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Rapid, Efficient Isolation of Murine Gonadotropes and Their Use in Revealing Control of Follicle-Stimulating Hormone by Paracrine Pituitary Factors

Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622

Address all correspondence and requests for reprints to: Dr. William L. Miller, Department of Molecular and Structural Biochemistry, Box 7622, North Carolina State University, Raleigh, North Carolina 27695-7622. E-mail: wlmiller{at}ncsu.edu.

FSH and LH are produced only in gonadotropes, which are reported to comprise 3–12% of mammalian pituitaries. Factors made within the pituitary are powerful regulators of FSH and also influence LH expression, but their identities and cellular origins are unknown because it is impossible to isolate and individually analyze different pituitary cell types. In this study FSH-producing gonadotropes were specifically tagged in vivo with a transgenic cell surface antigen (H-2K^k) so they could be purified in vitro using paramagnetic anti-H-2K^kmicrobeads. After enzymatic dispersion of pituitary cells, it took 1 h or less to extract 55 ± 5% of FSH-producing gonadotropes at 95 ± 0.5% purity, as judged by immunostaining for FSH or prolactin. Although this procedure selected for FSH expression, the isolated gonadotropes were also enriched 22-fold for LH-containing cells. For studies aimed at understanding factors that control FSH transcription, the purified gonadotropes were treated with activin A, which increased FSH expression 480% above basal levels (d 3 of culture). Coincubation of purified gonadotropes with pituitary nongonadotropes increased FSH expression 800% (d 3 of culture). Follistatin, an activin-binding protein, decreased FSH expression 35–50%, suggesting that gonadotropes make some activin and/or other follistatin-sensitive molecule(s) that induce FSH. These data show that paracrine factors from pituitary nongonadotropes can play a major role in controlling FSHß at the pituitary level. The study presented here describes a rapid, reliable, and efficient method for isolating any specialized cell type, including all cells that produce endocrine hormones.

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