A dynamic in vitro investigation of the characteristics of the granulosa cells hormone secretion.

OBJECTIVE: Basic characteristics of ovarian steroid production has largely been clarified by the use of human granulosa cell cultures in vitro. This technique made also possible to test different stimulatory and blocking hormones and chemicals in the cell cultures to clarify regulatory mechanisms of the granulosa cells. However, these cell cultures are static systems that do not give information about the dynamics of steroid production of the granulosa cells. Therefore, in the present study we have tried to establish a dynamic model using the so called superfusion method. METHODS: Granulosa cells were isolated from follicular fluid obtained by aspiration from patients undergoing in vitro fertilization program. The granulosa cells were packed into a closed Sephadex G10 column which was continuously kept at 37 degrees C and washed with McCoy's 5A culture medium. Samples of the medium leaving the column were collected in every 30 minutes with a fraction collector. Stimulation with LH and blocking with cycloheximide were carried out by adding the materials into the perfusing culture medium. Progesterone levels of the samples were measured by RIA-s. RESULTS: We found that the basal progesterone secretion is pulsatile even in the absence of any stimulation. After stimulation with LH there is a rapid, but a slight increase in the steroid levels, followed by a delayed and also pulsatile definite increase of progesterone levels starting at about 30 minutes. CONCLUSIONS: From the data obtained it is clear that human granulosa cells are storing progesterone in a small quantity that is LH releasable. The delayed progesterone increase is due to de novo synthesis of the hormones after the gonadotropin stimulation, which takes about 60 minutes.