@article{wu_su_safwat_sebastian_miller_2004, title={Rapid, efficient isolation of murine gonadotropes and their use in revealing control of follicle-stimulating hormone by paracrine pituitary factors}, volume={145}, ISSN={["1945-7170"]}, DOI={10.1210/en.2004-0257}, abstractNote={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-2Kk) so they could be purified in vitro using paramagnetic anti-H-2Kk microbeads. 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 FSHbeta 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.}, number={12}, journal={ENDOCRINOLOGY}, author={Wu, JC and Su, P and Safwat, NW and Sebastian, J and Miller, WL}, year={2004}, month={Dec}, pages={5832–5839} }
 @article{huang_sebastian_strahl_wu_miller_2001, title={The promoter for the ovine follicle-stimulating hormone-beta gene (FSH beta) confers FSH beta-like expression on luciferase in transgenic mice: Regulatory studies in vivo and in vitro}, volume={142}, ISSN={["0013-7227"]}, DOI={10.1210/en.142.6.2260}, abstractNote={Transgenic mice harboring the ovine FSHβ (oFSHβ) promoter plus first intron (from −4741 to +759 bp) linked to a luciferase reporter gene (oFSHβLuc) were generated to determine whether this promoter can direct tissue-specific expression in vivo and serve as a model for studying hormonal regulation of the FSHβ gene. Of six lines of transgenic mice analyzed, luciferase was detected uniquely in the pituitaries of five of them. Pituitary luciferase activity was decreased 51–99% by chronic GnRH treatment (Lupron depot). Orchidectomy caused a 2- to 8-fold increase, and ovariectomy caused a 2- to 27-fold increase in pituitary luciferase activity. Furthermore, pituitary luciferase expression was consistently higher on estrus than on diestrus (3- to 20-fold). These data strongly suggested that the transgene was expressed specifically in pituitary gonadotropes and regulated in the same way as the endogenous mouse FSHβ gene. Using primary pituitary cell cultures prepared from these transgenic mice, basal luciferase expression was maximal on day 3 and then decreased by day 6 of culture, a pattern reflected by endogenous mouse FSH secretion. In these pituitary cultures, basal oFSHβLuc expression was decreased 61–82% by follistatin or 59–79% by inhibin. Similarly, mouse FSH secretion was decreased 71% by follistatin or 65% by inhibin. Progesterone inhibited oFSHβLuc expression by 44–51%, but it had no effect on endogenous mouse FSH secretion. Estradiol lowered FSH secretion by 21%, but did not decrease oFSHβLuc expression significantly. In conclusion, these data demonstrated the ability of the oFSHβ promoter to direct expression of a reporter gene specifically to pituitary gonadotropes in transgenic mice. Studying oFSHβLuc expression in vivo and in cell cultures derived from pituitaries of these transgenic mice should prove useful for understanding many features of FSHβ regulation.}, number={6}, journal={ENDOCRINOLOGY}, author={Huang, HJ and Sebastian, J and Strahl, BD and Wu, JC and Miller, WL}, year={2001}, month={Jun}, pages={2260–2266} }
 @article{huang_sebastian_strahl_wu_miller_2001, title={Transcriptional regulation of the ovine follicle-stimulating hormone-beta gene by activin and gonadotropin-releasing hormone (GnRH): Involvement of two proximal activator protein-1 sites for GnRH stimulation}, volume={142}, ISSN={["0013-7227"]}, DOI={10.1210/en.142.6.2267}, abstractNote={Previous studies from our laboratory demonstrated that a transgene consisting of the promoter for the ovine FSH β-subunit gene and a luciferase reporter (wt-oFSHβLuc) was expressed and regulated like the FSHβ gene in vivo and in vitro. In the present study pituitary cultures were prepared from these transgenic mice as well as mice carrying mutated oFSHβLuc lacking two functional activator protein-1 (AP-1) sites at −120 and −83 bp (mut-oFSHβLuc). These AP-1 sites were reported necessary for induction of oFSHβLuc by GnRH in a HeLa cell system. To examine the importance of the two AP-1 sites in mediating GnRH and activin effects in primary gonadotropes, pituitary cultures derived from transgenic mice were pretreated with follistatin to remove activin or activin-like factors present in the cultures. Follistatin lowered luciferase expression in cultures carrying both wt-oFSHβLuc and mut-oFSHβLuc transgenes by 74–86%, and subsequent addition of activin induced luciferase expression of both wt- and mut-transgenes by 4- to 14-fold within 4 h, suggesting that these AP-1 sites are not involved in activin stimulation of FSHβ gene transcription. When GnRH was added along with activin, the wt-oFSHβLuc transgene was induced 200% compared with activin alone, but this effect was not observed with the mut-oFSHβLuc transgene. These data confirmed the HeLa cell studies showing that GnRH signals through two AP-1 sites to increase oFSHβ transcription in gonadotropes. However, as the mutation of both AP-1 sites had no apparent effect on the expression and regulation of the transgene in vivo (basal, castration, GnRH down-regulation, cycle stage, and GnRH immunoneutralization), it appears that these AP-1 sites have little influence over the major effect of GnRH observed in vivo. These data also showed that activin plays a major role in transcriptional regulation of the FSHβ gene, and the oFSHβ promoter contains the activin response element(s) that is as yet undefined.}, number={6}, journal={ENDOCRINOLOGY}, author={Huang, HJ and Sebastian, J and Strahl, BD and Wu, JC and Miller, WL}, year={2001}, month={Jun}, pages={2267–2274} }
 @article{gardner_sebastian_miller_2000, title={Estradiol induces and hyperglycosylates the receptor for ovine gonadotropin-releasing hormone}, volume={141}, ISSN={["1945-7170"]}, DOI={10.1210/en.141.1.91}, abstractNote={The crucial first link between GnRH and its pleiotropic stimulation of the reproductive system is its receptor (GnRHRec). In mammals, 17beta-estradiol is a major regulator of GnRH action, and part of its regulation occurs at the level of the GnRHRec. In ovine pituitary cultures, estradiol simultaneously increases GnRHRec and GnRH-stimulated LH secretion (the LH response), but after 6-15 h the effect of estradiol becomes paradoxical, and the LH response rapidly decreases to control levels (by 24 h), whereas GnRHRec remains elevated. A preliminary study used photoaffinity labeling of the GnRHRec to show that estradiol can induce 38- and 43-kDa GnRHRec. The photoaffinity technique has been used here to 1) further investigate estradiol-mediated induction of GnRHRec, 2) define the nature of the different sized GnRHRecs, and 3) determine whether the larger size is related to degradation of the LH response. The effect of estradiol is compared with that of inhibin, which only induces the 38-kDa GnRHRec and always increases the LH response to GnRH treatment. Receptors for GnRH in ovine pituitary cultures were photoaffinity labeled with [125I](azidobenzoyl-D-Lys6-des-Gly10)-GnRH-N-ethylamide and analyzed by SDS-PAGE. Treatment with estradiol or inhibin for 6-24 h induced a 38-kDa GnRHRec only. Further treatment with estradiol (>24 h), but not inhibin, shifted the apparent Mr of the GnRHRec to 43 kDa. Phosphatase treatment did not reverse this apparent Mr change. Analysis of receptor glycosylation using N-glycosidase F or tunicamycin showed that the 43-kDa GnRHRec was a hyperglycosylated form of the 38-kDa GnRHRec. The 38-kDa GnRHRec, in turn, was a glycosylated form of the 29-kDa GnRHRec. The studies presented here define several glycosylated intermediates of the ovine GnRHRec that are induced by estradiol and/or inhibin. The function of estrogen-mediated hyperglycosylation is unclear, but kinetic studies dissociate it from degeneration of the LH response to GnRH.}, number={1}, journal={ENDOCRINOLOGY}, author={Gardner, DB and Sebastian, J and Miller, WL}, year={2000}, month={Jan}, pages={91–99} }
 @article{strahl_huang_sebastian_ghosh_miller_1998, title={Transcriptional activation of the ovine follicle-stimulating hormone beta-subunit gene by gonadotropin-releasing hormone: Involvement of two activating protein-1-binding sites and protein kinase C}, volume={139}, ISSN={["1945-7170"]}, DOI={10.1210/en.139.11.4455}, number={11}, journal={ENDOCRINOLOGY}, author={Strahl, BD and Huang, HJ and Sebastian, J and Ghosh, BR and Miller, WL}, year={1998}, month={Nov}, pages={4455–4465} }