Corpus Luteum Slices Research Articles

Search for a progesterone-binding protein in secretory granules of the ovine corpus luteum.

The hypothesis that electron-dense granules present in the corpus luteum contain progesterone in a protein-bound form was examined using differential and sucrose gradient centrifugation. Ovine luteal tissue was fractionated by differential centrifugation at 1,000 X g (P1 pellet), 10,000 X g (P2), and 82,000 X g (P3; supernatant S3). Samples of P2, P3, and S3 were further fractionated o 20-40% (P2 and P3) or 5-25% (S3) sucrose gradients and examined for progesterone-binding activity by measuring the progesterone content and/or the specific binding of [3H]progesterone of sucrose gradient samples. In addition, saturation binding assays were performed with steroid-free samples of P2 and S3. Saturable binding of progesterone was not found in P2, the fraction containing electron-dense granules. In S3, two progesterone-binding proteins with sedimentation rates of 3.2S and 8.6S and an affinity of 7.1 X 10(5) M-1 for progesterone were detected. The sedimentation behavior of these proteins was distinct from that of ovine plasma transcortin, a 4S protein. The view that a binding protein is released into the interstitial fluid during the exocytosis of granules was examined by measuring the progesterone-binding activity of protein released by slices of corpus luteum in vitro. No binding activity was found. The results of this investigation do not support the hypothesis that putative progesterone-secreting granules observed in luteal tissue contain a binding protein

Demonstration of Luteinizing Hormone/Human Chorionic Gonadotrophin Receptor-Binding Inhibitor in Aqueous Extracts of Frozen Human Corpus Luteum

Aqueous extracts of frozen human corpora lutea were tested for the presence of an inhibitor of luteinizing hormone-receptor site binding (LHRBI) and for the subsequent effect on the stimulatory response of luteinizing hormone (LH) on progesterone synthesis by sheep ovarian cells. In the presence of human corpus luteum extract of normal menstrual cycle (30,000-g supernatant), the binding of 125I human chorionic gonadotrophin (hCG) to granulosa and luteal cells of sheep ovaries was markedly reduced, but the ability of rat testicular LH receptors to bind labelled hCG was less affected. However, extracts of corpora lutea of the first trimester of pregnancy appeared to be less inhibitory on the binding of LH/hCG to ovarian cells and had no effect on the binding of rat testicular cells compared to those of normal menstrual cycle. Addition of both extracts separately inhibited the LH-stimulated in vitro progesterone synthesis by granulosa cell cultures and by incubated sheep corpus luteum slices. These findings provide evidence for the presence of LHRBI in human corpus luteum.

Studies on Mitochondrial Protein Kinase Activity of Porcine Corpora Lutea*

Activation of mitochondrial protein kinase and its role in regulation of [4-14MC]cholesterol conversion to [4- 14C]pregnenolone and [4-14C]progesterone were studied using mitochondria prepared from porcine corpora lutea. The presence of a cAMP-dependent protein kinase in luteal mitochondria was confirmed by using highly purified samples prepared by large scale zonal centrifugation. Slices of corpora lutea secreted almost 3 times more progesterone when incubated for 30 min with 10 μg LH/ml. The LH treatment of luteal tissue also resulted in an increase in endogenous protein kinase activity (measured without cAMP) of mitochondria from 45.0 to 60.7 pmol 32P/mg protein min. However, the LH treatment of corpora lutea did not significantly change [4-14C]cholesterol conversion activity. When isolated intact luteal mitochondria were incubated with cAMP, almost all of themitochondrial protein kinase was activated, but there was no change in the [4-14C]cholesterol conversion activity of these mitochondria. The addi...

The metabolism of prostaglandins F 2α and E 2 by non-pregnant porcine endometrial and luteal tissue and early pregnant porcine endometrial tissue, luteal tissue and conceptuses [formula omitted

Slices of porcine endometrium and corpus luteum from both early pregnant and non-pregnant sows and conceptuses from early pregnant sows were incubated with radioactive PGF 2a and PGE 2 and the degree of metabolism of the prostaglandins measured. Prostaglandins and metabolites were separated by TLC, radioactive bands located with a Panax scanner and the radioactivity measured in a scintillation counter. Endometrial and luteal tissue from non-pregnant sows gave no significant metabolism at any stage of the oestrous cycle, and while similar tissue from pregnant sows metabolised both prostaglandins slightly, only the conceptuses gave any significant metabolism. The possible physiological significance of these results is discussed.

Reevaluation of the Role of Cyclic Adenosine 3',5'-Monophosphate and Protein Kinase in the Stimulation of Steroidogenesis by Luteinizing Hormone in Bovine Corpus Luteum Slices1

Incubation of bovine corpus luteum slices at 37 C with luteinizing hormone showed that 10.0 microng LH/ml caused a maximum rise in tissue cyclic AMP content within 15 min. Slices incubated with 1.0 or 0.1 microng LH/ml showed a much more gradual accumulation of this nucleotide. In the absence of added LH, a marked decline in the amount of cyclic AMP was observed during the first 60 min. The possible role of cyclic AMP in the action of LH was reexamined by studying the dose-response effect of LH on the stimulation of progesterone synthesis, cyclic AMP-dependent protein kinase activation, and cyclic AMP accumulation. After a 2-h incubatin, the results showed that the dose required to elicit a minimal significant stimulation of steroidogenesis was 0.01 microng/ml LH. At this and higher concentrations of LH, a concomitant stimulation of protein kinase activity and progesterone synthesis was also consistently observed. However, significant accumulation of cyclic AMP became consistently detectable only at 0.1 microng/ml LH. This report is the first to show a positive correlation between the activation of cyclic AMP-dependent protein kinase and the stimulation of steroidogenesis in the corpus luteum at the same minimal effective level of LH. These results indicate that cyclic AMP and the cyclic AMP-dependent protein kinase probably play important intermediary roles in the stimulation of steroidogenesis by LH in the bovine corpus luteum.

Stimulation by LH of cyclic AMP-dependent protein kinase activity in bovine corpus luteum slices

FEBS LettersVolume 63, Issue 1 p. 159-163 Full-length articleFree Access Stimulation by LH of cyclic AMP-dependent protein kinase activity in bovine corpus luteum slices First published: March 15, 1976 https://doi.org/10.1016/0014-5793(76)80216-5Citations: 9AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume63, Issue1March 15, 1976Pages 159-163 ReferencesRelatedInformation

Investigations on the ability of antisera produced by Pr-β-HCG-TT to neutralize the biological activity of HCG

The ability of antibodies generated by the vaccine Pr-β-HCG-TT in two species of animals to neutralize the biological activity of HCG has been investigated. The antisera from an immunized goat and serveral rhesus monkeys ( Macaca mulatta ) abolished the HCG induced rise in ventral prostate weight of immature male rats and of uterine weight in prepubertal mice. These were also found to be effective in preventing the binding of 125I-HCG to receptors in rat testes and goat corpora lutea preparations. The antisera also blocked the HCG induced synthesis and secretion of progesterone in corpus luteum slices in vitro . These investigations show that the antibodies elicited by the conjugate (Pr-β-HCG-TT) form a complex with HCG, which is biologically inactive.

LH does not increase cyclic AMP in the thyroid.

We previously reported that LH increased cyclic AMP concentrations in dog thyroid slices and that this effect could not be accounted for on the basis of the stated TSH contamination of the preparation (1). This conclusion now appears incorrect since anti-² subunit LH antibody did not inhibit the effect of 2 μg LH on the thyroid. An equivalent amount of the antibody completely abolished the effect of 2 μg LH on cyclic AMP concentrations in slices of pig corpus luteum. Therefore, the action of LH on the thyroid represented TSH contamination and LH does not have any intrinsic activity to increase cyclic AMP in the thyroid. The TSH contamination of LH appears to be underestimated on the basis of a bioassay dependent upon 32P incorporation into phospholipids in chick thyroid. Bioassay based on cyclic AMP accumulation in thyroid slices indicated a TSH contamination tenfold greater than that reported using 32P incorporation into phospholipids. Two other LH preparations also had greater TSH contamination based on...

Cyclic AMP accumulation and steroidogenesis in the human corpus luteum: effect of gonadotropins and prostaglandins.

ABSTRACT Prostaglandin E2 (PGE2) stimulates steroidogenesis in vitro in human corpus luteum slices, to a similar extent as hCG. This prostaglandin, as well as hCG and hLH also stimulates the incorporation of [8-3H] adenine into [8-3H] cyclic AMP in this in vitro system and this stimulation appears to be specific and dose related for these substances. The increase of [8-3H] cyclic AMP accumulation was found to be due to the stimulation of the synthetic adenyl cyclase enzyme system rather than the inhibition of the destructive phosphodiesterase enzyme system. Throughout the study, corpora lutea of the menstrual cycle were shown to be much more responsive to stimulation (hCG, LH, PGE2) in terms of steroid synthesis and cyclic AMP formation than corpora lutea of pregnancy. The reason for this difference between the two types of corpora lutea is not clear at this time but does not appear to be due to a deficiency in the adenyl cyclase system of pregnancy corpora lutea. The data of the present study brings our ...

Stimulation of progesterone biosynthesis in bovine corpora lutea by luteinizing hormone in the presence of an inhibitor of cholesterol synthesis.

Slices of corpora lutea from 4 normally cycling dairy cows have been incubated in the absence and in the presence of trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride (AY9944), an inhibitor of cholesterol biosynthesis. AY9944 (10−5M) decreased the in vitro incorporation of acetate-1-14C into cholesterol both in the absence and presence of LH, to 26 and 18% of control rates, in half-hour and 2-hour incubations, respectively. LH was equally effective in increasing the rate of net in vitro progesterone synthesis in the presence of AY9944 as in its absence. No evidence of a stimulatory action of LH upon cholesterol synthesis was observed; in fact, LH decreased markedly, the incorporation of acetate-1-14C into cholesterol. Neither LH nor AY9944 influenced incorporation of acetate-1-14C into fatty acids of triglycerides or of phospholipids. The ability of LH to increase rate of in vitro glycolysis in this tissue was not affected by AY9944. It is concluded that a stimulation, by LH, of in vitro synthesis of progesterone in bovine corpus luteum slices does not depend upon rate of concurrent synthesis of cholesterol, and that LH, in addition to stimulating conversion of cholesterol to progesterone, may exert an inhibitory action on cholesterol biosynthesis.

NEUTRAL STEROID METABOLITES OF DEHYDROEPIANDROSTERONE-7α-3H IN HUMAN OVARIAN TISSUES

Abstract The metabolism of dehydroepiandrosterone-7α- 3 H by human ovarian tissues was studied by incubating slices of cortical stroma and corpora lutea of pregnancy. In the stroma the major metabolites of DHA were other androgens. A larger turnover of DHA occurred with luteal tissues and estrogens were the major metabolites. The isolation of Δ5-androstenediol, androstenedione and testosterone in all incubations suggests alternate pathways for the metabolism of DHA in both cortical stroma and corpora lutea.

Control of progesterone biosynthesis in the bovine corpus luteum: effects of luteinizing hormone and of reduced nicotinamide-adenine dinucleotide phosphate in vitro.

The effects of luteinizing hormone (LH) and of reduced nicotinamide–adenine dinucleotide phosphate (NADPH) upon in vitro progesterone synthesis by bovine corpus luteum slices incubated for varying periods of time have been examined. The increased net synthesis of progesterone caused by LH was accompanied by markedly increased incorporation of acetate-1-14C into progesterone and decreased incorporation of acetate-1-14C into cholesterol. In most experiments, especially at longer incubation times, exogenous NADPH decreased incorporation of acetate-1-14C into progesterone. The effect of NADPH on incorporation of acetate-1-14C into cholesterol was variable, increasing cholesterol specific activity in some experiments and decreasing it in others. At all time intervals in all experiments, the specific activity of progesterone synthesized was greater than that of the cholesterol remaining in the tissue at the end of the interval. With increasing duration of incubations, specific activity of the progesterone synthesized became increasingly greater than that of cholesterol remaining at the end of the incubation period. These findings suggest that newly synthesized cholesterol is utilized for progesterone formation without coming into equilibrium with a large portion of the intracellular cholesterol pool. Experiments with radioactive cholesterol in vitro indicated that a significant amount of conversion of this precursor occurred in the incubation medium by enzymes which had leaked out of the tissue, and this conversion was strikingly stimulated by exogenous NADPH but not by LH. Rates of progesterone synthesis were greatest in shortest incubations, declining as incubation periods were extended. Highly significant correlations were observed between the increments, caused by LH, in net synthesis (mass) of progesterone, incorporation of acetate-1-14C into progesterone, and lactic acid production. Inhibition by puromycin of the stimulatory action of LH upon progesterone synthesis failed to prevent the stimulatory action of this gonadotropin upon glycolysis. These findings indicate that the stimulation of glycolysis is not a consequence of stimulated progesterone synthesis but may be related to the stimulatory action of LH upon 3′,5′-AMP production.

Some characteristics and optimum incubation conditions of in vitro progesterone synthesis by bovine corpora lutea.

In view of the widespread use of in vitro incubations of corpus luteum slices for a variety of purposes, experiments were conducted to establish optimum incubation conditions and to determine some of the sources of error variation observed in this system. A 2-hr incubation period was sufficient to detect an LH (luteinizing hormone) induced stimulation of progesterone synthesis; longer incubation times yielded higher levels of synthesis but did not increase the precision of the assay. The pH optimum for the incubation system was between 7.15 and 7.55. Addition of 1 μmole NADPH2 resulted in a consistent stimulation of progesterone synthesis, even in those groups already maximally stimulated with LH, suggesting that 2 different mechanisms are operative for the 2 stimulating agents. NADPH2 did not change the regression coefficient or the precision of the assay. Addition of nicotinamide (30 HIM) resulted in higher rates of progesterone synthesis, presumably by maintaining the integrity of the pyridine nucleoti...

Studies on the hydroxylation and metabolism of Δ 4-androstene-3,17-dione-7H 3 in the human corpus luteum tissue

Surviving slices of human corpus luteum, obtained from a patient on the 26th day of the menstrual cycle, were studied in vitro . The experimental data, based on partition column chromatography and isotope dilution analysis, demonstrate a conversion of Δ 4-androstene-3, 17-dione-3,17-7H 3 to radioactive 6β-hydroxy-Δ 4-androstene-3,17-dione and testosterone as well as estrone and 17 β-estradiol.

Formula omitted] inhibition of cholesterol synthesis by pregnenolone in bovine corpus luteum

Bovine corpus luteum slices and. minces were incubated with acetate-1- 14C in the presence of pregnenolone. There was a general inhibition in the utilization of acetate-1- 14C as manifested, by a decreased incorporation of radioactivity into digitonin precipitable sterols, cholesterol, progesterone, and the total lipid extract of the tissue. LH which alone would stimulate the incorporation of acetate-1- 14C into these substances was not able to reverse the pregnenolone-induced. inhibition. It is suggested that pregnenolone may have a role in the regulation of steroidogenesis in the corpus luteum.

The Stimulatory Effect of Luteinizing Hormone on Adenosine 3‘,5‘-Monophosphate Accumulation in Corpus Luteum Slices

The presence of adenosine 3‘,5‘-monophosphate was demonstrated in bovine luteal tissue. The addition of luteinizing hormone to incubating slices of a bovine corpus luteum caused a rapid accumulation of this cyclic nucleotide which preceded the increase in progesterone synthesis. This effect appeared to be specific for luteinizing hormone. Puromycin did not inhibit this action of luteinizing hormone on cyclic AMP accumulation. In view of the previous finding that exogenous cyclic AMP added to incubating slices can mimic the effect of luteinizing hormone on steroidogenesis, these results indicate that the cyclic nucleotide is a mediator of this action of luteinizing hormone. An increase in the concentration of cyclic AMP has also been demonstrated in a human corpus luteum stimulated in vitro by human chorionic gonadotropin.

SPECIFICITY OF GONADOTROPIN STIMULATION OF PROGESTERONE SYNTHESIS IN BOVINE CORPUS LUTEUM IN VITRO

The effect of varying amounts of LH and FSH on progesterone biosynthesis was studied in vitro in bovine corpus luteum slices. It was found that a stimulatory effect could be obtained with amounts of LH as low as 0.01-0.02 μg/g of luteal tissue, or approximately 1×10-10M in the incubation medium. The stimulation was found to increase with increasing amounts of added LH. FSH (NIHFSH-S-1) was found to have about 1/100th the stimulating activity of LH, a value which correlates with its LH content as determined in the ascorbic acid depletion test of Parlow. Urea treatment of this FSH preparation markedly reduced its stimulatory activity on luteal progesterone synthesis. It was concluded that the stimulation of progesterone synthesis by FSH is due to its small LH content. On the basis of these and other data, it appears that, among the anterior pituitary hormones, LH alone stimulates steroidogenesis in the corpus luteum. (Endocrinology 74: 664, 1964)

Conversion of Cholesterol to Progesterone by Corpus Luteum Slices

The conversion of cholesterol-7-3H to progesterone was studied in slices of bovine corpus luteum by measuring both the total amount of progesterone synthesized and the radioactivity in the final product derived from cholesterol-7-3H. The addition of luteinizing hormone to the incubating slices caused an increase in the amount of progesterone synthesized but only a slight increase in the conversion of radioactive cholesterol. The addition of NADP +glucose-6-phosphate, on the other hand, increased both the amount of progesterone and, to a greater extent, the conversion of added cholesterol-7-3H to progesterone. Neither added LH nor added NADP -fglucose-6-P influenced the penetration of the radioactive cholesterol into the slices. It was concluded that NADPH increases progesterone synthesis primarily through its action on the transformation of cholesterol, whereas LH affects the transformation of cholesterol to a slight extent only and acts mainly on precursors prior to cholesterol in the biosynthetic pathwa...

SPECIFICITY OF GONADOTROPIN STIMULATION OF PROGESTERONE SYNTHESIS IN BOVINE CORPUS LUTEUM IN VITRO.

The effect of varying amounts of LH and FSH on progesterone biosynthesis was studied in vitro in bovine corpus luteum slices. It was found that a stimulatory effect could be obtained with amounts of LH as low as 0.01-0.02 μg/g of luteal tissue, or approximately 1×10-10M in the incubation medium. The stimulation was found to increase with increasing amounts of added LH. FSH (NIHFSH-S-1) was found to have about 1/100th the stimulating activity of LH, a value which correlates with its LH content as determined in the ascorbic acid depletion test of Parlow. Urea treatment of this FSH preparation markedly reduced its stimulatory activity on luteal progesterone synthesis. It was concluded that the stimulation of progesterone synthesis by FSH is due to its small LH content. On the basis of these and other data, it appears that, among the anterior pituitary hormones, LH alone stimulates steroidogenesis in the corpus luteum. (Endocrinology74: 664, 1964)

EFFECTS OF PITUITARY HORMONES AND NADPH ON ACETATE UTILIZATION IN OVARIAN AND ADRENOCORTICAL TISSUES.

In the course of the biosynthesis de novo of progesterone by corpus luteum slices in vitro, acetate-l-14C is incorporated into the steroid. Both the amounts of steroid formed (measured photometrically) and the incorporation of radioactivity are increased by exogenous gonadotropin, LH. Although exogenous NADPH (as a generating system) increases the quantity of progesterone formed de novo, it does not increase the incorporation of acetate-l-14C. The same phenomenon occurs in the biosynthesis of corticosteroids by adrenal cortex slices, with NADPH. It is concluded that the tropic hormone stimulates steroid biosynthesis from small molecule precursors, and that exogenous NADPH does not. (Endocrinology 74: 599, 1964)