Peritubular myoid cells are not the migrating population required for testis cord formation in the XY gonad

Abstract

Cell migration is one of the earliest events required for development of the testis. Migration occurs only in XY gonads downstream of Sry expression and is required for the subsequent epithelialization of testis cords. Using organ culture experiments and tissue recombination, we and others speculated that peritubular myoid (PTM) cells were among the migratory cells and were likely the cell type required for cord formation. However, because no unique marker was found for PTM cells, their positive identification during or after migration remained unclear. alpha-Smooth Muscle Actin (alphaSma; approved gene symbol Acta2), a classic marker of adult PTM cells,is expressed broadly in testis interstitial cells at E12.5, and becomes highly enriched in PTM cells by E15.5-16.5. We used a novel transgenic line expressingEYFP under the control of an alphaSma promoter to determine whether alphaSma-EYFP positive cellsmigrate into the gonad. Surprisingly, mesonephroi expressing alphaSma-EYFP do not contribute any EYFP positive cells to XY gonads when used as donors in recombination cultures. These results indicate that alphaSma-EYFP cells do not migrate into the gonad during the critical window of sex determination and cannot be the migrating cell type required for testis cord formation. Our results suggest that PTM cells, and most other interstitial lineages, with the exception of endothelial cells, are induced within the gonad. These experiments suggest that endothelial cells are the migrating cell type required for epithelialization of testis cords.

Fig. 1

αSma-EYFP labels a subset of interstitial cells including peritubular myoid cells. A Optical confocal Z-sections of αSma-EYFP testes. EYFP is expressed throughout the interstitium but never within testis cords. At early (E12.5) and late (E15.5) stages of testis development, EYFP is enriched in cells immediately adjacent to the laminin (red) rich extracellular matrix (arrowheads). A’ Magnification of a small portion of A , as outlined by the white box, shows αSma-EYFP (green) expressed in presumptive peritubular cells immediately adjacent to the ECM (red). B High magnification images of αSma-EYFP gonads demonstrate that expression is excluded from PECAM-1 (red) positive endothelial cells (arrowheads).

Fig. 2

αSma-EYFP positive cells do not migrate into the testis. Control experiments confirmed robust migration of cells from the mesonephros (m) into the gonad (g) at E11.5 (A) and E12.5 (B). As previously reported, the vast majority of these cells co-label with the endothelial marker PECAM-1 (C). Identical recombinations using αSma-EYFP mesonephroi resulted in no migration when initiated at E11.5 (D) and E12.5 (E). In these experiments migration and testis cord formation occurred normally as indicated by PECAM-1 endothelial cells (arrowheads) and germ cell aggregation (asterisks). All images are maximum intensity projections of multiple optical sections. Migration of αSma-EYFP cells was never observed whereas pan-EGFP controls nearly always contributed cells to wild type XY gonads (F).