Expressions of VEGF and its receptors in rat corpus luteum during interferon alpha administration in early and pseudopregnancy

It is accepted that angiogenesis plays an important role in the development of the corpus luteum (CL) and is probably necessary for normal lutein cell function. A number of drugs currently being tested in clinical trials as possible angiogenesis inhibitors were not originally developed with the intention of suppressing tumor angiogenesis. Interferon alpha (IFN-alpha) is one of the notable examples of such 'accidental angiogenesis inhibitors' and daily administration of IFN-alpha is known to suppress tumor growth, tumor vascularization, and down-regulation of various growth factors. We investigated the effects of IFN-alpha treatment on the expression of vascular endothelial growth factor (VEGF), and its receptors KDR and Flt-1, and CD34 in CL during the first week of pseudopregnancy and pregnancy in hormonally induced rat ovaries by immunohistochemistry and Western blot techniques. Basal body temperatures of the drug-treated rats, as an indicator of treatment effect, were determined daily and were increased significantly when compared to controls (38.03 +/- 0.18 vs. 36.6 +/- 0.1 degrees C), respectively. The effect of IFN-alpha treatment was minimal when the entire week was evaluated, however, the expression of VEGF decreased at 3rd, 5th, and 7th days of both pregnancy and pseudopregnancy, when compared to the 1st day, whereas there was not a such alteration in the untreated rats regarding these days. The daily subcutaneous administrations of 672.500 U IFN-alpha2b had minimal effects on the expressions of VEGF, and its two receptors KDR and Flt-1 in either pregnant or pseudopregnant corpora lutea utilizing HSCORE.     

Expression of the vascular endothelial growth factor-receptor system in the porcine endometrium throughout the estrous cycle and early pregnancy

The objective of this study was to investigate the protein and mRNA expression of vascular endothelial growth factor (VEGF), VEGFR-1 (fms-like tyrosine kinase, Flt-1) and VEGFR-2 (fetal liver kinase-1/kinase insert domain-containing receptor, Flk-1/KDR) in the endometrium during the estrous cycle and early pregnancy in pigs. The VEGF-receptor system was localized in epithelial and stromal cells, blood vessels, and myometrium. Western blot analysis showed higher levels of VEGF protein during the periovulatory and periimplantation periods (P < 0.001, and P < 0.05, respectively). Constant expression of VEGF mRNA during the cycle and significant upregulation on Days 22-25 of gestation (vs. Days 9-17; P < 0.001) was observed. Stable levels of VEGFR-1 mRNA and protein were detected in the endometrium of cyclic animals. However, higher VEGFR-1 protein expression was found on Days 16-17 of the estrous cycle (P < 0.01) and Days 13-15 of gestation (P < 0.05). Protein expression of VEGFR-2 was elevated on Days 2-4 of the estrous cycle (P < 0.001), but mRNA levels were constant during the cycle. In pregnancy, VEGFR-2 protein expression started to increase after Day 15 (vs. Days 9-12; P < 0.05), but induction of VEGFR-2 mRNA expression occurred earlier on Days 13-15. It appears from the present study that the VEGF-receptor system is regulated in a temporal and spatial manner during the estrous cycle and early pregnancy in pigs. The results suggest that VEGF-A family members are probably involved in appropriate preparation of endometrium for implantation and in vascular events during implantation in pigs.     

Effect of local neutralization of basic fibroblast growth factor or vascular endothelial growth factor by a specific antibody on the development of the corpus luteum in the cow

Active angiogenesis and progesterone (P) synthesis occur in parallel during development of the corpus luteum (CL). Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are known to stimulate angiogenesis and P synthesis in vitro. The aim of the present study was to investigate the impact of bFGF or VEGF on the CL development in the cow by using a specific antibody against bFGF or VEGF. bFGF antibody, VEGF antibody, or saline as a control (n = 4 cows/treatment) were injected directly into the CL immediately after ovulation (Day 1), and the treatment was continued for 3 times/day over 7 days. Luteal biopsies were applied on Day 8 of the estrous cycle to determine the expression of genes associated with P synthesis and angiogenesis. Intraluteal injections with the bFGF antibody or the VEGF antibody markedly decreased the CL volume, plasma P concentration and StAR mRNA expression. bFGF antibody treatment decreased the mRNA expression of bFGF, FGF receptor-1, VEGF120, and angiopoietin (ANPT)-1, and increased ANPT-2/ANPT-1 ratio. However, VEGF antibody treatment decreased ANPT-2 mRNA expression and ANPT-2/ANPT-1 ratio. These results indicate that local neutralization of bFGF or VEGF changes genes regulating angiogenesis and P synthesis, and remarkably suppresses the CL size and P secretion during the development of CL in the cow, supporting the concept that bFGF and VEGF control the CL formation and function.     

Differences of microvascular endothelium in the bovine corpus luterum of pregnancy and the corpus luteum of the estrous cycle

Summary— The purpose of the present study was to investigate potential modulations of endothelial cells of the bovine corpus luteum (CL) during pregnancy. Luteal endothelia of pregnant and non-pregnant cows were isolated and purity of cultures was verified by flow cytometric quantification of three independent endothelial markers (von Willebrand factor, angiotensin converting enzyme, Bandeiraea simplicifolia agglutinin I ligands). Different cellular parameters including light and electron microscopical investigation of morphology and growth characteristics as well as quantification of cellular lectin binding sites were compared. Extensive heterogeneity between luteal endothelial cells in pregnant and non-pregnant animals could be demonstrated, reflected in functional attributes like angiogenic activity, ultrastructural characteristics and the quantitative expression of cellular carbohydrates. Two different morphological types of cells (‘cobblestone growth pattern’ and ‘arcuate growth pattern’) were isolated from the CL of pregnancy as well as from the cyclic CL. Spontaneous angiogenic activities, including cellular migration in band-like structures and formation of ring-like structures, were observed in endothelial cells isolated from the CL of pregnant cows exclusively. This strongly suggests that microvascular luteal endothelium of pregnant animals, in contrast to the one of non-pregnant animals, is able to produce quantitatively and/or qualitatively specific angiogenesis factor(s). Heterogeneity between luteal endothelial cells in the pregnant and non-pregnant animal could also be demonstrated by quantification of lectin (Bandeiraea simplicifolia agglutinin I, concanavalin A, Dolichos biflorus agglutinin, Ulex europaeus agglutinin I, wheat germ agglutinin) binding sites: quantitative expression of specific endothelial cell surface carbohydrates could be correlated to be status of pregnancy, thus emphasizing the actual need of quantification of lectin binding.     

Changes in expression of 11beta-hydroxysteroid dehydrogenase type-1, type-2 and glucocorticoid receptor mRNAs in porcine corpus luteum during the estrous cycle

The objective of the present study was to determine whether glucocorticoid (GC) and its receptor (GC-R) are expressed in the porcine corpus luteum (CL), and whether GC influences porcine luteal hormone production. The gene expressions of 11beta-hydroxysteroid dehydrogenase type 1 (11-HSD1), type 2 (11-HSD2), GC-R, and the concentrations of GC were determined in the CL of Chinese Meishan pigs during the estrous cycle. Moreover, the effects of GC on progesterone (P(4)), estradiol-17beta (E(2)), and prostaglandin (PG) F2alpha secretion by cultured luteal cells were investigated. Messenger RNAs of the 11-HSD1, 11-HSD2, and GC-R were clearly expressed in the CL throughout the estrous cycle. The 11-HSD1 mRNA level in the CL was higher at the regressed stage than at the other stages (P < 0.05), whereas 11-HSD2 mRNA was lower at the regressed stage than at the other stages (P < 0.05). GC-R mRNA level was higher at the regressed stages than at the other stages (P < 0.01). Concentrations of GC were lower in the regressed CL than in the other stages (P < 0.01). When the cultured luteal cells obtained from mid-stage CL (Days 8-11) were exposed to GC (50-5,000 ng/ml), P(4) and PGF2alpha secretion by the cells were reduced (P < 0.05), whereas GC had no effect on E(2) secretion by the cells. The overall results suggest that GC is regulated locally by 11-HSD1 and 11-HSD2 in the porcine CL. GC inhibits P(4) and PGF2alpha production from luteal cells via their specific receptors, implying GC plays some roles in regulating porcine CL function throughout the estrous cycle.     

Expression of mRNA and proteins for GnRH I and II and their receptors in primate corpus luteum during menstrual cycle

The differential expression of mRNA and protein of GnRH I, II and their receptors (RI and RII) in the monkey corpus luteum (CL) were measured during different stages of the luteal phase of the menstrual cycle as an initial step towards considering the role and regulation of GnRH (I and II) system during luteinization and luteolysis in primates. RT-PCR confirmed the sequence identity of PCR products and real time PCR quantified specific mRNA expressions. Proteins were localized by immunohistochemistry (IHC). Changes in mRNA expression patterns of GnRH I and II (increased) and GnRH RII (decreased) were maximal at mid-late to late stages, that is, at CL regression, where as GnRH RI was low during the entire luteal phase. However, RT-PCR and IHC studies confirmed the presence of GnRH RI at both mRNA and protein levels, respectively. IHC results showed the presence of GnRH I, II and their receptors in steroidogenic cells (granulose-luteal cells and thecal-luteal cells) across the luteal phase. Hence, GnRH I and II systems may have a role on both luteinization (from early to mid stages of CL) and luteolysis (from mid-late to very-late stages of CL). These novel findings suggest that monkey luteal GnRH system may have a role in fertility regulation in paracrine and/or autocrine manner.     

Vascular endothelial growth factor and its receptors

Vascular endothelial growth factor (VEGF) is a prime regulator of endothelial cell proliferation, angiogenesis, vasculogenesis and vascular permeability. Its activity is mediated by the high affinity tyrosine kinase receptors, KDR/Flk-1 and Flt-1. In this article, recently discovered structural, molecular and biological properties of VEGF are described. Among the topics discussed are VEGF and VEGF receptor structure and bioactivity, the regulation of VEGF expression, the role of VEGF and its receptors in vascular development, and the involvement of VEGF and its receptors in normal and pathological (ocular and tumor) angiogenesis.     

Expression of vascular endothelial growth factor and its receptors in the rhesus monkey (Macaca mulatta) endometrium and placenta during early pregnancy

Vascular endothelial growth factor (VEGF) is fundamental for development and maintenance of endometrial and placental vascular function during pregnancy. While there are a number of studies on VEGF in the human placenta, they are mostly restricted to late pregnancy. To further understand the role of VEGF in mediating angiogenesis during human early pregnancy, we employed a rhesus monkey early pregnancy model to study the temporal and spatial expression of VEGF and its receptors, fms-like tyrosine kinase (Flt)-1, and kinase-insert domain-containing receptor (KDR) mRNAs and proteins in the uteri on day 12, 18, and 26 of pregnancy using in situ hybridization, RT-PCR, and immunohistochemistry. VEGF mRNA had been identified in the luminal epithelium on day 12, in the glandular epithelium on day 12 and 18, and the highest expression was detected in the walls of some spiral arterioles adjacent to the implantation site on day 18, in the placental villi and in the fetal-maternal border on day 18 and 26. Besides, immunostaining of VEGF was detected in the placental villi and endometrial compartments including spiral arteries walls and the glandular epithelium. The localization of VEGF in the endothelium correlates with the presence of Flt-1 and KDR receptors on vascular structure. All the results above suggest that VEGF-VEGFR pairs were involved in the process of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development during the rhesus monkey early pregnancy. Expression of VEGF, Flt-1, and KDR in the epithelial cells also hints some additionally functional roles of VEGF during early pregnancy.     

假孕大鼠黄体VEGF表达的动态变化与NO的关系

目的:观察大鼠卵巢黄体血管内皮生长因子(VEGF)的变化规律,探讨其在黄体形成和退化中的作用及机制.方法:将未成年雌性SD大鼠用孕马血清促性腺激素(PMSG)和人绒毛膜促性腺激素(hCG)做成假孕模型,采用免疫组化方法观察黄体VEGF表达的动态变化,用ELISA法检测黄体VEGF的含量,同时用化学法测定黄体NO的含量.结果:在假孕大鼠第1 d黄体中VEGF已有微弱表达,第3 d至第5 dVEGF表达逐渐增强,第7 d达到最高,并维持到第9 d,11 d时开始下降,第13 d明显减弱,在15 d降至最低水平.VEGF含量的变化与VEGF表达的情况类似.卵巢NO的含量在整个黄体期呈双峰态势,从第1 d到第5 d逐渐增加,在第7 d达到第一个高峰,第9 d NO含量下降,第11 d有所恢复,13 d时NO含量升高明显,并且达到最高,第15 d NO含量又开始下降.结论:VEGF表达与黄体血管生成具有密切时间关系,VEGF通过NO介导促进血管新生参与黄体的形成,在黄体晚期NO作为溶黄体物质参与黄体的退化.     

Specific association of increased vascular endothelial growth factor expression and its receptors with macrophage differentiation of HL-60 leukemia cells

We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of ∼31 and ∼60 kDa in cell lysates, and the higher expression of ∼31 kDa band was further increased after stimulation with tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS). A ∼31 kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.     

Expression of prostaglandin synthesis pathway enzymes in the porcine corpus luteum during the oestrous cycle and early pregnancy

Prostaglandins (PGs) of luteal origin may have paracrine and/or autocrine actions on the functions of the corpus luteum (CL). Previously, we have shown that enzymes of PG synthesis pathway such as prostaglandin E synthase (mPGES-1), prostaglandin F synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1) are important in regulation of PG production in the conceptuses and endometrium of cyclic and pregnant pigs. Therefore, localization and expression patterns of these enzymes were determinated in porcine CL. The PGFS protein content was lower in metestrus and higher around luteolysis, and then decreased in late regressing CL. PGFS protein levels were lower on days 5-8 of pregnancy and did not differ between days 10 and 25. Elevated expression of mPGES-1 mRNA was found in early luteal phase. The mPGES-1 protein content, similarly to PGFS, was higher during luteolysis. mPGES-1 mRNA and protein levels were constant between days 5 and 25 of pregnancy. PGFS and mPGES-1 expression was down-regulated on days 16-17 of the oestrous cycle when compared to the corresponding days of pregnancy. Enhanced mPGES-1/PGFS ratio occurred during early luteal phase and days 5-8 of pregnancy. Expression of CBR1 mRNA and protein was constant during the cycle and pregnancy. Our studies revealed higher mPGES-1/PGFS ratios in the CL during early luteal phase and corresponding days of pregnancy that could favor PGE(2) synthesis and may be important in the control of luteal development. However, PG synthesis in the endometrium/conceptus rather than in the CL could be involved in luteolysis and maternal recognition of pregnancy in pigs.     

Lipoprotein lipase activity in the bovine corpus luteum during the estrous cycle and early pregnancy.

Lipolytic activity measured at pH 8.6 in bovine corpora lutea exhibited classical properties of lipoprotein lipase (LPL) in terms of serum and heparin stimulation and NaCl inhibition. LPL activity was measured in 23 corpora lutea collected at different stages of the estrous cycle and early pregnancy. The LPL activity in cyclic corpora lutea (mumole FA released/hr/100 mg acetone powder) was low at Days 4-8 of the estrous cycle (3.1 +/- 1.5: mean +/- SE) and at Days 19-20 (1.6 +/- 0.6). However, high activity of the enzyme was found at Days 12-15 of the cycle (11.8 +/- 1.8); these concentrations were significantly (P less than 0.01) elevated over those found at Days 4-8 and 19-20. The enzyme activity began to decline at Days 16-18 of the estrous cycle (5.1 +/- 1.7). Low enzyme activity was found in the corpora lutea removed from two cows at Day 22 of pregnancy. Progesterone concentrations were measured in 16 of the 23 corpora lutea and a good correlation (r = 0.75, P less than 0.01) was found between lipoprotein lipase and progesterone concentrations of the tissue. The data suggest that LPL may be involved in controlling the transfer of fatty acids, including arachidonic, from plasma lipoproteins to luteal tissue.     

Oxytocin receptors in the porcine endometrium during the estrous cycle and early pregnancy

Oxytocin (OT) receptors in the porcine endometrium were investigated at four stages of the estrous cycle (Days (D) 0, 5, 10 and 15, n = 3), and at two stages of early pregnancy (D5 and D15 after mating, n = 3) by a radioreceptor assay using ¹²⁵I-labeled OT antagonist [d(CH₂)₅,Tyr(Me)²,Thr⁴,Tyr-NH⁹₂]-vasotocin. Binding specificity was demonstrated by displacement with four peptides related to oxytocin ([Arg⁷]-vasopressin, [Thr⁴,Gly⁷]-OT, OVT, OT) and two peptides unrelated to oxytocin (luteinizing hormone-releasing hormone, [Ile³]-pressinoic acid (tocinoic acid)). The dissociation constant (K_d) of endometrial OT receptors on D0 (0.59 ± 0.10 nM) was similar to those on D10 and D15 (D10, 0.75 ± 0.21; D15, 0.60 ± 0.14 nM; mean ± SEM). In the early luteal stage (D5), K_d (2.41 ± 0.24 nM) was higher than on D0, D10 and D15 (P < 0.01). In early pregnancy, K_d values were 3.25 ± 0.29 nM on D5 and 2.44 ± 0.44 nM on D15. Binding site concentration (B_max) on D0 (910.0 ± 25.1 fmol mg⁻¹ protein) was significantly higher than on D5 and D10 (D5, 322.5 ± 71.7; D10, 147.5 ± 25.8 fmol mg⁻¹ protein; P < 0.01) of the estrous cycle and D5 and D15 (D5, 302.5 ± 82.6; D15, 315.0 ± 20.1 fmol mg⁻¹ protein; P < 0.01) of early pregnancy. In the two stages of early pregnancy, B_max values were constant and similar to that on D5 of the early luteal stage.Our results reveal the existence of specific OT binding sites in the porcine endometrium during the estrous cycle and early pregnancy. Furthermore, the fluctuation in the binding of OT to the endometrium during the different stages of the estrous cycle suggests that OT plays an important role in regulating the estrous cycle of the pig as seen in other animals.     

Interrelationships among morphology, echotexture, and function of the bovine corpus luteum during the estrous cycle

It has been suggested that ultrasound image attributes are a potential indicator of physiological and functional status of the corpus luteum (CL) in several species, including cattle. The aims of this study were to evaluate CL morphological, functional and echotextural characteristics, and also to investigate the hypothesis that those attributes are correlated and change similarly throughout an estrous cycle. Ovaries of crossbred (Bos taurus taurus × Bos taurus indicus) heifers were evaluated using ultrasonography daily throughout an interestrus interval using a B-mode, real-time ultrasound machine equipped with a 5 MHz linear-array rectal transducer, during a natural estrous cycle (Experiment 1; n = 12) or during a shortened cycle, with luteolysis induction 10 d after estrus (Experiment 2; n = 6). Blood samples were collected for assay of plasma progesterone concentrations. Corpora lutea areas were measured and daily images of each CL were videotaped and digitized for computer-assisted analysis using custom-developed software. In Experiment 1, area of luteal tissue increased until a maximum value 10 d after estrus (P < 0.001), followed by a plateau phase, and then a decline beginning 14 d after estrus. Luteal tissue area was highly correlated to plasma progesterone concentrations (r = 0.86; P < 0.001). When luteolysis was induced in Experiment 2, loss of CL function (decrease in plasma progesterone concentrations to metestrous values) preceded tissue regression by 48 h (24 h compared with 72 h; P < 0.001). The mean pixel value of ultrasound images did not change in Experiment 1 (P > 0.70), but a day effect on this attribute was observed in Experiment 2 (P = 0.052). In contrast, mean pixel value was correlated to plasma progesterone concentrations in Experiment 1 (r = −0.63; P < 0.05), but not in Experiment 2 (r = −0.28; P > 0.10). In regard to CL heterogeneity, defined as the standard deviation of the mean pixel value of the luteal tissue, a time effect was observed following both natural (Experiment 1; P < 0.009) and luteolysis-induced (Experiment 2; P < 0.05) estrous cycles (P < 0.05). Moreover, this variable was correlated with plasma progesterone concentrations (r = −0.71 and −0.58 in Experiments 1 and 2, respectively; P < 0.01), indicating that CL images were more heterogeneous during metestrus and after luteolysis (functional regression). In summary, morphological and echotextural attributes were correlated with CL function and underwent similar changes during the estrous cycle. Luteal tissue heterogeneity, assessed by ultrasonography, is considered a potential indicator of CL functional status, because it is correlated to circulating progesterone concentrations.