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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02667

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Infertility caused by retardation of follicular development in mice with oocyte-specific expression of Foxo3a

Lian Liu^1,2,*, Singareddy Rajareddy^1,*, Pradeep Reddy^1,*, Chun Du¹, Krishna Jagarlamudi¹, Yan Shen¹, David Gunnarsson³, Gunnar Selstam³, Karin Boman⁴ and Kui Liu^1,

¹ Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187, Umeå, Sweden.
² Qilu Hospital, Shandong University, Jinan, 250012, Shandong, China.
³ Department of Molecular Biology and Umeå University, SE-90187, Umeå, Sweden.
⁴ Department of Radiation Sciences, Umeå University, SE-90187, Umeå, Sweden.

View larger version (113K): [in this window] [in a new window]   Fig. 1. Expression of Foxo3a in WT mouse oocytes. (A-D) Immunostaining of Foxo3a in ovarian follicles of postnatal mice. Ovaries from 8-day-old mice were collected and embedded in paraffin, and sections were used for staining of Foxo3a and then counter-stained with Hematoxylin. The immunohistochemistry signals appear as a reddish-brown color and counter-stained background appears as a blue color. (B,C) Images at a higher magnification. (D) Negative control for immunostaining of Foxo3a, in which primary antibody against Foxo3a was not included. (E) Expression of Foxo3a in oocytes that were larger or smaller than 25 µm. Oocytes were isolated from 17- to 20-day-old female C57/BL6 mice and sorted as described in Materials and methods. Levels of ß-actin are shown as an internal control. All experiments were repeated at least three times, and representative results are shown; for each experiment, n=15-20.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Generation and characterization of Zp3-Foxo3a Tg mice. (A) DNA construct for generation of the Tg mice. The construct consists of a 6.5 kb Zp3 promoter, a 2 kb constitutively active Foxo3a cDNA (Foxo3a-TM) with a FLAG tag, and a 0.3 kb bovine growth hormone (BGH) poly-A tail. P1 and P2 indicate the positions from which the two genotyping PCR primers were derived. I-SceI, SmaI and NotI restriction sites are indicated. (B,C) The expression of constitutively active Foxo3a-FLAG in ovaries of Tg mice was confirmed by RT-PCR where a band of approximately 1.3 kb was detected (B), and by detection of the FLAG tag at the C-terminus of the exogenous Foxo3a protein in ovarian lysates of Tg mice (C). Ovaries from 15- to 17-day-old Zp3-Foxo3a Tg mice and WT littermates were homogenized and subjected to western blotting for FLAG tag and ß-actin expression. (D,E) Localization of Foxo3a antigen in ovaries of 8-day-old Tg mice and WT littermates. Immunohistochemistry confirmed Foxo3a expression in the nuclei and cytoplasm of oocytes from primary follicles (D, arrow) and secondary follicles (not shown) of Tg mice, but not in oocytes from similar follicles in WT ovaries (E, arrows). Nuclear expression of Foxo3a in oocytes of primordial follicles, which is believed to be endogenous expression, was also detected in both Tg mice and WT mice (D,E, arrowheads). The experiments were repeated three times and representative results are shown.

View larger version (78K): [in this window] [in a new window]   Fig. 3. Histological analysis of ovaries from postnatal Zp3-Foxo3a Tg mice and their WT littermates. Ovaries from 8-, 13- and 24-day-old, and 6-week-old Zp3-Foxo3a Tg mice and their WT littermates were isolated, fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Serial 8 µm sections were prepared and stained with Hematoxylin for morphological observation. (A-C) Total numbers of follicles (mean±s.e.m.; *P<0.01) of type 3b, type 4, type 5 and types 6-7 per ovary in Tg mice (Tg) and WT littermates (WT) at PD8, PD13 and PD24. For each genotype and each age, ovaries from 3-5 mice were used. (D-M) Representative images of ovaries from Zp3-Foxo3a Tg mice (Tg) and WT littermates (WT) at PD8, PD13, PD24 and 6 weeks (6-wk). At PD13, in WT mice the first wave of follicular growth had reached the type 4 and 5 stages (H, arrows), whereas the majority of follicles in Tg ovaries were still at the stages of types 3b or 4 (I, arrows). In 6-week-old WT mice, corpus luteum (CL; L, arrow) and developing follicles (L, arrowhead) were seen, whereas, in Tg mice, most of the follicles were types 3b and 4 (M, arrow), with occasionally a few follicles of type 5 (M, arrowhead).

View larger version (131K): [in this window] [in a new window]   Fig. 4. Arrested granulosa cell proliferation and reduced FSH receptor expression in Zp3-Foxo3a Tg mice. Twenty-five-day-old Tg or WT mice were used for ovarian staining of PCNA and FSH receptor, or for the BrdU incorporation assay. (A,B) Immunostaining of PCNA expression in ovarian sections from WT (A, arrow) and Tg (B, arrow) mice. Signals appear in brown using DAB as substrate. (C,D) Reduced incorporation of BrdU into granulosa cells of Tg mice (D, arrow) as compared with granulosa cells of WT littermates (C, arrow). Signals appear reddish-brown using AEC as substrate. (E,F) Reduced expression of FSH receptor mRNA in ovaries of Tg mice (F, arrows) as compared with that in ovaries of WT mice (E, arrows), as measured by in situ hybridization. Signals appear in black.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Expression of constitutively active Foxo3a in mouse oocytes leads to significantly reduced oocyte size. Zp3-Foxo3a Tg mice, or their WT littermates, were sacrificed at PD8 and PD13, and oocyte diameters measured. The mean diameter of oocytes from type 3b follicles was 24.27±3.06 µm for WT mice (WT3b), and 19.47±3.14 µm for Tg mice (Tg3b). Mean oocyte diameter in type 4 follicles was 34.36±4.21 µm for WT mice (WT4) and 24.08±6.29 µm for Tg mice (Tg4). Different lowercase letters (a, b, c) indicate significant differences (P<0.001). For each genotype and each follicle type, 100 oocytes were measured from ovarian sections.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Downregulation of Bmp15 expression and suppression of Smad pathway activation in ovaries of Zp3-Foxo3a Tg mice. Ovaries from 6-, 8- and 15- to 17-day-old Tg or WT mice were collected and used for RNA and protein extraction. (A-C) Levels of Bmp15 mRNA from Tg and WT ovaries at PD6, PD8 and PD15-17, as measured by quantitative real-time PCR. (D) Levels of Gdf9 mRNA from Tg and WT ovaries at PD15-17, as measured by quantitative real-time PCR. The relative levels of Bmp15 or Gdf9 mRNA were normalized against levels of ß-actin mRNA. The mRNA levels in WT mice were defined as 1.0. mRNA levels in Tg ovaries were calculated relative to those of WT mice and are shown as mean±s.e.m. (E-I) Western blot analysis for levels of activated Smad1/5/8 (E, p-Smad1/5/8), total Smad1 (F), ß-actin (G), activated Smad2 (H, p-Smad2), and total Smad2 (I) in WT and Tg ovaries at PD8 and PD15-17. All experiments were repeated at least three times. For each experiment with PD6 and PD8 mice, n=10-15; for experiments with PD15-17 mice, n=5-8. Representative results are shown.

View larger version (48K): [in this window] [in a new window]   Fig. 7. Reduced expression of connexin 37 and connexin 43 in Zp3-Foxo3a Tg mice. Ovaries from 8- and 15- to 17-day-old Tg mice or WT littermates were collected and used for western blot analysis. (A,D) Levels of connexin 37 in WT and Tg ovaries at PD8 and PD15-17; (B,E) levels of connexin 43 in WT and Tg ovaries at PD8 and PD15-17; (C,F) levels of ß-actin, used as a control for protein loading. Experiments were repeated at least three times. For each experiment with PD8 mice, n=10-15; for experiments with PD15-17 mice, n=5-8. Representative images are shown.

View larger version (54K): [in this window] [in a new window]   Fig. 8. p27 expression is arrested in oocyte nuclei of Zp3-Foxo3a Tg mice. Ovaries from 8- and 20-day-old WT mice and 20-day-old Tg mice were collected for immunohistochemistry for p27. (A) In 8-day-old WT mice (WT PD8), p27 was expressed in the nuclei of oocytes from primary/secondary (large arrows) and primordial follicles (small arrow), and also in the granulosa cells of these follicles (yellow arrows). (B) In the follicles of 20-day-old WT mice (WT PD20), p27 was only found to be expressed in the proliferating granulosa cells (yellow arrow), but not in oocytes of the developing follicles (black arrows). (C) In the ovaries of 20-day-old Tg mice (Tg PD 20), however, there was still expression of p27 in the nuclei of the oocytes (yellow arrows). The experiments were repeated three times and representative results are shown.

View larger version (48K): [in this window] [in a new window]   Fig. 9. Activation status of Akt, p70S6 kinase, mTOR and MAPK in oocytes of WT and Tg mice. Oocytes were isolated from 15- to 17-day-old WT or Tg mouse ovaries and lysed. Samples containing 50 µg of protein were analyzed by western blot. Levels of activated Akt (A), activated p70S6 kinase (B), activated mTOR (C), total mTOR (D), activated MAPK (E), total MAPK (F), and ß-actin (G) as an internal loading control, are shown. The experiments were repeated three times; for each experiment n=15-20 for each genotype. Representative results are shown.

View larger version (114K): [in this window] [in a new window]   Fig. 10. Defective COC expansion, anovulation and luteinization of unruptured follicles in adult Zp3-Foxo3a Tg mice. Adult female mice were checked by vaginal smears for the estrous stage, which indicates approaching ovulation. To obtain preovulatory follicles or newly formed corpus luteum (CL), ovaries were collected 2-4 hours before ovulation, or 36-48 hours after ovulation. (A) A developed follicle of preovulatory size in the Tg mice, with defective cumulus oocyte complex (COC) expansion. Note the tightly packed cumulus cells (arrow). (B) A CL with a trapped oocyte (Oo, arrow) in the Tg mice. In total, 15 adult female Tg mice were analyzed and representative results are shown. (C,D) Immunostaining for progesterone receptor (PR) in preovulatory follicles of WT (C, arrow) and Tg (D, arrow) mice. The experiments were repeated three times and representative results are shown.

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