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 123 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 3-hydroxysteroid dehydrogenase (3-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 3-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 meanSEM. 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. MeanSEM, ^cP < 0.01, compared with controls.

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

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

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

After 30-60 days of treatment the spermatozoa (Papanicolaou stain) showed deleterious changes, particularly in the mid-piece. Typical changes included bent mid-piece and coiled tail. At day 120 post-treatment, the sperm morphology reverted to the pretreatment level (Figure 1).

Figure 1. Sperm morphology of langur monkeys: <A> before treatment: normal spermatozoa; <B> after 30 days of treatment with chloroform extract of Carica papaya seeds: most spermatozoa showing bent mid piece and coiled tail; <C> after 60 days of treatment: a drastic reduction in number of spermatozoa, all spermatozoa showing bent mid piece; <D> after 120 days of treatment withdrawal: number and morphology of spermatozoa comparable to pretreatment level. Papanicolaou staining 400.

The pretreatment spermatozoa showed an oval head with distinct acrosomal and post-acrosomal region. The mid-piece was thick, encircled by spiral mitochondrial sheath. The annulus appeared as a constriction at the tail, which separated the mid-piece from the tail (Figure 2A). The spermatozoa of treated animals were well correlated with light microscopic studies, with bent mid-piece and coiled tail. In a few spermatozoa, head abnormalities were also evident, showing amorphous head and acrosome deformities (Figure 2B).

Figure 2. SEM study on sperm morphology of langur monkeys: <A> before treatment: normal morphology. 4000; <B> after 60 days of treatment with chloroform extract of Carica papaya seeds. 2000.

In all the sperm function tests, the first few ejaculates after treatment scored in the infertile range, which persisted in the whole treatment phase and early post-treatment phase. At 150 days post-treatment, they reached fertile scores comparable to the pretreatment values (Tables 2 a,b,c).

Table 2a. Sperm mitochondrial activity index test (%) in langur monkey after treatment with chloroform extract of Carica papaya seeds. Mean SEM, ^cP < 0.01, compared with controls.

Table 2b. Acrosome intactness test (%) in langur monkey after treatment with chloroform extract of Carica papaya seeds. MeanSEM, ^cP < 0.01, compared with controls.

Table 2c. Hypo-osmotic swelling test (%) in langur monkey after treatment with chloroform extract of Carica papaya seeds. Mean SEM, ^cP < 0.01, compared with controls.

The levels of fructose, ACP, GPC, LDH, protein, calcium and ALP in the seminal plasma were not significantly changed in the treatment animals, however, there was a tendency of reduction during the treatment phase (data not shown).

3.2 Histology of testis

The testis of control animals showed evidence of normal spermatogenesis with prominent Sertoli cells containing granular cytoplasm, spermatogonia, spermatocytes and spermatids and their differentiation into spermatozoa (Figure 3A).

After completion of treatment, the testis revealed shrunken tubules. Spermatogonia appeared to be normal. Although spermatocyte differentiation was observed, the cells were atrophic with nuclear and cytoplasmic vacuolization. There were only a few round spermatids which were atrophic. Leydig cells appeared normal (Figure 3B).

Figure 3. Testicular histology of langur monkey: <A> control: normal spermatogenesis; <B> treated with chloroform extract of Carica papaya seeds for 360 days: Tubules shrunken, showing disorganised germinal cells. Germ cell differentiation diminished at the level of spermatocytes. Leydig cells normal. 100.

3.2.2 Ultrastructure

In control animals, the ultrastructure of the Sertoli cells and germ cells showed characteristic nuclear and cytoplasmic features (Figure 4).

Figure 4. Testicular ultrastructure of control langur monkey: <A> showing normal Sertoli cell (Ser). Nucleus with deep indentation, Golgi bodies (G), mitochondria (M), smooth and rough endoplasmic reticulum (Rer), secretory granules (S) and coated vesicles (C) are well defined. Spermatids (Sp) with condensed nucleus, granular cytoplasm and peripheral mitochondria seen closely associated with Sertoli cells. 5,600; <B> showing primary spermatocyte. Nucleus containing condensed chromatin material. Cytoplasm granular with well defined mitochondria. 5,000. <C> showing spermatid at Golgi phase. Nucleus round with head cap. Golgi vesicles (Gv) concentrated around head cap. Mitochondria at the peripheral region. 4,600.

After completion of treatment, vacuolization was significant in the cytoplasm of Sertoli cells and germ cells. The Sertoli cells were ill defined with nucleus containing pale chromatin network and loss of cytoplasmic organ-elles. There were plenty of lipid droplets and few secretory granules. Loss of cytoplasmic organelles were also evident in the spermatocytes and spermatids; particularly in round spermatids the Golgi bodies and the peripheral mitochondria were virtually absent and the entire cytoplasm was vacuolated. Membrane damage was also evident (Figure 5).

Figure 5. Testicular ultrastructure of langur monkey after treatment with chloroform extract of Carica papaya seeds for 360 days: <A> showing Sertoli cell. Nucleus containing relatively pale chromatin network. Cytoplasm showing vacuolization. Secretory granules (S) few and mitochondria (M) vacuolated. 5.600. <B> showing primary spermatocyte. Nucleus pyknotic, cytoplasmic granules sparse. 5,000. <C> showing round spermatid. Note membrane damage in nucleus. Other cytoplasmic organelles sparse. 5, 600.

The results of the in vitro hCG-stimulated testosterone biosynthesis of the Leydig cells revealed that these cells were capable of synthesizing testosterone (Table 3). However, the steroidogenic capacity of the Leydig cells of the treated animals was slightly inhibited as inferred from the 3b-HSD histochemistry and the testosterone levels in the spent media of the treated animals which showed significant reduction, compared to the control levels.

Table 3. Effect of chloroform extract of Carica papaya seeds on hCG-stimulated testosterone biosynthesis (ng/mL) of Leydig cells in vitro. Mean SEM, ^cP < 0.01, compared with controls.

The levels of the blood RBC, WBC, Hb, PCV, MCV, MCH and MCHC and the serum protein, glucose, cholesterol, SGPT, SGOT, LDH, creatinine and CK showed wide fluctuations in the study period, but they were within the pretreatment range throughout the course of the investigation (data not shown).

3.5 Hormone assay

Serum testosterone levels of the control and treated animals fluctuated within the pretreatment range throughout the study period (data not shown).

4 Discussion

The seeds of Carica papaya have been proven to possess male antifertility property. We have conducted a series of experiments with various extracts of the seeds of Carica papaya at different dose and duration regimens. Among the various extracts tested, the chloroform extract, the benzene chromatographic fraction of the chloroform extract and its methanol and ethyl acetate sub-fractions have shown reversible antifertility activity in rats and rabbits without adverse toxicity [1]. It is pertinent to note that Carica papaya possesses species variation, but the seed extract tested all show sperm motility inhibitory action in rats [4,21] and azoospermia in rabbits [22,23], while the aqueous extract and the chloroform chromatographic fraction of the chloroform extract showing contraceptive efficacy in rats [3,24] but failed to elicit significant response in rabbits[23,25].

In the present investigation, the chloroform extract of the seeds of Carica papaya in langur monkeys leads to azoospermia without adverse toxicity after 90 days of treatment, in a manner similar to that in rats and rabbits [1]. The sperm functional tests indicated that the voided spermatozoa after 30 and 60 days of treatment were in the infertile range. The effect is reversible as all the semenology parameters returned to pretreatment levels 150 days after treatment withdrawal.

The mechanism by which the chloroform extract of the seeds of Carica papaya brings about sperm motility inhibition/spermatogenic arrest in animals is not clear. In our earlier studies [22], it has been suggested that the spermatogenic arrest could be attributable to the estrogenic [26], androgenic [27,28] or antiandrogenic [29-31] property of the seed extract. Although the chloroform extract and the benzene chromatographic fraction of the chloroform extract has been reported to possess a mild estrogenic property [4,32], it is not likely that this property causes azoospermia in monkeys without severe side effects and change in hormonal pattern and libido. Androgenic and antiandrogenic properties seem to be less probable, as the seminal plasma biochemistry and serum hormonal pattern were all within the normal range. The increase in abnormal sperm count and total inhibition of sperm motility during the initial period of treatment suggest that the action of the drug could target the internal milieu of the epididymis.

Huynh et al.[33] in the triptolide treated rats described two phenotypic effects on mature and maturing germ cells. The first action appears early on the epididymal sperm and the subsequent action is directly on the germ cells of testis. However, in the present investigation, the chloroform extract of Carica papaya seeds alter the testicular but not the epididymal histology. Even the ultrastructure of epididymis shows normal configuration with active protein synthetic machinery (unpublished observations), suggesting that the drug acts upon the testicular germ cells. Interestingly, in the triptolide treated rats, no ultrastructural differences in the epididymal epithelium observed between control and treated rats, but the treated rats exhibited total motility inhibition with severe sperm abnormalities and varied sperm concentration in the cauda epididymis [33-35].

Sertoli cells play an important role in germ cell maturation, but are highly susceptible to extraneous damage [36-39]. The chloroform extract of Carica papaya seeds induced cytoplasmic vacuolization and loss of cytoplasmic organelles in the Sertoli cells. Thus, degeneration and maturational arrest of germ cells, i.e., spermatocytes and spermatids, could be due to the Sertoli cell damage [37]. In the present study, a similar effect on the mitochondria of late spermatids and spermatozoa was also observed. It is therefore concluded that azoospermia could be due to a selective action of the drug on developing germ cells, possibly mediated via Sertoli cells, leading to inhibition of mitochondrial activity, which might alter the respiratory chain, generating a cytotoxic effect on the germ cell proliferation [40-43]. It is further evidenced by an increased mid-piece defects and vacuolization in the mitochondria of the spermatozoa. However, further evidences are needed to substantiate this view.

Available evidences indicate that the chloroform extract of Carica papaya seeds is an orally effective, safe and readily reversible antifertility agent that meets the essential criteria for a male contraceptive.

The investigation was supported by the Ministry of Health and Family Welfare, Government of India. Ultrastructural studies were carried out at the Regional Centre for Sophisticated Instrument Facility for Electron Microscopy of the Department of Anatomy, All India Institute of Medical Sciences (AIIMS), New Delhi. The authors are also thankful to Dr. Anand Kumar, Prof. & Head, Department of Reproductive Biology, AIIMS for his kind help in hormone assay.

References

[1] Lohiya NK, Manivannan B, Mishra PK, Pathak N. Prospects of developing a plant based male contraceptive pill. In: Chowdhury SR, Gupta CM, Kamboj VP, editors. Current status in fertility regulation: indigenous & modern approaches. Lucknow: Central Drug Research Institute; 2001. p 99-119.
[2] Lohiya NK, Ansari AS. Male contraceptive agents. In: Joy KP, Krishna A, Haldar C, editors. Comparative endocrinology and reproduction. New Delhi: Narosa Publishing House; 1999. p 260-77.
[3] Lohiya NK, Goyal RB, Jayaprakash D, Ansari AS, Srivastava S, Singh P. Antifertility effects of Carica papaya seeds in male rats. In: Puri CP, Van Look PFA, editors. Current concepts in fertility regulation and reproduction. New Delhi: Wiley Eastern Ltd.; 1994. p 177-92.
[4] Pathak N, Mishra PK, Manivannan B, Lohiya NK. Sterility due to inhibition of sperm motility by oral administration of benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in rats. Phytomedicine 2000; 7: 325-33.
[5] David GFX, Ramaswami LS. Reproductive system of North Indian langur (Presbytis entellus entellus Dufrense). J Morphol 1971; 135: 89-130.
[6] Lohiya NK, Sharma RS, Puri CP, David GFX, Anand Kumar TC. Reproductive exocrine and endocrine profile of female langur monkeys, Presbytis entellus. J Reprod Fertil 1988; 82: 485-92.
[7] Lohiya NK, Sharma RS, Manivannan B, Anand Kumar TC. Reproductive exocrine and endocrine profiles and their seasonality in male langur monkeys Presbytis entellus entellus. J Med Primatol 1998; 27: 15-20.
[8] Indian National Science Academy. Guidelines for Care and Use of Animals for Scientific Research. New Delhi: Indian National Science Academy; 1992.
[9] WHO: Laboratory manual for the examination of the human semen and sperm-cervical mucus interaction, 4th edition. New York: Cambridge University Press; 1999.
[10] Gopalkrishnan K. Standardized procedures in human semen analysis. Curr Sci 1995; 68: 353-62.
[11] Gopalkrishnan K, Hinduja IN, Anand Kumar TC. Assesment of mitochondrial activity of human spermatozoa with reference to motility & viability in fertile and infertile men. Mol Androl 1991; 3: 243-50.
[12] Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Cabro BG, Zanevald LJD. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984; 70: 219-28.
[13] Lohiya NK, Manivannan B, Mishra PK. Repeated vas occlusion and non-invasive reversal with styrene maleic anhydride for male contraception in langur monkeys. Int J Androl 2000; 23: 36-42.
[14] WHO: Laboratory manual for the examination of the human semen and sperm-cervical mucus interaction, 3rd edition. New York: Cambridge University Press; 1992.
[15] White IG. Studies on the estimation of glycerol, fructose and lactic acid with particular reference to semen. Aust J Expt Biol 1959; 37: 441-50.
[16] Bergmeyer HU. Methods of enzymatic analysis, New York and London: Academic Press; 1965.
[17] Klinefelter GR, Kelce WR, Hardy MP. Isolation and culture of Leydig cells from adult rats. Methods in Toxicol 1983; 3A: 166-81.
[18] Natelson S. Routine use of ultramicro methods in the clinical laboratory. Am J Clin Pathol 1951; 21: 1153-72.
[19] Crosby WH, Munn JI, Furth FW. Standardizing a method for clinical haemoglobinometry. US Armed Forces Med J 1954; 5: 693-703.
[20] Lynch MJ, Raphael SS, Mellor LD, Spare PD, Inwood MJH. Medical laboratory technology and clinical pathology. Philadelphia: WB Saunders Company; 1969.
[21] Lohiya NK, Goyal RB. Antifertility investigations on the crude chloroform extract of Carica papaya Linn. seeds in male albino rats. Indian J Exp Biol 1992; 30: 1051-55.
[22] Lohiya NK, Pathak N, Mishra PK, Manivannan B. Reversible contraception with chloroform extract of Carica papaya Linn. seeds in male rabbits. Reprod Toxicol 1999; 13: 59-66.
[23] Lohiya NK, Mishra PK, Pathak N, Manivannan B, Jain SC. Reversible azoospermia by oral administration of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in rabbits. Adv in Contracept 1999; 15: 141-61.
[24] Lohiya NK, Goyal RB, Jayaprakash D, Ansari AS, Sharma S. Antifertility effects of aqueous extract of Carica papaya seeds in male rats. Planta Med 1994; 60: 400-4.
[25] Lohiya NK, Pathak N, Mishra PK, Manivannan B. Contraceptive evaluation and toxicological study of aqueous extract of the seeds of Carica papaya in male rabbits. J Ethnophar-macol 2000; 70: 17-27.
[26] Bartke A, Williams KIH, Dalterio S. Effects of estrogen on testicular testosterone production in vitro. Biol Reprod 1977; 17: 645-9.
[27] Jayaprakash D, Sharma K, Ansari AS, Lohiya NK. Testosterone enanthate, a single entity male contraceptive agent in langur monkey. Eur Arch Biol 1994; 105: 103-10.
[28] Handelsman DJ. Hormonal male contraception. Int J Androl 2000; 23(Suppl): 8-12.
[29] Lohiya NK, Sharma OP. Effects of cyproterone acetate with combination of testosterone enanthate on seminal characteristics, androgenisity and clinical chemistry in langur monkey. Contraception 1983; 28: 575-86.
[30] Sharma RS, Rajalakshmi M, Pal PC, Roy S, Behal VK, Sharma DN, et al. Evaluation of efficacy, safety and reversi-bility of combination regimen of cyproterone acetate and testosterone buciclate in bonnet monkey. Contraception 2000; 62: 195-201.
[31] Rajalakshmi M, Sharma RS. Methods for the regulation of male fertility. In: Chowdhury SR, Gupta CM, Kamboj VP, editors; Current status in fertility regulation: indigenous & modern approaches. Lucknow: Central Drug Research Institute; 2001. p 179-209.
[32] Mishra PK, Pathak N, Manivannan B, Lohiya NK. Screening for the hormonal properties of the chloroform extract of Carica papaya Linn. seeds for antifertility investigation. Nat Prod Sci 2000; 6: 5-10.
[33] Huynh PN, Sinha Hikim AP, Wang C, Stefonovic K, Lue YH, Leung A, et al. Long-term effects of triptolide on spermato-genesis, epididymal sperm function, and fertility in male rats. J Androl 2000; 21: 689-99.
[34] Qian SZ, Xu Y, Zhang JW. Recent progress in research on Tripterygium: a male antifertility plant. Contraception 1995; 51: 121-9.
[35] Wang ZP, Gu ZP, Cao L, Xu Y, You GD, Mao BY, Qian SZ. Effects of tripchlorolide on the epididymides and testes of rats. Asian J Androl 1999; 1: 121-5.
[36] Flickinger CJ. Regional difference in synthesis, intracellular transport and secretion of protein in the mouse epididymis. Biol Reprod 1981; 25: 871-83.
[37] Pudney J. Fine structural changes in Sertoli and Leydig cells during the reproductive cycle of the ground squirrel, Citellus lateralis. J Reprod Fertil 1986; 77: 37-49.
[38] Ritzen EM, Boitani C, Parvinen M, French FS, Feldman M. Stage-dependent secretion of ABP by rat seminiferous tubules. Mol Cell Endocrinol 1982; 25: 25-34.
[39] Gerard H, Gerard A, EnNya A, Felden F, Gueant JL. Spermatogenic cells do internalize Sertoli androgen-binding protein: a transmission electron microscopy autoradiographic study in the rat. Endocrinology 1994; 134: 1515-27.
[40] Hoffer AP. Effects of gossypol on the seminiferous epithelium in the rat : a light and electron microscope study. Biol Reprod 1983; 28: 1007-20.
[41] Hoffer AP. Ultrastructural, biochemical and endocrine studies on the effects of gossypol and its isomeric derivatives on the male reproductive tract. In: Segal SJ, editor. Gossypol, a potential contraceptive for men. New York: Plenum Press; 1985. p 143-86.
[42] Hoffer AP, Agarwal A, Meltzer P, Herlihy P, Naqvi RH, Lindberg MC, Matlin SA. Ultrastructural, fertility, and spermicidal studies with isomers and derivatives of gossypol in male hamsters. Biol Reprod 1987; 37: 909-24.
[43] Kim IC, Waller DP. Specific inhibition of the testicular mitochondrial respiratory chain in vitro by gossypol. J Androl 1984; 5: 424-30.

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Correspondence to: Dr. N. K. Lohiya, Reproductive Physiology Section, Department of Zoology, University of Rajasthan, Jaipur -302 004, India.
Tel: +91-141-701 809, Fax: +91-141-510 880
E-mail: lohiyank@hotmail.com; lohiyank@rediffmail.com
Received 2002-01-10      Accepted 2002-02-19