The impact of various gonadotropic hormones on the growth and development of secondary follicles from primordial and primary follicles obtained by enzymatic dissociation of the ovaries of immature 14-day-old rats was studied in vitro. The substratum-adherent culture technique developed for studying folliculogenesis in the current study permitted direct visualization of follicular growth on a day to day basis by avoiding the cumbersome process of fixing and sectioning follicles in culture. The cultures were maintained in a serum-free modified McCoy's medium in a humidified atmosphere containing 5% CO2 at 37 C. Daily observation of the culture dishes under the phase contrast microscope revealed that the follicles grew and developed from primordial to primary and secondary follicular stages in the presence of FSH. Large antral follicles were able to secrete estradiol and progesterone into the medium, indicating that the follicles are not merely formed by cellular reorganization, but are physiologically functional competent units. The organized release of the oocyte with accompanying corona radiata was made possible in some secondary follicles with large antral structures by introducing LH into the culture medium. However, introduction of hCG (which has the biological properties of LH) into the cultures on day 1 resulted in follicular degeneration within 3-4 days of culture. Follicular organization was also disrupted when LH was introduced together with FSH into the medium on day 1 of culture. Primordial or primary follicles obtained from the ovaries could survive, but could not transform to secondary follicles in the absence of FSH. The results of our in vitro studies indicate, and therefore are in agreement with earlier in vivo studies, that FSH alone is essential for the progression of folliculogenesis to the preovulatory condition, and that LH is essential for the organized expulsion of the oocyte from a mature follicle. Our technique, described in the current study, for producing physiologically functional secondary follicles in culture not only allows progress in folliculogenesis to be monitored very closely, but also serves as a model for studying the various intrinsic factors that may be involved in the successful development of dominant mature Graafian follicles that can finally ovulate. It also facilitates access to the growing follicle along with its oocyte, which can, therefore, be used as a powerful model to study the effects of various test substances on follicular development.subjected to in vitro fertilization for producing animal species that could be used for research purposes, clinical trials, and restoring species that are on the brink of extinction.
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