Publication Date

1986

Document Type

Dissertation/Thesis

First Advisor

Ledwitz-Rigby, Florence

Degree Name

M.S. (Master of Science)

Legacy Department

Department of Biological Sciences

LCSH

Enzymes; Cytochemistry; Ovaries

Abstract

Progesterone (P₄) secretion by porcine granulosa cells (GC) from small (1-2 mm) ovarian follicles is enhanced by incubation with charcoal treated and filtered fluid from large (6-12 mm) porcine, ovarian follicles (LFF1). This study examines the enzymatic steps in the conversion of cholesterol to progesterone at which LFF1 acts to enhance P₄ secretion. The effect of nafoxidine, an anti-estrogen, on LFF1's enhancement of P₄ secretion and LFF1's effects on cellular DNA content were also studied. GC from small, antral follicles were incubated in monolayer for 3 days using an initial inoculum of 5 x 10⁵ cells. P₄ and progesterone (P₅) concentrations were determined by specific radioimmunoassay and cellular DNA content determined by a fluorometric assay. To study LFF1's effect on P₅ production, 0 to 5 x 10⁻⁵ M P₅ were added for the 3 day incubation with LFF1 or serum. P₄ secretion by LFF1 treated cells was not increased until 10 M P₅ was present, whereas 10 M P₅ enhanced P₄ secretion by controls. When 5 x 10 M P₅ was present, treated cells produced more P₄ than controls. This implies that LFF1 increased P₅ production and utilization. When 5 x 10⁴ viable GC were inoculated, 10⁻⁶ M P₅ saturated the enzymes of both treated and control cells. LFF1 treated cells secreted more P4 than controls at saturating concentrations of P₅. During a 3 hour incubation with 10⁻⁶ M P₅ following a 3 day incubation with LFF1 or serum, the rate of P₄ secretion by LFF1 treated cells and controls was similar over the first 1/2 hour. P₄ secretion by LFF1 treated cells remained linear for 2 hours while controls stopped secreting P₄ after 1 hour. This may be due to a difference in the subcellular compartment containing the enzyme. 10⁻⁶ M nafoxidine blocked LFF1's enhancement of P₄ secretion in 2 of 4 experiments. LFF1 increased cellular DNA content by 60+7% in 5 of 9 experiments. The interpretation of LFF1's effect on P₄ secretion is the same if data are expressed per culture, cell #, or ug DNA. 3B-hydroxysteroid dehydrogenase (3B-HSD) activity was measured in 10,000 x g supernatant fractions of GC after 3 day incubations with LFF1 or serum. There was no difference in Km but Vmax was increased 60% in LFF1 treated cells. These results imply that LFF1 increases P₅ production and the number of 3B-HSD molecules.

Comments

Bibliography: pages [57]-64.

Extent

vii, 64 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

Download

Share

COinS