‘Supported by grants from the Medical Research Council of Can-

991

0022-I 551/82/10099 108S02.75The Journal of Histochemistry and CytochemistryCopyright 1/: 1982 by The Histochemical Societ , Inc.

Vol. 30. NI). 10. pp. 991-998, 1982in (Si).

IIIntracellular Localization of Basement MembranePrecursors in the Endodermal Cells of the Rat ParietalYolk Sac.

III. Immunostaining for Laminin and Its Precursors’

G.W. LAURIE, C.P. LEBLOND, G.R. MARTIN, and M.H. SILVER

Department ofAnatomy (G.W.L.: C.P.L.. McGill Unizersity. Montreal. Quebec. Canada H3A 2B2 and National institute of DentalResearch !G.R.M.; M.H.Sj. National Institutes ofHealth. Bethesda. Maryland

Received for publication September 30, 1981 and in revised form March 3, 1982; accepted March 4, 1982 (OA 81-250)

Reichert’s membrane and the endodermal cells of the par-ietal yolk sac were examined for the presence of lamininantigenicity using anti-laminin antibodies and the per-oxidase-antiperoxidase sequence. Immunostaining was ob-served through the full width of Reichert’s membrane andwithin endodermal cells. In these cells immunostainingwas observed in rough endoplasmic reticulum (rER) cis-ternae and Golgi apparatus. The Golgi staining could oc-cur in any saccule, but predominated in components in-terpreted as the last saccule of the stack, the GERL element,and associated prosecretory granules. The secretory gran-ules found in the ectoplasm were also immunostained. Fi-nally, multivesicular bodies showed some staining. Theimmunostaining of Reichert’s membrane indicates the

IntroductionLaminin M. 0.8-1.0 x 10 (25)) is a glycoprotein (2,20,24)

comprised oftwo different polypeptide chains (9,24). By light

microscopic immunostaining laminin has been localized to

basement membranes in various organs ofthe body (6,12,17,23).In addition, laminin has been biochemically extracted fromReichert’s membrane (9,10,22) and from the matrix of base-

ment membrane tumors (2,24).

Biochemical studies have been carried out on the biosyn-

thesis of laminin using cultures of the endodermal cells from

the parietal yolk sac (9,10,21,22). However, no attention has

been paid to the cellular organelles involved. As well, it wasnot sure from light microscopic immunostaining reports whether

in Reichert’s membrane, laminin occurred only on the en-

dodermal cell side (27) or throughout its thickness (16,21). In

ada.

presence of laminin itself, while that of rER cisternae andthe Golgi apparatus is attributed to laminin precursors.Presumably the biosynthesis of laminin occurs along theusual protein pathway, that is, from rER through Golgisaccules and the GERL element to secretory granules, whichrelease their content into Reichert’s membrane. The lam-mm immunostaining of Reichert’s membrane and endo-dermal cells is similar to that of type IV collagen. It is,therefore, likely that the two substances are processed andsecreted simultaneously.KEY WORDS: Laminin; Anti-laminin antibodies; Reichert’s mem-brane; Endodermal cell; Rough endoplasmic reticulum; Golgiapparatus; GERL element; Prosecretory granules; Secretorygranules.

the present work, parietal endodermal cells and Reichert’smembrane ( 1 1) were immunostained with anti-laminin anti-bodies for light and electron microscopy.

Materials and MethodsPreparation of laminin and anti-laminin antibodies. Laminin

was extracted from the mouse EHS tumor with 0.5 M NaCI in Tris-HCI (pH 7.4), purified by ion exchange chromatography (24), andused to immunize New Zealand white rabbits. The antiserum obtainedwas then passed over columns of Sepharose-bound type IV collagen,fibronectin, and heparan sulfate proteoglycan to remove possible con-taminating antibodies. Anti-laminin antibodies were then extracted bySepharose-bound laminin.

To test binding specificity, 0.024 mg ofanti-laminin antibodies in

0.2 ml were incubated overnight at 4#{176}Cwith 0.28 mg oflaminin boundto Sepharose (0.2 ml). When the supernatant was exposed withoutdilution to frozen sections of the rat incisor tooth for immunostainingby the peroxidase-antiperoxidase (PAP) technique (4), there was noreaction of the basement membranes, whereas untreated antibodiesdiluted to an equivalent amount stained them strongly ( 12). When

. ‘

992 LAURIE, LEBLOND, MARTIN, SILVER

the antibodies were tested by enzyme-linked immunoassay (ELISA)(19), they showed a titer with laminin of 1:10,000 (as defined in ref.26 and no cross-reaction with type IV collagen, heparan sulfate, or

fibronectin.

Preparation of parietal yolk sac specimens and immuno-staining. Parietal yolk sacs were fixed in 5% formaldehyde (14). Forlight microscopy, frozen sections were exposed to a 1 : 1 5,000 dilutionof the antiserum followed by the PAP sequence (4).

For electron microscopy, pieces ofparietal yolk sac, fixed as above,were chopped into 25 jam slices, incubated overnight at 4#{176}Cwith anti-laminin antibodies (0.06 mg/ml), washed, and immunostained by theindirect PAP procedure. This procedure, performed at room tem-perature, involved a 15 mm incubation with goat anti-rabbit immu-noglobulin (Ig) G (1:50; Cappel Labs), a 40 mm wash, a 4 mm in-cubation in rabbit PAP (1:50; Cappel Labs), a 30 mm wash, and thenpostfixation and further treatment as described in the previous articlefor the direct method ( 14 ). For control, peroxidase-linked nonimmunerabbit IgG (0.01 mg IgG/ml; Cappel Labs) was used.

ResultsSections exposed to anti-laminin antiserum or antibodies dis-

played immunostaining throughout the entire width of Reich-ert’s membrane, as well as in parietal endodermal cells, butnot in trophoblast cells, as shown by light microscopy (Figure

1). In the electron microscope (Figure 2), the rough endo-

plasmic reticulum (rER) cisternae ofendodermal cells showed

Figure 2. Electron micrograph of anendodermal cell on Reichert’s mem-brane from the parietal yolk sac of arat at 1 3 days’ gestation. Formalde-hyde-fixed yolk sacs were cut into 25 am slices and exposed to anti-lamininantibodies, then passed through thePAP sequence, stained with diamino-benzidine-H2O2 and Epon embedded.Thin sections were made from the sur-face of the slices. Subsequent figures,except where otherwise noted, wereprepared in the same manner, usinganti-laminin antibodies and 13 days’pregnant rats.

Densities representing diamino-benzidine polymerized by the reactionof the peroxidase moiety of the PAPwith H2O2 are observed over Reich-ert’s membrane. In the cells, densitiesmay be seen within organelles that arepresumed to have been cut open bythe tissue chopper when slices weremade. Thus reaction may be seen withinmost rER cisternae, but not all (un-stained cisternae at top right). In theGolgi apparatus several stacks arestained, one ofwhich is labeled “Golgi.”The overall weak staining due to os-mium tetroxide helps in the resolutionof the nucleus (N), mitochondria, andlipid droplets (Li), none ofwhich con-tan reaction product. Original mag-nification x 10,000.

S.,s_i. .

Od . S TrophFigure 1. Light micrograph of a frozen section of parietal yolk sacimmunostained by incubation with anti-laminin antiserum (1:15,000)followed by the PAP method. The full width of Reichert’s membrane(R Mb) is intensely stained. The endodermal cells nearby show densedeposits of immunostain in the cytoplasm, but not the nucleus. Onlyslight staining is seen over trophoblast cells (Troph; 14 days’ gestation).Original magnification x 800.

LAM! NIN ANTIGENICITY IN ENDODERMAL CELLS 993

Figure 3. Anti-laminin immuno-staining of the Golgi region in an en-dodermal cell. The Golgi apparatus iscomposed of several stacks of circu-larly arranged saccules. The last sac-cule of the stack adjacent to the trans(tr) region is immunostained (arrow).Its immunostaining is irregular andtends to predominate along the innerwalls. In the trans region, twoimmunostained rER cisternae are seen.These are similar to the homogene-ously immunostained rER cisternae atupper left. The lack ofstaining in someGolgi saccules is possibly explained bynonpenetrance of antibodies, sinceimmunostaining is dependent on sac-cules being opened by the chopperknife. Original magnification x 48,000.

Figure 4. Uranyl acetate and lead cit-rate stained, glutaraldehyde-fixedpreparation of a portion of an endo-dermal cell at 1 3 days’ gestation forcomparison with the immunostainedGolgi stacks in Figures 5 and 6. Herethe stack is made up of five saccules,with the first saccule adjacent to thecis region being lightly stained. Theother saccules have a grey content. Acircular profile in the trans region (tr;arrowhead) is of the type described asputative prosecretory granule ( 1 1).Original magnification x 87,500.

Figure 5. Anti-laminin immuno-staining of a Golgi stack in an endo-dermal cell. Strong staining is seen inwhat appears to be the last saccule (ar-row) before the trans region (tr). Be-low this saccule is a stained circularprofile (arrowhead) similar to that ob-served in Figure 4 and considered tobe a prosecretory granule. At the topof the micrograph, portions ofimmunostained rER cisternae can beseen. Original magnification x 50,000.

evenly distributed dots of immunostaining (Figures 2,6). InGolgi saccules, the immunostaining predominated along theinner surface of the walls (Figure 3). Reaction could be found

in any of the saccules comprising a Golgi stack (Figure 8), but

was most common in those facing the trans region (Figures

3,9) and in GERL elements (Figures 6,9). The trans region

also included immunostained prosecretory granule-like pro-

files, which could be circular (Figure 5 versus Figure 4; Figure

6) or rectangular (Figure 6 versus Figure 7). Somewhat largercircular and rectangular secretory granule-like profiles were

found free in the ectoplasm (not shown) or fused with the

plasmalemma near Reichert’s membrane (Figures 10, 1 1). Their

content was darkly stained except for a light peripheral rim.In Figure 12, what appears to be an expansion of Reichert’s

membrane enclosed by cell membrane is attributed to thedischarge of several of these structures.

Multivesicular bodies were also immunostained (Figures

14-16 versus Figure 13).

DiscussionLaminin immunostaining of Reichert’s membrane was recently

described in the light microscope (16,21,27). In two of the

reports, the illustrations suggested that immunostaining ex-tended through the membrane (16,21), while in a third one,the membrane was said to be unstained, except for a thin linein contact with the endodermal cells (27). The present obser-vations demonstrated that immunostaining occurred through-

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LAMININ ANTIGEN1CITY IN ENDODERMAL CELLS 995

Figure 6. Anti-laminin immunostaining of the Golgi region in an en-dodermal cell. The Golgi stacks are circularly arranged, but cut obliquelyso that identification of the parts is difficult. However, the stack atright shows the cis and trans (tr) regions, with immunostaining atseveral levels (in part of the first saccule at the short black arrow, andin the intermediate and last saccules above and left). In the trans regionin the middle, several circular profiles (hollow arrows) are ofthe samevariety as those observed in Figures 4 and 5 and, like them, areidentified as prosecretory granules. Their immunostaining tends to belight in a narrow rim at the periphery and dark in the center. Juttinginto the trans region, are two parallel cylindrical structures (arrowhead)comparable to the pair in Figure 7 and, likewise, identified as prose-cretory granules. Adjacent to these at left is a weakly stained andobliquely cut GERL element. At bottom right are parallel rER cis-ternae in which immunostaining appears evenly distributed. At upperleft is the nucleus. Original magnification x 48,800.

Figure :7. Uranyl acetate and lead citrate stained preparation of aportion of the Golgi apparatus in a glutaraldehyde-fixed endodermalcell. In this stack of five saccules the saccule adjacent to the cis regionis characteristically fenestrated and lightly stained; the remaining sac-cules have a more darkly stained content. Of particular interest aretwo parallel cylindrical structures in the trans (tr) region; these havea narrow lightly stained rim surrounding a darkly stained content andmay be associated with coated vesicles (arrowhead). They are likelyto be prosecretory granules associated with a GERL element. Originalmagnification x 62,500.

out Reichert’s membrane. Since laminin had been biochemi-cally extracted from the membrane (9, 10,22), it was likely that

the antigenicity detected by immunostaining was due to lam-

mm itself. It is proposed, therefore, that laminin is presentthroughout the thickness of Reichert’s membrane.

Figure 9. Two continuous Golgi stacksin which reaction is seen along what isconsidered to be their last saccule (ar-row). At the arrowhead, a GERL ele-ment is weakly reactive. Other sac-cules show no reaction except for asmall portion of the first saccule. Thenucleus and a mitochondrion, both Un-stained, are seen at upper left. At cen-ter right is a stained rER cisterna. Orig-inal magnification x 60,000.

Figure 8. Anti-laminin immunostaining in portions of the Golgi ap-paratus in endodermal cells. Reaction is present in the fenestratedsaccule on the cis side, in the fourth, fifth, and sixth saccules and ina somewhat cylindrical structure in the trans (tr) region. The stack liesbetween an immunostained partially distended rER cisterna and theunstained nucleus (N). Original magnification x 45,000.

The parietal endodermal cells displayed immunostaining inthe organelles of the classical secretory pathway, that is, rER,Golgi apparatus, and secretory granules. Recently Cooper et

al. (3) observed that, within endodermal cells, the carbohy-

drate side chains were cleaved by /3-N-acetylglucosaminidase

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996 LAURIE, LEBLOND, MARTIN, SILVER

Figures 10, 1 1, and 12. Anti-laminin immunostaining in structureslocated in the ectoplasmic region of endodermal cells near Reichert’smembrane.

Figure 10. Circular immunostained structure whose membrane is fusedwith the membrane of the cell. The structure is distinguished by its

dark immunostained content and light periphery. Nearby, Reichert’smembrane (R Mb) is reactive. Original magnification x 33,200.

Figure 1 1. Structure fused with the cell membrane. Its content iswell-stained and is similar to Reichert’s membrane (R Mb) below.Original magnification x 48,800.

Figure 12. Large immunostained masscontinuous with Reichert’s membrane(R Mb), and surrounded by the cellmembrane, which includes severalsemicircular portions (arrows). Themass is presumably formed by thecombination of several secretory gran-ules (seen as semicircular remnants)opening at the surface as in Figures 10and 1 1 and releasing their content forthe build up of Reichert’s membrane.Original magnification x 25,800.

LAM! NIN ANTIGENICITY IN ENDODERMAL CELLS 997

Figures 13, 14, 15, and 16. Controland anti-laminin immunostaining ofmultivesicular bodies in endodermalcells. Nucleus, N.

Figure 1 3. In this control preparation,no reaction is seen in a multivesicularbody. Original magnification x 50,000.

Figure 14. In this experimental prep-aration, a multivesicular body showsimmunostaining in its content and onthe walls of small and large vesicles.Original magnification x 40,000.

Figure 15. Anti-laminin immuno-staining seen on the wall of small yes-ides and of tubules at the periphery ofa multivesicular body. Original mag-nification x 50,000.

Figure 16. Small multivesicular bodyin which immunostaining is observedprimarily on the wall of small vesicles.Nearby Reichert’s membrane (R Mb)is immunostained. Original magnifi-cation X 50,000.

in nascent, but not in 1-hr-old laminin polypeptides; the re-

action was presumed to be blocked by the addition of pe-

ripheral sugars in the Golgi apparatus (3). These results sug-gested that laminin side chains were of the asparagine-linked

type and acquired core sugars in rER cisternae and peripheral

ones in the Golgi apparatus, as shown for other glycoproteins( 1 , 1 5 ). Hence the immunostaining of endodermal cells might

be attributed to sugar-poor laminin precursors in rER cister-

nae, to partially completed laminin in Golgi saccules, and to

full-fledged laminin in the GERL element, prosecretory, and

secretory granules.

The staining of endodermal cell organelles with anti-lam-mm antibodies was similar to that observed with anti-type IV

collagen antibodies. It appeared, therefore, that both laminin

and type IV collagen precursors were simultaneously elabo-

rated along the same pathway of biosynthesis, with some di-version to multivesicular bodies. Indeed, the estimated celltransit times for laminin [50-60 mm; (3)) and type IV collagen

[60 mm; (8)J were the same. Recently, we found in duodenum

and incisor tooth that laminin immunostaining occupied thebasal lamina part of basement membranes (13), and not the

lamina lucida part as proposed by others (5,7,18). Moreover,

the layers of Reichert’s membrane were comparable to basallaminae ( 1 1 ). Accordingly, we propose that laminin associates

with type IV collagen to form the individual layers of Reich-

ert’s membrane in a manner similar to what may occur in thebasal lamina of conventional basement membranes ( 1 3).

In conclusion, the basement membrane component, lami-nm, occurs throughout Reichert’s membrane. Laminin is elab-

orated in parietal endodermal cells along a pathway extendingfrom rER cisternae through Golgi saccules and GERL elementsto secretory granules, which presumably release their contentto Reichert’s membrane. Laminin and type IV collagen appear

to be produced and deposited simultaneously to compose thesuccessive layers making up this membrane.

AcknowledgmentThe authors acknowledge the technical assistance of Ms S. Bujold.

998 LAURIE, LEBLOND, MARTIN, SILVER

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