The role of heat stress in sheep Sertoli cells and its effect on nitric oxide production and lipid peroxidation screening in vitro

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1 International Research Journal of Applied and Basic Sciences 2013 Available online at ISSN X / Vol, 6 (10): Science Explorer Publications The role of heat stress in sheep Sertoli cells and its effect on nitric oxide production and lipid peroxidation screening in vitro Forutan Salehi Nezhad *1, Abolghasem Lavvaf 1 and Shoukoh Karimi 2 1. Department of animal Science, Karaj Branch, Islamic Azad University, Karaj, Iran 2. Department of Agricultural science, Khozestan Branch, Islamic Azad University, Khozestan, Iran Corresponding author forutansalehi@yahoo.com ABSTRACT: In order to determine the effect of heat stress on the sheep's Sertoli cells under the specified condition, this study has been done to evaluate the role of heat stress on the sheep's Sertoli cells and its effect on production rate of Nitric Oxide and lipid peroxidation screening using the TUNEL method in Institute of Animal Embryo Technology, Shahrekord University. 9 testes of male sheep ranging 3 to 10 months were taken from the abattoir and the isolation and cell plant of Sertoli cells was carried on these samples,. In order to evaluate the effect of heat stress, Petri dishes containing Sertoli cells were divided into three categories, the first group was the control group, at the temperature of 32 C. The second group in which were under low heat stress for 6 h at 39 C and the third group were under severe heat stress of 42 C for 6 hours.four parameters related to oxidative under heat stress in control group and the groups under heat stress were performed as evaluating lipid peroxidation by measuring the concentration of malondialdehyde (TBARS), and measuring the amount of Nitric oxide in order to measure the total amount of Oxidation compound in Sertoli cells planting base. TBARS levels increased in higher temperature than the control group and difference is significant between the control group and the group of temperature 42 C (P < 0/05). However, differences between the control group and the group of 39 C was not significant. The result showed that antioxidant in the plant medium increased compared to control group, but this increase was not significant. On the other hand, heat stress increases SOD enzyme production levels in both groups, but this change was not significant compared to control. With increasing temperature amount of nitric oxide increases. More specifically, difference between the control group and the group of 42 C was significant (P < 0.05). Despite the increase it was determined that the level of nitric oxide in the group of 39 C was not significant compared to the control group. In the male gender gonads placed out of the abdomen, in the scrotum which is composed of skin and abdomen mucous membranes. Each testes is covered by a vaginal bag that is a part of peritoneum and testis is placed inside it and the vessels and the nerves reach to testis through that (Hafez, 2000). Testicular parenchyma tissue contains tubular sections called tiny tubules that has a round and long shape and has many complex spirals. This micro tubules contains a sets of reproductive cells that finally make up the male gametes. Key Words: Heat Stress, Oxidative Reaction, Nitric Oxide, Sheep's Sertoli Cells The internal coating or epithelium Micro tubules consists of growing reproductive cells and nurse or sertoli cells. Reproductive cells are undergoing divisions and changes regularly that results production of haploid cells called spermatozoa (Hafez, 2000). It is known that the temperature of testes should be lower than the body temperature to have proper function. The specifics of testes and the scrotum provide the proper temperature. The reactions which occur in heat receptors in the scrotum skin cause breathing and sweating and decreasing body temperature (Robertshaw and Vercoe, 1980). Sertoli cells are somatic cells that extends from the base to the tip of the tubular epithelium and directly contact with developing sexual cells in the process of spermatogenesis. Several studies have shown that these cells have an important role in the development of sexual cells (Russell et al., 1980) A large number of models have been created to study the effects of heat stress on testes. These models include exposing high temperatures (above 40 C) on the testes or placement the testes in abdomen area using surgical procedures and create cryptorchidism that makes the testes to be under body core temperature (37 C) for long-term. Both, methods cause changes including weight loss, increased apoptosis, and loss of sex

2 cells and change sperm fertilizing ability (Lue et al., 1999).Measurement of lipid peroxidation in the cell, is an important criteria to measure oxidative reaction. Oxidative reactions affect mainly cell membrane systems, in particular phospholipids of two-layer membrane. So, lipid peroxidation can be considered as lipid oxidative damage, including carbon double bonds. Body's anti oxidant defense system is including enzymatic and nonenzymatic defense system. In enzymatic defense system, enzymes like Reductase, Glutathione Peroxidase, Catalase, Superoxide Dismutase and some other enzymes participate. In the non-enzymatic systems, α- Tocopherol, Ascorbic Acid, Beta-Carotene and Selenium are involved. The damage caused by Ischemia reperfusion is the most common side effect occurs in the place of ischemia subsequent of oxidative retention of material and their sudden release (Xia et al., 2006).For a decade, it was thought the Endothelial vascular release a very unstable factorin which relaxes the adjacent smooth muscles. This substance has been known as of Endothelium- relaxing factor till But later it was found that this factor has the biological properties of Nitric Oxide and in fact is the same material and released from endothelial cells adequately and will participate in the control of vascular diameter (Li et al., 2009).Nitric oxide is a simple molecule with a single electron, which is known also as free radicals in which it makes the second Kinetics materials in the presence of oxygen, However, when Nitric Oxide levels are high, the oxidation will occurs. NO oxidation products are including Nitrite (NO2) in liquid solutions and Nitrate (NO3) is in the presence of oxy-hemoglobin (in blood). Nitric Oxide and its oxidation products including NO2 and NO3 are called Nox (Li et al., 2009). METHODS & MATERIALS This study shows the role of heat stress in sheep Sertoli cells and its effect on nitric oxide productionand lipid peroxidation screening in vitro. All stages was carried out in vitro in Institute of Animal Embryo Technology, Shahrekord University. Testicles of 9 male sheeps ranging 3 to 10-month-old prepared from Shahrekord abattoir located in Jonaqan and transferred to laboratory in vicinity of ice. After transferring the samples after 4-3 hours testis, washed and disinfected several times by 70% alcohol and then THE Tunica Alboujina layer was cut by a sterile scalpel blade. When required tissue was taken, testis was sliced with two scalpel within other sterile container. These pieces were transferred to a conical tube. If the sperm level is high in samples, it can be washed twice to reduce the sperm. Thus, the amount of PBS added to the tubes (5 ml), then centrifuged for a short time, so the sperm would seperate from the tube of deposition and suspended in PBS. PBS containing sperm was discarded and the tube contents were centrifuged again for 4 minutes by the speed of 400g. By the second centrifuge the remaining PBS was removed. Two-step enzymatic digestion: During this phase, first of Collagens 0/1% enzyme was added to the sample and incubated at 37 C for half an hour. For the best enzyme digestion, the enzymes with testis tissue samples was shaken every ten minutes. After this, the sample was taken out from the oven and was centrifuged for 4 min with the speed of 400g. When centrifuge finished, super liquid layer containing Leydig cells is digested by collagenase and were discarded. After this phase, the second phase of digestion began. In the next step, the enzyme containing 0/25% Trypsin and DNAse enzyme of 20 μl / ml respectively, were added. The sample containing these two enzymes was incubated for 20 min and was shaken once every ten minutes. After second enzymatic digestion, a sample enzyme was centrifuged for short time so that large pieces of tissue was deposited. At this stage, the culture super liquid layer was used to continue planting. The second set of enzymes are responsible for separating tubules.note that for deactivation of the second series of enzyme we use a medium containing 10% fetal bovine serum as the available calcium make the Trypsin ineffective. Samples were centrifuged for 4 min at the speed of 400g. The superliquid layer was discharged, and the deposited cells were used. Amount of 4/5 ml DMEM plant medium and 0/5 ml Serum was added to it to be smooth. The mixture was filtered through a diameter with 70 mm hole, then centrifuged at 60g for a minute. Again were centrifuged for 4 min at 400 g. The super liquid layer was discharged and the bottom cells were mixed with DMEM and 10% seru for the final plant. Then transferred to culture plates and was checked under the microscope on terms of the planting quality and then placed into an oven at 32 C. Nitric oxide assay using Griess The Serum Deproteinized Stage A) 250 micro liter zinc sulfate (mm75) was added to 300 micro liter of the sample B) 350 micro liter NaOH (mm 55) was then added to the above solution and has been vortexed then centrifuged at g for three minutes. C) The super layer of liquid was removed. D) 750 micro liters of super liquid layer with 350 micro liters of Bafer Glaysyn (ph = 9.7, 4.5 g /l) was added to the solution the third and then would be vortex.

3 Enabling Cadmium A) Two cadmium granules were chosen and washed three times with sterilized water. B) Cadmium was shaken with Copper Sulfate mm 5 and Bafer Glaysyn (ph = 9.7, 15 g /l) for 5 min. Nitrite Measuring A) 550 ml of the product of the first stage as well as two active cadmium were placed in one micro tubes and shaker for 10 min. B) 150 micro litter of 1% sulfanilamide was added to 350 micro liters of above sample and was placed in the dark for 5 minutes. C) 150 micro liter of NED (% 1/0) was added to the above solution and the optical absorbance read in spectrophotometer at 540 nm freq for ten minutes. Blank Preparation A) 750 micro liter of sterilized water was added to 350 micro liter Glycine Buffer and vortexed for short time. B) 150 micro liter of sulfanilamide was added to 350 micro liter of the above solution and placed in the dark for 5 min. C) 150 micro liter of NED was added to above solution in and then the spectrophotometer was set to zero. TBARS Test Preparation of Work Solution A) Hydrochloric acid solution 0.25 was prepared as normal. B) 0.375g of Tiobarbiotryk was added to hydrochloric acid. C) 15 g of Three-Chloro Acetic Acid was added to solution. D) Final volume was reached to 100 ml gradually using normal Hydrochloric acid TEST METHOD A) 200 micro liters of serum or sample with 800 micro liters of sterilized water were added to 2 micro liters of workingsolution. B) Blank Preparation; 1 ml of sterilized water was added to 2 ml of working solution. C) The Ben Mari was set on 100 C and samples were placed into 100 C bath for 15 minutes into the. D) Samples left to be cool at the room temperature and then were centrifuged for 10 min at 1000 rpm. E) The super layer of solution was taken and put into the Cote and optical absorbance was read against Blank at 535 nm. Statistical Calculations The amount of DNA damage and oxidative reactions in the control group, under low heat stress and extreme heat stress was investigated using Random Design method and statistical analysis "One way ANOVA" in SPSS statistical software. Gri ess Test to Measuring Nitric Oxide production Table 4-1 and Figure 4-5 shows the nitric oxide production in different temperature. As can be seen in Figure 4-5 and Table 4-1. Level of nitric oxide increases with increasing temperature. More specifically, there is a significant difference between the control group and the group of 42 C. However, despite of this increase, there is no significant difference in levels of nitric oxide between control group and the group of 39 C. Table 4-4 and Table 4-1 show the of SOD production in different temperature. As can be seen the production of this enzyme increased where there is h stress but this change was not significant compared to the control group. It is known that the temperature of testes should be lower than the body temperature to have proper function. Meanwhile, one on the major constituent cells of the testis are Sertoli cells which are developing in spermatogenesis process. Several studies have shown that these cells have an important role in the evolution of the sex cells (Russell, 1980). In an adult rat Sertoli cells from 17 to 19% of epithelial tubules (Wong and Russell, 1983). Sertoli cells have several roles that represent the role these cells in evolution of sex cells. Considering the important role of Sertoli cells in the sperm production in testes, cell damage can disturb the fertility in males. One of these harmful impacts, of heat stress. This study was has been done to investigate the effects of stress on Sertoli cells. Cell survival measuring is a very good criteria to evaluate the harmful impact of heat stress on cells. We have used Trepan blue color to study the effect of heat stress on the survival of Sertoli cells. 2939

4 It was found that the percentage of living Sertoli cells reduced as a result of heat stress, which shows the destructive effect of heat stress on Sertoli cells. Limited numbers of clinical studies that have been done in this regards on the Rams have confirmed these findings. In another study by Miost and colleagues (1992) found that increase in Rams scrotal temperature to 2/2 ºC for 16 hours per day and for 21 days based on every other day, causes considerable reduction the fetal survival rate from 17 to 65 days after inoculation compared with the control group. Tbars Test To Study Per Oxidation This test is method for lipid peroxidation screening and an important indicator to detect oxidative reactions. This test provides useful information about the production of free radicals and antioxidant activity of the mixtures. The results of these tests can be seen in Figure 4-2 and Table 4-1. As can be seen in Figure, TBARS level increases in the higher temperature than the control group. More specifically, there is significant difference between the control group and the group of 42 C (P <0.05) statistically. However, a there is not significant difference between the control group and the group of 39 C although the amount of TBARS was increased in this group compared to the control group. Table 1. The average ± standard error of the different test was evaluated in planted Sertoli cells in different tempratuers. 32 ºC 39 ºC 42 ºC Rate SOD ( IU/ml) 0.46 ±0.05a 0.64 ± 0.06ab 0.65 ± 0.06b Rate Nitrite ( M ) µ ± 2.84a 27.4 ± 3.84 a,b ± 3.7 b TBARS: Thiobarbituric Acid Reactive Substances The difference is the in each row indicate significant differences (P <0.05). Figure 1. Level of TBARS in plant of Sertoli cells sample in different temperature. Different letters indicate significant differences compared to the group of 32 C º (P <0.05).

5 Figure 2. Sample of Nitric Oxide production in Sertoli cells sample in different temperature. Different letters indicate significant differences compared to the group of 32 C º (05/0P <). REFERENCES Hafez ESE Reproduction in farm animals. Seventh ed. Lippincott Williams and Wilkins, Baltimore, MD. Li Y, Xian-zhong W, Meng-bo Y Effect of nitric oxide on Sertoli cell microtubule of piglets. Journal of Agricultural Biotechnology, 17: Lue YH, Hikim AP, Swerdloff RS, Im P, Taing KS, Bui T, Leung A, Wang C Single exposure to heat induces stage-specific germ cell apoptosis in rats: role of intratesticular testosterone on stage specificity. Endocrinology, 140: Mieusset R, Quintana Casares P, SanchezPartida LG, Sowerbutts SF, Zupp JL, Setchell BP Effects of heating the testes and epididymides of rams by scrotal insulation on fertility and embryonic mortality in ewes inseminated with frozen semen. J ReprodFertil, 94(2): Robertshaw D, Vercoe JE Scrotal thermoregulation of the bull (Bos sp). Aust J Agric Res, 31: 401. Russell LD, Sertoli-germ cell interrelations: a review. Gamete Res, 3: Wong V, Russell LD. 1983: Three-dimensional reconstruction of a rat stage V Sertoli cell. I. Methods, basic configuration, and di- mensions. Am J Anat 167: Xia T, Kovochich M, Brant J Comparison of the Abilities of Ambient and Manufactured Nanoparticles to Induce Cellular Toxicity According to an Oxidative Stress Paradigm. NanoLetter Journal, 6: