Introduction
Ovarian folliculogenesis in mammals is a complex process that is comprised of interactions between several autocrine, paracrine and endocrine factors. With respect to paracrine factors, the role of vascular endothelial growth factor (VEGF) is noteworthy. VEGF was initially identified and named vascular permeability factor (VPF). Subsequently, its angiogenic activity was described, and the renamed VEGF is now considered possibly the most potent angiogenic agent ever described. VEGF also stimulates the survival of endothelial cells in vessels through the inhibition of apoptosis, as well as promoting their proliferation, migration and differentiation, and causing changes in gene expression patterns and inhibition of senescence (Dvorak, Reference Dvorak2000).
The VEGF family is comprised of several members: VEGF-A, placental growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E. VEGF-A is the most studied subtype and has been detected in preantral follicles from several mammalian species such as humans (Otani et al., Reference Otani, Minami, Yamoto, Shikone, Otani, Nishiyama, Otani and Nakano1999; Harata et al., Reference Harata, Ando, Iwase, Nagasaka, Mizutani and Kikkawa2006), rats (Celik-Ozenci et al., Reference Celik-Ozenci, Akkoyunhlu, Kayisli, Arici and Demir2003), pigs (Barboni et al., Reference Barboni, Turriani, Galeati, Spinaci, Bacci, Forni and Mattioli2000), goats (Sharma and Sudan, Reference Sharma and Sudan2010) and cows (Greenaway et al., Reference Greenaway, Centry, Feige, Lamarre and Petrik2005). Moreover, regulatory effects of VEGF on mammalian folliculogenesis and luteogenesis have been observed (Quintana et al., Reference Quintana, Kopcow, Sueldo, Marconi, Rueda and Barañao2004; Roberts et al., Reference Roberts, Arbogast, Friedman, Cohn, Kaumaya and Danforth2007; Yang et al., Reference Yang, Lee, Lee, Ko and Kim2008).
For preantral folliculogenesis, the importance of VEGF for the survival and growth of early (Bruno et al., Reference Bruno, Celestino, Lima-Verde, Lima, Matos, Araújo, Saraiva, Martins, Name, Campello, Báo, Silva and Figueiredo2009) and advanced (Fisher et al., Reference Fisher, Zelinski, Molskness and Stouffer2009) follicles has been reported. Based on the observation of a positive correlation between follicle diameter and VEGF production, Fisher et al. (Reference Fisher, Zelinski, Molskness and Stouffer2009) demonstrated that this compound might play an important role during in vitro follicle development.
The involvement of VEGF in the regulation of the various phases of follicle development has been shown, however more studies are necessary for a better understanding of the mechanisms by which this factor (ligand and receptors) acts in mammalian ovarian folliculogenesis.
Features of the ovarian vascular system and follicular angiogenesis
The ovary in mammalian species is comprised of two distinct portions: (1) the cortex, which is the outermost part with a stroma of conjunctive tissue, and follicles and corpora lutea at several developmental stages; and (2) the medulla, the inner region, which contains loose conjunctive tissue highly vascularized and originating from ovarian arteries. Histologically, the limits between these two regions are not well defined.
The folliculogenesis process takes place within the cortex, from the formation of the primordial follicle to the development to the preovulatory stage, which comprises the preantral (primordial, primary and secondary follicles) and antral (tertiary and preovulatory follicles) phases. Despite the fact that preantral follicles do not possess their own vascular supply, the formation of the capillary network that surrounds the follicle is critical for growth beyond this phase. Angiogenesis begins within the stroma during early follicular development (Suzuki et al., Reference Suzuki, Sasano, Takaya, Fukaya, Yajima and Nagura1998). Up to this point, nutrition and oxygenation of primordial and primary follicles rely on passive diffusion from stromal blood vessels, which are thin and single layered at this time. At the secondary stage or later, stromal cells that surround the follicles become organized in thecal layers, in which the innermost part (theca internal) contains many blood vessels, whilst the outer layer (theca external) is composed mainly of fibrous conjunctive tissue. Thereafter, during the appearance of the antral cavity full of follicular fluid, follicles become surrounded by a capillary network, which promotes the nutrition of both these cells and granulosa cells. This vascular system is divided into two distinct parts that enters either the external and internal thecal cells layers (Stouffer et al., Reference Stouffer, Martínez-Chequer, Molskness, Xu and Hazzard2001), and both contribute to the production of follicular fluid (Van den Hurk & Zhao, Reference Van den Hurk and Zhao2005), which is rich in VEGF (Ferrari et al., Reference Ferrari, Pezzuto, Barusi and Coppola2006). The number and diameter of blood vessels increase as the follicle develops, but these never penetrate the basement membrane that separates theca interna and granulosa cells layers.
Structural characterization of VEGF and its receptors
VEGF is a cytokine and is a homodimeric glycoprotein that is expressed in several tissues as various types, with a molecular weight of about 45 kD (Ferrara & Henzel, Reference Ferrara and Henzel1989). Its structure forms an antiparallel homodimer that is linked covalently by two disulphide bridges between cystine residues. The cystine knot motif consists of an eight-residue ring formed by the disulphide bridges and is conserved in the same position by a third disulphide bond (Muller et al., Reference Muller, Li, Christinger, Wells, Cunningham and de Vos1997). VEGF-A is formed by two monomers that contain a cystine knot motif determined by three intrachain disulphide bridges, whilst the homodimer is assembled by two interchain disulphide bridges linking the monomers. Overall, the VEGF monomer resembles that of other cystine knot growth factors such as platelet-derived growth factor (PDGF), but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is similar to that of PDGF and is very different from that of transforming growth factor (TGF)-β. Mutational analysis of VEGF reveals that symmetrical binding sites for the receptor kinase domain receptor (KDR) are located at each pole of the VEGF homodimer (Muller et al., Reference Muller, Li, Christinger, Wells, Cunningham and de Vos1997).
In humans, the gene that encodes VEGF is comprised of eight exons that are separated by seven introns, and the coding region is approximately 14 kb (Tischer et al., Reference Tischer, Mitchell, Hartmann, Silva, Gospodarowicz, Fiddes and Abraham1991; Houck et al., Reference Houck, Ferrara, Winer, Cachianes, Li and Leung1991). VEGF mRNA undergoes alternative splicing events that lead to the production of mature homodimeric proteins. Each monomer is designated in accordance with the number of amino acids along their chains (VEGF110, VEGF111, VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189 and VEGF206; Fig. 1). The isoforms VEGF110 (Keyt et al., Reference Keyt, Berleau, Nguyen, Chen, Heinsohn, Vandlen and Ferrara1996) and VEGF121 (Park et al., Reference Park, Keller and Ferrara1993) do not bind to heparin as the carboxy-terminal domain located between amino acids 111 and 165 is not present, which makes both molecules freely diffusible within cells. In contrast, VEGF165 and VEGF189 bind to heparin with greater affinity. The use of heparinase either in vivo (Sasisekharan et al., Reference Sasisekharan, Moses, Nugent, Cooney and Langer1994) or in vitro (Rathjen et al., Reference Rathjen, Toth, Willis, Heath and Smith1990) indicates the potential of heparin molecules to be an important element of the binding complex VEGF receptor. In both cases, cell proliferation and neovascularization were inhibited. The absence of binding may not be due to a loss of VEGF receptors (Gitay-Goren et al., Reference Gitay-Goren, Sofer, Vlodavsky and Neufeld1992), as this activity could be recovered by the use of exogenous heparin (Rathjen et al., Reference Rathjen, Toth, Willis, Heath and Smith1990). Therefore, it was observed that successful signal transduction depends on the formation of a complex of VEGF, its receptors and heparin (VEGF–heparin–receptor) (Gitay-Goren et al., Reference Gitay-Goren, Sofer, Vlodavsky and Neufeld1992). These data suggest that the stability of VEGF–heparin–receptor complexes probably contributes to effective signal transduction and stimulation of endothelial cell proliferation (Keyt et al., Reference Keyt, Berleau, Nguyen, Chen, Heinsohn, Vandlen and Ferrara1996).
Figure 1 VEGF isoforms generated by alternative splicing. VEGF-A comprises monomers designated according to the number of amino acids in the polypeptide chain (VEGF110, VEGF111, VEGF121, VEGF145, VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183, VEGF189 and VEGF206). VEGF, vascular endothelial growth factor.
All transcripts contain exons one to five and exon eight, with diversity generated through the alternative splicing of exons six and seven, except for VEGF-A165b, which contains an alternative exon eight (Holmes & Zachary, Reference Holmes and Zachary2005). This variant is an endogenous inhibitory VEGF molecule that does not contain exon six, but possesses an alternative exon (eight) that encodes a new carboxy terminus that increases the chances of the occurrence of a family of isoforms with this novel carboxy-terminal end (Bates et al., Reference Bates, Cui, Doughty, Winkler, Sugiono, Shields, Peat, Gillatt and Harper2002).
In relation to the necessity of the above complex for the activity of VEGF, it is known that this molecule binds directly to three receptor types: VEGFR-1/Flt-1 (Fms-like tyrosine kinase-1; De Vries et al., Reference De Vries, Escobedo, Ueno, Houck, Ferrara and Williams1992), VEGFR-2/KDR (kinase insert domain containing region; Terman et al., Reference Terman, Dougher-Vermazen, Carrion, Dimitrov, Armellino, Gospodarowicz and Bohlen1992) and VEGFR-3/Flt-4 (Fms-like tyrosine kinase-4; Kaipainen et al., Reference Kaipainen, Korhonen, Mustonen, Van Hinsbergh, Fang, Dumont, Breitman and Alitalo1995; Karkkainen et al., Reference Karkkainen, Makinen and Alitalo2002) (Fig. 2). These receptors are members of the tyrosine kinase family, and have as common features the presence of seven immunoglobulin-like domains in the extracellular portion, a single transmembrane region and a tyrosine kinase sequence interrupted by the kinase insertion domain in its intracellular portion (Shibuya et al., Reference Shibuya, Yamaguchi, Yamane, Ikeda, Tojo, Matsushime and Sato1990). Nevertheless, VEGF binds with high affinity to only two of these three receptors (VEGFR-1/Flt-1 and VEGFR-2/KDR), whilst VEGFR-3/Flt-4 is involved in interactions with other VEGF forms (VEGF-C and VEGF-D; Neufeld et al., Reference Neufeld, Cohen, Gengrinovitch and Poltorak1999). The cleavage of VEGF165 by plasmin, which is an important process in the angiogenesis cascade (Mignatti et al., Reference Mignatti, Tsuboi, Robbins and Rifkin1989), releases an N-terminal fragment that is comprised of amino acids 111–165. This polypeptide binds to two of the VEGF receptors, VEGFR-1/Flt-1 and VEGFR-2/KDR, in the absence of heparin (Keyt et al., Reference Keyt, Berleau, Nguyen, Chen, Heinsohn, Vandlen and Ferrara1996). Once VEGF has bound, VEGFR-2 dimerizes and autophosphorylates, which in turn activates several signal transduction cascades (Byrne et al., Reference Byrne, Bouchier-Hayes and Harmey2005).
Figure 2 Binding complex VEGF-heparin-receptor involved in biological responses to VEGF in various cells and tissues. VEGF-A binds both to VEGFR-1 and VEGFR-2, whilst PIGF and VEGF-B interact only with VEGFR-1. VEGF-C and VEGF-D bind to receptors VEGFR-2 and VEGFR-3, and VEGF-E binds only to VEGFR-2. Flt, Fms-like tyrosine kinase-1; PIGF, placental growth factor; VEGF, vascular endothelial growth factor.
Expression, immunolocalization and mechanism of action of VEGF and its receptors
VEGF and its receptors VEGFR-1/Flt-1 and VEGFR-2/KDR are expressed in the ovary of mammals, and have been identified in several reproductive tissues such as the ovarian follicle, corpus luteum, endometrial vessels and at embryo implantation sites (Jakeman et al., Reference Jakeman, Armanini, Phillips and Ferrara1993; Shweiki et al., Reference Shweiki, Itin, Neufeld, Gitay-Goren and Keshed1993; Gordon et al., Reference Gordon, Messiano, Zaloudek and Jaffe1996; Neufeld et al., Reference Neufeld, Cohen, Gengrinovitch and Poltorak1999; Krussel et al., Reference Krussel, Berh, Milki, Hirchehain, Wen, Bielfeld and Polan2001; Al-zi'abi et al., Reference Al-zi'abi, Watson and Fraser2003). Previous studies have demonstrated the presence of VEGF and its respective mRNA expression in the endometrium of fertile women with normal uteri (Shifren et al., Reference Shifren, Tseng, Zaloudek, Ryan, Meng, Ferrara, Jaffe and Taylor1996). Both the protein and mRNA corresponding to VEGF and its receptors were also detected in the granulosa and in thecal cells of bovine secondary ovarian follicles (Yang & Fortune, Reference Yang and Fortune2007). In sows, expression of mRNA for VEGF was observed in granulosa cells of antral follicles (Shimizu et al., Reference Shimizu, Jiang, Sasada and Sato2002, Reference Shimizu, Kawahara, Abe, Yokoo, Sasada and Sato2003) during the early and mid-luteal phases (Kaczmarek et al., Reference Kaczmarek, Kowalczyk, Waclawik, Schams and Ziecik2007), whilst mRNA for receptors VEGFR-1 and VEGFR-2 were expressed especially within the layers of thecal cells (Shimizu et al., Reference Shimizu, Jiang, Sasada and Sato2002, Reference Shimizu, Kawahara, Abe, Yokoo, Sasada and Sato2003).
VEGF expression was also reported in granulosa and thecal cells of secondary follicles in rats, and could be enhanced in response to the gonadotropins follicle stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) (Koos, Reference Koos1995; Yang et al., Reference Yang, Lee, Lee, Ko and Kim2008). Conversely, at the end of the growth phase in porcine folliculogenesis, a progressive decrease in the production of VEGF is observed in response to the LH surge or to the administration of hCG (Barboni et al., Reference Barboni, Turriani, Galeati, Spinaci, Bacci, Forni and Mattioli2000).
Immunolocalization of VEGF in caprine ovarian tissue reveals the presence of VEGF in follicles at all developmental stages, with a progressive increase from the primary to the preovulatory stage, as well as in surrounding stroma cells (Sharma & Sudan, Reference Sharma and Sudan2010). With regard to the immunoreactivity of goat ovaries in relation to receptor 2 (VEGFR-2/KDR), Bruno et al. (Reference Bruno, Celestino, Lima-Verde, Lima, Matos, Araújo, Saraiva, Martins, Name, Campello, Báo, Silva and Figueiredo2009) observed the expression of this receptor in all follicle categories, but antral follicles displayed weak positive reactions. Furthermore, their study demonstrated the presence of this receptor in oocytes of primordial follicles, which indicated the involvement of VEGF in the growth and development of these cells (Bruno et al., Reference Bruno, Celestino, Lima-Verde, Lima, Matos, Araújo, Saraiva, Martins, Name, Campello, Báo, Silva and Figueiredo2009). Immunolocalization reactions for VEGF are stronger in cells of the theca interna in comparison with granulosa cells and stromal vascular tissue (Sharma & Sudan, Reference Sharma and Sudan2010). As VEGF levels rise whilst the respective receptors decline throughout follicle development, the role of this factor may rely more probably on its function of promoting cellular permeability.
VEGF and its receptors VEGFR-1/Flt-1 and VEGFR-2/KDR are detected in cells from the endothelium and during pregnancy corpora lutea in sows (Kaczmarek et al., Reference Kaczmarek, Kowalczyk, Waclawik, Schams and Ziecik2007), and also in endothelial cells of bovine ovaries (Berisha et al., Reference Berisha, Schams, Kosmann, Amselgruber and Einspanier2000). In humans, mRNA and proteins corresponding to these receptors, as well as to VEGF-A (protein), were expressed in oocytes, granulosa and stroma cells (Abir et al., Reference Abir, Ao, Zhang, Garor, Nitke and Fisch2010). In addition, VEGF was also immunolocalized in granulosa and theca interna cells of healthy follicles from rats (Koos, Reference Koos1995) and cows (Berisha et al., Reference Berisha, Schams, Kosmann, Amselgruber and Einspanier2000), as well as in luteinized granulosa cells in buffalos (Papa et al., Reference Papa, Moura, Artoni, Fátima, Campos, Marques, Baruselli, Binelli, Pfarrer and Leiser2007) and mares (Al-zi'abi et al., Reference Al-zi'abi, Watson and Fraser2003).
Biological activity and role of VEGF in mammalian folliculogenesis
The selective activation of each of the VEGF receptor types results in distinct biological responses. Binding to VEGFR-1/Flt-1 leads to organizational effects on vascular structures, which are important for the interaction of endothelial cells and for blood vessels formation. In contrast, activation of VEGFR-2/KDR induces the formation, migration and proliferation of vascular endothelial cells (Neufeld et al., Reference Neufeld, Cohen, Gengrinovitch and Poltorak1999; Ho & Kuo, Reference Ho and Kuo2007), as well as contributing to cellular survival. Binding to VEGFR-3/Flt-4, predominantly expressed in lymphatic vessels, resulted in lymphatic angiogenesis (Ho & Kuo, Reference Ho and Kuo2007). Some biological responses to VEGF binding to its receptors important for follicle development are described in more detail below (Fig. 3).
Figure 3 Biological activities of VEGF in the mammalian ovarian follicle. The expansion of the vascular network during follicle development enhances oxygenation and diffusion of several substances important for follicle cells, and leads to the discussed biological responses. GC, granulosa cell; TC, theca cell; VEGF, vascular endothelial growth factor; ZP, zona pellucida.
Angiogenic action of VEGF
VEGF was discovered originally as a compound that was capable of enhancing the permeability of vessels, thus enabling proteins and other molecules to exit blood vessels and enter perfused tissues (Senger et al., Reference Senger, Galli, Dvorak, Perruzzi, Harvey and Dvorak1983; Dvorak et al., Reference Dvorak, Brown, Detmar and Dvorak1995). With regard to the mammalian ovary, VEGF properties were demonstrated first in the bovine corpus luteum (Tischer et al., Reference Tischer, Gospodarowicz, Mitchell, Silva, Schilling, Lau, Crisp, Fiddes and Abraham1989), and later in the same tissue from ewes (Redmer et al., Reference Redmer, Dai, Li, Charnock-Jones, Smith, Reynolds and Moor1996). The cyclic changes during formation and regression of the corpus luteum comprise the formation of new blood vessels (Redmer & Reynolds, Reference Redmer and Reynolds1996; Wulff et al., Reference Wulff, Wiegand, Saunders, Scobie and Fraser2001) from pre-existing vessels, and is named angiogenesis.
Later studies have revealed that this factor was also involved in other processes such as the promotion of growth of vascular cells derived from arteries, veins and lymphatic vessels (Ferrara & Davis-Smyth, Reference Ferrara and Davis-Smyth1997; Ferrara & Alitalo, Reference Ferrara and Alitalo1999). Moreover, VEGF was found to induce a potent angiogenic response in a wide range of in vivo (Leung et al., Reference Leung, Cachianes, Kuang, Goeddel and Ferrara1989) and in vitro (Pepper et al., Reference Pepper, Ferrara, Orci and Montesano1992; Reference Pepper, Vassallim, Wilks, Schweigerer, Orci and Montesano1994) models.
In the ovary, angiogenesis facilitates oxygenation and nutrition of target cells, and secures an increasing supply of gonadotropins, growth factors, oxygen, steroid precursors, as well as other substances to the growing follicle (Kaczmarek et al., Reference Kaczmarek, Schams and Ziecik2005). Such rise in the delivery of nutrients can be a decisive factor for the selection of the dominant follicle (Zimmermann et al., Reference Zimmermann, Xiao, Husami, Sauer, Lobo, Kitajewski and Ferin2001). Therefore, there is evidence that thecal angiogenesis plays a pivotal role in follicle development (Tamanini & De Ambrogi, Reference Tamanini and De Ambrogi2004). Furthermore, granulosa cells are important for the angiogenic process, as these cells secrete several angiogenic factors that act on thecal cells.
VEGF and cell permeability
VEGF can also act indirectly through reorganization or formation of a primitive capillary plexus for supply of tissue needs, increase in vascular permeability and enabling a higher availability of growth factors, gonadotropins, steroids and oxygen, which are important for follicle growth. This fact was confirmed in vivo by Danforth et al. (Reference Danforth, Arbogast, Ghosh, Dickerman, Rofagha and Friedman2003) and Quintana et al. (Reference Quintana, Kopcow, Sueldo, Marconi, Rueda and Barañao2004) through direct injection of VEGF into the ovarian bursa in mice that enhanced neovascularization, increased the numbers of primary and secondary follicles and vascular permeability for developing follicles, and, as a consequence, reduced apoptosis. In vitro, Mattioli et al. (Reference Mattioli, Barboni, Turriani, Galeati, Zannoni, Castellani, Berardinelli and Scapolo2001) observed that VEGF production raised blood supply and activated primordial follicles.
The cellular permeability induced by VEGF is attributed to the appearance of fenestrations that, through a not well defined mechanism, enables a rise in the efflux of small solutes (Roberts & Palade, Reference Roberts and Palade1995). Dvorak (Reference Dvorak2000) observed that the interaction between VEGF and its receptors VEGFR-1 and VEGFR-2 triggers a cascade of events that includes an increase in microvascular permeability, leading to deposition of pro-angiogenic fibrin in the extracellular matrix and formation of new vessels. Furthermore, VEGF induces an increase in calcium influx, as well as a rise in the concentration of this ion within endothelial cells (Bates & Curry, Reference Bates and Curry1997).
In the ovarian follicle, the promotion of vascular permeability, vasodilation and development of endocrine function by theca cells resulted in a gradual rise in ovarian blood flux, and supported antrum formation and functional adaptation events for ovulation, which led to follicle rupture (Jiang et al., Reference Jiang, Macchiarelli, Tsang and Sato2003; Tamanini & De Ambrogi, Reference Tamanini and De Ambrogi2004). Thus, the establishment of an adequate vascular supply is possibly a limiting step in the selection and maturation of the one dominant follicle that will ovulate (Stouffer et al., Reference Stouffer, Martínez-Chequer, Molskness, Xu and Hazzard2001).
The formation of the antral cavity is a spontaneous event during the in vitro culture of advanced preantral follicles, however mitogenic factors such as VEGF may enhance rates of occurrence of this process (Araújo et al., unpublished data). One study showed that VEGF secretion is stage dependent and increases as the follicle grows, which reflects in the amounts of VEGF in the follicular fluid (Barboni et al., Reference Barboni, Turriani, Galeati, Spinaci, Bacci, Forni and Mattioli2000). VEGF is also produced by cells of preovulatory follicles, as well as by luteinized cells (Taylor et al., Reference Taylor, Hillier and Fraser2004).
VEGF and cell survival
The role of VEGF as a survival factor was observed either in vitro or in vivo with endothelial cells (Alon et al., Reference Alon, Hemo, Itin, Pe'er, Stone and Keshet1995; Yuan et al., Reference Yuan, Chen, Dellian, Safabakhsh, Ferrara and Jain1996), as well as with other cell types. VEGF inhibits apoptosis induced by absence of serum in culture medium (Gerber et al., Reference Gerber, Dixit and Ferrara1998a) or by injuries that result from cryopreservation (Shin et al., Reference Shin, Lee, Lee, Choi, Yoon, Bae and Choi2006). This property may be mediated via PI3kinase/Akt (Gerber et al., Reference Gerber, Dixit and Ferrara1998a), which is a signalling pathway fundamental for regulation of cell proliferation, survival, migration and metabolism, and also plays an important role in the activation of primordial follicles (Cantley, Reference Cantley2002). Moreover, VEGF induces the expression of anti-apoptotic proteins such as Bcl-2 and A1 in endothelial cells (Gerber et al., Reference Gerber, McMurtrey, Kowalski, Yan, Keyt, Dixit and Ferrara1998b). The addition of VEGF to in vitro culture supported the maintenance of viability and ultrastructure of goat early preantral follicles (Bruno et al., Reference Bruno, Celestino, Lima-Verde, Lima, Matos, Araújo, Saraiva, Martins, Name, Campello, Báo, Silva and Figueiredo2009).
Mitogenic action of VEGF
In addition to its angiogenic properties, VEGF is also a potent mitogenic factor that is secreted by many differentiated cells in response to several stimuli such as, for instance, hypoxia. Nonetheless, the loss of its carboxy-terminal domain reduces significantly the potency for induction of proliferation in endothelial cells (Keyt et al., Reference Keyt, Berleau, Nguyen, Chen, Heinsohn, Vandlen and Ferrara1996). VEGF exerts direct mitogenic effects on granulosa cells, and then acts on follicle growth in human ovaries (Otani et al., Reference Otani, Minami, Yamoto, Shikone, Otani, Nishiyama, Otani and Nakano1999). The presence of VEGF-A receptors, especially in granulosa cells, suggests that this factor may be involved in proliferation events, as well as in the onset of development of primordial follicles in humans (Abir et al., Reference Abir, Ao, Zhang, Garor, Nitke and Fisch2010). Furthermore, during the transition of these follicles to the primary stage, an increase in VEGF and its mRNA takes place in rats (Kezele et al. Reference Kezele, Ague, Nilsson and Skinner2005). Yang & Fortune (Reference Yang and Fortune2007) observed the transition of primary follicles to the secondary stage, and also the increase in follicle diameter, through the in vitro culture of ovarian tissue retrieved from bovine fetuses in medium supplemented with VEGF. Similarly, in addition to follicular growth, an increase in oocyte diameter could also be seen in early (Bruno et al., Reference Bruno, Celestino, Lima-Verde, Lima, Matos, Araújo, Saraiva, Martins, Name, Campello, Báo, Silva and Figueiredo2009) and advanced (Araújo et al., unpublished data) goat preantral follicles.
Role of VEGF on oocyte maturation
As VEGF expression increases progressively from the primary to the preovulatory stage, which is directly correlated to the expansion of vascularization and oxygenation of follicles (Sharma & Sudan, Reference Sharma and Sudan2010), selection of the dominant follicle depends on the formation and the differentiation of a rich vascular supply with an increment in the permeability of the respective vessels (Kawano et al., Reference Kawano, Hasan, Fukuda, Mine and Miyakawa2003). Such conditions are very important because hypoxia may reduce oocyte metabolism and cause changes in intracellular pH, which in turn affects organization and stability of the meiotic spindle (Gaulden, Reference Gaulden1992). Such an effect can result in chromosomal disorders (non-disjunction of chromosomes) (Van Blerkom et al., Reference Van Blerkom, Antczak and Schrader1997). Moreover, deficiencies in blood supply impair the delivery of substances that are essential for the development of follicles to the preovulatory phase (Zimmermann et al., Reference Zimmermann, Hartman, Kavic, Pauli, Bohlen, Sauer and Kitajewski2003). Therefore, VEGF is an important factor for the development of mammalian oocytes, and contributes to making these gametes competent for fertilization, embryo development and pregnancy.
The incomplete cytoplasmic maturation commonly observed after in vitro maturation of oocytes (First & Barnes, Reference First and Barnes1989) may explain the low rates of fertilization and extrusion of the first polar body (Trounson et al., Reference Trounson, Willadsen and Rowson1977). The use of VEGF in culture of bovine cumulus–oocyte complexes promoted nuclear (Einspanier et al., Reference Einspanier, Schönfelder, Müller, Stojkovic, Kosmann, Wolf and Schams2002; Luo et al., Reference Luo, Kimura, Aoki and Hirako2002) and cytoplasmic (Luo et al., Reference Luo, Kimura, Aoki and Hirako2002) maturation of the oocytes, and enhanced normal fertilization rates and the subsequent embryo development to the blastocyst stage. Moreover, Iijima et al. (Reference Iijima, Jiang, Shimizu, Sasada and Sato2005) observed that treatment of rats with VEGF promoted ovarian follicular angiogenesis, stimulated follicle development and increased the number of ovulated oocytes, which showed normal fertilization and developmental competence to term.
Despite the evidence that VEGF can contribute to oocyte maturation, the mechanisms by which this factor acts in this process are still unclear. It has been postulated that VEGF may exert its main paracrine effects directly on oocytes or indirectly via cumulus cells that express VEGF receptors type 2 (VEGFR-2/KDR) (Bruno et al., Reference Bruno, Celestino, Lima-Verde, Lima, Matos, Araújo, Saraiva, Martins, Name, Campello, Báo, Silva and Figueiredo2009) and are expanded in bovine (Einspanier et al., Reference Einspanier, Schönfelder, Müller, Stojkovic, Kosmann, Wolf and Schams2002; Luo et al., Reference Luo, Kimura, Aoki and Hirako2002) and caprine (Araújo et al., unpublished data) cumulus–oocytes complexes cultured with VEGF.
Conclusions
A full understanding of the role of VEGF on the modulation of ovarian physiology is very important as this growth factor controls vascularization and therefore the availability of oxygen and nutrients for the follicles. Studies have demonstrated that VEGF influences cell survival, proliferation and thus follicular development positively, along with the stimulation of secretion of some steroid hormones such as, for instance, progesterone. In spite of the recognized potential of VEGF for enhancing follicle and oocyte developmental processes, studies on the functions of this factor in folliculogenesis are still scarce. Therefore, more investigation is necessary in order to explore the various biological properties of VEGF and its receptors.
Acknowledgements
The authors thank Dr Anderson P. Almeida for the creation and editing of the images of this work. Valdevane R. Araújo is a recipient of a grant from the Coordination for Improvement of Graduate Personnel (CAPES-Brazil).
