Activation of dormant ovarian follicles to generate mature eggs

Abstract

Although multiple follicles are present in mammalian ovaries, most of them remain dormant for years or decades. During reproductive life, some follicles are activated for development. Genetically modified mouse models with oocyte-specific deletion of genes in the PTEN-PI3K-Akt-Foxo3 pathway exhibited premature activation of all dormant follicles. Using an inhibitor of the Phosphatase with TENsin homology deleted in chromosome 10 (PTEN) phosphatase and a PI3K activating peptide, we found that short-term treatment of neonatal mouse ovaries increased nuclear exclusion of Foxo3 in primordial oocytes. After transplantation under kidney capsules of ovariectomized hosts, treated follicles developed to the preovulatory stage with mature eggs displaying normal epigenetic changes of imprinted genes. After in vitro fertilization and embryo transfer, healthy progeny with proven fertility were delivered. Human ovarian cortical fragments from cancer patients were also treated with the PTEN inhibitor. After xeno-transplantation to immune-deficient mice for 6 months, primordial follicles developed to the preovulatory stage with oocytes capable of undergoing nuclear maturation. Major differences between male and female mammals are unlimited number of sperm and paucity of mature oocytes. Thus, short-term in vitro activation of dormant ovarian follicles after stimulation of the PI3K-Akt pathway allows the generation of a large supply of mature female germ cells for future treatment of infertile women with a diminishing ovarian reserve and for cancer patients with cryo-preserved ovaries. Generation of a large number of human oocytes also facilitates future derivation of embryonic stem cells for regenerative medicine.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Activation of the PI3K-Akt-Foxo3 pathway in oocytes and increased follicle development after treatment with bpV(pic) and 740Y-P. (A) Nuclear exclusion of Foxo3 in oocytes of primordial follicles at 6 h after treatment with bpV(pic) and 740Y-P (Insets, higher magnification). (Scale bar: 100 μm.) (B) Increases in the fraction of oocytes showing nuclear export of Foxo3. (C) AMH staining in granulosa cells of primary and secondary follicles at 2 days after in vitro culture. (D) BrdU incorporation into granulosa cells of activated follicles at 5 days after transplantation (Upper, hematoxylin and eosin staining; Lower, BrdU staining). (Scale bars: 100 μm.)

Fig. 2.

Activation of dormant primordial follicles after in vitro treatment with bpV(pic) and 740Y-P, followed by transplantation into the kidney capsule of ovariectomized hosts. (A) Ovaries at 14 days after transplantation. Isolated ovaries for control vs. bpV(pic), control vs. 740Y-P, and control vs. bpV(pic) plus 740Y-P groups. (Scale bars: 1 mm.) (B) Ovarian weight increases as fold changes relative to paired nontreated controls. Numbers of paired ovaries used for analyses are shown in parentheses. (C) Ovarian histology showing follicle development to the large antral stage (arrow) at 14 days after treatment with bpV(pic) and 740Y-P followed by transplantation. (Scale bars: 200 μm.) (D) Distribution of follicles in ovaries without and with exposure to bpV(pic) and 740Y-P. Follicle dynamics of day 3 ovaries before transplantation is included for comparison. s, secondary follicle. (E) Ovarian morphology at 14 days after transplantation of grafts treated with bpV(pic) and 740Y-P with or without inhibitors to Akt (SH5) or PI3K (Wortmannin). The symbol -/ indicates groups without the inhibitors. (Scale bar: 1 mm.)

Fig. 3.

Retrieval of mature mouse oocytes for epigenetic analyses, in vitro fertilization, and early embryonic development. (A) Histology of ovaries with or without hCG treatment at 18 days after transplantation after pretreatment with bpV(pic) and 740Y-P. (A Left) An ovary before hCG treatment showing preovulatory follicles with an oocyte at the GV stage. (A Right) An ovary after hCG treatment showing preovulatory follicles with an oocyte exhibiting germinal vesicle breakdown. (Scale bars: 100 μm.) (B) Methylation status of the differentially methylated regions (DMRs) of two maternal imprinted genes, Igf2r and Lit1, and one paternal imprinted gene H19 in mature oocytes retrieved from transplanted ovaries after in vitro activation (T) and those from superovulated control ovaries (C). Each row represents a unique methylation profile within the pool of clones sequenced with the frequency of that methylation state indicated on the right side of each row. Each circle within the row represents a single CpG site (open circles, nonmethylated cytosines; filled circles, methylated cytosines). n = 15–20 sequenced clones. (C) Early embryonic development of retrieved mature oocytes after in vitro fertilization. Representative figures for embryos reaching two-cell (24 h), four-cell (48 h), morula (72 h), and blastocyst (96 h) stages. (D) Efficiency of early embryonic development. Percentage of mature oocytes retrieved from transplanted grafts capable of developing into two-cell embryos and blastocysts as compared with mature oocytes obtained from ovaries from superovulated controls without transplantation.

Fig. 4.

Activation of human primordial follicles from patients with benign ovarian tumor. (A Left) Increased nuclear export of Foxo3 in primordial follicles after 1 h of treatment with 100 μM bpV(pic). Arrow, positive staining in diminished cytoplasmic space due to fixation-induced shrinkage. (Scale bars: 50 μm.) (A Right) Percentage of primordial oocytes showing Foxo3 nuclear export in control and bpV(pic)-treated groups. (B) Ovarian morphology at 6 months after xeno-transplantation into SCID mice (Upper, kidneys with ovarian grafts; Lower, in situ kidney picture of one host). (C) Distribution of follicles at different stages in grafts with or without bpV(pic) treatment. Follicle distribution in cortical cubes before xeno-grafting is provided for comparison. s, secondary follicles. (D) Representative sections showing the development of two large antral follicles in bpV(pic)-treated group with mature oocytes exhibiting germinal vesicle breakdown after hCG treatment (insets).