Vascular Endothelial Growth Factor (VEGF) plays an important in ovarian function from follicular development to ovulation. It is an angiogeneic factor that stimulates vessel formation and increases vascular permeability to supply nutrition and oxygen to developing follicle. During luteal phase, VEGF expression is required for corpus luteum (CL) development and function. Inhibition of VEGF expression results in abnormal angiogenesis and hence affects ovarian structure. Therefore, understanding the action of VEGF along with action of hormones influencing its expression is necessary to assimilate ovarian pathology.
In cattle, follicular development is characterized by recruitment of cohort of follicles, selection of dominant follicle and its growth and maturation till it reaches preovulatory stage and atresia of the other subordinate follicles (Berisha and Schams, 2005). Following ovulation, certain morphological and biochemical changes occur in theca and granulosa cells leading to formation of corpus luteum (CL). This complex mechanism is controlled by the pituitary hormones along with locally produced steroid hormones, peptides and growth factors (Fortune, 1994). Among the various growth factor, vascular endothelial growth factor (VEGF), a potent and specific angiogenic factor, is found essential for the follicular growth and development and CL formation (Zimmermann et al., 2003; Berisha and Schams, 2005). Several intraovarian factors play crucial role in modulation of VEGF expression. Cytokines such as Transforming Growth Factor (TGF) and Bone Morphogenic Proteins (BMP) induce expression of VEGF in endothelial cells (Kuo et al., 2011) as well as in granulosa cells (Akiyama et al., 2014) thereby stimulate vascularization in growing follicle. Similarly, upregulation of VEGF expression by polypeptides Nerve Growth Factor (NGF) (Julio-Pieper et al., 2006) and Insulin Growth Factor (Schams et al., 2001) has been reported. The coordination between intraovarian factors and VEGF thus regulate follicle growth and development. In this review, the function of VEGF in ovarian functioning is summarized.
Vascular endothelial growth factor, originally termed as vascular permeability factor (VPF) was described as a 34-42 kDa protein that increases vascular permeability in the skin (Byrne et al., 2005). The VEGF family consists of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta derived growth factor. Among these glycoproteins, VEGF-A is mainly involved in angiogenesis (Byrne et al., 2005). The VEGF-A exits in a five isoforms (120, 145, 164, 188 and 205) and VEGF-164 and VEGF-120 are highly expressed in mammalian ovary (Miyabayashi et al., 2006; Ng et al., 2006).
VEGF Receptor and Signaling
VEGF receptor contains an extracellular region with seven immunoglobulin-like loops and transmembrane tyrosine kinase domain (Ortega et al., 1999). Three VEGF receptors have been identified and are fms-like tyrosine kinase Flt-1 (VEGFR- 1/Flt-1), fetal liver kinase (VEGFR-2/KDR/Flk-1) and Flt-4 (VEGFR-3). Both VEGFR-1 and VEGFR-2 are expressed predominantly by vascular endothelial cells. While, VEGFR-3 is found in venous endothelium during early embryonic development, but remain confined to lymphatic endothelial cells later in fetal development (Hoeben et al., 2004; Shibuya, 2011). Although the affinity of VEGFR-2 for VEGF-A is tenfold lower than VEGFR-1 (Moghaddam et al., 2012), it is the main receptor in VEGF-induced mitogenesis and permeability (Waltenberger et al., 1994; Zachary, 1998). The VEGFR-1 plays an inhibitory role by sequestering VEGF and therefore prevents its binding with VEGFR-2 (Ferrara, 2004).
Binding of VEGF to its receptor (VEGFR-2) results in autophosphorylation of the tyrosine residues present in the domain. VEGF regulates various functions including proliferation and migration of endothelial cells, increase in vascular permeability, and cell survival by interacting with receptors and transmitting signals to various proteins (Byrne et al., 2005). Various sites for potential binding of molecules and phosphorylation have been identified but their role in VEGF stimulated response (increase vascular permeability, cell survival and regulation of angiogenesis) is unclear (Kaczmarek et al., 2005).
Physiological Regulation of VEGF in Ovarian Function
Growth and Development of Follicle
The primordial follicle is dependent on the proximity stromal vessel for uptake of nutrient and oxygen via passive diffusion as they do not have independent capillary network (Geva and Jaffe, 2000; Robinson et al., 2009). As the follicle grows, the antral follicle acquires two concentric vessels in thecal layer immediately outside the basement membrane and not penetrating the granulosa cells (Tamanini and De Ambrogi, 2004; Martelli et al., 2006). A period of rapid growth of the follicle coincides with the establishment of this vascular wreath (Geva and Jaffe, 2000). The extensive vascularization occurs due to stimulation of VEGF which might be expressed by oocyte derived factor (Robinson et al., 2009). This increased blood flow to a single follicle results in preferential delivery of gonadotropins, nutrients, oxygen and growth factors and plays necessary role in selection of follicle (Wulff et al., 2001). Also, it is observed that degeneration of capillaries in follicles is directly associated with follicular atresia (Macchiarelli et al., 1993) due to a low expression and secretion of VEGF. Similarly, inhibition of antral follicle development is observed on administration of an anti-VEGFR-2 (Zimmermann et al., 2003) indicating contribution of VEGF in follicular antrum formation (Kaczmarek et al., 2005; Ansari et al., 2017).
VEGF also has cytoprotective factor against follicular atresia by inducing expression of antiapoptotic protein in endothelial cells (Fraser et al., 2000). Follicles with high expression of VEGFR-2 exhibit low incidences of apoptosis while, the blockage of receptor inhibits the ability of cells to respond to VEGF endogenously or exogenously present and reduces protection against apoptosis (Greenaway et al., 2004).
VGEF appears to play an important role in growth, development and function of CL in bovines (Berisha et al., 2000; Chouhan et al., 2013). The intensive angiogenesis in the CL enable it to receive greatest amount of blood flow than any other tissue of the body (Neuvians et al., 2004). Following ovulation, reduction in local oxygen level stimulates expression of hypoxia-inducible factor (HIF-1α), a predominant stimulator for VEGF production. This HIF-1α/VEGF signaling may regulate development of CL (Wu et al., 2015). The VEGF induced hyperpermeability allows extravasation of fibrin, activation of plasminogen activators and additional growth factor to extravascular compartment for remodeling of ruptured follicle to CL (Pepper et al., 1991; Senger et al., 1993). It has been reported that about 85% of the proliferating cells in CL are derived from vascular origin (Reynolds and Redmer, 1999). Further, VEGF initiates establishment to intensive capillary network for nourishment of CL throughout its lifespan through invasion of vascular granulosa cells by endothelial cells (Kaczmarek et al., 2005).
The expression of VEGF has been found highest during early luteal phase and loss in mid phase with little expression during late luteal phase (Berisha et al., 2000; Fraser and Lunn, 2000). A decrease in VEGF expression together with dissolution of blood vessels and decline of blood flow indicate its involvement in functional and structural demise of CL (Fraser and Wulff, 2003). Further, during luteolysis, apoptosis of parenchymal cells of CL follows endothelial cells degeneration (Sawyer et al., 1990) indicate relation between regression of vasculature, functional and structural integrity of CL and VEGF expression (Berisha and Schams, 2005).
Hypoxia Intraovarian factors
HIF-1α TGF, BMP, IGF, NGF
Extensive vascularization Activation of antiapoptotic protein Extravasation of fibrin,
Activation of plasminogen
Growth factor activation
Follicular growth Cell survival Remodeling of follicle to CL
Fig. 1: Schematic presentation of regulation of VEGF in ovarian function
Ovarian angiogenesis is mainly regulated by luteinizing hormone (LH) (Hyder and Stancel, 1999) and LH-induced intense angiogenesis is associated with Follicle-CL transition (Dickson and Fraser, 2000). An increased VEGF expression has been reported during luteinization of granulosa cells at the time of ovulation (Yan et al., 1998; Hoeben et al., 2004). A decreased VEGF production by steroidogenic cells on treatment with GnRH antagonist is reported (Dickson and Fraser, 2000). Therefore, VEGF/VEGFR-2 pathway plays a critical role in the process follicular development mediated via. gonadotropin (Zimmermann et al., 2003). FSH stimulates expression of VEGF in granulosa cells but can influence VEGF isoform to be transcript (Shimizu et al., 2007; Ma et al., 2017). However, the mechanism regulating expression pattern of different VEGF isoforms by FSH is still unknown. Estrogen stimulates the expression of VEGF in bovine granulosa cells (Shimizu and Miyamoto, 2007) and is capable of inducing angiogenesis (Schnaper et al., 1996). Estrogen binds with specific estrogen response element present in VEGF gene and thus upregulates VEGF expression (Hyder et al., 2000) in bovine granulosa cells (Garrido et al., 1993). However, VEGF isoforms are differently expressed by the hormone. Estrogen causes upregulation of VEGF 164 expression while downregulates VEGF 120 expression in bovine granulosa cells (Shimizu & Miyamoto 2007).
Similarly, progesterone is involved in follicular angiogenesis by stimulating the expression of VEGF in granusola cells (Bailey et al., 2010). Further, the reduced luteal angiogenesis due to poor expression of VEGF has been demonstrated to result in infertility (Ebisch et al., 2008).
Ovarian follicular angiogenesis is initiated during the early stages of follicular development, and blood vessels in the thecal layer increase in number and size as the follicle develop. The strong association between angiogenesis and follicular development CL indicates critical role of VEGF in female reproduction. Several hormones and growth factors play critical role in expression of VEGF. Therefore, the study of their mechanism and interaction with each other is required for the treatment and management targeting ovarian pathology.