Isolation of thecal cells: an assessment of purity and steroidogenic potential.
In the present investigation, the responsiveness of rat thecal cells, prepared by means of an optimised discontinuous Percoll density gradient centrifugation procedure and cultured under serum-free cell culture conditions, to different concentrations of follitropin (FSH), basic fibroblast growth factor (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 simultaneously determined with aliquots collected after different times of exposure to these regulatory proteins, either individually or in combination. The results 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 preparations 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 production of P(4) in the Percoll-purified rat thecal cell cultures receiving different 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 was greater at higher LH concentration (50 ng/ml) (P<0.05). Concurrent treatment of LH (20 or 50 ng/ml) and FGF-2 (1-100 ng/ml) showed that FGF-2 inhibited the production of P(4) by LH-stimulated thecal cell cultures (P<0.05). The inhibition by FGF-2 was greater when LH was at a lower concentration (EC(50)<1 ng/ml at LH-20 ng/ml vs. EC(50)>1 ng/ml at LH-50 ng/ml). The results of the present study thus indicate that rat thecal cells isolated by this optimised Percoll density centrifugation procedure maintain a very high steroidogenic potential and specificity. Consistent with the absence of contaminating granulosa cells, these rat theca cell preparations do not respond to FSH treatment in terms of E(2) production. However, these rat theca cell preparations can be stimulated by LH to express their differentiated status in serum-free medium and respond to growth factors such as FGF-2.
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Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Victoria 3168, Clayton, Australia.
Hearn
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D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods; D016895:Culture Media, Serum-Free; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D066298:In Vitro Techniques; D007986:Luteinizing Hormone / Q000494:pharmacology; D011374:Progesterone / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013799:Theca Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*
D016895:Culture Media, Serum-Free; D016222:Fibroblast Growth Factor 2; D011374:Progesterone; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone