ULTRASTRUCTURAL AND IMMUNOCYTOCHEMICAL EVIDENCE FOR THE PRESENCE OF POLARIZED PLASMA-MEMBRANE H-ATPASE IN 2 SPECIALIZED CELL-TYPES IN THE CHICK-EMBRYO CHORIOALLANTOIC MEMBRANE()

The chick embryo, confined in the eggshell, has to dispose/buffer the acid generated by its metabolism, as well as to release calcium from t he shell which is used for growth. To localise H+-ATPase, electron mic roscope and immunocytochemical studies were conducted on chorioallanto ic membranes of 15-17 d chick embryos. Ultrastructural studies of the villus cavity (VC) cells in the chorionic epithelium demonstrated that their apical plasma membrane, juxtaposed with the shell membranes, co ntains microvilli as well as microplicae which possess 9-10 nm studs a t a density of 16700 particles/mu m(2), a characteristic feature of th e polarised H+-ATPase pump. Immunocytochemical staining, using a monoc lonal antibody to the 31 kDa subunit of H+-ATPase, confirmed the prese nce of large amounts of the vacuolar H+-ATPase in the VC shells with a distribution highly polarised towards the eggshell membranes. Immunoe lectronmicroscopic localisation studies using a rabbit antiserum to wh ole bovine Hi-ATPase and immunogold technique, confirmed the localisat ion of H+-ATPase at the apical microvilli/microplicae as well as in th e subapical vesicles. In the allantoic epithelium, the presence of mit ochondria-rich (MR) cells was confirmed; it-was shown that these cells extend through the full thickness of this epithelium. The MR cells al so contained large numbers of 9-10 nm studs, typical of proton secreti ng cells, in their apical plasma membrane. This was confirmed by immun ocytochemical staining which showed abundant localisation of H+-ATPase in these cells; this localisation was, however, diffuse rather than a pical. We conclude that VC cells in the chorionic epithelium, and most (if not all) MR cells in the allantoic epithelium are able to secrete acid, and hence contribute to the acid-base homeostasis in the chick embryo. Our localisation of H+-ATPase suggests that VC cells secrete H + towards the eggshell which is buffered by calcium carbonate, resulti ng in release of bicarbonate and calcium, which in turn will be used b y the chick embryo for further buffering of the generated acid and for bone mineralisation, respectively. The MR cells secrete H+, analogous to kidney intercalated cells, and may be responsible for the progress ive acidification of the embryonic urine.