Current techniques to study (i)in vitro(/i) the early stages of follicular development are handicapped by the spontaneous wholesale transition of primordial to primary follicles. Implantation of ovarian tissue into SCID mice is possible but expensive. We have, therefore, adapted a model first described in 1958. Fragments of ovarian cortex are implanted on the chorioallantoic membrane (CAM) of 5-6 day old fertilised chick eggs. The membrane is rich in blood vessels and results in vascularised fragments of ovarian tissue that develop in an apparently physiological manner. White fertilised chicken eggs were incubated at 37-38 degC and 55% relative humidity. On day 5-6, a triangular window was cut in the shell (Dremel handy tool), the shell membrane pierced, the tissue placed on the CAM and the window sealed. At day 10 of incubation the tissue was retrieved, fixed, embedded in paraffin and sectioned at 5-6 microns.
Overall fetal survival rates were 31.8%. Bacterial culture revealed that infection was not the cause of egg death. Successful vascularisation was heavily dependent on fetal survival. 57% of viable eggs contained successfully vascularised tissue. Average time taken to implant was 6 min 9 sec. At less than 5 and 6-7 minutes fetal survival rates were 80 and 23.5% respectively. Fetal survival rates with ovarian tissue from different species were 84.6% (mouse), 31.3% (rat), 36.4% (lamb), 35.1% (sheep), 39.1% (bovine) and 22.9% (human). Corresponding vascularisation rates were 42.9%, 15.0%, 8.3%, 32.1%, 21.1%, 12.1%. In vascularised tissue, both primordial and more mature follicles were observed after serial sections with haematoxylin and eosin staining.
This technique provides an inexpensive method of culturing intact ovarian cortical tissue, allowing research into the early phases of follicle growth.
03 - 05 Nov 2003
Society for Endocrinology