Effect of exosomes derived from bone marrow mesenchymal stem cells on maturation of preantral follicles of immature NMRI mice

Infertility is a life crisis that affects patients worldwide. Infertility is defined as failure to conceive after 12 months of sexual activity, affecting 15-17% of couples worldwide. and about 50% of them are related to the factors of female infertility. Activating the process of meiosis in the oocyte and its maturation has been one of the important therapeutic goals of infertility researchers. Today, inducing oocyte growth and development outside the body is one of the methods used in assisted reproduction technology. Bone marrow is a complex organ in which different lineages of hematopoietic and stromal cells support hematopoiesis. Extracellular vesicles (EVs) with a size of 20-100 nm are released by different types of cells in culture media. In addition to proteins, exosomes are enriched with an array of cytokines, specific lipid rafts such as phosphoglyceride, cholesterol, ceramide, fatty-acyl chains, as well as mRNAs, miRNAs, non-coding RNAs, tRNAs, rRNAs, and rarely DNA.  The purpose of this experimental research is to investigate the effect of exosomes derived from the bone marrow stem cells of small laboratory mice on the maturation of preantral follicles.

Exosomes were isolated and cultured from the mesenchymal stem cells of the bone marrow of small laboratory mice by the flushing method. Identification of stem cells was done by flow cytometry method and separation and purification of exosomes was done by ultracentrifuge, identification of exosome was also checked by atomic force microscope (AFM). The effects of exosomes on the viability of follicles were measured by the MTT method, and developmental parameters such as the diameter and the formation of the antrum cavity in the follicles were examined and the follicles were examined on days 0, 2, 3 and 4 of culture with 20 magnification and inverted microscope. Photographs were taken and the diameter of the follicles was measured in micrometers by Image J software. The expression of GDF-9, BMP-15 and BMP-7 genes as genes involved in the growth and maturation of follicles was investigated using Real Time-PCR method. GraphPad Prism 8 software and one way Anova statistical test were used to analyze the data of this research.

The evaluation of the viability of the follicles showed that compared to the control group, the follicles treated with exosome 25, 50 and 100 micrograms/ml showed an increase in viability, as well as the rate of antrum formation increased significantly in the group with the concentration 100 μg/ml showed a significant level (p<0.01**) compared to the control group. The diameter of the follicles increased with increasing the concentration of exosomes compared to the control group. GDF-9, BPM-15 and BMP-7 genes also increased in the treatment groups.

According to the findings of this experimental research, it can be stated that exosomes derived from bone marrow stem cells Small laboratory mice have a positive effect on survival and maturity, as well as the growth of ovarian follicles.