Expression of vascular endothelial growth factor (VEGF) receptor in goat ovaries and improvement of in vitro caprine preantral follicle survival and growth with VEGF.
ABSTRACT The aim of the present study was to evaluate the effect of vascular endothelial growth factor (VEGF) on the survival and growth of goat preantral follicles after in vitro culture and to verify the expression of VEGF receptor (VEGFR)-2 in goat ovaries. Ovarian fragments were cultured for 1 or 7 days in minimal essential medium (MEM) with different concentrations of VEGF (1, 10, 50, 100 or 200 ng mL(-1)). Non-cultured (fresh control) and cultured tissues were processed for histological and ultrastructural studies. The results showed that 200 ng mL(-1) VEGF resulted in a similar percentage of normal preantral follicles after 1 and 7 days of culture compared with control. Compared with basic culture medium alone, an increase in follicular and oocyte diameters was observed in the presence of 10 ng mL(-1) VEGF after 7 days culture. Ultrastructural analysis confirmed follicular integrity after 7 days culture in the presence of 200 ng mL(-1) VEGF. Immunohistochemical studies demonstrated the expression of VEGFR-2 in oocytes and granulosa cells of all follicular stages, except in granulosa cells of primordial follicles. In conclusion, the present study has shown that VEGF maintains follicular ultrastructural integrity and promotes follicular growth. In addition, VEGFR-2 is expressed in oocytes of caprine ovarian follicles at all developmental stages and in granulosa cells of developing follicles.
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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.Biology of Reproduction 10/2003; 69(4):1388-93. · 4.03 Impact Factor
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ABSTRACT: Ovarian follicular development in mammals is the complex process including endocrine, paracrine and autocrine. There is the development of four basic stages of ovarian follicles, i.e. the primordial, primary, secondary and tertiary or Graafian follicles. There are few blood vessels in the cortical area where primordial and primary follicles are assembled. The development of these follicles is stimulated by oocytes derived factor including growth differentiation factor 9 (GDF-9) or bone morphogenetic protein 15 (BMP-15). Porcine GDF-9 complementary DNA (cDNA) cloned, and then injected its gene into the ovary in gilts. The injection of porcine GDF-9 gene resulted in an increase in the number of primary, secondary and tertiary follicles, concomitant with a decrease in the number of primordial follicles, indicating that exogenous GDF-9 can promote early folliculogenesis in the porcine ovary. On the other hand, the development of antral follicles is associated with increased density of blood vessels within the theca cell layers surrounding the follicles. A recent study reported that vascular endothelial growth factor (VEGF) play an important role in the process of thecal angiogenesis during follicular development. To investigate whether additional induction of thecal angiogenesis would support subsequent follicular development, miniature gilts were directly injected VEGF gene into the ovary. Injection of VEGF gene increased the levels of mRNA expression of VEGF 120 and VEGF 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 compared with those treated with eCG alone, indicating that the regulation of thecal angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. This technique may be 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.Journal of Reproduction and Development 03/2006; 52(1):23-32. · 1.76 Impact Factor
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ABSTRACT: The authors evaluated the relationship between vascular endothelial growth factor (VEGF) production, blood vessel extension, and steroidogenesis in small (<4 mm), medium (4-5 mm), and large (>5 mm) follicles isolated from gilts treated with eCG. VEGF and estradiol levels were measured in follicular fluid by an enzyme immunoassay and radioimmunoassay, respectively, and then each follicle wall was used to evaluate VEGF mRNA content and for the immunohistochemical analysis of blood vessels. VEGF production was low in small follicles (<3 ng/ml), high in large follicles (>10 ng/ml), and markedly differentiated in medium follicles; 44% exhibited values up to 15 ng/ml, whereas the levels never exceeded 3 ng/ml in the remaining aliquot. Medium follicles were then used as a model to investigate angiogenesis. Reverse transcription-polymerase chain reaction for VEGF mRNA demonstrated that granulosa cells represent the main component involved in the production of VEGF. The follicle wall, which presents two distinct concentric vessel networks, showed a vascular area (positive stained area/percent of field area) that was significantly wider in high VEGF follicles than in low VEGF follicles (2.54% +/- 0.58% vs. 1.29% +/- 0.58%, respectively). Medium follicles with high VEGF levels and extensive vascularization accumulated high estradiol levels (150-300 ng/ml), whereas follicles with low VEGF levels had basal estradiol levels that never exceeded 30 ng/ml. Early atretic medium-size follicles had undetectable levels of VEGF and estradiol paralleled by a marked reduction in blood vessel. The data presented propose an improved model for follicle dynamics in which the production of VEGF, stimulated by gonadotropin, creates the vascular conditions required for follicle growth and activity.Biology of Reproduction 10/2001; 65(4):1014-9. · 4.03 Impact Factor