First published online 8 April 2003
doi: 10.1242/jcs.00430
Infertility in female mice with an oocyte-specific knockout of GPI-anchored proteins
Jennifer A. Alfieri1,
Arlan D. Martin1,
Junji Takeda3,
Gen Kondoh3,
Diana G. Myles1 and
Paul Primakoff2,*
1 Section of Molecular and Cell Biology, University of California – Davis,
One Shields Avenue, Davis, CA 95616, USA
2 Department of Cell and Human Anatomy, University of California – Davis,
One Shields Avenue, Davis, CA 95616, USA
3 Department of Social and Environmental Medicine, Osaka University, Suita,
Osaka, Japan
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Fig. 1. Generation of conditional Pig-a-knockout female mice by
Cre/loxP recombination. Male mice carrying a ZP3-Cre
transgene and wild-type Pig-a alleles were mated with females
carrying an eGFP-GPI transgene and two Pig-aflox
alleles. The eGFP-GPI transgene provides expression of eGFP-GPI as a marker to
confirm loss of GPI-APs in knockout animals. ZP3-Cre:eGFP-GPI:Pig-a f/y males
derived from this mating were crossed with eGFP-GPI:Pig-a f/f females to
generate conditional knockout females with the genotype ZP3-Cre:eGFP-GPI:Pig-a
f/f.
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Fig. 2. Surface expression of eGFP-GPI on wild-type or
Pig-a–/– oocytes. Oocytes from wild-type and
conditional knockout females carrying the eGFP-GPI transgene were stained with
anti-GFP to determine the level of eGFP expressed on the surface as a GPI-AP.
(A) Transmission and fluorescence images of wild-type oocytes. (B)
Transmission and fluorescence images of Pig-a–/–
oocytes.
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Fig. 3. In vivo fertilization rate as assessed by the percentage of fertilized eggs
retrieved from mated wild-type and conditional knockout females. Five
wild-type and three knockout females were treated with gonadotropins and mated
with wild-type males immediately following hCG injection. 40 hours post-hCG,
unfertilized eggs and two-cell embryos were released from oviducts and
observed under the light microscope to score for the number of two-cell
embryos. Error bars represent s.e.m.
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Fig. 4. In vivo fertilization assay. The fertilization rate, fertilization index
and sperm bound per egg were compared for wild-type and knockout eggs in six
experiments (n=6) using a total of 158 wild-type oocytes and 71
Pig-a–/– oocytes. (A) The fertilization rate was
54±12% in wild type eggs and 6±6% in
Pig-a–/– eggs; P=0.004. (B) The fertilization
index was 0.63±0.14 in wild-type eggs and 0.07±0.07 in
Pig-a–/– eggs; P<0.002. (C) The number of
sperm bound per egg in wild-type eggs is not significantly different from
wild-type and Pig-a–/– eggs (P=.09). Asterisks
indicate values significantly different from the control. Error bars represent
s.e.m.
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Fig. 5. Identification of CD55 on wild-type oocytes by indirect immunofluorescence.
Wild-type zona-free mouse oocytes stained with goat anti-mouse CD55 and
detected by Alexa-Fluor® 488-conjugated donkey anti-goat. (A) Wild-type
oocytes. (B) PI-PLC-treated oocytes.
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Fig. 6. Detection of CD55 on wild-type eggs by western blotting. Eggs,
PI-PLC-treated, +, (right lane) or untreated, –, (center lane), were
solubilized and separated by reducing SDS-PAGE. PVDF membranes of transferred
proteins were probed for CD55. The left lane is staining from a positive
control lysate of mouse CTLL-2 cells, a line known to express CD55. The
slightly larger size of the CD55 in the CTLL-2 cultured cells may reflect a
difference in CD55 glycosylation.
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© The Company of Biologists Ltd 2003
