Sialidases on mammalian sperm mediate deciduous sialylation during capacitation

Abstract

Sialic acids (Sias) mediate many biological functions, including molecular recognition during development, immune response, and fertilization. A Sia-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity. During capacitation, sperm lose a fraction of their Sias. We quantified shed Sia monosaccharides released from capacitated sperm and measured sperm sialidase activity. We report the presence of two sialidases (neuraminidases Neu1 and Neu3) on mammalian sperm. These are themselves shed from sperm during capacitation. Inhibiting sialidase activity interferes with sperm binding to the zona pellucida of the ovum. A survey of human sperm samples for the presence of sialidases NEU1 and NEU3 identified a lack of one or both sialidases in sperm of some male idiopathic infertility cases. The results contribute new insights into the dynamic remodeling of the sperm glycocalyx prior to fertilization.

FIGURE 1.

Release of Sia monosaccharides during sperm capacitation. A and B, released Sia (monosaccharides) from in vitro capacitated sperm. A, Neu5Gc and Neu5Ac from mouse sperm. B, Neu5Ac from human sperm. C and D, increase in exposed galactose as measured by lectins. C, E. crista-galli lectin (ECL; representative of five experiments). D, PNA lectin (representative of five experiments). Yellow circles, galactose; blue square, N-acetylglucosamine; yellow square, N-acetylgalactosamine. E and F, sialidase activity during sperm capacitation. E, sialidase activity of uncapacitated and in vitro capacitated mouse sperm with and without addition of the sialidase inhibitor DANA (representative of eight experiments). 4MU, 4-methylumbelliferyl. F, sialidase activity of uncapacitated and in vitro capacitated human sperm with and without addition of the sialidase inhibitor DANA (representative of eight experiments). G, sialidase activity in female mouse uterine fluid collected during estrous and 1.5 h after mating, capacitated mouse cauda epididymal sperm, and mouse sperm retrieved from the uterus 1.5 h after mating (representative of four experiments). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 versus the control.

FIGURE 2.

Neu1 and Neu3 on sperm and change during capacitation. A, fluorescent staining of Neu1 on mouse sperm head before and after capacitation and counterstained with DAPI and an unspecific IgG control. The change was quantified using FACS and could be partially inhibited by DANA. B, fluorescent staining of Neu3 on mouse sperm head before and after capacitation and counterstained with DAPI and an unspecific IgG control. The change was quantified using FACS and could be partially inhibited by DANA. A and B are representative of 12 experiments each. C, Neu1/3 on mouse sperm in the testes and the proximal and distal epididymis. Arrowheads show positive staining only in mature sperm of the cauda (distal epididymis). HE, hematoxylin and eosin. D, NEU1/3 on human ejaculated sperm. Expression was highest in the hind part of the sperm head. The counterstain is DAPI (blue). ALF488, Alexa Fluor 488. E, release of Neu1 and Neu3 from sperm into the supernatant and the effect of the sialidase inhibitor DANA. U, uncapacitated; C, capacitated; C+I, capacitated with the inhibitor DANA. F, sialidase activity in and release from sperm are both affected by protein inhibitor (PI). ***, p ≤ 0.001 versus the control.

FIGURE 3.

Function of neuraminidase during capacitation. A, zona pellucida (ZP) binding by uncapacitated sperm (U), capacitated sperm (C), and sperm capacitated in the presence of the sialidase inhibitor DANA (C+I). B, Western blot for phospho-ERK of mouse sperm. C, FACS histogram of mouse sperm stained for PY20. D, Western blot for phospho-ERK and PY20 of human sperm (C+Sia) capacitated in the presence of free Neu5Ac. **, p ≤ 0.01 versus the control.

FIGURE 4.

NEU1/3 in human idiopathic subfertility patients. A, FACS analysis of human ejaculated sperm samples stained for NEU1 and NEU3 with normal and abnormal expression levels (each representative of three experiments). B, summary panel of 40 human sperm samples, including 24 fertility patients and 16 normal controls (three independent measures each).

FIGURE 5.

Model of Sia loss during capacitation. Neu1 and Neu3 are responsible for cleaving terminal Sias and are themselves shed. Sialoglycoconjugates such as gangliosides and glycosylphosphatidylinositol (GPI)-anchored sialoglycoproteins are also shed. Unmasked glycoproteins come into action and allow cell signaling during capacitation.