Abstract
A fraction of sperm deposited at mating or insemination reaches the oviduct isthmus, where sperm are retained and thereby form a reservoir. This reservoir delays capacitation, prevents polyspermy, selects a fertile population of sperm, and, foremost, increases sperm lifespan. The molecular interactions underlying the formation of a sperm reservoir are becoming clearer in mammals. Sperm lectins bind to oviductal glycans to form the reservoir. Herein, we found that the highest percentage of bovine sperm bound to the 3'-O-sulfated form of Lewis A (suLeA) trisaccharide and sialylated Lewis A and that fluoresceinated versions of each localized to receptors on the anterior head of the sperm. Following capacitation, binding to suLeA decreased significantly, a potential explanation for sperm release from the reservoir. MS and immunohistochemistry analyses indicated that suLeA motifs were present predominantly on O-linked glycans initiated by GalNAc residues, but no sialylated Lewis A was detected. To determine whether sperm binding to isolated suLeAin vitro could mimic in vivo sperm binding to oviduct cells and increase sperm longevity, we immobilized suLeA and incubated it with sperm. Using free-swimming sperm and sperm bound to immobilized laminin as controls, we observed that over 96 h, the viability of free-swimming sperm decreased to 10%, and that of sperm bound to immobilized laminin decreased to about 50%, whereas viability of sperm bound to immobilized suLeA was highest throughout the incubation and 60% at 96 h. These results indicate that bovine sperm binding to oviduct suLeA retains sperm for reservoir formation and extends sperm lifespan.
Highlights
A fraction of sperm deposited at mating or insemination reaches the oviduct isthmus, where sperm are retained and thereby form a reservoir
Using free-swimming sperm and sperm bound to immobilized laminin as controls, we observed that over 96 h, the viability of free-swimming sperm decreased to 10%, and that of sperm bound to immobilized laminin decreased to about 50%, whereas viability of sperm bound to immobilized sulfated form of Lewis A (suLeA) was highest throughout the incubation and 60% at 96 h
Using Lewis A (LeA) covalently linked to a fluoresceinated 30-kDa polyacrylamide core to mimic glycan attachment to glycoproteins, we found that LeA bound to less than 10% of sperm, but a 3Ј-O-sulfated version of LeA bound to the apical ridge of more than 30% of the sperm (Fig. 1, A and B)
Summary
A fraction of sperm deposited at mating or insemination reaches the oviduct isthmus, where sperm are retained and thereby form a reservoir. Using free-swimming sperm and sperm bound to immobilized laminin as controls, we observed that over 96 h, the viability of free-swimming sperm decreased to 10%, and that of sperm bound to immobilized laminin decreased to about 50%, whereas viability of sperm bound to immobilized suLeA was highest throughout the incubation and 60% at 96 h These results indicate that bovine sperm binding to oviduct suLeA retains sperm for reservoir formation and extends sperm lifespan. We examined porcine sperm binding to 377 glycans using a high-throughput array and discovered that uncapacitated sperm bind with high affinity to glycans with either a Lewis X (LeX) trisaccharide motif or biantennary structures containing a mannose core with 6-sialylated lactosamine at one or more termini [24]. The abbreviations used are: LeX, Lewis X; LeA, Lewis A; MSn, sequential MS; PI, propidium iodide; GSL, glycosphingolipid; LacdiNAc, GalNAc14GlcNAc; TIM, total ion mapping; NL, neutral loss; DIC, differential interference contrast; NSI, nanospray ionization; Man, mannose; Lac, lactose; Hex, hexose; LacNAc, N-acetyl-D-lactosamine; HexNAc, N-acetylhexosamine
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