Effect of papaya seed extract on microenvironment of cauda epididymis

R.J. Verma,  N.J. Chinoy

Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad  380009, India

Asian J Androl  2001 Jun; 3:  143-146

Keywords: Carica papaya; microenvironment; epididymis; spermatozoa; protein; sialic acid

Abstract

Aim: To evaluate the effect of aqueous Carica papaya seed extract on microenvironment of cauda epididymis. Methods: Adult male albino rats were intramuscularly administered with 0 (control) or 0.5 mg papaya seed extract/kg body weight for 7 days. Cauda epididymal tubular content was collected by micropuncture technique; epididymal luminal fluid and sperm pellets were separately analyzed. Results: The results revealed that the extract treatment caused significant reduction, as compared with control, in total protein and sialic acid contents in both epididymal fluid and sperm pellet. As compared with control, significantly lowered acid phosphatase activity was recorded in sperm pellet but was higher in epididymal fluid after the treatment. The extract treatment also caused significant reduction in level of inorganic phosphorus in the epididymal fluid. Conclusion: It is concluded that the aqueous papaya seed extract alters cauda epididymal microenvironment.

1 Introduction

Preliminary study of Udoh and Kehinde^[1] revealed that oral administration of 100 mg/kg body weight of crude ripe pawpaw seeds (Carica papaya) in male rats for 8 weeks caused degeneration of germinal epithelium and germ cells, a reduction in the number of Leydig cells and presence of vacuoles in the tubules. The epididymis showed many empty vacuoles containing degenerated spermatozoa and cell debris in the lumen. However, at lower dose (50 mg/kg body weight) comparatively less effect was observed in the testis and epididymis. Pathak et al^[2] reported total suppression of crude epididymal sperm motility, count, viability and an increase in percent abnormal spermatozoa in male rats administered orally with 5 and 10 mg/animal/day of benzene chromatographic fraction of chloroform extract for 60-150 days.

Oral administration of crude aqueous Carica papaya seed extract in doses of 10 and 50 mg/animal/day orally for 30, 60 and 90 days and 0.1 and 1.0 mg/animal/day intramuscularly for 15 and 30 days in male albino rats caused a significant reduction in cauda epididymal sperm motility, count and fertility rate which returned to normal on cessation of the treatment^[3].  Chinoy et al^[4] have also shown that intramuscular administration of aqueous Carica papaya seed extract in 0.5 and 5.0 mg/kg body weight/day for 7 days caused a selective androgen deprivation effect on reproductive organs resulting in infertility with complete reversibility on withdrawal of the treatment. These results indicate that reversible sterility could be induced in male rats by papaya seeds aqueous extract treatment without adverse effects on libido and toxicologic profile.

2 Materials and methods

Shade-dried seeds of ripe papaya (Carica papaya) variety Honey dew were obtained from village development unit, Dehradun, India.  The extract was prepared according to WHO protocol CG-06^[7]. Air-dried papaya seeds were ground in with a mortar and pestle. An aqueous suspension of 5 mg of powder in 100 mL of distilled water was prepared and soxhlated for 1 hr. After cooling, the content was filtered successively through ordinary and then through whatman filter paper number 1. The filtrate and residue were collected separately. The residue was then resuspended in the same amount of distilled water and soxhlated three or four times for the complete extraction of water soluble contents. The filtrate was collected, pooled and evaporated on a water bath to dryness. The powder was stable at room temperature. The powder was resuspended in double distilled water and used for the treatment. The extract was completely soluble in water and a pale yellow solution was obtained.

Colony bred adult male rats of Charles Foster strain obtained from Cadila Health Care, Ahmedabad, were provided with animal feed and water ad-libitum and maintained under laboratory condition. 

Thirty adult male rats were divided into three groups. Group 1 (untreated control) animals were maintained without any treatment. Animals of Group 2 (vehicle control) were sham injected with only saline (0.9% NaCl) for 7 days. Animals of Group 3 were intramuscularly injected with aqueous Carica papaya seed extract (0.5 mg/kg body weight in saline) for 7 days. On day 8, the cauda epididymal luminal content was collected by micropuncture technique^[8]. Rats were an aesthesized by an intraperitoneal injection of sodium phenobarbitone (30 mg/kg body weight). A scrotal incision was made to expose the testis and epididymis unilaterally and positioned in a specially designed holder. The cauda epididymis was adjusted in a convenient position with the help of agar-agar (3% v/v in 0.9% saline). Epididymal tunica was removed from a required area to expose the tubule for inserting glass micropipette. Liquid paraffin oil was applied on the surface of epididymis to prevent dehydration and to increase the visibility.

The epididymal fluid was collected with the help of micropipettes filled with sudan black B stained mineral oil mounted on Leitz micromanipulator. A small electrode was used to stimulate contractions of epididymal ducts in order to collect more fluid. After sufficient volume of sample was withdrawn, the pipette was removed. After measurement and appropriate dilution of epididymal fluid, the samples were centrifuged at 1000g for 15 min to separate epididymal fluid and sperm pellet. The sperm pellet was further diluted and utilized for  microanalysis simultaneously along with the epididymal fluid.

The concentration of total protein was estimated by folin-phenol method of Lowry et al^[9] using bovine serum albumin as standard. The inorganic phosphorus content was measured by the method of Fiske and Subbarao^[10]. Acid phosphatase activity and sialic acid contents were estimated by the methods of Bessey et al^[11] and Jourdian et al^[12] respectively.

3 Results

Table 1.  Effect of papaya seed extract treatment on biochemical composition of epididymal luminal fluid in rats. meanSEM. n=10.^cP<0.01 as compared with Group 2.

Table 2.  Effect of papaya seed extract treatment on biochemical composition of sperm pellet  in rats. meanSEM. n=10. ^cP<0.01 as compared with Group 2.

4 Discussion

Sialic acids are also secreted by the epididymal epithelium and they are coated on spermatozoa as they pass through the epididymis. They are concerned with changing the membrane surface of maturing spermatozoa, coating of spermatozoa with certain antigens and in the development of their fertilizing capacity^[5,6]. It is likely that the extract might be interfering with the synthesis and/or their release from the principal cells of the epididymal epithelium.

Acid phosphatase activity was lowered in the sperm pellet of extract treated animals correlated with the resorption of dead and non-motile spermatozoa which might account for the decrease in sperm density in treated rats^[2-4]. An increase in acid phosphatase activity of epididymal fluid observed in present investigation might be due to release of enzyme from spermatozoa into the epididymal fluid.

Inorganic phosphates are also secreted largely in the head of the epididymis and changes along with it. It is also known that some inorganic phosphate and phosphate-containing compounds are lost along with epididymis probably because of reabsorption or metabolism by epididymis or by maturing spermatozoa^[14,15].

References

[1] Udoh P, Kehinde A. Studies on antifertility effect of pawpaw seeds (Carica papaya) on the gonads of male albino rats. Phytother Res 1999; 13: 226-8.
[2] 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.
[3] 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. 
[4] Chinoy NJ, D'Souza JM, Priya Padman. Effect of crude aqueous extract of Carica papaya seeds on male albino mice. Reprod Toxicol 1994; 8: 75-80.
[5] Hinton BT, Palladino MA. Epididymal epithelium: Its contribution to the formation of a luminal fluid microenvironment. Microsc Res Tech 1995; 30: 67-81.
[6] Turner  TT, Miller DW, Avery EA. Protein synthesis and secretion by the rat caput epididymidis in vivo influence of the luminal microenvironment. Biol Reprod 1995; 53: 1012-9. 
[7] WHO Protocol CG-06. Extraction and fractionation for biological and phytochemical studies. APJF/IP 1001 A, Geneva; World Health Organization, 1983.
[8] Chinoy NJ, Rao MV, Verma RJ. Micropuncture technique in studying microenvironment of epididymis. Indian J Med Res 1987;  88: 410-1.
[9] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-75.
[10] Fiske CH, Subbarao Y. The colorimetric determination of phosphorus. J Biol Chem 1925;  66: 375. 
[11] Bessey OA, Lowry OH, Brock NJ. A method for the determination of acid phosphatase with 5 cubic mm of serum. J Biol Chem 1946; 164: 321-9.
[12] Jourdian GW, Dean L, Roseman S. Sialic acid. XI Periodate-resorcinol method for the quantitative estimation of free sialic acids and their glycosides. J Biol Chem 1971; 246: 430-5. 
[13] Palladino MA, Hinton BT. Expression of multiple -glutamyl transpeptidase messenger ribonucleic acid transcripts in the adult rat epididymis is differentially regulated by androgens and testicular factors in a region specific manner. Endocrinology 1994; 135: 1146-56.
[14] Amann RP. Structure and function of the normal testis and epididymis. J Am Coll Toxicol 1989; 8: 457-71. 
[15] Verma RJ. Sperm quiescence in cauda epididymis. Acta Europaea Fertilitatis 2001; in press. 

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Correspondence to: Dr. R. J. Verma, Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380009, India.
Tel +91-079-630 2362 
E-mail: zooldeptgu@satyam.net.in
Received 2000-07-18     Accepted 2001-04-06