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From the * Department of Cell Biology and
Physiology, Biomedical Research Institute, National Autonomous University of
Mexico, Mexico City, Mexico; the Department of
Pharmacology, Section of Experimental Endocrinology, Universidade Federal de
São Paulo-Escola Paulista de Medicina, São Paulo, Brazil; the
Department of Reproductive Biology, Faculty of
Sciences, National Autonomous University of Mexico, Mexico City, Mexico; and
the Unit of Medical Investigation in
Neurological Diseases, XXI Century National Medical Center, Mexican Institute
of Social Security, Mexico City, Mexico.
| Correspondence to: Dr. Gabriel Gutiérrez-Ospina, Department of Cell Biology and Physiology, National Autonomous University of Mexico, Ciudad Universitaria, Mexico City, 04510 (e-mail: gabo{at}correo.biomedicas.unam.mx). |
| Received for publication February 1, 2006; accepted for publication September 13, 2006. |
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Abstract |
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51 kd and 48 kd) that
were immunoreactive for tryptophan hydroxylase. Chromatographic analyses
documented the presence of tryptophan hydroxylase in the caput, and showed
that both its activity and serotonin availability increased with sexual
maturation and decreased following p-chlorophenylalanine treatment,
an inhibitor of tryptophan hydroxylase activity. Interestingly, serotonin
concentration and tryptophan hydroxylase activity tended to be higher in
breeding males than in those with no mating experience. We think that these
results support the existence of a local serotoninergic system in the rat
caput epididymis that might regulate some aspects of male reproductive
function.
Key words: 5-hydroxytryptamine, tryptophan hydroxylase, neuroendocrine cells, male reproduction, serotonin receptor, spermatozoa
In the male reproductive system, serotonin plays an important role in the regulation of testicular blood flow (Collin et al, 1996) and in the secretion of corticotropin-releasing factor and testosterone from Leydig cells (Dufau et al, 1993; Tinajero et al, 1993; Frungieri et al, 1999). Serotonin also induces the contraction of the vas deferens (Hay and Wadsworth, 1982). Although the concentration of serotonin in the caput epididymis of some mammals is one of the highest in the body (Kormano and Penttila, 1968; Anderson and Paparo, 1977; Anderson et al, 1979), we know very little about the biology of serotonin in this organ. It is known, however, that serotonin regulates chloride secretion from epithelial cells in the cauda epididymis (Leung et al, 1999).
With respect to its origin, Anderson et al (1979) proposed that the epithelial, mast, and myoepithelial cells were most likely the sources of serotonin in the epididymis; this contention has not been confirmed. In fact, the epididymis contains a large number of mast cells that could well function as an important local source of serotonin (Leung et al, 1999), even though most of them lack the ability to synthesize it (Padawer, 1974). There are, however, a couple of reports that show the presence of the pineal isoform of tryptophan hydroxylase, the limiting-step enzyme in the serotonin synthesis pathway, in a subtype of mast cells located in the dura mater (Mathiau et al, 1994) and in a serotonin-producing RBL2H3 mast cell line (Kojima et al, 1998).
Another potential source of serotonin in the epididymis is constituted by neuroendocrine cells. Neuroendocrine cells are neuroectoderm-derived cells that have the ability of taking up, storing, and releasing serotonin, but they lack tryptophan hydroxylase activity, so they do not have the ability to synthesize this amine (Ibrahim and Koshayan, 1981; Gapp, 1987; for a recent review, see Fujita et al, 1995). A number of studies have identified neuroendocrine cells in various organs of the body and indeed in those of the male reproductive system, but not yet in the epididymis (Hanyu et al, 1987; Vittoria et al, 1990; Abrahamsson, 1999; Mayerhofer et al, 1999; Arrighi et al, 2004). Having this in mind, the main goals of the present work were 1) to identify serotonin cell sources and targets, 2) to document the possibility of local serotonin synthesis, and 3) to document changes in serotonin concentration associated with sexual maturation and reproductive status. Although descriptive in nature, our morphological and biochemical results provide guidelines to design future experiments aimed at evaluating directly the functions of serotonin in the caput epididymis of the male reproductive system.
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Materials and Methods |
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Immunohistochemistry
The epididymis of anesthetized animals (pentobarbital, 45 mg/kg of body
weight; Pfizer, New York, NY) was dissected and freed of fat. The caput
epididymis was isolated, included in tissue freezing medium (Leica
Instruments, Nussloch, Germany), frozen rapidly in 2-methyl butane prechilled
with dry ice, and stored at –75°C until use. Longitudinal sections
(8–15 µm) of the caput (zones II and III) were cut in a cryostat,
mounted onto gelatin-coated slides, and fixed in paraformaldehyde (4%)
dissolved in phosphate buffer (PB 0.1 mol, pH 7.4). Endogenous activity of
peroxidase was blocked by incubating the sections in a solution containing 3%
hydrogen peroxide. Sections intended for detecting tryptophan hydroxylase,
serotonin transporter, and serotonin receptors 5HT1A,
5HT1B, 5HT2A, and 5HT3 were immersion-fixed
for 20–30 minutes. We focused on these serotonin receptors because
5HT1B receptors have been identified in epithelial cells of the rat
cauda epididymis (Leung et al,
1999), whereas receptors of the 5HT1 and
5HT2 families appear to be located in invertebrate (sea urchin:
Bandivdekar et al, 1992;
Stephens and Prior, 1992) and
mammalian spermatozoa (hamster: Meizel and
Turner 1983; rabbit: Young and
Laing, 1990). In addition, Meizel and Turner
(1983) suggested the existence
of a serotonin responsive ionotropic receptor in the sperm of hamsters. The
ionotropic serotonin receptors are grouped in the 5HT3 subfamily
(Gaspar et al, 2003). Sections
used to stain neurofilaments 150 kd and serotonin also were immersion-fixed
for 4 hours at room temperature. Neurofilaments are currently used to identify
neuroendocrine cells (Davidoff et al,
1999; Mayerhofer et al,
1999; Frungieri et al,
2002). After a thorough wash, sections were incubated with
blocking solution (bovine albumin 3%, Triton X-100 0.3%, and sodium azide
0.025% in PB) for 4 hours at room temperature. Sections were then incubated
overnight at 4°C with one of the following primary antibodies. The
information about the specificity and cross-reactivity of each antibody was
provided by the suppliers:
All antibodies were diluted in blocking solution. After 3 washes (10–15 minutes each) with PB, sections were incubated with the corresponding biotinylated secondary antibodies (Vector Laboratories, Burlingame, Calif and Santa Cruz Biotechnologies, Austin, Tex), diluted 1:200 in blocking solution. This incubation lasted for 2 hours at room temperature. The avidin-biotin-peroxidase staining system (Vector Laboratories) was used to detect the biotinylated antibodies following the manufacturer's instructions (Vector Laboratories). The enzymatic reaction was stopped after 1–3 minutes by washing several times in PB. Air-dried slides were cover slipped with Cytoseal (Richard Allan Scientific, Kalamazoo, Mich). In control experiments, slides were incubated with preimmune serum or the incubation with primary antibodies was omitted. The sections were visualized and images acquired using a Nikon Optiphot-2 microscope equipped with a digital camera CoolPix 4300 (Nikon, Melville, NY) and an Olympus BX51 microscope equipped with a digital camera Olympus DP70 (Olympus American Inc).
Images were digitized and figures elaborated using Adobe Photoshop 5.5 (Adobe Systems Incorporated, San Jose, Calif).
Estimations of Cell Density
The density (cell number/750 µm2) of mast and neuroendocrine
cells displaying immunoreactivity for serotonin and of neuroendocrine cells
immunoreactive to neurofilaments was estimated in 7 alternate slices per
animal following the single-slice cell counting protocol
(Chang et al, 1993). Although
this is not an unbiased method, the results regarding cell density are fully
comparable with those obtained using the dissector method
(Wilson et al, 1998). We
analyzed caput epididymis from at least 6 animals per age (40, 60, 90, and 120
days). Neuroendocrine cells were clearly distinguishable from mast cells,
since the latter are larger, have secretory granules of considerable size, and
are located in the interstitial space. The average cell density was estimated
per animal and then per animal group. Because data did not pass the normality
test, we carried out a Kruskal-Wallis one-way ANOVA on ranks (significance
level set at P < .01) followed by a Dunn's method for multiple
comparisons (significance level set at P < .05).
Western Blot for Tryptophan Hydroxylase
Rats of different ages were anesthetized and the caput rapidly dissected,
frozen in 2-methylbutane prechilled with dry ice, and stored at
–75°C. Tissue samples were homogenized in a buffer containing Trizma
hydrochloride (Tris-HCl) (0.05 mol, pH 7.4), dithiothreitol (1 mmol), and
acetic acid (ethylenebis (oxyethylenenitrilo)) tetra-; ethylene glycol bis
(2-aminoethyl ether) N,N,NN-tetraacetic acid (EGTA) (1 mmol), supplemented
with a mixture of protease inhibitors (Complete, EDTA-free; Roche, Mannheim,
Germany). Samples (12.5 µg of protein/well) diluted in Laemmli solution
were electrophoresed, under reducing conditions (5% ß-mercaptoethanol),
through sodium dodecyl sulphate-polyacrylamide gels (12%) at 100–150 V
for 2 hours. Prestained molecular weight markers (Amersham-Pharmacia-Biotech,
Piscataway, NJ) were used to determine the relative mobility of proteins.
Following electrophoresis, the gels were equilibrated in a buffer containing
Tris 25 mmol, glycine 192 mmol, methanol 20% for 15 minutes. The proteins were
then transferred to nitrocellulose sheets (BIO-RAD) at 200 µA for 1 hour at
4°C. The membranes were blocked with nonfat milk (5%) dissolved in Tris
(20 mmol)-sodium chloride (500 mmol) buffer [TBS] for 2 hours at room
temperature. Membranes were then washed 3 times for 5 minutes with TBS
containing Tween-20 (0.05%; Tween-Tris sodium chloride [T-TBS]), and incubated
with a monoclonal antitryptophan hydroxylase (1:3000; Sigma) at room
temperature overnight. Membranes were washed 3 times with T-TBS and incubated
with goat anti-mouse secondary antibodies conjugated with horseradish
peroxidase (1:5000; Vector) for 2 hours at room temperature. Finally, after
washing the membranes with T-TBS 3 times for 5 minutes, peroxidase activity
was revealed by using a chemiluminescence-based detection kit according to the
protocol suggested by the manufacturer (ECL; Amersham-Pharmacia-Biotech,
Buckinghamshire, United Kingdom). Membranes were exposed to film sheets for 2
minutes at room temperature and the films developed (Dektol-19; Kodak,
Rochester, NY) and fixed (Rapid Fixer; Kodak). Images of these films were
captured, digitized, and analyzed through densitometry (Software Quantity One
4.4.1; BIORAD) by using a computer-based imaging analysis system (Fluor S
Multimager; BIORAD). All measurements were corrected based upon the average
value of each film's background. Statistical comparisons among different ages
were carried out with a one-way ANOVA (significance level set at P
< .05).
HPLC Analyses of Tryptophan Hydroxylase Activity and Serotonin Concentration
Caput epididymis samples were obtained of animals between the ages of 40
and 120 days to evaluate local variations of serotonin concentration during
sexual maturation. Caput samples were also collected from 170-day-old breeder
male rats and from those with no mating experience to analyze whether
serotonin concentration in the epididymis shifts in association with the
animal's reproductive status. Finally, we collected caput samples from
120-day-old rats treated or not with pCPA to further evaluate the
possibility of local synthesis. This agent was administered intraperitoneally
every 72 hours for 3 times, the animals were sacrificed 72 hours after the
last injection. Tissue samples from the 3 groups described were dissected on
an ice-chilled plate, weighed, frozen in liquid nitrogen, and stored at
–75°C until use. The activity of tryptophan hydroxylase in the caput
of the epididymis was estimated by measuring the production of
5-hydroxytryptophan following the protocol described previously
(Johansen et al, 1991).
Briefly, the samples were homogenized in Tris-HCl, pH 7.4, added to 1 mmol of
dithiothreitol and 1 mmol EGTA. After this procedure, the samples were
centrifuged at 29 000 g at 4°C for 16 minutes. The supernatant
was then collected and the protein concentration estimated using bovine
albumin to define the reference curve (Lowry et al, 1951). An aliquot of 300
µg of protein from the samples was used to measure the production of
5-hydroxytryptophan by incubating the samples with a working solution (200
µL total volume) containing 40 µL of 1 mmol EGTA, 30 µL of catalase 1
mg/mL, 20 µL of 1 mmol of pargyline, and 10 µL of 1 mmol ammonium ferric
sulfate in 0.05 mol buffer Tris-HCl), pH 7.4; L-Trp (200 µM) was added to
half of the samples. The mixture was incubated for 5 minutes at 37°C.
After this, 20 µL of 6-methyl-5,6,7,8-tetrahydrobiopterine (0.2 mmol) was
added and the reaction was allowed to continue for 10 minutes at the same
temperature. At the end of this period the reaction was stopped by adding 20
µL of a solution containing 5 mmol EDTA, 6 mmol ascorbic acid, and 0.6%
HClO4. The samples were then centrifuged at 10 000 RPM for 10
minutes. The supernatants were collected, filtered (0.45 µm acrodiscs), and
injected into the chromatograph. We used a symmetric C18 column in
a reverse phase mode (2000 psi, 1 mL/min). The mobile phase was sodium acetate
(95%) and acetonitrile (5%). 5-hydroxytryptophan was then quantified by using
a fluorescence detector (Waters Co, Milford, Mass). The standards used were
5-hydroxytryptophan (100, 2.5, and 0.0625 ng/20 µL) and L-Trp (1, 0.05, and
0.001 ng/20 µL). Results are expressed in nanomoles of product/milligram of
protein/hour (nmol/mg protein/h).
The detection of serotonin and 5-hydroxyindole acetic acid (5-HIAA) was determined by HPLC and the fluorescence method as described elsewhere (Peat and Gibb, 1983; Manjarrez-Gutiérrez et al, 1994). Briefly, tissue samples were homogenized in sodium metabisulfite (4 mmol) in a HClO4 (0.85%) solution and centrifuged at 18 000 x g for 16 minutes at 4°C. The supernatants were collected and protein concentration determined. Supernatant samples (20µL) were filtered (0.40-µm, acrodiscs) and injected into the HPLC (Waters Model 474 scanning fluorescence detector; Waters). We used a symmetric C18 column (5-µm particle size; Waters), 3.9 x 150 mm in a reverse phase mode (2000 psi, 1 mL/minute). The mobile phase was prepared with potassium monobasic phosphate (2 mmol, pH 3.50), containing 1 g/L of heptanesulfonic acid sodium salt and a mixture of methanol: double-distilled deionized water (3:2). Serotonin was then quantified by using a fluorescence detector (least detectable dose 5 pg/µL; Waters). The standards used were serotonin (100, 2.5, and 0.0625 ng/20 µL) and 5-HIAA. Chromatograms were recorded on line and the peak heights measured by using Millennium 32 Software (Waters). Results are expressed in nanograms of serotonin or 5-HIAA/milligram of tissue (ng/mg tissue). The statistical test used to analyze differences among groups when the data sets passed the normality test was ANOVA (significance level set at P < .01) followed by a Bonferroni's t test for multiple comparisons (significance level set at P < .05). For data sets that did not pass the normality test, we used a Kruskal-Wallis one-way ANOVA on ranks (significance level set at P < .01) followed by a Dunn's or Holm-Sidak's method for multiple comparisons (significance level set at P < .05).
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Besides presenting immunoreactivity to serotonin, neuroendocrine cells also display immunoreactivity to neurofilaments. In the caput, neurofilament-positive neuroendocrine cells showed fine processes running parallel to the tubular wall and/or between adjacent epithelial cells (Figure 1d). The density of these cells increased significantly from 40 to 60 days of age (Table 1). Another increase was observed between 60 and 90 days. There was a significant fall in the density of neurofilament positive neuroendocrine cells between 90 and 120 days.
Immunocytochemical studies evaluated the presence and distribution of the serotonin transporter and of serotonin receptors 5HT1A, 5HT1B, 5HT2A, and 5HT3. Serotonin transporter immunoreactivity was observed in the luminal border of putative principal epithelial (Figure 2a), endothelial (Figure 2b), and mast cells (Figure 2c). Interestingly, the staining to serotonin transporters in mast cells appears distributed in granules. One must remember that the majority of these cells take up and concentrate serotonin in these granules (Padawer, 1974; Wingren et al, 1983), so this pattern of staining is not unexpected.
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Tryptophan Hydroxylase and Serotonin in the Caput Epididymis of Males With No Mating Experience, in Breeders, and in pCPA-Treated Rats
To further document the ability of the caput epididymis to produce
serotonin, we injected a group of adult rats with pCPA and measured
tryptophan hydroxylase activity and serotonin concentration in the caput.
Table 3 summarizes these
results. pCPA treatment decreased both the activity of tryptophan
hydroxylase and serotonin concentration in adult rats. The administration of
pCPA did not affect body, epididymal, or testicular weight. To begin
evaluating whether reproductive functions affect epididymal serotonin content,
tryptophan hydroxylase activity and serotonin concentrations were determined
in the epididymis of male with no mating experience and breeder rats. The
activity of tryptophan hydroxylase tends to increase in breeder rats
(Table 4). The concentration of
serotonin was significantly higher in breeder males than in those with no
mating experience (Table 4).
Breeder rats displayed increased body but not epididymis and testes weight as
compared to males with no mating experience
(Table 4).
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Discussion |
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Our immunocytochemical studies revealed the presence of 5HT1B receptors and of serotonin transporters in endothelial cells of the caput epididymis. These observations agree with previous work showing the expression of both in endothelial cells in the nervous system (Daws, 2000) and in the lung (Wang, 2004). Serotonin transporters and the receptors 5HT1B in endothelial cells may control serotonin uptake, blood vessel remodeling, and permeability and blood flow (Lee and Fanburg, 1986; Collin et al, 1996; Nilsson et al, 1999; MacLean et al, 2000; Horschitz et al, 2001; Wakayama et al, 2002). Mast cells also displayed immunoreactivity for serotonin transporter and 5HT1B receptors. The presence of serotonin transporter in mast cells supports the notion that they take up serotonin (Vega and Rudolph, 2002). 5HT1B receptors function as autoreceptors that modulate serotonin release in serotoninergic neurons (Blier et al, 1998). This might also be their role in mast cells. Finally, we did not detect tryptophan hydroxylase immunoreactivity in mast cells as previously reported in a subtype of them located in the nervous system (Mathiau et al, 1994). This indicates that mast cells in the epididymis are of the subtype that does not have the ability to synthesize serotonin. This could explain why they displayed immunoreactivity to serotonin transporter.
Epithelial cells that appear to be apical showed immunoreactivity to 5HT1A receptors. On the other hand, epithelial cells likely of the principal type displayed immunoreactivity to serotonin transporters and 5HT3 receptors. Because the activation of 5HT1A receptors regulate cell proliferation (reviewed in Gaspar et al, 2003) and 5HT3 receptors stimulate enterocyte secretion (Liu et al, 2002; Costall and Naylor, 2004), we think that they might have similar functions in the caput epididymis. The presence of serotonin transporter in the luminal face of principal-like cells suggests that they either transfer serotonin between compartments or degrade this amine as described in neuronal systems (Horschitz et al, 2001).
It is interesting to note that immunoreactivity for various serotonin receptors, especially in the epithelial cells, appears cytoplasmic, with the exception of that observed for 5HT3 receptors. This pattern of immunocytochemical staining has been reported in neuronal and nonneuronal tissue (Langlois et al, 1995; Sari et al, 1997; Bonaventure et al, 1998; Huang et al, 1998; Haase et al, 2001; Potrebic et al, 2003; see also www.chemicon.com). Because transporters and receptors are subjected to a process of vesicular-mediated recycling (Melikian, 2004; Saxena et al, 2005), this may contribute to explaining the cytoplasmic distribution of immunocytological labels (see also Potrebic et al, 2003). Also, the observation that suggests that 5HT1A receptors do not colocalize with serotonin in the same type of epithelial cell is not surprising, since Kaya et al (2004) have reported a similar finding for taste bud receptors.
Previous studies conducted in invertebrates support the presence of 5HT1A, 5HT2A, and 5HT3 receptors in mature spermatozoa (Bandivdekar et al, 1992; Stephens and Prior, 1992) and of 5HT2 receptors in spermatozoa of some mammals (Meizel and Turner, 1983; Young and Laing, 1990). Our results support the existence of 5HT2A and 5HT3 receptors in rat spermatozoa, but not of 5HT1A receptors as shown for invertebrates. The reason for this discrepancy may be interspecies variations and/or differences in the maturation stage of the sperm. Because spermatozoa immunostained for 5HT2A and 5HT3 are observed in caput tubules, our observation suggests that both receptors may participate in the process of maturation of the spermatozoon. In support of this possibility, we found that the content of serotonin and tryptophan hydroxylase activity tends to increase in breeders as compared with males that had no mating experience. Also it is known that serotonin (50 µmol) activates motility in ejaculated, invertebrate spermatozoa (Parisi et al, 1984). This might result from the activation of flagella through a process of cAMP-dependent dynein phosphorylation (Stephens and Prior, 1992). Furthermore, in humans, hyperserotoninemia (120.8 ± 33.0 ng/mL relative to control values of 68.5 ± 5.3 ng/mL) leads to azoospermia (Gonzales et al, 1992).
One of the goals of this work was to evaluate whether some of the cellular elements of the caput epididymis have the ability to produce serotonin. This amine is synthesized through a metabolic pathway in which the limiting step is catalyzed by tryptophan hydroxylase. Hence, the presence of tryptophan hydroxylase in the epididymis would support local synthesis of serotonin. In agreement with this possibility, our morphological studies documented the presence of tryptophan hydroxylase and serotonin immunoreactivity in what appeared to be principal cells of the caput epididymis. Western blot analyses showed the presence of 2 bands (48 kd and 51 kd) positive to tryptophan hydroxylase in caput homogenates. Chromatographic studies revealed pCPA-sensitive tryptophan hydroxylase activity in the caput and a decreased concentration of serotonin in pCPA-treated rats. All these data, together with those discussed earlier, support the concept that the caput epididymis has the ability to produce serotonin locally, the principal epithelial cells being the likely source. Other sources, such as neuroendocrine, mast, and vascular cells as well as the tubular fluid incoming from the testicle, surely make a significant contribution to the total amount of serotonin in the caput. Finally, the presence of 2 isoforms of tryptophan hydroxylase in the caput is not surprising, because previous studies suggested the existence of 2 isoforms of tryptophan hydroxylase encoded by 2 distinct genes in the central nervous system and peripheral organs (Walther et al, 2003). It is clear, however, that more studies are needed before concluding that the caput epididymis indeed contains 2 active forms of tryptophan hydroxylase.
An interesting observation is that the activity of tryptophan hydroxylase increased with age despite the fact that both the intensity of the bands positive for tryptophan hydroxylase in the Western blots and that of the immunocytochemical staining did not vary much among different ages. This suggests that the mechanisms that regulate the activity of this enzyme in the epididymis change during sexual maturation. For instance, the levels of activity of tryptophan hydroxylase are modulated by the availability of cofactors such as that of tetrahydrobiopterine (Fitzpatrick, 1999); it is possible that the availability of this cofactor increases with age. Furthermore, caput serotonin concentration increased with age. However, the activity of tryptophan hydroxylase is much greater than that expected considering the concentration of serotonin. A relative excess of tryptophan hydroxylase activity with respect to the concentration of serotonin also occurs in the brain (Feldman et al, 1997). Although the excess of the enzymatic activity may indicate extra demands (Feldman et al, 1997), tryptophan hydroxylase might also be involved in other metabolic pathways in the epididymis, such as in the synthesis of melatonin, as described in the testis (Tijmes et al, 1996).
The concentration of serotonin in the caput increased with age, but that of 5-hydroxyindole acetic acid was essentially unchanged during sexual maturation. This finding suggests that serotonin synthesis increases as animals mature sexually, likely as a result of an increment in tryptophan hydroxylase activity, as discussed in the preceding paragraph. This conclusion contrasts with previous studies which conclude that increments of serotonin concentration in reproductive organs result from changes in the turnover rate in golden hamsters (Frungeri et al, 1999).
Finally, to evaluate whether serotonin might participate in the reproductive function of the epididymis, we measured the content of this amine and the activity of tryptophan hydroxylase in the caput of males with no mating experience and breeder rats subjected to a monogamous mating system. Both parameters were found increased in breeders, supporting the concept that sexual activity and serotonin concentration in the epididymis are somehow linked. Although the mechanism is yet unknown, evidence supports that serotonin modulates testosterone release from testicular Leydig cells (Tinajero et al, 1993; Frungieri et al, 1999) and that both testosterone and serotonin regulate sexual behavior by acting on the hypothalamic preoptic area (eg, Popova and Amstislavskaya, 2002). Interestingly, preliminary observations support that testosterone serum levels in breeders are 18% higher than those observed in male rats with no mating experience (Jiménez-Trejo and Gutiérrez-Ospina, unpublished observations). It might then be possible that testosterone serum levels match sexual behavior with serotonin concentration in the epididymis. In addition, based upon our results, we could speculate that serotonin released from epithelial cells might affect directly sperm maturation acting through 5HT2A and 5HT3 receptors. On the other hand, serotonin secreted from neuroendocrine and mast cells could affect sperm maturation by modulating local temperature, nutrient apportionment, and/or trophic support through controlling vasomotor and epithelial function. Clearly, more research is needed to evaluate the merit of each of these ideas.
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Acknowledgments |
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Footnotes |
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|| These authors contributed equally to the present work. 
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