Isolation of thecal cells: an assessment of purity and steroidogenic potential

In the present investigation, the responsiveness of rat thecal cells, prepa red by means of an optimised discontinuous Percoll density gradient centrif ugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth fac tor (FGF-2 or bFGF), and lutropin (LH) has been examined. The estradiol (E- 2) and progesterone (P-4) contents of the cell culture medium were simultan eously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The r esults confirm that no E-2 could be detected in the cell culture medium of the rat thecal cells prepared and cultured in this manner following all of these different treatments, and hence no contamination of the thecal cell p reparations by granulosa cells was evident. The effects of FGF-2 and LH on the steroidogenic and cytodifferentiational properties of these rat thecal cells under serum-free cell culture procedures were also examined. The prod uction of P-4 in the Percoll-purified rat thecal cell cultures receiving di fferent treatments of FSH, and/or FGF-2 did not differ from the basal cell cultures, and no E-2, was detected from the same culture media. In contrast , LH (20 or 50 ng/ml) was found to enhance the production of P-4 (P < 0.05) in the serum-free cell culture media. The stimulation of P-4 production wa s greater at higher LH concentration (50 ng/ml) (P < 0.05). Concurrent trea tment of LH (20 or 50 ng/ml) and FGF-2 (1-100 ng/ml) showed that FGF-2 inhi bited the production of P, by LH-stimulated thecal cell cultures (P < 0.05) . The inhibition by FGF-2 was greater when LH was at a lower concentration (EC50 < 1 ng/ml at LH-20 ng/ml vs. EC50 > 1 ng/ml at LH-50 ng/ml). The resu lts of the present study thus indicate that rat thecal cells isolated by th is optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of con taminating, granulosa cells, these rat theca cell preparations do not respo nd to FSH treatment in terms of E-2 production. However, these rat theca ce ll preparations can be stimulated by LH to express their differentiated sta tus in serum-free medium and respond to growth factors such as FGF-2. (C) 2 000 Elsevier Science BN. AII rights reserved.