Alejandro S. Mechaly

Identification of Two Isoforms of the Kisspeptin-1 Receptor (kiss1r) Generated by Alternative Splicing in a Modern Teleost, the Senegalese Sole (Solea senegalensis)

Abstract The KISSPEPTIN-1 receptor (KISS1R) and its ligands (KISSPEPTINS) are implicated in the regulation of the onset of puberty. We report the coding region and genomic structure of the kiss1r gene of a modern teleost, the Senegalese sole (Ss). Ss kiss1r cDNA contained an opening frame of 1137 bp, which results in a predicted 378 amino acid protein. Searching genomic databases allowed the identification of kiss1r orthologues in six new species belonging to three vertebrate groups and established the evolutionary relationships of all KISS1R sequences available to date. Analysis of Ss kiss1r revealed for the first time in any vertebrate KISS1R gene the presence of features that are characteristic of a mechanism of alternative splicing. This was confirmed by the identification of two transcripts, Ss kiss1r_v1 and Ss kiss1r_v2. The latter, arising from intron III retention, contained a 27 codons insert in transmembrane region 4 with two stop codons, suggesting it may lead to a truncated protein. The mRNA of the two variants was differently expressed in several tissues. In the brain, levels of the Ss kiss1r_v1 were higher than those of Ss kiss1r_v2. In the gonads, the opposite was observed. Both isoforms exhibited changes depending on sex and maturity stage. The presence of two variants may help to explain some discrepancies observed in past studies regarding KISS1R expression during puberty. Thus, the existence of alternative splicing for the KISS1R gene may contribute to our understanding of the many physiological functions suspected to be mediated by KISSPEPTIN-KISS1R signaling.

Gene structure of the Kiss1 receptor-2 (Kiss1r-2) in the Atlantic halibut: Insights into the evolution and regulation of Kiss1r genes

Kisspeptin and its receptor, Kiss1r, play an essential role in the control of the onset of puberty in vertebrates. We characterized the cDNA and genomic DNA encoding Kiss1r in Atlantic halibut (Hippoglossus hippoglossus). The 1146bp open reading frame predicts a 381 amino acid protein with high homology to the Kiss1r-2 of other teleost fish. Phylogenetic analysis of Kiss1r sequences suggests that the mammalian Kiss1r-1 form arose by way of a gene duplication prior to the emergence of amphibians. Synteny analysis demonstrated the highly conserved nature of the Kiss1r-2 region in teleosts, suggesting that flanking regulatory sequences are also likely to be conserved. Bioinformatic analysis identified six conserved regions in piscine Kiss1r-2 upstream sequences, providing potential targets for future in-depth investigation of Kiss1r-2 regulation. Kiss1r-2 expression in the brain increased coinciding with the onset of puberty. Expression levels in the gonads were two orders of magnitude lower than those of the brain, a characteristic apparently conserved in other fishes, and expression in gonads was only detected in immature fish.

Gene structure analysis of kisspeptin-2 (Kiss2) in the Senegalese sole (Solea senegalensis): Characterization of two splice variants of Kiss2, and novel evidence for metabolic regulation of kisspeptin signaling in non-mammalian species

Kisspeptin signaling in the brain is involved in the control of the onset of puberty in vertebrates. In this study, we present novel evidence indicating that kisspeptin may link energy balance and reproduction. For that purpose, we determined the complete gene structure of kisspeptin in a teleost fish, the Senegalese sole (Ss). In contrast to the situation evident in several fish, in this species only Kiss2 was found. Yet, two Ss Kiss2 isoforms generated by alternative splicing through intronic retention were detected: Ss Kiss2_v1, producing the functional protein, and Ss Kiss2_v2, coding for a truncated, non-functional protein. Specific qPCRs showed that the expression of these two isoforms varied differently in brain and gonads throughout maturation. In addition, and in contrast to what has been observed in mammals, fasting increased hypothalamic mRNA levels of Ss Kiss2_v1, which also caused a concomitant rise in pituitary Ss LH and Ss FSH mRNA. Together, these data indicate the impact of the nutritional status on Kiss mRNA expression as a potential regulatory mechanism for the metabolic control of reproduction in non-mammalian species, albeit with some significant differences with respect to the situation described in mammals.

The kisspeptin system genes in teleost fish, their structure and regulation, with particular attention to the situation in Pleuronectiformes

It is well established that Kisspeptin regulates the onset of puberty in vertebrates through stimulation of the secretion of gonadotropin-releasing hormones. However, the function of kisspeptin in peripheral tissues and in other functions is still poorly understood. Recently, the evolution and distribution of kisspeptin genes in vertebrates has been clarified. In contrast to placental mammals, which have a single gene for the ligand (Kiss) and for the receptor (Kissr), fish may have up to three Kiss genes and up to four Kissr genes because of genome duplications. However, information on the genomic structure of the piscine kiss and kissr genes is still scarce. Furthermore, when data from several species is taken together, interspecific differences in the expression of kiss and kissr during the reproductive cycle are found. Here, we discuss data gathered from several fish species, but mainly from two flatfishes, the Senegalese sole and the Atlantic halibut, to address general questions on kiss gene structure, regulation and function. Flatfish are among the most derived fish species and the two species referred to above have only one ligand and one receptor, probably because of the genome reduction observed in Pleuronectiformes. However, gene analysis shows that both species have an alternative splicing mechanism based on intron retention, but the functions of the alternative isoforms are unclear. In the Senegalese sole, sex-related differences in the temporal and spatial expression of kiss and kissr were observed during a whole reproductive cycle. In addition, recent studies suggested that kisspeptin system gene expression is correlated to energy balance and reproduction. This suggests that kisspeptin signaling may involve different sources of information to synchronize important biological functions in vertebrates, including reproduction. We propose a set of criteria to facilitate the comparison of kiss and kissr gene expression data across species.

Sex-specific changes in the expression of kisspeptin, kisspeptin receptor, gonadotropins and gonadotropin receptors in the Senegalese sole (Solea senegalensis) during a full reproductive cycle

Kisspeptin is thought to have a major role in the control of the onset of puberty in vertebrates. However, our current understanding of its function in fish and how it integrates with other hormones is incomplete due to the high diversity of this group of animals and a still limited amount of available data. This study examined the temporal and spatial changes in expression of kisspeptin, gonadotropins and their respective receptors in the Senegalese sole during a full reproductive cycle. Kiss2 and kiss2r expression was determined by qRT-PCR in the forebrain and midbrain while expression of fshβ and lhβ was determined in the pituitary and fshr and lhr in the gonads. Plasma levels of testosterone (T), 11-ketotestosterone (11-KT) and estradiol-17β were measured by ELISA and gonadal maturation was assessed histologically. In males, kiss2 and kiss2r expression in the brain areas examined was highest towards the end of winter, just before the spawning season, which took place the following spring. This coincided with maximum levels of pituitary fshβ and lhβ, plasma T and 11-KT and the highest number of maturing fish. However, these associations were not evident in females, since the highest expression of kiss2, kiss2r and gonadotropins were observed in the fall, winter or spring, depending upon the variable and tissue considered. Taken together, these data show not only temporal and spatial, but also sex-specific differences in the expression of kisspeptin and its receptor. Thus, while expression of kiss2 in Senegalese sole males agrees with what one would expect according to its proposed role as a major regulator of the onset of reproduction, in females the situation was not so clear, since kiss2 and kiss2r expression was highest either before or during the spawning season.

Stage-specific gene expression during spermatogenesis in the Senegalese sole (Solea senegalensis), a fish with semi-cystic type of spermatogenesis, as assessed by laser capture microdissection and absolute quantitative PCR

Spermatogenesis is a complex process where hormonal signals regulate the interaction of different cell types in a tight spatial and temporal fashion. The Senegalese sole (Solea senegalensis) is a marine flatfish that, in contrast to many fish, exhibits a semi-cystic, asynchronous pattern of spermatogenesis progression. This pattern is characterized by the release of spermatids into the tubule lumen, where they transform into spermatozoa. In this study, we used laser capture microdissection (LCM) to isolate cells from cysts containing spermatogonia, spermatocytes, spermatids or spermatozoa in order to investigate developmental patterns of gene expression. Furthermore, we also analyzed the stage-specific expression of the same set of genes throughout spermatogenesis (early-mid, late and maturing spermatogenic stages) in tissue fragments of the Senegalese sole testis. Genes analyzed by absolute qPCR in cysts isolated by LCM and stage-specific testis samples included genes involved in steroid synthesis and action (3β-hsd, 17β-hsd, 20β-hsd, star, star-like, progesterone receptor), gonadotropin action (fshr, lhr), the kisspeptin system (kiss2, kiss2r) and other genes important for the production of mature gametes (zona pellucida 2.2, claudin and clusterin). Our results show that, in general, steroidogenesis-related genes tended to increase with spermatogenesis progression and that 3β-hsd and 20β-hsd were expressed in germ cells but 17β-hsd was not. Our results also show that fshr is expressed in most testicular cell types, including germ cells. In contrast, lhr is expressed only in late spermatogenesis and is not expressed in any of the germ cell types examined, indicating that, in contrast to fshr, lhr may be primarily expressed in non-germinal cells (e.g. Leydig cells). Furthermore, kisspeptin and its receptor were expressed in all germ cell types examined and, as expected, gamete maturation-related genes were more expressed in mature stages. These results illustrate that key factors that participate in the hormonal regulation of spermatogenesis in the Senegalese sole testis show complex cell type- and stage-specific patterns of gene expression.

Evidence of Alternative Splicing as a Regulatory Mechanism for Kissr2 in Pejerrey Fish

Kisspeptin receptors are G-Protein-Coupled Receptors that regulate GnRH synthesis and release in vertebrates. Here, we report the gene structure of two kisspeptin receptors (kissr2 and kissr3) in pejerrey fish. Genomic analysis exposed a gene structure with 5 exons and 4 introns for kissr2 and 6 exons and 5 introns for kissr3. Two alternative variants for both genes, named kissr2_v1 and _v2, and kissr3_v1 and v2, were revealed by gene expression analyses of several tissues. For both receptors, these variants were originated by alternative splicing retaining intron 3 and intron 4 for kissr2_v2 and kissr3_v2, respectively. In the case of kissr2, the intron retention introduced two stop codons leading to a putatively truncated protein whereas for kissr3, the intron retention produced a reading shift leading to a stop codon in exon 5. Modeling and structural analysis of Kissr2 and Kissr3 spliced variants revealed that truncation of the proteins may lead to non-functional proteins, as the structural elements missing are critical for receptor function. To understand the functional significance of splicing variants, the expression pattern for kissr2 was characterized on fish subjected to different diets. Fasting induced an up-regulation of kissr2_v1 in the hypothalamus, a brain region implicated in control of reproduction and food intake, with no expression of kissr2_v2. On the other hand, fasting did not elicit differential expression in testes and habenula. These results suggest that alternative splicing may play a role in regulating Kissr2 function in pejerrey.