Doctoral Dissertations

Spring 1988

Dissertation

Program or Major

Animal and Nutritional Sciences

Degree Name

Doctor of Philosophy

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.