Date of Award

Spring 1988

Project Type

Dissertation

Program or Major

Animal and Nutritional Sciences

Degree Name

Doctor of Philosophy

First Advisor

William A Condon

Abstract

Luteal regression in the cow occurs every cycle in which pregnancy does not occur. The agent which induces luteolysis is a uterine prostaglandin, PGF$\sb{2\alpha}$, although the mechanism of its action remains unknown. The purpose of this study was to investigate the mode of action of PGF$\sb{2\alpha}$ in a long-term, serum-free culture system of bovine luteal cells. Experiment I investigated the influence of LH and PGF$\sb{2\alpha}$ on 3$\beta$-HSD presence in cultured luteal cells. Total numbers of cells dropped slightly throughout the 8 day culture. Numbers of 3$\beta$-positive cells also dropped in all treatment groups. 3$\beta$-HSD was best maintained in the presence of LH. PGF$\sb{2\alpha}$ treatment had no influence on numbers of 3$\beta$-positive cells. Experiment II investigated the role of Ca$\sp{++}$ and calmodulin in the regulation of P$\sb4$ steroidogenesis. The presence of extracellular Ca$\sp{++}$ is mandatory for LH stimulation of P$\sb4$ as is seen in EGTA treated medium. Ca$\sp{++}$-enriched medium (A23187) increased LH-stimulated P$\sb4$ production. P$\sb4$ in the PGF$\sb{2\alpha}$ treatment was not altered in either the presence or absence of elevated Ca$\sp{++}$. The production of endogenous PGF$\sb{2\alpha}$ was not influenced by Ca$\sp{++}$ environment. Ca$\sp{++}$ antagonists TMB-8 and CCCP had no effects on basal P$\sb4$ production but did inhibit LH-stimulated P$\sb4$. Calmodulin antagonists, TFP and W-7, were only able to slightly inhibit LH-stimulated P$\sb4$ and had no effect on basal P$\sb4$. Experiment III investigated the influence of E$\sb2$ and phenol red, a weak E$\sb2$, on luteal P$\sb4$ steroidogenesis. E$\sb2$ was shown to suppress LH-stimulated P$\sb4$ in phenol-containing medium but had no effect on basal or PGF$\sb{2\alpha}$-induced P$\sb4$. The use of phenol-free medium resulted in higher absolute P$\sb4$ levels for control and LH treatments. E$\sb2$ suppressed LH-stimulated P$\sb4$ early in the culture but was unable to cause an effect on day 8 and 10. No statistically significant cooperative effects of E$\sb2$ + PGF$\sb{2\alpha}$ were found but trends suggest lowered P$\sb4$ in the presence of both of these luteolytic agents. In conclusion, the presence of low levels of LH is suggested to maintain the number of steroidogenically active cells. PGF$\sb{2\alpha}$ does not mediate its action through a reduction in 3$\beta$-HSD. LH-stimulated P$\sb4$ production requires free, intracellular and extracellular Ca$\sp{++}$ whereas, the role of Ca$\sp{++}$ in PGF$\sb{2\alpha}$ action remains unknown. Also, free Ca$\sp{++}$ appears to be a more important regulator of luteal steroidogenesis than calmodulin-bound Ca$\sp{++}$. Estrogen appears to have a direct effect on LH-stimulated P$\sb4$ production. Phenol red appears to influence basal and LH-stimulated P$\sb4$ production. The complete mechanism of PGF$\sb{2\alpha}$ action remains to be elucidated.

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