Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment

Abstract

Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.

Keywords: CCN2; PTEN; YAP; aging; ovary.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Ovarian fragmentation and grafting promoted follicle growth in mice. Paired ovaries from juvenile mice were grafted into kidneys of adult ovariectomized mice (intact, IN; pieces, PI). Hosts were injected with FSH daily for 5 d before graft retrieval. (A) Morphology of paired ovarian grafts with or without fragmentation into three pieces. (A, Upper) Grafts inside kidney capsules. (A, Lower) Isolated paired grafts. (B) Weights of paired ovaries following fragmentation into 2–4 pieces from day 10 (D10) mice and incubated for 1–24 h before grafting. Ovarian weights before grafting (D10) and at 5 d after grafting with (D15 FSH) or without FSH treatment (D15) served as controls; n = 8–22. (C) Follicle dynamics before and after grafting of intact and fragmented (three pieces) ovaries from day 10 mice. (C, Left) Total follicle numbers. (C, Right) Follicle dynamics; n = 5. Pmd, primordial; Prm, primary; Sec, secondary; PO, preovulatory. (D) Weights of paired ovaries from mice at different ages following fragmentation into 3–4 pieces and grafting. Mean ± SEM; *P < 0.05; n = 8–22.

Fig. 2.

Fragmentation of murine ovaries increased actin polymerization, disrupted Hippo signaling, and increased CCN growth factors and apoptosis inhibitors. (A) Ovarian fragmentation increased F-actin levels. Paired ovaries from day 10 mice were cut into three pieces or kept intact before immunoblotting analyses of F- and G-actin levels (Upper). (A, Lower) F- to G-actin ratios; n = 6–11. (B) Ovarian fragmentation decreased pYAP levels and pYAP to total YAP ratios. Paired ovaries with or without cutting were incubated for 1 h, followed by immunoblotting. (B, Left) Representative immunoblots. (B, Right) Ratios of different antigens; n = 8 pairs. (C) Ovarian fragmentation increased expression of CCN growth factors and BIRC apoptosis inhibitors. Paired ovaries with or without cutting were incubated for 1 h with subsequent grafting before analyses of transcript levels normalized by GAPDH. Intact ovaries, solid lines; pooled three pieces, dashed lines; n = 10–15. (D) Ovarian fragmentation increased CCN2 proteins. Paired ovaries with or without cutting were incubated for 3–5 h before immunoblotting. (D, Upper) Representative blots. (D, Lower) Quantitative analyses; n = 3–5. (E) Treatment with CCN2, 3, 5, and 6 increased ovarian explant weights. Explants from day 10 mice were cultured with different CCN growth factors for 4 d before weighing; n = 5–6. Mean ± SEM; *P < 0.05. IN, intact; PI, pieces.

Fig. 3.

Additive effects of Hippo signaling disruption and Akt stimulation promoted secondary follicle growth. (A) Secondary follicles were isolated from juvenile mice and cultured with Akt stimulators; n = 30. (B) Additive increases in graft weights following ovarian fragmentation and/or Akt stimulation. Paired ovaries from juvenile mice were fragmented and incubated with or without Akt stimulators for 2 d followed by allo-transplantation for 5 d before graft weight determination; n = 8–10. (C) Follicle dynamics before and after grafting of intact and fragmented murine ovaries with or without treatment with Akt stimulators. (C, Left) Total follicle numbers. (C, Right) Follicle dynamics; n = 4. Same letter symbols indicate significant differences (P < 0.05). (D–F) Vitrified human cortical strips were thawed and fragmented into cubes before treatment with Akt stimulators for 2 d followed by grafting into immune-deficient mice for 4 wk. (D) Cortical strips before grafting. Arrow, a secondary follicle; arrowheads, primordial/primary follicles. (Scale bar, 100 μm.) (E) A kidney graft in situ (Left) and after isolation (Right), showing an antral follicle. (F) Histology of two large antral follicles with the side view of one showing an oocyte at the germinal vesicle stage (arrow). (Scale bar, 1 mm.) Mean ± SEM; *P < 0.05.

Fig. 4.

Ovarian fragmentation/Akt stimulation followed by autografting promoted follicle growth in POI patients to generate mature oocytes for IVF–embryo transfer, pregnancy, and delivery. (A) Under laparoscopic surgery, ovaries were removed and cut into strips. Ovarian strips from POI patients were vitrified. After thawing, strips were fragmented into 1–2 mm2 cubes, before treatment with Akt stimulators. Two days later, cubes were autografted under laparoscopic surgery beneath serosa of Fallopian tubes. Follicle growth was monitored via transvaginal ultrasound and serum estrogen levels. After detection of antral follicles, patients were treated with FSH followed by hCG when preovulatory follicles were found. Mature oocytes were then retrieved and fertilized with the husband’s sperm in vitro before cyropreservation of four-cell stage embryos. Patients then received hormonal treatments to prepare the endometrium for implantation followed by transferring of thawed embryos. (B) Transplantation of ovarian cubes beneath the serosa of Fallopian tubes. Arrow, fallopian tube; arrowheads, cubes. (C) Multiple cubes were put beneath serosa. (D) Serosa after grafting. Ovarian cubes are visible beneath serosa (arrow). (E) Detection of preovulatory follicles in grafts for oocyte retrieval. Following ultrasound monitoring, follicle growth was found in eight patients. After follicles reached the antral stage (>5 mm in diameter, right upward arrows), patients were treated with FSH followed by hCG for egg retrieval (upward arrows). Double circles represent preovulatory follicles, whereas single circles represent retrieved oocytes. Dashed lines depict ongoing observation.