The kisspeptin system in domestic animals: what we know and what we still need to understand of its role in reproduction.

The discovery of the kisspeptin (Kp) system stirred a burst of research in the field of reproductive neuroendocrinology. In the last 15 yr, the organization and activity of the system, including its neuroanatomical structure, its major physiological functions, and its main pharmacological properties, were outlined. To this endeavor, the use of genetic tools to delete and to restore Kp system functionality in a specific tissue was essential. At present, there is no question as to the key role of the Kp system in mammalian reproduction. However, easily applicable genetic manipulations are unavailable for domestic animals. Hence, many essential details on the physiological mechanisms underlying its action on domestic animals require further investigation. The potentially different effects of the various Kp isoforms, the precise anatomical localization of the Kp receptor, and the respective role played by the 2 main populations of Kp cells in different species are only few of the questions that remain unanswered and that will be illustrated in this review. Furthermore, the application of synthetic pharmacologic tools to manipulate the Kp system is still in its infancy but has produced some interesting results, suggesting the possibility of developing new methods to manage reproduction in domestic animals. In spite of a decade and a half of intense research effort, much work is still required to achieve a comprehensive understanding of the influence of the Kp system on reproduction. Furthermore, Kp system ramifications in other physiological functions are emerging and open new research perspectives.

Towards new strategies to manage livestock reproduction using kisspeptin analogs.

The discovery of the hypothalamic neuropeptide kisspeptin and its receptor (KISS1R) have dramatically improved our knowledge about the central mechanisms controlling reproduction. Kisspeptin neurons could be considered the hub where internal and external information controlling reproduction converge. The information is here elaborated and the command dispatched to GnRH neurons, the final output of the brain system controlling reproduction. Several studies have shown that in mammals administration of kisspeptin could finely modulate many aspects of reproduction from puberty to ovulation. For example in ewes kisspeptin infusion triggered ovulation during the non-breeding season and in prepubertal rat repeated injections advanced puberty onset. However, especially in livestock, the suboptimal pharmacological properties of endogenous kisspeptin, notably it short half-life and consequently its poor pharmacodynamics, fetters its use to experimental setting. To overcome this issue synthetic KISS1R agonists, mainly based on kisspeptin backbone, were created. Their more favorable pharmacological profile, longer half-life and duration of action, allowed to perform promising initial experiments for controlling ovulation and puberty. Additional experiments and further refinement of analogs would still be necessary to exploit fully the potential of targeting the kisspeptin system. Nevertheless, it is already clear that this new strategy may represent a breakthrough in the field of reproduction control.

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.

Effect of luteinizing hormone overstimulation on equine follicle maturation.

There is evidence in several species that high circulating LH concentrations can interfere with normal follicle development and ovulation. In the mare, high LH levels after induction of luteolysis with PGF(2α) have been temporally associated with an increased incidence of anovulatory follicles. We hypothesized that a premature increase in LH levels during a follicular wave in mares would disrupt normal follicle maturation leading to ovulatory dysfunction. In experiment 1, all follicles >10 mm were ablated at midestrous cycle in pony mares followed by twice daily administration of equine LH (eLH; 1.6 µg/kg body weight) or saline (vehicle; N = 8 mares per group). When a dominant follicle reached >32 mm, an ovulatory dose of hCG was given. Treatment with eLH had no effects on ovulatory responses or progesterone levels during the posttreatment luteal phase. In experiment 2, after follicle ablation, mares were treated with eLH or vehicle (as above) or were given a single injection of PGF(2α) (N = 7 mares per group), followed by aspiration of a dominant follicle when it reached >32 mm. Administration of eLH induced an increase in circulating LH levels similar to that after PGF(2α) injection. Neither PGF(2α) nor eLH administration had significant effects on follicle growth or total number of follicles in the postablation wave. However, compared with mares treated with vehicle, the preovulatory follicle in the eLH and PGF(2α) groups had lower levels of androstenedione (P = 0.03) and higher levels of insulin-like growth factor I (P = 0.03). Further, levels of prostaglandin E2 in preovulatory follicles tended to be lower in the eLH and PGF(2α) groups (P = 0.06). In conclusion, exposure of developing follicles to high LH in mares did not have apparent effects on ovulation but it induced changes in follicular fluid factor levels which might reflect a disruption in follicle and/or oocyte maturation, indicating the need to further study the implications of using PGF(2α) for the control of fertility in farm animals.

Kisspeptins and the reproductive axis: potential applications to manage reproduction in farm animals.

Kisspeptins (Kp) are a family of neuropeptides produced mainly by two hypothalamic neuronal cell populations. They have recently emerged as a major regulator of the gonadotropin axis and their action is located upstream of the gonadotropin-releasing hormone (GnRH) cell population. In less than 10 yr a growing body of literature has demonstrated the involvement of these peptides in most, if not all, aspects of reproductive axis maturation and function. In contrast to these abundant basic research studies, few experiments have evaluated the potential application of Kp as tools to manipulate reproduction in domestic animals. In mammals, exogenous Kp administration potently stimulates gonadotropin secretion. This action is exerted mainly, if not exclusively, through the stimulation of GnRH release. Intravenous, intraperitoneal, or subcutaneous administration of Kp induced a robust and rapid increase in plasma gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]). However, this stimulatory effect is of short duration. Prolonged LH and FSH release over several hours can be achieved only when Kp are given as repeated multiple bolus or as an infusion. Kp administration was used in two experimental models, ewe and pony mare, with the aim of inducing well-timed and synchronized ovulations. During the breeding season, progesterone-synchronized ewes were given an intravenous infusion of Kp starting 30 h after the removal of progesterone implants. An LH surge was induced in all Kp-treated animals within 2 h of infusion onset. In contrast, in pony mares a constant infusion of Kp for 3 d in the the late follicular phase was unable to induce synchronized ovulation. Another set of studies showed that Kp could be used to activate reproductive function in acyclic animals. Pulsatile administration of Kp in prepubertal ewe lambs was shown to activate ovarian function, leading to enhanced ovarian steroidogenesis, stimulation of LH preovulatory surge, and ovulation. In anestrous ewes, an intravenous infusion of a low dose of Kp induced an immediate and sustained release of gonadotropins, followed a few hours later by an LH surge. This hormonal pattern mimicked hormonal changes normally observed during the estrous cycle follicular phase and was associated with a high percentage of ovulating animals (80%). In summary, exogenous administration of Kp appears to be a new tool to manipulate reproduction. However, optimal doses and periods of treatment should be defined for each species, and the development of powerful analogs or long-term release formulations is necessary before large-scale applications in domestic animals could be envisaged.

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.

Modulation of Fas-induced apoptosis by p75 neurotrophin receptor in a human neuroblastoma cell line.

Fas and p75 neurotrophin receptors (p75(NTR)) are death receptors that alone induce apoptosis of SH-SY5Y neuroblastoma cell line respectively by Fas ligand or brain-derived neurotrophic factor (BDNF, a p75(NTR) ligand). We report on the modulation of Fas-mediated apoptosis by concomitant p75(NTR) activation. The exposure to both ligands suppressed the apoptotic effect. A co-localisation of Fas and p75(NTR) receptors was evidenced by co-capping and immunoprecipitation assays. Moreover, a caspase-8 inhibitor suppressed the protective effect of the concomitant BDNF and Fas ligand stimulation, suggesting that p75(NTR) and Fas receptors could share common signalling pathways.

Heavy and light chain primary structures control IgG3 nephritogenicity in an experimental model for cryocrystalglobulinemia.

Crystal formation by monoclonal immunoglobulins is a well-known but rare complication of B-cell neoplasia. We have designed an in vivo model of cryocrystalglobulinemia by grafting to mice hybridoma clones producing a pathogenic monoclonal immunogloblulin (Ig) G3kappa. One clone, 8A4, secreted a singular IgG3 that formed crystals both in the proliferating plasma cells and as mesangial and subendothelial deposits in the kidney glomeruli. Morphologic analysis of kidneys revealed neutrophil infiltration and endocapillary hyperplasia, while the morphology of deposits was reminiscent of those in cryocrystalglobulinemia patients. A variant clone that only differed from 8A4 by a 3-amino acid deletion in the V(kappa) CDR1 increased its secretion level by 7-fold and produced an abundant bona fide serum monoclonal cryoglobulin in mice, without crystal formation within tumoral cells; it yielded no subendothelial deposits but only amorphous precipitates in capillary lumens of kidney glomeruli, reminiscent of those seen in the human hyperviscosity syndrome, without other glomerular lesions. A limited variation in the V(kappa) domain thus proved able to increase secretion, to abrogate crystallization, and to modify patterns of glomerular lesions and deposits. Both the crystallizing and noncrystallizing IgG3kappa sequences were related to previously reported murine cryoglobulins, all including a gamma3 chain and canonical VH sequences. Two additional variants of 8A4 with identical VH and VL domains but having switched to IgG1 also lost crystal formation, further showing this feature of 8A4 to result from a unique 3-dimensional conformation of the complete immunoglobulin, relying on V and C domain primary structures of both chains.

Mutational analysis in murine models for myeloma-associated Fanconi's syndrome or cast myeloma nephropathy.

We have designed an in vivo model in which murine hybridoma cell clones producing human Ig light chains (LC) are administered to mice. Depending on which monoclonal LC is expressed, this model mimics either cast myeloma nephropathy or the pathological condition defined as myeloma-associated Fanconi's syndrome (FS) with LC crystallization. Morphological alterations of the kidney cells are thus obtained in mice. All studied LC are closely related human monoclonal VkappaI proteins, which differ by a limited number of substitutions within the variable region. In the case of an FS monoclonal LC, we show that limited changes introduced through site-directed mutagenesis in the variable domain may suppress formation of intracellular crystals within tubular cells. We also show that multiple peculiarities of the variable region are simultaneously needed to allow LC crystallization; this property thus likely results from a unique LC tridimensional conformation imposed by concomitant somatic mutations of a specific germinally encoded framework.

Age-related alterations of somatic hypermutation and CDR3 lengths in human Vkappa4-expressing B lymphocytes.

The lower avidity and/or affinity of antibodies generated by an aged immune system could be attributed to two major changes in the antibody repertoire: a shift in germline gene usage and a decrease in the rate of immunoglobulin hypermutation. In an attempt to identify the mechanisms involved in the observed humoral immune deficiency in the elderly, we studied whether differences in the somatic diversity of a particular Vkappa region occurred with ageing. By using the polymerase chain reaction and sequencing, we analysed and compared Vkappa4-Jkappa rearrangements isolated from young (mean age 21 years) and aged (mean age 83 years) healthy adults. Mutations in the Vkappa4 gene compared with the germline sequence were determined as well as the length and structure of the CDR3 sequence. We analysed in detail various mechanisms contributing to CDR3 and Vkappa variability in rearrangements involving the Vkappa4 gene. Our data revealed that, despite strong individual variations, significantly lower levels of somatic mutation were found in the aged group, both for complementarity-determining regions (CDRs) and framework regions (FRs) encoding Vkappa4 sequences. This decrease mostly affected mutations responsible for replacements and thus resulted in a lowered somatic diversification of the encoded Vkappa4 proteins in aged individuals. Moreover, comparison of the CDR3 regions of the Vkappa4-Ckappa cDNA revealed changes in light-chain junctional diversity that correlated with age. Altogether these data suggest an impaired light-chain somatic diversity in connection with human senescence.

Complete primary sequences of two lambda immunoglobulin light chains in myelomas with nonamyloid (Randall-type) light chain deposition disease.

We herein report on the first two primary sequences (BOU and RAC) of monoclonal light chains of the lambda type responsible for nonamyloid lambda light chain deposition disease. Both patients were affected with severe forms of myeloma complicated with renal failure. The pathological presentation typically featured Congo red-negative deposits along tubular basement membranes but differed somewhat from the typical "Randall-type" kappa light chain deposition disease: they lacked the prominent glomerulosclerosis pattern often featuring nonamyloid kappa deposits and were associated with cylinders or myeloma casts. Both protein sequences were deduced from those of the corresponding complementary DNAs in the bone marrow plasma cells. For each chain, products of three independent amplifications by polymerase chain reaction were sequenced and found to be identical. BOU and RAC lambda mRNAs had a normal overall structure consisting of Vlambda2 segments rearranged to Jlambda2Clambda2 but displayed a number of unusual features within their primary sequences. These substitutions are likely responsible for changes in light chain conformation that promote their aggregation and deposition along renal tubule basement membranes.

Structural peculiarities of a truncated V kappa III immunoglobulin light chain in myeloma with light chain deposition disease.

We report on the primary sequence of the monoclonal immunoglobulin light chain (LC) REV involved in myeloma-associated light chain deposition disease (LCDD). This sequence was deduced from that of the corresponding complementary (c)DNA in bone marrow plasma cells. Products of three independent amplifications by polymerase chain reaction (PCR) were sequenced and found to be identical. The kappa mRNA encoding this N-glycosylated LC showed an overall normal structure consisting of a V kappa III segment rearranged to J kappa II. Direct N-terminal amino acid sequencing of the circulating monoclonal IgA2, kappa showed identity with the bone marrow-derived sequence. The kappa-chain presented several unusual features affecting both the leader sequence and the variable (V) region. Four unique amino acid substitutions were found at positions -8, -3, -2 and -1 in the leader sequence and probably resulted in an unusual cleavage by signal peptidase, thus making the LC truncated by one residue and accounting for its unique hydrophobic N-terminus: Ile-Ile-Leu. Additional peculiarities were observed in the V region, including a Thr74-->Asn substitution creating a N-glycosylation site, and Thr53-->Ile, which was only reported once among human kappa III chains, in another LCDD case, and may be of special significance at a position usually harbouring a polar amino acid.

Secretory patterns of 1 alpha-hydroxycorticosterone in the isolated perifused interrenal gland of the dogfish, Scyliorhinus canicula.

An isolated in-vitro perifused interrenal gland preparation from the dogfish Scyliorhinus canicula was used to study production of quantitatively the major corticosteroid 1 alpha-hydroxycorticosterone (1 alpha-OH-B), measured by radioimmunoassay. Basal secretory rates were 877.1 +/- 145 (S.E.M.) fmol/mg per 15 min (n = 14) and the preparation remained viable for up to 22 h, as reflected in a brisk response to 10 microM cyclic AMP (cAMP) after this time. Steroid production responded in a dose-dependent manner to porcine ACTH, with 10 microM producing a maximum stimulation of 225% above the basal secretory rate. cAMP (10 microM) produced an increase of 278% above basal, while 1 microM forskolin increased basal secretory rates by 127%. [Val5]- and [Ile5]-angiotensin II (0.1 microM) increased 1 alpha-OH-B production by 120 and 372% respectively over basal secretory rates. Increasing the concentration of K+ in the perfusate from 8 mM to 12, 18, 28 and 40 mM produced a significant rise only at 28 mM. Alterations in the concentration of Na+ and osmolarity of the perifusion medium had inconsistent effects on steroid production. Increased concentrations of urea (from 360 to 720 mM) increased the basal secretory rate by 121%, whilst reducing the concentration of urea (from 360 to 90 mM) had no effect.