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

Hiroyuki KoizumiDagger , Noritaka YamaguchiDagger , Mitsuharu HattoriDagger §, Tomo-o Ishikawa||, Junken AokiDagger , Makoto M. Taketo**||, Keizo InoueDagger DaggerDagger, and Hiroyuki AraiDagger §§

From the Dagger  Department of Health Chemistry and ** Laboratory of Biomedical Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan and  Banyu Tsukuba Research Institute (Merck), Tsukuba, Ibaraki 300-0026, Japan

Received for publication, November 20, 2002, and in revised form, January 22, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intracellular type I platelet activating factor-acetylhydrolase is a phospholipase that consists of a dimer of two homologous catalytic subunits alpha 1 and alpha 2 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 the in vivo function of the catalytic subunits remains unclear. In this study, we generated alpha 1- and a2-deficient mice by targeted disruption. alpha 1-/- mice are indistinguishable from wild-type mice, whereas alpha 2-/- 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, alpha 2 is expressed in all seminiferous tubule cell types, whereas alpha 1 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 alpha 2-/- and double-mutant mice, suggesting that the catalytic subunits, especially alpha 2, are a determinant of LIS1 expression level.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Platelet-activating factor (PAF)1 is a potent signaling phospholipid involved in diverse physiological events, such as inflammation and anaphylaxis (1). In addition, PAF has been implicated in the central nervous system (2, 3) and the reproductive system (4, 5). 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 (6, 7) and a plasma type (8). Intracellular type I PAF-AH (PAF-AH (I)) is an oligomeric complex. It contains a dimer of two homologous catalytic subunits, alpha 1 and alpha 2, and a non-catalytic beta  subunit (6, 9-11). Interestingly, the beta  subunit was later found to be identical to LIS1, the product of the causative gene for type I lissencephaly (10, 12). 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 Lis1 null mutation die early in embryogenesis soon after implantation (13). Heterozygous and compound heterozygous mice have expression level-dependent defects in neuronal migration (13). 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 (14), cytoplasmic dynein (15, 16), and NUDE (17-20). 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. (21) have described a case of functional hemizygosity of alpha 1, possibly responsible for the resulting mental retardation, ataxia, and brain atrophy in this patient. Furthermore, Lecointe et al. (22) have proposed that deregulation of transcription of the human alpha 2 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 alpha 1 and alpha 2 catalytic subunits belong to a novel serine esterase family (9). These subunits, which show ~60% amino acid homology with each other, form homodimers and a heterodimer. Ho et al. (23) have reported the x-ray crystal structure of the alpha 1 homodimer. The folding is unique among known lipases and phospholipases. The structure unexpectedly resembles those of the G-protein family such as p21ras and Galpha . To elucidate the in vivo function of the catalytic subunits of PAF-AH (I), namely alpha 1 and alpha 2, we generated mice lacking either one or both of these two proteins.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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Generation of alpha 1, alpha 2 Mutant Mice-- alpha 1, alpha 2 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 the alpha 1 and alpha 2 genes, which include a translation initiation site and catalytic motif (GXSXV) with a PGKneobpA cassette (24). A PGKDTA (diphtheria toxin A 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.

Antibodies-- Mouse monoclonal antibodies against alpha 1 and alpha 2 were established as follows. alpha 1-/- and alpha 2-/- 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 µg of protein and established monoclonal antibody, producing hybridoma cell lines as previously described (11). Monoclonal antibody against LIS1 (clone 338, a kind gift from Dr. O. Reiner, Weizmann Institute, Rehovot, Israel) and alpha -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.

Histological and Immunohistochemical Analyses-- Testes were dissected and fixed overnight in Bouin's fixative at 4 °C. Paraffin sections (5 µm) 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 4% paraformaldehyde in PBS. Testes were dissected and refixed overnight in 4% paraformaldehyde at 4 °C. Paraffin sections (5 µm) 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 alpha 1 and alpha 2 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.

Western Blot Analysis-- 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 mM Na3PO4). 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 µg/lane) was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes or nitrocellulose membranes. The membranes were blocked with 5% (w/v) skim milk (Wako) in TTBS buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% (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.

Northern Blot Analysis-- Total RNA was extracted from mouse tissues using Isogen (Nippongene). Total RNA (10 µg/lane) was separated by 1% 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, 0.1% SDS at 65 °C. Probes for alpha 1, alpha 2, 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 dehydrogenase cDNA probe (Clontech) to ensure equal loading.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We established monoclonal antibodies against alpha 1 and alpha 2 by immunizing the respective knockout mice. A Western blot analysis of adult mouse tissue shows that alpha 1, alpha 2, and LIS1 were most abundantly expressed in brain and testis (Fig. 1). Expression of alpha 2 and LIS1 were observed in other tissues as well, whereas alpha 1 expression was restricted to embryonic brain and adult testis (Fig. 1). The expression levels of alpha 2 and LIS1 were observed to be essentially proportional to that of alpha -tubulin, a component of microtubules. Because LIS1 plays an important role in microtubule dynamics (14, 16), it can be postulated that both alpha  subunits are also involved in this process.


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Fig. 1.   Expression pattern of PAF-AH (I) subunits in mice. Expression of alpha 1, alpha 2, LIS1, and alpha -tubulin in various mouse tissues was examined by Western blotting using specific antibodies.

Immunohistochemical staining of adult mouse testes revealed that alpha 2 and LIS1 immunoreactivity was present in all seminiferous tubule cell types (Fig. 2, D and G). Intense staining of alpha 2 and LIS1 was observed in meiotically dividing spermatocytes and elongating spermatids. In contrast, alpha 1 staining was restricted to the cells lining the basal compartment of seminiferous tubules (Fig. 2A). Magnification revealed that alpha 1 was specifically localized in spermatogonia cytoplasm (Fig. 2B, arrow), whereas alpha 2 and LIS1 were expressed in the cytoplasm of all types of spermatogenic cells and Sertoli cells (Fig. 2, E and H), suggesting that alpha 1 is involved specifically in proliferation and/or differentiation of spermatogonia. LIS1 was also localized at meiotic spindles of spermatocytes (Fig. 2H, arrowhead) and manchettes of elongating spermatids (Fig. 2H, arrow), both of which are specific microtubule structures. No staining of alpha 1 or alpha 2 was detected in the seminiferous tubules of null mutant mice (Fig. 2, C and F).


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Fig. 2.   Localization of PAF-AH (I) subunits in mouse testis. Immunohistochemical staining of testis cross-sections of wild-type (A, B, D, E, G, and H), alpha 1-/- (C), and a2-/- (F) adult mice was performed using specific antibodies for alpha 1 (A-C), alpha 2 (D-F), and LIS1 (G and H). Hematoxylin was used for counterstaining. Scale bars: A, C, D, F, and G, 100 µm; B, E, and H, 25 µm.

We used homologous recombination in embryonic stem cells to generate mice lacking the alpha 1 (Pafah1b3) and a2 (Pafah1b2) genes. Parts of exon 2 and exon 3 of each gene, including the translation initiation site and the catalytic serine residue, were replaced with a neomycin-resistance gene (Fig. 3A). Targeted embryonic stem cell clones and subsequent germ line transmissions were detected by PCR and/or Southern blot analysis (Fig. 3B). Both alpha 1-/- and alpha 2-/- mice were born with the expected Mendelian frequencies, viable and apparently indistinguishable from their wild-type littermates. Western blot analysis of the brain and the testis homogenates showed no immunoreactive bands in either alpha 1-/- or alpha 2-/- mice (Fig. 6A). alpha 1-/-/alpha 2-/- mice were also viable and apparently indistinguishable from wild-type mice. However, alpha 1-/-/alpha 2-/- males were found to be infertile, whereas female fertility was not affected. Testes weights of 5-week-old alpha 1-/-/alpha 2-/- mice were significantly (~35%) smaller than those of wild-type mice (Table I). Testes weights were not noticeably reduced in alpha 1-/- mice, whereas they were reduced to 60% in alpha 2-/- mice (Table I). There was no significant difference in body weight among any of the genotypic combinations (Table I).


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Fig. 3.   Targeted disruption of alpha 1 (Pafah1b3) and a2 (Pafah1b2) genes. A, map of the alpha 1 and alpha 2 129/SvJ genomic clones and construction of the targeting vectors. Exons are represented as numbered boxes. Hatched boxes show coding regions. The translation initiation ATG codon is present in exon 2, and the catalytic site is coded in exon 3 of each gene. The positions of PGKneobpA cassette (Neo) and PGKDTA cassette (DTA) for positive and negative selection of the transfected embryonic stem cells are indicated. Arrowheads indicate the position of the PCR primers. Horizontal bars indicate the Southern hybridization probes. K, KpnI; B, BglII. B, Southern blot analyses of offspring from heterozygous matings. Hybridization of the probe-29 with KpnI-digested genomic DNA from alpha 1 mutant littermates yielded 7.2-kb (alpha 1 wild-type) and 8.0-kb (alpha 1 mutant) bands. Hybridization of the probe-30 with BglII-digested genomic DNA from alpha 2 mutant littermates yielded 4.7-kb (alpha 2 wild-type) and 5.8-kb (alpha 2 mutant) bands.


                              
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Table I
Reduced testis size in mutant mice
Comparison of mean (±S.E.) testes and body weight in wild-type and various mutant mice at 5 weeks of age (n = 8).

The histology of mutant testes was examined at age 5 weeks, the time when the first wave of murine spermatogenesis is completed. The seminiferous tubules of alpha 1-/-/alpha 2-/- mice showed a 50% reduction in diameter, and spermatogenic cells were dramatically decreased (Fig. 4F) when compared with wild-type mice (Fig. 4A). Spermatocytes beyond the pachytene stage and round spermatids were significantly reduced in number. Elongated spermatids were rare, and the few remaining spermatids had deformed nuclei. Some germ cells appeared to be detached from the Sertoli cells. No spermatozoa were observed in the epididymis (data not shown). In older mice, early germ cell stages were more severely affected, leading to increased depletion of spermatocytes and spermatogonia (data not shown). In TUNEL assays, apoptotic cells were rare in wild-type testes as previously reported (25) (Fig. 5A), whereas alpha 1-/-/alpha 2-/- testes had a significantly larger number of apoptotic cells (Fig. 5B). The cells undergoing apoptosis in alpha 1-/-/alpha 2-/- testes were predominantly spermatocytes. These results indicate that in the absence of the catalytic subunits of PAF-AH (I), the differentiation of prehaploid stages of spermatogenesis fails, leading to induction of programmed cell death in the germ cell compartment.


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Fig. 4.   PAS and hematoxylin staining of various 5-week-old mutant mice testis sections. Wild-type (A), alpha 1-/- (B), alpha 2-/- (C), alpha 1-/-/alpha 2+/- (D), alpha 1+/-/alpha 2-/- (E), and alpha 1-/-/alpha 2-/- (F) mice were examined. Note severe reduction of spermatogenic cells, especially after pachytene spermatocyte stage in alpha 1+/-/alpha 2-/- (E) and alpha 1-/-/alpha 2-/- (F) mice. Elongating spermatids were almost absent in the latter. Scale bar: 100 µm


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Fig. 5.   Increased apoptosis in spermatogenic cells of alpha 1-/-/alpha 2-/- testis. Sections of seminiferous tubules of wild-type (A) and alpha 1-/-/alpha 2-/- mice (B) at 5 weeks of age are shown. Apoptotic cells are labeled by TUNEL staining. Scale bar: 100 µm

Histologically, alpha 1-/- mice testes showed no apparent impairment of spermatogenesis (Fig. 4B). On the other hand, alpha 2-/- mice testes showed significant weight reduction and varying germ cell impairment (Fig. 4C and Table I). When comparing alpha 1-/-/alpha 2+/- mice testes with alpha 1+/-/alpha 2-/- mice testes from both the histological and weight points of view, impairment was more severe when both alpha 2 alleles were missing (Fig. 4, D and E, and Table I). These results indicate that alpha 2 plays a more important role in spermatogenesis than alpha 1 and that missing alpha 1 alleles can in part be compensated by the presence of alpha 2. However, it is evident that alpha 1 also plays a role in male fertility based on the observation that the absence of both alpha 2 alleles can be partly compensated by the presence of alpha 1 alleles (Fig. 4, C, E, and F, and Table I). The alpha 1 protein level was reduced to about 20% of the normal level in alpha 2-/- mice (Fig. 3C) even though there was no reduction of the mRNA level (data not shown), whereas the alpha 2 level was not changed in alpha 1-/- mice.

Because a large portion of LIS1 forms complexes with alpha 1 and/or alpha 2 in the cytosolic fraction, we examined the LIS1 protein levels in alpha 1 and/or alpha 2 mutant adult mice. In alpha 2-/- mice, LIS1 levels in both brain and testis were reduced to ~30% compared with wild-type mice (Fig. 6A). In contrast, no reduction of LIS1 was observed in alpha 1-/- mice (Fig. 6A). In alpha 1-/-/alpha 2-/- mice, as in alpha 2-/- mice, LIS1 levels were reduced to about 20% of the levels in the wild type mice. Lis1 mRNA expression in alpha 2-/- mice was either the same as or slightly higher than the expression in wild type mice (Fig. 6B). On the other hand, LIS1 expression in E14.5 (embryonic day 14.5) brain of each mutant mouse was not significantly less than that in wild-type mice (Fig. 6C).


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Fig. 6.   LIS1 protein expression is reduced in alpha 2-/- and alpha 1-/-/alpha 2-/- adult mice. A, Western blot analysis of PAF-AH (I) subunits in adult brains and testes of the wild-type mice and various alpha  mutant mice. Note the alpha 1 expression is reduced in both brain and testis of alpha 2-/- mice compared with wild-type mice (*), and LIS1 expression is reduced in both brain and testis of alpha 2-/- and alpha 1-/-/alpha 2-/- mice compared with wild-type mice (**). B, Northern blot analysis of mRNA expression of Lis1 in brains of the wild-type, alpha 2+/- and alpha 2-/- mice. Glyceraldehyde-3-phosphate dehydrogenase is included as a control for equal RNA loading. C, Western blot analysis of LIS1 protein in embryonic (E14.5) brain of wild-type and various alpha  mutant mice.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated that the catalytic subunits of intracellular PAF-AH (I) are involved in murine spermatogenesis. Our study gives new insights into the in vivo function of PAF-AH (I). We showed by Western blotting that in mice the catalytic subunits alpha 1 and alpha 2 as well as LIS1 are present at high levels in both brain and testis. Interestingly, it was noted that PAF-AH (I) subunits exhibit expression levels proportional to those of alpha -tubulin, a major component of intracellular microtubules. The above findings and the fact that LIS1 is a microtubule-associated protein lead us to speculate that the catalytic subunits are also involved to a major extent in microtubule dynamics. Microtubule structures undergo dramatic rearrangements in the process of spermatogenesis. Processes involving microtubule rearranging include mitotic division of spermatogonia, meiotic division of spermatocytes, manchette formation, and flagellar axoneme assembly in spermatids. The most severe degeneration occurs in primary spermatocytes of alpha 1-/-/alpha 2-/- mice, but degeneration also occurs in meiotically dividing spermatocytes (increased apoptosis) and in elongating spermatids (abnormal nuclear morphogenesis). Therefore, it can be stated that PAF-AH (I) catalytic subunits are involved in the several processes of spermatogenesis and not just in a specific stage of spermatogenesis.

Although the exact molecular mechanism and function of the PAF-AH (I) catalytic subunits in spermatogenesis are unclear, we found that depletion of both catalytic subunits leads to a major decrease in LIS1 protein levels, suggesting that the catalytic subunits are associated with LIS1. It is most likely that LIS1 protein levels are post-transcriptionally influenced by the catalytic subunits, because LIS1 mRNA levels are not altered in alpha 2-/- mice. Because LIS1 levels are crucial for cortical brain development (13), we speculate that LIS1 is involved in microtubule organization of spermatogenesis and that reduced LIS1 protein levels are responsible for the testicular defects occurring in alpha 1-/-/alpha 2-/- mice. However, our studies also revealed that the more severe testicular degeneration in alpha 1-/-/alpha 2-/- mice than in alpha 2-/- mice cannot be explained solely by the amount of reduction in the level of LIS1, because the reduction of LIS1 was not very different between alpha 2-/- and alpha 1-/-/alpha 2-/- mice. Given that PAF-AH (I) closely resembles trimeric G-proteins (23), the catalytic subunits may mediate a novel intracellular signaling to LIS1 in mammals, and depletion of this signaling pathway may result in severe impairment in spermatogenesis.

Because PAF-AH (I) catalytic subunits are predominantly expressed in brain as well as in testis and because haplo-insufficiency of LIS1 leads to severe brain malformation in both humans and mice (13), we expected that mice lacking the catalytic subunits would exhibit brain abnormalities. However, Nissl staining of adult alpha 1-/-/alpha 2-/- mice brain showed no obvious abnormalities in lamination of neurons in the cerebral cortex, hippocampus, or cerebellum (data not shown). To our surprise, in E14.5 brain of alpha 1-/-/alpha 2-/- mice, there was no significant reduction of LIS1 protein levels, suggesting that there is a mechanism to maintain LIS1 protein levels and the function of catalytic subunits in brain.

In alpha 2-/- mice, both alpha 1 and LIS1 protein levels were significantly reduced compared with wild-type mice, whereas in alpha 1-/- mice both alpha 2 and LIS1 levels were not changed markedly. In preliminary experiments, supernatants of mice testis or brain homogenates were subjected to DEAE-Sepharose ion exchange column chromatography. In the case of the alpha 1-/- homogenates, alpha 2 eluted in the same fraction as LIS1, whereas in the case of the alpha 2-/- homogenates alpha 1 and LIS1 eluted at the different positions.2 These results suggest that alpha 2, probably the alpha 2 homodimer, has a strong affinity for LIS1 and that the alpha 1 homodimer has a weak or negligible affinity for LIS1 in vivo. Considering the fact that alpha 1 mRNA levels are not changed in alpha 2-/- mice (data not shown), the present results also suggest that the alpha 1 protein is not stably expressed in the absence of alpha 2 in vivo. These observations are consistent with our previous report (26) that alpha 1/alpha 2 heterodimers and alpha 2 homodimers are the major PAF-AH (I) catalytic units present in vivo.

Immunostaining studies revealed that alpha 2 is expressed in all spermatogenic cells, whereas alpha 1 is expressed only in spermatogonia. This expression pattern parallels the finding that deletion of both subunits induces a marked loss of germ cells, even at an early spermatogenic stage. We have previously shown that alpha 1 is specifically expressed in migrating neurons in the embryonic and postnatal stages, whereas the alpha 2 expression level is almost constant from the fetal stages through adulthood (26, 27). As a result, the catalytic subunits change from the alpha 1/alpha 2 heterodimer to the alpha 2/alpha 2 homodimer in neurons during brain development. It is likely that the same type of alteration in the catalytic dimer occurs during differentiation from spermatogonia to spermatocytes. Interestingly, it has been shown that undifferentiated spermatogonia move to specific sites within the seminiferous tubule and spread their progeny laterally along the base of the tubule (28). Transplantation experiments demonstrated that spermatogonia are capable of moving along the length of the seminiferous tubule at a rate of more than 50 µm/day (29, 30). Although the biological significance of the change in the catalytic subunit combination is not known, it is interesting to speculate that alpha 1 mediates a common signaling pathway in migrating neurons and spermatogonia.

The cellular function of the enzyme activity and the physiological substrate of this enzyme are largely unknown. PAF has been detected in sperm from several mammalian species and has been shown to affect sperm motility and fertility (4). High-fertility spermatozoa, for example, have a substantially greater PAF content than low-fertility spermatozoa (31, 32). Exogenously added PAF increases the motility of human spermatozoa (33). Our study gives hints at the possibility that PAF is not only involved in spermatozoal maturation and penetration but is also involved in spermatogenesis itself. On the other hand, PAF-AH (I) shows striking substrate specificity for an acetyl group but hydrolyzes other types of acetyl-containing esters in vitro (34). Studies on the tertiary structure of the catalytic dimer suggest that the substrate of this enzyme is not necessarily a lipophilic substance (23). Because PAF-AH (I) shows similarities to trimeric G-proteins (23), the PAF-AH (I)-mediated novel intracellular signaling is likely operating in mammals, with PAF or a related substance as a GTP-like switch.

When measuring cytosolic PAF-AH activity of alpha 1-/-/alpha 2-/- mice in brain and testis, enzymatic reduction to ~65% of wild-type mice was seen in both tissues (data not shown). This phenomenon is likely because type I PAF-AH is the only affected subtype, whereas enzymatic activities of type II PAF-AH and further not yet identified PAF-AH subtypes are probably responsible for the remaining activity.

In conclusion, we found that the depletion of the PAF-AH (I) catalytic subunits induces reduction of LIS1 protein on the cellular level and severe testicular malformation on the phenotypic level. The next question to be considered is whether the catalytic activity of PAF-AH (I) is required for LIS1 protein stability and spermatogenesis. To answer these questions, we are planning to insert the catalytically inactive alpha 1 and alpha 2 subunit genes into our double-knockout mouse model.

    ACKNOWLEDGEMENTS

We thank O. Reiner for the LIS1 antibody and S. Ishikawa and staff for animal care.

    FOOTNOTES

* This work was supported by the Joint Research Fund between the University of Tokyo and Banyu Pharmaceutical Co.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Division of Molecular Neurobiology, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

|| Present address: Dept. of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.

Dagger Dagger Present address: Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa 199-0195, Japan.

§§ To whom correspondence should be addressed: Dept. of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4720; Fax: 81-3-3818-3173; E-mail: harai@mol.f.u-tokyo.ac.jp.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M211836200

2 H. Koizumi, N. Yamaguchi, J. Aoki, K. Inoue, and H. Arai, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PAF, platelet-activating factor; PAF-AH, platelet-activating factor acetylhydrolase; TUNEL, TdT-mediated dUTP nick-end labeling.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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