Grady, Sicilia Tatiana (2017-06). Effect of Mesenchymal Stem Cells on Equine Ovarian Follicular Development and Gene Expression. Doctoral Dissertation.
Thesis
Aging affects the reproductive efficiency of females. In rodent models, injection of mesenchymal stem cells (MSCs) improves function and induces trophic mRNA expression in ovaries compromised by chemotherapy. However, little information is available on the effect of MSCs in the aging ovary, an application which if effective in increasing fertility, would have an impact in both human and equine reproduction. The aim of the research outlined in this dissertation was to investigate this area. We hypothesized that injection of MSCs into the ovaries of old mares would increase follicle numbers and increase expression of genes related to follicle growth. We examined for the first time the use of fluorescent quantum dots (QDs) to label equine MSCs, to see if this would allow them to be tracked after injection. We found that QD-labeled MSCs retain their ability to proliferate and differentiate. The QDs were maintained in MSCs induced to differentiate into chondrocytes (non-proliferating), but the percentage of labeled cells and the fluorescence intensity decreased to essentially non-detectable within 3 to 5 days in rapidly proliferating cells. Co-culture of equine ovarian tissue with MSCs was not effective due to tissue degradation in culture; however, we used this trial to develop effective methods for isolation of RNA from the extremely fibrous equine ovarian tissue. In a preliminary study on intra-ovarian MSC injection, there was an apparent increase in follicle numbers 4 weeks after injection in 2 randomly-selected young mares. Based on these results, a more comprehensive study was performed on old and young mares to determine if MSC injection affects ovarian function. There was no significant increase in follicle numbers after MSC injection in either old or young mares, nor was there an increase in serum levels of anti-Mullerian hormone, a marker for numbers of growing follicles. Results of RNA sequencing of ovarian tissue showed that injection of MSCs in old mares was associated with a minor increase in gene expression of factors that might relate to ovarian function.
