Uterine dysfunction and genetic modifiers in centromere protein B-deficient mice

Abstract

Centromere protein B (CENP-B) binds constitutively to mammalian centromere repeat DNA and is highly conserved between humans and mouse. Cenpb null mice appear normal but have lower body and testis weights. We demonstrate here that testis-weight reduction is seen in male null mice generated on three different genetic backgrounds (denoted R1, W9.5, and C57), whereas body-weight reduction is dependent on the genetic background as well as the gender of the animals. In addition, Cenpb null females show 31%, 33%, and 44% reduced uterine weights on the R1, W9.5, and C57 backgrounds, respectively. Production of "revertant" mice lacking the targeted frameshift mutation but not the other components of the targeting construct corrected these differences, indicating that the observed phenotype is attributable to Cenpb gene disruption rather than a neighbouring gene effect induced by the targeting construct. The R1 and W9.5 Cenpb null females are reproductively competent but show age-dependent reproductive deterioration leading to a complete breakdown at or before 9 months of age. Reproductive dysfunction is much more severe in the C57 background as Cenpb null females are totally incompetent or are capable of producing no more than one litter. These results implicate a further genetic modifier effect on female reproductive performance. Histology of the uterus reveals normal myometrium and endometrium but grossly disrupted luminal and glandular epithelium. Tissue in situ hybridization demonstrates high Cenpb expression in the uterine epithelium of wild-type animals. This study details the first significant phenotype of Cenpb gene disruption and suggests an important role of Cenpb in uterine morphogenesis and function that may have direct implications for human reproductive pathology.

Figure 1

Strategies for production and screening of Cenpb disrupted and targeted control mice. (A) Intronless wild-type Cenpb gene showing the 1.8-kb coding region (solid box) and polyadenylation p(A) site. Recombination at the sites shown by the dotted crosses results in the incorporation of the targeting construct containing a translational frameshift oligonucleotide (D/TAA) at the 5′ coding region and an IRES-selectable marker cassette (shaded box) within the 3′-untranslated region (i). This recombination event results in the disruption of the Cenpb gene. An alternative recombination event at sites indicated by the solid crosses, one of which is 3′ of the D/TAA frameshift mutation, results in the introduction of the IRES-selectable marker cassette but not the frameshift mutation (ii). This targeted allele served as a control for any positional effect the inserted IRES-selectable marker cassette may have on the phenotype of the mice. The Cenpb wild-type gene (+), null allele (−), and targeted control allele (o) allele were detected as described previously (Hudson et al. 1998) as 5.3^-, 0.7^-, and 7-kb bands (broken lines), respectively, by DraI (D) digestion and Southern blot hybridization using a 5′-probe (open box) (see Hudson et al. 1998). Once the heterozygous ES cell lines carrying the + and o alleles were identified, they were used for the production of the targeted control (o/o) mice. Subsequent genotype screening for these mice was based on PCR analysis using primers B4top and B1comp, which gave a 281-bp product for the + allele, and primers Neo1 and B1comp which gave a 320-bp product for the o allele. (B) PCR analysis of mouse progeny from a +/o × +/o cross using a combination of the primers B4top, B1comp, and Neo1, showing the +/+ (lanes 5,7), +/o (lanes 2,3,4,6), and o/o (lane 1) genotypes.

Figure 2

Immunofluorescence analysis of fibroblast cell lines derived from +/+ (A) and o/o (B) mice. Metaphase chromosomes (DAPI-stained and pseudocolored red) were prepared from fibroblasts cultures of mouse tail tissues and stained with a monoclonal anti-CENP-B antibody (green). (1) Merged immunofluorescence signals; (2) split image for the anti-CENP-B (green) signals.

Figure 3

Total body weight of male (A,C,E) and female (B,D,F) mice in different genetic backgrounds: (A,B) RI; (C,D)W9.5; and C57 (E,F) C57. At least four animals were used for each time point. Age of the animals was determined from birth. (▴) +/+ animals; (○) −/− animals.

Figure 4

Uterine and pregnancy problems in Cenpb null mice. (A) Size comparison of uteri from 10-week-old R1^+/+ and R1^−/− mice, showing the left and right uterine horns. The sizes of the ovaries and oviducts attached to these horns were normal in both animals. (B) Nine-month-old, 1-week overdue W9.5^−/− pregnant female (first pregnancy), showing a full-term dead fetus (arrowhead) attributable to placental necrosis in the right horn (rh) and fetal growth arrest/resorption (arrow) in the left horn (lh; shown in circle). (C,D) Twelve-month-old, 10-day-overdue R1^−/− pregnant female (fourth pregnancy), showing external and internal views of a necrotic fetus in the right horn, and absence of fetuses in the left horn. (E,F) Nine-month-old, 4-day-overdue R1^−/− pregnant female (first pregnancy), showing external and internal views of decomposed fetal content in the left horn, and absence of fetuses in the right horn.

Figure 5

Histology (A–D) and in situ hybridization (E,F) of uterine sections. (A,C) Ten-week-old, hematoxylin-and eosin-stained C57^+/+ uterus (day 0.5 VP), showing normal morphology of the endometrial epithelium lining the uterine lumen (A) and endometrial glands (C). (B,D) ten-week-old, hematoxylin- and eosin-stained C57^−/− uterus (day 0.5 VP), showing highly disorganized and apoptotic (clear) epithelial cells lining the uterine lumen (B) and endometrial glands (D). Apoptosis of the clear cells (containing condensing chromatin) was confirmed by TUNEL assay (Gavrieli et al. 1992) (data not shown). Scale bar for A–D, 20 μm. (E,F) Bright-field (hematoxylin-stained) and dark-field views, respectively, of 10-week-old R1^+/+ uterus hybridized with a ³⁵S-labeled mouse Cenpb antisense riboprobe, showing mRNA signals (dark brown grains in E and white grains in F) throughout the endometrium (and the myometrium; not included in picture) with maximal mRNA expression in the epithelial lining of the uterine lumen and endometrial glands (selected examples of which are indicated by arrows). Scale bar for E and F, 50 μm. (en) Endometrium; (ep) epithelium, (l) uterine lumen.