title: Vascular endothelial growth factor and angiopoietin production by primate follicles during culture is a function of growth rate, gonadotrophin exposure and oxygen milieu.
abstract: OBJECTIVE
  What is the time course of production of vascular endothelial growth factor-A (VEGF-A), angiopoietin (ANGPT)-1 and ANGPT-2 by primate follicles during encapsulated three-dimensional culture, and what conditions affect their production?
  
  
  CONCLUSIONS
  Primate follicles produce VEGF-A and ANGPT-2 in vitro, particularly after developing to the antral stage, with VEGF production influenced by FSH concentration and O(2) tension.
  
  
  BACKGROUND
  Folliculogenesis, i.e. the development of primordial follicles into mature, antral follicles, requires the creation of a vascular network in the follicle wall via a process called angiogenesis. Angiogenic factors including VEGFs and ANGPTs have documented roles in angiogenesis. However, direct studies on the production and regulation of angiogenic factors by individual, growing follicles are limited.
  
  
  METHODS
  Ovaries (n = 9 pairs) were obtained from rhesus macaques during the early follicular phase of the menstrual cycle (cycle days 1-4). Secondary (125-225 µm) follicles were isolated mechanically, encapsulated into alginate (0.25% w/v) and cultured for 40 days.
  
  
  METHODS
  Individual follicles were cultured in a 5 or 20% O(2) environment in alpha minimum essential medium supplemented with recombinant human (h) FSH. Half of the follicles had recombinant hLH added to the media from Days 30 to 40. Follicle diameters were measured weekly. Follicles were categorized at Week 5 as no-grow (NG; <250 μm in diameter), slow-grow (SG; 251-499 μm) and fast-grow (FG; >500 μm). VEGF-A, ANGPT-1 and -2 concentrations in media were measured by ELISA.
  
  
  RESULTS
  VEGF concentrations were low throughout the culture for NG follicles. SG and FG follicles had detectable VEGF concentrations at Week 2, which continued to rise throughout culture. VEGF concentrations were distinct (P < 0.05) among all three follicle categories during Weeks 4 and 5. VEGF concentrations were higher (P < 0.05) in SG follicles in the presence of high/mid-dose FSH at 5% O(2). In contrast, there were no dose-dependent differences in VEGF production for FG follicles based on FSH concentrations or O(2) tension. At Week 5, follicles that produced metaphase II oocytes, following exposure to an ovulatory hCG dose, secreted higher concentrations of VEGF than those containing germinal vesicle-intact oocytes. Media concentrations of ANGPT-1 were low throughout culture for all three follicle categories. ANGPT-2 concentrations were low throughout culture for NG follicles. In contrast, ANGPT-2 concentrations of SG and FG follicles continued to rise from Weeks 1 to 4. During Weeks 2-4, ANGPT-2 concentrations in FG follicles were significantly higher than those of SG and NG follicles (P < 0.05).
  
  
  CONCLUSIONS
  This study reports VEGF-A, ANGPT-1 and -2 production by in vitro-developed individual primate (macaque) follicles, that is limited to the interval from the secondary to small antral stage. After VEGF and ANGPT-1 assays, the limited remaining samples did not allow assessment of the independent effects of gonadotrophin and O(2) on the ANGPT-2 production by cultured follicles. Findings await translation to human follicles.
  
  
  CONCLUSIONS
  The above findings provide novel information on the process of primate follicle maturation. We hypothesize that a symbiotic relationship between elevated concentrations of ANGPT-2 and VEGF allows FG antral follicles to excel in follicle maturation, e.g. by promoting its vascularization. Elevated ANGPT-2 may also offer possible insight into future oocyte quality as early as Week 2, compared with Week 4 for VEGF and follicle size.
  
  
  BACKGROUND
  The study was funded by the following grants: NIH U54 RR024347/HD058294/PL1-EB008542 (Oncofertility Consortium), NIH U54-HD018185 (SCCPIR), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), NIH FIC TW/HD-00668, ONPRC 8P51OD011092. There are no conflicts of interest to declare.
authors.affiliation: Division of Reproductive and Developmental Sciences, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006, USA.
authors.forename: T E
authors.initials: TE
authors.lastname: Fisher
authors.forename: T A
authors.initials: TA
authors.lastname: Molskness
authors.forename: A
authors.initials: A
authors.lastname: Villeda
authors.forename: M B
authors.initials: MB
authors.lastname: Zelinski
authors.forename: R L
authors.initials: RL
authors.lastname: Stouffer
authors.forename: J
authors.initials: J
authors.lastname: Xu
mesh_terms: D042683:Angiopoietin-1 / Q000096:biosynthesis; D042702:Angiopoietin-2 / Q000096:biosynthesis; D000818:Animals; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D008253:Macaca mulatta; D006080:Ovarian Follicle / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D010100:Oxygen / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*
chemical_list: D042683:Angiopoietin-1; D042702:Angiopoietin-2; D006063:Chorionic Gonadotropin; D042461:Vascular Endothelial Growth Factor A; D005640:Follicle Stimulating Hormone; D010100:Oxygen
keywords: VEGF; angiogenesis; angiopoietin; follicle culture; follicle development
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