|Cushman, Robert - Bob|
Submitted to: Human Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/20/2001
Publication Date: 1/20/2002
Citation: Cushman, R.A., Wahl, C.M., Fortune, J.E. 2002. Bovine ovarian cortical pieces grafted to chick embryonic membranes: a model for studies on the activation of primordial follicles. Human Reproduction. 17(1):48-54.
Interpretive Summary: There are two systems for increasing the number of oocytes collected from a cow during her reproductive life time, increasing ovulation rate (either by hormonal treatment or increased selection pressure) or culturing the microscopic follicles to grow them to a stage where the oocytes are competent to undergo fertilization. Cultures also provide information on the mechanisms controlling follicle growth and oocyte quality, and subsequently, this information can be used to develop new superovulatory treatments. Because current culture systems do not support growth of oocytes to competence, we used chick chorioallantoic membrane grafts. However, growth of follicles in the graft lagged behind that of follicles placed in culture, suggesting that an inhibitory factor was, likely, produced by the chick. Chicks of both genders produce Mullerian Inhibitory Substance (MIS or anti-Mullerian Hormone) and MIS has been shown to be an inhibitory hormone in the ovary, we theorized that MIS, produced by the chick gonads is the inhibitory substance that prevents follicle growth in the chorioallantoic membrane grafts. There will need to be further work with follicle culture systems before they can be used to grow bovine oocytes to competency.
Technical Abstract: 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.