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Testis |
Institute of Human Genetics, University of Goettingen, 37073 Goettingen, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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42.0-kDa nuclear protein, which is specifically expressed in spermatid cells. Its expression is upregulated by some unknown factor(s) from Sertoli cells. To elucidate the function of Theg protein and its role in spermatogenesis, we disrupted the Theg locus in mouse by homologous recombination. For functional dissection of the domain structure of the Theg protein, two different knockout approaches were undertaken. In the first knockout mouse (Th14), the C-terminal region of the Theg protein (amino acids 137–376) was deleted. Both Th14+/- and Th14-/- mice from genetic backgrounds of C57BL/6J x 129X1/SvJ hybrid and 129X1/SvJ inbred exhibited a normal phenotype and were fertile. The testes of Th14-/- mice were smaller than those of Th14+/- and Th14+/+ mice; however, the testicular morphology and the properties of sperm, including morphology and motility, from Th14-/- mice were similar to those of Th14+/- and Th14+/+ mice. These results demonstrate that the C-terminal region of Theg (amino acids 137–376) does not play an important role in progression of spermatogenesis. In the second knockout mouse (Th15), we deleted the N-terminal domain of the Theg protein, which resulted in complete loss of Theg transcripts. Both Th15+/- and Th15-/- mice from genetic backgrounds C57BL/6J x 129X1/SvJ hybrid, C3H/J congenic, and 129X1/SvJ inbred appeared normal and were fertile, with no gross abnormalities detected in testicular morphology or sperm properties. Our results from both knockout mouse model systems clearly illustrate that Theg is not essential for spermatogenesis in the mouse.
gamete biology, sertoli cells, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To date, the exact nuclear function(s) of Theg in spermatids are not fully understood. In a recent study, Yanaka et al. [14] reported that an insertional mutation deleting the Theg locus caused spermatogenic arrest in the insertional mutant mouse. They also suggested that Theg interacts with a cytoplasmic protein named CCT
. Because we previously demonstrated that Theg is localized predominantly in the nucleus of round spermatids [13], further investigations are necessary to define the exact role of Theg in spermatogenesis.
Here, we report targeted disruption of the Theg gene through two knockout (Th14 and Th15) mouse models. In the first knockout mouse (Th14), the C-terminal domain of Theg (amino acids 137–376) was deleted. In the second knockout mouse (Th15), we deleted the 5' end of Theg, which resulted in a complete loss of Theg transcripts. The results clearly show that Theg is not essential for spermatogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiments were performed with inbred mice reared at the Animal Facility of the Institute of Human Genetics (Goettingen, Germany). The animals were housed under a 12L:12D cycle with free access to standard mouse chow and tap water. All of the experimental procedures complied with national regulations for the Care and Use of Laboratory Animals (similar to the U.S. National Research Council guidelines).
Construction of Theg Gene Disruption Vectors
A lambda phage genomic clone carrying the complete mouse Theg gene was previously isolated from a FixII 129X1/SvJ (Stratagene, La Jolla, CA) genomic library [11]. The genomic clone was characterized by restriction enzyme mapping and sequencing. We first constructed a targeting vector using the plasmid pTKNeo3 [15], in which we replaced four exons encoding residues 161–298 of the Theg protein with a neomycin resistance gene under the control of a phosphoglycerate kinase promoter (Pgk-Neo), which we named Th14 construct (Fig. 1A). A 4.5-kilobase (kb) XbaI fragment containing the 5' flanking region of the Theg gene, including exons 1–3, was isolated from the genomic clone, blunted with PfuI (Stratagene, Heidelberg, Germany), and subcloned into the HincII site of pBluescript (Stratagene, Heidelberg, Germany). Thereafter, the fragment was excised out from pBluescript using SalI/ClaI enzymes and subcloned in a directional manner into the XhoI/ClaI sites of the pTKNeo3 vector (Fig. 1A). A 4.0-kb BamHI fragment containing exon 8 and the 3' flanking region of Theg was cloned into the BamHI site of the pTKNeo3 vector (which contained the 4.5-kb 5' fragment; Fig. 1A). The resulting targeting vector was linearized with NotI and electroporated into the R1 ES cell line [16].
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To generate the targeting vector for the deletion of the 5' end of the Theg gene (named Th15 construct), a 2.8-kb HindIII fragment containing the 5' flanking region of Theg was isolated from the lambda clone and subcloned into the HindIII site of pZERO2 vector (Invitrogen, Karlsruhe, Germany). Thereafter, a 1.6-kb SpeI fragment was excised from pZERO2, using one insert-specific SpeI site and another from the vector, and subcloned into the SpeI site of the pTKNeo3 plasmid. A 5-kb HincII fragment containing exons 5–8 and the 3' flanking region of Theg was isolated and subcloned into the HincII site of pBluescript. This fragment was excised from pBluescript by SalI/ClaI enzymes and subcloned into the XhoI/ClaI sites of the pTKNeo3 vector (containing the 1.6-kb 5' fragment).
ES Cell Culture and Generation of Theg Mutant Mice
The R1 ES cell line was cultured as described previously [16]. Confluent plates were washed in PBS buffer and trypsinized. The cells were suspended in PBS buffer at 2 x 107 cells/ml. One-milliliter aliquots were mixed with 50 µg of linearized targeting vector and electroporated at 250 V and 500°F using a Gene Pulser apparatus (Bio-Rad, Munich, Germany). Cells were plated onto nonselective medium in the presence of G418-resistant embryonic mouse fibroblasts. After an incubation step of 36 h, the medium was changed to a medium containing G418 (400 µg/ml) and ganciclovir (2 µM). After 10 days of selection, drug-resistant clones were picked and transferred into 24-well plates.
Genomic DNA was extracted from ES cell clones using standard methods [17] and digested with BamHI (in case of the Th14 construct) or with KpnI (in case of the Th15 construct), electrophoresed, and blotted onto Hybond-N membranes (Amersham, Braunschweig, Germany). Southern blot analyses were performed using 32P-labeled probes, a 1.2-kb fragment named ex1 (Fig. 1A) was used as an external probe for screening of homologous event for the Th14 construct and a 900-base pair (bp) fragment named ex2 was used as an external probe for screening of Th15 recombinant ES cell clones.
To confirm a correct homologous recombination and absence of additional random integration of the targeting constructs, Southern blots were rehybridzed with a Pgk-Neo probe. The ES cells from each cell line, Th14 and Th15, carrying the disrupted Theg alleles were injected into C57BL/6J blastocyts [18], and chimeric mice were generated. The chimeric males were mated to C57BL/6J and 129X1/SvJ females, and the resulting offspring were genotyped by polymerase chain reaction (PCR) analyses. Genomic DNA was extracted from mouse tails by using standard protocols [18]. PCR was carried out for 35 cycles under the following conditions: 30 sec at 95°C, 45 sec at 59°C, and 1 min at 72°C. For the knockout line Th14, the following primers were used: Th14f (5' GGG CTA TGC CTG GAT TTC CCC ACG 3'), Th14r (5' GGG ACC GTG ATG GTC AAC GTG G 3'), and NeoRI (5' AGG AGC AAG GTG AGA TGA CAG 3'). These primers amplified a 650-bp product of the wild-type allele and the 850-bp product of the mutant allele. For the knockout line Th15, the primers were Th15f (5' AAT CTG TGT TTC CCC TGG TG 3'), Th15r (5' GAT CCC ATT TGG GAA GGA AG 3'), and NeoRI, which amplified PCR products of 500 bp for the wild-type allele and 800 bp for the mutant allele. Heterozygous animals were intercrossed to obtain homozygous mutant mice.
To generate the Th15-/- congenic strain on a C3H/J background, Th15-/- male mice from the 129X1/SvJ inbred strain were backcrossed to strain C3H/J for seven generations to reach 97% genomic homogeneity for the C3H/J strain.
Reverse Transcription PCR and Northern blot Analysis
Total RNA was prepared from mouse testes using the Total RNA Isolation Reagent (Biomol, Hamburg, Germany) according to the manufacturer's instructions. For reverse transcription (RT) PCR analysis, total RNA (4 µg) was reverse transcribed into cDNA at 42°C for 50 min using a poly(dT)-oligonucleotide and the Superscript Reverse Transcriptase kit (Invitrogen). One microliter of the cDNA was then subjected to 30 cycles of PCR with primer pairs mTHEGf (5' GCT GAG GAG GGA GTG AGG TTA AAG 3') and NeoRI. The amplified products were analyzed by agarose gel electrophoresis, isolated from the gel, subcloned into the pGEMT-vector (Promega, Madison, WI), and sequenced on both strands using standard protocols [19]. The integrity of RNA in RT-PCR was checked using Tnp2 primers (accession no. NM_013694).
Northern blot experiments were performed according to standard protocols [19] using 32P-labeled cDNA probes: 3' Theg cDNA (nucleotide positions 734–1437) [11], full-length Theg cDNA, Pgk-Neo, and 
-actin.
Immunocytochemical and Green Fluorescent Protein Fusion Protein Analysis
For immunocytochemical staining, cellular suspensions were prepared from mouse testes by using the collagenase/trypsin method according to published procedures [20]. Testes from adult Th14+/+ and Th14-/- mice were collected aseptically in serum-free culture medium. The immunostaining (using purified antibody against Theg) on testicular cell suspensions was performed as described previously [13]. Immunostained slides were examined using a fluorescence-equipped microscope (BX60; Olympus, Hamburg, Germany).
For subcellular localization of green fluorescent protein (Gfp) fusion proteins, wild-type Theg cDNA and mutant Theg cDNA (generated in Th14 knockout mice) were cloned in frame into the mammalian expression vector pECFP-C1 containing the Gfp (Clontech, Heidelberg, Germany). For transfection experiments, NIH3T3 cells were cultured in Dulbecco modified Eagle medium containing 10% fetal calf serum and penicillin/streptomycin solution (Invitrogen), and 2 x 105 cells were plated per 6-cm culture dish 24 h prior to transfection. Expression vectors (2 µg/6-cm culture dish) were transfected using the Superfect reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Cells were harvested 16–24 h after transfection and replated on multiwell slides (Nunc, Wiesbaden, Germany). Transiently transfected NIH3T3 cells were replated on chamber slides, fixed with PBS containing 4% paraformaldehyde, and washed with PBS, and nuclei were counterstained with 4',6'-diamidino-2-phenylindole (DAPI; Vector, Burlingame, CA) and microscopically analyzed as described above.
Fertility Test and In Vitro Competition Fertilization Assay
To determine the fertility of Th14-/- and Th15-/- mice, five sexually mature male mice from genetic backgrounds were mated, each with two females, for 3 mo. Pregnant females were removed and put into holding cages to give birth. The number and size of litters sired by each group of males were determined in a 3-mo mating period.
For in vitro competition fertilization (IVCF) assay, adult female CD1 mice were superovulated by i.p. injection of 5 IU of eCG (Intergonan; Intervet, Toenisvorst, Germany) followed by 5 IU of hCG (Predalon; Organon, Oberschleissheim, Germany), and oocytes were collected after 10–12 h (after hCG administration). The cumulus cells were removed by hyaluronidase treatment, and the oocytes were washed in in vitro fertilization (IVF) medium (MediCult, Jyllinge, Denmark) and then maintained in this medium. Spermatozoa were isolated from the cauda epididymis and vas deferens of each male group (wild type and Th14-/-), mixed together in equal numbers, capacitated in Tyrode medium at 37°C for 1.5 h, and added to the oocytes in 400-µl drops of fertilization medium. The mixture was then incubated for 6 h at 37°C in 5% CO2 covered with mineral oil. Using a large-bore micropipette, eggs were washed in M16 (Sigma-Aldrich, Deisenhofen, Germany) and examined for the presence of male and female pronuclei. The eggs were cultured in M16 and then transferred to pseudopregnant mice. The genotypes of newborn animals were determined by PCR (as described above).
Sperm Count and Sperm Motility Analysis
To determine the total sperm counts from wild-type and mutant homozygous (Th14-/- and Th15-/-) mice, the epididymides were collected and dissected aseptically in Tyrode medium. For determination of sperm numbers in the uterus and oviduct, female wild-type mice were mated with wild-type and mutant homozygous (Th14-/- and Th15-/-) mice. The uteri and oviducts of those mice that were positive for a vaginal plug were dissected in Tyrode medium, and sperm were flushed out. The sperm numbers in cauda epididymis, uteri, and oviducts were determined using the Neubauer cell chamber (Schütt Labortechnik GmbH, Goettingen, Germany).
To investigate sperm motility properties, epididymides of wild-type and mutant homozygous mice were dissected in IVF medium. Spermatozoa were allowed to swim out of the epididymides and were incubated for 3.5 h at 37°C. A drop of the sperm suspension was transferred to the incubation chamber, which was set at a temperature of 37°C. Sperm movement was quantified in a way similar to that described previously [21] using a computer-assisted semen analysis (CASA) system (CEROS version 10; Hamilton Thorne Research, Beverly, MA).
Histological Studies
Fixation and subsequent treatment of mouse testicular tissue was performed as described previously [22]. Mouse testis tubule sections (10 µm) were first fixed for 20 min in 4% (w/v) paraformaldehyde (Sigma-Aldrich, Deisenhofen, Germany) and then stained with 1.6% (w/v) hematoxylin and 0.1% (w/v) eosin (Sigma-Aldrich) for 10 min. Sections were washed in water twice for 2 min, followed by sequential incubation in 70% (v/v), 90% (v/v), 100% (v/v), and 100% (v/v) ethanol for 2 min each. For dilapidation, the sections were then incubated in xylol twice for 2 min before being covered in Eukitt mounting solution (Sigma-Aldrich).
For sperm morphological analysis, spermatozoa were isolated from the cauda epididymis and vas deferens and smeared on glass slides. Air-dried smears were fixed in methanol:acetone (1:1) for 10 min and stained with DAPI mounting solution (Vector). Stained testicular sections and fixed sperm slides were examined using an Olympus BX60 microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To elucidate the functional role of the Theg protein in spermatogenesis, the mouse Theg gene was disrupted in ES cells by homologous recombination using a replacement-targeting vector containing Pgk-Neo and thymidine kinase expression cassettes. In the first approach, we deleted the C-terminal part of Theg (Fig. 1A). In the targeting construct, four exons (4–7) of Theg were replaced with the Pgk-Neo cassette. The linearized targeting vector was electroporated into the R1 ES cell line and selected for homologous recombination events. Drug-resistant ES clones containing the Theg-disrupted allele were identified using Southern blot analysis with an external probe (ex1) present upstream of the 5' flanking fragment of the targeting construct (Fig. 1A). The ex1 probe detected the wild-type allele as a 12-kb fragment and the recombinant allele as a 12.8-kb fragment in genomic DNA digested with the BamHI restriction enzyme. Two independent ES clones carrying the recombinant allele were identified from screening of 56 ES clones (Fig. 1B). The ES cells from one clone (Th14) were injected into C57BL/6J blastocysts to generate chimeric mice. Male chimeric mice transmitting the targeted mutation into the germ line were bred with female mice from C57BL/6J and 129X1/SvJ strains, respectively, to establish the Th14 allele in two different genetic backgrounds, the C57BL/6J x 129X1/SvJ hybrid and the 129X1/SvJ inbred. Heterozygous animals were identified by PCR analysis of DNA from mice tails (Fig. 1C) and were intercrossed to generate homozygous mice in the respective genetic backgrounds.
Analysis of Theg Expression in Knockout Mice
Northern blot analysis was performed on total testicular RNA isolated from Th14+/+, Th14+/-, and Th14-/- mice (129X1/SvJ) using a 703-bp 3' end Theg cDNA (nucleotide positions 734–1437) fragment as probe. A strong expression of Theg with a transcript size of 1.4 kb was observed in Th14+/+ mice, Theg expression was weaker in Th14+/- mice, and no expression was detected in testicular RNA isolated from Th14-/- mice (Fig. 2A). However, when complete Theg cDNA was used as a probe, weak expression was also detected in testicular RNA from Th14-/- mice (Fig. 2B). When Pgk-Neo cDNA was used as a probe in Northern blot, no transcript was observed in Th14+/+ mice, but two transcripts of approximately 1.2 kb and 1.4 kb were observed in both Th14+/- and Th14-/- mice (Fig. 2D). The additional transcript detected by the Pgk-Neo cDNA probe along with endogenous neomycin resistance transcript (1.2 kb) might be a fusion transcript between the 5' end of Theg and Pgk-Neo derived from the endogenous Theg promoter. To check whether in fact a fusion transcript exists in Th14-/- mice, RT-PCR was performed with a mTHEGf/NeoRI primer pair (Fig. 1A). A PCR product was amplified only with RNA of Th14+/- and Th14-/- mice (Fig. 2D). Sequence analysis of this PCR product (data not shown) revealed that exon 1 and exon 2 of the Theg transcript were fused with the Pgk-Neo transcript, thus producing a fusion Theg/Pgk-Neo transcript.
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To determine the subcellular localization of recombinant Theg protein, which arose due to fusion of exon 1 and exon 2 of Theg with the Pgk-Neo cassette, we undertook two different approaches. First, we performed immunostaining on testicular cell suspension derived from Th14+/+ and Th14-/- mice with a polyclonal antibody specific for a Theg epitope derived from exon 2 of Theg. The Theg fusion protein was localized predominantly in the cytosol of round spermatids isolated from Th14-/- mice (Fig. 3, D–F) in contrast to wild-type Theg, which was localized predominantly in the nucleus of round spermatids (Fig. 3, A–C). In a second approach, we generated a Gfp-tagged mutant Theg construct by subcloning the fusion cDNA generated in Th14-/- mice in frame with the pECFP-C1 vector and transfected this construct into NIH3T3 cells. The recombinant Theg showed a cytosolic localization in NIH3T3 cells (Fig. 3, J–L); however, Gfp-tagged wild-type Theg showed a nuclear localization (Fig. 3, G–I). These results suggest that the mutant Theg fusion protein is predominantly localized in the cytosol in contrast to wild-type Theg protein, which is mainly present in the nucleus of spermatid cells.
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Phenotypic Analysis of Th14 Knockout Mice
The mice heterozygous for the Th14 allele were phenotypically normal and fertile. Homozygous mice also appeared normal and were fertile. However statistical analysis of F2 breeding (Table 1) showed that mice in C57BL/6J x 129X1/SvJ hybrid genetic background deviate from the Mendelian mode of inheritance in the ratio of wild-type:homozygous progenies, with a 
2 value of 38.06 (P < 0.001). However, the mode of inheritance in mice from the 129X1/SvJ strains was in agreement with Mendelian segregation, with 2 values of 0.12 (P < 0.094). To determine whether Th14 sperm in C57BL/6J x 129X1/SvJ genetic background is inferior to wild-type sperm, we performed an IVCF assay. For this experiment, wild-type mouse eggs were inseminated with an equally mixed suspension of sperm from Th14+/+ and Th14-/- mice (C57BL/6J x 129X1/SvJ). The fertilized eggs with male and female pronuclei were allowed to develop into embryos and were genotyped for the Th14 locus. Nearly equal percentages of wild-type and heterozygous embryos were obtained (data not shown).
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The testes of Th14-/- mice (both genetic backgrounds: C57BL/6J x 129X1/SvJ and 129X1/SvJ) were smaller than those of Th14+/+ mice; therefore, the growth curve of testes during mouse development was determined for Th14+/+, Th14+/-, and Th14-/- mice (129X1/SvJ). Testes from different developmental stages were dissected under aseptic condition and weighed. For each developmental stage, testes of three mice were weighed, and the mean weight was obtained. The growth curve analysis (Fig. 2G) revealed a reduced testes weight of adult Th14-/- mice (76%) compared with the Th14+/+ mice.
Counting of sperm in the cauda epididymis and in uteri and oviducts of females inseminated by Th14+/+ and Th14-/- mice did not reveal any significant differences (Table 2).
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To measure sperm motility, sperm from Th14+/+ and Th14-/- mice were analyzed with the CASA system. Several motility parameters were determined; only straightness (straight line progressive movement of the sperm between the beginning and the end of the measurement) is shown in Table 2. We did not observe any significant difference in motility properties between sperm from Th14+/+ and Th14-/- mice.
Histological analysis of testicular tubule sections (Fig. 4B) and morphological analysis of sperm (data not shown) from Th14-/- mice also did not show any abnormalities, when compared with Th14+/+ mice (Fig. 4A).
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Generation of Knockout Mice Deleting the 5' End of the Theg Gene (Th15)
In our first Theg knockout mice (Th14), the N-terminal part of the Theg protein (amino acids 1–136) was still present, which resulted in a fusion protein between the N-terminal region of the Theg and neomycin resistance protein. To check whether this N-terminal domain of Theg plays any functional role, we deleted this domain in a second knockout mouse (Th15). A replacement-targeting construct was generated, where 2-kb upstream sequences of the Theg gene including the first four exons (1–4) were replaced with the Pgk-Neo cassette. The linearized targeting vector was electroporated into the R1 ES cell line and selected for homologous recombination events. Drug-resistant ES clones containing the Th15 allele were identified by using Southern blot analysis with an external probe (ex2) present upstream of the 5' flanking fragment of the targeting construct (Fig. 5A). The ex2 probe detected the wild-type allele as a 7-kb fragment and the recombinant allele as a 10.8-kb fragment in genomic DNA digested with the restriction enzyme KpnI. Seventeen independent ES clones carrying the recombinant allele were identified from screening of 48 ES clones (Fig. 5B). Mice heterozygous for the Th15 allele were generated in two different genetic backgrounds, C57BL/6J x 129X1/SvJ hybrid and 129X1/SvJ inbred, in a fashion similar to that described for Th14. Heterozygous mice in both backgrounds appeared normal and fertile. When heterozygous mice were mated, they produced 25% homozygous mice (data not shown) as determined by PCR genotyping (Fig. 5C). Thus, inheritance of the Th15 allele was in accordance with the Mendelian mode of segregation. Homozygous mice also appeared normal and were fertile.
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The male Th15-/- mice from the C3H/J congenic strain were also fertile, and we did not observe any defect in spermatogenesis (data not shown). The litter sizes of Th15-/- mice from the C3H/J congenic strain were also comparable to those of Th15+/+ C3H/J mice (Table 1).
When Northern blot analysis was performed on testicular RNA using full-length Theg cDNA as a probe, no Theg expression was detected in homozygous mice, and weaker expression was observed in heterozygous mice compared with wild-type mice (Fig. 5D).
To identify any testicular abnormalities present in Th15-/- male mice, we performed histological analysis on adult testicular sections and on mature spermatozoa. No gross abnormalities either in seminiferous tubule structure (Fig. 4C) or in morphology of sperm (data not shown) were detected. The total numbers of sperm in the cauda epididymis and in uteri and oviducts of wild-type females inseminated by Th15-/- male mice were equivalent to those for Th15+/+ mice (Table 2). Sperm motility for Th15-/- mice was comparable to that for wild-type mice (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The temporal-spatial nuclear expression of Theg in round spermatid cells [13] suggests that Theg might play an important role in successful differentiation of male germ cells. To elucidate the function of Theg in mice, a targeted mutation of the gene was generated by deleting the C-terminal part of the protein (amino acids 137–376). We deleted a genomic fragment of Theg that included a region spanning exon 4 to exon 7 of the gene, encompassing important nuclear localization signal sequences [11]. In our Theg-deleted (Th14) knockout mice, Northern blot and RT-PCR analysis revealed a fusion transcript comprised of exon 1 and exon 2 of Theg fused with the Pgk-Neo transcript. The plausible reason for the generation of this fusion transcript might be an inherent propensity of Theg to undergo alternative splicing. Under normal circumstances, Theg gives rise to four different spliced isoforms [11, 14]. In one instance, exon 3 is spliced out, resulting in a Theg isoform that only lacks exon 3. In the case of Theg deletion, a cryptic splice acceptor site present in the Pgk-Neo cassette may have generated a fusion Theg transcript isoform between exon 2 of Theg and Pgk-Neo. Because the Pgk-Neo cassette contains a polyadenylation sequence, the fusion transcript generated in this way is stable in vivo. The next obvious step was to determine the significance of this fusion transcript in Th14-/- mice. Because wild-type Theg protein is predominantly localized in the nucleus of round spermatids, we investigated the intracellular localization of the fusion Theg protein. Immunocytochemical staining of cellular suspension isolated from Th14-/- testes showed a cytosolic localization of Theg fusion protein (Fig. 3, D–F). This result was further supported by generating a Gfp-tagged Theg fusion protein, which is expressed only in the cytoplasm of transfected NIH3T3 cells (Fig. 3, J–L). These results clearly show that the Theg fusion protein in Th14-/- mice has lost the ability to localize predominantly in the nucleus of round spermatids.
Phenotypically, Th14-/- mice from different genetic backgrounds (inbred strain 129X1/SvJ and hybrid strain C57BL/6J x 129X1/SvJ) appeared normal and were fertile.
When a phenotype-genotype correlation was made, we discovered that in the genetic background of C57BL/6J x 129X1/SvJ, the Th14 allele showed deviation from the Mendelian mode of inheritance (Table 1). The relative percentages of Th14+/- and Th14-/- mice are reduced compared with that of Th14+/+ mice, suggesting that during segregation of alleles, the Th14- allele is disadvantageous compared with the wild-type allele. To determine whether Th14- sperm are inferior to Th14+ sperm, we performed IVCF, which indicated that both Th14- and Th14+ sperm are equally competent in their ability to fertilize oocytes. In contrast, the mode of inheritance of the Th14 allele in the genetic background 129X1/SvJ did not show any deviation from Mendelian inheritance. An inferior Th14- allele in the genetic background C57BL/6J x 129X1/SvJ could be due to genetic heterogeneity of the mice. There are numerous reports of genetic heterogeneity causing differences in the phenotype of knockout mice [21, 23].
We also observed a minor but significant reduction in testes weight (Fig. 2G) in Th14-/- mice. However, histological examination of the testes revealed that in Th14-/- mice the process of spermatogenesis was normal (Fig. 4B) and the numbers of spermatozoa present in the cauda epididymis, uteri, and oviducts were comparable to those of wild-type mice (Table 2). The morphological appearance of Th14- sperm was normal.
We also did not detect any significant defect in motility of Th14-/- sperm, as determined by the CASA system (Table 2). From the results of Th14 knockout mice, we conclude that the deleted part of Theg in Th14 mice is not essential for spermatogenesis. Although we observed some minor defects, those abnormalities are subtle and do not affect the ability of Th14- sperm to fertilize oocytes.
Yanaka et al. [14] reported an insertional mutant mouse in which a foreign DNA molecule (human PDE5A gene under the control of cytomegalovirus promoter [24]) was integrated into the Theg locus. In these mice, several copies of the foreign DNA were integrated into the Theg locus, thus producing a deletion of about 20 kb. They named this deleted transgenic locus kisimo (ki). This insertion deleted the complete open reading frame of the Theg gene and deleted an additional 5' fragment upstream from the Theg gene.
The male mice homozygous for the ki locus were sterile because of spermatogenic arrest. Histological analysis of cross sections of ki/ki testes showed that elongated spermatids were vacuolated in the vicinity of the lumina of seminiferous tubules and were occasionally phagocytosed by Sertoli cells [14]. In ki/ki mice, there were virtually no spermatozoa in the lumina of seminiferous and epididymal tubules. Yanaka et al. [14] reported that in elongated spermatids, Theg interacts with a cytoplasmic protein termed CCT, a subunit of CCT complex protein [14]. The CCT complex is required for the proper folding or assembly of cytoskeletal proteins under both in vitro and in vivo conditions [25–27]. In contrast to the report of Yanaka et al., our results clearly demonstrate that Theg is localized predominantly in the nucleus of round spermatids. From the results of our Th14 knockout mouse and the insertional mutant mouse of Yanaka et al., one can speculate that the N-terminal part of Theg (exon 1 and exon 2), which constitutes a domain of 136 amino acids, is essential for in vivo function of Theg. Therefore, a further investigation including the generation of second knockout mouse with complete deletion of Theg was necessary to define the exact role of Theg in spermatogenesis.
We generated the second Theg knockout (Th15) mouse by deleting exons 1–4 of Theg and replacing them with the Pgk-Neo cassette (Fig. 5A). The heterozygous mice in genetic backgrounds C57BL/6J x 129X1/SvJ and 129X1/SvJ inbred were phenotypically normal and fertile. The mice in both genetic backgrounds showed no deviation from Mendelian inheritance, and the homozygous mice were fertile. Northern blot analysis of testicular RNA showed that Theg transcript was lacking in Th15-/- mice. Thus, the Th15 deleted allele is a null mutation and Th15-/- is a loss-of-function mouse model system. We detected no abnormalities in testicular morphology of Th15-/- mice and in properties of Theg-deficient sperm.
With our first and second Theg knockout (Th14 and Th15) mouse model systems, we attempted to genetically dissect the functional domain(s) of Theg protein. Our results indicate that Theg is not essential for spermatogenesis.
One possible explanation for the difference in phenotype between our Theg knockout mice and the insertional mutant mouse of Yanaka et al. [14] could be a difference in the genetic background of the mouse. To determine whether a genetic factor that influences the phenotype of Theg deletion is involved, we generated Th15-/- congenic mice in C3H/J genetic background. The male Th15-/- congenic C3H/J mice were also fertile. Therefore, we concluded that genetic background does not influence the phenotype of Theg deletion in mice. However, Yanaka et al. generated insertional mutant mice by deleting a >20-kb region near the Theg locus. Thus, the effect on the phenotype may have resulted from another gene near this locus. Another possibile explanation for the different phenotype of the ki/ki mouse is that the deletion might affect a cis-acting effector(s) sequence, which could influence distant gene(s) involved in spermatogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Wolfgang Engel, Institute of Human Genetics, University of Goettingen, Heinrich-Dueker-Weg 12, D-37073 Goettingen, Germany. Fax: 49 551 399303; wengel{at}gwdg.de
Received: 24 March 2003.
First decision: 18 April 2003.
Accepted: 30 April 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  P. Grzmil, D. Boinska, K. C. Kleene, I. Adham, G. Schluter, M. Kamper, B. Buyandelger, A. Meinhardt, S. Wolf, and W. Engel Prm3, the Fourth Gene in the Mouse Protamine Gene Cluster, Encodes a Conserved Acidic Protein That Affects Sperm Motility Biol Reprod, June 1, 2008; 78(6): 958 - 967. [Abstract] [Full Text] [PDF]
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 U. Schlecht, P. Demougin, R. Koch, L. Hermida, C. Wiederkehr, P. Descombes, C. Pineau, B. Jegou, and M. Primig Expression Profiling of Mammalian Male Meiosis and Gametogenesis Identifies Novel Candidate Genes for Roles in the Regulation of Fertility Mol. Biol. Cell, March 1, 2004; 15(3): 1031 - 1043. [Abstract] [Full Text] [PDF]
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 N. Schultz, F. K. Hamra, and D. L. Garbers A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets PNAS, October 14, 2003; 100(21): 12201 - 12206. [Abstract] [Full Text] [PDF]
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