ENHANCEMENT OF FERTILIZATION BY PIPERINE IN HAMSTERS

Cell Biology International, 1997, Vol. 21, No. 7, 405–409

ENHANCEMENT OF FERTILIZATION BY PIPERINE IN HAMSTERS PAWINEE PIYACHATURAWAT* and CHUMPOL PHOLPRAMOOL Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Accepted 19 June 1997

The effect of piperine on the fertilization of eggs with sperm was investigated in female hamsters. They were intragastrically treated with piperine at doses of 50 and 100 mg/kg BW from day 1 through day 4 of the oestrous cycle. During piperine treatment, these females were superovulated and artificially inseminated (AI) with spermatozoa from untreated male hamsters at 12 h after hCG injection. The fertilization and growth of embryos were examined at various times after AI. In control hamsters, the percent fertilization increased with time, from 27.4&3.3% at 9 h after AI to 75.3&9.6 at 24 h after AI. Administration of piperine to the superovulated animals markedly enhanced the percent fertilization at 9 h after AI. It was increased to 85.4&4.1 and 82.8&4.8% by piperine at doses of 50 and 100 mg/kg BW, respectively. However, examination of the embryos retrieved 48 h after AI revealed no differences in the stage of embryonic development among different groups of animals. The possibility that this effect was due to the direct action of vanillic acid, a major piperine metabolite, was tested in vitro. Direct exposure of spermatozoa to vanillic acid at doses 25–100 mg% did not significantly affect their motility, percent acrosome reaction or fertilizing ability. This suggests that the enhancement of fertilization by piperine treatment was not related to the secretion of vanillic acid into the ? 1997 Academic Press Limited oviduct. K: enhanced fertilization; embryo development; piperine; vanillic acid

INTRODUCTION Piperine (1-piperoyl piperidine) is a major alkaloid of black pepper (Piper nigrum, family Piperaceae) which is used world-wide as a condiment, and as a major ingredient of indigenous medicine. This alkaloid has a wide spectrum of biological activity. It enhances activity of drugs by acting as a potent inhibitor of drug metabolizing enzymes (Atal et al., 1981, 1985). This compound suppresses convulsion by stimulating the release of 5-HT from the cerebral cortex (Mori et al., 1985) and catecholamine from the adrenal gland (Kawada et al., 1988). Piperine also has an antifertility activity in male mice and rats (Munshi and Roa, 1972; Piyachaturawat et al., 1991). It inhibits in vitro fertilization of sperm and eggs by decreasing calcium influx into sperm which leads to inhibition of the acrosome reaction (Piyachaturawat et al., 1991). In female rats, it inhibits implantation and To whom correspondence should be addressed: Pawinee Piyachaturawat, Ph.D., Department of Physiology, Faculty of Science, Mahidol University, Rama VI Rd, Payathai, Bangkok 10400, Thailand. 1065–6995/97/070405+05 $25.00/0/cb970167

causes abortion when administered at various times during gestation (Piyachaturawat et al., 1982). Since piperine consumption can impair fertility in both male and female animals, and there has been no report of the effect of piperine during the period of fertilization in females, the present study was undertaken to examine the effect of piperine on fertilization in vivo.

MATERIALS AND METHODS Animal treatment and artificial insemination Mature golden hamsters (8–12 weeks old) were maintained in a 12-h light/dark cycle (light on at 0600 h) and were provided with laboratory chow and tap water ad libitum. Female hamsters were divided into three groups of seven animals: control and piperine-treated groups at doses of 50 and 100 mg/kg BW, respectively. Piperine (Sigma Chem. Co. MO, U.S.A.) dissolved in 10% DMSO ? 1997 Academic Press Limited

406

and suspended in corn oil, was intragastrically administered from day 1 through day 4 of the oestrous cycle. Control animals were similarly treated except only vehicles were given. During piperine treatment, they were induced to superovulate by an intraperitoneal injection of 25–30 IU PMSG (Sigma Chem. Co., MO, U.S.A.) in the morning of the first day of the oestrous cycle (day 1). In the evening of the third day (day 3), 25–30 IU hCG (Sigma Chem. Co., MO, U.S.A.) was intraperitoneally injected between 2000–2200 h. Twelve hours after hCG injection each female was artificially inseminated (2–4 h prior to ovulation) with epididymal hamster sperm from untreated-males according to the method of Bavister (1973). One hundred microlitres of the cauda epididymal sperm suspension containing approximately 1.0#108 sperm/ml in modified Tyrode’s solution (Bavister, 1973) was injected into each uterine horn near the ovarian end. Eggs and embryos were collected from the oviduct at 9, 24 and 48 h after AI and were examined for the evidence of fertilization and for the development of embryos. An egg containing one or more swollen (decondensed) sperm-heads or male pronuclei was considered to be fertilized. Fertilization was expressed as percentage of eggs fertilized. Effect of vanillic acid on sperm capacitation and fertilization The effects of vanillic acid on the capacitating and fertilizing abilities of spermatozoa were tested and assessed as previously described (Piyachaturawat et al., 1991). Briefly, the cauda epididymal sperm sample was suspended in a mixture containing one part of modified Tyrode’s solution and one part heat-treated human serum (56)C for 1 h). The sperm suspension at a concentration of approximately 20#106 sperm/ml was incubated with 25– 100 mg% vanillic acid in a final volume of 200 ìl under paraffin oil for 5 h at 37)C. At 1-h intervals, small samples were removed from each dish for determination of percentage sperm motility and the percentage of acrosome-reacted sperm. The fertilizing ability of sperm was assessed by determining the percent fertilization with Zonaintact hamster eggs in vitro. Zona-intact eggs were obtained at 14–16 h after hCG injection, and 10–15 hamster eggs were inseminated with 10 ìl of the capacitated sperm which had been pre-incubated in the capacitation medium for 3 h. The concentration of sperm in the fertilization medium was 1.0#106 sperm/ml. To test the effect of vanillic

Cell Biology International, Vol. 21, No. 7, 1997

acid during fertilization, it was added at this stage in the same volume of 5 ìl to yield a final concentration of 25–50 mg%. The mixtures were then incubated at 37)C for 3 h. At the end of the incubation period the sperm were inactivated by the addition of 154 m sodium azide. The eggs were then washed and were examined under a phase contrast microscope (#400) for evidence of fertilization. Fertilization was expressed as percentage of eggs fertilized, and as a fertilization index (number of swollen sperm-heads/number of eggs). Statistical analysis All results were analysed for variance by using one way ANOVA. Significant differences between two groups of means were subsequently tested by the Student’s t-test. RESULTS Effect of piperine treatment on fertilization in vivo Figure 1 shows the effect of piperine treatment on the fertilization between hamster sperm and eggs at various times after AI. In the control group, fertilization progressively increased with time. The percentages of eggs fertilized at 9 and 24 h after AI were 27.4&3.3% and 75.3&9.6, respectively. Piperine treatment from day 1 through day 4 of the oestrous cycle markedly enhanced fertilization at 9 h after AI. It was significantly increased to 85.4&4.1 and 82.8&4.8% in the piperine-treated animals at doses of 50 and 100 mg/kg BW, respectively (P<0.05). After 9 h, percent fertilized eggs in the piperine-treated animals were not significantly changed but they remained significantly higher than those in the control group (Fig. 1). In addition, examinations of the embryos retrieved 48 h after AI revealed no differences in the stage of embryonic development among control and the two piperine-treated groups (Fig. 2). Most of the embryos were still at the one-cell stage. Effect of vanillic acid on sperm capacitation and fertilization Since vanillic acid is a major metabolite of piperine the possibility that vanillic acid is an active intermediate of piperine was tested. Table 1 shows the effect of direct exposure of vanillic acid on sperm capacitation and fertilization. In the control medium, the percentage of motile sperm was

Cell Biology International, Vol. 21, No. 7, 1997

407

120 *

Percentage fertilization

100 *

*

*

80

DISCUSSION

60

40

20

0

9h

24 h

Fig. 1. Effect of piperine on the percentage fertilization at various times after artificial insemination. Values are means& SEM obtained from 6–7 animals (200–250 ova/group). *P<0.05 Significantly different from control. , Control; , piperine (50 mg/kg); , piperine (100 mg/kg).

gradually decreased with time from 61.4&1.1% at the first hour of incubation (1 h) to 43.2&1.8% after 5 h. Addition of vanillic acid at concentrations of 25–100 mg% into the medium did not affect sperm motility observed at 1 h and 5 h (Table 1). Further, the acrosome reaction which began to appear after 2–3 h of incubation and become highly developed after 5 h was not altered by vanillic acid. After 3-h incubation of the capacitated sperm with eggs in the fertilization medium, the percentage of fertilized eggs was 85.1&5.5%. Inclusion of various doses of vanillic acid to either the capacitation or the fertilization medium did not significantly change percent fertilization. However, the 100

Embryo (% of total ova)

presence of vanillic acid in the capacitation medium tended to decrease the number of sperm which penetrated eggs (the fertilization index) whereas vanillic acid in the fertilization medium tended to increase the fertilization index (Table 1).

80

60

40

20

0

1-cell

2-cell

4-cell

Fig. 2. Effect of piperine on the embryo growth at 24 h after artificial insemination. Values are means&SEM obtained from 6–7 animals (200–250 embryos/group). , Control; , piperine (50 mg/kg); , piperine (100 mg/kg).

The results of the present study demonstrate the enhancing effect of piperine treatment on the early phase of in vivo fertilization. This finding is in contrast with our previous study which showed that piperine impaired in vitro fertilization by inhibiting calcium influx into sperm during capacitation (Piyachaturawat et al., 1991). The discrepancy is apparently due to differences in the methodology of piperine administration. In the present study, piperine was intragastrically administered to the females and the fertilization occurred in the oviduct whereas in the previous study fertilization occurred in vitro in which both eggs and sperm were directly exposed to piperine in a culture medium (Piyachaturawat et al., 1991). There are a number of possible mechanisms which might account for the effect of piperine on fertilization in this study. The enhancing effect might be due to (a) increased fertilizing ability of the spermatozoa and/or (b) increased number of spermatozoa available for fertilization in the oviduct. For mammalian spermatozoa, capacitation and acrosome reactions are known to be obligatory processes for fusion with oocytes. All these processes occur in the oviduct. In addition to being the site for fertilization, the oviduct also plays a very important role in sustaining embryo development. It has been demonstrated that secretion products from oviductal epithelium and mucosa transudates create a unique environment that is essential for sperm capacitation and for sustaining early embryo development (Smith and Yanagimachi, 1989; Hammar and Larsson-Cohn, 1978; Bavister, 1988). A number of substances have been found in the oviductal fluid including electrolytes, nutrients, enzymes, proteins and also embryotrophic factors (Lippes et al., 1981; Borland et al., 1980). Piperine, which has been reported to be rapidly absorbed and biotransformed after administration (Bhat and Chandrasekhara, 1986), as well as its metabolites might act on the oviduct and modify its luminal environment to enhance fertilization. One of its major metabolic products, vanillic acid (Bhat and Chandrasekhara, 1987), was evaluated in the present study. Direct exposure of spermatozoa to various concentrations of vanillic acid in the

408

Cell Biology International, Vol. 21, No. 7, 1997

Table 1. Effect of vanillic acid on motility, acrosome reaction and fertilizing ability of spermatozoa Parameters

Motility of sperm at 1 h (%)* Motility of sperm at 5 h (%)* Acrosome reaction (%)* Fertilization (%)* Fertilization index* Fertilization (%)† Fertilization index†

Vanillic acid (mg%) 0

25

50

100

61.4&1.1 43.2&1.8 64.0&5.0 85.1&5.5 3.5&0.6 86.2&4.8 3.4&0.7

64.6&1.9 46.1&2.8 72.1&3.1 87.4&5.9 3.2&0.9 92.2&3.2 4.2&1.0

62.2&2.4 46.8&2.1 71.7&4.1 88.7&6.6 3.0&0.5 94.3&4.3 4.4&0.9

62.2&2.2 44.1&2.5 66.9&5.1 84.2&6.8 2.0&0.3 95.5&3.3 5.8&1.3

Value is mean&SEM obtained from six animals. Vanillic acid was added into either the capacitation medium (*) or the fertilizing medium (†).

capacitation medium showed that this compound failed to alter sperm motility or the acrosome reaction. Similarly, addition of vanillic acid into the fertilizing medium had no effect, although some differences in the fertilization index were observed depending on whether vanillic acid was added to the capacitation or the fertilizing medium. The difference was probably due to a slight change in pH of the medium. However, it has been reported that pH lower than 7.2 is detrimental to the sperm activation and the acrosome reaction (Mahi and Yanagimachi, 1973). Thus the enhancing effect of piperine on fertilization is unlikely to be due to the secretion of the metabolite, vanillic acid, into the oviduct resulting in a reduction in pH. In addition, the possible changes in the oviductal factors for embryonic development are less likely since the embryos obtained from the piperine-treated animals were not in a more advanced stage than those of the controls. On the other hand, it is known that the circular smooth muscle at the isthmus of the oviduct is densely innervated by adrenergic fibers and relaxes upon administration of norepinephrine (Owman et al., 1976). The relaxation of this muscle allows more sperm to pass through and would facilitate fertilization at the ampulla since the isthmus serves as a filter for the transport of spermatozoa in the female reproductive tract (Smith et al., 1987). In the present study, it is suggested that the enhancement of in vivo fertilization by piperine occurred as a result of the relaxation of the isthmus circular smooth muscle allowing a higher number of spermatozoa at the fertilization site in the female oviduct. There are two lines of evidence in support of this hypothesis. Firstly, we (Piyachaturawat et al., 1982) and others (Takaki et al., 1990) have previously shown that piperine causes relaxation of

the uterine smooth muscle in mice and guinea-pig ileum, respectively. Secondly, piperine also induces a release of epinephrine (Kawada et al., 1988), which has been demonstrated to enhance fertilization (Bavister et al., 1976; Ball et al., 1983). However, these effects were not evaluated in our study. Further investigations on the number and fertilizing ability of sperm in different parts of the piperine-treated oviduct at various times after AI are required before the mechanism of piperine action can be clearly understood. In conclusion, the results indicate that female hamsters treated with piperine during the oestrous cycle had a higher percentage of fertilized eggs in the early phase of in vivo fertilization. Although the mechanism of action of piperine is not clear, this finding may be helpful in elucidating mechanisms of early fertilization in more detail. It will also be beneficial in searching for more effective culture media, as the current problem encountered in in vitro culture of preimplant embryos is the decrease in its viability, and the ability of embryos to develop over a long period in sub-optimal culture media (Bavister, 1988).

ACKNOWLEDGEMENTS The authors wish to thank Archan W. Sriwattana for his assistance in preparation of the manuscript. REFERENCES A CK, Z U, R PG, 1981. Scientific evidence on the role of Ayurvedic herbals on bioavailability of drugs. J Ethnopharmacol 4: 229–232.

Cell Biology International, Vol. 21, No. 7, 1997

A CK, D RK, S J, 1985. Biochemical basis of enhanced drug bioavailability of piperine: evidence that piperine is a potent inhibitor of drug metabolism. J Pharmacol Exp Ther 232: 258–262. B GD, L ML, L RW, A RL, B BD, F NL, 1983. Factors affecting successful in vitro fertilization of bovine follicular oocytes. Biol Reprod 28: 717–725. B BD, 1973. Capacitation of golden hamster spermatozoa during incubation in culture medium. J Reprod Fertil 35: 161–163. B BD, Y R, T RJ, 1976. Capacitation of human spermatozoa with adrenal gland extracts. Biol Reprod 14: 219–221. B BD, 1988. Role of oviductal secretions in embryonic growth in vivo and in vitro. Theriogenology 29: 143–154. B BG, C N, 1986. Studies on the metabolism of piperine: absorption, tissue distribution and excretion of urinary conjugates in rats. Toxicology 40: 83–92. B BG, C N, 1987. Metabolic disposition of piperine in the rats. Toxicology 44: 99–106. B RM, B JD, L CP, T ML, 1980. Element composition of fluid in the human fallopian tube. J Reprod Fertil 58: 479–482. H M, L-C U, 1978. Massive enlargement of occluded tubes after postmenopausal treatment with natural estrogens. Acta Obstet Gynecol Scand 57: 189–190. K T, S S, W T, Y M, I K, 1988. Some pungent principles of spices cause the adrenal medulla to secret catecholamine in anesthetized rats. Proc Soc Exp Biol Med 188: 229–233. L J, K J, A LA, D ED, L R, 1981. Human oviductal fluid proteins. Fertil Steril 36: 623–629. M CA, Y R, 1973. The effects of temperature, osmolarity and hydrogen ion concentration on the activa-

409

tion and acrosome reaction of golden hamster spermatozoa. J Reprod Fertil 35: 55–66. M A, K H, P YQ, 1985. Effect of piperine on convulsions and on brain serotonin and catecholamine levels in E1 mice. Neurochem Res 10: 1269–1275. M SR, R SS, 1972. Antifertility activity of an indigenous plant preparation (ROC-101). Effect on reproduction. Ind J Med Res 60: 1054–1060. O CL, F B, J EDB, R E, S NO, S B, S KG, W B, 1976. Autonomic nerves and related amine receptors mediating motor activity in the oviduct of monkey and man. Histochemical, chemical and pharmacological study. In: Harper MJK, Pauerstein CJ, Adam CE, Coutinho EM, Croxatto HB, Patton DM, eds. Ovum Transport and Fertility Regulation. Scriptor, Copenhagen, 256–275. P P, G T, P P, 1982. Postcoital antifertility effect of piperine. Contraception 26: 625–633. P P, S W, D P, P C, 1991. Effects of piperine on hamster sperm capacitation and fertilization in vitro. Int J Androl 14: 283–290. S TT, K F, Y R, 1987. Distribution and number of spermatozoa in the oviduct of the golden hamster after natural mating and artificial insemination. Biol Reprod 37: 225–234. S TT, Y R, 1989. Capacitation status of hamster spermatozoa in the oviduct at various times after mating. J Reprod Fertil 86: 255–261. T M, J JG, L YF, N S, 1990. Effect of piperine on the motility of the isolated guinea-pig ileum: comparison with capsaicin. Eur J Pharmacol 186: 71–77.