Biomedical subjects

J L Tilly

Publications and source records attributed to J L Tilly.

101 records · Page 6Linked to original sources

PubMed1990

Control of plasminogen activator activity in the thecal layer of the largest preovulatory follicle in the hen ovary.

Although the existence of plasminogen activator (PA) activity and the factors that regulate it in ovarian granulosa cells of both mammalian and avian species have been extensively documented, very little information has been generated concerning the control of PA activity in the adjacent thecal layer. This study was conducted to evaluate the effects of several physiological and pharmacological agents on PA activity in dispersed cells from the thecal layer of the largest preovulatory follicle in the hen ovary 17-16 h before ovulation. LH (50 and 100 ng) in the presence of 3-isobutyl-1-methylxanthine (0.01 mM) stimulated an approximate 25% increase in cell-associated PA activity, possibly via elevated levels of cAMP. Prostaglandin E1 and E2 (PGE1 and PGE2; 0.1 and 1 microM), but not PGI2 or PGF2 alpha (1 microM), enhanced PA activity and cAMP formation, effects that were potentiated by 0.01 mM 3-isobutyl-1-methylxanthine. Activation of Gs with cholera toxin (0.01-10 ng/tube) or adenylyl cyclase with forskolin (0.01-10 microM) stimulated cAMP formation and PA activity in a dose-dependent manner. Exposure of cells to the cAMP analog 8-bromo-cAMP (0.1-5 mM) caused similar increases in thecal cell PA activity. Incubation of cells with phorbol 12-myristate 13-acetate (PMA; 3.2-162 nM), an agonist known to activate protein kinase-C, resulted in a dose-dependent increase in PA activity. However, an equimolar concentration of phorbol 13-monoacetate (162 nM), an inactive analog of PMA that does not activate protein kinase-C, was without effect. Coincubation of cells with forskolin (1 microM) and PMA (32 nM) resulted in a synergistic stimulation of secreted PA activity, apparently via an enhancement of adenylyl cyclase activity. Treatment of cells with the calcium ionophore A23187 (0.01-1 microM) suppressed basal PA activity. However, PA activity stimulated by PMA (32 nM) was synergistically increased after coincubation with a 0.05-microM concentration of A23187, but was inhibited at doses of 0.5 and 1 microM. Taken collectively, the data indicate that PA activity is present in the thecal layer of the largest preovulatory follicle in the ovary of the domestic hen. Furthermore, several endocrine factors (i.e. LH and PGs) were found to stimulate PA activity, possibly via both the adenylyl cyclase-cAMP-protein kinase-A and phosphoinositide-protein kinase-C pathways. In light of these findings, we propose that the preovulatory increase in PGs and LH activates PA in the thecal layer of the largest preovulatory follicle, resulting in proteolytic degradation of the follicular connective tissue and, ultimately, ovulation.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Modulation of hen granulosa cell steroidogenesis and plasminogen activator activity hy transforming growth factor alpha.

Studies were conducted with chicken granulosa cells to evaluate the relative efficacy of human recombinant transforming growth factor alpha (TGF alpha) versus murine epidermal growth factor (EGF) to affect cyclic adenosine monophosphate (cAMP) accumulation and progesterone production stimulated by luteinizing hormone (LH) or steroid output induced by a cAMP analogue, and to modulate plasminogen activator (PA) activity. Increasing concentrations of EGF (33-328 nM) and TGF alpha (0.04-18 nM) were found to inhibit cAMP formation stimulated by LH in a dose-dependent manner, with calculated half-maximal inhibitory doses (ID50's) of 97.1 and 0.27 nM, respectively. Similarly, a 470-fold difference in the ability of TGF alpha (ID50 = 0.13 nM) versus EGF (ID50 = 61.3 nM) to half-maximally suppress LH-induced progesterone production was observed in the same cells. Progesterone production stimulated by a cAMP analogue (8-bromo-cAMP, 1 mM) was also attenuated by EGF (ID50 = 75.9 nM) and TGF alpha (ID50 = 0.08 nM), suggesting a post-cAMP site of inhibition by these growth factors on steroidogenesis. Finally, a 260-fold to 330-fold difference in the efficacy of TGF alpha versus EGF to half-maximally stimulate cell-associated and secreted PA activity was observed. From these data, we propose that TGF alpha may serve an important role in regulating follicular growth and maturation in the domestic hen via its ability to affect granulosa cell steroidogenesis and plasminogen activator activity.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

Effect of several growth factors on plasminogen activator activity in granulosa and theca cells of the domestic hen.

This study was conducted to evaluate the effects of several growth factors on plasminogen activator (PA) activity in granulosa and theca cells collected from the largest preovulatory follicle in the hen ovary and to determine the involvement of cyclic adenosine monophosphate (cAMP) or protein kinase C, or both, in mediating the actions of epidermal growth factor (EGF) on granulosa PA activity. The granulosa cells were treated with increasing concentrations of: EGF (.33 to 16.4 nM); insulin-like growth factor I (IGF-I, 2.61 to 131 nM); fibroblast growth factor (FGF, .15 to 7.5 nM); or platelet-derived growth factor (PDGF, .02 to 1 nM). The treatments resulted in a dose-dependent increase in both cell-associated and secreted enzyme activity. The stimulatory effects of IGF-I (131 nM), however, were not mimicked with an equimolar concentration of the related peptide, insulin-like growth factor II. By contrast, theca cell PA activity was not significantly altered by EGF (16.4 nM), IGF-I (131 nM), FGF (7.5 nM), or PDGF (1 nM). Accumulation of cAMP was measured following exposure of granulosa cells to luteinizing hormone (LH, 10 ng, used as a positive control) or to EGF (16.4 and 164 nM) in the presence of .1 mM isobutylmethylxanthine. A 5-fold increase in cAMP levels was observed in response to LH; however, granulosa cell cAMP production was not altered by the presence of EGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Mechanisms by which a phorbol ester and a diacylglycerol analog inhibit hen granulosa cell steroidogenesis.

We have previously reported that treatment of hen granulosa cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), or the diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), attenuates the steroidogenic response to luteinizing hormone (LH) at sites both prior and distal to the formation of cyclic 3',5'-adenosine monophosphate (cAMP). The present study was designed to determine the site(s) of inhibition within the steroidogenic pathway by evaluating the effects of OAG and PMA on key enzyme systems involved in hen granulosa cell steroidogenesis: adenylyl cyclase, phosphodiesterase, the cholesterol-side-chain-cleavage (CSCC) complex and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The adenylyl cyclase activator, forskolin (0.1 mM), stimulated a 3.3-fold increase in granulosa cell cAMP formation, and this increase was inhibited by the presence of OAG (2.5, 25 and 63 microM) in a dose-dependent manner. By contrast, a 1.8-fold increase in cAMP accumulation induced by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM), was not altered by OAG at any dose (2.5, 25 and 63 microM). Inclusion of 25-hydroxycholesterol (2500 ng/tube) in the incubation medium in the presence of 1.0 microM cyanoketone resulted in a 10-fold increase in pregnenolone production. Increasing concentrations of OAG (2.5, 25 and 63 microM) caused a dose-dependent suppression of the conversion of 25-hydroxycholesterol to pregnenolone. On the other hand, granulosa cells incubated with 200 ng/tube pregnenolone increased progesterone production 100-fold, but this increase was not inhibited by either PMA (3.2, 32, 8.1 and 162 nM) or OAG (2.5, 25 and 63 microM). The results indicate that activation of protein kinase C can suppress the function of at least two key enzymes involved in hen granulosa cell steroidogenesis. Inhibition of adenylyl cyclase greatly reduces the steroidogenic response of granulosa cells to endocrine factors that act via increasing levels of cAMP (i.e. LH). Furthermore, a reduction in CSCC activity limits the availability of precursor required for progesterone production. These data provide additional evidence of a role for protein kinase C in modulating ovarian function in the domestic hen.

Animals↗

PubMed1989

Regulation of androstenedione production by adenosine 3',5'-monophosphate and phorbol myristate acetate in ovarian thecal cells of the domestic hen.

Although factors that regulate cAMP and steroid production in granulosa cells of hen preovulatory follicles have been well studied, much less is known of the mechanisms that control steroidogenesis in the adjacent thecal layer. These studies were conducted to examine the involvement and interaction of cAMP and protein kinase-C in modulating androstenedione output from isolated ovarian thecal cells collected from the second largest preovulatory follicle. Treatment of thecal cells with ovine LH (0.01-100 ng/tube) caused a dose-dependent increase in androstenedione secretion. Although coincubation of cells with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1 mM) potentiated the effects of LH on steroid production, cAMP levels increased only in response to the higher doses of LH (10-100 ng/tube). Small but significant increases in cAMP accumulation and androstenedione production were observed in response to vasoactive intestinal peptide (0.1 and 1.0 microM), but not to 100 ng/tube chicken FSH, in the presence of 0.1 mM 3-isobutyl-1-methylxanthine. Treatment of thecal cells with cholera toxin (0.001-100 ng/tube) or forskolin (0.001-10 microM) resulted in a dose-dependent increase in cellular cAMP levels and androstenedione secretion. Thecal cell androstenedione production was also stimulated by the cAMP analog 8-bromo-cAMP (0.1-1.0 mM). Incubation of thecal cells with phorbol 12-myristate 13-acetate (PMA; 0.32-162 nM) or 1-oleoyl-2-acetylglycerol (OAG; 2.5-126 microM) increased basal steroidogenesis (progesterone and androstenedione production) in the absence of a rise in cAMP levels. By contrast, the stimulatory effects of 1 ng/tube LH on androstenedione, but not progesterone, production were attenuated by the presence of PMA (3.2-162 nM) or OAG (25-126 microM). Only a high concentration of OAG (126 microM) suppressed cAMP accumulation stimulated by LH (50 ng/tube). Phorbol ester treatment (32-162 nM PMA) also inhibited androstenedione production in thecal cells stimulated by the presence of 8-bromo-cAMP (1 mM), indicating a post-cAMP effect of protein kinase-C activity on steroidogenesis. In contrast to the effects of PMA, phorbol 13-monoacetate (162 nM), a nontumor-promoting analog of PMA which does not activate protein kinase-C, did not alter basal steroidogenesis, nor did it affect androstenedione secretion stimulated by LH or 8-bromo-cAMP. Data from the present studies indicate that the adenylyl cyclase-cAMP pathway can mediate the induction of thecal cell steroidogenesis by extracellular signals (i.e. LH and vasoactive intestinal peptide), whereas activated protein kinase-C can both stimulate and inhibit androstenedione production, depending upon the hormonal environment.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1988

Effects of vasoactive intestinal peptide on steroid secretion and plasminogen activator activity in granulosa cells of the hen.

Studies were conducted to evaluate the effects of vasoactive intestinal peptide (VIP) on steroidogenesis and plasminogen-activator (PA) activity in isolated granulosa cells of the largest preovulatory (F1) follicle of the hen. Vasoactive intestinal peptide, but not avian pancreatic polypeptide, the chicken VIP fragment (16-28) or the VIP congener, PHM-27, induced a dose-related increase in progesterone and androgen secretion, with an apparent median effective dose (ED50) of 5.9 X 10(-7) and 5.7 X 10(-7) M, respectively. The effects of VIP were, at least in part, mediated by the adenylyl cyclase system in that cotreatment of cells with VIP and the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), potentiated the steroidogenic effects. However, the time course of action for VIP on steroidogenesis was considerably slower than that for the gonadotropin, luteinizing hormone (LH), and this was attributed to a slower induction of cyclic adenosine 3',5'-monophosphate (cAMP) formation within granulosa cells. Finally, VIP was found to be a potent inhibitor of PA activity, and this inhibition was potentiated by coincubation of VIP with IBMX. We suggest that, in the hen, VIP has a direct and specific action on both steroidogenesis and PA activity, and that these actions are mediated, at least in part, by the adenylyl cyclase system. The comparatively slow induction of cAMP formation by VIP suggests that this peptide is involved in the control of cell differentiation and development rather than the ovulatory process.

1-Methyl-3-isobutylxanthine↗

PubMed1988

Attenuation of hen granulosa cell steroidogenesis by a phorbol ester and 1-oleoyl-2-acetylglycerol.

A series of studies was conducted to evaluate the effects of phorbol esters and a diacylglycerol analog on basal and hormone-stimulated steroidogenesis in granulosa cells from the largest preovulatory follicle of the domestic hen. Agents that previously have been shown to activate protein kinase C, such as the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the synthetic diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), suppressed luteinizing hormone (LH)-induced progesterone (PMA at levels of 10 and 100 ng/tube; OAG at levels of 10 and 25 micrograms/tube), and androgen (10 and 100 ng PMA; 25 micrograms OAG) production, but had no effect on basal levels of either steroid. Furthermore, PMA decreased the ability of vasoactive intestinal peptide to induce steroidogenesis, suggesting that protein kinase C activation may generally modulate the activity of hormones that act via the adenylyl cyclase/cyclic 3',5'-adenosine monophosphate (cAMP) second messenger system. In further support of this proposal was the finding that PMA and OAG decreased the production of cAMP in response to LH, and attenuated the steroidogenic response in granulosa cells exposed to 10 mM 8-bromo-cAMP. By contrast, the induction of calcium mobilization using a calcium ionophore (A23187; 0.5-2.0 microM) stimulated progesterone and androgen production without increasing intracellular levels of cAMP, and this stimulatory effect on steroidogenesis was not inhibited by the presence of 100 ng PMA/tube. From these data, we suggest that the activation of protein kinase C in granulosa cells of the hen may provide a physiological mechanism by which receptor-mediated steroidogenesis, involving the adenylyl cyclase second messenger system, is modulated.

Androgens↗

PubMed1988

Effects of a phorbol ester, a calcium ionophore, and 3',5'-adenosine monophosphate production on hen granulosa cell plasminogen activator activity.

Recent studies conducted in our laboratory have demonstrated that plasminogen activator (PA) is present in granulosa cells collected from the largest preovulatory follicle in the ovary of the domestic hen, and that its activity can be modulated by a variety of hormones in vitro. The present study was conducted to evaluate the intracellular mechanisms involved in the control of hen granulosa cell PA activity through the use of physiological and pharmacological agents. Treatment of granulosa cells with increasing doses (1, 10, and 50 ng/tube) of ovine LH resulted in a significant reduction of PA activity, which was accompanied by an increase in intracellular levels of cAMP. Furthermore, the effects of LH were potentiated by cotreatment with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1 mM). Exposure of cells to increasing concentrations of the adenylyl cyclase activator forskolin (0.005, 0.01, 0.05, and 0.1 mM) resulted in a significant reduction in PA activity at all doses given. Similarly, the presence of the cAMP analog 8-bromo-cAMP (0.005, 0.01, 0.05, 0.1, 0.5, 1.5, and 10 mM) caused a dose-dependent inhibition of PA activity from 0.005 to 1.0 mM, further suggesting the involvement of cAMP in the inhibitory regulation of hen granulosa cell PA activity. The induction of intracellular calcium mobilization through the use of the calcium ionophore A23187 (0.1, 0.5, 1, and 2 microM) resulted in a dose-dependent suppression of PA activity. By contrast, treatment of granulosa cells with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA; 0.5, 5, 10, 25, and 50 micrograms/tube), a compound that activates protein kinase-C, stimulated PA activity in a dose-dependent fashion; a non-tumor-promoting phorbol ester (phorbol 13-monoacetate; 0.5, 10, and 50 ng/tube) was without effect. Coincubation of granulosa cells with a submaximal dose of PMA (5 ng/tube) and low concentrations of A23187 (0.001, 0.005, 0.01, and 0.05 microM) could not significantly enhance the stimulatory effects of PMA on PA activity; however, higher concentrations of the ionophore (0.1, 0.5, and 1.0 microM) completely abolished PMA-stimulated PA activity. The stimulatory effects of PMA could also be eliminated by cotreatment with a protein kinase-C inhibitor (H-7; 100 microM), a mRNA transcription blocker (actinomycin-D; 5 micrograms/tube), or a protein synthesis inhibitor (cycloheximide; 50 micrograms/tube).(ABSTRACT TRUNCATED AT 400 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1987

Presence and hormonal control of plasminogen activator in granulosa cells of the domestic hen.

Plasminogen activator (PA) has been implicated in the control of ovarian cell differentiation and in the process of follicular rupture at the time of ovulation in rats. This study was conducted to evaluate whether PA activity is present in granulosa cells of the largest preovulatory (F1) follicle in the ovary of the domestic hen, and if so, to examine its hormonal regulation. An in vitro assay system, which measures the plasmin-mediated lysis of a chromogenic substrate, S-2251, was validated for use with granulosa cells of the hen to assess levels of both cell-associated and secreted PA. Chicken luteinizing hormone (cLH) suppressed PA activity in a dose-dependent fashion, with the highest dose (100 ng/ml incubation medium) resulting in levels that were 21.8% (cell-associated) and 50.9% (secreted) those of basal values (overall mean of 5 separate assays). Similarly, the ovarian steroid, testosterone, at the lowest dose administered (2 ng/ml medium), was found to inhibit both cell-associated and secreted PA activity (29.0% and 73.0% versus basal, respectively; N = 5 assays), whereas progesterone at the highest dose (50 ng/ml media) significantly depressed only cell-associated PA activity (71.9%), but not secreted PA, compared to basal levels (N = 5). By contrast, prostaglandin (PG) E1 or PGE2 at 10(-6) M, stimulated both cell-associated (a 20.9% and a 32.1% increase, respectively, compared to basal levels) and secreted (a 17.1% and a 28.3% increase, respectively, compared to basal) PA activity (N = 5); however, PGE2 at 10(-6) M, when combined with cLH at 100 ng/ml, was only partially effective in reversing the inhibitory action of cLH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Detection of apoptosis in human and rat ovarian follicles.

OBJECTIVE: The purpose of this study was to determine whether cells acquired from individual human preovulatory follicles undergo apoptosis (physiologic cell death) and, if so, to correlate the degree of apoptosis with characteristics of the follicles or the oocytes derived from the follicles. METHODS: We devised a sensitive nonradioactive method for detecting apoptotic DNA fragmentation in small numbers of cells derived from rat atretic follicles and follicular aspirates of patients undergoing assisted reproductive technologies. RESULTS: Using this method, apoptotic DNA was detected in rat atretic follicles, with optimal detection at 10-100 ng. Furthermore, apoptotic DNA was detected in some, but not all individual human follicular aspirates from several patients, and was found in follicles that produced oocytes that fertilized and developed into embryos. CONCLUSION: Apoptosis occurs in cells from human ovarian preovulatory follicles and may be a normal physiologic process of the follicle during luteinization.

Animals↗

PubMedDate not supplied

Models to elucidate the regulation of adrenal cell death.

Hypophysectomy-induced apoptosis in the rat adrenal gland is slow (not apparent until 12-24 h) and in-situ, 3'-end labeling (ISEL) of DNA strand breaks is restricted to a subpopulation of zona reticularis cells. In addition, it is completely blocked by ACTH. By contrast, apoptosis in the intact rat adrenal gland, cultured in the absence of trophic support, is extensive and rapid. Culture-triggered apoptosis (as determined by oligonucleosome formation) is attenuated by ACTH and is largely restricted to the zonae fasciculata and reticularis even at 3 h (as determined by ISEL and DAPI cytochemistry). Thus, this organ culture system may help elucidate factors that can acutely regulate adrenocortical cell survival. Quartered glands have a nearly 2-fold increase in oligonucleosome formation compared to intact glands at 3 h and are resistant to the antiapoptotic action of ACTH. In contrast, ACTH-induced corticosterone secretion is not attenuated. Angiotensin II (Ang II) enhances culture-triggered apoptosis, and its apoptotic action is attenuated by ACTH. These observations suggest that 1) acute hormonal modulation of apoptosis may require some level of gross adrenal structural integrity, and 2) ACTH and Ang II act in an antagonistic fashion to regulate adrenocortical apoptosis. The apoptotic effect of Ang II may be mediated via the type 2 receptor.

Adrenal Glands↗