Deleterious Effects of Progestagen Treatment in VEGF Expression in Corpora Lutea of Pregnant Ewes

Reprod Dom Anim 46, 481–488 (2011); doi: 10.1111/j.1439-0531.2010.01692.x ISSN 0936-6768

Deleterious Effects of Progestagen Treatment in VEGF Expression in Corpora Lutea of Pregnant Ewes CA Letelier1,2, MA Sanchez3, RA Garcia-Fernandez3, B Sanchez3, P Garcia-Palencia3, A Gonzalez-Bulnes2 and JM Flores3 1 Instituto de Ciencia Animal, Universidad Austral de Chile, Casilla, Valdivia, Chile; 2Departamento de Reproduccio´n Animal, INIA; 3Departamento de Medicina y Cirugı´a Animal, Facultad de Veterinaria, Madrid, Spain

Contents The aim of the current study was to determine the possible effects of progestagen oestrous synchronization on vascular endothelial growth factor (VEGF) expression during sheep luteogenesis and the peri-implantation period and the relationship with luteal function. At days 9, 11, 13, 15, 17 and 21 of pregnancy, the ovaries from 30 progestagen treated and 30 ewes cycling after cloprostenol injection were evaluated by ultrasonography and, thereafter, collected and processed for immunohistochemical evaluation of VEGF; blood samples were drawn for evaluating plasma progesterone. The progestagen-treated group showed smaller corpora lutea than cloprostenol-treated and lower progesterone secretion. The expression of VEGF in the luteal cells increased with time in the cloprostenol group, but not in the progestagen-treated group, which even showed a decrease between days 11 and 13. In progestagen-treated sheep, VEGF expression in granulosaderived parenchymal lobule capillaries was correlated with the size of the luteal tissue, larger corpora lutea had higher expression, and tended to have a higher progesterone secretion. In conclusion, the current study indicates the existence of deleterious effects from exogenous progestagen treatments on progesterone secretion from induced corpora lutea, which correlate with alterations in the expression of VEGF in the luteal tissue and, this, presumably in the processes of neoangiogenesis and luteogenesis.

Introduction In veterinary and human medicine, the administration of exogenous treatments based on progestative supply is used in the management of the reproductive cycle, promoting ovulation at progesterone withdrawal (Kusina et al. 2000; Yildirim et al. 2000; Fraser and Kovacs 2003; Rubianes and Menchaca 2003; GonzalezBulnes et al. 2004; Kaeoket 2008). The efficiency of such treatments for inducing ovulation is high; however, final fertility yields are lowered (Menchaca and Rubianes 2004). Mechanistic studies in humans are impeded by ethical reasons, but previous studies about progestagen treatments in domestic mammals have reported alterations in the development of corpora lutea and in the secretion of progesterone (Killian et al. 1985; Vin˜oles et al. 2001; Gonzalez-Bulnes et al. 2005). Inadequacy of luteal function is considered a major reproductive problem (Wathes et al. 2003) as progesterone is the key factor for pregnancy success, being essential both for endometrial development and for embryo survival (Mann and Lamming 1999; Mann et al. 1999; Webb et al. 2002). The development of a corpus luteum (i.e. luteogenesis) is characterized for rapid changes in the cells of the ovulatory follicle after ovulation, involving the  2010 Blackwell Verlag GmbH

processes of cell proliferation, differentiation and transformation (Zheng et al. 1994). The ovulatory follicle is compartmentalized in a highly vascular theca layer and a non-vascular granulosa layer, separated by a basement membrane (Redmer et al. 2001). The process of luteogenesis requires a well-developed vascular system to fulfil the increased metabolic demands of the proliferating cells (Reynolds et al. 1992; Redmer et al. 1996). Mature corpora lutea are so vascular, in fact, that the majority of steroidogenic cells are adjacent to one or more capillaries (Schams and Berisha 2004). At the same time, a dense capillary network in an endocrine organ like the corpus luteum is crucial for distribution of the secreted hormones throughout the body. A close relationship between vascularization and blood flow and hormonal production has already been reported in the bovine corpus luteum (Berisha et al. 2000; Kobayashi et al. 2001; Neuvians et al. 2004; Miyamoto et al. 2009); ovarian blood flow being found highly associated with the rate of progesterone secretion (Niswender et al. 2000; Reynolds et al. 2000; GrazulBilska et al. 2001). Thus, the developing corpus luteum is a site of intense formation of new blood vessels (angiogenesis), and factors regulating luteal angiogenesis were early proposed to play a major role in regulating luteal function (Wiltbank et al. 1990; Reynolds and Redmer 1999; Reynolds et al. 2000; Robinson et al. 2009). The sheep has been widely used for the study of angiogenic factors throughout the oestrous cycle and early pregnancy from 1990s (Reynolds et al. 1994; Redmer and Reynolds 1996). Cellular and molecular regulation of angiogenesis involves several regulating factors; however, vascular endothelial growth factor (VEGF) is considered the main effector (Reynolds et al. 2000; Webb et al. 2002; Fraser and Duncan 2005; Sugino et al. 2005; Fraser et al. 2006; Mariani et al. 2006). Vascular endothelial growth factor is known to be a specific stimulator of proliferation, migration and permeability of vascular endothelial cells, processes that are necessary for the establishment of angiogenesis (Kaczmarek et al. 2007). Vascular endothelial growth factor–dependent angiogenesis is decisive for follicular growth and corpus luteum formation and function (Duncan et al. 2008), and VEGF has been found in luteal cells (Mariani et al. 2006). Therefore, the objective of our current experiment was to determine in pregnant ewes the possible effects of progestagen treatments on the expression of VEGF during luteogenesis and peri-implantation phases and their relationship with luteal function.

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CA Letelier, MA Sanchez, RA Garcia-Fernandez, B Sanchez, P Garcia-Palencia, A Gonzalez-Bulnes and JM Flores

Materials and Methods Animals, treatment and sampling Sixty Manchega ewes, 3–8 years old, were used. Females were maintained at the experimental farm of the Animal Reproduction Department at the Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA, Madrid, Spain; latitude 4025¢N). These facilities meet the requirements of the European Union for Scientific Procedure Establishments. The current experiment was performed, after approval from the Scientific Ethics Committee of the INIA, during the natural breeding season (October–March) described for this breed at this latitude. Half of the animals (n = 30) were treated with progestagens impregnated in an intravaginal pessary (40 mg fluorogestone acetate, FGA, Chronogest; Intervet International, Boxmeer, The Netherlands) and maintained inside the vagina for 14 days. In the remaining ewes (n = 30), ovarian cyclic activity was synchronized by inducing luteolysis with cloprostenol, a potent prostaglandin F2a analogue (Estrumate, Mallinckrodt Vet GmbH, Friesoythe, Germany), in three i.m. doses of 100 lg given 10 days apart. Oestrous behaviour was determined every 6 h from 18 to 42 h after the third cloprostenol injection or the sponge withdrawal by using adult rams. When a ewe was detected in oestrus, mating was allowed to be repeated 6 h later for assuring fertilization. The day of first detection of oestrous signs and mating was considered day 0 for experimental purposes. Samplings were performed at days 9, 11, 13, 15, 17 and 21 from five animals per group each day. In order to avoid the misuse of non-ovulating animals, the presence of at least one corpus luteum was assessed, in each sheep, by 7.5 MHz transrectal ultrasonography (Aloka SSD-500; Ecotron, Madrid, Spain), as previously described (Schrick et al. 1993) and validated in our laboratory (Gonzalez-Bulnes et al. 1994). In order to avoid data from corpora lutea representing previous ovarian cycling activity, only corpora lutea resulting from the induced oestrus were assessed. The area of luteal tissue of the corpora lutea resulting from induced oestrus was also evaluated during the scanning, using the electronic calipers of the ultrasound machine. At this time, for the determination of progesterone secretion, a jugular blood sample was collected by using vacuum blood evacuation tubes with heparin (Vacutainer Systems Europe, Becton Dickinson, Meylan Cedex, France). Finally, ovaries were surgically retrieved under general anaesthesia with xylazine (Rompun, 6 mg IM; Bayer AG, Leverkusen, Germany) and ketamine (Imalgene1000, 130 mg IM; Merial, Lyon, France). Blood processing and progesterone assays Blood samples were centrifuged at 1500 · g for 15 min, and plasma was stored at )20C until assayed. Progesterone concentrations were measured using a Coat-A-Count solid phase radioimmunoassay kit (Coat-A-Count Progesterone; Diagnostic Products Corporation, Los Angeles, CA, USA). Sensitivity

for progesterone was 0.09 ng ⁄ ml, and the inter- and intra-assay variation coefficients were 5.1% and 4.2%, respectively. Ovarian tissue processing and immunohistochemistry After surgical removal, ovaries were immediately fixed in 4% paraformaldehyde for 24 h at 4C, washed in PBS for 24 h in a refrigerator and passed through a series of graded volumes of ethanol changed every 24 h at 4C (from 30% to 70% ethanol in deionized water, in the latter, samples can be stored indefinitely). Thereafter, ovaries were routinely processed, paraffin embedded and sectioned (3 lm). These sections were deparaffinated with xylene and rehydrated through a series of graded volumes of ethanol in distilled water and washed in distilled water (5 min). Immunohistochemistry of CL was performed at days 9, 11, 13, 15, 17 and 21 of gestation to localize the presence of VEGF protein. Antigen retrieval was undertaken in sodium citrate buffer (pH = 6) in a pressure cooker for 2 min at the highest pressure and cooled for 30 min. Endogenous peroxidase was blocked with 1.5% (v ⁄ v) hydrogen peroxidase in methanol for 15 min at room temperature; the slides were washed in distilled water and TRISbuffered saline (TBS). Sections were incubated with a mouse anti-VEGF monoclonal antibody (C-1: sc-7296; Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted 1 : 80 (2.5 lg ⁄ ml) overnight at 4C. After washing two times in TBS, slides were incubated with monoclonal horse anti-mouse biotinylated secondary antibody (Vector, Burlingame, CA, USA) 1 : 400 in TBS for 30 min at room temperature, washed again (twice) in TBS and incubated with streptavidin horseradish peroxidase (HRP) conjugate (Zymed, Invitrogen Co., Carlsbad, CA, USA) 1 : 400 in TBS for 30 min at room temperature. Immunoreactions were detected by incubating 10 min with diaminobenzidine (Vector, Burlingame, CA, USA). After washing in distilled water for 10 min, sections were counterstained with haematoxylin for 4 min, dehydrated in alcohol, cleared in xylene and mounted. Sections for negative controls were prepared using a non-immune mouse IgG antibody (Dakocytomation Cat. No. Z0259; BD Diagnostics, Sparks, MD, USA) instead of the primary antibody. Image analysis Four different compartments were considered for evaluating the expression of VEGF in each corpus luteum: peripheral blood vessels, thecal-derived compartments, granulosa-derived parenchymal lobules and luteal cells in agreement with established criteria (Redmer et al. 2001). Vascular endothelial growth factor was evaluated, by two observers who were not aware of which group of animals was observed, in different portions of the corpus luteum using a light microscope (Olympus DX50; Olympus America Inc., Center Valley, PA, USA) at 20· magnification. A subjective analysis was performed to estimate the intensity of staining of the cells that was scored as follows: (i) none, (ii) weak, (iii) strong and (iv) very strong.

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Statistical analysis Effects of treatment on corpora lutea size and plasma progesterone concentration were determined by analysis of variance (ANOVA). The expression frequencies of VEGF were assessed by Chi-Square distribution test. Statistical analyses for relationship between the different variables were performed by Pearson correlation analysis and linear regression procedures. All results were expressed as mean ± SEM, and statistical significance was set at p < 0.05.

In the latter group of animals, VEGF expression and total luteal size showed a positive correlation; in other words, bigger corpora lutea had higher levels of VEGF immunoexpression (r = 0.416, p < 0.01), mainly in capillaries situated in granulosa-derived parenchymal lobules. Similarly, corpora lutea with higher immunolocalization of VEGF in granulosa-derived parenchymal lobules capillaries tended to have higher progesterone secretion (r = 0.417, p = 0.054).

Discussion Results The evaluation of the size of corpora lutea and plasma progesterone concentration showed differences between groups from the first observations at day 9 (p < 0.01; Table 1). In this way, it was possible to see that the total area of luteal tissue per animal, equivalent to the sum of all corpora lutea in each ovary, was significantly larger in cloprostenol-treated sheep than in progestagen-treated ewes at day 9 (p < 0.05), while the progesterone secretion was significantly higher at days 11 (p < 0.005), day 15 (p < 0.05) and day 21 (p < 0.05), as detailed in Table 1. At the same time, when the individual size of each corpus luteum and plasma progesterone concentration was compared, a positive correlation between both parameters was found in each group (r = 0.427, p < 0.001 for cloprostenol-treated females and r = 0.166, p < 0.05 for progestagen-treated sheep). Vascular endothelial growth factor immunolocalization was always present in the corpora lutea throughout the different days of observation in the cell cytoplasm, both in luteal and in peri-endothelial cells (Table 2). In the cloprostenol group, the expression of VEGF in the luteal cells increased with time (r = 0.384, p < 0.05; Fig. 1); however, such relationship was not observed in the group treated with progestagens, which showed a decrease between days 11 and 13 (p = 0.05, Fig. 2), although a slight tendency to increase through the next days was observed (days 15, 17 and 21 pc). On the other hand, the expression of VEGF was higher in peripheral blood vessels (p < 0.01) and granulosa-derived parenchymal lobule capillaries (p < 0.05) in cloprostenol group than in progestagen-treated sheep at day 9 (Fig. 3).

A close relationship between luteal function and expression of VEGF in corpora lutea in sheep has been demonstrated with our results, which also indicate that such a relationship may be diminished when exogenous hormone therapies such as progestagens are administered. Differences in VEGF expression in CL were found between sheep treated with progestagens and females treated with cloprostenol. These dissimilarities were found to be correlated with variations in luteal morphology and function and thus may contribute to fertility failures previously reported to be associated with progestative treatments in ewes (Gonzalez-Bulnes et al. 2004). In the progestagen-treated sheep, the size of the corpora lutea and the amount of progesterone secreted to peripheral blood were lower than in cloprostenoltreated ewes; although in both groups, they showed a slight increase through the different days of observation. These results suggest deficiencies in luteal growth and function, agreeing with previous studies (Killian et al. 1985; Vin˜oles et al. 2001; Gonzalez-Bulnes et al. 2005). The growth and the secretory function of corpora lutea require an intense angiogenesis but also stabilization of blood vessels, with VEGF playing a key role (Reynolds et al. 2000; Webb et al. 2002; Fraser and Duncan 2005; Sugino et al. 2005; Fraser et al. 2006; Mariani et al. 2006). In the current study, increases in corpus luteum growth and progesterone secretion in cloprostenoltreated sheep were concomitant with increases in the expression of VEGF; on the contrary, in vitro studies showed that VEGF addition to culture plates does not increase the production of progesterone by the luteal cells (Tropea et al. 2006). This could be because in vivo

Table 1. Corpora lutea area and plasma progesterone concentration (mean ± SEM) in cloprostenol and progestagen-treated sheep for oestrous synchronization Day Treatment groups Cloprostenol Progesterone (ng ⁄ ml) Individual luteal tissue (cm2)* Total luteal tissue (cm2)** Progestagen Progesterone (ng ⁄ ml) Individual luteal tissue (cm2)* Total luteal tissue (cm2)**

9

11

13

15

17

21

5.0 ± 0.2a 0.9 ± 0.1a 1.3 ± 0.3c

5.5 ± 0.2e 1.0 ± 0.2a 1.2 ± 0.3

5.6 ± 0.2 0.9 ± 0.2 1.3 ± 0.2

6.1 ± 0.3c 1.0 ± 0.1 1.3 ± 0.1

6.2 ± 0.3 1.1 ± 0.1 1.3 ± 0.2

6.4 ± 0.3c 1.2 ± 0.2c 1.5 ± 0.2

4.6 ± 0.2b 0.8 ± 0.1b 0.9 ± 0.2d

4.7 ± 0.2f 0.9 ± 0.2b 1.0 ± 0.1

5.1 ± 0.2 0.9 ± 0.3 1.1 ± 0.1

5.1 ± 0.3d 0.9 ± 0.2 1.2 ± 0.2

5.4 ± 0.3 0.9 ± 0.2 1.1 ± 0.1

5.3 ± 0.3d 1.0 ± 0.1d 1.0 ± 0.2

a „ b : p < 0.01; c „ d: p < 0.05; e „ f: p < 0.005 means values within the same column. *Measurement of total luteal tissue area from a single CL present on each ewe. **Measurement of total luteal tissue area represented by the presence of ‡1 corpus luteum per female.

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CA Letelier, MA Sanchez, RA Garcia-Fernandez, B Sanchez, P Garcia-Palencia, A Gonzalez-Bulnes and JM Flores

Table 2. Immunolocalization and expression (mean ± SEM) of vascular endothelial growth factor (VEGF) in corpora lutea of cloprostenoltreated ewes and sheep treated with progestagens for oestrous management. We have to note that besides statistical differences identified by different superscripts, the expression of VEGF in the luteal cells increased with time only in the cloprostenol group (r = 0.384, p < 0.05) Day Immunostaining of VEGF

9

Cloprostenol Number of corpora lutea Luteal cells CL peripheral blood vessels Thecal-derived compartments Granulosa derived-parenchymal lobules Progestagen Number of corpora lutea Luteal cells CL peripheral blood vessels Thecal-derived compartments Granulosa derived-parenchymal lobules a „ b c „ d

,

11

13

15

17

21

0.4 2.6 1.2 1.6

10 ± 0.2 ± 0.2a ± 0.3 ± 0.3c

0.6 1.8 1.0 0.5

7 ± ± ± ±

0.3 0.3 0.3 0.2

0.6 1.5 0.7 0.4

6 ± ± ± ±

0.1 0.2 0.2 0.2

0.6 1.1 0.6 0.0

8 ± ± ± ±

0.3 0.1 0.2 0.0

1.3 1.3 0.9 0.0

7 ± ± ± ±

0.3 0.2 0.3 0.0

1.4 1.3 1.2 0.1

9 ± ± ± ±

0.3 0.2 0.2 0.0

0.6 1.6 0.6 0.4

8 ± ± ± ±

1.0 1.4 0.8 0.3

7 ± ± ± ±

0.3 0.3 0.3 0.1

0.5 1.3 0.8 0.3

6 ± ± ± ±

0.2 0.4 0.3 0.1

0.7 1.2 1.0 0.0

6 ± ± ± ±

0.2 0.2 0.3 0.0

0.9 1.1 0.8 0.0

8 ± ± ± ±

0.2 0.2 0.2 0.0

1.4 1.8 1.6 0.0

5 ± ± ± ±

0.4 0.2 0.2 0.0

0.3 0.3b 0.2 0.2d

: p < 0.005 mean values within the same column are different.

(a)

(b)

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(c)

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(d)

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50 µm

(e)

(f)

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the VEGF effect is indirect through an enhanced functionality of luteal cells by an increased vascular support. On the other hand, in progestagen-treated sheep, VEGF expression remained stable through time though seems to decrease between days 11 and 13; a critical period, as this is coincidental with the period of implantation in sheep (Bazer et al. 1991).

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Fig. 1. Vascular endothelial growth factor expression in luteal cells of cloprostenol-treated ewes. (a) Day 9 post-mating (pm), (b) day 11 pm, (c) day 13 pm, (d) day 15 pm, (e) day 17 pm and (f) 21 pm. Immunoreaction increased from day 13 post-mating onwards. Inset in (a) negative control. Streptavidin biotin peroxidase method. Haematoxylin counterstained. Scale bar = 50 lm

The analysis by CL cellular compartments showed that both groups of ewes expressed immunoreactive VEGF in the blood vessels of thecal- and granulosaderived parenchymal lobules, which is consistent with previous studies on neovascularization in developing corpora lutea (Mariani et al. 2006). In our study, the expression of VEGF in granulosa-derived parenchymal  2010 Blackwell Verlag GmbH

VEGF in Early Pregnancy Ovine CL

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(b)

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(c)

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(d)

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(e) Fig. 2. Vascular endothelial growth factor expression in luteal cells of progestagen-synchronized ewes. (a) Day 9 post-mating (pm), (b) day 11 pm, (c) day 13 pm, (d) day 15 pm, (e) day 17 pm, (f) day 21 pm. Immunoreaction decreased between days 11 (b) and 13 (c) post-mating. Inset in (a) negative control. Streptavidin biotin peroxidase method. Haematoxylin counterstained. Scale bar = 50 lm

Fig. 3. Higher vascular endothelial growth factor expression in corpora lutea at day 9 post-mating in peripheral blood vessels in cloprostenol (a) than in progestagen-synchronized groups (b) and in Granulosa-derived parenchymal lobules capillaries in cloprostenoltreated sheep (c) than in progestagen-synchronized groups (d). *Immunopositive blood vessel in granulosa-derived parenchymal lobules. Inset in (d): negative control. Streptavidin biotin peroxidase method. Haematoxylin counterstained. Scale bar = 40 lm

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lobules was low during the first days of study and absent from day 15 onwards in both groups, which is also consistent with the study previously described  2010 Blackwell Verlag GmbH

(f)

(Redmer et al. 2001). In brief, there are no blood vessels supplying granulosa-derived parenchymal lobules before or immediately after ovulation (Redmer

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et al. 2001); hence, this compartment exhibits all of the characteristics of hypoxia (Niswender and Nett 1994) considered a major stimulator of VEGF expression (Shweiki et al. 1992; Nishimura and Okuda 2010); thus, VEGF increased in response to hypoxia and stopped when hypoxia was circumvented by neovascularization. In our study, most of the VEGF protein was found in vessels located in the theca-derived compartment and in peripheral blood vessels, the highly vascularized structures in the pre-ovulatory follicles, supporting previous results (Reynolds et al. 2000; Robinson et al. 2009). Comparison of both groups showed not previously reported differences in VEGF expression in both granulosa- and theca-derived compartments. Progestagen-treated sheep showed a lower expression of such protein in those compartments derived from granulosa cell layers, especially in the first day of sampling (day 9 pc). Moreover, we found that cloprostenol-treated sheep showed an increased expression of VEGF in peripheral blood vessels, which was associated with an increased expression in theca-derived compartments that, in turn, was related to an increased expression in the luteal cells. The lower expression of VEGF and the lack of associations of increases in VEGF expression in blood vessels and luteal cells in progestagen-treated sheep may be considered as strong indicators of a compromised luteogenesis in this group. These features may be related to a lower vascularization and, thus, a lower secretory activity in progestagen-treated sheep, given the close relationship between vascularization and progesterone secretion (Reynolds et al. 2000; Grazul-Bilska et al. 2001; Fraser and Duncan 2005; Fraser et al. 2006; Niswender et al. 2007). Failures in luteal functionality and fertility in progestagen-treated sheep may be related to an inadequate development and maturation of pre-ovulatory follicles (White et al. 1987; Keisler and Keisler 1989), which has been found to be augmented in progestative treatments. Thus, it leads to deficiencies in the secretion of oestradiol during the pre-ovulatory phase and, thereafter, in the ability to ovulate (Gonzalez-Bulnes et al. 2005). Highly vascularized follicles usually reach the dominant ovulatory phase, while follicles with reduced vascularization enter the atresia stage (Redmer and Reynolds 1996; Augustin 2000; Reynolds et al. 2002). In addition, alteration of steroidogenesis decreases the quality of oocytes, a common feature in progestagen-treated cycles (Greve et al. 1995), that has been related to reductions in the vascularity of follicles (Borini et al. 2001; Merce´ et al. 2006). In conclusion, progestagen-treated sheep had a lower expression of VEGF correlated with a deleterious effect on progesterone secretion from induced corpora lutea that is likely associated with an altered neovascularization and luteogenesis of the CL, which may contribute to the decreases in fertility reported for progestagen treatments. Acknowledgements We thank P. Aranda for technical assistance and H. Page for her help with English language and editing.

Conflicts of interest None of the authors have any conflict of interest to declare.

Funding This work was supported by a grant CYCIT AGL2005-02669. C. Letelier was funded by a CONICYT (Chilean National Council of Science and Technology) doctoral fellowship.

Author contributions Claudia Letelier participated in study design, execution, analysis and interpretation of data, manuscript drafting and critical discussion. Rosa A. Garcia-Fernandez participated in acquisition of data, analysis and interpretation of data, manuscript drafting and critical discussion. Maria A. Sanchez participated in analysis and interpretation of data, manuscript critical discussion and final approval of the version to be published. Pilar Garcia Palencia participated in acquisition of data, analysis and interpretation of data, manuscript drafting and critical discussion. Belen Sanchez participated in acquisition of data, analysis and interpretation of data, and manuscript critical discussion. Antonio Gonzalez-Bulnes participated in study design, analysis and interpretation of data, manuscript drafting, critical discussion and final approval of the version to be published. Juana M. Flores participated in interpretation of data, manuscript critical discussion and final approval of the version to be published.

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Yildirim M, Noyan V, Tiras MB, 2000: Estrogen-progestagen pre-treatment before HMG induction in hypogonadotropic patients. Int J Gynaecol Obstet 71, 249–250. Zheng J, Fricke PM, Reynolds LP, Redmer DA, 1994: Evaluation of growth, cell proliferation, and cell death in bovine corpora lutea throughout the estrous cycle. Biol Reprod 51, 623–632. Submitted: 27 May 2010; Accepted 9 Aug 2010 Author’s address (for correspondence): Rosa Ana Garcia-Fernandez, Departamento de Medicina y Cirugı´ a Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avenida Puerta de Hierro sn. 28040, Madrid, Spain. E-mail: [email protected]

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