In vitro Secretion of Gonadotropin-Releasing Hormone (GnRH) and [Hydroxyproline⁹]GnRH from the Rat Hypothalamus Exhibits a Differential Sensitivity to Castration and Second Messengers

Abstract

The decapeptide [hydroxyproline⁹]GnRH (HypGnRH) has been characterized as an endogenous posttranslational product of the gonadotropin-releasing hormone (GnRH) precursor in a wide range of mammalian brains. Despite consistent biological effects, its secretion by the hypothalamus remains hypothetical. We report here in vitro secretion of HypGnRH and GnRH by the hypothalamus from intact and castrated male rats and provide evidence that they are differentially regulated. Both peptides were identified by two anti-GnRH antibodies of different specificities after separation under two high-performance liquid chromatography conditions. Calcium dependency of HypGnRH release was demonstrated under stimulation with KCl in the absence or presence of Ca²⁺, as well as with Bay K 8644, veratridine, methoxyverapamil, or tetrodotoxin. Activation of signaling pathways involving adenylate cyclase and protein kinases A and C (PKC) induced HypGnRH release. Expression of data as percentage of release over tissue stores revealed a two- to threefold higher release of HypGnRH than of GnRH under the different modes of stimulation used, except under PKC activation which triggered a comparable recruitment of both peptides. Castration selectively affected PKC-coupled GnRH secretion which showed a twofold lesser release than in intact rats, while the HypGnRH release was unaffected. We conclude that HypGnRH and GnRH are not secreted from the hypothalamus according to the same mechanisms.

Structure and Biological Activity of Gonadotropin-Releasing Hormone Isoforms Isolated from Rat and Hamster Brains

“…It is reported that [Hyp 9 ]GnRH is released from the hypothalamus by different mechanisms in adult [39]and developing rats [40]and can potentially act on the secretion of human chorionic gonadotropin secretion from perifused placental cells [41]. Nevertheless, its actions are not fully understood.…”

Differential In Vitro Secretion of Gonadotropin‐Releasing Hormone (GnRH) and [Hydroxyproline9]GnRH from the Rat Hypothalamus During Postnatal Development

“…Experiments were performed with the same weight of tissue (0.9–1.1 g) for each postnatal day examined. Preparation of tissues has been previously reported ( 2). Briefly, for one experiment, 15–60 tissues (according to the postnatal period examined) were mixed and placed into a single plastic flask (35 × 25 mm) positioned in a water bath (37 °C) under constant shaking.…”

“…We recently reported the in vitro cosecretion of gonadotropin‐releasing hormone (GnRH) and its hydroxylated molecular form, [hydroxyproline 9 ]GnRH (HypGnRH) ( 1) from the whole hypothalamus of adult intact and castrated male rats ( 2). Release of the two forms of GnRH was shown to be differentially regulated after either membrane depolarization or activation of different signalling pathways involving adenylate cyclase, protein kinases A and C (PKA, PKC).…”

“…In the hypothalamus, despite the recent detection of chicken GnRH‐II in the adult and foetal brain of mammals ( 6, 7), HypGnRH remains the major molecular form of the GnRH‐like material in the rat foetus ( 1), and is present at high levels during the first days of life ( 8). A specific role of HypGnRH is suggested by the demonstration of a preferential mobilization of the peptide in sustaining secretion in the adult rat ( 2). However, the HypGnRH secretion may be regulated differently in the immature rat than in the mature animal, since the onset of puberty involves important changes in the feedback of gonadal steroids ( 9) and in the effect of a number of neurotransmitters and neuromodulators on GnRH and gonadotrophin secretion ( 10–15).…”

Molecular Polymorphism of Native Gonadotropin-Releasing Hormone (GnRH) Is Restricted to Mammalian GnRH and [Hydroxyproline⁹] GnRH in the Developing Rat Brain

“…But, in addition to a previous study [34], we established here that both decapeptides are present at an early developmental stage in association with some of their catabolites and precursors which have been already identified in the adult animals [15, 16] and in the GT1 cell line [35, 36]. The chromatography of large amounts of tissues and the use of two combined HPLC steps made it possible to detect these additional metabolic compounds which mostly coelute with mHypGnRH or mGnRH on a single RP-HPLC [37]. Our data confirm that the hypothalamic GnRH-like content increased with advancing age in both sexes, reaching higher amounts in males than in females [38].…”