Desialylation of Spermatozoa and Epithelial Cell Glycocalyx Is a Consequence of Bacterial Infection of the Epididymis

Abstract

Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in humans. In men, pathogens can also spread to the genital tract via the continuous ductal system, eliciting bacterial prostatitis and/or epididymo-orchitis. Antibiotic treatment usually clears pathogens in acute epididymitis; however, the fertility of patients can be permanently impaired. Because a premature acrosome reaction was observed in an UPEC epididymitis mouse model, and sialidases on the sperm surface are considered to be activated via proteases of the acrosome, we aimed to investigate whether alterations of the sialome of epididymal spermatozoa and surrounding epithelial cells occur during UPEC infection. In UPEC-elicited acute epididymitis in mice, a substantial loss of N-acetylneuraminic acid residues was detected in epididymal spermatozoa and epithelial cells using combined laser microdissection/HPLC-ESI-MS analysis. In support, a substantial reduction of sialic acid residues bound to the surface of spermatozoa was documented in men with a recent history of E. coli-associated epididymitis. In vitro, such an UPEC induced N-acetylneuraminic acid release from human spermatozoa was effectively counteracted by a sialidase inhibitor. These findings strongly suggest a substantial remodeling of the glycocalyx of spermatozoa and epididymal epithelial cells by endogenous sialidases after a premature acrosome reaction during acute epididymitis.

Keywords: bacterial pathogenesis; glycosylation; glycosylation inhibitor; reproduction; sialic acid; sialidase; sperm; spermatozoa.

FIGURE 1.

UPEC infection causes pathological alterations in the cauda epididymis and a premature acrosome reaction in epididymal spermatozoa. A, Masson-Goldner-stained sections of cauda epididymal tissues from mice 3 days post-UPEC infection and PBS sham controls were assessed by light microscopy. Arrows indicate fibrotic remodeling (thickening of the smooth muscle cell layer, collagen accumulation). Circles demonstrate interstitial infiltrating leukocytes. B, sperm acrosomes were fluorescently stained with peanut agglutinin-FITC lectin (green), and nuclei were counterstained using TO-PRO-3 (red) in the cauda epididymides from mice 3 days post-UPEC treatment, PBS sham controls, and untreated controls. The insets in the second row were magnified digitally.

FIGURE 2.

In UPEC epididymitis, sialic acid labeling is decreased in spermatozoa. α2,6-linked sialic acid residues were visualized by SNA-FITC lectin (green), and nuclei were counterstained with TO-PRO-3 (red) in sections from mouse epididymis 3 days post-UPEC and a PBS sham control as well as an untreated control. In contrast to untreated and sham controls, in UPEC infection, a more diffuse sialic acid labeling is evident. The images in the second row were magnified digitally.

FIGURE 3.

Epididymal spermatozoa are desialylated following UPEC infection. A, spermatozoa from the epididymal lumen were isolated by laser microdissection from hematoxylin-stained, paraffin-embedded tissue sections. The excised area is indicated by the black arrow and green circle. Scale bar = 150 μm. B, the same section after the excision of luminal content. C, the obtained spermatozoa were subsequently used for sialic acid quantification. After hydrolysis and fluorescent labeling, DMB-sialic acid residues were separated using reverse-phase HPLC. A set of sialic acid standards was used for the generation of a calibration line and to control the retention times. ESI-MS spectra were recorded during the HPLC runs to verify the presence of different sialic acid species, as shown exemplary for Neu5Ac in UPEC-treated and PBS sham control epididymides. D, the amounts of Neu5Ac and the respective standard deviations were calculated from four epididymides obtained from PBS sham control and UPEC-injected mice, respectively (n = 6 animals). Given mean values were set to 100% for the sham control. The statistical evaluation was performed using Student's t test (unequal variances, two-tailed). *, p ≤ 0.05.

FIGURE 4.

Analysis of α-keto acids in UPEC. A, sialic acid standards and UPEC samples were hydrolyzed, and α-keto acids were fluorescently labeled with DMB for HPLC separation. B, the ESI-MS spectrum recorded during the elution time of the peak is highlighted by the red dotted frame. C, comparison of the retention time of KDO and Neu5Gc standards.

FIGURE 5.

Reduction of sialic acid content in epithelial cells in UPEC epididymitis. A, epithelial cells were isolated from paraffin-embedded mouse epididymis by laser microdissection. The excised area is indicated by the black arrow and green line. B, the same section after excision of epithelium. Scale bar = 150 μm. C, DMB-labeled sialic acid residues of the isolated cells were separated using RP-HPLC. A set of sialic acid standards was used for quantification and to control the retention times. The ESI-MS spectrum was recorded during the HPLC runs (data not shown). D, the amounts of Neu5Ac and the respective standard deviations of six different controls and infected mice (one epididymis from six animals each). Given mean values were set to 100% for the sham control. The statistical evaluation was performed using Student's t test (unequal variances, two-tailed). *, p ≤ 0.05.

FIGURE 6.

UPEC-dependent desialylation of human spermatozoa can be counteracted by sialidase inhibitor in vitro. A, spermatozoa from healthy donors prepared by swim-up were incubated with UPEC for 3 h, and the amount of Neu5Ac was compared with untreated samples using the DMB-HPLC approach as shown in Fig. 3. In parallel, UPEC-treated as well as untreated spermatozoa were co-incubated with the sialidase inhibitor DANA. One control was set as 100%, and the other controls were set in relation to it. Treatments were calculated relative to the mean of controls. Significance levels were calculated using one-way analysis of variance. *, p ≤ 0.05; **, p ≤ 0.01; N.S., not significant (n = 3). B, acrosomes were labeled by PSA-FITC lectin (green), and nuclei were counterstained with TO-PRO-3 (red). The presence of acrosomes was compared with the untreated control.

FIGURE 7.

Reduced levels of Neu5Ac in patients with a history of acute epididymitis. A, acrosomes of swim-up spermatozoa obtained from healthy men (control) and patients 14 days after diagnosis and treatment of acute epididymitis were visualized by PSA-FITC lectin (green). Nuclei were counterstained with TO-PRO-3 (red). B, sialic acid residues of isolated spermatozoa from these patient groups (n = 3, respectively) were released and fluorescently labeled with DMB for quantification. The statistical evaluation was performed using Student's t test (unequal variances, two-tailed). Given mean values were set to 100% for all sham controls. *, p ≤ 0.05.

FIGURE 8.

Summary and working model. Blue box, in the female reproductive tract, a capacitation/acrosome reaction leads to proteolytic activation and release of neuraminidases, resulting in desialylation of spermatozoa to allow sperm/egg interaction. Red box, the UPEC-induced acrosome reaction triggers premature desialylation of spermatozoa and the release of sialic acid residues from adjacent cells in the male reproductive tract. Consequently, pathological hyposialylation may negatively influence the protection of host cells against phagocytosis and the complement system. Green box, in vitro UPEC-induced hyposialylation of spermatozoa could be counteracted using neuraminidase inhibitors. Putatively, a further level of intervention could be upstream by blocking the proteolytic activation and release of neuraminidases using suitable protease inhibitors according to Ma et al. (10).