Andrea S. Cupp

Developmental and Hormonal Regulation of Transforming Growth Factor- β1 (TGFβ1), -2, and -3 Gene Expression in Isolated Prostatic Epithelial and Stromal Cells: Epidermal Growth Factor and TGFβ Interactions

Growth factors are postulated to mediate stromal-epithelial interactions in the prostate to maintain normal tissue physiology. Transforming growth factor-beta (TGFbeta) has been shown to influence the prostate and probably mediate stromal-epithelial interactions. TGFbeta1 messenger RNA (mRNA) expression is stimulated after castration and can be suppressed by in vivo treatment with androgens. The hypothesis tested is that TGFbeta is regulated not only by androgen, but also by a network of locally produced growth factors that influence prostatic growth and differentiation. Epithelial and stromal cells from 20-day-old rat ventral prostate were isolated and used to test this hypothesis. The expression of mRNA for TGFbeta1, -2, and -3 was analyzed by a quantitative RT-PCR procedure. Observations from this assay demonstrate that both epithelial and stromal cells express the mRNA for TGFbeta1, -2, and -3. TGFbeta1 mRNA expression was constant during development of the prostate. TGFbeta2 mRNA expression was elevated at birth, then declined and elevated again at 100 days of age. TGFbeta3 mRNA expression was high during puberty and young adult ages then declined at 100 days of age. TGFbeta2 and TGFbeta3 expression are inversely related during prostate development. After castration of 60-day-old rats, both TGFbeta1 and TGFbeta2 mRNA were enhanced. Interestingly, TGFbeta3 mRNA was significantly suppressed after castration. Epidermal growth factor (EGF) stimulated TGFbeta1 mRNA expression in stromal cells (6-fold increase), whereas keratinocyte growth factor stimulated TGFbeta2 mRNA in epithelial cells. TGFbeta inhibited both testosterone- and EGF-stimulated prostatic stromal and epithelial cell growth. EGF and TGFbeta also inhibited prostatic ductal morphogenesis and growth in organ culture. Immunocytochemical localization of TGFbeta in 20-day-old prostate demonstrated predominately stromal localization of the protein. These results indicate that the isoforms of TGFbeta2 and TGFbeta3 are differentially regulated during prostate development, suggesting distinct regulatory mechanisms. Testosterone did not affect TGFbeta expression in cultured prostatic cells. These observations suggest that the in vivo effects of castration on TGFbetas are regulated indirectly through a complex network of growth factors, not simply by direct androgen depletion. The ability of EGF to inhibit prostatic ductal morphogenesis and growth in organ culture is postulated to be in part mediated by the increase in TGFbeta1 expression. In summary, a network of growth factor-mediated stromal-epithelial interactions is needed to maintain prostate growth and development. TGFbeta is postulated to have an important role in this process.

Expression and Action of Neurotropin-3 and Nerve Growth Factor in Embryonic and Early Postnatal Rat Testis Development

Abstract The current study examines the expression and potential actions of neurotropin-3 (NT3), nerve growth factor (NGF), and their receptors during morphological sex determination (seminiferous cord formation) and perinatal rat testis development. The expression of neurotropins and their receptors was analyzed with immunohistochemistry. Cellular localization of neurotropin ligand and receptor proteins changed during embryonic testis development. Neurotropin-3 was localized to Sertoli cells at Embryonic Day 14 (E14), was present in gonocytes at Postnatal Day 0 (P0), and after birth became localized to the interstitium and Sertoli cells (P3–P5). The expression of trk C (the high affinity receptor for NT3) was localized to mesonephric ducts and cells surrounding the cords (E14–E18). In addition, Sertoli cells and preperitubular cells surrounding the cords at E14 also stained for trk C. Neurotropin-3 was expressed in gonocytes and Sertoli cells at P0–P5. Nerve growth factor was detected in Sertoli cells at E14, was clearly in Sertoli and interstitial cells at E16 and E18, and in Sertoli, germ, and interstitial cells from P0–P5. The expression of trk A (the high affinity receptor for NGF) was located in Sertoli and interstitial cells at E16–P5. To determine the actions of neurotropins during embryonic and perinatal testis development, experiments were conducted on E13 and P0 testis. Antisense oligonucleotide experiments with NT3 were used on E13 testis organ cultures to determine effects on seminiferous cord formation. Cord formation was inhibited in 40% of the organ cultures treated with the antisense NT3 oligonucleotides, while no inhibition was observed with sense oligonucleotides. In P0 testis cultures, both NT3 and NGF alone and in combination stimulated thymidine incorporation into DNA. Therefore, the neurotropins are involved in embryonic morphological events (cord formation; NT3) and in growth of the perinatal testis (P0; NT3 and NGF). To define further the growth effects of neurotropins on testis development, expression of transforming growth factor alpha and beta (TGFα and TGFβ) were examined in response to neurotropins. The P0 testis cultures were treated with neurotropins, and expression of mRNA for TGFα and TGFβ was analyzed utilizing a quantitative reverse transcription-polymerase chain reaction assay. Nerve growth factor and NT3 alone or in combination inhibited expression of mRNA for TGFα while NT3 increased mRNA expression of epidermal growth factor receptor. The combination treatment of neurotropins inhibited expression of TGFβ1 and increased expression of TGFβ3. In summary, observations suggest that NT3, NGF, trk A, and trk C are localized to cells critical to seminiferous cord formation and appear to be important regulators of morphological sex determination. In addition to these morphological effects, both NT3 and NGF stimulate P0 testis growth and may elicit their action through altering the expression of locally produced growth factors such as TGFα and TGFβ. Taken together these results suggest that neurotropins are regulators of paracrine cell-cell interactions that result in morphological sex determination and perinatal testis growth.

Role of Transforming Growth Factor-α and the Epidermal Growth Factor Receptor in Embryonic Rat Testis Development

Abstract 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-α (TGF-α) is involved in regulating embryonic testis growth. TGF-α has previously been shown to function in the postnatal testis. TGF-α 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-α 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-α and the EGFR were shown to be expressed in the embryonic and postnatal rat and mouse testis. Perturbation of TGF-α function using neutralizing antibodies to TGF-α on testis organ cultures dramatically inhibited the growth of both embryonic and neonatal testis. TGF-α antibodies had no effect on cord formation. The TGF-α antibody was found to be specific for TGF-α 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-α appears to influence embryonic testis growth but not morphogenesis (i.e., cord formation). Treatment of embryonic testis organ cultures with exogenous TGF-α also perturbed development, leading to an increased proliferation of unorganized cells. Testis from EGFR and TGF-α knockout mice were analyzed for testis morphology. TGF-α 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-α actions in the mutant mice. In summary, the results obtained suggest that TGF-α is an important factor in the regulation of embryonic testis growth, but other factors will also be involved in the process.

Chemotactic Role of Neurotropin 3 in the Embryonic Testis That Facilitates Male Sex Determination

Abstract 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.

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

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 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.

Testis Developmental Phenotypes in Neurotropin Receptor trkA and trkC Null Mutations: Role in Formation of Seminiferous Cords and Germ Cell Survival

Abstract The objective of the present study was to determine if the neurotropin receptors trkC and trkA are involved in embryonic testis development. These receptors bind neurotropin 3 and nerve growth factor, respectively. The hypothesis tested was that the absence of trkC or trkA receptors will have detrimental effects on testis development and morphology. The trkA and trkC homozygote knockout (KO) mice generally die either at or shortly after birth. Therefore, heterozygote mice were mated to obtain homozygote gene KO mice at Embryonic Day (E) 13, E14, E17, and E19 of gestation, with E0 being the plug date. Gonads from approximately 80 embryos were collected and fixed, and each embryo was genotyped. To determine gonadal characteristics for each genotype, the number of germ cells, number of seminiferous cords, seminiferous cord area, and interstitial area were calculated at each developmental age. Germ cell numbers varied in trkA gene KO mice from those of wild-type mice at each age evaluated. In trkC gene KO mice, differences were detected in germ cell numbers when compared to wild-type mice at E17 and E19. At E19, germ cell numbers were reduced in both trkA and trkC gene KO mice when compared to wild-type animals. Apoptosis was evaluated in testes of wild-type, trkC gene KO, and trkA gene KO mice to determine if the alteration in germ cell numbers at each developmental age was influenced by different patterns of germ cell survival or apoptosis. No differences were found in germ cell apoptosis during embryonic testis development. Interestingly, trkA gene KO mice that survived to Postnatal Day 19 had a 10-fold increase in germ cell apoptosis when compared to germ cells in wild-type mice. Evaluation of other morphological testis parameters demonstrated that trkC KO testes had reduced interstitial area at E13, reduced number of seminiferous cords at E14, and reduced seminiferous cord area at E19. The trkA gene KO testes had a reduction in the number of seminiferous cords at E14. Histology of both trkA and trkC gene KO testes demonstrated that these gonads appear to be developmentally delayed when compared to their wild-type testis counterparts at E13 during testis development. The current study demonstrates that both trkA and trkC neurotropin receptors influence germ cell numbers during testis development and events such as seminiferous cord formation.