Gene injections of vascular endothelial growth factor and growth differentiation factor-9 stimulate ovarian follicular development in immature female rats  Takashi Shimizu, Ph.D., Koji Iijima, Ph.D., Yoshinori Ogawa, M.S., Hitoshi Miyazaki, Ph.D., Hiroshi Sasada, Ph.D., Eimei Sato, Ph.D.  Fertility and Sterility  Volume 89, Issue 5, Pages 1563-1570 (May 2008) DOI: 10.1016/j.fertnstert.2007.06.043 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 VEGF gene injection inhibits the follicular atresia and increases the number of ovulatory oocytes. (A) Influence of site of injection of the VEGF gene fragments on ovulation. Immature rats were dorsally (n = 5) or abdominally (n = 5) injected with the VEGF gene (20 μg) at the age of 21 days. (B) Photograph of oocytes from animals that were dorsally injected with the VEGF gene (20 μg) at the age of 21 days. (C) Effect of dosage of VEGF gene on follicular development in immature female rats. Immature female rats were dorsally injected with the VEGF gene fragments at various dosages (n = 5 for each dosage) at the age of 21 days. (D) Relationship between ovulation and the age of rats injected with VEGF gene fragments. Immature rats were dorsally injected with the VEGF gene (20 μg) at the indicated ages (n = 5 for each day). Fertility and Sterility 2008 89, 1563-1570DOI: (10.1016/j.fertnstert.2007.06.043) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Injection of VEGF gene fragments stimulate follicular development in immature female rats. (A) Micrographs of control and VEGF gene fragment–injected ovaries of immature female rats. (B) Number of follicles and (C) percentage of atretic follicles at different developmental stages in ovaries of control rats (n = 5) and rats with dorsal VEGF gene fragments injection (n = 5) at 26 days (5 days after gene injection). Animals received direct ovarian injection of VEGF gene fragments (20 μg DNA) at 21 days after birth, and then the ovaries were removed at 26 days after birth. ∗Significantly different values (P<.05, Student's t-test). (D) Micrographs of eCG alone and VEGF + eCG–treated ovaries of immature female rats. (E) Number of follicles and (F) percentage of atretic follicles at different developmental stages in ovaries of control rats (n = 5) and rats with dorsal VEGF gene fragments injection (n = 5) at 28 days (2 days after eCG treatment) after birth. Animals received direct ovarian injection of VEGF gene fragments (20 μg DNA) at 21 days after birth, followed by administration of eCG (20 IU IP) at the age of 26 days, and then the ovaries were removed at 28 days after birth. ∗Significantly different values (P<.05, Student's t-test). Fertility and Sterility 2008 89, 1563-1570DOI: (10.1016/j.fertnstert.2007.06.043) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 VEGF gene fragments injection stimulates the vasculature around the antral follicles. Scanning electron microscopic images of the vascular formation of ovaries in immature female rats at the age of 22 days (a, b), 23 days (c, d), 24 days (e, f), 25 days (g–j) or 26 days (k–n) in control and VEGF gene injection groups. Higher-magnification views of thecal vasculature surrounding antral follicles are shown in i, j, m, and n. Bars = 300 μm in a–h, k, and l and 100 μm in i, j, m, and n. Fertility and Sterility 2008 89, 1563-1570DOI: (10.1016/j.fertnstert.2007.06.043) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Embryogenesis of ova ovulated after VEGF gene injection. (A) Photographs of two-cell and blastocyst-stage embryos after fertilization. (B) Percentages of fertilized oocytes and of the development of oocytes from the ovary with or without VEGF gene fragments injection. The oviduct was perfused with R1ECM medium, and the fertilized eggs were collected. The developmental stage of fertilized oocytes was observed in R1ECM medium in an in vitro culture system. Fertility and Sterility 2008 89, 1563-1570DOI: (10.1016/j.fertnstert.2007.06.043) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Direct ovarian injection of GDF-9 gene stimulates the early follicular development in immature female rats. Immature rats were injected with the GDF-9 gene into the ovary at ages 14 days (A), 16 days (B), or 18 days (C), and ovaries were removed at the age of 21 days. The number of follicles in the ovary with (GDF-9) or without (control) gene injection at several days (n = 4 for each day) after birth in rats were estimated. (D) Total number of follicles per ovary with or without GDF-9 gene. ∗Significantly different values (P<.05, Student's t-test). (E) The number of ovulated oocytes from immature rats with VEGF gene injection alone and with combined VEGF and GDF-9 gene fragments injection. Immature rats were injected with the GDF-9 gene fragments into the ovary at the age of 14 days or 16 days and then dorsally injected with the VEGF gene at age 21 days. Different letters represent a significant difference at P<.05. Fertility and Sterility 2008 89, 1563-1570DOI: (10.1016/j.fertnstert.2007.06.043) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions