Hormonal activation of adenylate cyclase in mouse melanoma metastatic variants.

The ability of melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), and prostaglandin E1 (PGE1) to stimulate the accumulation of cyclic AMP was examined in intact mouse melanoma cells of varying metastatic potential. F1 cells (low metastatic potential) had significantly greater cyclic AMP levels in response to all three hormones than F5 (intermediate metastatic potential) and F10 (high metastatic potential) cells. The ranking of the response was as follows: MSH, F1 greater than F5 greater than F10, ACTH, F1 greater than F5 greater F10, PGE, F1 greater than F10 greater F5. In contrast to the above, the degree of hormonal stimulation of adenylate cyclase in broken cell preparations was virtually identical in all three melanoma cell lines. Control enzyme activity was depressed in both F5 and F10 relative to F1. The conflicting results between studies of intact vs. broken cell preparations could not be explained by increased cyclic AMP phosphodiesterase activity in F5 and F10 cells. We conclude that as the melanoma cells increase in metastatic potential, there is a significant loss in the ability of their cyclic AMP system to respond appropriately to hormonal stimuli.     

Prostaglandin biosynthesis and stimulation of cyclic AMP in primary monolayer cultures of epithelial cells from mouse mammary gland.

Cyclic AMP levels in primary monolayer cultures of epithelial cells prepared from mid-pregnant mice are stimulated by prostaglandin E1 and E2. Prostaglandin F1alpha and F2alpha have only a slight effect upon cyclic AMP levels. In the absence of phosphodiesterase inhibitors the rise in cyclic AMP produced by PGE1 is only transient and the levels return to normal within 30 minutes. High concentrations (16 mM) of theophylline are needed to prevent this decline, suggesting that the phosphodiesterase activity of epithelial cells in culture is high. However, theophylline alone produced only a small increase in basal cyclic AMP levels even over a 2-hour period indicating that basal cyclic AMP is turned over more slowly than cyclic AMP produced in response to stimulation with PGE1. Both PGE and PGF synthesis were monitored using radioimmunoassay procedures previously reported. The observed levels were found to decrease as cell density increased and were sensitive to the addition of agents such as collagen and naproxen.     

Action of ACTH in the luteal ovary.

Oestrous rats and golden hamsters were anesthetized with pentobarbital, one of the femoral arteries and veins and one of the ovarian veins were cannulated. Blood fractions were collected from the ovary. After the first two fractions synthetic adrenocorticotropic hormone (ACTH) or human chorionic gonadotropin (hCG) was injected i.v. Blood pressures and ovarian blood flow were continuously recorded. Progesterone (P) and oestradiol-17 beta (E2) were determined from the ovarian venous blood by radioimmunoassay (RIA). ACTH induced a temporary elevation in the ovarian blood flow, P and E2 secretion both in rats and hamsters. In rats and hamsters hCG induced a continuous elevation in P secretion but the ovarian blood flow and E2 secretion remained unchanged. Luteal cells from pseudopregnant rats or oestrous hamsters were dispersed with collagenase and incubated with ACTH or hCG. A sample of the cells was preincubated with polymixin-B, indomethacin or ibuprofen. P and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) contents of the medium and cyclic 3,5 adenosine monophosphate (cAMP) content of the cells were determined by RIA. ACTH stimulated the release of 6-keto-PGF1 alpha and the secretion of P from the luteal cells of both species, which was inhibited by indomethacin or ibuprofen, but ACTH did not alter the cAMP content of luteal cells. The polymixin-B prevented ACTH to stimulate P secretion, but it did not elevate the 6-keto-PGF1 alpha release, while the cAMP content of the cells remained unchanged. It is supposed that the polyphosphoinositol-Ca(2+)-protein kinase-C second messenger system is involved in the ACTH induced stimulation of P secretion.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis.

The steroidogenic response to ACTH prostaglandin E2 (PGE2) was studied in cat adrenocortical cells dispersed with trypsin. The dose-response curves of both agents were qualitatively and quantitatively similar. Exposure to PGE2 or ACTH in the presence of labeled steroid precursor (acetate) resulted in the accumulation of comparable levels of steroid intermediates and end-product. Submaximal or maximal concentrations of ACTH and PGE-2 given simultaneously elicited a response which was no greater than that obtained with either stimulant alone. Although calcium was required for optimal PGE-2 stimulation of steroid production, this requirement with ACTH as the stimulant, but greater than with butyryl cyclic AMP. PGE-2-induced increase in the adrenal cyclic AMP was not statistically significant and was small in relation to that found with equipotent steroidogenic ACTH concentrations. The possible relationship between prostaglandins, cyclic AMP, and calcium in the action of ACTH is discussed.     

Correlation of the biosynthesis of prostaglandin and cyclic AMP in monolayer cultures of rabbit articular chondrocytes

We have utilized ionophores to test whether stimulation of chondrocyte prostaglandin biosynthesis is accompanied by an increase in cyclic nucleotide levels in these cells. Radioimmunoassay of prostaglandin E2, 6-oxo-prostaglandin F1 alpha (the stable metabolite of prostaglandin I2) and prostaglandin F2 alpha showed that synthesis of each was stimulated by the divalent-cation ionophore, A23187 after short-term incubation (1-7 min) in serum-free medium. No stimulation of thromboxane B2 was detected. Two monovalent ionophores, lasalocid and monensin failed to stimulate prostaglandin biosynthesis after short-term incubation. Ionophore A23187-stimulated prostaglandin biosynthesis was variably and partially inhibited by sodium meclofenamate, indomethacin and aspirin, but not by sodium salicylate. Ionophore A23187-stimulated prostaglandin biosynthesis was accompanied by a 7.5-fold increase in cyclic AMP levels after 15 min. Sodium meclofenamate, indomethacin and aspirin which inhibited prostaglandin E2 biosynthesis also reduced cyclic AMP levels. Exogenous prostaglandin E2 (1 microgram/ml) stimulated cyclic AMP biosynthesis, which was not inhibited by aspirin. These results indicated that prostaglandins can be considered as one of the local effectors controlling cyclic AMP production in articular cartilage.     

Induction of synthesis of bovine adrenocortical cytochromes P-450scc, P-45011 beta, P-450C21, and adrenodoxin by prostaglandins E2 and F2 alpha and cholera toxin

To further elucidate the mechanisms by which ACTH (adrenocorticotropin) exerts its long-term action to maintain normal levels of adrenocortical cytochromes P-450 and related enzymes, the abilities of cholera toxin and prostaglandins E2 and F2 alpha to induce the synthesis of cytochromes P-450scc, P-45011 beta, and P-450C21 and adrenodoxin have been examined. These effectors stimulate the production of cyclic AMP and thus steroidogenesis in the adrenal cortex. Using bovine adrenocortical cells in primary monolayer culture, we have shown that treatment with cholera toxin results in increased synthesis of cytochromes P-450scc and P-45011 beta and adrenodoxin, similar to the effect observed upon ACTH treatment. Prostaglandins E2 and F2 alpha are less effective at inducing the synthesis of the mitochondrial cytochromes P-450, and do not seem to induce the synthesis of adrenodoxin. Furthermore, cholera toxin was found to be less effective at inducing the synthesis of microsomal cytochrome P-450C21 than ACTH, and no more effective than the prostaglandins. Thus, while it appears that elevation of cyclic AMP levels is a necessary step leading to increased synthesis of adrenocortical forms of cytochrome P-450, the detailed mechanism of this induction will be found to be different for each of the different enzymes.     

Release of arachidonic acid and the effects of corticosteroids on steroidogenesis in rat testis Leydig cells.

The release of arachidonic acid by luteinizing hormone (LH) and the effects of inhibiting phospholipase A2 (PLA2) in vivo and in vitro on LH stimulated steroidogenesis in rat testis Leydig cells has been investigated. It was found that arachidonic acid is rapidly incorporated into phospholipids and is released within 1 min after addition of LH. The effects of treating adult rats with dexamethasone and human chorionic gonadotropin (hCG) in vivo on steroidogenesis and prostaglandin synthesis in Leydig cells isolated 6 h later were determined. It was found that hCG caused a marked increase in prostaglandin F2 alpha formation which was inhibited by treatment with dexamethasone. LH-stimulated testosterone production was inhibited in the hCG treated rats and dexamethasone caused a further decrease. Treatment with dexamethasone alone also caused a decrease in the response to LH. HCG, but not dexamethasone, had similar inhibitory effects on LH-stimulated cyclic AMP production. Similarly, the PLA2 inhibitors quinacrine, dexamethasone and corticosterone, added to the Leydig cells in vitro, inhibited LH-stimulated testosterone production but not cyclic AMP production. 11-Dehydrocorticosterone also inhibited LH-stimulated testosterone production, but higher concentrations were required to give 50% inhibition compared to corticosterone (50 and 25 microM, respectively). Ring A-reduced metabolites of corticosterone and progesterone were also found to inhibit LH-stimulated steroidogenesis. The results obtained in this and previous studies are consistent with the activation of PLA2, (either directly by LH and/or via cyclic AMP), which results in the release of arachidonic acid and the formation of leukotrienes, which stimulate steroidogenesis in the Leydig cell. This study also indicates that corticosteroids and their metabolites may exert inhibitory effects at other sites in the steroidogenic pathways, in addition to PLA2.     

Effect of steroids on basal and LH-stimulated prostaglandins F(2alpha) and E(2) release and cyclooxygenase-2 expression in cultured porcine endometrial stromal cells

Ovarian steroids modulate uterine receptivity in domestic species. Luteinizing hormone (LH) stimulates prostaglandin (PG)F(2alpha) release from the porcine endometrium. However, the combined action of LH and steroids on PGs secretion has not yet been studied in pigs. The aim of the present study was to examine the effect of estradiol (E(2)) and progesterone (P(4)) on basal and LH-stimulated PGF(2alpha) and PGE(2) secretion and cyclooxygenase-2 (COX-2) protein expression in porcine endometrial stromal cells obtained on days 12-13 of the estrous cycle. Cells were cultured for 48 h in a medium containing charcoal-stripped newborn calf serum alone or supplemented with 10 nM E(2) and/or 50 nM P(4). Then, the cells were incubated for 6 h in the presence or absence of LH (20 ng/ml). Long exposure of stromal cells to steroids had no effect on PGF(2alpha) secretion, but PGE(2) release increased in the presence of E(2) plus P(4) (p<0.05). Pre-incubation of cells with E(2) plus P(4) resulted in enhanced PGF(2alpha) (p<0.05) and PGE(2) (p<0.001) secretion. Moreover, LH increased PG(2alpha) secretion in control (p<0.05) and E(2)-treated stromal cells (p<0.01). LH tended (p=0.07) to elevate PGE(2) release only in cells pre-exposed to E(2) plus P(4). The expression of COX-2 protein was increased by LH (p<0.05), but not by steroids. These results confirm the stimulatory effect of LH on PGF(2alpha) secretion and COX-2 expression in porcine stromal cells before luteolysis. PG release from porcine endometrium seems to be controlled by ovarian steroids, however only E(2)-treated-treated cells responded to LH.     

Prostaglandin F2 alpha inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F2 alpha (PGF2 alpha). The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF2 alpha. A dose-dependent inhibition by PGF2 alpha (0.5-50 microM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165-165 microM). The stimulation by epinephrine on progesterone production was inhibited by PGF2 alpha (5 microM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF2 alpha can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF2 alpha shows in this respect the same age dependent inhibitory pattern as in relation to LH stimulation.     

Effect of prostaglandins on steroid secretion by human fetal adrenal tissue

In the present investigation we evaluated the effect of prostaglandins on the rate of steroid secretion by human fetal adrenal (HFA) tissue. Prostaglandins F2 alpha and E2 (10 micrograms/ml) were added to the culture medium in the presence or absence of ACTH (1 micrograms/ml). The medium was assayed for content of cortisol (F), dehydroepiandrosterone sulfate (DS) and pregnenolone sulfate (PS) by radioimmunoassay. When HFA tissue fragments were maintained in the absence of ACTH, F secretion was low; PGF2 alpha but not PGE2 suppressed F secretion by 60-65%. When ACTH was added to the culture medium, the secretion rate of F increased 15-fold, whereas DS and PS secretion was maintained at or near initial rates of secretion. The addition of PGF2 alpha to the culture medium containing ACTH resulted in a 80% decrease in F secretion, but PGE2 only suppressed F secretion by 50%. In contrast, PGE2 or PGF2 alpha had little effect on the rate of DS or PS secretion either in the presence or absence of ACTH. In conclusion, prostaglandins appear to inhibit the secretion of F, but not of DS or PS by the HFA.     

Prostaglandin synthesis in rat adrenocortical cells.

The biosynthesis of prostaglandins by isolated rat adrenocortical cells has been studied by determinations of products formed during incubations with labeled arachidonic acid and by radioimmunoassays. Analysis by thin-layer chromatographic separation of silicic acid column fractions indicated that PGE2, PGA2, (B2) and PGF2 alpha were the predominant prostaglandins formed by rat adrenocortical cells. Approximately 75% of the incorporated isotope was associated with the prostaglandins of the PGE pathway [PGE2 + PGA2 (B2)]. This was a consistent finding whether cells were incubated directly with arachidonic acid or with cells prelabeled with the substrate prior to study. ACTH did not affect the uptake or oxidation of [1-14C]-arachidonate, but did significantly increase incorporation of labeled substrate into [14C]prostaglandins. Of the ACTH-induced increase, 92% was accounted for by an increase in prostaglandins of the E pathway. Studies with prelabeled cells indicated that 77% of the prostaglandins synthesized in both control and ACTH-stimulated adrenocortical cells was released into the incubation medium during the 2-hr study. These had the same composition [88% PGE2 + PGA2 (B2)] as did the intracellular prostaglandins. Analysis by radioimmunoassays gave comparable data on the distribution of E- and F-type prostaglandins in control cells and cells incubated with ACTH or dibutyryl cyclic AMP. Thus, with these techniques, 88-92% of the increased prostaglandin synthesis due to ACTH or cyclic AMP was produced by the PGE2 rather than the PGF2 alpha pathway.     

A role for calmodulin in the regulation of steroidogenesis

Two approaches were used to study the possible role of calmodulin in the regulation of steroid synthesis by mouse adrenal tumor cells: trifluoperazine was used as an inhibitor of calmodulin and liposomes were used to deliver calmodulin into the cells. Trifluoperazine inhibits three steroidogenic responses to both ACTH and dibutyryl cyclic AMP: (a) increase in steroid production, (b) increased transport of cholesterol to mitochondria, and (c) increased side-chain cleavage by mitochondria isolated from cells incubated with ACTH or dibutyryl cyclic AMP. When calmodulin is introduced into the cells via liposomes, steroid synthesis is slightly stimulated. When calmodulin extensively dialyzed against EGTA, this stimulation is abolished. Ca(2+) introduced via liposomes was also without effect. However, when both calmodulin and Ca(2+) are introduced via liposomes (either in separate liposomes or in the same liposomes), steroid synthesis is stimulated. This stimulation does not occur when either anticalmodulin antibodies or EGTA is also present in the liposomes or when trifluoperazine is present in the incubation medium. Calmodulin and Ca(2+) presented together in liposomes to the cells stimulate transport of cholesterol to mitochondria, and side-chain cleavage activity is greater in mitochondria isolated from cells previously fused with liposomes containing calmodulin and Ca(2+) than in mitochondria from cells fused with liposomes containing buffer only. These observations suggest that calmodulin may be involved in regulating the transport of cholesterol to mitochondria, a process which is stimulated by ACTH and dibutyryl cyclic AMP and which may account, at least in part, for the increase in steroid synthesis produced by these agents.     

Effects of prostaglandins on the bovine corpus luteum: granules, lipid inclusions and progesterone secretion

Corpora lutea collected at 15, 30 and 60 min after prostaglandin F2 alpha (PGF2 alpha) treatment were compared to control corpora lutea at 60 min after saline treatment. There were decreases (P less than 0.05) in the relative percentages of cytoplasm occupied by granules in large luteal cells (LLC) by 30 min and in small luteal cells (SLC) by 60 min. Differences were not observed among the groups for lipid inclusions. Luteal progesterone was decreased at all post-PGF2 alpha treatment times when compared to 60-min controls (P less than 0.05). PGF2 alpha was then compared with prostaglandin F1 alpha (PGF1 alpha), prostaglandin E1 (PGE1), and 17-phenyl-18,19,20-trinor-prostaglandin F2 alpha (17-phenyl-PGF2 alpha) in 60-min trials with plasma progesterone and luteinizing hormone (LH) determined every 5 min. LH was not affected by these treatments. Like PGF2 alpha, 17-phenyl-PGF2 alpha induced a greater loss of granules from LLC then SLC. 17-phenyl-PGF2 alpha also induced an increase in the lipid content of LLC. Treatments with PGF2 alpha and 17-phenyl-PGF2 alpha were associated with decreased concentrations of luteal progesterone but PGF1 alpha and PGE1 were without effect on this variable. In contrast to PGF1 alpha, PGE1 increased both luteal progesterone and the area occupied by cytoplasmic granules. The latter effect was greater in LLC than SLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of prolactin synthesis and of adenosine 3'':5''-cyclic phosphate formation by prostaglandins and thyroliberin in cultured rat pituitary cells.

1. The effects of prostaglandins E2 and F2alpha on prolactin synthesis were examined in a clonal strain of rat pituitary tumour cells, and compared with those of thyroliberin. 2. The prostaglandins and thyroliberin gave a dose-related and time-dependent stimulation of prolactin synthesis. The maximal effects (about twofold increases) were observed after 54h of treatment with 25nM-prostaglandin E2 and 2.5nM-prostaglandin F2alpha. A similar stimulation of prolactin synthesis was observed after 250nM-thyroliberin. The combined treatment with prostaglandins and thyroliberin did not increase prolactin synthesis over and above that obtained with each compound alone. 3. After removal of prostaglandins E2 and F2alpha there was a complete reversal of prolactin synthesis to pre-stimulation values 18h later (t1/2less than or equal to 9h). The rapid reversible effect of prostaglandins was in contrast with that of thyroliberin, where prolactin synthesis returned to control values with a t1/2 of about 42 h. 4. Prostaglandin E2 (5mum) and thyroliberin (5mum) increased cellular concentrations of cyclic AMP eight- and four-fold respectively. Maximal effects were observed after 2-5min of incubation. The increases in cyclic AMP were biphasic; normal values were obtained 60 min after the start of incubation with prostaglandin E2 or thyroliberin. 5. The dose/response curve showed that prostaglandin E2 caused maximal increase of cyclic AMP at 50nM. Concentrations of prostagland in E2 that caused half-maximal stimulation of cyclic AMP accumulation and of prolactin synthesis were 4 and 5nM respectively. 6. Combined treatment with prostaglandin E2 and thyroliberin in concentrations that separately caused maximal cyclic AMP increases did not result in a further increase in this cyclic nucleotide. 7. These results are consistent with a role of cyclic AMP in mediating the effects or prostaglandins and thyroliberin on prolactin synthesis. However, if cyclic AMP is involved as a common intracellular mediator of prolactin synthesis, it cannot alone explain all the effects of prostaglandins and thyroliberin in this cell system.     

Role of cyclic AMP and protein kinase on the steroidogenic action of ACTH, prostaglandin E1 and dibutyryl cyclic AMP in normal adrenal cells and adrenal tumor cells from humans.

The role of the cyclic AMP-protein kinase system in mediating the steroidogenic effect of ACTH, prostaglandin E1 and dibutyryl cyclic AMP, induced similar stimulations of protein kinase activity, cyclic AMP was studied using human adrenal cells isolated from normal and adrenocortical secreting tumors. At high concentrations of ACTH, complete activation of protein kinase of normal adrenal cells was observed within 3 min, at the time when cyclic AMP production was slightly increased and there was still no stimulation of steroidogenesis. At supramaximal concentrations, ACTH, PGE1 and dibutyryl cyclic AMP and cortisol productions in adrenal cells isolated from normal and from one adrenocortical tumor. In one tumor in which the adenylate cyclase activity was insensitive to ACTH, the hormone was unable to stimulate protein kinase or steroidogenesis, but the cells responded to both PGE1 and dibutyryl cyclic AMP. In another tumor in which the adenylate cyclase was insensitive to PGE1, this compound also did not increase protein kinase activity or steroidogenesis, but both parameters were stimulated by ACTH and dibutyryl cyclic AMP. After incubation of normal adrenal cells with increasing concentrations of ACTH (0.01-100 nM) marked differences were found between cyclic AMP formation and cortisol production. However at the lowest concentrations of ACTH exerting an effect on steroid production a close linked correlation was found between protein kinase activation and cortisol production, but half-maximal and maximal cortisol production occurs at lower concentration of ACTH than was necessary to induce the same stimulation of protein kinase. Similar findings were found after incubating the adrenal cells with dibutyryl cyclic AMP (0.01-10 mM). The results implicate an important role of the cyclic AMP-protein kinase system during activation of adrenal cell steroidogenesis by low concentrations of steroidogenic compounds.     

Prostaglandin production by type II alveolar epithelial cells.

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.     

Feedback effects of ovarian steroids on the hypothalamic-hypophyseal axis in the rabbit

The feedback effects of two ovarian steroids, estradiol-17 beta (E2) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha OH), were examined in both intact (INT) and ovariectomized (OVEX) does. We measured steroid-induced alterations in endogenous gonadotropin-releasing hormone (GnRH) from sequential 10-min samples of hypothalamic perfusates, simultaneous changes in peripheral plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the modification of pituitary responsiveness, i.e., increments in plasma LH (delta LH) and plasma FSH (delta FSH), after 50 ng, 250 ng, and 1 microgram of exogenous GnRH in individual does of 6 treatment groups. The groups were: INT does, OVEX does, OVEX does receiving either one (1 E2) or two (2 E2) E2-filled Silastic capsules, OVEX does receiving a 20 alpha OH-filled capsule (20 alpha OH), and OVEX does receiving both capsules of E2 and 20 alpha OH (1 E2 + 20 alpha OH). Ovariectomy enhanced the pulsatile release of hypothalamic GnRH and pituitary LH and FSH, and increased the LH response (delta LH) to exogenous GnRH (OVEX vs. INT, p less than 0.05). Replacement of E2 at the time of ovariectomy prevented the increased GnRH and gonadotropin secretion as well as the enhanced delta LH that were observed in untreated OVEX does. The release of hypothalamic GnRH in the 20 alpha OH group was lower (p less than 0.05) than that in the OVEX group and not different from that in the INT group. The release of pituitary LH and FSH and the delta LH in the 20 alpha OH group was not different from that in the OVEX group, but these parameters were greater (p less than 0.05) than those in the INT group. The hypothalamic GnRH pulse frequency in the 1 E2 + 20 alpha OH group was lower (p less than 0.05) than that in either the 1 E2 or the 20 alpha OH group, but the delta LH in the 1 E2 + 20 alpha OH group was not different from that in either the 1 E2 or the 20 alpha OH group. The highest dose (1 microgram) of exogenous GnRH stimulated a modest increase in FSH in the OVEX, 20 alpha OH, 1 E2 + 20 alpha OH, and 1 E2 groups; but a steroid effect on delta FSH among these 4 groups was not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural changes induced by ACTH in normal adrenocortical cells in culture

The effects of ACTH, its o-nitrophenyl sulfenyl derivative (NPS-ACTH) and dibutyryl cyclic AMP (dbc AMP) on the ultrastructural morphology of adrenocortical cells of adult rats in monolayer culture have been investigated. NPS-ACTH, which has previously been shown to stimulate steroidogenesis but not cAMP synthesis in adrenal cells, induced the same characteristic transformation of mitochondrial architecture as produced by ACTH or high concentrations of dbcAMP. All three agents caused the disappearance of electron-opaque granules present in the mitochondria of unstimulated cells. It was found that these granules could be extracted with EGTA (ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetate). These results are discussed in the light of the known importance of calcium ions in the actions of ACTH.     

Functional and morphological characteristics of the first corpus luteum formed after parturition in ewes

The ability of sheep luteal cells from the first corpus luteum formed after parturition (Group F) to secrete progesterone in the presence or absence of LH was compared with that of luteal cells obtained from normal cyclic ewes (Group C). Luteal concentrations of receptors for LH and prostaglandins (PG) F-2 alpha (PGF-2 alpha) and the cellular composition of corpora lutea from Groups F and C were also compared. Luteal cells from Group F secreted less progesterone in either the presence or absence of LH (P less than 0.01). There was no difference in the number of receptors for LH or PGF-2 alpha per luteal cell between Groups F and C (P greater than 0.1), nor was there a difference in the number of large or small steroidogenic luteal cells (P greater than 0.1). It was concluded that, if short-lived corpora lutea are insensitive to gonadotrophins, this response is not mediated by decreased numbers of receptors for LH. In addition, if the first corpus luteum formed post partum in ewes is more sensitive to the luteolytic effects of PGF-2 alpha, this effect is not mediated by an increased number of receptors for PGF-2 alpha or an increased proportion of PGF-2 alpha-sensitive large luteal cells.