Fourier analysis of differential light scattering for the quantitation of FSH response associated with structural changes in immortalized granulosa cells.
We have established granulosa cell lines which express constitutively the rat FSH receptors by cotransfection of primary granulosa cells obtained from preovulatory follicles with SV40 DNA, Ha-ras oncogene and a plasmid expressing FSH receptors. These cells respond specifically to ovine and human FSH by cell rounding, intracellular cAMP accumulation, and progesterone secretion in a dose-dependent manner. A new method for the demonstration and quantitation of changes in cell shape-Small Angle Laser Light Scattering (SALLS) analysis-has been utilized for measurement of cell rounding in response to FSH stimulation in these cells. When cells were incubated with increasing doses of either ovine or human FSH, partial rounding of cells was observed at FSH concentrations as low as 24 pM, while complete rounding of cells was observed at a range of 0.24-2.4 nM of FSH. Following aldehyde fixation, hormone-treated cells were examined using the method of SALLS analysis. Histograms obtained by applying SALLS analysis on FSH stimulated GFSHR-17 cells were a reflection of the structural changes induced by the hormone. FSH- and forskolin-incubated cells yielded structured distributions with defined mean size and standard deviations. Moreover, the increase in sharpness of dominant peak in the histogram was correlated with elevated concentration of FSH in a dose dependent manner. In conclusion, cellular response to FSH is correlated with a specific pattern of light scattered in immortalized granulosa cells expressing functional FSH receptors. Therefore, SALLS analysis may serve as a useful tool for in vitro bioassay of the gonadotropic hormone. Moreover, this method may lend itself to in vitro bioassay of any hormone that induces specific morphological changes in target cells.
Schiffer
Z
Z
Physics Department, Schottenstein Cellscan Center for Early Detection of Cancer, Bar-Ilan University, Ramat-Gan, Israel.
Keren-Tal
I
I
Deutsch
M
M
Dantes
A
A
Aharoni
D
D
Weinerb
A
A
Tirosh
R
R
Amsterdam
A
A
D000818:Animals; D002452:Cell Count; D002460:Cell Line; D018914:Cells, Immobilized; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D005640:Follicle Stimulating Hormone / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D005583:Fourier Analysis; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008027:Light; D011374:Progesterone / Q000378:metabolism; D051381:Rats; D011962:Receptors, FSH / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity; D012756:Sheep
D011962:Receptors, FSH; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D011374:Progesterone; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP