Jin-Yi Jiang

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.

Changes of Messenger RNA Expression of Angiogenic Factors and Related Receptors During Follicular Development in Gilts

Abstract The interaction between angiogenic factors and related receptors is closely associated with follicular angiogenesis. The present study was performed to determine the relationships between the capillary network and mRNA expression of several angiogenic factors and related receptors during porcine follicular development. Ovaries in gilts were collected 72 h after eCG (1250 IU) treatment for histological observation. Granulosa cells and thecal tissues in small (diameter, <4 mm), medium (diameter, 4–5 mm), or large (diameter, >5 mm) individual follicles were collected for detection of mRNA expression of vascular endothelial growth factor (VEGF) 120, VEGF 164, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) in granulosa cells and fms-like tyrosine kinase (Flt-1), fetal liver kinase (Flk-1) or the murine homologue of kinase domain region (KDR), bFGF receptor (bFGF-R), and EGF receptor (EGF-R) in thecal tissue by semiquantitative reverse transcription-polymerase chain reaction. The eCG treatment resulted in the emergence of healthy preovulatory follicles (diameter, >6.0 mm) that possessed more capillaries in the thecal cell layer and a significant increase in the percentage of atretic follicles of 1.0–2.9 mm in diameter. The number of capillaries in the thecal cell layer increased significantly in healthy follicles larger than 3 mm in diameter in the eCG group compared with those in controls. The expression of VEGF 120, VEGF 164, and bFGF mRNAs increased in granulosa cells of medium and large follicles from ovaries of prepubertal gilts after eCG treatment. The Flt-1, Flk-1/KDR, and bFGF-R mRNA expression increased in theca cells of medium and large follicles after eCG treatment. The expression of EGF mRNA increased in granulosa cells of small, medium, and large follicles from ovaries after eCG treatment, but the mRNA expression of EGF-R in thecal tissue did not change. These data indicate that preovulatory follicles possessed a larger capillary network and expressed more mRNAs of angiogenic factors in granulosa cells and related receptors in thecal tissue. We concluded that VEGF 120, VEGF 164, bFGF, and EGF may be greatly involved in the angiogenic process of follicular development in prepubertal gilts with eCG treatment.

Characteristics of Infertility and the Improvement of Fertility by Thyroxine Treatment in Adult Male Hypothyroid rdw Rats

Abstract We previously reported that rdw rats were infertile in both sexes. The present study was conducted to determine whether hypothyroidism in adult male rdw rats induced infertility by impairing sexual behavior or testicular function, whether the infertility could be reversed by thyroxine (T4) treatment, and whether the mutant could be produced by infertile rdw rats via in vitro fertilization. The sexual behavior was analyzed by pairing with normal female rats. The fertility of epididymal sperm was determined by in vitro fertilization. The results indicated that the infertility resulted from both defective sexual behavior and testicular function. No untreated rdw rats mated. The weights of epididymides were significantly low, whereas those of testes were not different from those of untreated normal rats. Epididymal sperm with cytoplasmic droplets were observed at a significantly high frequency. No fertilization was detected either in vivo or in vitro. Thyroxine treatment markedly increased serum T4 levels and the weights of both epididymides and testes. Partial reversion of the impaired sexual behavior was observed, and the percentage of epididymal sperm with cytoplasmic droplets was markedly decreased after T4 treatment. Fertility of epididymal sperm was completely reversed when determined both in vivo and in vitro, and homozygous embryos developed to term after transfer without loss of viability.

Thyroxine treatment stimulated ovarian follicular angiogenesis in immature hypothyroid rats

The development of mature ovarian follicles is greatly dependent on healthy thecal angiogenesis. Recent experimental evidence showed that thyroxine (T4) treatment promoted ovarian follicle development in immature hypothyroid (rdw) rats. However, an involvement of thyroid hormone in ovarian follicular angiogenesis has not yet been demonstrated. By morphological and molecular approaches, the present studies demonstrated that antral follicles in untreated, T4- or equine chorionic gonadotropin (eCG)-treated rdw rats were mainly small and/or atretic, and presented a poorly developed thecal microvasculature with ultrastructural evidence of diffuse quiescent or degenerative thin capillaries. However, T4 together with eCG increased the number of large antral and mature follicles with numerous activated capillaries and ultra-structural evidence of rich and diffuse angiogenesis in the theca layer. While T4 alone significantly increased mRNA expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha), it decreased that of fetal liver kinase compared with those in the untreated group. Combined treatment of T4 and eCG markedly increased mRNA abundance of not only VEGF and TNFalpha, but also basic fibroblast growth factor. These data suggest that T4 may promote ovarian follicular angiogenesis in rdw rats by up-regulating mRNA expression of major angiogenic factors.