Vascular Endothelial Growth Factor Production in Growing Pig Antral Follicles1

Abstract

Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4-5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.

Follicle selection in cattle and horses: role of intrafollicular factors

“…An increase in VEGF, an angiogenic factor, occurred in the follicles of cattle (Berisha et al 2000) and pigs (Barboni et al 2000) as diameter increased. The synthesis and secretion of VEGF increased in cultures of granulosa cells in cattle (Schams et al 2001) and monkeys (Martinez-Chequer et al 2003) when exposed to IGF-I.…”

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

“…Ovulation was induced with hCG (Corulon; Intervet) treatment (750 IU, i.m.) carried out 60 h after eCG when more than 80% of the follicles have reached a preovulatory diameter (Barboni et al 2000), and have activated steroidogenesis (Mattioli et al 2001).…”

“…Ovaries were recovered by laparotomy from anaesthetized animals by an injection of azaperone (6 ml/gilt; Stresnil; Janssen, Geerse, Belgium) and atropine sodium salt (2 mg/gilt; Industria Galenica Senese, Siena, Italy), and maintained under thiopenthal sodium (1·5 g/gilt; pentothal sodium; Gellini, Aprilia, Italy) anaesthesia (Barboni et al 2000). All protocols had prior approval from the Ethical Committee of the University of Teramo.…”

“…VEGF has been shown to be a critical molecule involved in the regulation of follicle development in different species (Barboni et al 2000, Mattioli et al 2001, Berardinelli et al 2002, Hunter et al 2004. Recent studies have shown that it exerts a crucial role during the transition from preovulatory follicles to ovulation , Zimmermann et al 2003 but few data describe the production of VEGF during the passage from pre-to periovulatory follicles and what role it exerts.…”

Candidate inhibitors of porcine complement

“…We used porcine cytokine kits commercially available, but the numbers of such kits are limited compared to rodent and human kits. We used some human kits that cross-react with porcine epitopes, for instance the TCC ELISA (136), TAT (138), and VEGF (173). We also used kits, like HMGB-1, that had specificity for several species including pig and human.…”