Androgen responsiveness of the pituitary gonadotrope cell line LbetaT2

Abstract

Androgens have a profound effect on the hypothalamicpituitary axis by reducing the synthesis and release of the pituitary gonadotropin LH. The effect on LH is partly a consequence of a direct, steroid-dependent action on pituitary function. Although androgen action has been well studied in vivo, in vitro cell models of androgen action on pituitary gonadotropes have been scarce. Recently, an LH-expressing cell line, L T2, was generated by tumorigenesis targeted to the LH-producing cells of the mouse pituitary. The purpose of these studies was to determine the presence of androgen receptor (AR) and establish its function in this cell line. RT-PCR analysis indicated that the L T2 cell line expresses AR mRNA. Transient transfection assays, using the mouse mammary tumor virus (MMTV) promoter, showed that a functional AR is also present. Testosterone (TEST), dihydrotestosterone (DHT), 7 -methyl-19-nortestosterone (MENT), and fluoxymesterone (FLUOXY) increased reporter gene activity in the rank order of potencies MENT>DHT> TEST>FLUOXY. Additionally, activation of MMTV promoter activity by DHT in L T2 cells was diminished by the AR antagonists casodex and 2-hydroxy-flutamide, indicating that the effects of DHT are mediated through AR. In summary, these studies showed that the L T2 cell line is a useful model for the evaluation and molecular characterization of androgen action in pituitary gonadotropes.

Androgen Regulates Follicle-Stimulating Hormone β Gene Expression in an Activin-Dependent Manner in Immortalized Gonadotropes

“…gonadotrope cell line expresses endogenous FSH␤, and that expression of FSH␤ in L␤T2 cells is regulated by the same factors known to regulate FSH␤ in vivo, such as GnRH, activin, and FS (22). Previous studies had reported that L␤T2 cells, like normal gonadotropes, express ER (38) and AR (23). The data presented in the current study confirm that endogenous AR is functional and active in L␤T2 cells, demonstrating the relevance of the L␤T2 cell line as a model system to study the action of androgenic steroid hormones.…”

“…In this study, the L␤T2 mouse gonadotrope cell line serves as a model system to discriminate androgen action at the level of the isolated gonadotrope from that of the whole pituitary gland by eliminating the variables associated with whole-animal studies or complex primary pituitary cultures. The L␤T2 cells express GnRH, androgen, and activin receptors, and produce activin and FS, providing a model that allows investigation of the interactions of these hormones on the expression of gonadotropin genes (22,23). We find that androgens act directly within gonadotropes to stimulate FSH␤ gene transcription independent of GnRH, and that this androgen stimulation occurs through two conserved androgen response elements (AREs) within the FSH␤ gene, at least one of which specifically binds to AR in vitro.…”

Androgen Receptor Drives Transcription of Rat PACAP in Gonadotrope Cells

“…Cycling conditions were as follows: 94°C for 2 minutes, then 35 cycles at 94°C ϫ 30 seconds, 55°C ϫ 30 seconds, and 72°C ϫ 60 seconds and a final extension for 2 minutes at 72°C. Primers for mouse AR exon 1 and mouse ␣-tubulin have been previously described (39,43) and are detailed in Supplemental Table 1 published on The Endocrine Society's Journals Online web site at http://mend.endojournals.org. PCR products were separated on 1% agarose gel and photographed using a ChemiImager 4400 Imaging System (Alpha Innotech, Santa Clara, California).…”

“…They express AR mRNA but the expression of AR protein has been poorly characterized (35,43,47). Although early studies reported AR immunoreactivity comparable to that of mouse and rat pituitary extracts, more recent studies have reported difficulty detecting endogenous AR, concluding that the overall level of endogenous AR in L␤T2 cells is low (35,36,46).…”

Mechanisms of FSH synthesis: what we know, what we don't, and why you should care

“…However, androgens dose-and time-dependently stimulate murine Fshb promoter-reporter activity in LbT2 cells cotransfected with the androgen receptor (AR) (188). Although the cells express AR endogenously (188,234), the response is amplified in the presence of an exogenous wild-type, but not a DNA-binding-deficient, form of the receptor. Thus, the androgen effect appears to be mediated via AR binding to DNA.…”