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Discussion Of DNA Fragmentation Biology Essay

In recent years advanced methods for treatment of malignant disease have markedly improved the chances of long-term remission or cure. However, these treatments often cause permanent sterility as a result of the loss of spermatogonial stem cells (1). Hence, the preservation of male germline cells in cancer patients is an urgent clinical need (2). Nevertheless, with the advent of assisted reproductive technology and an improved understanding of cryobiology, strategies have been developed which allow the long-term storage of gametes and embryos. But, the gonadal tissue banking, in theory, is a practical alternative to gamete storage which can be utilized by both adults and children (4).

Even with the large number of studies on ejaculated spermatozoa, there was a lack of data on the influence of the freezing/thawing procedure on testicular tissue. Although the freezing of testicular tissue cannot be viewed as an alternative to cryobanking ejaculated sperm, the importance of freezing testicular tissue is increasing because of new developments within our field5. Cryopreservation of structurally intact tissues in certain situations is more desirable than cryopreservation of isolated cells. This is especially important for complex tissues in which preservation of the target cells' functionality depends on that of other cell types present within the tissue. In case of testicular tissue, not only germ cells but also the intra-tubular supporting - Sertoli - cells as well as androgen producing interstitial - Leydig - cells are of particular interest (6). In addition to that, the cryopreservation of testicular tissue as pieces is to avoid the negative influence of enzymatic or mechanical processing that influenced the viability of the cells. Also, to escape cell hypoxia that happen when preparing cell suspension, furthermore preservation of testicular tissue as a pieces could be prevent the loss of spermatogonia that occurs during the process of tissue disintegration (7).

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Despite of the tissue and cell are exposed to physical and chemical stress during cryopreservation and thawing cycle through osmotic changes and ice crystal formation, but these negative influences of cryopreservation could be minimized by use a chemical substance called cryoprotectant (CPA). Cryoprotectants are low-molecular weight and highly permeable chemicals used to protect spermatozoa from freeze damage by ice crystallization. There are four main well-known permeating cryoprotectants (glycerol, ethylene glycol, dimethyl sulphoxide, and 1,2-propanediol)(8), also there are non-permeating cryoprotectants such as sucrose and albumin(9). Cryoprotectants act by decreasing the freezing point of a substance, reducing the amount of salts and solutes present in the liquid phase of the sample and by decreasing ice formation within the spermatozoa (8).

Methodologies for measuring DNA damage differ between

laboratories and depend upon the DNA-damaging agents used, DNA repair

kinetics, the endpoint measured and ways to measure the endpoint (quantitatively or qualitatively). The immunohistochemical assay used in this study is adequate to detect significant differences in the oxidative DNA damage between cryopreserved groups and control group by using the 8OHdG as marker of oxidative DNA damage.

Clear advantages associated with immunohistochemical analysis include the facts that this method is relatively inexpensive, quick, highly sensitive and easy to perform, uses light microscopy, and is a well-established routine procedure in clinical laboratories (10). As well as, this method is appropriate for a remarkably wide range of applications. Any cell- or tissue-bound immunogenic molecule can, theoretically, be detected in situ using this technique. Immunohistochemical techniques detect antigens in tissues or in cells. Proteins (including immunoglobulins), carbohydrates, nucleic acids, lipids and other compounds can act as antigens (11).

In the present study, level of oxidative DNA damage (23.06%) as expressed by immunohistchemical assay was reported among healthy control mice. This is possible largely due to the process of spermatogenesis. Spermatogenesis is an extremely active replicative process capable of generating approximately 1,000 sperm a second (1). The high rates of cell division inherent in this process imply correspondingly high rates of mitochondrial oxygen consumption by the germinal epithelium. However, the poor vascularization of the testes means that oxygen tensions in this tissue are low1 and that competition for this vital element within the testes is extremely intense. Since both spermatogenesis" and Leydig cell steroidogenesis3''' are vulnerable to oxidative stress, the low oxygen tension that characterizes this tissue may be an important component of the mechanisms by which the testes protects itself from free radical-mediated damage. In addition, the testes contain an elaborate array of antioxidant enzymes and free radical scavengers to ensure that the twin spermatogenic and steroidogenic functions of this organ are not impacted by oxidative stress. Since, low levels of antioxidant during preparation of the testicular tissue of control group lead to increase in the levels of ROS and therefor resulting in oxidative stress causing oxidative DNA damage.

In addition to that, study done by (Magnus et al, 1999) showed that the 8-OHdG "which is a chemical modification of DNA caused by reactive oxygen species in the cell" seems to exist normally in the nuclear DNA at a level of -0.1-1 per 105 deoxyguanosines (dGs). Also, a study done by (Dai Nakae, et al. 2000)( 3780028a) revealed that the level of the 8-OHdG increase with age. They stated that the nuclear 8-OHdG levels were high (more than two lesions per 106 deoxyguanosines) from 3 to 52 weeks in the testis.

The increase in percentage sperm DNA fragmentation following cryopreservation has been well documented by many researchers (Thomson et al., 2009; Hammadeh et al., 1999; Donnelly et al., 2001; de Paula et al., 2006). Our study is to demonstrate an increase in the percentage of oxidative DNA damage as determined using 8OHdG as a biomarker of oxidative stress following cryopreservation.

In the current study, the central issue in the results of immunohistochemical assay showed highly significant increase of oxidative DNA damage (P<0.0001) in cryopreserved group (46.78%, 63.45% and 32.59% represent cryoprotectants glycerol, 1, 2 PrOH and DMSO; respectively) after six weeks of cryopreservation when compared with control group (19.27%).

This study suggests that cryopreservation provoke oxidative stress (generation of ROS overwhelms these antioxidant defenses) this aspect is in agreement with many researchers (Thomson et al., (121) and Zribi et al. ,hakeem,). Other groups have however, demonstrated an increase in ROS following cryopreservation / thawing cycle due to slow recovery of antioxidant activity. (Mazzilli et al., 1995; Wang et al., 1997, Calamera et al., (123)).

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In normal condition excessive production of ROS (superoxide, hydrogen peroxide and hydroxyl radical) that known as free radical; is limited by various regulatory systems, some enzymatic (catalase, superoxide dismutase, and glutathione reductase) and others, nonenzymatic (vitamin E, vitamin C, taurine, hypotaurine, and pyruvate) (124). It has since been postulated that the effects produced by ROS depend on their nature, quantity, length of exposure and on the time length of exposure (125) (126).

As well as attack from reactive oxygen species (ROS) on DNA is considered a major source of spontaneous damage to the DNA as well as other macromolecules such as proteins and lipids. There are various intra- and extracellular sources of oxygen radicals. The major intracellular source is believed to be electron leakage from the metabolism process in mitochondria but the main extracellular source ionizing and near-UV radiation (127).

Excessive production of ROS is potentially toxic to sperm quality and function (Saleh and Agarwal, 2002). This is because ROS are highly reactive oxidizing agents, among which are included hydrogen peroxide, superoxide and free radicals (Warren et al., 1987). Strong evidence suggests that high levels of ROS mediate the occurrence of high frequencies of single- and double-strand DNA breaks commonly observed in the spermatozoa (Fraga et al., 1996; Kodama et al., 1997; Sun et al., 1997; Aitken and Krausz, 2001). Furthermore, studies in which the sperm was exposed to artificially produced ROS resulted in a significant increase in DNA damage in the form of modification of all bases, production of base-free sites, deletions, frame shifts, DNA cross-links and chromosomal rearrangements (Twigg et al., 1998b; Duru et al., 2000).

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