J. Anat. (1999) 195, pp.

481–489, with 10 figures Printed in the United Kingdom 481

Colocalisation of neuropeptides, nitric oxide synthase and

immunomarkers for catecholamines in nerve fibres of the
adult human vas deferens

P. Y. P. J E N, J. S. D I X O N A N D J. A. G O S L I N G

Department of Anatomy, The Chinese University of Hong Kong, Shatin, Hong Kong

(Accepted 25 May 1999)


Single and double-label immunofluorescence methods were used to determine the distribution and patterns
of colocalisation of various neuropeptides and nitric oxide synthase (NOS) with the catecholamine
synthesising enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DβH) in nerve fibres
within specimens of adult human vas deferens obtained at vasectomy (age range 28 to 83 y). Cholinergic
nerve fibres were immunolabelled with an antiserum to vesicular acetylcholine transporter (VAChT). Using
the general nerve marker protein gene product 9.5 (PGP) the density of intramural nerve fibres was found to
be similar irrespective of age. Many of these axons, especially in the outer 2 muscle layers were TH and
DβH-immunoreactive (IR) and were thus confirmed as noradrenergic. Fewer such axons were seen in the
inner longitudinal muscle layer. All the noradrenergic nerve fibres also displayed NPY-immunoreactivity
with minor populations containing galanin (GAL) or somatostatin (SOM). Nerve fibres lacking TH and
DβH-IR were immunoreactive for VAChT and were sparsely distributed throughout the 2 outer muscle
layers but more numerous in the inner muscle layer. Nerves lacking TH and DβH were immunoreactive for
NPY and some also contained NOS, VIP or CGRP. These results have been compared with those obtained
previously from specimens of human neonatal and infant vas deferens where, in contrast to the present
results, NOS and VIP were shown to be colocalised with TH in many of the intramuscular nerve fibres. It
thus appears that NOS and VIP cease their coexistence with TH in intramuscular nerve fibres of the human
vas deferens between the pre- and postpubertal states. In addition to the intramuscular nerve fibres a
VAChT-IR subepithelial nerve plexus occurs in the vas deferens and may control the secretory activity of
the lining epithelium. Most of these subepithelial nerve fibres were immunoreactive for NPY and many also
contained VIP while minor populations were immunoreactive for NOS, GAL, SOM or SP although fibres
containing CGRP were not observed. The neuropeptide content of the subepithelial nerve plexus was similar
to that observed in the infant, except for an increased density of VIP-IR nerves, which may reflect greater
activity of the lining epithelial cells in the adult vas deferens.

Key words : Autonomic nervous system ; ageing

1973 ; Elbadawi & Goodman, 1980 ; Alm, 1982 ;


It has long been known that the smooth muscle coat
of the vas deferens of numerous species including the
human receives a dense noradrenergic innervation
(Sjo$ strand, 1965 ; Owman & Sjo$ strand, 1965). Early
studies used the specific Falck-Hillarp formaldehyde
induced fluorescence (FIF) method to demonstrate
catecholamines (Baumgarten et al. 1968 ; Shirai et al.
McConnell et al. 1982) while electron microscopic
studies have confirmed the presence of characteristic
small dense-cored vesicles in the majority of intramus-
cular nerve terminals in the human vas deferens
(Baumgarten et al. 1971 ; Popovic et al. 1973 ;
McConnell et al. 1982). More recent immunohisto-
chemical studies have revealed the presence of a
variety of biologically active substances within nerve

Correspondence to Professor J. S. Dixon, Department of Anatomy, The Chinese University of Hong Kong, Shatin, New Territories, Hong
Kong.
482 P. Y. P. Jen, J. S. Dixon and J. A. Gosling
terminals supplying the human vas deferens, including
various neuropeptides, neurotransmitter synthesising
enzymes and also nitric oxide synthase (NOS), an
enzyme involved in the formation of nitric oxide (NO)
(Alm, 1982 ; Gu et al. 1983 ; Vaalasti et al. 1986 ; Ehre! n
et al. 1994 ; Gosling & Dixon, 1994 ; Jen et al. 1995 ;
1996 a, 1997 ; Tainio, 1995 ; Dixon et al. 1998).
However, as yet, no attempt has been made to
determine the patterns of colocalisation of the various
neuropeptides with NOS and to relate these to the
noradrenergic and nonnoradrenergic nerves supplying
the adult human vas deferens using double-label
immunocytochemistry. Our own previous studies of
the human vas using this method were limited to
neonatal and infant specimens (Jen et al. 1996 a ; 1997)
while similar studies in various laboratory animals
have indicated the existence of distinct species varia-
tions in the neurochemical coding of nerve fibres
innervating this organ (for review see Kaleczyc, 1998).
In the present study, we have used single and
double-label immunofluorescence techniques to in-
vestigate the distribution and patterns of colocalis-
ation of neuropeptide Y (NPY), vasoactive intestinal
peptide (VIP), calcitonin gene related peptide
(CGRP), substance P (SP), galanin (GAL), somato-
statin (SOM) and NOS with the catecholamine
synthesising enzymes tyrosine hydroxylase (TH) and
dopamine beta-hydroxylase (DβH) in specimens of
human vas deferens removed at vasectomy from adult
males ranging in age from 28 to 83 y. In addition we
have used an antiserum to vesicular acetylcholine
transporter (VAChT) to immunolabel cholinergic
nerve terminals. The results obtained have been
compared with those from our previous studies of the
human prepubertal vas deferens and provide evidence
that changes occur to the neurochemical coding of the
noradrenergic nerves in this organ between pre- and
postpuberty.
  
Collection and processing of tissue
Specimens of human vas deferens were obtained from
12 males undergoing elective vasectomy (age range
28–83 y ; mean age 40 y). In each case consent from
the subject was obtained. The specimens were rapidly
frozen in 2-methylbutane precooled in liquid nitrogen
prior to storage in a refrigerator at k70 mC. Serial
sections (12–15 µm) were cut on a cryostat (Cryocut
1800, Reichert-Jung) at k20 mC, mounted on gelatin-
coated slides and air dried. Every 10th slide was
processed for routine histology using Masson’s tri-
chrome stain, the remaining slides from each specimen
being stored at k70 mC prior to immunohistochemical
processing as described below.
Immunofluorescence
The tissue sections were fixed on slides at room
temperature in acetone for 10 min and subsequently
washed in 3 changes of phosphate-buffered saline
(PBS). The sections were then incubated in 1.5 % goat
serum (ABC Kit, Vector Labs, USA) in PBS for
30 min followed without washing by incubation with
a primary antiserum for 4 h at room temperature (see
Table 1 for details of antisera and dilutions). All
antisera were diluted with PBS containing 0.05 %
sodium azide, 1 % bovine serum albumin and 0.3 %
Triton.
The sites of antigen-antibody reaction were revealed
by incubating each section for 30 min at room
temperature with biotinylated antirabbit antiserum
(BA-1000 dilution 1 : 200, Vector Labs), followed by
fluorescein isothiocyanate-labelled avidin D (FITC
dilution 1 : 200, Vector Labs) for 30 min at room
temperature. Both secondary and tertiary markers
were also reconstituted in 0.05 % sodium azide, 1 %
bovine serum albumin and 0.3 % Triton prior to
incubation. The preparations were then washed in 3
changes of PBS and mounted in glycerol\PBS
mountant.
In addition to the above, some sections from each
specimen were double-immunostained, a total of 12
combinations of primary antisera raised in different
species being used (Table 2). Following the addition
of FITC as described above to label the various
primary antisera these sections were incubated with
the second antiserum for 4 h at room temperature,
washed and transferred to Texas red labelled anti-
mouse or antisheep secondary antibody (IgG-TRSC
conjugate, Jackson Labs, USA) as appropriate for 3
h, followed by washing in 3 changes of PBS prior to
mounting and examination in a Zeiss microscope
equipped with epifluorescence and appropriate exciter
and barrier filters (exciter filter BP 450–490 ; barrier
filter LP 520 ; dichroic mirror FT 510). Fluorescence
photomicrographs were recorded using Fujichrome
(400 ASA) film.
Cross-reactivities of the primary antibody and
secondary antisera were tested and found to be
negative.
 Immunohistochemistry of human vas deferens innervation 483

Table 1. Primary antibody characteristics

Antigen Host species Dilution Source

Calcitonin gene related peptide (CGRP) Rabbit (P) 1 : 500 Serotec

Dopamine beta-hydroxylase (DβH) Rabbit (P) 1 : 200 Incstar
Galanin (GAL) Rabbit (P) 1 : 200 Peninsula
Neuropeptide Y (NPY) Rabbit (P) 1 : 800 Incstar
Neuropeptide Y (NPY) Sheep (P) 1 : 5000 Dr Blessing
Nitric oxide synthase (NOS) Rabbit (P) 1 : 400 Calbiochem
Protein gene product 9.5 (PGP) Rabbit (P) 1 : 400 Ultraclone
Somatostatin (SOM) Rabbit (P) 1 : 500 Serotec
Substance P (SP) Rabbit (P) 1 : 50 Zymed
Tyrosine hydroxylase (TH) Mouse (M) 1 : 200 Incstar
Vasoactive intestinal peptide (VIP) Rabbit (P) 1 : 200 Peninsula
Vesicular acetylcholine transporter (VAChT) Rabbit (P) 1 : 800 Eurodiagnostica

(M), monoclonal ; (P), polyclonal

Table 2. Double-label combinations employed in this study
CGRP (Rabbit) – TH (Mouse)
DβH (Rabbit) – TH (Mouse)
GAL (Rabbit) – TH (Mouse)
NOS (Rabbit) – TH (Mouse)
NPY (Rabbit) – TH (Mouse)
PGP (Rabbit) – TH (Mouse)
SP (Rabbit) – TH (Mouse)
VIP (Rabbit) – TH (Mouse)
VAChT (Rabbit) – TH (Mouse)
DβH (Rabbit) – NPY (Sheep)
NOS (Rabbit) – NPY (Sheep)
VIP (Rabbit) – NPY (Sheep)

Intramuscular nerve fibres
Immunostaining for protein gene product 9.5 (PGP),
a general nerve marker, revealed a dense plexus of
branching varicose nerve fibres throughout the
smooth muscle coat, which in the human vas deferens
comprises a thin inner longitudinal layer, together
with much thicker middle circular and outer longi-
tudinal layers. Irrespective of age, the density of the
intramuscular nerve fibres was similar in all specimens
examined and the majority were observed to be
immunopositive for both DβH and TH (Fig. 1 a, b).
While TH and DβH-IR nerve fibres occurred in
profusion in the 2 outer muscle layers, fewer such
fibres were detected in the inner longitudinal muscle
layer. Double-labelled sections showed that NPY was
colocalised with TH (Fig. 2 a, b) although a number of
NPY-IR nerve fibres which lacked coexistent TH were
also observed. NPY-IR nerve fibres without coexistent
TH were more common in the inner muscle layer.
Occasionally TH-IR nerve fibres were observed
throughout the muscle coat which contained colocal-
ised SOM or GAL (Fig. 3), although in some
specimens the latter type of fibre was quite numerous.
Both NOS-IR (Fig. 4) and VIP-IR nerve fibres were
only moderate in number, being observed within all 3
muscle layers although none appeared to coexist with
TH or DβH (Fig. 5 a, b). Immunostaining for VAChT
revealed a sparse distribution of varicosities through-
out the 2 outer muscle layers while a higher density
was observed in the innermost muscle layer (Fig. 6 a, b).
In some specimens VIP-IR nerve fibres were particu-
larly numerous in the innermost muscle layer (Fig. 7).
Very infrequent CGRP or SP-IR nerve fibres were
observed in all 3 layers of the muscle coat but none
were observed to contain coexistent TH.
Subepithelial nerve fibres
Using the general nerve marker PGP, a nerve plexus
composed of delicate, branching varicose fibres was
observed in the lamina propria at the base of the lining
epithelium of each specimen of vas deferens (Fig. 8).
These subepithelial PGP-IR nerve fibres followed the
epithelial infoldings and in some instances, single
varicose nerves were seen to penetrate between the
epithelial cells (Fig. 8). A subepithelial plexus of
similar density was seen following immunostaining
for NPY (Fig. 9) while VIP-IR nerve fibres formed a
subepithelial plexus which was less extensive. How-
ever, unlike NPY, the VIP-IR nerve fibres were
variable in density from section to section of the same
specimen, being quite numerous in many instances
(Fig. 10). Occasional NOS, SP, GAL and SOM-IR
nerves were also observed at the base of the epithelium
while TH and DβH-IR were extremely rare. Subepi-
thelial CGRP-IR nerve fibres were not observed in
any of the specimens included in this study although
VAChT-IR subepithelial varicose axons were nu-
484 P. Y. P. Jen, J. S. Dixon and J. A. Gosling

Fig. 1. Outer muscle layers of human vas deferens double immunostained for DβH (a) and TH (b) showing colocalisation (arrowheads). 42-
y-old subject. i90.
 Immunohistochemistry of human vas deferens innervation 485

Fig. 6. Double immunolabelling for VAChT (a) and TH (b). VAChT-IR varicosities are numerous in the inner muscle coat (arrowheads)
in contrast to TH-IR nerves. Note also that numerous VAChT but not TH-IR varicosities are present beneath the epithelium (E). 28-y-
old subject. i65
Fig. 7. Numerous VIP-IR nerves occur in the inner muscle layer of the human vas deferens. 58-y-old subject. i130.
Fig. 8. Immunostaining for PGP reveals a dense subepithelial plexus of nerve fibres. Arrow indicates a single nerve penetrating the epithelial
layer (E). 36-y-old human vas deferens. i200.
Fig. 9. NPY-IR nerves occur in profusion beneath the epithelium (E) of the human vas deferens. 28-y-old subject. i65.
Fig. 10. Subepithelial VIP-IR nerves are numerous in this specimen of human vas deferens from a 54-y-old subject. i350.

Fig. 2. Middle circular muscle layer of human vas deferens double immunostained for TH (a) and NPY (b) showing colocalisation
(arrowheads). 36-y-old subject. i130.
Fig. 3. GAL-IR nerves are particularly numerous in this specimen of human vas deferens from a 54-y-old subject. i130.
Fig. 4. Moderate numbers of NOS-IR nerves occur throughout the muscle coat of the human vas deferens. 58-y-old subject. i130.
Fig. 5. Double immunolabelling for DβH (a) and VIP (b) showing a profusion of DβH-IR nerves and sparse VIP-IR nerves. Note the absence
of colocalisation. Middle circular layer of human vas deferens from a 36-y-old subject. i65.
486 P. Y. P. Jen, J. S. Dixon and J. A. Gosling
merous in all sections (Fig. 6 a). Double immuno-
labelled sections showed colocalisation of VIP with
NPY and also NOS with NPY in some of the fine
varicose subepithelial nerve fibres.

Using PGP as a general nerve marker (Thompson et
al. 1983), the present study has confirmed that the
muscle coat of the human vas deferens is richly
innervated by autonomic nerves, the density of which
appeared to be similar throughout the wide age range
examined. As anticipated from previous histochemical
studies the majority of the intramuscular nerves of the
human vas deferens contain both TH and DβH,
enzymes involved in the synthesis of noradrenaline,
and may therefore be classified as noradrenergic in
type. It is generally accepted that noradrenergic nerves
are excitatory to the smooth musculature of the vas
(Anton & McGrath, 1977) although a major trans-
mitter of this type of nerve is likely to be a purine,
possibly ATP (Allcorn et al. 1986). A previous study
of the human vas deferens using the FIF technique
reported that fluorescent noradrenergic nerves were
quite pronounced in specimens from younger indiv-
iduals but there was a relative scarcity of such nerves
in older specimens (Baumgarten et al. 1968). From the
present observations using PGP as a nerve marker on
specimens which varied in age from 28 to 83 y it is
apparent that this previous observation was most
probably due to a reduction in the level of noradren-
aline with age rather than to an actual decrease in the
number of nerve fibres, since even in the oldest
specimens the innervation density was not diminished
when compared with young adults. Thus the density
of autonomic nerves in the human vas deferens is
apparently not affected by age, unlike that reported to
occur in the urinary bladder where the innervation
density of the detrusor muscle in elderly subjects is
around half that of a young adult (Dixon & Gosling,
1987).
Sections double-immunolabelled for NPY and
either TH or DβH revealed that all the TH and DβH-
IR (noradrenergic) nerve fibres throughout the muscle
coat of the adult human vas deferens contain NPY.
The presence of NPY in noradrenergic nerves has
been reported previously in numerous tissues where it
may have a prejunctional inhibitory effect on the
release of noradrenaline (Lundberg & Stja$ rne, 1984).
NPY has also been reported in the majority of
noradrenergic nerves supplying the rat vas deferens
(Fried et al. 1985 ; Keast, 1992) and in about two-
thirds of those in the guinea pig vas (Song et al.1994)
although curiously there is a paucity of NPY in
noradrenergic nerves supplying the pig vas deferens
(Kaleczyc et al. 1997). In a previous study we found
that about two-thirds of the intramuscular noradren-
ergic (TH-IR) nerves in the neonatal and infant
human vas deferens contained NPY (Jen et al. 1997),
and while this was only a subjective assessment, it
would appear from the present observations that a
change occurs between the pre- and postpubertal
states of the human vas deferens which results in NPY
being expressed in all the noradrenergic intramuscular
nerves in the adult organ.
Double-labelled sections showed that some TH-IR
nerve fibres within the muscle coat also contained
GAL, thus forming a subpopulation of the noradren-
ergic nerves. A similar observation was reported in the
infant and child vas deferens (Jen et al. 1997). Previous
workers reported the presence of GAL in the human
vas deferens using both immunohistochemical and
radioimmunoassay, suggesting the possibility that this
neuropeptide could play a role in the regulation of
smooth muscle tone (Bauer et al. 1986).
SOM was another neuropeptide found to occur in
a small proportion of the noradrenergic intramuscular
nerves of the adult human vas deferens in the present
study. SOM-IR nerve fibres have been reported
previously in the human vas deferens (Gu et al. 1983)
although a more recent study denied their existence
(Tainio, 1995). SOM-IR nerve fibres were occasionally
observed in the neonatal and infant vas deferens (Jen
et al. 1997) although the porcine vas deferens is well
supplied with nerves displaying SOM-immunoreac-
tivity where they form a large subpopulation of the
noradrenergic nerves (Kaleczyz et al.1997). In con-
trast, SOM-containing nerve fibres have been shown
in the present study to form a very minor population
of the noradrenergic nerves supplying the adult human
vas deferens.
In the present study specimens double-labelled for
PGP and TH showed that while PGP-IR nerves
occurred throughout all 3 muscle layers in similar
density, those containing TH were less frequent in the
narrow diameter inner smooth muscle layer. If nerves
lacking TH are assumed to be cholinergic in type then
this observation indicates that cholinergic (para-
sympathetic) nerves are more common in the inner
longitudinal muscle layer, a finding in agreement with
the present observation of numerous VAChT-IR
varicosities in this region. Furthermore this obser-
vation agrees with previous studies which used a
histochemical technique to demonstrate acetylcholin-
esterase (AChE), a presumed marker for cholinergic
nerves (Elbadawi & Goodman, 1980 ; Alm, 1982 ;
 Immunohistochemistry of human vas deferens innervation
McConnell et al. 1982). While the functional signif-
icance of cholinergic input to the innervation of the
muscle coat of the human vas remains unknown,
Sjo$ strand (1962) suggested that its main action may
be to suppress noradrenergic transmission.
This study has shown that the intramuscular nerves
which lack TH and DβH (i.e. nonnoradrenergic) are
immunopositive for NOS, VIP or CGRP and in this
regard the adult human vas is similar to the guinea pig
(Song et al. 1994) and the rat (Ventura et al. 1998).
However, the noncoexistence of NOS and VIP with
TH as reported in the present study of the adult
human vas deferens contrasts with our previous
observations using tissues from infants and children
(Jen et al. 1996 a, 1997). In these latter studies, not
only were NOS and VIP seen to be colocalised with
TH in some of the intramuscular nerve fibres but also
in many of the nerve cell bodies within the pelvic
plexus (Jen et al. 1996 b), a proportion of which are
known to project to the wall of the vas deferens.
Hence it appears that both NOS and VIP become
noncoexistent with TH within the human vas deferens
between infancy and adulthood. In the adult both
NOS and VIP reside in populations of nerve fibres
separate from those containing TH while in the child
there is a certain degree of coexistence. In this
context, it is known from previous studies that some
cholinergic neurons express the noradrenergic pheno-
type (i.e. TH) transiently during development (Landis
& Keefe,1983) and a similar phenomenon may occur
in the human vas deferens in the early postnatal
period. Furthermore, the presence or absence of
androgen has been shown to differentially affect the
levels of NOS in the epididymis, prostate and seminal
vesicles of the rat, suggesting a hormonal regulatory
influence on NOS (Chamness et al. 1995). The precise
roles of NO and VIP in the functional control of the
human vas deferens clearly require further invest-
igation. However VIP has been shown to have an
inhibitory effect on the smooth musculature of the
human genitourinary tract (Larssen et al. 1981) and in
other organs such as the urinary bladder, NO has
been shown to cause relaxation of smooth muscle cells
(Burnett, 1995). In contrast, in the rat vas deferens
NO has been proposed to play an excitatory role in
neurotransmission (Vladimirova et al. 1994) whereas
in the guinea pig vas deferens NO has been shown to
affect neurotransmission in a complex fashion without
altering noradrenaline release (Cederqvist & Gus-
tafsson, 1994).
When considering the role of neuropeptides and
NO on the functional activity of the vas deferens it
must be appreciated that the density of nerves and
487
their chemical coding may vary along the length of the
human organ. All the specimens of the vas presently
studied were obtained from a region anterior to the
body of the pubis and so we are unable to comment on
possible variations which may occur in other regions
of the vas deferens. However, a recent study of the rat
vas deferens demonstrated that NOS-IR nerves were
more numerous in the abdominal portion when
compared with the scrotal portion (Ventura &
Burnstock, 1996) although the distribution of neuro-
peptides in the pig vas deferens was reported to be
similar throughout its length (Kaleczyc et al. 1997).
The human vas deferens possesses a dense plexus of
nerve fibres in the lamina propria as presently
demonstrated by immunostaining for PGP and oc-
casional nerve fibres were seen to penetrate between
the epithelial cells although this has not been reported
in previous studies of the vas deferens in other
mammalian species. Previous neurohistochemical
studies of the human vas deferens have shown that the
subepithelial nerves are rich in AChE (Shirai et al.
1973 ; Alm, 1982 ; McConnell et al. 1982) and are
considered to be cholinergic in type, probably per-
forming a secretomotor function (Sjo$ strand, 1962).
The present observation of numerous VAChT-IR
subepithelial varicosities confirms that these nerves
are indeed cholinergic. We have also shown that the
subepithelial nerve fibres contain NPY while many
also contain VIP and some contain NOS. A few
subepithelial nerve fibres were also found to contain
SP, GAL or SOM. The physiological relevance of
these various neuropeptides remains to be elucidated
although curiously CGRP was never observed in a
subepithelial location. If sensory nerve fibres are
present beneath the epithelium one would expect to
observe CGRP together with SP in such nerves, since
these neuropeptides are consistent markers for sensory
neurons (Maggi, 1991). Subepithelial VIP-containing
nerve fibres also occur in other reproductive organs
such as the seminal vesicle and prostate (Ottesen &
Fahrenkrug, 1995) where they are considered to be
involved in the control of secretion. In addition VIP
may exert a trophic effect on the epithelial cells as
suggested for subepithelial nerves in the rat vas
deferens (Keast, 1992).
Comparing the present findings with those reported
previously for the neonatal and infant vas deferens
(Jen et al. 1997) it appears that while the density of
NPY-IR subepithelial nerves are similar, those con-
taining VIP are considerably more numerous in the
adult than the child, a finding which may reflect the
increased activity of the lining epithelial cells in the
adult organ. Interestingly it has been shown that sex
488 P. Y. P. Jen, J. S. Dixon and J. A. Gosling
hormones can influence VIP levels in autonomic
nerves (Ottesen & Fahrenkrug, 1995).
In conclusion the present study has provided data
on the neurochemical coding of autonomic nerves
supplying the adult human vas deferens and also
provided evidence that this coding changes between
infancy and adulthood.
              
We are indebted to Mr N. J. R. George of the
Department of Urology, Withington Hospital, Man-
chester for the specimens of human vas deferens, to
Dr Blessing, Flinders Medical Centre, Bedford Park,
Australia for his gift of sheep anti-NPY and to Mr C.
B. Wong and Mr Samuel Y. T. Wong for their expert
technical and photographic assistance respectively.

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