CHARACTERISTICS OF THE CYCLIC 3′, 5′-AMP FORMATION IN ISOLATED OVARIAN FOLLICLES FROM PMSG-TREATED IMMATURE RATS AFTER STIMULATION IN VITRO WITH GONADOTROPHINS AND PROSTAGLANDINS

1974 ◽  
Vol 77 (3) ◽  
pp. 559-574 ◽  
Author(s):  
Lars Nilsson ◽  
Sten Rosberg ◽  
Kurt Ahrén
Keyword(s):  
Binding Assay ◽  
Prostaglandin E ◽  
Maximal Effect ◽  
Bicarbonate Buffer ◽  
Protein Binding Assay ◽  
Immature Rats ◽  
Significant Release ◽  
Time Courses ◽  
Ovulatory Follicles

ABSTRACT Isolated pre-ovulatory follicles from PMSG-injected immature rats, described in a previous publication, have been used for the investigation of the pattern of cyclic 3′,5′-AMP synthesis after in vitro addition of gonadotrophins and prostaglandins. The follicles were incubated in a modified Krebs bicarbonate buffer for periods up to 4 h and the cyclic 3′,5′-AMP content in tissue and medium was analyzed with the protein binding assay of Gilman (1970). Addition of LH, FSH or prostaglandin E2 (PGE2) increased the cyclic 3′,5′-AMP level in follicular tissue dramatically. HCG and TSH, but not STH or prolactin, also induced a similar increase. The effect of FSH was blocked by a highly specific antiserum to LH. The effect of LH had a maximum after approximately 1 h of incubation, whereas the maximal effect of PGE2 was seen between 5 and 15 min of incubation. When LH was present in the incubation medium a significant release of cyclic 3′,5′-AMP into the medium occurred. Theophylline potentiated the effects of LH and PGE2. The different time-courses of the effects of LH and PGE2 on the cyclic 3′,5′-AMP production by follicular tissue are discussed in relation to the hypothesis of prostaglandins as obligatory mediators of all LH effects on the ovary (Kuehl et al. 1970).

OOCYTE MATURATION AND GLYCOLYSIS IN ISOLATED PRE-OVULATORY FOLLICLES OF PMS-INJECTED IMMATURE RATS

1976 ◽  
Vol 82 (4) ◽  
pp. 809-830 ◽  
Author(s):  
Torbjörn Hillensjö
Keyword(s):  
Oocyte Maturation ◽  
Time Course ◽  
Polar Body ◽  
Prostaglandin E ◽  
Germinal Vesicle Breakdown ◽  
Bicarbonate Buffer ◽  
Lh Surge ◽  
Immature Rats ◽  
Lactate Formation

ABSTRACT Graafian follicles were isolated by microdissection from the ovaries of PMS-injected immature rats killed at specific times on the day before ovulation. They were incubated in Krebs bicarbonate buffer containing 5 mm glucose and 1 % bovine serum albumin. The oocytes were recovered after incubation and examined by Nomarski interference contrast microscopy. The amount of lactate and glucose in the incubation medium was analysed enzymatically. Oocytes recovered from follicles extirpated in the morning, i. e. before the endogenous LH surge, and incubated for 2–10 h were in the dictyate stage, while addition of LH or FSH to the medium resulted in oocyte maturation as revealed by germinal vesicle breakdown (GVB) and polar body formation. The time-course of GVB in vitro was similar to that seen in vivo after exogenous LH. Both gonadotrophins markedly enhanced lactate accumulation in the medium and, as studied for LH, glucose uptake by the follicles. The effects of LH but not those of FSH, on GVB and lactate formation were prevented by the presence of an antiserum against the β-subunit of LH. The gonadotrophic effects on GVB could be mimicked by the addition of prostaglandin E2 to the medium. When the follicles were extirpated in the late afternoon, i. e. after the LH surge, and incubated in hormone-free medium for 4 h the oocytes showed GVB in a progressively increasing proportion depending on the time of follicle extirpation. Lactate formation by this group of follicles was markedly enhanced compared to that of "morning" follicles, but it could be even more stimulated by in vitro exposure to LH. A preliminary series of experiments on the effect of phosphodiesterase inhibitors showed that theophylline and isobutylmethylxanthine at certain concentrations completely blocked the LH effect on GVB, whereas a newly developed compound, ICI 63.197, in itself induced GVB.

Oestradiol synthesis by granulosa cells from immature rats treated with pregnant mare's serum gonadotrophin

1983 ◽  
Vol 104 (3) ◽  
pp. 372-380 ◽  
Author(s):  
Donald C. Johnson ◽  
Roger C. Hoversland
Keyword(s):  
Granulosa Cells ◽  
Steroid Synthesis ◽  
Bicarbonate Buffer ◽  
Immature Rats ◽  
Hypophysectomized Rats ◽  
System Inclusion ◽  
Ovulatory Follicles ◽  
Generating System

Abstract. Granulosa cells harvested from follicles in hypophysectomized or intact immature rats treated with 20 IU of pregnant mare's serum gonadotrophin (PMS) produced immunoreactive oestradiol (E2) when incubated in Krebs Ringer bicarbonate buffer containing an NADPH generating system; inclusion of steroid substrates in the medium increased the rate of synthesis. Further, tritiated E2 was synthesized when labelled progesterone was used as substrate. Granulosa cells removed from pre-ovulatory follicles on the morning of prooestrus in adult females also produced E2 in vitro. Although E2 synthesis was apparent by cells from immature hypophysectomized rats within 12 h of PMS treatment, it inceased greatly with longer in vivo exposure to the gonadotrophin. Production was linear with the number of cells incubated and with time, at least through the first 30 min; the production rate decreased slightly with longer incubations. Exposure of the cells in vivo to hCG or ovine LH, before incubation, destroyed most of their ability to synthesize E2 even if progesterone or pregnenolone was added to the medium, but conversion of testosterone to E2 was reduced by only about 50%. Inhibitors of steroid synthesis, i.e. 4-OH-androstenedione, SU-10603, cyanoketone, or aminoglutethimide, greatly reduced the amount of E2 synthesized by the cells. The results indicate that granulosa cells exposed in vivo to gonadotrophins can synthesize E2 without the addition of androgenic substrate provided that cofactors are supplied. This finding has important implications for the current 'two cell' theory for oestrogen production by the ovary. A deficiency in steroidogenic enzymes within the granulosa cell appears to be an inadequate basis for the theory. However, the total synthesis of E2 in vivo by granulosa cells has not been shown.

ACUTE EFFECTS OF GONADOTROPHINS AND PROSTAGLANDINS ON THE METABOLISM OF ISOLATED OVARIAN FOLLICLES FROM PMSG-TREATED IMMATURE RATS

1974 ◽  
Vol 77 (3) ◽  
pp. 540-558 ◽  
Author(s):  
Lars Nilsson
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Production ◽  
Isobutyric Acid ◽  
Bicarbonate Buffer ◽  
Immature Rats ◽  
Prostaglandin F ◽  
Protein Incorporation ◽  
Ovulatory Follicles

ABSTRACT Pre-ovulatory follicles from immature rats injected with a low dose of PMSG have been used for studies of carbohydrate and protein metabolism in vitro. The follicles were isolated by free-hand dissection and incubated in modified Krebs bicarbonate buffer for periods up to 4 h. The lactic acid content of the medium was analyzed after various incubation periods. In most experiments the tissue uptake of [14C]α-amino-isobutyric acid and [3H] leucine as well as the incorporation of [3H] leucine into follicular protein were determined. Addition of LH or FSH to the incubation medium markedly stimulated the production of lactic acid, whereas prostaglandins of the E-type were less effective. Prostaglandin F2α and dibutyryl cyclic 3′, 5′-AMP had no effect at all. No in vitro effects were seen on the uptake and incorporation af labelled amino acids. When LH or FSH was injected to the animals in vivo 1 or 2 h before incubation of the follicles, the lactic acid production was also stimulated, but only the injection of LH stimulated the protein incorporation of [3H] leucine.

Inhibition of LH-stimulated cyclic AMP formation in pre-ovulatory rat granulosa cells by follicular fluid

1980 ◽  
Vol 95 (1) ◽  
pp. 84-89 ◽  
Author(s):  
Knut Nordenström ◽  
Anita Sjögren ◽  
Lars Hamberger
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Phosphodiesterase Inhibitor ◽  
Inhibitory Effect ◽  
Female Rats ◽  
Bicarbonate Buffer ◽  
Ovulatory Follicles

Abstract. Immature female rats were injected sc with a single dose of PMSG to induce growth and maturation of ovarian follicles. In the morning of prooestrus the rats were given a single ip injection of LH (10 μg/rat) or 0.154 m NaCl, 2 h prior to sacrifice. Granulosa cells were isolated from the pre-ovulatory follicles and incubated in Krebs bicarbonate buffer, for 1 h with or without in vitro addition of various test substances. Following incubation the amounts of cAMP in tissue plus medium were determined. It was found that the isolated granulosa cells exposed to LH in vivo responded to the addition of LH in vitro with a production of high amounts of cAMP, i.e. these cells were not refractory to LH stimulation and in fact responded better than granulosa cells isolated from ovaries not exposed to LH in vivo. The addition to the incubation medium of follicular fluid (FFl) obtained from pre-ovulatory follicles decreased the effect of LH in vitro when added at a final concentration of 1% and completely abolished it at a concentration of 3%. Removal of steroids from the FFl did not influence the inhibitory effect and the addition of a phosphodiesterase inhibitor (IBMX) in vitro did not alter the results in principle. These results point to the existence of a factor in the FF1 which interacts with the sensitivity of the isolated preovulatory granulosa cells to repeated exposures to LH. Characterization of this factor is subject to further investigations.

In vitro RNA-protein Binding Assay by UV Crosslinking

BIO-PROTOCOL ◽  
2012 ◽  
Vol 2 (21) ◽  
Author(s):  
Hailong Zhang ◽  
Muxiang Zhou
Keyword(s):  
Protein Binding ◽  
Binding Assay ◽  
Uv Crosslinking ◽  
Protein Binding Assay

CORTICOSTEROID RELEASE BY ADRENAL TISSUE FROM FOETAL AND NEWBORN LAMBS IN RESPONSE TO CORTICOTROPHIN STIMULATION IN A PERIFUSION SYSTEM IN VITRO

1973 ◽  
Vol 58 (1) ◽  
pp. 75-87 ◽  
Author(s):  
DENISE MADILL ◽  
J. M. BASSETT
Keyword(s):  
Adrenal Glands ◽  
Binding Assay ◽  
Developmental Changes ◽  
Acth Stimulation ◽  
Adult Sheep ◽  
Adrenal Tissue ◽  
A Value ◽  
Protein Binding Assay ◽  
Perifusion System

SUMMARY Adrenal glands from 24 foetal lambs (100–145 days gestation) and eight postnatal lambs (birth to 25 days of age) were used to study developmental changes in the responsiveness of the lamb adrenal to stimulation by synthetic adrenocorticotrophin (ACTH). A continuous flow incubation system in vitro (perifusion) was used in these studies. Addition of ACTH to the perifusion medium (0·3 μg Synacthen/ml) significantly increased the rate of corticosteroid release (as measured by a protein-binding assay) by adrenal tissue from foetuses of all ages studied. With tissue from foetuses of 137 days gestation or less, corticosteroid release during ACTH stimulation was < 2 μg/100 mg tissue in the 3 h following addition of ACTH to the perifusion medium. With tissue from foetuses older than this, the rate of corticosteroid release stimulated by ACTH increased as foetal age increased. At 145 days gestation (just before birth) release was 6·8 μg/100 mg tissue and in postnatal lambs less than 1 day old it was 12·3 μg/100 mg tissue during the 3 h following the start of stimulation. This increase in the response of the foetal adrenal to ACTH stimulation paralleled the increases in adrenal weight and plasma corticosteroid concentration which occurred before birth. With tissue from older lambs ACTH-stimulated corticosteroid release was 5·5 μg/100 mg tissue/3 h, a value comparable to that of 5·6 μg/100 mg tissue/3 h obtained with adrenal tissue from adult sheep. Separation of corticosteroids in the perifusion medium by chromatography on LH 20 Sephadex indicated that adrenals from younger foetuses (< 137 days gestation) released approximately equal amounts of cortisol and corticosterone, whereas adrenals from older foetuses and postnatal lambs released mainly cortisol. Pituitary tissue from all foetuses stimulated corticosteroid release from adrenal tissue of adult sheep, implying that foetal pituitary stores of ACTH do not limit development of foetal adrenal cortex function during the last third of gestation. The results indicate that there is a marked increase during the last week of gestation in the responsiveness of the foetal adrenal to ACTH stimulation and that this increase is related to maturation of corticosteroid biosynthetic pathways during this period.

CYCLIC AMP IN ISOLATED CORPORA LUTEA OF THE RAT: INFLUENCE OF GONADOTROPHINS AND PROSTAGLANDINS

1974 ◽  
Vol 77 (4) ◽  
pp. 737-752 ◽  
Author(s):  
Hans Herlitz ◽  
Lars Hamberger ◽  
Sten Rosberg ◽  
Kurt Ahrén
Keyword(s):  
Corpus Luteum ◽  
Incubation Medium ◽  
Time Course ◽  
First Generation ◽  
Prostaglandin E ◽  
Corpora Lutea ◽  
Bicarbonate Buffer ◽  
Camp Content ◽  
Camp Levels

ABSTRACT Corpora lutea were isolated from 34–39 day-old rats injected with 10 IU PMSG when 30 days old leading to ovulation early in the morning of day 33. Three day-old corpora lutea were incubated in Krebs bicarbonate buffer containing 5.5 mmol/l glucose. Addition of LH to the incubation medium increased accumulation of cyclic 3′, 5′-AMP (cAMP) in a dose-dependent way in the isolated tissue and caused a release of cAMP to the incubation medium. cAMP was determined by the protein binding technique of Gilman (1970). There was a continuous rise in the levels of cAMP in both tissue and medium with increasing incubation time in presence of LH. Neither prolactin nor FSH could influence the cAMP levels in the corpus luteum tissue in vitro. Prostaglandin E2 (PGE2) caused a stimulation with respect to cAMP formation. The time-course of the PGE2 effect was quite different from that of LH: PGE2 gave maximal tissue cAMP content within 5–15 min with decreasing levels thereafter, compared to the continuous rise with LH stimulation for at least 1 h. In experiments performed on corpora lutea of different ages (1–7 day-old), tissue levels of cAMP were determined after incubations with LH or PGE2. There was a dramatic increase of tissue cAMP in the very young corpus luteum and a marked decrease in sensitivity to LH with increasing age of the corpus luteum with a very small but still significant effect on the old corpora lutea. The stimulatory effect of PGE2 on cAMP levels on the other hand showed only a minimal change. A few experiments on the first generation of corpora lutea from mature cycling rats are also reported for comparison.

Determination of erythrocyte folate by competitive protein binding assay preceded by extraction.

1976 ◽  
Vol 22 (7) ◽  
pp. 982-992 ◽  
Author(s):  
E Mortensen
Keyword(s):  
Protein Binding ◽  
Binding Assay ◽  
Extraction Procedure ◽  
Molecular Configuration ◽  
Protein Binding Assay ◽  
Competitive Protein Binding Assay ◽  
Competitive Protein Binding ◽  
Erythrocyte Folate

Abstract Determination of the concentration of erythrocyte folate by means of competitive protein binding assay critically depends on the extraction procedure applied. Results will be influenced by variable factors such as the in vitro age of the blood samples, the degree of hemolysis, the presence of ascorbic acid, and the pH during extraction and elimination of proteins. The radioassay is strongly influenced by the pH of the final reaction mixture, the method used to separate free and protein-bound molecules, and the molecular configuration of the folates present. Based on experimental results presented, I describe a method for the determination of erythrocyte folate.

Relaxin-induced changes in adenosine 3',5'-monophosphate levels in the human cervix

1985 ◽  
Vol 109 (1) ◽  
pp. 122-125 ◽  
Author(s):  
Anders Norström ◽  
Ingrid Wiqvist
Keyword(s):  
Pregnant Women ◽  
Needle Biopsy ◽  
Binding Assay ◽  
First Trimester ◽  
Cervical Tissue ◽  
Protein Binding Assay ◽  
Induced Changes ◽  
Camp Levels ◽  
Human Cervix

Abstract. The effects of porcine relaxin on the levels of cAMP in human cervical tissue were studied in vitro. The specimens were obtained by needle biopsy from women undergoing hysterectomy, legal abortion in the first trimester or elective Casearean section at term, and were incubated in Krebs-Ringer buffer for 15 min in the presence of porcine relaxin (5 μg/ml, 3000 GPU/mg). cAMP was determined using a modified protein binding assay. The concentration of cAMP was higher in pregnant than in non-pregnant women. Relaxin stimulated the production of cAMP in the 7th–8th week of gestation and at term but did not significantly alter the cervical cAMP levels in neither non-pregnant women nor in women in the 10th–12th week of pregnancy. Previous studies have shown that porcine relaxin reduces collagen synthesis in tissue from the human cervix and lower uterine segment. The present observations indicate that these effects can be mediated by cAMP.

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