TBP2 binds to promoters of active genes in the oocyte. (A–B) ChIP using anti-TBP2 or anti-H3K4me3 antibodies as indicated and promoter regions of oocyte-specific genes were analyzed by real-time qPCR. Data is represented as the enrichment over the input (in percentage). IRs are shown as negative controls. ChIP with a mouse or rabbit IgG antibody showed no enrichment above background levels. Shown are representative results of at least three independent experiments. Asterisk (*) indicates significant enrichment (P ≤ 0.05). Note that although Bmp15 mRNA accumulation in the oocyte is high (Supplemental Fig. S2), H3K4me3 levels on its promoter are not significant, suggesting that H3K4me3 regulation on Bmp15 promoter is different or that Bmp15 mRNA is relatively stable and that actual rate of active transcription of this gene is low.

RNA Pol II activity and chromatin structure are altered in oocytes lacking TBP2. (A,B) Immunofluorescence analysis of control and Tbp2^−/− ovarian sections from 6-wk-old females using antibodies recognizing the Ser2-phophorylated CTD of Pol II (A) or all forms of the CTD of Pol II (B). Pol II staining is shown in red. DNA (blue) was counterstained with DAPI. Follicular stages are indicated. (A) The arrow points to the follicle at the stage indicated at the top. Bars: for primordial stages, 10 μm; for later stages, 50 μm. (B) Bar, 50 μm. (C) Tbp2^−/− oocytes show reduced H3K4me3 levels from the primary follicular stage. Immunostaining analysis for H3K4me3 in oocytes at different stages of follicular development from 6-wk-old females. DNA (blue) was stained with DAPI. Bar, 10 μm. Confocal acquisition was done using identical parameters to allow comparison. Hence, H3K4me3 levels in preovulatory stage oocyte in the control appear saturated. Note that Tbp2^−/− oocytes degenerate after the secondary follicular stage, as seen from the presence of follicular cells within the follicle. The dashed white line demarcates the oocyte membrane. (D) Altered chromatin configuration in Tbp2^−/− oocytes. Sections from control and Tbp2^−/− mice were stained with DAPI and analyzed under confocal microscopy. (NSN) Nonsurrounded nucleolus; (PSN) partially surrounded nucleolus; (SN) surrounded nucleolus. Representative sections are shown. (ND) Not determined.

Characterization of Tbp2-deficient mice. (A) RNA from ovaries derived from females of the indicated genotype was reverse-transcribed and amplified with primers specific for TBP2. PCR products were transferred and hybridized with a radioactive Tbp2-specific probe to confirm the specificity of the products and to allow quantification. Histone H2A was used as internal control. Quantification of TBP2 mRNA levels in Tbp2^+/+, Tbp2^+/−, and Tbp2^−/− ovaries is shown on the graph. Data are normalized to H2A expression and calculated relative to TBP2 mRNA expression in wild-type ovaries. Average ± SEM of two ovaries per genotype are represented. (B) TBP2 protein is absent in Tbp2^−/− ovaries. Western blot analysis on total extracts from ovaries of females of the indicated genotype using the α-TBP2 antibody. α-Tubulin was used as a loading control. Shown is a representative experiment of three biological replicates. (C) Tbp2^−/− females do not ovulate upon hormonal stimulation. Control and Tbp2^−/− 4- to 6-wk-old females were induced for superovulation with intraperitoneal injection of PMSG followed by administration of hCG 48 h later. The number of ovulated oocytes was determined after dissection for each female. The average number of oocytes ovulated per female is indicated. (D) Increased number of immature follicles and abnormal oocytes in Tbp2^−/− females. Examples of follicles dissected from control and Tbp2^−/− ovaries observed under DIC microscopy. While GV stage (preovulatory) oocytes are easily distinguishable in control females, Tbp2^−/− oocytes contain mainly immature follicles or oocytes of abnormal morphology and devoid of zona pellucida. (E) Gross morphology of ovaries from 6-wk-old control and Tbp2^−/− females. While control ovaries show clear mature follicles (arrows), such structures are absent in the Tbp2^−/− ovaries.

TBP is dispensable for oocyte maturation, ovulation, and subsequent fertilization. (A) Genetic approach used to delete TBP in the maternal germline. Homozygous females for the floxed Tbp allele were mated to homozygous males for the ZP3-Cre transgene. Females express Cre in their oocytes. Haploid genotype of 50% of their oocytes is thus Tbp⁻ (deleted) and the other 50% have a Tbp⁺ (wild-type) genotype. To determine the competence of these oocytes, females were mated with wild-type males as indicated. Male littermates of the F1 were used as controls. (B) Table summarizing the results obtained from the crosses between transgenic F1 females (Tbp^lox/+;ZP3-Cre^tg/+) or males and their respective wild-type partner. (nd) Not determined. (C) Oocytes carrying the maternal Tbp⁻ (deleted) allele can be fertilized in vivo and develop normally until the blastocyst stage. Zygotes derived from F1(Tbp^lox/+;ZP3-Cre^tg/+) females mated with wild-type males were collected at fertilization and were cultured until the blastocyst stage. Single embryo genotyping was performed subsequently. (D) Efficient deletion of TBP in oocytes ovulated from F1(Tbp^lox/+;ZP3-Cre^tg/+) females upon Cre expression driven from the Zp3 promoter. Zygotes were collected from F1 X wild-type male crosses immediately after fertilization, stained with anti-TBP antibody (3G3), and analyzed using confocal microscopy. Individual embryos were genotyped after acquisition. Shown are representative embryos derived from oocytes inheriting the deleted Tbp allele maternally (Tbp^−/+) and those inheriting the Tbp wild-type allele (Tbp^+/+). Note that in the Tbp^+/− embryo, the two pronuclei mask each other. (E) TBP2 overexpression results in altered embryonic development and in reduced cell proliferation. Experimental design for TBP2 overexpression is shown on the top. Zygotes were microinjected with mRNA for DsRed alone (control) or in combination with mRNA for TBP2 and cultured until control embryos reached the blastocyst stage. (F) Zygotes overexpressing TBP2 cleave to the two-cell stage normally, but show reduced development until the blastocyst stage.

TBP2 ablation results in altered oocyte-specific transcriptional profile in 2-wk-old ovaries. (A) Distribution of the number of probes on the microarray with respect to their FDR and fold change in 2-wk-old the Tbp2^−/− ovaries compared with the control. (Top panel) In the histogram, bars of different colors represent the number of probes differentially hybridized at the indicated FDR threshold. (Bottom panel) The exact number of probes is displayed in the table. At FDR = 0.12 (yellow bars), there are as many probes in the down-regulated class (negative fold change) as there are in the up-regulated one (positive fold change). (B) Microarray validation in 2-wk-old Tbp2^−/− and Tbp2^+/+ ovaries. Up-regulated and down-regulated genes with different FDR and fold change values (ranging from −0.22-fold to 2.5-fold) were randomly selected and their mRNA levels were analyzed by RT-qPCR. mRNA levels are shown relative to 18S RNA and are expressed relative to the control, for which mRNA levels were set at 1 (black line). Microarray (black bars) and RT-qPCR (gray bars) fold changes are average of three independent biological replicates, respectively. Error bars are SD. (C) Most enriched functional categories among the down-regulated and up-regulated genes according to Ingenuity Pathways. (D) Genes down-regulated upon TBP2 loss are enriched in oocyte-specific genes. Comparison of the up-regulated and down-regulated genes with an FDR of 0.12 with genes that are specifically expressed in the oocyte. P values for the intersection of the up-regulated and down-regulated genes are shown at the bottom. Note that only the intersection of down-regulated genes is statistically significant. (E) Sequence composition of the promoters belonging to the two classes of genes according to their nucleotide content. The percentage of AT content of the up-regulated (red) and down-regulated (green) gene promoters within a region from −100 to +100 bp around TSS is shown. The average AT content for the two promoter populations is also indicated. Comparison of the two groups of genes using a Student's t-test indicates that the two populations of promoters differ significantly. The TSS positions follow the current NCBI annotation.

TBP2 loss results in ovarian failure due to defective folliculogenesis. (A–C) Histological analysis of ovaries from Tbp2^−/− mice reveals a block in folliculogenesis around the primary and secondary follicular stage. In A, sections from 2-wk-old and 6-wk-old control (panels a–b,e–f) and Tbp2^−/− (panels c–d,g–h) ovaries stained with hematoxylin-eosin are shown at two different magnifications. In control 6-wk-old ovaries, primary (Prf), secondary (SF), and antral follicles (AnF) as well as corpus lutei (CL) are indicated. Tbp2^−/− oocytes lack antral secondary follicles and large graafian follicles; instead, intersticial proliferation (IP) is visible in panel g. Data are representative of three biological replicates. Bar, 100 μm. (B) Higher magnifications of oocytes at different stages of follicular development from 6-wk-old control (panels a–d) and Tbp2^−/− (panels e–h) ovaries. (Panels a,e) Arrows point to two different primordial follicles, which appear normal when compared with control ovaries. Primary and secondary follicles can be found in null ovaries, albeit in reduced numbers (see C). (Panel h) Following the secondary follicle stage, the few Tbp2^−/− oocytes that survive fail to undergo antrum formation and instead show disorganized follicular structure. Note the absence of zona pellucida in these oocytes as seen from the lack of regular white space surrounding the oocyte (arrow). (C) Increased number of underdeveloped oocytes in Tbp2^−/− females. Analysis of oocyte counts comparing Tbp2^−/− and control 2-wk-old and 6-wk-old ovaries. Oocytes within primordial (PF), primary (PrF), and secondary (SF) follicles were counted on ovarian sections. Numbers represent average of counts of three sequential sections from serially sectioned ovaries. Antral oocytes are only present in the controls and were therefore not included in the counts. P values were calculated using an unpaired t-test for triplicates. (D) TUNEL assay on sections from control and Tbp2^−/− ovaries. Apoptotic nuclei were labeled using a modified TUNEL protocol with fluorescein detection. DNA was stained with DAPI. In 6-wk-old control sections, an artetic follicle (AtF) and nuclei within the CL show normal signs of apoptosis. A healthy antral follicle (AnF) is also depicted. Representative merge (panels a,b,e,f) and green (panels c,d,g,h) channel images of lower (panels a,c,e,g) and higher (panels b,d,f,h) magnifications are shown.