Role of Neurotropins in Rat Embryonic Testis Morphogenesis (Cord Formation)1

Abstract

The process of seminiferous cord formation is the first morphological event that differentiates a testis from an ovary and indicates male sex determination. Cord formation occurs by embryonic Day 14 (Day 0 = plug date; E14) in the rat. A series of experiments were conducted to determine if neurotropins and their receptors are important for the process of rat embryonic cord formation. The expression of low affinity neurotropin receptor (p75/LNGFR) was determined by immunohistochemistry on sections of both testis and ovary from E13 through birth (Day 0, P0) with an antibody to p75/LNGFR. The staining for p75/LNGFR was present in the mesonephros of E13 gonads and in a sex-specific manner appeared around developing cords at E14 in the embryonic testis. At birth, staining for p75/LNGFR was localized to a single layer of cells (i.e., peritubular cells) that surrounded the seminiferous cords. The genes for both neurotropin 3 (NT3) and for corresponding high affinity neurotropin trkC receptor were found to be expressed in the E14 rat testis, as well as other neurotropins and receptors. Immunocytochemical analysis of E14 rat testis demonstrated that NT3 was localized to the Sertoli cells and trkC was present in individual cells of the interstitium at E16 and in selected preperitubular cells at E18. Previously, the peritubular cells adjacent to the cords were demonstrated to be derived from migrating mesonephros cells around the time of cord formation. To determine if neurotropins were involved in cord formation, the actions of neurotropins were inhibited. A high affinity neurotropin receptor (trk)-specific kinase inhibitor, K252a, was used to treat organ cultures of testes from E13 rats prior to cord formation. Treatment of E13 testis organ cultures with K252a completely inhibited cord formation. K252a-treated organ cultures of E14 testis that contained cords did not alter cord morphology. A second experiment to inhibit neurotropin actions utilized a specific antagonist trk-IgG chimeric fusion protein and E13 testis organ cultures. The trk-IgG molecules dimerize with endogenous trk receptors and inhibit receptor signaling and activation of ligand function. Forty percent of E13 testis organ cultures treated with trkC-IgG had significantly reduced cord formation. TrkA-IgG had no effect on initiation of cords; however, in fifty percent of the treated organs, a "swollen" appearance of the cord structures was observed. Experiments using trkB-IgG chimeric protein on E13 organ cultures had no effect on cord formation or cord morphology. The testes from trkC and NT3 knockout mice were examined to determine if there were any morphological differences in the testis. NT3 knockouts appeared to have normal cord morphology in E15 and E17 testis. TrkC knockout mice also had normal cord morphology in E14 and P0 testis. Both NT3 and trkC knockout-mice testis had less interstitial area than wild-type controls. In addition, the trkC knockout mice have an increased number of cells expressing p75LNGFR within the cords when compared to control...

The hepatocyte growth factor system as a regulator of female and male gonadal function

“…Additional paracrine factors with important functions in the developing testis are neurotrophins , Levine et al 2000, TGF-b (Cupp et al 1999), VEGF (Bott et al 2006), kit ligand (Vincent et al 1998), and glial cell line-derived neurotropic factor (GDNF; Meng et al 2000). The roles of these growth factors in promoting testicular differentiation and development have been extensively reviewed elsewhere (Brennan & Capel 2004, Mackay & Smith 2007.…”

Regulation of the gonadal transcriptome during sex determination and testis morphogenesis: comparative candidate genes

“…Sertoli cell precursors form aggregates with the primordial germ cells , Karl et al 1998. Mesenchymal peritubular myoid precursor cells migrate from the mesonephros into the gonad in a male specific manner and surround Sertoligerm cell aggregates to promote formation of seminiferous cords around E14 in the rat , Jost et al 1981, Buehr et al 1993, Martineau et al 1997, Levine et al 2000, Schmahl et al 2000, Cupp et al 2003. Once peritubular myoid cells and Sertoli cells are in contact, an extracellular matrix is secreted and separates the testis cords from the interstitial tissue (Tung et al 1984, Kanai et al 1992.…”

“…During this embryonic period Leydig cells, which are later responsible for testosterone production, arise from the mesenchymal interstitial cells (Merchant-Larios & Moreno-Mendoza 1998, Capel 2000, Nishino et al 2001. After seminiferous cord formation, there is sexspecific growth in the male gonad that includes proliferation of Sertoli, germ, peritubular, and interstitial cells (Mittwoch et al 1969, Chubb 1992, Levine et al 2000.…”

“…Fibroblast growth factor 9 (Fgf9) is expressed in male gonads early in testis development and Fgf9 null mutants show some sex reversal (Colvin et al 2001). In addition, the neurotropic growth factor NT3 has been shown to be important for testis cord formation (Levine et al 2000, Cupp et al 2003. NT3 is a Sertoli cell product that acts as a chemotactic agent (Cupp et al 2003) to promote cell migration of peritubular cell precursors from the mesonephros by binding its receptor TRKC (Levine et al 2000, Cupp et al 2002, Cupp et al 2003.…”

“…The gene expression in E13, E14, and E16 male and female gonads were compared in order to identify sex differences during this developmental period. In addition, rat E13 testis organ cultures, which develop seminiferous cords in vitro, were used and compared with in vivo testis development (Martineau et al 1997, 2003, Levine et al 2000, Uzumcu et al 2002. Comparative microarray analysis was also used to narrow the candidate list of potential regulatory factors for testis development.…”