PGAP1 Knock-out Mice Show Otocephaly and Male Infertility

Eiichi Morii,Taroh Kinoshita,Masahito Ikawa,Masaru Okabe,Ryo Yamaguchi,Yasutaka Ueda,Yusuke Maeda

doi:10.1074/jbc.m705601200

Abstract

A palmitate linked to the inositol in glycosylphosphatidylinositol (GPI) is removed in the endoplasmic reticulum immediately after the conjugation of GPI with proteins in most cells. Previously, we identified PGAP1 (post GPI attachment to proteins 1) as a GPI inositoldeacylase that removes the palmitate from inositol. A defect in PGAP1 caused a delay in the transport of GPI-anchored proteins (GPI-APs) from the endoplasmic reticulum to the cell surface in Chinese hamster ovary cells, although the cell-surface expression of GPI-APs in the steady state was normal. Nevertheless, in most cells, GPI-APs undergo deacylation. To elucidate the biological significance of PGAP1 in vivo, we established PGAP1 knock-out mice. Most PGAP1 knock-out mice showed otocephaly, a developmental defect, and died right after birth. However, some survived with growth retardation. Male knock-out mice showed severely reduced fertility despite the capability of ejaculation. Their spermatozoa were normal in number, motility, and ability to ascend the uterus, but were unable to go into the oviduct. In vitro, PGAP1-deficient spermatozoa showed weak attachment to the zona pellucida and a severely diminished rate of fertilization. Therefore, an extra acyl chain in GPI anchors caused severe deleterious effects to development and sperm function.

Highlights

Many eukaryotic cell-surface proteins with various functions are anchored to the membrane via glycosylphosphatidylinositol (GPI)2 [1,2,3]
Phosphatidylinositol glycan A is the enzyme involved in the early step of GPI synthesis, and its disruption in mice causes complete loss of GPI synthesis resulting in embryonic lethality with defective gastrulation and neural development indicating the importance of GPI-anchored proteins (GPI-APs) for normal development [7]
GPI-APs accumulate in specific microdomains in the plasma membrane called lipid rafts, which are enriched in sphingolipids and cholesterol [11,12,13]

Summary

EXPERIMENTAL PROCEDURES

Generation of PGAP1-deficient Mice—The targeting vector for deletion of PGAP1 exon 5 contains an expression cassette of the neomycin resistance gene (neo) for positive selection and diphtheria toxin for negative selection. Superovulated females were mated with PGAP1 mutant males of each genotype 12 h after human chorionic gonadotropin injection, and the formation of a vaginal plug was observed every 30 min. In Vitro Fertilization—The capacitation was induced in vitro by incubating sperm from PGAP1 homozygous mutant and wild-type littermates in TYH medium (modified Krebs-Ringer bicarbonate solution containing glucose, sodium pyruvate, bovine serum albumin, and antibiotics) [20]. For the sperm-zona binding assay, egg masses were treated with bovine testicular hyaluronidase (175 units/ml, Sigma) for 5 min to remove the cumulus cells. After 30 min of incubation, sperm bound to the zona pellucida of the eggs were observed using an IX-70 microscope (Olympus). The analyses of PGAP1 knock-out mouse in this report are basically based on the first PGAP1Ϫ/Ϫ line, and the phenotypes observed in the first line were examined in other lines derived from different embryonic stem cells.

High Frequency of Perinatal

Findings

DISCUSSION