INK4d-deficient mice are fertile despite testicular atrophy - PubMed (original) (raw)
INK4d-deficient mice are fertile despite testicular atrophy
F Zindy et al. Mol Cell Biol. 2000 Jan.
Abstract
The INK4 family of cyclin-dependent kinase (CDK) inhibitors includes four 15- to 19-kDa polypeptides (p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d)) that bind to CDK4 and CDK6. By disrupting cyclin D-dependent holoenzymes, INK4 proteins prevent phosphorylation of the retinoblastoma protein and block entry into the DNA-synthetic phase of the cell division cycle. The founding family member, p16(INK4a), is a potent tumor suppressor in humans, whereas involvement, if any, of other INK4 proteins in tumor surveillance is less well documented. INK4c and INK4d are expressed during mouse embryogenesis in stereotypic tissue-specific patterns and are also detected, together with INK4b, in tissues of young mice. INK4a is expressed neither before birth nor at readily appreciable levels in young animals, but its increased expression later in life suggests that it plays some checkpoint function in response to cell stress, genotoxic damage, or aging per se. We used targeted gene disruption to generate mice lacking INK4d. These animals developed into adulthood, had a normal life span, and did not spontaneously develop tumors. Tumors did not arise at increased frequency in animals neonatally exposed to ionizing radiation or the carcinogen dimethylbenzanthrene. Mouse embryo fibroblasts, bone marrow-derived macrophages, and lymphoid T and B cells isolated from these animals proliferated normally and displayed typical lineage-specific differentiation markers. Males exhibited marked testicular atrophy associated with increased apoptosis of germ cells, although they remained fertile. The absence of tumors in INK4d-deficient animals demonstrates that, unlike INK4a, INK4d is not a tumor suppressor but is instead involved in spermatogenesis.
Figures
FIG. 1
Mouse INK4d locus, targeting vector, and targeted allele. The upper line shows a map of the INK4d locus with exons 1 and 2 defined by filled bars and various restriction sites indicated. R1, _Eco_RI; HIII, _Hin_dIII; Bg, _Bgl_II; RV, _Eco_RV. The unique _Spe_I site in exon 2 is circled. A β-geo gene flanked by an internal ribosome entry site (IRES) was inserted into the _Spe_I site in exon 2 to generate the targeting cassete. The map of the recombined locus is shown by the lower line. Probes A and B corresponding to restriction fragments flanking INK4d coding sequences (top) are predicted to differentially hybridize to the _Hin_dIII and _Bgl_II fragments defined at the top (wild-type allele) and at the bottom (mutant allele).
FIG. 2
INK4d genotype and expression. (A) Southern blotting analysis of tail DNA taken from littermates derived from interbred INK4d+/− hemizygotes. DNA was digested with _Bgl_II, was transferred to nitrocellulose, and was hybridized with probe B (Fig. 1, top). (B) p19INK4d protein expression in testes from mice of the indicated genotypes, as determined by sequential immunoprecipitation and immunoblotting. (C) INK4 protein expression in kidney, spleen, brain, and testis from 8-week-old wild-type (+/+) and _INK4d_-null (−/−) mice determined as for panel B. Mouse erythroleukemia (MEL) cells were used as a positive control for p16INK4a and p18INK4c expression.
FIG. 3
Testicular atrophy in _INK4d_-deficient males. (A) Testes from 4-month-old wild-type (+/+) and _INK4d_-deficient (−/−) mice. (B) Comparison of the weights of wild-type (squares) and _INK4d_-deficient (triangles) testes at different ages of development. Additional data and statistical parameters are summarized in Table 1.
FIG. 4
Deficient INK4d expression and apoptosis in testis. (Panels A to D) In situ hybridization, using an INK4d antisense riboprobe, was performed on testis sections from 6-week-old wild-type (A and B) and _INK4d_-deficient (C and D) mice. Panel A shows a toluidine blue-stained bright field of panel B, and panel C shows a bright field of panel D, all at equal magnification. (Panels E to G) Because the β-geo cassette is encoded by the disrupted INK4d gene, tissues containing the mutant allele express β-galactosidase, which was detected in tissues from hemizygous (F) and _INK4d_-null mice (G). Wild-type mice that lack the β-geo cassette exhibit only background staining (E). All tissues were counterstained with neutral red dye. INK4d expression is heterogeneous throughout the tubules and is reduced by about 50% in heterozygous animals (cf. Fig. 2B). Under matched staining conditions, more intense and more uniform staining for β-galactosidase is therefore observed in tubules from _INK4d_-deficient mice (G) than in testes from hemizygous animals (F). (Panels H to K) Sections from testes of wild-type (panels H and J) and _INK4d_-deficient mice (panels I and K) were photographed and reproduced at equal magnification and were stained with toluidine blue (H and I) or subjected to TUNEL assay (J and K). Increased apoptosis in _INK4d_-deficient animals correlated with testicular atrophy.
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