Human Reproduction, Vol.31, No.2 pp. 436–444, 2016 Advanced Access publication on January 5, 2016 doi:10.1093/humrep/dev320

ORIGINAL ARTICLE Reproductive biology

Prostaglandin E2 and vascular endothelial growth factor A mediate angiogenesis of human ovarian follicular endothelial cells Heidi A. Trau 1, Mats Bra¨nnstro¨m2, Thomas E. Curry Jr 3, and Diane M. Duffy 1,* 1

Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA 23501, USA 2Department of Obstetrics and Gynecology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 3Department of Obstetrics and Gynecology, University of Kentucky College of Medicine, Lexington, KY, USA *Correspondence address. Eastern Virginia Medical School, PO Box 1980, Norfolk, VA 23501, USA. Tel: +1-757-446-5705; Fax: +1-757-624-2269; E-mail: [email protected]

Submitted on September 24, 2015; resubmitted on November 17, 2015; accepted on November 26, 2015

study question: Which receptors for prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) mediate angiogenesis in the human follicle around the time of ovulation? summary answer: PGE2 and VEGFA act via multiple PGE2 receptors (PTGERs) and VEGF receptors (VEGFRs) to play complementary roles in follicular angiogenesis. what is known already: Production of PGE2 and VEGFA by the follicle are prerequisites for ovulation. PGE2 is an emerging regulator of angiogenesis and has not been examined in the context of the human ovulatory follicle. VEGFA is an established regulator of follicular angiogenesis.

study design, size, duration: Ovarian biopsies containing the ovulatory follicle were obtained from 11 women of reproductive age (30 –45 years) undergoing surgery for laparoscopic sterilization. In some cases, women received hCG to substitute for the ovulatory LH surge before ovarian biopsy. In addition, aspirates from four women of reproductive age (18 –31 years) undergoing gonadotrophin stimulation for oocyte donation were obtained for isolation of human ovarian microvascular endothelial cells (hOMECs). participants/materials, setting, methods: Ovarian biopsies were utilized for immunocytochemical detection of von Willebrand factor to identify endothelial cells. hOMECs were cultured with PGE2, PTGER receptor selective agonists, VEGFA, or VEGFR selective agonists. hOMECs were assessed for proliferation by Ki67 immunocytochemistry. hOMEC migration was determined by counting cells which migrated through a porous membrane in vitro. Sprout formation was quantified by determining sprout number and length from photographs take after culture of hOMECs in a 3-dimensional matrix.

main results and the role of chance: Endothelial cells were not observed within the granulosa cell layer of human ovulatory follicles prior to an ovulatory dose of hCG and were first seen amongst granulosa cells 18 – 34 h after hCG. In vitro, PGE2 enhanced migration and sprout formation but did not alter hOMEC proliferation. Agonists selective for each PTGER increased migration with no change in proliferation. PTGER1 and PTGER2 agonists increased the number of sprouts, while only PTGER1 affected sprout length. VEGFA increased hOMEC proliferation, migration, and formation of structures resembling capillary sprouts. Signaling through VEGFR1 promoted hOMEC migration, proliferation, and the formation of few, long endothelial cell sprouts, while VEGFR2 stimulation promoted hOMEC migration and the formation of many, short sprouts. All effects of treatments in vitro were considered significant at P , 0.05. limitations, reasons for caution: While primary cultures of hOMECs respond to PGE2 and VEGFA differently than other cultured endothelial cells, hOMECs may not respond to PGE2 and VEGFA in vivo as they do in vitro. wider implications of the findings: Agonists and antagonists selective for PTGER1, PTGER2, VEGFR1, or VEGFR2 may have therapeutic value to promote or prevent ovulation in women. & The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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study funding/competing interest(s): This research was supported by grant funding from the Eunice Kennedy Shriver National Institutes of Child Health and Human Development (HD071875 to D.M.D., T.E.C., M.B.). The authors have no conflicts of interest to disclose. Key words: endothelial cell / PGE2 receptor / VEGF receptor / ovulation / ovary / follicle / prostaglandin / vascular endothelial growth factor

Introduction

Materials and Methods

Angiogenesis of the ovulatory follicle is initiated by the LH surge. Prior to the LH surge, vessels are restricted to the stroma of the follicle. In macaques, new endothelial cell networks are observed within the granulosa cell layer as early as 24 h after the LH surge, well before ovulation which occurs 37 – 40 h after the LH surge (Trau et al., 2015). The LH surge initiates production of prostaglandins and angiogenic factors by granulosa cells (Jeremy et al., 1987; Christenson and Stouffer, 1997; Hazzard et al., 1999; Duffy and Stouffer, 2001; Gutman et al., 2008; Wissing et al., 2014). Growth factors typically form a gradient, which is predicted to be highest in the granulosa cells and lower in the stroma surrounding the follicle. Endothelial cells of the stromal vasculature respond to growth factor gradients (Fraser and Duncan, 2005). In the ovulatory follicle, the resulting angiogenesis creates new vascular sprouts which branch from existing stromal vessels and grow towards the luteinizing granulosa cells (Hansen-Smith, 2000). Blockade of either prostaglandin E2 (PGE2) or vascular endothelial growth factor A (VEGFA) action within the non-human primate follicle prevents both follicular angiogenesis and oocyte release, suggesting that both PGE2 and VEGFA signaling are essential for successful ovulation and formation of the corpus luteum (Duffy and Stouffer, 2002; Hazzard et al., 2002; Wulff et al., 2002). PGE2 and VEGFA exert their actions through specific receptors with well-defined signal transduction pathways. PGE2 acts via four G-protein coupled receptors: prostaglandin E receptors 1– 4 (PTGER1, PTGER2, PTGER3 and PTGER4) (Bos et al., 2004). Each PTGER couples to different G proteins and regulates distinct downstream signaling pathways. All four PTGERs are expressed and functional in monkey ovarian endothelial cells (Trau et al., 2015). VEGFA acts via VEGF receptors (VEGFRs) 1 and 2 (deVries et al., 1992; Terman et al., 1992). VEGFA binding to these receptor tyrosine kinases induces dimerization and phosphorylation, leading to activation of multiple signaling cascades that affect numerous processes including endothelial cell proliferation, actin remodeling, cell migration, and vascular permeability (reviewed in (Herbert and Stainier, 2011)). To determine which PGE2 and VEGFA receptors mediate follicular angiogenesis in human ovulatory follicles, human ovarian follicles were collected before and immediately after ovulation at specific times spanning the interval between the administration of an ovulatory gonadotrophin stimulus and the post-ovulatory period. These biopsied follicles were used to determine if endothelial cells were present in the granulosa cell layer of human ovarian follicles prior to ovulation and consistent with the timing of PGE2 and VEGFA synthesis by follicular granulosa cells. Human endothelial cells were obtained from follicular aspirates and treated in vitro with PGE2, VEGFA, and agonists selective for a single PTGER or VEGFR to determine if endothelial cells responded to PTGER and VEGFR selective agonists in a manner consistent with the concept that each PTGER and VEGFR plays a role in follicular angiogenesis, a critical component of successful ovulation.

Human ovarian biopsies Women with proven fertility, regular menstrual cycles, and without hormone medication for at least 3 months were candidates for follicular biopsy. This study was approved by the regional human ethics committee of Gothenburg University, and informed consent was obtained from all patients. Detailed information regarding women who participated in follicular biopsy have been reported previously (Rosewell et al., 2014). Women experiencing natural menstrual cycles and undergoing laparoscopic tubal sterilization had their surgery timed to allow collection of the single, dominant follicle (≥14 mm and ≤20 mm) either in the absence of human chorionic gonadotrophin (hCG; pre-ovulatory, N ¼ 2) or 12 – 18 h (early ovulatory, N ¼ 4), 18 – 34 h (late ovulatory, N ¼ 4), or 44 – 70 h (postovulatory, N ¼ 1) after administration of hCG (250 mg, Ovitrelle, Merck Serono). Each woman provided a single biopsy for this study. The entire intact dominant follicle was excised from surrounding ovarian tissue, fixed in 4% (w/v) paraformaldehyde overnight, and paraffin embedded for immunohistochemistry. Serum estrogen and progesterone at the time of surgery were determined to confirm placement within the assigned ovulatory category.

Human ovarian microvascular endothelial cells Human ovarian microvascular endothelial cells (hOMECs) were isolated from follicular aspirates obtained during oocyte retrieval from healthy young women (aged 18–31 years) undergoing ovarian stimulation for oocyte donation at the Jones Institute for Reproductive Medicine, Eastern Virginia Medical School (EVMS). This use of discarded human aspirates does not constitute human subjects research as determined by the EVMS Institutional Review Board. While no specific patient data are available, follicular aspirates are routinely obtained 34–36 h after administration of an ovulatory dose of hCG (Arslan et al., 2005). Aspirated cells were plated in fibronectin-coated flasks in EGM2 media (Lonza, Walkersville, MD), which is optimized for microvascular endothelial cells. Once cells were confluent, endothelial cells were isolated using CD31 Dynabeads (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol (Gillies et al., 2015; Trau et al., 2015). A second Dynabead isolation was performed at the second passage, resulting in a proliferating primary cell population of .99% endothelial cells (Supplementary Fig. S1), which were assessed as described below. This is similar to the approach used by Ratcliffe et al. to culture endothelial cells isolated from follicular fluid (Ratcliffe et al., 1999). This approach is optimal for selection of vascular endothelial cells, and the vast majority of selected cells are anticipated to be vascular endothelial cells (Bohgaki and Kitaguchi, 2007). However, macrophages may also express CD31. Any CD31+ macrophages isolated during this procedure are expected to quickly become functionally and molecularly indistinguishable from vascular endothelial cells (Bohgaki and Kitaguchi, 2007). A total of four hOMEC lines were established, each from an individual woman.

Human umbilical vein endothelial cells Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC (Manassas, VA). HUVECs were cultured and assessed as described for hOMECs.

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Immunohistochemistry

Migration assay

Paraffin-embedded human ovary sections were immunostained essentially as previously described (Trau et al., 2015). Briefly, tissue sections were heated, deparaffinized, underwent acidic antigen retrieval in sodium citrate buffer, and were blocked with 5% (v/v) non-immune serum in PBS containing 0.1% (v/v) Triton X-100. Slides were incubated overnight with primary antibody against von Willebrand Factor (VWF) (7.75 mg/ml, Dako, Carpinteria, CA) and color developed using a rabbit Vectastain ABC kit (Vector Laboratories, Burlingame, CA). hOMECs were grown to confluence on chamber slides for characterization using primary antibodies against the endothelial cell protein VWF (7.75 mg/ml, Dako), the steroidogenic enzyme 3b-hydroxysteroid dehydrogenase (HSD3B) (1:2000, antibody of Dr Ian Mason, University of Edinburgh, MRC Center for Reproductive Health), or smooth muscle actin ACTA2 (0.067 mg/ml, Thermo Fisher, Hudson, NH) as previously described (Trau et al., 2015). Ki67 immunodetection in hOMECs and HUVECs was performed as previously described (Trau et al., 2015). Cells were treated for 24 h with basal media (EBM2, Lonza) with the addition of 1 mM PGE2 (Cayman, Ann Arbor, Michigan), an individual PTGER receptor agonist (10 mM 17-phenyl trinor prostaglandin E2 (17-PTP), 10 mM butaprost, 1 mM sulprostone, 1 mM PGE1-OH, Cayman), 5 ng recombinant human VEGFA165/ml (R&D Systems, Minneapolis, MN), or an individual VEGFA receptor agonist (5 ng VEGFE/ml, Fitzgerald Industries International, Acton, MA; 5 ng Placental Growth Factor (PlGF)/ml, R&D Systems). Optimal agonist concentrations used in these experiments were selected based on the results of dose – response curves (not shown). Additional cultures received either basal medium or EGM2 medium (optimal growth medium; contains vascular growth factors including VEGFA) as negative or positive controls, respectively. Cells were fixed and stained as described above using a primary antibody directed against Ki67 (0.35 mg/ml, Dako). Four images of each treatment were assessed to determine the percentage of proliferating cells. HUVECs and hOMECs were grown to confluence in EGM2 medium, then fixed as previously described (Trau et al., 2015). hOMECs cultured on chamber slides for the analysis of VEGFR1 and VEGFR2 were exposed to acidic antigen retrieval (described above). Slides were incubated with an antibody directed against a single PGE2 receptor (PTGER1 (2 mg/ml), PTGER2 (10 mg/ml), PTGER3 (4 mg/ml), PTGER4 (6 mg/ml); Cayman) or a single VEGFA receptor (VEGFR1 (10 mg/ml), R and D Systems; VEGFR2 (20 mg/ml), Sigma-Aldrich, St Louis, MO), followed by Alexa Fluor 488-conjugated anti-rabbit secondary antibody (1:1000; Thermo Fisher). Slides were incubated in 1% (w/v) Sudan Black in 70% (v/v) methanol and counterstained with DAPI (Invitrogen). All images were obtained using an Olympus BX41 microscope fitted with a DP70 digital camera and associated software (Olympus, Melville, NY). Omission of the primary antibody served as a negative control.

Migration was assessed as previously described (Trau et al., 2015). Briefly, hOMECs were cultured for 24 h on 6-well plate inserts with 8 mm pores (BD Biosciences, San Jose, CA) in basal media with or without the addition of PGE2, VEGFA, PTGER agonists, and VEGFR agonists (concentrations listed above); EGM2 medium was used as a positive control. Cells migrating to the opposite side of the porous membrane were photographed, and four images of each membrane were used to determine the number of migrated cells.

RNA isolation, amplification, and quantitative PCR HUVECs and hOMECs were grown to confluence, then switched to basal media for 4 h. Total RNA was harvested using Trizol (Invitrogen) and reverse transcribed as previously described (Trau et al., 2015). Quantitative PCR (qPCR) was performed using a Roche LightCycler and FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Indianapolis, IN). Primers and reaction conditions for amplification of PTGER1, PTGER2, and PTGER4 were previously described (Markosyan et al., 2006). Primers for VEGFR1, VEGFR2, and PTGER3 were designed based on human sequences and span an intron to prevent undetected amplification of genomic DNA (Supplementary Table SI). PCR products were sequenced (Genewiz, South Plainfield, NJ) to confirm amplicon identity.

Sprouting assay Cytodex microcarrier beads (GE Healthcare, Uppsala, Sweden) were coated with hOMECs (500 – 1000 cells per bead) and embedded in a fibrin matrix as previously described (Trau et al., 2015). Basal media with or without the addition of PGE2, VEGFA, PTGER agonists or VEGFR agonists (concentrations listed above) was added on top of matrixes; EGM2 medium was used as a positive control. For each well, five areas were photographed daily for 2 days; the number of sprouts per bead and length of sprouts were determined.

Data analysis Data were assessed for heterogeneity of variance by Bartlett’s test. Data were log transformed when Bartlett’s test yielded P , 0.05; log-transformed data were subjected to Bartlett’s test to confirm that P . 0.05. As indicated in the figure legends, data sets were assessed by 2-tail paired t-test or ANOVA (without or with repeated measures) following by Duncan’s multiple range test (StatPak version 4.12 software; Northwest Analytical, Portland, OR). Significance was assumed at P , 0.05. Data are expressed as mean + SEM.

Results The midcycle gonadotrophin surge stimulates angiogenesis in the human ovulatory follicle Biopsies of human follicles collected from women experiencing natural menstrual cycles before and at specific times after administration of hCG were sectioned and immunostained for the vascular endothelial cell marker VWF. In the absence of hCG and for at least 18 h following hCG administration, endothelial cells were restricted to the theca layer and surrounding stroma and were never observed within the granulosa layer of the ovulatory follicle (Fig. 1A, C and D). Endothelial cells were observed within the granulosa cell layer of follicles obtained 18– 34 h after hCG administration, prior to ovulation (Fig. 1E and F). Endothelial cells were also observed among the granulosa cells of the developing corpora lutea collected 44–70 h after hCG (Fig. 1G and H).

PTGER and VEGFR expression Primary cultures of hOMECs were established to determine if PGE2 and VEGFA act directly at these endothelial cells to promote angiogenic processes. PTGER1, PTGER2, PTGER3, PTGER4, VEGFR1 and VEGFR2 were detected in hOMEC cDNA (Supplementary Fig. S2). PTGER and VEGFR proteins were detected by immunofluorescence in hOMECs maintained in vitro (Fig. 1I –N).

PGE2 and VEGFA promote endothelial cell migration In hOMECs cultured on porous membrane inserts, PGE2 stimulated hOMEC migration (Fig. 2A). To determine the importance of PGE2

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Figure 1 Endothelial cells of the ovulatory follicle are present in the granulosa cell layer prior to ovulation and express prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) receptors. (A – H) Human follicular biopsies obtained prior to (no hCG, A) or 12 – 18 h (C and D), 18– 34 h (E and F), or 44– 70 h (G and H) after hCG were immunostained for von Willebrand Factor (VWF) (brown); nuclei are blue. VWF+ cells in the stroma (arrows), VWF+ cells in large stromal vessels (large arrowheads), and VWF+ cells in the granulosa cell layer (small arrowheads) are indicated. (A) – (H) are oriented with antrum (an) in upper right, granulosa cells (gc) central, and stroma (st) in lower left. (B) shows staining when primary antibody was omitted and bar ¼50 mm. Images are representative of biopsies obtained prior to hCG (n ¼ 2), 12 – 18 h after hCG (n ¼ 4), 18 – 34 h after hCG (n ¼ 4), or 44 – 70 h after hCG (n ¼ 1). (I – P) Immunodetection (green) of PTGER1 (I), PTGER2 (J), PTGER3 (K), PTGER4 (L), VEGFR1 (M) and VEGFR2 (N) in human ovarian microvascular endothelial cells (hOMECs) in vitro; nuclei are blue. For (I), upper shows merged image and lower shows PTGER1 only. No staining was seen when primary antibody was omitted under conditions used for PTGER (P) and VEGFR (O) immunodetection. Representative of n ¼ 4 hOMEC lines. (PTGER, PGE2 receptor; VEGFR, VEGF receptor.)

signaling through its receptors, agonists specific for each of the four PGE2 receptors were utilized. Each PGE2 receptor agonist induced hOMECs to migrate at similar levels to PGE2 (Fig. 2B). VEGFA induced robust migration above basal levels (Fig. 2A). PlGF and VEGFE were utilized as agonists selective for VEGFR1 and VEGFR2,

respectively (Ogawa et al., 1998; Bellik et al., 2005). Each VEGFR agonist induced migration above basal levels but at levels lower than observed in response to VEGFA (Fig. 2C). Treatment with both PGE2 and VEGFA did not increase the number of cells that migrated in response to either PGE2 or VEGFA alone (Fig. 2A).

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Figure 2 Prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) stimulate human ovarian microvascular endothelial cell (hOMEC) migration. (A) Migration during culture with basal medium alone or containing PGE2, VEGFA, or both PGE2 and VEGFA. (B) Migration during culture with PGE2 and PTGER selective agonists. (C) Migration during culture with VEGFA and VEGFR receptor selective agonists. Representative migration in basal (D), PGE2 (E), or VEGFA (F) treatment groups; representative pore (arrowhead) and migrated cell (arrow) are indicated in (D). For each Panel, groups with different superscripts are different by repeated measures ANOVA and Duncan’s post hoc test, P , 0.05, n ¼ 4 hOMEC lines.

Figure 3 Vascular endothelial growth factor A (VEGFA), but not prostaglandin E2 (PGE2), stimulates human ovarian microvascular endothelial cell (hOMEC) proliferation. (A) Percentage of cells expressing Ki67 after culture with basal media alone or with addition of PGE2, VEGFA, or both PGE2 and VEGFA. (B) Proliferation after culture with PGE2 or PTGER selective agonists. (C) Proliferation in response to VEGFA and VEGFR selective agonists. Representative Ki67 staining in basal (D), PGE2 (E), or VEGFA (F) treatment groups are shown; representative Ki67+ (arrowhead) and Ki672 (arrow) nuclei are indicated in (D). For each Panel, groups with different superscripts are different by repeated measures ANOVA and Duncan’s post hoc test, P , 0.05, n ¼ 4 hOMEC lines.

VEGFA, but not PGE2, stimulates endothelial cell proliferation PGE2 and individual PTGER agonists had no effect on hOMEC proliferation as determined by the percentage of Ki67-positive hOMEC nuclei (Fig. 3A and B). VEGFA treatment increased proliferation above basal levels (Fig. 3A). The VEGFR1 agonist PlGF also increased proliferation, similar to that seen with VEGFA (Fig. 3C). In contrast, the VEGFR2 agonist VEGFE did not alter proliferation (Fig. 3C). Treatment with the combination of PGE2 and VEGFA resulted in a hOMEC proliferation rate similar to that observed with VEGFA alone (Fig. 3A).

PGE2 and VEGFA regulate vascular sprout formation in vitro The ability of PGE2 and PTGER selective agonists to regulate hOMEC sprouting was assayed by culturing hOMEC-coated beads with PGE2

or agonist for up to 2 days in vitro (Fig. 4A –F). After 1 day in culture, PGE2 induced the formation of numerous, longer sprouts when compared with basal medium. PTGER1 agonist treatment increased the number and length of sprouts. PTGER2 agonist treatment resulted in an increased number of sprouts, but this agonist did not alter sprout length. Both PTGER3 and PTGER4 agonists yielded sprouts that were not different in number and length from those observed with basal medium. By Day 2 of culture, sprouts were longer in PGE2-treated cultures, but the number of sprouts was similar to that observed after culture in basal medium. Sprout formation in response to VEGFA and VEGFR-selective agonists was also examined (Fig. 4G –L). No significant differences were observed between treatment groups on Day 1. By Day 2, short sprouts were observed on most beads cultured with basal media. VEGFA treatment resulted in the formation of longer, more numerous sprouts. Treatment with the VEGFR1 agonist PlGF yielded a similar number of sprouts when compared with basal medium. However, PlGF treatment yielded sprouts that were as long as those observed after VEGFA treatment. Treatment

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Figure 4 Prostaglandin E2 (PGE2) and vascular endothelial growth factor A (VEGFA) promote sprout formation by human ovarian microvascular endothelial cell (hOMECs). (A – F) Sprout counts (A) and sprout lengths (C) after culture with basal media with or without PGE2 at 1 and 2 days in vitro. Sprout number (B) and length (D) after 1 day in vitro in response to PGE2 and PTGER agonists are also shown. Images show hOMEC sprouting in response to basal (E) and PGE2 (F). (G– L) Sprout counts (G) and sprout lengths (I) after culture with basal media with or without VEGFA at 1 and 2 days in vitro. Sprout number (H) and length (J) after 2 days in vitro in response to VEGFA and VEGFR agonists are also shown. Images show hOMEC sprouting in response to basal (K) and VEGFA (L). For (A, C, G and I) treatment effects were determined by 2-tailed paired t-test; asterisk (*) indicates P , 0.05, n ¼ 4 hOMEC lines. For (B, D, H and J) groups with different superscripts are different by repeated measures ANOVA and Duncan’s post hoc test, P , 0.05. n ¼ 4 hOMEC line. Images in (E), (F), (K) and (L) are representative of four hOMEC lines.

with the VEGFR2 agonist VEGFE yielded very abundant sprouts, similar to the number of sprouts observed after VEGFA treatment. However, VEGFE treatment did not alter sprout length when compared with sprouts formed in basal medium.

combined treatment of PGE2 and VEGFA was not different from the basal level of proliferation. While hOMECs responded to PGE2 and VEGFA with increased migration and sprout formation, these treatments did not alter HUVEC migration or sprouting when compared with HUVECs maintained in basal medium.

HUVECs and hOMECs respond differently to PGE2 and VEGFA

Discussion

To explore the concept that hOMEC responses are unique to human endothelial cells from the ovulatory follicle, HUVECs were also examined (Supplementary Fig. S3). As with hOMECs, HUVECs expressed PTGER1-4 and VEGFR1-2 (data not shown), and each PGE2 and VEGF receptor protein was detected by immunofluorescence. hOMECs appeared to uniformly express all PTGERs and VEGFRs. In contrast, HUVEC cultures uniformly expressed PTGER2, PTGER4 and VEGFRs but contained cells staining positive and negative for PTGER1 and PTGER3. In contrast to hOMECs, PGE2 reduced the number of proliferating HUVECs. Similar to hOMECs, HUVECs responded to VEGFA with increased proliferation. Proliferation in response to the

Human ovarian endothelial cells and ovarian tissues were used to explore how PGE2 and VEGFA receptors mediate angiogenesis around the time of ovulation. hOMECs expressed all four PTGERs in vitro. While PTGER2, PTGER3, and PTGER4 were not localized to a specific region of the cell, PTGER1 appeared to be concentrated in the cell nucleus. Localization of functional PTGERs to the nuclear envelope and intracellular membranes, as well as the plasma membrane, has been previously described (Narumiya et al., 1999). All four PTGERs were previously found to be expressed by monkey ovarian microvascular endothelial cells and generated appropriate downstream signals when stimulated with specific PTGER agonists (Trau et al., 2015). VEGFR1

442 and VEGFR2 were also expressed by hOMECS in vitro. These receptors are well characterized tyrosine kinases (deVries et al., 1992; Terman et al., 1992) and function as critical mediators of angiogenesis in a wide variety of tissues, including the ovulatory follicle (Hazzard et al., 2002; Wulff et al., 2002). The presence of these receptors on endothelial cells of the ovulatory follicle indicates that they may mediate follicular angiogenesis as follicular levels of PGE2 and VEGFA increase after the ovulatory gonadotrophin surge. PGE2 is a key regulator of follicular angiogenesis in non-human primates (Trau et al., 2015). In hOMECs, PGE2 and all PTGER agonists induced migration. These findings are similar to observations in monkey ovarian microvascular endothelial cells, where PGE2, acting via all four PTGERs, increased migration in vitro (Trau et al., 2015). In contrast, studies in pulmonary endothelial cells showed that PTGER2 and PTGER4, but not PTGER1, promoted migration (Kamiyama et al., 2006; Rao et al., 2007), highlighting the distinctive nature of ovarian endothelial cells. PGE2 and PTGER agonists had no apparent influence on hOMEC proliferation. This is consistent with our previous report in monkey ovarian microvascular endothelial cells (Trau et al., 2015). However, there are conflicting reports on the ability of PGE2 to induce pulmonary endothelial cell proliferation in vitro, with separate studies reporting increased and decreased proliferation in response to PGE2 (Kamiyama et al., 2006; Rao et al., 2007). In hOMECs, PGE2 and agonists selective for PTGER1 and PTGER2 increased the formation of sprouts, while PTGER3 and PTGER4 did not promote sprout formation. Previous reports found that PTGER1, PTGER2 and PTGER4 induced endothelial cell sprouting (Rao et al., 2007; Sakurai et al., 2011; Zhang and Daaka, 2011; Trau et al., 2015), while PTGER3 was reported as failing to induce sprout formation (Sakurai et al., 2011) or inhibiting sprouting (Trau et al., 2015). Overall, there is general consensus that PGE2, as well as PTGER1 and PTGER2 agonists, promote angiogenesis. PTGER4 appears to play less of a role in mediating sprout formation in the ovulatory follicle than in other systems. The current results also provide insight into the mechanism of cell sprouting as both PGE2 and PTGER1 promoted the formation of long sprouts whereas PTGER2 stimulated the formation of short sprouts. Importantly, our study identifies both PTGER1 and PTGER2 as likely mediators of PGE2-stimulated vascular sprouting in the human ovulatory follicle. VEGFA is well established as a critical regulator of angiogenesis in the ovulatory follicle (Hazzard et al., 2002; Wulff et al., 2002). Because VEGFRs mediate different responses to VEGFA, specific VEGFR agonists were used in the current experiments to examine the response of hOMECs to signaling through an individual VEGFR. VEGFR1 agonist increased hOMEC proliferation, while VEGFR2 agonist had no effect on proliferation. VEGFR1-mediated proliferation has been previously shown in HUVECs (Nishi et al., 2008); VEGFR2 increased proliferation of both aortic endothelial cells and HUVECs (Bernatchez et al., 1999; Miao et al., 2006; Nieminen et al., 2014). This contrasts with our observations in hOMECs, and this differential response to VEGF receptor stimulation supports the concept that hOMECs are different from other endothelial cells in other organs. In addition to proliferation, VEGFA promoted hOMEC migration. Stimulation of VEGFR1 and VEGFR2 each increased hOMEC migration, though not to the extent of VEGFA, which activates both VEGFR1 and VEGFR2. It is therefore likely that signaling through VEGFR1 and VEGFR2 has an additive effect on migration. The role of individual VEGFRs to mediate VEGFA promotion of migration has received little attention. In aortic endothelial cells,

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VEGFR2 has been reported to induce migration, but few reports have examined the role of VEGFR1 in this process (Bernatchez et al., 1999; Endo et al., 2003; Miao et al., 2006). These findings suggest that coordinated signaling through both VEGF receptors may be needed for maximal migration of these ovarian specific endothelial cells. Signaling through each VEGFR promotes distinct aspects of capillary sprout formation. The VEGFR2 agonist increased the number of sprouts that formed but did not increase sprout length, which is consistent with a role for VEGFR2 to initiate sprout formation. The VEGFR1 agonist promoted increased sprout length but did not increase the number of sprouts that formed; this is consistent with VEGFR1-induced sprout elongation. Overall, these findings in our novel human ovarian endothelial cells are consistent with previous reports in other systems that signaling through both receptors is required for capillary formation (Endo et al., 2003; Nishi et al., 2008; Buharalioglu et al., 2011). VEGFR1 and VEGFR2 likely have complementary roles to promote these angiogenic events in hOMECs in vitro and co-operate to promote capillary formation in the human ovulatory follicle in vivo. Angiogenesis of the luteinizing follicle is initiated by the ovulatory LH surge (Acosta and Miyamoto, 2004). Prior to the LH surge, vessels are restricted to the stroma and theca surrounding the ovulatory follicle (Corner, 1945; Koering, 1969). In a recent study by our laboratory, endothelial cells were detected within the granulosa cell layer of nonhuman primate follicles as early as 24 h after an ovulatory dose of hCG, and endothelial cell networks suggestive of developing capillaries were present 36 h after hCG in ovarian follicles collected just prior to follicle rupture (Trau et al., 2015). In the present study, carefully staged human follicular biopsies were used to demonstrate that endothelial cells were present in the granulosa layer 18 –34 h following an ovulatory dose of hCG, with ovulation anticipated 37– 40 h after hCG (Weick et al., 1973). Extensive endothelial cell networks were present 44– 70 h after hCG, consistent with corpus luteum formation. In response to the LH surge, PGE2 and VEGFA production by granulosa cells dramatically increase (Jeremy et al., 1987; Christenson and Stouffer, 1997; Hazzard et al., 1999; Duffy and Stouffer, 2001; Gutman et al., 2008; Wissing et al., 2014). The timing of endothelial cell entry into the granulosa cell layer, taken together with observations of PGE2 and VEGFA action on endothelial cells presented here, supports the concept that initiation of angiogenesis in human follicles is mediated by rising levels of PGE2 and VEGFA during the ovulatory interval and mediated by PTGERs and VEGFRs. To explore the concept that ovarian microvascular endothelial cells may have properties which are different from properties of endothelial cells from other sources, a well characterized venous cell line, HUVECs, were studied in parallel to hOMECs. Similar to hOMECs, HUVECs expressed receptors for all four PTGERs, as well as VEGFR1 and VEGFR2. In contrast to hOMECs, HUVECs did not migrate or form sprouts in vitro in response to either PGE2 or VEGFA. Treatment with PGE2 decreased HUVEC proliferation, which was not observed in hOMECs. Both hOMECs and HUVECs responded to VEGFA with increased proliferation. Overall, major differences were noted between hOMECs and HUVECs. Other groups have also reported that angiogenic responses to VEGFA differ depending on the source of the endothelial cells (Herve et al., 2005). Since hOMECs and endothelial cells of different origins respond in unique ways to VEGFA and other stimuli, it may be possible to target selectively ovarian endothelial cells for therapeutic purposes.

Angiogenesis in the human ovulatory follicle

Our data support the concept that angiogenesis is well underway at the time of ovulation in the human follicle. While VEGFA is widely regarded as an essential angiogenic factor, these data establish PGE2 as an additional significant regulator of follicular angiogenesis and support further examination of PTGER1, PTGER2, VEGFR1, and VEGFR2 as important mediators of these angiogenic signals. Our previous work in the monkey ovulatory follicle identified PTGER3 and PTGER4 as the primary PTGERs expressed by endothelial cells of established vessels in the ovarian stroma (Trau et al., 2015). It is interesting to note that PTGER3 and PTGER4 receptors require lower levels of PGE2 to achieve maximal activation (Narumiya et al., 1999), so PTGER3 and PTGER4 may be key receptors at low PGE2 concentrations to maintain healthy stromal vasculature. In contrast, PTGER1 and PTGER2 require higher PGE2 concentrations for maximal stimulation (Narumiya et al., 1999), so rising follicular PGE2 concentrations after the LH surge may be key to the initiation of follicular angiogenesis. Since angiogenesis appears to be a requirement for successful ovulation, the use of selective PTGER or VEGFR agonists or antagonists may prove effective as novel, non-hormonal contraceptives. Similarly, ligands which regulate PTGER or VEGFR activity may be useful for the treatment of conditions associated with dysregulated vascularization, such as polycystic ovarian syndrome and ovarian hyperstimulation syndrome (Delgado-Rosas et al., 2009; Scotti et al., 2014).

Supplementary data Supplementary data are available at http://humrep.oxfordjournals.org/.

Acknowledgements The authors would like to acknowledge the assistance of the Jones Institute for Reproductive Medicine at EVMS for providing human ovarian aspirates. The assistance of Kathy Rosewell in the preparation of the human follicle sections is gratefully acknowledged.

Authors’ roles H.A.T. data generation, drafted and reviewed final manuscript. M.B. obtained funding, data generation, drafted and reviewed final manuscript. T.E.C. obtained funding, data generation, drafted and reviewed final manuscript. D.M.D. obtained funding, data generation, drafted and reviewed final manuscript.

Funding This research was supported by grant funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD071875 to D.M.D., T.E.C., M.B.).

Conflict of interest None declared.

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