Ursolic Acid Generates Symplasts in Rat Spermatogenic Clones M. A. Akbarsha,  M. Palanisamy, P. Murugaian and P. N. Lakshmi Latha Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli-620 024, India Treatment of ursolic acid to Wistar strain male albino rats resulted in severe disruption of spermatogenesis. The most diagnostic change in the seminiferous epithelium was the opening up of the intercellular bridges between the male germ cell clones, resulting in the formation of symplasts. In this property, ursolic acid is comparable to cytochalasin D. Symplasts are exfoliated from the Sertoli cell. The Leydig cells are hypertrophied. Cauda epididymidal sperm motility was impaired, and several sperm exhibited abnormalities. Among the epididymal epithelial cell types, the clear cells of the caput as well as the cauda appeared to be increased in abundance and were rounded-up. The results indicate that from the male reproductive toxicological point of view, caution is required in using ursolic acid as a curative/protective agent. However, the testicular and epididymal effects of ursolic acid may be applied in male antifertility/contraception. © 1997 John Wiley & Sons, Ltd. Phytother. Res. 12, 32–36 (1998) Keywords: ursolic acid; spermatogenic clones; symplasts; epididymal clear cells; ventral prostate. INTRODUCTION Ursolic acid is a triterpene isolated from the leaves of several plants such as Arctostaphylus uva-ursi, Vaccinium macrocarpon, Rhododendron hymenanthes etc., (Budavari, 1983). This compound has been reported to inhibit arachi- donic acid metabolism in mouse peritoneal macrophages, human platelets and differentiated leukaemic cells; it has also been shown to exhibit lipoxygenase and cyclo- oxygenase inhibition, reflecting antiinflammatory character- istics (Najid et al., 1992). It markedly decreases the liver injury caused by hepatotoxicants like carbon tetrachloride, acetaminophen and cadmium chloride; it also increases hepatic metalothyanin levels 48-fold, and thus protects the liver from injury (Liu et al., 1994). Interestingly, this compound has been reported to inhibit tumorigenesis (Simon et al., 1992; Huang et al., 1994). Theoretically, inhibition of tumorigenesis would mean arrest of cell proliferation. Spermatogenesis involves extensive cell pro- liferation (Russell et al., 1995). Therefore, hypothetically, ursolic acid would arrest spermatogenesis and, hence, could be applied in male antifertility. In order to control population growth, several devices/ drugs are being tested in the female as well as male systems, but none so far has become totally acceptable (von Hertzen, 1994; Waites, 1994); the current thinking in this regard is to find a male contraceptive which would either inhibit spermatogenesis or the post-testicular mechanisms of sperm maturation, without affecting the steroidogenic machinery and the libido (Nieschlag et al., 1992; Coyaji, 1994; Waites, 1994). It is believed that such a contraceptive could be found from plants (Kamboj and Dhavan, 1989; Akbarsha et al., 1995). Whether ursolic acid would be one such phytochemical was tested in the present study and the paper reports some positive results. MATERIALS AND METHODS Ursolic acid (Sigma, USA) was quantitatively dissolved in a minimum quantity of ethanol and, then diluted in physiological saline (0.9% NaCl). Wistar strain male albino rats (3 months old) were administered ursolic acid intra- peritoneally at a daily dose of 5 mg/kg body weight in 0.5 mL of the vehicle for 15 days. Control rats received saline. Each group consisted of 10 rats. Rats were fed on pellet feed (Gold Mohur Animal Feeds, Lipton India Ltd., Bangalore, India) and water ad libitum. At the end of the experiment, rats were killed by decapitation and slices of testis, epididymis, ventral prostate and seminal vesicles were fixed in Bouin-Hollande fixative, embedded in paraffin and serially sectioned at 3 m thickness in a Leica (Germany) microtome; sections were stained in Ehrlich haematoxylin and eosin (Humason, 1979) and observed under a Leitz diaplan microscope (Leica, Germany). Cauda epididymidal sperm were suspended in phosphate-buffered saline; a hanging drop preparation was used to observe sperm motility, and Giemsa’s stained smears were used to observe sperm morphology. The experiment was repeated once. RESULTS In the testis of the control rat the seminiferous tubules were normal, and the epithelium was intact (Fig. 1) In the ursolic acid treated rat, in general, deep in the seminiferous epithelium there were spherical masses separated out from the rest of the epithelium and the Sertoli cell (Fig. 2). Such masses contained several nuclei (up to 72) in a common  Correspondence to: M. A. Akbarsha, Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, India. Contract grant sponsor: University Grants Commission, Government of India. CCC 0951–418X/98/010032–05 $17.50 Accepted 22 July 1997 © 1998 John Wiley & Sons, Ltd. PHYTOTHERAPY RESEARCH, VOL. 12, 32–36 (1998)