Date of Award


Degree Type


Degree Name

Doctor of Philosophy


The purposes of this study were: (1) to develop techniques for in vitro maturation of rat oocytes which would permit fertilization and normal embryonic development, and (2) to investigate the role of somatic cell-germ cell interactions, and hormonal influences, in the physiological regulation of oocyte maturation as assessed by these techniques.;Procedures for in vitro fertilization (IVF) of rat oocytes were developed and validated by transferring the resulting embryos to recipient females. Although embryos resulting from IVF were less successful in establishing pregnancy than appropriate in vivo fertilized controls, optimization of culture and transfer techniques enabled minimization of embyronic losses and permitted valid use of these techniques.;Oocytes matured in the presence of their cumulus cells and serum were as capable of IVF, embryonic and fetal development as ovulated oocytes. Oocytes matured in the absence of cumulus cells showed a high incidence of abnormal pronuclear formation during fertilization, indicating that the cumulus cells played a role in ensuring normal cytoplasmic maturation.;Oocytes matured in the absence of cumulus cells, serum or follicular fluid had an increased resistance to sperm penetration. When this penetration problem was overcome by drilling a hole in the zona pellucida, cumulus-free oocytes continued to show a high incidence of abnormal fertilization, verifying a role for cumulus cells in cytoplasmic maturation.;Immature oocytes obtained from prepubertal rats were capable of spontaneous nuclear maturation, and a small proportion was capable of being fertilized. Administration of pregnant mares' serum gonadotropin prior to oocyte collection increased the proportion of oocytes which subsequently underwent fertilization. Follicle-stimulating hormone (FSH) stimulation of in vitro maturing oocytes delayed nuclear maturation but did not affect the proportion of oocytes capable of undergoing fertilization. Conditioned media from cultures of FSH- or LH-stimulated granulosa cells could substitute for serum or follicular fluid in preventing zona hardening.;The results of this research indicate that a granulosa cell product(s), present in follicular fluid and serum, helps to maintain the penetrability of in vitro matured oocytes. Cumulus cells play an essential role in ensuring normal cytoplasmic maturation and, therefore, normal pronuclear formation in oocytes during fertilization.



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