Development of new methods to assess the quality of zebrafish (Danio rerio) ovarian follicles

A thesis submitted for the degree of Doctor of Philosophy of the University of BedfordshireHigh quality fish oocytes are essential for in vitro maturation (IVM), in vitro fertilization (IVF) protocols, and for use in cryopreservation. It is important to develop methods for assessing oocyte quality for applications in aquaculture, the preservation of endangered species and managing fish models used in biomedical research. The lack of reliable methods of evaluating oocyte quality limits progress in these areas. The present study was undertaken to develop new methods to assess ovarian follicle viability and quality of stage III zebrafish (Danio rerio) ovarian follicles. The methods developed were then applied to study the impact of cryoprotectant and/or cryopreservation procedures. A vital staining procedure, not previously used with zebrafish oocytes, has been investigated. FDA-PI (Fluorescein diacetate-Propidium Iodide) staining was found to be a more sensitive then currently used viability tests and it could also be applied to all ovarian follicles developmental stages. Mitochondrial activity and distribution as biological markers was investigated with the mitochondrial membrane potentialsensitive dye JC-1- (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide). Confocal microscopy, Cryo-scanning and electron microscopy studies were undertaken to determine mitochondria distributional arrangement within the ovarian follicle. This provided new information on zebrafish ovarian follicle structure, and showed that mitochondria exhibited a contiguous distribution at the margin of the granulosa cell layer surrounding stage III zebrafish oocytes. Cryoscanning results showed a polygonal structure of the vitelline envelope, which is reported here for the first time with the mitochondrial distributional arrangement in the granulosa cell layer. Mitochondrial distribution and the evaluation of mitochondrial activity proved to be sensitive markers for ovarian follicle quality, providing more detailed information on cryoprotectant impact. The measurement of ATP levels, ADP/ATP ratio and mtDNA copy number were also undertaken following cryoprotectant exposure. These findings, together with the observation of mitochondrial distribution, suggested that even cryoprotectant treatments that are considered to have little or no toxicity can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development. Therefore, a further optimization of the currently used protocol may need to be considered. The study of organelle distribution and organisation would support in vitro maturation and oocyte development fields, as well as their use as biological markers for quality determination. These findings will contribute to a better understanding of oogenesis/folliculogenesis processes in fish

Cytoskeleton proteins F-actin and tubulin distribution and interaction with mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes

The distributional arrangement of mitochondria in the granulosa cells surrounding stage III zebrafish oocyte has been reported as a contiguous aggregation of mitochondria at the margin of the each granulosa cell. The aim of the present study was to further investigate the mitochondrial distribution in the granulosa cell layer in stage III ovarian follicles and the interaction between mitochondria and cytoskeleton elements actin and tubulin. To determine mitochondrial distribution/transport, immunocytochemistry analysis of tubulin and mitochondrial COX-I was carried out along with phalloidin staining of polymerised F-actin. The follicles were also exposed to a range of conditions that are known to affect mitochondria and the cytoskeleton proteins actin and tubulin. The mitochondrial inhibitor FCCP, the anti-mitotic drug nocodazole, and actin polymerisation inhibitor cytochalasin B were used. Levels of ATP, mtDNA copy number, and viability assessed by Trypan blue were also studied after exposure to inhibitors in order to determine the relationship between mitochondrial distribution/activity and ATP production. F-actin showed a hexagonal-polygonal distribution surrounding the mitochondria in granulosa cells, with the F-actin network adjacent to the plasma membrane of each granulosa cell. Tubulin structure presented a less organised distribution than F-actin, it was sparse in the cytosol. Interaction between mitochondria and tubulin was found indicating that mitochondria and tubulin are colocalised in zebrafish ovarian follicles. The exposure of ovarian follicles to inhibitors induced the loss of mitochondrial structural integrity showing that mitochondria distribution in granulosa cells of stage III zebrafish ovarian follicles is determined by the microtubules network

Effect of methanol and Me2SO exposure on mitochondrial activity and distribution in stage III ovarian follicles of zebrafish (Danio rerio).

In this study the effect of cryoprotectants that have been shown to be the least toxic to zebrafish ovarian follicles (methanol and Me(2)SO), on mitochondria of stage III ovarian follicles was evaluated. The mitochondrial distributional arrangement, mitochondrial membrane potential, mtDNA copy number, ATP levels and ADP/ATP ratios were assessed following exposure to cryoprotectants for 30 min at room temperature. Results obtained by confocal microscopy showed that 30 min exposure to 2M methanol induced a loss of membrane potential, although viability tests showed no decrease in survival even after 5h post-exposure incubation. Higher concentrations of methanol (3 and 4M) induced not only a decrease in mitochondrial membrane potential but also the loss of mitochondrial distributional arrangement, which suggested a compromised mitochondrial function. Furthermore 3 and 4M treatments resulted in a decrease in viability assessed by Fluorescein diacetate-Propidium iodide (FDA-PI) and in a decrease in mtDNA copy number and ADP/ATP ratio after 5h incubation following methanol exposure, indicating a delayed effect. The use of Me(2)SO, which is considered to be a more toxic CPA to zebrafish ovarian follicles than methanol, caused a decrease in viability and a sustained decrease in ATP levels accompanied by failure to maintain mtDNA copy number within 1h post-exposure incubation. These results indicated that even CPAs that are considered to have no toxicity as determined by Trypan blue (TB) and FDA-PI tests can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development

Effect of methanol on mitochondrial organization in zebrafish (Danio rerio) ovarian follicles.

Successful cryopreservation is usually measured in terms of cell survival. However, there may also be more subtle effects within cells that survive. Previous studies on zebrafish have produced evidence of mitochondrial DNA (mtDNA) damage in cryopreserved embryonic blastomeres and, after exposure to cryoprotectants, alterations in mtDNA replication in embryos and decreased mitochondrial membrane potential, mtDNA and ATP production in ovarian follicles. This study shows that the decreased ATP levels previously observed in stage III zebrafish ovarian follicles exposed to ≥3 M methanol persisted in those follicles that subsequently developed to stage IV. However, the decreased mtDNA levels were restored in those follicles. In order to determine whether mitochondrial distribution and/or their transport network was affected by the methanol exposure, immunocytochemistry analysis of tubulin and mitochondrial cytochrome c oxidase I (COX-I) was performed, along with phalloidin staining of polymerized actin. Neat arrangements of all proteins were observed in control follicles, with COX-I and tubulin being colocalized near granulosa cell nuclei, while actin formed hexagonal and/or polygonal structures nearer granulosa cell membranes and projected into the oocyte surface. Exposure to methanol (2 to 4 M) disrupted the COX-I and tubulin arrangements and the hexagonal and/or polygonal actin distribution and actin projections into the oocyte. These effects were still observed in those follicles that developed to stage IV, although the severity was reduced. In summary, the disruption to function and distribution of mitochondria in ovarian follicles exposed to >2 M methanol may be mediated via disruption of the mitochondrial transport system. Some recovery of this disruption may take place after methanol removal and subsequent follicle maturation

Impact of cryoprotectants and cryopreservation on metabolic activity and cytoskeleton proteins of zebrafish (Danio rerio) ovarian fragments.

Cryopreservation of reproductive cells and tissues of aquatic species offers many benefits to the field of conservation, aquaculture and biomedicine. Although cryopreservation of fish sperm has been successfully achieved, cryopreservation of embryos and oocytes remains unsuccessful. Several studies have been undertaken on cryopreservation of isolated fish ovarian follicles at different stages, although the protocols used lead to a compromised viability. The present study investigates the effect of cryoprotectants and cryopreservation on the viability of ovarian tissues of zebrafish (Danio rerio). The effect of permeating cryoprotectants (CPAs) methanol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) on ovarian tissues were investigated in a series of toxicity tests. Controlled slow cooling of ovarian tissues using 1M and 4M methanol was also carried out. Ovarian tissue viability was assessed by trypan blue (TB) and fluorescence diacetate (FDA)-propidium iodide (PI) tests. In addition, the effect of methanol exposure and cryopreservation on ovarian follicle ATP level, mitochondria, actin and tubulin distribution were also investigated. Results showed that cryoprotectant toxicity to ovarian fragments increased in the order of methanol, DMSO and EG. The results from controlled slow cooling showed that 1M methanol was more effective than 4M methanol although subsequent cryopreservation induced decreases in ATP levels. Immunocytochemistry and actin staining results showed impacts of cryopreservation on mitochondria and cytoskeleton proteins distribution

A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles

AbstractAttempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25ml plastic straw, vitrification block and fibreplug™). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9±18.4% when fibreplug and V16 (1.5M methanol+4.5M propylene glycol) solution were employed. When vitrified in V2 (1.5M methanol+5.5M Me2SO) the membrane integrity decreased to 42.0±21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance 2h post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes

A multi-element psychosocial intervention for early psychosis (GET UP PIANO TRIAL) conducted in a catchment area of 10 million inhabitants: study protocol for a pragmatic cluster randomized controlled trial

Multi-element interventions for first-episode psychosis (FEP) are promising, but have mostly been conducted in non-epidemiologically representative samples, thereby raising the risk of underestimating the complexities involved in treating FEP in 'real-world' services