Kisspeptin immunoreactive neurons in the equine hypothalamus: Interactions with GnRH neuronal system

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Abstract

To determine if kisspeptin could be implicated in the control of reproduction in equine species, we studied the distribution of kisspeptin neurons and their anatomical interactions with GnRH neurons in the hypothalamus of pony mares. Brains were collected in three pony mares between 2 and 4 h after ovulation. One major population of kisspeptin immunoreactive cell bodies was found in the arcuate nucleus (ARC), where they extended from the middle of the nucleus to the premammillary recess. Kisspeptin immunoreactive varicose fibers extended from the preoptic area to the mammillary nuclei, with important densities especially in the anterior periventricular area and the median eminence (ME). Rare close appositions of kisspeptin fibres on GnRH cell bodies were observed in the ARC. Close appositions between kisspeptin and GnRH fibres were also confirmed at a low incidence in the anterior basal periventricular area and at a high incidence in the ME. This work provides neuroanatomical bases for further investigations into the role of kisspeptin in equine reproduction.

Introduction

KiSS-1 was originally identified as a metastasis suppressor gene encoding an array of structurally related peptides, namely kisspeptins which act through the G protein-coupled receptor GPR54. These peptides include metastin, also known as kisspeptin-54, and shorter peptide fragments of the common KiSS-1 precursor, such as kisspeptin-14, kisspeptin-13 and kisspeptin-10 (kp10). All kisspeptins share the 10 amino acid C-terminal region and have the same potency in terms of receptor activation (Kotani et al., 2001).

Recently, the role of the KiSS-1/GPR54 system in the reproductive function has emerged, through neuroendocrine control of GnRH and gonadotropin secretion. Several studies have reported the marked ability of kisspeptin to elicit LH secretion in rodents (Matsui et al., 2004, Gottsch et al., 2004), sheep (Caraty et al., 2007) and primates (Shahab et al., 2005). As a result of this effect on gonadotropins, kisspeptin can induce ovulation in the rat (Matsui et al., 2004) and the cyclic ewe (Caraty et al., 2007), and is also able to synchronize cycles with ovulations in anoestrus ewes (Caraty et al., 2007).

Kisspeptins have been identified in the brains of rodents (Smith et al., 2005), ewes (Pompolo et al., 2006, Franceschini et al., 2006), monkeys and women (Rometo et al., 2007). Their distribution varies between species and according to physiological state. In female mice, two main populations of kisspeptin-expressing neurons have been identified by in situ hybridization (Smith et al., 2005): in the arcuate nucleus (ARC) and in the anteroventral periventricular nucleus (AVPV), a well-defined sexually dimorphic nucleus located in the preoptic area (POA) in this species. It seems that these two populations play an important role in relaying the negative and positive (preovulatory GnRH/LH surge) feedback effects of estrogens on GnRH neurons, respectively (Smith et al., 2005). In female rats, most of the KiSS-1 mRNA-expressing cells contain estrogen receptor (ER) alpha immunoreactivity in the AVPV and ARC (Adachi et al., 2007). Kisspeptin immunoreactive fibres have also been found in the preoptic area in close apposition to GnRH neuronal cell bodies or fibres (Clarkson and Herbison, 2006, Kinoshita et al., 2005).

In the ewe, kp10 immunoreactive cells have been found predominantly in the ARC and POA (Pompolo et al., 2006, Franceschini et al., 2006). Numerous immunoreactive fibres were observed in the median eminence (ME), with the highest density in the external zone (Franceschini et al., 2006). Fibres were also seen running parallel to the walls of the third ventricle (Franceschini et al., 2006). Kp10 neurons co-express ER alpha (Franceschini et al., 2006) and progesterone receptors (Smith et al., 2007), particularly in the ARC. In the ARC, KiSS1-mRNA is negatively regulated by chronic elevated levels of E2 in ovariectomized ewes (Smith et al., 2007) and up-regulated by transient elevated levels of E2, in the caudal portions of the ARC during the late follicular phase (Estrada et al., 2006).

In humans and monkeys, neurons expressing KiSS1-mRNA are localized predominantly in the ARC and are negatively regulated by steroids (Rometo et al., 2007, Shibata et al., 2007).

In equine species, efficient treatments to synchronize ovulation would be very useful, as it is not currently possible to predict the day of ovulation in advance. In view of kisspeptin’s potency to induce cycles with synchronized LH surges and ovulations in cyclic or anoestrus ewes, it appears interesting to investigate this molecule in mares. Therefore, the aim of the present experiment was to study the anatomical distribution of kisspeptin immunoreactive neurons, in the hypothalamus of pony mares, and their potential interactions with GnRH neuronal system. Brains were sampled in pony mares at a specific time of their ovarian cycle, when kisspeptin is known to play an important role in LH secretion. Thus, brains were collected at the moment when the LH secretion dramatically increases, on the day they ovulated, and specifically between 2 and 4 h after ovulation.

Section snippets

Animals

All animal procedures were performed in accordance with the French law on experimental procedures (authorizations 37-118 and C37-175-2 of the French Ministry of Agriculture).

Three cyclic Welsh pony mares from the INRA herd of Nouzilly were used (body weight ± SEM: 315 ± 19 kg; age ± SEM: 11 ± 1.2 years). Cycles were synchronized with an injection of prostaglandin F2α (cloprostenol, 250 mg, Estrumate™, IM, Shering Plough, Levallois-Perret, France) between 5 and 8 days after their previous ovulation. To

Results

The distribution of kisspeptin immunoreactivity was clearly cytoplasmic, detectable in both cell bodies and fibres. The distribution of kisspeptin immunoreactive cell bodies is presented in Fig. 1, Fig. 2. Although the number of immunoreactive cells appeared greater for the mare whose head was perfused, their pattern of distribution was similar between the three mares. Kisspeptin immunoreactive cell bodies were located predominantly in the ARC and a few scattered cells were seen in the

Discussion

To the best of our knowledge, this study is the first to describe the distribution of kisspeptin neurons and their morphological interactions with GnRH neurons in the equine hypothalamus. In pony mares, at the time of the ovulatory LH surge, the majority of kisspeptin immunoreactive cell bodies are distributed in the ARC. This finding is consistent with results obtained in all studied species: rodents (Smith et al., 2005), sheep (Pompolo et al., 2006, Franceschini et al., 2006) and primates (

Acknowledgements

We greatly thank Gaël Ramé and Jean-Philippe Dubois for their help to collect brains, Maryline Cateau Stéphanie Martinet and Samia Ben-Saïd for their technical assistance, Gilles Bruneau for his help to use genome data bases, Guy Duchamp and his team from the UPEA PRC for taking care of the mares and the French National Studs for their financial support.

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