ABSTRACT
The study was to evaluate the testicular and epidydimal effects of Smart Herbal Purifier® (SHP) - a poly herbal supplement with a composition of Cassia alata 30 %, Morinda Lucida 30 %, and Nuclea Blend 40 % , in a 90 day repeated dose toxicity test in male Sprague-Dawley(SD) rats. The study was with control,48,240 and 480 mg/kg SHP treated rats. Sperm motility, debris generation, abnormal sperm morphology, sperm viability, testosterone, Follicular stimulating Hormone (FSH), luteinizing Hormone (LH), Prolactin, and malondialdehyde (MDA) levels as a measure of oxidative stress in the testes were analyzed. Histology of the testes was also examined. There was no significant difference in both Testicular Sperm Number (TSN), Epididymal sperm Number (ESN), the abnormal sperm morphology of both the control and SHP treated groups. A significant increase was observed in the debris generated in the testes and epididymis when control was compared to SHP treated groups. There was also a significant decrease in viability and motility in the testes and epididymis when control was compared to SHP treated group. Testosterone, FSH, LH and Prolactin were not significantly different when control was compared to SHP treated group. MDA was significantly increased when control was compared to the treated groups. Histology of the testes of the treated group showed necrosis of the seminiferous tubules. SHP may have toxicological concerns in the male reproductive system.
ABSTRACT
PURPOSE: Recently, in assisted reproductive technologies(ART) programs theme Is an increasing Interest in the use of agents for the enhancement of sperm motility for assisted fertilization. In an attempt to improve the motility at the cryopreserved human semen and hence the fertilizing capacity of asthenospermic semen samples, different semen preparation techniques have been attempted and the effects of chemical stimulants as nitric oxide(NO) have been studied extensively. Superoxide anions cause lipid peroxidative damage to cell membrane phospholipids, and sperm are known to be particularly susceptible to lipid peroxidation. Such sperm with damaged membranes are impaired functionally. Recently, peroxynitrite, an anion and a potent oxidant, generated by the interaction of nitric oxide and superoxide anions has been demonstrated In macrophages and other cellular systems. Also this anion cause lipid peroxidative damage to cell membrane phospholiplds. We therefore Investigated whether NO and peroxynitrite have the roles to modulate sperm motility and to affect Its viability. MATERIALS AND METHODS: Normal human semen samples(as per World health Organization (WHO) criteria) were obtained after 3day period of abstinence by donors. The samples(n=5) were incubated with either sodium nitroprusside(SNP; 0.1, 05, 1 or 2mM) or peroxynitrite (10, 50 or 100 micrometer ) and the percent viability and motility were assessed at various time inteval up to 4hr. The human semen samples were treated with N-acetyl-L-cystein(NAC;10mM), SNP (0.5mM), phorbol myristate acetate(PMA; 100nM), of SNF plus PMA. Both superoxide and peroxynitrite release were measured directly by chemiluminometer. Percent viability and motility were assessed at 4hr of Incubation. A sample of each aliquot was placed in a Mauler chamber for videomicrography Percent motility were analyzed by using the sperm analysis imaging system. The sperm vlability was assessed by flow cytometer using LIVE/DEAD sperm viability kIt. The production of superoxide and peroxynitrite were measured by the method of chemiluminescence assay. Result : All results represent a mean +/-SEM, n=5. Treatment of human semen samples for 4hr with SNP, a NO generating agent, significantly decreased sperm motility and viability in high concentration [relative motility(% of control); 38 +/-4 and 30 +/-5, relative viability; 42 +/-4 and 30 +/-3 by 1 and 2mM of SNP]. In the presence of low concentration SNP(0.5mM), the sperm viability was not significantly affected(82 +/-3), whereas the sperm motility was affected(64 2). SNP(0.5mM) also decreased sperm motility(80 +/-2 at 2hr 64 +/-3 at 4hr, 44 +/-3 at 6hr, and 38 +/-4 at 8hr) in a time dependent manner. Since it was demonstrated that superoxide anions are one of the common source of lipid peroxidation, we investigated whether superoxide anions produced by human semens could Interact wlth NO to generate peroxynitrite. Adding N-acetyl-L-cystein(NAC) to the human semen samples partially blocked spontaneous release of superoxide, whereas PMA augmented the release of superoxide from human semen samples (control:0.9 106 0.3, NAC: 0.5 106 +/-0.4, and PMA: 2.5 106 +/-0.4photons/60min). The production of superoxide was corresponded with the production of peroxynltrite(control: 1.0 104CPM, SNP: 3.8 106CPM, SNP plus PMA. 12chi106CPM). In addition, SNP in combination with PMA(65 +/-3) markedly decreased sperm motility than that of SNP alone(77 +/-2.5) at 4hr, implying that nitric oxide might inhibit sperm motility via the formation of peroxynitrite In human semen samples. Exogenous peroxynitrite also decreased sperm motility in a dose dependent manner(10 micrometer : 64 +/-2, 50rM: 53 +/-3, and 1 0 micrometer of peroxynitrite: 23 4). CONCLUSIONS: These results suggest that NO inhibits sperm motility via the formation of peroxynltrite and further demonstrate that NO-induced inhibition of sperm motility is depended on the production of superoxide from human semens because peroxynitrite is generated by the interaction of NO and superoxide.