Cyclic expression of endothelin-converting enzyme-1 mediates the functional regulation of seminiferous tubule contraction.

The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.

FLIP is expressed in mouse testis and protects germ cells from apoptosis.

Apoptosis control in adult testis is crucial to achieve normal spermatogenesis. In this study c-FLIP, an apoptosis-modulating protein, was investigated. In Western blot and immunohistochemical analyses, the 55 KDa c-FLIP long isoform (c-FLIP(L)) was found to be expressed strongly in spermatocytes and spermatids, at low levels in spermatogonia and at almost undetectable levels in Sertoli cells. This expression pattern was confirmed by Northern blot analyses. Further experiments carried out on GC-1spg germ cell line revealed that reducing c-FLIP(L) expression increases Fas-dependent apoptosis. Conversely, restoring c-FLIP(L) expression reduces this response to control levels. Caspase-10 expression was found to match c-FLIP(L) expression pattern; further, caspase-10 activation upon anti-Fas treatment inversely correlated with c-FLIP(L) expression. Finally, TUNEL staining of seminiferous tubules incubated with anti-Fas antibody showed that apoptosis occurs mostly in basally located germ cells, indicating that such cells, expressing low levels of c-FLIP(L), are sensitive to Fas-mediated apoptosis. These data indicate for the first time that c-FLIP(L) might control germ cell apoptosis and caspase activity in the adult testis.

The endogenous caspase-8 inhibitor c-FLIPL regulates ER morphology and crosstalk with mitochondria.

Components of the death receptor-mediated pathways like caspase-8 have been identified in complexes at intracellular membranes to spatially restrict the processing of local targets. In this study, we report that the long isoform of the cellular FLICE-inhibitory protein (c-FLIP(L)), a well-known inhibitor of the extrinsic cell death initiator caspase-8, localizes at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). ER morphology was disrupted and ER Ca(2+)-release as well as ER-mitochondria tethering was decreased in c-FLIP(-/-) mouse embryonic fibroblasts (MEFs). Mechanistically, c-FLIP ablation resulted in enhanced basal caspase-8 activation and in caspase-mediated processing of the ER-shaping protein reticulon-4 (RTN4) that was corrected by re-introduction of c-FLIP(L) and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIP(L) emerges as a component of the MAMs signaling platforms, where caspases appear to regulate ER morphology and ER-mitochondria crosstalk by impinging on ER-shaping proteins like the RTN4.

Ontogenesis of the nuclear 3,5,3'-triiodothyronine receptor in the rat testis.

The aim of this study was to investigate the ontogenesis of the nuclear T3 receptor among the different cell types in the rat testis from fetuses at the 19th day of gestation and animals 1, 5, 15, 20, and 60 days after birth. Whole testis, tubular fraction, nontubular fractions, and Sertoli cells cultured in vitro or enriched in vivo by irradiation were used. The results demonstrate that high affinity, low capacity T3-binding sites are localized only in Sertoli cells; the binding specificity and affinity (Kd ranges from 0.8 +/- 0.2 to 2.6 +/- 0.4 nM) do not change significantly with the age of the animals and are comparable to those observed for T3 receptors in other mammalian tissues. In the whole testis, the concentration of receptors changes during gonadal development, being maximally expressed in the fetus (154.3 +/- 8.1 fg T3 bound/10(6) nuclei) and from 1 (203.4 +/- 10.9 fg T3 bound/10(6) nuclei) to 5 (185.3 +/- 15.1 fg T3 bound/10(6) nuclei) days of postnatal life, decreasing significantly at 15 and 20 days (65.4 +/- 2.0 and 57.9 +/- 1.9 fg T3 bound/10(6) nuclei, respectively) and being virtually absent in the adult. The same change in receptor concentration was found in Sertoli cells obtained by different techniques. This ontogenetic profile coincides with the pattern of Sertoli cell proliferation and differentiation, thus suggesting a role of thyroid hormones in the regulation of growth and maturation of the somatic cells of the seminiferous epithelium.

Rat testicular myoid cells respond to endothelin: characterization of binding and signal transduction pathway.

The presence of endothelin (ET), a vasoconstrictor peptide, in the testis suggests that it may regulate nonvascular target cells. We investigated binding ability, regulation of inositol phosphate metabolism, changes in cytosolic free Ca2+ concentrations ([Ca2+]i), and induction of morphological changes by ET-1 in rat primary testicular myoid cell cultures. ET-1 specifically bound to highly purified rat testicular myoid cells in a time- and temperature-dependent manner. Scatchard analysis of the binding studies indicated the presence of a single class of high affinity binding sites, with an apparent Kd of 3 x 10(-10) M and a maximal binding capacity of 10(5) sites/cell. ET-1 induced both rapid production of inositol triphosphate and mobilization of [Ca2+]i in a concentration-dependent fashion. By contrast, inositol lipid metabolism was slightly affected by ET-1 in the total peritubular cell population. Purified Sertoli cells failed to show either ET-1 binding or ET-1-induced phosphatidylinositol hydrolysis. Mobilization of [Ca2+]i mostly depended upon the release of Ca2+ from thapsigargin-sensitive intracellular Ca2+, whereas it was not affected by abolishment of the Ca2+ gradient through the plasma membrane or by inhibition of L-type voltage-sensitive Ca(2+)-channels by nifedipine. These findings together with the fact that Sertoli cells are unable to respond to and bind ET-1 indicate that ET is a specific agonist of myoid cells in the seminiferous tubule and suggest a role for ET-1 in the autocrine/paracrine regulation of testicular function.

Activation of inositol phospholipid turnover and calcium signaling in rat Sertoli cells by P2-purinergic receptors: modulation of follicle-stimulating hormone responses.

To study the role of extracellular nucleotides in the regulation of Sertoli cells, the effects of ATP and its analogs on the Ca(2+)-phospholipid- and cAMP-dependent pathways were tested. Cultured Sertoli cells from immature animals were incubated with ATP or structurally related compounds, and phosphoinositide (PI) turnover or cAMP accumulation was measured. Among the several nucleotide phosphate analogs tested, adenosine 5'-O-(3-thiotriphosphate) was the agonist most potent in stimulating inositol phosphate accumulation. The effects of purine nucleotides on PI turnover were time and concentration dependent. Because nonhydrolizable ATP analogs also stimulated PI turnover, ATP metabolites or metabolic products are not responsible for the observed stimulation. The order of potency of the different ATP analogs [adenosine 5'-O-(3-thiotriphosphate) > ATP approximately equal to UTP > beta, gamma-methyleneadenosine 5'-triphosphate, 2-methylthio-ATP > adenosine] was consistent with the presence of P2U receptors (nucleotide receptors) on the surface of the Sertoli cell. Augmented PI turnover was accompanied by a transient increase in Ca2+ concentration, measured in single Sertoli cells loaded with the intracellular Ca2+ indicator fura-2. When used alone, ATP and its analogs did not have a direct effect on cAMP levels in the Sertoli cell. However, ATP or its analogs inhibited FSH-dependent cAMP accumulation by more than 70%. Purine nucleotides also efficiently blocked the effects of FSH distal to cAMP accumulation, because extracellular ATP completely reversed the changes in Sertoli cell shape induced by FSH. The nucleotide-dependent inhibition of cAMP accumulation was blocked by pertussis toxin to a different degree depending on the purine or pirimidine nucleotide used. This indicated that more than one mechanism contributes to the purine nucleotide-dependent inhibition of cAMP accumulation. These data provide evidence that purine nucleotide receptors coupled to multiple pathways are present on the Sertoli cell in culture, and that extracellular ATP has profound biological effects on the FSH responsiveness of the Sertoli cell.

Murine Sertoli cells: major histocompatibility antigens and glycoconjugates.

Sertoli cells, cultured from testes of 2-3-week-old Balb/c mice, contain tripartite nucleoli, exhibit phagocytic function, and have the typical morphologic appearance of Sertoli cells by light microscopy, transmission and scanning electron microscopy. Fluorescence-activated cell sorter analysis indicated the presence on mouse. Sertoli cells of H-2 but not Ia antigens. Alpha-D-mannose and N-acetyl-D-glucosamine determinants are detected on Sertoli cell surface by inhibition of lectin binding using appropriate sugars. Interaction of Sertoli cells with concanavalin A (Con A) or wheatgerm agglutinin (WGA) results in rapid patching of the labeled lectin, which become internalized as perinuclear vesicles. These changes are accompanied by rounding of the Sertoli cell, mimicking cellular changes known to occur when fat Sertoli cells are stimulated in vitro by FSH or cyclic AMP. Thus, Sertoli cells have surface alloantigens that permit them to serve as target to cytotoxic T lymphocytes, but not as antigen presenting cells.

Testicular germ cells of HIV-seropositive asymptomatic men are infected by the virus.

In situ PCR hybridization studies in the testis of infected asymptomatic subjects detected the presence of HIV-1 proviral DNA in the nuclei of germ cells at all stages of differentiation suggesting that HIV-seropositive men produce infected spermatozoa that are released in the genital tract. In all subjects studied spermatogenesis was normal, the presence of provirus was not associated with germ cell damage and a very mild local immune response was observed. The HIV hybridization pattern observed in germ cells supports the hypothesis of a clonal infection. It is suggested the possibility of a direct infection of the germ cells by cell-free virus and that the testis might represent a site of early viral localization, well tolerated because of the immune privilege of this organ.

Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells.

In addition to act as an antiapoptotic protein, B-cell lymphoma (bcl)-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia-inducible factor-1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes, such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1α protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1α protein half-life impairing HIF-1α protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia. Finally, we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2.

c-Flip overexpression affects satellite cell proliferation and promotes skeletal muscle aging.

This study shows that forcing c-Flip overexpression in undifferentiated skeletal myogenic cells in vivo results in early aging muscle phenotype. In the transgenic mice, adult muscle histology, histochemistry and biochemistry show strong alterations: reduction of fibers size and muscle mass, mitochondrial abnormalities, increase in protein oxidation and apoptosis markers and reduced AKT/GSK3ß phosphorylation. In the infant, higher levels of Pax-7, PCNA, P-ERK and active-caspase-3 were observed, indicating enhanced proliferation and concomitant apoptosis of myogenic precursors. Increased proliferation correlated with NF-κB activation, detected as p65 phosphorylation, and with high levels of embryonic myosin heavy chain. Reduced regenerative potential after muscle damage in the adult and impaired fiber growth associated with reduced NFATc2 activation in the infant were also observed, indicating that the satellite cell pool is prematurely compromised. Altogether, these data show a role for c-Flip in modulating skeletal muscle phenotype by affecting the proliferative potential of undifferentiated cells. This finding indicates a novel additional mechanism through which c-Flip might possibly control tissue remodeling.

Presence of membrane and soluble forms of Fas ligand and of matrilysin (MMP-7) activity in normal and abnormal human semen.

BACKGROUND: The aim of this study is to shed some light on the role of the Fas system in human semen, by investigating whether there is an association between the expression of the molecules regulating the Fas system [membrane-bound Fas ligand (mFasL), soluble Fas ligand (sFasL) and matrilysin, the metalloprotease cleaving mFasL to sFasL] and sperm parameters. METHODS: We investigated, by flow cytometric analysis, the presence of FasL on spermatozoa from normozoospermic and teratozoospermic subjects and, by western blot, the presence of sFasL and matrilysin in the seminal plasma of the same samples as well as on samples from azoospermic subjects. The enzymatic activity of matrilysin was examined by gel zymography. RESULTS: We observed that sperm cells expressed mFasL in 22% of normozoospermic men, whereas it was absent from spermatozoa from teratozoospermic patients. Higher levels of sFasL and augmented enzymatic activity of matrilysin were found in azoospermic samples. CONCLUSIONS: The presence of mFasL on sperm from normozoospermic men and its absence in pathological samples emphasize the role of the Fas system in human semen. Moreover, the presence of both sFasL and matrilysin in seminal plasma implies a fine regulation of the function of the Fas system and, consequently, of the apoptotic process in the human genital tract.

The distribution of spectrin along the membranes of normal and echinocytic human erythrocytes.

Spectrin molecules are distributed uniformly throughout the submembranous regions of intact human erythrocytes. Spectrin does not appear to extend into the red blood cell cytoplasm to any significant extent. Thus, it does not form a recognizable internal scaffolding nor does it seem to connect distant segments of the cell membrane. Spectrin retains its submembranous location in the spiny processes of echinocytes produced by ATP depletion. Thus, these processes do not seem to form by a simple extrusion mechanism powered by contraction of the spectrin network. Spectrin seems to be important for the stability of the lipid bilayer of the red cell membrane, and it probably also plays a role in regulating red cell shape. How it performs either function is still unknown.

Spectrin, fodrin and protein 4.1-like proteins in differentiating rat germ cells.

The presence and the distribution of proteins of the membrane skeleton in differentiating germ cells of the rat has been investigated. Immunofluorescence and immunoblotting analysis, performed using monoclonal and polyclonal antibodies to human erythroid alpha-spectrin and protein 4.1 and to brain spectrin (fodrin), demonstrated the presence of analogues of spectrin and fodrin in spermatocytes and round spermatids and of protein 4.1-like molecules in spermatocytes, spermatids and spermatozoa. Spectrin and fodrin showed molecular weights comparable to those of their analogues in erythrocytes but a distinct intracellular distribution. Fodrin was localized along the plasma membrane while spectrin appeared associated with the regions of the Golgi apparatus and of the developing acrosome. Antibodies to protein 4.1 recognized molecules with a molecular weight not comparable with that in erythrocytes, and their presence in spermatozoa was confined to specific regions of the head and of the tail.

Dual control of seminiferous tubule contractility mediated by ETA and ETB endothelin receptor subtypes.

Testicular myoid cells surrounding the seminiferous tubule are contractile cells responsible for peritubular contractility and for the propulsion of tubular fluid and spermatozoa. We have investigated the contractile response of rat myoid cells to endothelins (ETs) in cell and organ culture and analyzed the cell signaling involved. ET-2, ET-3, and IRL 1620, a highly selective agonist of ETB receptor, elicit [Ca2+]i increases, though with dissimilar potencies and kinetics. Competition binding assays using [125I]ET-1, [125I]ET-3 and [125I]IRL 1620 show that myoid cells express both ETA and ETB receptors with high affinity for ET-1 and ET-1/ ET-3, respectively. All endothelin isoforms activate phosphoinositide (PI) turnover, but only stimulation of the ETA receptor mediates both PI turnover and mobilization of [Ca2+]i. Although stimulation of the ETB receptor with IRL 1620 fails to produce a significant effect on inositol phosphate (IP) production, it induces mobilization of a thapsigargin-sensitive intracellular Ca2+ pool in the absence of any measurable IP production. We also studied the effect of U-73122 [1-(6-[17-beta-3-methoxyestra-1,3,5 (10)-trien-17-yl] amino/hexyl)-1H-pirrole-2,5-dione] and its inactive analog, U-73343, on Ca2+ mobilization and IP production after selective stimulation of ET receptors. U-73122 (1 microM) completely inhibited the effect of ETA-mediated ET-1 stimulation of IP production, whereas U-73343 was inactive. However, in the presence of U-73122, the selective stimulation of ETB receptors induced the mobilization of a thapsigargin-sensitive and inositol phosphate-independent intracellular Ca2+ pool. The ETB receptor-dependent mobilization of [Ca2+]i resulted mainly from Ca2+ release from intracellular Ca2+ stores. This paper illustrates contraction of myoid cells in the seminiferous tubule in response to selective activation of either ET receptor. Scanning electron microscopy of the peritubular tissue demonstrates that the contractile response to ET was inhibited by a combination of BQ-123 and BQ-788, but not by either antagonist alone. Moreover, the observation that selective stimulation of ETB receptor with IRL 1620 also resulted in cell contraction strongly suggests that stimulation of either ETA or ETB receptors alone may be sufficient to elicit seminiferous tubule contractility. Two types of receptors account for the actions of endothelin on contractile activity of seminiferous tubule: 1) an ETA receptor that is positively coupled to phospholipase C (PLC) and Ca2+ mobilization; and 2) an ETB receptor that induces the mobilization of a thapsigargin-sensitive intracellular Ca2+ pool in a manner independent of the formation of inositol phosphates. ET may play a complex role in regulating the flux of spermatozoa along the seminiferous tubule through its contractile effect on peritubular myoid cells.

Identification and characterization of an ecto-ATPase activity in rat Sertoli cells.

A membrane associated extracellular ATPase (ecto) has been identified on rat Sertoli cells. Sertoli cell ecto-ATPase demonstrated a Km for ATP of 114 muM and a V(max) of 1.79 mumol/min/2 x 10(5) cells and was activated by either Mg2+ or Ca2+. This ecto-ATPase hydrolyzes other nucleoside triphosphates, but is inactive with ADP. The effects of some possible inhibitors of ectonucleotidases on the breakdown of extracellular ATP by Sertoli cells were also investigated.

Surface interaction in vitro between Sertoli cells and germ cells at different stages of spermatogenesis.

The presence of surface-recognition mechanisms between somatic and germ cells of the seminiferous epithelium has been studied in the rat by an assay in vitro based on the ability of homogeneous populations of spermatogenic cells, at specific differentiative stages, to adhere to monolayers of cultured Sertoli cells. The results show that germ cells adhere specifically to Sertoli cells and that the adhesion is dependent on the differentiative stage of the germ cells. Pachytene spermatocytes show the highest ability to adhere and form typical junctional specializations with the underlying Sertoli cells, while round spermatids adhere much less to the substrate. The possible regulative role of a somatic cell-germ interaction is discussed.

Proteins of the membrane skeleton in rat Sertoli cells.

Analogues of the alpha, beta and gamma subunits of human spectrin and erythroid protein 4.1 have been detected in rat Sertoli cell primary cultures. Immunofluorescence of permeabilized cells showed that erythroid type spectrin, protein 4.1 and actin co-distribute within the cells as filamentous structures. Fodrin-like molecules were distributed in a diffuse manner, mostly associated with the plasma membrane. Immunoprecipitation and immunoblotting experiments indicated that the polypeptides present in rat Sertoli cells are immunologically related and display molecular weights similar to their analogues in the human erythroid and non-erythroid membrane skeleton.