Male Mice That Do Not Express Group VIA Phospholipase A2 Produce Spermatozoa with Impaired Motility and Have Greatly Reduced Fertility

Shunzhong Bao,David J Miller,Zhongmin Ma,Mary Wohltmann,Grace Eng,Sasanka Ramanadham,Kelle Moley,John Turk

doi:10.1074/jbc.m406489200

Abstract

The Group VIA Phospholipase A(2) (iPLA(2)beta) is the first recognized cytosolic Ca(2+)-independent PLA(2) and has been proposed to participate in arachidonic acid (20:4) incorporation into glycerophosphocholine lipids, cell proliferation, exocytosis, apoptosis, and other processes. To study iPLA(2)beta functions, we disrupted its gene by homologous recombination to generate mice that do not express iPLA(2)beta. Heterozygous iPLA(2)beta(+/-) breeding pairs yield a Mendelian 1:2:1 ratio of iPLA(2)beta(+/+), iPLA(2)beta(+/-), and iPLA(2)beta(-/-) pups and a 1:1 male:female gender distribution of iPLA(2)beta(-/-) pups. Several tissues of wild-type mice express iPLA(2)beta mRNA, immunoreactive protein, and activity, and testes express the highest levels. Testes or other tissues of iPLA(2)beta(-/-) mice express no iPLA(2)beta mRNA or protein, but iPLA(2)beta(-/-) testes are not deficient in 20:4-containing glycerophosphocholine lipids, indicating that iPLA(2)beta does not play an obligatory role in formation of such lipids in that tissue. Spermatozoa from iPLA(2)beta(-/-) mice have reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo, and inhibiting iPLA(2)beta with a bromoenol lactone suicide substrate reduces motility of wild-type spermatozoa in a time- and concentration-dependent manner. Mating iPLA(2)beta(-/-) male mice with iPLA(2)beta(+/+), iPLA(2)beta(+/-), or iPLA(2)beta(-/-) female mice yields only about 7% of the number of pups produced by mating pairs with an iPLA(2)beta(+/+) or iPLA(2)beta(+/-) male, but iPLA(2)beta(-/-) female mice have nearly normal fertility. These findings indicate that iPLA(2)beta plays an important functional role in spermatozoa, suggest a target for developing male contraceptive drugs, and complement reports that disruption of the Group IVA PLA(2) (cPLA(2)alpha) gene impairs female reproductive ability.

Highlights

The Group VIA Phospholipase A2 is the first recognized cytosolic Ca2؉-independent PLA2 and has been proposed to participate in arachidonic acid (20:4) incorporation into glycerophosphocholine lipids, cell proliferation, exocytosis, apoptosis, and other processes
Female struct was introduced into mouse embryonic stem cells, and those that incorporated it by homologous recombination, which disrupts the iPLA2␤ gene coding sequence (Fig. 1B), were introduced into mouse blastocysts that were implanted into pseudo-pregnant female mice
Inhibition of iPLA2␤ with bromoenol lactone (BEL) reduces motility of wild-type spermatozoa in a concentration- and time-dependent manner that resembles the effect of eliminating iPLA2␤ by gene disruption

Summary

Greatly Reduced Fertility*

Spermatozoa from iPLA2␤؊/؊ mice have reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo, and inhibiting iPLA2␤ with a bromoenol lactone suicide substrate reduces motility of wild-type spermatozoa in a time- and concentration-dependent manner. Mating iPLA2␤؊/؊ male mice with iPLA2␤؉/؉, iPLA2␤؉/؊, or iPLA2␤؊/؊ female mice yields only about 7% of the number of pups produced by mating pairs with an iPLA2␤؉/؉ or iPLA2␤؉/؊ male, but iPLA2␤؊/؊ female mice have nearly normal fertility These findings indicate that iPLA2␤ plays an important functional role in spermatozoa, suggest a target for developing male contraceptive drugs, and complement reports that disruption of the Group IVA PLA2 (cPLA2␣) gene impairs female reproductive ability. Testes of wild-type mice express the highest iPLA2␤ levels, and male iPLA2␤Ϫ/Ϫ mice produce spermatozoa with reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo. Our findings indicate that iPLA2␤Ϫ/Ϫ plays an important functional role in spermatozoa

EXPERIMENTAL PROCEDURES

TABLE I

DISCUSSION