Influence of follicular maturation upon the effect of ovine follicle stimulating hormone and luteinizing hormone upon cyclic AMP accumulation by isolated porcine granulosa cells

The ability of ovine follicle stimulating hormone (oFSH) and ovine luteinizing hormone (oLH) to stimulate cyclic AMP accumulation was examined in granulosa cells harvested from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) porcine follicles. The oFSH was pretreated with antiserum to LH to neutralize contaminating LH. The cells were incubated in Eagle’s medium plus 1% BSA for varying periods of time ranging from 1-60 min. Cyclic AMP accumulation in the cells and in the incubation medium was measured by a protein binding assay. In cells harvested from small follicles, 10 µg of oFSH produced a significant stimulation in intracellular cyclic AMP (791 ± 121%; n = 6) after 1 min of incubation and produced a maximal 23-fold (2340 ± 347%; n = 6) stimulation of intracellular cyclic AMP after 5 min of incubation. When cells harvested from small, medium and large follicles were incubated for 15 min with 10 µg oFSH, a 1802 ± 109% (n = 11), 520 ± 97% (n = 11) and a 151 ± 20% (n = 8) stimulation of intracellular cyclic AMP accumulation, respectively, was observed. When FSH was present throughout an incubation period of 60 min, there was an initial stimulation of intracellular cyclic AMP accumulation followed by a decline in intracellular cyclic AMP between 30 and 60 min of incubation in the case of cells from all 3 types of follicles. After 30 and 60 min of incubation with cells obtained from small and medium follicles, 10 µg of oFSH resulted in an increased accumulation of cyclic AMP in the incubation medium. The responsiveness of granulosa cells to oLH was dissimilar to the response to FSH. Addition of 1.0 µg oLH for a 15 min incubation period led to a 153 ± 19% (n = 9), 410 ± 65% (n = 11) and a 1139 ± 90% (n = 8) stimulation of intracellular cyclic AMP accumulation in granulosa cells from small, medium and large follicles, respectively. During 30 and 60 min incubation periods, LH stimulation of cells obtained from medium and large follicles resulted in accumulation of cyclic AMP in the incubation medium.