The effect of intracerebroventricular infusions of leptin on the immunoreactivity of neuropeptide Y and gonadotrophin releasing hormone neurons in the hypothalamus of prepubertal sheep in conditions of short fasting

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Abstract

In the study we evaluated the effects of infusion of exogenous leptin to the third ventricle of the brain on the expression of immunoreactive (ir) neuropeptide Y (NPY) neurons in the hypothalamus and ir gonadotrophin releasing hormone (GnRH) nerve terminals in the median eminence of prepubertal lambs in the conditions of short fasting. Merino female sheep (n = 16) were randomly divided into four groups, two fed with standard feeds and two fasted for 72 h. One standard and one fasted groups were infused with Ringer saline (controls), remaining standard and fasted groups with leptin (25 μg/120 μl/h), for 4 h during three consecutive days, and then slaughtered. Ir NPY and ir GnRH were localized by immunohistochemistry using specific polyclonal antibodies. Detection of both hormones was followed by the image analysis and expressed as the percent area stained and integral density of immunostaining. In the hypothalami from all groups the ir NPY perikarya and varicose nerve fibers were localized in three distinct sub-areas, in the arcuate (ARC), paraventricular and periventricular nuclei. In fasted sheep the percent area and integral density for immunoreactivity of NPY increased significantly (P < 0.001) in three sub-areas compared to the standard-fed animals. Leptin infusion lowered the both parameters (P < 0.001) but solely in the ARC NPY population of fasted sheep. The percent area and integral density of immunostaining for ir GnRH in fasted sheep revealed the augmentation (P < 0.001) compared to standard-fed sheep. Leptin infusions diminished (P < 0.001) both parameters in fasted, without effects in standard-fed lambs. In conclusion, the enhanced by fasting immunoreactivity of the ARC NPY perikarya and varicose nerve fibers and restrained immunoreaction of GnRH terminals in the median eminence were reversed by exogenous leptin. It is suggested that leptin can affect GnRH release via ARC NPY neurons in conditions of deficit of nutrients in prepubertal, female lambs.

Introduction

Leptin, the product of the ob gene, implicated as one of the major peripheral signals that participate in the control of a food intake and reproductive processes, is a candidate for an endocrine link between nutrition and reproduction (reviewed from Cunningham et al., 1999). There are no data regarding the direct effect of leptin on GnRH secretion, but the possible interactions between leptin and reproductive axis are based on data concerning influence of exogenous leptin on LH pulsatility in the peripheral blood (Foster and Nagatani, 1999). Leptin treatment of fasting animals maintains oestrous cyclicity in mice and hamster (Ahima et al., 1996, Schneider et al., 1998) and prevents the decrease of LH pulsatility evoked by fasting in rats and monkeys (Nagatani et al., 1998, Finn et al., 1998). In ruminants however, the effect of leptin on LH secretion is less known. In well-nourished castrated sheep intracerebroventricular (icv) infusion of leptin does not affect LH secretions (Henry et al., 1999) but restores the LH secretion diminished by food restriction (Henry et al., 2001, Nagatani et al., 2000).

The site of leptin action in modulating neuroendocrine effects is hypothalamus (Campfield et al., 1995) and the prime candidate for the mediation of its actions is neuropeptide Y (NPY), because of its role in the control of feeding and body weight regulation which has been firstly estimated in rodents (Morley, 1987, Sahu and Kalra, 1993). In NPY neurons, the leptin receptors have been localised (Mercer et al., 1996) suggesting a direct action of leptin on these neurons. Neuropeptide Y except of its role in food intake is also involved in the control of reproductive processes, and more precisely in the regulation of GnRH neuronal function (Kalra, 1993). In the rat, NPY stimulates GnRH release from the hypothalamus in the presence of oestrogen, whereas inhibits GnRH under conditions of oestrogen deficiency (Kalra et al., 1992). In the sheep, the role of NPY in the regulation of GnRH/LH axis is less clear, because most of data are based only on estimation of LH concentration in the blood plasma. Exogenous NPY suppress release of LH in both ovariectomized and ovariectomized oestrogen treated sheep (Malven et al., 1992, McShane et al., 1992), however Porter et al. (1993) suggested that NPY can play stimulatory action in the preovulatory surge of LH. The results of our previous study confirmed, that NPY can stimulate GnRH and LH release only during the period of high ovarian activity (Wójcik-Gładysz et al., 2003).

The role of NPY in reproductive functions is attributed to the population of neurons located in the hypothalamus (Kalra and Crowley, 1992). NPY perikarya have been localized in the arcuate (infundibular) nucleus, from where they innervate several sites in the diencephalon implicated in the control of the pituitary LH secretion. The presence of NPY fibers in the preoptic area (Chaillou et al., 2002), the median eminence (Contijoch et al., 1993) and the presence of synaptic contacts between NPY containing fibers and GnRH containing perikarya (Tillet et al., 1989, Norgren and Lehman, 1989) suggests that they may regulate LH secretion by direct modulation of the GnRH neurones on the levels of GnRH synthesis and release.

It has been shown that NPY neurons in the hypothalamus of female intact or ovariectomized sheep can be affected by food manipulation. Long-term protein undernutrition or generally restriction of food supply resulted in higher expression of the hypothalamic population of NPY neurons (Barker-Gibb and Clarke, 1996, Chaillou et al., 2002, Polkowska and Gładysz, 2001). On the other hand, undernutrition suppresses the frequency and amplitude of the GnRH pulses in ovariectomized sheep (I’Anson et al., 2000) and restrains the release of GnRH from the nerve terminals in prepubertal ewes (Polkowska and Przekop, 1993). Taking into consideration the presented above data, it should be hypothesized that in conditions of restricted feeding, leptin could change release of GnRH through its action on NPY neurons.

To verify this hypothesis we performed the experiment on the model of prepubertal female lamb undergo the short starvation. Using the immunohistochemistry we examined the effect of administration of leptin to the third ventricle of the brain on the expression of ir NPY-neurons in the three sub-areas of the hypothalamus (the arcuate, paraventricular and periventricular nuclei) and on the GnRH nerve terminals in the median eminence.

Section snippets

Animals

The experiment was performed on the prepubertal (7-month-old) female, lambs (Polish Merino sheep), weighted 35 ± 3 kg. The animals were maintained indoor under natural lighting conditions (52°N, 21°E). Beginning with the 6 months of age the progesterone level in the blood plasma was monitored to determine the sexual maturity of animals. Ovaries were examined postmortem. Merino female sheep (n = 16) were randomly divided into four groups, two fed with standard diets with commercial concentrates, with

Results

In the sheep from standard fed and fasted groups, the ir NPY neurons were localized in the three distinct sub-areas of the hypothalamus. The network of ir NPY varicose nerve fibres was seen in the periventricular (PEV) nucleus beginning from the suprachiasmatic area, through the anterior hypothalamic area (AHA) up to the medial basal hypothalamus (MBH). Next area, where the network of ir NPY varicose nerve fibres was observed was the MBH, namely the periventricular subdivision of the

Discussion

The results of presented study show that short starvation of prepubertal female lambs influence the response to icv infusion of leptin with regard to the immunoreactivity of the hypothalamic NPY varicose nerve fibers and perikarya located in the ARC nucleus and GnRH-nerve terminals in the ME.

In the state of fast, the enhanced density of NPY varicose nerve fibers within three hypothalamic sub-area investigated and visualization of NPY perikarya in the ARC nucleus have been found. The similar

Acknowledgments

Research was supported by the State Committee for Scientific Research as a Solicited Project, PBZ-KBN-084/P06/2002 from 2003 to 2005 year.

We wish to thank Prof. Arieh Gertler for recombinant ovine leptin used in this experiment.

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