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From the * Department of Pharmacology and the
Department of Neurology and Neurosurgery,
Universidade Federal de São Paulo-Escola Paulista de Medicina,
São Paulo, São Paulo, Brazil; and the
Department of Cell Biology, Biomedical
Research Institute, National University of Mexico, Mexico City, Mexico.
| Correspondence to: Dr Maria Christina W. Avellar, Section of Experimental Endocrinology, Department of Pharmacology, UNIFESP-Escola Paulista de Medicina, Rua 03 de maio 100, São Paulo, SP, Brazil 04044020 (e-mail: avellar.farm{at}infar.epm.br ). |
| Received for publication July 6, 2001; accepted for publication November 28, 2001. |
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Abstract |
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Key words: Autonomic innervation, male reproductive tract, development, neurons, age
Studies using both surgical and guanethidine-induced denervation have shown that the decreased contractility, observed in the rat epididymis with the loss of adrenergic innervation, induces a delay in cauda luminal transit, with a significant increase in the number of spermatozoa present in the cauda epididymis (Billups et al, 1990; Ricker et. al, 1996; Kempinas et al, 1998a,b). The consequences of the loss of innervation to the quality of sperm are, however, contradictory. Billups et al (1990) reported changes in sperm motion parameters after removal of the rat inferior mesenteric ganglion. Ricker et al (1996) also found significant decreases in the fertility of cauda epididymis sperm 1 and 4 weeks after surgical denervation. Kempinas et al (1998a,b), on the other hand, observed that either surgical or chemical sympathectomy, in this case induced by a low level of guanethidine exposure, resulted in a prolonged transit time of the sperm within the epididymis, with no effects on the quality and fertility of the sperm collected from the distal cauda epididymis.
Pharmacological and surgical denervation experiments have also documented the importance of autonomic, and especially adrenergic, innervation for sustaining both the normal growth pattern and the rate of development of testes and other male reproductive organs (Nagai et al, 1982; Gerendai et al, 1984, 1989; Bergh et al, 1987; Zhu et al, 1998; Chow et al, 2000). In fact, autonomic innervation is necessary to maintain mature ovary (Burden and Lawrence, 1977; Gerendai et al, 1978; Burden et al, 1981, 1983) and testicular functional and structural integrity (Hodson, 1965; Nagai et al, 1982; Bergh et al, 1987; Lamano-Carvalho et al, 1996; Zhu et al, 1998; Chow et al, 2000). It has also been suggested that autonomic innervation is even required for the process of gonadal sex determination and/or differentiation in some vertebrate species (Gutiérrez-Ospina et al, 1999). Taken together, these observations suggest that the autonomic innervation might control cell differentiation and development throughout the male reproductive tract.
In the present work, we combined histochemical and biochemical techniques to illustrate how the epididymal innervation pattern might change during the rat sexual maturation. We focused our study at this developmental period because of our interest in understanding the possible role of neuron—target-cell reciprocal trophic interactions on male reproduction and fertility.
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Materials and Methods |
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Glyoxylic Acid Histochemistry
Anesthetized rats were placed on an ice tray and perfused through the left
ventricle with ice-cold saline, followed by an ice-cold phosphate-buffered (61
mM, pH 7.4) fixative solution containing paraformaldehyde (0.5%) and glyoxylic
acid (2%). The caput and cauda epididymis were each isolated, dissected, and
frozen each in dry-ice prechilled 2-methyl butane. The tissue samples were
then cryostat cut (20 µm), mounted onto silanecoated slides, and incubated
(60 seconds) in an ice-cold phosphate-buffered solution containing glyoxylic
acid (2%) for 1 minute. Slides were air dried and placed in an oven at
100°C for 10 minutes. The slides were coverslipped with glycerol and
visualized using a Zeiss epifluorescence microscope (Carl Zeiss, Jena,
Germany) with a 395- to 440-nm excitation filter.
Immunohistochemistry
The caput and cauda epididymides from each rat were dissected, embedded in
Jung tissue freezing medium (Leica Instruments, Nussloch, Germany), rapidly
forzen in dry-ice-prechilled 2-methyl butane, and stored at -75°C until
use. Cryostat caput and cauda epididymis transverse sections (8 µm) were
fixed in formalin (4%) in phosphate buffer (0.1 M, pH 7.4) for 30 minutes. The
sections were then incubated with blocking solution (albumin 3% and Triton
X-100 0.3% in phosphate buffer 0.1 M, pH 7.4) for 1 hour at room temperature.
After several washes with phosphate buffer, sections were then incubated with
primary goat polyclonal antibodies raised against rat dopamine
ß-hydroxylase (DßH), acetylcholinesterase (AChE) (1:25 each, Santa
Cruz Biotechnologies, Santa Cruz, Calif), and microtubule-associated protein
1B (MAP 1B 1:500, Santa Cruz Biotechnologies) diluted in blocking solution,
overnight at 4°C. Following three 10-minute washes in blocking solution,
sections were incubated for 90 minutes at room temperature with rabbit
anti-goat secondary antibody conjugated to biotin (1:200) diluted in blocking
solution. An avidin-biotin complex (ABC) staining system (Santa Cruz
Biotechnologies) was used to localize the biotinylated antibody according to
manufacturer's instructions. Peroxidase activity was revealed by using a
phosphate buffer containing 3,3-diaminobenzidine (0.05%) and hydrogen peroxide
(0.01%) for 3 minutes at room temperature. The enzyme reaction was stopped by
washing several times in phosphate buffer. Air-dried slides were then
coverslipped with entellan. Controls for DßH and AChE
immunohistochemistry included preadsorption of the primary antibody for 2
hours with a fivefold excess of the corresponding blocking peptide (Santa Cruz
Biotechnologies). Thus, the immunostaining obtained with the preadsorbed
antibody was always compared to the nonpreadsorbed primary antibody in serial
sections, in order to analyze specific staining. Negative controls, in the
absence of the primary antibody, were also processed. Regional differences in
the intensity of staining in epithelial cells, being nonexistent in the
efferent ducts, intermediate in the initial segment and caput region, and
highest in the cauda epididymis at all ages analyzed, were observed in
experiments done in the absence of primary and secondary antibodies. The
incubation of an excess of unlabeled avidin (1 µg) for 90 minutes before
detection of peroxidase activity in these experiments prevented the epithelial
staining, indicating a nonspecific epithelial staining associated with the
ABC. The sections were visualized with a Nikon E800 microscope (Nikon,
Melville, NY). Images were processed using Image-Pro Express Software Program
(Media Cybernetics, Silver Spring, Md).
High-Performance Liquid Chromatography for Monoamine
Determination
High-performance liquid chromatography (HPLC) for detection of monoamines
(noradrenaline and adrenaline) was carried out according to the protocol
described by Cavalheiro et al
(1994). Briefly, the caput and
cauda epididymis from rats of different ages were dissected on an ice-chilled
plate, snap frozen in liquid nitrogen, weighted, and stored at -75°C until
use. The tissue samples were ultrasonically homogenized (15 µL/mg tissue)
in a solution containing HClO4 (0.1 M),
Na2S2O5 (0.02%), and dihydroxybenzylamine
(0.7 µM), the latter used as a monoamine internal standard. Samples were
then centrifuged at 11 000 x g at 4°C for 40 minutes, and
the supernatant was filtered and injected into the HPLC system. An isocratic
system consisting of an LKB pump, a clamper, and a column oven fitted with a
rheodyme loop injector (20 µL) was used. Electrochemical detection of
monoamines was performed with an LKB detector with an electrode potential of
-0.5 V. An LKB 2-channel recorder was used, and the chromatographic peak
height was measured. An OD-224 Spheri-5 RP-18 (220 by 4.6 mm) column (Brownlee
Precision Co, San Jose, Calif) with a flow rate of 0.8 mL/min was used. The
phosphate/citrate buffer (0.02 M, pH 2.64) mobile phase contained methanol
(90/10 [vol/vol]), disodium EDTA (0.12 mM), and heptanesulfonic acid (0.06%).
Standard monoamine mixtures were injected at the beginning and end of each set
of experiments to control the performance of the system. Monoamine recoveries
and epididymal concentration calculations obtained after acid treatment were
made as described by Mazzacoratti et al
(1990). Chromatograms were
computer recorded, and the peak height was measured. Results were expressed in
picograms of monoamines per total tissue weight or per milligram of
tissue.
Extraction of Total AChE and Enzyme Activity Assay
The caput and cauda epididymis were dissected, snap frozen in liquid
nitrogen, weighted, pooled in a tube, and stored at -75°C until use. The
tissue samples were homogenized in 1 mL of borate extraction buffer (20 mM, pH
9.0) containing NaCl (1 M), EDTA (5 mM), Triton X-100 (0.5%),
n-ethylmaleimide (5 mM), benzamidine (2 mM), and bacitracin (0.7 mM),
with an Ultra-Turrax homogenizer (T-25, Ika Labortechnik, Stanfeni, Germany).
Each homogenate was centrifuged for 30 minutes (20 000 x g,
4°C), and total AChE activity from the supernatant was assayed by a
radiometric procedure (Johnson and
Russell, 1975), as described by Rotundo and Fambrough
(1979), using 0.1 µCi
[3H]-acetylcholine iodide (2.0 GBq/mmol, 55.2 µCi/mmol, New
England Nuclear, Boston, Mass) as substrate. The enzyme activity was assayed
in the presence of 10 µM butyrylcholinesterase inhibitor tetraisopropyl
pyrophosphoramide (Iso-OMPA; Sigma Chemical Co, St Louis, Mo), and the total
AChE activity (dpm/min) was calculated as arbitrary units (AU). Results were
expressed as AU per total tissue weight or per milligram of tissue.
Statistics
Data were expressed as mean plus or minus standard error of the mean.
Statistical analysis was determined by analysis of variance, followed by
Bonferroni multiple range analysis, using the Instant program (GraphPad
Software, San Diego, Calif). P values less than.05 were accepted as
significant.
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Results |
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AChE-positive neurons, nerve fibers, and puncta were identified in the caput epididymis interstitial space (Figure 2a and b). Qualitative observations demonstrated no obvious variations in the few numbers of AChE-positive staining in the caput, as animals matured (Figure 2a and b). Although no major differences were observed in the number of neurons, fibers, and puncta positively labeled for AChE when 40- and 60-day-old rat cauda epididymides were compared (data not shown), a significant reduction in the amount and intensity of these AChE-positive neuronal elements occurred in the adult cauda epididymis when compared to immature rats (Figure 2c and d). The number and density of staining of AChE-positive neuronal elements were greater in the cauda (Figure 2c and d) than in the caput epididymis (Figure 2a and b) in all ages analyzed.
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Numerous AChE-positive nerve fibers were found sorting out among epithelial cells and ending free upon the epithelial surface or into the tubular lumen in the cauda region of adult animals (Figure 2d, insert). AChE-positive staining of epididymal epithelial cells was currently observed in both the caput and cauda epididymis of 40-, 60-, and 120-day-old rats (Figure 2). All the AChE-positive staining observed in the rat epididymis was blocked when experiments were performed in the presence of the respective blocking peptide (Figure 2e and f).
Immunohistochemical studies against MAP 1B, a neuronal cytoskeletal marker, were used to identify neuronal processes in the caput and cauda epididymis of maturing animals (Figure 3). The amount of fibers labeled in the caput (Figure 3a and b) was lower than that in the cauda epididymis (Figure 3c and d) regardless of animal age. MAP 1B immunoreactive fibers in the caput epididymis did not change with progression of sexual maturation. However, qualitative observations indicated that the amount of MAP 1B-positive elements in the cauda epididymis did not change from 40- to 60-day-old rats (data not shown) but decreased in the adult rats when compared to younger animals (Figure 3c and d).
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Rat Body and Tissue Weight
The effect of sexual maturation on rat body weight and on caput and cauda
epididymis wet weight is shown in Table
1. Rat body weight significantly increased with age. Caput and
cauda epididymis wet weight also increased significantly with progression of
sexual maturation.
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Monoamine Determination in the Caput and Cauda Epididymis of Sexually
Maturing Rats
The caput and cauda epididymis presented a gradual increase in
noradrenaline content, expressed per total tissue weight, with sexual
maturation (Table 2).
Noradrenaline concentration, expressed per milligram of tissue, did not change
in the caput epididymis with increasing age. Although there was no difference
in noradrenaline concentration between 40- and 60-day-old rats, a marked
decrease in noradrenaline concentration occurred in the cauda region of adult
rats (Table 2). Noradrenaline,
either expressed per total tissue weight or per milligram of tissue, was
higher in the cauda than in the caput epididymis in all ages studied
(Table 2).
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Adrenaline was detected in the caput and cauda epididymis of 40-, 60-, and 120-day-old rats within the low range of 12.67-58.33 pg/mg tissue, indicating that nor-adrenaline is the main catecholamine in both regions of the rat epididymis.
AChE Activity in the Caput and Cauda Epididymis of Sexually Maturing
Rats
The caput and cauda epididymis presented an increase in AChE activity per
total tissue weight with sexual maturation
(Table 2). When results were
expressed per milligram of tissue, AChE activity showed a biphasic profile in
the caput epididymis since the activity increased from 40- to 60-day-old rats
and then dropped from 60- to 120-day-old rats to similar values observed in
the immature animals. In the cauda epididymis, on the other hand, there was a
significant progressive decline of AChE activity with increasing age. AChE
activity was higher in the cauda than in the caput epididymis, regardless of
animal age (Table 2).
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Discussion |
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Glyoxylic acid histochemistry and immunohistochemical studies against DßH and AChE indicated that catecholaminergic and AChE-positive neuronal elements were more abundant in the cauda than in the caput epididymal region, as previously described (El-Badawi and Schenk, 1967; Baumgarten et al, 1968). Furthermore, qualitative observations demonstrated a sensible reduction in catecholaminergic and AChE-positive neurons, fibers, and puncta detected in the cauda epididymis of 120-day-old rats when compared to immature (40 days) and young adult (60 days) animals. No obvious variations in the few catecholaminergic and AChE-positive fibers and puncta were observed in the caput region with age.
In accordance with the results obtained with glyoxylic acid histochemistry and immunohistochemical studies, a decrease in the cauda epididymis noradrenaline concentration, as well as in the AChE activity as rats mature sexually, was observed. No obvious shifts in these parameters were observed in the caput epididymis with age, suggesting that noradrenaline and AChE maturational variations are segment-specific. These results support the hypothesis that autonomic nerve remodeling through trophic factor occurs in the cauda epididymis with sexual maturation.
A reduction with age in the number of innervating adrenergic fibers has been reported in other organs of the male reproductive tract of rats (Zieher et al, 1971), macaques (Mayerhofer et al, 1996), and humans (Baumgarten et al, 1968). The results of the present work add, however, that such maturational changes also affect the cholinergic system. It is important to emphasize that, although most AChE-positive staining observed in the present work is generally associated with cholinergic innervation, AChE-positive fibers can also be related to noncholinergic nerves (Papka et al, 1981, 1985), such as neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), calcitonin generelated peptide (CGRP), and substance P-immunoreactive, reported to be present in the cauda epididymis (Lamano-Carvalho et al, 1986). It is worth mentioning that the immunohistochemical studies performed in the present work with the antibody against MAP 1B, a cytoskeletal protein expressed in nerve cells only while remodeling (Schoenfeld et al, 1989; Viereck et al, 1989), also indicated a reduction in the number of MAP 1B-positive nerves in the cauda epididymis. Thus, the results suggest that nerve refinement might also occur in other types of fibers reaching the epididymis or in fibers rising from intrinsic neurons.
The development of the epididymis can be divided into 3 distinct postnatal phases: a proliferative phase, in which undifferentiated cells undergo mitotic activity (days 0-15); a period of differentiation when the blood epididymal barrier is formed, and the columnar cells differentiate into principal cells (days 16-44); and a phase of expansion in which spermatozoa enter the epididymis and are stored in the lumen of the cauda epididymis (days 44-91) (Sun and Flickinger, 1979, 1982). Histological and functional differentiation of the caput during postnatal development precedes that of the cauda epididymis (Rajalakshmi, 1985; Limanowski et al, 2001), mainly due to varying dependence on testicular fluid and the age-dependent and segment-specific role of testosterone during epididymal development (Sun and Flickinger, 1982; Viger and Robaire, 1994). Our results suggest that the age-related reduction in the cauda noradrenaline concentration and AChE activity might be associated in part with a decrease in the number of cauda autonomic neuronal elements. Possible, however, is the involvement of a nerve fiber dilution effect associated with the process of growth of the epididymal structure on these observations. Interestingly, the cauda epididymal weight increased 14-fold between immature and adult animals, while noradrenaline concentration and AChE activity decreased 1.5- and 4.5-fold (or increased 4.3- and 3.2-fold per total tissue weight), respectively. Thus, these results indicate that the availability of noradrenaline and AChE within the tissue does not keep up with the changes in epididymal mass that occur with progression of sexual maturation. Although the biochemical data appear to support the existence of segment-specific remodeling of autonomic innervation through afferent elimination, changes in catecholamine and acetylcholine synthesis and/or degradation as animals mature sexually can not be ruled out. Both processes are not mutually exclusive.
Androgen concentration in epididymal tissue extracts is high relative to plasma, especially in the caput epididymis (Vreeburg, 1975; Pujol et al, 1976; Turner et al, 1989). The circulating levels of testosterone modify in different ways the monoaminergic activity in the central and peripheral nervous systems (Battaner et al, 1987; Siddiqui and Shah, 1997; Kritzer, 2000). It is then plausible to raise the possibility that increased testosterone availability induces epididymal autonomic, especially adrenergic, nerve refinement. Against this possibility, however, is the fact that testosterone serves as a potent trophic signal for the somas of pelvic ganglion neurons supplying adrenergic innervation to vas deferens, urinary bladder, and rectum and for cholinergic neurons supplying innervation to the penis, vas deferens, and prostate gland (Keast and Saunders, 1998). In our study, no major differences were observed in the number of catecholaminergic fibers and the concentration of monoamines when 40- and 60-day-old rat cauda epididymides were compared, although the testosterone levels in the plasma of these animals significantly increased during this period (Queiróz et al, 2001).
In 120-day-old rats, numerous AChE-positive fibers were found sorting out among epithelial cells and ending free upon the epithelial surface or into the tubular lumen of the cauda region. These findings closely resemble those previously reported for histochemically stained AChE in the adult dog and the rat epididymis (El-Badawi and Schenk, 1967). Although the role of these nerves is currently unknown, they have been thought to serve sensory functions (El-Badawi and Schenk, 1967). They might also be the source of minute amounts of acetylcholine that could in part explain the presence of cholinergic receptors (Florman and Storey, 1982; Ward et al, 1994; Baccetti et al, 1995), as well as AChE activity in spermatozoa (Egbunike, 1980). Immunohistochemical studies also revealed the localization of AChE in the epithelial cells of the caput and cauda epididymis in all ages studied. There is speculation in the literature that acetylcholine might be metabolized by an epithelium-dependent AChE activity in guinea pig airways (Small et al, 1990; Koga et al, 1992; Folkerts et al, 2001). Further experiments will be necessary to confirm if the presence of AChE in the epididymal epithelium compartment is correlated with enzyme activity.
Why reduce autonomic innervation during cauda epididymis sexual maturation? We have no definitive answer to this fundamental question. Increased availability of catecholamines at least in the adult testis has deleterious effects on the germinal epithelium growth and differentiation (Chow et al, 2000). In fact, a possible relation between increased number of catecholaminergic neural elements and testicular pathologies has been suggested (Mayerhofer et al, 1999). Also, El-Badawi and Schenk (1967) discussed the inverse relation between innervation density and epithelial cell secretory function. Autonomic innervation growth-promoting actions might then be restricted to certain points along epididymal development. Also, decreased innervation may improve secretory processes in the maturing cauda epididymis, while the lack of innervation may keep ongoing secretion high in the caput epididymis.
It is known that neurotrophic factor-dependent peripheral nerve elimination is a common process during maturation (for a review, see Purves, 1988). In this regard, it would be instructive to evaluate whether the expression of target-derived neurotrophic signals by cells within the reproductive organs (eg, neurotrophins; Russo et al, 1999) covary with the amount of innervation they receive at different times during sexual maturation. Also, it is known that neurotrophic signals may have deleterious effects on the development of neuronal processes depending on the type of the neuron and the time that developing cells and cell factors interact with one another (McAllister et al, 1999). Epididymal autonomic nerve retraction might thus also result from "negative" interactions with neurotrophic signals.
Finally, the present work and previous data (Lamano-Carvalho et al, 1986; Lakomy et al, 1997) show that catecholaminergic, cholinergic, and peptidergic innervation is mainly concentrated in the cauda epididymis. This condition appears permanent throughout development since the present work and others (Properzi et al, 1992) have failed to demonstrate the appearance of significant innervation in the caput epididymis with age. Although the reason for these segmental differences in the epididymal autonomic innervation pattern is unknown, it might reflect the expression of segment-specific morphogenes (or the lack of them; for reviews, see Viger and Robaire, 1995; Serre and Robaire, 1998; Kirchhoff, 1999), whose translation products allow or prevent nerves from growing into the cauda or caput, respectively. Protein families such as netrins, ephrins, semaphorins/collapsins, and slit, known to be involved in nervous system axon guidance (Mark et al, 1997), are just a few potential candidates among others to be considered in the search for epididymal chemoattractant or chemorepellant molecules.
Thus, in this work, we present data showing a segment-specific nerve refinement during rat sexual maturation in the cauda epididymis.
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Footnotes |
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