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Effect of Prostaglandin F2 Alpha on Local Luteotropic and Angiogenic

Factors During Induced Functional Luteolysis in the Bovine Corpus Luteum

Bajram Berisha1,2

, Heinrich H.D. Meyer1, Dieter Schams

1

1)Physiology Weihenstephan, Technical University Munich, Weihenstephaner Berg 3, D-85354

Freising, Germany

2)Faculty of Agriculture and Veterinary, University of Prishtina, Bulevardi Bill Clinton p.n.,

10000 Prishtin, Kosovo

Short title: Luteotropic and angiogenic factors during luteolysis

Grant sponsor: German Research Foundation (DFG).

Correspondence to Bajram BerishaPhysiology Weihenstephan, Technical University Munich

Weihenstephaner Berg 3.

D-85354 Freising - WeihenstephanGermany

E-mail: berisha@wzw.tum.de

Key words: angiogenic factors, luteotropic factors, gene regulation, corpus luteum function,

luteolysis

BOR Papers in Press. Published on January 7, 2010 as DOI:10.1095/biolreprod.109.076752

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ABSTRACT

The essential role of endometrial prostaglandin F2 alphaPTGF for induction of the corpus luteum(CL) regression is well documented in the cow. However, the acute effects of PTGF on known

local luteotropic factors (oxytocin and its receptor, IGF1 and progesterone and its receptor), the

principal angiogenic factor VEGFA and the capillary destabilization factor angiopoietin(ANGPT)-2 were not thoroughly studied in detail. The aim of this study was therefore to

evaluate the tissue concentration of these factors during PTGF induced luteolysis. In addition the

mRNA expression of progesterone receptor (PGR), oxytocin receptor (OXTR),IGF1,IGFBP1,

ANGPT1 andANGPT2 was determined at different times after PTGF treatment. Cows (n=5 per

group) in the mid-luteal phase (days 8-12, control group) were injected with the PTGF analogue(cloprostenol) and CL were collected by transvaginal ovariectomy at 0.5, 2, 4, 12, 24, 48 and 64h after injection. The mRNA expression was analyzed by a quantitative real-time PCR, and the

protein concentration was evaluated by enzyme immunoassay or radio immunoassay.

Progesterone concentrations as well as mRNA expression ofPGR in CL tissue were significantlydown-regulated by 12 h after PTGF. Tissue OXT peptide and OXTR mRNA decreased

significantly latest after 2 h followed by a continuous decrease ofOXTmRNA. IGF1 and

VEGFA protein decreased already after 0.5 h. By contrast theIGFBP1 mRNA was up-regulated

significantly after 2 h to a high plateau. ANGPT2 protein and mRNA significantly increasedduring the first 2 h followed by a steep decrease after 4 h. The acute decrease of local luteotropic

activity and acute changes of ANGPT2 and VEGFA suggest that modulation of vascular stability

may be a key component in the cascade of events leading to functional luteolysis.

INTRODUCTION

In ruminants, luteal regression is stimulated by episodic release of prostaglandin F2 alpha(PTGF) from the uterus which reaches the corpus luteum (CL) through a counter-current systembetween uterine vein and ovarian artery. Luteolysis is characterized by a rapid decline in

progesterone secretion, which is followed by degeneration of vasculature and apoptosis of

steroidogenic cells [1]. Several researchers suggested that reduced ovarian blood flow anddecrease in luteal vascularity play a critical role in initiation of luteal regression [2-3]. This is

followed by a decrease in blood vessel density and then the degeneration and disappearance of

luteal cells [4]. The growth, differentiation and regression of the CL depends on the balance

between luteotropic and luteolytic as well as angiogenic and anti-angiogenic factors. These inturn regulates the cyclical generation and regression of its vasculature. Thus, the life span of the

CL involves several tightly regulated and transient proteolytic processes which include tissue

remodeling during angiogenesis and tissue degradation during angioregression [1, 5-9]. In thecow, the major luteotropic factor controlling luteal function is luteinising hormone (LH),

whereas the primary inducer of luteolysis is PTGF. Recently, we have shown that there is a very

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insulin like growth factor (IGF) [24-26], nitric oxide [27-28], angiotensin and endothelin systems

[5, 29-31], extracellular matrix degrading proteases and different cytokines, apoptotic factors and

enzymes [10, 32-35]. Most of these studies have been done at the end of functional luteolysis(about 12 h after PTGF administration) or during structural luteolysis (after 12 h). However,

there is limited information on the early cascade of events directly after the PTGF administration

surge.Our hypothesis was that PTGF acting on luteal cells directly, acutely regulates the

production of factors which have luteotropic, angiogenic or vascular-stabilizing effects. These

then play a critical role in functional luteolysis. Therefore, the aim of this study was to evaluatethe mRNA expression and the protein concentration for IGF1, oxytocin (OXT) and progesterone

as well as VEGFA and ANGPT2 in the CL at different times immediately following PTGFinduced luteolysis.

MATERIALS AND METHODS

Collection of Bovine Corpora Lutea (CL) During Induced LuteolysisPreviously estrus-synchronized cows (Holstein-Friesians and Brown Swiss), were

injected i.m. with 500 g of the PTGF analog Cloprostenol (Estrumate, Intervet, Germany) at the

mid-luteal phase (Days 812). The CL were collected by transvaginal ovariectomy at 0.5, 2, 4,

12, 24, 48, and 64 h (n=5 per time point) after PTGF injection [29]. The experiments wereconducted according to the rules of the German animal welfare law and were licensed by the

local authorities. This is in accordance with the International Guiding Principles for Biomedical

Research Involving Animals. Control bovine CL were collected from the slaughterhouse in themid-luteal phase (Days 812, n=5/group). All CL were immediately frozen in liquid nitrogen and

stored at 80 C until RNA and protein extraction prior to analysis). Blood samples for

progesterone determination were taken from the jugular vein.

Hormone DeterminationCL tissue extraction.One gram of tissue was transferred into ten volumes of PBS containing one

complete mini tablet of bovine serum albumin (BSA, Boehringer, Mannheim, Germany). Thistablet contains both reversible and irreversible protease inhibitors, and inhibits a broad spectrum

of serine-, cysteine- and metallo-proteases. The mixture was homogenized for 1 min using Ultra

Turrax equipment (Jahnke and Kunkel, Staufen, Germany) on ice and then kept in iced water for

1 h. After centrifugation for 10 min at 3500 g, the total protein content in the supernatant wasdetermined by a BCA test (Sigma-Aldrich GmbH, Taufkirchen, Germany).

Enzyme Immunoassay (EIA) for Progesterone and ANGPT2 DeterminationProgesterone determination in blood and CL tissue.The concentration of progesterone

in blood plasma was measured after extraction with petroleum ether using an EIA technique

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the assay was 750 pg/ml. The intra and inter-assay CV values were below 6 and 10%

respectively.

Radioimmunoassay (RIA) for VEGFA, IGF1, and Oxytocin (OXT) in CL TissueVEGFA determination.The concentration of VEGFA in homogenate supernatant of CL

tissue was determined by RIA as previously described by Berisha et al. [37]. It utilized a rabbit

antiserum raised in our laboratory against recombinant bovine VEGF164 (kindly supplied by D.

Gospodarowicz, Chiron Corp., Berkeley, CA). The antibody cross-reacts with all four humanisoforms of VEGFA (VEGF121, VEGF165, VEGF189 and VEGF206). The cross-reactivity of

the antibody to other growth factors, platelet-derived growth factor (PDGF)-AA, PDGFBB,PDGFAB, FGF1, FGF2 and transforming growth factor alpha was below 0.1%. Briefly, thestandard curve for VEGFA ranged from 0.062 to 8 ng/ml with an ED50 of 0.6 ng/ml. The intra-

and inter-assay coefficients of variation were below 6% and 14%, respectively.IGF1 determination. IGF1 was evaluated by RIA as described by Einspanier et al. [38]

in tissue extract supernatant. The standard curve for IGF1 ranged from 0.078 to 10 ng/ml and the

ED50 for the assay was 2.05 ng/ml. The intra and inter-assay values were below 6 and 14%

respectively.OXT determination. The concentration of OXT in homogenate supernatant of CL tissue

was determined by RIA using arabbit antiserum raised against OXT [39]. The RIA for OXT was

evaluated in different dilutions of extracts. The standard curve for OXT ranged from 0.125 to 64

pg/0.1 ml, and the ED50 of the assay was 3.7 pg/ml. The intra and inter-assay CV were 5.8-7.4%and 10.5-15.4% respectively.

Isolation of Total RNA

Total RNA was prepared from CL tissue according to the methods described byChomczynski and Sacchi [40] using the TriPure isolation reagent (Roche Diagnostics,Mannheim, Germany). Possible DNA contamination was eliminated by additional DNase

(Promega, Madison, WI, USA) digestion according to the manufacturers instructions. Total

RNA was finally purified using Nucleospin RNA II (Macherey & Nagel, Dren, Germany) andchecked for quantity and quality using a biophotometer at wavelengths of 260 and 260/280 nm,

respectively (Eppendorf, Hamburg, Germany).

Degradation of the RNA was measured with the Agilent 2100 bioanalyzer (Agilent

Technologies, Deutschland Gmbh, Waldbronn, Germany) in conjunction with the RNA 6000Nano Assay according to the manufacturers instructions. The bioanalyzer enables the

standardisation of RNA quality control. RNA samples are electrophoretically separated on a

microfabricated chip and subsequently detected with laser-induced fluorescence induction. TheBioanalyzer electropherogram of total RNA shows two distinct ribosomal peaks corresponding

to either 18S or 28S for eukaryotic RNA and a relatively flat baseline between the 5S and 18S

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murine leukemia virus (M-MLV) reverse transcriptase (200 U/L; Promega) as described

previously by Pfaffl et al. [41]. A negative control reverse transcription reaction (in which the

reverse transcription enzyme was replaced by water) was performed to detect residual DNAcontamination.

Real-Time Polymerase Chain ReactionPrimers for housekeeping genes ubiquitin, glycerolaldehyde-3-phosphate-dehydrogenase

(GAPDH) and examined factors (PGR, OXT, OXTR, IGF1, IGFBP1, ANGPT1, ANGPT2) weredesigned using the EMBL database or used according to the literature (Table 1). Quantitative

fluorescence real-time RT-PCR analysis was performed with the Rotor-Gene 3000 system(Corbett Research, Sydney, Australia). Online PCR reactions were carried out using aLightCycler DNA Master SYBR Green I kit (Roche Diagnostics, Mannheim, Germany) with 1

l of each cDNA (16.66 ng) in a 10 l reaction mixture (3 mmol/l MgCl2, 0.4 mol/l of each

forward and reverse primer, 1x LightCycler DNA Master SYBR Green I). After initialincubation at 95C for 10 min to activate Taq DNA polymerase, templates of all specific

transcripts were amplified for 40 cycles at 95C for 10 s followed by annealing temperatures

between 59C - 63C according to the used primer, each 10 s, and elongation at 72C for 15 s.

Fluorescence data were acquired after each elongation step by SYBR Green binding to theamplified dsDNA at 72C for 5 s. The specificity of each PCR product was determined by

melting curve analysis (Rotor-Gene 3000 software, version 5.03) immediately after PCR

completion by heating at 95C for 5 s, followed by cooling to 65C for 5 s and continuousheating to 99C at 0.5C/s under permanent fluorescence detection. Confirmation of PCR

product identity was obtained through melting curve analysis (Rotor-Gene 3000TM) and

subsequent gel electrophoresis separation. Data were analyzed using the Rotor-Gene 3000software (version 5.03). The relative level of each target gene was calculated using the

comparative quantification method (Takeoff points). The changes in mRNA level ofexamined factors were assayed by normalization to the GAPDHinternal control [42]. In order to

obtain the CT (cycle threshold) difference the GAPDH-normalized data were analyzed using theCT method described previously by Livak and Schmittgen [43]. Thereby CP was notsubtracted from a control group, but from the value 40 (arbitrary value), so that a high 40 minus

CP value indicated a high-gene expression level and vice versa [32].

Statistical AnalysesThe study was conducted on 40 cows which were divided into eight groups (n=5 per

group). All experimental data presented are shown as the mean SEM. Statistical differences in

mRNA levels and protein concentrations of the factors examined were analyzed by ANOVAfollowed by Fishers l.s.d. test as a multiple-comparisons test. Differences were considered

significant if P

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basal levels and are considered to reflect luteolysis or the absence of a functional CL. Thus, the

measured progesterone levels demonstrate the efficiency of induced luteolysis.

RNA Quality DeterminationRIN values of the samples collected during induced luteolysis ranged between 6.3 and

9.4. All samples revealed two distinct ribosomal peaks corresponding to the 18S and 28S foreukaryotic RNA. Only one sample (from a total of 45 samples) showed slight degradation, which

was for us without concern for the gene expression level of all investigated factors [32].

Confirmation of Primer Specificity and Sequence Analysis

The mRNA expression was analysed by real-time RT-PCR (Rotor Gene 3000). InitialRT-PCR experiments verified specific transcripts for all factors in bovine CL. For exact lengthverification, RT-PCR products were separated on 2% high-resolution agarose gel

electrophoresis. PCR products were verified by commercial DNA sequencing (TopLab, Munich,

Germany). Each PCR product (Table 1) showed 100% homology to the known genes aftersequencing.

Housekeeping Gene Expression

To evaluate equal quantity and quality of the preceding reverse transcription reaction ineach sample, the housekeeping genes ubiquitin and glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) were examined in all samples. As both housekeeping genes were constantly expressed

in all samples, we chose GAPDHas normalizer. The results of mRNA expression of examinedfactors (Fig. 1, 2, 3, 4) are presented as changes (40 minus CTSEM from 5 CL per group) in

the target gene expression, normalised to GAPDH.

Expression of mRNA and Peptide Concentration of Examined FactorsLuteotropic factors. Luteal progesterone concentrations and progesterone receptor (PGR)

mRNA expression levels are shown in Fig. 1. Progesterone concentrations (Fig. 1a) as well as

mRNA expression ofPGR in CL tissue (Fig. 1b) were significantly down-regulated by 12 h after

PTGF administration.

Concentrations of luteal OXT rapidly declined and were significantly lower at 0.5 h

before declining further at 2 h post PTGF and remained at these low levels thereafter (Fig. 2a).

While, its receptor (OXTR) mRNA expression declined after 2 h to a significant lower plateau

(Fig. 2c), and remained at these low levels thereafter.IGF1 peptide concentrations rapidly declined after 0.5 h and remained at this low level

until the end of experiment (Fig. 3a). In contrast,IGF1 mRNA was up-regulated at 0.5 h, before

decreasing at 2 h and then remained at that low level for the remaining time (Fig. 3b). Bycontrast theIGFBP1 mRNA was upregulated significantly after 2 h to a high plateau for 24 h but

was rapidly down-regulated thereafter (Fig. 3c).

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found forANGPT2 mRNA (Fig. 4c). By contrast, the level ofANGPT1 mRNA did not change

(data not shown), and therefore the ratio forANGPT2/ANGPT1 showed a similar pattern as for

ANGPT2 mRNA (Fig. 4d).

DISCUSSION

The luteolytic process in cow induced by PTGF surge, consists of two phases, functional(first 12 h) and structural luteolysis (time after 12 h), which are reflected predominantly by a

decrease in progesterone and a decrease in luteal size, respectively [44]. The decrease in

progesterone blood levels in our experiment confirmed the occurrence of induced luteolysis.PTGF treatment showed acute effects on the investigated luteotropic factors (progesterone, PGR,

OXT, OXTR, IGF1) and anti-luteotropic factor IGFBP1 as well as capillary maintaining factors(VEGFA, ANGPT1 and ANGPT2) during functional luteolysis of the CL.

In the present study, progesterone concentration in blood serum and in CL tissue as well

as mRNA expression ofPGR in CL tissue, were significantly down-regulated at 12 h after

PTGF. As shown in an earlier study [45] blood progesterone decreased already significantly 4 hafter PTGF. Similarly, luteal progesterone tissue concentration, in the present study, also tended

to decrease after 4 h. It is well established that progesterone concentrations decrease during the

first 12 h after PTGF but progesterone may have also direct effects on its own production [46].

For example, Rueda et al. [47] showed that decreased progesterone level and PGR antagonistspromote apoptotic cell death in bovine luteal cells. Progesterone seems to act on the secretory

function of bovine CL via genomic (intracellular) receptors (well documented) but also non-

genomically (by membrane receptors). The nature of non-genomic action of progesterone has notbeen fully understood [48-49].

OXT peptide concentrations in the present study rapidly declined immediately after 0.5 h

and 2 h by nearly 90%, while its receptor (OXTR) mRNA expression decreased 2 h after PTGFadministration to a low level afterwards. PTGF might also have stimulated the release of OXT

from the CL as part of the luteolytic positive feedback loop [45]. In contrast it took longer forOXTmRNA expression before it started to decrease (only after 12 h). But the low level of luteal

OXT protein at this time suggests there is a decrease in protein stability or no new translation

into protein. There are clear indications that OXT has intra-ovarian direct effects onsteroidogenesis [50]. For example, OXT had the greatest effect on progesterone secretion after

perfusion in the microdialysis system (MDS) of early luteal phase (days 57) CL and decreased

continuously from days 812 to days 1518 [50]. Furthermore, OXT had a modulating action on

LH-stimulation of progesterone secretion in vitro [51]. Namely, in the MDS, administration ofLH stimulated the release of progesterone throughout the luteal phase with maximal effects on

days 1518. By contrast, pre-stimulation with OXT prior to LH perfusion increased the

stimulative effect of LH in the early luteal phase. The results support the assumption that OXTmay be a potent luteotropic factor in bovine CL especially during the developing phase [52].

In the present study, IGF1 peptide concentrations rapidly declined after 0.5 h and

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localized in large luteal cells, the regulation seems to be, at least in part, an autocrine action. As

described by ourselves [24], there is clear evidence for mRNA expression for all sixIGFBPs in

CL withIGFBP1 mRNA expression being relatively low during most of the oestrous cycle.However, during PTGF induced luteolysis Sayre et al. [55] found a dramatic increase inIGFBP1

mRNA and protein in the cow. This is in strong agreement with our previous observations [25]

and the present study, which noted an even earlier (0.5 h) up-regulation. From allIGFBPsstudied onlyIGFBP1 showed such a large and early up-regulation whileIGFBP3 mRNA

declined after 12 h andIGFBP5 mRNA increased 12 h onwards. Also theIGF1R mRNA

decreased shortly after PTGF and again after 12 h [25]. The increase (about 34 fold) ofIGFBP1may cause further reduction of bioactive free IGF1 in CL tissue. The acute decrease in the

concentration of the local luteotropic factors, OXT and IGF1 as well as the massive increase inIGFBP1 in the CL within 0.5 h of luteolysis being induced may represent one of the importantinitial luteolytic actions of PTGF.

The described actions of PTGF on local luteotropic factors suggest negative effects on

luteal cells. The survival of these cells depends on a full functioning capillary network. One ofthe most important factors regulating new capillary formation, survival and endothelial cell

permeability is VEGFA [2, 4, 15-16]. Previously, we have shown [17] that the mRNA

expression starts to decline 12 h after PTGF. However, in the present study VEGFA protein

concentrations had already declined (about 60%) by 0.5 h after the PTGF injection. This rapiddecline of VEGFA protein appears to be an acute (releasing) effect of PTGF. The remaining low

protein level in tissue suggests decrease of protein stability or no new translation of protein even

when the mRNA levels remain high up to 12 h.A very interesting observation was the up-regulation ofANGPT2 mRNA and protein

indicating a very rapid translation of the protein. The mRNA data for bothANGPTs and

receptors were already published in part by Tanaka et al. [21]. Both ANGPT are important forblood capillary stabilization which depends on the ratio of ANGPT2:ANGPT1. If the

ANGPT2:ANGPT1 ratio is high, as 2 h after PTGF, and VEGFA is either absent or at low levels,then this will lead to blood vessel destabilization and regression [56]. This appears to be the case

during early luteolysis after exogenous PTGF. Recently, mRNA expression ofANGPT1 was

unaffected butANGPT2 increased at 8 h after PTGF-induced luteolysis in sheep [57]. Then, 24 hpost PTGF injection,ANGPT2 mRNA decreased when compared to the 8 h levels. This may

indicate a different time frame for luteolysis in bovine and sheep.

Taking together the very acute decrease of protein for the luteal cell luteotropic and anti-

apoptotic factors (OXT, IGF1, progesterone) as well as for the main survival factor forendothelial cells (VEGFA) suggest that the decrease may be one of the earliest steps for

functional luteolysis after exogenous PTGF. As shown in sheep [57] the number of cells

undergoing apoptosis increased at 12 h after PTGF treatment, which was associated with thedecrease in the number of endothelial cells. Sawyer et al. [58] postulated that during luteal

regression, endothelial cell number are reduced first, following by a decline of parenchymal

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In conclusion, the acute changes of vasoactive key factors suggest that modulation of

vascular stability is a key component in the cascade of events leading to functional luteolysis.

This process is accompanied parallel or slightly delayed apoptosis signalling cascades and theinitiation of factors associated with inhibition of capillary function. These changes culminate at

the end of functional luteolysis (12 h) or during early structural luteolysis.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Fritz F. Parl, Vanderbilt University Medical Care and Dr.Bob Robinson, University of Nottingham for critical reading and language editing of the

manuscript. The expert technical assistance by Mrs. G. Schwentker and Mrs. I. Celler is alsohighly appreciated. The authors declare that there is no conflict of interest that would prejudicethe impartiality of this scientific work.

REFERENCES1. Niswender GD, Juengel JL, Silva PJ, Rollyson MK, McIntush EW. Mechanisms controlling the function and life

span of the corpus luteum. Physiol Rev 2000; 80:1-29.

2. Reynolds LP, Grazul-Bilska AT, Killilea SD, Redmer DA. Mitogenic factors of corpora lutea. Prog Growth

Factor Res 1994; 5:159-175.

3. Acosta TJ, Yoshizawa N, Ohtani M, Miyamoto A. Local changes in blood flow within the early and midcycle

corpus luteum after prostaglandin F(2) injection in the cow. Biol Reprod 2002; 66:651-658.

4. Augustin HG, Braun K, Telemenakis I, Modlich U, Kuhn W. Ovarian Angiogenesis: Phenotypic characterization

of endothelial cells in a physiological model of blood vessel growth and regression. Am J Pathol 1995; 147:339-

351.

5. Hansel W, Blair RM. Bovine corpus luteum: a historic overview and implications for future research.Theriogenology 1996; 45:1267-1294.

6. Milvae RA, Hinckley ST, Carlson JC. Luteotropic and luteolytic mechanisms in the bovine corpus luteum.

Theriogenology 1996; 45:1327-1350.

7. Schams D, Berisha B. Regulation of corpus luteum function in cattle an overview. Reprod Domest Anim 2004;

39:241-251.

8. Hayashi K, Acosta TJ, Berisha B, Kobayashi S, Ohtani M, Schams D, Miyamoto A. Changes in prostaglandin

secretion by the regressing bovine corpus luteum. Prostaglandins Other Lipid Mediat 2003; 70:339-349.

9. Yamashita H, Kamada D, Shirasuna K, Matsui M, Shimizu T, Kida K, Berisha B, Schams D, Miyamoto A. Effectof 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. Mol Reprod Dev 2008; 75:1449-1456.

10. Berisha B, Schams D. Ovarian function in ruminants. Domest Anim Endocrinol 2005; 29:305-317.

11 R bi RS H d AJ M GE H t MG A l h i l i l lt t th t i i l t l

8/8/2019 biolreprod.109.076752

10/19

15. Ferrara N, Hauk K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial

growth factor family of proteins. Endocr Rev 1992; 13:18-32.

16. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors.FASEB J 1999; 13:9-22.

17. Neuvians TP, Berisha B, Schams D. Vascular endothelial growth factor (VEGF) and fibroblast growth factor(FGF) expression during induced luteolysis in the bovine corpus luteum. Mol Reprod Dev 2004; 67:389-395.

18. Sato TN, Qin Y, Kozak CA, Audus KL. Tie-1 and tie-2 define another class of putative receptor tyrosine kinase

genes expressed in early embryonic vascular system. PNAS 1993; 90:9355-9358.

19. Wulff C, Wilson H, Largue P, Duncan WC, Armstrong DG, Fraser HM. Angiogenesis in the human corpus

luteum: localization and changes in angiopoietins, tie-2, and vascular endothelial growth factor messenger

ribonucleic acid. J Clin Endocrinol Metab 2000; 5:4302-4309.

20. Hayashi KG, Acosta TJ, Tetsuka M, Berisha B, Matsui M, Schams D, Ohtani M, Miyamoto A. Involvement of

angiopoietin-tie system in bovine follicular development and atresia: messenger RNA expression in thecainterna and effect on steroid secretion. Biol Reprod 2003; 69: 2078-2084.

21. Tanaka J, Acosta TJ, Berisha B, Tetsuka M, Matsui M, Kobayashi S, Schams D, Miyamoto A. Relative changes

in mRNA expression of angiopoietins and receptors tie in bovine corpus luteum during estrous cycle andprostaglandin F2alpha-induced luteolysis: a possible mechanism for the initiation of luteal regression. J Reprod

Dev 2004; 50:619-626.

22. Klagsbrun M, DAmore PA. Regulators of angiogenesis. Annu Rev Physiol 1991; 53:217-239.

23. Neuvians TP, Schams D, Berisha B, Pfaffl MW. Involvement of pro-inflammatory cytokines, mediators of

inflammation, and basic fibroblast growth factor in prostaglandin F2alpha-induced luteolysis in bovine corpus

luteum. Biol Reprod 2004; 70:473-480.

24. Schams D, Berisha B, Kosmann M, Amselgruber WM. Expression and localization of IGF family members inbovine antral follicles during final growth and in luteal tissue during different stages of estrous cycle and

pregnancy. Domest Anim Endocrinol 2002; 22:51-72.

25. Neuvians TP, Pfaffl MW, Berisha B, Schams D. The mRNA expression of the members of the IGF-system in

bovine corpus luteum during induced luteolysis. Domest Anim Endocrinol 2003; 25:359-372.

26. Neuvians TP, Pfaffl MW, Berisha B, Schams D. The mRNA expression of insulin receptor isoforms (IR-A andIR-B) and IGFR-2 in the bovine corpus luteum during the estrous cycle, pregnancy, and induced luteolysis.

Endocrine 2003; 22:93-100.

27. Motta AB, Estevez A, Tognetti T, Gimeno MA, Franchi AM. Dual effects of nitric oxide in functional and

regressing rat corpus luteum. Mol Hum Reprod 2001; 7:43-47.

28. Al-Gubory KH, Ceballos-Picot I, Nicole A, Bolifraud P, Germain G, Michaud M, Mayeur C, Blachier F.

Changes in activities of superoxide dismutase, nitric oxide synthase, glutathione-dependent enzymes and theincidence of apoptosis in sheep corpus luteum during the estrous cycle. Biochim Biophys Acta 2005; 1725:348-

357.

29. Schams D, Berisha B, Neuvians T, Amselgruber W, Kraetzl WD. Real-time changes of the local vasoactive

peptide systems (angiotensin, endothelin) in the bovine corpus luteum after induced luteal regression. Mol

Reprod Dev 2003; 65:57-66.

8/8/2019 biolreprod.109.076752

11/19

33. Berisha B, Bridger P, Toth A, Kliem H, Meyer HHD, Schams D, Pfarrer C. Expression and localisation of gap

junctional connexins 26 and 43 in bovine periovulatory follicles and in corpus luteum during different

functional stages of oestrous cycle and pregnancy. Reprod Domest Anim 2009; 44:295-302.

34. Kliem H, Berisha B, Meyer HHD, Schams D. Regulatory changes of apoptotic factors in the bovine corpus

luteum after induced luteolysis. Mol Reprod Dev 2009; 76:220-230.

35. Shirasuna K, Shimizu T, Sayama K, Asahi T, Sasaki M, Berisha B, Schams D, Miyamoto A. Expression and

localization of apelin and its receptor APJ in the bovine corpus luteum during the estrous cycle and

prostaglandin F2alpha-induced luteolysis. Reproduction 2008; 135:519-525.

36. Prakash BS, Meyer HHD, Schallenberger E, van de Wiel DF. Development of a sensitive enzymeimmunoassay

(EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique. J

Steroid Biochem 1987; 28:623-627.

37. Berisha B, Schams D, Kosmann M, Amselgruber W, Einspanier R. 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 2000; 63:1106-1114.

38. Einspanier R, Miyamoto A, Schams D, Mller M, Brem G. Tissue concentration, mRNA expression and

stimulation of IGF-I in luteal tissue during the oestrous cycle and pregnancy of cows. J Reprod Fertil 1990;90:439-445.

39. Schams D. Oxytocin determination by radioimmunoassay. III. Improvement to subpicogram sensitivity and

application to blood levels in cyclic cattle. Acta Endocrinol (Copenh). 1983; 103:180-183.40. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-

chloroform extraction. Anal Biochem 1987; 162:156-159.

41. Pfaffl MW, Georgieva TM, Georgiev IP, Ontsouka E, Hageleit M, Blum JW. Real-time RT-PCR quantificationof insulin-like growth factor (IGF)-1, IGF-1 receptor, IGF-2, IGF-2 receptor, insulin receptor, growth hormone

receptor, IGF-binding proteins 1, 2 and 3 in the bovine species. Domest Anim Endocrinol. 2002; 22:91-102.

42. Berisha B., Steffl M, Amselgruber W. Schams D. Changes in fibroblast growth factor 2 and its receptors in

bovine follicles before and after GnRH application and after ovulation. Reproduction 2006; 131:319-329.

43. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-

Delta Delta C(T)) Method. Methods 2001; 25:402-408.

44. Niswender GD, Nett TM. Corpus luteum and its control in infraprimate species. In: Knobil E, Neil JK (eds.),

The Physiology of Reproduction, New York: Raven Press Ltd; 1994; 781-816

45. Schallenberger E, Schams D, Bullermann B, Walters DL. Pulsatile secretion of gonadotrophins, ovarian steroids

and ovarian oxytocin during prostaglandin-induced regression of the corpus luteum in the cow. J Reprod Fertil

1984; 71:493-501.

46. Kotwica J, Rekawiecki R, Duras M. Stimulatory influence of progesterone on its own synthesis in bovine corpusluteum. Bull Vet Inst Pulawy 2004; 48: 139-145.

47. Rueda BR, Hendry IR, Hendry III WJ, Stormshak F, Slayden OD, Davis JS. Decreased progesterone levels and

progesterone receptor antagonists promote apoptotic cell death in bovine luteal cells. Biol Reprod 2000; 62:269-

276.

48. Rekawiecki R, Kowalik MK, Slonina D, Kotwica J. Regulation of progesterone synthesis and action in bovine

8/8/2019 biolreprod.109.076752

12/19

52. Okuda K, Korzekwa A, Shibaya M, Murakami S, Nishimura R, Tsubouchi M, Woclawek-Potocka I, Skarzynski

DJ. Progesterone is a suppressor of apoptosis in bovine luteal cells. Biol Reprod 2004; 71:2065-2071.

53. Einspanier R, Miyamoto A, Schams D, Mller M, Brem G. Tissue concentration, mRNA expression andstimulation of IGF-I in luteal tissue during the oestrous cycle and pregnancy of cows. J Reprod Fertil 1990;

90:439-445.

54. Liebermann J, Schams D, Miyamoto A. Effects of local growth factors on the secretory function of bovine

corpus luteum during the oestrous cycle and pregnancy in vitro. Reprod Fertil Dev 1996; 8:1003-1011.

55. Sayre BL, Taft R, Inskeep EK, Killefer J. Increased expression of insulin-like growth factor binding protein-1

during induced regression of bovine corpora lutea. Biol Reprod 2000; 63:21-29.

56. Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J,

Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD. Angiopoietin-2, a naturalantagonist for Tie2 that disrupts in vivo angiogenesis. Science 1997; 277:55-60.

57. Vonnahme KA, Redmer DA, Borowczyk E, Bilski JJ, Luther JS, Johnson ML, Reynolds LP, Grazul-Bilska AT.

Vascular composition, apoptosis, and expression of angiogenic factors in the corpus luteum during

prostaglandin F2alpha-induced regression in sheep. Reproduction 2006; 131:1115-1126.

58. Sawyer HR, Niswender KD, Braden TD, Niswender GD. Nuclear changes in ovine luteal cells in response toPGF2 alpha. Domest Anim Endocrinol 1990; 7:229-237.

59. Shirasuna K, Watanabe S, Asahi T, Wijayagunawardane MP, Sasahara K, Jiang C, Matsui M, Sasaki M,

Shimizu T, Davis JS, Miyamoto A. Prostaglandin F2alpha increases endothelial nitric oxide synthase in theperiphery of the bovine corpus luteum: the possible regulation of blood flow at an early stage of luteolysis.

Reproduction. 2008; 135:527-539.

60. Watanabe S, Shirasuna K, Matsui M, Yamamoto D, Berisha B, Schams D, Miyamoto A. Effect of intralutealinjection of endothelin type A receptor antagonist on PGF2alpha-induced luteolysis in the cow. J Reprod Dev

2006; 52:551-559.

61. Berisha B, Pfaffl MW, Schams D. Expression of estrogen and progesterone receptors in the bovine ovary during

estrous cycle and pregnancy. Endocrine 2002; 17:207-214.

62. Berisha B, Steffl M, Welter H, Kliem H, Meyer HHD, Schams D, Amselgruber W. Effect of the luteinising

hormone surge on regulation of vascular endothelial growth factor and extracellular matrix-degradingproteinases and their inhibitors in bovine follicles. Reprod Fertil Dev 2008; 20:258-268.

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FIGURE LEGENDS

Figure 1 (a) Concentration of progesterone in CL tissue (g/g wet tissue) after induced

luteolysis; Results are presented as the meanSEM (n=5 CL per class). (b) mRNA expression ofprogesterone receptor (PGR) in CL tissue after induced luteolysis; mRNA data are shown after

normalization as 40-CPSEM (n=5 CL per class). Different superscript letters indicate

significant differences (P

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Table 1 Primer sequences ofANGPT1, ANGPT2, IGF1, IGFBP1, oxytocin (OXT), oxytocin

receptor (OXTR), progesterone receptor (PGR), housekeeping genes ubiquitin andglycerolaldehyde-3-phosphate-dehydrogenase (GAPDH), RT-PCR product length, and reference

of the investigated factors or of the according accession number (Acc. No.) in the EMBL

database.

Target Sequence of Fragment EMBL/ Nucleotide* Size (bp) Reference**

ANGPT1 For 5'-CAGACCAGAAAGCTGACAGATG-3' 806 [21]

Rev 5'-CCAGTGTGACCCTTCAAATACA-3'

ANGPT2 For 5'-GAGAAAGATCAGCTCCAGGTGT-3' 507 [21]

Rev 5'-ATTCCCTTCCCAGTCTCTTAGG-3'

IGF1 For 5'-TCGCATCTCTTCTATCTGGCCCTGT-3' 240 [41]

Rev 5'-GCAGTACATCTCCAGCCTCCTCAGA-3'

IGFBP1 For 5'-TCAAGAAGTGGAAGGAGCCCT-3' 123 [41]

Rev 5'-AATCCATTCTTGTTGCAGTTT-3'

OXT For 5'-ACCTCCGCCTGCTACATTC-3' 200 NC_007311

Rev 5'-GGCAGGTAGTTCTCCTCTTGG-3'

OXTR For 5'-ACGGTGTCTTCGACTGCTG-3' 100 NM_174134

Rev 5'-GGGGCAAGGACGATGAC-3'

PGR For 5'-GAGAGCTCATCAAGGCAATTGG-3' 227 [61]

Rev 5'-CACCATCCCTGCCAATATCTTG-3'

Ubiquitin For 5'-AGATCCAGGATAAGGAAGGCAT-3' 198 [62]Rev 5'-GCTCCACCTCCAGGGTGATT-3'

GAPDH For 5'-GTCTTCACTACCATGGAGAAGG-3' 197 [61]

Rev 5'-TCATGGATGACCTTGGCCAG-3'

* For, forwards; Rev, reverse.** EMBL accession number or reference of published sequence.

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a

a

aab

bc

cc c

0

2

4

6

8

control 0.5 2 4 12 24 48 64

P4(

g/gwettissue)

(a) Progesterone concentration in CL tissue

a a aa

b b b

b

34

35

36

37

38

39

40-CP

(b) PGR mRNA expression in CL tissue

FIG. 1. (Berisha et al.)

a

a

aab

bc

cc c

0

2

4

6

8

control 0.5 2 4 12 24 48 64

P4(

g/gwettissue)

(a) Progesterone concentration in CL tissue

a a aa

b b b

b

34

35

36

37

38

39

control 0.5 2 4 12 24 48 64

40-CP

Time in hours after induced luteolyses

(b) PGR mRNA expression in CL tissue

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a

b

cc

c cc c

0

10

20

30

40

control 0.5 2 4 12 24 48 64

OT(ng/gwettissue)

(a) Oxytocin concentration in CL tissue

aab

abc abc

bc cd cd

d

34

36

38

40

42

44

40-CP

(b) Oxytocin mRNA expression in CL tissue

a

b

cc

c cc c

0

10

20

30

40

control 0.5 2 4 12 24 48 64

OT(ng/gwettissue)

(a) Oxytocin concentration in CL tissue

aab

abc abc

bc cd cd

d

34

36

38

40

42

44

control 0.5 2 4 12 24 48 64

40-CP

(b) Oxytocin mRNA expression in CL tissue

ab

a

b bb

b b b

30

31

32

33

34

35

contol 0.5 2 4 12 24 48 64

40-CP

Time in hours after induced luteolysis

(c) OXTR mRNA expression in CL tissue

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a

bb

b bb

bb

0

20

40

60

80

control 0.5 2 4 12 24 48 64

IGF1(ng/gwettissue)

(a) IGF1 protein concentration in CL tissue

b

a

bcc

bc bc bcc

32

34

36

38

40

40-CP

(b) IGF1 mRNA expression in CL tissue

a

bb

b bb

bb

0

20

40

60

80

control 0.5 2 4 12 24 48 64

IGF1(ng/gwettissue)

(a) IGF1 protein concentration in CL tissue

b

a

bcc

bc bc bcc

32

34

36

38

40

control 0.5 2 4 12 24 48 64

40-CP

(b) IGF1 mRNA expression in CL tissue

bb

aa a

a

b

b

30

32

34

36

38

control 0.5 2 4 12 24 48 64

4

0-CP

Time in hours after induced luteolysis

(c) IGFBP1 mRNA expression in CL tissue

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a

b b b bc

c c

0

5

10

15

20

25

control 0.5 2 4 12 24 48 64

VEGF

(ng/gwettissue)

(a) VEGF protein concentration in CL tissue

b

a a

bb

b bb

0

5

10

15

ANPT2(ng/gwetti

ssue)

(b) ANGPT2 protein concentration in CL tissue

a

b b b bc

c c

0

5

10

15

20

25

control 0.5 2 4 12 24 48 64

VEGF

(ng/gwettissue)

(a) VEGF protein concentration in CL tissue

b

a a

bb

b bb

0

5

10

15

control 0.5 2 4 12 24 48 64

ANPT2(ng/gwetti

ssue)

(b) ANGPT2 protein concentration in CL tissue

b

b

a

b

b

b

b b

34

36

38

40

42

control 0.5 2 4 12 24 48 64

40-CP

(c) ANGPT2 mRNA expression in CL tissue

(d) ANGPT2:ANGPT1 ratio (mRNA) in CL tissue

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PTGF

Blood flow TNF (m) FAS (m) IGF1 (m, p) VEGFA (m, p)

Blood Inflammatory Apoptotic, Luteotropic, Angiogenic, ECM

Flow Cytokines Antiapoptotic Antiluteotropic Vasoactive proteases

MMP1 (m)

4 h

[27-28, 59]Func

tio

[23, 34] [34]

IGFBP1 (m)

[25-26, 55]

EDNRA (m)

EDNRB (m)

[17, 21, 29, 60] [32]

NOS2 (m)

TNF (p)

CASP3 (m)

CASP6 (m)

CASP7 (m)

BAX (m)

LHR (m)

GHR (m)

CYP11A1 (m)

IGF2 (m)

VEGFA (m, p)

VEGFR1 (m)

VEGFR2 (m)

MMP2 (m)

MMP9 (m)

MMP14 (m)

MMP19 (m)

[23, 34]

[34]

IGFBP3 (m)

IGFBP4 (m)

[25-26]

ANGII (m, p)

EDN1 (m, p)

[17, 29]

TIMP2 (m)

[32]

Fig. 5. (Berisha et al.)