLL/lymphoma 2 (BCL2), BCL2-associated X protein (BAX), superoxide dismutase (SOD), and Heat shock proteins (HSP70) in matured oocytes were assessed by quantitative Real-time polymerase chain reaction (qRT-PCR) and compared with fresh and vitrified in vivo matured oocytes, which were used as fIVM and vIVM controls, respectively. The highest maturation rate was found in hUCM in fIVM, and the lowest maturation rate was found using α-MEM in vIVM (85.18% and 71.42%, respectively). The cleavage rate in fIVM was higher than that in vIVM (83.4% vs. 72.0%). In addition, the cleavage rate in α-MEM was lower than that in the hUCM (66.0% vs. 89.4%). Furthermore, the difference between parthenote embryo arrested in 4-8 cells (p < 0.04) and the quality of embryo arrested in 8-cell (p < 0.007) were significant. The developmental stages of parthenote embryos in hUCM versus α-MEM were as follows: 2-4 cell (89.45% vs. 66.00%, respectively), 4-8 cell (44.31% vs. 29.11%, respectively), morula (12.27% vs. 2.63%, respectively), and blastocysts (2.5% vs. 0%, respectively). The messenger RNA (mRNA) expression levels of BCL2, BAX and SOD were significantly different (p < 0.05) between the matured IVM oocytes. Overall, hUCM showed potential efficacy in terms of ameliorating oocyte maturation and in promoting the development and mRNA expression of BAX, BCL2, and SOD.

Keywords: conditioned medium; gene expression; in vitro oocyte; mesenchymal stem cells; oocytes.

Figure 1

Microscopic observation of the cleavage rate of activated oocytes. About 11–18 h after mature oocyte activation, the fertilization pronucleus (a) develops; about 30 h into the activation process, the first embryonic cell is formed. 2–4 cells embryo, were formed on the second day after fertilization (b); and 4–8 cells embryo, were formed on the third day after fertilization (c). Compaction occurred 68 h after activation (d); expanded blastocysts formed on day five after oocyte fertilization (through activation or insemination). Parthenote embryos were scored according to the A, B, and C scale described above (e–g); the embryo formation of 2–4 cells was scored as A (e); the embryo formation of 4–8 cells was scored as B (f); and the embryo formation of 4–8 cells was scored as C (g).

Figure 2

Expression of apoptotic genes (BAX and BCL2) and stress genes (HSP70 and SOD) in IVM oocytes. Each value represents the mean ± standard error of the mean (SEM) of 17 oocytes per group. The IVM groups were compared with the control groups (fresh and vitrified in vivo matured oocytes obtain from cancelled intracytoplasmic sperm injection (ICSI). 1: Control F represents the fresh in vivo matured oocytes; 2: Control V represents the vitrified in vivo matured oocytes; 3: fIVM: Fresh GV oocytes were placed in α-MEM or hUCM and then matured; 4: vIVM: vitrified GV oocytes were placed in α-MEM or hUCM and then matured; * represents a significant difference (p < 0.05) with the hUCM in vIVM group; #: represents a significant difference (p < 0.05) with the hUCM in fIVM group.