Sertoli cell-specific expression of rat androgen-binding protein in transgenic mice: effects on somatic cell lineages

The Sertoli cells of many species produce an androgen binding protein (ABP) which carries testicular androgens to the epididymis and is thought to play a role in sperm maturation. In the present report we analyzed the morphological modifications present in Leydig, Sertoli, and peritubular cells of the testis of young adult male mice transgenic for ABP gene, which overproduce ABP in testis. By in situ hybridization we demonstrated that ABP is specifically produced by Sertoli cells. Using light and electron microscopy, we detected scattered alterations of the seminiferous tubule cells which include cell degeneration and vacuolization. Leydig and Sertoli cells present morphological signs of hyperfunctioning compensatory mechanisms which include increased amounts of lipid droplets probably due to the existence of a stimulated steroid synthesis that in turn could be a consequence of the decreased unbound testosterone and/or a direct paracrine effect of ABP. Peritubular cells also present numerous signs of hyperstimulation.     

Endocytosis of androgen-binding protein (ABP) by spermatogenic cells

To test whether Sertoli cell-secreted ABP could serve as steroid carrier to the germ cell (GC) lineage, radiolabeled ABP and SHBG and gold SHBG were used for binding studies and for internalization studies based on transmission electron microscope analyses and autoradiography of the radiolabeled samples. The data clearly showed that: (1) rat and human germ cells possess a single class of binding sites for rat ABP and human SHBG respectively (Kd of 0.78 and 0.56 nM); (2) 1.7 x 10(10) and 2.7 x 10(10) sites/mg protein was found in the corresponding plasma membrane preparations; (3) the receptor peak was eluted in the same position as dextran blue: 2000 kDa (M(r) = 2 x 106) for labeled rat ABP; (4) in the whole GC lineage, the labeled ligand was internalized through an endocytic pathway involving clathrin coated structures and the distribution was similar throughout the maturation step, however striking differences in the internalization rate were revealed with regard to the maturation step; and (5) this internalization occurred even in ligated seminiferous tubules, via the Sertoli cells cytoplasm. When isolated rat GC were incubated in the presence of ABP, a dose dependent increase in labeled secreted protein was observed for spermatocytes (50-250%) whereas ABP had no effect on spermatids. Addition of steroids and ABP caused a 200 and 50% increase in labeled secreted proteins for spermatocytes and spermatids respectively. 2-D SDS-PAGE analysis revealed that ABP alone increased the secretion of specific spermatocyte proteins whereas steroids in the presence of ABP resulted in the synthesis of new spermatocyte secreted proteins. Taken together these results strongly suggest that ABP may be required for spermatogenesis either as a steroid transmembrane carrier or on its own.     

Growth response of adult germ cells to rat androgen-binding protein and human sex hormone-binding globulin

Androgen-binding protein (ABP) is produced by Sertoli cells depending on the development and the stage of the spermatogenic cycle. Germ cell proliferation is at its peak when ABP is at its peak and secreted towards the testicular basal compartment containing spermatogonia and premeiotic spermatocytes. Rat isolated adult germ cell DNA synthesis was studied in vitro in the presence of ABP with and without steroids and in the presence of pure or recombinant sex steroid hormone-binding globulin (SHBG) using thymidine incorporation. Results are: SHBG is able to promote DNA synthesis in the absence of cofactors. Testosterone reacted negatively to the stimulatory effect of SHBG. We conclude that ABP, the physiological steroid-binding protein, should be considered as a paracrine regulator of spermatogenic DNA synthesis in the adult rat.     

A mutation in zebrafish affecting a localized cellular function required for normal ear development

Tropomyosin is an actin-associated cytoskeletal protein expressed in muscle and non-muscle cells. There are several tropomyosin isoforms, and their cellular expression is known to be associated with transformation events caused by retroviral infection and chemical mutagens. We found that expression of a low-molecular weight tropomyosin isoform, TM5/TM30nm, was higher in a high-metastatic B16 mouse melanoma cell line, B16-F10, than in B16-F1, a low-metastatic mouse melanoma cell line. In order to determine whether this elevated level of TM5/TM30nm plays a role in malignant phenotype, B16-F10 cells were transfected with recombinant DNA containing antisense rat TM5/TM30nm cDNA linked to the human metallothioneinIIa promoter, which is inducible by heavy metals such as zinc and cadmium. When the stably transfected clones were treated with ZnSO4, decreased expression of TM5/TM30nm and reduction in cell motility, which is thought to be an indicator of cellular malignancy were observed. These findings suggest that TM5/TM30nm plays a fundamental role in regulating cell motility, which is essential for metastasis and invasion of tumor cells.     

Mice with mottled mutation--a model for defective copper metabolism in humans

Androgen binding protein (ABP) has been shown to be secreted by Sertoli cells and to be actively taken up by the efferent ducts and proximal caput epididymidis and, yet, to be present at high concentrations in epididymal fluids. In the present study, ABP was immunolocalized by light microscopy in epithelial cells of the efferent ducts and epididymis of adult rats and during postnatal development and by electron microscopy in specific organelles within these cells. In adults, the efferent ducts actively endocytosed Sertoli cell-derived ABP. In the epididymis, principal cells displayed a variable staining reminiscent of a checkerboardlike pattern, with cells being intensely, moderately, or weakly reactive throughout their cytoplasm or unreactive. In the electron microscope, reactive cells displayed a labeling of their Golgi apparatus and secretory vesicles indicative of an epididymal-secreted form of ABP. However, labeling was also noted over endosomes of principal cells, but only of the initial segment and intermediate zone, which, along with labeling of coated pits and vesicles, indicated that ABP was also endocytosed by principal cells of these regions. The postnatal study revealed that principal cells attained an adultlike staining pattern indicative of secretion in a region-specific manner at different ages, suggesting that ABP secretion is regulated by different factors. Ligation of the efferent ducts of 15-day-old animals revealed no reaction along the entire epididymis in animals sacrificed at later ages, suggesting the importance of luminal testicular factors in its regulation during development. In addition, as in the adult, ABP was also endocytosed by principal cells, but only in the initial segment and intermediate zone. Taken together, the present results indicate that secretion of ABP occurs along the entire epididymis, whereas endocytosis is region specific. The functional role of ABP in the epididymis in relation to sperm maturation is discussed.     

Variations in mental health needs and fee-for-service reimbursement for physicians in Ontario

In order to evaluate the intracellular function of glia maturation factor (GMF), we overexpressed GMF in C6 rat glioma cells using two methods: stable transfection using the pcDNA3 plasmid, and transient transfection using replication-defective human adenovirus. With both methods, C6 cells transfected with GMF and overexpressing the protein exhibit a lower saturation density in culture compared to non-transfected or vector alone controls. Transfected cells also exhibit morphological differentiation as shown by the outgrowth of cell processes. When inoculated into nude mice, transfected cells are less tumorigenic than controls, and express the mature astrocytic marker glial fibrillary acidic protein. In tissue culture, transfected cells show a 3.5-fold increase in CuZn-dependent superoxide dismutase (CuZnSOD) activity. Western blot analysis reveals a 3.5-fold increase in CuZnSOD protein, suggesting an induction of the enzyme. In view of recent findings that reactive oxygen species (ROS) and the antioxidant enzymes are intricately involved in key physiologic processes such as proliferation, differentiation and apoptosis, the study raises the possibility that CuZnSOD may be a mediator of GMF function.     

Induction of nest-material collecting in male Barbary doves by intracerebral androgen

The formation of androgen-binding protein (ABP) by cultured Sertoli cells, prepared from testes of immature rats, is increased when androgens or follicle-stimulating hormone (FSH) are present in the medium. Testosterone and 5alpha-dihydrotestosterone are equally effective in stimulating the synthesis and secretion of ABP, but non-androgenic steroids examined (progesterone, 17beta-estradiol and corticosterone) are without influence. Maximal increases are observed when androgens are added at the time of cell plating. Cells maintained in culture medium devoid of hormones become progressively less sensitive to subsequent addition of testosterone or FSH. Data are discussed in relation to the sites of androgen requirements for spermatogenesis.     

Identification and regulation of testicular interferon-gamma (IFNgamma) receptor subunits: IFNgamma enhances interferon regulatory factor-1 and interleukin-1beta converting enzyme expression

Interferon-gamma (IFNgamma) transmits its signal through a specific cell surface receptor (IFNgammaR), which consists of a primary ligand binding alpha-chain (IFNgammaR alpha) and a signaling beta-chain (IFNgammaR beta). Recent studies identified the cytokines IFNgamma, interleukin-6 (IL-6), IL-1alpha, and tumor necrosis factor-alpha in testicular cells. Therefore, we: 1) examined the expression of IFNgammaR alpha and IFNgammaR beta subunits in freshly isolated and purified rat testicular cells; 2) examined the differential regulation of receptor components by cytokines using primary cultures of Sertoli cells; 3) identified the cell signaling pathway components of testicular IFNgammaR; and 4) characterized the functional role of testicular IFNgamma using primary Sertoli cells. We demonstrated the messenger RNAs for both chains of IFNgammaR in rat testicular cells using Northern hybridization analysis. Western blot analysis and immunocytochemistry showed that both specific IFNgammaR protein subunits were present in cultured primary Leydig and Sertoli cells prepared from the testes of immature rats. The expression of both IFNgammaR component messenger RNAs in cultured Sertoli cells was increased by its specific ligand (IFNgamma), as well as IL-1alpha and tumor necrosis factor-alpha, in both a time- and dose-dependent manner. IFNgamma-activation of the Janus (JAK) tyrosine kinases, JAK1 and JAK2 proteins, indicate that IFNgammaR, expressed in the Sertoli cell, is functional. Moreover, IFNgamma modulates the expression of interferon regulatory factor (IRF)-1 and IL-1beta converting enzyme genes in Sertoli cells. Thus, our data are suggestive of a role(s) for IFN-gamma in the regulation of distinct gene expression and cell-specific sensitivity to apoptosis in the testis.     

Antisense insulin-like growth factor I transferred into a rat hepatoma cell line inhibits tumorigenesis by modulating major histocompatibility complex I cell surface expression

We have established a hepatocarcinoma cell line (LFCI2 A) that produces voluminous tumors when injected subcutaneously into syngeneic Commentry rats. These neoplastic cells express both insulin-like growth factors (IGF) I and II. When transfected with an episomal cassette-expressing IGF-I antisense RNA, the modified LFCI2 A cell lines become poorly tumorigenic and, when injected subcutaneously, are associated with inhibition of the growth of the parental tumoral cells and/or induction of regression of established tumors. By contrast, cell lines isolated after transfection with the IGF-II antisense-expressing vector were as tumorigenic as the parental cell lines. The results are discussed in terms of protective immunity induced by the tumoral cells transfected by the IGF-I antisense vector. In the transfected hepatocarcinoma cells that do not produce IGF-I, the expression of major histocompatibility complex class I antigen was increased at least 4-fold compared with parental cells. The introduction of these cells in vivo induced a tumor-specific immunity that was associated with CD8 T cells.     

Reduced left ventricular relaxation velocity after acute myocardial infarction

The full length porcine granulocyte/macrophage colony stimulating factor (GM-CSF) cDNA, including secretion signal peptide coding region was recloned into baculovirus transfer vector pAcYM1. The vector was then transfected with Autographica californica nuclear polyhedrosis virus (AcNPV) DNA into SF21AE cells and the recombinant virus AcPGM was recovered. Recombinant porcine GM-CSF (rpGM-CSF) was obtained from the serum-free culture medium of Tn5 cells infected with the AcPGM virus, and was shown to be a glycosylated 21 kDa protein as confirmed by tunicamycin treatment and [3H]-glucosamine uptake. The biological activities of rpGM-CSF in AcPGM-infected cell culture supernatants were demonstrated by porcine bone marrow cell proliferation and haematopoietic cell colony formation assays. The use of rpGM-CSF enabled us to culture porcine monocytes/macrophage and dendritic-like cells, derived from either porcine bone marrow or peripheral blood, for up to 4 months.     

Haptoglobin is a Sertoli cell product in the rat seminiferous epithelium: its purification and regulation

Using multiple HPLC steps, a protein of 67 kDa (estimated by gel permeation HPLC) was purified from Sertoli cell-enriched culture medium that consisted of two dissimilar subunits of 9 (alpha chain) and 24 (beta chain) kDa on SDS-polyacrylamide under reducing conditions. Direct protein sequence analysis of the 9-kDa subunit revealed a sequence of NH2-VELGNDATDIEXD, which is identical to the alpha subunit of the rat haptoglobin (Hp). Hp is a 67-kDa tetrameric serum acute-phase protein consisting of two alpha and two beta subunits (alpha2beta2) of 8.5 kDa and 24.5 kDa, respectively. Using a 351-bp cDNA coding for Hp for northerns and two Hp primers for RT-PCR, we have demonstrated the expression of Hp in Sertoli and Leydig cells, germ cells, and the testis, but not in the epididymis. In contrast to the hepatic haptoglobin, an acute-phase protein whose steady-state mRNA level increased by as much as fivefold during induced inflammation, the testicular homolog reduced by fourfold within 24 hours following induced inflammation, suggesting that this gene is regulated differently in the testis and in the liver. Moreover, the testicular steady-state Hp mRNA level increased steadily after birth during maturation, suggesting its involvement in spermatogenesis. Using primary Sertoli cell cultures in vitro, it was found that the Sertoli cell Hp expression was not regulated by either FSH, testosterone, estradiol, dexamethasone, interleukin-1beta (IL-1beta), IL-6, interferon-gamma (INF-gamma), transforming growth factor-beta (TGF-beta), lymphocyte inhibitory factor (LIF), or germ-cell-conditioned medium (GCCM). Since transferrin secreted by Sertoli cells is an important molecule in maintaining the crucial iron level necessary for spermatogenesis, the identification of haptoglobin as a Sertoli and germ cell product adds a new member to the growing family of metal transporters in the testis that are likely to play an important role in iron metabolism in the testis.     

Stimulatory effect of follicle-stimulating hormone on basal and luteinizing hormone-stimulated testosterone secretions by the fetal rat testis in vitro

The in vitro effect of FSH on testosterone secretion by the fetal rat testis was studied. Testes were cultured in the presence or absence of either commercial human (h) FSH (Metrodine; 200 mIU/ml) or recombinant hFSH (200 mIU/ml) for 3 days and with 100 ng/ml ovine LH during the last 4 h of culture. To avoid a stimulatory effect by the 0.4% LH that contaminates Metrodine, the cultures were performed in the presence of a monoclonal anti-hLH beta antibody and with a concentration of Metrodine that had no short term stimulatory effect on testosterone production by the fetal testes in vitro. Metrodine treatment had a positive long term effect on both basal and LH-stimulated testosterone secretion by fetal testes explanted on days 18.5, 20.5, and 22.5 postconception, which was abolished by the addition of a monoclonal anti-hFSH beta antibody. LH-free recombinant FSH also augmented basal and LH-stimulated testosterone secretion of testes explanted on days 13.5, 14.5, and 18.5 postconception. The positive effect of recombinant hFSH appeared during the second day of treatment with day 14.5 and 18.5 testes and on the third day of treatment with day 13.5 testes. As it is widely accepted that FSH receptors are exclusively localized on Sertoli cells, these results suggest that on or before day 15.5 of fetal life, 1) Sertoli cells are able to respond to FSH, 2) Sertoli cells can produce factors that are able to act on Leydig cell function, and 3) Leydig cells are sensitive to FSH-induced Sertoli cell factors. In conclusion, this study points out a potential paracrine control of fetal Leydig cell function and/or differentiation by fetal Sertoli cells as soon as fetal Leydig cells differentiate.     

Pituitary tumor-transforming gene protein associates with ribosomal protein S10 and a novel human homologue of DnaJ in testicular cells

Pituitary tumor-transforming gene (PTTG) is a recently characterized proto-oncogene that is expressed specifically in adult testis. In this study, we have used in situ hybridization and developmental Northern blot assays to demonstrate that PTTG mRNA is expressed stage-specifically in spermatocytes and spermatids during rat spermatogenic cycle. We have used the yeast two-hybrid system to identify proteins that interact with PTTG in testicular cells. Two positive clones were characterized. One of the clones is the ribosomal protein S10, the other encodes a novel human DnaJ homologue designated HSJ2. Northern blot analysis showed that testis contains higher levels of HSJ2 mRNA than other tissues examined, and the expression pattern of HSJ2 mRNA in postnatal rat testis is similar to PTTG. S10 mRNA levels do not vary remarkably among different tissues and remains unchanged during testicular germ cell differentiation. In vitro binding assays demonstrated that both S10 and HSJ2 bind to PTTG specifically and that PTTG can be co-immunoprecipitated with S10 and HSJ2 from transfected cells. Moreover, the binding sites for both proteins were located within the C-terminal 75 amino acids of the PTTG protein. These results suggest that PTTG may play a role in spermatogenesis.     

Down-regulation of E-cadherin in mouse skin carcinoma cells enhances a migratory and invasive phenotype linked to matrix metalloproteinase-9 gelatinase expression

To assess the role of gelatinases in mouse skin tumor progression and their link to the expression of E-cadherin (E-CD), the cell-cell adhesion protein, we used the highly metastatic squamous HaCa4 cell line and several HaCa4-derived clones obtained by transfection of the mouse E-CD cDNA. Expression of matrix metalloproteinase-9 (MMP-9) mRNA and protein activity were present in E-CD (-) HaCa4 and control clones in culture, but they were strongly diminished in E-CD (+) clones (E24 and E62) at subconfluence. To explore the suppressive effect of the cell-cell contacts mediated by E-CD on MMP-9 expression, we introduced a plasmid encoding mouse E-CD antisense cDNA into the E24 cell clone. The transfectant P1-clones obtained with reduced or absent E-CD expression showed increased levels of MMP-9 gelatinase, motility in vitro, and metastatic potential in vivo. Expression of MMP-9 in the various cell clones was also negatively modulated by cell density, but this effect was much stronger in E-CD (+) cells, despite the fact that all of the cell clones analyzed maintained the expression of P-cadherin and made cell-cell contacts at high cell density. Our results indicate that in this cell system, the E-CD-mediated cell-cell contacts are involved in the down-regulation of MMP-9 expression. Thus, the loss of E-CD triggers a migratory and invasive phenotype in mouse squamous carcinoma cells.