Revealing Spermatogenesis in Smooth-Hound Sharks Mustelus mustelus: Insights into the Morphological and Macromolecular Composition of Spermatogenic Cells.

Elasmobranchs have an ancestral reproductive system, which offers insights into vertebrate reproductive evolution. Despite their unchanged design over 400 million years, they evolved complex mechanisms ensuring reproductive success. However, human activities induced a significant decline in elasmobranch populations worldwide. In the Mediterranean basin, the smooth-hound shark (Mustelus mustelus) is one of the species that are considered vulnerable to human activities. Conservation efforts necessitate a thorough understanding of its reproductive strategy. This study focused on mature male specimens of smooth-hound sharks that were captured in the Adriatic area and successively analyzed to provide, for the first time, a histologically detailed description of testicular development in the species. Seven phases of the spermatogenesis process were identified, along with the macromolecular characterization of cells obtained using Fourier-transform infrared imaging. Histological analysis showed structural and cellular features similar to those documented in the spermatocysts of other elasmobranchs. The examination of the evolution and migration of both germinative and Sertoli cells at each phase revealed their close connection. Furthermore, different expression levels of lipids, proteins, and phosphates (DNA) at each spermatogenesis stage were observed. This research provided new information on spermatogenesis in the common smooth-hound shark, which is crucial for conservation efforts against population decline and anthropogenic pressures.

First In Vivo Insights on the Effects of Tempol-Methoxycinnamate, a New UV Filter, as Alternative to Octyl Methoxycinnamate, on Zebrafish Early Development.

The demand for organic UV filters as active components in sunscreen products has rapidly risen over the last century, as people have gradually realized the hazards of overexposure to UV radiation. Their extensive usage has resulted in their ubiquitous presence in different aquatic matrices, representing a potential threat to living organisms. In this context, the need to replace classic UV filters such as octyl methoxycinnamate (OMC), one of the most popular UV filters reported to be a potential pollutant of aquatic ecosystems, with more environmentally friendly ones has emerged. In this study, using zebrafish, the first in vivo results regarding the effect of exposure to tempol-methoxycinnamate (TMC), a derivative of OMC, are reported. A comparative study between TMC and OMC was performed, analyzing embryos exposed to similar TMC and OMC concentrations, focusing on morphological and molecular changes. While both compounds seemed not to affect hatching and embryogenesis, OMC exposure caused an increase in endoplasmic reticulum (ER) stress response genes, according to increased eif2ak3, ddit3, nrf2, and nkap mRNA levels and in oxidative stress genes, as observed from modulation of the sod1, sod2, gpr, and trx mRNA levels. On the contrary, exposure to TMC led to reduced toxicity, probably due to the presence of the nitroxide group in the compound's molecular structure responsible for antioxidant activity. In addition, both UV filters were docked with estrogen and androgen receptors where they acted differently, in agreement with the molecular analysis that showed a hormone-like activity for OMC but not for TMC. Overall, the results indicate the suitability of TMC as an alternative, environmentally safer UV filter.

Gestational Diabetes Mellitus and Small-for-Gestational-Age: An Insight into the Placental Molecular Biomarkers.

Gestational diabetes mellitus (GDM) and small-for-gestational-age (SGA) are two metabolic-related diseases that could affect women during pregnancy. Considering that the chorionic villi (CVs) are crucial structures for the feto-maternal exchange, the alterations in their conformation have been linked to an imbalanced metabolic environment of placenta. In this study, a multidisciplinary approach has been carried out to describe the changes occurring in the placental CVs of GDM and SGA patients. The results revealed higher levels of superoxide dismutase 1 (SOD-1) and catalase (CAT), especially in the GDM placentae, which could be correlated with the hyperglycemic environment characteristic of this pathology. Furthermore, spectroscopy and histologic analyses revealed that both pathologies modify the placental lipid composition altering its structure. However, SGA induces lipid peroxidation and reduces collagen deposition within the CVs. Since the endocannabinoid system (ECS) is involved in placentation and different metabolic activities, the cannabinoid receptor 1 (CB1) and transient receptor potential cation channel subfamily V member 1 (TRPV-1) were analyzed. No changes have been observed either at general or specific levels in the CVs comparing control and pathological samples, suggesting the non-involvement of the cannabinoid system in these two pathologies.

Metabolomic and Transcript Analysis Revealed a Sex-Specific Effect of Glyphosate in Zebrafish Liver.

Glyphosate is a component of commonly used herbicides for controlling weeds in crops, gardens and municipal parks. There is increasing awareness that glyphosate-based herbicides, in addition to acting on plants, may also exert toxicity in wildlife and humans. In this study, male and female adult zebrafish were exposed to 700 µg/L of glyphosate (GLY), for 28 days. We used the metabolomic approach and UHPLC-ESI-MS to analyze liver samples to investigate the adverse effects of glyphosate on hepatic metabolism. The impact of GLY was found to be sex-specific. In female, GLY exposure affected purine metabolism by decreasing the levels of AMP, GMP and inosinic acid, consequently increasing uric acid levels with respect to the control (CTRL). Exposure to GLY also caused a decrease of UMP levels in the pyrimidine metabolism pathway. In male, GLY exposure decreased the aminoadipic acid within the lysine degradation pathway. Transcript analysis of genes involved in stress response, oxidative stress and the immune system were also performed. Results demonstrated an increased stress response in both sexes, as suggested by higher nr3c1 expression. However, the hsp70.2 transcript level was increased in female but decreased in male. The results demonstrated reduced sod1, sod2, and gpx1a in male following exposure to GLY, indicating an impaired oxidative stress response. At the same time, an increase in the cat transcript level in female was observed. mRNA levels of the pro-inflammatory interleukins litaf and cxcl8b.1 were increased in female. Taken together, the results provide evidence of disrupted nucleotide hepatic metabolism, increased stress inflammatory response in female and disruption of oxidative stress response in male.

Preeclampsia Correlates with an Increase in Cannabinoid Receptor 1 Levels Leading to Macromolecular Alterations in Chorionic Villi of Term Placenta.

Preeclampsia is a human pregnancy-specific disease characterized by abnormal placentation that usually presents with maternal hypertension and proteinuria. The main hallmark of preeclampsia, impaired trophoblast migration, and the subsequent disruption of uterine arteries remodeling lead to several molecular alterations in the placental compartments with those occurring in the chorionic villi being of the utmost importance. Given the essential role of the endocannabinoid system during preimplantation and trophoblast migration, we have combined the histological and hyperspectral imaging analyses to shed light on the involvement of two cannabinoid receptors in the macromolecular alterations related to preeclampsia. The results obtained by immunohistochemistry showed a significant increase in the protein levels of cannabinoid receptor 1 (CB1) in the preeclamptic chorionic villi. However, no changes were reported regarding transient receptor potential vanilloid 1 (TRPV-1) levels either in the bulk placental samples or chorionic villi when comparing control and preeclamptic patients. Histological analysis and Fourier-transform infrared spectroscopy (FTIRI) showed an increase in collagen deposition together with higher levels of lipid peroxidation and phosphorylated compounds in the pathological villi. Since CB1 enhancement has been described as promoting fibrosis and oxidative stress in several tissues, we proposed that the higher receptor abundance in preeclampsia could be triggering similar molecular effects in preeclamptic term placentas.

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.

Knockout of the Glucocorticoid Receptor Impairs Reproduction in Female Zebrafish.

The pleiotropic effects of glucocorticoids in metabolic, developmental, immune and stress response processes have been extensively investigated; conversely, their roles in reproduction are still less documented. It is well known that stress or long-lasting therapies can cause a strong increase in these hormones, negatively affecting reproduction. Moreover, the need of glucocorticoid (GC) homeostatic levels is highlighted by the reduced fertility reported in the zebrafish glucocorticoid receptor mutant (nr3c1ia30/ia30) line (hereafter named gr-/-). Starting from such evidence, in this study, we have investigated the role of glucocorticoid receptor (Gr) in the reproduction of female zebrafish. Key signals orchestrating the reproductive process at the brain, liver, and ovarian levels were analyzed using a multidisciplinary approach. An impairment of the kiss-GnRH system was observed at the central level in (gr-/-) mutants as compared to wild-type (wt) females while, in the liver, vitellogenin (vtg) mRNA transcription was not affected. Changes were instead observed in the ovary, particularly in maturing and fully grown follicles (classes III and IV), as documented by the mRNA levels of signals involved in oocyte maturation and ovulation. Follicles isolated from gr-/- females displayed a decreased level of signals involved in the acquisition of competence and maturation, causing a reduction in ovulation with respect to wt females. Fourier transform infrared imaging (FTIRI) analysis of gr-/- follicle cytoplasm showed major changes in macromolecule abundance and distribution with a clear alteration of oocyte composition. Finally, differences in the molecular structure of the zona radiata layer of gr-/- follicles are likely to contribute to the reduced fertilization rate observed in mutants.

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.

Dietary diisononylphthalate contamination induces hepatic stress: a multidisciplinary investigation in gilthead seabream (Sparus aurata) liver.

In this study, adult gilthead seabream (Sparus aurata) were exposed for 21 days to Di-iso-nonylphthalte (DiNP at 15 and 1500 µg kg-1 bw day-1) via the diet. This plastic additive has been recently introduced to replace the di-(2-ethylhexyl)phthalate, the toxicity of which has been demonstrated conclusively both in vivo and in vitro trials. An analysis of a set of biomarkers involved in stress and immune response provides evidence of hepatic toxicity by DiNP in the present study. Both hsp70 and gr mRNA levels were upregulated significantly by DiNP, while plasma cortisol increased only in fish fed with the lowest DiNP dose. The oxidative stress markers g6pdh, glut red, gpx1 and CAT were upregulated by DiNP; gst mRNA was induced by the high dose and gck mRNA was downregulated significantly by the low dose. The mRNA levels of genes involved in the immune response, such as pla2, 5-lox, tnfa and cox2, were upregulated significantly only by the high dose of DiNP, while il1 mRNA increases in both doses. These molecular evidences were complemented with features obtained by Fourier Transform Infrared Imaging (FTIRI) analysis regarding the hepatic distribution of the main biological macromolecules. The FTIRI analysis showed an alteration of biochemical composition in DiNP samples. In particular, the low dose of DiNP induced an increase of saturated and unsaturated lipids and phosphorylated proteins, and a decrease of glycogen levels. The levels of caspase did not change significantly in the study, suggesting that DiNP does not activate apoptosis. Finally, the results also suggested the onset of hepatic oxidative stress and the activation of immune response, adding new knowledge to the already described hepatic DiNP toxicity.

Effects of Dietary Bisphenol A on the Reproductive Function of Gilthead Sea Bream (Sparus aurata) Testes.

Bisphenol A (BPA), a known endocrine disrupting chemical (EDC), was administered by diet to gilthead sea bream (Sparus aurata) in order to study its effects on the endocannabinoid system (ECS) and gonadal steroidogenesis. 2-year-old male gilthead sea bream were fed with two different concentrations of BPA (LOW at 4 and HIGH at 4000 µg/kg body weight for 21 days during the reproductive season. Exposure to 4000 µg BPA/kg bw/day (BPA HIGH) reduced sperm motility and altered the straight-line velocity (VSL) and linearity (LIN). Effects on steroidogenesis were evident, with testosterone (T) being up-regulated by both treatments and 11-ketotestosterone (11-KT) down-regulated by BPA HIGH. Plasma levels of 17ß-estradiol (E2) were not affected. The Gonadosomatic Index (GSI) increased in the BPA HIGH group. Interestingly, the levels of endocannabinoids and endocannabinoid-like compounds were significantly reduced after both treatments. Unpredictably, a few changes were noticed in the expression of genes coding for ECS enzymes, while the receptors were up-regulated depending on the BPA dose. Reproductive markers in testis (leptin receptor (lepr), estrogen receptors (era, erb), progesterone receptors (pr) and the gonadotropin releasing hormone receptor (gnrhr)) were up-regulated. BPA induced the up-regulation of the hepatic genes involved in oogenesis (vitellogenin (vtg) and zona pellucida 1 (zp1)).

Endocrine-disrupting chemicals in aquatic environment: what are the risks for fish gametes?

Over the past 25 years, extensive research in vertebrate species has identified several genomic pathways altered by exposures to anthropogenic chemicals with hormone-like activity mediated by their interaction with nuclear receptors. In addition, many pollutants have been shown to interfere with non-genomic (non-classical) pathways, but this mechanism of endocrine disruption is still poorly understood. Recently, the number of publications describing the effects of Endocrine disrupting chemicals (EDCs) on fish reproduction, focusing on the deregulation of the hypothalamus-pituitary-gonadal axis as well as on gamete quality, significantly increased. Depending on their ability to mimic endogenous hormones, the may differently affect male or female reproductive physiology. Inhibition of gametogenesis, development of intersex gonads, alteration of the gonadosomatic index, and decreased fertility rate have been largely documented. In males, alterations of sperm density, motility, and fertility have been observed in several wild species. Similar detrimental effects were described in females, including negative outcomes on oocyte growth and maturation plus the occurrence of apoptotic/autophagic processes. These pathways may affect gamete viability considered as one of the major indicators of reproductive endocrine disruption. Pollutants act also at DNA level producing DNA mutations and changes in epigenetic pathways inducing specific mechanisms of toxicity and/or aberrant cellular responses that may affect subsequent generation(s) through the germline. In conclusion, this review summarizes the effects caused by EDC exposure on fish reproduction, focusing on gametogenesis, giving a general overview of the different aspects dealing with this issue, from morphological alteration, deregulation of steroidogenesis, hormonal synthesis, and occurrence of epigenetic process.

Effects of BPA on female reproductive function: The involvement of epigenetic mechanism.

Epigenetic modifications are classified as heritable and reversible chemical modifications of chromatin that do not cause changes in DNA sequence. Changes in epigenetic modifications can be caused by exposure to certain environmental factors, such as contaminants like bisphenol A (BPA). Bisphenol A is ubiquitous in the environment and produced in large quantities, and known to have hormone-like activity, whereby disrupting endocrine function. Because of evidence for disruption of sex steroid mediated pathways, there is a concern that BPA could have adverse effects on female reproduction. The purpose of this review is to summarize the effects of BPA on adult female reproduction with focus on epigenetic changes that can be heritable.

Beneficial bacteria affect Danio rerio development by the modulation of maternal factors involved in autophagic, apoptotic and dorsalizing processes.

BACKGROUND/AIMS: Probiotic strains have been recognized to exert important roles in many biological systems, including immune response, growth, development and reproduction. However, to date, no studies have focused either on the relation among probiotics and maternal factors or on probiotics' ability to qualitatively and/or quantitatively modulate maternal transcripts. METHODS: In this study, the effects of Lactobacillus rhamnosus administered to parental fish on the control of maternal factors involved in autophagic, apoptotic and dorsalizing processes during zebrafish embryo development were assessed through q-PCRs, WMISH and TUNEL assay. RESULTS: The results we obtained show that probiotic induced significant changes in both maternal and zygotic mRNA levels involved in embryo development. The maternal autophagy-regulating genes herein investigated--ambra1a, ambra1b, beclin, lc3-, as well as those involved in the apoptotic process--caspase3, bcl2, bax--were modulated in disfavor and favor of the treated group, respectively. Also, the key transcripts ruling the dorsalizing process--goosecoid and chordin--were subject to a significant regulation of their gene expression. CONCLUSION: The results we acquired demonstrated that parentally administered Lactobacillus rhamnosus is able to modulate important physiological processes involved in zebrafish embryo development.

First Feeding of Cuttlefish Hatchlings: Pioneering Attempts in Captive Breeding.

In the last few decades, the cuttlefish market has grown to approximately 14% of the world's fisheries, and operators have begun to express concerns about the decline of this resource. In this context, the production of cuttlefish through aquaculture could offer a diversifying and valuable response to the increasing market demand and help alleviate the environmental pressure on this species. Therefore, the aim of this study is to identify a dry, cost-effective, and easy-to-administer diet that can successfully support the initial phases of cuttlefish growth and provide a similar performance to a krill-based diet, which closely mimics their natural diet. To achieve this objective, cuttlefish hatchlings were distributed among different experimental tanks, each receiving one of the five different diets (namely Diets A to E). Mortality and morphological parameters were monitored until day 10 post hatching, and the two most effective diets (Diets A and B) were chosen for further trials. The results indicated that Diet B had similar survival and growth rates to Diet A, which was based on frozen krill. Histological analysis revealed a comparable degree of gut maturity between the organisms fed the two diets. Likewise, levels of amylase and trypsin enzymes and hsp70, cat, and sod mRNA did not exhibit significant differences between the two groups. In conclusion, our findings provide preliminary evidence supporting the possibility of cultivating cuttlefish in captivity using a pelleted diet, representing a promising starting point for larger-scale breeding efforts.

The probiotic SLAB51 as agent to counteract BPA toxicity on zebrafish gut microbiota -liver-brain axis.

A plethora of studies have so far described the toxic effects of bisphenol A (BPA) on organism health, highlighting the urgent need to find new strategies not only to reduce the presence of this toxicant but also to counteract its adverse effects. In this context, probiotics emerged as a potential tool since they promote organism welfare. Using a multidisciplinary approach, this study explores the effects of SLAB51 dietary administration to counteract BPA toxicity using zebrafish as a model. Adult males and females were maintained under standard conditions (control group; C), exposed for 28 days via the water to an environmental relevant dose of BPA (10 µg/L; BPA), dietary treated with SLAB51 (109 CFU/g of body weight; P) and co-treated with BPA plus SLAB51 (BPA + P). In the gut, exposure to BPA resulted in altered architecture in both males and females, with females also experiencing an increase of pathogenic bacterial species. Co-administration of BPA + P led to the restoration of normal gut architecture, favored beneficial bacteria colonization, and decreased the abundance of pathogenic species. In the liver, male BPA exposure led to steatosis and glycogen depletion, which was partially mitigated by SLAB51 co-administration. In contrast, in females exposed to BPA, the lack of steatosis along with the greater glycogen depletion, suggested an increase in energy demand as supported by the metabolomic phenotype. The analysis of liver metabolites in BPA + P males revealed increased levels of anserine and reduced levels of glutamine, which could lie behind the counteraction of the brain histopathological damage caused by BPA. In BPA + P females, a reduction of retinoic acid was found in the liver, suggesting an increase in retinoids responsible for BPA detoxification. Overall, these results demonstrate that SLAB51 exerts its beneficial effects on the gut microbiota-brain-liver axis through distinct molecular pathways, effectively mitigating the pleiotropic toxicity of BPA.

Bacillus subtilis Modulated the Expression of Osteogenic Markers in a Human Osteoblast Cell Line.

Several in vivo trials have previously demonstrated the beneficial effects of the administration of various probiotic forms on bone health. In this study, we explored the potency of two probiotics, Bacillus subtilis and Lactococcus lactis, alone or in combination with vitamin D (VD), to modulate the transcription of genes involved in the ossification process in a human osteoblast cell line. Genes that mark the "osteoblast proliferation phase", such as RUNX2, TGFB1, and ALPL, "extracellular matrix (ECM) maturation", such as SPP1 and SPARC, as well as "ECM mineralization", such as BGN, BGLAP, and DCN, were all highly expressed in osteoblasts treated with B. subtilis extract. The observed increase in the transcription of the ALPL mRNA was further in agreement with its protein levels as observed by Western blot and immunofluorescence. Therefore, this higher transcription and translation of alkaline phosphatase in osteoblasts treated with the B. subtilis extract, indicated its substantial osteogenic impact on human osteoblasts. Although both the probiotic extracts showed no osteogenic synergy with VD, treatment with B. subtilis alone could increase the ECM mineralization, outperforming the effects of L. lactis and even VD. Furthermore, these results supported the validity of employing probiotic extracts rather than live cells to investigate the effects of probiotics in the in vitro systems.

Sustainable Ornamental Fish Aquaculture: The Implication of Microbial Feed Additives.

Ornamental fish trade represents an important economic sector with an export turnover that reached approximately 5 billion US dollars in 2018. Despite its high economic importance, this sector does not receive much attention. Ornamental fish husbandry still faces many challenges and losses caused by transport stress and handling and outbreak of diseases are still to be improved. This review will provide insights on ornamental fish diseases along with the measures used to avoid or limit their onset. Moreover, this review will discuss the role of different natural and sustainable microbial feed additives, particularly probiotics, prebiotics, and synbiotics on the health, reduction in transport stress, growth, and reproduction of farmed ornamental fish. Most importantly, this review aims to fill the informational gaps existing in advanced and sustainable practices in the ornamental fish production.

Melatonin effects on Fundulus heteroclitus reproduction.

This study aimed to investigate the effects of two different doses (100nM (M1) and 1µM (M2)) of exogenous melatonin on the reproductive capacity of Fundulus heteroclitus. Eight days of melatonin exposure significantly increased the fecundity and embryo survival of F. heteroclitus only in the M2 group compared with the control; the hatching rate was unaffected. Moreover, increases in the local expression of the melatonin receptor (mtnr) gene during follicle maturation were found; however, there were no differences between the experimental groups. Furthermore, in vitro melatonin-treated follicles showed a significantly higher germinal vesicle break down percentage compared with the control, while SDS-PAGE showed no difference in the electrophoretic pattern of the major yolk proteins. Nevertheless, densitometry revealed a greater intensity of the 118-, 95- and 40-kDa components in groups treated with melatonin. Finally, Fourier transform infrared microspectroscopy was applied to classify the different stages of oocyte development (Stages I-II, III and IV) on the basis of their macromolecular composition. The effects induced by melatonin on oogenesis were investigated by comparing vibrational spectra of females exposed to melatonin with those of controls. Changes to the Amide I band, corresponding to an increase in ß-structure, were found in oocytes of females exposed to the highest melatonin dose. These results highlight the positive role of melatonin, which is able to enhance the reproductive capacity of F. heteroclitus. Further studies are in progress to better explain the molecular mechanisms by which melatonin treatment affects reproduction in this marine species.

First Evidence of Microplastics in the Yolk and Embryos of Common Cuttlefish (Sepia officinalis) from the Central Adriatic Sea: Evaluation of Embryo and Hatchling Structural Integrity and Development.

Once they reach the aquatic environment, microplastics (MPs) are accidentally ingested by aquatic biota, thus entering the food chain with possible negative effects. The present study investigated, for the first time, MP presence in cuttlefish (Sepia officinalis) eggs and their association with embryonic development. Cuttlefish eggs were sampled from four different sites along the Marche region (Senigallia, Ancona, Numana, and San Benedetto del Tronto). Embryo and hatchling biometric parameters were evaluated and the internal structural integrity was examined through histological analysis. MPs were detected and characterized in embryos and yolk samples. MPs were identified in all sites (size < 5 µm), however, their presence has not been associated with an impairment of either embryo or hatchling internal structures. Noteworthy, the highest number of MPs (in both yolk and embryo samples) were found in Numana (37% of the total amount), where the lowest hatchling size was observed. On the other hand, the highest embryo mantle length was associated with the lowest number of MPs detected (9%) in Ancona. Overall, only MP fragments and sphere types (74 and 26%, respectively) were observed, and the most frequent polymers were Polyvinyl chloride (52%), Polypropylene, and Cellulose acetate (15% both). Further studies are needed to assess the possible MP effects on the yolk quality and assimilation.

Zebrafish as a Model to Unveil the Pro-Osteogenic Effects of Boron-Vitamin D3 Synergism.

The micronutrient boron (B) plays a key role during the ossification process as suggested by various in vitro and in vivo studies. To deepen our understanding of the molecular mechanism involved in the osteogenicity of B and its possible interaction with vitamin D3 (VD), wild-type AB zebrafish (Danio rerio) were used for morphometric analysis and transcriptomic analysis in addition to taking advantage of the availability of specific zebrafish osteoblast reporter lines. First, osteoactive concentrations of B, VD, and their combinations were established by morphometric analysis of the opercular bone in alizarin red-stained zebrafish larvae exposed to two selected concentrations of B (10 and 100 ng/ml), one concentration of VD (10 pg/ml), and their respective combinations. Bone formation, as measured by opercular bone growth, was significantly increased in the two combination treatments than VD alone. Subsequently, a transcriptomic approach was adopted to unveil the molecular key regulators involved in the synergy. Clustering of differentially expressed genes revealed enrichment toward bone and skeletal functions in the groups co-treated with B and VD. Downstream analysis confirmed mitogen-activated protein kinase as the most regulated pathway by the synergy groups in addition to transforming growth factor-ß signaling, focal adhesion, and calcium signaling. The best-performing synergistic treatment, B at 10 ng/ml and VD at 10 pg/ml, was applied to two zebrafish transgenic lines, Tg(sp7:mCherry) and Tg(bglap:EGFP), at multiple time points to further explore the results of the transcriptomic analysis. The synergistic treatment with B and VD induced enrichment of intermediate (sp7+) osteoblast at 6 and 9 days post fertilization (dpf) and of mature (bglap +) osteoblasts at 15 dpf. The results obtained validate the role of B in VD-dependent control over bone mineralization and can help to widen the spectrum of therapeutic approaches to alleviate pathological conditions caused by VD deficiency by using low concentrations of B as a nutritional additive.