Enhancement of long-term testosterone secretion and steroidogenic enzyme expression in human Leydig cells by co-culture with human Sertoli cell-enriched preparations

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Abstract
We have shown previously that long-term testosterone secretion, which decreases when human Leydig cells are cultured alone, increases when purified human Leydig and Sertoli cells are cultured together. In this work, human Leydig cell functions were studied further during in vitro culture, either alone or with human Sertoli cells, on a basal membrane derived from bovine corneal endothelial cells. The secretion of testosterone increased during the first week of co-culture and remained elevated up to day 12 of culture. In one prolonged co-culture, testosterone secretion decreased progressively after day 12 and, after 1 month of culture, was at a level similar to that observed during the first 48 h. After culture for 48 h, testosterone secretion in the co-culture was enhanced by 162 +/- 5% (p < 0.0001) compared with values observed when Leydig cells were cultured alone (42.6 +/- 10.6 ng/10(6) Leydig cells/48 h; mean +/- SEM). This change was associated with increase in mRNA levels for 3 beta-hydroxysteroid dehydrogenase delta 5-delta 4-isomerase (2.49 +/- 0.58-fold), cytochrome P450c17 (2.81 +/- 0.99-fold), cytochrome P450scc (5.20 +/- 0.13-fold) and cytochrome P450 aromatase (1.73 +/- 0.21-fold) when Leydig cells were co-cultured with Sertoli cells (p < 0.05 for each enzyme). IGF-I mRNA levels were higher (2.77 +/- 0.72-fold for 7.6 kb and 1.41 +/- 0.07-fold for 1.1-1.3 kb transcripts) in the Leydig-Sertoli cell co-cultures than the sum of the levels in Leydig and in Sertoli cells cultured alone. Both basal and hCG-induced testosterone secretion were enhanced by treatment of the co-culture with human recombinant FSH (50 mIU/mL). For basal testosterone secretion, this increase amounted to 163 +/- 5% compared with Leydig cells cultured alone (p < 0.0001) and by 112 +/- 4% compared with non-FSH treated co-cultures (p < 0.01); for hCG-stimulated testosterone secretion this increase was 220 +/- 12% compared with Leydig cells cultured alone (p < 0.0001) and 132 +/- 8% compared with non-FSH treated co-cultures (p < 0.01). This study confirms the enhancement of long-term testosterone secretion by adult human Leydig cells by co-culture with adult human Sertoli cells and shows that this effect is associated with an enhancement of the expression of several steroidogenic enzymes; it might be mediated, as in other species, through increased production of IGF-I by co-culture.