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

Abstract

Vascular endothelial growth factor (VEGF) expression pattern and blood vessel remodelling were evaluated during the transition from the preovulatory follicle to the corpus luteum (CL). To this end, prepubertal gilts were treated with equine chorionic gonadotrophin (eCG) to collect preovulatory follicles (60 h after eCG) and with human chorionic gonadotrophin (hCG) to obtain periovulatory follicles 18 h and 36 h later. The VEGF mRNA content was analysed by in situ hybridization, while protein localization in follicular fluid (FF) and in granulosa and theca compartments was evaluated by ELISA, immunohistochemistry or western blot. Blood vessel architecture and vascular area (VA) were investigated using immunohistochemistry for von Willenbrand Factor, a specific endothelial marker. Vascular remodelling was finally tested using Ki-67 immunocytochemistry as a proliferation marker, or -smooth muscle actin ( -SMA) as a specific mural cell marker. eCG-treated follicles showed high VEGF levels and two concentric blood vessel networks composed of proliferating endothelial cells without any association with mural components. hCG injection inhibited VEGF synthesis in the granulosa compartment and, as a consequence, the protein fell within the FF. In parallel, endothelial cell proliferation stopped and the VA decreased. Close to ovulation, VEGF production restarted in both follicular compartments and VEGF mRNA content significantly increased in the theca layer. Changes in follicular VEGF secretion were observed; the protein disappeared from FF and was observed in the extracellular matrix. An active angiogenesis characterized the follicle; endothelial cell proliferation was associated with a recruitment of -SMA-positive mural cells. The data presented in this work showed that, in the phases preceding ovulation, a complete vascular remodelling occurs, characterized by both an evident neovascularization and the appearance of blood vessels presenting smooth musculature which could be involved in CL formation after ovulation.

Vascular supply as a discriminating factor for pig preantral follicle selection

“…Similar to other molecules, VEGFA could be included among the signals involved in the bidirectional control of oocyte-follicle function (Diaz et al 2007, Hutt & Albertini 2007, by guaranteeing that follicle development can be synchronized with the growth of the metabolically active germinal cells. Even if it has been demonstrated that there are also post-transcriptional and post-translational mechanisms involved in VEGFA expression in the ovary and other tissues (Hazzard et al 1999, Neufeld et al 1999, Martelli et al 2006, in this research all preantral follicles showed a relationship between VEGFA mRNA and protein production. In fact, the follicles and the oocytes that expressed the transcript were also positive Vasculature during preantral follicle development 51 to the related protein.…”

“…In addition, morphological adjustments of the vascular supply may be crucial to regulate follicle growth and differentiation, although such a cause effect has yet to be established (Redmer & Reynolds 1996, Barboni et al 2000, Berardinelli et al 2002. In particular, during the final antral stages, follicle growth is coupled with an increased blood vessel area , close to ovulation, changes its organization into a mature vascular network (Martelli et al 2006). In fact, until the periovulatory phase, the vascular network is represented by capillaries that, a few hours before ovulation, become covered by pericytes, and new large blood vessels appear in the follicular wall (Zimmermann et al 2003, Martelli et al 2006.…”

“…In particular, during the final antral stages, follicle growth is coupled with an increased blood vessel area , close to ovulation, changes its organization into a mature vascular network (Martelli et al 2006). In fact, until the periovulatory phase, the vascular network is represented by capillaries that, a few hours before ovulation, become covered by pericytes, and new large blood vessels appear in the follicular wall (Zimmermann et al 2003, Martelli et al 2006. The acquisition of a specific vascular supply seems to represent a limiting step in the selection of a dominant follicle destined to ovulation (Mattioli et al 2001, Stouffer et al 2001, while its disappearance is correlated with the process of early follicular atresia (Moor & Seamark 1986).…”

“…In particular, the vascular endothelial growth factor A (VEGFA) seems to play a major role in ovarian angiogenesis as demonstrated recently by several pieces of evidence (Wulff et al 2001, Zimmermann et al 2003, Kaczmarek et al 2005, Shimizu & Sato 2005, Abramovich et al 2006, Shimizu 2006. Even if relevant progress has been made in understanding the mechanisms involved in the angiogenic events during the dominant final phase of tertiary follicle development (Fraser 2006, Martelli et al 2006, the role of blood vessel remodelling in early follicular stages is unclear. In regard to preantral follicles, different reports indicate that these are able to synthesize VEGFA and are sensitive to its stimulatory effect (Danforth et al 2003).…”

A novel physiological culture system that mimics luteal angiogenesis

“…After 2, 5 and 9 days, the medium was collected and the cells fixed in acetone:methanol (1:1) at room temperature for 5 min. The endothelial cells were then immunostained using VWF as an established endothelial cell marker in order to assess the formation of tubule-like structures over time (Al-zi'abi et al 2003, Martelli et al 2006.…”