Oocyte-specific deletion of Pten in mice reveals a stage-specific function of PTEN/PI3K signaling in oocytes in controlling follicular activation

Abstract

Immature ovarian primordial follicles are essential for maintenance of the reproductive lifespan of female mammals. Recently, it was found that overactivation of the phosphatidylinositol 3-kinase (PI3K) signaling in oocytes of primordial follicles by an oocyte-specific deletion of Pten (phosphatase and tensin homolog deleted on chromosome ten), the gene encoding PI3K negative regulator PTEN, results in premature activation of the entire pool of primordial follicles, indicating that activation of the PI3K pathway in oocytes is important for control of follicular activation. To investigate whether PI3K signaling in oocytes of primary and further developed follicles also plays a role at later stages in follicular development and ovulation, we conditionally deleted the Pten gene from oocytes of primary and further developed follicles by using transgenic mice expressing zona pellucida 3 (Zp3) promoter-mediated Cre recombinase. Our results show that Pten was efficiently deleted from oocytes of primary and further developed follicles, as indicated by the elevated phosphorylation of the major PI3K downstream component Akt. However, follicular development was not altered and oocyte maturation was also normal, which led to normal fertility with unaltered litter size in the mutant mice. Our data indicate that properly controlled PTEN/PI3K-Akt signaling in oocytes is essential for control of the development of primordial follicles whereas overactivation of PI3K signaling in oocytes does not appear to affect the development of growing follicles. This suggests that there is a stage-specific function of PTEN/PI3K signaling in mouse oocytes that controls follicular activation.

Figure 1. Generation of mutant mice with oocyte-specific deletion of Pten.

A schematic representation of deletion of Pten exon 5 in oocytes of primary and further developed follicles by using the Zp3 promoter-mediated Cre transgenic mice. The developmental stages at which the Gdf-9 promoter and the Zp3 promoter become active are indicated above the illustration of follicles in the figure.

Figure 2. Characterization of Pten deletion by western blot and PCR.

(A) Oocytes were prepared and lysed for western blot as described in Materials and Methods . PTEN expression was found to be completely absent in Pten^loxP/loxP; Zp3-Cre+ oocytes. For each lane, 150 oocytes were used. β-actin was used as internal control. (B) PCR analysis showing the complete deletion of Pten exon 5 (Pten Δ5) in one allele of the genomic DNA of pups from Pten^loxP/loxP; Zp3-Cre+ females.

Figure 3. Enhanced Akt signaling in oocytes of Pten^loxP/loxP; Zp3-Cre+ mice.

Oocytes were prepared from ovaries of 3–4 week old mice that were treated with PMSG, as described in Materials and Methods . Signaling studies in Pten^loxP/loxP; Zp3-Cre+ oocytes showed elevated levels of p-Akt (Ser473), p-Akt (Thr308), and p-Tsc2 (Thr1462) as compared to Pten^loxP/loxP oocytes. Levels of total Akt, Tsc2, and β-actin were used as internal controls. 100–150 oocytes were used for each lane. All experiments were repeated at least three times and representative results are shown.

Figure 4. Normal follicular development in Pten^loxP/loxP; Zp3-Cre+ mice.

Morphological analysis of ovaries from 13- and 23-day-old, and 16-week-old Pten^loxP/loxP; Zp3-Cre+ mice, Pten^loxP/loxP; GCre+ mice, and control Pten^loxP/loxP mice. Ovaries were embedded in paraffin and sections of 8-µm thickness were prepared and stained with hematoxylin. Note the overactivation of primordial follicles in Pten^loxP/loxP; GCre+ ovaries (C, F, and I, arrows) and the normal follicular development and CL in Pten^loxP/loxP; Zp3-Cre+ ovaries (B, E, H and K), which is comparable to the control Pten^loxP/loxP ovaries (A, D, G, and J). CL, corpora lutea.

Figure 5. Normal GVBD, ovulation, fertilization, and fertility in Pten^loxP/loxP; Zp3-Cre+ mice.

(A) Oocytes from 3–4-week-old PMSG-primed Pten^loxP/loxP; Zp3-Cre+ mice or control Pten^loxP/loxP mice were collected in M2 medium. They were cultured further in M16 medium for GVBD analysis. GVBD rate was scored after a culture period of 6 h. Normal GVBD rates were observed in Pten mutant oocytes as compared to control oocytes. The numbers of oocytes used (n) are shown. (B) Two-cell embryos were collected at E1.5 and were cultured in KSOM medium supplemented with amino acids. Numbers of 2-cell embryos per female (mean±SEM) mouse were counted. The numbers of mice used (n) and the results of statistical analysis are shown. No significant difference (NS) was seen between Pten^loxP/loxP; Zp3-Cre+ mice and control mice. (C) Normal litter size of Pten^loxP/loxP; Zp3-Cre+ females as compared to Pten^loxP/loxP control mice (mean±SEM). The numbers of litters (n) from 10 mice of each genotype and results of statistical analysis are shown. NS, not statistically significant.