1 Introduction

2 Materials and methods

2.1 Test materials

The seeds of Carica papaya (Caricaceae,Voucher No. RUBL 16590) of honey dew variety, were examined using the extraction procedures described earlier^[6]. Briegly, the seeds of Carica papaya were shade-dried, powdered and soxhalated in chloroform at 58℃ for 12×3 hours. The soxhalated material was concentrated under reduced pressure and further purified by silica gel column chromatography using benzene, chloroform and ethyl acetate as eluents. Following partial purification, the benzene chromatographic fraction, which showed significant effect on semen parameters^[4,6], was subjected to methanol and ethyl acetate sub-fractionation and the residues after evaporation of the solvents were used for further purification by Thin Layer Chromatography (TLC). Two compounds were obtained from each sub-fraction on TLC viz., MCP 1 & 2 and ECP 1 & 2, respectively. The compounds were scraped, extracted with the corresponding solvents, evaporated to dryness under reduced pressure and the residues were separated. The chloroform extract, the benzene chromatographic fraction of the chloroform extract and its methanol and ethyl acetate sub-fractions and the isolated compounds MCP 1 & 2 and ECP 1 & 2 which showed significant effects on sperm parameters by oral administration in rats and rabbits^[3,5,6], were used in the present investigation for in vitro sperm motility assessment. The extract, fractions and compounds were dissolved in Biggers Whitten and Whittingham (BWW) medium, supplemented with sodium bicarbonate (210 mg/L), glucose (100 mg/L), sodium pyruvate (3 mg/mL), bovine serum albumin (300 mg/L), sodium lactate (0.37 mL), benzyl penicillin (10,000 U/mL) and HEPES buffer (2 mL) at concentrations 2%, 1%, 0.5% and 0.1%.

2.2 Sample preparation

Semen samples (n=15) from normal subjects after 48 hours of sexual abstinence were subjected to routine semen analysis^[8] following liquefaction at 37℃. Sperm counts above 50 million/mL with normal morphology, rapid and linear and progressive motility, and viability above 50% was considered for the in vitro test. WHO's strict evaluation criteria were followed for assessing sperm morphology^[8]. Spermatozoa free of seminal plasma were obtained by centrifugation, washed once in BWW media and adjusted to the final concentration of 50 million/mL.

2.3 Experimental procedure

Prior to experiment, semen samples and the seed extract/product preparations were prewarmed to 37℃; 250 μL semen sample containing 15 million spermatozoa was added to 250 μL of the extract preparation at various concentrations. Sperm motility (a, b, c & d grades)^[8], was assessed immediately after addition of the semen samples and thereafter every 5 min for up to 30 min in a phase contrast microscope along with placebo control. Following completion of the experiment, sperm viability and morphological assessments were carried out by nigrosin-eosin and papanicolaou staining methods, respectively^[8].  

2.4 Ultrastructural studies

Following completion of motility assessment, the spermatozoa were separated by centrifugation, washed with phosphate buffer (0.1 mol/L; pH 7.2) and pelleted by centrifugation. The pellets were immediately fixed in 2.5% glutaraldehyde in phosphate buffer for 30 min and washed thrice in phosphate buffer. For scanning electron microscopy (SEM), a thin film of spermatozoa was smeared on an SEM stub with silver paint, air dried, sputter coated with gold and observed under the scanning electron microscope (Leo SEM 435 VP). For transmission electron microscopy (TEM), the spermatozoa pellet was post-fixed in OsO₄ for 30 min, washed in phosphate buffer, followed by distilled water, dehydrated in acetone, embedded in low viscosity spur medium and polymerized at 60℃ for 48 h. The ultrathin sections were stained with uranyl acetate and lead citrate and observed under the transmission electron microscope (Philips model CM-10).

2.5 Sperm revival test

Following completion of the experiment, the spermatozoa were washed twice with BWW medium and incubated once again in the same medium free of extract/products at 37℃ for 30 min to observe revival of sperm  motility, if any. Immotile spermatozoa showing vibratory movement to progressive motility after incubation were considered revived.

2.6 Statistical analysis

3 Results

Sperm motility from its initial level of 55.07%±3.30% dropped immediately following the addition of the chloroform extract, benzene chromatographic fraction and its sub-fractions as well as the isolated compounds, ranged between 8.3%±0.7% and 31.2%±1.5%. The effects were dose-dependent and more pronounced effects were observed with the compounds isolated from the ethyl acetate sub-fractions (ECP 1 & 2). Although many of the spermatozoa showed only vibratory movement after 5 min of addition of the drugs, particularly at 2% concentration, total inhibition of motility was observed after 10 min in the chloroform extract, benzene chromatographic fraction of the chloroform extract and ECP 1 & 2 treated samples. None of the spermatozoa showed motility, 25 min after addition of the extract/fractions/compounds (Tables 1 & 2).

The viability of spermatozoa, following completion of the experiment, fell significantly (P<0.001; Range 22.8%±0.6% to 45.4%±1.2% as against 54.9%±1.2% in control) and the effects were more pronounced in the ECP 1 & 2 treated samples at 2% concentration. Sperm abnormality showed a significant increase in its pattern (P<0.001; Range 26.7%±1.1% to 46.9%±1.3% as against 16.6%±1.8% in  control samples). Most abnormalities were found in MCP 1 & 2 treated samples at 2% concentration (Table 3). 

Table 1.Human sperm motility (%) following incubation with chloroform extract, bezene chromatographic fraction and its sub-fractions of the chloroform extract of the seeds of Carica papaya in vitro (mean±SEM of 15 samples)^*.