Alain Caraty

Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene.

The G protein-coupled receptor GPR54 (AXOR12, OT7T175) is central to acquisition of reproductive competency in mammals. Peptide ligands (kisspeptins) for this receptor are encoded by the Kiss1 gene, and administration of exogenous kisspeptins stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release in several species, including humans. To establish that kisspeptins are the authentic agonists of GPR54 in vivo and to determine whether these ligands have additional physiological functions we have generated mice with a targeted disruption of the Kiss1 gene. Kiss1-null mice are viable and healthy with no apparent abnormalities but fail to undergo sexual maturation. Mutant female mice do not progress through the estrous cycle, have thread-like uteri and small ovaries, and do not produce mature Graffian follicles. Mutant males have small testes, and spermatogenesis arrests mainly at the early haploid spermatid stage. Both sexes have low circulating gonadotropin (luteinizing hormone and follicle-stimulating hormone) and sex steroid (β-estradiol or testosterone) hormone levels. Migration of GnRH neurons into the hypothalamus appears normal with appropriate axonal connections to the median eminence and total GnRH content. The hypothalamic–pituitary axis is functional in these mice as shown by robust luteinizing hormone secretion after peripheral administration of kisspeptin. The virtually identical phenotype of Gpr54- and Kiss1-null mice provides direct proof that kisspeptins are the true physiological ligand for the GPR54 receptor in vivo. Kiss1 also does not seem to play a vital role in any other physiological processes other than activation of the hypothalamic–pituitary–gonadal axis, and loss of Kiss1 cannot be overcome by compensatory mechanisms.

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.

The kisspeptin system of the human hypothalamus: sexual dimorphism and relationship with gonadotropin-releasing hormone and neurokinin B neurons.

Kisspeptin signaling via the kisspeptin receptor G‐protein‐coupled receptor‐54 plays a fundamental role in the onset of puberty and the regulation of mammalian reproduction. In this immunocytochemical study we addressed the (i) topography, (ii) sexual dimorphism, (iii) relationship to gonadotropin‐releasing hormone (GnRH) neurons and (iv) neurokinin B content of kisspeptin‐immunoreactive hypothalamic neurons in human autopsy samples. In females, kisspeptin‐immunoreactive axons formed a dense periventricular plexus and profusely innervated capillary vessels in the infundibular stalk. Most immunolabeled somata occurred in the infundibular nucleus. Many cells were also embedded in the periventricular fiber plexus. Rostrally, they formed a prominent periventricular cell mass (magnocellular paraventricular nucleus). Robust sex differences were noticed in that fibers and somata were significantly less numerous in male individuals. In dual‐immunolabeled specimens, fine kisspeptin‐immunoreactive axon varicosities formed axo‐somatic, axo‐dendritic and axo‐axonal contacts with GnRH neurons. Dual‐immunofluorescent studies established that 77% of kisspeptin‐immunoreactive cells in the infundibular nucleus synthesize the tachykinin peptide neurokinin B, which is known to play crucial role in human fertility; 56 and 17% of kisspeptin fibers in the infundibular and periventricular nuclei, respectively, contained neurokinin B immunoreactivity. Site‐specific co‐localization patterns implied that kisspeptin neurons in the infundibular nucleus and elsewhere contributed differentially to these plexuses. This study describes the distribution and robust sexual dimorphism of kisspeptin‐immunoreactive elements in human hypothalami, reveals neuronal contacts between kisspeptin‐immunoreactive fibers and GnRH cells, and demonstrates co‐synthesis of kisspeptins and neurokinin B in the infundibular nucleus. The neuroanatomical information will contribute to our understanding of central mechanisms whereby kisspeptins regulate human fertility.

Mapping of Kisspeptin Fibres in the Brain of the Pro-Oestrous Rat

Kisspeptins are a family of small peptides that play a key role in the neuroendocrine regulation of the reproductive function through neural pathways that have not yet been completely identified. The present study aimed to investigate the distribution of kisspeptin neurone fibres in the female rat brain by comparing precisely the immunoreactive pattern obtained with two antibodies: one specifically directed against kisspeptin‐52 (Kp‐52), the longest isoform, and the other directed against kisspeptin‐10 (Kp‐10), whose sequence is common to all putative mature isoforms. With both antibodies, immunoreactive cell bodies were exclusively observed in the arcuate nucleus, and immunoreactive fibres were confined to the septo‐preoptico‐hypothalamic continuum of the brain. Fibres were observed in the preoptic area, the diagonal band of Broca, the septohypothalamic area, the anteroventral periventricular, suprachiasmatic, supraoptic, paraventricular and periventricular nuclei, the dorsal border of the ventromedian nucleus, the dorsomedial and arcuate nuclei, and the median eminence. In the latter structure, varicose fibres were mainly distributed in the internal layer and were detected to a lesser extent throughout the external layer, including around the deeper part of the infundibular recess. Most regions of immunoreactive cells and fibres matched perfectly for the two antibodies. However, fibres in the dorsolateral septum, anterior fornix, accumbens nucleus and the lateral bed nucleus of the stria terminalis were only recognised by antibody anti‐Kp‐10, suggesting that anti‐Kp‐10 may recognise a wider range of kisspeptin isoforms than anti‐Kp‐52 or cross‐react with molecules other than kisspeptin in rat tissue. Overall, these results illustrate the variety of projection sites of kisspeptin neurones in the rat and suggest that these peptides play a role in different functions.

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

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 4h 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.

Short-Term Effect of Oestradiol on Neurokinin B mRNA Expression in the Infundibular Nucleus of Ewes

In sheep, essentially all the neurokinin B (NKB) neurones of the infundibular nucleus express oestradiol receptor α, and analysis of female and male brains has revealed an exceptionally marked female‐dominant sex difference in the numbers of NKB neurones in the infundibular nucleus. This neuronal population is located in an oestradiol‐sensitive brain area involved in the control of gonadotropin‐releasing hormone (GnRH) secretion and oestrous behaviour, but its physiological role is poorly documented. The aim of the present study was to analyse NKB mRNA expression at a crucial time when the steroid has stimulated the pathways leading to the induction of these two events. After cloning a specific ovine NKB antisense riboprobe, we examined the effects of a short oestradiol treatment (4u2003h subcutaneously) on the expression of NKB mRNA in the caudal part of the infundibular nucleus of progesterone‐primed ovariectomized ewes. We demonstrated that oestradiol decreased both the level of NKB mRNA expression (34%) and the number of cells containing NKB mRNA (43%). Oestradiol acts strongly on these NKB cells in the short term. We suggest that this early change in NKB mRNA expression during the preovulatory period might be involved in the control of the induction of GnRH secretion or oestrous behaviour.

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 50–450u2003nmol) 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 12.4u2003μmol per ewe) or as a constant i.v. infusion (2.1, 6.2, 12.4 and 18.6u2003μmol/h per ewe) to anoestrous acyclic ewes induced a sustained increase in LH plasma concentrations. A pharmacokinetic study showed that RF9 (12.4u2003μmol bolus i.v.) has an effective half life of 5.5u2003h 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 1.3u2003μmol 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.

Kisspeptin and Seasonality in Sheep

Sheep are seasonal breeders, experiencing a period of reproductive quiescence during spring and early summer. During the non-breeding period, kisspeptin expression in the arcuate nucleus is markedly reduced. This strongly suggests that the mechanisms that control seasonal changes in reproductive function involve kisspeptin neurons. Kisspeptin cells appear to regulate GnRH neurons and transmit sex-steroid feedback to the reproductive axis. Since the non-breeding season is characterized by increased negative feedback of estrogen on GnRH secretion, the kisspeptin neurons seem to be fundamentally involved in the determination of breeding state. The reduction in kisspeptin neuronal function during the non-breeding season can be corrected by infusion of kisspeptin, which causes ovulation in seasonally acyclic females.

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.