Marta F. Riesco

Cryopreservation Causes Genetic and Epigenetic Changes in Zebrafish Genital Ridges.

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 (cxcr4b, pou5f1, vasa and sox2) and upregulation of heat shock proteins (hsp70, hsp90). The observed downregulation could not always be explained by promoter hypermethylation (only the vasa promoter underwent clear hypermethylation). To corroborate this, we used human spermatozoa (transcriptionally inactive cells) obtaining a reduction in some transcripts (eIF2S1, and LHCGR). 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.

Analysis of DNA damage after human sperm cryopreservation in genes crucial for fertilization and early embryo development

Sperm cryopreservation is widely used in clinic for insemination, in vitro fertilization and other procedures such as intracytoplasmic sperm injection. The assessment after freezing/thawing of spermatozoa viability, motility and sometimes DNA integrity (mainly using fragmentation assays) has been considered enough to guarantee the safety and effectiveness of the technique. However, it is known that, even when fragmentation is absent, a significant DNA damage could be detected in some genome regions. This is particularly important considering that, during the last years, several studies have pointed out the importance of key paternal genes in early embryo development. In this study, using normozoospermic donors, we present a candidate gene approach in which we quantify the number of lesions produced by freezing/thawing over key genes (PRM1, BIK, FSHB, PEG1/MEST, ADD1, ARNT, UBE3A, SNORD116/PWSAS) using quantitative PCR. Our results demonstrated that the cryopreservation protocol used, which is routinely employed in clinic, produced DNA lesions. The genes studied are differentially affected by the process, and genome regions related to Prader‐Willi and Angelman syndromes were among the most damaged: SNORD116/PWSAS (4.56 ± 1.84 lesions/10 kb) and UBE3A (2.22 ± 1.3 lesions/10 kb). To check if vitrification protocols could reduce these lesions, another experiment was carried out studying some of those genes with higher differences in the first study (FSHB, ADD1, ARNT and SNORD116/PWSAS). The number of lesions was not significantly reduced compared to cryopreservation. These results could be relevant for the selection of the most adequate available cryopreservation protocol in terms of the number of lesions that produced over key genes, when no differences with other traditional techniques for DNA assessment could be detected.

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage.

In Vitro Generation of Zebrafish PGC-Like Cells

ABSTRACT The possibility of generating primordial germ cells (PGCs) in vitro from noncommitted embryonic cells represents an extremely useful tool in current research. Primordial germ cell in vitro differentiation has been successfully reported in mammals. However, contrary to fish, PGC specification in mammals is an inductive mechanism. This study is the first to date to describe a rapid method for PGC in vitro differentiation in teleosts. Primordial germ cell-like cells were characterized by several lines of evidence, including gene expression, cell complexity, size, and image analysis for the quantification of fluorescence under vasa promoter. Moreover, differentiated cells were able to colonize the genital ridge after transplantation. Differentiation treatments increased the number of PGCs in culture, causing differentiation of cells rather than inducing their proliferation. These results open up the possibility of differentiating genetically modified embryonic cells to PGC-like cells to ensure their transmission to the progeny and could be crucial for an in-depth understanding of germline differentiation in teleosts.

Molecular basis of spermatogenesis and sperm quality

Spermatozoan quality can be evaluated in different ways, here we focus on the analysis of DNA, RNA and epigenetic status of germ cells. These characterizations also can be the bases for explaining sperm quality at other levels, so we will see how some of these molecules could affect other sperm quality markers. Moreover, we consider the possibility of using some of these molecules as predictors of sperm quality in terms of the ability to produce healthy offspring. The relevant effect of different types of RNA molecules in germ line specification and spermatogenesis and the importance of germ cell DNA integrity and a proper epigenetic pattern will be also discussed. Although most studies at this level have been performed in mammals, some information is available for fish; these recent discoveries in fish models are included. We provide a general overview on how these molecules could have a deep influence in the final sperm quality.

First study in cryopreserved Crassostrea angulata sperm

Sperm cryopreservation is a widely employed technique that promotes alternative techniques to contribute to broodstock management or restoration programs for species of commercial interest, endangered species or species with an interesting genotype. The preservation of genetic material from improved stocks or from the original population is extremely important for the oyster aquaculture industry to prevent the potential impacts of epidemic diseases and natural disasters. The Portuguese oyster, Crassostrea angulata, was the most important species commercialized by the shellfish industry. However, inadequate management of this industry and pathology occurrences resulted in a significant decrease in natural populations. For this reason, in this work a successful sperm cryopreservation protocol for this important species has been developed for the first time. Different internal cryoprotectants (DMSO, ethylene glycol, polyethylene glycol and methanol) at several concentrations (5, 10, 20%), containers (straws vs cryovials) and freezing rates (slow and fast rates) were tested. Cryoprotectant toxicity tests corroborated that this assay did not take into account the following steps of cryopreservation protocol as sperm agglutination. A fast freezing rate of cells diluted in10% DMSO and the use of straws as containers were the best cryopreservation conditions for Portuguese oyster sperm. Finally, fertilization assays confirmed the efficiency of the cryopreservation protocol in oyster sperm. These results demonstrated that different susceptibilities have been detected concerning sperm cryopreservation depending on oyster species or genetic material composition.

Cryobiology of cephalopod (Illex coindetii) spermatophores

Cephalopod culture is expected to increase in the near future and sperm cryopreservation would be a valuable tool to guarantee sperm availability throughout the year and to improve artificial insemination programs. We have studied the tolerance of spermatophores from the oceanic squid Illex coindetii to several cryoprotectants, in two toxicity experiments and a cryopreservation test. Five permeating cryoprotectants were tested: Dimethyl sulfoxide (Me2SO), methanol, glycerol, propylene glycol and ethylene glycol. In the first experiment, spermatophores were exposed to the five cryoprotectants at 5% (v/v) and 15% (v/v) at 4 °C for 5 min. In the second experiment, spermatophores were exposed to the cryoprotectants at 15% using different exposure times: 5, 15 and 30 min. In a third experiment, we tested two cryopreservation protocols: LN₂ vapor or -80 °C freezer, using a 15% cryoprotectant and 15 or 30 min of exposure. Viability and mitochondrial activity were assessed using Mitotracker deep red, YOPRO1 and Hoechst 33342, by flow cytometry. Spermatozoa in this species remain viable after cryoprotectant exposure but their quality decreased considerably after cryopreservation, only 5-10% of spermatozoa being motile. Flow cytometry demonstrated that Me2SO may be the most appropriate cryoprotectant for I. coindetii spermatozoa, and shows a first approach on cephalopod sperm cryopreservation, opening new possibilities for the research and culture of this group of molluscs.

Solea senegalensis sperm cryopreservation: New insights on sperm quality

Cryopreservation of Senegalese sole sperm can represent an alternative to overcome some reproductive problems of this species. However, it is important to guarantee the safe use of cryopreserved sperm by selecting an appropriate protocol according to a high demand quality need to be ensured. It has been demonstrated that traditional assays such as motility and viability do not provide enough information to identify specific damage caused by cryopreservation process (freezing and thawing). Specific tests, including lipid peroxidation and DNA damage, should be performed. In the present study, motility and lipid peroxidation were performed as specific tests allowing us to discard cryopreservation conditions such as methanol as internal cryoprotectant and bovine serum albumin as external cryoprotectant. In addition, a caspase 3/7 detection by flow cytometry was performed to analyze apoptosis activity in the best selected conditions. Moreover, new highly sensitive tests based on transcript number detection have recently been described in fish sperm cryopreservation. For this reason, a transcript level detection assay was performed on certain oxidative and chaperone genes related to fertilization ability and embryo development (hsp70, hsp90BB, hsp90AA, gpx) to select the best cryopreservation conditions. DMSO+ egg yolk proved to be the best cryoprotectant combination in terms of transcript level. This study describes an optimized cryopreservation protocol for Solea senegalensis sperm demonstrating for the first time that transcript degradation is the most sensitive predictor of cell status in this species after cryopreservation.

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 VK1) 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.

Chapter 19 Cryopreservation Effect on Genetic Function: Neonatal Outcomes

Cryopreservation is a well-established technique commonly used in clinical practice. It is used widely for the conservation of gametes and embryos that will be used later for insemination or in vitro fertilization. However, several studies have shown that this technique can produce changes in messenger RNA levels, in the epigenome and induce DNA damage. Although the embryo has potent mechanisms for DNA repair, and molecular changes in spermatozoa are not necessarily reflected in the embryo, it is important to explore new molecular tests and diagnostic tools to design optimal cryopreservation protocols and avoid undesirable molecular alterations. This chapter describes a protocol to quantify the lesions produced by cryopreservation using a protocol previously published by Rothfuss.