Angiogenesis and vascular endothelial growth factor expression in the equine corpus luteum

Precise pharmacological control of the corpus luteum is important in the manipulation of the oestrous cycle in mares. Angiogenesis plays a key role in the growth and regression of the corpus luteum; therefore, influencing the vasculature of the corpus luteum may offer a novel method for controlling its lifespan. In the present study, changes in angiogenesis and vascular expression of endothelial growth factor (VEGF) were evaluated throughout the luteal phase and after PGF(2alpha)-induced luteolysis. Corpora lutea were collected from mares in the early luteal phase (days 3-4), mid-luteal phase (day 10), early regression (day 14), late regression (day 17), and at 12 and 36 h after administration of PGF(2alpha) on day 10 of the oestrous cycle. Immunohistochemistry was used to localize Von Willebrand factor and Ki67 in endothelial and proliferating cells, respectively. VEGF mRNA and protein were localized by in situ hybridization and immunohistochemistry. The proliferation index of endothelial cells was intense in the early luteal phase. The early and mid-luteal phases were characterized by a dense network of capillaries. The microvasculature started to regress by day 14. After administration of PGF(2alpha), vasodilation was observed after 12 h, but after 36 h, luteal degeneration was accompanied by a significant decrease in vascularity. VEGF mRNA and protein were expressed mainly in the luteal cells during the early and mid-luteal phases and expression declined at early regression (day 14). However, immunostaining for VEGF protein was high in late luteal regression (day 17) and 36 h after PGF(2alpha) administration. These findings indicate a close temporal association between VEGF expression and angiogenesis in the equine corpus luteum during its functional lifespan.

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Angiogenesis in the Human Corpus Luteum: Localization and Changes in Angiopoietins, Tie-2, and Vascular Endothelial Growth Factor Messenger Ribonucleic Acid1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.85.11.6942 ◽

2000 ◽

Vol 85 (11) ◽

pp. 4302-4309 ◽

Cited By ~ 3

Author(s):

Christine Wulff ◽

Helen Wilson ◽

Pawlina Largue ◽

W. Colin Duncan ◽

David G. Armstrong ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Growth Factor ◽

Corpus Luteum ◽

Luteal Phase ◽

Corpora Lutea ◽

Vascular Endothelial ◽

Vegf Mrna ◽

Messenger Ribonucleic Acid ◽

Endothelial Growth Factor

In the menstrual cycle, extensive angiogenesis accompanies luteinization. During luteolysis, endothelial cells die, whereas in a conceptual cycle, the corpus luteum (CL) persists, and endothelial cell survival is extended. A main stimulator for angiogenesis is vascular endothelial growth factor (VEGF), while the angiopoietins (Ang-1 and Ang-2) may be important modulators. The aim of this study was to investigate the localization of Ang-1, Ang-2, their common receptor Tie-2, and VEGF messenger ribonucleic acid (mRNA) at the different stages of the functional luteal phase and after rescue by hCG. Ang-1 mRNA was uniformly expressed at a low level throughout the CL. The signal was highest during the early luteal phase. In contrast, Ang-2 mRNA expression was localized strongly to individual granulosa and thecal luteal and endothelial cells. Administration of hCG was associated with an increase in the Ang-2 mRNA area of expression and grain density in individual luteal and endothelial cells. The Tie-2 receptor mRNA was localized in endothelial cells, and the area of expression was highest during the early luteal phase and during luteal rescue. VEGF mRNA was found exclusively in granulosa luteal cells, and the area of expression was highest in corpora lutea during simulated pregnancy. These results begin to characterize the molecular regulation of the divergent processes involved in luteal angiogenesis during luteinization, luteolysis, and rescue in the human and imply that the angiopoietins are involved during the initial angiogenic phase and in luteal rescue.

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Expression and localization of vascular endothelial growth factor and its receptors in pig corpora lutea during the oestrous cycle

Reproduction ◽

10.1530/rep.0.1260393 ◽

2003 ◽

pp. 393-405 ◽

Cited By ~ 16

Author(s):

U Boonyaprakob ◽

JE Gadsby ◽

V Hedgpeth ◽

P Routh ◽

GW Almond

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Northern Blot ◽

Luteal Phase ◽

Northern Blot Analysis ◽

Corpora Lutea ◽

Vegf Receptor ◽

Vascular Endothelial ◽

Vegf Mrna ◽

Endothelial Growth Factor

Expression and localization of mRNAs for vascular endothelial growth factor (VEGF), VEGF receptor 1 (Flt) and VEGF receptor 2 (KDR) (VEGFR-1 and VEGFR-2, respectively) were investigated in pig corpora lutea. Northern blot analysis of total RNA indicated hybridization of pig VEGF, VEGFR-1 and VEGFR-2 cDNA probes to mRNA transcripts of approximately 3.9, 7.0 and 5.0 kb, respectively. The expression of mRNAs for VEGF and its receptors during the luteal phase (days 4, 7, 10, 13 and 15 after the onset of oestrus) were assessed by northern blot analysis, and hybridization signals were normalized to expression of pig 18S rRNA. Relative hybridization signals of expression of VEGF mRNA appeared to be constant; however, expression of VEGFR-1 mRNA was low on day 4, increased on day 7, and was higher on days 10, 13 and 15 (P<0.05, compared with day 4). In contrast, no changes in expression of mRNA for VEGFR-2 were evident on days 4-13, but a decrease was detected (P<0.05) at day 15. In situ hybridization revealed that VEGF mRNA was localized predominantly in large luteal cells, whereas both VEGFR-1 and VEGFR-2 were localized to small cells. These data indicate that the VEGF system may be involved in the regulation of luteal vasculature throughout the lifespan of the corpus luteum. Although the expression of VEGF mRNA was unchanged during the luteal phase, variations in the expression of VEGFR-1 and VEGFR-2 mRNAs indicate that differential regulation of expression of the VEGF receptors may play a role in the control of VEGF-mediated vascular growth at different phases of development and maturation of the pig corpus luteum.

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Localization and Quantification of Cyclic Changes in the Expression of Endocrine Gland Vascular Endothelial Growth Factor in the Human Corpus Luteum

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2004-0843 ◽

2005 ◽

Vol 90 (1) ◽

pp. 427-434 ◽

Cited By ~ 74

Author(s):

Hamish M. Fraser ◽

Julie Bell ◽

Helen Wilson ◽

Paul D. Taylor ◽

Kevin Morgan ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Corpus Luteum ◽

Luteal Phase ◽

Endocrine Gland ◽

Vascular Endothelial ◽

Vegf Mrna ◽

Prokineticin 2 ◽

Endothelial Growth Factor ◽

Human Corpus Luteum

Abstract Angiogenesis is essential for normal growth and function of the corpus luteum. The roles of various angiogenic factors in these events are being elucidated. Endocrine gland vascular endothelial growth factor (EG-VEGF) has recently been described in the human ovary. To define the localization of EG-VEGF mRNA in the corpus luteum and determine changes in its expression, dated human corpora lutea were studied at the early, mid-, and late luteal phases. Quantitative RT-PCR was employed to determine changes in EG-VEGF mRNA and compare expression to its related factor prokineticin-2 and the established angiogenic factor, VEGF. In situ hybridization was used to localize sites of production of EG-VEGF. To investigate whether expression of EG-VEGF was under the influence of LH or progesterone, luteinized granulosa cells were stimulated with human chorionic gonadotropin in the presence or absence of a progesterone synthesis inhibitor. EG-VEGF mRNA increased throughout the luteal phase, whereas there was no change in VEGF mRNA. The relative abundance of RNAs based upon PCR signal intensity showed that VEGF and EG-VEGF were highly expressed, whereas expression of prokineticin-2 was low. EG-VEGF mRNA was localized predominantly to granulosa-derived cells of the corpus luteum. Human chorionic gonadotropin stimulated both VEGF and EG-VEGF mRNA in vitro, but the level of expression was not influenced by progesterone. These results establish that in the human corpus luteum EG-VEGF is principally derived from granulosa lutein cells and that its synthesis is highest during the mid- to late luteal phase.

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Mid-luteal angiogenesis and function in the primate is dependent on vascular endothelial growth factor

Journal of Endocrinology ◽

10.1677/joe.0.1680409 ◽

2001 ◽

Vol 168 (3) ◽

pp. 409-416 ◽

Cited By ~ 50

Author(s):

SE Dickson ◽

R Bicknell ◽

HM Fraser

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Luteal Phase ◽

Smooth Muscle Actin ◽

Proliferation Index ◽

Endothelial Cell Proliferation ◽

Vascular Endothelial ◽

Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is essential for the angiogenesis required for the formation of the corpus luteum; however, its role in ongoing luteal angiogenesis and in the maintenance of the established vascular network is unknown. The aim of this study was to determine whether VEGF inhibition could intervene in ongoing luteal angiogenesis using immunoneutralisation of VEGF starting in the mid-luteal phase. In addition, the effects on endothelial cell survival and the recruitment of periendothelial support cells were examined. Treatment with a monoclonal antibody to VEGF, or mouse gamma globulin for control animals, commenced on day 7 after ovulation and continued for 3 days. Bromodeoxyuridine (BrdU), used to label proliferating cells to obtain a proliferation index, was administered one hour before collecting ovaries from control and treated animals. Ovarian sections were stained using antibodies to BrdU, the endothelial cell marker, CD31, the pericyte marker, alpha-smooth muscle actin, and 3' end DNA fragments as a marker for apoptosis. VEGF immunoneutralisation significantly suppressed endothelial cell proliferation and the area occupied by endothelial cells while increasing pericyte coverage and the incidence of endothelial cell apoptosis. Luteal function was markedly compromised by anti-VEGF treatment as judged by a 50% reduction in plasma progesterone concentration. It is concluded that ongoing angiogenesis in the mid-luteal phase is primarily driven by VEGF, and that a proportion of endothelial cells of the mid-luteal phase vasculature are dependent on VEGF support.

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A comparative study of the corpus luteum

Reproduction Fertility and Development ◽

10.1071/rd9950303 ◽

1995 ◽

Vol 7 (3) ◽

pp. 303 ◽

Cited By ~ 26

Author(s):

RT Gemmell

Keyword(s):

Corpus Luteum ◽

Luteal Phase ◽

Secretory Granules ◽

Hormonal Control ◽

Egg Laying ◽

Oestrous Cycle ◽

Corpora Lutea ◽

Embryonic Diapause ◽

Alternative Reproductive Strategy ◽

Uterine Development

The corpus luteum (CL) is a transitory organ which has a regulatory role in reproduction. Sharks, amphibians and reptiles have corpora lutea that produce progesterone which influences the rate of embryonic development. The egg-laying monotremes and the two major mammalian groups, eutherian and marsupial, have a CL that secretes progesterone. Most eutherians have allowed for the uterine development of their young by extending the length of the oestrous cycle and the CL or placenta actively secretes progesterone until birth. Gestation in the marsupial does not extend beyond the length of an oestrous cycle and the major part of fetal development takes place in the pouch. Where the extension of the post-luteal phase in the eutherian has allowed for the uterine development of young, the marsupial has extended the pre-luteal phase of the oestrous cycle and has evolved an alternative reproductive strategy, embryonic diapause. The mechanism for the secretion of hormones from the CL has been controversial for many years. Densely-staining secretory granules have been observed in the CL of sharks, marsupials and eutherians. These granules have been reported to contain relaxin, oxytocin or mesotocin, and progesterone. A hypothesis to suit all available data is that all hormones secreted by the CL are transported within such granules. In conclusion, although there are obvious differences in the mode of reproduction in the two main mammalian groups, it is apparent that there is a great deal of similarity in the hormonal control of regression of the CL and parturition.

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Expression of vascular endothelial growth factor (VEGF) receptors in rat corpus luteum: regulation by oestradiol during mid-pregnancy

Reproduction ◽

10.1530/rep.0.1220875 ◽

2001 ◽

pp. 875-881 ◽

Cited By ~ 17

Author(s):

N Sugino ◽

S Kashida ◽

S Takiguchi ◽

A Karube-Harada ◽

H Kato

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cells ◽

Growth Factor ◽

Corpus Luteum ◽

Vascular Endothelial Cells ◽

Corpora Lutea ◽

Vascular Endothelial ◽

Vegf Receptors ◽

Luteal Cells ◽

Endothelial Growth Factor

The aim of this study was to investigate the expression of vascular endothelial growth factor (VEGF) receptors, the fms-like tyrosine kinase (flt-1) and kinase insert domain-containing region (KDR), in corpora lutea obtained at different stages of the oestrous cycle and during pregnancy in rats. Immunohistochemistry revealed that both flt-1 and KDR were localized in luteal cells in addition to vascular endothelial cells, and that the intensity of staining was stronger in pregnant rats than in cyclic rats. Rats undergoing hypophysectomy-hysterectomy on day 12 of pregnancy were treated with oestradiol until day 15 of pregnancy to determine whether oestradiol is involved in expression of flt-1 and KDR mRNA in the corpus luteum during mid-pregnancy. The flt-1 and KDR mRNA contents in the corpus luteum were decreased significantly by hypophysectomy-hysterectomy, and these decreases recovered significantly after oestradiol treatment. Changes in the mass of the corpus luteum and serum progesterone concentrations paralleled the changes in expression of flt-1 and KDR mRNA. Developmental studies indicated that flt-1 and KDR mRNA contents in the corpus luteum were constant until day 15 of pregnancy but decreased significantly on day 21 of pregnancy. In conclusion, both flt-1 and KDR were expressed in luteal cells in addition to vascular endothelial cells, and expression was upregulated by oestradiol during mid-pregnancy. flt-1 and KDR may play a role in development of the corpus luteum and in production of progesterone during mid-pregnancy in rats.

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Vascular endothelial growth factor increases messenger RNAs encoding cyclooxygenase-II and membrane-associated prostaglandin E synthase in rat luteal cells

Journal of Endocrinology ◽

10.1677/joe.1.05629 ◽

2004 ◽

Vol 183 (3) ◽

pp. 527-533 ◽

Cited By ~ 25

Author(s):

T Sakurai ◽

K Tamura ◽

H Kogo

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Mrna Expression ◽

Corpus Luteum ◽

Prostaglandin E ◽

Vascular Endothelial ◽

Vegf Mrna ◽

Luteal Cells ◽

Endothelial Growth Factor ◽

Vegf Mrna Expression

Vascular endothelial growth factor (VEGF) is known to be necessary for the vascularization of the developing corpus luteum. Our recent data suggested that cyclooxygenase-II (COX-II) may play a role in the formation of vascular plexuses in developing corpora lutea of the rat. Here we examined the relationship between VEGF and the expression of prostaglandin (PG)- metabolizing enzymes in rat ovarian luteal cells. VEGF treatment caused a dose-dependent increase in the expression of COX-II and membrane-associated PGE synthase (mPGES) mRNA in cultured rat luteal cells. However, pretreatment of the luteal cells with a selective COX-II inhibitor, NS-398, abolished the VEGF-enhanced mPGES mRNA expression. VEGF also increased PGE2 secretion. Conversely, PGE2 dose-dependently stimulated VEGF mRNA expression. Furthermore, VEGF induced VEGF mRNA expression, but this effect was abolished by NS-398 pretreatment. These findings suggest that VEGF enhances PGE2 production by stimulating COX-II and mPGES expression in rat corpus luteum and that the effect of VEGF on luteal cells may be partially mediated by this stimulation of PGE2 production.

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Administration of vascular endothelial growth factor Trap during the ‘post-angiogenic’ period of the luteal phase causes rapid functional luteolysis and selective endothelial cell death in the marmoset

Reproduction ◽

10.1530/rep.1.01064 ◽

2006 ◽

Vol 132 (4) ◽

pp. 589-600 ◽

Cited By ~ 39

Author(s):

Hamish M Fraser ◽

Helen Wilson ◽

Christine Wulff ◽

John S Rudge ◽

Stanley J Wiegand

Keyword(s):

Vascular Endothelial Growth Factor ◽

Endothelial Cell ◽

Corpus Luteum ◽

Luteal Phase ◽

Caspase 3 ◽

Corpora Lutea ◽

Vascular Endothelial ◽

Late Luteal Phase ◽

Endothelial Growth Factor ◽

Vegf Trap

The intense angiogenesis characteristic of early corpus luteum development is dependent upon vascular endothelial growth factor (VEGF) as inhibitors of VEGF administered at the peri-ovulatory period suppress endothelial cell proliferation and progesterone secretion. We now report that administration of VEGF Trap, a soluble decoy receptor-based inhibitor, at the mid- or the late luteal phase in the marmoset results in a rapid decline in plasma progesterone. Since vascularisation of the corpus luteum is largely complete by the mid-luteal phase, it suggested that this functional luteolysis involved mechanisms other than inhibition of angiogenesis. A second experiment investigated the role of VEGF in maintaining the integrity of the luteal vasculature and hormone-producing cells. VEGF Trap was administered to marmosets in the mid-luteal phase and ovaries were obtained 1, 2, 4 or 8 days later for localisation of activated caspase-3 staining in the corpus luteum and compared with those obtained 2, 4 and 8 days after administration of control protein. The number of cells with activated caspase-3 staining was significantly increased after administration of VEGF Trap. Dual staining of activated caspase-3 with the endothelial cell marker CD31 showed that at 1 day post-treatment, more than 90% caspase-3-stained cells were vascular endothelium, prior to detection of an increasing incidence in death of hormone-producing cells on days 2 and 4. Staining with CD31 showed that the endothelial cell area was decreased after treatment. By 8 days after treatment, corpora lutea had regressed to varying degrees, while all control corpora lutea remained healthy. These results show that VEGF inhibition in the mid- or the late luteal phase induces functional luteolysis in the marmoset that is associated with premature and selective death of endothelial cells.

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Effects of local growth factors on the secretory function of bovine corpus luteum during the oestrous cycle and pregnancy in vitro

Reproduction Fertility and Development ◽

10.1071/rd9961003 ◽

1996 ◽

Vol 8 (6) ◽

pp. 1003 ◽

Cited By ~ 33

Author(s):

J Liebermann ◽

D Schams ◽

A Miyamoto

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Corpus Luteum ◽

Luteal Phase ◽

Late Pregnancy ◽

Oestrous Cycle ◽

Tnf Alpha ◽

Igf I ◽

Factor Alpha

The impact of insulin-like growth factor-I (IGF-I) basic fibroblast growth factor (bFGF), endothelin-1 (ET-1), tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-alpha (TGF-alpha) and platelet-derived growth factor (PDGF) on the release of progesterone (P4) and oxytocin (OT) from individual bovine corpora lutea at different stages of the oestrous cycle and pregnancy was evaluated with a microdialysis system (MDS) in vitro. IGF-I (1 microgram mL-1) induced significantly the acute effects on P4 release at the late luteal stage (Days 15-18) and early pregnancy (Days 60-120), whereas bFGF (100 ng mL-1) was extremely effective in stimulating P4 release particularly during the mid-luteal stage (Days 8-12). Both peptides stimulated (P < 0.05) the release of OT throughout the three luteal stages and during early and late pregnancy (Days 30-60 and Days 150-210). ET-1 (100 ng mL-1) clearly inhibited P4 release during the early (Days 5-7) and mid-luteal phase and stimulated OT release only during the mid-luteal stage (P < 0.001). TNF-alpha (100 ng mL-1) stimulated the release of P4 exclusively at the early luteal phase (P < 0.05), whereas OT secretion was increased by TNF-alpha during all stages of the oestrous cycle (P < 0.001). TGF-alpha and PDGF (100 ng mL-1) were effective in stimulating P4 release particularly during late pregnancy (P < 0.05). In contrast, stimulation of OT secretion by TGF-alpha was maximal during the late-luteal stage (P < 0.001), whereas PDGF significantly increased OT secretion during the oestrous cycle (except the early luteal stage) and pregnancy (P < 0.001). The data demonstrate distinct and stage-specific effects of growth factors on P4 and OT secretion in vitro. IGF-I, bFGF and TGF-alpha may play an important role in corpus luteum (CL) function during the oestrous cycle and pregnancy since they are locally expressed and synthesized, there are receptors for these growth factors, and they have been demonstrated to exert biological effects on the CL.

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Insulin-like growth factor I receptor mRNA and protein expression in pig corpora lutea

Reproduction ◽

10.1530/reprod/120.1.109 ◽

2000 ◽

pp. 109-114 ◽

Cited By ~ 9

Author(s):

Z Ge ◽

WE Nicholson ◽

DM Plotner ◽

CE Farin ◽

JE Gadsby

Keyword(s):

Growth Factor ◽

Corpus Luteum ◽

Oestrous Cycle ◽

Receptor Protein ◽

Corpora Lutea ◽

Western Blots ◽

Luteal Cells ◽

Factor I ◽

Receptor Mrna ◽

Igf I

Insulin-like growth factor I (IGF-I) is believed to play a luteotrophic role in the pig corpus luteum during the oestrous cycle. Since the actions of IGF-I in target tissues are mediated by the type I IGF receptor, the concentrations of IGF-I receptor mRNA and protein were examined in pig corpora lutea at different stages of the oestrous cycle. Corpora lutea were collected from normally cyclic gilts on days 4, 7, 10, 13, 15 and 16 of the oestrous cycle (n = 4 animals per day). Corpora lutea on days 7, 10 and 13 were dissociated with collagenase, and large and small luteal cell sub-populations were separated by elutriation. Northern and slot blots were used to examine mRNA, and western blots were used to measure the concentrations of IGF-I receptor protein in the pig corpus luteum. On northern blots, luteal IGF-I receptor mRNA was present as a single 11 kb transcript. The slot blots showed that the steady state expression of IGF-I receptor mRNA increased significantly (P < 0.05) from its lowest value on day 4, to reach a maximum on days 13-16. IGF-I receptor mRNA was also expressed to a greater extent in large compared with small luteal cells (P < 0.05). On western blots, IGF-I receptor appeared as a 95 kDa protein band (beta-subunit) and IGF-I receptor protein concentrations were significantly higher (P < 0.05) on days 4-10 than on days 13-16. Finally, large luteal cells appeared to contain more IGF-I receptor protein than the small luteal cells. In conclusion, since IGF-I receptor was detected in the pig corpus luteum, it is a likely target tissue for IGF-I, especially during the early luteal phase. Furthermore, IGF-I receptor was localized primarily on large luteal cells, thus it is hypothesized that IGF-I may play a paracrine role in the pig corpus luteum.

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