The preparation and purification of isolated rat corpus-luteum cells and their use in studying the relationship between cholesterol biosynthesis and the lutropin-stimulated formation of progesterone

Abstract

Isolated luteal cells, prepared from superovulated rat ovaries by digestion with collagenase, were subjected to density-gradient centrifugation on Percoll to give a more highly purified preparation of luteal cells than has been reported previously. The cells formed progesterone when incubated in vitro; lutropin stimulated this steroidogenesis. Progesterone formation was linear for at least 2 h; a minimal lutropin concentration of 1.0 ng/ml was needed for stimulation and concentrations of 3.0 and 100 ng/ml gave half-maximal and maximal responses respectively. The cells were unresponsive towards hormones other than lutropin. Exposure to lutropin raised the cellular cyclic AMP concentration, and dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, was as effective in stimulating steroidogenesis as was lutropin. Aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, completely blocked progesterone formation by the cells, showing cholesterol side-chain cleavage to be an obligatory step in steroidogenesis by these cells. Neither the activity of 3-hydroxy-3-methylglutaryl-CoA reductase nor the incorporation of radioactively labelled acetate or mevalonate into cholesterol by cells incubated in vitro were detectable unless the rats had been treated previously with 4-aminopyrazolo[3,4-d]pyrimidine. In cells from rats so treated, compactin was found to block almost completely the incorporation of radioactively labelled acetate, but not of mevalonate, into cholesterol, indicating that this inhibitor acts in corpus luteum in the same way as it does in other tissues. In cells from rats not treated with 4-aminopyrazolo[3,4-d]pyrimidine compactin had no effect on progesterone formation in vitro, showing cholesterol biosynthesis to be unnecessary for the rapid steroidogenic response by luteal cells to lutropin.

Mechanism of inhibitory action of TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] on aldosterone secretion in adrenal glomerulosa cells

“…5 mmcalcium and 5.5 mM-glucose (KRBG buffer) unless otherwise mentioned. For calcium influx measurement, 'purified glomerulosa cells' were prepared by using a Percoll density gradient (McNamara et al, 1980). Static incubations were done as described previously (Kojima et al, 1984a).…”

Luteotrophic factors in hyperstimulated pseudopregnant rabbit: I – Evidence for aromatase activity in luteal tissue and luteal cells

“…Fractions of large and small luteal cells were prepared by equilibrium centrifugation on a stepwise gradient of Percoli (which separates cells according to their density) by a modified procedure described by McNamara et al (1980) for rat purified luteal cells. Briefly, luteal cell suspensions (10-20 x 106 cells/500 µ ) were placed in 15 ml polystyrene tubes, the Percoli solutions (Percoli/ MEM-BSA (10-X), 9v/lv) were prepared in MEM containing 0-1% BSA in solutions of 60, 40, 20, 15, 10 and 5%, and were sequentially overlayered in that order, then the tubes were centrifuged at 500 g for 10 min.…”

The contribution of the cholesterol biosynthetic pathway to intermediary metabolism and cell function

“…(ii) Endocrine tissues have an additional demand for sterol, for steroid hormone production, which can be satisfied by uptake of HD and LD lipoproteins. In both adrenal gland (Andersen & Dietschy, 1978) and ovary (McNamara et al, 1980) steroid hormone biosynthesis is dependent on cholesterol uptake rather than endogenous synthesis.…”