Pattern Of Immunolocalization Research Articles

Molecular Characterization of Trypanosoma cruzi Tc8.2 Gene Indicates Two Differential Locations for the Encoded Protein in Epimastigote and Trypomastigote Forms.

This report describes the molecular characterization of the Tc8.2 gene of Trypanosoma cruzi. Both the Tc8.2 gene and its encoded protein were analyzed by bioinformatics, while Northern blot and RT-PCR were used for the transcripts. Besides, immunolocalization of recombinant protein was done by immunofluorescence and electron microscopy. Analysis indicated the presence of a single copy of Tc8.2 in the T. cruzi genome and 2-different sized transcripts in epimastigotes/amastigotes and trypomastigotes. Immunoblotting showed 70 and 80 kDa polypeptides in epimastigotes and trypomastigotes, respectively, and a differential pattern of immunolocalization. Overall, the results suggest that Tc8.2 is differentially expressed during the T. cruzi life cycle.

Cell Proliferation Index Determination by Immunohistochemical Detection of hCDC47 Protein

A member of the human minichromosome maintenance complex protein family, hCDC47 (alias MCM7) has been identified as a component of the regulatory mechanism in cell proliferation. The expression of this protein, as determined by immunohistochemistry, was investigated to determine its application as a proliferation marker. A mouse monoclonal antibody (Clone 47DC141, NeoMarkers, Fremont CA) raised against recombinant hCDC47 protein was tested against a wide range of tissues. Immunoreaction patterns were determined in normal and neoplastic, human tissues, including skin, tonsils and lymph nodes, primary, and metastatic brain tumors. The protein was detected in the nuclei of both, normal and neoplastic proliferating cells. Similarly, we also examined the distribution of hCDC47 in normal rat and mouse tissues, and rodent and human tumors grown in nude mice.The pattern of immunolocalization was identical to that seen in human tissue, with positive nuclear immunoreaction readily identified in proliferating cells. Western immunoblot was carried out on extracts from PANC cells (human pancreatic adenocarcinoma cell line) to confirm the specificity of the protein. To correlate Ki67 protein immunoexpression with hCDC47 antibody reactivity, semiquantitative comparisons were carried out on parallel tissue sections. There was excellent correlation in the distribution pattern of the 2 markers, although hCDC47 was more sensitive.Thus this marker may have important clinical and research applications because of its activity in formalin-fixed, paraffin-embedded, proliferating, normal, and neoplastic tissue. More significantly, its application to animal tissue makes it a reliable and easy to use, proliferation marker for experimental studies.

Immunolocalization of corticotropin-releasing factor (CRF) and corticotropin-releasing factor receptor 2 (CRF-R2) in the developing gut of the sea bass (Dicentrarchus labrax L.)

Our previous data indicated an important role for adrenocorticotropic (ACTH)-like molecules co-operating with macrophages to control the modifications in body homeostasis during the first period of the life of sea bass (up to 30 days post-hatching) before the lymphoid cells have reached complete maturation. The aim of the study was to determine the immunolocalization of corticotropin-releasing factor (CRF), which is a very important mediator of stress-related responses. Our data showed that immunostaining for CRF is localized already at 8 days after hatching in nerve fibers of the gastrointestinal tract wall from the pharynx to the anterior gut, when the larvae are still feeding on yolk. This pattern of immunolocalization appeared similar to that in 24-day-old larvae, but at this stage there were also large cells immunopositive to CRF located in the wall of the midgut and hindgut. Lipopolysaccharide (LPS) treatment, which is a known stimulator of stress hormone responses, did not modify the CRF immunostaining pattern, though it did affect the immunolocalization of the peripheral CRF receptor, i.e. CRF-R2. Immunolocalization of CRF-R2 appeared in nerve fibers of the gut wall in larvae fixed 1h after the end of lipopolysaccharide (LPS) treatment. The present results suggest that CRF plays important autocrine and/or paracrine roles in the early immune responses at the gut level in the larval stages of sea bass (Dicentrarchus labrax L.) as already proposed for ACTH. Moreover, our studies taken together with other research on fish, in comparison with mammals, suggest a phylogenetically old role of CRF in immune–endocrine interactions.

Late expression of FosB transcription factor in 4-aminopyridine-induced seizures in the rat cerebral cortex

In this study, the immunolocalization of FosB transcription factor was investigated in acute and chronic experimental models of seizures induced by 4-aminopyridine. Wistar rats were injected intraperitoneally daily with 5mg/kg 4-aminopyridine for 1, 4, 8 and 12 days and sacrificed 24h after the last injection. Corresponding control groups received the solvent of 4-aminopyridine. Immunohistochemistry revealed an increase in FosB immunolabelling in the frontal cortex in 4-aminopyridine-treated animals compared to controls, both in acute and chronic time course groups. The dentate gyrus displayed elevated FosB immunopositivity only after repeatedly applied convulsant (4-aminopyridine), i.e. following 4, 8 and 12 days of treatment, but no significant immunolocalization was observed in the hippocampus proper. The neuronal localization of FosB after 12 days of 4-aminopyridine-induced convulsions was analysed by means of FosB-parvalbumin double immunolabelling. The increased number of double-labelled cells was significant in the frontal cortex, hilum of the dentate fascia and region CA1 of the hippocampus. We conclude that the studied neocortical and allocortical areas showed a different pattern of FosB immunolocalization, which suggests a relative deficiency of transcriptional regulation in the Ammon's horn and may be responsible for distinct response to seizure-induced cellular insult.

The changed immunolocalization of START-domain-containing 6 (StarD6) during the development of testes in rat perinatal hypothyroidism

Thyroid hormones have an essential role in maintaining the normal developmental structure of testes during the neonatal stage. START-domain-containing 6 (StarD6) is exclusively expressed in germ cells during spermatogenesis; however, its biological role in rat perinatal hypothyroidism is not clear. After hypothyroidism was induced by daily administration of 0.05% 6-propyl-2-thiouracil (PTU), the pattern of StarD6 immunolocalization was examined from gestation day 15 to postnatal day 49. In normal rats, the labelling of StarD6 was confined to the germ cells from the third-week postpartum. In contrast, its immunoreactivity in hypothyroidal rats was not detected until the fourth-week postpartum. The immunolocalization pattern of StarD6 differed from that of normal adult rats during the seventh-week postpartum. StarD6 was clearly detected in the Leydig cells of the perinatal hypothyroid rats from the fifth-week postpartum. Therefore, StarD6 may play a pivotal role, not only in the spermatogenesis of normal rats, but also in the steroidogenesis of Leydig cells under perinatal hypothyroidism.

Wheat nitrogen metabolism during grain filling: comparative role of glumes and the flag leaf

The mobilization of nitrogen (N) compounds and the roles played by glumes and the flag leaf during grain filling were studied in bread wheat (Triticum aestivum L. cv. Florida) grown under field conditions. Glumes lost twice as much of their total N content as that lost by the flag leaf between the milk and early dough stages. In the flag leaf, glumes and grains, Glu, Asp, Ser and Ala accounted for 85% of all the reductions in the free amino acid pool. Principal component analysis of free amino acid pools separated grains from the glumes and the flag leaf, suggesting grain specific regulations in the use of free amino acids in protein synthesis. In all three organs, no decrease in Gln was detected, probably due to steady glutamine synthetase (GS; EC 6.3.1.2) activities per soluble protein in both the flag leaf and glumes. Compared with the flag leaf, glumes presented relatively smaller amounts of the chloroplast GS associated isoform. This we show is due to a lower relative number of mesophyll cells in glumes as supported by the different anatomy and the cellular pattern of the GS immunolocalization. We argue that cellular distribution plays a key role in supporting metabolism to enable the various functions undertaken by glume tissue.

Seasonal expression and pattern of SP22 immunolocalization in stallion semen

The objective of this investigation was to examine the effects of 6-methoxy-benzoxazolinone (MBOA), a plant compound that resembles melatonin and alters ovarian function in rodents, in combination with PMSG on superovulatory responses in the cycling ewe. In Experiment I, St. Croix White ewes (n = 44) were synchronized (intra-vaginal progestin sponge) for 14 days followed by hCG (750 IU) at 1 day after sponge removal (day 0). Ewes were assigned to one of six treatments administered on day −1: Control (no PMSG or MBOA; n = 7); PMSG (1000 IU i.m.; n = 7); Low MBOA (0.43 mg/kg i.m.; n = 7); High MBOA (1.15 mg/kg i.m.; n = 7); Low MBOA + PMSG (n = 8); High MBOA + PMSG (n = 8). In Experiment II, St. Croix White ewes (n = 24) were synchronized (progestin CIDR) for 14 days followed by hCG on day 1 after CIDR removal (day 0). Ewes were assigned to one of three treatments administered on day −1: Control (n = 8); PMSG (n = 8); Low MBOA + PMSG (n = 8). Laparoscopy was performed on day 9 to assess numbers of corpora lutea (CL) and visible follicles on each ovary. Blood samples were collected on day −13, −1, 0, 1, and days 6 or 7–12 for analysis of serum progesterone (P4) by RIA. Treatment groups receiving PMSG (alone or with MBOA) exhibited greater (P < 0.05) serum concentrations of P4 post-synchrony than Control and MBOA-only groups. Ovulation rate was lower (P < 0.05) for Control and MBOA-only treated ewes than ewes receiving PMSG. Ovulation rate in ewes treated with MBOA alone was similar (P > 0.10) to Controls, and PMSG treatment alone did not differ (P > 0.10) from MBOA + PMSG treatment. Ewes treated with PMSG alone did not differ (P > 0.10) in follicle number from High MBOA + PMSG treated ewes, however, Low MBOA + PMSG treated ewes had greater numbers of follicles at day 9 (P < 0.05) than the PMSG or High MBOA + PMSG groups in Experiment I; although, this was not replicated in Experiment II with numbers of follicles in the Low MBOA + PMSG group being similar (P > 0.10) to PMSG alone. In summary, the addition of MBOA in combination with PMSG as part of a synchronization–superovuation protocol in the ewe did not increase ovulation rate.

HRPT2 gene alterations in ossifying fibroma of the jaws

Ossifying fibroma (OF) is a benign neoplasm related to bone characterized by a progressive enlargement of the affected jaw. Recently, the candidate tumor suppressor gene HRPT2 was identified and alterations in this gene were related with the Hyperparathyroidism-jaw tumor syndrome that is characterized by parathyroid adenoma or carcinoma, fibro-osseous lesions (mainly OF) of the jaws, and renal lesions. The purpose of the present study was to evaluate the HRPT2 gene in OF. Tumour and blood samples were obtained from 3 patients with OF and one with juvenile ossifying fibroma (JOF). The results demonstrated three novel mutations in two out of three genotyped OF's. Interestingly, one of these patients showed a germ-line mutation after blood analysis. RT-PCR amplification was performed to analyze HRPT2 mRNA expression and only wild-type HRPT2 transcript was found in all tumours. Investigation of the parafibromin protein by immunohistochemistry showed a similar pattern of immunolocalization with strong nuclear and cytoplasmic staining in all cases. In conclusion, the present study shows for the first time mutations of HRPT2 gene in OF and suggests that OF may arise due to haploinsufficiency of the HRPT2 gene.

Production and characterization of a monoclonal antibody against 28.5 kDa tegument antigen of Fasciola gigantica

A monoclonal antibody (MoAb) against the 28.5 kDa tegumental antigen of Fasciola gigantica was produced by the hybridoma technique using spleen cells from BALB/c mice immunized with the tegumental extract from adult F. gigantica. This MoAb was found to be of the isotype IgG 1, κ-light chain, and shown by immunoblotting to specifically react with the 28.5 kDa antigen present in the tegument, excretion–secretion material of the adult, whole-body extracts of newly excysted juveniles, 5-week-old juvenile and adult parasites. It did not cross-react with antigens from other trematode parasites, including Schistosoma mansoni, Eurytrema pancreaticum and Paramphistomum spp. Immunolocalization of this antigen by indirect immunofluorescence indicated that it was present as a major component of the adult tegument, particularly in its outer rim, tegumental cells, and their processes. Furthermore, the epithelium linings of the oral sucker, buccal tube, pharynx, caecal bifurcation, both male and female genital canals, which were the continuation of the tegumental-type epithelium, were also positively stained with this MoAb. A similar pattern of immunolocalization, but with weaker staining intensity, was observed in newly excysted, 5- and 7-week-old juveniles. Thus this antigen is expressed in all developmental stages of the parasite, and it could be a strong candidate for immunodiagnosis and vaccine development.

Corticotropin-releasing hormone signaling in synovial tissue vascular endothelium is mediated through the cAMP/CREB pathway.

Modulation of locally produced corticotropin-releasing hormone (CRH) is a component of the cytokine network in human inflammatory arthritis. CRH signaling, through the CRH-receptor subtype R1alpha, may play a role in both vascular changes and pathologic mechanisms associated with joint inflammation. Furthermore, the peripheral actions of CRH may be mediated in part through the NURR subfamily of nuclear orphan receptors. The aim of this study was to establish the signaling mechanisms through which CRH receptor-mediated responses contribute to gene regulation in inflamed synovial vasculature. Immunohistochemical analysis of serial rheumatoid arthritis (RA) tissue sections demonstrates CRH and NURR1 expression in the synovial lining layer, subsynovial lining layer, and the vascular endothelium. The identical pattern of immunolocalization confirms that NURR1 is produced at the same synovial sites shown to produce CRH. The distribution of specific NURR1 staining on the synovial vasculature parallels that observed for CRH-R1 expression. Using primary synovial tissue endothelial cells, we demonstrate that CRH induces specific CREB-1 and ATF-2 binding to the NURR1 promoter. We further provide evidence that CRH signaling can be mimicked by activation of cAMP/PKA/CREB using forskolin in primary human microvascular endothelial cells. These data indicate that the CRH receptor-dependent inflammatory response in synovial tissue endothelium is mediated through the cAMP/CREB signaling pathway.

An immunohistochemical study of matrix molecules associated with barrier membrane-mediated periodontal wound healing.

Guided tissue regeneration (GTR) is a clinical procedure developed to facilitate periodontal regeneration by using barrier membranes to selectively promote the repopulation of a periodontal defect by periodontal ligament and bone cells at the expense of epithelial and gingival connective tissue cells. The aim of this study was to gain insight into the biological events occurring during membrane mediated periodontal wound healing by examining the immunohistochemical expression of a number of extracellular matrix components in tissues treated via the GTR technique. Experimental periodontal defects were created around the second premolar tooth in 4 dogs and wound closure was achieved by application of expanded polytetrafluoroethylene membranes around each tooth and flap positioning coronal to the cementoenamel junction. The dogs were sacrificed after a 4-wk healing period, block dissections of the part of the mandible containing the experimental tooth were obtained and paraffin sections were prepared. Using standard immunohistochemical techniques, the sections were stained with a monoclonal antibody against bone morphogenetic proteins 2 and 4 (BMP-2 and -4) and polyclonal antibodies against collagen I, collagen II, decorin, biglycan, bone sialoprotein, osteopontin and osteocalcin. Collagen I was predominantly localized within the regenerating bone, whereas collagen III staining was more abundant in the soft connective tissues of the defect. Decorin and biglycan staining was faint within the extracellular matrix of the regenerating defect, although both proteoglycans exhibited intense intracellular localization within some of the cells inhabiting the defect. The staining for BMP-2 and -4 was weak within the bone but strong within the extracellular matrix of the regenerating soft tissue. Osteopontin and bone sialoprotein were strongly localized in the regenerating bone and cementum found within the defect. Osteocalcin staining was present in both the regenerating and mature cementum and associated cementoblasts, and it was relatively weaker in the regenerating bone compared to the mature bone. The observed pattern of immunolocalization of the extracellular matrix macromolecules suggests that the heterogeneous cell population filling the GTR wound had created an environment that was conducive to periodontal regeneration.

Immunocytochemical localization of Na+,K(+)-ATPase in the calcium-transporting sternal epithelium of the terrestrial isopod Porcellio scaber L. (Crustacea).

Terrestrial isopods store large amounts of calcium carbonate between the epithelium and the old cuticle of the first four anterior sternites before molt. During the formation of these sternal CaCO3 deposits, large amounts of calcium are transported across the anterior sternal epithelium from the base to the apical side of the integument, and in the reverse direction during resorption of the deposit. A monoclonal antibody against the avian alpha-subunit of Na+,K(+)-ATPase was used to localize Na+,K(+)-ATPase in the anterior and the posterior sternal epithelium of Porcellio scaber. Semithin cryosections 0.5 micron thick were used for immunofluorescence microscopy and ultrathin cryosections for immunogold electron microscopy. The Na+,K(+)-ATPase was localized in the basolateral plasma membrane of the posterior and anterior sternal epithelium. The apical plasma membrane, including cytoplasmic extensions into the newly secreted cuticle, was virtually devoid of the enzyme. This pattern of immunolocalization was not affected by the direction of transepithelial calcium transport associated with the deposition and resorption phases of the molt cycle.

Brain microvascular and astrocyte localization of P-glycoprotein.

The hypothesis that P-glycoprotein plays a functional role at the brain capillary endothelium, which makes up the blood-brain barrier in vivo, is based largely on immunocytochemical studies showing immunoreactive P-glycoprotein localized to either isolated brain microvessels or microvessels within tissue sections. The present studies use the MRK16 monoclonal antibody to human P-glycoprotein to demonstrate that the pattern of immunolocalization of P-glycoprotein in microvessels of human or primate brain is similar to the pattern of immunolocalization of an astrocyte protein, glial fibrillary acidic protein. In contrast, the discontinuous staining pattern of MRK16 is not colocalized with the continuous immunostaining of the brain endothelial GLUT1 glucose transporter. The MRK16 antibody was radiolabeled with [125I]-iodine, and 125I-MRK16 avidly bound isolated human brain capillaries via a saturable mechanism. However, the 125I-MRK16 antibody was not taken up by primate brain capillaries in vivo following intravenous injection. In conclusion, these studies provide evidence that P-glycoprotein does not play a functional role at the luminal membrane of the brain capillary endothelium in vivo, and that a principal site of immunoreactive P-glycoprotein in brain microvasculature is localized to astrocyte foot processes.

In vivo presence of the high affinity nerve growth factor receptor Trk-A in the rat pancreas: differential localization during pancreatic development.

It is now established that the pancreatic islet cells derive from precursors present in the pancreatic ducts. These precursor cells as well as the factors that influence their differentiation to mature insulin-secreting beta-islet cells are nevertheless not elucidated yet. However, the large number of similarities existing between beta-cells and neuronal cells led to the suggestion that these two different cell types may be sensitive to the same growth and differentiation factors, for example, nerve growth factor (NGF), which is important for the differentiation and survival of several neurons. It was previously demonstrated that the high affinity NGF receptor, Trk-A, which is sufficient for NGF signal transduction, is expressed in different beta-cell lines and in normal rat islet cells in primary culture. The aim of this study was therefore to examine whether the Trk-A receptor is present in vivo in the rat pancreas during development and in adult life. By double immunofluorescence analysis using anti-Trk-A/antiinsulin or anti-Trk-A/antiglucagon antibodies on pancreatic sections of adult rats, it is demonstrated that the Trk-A protein is present in the beta- and not in the alpha-islet cells of adult rat pancreas. No Trk-A immunostaining was observed in the exocrine pancreas or in the pancreatic ducts of adult pancreas. Interestingly, the pattern of Trk-A immunolocalization in the pancreas is different during fetal life, when Trk-A immunostaining is observed in the pancreatic ductular cells and undetectable in both beta- and alpha-islet cells. During late prenatal and early postnatal life, Trk-A immunostaining is present in the islet cells, although weaker than in adult rats, and progressively decreases in the pancreatic ducts, as pancreatic maturation progresses. The intensity and localization of the Trk-A immunostaining in the rat pancreas are therefore developmentally regulated.

In vivo presence of the high affinity nerve growth factor receptor Trk- A in the rat pancreas: differential localization during pancreatic development

It is now established that the pancreatic islet cells derive from precursors present in the pancreatic ducts. These precursor cells as well as the factors that influence their differentiation to mature insulin-secreting beta-islet cells are nevertheless not elucidated yet. However, the large number of similarities existing between beta-cells and neuronal cells led to the suggestion that these two different cell types may be sensitive to the same growth and differentiation factors, for example, nerve growth factor (NGF), which is important for the differentiation and survival of several neurons. It was previously demonstrated that the high affinity NGF receptor, Trk-A, which is sufficient for NGF signal transduction, is expressed in different beta-cell lines and in normal rat islet cells in primary culture. The aim of this study was therefore to examine whether the Trk-A receptor is present in vivo in the rat pancreas during development and in adult life. By double immunofluorescence analysis using anti-Trk-A/antiinsulin or anti-Trk-A/antiglucagon antibodies on pancreatic sections of adult rats, it is demonstrated that the Trk-A protein is present in the beta- and not in the alpha-islet cells of adult rat pancreas. No Trk-A immunostaining was observed in the exocrine pancreas or in the pancreatic ducts of adult pancreas. Interestingly, the pattern of Trk-A immunolocalization in the pancreas is different during fetal life, when Trk-A immunostaining is observed in the pancreatic ductular cells and undetectable in both beta- and alpha-islet cells. During late prenatal and early postnatal life, Trk-A immunostaining is present in the islet cells, although weaker than in adult rats, and progressively decreases in the pancreatic ducts, as pancreatic maturation progresses. The intensity and localization of the Trk-A immunostaining in the rat pancreas are therefore developmentally regulated.

Immunocytochemical localization of 4-ene steroid 5 alpha-reductase type 1 along the rat epididymis during postnatal development

Dihydrotestosterone (DHT), the active androgen in many tissues, is synthesized from testosterone by the enzyme 4-ene steroid 5 alpha-reductase (5 alpha-reductase; EC 1.3.1.22). In the epididymis, the maturation and storage of spermatozoa are dependent on the presence of 5 alpha-reduced androgens. The regulation of epididymal 5 alpha-reductase is complex. To date, the regulation of this enzyme has been studied extensively at the level of enzymatic activity and more recently at the mRNA level. Regulation at the level of the protein, however, remains poorly understood. We have raised rabbit polyclonal antibodies to a 24-mer synthetic peptide whose sequence was determined from the predicted amino acid sequence for rat 5 alpha-reductase type 1 to immunolocalize the 5 alpha-reductase type 1 protein in the rat epididymis during postnatal development. Western blot analysis revealed a specific immunoreactive band of 26 kilodaltons in male rat liver, epididymis, and prostate; this apparent molecular size is identical to that obtained when the 5 alpha-reductase type 1 cDNA is expressed in mammalian cells. Furthermore, the relative protein levels, liver > epididymis > prostate, were consistent with the mRNA levels for type 1 rat 5 alpha-reductase. Perfusion-fixed paraffin-embedded epididymal tissue sections were used to immunolocalize type 1 5 alpha-reductase. In the adult rat epididymis, the most intense immunoperoxidase reaction was observed in a discrete lobule of the initial segment of the epididymis. A progressive decrease in staining intensity occurred distally along the tissue to the cauda epididymis. The staining reaction was specific to cytoplasmic elements of epithelial principal cells; no reaction was evident over nuclei. However, specifically in the initial segment, very intense staining was seen in the infranuclear region of the principal cells. In the proximal caput epididymidis, the staining was primarily confined to an oval region above the nuclei, whereas in the remaining epididymal regions, weak staining was seen throughout the cytoplasm. Thus, the intracellular localization of the 5 alpha-reductase type 1 protein changed as one moved down the epididymis. Finally, the pattern of immunolocalization of 5 alpha-reductase type 1 protein was different in the epididymis of rats of different postnatal ages. On day 7, no reactivity was noted; by day 28, a weak apical staining of principal cells was seen throughout the epididymis; by day 47, the adult pattern of staining had been established. Our results revealed that the expression and intracellular localization of the 5 alpha-reductase type 1 protein are both age dependent and epididymal segment specific.

Immunocytochemical localization of 4-ene steroid 5 alpha-reductase type 1 along the rat epididymis during postnatal development.

Dihydrotestosterone (DHT), the active androgen in many tissues, is synthesized from testosterone by the enzyme 4-ene steroid 5 alpha-reductase (5 alpha-reductase; EC 1.3.1.22). In the epididymis, the maturation and storage of spermatozoa are dependent on the presence of 5 alpha-reduced androgens. The regulation of epididymal 5 alpha-reductase is complex. To date, the regulation of this enzyme has been studied extensively at the level of enzymatic activity and more recently at the mRNA level. Regulation at the level of the protein, however, remains poorly understood. We have raised rabbit polyclonal antibodies to a 24-mer synthetic peptide whose sequence was determined from the predicted amino acid sequence for rat 5 alpha-reductase type 1 to immunolocalize the 5 alpha-reductase type 1 protein in the rat epididymis during postnatal development. Western blot analysis revealed a specific immunoreactive band of 26 kilodaltons in male rat liver, epididymis, and prostate; this apparent molecular size is identical to that obtained when the 5 alpha-reductase type 1 cDNA is expressed in mammalian cells. Furthermore, the relative protein levels, liver > epididymis > prostate, were consistent with the mRNA levels for type 1 rat 5 alpha-reductase. Perfusion-fixed paraffin-embedded epididymal tissue sections were used to immunolocalize type 1 5 alpha-reductase. In the adult rat epididymis, the most intense immunoperoxidase reaction was observed in a discrete lobule of the initial segment of the epididymis. A progressive decrease in staining intensity occurred distally along the tissue to the cauda epididymis. The staining reaction was specific to cytoplasmic elements of epithelial principal cells; no reaction was evident over nuclei. However, specifically in the initial segment, very intense staining was seen in the infranuclear region of the principal cells. In the proximal caput epididymidis, the staining was primarily confined to an oval region above the nuclei, whereas in the remaining epididymal regions, weak staining was seen throughout the cytoplasm. Thus, the intracellular localization of the 5 alpha-reductase type 1 protein changed as one moved down the epididymis. Finally, the pattern of immunolocalization of 5 alpha-reductase type 1 protein was different in the epididymis of rats of different postnatal ages. On day 7, no reactivity was noted; by day 28, a weak apical staining of principal cells was seen throughout the epididymis; by day 47, the adult pattern of staining had been established. Our results revealed that the expression and intracellular localization of the 5 alpha-reductase type 1 protein are both age dependent and epididymal segment specific.

Immunolocalization of basic fibroblast growth factor and its receptor in adult goldfish retina

The neural retina of teleost fish can regenerate following surgical or neurotoxic lesions. As a first attempt to uncover the factors important for the regenerative response, we used immunocytochemistry to demonstrate the presence of basic fibroblast growth factor (bFGF) and its receptor in the goldfish retina. The bFGF-immunoreactivity was present throughout the retina, but was most intense in photoreceptor cells, especially cones, and Müller glia. Immunoreactivity for the bFGF receptor was strongest in the axon terminals of photoreceptors, both rods and cones. This pattern of immunolocalization is especially interesting since the proliferating cells that are thought to be responsible for generating the neural regenerate are located among the photoreceptor axon terminals. These proliferating cells have been identified as rod precursors because in the intact retina they give rise only to rod photoreceptors. When the neural retina is damaged, however, rod precursors are thought to be the source of proliferating neuroepithelial cells responsible for generating the retinal regenerate. The role played by bFGF in normal neurogenesis, cell differentiation, and/or neuronal regeneration in the fish retina has yet to be determined.