Cryosensitivity Of Zebra Fish Ovarian Follicles Biology Essay


The process of cryopreservation has been universally accepted for preserving the biological materials for infinity years (Chen, 1988). In theory, the viability of the cryopreserved samples could be retained with no structural changes. Being the natural element water plays an essential role by bearing the biochemical activities within the cells. Ever since, water has been proved to have a tremendous role in degradation of stored materials (Adams, 2007) the studies on conversion of water in to ice and its lethal effects has also received enough attention.

The principle of cryopreservation is to apply ultra low temperature to suspend the biochemical reaction of the samples. Generally, cryopreservation employs using liquid nitrogen temperature (-196C) and at this temperature no biochemical reaction takes place. However, it is not possible to reduce the temperature successfully to such low temperature because water inside the tissues is forced to form ice when its reaches its freezing point. Although, the freezing point of water is not exactly 0C, and the process of super cooling tend to be an important phenomenon in most of the samples surviving low temperature. Super cooling is the process by which the cellular water is seized from freezing and when the temperature lowers any further may result in forced ice formation.

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In cryobiology, the great challenge is to lower the temperature to glass transition temperature (Tg) because most of the samples have no activity when the water inside the samples are subsequently converted to glass. Glass is an amorphous solid, and there has been debates concerning the smaller activity of the samples that could lead to sample degradation over a period of time. In wider context, most of the samples posses glass transition around temperatures -130C and to be very precise any temperature below the glass transition temperature could give longer viability than the samples held at liquid nitrogen vapor phase temperature i.e. -130C. This is the reason for choosing liquid nitrogen temperature whilst establishing longer cryobiological protocols.

Clearly, not all samples amend to the cryo-banking and for reducing the temperature to the temperature of interest; cryoprotectants become an essential role (Watson et al., 1985). It has been highly recognized that the simple freezing and thawing of the cells leads to cell damage and this is believed to occur because of the ice nucleation and dehydration (Mazur, 1970). The objective of using the cryoprotectant during freezing is to hold the water with out forming ice until the glass transition temperature is reached. Although, such additives cannot result in 100% survival after freezing and thawing, but in majority of the cases the freezing injury tend to be suppressed (Fahy, 1985). The cryoprotectants used must sufficiently concentrate to supercool and solidify into a glass and the water must not be converted to ice readily and instead it would be converted to glass by the process of vitrification (Rall, 1985). There are two different type of cryoprotectants being used for cryopreservation technique. The permeating cryoprotectant commonly used for sperm cryopreservation could possibly permeate in to the cells and produce significant effect. The most commonly used penetrating cryoprotectant includes glycerol and dimethyl sulfoxide (DMSO) is the best example (Li et al., 2005). The other type of cryoprotectant has the tendency not to enter inside the membranes and it remains in the extracellular space (Sztein et al., 2001).

In most of the cryopreservation techniques the permeating cryoprotectant are permeated in to the cells. The entry of the cryoprotectant is believed to happen by thermodynamic equilibration and it also enhances the removal of permeating cryoprotectants during thawing (Elmoazzen et al., 2005). Ethylene glycol finds its important application in cryopreserving mammalian embryo freezing because it carries low formula weight and high permeation in to cells when compared with other cryoprotectant (Chi et al., 2002).

Urgent requirements for both plant and animal conservation have attracted lot of the conservation scientists to focus on cryopreservation. Indeed, the cryopreservation is a reliable method for conservation and requires less money and less time consuming. The field expanded in conserving the endangered fish species through out the world, and the aquatic species number has been declining gradually. The sperm cryopreservation of zebra fish has been proved to be possible ensuring the species conservation, the cryopreservation of oocyte and embryo still remains a greater challenge (Guan et al., 2008). Recently the zebra fish and other aquarium fish have suit to be the most vital model system species for the studies (Robles et al., 2009). In zebra fish creation of cryobanks of viable fish sperm, oocytes and embryos are of important demand. Although the storage time of cryobanks is unpredictable the liquid nitrogen storage of fish cells could last between 200 and 32,000 years (Robles et al., 2009).

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Despite of successful cryopreservation of spermatozoa of many species of fish (e.g. cyprinids, salmonid etc.) are in practice, the systemic studies on fish oocytes cryopreservation is still lacking (Chen and Tian, 2005). However, few embryos have been concluded to with stand short period of reduction in temperature to liquid nitrogen temperature and the consistency in producing the same results remains indefinable (Isayeva et al., 2004). The maternal genome cryopreservation of the humans has been shown to be possible; the successful survival of maternal genome of fish still remains challenging. Moreover, maternal genome cryopreservation seems to be very crucial in aquatic species because there are several genetic factors that are being transformed maternally through oocyte cytoplasm including mitochondrial DNA and mRNS’s that plays a vital role in early embryonic development (Zhang et al., 2008). Preserving such fundamental genes can also improve the chance of conserving the species where the oocyte and embryo cryopreservation fails like in zebrafish.

The availability of eggs that are located in the cortex with in the primordial follicles, can be isolated easily because it is very close to the surface (Lass et al., 1996). The oarian cortex encloses most of the primordial follicles and it characterizes the resting stockpile of germ cells. Nevertheless, not all the follicles undergo maturation and ovulation, most of the cells die through a process known as atresia. Folliclular atresia is a degenerative procedure through which the ovarian follicles lose their integrity and this process takes before the ovulation. Furthermore, the ovary of Danio rerio is asynchoronous in nature and the development takes place in four different stages, and it includes primary oocyte growth, cortical alveolus stage, vitellogenesis and maturation (Ucuncu and Cakici, 2009). In fact, more consideration has been given to cryopreserving follicular cells with using those cells for both in vitro and/or in vivo vivo maturation. Since the morphological studies after freezing and thawing are inadequate and it is not necessary that the morphology correlates to the follicular development.

The problems associated with embryo cryopreservation of zebrafish are generally high yolk content and the chilling injury (Isayeva et al., 2004).The injury could occur either during freezing or thawing. Evidently, the intracellular ice crystal formation breaks the organelles and cells, Before considereing a cryopreservation technique the equilibrium rate along with freezing and thawing rates are to be considered very crucially. However, the oocytes are smaller in size when compared to the eggs and it constitute single section and have a reduced risk because of the absence of yold syncytical layer that are seen only in embryos (Prescott, 1995). Also, the fish oocytes possess greater cryoprotectant tolerance limit when compared to embryos (Plachinta et al., 2004) and the permeability of the cryoprotectant to the oocytes seems to be more in oocytes and the embryos often show lesser permeation (). The temperature of ice crystal formation and the intensity of the cryoprotectant permeation and the capacity to prevent crystal formation must be critically reviewed before using the cryoprotectant. Furthermore, the cryoprotectant toxicity and the extent to prevent the ice crystal formation may vary for each cells and tissue types.

There are two methods noted so far in cryobiology techniques for reducing the temperature from room temperature to liquid nitrogen temperature. The oldest method that still finds a competitive role in cryopreservation is the controlled slow cooling. Such method uses slow cooling rate of about 1 or 2C/hr until the glass transition temperature is reached and this step is followed is followed by plunging the samples in to liquid nitrogen. This method may perhaps require cryoprotectant and many protocols defined previous has used less cryoprotectant (Zhang and Rawson, 1995). This is because the slow freezing desiccates the intracellular water and thus emphasis higher survival. On the other hand, the other method that has wider application and recently accepted is the vitrification. In contrast to controlled slow cooling the vitrification protocols employ using faster cooling rate and this requires using more concentration of cryoprotectant. The cooling rate generally performed for vitrification ranges from 40C/hr to 700C/hr (Kasai et al., 1990; Fahy, 1985). Since the vitrification uses rapid cooling rates and this results in solidification of the solution thus the samples escape from forming ice-crystals and subsequent cellular damage. However, using extreme concentration of cryoprotectant may be particularly toxic to one cell embryos and oocytes (Mukaida et al., 1998). Although, both the method finds equal importance in cryopreservation, in strict sense, slow cooling rates result in higher survival for fish embryos than high cooling rates with successful rates ranging from 0.01 to 0.75C/min (Harvey and Ashwood-Smith, 1982).

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Furthermore, in corresponding to the success rates of cryopreservation of specific organs of endangered species, the problems associated with cryodamage has known to cause wide damage to the cellular membrane. In addition, the metabolic activity of the cells seems to be reduced and interrupt the mitochondrial bioenergetical processes with in the cells (Kopeika, 2004). Although recent reviews clearly project the picture of reproductive cells and its cryodamage in terms of structural alterations, the biochemical alterations of the cryopreserved reproductive cells still remains unknown (Tatone et al., 2010). The cryopreservation of gamete and embryo has developed greatly by using glycerol as a cryoprotectant, the knowledge in enhancing the morphological and fuctional survival has been more. In contrast, the information on the molecular level changes of the cryopreserving the oocytes and embryos are still lacking.

Oxidative stress:

Most of the cells undergoing cryopreservation need to be thawed once required. Most of the cells experience biochemical damage including oxidative stress by reactive oxygen species formed upon thawing (Park, 1998). Oxidative stress seems to be a limiting factor in obtaining successful cryopreservation protocol, nonetheless, heat shock treatment enhances the cells resistant to damage that occurs from freezing and thawing (Komatsu, 1990). Heat shock proteins may save damaged cells. GSH1, GSH2, and trehalose seem to have an important role in eradicating oxidative stress.

The production and accumulation of reduced oxygen intermediates including superoxide radicals, singlet oxygen, hydrogen peroxide and hydroxyl radical are often grouped under oxidative stress. Such stress can affect the physiological state of the cryopreserved cells including lipid damages, protein and DNA damages (Lesser, 2006). The formation of oxidative stress has become more common in world’s oceans and this stress has been witnessed to be a result of environmental alteration covering thermal stress, ultraviolet radiation exposure and pollution effects. In addition to natural stresses, the cryopreservation also tends to increase the oxidative stress. In Saccharomyces cerevisiae (Yeast), Ty1 is a retrotransposon and the transposition at new locations in the host geneome can be efficiently activated by exposure to UV light, X-ray, and nitrogen starvation. However recent report by Stamenova et al. (2008) showed that controlled step wise cooling of the cells also resulted in increased Ty1 levels. The cells were cooled for 2 hours at 4C and this step was followed by freezing for 1 hour at -10C and subsequent cooling by 16 hours at -20C. Clearly, the freeze-induced Ty1 transposition did not took place in mitochondrial mutants and this gives good evidence that the Ty1 transposition resulted on cooling depends greatly on mitochondrial oxidative phosphorylation. However, the increased level of reactive oxygen species possesses a significant role in activating the Ty1 retrotransposon transposition in the frozen yeast cells. Reactive oxygen species has been given great concerns and it accumulates while performing the cryopreservation techniques.

The induction of oxidative stress by a pesticide called atrazine (ATZ) has been reproted in the liver and ovary samples of the female zebrafish (Jin et al., 2010). The GSH and MDA content of the liver was also found to be altered. The antioxidant enzyme activity also seems to be reduced. Fish carry similar defense against toxic chemicals like mammals, and to wider context this covers the oxidative stress generation, the transcription of certain protective genes has been related to be induced by specific DNA motifs often reffered to as electrophile response elements (EPREs) (Carvan et al.,2001).

However, blastomeres cryopreserved using phosphate buffered saline and in another attempt made by equilibration of blastomeres with 2M dimethyl sulfoxide (Me2SO) for 1 hour and the other method involved using dimethyl sulfoxide as a cryoprotectant resulted in some mitochondrial DNA mutation (Kopeika et al., 2005). The Polymerase Chain Reaction (PCR) frequently used in the gene mutation analysis has become a revolutionised method and it was used to anlayse the mitochondrial DNA. The loci of the mt DNA were amplified and the comparison between the standard and experimental results revealed the increase in mutation for phosphate buffered saline. However, no significant mutation has been increased in the result of using 2 M Me2SO4.

Significant effect of increased percentage of DNA fragmentation has been noted during the cryopreservation of sperm. Oxidative stress biomarker has been used to identify the impacts, 8-oxo-7, 8-dihydro-2’deoxyguanosine (8OHdG) is the most common biomarker. Apoptotis indicator would give the percentage caspase of positive cells (Zribi et al., 2008). According to Perez-Cerezales et al. (2008) the damage in the DNA which has been often described as a consequence of frozen storage may be result from oxidative stress. The production of DNA damage by oxidative stress during sperm cryopreservation has been concluded as a result of free radical generation. Moreover, the oxidation stress can affect the strands of the DNA and the nitrogen bases may get altered.

Apoptosis is an important phenomenon in higher organism and it is fundamental biological process necessary for normal development and tissue maintenance (Kerr et al., 1995). Significant relation between oxidative stress altering the mediators of cellular processes including apoptosis and necrosis in marine organisms has been noted (Lesser, 2006). Cooling stress as an trigger to cell stress response and subsequently activating the process the apoptosis which leads to necrosis is also clearly understood (Sonna et al., 2002). Moreover, the response of apoptosis as a result of low temperature seems to have little influence on both the temperature exposed and the duration of the exposing time. The cooling stress also results in affecting some of the genes which also includes apoptotic specific protein gene (ASP). In order to analyse the effect of oxidative stress in cryopreservation, measuring the particular genes will become more responsive in terms of molecular level study. In addition the cold stress also seems to increase E selection (Cell adhesion gene), HSP gene, p53 (Cell-cycle gene) and IL-8 (Cytokine gene) (Boonkuson et al., 2006).

Methanol will be used as a cryoprotectant for this experiment because early reports suggested the toxicicity of using DMSO in mouse oocytes (Vincent et al., 1990). Since there has been no report suggesting the initiation of oxidative stress as a limiting factor for oocyte cryopreservation in zebrafish, the study is intended to analyse the effect of oxidative stress during cryopreservation of zebrafish oocyte. As mentioned earlier, the genes are susceptible to cold stress and undergo slight to dramatic change, PCR gene expression gives the clear picture of whether or not the gene is altered after cryopreservation. It is not known any difference between vitrification and slow cooling methods result in different results, however, as an initial attempt the oocyte of zebrafish is extracted and using methanol as a cryoprotectant, the oxidative stress if any induced for controlled slow cooling will be reviewed. However, vitrification requires using high concentration of cryoprotectant, although, the intracellular ice is avoided there are plenty of risk like osmotic shock and toxicity of cryoprotectant.

Viability test of cryopreseved cells

The viability of the cells after cryopreservation is also essential to report the extent of oxidative stress. Most of the oocyte cryopreservation tests use staining as a standard viability test. Despite of many standard dyes available for staining, the tryptan blue has become the most common dye used to assess the viability (Chen et al., 2006). In particular, ovarian cortex staining with vital dye or fluorescent live or dead probes. The advantage upon tissue warming follicles is released from cortical tissue by mechanical dissection or enzyme lysis gives easy access to assess the viability. However, according to santos et al. () the viability status of the thawed preantral follicles immediately after cryopreservation could not be determined accurately.

Review of all the literature suggests the cryopreservation protocol requires specific attention about the metabolic stresses the cells undergo during cryopreservation. More concerns about loss of viability percentage because of the metabolic stress both during and after cryopreservation should be considered. It is not known in particular use of one cryoprotectant may have less impact over the other cryoprotectant. If the cryoprotectant produces less stress the successful reduction of the temperature below the glass transition temperature may not be achieved. Therefore, optimization of cryopreservation protocols possibly to have diminished stress and the extent to which it affect the overall cellular activity need to be analyzed. Although cryopreservation has become the routine method for preserving the endangered species for number of years, the possibilities of loss of its viability over the time has to be major concern and emphasized studies on such variables are highly required.