Comparative Biochemistry and Physiology, Part A 172 (2014) 31–38

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Comparative Biochemistry and Physiology, Part A

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Melatonin-mediated effects on killifish reproductive axis

Francesco Lombardo a, Giorgia Gioacchini a, Adele Fabbrocini b, Michela Candelma a,Raffaele D'Adamo b, Elisabetta Giorgini a, Oliana Carnevali a,⁎a Dipartimento di Scienze della Vita e dell'Ambiente, Università Politecnica delle Marche, Via Brecce Bianche, Ancona, Italyb Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, UOS Lesina, FG, Italy

⁎ Corresponding author at: Dipartimento di ScienzUniversità Politecnica delle Marche, Via Brecce BiancTel.: +39 071 2204990; fax: +39 071 2204650.

E-mail address: o.carnevali@univpm.it (O. Carnevali).

http://dx.doi.org/10.1016/j.cbpa.2014.02.0081095-6433/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 November 2013Received in revised form 30 January 2014Accepted 4 February 2014Available online 15 February 2014

Keywords:mtnrFecunditySperm motilityRT-PCRFT-IRSperm Class Analyzer®F. heteroclitus

The aim of this study was to investigate the melatonin-mediated effects upon the neuroendocrine axis of thebrackish killifish (Fundulus heteroclitus), a suitable experimental model to study reproductive events. The abilityof melatonin to enhance reproductive capacity (fecundity, embryo survival and hatching rate) inducing thetranscriptional activity of gonadotropin releasing hormone (gnrh), luteinizing hormone receptor (lhr) andmelatonin receptor (mtnr) was investigated in adult females. Moreover, the melatonin-mediated enhancementof killifish sperm motility and velocity was found consistent with higher fecundity of melatonin-exposed fishes.As a further extent, Fourier Transform Infrared (FT-IR) microspectroscopy evidenced a reduction of lipidunsaturation level on isolated spermatozoa from treatedmales.Moreover, the reduction ofmtnr gene expressionduring embryo development and lower biometric parameters documented in the larvae from melatonin-exposed parents suggest that melatonin acts as a hormonal mediator able to transfer the environmental signalto oocytes and then to embryos as inheritance of adaptive environmental changes. These results supportthe positive role of melatonin on killifish reproduction and its role as a maternal factor on embryo andlarval development.

© 2014 Elsevier Inc. All rights reserved.

1. Introduction

In all vertebrates, the photo-neuro-endocrine structure involved inenvironmental stimuli perception is the pineal organ and, becausephotoperiod is the most reliable cue signalling the changing seasons,interpretation and transduction of this message are one of the mostessential events in the regulation of reproduction and thus in theprogress of life cycle (Maitra and Chattoraj, 2007).

The Kiss system, under the photoperiodic control of the pinealmelatonin, acts on the hypothalamus working as central processorand neuroendocrine conduit for conveying the environmental infor-mation onto brain reproductive centre, the gonadotropin releasinghormone gnrh neurones. Thus, during puberty or during annualrecrudescence, the gnrh-dependent gonadotropins (fsh, folliclestimulating hormone; lh, luteinizing hormone) released from theanterior pituitary gland regulate in turn the steroid-signalling path-way involved in the gonadal biosynthesis and metabolism of steroidhormones. The latter, in turn, mediate the physiological processes

e della Vita e dell'Ambiente,he, 60131 Ancona, AN, Italy.

that regulate oogenesis and spermatogenesis (Shahab et al., 2005;Filby et al., 2008).

The environmental message by which animals regulate theirlife cycle is translated into a rhythmic endocrine signal, the pinealindoleamine (N-acetyl-5-methoxytryptamine) melatonin, exclusivelysecreted at night. This hormone plays a central role in transmittingday-length information to the whole organism, and particularly to theneuroendocrine-gonadal axis (Malpaux et al., 2001), therefore, animalsadjust their bio-clock/bio-calendar with their reproductive activities forthe best time for spawning (Hofman, 2004; Carnevali et al., 2010) toensure a better survival of the new progeny.

Melatonin is a highly conserved molecule and its presence can betraced in all evolutionary life forms from the simplest bacterium to thehuman being. A primitive and primary function of melatonin was toprotect against oxidative stress. Later in evolution, melatonin producedby the pineal gland evolved to be a chemical signal of dark/light, tomediate seasonal physiological functions, immunostimulation andother receptor-mediated functions in multicellular organisms, suchas body weight and energy balance (Piccinetti et al., 2010; Tanet al., 2010).

In all vertebrates, melatonin exerts its biological effects via twospecific G protein-coupled seven transmembrane-spanning domainreceptors: mt1 (mel1a) and mt2 (mel1b), all these receptors areexpressed both singly and together in various areas of the central

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nervous system and in peripheral organs. An additional form mt3(mel1c) is expressed only in extrapineal tissues of non-mammalianspecies, such as the kidney, brain, heart, lung, intestine, muscle,brown adipose tissue and eye (Pandi-Perumal et al., 2008; Ikegamiet al., 2009).

The link between photoperiod, and thus melatonin, and the regula-tion of reproductive functions are still little explored in teleosts(Falcón et al., 2007; Carnevali et al., 2011; Lombardo et al., 2012) asopposed to higher vertebrates (mainly mammals) where clear linksbetween reproduction and melatonin have been reported (Arendt,1998). However, its molecular mechanisms of action (gonadotropicor anti-gonadotropic) are still not well comprehended and the resultsdiffer between species (Falcón et al., 2007; Tamura et al., 2009;Carnevali et al., 2010, 2011). This is because these studies used differentexperimental procedures (the time of the year at which the experi-ments were done was crucial), different species or, within a same spe-cies, animals of different sex and historical status. However, evidenceis now coming to light that indicates melatonin mediates the effects ofphotoperiod on several neuroendocrine and behavioural functions.Depending on the species (long-day/short day seasonal breeders),melatonin may induce or inhibit the Kiss-HPG-reproductive axis, thus,interruption of the neural pathways anywhere between the suprachias-matic nuclei and the pineal, turns this gland non-functional in terms ofits capacity to alter the reproductive system (Kauffman et al., 2007; Roaet al., 2008; Castellano et al., 2009; Ansel et al., 2010).

Considering this discrepancy in the melatonin effects on vertebratereproduction and despite the increasing interest in its physiologicalrole in reproduction (Tamura et al., 2009), the aim of the currentstudy was to prove the melatonin-mediated effects upon Fundulusheteroclitus reproduction. In this light, the effects of melatonin adminis-tration on gametes quality were investigated by reproductive perfor-mance and sperm motility parameters assessment. More in detail, theeffects of melatonin treatment, at central (brain) and peripheral level(ovary),were analysed taking into accountmtnr aswell as genes codify-ing for key factors involved in the regulation of gametogenesis and thusin the reproductive axis, such as gnrh and lhr.

In addition, sperm motility and the antioxidant effects in the spermof melatonin-exposed fishes were also studied together with theexpression ofmtnr gene during, embryo development and larval growthfrom melatonin-treated parents.

2. Materials and methods

2.1. Animals

The mummichog F. heteroclitus (Cyprinodontidae) or killifish is aeuryhaline oviparous teleost that in nature displays a semi-lunarspawning cycles synchronized with new and full moon tides. Sucha semi-lunar pattern of reproduction is free-running under properlaboratory conditions and can be monitored by daily egg collectionor regular ovarian sampling. The fish utilized in this study didn'tdisplay a semilunar spawning rhythm. Mature killifish, F. heteroclitusL. TL = 70–90 mm were maintained in our facility under controlledcondition (24 °C; 14 L/10 D, 30‰) and fed ad libitum with commercialdiet (Tetramin®, Tetra, Melle, Germany) twice a day. In addition,the spawning tanks were equipped with a vertical plastic screen(5 mm diameter) since these fishes tend to spawn against the screen(Hsiao et al., 1994). Procedures were performed in accordance withthe Guidelines on the handling and Training of Laboratory Animals bythe Universities Federation for Animal Welfare (UFAW) and with theItalian animal welfare legislation (D.L. 116/92).

2.2. Melatonin exposure

Two experimental groups were set up with a sex ratio of 3females:2 males, a control group (CTRL) and a treated group (MEL)

exposed via water, for 8 days, to 1 μM melatonin (Sigma Aldrich CoMilan, Italy) following Zhdanova et al. (2008). The experiment wasrepeated three times. During the treatment, melatonin was addeddaily at 11 a.m. and the concentration in the tankwaterwasmaintainedby renewing water every 24 h in each tank.

2.3. Killifish reproductive performance assay

To evaluate the effects of melatonin on F. heteroclitus reproductiveactivity, daily spawning assessment during the 8 days treatment wasperformed at 10 a.m. by siphoning out the spawning tank bottom. Thefertilized eggs from each experimental group were then deposited ona piece of blotting paper in a 90 mm plastic Petri dish moistened with30‰ artificial seawater and incubated, at room temperature (24 °C)for 8 days; thus, at the end of the embryo development (8 dpf) embryosurvival and number of hatched embryo were assessed.

At the end of the experiment, females were sacrificed with a lethaloverdose of anaesthesia [500 mg L−1 MS-222 (3-aminobenzoic acidethyl ester) buffered to pH 7.4] and the brain and ovary were sampledand kept at −80 °C for future molecular analyses. Sperm samples,obtained by stripping live males, were utilized for sperm motility andFT-IR assays. During embryo incubation period, 6 embryos from eachexperimental group were sampled at 3 (stage 24: Prominent finbud),6 (stage 29: circulation in pectoral fin), 8 (stage 31: formation of swimbladder) dpf and at 0 (stage 32: hatching) dph (day post-hatching)(Oppenheimer, 1937) for future molecular analyses.

2.4. Killifish progeny biometric parameters assessment

To assess the potential effects of melatonin treatment on theprogeny of treated broodstocks, 50 ± 2 larvae (9 replicates each),hatched fromCTRL andMEL groupswere reared in 5 L tanks for a periodof 30 days. Both groups were maintained in the same rearing conditionas the broodstocks and fed with Artemia salina nauplii (at 9 a.m.) andcommercial dry diet (Tetramin®, Tetra, Melle, Germany) (at 5 p.m.).Larval survival was monitored during the experiment and larvalbiometrical parameters, such as BW (mg) and TL (mm), were recordedduring sampling at 0 (start), 7, 14 and 21 (end) dph. At each sampling,10 larvae from each experimental group were captured by nettingand sacrificed by a lethal overdose of anaesthesia [200 mg L−1

MS-222 (3-aminobenzoic acid ethyl ester) buffered to pH 7.4],dried on blotting paper and weighed using an analytical balanceaccurate to 0.1 mg (OHAUS Explorer E11140). Subsequently, larvaewere measured in length using a sliding micrometer microscope(STEMI 2000).

2.5. RNA extraction and cDNA synthesis

Total RNA was extracted from embryos in toto and from the adultbrain and ovary samples with RNeasy Mini Kit (Qiagen) and eluted in15 μL of RNase-free water. Final RNA concentrations were determinedby a Thermo Scientific NanoDropTM 1000 Spectrophotometer and theRNA integrity was verified by ethidium bromide staining of 28S and18S ribosomal RNA bands on 1% agarose gel. Total RNA was treatedwith DNAse (10 UI at 37 °C for 10 min, MBI Fermentas), and a totalamount of 1 μg of RNA was used for cDNA synthesis, employing iScriptcDNA Synthesis Kit (Bio-Rad).

2.6. Real time PCR

Specific amplification primers were designed using PCR designersoftware PRIMER3 (http://frodo.wi.mit.edu/primer3/). The sequencesof primers used at a final concentration of 200 nM were reported inTable 1. Triplicate PCR reactions were carried out for each sampleanalysed. After real-time condition optimization, PCRs were performedwith the SYBR greenmethod in an iQ5 iCycler thermal cycler (Bio-Rad).

Table 1Primers used in the study.

Gene name Forward primer Reverse primer GenBank accession no.

β-Actin 5′-CGACATCCGTAAGGACCTGT-3′ 5′-ACATCTGCTGGAAGGTGGAC-3′ AF43509218s 5′-TTTCTCGATTCTGTGGGTGGTGGT-3′ 5′-TAGTTAGCATGCCGGAGTCTCGTT-3′ M91180gnrh 5′-GCTGAGTCTGTGGCTTCTCC-3′ 5′-GCCCACATGTGCTAAACCTT-3′ AB302265lhr 5′-GCTGTCGTCTGCGTTTGTTA-3′ 5′-CAGGAGTTGATGGCCAAGAA-3′ AB295491mtnr 5′–GCTCACCATCGTAGCCATC-3′ 5′-TGCGCAGGTAGCAGTAGGT-3′ JQ029964

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The reactions were set on a 96-well plate by mixing, for each sample,1 μL of diluted (1⁄10) cDNA, 5 μL of 2× concentrated iQ™ SYBRGreen Supermix (Bio-Rad), containing SYBR Green as a fluorescentintercalating agent, 0.2 μM forward primer and 0.2 μM of reverseprimer. The thermal profile for all reactions was 15 min at 95 °Cand then 45 cycles of 20 s at 95 °C, 20 s at 60 °C and 20 s at 72 °C.Fluorescence monitoring occurred at the end of each cycle. Addition-al dissociation curve analysis was performed and showed in all casesone single peak. β-Actin and 18S were used as reference genesin each sample in order to standardize the results by eliminatingvariation in mRNA and cDNA quantity and quality. No amplificationproduct was observed in negative control and no primer-dimerformation was observed in the control templates. The data obtainedwere analysed using the iQ5 optical system software version 2.0(Bio-Rad) including GeneEx Macro iQ5 Conversion and genexMacro iQ5 files.

2.7. Sperm motility evaluation

For each assessment of motility, semen was collected from controland 8-days exposed male fishes by abdominal stripping and activatedin a small polyethylene vial (Eppendorf) by dilution in filtered seawater(15‰, 440 mOsm) at a rate of 1:50. The motility activation protocol(modified from Khan andWeis, 1987; Fabbrocini et al., 2012) was stan-dardized in preliminary trials, where ranges of salinity and of dilutionrates were tested in order to identify the best conditions for spermmotility activation and recording. Aliquots of semen (1.5 μL) wereimmediately pipetted on a multichamber counting slide (10 μmthick; Leja, The Netherlands). Sperm movement was recorded usinga 100 frame s−1 camera (Basler A602f-2, 782 × 582 resolution) at-tached to a microscope (Nikon Eclipse E600) with a phase-contrastobjective (10 × 10 magnification) and connected to a computerisedmotion analysis system, the Sperm Class Analyzer® (SCA,Microptic, s.l., Spain). The SCA® acquisition parameters were setas follows: maximum area = 400 μm2; minimum area = 50 μm2;frame rate = 100 s−1; and total captured images = 100.

Spermmotility evaluationswere performed 15 s after activation (t0)and up to the decay of motility (15 min after activation).

The following parameters were evaluated (Kime et al., 2001):percentage of total motile sperm; VCL — curvilinear velocity (μm s−1);VSL — straight-line velocity (μm s−1); VAP — average path velocity(μms−1). Only spermatozoa having a VCL N 35 μms−1 were consideredto bemotile in order to discern them from local vibrating ones. The con-sistency of the sub-population of rapid sperm, i.e. the sperm showingthe best motility performance (Beirão et al., 2011), was also assessed:the percentage of rapid sperm, their relative velocities (VCL-r, VSL-r,VAP-r), their ALH-r — amplitude of lateral head displacement (μm)and BCF-r — beat/cross frequency (beats s−1) were therefore evalu-ated. For this species we considered as rapid sperm those having VCLN 200 μm s−1, as in the control sample on activation about 50% ofsperm showed a VCL higher than this value, and this subpopulationwas significantly affected by melatonin treatment.

For each semen sample, 3 motility records were taken in 3 differentmicroscopic fields; each record consisted of the mean of threereplicates, each analysing from 250 to 500 sperm tracks. Records

were carefully checked for sample drifting. Bovine Serum Albumin(BSA, SIGMA, Milan, Italy) was used as an anti-sticking agent (Beirãoet al., 2011).

2.8. Fourier Transform Infrared microspectroscopy (FT-IR)

FT-IR microspectroscopic analyses were carried out in reflectancemode, on centrifuged sperm samples (400 g for 10 min) to eliminateseminal liquid interference, so sperm cells were deposed on steelsupports.

By means of the microscope television camera, on each sample,a specific areawas selected (ca. 2000× 2000 μm)onwhich the chemicalmap, representing the total intensity of the infrared absorption,was acquired; each pixel corresponds to a single spectrum. Reflectionspectra were performed at room temperature by using a Perkin ElmerGXI spectrometer, equippedwith a Perkin Elmer Autoimagemicroscopewith a photoconductive HgCdTe, MCT, array detector, operating atliquid nitrogen temperature which covers the entire IR spectral rangefrom 4000 to 700 cm−1. The spectral resolution was 4 cm−1 and thespatial resolution was 40 × 40 μm (128 scans). Background scanswere acquired and rationed against the sample spectrum. For datahandling, the following software packages were used: Spectrum Image1.6.1 and Spectrum 6.3.4 (Perkin Elmer), Grams AI 7.02 (Galactic, USA).Average spectra, calculated by co-added procedure, were convertedin absorbance, two points baseline linear fitted (4000–700 cm−1) andvector normalised. At the occurrence, SecondDerivative (5-point smooth-ing, Savitzky–Golaymethod) andPeak Fitting (Gaussian character) proce-dures were adopted to determine position and absorbance intensity ofbands. Peak fitting was performed on average spectra, previouslyinterpolated in the ranges of 1780–1480 and 1330–980 cm−1 andtwo points baseline linear fitted; to identify the underlying compo-nent bands, the number of peaks together with their centre valueswere carefully individuated according to second derivative resultsand fixed before running the iterative process, to obtain the bestreconstructed curve (residual near to zero). The mean values ofarea and wavelength were performed for each component peak.Attribution of the bandswas done according to literature (Stuart, 2004).

2.9. Statistical analysis

All data are presented as mean ± SD. One-way ANOVA followedby t-test were used for comparison between each experimentalgroup using a statistical software package, GraphPad Prism (GraphPadSoftware Inc., USA). P b 0.05 was considered significant.

Prior to analysis semen motility data were arcsine transformedand tested for normality using Cochran's test and for homogeneity ofvariance using Shapiro–Wilk's test. As in some cases they did not meetthe assumption of homogeneity of variance, the non-parametric UMann–Whitney test was performed to evaluate the differences insperm motility parameters on activation (t0) between exposed (MEL)and control (CTRL) groups. P b 0.05 was considered significant.STATISTICA (Vers. 8.0, 2008, StatSoft, Inc.) software system wasused for these statistical analyses.

Fig. 1. Killifish reproductive performance. (A) number of daily spawned eggs, (B) embryosurvival and (C) number of hatched embryo. Control group (CTRL); group treated with1 μM melatonin (MEL).

Fig. 2.Melatonin receptor in female killifish brain.mtnr (A) and gnrh (B) gene expressionin the killifish brain. Control group (CTRL); group treated with 1 μM melatonin (MEL).

Fig. 3. Melatonin receptor in killifish ovary. mtnr (A) and lhr (B) gene expression in thekillifish ovary. Control group (CTRL); group treated with 1 μM melatonin (MEL).

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3. Results

Eight days of melatonin exposure increased the number of dailyspawned eggs (~30%) and the effects resulted significantly (P b 0.05)different with respect to the control group starting from day 4 untilthe end of the experiment (Fig. 1A). Embryo survival (Fig. 1B) and thenumber of hatched embryo (Fig. 1C) were enhanced (~25% and 20%,respectively) with significant differences (P b 0.05) only in embryosfrom 8 days melatonin-treated parents.

At molecular level, melatonin treatment significantly (P b 0.05)increased mtnr (Fig. 2A) and gnrh (Fig. 2B) expression in the brainof treated females with respect to control. The mtnr (Fig. 3A) andlhr (Fig. 3B) expression levels resulted significantly (P b 0.05) higherin the ovary of treated fish with respect to control. Moreover, mtnrexpression levels resulted significantly (P b 0.05) reduced in embry-os from melatonin-treated parents at 3, 6 and 8 dpf, while no differ-ence was observed in the larvae (0 dph, Fig. 4).

On the other hand, melatonin exposure increased the killifish spermmotility parameters analysed by the SCA® system. On activation,both the percentages of total motile (VCL N 35 μm s−1) and rapid(VCL N 200 μm s−1) spermatozoa were higher in semen frommelatonin-exposed specimens, even if the difference was significantonly for rapid sperm (Fig. 5A). The levels of curvilinear (VCL),straight-line (VSL) and average path (VAP) velocities were alwayssignificantly higher in MEL samples with respect to CTRL ones(Fig. 5B); as regard the velocities of the sub-population of rapid sper-matozoa, no differences were recorded for VCL-r, while VSL-r andVAP-r levels were again significantly higher in sperm from exposed

Fig. 4. Melatonin receptor (mtnr) in killifish embryos. mtnr in embryos (3, 6 and 8 dpf)and in newly hatched larvae (0 dph). Control group (CTRL); group treated with 1 μMmelatonin (MEL).

Fig. 6. FT-IR average spectra of killifish sperm. Representative spectra of CTRL (dotted line)and MEL (continuous line) experimental groups in the spectral range of 4000–700 cm−1.

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killifish (Fig. 5C). The amplitude of lateral head displacement of rapidsperm (ALH-r) did not change between samples coming from controland exposed specimens; on the contrary the beat cross frequency(BCF-r) was significantly higher in MEL samples (Fig. 5C). Furthermore,both the percentages of total motile and rapid spermatozoa quicklydecreased and 10 min after activation no rapid spermatozoa wererecorded in both MEL and CTRL groups (Fig. 5D). In all cases, 15 min

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Fig. 5.Motility parameters of killifish sperm. Spermmotility parameters on activation inmelatoninand rapid (VCL N 200 μm s−1) spermatozoa; (B) VCL, VSL and VAP (μm s−1) of the total motilesubpopulation of rapid sperm. (D) Percentages of total motile and rapid spermatozoa up to 15 mafter activation.

after activation all spermatozoa were immotile and a similar rapiddecreasing trend was observed also for the velocity parameters, VCL,VSL and VAP (Fig. 5E).

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exposed (MEL) and control (CTRL) groups. (A) Percentages of totalmotile (VCL N 35 μms−1)spermatozoa. (C) VCL-r, VSL-r, and VAP-r (μm s−1), ALH-r (μm) and BCF-r (beats/s) of thein after activation; (E) VCL, VSL and VAP (μm s−1) of the motile spermatozoa up to 15 min

Fig. 8. Killifish larval biometric parameters. (A) Bodymass, (B) total length. Control group(CTRL); group treated with 1 μM melatonin (MEL).

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By FTIR microspectroscopy, average spectra of spermatozoa fromcontrol (CTRL) and melatonin exposed (MEL) males were acquired(Fig. 6), in which meaningful spectral regions were analysed as follow:

- 3100–2800 cm−1 (CH stretching region): the bands at 2957, 2925,2873 and 2852 cm−1 appeared unchanged in the position andabsorption intensity. On the contrary, the band at 3015 cm−1

decreased in intensity in MEL, as confirmed by the absorption bandratio of 3015/2957, which showed a lower value in samples treatedwith melatonin (Fig. 6);

- 1780–1470 cm−1 (proteic pattern): by applying peak fittingprocedure, the different components of Amide I bandwere detected:β-turn, 1698 ± 1 and 1617 ± 1 cm−1 (intermolecular β-sheet);1683 ± 2 and 1630 ± 1 cm−1 (intramolecular β-sheet); 1670 ±2 cm−1 (3-turn helix); 1659 ± 2 cm−1 (α-helix), and 1642 ±1 cm−1 (random coil) (data not shown). The percentage amountsof the different structures were calculated by considering the areasof the above cited peaks;

- 1320–800 cm−1 (nucleic acid spectral region): by applying peakfitting procedure, all the component bands were evaluated. In MELspectra, a higher value was observed in the absorption band arearatio 1046/1087 cm−1 (ν C\O carbohydrates/νsym PO2−) (Fig. 7).

From hatching time (0 dph) until 14 dph, biometric parameters oflarvae hatched from CTRL andMEL groups showed no significant dif-ferences (P N 0.05) in terms of BW (Fig. 8A) and TL (Fig. 8B).However, at 21 dph larvae hatched fromMEL group showed a signif-icant (P b 0.05) lower BW and TL than larvae from CTRL group.On the other hand, larval survival was not affected by broodstocks'melatonin exposure (data not shown).

4. Discussion

Gametogenesis is an evolutionary conserved process under the con-trol of the HPG system in vertebrates that obviously uses the seasonallydependent melatonin rhythm to adjust both testicular and ovarianphysiology accordingly (Roa et al., 2008; Castellano et al., 2009). Theseactions ofmelatonin aremediated bymelatonin receptors and the dem-onstration ofmelatonin receptors in the ovary and in the testis indicatesmultiple sites at which melatonin may interfere with the reproductivesystem (Yie et al., 1995; Izzo et al., 2010). However, conflictingconclusions on the role of melatonin in the neuroendocrine control ofgametogenesis of teleost have been achieved by experiments dealingwith photoperiod manipulation, pinealectomy and melatonintreatment (Mayer, 2000; Ansel et al., 2010; Chalivoix et al., 2010;Lombardo et al., 2012). Recently, studies in our laboratory demonstrat-ed that melatonin administration significantly affects Danio rerio

Fig. 7. Lipid peroxidation on killifish sperm. Histograms representing the following parameters fband area ratios; (B) 1046/1087 (ν C\O carbohydrates/νsym PO2−). Data were presented as m

(Carnevali et al., 2011) and F. heteroclitus (Lombardo et al., 2012)mean number of spawned eggs. On the other hand, the first experimen-tal evidence that melatonin plays a significant role in the regulation ofannual testicular events was obtained from studies in a sub-tropicalsurface-dwelling carp, Catla catla, but the influence of this pineal hor-mone on the seasonal activity of testis varies in relation to the reproduc-tive status of the concerned fish (Bhattacharya et al., 2007). In thepresent study, melatonin administration increases the number ofspawned eggs in agreement with previous results obtained in zebrafish(Carnevali et al., 2011). Melatonin-mediated enhancement of spawnedeggs result let us speculate that this hormone, both endogenouslysynthesised or exogenously added, has been useful during oogenesispossibly acting on gonadal steroidogenesis by regulating steroidogenicenzyme activities or their gene expression in theca and granulosa cellsas in mammals (Tamura et al., 2009) and/or acting as maturation-inducing hormone (MIH) stimulating the final follicle maturation, aspreviously demonstrated in killifish (Lombardo et al., 2012). Moreover,melatonin may function as antioxidant molecule capable to protectgametes from ROS damage preventing atresia and increasing ovulation(Tamura et al., 2009). Thus, the increased embryo survival suggests a

or CTRL andMEL experimental groups: (A)=CHstretching-to-CH3 asymmetric stretchingean ± S.D.

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powerful role of melatonin on killifish gamete/embryo qualitysuggesting the involvement of melatonin in the complex biologicaland molecular processes that govern embryogenesis overall.

The mechanism by which melatonin acts on the HPG axis is stillunclear. In the female killifish brain, melatonin receptors mediatemelatonin effects on circadian rhythms, photoperiodic entrainmentand reproduction; the increment of melatonin receptor (mtnr) andgnrh expression in the brain suggests a receptor-mediated action ofmelatonin at central level indicating the melatonin capability to stimu-late the hypothalamic Gnrh release, probably involving the Kiss/Gpr54system in the Gnrh neuronsmodulation, as observed in Syrian hamster,sheep, ewe and zebrafish (Kauffman et al., 2007; Chalivoix et al., 2010;Carnevali et al., 2011). Moreover, at ovary level, melatonin-mediatedincrease of mtnr and lhr expression confirmed the role of melatonin inthe final step of oocyte maturation and ovulation, supporting our earlyresults on in vitro follicle studies (Lombardo et al., 2012). It is reasonableto hypothesize that melatonin exposure regulated lhr transcription inthe ovary or throughout a Gnrh-stimulated Lh pituitary release thatincreases its gonadal receptor.

On the other hand, melatonin enhanced the sperm motility, both interms of percentage of motile spermatozoa and velocity, and the resultswere consistent with the increase of killifish fecundity and embryo sur-vival. The effect of melatonin exposure on sperm motility parametersmay be due, besides by melatonin properties as a circadian rhythmtransducer, probably to its direct free radical scavenging and broad-spectrum antioxidant activity against peroxidative damage induced byROS (Sikka et al., 1995; Tan et al., 2010) as confirmed by the decreaseof lipid unsaturation levels obtained by the vibrational analysis. In fact,the band at 3015 cm−1 (ν_CH) is usually considered a marker ofperoxidative processes, being correlated to the level of fatty acyl chainperoxidation (Severcan et al., 2005).

The antioxidant systems in mammal semen have been widely stud-ied. Spermatozoa are very sensitive to ROS: asmost of sperm cytoplasmis removed during the final stages of spermatogenesis, they have almostno enzymes involved in the protection from peroxidative damage in-duced by ROS (du Plessis et al., 2010); therefore, reactive oxygen speciesmay cause lipid peroxidation of sperm cell membranes due to the highcontent of polyunsaturated fatty acids within their plasma membraneand damage axonemal structure causing loss of motility and infertility(Ortiz et al., 2011). For this reason, due to their scavenging activity onROS, molecules such as melatonin may protect spermatozoa from freeradical damages, with positive effects on sperm motility and fertility(Lahnsteiner et al., 2011). Recently, the presence of antioxidant systemshas been demonstrated also in seminal plasma and sperm cells of teleostspecies (Metwally and Fouad, 2009), and improvement in spermmotil-ity and fertility has been recorded when antioxidants were suppliedwith diet to Arctic char and grass carp (Mansour et al., 2006; Metwallyand Fouad, 2009). The higher sperm motility and velocity of killifishspermatozoa herein obtained could be referable to the protectiveefficiency of melatonin against free radical-induced macromoleculardamages during spermatogenesis process beside the higer amount ofcarbohydrates found on spermatozoa from melatonin treated malesmay be related to an higer amount of available energy (Wong et al.,1993; Ci et al., 1999).

The mtnr expression pattern observed in killifish embryos suggeststhe receptor-mediated action of melatonin also in cell proliferation,developing tissues and organogenesis suggesting that the onset ofthese effects coincides in time with the activation of endogenous mela-tonin production and with the increase of melatonin receptor duringembryo development, as reported in zebrafish (Danilova et al., 2004).

Embryos derived from melatonin-treated broodstock showed re-duced mtnr gene transcription levels during development suggesting arole of melatonin as a mediator of epigenetic inheritance system (EIS):parents' melatonin can transfer the environmental information togametes and this information is used to influence embryo and juveniledevelopment. Melatonin present in the oocyte may act as a mediator

to produce epigenetic modifications and contribute to the inheritanceof adaptive changes (biological adaptation) to the next generation(Irmak et al., 2005) However, this molecular data needs additionalstudies to better explain the relationship between melatonin andembryo development.

In this study, the effects of maternal-melatonin inheritance on larvalgrowth, assessed by biometric parameters of the progeny records,suggest no melatonin effect on the early larval development (within14 dph) while, at longer extent (21 dph), a reduction of BW and TLwas induced in the progeny derived from melatonin-treated parents,suggesting a control of food intake as previously demonstrated inzebrafish (Piccinetti et al., 2010, 2013).

5. Conclusion

These results support the positive effect ofmelatonin on F. heteroclitusreproduction proving its role on the activation of the photo-neuro-endocrine system that regulates reproduction, in addition to gametequality and embryo and larval development.

Author contributions

FL: concept/design, acquisition of RT-PCR and fecundity, embryosurvival and hatching rate data, drafting of the manuscript; GG: FT-IRand RT-PCR data analysis/interpretation, critical revision of themanuscript; AF: acquisition and analysis/interpretation of spermmotil-ity and velocity data, drafting of the manuscript; MC: acquisition offecundity, embryo survival and hatching rate data; DAR acquisitionand analysis/interpretation of spermmotility and velocity data; EG: ac-quisition of FT-IR data; OC: concept/design data analysis/interpretation,critical revision of the manuscript.

Acknowledgements

The authors wish to thank OceAN soc coop for the technical supportat the facilities.

FA to OC supported this study.

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