1. Dopamine receptor 2 activation inhibits ovarian vascular endothelial growth factor secretion in an ovarian hyperstimulation syndrome (OHSS) animal model: implications for treatment of OHSS with dopamine receptor 2 agonists 
Hortensia Ferrero, Ph.D., Carmen M. García-Pascual, Ph.D., María Gaytán, Ph.D., Concepción Morales, Ph.D., Carlos Simón, M.D., Francisco Gaytán, M.D., Antonio Pellicer, M.D., Raúl Gómez, Ph.D. 
Fertility and Sterility 
Volume 102, Issue 5, Pages e1 (November 2014)
DOI: /j.fertnstert
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Identification of D2 expression in rat CL. Immunohistochemical detection of D2 in ovarian sections of PMSG + hCG-treated immature female rats. (A) The primary antibodies were omitted in the negative control. (B–D) D2 expression was detected by immunohistochemistry using DAB as a chromogen to reveal D2 staining, and slides were counterstained with hematoxylin. Notice the very prominent staining for D2 in TCs (red arrows) and a more diffuse but clear positive staining in LGCs (asterisks) with faint or no staining in preovulatory or atretic follicle GCs (black arrows). Magnifications ×100 (A–C) and ×400 (D).
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Dose-response effects of D2-ag administration in PRL-supplemented OHSS rats. To find the optimal D2-ag (Cb2) dose able to decrease the VP and VEGF secretion in OHSS without affecting luteolysis, dose-response experiments with different doses of Cb2 were performed in PRL-supplemented OHSS model animals. (A) VP (as micrograms of extravasated EB dye per 100 g animal weight). (B) Intracellular VEGF protein levels (normalized to the GAPDH housekeeping) and (C) VEGF mRNA expression (as arbitrary units of intensity resulting from the ratio of VEGF normalized to β-actin expression) 48 hours after hCG injection in OHSS rats supplemented with 5 mg PRL and treated with Cb2 at 0- (control), 50-, 100-, or 500-μg/kg doses. The image on the right corresponds to VEGF and GAPDH protein bands from a lysate pool corresponding to four animals in each group (0- [control], 50-, 100-, or 500-μg/kg Cb2 doses) detected by Western blot (n = 16). Arrowheads denote the position of VEGF-reduced protein ∼20 kDa and GAPDH protein ∼37 kDa. Note that with 100- and 500-μg/kg doses of Cb2, both VP and intracellular VEGF protein levels decreased significantly when compared with the control. ∗∗P<.01, comparing 0 μg/kg (control group) with the experimental (50, 100, or 500 μg/kg) groups.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
The effects of low-dose (100 μg/kg) D2-ag and D2-ant treatment on VP, VEGF protein, and mRNA levels in PRL-supplemented OHSS rats. (A) VP (as micrograms of extravasated EB dye per 100 g body weight) in OHSS rats treated with different regimes of D2 compounds, (B) intracellular VEGF protein levels (normalized to the GAPDH housekeeping) in control and D2-ag-/ant-treated OHSS rats, and (C) VEGF mRNA expression (as arbitrary units of intensity resulting from the ratio of VEGF normalized to β-actin expression) on the ovaries of control and experimental OHSS rats, 48 hours after hCG administration. The image to the right corresponds to the VEGF and GAPDH protein levels detected by Western blot. Lanes 1 and 2: control group (CT; 0 μg/kg D2-ag). Lanes 3 and 4: experimental group 1 (D2-ag; 100 μg/kg D2-ag). Lanes 5 and 6: experimental group 2 (D2-ag + D2-ant; 100 μg/kg D2-ag + 100 μg/kg D2-ant). Each lane contains a lysate pool from four animals. Arrowheads denote the position of VEGF-reduced protein ∼20 kDa and GAPDH protein ∼37 kDa. Note that administration of a 100-μg/kg D2-ag dose significantly inhibited increased VP in PRL-supplemented OHSS rats and that VP was reverted to control values when the D2-ant (100 μg/kg) was concomitantly administered with the D2-ag (100 μg/kg). Also note the parallel decrease in VEGF protein levels when the D2-ag was added and how this inhibition was reverted when the D2-ag and D2-ant were combined. However, no differences were found after comparison of VEGF mRNA levels in the control group versus the experimental groups. ∗P<.05 and ∗∗P<.01 compared with the CT.
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Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
The effects of low-dose (100 μg/kg) D2-ag and D2-ant on luteal function. Graphs correspond to (A) serum PRL levels (ng/mL), (B) serum P4 levels (ng/mL), and (C) luteal vascular density (as the percentage of PECAM, a specific EC marker, stained area per total area) in OHSS rats supplemented with 5 mg PRL and treated with 0 μg/kg D2-ag (control group [CT]), 100 μg/kg D2-ag (experimental group 1 [D2-ag]), or 100 μg/kg D2-ag + 100 μg/kg D2-ant (experimental group 2 [D2-ag + D2-ant]), 48 hours after hCG injection. Photographs at the side of graph C correspond to representative ovarian sections from the different groups, immunostained for PECAM to detect blood vessels. C− is the negative control. Note how exogenous PRL supplementation maintains similar circulating PRL levels in treated animals versus controls. Also note how the different treatments do not affect luteal function (as assessed by P4 levels and ovarian vascularization).
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

6. Supplemental Figure 1
Dose-response effects of D2-ag (Cb2) administration on the luteal function in PRL-supplemented OHSS rats. To find the optimal Cb2 dose able to decrease the VP and VEGF secretion in OHSS PRL-supplemented animals without affecting luteal function, dose-response experiments with different doses of the D2-ag were performed. (A) Serum PRL levels (ng/mL), (B) serum P4 levels (ng/mL), (C) luteal vascular density (as the percentage of PECAM, a specific EC marker, stained area per total area) 48 hours after hCG injection in OHSS rats supplemented with 5 mg PRL and treated with Cb2 at 0- (control), 50-, 100-, or 500-μg/kg doses. Note that 500 μg/kg of Cb2 produced a significant decrease in P4 and luteal vascular density, indicating that this dose affects luteolysis. ∗∗P<.01, comparing 0 μg/kg (control group) against the experimental (50, 100, or 500 μg/kg) groups.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions