ENHANCEMENT OF TESTOSTERONE SECRETION BY NORMAL ADULT HUMAN LEYDIG-CELLS BY COCULTURE WITH ENRICHED PREPARATIONS OF NORMAL ADULT HUMAN SERTOLI CELLS

An in-vitro method was developed to study Sertoli - Leydig cell interactions in man, using testes removed at the time kidneys were removed for transplantation from 6 young adult men (aged 17-45 years) after cerebral death. After collagenase digestion of testicular tissue, Leydig cells were purified on discontinuous Percoll gradients. Two fractions of Leydig cells, 'L2' and 'L3' which differed in their buoyant density (1.05 g < L2 < 1.06 g and 1.06 g < L3 < 1.08 g), were obtained. The Sertoli cell-enriched preparation was obtained from seminiferous tubular fragments after sequential treatment with glycine buffer to remove peritubular-myoid cells, a second collagenase digestion, mechanical fragmentation and washes to remove germ cells. Purified Leydig cells were then cultured either alone or together with Sertoli cells in culture dishes coated with collagen, fibronectin and laminin in a chemically defined medium without serum. The influence of co-culture on basal testosterone secretion was examined in 3 successive 48 h periods. During culture for 0-48 h, 48-96 h or 96-144 h, testosterone secretion (ng/10(6) Leydig cells, means +/- SEM) was: 9.7 +/- 2.1, 6.1 +/- 2.1, and 3.4 +/- 1.3, by L2 Leydig cells; 81.6 +/- 16.3, 82.6 +/- 23.1, and 39.1 +/- 13.6, by L3 Leydig cells; 19.0 +/- 4.6, 44.0 +/- 18.0, and 154 +/- 69.3 by L2 + Sertoli cell co-cultures; and 124.4 +/- 13.2, 199.2 +/- 14.7, and 346.1 +/- 78.4, by L3 + Sertoli cell co-cultures, respectively, showing that: (i) L3 Leydig cells secrete more testosterone than L2 Leydig cells, irrespective of whether they were cultured alone or with Sertoli cells; (ii) During all 3 time periods, co-culture with Sertoli cells enhanced testosterone secretion by L2 and L3 Leydig cells; and (iii) during the course of culture, testosterone secretion decreased when Leydig cells were cultured alone and increased when Leydig cells were co-cultured with Sertoli cells. When cultured in the presence of hCG (10(-10) M), testosterone secretion was enhanced during the first 48 h of culture, but not there after, in all types of culture. These data show a reproducible, time-dependent, stimulatory effect of adult human Sertoli cells on basal testosterone secretion by adult human Leydig cells. It is concluded that culture of normal adult human Sertoli and Leydig cells can be performed with testicular tissue removed from young men, and in this in-vitro system, Sertoli cells stimulate long-term Leydig cell testosterone secretion. This study supports the concept of the control of Leydig cell function by Sertoli cells in the human testis.