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U-cadherin is a member of the cadherin family in Xenopus that participates in interblastomere adhesion in the early embryo from the first cleavage onwards. Though a maternal pool of U-cadherin is available in the egg, it is not present on the egg membrane (Angres et al., 1991. Development 111, 829-844). To assess the origin of this unexpected distribution in the egg, the accumulation and localization of the cadherin during oogenesis and oocyte maturation were investigated. We report here that U-cadherin is present in Xenopus oocytes throughout oogenesis. It is localized at the oocyte-follicle cell contacts suggesting that it functions in the adhesion of the two cell types. When oocytes mature and the contacts to the follicle cells break, U-cadherin disappears from the oocyte surface. Evidence for a translocation of U-cadherin from the membrane to the inside of the oocyte was obtained when the fate of membrane-bound U-cadherin, which was labelled on the surface of oocytes prior to maturation, was followed through maturation. The total U-cadherin content of the oocyte increases during maturation. Metabolic labelling experiments indicate that at maturation the translation of U-cadherin is elevated well above the level that one would expect from the general increase in protein synthesis is presumably the main source of the maternal pool of U-cadherin in the egg.
Fig. 1. Localization of U-cadherin in an early oocyte. Small
fragments of ovary were immunostained with mAb 6D5 as
described in Material and Methods. The immunostaining of
a stage I oocyte within the ovarian tissue is shown.
Arrowheads indicate the autofluorescence of red blood
cells in blood vessels of the theca layer. Bar represents 25
um.
Fig. 2. Localization of U-cadherin at intercellular contacts
of oocytes and follicle cells. Follicles of stage VI oocytes
were manually dissected from the ovary and
immunostained with mAb 6D5 (A,C and D) or the inert
IgG P3 as a control (B). Whole mounts were either
embedded in glycolmethacrylate and sectioned (A and B)
or analyzed directly with the confocal microscope (C and
D). The arrows in A indicate transversally sectioned cell
contacts between adjacent follicle cells. The intercellular
contacts of the follicle cells are in focus in C and the
oocyte surface in D. Bars represent 25 um.
Fig. 3. Absence of U-cadherin
from the intercellular contacts
of oocytes and follicle cells
after in vitro maturation.
Isolated follicles were treated
with progesterone (2 jig/ml)
and immunostained with mAB
6D5 after oocyte maturation.
In A, the oocyte periphery in
the equatorial region is shown.
Arrows indicate intercellular
contacts of the follicle cells. B
and C show a pair of confocal
scanning images in different
focus planes. The follicle cell
contacts are depicted in B and
the oocytes surface in C. Bars
represent 25um.
Fig. 4. Restoration of U-Cadherin on the plasma membrane of oocytes defoUiculated by collagenase treatment. Oocytes
were defoUiculated with collagenase and cultured in OR-2 medium. Oocytes were fixed at 0 hour (A), 12 hours (B) and 24
hours (C) of culture and immunostained with mAb 6D5. The equatorial regions of oocytes are depicted in A and B. A
transversal section from the animal to the vegetal pole is shown in C. Bars represent 50 /nn in A and B, 100 /an in C. The
method of whole mount staining applied here causes an autofluorescence of the yolk platelets which varies between
different specimens. This autofluorescence seems to depend in an uncontrollable way on the degree of dehydration of the
specimen. Due to its different colour, the autofluorescence is easily distinguished from FTTC label. Only the plasma
membranes and some gtanular structures in the subcortical region exhibit true FTTC staining.
Fig. 5. Disappearance of U-cadherin from the oocyte plasma membrane during in vitro maturation. Stage VI oocytes were
defoUiculated with collagenase and cultured in OR-2 overnight. Maturation was triggered by progesterone. Oocytes were
fixed at different stages of GVBD and stained with mAb 6D5. An early stage of GVBD is shown in A and a later stage in
C. Corresponding phase-contrast micrographs are shown in B and D. Bars represent 100um.
Fig. 6. Immunoblot analysis of surface-bound U-cadherin
during oocyte maturation. Defolliculated oocytes were
prepared and cultured in OR-2 medium for 12 hours.
Oocytes were induced to mature with progesterone and
labelled with Biotin-X-NHS. Biotinylated proteins were
precipitated with Streptavidin (SAv) agarose as described
in Materials and methods and the presence of U-cadherin
in the biotinylated protein fractions was analyzed by
immunoblotting with mAb 6D5. The position (bars) and
size (Mr x 10 ) of molecular weight markers is indicated.
Lane a, oocytes labelled and processed prior to
maturation; lane b, unlabelled oocytes were processed;
lane c, SAv was preabsorbed with biotin; lane d, oocytes
labelled prior to maturation and processed after
maturation; lane e, oocytes labelled and processed after
maturation.
Fig. 7. Immunoblot analysis of U-cadherin during
maturation. The amount of U-cadherin in total detergent
soluble fraction of oocytes was analyzed by immunoblotting
with mAb 6D5. The position (bars) and size (MT x 1CT3)
of molecular weight markers is indicated. Lane a, Ucadherin
in total follicles manually dissected from ovary;
lane b, U-cadherin in collagenase-treated oocytes; lane c,
U-cadherin in matured oocytes taken at a time when 50%
of the population had undergone GVBD; lane d, Ucadherin
in controls not treated with progesterone.
Fig. 8. Pulse labelled Ucadherin
in oocytes during
maturation. Oocytes were
incubated with
progesterone for 2.5 hours
and labelled with [35S]
methionine for additional
1.5 hours. U-cadherin was
immunoprecipitated from
detergent extracts,
electrophoresed and
detected by fluorography.
Lane a, non-stimulated
controls; lane b, maturing
oocytes; lane c, P3
control.
