DEVELOPMENT OF A TWO-SITE IMMUNORADIOMETRIC ASSAY FOR DIMERIC INHIBIN USING ANTIBODIES AGAINST CHEMICALLY SYNTHESIZED FRAGMENTS OF THE α AND β SUBUNIT

1991 ◽  
Vol 129 (2) ◽  
pp. R9-R12 ◽  
Author(s):  
P.G. Knight ◽  
N. Groome ◽  
A.J. Beard
Keyword(s):  
Synthetic Peptide ◽  
Peripheral Circulation ◽  
Cross Reaction ◽  
Immunoradiometric Assay ◽  
Ovarian Vein ◽  
In Vitro Bioassay ◽  
Α Subunit ◽  
N Terminus ◽  
Rat Ovary

ABSTRACT A two-site (liquid-phase) immunoradiometric assay (IRMA) for dimeric inhibin has been developed using antibodies raised against synthetic peptide sequences corresponding to the N-terminus (1-32) of the α subunit and the C-terminal region (82-114) of the βA subunit of Mr ∼30,000 human inhibin. Highly-purified Mr 32,000 bovine inhibin (standard) gave a dilution curve parallel to those for bovine follicular fluid (bFF), human (h)FF and rat ovary extract. Whilst the assay detected both Mr 56,000 and 32,000 inhibin forms in bFF, little reaction with higher Mr forms was evident. Cross-reaction of 'free' inhibin subunit (Mr 25,000 form) and recombinant human activin A in the IRMA were minimal (< 0.1 and < 2% respectively). Although the detection limit of the IRMA (∼ 50 pg/tube) was similar to that of several reported radioimmunoassays (RIA), the IRMA was unable to detect dimeric inhibin in jugular or utero-ovarian vein plasma of heifers. Similarly, when assayed by IRMA, bFF, hFF and rat ovary extract contained 8-58 times less inhibin than was indicated by RIA. These observations are consistent with earlier evidence that the ovary secretes a substantial excess of 'free' inhibin α subunit and that this material reaches the peripheral circulation. Surprisingly, however, the inhibin contents of bFF, hFF and rat ovary extract determined by in vitro bioassay were 8-23 times greater than the corresponding IRMA values, being similar to those derived by RIA. It is suggested that this quantitative discrepancy between inhibin contents estimated by IRMA and bioassay may be due to (1) loss of bioactivity of the inhibin standard during its purification and/or storage, (2) failure of the IRMA to detect high Mr forms of bioactive inhibin and/or (3) cross reaction of follistatin and other FSH-suppressing substances in the in vitro bioassay.

Inhibin immunoneutralization by antibodies raised against synthetic peptide sequences of inhibin α subunit: effects on gonadotrophin concentrations and ovulation rate in sheep

1990 ◽  
Vol 124 (1) ◽  
pp. 167-176 ◽  
Author(s):  
J. H. M. Wrathall ◽  
B. J. McLeod ◽  
R. G. Glencross ◽  
A. J. Beard ◽  
P. G. Knight
Keyword(s):  
Plasma Levels ◽  
Synthetic Peptide ◽  
Plasma Concentrations ◽  
Ovulation Rate ◽  
Control Groups ◽  
Α Subunit ◽  
Blood Samples ◽  
Antigenic Properties ◽  
N Terminus ◽  
The Mean

ABSTRACT Two experiments were conducted to explore the effectiveness of synthetic peptide-based vaccines for active and passive autoimmunization of sheep against inhibin. In the first experiment, adult Romney ewes (n = 20) were actively immunized against a synthetically produced peptide that corresponded to the N-terminus of the α-subunit of bovine inhibin (bIα(1–29)-Tyr30). This peptide was conjugated to tuberculin purified protein derivative (PPD) to increase its antigenic properties. Control groups comprised non-immunized (n = 10) and PPD-immunized (n = 10) ewes. Primary immunization (400 μg conjugate/ewe) was followed by two booster immunizations (200 μg conjugate/ewe), given 5 and 8 weeks later. Following synchronization of oestrus using progestagen sponges, ovulation rates were assessed by laparoscopy. Weekly blood samples were taken throughout the experiment. All inhibin-immunized ewes produced antibodies which bound 125I-labelled bovine inhibin (Mr 32 000), and ovulation rate in inhibin-immunized ewes (2·15 ± 0·22; mean ± s.e.m.) was significantly (P<0·01) greater than in both non-immunized (0·90 ± 0·23) and PPD-immunized (1·20 ± 0·13) control groups. Immunization against the peptide, but not against PPD alone, resulted in a modest rise in plasma FSH, with mean levels after the second boost being significantly (P<0·025) higher (22%) than those before immunization. Moreover, when blood samples were taken (2-h intervals) from randomly selected groups of control (n = 7) and inhibin-immunized (n = 7) ewes for an 84-h period following withdrawal of progestagen sponges, the mean plasma concentration of FSH during the 48 h immediately before the preovulatory LH surge was 37% greater (P< 0·025) in immunized than in control animals. However, more frequent blood sampling (every 15 min for 12 h) during follicular and mid-luteal phases of the oestrous cycle revealed no significant differences between treatment groups in mean plasma concentrations of FSH. In addition, neither mean concentrations of LH nor the frequency and amplitude of LH episodes differed between immunized and control ewes. However, the mean response of LH to a 2 μg bolus of gonadotrophin-releasing hormone, given during the luteal phase, was significantly (P<0·05) less in immunized than in control ewes. These findings indicate that active immunization of Romney ewes against a synthetic fragment of inhibin can promote a controlled increase in ovulation rate, but this response cannot be unequivocally related to an increase in plasma levels of FSH. In the second experiment, passive immunization of seasonally anoestrous ewes (mule × Suffolk crossbred; n = 6 per group) against inhibin, using an antiserum raised in sheep against a synthetic peptide corresponding to the N-terminus of the α-subunit of human inhibin promoted a rapid (<3 h), dose-dependent rise in plasma levels of FSH which remained increased (2·5-fold; P<0·001) for up to 30 h. Plasma concentrations of LH, however, were unaffected by treatment with the antiserum. It is deduced from this observation that, even in the seasonally anoestrous ewe, the ovary secretes physiologically active levels of inhibin, which exert an inhibitory action on the synthesis and secretion of FSH. Journal of Endocrinology (1990) 124, 167–176

scholarly journals Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

2008 ◽  
Vol 40 (4) ◽  
pp. 185-198 ◽  
Author(s):  
Sébastien Legardinier ◽  
Jean-Claude Poirier ◽  
Danièle Klett ◽  
Yves Combarnous ◽  
Claire Cahoreau
Keyword(s):  
Thermal Stability ◽  
Chorionic Gonadotropin ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

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

Preovulatory follicle development and luteal function in ewes immunized against a synthetic peptide sequence of the α subunit of bovine inhibin

1992 ◽  
Vol 133 (3) ◽  
pp. 413-419 ◽  
Author(s):  
B. J. McLeod ◽  
M. G. Hunter ◽  
E. C. L. Bleach ◽  
R. G. Glencross ◽  
J. H. M. Wrathall
Keyword(s):  
Synthetic Peptide ◽  
Ovulation Rate ◽  
Peptide Sequence ◽  
Corpora Lutea ◽  
Follicle Development ◽  
Preovulatory Follicle ◽  
Α Subunit ◽  
Luteal Function ◽  
The Mean

ABSTRACT Immunization against inhibin consistently results in an increase in ovulation rate in sheep, but the effects that this treatment has on follicle development are unknown. In order to determine the influence of inhibin, parameters of follicle development were assessed in ewes that had been actively immunized against a synthetic peptide homologous to the N-terminal sequence (α1–29, Tyr30) of the a subunit of bovine inhibin, a treatment that neutralizes the biological activity of endogenous inhibin. The final stages of preovulatory follicle development that culminate in ovulation were induced in seasonally anoestrous ewes, and follicles were recovered prior to the predicted time of ovulation. After priming with progestagen, inhibin-immunized and control ewes were treated with gonadotrophin-releasing hormone (GnRH) by continuous infusion (200 ng/h). The ovaries were recovered at slaughter 24 h after the start of GnRH treatment and all follicles ≥ 2·0 mm diameter were dissected out and their capacity to produce oestradiol in vitro was assessed. Further groups of similarly treated animals were blood-sampled daily to determine luteal function following GnRH-induced ovulation. The ovaries were recovered from these ewes at slaughter 10 days after the start of GnRH treatment, the corpora lutea were dissected out and their progesterone content was assessed. There were more (P < 0·01) follicles of 5–6 mm diameter (3·2 ± 0·45 (s.e.m.) compared with 1·1 ± 0·25 follicles/ewe) and more (P < 0·001) follicles of > 6 mm diameter (2·8 ± 0·56 compared with 0·9 ± 0·17 follicles/ewe) in inhibin-immunized than in control ewes. In addition, the mean number of the antral follicles that were oestrogenic was greater (P < 0·05) in immunized than in control ewes (2·8 ± 0·66 compared with 1·3 ± 0·25 follicles/ewe). In animals slaughtered 10 days after the start of GnRH treatment, mean ovulation rate was greater (3·17 ± 0·65 and 1·14 ± 0·14, P < 0·01) in inhibin-immunized ewes. Although there was more (P < 0·01) total luteal tissue/ewe in the immunized group, both the mean weight and progesterone content (ng/mg tissue) of individual corpora lutea were similar between treatment groups. Mean plasma progesterone levels increased earlier and reached higher (P < 0·01) mean concentrations in immunized than in control ewes. These results demonstrate that immunization against inhibin increases the number of preovulatory follicles during the follicular phase, and that steroidogenesis within these follicles and the resultant corpora lutea appears to be normal. Journal of Endocrinology (1992) 133, 413–419

Measurement of exogenous and endogenous inhibin in sheep serum using a new and extremely sensitive bioassay for inhibin based on inhibition of ovine pituitary FSH secretion in vitro

1986 ◽  
Vol 110 (2) ◽  
pp. 341-352 ◽  
Author(s):  
C. G. Tsonis ◽  
A. S. McNeilly ◽  
D. T. Baird
Keyword(s):  
Follicular Fluid ◽  
Half Life ◽  
Bolus Injection ◽  
Peripheral Circulation ◽  
Biologically Active ◽  
Ovarian Vein ◽  
Minimum Sensitivity ◽  
Rebound Increase ◽  
First Time

ABSTRACT An extremely sensitive and reliable bioassay for inhibin based on inhibition of ovine pituitary FSH secretion in vitro was developed and used to measure exogenous and endogenous inhibin activity in the ewe. The sheep inhibin bioassay is 30- to 40-fold more sensitive than conventional rat inhibin bioassays. The minimum sensitivity of each bioassay in the measurement of inhibin activity in 1 ml of sheep serum is 220 mu. and 4080 mu. in the sheep and rat bioassays respectively. This sensitive inhibin bioassay has permitted, for the first time, the measurement of endogenous inhibin in the peripheral and ovarian vein blood of the sheep, as well as exogenously administered inhibin. The half-life of exogenously administered ovine inhibin (in follicular fluid) in the sheep was calculated as two components (18–24 and 50–60 min) from the inhibin profiles of six ewes. Inhibin contained in the ovine follicular fluid, given as a bolus i.v. injection, increased to maximum levels after 5 min and then remained increased for 10–32 min depending upon the dose administered, before exponentially decaying. The time for inhibin to exert its effect ranged from 3 to 6 h after injection and appeared to be dose-related. The bolus injection of inhibin, apart from causing suppression of FSH, evoked a large rebound increase of FSH up to 400% of preinjection levels. The development of the sheep bioassay will allow the measurement of biologically active inhibin in the peripheral circulation and ovarian vein blood of sheep with the possibility of extending this to man. J. Endocr. (1986) 110, 341–352

RELATIONSHIP BETWEEN IN VITRO BIOASSAY AND RADIOIMMUNOASSAY ESTIMATES OF LH IN HUMAN SERUM DEPENDING ON DIFFERENT STANDARD PREPARATIONS.

1979 ◽  
Vol 92 (2_Supplb) ◽  
pp. S129-S157 ◽  
Author(s):  
V. Lichtenberg ◽  
S. Elsner ◽  
V.G. Pahnke
Keyword(s):  
Human Serum ◽  
In Vitro Bioassay

ÉTUDE DE L'ACTIVITÉ BIOLOGIQUE IN VITRO DES HORMONES GONADOTROPES

1960 ◽  
Vol XXXIII (III) ◽  
pp. 444-450 ◽  
Author(s):  
Maria de la Luz Suarez Soto ◽  
Jean Legault Démare
Keyword(s):  
Paper Chromatography ◽  
Incubation Medium ◽  
Ultraviolet Absorption ◽  
Rat Ovary

ABSTRACT Serum gonadotrophin (PMS) when added to the incubation medium of rat ovary slices increases the amount of Δ4-3-ketosteroids produced. This enhancement is proportional to the logarithm of dose. The ketosteroids were determined by their ultraviolet absorption; paper chromatography has shown that only androst-4-en-3,17-dione is present.

scholarly journals RNA Helicase A Regulates the Replication of RNA Viruses

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 361
Author(s):  
Rui-Zhu Shi ◽  
Yuan-Qing Pan ◽  
Li Xing
Keyword(s):  
Virus Replication ◽  
Rna Binding ◽  
Rna Viruses ◽  
Rna Helicase ◽  
Rna Helicase A ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
N Terminus

The RNA helicase A (RHA) is a member of DExH-box helicases and characterized by two double-stranded RNA binding domains at the N-terminus. RHA unwinds double-stranded RNA in vitro and is involved in RNA metabolisms in the cell. RHA is also hijacked by a variety of RNA viruses to facilitate virus replication. Herein, this review will provide an overview of the role of RHA in the replication of RNA viruses.

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