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Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development
Published on: February 9, 2015
The developing Xenopus oocyte specifies the type of gonadotropin-stimulated steroidogenesis performed by its
1Department of Anatomy, Faculty of Science, Mahidol University, Rajathevi, Bangkok, Thailand.
Abstract:
As a response to gonadotropin, amphibian ovarian follicles primarily synthesize and secrete estradiol-17 beta (E2) during vitellogenesis and progesterone (P) when fully grown. Stage IV (vitellogenic) and stage VI (full-grown) ovarian follicles from Xenopus laevis, as well as intermediate sizes, were used to explore this change in steroidogenesis. Optimum steroidogenesis occurred in both stage IV and stage VI follicle exposed for 6 h to 20 IU human chorionic gonadotropin/mL. Although the total amounts of steroid fund were about the same, the E2/P ratios ranged from 26 to 35 for intact stage IV follicles, but only 0.02-0.03 for intact stage VI follicles. Steroid-producing follicle cells were isolated from stage IV and stage VI follicles by non-enzymatic procedures, were washed and were tested for steroidogenic activity in the absence of oocytes. In both cases, P was the predominant steroid produced (E2/P = 0.004-0.04), so the presence of stage IV, but not stage VI, oocytes appears to be necessary for E2 production as a response to gonadotropin. Octanol had no significant effect on the E2/P ratio of intact stage IV follicle. Dissected oocyte/follicle cell preparations from stage IV follicles were also periodically challenged with gonadotropin over 72 h, during which time most follicle cells detached from the oocyte and formed a monolayer over the bottom of the culture dish. The relatively high E2/P ratios for such preparations showed no significant change when stimulated with gonadotropin at various times over the 72 h, as long as the medium was not replaced. We conclude that the estrogenic effect of stage IV oocytes is most likely mediated by a secretory product rather than by gap junctions or by cell contact. Because the X. laevis oocyte has been shown to be a self-differentiating cell, the steroidogenic shift that occurs in developing ovarian follicles appears to be fundamentally regulated by the growing oocyte as if undergoes a physiological change rather than by different gonadotropins.
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