Didier Lomet

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.

RF9 Powerfully Stimulates Gonadotrophin Secretion in the Ewe: Evidence for a Seasonal Threshold of Sensitivity

GPR147 and its endogenous ligands, RFRPs, are emerging as important actors in hypothalamic‐pituitary axis control. The role of this system would be to inhibit gonadotrophin secretion. However, data on the subject are contradictory. The discovery of RF9 (adamantanecarbonyl‐RF‐2‐NH2), a GPR147 antagonist, prompted us to use this new tool to further investigate this system in the ewe. Accordingly, we tested the effect of i.c.v. administration of RF9 on gonadotrophin secretion in the ewe during anoestrous and the breeding season. Intracerebroventricular injections of RF9 (from 50–450 nmol) caused a clear elevation in peripheral blood plasma luteinising hormone (LH) concentrations. The effect of RF9 on LH was more pronounced during the anoestrous season. Furthermore, peripheral administration of RF9 as a bolus (2.1, 6.2 and 12.4 μmol per ewe) or as a constant i.v. infusion (2.1, 6.2, 12.4 and 18.6 μmol/h per ewe) to anoestrous acyclic ewes induced a sustained increase in LH plasma concentrations. A pharmacokinetic study showed that RF9 (12.4 μmol bolus i.v.) has an effective half life of 5.5 h in the plasma. Conversely, RF9 is not detectable in the cerebrospinal fluid, suggesting that it does not cross the blood–brain barrier. The increase in LH plasma concentrations induced by RF9 was blocked by previous administration of 1.3 μmol per ewe of gondotrophin‐releasing hormone (GnRH) antagonist Teverelix. This suggests that GnRH is involved in the stimulatory effect of RF9 on gonadotrophin secretion. Finally, no variation in LH plasma concentrations could be detected in ovariectomised ewes injected either i.c.v. or i.v. with RFRP3 (VPNLPQRF‐NH2). The lack of effect of RFRP3 in our experimental setting suggests that the mechanisms involved in RF9 action are probably more complex than previously assumed. Our results indicate that delivery of RF9 in the ewe greatly increases gondadotrophin secretion in both the oestrus and anoestrus season, suggesting a potential new way of controlling reproduction in mammals.

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.

Differential Estradiol Requirement for the Induction of Estrus Behavior and the Luteinizing Hormone Surge in Two Breeds of Sheep

Abstract 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.

Rational Design of Triazololipopeptides Analogs of Kisspeptin Inducing a Long-Lasting Increase of Gonadotropins

New potent and selective KISS1R agonists were designed using a combination of rational chemical modifications of the endogenous neuropeptide kisspeptin 10 (KP10). Improved resistance to degradation and presumably reduced renal clearance were obtained by introducing a 1,4-disubstituted 1,2,3-triazole as a proteolysis-resistant amide mimic and a serum albumin-binding motif, respectively. These triazololipopeptides are highly potent full agonists of KISS1R and are >100 selective over the closely related NPFF1R. When injected in ewes with a quiescent reproductive system, the best compound of our series induced a much prolonged increase of luteinizing hormone release compared to KP10 and increased follicle-stimulating hormone plasma concentration. Hence, this KISS1R agonist is a new valuable pharmacological tool to explore the potential of KP system in reproduction control. Furthermore, it represents the first step to develop drugs treating reproductive system disorders due to a reduced activity of the hypothalamo-pituitary-gonadal axis such as delayed puberty, hypothalamic amenorrhea, and hypogonadotropic hypogonadism.

Concentrations of estradiol in ewe cerebrospinal fluid are modulated by photoperiod through pineal‐dependent mechanisms

Abstract:  In the ewe, seasonal anestrus results from an increased responsiveness of the hypothalamus to the negative feedback of estradiol (E2) on the gonadotropic axis under long‐day conditions. However, this seasonal effect could also depend upon variable uptake of steroids by the brain. The aim of the present experiment was to compare the concentration of E2 in the blood plasma and in the cerebrospinal fluid (CSF) from the third ventricle in groups of ovariectomized, estradiol treated ewes maintained under short day (SD) or long day (LD) conditions and to study the involvement of the pineal gland in this photoperiodic regulation. Pinealectomized and sham‐operated ewes were equipped with an intracerebral cannula to sample the CSF. The plasma E2 concentrations showed no difference between LD and SD in sham‐operated and pinealectomized animals. In contrast, in the CSF, E2 concentration was higher in the LD than the SD group, and pinealectomy suppressed this effect of photoperiod. Concomitantly, the stimulatory effect of SD on luteinizing hormone levels observed in sham‐operated ewes was abolished by pinealectomy. The results demonstrate that LD increases the E2 concentration in the CSF by a mechanism involving the pineal gland.

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.

Turnover rate of cerebrospinal fluid in female sheep: changes related to different light-dark cycles

BackgroundSheep are seasonal breeders. The key factor governing seasonal changes in the reproductive activity of the ewe is increased negative feedback of estradiol at the level of the hypothalamus under long-day conditions. It has previously been demonstrated that when gonadotropin secretions are inhibited during long days, there is a higher concentration of estradiol in the cerebrospinal fluid (CSF) than during short days. This suggests an involvement of the CSF and choroid plexus in the neuroendocrine regulatory loop, but the mechanisms underlying this phenomenon remain unknown. One possible explanation of this difference in hormonal content is an effect of concentration or dilution caused by variations in CSF secretion rate. The aim of this study was thus to investigate changes in the CSF turnover rate related to light-dark cycles.MethodsThe turnover rate of the CSF was estimated by measuring the time taken for the recovery of intraventricular pressure (IVP) after removal of a moderate volume (0.5 to 2 ml) of CSF (slope in mmHg/min). The turnover rate was estimated three times in the same group of sheep: during a natural period of decreasing day-length corresponding to the initial period when gonadotropin activity is stimulated (SG1), during a long-day inhibitory period (IG), and finally during a short-day stimulatory period (SG2).ResultsThe time taken and the speed of recovery of initial IVP differed between groups: 8 min 30 sec, 0.63 ± 0.07 mmHg/min(SG1), 11 min 1 sec, 0.38 ± 0.06 mmHg/min (IG) and 9 min 0 sec, 0.72 ± 0.15 mmHg/min (SG2). Time changes of IVP differed between groups (ANOVA, p < 0.005, SG1 different from IG, p < 0.05). The turnover rate in SG2: 183.16 ± 23.82 μl/min was not significantly different from SG1: 169. 23 ± 51.58 μl/min (Mann-Whitney test, p = 0.41), but was significantly different from IG: 71.33 ± 16.59 μl/min (p = 0.016).ConclusionThis study shows that the turnover rate of CSF in ewes changes according to the light-dark cycle; it is increased during short day periods and reduced in long day periods. This phenomenon could account for differences in hormonal concentrations in the CSF in this seasonal species.

The effect of an intracerebroventricular injection of metformin or AICAR on the plasma concentrations of melatonin in the ewe: potential involvement of AMPK?

BackgroundIt is now widely accepted that AMP-activated protein kinase (AMPK) is a critical regulator of energy homeostasis. Recently, it has been shown to regulate circadian clocks. In seasonal breeding species such as sheep, the circadian clock controls the secretion of an endogenous rhythm of melatonin and, as a consequence, is probably involved in the generation of seasonal rhythms of reproduction. Considering this, we identified the presence of the subunits of AMPK in different hypothalamic nuclei involved in the pre- and post-pineal pathways that control seasonality of reproduction in the ewe and we investigated if the intracerebroventricular (i.c.v.) injection of two activators of AMPK, metformin and AICAR, affected the circadian rhythm of melatonin in ewes that were housed in constant darkness. In parallel the secretion of insulin was monitored as a peripheral metabolic marker. We also investigated the effects of i.c.v. AICAR on the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a downstream target of AMPK, in brain structures along the photoneuroendocrine pathway to the pineal gland.ResultsAll the subunits of AMPK that we studied were identified in all brain areas that were dissected but with some differences in their level of expression among structures. Metformin and AICAR both reduced (p < 0.001 and p < 0.01 respectively) the amplitude of the circadian rhythm of melatonin secretion independently of insulin secretion. The i.c.v. injection of AICAR only tended (p = 0.1) to increase the levels of phosphorylated AMPK in the paraventricular nucleus but significantly increased the levels of phosphorylated ACC in the paraventricular nucleus (p < 0.001) and in the pineal gland (p < 0.05).ConclusionsTaken together, these results suggest a potential role for AMPK on the secretion of melatonin probably acting trough the paraventricular nucleus and/or directly in the pineal gland. We conclude that AMPK may act as a metabolic cue to modulate the rhythm of melatonin secretion.

Seasonal breeding in mammals: From basic science to applications and back.

Seasonal breeding is a remarkable adaptive feature, which allows animals to coordinate physiological functions throughout the year. However, in the context of animal production, it becomes an undesirable complication, which needs to be circumvented. Therefore, eco-friendly methods based on photoperiodic treatments and the use of the male effect have been developed to control seasonal reproduction in small ruminants. In practice, such treatments are hardly used and hormonal treatments constitute the benchmark, but practicality of hormonal treatments comes at a high cost for human health and the environment. Here, we summarize our current understanding of the molecular and neuroendocrine mechanisms underlying seasonal breeding in small ruminants. We then move on to describe current methods to control reproduction and detail why such methods are not sustainable. Finally, using the neuropeptide kisspeptin as an example, we show that an improved understanding of the molecular and neuroendocrine mechanisms that underlie photoperiodism might help design novel strategies for the development of improved and sustainable breeding schemes.