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 gonadotroph 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 VEGF 164) in granulosa cells and expression of fms-like tyrosine kinase (Flt-1), expression of 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 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, >4 mm) were collected to detect the mRNA expression of VEGF isoforms in the granulosa cells and of Flt-1 and Flk-1 in the thecal tissues by semiquantitative reverse transcription-polymerase chain reaction. The VEGF levels were measured in the follicular fluid by enzyme immunoassay. Injection of VEGF gene fragments 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 fragments compared with those treated with eCG alone. The Flt-1, but not the Flk-1, mRNA expression show a tendency toward increasing in the thecal tissues of antral follicles in the ovaries injected with VEGF gene fragments. 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 suggest 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.

Spatio-temporal analysis of vascular endothelial growth factor expression and blood vessel remodelling in pig ovarian follicles during the periovulatory period

“…A 96-well plate reader (Biomek 1000; Beckman Instruments, Fullerton, CA, USA) set to read at an emission of 450 nm was used to quantify the results. Since the VEGF kit was manufactured to detect human VEGF, a test of parallelism was carried out to investigate whether the assay could also be reliably used to measure pig soluble VEGF isoforms, such as VEGF 164 , which is the prevalent isoform demonstrated in porcine ovaries (Barboni et al 2000, Boonyaprakob et al 2003, Shimizu et al 2003. To this end, the standard curve generated with a highly purified Sf-21-expressed recombinant human VEGF 165 (R&D Systems;0,62·5,125,250,500 and 1000 pg/ml) was compared with a curve that was obtained from a sample of pig FF diluted with the appropriate calibrator solution provided with the kit, in order to produce samples with values that fell within the dynamic range of the assay.…”

“…Human recombinant VEGF 165 (R&D Systems) as well as mouse recombinant VEGF 164 (R&D Systems) were used to identify the band of the angiogenic factor and to co-localize the porcine VEGF that has a similar molecular weight (Shimizu et al 2003).…”

Hormonal regulation and differential expression of neuropilin (NRP)-1 and NRP-2 genes in bovine granulosa cells

“…The impact of vascular endothelial growth factor (VEGF) as a central regulator of angiogenesis in the ovary was demonstrated in studies using the techniques of gene or protein injection (Hazzard et al 2002, Shimizu et al 2003, Xu et al 2005. VEGF induces signal transduction in the cells via VEGF receptors that belong to the tyrosine-kinase receptor family.…”

Development of a technique for efficient gene transfer to antral follicular cells in the mouse ovary

“…In this context, an ovary appears to be a good target for gene delivery to test the function of a GOI, as previously shown by others [Matsumoto et al 1999;Shimizu et al 2003;Shimizu et al 2004;Shimizu et al 2008;Ghadami et al 2010].…”

“…Several laboratories have reported the use of this approach. Shimizu et al [2003;Shimizu et al 2004;Shimizu et al2008] demonstrated that overexpression of growth differentiation factor-9 (GDF-9), a molecule expressed in an oocyte-specific manner, and vascular endothelial growth factor (VEGF), a molecule enhancing vasculogenesis, stimulated follicular development in rats and pigs. They employed direct injection of liposome-encapsulated plasmid DNA into the medulla of an ovary.…”