Lauffenburger:PamKreeger

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Pamela K. Kreeger (BE postdoctoral) In vitro systems for ovarian follicle maturation will provide an important link for clinicians to help women and young girls preserve their fertility, which may be compromised due to cancer or other disorders. Cryopreservation of ovaries, and thus the follicles which contain the egg, has been proposed; however, a critical limitation is the insufficient supply of meiotically competent oocytes obtained. The majority of follicles that survive freezing and thawing are immature follicles which will require further development prior to fertilization of the egg. Therefore, systems must be developed to allow these follicles to mature in vitro. By merging principles from tissue engineering with those from follicle biology, I developed synthetic matrices that promote follicle maturation to produce meiotically competent oocytes. Ovarian follicles are isolated from immature mice and encapsulated and cultured within alginate matrices to maintain an in vivo-like architecture, resulting in follicle maturation and development of meiotically competent oocytes. Utilizing the alginate scaffold, I examined the regulation of granulosa cell and follicle development by the extracellular matrix (ECM; 1 & 2) and follicle stimulating hormone (FSH, 3) in a three-dimensional culture system. The emerging picture of follicle development from these studies is that as the follicle progresses through development, it has distinct responses to these stimuli. It is important to note that these distinct signals integrate to regulate the quality of the oocyte, the critical determinant of whether a culture system can be utilized for embryo production. These studies indicate that the extra-follicular ECM milieu and endocrine signals such as FSH both direct this developmental process in vitro, providing a new and exciting method to regulate follicle development for fertility restoration. Follicles cultured in this system can be fertilized in vitro at rates similar to in vivo grown oocytes, and live pups which are healthy and fertile have been produced (4). Additionally, in collaboration with Sarah Bristol-Gould, I examined factors that contribute to the development of the initial follicle pool in the neonatal mouse ovary. Our studies indicate that exogenous treatment with activin, a member of the TGF-beta superfamily of growth factors, can induce germ cell proliferation and increase the number of follicles formed in the ovary 5. However, this increase is not without a cost - oocytes retrieved around puberty are significantly smaller and display tubulin abnormalities. By the onset of puberty, the number of follicles returns to untreated levels and oocytes retrieved from adult mice are normal, indicating that a compensatory mechanism exists to preserve fertility. We next examined follicle numbers throughout the fertile lifespan of the mouse, and utilizing a series of differential equations, determined that the initial pool is sufficient to account for adult fertility with no evidence to suggest a need a for stem cell mediated supplementation of this pool 6. Together, these studies highlight the critical window in which the initial follicle pool is formed in the ovary and provide evidence that this pool serves as the only source of oocytes for adult fertility. This work was conducted at Northwestern University under Dr. Lonnie Shea (Dept. of Chemical and Biological Engineering) and Dr. Teresa Woodruff (Dept. of Neurobiology and Physiology). 1. Kreeger, Woodruff, Shea, Mol Cell Endocrinol 205, Jul 2003. 2. Kreeger, Deck, Woodruff, Shea, Biomaterials 27, Feb 2006. 3. Kreeger, Fernandes, Woodruff, Shea, Biol Reprod 73, Nov 2005. 4. Xu, Kreeger, Shea, Woodruff, Tissue Engineering, Oct 2006. 5. Bristol-Gould, Kreeger, Selkirk, Kilen, Cook, Kipp, Shea, Mayo, and Woodruff, Developmental Biology, Oct 2006. 6. Bristol-Gould, Kreeger, Selkirk, Kilen, Mayo, Shea, and Woodruff, Developmental Biology, Oct 2006.