Vanesa Robles

Regeneration and reprogramming compared.

BackgroundDedifferentiation occurs naturally in mature cell types during epimorphic regeneration in fish and some amphibians. Dedifferentiation also occurs in the induction of pluripotent stem cells when a set of transcription factors (Oct4, Sox2, Klf4 and c-Myc) is over expressed in mature cell types.ResultsWe hypothesised that there are parallels between dedifferentiation or reprogramming of somatic cells to induced pluripotent stem cells and the natural process of dedifferentiation during epimorphic regeneration. We analysed expression levels of the most commonly used pluripotency associated factors in regenerating and non-regenerating tissue and compared them with levels in a pluripotent reference cell. We found that some of the pluripotency associated factors (oct4/pou5f1, sox2, c-myc, klf4, tert, sall4, zic3, dppa2/4 and fut1, a homologue of ssea1) were expressed before and during regeneration and that at least two of these factors (oct4, sox2) were also required for normal fin regeneration in the zebrafish. However these factors were not upregulated during regeneration as would be expected if blastema cells acquired pluripotency.ConclusionsBy comparing cells from the regeneration blastema with embryonic pluripotent reference cells we found that induced pluripotent stem and blastema cells do not share pluripotency. However, during blastema formation some of the key reprogramming factors are both expressed and are also required for regeneration to take place. We therefore propose a link between partially reprogrammed induced pluripotent stem cells and the half way state of blastema cells and suggest that a common mechanism might be regulating these two processes.

Cryopreservation of rainbow trout sperm in large volume straws: application to large scale fertilization

Abstract Traditional 0.25- and 0.5-ml straws have been successfully applied to the cryopreservation of rainbow trout sperm. In order to facilitate the fertilization of large egg batches, and with the purpose of transferring this technique to commercial hatcheries, the suitability of 1.8 and 5 ml straws was studied. Sperm was diluted at a 1:3 ratio in #6 Erdahl and Graham extender with 7% DMSO, 10% egg yolk and 7.5 mg/ml Promine. Freezing was performed at different levels above the surface of liquid nitrogen using a styrofoam box and thawing was carried out in a water bath at different temperatures. Freezing and thawing rates inside the straws were registered with a thermocouple. The success of sperm cryopreservation was checked in vitro using several sperm quality parameters: motility, viability and membrane functionality. Fertility tests were also performed on small batches using a ratio of 200 eggs/ml of diluted sperm. Freezing and thawing rates were similar in 1.8- and 0.5-ml straws. However, lower freezing and thawing rates were achieved with 5-ml straws even at lower freezing and higher thawing temperatures. Motility analysis gave similar results in sperm cryopreserved in all the tested straws (0.5, 1.8 and 5 ml). However, cell viability (61.9%) and fertility (73.2%) were slightly lower in large volumes than in 0.5-ml straws (77.4% and 84%, respectively). Fertilization of larger egg batches (1600–2000 eggs) with sperm frozen in 5-ml straws provided similar fertilisation rates (73%), demonstrating that the slight loss in fertility is compensated by the benefits of 5-ml straws. Our results showed that cryopreservation of large sperm volumes could be useful for hatchery purposes.

Methods in reproductive aquaculture : marine and freshwater species

GAMETE EXTRACTION TECHNIQUES Reproduction and control of ovulation, spermiation and spawning in cultured fish, E. Mananos, N. Duncan, and C. Mylonas Methods for sperm collection, J-C. Gwo SPERM AND EGG QUALITY EVALUATION Sperm quality assessment, E. Cabrita, V. Robles, and P. Herraez Egg quality determination in teleost fish, F. Lahnsteiner, F. Soares, L. Ribeiro, and M.T. Dinis ARTIFICIAL FERTILIZATION IN AQUACULTURED SPECIES:FROM NORMAL PRACTICE TO CROMOSOME MANIPULATION Artificial fertilization in aquaculture species: from normal practice to chromosome manipulation, B. Urbanyi , A. Horvath, and Z. Bokor SPERM AND EGG CRYOPRESERVATIONChilled storage of sperm and eggs, J. Bobe and C. Labbe Basic principles of fish spermatozoa cryopreservation, J. Cloud and S. Patton Cryopreservation of fish oocytes, T. Zhang and E. Lubzens Embryo cryopreservation: what we know until now, V. Robles, E. Cabrita, J.P. Acker, and P. Herraez Cryobiological material and handling procedures, F. Martinez-Pastor and S.L. Adams PROTOCOLS FOR SPERM CRYOPRESERVATION Cryopreservation of sperm of loach (Misgurnus fossilis), J. Kopeika, E. Kopeika, T. Zhang, and D. Rawson Zebrafish Sperm Cryopreservation with N, N-Dimethylacetamide, J.P. Morris IV, A. Hagen, and J.P. Kanki Sperm cryopreservation of live-bearing fishes of the Genus Xiphophorus, Q. Dong, C. Huang, L. Hazlewood, R.B. Walter, and T.R. Tiersch Use of post-thaw silver carp (Hypophtalmichthys molitrix) spermatozoa to increase hatchery productions, B. Alvarez, A. Arenal, R. Fuentes, R. Pimentel, Z. Abad, and E. Pimentel Cryopreservation of common carp sperm, B. Urbanyi and A. Horvath Sperm cryopreservation from sea perch, Lateolabrax japonicas, S-L. Chen, X.S. Ji, and Y-S. Tian Semen cryopreservation of piracanjuba (Brycon orbignyanus), an endangered Brazilian species, A.T.M. Viveiros and A.N. Maria Cryopreservation of endangered Formosan landlocked salmon (Oncorhynchus masou formosanus) semen, J-C. Gwo, H. Ohta, K. Okuzawa, L-Y. Liao, and Y-F. Lin Protocols for the cryopreservation of Salmonidae semen, Lota lota (Gadidae) and Esox lucius (Esocidae), F. Lahnsteiner and N. Mansour Rainbow trout (Oncorhynchus mykiss) sperm cryopreservation, P. Herraez, V. Robles, and E. Cabrita Sperm cryopreservation of sex-reversed rainbow trout (Oncorhynchus mykiss), V. Robles, E. Cabrita, and P. Herraez Cryopreservation of testicular sperm from European catfish (Silurus glanis), G. Maisse, B. Ogier de Baulny, and C. Labbe Semen Cryopreservation of the African catfish, Clarias gariepinus, A.T.M. Viveiros and J. Komen Cryopreservation of sperm from species of the order Acipenseriformes, A. Horvath, B. Urbanyi, and S.D. Mims Different protocols for the cryopreservation of European eel (Anguilla anguilla) sperm, J.F. Asturiano Cryopreservation of Striped Bass Morone saxatilis Spermatozoa, L.C.Woods III, S. He, and K. Jenkins-Keeran Cryopreservation of semen from striped trumpeter (Latris lineata), A.J. Ritar Cryopreservation of Atlantic halibut sperm, Hippoglossus hippoglossus, I. Babiak Sperm cryopreservation: an actual tool for Seabass (Dicentrarchus labrax) reproduction and breeding control in research and production, C. Fauvel and M. Suquet Notes on Diplodus puntazzo sperm cryopreservation, F. Barbato, S. Canese, F. Moretti, A. Taddei, A. Fausto, L. Abelli, M. Mazzini, and K.J. Rana Sperm Cryopreservation from the Marine Teleost, Sparus aurata, E. Cabrita, V. Robles, C. Sarasquete, and P. Herraez Cryopreservation of seabream semen, J-C. Gwo Cryopreservation of sperm from winter flounder, Pseudopleuronectes americanus, R.M. Rideout, I.A.E. Butts, and M.K. Litvak Cryopreservation of sperm in turbot (Psetta maxima), M. Suquet, O. Chereguini, and C. Fauvel Cryopreservation of grouper semen, J-C. Gwo and H. Ohta Cryopreservation of sperm from Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus), R.M. Rideout and D.L. Berlinsky Cryopreservation of small abalone (Haliotis diversicolor supertexa) semen, J-C. Gwo Cryopreservation of Pacific Oyster Sperm, Q. Dong, C. Huang, B. Eudeline, and T. R. Tiersch A simple method for freezing Pacific oyster (Crassostrea gigas) sperm in quantities suitable for commercial hatcheries, S.L. Adams, J.F. Smith, R.D. Roberts, A.R. Janke, H.F. Kaspar, H.R. Tervit, P.A. Pugh, S.C. Webb, and N.G. King Cryopreservation of Eastern Oyster Sperm, C.G. Paniagua-Chavez and T.R. Tiersch Cryopreservation of sea urchin (Evechinus chloroticus) sperm, S.L. Adams, P.A. Hessian, and P.V. Mladenov Protocol for cryopreservation of Penaeus vannamei sperm cells, M. Salazar, M.Lezcano, and C.Granja Techniques for cryopreservation of spermatophores of the giant freshwater prawn, Macrobrachium rosenbergii (de Man), P. Damrongphol and K. Akarasanon Protocol development for the cryopreservation of spermatozoa and spermatophores of the mud crab, Scylla serrata, S. Bhavanishankar and T. Subramoniam Index

Vitrification of turbot embryos: preliminary assays

Successful fish embryo cryopreservation is still far from being achieved. Vitrification is considered the most promising option. Many factors are involved in the success of the process. The choice of a proper vitrification solution, the enzymatic permeabilization of embryos to increase cryoprotectant permeability, the adequate container for embryo loading, and the temperature for thawing, were the parameters considered at different developmental stages in the present study. After vitrification, embryo morphology was evaluated under stereoscopic microscopy, establishing the percentage of intact embryos. Two of the studied parameters yielded differences in this percentage, the volume of straw used for embryo loading (1 ml straws were significantly better than 0.5 ml straws, with regard to post-thawed embryo morphologies), and the thawing temperature, achieving 49% of embryos with intact morphology after thawing at 0 degrees C. After thawing, the intact embryos were incubated and periodically observed to detect morphological changes. Changes in the perivitelline space, shrinkage of the yolk and chorion ruptures as well as a progressive whitening of the embryo and yolk were observed. After 8 h all embryos showed clear signs of degradation and during this incubation period no embryo showed any developmental ability.

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.

Sperm cryopreservation of sex-reversed rainbow trout (Oncorhynchus mykiss): parameters that affect its ability for freezing

Abstract Sex control profits the aquaculture industry allowing the obtaining of “all female” rainbow trout populations. Female production is highly profitable since they become sexually mature 1 year later than males, reaching their marketable size before maturation. Sex-reversed rainbow trouts have similar external morphology to normal males but lack sperm ducts, meaning that the animals must be sacrificed to obtain the milt. The peculiarities of the sperm, obtained directly from the testicle, make necessary the development of a specific cryopreservation protocol. In this study, several factors that could affect the freezability of these spermatozoa have been studied: the season of sperm extraction, the method of sperm extraction, and the activation with motility stimulators have been considered. Our results showed that seasonality clearly affects the success of the cryopreservation process, which should always be carried out with sperm obtained in winter season, the natural breeding period. The development of a clean sperm extraction method improved significantly the fertility rates obtained with cryopreserved sperm. The addition of methylxanthines as motility stimulators usually increased motility and fertility rates, but they did not provide significant improvements.

Transgenerational inheritance of heart disorders caused by paternal bisphenol A exposure

Bisphenol A (BPA) is an endocrine disruptor used in manufacturing of plastic devices, resulting in an ubiquitous presence in the environment linked to human infertility, obesity or cardiovascular diseases. Both transcriptome and epigenome modifications lie behind these disorders that might be inherited transgenerationally when affecting germline. To assess potential effects of paternal exposure on offspring development, adult zebrafish males were exposed to BPA during spermatogenesis and mated with non-treated females. Results showed an increase in the rate of heart failures of progeny up to the F2, as well as downregulation of 5 genes involved in cardiac development in F1 embryos. Moreover, BPA causes a decrease in F0 and F1 sperm remnant mRNAs related to early development. Results reveal a paternal inheritance of changes in the insulin signaling pathway due to downregulation of insulin receptor β mRNAs, suggesting a link between BPA male exposure and disruption of cardiogenesis in forthcoming generations.

Dimethyl sulfoxide influx in turbot embryos exposed to a vitrification protocol

The particular characteristics of fish embryos require the development of specific methods for cryopreservation. One of the main obstacles is related to the presence of membranes and compartments with different water and cryoprotectant permeability. To assess dimethyl sulfoxide (Me2SO4) permeability, we exposed turbot embryos (Scophthalmus maximus) at F stage (tail bud) to the cryoprotectant solutions used in a vitrification protocol and then evaluated the Me2SO4 content inside the embryo using high-performance liquid chromatography (HPLC). The Me2SO4 influx was analyzed in normal embryos and in embryos treated with pronase (2mg/ml) in order to increase chorion permeability. The evaluation was made after each step of cryoprotectant incorporation and removal. Three embryo compartments were distinguished: the perivitelline space (PVS), the yolk sac (YS) and the cellular compartment (CC), and the relative volumes of each, estimated using stereoscopic microscopy imaging, were 11.37, 81.23 and 7.40%, respectively. The Me2SO4 concentration inside the embryos was calculated based on their entrance into one, two or three compartments. Results suggest high entrance of Me2SO4 into the PVS and a low concentration of this cryoprotectant inside the other compartments. Pronase did not significantly increase Me2SO4 influx, but facilitated its elimination during the washing steps.

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.

Germplasm Cryobanking in Zebrafish and Other Aquarium Model Species

During the last few years zebrafish and other aquarium fish have become more important model system species. In zebrafish valuable biotechnological lines are constantly emerging, and the creation of cryobanks of viable fish sperm, oocytes, and embryos from those lines is a constant and important demand. Cryopreservation is an essential tool for storing specific genetic material and would significantly help in the conservation of such lines, facilitating daily procedures in labs and companies working with this species. In this review, all advances achieved in this field are presented. Protocols for sperm cryopreservation are described, and the new directions of approaches in embryo and oocyte cryopreservation and also in blastomeres, and primordial germ cell cryopreservation are discussed.