Androgen Suppresses In Vivo and In Vitro LH Pulse Secretion and Neural Kiss1 and Tac2 Gene Expression in Female Mice

Abstract

Androgens can affect the reproductive axis of both sexes. In healthy women, as in men, elevated exogenous androgens decrease gonad function and lower gonadotropin levels; such circumstances occur with anabolic steroid abuse or in transgender men (genetic XX individuals) taking androgen supplements. The neuroendocrine mechanisms by which endogenous or exogenous androgens regulate gonadotropin release, including aspects of pulsatile luteinizing hormone (LH) secretion, remain unknown. Because animal models are valuable for interrogating neural and pituitary mechanisms, we studied effects of androgens in the normal male physiological range on in vivo LH secretion parameters in female mice and in vitro LH secretion patterns from isolated female pituitaries. We also assessed androgen effects on hypothalamic and gonadotrope gene expression in female mice, which may contribute to altered LH secretion profiles. We used a nonaromatizable androgen, dihydrotestosterone (DHT), to isolate effects occurring specifically via androgen receptor (AR) signaling. Compared with control females, DHT-treated females exhibited markedly reduced in vivo LH pulsatility, with decreases in pulse frequency, amplitude, peak, and basal LH levels. Correlating with reduced LH pulsatility, DHT-treated females also exhibited suppressed arcuate nucleus Kiss1 and Tac2 expression. Separate from these neural effects, we determined in vitro that the female pituitary is directly inhibited by AR signaling, resulting in lower basal LH levels and reduced LH secretory responses to gonadotropin-releasing hormone pulses, along with lower gonadotropin gene expression. Thus, in normal adult females, male levels of androgen acting via AR can strongly inhibit the reproductive axis at both the neural and pituitary levels.

Keywords: DHT; GnRH; Kiss1; Tac2; androgen; androgen receptor; gonadotrope; kisspeptin; neurokinin B; pituitary.

Figure 1.

Schematic of experimental paradigms for testing androgen effects on LH levels in young adult male mice (Experiment 1) or LH pulse secretion and gene expression in young adult female mice (Experiments 2-4).

Figure 2.

Dose-response study comparing effects of different DHT implant doses, given for 2 weeks, on LH levels and BW in young adult male mice. A) Mean seminal vesicle (SV) weights of males after 2 weeks of DHT treatment. B) Mean % increase in BW of males during the 2-week DHT treatment period between 5 and 7 weeks of age. C) LH levels in gonad-intact males compared to GDX males with or without 2-week DHT treatment. Bars with different letters are significantly different from each other (P < 0.05).

Figure 3.

DHT treatment reduces endogenous LH pulse secretion in young adult OVX females. (A) Representative profiles of in vivo LH secretion in DHT-treated OVX female mice (middle and right columns), and control OVX littermates (left column). LH was measured in serial tail-tip bleeds from awake, unrestrained females every 6 minutes for 2 hours. Identified pulses are indicated by *. Mean LH pulse frequency (pulses/hour) (B), interpulse interval (IPI; the number of minutes between pulses) (C), basal LH level (D), pulse amplitude (E), pulse peak (zenith value of a pulse) (F), and mean LH across the entire sampling period (G) were all significantly different in OVX+DHT vs control OVX females. Different letters above bars indicate significantly different (P < 0.05) from each other.

Figure 4.

Androgens suppress Kiss1 levels in the arcuate nucleus of young adult female mice. A) Representative microscope images of Kiss1 mRNA expression in the ARC nucleus, determined with in situ hybridization, in OVX females with and without 2-week DHT treatment. Mean Kiss1 cell number (B), Kiss1 mRNA per cell (C), and total Kiss1 mRNA levels (D) in the ARC of females with and without 2-week DHT treatment. *, significantly different from OVX control group (P < 0.05).

Figure 5.

Androgens suppress Tac2 levels in the arcuate nucleus of young adult female mice. A) Representative microscope images of Tac2 mRNA expression (encoding NKB) in the ARC nucleus, determined with in situ hybridization, in OVX females with and without 2-week DHT treatment. Mean Tac2 cell number (B), Tac2 mRNA per cell (C), and total Tac2 mRNA levels (D) in the ARC of females with and without 2-week DHT treatment. *, significantly different from OVX control group (P < 0.05).

Figure 6.

Effects of androgen on in vitro LH secretion from isolated female pituitaries in response to GnRH pulse input. A, B) Mean LH secretion measured in 5-minute fractions from DHT-treated pituitaries (1 nM or 10 nM DHT) before and after brief (2-minute) GnRH pulses. The first 2 fractions show basal LH levels prior to any GnRH input, and subsequent fractions show mean LH secretion immediately following GnRH pulses. For better viewing, 1 nM (A) and 10 nM (B) DHT groups are graphed separately with vehicle (VEH) controls. Analyses of various pulse parameters are shown in subsequent panels, including mean basal LH (C), mean pulse peak (D), and mean pulse amplitude (E). *, significantly different from VEH control group (P < 0.05).

Figure 7.

Androgen lowers gonadotropin gene expression levels in isolated pituitaries of adult female mice. DHT treatment significantly lowers mean Lhβ mRNA (A) and Fshβ  (B) levels, but not Egr1  (C) levels in GnRH-pulsed female pituitaries, as measured by qPCR and normalized to Gapdh expression. *, significantly different from VEH control group (P < 0.05).