Role of transforming growth factor-alpha and the epidermal growth factor receptor in embryonic rat testis development

Embryonic testis development requires the morphogenesis of cords and growth of all cell populations to allow organ formation. It is anticipated that coordination of the growth and differentiation of various cell types involves locally produced growth factors. The current study was an investigation of the hypothesis that transforming growth factor-alpha (TGF-alpha) is involved in regulating embryonic testis growth. TGF-alpha has previously been shown to function in the postnatal testis. TGF-alpha and other members of the epidermal growth factor (EGF) family act through the epidermal growth factor receptor (EGFR) to stimulate cell proliferation and tissue morphogenesis. To understand the potential actions of TGF-alpha in the embryonic testis, general cell proliferation was investigated. Characterization of cell proliferation in the rat testis throughout embryonic and postnatal development indicated that each cell type has a distinct pattern of proliferation. Germ cell growth was transiently suppressed around birth. Interstitial cell growth was high embryonically and decreased to low levels around birth. A low level of Sertoli cell proliferation was observed at the onset of testis cord formation. Sertoli cell proliferation in early embryonic development was low; the levels were high later in embryonic development and remained high until the onset of puberty. Both TGF-alpha and the EGFR were shown to be expressed in the embryonic and postnatal rat and mouse testis. Perturbation of TGF-alpha function using neutralizing antibodies to TGF-alpha on testis organ cultures dramatically inhibited the growth of both embryonic and neonatal testis. TGF-alpha antibodies had no effect on cord formation. The TGF-alpha antibody was found to be specific for TGF-alpha in Western blots when compared to EGF and heregulin. Testis growth was also inhibited by perturbation of EGFR signaling using an EGFR kinase inhibitor. Therefore, TGF-alpha appears to influence embryonic testis growth but not morphogenesis (i.e., cord formation). Treatment of embryonic testis organ cultures with exogenous TGF-alpha also perturbed development, leading to an increased proliferation of unorganized cells. Testis from EGFR and TGF-alpha knockout mice were analyzed for testis morphology. TGF-alpha knockout mice had no alterations in testis phenotype, while EGFR knockout mice had a transient decrease in the relative amount of interstitial cells before birth. Observations suggest that there may be alternate or compensatory factors that allow testis growth to occur in the apparent absence of TGF-alpha actions in the mutant mice. In summary, the results obtained suggest that TGF-alpha is an important factor in the regulation of embryonic testis growth, but other factors will also be involved in the process.     

Inhibition of platelet-derived growth factor actions in the embryonic testis influences normal cord development and morphology

Platelet-derived growth factors (PDGFs) are paracrine factors with roles in mesenchymal-epithelial interactions during normal and pathologic processes. Previously, PDGF and its receptor (PDGFR) have been shown to be present in perinatal, peripubertal, and adult rat testes. The role of PDGF in embryonic testicular cord formation is not known. The hypothesis tested is that PDGFs and PDGFRs are expressed during cord formation and that inhibition of their action influences normal cord formation during embryonic testis development. Embryonic Day (E) 13 gonadal organ cultures were used. Organs were cultured for 3 days and treated daily with vehicle or a PDGFR-specific tyrosine phosphorylation inhibitor (i.e., the tyrphostin AG1295 or AG1296). Vehicle-treated testes formed normal cords, whereas tyrphostin-treated testes formed "swollen cords," a phenomenon characterized by a significant decrease in the number of cords per testis area and increased cord diameter due to fusion of cords. Expression of PDGF and PDGFR in E13, E14, E16, Postnatal Day (P) 0, and P20 testes was examined. Messenger RNAs for PDGF-A and -B and PDGF alpha- and beta-receptors were expressed in isolated testes during all developmental periods examined. Immunoreactivity for PDGF was present throughout the testicular compartment at E14, restricted primarily to testicular cords at E16, and present in cells of the testicular cords with a stronger immunoreactivity in certain interstitial cell types of P0 testis. PDGFR beta-receptor immunoreactivity was primarily localized to the mesonephros of E14 organs and the testicular interstitium of E16 and P0 testes. Tyrphostins did not affect apoptotic cell number in the testis. PDGF had no effect on cell growth in P0 testis cultures. The results show that PDGFs and PDGFRs are expressed in embryonic testis during cord formation in a tissue-specific manner. Inhibition of PDGF actions does not inhibit cord formation but does alter normal cord development and morphology. The observations provide insight into the factors involved in male sex differentiation and embryonic testis development.     

Effects of FGF9 on embryonic Sertoli cell proliferation and testicular cord formation in the mouse

Proliferation and cord formation by embryonic Sertoli cells are pivotal events involved in testis morphogenesis. A number of growth factors have been implicated in mediating these events. However, the exact level of involvement and importance of each as yet remains elusive. We have adopted an in vitro approach to assess developing mouse Sertoli cells, whereby they are cultured in the presence or absence of fibroblast growth factor (FGF9) and/or extracellular matrix (ECM) gel, since previous studies have shown that ECM gel aids Sertoli cell differentiation. The present findings corroborate this effect, but in addition demonstrate that in the presence of FGF9 (10 ng/ml), cells undergo greater proliferation than those cultured on gel alone. They also display a differentiated epithelial phenotype, with appositional contact of cell membranes in cord-like aggregations. In addition we have shown that cultured Sertoli cells generally express a smaller truncated, nuclear form of the FGFr3, although in the presence of FGF9 and absence of gel, the larger, cytoplasmic form of the receptor is also expressed. Immunolocalisation of FGFr3 in Sertoli cells of whole testes revealed a temporal expression pattern profile, with high levels being abundant in the embryonic testicular cords and at puberty, but an absence in adult Sertoli cells. Our findings suggest that FGF9 plays an important role in proliferation and organisation of embryonic Sertoli cells during testis morphogenesis.     

Chemotactic role of neurotropin 3 in the embryonic testis that facilitates male sex determination

The first morphological event after initiation of male sex determination is seminiferous cord formation in the embryonic testis. Cord formation requires migration of pre-peritubular myoid cells from the adjacent mesonephros. The embryonic Sertoli cells are the first testicular cells to differentiate and have been shown to express neurotropin-3 (NT3), which can act on high-affinity trkC receptors expressed on migrating mesonephros cells. NT3 expression is elevated in the embryonic testis during the time of seminiferous cord formation. A trkC receptor tyrophostin inhibitor, AG879, was found to inhibit seminiferous cord formation and mesonephros cell migration. Beads containing NT3 were found to directly promote mesonephros cell migration into the gonad. Beads containing other growth factors such as epidermal growth factor (EGF) did not influence cell migration. At male sex determination the SRY gene promotes testis development and the expression of downstream sex differentiation genes such as SOX-9. Inhibition of NT3 actions caused a reduction in the expression of SOX-9. Combined observations suggest that when male sex determination is initiated, the developing Sertoli cells express NT3 as a chemotactic agent for migrating mesonephros cells, which are essential to promote embryonic testis cord formation and influence downstream male sex differentiation.     

Identification of Hedgehog signaling outcomes in mouse testis development using a hanging drop-culture system

The Hedgehog (Hh) signaling pathway affects fetal testis growth. Recently, we described the dynamic cellular production of Hh signaling pathway components in juvenile and adult rodent testes. The Hh signaling is understood to regulate cord formation in the fetal testis, but minimal knowledge exists regarding how Hh signaling impacts the postnatal testis. To investigate this, we employed hanging drop cultures, which are used routinely in embryoid body formation. This approach has the advantage of using small media volume, and we examined its suitability for short-term culture of both murine embryonic gonads and adult testis tubules. The effects of cyclopamine, a specific Hh signaling inhibitor, were examined following culture of Embryonic Day 11.5 urogenital ridges (as control) and adult seminiferous tubule fragments for 24-48 h using histological, cell proliferation, and gene expression analyses. Cultured embryonic testes displayed generally normal cord structure, anti-Müllerian hormone (Amh) expression, and cell proliferation; known Hh target gene expression (Gli1, osteopontin, official symbol Spp1, and Amh) was altered in response to cyclopamine. Cultured adult tubules exhibited some loss of seminiferous epithelium organization over 48 h. Spermatogonia continued to proliferate, however, and no significant loss of viability was noted overall. Addition of cyclopamine significantly affected levels of Gli1, Igfbp6, Ccnd2 (cyclin D2), Ccnb1 (cyclin B1), Spp1, Kit, and Amh mRNAs; these genes have been shown previously to be expressed in Sertoli and germ cells. These novel results identify Hh target genes in the testis and demonstrate this signaling pathway likely affects cell survival and differentiation in the context of normal adult testis.     

Role of neurotropins in rat embryonic testis morphogenesis (cord formation)

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 controls or NT3 knockout mice. Combined observations suggest compensation between the different neurotropin ligands, receptors, and/or possibly different growth factors for this critical biological process. In summary, results suggest a novel nonneuronal role for neurotropins in the process of cord formation during embryonic rat testis development. The hypothesis developed is that neurotropins are involved in the progression of male sex differentiation and are critical for the induction of embryonic testis cord formation.     

The oocyte lamin persists as a single major component of the nuclear lamina during embryonic development of the surf clam

Nuclei and nuclear lamina-enriched fractions, isolated from 1 to 5-day-old embryos of the surf clam, Spisula solidissima, contain only one major lamin protein, which appears to be identical to the oocyte lamin (L67), as judged by 2D IEF/SDS PAGE, reactivity with a polyclonal antibody directed against L67 and 125I tryptic peptide mapping. The same protein is also present in liver, muscle, nerve and testis from adult animals. No proteins--recognized by several poly- and monoclonal antibodies, specific for somatic lamins from different vertebrate species or the oocyte lamin LIII of Xenopus- have been detected in nuclei or NL-enriched preparations, isolated from embryos or adult tissues. Synthesis of L67 is detectable in embryos 2h after fertilization; it reaches a maximum in 6h-old embryos and gradually declines thereafter. These results argue that the composition of the NL bears no obvious relationship to the structural and functional changes that take place during the embryonic development of this invertebrate.     

The murine seminiferous epithelial cycle is pre-figured in the Sertoli cells of the embryonic testis

The seminiferous epithelial cycle and spermatogenic wave are conserved features of vertebrate spermatogenic organisation that reflect the need for the rigorous maintenance of sperm production. Although the cycle and the wave of the adult seminiferous epithelium have been well characterised, particularly in rodent species, their developmental origins are unknown. We show that the Sertoli cells of the pre-pubertal mouse, including those of the germ cell-deficient XXSxra mutant, exhibit coordinated, cyclical patterns of gene expression, presaging the situation in the adult testis, where Sertoli cell function is coupled to the spermatogenic cycle. In the case of the galectin 1 gene (Lgals1), localised differential expression in the Sertoli cells can be traced back to neonatal and embryonic stages, making this the earliest known molecular marker of functional heterogeneity in mammalian testis cords. In addition, the timing of germ cell apoptosis in normal pre-pubertal testes is linked to the temporal cycle of the Sertoli cells. These data show that the cycle and wave of the murine seminiferous epithelium originate at a much earlier stage in development than was previously known, and that their maintenance in the early postnatal cords depends exclusively on the somatic cell lineages.     

Action of embryonic thymus and liver cells on the development of the ascitic form of Ehrlich's carcinoma

The influence of the cells of embryonic thymus and liver on the development of Ehrlich carcinoma was studied. The intraperitoneal injection of the embryonic cells in the adult mice infested by the Ehrlich carcinoma resulted in a marked lengthening of the life time of animals and an increase of the survival percentage. The embryonic cells of thymus and liver inhibited sharply the growth of carcinoma cells in the diffusion chambers as well. In contrast to this, the thymus and bone marrow cells of adult animals, taken in the same concentrations as the embryonic cells, exhibited only a slight inhibiting effect on the growth of tumour cells. On the basis of these data a suggestion is put forward to the effect that the embryonic immunocompetent cells determine the stronger inhibition of tumour growth in the embryos as compared with the adult animals.