A synthetic kisspeptin analog that triggers ovulation and advances puberty.

The neuropeptide kisspeptin and its receptor, KiSS1R, govern the reproductive timeline of mammals by triggering puberty onset and promoting ovulation by stimulating gonadotrophin-releasing hormone (GnRH) secretion. To overcome the drawback of kisspeptin short half-life we designed kisspeptin analogs combining original modifications, triazole peptidomimetic and albumin binding motif, to reduce proteolytic degradation and to slow down renal clearance, respectively. These analogs showed improved in vitro potency and dramatically enhanced pharmacodynamics. When injected intramuscularly into ewes (15 nmol/ewe) primed with a progestogen, the best analog (compound 6, C6) induced synchronized ovulations in both breeding and non-breeding seasons. Ovulations were fertile as demonstrated by the delivery of lambs at term. C6 was also fully active in both female and male mice but was completely inactive in KiSS1R KO mice. Electrophysiological recordings of GnRH neurons from brain slices of GnRH-GFP mice indicated that C6 exerted a direct excitatory action on GnRH neurons. Finally, in prepubertal female mice daily injections (0.3 nmol/mouse) for five days significantly advanced puberty. C6 ability to trigger ovulation and advance puberty demonstrates that kisspeptin analogs may find application in the management of livestock reproduction and opens new possibilities for the treatment of reproductive disorders in humans.

No Evidence That RFamide-Related Peptide 3 Directly Modulates LH Secretion in the Ewe.

The neuropeptide RFamide-related peptide 3 (RFRP-3) has been implicated in the control of gonadotropin secretion in both birds and mammals. However, in mammals, depending on species, sex and photoperiod, inhibitory, excitatory, or no effect of RFRP-3 on the plasma concentration of LH has been reported. In the ewe, treatment with RFRP-3 either reduced LH concentration or had no effect, and treatment with an RFRP-3 receptor antagonist (ie, RF9) resulted in increased concentration of plasma LH. To clarify these conflicting results in the present study, a set of experiments was performed in ewes. Multiple iv injections of RFRP-3 (6 × 50 µg) in ovariectomized ewes had no effect on plasma LH pulsatility. In intact ewes a bolus injection (500 µg) or an injection (250, 500, or 1000 µg) followed by a 4-hour perfusion (250, 500, or 1000 µg · h(-1)) of RFRP-3 had no effect on the LH pulse induced by kisspeptin (6.5 µg). In ovariectomized, estrogen-replaced ewes, the LH surge induced by estradiol benzoate was not modified by a 24-hour perfusion of RFRP-3 (500 µg h(-1)). Finally, although treatment with RF9 induced a robust release of LH, treatment with a more selective RFRP-3 receptor antagonist, GJ14, resulted in no evident increase of LH. In contrast to the inhibitory effect previously suggested, our data are more consistent with the concept that RFRP-3 has no direct effect on LH secretion in ewes and that RF9 effect on LH release is likely not RFRP-3 receptor mediated. Hence, RFRP-3 probably has a minor role on the control of LH secretion in the ewe.

The Content of Thyroid Hormone Receptor α in Ewe Kisspeptin Neurones is not Season-Dependent.

Seasonal reproduction is grounded in several mechanisms, among which are plasticity in both hormone synthesis and neuronal networks. Increased daylength on long days (LD) translates into local tri-iodothyronin (T3) production in the mediobasal hypothalamus that will enable the transition to the anoestrus season in sheep. The photoperiod also strongly affects the content of kisspeptin (Kiss), a hypothalamic neuropeptide exerting a potent stimulatory effect on gonadotrophin-releasing hormone release. Our hypothesis was that T3 directly inhibits Kiss release during LD. Using double immunocytochemistry, we first searched for coexpression of thyroid hormone receptor (THR)α in Kiss neurones in ewes with an active or inactive gonadotrophic axis. In both the preoptic area and the arcuate nucleus, most Kiss neurones were labelled by THR antibody under both physiological/photoperiodic conditions. These results suggest thyroid hormones may affect Kiss synthesis and release all through the year. We then attempted to assess the influence of T3 on Kiss content in hypothalamic explants sampled from ewes with an active gonadotrophic axis. Kiss produced by hypothalamic explants cultured with different doses of T3 (300 or 600 pg) and subjected to different times of incubation (2 or 24 h) was measured. No significant effects of T3 on Kiss tissular content were observed for the two doses of T3 and for the two incubation times. In light of these findings, potential reasons for the divergent effects of thyroid hormones on Kiss content are discussed. Our data emphasise that the effects of thyroid hormone on Kiss synthesis are not one-sided and may affect a wide range of functions.

Gonadotrophin-releasing hormone release into the hypophyseal portal blood of the ewe mirrors both pulsatile and continuous intravenous infusion of kisspeptin: an insight into kisspeptin's mechanism of action.

Recent studies have demonstrated that kisspeptin (Kp) administration, given as a slow constant infusion of Kp10 (the shortest endogenous form of the Kp molecules which carries biological activity), is able to stimulate gonadotrophin secretion and induce ovulation in anoestrus acyclic ewes. Detailed analysis of peripheral luteinising hormone (LH) concentrations, obtained at 10-min intervals, suggested that this Kp10 treatment induced the continuous release of gonadotrophins. Whether this apparent constant secretion of LH resulted from a continuous elevation of GnRH or discrete high-frequency pulses could not be determined. In the present study, we monitored the patterns of gonadotrophin-releasing homrone (GnRH) secreted into hypophyseal portal blood (HPB) and LH in the peripheral circulation when Kp10 was administered either as discrete pulses or by means of a continuous infusion. Samples of HPB and peripheral blood were obtained at 2 and 10-min intervals, respectively, over a 6-h period, from anoestrous acyclic ewes that received an i.v. bolus injection of Kp10 at 1 h and an infusion of Kp10 between hours 2 and 6. GnRH release following Kp10 administration appeared to be dose-dependent, with larger responses being seen to the 20 µg bolus and 20 µg/h infusion than to the 10 µg bolus and 10 µg/h infusion, with the latter being marginally effective in inducing LH release. Bolus injections of Kp10 (either 20 or 10 µg) induced a sharp GnRH pulse in HPB and a discrete LH pulse in peripheral blood. By contrast, constant infusion of Kp10 (either 20 or 10 µg/h for 4 h) induced a sustained increase in baseline GnRH secretion with no convincing evidence of strictly episodic release. Values remained continuously elevated in HPB. No sign of pituitary desensitisation was observed at either concentration. Finally, i.v. injection of a large bolus (500 µg) of Kp10 produced immediate pharmacological concentrations of Kp10 in the peripheral circulation but were not associated with detectable levels of the peptide in the cerebrospinal fluid. In summary, our results demonstrate that the mode of Kp10 administration (pulsatile versus continuous) is important in shaping the pattern of GnRH secretion and suggests that this regulatory effect is most likely exerted at the level of the terminals of GnRH neurones. Moreover our data also suggest that Kp is involved in, rather than having a permissive role in, the control of endogenous GnRH pulsatility.

Insights into the mechanism by which kisspeptin stimulates a preovulatory LH surge and ovulation in seasonally acyclic ewes: potential role of estradiol.

We have previously demonstrated that a constant intravenous infusion of kisspeptin (Kp) for 48 h in anestrous ewes induces a preovulatory luteinizing hormone (LH) surge followed by ovulation in approximately 75% of animals. The mechanisms underlying this effect are unknown. In this study, we investigated whether Kp-induced preovulatory LH surges in anestrous ewes were the result of the general activation of the whole gonadotropic axis or of the direct activation of central GnRH neurons required for the GnRH/LH surge. In the first experiment, a constant iv infusion of ovine kisspeptin 10 (Kp; 15.2 nmol/h) was given to 11 seasonally acyclic ewes over 43 h. Blood samples were taken every 10 min for 15 h, starting 5h before the infusion, and then hourly until the end of the infusion. We found that the infusion of Kp induced a well-synchronized LH surge (around 22 h after the start of the Kp infusion) in 82% of the animals. In all ewes with an LH surge, there was an immediate but transient increase in the plasma concentrations of LH, follicle-stimulating hormone (FSH), and growth hormone (GH) at the start of the Kp infusion. Mean (+/- SEM) concentrations for the 5-h periods preceding and following the start of the Kp infusion were, respectively, 0.33 +/- 0.09 vs 2.83 +/- 0.49 ng/mL (P = 0.004) for LH, 0.43 +/- 0.05 vs 0.55 +/- 0.03 ng/mL (P = 0.015) for FSH, and 9.34 +/- 1.01 vs 11.51 +/- 0.92 ng/mL (P = 0.004) for GH. In the first experiment, surges of LH were observed only in ewes that also had a sustained rise in plasma concentrations of estradiol (E(2)) in response to Kp. Therefore, a second experiment was undertaken to determine the minimum duration of Kp infusion necessary to induce such a pronounced and prolonged increase in plasma E(2) concentration. Kisspeptin (15.2 nmol/h) was infused for 6, 12, or 24h in seasonally acyclic ewes (N = 8), and blood samples were collected hourly for 28 h (beginning 5h before the start of infusion), then every 2h for the following 22 h. Kisspeptin infused for 24h induced LH surges in 75% of animals, and this percentage decreased with the duration of the infusion (12h = 50%; 6h = 12.5%). The plasma concentration of E(2) was greater in ewes with an LH surge compared to those without LH surges; mean (+/- SEM) concentrations for the 5-h period following the Kp infusion were, respectively, 2.23 +/- 0.16 vs 1.27 +/- 0.13 pg/mL (P < 0.001). Collectively, our results strongly suggest that the systemic delivery of Kp induced LH surges by activating E(2)-positive feedback on gonadotropin secretion in acyclic ewes.

The effect of AMP-activated kinase activation on gonadotrophin-releasing hormone secretion in GT1-7 cells and its potential role in hypothalamic regulation of the oestrous cyclicity in rats.

Hypothalamic AMP-activated kinase (AMPK) is a key regulator of food intake in mammals. Its role in reproduction at the central level and, more precisely, in gonadotrophin-releasing hormone (GnRH) release has never been investigated. We showed that each subunit of AMPK is present in immortalised GnRH neurones (GT1-7 cells). Treatment with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) and metformin, two activators of AMPK, increased dose-dependent and time-dependent phosphorylation of AMPKalpha atThr172 in GT1-7 cells. Phosphorylation of acetyl-coenzyme A carboxylase at ser79 also increased. Treatment with AICAR (5 mM) or metformin (5 mM) for 4 h inhibited GnRH release in the presence or absence of GnRH (10(-8) M). Specific AMPK inhibitor compound C completely eliminated the effects of AICAR or metformin on GnRH release. Finally, we determined the central effects of AICAR in vivo on food intake and oestrous cyclicity. Ten-week-old female rats received a 50 microg AICAR or a saline i.c.v. injection. We detected increased AMPK and acetyl-CoA carboxylase phosphorylation, specifically in the hypothalamus, 30 min after AICAR injection. Food intake was significantly higher (P < 0.05) in animals treated with AICAR than in animals injected with saline, 24 h after injection. This effect was abolished after 1 week. Moreover, during the 4 weeks following injection, the interval between two oestrous stages was significantly lower in the AICAR group than in the saline group. Our findings suggest that AMPK activation may act directly at the hypothalamic level to affect fertility by modulating GnRH release and oestrous cyclicity.

Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes.

We determined whether kisspeptin could be used to manipulate the gonadotropin axis and ovulation in sheep. First, a series of experiments was performed to determine the gonadotropic responses to different modes and doses of kisspeptin administration during the anestrous season using estradiol-treated ovariectomized ewes. We found that: 1) injections (iv) of doses as low as 6 nmol human C-terminal Kiss1 decapeptide elevate plasma LH and FSH levels, 2) murine C-terminal Kiss1 decapeptide was equipotent to human C-terminal Kiss1 decapeptide in terms of the release of LH or FSH, and 3) constant iv infusion of kisspeptin induced a sustained release of LH and FSH over a number of hours. During the breeding season and in progesterone-synchronized cyclical ewes, constant iv infusion of murine C-terminal Kiss1 decapeptide-10 (0.48 mumol/h over 8 h) was administered 30 h after withdrawal of a progesterone priming period, and surge responses in LH occurred within 2 h. Thus, the treatment synchronized preovulatory LH surges, whereas the surges in vehicle-infused controls were later and more widely dispersed. During the anestrous season, we conducted experiments to determine whether kisspeptin treatment could cause ovulation. Infusion (iv) of 12.4 nmol/h kisspeptin for either 30 or 48 h caused ovulation in more than 80% of kisspeptin-treated animals, whereas less than 20% of control animals ovulated. Our results indicate that systemic delivery of kisspeptin provides new strategies for the manipulation of the gonadotropin secretion and can cause ovulation in noncyclical females.

Differential estradiol requirement for the induction of estrus behavior and the luteinizing hormone surge in two breeds of sheep.

For a better understanding of the mechanisms that lead to the preovulatory GnRH/LH surge and estrus behavior, the minimum estradiol (E) requirements (dose and duration) to induce each of these events were determined and compared between two breeds of ewes having either single (Ile de France) or multiple (Romanov) ovulations. The ewes were initially studied during a natural estrus cycle, and were then ovariectomized and run through successive artificial estrus cycles. For these artificial cycles the duration and amplitude of the follucular phase E increase were manipulated by E implants. Under all conditions, the onset of estrus behavior was similar in the two breeds, although its duration was longer in Romanov ewes. While a moderate E signal (6 cm for 12 h) induced an LH surge in 10/10 Ile de France ewes, a larger E signal (12 cm for 12 h) was minimally effective in Romanov ewes (4/10). Additional studies revealed that a small E signal (3 cm for 6 h) induced full estrus behavior in all Romanov ewes but was completely ineffective in Ile de France animals (0/10). Higher doses and mostly longer durations of the E signal (12 cm for 24 h) were required to induce a surge in all the Romanov ewes. These results demonstrate a clear difference in the E requirement for the induction of estrus behavior and the LH surge between breeds of ewes that have different ovulation rates. These data provide compelling evidence that, in one breed, the neuronal systems that regulate both events require different estrogen signals.

Kisspeptin immunoreactive cells of the ovine preoptic area and arcuate nucleus co-express estrogen receptor alpha.

Kisspeptins are peptide ligands of the G protein-coupled receptor GPR54, recently shown to be essential to reproductive function. We have raised specific rabbit antisera against a highly conserved 10 amino acid-amidated peptide (kp10) common to all kisspeptin isoforms isolated so far and mapped the distribution of kp10-immunoreactive (ir) cells in the ovine hypothalamus. Kp10-ir cells were predominant in the caudal arcuate nucleus, the dorsomedial nucleus and the medial preoptic area. Numerous varicose kp10-ir fibers were found in the preoptic area where GnRH neurons reside and in the median eminence, seemingly projecting around small capillaries in its external zone. Within the caudal arcuate nucleus, nearly all kp10-ir cells showed an intense estradiol receptor alpha immunofluorescent signal compared with approximately half of kp10-ir cells in the preoptic area. The pattern of distribution of kp10 immunoreactivity in the hypothalamus suggests a role for kisspeptin in the estrogen-dependent regulation of GnRH and LH secretion in the ewe.