A Testicular Germ Cell-Associated Serine-Threonine Kinase, MAK, Is Dispensable for Sperm Formation

Establishment of Mak knockout ES cells and mice.A DNA fragment containing the mouse Mak gene was isolated from a 129 genomic DNA library. A 1.1-kb HpaI fragment and a 4.5-kb XhoI-NotI (vector origin) fragment 5′ to exon 5 and 3′ to exon 8 of Mak, respectively, were subcloned into the PLN-tk plasmid (5). The numbering of these exons was based on our mouse genomic DNA analyses and a published human Mak gene exon/intron structure (EMBL accession no. AL024498 ). Exons 5 to 8 of the mouse Mak gene were estimated to encode Lys¹²⁷-Val³⁸¹ of MAK protein.

Twenty micrograms of the linearized targeting construct, Tk2p102, was transfected into 2 × 10⁷ TT2 ES cells (23), and the targeted clones were enriched with 0.3 mg of G418/ml and 1.5 μM of ganciclovir. The drug-resistant colonies were then screened by PCR with the following primers: SCR4, 5′-AGCCATCTCGCCAGCACCTCATA-3′, and G3, 5′-GGGCCAGCTCATTCCTCCACTC-3′. The positions of these primer sequences are shown in Fig. 1A. The correctly targeted clones were further confirmed by Southern blot analysis (described below) and then were injected into ICR mice embryos in the morula stage. Established chimeric male mice were bred with C57BL/6 (B6) females, resulting in germ line transmission. To minimize the effect of the mixed genetic background, the Mak ^+/− females were backcrossed with either B6 or CBA/J (CBA) males over five generations. Each strain of F₆ animals from the F₅ Mak ^+/− interbreeding or their descendents were used in some experiments, including mating experiments. B6, CBA, and ICR mice were purchased from Japan SLC Inc. (Hamamatsu, Japan), and all the animals were maintained and bred at the animal facility in our institute under specific pathogen-free conditions.

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FIG. 1.

Generation of a mouse line bearing a targeted disruption of the Mak gene. (A) Schematic representation of homologous recombination of the targeting vector, TK2p102, with the endogenous locus. TK2p102 was designed to replace the regions of exons 5 to 8 of the Mak gene, encoding a catalytic kinase domain (the activation loop region) and a PQ-rich domain with a PGK-Neo^r selection cassette (neo). Detection of homologous recombinants in ES cells was performed by PCR assay with SCR4 and G3 primers (small arrows). (B) Genotyping of Mak ^−/− mice by Southern blotting. DNA was digested with SacI and hybridized with the probe shown in panel A. The 5.5-kb SacI fragment corresponding to the wild-type allele is increased to 9 kb upon disruption of the SacI recognition site located between exons 4 and 5 by integration of the Neo^r gene. (C) Northern blotting of testicular total RNA probed with the entire coding region of the Mak cDNA. Two distinct Mak transcripts (3.8 to 3.9 kb and 2.6 to 2.8 kb) were detected in the Mak ^+/+ and Mak ^+/− testicular cell RNA. However, in the Mak ^−/− testicular RNA sample, the amount of these two transcripts was considerably diminished and sizes of both transcripts were reduced a little bit, probably due to the deletion of exons 5 to 8 in the targeted allele. The lower panel shows 18S and 28S rRNA stained with ethidium bromide on the same blot before probing. (D) Immunoblot of testicular cell lysates probed with a polyclonal Ab against a MAK carboxy terminus peptide (10). Both the p60 and p66 MAK proteins were missing from the Mak ^−/− testicular cell lysates. (E) Absence of MAK-associated protein kinase activity in the Mak ^−/− mice. Testicular cytoplasmic lysates were prepared as described in Materials and Methods and were incubated with the control Ab, Ab against MAK (α-MAK), or Ab against MST, a STE20-like serine/threonine kinase (α-MST). The immunoprecipitates (IP) were then applied to the in vitro kinase assay, with MBP as a substrate. p210 is a MAK-associated molecule and is efficiently phosphorylated by MAK (10).

Western blotting.Eighty micrograms of total testicular cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred to a nitrocellulose membrane, and probed with rabbit polyclonal antibody (Ab) against MAK (amino acid residues 603 to 622) (10).

Southern and Northern blotting.Ten micrograms of mouse genomic DNA was digested with SacI, separated by 0.7% agarose-Tris-acetate-EDTA gel electrophoresis, transblotted to a nylon membrane, and probed with the ³²P-labeled 1.1-kb HpaI fragment used in the Mak targeting vector. Eight micrograms of total RNAs from various mouse organs were separated by 1% agarose-formaldehyde gel electrophoresis, transblotted to a nylon membrane, and probed with the ³²P-labeled Mak cDNA (entire coding region). All the hybridized images were visualized with a BAS5000 Bio-Image Analyzer (Fuji Film, Tokyo, Japan).

Protein kinase assay.Testicular cell lysates were prepared based on the procedure described previously (10). Briefly, the testis was homogenized in modified LBK buffer containing 20 mM HEPES-NaOH (pH 7.5), 10% glycerol, 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM β-glycerophosphate, 50 mM NaF, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor cocktails (Boehringer Mannheim, GmbH, Germany), and they were centrifuged at 16,000 × g for 10 min at 4°C to obtain soluble cytoplasmic proteins. Cytosolic proteins (750 μg) were incubated with 1 μg of control Ab, Ab against MAK, or Ab against MST, a STE20-like serine/threonine kinase (14); the immune complexes were precipitated with protein A-Sepharose beads. The immunoprecipitates were suspended in 15 μl of the kinase assay buffer, which contained 20 mM HEPES-NaOH (pH 7.5), 10 mM MnCl₂, 0.25 mM β-glycerophosphate, 25 μM ATP, 5 to 10 μCi of [γ-³²P]ATP (3,000 Ci/mmol), and 1 μg of myelin basic protein (MBP), and they were incubated for 30 min at 23°C. Protein phosphorylation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography.

Histochemical analysis.Tissues from Mak ^−/− mice and their Mak ^+/+ or Mak ^+/− littermates or age-matched control mice were rapidly dissected and placed in Bouin's fixative overnight. Excess fixative was removed with a 70% ethanol-NH₄OH solution. Tissues were then dehydrated and embedded in paraffin for microtome sections. Hematoxylin-eosin staining procedures were performed by a standard protocol.

Mating experiment.Two- to 5-month-old male Mak ^−/− mice and their Mak ^+/+ or Mak ^+/− littermates or age-matched control mice were crossed with B6 females for at least 2 weeks. One male was bred to one or two reproductively active females.

Sperm motile activity assay.Mature sperm were prepared as described previously (8). Epididymides were removed from Mak ^−/− mice and their Mak ^+/+ or Mak ^+/− littermates or age-matched control mice, and the vas deferens was cut close to the cauda epididymis. Mature sperm were expressed from the epididymis with forceps and were placed into human tubal fluid medium (1 epididymis per ml). After 30 min of incubation, sperm motility was monitored with a Sperm Quality Analyzer (Medical Electonic Systems Ltd., Santa Ana, Calif.) according to the manufacturer's instruction. The results are reported as the sperm motility index (SMI); SMI is the total value of sperm motility times sperm concentration (2, 11).

Statistical analysis.Data obtained from mating experiments were analyzed by the Mann-Whitney U test. The Student's t test was used to examine the significance of differences in the values of SMI. A probability of P < 0.05 was considered statistically significant.

Establishment of Mak knockout mice.To establish MAK-deficient mice, the Mak targeting construct Tk2p102 was transfected into the (B6 × CBA) F₁ ES cell line, TT2. Tk2p102 potentially introduced a deletion of exons 5 to 8 of mouse Mak that encodes about 45% of MAK amino acids, including a catalytic kinase domain (the activation loop region) and a PQ-rich domain (Fig. 1A). Of approximately 500 TT2 clones screened, 5 were identified as being targeted. One of the targeted clones, number 50, was germ line transmitted, and mice homozygous for the Mak deletion (Mak ^−/−) were established by being mated with heterozygous mutant (Mak ^+/−) mice. Southern blot analysis shows the status of the Mak deletion in the wild-type (Mak ^+/+), Mak ^+/−, and Mak ^−/− mice (Fig. 1B). Mak ^−/− mice were born at the expected Mendelian ratio and did not show any gross abnormalities (data not shown).

Development and function of male germ cells in the Mak ^−/− mice.To confirm the absence of MAK protein in the Mak ^−/− mice, we performed Northern and Western blot analyses. As shown previously (3, 10, 21) and in Fig. 1C, two distinct Mak transcripts (3.8 to 3.9 kb and 2.6 to 2.8 kb) were detected in the Mak ^+/+ and Mak ^+/− testicular cells. In the Mak ^−/− testicular RNA sample, the amount of these two transcripts was severely diminished (the sizes of the two transcripts from the targeted allele were a little bit smaller, probably due to the deletion of exons 5 to 8 of Mak). Ab against MAK carboxy-terminus peptides (kindly provided by M. Shibuya) detected the two different sizes of Mak products, p66 and p60, in the Mak ^+/+ and Mak ^+/− testicular cell lysates (Fig. 1D and reference 10). However, both products were totally absent in the Mak ^−/− testicular samples. In addition, we examined the protein kinase activity of immunoprecipitates from the Mak ^−/− testicular cell lysates with this anti-MAK Ab. As shown in Fig. 1E, kinase activity of the control serine/threonine kinase, MST (14), was present in both Mak ^+/+ and Mak ^−/− testicular cell lysates, and MST itself and an artificial substrate, MBP, were phosphorylated. However, there was no MAK kinase activity in the Mak ^−/− testicular samples (no phosphorylation of MBP and a MAK natural substrate, p210 [10]).

To evaluate the impact of the Mak knockout on the development and function of male germ cells, we initially performed a histochemical analysis of the male reproductive organs. As shown in Fig. 2, spermatogenesis in most of the Mak ^−/− testes was normal, and mature sperm were present in the cauda (of 8 out of 9 animals). No significant differences were apparent with regard to size and weight of testis and epididymis among the age-matched Mak ^+/+, Mak ^+/−, and Mak ^−/− littermates (data not shown). These results allowed us to conclude that the Mak products are deficient in the Mak ^−/− mice, even though minor truncated transcripts are present. The presence of spermatogenesis and viable sperm indicate that MAK is not essential for these processes.

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FIG. 2.

Histochemical analysis of the Mak ^−/− mouse testis and epididymis. Hematoxylin-eosin staining of the testicular (A to C) and cauda of epididymal (D and E) tissue sections and a smear of the epididymal sperm are shown (F). (A) Mak ^+/+; (D) Mak ^+/−; (B, C, E, and F) Mak ^−/−. The sizes of the scale bars in the panels are the following: 100 μM (A and B), 50 μM (C), 70 μM (D and E), and 10 μM (F).

Next we examined the fertility of the male Mak ^−/− mice in vivo. Due to the B6 × CBA mixed genetic background of the established animals, we performed two different mating experiments. As the initial experiment, we used these mixed-background Mak ^−/− males and their Mak ^+/− littermates. In other experiments we back-crossed Mak ^+/− mice with either B6 or CBA mice and then used F₆ male mice established from F₅ B6 or CBA Mak ^+/− animals. In either mixed-background or F₆ (B6 or CBA) animals, most of male Mak ^−/− mice were fertile (7 out of 9 for mixed background Mak ^−/− male mice and 3 out of 3 and 4 out of 4 for B6 and CBA F₆ Mak ^−/− male mice, respectively).

The data indicate that the fertilizing ability of Mak ^−/− males is within normal limits. To confirm this observation, another set of mating experiments was designed to examine the average litter size from the Mak ^−/− male × B6 female breeding pairs. Results obtained from these in vivo experiments demonstrated that the average litter size from the Mak ^−/− male × B6 female breeding pairs (7 male mice, 12 replicates; litter size, 5.4 ± 2.8 [P = 0.017]) was approximately 35% reduced compared to that from the control pairs (8 male mice, 14 replicates; litter size, 8.3 ± 2.6).

The motility of mature sperm obtained from the cauda epididymis in the B6 background Mak ^−/− mice was studied in vitro. Sperm motility of the Mak ^−/− mice was mildly reduced compared to that of Mak ^+/+ mice, and the SMI for the Mak ^−/− males was about 80% (for experiment 1, n = 2 and SMI = 174 ± 24 [this result was inconclusive due to the small number of replicates]; for experiment 2, n = 5 and SMI = 220 ± 24 [P = 0.039]) that of the control Mak ^+/+ males (for experiment 1, n = 2 and SMI = 225 ± 19; for experiment 2, n = 6 and SMI = 266 ± 20).