Ovary

Induction of Follicular Development by Direct Single Injection of Vascular Endothelial Growth Factor Gene Fragments into the Ovary of Miniature Gilts

Abstract

Perifollicular angiogenesis is closely associated with ovarian follicular development. To investigate whether additional induction of perifollicular angiogenesis would support subsequent follicular development, we directly injected vascular endothelial growth factor (VEGF) gene fragments into the ovaries of miniature gilts, followed by gonadotrophin treatment to stimulate follicle growth. In addition, to confirm extraexpression of the VEGF gene after injection, we assessed the expression of two isoforms of VEGF (VEGF 120 and VEGF164) in granulosa cells, and expression of fms-like tyrosine kinase (Flt-1), fetal liver kinase (Flk-1) and density of capillary networks in theca cells. Direct injection of VEGF gene fragments into the ovaries was performed 7 days before equine chorionic gonadotropin (eCG) treatment. The ovaries in miniature gilts were removed 72 h after eCG treatment for histological examination. Granulosa cells and thecal tissues in the antral follicles (diameter, > 4mm) were collected to detect the mRNA expression of VEGF isoforms in the granulosa cells, and Flt-1 and Flk-1 in the thecal tissues by semiquantitative reverse transcription-polymerase chain reaction. VEGF levels were measured the in follicular fluid by enzyme immunoassay. Injection of VEGF gene fragment increased the level of mRNA expression of VEGF 120 and 164 isoforms in the granulosa cells and VEGF protein contents in the follicular fluid. The number of preovulatory follicles and the capillary density in the theca interna increased significantly in the ovaries injected with VEGF gene fragment compared with those treated with eCG alone. The Flt-1, but not Flk-1, mRNA expression show a tendency toward increasing in the thecal tissues of antral follicles in the ovaries injected with VEGF gene fragment. These results demonstrate that Flt-1 may be predominantly involved in the regulation of the capillary network in the theca interna during follicular development. Our data suggested that the regulation of perifollicular angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. Our findings may offer an innovative technique for enhanced induction of follicular development in the ovary through gene and hormonal treatment, which may lead to prevention of infertility caused by ovarian dysfunction.