Establishment of an in vitro bioassay and radio receptor assay for LH/CG in human sera using immortalized granulosa cells transfected with LH/CG receptor

Endocrine ◽  
1996 ◽  
Vol 5 (3) ◽  
pp. 275-283 ◽  
Author(s):  
Natarajagounder Selvaraj ◽  
Ada Dantes ◽  
Roma Limor ◽  
Avraham Golander ◽  
Abraham Amsterdam
Keyword(s):  
Granulosa Cells ◽  
In Vitro Bioassay ◽  
Receptor Assay ◽  
Human Sera

The International Standard for Pituitary FSH: Collaborative study of the Standard and of four other purified human FSH preparations of differing molecular composition by bioassays, receptor assays and different immunoassay systems

1989 ◽  
Vol 123 (2) ◽  
pp. 275-293 ◽  
Author(s):  
P. L. Storring ◽  
Gaines Das R. E.
Keyword(s):  
Geometric Mean ◽  
In Vitro Bioassay ◽  
International Standard ◽  
Receptor Assay ◽  
Specific Activities ◽  
In Vivo Bioassay ◽  
Ranking Order ◽  
Receptor Assays

ABSTRACT The International Standard for Pituitary FSH (IS; in ampoules coded 83/575) was assayed in terms of the Second International Reference Preparation of Human Pituitary FSH and LH for Bioassay (IRP 78/549) by 27 laboratories in 13 countries using bioassays, receptor assays and immunoassays. Estimates of the FSH content of the IS by in-vivo bioassay were homogeneous both within and between laboratories and gave a combined geometric mean (with 95% fiducial limits) of 79·9 (74·6–85·4) i.u./ampoule. Estimates by different in-vitro bioassays and receptor assays were also homogeneous between assays and laboratories, and gave a combined geometric mean (with 95% fiducial limits) of 31·2 (28·8–33·9) i.u./ampoule. However, estimates by the 19 different immunoassay systems were heterogeneous and varied between 5 and 31 i.u./ampoule. The material in ampoules coded 83/575 was established by the World Health Organization as the International Standard for Pituitary FSH. It was assigned a unitage of 80 i.u./ampoule on the basis of its calibration by in-vivo bioassay, because this assay best identifies and defines the hormone. However, the introduction of the new IS will necessitate the recalibration of immunoassay kits. FSH 84/530, prepared in the same way as the IS from the same FSH preparation, did not differ significantly from the IS in any of the assay systems studied and appeared to be equally suitable as a standard. Four highly purified preparations of human FSH (FSH A–D), differing in their isoform compositions and in their in-vivo: in-vitro bioactivity ratios, were also studied. The ranking order of the specific activities of FSH A–D by in-vitro bioassays paralleled their order by receptor assays and the order of their content of FSH isoforms with isoelectric points > 4·5. (Potency estimates of FSH B and C in terms of the IS were greater by receptor assay than by in-vitro bioassay.) The overall ranking order of the specific activities of FSH A–D by immunoassays was different. Contrary to expectation, estimates in terms of the IS of specific activities by immunoassay differed more between preparations than those by in-vitro bioassay or receptor assay. Differences in specificity between immunoassay systems were demonstrated not only in the calibration of the IS in terms of the crude FSH of IRP 78/549 but also in the comparisons of the highly purified FSH in the IS and FSH A–D. The differences in the immunoreactivities and bioactivities of FSH preparations differing in their isoform compositions greatly complicate the standardization of assays for FSH. Journal of Endocrinology (1989) 123, 275–293

Inhibin-like activity in media from cultured rat granulosa cells collected throughout the oestrous cycle

1984 ◽  
Vol 103 (1) ◽  
pp. 77-84 ◽  
Author(s):  
H. J. Sander ◽  
E. C. M. van Leeuwen ◽  
F. H. de Jong
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Oestrous Cycle ◽  
The Other ◽  
Pituitary Cells ◽  
In Vitro Bioassay ◽  
Cell Conditioned Medium

ABSTRACT Granulosa cells of antral ovarian follicles from adult 5-day cyclic rats were cultured on each day of the cycle. The rat granulosa cell conditioned medium (rGCCM) was harvested and renewed on each day of a 4-day culture period. Inhibin-like activity and progesterone were estimated in rGCCM using an in-vitro bioassay system with dispersed rat anterior pituitary cells and radioimmunoassay respectively. Removal of steroids from rGCCM with dextran-coated charcoal was effective and did not significantly change the inhibin-like activity of the treated samples. On day 1 of culture the inhibin-like activity of rGCCM for each day of the oestrous cycle was 20–90% higher than on days 2, 3 and 4 of culture when low and constant levels were observed. Media collected after culture on days 1 and 2 from pro-oestrous cells contained larger amounts of inhibin-like activity than media collected on the other days of the cycle. On day 1 of culture, rGCCM from pro-oestrous cells contained higher concentrations of progesterone than that from cells collected on the other days of the cycle. On days 2, 3 and 4 progesterone levels in rGCCM were undetectable (< 320 pmol/l) except in media from pro-oestrous cultures on day 2. Addition of FSH (62 μg/l) to granulosa cell cultures in medium with or without 10% fetal calf serum (FCS) did not alter the inhibin-like activity of rGCCM from pro-oestrous cells. The presence of FCS maintained the production of inhibin-like activity since rGCCM from cells cultured without FCS was devoid of FSH-suppressing activity after 3 days of culture. It is concluded that material with inhibin-like activity is secreted at a higher rate by granulosa cells collected on the day of pro-oestrus than on any other day of the cycle. However, on the day of pro-oestrus, transport of inhibin-like activity from the follicle to the circulation is apparently impeded since this increased inhibin production is not reflected in a reduction in plasma FSH concentration. J. Endocr. (1984) 103, 77–84

Biopotency in vitro and metabolic clearance rates of five pituitary preparations of follicle stimulating hormone

1993 ◽  
Vol 5 (2) ◽  
pp. 181 ◽  
Author(s):  
DJ Phillips ◽  
NL Hudson ◽  
S Lun ◽  
LA Condell ◽  
KP McNatty
Keyword(s):  
Clearance Rate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Follicle Stimulating Hormone ◽  
Metabolic Clearance ◽  
Clearance Rates ◽  
Metabolic Clearance Rate ◽  
In Vitro Bioassay ◽  
Receptor Assay ◽  
Stimulating Hormone

Five pituitary preparations of follicle stimulating hormone (FSH), namely NIDDK-oFSH-17, Bioscan oFSH, Ovagen, Folltropin-V and F.S.H.-P., were examined for biological activity in terms of their potency in an in vitro bioassay, receptor assay and heterologous radioimmunoassay and in terms of their metabolic clearance rates. In the three assays, Bioscan oFSH was the most potent (P < 0.05) (3- to 5-fold the potency of NIDDK-oFSH-17), with Ovagen being 25-50% the potency of the NIDDK standard (P < 0.05). Folltropin-V and F.S.H.-P. had the lowest potencies in all three assays. For each preparation, the ratio of activities between the assays was not consistent, suggesting that the preparations behaved differently in each assay. In 9 of 10 cases, potency estimates in the heterologous radioimmunoassay were greater than those in the in vitro bioassay or receptor assay. Polyacrylamide gel electrophoresis of the preparations showed banding consistent with the molecular weight of FSH, but also indicated that the preparations were contaminated with other proteins to varying extents. The half-lives of these preparations when injected into the bloodstream of mature female mice were 28.0, 8.6, 13.4, 11.6 and 17.4 min for NIDDK-oFSH-17, Bioscan oFSH, Ovagen, Folltropin-V and F.S.H.-P. respectively. The slopes of the decay rates were significantly different from each other (P < 0.05) except between Ovagen and Folltropin-V. The results of these studies show that a number of widely available FSH preparations have differing biopotencies. Moreover, the biopotency of a preparation in vitro is not related to its metabolic clearance rate, and not all FSH preparations behave identically in different assays. Measures of biopotency in vitro combined with those of metabolic clearance rate may provide useful information on the properties of FSH preparations used for research purposes and for superovulation of farmed livestock.

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

Regulatory effect of vasopressin and its analogs on the steroidogenesis of human granulosa cells (GC) in vitro

1989 ◽  
Vol 120 (3_Suppl) ◽  
pp. S183-S185
Author(s):  
H. MUELLER ◽  
T. RABE ◽  
B. HAUFF ◽  
L. KIESEL ◽  
B. RUNNEBAUM
Keyword(s):  
Granulosa Cells ◽  
Regulatory Effect ◽  
Human Granulosa Cells
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