Porins and Lipopolysaccharide Induce Apoptosis in Human Spermatozoa

Abstract

Treatment of human spermatozoa with porins or lipopolysaccharide (LPS) increases spontaneous apoptosis in these cells. Porins and LPS were extracted from Salmonella enterica serovar Typhimurium and Pasteurella multocida and were mixed with human spermatozoa for detection of levels of apoptosis.A relationship between genitourinary tract infections in women and impaired fertility has been postulated for some time. It has already been demonstrated that the endotoxin of Vibrio fetus, which is a common pathogen in rams and bulls (9), is able to immobilize ram and bull spermatozoa. In other studies, a factor from Escherichia coli that immobilizes human spermatozoa was isolated and identified (19). E. coli strains obtained from urinary tract and cervical swab specimen cultures produce a strong depression in the motility and viability of human spermatozoa in vitro. Teague et al. (21) showed that high concentrations of E. coli interfere with the motility of spermatozoa by the attachment of E. coli to the spermatozoa (1). It has also been demonstrated that Ureaplasma urealyticum (12) and Neisseria gonorrhoeae (13) attach themselves to spermatozoa. Gram-negative bacteria release structural components from the outer membrane into the surrounding microenvironment by both cell lysis and secretion during active growth (23). The vaginal tracts of healthy women harbor a wide variety of microbial species. The prevalence of gramnegative bacteria consisting of potentially pathogenic enterobacteria can represent a more or less frequent event.In previous studies lipopolysaccharide (LPS), porins, and peptidoglycan fragments have been reported to be toxic for human spermatozoa (11) and may lead to reduced fertility or sterility. In recent years several research teams (3,14,17) demonstrated the presence of apoptosis in rodent testes. This phenomenon was found essentially in spermatogenic stages VII to XIV and was also found in the postmeiotic stages of dogfish (7). In the ejaculates of infertile men, many spermatozoa present signs of apoptosis (2).On the basis of these considerations, the purpose of the present study was to verify the probable effects of porins and LPS in increasing the rate of naturally occurring apoptosis of spermatozoa.Porin was extracted from Salmonella enterica serovar Typhimurium SH 5014 and Pasteurella multocida ATCC 6533 by the method of Nurminen (18). Briefly, 1 g of cell envelopes was suspended in 2% Triton X-100 in 0.01 M Tris-HCl (pH 7.5) containing 10 mM EDTA; after the addition of trypsin (10 mg/1 g of cell envelopes), the pellet was dissolved in sodium dodecyl sulfate (SDS) buffer (4% [wt/vol] in 0.1 M sodium phosphate; pH 7.2) and the solution was applied to an Ultragel ACA 34 column (Pharmacia, Uppsala, Sweden) equilibrated with 0.25% SDS buffer. The fraction containing proteins, identified by determination of the absorption at 280 nm (A 280 ), was extensively dialyzed and checked by SDS-polyacrylamide gel electrophoresis in slabs as described by Laemmli (15). The protein content of the porin preparati...

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