Non-muscle cofilin is a component of tubulobulbar complexes in the testis

Tubulobulbar complexes are finger-like structures that form at the interface between maturing spermatids and Sertoli cells prior to sperm release and at the interface between two Sertoli cells near the base of the seminiferous epithelium. They originate in areas previously occupied by actin filament-associated intercellular adhesion plaques known as ectoplasmic specializations. Actin filaments also are associated with tubulobulbar complexes where they appear to form a network, rather than the tightly packed bundles found in ectoplasmic specializations. Cofilin, a calcium-independent actin-depolymerizing protein, previously has been identified in the testis, but has not been localized to specific structures in the seminiferous epithelium. To determine if cofilin is found in Sertoli cells and is concentrated at actin-rich structures, we reacted fixed frozen sections of rat testis, fixed fragmented tissue, and blots of seminiferous epithelium with pan-specific and non-muscle cofilin antibodies. In addition, GeneChip microarrays (Affymetrix, Santa Clara, CA) were utilized to determine the abundance of mRNA for all cofilin isoforms in Sertoli cells. Using the monoclonal pan-specific cofilin antibody, we found specific labeling exclusively at tubulobulbar complexes and not at ectoplasmic specializations. On one-dimensional (1D) Western blots this antibody reacted monospecifically with one band, and on 2D blots reacted with two dots, which we interpret as phosphorylated and nonphosphorylated forms of a single cofilin isotype. Messenger RNA for non-muscle cofilin in Sertoli cells is about 8.5-fold higher than for muscle-type cofilin. To confirm that the non-muscle isoform of cofilin is present at tubulobulbar complexes, we used antibodies specific to non-muscle cofilin for immunofluorescent localization. As with the pan-specific antibody, we found that the non-muscle cofilin antibody exclusively labeled tubulobulbar complexes. Results presented here indicate that non-muscle cofilin is concentrated at tubulobulbar complexes. Our results also indicate that cofilin is not concentrated at ectoplasmic specializations.     

Molecular weight forms of inhibin a and inhibin B in the bovine testis change with age

To investigate alterations in the molecular weight forms of inhibin in bull testis from the infantile (4-5 wk of age) to postpubertal (49-56 wk of age) periods, testicular homogenates were obtained from animals of various ages and fractionated by a combination of immunoaffinity chromatography and SDS-PAGE. Subsequently, the fractions eluted from the SDS gels were assayed for total inhibin, inhibin A, and inhibin B by fluoroimmunoassay or immunofluorometric assays (IFMAs) and for inhibin bioactivity by an in vitro bioassay. The molecular mass patterns of inhibin A and inhibin B in the testis, as determined by the dimer-specific IFMAs, showed the presence of a peak of approximate 47 kDa until 21-26 wk of age. However, the peak disappeared after 31-32 wk of age. As bulls aged, especially after 31-32 wk of age, inhibin A and inhibin B levels increased in the molecular mass region of 27-34 kDa. Total inhibin showed two peaks, of between 20 and 26 kDa and at approximately 47 kDa, until 21-26 wk of age and a single peak between 20 and 30 kDa after 31-32 wk of age. The eluted fractions corresponding to 29, 31, or 47 kDa gave a dose-response curve that was parallel to the curve generated with 32-kDa inhibin A or 29-kDa inhibin B standard in the IFMA for inhibin A or inhibin B. The fractions corresponding to 29 and 31 kDa suppressed basal release of FSH from rat pituitary cells, but the 47-kDa fraction had a lower FSH-suppressing activity. In the testes of older bulls, immunoblot analysis revealed the presence of a 29-kDa band cross-reacting with inhibin alpha and inhibin betaB antibodies and of a 31-kDa band cross-reacting with inhibin alpha and inhibin betaA antibodies. The 47-kDa band was recognized by the alpha, betaA, and betaB antibodies. Immunohistochemisty of the testis at each age showed that inhibin alpha subunits were found exclusively in Sertoli cells, but the intensity of immunostaining diminished in older bulls, in parallel with the decrease in the testicular concentrations of total inhibin. We conclude that 1) bovine Sertoli cells produce both inhibin A and inhibin B, 2) inhibin production in Sertoli cells during the prepubertal period is characterized by the 47 kDa inhibin-related material that contains precursor forms of inhibin A and inhibin B, and 3) the proportion of the mature forms of inhibin A and inhibin B increases as bulls age, although total inhibin production in Setroli cells decreases.     

Immunolocalization and concentrations of inhibin alpha in the ovine testis and excurrent duct system

Inhibin was localized in the ovine testis, excurrent ducts, and accessory sex glands by using a rabbit antiserum against a synthetic polypeptide representing the first 30 amino acids of porcine inhibin alpha-subunit. Concentrations of inhibin in fluids entering and leaving the epididymis also were determined in a radioimmunoassay using the same antibody. In the testis, immunostaining of inhibin was conspicuous in the seminiferous epithelium. Leydig cells occasionally were stained and the tunica media of blood vessels always was stained. Intense staining was observed in the epithelia lining the rete testis and ductuli efferentes. Staining also was intense in the epithelium of the initial segment and proximal caput epididymidis, and became less intense along the length of the epididymis. These observations were consistent with concentrations of inhibin in rete testis fluid (8.2 pmol/ml) entering the ductuli efferentes and in cauda epididymal plasma (0.67 pmol/ml) leaving the epididymis. Epithelia of ampullary and vesicular glands and of some prostatic acini were positively stained, but bulbourethral glands were never stained. Adrenal cortex, some proximal convoluted tubules in the kidney, and transitional epithelium of the urethra also were stained. Based on radioimmunoassay data and fluid flow rates for the ram, it was concluded that almost all of the 328 pmol inhibin that enters the ductuli efferentes daily is endocytosed in the proximal parts of the excurrent duct system. The physiological role(s) for inhibin, or inhibin-like peptides, in the excurrent duct system remains speculative.     

Nitric oxide is a central component in neuropeptide regulation of appetite

In recent years, there have been a large number of neuropeptides discovered that regulate food intake. Many of these peptides regulate food intake by increasing or decreasing nitric oxide (NO). In the current study, we compared the effect of the food modulators ghrelin, NPY and CCK in NOS KO mice. Satiated homozygous and heterozygous NOS KO mice and their wild type controls were administered ghrelin ICV. Food intake was measured for 2 h post injection. Ghrelin did not increase food intake in the homozygous NOS KO mice compared to vehicle treated NOS KO mice, whereas food intake was increased in the wild type controls compared to vehicle treated wild type controls. NPY was administered ICV and food intake measured for 2 h. Homozygous NOS KO mice showed no increase in food intake after NPY administration, whereas the wild type controls did. In our final study, we administered CCK intraperitoneally to homozygous and heterozygous NOS KO mice and their wild type controls after overnight food deprivation. Food intake was measured for 1 h after injection. CCK inhibited food intake in wild type mice after overnight food deprivation, however, CCK failed to inhibit food intake in the NOS KO mice. The heterozygous mice showed partial food inhibition after the CCK. The current results add further support to the theory that NO is a central mediator in food intake.     

Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

Background  

Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA.     

The cancer/testis antigen CAGE-1 is a component of the acrosome of spermatids and spermatozoa

Cancer/testis antigens (CTAs) are characterized by their restricted expression pattern. In normal individuals their expression is largely restricted to the testis. In the case of cancer patients, CTA expression has also been frequently observed in the tumoral cells. CTAs are considered to be promising targets for immunotherapy. However, almost nothing is known about the properties defined by the vast majority of CTAs. Here, we have investigated the expression pattern and localization of the CTA CAGE-1 during mouse spermatogenesis. We show that protein CAGE-1 is 849 amino acids long. Analysis of the first spermatogenic wave of pubertal mice by RT-PCR and immunoblotting showed that CAGE-1 is predominantly expressed during postmeiotic stages. CAGE-1 localizes to the acrosomal matrix and acrosomal granule, as demonstrated by immunocytochemistry at the light and electron microscopic level. Taken together, our results allowed to define protein CAGE-1 as a novel component of the acrosome of mammalian spermatids and spermatozoa.     

Changes in expression of inhibin subunits in the cyclic golden hamster (Mesocricetus auratus) and the regulation of inhibin alpha subunit production by luteinizing hormone

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin alpha subunit in the ovary was also investigated. Inhibin alpha subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin alpha subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin alpha subunit on days 1 and 2. On the other hand, positive reactions for inhibin beta A and beta B subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin beta B subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin alpha subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin beta A from beta B subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin alpha subunit in interstitial cells of the cyclic hamster.     

GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop

Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5'-phosphate-dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.     

Adrenomedullin in the rat testis. II: Its production, actions on inhibin secretion, regulation by follicle-stimulating hormone, and its interaction with endothelin 1 in the Sertoli cell

The present study demonstrates the expression of adrenomedullin (ADM) in the rat Sertoli cells and its effect on inhibin production. The regulation of ADM by FSH and its interaction with endothelin 1 (EDN1) in the rat Sertoli cells have also been established. Primary culture of Sertoli cells secreted 414+/-27 pg immunoreactive ADM per 10(6) cells in 24 h and expressed Adm mRNA. In addition, the Sertoli cell was shown to co-express mRNAs encoding for the calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs) 1-3. These may account for the specific binding of ADM to the Sertoli cells. Administration of ADM to Sertoli cells resulted in an enhancement of basal and FSH-stimulated inhibin B production. On the other hand, the production of ADM and the mRNA levels of Calcrl and Ramp2 in the Sertoli cells were suppressed by FSH. The results suggest that ADM, via its control in the secretion of inhibin B, may play a role in regulating spermatogenesis as well as the hypothalamus-pituitary-gonad feedback system. In addition, like in the Leydig cell, ADM and EDN1 were found to regulate the production of each other in opposite directions in the Sertoli cells, suggesting the presence of yet another local regulatory mechanism in the rat testis that may be important in modulating testicular functions regulated by gonadotropins.     

Differing effects of endocrine-disrupting chemicals on basal and FSH-stimulated progesterone production in rat granulosa-luteal cells

Previous studies have shown that the phytoestrogen, genistein, inhibits basal and forskolin-stimulated progesterone synthesis in rat granulosa-luteal cells. Genistein, however, not only binds and activates the estrogen receptor (ER), but is also a potent inhibitor of tyrosine kinase. In these studies we have compared the effects of estradiol, two other phytoestrogens, apigenin and coumarin, the pesticide, [2-(chlorphenyl)-2-(4-chlorphenyl)-1,1,1-trichlorethan] (2,4'DDT), and the industrial chemical, 4-octyl-phenol, on basal and follicle stimulating hormone (FSH)-stimulated progesterone production in the same experimental system. Only a supraphysiological dose of estradiol (10(-5) M) significantly inhibited basal and forskolin-stimulated progesterone production in granulosa-luteal cells, but had no effect on FSH-stimulated production. In contrast, apigenin, DDT, and octyl-phenol stimulated basal progesterone production at doses around 10(-8) to 10(-7) M, but this effect was reversed at higher doses. Coumarin was without effect. Like basal production, the two phytoestrogens had opposing effects on FSH-stimulated progesterone production. Genistein at 10(-5) M was inhibitory, while apigenin significantly potentiated the response at 19(-7) M. In contrast, DDT had no effect on the FSH-induced response, though 10(-7) M octyl-phenol nearly doubled the response. While all these chemicals are known to interact with the estrogen receptor to a greater or lesser extent, these studies suggest that like genistein, these different endocrine-disrupting chemicals may have other actions apart from those on the estrogen receptor.     

Hormonal regulation of beta-endorphin in the testis

It is well established that beta-endorphin has a regulatory influence on the reproductive function at the level of the hypothalamic-pituitary axis. However, recent immunohistochemical evidence demonstrated that beta-endorphin is also present in the Leydig cells of fetal, neonatal and adult mice and hamsters. In addition, beta-endorphin synthesis was localized in the Leydig cells of adult rats, leading to the hypothesis of a direct function of the peptide in the reproductive organs. Our interest was to investigate the role of beta-endorphin at testicular level. We have demonstrated the presence of high-affinity opioid binding sites (Kd in the nanomolar range) in tubular homogenates and Sertoli cells in culture of adult (50 days) and immature (18 days post-natal) rat testes. Also, chronic beta-endorphin treatment of the Sertoli cells significantly inhibited basal and FSH-stimulated androgen-binding protein production, this effect being prevented by the universal opiate antagonist naloxone. No opiate binding was observed on Leydig cell cultures. Furthermore, we have demonstrated that acute or chronic beta-endorphin treatment does not affect testosterone production by Leydig cells in vitro, consistent with the absence of receptors on these cells. On the other hand, fetal Leydig cells (21 days fetal life) in culture produced considerable amounts of beta-endorphin. Also, fetal Leydig cells represented a preferred in vitro system to study beta-endorphin release since in adult cell culture a marked degradation of the peptide was detected (greater than 50%). beta-endorphin accumulation for 3 and 5 days was markedly increased by inhibitors of steroid biosynthesis (1.5-fold); a significant reduction by GnRH at both days (by 50-30%) was observed, while by dexamethasone the reduction was only noted after 5 days of treatment (by 50%). Acute stimulation (3 h) of control cells with hCG enhanced by 10-12-fold the beta-endorphin secretion. The hormone stimulation of beta-endorphin production was not mediated by testosterone. On the contrary, inhibition of Leydig cells steroid biosynthesis markedly increased basal and hCG-stimulated beta-endorphin production (150-200%), suggesting autocrine negative modulation of Leydig cell beta-endorphin by androgen and/or its metabolites. In contrast, dexamethasone reduced basal and hCG-stimulated beta-endorphin production (by 50%). Altogether these findings indicate that beta-endorphin produced within the Leydig cells may behave as a paracrine inhibitor of the Sertoli cell function and demonstrate that the peptide production is under direct control by gonadotropins and is modulated by steroids.     

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

Human Cytomegalovirus (HCMV), an ubiquitous β-herpesvirus, is a significant pathogen that causes medically severe diseases in immunocompromised individuals and in congenitally infected neonates. RhoB belongs to the family of Rho GTPases, which regulates diverse cellular processes. Rho proteins are implicated in the entry and egress from the host cell of mainly α- and γ-herpesviruses, whereas β-herpesviruses are the least studied in this regard. Here, we studied the role of RhoB GTPase during HCMV lytic infection. Microscopy analysis, both in fixed and live infected cells showed that RhoB was translocated to the assembly complex/compartment (AC) of HCMV, a cytoplasmic zone in infected cells where many viral structural proteins are known to accumulate and assembly of new virions takes place. Furthermore, RhoB was localized at the AC even when the expression of the late HCMV AC proteins was inhibited. At the very late stages of infection, cellular projections were formed containing RhoB and HCMV virions, potentially contributing to the successful viral spread. Interestingly, the knockdown of RhoB in HCMV-infected cells resulted in a significant reduction of the virus titer and could also affect the accumulation of AC viral proteins at this subcellular compartment. RhoB knockdown also affected actin fibers'' structure. Actin reorganization was observed at late stages of infection originating from the viral AC and surrounding the cellular projections, implying a potential interplay between RhoB and actin during HCMV assembly and egress. In conclusion, our results demonstrate for the first time that RhoB is a constituent of the viral AC and is required for HCMV productive infection.     

Parasympathetic component of the regulation of the kallikrein-kinin system in experimental pneumonia

Experimental vagus-bacterium pneumonia was modeled on 53 rabbits. Influence of nervous vagus on kallikrein-kinin system (KKS) was studied on 23 rats. It is obvious that disturbance of parasympathetic regulation may be an additional factor of KKS activation. Lowering of kinase II under experimental pneumonia was discovered both in the blood and in the lung. Activation of kinase II in the blood was after vagotomy. So parasympathetic regulation of kinase activity is of no significance in experimental pneumonia, and lowering of kinase activity is connected with bacterial factors.     

The trophoblast is a component of the innate immune system during pregnancy

Systemic infection with Listeria monocytogenes, a Gram-positive intracellular bacterium, has been used extensively to analyze the innate immune response. Macrophages are central to this response, acting as both the host for and principal defense against this bacterium. During pregnancy L. monocytogenes has a predilection for replication at the maternal-placental interface and consequently is an important cause of fetal morbidity and mortality. However, macrophages are mostly excluded from the murine placenta with neutrophils acting as the main immune effector cell against this bacterium. Colony stimulating factor (CSF)-1, a macrophage growth factor, is synthesized in high concentrations by the uterine epithelium during pregnancy, where it is targeted to trophoblast bearing CSF-1-receptors. To define the involvement of CSF-1 in placental immunity, we infected pregnant mice either homozygous or heterozygous for an inactivating recessive mutation in the gene for CSF-1 (osteopetrotic; Csfmop) with L. monocytogenes. CSF-1 was required to recruit neutrophils to the site of listerial infection in the decidua basalis, and infection by Listeria remained unrestrained in its absence. CSF-1 acted by inducing the trophoblast to synthesize the neutrophil chemoattractants (KC) and macrophage inflammatory protein (MIP)-2. Thus, during pregnancy, trophoblast responsive to CSF-1 acts to organize the maternal immune response to bacterial infection at the utero-placental interface. This previously unknown function indicates that the trophoblast acts as a pregnancy-specific component of the innate immune system.     

Immunoactive inhibin as a marker of Sertoli cell function following cytotoxic damage to the human testis

Sertoli and Leydig cell functions were evaluated in men with testicular damage due either to cytotoxic chemotherapy (CCT) or radiotherapy (XRT). Serum immunoactive inhibin, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone concentrations were measured in 15 men (19-50 years) who had received 6-10 courses of combination CCT (mustine, vinblastine, procarbazine and prednisolone) for Hodgkin's disease 1-8 years earlier and 18 men (21-49 years) who had undergone unilateral orchidectomy for testicular seminoma followed by XRT (30 Gy) to the remaining testis, 1-4 years earlier. Normal men (n = 16, 19-36 years) acted as controls. Median inhibin (422 U/l) and testosterone (16.0 nmol/l) levels in the CCT-treated group were not significantly different from controls, whereas median FSH (14.5 IU/l) and LH (10.0 IU/l) levels were higher (p less than 0.0001 and p less than 0.001) than normal (2.9 and 5.5 IU/l). The median inhibin/FSH (I/FSH) ratio in the patients was lower (p less than 0.0001) than in the controls (33.8 vs. 187.0) as was the testosterone/LH (T/LH) ratio (1.7 vs. 3.8, p less than 0.001). In the XRT-treated group, both median inhibin (194.5 U/l) and testosterone (12.7 nmol/l) levels were lower (p less than 0.0001 and p less than 0.01) than normal (532.8 U/l and 20.0 nmol/l) in the presence of greatly elevated FSH (26.0 IU/l) and LH (14.5 IU/l) levels. In conclusion, CCT-induced testicular damage is associated with subtle Sertoli and Leydig cell dysfunction demonstrated by the reduced I/FSH and T/LH ratios; however, compensatory mechanisms maintain normal testosterone and inhibin levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop

No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it.