Targeted Disruption of Intracellular Type I Platelet Activating Factor-acetylhydrolase Catalytic Subunits Causes Severe Impairment in Spermatogenesis

Abstract

Intracellular type I platelet activating factor-acetylhydrolase is a phospholipase that consists of a dimer of two homologous catalytic subunits α1 and α2 as well as LIS1, a product of the causative gene for type I lissencephaly. LIS1 plays an important role in neuronal migration during brain development, but thein vivo function of the catalytic subunits remains unclear. In this study, we generated α1- anda2-deficient mice by targeted disruption.α1−/− mice are indistinguishable from wild-type mice, whereas α2−/− male mice show a significant reduction in testis size. Double-mutant male mice are sterile because of severe impairment of spermatogenesis. Histological examination revealed marked degeneration at the spermatocyte stage and an increase of apoptotic cells in the seminiferous tubules. The catalytic subunits are expressed at high levels in testis as well as brain in mice. In wild-type mice, α2 is expressed in all seminiferous tubule cell types, whereas α1 is expressed only in the spermatogonia. This expression pattern parallels the finding that deletion of both subunits induces a marked loss of germ cells at an early spermatogenic stage. We also found that the LIS1 protein levels, but not the mRNA levels, were significantly reduced in α2−/− and double-mutant mice, suggesting that the catalytic subunits, especially α2, are a determinant of LIS1 expression level. Intracellular type I platelet activating factor-acetylhydrolase is a phospholipase that consists of a dimer of two homologous catalytic subunits α1 and α2 as well as LIS1, a product of the causative gene for type I lissencephaly. LIS1 plays an important role in neuronal migration during brain development, but thein vivo function of the catalytic subunits remains unclear. In this study, we generated α1- anda2-deficient mice by targeted disruption.α1−/− mice are indistinguishable from wild-type mice, whereas α2−/− male mice show a significant reduction in testis size. Double-mutant male mice are sterile because of severe impairment of spermatogenesis. Histological examination revealed marked degeneration at the spermatocyte stage and an increase of apoptotic cells in the seminiferous tubules. The catalytic subunits are expressed at high levels in testis as well as brain in mice. In wild-type mice, α2 is expressed in all seminiferous tubule cell types, whereas α1 is expressed only in the spermatogonia. This expression pattern parallels the finding that deletion of both subunits induces a marked loss of germ cells at an early spermatogenic stage. We also found that the LIS1 protein levels, but not the mRNA levels, were significantly reduced in α2−/− and double-mutant mice, suggesting that the catalytic subunits, especially α2, are a determinant of LIS1 expression level. platelet-activating factor platelet-activating factor acetylhydrolase TdT-mediated dUTP nick-end labeling Platelet-activating factor (PAF)1 is a potent signaling phospholipid involved in diverse physiological events, such as inflammation and anaphylaxis (1Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. Biochem. 2000; 69: 419-445Google Scholar). In addition, PAF has been implicated in the central nervous system (2Bazan N.G. Prog. Brain Res. 1998; 118: 281-291Google Scholar, 3Maclennan K.M. Smith P.F. Darlington C.L. Prog. Neurobiol. 1996; 50: 585-596Google Scholar) and the reproductive system (4Levine A.S. Kort H.I. Toledo A.A. Roudebush W.E. J. Androl. 2002; 23: 471-476Google Scholar,5Minhas B.S. Ripps B.A. Zhu Y.P. Kim H.N. Burwinkel T.H. Gleicher N. Am. J. Reprod. Immunol. 1996; 35: 267-271Google Scholar). PAF is hydrolyzed to an inactive metabolite by a specific enzyme called PAF-acetylhydrolase (PAF-AH). At least three types of PAF-AH exist in mammals, namely the intracellular types I and II (6Hattori M. Arai H. Inoue K. J. Biol. Chem. 1993; 268: 18748-18753Google Scholar, 7Hattori K. Hattori M. Adachi H. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1995; 270: 22308-22313Google Scholar) and a plasma type (8Tjoelker L.W. Wilder C. Eberhardt C. Stafforini D.M. Dietsch G. Schimpf B. Hooper S. Le T.H. Cousens L.S. Zimmerman G.A. Yamada Y. McIntyre T.M. Prescott S.M. Gray P.W. Nature. 1995; 374: 549-553Google Scholar). Intracellular type I PAF-AH (PAF-AH (I)) is an oligomeric complex. It contains a dimer of two homologous catalytic subunits, α1 and α2, and a non-catalytic ॆ subunit (6Hattori M. Arai H. Inoue K. J. Biol. Chem. 1993; 268: 18748-18753Google Scholar, 9Hattori M. Adachi H. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1994; 269: 23150-23155Google Scholar, 10Hattori M. Adachi H. Tsujimoto M. Arai H. Inoue K. Nature. 1994; 370: 216-218Google Scholar, 11Hattori M. Adachi H. Aoki J. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1995; 270: 31345-31352Google Scholar). Interestingly, the ॆ subunit was later found to be identical to LIS1, the product of the causative gene for type I lissencephaly (10Hattori M. Adachi H. Tsujimoto M. Arai H. Inoue K. Nature. 1994; 370: 216-218Google Scholar, 12Reiner O. Carrozzo R. Shen Y. Wehnert M. Faustinella F. Dobyns W.B. Caskey C.T. Ledbetter D.H. Nature. 1993; 364: 717-721Google Scholar). Type I lissencephaly is a genetic brain malformation showing a smooth cerebral surface without gyri, caused by abnormal neuronal migration at early developmental stages. Mice homozygous for the Lis1null mutation die early in embryogenesis soon after implantation (13Hirotsune S. Fleck M.W. Gambello M.J. Bix G.J. Chen A. Clark G.D. Ledbetter D.H. McBain C.J. Wynshaw B.A. Nat. Genet. 1998; 19: 333-339Google Scholar). Heterozygous and compound heterozygous mice have expression level-dependent defects in neuronal migration (13Hirotsune S. Fleck M.W. Gambello M.J. Bix G.J. Chen A. Clark G.D. Ledbetter D.H. McBain C.J. Wynshaw B.A. Nat. Genet. 1998; 19: 333-339Google Scholar). A series of recent studies has suggested that LIS1 interacts not only with PAF-AH (I) catalytic subunits but also with a number of proteins, including tubulin (14Sapir T. Elbaum M. Reiner O. EMBO J. 1997; 16: 6977-6984Google Scholar), cytoplasmic dynein (15Faulkner N.E. Dujardin D.L. Tai C.Y. Vaughan K.T. O'Connell C.B. Wang Y. Vallee R.B. Nat. Cell Biol. 2000; 2: 784-791Google Scholar, 16Smith D.S. Niethammer M. Ayala R. Zhou Y. Gambello M.J. Wynshaw B.A. Tsai L.H. Nat. Cell Biol. 2000; 2: 767-775Google Scholar), and NUDE (17Kitagawa M. Umezu M. Aoki J. Koizumi H. Arai H. Inoue K. FEBS Lett. 2000; 479: 57-62Google Scholar, 18Feng Y. Olson E.C. Stukenberg P.T. Flanagan L.A. Kirschner M.W. Walsh C.A. Neuron. 2000; 28: 665-679Google Scholar, 19Niethammer M. Smith D.S. Ayala R. Peng J. Ko J. Lee M.S. Morabito M. Tsai L.H. Neuron. 2000; 28: 697-711Google Scholar, 20Sasaki S. Shionoya A. Ishida M. Gambello M.J. Yingling J. Wynshaw B.A. Hirotsune S. Neuron. 2000; 28: 681-696Google Scholar). Through interaction with these proteins, LIS1 plays important roles in microtubule-associated cellular functions such as mitotic cell division, chromosomal segregation, and neuronal migration. In contrast, the biological role of the catalytic subunits of PAF-AH (I) remains a complete enigma. Nothwang et al. (21Nothwang H.G. Kim H.G. Aoki J. Geisterfer M. Kubart S. Wegner R.D. van Moers A. Ashworth L.K. Haaf T. Bell J. Arai H. Tommerup N. Ropers H.H. Wirth J. Hum. Mol. Genet. 2001; 10: 797-806Google Scholar) have described a case of functional hemizygosity of α1, possibly responsible for the resulting mental retardation, ataxia, and brain atrophy in this patient. Furthermore, Lecointe et al. (22Lecointe N. Meerabux J. Ebihara M. Hill A. Young B.D. Oncogene. 1999; 18: 2852-2859Google Scholar) have proposed that deregulation of transcription of the human α2 gene is associated with the development of a certain lymphoma. Therefore, the PAF-AH (I) catalytic subunit is also likely to play an important role in some pathological conditions. The α1 and α2 catalytic subunits belong to a novel serine esterase family (9Hattori M. Adachi H. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1994; 269: 23150-23155Google Scholar). These subunits, which show ∼607 amino acid homology with each other, form homodimers and a heterodimer. Ho et al.(23Ho Y.S. Swenson L. Derewenda L.S. Wei Y. Dauter Z. Hattori M. Adachi T. Aoki J. Arai H. Inoue K. Derewenda Z.S. Nature. 1997; 385: 89-93Google Scholar) have reported the x-ray crystal structure of the α1 homodimer. The folding is unique among known lipases and phospholipases. The structure unexpectedly resembles those of the G-protein family such as p21ras and Gα. To elucidate the in vivofunction of the catalytic subunits of PAF-AH (I), namely α1 and α2, we generated mice lacking either one or both of these two proteins. α1,α2 genomic clones were isolated from a mouse 129/SvJ genomic library in the Lambda FIXII vector (Stratagene). Targeting vectors were constructed for replacing part of exon 2 and 3 of theα1 and α2 genes, which include a translation initiation site and catalytic motif (GXSXV) with a PGKneobpA cassette (24Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Google Scholar). A PGKDTA (diphtheria toxin Α fragment) cassette was inserted at the 3′-end of the short arm for negative selection. The targeting vectors were linearized and electroporated into ES cell line RW4 (Genome Systems), which was cultured on neomycin-resistant mouse embryonic fibroblasts. G418-resistant colonies were screened for homologous recombinants by PCR. Candidates of homologous recombinants were verified by Southern analysis using fragments at the 3′-ends of the genes, external to the targeting vectors as probes. Chimeric mice were generated by injection of the ES cells into C57BL/6N blastocysts, followed by transfers to foster mothers, and backcrossed to C57BL/6N mice. Genotypes were determined by PCR and/or Southern analysis of the tail DNA samples. Mouse monoclonal antibodies against α1 and α2 were established as follows. α1−/− andα2−/− female mice were immunized with each purified recombinant rat protein with Freund's complete adjuvant (DIFCO), followed by six boosters at 2-week intervals with 20 ॖg of protein and established monoclonal antibody, producing hybridoma cell lines as previously described (11Hattori M. Adachi H. Aoki J. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1995; 270: 31345-31352Google Scholar). Monoclonal antibody against LIS1 (clone 338, a kind gift from Dr. O. Reiner, Weizmann Institute, Rehovot, Israel) and α-tubulin (clone DM1A, Sigma) were used for a Western blot analysis. Polyclonal antibody against LIS1 (N-19, Santa Cruz Biotechnology) was used for an immunohistochemical analysis. Testes were dissected and fixed overnight in Bouin's fixative at 4 °C. Paraffin sections (5 ॖm) were prepared and stained with Periodic Acid-Schiff (PAS) and hematoxylin. For immunohistochemistry, mice under anesthesia were perfused with phosphate-buffered saline (PBS) and then with 47 paraformaldehyde in PBS. Testes were dissected and refixed overnight in 47 paraformaldehyde at 4 °C. Paraffin sections (5 ॖm) were boiled in a microwave oven in 10 mm sodium citrate buffer (pH 6.0) for antigen retrieval. Subsequent immunodetection was performed using a Vector M.O.M. immunodetection kit (Vector Laboratories) for α1 and α2 and Vectastain ABC kit (Vector Laboratories) for LIS1. Immunostaining was visualized using diaminobenzidine and counterstained with hematoxylin. For detection of apoptotic germ cells, Bouin's-fixed, paraffin-embedded testis sections were subjected to TUNEL staining using an in situ cell death detection kit, POD (Roche Molecular Biochemicals) according to the manufacturer's instructions. Tissues were homogenized in quadruple volumes (w/v) of SET buffer (10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 250 mm sucrose) with protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin, 10 mg/ml leupeptin, 10 mg/ml aprotinin) and phosphatase inhibitors (50 mm NaF, 10 mmNa3PO4). After centrifugation at 1,000 ×g at 4 °C, the supernatants were used as the total protein lysates. The protein concentrations of samples were determined by the BCA assay (PIERCE). Each total protein lysate (50 ॖg/lane) was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes or nitrocellulose membranes. The membranes were blocked with 57 (w/v) skim milk (Wako) in TTBS buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.057 (w/v) Tween 20) and incubated with antibodies in TTBS. Chemiluminescence (ECL kit, Amersham Biosciences) was used for analyzing levels of protein according to the manufacturer's instructions. Total RNA was extracted from mouse tissues using Isogen (Nippongene). Total RNA (10 ॖg/lane) was separated by 17 agarose-formaldehyde gel electrophoresis and transferred to Hybond-N membranes (Amersham Biosciences) in 20× SSC. The membranes were hybridized in Rapid-hyb buffer (Amersham Biosciences) at 65 °C and washed with 0.5× SSC, 0.17 SDS at 65 °C. Probes for α1, α2, and LIS1 were obtained by RT-PCR from mouse RNA and labeled by 32P[dCTP] (Amersham Biosciences) using the Rediprime II DNA labeling system (Amersham Biosciences). Membranes were stripped and rehybridized with human glyceraldehyde-3-phosphate to We established monoclonal antibodies against α1 and α2 by the mice. A Western blot analysis of mouse that α1, α2, and LIS1 were expressed in brain and testis of α2 and LIS1 were in tissues as whereas α1 expression was to embryonic brain and testis The expression levels of α2 and LIS1 were to be to that of a of LIS1 plays an important role in (14Sapir T. Elbaum M. Reiner O. EMBO J. 1997; 16: 6977-6984Google Scholar, 16Smith D.S. Niethammer M. Ayala R. Zhou Y. Gambello M.J. Wynshaw B.A. Tsai L.H. Nat. Cell Biol. 2000; 2: 767-775Google Scholar), be that both subunits are also involved in this staining of mouse revealed that α2 and LIS1 was in all seminiferous tubule cell types staining of α2 and LIS1 was in and In contrast, α1 staining was to the cells the of seminiferous revealed that α1 was in whereas α2 and LIS1 were expressed in the of all types of spermatogenic cells and cells and suggesting that α1 is involved in and/or of spermatogonia. LIS1 was also at of and of both of which are specific staining of α1 or α2 was in the seminiferous of mice and We used homologous in embryonic cells to mice lacking the α1 of exon 2 and exon 3 of each including the translation initiation site and the catalytic serine were with a gene embryonic cell clones and germ line were by PCR and/or Southern blot analysis and were with the and indistinguishable from wild-type Western blot analysis of the brain and the testis in either α1−/− mice mice were also and indistinguishable from wild-type mice. were found to be whereas female was not Testes of mice were significantly those of wild-type mice Testes were not reduced in α1−/− mice, whereas were reduced to in α2−/− mice was significant in among of the protein expression is reduced mice. Western blot analysis of PAF-AH (I) subunits in and of the wild-type mice and mice. the α1 expression is reduced in both brain and testis mice with wild-type mice and LIS1 expression is reduced in both brain and testis mice with wild-type mice blot analysis of mRNA expression of in of the and is as a for RNA Western blot analysis of LIS1 protein in embryonic brain of wild-type and testis in of and in wild-type and mice at of in a of and in wild-type and mice at of The of was at the the of is The seminiferous mice a reduction in and spermatogenic cells were with wild-type mice the stage and were significantly reduced in were and the germ cells to be from the were in the not In mice, early germ cell were to of and not In TUNEL apoptotic cells were in wild-type as previously reported C. N. M. S. K. 1997; Scholar) a significantly number of apoptotic cells The cells were These that in the of the catalytic subunits of PAF-AH (I), the of of to of cell death in the germ cell in spermatogenic cells of seminiferous of wild-type mice at of are cells are labeled by TUNEL α1−/− mice impairment of the α2−/− mice significant reduction and germ cell impairment and mice mice from both the and of impairment was severe both α2 were and and These that α2 plays a important role in α1 and that α1 in part be by the of is that α1 also plays a role in male on the that the of both α2 be by the of α1 and and The α1 protein was reduced to of the mice was reduction of the mRNA not whereas the α2 was not in a of LIS1 with α1 and/or α2 in the we the LIS1 protein levels in α1 and/or α2 mice. In LIS1 levels in both brain and testis were reduced to with wild-type mice In contrast, reduction of LIS1 was in α1−/− mice mice, as in α2−/− mice, LIS1 levels were reduced to of the levels in the type mice. expression in α2−/− mice was either the as or the expression in type mice the LIS1 expression in brain of each mouse was not significantly that in wild-type mice In this study, we that the catalytic subunits of intracellular PAF-AH (I) are involved in spermatogenesis. into the in vivo function of PAF-AH We by Western that in mice the catalytic subunits α1 and α2 as well as LIS1 are at high levels in both brain and Interestingly, was that PAF-AH (I) subunits expression levels to those of a of intracellular The and the that LIS1 is a microtubule-associated protein to that the catalytic subunits are also involved to a in in the of spermatogenesis. include mitotic of of and in The severe degeneration in mice, but degeneration also in and in Therefore, be that PAF-AH (I) catalytic subunits are involved in the of and not in a specific stage of spermatogenesis. the and function of the PAF-AH (I) catalytic subunits in are we found that of both catalytic subunits to a in LIS1 protein levels, suggesting that the catalytic subunits are associated with LIS1. It is likely that LIS1 protein levels are by the catalytic subunits, because LIS1 mRNA levels are not mice. LIS1 levels are for brain development (13Hirotsune S. Fleck M.W. Gambello M.J. Bix G.J. Chen A. Clark G.D. Ledbetter D.H. McBain C.J. Wynshaw B.A. Nat. Genet. 1998; 19: 333-339Google Scholar), we that LIS1 is involved in of and that reduced LIS1 protein levels are responsible for the defects mice. studies also revealed that the severe degeneration mice in α2−/− mice be by the of reduction in the of LIS1, because the reduction of LIS1 was not mice. that PAF-AH (I) resembles Y.S. Swenson L. Derewenda L.S. Wei Y. Dauter Z. Hattori M. Adachi T. Aoki J. Arai H. Inoue K. Derewenda Z.S. Nature. 1997; 385: 89-93Google Scholar), the catalytic subunits a novel intracellular signaling to LIS1 in mammals, and of this signaling in severe impairment in spermatogenesis. PAF-AH (I) catalytic subunits are expressed in brain as well as in testis and because of LIS1 to severe brain malformation in both and mice (13Hirotsune S. Fleck M.W. Gambello M.J. Bix G.J. Chen A. Clark G.D. Ledbetter D.H. McBain C.J. Wynshaw B.A. Nat. Genet. 1998; 19: 333-339Google Scholar), we that mice lacking the catalytic subunits brain staining of mice brain in of in the cerebral or not To in brain mice, was significant reduction of LIS1 protein levels, suggesting that is a to LIS1 protein levels and the function of catalytic subunits in In α2−/− mice, both α1 and LIS1 protein levels were significantly reduced with wild-type mice, whereas in α1−/− mice both α2 and LIS1 levels were not In supernatants of mice testis or brain were subjected to In the case of α2 in the as LIS1, whereas in the case of α1 and LIS1 at the N. J. K. and H. that α2, the α2 has a for LIS1 and that the α1 has a or for LIS1 in the that α1 mRNA levels are not mice not the also that the α1 protein is not expressed in the of α2 in These are with H. Aoki J. M. Adachi T. H. Inoue Y. Arai H. Inoue K. J. Biol. Chem. 1998; Scholar) that and α2 homodimers are the PAF-AH (I) catalytic Immunostaining studies revealed that α2 is expressed in all spermatogenic cells, whereas α1 is expressed only in spermatogonia. This expression pattern parallels the finding that deletion of both subunits induces a marked loss of germ cells, at an early spermatogenic stage. We have previously that α1 is expressed in in the embryonic and whereas the α2 expression is from the H. Aoki J. M. Adachi T. H. Inoue Y. Arai H. Inoue K. J. Biol. Chem. 1998; Scholar, B. Hattori M. Aoki J. Arai H. Inoue K. G. Biol. 1996; Scholar). a the catalytic subunits from the to the in during brain It is likely that the type of in the catalytic dimer during from to Interestingly, has been that to specific the seminiferous tubule and the of the tubule Biol. Reprod. 2001; Scholar). that are of the of the seminiferous tubule at a of M. Biol. Reprod. 1999; Scholar, T. Biol. Reprod. 1998; Scholar). the biological of the in the catalytic subunit is not is to that α1 a signaling in and spermatogonia. The cellular function of the enzyme and the physiological of this enzyme are PAF has been in from and has been to and (4Levine A.S. Kort H.I. Toledo A.A. Roudebush W.E. J. Androl. 2002; 23: 471-476Google Scholar). for have a PAF W.E. 2000; Scholar, W.E. 2000; Scholar). PAF the of human B.S. R. Scholar). at the that PAF is not only involved in and but is also involved in the PAF-AH (I) for an but types of in Wilder C. Aoki J. Arai H. Inoue K. L. 2001; Scholar). on the structure of the catalytic dimer that the of this enzyme is not a Y.S. Swenson L. Derewenda L.S. Wei Y. Dauter Z. Hattori M. Adachi T. Aoki J. Arai H. Inoue K. Derewenda Z.S. Nature. 1997; 385: 89-93Google Scholar). PAF-AH (I) to Y.S. Swenson L. Derewenda L.S. Wei Y. Dauter Z. Hattori M. Adachi T. Aoki J. Arai H. Inoue K. Derewenda Z.S. Nature. 1997; 385: 89-93Google Scholar), the PAF-AH novel intracellular signaling is likely in mammals, with PAF or a as a PAF-AH mice in brain and reduction to of wild-type mice was in both tissues not This is likely because type I PAF-AH is the only whereas of type II PAF-AH and not PAF-AH are responsible for the In we found that the of the PAF-AH (I) catalytic subunits induces reduction of LIS1 protein on the cellular and severe malformation on the level. The to be is the catalytic of PAF-AH (I) is for LIS1 protein and spermatogenesis. To these we are to the inactive α1 and α2 subunit into mouse We O. Reiner for the LIS1 antibody and S. and for