Vasoconstriction At The Apex Of The Follicle Is Essential For Follicle Rupture At Ovulation: Findings From Studies In Vivo Using Multiphoton Microscopy And Power Doppler Ultrasound In Anovulatory Mouse Models
In the effort to elucidate the role of the hedgehog (HH) signaling pathway in reproduction, mice were created in which HH signaling was overactivated in the gonads and in the reproductive tract. These mice expressed a dominant active allele of the signal transducer SMOM2 that was directed by Amhr2cre-mediated recombination. Amhr2cre/+SmoM2 mutant females were infertile due to a failure in follicle rupture and abnormal development of the reproductive tract. Amhr2cre/+SmoM2 mutant males were subfertile due to abnormal development of the reproductive tract and persistence of the Müllerian duct that led to reduced sperm numbers in the ejaculate. In Amhr2cre/+SmoM2 mutant females, vessels within the theca were deficient in vascular smooth muscle. It was hypothesized that this defect would prevent constriction of blood vessels in response to physiologic stimuli and contribute to anovulation in this model. Therefore, mutant females were used as a model to determine the importance of vasoconstriction within the follicle in ovulation. To pursue this goal, a procedure was devised in which vessel characteristics such as velocity of red blood cells and vessel diameter could be studied in vivo. In this procedure, vessels were fluorescently labeled by systemic intravascular injection of rhodamine-labelled dextran. Leakage of dextran from the vessels provided enough contrast to identify structures within the follicle and as a result the thickness of the follicle wall could be determined. Ovaries of anesthetized mice were imaged using multiphoton microscopy. Initial studies with mutant and control mice revealed an association between vasoconstriction, thinning of the follicle wall and follicle rupture. Therefore additional experiments were performed to gain further insight in the role of vasoconstriction in ovulation. An experimental setting was devised in which thinning of the follicle wall was prevented, in this experiment, it was demonstrated that vasoconstriction was not a result of thinning of the follicle wall. In wild-type mice, vasoconstriction was experimentally blocked and then vasoconstrictors were infused into the bursa. While inhibition of vasoconstriction impaired ovulation, infusion with vasoconstrictors rescued ovulation demonstrating that vasoconstriction is essential for ovulation. Using Doppler ultrasound, it was determined that in the mouse, the vasoconstriction detected by multiphoton microscopy only occurred at the outer surface of the follicle and not at the base. In Amhr2cre/+SmoM2 mutant males, subfertility was likely due to reduced sperm numbers in the ejaculate. Reduced sperm numbers could be explained by the presence of persistent Müllerian tissue that impedes normal development of the vas deferens or impairs its contractions at the time of ejaculation. In addition, the vas deferens had localized narrowing of the lumen and a tortuous trajectory which could impair contractions at ejaculation. Subfertility could also be explained by the reduced weight and spermatid content of the mutant testes that is associated with a reduction in sperm numbers in the cauda epididymis.
Ovulation; Ovary; Vasoconstriction
Quirk, Susan MaryQuirk, Susan Mary
Schaffer, Chris; Fortune, Joanne Elizabeth; Schimenti, John C.; Schimenti, John C.; Zipfel, Warren R.; Fortune, Joanne Elizabeth
Ph. D., Animal Science
Doctor of Philosophy
dissertation or thesis