Reproductive Biology of Male European Hake (Merluccius merluccius) in Central Mediterranean Sea: An Overview from Macroscopic to Molecular Investigation.

The improvement of scientific knowledge about overexploited fishery resources allow us to provide scientific advice for their management and stock protection. By using a multidisciplinary approach, the aim of the study was to characterize, for the first time in the Central Mediterranean Sea (GSA 17), the reproductive biology of males of M. merluccius, currently highly exploited. A multi-year sampling from January 2017 to December 2019 was performed to exhaustively evaluate the sex ratio of the stock, while the 2018 annual sampling was selected to investigate the reproductive pattern of males. Individuals in spawning conditions were found every month, proving that M. merluccius is an asynchronous species, reproducing all year round, with a seasonal reproductive peak in spring/summer, as indicated by GSI. Five gonadal development stages were defined to fully describe the reproductive cycle of males. The macroscopic and histological L50, respectively 18.6 cm and 15.4 cm, were both below the Minimum Conservation Reference Size (MCRS). According to the mRNA levels, fsh and lh played a significant role during spermiation, whereas the gnrhr2a was involved at the beginning of sexual maturity. In the testis, fshr and lhr reached maximum expression levels before the spermiation. The hormonal stimuli of 11-ketotestosterone and its receptor were significantly higher when the specimen was in reproductive activity.

Probiotic Administration Mitigates Bisphenol A Reproductive Toxicity in Zebrafish.

Although the use of bisphenol A (BPA) has been banned in a number of countries, its presence in the environment still creates health issues both for humans and wildlife. So far, BPA toxicity has been largely investigated on different biological processes, from reproduction to development, immune system, and metabolism. In zebrafish, Danio rerio, previous studies revealed the ability of environmentally relevant concentrations of this contaminant to significantly impair fertility via epigenetic modification. In addition, several studies demonstrated the ability of different probiotic strains to improve organism health. This study provides information on the role of the probiotic mixture SLAb51 to counteract adverse BPA effects on reproduction. A 28-day trial was set up with different experimental groups: BPA, exposed to 10 µg/L BPA; P, receiving a dietary supplementation of SLAb51 at a final concentration of 109 CFU/g; BPA+P exposed to 10 µg/L BPA and receiving SLAb51 at a final concentration of 109 CFU/g and a C group. Since oocyte growth and maturation represent key aspects for fertility in females, studies were performed on isolated class III (vitellogenic) and IV (in maturation) follicles and liver, with emphasis on the modulation of the different vitellogenin isoforms. In males, key signals regulating spermatogenesis were investigated. Results demonstrated that in fish exposed to the combination of BPA and probiotic, most of the transcripts were closer to C or P levels, supporting the hypothesis of SLAb51 to antagonize BPA toxicity. This study represents the first evidence related to the use of SLAb51 to improve reproduction and open new fields of investigation regarding its use to reduce endocrine disrupting compound impacts on health.

Opsins and gonadal circadian rhythm in the swordfish (Xiphias gladius) ovary: Their potential roles in puberty and reproductive seasonality.

Circadian rhythm is well known to play a pivotal role in reproduction but the presence of a gonadal circadian rhythm is opening a lot of questions about a local regulation of reproduction. In the present study, we first set to identify the key genes driving circadian rhythmicity from the gonadal transcriptome of the swordfish, a commercially relevant species undergoing overfishing, and we then investigated whether their transcriptional activity was influenced by the maturation stage. Finally, we explored whether seasonality had the ability to modulate the expression of these genes. Interestingly, we identified a wide number of circadian rhythm related genes in the transcriptome of the swordfish gonad including, among the others, clock, bmal1, cry2 and per2, which have been found to be differentially expressed between sexually immature and mature individuals sampled during the breeding season. This differential modulation was also found for melatonin biosynthesis genes (mel1b, asmt and tph1) as well as opsin genes (opsin4, tmt opsin, parapinopsin, VA opsin, rho and sws), known to be the primary receptors of light stimuli. These differences were not observed between mature and immature individuals when considering the non-breeding season suggesting that, despite the molecular machinery of mature gonads is able to respond to signals driving ovarian maturation, these signals are not present hence highlighting the potential role of seasonality in modulating the gonadal circadian rhythm. These results confirm the presence of a gonadal circadian rhythm in the swordfish and open new interesting questions about its role in driving puberty onset.

Mediterranean swordfish (Xiphias gladius Linnaeus, 1758) population structure revealed by microsatellite DNA: genetic diversity masked by population mixing in shared areas.

BACKGROUND: The Mediterranean swordfish stock is overfished and considered not correctly managed. Elucidating the patterns of the Mediterranean swordfish population structure constitutes an essential prerequisite for effective management of this fishery resource. To date, few studies have investigated intra-Mediterranean swordfish population structure, and their conclusions are controversial. METHODS: A panel of 20 microsatellites DNA was used to investigate fine-scale population structuring of swordfish from six main fishing areas of the Mediterranean Sea. RESULTS: This study provides evidence to reject the hypothesis of a single swordfish population within the Mediterranean Sea. DAPC analysis revealed the presence of three genetic clusters and a high level of admixture within the Mediterranean Sea. Genetic structure was supported by significant F ST values while mixing was endorsed by the heterozygosity deficit observed in sampling localities indicative of a possible Wahlund effect, by sampling admixture individuals. Overall, our tests reject the hypothesis of a single swordfish population within the Mediterranean Sea. Homing towards the Mediterranean breeding areas may have generated a weak degree of genetic differentiation between populations even at the intra-basin scale.

Insights on the seasonal variations of reproductive features in the Eastern Atlantic Bluefin Tuna.

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.

Macromolecular Characterization of Swordfish Oocytes by FTIR Imaging Spectroscopy.

During folliculogenesis, primary oocytes of teleosts grow by several orders of magnitude by-self synthesizing proteins and mRNA, or sequestering from blood specific macromolecular components, such as fatty acids and vitellogenin. All these materials are stored into cortical alveoli, yolk globules or oil droplets during oocyte development. The proper synthesis, storage and displacement of these macromolecular components inside the oocyte play a key role for a successful fertilization process and for the subsequently correct embryo development. In this study, for the first time, the FTIR Imaging (FTIRI) spectroscopy has been applied to characterize the chemical building blocks of several cellular components of swordfish oocytes at different developmental stages. In particular, the spectral features of previtellogenic (PV), vitellogenic (VTG), mature (M) and atretic (A) follicles as well as and of cortical alveoli (CA), yolk vesicles (YV), oil droplets (OD) and Zona Radiata (ZR) have been outlined, providing new insights in terms of composition and topographical distribution of macromolecules of biological interest such as lipids, proteins, carbohydrates and phosphates. The macromolecular characterization of swordfish oocytes at different developmental stages represents a starting point and a useful tool for the assessment of swordfish egg quality caught in different conditions, such as periods of the year or different fishing area.

A de novo transcriptome assembly approach elucidates the dynamics of ovarian maturation in the swordfish (Xiphias gladius).

The Mediterranean swordfish (Xiphias gladius) has been recently classified as overfished and in 2016, the International Commission for the Conservation of the Atlantic Tunas (ICCAT) established a multi-annual management plan to recover this stock. To successfully achieve this goal, knowledge about swordfish biology is needed. To date, few studies on swordfish have been performed and none of them has provided useful insights into the reproductive biology at molecular level. Here we set to characterise the molecular dynamics underlying ovarian maturation by employing a de novo transcriptome assembly approach. Differential gene expression analysis in mature and immature ovaries identified a number of differentially expressed genes associated with biological processes driving ovarian maturation. Focusing on ovarian steroidogenesis and vitellogenin uptake, we depict the molecular dynamics characterizing these processes while a phylogenetic analysis let us identify a candidate vitellogenin receptor. This is the first swordfish transcriptome assembly and these findings provide in-depth understanding of molecular processes describing ovarian maturation. Moreover, the establishment of a publicly available database containing information on the swordfish transcriptome aims to boost research on this species with the long-term of developing more comprehensive and successful stock management plans.

Cloning, characterization, and molecular expression of gonadotropin receptors in European hake (Merluccius merluccius), a multiple-spawning species.

Teleosts have many spawning strategies and the hormonal control of gametogenesis is not well defined among the species or even, between sexes. To increase the knowledge of gonadotropin hormones, we studied the trend by gene expression of gonadotropin receptors in the follicles and testis at different maturity stages in the European hake (Merluccius merluccius), a multiple-spawning species. With this aim, fshr and lhr were sequenced, characterized, and their gene expression was quantified in oocytes and in testes at different maturity stages. The deduced amino acid sequences were used to phylogenetic studies and evidenced that both receptors are phylogenetically closed to other gadoid species. The gene expression of both receptors was poorly expressed in primary follicles, increased in vitellogenic follicles and to later decrease in hydrated oocytes. In testis, highest levels of lhr were detected during spermiation, while levels of fshr were constant. For the first time, a histological analysis was performed in European hake testes showing an unrestricted lobular testis. To better elucidate the mechanisms involved in the oogenesis of the European hake, the expression of estrogen receptor and cyp19a was also investigated displaying high levels in all classes of follicles. All these data allow to increase the knowledge on reproductive physiology of an important socioeconomical species and it seeks to shed more light on the role of the receptors here studied during gametogenesis of multiple-spawning fish.

Gonadotropin characterization, localization and expression in the European hake (Merluccius merluccius).

In vertebrates, the regulation of gametogenesis is under the control of gonadotropins (Gth), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh). In fish, the physiological role of Gths is not fully understood, especially in species with asynchronous ovarian development. To elucidate the role of Gths in species with asynchronous ovary, we studied European hake (Merluccius merluccius) during the reproductive season. For this aim, we first cloned and sequenced both hormones. Then, we characterized their amino acid sequence and performed phylogenetic analyses to verify the relationship to their orthologues in other species. In addition, the quantification of gene expression during their natural reproductive season was analyzed in wild-caught female hake. Our results revealed that fshb peaked during the vitellogenic phase, remaining high until spawning. This is in contrast to the situation in species with synchronous ovary. lhb, on the other hand, peaked during maturation as it is also common in species with synchronous ovarian development. Finally, combining double-labeling fluorescent in situ hybridization (FISH) for Gth mRNAs with immunofluorescence for Lh protein, we evidenced the specific expression of fshb and lhb in different cells within the proximal pars distalis (PPD) of the pituitary. In addition to gonadotrope cells specific to expression of either fshb or lhb, some cells showed co-expression of both genes. This suggests either that gonadotropes with co-expression are not yet specified or they could have a plasticity that permits changes from one cell phenotype to another during certain life stages and in turn during different physiological states.

Melatonin-mediated effects on killifish reproductive axis.

The aim of this study was to investigate the melatonin-mediated effects upon the neuroendocrine axis of the brackish killifish (Fundulus heteroclitus), a suitable experimental model to study reproductive events. The ability of melatonin to enhance reproductive capacity (fecundity, embryo survival and hatching rate) inducing the transcriptional activity of gonadotropin releasing hormone (gnrh), luteinizing hormone receptor (lhr) and melatonin receptor (mtnr) was investigated in adult females. Moreover, the melatonin-mediated enhancement of 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 lipid unsaturation level on isolated spermatozoa from treated males. Moreover, the reduction of mtnr gene expression during 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 signal to oocytes and then to embryos as inheritance of adaptive environmental changes. These results support the positive role of melatonin on killifish reproduction and its role as a maternal factor on embryo and larval development.