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First published online 19 April 2006
doi: 10.1242/dev.02371

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Basonuclin: a novel mammalian maternal-effect gene

¹ Department of Dermatology, University of Pennsylvania, PA 19104, USA.
² Department of Biology, University of Pennsylvania, PA 19104, USA.
³ Shanghai Institute of Medical Genetics, Shanghai Jiao Tong University, Shanghai 200040, People's Republic of China.
⁴ Center for Research on Reproduction and Women's Health, University of Pennsylvania, PA 19104, USA.
⁵ Department of Cell and Developmental Biology, University of Pennsylvania, PA 19104, USA.

View larger version (43K): [in a new window]   Fig. 1. Basonuclin expression profile during oogenesis and pre-implantation development. The relative abundance of basonuclin mRNA was plotted using a log scale and data from a previous microarray analysis (Zeng et al., 2004) (unbroken line) and real-time PCR (broken line). Immunocytochemical staining of oocytes and pre-implantation embryos are shown in the insets in the graph. The arrowheads indicate the positions of the germinal vesicle, the pronuclei and the nuclei of the respective developmental stages. An immunoblot of basonuclin at the corresponding developmental stages is shown below the graph (75 oocytes/embryos per lane). Developmental stages: GV, germinal vesicle-intact oocyte; Egg, metaphase II-arrested egg; 1C, one-cell embryo; 2C, two-cell embryo; M/B, morulae and blastocyst.

View larger version (42K): [in a new window]   Fig. 2. Basonuclin-RNAi transgene and its effect on female fertility, levels of basonuclin mRNA and protein in oocytes. (A) Structures of the two transgene constructs with different vector backbones, splicing sequences and basonuclin cDNA sequences. The nucleotide coordinates of mouse basonuclin cDNA are shown above one copy of the inverted repeats (black arrows). (B,C) Fertility of the transgenic RNAi females was inferred by the average litter-size. Transgenic females (founder, B, or out-crossed progeny of the founder males, C) were mated with three normal males in a randomized sequence. (B) The data are based on three matings for each transgenic founder, and the control (non-TG) is the average litter size of the seven non-transgenic females. (C) The mating/pregnancy number (n) is shown above each bar. *P<0.001. Basonuclin mRNA was knocked-down in transgenic GV-stage oocytes as shown by real-time PCR (D), immunocytochemistry (F) and immunoblot (G). (D) The mRNA for the upstream-binding factor (UBF), a ubiquitous Pol I transcription factor, was used as a control. (E) Real-time PCR data were converted into the percentage of wild-type basonuclin mRNA level and plotted against the fertility data from C. (F) Oocytes were stained with anti-basonuclin antibody (red) and DAPI (blue), and the two images are overlaid to indicate the position of germinal vesicle in the transgenic oocytes. Scale bar: 10 µm. (G) Two duplicate samples were analyzed for each transgenic line. Each lane contains protein extracted from 50 oocytes. After probing for basonuclin, the membrane was stripped and re-probed with antibody against ß-tubulin. TG, transgenic oocytes; non-TG, non-transgenic oocytes.

View larger version (66K): [in a new window]   Fig. 3. Basonuclin-RNAi transgene affected the morphology of ovary and oocytes. Histology of non-transgenic (A,B) and T50 transgenic (C,D) ovaries are shown at two magnifications: the entire ovary (A,C) and an enlarged region (B,D). Arrows indicate follicles that contain abnormal oocytes; arrowheads indicate remnants of degenerating follicles. Follicles and oocytes are apparently normal in the transgenic ovary (*). cl, corpus luteum. GV-stage oocytes were isolated and observed under the phase-contrast (E,F) and differential interference contrast (DIC) (G,H) microscopy. Non-transgenic oocytes are shown in E,G, and transgenic ones in F,H. Arrowheads indicate examples of dark granules in the transgenic cytoplasm (F) and the bumps on transgenic cell surface (H). The transgenic population is heterogenous: both morphologically normal and abnormal oocytes are present. Scale bars: 50 µm in E; 10 µm in H.

View larger version (73K): [in a new window]   Fig. 4. Pol I transcription is reduced in basonuclin-RNAi transgenic oocytes. Run-on assays were performed on growing oocytes (14 days) from control (A,C,F) and line T50 (B,D,G) in the absence (A,B) and presence (C,D) of 100 µg/ml -amanitin. The incorporated BrdU was visualized by AlexFluor-488-labeled anti-BrdU antibody; nucleoplasm staining is indicated by arrowheads and that of nucleolus by arrows. The fluorescence intensity of nucleolus in run-on with -amanitin was measured from micrographs with ImageJ (non-T, n=14; T50, n=10), and the average in arbitrary units is shown (E). Nucleoli of 20 oocytes from each group were optically sectioned (F, non-T control; G, T50), and the transcription foci (brightly labeled spots) were scored. The average number of foci per oocyte is shown (H). *P<0.05; **P<0.001.

View larger version (54K): [in a new window]   Fig. 5. Microarray analysis of basonuclin-deficient oocytes. Oocyte RNAs from four non-transgenic and six transgenic mice were analyzed using Affymetrix Mouse Genome GeneChip MOE 430 v2. Raw microarray data were processed by the GC-RMA or MAS 5 program and analyzed with SAM, GeneSpring and EASE software. (A) A sample clustering analysis showed a high degree of association within the control and transgenic groups. (B) Pathway analysis grouped the upregulated genes into four processes, and that of the downregulated into three. The percentage of each group in the up- or downregulated category is indicated by the length of the bar (black for upregulated and white for downregulated).

View larger version (93K): [in a new window]   Fig. 6. Pre-implantation development of basonuclin-RNAi transgenic embryos. Eggs fertilized in vivo were collected and cultured in vitro for 84 hours (3.5 days). (A) Development of the transgenic (T50) embryos was arrested at the two-cell stage. (B) At 36, 60 and 84 hours post-coitum, the developmental stages of the embryos were scored and expressed as a percentage (±s.d.) of the population. The data are the average of three experiments, in which a total of 40-60 embryos for each transgenic line were scored. The dead category includes collapsed, degenerated or fragmented embryos; 1C, one cell, 2C, two cell (etc.); M/B, morula/blastocyst.

View larger version (66K): [in a new window]   Fig. 7. Abnormalities of basonuclin-RNAi transgenic embryos. Fertilized eggs were examined under DIC (Normaski) optics (A-D) or fluorescence (DAPI) (E-G). (H-L) Merged images. Compared with non-transgenic one-cell embryos (A), the transgenic T50 embryos had a much rougher cell surface (B-D) caused by numerous `bumps' (B-D,L, black arrowheads). At the same one-cell stage, the transgenic pronuclei, as revealed by DAPI staining, were smaller (F-H) and contained irregular DNA distribution (white arrow in F) when compared with the control (E). In some transgenic embryos, additional nucleus/chromatin aggregates were seen (white arrowhead in G,H), some of which were probably derived from polyspermy (arrow indicates the head of an attached sperm). At the two-cell stage and beyond, additional abnormalities were seen in transgenic embryos but not in control (I); some examples were collapsed embryos with disorganized chromosomes (J), incomplete chromosome segregation during mitosis (K, arrowhead indicates the two chromatin mass was still linked by a thread of DNA) and cytoplasmic fragmentation (L, white arrow). (M) A summary of the defects of transgenic T50 embryos during the early pre-implantation development in culture. The observed defects were indicated above the time line (thick black line) of one-cell to two-cell embryonic development (Moore et al., 1996). The cell-cycle stages (G1, S, G2 and M) were shown above the time line and the hours post copulation (hpc) below. The black dots indicate that the timing of the observation could be determined to a particular stage of the cell cycle; the short thick lines imply that the measurements were made on pooled one- or two-cell embryos at varying stages of the cell cycle. The autoradiogram of a PAGE gel shows the presence of TRC in both the control and transgenic embryos. Scale bars: 50 µm in C; 10 µm in E,I.