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After mating, many female mammals store a subpopulation of sperm in the lower portion of the oviduct, forming a reservoir. The reservoir lengthens sperm lifespan, regulates sperm capacitation, controls polyspermy, and selects normal sperm. It is believed that sperm bind to glycans on the oviduct epithelium to form the reservoir, but the specific adhesion molecules that retain sperm are unclear. Herein, using a glycan array to test 377 glycans for their ability to bind porcine sperm, we found two glycan motifs in common among all glycans with sperm-binding ability: the Lewis X trisaccharide and biantennary structures containing a mannose core with 6-sialylated lactosamine at one or more termini. Binding to both motifs was specific; isomers of each motif did not bind sperm. Further work focused on sialylated lactosamine. Sialylated lactosamine was found abundantly on the apical side of epithelial cells collected from the oviduct isthmus, among N-linked and O-linked glycans. Sialylated lactosamine bound to the head of sperm, the region that interacts with the oviduct epithelium. After capacitation, sperm lost affinity for sialylated lactosamine. Receptor modification may contribute to release from the reservoir so that sperm can move to the site of fertilization. Sialylated lactosamine was required for sperm to bind oviduct cells. Simbucus nigra agglutinin or an antibody specific to sialylated lactosamine with a preference for Neu5Acalpha2-6Gal rather than Neu5Acalpha2-3Gal reduced sperm binding to oviduct isthmic cells, as did occupying putative receptors on sperm with sialylated biantennary glycans. These results demonstrate that sperm binding to oviduct 6-sialylated biantennary glycans is necessary for normal adhesion to the oviduct.

The Bacillus megaterium protein production system based on the inducible promoter of the xyl operon (P xylA ) was systematically optimized. Multiple changes in basic promoter elements, such as the ؊10 and ؊35 region and the ribosome-binding site, resulted in an 18-fold increase of protein production compared to the production of the previously established system. The production in shaking-flask culture of green fluorescent protein (Gfp) as a model product led to 82.5 mg per g cell dry weight (g CDW ) or 124 mg liter ؊1 . In fed-batch cultivation, the volumetric protein yield was increased 10-fold to 1.25 g liter ؊1 , corresponding to 36.8 mg protein per g CDW . Furthermore, novel signal peptides for Sec-dependent protein secretion were predicted in silico using the B. megaterium genome. Subsequently, leader peptides of Vpr, NprM, YngK, YocH, and a computationally designed artificial peptide were analyzed experimentally for their potential to facilitate the secretion of the heterologous model protein Thermobifida fusca hydrolase (Tfh). The best extracellular protein production, 5,000 to 6,200 U liter ؊1 (5.3 to 6.6 mg liter ؊1 ), was observed for strains where the Tfh export was facilitated by a codonoptimized leader peptide of YngK and by the signal peptide of YocH. Further increases in extracellular protein production were achieved when leader peptides were used in combination with the optimized expression system. In this case, the greatest extracellular enzyme amount of 7,200 U liter ؊1 , 7.7 mg liter ؊1 , was achieved by YocH leader peptide-mediated protein export. Nevertheless, the observed principal limitations in protein export might be related to components of the Sec-dependent protein transport system.

In many mammals, after semen deposition, a subpopulation of the sperm is transported to the lower oviduct, or isthmus, to form a functional sperm reservoir that provides sperm to fertilize oocytes. The precise molecular interactions that allow formation of this reservoir are unclear. It is proposed that binding of sperm receptors (lectins) to their oviductal cell ligands is accomplished by glycans. Previous results indicated that Lewis trisaccharides are present in glycosphingolipids and O- and N-linked glycans of the porcine isthmus and that Le(X)-containing molecules bind porcine sperm. Immunohistochemistry indicated that the Lewis structures identified by mass spectrometry were, in fact, Lewis X (Le(X)) trisaccharides. These motifs were localized to the luminal border of the isthmus. Assays using fluoresceinated glycans showed that 3-O-sulfated Le(X) (suLe(X)) bound to receptors localized on the head of nearly 60% of uncapacitated boar sperm but that the positional isomer 3-O-sulfo-Le(A) (suLe(A)) bound to <5% of sperm. Sperm also bound preferentially to suLe(X) made insoluble by coupling to beads. Capacitation reduced the ability of suLe(X) to bind sperm to <10%, perhaps helping to explain why sperm are released at capacitation. Pretreatment of oviduct cell aggregates with the Le(X) antibody blocked 57% of sperm binding to isthmic aggregates. Blocking putative receptors on sperm with soluble Le(X) and suLe(X) glycans specifically reduced sperm binding to oviduct cells up to 61%. These results demonstrate that the oviduct isthmus contains Le(X)-related moieties and that sperm binding to these oviduct glycans is necessary and sufficient for forming the sperm reservoir.

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