Notochordal stimulation of in vitro somite chondrogenesis before and after enzymatic removal of perinotochordal materials☆

[摘要] Inthepresentinvestigation,evidenceispresenteddirectlyimplicatingproteoglycansproducedbytheembryonicnotochordinthecontrolofsomitechondrogenesis.Ithasbeendemonstratedbyseveralhistochemicaltechniquesthatduringtheperiodofitsinteractionwithsomites,thenotochordsynthesizesperinotochordalproteoglycans,andtheseproteoglycanshavebeenshowntocontainchondroitin4-sulfate(40%),chondroitin6-sulfate(40%),andheparansulfate(20%).Dissectionofnotochordsfromembryoswiththeaidofabrieftreatmentwithtrypsinresultsintheremovalofperinotochordalextracellularmatrixmaterialsincludingproteoglycans,whiledissectionofnotochordswithouttheaidofenzymetreatmentorwithalowconcentrationofcollagenaseresultsintheirretention.Thereisaconsiderableincreaseintherateandamountofcartilageformationandacorresponding2to3-foldincreaseintheamountofsulfatedglycosaminoglycanaccumulatedbysomitesculturedinassociationwithnotochordsdissectedunderconditionsinwhichperinotochordalmaterialsareretained.Treatmentofcollagenase-dissectedorfreelydissectednotochordswithhighlypurifiedenzymes(chondroitinaseABC,AC,andtesticularhyaluronidase)whichspecificallydegradeproteoglycanscausesalossofhistochemicallydetectableperinotochordalproteoglycans.Thesenotochordsareconsiderablyimpairedintheirabilitytosupportinvitrosomitechondrogenesis.Inaddition,whentrypsin-treatednotochordsarecultured(“precultured”)for24hronnutrientagar(intheabsenceofsomites),perinotochordalmaterialreaccumulates.Somitesculturedinassociationwithsuch“precultured”notochordsexhibitconsiderableincreaseintheamountofcartilageformedanda2-to3-foldincreaseintheamountofsulfatedglycosaminoglycanaccumulatedascomparedtosomitesculturedinassociationwithtrypsin-treatednotochordswhichhavenotbeen“precultured.”Thisobservationindicatesthattrypsin-treatednotochordsreacquiretheirabilitytomaximallystimulateinvitrosomitechondrogenesisbyresynthesizingandaccumulatingperinotochordalmaterial.Finally,“precultured”notochordstreatedwithchondroitinasetoremoveperinotochordalproteoglycansareconsiderablyimpairedintheirabilitytosupportinvitrosomitechondrogenesis.Theseobservationsareconsonantwiththeconceptthatproteoglycansproducedbytheembryonicnotochordplayanimportantroleinsomitechondrogenesis.

[学科分类] 生物科学（综合）