INTRODUCTION Infertility is one of the major economic issues challenging the dairy industry today [1]. Currently in the United States, expenses attributed to dairy cow infertility are estimated to exceed $350M per year [2]. Infertility is the absence of pregnancy after 12 months of breading attempt [3]. There are numerous causes of infertility in cattle, including infectious diseases, anatomical abnormalities, physical trauma, environmental toxins, and anovulation [4]. Among these, anovulation, or the failure to ovulate, is one of the most significant causes of infertility [5].
Ovulation is a complex biological process that is initiated by a surge of luteinizing hormone secreted from the anterior pituitary that ultimately triggers the rupture of a follicle at the surface of the ovary and the release of oocyte. Ovulation is analogous to an inflammatory response. The ovulatory process and inflammation have been equated with one another because many inflammatory mediators present during ovulation are also present during an inflammatory response in the tissue. The recruitment of immune cells or leukocytes to the ovary, for instance, follows a very specific series of events.
During ovulation, there is a large surge of luteinizing hormone (LH) released by the pituitary gland that triggers a cascade of actions within the ovary, including the recruitment of leukocytes from the bloodstream to the ovary as shown below [6]. [LH-mediated upregulation of adhesion molecule expression on ovarian endothelial cells and migration of leukocytes through the interstitial space towards the ovary. ] Leukocytes are circulating immune cells that become attracted to certain locations by a variety of chemical signals such as adhesion olecules and chemokines [7] In the rat, approximately half a million leukocytes infiltrate the ovary within an hour of the LH surge and ovulation [6]. Additionally, the majority of leukocytes that infiltrate the ovary are macrophages and neutrophils. The large number of macrophages and neutrophils present likely produce large quantities of proteases, which facilitate the degradation of connective tissue and extracellular matrix, leading to follicular rupture and ovulation [8].
Macrophages not only produce proteases, but VEGF as well [9], which is essential for angiogenesis within the ovary before and after the occurrence of ovulation. Angiogenesis is defined as the generation of new blood via the sprouting of existing blood vessels [20]. Chiefly, endothelial cells are responsible for angiogenesis and there are at least three steps to angiogenesis. First, the basement membrane of existing blood vessels breakdown via MMPs and other proteases, second, endothelial cells migrate toward an angiogenic stimulus and third, endothelial cells proliferate and organize to establish a new network of blood vessels [21].
The formation of new blood vessels from a pre-existing vascular network in the ovary is regulated by a variety of factors such as vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF), and angiopoeitins (Ang). Among these, the first factor to be characterized on the basis of its ability to promote endothelial cell proliferation and increase vascular permeability is VEGF [10]. Vascular endothelial growth factor (VEGF), a potent mitogenic factor essential for growth and migration endothelial cells has at least six members, VEGF A-F.
Furthermore, VEGF-A harbors five different isoforms as a result of alternative splicing of the gene. Among five isoforms of VEGF-A, three are soluble, while remaining two are membrane bound [10]. VEGFs exert their functions by binding to a family of tyrosine kinase receptors (VEGF-receptors); VEGFR-1, VEGFR-2 and VEGFR-3. The VEGFR-2 is mainly expressed by angiogenic endothelial cells and regulates endothelial cell migration, proliferation and differentiation [11].
In general, binding of VEGF to VEGE-R causes dimerization and autophosphorylation of the receptor. This leads to phosphorylation of intracellular proteins such as PLCgamma, VEGFR-associated protein (VRAP) and Sck. Now, the phosphorylated intracellular proteins eventually lead to increased proliferation, survival, permeability and migration of endothelial cells as shown below [12]. All isoforms of VEGF-A as well as VEGFR-1 and VEGFR-2 have been detected in the ovaries of rodent and primates [13-14].
Furthermore, in the primate ovary, VEGF mRNA and protein expression were observed in the theca cells of the pre-ovulatory follicles, exemplifying an integral role of VEGF in folliculogenesis [15]. Even though VEGF expression is largely associated with tumorigenic potential, VEGF confers physiological functions as well [16]. The process of angiogenesis is restricted to pathological conditions such as tumor growth and metastasis and physiological conditions such as wound healing, inflammation and female reproductive processes such as, placentation, follicular development and corpus luteum formation [22-24].
The formation of blood vessels plays an important role in the female reproductive system throughout its functional lifespan. For instance, in order to accommodate the follicular growth that occurs before and after ovulation, the ovary develops intricate vascularity and receives some of the highest blood flow in the entire body [17]. Angiogenesis and neovascularization occur simultaneously within the ovary and VEGF expression is closely connected with these dynamic changes.
VEGF expression is a critical component of follicular growth, ovulation, and the development and maintenance of the corpus luteum [13]. Prior to ovulation, follicles within the ovary destined to grow and potentially ovulate begin to develop more than one arteriole and acquire a vascular ring within the theca layer surrounding the follicle. The establishment of this vascular ring facilitates rapid growth and differentiation of the tissue, which is critical to maturation of both the follicle and ocyte before ovulation [18]. Following ovulation, the ruptured follicle establishes a temporary endocrine structure called the corpus luteum, which consists of steroidogenic cells, connective tissue, an extensive capillary network, all of which form as a consequence of extensive VEGF expression as shown below [19]. As described above, ovulation is likened to an immune response in which a large number of leukocytes, especially macrophages, accumulate within the ovary during the ovulatory process.
Macrophages are one of the major leukocytes associated with ovulation, so it is conceivable that angiogenesis within the ovary is in part influenced by macrophages. The cellular source(s) of VEGF that drives angiogenesis within the ovary has not yet been clearly determined. Some reports [9& 20] indicate macrophages, monocytes and neutrophils are the source of VEGF within the ovary, but this question warrants further study. Since immune cells are associated with VEGE secretion with the ovaries, it is logical that if we downregulate VEGF secretion then it would alter follicular development and therefore ovulation.
One of the ways to downregulate VEGE secretion is by downregulating the cellular sources of VEGF. In other words, we can downregulate VEGF at its source by downregulating immune cells, such as macrophages via immunosuppressant such as dexamethasone (DEX), a glucocorticoid. Glucocorticoids, a group of corticosteroid exerts inhibitory effects on the various constituents of the immune system, ranging from B-cells and T-cells to macrophages and neutrophils [21]. Since its discovery, much has been learned about the mechanistic workings of DEX, but it is yet to be fully understood.
The commonly accepted theory is that, glucocorticoids exert their effect at the molecular level by altering gene expression. Glucocorticoids such as DEX bind with its receptor (glucocorticoid receptor), which is a transcription factor that influences gene expression by either upregulating or downregulating certain genes [22]. DEX in particular suppresses the immune system by binding to its receptors, which in turn downregulates the expression of proinflammatory genes by blocking nuclear factor kappa B (NF-kB) and upregulates the expression of anti-inflammatory genes [23-24].
Furthermore, DEX can downregulate the extravasation of monocytes, macrophages and other innate immune cells to tissues [25]. In this study, we hypothesize that leukocytes, especially macrophages are the source of VEGF within the ovary, and we tested this hypothesis by suppressing the number of leukocytes in the ovary by treating with the immune-suppressant dexamethasone (DEX) and quantifying leukocytes activity and VEGF expression within the ovarian tissue.
We anticipated that DEX-induced suppression of leukocytes within the ovary would decrease the number of macrophages and thereby reduce VEGE expression within the ovary. VEGF is a potent angiogenic factor in the ovary, so it is essential to know the source(s) of VEGF production. Identifying the source(s) could potentially address problems with anovulation, infertility, and pregnancy loss attributable to defects in ovarian angiogenesis [26].