The female reproductive tract contains multiple innate sialic acid-binding immunoglobulin-like lectins (Siglecs) that facilitate sperm survival

Abstract

A sperm that fertilizes an egg has successfully survived multiple checkpoints within the female reproductive tract, termed pre-fertilization events. The leukocytic response is a pre-fertilization event in which sperm trigger an immune response that promotes homing of circulating leukocytes to the uterine lumen to destroy most sperm. Various glycoconjugates decorate the sperm surface, including sialic acids, which are abundant at the sperm surface where they cap most glycan chains and regulate sperm migration through cervical mucus, formation of the sperm oviductal reservoir, and sperm capacitation. However, the role of sperm-associated sialic acids in the leukocytic reaction remains unknown. The cognate endogenous binding partners of sialic acids, sialic acid-binding immunoglobulin-like lectins (Siglecs) play a pivotal role in regulating many immune responses. Here we investigated whether sperm-associated sialic acids inhibit activation of neutrophils, one of the major immune cells involved in the leukocytic reaction. We used in vitro interactions between sperm and neutrophils as well as binding assays between sperm and recombinant Siglec-Fc chimeric proteins to measure interactions. Moreover, we examined whether Siglecs are expressed on human and mouse endometria, which have a role in initiating the leukocytic reaction. Surprisingly less sialylated, capacitated, sperm did not increase neutrophil activation in vitro However, we observed expression of several Siglecs on the endometrium and that these receptors interact with sialylated sperm. Our results indicate that sperm sialic acids may interact with endometrial Siglecs and that these interactions facilitate sperm survival in the face of female immunity.

Keywords: glycan; innate immunity; lectin; reproduction; sialic acid; sialic acid-binding immunoglobulin-like lectins (Siglec); spermatozoa.

Figure 1.

The sperm glycocalyx and siglec signaling. a, a schematic of the glycocalyx of mammalian ejaculated sperm. Sialic acid is presented as purple diamonds. Modified from Ref. . b, Siglecs and downstream signaling. A schematic of an inhibitory Siglec (left) and activating Siglec (right) are shown. After binding a sialic acid ligand, via the V-set domain, Siglecs can modulate an immune response by downstream signaling pathways. Phosphorylation the ITIM and ITIM-L domains of inhibitory Siglecs by SHP-1 initiates a signaling cascade resulting in immune inhibition. Activating Siglecs bind to DAP12 via their transmembrane ITAM domains and initiate a signaling cascade resulting in immune activation.

Figure 2.

Confirmation of sperm capacitation and sialic acid shedding. Sperm co-incubated with the fluorogenic sialic acid analog 4-MU-NANA were exposed to a noncapacitating or capacitating buffer. Only sperm in the capacitating buffer show an immediate increase in fluorescence (a), indicative of rapidly activated sperm sialidases. Sperm capacitation was confirmed by using the fluorescent molecule JC-1 to probe for increased mitochondrial activity. The majority of sperm incubated under capacitating conditions show an increase in JC-1 staining (b and c), compared with sperm in noncapacitating conditions (∼81 versus ∼10%, respectively). Staining with anti-phosphotyrosine antibody on Western blotting shows an increase of phosphorylation between low motility (LM) and high motility (HM) human sperm and with increased duration (1 versus 4 h) of in vitro exposure to capacitating conditions (d).

Figure 3.

Impact of sperm on neutrophil activation. Flow cytometry analysis of the expression of several neutrophil reactivity markers (a–c). Neutrophils co-incubated with NC or C sperm were stained with antibodies targeting several markers of neutrophil activation and measured using flow cytometry. Co-incubation of isolated human leukocytes with NC or C sperm show no additional shedding of the neutrophil quiescence marker L-selection when compared with leukocytes alone (a). Isolated human neutrophils co-incubated with C or NC sperm show similar CD11b expression compared with isolated leukocytes alone (b). Isolated human neutrophils co-incubated with C sperm show a nonsignificant decrease in the percentage of OxyBURST-positive cells (positivity indicates an increase in intracellular reactive oxygen species) compared with isolated neutrophils alone or neutrophils co-incubated with NC sperm (c). PMA-induced isolated human neutrophils trigger NET formation and capture live NC and C sperm (d). Captured sperm are indicated by white arrowheads. e, sperm from two different donors stained with Siglec-Fc chimeric recombinant protein: black is control, blue is Siglec-5::FC, red is Siglec-9::FC.

Figure 4.

Siglec-10 is expressed in the human endometrium. Immunofluorescence staining of Siglec-10 (orange) and all nuclei (4′,6-diamidino-2-phenylindole, DAPI). Siglec-10 expression is observed exclusively in the endometrium and not in the underlying stroma. Asterisks indicate the uterine lumen. Images are representative for a sample of n = 7.

Figure 5.

Siglecs and their downstream signaling proteins of Siglecs are present in endometrial cancer cell lines. Flow cytometry analysis of the expression of select Siglec and their signaling molecules in endometrial cell lines. Both HEC-1B (a) and Ishikawa (b) cell lines express Siglec-10 and Siglec-16. Both cell lines also express SHP-1 and DAP12, the downstream signaling molecules of Siglec-10 and Siglec-16, respectively.

Figure 6.

Human spermatozoa carry sialylated ligands for Siglec-10. Flow cytometry analysis indicates that human sperm do not bind Siglec-11-Fc (a) and Siglec-16-Fc (b) (black line is control and red is the Siglec-Fc fusion protein). Human sperm do bind Siglec-9 (c) and Siglec-10-Fc (d) in a sialic acid-dependent manner (black is control, red is WT Siglec-10-Fc, blue is Siglec-10(R120K)-Fc). A subset of sperm also express CD24 (e), a known sialylated ligand of Siglec-10.

Figure 7.

Siglec-3 is expressed in the mouse uterine epithelium. qPCR analysis (a) of the whole mouse uteri indicates that Siglec-3 expression is higher than Siglec-E and -F. Immunofluorescence staining for Siglec-3 (b) indicates Siglec-3 expression is enriched in the epithelium and epithelium glands as compared with the underlying stroma (asterisks indicate the uterine lumen). c, quantification of Siglec-3 antibody staining.

Figure 8.

Sperm sialic acid and endometrial Siglecs may interact to modulate the female immune response to sperm. Both human and the mouse endometrium express inhibitory Siglecs (Siglec-10 and Siglec-3, respectively). When bound by sialic acid on sperm, the expressed inhibitory Siglecs may inhibit the immune response of the endometrium by inhibiting the release of cytokines, complement or other pro-inflammatory proteins via SHP-1 signaling. Sperm may further directly interact with neutrophils and macrophages and inhibit their immune response.