Initiation of testicular tubulogenesis is controlled by neurotrophic tyrosine receptor kinases in a three-dimensional Sertoli cell aggregation assay

Abstract

The first morphological sign of testicular differentiation is the formation of testis cords. Prior to cord formation, newly specified Sertoli cells establish adhesive junctions, and condensation of somatic cells along the surface epithelium of the genital ridge occurs. Here, we show that Sertoli cell aggregation is necessary for subsequent testis cord formation, and that neurotrophic tyrosine kinase receptors (NTRKs) regulate this process. In a three-dimensional cell culture assay, immature rat Sertoli cells aggregate to form large spherical aggregates (81.36G7.34 mm in diameter) in a highly organized, hexagonal arrangement (376.95G21.93 mm average distance between spherical aggregates). Exposure to NTRK inhibitors K252a and AG879 significantly disrupted Sertoli cell aggregation in a dose-dependent manner. Sertoli cells were prevented from establishing cell-cell contacts and from forming spherical aggregates. In vitro-derived spherical aggregates were xenografted into immunodeficient nude mice to investigate their developmental potential. In controls, seminiferous tubule-like structures showing polarized single-layered Sertoli cell epithelia, basement membranes, peritubular myoid cells surrounding the tubules, and lumen were observed in histological sections. By contrast, grafts from treatment groups were devoid of tubules and only few single Sertoli cells were present in xenografts after 4 weeks. Furthermore, the grafts were significantly smaller when Sertoli cell aggregation was disrupted by K252a in vitro (20.87 vs 6.63 mg; P!0.05). We conclude from these results that NTRKregulated Sertoli-Sertoli cell contact is essential to the period of extensive growth and remodeling that occurs during testicular tubulogenesis, and our data indicate its potential function in fetal and prepubertal testis differentiation.

Building the mammalian testis: origins, differentiation, and assembly of the component cell populations

“…Their aggregation can be reproduced in vitro by culturing them on reconstituted basement membrane gels (Hadley et al 1985) and is impeded by inhibitors of neurotrophic tyrosine kinase receptors (NTRKs) (Gassei et al 2008). It is known that Ntrk3 is expressed by pre-Sertoli cells at the onset of testis cord formation (Russo et al 1999;Cupp et al 2000;Levine et al 2000), as is the NTRK ligand NTF3, and both seem to be involved in forming adhesive cell contacts (Cupp et al 2002;Gassei et al 2008). However, knockout mice for both Ntrk1 and Ntrk3 show disruption, but not a complete absence, of seminiferous tubules (Cupp et al 2002), and the story is far from complete.…”

Xenografting of human fetal testis tissue: a new approach to study fetal testis development and germ cell differentiation

“…Much of our understanding of normal and disrupted testicular development derives from rodent models (Wilhelm et al ., 2007; Gassei et al ., 2008), because only retrospective studies are usually possible in patients with DSDs or TDS disorders, preventing identification of the mechanisms linking fetal testis maldevelopment to altered cell development/function. However, there are major fundamental differences in the pace and duration of fetal testis development between primates and rodents (Ehmcke et al ., 2006), especially regarding fetal germ cell differentiation (Mitchell et al ., 2008).…”

Xenografting of Human Fetal Testis Tissue: A New Approach to Study Fetal Testis Development and Germ Cell Differentiation

“…Much of our understanding of normal and disrupted testicular development derives from rodent models (Wilhelm et al, 2007;Gassei et al, 2008), because only retrospective studies are usually possible in patients with DSDs or TDS disorders, preventing identification of the mechanisms linking fetal testis maldevelopment to altered cell development/function. However, there are major fundamental differences in the pace and duration of fetal testis development between primates and rodents , especially regarding fetal germ cell differentiation (Mitchell et al, 2008).…”