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].
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,
6,
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.
3. Discussion
In the present study, we demonstrated that a single dose treatment with kisspeptin affected the HPG axis at different levels, indicating the promising potential of this treatment to solve Senegalese sole reproductive dysfunctions. In addition, this is the first study reporting an effect of hormonal treatment on the levels of circulating miRNAs in fish, widening the previously reported identification of miRNAs as potential biomarkers in fish physiology, particularly for nutritional and reproductive status [
20], and providing new clues on the mechanisms by which kisspeptin might impact the HPG axis in teleosts.
The KISS2-10 treatment administrated to Senegalese sole females induced a positive response on gonadotropin levels. Fsh elevation was sufficient to elicit a response from T at 2 d, but not to induce E2 production. The increase in Lh levels seemed to induce oocyte maturation as well, since it was accompanied by an increase in the number of females in stage IV of gonadal maturation. In males, the single kisspeptin dose elicited a similar response. In this case, Fsh was significantly elevated at 2 and 4 h after administration and Lh was higher at 2 d post-treatment. The Fsh elevation influenced T, but this time a bit later, 4 days after treatment. The levels of 11-KT, though, were not altered by the treatment. Sperm motility parameters (TM and VCL) increased at 2 and 4 days after the treatment with kisspeptin, with a minor rise also observed in the control group, suggesting that the surge induced by the treatment could have an adjuvant like natural oscillations in water temperature or sperm renovation after first collection, inducing the production of fresh and high-quality sperm. This is likely related to the type of spermatogenesis in this species, semi-cystic and asynchronous, which happens gradually in successive batches [
37]. Probably, multiple injections, higher doses and/or additional signals might be needed to fully trigger the reproductive axis, increasing sperm production and its quality. Consequently, also the reproductive courtship display in this species could be improved.
Actually, in two species of the genus
Morone, multiple injections of kisspeptins increased spermatozoa production in juveniles and elicited gonadal development in sexually mature fish [
38]. Although this hormonal therapy is far more advanced in humans [
1,
4], previous studies in fish have also reported positive effects on the HPG axis. In European seabass (
Dicentrarchus labrax) a single intramuscular injection of KISS2-10 decapeptide increased the production of gonadotropins, both in pre-pubertal and pubertal fish [
9]. In line with these results, KISS1–10 or KISS2–10 treatment with slow release implants produced an upregulation of pituitary expression of
fshβ and
lhβ and stimulated gonadal development in yellowtail kingfish (
Seriola lalandi) pre-pubertal males [
39], both within and outside the breeding season. Other positive example of exogenous kisspeptin administration was seen in the chub mackerel (
Scomber japonicus) after treatment with KISS1 through subcutaneous injections [
40]. In this case, gonadosomatic index (GSI), spermatozoa concentration and plasma sex steroids levels significantly increased in treated fish. Although the effects of kisspeptin treatment depend on the gonad stage and the mode of administration [
7,
9,
41,
42], altogether the reported results highlight its potential use to induce maturation in fish species with reproductive problems, representing a valuable tool to optimize hormonal induction and breeding protocols.
In vertebrates, kisspeptin seems to act upstream of HPG axis (reviewed in Beato
, et al. [
43], inducing the release of GnRH at the hypothalamus and stimulating the synthesis and secretion of Lh and Fsh in the anterior pituitary. Lh and Fsh released to the circulatory system reach the gonads and regulate steroidogenesis in the Leydig and theca cells and support gametogenesis in Sertoli and granulosa cells, respectively. In vertebrates, there is a complex and tight regulation of the HPG axis, with several negative feedback regulations from gonads, as well as a tight control of the KISS signaling through neurotransmitters and neuropeptides (reviewed in Beato
, et al. [
43]). Although kisspeptin was found to be essential for mammalian reproduction as a stimulator of hypothalamic GnRH and a regulator of puberty onset [
44], it seems that in fish, kisspeptin actions appear not to be mediated by GnRH neurons as in mammals (reviewed in Somoza
, et al. [
8]). Despite of all these positive results of kisspeptin injection on fish HPG axis and gonadal maturation, the underlying mechanism remain to be elucidated. Moreover, since fish with
kiss and/or
kissr mutated genes are able to reproduce relatively normally [
45,
46,
47], kisspeptin might act as an enhancer of gonadotropin synthesis and release during fish reproduction through a mechanism that remains to be discovered. Furthermore, although the lack of kisspeptin receptors in GnRH neurons in some teleost species suggests there is no direct neuronal action [
47], KISS1 increased spike frequency and depolarized membrane potential of hypophysiotropic GnRH3 neurons, while KISS2 suppressed it, in the brain of adult zebrafish [
48].
In the present study, the circulating miRNAs let-7e and miR-199a-3p were up-regulated in Senegalese sole females treated with kisspeptin in comparison with control females, while miR-100-5p was down-regulated. The let-7 family was found to be highly expressed in the gonads of olive flounder (
Paralichthys olivaceus) [
49], pompano (
Trachinotus ovatus) [
50], and roundworm (
Caenorhabditis elegans) [
51], suggesting a functional conservation of its crucial role in reproductive physiology [
52]. Let-7e from blood plasma was also found to be up-regulated in Senegalese sole breeders with improved sperm quality [
20]. miR-199a-3p has also been correlated with reproduction. While miR-199a, was down-regulated in mature testis of rainbow trout (
Oncorhynchus mykiss), miR-199a-5p was particularly up-regulated in the brain of mature fish [
53]. In contrast to previous reports, where miR-100-5p was more abundant during later stages of gonadal development in ovaries and testis of zebrafish [
54], and shown to have putative functions of promoting cell differentiation [
55]; we found that it was down-regulated in the plasma of kisspeptin-treated females. Nevertheless, previous studies also link the expression of this miRNA with reproduction in oriental river prawn (
Macrobrachium nipponense) [
56] and the pompano [
50]. Indeed, this miRNA was also observed to be involved in oocyte maturation [
57], and its over-expression has been associated with the inhibition of T release in mammals [
58]. These last results were in concordance with the present study where the application of kisspeptin treatment increased the T plasma levels at 2 d post-injection in females and down-regulated the circulating miR-100.
Only one circulating mature miRNA (miR-1-3p) was found DE in Senegalese sole males, being more highly expressed in males treated with kisspeptin hormone in comparison with control males. miR-1 has been found to be expressed in both skeletal and cardiac muscle lineages, where its main biological roles are cardiogenesis, myogenesis and skeletal muscle hypertrophy. Humans with cardiac injury have higher circulating levels of this miRNA after acute myocardial infarction [
59]. In fish, this miRNA has been reported to show highly conserved tissue-specific expression patterns [
60,
61] and potentially playing an important role in regulation of muscle gene expression in fish species [
60].
Only 4 mRNAs potentially targeted by miR-1-3p were identified (
dnmt3ab,
sptlc1,
pqlc3, and
tpm4a). DNA methylation is crucial for normal development and cellular differentiation in many large-genome eukaryotes [
62]. In mammals, both Dnmt3a and Dnmt3b are primarily responsible for the
de novo DNA methylation. Dnmt3 morpholino-injected zebrafish embryos exhibited small brains, defective pharyngeal arch formation, and abnormal retinal neural epithelial differentiation [
63]. Interestingly, although
dnmt3b transcript levels were higher in metamorphosed Senegalese sole specimens reared at 15 °C than at either 18 or 21 °C,
dnmt3a paralogue had a uniform expression profile across temperatures [
64]. Nevertheless, the impact of thermal regime (comparing 16 °C and 20 °C) on the expression of
dnmt3aa and
dnmt3ab was reported in Senegalese sole lecithotrophic larval stages, suggesting they might be involved in thermal programming [
65].
Sptlc1 gene mutations cause a neuropathy known as hereditary sensory neuropathy type I [
66], which might be related with its reported role in the sphingolipid metabolism pathway [
67]. Sphingolipid metabolism was also previously associated with the Senegalese sole sperm quality improvement through dietary vitamin K supplementation by the analysis of DE sncRNAs in circulation [
20]. While nothing is known about the potential role of Pqlc3, Tpm4 has been reported to be involved in the fine tuning of the cellular contraction, and an association with the development of cardiac hypertrophy was suggested [
68], in line with the cardiac injury observed in humans with higher circulating levels of miR-1 after acute myocardial infarction [
59].
Among the potentially targeted mRNAs by DE miRNAs in females, cell junction protein class, and plasma membrane and cell periphery GO-Slim cellular components were significantly overrepresented. Genes coding for gap junction proteins (or connexins) such as
connexin 32.3 (
cx32.3) and
connexin 28.9 (
cx28.9), and tight junction proteins such as
tight junction protein ZO-3 (
tjp3) and
occludin a (
oclna) were found. Connexins form channels between the cells and facilitate the cellular cross talk, connecting the cytoplasm of adjacent cells together allowing ions, small molecules (<1 kDa) and secondary metabolites to be diffused [
69]. In mammals, several connexins are known to play an essential role in female reproductive health [
70,
71]. Although little is known regarding the potential role of connexins in fish reproduction, their role on controlling meiotic arrest of oocytes have been evidenced [
72]. In addition to connexins, tight junctions (TJ) are also protein structures that control paracellular permeability to various molecules, including water, ions, and macromolecules, across cell layers; and play a key role in ovarian follicle development (reviewed in Zhang
, et al. [
73]), among other biological processes. Indeed, abundance of
ocln,
claudins (
cldns), and
tjp1 mRNAs changed during follicular growth and are hormonally regulated. Recent reports further suggest that hormonal down-regulation of TJ proteins during ovarian follicular development could reduce barrier function (i.e., allowing the paracellular passage of water and molecules) and allow the increase of follicular fluid volume as well as serum factors going into the follicle [
73]. Nevertheless, since the expression domains of genes encoding TJ proteins are highly diverse, including different normal but also neoplastic tissues [
74], it is difficult to address whether this bioinformatically prediction is specifically linked to TJ proteins from the female gonadal (granulosa and theca) cells. Besides genes encoding TJ proteins,
slc6a22 solute carrier was also found to be predicted as targets of DE ncRNAs. Slc6 transporters include the serotonin, dopamine, norepinephrine, taurine, creatine, as well as amino acid transporters [
75]. While serotonin can control oocyte maturation [
76], dopamine is a catecholamine implicated in many functions, mediating sexual motivation in mice [
77] and gonadotropin inhibition in fish [
78]. Furthermore, it has been recently hypothesized that dopamine might be also involved in the reproductive dysfunction of Senegalese sole [
79].
In addition to all these proteins regulating cell to cell communication, several genes (including
snx1 and
snx27 as well as
dopey2 and
track1) encoding intracellular trafficking proteins were also predicted to be targeted by DE miRNAs. Sorting nexins (SNXs) is a growing family of proteins involved in vesicular trafficking between cellular compartments [
80]. Furthermore, while Dopey2 has been shown to be involved on normal neural development and functioning [
81], Trak1 is involved in the regulation of endosome-to-lysosome trafficking, essential for mitochondria motility [
82]. In neurons, the efficient and regulated transport of mitochondria along axons to synapses is crucial for maintaining function. Indeed, gene silencing by targeted shRNAi and dominant negative approaches resulted in impairing mitochondrial mobility in axonal processes [
83].
Altogether, the in silico prediction of mRNAs targeted by DE miRNAs suggests that the kisspeptin treatment of Senegalese sole females might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking. In males, kisspeptin therapy might induce an altered profile of DNA methylation and sphingolipid metabolism. Therefore, the analysis of miRNAs from blood plasma revealed kisspeptin may affect the HPG axis in fish through previously unexpected molecular pathways.