Androgen receptor positively regulates gonadotropin-releasing hormone receptor in pituitary gonadotropes.

Within pituitary gonadotropes, the gonadotropin-releasing hormone receptor (GnRHR) receives hypothalamic input from GnRH neurons that is critical for reproduction. Previous studies have suggested that androgens may regulate GnRHR, although the mechanisms remain unknown. In this study, we demonstrated that androgens positively regulate Gnrhr mRNA in mice. We then investigated the effects of androgens and androgen receptor (AR) on Gnrhr promoter activity in immortalized mouse LßT2 cells, which represent mature gonadotropes. We found that AR positively regulates the Gnrhr proximal promoter, and that this effect requires a hormone response element (HRE) half site at -159/-153 relative to the transcription start site. We also identified nonconsensus, full-length HREs at -499/-484 and -159/-144, which are both positively regulated by androgens on a heterologous promoter. Furthermore, AR associates with the Gnrhr promoter in ChIP. Altogether, we report that GnRHR is positively regulated by androgens through recruitment of AR to the Gnrhr proximal promoter.

Msx1 homeodomain protein represses the αGSU and GnRH receptor genes during gonadotrope development.

Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LßT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at -114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program.

Kisspeptin regulates gonadotropin genes via immediate early gene induction in pituitary gonadotropes.

Kisspeptin signaling through its receptor, Kiss1R, is crucial for many reproductive functions including puberty, sex steroid feedback, and overall fertility. Although the importance of Kiss1R in the brain is firmly established, its role in regulating reproduction at the level of the pituitary is not well understood. This study presents molecular analysis of the role of kisspeptin and Kiss1R signaling in the transcriptional regulation of the gonadotropin gene ß-subunits, LHß and FSHß, using LßT2 gonadotrope cells and murine primary pituitary cells. We show that kisspeptin induces LHß and FSHß gene expression, and this induction is protein kinase C dependent and mediated by the immediate early genes, early growth response factor 1 and cFos, respectively. Additionally, kisspeptin induces transcription of the early growth response factor 1 and cFos promoters in LßT2 cells. Kisspeptin also increases gonadotropin gene expression in mouse primary pituitary cells in culture. Furthermore, we find that Kiss1r expression is enhanced in the pituitary of female mice during the estradiol-induced LH surge, a critical component of the reproductive cycle. Overall, our findings indicate that kisspeptin regulates gonadotropin gene expression through the activation of Kiss1R signaling through protein kinase C, inducing immediate early genes in vitro, and responds to physiologically relevant cues in vivo, suggesting that kisspeptin affects pituitary gene expression to regulate reproductive function.

Prenatal exposure to low levels of androgen accelerates female puberty onset and reproductive senescence in mice.

Sex steroid hormone production and feedback mechanisms are critical components of the hypothalamic-pituitary-gonadal (HPG) axis and regulate fetal development, puberty, fertility, and menopause. In female mammals, developmental exposure to excess androgens alters the development of the HPG axis and has pathophysiological effects on adult reproductive function. This study presents an in-depth reproductive analysis of a murine model of prenatal androgenization (PNA) in which females are exposed to a low dose of dihydrotestosterone during late prenatal development on embryonic d 16.5-18.5. We determined that PNA females had advanced pubertal onset and a delay in the time to first litter, compared with vehicle-treated controls. The PNA mice also had elevated testosterone, irregular estrous cyclicity, and advanced reproductive senescence. To assess the importance of the window of androgen exposure, dihydrotestosterone was administered to a separate cohort of female mice on postnatal d 21-23 [prepubertal androgenization (PPA)]. PPA significantly advanced the timing of pubertal onset, as observed by age of the vaginal opening, yet had no effects on testosterone or estrous cycling in adulthood. The absence of kisspeptin receptor in Kiss1r-null mice did not change the acceleration of puberty by the PNA and PPA paradigms, indicating that kisspeptin signaling is not required for androgens to advance puberty. Thus, prenatal, but not prepubertal, exposure to low levels of androgens disrupts normal reproductive function throughout life from puberty to reproductive senescence.

Inhibition of renin-angiotensin system (RAS) reduces ventricular tachycardia risk by altering connexin43.

Renin-angiotensin system (RAS) activation is associated with arrhythmias. We investigated the effects of RAS inhibition in cardiac-specific angiotensin-converting enzyme (ACE) overexpression (ACE 8/8) mice, which exhibit proclivity to ventricular tachycardia (VT) and sudden death because of reduced connexin43 (Cx43). ACE 8/8 mice were treated with an ACE inhibitor (captopril) or an angiotensin receptor type-1 blocker (losartan). Subsequently, electrophysiological studies were performed, and the hearts were extracted for Cx43 quantification using immunoblotting, immunohistochemistry, fluorescent dye spread method, and sodium current quantification using whole cell patch clamping. VT was induced in 12.5% of captopril-treated ACE 8/8 and in 28.6% of losartan-treated mice compared to 87.5% of untreated mice (P < 0.01). Losartan and captopril treatment increased total Cx43 2.4-fold (P = 0.01) and the Cx43 phosphorylation ratio 2.3-fold (P = 0.005). Treatment was associated with a recovery of gap junctional conductance. Survival in treated mice improved to 0.78 at 10 weeks (95% confidence interval 0.64 to 0.92), compared to the expected survival of less than 0.50. In a model of RAS activation, arrhythmic risk was correlated with reduced Cx43 amount and phosphorylation. RAS inhibition resulted in increased total and phosphorylated Cx43, decreased VT inducibility, and improved survival.

Cardiac-restricted angiotensin-converting enzyme overexpression causes conduction defects and connexin dysregulation.

Renin-angiotensin (RAS) system activation is associated with an increased risk of sudden death. Previously, we used cardiac-restricted angiotensin-converting enzyme (ACE) overexpression to construct a mouse model of RAS activation. These ACE 8/8 mice die prematurely and abruptly. Here, we have investigated cardiac electrophysiological abnormalities that may contribute to early mortality in this model. In ACE 8/8 mice, surface ECG voltages are reduced. Intracardiac electrograms showed atrial and ventricular potential amplitudes of 11% and 24% compared with matched wild-type (WT) controls. The atrioventricular (AV), atrio-Hisian (AH), and Hisian-ventricular (HV) intervals were prolonged 2.8-, 2.6-, and 3.9-fold, respectively, in ACE 8/8 vs. WT mice. Various degrees of AV nodal block were present only in ACE 8/8 mice. Intracardiac electrophysiology studies demonstrated that WT and heterozygote (HZ) mice were noninducible, whereas 83% of ACE 8/8 mice demonstrated ventricular tachycardia with burst pacing. Atrial connexin 40 (Cx40) and connexin 43 (Cx43) protein levels, ventricular Cx43 protein level, atrial and ventricular Cx40 mRNA abundances, ventricular Cx43 mRNA abundance, and atrial and ventricular cardiac Na(+) channel (Scn5a) mRNA abundances were reduced in ACE 8/8 compared with WT mice. ACE 8/8 mice demonstrated ventricular Cx43 dephosphorylation. Atrial and ventricular L-type Ca(2+) channel, Kv4.2 K(+) channel alpha-subunit, and Cx45 mRNA abundances and the peak ventricular Na(+) current did not differ between the groups. In isolated heart preparations, a connexin blocker, 1-heptanol (0.5 mM), produced an electrophysiological phenotype similar to that seen in ACE 8/8 mice. Therefore, cardiac-specific ACE overexpression resulted in changes in connexins consistent with the phenotype of low-voltage electrical activity, conduction defects, and induced ventricular arrhythmia. These results may help explain the increased risk of arrhythmia in states of RAS activation such as heart failure.