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Stimulation of Xenopus oocyte maturation by inhibition of the G-protein alpha S subunit, a component of the plasma membrane and yolk platelet membranes.
Gallo CJ
,
Hand AR
,
Jones TL
,
Jaffe LA
.
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Oocytes of Xenopus laevis undergo maturation when injected with an affinity-purified antibody against the COOH-terminal decapeptide of the alpha subunit of the G-protein Gs, an antibody that inhibits Gs activity. Germinal vesicle breakdown, chromosome condensation, and polar body formation occur, with a time course similar to that for oocytes treated with progesterone. The alpha S antibody-injected oocytes also acquire the ability to be activated by sperm. Coinjection of the catalytic subunit of cAMP-dependent protein kinase, or incubation with cycloheximide, inhibits maturation in response to injection of the alpha S antibody; these experiments show that the alpha S antibody acts at an early point in the pathway leading to oocyte maturation, before formation of maturation promoting factor, and like progesterone, its action requires protein synthesis. Immunogold electron microscopy shows that alpha S is present in the yolk platelet membranes as well as the plasma membrane. These results support the hypothesis that progesterone acts by inhibiting alpha S, and suggest that the target of progesterone could include yolk platelet membranes as well as the plasma membrane.
Figure 1. Immunoblots of oocyte cortices (A), membranes (B),
and yolk platelets (C) with antibodies against G-protein ~ subunits.
For each: (lane 1) RM antibody against as, (lane 2) EC antibody
against oti3. The amount of protein loaded per gel lane:
cortices, 50 p~g; membranes, 70 p~g; and yolk platelets, 100 Ixg.
Figure 2. White spot formation in oocytes injected with as antibody.
Oocytes were injected with 50 nl of i mg/ml antibody solution
(0.3 p.M cytoplasmic concentration). White spots formed at
11.5 h after injection; the photograph was taken at 2.5 h after
white spot formation. Bar, 1.0 ram.
Figure 3. Concentration dependence of as antibody stimulation
of GVBD. The x-axis indicates the antibody concentrations in the
cytoplasm. The y-axis indicates the % of oocytes undergoing
GVBD, as indicated by formation of a white spot at the animal
pole or by the absence of a germinal vesicle in TCA-fixed oocytes.
Oocytes were scored for GVBD at 24 h after injection. A,
RM antibody against as (n = 273 oocytes, 21 animals); A, RM antibody
against a s preincubated with an equal amount (wt/wt) of
the RM peptide (n = 10 oocytes, 1 animal). O, EC antibody
against oti3 (n = 38 oocytes, 4 animals); II, nonimmune IgG (n = 7
oocytes, 2 animals).
Figure 4. Time course of GVBD in oocytes injected with ct s antibody
(RM) or exposed to progesterone. Oocytes were injected
with 0.3 0,M RM antibody (&) (n = 15 oocytes), or exposed to 3
~M progesterone (O) (n = 20 oocytes). The time of GVBD was
counted as the time of appearance of the white spot at the animal
pole. These results are typical of seven similar experiments, using
RM antibody concentrations ranging from 0.3 to 2.0 txM.
Figure 5. DNA staining of live oocytes
that had been exposed to progesterone
(3 ixM) (A and A'), or injected with the
RM antibody against as (0.3 p~M) (B
and B'). In A and B, the microscope
was focused at the level of the second
metaphase chromosomes. In A' and B',
the microscope was focused at the level
of the tightly condensed chromatin in
the polar body (same oocytes as in A
and B). Bar, 50 p.m.
Figure 6. Immunogold labeling
of as in oocyte plasma
membrane and yolk platelet
membranes. (A) A section
showing labeling of the
plasma membrane and a
yolk platelet membrane by
the RM antibody against as.
(B) A section showing labeling
of a yolk platelet membrane
by the RM antibody
against eq. (C) A control section
showing the absence of
labeling by nonimmune IgG.
Bars, 100 nm.
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