Introduction: Orexin A is a neuropeptide synthesized in the lateral hypothalamus. Orexin A immunoreactive fibers overlap distribution with GnRH neurons. In adult rats, orexin A is known to affect LH secretion via GnRH release modulation. Since data concerning orexin A impact on the hypothalamo-pituitary axis activity are limited, we focused on orexin A and receptors of NPY involvement in the modulation of LH release and LH subunit β mRNA expression in prepubertal female rats.
Material and methods: Forty immature female Wistar rats were divided into four groups and received two intracerebroventricular (icv) microinjections of 1. artificial cerebrospinal fluid - CSF (controls) 2. CSF followed by orexin A; 3. Selective NPY receptor antagonist (BIBP) followed by CSF; 4. BIBP followed by orexin A. One hour after the last microinjection, all rats were decapitated. Trunk blood was collected and serum was stored at -20°C for the LH RIA examination. The adenohypophysis was immediately excised, flash-frozen, and kept at -80°C for RNA extraction. Real-time PCR amplification was carried out and relative LHβ gene expression was calculated.
Results: In comparison to the CSF-treated controls with mean LH serum concentration of 0.40±0.02 ng/ml, mean LH serum level was diminished both after orexin A (0.27±0.01 ng/ml) or BIBP (0.30±0.02 ng/ml), icv microinjections. In the presence of BIBP, orexin A more effectively inhibited LH release (0.20±0.01 ng/ml) when compared to the BIBP-treated group. Orexin A and BIBP exerted a consistent inhibitory effect on Lhb mRNA expression levels in the anterior pituitary gland. In comparison to the CSF-treated controls, orexin A and BIBP-treated females responded with, respectively, 35% and 40% reduction of Lhb mRNA expression. Orexin A and BIBP co-administration evoked a further reduction of LHβ gene transcriptional activity.
Conclusions: Orexin A exerts a down-regulatory effect on LH synthesis and release in immature female rats. Considering that Y1R-oriented down-regulation of endogenous NPY activity did not reverse the suppressive effect of exogenous orexin A, it might be suggested that NPY and orexin A systems can operate independently to affect gonadotropin activity in the anterior pituitary of the immature female rats.
Keywords: LH; Lhb mRNA; NPY Y1 receptor antagonist; Orexin A; prepubertal female rats.