Effects of Ovarian Varicose Vein on Mitochondrial Structure, Malondialdehyde and Prooxidants Antioxidants Balance in Rat Ovaries/Efectos de las Venas Ovaricas Varicosas sobre la Estructura Mitocondrial, Niveles de Malondialdehido y Balance Prooxidantes-antioxidantes en Ovarios de Ratas.
By definition a free radical is any atom with at least one unpaired electron in the outermost shell, which is capable of independent existence (Da Silva et al, 2010). They become stable by acquiring electrons from nucleic acids, lipids, proteins, carbohydrates or any close molecule causing a cascade of chain reactions resulting in cellular damage and disease (Agarwal et al, 2005).
Reactive oxygen species (ROS) are formed endogenously during aerobic metabolism and as a result of various metabolic pathways of oocytes and embryos or as part of the body's defense mechanisms (Sekhon et al, 2010). Oxidative stress occurs as a consequence of exceeding formation of ROS and impaired antioxidant defense systems (Sugino, 2005). ROS can affect a variety of physiological functions in the reproductive tract, and excessive levels may result in precipitous pathologies affecting female reproduction (Sekhon et al, 2010). Some amount of ROS is needed in the ovarian follicle and also for normal spermoocyte interaction and sperm capacitation. Although, raised levels of ROS have a harmful effect on cell membranes, cellular DNA, mitochondria and eventually accelerates cell death either by apoptosis or necrosis (Gupta et al, 2006). Several theories have been reported to clarify the correlation between varicocele and infertility (Ozturk et al, 2012). A variety of designations has been used to describe the presence of ovarian and pelvic varices, also known as pelvic varicocele (Gandini et al, 2008).
ROS cause oxidation of lipid membranes (Guzik et al, 2011). The lipid peroxidation product, malondialdehyde (MDA) is commonly used as a measure of the oxidative stress in cells (Pandey et al., 2012). The amount of MDA, which is formed from the breakdown of polyunsaturated fatty acids, perhaps taken as an indicator of peroxidation reaction (Motta et al., 2001). Along increased MDA, the balance of oxidants and antioxidants disturb so prooxidant-antioxidant balance (PAB) levels is expected to increase. The aim of this study was to evaluate the effects of varicocele induction on ovarian MDA and PAB levels.
MATERIAL AND METHOD
Adult wistar female rats (mean weight, 300 [+ or -] 55 g) were maintained under standard laboratory conditions. The rats were kept throughout the study on a 12h light-dark cycle (lights on at 07:00, 4OW), room temperature of 22 [+ or -] 2 [degrees]C. Two-month-old rats were randomly divided into 3 groups: Unilateral Varicose Vein (A), sham (B) and Control (C). Rats were weighted and anaesthetized with an i.p. injection of ketamine (100 mg/kg) and xylazine (1 mg/kg). The experimental left varicocele was induced in group A according to the method of Turner (2001). In brief, through a midline laparotomy incision, the upper left abdominal quadrant was approached. Left ovarian vein was dissected carefully medial to the insertion of the ovarian vein, and a 4-0 silk suture was tied around the ovarian vein over a 20-gauge needle. Then the needle was carefully removed, and approximately 50% reduction in the diameter of the left ovarian vein was achieved. In group B, rats underwent a similar procedure but without ligation of ovarian vein, and group C served as control. In 4 months, the animals were killed in proestrus phase. Ovariectomy was performed for examination.
The Stages of Estrus. Estrous cycles were monitored by daily vaginal smears, taken between 0800 and 1000 hr. Images are provided of unstained "wet" sample. The different cell population across the cycle were quantified and ratios determined to show trend between the predominant and other types in each stage of the estrus cycle (Hubscher et al., 2005) (Fig. 2). The rats were selected by proestrus stage of estrus cycle.
Electron microscope study: All rats were subjected to perfusion fixation under ether anesthesia on 60th day after the varicocele induction. Perfusion fixation was performed by immersion in 2.5% gluteraldehyde (Sigma, America) in 0.1 M phosphate buffer (Sigma, America) (pH 7.4) at room temperature for 4 hr and post-fixed in 1% phosphate-buffered osmium tetroxide (Sigma, America) for 2 h. Samples were dehydrated by being passed through the graded ethanol series and embedded in araldite-epoxy resin (TAAB, England). Semi-thin sections were stained with toluidine blue (Sigma, America). Ultrathin sections were contrast stained with uranyl acetate (Sigma, America) and lead citrate (Sigma, America) and examined using a transmission electron microscope (Leo 906 E) (Cheville & Stasko, 2014).
Malondialdehyde (MDA) Measurement Method: The
ovaries were eliminated quickly. Each ovary was weighed and tissue samples of 50 mg were frozen immediately in liquid nitrogen and stored at -80[degrees]C. The tissue sample was mixed with 2 volumes of cold 10% trichloroacetic acid (TCA) (mark, Germany) to precipitate protein. The precipitate is pelleted by centrifugation (3,000 g for 10 min), and 500 ml of the supernatant is reacted with an equal volume of 0.67% thiobarbituric acid (TBA) (Sigma, America) in a boiling water bath for 10 min. After cooling the absorbance is read at 532 nm (Esterbauer & Cheeseman, 1990). MDA assay was performed with a spectrophotometric assay. Data were expressed in nmol of MDA per ml of that tissue (Esterbauer & Cheeseman).
Determination-Prooxidants-Antioxidants Balance (PAB)
Systems: Homogenization. The ovaries were eliminated quickly. Each ovary was weighed and tissue samples of 40 mg were frozen immediately in liquid nitrogen and stored at -80 [degrees]C. The ovary fragments were homogenized using tissue homogenizer in 1.25 mL cold 0.1 M phosphate buffer, pH 7.4, including 1 mM ethylenediaminetetraacetic acid (EDTA) (Riedel-deHaen, America). The homogenate was centrifuged at 10,000 g for 15 min. Then the supernatant fraction was removed for the definition of hydroperoxide, and total antioxidant substances (Chuffa et al, 2011).
Prooxidants-Antioxidants-Balance (PAB) Method: Tetramethylbenzidine 3, 3', 5.5' (TMB) (Sigma, America) and TMB cations were used as oxidation-decrease index, due to their optical and electrochemical features. With This method, it is possible to measure the oxidant-antioxidant balance simultaneously in one experiment by two different reaction types: one enzymatic reaction where the chromogen TMB is oxidized to a color cation by peroxides and one chemical reaction where the TMB cation is reduced to a colorless compound by antioxidants. After that the photometric absorbency was compared with the given absorbencies of a series of standard solutions that are mixtures of uric acid and various proportions (0% to 100%) of hydrogen peroxide. They measured in an ELISA reader at 450 nm. PAB was arbitrarily expressed in HK units (Alamdari et al., 2009).
Statistical Analysis . All data are expressed as Means[+ or -]SD. Statistical analysis was performed by analysis Kruskal-Wallis test and the significance between two means was determined by Mann-Whitney post-hoc test. A value of p[less than or equal to] 0.05 was considered statistically significant.
After 60 days of varicocele induction, the mitochondrial structure of the ovarian tissue during proestrus is shown in Figure 3. Mitochondrial structure was denatured just in the left ovarian tissue.
The oxidative status of the ovarian tissue during proestrus is exhibited in Table I. The levels of the lipid peroxidation (MDA) content was significantly higher in A group (P[less than or equal to] 0.05) than C group (Fig. 4A) and the group A showed a significant increase in PAB levels comparison with the C group (P[less than or equal to] 0.05) (Fig. 5A). The results for Sham (B) and Control (C) groups did not differ significantly (P>0.05). Data from the concentration of PAB at right ovarian tissue are shown in Figure 5B. There was no significant difference between the groups A and C in PAB levels (P>0.05). However, the concentration of MDA significantly increased in the varicocele induction group (P[less than or equal to] 0.05) (Fig. 4B).
In this study, it was investigated whether denaturation of mitochondrial structure and increasing ROS occurs in ovarian tissue by varicocele induction or not. In addition, biochemical examination was performed in unilateral varicocele induction, sham and control groups. Mitochondrial structure and amounts of MDA and PAB were investigated in ovarian tissue of all the rats.
The present results demonstrated a significant increase in PAB levels at 2 months after varicocele induction in left ovarian tissue. MDA also increase within this period, which was significant and mitochondrial structure was damaged. In right ovarian tissue, there was a significant increase in MDA levels but the increase in PAB levels was not significant in comparison to the other groups. This could be due to antioxidant increases; the prooxidantantioxidant balance in the ovarian tissue is not affected significantly. Conversely, the above parameters of ovarian tissue at the right side, there were no significant alterations compared with the control group.
ROS induce lipid peroxidation with related influences in mitochondrial dysfunction (Da Silva et al) so we evaluated mitochondrial structure in follicle's cells and observed denature of mitochondrial structure in the left ovarian tissue.
We have applied a previously described animal model of male varicocele to female rats (Gokdeniz, 1999). Physiologically, ROS are increased in ovary after the preovulatory gonadotrophin surge (Yener et al., 2013) so, in order to the same conditions of ovaries; In the present study, we selected proestrus phase.
Ovarian varicosities were first described by Richet in 1857 (Venbrux et al., 2012). In 1991, Galkin et al., hypothesized that longlasting ovarian varicocele might cause hypo function of the ovaries and, similar to testicular varicocele, could be a cause of infertility (Galkin et al., 1991) but the significance of varicocele in female patients is not yet documented.
It has been experimentally shown that the severity of clinical changes is dependent on the efficiency of ROS scavenging (Krzysciak & Kozka, 2011), which additionally proves the participation of free radicals in the pathogenesis of varicocele induction, also increased ROS levels have been implicated in reduced fertility in patients with varicocele (Smith et al., 2006). We feel that there is no evidence in the literatures to date to state that oxidants play an important role as cause for infertility in female varicocele. Our study showed an increase in MDA and PAB after induction of varicocele, therefore infertility in varicocele patient may be caused by increased of these markers.
About male varicocele, reported that semen ROS levels correlated positively with varicocele grade. In addition, some researchers reported that men with varicocele grade II or III had significantly higher semen ROS levels than men with varicocele grade I (Cocuzza et al., 2008). According to a study performed in the field of classification for ovarian varices vein (Hiromura et al., 2004), since all the changes of our study are limited to the left side, subsequently after two months, varicocele had probably progressed to grade II.
Our data suggesting an enhanced oxidative stress in varicocele induction and suggests that damages by varicocele induction may have negative effects on the process of fertility in female rat so in addition to the removal of varicose veins, using antioxidants may reduce the effects of varicose veins that which require further investigation.
This work was supported by Tehran University of Medical Science.
Agarwal, A.; Gupta, S. & Sharma, R. K. Role of oxidative stress in female reproduction. Reprod. Biol. Endocrinol., 3:28, 2005.
Alamdari, D. H.; Ordoudi, S. A.; Nenadis, N.; Tsimidou, M. Z.; Koliakos, G.; Parizadeh, S. M. R.; Safarian, M.; Karimian, M. S. & Nobakht M., B. F. Gh. Comparison of prooxidantantioxidant balance method with crocin method for determination of total prooxidant-antioxidant capacity. Iran. J. Basic Med. Sci., 12(2) :93-9, 2009.
Cheville, N. F. & Stasko, J. Techniques in electron microscopy of animal tissue. Vet. Pathol., 51(1):28-41, 2014.
Chuffa, L. G.; Amorim, J. P.; Teixeira, G. R.; Mendes, L. O.; Fioruci, B. A.; Pinheiro, P. F.; Seiva, F. R.; Novelli, E. L.; Mello Junior, W.; Martinez, M. & Martinez, F. E. Long-term melatonin treatment reduces ovarian mass and enhances tissue antioxidant defenses during ovulation in the rat. Braz. J. Med. Biol. Res., 44(3):217-23, 2011.
Cocuzza, M.; Cocuzza, M. A.; Bragais, F. M. & Agarwal, A. The role of varicocele repair in the new era of assisted reproductive technology. Clinics (Sao Paulo), 63(3):395-404, 2008.
Da Silva, F.; Marques, A. & Chaveiro, A. Reactive oxygen species: a double-edged sword in reproduction. Open Vet. Sci. J., 4:127-33, 2010.
Esterbauer, H. & Cheeseman, K. H. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4 hydroxynonenal. Methods Enzymol., 186:407-21, 1990.
Galkin, E. V.; Grakova, L. S. & Naumova, E. B. Roentgenoendovascular surgery of hypofunctional ovaries in varicosities of the ovarian veins. Vestn.Rentgenol.Radiol., (5):51-9, 1991.
Gandini, R.; Chiocchi, M.; Konda, D.; Pampana, E.; Fabiano, S. & Simonetti, G. Transcatheter foam sclerotherapy of symptomatic female varicocele with sodium-tetradecyl-sulfate foam. Cardiovasc. Intervent. Radiol., 31(4):778-84, 2008.
Gokdeniz, R.; Ozbek, E.; Mizrak, B. & Ozen, S. Female varicocele: A rat model. Turgut Ozal Tip Merkezi Dergisi, 6(3):183-6, 1999.
Gupta, S.; Banerjee, J. & Agarwal, A. The impact of reactive oxygen species on early human embryos: A systematic review of the literature. Embryo Talk, 1:87-94, 2006.
Guzik, B.; Chwala, M.; Matusik, P.; Ludew, D.; Skiba, D.; Wilk, G.; Mrowiecki, W.; Batko, B.; Cencora, A.; Kapelak, B.; Sadowski, J.; Korbut, R. & Guzik, T. J. Mechanisms of increased vascular superoxide production in human varicose veins. Pol. Arch. Med. Wewn., 121(9):279-86, 2011.
Hiromura, T.; Nishioka, T.; Nishioka, S.; Ikeda, H. & Tomita, K. Reflux in the left ovarian vein: analysis of MDCT findings in asymptomatic women. A. J. R. Am. J. Roentgenol., 183(5):1411-5, 2004.
Hubscher, C. H.; Brooks, D. L. & Johnson, J. R. A quantitative method for assessing stages of the rat estrous cycle. Biotech. Histochem., 80(2):79-87, 2005.
Krzysciak, W. & Kozka, M. Generation of reactive oxygen species by a sufficient, insufficient and varicose vein wall. Acta Biochim. Pol, 58(1):89-94, 2011.
Motta, A.; Estevez, A.; Franchi, A.; Perez-Martinez, S.; Farina, M.; Ribeiro, M. L.; Lasserre, A. & Gimeno, M. F. Regulation of lipid peroxidation by nitric oxide and PGF2alpha during luteal regression in rats. Reproduction, 121(4):631-7, 2001.
Ozturk, M. I.; Koca, O.; Keles., M. O.; Yilmaz, S. & Karaman, M. I. Increased sperm DNA damage in experimental rat varicocele model and the beneficial effect of varicocelectomy. Int. J. Fertil. Steril., 6(2):95-100, 2012.
Pandey, M. K.; Mittra, P. & Maheshwari, P. K. The lipid peroxidation product as a marker of oxidative stress in epilepsy. J. Clin. Diagn. Res., 6(4):590-2, 2012.
Sekhon, L. H.; Gupta, S.; Kim, Y & Agarwal, A. Female infertility and antioxidants. Curr. Women Health Rev., 6(2):84-95, 2010.
Smith, R.; Kaune, H.; Parodi, D.; Madariaga, M.; Rios, R.; Morales, I. & Castro, A. Increased sperm DNA damage in patients with varicocele: relationship with seminal oxidative stress. Hum. Reprod, 21(4):986-93, 2006.
Sugino, N. Reactive oxygen species in ovarian physiology. Reprod. Med. Biol., 4(1,1:31-44, 2005.
Turner, T. T. The study of varicocele through the use of animal models. Hum. Reprod. Update, 7(1,1:78-84, 2001.
Venbrux, A. C.; Sharma, G. K.; Jackson, E. T.; Harper, A. P. & Hover, L. Pelvic Varices Embolization. In: Ignacio, E. A. & Venbrux, A. C. (Eds.). Women's Health in Interventional Radiology. New York, Springer, 2012. pp. 37-59.
Yener, N. A.; Sinanoglu, O.; Ilter, E.; Celik, A.; Sezgin, G.; Midi, A.; Deveci, U. & Aksungar, F. Effects of spirulina on cyclophosphamide-induced ovarian toxicity in rats: biochemical and histomorphometric evaluation of the ovary. Biochem. Res. Int., 2013:764262, 2013.
Department of Embryology
Faculty of Medicine
Tehran University of Medical Sciences
Leila Heydari *; Seyed Mohamad Hossein Noori Mugahi *; Simin Fazelipour **; Morteza Koruji ***; Rafieh Alizadeh ***; Melika izadpanah *; Niloufar Abbasi **** & Mehdi Abbasi ***
* Department of Embryology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
** Department of Anatomy, Faculty of Medicine, Azad University of Medical Sciences, Tehran, Iran.
*** Department of Anatomy, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
**** Department of medicine, Faculty of Medicine, Azad University of Medical Sciences, Tehran, Iran.
Caption: Fig 1. A, B: Distended ovarian vein in varicocele. C: Normal Ovarian Vein (V= Ovarian Vein, O= Ovary, K: Kidney).
Caption: Fig 2. Vaginal smear in proestrus phase. Arrow refers to epithelial cell (E) as proestrus phase marker (Round or oval cells with a nucleus in the center).
Caption: Fig 3. Semi-thin sections of the primordial follicle from the ovary of a rat. Arrows: Mitochondria. A, B: Denature of mitochondrial structure. C: Normal of mitochondrial structure.
Caption: Fig. 4. Biochemical analysis of the ovarian MDA levels (A: left side, B: right side). Values are expressed as Mean[+ or -]SD. Statistically significant difference between the groups (P<0.05). Statistically significant difference was found between the groups for MDA activity in Varicose Vein group (n= 5 in each group).
Caption: Fig. 5. Biochemical analysis of the ovarian PAB levels (A: left side, B: right side). Values are expressed as Mean[+ or -]SD. Statistically significant difference between the groups (P<0.05). Statistically significant difference was found between the groups for PAB activity in left side (n= 5 in each group).
Table I. Malondialdehyde (MDA) and Prooxidants-Antioxidants Balance (PAB) parameters of female rats after 60 days of varicocele induction. Groups MDA (ng/ml) PAB (UK unit) Left- varices 4.32 [+ or -] 0.87* 85.11 [+ or -] 34.80** Left- Control 1.55 [+ or -] 0.4 51.148 [+ or -] 2.21 Left-- Sham 2.47 [+ or -] 1.58 58.44333 [+ or -] 6.05 Right- varices 2.79 [+ or -] 0.15* 62.63333 [+ or -] 6.93 Right- Control 1.86 [+ or -] 0.4 54.48 [+ or -] 4.46 Right- Sham 2.08 [+ or -] 0.62 53.74333 [+ or -] 6.46 * shows significant increase in MDA levels compared with control and sham groups (P[less than or equal to] 0.05). **shows significant increase in PAB levels compared with control and sham groups (P[less than or equal to] 0.05).