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First published online 8 April 2003
doi: 10.1242/jcs.00430

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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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