International Journal of Molecular Sciences Article Kisspeptin Influences the Reproductive Axis and Circulating Levels of microRNAs in Senegalese Sole Catarina C. V. Oliveira 1, *,† , Elvira Fatsini 1,† , Ignacio Fernández 2 , Catarina Anjos 1 , François Chauvigné 3 , Joan Cerdà 3 , Robin Mjelle 4 , Jorge M. O. Fernandes 4 and Elsa Cabrita 1, * 1 Center of Marine Sciences-CCMAR, University of Algarve, 8005-139 Faro, Portugal; effernandez@ualg.pt (E.F.); cmanjos@ualg.pt (C.A.) 2 Aquaculture Research Center, Agrarian Technological Institute of Castile and Leon, Ctra. Arévalo, s/n, 40196 Segovia, Spain; FerMonIg@itacyl.es 3 IRTA-Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; francois.chauvigne@irta.cat (F.C.); joan.cerda@irta.cat (J.C.) 4 Faculty of Bioscience and Aquaculture, Nord University, 8049 Bodø, Norway; robin.mjelle@ntnu.no (R.M.); jorge.m.fernandes@nord.no (J.M.O.F.) * Correspondence: ccoliveira@ualg.pt (C.C.V.O.); ecabrita@ualg.pt (E.C.) † Both authors contributed equally to this work.  Received: 13 November 2020; Accepted: 26 November 2020; Published: 28 November 2020  Abstract: Kisspeptin regulates puberty and reproduction onset, acting upstream of the brain–pituitary–gonad (HPG) axis. This study aimed to test a kisspeptin-based hormonal therapy on cultured Senegalese sole (G1) breeders, known to have reproductive dysfunctions. A single intramuscular injection of KISS2-10 decapeptide (250 µg/kg) was tested in females and males during the reproductive season, and gonad maturation, sperm motility, plasma levels of gonadotropins (Fsh and Lh) and sex steroids (11-ketotestosterone, testosterone and estradiol), as well as changes in small non-coding RNAs (sncRNAs) in plasma, were investigated. Fsh, Lh, and testosterone levels increased after kisspeptin injection in both sexes, while sperm analysis did not show differences between groups. Let7e, miR-199a-3p and miR-100-5p were differentially expressed in females, while miR-1-3p miRNA was up-regulated in kisspeptin-treated males. In silico prediction of mRNAs targeted by miRNAs revealed that kisspeptin treatment might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking in Senegalese sole females; also, DNA methylation and sphingolipid metabolism might be altered in kisspeptin-treated males. Results demonstrated that kisspeptin stimulated gonadotropin and testosterone secretion in both sexes and induced an unanticipated alteration of plasma miRNAs, opening new research venues to understand how this neuropeptide impacts in fish HPG axis. Keywords: Solea senegalensis; G1 breeders; hormonal treatment; KISS2; gonadotropins; sex steroids 1. Introduction The kisspeptin system is widely known to control puberty and to be involved in the onset of reproduction in mammals, acting centrally via the kisspeptin receptor and stimulating the secretion of gonadotropin-releasing hormone (GnRH) [1,2]. In humans, kisspeptin treatments have been proven to be a very promising therapeutic in the treatment of fertility disorders, as it stimulates the release of gonadotropins [1]. In subfertile women, it has been observed to induce oocyte maturation [3] and in men, intravenous administration of two different kisspeptin isoforms stimulated the levels of pituitary gonadotropins luteinizing (Lh) and follicle-stimulating (Fsh) hormones in serum [4]. Int. J. Mol. Sci. 2020, 21, 9051; doi:10.3390/ijms21239051 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9051 2 of 21 In fish, as in mammals, the kisspeptin system seems to play a major role in the regulation of the gonadotropic axis, especially in the timing of puberty and control of gonadotropin secretion [5–7]. In most teleost fish, the kisspeptin system is composed of two ligands, KISS1 and KISS2, and two receptors, KISS2r and KISS3r (reviewed in Somoza, et al. [8]). Although KISS2 appears to have a predominant role in the control of reproduction [9,10], both kisspeptin peptides have been demonstrated to stimulate gonadotropin synthesis and secretion in different fish species, accelerating puberty in juveniles or gametogenesis in adults [9]. However, their actions do not appear to be mediated by GnRH neurons as in mammalian models (reviewed in Somoza, et al. [8]). Acting upstream of the hypothalamus–pituitary–gonad (HPG) axis, this hormonal treatment could represent a valuable tool to optimize current breeding protocols in commercial cultured fish. The Senegalese sole, Solea senegalensis, is an emerging and promising species for European aquaculture [11]. However, the lack of fertilization of spawned eggs from broodstock bred and reared in captivity (first generation, G1) is one of the major constraints [12,13]. Such reproductive failure has been attributed in part to males, based on the fact that G1 males lack the courtship behavior observed in wild breeders [14]. However, lower levels of Lh [15] and of sperm volume and quality [16], with respect to wild individuals, are also factors involved. Some advances have been achieved to temporarily solve these problems, namely the development of artificial fertilization [17,18] or cryopreservation protocols [19], as well as the improvement of the reproductive status through nutritional approaches [20]. Hormonal induction in G1 sole has shown partially successful results; in females with gonadotropin-releasing hormone agonists (GnRHa) slow-release implants [18,21] and in males with human chorionic gonadotropin (hCG) [22] or homologous recombinant gonadotropins [23,24] injections. Hormonal therapies could provide a solution for the reproductive dysfunctions, but improvement is still insufficient and further research is still needed. In Senegalese sole, the expression of only kiss2 gene (Sskiss2) has been identified so far, although two sskiss2 mRNA splice forms are detected: Sskiss2_v1, which produces a functional protein, and Sskiss2_v2, which encodes for a truncated, non-functional protein [25]. In this species, not only temporal and spatial, but also sex-specific differences in transcript levels were found. In males, kiss2 and its receptor were more expressed in the brain towards the end of winter, just before the spawning season, coinciding with the highest levels of fshb and lhb subunit mRNAs in the pituitary, and of plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) [26]. Over the last years, several studies have reported the key role of non-coding RNAs (ncRNAs) in the regulation of gene expression and translation in multicellular organisms [27]. MicroRNAs (miRNAs) are small single-stranded ncRNAs (21–25 nucleotides (nt), reviewed in [28]) playing crucial roles in response to environmental changes, specific treatment and/or disease [29,30] and are involved in different fundamental biological processes, including reproduction [31,32]. Many miRNAs have been found in the blood plasma of various organisms, including mammals and fish [20,33]. Moreover, the blood plasma miRNAs might be used as timely, sensitive biomarkers for several biological processes [34]. In fish, differentially expressed (DE) miRNAs related to sex were observed in serum in the tongue sole (Cynoglossus semileavis) [35]. In common carp (Cyprinus carpio), several circulating miRNAs were expressed after exposure to a potent herbicide [36]. Specifically, in case of the Senegalese sole, there is evidence that vitamin K supplementation improves sperm quality and an additional complex tissue crosstalk along the HPG axis might take place through some small non-coding RNAs (sncRNAs) in blood plasma [20]. Currently, to our knowledge, there is no information on how the treatment with kisspeptin may affect sole reproduction and which levels of the HPG axis it would affect, including the regulation of gene expression through sncRNAs. Hence, we performed an integrative analysis, including gonadotropins and sex steroids plasma concentrations, gonadal development, sperm quality, and circulating miRNAs in blood plasma, after a kisspeptin treatment. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 21 Int. J. Mol. Sci. 2020, 21, 9051 3 of 21 2. Results 2. Results 2.1. Kisspeptin Treatment Affects the Reproductive axis 2.1. Kisspeptin During Treatment Affects the experimental the Reproductive period, axis several alterations on the gonad development, gonadotropins and sex steroids During plasma levels, asperiod, the experimental well several as on alterations the expression levelsdevelopment, on the gonad of blood plasma miRNAs were gonadotropins observedand in sex both genders. steroids At the plasma beginning levels, as well asofonthe the experiment (T0),ofboth expression levels bloodfemale plasmagroups miRNAs(Ccontrol were and observed in both genders. At the beginning of the experiment (T0), both female groups kisspeptin treatments) showed a similar percentage of maturation stages with the presence of more (Ccontrol and kisspeptin treatments) showed a similar percentage of maturation stages with the presence of more females in stages III and IV (Figure 1). Nevertheless, the group treated with kisspeptin seemed to females in stages III and IV (Figure 1). Nevertheless, the group treated with kisspeptin seemed to present present higher higher percentage of females percentage of females ininstage stageIVIV than control than control group group at 2 at 2 days days post-treatment post-treatment (2 d). (2 d). However, at 4 days However, at 4 post-treatment days post-treatment (4 (4 d)d)no nofemales females ininstage stage IV IV were were observed observed in bothin both groups. groups. Figure 1. Percentage of gonad maturation stages (0 to IV) in Senegalese sole females treated with Figure 1. Percentage of gonad maturation stages (0 to IV) in Senegalese sole females treated with kisspeptin hormone (n = 15) and untreated controls (n = 17) before (T0) and after (2 d: 2-days, 4 d: kisspeptin hormone 4-days) (n =injection. kisspeptin 15) and untreated controls (n = 17) before (T0) and after (2 d: 2-days, 4 d: 4- days) kisspeptin injection. Regarding Fsh plasma levels, they were significantly increased from T0 (9.22 ± 1.62 ng/mL), reaching a peak at 4 h post-injection (4 h) (24.11 ± 2.53 ng/mL, p = 0.0435, Figure 2A) in females treated Regarding Fsh plasma levels, they were significantly increased from T0 (9.22 ± 1.62 ng/mL), with kisspeptin. This variation was not observed in the control group, and significantly lower levels of reachingFsha peak at 4 h post-injection (4 h) (24.11 ± 2.53 ng/mL, p = 0.0435, Figure 2A) in females treated in plasma was noted at 4 h post-injection (11.10 ± 2.18 ng/mL) in comparison with kisspeptin treated with kisspeptin. females (p This variation = 0.002, was Figure 2A). not observed Afterwards, in the Fsh plasma control levels group, decreased, still and beingsignificantly lower levels significantly higher of Fsh ininplasma was noted kisspeptin-treated at 4 (16.16 females h post-injection ± 5.37 ng/mL)(11.10 than in ± 2.18 group control ng/mL) 1.25 ng/mL, p =with in±comparison (5.47 0.03) kisspeptin at 2 d, but not at 4 d (Figure 2A). Concerning Lh plasma levels, although treated females (p = 0.002, Figure 2A). Afterwards, Fsh plasma levels decreased, still beingvalues in the kisspeptin group tend to increase from T0 (37.10 ± 4.47 ng/mL: Figure 2B) to 2 h (60.33 ± 6.76 ng/mL), no statistical significantly higher in kisspeptin-treated females (16.16 ± 5.37 ng/mL) than in control group (5.47 ± differences were found along the sampling points. Nevertheless, at 2 d post-treatment, values in 1.25 ng/mL, p = 0.03) at 2 d, but not at 4 d (Figure 2A). Concerning Lh plasma levels, although values the kisspeptin group were significantly higher than in control group females (36.03 ± 5.13 ng/mL: in the kisspeptin Figure 2B, pgroup = 0.014).tend to increase The levels from exhibited of T in females T0 (37.10the±same4.47profile ng/mL: Figure of Lh, being 2B) only to 2 h (60.33 ± 6.76 significantly ng/mL), higher no statistical differencesfemales in kisspeptin-treated were (1.12 found along ± 0.27 thethan ng/mL) sampling points. in control females Nevertheless, at 2 d post- (0.55 ± 0.09 ng/mL) at 2values treatment, d (Figure thep =kisspeptin in3A, 0.03). The levels group of estradiol (E2 ) decreasedhigher were significantly in boththan female ingroups controlfrom T0 to females group 4 d (p = 0.0127) and no differences were found between experimental groups at any sampling time (36.03 ± 5.13 ng/mL: Figure 2B, p = 0.014). The levels of T in females exhibited the same profile of Lh, (Figure 3B). being only significantly higher in kisspeptin-treated females (1.12 ± 0.27 ng/mL) than in control females (0.55 ± 0.09 ng/mL) at 2 d (Figure 3A, p = 0.03). The levels of estradiol (E2) decreased in both female groups from T0 to 4 d (p = 0.0127) and no differences were found between experimental groups at any sampling time (Figure 3B). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 21 Int. J. Mol. Sci. 2020, 21, 9051 4 of 21 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 21 Figure 2. Gonadotropin plasma levels in ng/mL (mean ± standard error of the means, SEM) from Figure 2. Gonadotropin Senegalese plasmawith sole females treated levelskisspeptin (mean ± standard in ng/mLhormone error of the(nmeans, SEM) Fshfrom Figure 2. Gonadotropin plasma levels in ng/mL (mean ±(nstandard= 15) and controls error = 17). (A) of the means, SEM) and from Senegalese (B) Lh sole levels females before (T0) treated and with after (2 kisspeptin h: 2-h; 4 h: hormone 4-h; 2 d: (n = 2-days;15)4 and d: controls 4-days) the(n = 17). (A) kisspeptin Fsh and injection. Senegalese sole females treated with kisspeptin hormone (n = 15) and controls (n = 17). (A) Fsh and (B) The Lh levels before asterisk (T0) and afterdifferences (2 h: 2-h; 4 h: 4-h; 2 d: 2-days; 4 d: groups 4-days) theeach kisspeptin injection. (B) Lh levels denotes before (T0) significant and after (2 h: 2-h; 4 between experimental h: 4-h; 2 d: 2-days; 4 d: 4-days) at sampling the kisspeptin point injection. The asterisk (Student’s denotes t-test; * p significant < 0.05; ** pdifferences < 0.002); between different experimental letters indicate groups at significanteach sampling differences point among The asterisk denotes significant differences between experimental groups at each sampling point (Student’s sampling < 0.05;each t-test; * pwithin ** p