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Intermediate-size filaments in a germ cell: Expression of cytokeratins in oocytes and eggs of the frog Xenopus.
Franz JK
,
Gall L
,
Williams MA
,
Picheral B
,
Franke WW
.
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Vitellogenic oocytes and eggs of the frog Xenopus laevis contain intermediate-size filaments that are resistant to extractions in high-salt buffers and Triton X-100 and are specifically stained with antibodies to cytokeratins. Gel electrophoresis of cytoskeletal proteins from Xenopus oocytes shows a specific enrichment of three polypeptides designated components 1 [Mr, 56,000; IEP (pI obtained by two-dimensional gel electrophoresis in the presence of 9.5 M urea), ca. 5.9], 2 (Mr, 46,000; IEP, 5.38), and 3 (Mr, 42,000; IEP, ca. 5.3). The same three cytoskeletal polypeptides are found in eggs and early embryos, in intestinal mucosa of adult frogs, and in cultured kidney epithelial cells. They are different from amphibian vimentin and desmin and from the keratins present in the epidermis of adult frogs. Peptide mapping and immunoblotting experiments indicate that Xenopus cytokeratin component 1 is related to cytokeratin A of higher vertebrates but is different from the two smaller cytoskeletal polypeptides 2 and 3. Incorporation of [35 S]methionine shows that all three polypeptides are synthesized in both oocytes and embryos. Our observations show that maternal storage is not only restricted to proteins serving basic cellular functions but also can extend to proteins related to a specific form of cell differentiation (i.e., epithelial formation) in the early embryo. The data suggest that mechanisms of epithelial differentiation in Xenopus embryogenesis are different from those of early mammalian embryos in which no such intermediate-size-filament storage pool has been detected.
FIG. 1. Immunofluorescence
(a, b, e, and h) and electron (c, d, f,
and g) microscopy showing cytokeratin
filaments in oocytes (a-e)
and early embryos (f-h) of X. laevis.
(a) Frozen section of ovary
stained with antibodies to bovine
cytokeratin. Note staining of follicle
cells (F) and offibrillar arrays
inthreeadjacentstage6oocytes(O°,
02, and 03). (Bar = 100 ,um.) (b)
Higher magnification of cytokeratin
fibril arrays (same preparation
as in a). (Bar = 50 ,um.) (c) Thin
sections through cytoskeleton from
oocyte cortex (stage 6) showing a
bundle of loosely fasciated cytokeratin
IFs terminating at a dense
aggregate (arrow). (Bar = 0.5 ,um.)
(d) Same preparation showing the
more frequent irregular meshwork
arrays of cytokeratin IFs. (Bar
= 0.2 um.) Insert shows electrontranslucent
cores (arrows) typical
of cross-sectioned IFs (Bar = 40
nm.) (e) Same ovary as in a but
stained with antibodies to vimentin.
Note that only interfollicular
mesenchymal cells are stained,
whereas oocytes (01, 02, and 03) are
negative. (Bar = 50 ,um.) (f) Thin
section through outer cells (S, surface)
of stage 7 embryo (early blastula)
showing an IF bundle (arrows)
in subapical position. Bracket
denotes the actin-rich cortex. (Bar
= 0.5 Pm.) (g) Same stage showing
association of cytokeratin IFs (arrows)
to typical desmosome (D).
(Bar = 0.2 /Lm.) (h) Immunofluorescence
microscopy of outer cells
of blastula (5, surface) showing
positive cortical staining with antibodies
to bovine cytokeratin. (Bar
=25 um.)
FIG. 2. Two-dimensional gel electrophoresis of cytoskeletal proteins
from stage 6Xenopus oocytes (a), intestinal mucosa of adult frogs
(b), cultured epithelial cells of Ar line (c), epidermis (d), leg skeletal
muscle (e), and stomach wall (f) of adult frogs. First-dimension electrophoresis
was by isoelectric focusing (IEF) ornonequilibrium pH gradient
electrophoresis (NEPHGE). Reference proteins in coelectrophoresis
are bovine serum albumin (BSA), phosphoglycerokinase (PGK),
and 0- and a-actin (A; only a-actin is denoted in c-f). Major cytoskeletal
polypeptides are designated 1, 2, and 3 (a-c andf). Arrowhead in
a denotes residual P-tubulin; bracket in c, a cytoskeletal polypeptide
notfurthercharacterized; bracketsin d, epidermal keratins; and brackets
in e and f, cytoskeletal polypeptides of muscle and stomach (one of
these, D, has been identified as desmin).
FIG. 3. Polypeptide identification by NaDodSO4/polyacrylamide
gel electrophoresis (a) and binding of cytokeratin antibodies (b) as well
as by two-dimensional coelectrophoresis of unlabeled cytoskeletal proteins
ofA6 cells (Coomassie blue stain) (c) with [35S]methionine-labeled
cytoskeletal proteins from stage 6 oocytes (d). (a) Coomassie blue staining
of reference proteins (lane R from top to bottom: f3galactosidase,
phosphorylase a, bovine serum albumin, actin, and chymotrypsinogen),
cytoskeletal proteins from frog epidermis (lane 1), laid eggs (lane 2),
and stage 6 oocytes (lane 3). Dots, IF polypeptides; bars, an unidentified
minor cytoskeletal polypeptide of Mr 66,000 that often exhibits immunological
crossreaction with IF proteins (cf. ref. 1); asterisks, major
yolk components. (b) Fluoroautoradiography of a gel parallel to a after
blotting on nitrocellulose paper and reaction with antibodies to bovine
epidermal cytokeratins, followed by washes and reaction with l25I-labeled
protein A. Note antibody reaction with all keratins of frog epidermis
(lane 1') but with only one cytokeratin (component 1) of oocytes
(lane 3') and eggs (lane 2'). Coomassie blue staining (c) and fluoroautoradiography
(d) of the same gel showing comigration of cytokeratins
1-3 in both cells. X in c, cytoskeletal polypeptide not further characterized.
Designations are as in Fig. 2.
FIG. 4. Autoradiographs showing tryptic peptide maps of cytoskeletal
polypeptides excised after separation on two-dimensional gel electrophoresis
followed by radioiodination. (a) Component 1 fromXerwpus
oocytes. (b) Component 1 from Xenopus intestinal cells. (c) Mixture of
peptides shown in a and b. (d) Cytokeratin A from rat intestinal cells
(cf. ref. 37). (e) Component 2 from Xenopus oocytes. (f) Component 3
from Xenopus oocytes. Brackets and arrows in a and c denote identical
peptide spots; in d they indicate some characteristic peptide spots of
rat cytokeratin A similar to peptides obtained from Xenopus cytokeratin
1. Note that components 2 (e) and 3 are different from component
1 (a-c).
FIG. 5. Two-dimensional electrophoresis of- [3S]methionine-labeled
cytoskeletal polypeptides of Xenopus oocytes [Coomassie blue
staining (a) and fluoroautoradiograph (b)] in comparison with flubroautoradiographs
of cytoskeletal polypeptides from laid eggs (c) and
from embryos of'stages9 (d) (component 3 was identified only after long
exposure) and 17 (e). Designations are as in Fig. 2..
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