Solid Phase Synthesis and Circular Dichroism Analysis of (i → i + 4) Cyclic Lactam Analogues of Kisspeptin

The α-helix is one of the most common secondary structure elements adopted by proteins and is commonly stabilized in synthetic peptides via the formation of a covalent side-chain to side-chain lactam bridge. In this study, we explored the application of various side-chain to side-chain lactam bridges to helix stabilization of kisspeptin analogues, an interesting candidate for ligand-based drug discovery with potential as anti-metastatic agents. We successfully synthesised a series of Asp/Lys, Lys/Asp, Glu/Lys and Lys/Glu lactams, finding peptide (1) cyclo(4,8)Tyr-Asn-Trp-Glu-Ala-Phe-Gly-Lys-Arg-Phe-NH₂, to exhibit characteristic α-helical activity in aqueous buffer, in comparison to the linear native peptide, which showed no helical character.     

The value of comparative approaches to our understanding of puberty as illustrated by investigations in birds and reptiles

This article is part of a Special Issue "Puberty and Adolescence". Studies of birds and reptiles have provided many basic insights into the neuroendocrine control of reproductive processes. This research has elucidated mechanisms regulating both early development, including sexual differentiation, and adult neuroendocrine function and behavior. However, phenomena associated with the transition into sexual maturation (puberty) have not been a focus of investigators working on species in these taxonomic classes. Research is complicated in birds and reptiles by a variety of factors, including what can be extended times to maturation, the need to reach particular body size regardless of age, and environmental conditions that can support or inhibit endocrine responses. However, careful selection of model systems, particularly those with available genetic tools, will lead to important comparative studies that can elucidate both generalizability and diversity of mechanisms regulating the onset of reproductive maturity.     

Expression of KiSS-1 in rat oviduct: possible involvement in prevention of ectopic implantation?

The mammalian oviduct is a crucial site for essential postovulatory events in the female reproductive system. These events are, in part, accomplished by clear-cut oviductal segmentation, which helps to provide appropriate epithelial and fluid microenvironments. Early embryonic development and the timely transport of the embryo to the uterus must be promoted, but implantation within the oviduct itself must be avoided. Indeed, the rarity of extra-uterine pregnancies in laboratory animals strongly suggests that active mechanisms operate to prevent ectopic implantation. Kisspeptins, products of the KiSS-1 gene, have been proposed as physiological regulators of uterine implantation by limiting the invasion of the trophoblast into the maternal decidua. We describe here the patterns of expression of the KiSS-1 gene and of kisspeptin immunoreactivity (IR) in the rat oviduct. KiSS-1 mRNA is readily detectable in oviduct samples from all phases of the estrous cycle, whereas kisspeptin-IR is detected in rat oviduct with a regionalized pattern of distribution, viz., strong expression in the isthmus, faint signals in the proximal ampulla, and a lack of immunostaining in the fimbriated infundibulum and interstitial portion. When positive, IR has been localized at the adluminal surface and the cytoplasmic domain of secretory cells. Of note, KiSS-1 expression (at the mRNA and protein levels) shows cycle-related changes with peak expression in proestrus/estrus and lower levels at metestrus/diestrus. This knowledge of the regional- and cycle-specific pattern of expression of KiSS-1 in rat oviduct should open up the possibility of a physiological role of kisspeptins in the prevention of ectopic (tubal) implantation. This work was supported by grants BFU 2005-01443 and BFU 2005-07446 (Ministerio de Educacion y Ciencia, Spain), by funds from the Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain), and by EU research contract EDEN QLK4-CT-2002-00603. M.T.-S. is also supported by grants from the CIBER Fisiopatología de la Obesidad y Nutrición (CB06/03/0003; Instituto de Salud Carlos III) of which he is a member.     

Molecular interaction between kisspeptin decapeptide analogs and a lipid membrane

Kisspeptin-10 is the C-terminal decapeptide amide of kisspeptin, an endogenous ligand for GPR54, and exhibits the same binding and agonist activity as the parent molecule. Although GPR54 is a membrane-embedded protein, details of the molecular interaction between kisspeptin-10 and lipid membranes remain unclear. Here, we performed a series of structural analyses using alanine-scanning analogs of kisspeptin-10 in membrane-mimetic medium. We found that there is a close correlation between lipid membrane binding and agonist activity. For instance, the F10A and non-amidated (NH₂ → OH) analogs showed little or no GPR54-agonist activity and elicited no blue shift in tryptophan fluorescence. NMR analysis of kisspeptin-10 analog in DPC micelles revealed it to contain several tight turn structures, encompassing residues Trp3 to Phe10, but no helical conformation like that seen previously with SDS micelles. Together, our results suggest that kisspeptin-10 may activate GPR54 via a ligand transportation pathway incorporating a lipid membrane.     

Inhibitory action of tongue sole LPXRFa,the piscine ortholog of gonadotropin-inhibitory hormone,on the signaling pathway induced by tongue sole kisspeptin in COS-7 cells transfected with their cognate receptors

Kisspeptin (Kiss) acts as a positive regulator of reproduction by acting on gonadotropes and gonadotropin-releasing hormone (GnRH) neurons. Despite its functional significance, the intricate web of intracellular signal transduction pathways in response to Kiss is still far from being fully understood in teleosts. Accordingly, we investigated the molecular mechanism of Kiss action and its possible interaction with LPXRFa signaling in this study. In vitro functional analysis revealed that synthetic tongue sole Kiss2 decapeptide increased the cAMP responsive element-dependent luciferase (CRE-luc) activity in COS-7 cells transfected with its cognate receptor, while this stimulatory effect was markedly reduced by two inhibitors of the adenylate cyclase (AC)/protein kinase A (PKA) pathway. Similarly, Kiss2 also significantly stimulated serum responsive element-dependent luciferase (SRE-luc) activity, whereas this stimulatory effect was evidently attenuated by two inhibitors of the phospholipase C (PLC)/protein kinase C (PKC) pathway. In addition, LPXRFa-2 suppressed Kiss2-elicited CRE-luc activity in a dose-dependent manner. Taken together, Kiss2 utilizes both AC/PKA and PLC/PKC pathways to exert its functions via its cognate receptor and LPXRFa may antagonize the action of Kiss2 by inhibiting kisspeptin signaling. As far as we know, this study is the first to characterize the half-smooth tongue sole kisspeptin and LPXRFa signaling pathway in COS-7 cells transfected with their cognate receptors and provides novel information on the interaction between LPXRFa system and kisspeptin system in teleosts.     

Comparative analysis of kisspeptin-immunoreactivity reveals genuine differences in the hypothalamic Kiss1 systems between rats and mice

Kiss1 mRNA and its corresponding peptide products, kisspeptins, are expressed in two restricted brain areas of rodents, the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC). The concentration of mature kisspeptins may not directly correlate with Kiss1 mRNA levels, because mRNA translation and/or posttranslational modification, degradation, transportation and release of kisspeptins could be regulated independently of gene expression, and there may thus be differences in kisspeptin expression even in species with similar Kiss1 mRNA profiles. We measured and compared kisspeptin-immunoreactivity in both nuclei and both sexes of rats and mice and quantified kisspeptin-immunoreactive nerve fibers. We also determined Kiss1 mRNA levels and measured kisspeptin-immunoreactivity in colchicine pretreated rats. Overall, we find higher levels of kisspeptin-immunoreactivity in the mouse compared to the rat, independently of brain region and gender. In the female mouse AVPV high numbers of kisspeptin-immunoreactive neurons were present, while in the rat, the female AVPV displays a similar number of kisspeptin-immunoreactive neurons compared to the level of Kiss1 mRNA expressing cells, only after axonal transport inhibition. Interestingly, the density of kisspeptin innervation in the anterior periventricular area was higher in female compared to male in both species. Species differences in the ARC were evident, with the mouse ARC containing dense fibers, while the rat ARC contains clearly discernable cells. In addition, we show a marked sex difference in the ARC, with higher kisspeptin levels in females. These findings show that the translation of Kiss1 mRNA and/or the degradation/transportation/release of kisspeptins are different in mice and rats.     

Effect of estrogen on the expression of GnRH and kisspeptin in the hypothalamus of rats during puberty

The aim of this study was to assess whether changes in kisspeptin and GnRH levels could be attributed to sex steroids at puberty onset. We used the ovariectomy (OVX) model in rats treated with 17β-estradiol (E₂; OVX + E₂), or oil (OVX + oil), and in intact rats treated with E₂ (intact + E₂) or oil only (intact + oil) to determine gene expression changes of Kiss1 and Gnrh1 in the hypothalamus and protein expression of kisspeptin and GnRH in the different areas of the hypothalamus. In the intact + E₂ and OVX + E₂ rats on the day of the onset of puberty, GnRH-immunoreactive (ir) cell numbers decreased (P < 0.05) in the arcuate nucleus but were increased in the preoptic area; Kisspeptin-ir cells increased (P < 0.05) in the arcuate nucleus, periventricular nucleus, and preoptic area; no difference (P > 0.05) was found in the paraventricularis nucleus for GnRH-ir or kisspeptin-ir cells. Additionally, levels of Kiss1 and Gnrh1 messenger RNA in the hypothalamus were significantly higher (P < 0.05) in the OVX + E₂ or intact + E₂ rats than in the OVX + oil or intact + oil animals, respectively. In the OVX + oil rats, OVX significantly increased (P < 0.05) levels of Gnrh1 and Kiss1 messenger RNA and the expression of GnRH and kisspeptin in the hypothalamus compared to intact + oil animals. These results suggest that kisspeptin and GnRH play major roles in modulating the activity of estrogen circuits at the onset of puberty.     

phoenixin(smim20), a gene coding for a novel reproductive ligand,is expressed in the brain of adult zebrafish

Gonadotropin-releasing hormone (GnRH) is a highly conserved neuroendocrine decapeptide that is essential for the onset of puberty and the maintenance of the reproductive state. In addition to its role as hypothalamic releasing hormone, GnRH has multiple functions including modulator of neural activity within the nervous system and of resulting behaviors. These multiple functions are reflected by the existence of multiple isoforms. Despite its importance as a critical hypothalamic releasing hormone, the gnrh1 gene has been lost in zebrafish, and its reproductive function is not compensated for by other GnRH isoforms (GnRH2 and GnRH3), suggesting that, surprisingly, zebrafish do not use any of the GnRH peptides to control reproduction and fertility. Previously we proposed that Phoenixin/SMIM20, a novel peptide identified in mammals and the ligand for the orphan GPR173, is a potential candidate to control the initiation of sexual development and fertility in the zebrafish. Here we confirm the sequence of the zebrafish phoenixin/smim20 gene and by RT-PCR show that it is expressed early in development through adulthood. Subsequently we show that phoenixin/smim20 is expressed in the adult brain including the regions of the hypothalamus important in the control of fertility and reproduction.     

Thermoneutral conditions correct the obese phenotype in female,but not male,Kiss1r knockout mice

Kisspeptin, a neuropeptide that activates gonadotropin-releasing hormone (GnRH) neurons, has also been implicated as a regulator of energy balance. Kisspeptin receptor (Kiss1r) knockout (KO) mice display an obese phenotype in adulthood compared to wild-type (WT) controls due to reduced energy expenditure. Additionally, experimental evidence shows that the temperature of typical rodent housing conditions (22 °C) increases the metabolism of mice above basal levels. Female Kiss1r KO mice show reduced core temperature and impaired temperature adaptation to an acute cold challenge, suggesting their temperature homeostasis processes are altered. The present study examined the phenotype of gonadectomised Kiss1r KO mice at both sub-thermoneutral and thermoneutral temperature (22 °C and 30 °C). Our results confirmed the obese phenotype in Kiss1r KO mice at 22 °C, and revealed a sexually dimorphic effect of thermal neutrality on the phenotype. In female KO mice, the obesity observed at 22 °C was attenuated at 30 °C. Plasma leptin levels were higher in KO than WT female mice at 22 °C (P < 0.001) but not at 30 °C. Importantly, the expression of Ucp1 mRNA in brown adipose tissue was lower in KO mice compared to WT mice at 22 °C (P < 0.05), but not different from WT at 30 °C. In male KO mice, a metabolic phenotype was observed at 22 °C and 30 °C. These results provide further evidence for kisspeptin-mediated regulation of adiposity via altered energy expenditure. Moreover, thermoneutral housing alleviated the obese phenotype in female Kiss1r KO mice, compared to WT, indicating the impairment in these mice may relate to an inability to adapt to the chronic cold stress that is experienced at 22 °C.     

Maternal hypothyroidism reduces the expression of the kisspeptin/Kiss1r system in the maternal-fetal interface of rats

Alterations of circulating and placental levels of kisspeptin have been associated with gestational diseases. However, there are still no studies on the placental and decidual expression of Kiss1 and its receptor Kiss1r in maternal hypothyroidism, which is the aim of this work. We demonstrate that the fetoplacental restriction caused by hypothyroidism in rats is associated with a reduction in the Kiss1r expression and reduced Kiss1 and Kiss1r mRNA levels in the decidua and/or placenta. This demonstrate that fetoplacental restriction in hypothyroid rats is linked with a suppression of the kisspeptin/Kiss1r system at the maternal-fetal interface.