Effect of Androgens on Sexual Differentiation of Pituitary Gamma-Aminobutyric Acid Receptor Subunit GABA_B Expression

Abstract

Previous work demonstrated a sexually dimorphic ontogenic expression of γ-aminobutyric acid receptors (GABA_BR) in rat pituitary. As sex steroids determine sex-specific expression patterns, we now studied the effect of sex hormones on pituitary GABA_BR expression. GABA_BR subunits, measured by Western blot and by semi-quantitative RT-PCR and luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone measured by RIA were determined in two experimental designs: First experimental design: 8- and 15-day-old females (8F, 15F); 8F and 15F treated with 100 µg testosterone propionate (TP) on day 1 of life (8F100TP, 15F100TP), 8- and 15-day-old males (8M, 15M) and 8M and 15M castrated on day 1 (8MC, 15MC). Second experimental design: 8-day-old female and male animals: 8F, 8F100TP, 8F treated with 1 µg/day TP on days 1–4 (8F1TP), 8F treated with the androgen antagonist Flutamide (Flut: 2.5 mg/100 g BW of pregnant mother on days E17-E23) (8F-Flut), 8M, 8MC, 8M treated with Flut as above (8M-Flut) and 8MC-Flut. In these animals, in addition, GABA, glutamate, aspartate and taurine were measured by HPLC in hypothalami and cortex. In the first set of experiments, GABA_B1R mRNA/protein expression was higher in 8F than in 15F, 8M or 15M. In 8F100TP, GABA_B1R mRNA/protein decreased to male levels. TP treatment did not alter GABA_B1R expression in 15F. There was no difference in GABA_B1R expression between 8M and 15M and neonatal castration did not modify its expression. In the second set of experiments, TP (1 µg) or Flut did not modify GABA_B1R in 8F, while 100 µg TP continued to decrease GABA_B1R expression. In 8M, Flut, alone or with castration, increased GABA_B1R mRNA/protein expression to 8F. Hypothalamic GABA content followed the same pattern as pituitary GABA_BR expression in 8-day-old animals, suggesting a cross-regulation. With regard to hormonal levels, 100 µg, but not 1 µg TP altered gonadotropins at 8 days, although both treatments effectively androgenized females as evidenced by lack of cycling. We conclude that androgens, acting pre- and postnatally, decrease pituitary GABA_BR subunit expression.

Lack of functional GABAB receptors alters GnRH physiology and sexual dimorphic expression of GnRH and GAD-67 in the brain

“…Functional GABA B Rs are obligate heterodimers composed of a GABA B1 and a GABA B2 subunit (12,48). In this regard, we showed that GABA B R subunits have particular ontogenic expression patterns in the rat hypothalamus and the pituitary (9,10) and that their expressions show sexual dimorphisms at early stages of development (8,9). In GABA B1 knockout (GABA B1 KO) mice, which lack functional GABA B Rs (79), we demonstrated that GABA B Rs are involved in regulating basal prolactin (PRL) release in males and that the hypothalamic-hypophyseal-ovarian axis is compromised in females, with significant alterations in cyclicity, postcastration LH increase, and reproduction indexes (14).…”

“…The anterior limit of the fragments was a plane at the height of the anterior commissure, lateral limits were the hypothalamic fissures, the posterior limit was behind the mammilary bodies, and the in-depth limit was the subthalamic sulcus, as previously described (8). The olfactory bulbs (OB) and frontoparietal cortex (CT) were also rapidly dissected and frozen.…”

“…GABA, glutamate, and taurine contents were determined by HPLC as previously described (8). Briefly, aminoacidic dansyl derivatives were measured using a HPLC system, which consisted of a model 125 Programmable Solvent Module (Beckman Instruments, Fullerton, CA), a 5-m 25 cm ϫ 4.6 mm Ultrasphere ODS reversed-phase column (Beckman), a model 166 programmable UV detector module (254 nm; Beckman), and System Gold software (Beckman).…”

Hypothyroid Phenotype Is Contributed by Mitochondrial Complex I Inactivation Due to Translocated Neuronal Nitric-oxide Synthase

“…Rats were divided into five groups (n ϭ 12/group): hypothyroid, (0.02% methimazole (w/v) in drinking water for 28 days); hypothyroidϩT 3 , (after 25 days of methimazole treatment received 15 g of T 3 /kg of body weight for 3 days by intraperitoneal injection); hypothyroidϩL-NAME, (0.75 mg/ml L-NAME in drinking water in the last 21 days of methimazole treatment); hyperthyroid, (intraperitoneal injection of 60 g of T 3 /kg of body weight for 3 days); and control group. Blood samples were collected at the time the animals were killed for estimation of thyrotropin (TSH) level by radioimmunoassay (20).…”

GABA_B1 Knockout Mice Reveal Alterations in Prolactin Levels, Gonadotropic Axis, and Reproductive Function

“…Results from our laboratory [18, 19] established that GABA B R subunits have particular ontogenic expression patterns in hypothalamus and pituitary, decreasing throughout postnatal development in both sexes and being more abundant in female than in male infantile rats. This dimorphic sexual expression pattern in the pituitary is the consequence of early testosterone imprinting in males [20]. In addition, we have demonstrated [21] that pituitary GABA B Rs are coupled Gi/0-type G-proteins and that their activation initiates the same intracellular signaling pathways as in the CNS.…”