Antisense oligonucleotide targeting the transforming growth factor β1 increases expression of specific genes and functions of Leydig cells

Transforming growth factor β1 (TGFβ1) has been reported to be a potent inhibitor of differentiated functions of many steroidogenic cells. Porcine Leydig cells (LC), as well as Sertoli cells (SC), express TGFβ1 mRNA and secrete this peptide, suggesting that it might play an autocrine role. Moreover, many studies have suggested a possible paracrine regulation of LC by SC‐secreted factors. To assess whether TGFβ1 plays an autocrine/paracrine role on these steroidogenic cells, we attempted to inhibit TGFβ1 protein synthesis by transfecting LC, SC and LC+SC for 24 h with 10 μM of an unmodified antisense oligonucleotide (AON) complementary to the translation‐initiation region of the TGFβ1 mRNA and, as controls, with the corresponding sense (SON) or scrambled (SCRON) oligonucleotides. First, we determined at which level, transcriptional or translational, the TGFβ1 AON acts. Neither TGFβ1 AON, SON nor SCRON modified TGFβ1 mRNA levels in LC, SC or LC+SC. However, TGFβ1 AON caused the disappearance of TGFβ1 immunoreactivity in both cell types. In addition, TGFβ1 AON reduced the attachment of TGFβ1 mRNA in ribosomal and polyribosomal fractions. Then, we showed that the decrease of the TGFβ1 protein induced by the AON results in an increase of the expression of LC specific genes and of LC steroidogenic capacity. In LC and LC+SC, TGFβ1 AON increased the mRNA levels of both LH/hCG receptor (1.9‐fold and 3.5‐fold, respectively) and P450 c17 (5‐fold and 8‐fold, respectively). This was associated with an enhancement of hCG‐induced testosterone production by both LC and LC+SC (1.6‐fold and 2.2‐fold, respectively) when compared with untransfected cells. The TGFβ1 AON effects were always more pronounced on LC+SC than on LC. The present findings show that TGFβ1 has an autocrine/paracrine inhibitory effect on cultured porcine Leydig cells, an effect that can be overcome by TGFβ1 AON.