Regulation of the VEGF-System in the Endometrium during Steroid-Replacement and Early Pregnancy of Pigs

H. Welter; K. Wollenhaupt; U. Tiemann; R. Einspanier

doi:10.1055/s-2003-37498

Experimental and Clinical Endocrinology & Diabetes, Inhaltsverzeichnis

Exp Clin Endocrinol Diabetes 2003; 111(1): 33-40
DOI: 10.1055/s-2003-37498

Article

J. A. Barth Verlag in Georg Thieme Verlag Stuttgart · New York

Regulation of the VEGF-System in the Endometrium during Steroid-Replacement and Early Pregnancy of Pigs

H. Welter¹ , K. Wollenhaupt² , U. Tiemann² , R. Einspanier¹

¹Institute of Physiology, Technical University of Munich, Freising, Germany
²Research Institute for the Biology of Farm Animals, Reproductive Biology, Dummerstorf, Germany

Abstract

Vascular endothelial growth factor (VEGF) and its specific receptors FLT-1 and FLK-1 represent an important ligand-receptor system involved in angiogenesis and permeability. These factors are supposed to be influenced by ovarian steroids involved in developmental changes in female reproductive tissue as oviduct and uterus. The aims of this study were to assess the expression of VEGF and its receptor mRNAs during the early implantation period in porcine endometrium using real-time RT-PCR. Furthermore, effects of estradiolbenzoate (EB) and progesterone (P) on endometrium of ovariectomized (ovx) pigs were examined by RT-PCR and immunohistochemistry. A complete VEGF system was found in endometrial tissue using RT-PCR detecting the main VEGF isoform 188 aa, FLT-1 and FLK-1. A significant upregulation of the mRNAs of VEGF and its receptors was observed in the endometrium during the peri-implantation when compared with the pre-implantation period. Regarding endometrium of non-pregnant ovx-pigs an application of P led to elevated transcript levels of VEGF whereas mRNA-expression was reduced after EB treatment compared to non-treated ovx-animals. When pigs were administrated EB and P simultanously, a decrease in VEGF mRNA concentration was recorded. For FLT-1, none of the steroids increased mRNA expression compared to the ovx-group. Analysis of FLK-1 receptor mRNA demonstrated that only after EB + P treatment mRNA-expression was stimulated but stayed unchanged after P and EB when compared with the ovx-group. Immunohistochemistry revealed FLK-1 and VEGF proteins in glandular and luminal epithelia of the endometrium with emphasized staining after P and P + EB treatment of ovx-pigs. Summarized, altered VEGF and FLK-1 expression during the implantation period as well as under steroid hormones suggest this growth factor as a potent regulator of hyperpermeability supporting the angiogenic process in porcine endometrium.

Key words

VEGF - Steroids - Endometrium - Pregnancy - Pig

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References

1 Ancelin M, Buteau-Lozano H, Meduri G, Osborne-Pellegrin M, Sordello S, Plouet J, Perrot-Applanat M. A dynamic shift of VEGF isoforms with a transient and selective progesterone-induced expression of VEGF189 regulates angiogenesis and vascular permeability in human uterus. Proc Natl Acad Sci USA. 2002; 99 6023-6028
2 Barleon B, Hauser S, Schollmann C, Weindel K, Marme D, Yayon A, Weich H A. Differential expression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells. J Cell Biochem. 1994; 54 56-66
3 Berisha B, Schams D, Kosmann M, Amselgruber W, Einspanier R. Expression and tissue concentration of vascular endothelial growth factor, its receptors and localisation in the bovine corpus luteum during estrous cycle. Biol Reprod. 2000; 63 1106-1114
4 Bogic L V, Brace R A, Cheung C Y. Developmental expression of vascular endothelial growth factor (VEGF) receptors and VEGF binding in ovine placenta and fetal membranes. Placenta. 2001; 22 265-275
5 Brown L F, Detmar M, Tognazzi K, Abu-Jawdeh G, Iruela-Arispe M L. Uterine smooth muscle cells express functional receptors (flt-1 and KDR) for vascular permeability factor/vascular endothelial growth factor. Lab Invest. 1997; 76 245-255
6 Chakraborty I, Das S K, Dey S K. Differential expression of vascular endothelial growth factor and its receptor mRNAs in the mouse uterus around the time of implantation. J Endocrinol. 1995; 147 339-352
7 Charnock-Jones D S, Sharkey A M, Rajput-Williams J, Burch D, Schofield J P, Fountain S A, Boocock C A, Smith S K. Identification and localisation of alternately spliced mRNAs for vascular endothelial growth factor in human uterus and estradiol regulation in endometrial carcinoma cell lines. Biol Reprod. 1993; 48 1120-1128
8 Charnock-Jones D S, Sharkey A M, Boocock C A, Ahmed A, Pelvin R, Ferrara N, Smith S K. Vascular endothelial growth factor receptor localisation and activation in human trophoblast and choriocarcinoma cells. Biol Reprod. 1994; 51 524-530
9 Charnock-Jones D S, Clark D E, Licence D, Day K, Wooding F BP, Smith S K. Distribution of vascular endothelial growth factor (VEGF) and its binding sites at the maternal-fetal interface during gestation in pigs. Reproduction. 2001; 122 753-760
10 Cheung C Y, Singh M, Ebaugh M J, Brace R A. Vascular endothelial growth factor gene expression in ovine placenta and fetal membranes. Am J Obstet Gynecol. 1995; 173 753-759
11 Chomczynski P, Sacchi N. Single Stepp method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162 156-159
12 Clark J H, Markaverich B M. Activation of ovarian steroids. Knobil E, Neill J The Physiology of Reproduction. New York; Raven Press 1988: 675-724
13 Clarke C L, Sutherland R L. Progestin regulation of cellular proliferation. Endocr Rev. 1990; 11 266-301
14 Cullinan-Bove K, Koos R D. Vascular endothelial growth factor/permeability factor expression in the rat uterus: Rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth. Endocrinology. 1993; 133 829-837
15 Das S K, Chakraborty I, Wang J, Dey S K, Hoffman L H. Expression of vascular endothelial growth factor (VEGF) and VEGF-messenger ribonucleic acids in the peri-implantation rabbit uterus. Biol Reprod. 1997; 56 1390-1399
16 Fong G H, Rossant J, Gerstenstein M, Breitman M L. Role of Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature. 1995; 376 66-70
17 Gabler C H, Einspanier A, Schams D, Einspanier R. Expression of vascular endothelial growth factor (VEGF) and its corresponding receptors (flt-1 and flk-1) in the bovine oviduct. Mol Reprod Dev. 1999; 53 376-383
18 Garrido C, Saule S, Gospodarowicz D. Transcriptional regulation of vascular endothelial growth factor gene expression in ovarian bovine granulosa cells. Growth Factors. 1993; 8 109-117
19 Gosh D, Sharkey A M, Charnock-Jones D S, Dhawan L, Dhara S, Smith S K, Sengupta J. Expression of vascular endothelial growth factor and placental growth factor (PIGF) in conceptus and during implantation in the rhesus monkey. Mol Hum Reprod. 2000; 6 935-941
20 Greb R R, Heikinheimo O, Williams R F, Hodgen G D, Godman A L. Vascular endothelial growth factor in primate endometrium is upregulated by oestrogen-receptor and progesterone-receptor ligands in vivo. Hum Reprod. 1997; 12 1280-1292
21 Halder J B, Zhao X, Paria B C, Klagsbrun M, Das S K, Dey S K. Differential expression of VEGF isoforms and VEGF 164-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF 164 in vascular permeability and angiogenesis during implantation. Genesis. 2000; 26 213-224
22 Houck K A, Ferrara N, Winer J, Cachianes G, Li B, Leung D W. The vascular endothelial growth factor family: identification of a fourth molecular species and characterisation of alternative splicing of RNA. Mol Endocrinol. 1991; 5 1806-1814
23 Houck K A, Leung D W, Rowland A M, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem. 1992; 267 26031-26037
24 Huang J C, Liu D Y, Dawood M Y. The expression of vascular endothelial growth factor isoforms in cultured human endometrial stromal cells and its regulation by 17 beta estradiol. Mol Hum Reprod. 1998; 4 603-607
25 Jakeman L B, Armanini M, Phillips H S, Ferrara N. Developmental expression of binding sites and messenger ribonucleic acid for vascular endothelial growth factor suggests a role for this protein in vasculogenesis and angiogenesis. Endocrinology. 1993; 133 848-859
26 Kendall R L, Thomas K A. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci USA. 1993; 90 10705-10709
27 Kendall R L, Wang G, Thomas K A. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem Biophys Res Commun. 1996; 226 324-328
28 Keys J L, King G J, Kennedy T G. Increased uterine vascular permeability at the time of embryonic attachment in the pig. Biol Reprod. 1986; 34 405-411
29 Kimberly A, Vonnahme K A, Wilson M E, Ford S P. Relationship between placental vascular endothelial growth factor expression and placental/endometrial vascularity in the pig. Biol Reprod. 2001; 64 1821-1825
30 Li X F, Gregory J, Ahmed A. Immunolocalisation of vascular endothelial growth factor in human endometrium. Growth Factors. 1994; 11 277-282
31 Mathwes W, Jordan C T, Gavin M, Jenskin N A, Copeland N G, Lemischka I R. A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit. Proc Natl Acad Sci USA. 1991; 88 9026-9030
32 Meduri G, Bausero P, Perrot-Applanat M. Expression of vascular endothelial growth factor receptors in the human endometrium: Modulation during the menstrual cycle. Biol Reprod. 2000; 62 439-447
33 Millauer B, Witzigmann-Voos S, Schnurch H, Martinez R, Moller N PH, Risau W, Ullrich A. High affinity VEGF binding and developmental expression suggest FLK-1 as a major regulator of vasculogenesis and angiogenesis. Cell. 1993; 72 835-846
34 Park J, Keller G A, Ferrara N. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular-matrix bound VEGF. Mol Biol Cell. 1993; 4 1317-1326
35 Perry J S, Rowlands I W. Early pregnancy in the pig. J Reprod Fertil. 1962; 4 175-188
36 Psychoyos A. Endocrine control of egg implantation. Greep RO, Astwood EG, Geiger SR Handbook of Physiology. Washington DC; American Physiology Society 1973: 187-215
37 Rabbani (Lofti-Miri) M, Rogers P AW. Role of vascular endothelial growth factor (VEGF) in endometrial vascular events before implantation in rats. Reproduction. 2001; 122 85-90
38 Reynolds L P, Kirsch J D, Kraft K C, Redmer D A. Time-course of the uterine response to estradiol-17β in ovariectomized ewes: expression of angiogenic factors. Biol Reprod. 1998; 59 613-620
39 Shalaby F, Rossant J, Yamaguchi T P, Gertsenstein M, Wu X F, Breitman M L, Schuh A C. Failure of blood-island formation and vasculogenesis in flk-1 deficient mice. Nature. 1995; 376 62-66
40 Shiraishi S, Nakagawa K, Kinukawa N, Nakano H, Sueishi K. Immunohistochemical localisation of vascular endothelial growth factor in the human placenta. Placenta. 1996; 17 111-121
41 Sharkey A M, Charnock-Jones D S, Boocock C A, Brown K D, Smith S K. Expression of mRNA for vascular endothelial growth factor in human placenta. J Reprod Fertil. 1993; 99 609-615
42 Shifren J L, Tseng J F, Zaloudek C J, Ryan I P, Meng Y G, Ferrara N, Jaffe R B, Taylor R N. Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab. 1996; 81 3112-3118
43 Shweiki D, Itin A, Neufeld G, Gitay-Goren H, Keshet E. Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogensis. J Clin Invest. 1993; 91 2235-2243
44 Spaziani E. Relationship between early vascular responses and growth in the rat uterus: stimulation of cell divison by estradiol and vasodilating amines. Endocrinology. 1963; 72 180-191
45 Stroband H W, Van der Lende T. Embryonic and uterine development during early pregnancy in pigs. J Reprod Fertil. 1990; 40 (Suppl) 261-277
46 Suzuma I, Mandai M, Tagaki H, Suzuma K, Otani A, Oh H, Kobayashi K, Honda Y. 17 beta-estradiol increases VEGF-receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells. Invest Ophthalmol Vis Sci. 1999; 40 2122-2129
47 Winther H, Ahmed A, Dantzer V. Immunohistochemical localisation of vascular endothelial growth factor (VEGF) and its two specific receptors, FLT-1 and KDR, in the porcine placenta and non pregnant uterus. Placenta. 1999; 20 35-43
48 Wollenhaupt K, Kettler A, Brussow K P, Schneider F, Kanitz W, Einspanier R. Regulation of the expression and bioactivation of the epidermal growth factor system by estradiol in pig oviduct and endometrium. Reprod Fertil Dev. 2001; 13 167-176
49 Yi X J, Jiang H Y, Lee K K, O WS, Tang P L, Chow P H. Expression of vascular endothelial growth factor and its receptors during embryonic implantation in the golden hamster (Mesocricetus auratus). Cell Tissue Res. 1999; 296 339-349

Ralf Einspanier

Institute of Physiology · Technical University of Munich

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85350 Freising

Germany

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