De novo morphogenesis of seminiferous tubules from dissociated immature rat testicular cells in xenografts

Testicular development is initiated with the differentiation of Sertoli cells in the embryonic gonad. The aggregation of Sertoli cells is crucial for the generation of testicular cords and thus for the first sign of male gonadal development. To date, functional testicular tissue has not yet been generated in vitro. The objective of this study was to explore the de novo morphogenesis of testicular tissue from isolated postnatal rat testicular cells using a combination of in vitro culture and ectopic xenografting. Immature rat testicular cells were cultured in either a 2-dimensional (laminin-coated cover-glass) or a 3-dimensional (extracellular matrix gel) culture system. Whereas testicular cells cultured on laminin showed a slow morphogenetic cascade resulting in cord formation after about 10 days of culture, cells cultured on extracellular matrix gel assembled to a network of cordlike structures within several hours after plating and formed spherical cell aggregates at day 3. Further progression of the morphogenetic cascade was not obtained in either the 2- or the 3-dimensional culture system. In contrast, structures resembling immature testicular tissue were obtained after xenografting of extracellular matrix gel-enclosed spherical testicular cell aggregates. The grafts were vascularized and contained elongated seminiferous tubules. Histologic analysis revealed the presence of a basement membrane, a histologically normal interstitium containing putative Leydig cells, the establishment of tubule lumen, and the integration of few putative spermatogonia into the seminiferous epithelium. We conclude that immature rat testicular cells carry the full potential to generate all somatic components of a testis in xenografts, thus opening fascinating pathways to study testicular organogenesis.