Circulating small non-coding RNAs provide new insights into vitamin K nutrition and reproductive physiology in teleost fish

Background: Vitamin K (VK) is a fat-soluble vitamin known for its essential role in blood coagulation, but also on other biological processes (e.g. reproduction, brain and bone development) have been recently suggested. Nevertheless, the molecular mechanisms behind its particular function on reproduction are not yet fully understood. Methods: The potential role of VK on reproduction through nutritional supplementation in Senegalese sole (Solea senegalensis) was assessed by gonadal maturation and 11-ketosterone, testosterone and estriol plasma levels when fed with control or VK supplemented (1250 mg kg(-1) of VK,) diets along a six month trial. At the end, sperm production and quality (viability and DNA fragmentation) were evaluated. Circulating small non-coding RNAs (sncRNAs) in blood plasma from males were also studied through RNA-Seq. Results: Fish fed with dietary VK supplementation had increased testosterone levels and lower sperm DNA fragmentation. SncRNAs from blood plasma were found differentially expressed when nutritional and sperm quality conditions were compared. PiR-675//676//4794//5462 and piR-74614 were found up-regulated in males fed with dietary VK supplementation. Let-7g, let-7e(18nt), let-7a-1, let-7a-3//7a-2//7a-1, let-7e(23nt) and piR-675//676//4794//5462 were found to be up-regulated and miR-146a and miR-146a-1//146a-2//146a-3 down-regulated when fish with low and high sperm DNA fragmentation were compared. Bioinformatic analyses of predicted mRNAs targeted by sncRNAs revealed the potential underlying pathways. Conclusions: VK supplementation improves fish gonad maturation and sperm quality, suggesting an unexpected and complex regulation of the nutritional status and reproductive performance through circulating sncRNAs. General significance: The use of circulating sncRNAs as reliable and less-invasive physiological biomarkers in fish nutrition and reproduction has been unveiled.European Regional Development Funds (ERDF) through the Portuguese "Programa Operational" (PROMAR)European Union (EU) [31-03-05-FEP-0073]COST Office (Food and Agriculture COST Action) [FA1205: AQUAGAMETE]network LARVAplus "Estrategias de desarrollo y mejora de la production de larvas de peces en Iberoamerica" - Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo (CYTED) [117RT0521]CYTED [318RT0549][CCMAR/SC/BPD/09/2016]info:eu-repo/semantics/publishedVersio

Larval Development in Tropical Gar (Atractosteus tropicus) Is Dependent on the Embryonic Thermal Regime: Ecological Implications under a Climate Change Context

In ectotherm species, environmental temperature plays a key role in development, growth, and survival. Thus, determining how temperature affects fish populations is of utmost importance to accurately predict the risk of climate change over fisheries and aquaculture, critical to warrant nutrition and food security in the coming years. Here, the potential effects of abnormal thermal regimes (24, 28 and 32 C; TR24, TR28, and TR32, respectively) exclusively applied during embryogenesis in tropical gar (Atractosteus tropicus) has been explored to decipher the potential consequences on hatching and growth from fertilization to 16 days post-fertilization (dpf), while effects on skeletal development and body morphology were explored at fertilization and 16 dpf. Egg incubation at higher temperatures induced an early hatching and mouth opening. A higher hatching rate was obtained in eggs incubated at 28 C when compared to those at 24 C. No differences were found in fish survival at 16 dpf, with values ranging from 84.89 to 88.86%, but increased wet body weight and standard length were found in larvae from TR24 and TR32 groups. Thermal regime during embryogenesis also altered the rate at which the skeletal development occurs. Larvae from the TR32 group showed an advanced skeletal development, with a higher development of cartilaginous structures at hatching but reduced at 16 dpf when compared with the TR24 and TR28 groups. Furthermore, this advanced skeletal development seemed to determine the fish body morphology. Based on biometric measures, a principal component analysis showed how along development, larvae from each thermal regime were clustered together, but with each population remaining clearly separated from each other. The current study shows how changes in temperature may induce craniofacial and morphological alterations in fish during early stages and contribute to understanding the possible effects of global warming in early development of fish and its ecological implications.Versión del edito

Natural feed after weaning improves the reproductive status of Solea senegalensis breeders

The aim of this study was to evaluate the effect of long term natural feeding in the subsequent reproductive status of 4 years old cultured Solea senegalensis, and to determine if the potential changes were structural or feeding dependent. To this aim, two different feeding regimes were used from one year after weaning and during the following 3 years; 1) a commercial dry food diet and 2) a natural feeding regime. After this period, the proportion of fluent males and the evolution of maturity stages of females over a breeding season were studied. A complete sperm quality analysis assessment was carried out, including individual volumes, motility, density and curvilinear, rectilinear and mean velocities of spermatozoa. Moreover, viability and apoptosis indexes were analyzed as indicator of molecular sperm membrane integrity. Additionally, a morphological characterization of the testes during the spawning season was conducted. Finally, both groups were fed with the same commercial pellets during one year to evaluate the effect of the diet of previous years on sperm quality. The results of this study showed how feeding can improve not only sperm quality and quantity, but also the proportion of fluent males and females in advanced maturity stages. All the sperm quality parameters resulted significantly higher in the group fed with a natural diet. Moreover, the number of apoptotic cells was significantly higher in the group fed with a commercial diet. According to the morphological features of the testes, the animals fed with a natural diet presented more basal position, less protuberances and irregular edges when compared with the animals fed with commercial diet. Interestingly, the progression of the spermatogenesis determined by the proportion of germ cells and the production of spermatozoa determined by the wider of the ducts system was also significantly larger in the natural diet group. After the standardization of the diets, mean volume per male and production of total motile cells were sig- nificantly higher in the group that was previously fed a natural diet, confirming structural improvements.Versión del editor2,04

A facility and community-based assessment of scabies in rural Malawi.

Background Scabies is a neglected tropical disease of the skin, causing severe itching, stigmatizing skin lesions and systemic complications. Since 2015, the DerMalawi project provide an integrated skin diseases clinics and Tele-dermatology care in Malawi. Clinic based data suggested a progressive increase in scabies cases observed. To better identify and treat individuals with scabies in the region, we shifted from a clinic-based model to a community based outreach programme. Methodology/principal findings From May 2015, DerMalawi project provide integrated skin diseases and Tele-dermatological care in the Nkhotakota and Salima health districts in Malawi. Demographic and clinical data of all patients personally attended are recorded. Due to a progressive increase in the number of cases of scabies the project shifted to a community-based outreach programme. For the community outreach activities, we conducted three visits between 2018 to 2019 and undertook screening in schools and villages of Alinafe Hospital catchment area. Treatment was offered for all the cases and school or household contacts. Scabies increased from 2.9% to 39.2% of all cases seen by the DerMalawi project at clinics between 2015 to 2018. During the community-based activities approximately 50% of the population was assessed in each of three visits. The prevalence of scabies was similar in the first two rounds, 15.4% (2392) at the first visit and 17.2% at the second visit. The prevalence of scabies appeared to be lower (2.4%) at the third visit. The prevalence of impetigo appeared unchanged and was 6.7% at the first visit and 5.2% at the final visit. Conclusions/significance Prevalence of scabies in our setting was very high suggesting that scabies is a major public health problem in parts of Malawi. Further work is required to more accurately assess the burden of disease and develop appropriate public health strategies for its control

Cryopreservation Causes Genetic and Epigenetic Changes in Zebrafish Genital Ridges

<div><p>Cryopreservation is an important tool routinely employed in Assisted Reproduction Technologies (ARTs) and germplasm banking. For several years, the assessment of global DNA fragmentation seemed to be enough to ensure the integrity of genetic material. However, cryopreservation can produce molecular alterations in key genes and transcripts undetectable by traditional assays, such modifications could interfere with normal embryo development. We used zebrafish as a model to study the effect of cryopreservation on key transcripts and genes. We employed an optimized cryopreservation protocol for genital ridges (GRs) containing primordial germ cells (PGCs) considered one of the best cell sources for gene banking. Our results indicated that cryopreservation produced a decrease in most of the zebrafish studied transcripts (<i>cxcr4b, pou5f1, vasa</i> and <i>sox2</i>) and upregulation of heat shock proteins (<i>hsp70, hsp90</i>). The observed downregulation could not always be explained by promoter hypermethylation (only the <i>vasa</i> promoter underwent clear hypermethylation). To corroborate this, we used human spermatozoa (transcriptionally inactive cells) obtaining a reduction in some transcripts (<i>eIF2S1</i>, and <i>LHCGR</i>). Our results also demonstrated that this effect was caused by freezing/thawing rather than exposure to cryoprotectants (CPAs). Finally, we employed real-time PCR (qPCR) technology to quantify the number of lesions produced by cryopreservation in the studied zebrafish genes, observing very different vulnerability to damage among them. All these data suggest that molecular alterations caused by cryopreservation should be studied in detail in order to ensure the total safety of the technique.</p> </div

Sperm Physiology and Artificial Fertilization

Sperm is composed by spermatozoa and seminal fluid which, in teleost fish, maintains the cell in a quiescent stage within the testes, providing all the metabolic resources needed for maintenance. Most fish species have external fertilization and for that they have an aquasperm cell type, characterized by the loss of acrosome membrane as a result of evolution. Seminal plasma plays a crucial role supporting spermatozoa and has physiological and endocrinological functions during the release of sperm from the testis into the sperm duct, and subsequently to the aquatic environment. The cell plasma membrane is composed of a thin bilayer of amphipathic lipids that separates and protects the cell from the extracellular environment, and it is responsible, as in other cells, for the control of substance traffic through the cell. In a general cell, in the chromatin structure, DNA is associated with proteins, mainly histones, forming a nucleoprotein complex in eukaryotic organisms.The authors are grateful to projects financed by programme MAR2020, Direção Geral das Pescas e Agricultura (ReproF1 16-02-01-FMP-0059 and Algasole 16-02-01-FMP0058); EBB (EAPA_501/2016), Interreg Atlantic area and Assemble plus JRA2 (H2020- INFRAIA-2016-2017). The work conducted received Portuguese national funds from FCT - Foundation for Science and Technology through project UID/Multi/04326/2019, and from the operational programmes CRESC Algarve 2020 and COMPETE 2020 through project EMBRC.PT ALG-01-0145-FEDER-022121

The Use of Antifreeze Proteins in the Cryopreservation of Gametes and Embryos

The cryopreservation of gametes and embryos is a technique widely used in reproductive biology. This technology helps in the reproductive management of domesticated animals, and it is an important tool for gene banking and for human-assisted reproductive technologies. Antifreeze proteins are naturally present in several organisms exposed to subzero temperatures. The ability for these proteins to inhibit ice recrystallization together with their ability to interact with biological membranes makes them interesting molecules to be used in cryopreservation protocols. This mini-review provides a general overview about the use of antifreeze proteins to improve the short and long term storage of gametes and embryos

Non-coding RNA regulation in reproduction: Their potential use as biomarkers

Non-coding RNAs (ncRNAs) are crucial regulatory elements in most biological processes and reproduction is also controlled by them. The different types of ncRNAs, as well as the high complexity of these regulatory pathways, present a complex scenario; however, recent studies have shed some light on these questions, discovering the regulatory function of specific ncRNAs on concrete reproductive biology processes. This mini review will focus on the role of ncRNAs in spermatogenesis and oogenesis, and their potential use as biomarkers for reproductive diseases or for reproduction success. Keywords: Non-coding RNAs, Spermatogenesis, Oogenesis, Reproductive transgenerational effects, Biomarker

Relative expression of human genes after cryopreservation (Cryo).

<p>Cryopreservation affects expression of some transcripts in human sperm. Analysis was carried out in human spermatozoa, transcriptionally inactive cells. Expression levels for each gene relative to <i>ACTB</i> were calculated for all samples using the 2^−ΔΔCt method. All results were expressed as the means ± SE of the 2^−ΔΔCt method of three independent experiments with three replicates for each. The Student’s t-test (μ=1) was performed to identify changes in gene expression levels after cryopreservation. Asterisks showed significant (p< 0.05) downregulation after cryopreservation (Cryo).</p

Relative expression of zebrafish genes after CPAs exposure (CPAs) and cryopreservation (Cryo).

<p>Cryopreservation affects gene expression levels in zebrafish genital ridges. Analysis was carried out in zebrafish genital ridges (GRs) containing primordial germ cells (PGCs). Expression levels for each gene relative to <i>actb2</i> were calculated for all samples using the 2^−ΔΔCt method. All results were expressed as the means ± SE of the 2^−ΔΔCt method of three independent experiments with three replicates for each. The Student’s t-test (μ=1) was performed to identify changes in gene expression levels after cryopreservation. Asterisks showed significant (p< 0.05) downregulation after cryopreservation (Cryo).</p