BIOLOGY OF REPRODUCTION 55, 291-297 (1996)
Light and Electron Microscopic Immunohistochemical Localization of Protein Gene
Product 9.5 and Ubiquitin Immunoreactivities in the Human Epididymis and Vas
Deferens'
B. Fraile, 3 R. Martin, s M.P. De Miguel, 3 M.I. Arenas, 3 F.R. Bethencourt, 4 F. Peinado, 6 R. Paniagua, 3 and
L. Santamaria2 7
Department of Cell Biology and Genetics3 and Department of Urology,4 University of Alcala' de Henares,
E-28871 Alcala de Henares (Madrid), Spain
Department of Histopathology (Hospital de La Princesa),5s Department of Urology (Hospital de La Princesa), 6 and
Department of Morphology (Histology),7 School of Medicine, Autonomous University, E-28029 Madrid, Spain
associate with cytoskeletal components such as microtubules and intermediate filaments, regulating their degradation and turnover [11-13].
Recently, the occurrence of both PGP and ubiquitin immunoreactivities have been reported at light microscopic
and ultrastructural levels in the epithelium from rat epididymis, and differences in the immunoexpression of both proteins were observed along the length of the ductus epididymidis [14, 15]. Immunoreactivity to PGP in the human
male reproductive system was described in spermatogonia
and Leydig cells [16]. The epididymis is an organ with
intense protein traffic between the epithelium and the lumen, and it is plausible that ubiquitin and its carboxylterminal hydrolase (PGP 9.5), which are involved in protein
degradation and turnover, take part in this traffic. At present, ubiquitin functions have been studied in the intracellular space. However, the detection of ubiquitin in human
seminal plasma [17] suggests that this protein is secreted
by some epithelial cells of the excurrent duct system or
accessory male glands, or both. Previous studies of PGP
and ubiquitin immunoreactions in the rat epididymis support the hypothesis that these proteins are secreted by epithelial cells from specific regions of the rat epididymis and
become linked to spermatozoa [14, 15]. Ubiquitin binding
to spermatozoa has also been observed in men [17], and a
relaxing action of this protein on the uterus has been demonstrated in women [18]. Although the function of ubiquitin
in seminal plasma is still open to speculation, these findings
suggest that this protein might have effects in reproduction,
including playing a role in spermatozoon maturation. For
all these reasons, it is of interest to study the presence and
distribution of epithelial immunoreactive cells to ubiquitin
and to its carboxyl-terminal hydrolase (PGP) along the ductus epididymidis and ductus deferens in humans. The aim
of this report was to study the distribution of PGP and
ubiquitin immunoreactivities in the epithelium of the ductuli efferentes, the different regions of the ductus epididymidis (caput, corpus, and cauda), and the ductus deferens of
normal, adult men, by means of Western blotting analysis,
light microscopic immunohistochemistry, and electron microscopic immunogold labeling of both antigens.
ABSTRACT
The distribution of protein gene product 9.5 (PGP) and ubiquitin immunoreactivities in the ductuli efferentes, ductus epididymidis, and ductus deferens of humans was studied by Western blot analyses and light and electron microscopic immunocytochemistry. PGP immunoreactivity was intense in the ductuli
efferentes and weak in the ductus epididymidis and ductus deferens, while ubiquitin immunoreactivity was intense in the ductuli efferentes and ductus epididymidis and very weak in the
ductus deferens. In the ductuli efferentes epithelium, PGP immunolabeling was observed in the cytoplasm of principal cells,
whereas ubiquitin immunoreactivity was found in the nucleus
and cytoplasm of principal cells and ciliated cells. In the ductus
epididymidis epithelium, only scattered cells (mitochondria-rich
cells) showed PGP immunoreaction in their cytoplasm, whereas
ubiquitin immunostaining was detected in the nucleus and cytoplasm of most epithelial cells, except for the cauda, where
ubiquitin immunolabeling was observed only in the nuclei. The
ductus deferens showed no immunostaining for PGP, and only
nuclear immunoreactivity to ubiquitin. The ultrastructural localization of PGP immunoreactivity was in the apical cytosol
and microvilli. In addition to these locations, ubiquitin immunoreactivity was also found in the nucleus of all cell types and
cilia of ciliated cells. Although the distribution of PGP and ubiquitin immunoreactivities in humans differs from that reported
in rats, it seems that PGP and ubiquitinated proteins are secreted
into the epididymal lumen in both species.
INTRODUCTION
Protein gene product 9.5 (PGP) is a normal component
of neuronal cytosol [1, 2]. This protein was detected by
immunohistochemistry in neurons and neuroendocrine cells
of several mammalian species [3-6]. It has been stated that
PGP is a ubiquitin carboxyl-terminal hydrolase [7] and that
its L1 isozyme (neuronal PGP 9.5) can be detected by
Western blotting in testicular tissues [8]. Ubiquitin is a normal component of most eukaryotic cells, and it is assumed
that this protein plays an important role in intracellular proteolysis [9] and that it is probably involved in many other
processes of cell metabolism, including extra-lysosomal
protein degradation, modulation of some cytoplasmic and
nuclear proteins and membrane receptors, and response to
heat shock [10]. It has also been reported that ubiquitin can
MATERIALS AND METHODS
Accepted April 9, 1996.
Received November 29, 1995.
'This work was partially supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Madrid, Spain.
2
Correspondence: Dr. Luis Santamarfa, Department of Morphology
(Histology), Autonomous University, Calle Arzobispo Morcillo, 2,Madrid,
E-28029 Madrid, Spain. FAX: 34-1-3975353.
291
The epididymides and deferent ducts from 16 men (between 40 and 75 yr of age) were obtained in autopsies
carried out in the hospitals "Principe de Asturias" (Alcala
de Henares) and "La Princesa" (Madrid). The conditions
for selection were the absence of reproductive, endocrine,
or related diseases in the patient histories and the presence
292
FRAILE ET AL.
of complete spermatogenesis in their testes. The specimens
were collected between 8 and 10 h after death. To evaluate
postmortem changes in the autopsy specimens, three epididymides obtained from testicular tumor surgery were immediately fixed and processed in the same way.
For Western blotting analysis, unfixed fragments of the
ductuli efferentes, ductus epididymidis, and ductus deferens
from each specimen were homogenized in 0.5 M Tris-HCI
buffer (pH 7.4) containing 1 mM EDTA, 12 mM 2-mercapto-ethanol, and 1 mM PMSFE The homogenates were
centrifuged at 10 000
g for 30 min. After boiling for 2
min at 98°C, 25-plg aliquots of protein extracts were separated in SDS-polyacrylamide (15% w:v) slab minigels, according to the procedure of Laemmli [19]. Electrophoretic
blotting onto nitrocellulose and immunological protein detection were carried out as described by Towbin et al. [20],
using either rabbit polyclonal anti-PGP (Ultraclone, Cambridge, UK) or rabbit polyclonal anti-ubiquitin (Dako,
Silkeborg, Denmark) as first antibodies, and the rabbit extra-avidin peroxidase staining kit (Sigma, Barcelona, Spain)
as the second antibody. The purity and specificity of primary antibodies had been tested by the manufacturers and
corroborated in previous studies [2, 3, 14]. The filters were
developed by an enhanced chemiluminescence (ECL)
Western blotting analysis, according to the procedure described by the manufacturer (Amersham, Buckinghamshire,
UK). Negative controls were obtained by omitting the primary antibody.
For light microscopic immunohistochemical study,
2-mm-wide slices from each epididymis and ductus deferens were fixed for 10 h in 4% paraformaldehyde in PBS
(pH 7.4), paraffin-embedded, and sectioned 5 m thick.
Sections from each region (ductuli efferentes; caput, corpus
and cauda of the ductus epididymidis; and ductus deferens)
were immunostained according to the avidin-biotin peroxidase method. Before incubation with antisera, endogenous
peroxidase activity of the tissues was blocked with 0.3%
hydrogen peroxide in PBS. The tissues were incubated
overnight at 4°C with the same primary antibodies used for
Western blotting-rabbit polyclonal antibody against PGP
at 1:1000 and rabbit polyclonal antibody to ubiquitin at 1:
400 dilutions. Further 1-h incubations were carried out using a goat anti-rabbit biotinylated immunoglobulin (Dako).
The sections were subsequently treated with an avidin-biotin peroxidase complex (Dako) and developed with diaminobenzidine (DAB) using the glucose oxidase-DABnickel intensification method [21]. The sections were then
counterstained with Harris' hematoxylin, dehydrated in ethanol, and mounted in Depex.
For the electron microscopic immunocytochemical
study, small fragments of the ductuli efferentes, all the
regions of the ductus epididymidis, and the deferent ducts
were fixed by immersion in 1:1 (v:v) 1% glutaraldehyde3% paraformaldehyde for 6 h and embedded in Lowicryl
K4M (Chemische Werke Lowi, Waldkraiborg, Germany).
Ultrathin sections were mounted onto nickel grids and incubated for 2 h at room temperature with the same primary
antibodies used in the light microscopy method at 1:500
dilution to anti-PGP antibody and at 1:200 dilution to antiubiquitin antibody. Then all the sections were treated with
15 nm gold-labeled IgG goat anti-rabbit antibody (Biocell,
Cardiff, UK) for 2 h at room temperature and counterstained with uranyl acetate. The background immunoreaction was determined in each subject and epididymal region
by counting the number of immunogold particles in extracellular locations in at least 15 micrographs (at a final mag-
FIG. 1. Western blotting in extracts from ductuli efferentes (EfD), ductus
epididymidis (EpD), and ductus deferens (DD), after 15% SDS-polyacrylamide gel electrophoresis. MW, molecular weight standards (x 10 3):
protein markers stained with Coomassie blue; TP, total protein stained
with Coomassie blue; C, negative controls obtained by omitting the primary antibody; PGP, Western blot stained with anti-protein gene product
9.5 antibodies; Ub, Western blot stained with anti-ubiquitin antibodies.
nification of 15 000) of three nonconsecutive ultrathin
sections (five micrographs per section). The intensity of
background was scored according to the following criteria:
intense (> 10 particles per pxm 2); medium (2-10 particles);
and low (< 2 particles).
To assess the immunostaining specificity in both light
and electron microscope immunocytochemical methods,
negative controls either omitting primary antibodies or using these antibodies preabsorbed with an excess of purified
antigens were also analyzed.
RESULTS
Comparison of the epididymides obtained during surgery
with the autopsy specimens showed neither histological nor
histochemical changes.
Western Bloting Analysis
Western blotting analysis revealed a PGP immunoreactive band, at approximately 27 kDa, in the extracts of all
sperm excretory duct segments studied. This band was intense for the ductuli efferentes and weak for the ductus
epididymidis and ductus deferens. A ubiquitin immunoreactive band, at -60 kDa, was also found for all these
FIG. 2. Negative control (primary antibody omitted) of ductuli efferentes
(star) counterstained with Harris' hematoxylin. No immunostaining is observed. 250.
FIG. 3. Caput epididymidis immunostained for PGP and counterstained
with Harris' hematoxylin. Intense, granular, and diffuse immunoreactivities are observed in some epithelial cells from ductuli efferentes (large
arrows). Several periductal myoid cells (small arrow) and nerve fibers
(arrowhead) are also immunostained. 400.
FIG. 4. Corpus epididymidis immunostained for PGP and counterstained
with Harris' hematoxylin. Only scattered epithelial cells are immunostained (arrows). 250.
FIG. 5. Ductus deferens immunostained for PGP and counterstained
with Harris' hematoxylin. No immunoreactivity is detected in the epithelial lining (star). Nerve fibers around the duct are immunostained (arrowhead). x250.
FIG. 6. Ductuli efferentes immunostained for ubiquitin. Columnar cells
show a granular immunostaining in their apical cytoplasm (arrow). X500.
FIG. 7. Negative control of the corpus epididymidis counterstained with
Harris' hematoxylin. No immunostaining is observed. 250.
PGP 9.5 AND UBIQUITIN IN HUMAN EPIDIDYMIS AND VAS DEFERENS
293
FIG. 8. Corpus epididymidis immunostained for ubiquitin and counterstained with Harris' hematoxylin. A weak and diffuse immunoreactivity is
observed in the cytoplasm from most epithelial cells lining the duct (large arrow). The nuclei are also immunostained (small arrow). x250.
FIG. 9. Ductus deferens immunostained for ubiquitin and counterstained with Harris' hematoxylin. The nuclei of many epithelial cells (arrow) and
some connective tissue cells (arrowhead) are immunoreactive. Only a slight immunostaining is observed in the cytoplasm of some epithelial cells.
x250.
294
FRAILE ET AL.
segments. This band was intense for the ductuli efferentes
and ductus epididymidis and weak for the ductus deferens.
No band was observed in negative controls (Fig. 1).
Light Microscopy Immunocytochemistry
By light microscopy, granular and diffuse PGP immunostaining was specifically detected in many epithelial cells
of the ductuli efferentes (Figs. 2 and 3), and a diffuse immunostaining was observed in isolated epithelial cells of
the ductus epididymidis in the caput and corpus (Fig. 4).
No PGP immunostaining was found in the epithelial cells
of the ductus deferens (Fig. 5). In all these locations, nerve
fibers and some periductal myoid cells and connective tissue cells were also immunostained for PGP (Figs. 3-5).
A granular immunoreactivity to ubiquitin was seen in
the apical cytoplasm of the ductuli efferentes epithelial cells
(Fig. 6). A diffuse cytoplasmic immunostaining for this
protein was also observed in most columnar cells of the
ductus epididymidis at the level of the caput and corpus
(Figs. 7 and 8). Cytoplasmic immunostaining for ubiquitin
was very slight or absent in the epithelial lining of the
cauda epididymidis and ductus deferens (Fig. 9). In all the
regions studied, a variable degree of ubiquitin immunostaining was observed in the nuclei of both epithelial cells
and connective tissue cells (Figs. 6-9).
Electron Microscopy Immunocytochemistry
In the ductuli efferentes, PGP immunoreactivity was observed in the principal cell cytoplasm but not in the ciliated
cells (Figs. 10-13). In the ductus epididymidis, ultrastructural PGP immunolabeling was found in some columnar
cells (the mitochondria-rich cells) of the caput and corpus
epididymidis (Fig. 14). Neither the principal cells nor the
basal cells immunoreacted to PGP in the ductus epididymidis. In all immunolabeled epithelial cell types, gold particles were very abundant in the apical cytosol and microvilli
(Fig. 11) and, to a lesser degree, in the basal cytoplasm
(Fig. 12). In addition, the mitochondria of the mitochondria-rich cells also appeared to be immunolabeled (Fig. 14).
A low number of immunogold particles (2-3 particles/
~xm 2), slightly higher than that of background, were also
seen on the filaments of the periductal myoid cells located
along the ductus epididymidis (Fig. 15). In 95% of the electron microscopic fields examined, the background immunogold particles was scored as low.
At the ultrastructural level, ubiquitin immunolabeling
was found in both the ciliated cells and the principal cells
of the ductuli efferentes (Fig. 16) and in both the principal
cells and the mitochondria-rich cells of the caput and corpus epididymidis (Fig. 17). Immunolabeling was mainly located in the nucleus, apical cytosol, cilia, and microvilli
(Figs. 16 and 17). The cytoplasm of the basal cells in the
caput and corpus epididymidis, and that of all epithelial cell
types in the cauda epididymidis (Fig. 18) and ductus deferens showed no immunolabeling to ubiquitin. Gold particles were also observed in the nucleus, cytosol, and filaments of some periductal myoid cells along the ductus epididymidis length. In 96% of the electron microscopic fields
examined, the background immunogold particles was
scored as low.
that reported in the rat epididymis [14, 15], although proteins labeled to PGP and ubiquitin seem to be secreted into
the epididymal lumen in both species. PGP immunolabeling
in the rat epididymis was positive in the principal cells of
the caput and cauda epididymidis and in scattered cells of
the ductuli efferentes and was negative in the corpus epididymidis. However, PGP immunolabeling in men was
found in the principal cells of the ductuli efferentes, and
only in scattered cells (mitochondria-rich cells) from the
ductus epididymidis. In addition, whereas ubiquitin immunolabeling was only positive in scattered epithelial cells
of the rat epididymis [14], ubiquitin immunoreactivity in
men was intense in most epithelial cells from both the ductuli efferentes and ductus epididymidis (with the exception
of the cauda).
In the present study, the immunocytochemical localization of PGP and ubiquitin-like proteins agrees with the results of Western blotting analysis: the PGP immunoreactive
band was intense in the ductuli efferentes and weak in both
the ductus epididymidis and the ductus deferens, whereas
ubiquitin immunoreactivity was intense in the ductuli efferentes and the ductus epididymidis and very weak in the
ductus deferens. Although no immunoreaction to either antigen was histochemically detected in the cytoplasm of epithelial cells in the ductus deferens, the weak PGP immunoreactive band found in the Western blotting analysis of
the ductus deferens may be attributable to immunoreaction
in nerve fibers and connective tissue cells, while the ubiquitin band may be due to nuclear immunoexpression in
most epithelial cells and connective tissue cells.
The ultrastructural study has revealed the cell types that
are immunostained and the intracellular localization of both
antigens. The observation of PGP immunoreactivity in the
cytosol agrees with the PGP location described in nerve
cells [22, 23] and in the epithelial cells of rat epididymis
[15]. In the ductuli efferentes, the observation of PGP immunolabeling only in the principal cells might be related
to the secretion of a PGP immunoreactive protein to the
FIG. 10. Negative control (omission of the primary antibody) of a principal cell of the ductuli efferentes. No immunolabeling is observed.
x35 000.
FIG. 11. Apical cytoplasm of a principal cell of the ductuli efferentesimmunolabeled to PGP. Gold particles are located in the cytosol (star)
and microvilli (arrow). x35 000.
FIG. 12. Sparse PGP immunogold labeling in the basal cytoplasm (star)
of a principal cell of the ductuli efferentes. BM, basal membrane.
x35 000.
FIG. 13. PGP immunogold labeling in a ciliated cell of the ductuli efferentes. No immunogold labeling to PGP is observed in the apical cytoplasm (star) and cilia (arrow). x35 000.
FIG. 14. Mitochondria-rich cell of the caput epididymidis immunolabeled to PGP. Immunogold particles appear in the cytosol (star) and mitochondria (M). J, intercellular junction. x35 000.
FIG. 15. Periductal myoid cell of the corpus epididymidis showing immunolabeling to PGP in its filaments (arrow). x35 000.
FIG. 16. Immunogold labeling to ubiquitin in a ciliated (star) and a principal cell (asterisk) of the ductuli efferentes. Cilia (large arrow), microvilli
(small arrow), and the cytosol are labeled. J, intercellular junction.
x35 000.
DISCUSSION
FIG. 17. Principal cell in the corpus epididymidis showing immunogold
labeling to ubiquitin in the cytosol (star) and microvilli (arrow). x35 000.
The distribution of PGP and ubiquitin immunoreactivities in the human epididymis differs in some aspects from
FIG. 18. Principal cell in the cauda epididymidis showing no immunolabeling to ubiquitin. x35 000.
PGP 9.5 AND UBIQUITIN IN HUMAN EPIDIDYMIS AND VAS DEFERENS
295
296
FRAILE ET AL.
ductal lumen, as has been reported in the rat [15]. The
secretion of this protein seems to be limited to the efferent
ducts in humans, and it might characterize one of the acidic
glycoproteins that have been detected in epididymal fluid
and that are involved in spermatozoon maturation [24, 25].
Since no immunolabeling was observed within cytoplasmic
membranes, a question arises concerning the way in which
the immunolabeled proteins are exported to the lumen. The
preferential localization of PGP and ubiquitin immunoreactivities in cytoplasmic apical regions of secretory cells suggests that the secretory mechanism is not exocytosis but an
apocrine-like secretion process similar to that recently reported in the mouse vas deferens [26].
The only epithelial cells that immunoreacted to PGP in
the human ductus epididymidis were the mitochondria-rich
cells (also called narrow cells, apical cells, or flask cells
according to their variable shape), which form a minor population of epididymal epithelial cells in humans and most
mammalian species studied [27]. These cells are usually
present only in the initial epididymal segments and are
characterized by many ultrastructural, enzymohistochemical, and histochemical features [28]. The morphological
patterns of these cells vary, even in the same epithelium,
from slender cells (narrow cells) to round apical cells without apparent connection with the epithelial basal lamina
(apical cells); but all of them have in common the presence
of short microvilli and an electron-dense cytoplasm with
abundant mitochondria (mitochondria-rich cells). Histochemical and biochemical studies have also shown differences between these cells and the epididymal principal cells
with respect to enzymatic activities, intermediate filament
types [28], and lectin affinity [29]. Two possible functions
have been suggested for the epididymal mitochondria-rich
cells: cooperation with the principal cells in reabsorption of
testicular fluid [30], and acidification of epididymal fluid
[31]. Although the significance of the apical mitochondriarich cells remains still unknown, it is generally assumed
that the function of these cells would be similar to that of
other mitochondria-rich cells, including those of the distal
convoluted tubules from the kidney [28]. These renal cells
also show PGP immunoreaction and seem to be involved
in endocytosis and transport of electrolytes and water [16].
The presence of a PGP immunoreactive protein in the cytosol and mitochondria of these cells could probably indicate involvement in the specific function of these cells. Although mitochondrial labeling is usually unspecific in immunoelectron microscopy, the only mitochondrial immunoreactivity observed in the present study was for PGP in
the apical mitochondria-rich cells, and this supports the
specificity of mitochondrial immunolabeling in this case.
Immunolabeling to PGP in the periductal myoid cells is
doubtful because the number of immunogold particles was
only slightly higher than that found in the background.
However, the location of these particles, limited to cellular
filaments, and the positive immunostaining of these cells
by light microscopy suggest the specificity of this immunostaining.
Immunolabeling to ubiquitin was observed in the nuclei
of all cell types, including connective tissue cells, in all the
regions studied. Ubiquitin has been shown to be involved
in the proteolysis of some nuclear proteins, and there is
also evidence that ubiquitinated histones may prevent the
formation of higher-order chromosomal structures and regulate cell cycle and gene expression [32]. Cyclins, which
lead to inactivation of M-phase promoting factors, seem
also to be degraded by the ubiquitin pathway [33]. Since
androgen receptors have been demonstrated in the epididymis [34], it is interesting that testosterone and LH-releasing hormone directly affect the occurrence of ubiquitin in
the nuclei of the epididymal epithelial cells [35].
Ubiquitin immunoreactivity was also present in the cytosol of most columnar epithelial cells in the ductuli efferentes and ductus epididymidis, but it appeared neither in
the basal cells of the ductus epididymidis nor in any cell
type of the ductus deferens. Cytosolic localization of ubiquitin has also been reported in other cell types [36]. The
association of free ubiquitin and PGP 9.5 with the slow
component b, which transports cytoplasmic enzymes but
not membranous structures through the axon [37], also is
consistent with the cytosolic location of these proteins.
Whereas ubiquitin immunolabeling in the principal cells
of the ductuli efferentes and those of the ductus epididymidis might be associated with proteins secreted into the lumen, in agreement with the presence of ubiquitin in human
seminal plasma [17], the cytoplasmic ubiquitinated proteins
observed in the ciliated cells of the ductuli efferentes are
probably not secreted, because these cells do not have a
secretory function. This ubiquitin might be associated with
extra-lysosomal degradation of denatured, misfolded, or
otherwise abnormal proteins [9], or play a structural role in
relation to cytoskeletal components, as described in other
locations [11-13]. The observation of ubiquitin immunolabeling in cell filaments of periductal and connective tissue
cells in the epididymis supports this hypothesis. In this
study, ubiquitin immunolabeling was not detected in the
vacuoles and residual bodies, and thus an association of
ubiquitin with digestive vacuoles, as reported in other cell
types such as hepatoma cells [36], neutrophil leukocytes
[38], cultured fibroblasts [39], and nerve tissue [40], is improbable in this case.
ACKNOWLEDGMENTS
We are indebted to Prof. J.M. Polak, Department of Histochemistry,
Hammersmith Hospital, London, UK, who supplied the PGP antibody
manufactured by Ultraclone.
REFERENCES
1. Doran JF, Jackson P, Kynoch PAM, Thompson RJ. Isolation of PGP
9.5, a new human neuron-specific protein detected by high resolution
of two-dimensional electrophoresis. J Neurochem 1983; 40:15421547.
2. Jackson P, Thompson VM, Thompson RJ. A comparison of the evolutionary distribution of the two neuroendocrine markers, neuron-specific enolase and protein gene product 9.5. J Neurochem 1985; 45:
185-190.
3. Rode J, Dhillon AP, Doran JF, Jackson P, Thompson RJ. PGP 95 a
new marker for human neuroendocrine tumors. Histopathology 1985;
9:147-158.
4. Kent C, Clarke PJ. The immunolocalisation of the neuroendocrine
specific protein PGP 9.5 during neurogenesis in the rat. Dev Brain
Res 1991; 58:147-150.
5. Kent C, Rowe HL. The immunolocalisation of ubiquitin carboxylterminal hydrolase, PGP 9.5, in developing paraneurons in the rat.
Dev Brain Res 1992; 68:241-246.
6. Properzi G, Cordeschi G, Francavilla S. Post-natal development and
distribution of peptide containing nerves in the genital system of the
male rat. An immunohistochemical study. Histochemistry 1992; 97:
61-69.
7. Wilkinson KD, Lee K, Deshpande S, Duerken-Hughes P, Boss JM,
Pohl J. The neuron-specific protein PGP 9.5 is a ubiquitin carboxylterminal hydrolase. Science 1989; 246:670-673.
8. Wilkinson KD, Deshpande S, Larsen CN. Comparisons of neuronal,
PGP 9.5 and non-neuronal ubiquitin C-terminal hydrolases. Biochem
Soc Trans 1992; 20:631-637.
9. Fried V, Smith HT, Hildebrandt E, Weiner K. Ubiquitin has intrinsic
PGP 9.5 AND UBIQUITIN IN HUMAN EPIDIDYMIS AND VAS DEFERENS
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
proteolytic activity: implication for cellular regulation. Proc Natl Acad
Sci USA 1987; 84:3685-3689.
Crooke ST, Mirabelli CK, Ecker DJ, Butt TR, Jonnalagadda S, Dixon
S, Muller L, Brown F, Weber P Monia BE Studies on the structure
and function of ubiquitin In: Hook JB, Poste G (eds.), Protein Design
and the Development of New Therapeutics and Vaccines. New York:
Plenum Press; 1990: 425-445.
Murti KG, Smith HT, Fried VA. Ubiquitin is a component of the
microtubule network. Proc Natl Acad Sci USA 1988; 85:3019-3023.
Ohta M, Marceau N, Perry G, Manetto V, Gambetti P. Autilio-Gambetti L, Metuzls J, Kawahara H, Cadrin M, French SW Ubiquitin is
present in cytokeratin intermediate filaments and Mallory bodies of
hepatocytes. Lab Invest 1988; 59:848-856.
Manetto V, Abdul-Karim FW, Pery G, Tabton M, Autilio-Gambetti P.
Selective presence of ubiquitin in intracellular inclusions. Am J Pathol
1989; 134:505-513.
Santamaria L, Martin R, Paniagua R, Fraile B, Nistal M, Terenghi G,
Polak JM. Protein gene product 9.5 and ubiquitin immunoreactivities
in rat epididymis epithelium. Histochemistry 1993; 100:131-138.
Martin R, Santamaria L, Fraile B, Paniagua R, Polak JM. Ultrastructural localization of PGP 9.5 and ubiquitin immunoreactivities in rat
ductus epididymidis epithelium. Histochem J 1995; 27:431-439.
Wilson POG, Barber PC, Hamid QA, Power BE Dhillon AP, Rode J,
Day INM, Thompson RJ, Polak JM. The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal
antibodies. Br J Exp Pathol 1988; 69:91-104.
Lippert TH, Seeger H, Schieferstein G, Voelter W. Immunoreactive
ubiquitin in human seminal plasma. J Androl 1993; 14:130-131.
Lippert TH, Schreisler-Zeh S, Voelter W, Gehrke P, Jemsson HP. uber
die uterusrelaxierenden eigenschaften von ubiquitin und relaxin. Arch
Gynecol Obstet 1987; 242:724-725.
Laemmli UK. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 1970; 227:680-685.
Towbin H, Staehlin T, Gordon J. Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure and some
applications. Proc Natl Acad Sci USA 1979; 76:4350-4354.
Hsu SM, Soban E. Colour modification of diaminobenzidine, DAB
precipitation by metallic ions and its application to double immunohistochemistry. J Histochem Cytochem 1982; 30:1079-1082.
Gulbenkian S, Wharton J, Polak JM. The visualisation of cardiovascular innervation in the guinea pig using an antiserum to protein gene
product 9.5, PGP 9.5. J Auton Nerv Syst 1987; 18:235-247.
Anderson VER, Hajimohammadreza I, Gallo JM, Anderson BH, Uney
J, Brown AW, Nolan CC, Cavanagh JB, Leigh PN. Ubiquitin, PGP
9.5 and dense body formation in trimethyltin intoxication: differential
neuronal responses to chemically induced cell damage. Neuropathol
Appl Neurobiol 1992; 1:360-375.
Cooper TG. Secretory proteins from the epididymis and their clinical
relevance. Andrologia 1990; 22(suppl):155-165.
Turner TT. Spermatozoa are exposed to a complex microenvironment
as they traverse the epididymis In: Robaire E (ed.), The Male Germ
Cell: Spermatogonium to Fertilization. New York: Nyas; 1991: 364387.
Manin M, Lecher P, Martinez S, Tournadre S, Jean CL. Exportation
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
297
of mouse vas deferens protein without a signal peptide, from mouse
vas deferens epithelium: a model of apocrine secretion. Biol Reprod
1995; 52:50-62.
Martinez-Garcia P, Regadera J, Cobo P, Paniagua R, Nistal M. The
apical mitochondria-rich cells of the mammalian epididymis. Andrologia 1995; 27:195-206.
Palacios J, Regadera J, Nistal M, Paniagua R. Apical mitochondriarich cells in the human epididymis: an ultrastructural, enzymohistochemical, and immunohistochemical study. Anat Rec 1991; 231:8288.
Arya M, Vanha-Perttula T. Comparison of lectin-staining pattern in
testis and epididymis of gerbil, guinea pig, mouse, and nutria. Am J
Anat 1986; 175:449-469.
Sun EL, Flickinger CJ. Morphological characteristics of cells with
apical nuclei in the initial segment of the adult rat epididymis. Anat
Rec 1980; 196:285-293.
Kierszenbaum AL, Lea O, Petrusz P, French FS, Tress LL. Isolation,
culture and immunohistochemical characterization of epididymal epithelial cells from pubertal and adult rats. Proc Natl Acad Sci USA
1981; 78:1675-1679.
Mueller RD, Yasuda H, Hatch CL, Bonner WM, Bradbury EM. Identification of ubiquitinated histones 2A and 2B in Physarum polycephalum. J Biol Chem 1985; 260:5147-5153.
Glotzer M, Murray AW, Kirschner MW Cyclin is degraded by the
ubiquitin pathway. Nature 1991; 349:132-138.
Schleicher G, Drews UM, Stumpf WE, Sar M. Differential distribution
of dihydrotestosterone and estradiol binding sites in the epididymis of
the mouse. An autoradiographic study. Histochemistry 1984; 81:139147.
Ohtani-Kaneko R, Wada Y, Hattori A, Takada K, Hara M, Ligo M,
Imai K, Suzuki T Effects of testosterone and luteinizing hormonereleasing hormone on occurrence of ubiquitin in the nucleus of rat LH
cells in vitro. Endocrinol J 1994; 2:69-73.
Schwarzt AL, Ciechanover A, Brandt RA, Greuze RJ. Immunoelectron microscopic localization of ubiquitin in hepatoma cells. EMBO
J 1984; 7:2961-2966.
Bizzi A, Schoetzle B, Patton A, Gambetti P, Autilio Gambetti L. Axonal transport of two major components of the ubiquitin system: free
ubiquitin and ubiquitin carboxyl-terminal hydrolase PGP 9.5. Brain
Res 1991; 548:292-299.
Laszlo L, Doherty FJ, Watson A, Self T, Landon N, Lowe J, Mayer
J. Immunogold localization of ubiquitin-protein conjugates in primary,
azurophilic. granules of polymorphonuclear neutrophils. FEBS Lett
1991; 279:175-178.
Mayer RJ, Lowe J, Landon M, McDermott H, Tuckwell J, Doherty F,
Laszlo L. Ubiquitin and lysosome system: molecular immunopathology reveals connection. Biomed Biochim Acta 1991; 50:4-6.
Lowe J, Blanchard A, Morrell K, Lennox G, Reynolds L, Billett M,
Landon M, Mayer J. Ubiquitin is a common factor in intermediate
filament inclusion bodies of diverse type in man including those of
Parkinson's disease, Pick's disease, and Alzheimer's disease, as well
as Rosenthal fibers in cerebellar astrocytomas, cytoplasmic bodies in
muscle, and Mallory bodies in alcoholic liver disease. J Pathol 1988;
155:9-15.