Mouse germ cell-less as an essential component for nuclear integrity

Abstract

A mouse homologue of the Drosophila melanogaster germ cell-less (mgcl-1) gene is expressed ubiquitously, and its gene product is localized to the nuclear envelope based on its binding to LAP2 beta (lamina-associated polypeptide 2 beta). To elucidate the role of mgcl-1, we analyzed two mutant mouse lines that lacked mgcl-1 gene expression. Abnormal nuclear morphologies that were probably due to impaired nuclear envelope integrity were observed in the liver, exocrine pancreas, and testis. In particular, functional abnormalities were observed in testis in which the highest expression of mgcl-1 was detected. Fertility was significantly impaired in mgcl-1-null male mice, probably as a result of severe morphological abnormalities in the sperm. Electron microscopic observations showed insufficient chromatin condensation and abnormal acrosome structures in mgcl-1-null sperm. In addition, the expression patterns of transition proteins and protamines, both of which are essential for chromatin remodeling during spermatogenesis, were aberrant. Considering that the first abnormality during the process of spermatogenesis was abnormal nuclear envelope structure in spermatocytes, the mgcl-1 gene product appears to be essential for appropriate nuclear-lamina organization, which in turn is essential for normal sperm morphogenesis and chromatin remodeling.

FIG. 1.

Two mgcl-1-null mice. The left and right panels show the respective data for mgcl-1^−/− mice that were produced by homologous recombination and for mgcl-1^{OST6847/OST6847} mice that were produced by retroviral insertional mutagenesis. (A and D) Maps of the wild-type mgcl-1 locus, targeting vector, and disrupted loci. The diagnostic restriction enzyme digestion patterns are indicated. Abbreviations: E, EcoRI; Sp, SpeI. (B and E) Southern blot analysis. The genomic DNA from each genotype was digested with EcoRI and hybridized with the 3′-terminal probes b (B) and c (E). (C and F) Western blot analysis. Extracts from the testes were separated on SDS-polyacrylamide gels and analyzed with an affinity-purified, anti-mGCL-1 polyclonal antibody.

FIG. 2.

Normal cell growth and E2F-DP target gene expression. (A) Growth rates and saturation densities of MEFs. Primary MEFs were seeded at 10³ cells/well at day 0, and cells were counted every other day. (B) 3T3 analysis. MEF cultures were passaged according to the 3T3 protocol, and the growth rate was calculated as described in the Materials and Methods section. (C) Expression of the E2F target genes in MEFs and testes. Total RNA was extracted and subjected to Northern blotting or semiquantitative RT-PCR analysis.

FIG. 3.

Abnormal nuclear morphology in cells of the liver and exocrine pancreas of mgcl-1^−/− mice. (A) Liver sections fixed with 4% paraformaldehyde. The nuclei were stained with hematoxylin. Liver sections that were fixed in Carnoy's solution showed the same abnormalities. (B) Liver sections stained with the anti-NPC antibody. (C and D) Pancreas sections stained with the anti-lamin B antibody. (B to D) Sections were analyzed using a confocal laser scanning microscope with 0.7-μm-thick optical sections. Scale bars, 10 μm.

FIG. 4.

Abnormal nuclear organization of spermatocytes in mgcl-1^−/− mice. (A) The testis sections of wild-type and mgcl-1^−/− mice were stained with antibodies against lamin B, LAP2, and NPC, and 1.0-μm-thick optical sections were analyzed by confocal microscopy. The sections were counterstained with propidium iodide, and the merged images are shown. Scale bar, 5 μm. (B) Electron micrographs of pachytene spermatocytes of wild-type and mgcl-1^−/− mice. P, pachytene spermatocyte; 8t, step 8 spermatid. Scale bar, 5 μm.

FIG. 5.

Morphological and functional abnormalities in the sperm of mgcl-1^−/− mice. (A) Sperm from the cauda epididymis of wild-type and mgcl-1^−/− mice were stained with HE, and their morphology was classified into four categories (see text). Data are means (error bars, standard deviations) of results for four mice (symbols for statistical significance [Student's t test]: *, P < 0.00001; †, P < 0.005; ‡, P < 0.05). (B) Morphology of sperm heads. Typical abnormalities observed in mgcl-1^−/− mice included blunt acrosomes, ectopic attachment of flagella, round heads, and narrowed heads (from left to right in the −/− panel). The sperm nuclei of mgcl-1^−/− mice are relatively well stained with hematoxylin, which suggests incomplete condensation. Scale bar, 10 μm. (C) Electron micrographs of sperm heads. Electron-dense and highly condensed nuclei are observed in the wild-type mice, whereas nuclear condensation is incomplete in the mgcl-1^−/− mice (arrows). The acrosomes of the sperm of mgcl-1^−/− mice are curled. The cytoplasmic components, such as mitochondria, are retained in an abnormal fashion around the acrosome (arrowhead). Abbreviations; a, acrosome; f, flagellum; n, nucleus. Scale bar, 5 μm. (D and E) Giant sperm with multiple heads and flagella in mgcl-1^−/− (D) and mgcl-1^{OST4876/OST4876} (E) mice. Scale bars, 10 μm. (F) Electron micrographs of giant sperm in mgcl-1^−/− mice. Three nuclei are embedded in a large section of the cytoplasm (left), and the cytoplasm is retained in the middle portion of the flagellum with two heads (right, arrowhead). Note the invaginated cytoplasm that is retained within the nuclei (left, arrowhead). Scale bar, 5 μm. (G) The percentage of motile sperm and the path velocity of motile sperm. Data represent the means (error bars, standard deviations) of four wild-type and six mgcl-1^−/− mice (symbols for statistical significance [Student's t test]: *, P < 0.005; †, P < 0.05).

FIG. 6.

Abnormal spermiogenesis in mgcl-1^−/− mice. Shown are electron (A to C and G to I) and light (D to F) micrographs of adult testes. (A and B) Step 10 spermatids of wild-type (A) and mgcl-1^−/− (B) mice. The arrowhead indicates the invaginated cytoplasm that accompanies the bundles of microtubules. (C) Step 15 spermatid of mgcl-1^−/− mice. Arrowheads indicate nucleoplasm extrusions from the condensed nucleus. (A to C) Scale bars, 2 μm. (D to F) The stage VIII tubules of wild-type (D) and mgcl-1^−/− (E and F) mice. Arrowheads indicate multinucleated cells. Scale bar, 20 μm. (G to I) Step 16 spermatids during spermiation in the wild-type (G) and mgcl-1^−/− (H and I) mice. Incomplete separation of the nucleus from the cytoplasm (H), and nuclear embedding in the cytoplasm (I) are frequently observed in mgcl-1^−/− mice. Arrows indicate the widening of intercellular bridges. Abbreviations: n, nucleus; f, flagellum; c, cytoplasm; ib, intercellular bridge. Scale bars, 2 μm.

FIG. 7.

Abnormal chromatin-remodeling proteins in mgcl-1^−/− mice. (A) The expression of mRNAs for Prms, TPs, Tarbp2, Camk4, and Csnk2a2 was examined by Northern blotting or RT-PCR analysis. (B) Prms from the sperm of wild-type and mgcl-1^−/− mice were separated on an acid urea polyacrylamide gel and either stained with Coomassie brilliant blue (CBB) or subjected to Western blot analysis with antibodies to Prm-1 (αPrm-1) or Prm-2 (αPrm-2). Protein from the same number of sperm was loaded in each lane. The top bands in the CBB-stained gels are precursor forms of Prm-2, and the lower band is a mixture of Prm-1 and Prm-2. (C) Immunohistochemical analysis of TP-2 in the testes of mgcl-1^+/− and mgcl-1^−/− mice. TP-2 was retained in an abnormal manner in late spermatids of mgcl-1^−/− mice at stage VIII (arrowheads). Nuclei are counterstained with methyl green. Scale bar, 10 μm.