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Prof. Yi-Fei WANG,

Chloroform extract of Carica papaya seeds induces long-term reversible azoospermia in langur monkey

N. K. Lohiya, B. Manivannan, P. K. Mishra, N. Pathak, S. Sriram, S. S. Bhande, S. Panneerdoss

Reproductive Physiology Section, Department of Zoology, University of Rajasthan, Jaipur 302 004, India

Asian J Androl  2002 Mar; 4:  17-26              

Keywords: Carica papaya; fertility; azoospermia; langur monkey

Abstract

Aim: To evaluate the antifertility activity of the chloroform extract of Carica papaya seeds by oral administration in langur monkey, Presbytis enellus entellus. Methods: The chloroform extract of Carica papaya seeds, 50 mg/kg/day, was administered orally for 360 days to adult male langur monkeys. The sperm characteristics by light and electron microscopy, the sperm functional tests, the semen biochemistry, the serum testosterone level, the Leydig cell function, and the histology and ultrastructure of testis were determined to evaluate the antifertility activity and the blood biochemistry and hematology, to evaluate the toxicology. Results: The extract gradually decreased the sperm concentration since days 30-60 of treatment with a total inhibition of sperm motility, a decrease in sperm viability and increase in sperm abnormality. Azoospermia was observed after day 90 of treatment and continued during the whole treatment period. Treatment withdrawal resulted in a gradual recovery in these parameters and 150 days later they reverted to nearly the pretreatment values. Morphological observation of the ejaculated sperm by light and scanning electron microscopy showed deleterious changes, particularly on the mid-piece. Sperm functional tests, viz., sperm mitochondrial activity index, acrosome intactness test and hypo-osmotic swelling test scored in the infertile range during treatment and returned to the fertile values 150 days after drug withdrawal. Histology of the testis revealed shrunken tubules, germ cell atrophy and normal Leydig cells. Ultrastructure of the testis showed vacuolization in the cytoplasm of Sertoli cells and germ cells. Loss of cytoplasmic organelles were evident in the spermatocytes and spermatids. Round spermatids showed loss of Golgi bodies, peripheral mitochondria and vacuolated cytoplasm, indicating maturational arrest. Leydig cell functional test indicated a mild inhibition of steroidogenic function. Haematology and serum biochemistry study disclosed no significant toxicological effect and the serum testosterone level was not affected. Conclusion: Carica papaya seed extract may selectively act on the developing germ cells, possibly mediated via Sertoli cells, leading to azoospermia.

1 Introduction

The contraceptive efficacy, reversibility and toxicity of the Carica papaya seed products have been investigated in rats and rabbits [1]. Oral administration of the aqueous, methanol, ethanol, ethyl acetate and chloroform extracts in rats revealed reversible contraceptive efficacy, but only the aqueous and chloroform extracts were without significant toxicity and impairment in libido [2]. Partial purification of the chloroform extract through silica gel column chromatography, eluted with benzene, chloroform and ethyl acetate in the order of polarity, showed total motility inhibition of cauda epididymal spermatozoa in rats with benzene and chloroform fractions [3,4]. Methanol and ethyl acetate sub-fractions of the benzene chromatographic fraction yielded similar results [1].

In rabbits, treatment with chloroform extract, benzene chromatographic fraction of the chloroform extract and its methanol and ethyl acetate sub-fractions leads to azoospermia, while aqueous extract and the chloroform and ethyl acetate chromatographic fractions of the chloroform extract were ineffective, thus showing differential effect among species [1].

Owing to the proven contraceptive efficacy of chloroform extract of the Carica papaya seeds and its further purified products in rats and rabbits, a pre-clinical investigation has been initiated in langur monkey, a non-human primate close to the human anatomically [5] and in its reproductive exocrine and endocrine profiles [6,7], with a view to extrapolate the findings to the human. The present investigation reports the contraceptive efficacy, reversibility and toxicity, if any, of the chloroform extract of the seeds of Carica papaya.

2 Materials and methods

Eight adult male langur monkeys (Presbytis entellus entellus Dufresne), 6-7 years old, as identified by the musculature, sex skin in the rump and dentition [5] were procured from places around Jaipur and kept in individual metallic cages in the Department Primate House Facility. The animals were fed with roasted wheat cakes, seasonal vegetables and fruits and tap water was provided ad libitum. Routine pathological tests and semen analysis were carried out to assess the health and reproductive status of the animals and only healthy animals were selected for the investigation. The experiments were conducted in accordance with accepted humane practices as approved by the Departmental Research/Ethical Committee. Complete veterinary care and supervision were provided to the animals throughout the course of the investigation. The "guidelines for care and use of animals for scientific research" [8] were strictly followed.

2.2 Test materials

The seeds of Carica papaya Linn. (Caricaceae; Voucher No. RUBL 16590) of honey dew variety, were obtained commercially, shade dried and coarsely pow-dered. The powdered material was soxhleted with chloroform at 58℃ for 12×3 h. The soxhleted material was concentrated under reduced pressure and the residue was used in the investigation.

Three pretreatment semen samples were collected at 10-day intervals for routine analysis and semen biochemistry. Blood samples were collected from the great saphenous vein for haematology and serum biochemistry and testosterone level determination.

Five animals were orally fed with the chloroform extract of the Carica papaya seeds, 50 mg/kg/day for 360 days, along with vegetables. Care was taken to ensure that the animal consumed the entire dose. Three animals served as control.

Following completion of the treatment, semen analysis was carried out at regular intervals to assess the recovery pattern.

Semen samples were collected every 10 days before treatment and every 15 days during and after treatment by penile electrostimulation for the following analyses:

Semen volume, ejaculation time, pH, colour, consistency, and sperm concentration, motility, viability and morphology were assessed according to the WHO manual [9].

Spermatozoa were washed with phosphate buffer (0.01 mol/L, pH 7.2) and pelleted by centrifugation. The sperm pellets were fixed in 2.5% glutaraldehyde for 30 min and washed thrice in phosphate buffer followed by distilled water. A thin film of spermatozoa was smeared on a clean glass slide, air dried and mounted on SEM stub with silver paint, sputter coated with gold and observed under SEM (Leo 435 VP).

Washed spermatozoa were used for assessment of the acrosome intactness test [10], mitochondrial activity index test [11] and hypoosmotic swelling test [12]. Scores below 50% in the acrosome intactness and mitochondrial activity index tests and below 60% in the hypo-osmotic swelling test were considered subfertile or infertile [13].

Sperm free seminal plasma was used for the quantitative determination of fructose and acid phosphatase (ACP) [14], glycerophosphocholine (GPC) [15], lactate dehydrogenase (LDH) [16], protein, calcium and alkaline phosphatase (ALP) (Reagent kits; Ranbaxy Laboratories Ltd, Mumbai).

Testicular biopsy was performed after completion of 360 days study period. For histology, the tissues were fixed in Bouin's solution, dehydrated in ethanol and embedded in paraffin wax. The sections cut at 5 mm were stained with haematoxylin and eosin.

The remaining portion of the testis was fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO₄, dehydrated in acetone and embedded in araldite for ultrastructural studies. The ultrathin sections were stained with uranyl acetate and lead citrate and viewed under Philips transmission electron microscope (CM-10).

The response of Leydig cells to 3β-hydroxysteroid dehydrogenase (3β-HSD) and the hCG-stimulated testosterone biosynthesis in vitro have been used as functional indicators for Leydig cells. Crude Leydig cell population was obtained by non-enzymatic dispersion and percoll density gradient centrifugation [17]. Purification of the Leydig cells was confirmed by 3β-HSD staining method. To 100 µL suspension of Leydig cell fraction, equivalent to 50,000 viable Leydig cells as identified by trypan blue staining, hCG 50 µL (mg/mL) was added and cultured for 6 h at 37℃ in a humidified atmosphere. Following completion of the culture, the spent media obtained by centrifugation was used for testosterone assay by RIA using NIDDK kits (Bethesda, USA), along with a parallel control without hCG. The sensitivity of the assay was 0.01 ng/mL.

Blood samples were collected monthly and used for the analyses of red blood corpuscles (RBC), white blood corpuscles (WBC), haemoglobin (Hb), haematocrit (packed cell volume, PCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) [18-20].

Serum glucose, protein, cholesterol, glutamate pyruvate transaminase (SGPT), glutamate oxalate transaminase (SGOT), lactate dehydrogenase (LDH), creatinine and creatine kinase (CK) were estimated colorimetrically using reagent kits (Ranbaxy Laboratories Ltd., Mumbai).

Serum testosterone levels were assayed from frozen samples by RIA using NIDDK kits (Bethesda, USA). The sensitivity of the assay was 0.01 ng/mL.

Data were expressed in mean±SEM. Student's 't' test was employed for statistical comparison. P<0.05 was considered significant

3 Results

Carica papaya did not significantly change the ejaculation time, semen volume, colour, consistency and pH of semen. Sperm concentration showed a gradual decline and azoospermia resulted in all treated monkeys after 90 days treatment that continued over the one year study period. On day 60 there was a total inhibition of sperm motility and a concomitant decrease in sperm viability; the per cent abnormal spermatozoa showed a gradual increase.

Following withdrawal of treatment, azoospermia continued for 30 days, severe oligospermia with sperm count less than 20 million/mL was observed up to day 60; a little later the sperm motility and viability increased gradually with a simultaneous increase in motility and viability and a decrease in per cent abnormal spermatozoa. At day 150 all these parameters, although at slightly lower levels, were comparable to pretreatment values (Tables 1 a,b,c,d).

Table 1a. Sperm concentration (10⁶/mL) of langur monkeys after treatment with chloroform extract of Carica papaya seeds. Mean±SEM, ^cP < 0.01, compared with controls.

Table 1b. Sperm motility (%) of langur monkeys after treatment with chloroform extract of Carica papaya seeds. Mean±SEM, ^cP < 0.01, compared with controls.

Table 1c. Sperm viability of langur monkeys after treatment with chloroform extract of Carica papaya seeds. Mean±SEM, ^cP <0.01, compared with controls.

Table 1d. Abnormal spermatozoa (%) of langur monkeys after treatment with chloroform extract of Carica papaya seeds. Mean±SEM, ^cP < 0.01, compared with controls.