Effect of culture-medium supplementation with α-mannosidase and/or β-N-acetyloglucosaminidase on in vitro bovine embryonic development

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  Effect of culture-medium supplementation with α-mannosidase and/or β-N-acetyloglucosaminidase on in vitro bovine embryonic development
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  Animal Reproduction Science 99 (2007) 208–212 Short communication Effect of culture-medium supplementation with  -mannosidase and/or   -  N  -acetyloglucosaminidase onin vitro bovine embryonic development Theodora Tsiligianni a , ∗ , Leen Vandaele b ,Aart de Kruif  b , Ann Van Soom b a  NAGREF, Veterinary Research Institute of Thessaloniki, Ionia, Thessaloniki 57008, Greece b  Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University,Salisburylaan 133, B-9820 Merelbeke, Belgium Received 11 February 2006; received in revised form 6 June 2006; accepted 12 June 2006Available online 25 July 2006 Abstract Glycosidases are enzymes with a potential role in embryonic development. The objectives of this studywere to assess: (a) whether in vitro bovine embryonic development is affected by the addition of    -  N  -acetyloglucosaminidase (  -NAGASE) and/or   -mannosidase to the culture medium and (b) whether theseenzymes are utilized by bovine embryos during their development in vitro. Bovine embryos were producedusing standard methods of IVM, IVF and IVC. Presumptive zygotes were cultured in groups of 20 in 50  ldropsofSOFmedium(plus5%FBSafter24hculture)incubatedin5%CO 2 ,5%O 2  and90%N 2  at38.5 ◦ C.Thegroupsofzygoteswereallocatedtofourtreatmentsinwhichtheculturemediumwassupplementedwith:(1)  -NAGASE, (2)  -mannosidase, (3)  -NAGASE plus  -mannosidase, and (4) control (no supplement).Embryoswereevaluatedandsamplesofculturemediumcollectedandfrozenpriortoassayforglycosidasesatday7ofculture.Theexperimentaldesignwasarandomisedblockarrangementof4treatments × 7replicateswith 20 zygotes per plot (culture droplet). Data were analysed by ANOVA and presented as mean ± S.E.M.The osmolarity of the control culture medium was 272mOsm. This was increased to 279mOsm by the addi-tion of   -mannosidase, 424mOsm by  -NAGASE and 337mOsm with a combination of the two enzymes.The   -NAGASE supplemented medium and the combined supplement reduced (0%) the development of zygotes to morula or blastocyst stages ( P <0.002) relative to control medium (35.7 ± 8.4%). Embryo devel-opment was also reduced to 21.9 ± 3.2 ( P <0.002), relative to control, by   -mannosidase supplementation.The reduced embryo development in the   -NAGASE-supplemented medium was attributed to increasedosmolarity of the culture medium. Embryos appeared to utilize   -mannosidase because its concentrationdecreasedfrom600.95 ± 174.03IU/lindropswithoutzygotes/embryosto211.01 ± 71.59IU/lindropswith ∗ Corresponding author. Tel.: +30 2310310536; fax: +30 2310781161.  E-mail address:  tsiligianni@nagref.gr (T. Tsiligianni).0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.anireprosci.2006.06.002  T. Tsiligianni et al. / Animal Reproduction Science 99 (2007) 208–212  209 zygotes/embryos. Other culture media supplementation showed no significant differences between droplets,withorwithoutzygotes/embryos.Itwasconcludedthat  -NAGASEincreasedmediumosmolarity,embryosutilized  -mannosidaseandbothglycosidases(singlyorincombination)inhibitedthedevelopmentofbovinezygotes to morulae/blastocysts.© 2006 Elsevier B.V. All rights reserved. Keywords:  Glycosidases; Embryos; In vitro; Bovine; Development 1. Introduction Traditionally,glycosidasesareconsideredintracellularenzymesthatdegradeoligosaccharideson glycoconjugates destined for the endocytic/lysosomal pathway (Miller et al., 1993). However, glycosidases can also be secreted extracellularly, where they may facilitate tumor cell metastasis(rapid division) in collaboration with protease activities (Liotta et al., 1986). It is not known whether the modified structure of glycosidases in tumors is a consequence of rapid growth orwhether this is a primary cause of the rapid growth, possibly by altering membrane permeability,thusallowingentryofimportantmetabolites(Holley,1972).Embryosalsodeveloprapidlyduring earlypregnancyandthepresenceoftheseglycosidasesintheculturemediummayhavesignificanteffects on early embryonic development.During embryogenesis, carbohydrate moieties may influence cell–cell adhesion and importantdevelopmentalprocesses,suchascompactionandblastulation,ashasbeenshownintheseaurchin(Khurrumetal.,2004),butdatainmammalsarescarce.Theexactroleoftheseenzymesinembryo development is not known. The cell coat or glycocalyx is composed of cell surface glycans, suchas glycoproteins and glycolipids, which encode information that modulates interactions betweencells by specifically regulating the binding to cell surface-associated or soluble carbohydrate-bindingreceptors,suchaslectins.Closeinteractionofcellscanbeimportantatfertilization,whenthe sperm meets the oocyte, and the involvement of carbohydrates during fertilization in cattlehasbeenwellestablished(Tangheetal.,2004).Lectinsspecificfor  - d -methyl-mannopyranoside(ConA)and  - d -mannose(LCA)havebeendetectedinplasmalemma,theperivitellinespaceandzona pellucida of fertilized oocytes but not in unfertilized oocytes (Hoodbhoy and Talbot, 2001).However, lectins specific for  N  -acetylglucosamine (WGA) were present on the zona pellucida of unfertilized oocytes but not on fertilized oocytes (Hoodbhoy and Talbot, 2001).It is not known: (a) if embryos utilize   -NAGASE and   -mannosidase during development,or (b) if, in embryonic development,  -NAGASE and  -mannosidase act as putative inhibitors orpromoters. The aims of the present study were: (a) to investigate the effects of these glycosidaseson in vitro embryonic development and (b) to determine if embryos utilize these enzymes duringdevelopment. 2. Material and methods 2.1. In vitro production of embryos Bovine embryos were produced by routine in vitro methods, as described previously(Tsiligianni et al., 2006). Washed zygotes were transferred to SOF and cultured in 50-  l dropletsunder mineral oil in groups of 20. Five percent FCS was added to each droplet 24h after transferto SOF. Embryos were cultured in an atmosphere of 5% CO 2 , 5%O 2  and 90% N 2  at 38.5 ◦ C.  210  T. Tsiligianni et al. / Animal Reproduction Science 99 (2007) 208–212 2.2. Experimental design Embryos were cultured in SOF medium supplemented with 5% FCS and with two differentglycosidase (  -NAGASE and   -mannosidase) preparations from  Canavalia ensiformis  (Jack bean) (Sigma, Bornem, Belgium). After IVM and IVF, presumed zygotes were divided intofour treatment groups (each replicated seven times with 20 zygotes per plot or culture droplet),18–24hafterfertilization.Ingroup1,embryoswereculturedinSOFwith100  g/ml  -NAGASE.Group 2 consisted of SOF with 2  g/ml   -mannosidase. In group 3, a combination of 50  g/ml  -NAGASE and 1  g/ml   -mannosidase was used. Group 4 was control without glycosidases.The concentration of enzymes added was based on previous results obtained during in vivoexperiments (Tsiligianni et al., 2005). All embryos were cultured in 50-  l droplets under mineraloil in groups of 20. Samples were collected at day 7 and embryonic development assessed.Osmolarity of culture media was measured immediately after the addition of   -NAGASE and  -mannosidase. 2.3. Measurement of glycosidase activity Aliquots of 5  l of culture medium were diluted in 495  l distilled water and 0.1ml of thissample used for assay of each glycosidase. Glycosidase activity was determined in all samples asdescribed previously (Tsiligianni et al., 2006). Results were expressed in IU/l. 2.4. Statistics Singledegreeoffreedomorthogonalcontrastswithanalysisofvariance(ANOVA)wasusedtoevaluatesignificantdifferencesamonggroups.DistributionwasnormalizedbyarcsinesquarerootpriortoANOVA.Untransformedmean ± S.E.M.arepresentedbutstatisticaltestswerecarriedouton transformed data. Furthermore,  t  -test for independent samples was used to compare enzymesactivity between groups. The results were expressed as mean ± S.E.M. 3. Results The development of embryos was negatively affected ( P <0.002) by addition of    -NAGASEsince no embryos developed when this enzyme was present, either alone or in combination, inthe culture medium compared to controls (35.7 ± 8.4%). Furthermore, fewer ( P <0.002) goodembryos (21.9 ± 2.0% morulae and blastocysts) developed when   -mannosidase was added toculture medium compared to control group.It appears that embryos utilize   -mannosidase during development, since it was obvious ingroup 2 that the presence of embryos reduced the amount of added   -mannosidase significantly(600.95 ± 174.03IU/l in droplets without embryos compared to 211.01 ± 71.59IU/l in dropletswith embryos). For the other combinations, no significant differences were detected (data notshown).Osmolarity of culture medium increased to 424mOsm after the addition of    -NAGASEand to 337mOsm after the addition of a combination of the two enzymes, while the additionof    -mannosidase (279mOsm) did not affect osmolarity compared to control SOF medium(272mOsm).  T. Tsiligianni et al. / Animal Reproduction Science 99 (2007) 208–212  211 4. Discussion Rapid modification of exposed carbohydrate moieties by glycosidases and glycosyltrans-ferases, and the equally dynamic patterns of their receptor expression during early development,suggest that both play important roles during embryogenesis, which has been studied in zebrafish(Vasta et al., 2004). In mammals, glycosyl transferases are associated with the free surface of the cell (Schlafke and Enders, 1975) and play a role in embryo implantation, since, to change from a non-adhesive to an adhesive state, the embryo must undergo a shift in the number, distributionand position of carbohydrate molecules on the cell surface coat (Schlafke and Enders, 1975). The mechanism by which glycosidases influence mammalian embryonic development is unclear asyet.Thus,glycosidaseswereaddedtotheculturemediumtostudytheeffectofthepresenceoftheseenzymes on embryo development. When culture medium was supplemented with  -mannosidasefewer good embryos developed compared to controls, which is in contrast to earlier findingswith rabbit embryos (Kane, 1986). Khurrum et al. (2004) found that the inhibitors of glyco- protein/proteoglycan synthesis, tunicamycin and sodium selenate, and the specific glycosidases,  -amylase,  -glycosidaseand  -mannosidase,allinhibitarchenteronorganization,elongationandattachment to the blastocoel roof in viable, swimming sea urchin (  Lytechinus pictus ) embryos.They suggested that the supernatant contains ligands and/or receptors, mediating archenterondevelopment and attachment to the blastocoel roof, which are released when embryos are disag-gregated into single cells. Thus, the presence of   -mannosidase in culture medium could mediatearchenteron development and have a negative effect on embryo development in invertebrates.In the present study, we found that the addition of    -NAGASE and a combination of bothglycosidases to culture medium inhibited the development of all embryos. To ascertain the reasontheosmolarityoftheculturemediumwasmeasuredimmediatelyafteradditionoftheglycosidases.The addition of    -NAGASE or a combination of the two enzymes increased osmolarity of theculture medium substantially, while the addition of    -mannosidase did not. Thus, the negativeeffect of    -NAGASE on embryo development is due to increased osmolarity of the medium.We are confident that the negative effect on embryonic development is due to high osmolaritycaused by the addition of    -NAGASE. However, the reason for the increase in osmolarity isunknown:itwasnotduetoanerrorindilutionandremainstobeclarified.Increasedosmolaritycannegativelyaffectembryonicdevelopment.McKiernanandBavister(1990)f oundthatdevelopment to blastocysts was reduced to 11% at an osmolarity of 350mOsm compared to 56.5% for anosmolarity of 275mOsm. The change in osmolarity observed after addition of    -NAGASE toSOF medium containing FCS is not due directly to the osmolarity-producing effects of enzymes.Itisknownthatosmolaritydependsonthenumberofparticlesandhighosmolaritiesareproducedby low-molecular weight (MW) compounds, such as Na, Cl, glucose and amino acids. Thus,largeweightconcentrationsofproteinswouldonlyproduceariseinosmolarityofabout1mOsm.However, the observed increase in osmolarity may arise from enzyme break-down of large MWcompounds in the FCS to low MW compounds.Theotherinterestingfindingwasthattheconcentrationof   -mannosidasedecreasedindropletscontaining embryos. Therefore, embryos might utilize or metabolize  -mannosidase during theirdevelopment. The activity of   -mannosidase exposed to 20 embryos was 300–400IU/l, but it wasnotpossibletomeasuretheexactenzymeconcentration.About2  gof   -mannosidaseand100  gof   -NAGASEwereaddedtoculturemediumandenzymeactivitymeasured(IU/l).Thedecreasein  -mannosidase activity in droplets with embryos, compared to droplets without embryos, maybe due to inactivation of the enzyme by the embryos during development.  212  T. Tsiligianni et al. / Animal Reproduction Science 99 (2007) 208–212 5. Conclusions  -NAGASEincreasesosmolarityandinhibitsthedevelopmentofembryoswhenaddedtocul-turemedium.Furthermore,  -mannosidaseinhibitsembryonicdevelopmentbutdoesnotincreaseosmolarity. Embryos may metabolize   -mannosidase during development. However, the exactpathway of the action of the glycosidases it is unknown and remains to be clarified. Acknowledgement Collaborator, via a fellowship under OECD Co-operative Research Programme: BiologicalResource Management for Sustainable Agriculture Systems, is acknowledged. References Holley, R.W., 1972. A unifying hypothesis concerning the nature of malignant growth. Proc. Natl. Acad. Sci. U.S.A. 69,2840.Hoodbhoy, T., Talbot, P., 2001. Characterization, fate and function of hamster cortical granule components. Mol. Reprod.Dev. 58, 223–235.Kane, M.T., 1986. A survey of the effects of protease and glycosidases on culture of rabbit morulae to blastocysts. J.Reprod. Fertil. 78, 225–230.Khurrum, M., Hernandez, A., Eskalaei, M., Badali, O., Coyle-Thompson, C., Oppenheimer, S.B., 2004. Carbohydrateinvolvement in cellular interactions in sea urchin gastrulation. Acta Histochem. 106, 97–106.Liotta, L.A., Rao, C.N., Wewer, U.M., 1986. Biochemical interactions of tumor blocks human sperm penetration of thezona pellucida. Biol. Reprod. 48, 340–348.McKiernan, S.H., Bavister, B.D., 1990. Environmental variables influencing in vitro development of hamster 2-cellembryos to the blastocyst stage. Biol. Reprod. 43, 404–413.Miller, D.J., Gong, X., Shur, B.D., 1993. Sperm require   -  N  -acetyloglucosaminidase to penetrate through the egg zonapellucida. Development 118, 1279–1289.Schlafke, S., Enders, A.C., 1975. Cellular basis of interaction between trophoblast and uterus at implantation. Biol.Reprod. 12, 41–65.Tanghe, S., Van Soom, A., Duchateau, L., Nauwynck, H., de Kruif, A., 2004. Carbohydrates and glycoproteins involvedin bovine fertilization in vitro. Mol. Reprod. Dev. 68, 492–499.Tsiligianni, Th., Amiridis, G.S., Vainas, E., 2005. Glycosidases activity in the uterine luminal fluid of cows after super-ovulation. Reprod. Domest. Anim. 40, 361 (abstract).Tsiligianni, T., Vandaelen, L., de Kruif, A., Van Soom, A., 2006. Role of two glycosidases (  -mannosidase and   -  N  -acetylglucosaminidase) on in vitro bovine embryonic development. Reprod. Domest. Anim. 41, 149–152.Vasta, G.R., Ahmed, H., Du, S., Henrikson, D., 2004. Galectins in teleost fish: Zebrafish (  Danio rerio ) as a model speciesto address their biological roles in development and innate immunity. Glycoconj. J. 21, 503–521.
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