Objective: 

Three-b-hydroxysteroid dehydrogenase (3b-HSD) is the enzyme responsible for progesterone production. This study aimed to determine whether 3b-HSD can be shown to reflect progesterone production by cultured granulosa cells under different serum, gonadotrophin and IGF-I conditions.

Methods: 

Large bovine follicles were dissected from abattoir ovaries for granulosa cells recovery. Cells were washed, stained for viability, and plated for 48 hours in basic medium with or without 5% fetal calf serum (FCS). Subsequently, cells were exposed to FSH, LH or FSH + IGF-I in serum-free medium for another 96 hours, predicted to cause different degrees of luteinisation. Before and after incubation, granulosa cells were stained for 3b-HSD activity using a previously published staining solution (Payne et al., 1980) [0.1M phosphate buffered saline (PBS) containing 0.1% BSA, 1.5mM NAD, 0.25mM nitro blue tetrazolium and 0.2mM 5a-androstene-3b-ol-17 one].

Results: 

In cells pre-incubated with FCS, the high dose of IGF-I increased (p<0.05) progesterone secretion over 3-fold compared with FSH alone or the low dose of IGF-I. This was reflected in the degree of 3-b HSD staining, with cells exposed to high IGF-I stained much darker. In addition, 3-b HSD staining showed that cells pre-incubated with FCS became firmly attached with the typical phenotype of luteinised granulosa cells.

Conclusions: 

Measuring 3-b HSD activity before and after in vitro culture appears to be an accurate indicator of progesterone producing capacity of live bovine granulosa cells. If quantifiable, this method has potential to detect the in situ luteinisation status and luteinising effect of hormones used in granulosa cell culture.