**3. Extraction and purification of sperm agglutinating factor (SAF) from**  *S. aureus*

Sperm agglutinating factor was extracted and purified from 72 h old cell culture of *S. aureus* by the method standardized in the laboratory [16]. The cell culture was centrifuged at 10,000 g for 20 minutes and the clear supernatant and the cells obtained were washed twice with sterile phosphate buffered saline (PBS) (50 mM, pH 7.2) and resuspended in the same buffer. When washed cells and cell free supernatant were studied for their sperm agglutinating property, the results showed that only washed cells were able to agglutinate the spermatozoa whereas culture supernatant failed to do so indicating that the agglutination of sperm might be associated with bacterial cells and not their metabolites. Pretreatment of *S. aureus* by sonication produced bacterial fragments that were unable to agglutinate sperm. Centrifugation of *S. aureus* fragments at 10,000 g for 5 min did not eliminate sperm agglutinating elements from the solution, indicating the sperm agglutinating factor to be present in sonicated supernatant. Based on its sperm agglutinating activity, the bioactive molecule from the sonicated supernatant was purified by ammonium sulphate precipitation, gel permeation chromatography and ion exchange chromatography. The sonicated supernatant was fractionated with ammonium sulphate so as to get 20, 40, 60 80 and 100% saturation. The flasks were kept at 4°C overnight and next day the precipitates were collected by centrifugation at 10,000 g for 15 min at 4°C and were redissolved in minimum amount of PBS. SAF was precipitated by ammonium sulphate at 40% saturation. The protein was dialyzed in preactivated dialysis bags against PBS under cold conditions and concentrated against polyethylene glycol (PEG) 6000 at 4°C. Further purification was done using Sephadex G-200 and DEAE cellulose column chromatography. The fractions containing approximately 1mg protein were applied on Sephadex G-200 (Pharmacia Fine Chemicals, Uppsala), column (2 cm X 31 cm) equilibrated and eluted with PBS. The aspirator bottle with PBS was joined with a fine capillary tube to maintain constant head pressure and allowed to run for 24 h. Fractions of 3 ml each were collected and each fraction was read at 280 nm on U.V. spectrophotometer. The fractions showing spermagglutinating activity i.e. fraction (4-7) with a peak value in fraction 5 were pooled and concentrated against PEG 6000 (Figure 1a) and applied to DEAE cellulose (Hi Media Laboratories Ltd., Mumbai, India) column. A column of 2 cm x 15 cm was made with activated slurry of DEAE cellulose, an anion exchanger with a pressure of about 1 cm water/cm of height of gel bed. The column was washed with PBS until the ion exchanger reached ionic equilibrium with the starting buffer. The aspirator bottle with PBS was joined with a fine capillary tube to maintain a constant head pressure and allowed to run. Final elution was done with PBS containing 0.05, 0.1, 0.2, 0.4 and 0.6 M NaCl. Fractions of 4 ml each were collected and read at 280 nm on U.V. spectrophotometer (Hitachi U-2900). All fractions were again checked for agglutination of spermatozoa. The fractions showing agglutination of sperm (35-39) were pooled and concentrated (Figure 1b). The purified pooled fractions were dialyzed, lyophilized and then subjected to protein estimation. The purification status of SAF was checked by gel electrophoresis (PAGE). Molecular weight estimation was done by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% resolving gel containing SDS [17]. 10µl of the pooled and concentrated sample obtained from DEAE cellulose column was mixed with loading dye (Bromophenol blue: glycerol) in the ratio of 1:50. The sample was applied to the gel and subjected to a current of 15 mA and run initially at 50V and thereafter at 100V. The gel was visualized by staining with Coomassie Brilliant Blue R-250. The molecular weight of SAF was estimated to be approximately, 65 kDa (Figure 1c).

296 Chromatography – The Most Versatile Method of Chemical Analysis

between spermatozoa and bacteria.

spermatozoa by *S. aureus* and *E. coli*.

Research (PGIMER), Chandigarh.

**2. Microorganisms** 

*S. aureus* 

agglutination and immobilization by *E. coli* [10,11]. Paulson & Polakoski [12] investigated the mechanism of how *E. coli* immobilizes spermatozoa and they reported a factor, apparently excreted by the bacteria which immobilizes spermatozoa without agglutinating it. However, Diemer et al. [13] reported that *E. coli* inhibits sperm motility by directly adhering to and agglutinating spermatozoa. Rapidity and extent of sperm–*E. coli* agglutination indicated strong adhesive forces. Bartoov et al*.* [8] proposed that mannose plays a critical role in adherence of *E. coli* to sperm. Although, a number of studies have evaluated the ability of *E. coli* to affect sperm motility by adherence, agglutination and dialyzable factors, however, none have identified the exact mechanism of interaction

In addition to *E. coli*, *Staphylococcus aureus,* the predominant flora in infertile men, has also been reported to cause a significant decrease in sperm motility [14]. Emokpae et al. [15] while studying the contribution of seminal tract infection to sperm density, asthenozoospermia and teratozoospermia, observed *S. aureus* as the causative organism accounting for 68.2% of seminal infections. Most practitioners dismiss this infection as mere contamination, which is assumed to have no significance. Semen that passes through the genital tract is routinely contaminated with staphylococci. However, since the prevalence of abnormal sperm indexes is high, it was suggested that *S. aureus* infection should be treated and no longer ignored when managing male factor infertility [15]. *S. aureus* is known to produce various toxins and enzymes that may be exerting damaging effect on human sperm, but its mechanism of action also needs further investigation. Therefore, the present work was undertaken to study the mechanism of immobilization/agglutination of

The bacterial isolates *Staphylococcus aureus* either showing immobilization or agglutination of human spermatozoa, were isolated from the cervices of women suffering from unexplained infertility, attending the Department of Obstetrics and Gynecology, Government Multi Speciality Hospital, Sector-16, Chandigarh, India. The isolates of *Escherichia coli* were isolated from semen samples of infertile males attending the infertility clinic at the Department of Urology, Post Graduate Institute of Medical Education and

**3. Extraction and purification of sperm agglutinating factor (SAF) from** 

Sperm agglutinating factor was extracted and purified from 72 h old cell culture of *S. aureus* by the method standardized in the laboratory [16]. The cell culture was centrifuged at 10,000 g for 20 minutes and the clear supernatant and the cells obtained were washed twice with sterile phosphate buffered saline (PBS) (50 mM, pH 7.2) and resuspended in the same buffer. When washed cells and cell free supernatant were studied for their sperm agglutinating property, the results showed that only washed cells were able to agglutinate the spermatozoa whereas culture
