Anatomia, Histologia, Embryologia
ORIGINAL ARTICLE
Angiogenesis in The Ovary – The Most Important Regulatory Event for Follicle and Corpus Luteum Development and Function in Cow – An Overview B. Berisha1,2*, D. Schams1, D. Rodler3 and M. W. Pfaffl1 1 2 3
€t Mu €nchen, Freising, Germany; Physiology Weihenstephan, Technische Universita Faculty of Agriculture and Veterinary, University of Prishtina, Prishtina, Kosovo; Department of Veterinary Sciences, Ludwig-Maximilians-University Munich, Munich, Germany
*Correspondence: Tel.: +49 8161-713509 fax: +49 8161-714204 e-mail: [email protected] With 3 figures Received February 2015; accepted for publication February 2015 doi: 10.1111/ahe.12180
Summary In the ovary, the development of new capillaries from pre-existing ones (angiogenesis) is a complex event regulated by numerous local factors. The dominant regulators of angiogenesis in ovarian follicles and corpora lutea are the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), angiopoietin (ANPT) and hypoxia-inducible factor (HIF) family members. Antral follicles in our study were classified according to the oestradiol-17-beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5–5, 5–20, 20–180 and >180 ng/ml FF). The corresponding sizes of follicles were 5–7, 8–10, 10–13, 12–14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The corpora lutea (CL) in our study were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12 13–16 and >18 of the oestrous cycle and months 1–2, 3–4, 6–7 and >8 of pregnancy. The dominant regulators were measured at mRNA and protein expression levels; mRNA was quantified by RT-qPCR, hormone concentrations by RIA or EIA and their localization by immunohistochemistry. The highest expression for VEGF-A, FGF-2, IGF-1 and IGF-2, ANPT-2/ANPT-1 and HIF-1-alpha was found during final follicle maturation and in CL during the early luteal phase (days 1–4) followed by a lower plateau afterwards. The results suggest the importance of these factors for angiogenesis and maintenance of capillary structures for final follicle maturation, CL development and function.
Introduction The objective of this overview was to discuss recent findings, which are important for angiogenesis during final maturation of antral follicles and corpus luteum (CL) formation and function in the bovine ovary. Angiogenesis and ovarian function Angiogenesis is the preferred term for processes leading to the generation of new blood vessels through sprouting from already existing blood vessels in a process © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol.
involving the migration and proliferation of endothelial cells from pre-existing vessels (Abulafia and Sherer, 2000). Angiogenesis in the reproductive tract, and especially in the ovary, is nowadays well established to be of great importance for its development and function (Reynolds and Redmer, 1999; Fraser and Lunn, 2000; Berisha et al., 2010, 2013). The ovarian cycle by ruminants is characterized by repeated patterns of specific cellular proliferation, differentiation and transformation that accompanies follicular development and the formation and function of the CL. Formation of the CL is initiated by a series of morphological and biochemical
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changes in cells of the theca interna and granulosa (luteinization) of the preovulatory follicle (Schams and Berisha, 2002; Berisha and Schams, 2005). The growth of follicle or new CL tissue depends upon growth of new blood vessels and establishment of a functional blood supply. In the mature CL, nearly every parenchymal cell is in contact with one or more capillaries (Redmer and Reynolds, 1996). An improved understanding of the role of angiogenesis during follicular growth and corpora lutea formation and function obviously has important implications for the regulation of fertility in mammals. Due to the dynamic regulation, the CL provides an ideal model for studying the regulation of the very complex process of angiogenesis, as the most important regulatory event for follicle and corpus luteum development and function. Angiogenic factors in the ovary In the last years, relatively few studies have evaluated the effects of growth factors and cytokines on proliferation of ovarian tissues (follicular or luteal cells). The ovary was among the first organs in which local produced growth factors with angiogenic activity were detected (Gospodarowicz and Thakral, 1978). Of the numerous promoters of angiogenesis that have been identified, the most important factors appear to be vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), angiopoietins (ANPT) and hypoxia-inducible factor (HIF) family members. Material and Methods Entire reproductive tracts from cows were collected at a local slaughterhouse. The stage of the oestrous cycle was defined by macroscopic observation of the ovaries (follicles and corpus luteum). Follicles were classified according to the oestradiol-17-beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5–5, 5–20, 20–180 and >180 ng/ml FF). The corresponding sizes of follicles were 5– 7, 8–10, 10–13, 12–14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The CL was accordingly assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–18 and >18 (after regression) of oestrous cycle and months 1–2, 3–4, 6–7 and >8 of pregnancy. The expression of mRNA for the ligands and receptors was evaluated by means of RT-qPCR and ribonuclease protection assay (RPA). The hormone concentrations were quantified by RIA or EIA and protein localization by immunohistochemistry (Berisha et al., 2000a,b; Hayashi et al., 2003).
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Results and Discussion Vascular endothelial growth factors (VEGFs) in the bovine ovary VEGF family members VEGF-A, VEGF-B, VEGF-C and VEGF-D bind their specific VEGF receptors. It is now well established that alternative exon splicing of a single VEGF-A gene results in the generation of different molecular species, having, respectively, 121, 165, 189 and 206 amino acids after signal sequence cleavage (VEGF121, VEGF165, VEGF189, VEGF206). Bovine VEGF-A is one amino acid shorter than the human factor. VEGF-A expression is influenced by numerous factors including hypoxia, hormones, growth factors and cytokines. The biological activities of VEGF-A are mediated through high-affinity receptor tyrosine kinases (VEGFR-1, VEGFR-2 and VEGFR-3) genes, largely restricted to the vascular endothelium. VEGF-A has been shown to regulate most steps of the angiogenic process, including extracellular matrix degradation, endothelial cell migration, proliferation and tube formation (Ferrara and Davis-Smyth, 1997). In our study, VEGF-A mRNA expression in both follicle compartments (TI and GC), protein concentration in total follicle tissue and protein concentration in follicular fluid (FF) increased significantly with developmental stage of follicle growth (Fig. 1). As shown by immunohistochemistry (Fig. 3a), VEGF-A protein was clearly localized in TI (endothelial and theca cells) and stronger in GC of preovulatory follicles (Berisha et al., 2000b). The relative mRNA expression data for VEGF-A and its receptors in bovine CL during oestrous cycle are shown in Fig. 2. The highest VEGF-A mRNA expression in CL was detected during the early luteal phase (period of angiogenesis).
Fig. 1. Schematic presentation of expression profiles of VEGF, FGF, IGF, ANPT and HIF system members in bovine antral follicles during folliculogenesis.
© 2015 Blackwell Verlag GmbH Anat. Histol. Embryol.
B. Berisha et al.
Fig. 2. Schematic presentation of expressions profiles of VEGF, FGF, IGF, ANPT and HIF system members in bovine corpus luteum during corpus luteum formation, function and luteolysis.
The expression intensity of VEGF-A decreased significantly after day 7 to a lower plateau and further after regression of CL. A strong staining by immunochemistry is seen for VEGF-A in large luteal cells, but as shown in Fig. 3b, macrophages, endothelial cells and smooth muscle cells stain positive as well. The VEGF-A protein concentration in CL extracts by RIA showed a similar trend as for the mRNA expression (Berisha et al., 2000a). Comparable results were obtained in sheep CL (Redmer et al., 1996). Fibroblast growth factors (FGFs) in the bovine ovary Acidic FGF (FGF-1) and basic FGF (FGF-2) are members of a large family of 25 structurally related proteins controlling normal growth and differentiation of mesenchymal, epithelial and neuroectodermal cell types (Powers et al., 2000). The biological activities of FGFs are mediated through high-affinity tyrosine kinase receptors (FGFR-1 to FGFR-4). Alternative splicing in the extracellular region of FGFR-1 to FGFR-3 generates receptor variants (IIIb and IIIc) with different ligand binding affinities. FGF-2 has pleiotropic roles in many cell types and tissues; it is a mitogenic, angiogenic and survival factor which is involved in cell migration, in cell differentiation and in a variety of development processes. In our preovulatory follicles, FGF-2 and FGF receptor (FGFR) mRNA expression in TI increased significantly during final growth of follicles (Fig. 1). Histological observation revealed that FGF-2 protein was localized in theca tissue (cytoplasm of endothelial cells and pericytes) but not in GC (Fig. 3c). Our results suggest that VEGF and FGF families are involved in sprouting and migration of capillaries that accompanies the selection of the © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol.
Angiogenesis in the Ovary
preovulatory follicle resulting in an increased supply of nutrients and precursors and, therefore, supporting the growth of the dominant follicle (Berisha et al., 2000b). The mRNA expression of FGF-2 was high during the early luteal phase, decreased significantly on days 5–7 to a lower plateau and remained at that level during pregnancy. Immunohistochemical analysis showed specific labelling of endothelial cells, smooth muscle cells and luteal cells in CL (Schams et al., 1994). During the early stages (days 1–4), FGF-2 was detected intensely in cytoplasm of capillary endothelial cells and in smooth muscle cells of arteries (Fig. 3e). From days 6–7 onwards, FGF-2 was localized in the cytoplasm of large and small luteal cells during mid- and late luteal phase and during pregnancy in bovine CL (Fig. 3d). This is in accord with earlier observations in cows (Zheng et al., 1993). The FGF-1 protein could be localized in the cytoplasm of GC, predominantly in smooth muscle cells of blood vessels and weaker in theca cells. FGF-7 in follicles (Fig. 3f) was found immunohistochemically in GC with decreasing intensity, in the smooth muscle cells and the TI of preferably tertiary follicles (Berisha et al., 2004). Insulin-like growth factors (IGFs) in the bovine ovary The components of the IGF system play important roles during bovine follicle development and CL function. IGF1 and IGF-2 have stimulatory effects on progesterone secretion in pigs and cattle (Ge et al., 2000; Schams et al., 2001). IGF-1 and IGF-2 have also been implicated in neovascularization occurring in response to injury and localization in the bovine CL (Amselgruber et al., 1994). The interaction of the IGF receptors with IGF-1 or IGF-2 protects different types of cells from apoptosis including ovarian cells (Chun et al., 1994). The biological actions of IGF-1 and IGF-2, which include proliferation and cellular differentiation, are regulated by six binding proteins (IGFBP1-6). The IGF-1 in TI of our preovulatory follicles shows a relatively high expression in small follicles (low oestradiol levels) with the tendency of a decrease in mature follicles (Fig. 1). The IGF-2 mRNA expression in GC was very low, with no clear regulatory change in all classes examined. In contrast, IGF-2 is much stronger expressed in TI of all follicle classes. The IGFR-1 expression is relatively low in GC and TI in small follicle classes with a clear upregulation in the following classes for both tissues to a higher plateau. There was clear evidence for the local expression of IGFBP1-6 in TI and GC compartment with clear regulatory differences (Schams et al., 2002). In our CL samples, the highest mRNA expression for IGF-1, IGF-2 and IGFR-1 was observed during the early luteal phase (days 1-4), followed by a decrease to a lower level
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(a)
(b)
(c)
(d)
(e)
(f)
Fig. 3. Immunohistochemical localization of VEGF-A, FGF-1, FGF-2 and FGF-7 in the bovine ovary. (a) VEGF-A, preovulatory follicle. The immunohistochemical reaction is dominant in the granulosa cells (GC) and with weaker intensity in the theca interna (TI) and the endothelium of the thecal vessels. Scale bar = 20 lm (slightly modified from Berisha et al., 2000b). (b) VEGF-A, corpus luteum graviditatis. A distinct expression in the macrophages (arrows) can be found, also in the endothelium and the smooth muscle cells of vessels (V). Scale bar = 10 lm. (c) FGF-2, preovulatory follicle. While the granulosa cells (GC) and the theca interna (TI) remain unstained, the endothelium of the theca vessels (arrow) shows a strong reaction. Scale bar = 20 lm. (d) FGF-2, corpus luteum cyclicum in luteal phase. In particular, the cytoplasms of the large luteal cells (LLC) display strong reaction with the FGF-2 antibody. Scale bar = 10 lm. (e) FGF-1, corpus luteum cyclicum. A strong reaction with the FGF-1 antibody can be seen in the smooth muscle cells of blood vessels (V) and a distinct immunostaining within the endothelium (arrow). A moderate staining can be found especially in the cytoplasm of the large luteal cells (LLC). Scale bar = 10 lm. (f) FGF-7, tertiary follicle. The FGF-7 reaction in the granulosa cells (GC) is decreasing during follicular maturation. A continuous staining can be observed at the theca interna (TI) and the smooth muscle cells of the thecal vessels (V). Scale bar = 100 lm. (slightly modified from Berisha et al., 2004).
of the cyclic CL (Fig. 2). The pronounced mRNA expression for IGFR-1 and localization in luteal cells agrees with results in pigs CL (Ge et al., 2000). Based on our data, it can be assumed that the IGF system plays an important role especially for the development of the early CL by actions on luteinization of the large luteal cells and stimulation of oxytocin and progesterone production (Schams, 1987). The IGF system may have rather indirect effects on angiogenesis in the early CL by stimulatory actions for VEGF-A production in luteal cells as well as by stimulation of proliferation and differentiation of luteal and endothelial cells (Schams et al., 2001). In con-
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trast to IGF-1, the IGF-2-positive immunoreactivity was restricted to the perivascular fibroblasts of large blood vessels and to the pericytes of capillaries (Amselgruber et al., 1994). This supports our assumption that IGFs act as an autocrine/paracrine growth factor, affecting the proliferation and differentiation of these cells (Schams et al., 2002). Angiopoietins (ANPTs) in the bovine ovary A recent finding suggests that ANPT-1, ANPT-2 and their receptor kinases Tie1 and Tie2 may play an important © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol.
B. Berisha et al.
role for the modulation of angiogenesis and angiolysis in the CL during the oestrous cycle (Goede et al., 1998; Hayashi et al., 2004). Generally, the ANPT-Tie system is thought to act in concert with growth or survival factors such as VEGF-A. The ANPT-1 is necessary to maintain and stabilize blood vessels (Yancopoulos et al., 2000). On the contrary, ANPT-2, which acts as natural antagonist for ANPT-1, appears to cause endothelial cells (EC) to undergo active remodelling; thus, it destabilizes vascular structure. As ANPT-1 and ANPT-2 bind to the same receptor Tie2, the ratio of ANPT-2/ANPT-1 appears to play a crucial role for vascular stability. The high ANPT2/ANPT-1 ratio in microenvironment induces destabilization of blood vessels, which is a prerequisite for vascular formation and regression. In such conditions, the presence of angiogenic factor such as VEGF-A could determine the fate of destabilized blood vessel. When VEGF-A is high, a destabilization of blood vessels results in the formation of a new vascular network, whereas a lack of VEGF-A results in a regression of blood vessels (Yancopoulos et al., 2000). The RT-PCR analyses of our bovine follicles showed that both ANPT-1 and ANPT-2 were expressed in TI. The expression of ANPT-2 mRNA was decreased in both TI and GC of the mature follicles (Hayashi et al., 2004). This resulted in a decrease in the ANPT-2/ANPT-1 ratio, indicating that the blood vessels became more stable or mature. The increase of the ANPT-2/ANPT-1 ratio during the LH surge in our experiments seems to be a basic mechanism of vascular remodelling in follicles during periovulation (Hayashi et al., 2004). The results of ANPT expression during angiogenesis are schematically presented and summarized in Figs 1 and 2, and suggest an important role of these growth factor systems in angiogenesis during follicular development and CL formation and function. In early and regressing CL, the ANPT-2/ANPT-1 ratio was higher than those of mid-CL and pregnancy CL. Our data support the earlier results of Goede et al. (1998). Hypoxia-inducible factors (HIFs) in the bovine ovary HIFs bind hypoxia responsive element (HRE) and play an important role in the transcriptional regulation of VEGFA by hypoxia (Forsythe et al., 1996). The regulatory role of HIFs in VEGF-A expression and angiogenesis was demonstrated in several tumour tissues (Zhong et al., 1999). Expression of the HIF-1-alpha, as well as its association with VEGF-A expression and angiogenesis, was reported to occur in ovarian cancer cells (Horiuchi et al., 2002). Few data exist on the expression of HIF-1-alpha mRNA and its localization in the normal adult ovary. In our preovulatory follicles, HIF-1-alpha mRNA expression in TI and GC increased significantly during © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol.
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follicle maturation (Fig. 1). Expression of HIF-1-alpha mRNA in pig follicles appeared to be more evident in GC than in the theca compartment (Boonyaprakob et al., 2005). Other studies demonstrated that production of VEGF-A or IGFs by hypoxia is primarily mediated by HIFs (Zelzer et al., 1998). In our study, the mRNA expression of HIF-1-alpha was high during CL formation (period of angiogenesis), followed by a decrease to a constantly lower level during the oestrous cycle and pregnancy (Fig. 2). The highest mRNA expression levels in pig CL were observed during early luteal phase (Boonyaprakob et al., 2005), which agrees with our HIF-1-alpha mRNA expression data in the cow. A variety of factors including oxygen tension, gonadotropins and growth factors may regulate the HIF-1-modulated VEGF-A transcription and angiogenesis (Boonyaprakob et al., 2005). Our results provide additional evidence for the important role of HIF-1-alpha in the development and angiogenesis during final follicular growth and CL formation and function in cow (Figs 1 and 2). General conclusions The schematically presented data for expression of VEGF, FGF, IGF, ANPT and HIF family members and protein localization of VEGF and FGF family members in preovulatory follicles and bovine CL are summarized in Figs 1, 2 and 3. These data suggests an important role of these systems in angiogenesis of growing follicles and CL formation. Furthermore, additional maintenance functions of these factors for capillary endothelial cells have been postulated. Alan et al. (1995) showed that a certain threshold concentration of VEGF-A is required to inhibit apoptosis of the endothelial cells and is essential for the stabilization of the newly formed blood vessels. FGF-2 may act as a differentiation and amplification system for VEGF. When added simultaneously, VEGF-A and FGF-2 induced an in vitro angiogenic response that was far greater than an additive action and occurred with greater rapidity than the response to either cytokine alone (Pepper et al., 1992; Gabler et al., 2004). This synergism was confirmed recently under in vivo conditions (Asahara et al., 1995). A similar synergistic system of VEGF-A/FGF-2 seems to exist in dominant bovine follicles (Berisha et al., 2000b). Not all the factors identified may have direct effects, but may act together in a complex way by synergistic or antagonistic mechanisms. Acknowledgements The authors gratefully acknowledge the technical assistance of Mrs Inge Celler, Mrs Gabi Russmeier, Mrs Monica Settles, Mrs Angela Servatius and Mr Y. G€ ock.
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References Abulafia, O., and D. M. Sherer, 2000: Angiogenesis of the ovary. Am. J. Obstet. Gynec. 182, 240–246. Alan, T., I. Hemo, A. Itin, J. Peer, J. Stone, and E. Keshet, 1995: Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat. Med. 1, 1024–1028. Amselgruber, W., F. Sinowatz, D. Schams, and A. Skottner, 1994: Immunohistochemical aspects of insulin-like growth factors I and II in the bovine corpus luteum. J. Reprod. Fertil.. 101, 445–451. Asahara, T., C. Bauters, and L. P. Zheng, 1995: Synergistic effect of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in vivo. Circulation. 91, II365–II371. Berisha, B., and D. Schams, 2005: Ovarian function in ruminants. Domest. Anim. Endocrinol. 29, 305–317. Berisha, B., D. Schams, M. Kosmann, W. Amselgruber, and R. Einspanier, 2000a: Expression and tissue concentration of vascular endothelial growth factor, its receptors and localization in the bovine corpus luteum during estrous cycle and pregnancy. Biol. Reprod. 63, 1106–1114. Berisha, B., D. Schams, M. Kosmann, W. Amselgruber, and R. Einspanier, 2000b: Expression and localisation of vascular endothelial growth factor and basic fibroblast growth during the final growth of bovine ovarian follicles. J. Endocrinol.. 167, 371–382. Berisha, B., F. Sinowatz, and D. Schams, 2004: Expression and localization of fibroblast growth factor (FGF) family members during the final growth of bovine ovarian follicles. Mol. Rep. Dev. 67, 162–171. Berisha, B., H. H. Meyer, and D. Schams, 2010: Effect of prostaglandin F2 alpha on local luteotropic and angiogenic factors during induced functional luteolysis in the bovine corpus luteum. Biol. Reprod. 82, 940–947. Berisha, B., S. Schilffarth, R. Kenngott, F. Sinowatz, H. H. Meyer, and D. Schams, 2013: Expression of lymphangiogenic vascular endothelial growth factor family members in bovine corpus luteum. Anat. Histol. Embryol.. 42, 292–303. Boonyaprakob, U., J. E. Gadsby, V. Hedgpeth, P. A. Routh, and G. W. Almond, 2005: Expression and localization of hypoxia inducible factor-1alpha mRNA in the porcine ovary. Can. J. Vet. Res.. 69, 215–222. Chun, S. Y., H. Billig, and J. L. Tilly, 1994: Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor I. Endocrinology. 135, 1845–1853. Ferrara, N., and T. Davis-Smyth, 1997: The biology of vascular endothelial growth factor. Endocr. Rev. 18, 4–25. Forsythe, J. A., B. H. Jiang, and N. V. Iyer, 1996: Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol.. 16, 4604–4613. Fraser, H. M., and S. F. Lunn, 2000: Angiogenesis and its control in the female reproductive system. Br. Med. Bull.. 56, 787–797.
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Gabler, C., A. Plath-Gabler, G. J. Killian, B. Berisha, and D. Schams, 2004: Expression pattern of fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) system members in bovine corpus luteum endothelial cells during treatment with FGF-2, VEGF or oestradiol. Reprod. Domest. Anim.. 39, 321–327. Ge, Z., W. E. Nicholson, D. M. Plotner, C. E. Farin, and J. E. Gadsby, 2000: Insulin-like growth factor I receptor mRNA and protein expression in pig corpora lutea. J. Reprod. Fertil.. 120, 109–114. Goede, V., T. Schmidt, S. Kimmina, D. Kozian, and H. G. Augustin, 1998: Analysis of blood vessel maturation processes during cycle ovarian angiogenesis. Lab. Invest.. 78, 1385– 1394. Gospodarowicz, D., and K. K. Thakral, 1978: Production of a corpus luteum angiogenic factor responsible for proliferation of capillaries and neovascularization of the corpus luteum. Proc. Natl Aacd. Sci. USA. 75, 847–851. Hayashi, K. G., T. J. Acosta, M. Tetsuka, B. Berisha, M. Matsui, D. Schams, M. Ohtani, and A. Miyamoto, 2003: Involvement of angiopoietin-tie system in bovine follicular development and atresia: messenger RNA expression in theca interna and effect on steroid secretion. Biol. Reprod.. 69, 2078–2084. Hayashi, K. G., B. Berisha, M. Matsui, D. Schams, and A. Miyamoto, 2004: Expression of mRNA for the angiopoietin-tie system in granulosa cells during follicular development in cows. J. Reprod. Dev. 50, 477–480. Horiuchi, A., T. Imai, and M. Shimizu, 2002: Hypoxia-induced changes in the expression of VEGF, HIF-1alpha and cell cycle-related molecules in ovarian cancer cells. Anticancer Res.. 22, 2697–2702. Pepper, M. S., N. Ferrara, L. Orci, and R. Montesano, 1992: Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem. Biophys. Res. Commun.. 189, 824–831. Powers, C. J., S. W. McLeskey, and A. Wellstein, 2000: Fibroblast growth factors, their receptors and signalling. Endocrin. Rel. Cancer. 7, 165–197. Redmer, D. A., and L. P. Reynolds, 1996: Angiogenesis in the ovary. Rev. Reprod. 1, 182–192. Redmer, D. A., Y. Dai, J. Li, D. S. Charnock-Jones, S. K. Smith, L. P. Reynolds, and R. M. Moor, 1996: Characterization and expression of vascular endothelial growth factor (VEGF) in the ovine corpus luteum. J. Reprod. Fert. 108, 157–165. Reynolds, L. P., and D. A. Redmer, 1999: Growth and development of the corpus luteum. J. Reprod. Fertil. Suppl.. 54, 181–191. Schams, D., 1987: Luteal peptides and intercellular communication. J. Reprod. Fertil. Suppl.. 34, 87–99. Schams, D., and B. Berisha, 2002: Angiogenic Factors (VEGF, FGF and IGF) in the bovine corpus luteum. J. Reprod. Dev. 48, 233–242.
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Schams, D., W. Amselgruber, R. Einspanier, F. Sinowatz, and D. Gospodarowicz, 1994: Localization and tissue concentration of basic fibroblast growth factor in the bovine corpus luteum. Endocrine. 2, 907–912. Schams, D., M. Kosmann, B. Berisha, W. M. Amselgruber, and A. Miyamoto, 2001: Stimulatory and synergistic effects of luteinising hormone and insulin like growth factor 1 on the secretion of vascular endothelial growth factor and progesterone of cultured bovine granulosa cells. Exp. Clin. Endocrinol. Diabetes. 109, 155–162. Schams, D., B. Berisha, M. Kosmann, and W. M. Amselgruber, 2002: Expression and localization of IGF family members in bovine antral follicles during final growth and in luteal tissue during different stages of estrous cycle and pregnancy. Domest. Anim. Endocrinol.. 22, 51–72.
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Yancopoulos, G. D., S. Davis, N. W. Gale, J. S. Rudge, S. J. Wiegand, and J. Holash, 2000: Vascular-specific growth factors and blood vessel formation. Nature. 14, 242–248. Zelzer, E., Y. Levy, C. Kahana, B. Z. Shilo, M. Rubinstein, and B. Cohen, 1998: Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT. EMBO J.. 17, 5085–5094. Zheng, J., D. A. Redmer, and L. P. Reynolds, 1993: Vascular development and heparin-binding growth factors in the bovine corpus luteum at several stages of the estrous cycle. Biol. Reprod.. 49, 1177–1189. Zhong, H., A. M. De Marzo, and E. Laughner, 1999: Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res.. 59, 5830– 5835.
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ORIGINAL ARTICLE
Angiogenesis in The Ovary – The Most Important Regulatory Event for Follicle and Corpus Luteum Development and Function in Cow – An Overview B. Berisha1,2*, D. Schams1, D. Rodler3 and M. W. Pfaffl1 1 2 3
€t Mu €nchen, Freising, Germany; Physiology Weihenstephan, Technische Universita Faculty of Agriculture and Veterinary, University of Prishtina, Prishtina, Kosovo; Department of Veterinary Sciences, Ludwig-Maximilians-University Munich, Munich, Germany
*Correspondence: Tel.: +49 8161-713509 fax: +49 8161-714204 e-mail: [email protected] With 3 figures Received February 2015; accepted for publication February 2015 doi: 10.1111/ahe.12180
Summary In the ovary, the development of new capillaries from pre-existing ones (angiogenesis) is a complex event regulated by numerous local factors. The dominant regulators of angiogenesis in ovarian follicles and corpora lutea are the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), angiopoietin (ANPT) and hypoxia-inducible factor (HIF) family members. Antral follicles in our study were classified according to the oestradiol-17-beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5–5, 5–20, 20–180 and >180 ng/ml FF). The corresponding sizes of follicles were 5–7, 8–10, 10–13, 12–14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The corpora lutea (CL) in our study were assigned to the following stages: days 1–2, 3–4, 5–7, 8–12 13–16 and >18 of the oestrous cycle and months 1–2, 3–4, 6–7 and >8 of pregnancy. The dominant regulators were measured at mRNA and protein expression levels; mRNA was quantified by RT-qPCR, hormone concentrations by RIA or EIA and their localization by immunohistochemistry. The highest expression for VEGF-A, FGF-2, IGF-1 and IGF-2, ANPT-2/ANPT-1 and HIF-1-alpha was found during final follicle maturation and in CL during the early luteal phase (days 1–4) followed by a lower plateau afterwards. The results suggest the importance of these factors for angiogenesis and maintenance of capillary structures for final follicle maturation, CL development and function.
Introduction The objective of this overview was to discuss recent findings, which are important for angiogenesis during final maturation of antral follicles and corpus luteum (CL) formation and function in the bovine ovary. Angiogenesis and ovarian function Angiogenesis is the preferred term for processes leading to the generation of new blood vessels through sprouting from already existing blood vessels in a process © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol.
involving the migration and proliferation of endothelial cells from pre-existing vessels (Abulafia and Sherer, 2000). Angiogenesis in the reproductive tract, and especially in the ovary, is nowadays well established to be of great importance for its development and function (Reynolds and Redmer, 1999; Fraser and Lunn, 2000; Berisha et al., 2010, 2013). The ovarian cycle by ruminants is characterized by repeated patterns of specific cellular proliferation, differentiation and transformation that accompanies follicular development and the formation and function of the CL. Formation of the CL is initiated by a series of morphological and biochemical
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changes in cells of the theca interna and granulosa (luteinization) of the preovulatory follicle (Schams and Berisha, 2002; Berisha and Schams, 2005). The growth of follicle or new CL tissue depends upon growth of new blood vessels and establishment of a functional blood supply. In the mature CL, nearly every parenchymal cell is in contact with one or more capillaries (Redmer and Reynolds, 1996). An improved understanding of the role of angiogenesis during follicular growth and corpora lutea formation and function obviously has important implications for the regulation of fertility in mammals. Due to the dynamic regulation, the CL provides an ideal model for studying the regulation of the very complex process of angiogenesis, as the most important regulatory event for follicle and corpus luteum development and function. Angiogenic factors in the ovary In the last years, relatively few studies have evaluated the effects of growth factors and cytokines on proliferation of ovarian tissues (follicular or luteal cells). The ovary was among the first organs in which local produced growth factors with angiogenic activity were detected (Gospodarowicz and Thakral, 1978). Of the numerous promoters of angiogenesis that have been identified, the most important factors appear to be vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), angiopoietins (ANPT) and hypoxia-inducible factor (HIF) family members. Material and Methods Entire reproductive tracts from cows were collected at a local slaughterhouse. The stage of the oestrous cycle was defined by macroscopic observation of the ovaries (follicles and corpus luteum). Follicles were classified according to the oestradiol-17-beta (E2) content in follicular fluid (FF) and were divided into five classes (E2 < 0.5, 0.5–5, 5–20, 20–180 and >180 ng/ml FF). The corresponding sizes of follicles were 5– 7, 8–10, 10–13, 12–14 and >14 mm, respectively. Follicle tissue was separated in theca interna (TI) and granulosa cells (GC). The CL was accordingly assigned to the following stages: days 1–2, 3–4, 5–7, 8–12, 13–18 and >18 (after regression) of oestrous cycle and months 1–2, 3–4, 6–7 and >8 of pregnancy. The expression of mRNA for the ligands and receptors was evaluated by means of RT-qPCR and ribonuclease protection assay (RPA). The hormone concentrations were quantified by RIA or EIA and protein localization by immunohistochemistry (Berisha et al., 2000a,b; Hayashi et al., 2003).
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Results and Discussion Vascular endothelial growth factors (VEGFs) in the bovine ovary VEGF family members VEGF-A, VEGF-B, VEGF-C and VEGF-D bind their specific VEGF receptors. It is now well established that alternative exon splicing of a single VEGF-A gene results in the generation of different molecular species, having, respectively, 121, 165, 189 and 206 amino acids after signal sequence cleavage (VEGF121, VEGF165, VEGF189, VEGF206). Bovine VEGF-A is one amino acid shorter than the human factor. VEGF-A expression is influenced by numerous factors including hypoxia, hormones, growth factors and cytokines. The biological activities of VEGF-A are mediated through high-affinity receptor tyrosine kinases (VEGFR-1, VEGFR-2 and VEGFR-3) genes, largely restricted to the vascular endothelium. VEGF-A has been shown to regulate most steps of the angiogenic process, including extracellular matrix degradation, endothelial cell migration, proliferation and tube formation (Ferrara and Davis-Smyth, 1997). In our study, VEGF-A mRNA expression in both follicle compartments (TI and GC), protein concentration in total follicle tissue and protein concentration in follicular fluid (FF) increased significantly with developmental stage of follicle growth (Fig. 1). As shown by immunohistochemistry (Fig. 3a), VEGF-A protein was clearly localized in TI (endothelial and theca cells) and stronger in GC of preovulatory follicles (Berisha et al., 2000b). The relative mRNA expression data for VEGF-A and its receptors in bovine CL during oestrous cycle are shown in Fig. 2. The highest VEGF-A mRNA expression in CL was detected during the early luteal phase (period of angiogenesis).
Fig. 1. Schematic presentation of expression profiles of VEGF, FGF, IGF, ANPT and HIF system members in bovine antral follicles during folliculogenesis.
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Fig. 2. Schematic presentation of expressions profiles of VEGF, FGF, IGF, ANPT and HIF system members in bovine corpus luteum during corpus luteum formation, function and luteolysis.
The expression intensity of VEGF-A decreased significantly after day 7 to a lower plateau and further after regression of CL. A strong staining by immunochemistry is seen for VEGF-A in large luteal cells, but as shown in Fig. 3b, macrophages, endothelial cells and smooth muscle cells stain positive as well. The VEGF-A protein concentration in CL extracts by RIA showed a similar trend as for the mRNA expression (Berisha et al., 2000a). Comparable results were obtained in sheep CL (Redmer et al., 1996). Fibroblast growth factors (FGFs) in the bovine ovary Acidic FGF (FGF-1) and basic FGF (FGF-2) are members of a large family of 25 structurally related proteins controlling normal growth and differentiation of mesenchymal, epithelial and neuroectodermal cell types (Powers et al., 2000). The biological activities of FGFs are mediated through high-affinity tyrosine kinase receptors (FGFR-1 to FGFR-4). Alternative splicing in the extracellular region of FGFR-1 to FGFR-3 generates receptor variants (IIIb and IIIc) with different ligand binding affinities. FGF-2 has pleiotropic roles in many cell types and tissues; it is a mitogenic, angiogenic and survival factor which is involved in cell migration, in cell differentiation and in a variety of development processes. In our preovulatory follicles, FGF-2 and FGF receptor (FGFR) mRNA expression in TI increased significantly during final growth of follicles (Fig. 1). Histological observation revealed that FGF-2 protein was localized in theca tissue (cytoplasm of endothelial cells and pericytes) but not in GC (Fig. 3c). Our results suggest that VEGF and FGF families are involved in sprouting and migration of capillaries that accompanies the selection of the © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol.
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preovulatory follicle resulting in an increased supply of nutrients and precursors and, therefore, supporting the growth of the dominant follicle (Berisha et al., 2000b). The mRNA expression of FGF-2 was high during the early luteal phase, decreased significantly on days 5–7 to a lower plateau and remained at that level during pregnancy. Immunohistochemical analysis showed specific labelling of endothelial cells, smooth muscle cells and luteal cells in CL (Schams et al., 1994). During the early stages (days 1–4), FGF-2 was detected intensely in cytoplasm of capillary endothelial cells and in smooth muscle cells of arteries (Fig. 3e). From days 6–7 onwards, FGF-2 was localized in the cytoplasm of large and small luteal cells during mid- and late luteal phase and during pregnancy in bovine CL (Fig. 3d). This is in accord with earlier observations in cows (Zheng et al., 1993). The FGF-1 protein could be localized in the cytoplasm of GC, predominantly in smooth muscle cells of blood vessels and weaker in theca cells. FGF-7 in follicles (Fig. 3f) was found immunohistochemically in GC with decreasing intensity, in the smooth muscle cells and the TI of preferably tertiary follicles (Berisha et al., 2004). Insulin-like growth factors (IGFs) in the bovine ovary The components of the IGF system play important roles during bovine follicle development and CL function. IGF1 and IGF-2 have stimulatory effects on progesterone secretion in pigs and cattle (Ge et al., 2000; Schams et al., 2001). IGF-1 and IGF-2 have also been implicated in neovascularization occurring in response to injury and localization in the bovine CL (Amselgruber et al., 1994). The interaction of the IGF receptors with IGF-1 or IGF-2 protects different types of cells from apoptosis including ovarian cells (Chun et al., 1994). The biological actions of IGF-1 and IGF-2, which include proliferation and cellular differentiation, are regulated by six binding proteins (IGFBP1-6). The IGF-1 in TI of our preovulatory follicles shows a relatively high expression in small follicles (low oestradiol levels) with the tendency of a decrease in mature follicles (Fig. 1). The IGF-2 mRNA expression in GC was very low, with no clear regulatory change in all classes examined. In contrast, IGF-2 is much stronger expressed in TI of all follicle classes. The IGFR-1 expression is relatively low in GC and TI in small follicle classes with a clear upregulation in the following classes for both tissues to a higher plateau. There was clear evidence for the local expression of IGFBP1-6 in TI and GC compartment with clear regulatory differences (Schams et al., 2002). In our CL samples, the highest mRNA expression for IGF-1, IGF-2 and IGFR-1 was observed during the early luteal phase (days 1-4), followed by a decrease to a lower level
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(a)
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Fig. 3. Immunohistochemical localization of VEGF-A, FGF-1, FGF-2 and FGF-7 in the bovine ovary. (a) VEGF-A, preovulatory follicle. The immunohistochemical reaction is dominant in the granulosa cells (GC) and with weaker intensity in the theca interna (TI) and the endothelium of the thecal vessels. Scale bar = 20 lm (slightly modified from Berisha et al., 2000b). (b) VEGF-A, corpus luteum graviditatis. A distinct expression in the macrophages (arrows) can be found, also in the endothelium and the smooth muscle cells of vessels (V). Scale bar = 10 lm. (c) FGF-2, preovulatory follicle. While the granulosa cells (GC) and the theca interna (TI) remain unstained, the endothelium of the theca vessels (arrow) shows a strong reaction. Scale bar = 20 lm. (d) FGF-2, corpus luteum cyclicum in luteal phase. In particular, the cytoplasms of the large luteal cells (LLC) display strong reaction with the FGF-2 antibody. Scale bar = 10 lm. (e) FGF-1, corpus luteum cyclicum. A strong reaction with the FGF-1 antibody can be seen in the smooth muscle cells of blood vessels (V) and a distinct immunostaining within the endothelium (arrow). A moderate staining can be found especially in the cytoplasm of the large luteal cells (LLC). Scale bar = 10 lm. (f) FGF-7, tertiary follicle. The FGF-7 reaction in the granulosa cells (GC) is decreasing during follicular maturation. A continuous staining can be observed at the theca interna (TI) and the smooth muscle cells of the thecal vessels (V). Scale bar = 100 lm. (slightly modified from Berisha et al., 2004).
of the cyclic CL (Fig. 2). The pronounced mRNA expression for IGFR-1 and localization in luteal cells agrees with results in pigs CL (Ge et al., 2000). Based on our data, it can be assumed that the IGF system plays an important role especially for the development of the early CL by actions on luteinization of the large luteal cells and stimulation of oxytocin and progesterone production (Schams, 1987). The IGF system may have rather indirect effects on angiogenesis in the early CL by stimulatory actions for VEGF-A production in luteal cells as well as by stimulation of proliferation and differentiation of luteal and endothelial cells (Schams et al., 2001). In con-
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trast to IGF-1, the IGF-2-positive immunoreactivity was restricted to the perivascular fibroblasts of large blood vessels and to the pericytes of capillaries (Amselgruber et al., 1994). This supports our assumption that IGFs act as an autocrine/paracrine growth factor, affecting the proliferation and differentiation of these cells (Schams et al., 2002). Angiopoietins (ANPTs) in the bovine ovary A recent finding suggests that ANPT-1, ANPT-2 and their receptor kinases Tie1 and Tie2 may play an important © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol.
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role for the modulation of angiogenesis and angiolysis in the CL during the oestrous cycle (Goede et al., 1998; Hayashi et al., 2004). Generally, the ANPT-Tie system is thought to act in concert with growth or survival factors such as VEGF-A. The ANPT-1 is necessary to maintain and stabilize blood vessels (Yancopoulos et al., 2000). On the contrary, ANPT-2, which acts as natural antagonist for ANPT-1, appears to cause endothelial cells (EC) to undergo active remodelling; thus, it destabilizes vascular structure. As ANPT-1 and ANPT-2 bind to the same receptor Tie2, the ratio of ANPT-2/ANPT-1 appears to play a crucial role for vascular stability. The high ANPT2/ANPT-1 ratio in microenvironment induces destabilization of blood vessels, which is a prerequisite for vascular formation and regression. In such conditions, the presence of angiogenic factor such as VEGF-A could determine the fate of destabilized blood vessel. When VEGF-A is high, a destabilization of blood vessels results in the formation of a new vascular network, whereas a lack of VEGF-A results in a regression of blood vessels (Yancopoulos et al., 2000). The RT-PCR analyses of our bovine follicles showed that both ANPT-1 and ANPT-2 were expressed in TI. The expression of ANPT-2 mRNA was decreased in both TI and GC of the mature follicles (Hayashi et al., 2004). This resulted in a decrease in the ANPT-2/ANPT-1 ratio, indicating that the blood vessels became more stable or mature. The increase of the ANPT-2/ANPT-1 ratio during the LH surge in our experiments seems to be a basic mechanism of vascular remodelling in follicles during periovulation (Hayashi et al., 2004). The results of ANPT expression during angiogenesis are schematically presented and summarized in Figs 1 and 2, and suggest an important role of these growth factor systems in angiogenesis during follicular development and CL formation and function. In early and regressing CL, the ANPT-2/ANPT-1 ratio was higher than those of mid-CL and pregnancy CL. Our data support the earlier results of Goede et al. (1998). Hypoxia-inducible factors (HIFs) in the bovine ovary HIFs bind hypoxia responsive element (HRE) and play an important role in the transcriptional regulation of VEGFA by hypoxia (Forsythe et al., 1996). The regulatory role of HIFs in VEGF-A expression and angiogenesis was demonstrated in several tumour tissues (Zhong et al., 1999). Expression of the HIF-1-alpha, as well as its association with VEGF-A expression and angiogenesis, was reported to occur in ovarian cancer cells (Horiuchi et al., 2002). Few data exist on the expression of HIF-1-alpha mRNA and its localization in the normal adult ovary. In our preovulatory follicles, HIF-1-alpha mRNA expression in TI and GC increased significantly during © 2015 Blackwell Verlag GmbH Anat. Histol. Embryol.
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follicle maturation (Fig. 1). Expression of HIF-1-alpha mRNA in pig follicles appeared to be more evident in GC than in the theca compartment (Boonyaprakob et al., 2005). Other studies demonstrated that production of VEGF-A or IGFs by hypoxia is primarily mediated by HIFs (Zelzer et al., 1998). In our study, the mRNA expression of HIF-1-alpha was high during CL formation (period of angiogenesis), followed by a decrease to a constantly lower level during the oestrous cycle and pregnancy (Fig. 2). The highest mRNA expression levels in pig CL were observed during early luteal phase (Boonyaprakob et al., 2005), which agrees with our HIF-1-alpha mRNA expression data in the cow. A variety of factors including oxygen tension, gonadotropins and growth factors may regulate the HIF-1-modulated VEGF-A transcription and angiogenesis (Boonyaprakob et al., 2005). Our results provide additional evidence for the important role of HIF-1-alpha in the development and angiogenesis during final follicular growth and CL formation and function in cow (Figs 1 and 2). General conclusions The schematically presented data for expression of VEGF, FGF, IGF, ANPT and HIF family members and protein localization of VEGF and FGF family members in preovulatory follicles and bovine CL are summarized in Figs 1, 2 and 3. These data suggests an important role of these systems in angiogenesis of growing follicles and CL formation. Furthermore, additional maintenance functions of these factors for capillary endothelial cells have been postulated. Alan et al. (1995) showed that a certain threshold concentration of VEGF-A is required to inhibit apoptosis of the endothelial cells and is essential for the stabilization of the newly formed blood vessels. FGF-2 may act as a differentiation and amplification system for VEGF. When added simultaneously, VEGF-A and FGF-2 induced an in vitro angiogenic response that was far greater than an additive action and occurred with greater rapidity than the response to either cytokine alone (Pepper et al., 1992; Gabler et al., 2004). This synergism was confirmed recently under in vivo conditions (Asahara et al., 1995). A similar synergistic system of VEGF-A/FGF-2 seems to exist in dominant bovine follicles (Berisha et al., 2000b). Not all the factors identified may have direct effects, but may act together in a complex way by synergistic or antagonistic mechanisms. Acknowledgements The authors gratefully acknowledge the technical assistance of Mrs Inge Celler, Mrs Gabi Russmeier, Mrs Monica Settles, Mrs Angela Servatius and Mr Y. G€ ock.
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Yancopoulos, G. D., S. Davis, N. W. Gale, J. S. Rudge, S. J. Wiegand, and J. Holash, 2000: Vascular-specific growth factors and blood vessel formation. Nature. 14, 242–248. Zelzer, E., Y. Levy, C. Kahana, B. Z. Shilo, M. Rubinstein, and B. Cohen, 1998: Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT. EMBO J.. 17, 5085–5094. Zheng, J., D. A. Redmer, and L. P. Reynolds, 1993: Vascular development and heparin-binding growth factors in the bovine corpus luteum at several stages of the estrous cycle. Biol. Reprod.. 49, 1177–1189. Zhong, H., A. M. De Marzo, and E. Laughner, 1999: Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res.. 59, 5830– 5835.
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