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M Shemesh

Publications and source records attributed to M Shemesh.

At least 37 records · Page 2Linked to original sources
PubMed1990

Effect of beta-endorphin on steroidogenesis by bovine luteal cells.

To determine if opioid peptides have a local effect on the modulation of progesterone (P4) synthesis, a study was made of the effect of beta-endorphin and leu-enkephalin on P4 production by pure preparations of small luteal cells and dissociated luteal cells comprising both small and large cells from cows 2-3 months pregnant. Corpora lutea were dispersed by collagenase, and the large and small luteal cells were separated using Percoll gradients. Viable luteal cells (5 x 10(5)) were incubated in 0.5 mL of Eagle medium for 2 h at 37 degrees C, in an atmosphere of 5% CO2. Cells were treated with 8-bromoadenosine 3',5'-monophosphate (8Br-cAMP), hCG, beta-endorphin (BE) and leu-enkephalin (LE) alone or in combination. When small luteal cells were used, P4 synthesis was significantly enhanced in the presence of opioid peptides alone (P less than 0.01); there was an additive effect with 8Br-cAMP and with hCG. For dissociated luteal cells, opioid peptides alone had no effect on P4 production but the stimulation of P4 production induced by 8Br-cAMP or hCG was significantly (P less than 0.01) inhibited in the presence of opioid peptides. In contrast, dissociated luteal cells that were preincubated with PGF2 alpha (degranulation) responded to the presence of BE with increased P4 synthesis similar to that seen with the pure preparation of small luteal cells. It is concluded that opioid peptides play an auto/paracrine role in both basal and tropic hormone-induced stimulation of steroidogenesis by the bovine luteal cell.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Ultrastructural localization of cytochrome P-450scc in the bovine placentome using protein A-gold technique.

We have previously reported that the steroidogenic activity of the bovine placentome is stimulated by a calcium-mediated, cyclic nucleotide-independent mechanism and that this steroidogenesis is limited by the availability of sterol substrate to the side-chain cleavage enzyme. We have recently established that the antibody against bovine adrenal cytochrome P-450 cholesterol side-chain cleavage enzyme (P-450scc) can be used to specifically detect P-450scc in both bovine placentome and corpus luteum. In the present study, we used an immunogold technique to localize the P-450scc in the bovine placentome by electron microscopy. The mononucleate cell of the cotyledon showed both giant and normal-sized mitochondria, with the latter, predominating. Both mitochondrial types found in the mononucleate cells clearly displayed gold particles located on the cristae; in contrast, these particles were absent in the binucleate cells. It is worth noting that giant mitochondria were found exclusively in the placental mononucleate cells in both the fetal and maternal sites but not in the binucleate cells. These findings suggest that the cholesterol side-chain cleavage enzyme is present in bovine cotyledon cells, primarily in mononucleate cells. The variations in P-450scc immunoreactivity among different cells of the placenta are suggestive of different steroidogenetic capacities of the cells.

Animals↗
PubMed1989

Regulation of side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase by Ca2+ second messenger and protein kinase C systems in the placenta of the cow.

The steroidogenic activity of the bovine placenta is not modulated by cyclic nucleotide-mediated mechanisms. However, both translocation of intracellular Ca2+ and influx of extracellular Ca2+ activate the side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase. Protein kinase C activation in concert with Ca2+ mobilization also activates the side-chain cleavage enzyme. Cholesterol availability is a rate-limiting factor. Using polyclonal antibodies against bovine adrenal cytochrome P-450scc, the presence of P-450scc was demonstrated in both placental and luteal tissues. The cytochrome P-450scc was then localized, using gold-staining electron microscopy, in the mononuclear cells but not the binuclear cells of the placentome. The results suggest that cholesterol is metabolized by the mononuclear cell to pregnenolone, where it is further metabolized to progesterone by the mononuclear and binuclear cells.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Partial purification of a luteotrophic substance from bovine fetal cotyledon granules.

To determine whether luteotrophic activity is present in the bovine placental granules, fetal cotyledons from fetuses of 50-100 days of gestation were used. Enriched granules were prepared using a Percoll gradient. Active substances were obtained from the granules by freeze-thawing. The extracts thus obtained were then eluted on a Sephacryl S-300 column. The resultant fractions were then analysed by (1) a radioreceptor assay for hCG-like substances and (2) a bioassay using progesterone production by bovine luteal cells. There were two peaks of activity, one indicative of a high molecular weight substance and the second of a low molecular weight substance. Higher molecular weight substances were eliminated by using acidic extracts. The low molecular weight fraction was further analysed using reverse phase h.p.l.c. (acetonitrile:water gradient). The elution of this substance at 45% acetonitrile resulted in a 100-fold increase in luteotrophic activity in the bioassay compared to the Sephacryl fraction. The small molecular weight substance is heat-stable and not extracted to the organic phase when partitioned between methanol and chloroform.

Animals↗
PubMed1988

Control of bovine placental progestin synthesis: calcium dependent steroidogenesis is modulated at the site of the cholesterol side chain cleavage enzyme.

We have previously reported that progesterone synthesis in the bovine placenta is regulated by Ca2+ dependent and cyclic nucleotide independent mechanism. In studies conducted to further define the role of Ca2+ in the synthesis of progestins in bovine placental tissue, it was found that both protein kinase C (PKC), as determined by phosphorylation, and cytochrome P-450 side chain cleavage, as determined by Western blot analysis, were detectable in the steroidogenetically active portion of the placentome. To determine the site of action of PKC, fetal cotyledon cells were incubated in media containing 25-hydroxycholesterol in the absence or or presence of 10 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (TPA). It was found that TPA significantly (P less than 0.05) increased the conversion of the exogenous cholesterol analog to progesterone. To determine if the TPA could act synergistically with calcium activators, fetal cotyledon cells were incubated with either methyl isobutyl xanthine (MIX), an activator of intracellular calcium, or the calcium ionophore, A23187, which increases extracellular calcium influx, or both of these agents, in the presence or absence of TPA. It was found that TPA synergistically increased the conversion of sterol to progestins induced by submaximal concentrations of either MIX or A23187. In the presence of both compounds, TPA induced an even more dramatic increase in progestin synthesis. In experiments in which cyanoketone, an agent that inhibits the conversion of pregnenolone to progesterone, was added, TPA addition resulted in increased pregnenolone production, indicating that side chain cleavage of cholesterol is the site of action. The data, therefore, suggest that: (a) Ca2+ affects mechanisms regulating placental steroidogenesis; (2) one locus of Ca2+ is the cholesterol side chain cleavage reaction; and (3) PKC found in this tissue has a role in the Ca activated progestin production.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Control of bovine placental progesterone synthesis: roles of cholesterol availability and calcium-activated systems.

It was previously reported that dispersed bovine placentome secretes progesterone and that the steroidogenic activity of these cells is stimulated by a calcium-mediated, cyclic nucleotide independent mechanism. In the present study, the influence of substrate availability was explored and the roles of calmodulin and protein kinase C in progestin production examined. Incubation of dispersed fetal cotyledon cells with 25-hydroxycholesterol (25-OH-C), a soluble sterol which readily enters cells and is metabolized to steroid hormones, increased progesterone secretion in a dose-dependent manner. The response to 25-OH-C was dependent on the extracellular calcium concentration. Methyl isobutyl xanthine (MIX) alone also increased pregnenolone as well as progesterone secretion, and the combination of 25-OH-C and MIX stimulated progesterone secretion was inhibited by trifluoperazine. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused no major effects on steroidogenesis but the stimulatory effects of MIX or the ionophore A23187 were enhanced in its presence. These findings suggest that (1) basal progesterone secretion by fetal cotyledon cells is limited by cholesterol availability; (2) MIX increases steroidogenesis in part by increasing the synthesis of pregnenolone, but its actions are expressed independently of cholesterol availability; (3) both calmodulin and protein kinase C may participate in the modulation of bovine placental steroidogenesis.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Calcium-dependent, cyclic nucleotide-independent steroidogenesis in the bovine placenta.

Dispersed bovine placental cells (fetal cotyledon and maternal caruncle) were shown to synthesize progesterone. To determine if their steroidogenic activity could be modulated by a cyclic nucleotide-mediated process, we added luteinizing hormone, 8-bromoadenosine 3',5'-monophosphate, 8-bromoguanosine 3',5'-monophosphate, adenosine, or cholera toxin to dispersed cells from placentomes of 100-283 days gestational age and examined progesterone synthesis during 3-to 16-hr incubation periods. Net progesterone production, defined as the amount of progesterone released in excess of the zero-time cellular progesterone content, was determined by using a specific RIA. None of these agents significantly affected progesterone synthesis. In contrast, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (MIX; 0.2-0.5 mM) caused a marked increase in progesterone formation. In time course studies it was found that MIX produced a 5-fold increase in progesterone production in 16 hr, with steroid production increasing linearly during this time. MIX also increased the conversion of exogenous pregnenolone to progesterone by placental cells. In view of the failure of cyclic nucleotide analogues and activators of adenylate cyclase to stimulate steroidogenesis, it was necessary to consider other modes of action of MIX. Since MIX is known to affect intracellular calcium translocation, we examined the effects of the calcium ionophore A23187 on progesterone formation. This drug enhanced progesterone formation and augmented the stimulatory effects of MIX. The stimulatory action of A23187 was not affected by cyclic nucleotide analogues. Our data suggest that progesterone synthesis in the bovine placentome is calcium dependent and cyclic nucleotide independent.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Foetal testicular steroidogenesis and responsiveness to LH in freemartins and their male co-twins.

Foetal gonads were obtained from 1) 8 male foetuses that were co-twin to freemartins, 2) 8 isosexual twins, 3) 59 singletons of 45-75 days of gestations, 4) 5 isosexual and 5 male co-twins of 90-120 days and 5) 5 freemartins at 70-120 days of gestation. The gonads were incubated in supplemented medium 199 for 24 h in the absence or presence or LH and the testosterone and progesterone produced measured by RIA. During the time of sexual differentiation (45-75 days) the testes of the male co-twins produced significantly less testosterone than testes from isosexual or singletons of the same age. Testes of foetuses older than 90 days were refractory to LH-stimulation, but freemartin ovaries and testes from their male co-twins continued to respond to LH with increased testosterone production. No significant differences in progesterone production were detected amount co-twins, isosexual twins or singletons at any age. We conclude that the testes of the male twin that is co-twin to a freemartin displays abnormal steroidogenesis. This may be related to reports of abnormal testes after birth in males co-twin to freemartins.

Animals↗
PubMed1984

Modulation of bovine placental prostaglandin synthesis by an endogenous inhibitor.

An endogenous, heat-labile, inhibitor of prostanoid synthesis in maternal caruncle tissue of bovine placentomes was studied. Inhibitory activity was present in caruncle extracts from days 120-250 of gestation, but was not detectable at term (260-280 days). The disappearance of inhibitory activity coincided with an increase in the secretion of prostanoids by dispersed caruncle cells in culture. Coculture of caruncle cells from placentomes of 120-day gestation with fetal cotyledon cells resulted in suppression of prostanoid synthesis by the cotyledon cells. However, this inhibition was not observed in cocultures of dispersed caruncle cells and fetal cotyledon cells from term placentomes. Our findings indicate that an endogenous inhibitor modulates bovine placental prostaglandin synthesis. A decline in the level of this inhibitor at term may be one factor triggering increased prostanoid synthesis required for parturition.

Animals↗
PubMed1984

The role of 17 beta-estradiol in the recovery from oviductal prolapse in layers.

Plasma 17 beta-estradiol concentrations, thecal estrogen content, and uterine prostaglandin synthetase activity were measured in healthy and prolapsed hens as well as in layers that recovered after exposure to low intensity lighting (250 or 50 lx). The effect of estradiol benzoate injections (100 ng, 3 X per week) in hens exposed to high intensity light (greater than 500 lx) was also studied. Prolapsed hens had significantly lower plasma 17 beta-estradiol concentrations (60 +/- 12 pg/ml; mean +/- SEM) than recovered (374 +/- 40 pg/ml) or healthy hens (475 +/- 45 pg/ml). Theca cells from recovered hens had a significantly higher content of 17 beta-estradiol (.7 ng/5 X 10(5) cells) than theca cells from normal or prolapsed birds (.3 ng/5 X 10(5) cells). Microsomes prepared from the uteri of prolapsed hens converted significantly less arachidonic acid to prostaglandin metabolites (4.4%) than did microsomes from healthy or recovered birds (9.0%). Treatment of prolapsed hens with estradiol benzoate resulted in 89% of the birds recovering within 3 weeks compared to a 4% recovery rate in the controls. We conclude that restoration of peripheral 17 beta-estradiol concentrations to normal levels was concomitant with recovery in prolapsed birds, and suggest that the estrogen exerts its effect by raising the level of prostaglandin synthetase activity in the uterus.

Animals↗
PubMed1983

Partial purification of a chorionic gonadotropin-like protein from bovine cotyledons.

The presence of placental gonadotropin activity in early pregnancy was studied in the cow. Cotyledons from the first trimester of gestation were used to isolate a chorionic gonadotropin-like (CG-like) protein by means of ammonium sulfate precipitations (0.17-4.5 M) and gel filtration on Sephadex G-100 and DEAE-Sephadex. The CG-like activity was measured by a radioreceptor assay (RRA) specific for human chorionic gonadotropin (hCG). Luteotrophic activity was also assessed in two separate in vitro bioassays using cultures of bovine granulosa and rat Leydig cells. It was found that the 3 M ammonium sulfate precipitate of cotyledon extracts contained a CG-like activity of 179 mIU/mg protein as measured by RRA. The precipitate inhibited 125I-hCG binding in a linear fashion. A ninefold increase in specific activity was achieved by further purification on Sephadex G-100 (800 mIU/mg protein) and Sephadex-DEAE (1600 mIU/mg). In experiments with cell cultures, it was found that 20 micrograms of protein eluted from Sephadex-DEAE (equivalent to 16 mIU of hCG as determined by RRA) added to the culture medium significantly enhanced (P less than 0.05) progesterone production by the granulosa cells and testosterone production by the Leydig cells. We conclude that the bovine placenta contains a chorionic gonadotropin which may serve to maintain the corpus luteum of pregnancy.

Animals↗
PubMed1983

Bovine placentomes contain factors which decrease progesterone secretion.

We examined the effects of 20% ammonium sulfate precipitates from cytosolic extracts of whole placental tissue collected between 100-150 days of gestation on progesterone secretion by bovine granulosa cells and dispersed bovine luteal cells. These extracts produced a dose-dependent inhibition (23-92%) of progesterone synthesis by bovine granulosa cells. However, no inhibitory activity could be demonstrated in similarly prepared extracts from term placentae. Inhibitory activity could be extracted from both maternal caruncles and fetal cotyledons. In the presence of 2 mg/ml of maternal caruncle extract, basal progesterone secretion was dramatically reduced (90%), as was steroidogenesis in the presence of bovine lutenizing hormone (bLH) and 8 bromocyclic (Br)-cAMP. Moreover, coincubation of dispersed luteal cells and dispersed fetal or maternal placental cells from 100- to 150-day placentae produced a significant (50%) reduction in progesterone content of the medium. The addition of 2 mg/ml of caruncle or fetal cotyledon extract from 100- to 150-day placentae also produced 100% and 50% inhibitions, respectively, of progesterone secretion by dispersed placental cells. Thus, the inhibitory factor appears to be produced by cells of both the maternal and fetal placenta. It is heat-stable and not extractable by ether. The inhibitory substance eluted was two distinct peaks from Sephadex G-100 columns, one with a molecular weight of about 60,000 daltons and the other about 30,000 daltons. Using isoelectric focusing, several peaks of inhibitory activity were obtained, one with a pI of 3-5, the others having pIs between 6 and 9.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Isolation and characterization of a 94,000-dalton protein with thyrotropic activity from early bovine placenta.

A thyrotropic protein was extracted and purified from the placenta of early bovine gestations. After protein extraction, the 45-60% ammonium sulfate precipitate of maternal and fetal bovine cotyledons was found to compete with thyroid stimulating hormone (TSH) for binding to thyroid cell membranes and to mediate TSH specific biological effects including the stimulation of cyclic AMP production, iodide uptake, and thyroxine secretion. The placental thyrotropin was further purified by gel and anion exchange chromatography, followed by binding to thyroid cell membranes and elution by mild acid treatment. 400 micrograms of isolated protein with 4.5 units of TSH-like binding activity/mg of protein was recovered from the placenta of a 90-day-old bovine gestation, representing 2 X 10(-4%) of its original wet weight. The placental thyrotropin appeared to be a 94,000-dalton protein with pI 6.0 and composed of two noncovalently associated chains of 50,000 and 44,000 daltons. The placental 94,000-dalton thyrotropin bound to TSH membrane receptors and induced specific TSH-mediated biological effects, but was structurally and immunologically distinct from TSH and hypophysical or placental gonadotropins.

Animals↗
PubMed1982

The interaction of cultured thyroid cells of early bovine embryos with thyrotropin and thyroglobulin.

We have prepared primary thyroid cell cultures of early bovine embryos from the first trimester of pregnancy in order to study the ontogeny of their interaction with TSH and thyroglobulin (Tg). The ability of these cells to synthesize and secrete Tg, as well as the trophic effect of TSH on the organization of the thyroid cells, were also investigated. To determine the maturation of these functions we prepared fluorescent conjugates of TSH, Tg, and anti-Tg antibodies, and visualized their interaction with the thyroid epithelial cells. Our study shows that the ability to bind TSH and Tg exists as early as the gestational age of 3 cm crown-rump length (CRL; 40 days) but does not develop linearly with embryonic age. Thus, there is a significant increase in the percentage of Tg-binding cells at 12 cm CRL, when colloid is first noticed in vivo, and a considerable elevation in TSH-binding cells around 15 cm CRL, when thyrotropic cells and TSH secretion from the fetal pituitary are first evident. Tg-containing cells and the ability to secrete Tg are observed at about 20 intrauterine days. The three thyroidal properties probably develop independently since only part of the Tg-containing cells bind Tg or TSH, and a significant proportion of the cells that exhibit Tg-binding do not bind TSH. The results support the notion that TSH is essential for the formation of follicle-like structures and effects the organization of thyroid cells into a functional structure in vitro from the late precolloidal stage.

Animals↗
PubMed1981

The ontogeny of the thyrotropin-thyroid axis in early bovine embryos.

T4 and T3 formation and their response to TSH, cAMP, and prostaglandin E2 (PGE2) stimulation were studied by RIA in cultured bovine fetal thyroids from 130 embryos of 1.2-25.0 cm crown-rump length (CRL). T4 and T3 were found in all of the freshly isolated glands studied, and their concentrations increased significantly (P less than 0.05) during a 24-h incubation of glands from fetuses with a CRL greater than 8.0 cm. The release of T4 (nanograms per mg tissue), but not of T3, increased consistently with CRL (r = 0.64; P less than 0.05). The addition of TSH (0.5 mU) to the culture medium induced a 2- to 3-fold increase in the secretion of T4, but not of T3, by glands of fetuses with a CRL of 3.0-25.0 cm (P less than 0.05). Dibutyryl cAMP (10(-4) M) and PGE2 (10(-4) M) had comparable effects. A combination of TSH and theophylline (1 mM) or of TSH and dibutyryl cAMP significantly enhanced the T4 released by cultured tissue into the medium (P less than 0.05) over that induced by either agonist, but the combined effect was not fully additive. The total PGE2 in the tissue and medium was not changed by the addition of 0.5 mU TSH, and indomethacin had no effect on TSH-induced T4 secretion. The data show that thyroids of fetuses that have reached a CRL of 3.0 cm have the enzymatic capacity to produce both T4 and T3, and T4 is the dominant product formed. While exogenous PGE2 at high concentrations stimulates fetal T4 secretion, it does not mediate the actions of TSH and cAMP on the fetal thyroid.

Adenosine↗
PubMed1981

Iodine metabolism and the effect of TSH in thyroid glands of early bovine embryos.

The ontogeny of the thyrotrophin-thyroid axis during the first trimester was studied in 104 bovine embryonic thyroids taken from foetuses of crown-rump length 1.4 to 19.2 cm (25-120 days). The uptake of labelled iodine in vitro in the absence or presence of TSH was measured. The per cent incorporation of radioiodine into iodotyrosines and iodothyronines in the presence and absence of TSH was also studied. It was found that the foetal tissue displayed radioiodine uptake by 25 days of foetal life and the uptake increased with age. TSH caused a further increase in radioiodine uptake in foetuses of 40 days or older. Incorporation of radioiodine into MIT and DIT was apparent at 25 days and into T3 and T4 by 40 days of foetal life. Addition of TSH increased the proportion of total radioiodine found as DIT and thyroxine in foetuses of 40 days older. This TSH stimulation of radioiodine incorporation increased with age. However, the proportion of radioiodine found as MIT and T3 was not affected before 120 days of foetal life. This was in marked contrast to the adult thyroid where the proportion of radioiodine found as T3 was increased by the addition of TSH. It is concluded that the foetal thyroid can respond to TSH by at least 40 days of foetal life and that this response differs from that seen in the adult.

Animals↗