Sperm membrane modulation by Sapindus mukorossi during sperm maturation
Manish Nivsarkar, Neeta Shrivastava, Manoj Patel, Harish Padh, Cherian Bapu
B.V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej-Gandhinagar Highway, Thaltej, Ahmedabad-380 054, Gujarat, India
Asian J Androl 2002 Sep; 4: 233-235
Keywords: Sapindus mukorossi; sperm membrane; contraceptive; sperm motility
Aim: To observe the alterations in the biochemical and biophysical changes in the sperm membrane during sperm maturation in male rats treated with the water extract of the fruit pericarp of S. mukorossi. Methods: Adult male Sprague-Dawley rats were gavaged the aqueous extract of the fruit pericarp of S. mukorossi at a dose of 50 mg/kg/d for 45 days. On day 46, the sperm parameters were observed in different sections of the epididymis and the sperm superoxide dismutase and the lipid peroxidation was determined and compared with the controls. The testis and epididymis were routinely prepared for histological examination under the light microscope. Results: No significant differences in the sperm number and morphology were observed between the control and treated groups. However, a significant inhibition (P<0.05-0.01) of sperm motility in the caput, corpus and cauda regions of the epididymis was seen in the treated group. No significant histopathological changes were found in the testis and epididymis. The important finding was that in the treated animals, the spermatozoa showed an abnormal distribution of the superoxide dismutase activity, being minimum in the caput and maximum in the corpus, which was just opposite to that of the controls. Conclusion: The study provides a unique observation where the plant extract alters the sperm membrane physiology without change the testicular and epididymal morphology.
Many plants have been known to possess antifertility activity, but limited attempts have been made to scientifically evaluate these claims . Sapindus mukorossi Gaerten (family sapindaceae), commonly known as Ritha or Aritha is found throughout India. The major chemical constituents of its fruit are saponins (10-11.5 %), sugars (10 %) and mucilages . The fruit of the plant is reported to have expectorant, emetic, alexipharmic and abortificiant effects. It is also used in excessive salivation, epilepsy and chlorosis [2,3]. Saponins from this plant are known to be spermicidal in vitro . This spermicidal property has been used in contraceptive cream . The alcoholic extract of Ritha (Sapindus trifoliatus Linn) is reported to possess anti-implantation activity .
Mammalian spermatozoa are known to possess both superoxide anion radical and superoxide dismutase activity and there is an increase in superoxide anion radical and a decrease in superoxide dismutase activity in the sperm membrane during its transit from caput to cauda . Crucial maturation-related changes in spermatozoa take place during their transit through the epididymis, which include alteration both in the head, tail, surface properties and constituents and acquisition of sustained forward motility . This paper reports alteration in the biochemical and biophysical levels in sperm membrane during sperm maturation when male rats are treated with water extract from fruit pericarp of S. mukorossi.
2 Materials and methods
Authentic samples of S. mukorossi fruit were obtained from authorized supplier. Fruit pericarp powder was extracted with double distilled water (overnight cold extraction). The extract was filtered and evaporated to dryness in a boiling water bath. The dried extract was stored in an air-tight container at 4-8 till further use.
2.2 Animals and treatment
Adult male Sprague-Dawley rats, aged 3-4 months, were used for the study. Animals were divided into two groups. Animals in group 1 (n=6) were gavaged 50 mg/kg/d of the extract for 45 days and animals in group 2 (control; n=6) received a similar amount of water for same duration. Animals were sacrificed on day 46 and the spermatozoa were collected from different parts of epididymis viz., caput, corpus and cauda by mincing and filtering through nylon screen. The sperm suspension was made up to 5 ml using freshly prepared Hanks Balanced Salt Solution (HBSS, pH 7.4). Sperm were counted and suspended in either HBSS (pH 7.4, 37) or Tris buffer (pH 8.2, 4) for further analysis. Superoxide dismutase activity assay was performed according to Marklund and Marklund  and the lipid peroxidation was measured in terms of malonaldehyde (MDA): thiobarbituric acid (TBA) reaction as reported by Okawa et al. . The testis and epididymis were routinely prepared for histological examination under the light microscope.
3.1 Testis and epididymis
After 45 days of treatment with S. mukorossi water extract no significant differences in the sperm number and morphology were observed between the control and treated groups. However, a significant inhibition (P< 0.05-0.01) of sperm motility in the caput, corpus and cauda regions of the epididymis was seen in the treated group (Table 1). No significant histopathological changes were found in the testis and epididymis.
Table 1. Percentage inhibition and the significant limit of sperm motility of treated spermatozoa compared to controls.
3.2 Superoxide dismutase and lipid peroxidation
Figure 1,2 show that the control spermatozoa showed an increase in lipid peroxidation and a reduction in superoxide dismutase activity during their passage from the caput to the cauda. However, the treated animals showed an abnormal superoxide dismutase activity which was minimum in the caput and maximum in the corpus. No significant changes were observed in the lipid peroxi-dation levels, where the MDA levels remained the same in all the three parts of epididymis.
Figure 1. Malonaldehyde levels in different parts of epididymis in control and treated animals. An increase in the levels of MDA levels was seen through caput to cauda in control animals however treated animals showed no changes in MDA levels.
Figure 2. Superoxide dismutase activity in the epididymis in control and treated animals. A normal decline was seen in control animals through caput to cauda, however, abnormal levels were seen in treated animals.
Reduction in sperm motility in the treated group motivated us to look for biochemical and biophysical levels in sperm membrane during maturation in different parts of epididymis.
Increase in superoxide anion radical and decrease in superoxide dismutase activity are prerequisites for sperm maturation as high levels of free radicals have been shown to be involved in the maintenance of membrane fluidity  and oxidative changes including an increase in the levels of unsaturated phospholipids of the sperm mem-brane, oxidation of thiol groups to disulphides and conversion of saturated phospholipids to unsaturated intermediates culminating the phospholipid bilayer of spermatozoa into a more fluid system, thereby facilitating the sperm membrane capable of sperm-egg interaction . Our results demonstrate the alterations in the superoxide anion radical and superoxide dismutase levels in different parts of epididymis. The most important observation is that no histopathological changes were observed in the testes and no morphological changes in the spermatozoa were noted. This suggests that the water extract may not have crossed the blood testis barrier but certainly affects the sperm maturation in the epididymis.
Antifertility effects have been shown by many plants, like extract of Andrographis paniculata, where after 48 days of dosing a decrease in sperm number and motility and increase in morphological abnormalities were seen in the spermatozoa. Minor histopathological changes were also observed . Ethanolic leaf extract of Colebroo-kia oppositifolia at a dose level of 100 and 200 mg/kg/d for 8-10 weeks did not cause any body weight loss, but significantly decreased the weights of testes and epididymis and notably reduced the sperm count . Petroleum ether extract of the leaves of Mentha arvensis showed antifertility effects in male mice when dosed orally for 60 days; A significant decrease in testicular weight and caudal sperm count was observed . Several other plants like Curcuma longa and Mondia whitei L., have been shown to possess such antifertility activities where reduction in sperm number and motility along with degenerative changes in testes have been reported [12-13].
The water extract of the fruit pericarp of Sapindus mukorossi does not show any antispermatogenic activity when given orally for 45 days, but alters the sperm membrane physiology. The exact mechanism could not be established by now, but a decrease in lipid peroxidation suggests an increase in the rigidity of the membrane and an abnormal superoxide dismutase level supports this hypothesis. Our results provide a unique observation where alteration in sperm membrane physiology has been observed with no histopathological or morphological changes. It is suggested that this fruit pericarp is worthy of further study to clarify its mechanism of action and to develop into a male contraceptive.
Authors are thankful to the Industries Commissiona-rate of Gujarat State, India for financial support towards instrumentation facilities.
Pandey G. Dravyaguna Vijanana; v I. Varanasi: Krishnadas Academy; 1998.
to: Dr. Neeta Shrivastava, B.V. Patel Pharmaceutical Education and Research
Development (PERD) Centre, Thaltej-Gandhinagar Highway, Thaltej, Ahmedabad
380 054, Gujarat, India.