Expression and regulation of the beta-subunit of ovine follicle-stimulating hormone relies heavily on a promoter sequence likely to bind Smad-associated proteins.

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.

Conditional induction of ovulation in mice.

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.

Rapid, efficient isolation of murine gonadotropes and their use in revealing control of follicle-stimulating hormone by paracrine pituitary factors.

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.