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Effect of monosaccharides on the interaction of equine spermatozoa with oviductal epithelial cells in vitro
198
Theriogenology
EFFECT OF MONOSACCHARIDES ON THE INTERACTION OF EQUINE SPERMATOZOA WITH OVIDUCTAL EPITHELIAL CELLS IN VITRO I. Dobrinski, B.A. Ball, P.G.A. Thomas Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Spermatozoa attach to epithelial cells in the isthmus of the oviduct, forming a sperm reservoir in the female genital tract of several species. This interaction appears to promote sperm longevity and may induce capacitation. Cell surface glycoproteins play an important role in cell to cell contactsWe tested the hypothesis that the attachment of equine sperm to oviductal epithelial cells (OEC) in vitro is mediated by glycoproteins and can therefore be competitively inhibited by monosaccharides. Monolayers of frozen-thawed equine OEC were grown on Matrigel coated wells. Oviductal epithelial cells were washed with modified Tyrode’s solution (TALP) and medium was replaced by either TALP (control) or TALP containing 100 mM of glucose, fructose, galactose, mannose, Nacetvl glucosamine (GlucNAc), N-acetvl galactosamine (GalNAc) or 50 mM N-acetvl neuraminic acid (NANA). Twdejaculates .from each-of 3 stallions (n=6) were washed and stained with the f Jorescent dye Hoechst 33342 (5pg/ml). Co-cultures were established by adding 1 x 106 sperm per culture well (4 wells/treatment), incubated at 38.5”C in 5% CO2 in air, and washed after 30 min and after 2 h of incubation to remove unbound sperm. After 2 h of co-culture the number of attached sperm per field (0.25 mm2) was counted-by fluorescence microscopy and analysis of digitized images. Treatments were applied within eiaculates and data blocked by stallion for ANOVA.The&mber of sperm attached to OEC was reduced (PcO.05) in all treatments except GlucNac when compared to control (Figure 1) and the lowest number of sperm was bound in cultures incubated with galactose and mannose. r To investigate whether the observed results were lkeatment due to an effect of the monosaccharides on suerm NANA cd function, 2 ejaculates from each of 3 stallions (n=6) GalNAc bc were sulit and incubated for 2 h at 38.5”C and 5% ab GlucNAc C 0 2 &in air in the solutions described above. lXi”tlOSe Comparison of motility (by phase contrast galactose microscopy), membrane integrity and acrosomal fructose status (bv simultaneous Hoechst 33258 dve exclusion glucose and staining with FITC-coupled Pisum sativum control agglutinin), and capacitation (by chlortetracycline 0 50 100 150 200 2x staining) were made before and after incubation. No. of sperm bound/O.25 mm2 Treatments were applied within ejaculates and data (mean k SEM) blocked by stallion for ANOVA. Progressive motility Tigure 1. Sperm bound to OEC in vitro and the percentage of viable, acrosome intact sperm means with different letters are was not different between treatments and controls significantly different (P
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Theriogenology
EFFECT OF MONOSACCHARIDES ON THE INTERACTION OF EQUINE SPERMATOZOA WITH OVIDUCTAL EPITHELIAL CELLS IN VITRO I. Dobrinski, B.A. Ball, P.G.A. Thomas Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Spermatozoa attach to epithelial cells in the isthmus of the oviduct, forming a sperm reservoir in the female genital tract of several species. This interaction appears to promote sperm longevity and may induce capacitation. Cell surface glycoproteins play an important role in cell to cell contactsWe tested the hypothesis that the attachment of equine sperm to oviductal epithelial cells (OEC) in vitro is mediated by glycoproteins and can therefore be competitively inhibited by monosaccharides. Monolayers of frozen-thawed equine OEC were grown on Matrigel coated wells. Oviductal epithelial cells were washed with modified Tyrode’s solution (TALP) and medium was replaced by either TALP (control) or TALP containing 100 mM of glucose, fructose, galactose, mannose, Nacetvl glucosamine (GlucNAc), N-acetvl galactosamine (GalNAc) or 50 mM N-acetvl neuraminic acid (NANA). Twdejaculates .from each-of 3 stallions (n=6) were washed and stained with the f Jorescent dye Hoechst 33342 (5pg/ml). Co-cultures were established by adding 1 x 106 sperm per culture well (4 wells/treatment), incubated at 38.5”C in 5% CO2 in air, and washed after 30 min and after 2 h of incubation to remove unbound sperm. After 2 h of co-culture the number of attached sperm per field (0.25 mm2) was counted-by fluorescence microscopy and analysis of digitized images. Treatments were applied within eiaculates and data blocked by stallion for ANOVA.The&mber of sperm attached to OEC was reduced (PcO.05) in all treatments except GlucNac when compared to control (Figure 1) and the lowest number of sperm was bound in cultures incubated with galactose and mannose. r To investigate whether the observed results were lkeatment due to an effect of the monosaccharides on suerm NANA cd function, 2 ejaculates from each of 3 stallions (n=6) GalNAc bc were sulit and incubated for 2 h at 38.5”C and 5% ab GlucNAc C 0 2 &in air in the solutions described above. lXi”tlOSe Comparison of motility (by phase contrast galactose microscopy), membrane integrity and acrosomal fructose status (bv simultaneous Hoechst 33258 dve exclusion glucose and staining with FITC-coupled Pisum sativum control agglutinin), and capacitation (by chlortetracycline 0 50 100 150 200 2x staining) were made before and after incubation. No. of sperm bound/O.25 mm2 Treatments were applied within ejaculates and data (mean k SEM) blocked by stallion for ANOVA. Progressive motility Tigure 1. Sperm bound to OEC in vitro and the percentage of viable, acrosome intact sperm means with different letters are was not different between treatments and controls significantly different (P
F