Isolation, characterization and radioimmunoassay of luteinizing hormone in the brushtail possum

1997 ◽  
Vol 9 (4) ◽  
pp. 419 ◽  
Author(s):  
L. G. Moore ◽  
W. Ng Chie ◽  
S. Lun, S. B. Lawrence ◽  
D. A. Heath ◽  
K. P. McNatty
Keyword(s):  
Amino Acid ◽  
Luteinizing Hormone ◽  
Plasma Concentrations ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Cross Reactivity ◽  
Brushtail Possum ◽  
Corpora Lutea ◽  
Rate Of Increase ◽  
Pituitary Glands

Luteinizing hormone (LH) was purified from brushtail possum (Trichosurus vulpecula) pituitary glands. The purification procedure consisted of ammonium sulfate precipitation followed by triazinyl-dye chromatography, hydrophobic interaction chromatography and gel filtration. A yield of 10 µg LH g-1 pituitary with a recovery of 20% was obtained from 1400 pituitary glands (20·3 g). Contamination with possum follicle-stimulating hormone (FSH) was ≤0.05%. The amino acid analysis and the N-terminal sequencing for 10 cycles revealed close homology with LH from other mammals. Minor amounts of LH that had been truncated near the N-terminal were also detected. No contaminating proteins were found by amino acid sequencing. The potency of possum LH was 20% that of ovine LH in a receptor assay using possum testicular receptors and 4% that of ovine LH when bovine corpora lutea receptors were used. Possum LH was able to stimulate production of cyclic adenosine 3′ ,5′-monophosphate by bovine granulosa cells. A radioimmunoassay (RIA) for possum LH using 125I-possum LH and an antiserum raised against ovine LH was developed. The RIA has a sensitivity of 0·15 ng mL-1 , a 50% displacement of 1·9 ng mL-1 and a cross-reactivity of <0 · 02% against possum FSH. Plasma concentrations were 0·24 ± 0· 04 ng mL-1 (n = 8) and 0·39 ± 0·12 ng mL-1 (n = 8) in female and male possums respectively. Administration of mammalian gonadotrophin-releasing hormone (GnRH) and chicken GnRH II stimulated increases in plasma LH concentrations in male and female possums. When comparing LH responses with administration of mammalian GnRH or chicken GnRH II, plasma LH concentrations remained elevated for a longer period of time in males than in females (P < 0· 01); plasma LH concentrations also remained elevated for longer after mammalian GnRH than after chicken GnRH II (P < 0· 01). Gonadectomy stimulated an increase in plasma concentrations of LH in both male (P < 0· 01) and female (P < 0· 05) possums. The rate of increase in plasma LH concentrations in males was faster than that in females. In summary, we have purified, partially characterized, and developed a RIA for possum LH.

The use of heterologous radioimmunoassays for the measurement of follicle-stimulating hormone and luteinizing hormone concentrations in horse and donkey serum

1983 ◽  
Vol 99 (2) ◽  
pp. 199-209 ◽  
Author(s):  
Valerie Urwin
Keyword(s):  
Luteinizing Hormone ◽  
Follicle Stimulating Hormone ◽  
Cross Reactivity ◽  
Chorionic Gonadotrophin ◽  
Cross Reaction ◽  
The Other ◽  
Corpora Lutea ◽  
Serum Concentrations ◽  
Study Support ◽  
Baseline Levels

Heterologous double-antibody radioimmunoassays were developed for the measurement of FSH and LH concentrations in the serum of both horses and donkeys. The FSH assay employed a rabbit anti-ovine FSH serum which showed a complete lack of cross-reaction with equine chorionic gonadotrophin (eCG) and negligible cross-reaction with equine LH. The LH assay utilized an antiserum raised against highly purified eCG. This similarly showed negligible cross-reaction with equine FSH but its high cross-reactivity with eCG prevented the measurement of equine LH concentrations in serum when eCG was also present. In both assays serial dilutions of horse and donkey serum were parallel to the standard. The assays were used to monitor changes in serum concentrations of FSH and LH during the first 100 days of pregnancy in pony mares and jenny donkeys. In both species during pregnancy LH levels reached a peak 1–2 days after ovulation. They then decreased rapidly to baseline levels where they remained until days 35–40 when the commencement of eCG production prevented their further measurement. Serum FSH concentrations, on the other hand, continued to fluctuate markedly throughout the first 100 days of pregnancy in both the ponies and donkeys. Pronounced surges in FSH levels occurred at regular intervals in some animals but the pattern of release was quite irregular in the others. The results of this study support the concept that it is continued pituitary FSH release, not eCG secretion, which is responsible for stimulating the secondary follicles which develop during early equine pregnancy. However, it appears likely that it is the LH-like activity of eCG which causes the subsequent ovulation and/or luteinization of these secondary follicles to produce accessory corpora lutea.

Isolation and partial characterization of tammar wallaby luteinizing hormone and development of a radioimmunoassay

1997 ◽  
Vol 9 (4) ◽  
pp. 475 ◽  
Author(s):  
James R. McFarlane ◽  
Carl D. Rudd ◽  
Lynda M. Foulds ◽  
Terry P. Fletcher ◽  
Marilyn B. Renfree
Keyword(s):  
Luteinizing Hormone ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Tammar Wallaby ◽  
Exchange Chromatography ◽  
Significant Rise ◽  
Brushtail Possum ◽  
Response Curves ◽  
Antechinus Stuartii

Tammar wallaby (Macropus eugenii) luteinizing hormone (LH) was purified from pituitaries collected from wild and captive populations by salt sequential precipitation, ion exchange chromatography and gel filtration. Pituitary tissue (5 g) yielded 1·8 mg of purified wallaby luteinizing hormone (ME-14B), as verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A heterologous radioimmunoassay has been developed for measurement of LH in plasma of marsupials using a monoclonal antibody raised against bovine LH (518B7). This assay system was able to measure basal LH concentrations in male and female tammars and detected a significant rise in plasma LH in response to oestradiol benzoate in female tammars and luteinizing hormone-releasing hormone (LHRH) in males. Parallel dose–response curves were also obtained from pituitary extracts from four other species of marsupial (brushtail possum, Trichosurus vulpecula; brown antechinus,Antechinus stuartii; kowari, Dasyuroides byrnei; and Eastern pygmy possum,Cercartetus nanus) in this assay, which suggests its usefulness in the measurement of LH in other marsupial species.

Luteolytic and antiluteolytic effect of the antiprogestagen RU486 in pseudopregnant rats

1995 ◽  
Vol 145 (3) ◽  
pp. 449-454 ◽  
Author(s):  
J Th J Uilenbroek ◽  
P van der Schoot ◽  
J A M Mattheij ◽  
J J M Swarts
Keyword(s):  
Plasma Concentrations ◽  
Early Morning ◽  
Prolactin Secretion ◽  
Female Rats ◽  
Corpora Lutea ◽  
Serum Progesterone ◽  
Early Afternoon ◽  
Two Generations ◽  
Pituitary Glands ◽  
High Concentrations

Abstract To study the effects of the antiprogestagen RU486 on luteal activity in pseudopregnant rats, adult female rats made pseudopregnant by sterile copulation were given daily injections with oil vehicle or with RU486 (2 mg/day) either during the entire period of pseudopregnancy (day 1 till day 14) or during the second half of pseudopregnancy (day 8 till day 14). Blood was taken every other day to measure serum concentrations of progesterone. At autopsy, on day 15, the weights of ovaries, isolated corpora lutea and pituitary glands were recorded. In a second study using the same experimental protocol, blood was taken via a jugular vein cannula on days 8, 9, 10 and 11 after induction of pseudopregnancy; on each of these days blood samples were taken at 0700, 0800 and 0900 h, and at 1700, 1800 and 1900 h to measure plasma concentrations of prolactin, LH and progesterone. Administration of RU486 from day 1 of pseudopregnancy onwards had no effect on the increasing concentrations of serum progesterone during the first half of pseudopregnancy. Thereafter progesterone concentrations increased further in RU486-treated rats whereas they decreased in oil-treated pseudopregnant rats. Administration of RU486 from day 8 of pseudopregnancy onwards resulted in a decline in progesterone concentrations in serum on day 10 followed by ovulation on day 11. Plasma LH concentrations in rats treated with RU486 from day 1 of pseudopregnancy were higher than those in oil-treated rats on days 8, 9, 10 and 11. Treatment from day 8 of pseudopregnancy resulted in low LH concentrations at days 8 and 9 and the presence of a preovulatory surge of LH on the afternoon of day 10 (day of pro-oestrus). Plasma concentrations of prolactin measured in oil-treated rats showed two daily surges of similar magnitude in the morning and afternoon of days 8, 9, 10 and 11. In animals treated with RU486 from day 8 onwards, the afternoon surge on day 9 and the morning surge on day 10 were absent. This demonstrated that the luteolytic effect of RU486 when given during the second part of pseudopregnancy is due to a blockade in the afternoon surge of prolactin on day 9. In animals treated with RU486 from day 1 of pseudopregnancy onwards, prolactin in the early morning samples was low, while prolactin in the afternoon samples was highly elevated. At autopsy on day 15, the weights of ovaries, corpora lutea and pituitary glands in animals treated with RU486 from day 1 were larger than those in oil-treated rats; this is in line with an increased secretion of prolactin. In contrast, in animals treated with RU486 from day 8, pituitary weight was not elevated and the increase in ovarian weight was due to the presence of two generations of corpora lutea. In conclusion, whether or not RU486 is luteolytic in pseudopregnant rats depends on the time of administration: injection during the second half of pseudopregnancy inhibits prolactin secretion and induces luteolysis, while administration during the early phase of pseudopregnancy results in high concentrations of prolactin in the early afternoon and therefore prevents luteolysis. Journal of Endocrinology (1995) 145, 449–454

scholarly journals Purification and properties of bovine pituitary follitropin

1976 ◽  
Vol 159 (3) ◽  
pp. 651-659 ◽  
Author(s):  
K W Cheng
Keyword(s):  
Amino Acid ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Elution Volume ◽  
Purification And Properties ◽  
Receptor Assay ◽  
Ion Exchange Column ◽  
Pituitary Glands ◽  
Radioligand Receptor Assay

A reproducible procedure was developed for the purification of follitropin from frozen bovine pituitary glands. The method involved precipitation with (NH4)2SO4 and acetone, followed by ion-exchange column chromatography on CM-cellulose and DEAE-cellulose and gel filtration on Sephadex G-100. A specific radioligand-receptor assay for follitropin was used to locate the activity in eluates after column chromatography and gel filtration. The potency of the highly purified bovine follitropin as measured by Steelman-Pohley bioassay was 164 times that of NIH-FSH-S1 standard preparation. They yield of bovine follitropin was 2.9 mg/kg of frozen pituitary glands. Electrophoretically, bovine follitropin was more acidic in nature and migrated further towards the anode than lutropin and thyrotropin. The elution volume of bovine follitropin by gel filtration on Sephadex G-100 was very similar to that of bovine lutropin. The amino acid composition of bovine follitropin was similar to that of sheep and human follitropin, being rich in lysine, aspartic acid, threonine, serine, glutamic acid and half-cystine.

Isolation of FSH from bovine pituitary glands

1993 ◽  
Vol 137 (1) ◽  
pp. 59-68 ◽  
Author(s):  
J.-B. Wu ◽  
P. G. Stanton ◽  
D. M. Robertson ◽  
M. T. W. Hearn
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
High Yield ◽  
Purification Procedure ◽  
In Vitro Bioassay ◽  
Α Subunit ◽  
Pituitary Glands

ABSTRACT An improved method is described for the isolation of FSH from bovine pituitary glands. The purification procedure consisted of an initial ammonium sulphate precipitation step followed by triazine-dye chromatography, immobilized metal affinity chromatography, high-performance anion-exchange chromatography and gel filtration. Three highly purified bovine FSH preparations (designated bFSH-A, -B and -C) were obtained, giving yields of approximately 5·7 mg FSH/kg bovine pituitary glands (wet weight), with specific radioreceptor activities for bFSH-A, -B and -C of 61, 25 and 29 units (NIH-FSH-S1)/mg protein respectively. The corresponding biological activities were 217 (bFSH-A), 62 (bFSH-B) and 86 (bFSH-C) units/mg, as measured by an FSH in-vitro bioassay. LH levels were found to be < 1% (w/w) as determined by an LH in-vitro bioassay. SDS-PAGE of these bFSH preparations under reducing conditions in 16% polyacrylamide gels showed two major silver-staining bands of apparent molecular masses 19·5 kDa and 15·8 kDa. Their amino acid compositions were in close agreement with the expected composition, based on the bFSH cDNA sequence and results reported by other investigators. N-terminal sequencing of the bFSH-A preparation yielded two major sequences consistent with α- and β-subunits, and a third minor (< 20%) sequence consistent with the α-subunit clipped at amino acid residue 6. It was concluded that the bFSH purification procedure reported here is a rapid method which produces bFSH in high yield and high purity, with radioreceptor and in-vitro specific activities comparable with those previously reported by other investigators. Journal of Endocrinology (1993) 137, 59–68

CHANGES IN THE PLASMA CONCENTRATIONS OF LUTEINIZING HORMONE, PROGESTERONE, OESTRADIOL AND TESTOSTERONE AND IN THE BINDING OF FOLLICLE-STIMULATING HORMONE TO THE THECA OF FOLLICLES DURING THE OVULATION CYCLE OF THE HEN (GALLUS DOMESTICUS)

1981 ◽  
Vol 91 (1) ◽  
pp. 11-22 ◽  
Author(s):  
R. J. ETCHES ◽  
K. W. CHENG
Keyword(s):  
Luteinizing Hormone ◽  
Follicle Stimulating Hormone ◽  
Plasma Concentrations ◽  
Gallus Domesticus ◽  
Interstitial Tissue ◽  
Follicular Maturation ◽  
Ovarian Stroma ◽  
Pituitary Glands ◽  
Two Peaks ◽  
Second Peak

The changes in the binding of FSH during follicular maturation were examined in the hen using 125I-labelled bovine FSH (bFSH) and unlabelled bFSH. The binding of 125I-labelled bFSH was not inhibited by bovine LH or chicken LH but was inhibited by extracts of chicken pituitary glands. The ovarian stroma, which contained both interstitial tissue and small follicles, bound the greatest amount of FSH. As the follicles progressed through the yolk-filled hierarchy of maturation, they bound decreasing amounts of FSH. In the two largest follicles of the hierarchy, there was a significant increase in the binding of FSH 12–16 h before ovulation. There were two peaks in the concentrations of LH; a preovulatory peak occurred 4–6 h before ovulation and a second peak occurred 14–16 h before ovulation. Plasma concentrations of testosterone, oestradiol and progesterone began to rise 9, 8 and 6 h, respectively, before ovulation. These data are consistent with the hypothesis that changes in the gonadotrophin concentration and binding regulate the order of the follicular hierarchy and the onset of preovulatory steroidogenesis in the hen.

INFLUENCE OF THE PURITY OF THE IODINATED TRACER ON THE SPECIFICITY OF THE RADIOIMMUNOASSAY OF HUMAN LUTEINIZING HORMONE

1978 ◽  
Vol 89 (3) ◽  
pp. 506-520 ◽  
Author(s):  
H. Suginami ◽  
D. M. Robertson ◽  
E. Diczfalusy
Keyword(s):  
High Resolution ◽  
Luteinizing Hormone ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Biologically Active ◽  
Immunological Activity ◽  
Subunit A ◽  
Adsorption Step ◽  
Reference Preparation

ABSTRACT Human luteinizing hormone (HLH) iodinated with 125I by the use of a lactoperoxidase method, was fractionated by either cellulose adsorption, gel filtration or by the combination of these methods. The products of iodination were characterized by their in vitro biological LH activity and by their binding profiles with antisera to HLH, HLHα and HLHβ subunits. Several radioactive components were obtained after gel filtration with or without an initial cellulose adsorption step. One of these fractions was identified as biologically active HLH and another as the HLHα subunit. Radioimmunoassay studies were conducted with different iodinated fractions as tracers, using three well defined and widely available antisera to HLH. The standard used was the HLH International Reference Preparation for immunoassay (68/40). Cross-reactivity was examined with several purified pituitary preparations, such as HFSH, HTSH, HLHα and HLHβ subunit. A significantly higher cross-reactivity with HLHα, HFSH and HTSH was obtained with the [125I]HLHα fraction as tracer than with biologically active [125I]HLH. Furthermore, in the radioimmunoassay of HLH preparations of varying purity, significantly higher estimates of immunological activity were obtained with the [125I]HLHα tracer than with the biologically active [125I]HLH. It is concluded that the presence of [125I]HLHα in the [125I]HLH tracer can result in serious overestimates of the immunological activity in the measurement of LH. Therefore [125I]HLHα should be separated from [125I]HLH prior to radioimmunoassay. Many of the fractionation methods commonly used (cellulose adsorption and short column gel filtration systems) are inadequate for this purpose. However, an adequate separation can be achieved by the use of high resolution gel filtration systems.

ISOLATION AND PURIFICATION OF LUTEINIZING HORMONE FROM PORCINE PITUITARY GLANDS

1972 ◽  
Vol 52 (3) ◽  
pp. 507-515 ◽  
Author(s):  
F. B. ANDERSON ◽  
J. E. O'GRADY
Keyword(s):  
Luteinizing Hormone ◽  
Isoelectric Focusing ◽  
Gel Filtration ◽  
High Potency ◽  
Fractional Precipitation ◽  
Isolation And Purification ◽  
Pituitary Glands ◽  
Short Time

SUMMARY A method is described for the isolation of a purified luteinizing hormone (LH) from the anterior lobes of porcine pituitary glands. This includes fractional precipitation, gel filtration and isoelectric focusing. The preparation, which can be recovered in a fairly short time, is of high potency (equivalent to 3·4 mg standard NIH-LH-B7/mg and 6800 i.u. LH/mg).

scholarly journals Concentrations of amino acids and urea in the plasma of the ruminating calf and estimation of the amino acid requirements

1974 ◽  
Vol 32 (2) ◽  
pp. 421-433 ◽  
Author(s):  
A. P. Williams ◽  
R. H. Smith
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dry Matter ◽  
Plasma Concentrations ◽  
Live Weight ◽  
Progressive Increase ◽  
Protein Supplement ◽  
Individual Amino Acid ◽  
Rate Of Increase ◽  
Amino Acid Concentrations

1. A study was made of factors affecting the plasma concentrations of free amino acids (PAA) and urea (PU) in calves receiving approximately equal daily amounts of concentrates (flaked maize and protein supplements) and straw, the former at 10.00 and 17.00 hours, the latter at 17.00 hours only.2. For calves receiving a diet containing 20 g nitrogen/kg dry matter in which the protein supplement was decorticated, extracted groundnut meal (DCGM) (diet A) there were marked increases in PAA and PU about 1–2 h after a morning feed, then a fall in these values 2 h later to a level which was maintained for the next 3 h. No similar changes occurred after the evening feed. Samples taken 3 h after the morning feed were used in subsequent comparative experiments. There was much more variation between animals than within animals in total PAA, PU and the concentrations of most individual amino acids in these samples.3. Total PAA and most individual amino acid concentrations were not appreciably affected when the DCGM intake was reduced to give 10 g N/kg dry matter in the diet (diet C), but PU was halved. When maize gluten replaced DCGM as the protein supplement at the higher N intake (diet B) then PU doubled, but again total PAA and most individual amino acid concentrations were little affected. Exceptions were arginine, which was halved, and leucine, which was doubled.4. Infusions of more than 4·4 g L-methionine/d into the abomasums of calves (110–160 kg live weight) receiving diet A led to a marked increase in plasma methionine concentration. This was considered to correspond with the point at which methionine requirements were met. Using a chromic oxide marker to estimate flows of methionine and cystine from the rumen to the duodenum, it was calculated that under these conditions the methionine requirement was 9·8 g/d, with a cystine flow of 4·9 g/d. Similar calculations showed the corresponding value to be 7·5 g/d with a cystine flow of 2·8 g/d for calves receiving diet C.5. Infusion of increasing levels of L-lysine into the abomasums of calves (110–160 kg live weight) receiving diet B led to a progressive increase in plasma lysine concentration. There was no consistent change in the rate of increase with increasing amounts infused. Estimated lysine requirement appeared therefore to be less than the flow of lysine from the rumen to the duodenum under these conditions (18·8 g/d).

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