Background
The corpus luteum (CL) is a unique endocrine organ that develops from the ovulated follicle during the sexual cycle. The main function of the CL is to secrete a large amount of progesterone (P), which is essential for establishment and maintenance of pregnancy. After ovulation, inadequate function of the CL is one of major causes of infertility in cows [
1]. Angiogenesis is fundamental to the normal development of the CL in many species. One of the major angiogenic factors, basic fibroblast growth factor (FGF2), is generally involved in cell growth, differentiation, transformation and angiogenesis. Gospodarowizc et al. [
2] have been shown that FGF2 is produced in the bovine CL and stimulates neovascularization and proliferation of a wide variety of cells, such as vascular smooth muscle cells, granulosa cells and endothelial cells. Additionally, vascular endothelial growth factor A (VEGFA, mainly localized in the cytoplasm of luteal cells) also is major player for angiogenesis and plays a fundamental role in maintenance of the vasculature in the CL when it is no longer undergoing active angiogenesis [
3].
Cytokines and chemokines, such as prostaglandins (PGs) [
4], tumor necrosis factor α (TNFα) [
5], interleukin (IL)-1 [
6] and IL-8 [
7,
8] are found in high concentrations in the follicular fluid in the pre-ovulatory phase. Consequently, high numbers of neutrophils and macrophages infiltrate in the pre-ovulatory follicle at the time of ovulation [
9]. Indeed, neutrophil depletion by administration of a monoclonal antibody against neutrophils reduced the ovulation rate in rats, indicating a critical role for neutrophils in ovulation [
10]. After ovulation, a multitude of leukocytes, such as macrophages and eosinophils are located in the luteinizing theca area in the developing CL [
11‐
13]. This series of phenomena from ovulation to luteal development involves bleeding, immune cell infiltration, tissue remodeling and angiogenesis, implying that the development of the CL following ovulation is a kind of physiological injury with an inflammatory response. In general, polymorphonuclear neutrophils (PMN) are the first cells recruited to inflammatory sites, providing cytokines and proteolytic enzymes. Moreover, PMNs secrete VEGFA [
14] and induce the sprouting of capillary-like structures of endothelial cells
in vitro [
15], suggesting that PMNs have a potential role, not only in phagocytosis, but also in the regulation of angiogenesis.
IL-8 is a small protein (8.4 kDa) and produced by macrophages, endothelial cells and neutrophils [
15,
16]. IL-8 has been shown to be a neutrophil-specific chemoattractant [
16], and IL-8 is involved in angiogenesis, cell proliferation, and apoptosis [
16,
17]. In the ovary, IL-8 is detected in theca, granulosa, granulosa-lutein, and vascular endothelial cells in humans [
18,
19] and rabbits [
20]. Goto
et al, [
21] observed that IL-8 injection increased follicular growth and capillary vessel densities around the follicles in rats. Furthermore, treatment of anti-IL-8 antibody inhibited the hCG-induced ovulation rate [
22], suggesting that IL-8 participates in the regulation of ovarian function to induce ovulation. However, no evidence has been shown regarding the role of neutrophils and IL-8 in the life span of the bovine CL.
We hypothesized that PMNs infiltrate in the developing CL from just after ovulation and may play a role in angiogenesis of the CL. Therefore, in the present study, we investigated the localization of PMNs and their chemoattractant, IL-8, in the bovine CL during the estrous cycle and effects of PMNs and IL-8 on the function of luteal endothelial cells in vitro.
Discussion
This study demonstrated the presence of PMN populations and IL-8 expression (mRNA and protein) within the CL during the estrous cycle in cows. Particularly, PMNs and IL-8 expression were predominant in the very early stage of the luteal phase. The early bovine CL induced PMN migration at least in part via IL-8 in vitro, and PMNs as well as IL-8 stimulated capillary-like structures of LECs in vitro. These findings suggest that PMNs and IL-8, a neutrophil-specific chemoattractant, have important roles in CL angiogenesis in cows.
The CL formation occurs by the rapidly induced terminal differentiation of granulosa and theca cells into luteal cells and promotes tissue transformation including break-down of the follicular basal lamina and migration of various cell types, such as leukocytes and endothelial cells into the granulosa layer, which eventually leads to neovascularization. In the present study, the early bovine CL, specifically days 1-4 of the estrous cycle possessed a large number of PMNs together with high levels of IL-8 expression (Figure
1). Therefore, we hypothesized that PMNs infiltrated
via IL-8 in the developing CL may play a role in angiogenesis of the CL.
To investigate the role of PMNs and IL-8 in CL development, we focused on the mechanism of PMN migration in the bovine CL. In the present study, we investigated the factors secreted from the CL to induce migration of PMNs. The early CL produced a greater quantity of IL-8, a good candidate of neutrophil-specific chemoattractant compared to the mid CL. Indeed, PMN migration was stimulated by the supernatant from the early CL but not from the mid CL, and this active PMN migration in the early CL was partially inhibited by pre-treatment with anti-IL-8 antibody (Figure
4D). However, treatment of IL-8 antibody could not completely inhibited (only 40%) the PMN migration by the supernatant of the early CL. Interestingly, as in the case of IL-8, FGF2 and VEGFA (10 and 100 ng/ml) stimulated PMN migration
in vitro (our preliminary study, data not shown), and FGF2 and VEGFA are also expressed at high levels within the developing CL. Indeed, Ancelin et al. demonstrated that VEGFA was chemotactic for neutrophils in humans and that a neutralizing with anti-VEGF antibody blocked this effect [
14]. FGF2 also enhanced recruitment of neutrophils in rats [
33]. Therefore, we speculate that IL-8, VEGFA, and FGF2 are acting synergistically as stimulators of PMN recruitment in the early CL in cows. We have to continuously investigate about this hypothesis in future study.
To elucidate our hypothesis that PMNs and IL-8 may play a role in angiogenesis of the CL, we investigated the effects of PMNs and IL-8 on the function of LECs
in vitro. Similar to FGF2 as a strong angiogenic factor, IL-8 increased proliferation of LECs and stimulated sprouting of capillary-like structures of LECs in
in vitro matrigel assay. On the other hand, it has been reported that human PMNs stimulated by 100 nM fMLP secretes substantial amount of IL-8 at 1 h after stimulation and is able to induce sprouting of capillary-like structures [
15,
29]. As referred these previous study, the supernatant of activated-PMNs by fMLP as well as FGF2 in the present study, stimulated sprouting of capillary-like structures of LECs in
in vitro matrigel assay, suggesting the crucial role of PMNs-IL-8 axis in the developing CL in cows. Actually, PMNs and IL-8 can induce angiogenesis
in vivo [
16,
21] and
in vitro [
15,
29], suggesting that the PMNs-IL-8 duo may function not only in the induction of tissue inflammation and wound healing, but also in the regulation of angiogenesis.
Angiogenesis is a critical component of normal luteal function, and FGF2 and VEGFA are major angiogenic factors in the bovine CL [
34‐
37]. Indeed, Robinson et al., demonstrated that in a luteal angiogenesis culture system (involves luteal cells, endothelial cells and smooth muscle cells), a physiological dose (1 ng/ml) of FGF2 and VEGFA stimulates the degree of the endothelial cell network [
35]. In addition, we recently showed that treatment with FGF2 and VEGFA antibodies markedly suppressed angiogenesis in the early luteal phase of the bovine CL
in vivo [
37]. VEGFA stimulated the mRNA expression of IL-8 in bovine theca [
38] and human endothelial cells [
15]. These findings suggest that PMNs and IL-8 together with FGF2 and VEGFA actively contribute to induce angiogenesis during the early CL development.
Similarly to PMNs in the present study, macrophages and lymphocytes have been identified in the developing CL in several species [
11,
39‐
41], and have been suggested to be putative intraovarian regulators. Indeed, peripheral blood lymphocytes and macrophages indicated a luteotrophic effect as these cells stimulated P secretion upon co-culture with granulosa cells [
42‐
44]. Meanwhile, Furukawa et al. revealed that platelets are novel regulators of neovascularization and luteinization during the early luteal phase in humans [
45]. An increased number of platelets localized to extravascular sites among luteinizing granulosa cells after ovulation and gradually decreased toward the mid luteal phase [
45], that are similar to the PMN profile observed in the present study. Moreover, platelets stimulated P secretion in luteinizing granulosa cells and endothelial cell migration [
45]. Taken together, these findings suggest that immune and blood cells are active components in CL development including angiogenesis, luteinization and P secretion.
Acknowledgements
The authors thank Dr. K Okuda, Okayama University, Japan, for the P antiserum. This study was supported by a Grant-in-Aid for Scientific Research of the Japan Society for the Promotion of Science (JSPS) and the Global COE Program, Ministry of Education, Culture, Sports, Science and Technology, Japan. This manuscript was reviewed by a professional service (KUSR002, Editage Cactus Communications Inc., Trevose, PA, USA) prior to submission.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SJ participated carried out all experiments and drafted the manuscript. KS participated in the design of the study, experiments of in vitro angiogenesis and drafted the manuscript. AN and AK collected the materials, and helped to immunohistochemistry and in vitro cell culture experiments. HJS and TS helped the design of the study and instructed technique of the experiments. AM supervised the study and helped to draft the manuscript. All authors read and approved the final manuscript.