Mechanism for control of hydroxymethylglutaryl-coenzyme A reductase and cytochrome P-450 side chain cleavage message and enzyme in the corpus luteum.

The present study was undertaken to investigate the mechanism for control of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase and cytochrome P-450 side chain cleavage (P-450scc), enzymes involved in the synthesis and processing of cholesterol in the corpus luteum of pregnant rats. The role of sterol and estradiol were investigated by administering 4-aminopyrazolo-[3,4d]pyrimidine (4-APP), aminoglutethimide and/or estradiol to day 12 hypophysectomized-hysterectomized pregnant rats. Estradiol treatment markedly increased mRNA levels of HMG-CoA reductase in the corpus luteum. This stimulatory effect of estradiol was specific for the reductase since the mRNA for luteal P-450scc did not increase following estradiol treatment. To determine whether estradiol's action on HMG-CoA reductase mRNA was mediated by changes in cellular free cholesterol, the separate and combined action of estradiol and either 4-APP or aminoglutethimide on both sterol content and HMG-CoA reductase expression was examined. The estradiol induced rise in HMG-CoA reductase was not accompanied by a decrease in luteal cholesterol. 4-APP treatment depleted cholesterol in the corpus luteum and induced a greater increase in HMG-CoA reductase mRNA than estradiol. Combined treatment with estradiol and 4-APP resulted in a synergistic increase in HMG-CoA reductase mRNA despite the fact that estradiol did not further reduce the 4-APP induced decrease in luteal sterol content. This indicates that luteal cells possess an estradiol-mediated mechanism for up-regulation of HMG-CoA reductase gene expression which is distinct from the cholesterol negative feedback regulation. However, estradiol stimulation of HMG-CoA reductase was totally inhibited when sterol content was elevated by aminoglutethimide, suggesting that estradiol stimulation of HMG-CoA reductase expression is not sufficient to overcome the negative regulatory effect of cholesterol. In contrast to HMG-CoA reductase mRNA which appears to be controlled by sterol and estradiol, P-450scc mRNA levels remained constant in corpora lutea despite wide changes in sterol and steroid content induced by either 4-APP, aminoglutethimide or estradiol treatment. However, interestingly, aminoglutethimide increased substantially the content of P-450scc protein. This increase in P-450scc enzyme was not due to a greater amount of translatable message since similar levels of immunoprecipitable 35S-P-450scc were translated in vitro from luteal mRNA of rats treated with or without aminoglutethimide. In summary, results of this investigation have revealed that the expression of HMG-CoA reductase and cytochrome P-450scc are controlled by totally different mechanisms in the rat corpus luteum.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction and repression of specific estradiol sensitive proteins in the rat corpus luteum.

In the pregnant rat estradiol causes luteal cell hypertrophy and a dramatic increase in overall protein synthesis. The aim of this investigation was to determine whether estradiol induces the synthesis of specific proteins in luteal cell subcellular fractions and to examine estradiol-regulated translational products in order to identify a specific protein marker of estradiol action in the corpus luteum. Corpora lutea obtained from day 12 hypophysectomized-hysterectomized pregnant rats treated with or without estradiol (2 cm implant) for 3 days were incubated with Trans35S-label for 8 hours at 37 degrees C in an atmosphere of 95% O2 - 5% CO2. Nuclear, mitochondrial, microsomal and cytosolic subcellular compartments were obtained by differential centrifugation. RNA was extracted and translated using a rabbit reticulocyte lysate reaction mixture. Radiolabeled proteins were separated by SDS-PAGE, visualized by fluorography and quantified by densitometry. Estradiol-treatment significantly increased 35S-amino acid incorporation into luteal proteins. Specific estradiol-induced proteins within cellular fractions included mitochondrial 80, 50, and 32 x 10(-3) Mr proteins; microsomal 100, 80, 32 x 10(-3) Mr proteins; and cytosolic 80, 60, 50 and 14 x 10(-3) Mr proteins. Estradiol-treatment increased the secretion of two proteins (60 and 45 x 10(-3) Mr) while no major estradiol-induced proteins were noted in the nuclear fraction. In vitro translation of mRNA indicated that estradiol markedly enhances the message for a protein with apparent Mr 32 x 10(-3) Mr. The 32 x 10(-3) Mr translated protein appeared as the major estradiol-induced protein in mitochondrial and microsomal fractions. Estradiol also reduced the expression of a 38 x 10(-3) Mr protein (pI 7.0) in the cytosolic compartment. The major estradiol sensitive protein observed in luteal particulate and translation profiles was a 32 x 10(-3) Mr protein with a pI greater than or equal to 8.5. This protein may serve as a potential protein marker of estradiol action in the rat corpus luteum.

Estradiol regulation of sterol carrier protein-2 independent of cytochrome P450 side-chain cleavage expression in the rat corpus luteum.

A major action of estradiol in the corpus luteum of the pregnant rat is to increase the supply of cholesterol substrate for progesterone production by stimulating both cholesterol synthesis and uptake. To determine whether this steroid also affects cholesterol metabolism and transport, estradiol's action on the expression of cytochrome P450 side-chain cleavage enzyme (P450scc) and the cholesterol transport protein, sterol carrier protein-2 (SCP2), was examined. Mitochondria isolated from corpora lutea of estradiol-treated rats secreted significantly more progestagen than mitochondria of control corpora lutea. Several findings indicate that estradiol enhances cholesterol transport and availability to the P450scc rather than affects the expression of this enzyme: 1) the difference in mitochondrial progestagen synthesis induced by estradiol was obliterated by the presence of 25-hydroxycholesterol; 2) immunoblotting of P450scc indicated no stimulatory effect of estradiol on the amount of enzyme; and 3) levels of P450scc mRNA were not increased by estradiol. Whereas estradiol had no stimulatory effect on P450scc it caused a mark (3-fold) increase in the mitochondrial content of SCP2. Thus, the increase in luteal progestagen synthesis stimulated by estradiol appears to be associated with an increase in mitochondrial SCP2 and is independent of luteal P450 content or message.

Protein secretion by mesometrial and antimesometrial rat decidual tissue: evidence for differential gene expression.

Our recent finding that decidual luteotropin, a PRL-like hormone secreted by the rat decidua, is found primarily in the antimesometrial cells suggests strongly that the synthesis of this hormone may well be an important function of the antimesometrial tissue. The objective of this investigation was, therefore, 1) to determine whether antimesometrial tissue expresses mRNA for and actively secretes a protein(s) with PRL-like activity, and 2) to examine the pattern of protein production by the antimesometrial and mesometrial zones throughout decidual development. RNA obtained from decidual tissue of day 8 pseudopregnant rats was translated in a cell-free system. The translated products were subjected to PRL receptor affinity chromatography in the presence or absence of ovine PRL to assess binding specificity. The eluted 35S-labeled proteins were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. A major 28K protein bound specifically to and was eluted from PRL receptor-enriched luteal membranes. This protein also cross-reacted with antibodies to human PRL. To determine where the mRNA for this 28K protein is expressed and whether this protein represents a prohormone, RNA isolated from both antimesometrial and mesometrial tissue was translated in the presence or absence of microsomal membranes. The 28K protein was synthesized specifically by RNA isolated from the antimesometrial zone. No apparent change in the relative mol wt of the 28K protein was observed when translation was performed in the presence of microsomal membranes. To determine whether this protein is a secreted product and to investigate the pattern of protein secretion by the mesometrial and antimesometrial decidua, tissue explants obtained from both zones between days 9-13 of pseudopregnancy were cultured in the presence of [35S]methionine. Antimesometrial tissue secreted one major 28K protein which was capable of binding to PRL receptors on luteal membranes and was immunoprecipitated by antibodies to human PRL, whereas the mesometrial tissue primarily secreted an approximately 180K protein. The overall pattern of protein synthesis and release not only differed between the mesometrial and antimesometrial tissues but also differed with each day of pseudopregnancy. The secretion of several proteins decreased with advancing gestational age, while the secretion of other proteins began abruptly after day 11, coincident with regression of the antimesometrial tissue. In summary, results of this investigation have established that rat decidual tissue synthesizes and selectively secretes proteins, and the specificity and rate of production of these distinct