Inactivation of a Testis-specific Lis1 Transcript in Mice Prevents Spermatid Differentiation and Causes Male Infertility
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m309583200
ISSN1083-351X
AutoresKarim Nayernia, Franz Vauti, Andreas Meinhardt, Christina Cadenas, Stephan Schweyer, Barbara I. Meyer, Iris Schwandt, Kamal Chowdhury, Wolfgang Engel, Hans-Henning Arnold,
Tópico(s)Ubiquitin and proteasome pathways
ResumoLis1 protein is the non-catalytic component of platelet-activating factor acetylhydrolase 1b (PAF-AH 1B) and associated with microtubular structures. Hemizygous mutations of the LIS1 gene cause type I lissencephaly, a brain abnormality with developmental defects of neuronal migration. Lis1 is also expressed in testis, but its function there has not been determined. We have generated a mouse mutant (LIS1GT/GT) by gene trap integration leading to selective disruption of a Lis1 splicing variant in testis. Homozygous mutant males are infertile with no other apparent phenotype. We demonstrate that Lis1 is predominantly expressed in spermatids, and spermiogenesis is blocked when Lis1 is absent. Mutant spermatids fail to form correct acrosomes and nuclei appear distorted in size and shape. The tissue architecture in mutant testis appears severely disturbed displaying collapsed seminiferous tubules, mislocated germ cells, and increased apoptosis. These results provide evidence for an essential and hitherto uncharacterized role of the Lis1 protein in spermatogenesis, particularly in the differentiation of spermatids into spermatozoa. Lis1 protein is the non-catalytic component of platelet-activating factor acetylhydrolase 1b (PAF-AH 1B) and associated with microtubular structures. Hemizygous mutations of the LIS1 gene cause type I lissencephaly, a brain abnormality with developmental defects of neuronal migration. Lis1 is also expressed in testis, but its function there has not been determined. We have generated a mouse mutant (LIS1GT/GT) by gene trap integration leading to selective disruption of a Lis1 splicing variant in testis. Homozygous mutant males are infertile with no other apparent phenotype. We demonstrate that Lis1 is predominantly expressed in spermatids, and spermiogenesis is blocked when Lis1 is absent. Mutant spermatids fail to form correct acrosomes and nuclei appear distorted in size and shape. The tissue architecture in mutant testis appears severely disturbed displaying collapsed seminiferous tubules, mislocated germ cells, and increased apoptosis. These results provide evidence for an essential and hitherto uncharacterized role of the Lis1 protein in spermatogenesis, particularly in the differentiation of spermatids into spermatozoa. Type 1 lissencephaly is an autosomal dominant congenital disorder in humans, characterized by a smooth surface of the brain due to abnormal neuronal migration during early development (1Dobyns W.B. Neurol. Clin. 1989; 7: 89-105Abstract Full Text PDF PubMed Google Scholar). Humans afflicted by isolated lissencephaly carry hemizygous mutations of the lissencephaly 1 gene (LIS1), suggesting that haplo-insufficiency of LIS1 causes the disease (2Reiner O. Carrozzo R. Shen Y. Wehnert M. Faustinella F. Dobyns W.B. Caskey C.T. Ledbetter D.H. Nature. 1993; 364: 717-721Crossref PubMed Scopus (909) Google Scholar, 3Hattori M. Adachi H. Tsujimoto M. 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The protein has been identified biochemically as the non-catalytic subunit of the trimeric type I platelet-activating factor acetylhydrolase (PAF-AH 1B) 1The abbreviations used are: PAF-AH 1Bplatelet-activating factor acetylhydrolase type IMops4-morpholinepropanesulfonic acidRTreverse transcriptionPBSphosphate-buffered salineTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick end labelingTBSTris-buffered salineFCSfetal calf serumPpostnatal daysEembryonic daysEMelectron microscopy. that inactivates platelet-activating factor (PAF) (3Hattori M. Adachi H. Tsujimoto M. Arai H. Inoue K. Nature. 1994; 370: 216-218Crossref PubMed Scopus (456) Google Scholar). PAF is a potent signaling phospholipid in various tissues (6Venable M.E. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Lipid Res. 1993; 34: 691-702Abstract Full Text PDF PubMed Google Scholar, 7Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. 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Cell Biol. 2000; 2: 784-791Crossref PubMed Scopus (378) Google Scholar). Whether the apparently different biochemical interactions of Lis1 are functionally related remains to be seen. platelet-activating factor acetylhydrolase type I 4-morpholinepropanesulfonic acid reverse transcription phosphate-buffered saline terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling Tris-buffered saline fetal calf serum postnatal days embryonic days electron microscopy. Proteins homologous to vertebrate Lis1 have been identified in several organisms, including Saccharomyces cerevisiae (29Geiser J.R. Schott E.J. Kingsbury T.J. Cole N.B. Totis L.J. Bhattacharyya G. He L. Hoyt M.A. Mol. Biol. Cell. 1997; 8: 1035-1050Crossref PubMed Scopus (173) Google Scholar), Aspergillus nidulans (30Xiang X. Osmani A.H. Osmani S.A. Xin M. Morris N.R. Mol. Biol. Cell. 1995; 6: 297-310Crossref PubMed Scopus (290) Google Scholar), Caenorhabditis elegans (31Dawe A.L. Caldwell K.A. Harris P.M. Morris N.R. 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Ovarian mutant clones of Lis1 in the fly indicated that it is required for germ line cell division and oocyte differentiation, supporting the notion that Lis1 interacts with the dynein complex to regulate the function of the membrane skeleton, necessary for nuclear and neuronal migration. Lis1 also functions in dendritic elaboration and axonal transport in Drosophila and in cultured neurons (38Liu Z. Steward R. Luo L. Nat. Cell Biol. 2000; 2: 776-783Crossref PubMed Scopus (181) Google Scholar). Collectively, these data argue that Lis1 has been highly conserved during evolution and may exert similar cellular functions in various developmental processes. Murine Lis1 mRNA is widely expressed in many cell types, but certain splicing and polyadenylation variants are differentially found in adult brain, heart, and testis (39Peterfy M. Gyuris T. Grosshans D. Cuaresma C.C. Takacs L. Genomics. 1998; 47: 200-206Crossref PubMed Scopus (15) Google Scholar). Particularly in testis, an alternatively spliced transcript exists that contains the additional exon 2a as part of the 5′-untranslated leader sequence. The functional role of Lis1 in organs other than brain has not been determined, partly due to early embryonic lethality of Lis1-null mutants. We have taken advantage of a gene trap mouse line that carries a mutagenic insertion within the LIS1 gene causing selective disruption of a testis-specific Lis1 transcript. This permitted us to explore the role of Lis1 in the male germ line. Here we report that Lis1-deficient male mice are infertile, whereas females show normal fertility. The defect in spermatogenesis leads to a blockade of spermatid differentiation and severely distorted tissue architecture of the seminiferous tubules. Mutant spermatids fail to form correct acrosomes and frequently do not undergo appropriate nuclear condensation. The mutant phenotype also presents significantly increased apoptosis of germ cells in adult testis. Thus, our results reveal a unique and novel function of Lis1 in spermatogenesis. Generation of the LIS1 Gene Trap Mouse—The exon gene trap vector pKC421 was constructed from the plasmid pGT1.8 IRESβgeo (52Mountford P. Zevnik B. Duwel A. Nichols J. Li M. Dani C. Robertson M. Chambers I. Smith A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4303-4307Crossref PubMed Scopus (284) Google Scholar) by deletion of the engrailed 2 (En-2) splice acceptor site with BamHI/BglII digestion and by removal of an SalI site downstream of theSV40 poly (A) addition signal. The ES cell line 2A-53 containing an insertion of the gene trap vector within the LIS1 gene was generated as described previously (53Chowdhury K. Bonaldo P. Torres M. Stoykova A. Gruss P. Nucleic Acids Res. 1997; 25: 1531-1536Crossref PubMed Scopus (80) Google Scholar). The 129/Sv-derived ES cell clone was used in morula aggregations according to published procedures to obtain chimeric male founders that were mated with outbred NMRI females (53Chowdhury K. Bonaldo P. Torres M. Stoykova A. Gruss P. Nucleic Acids Res. 1997; 25: 1531-1536Crossref PubMed Scopus (80) Google Scholar). Heterozygous progeny was mated to maintain the allele. For genotyping by Southern blot analysis, DNA was extracted from tail biopsies and digested with SacI restriction enzyme. Blots on Hybond N membranes (Amersham Biosciences, Freiburg, Germany) were hybridized in 6× SSC, 5× Denhardt's, 0.1% SDS, 100 μg/ml denatured salmon sperm DNA at 65 °C overnight using an SacI/fragment of the second intron of the LIS1 gene as probe (see Fig. 1). Hybridized filters were washed twice at 65 °C with 0.2× SSC containing 0.1% SDS. Wild type and mutant alleles are represented by 4.3- and 8-kb SacI fragments, respectively. Construction of Genomic Phage Library—To clone the site of vector integration, a genomic phage library of a heterozygous mouse was generated in the λDASH-II vector (Stratagene) according to standard procedures. Two independent recombinant phage clones carrying inserts of 21 and 17.2 kb were isolated with LacZ- and neo-specific hybridization probes. Regions flanking the integrated vector were sequenced and searched using Blast against mouse genome databases. Northern Blot Analysis—Total RNA was isolated from testis and brain of wild type and mutant mice at various postnatal stages using the Total RNA Isolation Reagent (Biomol) according to the manufacturer's instructions. RNA samples (30 μg) were denatured at 65 °C for 10 min in loading buffer (50% formamide, 13 Mops buffer, 6.5% formaldehyde) and run on 0.8% agarose gels containing 0.12% formaldehyde. Electrophoresis and RNA blotting was performed according to standard procedures (54Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, Second Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Blots were hybridized with radioactively labeled testis-specific Lis1 cDNA. The human elongation factor 2 cDNA probe was used as loading control (55Rapp G. Klaudiny J. Hagendorff G. Luck M.R. Scheit K.H. Biol. Chem. Hoppe.-Seyler. 1989; 370: 1071-1075Crossref PubMed Scopus (66) Google Scholar). RT-PCR Analysis—Total RNA (1 μg) was reverse-transcribed in a final volume of 20 μl containing 200 units of Superscript reverse transcriptase (Invitrogen, Karlsruhe, Germany), 40 units of RNasin (Roche Applied Science, Mannheim, Germany), and oligo-dT primer. PCR was carried out with 2 μl of cDNA, 10 pmol each of forward and reverse primers, and 3 units of Taq polymerase. Cycling condition were 94 °C, 58 °C, and 68 °C for 30 s each. Glyceraldehyde-3-phosphate dehydrogenase was used as internal control. The glyceraldehyde-3-phosphate dehydrogenase 5′ primer was 5′-ACC ACA GTC CAT GCC ATC AC-3′; the glyceraldehyde-3-phosphate dehydrogenase 3′ primer was 5′-TCC ACC ACC CTG TTG CTG TA-3′. Western Blot Analysis—Protein extracts from testis and brain were prepared in SE buffer containing 0.32 mol/liter sucrose, 1 mmol of EDTA, and 0.1% β-mercaptoethanol. For electrophoresis samples (50 μg/lane) were boiled for 10 min, briefly spun down at 500 rpm, and loaded onto an 8% polyacrylamide gel. Proteins were blotted onto a polyvinylidene difluoride membrane (Roche Applied Science) and probed with goat polyclonal anti-Lis-1 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and rabbit polyclonal α-tubulin antibody (Sigma-Aldrich) as internal reference. Antibody reactions were visualized on filters with a 1:10,000 dilution of alkaline phosphatase-conjugated anti-goat or anti-rabbit immunoglobulin (Sigma-Aldrich) and incubated in 0.35 mg/ml nitro blue tetrazolium and 0.18 mg/ml 5-bromo-4-chloro-indolylphosphate substrate. Analysis of Fertility—Reproductive capacity of Lis-1GT/GT and Lis-1+/GT males was determined by breeding with wild type and mutant females. Ten males of each genotype were mated with females for 6 months. Females were checked for vaginal plugs every day and separated for 21 days when positive. The total number of vaginal plugs and born offspring was counted for each male during the entire investigation period. Histology—Testis and epididymis from mice of different postnatal age were fixed in Bouin's fixative for 48 h at room temperature. The fixative was removed with 70% ethanol for 2-3 days, and tissues were embedded in paraffin. Mounted sections (4-6 mm) were deparaffinized, rehydrated, and stained with hematoxylin-eosin, or used for immunohistochemistry. Spermatozoa were prepared and resuspended in PBS. Air-dried smears were fixed in methanol/acetone (1:1) for 5 min and used for immunohistochemistry. Immunohistochemistry—Spermatozoa smears and sections of testis were incubated for 16-18 h at 4 °C with one of the following antibodies in a 1:200 dilution: goat anti-Lis-1 antibody (Santa Cruz Biotechnology), rabbit anti-OAM antibody (outer acrosomal membrane), rabbit anti-acrosin antibody, rabbit anti-Tnp-2 antibody, goat anti-HSP90α antibody (Santa Cruz Biotechnology). For co-localization studies, sperm smears were incubated with a 1:200 dilution of anti-Lis-1 antibody and a 1:300 dilution of rabbit anti-OAM or rabbit anti-acrosin antibodies. Slides were then washed three times with PBS and incubated for several hours at room temperature with 1:100 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Sigma-Aldrich Chemie, Deisenhofen, Germany). Double immunostainings were performed with a mixture of 1:100 fluorescein isothiocyanate-conjugated goat anti-mouse IgG and Cy3-conjugated anti-rabbit IgG (Sigma-Aldrich). After three further washes with PBS, slides were mounted in 4′,6-diamidino-2-phenylindole mounting solution (Vector Laboratories Inc., Burlingame, CA). Electron Microscopy—For conventional electron microscopy, mouse testis was fixed with 1% paraformaldehyde and 3.5% glutaraldehyde in 0.1 m cacodylate buffer (pH 7.4) for 8-12 h. Fixed testes were cut into small pieces and thoroughly washed over 3-4 days at 4 °C in 0.1 m cacodylate buffer containing 0.1 m saccharose. Tissue fragments were then treated with 1% OsO4 in cacodylate buffer for 2 h, washed three times, dehydrated, and embedded in epoxy resin. Ultrathin sections were contrasted using uranyl acetate and lead citrate and examined with a Leo 906 electron microscope. For immunoelectron microscopy wild type testes fragments were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 m phosphate buffer for 4 h. Specimens were washed in 0.1 m phosphate buffer supplemented with 7% sucrose, subsequently dehydrated in increasing concentrations of ethanol, and embedded in LR White resin. Ultrathin sections were blocked in 2% bovine serum albumin and 0.04 m glycine in phosphate-buffered saline (PBS, pH 7.4) for 1 h at room temperature. The anti-Lis-1 antibody was incubated at room temperature for 1 h at a 1:200 dilution in 2% bovine serum albumin in PBS. After thorough washing, specimens were decorated with the rabbit anti-goat secondary antibody coated on 15-nm colloidal gold particles (dilution 1:20; Biocell) for 1 h. Finally, the sections were contrasted with 10% uranyl acetate for 20 min in the dark. Omission of the primary antibody served as a negative control. Nuclear DNA Fragmentation Labeling (TUNEL)—Testes were removed from 90-day-old wild type and Lis-1GT/GT mice, fixed in formalin solution, embedded in paraffin, and cut into 5-μm sections on which hematoxylin-eosin stainings and TUNEL assays were performed. To determine apoptotic cells sections were dewaxed prior to digestion with 0.7 unit/ml proteinase K (Sigma, Deisenhofen/Germany) in Tris-buffered saline (50 mm Tris-HCl, 150 mm NaCl, pH 7.5, TBS), supplemented with 2 mm CaCl2. Sections were then incubated in TBS containing 10% fetal calf serum (FCS) and 0.3% H2O2 to block endogenous peroxidase activity. Subsequently, slides were rinsed with TBS and incubated for 60 min at 37 °C in reaction buffer for terminal transferase (Roche Applied Science, Germany), containing 50 μl of labeling buffer (250 units/ml terminal transferase, 20 μl/ml 10× digoxigenin-DNA labeling mix, and 1 mm CoCl2). After labeling, sections were washed in TBS, blocked with 10% FCS (Roche Applied Science) for 15 min, and then incubated with a rabbit horseradish peroxidase-conjugated F(ab)2 fragment against digoxigenin (Dako/Hamburg, Germany) for 60 min. The horseradish peroxidase-conjugated F(ab)2 fragment was applied in a 1:200 dilution with TBS containing 10% FCS. Nuclear signals were visualized using 3,3′-diaminobenzidine. Negative controls were performed without terminal transferase. Lymph nodes with reactive follicular hyperplasia were used as positive control. Statistical significance of the obtained data was determined using the Man Whitney U test with p < 0.05 considered to be significant. Integration of a Gene Trap Vector in the LIS1 Locus Results in Male Infertility—In a gene trap approach using an exon trap vector, we isolated the embryonic stem (ES) cell clone 2A-53 from which a stable mouse line was generated. The trapped gene was most abundantly expressed in heart, neural tube, brain, and dorsal root ganglia during embryogenesis (Fig. 1C). 5′ Rapid amplification of cDNA ends from known vector sequences failed to identify a fusion transcript in RNA from heart and brain. Therefore, the gene trap vector, including flanking genomic sequences, was cloned. Sequence analysis of two independently isolated phage clones showed that the exon trap vector had unintentionally integrated in the second intron of the mouse LIS1 gene (Fig. 1A). RT-PCR on RNA from the ES cell clone 2A-53 identified a transcript containing the second exon of the LIS1 gene spliced to a cryptic splice acceptor site present within the internal ribosomal entry site sequence of the vector. This aberrant splicing event generates the β-Geo mRNA and explains why the trapped ES cell clone could be obtained under Geneticin selection. We next generated mice homozygous for the gene trap integration (Lis1GT/GT). Offspring of heterozygous parents displayed normal Mendelian distribution of genotypes and no apparent pathological phenotype (Fig. 1B). In particular, Nissl staining of brains from adult mutants failed to show any signs of lissencephaly and compound heterozygotes with a Lis1-defective allele had no augmentation of the brain phenotype (data not shown). These observations suggested that the gene trap insertion had not generally disrupted the LIS1 gene. However, subsequent breeding indicated that homozygous mutant males were consistently infertile, whereas mutant females reproduced normally (Table I). Mating with homozygous males yielded vaginal plugs, but sperm could not be recovered from the uterus and no pregnancies were recorded. In line with this result, we found that testes of homozygous males were considerably smaller (∼50%) and the epididymis contained essentially no spermatozoa (Fig. 2A). To determine the onset of the phenotype in mutant mice, we histologically evaluated testes from postnatal days 15-90 (P15-P90). Sections of mutant testis initially (P14-P45) exhibited intact seminiferous tubules of normal diameter and germ cells as well as Sertoli and Leydig cells present at the appropriate locations (Fig. 2B). Notably, spermatogonia at the lamina propria, spermatocytes of the post-pachytene stage, and round spermatids at the luminal side of seminiferous tubules were observed until P45 when elongating spermatids begin to accumulate in the inner lining of the seminiferous epithelium. At postnatal P90, however, round and elongating spermatids were released prematurely from the epithelium and located inside the lumen of tubules, in contrast, to wild type spermatids assembling correctly at the luminal surface of seminiferous tubules. At this postnatal stage also the tubular structure seemed to have collapsed in the mutant, lacking the epithelial architecture and a clearly visible lumen. Moreover, only very few spermatozoa were present in mutant testis as compared with wild type (Fig. 2B) and the residual ones appeared in small clusters and frequently abnormal (data not shown). These results then suggested that the gene trap mutation affected the maintenance phase of spermatogenesis, including terminal differentiation of spermatids, but apparently did not interfere with the initiation phase up to the generation of spermatids. To determine the distribution of sperm progenitor cells in a more quantitative fashion, we investigated the expression of the spermatocyte-specific marker transcripts, testis-specific phosphoglycerate kinase (Pgk-2) (40Boer P.H. Adra C.N. Lau Y.F. McBurney M.W. Mol. Cell. Biol. 1987; 7: 3107-3112Crossref PubMed Scopus (147) Google Scholar), and proacrosin (ACR) (41Kremling H. Keime S. Wilhelm K. Adham I.M. Hameister H. Engel W. Genomics. 1991; 11: 828-834Crossref PubMed Scopus (58) Google Scholar), as well as the spermatid-specific markers, transition protein 2 (Tnp2) (42Adham I.M. Nayernia K. Burkhardt-Gottges E. Topaloglu O. Dixkens C. Holstein A.F. Engel W. Mol. Hum. Reprod. 2001; 7: 513-520Crossref PubMed Scopus (116) Google Scholar), protamin 2 (Prm-2) (43Schluter G. Celik A. Obata R. Schlicker M. Hofferbert S. Schlung A. Adham I.M. Engel W. Mol. Reprod. Dev. 1996; 43: 1-6Crossref PubMed Scopus (45) Google Scholar), and Hook1 (44Strausberg R.L. Feingold E.A. Grouse L.H. 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A. 2002; 99: 16899-16903Crossref PubMed Scopus (1559) Google Scholar) in testis of wild type, heterozygous, and homozygous mice at 45 days of age. As shown in Fig. 2C, all transcripts were expressed at similar levels in wild type and mutant animals confirming that germ cell progenitors, including spermatids, were normally formed. From these results we concluded that the gene trap mutation causes a blockade of late spermatid differentiation resulting in a severe reduction of mature gametes. However, Lis1 is not essential for testis development, because testis histology during early postnatal stages appeared normal in the mutant and spermatogonia, spermatocytes, and round spermatids were formed in seminiferous tubules, whereas the late transition to spermatozoa essentially failed to occur.Table IFertility of wild type, LIS1+/GT, and LIS1GT/GT mutant miceMating of genotypesMaleaAge-matched 3- to 9-month-old animals were used for crossingFemaleaAge-matched 3- to 9-month-old animals were used for crossingMice bornLitter sizebMean of more than 20 littersLIS+/+LIS+/+1687.3LIS+/+LIS1+/GT1766.6LIS+/+LIS1GT/GT1917.6LIS1+/GTLIS+/+1597.1LIS1GT/GTLIS+/+00a Age-matched 3- to 9-month-old animals were used for crossingb Mean of more than 20 litters Open table in a new tab
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