A technique for superfusion of isolated granulosa cells. Dynamics of cAMP production and steroidogenesis in response to gonadotropins.
A superfusion model for isolated ovarian cells was developed and characterized in detail. Granulosa cells isolated from pre-ovulatory rat ovarian follicles were placed in superfusion (perifusion) chambers with a volume of 125 microliters. Culture medium was pumped through the chambers, collected in 20-min fractions of 600 microliters and analysed for cAMP and steroids. Viability was confirmed by morphological examination. The use of polycarbonate membranes to retain the cells in the chambers was abandoned since the membranes caused severe cell damage. The temporal relationships between gonadotropic stimuli and the release of cyclic 3':5'-adenosine monophosphate (cAMP) and steroids was investigated. Within 10 min FSH elicited transient increase in the release of cAMP and progesterone but had no effect on testosterone or estradiol-17 beta release. Amplitude and duration of the response in cAMP and progesterone release were correlated to concentration and length of the FSH pulse when these parameters were varied within the ranges 1-100 micrograms/l and 30-270 min, respectively. Compared with the cAMP response, the progesterone response peaked up to 30 min later and lasted 1 to 2 h longer but could not be extended to more than approximately 6 h, not even with longer FSH pulses. These results could indicate a development of desensitization.
Johanson
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Department of Physiology, University of Gothenburg, Sweden.
Johanson
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D000818:Animals; D002452:Cell Count; D002469:Cell Separation; D000242:Cyclic AMP / Q000096:biosynthesis*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006064:Gonadotropins, Equine / Q000494:pharmacology*; D006107:Granulosa Cells / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D010477:Perfusion / Q000379:methods*; D011374:Progesterone / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D006064:Gonadotropins, Equine; D011374:Progesterone; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP