Supplementary MaterialsJMCB-2019-0052_R2_Supplementary_Material_mjz105. H&E staining of ovaries. H&E-stained ovary sections were obtained from P9 mice. Mice were injected with a single dose of Cs (5?mg/kg body weight) or 0.9% NaCl at P5. Black arrowheads indicate the primordial follicles. (B) Quantification of the numbers of primordial, primary, and secondary follicles. Data are presented as mean??SD (experiments. Open in a separate window Physique 2 hUCMSC-CM reduces primordial follicle depletion and RGS2 preserves ovarian reserve and fertility AZ3451 after Cs treatment. (A) Analysis of ovarian follicles. Ovary sections used for H&E staining and DDX4 immunofluorescence (cytoplasm, green) were obtained from P9 mice. Cs (5?mg/kg body weight) was administered via intraperitoneal injection at P5 and hUCMSC-CM was AZ3451 injected daily from P5 to P9. Black arrowheads indicate the primordial follicles. Nuclei were stained with DAPI. Scale bar, 50?m. (B) Quantification of the numbers of primordial, primary, and secondary follicles. Data are AZ3451 presented as mean??SD ((2013) compared the RNA expression patterns of the ovaries in the hUCMSC transplantation group with the POF model and wild-type control groupings using RNA array evaluation. They discovered that the RNA appearance design in the hUCMSC-treated group was even more like the wild-type group (Wang et al., 2013). Inside our research, the RNA appearance pattern from the Cs?+?CM group clustered nearer to the CM and control groupings, as the Cs group was different during 12 significantly?h. The protective ramifications of hUCMSC-CM were obvious at the proper time of 6?h. As a result, we consider that hUCMSC-CM exerts defensive effects at the first stage. In order to discover the initial elements that inspired cell destiny decision, AZ3451 we centered on previously stage to choose the comprehensive research target for the next research. KEGG evaluation showed the fact that differentially portrayed genes at the proper period of 6?h were enriched in cytokineCcytokine receptor relationship pathway. Within this pathway, G-CSF, granulocyte-macrophage colony-stimulating aspect (GM-CSF), and Ccl2 have already been reported as critical indicators in regulating follicular advancement and steroidogenic capability. G-CSF and GM-CSF are glycoproteins made by many different cell types and also have an array of physiological features. G-CSF plays AZ3451 essential jobs in ovulation, oocyte maturation, advancement of preimplantation embryos, and trophoblast invasion (Eftekhar et al., 2018). Regarding to Akdemir et al. (2014), G-CSF can decrease follicle loss within a Cs-induced rat model. In the ovary, GM-CSF mRNA and proteins synthesis are happened in theca layers and follicular liquid mainly. GM-CSF exerts natural activity through GM-CSF receptor (Wang et al., 2005). Ccl2 can be an essential regulatory aspect of BMP15 in stopping cumulus cell apoptosis (Zhai et al., 2013). Among these six genes, the flip switch of G-CSF expression is most significant. Thus, our study focused on the effects of G-CSF. We found that hUCMSC-CM can upregulate G-CSF expression in granulosa cells and decrease granulosa cell apoptosis. Anti-apoptotic effects of G-CSF were reported in vascular endothelial cells, cardiomyocytes, and neuronal cells (Kojima et al., 2011). KEGG analysis showed that this differentially expressed genes at the time of 12?h were enriched in the PI3K/Akt pathway. The PI3K/Akt pathway was activated in granulosa cells after the hUCMSC-CM or recombinant G-CSF treatment in the present study. After G-CSF downregulation, recombinant G-CSF restored the levels of p-PI3K and p-Akt. These results indicate that G-CSF is usually a mediator of hUCMSC-CM in protecting granulosa cells from apoptosis through the PI3K/Akt pathway. In conclusion, we confirmed that hUCMSCs exert protective effects on Cs-induced ovarian damage via the paracrine pathway. We expect the obtaining can promote the application of CM in clinical treatment, and we hope infertile patients can benefit from hUCMSC-CM treatment in the future. Materials and Methods Animals CD-1 mice were purchased from SPF Biotechnology Co., Ltd. Mice were housed under standard laboratory conditions in an environmentally controlled room with free access to water and food. Light was provided between 07:00 and 19:00. All procedures involving mice were approved by the Animal Research Committee of the Institute.