Cryopreservation of fish ovarian tissue fragments can be a viable alternative to cryopreservation of oocytes and embryos. The ability to cryopreserve both maternal and paternal gametes would provide a reliable source of fish genetic material for scientific and aquaculture purposes. The main aim of the present study was to develop an in-vitro culture protocol and cryopreservation protocol for zebrafish ovarian tissue fragments. In-vitro culture protocol for the tissue fragments containing stage I and stage II follicles were developed and the growth assessment of follicles were evaluated using biomarkers. To develop the cryopreservation protocol using control slow cooling method, the effect on freezing medium, cryoprotectants and cooling rate on the tissue fragments were investigated. The in-vitro culture experiments showed that L-15 medium (pH 9) containing 100mIU/ml FSH along with 20% FBS was effective for tissue fragments containing stage I and II follicles to grow in-vitro. The growth of the ovarian follicle stages was confirmed by the level of expression of p450aromA and vtg1 gene. The optimal cryopreservation protocol for the ovarian tissue fragments was found as 2M methanol+ 20%FBS in 90% L-15 medium with the cooling rate of 4°C/min. Although the highest survival rate obtained for stage II follicles within the fragments was 68.2±1.9% and stage I follicles within the fragments was 55.4±2.3% using TB staining, it showed a significant decrease in their ATP levels. This is the first study carried out on the zebrafish ovarian tissue fragments. Study on cryopreservation of the ovarian tissue fragments and development of the in-vitro culture protocol and use of biomarkers for the ovarian tissue fragments were reported here for the first time. The outcomes of this study have provided useful information for future cryopreservation protocol development.

In recent years, numerous studies have linked cryopreservation with increased occurrence of mutations, DNA fragmentation and the event of apoptosis in biological objects. However, the evidence emerged from such studies is somewhat inconclusive. The current study, therefore, aimed to analyse the DNA damage response (DDR) from the cryopreserved cells in order to characterise the nature of the putative DNA damage. The study set out to investigate the effects of different cryopreservation parameters on the genome in terms of double strand breaks (DSBs), single strand breaks (SSBs), and various forms of sequence alteration using 5¼ hour post fertilisation (hpf) zebrafish (Danio rerio) embryos. The experimental conditions under which the investigation was carried out were short term chilling at 0˚C, treatment with two cryoprotective agents (CPA), namely, MeOH and Me2SO, and cooling to -35˚C. Assays for detecting DSB-activated DDR proteins and SSB-activated DDR proteins in 5¼ hpf zebrafish (Danio rerio) were developed and then utilised to investigate the occurrence of DSBs and SSBs in the genome of the embryos treated with the experimental conditions. The study then analysed the expression profiles of a set of genes unique to the base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) pathways as indicators of the occurrence of various forms of sequence alterations in the genome of the embryos treated with the experimental conditions. It was found that chilling and CPA treatment did not induce DSBs or SSBs but up-regulated the MMR and BER, respectively. CPA treatment also down-regulated the NER and the MMR mechanisms. Cooling, on the contrary, did not induce DSBs but induced SSBs in the genome, which were repaired when the embryos were provided with a recovery time. Cooling also up-regulated the NER and the BER mechanisms in the embryos. The overall finding of the study indicated that the experimental conditions increased the occurrence of various single stranded DNA lesions in the genome of the embryos. The present study provided important insights into how eukaryotic cells respond to different cryopreservation parameters, which will significantly enhance the current knowledge of the effects of cryopreservation on the genome of biological objects.

Cryopreservation of fish germ cells has important applications in aquaculture, conservation of endangered species and human genomic studies. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. The objective of the present study was to develop successful cryopreservation protocol for zebrafish oocytes at temperature of liquid nitrogen (-196°C), or if unachieved, to investigate the limiting factors associated with fish oocytes cryopreservation. In this study, the effects of cryoprotectants exposure and enzymatic treatments on oocytes survival were studied, and new viability tests for zebrafish oocytes were developed. The effects of controlled slow cooling with different cryoprotective agents, in different freezing media and at different cooling rates on cryosurvival of zebrafish (D. rerio) oocytes were investigated. Cryomicroscopic observations on zebrafish oocytes were also carried out. Three reliable vital tests -trypan blue (TB) staining, ATP assay, and in vitro maturation followed by germinal vesicle breakdown observation (GVBD) were found suitable for assessment of oocytes viability. Vitellogenesis (stage III) was found to be the optimal developmental stage for cryopreservation. Methanol was found to be the best CPA for zebrafish oocytes. Combination of 4M methanol and 0.2M glucose in potassium chloride (KCI) buffer was found to be the optimal cryoprotective solution. Controlled slow cooling at 0.3°C/min rate, combined with seeding at -12.5°C and plunge to liquid nitrogen (LN) at-40°C were found to be the optimal conditions for cryopreservation of stage III oocytes. However, even with the optimal protocol, TB-assessed viability, Le. the ratio of oocytes with intact plasma membrane after cooling to -196°C was 19.6±8%. Furthermore, GVBD experiments showed that none of the cryopreserved oocytes can be matured in vitro, and their ATP levels were decreased dramatically, indicating that successful cryopreservation of fish oocytes at liquid nitrogen temperature still remains elusive. Cryomicroscopic observations demonstrated, that the damages of oocytes are associated with intracellular ice formation (lIF). IIF occurred simultaneously with extracellular ice formation (ElF) in nearly 100% of the cases, and formation of lethal hexagonal type of ice was observed. This study was the first systematic attempt to cryopreserve fish oocytes at liquid nitrogen temperature. The results provided will undoubtedly assist successful protocol design for cryopreservation of fish oocytes in the future.

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programs for endangered species. Many studies have been devoted to the mechanisms of cryodamage. However, in spite of growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells often remains poor. It is known that cryopreservation causes extensive damage to membrane, results in decreased metabolism of cells, and disturbs the bioenergetical processes of cells by damage to mitochondria. Nonetheless, it has not yet been identified clearly if cryopreservation causes some disruption in the genetic integrity of reproductive cells and the safety of this approach still needs to be confirmed. The present study was undertaken on the spermatozoa of weather loach (Misgurnus tassilis) and blastomeres cells of zebrafish (Danio rerio). It was shown that survival was decreased for embryos derived from sperm after cryoprotectant treatment or cryopreservation. Some evidence has emerged that this decrease is more likely to reflect some genetic instability caused by cryopreservation of sperm. The present study showed for the first time that the DNA repair system of oocytes was activated after fertilisation with cryopreserved sperm. The effect of DNA repair system was also studied. It was found that incubation of fertilised eggs in caffeine could reverse the detrimental effects of cryopreservation of loach sperm on subsequent embryo development. On the other hand incubation of fertilised eggs with 3-aminobenzamide - inhibitor of the poly (ADP-ribose) polymerase (PARP)- brought further decrease in the survival of embryos derived from cryopreserved sperm. The effect of individual donors of sperm and eggs on overall embryo survival was also studied and these investigations revealed significant differences between different donors. Effect of cryopreservation on zebrafish blastomeres was studied at the DNA molecular level. Mitochondrial DNA was sequenced after cryopreservation and increased level of frequency of the mutation was observed. This finding showed that cryopreservation might potentially increase the instability of mtDNA genome. The significance of these changes on the subsequent function of the cells is to be elucidated. Meanwhile this study suggests that it is important to be cautious in making judgements on the safety of cryopreservation techniques in reproduction.

Zebrafish (Danio rerio) have been extensively used over the past two decades to study muscle development, human myopathies and dystrophies, due to its higher degree of homology with human disease causing genes and genome. Despite its unique qualities, zebrafish have only been used as an in-vivo model for muscle development research, due to the limitations surrounding lack of a consistent isolation and culture protocol for zebrafish muscle progenitor cells in-vitro. Using different mammalian myoblast isolation protocols, a novel and robust protocol has been developed to successfully isolate and culture zebrafish skeletal muscle cells repeatedly and obtain differentiated long multi nucleated zebrafish myotubes. Commitment to myogenic lineage was confirmed by immuno-staining against muscle specific protein desmin, and expression pattern of different genetic markers regulating myogenesis. In order to recapitulate the in-vivo bio-physiological environment for zebrafish skeletal muscle cells in-vitro, these cells were successfully cultured in tissue engineered three dimensional (3D) constructs based on fibrin and collagen models. Maturation of tissue engineered collagen and fibrin based constructs was confirmed using the basic parameters described in the literature i.e. collagen three times greater contraction from the original width (Mudera, Smith et al. 2010) and fibrin constructs tightly coiled up to 4mm of diameter (Khodabukus, Paxton et al. 2007). In-vitro characterisation of zebrafish skeletal muscle cells showed hypertrophic growth of muscle mass compared to hyperplasic growth in-vivo as suggested for fish species in literature (Johnston 2006), which is different from human and other mammals. Comparative analysis of zebrafish muscle cells cultured in monolayer against cultured in 3D tissue engineered constructs showed significant increase in fusion index, nuclei per myotube (two-fold) and myotubes per microscopic frame (two-fold). Cells cultured in tissue engineered construct closely resembled in-vivo muscle in terms of their unidirectional orientation of myotubes. These tissue engineered 3D zebrafish skeletal muscle models could be used for various purposes such as drug screening, effect of different temperature extremes, studying underlined pathways involved in human diseases; and with further refinements it would potentially replace the need for studies on live fish in these areas.

In aquaculture, short term chilled storage has been used to transport brood stock fish embryos for genetic improvement programmes. It is therefore important to understand the effect of chilling on embryos at both developmental and molecular levels. In the present study, gene expression patterns in zebrafish embryos were studied before investigations were carried out on the effect of chilling on gene and protein expression in these embryos. The gene expression results obtained in different developmental stages using conventional PCR showed that, only sox genes were expressed throughout the tested developmental stages from 30% epiboly to 6 somites. Quantitative RT-PCR was then used to investigate sox gene expression patterns during chilling of 50% epiboly stage embryos at 0°C for up to 180 min and also after warming. Significant decreases in sox2 and sox3 expressions were observed when compared to those of controls following chilling whilst significant increases of expressions of the two genes were observed after warming in the embryos chilled for 30 and 60 min. Studies on the impact of cryoprotectant MeOH on sox genes and protein expression showed that 50% epiboly stage zebrafish embryos could tolerate chilling for up to 6 h with or without MeOH. It was observed that expression of all three sox genes were significantly decreased following chilling for 3 h at 0°C. However the degree of decrease was less pronounced in embryos chilled with different concentrations of MeOH. Significant increases in sox genes were observed in hatching stage embryos chilled with 1 M MeOH for 3h but subsequent sox2 and sox19a protein expression was not affected. The effect of long term chilling (18h) on sox gene and protein expression in 50% epiboly stage embryos was also investigated. Improved hatching rates (56% ± 5) were achieved when embryos were chilled with 1 M MeOH + 0.1 M sucrose. Results from gene expression studies showed a stable sox2 gene expression in 18 h chilled embryos in cryoprotectant mixture when compared to that of embryos chilled without cryoprotectant mixture. Similar patterns were observed when the expression of sox2 and sox3 protein was investigated. This is the first study carried out on the effect of chilling in early stage zebrafish embryos at the molecular level. The results obtained from the present study provided useful information on the molecular mechanisms of the effect of chilling on zebrafish embryos and will have important implications in designing chilled storage protocols for fish embryos.

Heat shock proteins (hsp) and heat shock transcription factors (HSF) have important roles in the development of the eye lens. Our lab previously demonstrated that knockdown of hsp70 gene expression using morpholino antisense technology (MO) resulted in a small lens phenotype in zebrafish (Danio rerio) embryos. A less severe phenotype was seen with knockdown of hsf1, suggesting other factors that regulate hsp70 are involved during lens formation. Both HSF1 and HSF4 are known to play a role in mammalian lens development. An expressed sequence tag encoding zebrafish HSF4, named hsf4a, has been identified and a second splice variant, hsf4b, has been predicted in the Ensembl database. The objectives of this thesis were to characterize the zebrafish HSF4 and compare its expression to other HSFs as well as investigate its role in lens development. Analysis of zebrafish HSF4 sequence was performed using standard in silico analytical software. The deduced amino acid sequence of HSF4a shares structural similarities with mammalian HSF4 including the lack of an HR-C domain. This domain is absent due to a C-terminal truncation within zebrafish HSF4a relative to the mammalian protein. HSF4b is identical to the HSF4a sequence with the exception of an additional 155 amino acids at the carboxyl end of the protein which contains an HR-C domain, unlike mammalian HSF4. Surprisingly, electrophoretic mobility shift assays (EMSA) demonstrated that the binding affinity of zebrafish HSF4 to discontinuous HSEs is more similar to HSF1 than to other HSF4 proteins. The amino acid sequence of zebrafish HSF4 DNA binding domain was also more similar to HSF1 than other HSF4 proteins. These results, along with a phylogenetic analysis of HSF proteins from eleven species, suggest that HSF1 was an evolutionary precursor of HSF4 and that functions of this protein may differ between zebrafish and mammals. The expression level for each of the three zebrafish HSFs was determined in adult tissues and in developing embryos by quantitative reverse transcription polymerase chain reaction (qPCR) analysis. Expression of both hsf4 transcripts was observed predominantly in the eye but only observed in developing embryonic tissue at 60 hours post fertilization or later. This, together with the lack of an observable phenotype following MO knockdown of hsf4, suggests that HSF4 likely has a role in later stages of lens development. Additionally, hsf1 and hsf2 expression were detected in all tissues and in all stages of development as well as being present as maternal transcripts in zebrafish eggs. The results presented in this thesis demonstrate that while zebrafish HSFs share some similarity with HSF proteins from other species, they also have structural characteristics and expression patterns unique to the zebrafish.

Caveolae are small plasma membrane invaginations present in many different cell types, which have been linked to diverse cellular functions, including cell signalling, membrane rearrangements and lipid regulation. The caveolae markers, members of the caveolin family of proteins, are essential for caveola formation and function. Recently, however, a protein named PTRF (Polymerase I and Transcript Release Factor) or cavin, originally identified as a nuclear factor that regulates transcription in vitro, was shown to be associated with caveolae in adipocytes. In the first chapter of this thesis, I have used the zebrafish Danio rerio to investigate the relation of PTRF/cavin to caveolae as well as caveola function in vivo. During zebrafish development, PTRF/cavin was highly expressed in the notochord in 18 h, 24 h and 35 h post-fertilization embryos, as detected by in situ hybrydization. Analysis of later development stages showed that PTRF/cavin is also present in the otic vesicle, brachial arches, and periderm. Disruption of PTRF/cavin expression, via morpholino-mediated inhibition, caused severely defective development of the notochord as well as heart edema, in a dose-dependent manner. PTRF/cavin knockdown embryos had curved notochords and were shorter than the controls. Examination of the notochord by electron microscopy showed that the number of caveolae was greatly reduced in PTRF/cavin-morpholino-injected embryos. Similar effects were observed when caveolin-1, the major protein of caveolae in non-muscle cells, was down-regulated. Altogether, these results indicated a role for PTRF/cavin during formation and/or stabilization of caveolae as well as an essential role for caveolae during zebrafish embryo development. Combined with results obtained in mammalian cells, these findings identify PTRF/cavin as the first component of a caveolar coat, required for caveola formation and function (Hill et al., 2008). We subsequently identified a family of PTRF/cavin-related proteins, the cavins, that all associate with caveolae. Using biochemistry, light microscopy, and FRET-based approaches we characterised PTRF/cavin and the new members of this family of proteins SDR/cavin-2, SRBC/cavin-3 and MURC/cavin-4. We have shown that the four members of the cavin family form a multi-protein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and then associate with caveolin at the plasma membrane caveolae; interestingly, caveolin is essential for the plasma membrane translocation of the cavin complex, and in caveolin-1 knockout cells the four cavin proteins are restricted to the cytosol. PTRF/cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The four cavin proteins present distinct patterns of tissue expression, which suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. MURC/cavin-4 is expressed predominantly in muscle and its distribution is perturbed in human muscle disease associated with caveolin-3 dysfunction, identifying MURC/cavin-4 as a novel muscle disease candidate caveolar protein. To functionally investigate the relation of cavins and caveolae, we explored a caveolar function in mechanosensation. Through the use of hypo-osmotic media, we induced membrane-stretch and showed that the increased membrane tension leads to dissociation of the caveolin-cavin module and caveola disassembly as observed by immunofluorescence and FLIM/FRET techniques. Once released from caveolae, caveolin was seen internalized in late endosomes and lysosomes. Cavin-1, on the other hand, was found to be diffused in the cytosol and from there it was translocated to the nuclear compartment. The nuclear translocation was observed in several different cell types, which suggests a universal role for nuclear cavin-1, and was independent of caveolin expression. Analysis of live cells using real-time FLIM/FRET showed that cells quickly respond to variations in membrane tension by dissociation/re-association of caveolin and cavin-1. Altogether, in the course of this project, I was able to show that cavin-1 is an essential regulator of caveola biogenesis in cultured cells and in vivo. Cavin-1 and the other members of the PTRF/Cavin family form a multiprotein complex that is recruited to caveolae by caveolin and coats plasma membrane caveolae. The association between cavin-1 and caveolin is crucial for caveolae assembly and this interaction has a role in the cellular sensation of plasma membrane tension. Under high membrane tensions, caveolin and cavin-1 dissociate with the consequent flattening of caveolae. Under these circumstances, caveolin is internalized into enlarged endosomes and lysosomes while cavin-1 is translocated to the nucleus, identifying for the first time a caveola- to nucleus signalling pathway. The exact role of nuclear cavin-1 under plasma membrane stretch is now amenable to analysis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Currently, the zebrafish (Danio rerio) is widely used in research related to anxiolytic properties and pharmacological mechanisms of alcoholism. Previous research has shown its effects on several behavioral parameters, similar to that already observed in exposure to anxiolytic drugs, as well as attenuation of fear responses against the predator. The detection of the presence of predator can occur in a visual way, and threatened fish has the ability to communicate about risk to other individuals through the release of a chemical called disturbance substance. The predatory threat causes physiological adjustments as a result of the stress response, which aims to maintain the homeostasis of the animal, and the secretion of the hormone cortisol, the main endocrine indicator of this response. As ethanol can affect the reactions of fear, we evaluated its possible effects on cortisol levels in zebrafish as a result of exposure to the visual perception of predator and disturbance substance. Finally, our study demonstrated for the first time the interference of low doses of ethanol (0.25%, 0.50%, and 1%) for acute exposure on the stress response triggered by the presence of the predator, blocking cortisol secretion in both fish: donors and receivers. In the absence of ethanol, we detected stress response with an increase in cortisol levels in fish tissue disturbance substance of donor fish due to the presence of predators, as well as in fish receptors due to the perception of chemical information. Therefore, we postulate that ethanol may interfere with the visual perception of both predator and/or secretion of disturbance substance, as well as the ability to detect this substance. / Recentemente o peixe zebra (Danio rerio) tem sido amplamente utilizado em pesquisas ligadas as propriedades ansiolíticas e mecanismos farmacológicos do alcoolismo. Pesquisas anteriores tem demonstrado seus efeitos em diversos parâmetros comportamentais, similares aos já verificados na exposição a drogas ansiolíticas. Atenuação das reações de medo, ocasionadas pela presença do predador também tem sido demonstradas, em decorrência da exposição ao etanol. A detecção da presença do predador pode ocorrer de maneira visual, onde o peixe ameaçado tem a capacidade de comunicar demais indivíduos sobre o risco, através da liberação de substância química, denominada substância distúrbio. A ameaça predatória ocasiona ajustes fisiológicos em consequência da resposta de estresse, que tem por objetivo manter a homeostase do animal, sendo a secreção do hormônio cortisol o principal indicador desta resposta. Uma vez que o etanol pode afetar as reações de medo, avaliamos seus possíveis efeitos no aumento dos níveis de cortisol de peixes zebra, em consequência da exposição visual ao predador e percepção da substância distúrbio. Finalmente, nosso trabalho demonstrou por primeira vez a interferência de baixas doses de etanol (0,25%, 0,50% e 1%) em exposição aguda, na resposta de estresse desencadeada pela presença do predador, bloqueando a secreção de cortisol tanto em peixes zebra doadores como peixes zebra receptores da substância distúrbio. Na ausência de etanol, detectamos resposta de estresse com aumento dos níveis de cortisol tecidual em peixes doadores de substância distúrbio devido a presença do predador, assim como em peixes receptores devido a percepção da informação química. Tendo isto em conta, postulamos que o etanol pode interferir na percepção visual do predador e/ou a secreção de substância distúrbio, assim como na capacidade de detecção desta substância por peixes receptores.

The potential for environmental toxicants to cause genetic damage (genotoxicity) in organisms is a prominent concern because effects on DNA can compromise reproductive success and survival in organisms. Genotoxicity in male germ cells is of particular concern because damage to DNA in sperm may not be repaired and the consequences of damaged genetic material may be transgenerational (from parent to offspring). An integrated approach across multiple levels of biological organization is necessary to establish linkages between exposure to genotoxicants and subsequent effects at molecular and higher levels of biological organization. This thesis addresses the relation between toxicant-induced genotoxicity and reproductive success in zebrafish, and focuses on a model genotoxicant (hydrogen peroxide) and dissolved metals (radionuclide or non-radioactive forms) under controlled laboratory conditions. Uptake and depuration kinetics of a mixture of radionuclides (54Mn, 60Co, 65Zn, 75Se, 109Cd, 110mAg, 134Cs, and 241Am) were investigated, and radiation dose estimations were computed to link exposure and bioaccumulation with radiation dose. Cobalt (Co, non-radioactive) was selected as an environmentally relevant toxicant for investigation of genotoxicity and effects on reproductive success with a focus on male fish. Chronic exposure (12-d) to 0 – 25 mg l-1 Co resulted in reduced numbers of spawned eggs, lower fertilization success, and reduced survival of larvae to hatching. In male fish, DNA damage was detected in sperm and genes involved in DNA repair (xrcc5, xrcc6, and rad51) were induced in testes from some Co treatments, generally consistent with reduced reproductive success. No change in expression of repair genes in larvae spawned from parents exposed to Co was observed. Overall, results indicate that DNA damage and induction of DNA repair genes can occur rapidly after exposure to genotoxicants and that, if exposure levels are elevated, negative effects on reproduction can occur. Results are considered with particular focus on implications of male genotoxicity on reproductive success and the potential for transgenerational effects of toxicants.