Testis formation in the fetal mouse: dynamic and complex de novo tubulogenesis

Recombination culture assays provide evidence for cell migration into the gonad. (a) To test whether cells from the mesonephros migrate into the gonad, recombination cultures were assembled and cultured. The gonad was separated from the closely apposed mesonephros at E11.5. A mesonephros from a transgenic mouse ubiquitously expressing green fluorescent protein (GFP) was assembled with a gonad from an XX or XY wild type mouse and cultured in an agar block. After 48 h, GFP‐positive cells from the mesonephros had migrated into the XY, but not the XX, gonad. (b) To investigate whether migration of cells from the mesonephros is required for testis cord formation, a membrane filter was placed between the gonad (g) and mesonephros (m) in agar block cultures. Under conditions where the membrane barrier permitted diffusion of small molecules, but blocked cell migration, cords (cyan circles) failed to form.

Mesenchymal cells cooperate to organize Sertoli cells into nodules in vitro. To explore the contribution of various cell types to cord formation, cells of the juvenile rat testis were separated in density gradient centrifugation to obtain relatively pure populations of Sertoli cells, mesenchymal (interstitial) cells, and germ cells at various stages. When Sertoli cells were cultured alone, they grew as monolayers. However, when a population of mesenchymal cells was cocultured with Sertoli cells (bottom), cord‐like structures are formed.

A model for the initiation of testis cord formation. (1) Sertoli progenitors (cyan), at least in part derived from divisions in the coelomic epithelium (gray) of the gonad, begin to cluster around germ cells (green), forming the Sertoli‐Germ Cell Mass (SGCM) at E11.5. (2) V‐shaped wedges of interstitial tissue (arrowheads) partition the SGCM into cord‐forming units (dashed outlines) starting around E11.75–E12.0. (3) Testis cords (tc) form as solid tubes each of which is connected at both ends to the rete testis (rt) near the mesonephros border (E12.5–E13.5).

The fetal testis undergoes rapid morphological changes during the initial 24 h of sexual differentiation. Immunofluorescent images of fetal XY gonads. Images throughout this review are oriented so that the coelomic surface is up and the gonad‐mesonephros border is toward the bottom. Dashed lines indicate gonad boundaries. When the gonad initially forms at E11.5, germ cells (green) and somatic cells are randomly mixed (left). Within 24 h of the peak of Sry expression (by E12.5), Sertoli progenitors (cyan) organize de novo into testis cord (tc) structures enclosing germ cells (right). This process is coincident with the appearance of a testis‐specific vasculature (red) characterized by a prominent coelomic vessel (cv) within the interstitial space (i).

Cord morphology shows strain‐specific differences. CDH1 (E‐cadherin) staining (germ cells, red) reveals that E13.5 gonads from the 129S1.Svlmj inbred strain have large testis cords (tc) containing many germ cells. In contrast, E13.5 gonads from the C57BL/6J inbred strain have many more cords, each containing fewer germ cells. Dashed lines indicate gonad–mesonephros boundary. g, gonad; m, mesonephros. Staining within mesonephros represents E‐cadherin‐positive mesonephric tubules/ducts.

A model integrating vascular formation with cord formation. Before sex determination the XY gonad contains rudimentary vascular sprouts (red) that emerge from a large vascular plexus (vp) along the gonad–mesonephros border. Microvasculature is interspersed among the germ cells (green) and presumptive Sertoli cells (cyan). Shortly after initiation of the testis program, the large vascular plexus breaks down and endothelial cells migrate into the gonad. Vascular migration results in the establishment of the male‐specific coelomic vessel (cv) and induces proliferation of interstitial mesenchyme (tan) around vascular branches (vb). The expanding mesenchyme around vascular branches forms interstitial wedges (iw) that subdivide the Sertoli‐germ cell mass (SGCM) and partition the SGCM into cord forming units. g, gonad; i, interstitium; m, mesonephros; tc, testis cord.

The invasion of endothelial cells precedes and pioneers the subdivision of the SGCM into cord‐forming units. Time‐lapse live imaging of fetal testes. Dashed lines indicate gonad–mesonephros border. As endothelial cells (marked by Kdr‐mCherry, red) enter the gonad, Sox9‐positive Sertoli cells (Sox9‐ECFP,39 cyan) begin to segregate into discrete cord‐forming units. After 24 h of culture, Sertoli cells have segregated into looping cords separated by blood vessels. See Movie S2. g, gonad; m, mesonephros.

Time‐lapse live imaging allows visualization of male‐specific vascularization. XY‐specific vascular remodeling occurs between E11.5 and E12.5. Starting at E11.5 endothelial cells (marked by Kdr‐mCherry38) begin to migrate into XY gonads (dashed line indicates gonad‐mesonephros border). Migratory cells are highly directed toward the coelomic domain (top). As increasing numbers of endothelial cells reach the coelomic domain, they begin to aggregate into a large vessel with branches extending down into the gonad. See Movie S1. g, gonad; m, mesonephros.