This dissertation, "An in Vitro Study of Ovarian Folliculogenesis in Galactosemic Rats" by Ka-wai, Lai, 黎嘉慧, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled An in vitro study of ovarian folliculogenesis in galactosemic rats Submitted by LAI KA WAI for the degree of Doctor of Philosophy at The University of Hong Kong in December 2004 Galactosemia is an autosomal recessive disorder of galactose metabolism and is caused by the absence of enzymes in the Leloir pathway. As a result, galactose metabolites accumulate in the blood and tissues. Clinical consequences include mental retardation, cataract and ovarian dysfunction. In this study, it was postulated that galactitol, from the alternate pathway of galactose metabolism, may accumulate in the follicles and thus affect folliculogenesis in the galactosemic rats. In our previous study, a galactosemic rat model had been established by feeding rats with high galactose diet for 4 weeks. Preantral follicles were isolated from these galactosemic rat ovaries by combined enzymes digestion and mechanical methods and were cultured to study the cause of ovarian failure in these animals. Using different concentrations of recombinant follicle stimulating hormone and luteinizing hormone (rFSH and rLH) in ovarian follicle culture, it was found that rFSH alone, rLH alone and a 1:1 ratio of rFSH and rLH could not sustain the growth of preantral follicles to preovulatory volumes and the in vitro ovulation rates were very low. However, when a 100:1 ratio of rFSH to rLH (0.5 IU/ml: 0.005 IU/ml) was used, more preantral follicles grew to the preovulatory stage and eventually ovulated. At this ratio of recombinant gonadotrophins, the in vitro growth and ovulation rate of the galactosemic follicles were significantly lower than that of the controls. Western Blot analysis studies showed that the aldose reductase protein expression in the galactosemic ovaries and isolated follicles were significantly higher than that of the controls. Its expression in the galactosemic follicles remained high throughout the culture period and was comparable to that of in vivo ones. The high aldose reductase was thought to lead to the accumulation of galactitol and hence affect folliculogenesis. Applying aldose reducatase antibody (1:1000) in follicular culture improved the follicular growth and in vitro ovulation rate in the galactosemic follicles. Although the treated follicles could not attain growth and ovulation rate to the same level as that in the controls, the aldose reductase antibody could neutralize partly the toxicity in the galactosemic follicles. Measurement of growth differentiation factor 9 (GDF-9) and kit receptor (c-kit) mRNA from single oocytes by RT-PCR showed that the expression of these two molecules were comparable in oocytes ovulated in vitro and in vivo, but was significantly higher than that in unovulated oocytes. Both GDF-9 and c-kit mRNA expression were significantly lower in oocytes ovulated from galactosemic follicles with or without aldose reductase antibody treatment. These two molecules could therefore serve as molecular markers to reflect the development of oocytes within the follicles. Results in this study suggested that the high expression of aldose reductase protein in the galactosemic follicles could account for compromised folliculogenesis. Although the toxicity of galactitol could be partially neutralized using aldose reductase antibody, the follicular growth, ovulation rate and storage of GDF-9 and c-kit could not be totally rest