Identification of Vascular Changes Required for Ovulation Using Multiphoton Microscopy.

Abstract

Mice in which the hedgehog signaling pathway is conditionally activated in the ovary were used as a model to determine vascular events required for ovulation. In Amhr2cre/+SmoM2 mutant mice, CRE-mediated recombination induces expression of a dominant active allele of the hedgehog signal transducer smoothened, beginning in the neonatal ovary. Preovulatory follicles develop in these mice and undergo many of the expected changes during the periovulatory period, but they fail to rupture. Vessels in the theca of growing follicles do not become appropriately associated with vascular smooth muscle. This defect appears to be due to changes elicited by over-activation of hedgehog signaling during neonatal development, leading to persistent failure throughout life of vessels within the theca to undergo maturation. These previous findings are consistent with the known importance of hedgehog signaling in vascular development. In the current study, multiphoton microscopy was used to image vascular changes in preovulatory follicles in vivo. Prepubertal mutant and genotype-matched control mice were primed with eCG followed 48h later by treatment with hCG. In this model, ovulation normally occurs 12-13h post hCG. An ovary with intact bursa was exteriorized and immobilized, and the entire preparation, including the abdomen of the mouse, was bathed with warm Ringer's solution. Rhodamine-labeled 2x106 MW dextran was injected retro-orbitally, and individual vessels were imaged over a four hour time period using a submersion objective. Vessel diameter and red blood cell velocity were measured repeatedly in individual capillary-scale vessels, and blood flow was calculated. At each time point, multiple vessels in the apical and lateral areas of preovulatory follicles from 3 control and 3 mutant mice were studied. At 48h after eCG, apical and lateral vessels did not differ in diameter or blood flow between control and mutant mice. The apex of mutant and control follicles had equivalent density of blood vessels. Following hCG treatment in control mice, blood flow in apical and lateral vessels decreased at 12h and 13h and was <5% of that observed at 11h (P<0.05). By 13h, 73% of the apical vessels and 20% of the lateral vessels were no longer detectable, and flow rates in the vessels that remained visible were low. Vessels in which blood flow slowed dramatically or stopped at 12h were undetectable one hour later, suggesting a process of vasoconstriction or remodeling. By 14h, the remodeling associated with ovulation prevented identification of previously measured vessels. In mutant mice, the flow rate in apical vessels did not change between 11h and 12h after hCG, but it decreased moderately at 13h to levels 44% of that at 11h (P<0.05). The apparent disappearance of vessels from the apex that was observed in control mice did not occur in mutants. In lateral vessels of mutant mice, flow was constant between 11h and 13h. In summary, the diameter and flow rate of thecal vessels in preovulatory follicles decreased during the periovulatory period in control mice. Thecal vessels in mutant mice failed to undergo these changes, likely due to lack of maturation of the vessels by association with smooth muscle. These findings suggest that vascular changes such as vasoconstriction are required for follicle rupture. Supported by NICHD R03HD057648, NIH/NIBIB P41 EB001976-20, Cornell Center for Vertebrate Genomics, and NSF ADVANCE 0547373