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M R Luck

Publications and source records attributed to M R Luck.

At least 37 records · Page 2Linked to original sources
PubMed1991

Beta adrenoceptors mediate the catecholamine-induced stimulation of oxytocin secretion from cultured bovine granulosa cells.

Bovine granulosa cells were treated in culture with alpha- and beta-adrenoceptor ligands to determine the receptor subtype mediating their response to catecholamines. The secretion of oxytocin by granulosa cells in serum-free medium was measured on the fourth day of culture (during the period of acquisition of a luteal phenotype). Cultures were performed in the presence of 0.5 mM ascorbic acid, which increased hormone output and potentiated the response to catecholamines. The effects of adrenaline and noradrenaline on oxytocin secretion were concentration-dependent; maximum stimulation was over 700% with adrenalin (EC50 92 nM) and 500% with noradrenaline (EC50 87 nM). The response to noradrenaline (10(-6) M) and adrenaline (10(-6) M) could be blocked by propranolol but not by phentolamine, suggesting that beta- rather than alpha-adrenoceptors were involved. Blockade by metoprolol and practolol (beta 1-adrenoceptor antagonists) was poor and dobutamine (beta 1-agonist) was weakly stimulatory. A concentration-dependent stimulatory response (EC50 200 nM) was obtained with salbutamol (beta 2-adrenoceptor agonist) and stimulation by adrenaline or salbutamol could be blocked by a selective beta 2-adrenoceptor antagonist (ICI 118,551). It is concluded that, during luteinization, the long-term response of bovine granulosa cells to stimulation induced by catecholamines is mediated through beta- rather than alpha-adrenoceptors. Although the beta 2-subtype is probably involved, the similar potencies of adrenaline and noradrenaline are uncharacteristic of beta 2-adrenoceptors and may be peculiar to the long-term response shown by these cells.

Adrenergic alpha-Antagonists↗
PubMed1990

Secretion and gene expression of inhibin, oxytocin and steroid hormones during the in vitro differentiation of bovine granulosa cells.

Bovine granulosa cells were cultured under defined conditions to examine (1) their secretion of immunoreactive inhibin, oxytocin, progesterone and oestradiol during differentiation in vitro; (2) their expression, by Northern analysis, of specific mRNAs for inhibin and oxytocin as compared with uncultured cells; (3) possible interrelationships between the four secreted hormones; and (4) the hypothesis that androgens and steroidogenesis influence the secretion of inhibin. The secretion of inhibin and oestradiol fell rapidly over the first few days of culture but remained at detectable levels for at least 7 days. Conversely, the secretion of oxytocin and progesterone rose steadily as culture progressed. These changes occurred spontaneously (i.e. without gonadotrophin treatment) and were not dependent on the addition of serum to the culture medium. Messenger RNAs for the inhibin alpha- and beta A-subunits were present in uncultured cells but barely detectable or undetectable in cells cultured for 4 days. Conversely, the mRNA for oxytocin, which was not detectable in uncultured cells, was present in cultured cells and increased in quantity as culture progressed. Treatment of cells with testosterone (5 nM-5 microM), in the presence or absence of serum (10% FCS), had no effect on the secretion of inhibin but stimulated the declining oestradiol secretion. Treatment with ascorbic acid (0.5 mM) increased the secretion of oxytocin and progesterone, as previously described, but not that of inhibin. Treatment with aminoglutethimide (0.5 mM), an inhibitor of steroidogenesis, substantially inhibited progesterone secretion and the response of oestradiol secretion to testosterone, but had no effect on the secretion of either inhibin or oxytocin. We conclude that bovine granulosa cells differentiate spontaneously in defined culture in a manner that, as defined by the secretion of steroid and peptide hormones, closely resembles their luteinization in vivo. The switch in protein hormone secretion from inhibin to oxytocin is accompanied by a corresponding change in mRNA expression. The changes in steroid and peptide hormone secretions that take place in culture appear to occur independently of one another although their absolute cause remains to be determined. In contrast to previous studies, we could find no evidence for the regulation of inhibin secretion by either androgens or steroidogenesis.

Animals↗
PubMed1990

Cholinergic stimulation, through muscarinic receptors, of oxytocin and progesterone secretion from bovine granulosa cells undergoing spontaneous luteinization in serum-free culture.

Bovine granulosa cells were cultured in a defined serum-free system to examine their responsiveness to acetylcholine (ACh). Continuous exposure to concentrations of ACh between 10(-8)-10(-4) M resulted in dose-dependent increases (up to 6.7-fold) in the secretion of oxytocin and progesterone, with an ED50 of 6.6 microM. Ascorbic acid (0.5 mM), a known stimulator of granulosa secretion, synergized with ACh, resulting in an increase in the amounts of hormone secreted and a 7-fold increase in cellular sensitivity to ACh (ED50 = approximately 0.9 microM). Treatment of cells with ACh for 24 h at various times during a typical 5-day culture resulted in a stimulation that persisted for up to 4 days after removal of ACh. Carbachol (10(-8)-10(-4) M), a receptor antagonist with both antimuscarinic and antinicotinic actions, had no distinct effect on hormone secretion by the cells, but the effects of 10(-5) M ACh could be completely abolished by equimolar or hypomolar concentrations of the specific muscarinic receptor antagonists atropine and scopolamine. Nicotine bitratrate (10(-8)-10(4) M), a dose-dependent nicotinic receptor agonist/antagonist, had no effect on the cells. It is concluded that bovine granulosa cells, exhibiting a luteinized phenotype in culture, are responsive to cholinergic agonists in a specific and saturable manner. The response of the cells is probably mediated through muscarinic receptors and has both medium and long term (persistent) components. These results indicate that cholinergic neurotransmitters may play a direct role in the regulation of ovarian function in the ruminant.

Acetylcholine↗
PubMed1989

Greatly elevated and sustained secretion of oxytocin by bovine granulosa cells in serum-free culture.

Bovine granulosa cells were cultured in collagen (Vitrogen 100)-pretreated wells using defined medium to study the secretion of oxytocin and progesterone under serum-free conditions. Secretion of oxytocin began spontaneously after the first day and was maintained at a high level during a 1 week culture period. Addition of serum to the medium reduced oxytocin concentrations by up to 90%. There were positive exponential relationships between oxytocin and progesterone concentrations and the inoculated cell density (range, 1.67 to 23.4 X 10(5) cells/ml/well). The results indicate that neither serum nor gonadotrophins are required for in vitro differentiation of bovine granulosa cells and that addition of serum may attenuate subsequent hormone secretion. This culture system should provide a better in vitro model for the study of ovarian oxytocin secretion than those previously described.

Animals↗
PubMed1989

Complete sequence of the coding region of human elongation factor 2 (EF-2) by enzymatic amplification of cDNA from human ovarian granulosa cells.

The use of two primers allowed the specific enzymatic amplification of elongation factor 2 starting with total double-stranded cDNA from human ovarian granulosa cells. The amplified DNA fragment with a length of 1765 bp was restricted and sequenced by the shot gun approach. From the sequences obtained from the amplified fragment and the cDNA insert of pHGR81 [Rapp et al. (1988) Biol. Chem. Hoppe-Seyler 369, 247-250] respectively, the DNA sequence containing the complete coding as well as the 3'-untranslated region was assembled.

Animals↗
PubMed1988

Ovarian oxytocin and progesterone are secreted independently of one another.

Experiments were performed with cultured bovine granulosa cells to examine the relationship between the secretions of oxytocin and progesterone and to determine whether progesterone could be responsible for the progressive refractoriness of these cells to stimulation by ascorbic acid. Aminoglutethimide suppressed progesterone secretion by 95% but it neither reduced oxytocin secretion nor restored the cellular response to delayed ascorbate treatment. Addition of a high concentration of progesterone to the culture medium also failed to affect oxytocin secretion, its stimulation by ascorbate, or the endogenous secretion of the steroid. It is concluded that oxytocin and progesterone can be independently secreted and that progesterone regulates neither its own secretion nor that of oxytocin.

Aminoglutethimide↗
PubMed1988

Enhanced secretion of oxytocin from bovine granulosa cells treated with adrenal steroids.

Bovine granulosa cells were exposed in vitro to various adrenal steroids (cortisol, cortisone, corticosterone, aldosterone; 1 mumol/l), in the presence and absence of stimulation by ascorbic acid (0.5 mmol/l), to determine the possible effects of these hormones on ovarian oxytocin and progesterone secretion. Only cortisol produced a consistent stimulation of the cells; the response was dose-related over the range 0.01 to 1.0 mumol/l and was greatly enhanced in the presence of ascorbate. The secretion of oxytocin was stimulated to a greater extent and with more consistency than was that of progesterone. Although the secretion of oxytocin could be stimulated by cortisol on the day of treatment, the cells also showed a delayed and persistent response to exposure earlier in the culture. It is concluded that cortisol may directly stimulate the secretion of ovarian oxytocin in the cow and that granulosa cells may respond in such a way as to smooth out the effects of short-term fluctuations in cortisol concentration.

Adrenal Cortex Hormones↗
PubMed1988

Stimulation of progesterone secretion by cultured human granulosa cells with melatonin and catecholamines.

Granulosa cells, aspirated from the follicles of patients undergoing treatment for in-vitro fertilization, were cultured in serum-supplemented medium. Adrenaline and noradrenaline stimulated a dose-related increase in progesterone secretion with a maximum stimulation at 10(-5) M, a response that was prevented by the beta-antagonist, propranolol. Adrenaline and hCG showed similar characteristics in their stimulation of progesterone secretion but there was no further increase in progesterone when the 2 compounds were added together. Melatonin stimulated progesterone secretion and, like adrenaline, this stimulation was prevented by propranolol. The ability of both adrenaline and melatonin to increase progesterone secretion was dependent on the degree of follicular development, as determined by peripheral oestradiol concentrations, on the day of laparoscopy. These results suggest that adrenaline and melatonin may have a physiological role in modulating luteal function and that melatonin may act by a beta-adrenergic-related mechanism.

Cells, Cultured↗
PubMed1988

The time-course of oxytocin secretion from cultured bovine granulosa cells, stimulated by ascorbate and catecholamines.

Bovine granulosa cells secrete oxytocin when cultured in a serum-supplemented medium. The time-course of secretion is similar to that in the early corpus luteum in vivo, with a delay of 1 to 2 days followed by a peak and decline over the first 5 days of culture. We have investigated the basis of this time-course in vitro and studied the temporal characteristics of the stimulatory actions of ascorbic acid and adrenaline on this process. Cells cultured on stirred microcarriers showed a similar pattern of secretion of oxytocin to those cultured on conventional flat plates, despite continuing and rapid mitosis. This indicated that the secretion profile in conventional culture was not an artifact related to the cessation of mitosis. Furthermore, secretion of oxytocin and progesterone by cells on microcarriers was stimulated without a corresponding change in mitotic rate, showing that the secretion per cell had been increased. In conventional culture, addition of ascorbic acid to culture media (0.5 mmol/l) increased the secretion of oxytocin (up to 4.5-fold) but only if ascorbic acid was present during the first day of culture. The cells showed a progressive refractoriness to stimulation after 12 h. Since the time-course of secretion was unaltered by treatment, this resulted in a delay of 1 to 2 days before the action of the ascorbate was seen. The secretion of progesterone was similarly affected but with less stimulation and less consistency. In contrast, cells treated with adrenaline (10 mumol/l) secreted more oxytocin on the day of treatment and did so at any time during culture provided that there was sufficient basal secretion of hormone. Adrenaline also failed to alter the time-course of secretion but treated cells showed a persistent response, maintaining enhanced secretion for up to 3 days after the adrenaline had been removed. Ascorbate and adrenaline were highly synergistic in their effects, provided that the ascorbate was present from the start of culture; the response to adrenaline strongly reflected the degree of ascorbate stimulation. We conclude that granulosa cells secrete oxytocin according to an inherent time-schedule and that there is a limited period during which they can respond to ascorbate. Since ascorbate is required for the biosynthesis of oxytocin, this suggests that the availability of ascorbate during corpus luteum formation may determine the amount of oxytocin which can be released subsequently in response to catecholamines.

Animals↗
PubMed1987

Catecholamines and ascorbic acid as stimulators of bovine ovarian oxytocin secretion.

The effects of catecholamines and ascorbic acid on cultured bovine granulosa cells have been examined to assess their possible role in the initiation and maintenance of luteal oxytocin secretion. The actions of these agents have also been compared with the previously reported ability of follicular theca tissue to enhance oxytocin secretion. Using granulosa cells cultured in serum-supplemented medium, we observed a highly significant enhancement of oxytocin secretion in the presence of adrenaline and noradrenaline, particularly over the concentration range 1-10 mumol/l. This effect was accompanied by smaller and less consistent changes in progesterone secretion and did not involve any change in the time-course of oxytocin secretion. Acetylcholine was without effect. Ascorbic acid stimulated oxytocin secretion when used alone over a range of concentrations, but was also able to synergize with adrenaline. Lactic acid was ineffective. The stimulation of oxytocin secretion by adrenaline could be blocked by equimolar propranolol, but the stimulation of progesterone was not blocked. Propranolol had a variable effect on the ability of theca tissue to stimulate oxytocin secretion by granulosa cells but the results also suggested the presence of some beta-agonistic activity in the culture medium. We conclude, first, that catecholamines may be involved in the regulation of ovarian oxytocin secretion, secondly, that ascorbate may regulate oxytocin secretion through its involvement in the biosynthesis of oxytocin but also through interaction with catecholamines and, thirdly, that the stimulatory action of theca tissue probably does not involve the action of beta-agonists.

Animals↗
PubMed1986

Melatonin directly stimulates the secretion of progesterone by human and bovine granulosa cells in vitro.

Melatonin, at concentrations and periods of exposure reflecting those present during the circadian cycle, was investigated for its influence on steroid production by granulosa cells cultured in serum-supplemented medium. At high (200 pg/ml) but not low (20 pg/ml) physiological concentrations, melatonin significantly stimulated progesterone production by human granulosa cells. This response was independent of the overall level of cell activity and was seen under the different culture conditions associated with different culture media. Exposure to melatonin for 8 h significantly stimulated progesterone secretion to a level similar to that achieved under continuous exposure, and the effect was reduced to control levels during subsequent periods in which no melatonin was added. Melatonin had no consistent effect on aromatase activity in the conversion of stored or serum-available androgen to oestradiol. Melatonin significantly stimulated progesterone production by bovine granulosa cells in vitro, at concentrations similar to those present during the endogenous nocturnal rise (100-400 pg/ml). This response to physiological conditions by human and bovine cells suggests a role for melatonin in the regulation of progesterone production by the ovary.

Animals↗
PubMed1986

Evidence for granulosa-theca interaction in the secretion of oxytocin by bovine ovarian tissue.

We have examined the ability of granulosa cells, from carefully selected preovulatory bovine follicles, to secrete oxytocin in vitro. Although cells from 83% of follicles underwent functional luteinization (greatly increased progesterone secretion) in serum-supplemented culture, only 69% had cells capable of oxytocin secretion. Secretion followed a similar time course in all cultures, with the peak appearing on day 3. Oxytocin, but not progesterone, output could be consistently increased by addition of pieces of theca interna tissue, or theca conditioned medium, to the cultures. The effect could be achieved by exposure to theca tissue at any time prior to peak output without altering the time course of secretion. Oxytocin could not be detected in follicular fluid from any of the selected follicles, nor in medium from theca cultured alone. We conclude that the potential for oxytocin secretion is a feature of follicular maturation which is lost during atresia and that the stimulus to secretion is associated with luteinization but not with progesterone output. Finally, the intermixing of follicular cells during corpus luteum formation may provide a mechanism for the enhancement of oxytocin secretion within a predetermined time frame.

Animals↗
PubMed1982

Effects of an anti-androgen in the laying hen (Gallus domesticus).

The daily injection of the anti-androgen, cyproterone acetate, into regularly laying hens failed to prevent ovulation immediately. The delayed response suggested that testosterone is not part of the ovarian positive feedback stimulus resulting from the presence of an ovulable follicle and leading to ovulation. Ovarian changes in treated birds, and their unimpaired response to LH-RH, suggested that the drug might be acting by altering ovarian steroid metabolism.

Animals↗
PubMed1979

The relationship between reproductive activity and blood calcium in the calcium-deficient hen.

1. The dependence of reproductive activity in the laying hen upon adequate calcium intake has been investigated. 2. The response of plasma luteinising hormone concentration in calcium-deficient, as compared with calcium-replete hens, to injections of luteinising hormone releasing hormone and progesterone suggested that the primary site of reproductive dysfunction is the hypothalamus rather than the pituitary gland. 3. Laying hens, when presented with a calcium-deficient diet, ceased to lay as the plasma ionised calcium concentration decreased to less than 1.0 mM, supporting the view that there is a threshold of blood calcium activity below which reproductive activity ceases. 4. Supplementation of the diet with calcium produced an immediate restoration of plasma ionised calcium concentration to normal, despite an interval of a few days before the plasma total calcium returned to normal and egg laying resumed.

Animals↗
PubMed1979

The adverse effects of nicarbazin on reproductive activity in the hen.

The effects of the anticoccidial agent nicarbazin on reproductive activity in the female fowl have been studied. 2. The drug had no effect on the plasma concentration of luteinising hormone, but treated hens showed a reduced hypothalamic sensitivity to exogenous progesterone, whilst the capacity of the pituitary to respond to luteinising hormone releasing hormone was unimpaired. 3. It is suggested that nicarbazin not only prevents yolk deposition within the ovary but also adversely affects the stimulatory function of the hypothalamus, possibly through an unsuitable hormonal environment.

Animals↗