Cell cycle regulation in Cib1^−/− MEFs is altered compared to that in Cib1^+/+ and Cib1^+/− MEFs. (A) Growth curves of early-passage MEFs derived from cells of Cib1^+/+, Cib1^+/−, and Cib1^−/− mice of various passages (P2, P4, and P6) were plated at 0.2 × 10⁵ cells/well in duplicate (day 0). For each passage (P2, P4, and P6), cells were then harvested at the indicated days and viable cells were counted. Cib1^+/+ MEFs, closed square; Cib1^+/− MEFs, open square; and Cib1^−/− MEFs, closed and open triangles (representing two separate knockout MEF cell lines). Data represent means ± standard errors of the means of duplicates (n = 2). (B) Early-passage Cib1^−/− MEFs (representative P4 is shown) showed similar morphology compared to Cib1^+/+ and Cib1^+/− MEFs (phalloidin, green; DAPI, blue). Scale bar, 20 μm. WT, wild type.

Cdc2 is up-regulated in Cib1^−/− mouse testis. (A) Various spermatogenesis stage-specific mRNA messages were tested in a one-step RT-PCR approach. Most pre- and postmeiotic gene expression levels were comparable in Cib1^+/+ and Cib1^−/− mice, except for a consistently increased level of Cdc2 mRNA in the testis of Cib1^−/− mice. (B) Cdc2 protein expression is up-regulated in adult Cib1^−/− mice. Testes of adult mice were homogenized, lysed in CHAPS buffer, and subjected to Western blotting, which indicated an increase in Cdc2 but not PP2A protein expression. In addition, PP1cγ protein levels were reduced in adult Cib1^−/− mice. (C) Cdc2 protein expression is up-regulated in juvenile Cib1^−/− mice during testis development. Day 8 and day 17 (17d) testes of juvenile mice as well as adult mice (60 days [60d]) were prepared for Western blotting as described for panel B. An increase in Cdc2 protein expression was detected in these juvenile stages of Cib1^−/− mice compared to that of Cib1^+/+ littermates, while CIB1 protein expression was maintained at a constant level throughout testis development. The loading control was α-tubulin. (D) Cdc2 protein expression is up-regulated in isolated testis cells of 40- and 60-day-old Cib1^−/− mice. Isolated testis cells (mainly germ cells and Sertoli cells, containing no tubular structures, in contrast to whole-testis lysates) were lysed in CHAPS buffer and subjected to Western blotting, with α-tubulin used as a loading control. The Cdc2 expression levels in isolated testis cells from Cib1^−/− mice were normalized to α-tubulin and are presented as fold increase relative to cells from Cib1^+/+ mice (n = 4). (E) Passage 4 Cib1^−/− MEFs showed similar Cdc2 protein expression and phosphorylation at Tyr15 compared to that of MEFs of Cib1^+/+ and Cib1^+/− mice.

Cib1-deficient males exhibit a disruption of spermatogenesis with an increase in apoptosis. Testis and epididymis tissues were fixed overnight in either 4% paraformaldehyde or Bouin's solution, and sections were stained with HE or PAS. Unstained sections were subjected to a fluorometric TUNEL assay. (A to D) PAS-stained sections from Bouin's fixed adult testis from Cib1^+/+ mice (A and C) and Cib1^−/− mice (B and D) are shown. Spermatogonia (black arrow head), spermatocytes (white arrow head), and round spermatids (white asterisk) are present in the testis of Cib1^−/− mice. However, seminiferous tubules in Cib1^−/− animals lack typical advanced spermatids (white arrow in panel C) but have multinucleated spermatids near the tubule lumen (black arrows in panels B and D) and accumulation of PAS-positive secretory material (black broken arrows in B and D). (E and F) HE-stained cross-sections of the cauda epididymis from adult mice show dark blue-stained sperm heads (black arrow) in the lumen of Cib1^+/+ epididymis (E) but no sperm in the Cib1^−/− epididymis (F). (G and H) TUNEL staining of testes sections from adult Cib1^−/− mice (H) reveals increased apoptosis (green) compared to that of Cib1^+/+ mice (G). Testes sections were also stained with propidium iodide (red) to delineate the cross-section contours. (I to K) HE-stained cross-sections of testes from postnatal day-20 (I and J) and day-25 (K and L) mice. Day-20 testes of Cib1^+/+ (I) and Cib1^−/− (J) mice show similar morphologically identifiable round spermatids (black star). By postnatal day 25, testis of Cib1^+/+ mice (K) continued to exhibit normal morphological development, whereas testis from Cib1^−/− mice (L) show the presence of multinucleated cells similar to those present in adult Cib1^−/− mice shown in panels B and D. Bars, 50 μm.

Targeted disruption of the mouse Cib1 gene. (A) Graphic representations of the genomic Cib1 allele, targeting vector, and mutant allele. Correct targeting would lead to the insertion of a new BamHI restriction enzyme site, resulting in a new 3-kb BamHI digestion band (B, BamHI; Bc, BclI; N, NotI; P, PvuII; X, XhoI; TK, thymidine kinase). (B) Verification of correct targeting in the Cib1 null mice. Genomic DNA was extracted from mouse tails and digested with the BamHI restriction enzyme. Southern blotting with the indicated probe verified correct targeting in Cib1^−/− mice with a new 3-kb BamHI digestion fragment (nontargeting genomic DNA shows a 15-kb band in a Cib1^+/+ sample). (C) CIB1 protein is absent in Cib1^−/− testes and Cib1^−/− MEFs. Western blotting with an anti-CIB1 polyclonal chicken antibody showed that CIB1 protein is not expressed in testis and MEF lysates derived from Cib1^−/− mice but was expressed in testis and sperm of Cib1^+/+ mice. (D) Expression of CIB1 protein in isolated testis cells. Lysates from various cell types isolated from Cib1^+/+ testes were probed with an anti-CIB1 polyclonal antibody. Western blotting analysis shows that CIB1 protein is present in round and condensing spermatids and is at much higher levels in cultured Sertoli cells. The loading control is α-tubulin. CIB1 expression in pachytene spermatocytes was visible upon extended exposure. (E) Testis weight curve during testis development. Cib1^−/−, Cib1^+/+, and Cib1^+/− mouse testes of various ages (8 days to 4 months old; n ≥ 3 for each data point) were dissected, weighed, and graphed. (F) Adult seminal vesicle weight is comparable between Cib1^−/−, Cib1^+/+, and Cib1^+/− mice. Adult mouse testes (3 to 4 months old) of different genotypes were dissected, and the average weight of seminal vesicles was graphed (n = 6 for all genotypes; P > 0.6 when seminal vesicle mass of Cib1^−/− mice is compared to that of Cib1^+/+ or Cib1^+/− mice; all values represent means ± standard deviations).