Semester

Summer

Date of Graduation

2009

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

E. Keith Inskeep

Committee Co-Chair

Jorge A. Flores

Abstract

Three experiments were carried out in order to examine the role of endothelin in follicular steroid secretion in vitro and in spontaneous and PGF2alpha-induced luteolysis in sheep.;In experiment 1, it was hypothesized that endothelin 1 (END1) acting through its receptors ENDRA and/or ENDRB, on granulosal cells of ovine preovulatory follicles would inhibit steroid production, and therefore, prevent the premature luteinization of granulosal cells of those follicles. Data from estrogen-inactive 20 follicles collected from 13 ewes were analyzed. Accumulation of E 2 and P4 by cultured granulosal cells was not affected by either LH or END1 or the END receptor blockers. Granulosal cells from follicles with the greatest concentration of E2 in FF produced on the average significantly more E2 and P4 than follicles with lower concentrations of E2 in FF. In conclusion, END1 did not affect steroid production by granulosal cells from preovulatory follicles in sheep, and therefore the working hypothesis was not supported.;In the second experiment, the ENDRA antagonist BQ-610 was delivered into the CL via an osmotic minipump to examine the expression of genes related with P4 production and structural luteolysis at 6 and 24 h after exogenous PGF2alpha. It was hypothesized that sustained blockade of ENDRA would prevent upregulation of genes stimulated by PGF2alpha during structural luteolysis. Data for luteal weight, and concentrations of serum and luteal P4, mostly at 24 h after PGF2alpha, indicated that in 9 of 10 treated ewes, BQ-610 did not prevent luteolysis, and the pattern of gene expression confirmed this finding. Exogenous PGF2alpha downregulated expression of StAR, 3betaHSD, VEGF, TIMP-1 and eNOS (P ≤ 0.02), whereas it upregulated 20alphaHSD, MMP-14 Bcl-2 (P ≤ 0.03). Expression of Bcl-2 and Bax were reduced and Fas-R was increased from 6 to 24 h after PGF2alpha. In BQ-610-treated ewes, mRNA expression was upregulated more for 20alphaHSD, and caspases 3 and 8 than in vehicle-treated and control ewes. In conclusion, PGF2alpha induced luteolysis in both vehicle-treated and BQ-610-treated ewes.;In the third experiment, it was hypothesized that chronic delivery of BQ-610 into the CL would prevent luteal regression induced by endogenous PGF 2alpha, thereby increasing length of the estrous cycle and maintaining functional and structural characteristics of the CL. Ewes were assigned receive an osmotic mini-pump containing 2 mg of BQ-610 (n = 12) or vehicle (n = 9), implanted surgically on d 9 of the estrous cycle. Corpora lutea were collected 12 h after onset of estrus, or on the afternoon of d 21 in ewes that had not returned to estrus, and from untreated ewes on d 10 to 12 of the estrous cycle (mid-phase CL). Three of 12 BQ-610-treated ewes did not show estrus before d 21 compared to 0 of 9 vehicle-treated ewes (P = 0.33); CL of the remaining nine ewes treated with BQ-610 were excluded from further analysis. In conclusion, chronic infusion of BQ-610 blocked luteolysis and lengthened the estrous cycle in 3 of 12 ewes. Furthermore, functional features of CL of those 3 ewes were similar to mid-phase CL. Overall, these results supported a role for END1 in luteal regression in sheep. (Abstract shortened by UMI.).;Keywords. luteolysis, progesterone, END1, endothelin receptor type A, BQ-610, gene expression, sheep.

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