Human prepubertal testicular cells in culture: steroidogenic capacity, paracrine and hormone control

The neonatal human Leydig cell undergoes a transient period of activation during the first months of life. The biological significance of this activation is unknown. Furthermore, little is known about the hormonal regulation of this biological process, even though it coincides with an elevation of LH levels in serum. In order to study the function of human prepubertal testicular culture cells, obtained during the neonatal period, a method for maintaining primary culture cells (isolated from testes collected at necropsy) in culture was developed. Within 24 h after death, testes were collected from 1-36-month-old subjects. Subjects were divided into two age groups, based on the presence or absence of fetal Leydig cells: 1-7-month-old infants (group 1) and 12-36-month-old children (group 2). Testes were digested with collagenase, and cells were seeded in multi-well dishes. Cells were grown in serum-free conditioned media supplemented with 5 mg/l vitamin C, 0.2 IU/l vitamin E and 10% fetal bovine serum for 2 days. Cells were then grown for an additional 4 days in serum-free media in the presence or absence of hLH (40 IU/l), hCG (135 IU/l), rh FSH (1.5 IU/l), rhGH (0.12 IU/l) or insulin (0.9 mumol/l). Concentrations of steroids in media were determined by RIA on day 6 of culture. In basal conditions cells of group 1 (n = 11) secreted more testosterone, androstendione, 17-hydroxyprogesterone, progesterone and dehydroepiandrosterone (mean +/- SE: 6.76 +/- 1.86, 7.37 +/- 1.82, 61.9 +/- 1.86, 5.75 +/- 1.74 and 8.51 +/- 3.23 pmol/10(6) cells/24 h, respectively) than cells of group 2 (n = 5) (2.95 +/- 1.15, 1.50 +/- 2.75, 1.44 +/- 2.75, 0.78 +/- 1.74 and 3.23 +/- 1.32, respectively). Under hLH stimulation, cells of group 1 increased testosterone, androstendione and 17-hydroxyprogesterone secretions (to 38.2 +/- 0.89, 13.5 +/- 1.17 and 51.7 +/- 3.23), while progesterone secretion remained unchanged (2.82 +/- 1.20). Cell response to rhFSH and rhGH was similar to that of hLH. On the other hand, medium collected from cultures of cells isolated from a Sertoli cell tumor was able to stimulate testosterone secretion in subcultures of control testicular cells in a way similar to that of hCG. In conclusion, (1) these prepubertal human testicular cells can be maintained in primary culture for several days keeping their in vivo steroidogenic potential; (2) cells isolated from young infants can respond to hLH in culture; (3) response to rhFSH is probably mediated by a paracrine factor; (4) response to rhGH is observed in the absence of gonadotropins.(ABSTRACT TRUNCATED AT 400 WORDS)