Distribution Of Α-tubulin Research Articles

P–113 α-tubulin and tyrosine phosphorylation immunolocalization on human sperm from a globozoospermic patient with proper embryo development after ICSI

Abstract Study question Can novel sperm biomarkers, such as α-tubulin and tyrosine phosphorylation, help to predict the fertilizing potential osf a globozoospermic sample? Summary answer The characterization of α-tubulin and tyrosine phosphorylation provide additional information that could be useful to determine the fertilizing capacity of the globozoospermic samples. What is known already Globozoospermia is a severe disorder characterized by the acrosome absence and other sperm alterations such as tail structural disorders and chromatin condensation abnormalities. This set of defects makes globozoospermia correlates with primary infertility and low fertilisation rates. Therefore, additional studies are necessary to know how the globozoospermia affects the disposition of α-tubulin and tyrosine phosphorylation and the relationship between embryo development and pregnancy. In this context, previous studies have independently described different patterns of α-tubulin and tyrosine phosphorylation in normozoospermic samples. Specifically, the continuous α-tubulin distribution along the flagellum and tyrosine phosphorylation have been recently linked to proper sperm functionality. Study design, size, duration We conducted a prospective study. The sample was obtained from a globozoospermic patient in October 2019. This sample was divided into two fractions, one proceeded to ICSI and the another was fixed to characterize α-tubulin and tyrosine phosphorylation using confocal microscopy. A total of 200 sperm were analyzed for each biomarker. Participants/materials, setting, methods A 38-year-old man requested assisted reproduction in IVF Spain after a failed treatment with no fertilized oocytes. The clinical procedure was performed at IVF Spain and the characterization studies were made at the University of Alicante. The flagellar cytoskeleton was assessed using anti-α-tubulin antibody (Sigma-Aldrich) at a 1:600 dilution. Besides, tyrosine phosphorylation was detected using anti-phosphotyrosine primary antibody (PY20, Sigma-Aldrich) at a 1:500 dilution. Spermatozoa were evaluated using a confocal microscope (Zeiss LSM 800). Main results and the role of chance Regarding the α-tubulin characterization, only the 33% of spermatozoa showed continuous labelling in the tail and in the 67% the fluorescence appeared in the terminal piece of the flagellum. Otherwise, we only observed 0.5% of positive tyrosine phosphorylation in the studied cells, whereas the 99.5% of sperm analyzed did not show positive fluorescence. The clinical parameters showed a fertilization rate of 20% with only one embryo after the MII oocyte artificial activation by calcium ionophore. The embryo development was further adequate, and it acquired Bt5AA status on day five. Unfortunately, the pregnancy did not ensue after the embryo transfer. Limitations, reasons for caution The main limitation of this study was that due to the very low frequency of globozoospermia (<0.01%) this research was conducted only in one patient. Thus, the present results are preliminary. We need to characterize the aforementioned biomarkers in additional globozoospermic samples to establish relationships with clinical parameters. Wider implications of the findings: The study of potential candidate biomarkers like α-tubulin and tyrosine phosphorylation in globozoospermia and the linkage with clinical parameters would facilitate the diagnosis and improve the selection of more effective treatment techniques. Trial registration number Not applicable

Melatonin Restores the Developmental Competence of Heat Stressed Porcine Oocytes, and Alters the Expression of Genes Related to Oocyte Maturation.

Simple SummaryMelatonin improves the quality and in vitro maturation (IVM) of oocytes under heat stress. Melatonin treatment counteracts the adverse effects induced by heat stress (HS), such as the poor survival rate and maturation rate, distribution of α-tubulin and F-actin, expression of NRF2 and GDF9 mRNA. However, HS and melatonin have similar effects on increasing expression of HSP70 and NRF2 mRNA. Furthermore, HS inhibits expression of GDF9 mRNA.Melatonin enhances the quality and in vitro maturation (IVM) of oocytes under heat stress (HS), but the mechanism of melatonin in reducing HS injury on oocytes is not fully understood. In this study, porcine cumulus-oocyte complexes (COCs) were randomly divided into three groups. The COCs of the control group were cultured at 38.5 °C for 42 h, and the COCs of the HS group were cultured at 41.5 °C for 4 h, and then transferred into 38.5 °C for 38 h. The COCs of the HS + melatonin group were cultured with 10−9 M melatonin under the same conditions as the HS group. The survival rate, maturation rate, distribution of α-tubulin and F-actin of the oocytes were assessed. In addition, the expression profiles for genes related to the oocyte maturation, including heat shock protein 70 (HSP70), nuclear factor erythroid 2-related factor 2 (NRF2), cyclin-dependent kinase 1 (CDK1), growth differentiation factor 9 (GDF9) were analyzed by real-time quantitative PCR. The results showed that HS decreased the survival rate and maturation rate, distribution of α-tubulin and F-actin, but melatonin treatment could partly counteract these adverse effects. In addition, HS increased expression of HSP70 and NRF2 mRNA, and melatonin treatment had a similar effect on HSP70 expression, but had a contrary effect on NRF2 expression. Furthermore, HS inhibited expression of CDK1 and GDF9 mRNA, but melatonin treatment could weaken the effect on GDF9 expression induced by HS. In summary, melatonin treatment could attenuate the unfavorable effects induced by HS to enhance developmental competence of porcine oocytes during IVM.

Proper cytoskeleton α-tubulin distribution is concomitant to tyrosine phosphorylation during in vitro capacitation and acrosomal reaction in human spermatozoa.

Spermatozoa motility is a key parameter during the fertilization process. In this context, spermatozoa tyrosine protein phosphorylation and an appropriate cytoskeleton α-tubulin distribution are some of the most important physiological events involved in motility. However, the relationship between these two biomarkers remains poorly defined. Here, we characterized simultaneously by immunocytochemistry the α-tubulin (TUBA4A) distribution and the tyrosine phosphorylation at flagellum before capacitation, during different capacitation times (1 and 4 hr), and after acrosome reaction induction in human spermatozoa. We found that the absence of spermatozoa phosphorylation in tyrosine residues positively and significantly correlated (p < 0.05) with the terminal piece α-tubulin flagellar distribution in all physiological conditions. Conversely, we observed a positive significant correlation (p < 0.01) between phosphorylated spermatozoa and continuous α-tubulin distribution in spermatozoa flagellum, independently of the physiological condition. Similarly, the subpopulation of spermatozoa with tyrosine phosphorylated and continuous α-tubulin increases with longer capacitation times and after the acrosome reaction induction. Overall, these findings provide novel insights into the post-transcriptional physiological events associated to α-tubulin and the tyrosine phosphorylation during fertilization, which present potential implications for the improvement of spermatozoa selection methods.

Effect of melatonin on actin and α-tubulin distribution related to in vitro capacitation in ram sperm

Effect of melatonin on actin and α-tubulin distribution related to in vitro capacitation in ram sperm

Immunocytochemical Analysis of α-Tubulin Distribution Before and After Rapid Axopodial Contraction in the Centrohelid Raphidocystis contractilis

The centrohelid Raphidocystis contractilis is a heliozoan that has many radiating axopodia, each containing a bundle of microtubules. Although the rapid contraction of the axopodia at nearly a video rate (30 frames/s) is induced by mechanical stimuli, the mechanism underlying this phenomenon in R. contractilis has not yet been elucidated. In the present study, we described for the first time an adequate immunocytochemical fixation procedure for R. contractilis and the cellular distribution of α-tubulin before and after rapid axopodial contraction. We developed a flow-through chamber equipped with a micro-syringe pump that allowed the test solution to be injected at a flow rate below the threshold required to induce rapid axopodial contraction. Next, we used this injection method for evaluating the effects of different combinations of two fixatives (paraformaldehyde or glutaraldehyde) and two buffers (phosphate buffer or PHEM) on the morphological structure of the axopodia. A low concentration of glutaraldehyde in PHEM was identified as an adequate fixative for immunocytochemistry. The distribution of α-tubulin before and after rapid axopodial contraction was examined using immunocytochemistry and confocal laser scanning fluorescence microscopy. Positive signals were initially detected along the extended axopodia from the tips to the bases and were distributed in a non-uniform manner within the axopodia. Conversely, after the induction of a rapid axopodial contraction, these positive signals accumulated in the peripheral region of the cell. These results indicated that axopodial microtubules disassemble into fragments and/ or tubulin subunits during rapid axopodial contraction. Therefore, we hypothesize that the mechanism of extremely rapid axopodial contraction accompanied by cytoskeletal microtubule degradati

Promotion of microtubule acetylation plays an important role in degranulation of antigen-activated mast cells.

The aim of this study was to investigate whether microtubule acetylation is triggered by antigen stimulation and how it affects mast cell degranulation. The RBL-2H3 cell line was used as a model for mast cells. Acetylation of α-tubulin was analyzed by Western blotting. Intracellular distribution of α-tubulin and acetylated α-tubulin was observed by immunostaining. Degranulation was monitored by measuring the activity of β-hexosaminidase secreted into cell supernatants. Tukey-Kramer test was used to compare differences between groups. Microtubule acetylation proceeds globally in mast cell cytoplasm after antigen stimulation in addition to accelerated formation of microtubule-organizing centers. Pretreatment with 5Z-7-oxozeaenol (5µmol/l), an inhibitor of TGF-β-activated kinase 1, which is a key activator of α-tubulin acetyltransferase 1, did not affect the distribution and acetylation of microtubules in resting cells; however, it significantly suppressed antigen-evoked microtubule acetylation and their reorganization, and subsequent degranulation (95.0 ± 1.2% inhibition, n = 3, P < 0.01). These results provided new insight into the post-translational modifications of microtubule to regulate mast cell degranulation.

Plk1 inhibition leads to a failure of mitotic division during the first mitotic division in pig embryos.

This study was conducted to examine the dynamic distribution of polo-like 1 kinase (Plk1) and the possible role it plays in first mitotic division during early porcine embryo development. Indirect immunofluorescence and confocal microscopy imaging techniques combined with western blot analyses were used to study the dynamic expression and subcellular localization of Plk1 protein in pig parthenogenetic embryos. Finally, a selective Plk1 inhibitor, GSK461364, was used to evaluate the potential role of Plk1 during this special stage. The results showed that Plk1 upon expression exhibited specific dynamic intracellular localization, which closely correlated with the α-tubulin distribution during the first mitotic division. GSK461364 treatment resulted in cleavage failure, with majority of the GSK461364-treated embryos being arrested in prometaphase. Further results of the subcellular structure examination showed that GSK461364 treatment led to a significantly higher proportion of the treated embryos having abnormal spindles and misarranged chromosomes at the prometaphase stage. Thus, these results indicated that Plk1 is essential for porcine embryos to complete the first mitotic division. Furthermore, Plk1 regulation was associated with effects on spindle assembly and chromosome arrangement.

The inhibition of aromatase alters the mechanical and rheological properties of non-small-cell lung cancer cell lines affecting cell migration

Tumor invasion and metastasis are key aspects of non-small cell lung cancer (NSCLC). During migration, cells undergo mechanical alterations. The mechanical phenotype of breast cancer cells is correlated with aromatase gene expression. We have previously shown that targeting aromatase is a promising strategy for NSCLC. The aim of this study was to examine morphological and mechanical changes of NSCLC cells, upon treatment with aromatase inhibitor and correlate their ability to migrate and invade. In vitro experiments were performed using H23 and A549 NSCLC cell lines and exemestane was used for aromatase inhibition. We demonstrated that exemestane reduced H23 cell migration and invasion and caused changes in cell morphology including increased vacuolar structures and greater pleomorphism. In addition, exemestane changed the distribution of α-tubulin in H23 and A549 cells in a way that might destabilize microtubules polymerization. These effects were associated with increased cell viscosity and decreased elastic shear modulus. Although exemestane caused similar effects in A549 cells regarding viscosity and elastic shear modulus, it did not affect A549 cell migration and caused an increase in invasion. The increased invasion was in line with vimentin perinuclear localization. Our data show that the treatment of NSCLC cells with an aromatase inhibitor not only affects cell migration and invasion but also alters the mechanical properties of the cells. It suggests that the different origin of cancer cells is associated with different morphological characteristics and mechanical behavior.

Sperm-associated antigen 6 (SPAG6) deficiency and defects in ciliogenesis and cilia function: polarity, density, and beat.

SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.

Reelin promotes microtubule dynamics in processes of developing neurons

The extracellular matrix protein reelin controls radial migration and layer formation of cortical neurons, in part by modulation of cytoskeletal dynamics. A stabilizing effect of reelin on the actin cytoskeleton has been described recently. However, it is poorly understood how reelin modulates microtubule dynamics. Here, we provide evidence that reelin increases microtubule assembly. This effect is mediated, at least in part, by promoting microtubule plus end dynamics in processes of developing neurons. Thus, we treated primary neuronal cultures with nocodazole to disrupt microtubules. After nocodazole washout, we found microtubule reassembly to be accelerated in the presence of reelin. Moreover, we show that reelin treatment promoted the formation of microtubule plus end binding protein 3 (EB3) comets in developing dendrites, and that EB3 immunostaining in the developing wild-type neocortex is most intense in the reelin-rich marginal zone where leading processes of radially migrating neurons project to. This characteristic EB3 staining pattern was absent in reeler. Also reassembly of nocodazole-dispersed dendritic Golgi apparati, which are closely associated to microtubules, was accelerated by reelin treatment, though with a substantially slower time course when compared to microtubule reassembly. In support of our in vitro results, we found that the subcellular distribution of α-tubulin and acetylated tubulin in reeler cortical sections differed from wild-type and from mice lacking the very low density lipoprotein receptor (VLDLR), known to bind reelin. Taken together, our results suggest that reelin promotes microtubule assembly, at least in part, by increasing microtubule plus end dynamics.

PKCα regulates vasopressin-induced aquaporin-2 trafficking in mouse kidney collecting duct cells in vitro via altering microtubule assembly

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel located in the collecting tubule and collecting duct cells of mammalian kidney. The aim of this study is to investigate whether PKCα plays a role in vasopressin-induced AQP2 trafficking in mouse inner medullary collecting duct 3 (mIMCD3) cells. AQP2-mIMCD3 stable cell line was constructed by transfection of mouse inner medullary collecting duct 3 (mIMCD3) cells with AQP2-GFP construct. Then the cells were transfected with PKCα shRNA, PKCα A/25E, or PKCα scrambled shRNA. The expression levels of PKCα, AQP2, and phospho-S256-AQP2 were analyzed using Western blot. The interaction between AQP2 and PKCα was examined using immunoprecipitation. The distribution of AQP2 and microtubules was studied using immunocytochemistry. The AQP2 trafficking was examined using the biotinylation of surface membranes. Treatment of AQP2-mIMCD3 cells with 100 μmol/L of 1-desamino-8-D-arginine vasopressin (DdAVP) for 30 min stimulated the translocation of AQP2 from the cytoplasm to plasma membrane through influencing the microtubule assembly. Upregulation of active PKCα by transfection with PKCα A/25E plasmids resulted in de-polymerization of α-tubulin and redistributed AQP2 in the cytoplasm. Down-regulation of PKCα by PKCα shRNA partially inhibited DdAVP-stimulated AQP2 trafficking without altering α-tubulin distribution. Although 100 μmol/L of DdAVP increased AQP2 phosphorylation at serine 256, down-regulation of PKCα by PKCα shRNA did not influence DdAVP-induced AQP2 phosphorylation, suggesting that AQP2 phosphorylation at serine 256 was independent of PKCα. Moreover, PKCα did not physically interact with AQP2 in the presence or absence of DdAVP. Our results suggested that PKCα regulates AQP2 trafficking induced by DdAVP via microtubule assembly.

Steps towards a centralized nervous system in basal bilaterians: Insights from neurogenesis of the acoel Symsagittifera roscoffensis

Due to its proposed basal position in the bilaterian Tree of Life, Acoela may hold the key to our understanding of the evolution of a number of bodyplan features including the central nervous system. In order to contribute novel data to this discussion we investigated the distribution of α-tubulin and the neurotransmitters serotonin and RFamide in juveniles and adults of the sagittiferid Symsagittifera roscoffensis. In addition, we present the expression pattern of the neuropatterning gene SoxB1. Adults and juveniles exhibit six serotonergic longitudinal neurite bundles and an anterior concentration of serotonergic sensory cells. While juveniles show an "orthogon-like" arrangement of longitudinal neurite bundles along the anterior-posterior axis, it appears more diffuse in the posterior region of adults. Commissures between the six neurite bundles are present only in the anterior body region of adults, while irregularly distributed individual neurites, often interconnected by serotonergic nerve cells, are found in the posterior region. Anti-RFamide staining shows numerous individual neurites around the statocyst. The orthogon-like nervous system of S.roscoffensis is confirmed by α-tubulin immunoreactivity. In the region of highest neurotransmitter density (i.e., anterior), the HMG-box gene SrSoxB1, a transcription factor known to be involved in neurogenesis in other bilaterians, is expressed in juvenile specimens. Accordingly, SoxB1 expression in S.roscoffensis follows the typical pattern of higher bilaterians that have a brain. Thus, our data support the notion that Urbilateria already had the genetic toolkit required to form brain-like neural structures, but that its morphological degree of neural concentration was still low.

Centrosome Size Sets Mitotic Spindle Length in Caenorhabditis elegans Embryos

Just as the size of an organism is carefully controlled, the size of intracellular structures must also be regulated. The mitotic spindle is a supramolecular machine that generates the forces which separate sister chromatids during mitosis. Although spindles show little size variation between cells of the same type, spindle length can vary at least 10-fold between different species. Recent experiments on spindle length showed that in embryonic systems spindle length varied with blastomere size. Furthermore, a comparison between two Xenopus species showed that spindle length was dependent on some cytoplasmic factor. These data point toward mechanisms to scale spindle length with cell size. Centrosomes play an important role in organizing microtubules during spindle assembly. Here we use Caenorhabditis elegans to study the role of centrosomes in setting spindle length. We show that spindle length correlates with centrosome size through development and that a reduction of centrosome size by molecular perturbation reduces spindle length. By systematically analyzing centrosome proteins, we show that spindle length does not depend on microtubule density at centrosomes. Rather, our data suggest that centrosome size sets mitotic spindle length by controlling the length scale of a TPXL-1 gradient along spindle microtubules.

Upregulation of SEPT7 Gene Inhibits Invasion of Human Glioma Cells

ABSTRACTObjectives: To explore the role of SEPT7 in glioma cell invasion. Methods: SEPT7 was transfected into human glioma cell lines U251 and TJ899, the invasive abilities were evaluated by transwell assay, scratch assay, and 3-D/2-D Matrigel growth. The expression of MMP2/9, MT1-MMP, integrin αvβ3, and TIMP1/2 was detected by immunohistochemistry, immunofluorescence, and Western blot analyses. Distribution of α-tubulin was examined by laser scanning confocal analysis. Result: After SEPT7 trasfection, cell invasion was inhibited, expression of MMP2/9, MT1-MMP, and integrin αvβ3 was decreased, while TIMP1/2 was increased, and α-tubulin was redistributed. Conclusion: These results suggest that SEPT7 plays an important role in the glioma cell invasion.

Glutamate receptor signaling pathway in melanoma cells and its effect on cytoskeleton protein.

Objective To investigate the effects of glutamate receptor signaling on melanoma cell dendrite morphology and cytoskeleton protein. Methods A metastatic human malignant melanoma cell line WM451LU was cultured and transfected by recombinant adenovirus vector carrying a cDNA encoding microtubule-associated protein 2a (MAP2a). MK-801, an antagonist of N-methyl-D-aspartate receptor (NMDAR), and CPCCOEt, an antagonist of metabotropie glutamate receptor 1 (mGluR1), were used to treat transfected or untrausfeeted WM451LU cells. Confocal microscopy and three dimensional atomic force microscopy were used to assess subcellular location of NMDAR2A, mGluR1 and MAP2a as well as the dis-tribution of α-tubulin in and dendrite morphology of WM451LU cells. The proliferation of WM451LU cells was estimated by cell survival growth curve. Results Confocal laser microscopy revealed that NMDAR2A, mGluRl and MAP2a were mainly co-localized in melanoma cell dendrites. Both MK-801 and CPCCOEt increased the density of microtubules in cell dendrites and dendritic branching of WM451LU cells, and both effects of MK-801 and CPCCOEt were enhanced by the expression of MAP2a. Furthermore, the proliferation of WM451LU cells was significantly inhibited by MK-801 of 100 μmol/L and CPCCOEt of 10 μmol/L. Conclusions In melanoma cells, glutamate receptors may participate in the development of dendrites, and anta- gonists of glutamate receptors could inhibit the proliferation of melanoma cells. Key words: Melanoma; Dendrite; Receptors, glutamate; Nuclear matrix-associated proteins

The effect of VEGF on the temporal–spatial change of α-tubulin and cortical granules of ovine oocytes matured in vitro

The effects of vascular endothelial growth factor (VEGF) on the temporal–spatial change of α-tubulin and cortical granules (CGs) in ovine oocytes matured in vitro were studied using human recombinant VEGF 165 at 5 ng/ml in maturation media. Immunofluorescence, confocal microscopy and orcein staining were used to evaluate cell cycle-dependent modifications in nuclear configuration, quality of the metaphase II oocytes, microtubule organizing centers (MTOCs) translocation, temporal and spatial redistribution of α-tubulin and CGs in ovine oocytes undergoing in vitro maturation. The percentage of oocytes that reached metaphase II (M-II) in the VEGF-treated and the control groups were 87.08% and 80.03% ( P = 0.077) at 18 h and 87.42% and 83.89% ( P = 0.28) at 24 h, respectively. The percentages of oocytes displaying a normal distribution of α-tubulin and chromosomes in M-II increased significantly ( P = 0.015) in the VEGF group (77.50%) compared to the control (62.60%). The percentage of oocytes with CGs transfering completely in cortex was significantly higher ( P = 0.002) in the VEGF group (79.24%) than in the control group (60.97%). VEGF promoted the MTOCs domains to disappear from the cortex and stimulated assembly of α-tubulin around chromosome domains when germinal vesicle breakdown (GVBD) was commencing. In conclusion, VEGF may improve the quality of nuclear and cytoplasmic maturation of ovine oocytes in vitro by its effect on temporal and spatial translocation or redistribution of α-tubulin and CGs and on the normal distribution of nuclear configuration.

Translocation of dna polymerase-α during the mitotic cycle of Physarum polycephalum

The location of DNA polymerase-α within nuclei of a macroplasmodium of Physarum polycephalum was identified at successive stages of the highly synchronous mitotic cycle by indirect immunofluorescence. The distribution of α-tubulin, the nucleolar protein fibrillarin and DNA were also identified. In G2-phase the polymerase-α was found mainly in the region of the nucleolus. In prophase the polymerase became dispersed into the nucleus with the breakdown of the nucleolus. In metaphase, the polymerase-α was located in two zones separated by the metaphase plate. In S-phase, which follows immediately after mitosis, the distribution of the polymerase was initially punctate but later segments were noted during nucleolar reconstruction. We infer that DNA polymerase-α is stored mainly in the region of the nucleolus (or the reconstructing nucleolus) when it is not needed for DNA replication.

Immunohistochemistry of the distribution of .ALPHA.-tubulin in rat-incisor odontoblasts and ameloblasts.

Distribution of α-tubulin in rat-incisor odontoblasts and ameloblasts was observed by means of the immunohistochemical method. Wistar rats were fixed by perfusion with formalin solution or glutar-paraformaldehyde, and their incisors were dissected out, demineralized with either a solution of formic-acid formalin or EDTA, and prepared into 3μm-thick paraffin sections. The sections were incubated with a primary antibody (an anti-α-tubulin mouse monoclonal antibody) and then with a secondary antibody (a horseradish peroxidase-labeled sheep antimouse antibody).Intense and homogeneous α-tubulin immunostaining occurred in the cytoplasm of odontoblasts, inner enamel epithelial cells, and ameloblasts. In the odontoblast processes, however, the staining decreased gradually until it disappeared at a certain level in the dentin. The intense staining was noticed in the distal portions and Tomes' processes of differentiating and secreting ameloblasts. The staining generally decreased in the ameloblasts at an early stage of enamel maturation, and the cells frequently showed either slight or no staining. In reduced ameloblasts, the staining increased and returned to the level of the inner enamel epithelial cells.These results suggest that changes in their α-tubulin distribution reflect functional and morphological changes in odontoblasts and ameloblasts.

Localization of mRNAs in mouse testes by in situ hybridization: Distribution of α-tubulin and developmental stage specificity of pro-opiomelanocortin transcripts

Gene expression of mouse testicular germ cells and surrounding somatic cells during spermatogenesis was examined by RNA:cDNA hybridization in situ and concomitant Northern and dot blot analysis. Particular attention was paid to obtaining fixation and hybridization procedures for use with mouse testes to accomplish sensitive and precise localization with the in situ technique. α-Tubulin mRNAs were localized in virtually all testis cell types. In elongating spermatids, a unique labeling pattern was visualized; possibly corresponding to the position of the asymmetrically displaced cytoplasm. Pro-opiomelanocortin (POMC) transcripts in the adult mouse testis detected by Northern blot analysis were shown to be of a smaller size (∼600–800 nt) than the pituitary POMC transcripts, similar to what has been reported recently for POMC expression in the rat testis and mouse Leydig cell lines. Localization of POMC mRNAs by in situ hybridization was primarily to Leydig cells, although some labeling of spermatogonia and spermatocytes within the seminiferous epithelium was detected. A cellular or developmental specificity of the pattern of POMC localization was observed: obvious labeling of Leydig cells was limited to interstitial regions which were surrounded completely or in part by tubules in stages IX to XII of the cycle of the seminiferous epithelium.