IN-VITRO FOLLICULOGENESIS OF RAT PREANTRAL FOLLICLES

The impact of various gonadotropic hormones on the growth and developm ent of secondary follicles from primordial and primary follicles obtai ned by enzymatic dissociation of the ovaries of immature 14-day-old ra ts was studied in, vitro. The substratum-adherent culture technique de veloped for studying folliculogenesis in the current study permitted d irect visualization of follicular growth on a day to day basis by avoi ding the cumbersome process of fixing and sectioning follicles in cult ure. The cultures were maintained in a serum-free modified McCoy's med ium in a humidified atmosphere containing 5% CO2 at 37 C. Daily observ ation of the culture dishes under the phase contrast microscope reveal ed that the follicles grew and developed from primordial to primary an d secondary follicular stages in the presence of FSH. Large antral fol licles were able to secrete estradiol and progesterone into the medium , indicating that the follicles are not merely formed by cellular reor ganization, but are physiologically functional competent units. The or ganized release of the oocyte with accompanying corona radiata was mad e 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. Folli cular organization was also disrupted when LH was introduced together with FSH into the medium on day 1 of culture. Primordial or primary fo llicles obtained from the ovaries could survive, but could not transfo rm to secondary follicles in the absence of FSH. The results of our in vitro studies indicate, and therefore are in agreement with earlier i n vivo studies, that FSH alone is essential for the progression of fol liculogenesis 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 physiological ly 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 t hat can finally ovulate. It also facilitates access to the growing fol licle along with its oocyte,, which can, therefore, be used as a power ful model to study the effects of various test substances on follicula r development. Additionally, the oocyte may be experimentally manipula ted and subjected to in vitro fertilization for producing animal speci es that could be used for research purposes, clinical trials, and rest oring species that are on the brink of extinction.