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

Abstract

The somatic (Sertoli cell (SC), Leydig cell (LC), and peritubular myoid (PTM) cell) cells play key roles in development of the fetal testis. We established monolayer cultures from second trimester human testes and investigated the pattern of expression of cell-lineage characteristic mRNAs. Expression of some SC-associated genes (SRY, SOX9, WT1, GATA4, and SF1) was detectable up to and including passage 3 (P3), while others (anti-Mü llerian hormone; desert hedgehog) present prior to dissociation were not expressed in the cultured cells. Transcripts encoding the androgen receptor were expressed but addition of dihydrotestosterone (DHT) had no impact on expression of mRNAs expressed in SC or LC. Total concentrations of mRNAs encoding smooth muscle actin (ACTA2) and desmin increased from P1 to P3; an increasing proportion of the cells in the cultures were immunopositive for ACTA2 consistent with proliferation/differentiation of PTM cells. In conclusion, somatic cell monolayer cultures were established from human fetal testes; these cultures could form the basis for future studies based on isolation of purified populations of somatic cells and manipulation of gene expression that is difficult to achieve with organ culture systems. Our results suggest that fetal SC do not maintain a fully differentiated phenotype in vitro, yet PTM (ACTA2 positive) cells readily adapt to monolayer culture conditions in the presence of DHT. This culture system provides an opportunity to study the impact of regulatory factors on gene expression in PTM cells, a population thought to play a key role in mediating androgen action within the developing testis.

Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics

“…Indeed, a higher somatic cell/SSC proliferation ratio could have resulted in overgrowth of certain somatic cells in culture at the expense of SSCs. This observation was also made in long-term cultures of testicular cells from immature marmoset, human fetal, and human adult origin (5,14,15,28,29).…”

Retinoic Acid Signalling and the Control of Meiotic Entry in the Human Fetal Gonad

“…For the assessment of gene expression in non-cultured tissues, first strand cDNA was generated from total RNA using RT Kit (Applied Biosystems, Life Technologies, Carlsbad, CA) and real-time quantitative PCR (qRT-PCR) was performed using the Roche Universal Probe Library (Roche Applied Science, Burgess Hill, UK) on an ABI7900HT thermal cycler (Applied Biosystems) as described previously [35]. Primer sequences along with corresponding probe numbers are listed in Table S1.…”

Comparative Marker Analysis after Isolation and Culture of Testicular Cells from the Immature Marmoset

“…The low expression levels of the Sertoli cell marker genes SOX9 and AMH could be due to the low Sertoli cell numbers. However, a reduced marker gene expression has previously been observed in somatic cells isolated from human fetal testicular tissue, indicating that Sertoli cells may change their expression under long-term culture conditions [Cowan et al, 2010].…”