BMCB753 Primary Cultures of Steroid-Producing Cells and Radioimmunoassay

The Bovine Ovary

Two functional structures: Follicle and corpus luteum

Follicle

Houses the egg

Corpus luteum

produces progesterone, the hormone of pregnancy

Non-pregnant cow estrous cycle

about 21 days long

Divided into luteal phase and follicular phase

Majority is luteal

Luteal cells in culture

Terminally differentiated, have granules in the cytoplasm

Lipid droplets in cytoplasm

Produce progesterone, secreted into conditioned medium

Why should you not use serum with primary culture

serum is a source of hormones which confounds studies on the regulation progesterone production by luteal cells

Biosynthesis of steroid hormones metabolism of cholesterol

All steroid hormones come from cholesterol

Pregnenolone is the first synthesized, precursor of progesterone

Luteinizing hormone (LH)

Primary regulator of progesterone biosynthesis in the corpus luteum

RIA

Radioimmunoassay

Theory of RIA

Requires non-radiolabeled analyte (hormone, variable), radiolabeled analyte (hormone, fixed), and antibody against analyte (hormone/antigen, fixed)

Competitive protein binding involving antigen/antibody interactions

Competitive protein binding RIA

Radiolabeled hormone competes with the non-radiolabeled hormone for the antibody

System detects radiolabeled hormone bound antibody complexes

As more non-radiolabeled hormone is introduced, there are fewer radiolabeled hormone bound antibody complexes

Scintillation counting

Tritium is a beta emitter that is relatively low energy

Scintillation counting amplifies the signal from tritium using solvents and fluors excited by tritium