Bovine ovarian cortical pieces grafted to chick embryonic membranes: A model for studies on the activation of primordial follicles

BACKGROUND: Little is known about the factors that control the initiation of growth of primordial follicles. Primordial follicles in pieces of fetal bovine ovarian cortex spontaneously activate in vitro and develop to the primary stage, but few follicles develop further. For decades, embryologists have grafted tissue to the chorioallantoic membrane (CAM) of chick embryos to study the development of various organs and structures. METHODS AND RESULTS: To test the hypothesis that grafting cortical pieces beneath the CAM membrane of 6-day-old chick embryos (`in ovo') would support the activation of primordial follicles and the growth of activated follicles to the secondary stage, ovarian cortical pieces from six bovine fetuses (6-8 months gestation) were placed either in ovo or in organ culture in serum-free medium (in vitro). Cortical pieces were retrieved after 0, 2, 4, 7, or 10 days in ovo or in vitro. Histological examination revealed a dramatic infiltration of the CAM-grafted cortical pieces with blood vessels. By day 2 in vitro, the number of primordial follicles had declined by 87% concomitant with a 3.5-fold increase in primary follicles (P < 0.01), providing evidence of the expected activation of primordial follicles. Unexpectedly, primordial follicles were not activated in CAM-grafted tissue, as shown by maintenance of their numbers and lack of increase in primary follicles during 10 days in ovo. In experiment 2, a subset of pieces was switched from culture to CAM grafts and from CAM grafts to culture on day 2. The CAM did not support the growth of primary follicles activated in vitro, apparently because the activated follicles did not survive the transfer (P < 0.05). However, primordial follicles maintained in ovo retained their ability to activate; after their removal from the CAM into culture, primordial follicles decreased in number and primary follicles increased in number within 2 days (P < 0.05). CONCLUSIONS: The CAM graft will provide a useful model for studying the factors involved in activation of primordial follicles.