Zebra fish has become the most common laboratory model for studying the aquatic animals at both structural level and molecular level (Harvey et al., 1982). There are plenty of reasons for using this species as wide source in studies and many reports has been published with this species in the past two decades. This includes easy availability and easy maintenance in aquarium like guppies (Briggs, 2002). The reason for studying the zebrafish in terms of cryopreservation is to avoid the problems of increasing loss of many fish species from their natural habitat. The ability to cryopreserve the embryo for preserving the species diversity for aquaculture has become a reliable option (Hagedorn et al., 2002). Cryopreservation of fish gametes allows the storage of genetic material of embattled species and this technique is highly demanded in aquaculture, conservation and also in human genomic research (Tsai et al., 2009).
The zebrafish is a tropical fresh water fish and it belongs to the Kingdom: Animalia and it has been grouped under the family Cyprinidae. This fish extremely reproduces like all other fish and it requires the ovulation. The female fishes can lay nearly hundreds of eggs during each clutch. The embryonic development of the eggs initiate with the help of cell divisions. The eggs after fertilization become transparent. Furthermore, the DNA of the offspring's is inherited from the parents and this may perhaps include nearly thousands of mtDNA.
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The decline in both fresh and marine fish has created lot of attention to cryopreserve fish gametes, embryo and oocyte. This is because retention of genetic resource possibly opens enough species survival after longer period (Rawson and Zhang, 2005). Having successfully cryopreserved the fish sperms, the low membrane permeability (Ledda et al., 2006) and large yolk mass (Isayeva et al., 2004) of the oocyte and early embryo makes the cryopreservation relatively unsuccessful (Yang et al., 2008. In addition, the lack of oocyte cryopreservation also has greater influence with chilling injury (Tsai et al., 2009). Besides, the mitochondrial DNA present in the embryonic cells of fish has been shown to have an enhanced frequency of base pair mutation upon cryo-treatment (Rawson and Zhang, 2005).
The field of cryopreservation has been debated as the highly reliable method with obvious benefits including preserving the genetic materials for years (Pegg, 2007) that are multiples of hundred (Morris et al., 2009). The cryopreservation of oocytes has noticeable benefit when judged with embryo. One of the vital advantages of using oocytes for cryopreservation is the size. Since the oocytes are smaller in size when compared to embryo along with the tendency to tolerate much lower temperature than embryos. The reduction of the temperature below the temperature in which it forms ice can be achieved by two different methods. The older method called controlled slow cooling uses a comparatively slower cooling rate when compared with the newly emphasized method called vitrification (Rall and Fahy, 1985).
Considerable reduction of temperature to lower levels is not at all easy, because the samples may meet intracellular ice which is often lethal to the viable cells and hence the viability seems to lost dramatically (reviewed by Muldrew et al., 2003). In order to avoid the lethal ice formation, the addition of concentrated solution of cryoprotective additive (CPA) before subjecting the samples directly in to liquid nitrogen temperature is suggested. The advantage of plunging the samples directly in to liquid nitrogen temperature is the solidification of the samples without crystallization is attained as a result of higher cooling rate (Martino et al., 1996). Most of the cryopreservation techniques require cryoprotectants (Libermann et al., 2002) and the purpose is the avoid ice formation. The examples of cryoprotectants include Dimethyl sulfoxide (DMSO), methanol, glycerol, Glucose, raffinose and sucrose (Sztein et al., 2001). The selection of cryoprotectant may be crucial because different compartment of same cell may have different permeability to water and cryoprotectants (Hagedorn et al., 1998). For vitrification, a mixture of cryoprotectant is often used and the water in the extracellular space solidifies instead of crystallization and therefore it reaches ultralow temperature (Rall, 1993).
Since no successful protocol has been proved to be successful in cryopreserving either the oocytes or embryos of zebra fish, recent studies have concentrated densely on studying the role of mitochondria. The mitochondria play a significant role in oocyte maturation, fertilization and embryo development (Zampolla et al., 2009). However, the behavior of mitochondria during early oogenesis is unfamiliar. Although, attempts by Zhang et al. (2008) to analyse the distribution and aggregation of mitochondria with the help of JC-1 dye and confocal microscope revealed the mitochondrial movement during oocyte maturation. Regulation of cytochrome c is controlled with the Bcl-2 family of proteins from mitochondria which directly relates to the apoptotic nature of the cells (Martin and Renshaw, 2009).
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The crucial role of miochondria in oxidative phosphorylation and ATP production makes it extremely dynamic in germ plasm. Mitochondria also act as a key regulator in apoptosis. Since the apoptosis involves multiple germ cell maturation steps the process is very complicated. Moreover, the process takes place under the control of endocrine and paracrine and autocrine signals (Matzuk and Lamb, 2002). Mitochondria being the power house of the cells are believed to have major role in oxidative phosphorylation and ATP production. Furthermore, they possess sequence of functions that contribute to redox and Ca2++ homeostasis, provide intermediary metabolites, store pro-apoptotic factors and organize and transport germ plasm during oogenesis (Zhang et al., 2008). The process of both spermatogenesis and oogenesis are intricate in most of the animals. Despite, the role of mitochondria in germ plasm processes is not certain; it has been significantly known that mitochondria participate in germ plasm formation and translocation (Wilding et al., 2001). The rigidity in maintaining its web-shaped network in cells with the help of a balance between fusion and fission makes mitocondria an important aspect of study during cryopreservation. It has been known that the balance can be violated and this results in morphological alteration of mitochondria.
The process of oogenesis, plays an important role in zebrafish, like all other animals and fish through which the primordial germ cells (PGCs) develop into ova. Since the formation of ova is highly required for the fertilization (Patino and Sullivan, 2002). During the time of birth, oogonia stop proliferating and the oocytes tend to hold at their diplotene stage of the first meiotic division. The oocytes of this stage have the tendency to be surrounded by a layer of granulose-like cells and later this would lead to form primordial follicles. The entire oocyte development in zebrafish has been divided into five different stages which are based on morphological criteria and on physiological and biochemical events. The initial stage which has been termed as stage I (primary growth stage) in which the oocytes live with other oocytes and later within a definitive follicle. In this stage the follicle develops greatly and increases in size as well. In stage II (cortical alveolus stage), the look of the oocytes get changed and cortical alveoli and the vitelline envelope turn into appropriate. During the next stage, i.e stage III (Vitellogenesis), the yolk proteins forms in the oocyte and the yolk grows very well. The next stage is the oocyte maturation stage (Stage IV) where oocytes increase to its maximum size and turn in to translucent and the yolk becomes non-crystalline and the process of final meiotic maturation in vivo is being taken place. In stage V (mature eggs) the eggs are ovulated into the ovarian lumen. These eggs can actively participate in fertilization (Selman et al., 1993).
The development of the oocyte in zebrafish takes place under four different steps. The first step is the primary oocyte growth step where the initial primary oocytes are formed. This stage consists of 2-4 nucleoli present in the centre of the nuclei plasma. The development of this stage is highly linked to the early prophase. The second step is the cortical alveolus stage; this stage contains yolk vesicles in the cytoplasm. The third step is the vitellogenesis. The fourth step is the maturation, and the nuclear membrane seems to be dissolved and the subsequent migration of the nucleus takes place. Such movement, results in bulging the germinal vesicle cells. All the four stages can be clearly distinguishable because the oocytes increase in size at every stage. Nonetheless, mature oocyte phase, makes the cell shape irregular, and hence the division of nucleoli suit to be abundant. The stage also forms tripartite vitelline envelope and the formation of second layer between the oolemma and the first layer takes place. The formation of the second layer looks to be inevitable because the outer layer is more electrons transparent, unlike the newly formed second layer.
The development of embryo in zebrafish takes place in sequence of phases depending upon the morphological characters. For easy identification the steps has been divided in to zygote period, cleavage period, blastula period, gastrula period, segmentation and pharyngula. The zygote period, as the name suggests the formation of zygote takes place and being the first stage during the development the size of the zygote will be relatively small when compared with normal zygote. The fertilization of eggs may require the egg being moved out from chorine (Charles et al., 1995) and it results in formation of single cell. Since the purpose of development is to replicate the single cell formed, and this is more likely to take place during the cleavage period. During the cleavage period, the blastomeres are divided continuously and produces number of cells. The division of cells takes place with the help of zygotic cycle. The single cell is generally encountered by a cleavage furrow and the furrow seems to grow from animal pole to vegetal pole. Once the furrow deepens enough, the cell divides itself in to two. The concept of furrow creating problems to blastodisc has been disproved (Kimmel and Law, 1958). Moreover, the division tends to be meroblastic and therefore, they have the tendency to incompletely undercut the blastodisc. Thus the marginal blstomeres are connected to the yolk cell and this is believed to be a result of cytoplasmic bridges. Since the single cell is divided in to two this stage can be subdivided in to two-cell stage. This sub stage is followed by four-cell stage, where the two cells formed are convered in to four cells, i.e each of the formed two cells is divided in to two more. Furthermore the cleavage furrow imitated is vertically oriented and similar pattern of furrow initiation continues until the 32-cell stage. Thus at the end of the cleavage period with the help of sub stages namely, one-cell stage (0 h), two-cell stage(3/4 h), four-cell stage (1 h), eight-cell stage (11/4 h), 64 cell stage (2 h), totally 64 cells are formed and the cleavage period ends here. The blastodisc cut in to a 2 x 4 array of blastomeres. The dechorionated embryo lies in the dish of four cell portion (Kimmel and Law, 1985).
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The following stage is the blastula period (21/4 - 51/4 h). The blastula period gives rise to 128 cells (128 cell stage) and since it is the eighth zygotic cell stage, this stage can also be referred to as eighth zygotic cell stage. During this stage, the blastodisc looks like a clear ball. In the blastula period, the embryo piereces the midblastula transition (MBT). The cleavage furrow occurring initially seems to be synchronous. The precise term may be metasynchronous. In addition, the marginal blastomeres subside and it loses the contact with the yolk cells and hence giving rise to Yolk Syncytial Layer (YSL). The epiboly is initiated. Epiboly, expands the YSL lineages vegetally and it covers the yolk sphere completely (). The cycle goes on until the division of cell reaches the gastrulation cycle. The beginning of the 14 cycle is the gastrulation stage. Since this stage is the 128 cell stage or the eighth zygotic cell stage, the blastodisc appears like a clear ball. The cycle continues until the gastrulation cycle begins at cycle number 14.
The so formed 128 cells, i.e 128 cell stage (21/4 h) brings the blastula stage to an end. All the 128 blastomeres resulted from continuous furrow development are arranged in a high mound of cells. When there are more number of cells the initiation of furrow takes place like a wave and therefore, the development of furrow may be irregular, the ancestry from its position of blastomere may be crucial. The cleavage of cells at the end of 128 cell stage results in 256 cell stages (21/2 h), and the ninth cleavage step is the highly synchronous/ metasynchronous stage occurring in the whole cell development periods. The next stage is the 512-cell stage and the midblastula trasition occurs gradually. The EVL cells can move away from their boundary and connect to the yolk cells. This step is continuously followed by 1k cell stage (3h). This step consists of 1,024 (though the number is significantly less).The 11th mitoses occur with the last wave passing through the blastodisc. The cycle of the cell division ends with the final step called high stage (31/2 h), the marginal cells convene the YSL. The YSL at this stage is also in the form of a thin ring surrounding the blastodisc. This step is followed with sphere stage (4h), Dome stage (41/3 h) and 30%-epilboly stage (42/3 h). There may be a sequence of conversions over these periods including the blastoderm surrounding the yolk cell and the epiboly fractioning the yolk cell which covers the blastoderms.The epiboly seems to be highly microtublule fuctional dependent and the influence of early-acting zygotic gene (Kane, 1991). However, the coverage of blastoderms with equal thickness across the embryo is not possible at all at this stage.
Epiboly grows during the gastrula period (51/4 -10 h) and resulting in production of primary germ layers and formation of embryonic axis. Since the morphological movement at the blastoderm margin is crucial, like in zebrafish, a small teleost develops. The development further takes place by segmentation period (10-24 h), pharyngula period (24-48 h), hatching period (48-72 h) and early larval period respectively (Charles et al., 1995).
The participation of active mitochondria to relocate during oocyte maturation or fertilization is many species has been noted. However, the importance of this relocation in terms of functional aspect is still not known (Bavister and Squirrell, 2000). As the mitochondria provides the primary cellular ATP through oxidative phosphorylation, the extranuclear mitochondria genome are circular and code for 13 subunits of oxidative phosphorylation and also codes for two rRNA subunits and 22 tRNA's. The mitochondrial DNA (mtDNA's) codes the nucleotides of mitochondria. Many mitochondria copies of mtDNAs are inherited maternally through the cytoplasm of the oocyte (Lightowlers et al., 1997). In the process of oogenesis the mitochondria results in intermediary metabolites. Moreover, the introns are absent in mitochondrial gene. In early oogenesis, the transfer of mitochondria from perinuclear space to vegetal cortex has been observed. Micro RNA's are described as the small non coding RNA's and they regulate the gene expression during embryonic development. This is believed to happen by the micro RNA's paring with partially complementary mRNA's.
The study of mitochondria in zebrafish using real time and in vivo study during the development is relatively an easy method when compared with mammals (). There are plenty of dyes being used in the fluorescent microscopy and recently the use of JC-1 dye received significant attention. The distribution of mitochondria during the oogenesis may be stained with this dye and the advantage of using JC-1 in mitochondrial localization has been linked to the mitochondrial membrane potential sensitivity. However, there seems to be problem with mitochondrial probes penetrating the oocyte cytoplasm. The granulose cells surrounding the oocyte of zebra fish gives a good comparison of result in the mitochondrial arrangement during the ovarian follicle development.
The mitochondrial genome of zebrafish consists of 16,596 bp in length and the gene order and the content is identical to the common vertebrate form. Early evidences suggest that mitochondrial movement during the oogenesis is a critical step and it is not known the mitochondria are active or not. The activity of the mitochondria may produce significant effect when compared to the inactive mitochondria. Furthermore, the activity of the mitochondria may vary from stage to stage. The replication of the mitochondrial DNA in the zebrafish, seems to beigin with protein coding genes with orthodox ATG start codon. There are many stop codons including seven TAA and three TAG. The expression of the COI may act as an start codon, however, the COII, ND4 and CYtb may not act as a stop codon (Broughton et al., 2001). According to Zhang (2008), although the distribution of the mitochondria is unique, during stage I of the oocyte development, the mitochondria seems to be in large clusters and in late stage I it seems to occupy a threadlike state. The cluster may be highly dense or may carry lower density and it also seems to have some correlation with germinal vesicle. The mitochondrial genome also possesses some important characters in producing the ATP. Moreover, the replication of the mtDNA needs the translocation of nuclear-encoded transcription and replication factors. The requirement of the primer that has been generated with the help of transcription by mitochondrial transcription factor A (TFAM) and also the mitochondrial-specific polymerase gamma, which includes catalytic (PolGA) and accessory (PolGB subunits) (Spikings et al., 2010).
Moreover, the use of cryoprotectant during cryopreservation seems to affect the activity of mitochondria and it has been not known if the cryoprotectant selection does not alter the mitochondrial activity then the chances of producing cryopreserved oocyte is possible. As described by Tsai et al (2009), the early stage oocyte cryopreservation seems to be possible and therefore the mitochondrial study at this stage may be less significant, although essential. However, careful characterization of the late stage oocytes and the role of mitochondrial movement during the development are much needed.
Although, literature suggests the movement of mitochondria during the ovarian follicle development in zebrafish may be true, it is not known how the movement takes place. In strict sense, the reason for mitochondrial movement is still an unknown parameter. Perhaps, elucidating the pattern of mitochondrial movement may suggest the reason behind the mitochondrial movement. The role of mitochondria during the different stages of the ovarian follicle tends to vary and this is because of the oocyte increasing in size. Therefore, screening of the ovarian follicles at different stages and staining for the mitochondria using the fluorescent dye and fluorescent microscope to trace the location of mitochondria at different stages of the development of the ovarian follicle will be carried out. Furthermore, the movement of mitochondria with in the cells may also be noted. This experiment is particularly designed to study the role of mitochondria during the development of ovarian follicles in the zebrafish. Therefore, the ovarian follicles can be collected by sacrificing a fresh zebrafish using trycaine solution and the ovarian follicle can be collected from ovary.
To understand the significant role of mitochondrial movement during the ovarian follicle development in zebrafish.