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First published online 18 October 2006
doi: 10.1242/dev.02651

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CPEB controls oocyte growth and follicle development in the mouse

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

View larger version (33K): [in a new window]   Fig. 1. CPEB activity during oogenesis and oocyte maturation. Migrating and mitotically dividing PGCs begin to populate the genital ridges. Around E12.5-13.5, they arrest and enter meiosis prophase I (E14.5). Homologous chromosome pairing begins at zygotene [and full synaptonemal complex formation and homologous recombination occurs at pachytene (E17.5)]. At the zygotene to pachytene transition, CPEB is phosphorylated by the kinase Aurora A on T171, which activates the cytoplasmic polyadenylation machinery. Two substrates of CPEB are mRNAs encoding synaptonemal complex proteins 1 and 3 (SCP1 and SCP3), which contain CPEs. In Cpeb KO mice, these two proteins are not synthesized and the synaptonemal complex is not formed; the oocytes do not develop beyond pachytene. When the wild-type oocytes progress to diplotene, PP1 dephosphorylates and inactivates CPEB. Oocytes then enter the dictyate stage, which begins before birth and is characterized by oocyte growth and follicle formation. The oocyte synthesizes and secretes the zona pellucida and is surrounded by granulosa cells. In the large preovulatory follicle, the oocyte and granulosa cells reside in the fluid-filled antrum. Upon hormonal signaling, an oocyte enters the meiotic divisions (maturation), which is characterized by germinal vesicle (GV) breakdown, chromosome condensation and polar body extrusion. At this stage, CPEB is re-phosphorylated on T171 by Aurora A, again activating the cytoplasmic polyadenylation machinery that promotes the translation of Mos and cyclin B1 RNAs. The Zp3 promoter becomes active during the dictyate stage.

View larger version (82K): [in a new window]   Fig. 2. Cpeb knockdown mice. (A) Schematic representation of the transgene construct. The Zp3 promoter was inserted upstream of the Egfp open reading frame, which was followed by 1301 bases of the Cpeb 3' UTR in an inverted repeat structure separated by irrelevant loop sequence. The Cpeb inverted repeats would form a double-stranded stem and be cleaved by DICER, thus yielding siRNAs that would anneal to the endogenous Cpeb 3' UTR and induce RNA destruction. (B) RT-PCR of Egfp, Cpeb, Mos, Zp3 and -tubulin mRNAs from ovary extracts derived from 2-month-old animals. (C) Ovaries from 8-week, 2-, 4- and 10-month-old animals were fixed, embedded and stained with hematoxylin and eosin. The Cpeb knockdown ovaries displayed a progressive decrease in oocyte number and follicles. (D) TUNEL labeling of 6-week-old ovaries from wild-type and TG mice. Note that while the wild-type ovary displayed little labeling, the TG ovary was heavily labeled, indicating substantial granulosa cell apoptosis. Scale bars: 100 µm in C,D.

View larger version (175K): [in a new window]   Fig. 3. Post-pachytene knockdown of Cpeb results in oocyte abnormalities. Ovaries collected from animals at 2, 4 and 6 months of age were processed for histology as described above. (A) An oocyte in a follicle (stage 5-6) that was detached from the cumulus granulosa cells (*) with abnormal and improperly positioned tripolar spindles (arrow). (B) The oocyte had a disorganized rosette of chromosomes (arrow) at MI that were spread randomly among three spindle poles (bold arrow). (C) This oocyte, enclosed in the asymmetrically developed granulosa cells (one to five layers), in a stage 5a follicle, contained a monopolar spindle; the granulosa cells were also starting to detach. (D-F) These pre-antral stage follicles contained MI and MII stage oocytes that displayed apparent granulosa cell apoptosis. (D) This oocyte had improperly extruded polar bodies at opposite sides, and displayed premature antrum formation (*). (E) This oocyte contained a polar body with condensed chromosomes forming a metaphase plate and a spindle. (F) The oocyte, enclosed in a pre-antral follicle, contained a polar body with three pronucleus-like structures. (G-I) These oocytes contained poorly structured nuclei and/or nuclear membranes. (G) This stage 3a oocyte lacked a nuclear membrane and contained partially condensed chromatin. Notice the chromatin particles dispersed in the oocyte cytoplasm. (H) This fully developed oocyte lacked normal nuclear membrane and contained several cytoplasmic structures that resemble nuage or perhaps nucleoli (empty arrowheads, also in I). (I) This fully developed oocyte had a disassembled nuclear envelope with condensed chromatin in the cytoplasm. Scale bars: 10 µm in G; 20 µm in all other panels.

View larger version (185K): [in a new window]   Fig. 4. Oocytes with reduced levels of CPEB undergo parthenogenetic activation. (A) This MI oocyte displayed two spindles and appeared to be undergoing asymmetric division with the cleavage furrow oriented longitudinally near of the spindle poles. (B) This four-cell embryo was enclosed in an abnormal follicle that apparently contained a polar body. The follicle contained undifferentiated and possibly apoptotic squamous granulosa cells (arrow). (C) This parthenote resided in an immature follicle (stage 5a-5b) and was detached from the cumulus granulosa cells. (D) This 7-8-cell parthenote resided in a 5a stage follicle; note the mitotic spindle in the bottom blastomere (arrow). (E,F) Multicell parthenotes enclosed in abnormal follicles (stage 4 to 5a); notice abnormally compacted granulosa cells. (G,H) Oocytes in stage 4-5b follicles with fragmented or multiple nuclei and nucleoli. (I) This irregularly shaped overgrown oocyte contains a number of structures in the cytoplasm that resemble nucleoli (empty arrowheads); notice two polar bodies. Scale bar: 20 µm. pb, polar bodies.

View larger version (111K): [in a new window]   Fig. 5. Cpeb knockdown results in ovarian abnormalities. (A) This follicle contained two oocytes. (B) Atretic oocytes in an undifferentiated follicle composed of a single layer of the squamous granulosa cells. (C) This atretic follicle contained an atretic oocyte with abnormal and compacted granulosa cells (compare with Fig. 2D bottom right). (D,E) These panels show ovarian cysts in the ovaries of a 4-month-old and a 12-month-old mouse, respectively. Scale bars: 20 µm in A-C; 50 µm in D; 100 µm in E.

View larger version (46K): [in a new window]   Fig. 6. RNA binding and translational control by CPEB. (A) Extracts from 3-week-old mice were incubated with CPEB antibody or IgG and subjected to immunoprecipitation followed by RNA extraction and RT-PCR for specific mRNAs. Two to 10% of the RNA was used for the loading control; RT refers to no reverse transcription. The sequence of the putative CPE and its distance from the AAUAAA is indicated for each RNA, as is the function of the encoded protein. (B) The upper panel shows the scheme of the chromatography procedure on poly (U) Sepharose followed by thermal elution. RNAs with shorter poly(A) tails elute at lower temperatures than those mRNAs with longer tails, and are detected by RT-PCR. The lower panel shows the distribution of the RT-PCR products following chromatography. In wild-type ovaries, all the RNAs eluted mostly at 45°C, but also at 60°C, indicating that the poly(A) tails were heterogeneous in length and some were long enough to elute only at the higher temperature. By contrast, all the RNAs from the TG ovaries except -tubulin, Zp1 and Mater that lack a CPE eluted completely at 45°C. These results suggest that the poly(A) tails of these RNAs were short in the TG ovaries because they eluted at a lower temperature. (C) Western blot shows that GDF9 (preprocessed) levels were substantially reduced in all TG lines compared with wild type. NS, a non-specific loading control.

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