Caused By Womens Problems Biology Essay


About 50 of infertility cases are related to mens problems and other 50 are caused by womens problems. In addition, anatomical abnormalities, endocrine disturbances, infections, psychological, immunological, environmental, and genetic factors play important roles in infertility(1, 2). Other important factor that has been studied extensively in recent years and can affect fertility is oxidative stress (OS). OS is a condition with excessive production of Reactive Oxygen Species (ROS) and antioxidant system failure (3). ROS include a wide range of molecules contain free radicals, non-radicals and oxygen derivatives (4). Physiological levels of ROS are necessary for normal sperm functions such as acrosome reaction, capacitation, sperm-oocyte interaction, implantation and embryo early development (5-7). About 40% of infertile men have increased amounts of ROS in their semen(8). All cellular components such as nucleic acids, sugars, proteins and lipids are potential targets of excessive production of ROS (9). Double bonds polyunsaturated fatty acid molecules in the sperm plasma membrane are vulnerable to free radicals attack leading to lipid peroxidation (10). During lipid peroxidation, free radicals attack to fatty acid chains to form lipid free radicals, which may react with molecular oxygen and generate the lipid peroxyl radical. Peroxyl radicals also can react with fatty acid chain, so reaction is continued (11). Lipid peroxidation reduces membrane integrity, causing increased cell permeability to electrolytes such as calcium and sodium ions that harmful to cell's energy forming mechanism. Protease and phospholipase activation occur due to increased intracellular calcium that can leads to further damage to proteins and lipids (12). It also shown that excessive ROS causes DNA fragmentation, which has a negative effect on fertility (12, 13). This damage occurs by changing in organic bases, creation of base-free sites, deletions, frame shifts, DNA cross-links, and chromosomal rearrangements (14). Continuous production of ROS also causes telomere shortening and reduced telomerase activity, which ultimately lead to cell senescence (15-17). DNA tight packaging is one of the major sperm defensive barrier against oxidative stress (18). During spermiogenesis phase, histones are replaced by protamines, which are rich in cysteine. During epididymal transition, disulphide bonds (S-S) are formed in protamine molecules, which are necessary for protection of sperm chromatin against acids, proteases, DNase and detergents (19, 20). Spermatozoa, also protected against oxidative stress by various antioxidants there are in seminal plasma and in spermatozoa (18, 21). Antioxidants inhibit, scavenge and suppress the formation of ROS or invert their actions (22). Some antioxidants are known as enzymatic (natural) antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR) which reduce hydrogen peroxides to water and alcohol. Other antioxidants are considered as non-enzymatic antioxidants also known as synthetic antioxidants or dietary supplements include vitamins (vitamin C, E,…) and minerals (zinc, taurine, glutathione,…) (23). Vitamin E (alpha-tocopherol) residing mainly on the cell membrane and inside the cell, interrupt with lipid peroxidation by scavenge the free radicals, so it is suggested as major chain breaking antioxidant(24). Saffron is the dried stigma of flowers of saffron (Crocus sativus L.) that belongs to Iridaceae family. Saffron is cultivated in Iran, Spain, Italy, Greece and India. Because of its color, taste and smell it is used as a food additive. Saffron has traditionally been considered as a medicinal plant. Saffron is used in folk medicine for various purposes such as antispasmodic, aphrodisiac (sexual stimulant) and expectorant. previous studies have demonstrated a wide variety of pharmacological effects of saffron and its active constituents such as curing nervous pains, insomnia, paroxysm, asthma, rheumatism, gingivitis, cough, gastric disorders, sleeplessness, uterus chronic hemorrhage, femininity disorders, scarlet fever, influenza, cardiovascular disorders, brain damages (25-31), sexual potential stimulant (32), anti-spasm (25, 33), anti-depression (34), sedative and anti-inflammation (28), memory power improvement, free radical scavenger, anti-tumor (35-37), blood pressure declining (26), anti-diabetic effects, blood's sugar and fat amount reduction (25, 27, 30, 38, 39), anti-mutation (40), the nucleic acid synthesis prevention in humans malignant cells (30, 35, 37).

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Due to increasing use of medicamental plants and considering that so far the effects of saffron as antioxidant on Sperm DNA structure and membrane integrity has not been investigated, we decided to design this study to examine the influence of saffron (Crocus sativus L. stigma) as an antioxidant and vitamin E as a positive control on DNA damage and membrane integrity in rat sperm. We used Acridin orange and CMA3 staining to evaluate DNA damage and HOS test to assess membrane integrity.

Materials and methods:

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Experimental animals:

This study was carried out on 15 mature (12 weeks old) male Wistar rats (180-200 g) obtained from Pasteur Institute, Tehran, Iran. Animals were housed in colony at 21±2°c temperature and under12 hours light and 12 hours dark conditions and with access to laboratory pellet chow and water at libitum. Rats were divided randomly to three following groups: saffron (n=10), vitamin E (n=10), and control group (n=10). Rats received saffron 100 mg/kg/day, vitamin E 100 mg/kg/day and distilled water 0/5cc /day via ora-gastric catheter, in saffron, vitamin E and control group respectively. Animals were acclimatized for one week prior to experimentation.

Saffron preparation:

Saffron purchased from Ghaen - Khorasan - Iran. 1000 mg grounded saffron was soaked and stirred in 20 mL warmed distilled water for at least two hours. The solution was stored at 4 °C until further use. This solution were used daily for treatment of saffron group rats (41) {Premkumar, 2003 #82}.

Vitamin E:

Vitamin E purchased from Sigma Chemical Co, St Louis, Mo. Previous studies showed that 100 mg vitamin E/kg body weights in rats were effective dose (42-44).

Epididymal sperm preparation:

After 60 days, rats first were anesthetized with chloroform and then cervical dislocation was done. Epididymis was removed immediately. The distal end of the cauda epididymis was separated and placed in petri dish contain 1 mL of prewarm normal saline (PBS). Several incisions were made in the tissue; sperm were allowed to swim out into the dish. The sperm suspension put in incubator for 10 min.

Rat Sperm DNA integrity:

Acridine orange staining:

We carried out this protocol according to Tejada et al (1984) with modification. After preparing a suitable concentration of sperm suspension, smears were prepared as for CMA3 staining and then fixed overnight in Carnoy's solution. Before staining each smear was incubated with acid-detergent solution pH 1.2 [0.08 N HCl, 0.15 M NaCl, 0.1% Triton-X 100], then was stained for 10 min with freshly prepared acridine orange (0.19 mg/mL) in citrate phosphate buffer (80 mL citric acid 0.1 M + 5 mL NaH2PO4 0.3 M, pH 2.5) Smears were evaluated on the same day with the aid of fluorescent microscope (نوع میکروسکوپ) (460-nm filter). The duration of illumination was limited to 40 second/field. The percentage of green (normal double stranded DNA) and orange/red (abnormally denatured DNA) fluorescence spermatozoa per sample were calculated (45) (Figure 1).

Chromomycin A3 (CMA3) staining:

CMA3 staining was performed based on Lolis et al (1996) with modification. After preparing a suitable concentration of sperm suspension, Smears were first prepared, air dried, then were fixed in Carnoy's solution (methanol/glacial acetic acid, 3:1) at 4ËšC for 10 min. Before staining each smear was incubated with acid-detergent solution pH 1.2 [0.08 N HCl, 0.15M NaCl, 0.1% Triton-X 100], then each slide was treated with 100 mL of CMA3 (Sigma, St Louis, MO, USA) (0.25 mg/mL in McIlvain buffer; 7 mL citric acid 0.1+ 32.9 mL Na2HPO4 7H2O 0.2 M, pH 7.0 containing 10 mM MgCl2) for 20 min. At the end, the slides were washed in buffer and mounted with buffered glycerol (1:1). Microscopic analysis of the slides was performed on a fluorescent microscope, with an appropriate filter (460-470 nm). Evaluation of CMA3 staining was done by distinguishing between bright yellow stained spermatozoa (CMA3 positive) and yellowish- green stained spermatozoa (CMA3 negative) (46) (Figure 1).

Hypo-osmotic swelling test (HOST):

To assess rat sperm plasma membrane integrity, the hypo-osmotic swelling test (HOST) was used. The hypo-osmotic solution (150 mosm/L) was prepared by dissolving 7.35g of sodium citrate dehydrate (C6H5Na3O7.2H2O) and 13.5 g fructose in 10 ml of distilled water. A 0/1 ml of hypo-osmotic solution was mixed with 0/1 ml of cauda epididymis sperm suspension and incubated at 37ËšC for 1 hour. 200 spermatozoa per each sperm were counted in different fields at X400 under phase contrast microscope (Nikon TE2000-U Japan) and HOST positive spermatozoa with coiled tails were determined (47) (Figure 1).

Statistical Analysis:

The Statistical Package for the Social Sciences (SPSS) 17.0 software was used to analyze data. All data are expressed as mean ± standard error (mean±S.E.) of the mean. Statistical analysis was performed using Independent sample t-test. Data were considered statistically significant when p < 0.05.


Acridine orange (AO) staining:

The mean percentage of AO-reacted spermatozoa (AO+) in control group is 26.98±2.46 that decrease to 6.81±1.39 in saffron groups, while the mean percentage of CMA3‏+ sperm cells in vitamin E group is 5.34±1.49. There are significant difference between control and experimental groups (P < 0.001), but no significant difference (P > 0.05) observed between saffron and vitamin E groups (Table1).

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Chromomycin A3 (CMA3) staining:

Significant differences in CMA3 staining are similar to AO staining. The mean percentage of CMA3‏+ sperm cells in control group was 8.16±0.54 that decreased to 2.51±0.64 and 1.98±0.74 in saffron and vitamin E groups respectively. There are significant difference between control and experimental groups (P < 0.001), but no significant difference (P > 0.05) observed between saffron and vitamin E groups (Table1).

Hypo-osmotic swelling test (HOST):

Saffron and vitamin E improve integrity of sperm cell membrane.

Treatment of spermatozoa with saffron and vitamin E resulted in significant improvement compared to the control group (P<0.001). The mean percentage of HOST+ spermatozoa in control group was 41.00±2.92 that decreased to 18.02±0.97and 9.53±0.80 in saffron and vitamin E groups respectively. Also there is significant difference (P<0.001) between vitamin E and saffron groups (Table 1).