Studies of gonadotropin synthesis and regulation, especially those involving the follicle-stimulating hormone (FSH), would benefit greatly by having a ready supply of gonadotropes in culture that accurately reflect gonadotrope function in vivo. In the past, dispersed primary gonadotropes have been prohibitively difficult to purify for such studies; also, transformed gonadotrope cell lines may not fully reflect characteristics of adult gonadotropes since they come from cells transformed at unknown stages of their development. Herein the applicant proposes isolating gonadotropes from the ImmortoMouse. All cells of the ImmortoMouse can express a thermolabile SV4O large T antigen (TLLTag) already known to transform mouse gonadotropes. Isolated gonadotropes from the ImmortoMouse should divide rapidly under permissive conditions (33 C with interferon-gamma) but revert to fully differentiated gonadotropes at 39 C which destabilizes the TLLTag. Numerous highly differentiated cell types have been amplified using this strategy and proliferated more than 50 generations without loss of differentiation. Effective isolation of ImmortoMouse gonadotropes will involve tagging them with the H2Kk cell surface antigen followed by isolation using magnetic anti-H2Kk-microbeads. The success of this procedure has recently been established in the applicant's laboratory using dispersed pituitary cells from transgenic mice that use the ovine FSH beta promoter to target H2Kk uniquely to gonadotropes. If conditional immortalization causes some gonadotropes to dedifferentiate, these cells can be eliminated by subsequent isolation based on H2Kk selection since H2Kk mimics FSH production. All gonadotrope cell lines will be monitored for production of FSH, LH and receptors for gonadotropin releasing hormone (GnRH) as well as regulation by ovarian steroids, GnRH, inhibin and other TGF beta family members such as activin. These cell lines will be available to the reproductive biology community for studies on gonadotropin regulation and synthesis.
