Recovery of sertoli cells by Allium cepa in Toxoplasma gondii infected rats

Women’s Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Faculty of Medicine Tabriz Branch, Islamic Azad University, Tabriz, Iran. Department Of Radiology And Nuclear Medicine Keck School of Medicine UNIVERSITY OF Southern California Los Angeles, California –USA. Department of Histology, Science and Research Campus Branch, Islamic Azad University, Tehran, Iran.


INTRODUCTION
Toxoplasmosis is a parasitic disease caused by the protozoan Toxoplasma gondii (Ryan and Ray, 2004).The parasite affects most genera of warm-blooded animals, including humans, but the primary host is the felid (cat) family.Although, cats are often blamed for spreading toxoplasmosis, contacting with raw meat is a more significant source of human infections in many countries, and fecal contamination of hands is a greater risk factor (Torda, 2001).Up to one third of the world's human population is estimated to carry a Toxoplasma infection (Montoya and Liesenfeld, 2004).The centers for disease control and prevention noted that the overall seropreva-*Corresponding author.E-mail: arashkhaki@yahoo.com.lence in the United States, as determined with specimens collected by the National Health and Nutritional Examination Survey (NHANES) between 1999 and 2004, was found to be 10.8%, with 11% seroprevalence among women of childbearing age (15 to 44 years) (Jones et al., 2007).
Several conditions can interfere with spermatogenesis and reduce sperm quality and production.Factors such as drug treatment, chemotherapy, toxins, infections, air pollutions, insufficient vitamins intake, and parasites like T. gondii tachyzoites, have harmful effects on spermatogenesis and sperm's normal production (Mosher and Pratt, 1991;Santana et al., 2010).Several studies have reported that antioxidants and vitamins A, B, C, and E can protect sperm's DNA from free radicals, and also, increase barrier stability of blood testis (Jedlinska- krakowska et 2006).Evidence suggests that Allium cepa (onion juice) has antioxidative and androgenic effects in rats, and can promote spermatogenesis cycle (Khaki et al., 2009).Antioxidants protect DNA and other important molecules from oxidation and damage, improve sperm quality, and consequently, increase fertility rate in men (Yang et al., 2006).Therefore, the role of nutritional and biochemical factors in reproduction and sub-fertility treatment is very important.The present study was planned to assess the ability of A. cepa to promote sertoli cells parameters and testosterone concentration in T. gondii infected rats.The results obtained will provide further insights into appropriate treatment of infertile male patients using herbals to improve spermatogenesis.

Preparation of onion juice
The underground yellowish-white bulbs of A. cepa (onion) were collected in August 2007 from Ilkhchi in the province of East Azerbaijan-Iran.Before the experiments, the skin was removed and fresh juice of onions was prepared using a Tefal fruit juice extracting machine.

Analysis of onion juice
The onion juice was tested to determine flavonoids using the Shinoda test (Yousef, 2005).Qualitative thin-layer chromatography (TLC) was employed for determining quercetin as a main flavonoid in onion.For TLC, 10 mL of fresh onion juice were dried in a vacuum and the resulting residue dissolved in 1 mL of methanol.20 mL of methanolic solution were spotted on a silica gel plate (10 × 20 cm, silica gel 60 GF254, Merck, Darmstadt, Germany) with a solvent system of EtOAc/MeOH (80:20).Quercetin, Sigma chemical Co. (St.Louis, MO, USA), was used as a control.After developing and drying, the TLC plate was sprayed with a 2% AlCl solution in methanol.Quercetin in the onion samples appeared as a yellow spot at RF = 0.6.Separation of quercetin was performed with further purification by preparative TLC on silica gel; quantitative determination of quercetin carried out on a Model 2100 Spectrophotometer (Shimadzu, Japan) in 370 nm comparing to a pure quercetin standard curve.The amount of quercetin in fresh onion was found to be 12 mg/100 g (Khaki etal., 2009).

T. gondii infection
T. gondii strain RH was maintained by passage in mice every 2 days.Tachyzoites were collected from the peritoneal cavity of infected mice and used to inoculate rats.The rats were intraperitoneally injected with 107 tachyzoites of T. gondii at the Department of Veterinary Pathology, Islamic Azad University, Tabriz Branch-Iran (Berdoy et al., 2000).

Experimental animals
Adult Wistar albino male rats (n=40) were included in the present study.The rats were 8 weeks old and weighing 250±10 g.They were obtained from animal facility of Pasture Institute of Iran.Male rats were housed in rooms with controlled temperature (25°C), constant humidity (40-70%), and 12h/12h light/ dark cycles prior to experimental protocols.All animals were treated in accordance with the Principles of Laboratory Animal Care [NIH].All rats were fed with a standard diet and water.The daily intake of water was monitored at least one week prior to the start of treatments in order to determine the amount of water needed fire very experimental animal.Thereafter, the rats were randomly divided into control (n=10) and experimental (n=30) groups.The control group just received 4cc distilled water daily.However, the experimental infected rats (n =20) were split into two infected T. gondii groups; one of these groups was T. gondii test group (n=10) and the other was T. gondii group (n=10) received 1cc of fresh onion juice daily.The fourth experimental group (n=10) received 1cc of fresh onion juice daily (Khaki et al., 2009) this group was onion test group.At the end of the study, the rats were killed by carbon dioxide.

Surgical procedure
the thirtieth day, the Pentobarbital sodium (40 mg/kg) was administered intra peritoneal for anesthesia, and the peritoneal cavity was opened through a lower transverse abdominal incision.Then, testisin control and experimental groups was immediately removed.The weights of testis in each group were registered.The animals were decapitated between 9:00 AM and 11:00 AM, and blood samples were obtained.Blood samples were centrifuged at 4°C for 10 min at 250 g, and the serum obtained was stored at −20°C until the time it was assayed.

analysis
To compare data in control and experimental groups, ANOVA test was applied.The results were expressed as mean ± S.E.M (standard error of measurements).Significant differences are written in parentheses.

Weight of individual male testis
The obtained results in this study are illustrated in Table 1.There was significant difference in testes weights between T. gondii groups, as compared to the other groups (p<0.05).

Results of testosterone levels
Levels of testosterone were significantly increased in fresh onion juice group comparing to control and T. gondii groups (p<0.05).This result is higher in infected rats with T. gondii that received 1cc fresh onion juice, as compared to T.gondii group.

DISCUSSION
T. gondii infection is associated with a wide spectrum of clinical pictures in men.Onion and garlic contain a wide variety of phytochemicals and micro constituents, such as trace elements, vitamins, fructans, flavonoids and sulphur compounds, which may have a protective effect against free radicals.The present results clearly indicate that A. cepa (onion) has a good effect on spermatogenesis in rats.Our results showed that administration of onion juice (1 g/rat/day) for 20 consecutive days caused a marked increase in sperm number, viability, and mobility, as compared to respective controls; this agrees with our previous research (Khaki et al., 2009).These effects could be related to vitamins, vitamin C, and flavonoids of onion such as quercetin.Oxidative damage was as certained by measuring malondialdehyde levels, reactive oxygen species (ROS) generation, alterations in antioxidant defences, and the extent of protein oxidation.Quercetin, an important flavonoid, has a beneficial effect on health due to its antioxidant function.Studies on the effect of quercetin on oxidative damage in cultured chicken spermatogonial cells showed that quercetin has no deleterious effect on spermatogonial cells at doses of 1 and 10 mg/mL.Quercetin (1 mg/mL) increased the number of spermatogonial cells and decreased the mortality of Aroclor-induced oxidative damage.In this study, the effect of quercetin on serum MDA was determined, but the results indicated no obvious effect of quercetin on MDA production (Mi and Zhang, 2005;Mi et al., 2007).In the present study, T. gondii significantly reduced sperm amount and mobility; on the other hand, our research showed that onion fresh juice can enhance both the number of sperm and mobility in group of animals infected with T. gondii.These results are in agreement with other finding.They showed that Toxoplasma infection was related to infertility, so it was possibly related to the antisperm antibodies being involved in the pathogenesis of infertility (Zhou et al., 2002;Aral et al., 2011).In their study regarding to mice, Sun et al. (2008) reached to the same results.These researchers found out that acute T. gondii infection affects the reproductive function of male mice.Khaki et al. 3 T. gondii infections have the ability to change the behavior of rats and mice, making them drawn to, rather than fearful of, the scent of cats.This effect is advantageous to the parasite, which will be able to sexually reproduce if its host is eaten by a cat (Berdoy et al., 2000).The infection is highly precise, as it does not affect a rat's other fears such as the fear of open spaces or of unfamiliar smells.Studies have also shown behavioral changes in humans, including slower reaction times and a six fold increased risk of traffic accidents among infected males (Flegr et al., 2002), as well as links to schizophrenia, such as hallucinations and reckless behavior.

Conclusion
In our study, T. gondii had a significant effect on sperm parameters and serum total testosterones.On the other hand, freshly prepared onion juice significantly affected the sperm number, percentage of viability, and mobility.Onion juice can both reduce and treat this malevolent effect, so it is suggested that eating of onion is useful in infected men.

Table 1 .
The effect of 1cc fresh onion juice /rat on sperm parameters, testosterone, apopetosis, and testis weight of control and T. gondii groups.