Ultrastructural localization of PGP 9.5 and ubiquitin immunoreactivities in rat ductus epididymidis epithelium

Summary The distribution of protein gene product 9.5 (PGP) and ubiquitin in the spermatozoa and epithelial cells in the different regions of the rat duetus epididymidis (proximal caput, distal caput, corpus and cauda) was studied by Western blotting analyses and electron microscopical immunogold labelling. Western blotting analyses showed that the PGP immunoreactive band was very intense in the caput and cauda epididymidis and almost irrelevant in the corpus, while the ubiquitin immunoreactive band was intense in the distal caput and cauda. No ubiquitin immunoreactive band was observed in the proximal caput and only a very weak band was seen in the corpus. The results of electron microscopical immunogold labelling varied from one epididymal region to another. The proximal caput epididymidis presented immunoreaction to PGP in the rough endoplasmic reticulum, cytosol, mitochondria and microvilli of most principal cells, and in the cytosol, rough endoplasmic reticulum and mitochondria of most basal cells. No ubiquitin immunoreaction was observed in this epididymal region. In the distal caput epididymidis, PGP immunoreactivity was detected in some principal and basal cells in the same intracellular locations as described in the proximal caput. In this region, ubiquitin immunoreactivity appears in the apical cytosol and mitochondria of principal cells. The corpus epididymidis showed no immunoreaction to PGP or ubiquitin. In the cauda epididymidis, immunostaining to PGP was observed in most clear cells and in isolated principal cells. The intracellular location of PGP in both cell types was the cytosol, mitochondria and microvilli. Ubiquitin immunoreactivity was detected in the perinuclear cytosol and mitochondria — but not in the digestive vacuoles — of some clear cells. Scanty ubiquitin immunolabelling was also found in the microvilli, cytosol and mitochondria of some principal cells. The head of the spermatozoa present in the ductal lumen in all epididymal regions immunoreacted intensely to PGP. Ubiquitin was detected in the intermediate piece and residual cytoplasm of intraluminal spermatozoa present in the corpus and cauda epididymidis. These findings suggest that a non-ubiquitinated PGP irnrnunoreactive protein is secreted by the principal cells in caput epididymidis and binds the spermatozoon heads. It is possible that the clear cells of the cauda epididymidis secrete the ubiquitin that binds to spermatozoon tail.     

Protein gene product 9.5 (PGP 9.5)

Summary The guinea pig uterus is supplied by different populations of nerves which can be demonstrated by specific immunocytochemical and histochemical techniques. So far, there has been no single marker displaying entire peripheral innervation patterns. Recently, protein gene product (PGP) 9.5, a cytoplasmic protein in neurons and neuroendocrine cells, was found to visualize both different populations and subtypes of nerves. This prompted the present study of using PGP 9.5 for visualization of the whole uterine innervation. This was performed by the indirect immunofluorescence method using antiserum to PGP 9.5 raised in rabbits.PGP-immunoreactivity was present in all neuronal parts of the extrinsic and intrinsic uterine innervation, including different subpopulations of nerves. This was verified by chemical sympathectomy and sensory denervation with 6-hydroxydopamine and capsaicin-treatment respectively, and double immunostaining.By term a disappearance of uterine PGP-nerve-immunoreactivity was observed which was almost complete in fetus-bearing uterine tissue and further strengthens previous assumptions of a general, pregnancy-induced uterine neuronal degeneration.The developmental time-course and morphology of PGP-immunoreactive nerve structures was similar to that for other neuronal markers and support the suggestion of PGP-immunoreactivity as a general marker for the entire uterine innervation, and suggests that the presence of PGP 9.5-immunoreactivity may coincide with functional maturation of uterine innervation.     

Protein gene product 9.5 expression in the human fetal cochlea

Summary The distribution of protein gene product (PGP) 9.5 was analyzed in the human fetal cochlea using the indirect immunofluorescence method. In the 12- and 14-week-old human fetuses, the cells of the greater epithelial ridge and the lesser epithelial ridge were overall labelled with PGP 9.5, while the stria vascularis and the Reissner's membrane did not exhibit any staining. Spiral ganglion cells and cochlear nerve fibers were labelled with PGP 9.5 and PGP 9.5-positive nerve fibers made contact with the basement membrane of the Corti primordium in the 12-week-old human fetus. These results suggest that PGP 9.5 might be used as a histological marker of maturation and innervation in the human cochlea.     

Immunohistochemical localization of protein gene product 9.5, ubiquitin, and neuropeptide Y immunoreactivities in epithelial and neuroendocrine cells from normal and hyperplastic human prostate.

This study was designed to investigate (a) the presence of protein gene product 9.5 (PGP 9.5), ubiquitin, and neuropeptide Y (NPY) in the neuroendocrine and secretory epithelium of the human normal prostate and its secretions, and (b) the changes in immunoreactivity to these proteins in men with benign prostatic hyperplasia. Western blotting and light microscopic immunohistochemistry techniques were used and the numerical density of immunoreactive neuroendocrine cells, and the volume fractions of immunostained secretory epithelium were evaluated. Western blotting revealed the presence of the three antigens in both tissue homogenates and prostate secretion. Some neuroendocrine cells immunoreacted to PGP 9.5 and NPY in all the prostate regions of control specimens. Ubiquitin immunoreactivity was detected in the nuclei from both basal cells and secretory epithelial cells. The cytoplasm of the secretory cells and the glandular lumen also showed immunostaining for the three proteins. The numerical densities of both PGP 9.5 and NPY neuroendocrine cells were lower in hyperplasia than in controls. No differences in the volume fraction occupied by epithelial immunostaining to both proteins was found between hyperplastic and control prostates. We concluded that (a) PGP 9.5 and NPY, but not ubiquitin, are common antigens in both neuroendocrine and secretory prostate cells, (b) the three immunoreactive proteins contribute to the prostate secretions, and (c) the secretion of ubiquitin is markedly diminished in the hyperplastic epithelium.(J Histochem Cytochem 48:1121-1130, 2000)     

Protein gene product 9.5 expression in the human fetal cochlea.

The distribution of protein gene product (PGP) 9.5 was analyzed in the human fetal cochlea using the indirect immunofluorescence method. In the 12- and 14-week-old human fetuses, the cells of the greater epithelial ridge and the lesser epithelial ridge were overall labelled with PGP 9.5, while the stria vascularis and the Reissner's membrane did not exhibit any staining. Spiral ganglion cells and cochlear nerve fibers were labelled with PGP 9.5 and PGP 9.5-positive nerve fibers made contact with the basement membrane of the Corti primordium in the 12-week-old human fetus. These results suggest that PGP 9.5 might be used as a histological marker of maturation and innervation in the human cochlea.     

Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin

Summary Sections of human skin were processed according to the indirect immunofluorescence technique with a rabbit antiserum against human protein gene product 9.5 (PGP 9.5). Immunoreactivity was detected in intraepidermal and dermal nerve fibres and cells. The intraepidermal nerves were varicose or smooth with different diameters, running as single processes or branched, straight or bent, projecting in various directions and terminating in the stratum basale, spinosum or granulosum. The density of the intraepidermal nerves varied between the different skin areas investigated. PGP 9.5-containing axons of the lower dermis were found in large bundles. They separated into smaller axon bundles within the upper dermis, entering this portion of the skin perpendicular to the surface. Then they branched into fibres mainly arranged parallel to the epidermal-dermal junctional zone. However, the fibres en route to the epidermis traversed the upper dermis more or less perpendicularly. Furthermore, immunoreactive dermal nerve fibres were found in the Meissner corpuscles, the arrector pili muscles, hair follicles, around the eccrine and apocrine sweat glands and around certain blood vessels. Such fibres were also observed around most subcutaneous blood vessels, sometimes heavily innervating these structures. Numerous weakly-to-strongly PGP 9.5-immunoreactive cells were found both in the epidermis and in the dermis.     

The structure of the human gene encoding protein gene product 9.5 (PGP9.5), a neuron-specific ubiquitin C-terminal hydrolase.

Database search using a bovine thymus ubiquitin C-terminal hydrolase sequence indicated 54% sequence identity with the abundant human neuron-specific protein gene product 9.5 (PGP9.5), which was then shown to possess the same activity [Wilkinson, Lee, Deshpande, Duerksen-Hughes, Boss & Pohl (1989) Science 246, 670-673]. A yeast counterpart of the enzyme is also known. The human PGP9.5 gene, described here, spans 10 kb, contains nine exons and displays 5' features some common to many genes and some common with neurofilament neuron-specific enolase and Thy-1-antigen gene 5' regions.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

Cutaneous innervation in man visualized with protein gene product 9.5 (PGP 9.5) antibodies

Summary Using antibodies to the neuronal cytoplasmic protein, protein gene product 9.5 (PGP 9.5) the cutaneous innervation in man was investigated. The distribution of PGP 9.5 immunoreactive nerve fibers was compared with the distribution of nerve fibers immunoreactive to neuron specific enolase, neurofilament proteins, calcitonin gene related peptide, vasoactive intestinal polypeptide and neuropeptide Y. PGP 9.5 immunoreactive nerve fibers were found in the epidermis, dermis, in Meissner's corpuscles, innervating Merkel cells, around blood vessels, sweat glands and hair follicles. Merkel cells were also PGP 9.5 positive. The labelled nerve fibers included sensory and autonomic fibers, visualizing the whole innervation of the human skin. The number of positive fibers and the intensity of the fluorescence was greater with PGP 9.5 antibodies than with any of the other markers included. Thus, PGP 9.5 antibodies may serve as a tool for investigations of cutaneous innervation, reinnervation and nerve regeneration in different clinical conditions.     

Immunohistochemical analysis of protein gene product 9.5, a ubiquitin carboxyl-terminal hydrolase, during placental and embryonic development in the mouse.

Protein gene product 9.5 (PGP9.5) is expressed at high level in the neural and neuroendocrine systems. We investigated the localization and degree of expression of PGP9.5 in the developing mouse placenta and embryo at 6.5, 10.5 and 14 days of gestation using an immunohistochemical technique. At 6.5 days of gestation PGP9.5 was detected at various levels in decidual and primary trophoblast giant cells in the placenta, and in embryonic ectodermal cells in the embryo. At 10.5 and 14 days of gestation PGP9.5 was expressed at moderate to strong levels in neurons in the embryo, but rarely in the placenta. These findings suggest that the protein may play a significant role in implantation and placental development, and differentiation of embryonic ectoderm.     

cDNA cloning and tissue distribution of a rat ubiquitin carboxyl-terminal hydrolase PGP9.5.

We have isolated a cDNA clone encoding ubiquitin carboxyl-terminal hydrolase PGP9.5 from a rat brain cDNA library and examined the tissue distribution. The primary structure of the cDNA consists of 856 nucleotides including the entire coding region for 223 amino acids, and the calculated molecular mass is 24,782 Da. The rat PGP9.5 is strikingly homologous to the human PGP9.5, 75.2% of nucleic acids and 95.1% of amino acids being identical. The mRNA of PGP9.5 is most abundant in the rat brain and to a lesser degree in the testis. In other peripheral tissues we tested, the mRNA was undetectable. Western blotting using an anti-rat PGP9.5 antibody revealed the parallel distribution of mRNA and protein in various brain regions and testis. The availability of the rat PGP9.5 clone provides a new approach to examine the function of PGP9.5 and the role that it plays in the pathology of neurodegenerative diseases.     

Expression of protein gene product 9.5, a neuronal ubiquitin C-terminal hydrolase, and its developing change in sertoli cells of mouse testis.

Protein gene product 9.5 (PGP9.5), originally isolated as a neuron-specific protein, belongs to a family of ubiquitin carboxyl-terminal hydrolases that play important roles in the nonlysosomal proteolytic pathway. Antibodies against PGP9.5 have been used for immunohistochemical detection of neural elements, although some non-neuronal cells are also immunoreactive for PGP9.5. In the present study, developing testes of the mouse were immunostained after autoclave pretreatment of sections. In the testes of days 8 and 16, PGP9.5 was only localized on the spermatogonia, whereas on day 30 and in adults it appeared not only on spermatogonia, but also on Sertoli cells. In the testis of the male sterile W/W(v) mutant, very little, but strong, immunoreactivity was detected at some Sertoli cells, which were phagocytizing Sertoli cell aggregations that had fallen from the basal membrane. Additionally, it was confirmed that the nucleotide sequence of PGP9.5 in mice was highly conserved, like that in other mammals. These results suggest that PGP9.5 is a useful marker for activated Sertoli cells, playing an important role in degradation of abnormal proteins.     

Protein gene product 9.5 is a spermatogonia-specific marker in the pig testis: application to enrichment and culture of porcine spermatogonia

Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, genetic manipulation, and/or transplantation research. In this study, we established that expression of PGP 9.5 is a spermatogonia-specific marker in porcine testes. The expression pattern of PGP 9.5 in spermatogonia was compared to cell type-specific protein (GATA-4 or PLZF) expression in seminiferous tubules at different ages, and expression levels of PGP 9.5, Vasa, and Oct-4 were compared in different cell fractions. Enrichment of spermatogonia from 2-week-old (2wo) and 10-week-old (10wo) boars by adhesion to laminin, differential plating, or velocity sedimentation followed by differential plating was assessed by identification of spermatogonia using expression of PGP 9.5 as a marker. Compared to the initial samples, spermatogonia were enriched twofold in laminin-selected cells (P < 0.05), and fivefold either in cells remaining in suspension (fraction I) or in cells slightly attached to the culture dish (fraction II) (P < 0.05) after differential plating. Cells in fraction II appeared to be superior for future experiments due to higher viability (>90%) than in fraction I ( approximately 50%). Velocity sedimentation plus differential plating achieved cell populations containing up to 70% spermatogonia with good viability (>80%). Enriched spermatogonia from 2wo and 10wo testes could be maintained in a simple culture medium without additional growth factors for at least 2 weeks and continued to express PGP 9.5. These data provide the basis for future studies aimed at refining conditions of germ cell culture and manipulation prior to germ cell transplantation in pigs.     

Protein D is differentially expressed and regulated in the rat epididymis.

We report the use of a sensitive and specific enyzme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) to study the expression of protein D, a major androgen-regulated sperm-binding glycoprotein at the protein and mRNA level in different anatomical regions of the rat epididymis. The concentration of protein D in the caput, corpus and cauda region of the epididymis was 10.2 +/- 0.67, 7.3 +/- 0.61 and 22.8 +/- 1.34 ng/micrograms total protein, respectively. The total RNA extracted from the caput, corpus and cauda regions of the rat epididymis was amplified by PCR with oligonucleotide primers specific for the 5' and 3' portion of protein D cDNA. Compared to the caput and cauda region, a significant reduction (greater than 82 +/- 3%) in the expression of protein D mRNA levels was observed for corpus epididymal RNA. This data demonstrates regional differences in the concentration of protein D and suggests that protein D expression may be regulated at the level of mRNA within the corpus epididymidis.     

Protein gene product (PGP) 9.5 immunoreactivity in nerve fibres and pinealocytes of guinea-pig pineal gland: interrelationship with tyrosine-hydroxylase- and neuropeptide-Y-immunoreactive nerve fibres

This light-microscopic (LM) immunohistochemical study has evaluated the presence and distribution of the pan-neural and neuroendocrine marker protein gene product (PGP) 9.5 in pinealocytes and nerve fibres of guinea-pig pineal gland. The pattern of PGP 9.5-immunoreactive (ir) nerve fibres has been compared with that of fibres staining for tyrosine hydroxylase (TH) or neuropeptide Y (NPY). The vast majority of pinealocytes stained for PGP 9.5, although with variable intensity. PGP 9.5 immunoreactivity was localized in pinealocytic cell bodies and processes. Double-immunofluorescence revealed that PGP 9.5 immunoreactivity was absent from glial cells identified with a monoclonal antibody against glial fibrillary acidic protein (GFAP), PGP 9.5 immunoreactivity was also present in a large number of nerve fibres and varicosities distributed throughout the pineal gland. The number of TH-ir and NPY-ir nerve fibres was lower compared with those containing PGP 9.5 immunoreactivity. All fibres staining for NPY also stained for TH. NPY-ir nerve fibres were found to be much more numerous than previously reported for this species. The double-immunofluorescence analysis indicated that almost all TH-ir nerve fibres of the pineal gland contained PGP 9.5 immunoreactivity. However, few PGP 9.5-ir nerve fibres, located in the periphery and the central part of the gland, were TH-negative. A large number of PGP 9.5-ir fibres was concentrated in the pineal stalk. In contrast, TH-ir and NPY-ir nerve fibres were rare in this part of the pineal gland. Our data provide evidence that immunohistochemistry for PGP 9.5 may be a useful tool further to differentiate central and peripheral origins of pineal innervation. Furthermore, the staining of pinealocytes for PGP 9.5 may be exploited to study the three-dimensional morphology and the architecture of pinealocytes and their processes under various experimental conditions.