Somatostatin inhibits follicle-stimulating hormone-induced adenylyl cyclase activity and proliferation in immature porcine Sertoli cell via sst2 receptor

The potential involvement of somatostatin (SRIF) in testicular function was studied by using as a model primary cultures of purified immature porcine Sertoli cells. In the present report we show that Sertoli cells express mRNA for sst2 SRIF receptor and display SRIF-sensitive adenylyl cyclase. Sensitivity of adenylyl cyclase to SRIF and its analogues is compatible with the pharmacological profile of this receptor type. Relevant cAMP production is similarly inhibited by SRIF in both basal and stimulated (by gonadotropin FSH or by forskolin) conditions. Moreover, the observed SRIF actions on Sertoli cells require functional coupling of specific membrane receptors to adenylyl cyclase via Gi proteins because pertussis toxin prevents SRIF-dependent inhibition of adenylyl cyclase in either basal or FSH-stimulated conditions. Given the potent antiproliferative actions of SRIF in other cell types, we further assessed the possible SRIF-dependent modulation of [(3)H]thymidine incorporation by Sertoli cells. Our data point to SRIF-mediated inhibition of both basal and FSH-stimulated [(3)H]thymidine uptake. This inhibition of Sertoli cell proliferation is, at least in basal conditions, also blocked by pertussis toxin pretreatment. Altogether, these data suggest that SRIF may play a role as an (local) inhibitor of FSH actions in testicular development.     

Somatostatin-dependent adenylyl cyclase activity in nonactivated and mitogen-activated human T cells: evidence for uncoupling of sst3 receptor from adenylyl cyclase

Neuropeptide somatostatin (SRIF) has been shown to modulate interleukin-2 (IL-2) secretion by mitogen-activated T cells. In this study, we further analyzed the transduction pathways underlying SRIF actions on human Jurkat T cells and compared SRIF signaling between nonactivated and mitogen-activated cells. SRIF effects on adenylyl cyclase activity in the absence and presence of mitogens were addressed by using three different analogs: SRIF14, SRIF28, and SMS 201-995. In semipurified membrane preparations obtained from nonactivated cells, all analogs inhibited adenylyl cyclase. However, in membrane preparations obtained from mitogen-activated cells, the maximal inhibition of adenylyl cyclase mediated by SRIF14 and SRIF28 equaled only one third of that measured in the absence of mitogens, whereas SMS 201-995 was completely inactive. To assess the relevant mechanisms associated with different effects of SRIF on adenylyl cyclase activity in nonactivated and mitogen-activated T cells, we performed binding assays by using iodinated SRIF as a radioligand. These experiments suggested that both the number of receptors and their affinities were almost identical in either nonactivated or activated cells. RT-PCR analysis of the pattern of SRIF receptor expression showed that nonactivated as well as activated Jurkat cells expressed only mRNA corresponding to the sst3 receptor subtype. Altogether, these data point to a functional activation-associated uncoupling of sst3 receptors from adenylyl cyclase in human T cells, indicating a T-cell activation-induced alteration in the sst3 receptor transduction pathway.     

Somatostatin analog SMS 201995 inhibits proliferation in human leukemia T-cell line: relevance of the adenylyl cyclase stimulation

Octreotide SMS 201995 is a stable somatostatin (SRIF) analog with potent antiproliferative actions in numerous cell types including normal T lymphocytes. It is currently used in the clinical treatment of different malignancies. However, the possible beneficial actions of octreotide in T-cell leukemia have not been addressed before, although these cells express SRIF receptors. For instance, human leukemia Jurkat T cells have been shown to express a single SRIF receptor isotype: sst3 that can be pharmacologically targeted by octreotide. In this study, we therefore studied SMS 201995 effects on in vitro [(3)H-CH3]thymidine incorporation in Jurkat T cells. Our data show that octreotide inhibits the proliferation of Jurkat cells both in the absence and in the presence of mitogens. By contrast, SRIF28, an endogenous SRIF analog sharing with SMS 201995 an almost identical affinity for somatostatin sst3 receptors, increases [(3)H-CH3]thymidine uptake in both mitogen-activated and nonactivated cells. To assess the mechanisms of the opposite actions of these two analogs on leukemia T-cell proliferation, we next studied their effects on adenylyl cyclase activity in whole Jurkat cells. At least in the presence of mitogens, SMS 201995 significantly enhances the adenylyl cyclase activity whereas SRIF28 inhibits it. Taken together these data are in accordance with the current hypothesis according to which increase and decrease in cAMP production are required to allow the inhibition and stimulation of T-cell proliferation, respectively. They also point to a potential therapeutic benefit of SMS 201995 in the management of human T-cell leukemia.     

sst5 somatostatin receptor mRNA induction by mitogenic activation of human T-lymphocytes.

SRIF has neuro-immunomodulatory actions on immune cells, including T-lymphocytes. Molecular mechanisms involved in these actions were studied by RT-PCR analysis of SRIF receptor expression in resting and initogen-activated human T-lymphocytes. Our results point to the mitogen-associated induction of sst5 receptor subtype. Conversely, sst3 receptor appears constitutively expressed in both activity states. Assessment of biologic actions of SRIF14 in activated T-lymphocytes indicates that, in nanomolar concentration range, this peptide moderately inhibits mitogen-induced IL-2 secretion. Nevertheless, T-lymphocyte proliferation is not inhibited in the presence of SRIF14 but is even slightly increased. Altogether these data suggest a complex mechanism of SRIF neuro-immunomodulatory actions.     

Adenylyl Cyclase/cAMP System Involvement in the Antiangiogenic Effect of Somatostatin in the Retina. Results from Transgenic Mice

Neoangiogenesis is a response to retinal hypoxia that is inhibited by somatostatin (SRIF) through its subtype 2 receptor (sst2). Using a mouse model of hypoxia-induced retinopathy, we investigated whether inhibition of adenylyl cyclase (AC) is involved in SRIF anti-angiogenic actions. Hypoxia increased AC responsiveness in wild type (WT) retinas and in retinas lacking sst2, but not in sst2-overexpressing retinas. Hypoxia also altered AC isoform expression with different patterns depending on sst2 expression level. The AC VII isoform mRNA and protein resulted the most affected. Indeed, in hypoxia AC VII expression was enhanced in WT retinas and it was further increased in sst2-lacking retinas, whereas in sst2 overexpressing retinas the increase of AC VII was lower than in WT retinas. These data suggest an involvement of AC/cAMP in mediating both hypoxia-evoked retinal neoangiogenesis and SRIF protective actions. The AC VII isoform is a candidate to a main role in these mechanisms.     

Nuclear testicular 1,25-dihydroxyvitamin D3 receptors in Sertoli cells and seminiferous tubules of adult rodents

1,25(OH)2D3 receptors were studied in whole testes, Sertoli cells, seminiferous tubules, Leydig cells and spermatogonia of adult NMRI mice and SD rats. Specific reversible high affinity binding (KD 1.4 x 10(-10)M; Nmax 72 fmol/mg protein) by a 3.5 S macromolecule was demonstrated in whole testes, Sertoli cells and seminiferous tubules. With identical techniques, no receptors were found in Leydig cells despite previous reports of 1,25(OH)2D3 actions on Leydig cell function.     

Somatostatin increases mitogen-induced IL-2 secretion and proliferation of human jurkat T cells via sst3 receptor isotype

The neuropeptide somatostatin (SRIF) modulates normal and leukemia T cell proliferation. However, neither molecular isotypes of receptors nor mechanisms involved in these somatostatin actions have been elucidated as yet. Here we show by using RT-PCR approach that mitogen-activated leukemia T cells (Jurkat) express mRNA for a single somatostatin receptor, sst3. This mRNA is apparently translated into protein since specific somatostatin binding sites (K_I1 = 78 ± 3 pM) were detected in semipurified plasma membrane preparations by using ¹²⁵I-Tyr¹-SRIF14 as a radioligand. Moreover, somatostatin inhibits adenylyl cyclase activity with similar efficiency (IC₅₀ = 23 ± 4 pM) thus strongly suggesting a functional coupling of sst3 receptor to this transduction pathway. The involvement of sst3 receptor in immuno-modulatory actions of somatostatin was assessed by analysis of neuropeptide effects on IL-2 secretion and on proliferation of mitogen-activated Jurkat cells. Our data show that in the concentrations comprised between 10 pM and 10 nM, somatostatin potentiates IL-2 secretion. This effect is correlated with somatostatin-dependent increase of Jurkat cell proliferation since the EC₅₀ concentrations for both actions were almost identical (EC₅₀ = 22 ± 9 pM and EC₅₀ = 12 ± 1 pM for IL-2 secretion and proliferation, respectively). Altogether, these data strongly suggest that in mitogen-activated Jurkat cells, somatostatin increases cell proliferation through the increase of IL-2 secretion via a functional sst3 receptor negatively coupled to the adenylyl cyclase pathway. J. Cell. Biochem. 68:62–73, 1998. © 1998 Wiley-Liss, Inc.     

Expression of Connexin 43 in normal canine testes and canine testicular tumors

In human testis, gap junctions containing connexin(Cx)43 are located within the seminiferous epithelium between Sertoli cells and between Sertoli and germ cells. Cx43 is known to play a role in the differentiation and proliferation of these cell types. It can further be associated with human seminoma development. The dog has been proposed as a model for studies of the male reproductive system, because of the frequent occurrence of testicular neoplasms. Thus, we investigated Cx43-mRNA and -protein expression in testes of normal prepubertal dogs, adult dogs, and in canine testicular tumors. Sertoli cells in prepubertal cords express Cx43 mRNA, but do synthesize only less Cx43 protein. Within the seminiferous tubules, Cx43 mRNA was detected in Sertoli cells, spermatogonia, and spermatocytes. Cx43 protein was mainly present in the basal compartment. In canine testicular tumors Cx43 mRNA was detectable in both seminoma and neoplastic Sertoli cells, whereas Cx43 protein was only found in neoplastic Sertoli cells. Our data indicate that Cx43 is regulated differentially in testicular tumors and that alterations of Cx43 expression may be involved in the pathogenesis of canine testicular malignancies. This study represents the first morphological work on the spatiotemporal expression pattern of Cx43 in normal and neoplastic canine testis.     

Crlz-1 Is Prominently Expressed in Spermatogonia and Sertoli Cells during Early Testis Development and in Spermatids during Late Spermatogenesis

The expression of the Crlz-1 gene in mouse testis, where it was found to be expressed most highly among the tested mouse organs, was analyzed spatiotemporally by employing RT-PCR and in situ hybridization techniques with the aid of immunohistochemistry and/or immunofluorescence methods. In 1-week-old neonatal testis, Crlz-1 was strongly expressed in the spermatogonia and Sertoli cells in its seminiferous cord. In 2- to 3-week-old prepubertal testis, where Sertoli cells cease to proliferate, Crlz-1 expression dropped and remained weakly at the rim layer of seminiferous cords and/or tubules, where spermatogonia are present. In the adult testis at 12 weeks after birth, Crlz-1 was expressed mainly in the spermatids near the lumen of seminiferous tubules. In a further in situ hybridization of Crlz-1 in the 12-week-old adult testis with hematoxylin nuclear counterstaining, Crlz-1 was mainly expressed at step 16 of spermatids between stages VII and VIII of seminiferous tubules as well as in their residual bodies at stage IX of seminiferous tubules.     

Ultrastructural study of the Sertoli cell and the limiting membrane in the seminiferous tubule of the adult cryptorchid rat

Cryptorchidism was simulated in 13-15-day-old rats by severing the gubernaculum testis and fixing the testis to the abdominal wall. Ultrastructural examination of the testis was made 100 days after birth when a number of modifications to the seminiferous tubules were noted. Germ cells were scanty, with only occasional spermatogonia and primary spermatocytes persisting. The nuclei of Sertoli cells were regular and oval or indented in shape. Their cytoplasm was characterized by a rich smooth endoplasmic reticulum, lipid inclusions and mitochondria with tubulo-vesicular cristae indicative of stero?dogenic activity. The decrease in the number of the germ cells induced a membrane rearrangement with numerous tight junctions and interdigitations between the Sertoli cells. Sertoli cell-specific junctional complexes were very extensive. The lamina propria of the seminiferous tubule appeared thickened and folded and the multilayered basal lamina had complex folds. After fixation with glutaraldehyde containing lanthanum, the latter substance was identified in the basal intercellular spaces of the seminiferous tubules indicating that the blood-testis barrier remains functional in the intra-abdominal testis.     

Spent media from immature seminiferous tubules and Sertoli cells inhibit adult rat Leydig cell aromatase activity

Adult rat Leydig cell aromatase activity is stimulated 2.5 fold by LH or dbcAMP. Spent media prepared from seminiferous tubules or Sertoli cells of immature rats depress both the basal and the LH stimulated estradiol syntheses (25 and 20% decreases, respectively). These inhibitory effects are further enhanced when FSH is added to the culture medium of seminiferous tubules or Sertoli cells. Rat serum as well as culture media from other cell lines are ineffective while seminiferous tubule media from other immature animals (mouse, guinea-pig, calf) inhibit the aromatase activity. This Sertoli cell factor is a heat stable protein (molecular weight greater than 10 kDa), different from the LHRH-like Sertoli cell compound, which acts on the aromatase activity at a step beyond the adenylate cyclase.     

Somatostatin regulates intracellular signaling in human carotid endothelial cells

Somatostatin (somatotropin release inhibitory factor; SRIF) is an endogenous peptide produced at sites of inflammation, making the SRIF a candidate in regulating vascular inflammation. We have used primary human coronary artery endothelial cells (hCAEC) as a model to study SRIF's vascular actions. RT-PCR analysis of hCAEC total mRNA demonstrated the presence of the sst(4) receptor subtype, providing a target for SRIF intracellular signaling. Western blotting with phospho-specific ERK1/2 antibodies showed that SRIF-14 acutely inhibited basal phosphorylation of the extracellular regulated kinases (ERK1/2) by 80%. In addition, SRIF-14 treated hCAEC cell lysates showed a 2.6-fold increase in phosphatase activity, which was inhibited by sodium vanadate. Furthermore, SRIF-14 appeared to be anti-inflammatory in hCAEC as IL-1beta-induced adhesion molecule expression was reduced by 50%. Together, these results show that the coronary artery endothelium is a direct target of SRIF action.     

Testicular involution following optic enucleation

Summary The testes of adult male Syrian hamsters underwent involution within six weeks after optic enucleation. The diameter of the seminiferous tubules was 39% less than controls. Sertoli cells, spermatogonia, and primary spermatocytes were still present, but all steps of spermatids were completely absent from the involuted testes. Lipid droplets filled the Sertoli cell cytoplasm and often encroached upon the nucleus. Sertoli cells had sparse mitochondria and smooth endoplasmic reticulum, but Golgi cisternae were abundant. Typical SertoliSertoli junctions attached contiguous Sertoli cells. With lanthanum tracers it was demonstrated that these junctions were impenetrable; therefore, the bloodtestis barrier was deemed intact. Irregularly shaped protrusions often arose from the peritubular tissue and extended inward toward the seminiferous epithelium, often displacing the cytoplasm of the Sertoli cells and spermatogonia. The core of these protrusions consisted of irregular extensions of myoid cell cytoplasm surrounded by the myoid cells' basal lamina. External to the myoid cell basal lamina were bundles of collagen filaments with the basal lamina of the seminiferous epithelium forming the outermost layer of these protrusions. The apices of the Sertoli cells gave rise to numerous leaf-like processes that extended into and obliterated the lumen of the tubules. The Sertoli cell basal cytoplasm often contained phagocytized degenerating germ cells that appeared to give rise to the lipid droplets that filled the Sertoli cell cytoplasm. Acid phosphatase rich lysosome-like organelles were seen fusing with the degenerating germ cells and lipid droplets. The degenerating germ cells also were shown to contain acid phosphatase activity.     

Gap junctions with varied permeability properties establish cell-type specific communication pathways in the rat seminiferous epithelium

Dye coupling experiments were performed to determine whether the gap junctions connecting Sertoli cells with other Sertoli cells and different germ cell stages in rats showed functional variations. Chop loading of adult rat seminiferous tubules was conducted using fluorescent dextran controls and a variety of low-molecular-weight tracers (lucifer yellow, biotin-X-cadaverine, biotin cadaverine, and neurobiotin) to evaluate dye coupling in situ, and scrape loading was used to study dye coupling in Sertoli-germ cell cocultures established using prepuberal rats. Sertoli-Sertoli coupling is relatively short range and nonselective in situ, whereas coupling between Sertoli cells and chains of spermatogonia is strongly selective for the positively charged biotin tracers relative to negatively charged lucifer yellow. Coupling between Sertoli cells and spermatogonia was also asymmetric; lucifer yellow in germ cells never diffused into Sertoli cells, and biotinylated tracers only weakly diffused from spermatogonia to Sertoli cells. Asymmetric coupling would facilitate the concentration in germ cells of molecules diffusing through junctions from Sertoli cells. Dye coupling between Sertoli cells and adluminal germ cells was too weak to detect by fluorescence microscopy, suggesting that the junctional communication between these cells may be functionally different from that between Sertoli and basal germ cells. The results show that there are multiple routes of gap junction communication in rat seminiferous tubules that differ in permeability properties and show alternative gating states. Functional diversity of gap junctions may permit regulated communication among the many interacting Sertoli cells and germ cell stages in the seminiferous epithelium.     

Functional analysis of the p53 gene in apoptosis induced by heat stress or loss of stem cell factor signaling in mouse male germ cells

Apoptosis plays an important role in controlling germ cell numbers and restricting abnormal cell proliferation during spermatogenesis. The tumor suppressor protein, p53, is highly expressed in the testis, and is known to be involved in apoptosis, which suggests that it is one of the major causes of germ cell loss in the testis. Mice that are c-kit/SCF mutant (Sl/Sld) and cryptorchid show similar testicular phenotypes; they carry undifferentiated spermatogonia and Sertoli cells in their seminiferous tubules. To investigate the role of p53-dependent apoptosis in infertile testes, we transplanted p53-deficient spermatogonia that were labeled with enhanced green fluorescence protein into cryptorchid and Sl/Sld testes. In cryptorchid testes, transplanted p53-deficient spermatogonia differentiated into spermatocytes, but not into haploid spermatids. In contrast, no differentiated germ cells were observed in Sl/Sld mutant testes. These results indicate that the mechanism of germ cell loss in the c-kit/SCF mutant is not dependent on p53, whereas the apoptotic mechanism in the cryptorchid testis is quite different (i.e., although the early stage of differentiation of spermatogonia and the meiotic prophase is dependent on p53-mediated apoptosis, the later stage of spermatids is not).