@article{su_shafiee-kermani_gore_jia_wu_miller_2007, title={Expression and regulation of the beta-subunit of ovine follicle-stimulating hormone relies heavily on a promoter sequence likely to bind smad-associated proteins}, volume={148}, ISSN={["1945-7170"]}, DOI={10.1210/en.2006-1635}, abstractNote={FSH is essential for normal gonadal function in mammals. Expression of its beta-subunit (FSHB) controls overall production/secretion of FSH and is induced by activin. Studies with ovine FSHB promoter/reporter constructs in L beta T2 gonadotropes show that induction by activin requires a putative Smad binding element in the ovine FSHB promoter (-162AGAC-159). Similar studies reported here show that another site, juxtaposed to the Smad binding element, was also required for 81% activin induction in L beta T2 cells. This site was similar to several that bind proteins known to partner with Smads. When this site (-171ACTgcgtTT-163) was mutated by changing the nucleotides shown in lowercase letters, the resulting ovine-derived construct (mut-oFSHBLuc) was expressed poorly as a transgene in primary mouse gonadotropes (<0.001 times compared with ovine wild-type transgenes). This decrease in expression demonstrated the importance of this site for activin induction and, perhaps, basal expression, although studies with L beta T2 cells did not suggest this latter possibility. Expression of mut-oFSHBLuc in male mouse gonadotropes in vivo was at least 644 times greater than expression in all but one nongonadotrope tissue tested, indicating that mut-oFSHBLuc retained significant gonadotrope-specific expression. An increase in FSHB expression occurs during estrus in mice and is faithfully reproduced with wild-type ovine FSHBLuc transgenes, but not with mut-oFSHBLuc, indicating that the mutated site is needed for this secondary FSH surge. These data suggest that activin gathers Smads and Smad-associated proteins at the -171/-159 promoter region to regulate expression of the ovine FSHB and overall FSH production.}, number={9}, journal={ENDOCRINOLOGY}, author={Su, Pei and Shafiee-Kermani, Farideh and Gore, A. Jesse and Jia, Jingjing and Wu, Joyce C. and Miller, William L.}, year={2007}, month={Sep}, pages={4500–4508} }
 @article{su_wu_sommer_gore_petters_miller_2005, title={Conditional induction of ovulation in mice}, volume={73}, ISSN={["1529-7268"]}, DOI={10.1095/biolreprod.104.039164}, abstractNote={Abstract Follicle-stimulating hormone controls the maturation of mammalian ovarian follicles. In excess, it can increase ovulation (egg production). Reported here is a transgenic doxycycline-activated switch, tested in mice, that produced more FSHB subunit (therefore more FSH) and increased ovulation by the simple feeding of doxycycline (Dox). The transgenic switch was expressed selectively in pituitary gonadotropes and was designed to enhance normal expression of FSH when exposed to Dox, but to be regulated by all the hormones that normally control FSH production in vivo. Feeding maximally effective levels of Dox increased overall mRNA for FSHB and serum FSH by over half in males, and Dox treatment more than doubled the normal ovulation rate of female mice for up to 10 reproductive cycles. Lower levels of Dox increased the number of developing embryos by 30%. Ovarian structure and function appeared normal. In summary, gene switch technology and normal FSH regulation were combined to effectively enhance ovulation in mice. Theoretically, the same strategy can be used with any genetic switch to increase ovulation (or any highly conserved physiology) in any mammal.}, number={4}, journal={BIOLOGY OF REPRODUCTION}, author={Su, P and Wu, JC and Sommer, JR and Gore, AJ and Petters, RM and Miller, WL}, year={2005}, month={Oct}, pages={681–687} }
 @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_wu_su_zhirnov_miller_2001, title={A novel role for bone morphogenetic proteins in the synthesis of follicle-stimulating hormone}, volume={142}, ISSN={["1945-7170"]}, DOI={10.1210/en.142.6.2275}, abstractNote={FSH is produced in pituitary gonadotropes as an α/β heterodimer, and synthesis of the β-subunit is the rate-limiting step in overall FSH production. Synthesis of FSHβ can be regulated by activin and inhibin, both of which are members of the transforming growth factor-β superfamily. Bone morphogenetic proteins (BMPs) also belong to the transforming growth factor-β family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis, including regulation of FSH action in the ovary. Here we report a novel function for BMP-7 and BMP-6 in regulating FSH synthesis in the pituitary. Using primary pituitary cell cultures derived from transgenic mice that carry the ovine FSHβ promoter linked to a luciferase reporter gene (oFSHβLuc), BMP-7 or BMP-6 was found to stimulate oFSHβLuc expression by 6-fold. Transient expression of the oFSHβLuc in a transformed gonadotrope cell line, LβT2, was induced 4-fold by BMP-7 or BMP-6 treatment. More importantly, BMP-7 and BMP-6 increased endogenous FSH secretion by 10- and 14-fold, respectively, from LβT2 cells, demonstrating for the first time that a functional signaling BMP system is present in gonadotropes. Two bioneutralizing antibodies to BMP-7, which cross-react with BMP-6, but not with activin A, decreased basal oFSHβLuc expression and FSH secretion from transgenic mouse pituitary cultures by 83–88% and 47–48%, respectively, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH synthesis. Neither bioneutralizing antibody to activin A or activin B decreased basal oFSHβLuc expression or mouse FSH secretion significantly. Dose-dependent inhibition of FSH synthesis by anti-BMP7 was also observed in rat and sheep pituitary cultures. These results combined with the fact that the messenger RNAs for BMP-7 and BMP-6 were detected in mouse pituitaries and LβT2 cells indicate that BMP-7 and/or BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression in vivo.}, number={6}, journal={ENDOCRINOLOGY}, author={Huang, HJ and Wu, JC and Su, P and Zhirnov, O and Miller, WL}, year={2001}, month={Jun}, pages={2275–2283} }
 @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{strahl_huang_pedersen_wu_ghosh_miller_1997, title={Two proximal activating protein-1-binding sites are sufficient to stimulate transcription of the ovine follicle-stimulating hormone-beta gene}, volume={138}, ISSN={["1945-7170"]}, DOI={10.1210/en.138.6.2621}, abstractNote={FSH is an important regulator of mammalian gametogenesis and the female reproductive cycle. Although little is known about the transcriptional regulation of the β-subunit (the rate-limiting subunit of FSH synthesis), sequence analysis of the ovine FSHβ promoter has revealed a number of potential activating protein-1 (AP-1; Jun/Fos)-binding sites. To determine whether the gene encoding theβ -subunit of ovine FSH (oFSHβ) is responsive to AP-1 transcriptional complexes, chimeric constructs containing deleted portions of the oFSHβ promoter fused to a luciferase reporter were transiently transfected along with c-Jun and c-Fos expression constructs into JAR cells. Analysis of these deletion constructs revealed that the proximal promoter of oFSHβ is highly stimulated by c-Jun and c-Fos proteins (typically 20-fold with a reporter construct containing oFSHβ sequences from −215 to +759 bp). This stimulation was lost when a similar construct containing sequences from −84 to+ 759 bp was tested. Transcriptional start site analysis using reverse transcription-PCR verified that the transcriptional initiation of the− 215-bp deletion construct, with or without cotransfected c-Jun/c-Fos, was the same as that observed in vivo. Computer analysis of oFSHβ sequences from −215 to +1 bp identified four putative AP-1-like elements, located at −155, −120, −83, and −10 bp. Gel retardation experiments using oligonucleotides corresponding to the four putative AP-1-like sites revealed that only −120 and −83 sites in oFSHβ bound AP-1 proteins in vitro. Site-directed mutagenesis of the −120 and −83 sites showed that each element was required for stimulation by c-Jun and c-Fos proteins as well as 12-O-tetradecanoyl phorbol-13-acetate in transient transfection assays. Finally, immunocytochemical dual labeling was used to show that more than 75% of all FSHβ-containing cells in ovine pituitary sections from cycling ewes contained nuclear c-Jun, JunB, JunD, and Fos proteins. These data, taken together, show that oFSHβ transcription can be stimulated by c-Jun and c-Fos proteins via two functionally linked AP-1-like sites in the oFSHβ proximal promoter and that these sites are likely to be important regulators of FSH production in vivo.}, number={6}, journal={ENDOCRINOLOGY}, author={Strahl, BD and Huang, HJ and Pedersen, NR and Wu, JC and Ghosh, BR and Miller, WL}, year={1997}, month={Jun}, pages={2621–2631} }
 @article{line_wu_arnold_jennings_rubin_1997, title={Water quality of first flush runoff from 20 industrial sites}, volume={69}, ISSN={["1061-4303"]}, DOI={10.2175/106143097X125489}, abstractNote={A sampling program was conducted to assess the quality of first flush storm water runoff from 10 industrial groups typical of many businesses located in North Carolina. Analysis of samples collected during the first 30 min of runoff (first flush) indicated that zinc and copper were the most common of the eight metals measured in runoff from the 20 industrial sites monitored. Ten volatile organic, semivolatile organic, or pesticide compounds were found at eight different sites, with the most common being methylene chloride (three sites). Conventional pollutants such as nutrients and solids were measured at varying levels at every site, but were generally the highest where a significant amount of biological waste or exposed soil was present.}, number={3}, journal={WATER ENVIRONMENT RESEARCH}, author={Line, DE and Wu, J and Arnold, JA and Jennings, GD and Rubin, AR}, year={1997}, pages={305–310} }
 @article{wu_sealfon_miller_1994, title={GONADAL-HORMONES AND GONADOTROPIN-RELEASING-HORMONE (GNRH) ALTER MESSENGER-RIBONUCLEIC-ACID LEVELS FOR GNRH RECEPTORS IN SHEEP}, volume={134}, ISSN={["1945-7170"]}, DOI={10.1210/en.134.4.1846}, number={4}, journal={ENDOCRINOLOGY}, author={WU, JC and SEALFON, SC and MILLER, WL}, year={1994}, month={Apr}, pages={1846–1850} }