A technique for superfusion of isolated granulosa cells. Dynamics of cAMP production and steroidogenesis in response to gonadotropins

Abstract

Abstract. 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 μl. Culture medium was pumped through the chambers, collected in 20-min fractions of 600 μl 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-17β 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 μg/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.

Differential Effects of Pulsatile Versus Continuous Follicle-Stimulating Hormone Delivery on the Production of Cyclic Adenosine 5′-Monophosphates and the Accumulation of Cytochrome P450 Cholesterol Side-Chain Cleavage Enzyme mRNA in Perifused Porcine Granulosa Cells1

“…Less frequent pulsatile administration of FSH at either concentration did not enhance SCC mRNA accumulation above basal. Our inferences regarding this molecular endpoint of FSH action are in agreement with previous functional studies that demonstrated that pulses of gonadotropins are more efficacious than continuous hormone exposure in increasing ovarian steroid production, follicular development, and luteinization [26][27][28][29][30]35]. The fractional increases in SCC mRNA accumulation observed here under perifusion conditions are consistent with the magnitude of the increases reported during static culture of porcine granulosa cells [36].…”

“…This sustained responsiveness to FSH action contrasts sharply with the clear downregulation of cAMP release by cells perifused similarly but at 37 0 C. Furthermore, at 37 0 C the magnitude of the maximal rate of cAMP release decreased in granulosa cells receiving both the low-(100 ng/ml) and high-(600 ng/ml) concentration pulses of FSH. Other investigators have seen similar down-regulation of cAMP production after constant or repeated exposures to LH [34,35]. Woody and LaBarbera [32] and Ford and LaBarbera [48] reported that porcine granulosa cells remained responsive to pulses of FSH but desensitized after 4 h of constant administration of FSH.…”

“…Pulsatile LH administration in the presence of a constant FSH stimulus increased both steroidogenesis and morphological luteinization in rabbit follicles in a dose-dependent manner [31] and produced greater cumulative cAMP responses than a tonic presentation [32,33]. Other investigators [34,35] have reported dose-related increases in cAMP and steroid hormone production in response to gonadotropin stimulation, which decreased or increased with successive stimuli depending on the concentrations used. However, at present very little is known about the importance of the temporal pattern of gonadotropin secretion or cAMP generation on the regulation of specific gene expression by gonadal target cells.…”

Perifusion of Human Granulosa‐luteal Cells: Response to Lh Stimulation

“…An experimental model based on a perifusion technique offers advantages over static culture where frequent sampling of cellular secretions is difficult, substrates may become depleted, and metabolic products may accumulate and inhibit cellular activity. While other approaches to granulosa cell perifusion have been described (Johanson & Johanson, 1988), the use of cells attached to microcarriers reduces the technical difficulties associated with this technique. In this study, granulosa-luteal cells were perifused for periods of up to 14 h and still retained the ability to secrete progesterone in response to acute LH stimulation.…”