Effects of prostaglandins E-2 and F-2 alpha on the metabolism of [U-14C]glucose by mouse morulae-early blastocysts in vitro

During 5-h culture in the presence of radioactive glucose, PGE-2 (10 micrograms/ml) significantly inhibited incorporation of glucose into the acid-soluble glycogen pool. PGE-2 at 1 and 10 micrograms/ml and PGF-2 alpha at 1 microgram but not 10 micrograms/ml stimulated incorporation of glucose into non-glycogen macromolecules during culture. However, the utilization of acid-soluble glycogen and other biochemical pools was not affected by the presence of PGs in the medium during 24-h chase culture of pulse-labelled embryos. Carbon dioxide production was significantly suppressed in the presence of PGs but accumulation of lactate was not affected. The results indicate that PGE-2 and PGF-2 alpha, in physiological concentrations, directly influence the metabolism of glucose by preimplantation embryos.     

Response of porcine theca and granulosa cells to GH during short-term in vitro culture

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.     

Regulation of progesterone receptor mRNA by oestradiol and antioestrogens in breast cancer cell lines

The induction of progesterone receptor mRNA by oestradiol and antioestrogens has been characterised in the MCF-7 breast cancer cell line. Progesterone receptor mRNA was induced more than 100-fold by oestradiol. The induction was half-maximal in the presence of 10(-10) M oestradiol and maximum levels were reached after 24 h treatment. Progesterone receptor mRNA was induced to 10% of the oestrogen-induced level by tamoxifen and its metabolite 4'-hydroxytamoxifen. The increase was half-maximal in the presence of 5 X 10(-8) M tamoxifen or 5 X 10(-10) M 4'-hydroxytamoxifen. In contrast, neither the benzothiophene antioestrogen LY117018 nor the 7 alpha-alkyl steroidal antioestrogen ICI 164,384 had any effect on progesterone receptor mRNA. The progesterone receptor mRNA was also induced by oestrogen in a T47D subline and in two other oestrogen-responsive breast cancer cell lines (ZR-75, EFM-19). Tamoxifen was a partial oestrogen for progesterone receptor mRNA induction in each of these cell lines. The large induction of the progesterone receptor mRNA by oestrogen in all 4 breast cancer cell lines supports the contention that the progesterone receptor may be a good predictive marker of hormonal response in human breast cancer.     

Effects of coculture with uterine epithelial cells on the metabolism of glucose by mouse morulae and early blastocysts

Coculture of mouse morulae/early blastocysts with isolated endometrial epithelial cells reduced incorporation of glucose carbon into embryonic glycogen but had no significant effect on incorporation into other internal carbon pools during a 5-h culture in serum-supplemented Dulbecco's modification of Eagle's minimum essential medium. Turnover of glycogen pools during 24-h chase culture of pulse-labelled embryos was unaffected by the presence of uterine epithelial cells recovered from day-4 pregnant or non-pregnant mice. However, significantly more label was retained in non-glycogen macromolecules during chase in the presence of endometrium recovered from non-pregnant than from pregnant uteri.     

Non-steroidal inhibition of granulosa cell aromatase activity in vitro

Treatment of cyclic rats with the substituted triazole R151885 (1,1-di (4-fluorophenyl)-2-(1,2,4-triazol-1-yl)-ethanol causes delayed ovulation with suppressed blood oestradiol levels. To determine if R151885 can exert a direct action on ovarian oestrogen biosynthesis, we studied its effect on steroidogenesis in granulosa cell cultures from prepubertal rat ovaries. The cells were incubated for 48 h in medium containing 100 ng human FSH/ml and 10(-7) M testosterone to induce steroidogenic enzymes. When R151885 was also present in the culture medium, there was a marked and concentration-dependent reduction in granulosa cell oestradiol production. Inhibition was half-maximal at approx 3 X 10(-7) M and almost complete at 10(-5) M R151885. Progesterone and 20 alpha-hydroxy-4-pregnen-3-one production were unaffected except by the highest concentration of the substituted triazole (36% inhibition at 10(-5) M). Direct assessment of aromatase activity in the 48-h cultured monolayers (oestradiol formation during a 3-h incubation with 10(-7) M testosterone) was made to determine if the inhibitory effect of R151885 was due to reduced aromatase induction/activation. This was not the case, since cells cultured in the presence of 10(-6) or 10(-5) M R151885 had levels of aromatase up to 60% higher than those cultured in its absence. To determine acute effects of R151885 on testosterone (10(-7) M) aromatization, 3-h incubations were carried out using granulosa cell suspensions with high extant aromatase activity due to stimulation by ovine FSH (100 micrograms sc, twice daily for 2 days) in vivo. The triazole acted as an apparent competitive aromatase inhibitor (apparent Km for testosterone 2.5 X 10(-8) M in the absence of R151885 rising to 4.4 X 10(-8) M in the presence of 10(-7) M R151885). Its potency as an aromatase inhibitor was approximately 10 times greater than that of the naturally occurring steroidal aromatase inhibitor 5 alpha-dihydrotestosterone. Various structurally related substances proved to be even more potent aromatase inhibitors than R151885. The most active were also substituted 4,4'-difluorophenyl derivatives containing an imidazolyl or pyridyl moiety instead of the 1,2,4-triazolyl substituent in R151885. This study has identified a novel series of nonsteroidal substances which have the characteristics of potent and specific inhibitors of testosterone aromatization by rat granulosa cells in vitro.     

Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture

Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1-5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 micrograms progesterone or estradiol-17 beta in serum-free defined medium and exposed to 1 muCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1-4 (1-4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5-6 layers granulosa cells and developing theca) and 6 (7-8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1-5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7-8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1-4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.     

Hormonal regulation of oestrogen and progesterone receptors in cultured bovine endometrial cells.

Changes in the number of progesterone and oestradiol receptors in the endometrium are thought to play a role in the induction of luteolysis. The effect of oestradiol and progesterone on the regulation of their receptors in cultured bovine uterine epithelial and stromal cells was examined to determine the mechanisms involved in this process. Cells were obtained from cows at days 1-3 of the oestrous cycle and were cultured for 4 or 8 days in medium alone (RPMI medium + 5% (v/v) charcoal-dextran stripped newborn calf serum) or with oestradiol, progesterone or oestradiol and progesterone. At the end of culture, receptor binding was measured by saturation analysis. Specific binding of both [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor (6,7-3H) pregn-4-ene-3,20-dione) and [3H]oestradiol to epithelial and stromal cells showed high affinities (Kd = 1.1 x 10(-9) and 6 x 10(-10) mol l-1, respectively, for progesterone receptors; Kd = 5.5 x 10(-9) and 7 x 10(-10) mol l-1, respectively, for oestradiol receptors). In the stromal cells, oestradiol (0.1-10 nmol l-1) increased the number of oestradiol receptors from 0.21 +/- 0.06 to 0.70 +/- 0.058 fmol microgram-1 DNA and the number of progesterone receptors from 1.4 +/- 0.83 to 6.6 +/- 0.70 fmol microgram-1 DNA in a dose-dependent manner after 4 days of culture (P < 0.01). After culture for 8 days, the stimulatory effect of oestradiol increased. Progesterone (50 nmol l-1) had no effect on the number of oestradiol or progesterone receptors (P > 0.05). However, progesterone inhibited the stimulatory effect of oestradiol. In epithelial cells, the lower concentrations of oestradiol (0.1 and 1 nmol l-1) stimulated the number of progesterone receptors (P = 0.05) after 4 days culture, whereas the highest concentration of oestradiol (10 nmol l-1), progesterone (50 nmol l-1) and progesterone (50 nmol l-1) plus oestradiol (1 nmol l-1) had no effect. After culture for 8 days, the stimulatory effect of oestradiol decreased. In contrast to progesterone receptors, the number of oestradiol receptors increased with oestradiol concentration (P < 0.01). These data show that the number of progesterone receptors was higher in the stromal cells than in epithelial cells, whereas the number of oestradiol receptors was higher in the epithelial cells than in stromal cells. Oestradiol upregulates its own receptor and increases the number of progesterone receptors in both cell types in vitro, whereas progesterone has little effect, but inhibits the effects of oestradiol on progesterone receptors.     

Hormonal regulation of progesterone secretion by cultured mouse granulosa cells

Secretion of progesterone by granulosa cells from preovulatory follicles of mice was determined during 2 weeks of cell culture in the presence of androgens, estrogen and pituitary gonadotropins. Androstenedione (10(-7) M) and dihydrotestosterone (10(-7) M) stimulated (P less than 0.05) progesterone secretion during the first 11 days of culture. In contrast, 17 beta-estradiol (10(-11)-10(-7) M) did not alter (P greater than 0.10) progesterone secretion throughout the culture period. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) stimulated (P less than 0.01) the granulosa cells in a dose-dependent manner during the first few days of culture. This luteotropic effect was rapidly lost and at later times when FSH was not effective, LH suppressed (P less than 0.05) progesterone secretion. In the presence of prolactin (Prl) (1 microgram/ml), granulosa cells progressively secreted more progesterone during the first week of culture. After maximal stimulation on Days 7-9, progesterone secretion by Prl-treated cells began to decline, but the amount of steroid produced on Day 13 was still higher (P less than 0.05) than in control cultures. Androstenedione and Prl gave an additive effect on progesterone secretion during Days 3-5 of culture. Thereafter, the androgen, although stimulatory by itself, did not influence the luteotropic action of Prl. Unlike the early effect of androgens, 17 beta-estradiol acted synergistically with Prl to maintain maximal secretion of progesterone during the last 4 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of epinephrine and norepinephrine on glucose release from chinook salmon (Oncorhynchus tshawytscha) liver incubated in vitro

The effects of two catecholamines, epinephrine (EP) and norepinephrine (NE), on carbohydrate metabolism were studied by incubating chinook salmon liver in vitro. Basal release of glucose over the course of a 5-h incubation was 7.93 +/- 1.70 mumol/g dry weight. Both EP and NE (2 X 10(-7) M) stimulated glucose release rapidly during the first hour. After 5 h, EP and NE significantly increased glucose release over basal levels to 43.55 +/- 9.01 and 32.75 +/- 6.17 mumol/g dry weight, respectively. Epinephrine- and NE-stimulated glucose release was dose dependent, with a minimum effective dose of 10(-9) M. ED50 for both agents was approximately 2 X 10(-7) M; maximal stimulation occurred at 10(-5) M. No difference in potency between the two catecholamines was found. The effects of adrenergic agonists and antagonists were also studied. Alpha-agonists, methoxamine and phenylephrine, had no effect on glucose release. Isoproterenol, a beta-agonist, stimulated glucose release in a manner similar to EP. The beta-antagonist, propranolol, inhibited both catecholamine- and isoproterenol-stimulated glucose release. Alpha-antagonists (phentolamine, prazosin, and yohimbine) had no effect on either catecholamine- or isoproterenol-stimulated glucose release. Epinephrine and NE stimulate glycogen phosphorylase activity; propranolol inhibits catecholamine-stimulated phosphorylase activity. These results indicate that catecholamines stimulate glucose mobilization in salmon liver by promoting glycogenolysis mediated through beta-adrenergic receptors.     

Effects of clofibrate on the metabolism of progesterone and oestradiol in rat liver microsomal fraction

1. The substrate conversion of [4-(14)C]progesterone and [4-(14)C]oestradiol during incubation with the liver microsomal fraction from both control and clofibrate-treated rats amounted to about 10-15 and 20-25% respectively. 2. The metabolites of progesterone formed by preparations from control rats were hydroxylated in the 16alpha-position (14%), the 6beta-position (12%) and the 2alpha-position (7%). Of the products formed from oestradiol 12% were recovered as a 16alpha-hydroxylated derivative whereas 5% had a 6beta- and 2% a 6alpha-hydroxyl group. 3. Clofibrate affected the microsomal metabolism of both progesterone and oestradiol. It induced 7alpha-hydroxylation of both compounds, metabolic conversions not found in control rats. The 6beta-hydroxylation of progesterone and the 6alpha-hydroxylation of oestradiol were enhanced by a factor of 2 and 3.5 respectively. The 2alpha-hydroxylation, and the 20alpha- and 20beta-hydroxy steroid reduction of progesterone were significantly decreased as were the 16alpha- and the 6beta-hydroxylation of oestradiol.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

Development of a long-term serum-free culture system for immature granulosa cells from diethylstilboestrol-treated prepubertal rabbits: influence of androstenedione and fibronectin on FSH-induced cytodifferentiation

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were cultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibronectin-coated plates with or without androstenedione to define the time course profile of oestradiol and progesterone secretion, and the possible modulator role of androstenedione and fibronectin during FSH-induced rabbit granulosa cell differentiation. Every 48 h, cultures were photographed and samples of medium were collected and assayed by ELISA for oestradiol and progesterone. FSH increased oestradiol secretion in a dose-dependent manner. Androstenedione augmented FSH-stimulated oestradiol secretion, and led to a decrease in secretion of oestradiol with time in culture. FSH stimulated progesterone secretion in a dose-dependent manner. This was increased by androstenedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH-stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fibronectin prevented this decrease, without affecting oestradiol or progesterone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodifferentiation and reaggregation of granulosa cells. Androstenedione appeared to act synergistically with FSH to promote steroidogenesis. Fibronectin sustained progesterone secretion during differentiation.     

Endometrial protein secretion during early pregnancy in entire and ovariectomized ewes

The secretion and synthesis of protein in vitro by explants of endometrium were examined in entire ewes during the first 10 days of the oestrous cycle and during an equivalent interval in ovariectomized ewes which received injections of oestradiol and progesterone. The schedule of steroid injections given was designed to simulate endogenous ovarian secretion of progesterone during the luteal phase before oestrus, of oestradiol around oestrus and of progesterone during the luteal phase after oestrus. The rate of protein synthesis and tissue RNA:DNA and protein:DNA ratios in intercaruncular and caruncular endometrium were generally higher in entire than in ovariectomized ewes. In ovariectomized ewes oestradiol increased these activities at 2-4 days after oestrus, whereas progesterone preceding oestradiol caused increases at oestrus, but not thereafter. In entire ewes and in ovariectomized ewes receiving the full steroid treatment regimen, protein secretion was high at oestrus and declined markedly during the next 4-6 days. In ovariectomized ewes not receiving progesterone before oestradiol, secretion increased between 4 and 6 days after oestrus, or during the equivalent stage of treatment in ewes which did not show oestrus. The omission of this progesterone did not modify secretion by caruncular endometrium. Oestradiol increased protein secretion by both tissues. The data suggest that progesterone given before oestradiol (or its equivalent in entire ewes) inhibits the secretion, at about 4-7 days after oestrus, of uterine proteins which may impair embryo development in ovariectomized ewes which do not receive this progesterone.     

Inhibition by 2-Deoxyglucose and 1,5-Gluconolactone of Glycogen Mobilization in Astroglia-Rich Primary Cultures

Abstract: The presence of glycogen in astroglia-rich primary cultures derived from the brains of newborn rats depends on the availability of glucose in the culture medium. On glucose deprivation, glycogen vanishes from the astroglial cultures. This decrease of glycogen content is completely prevented if 2-deoxyglucose in a concentration of > 1 m M or 1,5-gluconolactone (20 m M ) is present in the culture medium. 2-Deoxyglucose itself or 3- O -methylglucose, a glucose derivative that is not phosphorylated by hexokinase, does not reduce the activity of glycogen phosphorylase purified from bovine brain or in the homogenate of astroglia-rich rat primary cultures. In contrast, deoxyglucose-6-phosphate strongly inhibits the glycogen phosphorylase activities of the preparations. Half-maximal effects were obtained at deoxyglucose-6-phosphate concentrations of 0.75 (phosphorylase a, astroglial culture), 5 (phosphorylase b, astroglial culture), 2 (phosphorylase a, bovine brain), or 9 m M (phosphorylase b, bovine brain). Thus, the block of glycogen degradation in these cells appears to be due to inhibition of glycogen phosphorylase by deoxyglucose-6-phosphate rather than deoxyglucose itself. These results suggest that glucose-6-phosphate, rather than glucose, acts as a physiological negative feedback regulator of the brain isoenzyme of phosphorylase and thus of glycogen degradation in astrocytes.     

Concentration of steroids in bovine peripheral plasma during the oestrous cycle and the effect of betamethasone treatment.

Testosterone, oestradiol and progesterone were measured in peripheral plasma during the oestrous cycle of 6 heifers. Oestradiol and progesterone results confirmed earlier reports. Concentration of testosterone on the day of oestrus was 40+/-3 pg/ml (mean+/-S.E.M.), and two peaks were detected during the cycle, one 7 days before oestrus (1809+/-603 pg/ml) and the other (78+/- 7 pg/ml) on the day before the onset of oestrus. The concentration of progesterone declined in most cases 1 day after the maximum concentration of testosterone. Betamethasone treatment in 5 heifers extended luteal function by an average of 10 days: plasma androstenedione and oestradiol concentrations were unaltered; cortisol values were depressed for at least 16 days after treatment; testosterone concentrations were lowered by 13+/-2-4% during treatment, and except in one heifer the peak on Day -7 was abolished.     

Resumption of follicular activity in the early post-partum period of dairy cows

Lactating Friesian dairy cows (2nd-4th parity) which calved in spring (N = 7) or autumn (N = 15) were used. Their ovaries were examined by ultrasound scanning and blood samples were obtained daily for progesterone and oestradiol concentrations from the 5th day after calving until the first post-partum ovulation occurred. Five autumn-calving cows selected at random were bled every 15 min over a 6-h period on 1 day each week for 4 weeks after calving to assess the patterns of LH secretion. Follicular development during the post-partum anoestrous period was characterized by the growth and regression of small (less than or equal to 4 mm) and medium-sized (5-9 mm) follicles, until a dominant follicle (greater than 10 mm) was detected. The first detected dominant follicle ovulated in 14 cows, became cystic in 4 cows (all in autumn), and failed to ovulate in 1 cow. It was not possible to detect a dominant follicle in 3 cows due to scanning difficulties. The post-partum interval to detection of the first dominant follicle (mean +/- s.d.) was shorter (P less than 0.05) in autumn (6.8 +/- 1.8 days) than in spring (20 +/- 10.1 days). However, there was no significant difference between the respective intervals to first ovulation (autumn 27.4 +/- 25.9 and spring 27.3 +/- 18.9 days). Autumn-calved cows which had cysts had longer (P less than 0.001) intervals to first ovulation (58.2 +/- 23.5 days) than did normal cows (12.0 +/- 2.5 days). All cows with cysts had twin ovulations at their first post-partum ovulation. A pulsatile pattern of LH secretion was detected in the first week post-partum and LH pulse frequency was 2-3 per 6-h period in Weeks 1 and 2 post partum and increased to 5-7 pulses per 6-h period in the presence of a dominant or cystic follicle. Concentrations of progesterone in plasma during post-partum anoestrus were usually low (less than 0.2 ng/ml); oestradiol concentrations were also low (less than 5 pg/ml), but higher values (5-110 pg/ml) were observed in cows that had a dominant or a cystic follicle.     

Role of progesterone and oestradiol in the regulation of uterine oxytocin receptors in ewes.

The interaction between oestrogen and progesterone in the regulation of the uterine oxytocin receptor in sheep was evaluated by measuring the binding of oxytocin to membrane preparations of caruncular and intercaruncular endometrium and myometrium. Ovariectomized ewes were assigned in groups of five to each cell of a 4 x 2 factorial design. The four treatments were (a) vehicle (maize oil) for 12 days, (b) progesterone (10 mg day-1) for 9 days, (c) progesterone for 9 days followed by maize oil until day 12 and (d) progesterone for 12 days. The two oestradiol treatments consisted of the administration of implants in the presence or absence of oestradiol. The ewes were killed on day 10 (group b) or day 13 (groups a, c and d) for collection of uterine tissues. The response of the caruncular and intercaruncular endometrium to the treatments was similar. In the absence of oestradiol, treatment with progesterone continuously for either 9 or 12 days reduced the concentration of the oxytocin receptor in comparison with both the control and the progesterone withdrawal group (in which values were similar). The presence of oestradiol reduced the receptor concentrations in control and both 9- and 12-day continuous progesterone treatment groups, but enhanced the concentration in the progesterone withdrawal group. The myometrial oxytocin receptors responded in a similar way to those in the endometrium to progesterone treatment alone, but the addition of oestradiol produced no further effect. In conclusion, progesterone and oestradiol caused downregulation of the endometrial oxytocin receptor. On the other hand, progesterone withdrawal, similar to that which occurs during luteolysis, increased receptor density in the presence of oestradiol. Progesterone may influence the response of the myometrium to oxytocin by causing a reduction in receptor density.     

Paracrine interactions between platelet-activating factor and prostaglandins in hormonally-treated human luteal phase endometrium in vitro

Stromal cells and epithelial glands were separated after enzymic digestion of specimens obtained from 27 women at hysterectomy or endometrial biopsy during the luteal phase, and then cultured to confluence in vitro. PGE release into the culture medium (mean +/- s.e.m.: ng/mg protein/24 h) from gland cell cultures was not changed by oestradiol (17.6 +/- 1.3 for control and 25.5 +/- 2.8 for oestradiol, respectively). However, in the presence of oestradiol, PAF (5 ng/ml) significantly elevated PGE release to 44.2 +/- 5.8. No stimulation was observed in the presence of progesterone. Stromal cell medium had no effect on PGE release in gland cell cultures. PGE release was always much lower in stromal cell cultures than in glands (control: 4.7 +/- 0.6). PAF stimulated PGE release in the presence of oestradiol in these cells also; gland cell medium was without effect. In co-cultures of glandular and stromal cells, PGE release was more similar to that seen in gland cell cultures, with PAF being stimulatory under the influence of oestradiol. PGF release into the medium from the same gland cell cultures was significantly elevated by hormonal treatment, being greatest (62.0 +/- 11.3) with oestradiol alone, and was strongly inhibited in all wells by addition of PAF and stromal cell medium. In stromal cell cultures without hormonal addition, PGF levels (15.0 +/- 2.4) were similar to those seen in glands (18.1 +/- 3.1), and no stimulation was achieved by oestradiol (29.6 +/- 5.9). PAF was inhibitory on PGF release, while gland cell medium was without effect. Co-cultures gave PGF values generally similar to those of stromal cells; oestradiol was again stimulatory (55.0 +/- 9.3). PAF was significantly inhibitory in the presence of oestradiol. PAF (mean +/- s.e.m.: pmol/mg protein/24 h using a platelet serotonin release assay) in stromal cells was significantly increased from control [M199 alone] (0.31 +/- 0.12) by progesterone (1.00 +/- 0.17). Addition of PGE-2 (7.5 ng/ml) to progesterone-treated wells further increased PAF concentration (5.34 +/- 0.09), but was without effect in wells receiving oestradiol alone. Wells exposed to both hormones exhibited an intermediate response. Similar results were obtained with addition of gland cell culture medium, presumably due to its endogenous PGE content. In co-cultures, PAF concentrations were significantly elevated by progesterone alone (4.78 +/- 0.78) or when combined with oestradiol (2.38 +/- 0.51), but not by oestradiol alone. Treatment with PGE-2 caused no additional stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of glucose by preimplantation mouse embryos in the presence of glucagon, insulin, epinephrine, cAMP, theophylline and caffeine

Neither insulin nor epinephrine influenced the incorporation of glucose into the acid-soluble or acid-insoluble glycogen pool of mouse embryos at the morula-early blastocyst stage during 5 h culture in the presence of radiolabelled glucose. During a 5 h chase culture of pulse-labelled embryos at this stage of development, acid-soluble glycogen labelled during the pulse was not utilized by the embryo but acid-insoluble glycogen was reduced. Addition of glucagon, insulin, epinephrine, cAMP, theophylline or caffeine during chase culture had no effect on the turnover of label in the glycogen pools of the embryo. These results indicate that the turnover of embryonic glycogen observed in vivo is not due to the direct effect of the hormones that regulate glycogen metabolism in the mother. Insulin was found to stimulate incorporation of glucose into non-glycogen macromolecules during both pulse and chase culture. Thus, whilst an effect of insulin on glycogen metabolism was absent, the anabolic effects of this hormone appear to have been expressed in the embryo at this stage of development.