Hypothalamic Gonadotropin‐Releasing Hormone Secretion: Methodological Aspects of in vitro Systems

Abstract

The regulation of gonadotropin-releasing hormone (GnRH) secretion from the medial basal hypothalamus (MBH) of adult male rats has been studied using two types of in vitro methods, the dynamic superfusion and static incubation systems. We compared both systems for methodological features including stability of baseline GnRH release and technical limitations or artefacts as well as for response to norepinephrine and naloxone, an opiate antagonist, which represent the two major physiological neuromodulators regulating the GnRH neuron. Baseline GnRH release rate per MBH was similar in both systems for at least 6 h, although owing to dilution of effluent samples by flow-through medium, the dynamic superfusion system required six MBH per chamber compared with the static incubation system which required only one MBH per chamber. Procedural losses of GnRH were low with either system without the need for proteolytic enzyme inhibitors and were mainly due to adhesion to plastic surfaces of chambers and tubings in the dynamic superfusion system. Mechanical agitation of chambers by either manual swinging or continuous shaking increased GnRH release rate. Addition of hypertonic KCI (final 50 mM) or equivalent NaCl or mannitol solution in a minimal volume directly into the tissue chambers, induced a non-specific GnRH release due to osmotic effects in the dynamic superfusion system. In contrast, the static incubation system, where hypothalamic tissue is exposed to the exact final concentration of solute by complete change of media, was more resistant to GnRH release by hypertonic stimulus. Thus, only in the static incubation system did 50 mM KCI concentrations cause GnRH release through depolarization alone. Using systems optimized to avoid technical artefacts, stimulation with naloxone demonstrated dose-dependent GnRH release in both systems whereas norepinephrine gave a clear dose-dependent GnRH release only in the static system.Thus both incubation systems produce stable and similar baseline GnRH release for at least 6 h. The static incubation system proved superior requiring fewer hypothalami, a shorter preincubation stabilization period, allowing more accurate concentration of additives and was less prone to mechanical and osmotic artefacts while preserving responsiveness to physiological stimuli. The static incubation system, being technically simpler, more robust and accurate, provided a more valid approach to the in vitro study of GnRH release and its regulation by neurochemicals.

Effects of Rat Prolactin on Gonadotropin-Releasing Hormone Secretion by the Explanted Male Rat Hypothalamus

“…First, Azad incu bated a hypothalamus divided in six pieces by 1 longitu dinal and 2 transversal cuts, this might have helped the penetration of the immunoglobulins within the tissue; and, second, they incubated their tissues in a metabolic shaker. Mechanical agitation has been reported to in crease basal GnRH output [13]. indeed, they reported basal values more than 20-fold higher than those reported in the present study.…”

“…This system was selected because it has been reported to have some advantages over a perfusion system [ 13]. Moreover, based on the observation that PRL exerts a more pro nounced suppressive effect on plasma gonadotropin lev els in female than in male rats [6. 14], we also examined whether the addition of testosterone (T), dihydrotestos terone (DHT).…”

Regulation of Hypothalamic Gonadotropin-Releasing Hormone Secretion in Experimental Uremia: In vitro Studies

“…GnRH secretion from MBH was studied for 6 h using an in vitro system as described and validated in detail elsewhere [12]. Briefly, the static incubation system consisted o f a single MBH seated in a 5-ml glass scintillation vial and bathed in 1 ml of incubation medium con sisting of Earle's balanced salt solutions (pH 7.4) with added 5 mM HF.PES (Hoechst) and 0.25% gelatin was gassed continuously with 95% 0 '/5% COj.…”

Release of chicken luteinising hormone-releasing hormone-I (cLHRH-I) by mediobasal hypothalamus in the cockerel: validation of an incubation system and effect of excitatory amino acids

“…Thus, several in vivo (Levine and Ramirez, 1986, in the rat; Caraty and Locatelli, 1988, in the ram) and in vitro (review in Wibullaksanakul and Handelsman, 1991) techniques have been developed. The in vitro approach has the advantage of allowing the direct testing of the effect of various substances on the secretion.…”

“…Two types of in vitro methods have been described: superfusion (Gallardo and Ramirez, 1977, in the rat; Knight, 1983, in the chicken) and static incubation (Rotsztejn et al, 1976, in the rat; Katz et al, 1990, in the chicken). Wibullaksanakul and Handelsman (1991) compared both systems in a systematic study and concluded the superiority of static incubation, which they judge technically simpler, more robust and accurate than superfusion. With the aim of studying the regulation of LHRH secretion in the chicken, we have developed an incubation system for chicken mediobasal hypothalamus (MBH) and a radioimmunoassay sensitive enough for reliable measurement of the amounts of chicken LHRH-I released in vitro.…”