Placental Growth Factor Is Required for Ovulation, Luteinization, and Angiogenesis in Primate Ovulatory Follicles

Abstract

Placental growth factor (PGF) is member of the vascular endothelial growth factor (VEGF) family of angiogenesis regulators. VEGFA is an established regulator of ovulation and formation of the corpus luteum. To determine whether PGF also mediates aspects of ovulation and luteinization, macaques received gonadotropins to stimulate multiple follicular development. Ovarian biopsies and whole ovaries were collected before (0 hours) and up to 36 hours after human chorionic gonadotropin (hCG) administration to span the ovulatory interval. PGF and VEGFA were expressed by both granulosa cells and theca cells. In follicular fluid, PGF and VEGFA levels were lowest before hCG. PGF levels remained low until 36 hours after hCG administration, when PGF increased sevenfold to reach peak levels. Follicular fluid VEGFA increased threefold to reach peak levels at 12 hours after hCG, then dropped to intermediate levels. To explore the roles of PGF and VEGFA in ovulation, luteinization, and follicular angiogenesis in vivo, antibodies were injected into the follicular fluid of naturally developed monkey follicles; ovariectomy was performed 48 hours after hCG, with ovulation expected about 40 hours after hCG. Intrafollicular injection of control immunoglobulin G resulted in no retained oocytes, follicle rupture, and structural luteinization, including granulosa cell hypertrophy and capillary formation in the granulosa cell layer. PGF antibody injection resulted in oocyte retention, abnormal rupture, and incomplete luteinization, with limited and disorganized angiogenesis. Injection of a VEGFA antibody resulted in oocyte retention and very limited follicle rupture or structural luteinization. These studies demonstrate that PGF, in addition to VEGFA, is required for ovulation, luteinization, and follicular angiogenesis in primates.

Copyright © 2018 Endocrine Society.

Figures

Figure 1.

Granulosa cell expression of VEGF family members. Granulosa cells obtained from monkey ovulatory follicles after ovarian stimulation in the absence of hCG (0 hours) and 12, 24, or 36 hours after hCG were assessed by quantitative PCR for (A) PGF mRNA, (D) total VEGFA mRNA, (G) VEGFA165 mRNA, (H) VEGFA121 mRNA, and (I) VEGFB mRNA. mRNA levels are expressed relative to ACTB mRNA in each sample. Granulosa cell concentration of protein for (B) PGF and (E) VEGFA were determined by ELISA and normalized to total protein for each sample. Follicular fluid obtained from monkeys by follicle aspiration before and after hCG was assessed for concentration of protein for (C) PGF and (F) VEGFA and normalized to total protein for each sample. For each panel, data were assessed by ANOVA and Duncan post hoc test. Data are expressed as mean ± standard error of the mean. Groups with no common letters are different, P < 0.05. n = 3 to 6 samples per group.

Figure 2.

PGF and VEGFA immunodetection in monkey ovulatory follicles. Monkey ovaries obtained after ovarian stimulation in (A and E) the absence of hCG (0 hours) and (B and F) 12, (C and G) 24, or (D and H) 36 hours after hCG were used for immunodetection of (A–D) PGF or (E–H) VEGFA as indicated by presence of brown precipitate. Additional serial sections from an ovary obtained after ovarian stimulation and 36 hours hCG were used for immunodetection of (J) PGF, (K) the theca cell enzyme CYP17, and (L) VEGFA to confirm colocalization of PGF and VEGFA with CYP17 (arrows). Tissue sections were counterstained with hematoxylin (blue). Insets show absence of brown stain was confirmed when primary antibodies were omitted for (A) PGF, (H) VEGFA, and (K) CYP17. All images are at same magnification and use bar in panel H (50 µm). All images are oriented as in panel A, with stroma (st) in lower left, granulosa cells (gc) central, and antrum (an) in upper right. Images are representative of n = 3 to 4 monkeys per group.

Figure 3.

Inhibition of follicular prostaglandin synthesis with celecoxib does not alter PGF or VEGFA protein levels in monkey ovulatory follicles obtained 36 hours after hCG treatment. Celecoxib (36+C) reduced granulosa cell mRNA for (A) PGF but did not alter PGF protein in (B) granulosa cell lysates or (C) follicular fluid. Celecoxib increased granulosa cell mRNA for (D) total VEGFA, (G) VEGFA165, and (H) VEGFA121 but did not alter VEGFA protein in (E) granulosa cell lysates or (F) follicular fluid. Granulosa cell mRNA for (I) VEGFB, (J) VEGFC, and (K) VEGFD was not altered by celecoxib treatment. Data are expressed as mean ± standard error of the mean. Within each panel, groups different by unpaired t test where indicated, P < 0.05. n = 3 to 6 monkeys/group. Data for VEGFC mRNA 36 hours after hCG and VEGFD mRNA 36 hours after hCG were previously published (27) and are used here by permission.

Figure 4.

Follicle rupture and retained oocytes after intrafollicular injection of antibodies against PGF or VEGFA. (A–E) Ovulatory stigmata and (I–M) histological views of rupture sites are shown after injection with (A and I) control IgG antibody, (B, C, J, and K) PGF antibody, or (D, E, L, and M) VEGFA antibody. (F) Graph shows area of rupture site (mean ± standard error of the mean) for each treatment group. Absence of rupture was set equal to 0 mm2. Number of ruptured follicles/total follicles is given above each bar. Examples of oocytes found trapped in follicles after injection with (G) PGF antibody or (H) VEGFA antibody are also shown. All histological images of rupture sites are at the same magnification; scale bar in panel M = 200 µm. Each oocyte is shown at approximately maximal diameter of about 100 µm.

Figure 5.

Structural luteinization and angiogenesis in follicles after intrafollicular injection of antibodies against PGF or VEGFA. Top panels show histology of follicle wall after injection with (A) control IgG antibody, (B) PGF antibody, or (C) VEGFA antibody. All images are in same orientation, with stroma (st) lower left, granulosa cells (gc) central, and antrum (an) upper right as indicated in panel A. Endothelial cells are identified as vWF+ cells (brown); nuclei are stained blue. Graphs show morphometric analysis of (D) granulosa cell layer thickness and (E) endothelial cell invasion into the granulosa cell layer as distance from granulosa cell basement membrane to outermost edge of granulosa or endothelial cells, (G) as indicated by red lines on the accompanying image. (F) The ratio of endothelial cell invasion to granulosa cell layer thickness is also shown. For each graph, data are shown as mean ± standard error of the mean and were assessed by ANOVA and Duncan post hoc test, groups with no common superscripts are different, P < 0.05. n = 3 to 5 ovaries per group. All histological images are at the same magnification; scale bar in panel (C) = 100 µm. (H and J) 3D modeling of endothelial cells (white on black background) is shown alongside (I and K) representative histological images of vWF immunostained ovarian tissues after injection with (H and I) control IgG antibody or (J and K) PGF antibody used for 3D modeling of endothelial cells. Red arrows indicate stromal vessels, red arrowheads indicate capillary-like structures that connect to a stromal vessel, and blue arrowheads indicate endothelial cells within the granulosa cell layer that lack connection to a stromal vessel. Images from control IgG antibody show four small vessels within the granulosa cell layer; all four small vessels connect to stromal vessel indicated with the red arrow in adjacent tissue sections not used for 3D model construction.