Effects of a purified fraction from Echeveria gibbiflora aqueous crude extract on guinea‐pig spermatozoa

PHYTOTHERAPY RESEARCH Phytother. Res. 13, 46–49 (1999)

Effects of a Purified Fraction from Echeveria gibbiflora Aqueous Crude Extract on Guinea-pig Spermatozoa Ne´stor M. Delgado,1 Javier Taboada Ramı´rez,2* Alfredo Ortega Herna´ndez,2 Horacio Merchant-Larios,3 Marı´a Luisa Sa´nchez-Va´zquez´,1 Guillermo Ramı´rez1 and Rosalina Reyes1 1 Divisio´n de Biologı´a de la Reproduccio´n. Centro de Investigacio´n Biome´dica del Sur, IMSS. Argentia No 1, Xochitepec, Morelos, Mexico C.P. 62790 2 Instituto de Quı´mica, Universidad Nacional Auto´noma de Me´xico, Circuito Exterior, C.P. 04510, Me´xico, D.F. Mexico 3 Instituto de Investigaciones Biome´dicas, Universidad Nacional Auto´noma de Me´xico, Ciudad Universitaria. Me´xico, D.F. Mexico. C.P. 04518

Guinea-pig spermatozoa in the presence of a purified fraction from Echeveria gibbiflora aqueous crude extract suffer a hypotonic-like effect. The phenomena exhibited included a distension of the plasma membrane over the acrosome region, inducing the formation of a huge ‘head-bubble’. The agglutination effect was so enhanced that instead of inducing sperm clusters, it produced cane-like ‘stalk’ structures. The immobilizing activity was induced instantaneously after the addition of the purified fraction. At electron microscope level it was possible to observe a heavy amount of electron dense material of the purified fraction embedded or intercalated along the plasma membrane. It was also possible to corroborate the dispersion of the acrosomal content and the disappearance of the external acrosome membrane. The purified fraction induced loosening of the plasma membrane all along the sperm cell, however, the distension of the membrane was only produced in the apical portion of the sperm head and not in the post equatorial region. The results suggest that the plant may yield a compound suitable for use as a vaginal barrier or male contraceptive agent. # 1999 John Wiley & Sons, Ltd. Keywords: hyperosmotic; acrosome; agglutination; immobilization

INTRODUCTION

The world population is currently expanding at a striking rate of nearly one billion per decade. This dramatic increase calls for new and improved methods of fertility regulation, including the development of male contraceptives. During the second half of the 20th century, progress in developing novel, practical contraceptive methods for men has lagged significantly behind developments for women. Male contraceptive agents should ideally be safe and effective without impairing libidow potency, and have a low failure rate and few adverse effects. Ideally, such agents should be easy to use, readily available and inexpensive. We have obtained encouraging results using the aqueous crude extract of a plant from the Crassulaceae family named Echeveria gibbiflora, a well-known plant in Me´xico, which is popularly used as a vaginal postcoital rinse without any reports of adverse side effects. Agglutination and immobilization effects in seven different species of mammalian spermatozoa, including human sperm have been shown in preliminary experiments (Huacuja et al., 1985). * Correspondence to: J. Taboada Ramı´rez, Instituto de Quı´mica, Universidad Nacional Auto´noma de Me´xico. Circuito Exterior, C.P. 04510, Me´xico, D.F. Me´xico

CCC 0951–418X/99/010046–04 $17.50 # 1999 John Wiley & Sons, Ltd.

Although both effects are irreversible, the viability and oxygen uptake after 3 h of incubation time were the same as in the control, showing no spermaticidal effect. When male D-1 mice received 1.25 mg/kg of OBACE i.d., every 4 days over 40 days, spermatogenesis showed large modifications. Two days after the first doses, at testicular level it was observed that sperm heads were swollen and in practically all of them the tail was separated from the head; and spermatogonias, spermatocytes and spermatid nuclei showed diffuse chromatin (Ma´rquez-Orozco et al., 1985). After six doses, the epithelium of seminiferous tubules became thin, and all the sperm tails were seen to be in the tubule (Ma´rquez-Orozco et al., 1986). Therefore, to extend the chemical and pharmacological investigation of Echeveria gibbiflora, the purified fractions were evaluated for their effects on guinea-pig spermatozoa.

MATERIAL AND METHODS Plant material and extraction. Plant specimens were collected in different batches at several locations in the Me´xico valley. Voucher specimens documenting these collections have been deposited in the IMSSM Herbarium under the code number 12213. Echeveria gibbiflora aqueous crude extract was prepared by grinding 300 g of fresh plant leaves to give 250 mL of aqueous crude Accepted 20 June 1998

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intact. To each separate aliquot of 5  107 sperm cells, 50, 100, 150 and 200 mL of purified fraction was added plus salt medium to give a total volume of 0.5 mL. The suspensions were incubated at 37°C and aliquots evaluated at preestablished times. The viability and agglutination pattern of the studied sperm cells were measured using techniques previously described (Jones and Senft, 1985; Toullet and Voisin, 1974). Electron microscopy. Treated spermatozoa were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 1 h (Zetterqvist, 1956). The samples were then washed for 20 min with 0.1M cacodylate buffer alone and post-fixed in 1% Os04 for 2 h. This was followed by dehydration in ascending ethanol concentrations and embedding in Epon (Luft, 1981). Thin sections were stained with uranyl acetate and lead citrate (Reynolds, 1963) and observed using the electron microscope.

RESULTS AND DISCUSSION

Figure 1. Phase contrast micrographs of guinea-pig spermatozoa treated with the puri®ed fraction of Echeveria gibbi¯ora aqueous crude extract. (A) Distenstion of the plasma membrane over the acrosome region, inducing a huge `head-bubble'. v, head-bubble; a, acrosomeless sperm; c, cytoplasmic droplet. 400. (B) Aspect of the agglutination pattern of guinea-pig spermatozoa in cane-like `stalk' structures. 200

extract which was filtered through gauze and centrifuged at 3000 rpm for 10 min. In order to get rid of ions that are extremely deleterious to sperm cells, the supernatant was dialysed against two changes of deionized water (20 L each). The active fraction remained inside the dialysis bag and it was lyophilized (Huacuja et al., 1985) for pharmacological and other studies. Animals. Mature guinea-pigs were killed by cervical dislocation, testes were excised and the epididymis removed and repeatedly washed in salt medium. Spermatozoa were obtained from the cauda epididymal region by the procedure described by Koehler et al., (1983). The collected spermatozoa were washed by gentle centrifugation (600 g for 10 min) in the following salt medium 113 mM KCl, 12.5 mM KH2PO4, 2.5 mM K2HPO4, 2.5 mM MgCl2, and 20 mM Tris, all adjusted to pH 7.4 (Keyhani and Storey, 1973). To reduce intersample variability, pools of sperm cells (obtained from several animals) were used for each experiment. These were washed by centrifugation, resuspended, counted and diluted if necessary to obtain a final concentration of 108 spermatozoa/0.1 mL. Bioassay procedure. In order to avoid the acrosome separating from the sperm head (acrosomeless), all sample manipulations were done with extreme care, to ensure at least 50% to 75% of sperm cells remained # 1999 John Wiley & Sons, Ltd.

One of the striking results was the unusual behaviour of guinea-pig sperm cells in acting as a sensitive hypotoniclike cell model in the presence of the purified fraction obtained from the aqueous crude extract of Echeveria gibbiflora. These effects include distension of the plasma membrane over the acrosome region, inducing the formation of a huge ‘head-bubble’ (Fig. 1A). This hypotonic-like effect was produced exclusively by the purified fraction and not by any of the other reagents employed in the incubation mixture. With phase contrast microscopy it was possible to observe a Brownian movement inside the ‘head-bubble’ of the particles of the acrosome content, an observation that indirectly confirms the lack of pores or holes along the plasma membrane of the ‘head-vesicle’, With respect to the agglutination effect, it was not possible to define the agglutination pattern because the activity was so enhanced that the sperm cells formed cane-like ‘stalk’ structures (Fig. 1B), instead of the sperm clusters already reported (Huacuja et al., 1985). Thin sections taken from guinea-pig spermatozoa treated with the purified fraction, under optimal experimental conditions, revealed no morphological changes in the tail, middle piece and nucleus (Fig. 2A) of the spermatozoon. Meanwhile at the sperm head level the purified fraction induced the formation of a huge bubble, by the distension of the plasma membrane (Fig. 2B). It is also possible to corroborate the dispersion of the acrosome content with the disappearance of the external acrosome membrane, leaving the nuclear and the inner acrosome membranes (Fig. 2B,C). Moreover, it is clearly seen that the distension of the plasma membrane was produced only above the acrosome region and not below the equatorial region of the sperm head (Fig. 2B,C). We may be able to explain this by a close interrelationship between the protein matrix and/or reticular membranouslike system, with the plasma membrane of the post equatorial region (Reyes et al., 1985). This interrelationship appears to be species specific, since in the rat spermatozoa no ‘head-bubble’ or membrane distension was produced, at least on phase contrast microscopy (data not shown). Phytother. Res. 13, 46–49 (1999)

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Figure 2. Electron microphotographs of thin sections of guinea-pig spermatozoa after treatment with the puri®ed fraction of Echeveria gibbi¯ora aqueous crude extract. (A) Control. Sperm heads appear highly condensed, when incubated for up to 72 h in the salt medium alone. a, acrosome; n, nucleus; Bar = 500 nm. (B) Distension effect of the plasma membrane (pm), over the acrosome region, inducing a huge `head-bubble' (Hv). Puri®ed fraction discloses a heavy amount of electron dense material embedded along the plasma membrane. am, acrosome material; n, nucleus. Bar = 1 mm (C) High magni®cation micrograph showing the dispersion of the acrosomal material (am), the presence of the inner acrosome membrane (iam) and the nuclear membrane (nm). It is also possible to see the heavy amount of electron dense material embedded along the plasma membrane (pm). The sperm nucleus (n) appears highly condensed. Bar = 500 nm. (D) Transverse section of the middle piece (mp) of the sperm cell, where the separation of the plasma membrane (pm) and the deposit of electron dense material embedded in the plasma membrane are clearly shown. Bar = 500 nm

These electron microphotographs show that the purified fraction results in deposition of electron dense material along the plasma membrane, making it impossible to observe its characteristic lamellar organization. These deposits suggest that the purified fraction itself might be embedded or intercalated in the plasma membrane (Figs. 2B,C,D), as well as causing a

# 1999 John Wiley & Sons, Ltd.

loosening of the plasma membrane along the cell (Figs. 2B,C). The increased activity of the purified fraction compared with the aqueous crude extract is large, making, it possible that in the future, this fraction might be useful as a possible vaginal barrier contraceptive or as a male contraceptive agent.

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ratoÂn. XXVIII Congreso Nacional de Ciencias FisioloÂgicas. Puebla, Pue. MeÂxico. 11±16 de Agosto. MaÂrquez-Orozco, M. C., MaÂrquez-Orozco, A., TaboadaRamõÂrez, J. et al. (1986). Cambios histoloÂgicos del testõÂculo de ratones, producidos por el extracto de una crasulaÂcea. XI Congreso Nacional de FarmacologõÂa. Taxco, Gro. MeÂxico. 2±6 de Marzo. Reyes, R., Magdaleno, V. M., Villalpando-Fierro, I., Merchant, H., and Delgado, N. M. (1985). Tubuline and microtubules-like structures after inducing sperm acrosome reaction with glycosamineglycan-sulfate. Adv. Contracept. Deliv. Syst. Monograph 1 150±157. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol. 17 208±212. Toullet, F., and Voisin, G. A. (1974). Spermatotoxic, spermagglutinating and cytotoxic activities of guinea-pig autoantibodies to sperm autoantigen. J. Reprod. Fertil. 37 299±313. Zetterqvist, H. (1956). The Ultrastructural Organization of the Columnar Absorbing Cells of the Mouse Jejunum. Thesis, Karolinska Institute, Stockholm.

Phytother. Res. 13, 46–49 (1999)