Reproduction Abstracts

The TGFβ superfamily consists of more than 30 ligands, which exert their multiple functions during early embryonic development and in adult tissues. During spermatogenesis, several TGFβ superfamily ligands are present simultaneously to regulate critical stages of development through shared receptors and overlapping transduction molecules. However, the understanding of this promiscuity of signalling pathways remains unfortunately incomplete. Our aim is to study the mechanisms and physiological outcomes of signalling interactions between several TGFβ superfamily ligands on testicular differentiation.

To study the relevance of TGFβ crosstalk in testes, our lab established a mechanical method to isolate and culture tubule fragments in vitro. Seminiferous tubule fragments were isolated from 6 to 11 weeks testes of rats. Testes were decapsulated and dissected into small fragments, which were further disaggregated with Medimachine. The tubule fragments were obtained by centrifugation for 5 min at 500 r.p.m. and plated into 24-well culture plates.

Our first attempt was to examine the interaction between TGFβ1/2 and BMP signalling pathways in testis. We found induction of Smad1 phosphorylation by TGFβ1/2 in a time-dependent manner in tubule fragments. Of note, induction of phosphorylation can be eliminated specifically by a BMP receptor inhibitor LDN193189. These results indicated that TGFβs can additionally phosphorylate Smad1 via BMP receptors. To further define which BMP-type receptors are possibly involved in induction of p-Smad1 by TGFβs, the tubule fragments were treated TGFβ1 with individual ALK2, ALK3, and ALK6 receptor inhibitors. We found that inhibition of the ALK2 receptor caused stronger effects compared to the ALK6 and ALK3 receptors. In summary, this is the prior report showing that TGFβ signal also via BMP receptors and Smad1 in testicular cells.