Activation And Survival Of Ovarian Follicles In Vitro: The Effects Of Bone Morphogenetic Proteins (Bmps) And/Or Cryopreservation On Bovine And Feline Ovarian Tissue.

Abstract

Assisted reproductive techniques, including in vitro development of follicles, cryopreservation and in vitro fertilization, are used to preserve female fertility. Ovarian tissue cryopreservation is a promising tool for maintaining fertility in women undergoing cancer treatment and for gamete rescue in wildlife species. Primordial follicles, the basic units of folliculogenesis that represent a female's reproductive potential, should be the priority when preserving fertility. Thus, a thorough understanding of primordial follicle activation, the first step of folliculogenesis, is paramount. The aims of this dissertation were to advance understanding of primordial follicle activation, examine the effects of ovarian tissue cryopreservation on activation, and identify the molecular impact of cryopreservation on follicular health and function. In a fetal bovine model, bone morphogenetic protein 4 (BMP4) stimulated primordial follicle activation in cultured ovarian tissue. Co-culture of ovarian issue with BMP4 and a blocker of KIT suggested that BMP4 stimulates activation by regulating the KIT ligand/KIT pathway. Analysis by qPCR, however, revealed that BMP4 did not regulate expression of mRNA for KIT ligand or KIT. Cryopreservation of fetal bovine ovarian tissue using needle immersion vitrification demonstrated that exposure to cryoprotectants and ultra-fast cooling did not impact primordial follicle activation. Furthermore, though the ovarian tissue sustained some damage following exposure to cryoprotectants and vitrification, normal follicular morphology was recovered after seven days of culture. The addition of sucrose to the cryoprotectants, however, did not improve follicular morphology. The protocol for needle immersion vitrification was optimized for peri-pubertal feline ovarian tissue. Cryoprotectant exposure temperature affected follicular health and function, with 4[MASCULINE ORDINAL INDICATOR]C being preferable to room temperature. However, the damage sustained due to vitrification was not repaired during culture, as it was in the fetal bovine tissue. Once again, sucrose did not improve follicular health or function. Nascent RNA transcription was found to be an early marker of follicular health. These new findings for primordial follicle activation and ovarian tissue vitrification can be used to improve assisted reproductive techniques, thereby advancing fertility preservation in women and wildlife species.