Secretion of inhibin B by human prepubertal testicular cells in culture

Abstract

Objective: Inhibin B is a secretory product of Sertoli cells of the human testis. It has been reported that serum levels of inhibin B in infant boys, peaking at 3 months of age, exceed levels in adult men. The aim of this study was to evaluate inhibin B secretion in primary prepubertal mixed testicular cell cultures, prepared from testes collected at necropsy. Design and Methods: Cell cultures were divided into three age groups on the basis of differences in testicular histology: group 1 (n ¼ 7), 1-to 10-day-old newborns, group 2 (n ¼ 7), 1-to 9-month-old infants, and group 3 (n ¼ 8), 12-to 84-month-old children. Cells were maintained in culture for 6 days, harvested and counted. In some samples, during the last 4 days, cells were stimulated with 10 ng/ml highly purified human (h) LH (n ¼ 9), 2 ng/ml recombinant human (rh) FSH (n ¼ 9) or 50 ng/ml rhGH (n ¼ 4). On day 6, the secretion of inhibin B and testosterone into the medium was estimated in triplicate. Inhibin B was determined by ELISA and testosterone by RIA. Results: Median (range) inhibin B secretion was 465 (225-1007), 275 (107-298), and 58 (15-184) pg/million cells.24 h in groups 1, 2 and 3 respectively. A logarithmic transformation of these values was performed to normalize data. Mean Ϯ S.D. of transformed inhibin B secretion in group 1 was significantly higher than in group 2 or 3 (P < 0:005) and the values for groups 1 and 2 were significantly higher than that for group 3 (P < 0:005). No significant correlation between testosterone and inhibin B secretion into the medium was found when the 22 culture samples were analyzed as a whole. Inhibin B secretion was significantly increased after stimulation with highly purified hLH, rhFSH and rhGH (P < 0:05) and a significant positive correlation between inhibin B and testosterone was found under both hLH and rhFSH stimulation. Conclusions: It is concluded that cells collected from newborns have the highest capacity to secrete inhibin B in vitro, and that this capacity decreases with age during the first years of life. Since no data are available on serum inhibin levels in newborns, it is possible that concentrations at 3 months of age do not represent a post-natal peak but a declining level of high newborn values. As expected, FSH stimulated inhibin B secretion in culture. LH stimulation was probably mediated by paracrine factors secreted by interstitial cells. Finally, our results add new evidence of the involvement of GH in testicular maturation.

Inhibin B in male reproduction: pathophysiology and clinical relevance

“…For example, in the foetal testis, aand b B subunits, but not b A were immunolocalised in the Sertoli and Leydig cells (20). In cultured testicular cells from prepubertal boys, both highly puri®ed luteinizing hormone (LH) and recombinant FSH stimulated inhibin B secretion (21). In the adult human testis, b A and b B subunits were found in Sertoli and Leydig cells but not in germ cells (22).…”

Establishment of long-term monolayer cultures of somatic cells from human fetal testes and expansion of peritubular myoid cells in the presence of androgen

“…While establishment of shortterm cultures has been described previously, we believe this is the first paper to document the impact of long-term culture on somatic cells obtained from the human fetal testis. Previous studies described monolayer cultures of human fetal testicular cells maintained for up to 6 days (Berensztein et al 2000) or 20 days (Ahmed et al 2009). …”

Role of IGFs and Insulin in the Human Testis During Postnatal Activation: Differentiation of Steroidogenic Cells

“…A sample of the testicular testes was dissected and double digested as previously described (21). Cells were seeded in DMEM/ Ham F12 supplemented with 5 mg/mL vitamin C, 0.2 g/mL vitamin E, and 10% FCS during the first 48 h. Germ cells are lost during this procedure.…”

“…On d 1 of culture, cells are organized in clusters surrounded by isolated dispersed cells (21). During the following days, a layer of cells grows out from cell aggregates to reach an almost confluent layer, spreading among clusters.…”

“…During the following days, a layer of cells grows out from cell aggregates to reach an almost confluent layer, spreading among clusters. Specific staining indicated that the intercluster layer was composed of IC (21).…”