**1. Introduction**

294 Chromatography – The Most Versatile Method of Chemical Analysis

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For successful fertilization, motility is the most obvious and most essential sperm function and has been repeatedly shown to be predictive of fertilization in vitro [1]. Several studies have shown that the motility characteristics of spermatozoa are of the utmost importance for the men's fertility [2]. Spermatozoa dysfunction is the single most important cause of infertility. A decrease in spermatozoa motility with time is universal phenomenon. This reduction differs from species to species and also among individuals of the same species, as in the human male. Most investigators agree that the majority of spermatozoa cease to move within the first 24 hours. The survival of spermatozoa after ejaculation is dependent on the environmental conditions under which they are kept. In the female genital tract they may remain active for several days [3], but their activity is of much shorter duration if they remain in the seminal fluid outside the body.

Male genital infections are relevant cause in the etiology of infertility due to abnormalities in sperm quality [4,5], affecting spermatozoal motility. The comparison of semen characteristics between infected and non-infected men show that motile spermatozoa are lower when the microorganisms are present in the semen [6]. It appears that bacteria have a direct effect on sperm motility with negative consequences in fertility. Among bacterial species that interact with spermatozoa are well-known causative pathogens of genitourinary infections such as *Escherichia coli, Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis* [7]. Of the various bacteria, *E. coli* is the most extensively studied microorganism in relation to infertility as a result of interaction with spermatozoa [8]. It is also the primary bacteria associated with prostatitis and epididymitis [9]. Several authors describe sperm

© 2012 Prabha and Kaur, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Prabha and Kaur, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 between spermatozoa and bacteria.

Isolation and Purification of Sperm Immobilizing/Agglutinating Factors from Bacteria and Their Corresponding Receptors from Human Spermatozoa 297

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).

*S. aureus* 

**4. Extraction and purification of sperm immobilization factor (SIF) from** 

For extraction of SIF, *S. aureus* was grown in Brain Heart Infusion (BHI) broth for 72 h at 37°C under shaking conditions (150 rpm). The cell culture was subjected to centrifugation at

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