ARTICLE |
Correspondence to: Maria V.T. Lobo, Servicio de Neurobiología, Departamento de Investigación, Hospital Ramón y Cajal, Ctra. de Colmenar Km 9, 28034 Madrid, Spain. E-mail: rafael.martin@hrc.es
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Summary |
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The amino acid taurine has been implicated in several aspects of reproductive system physiology. However, its localization in these organs has not been previously analyzed. The aim of this study was to characterize its distribution in male rat reproductive organs by immunohistochemical methods. Taurine was localized in the smooth muscle cells of the tissues studied and in the skeletal fibers of the cremaster muscle. In the testis, taurine was found in Leydig cells, vascular endothelial cells, and other interstitial cells. No immunoreactivity was observed in the cells of the seminiferous tubules, either in germ cells at all spermatogenic stages or in Sertoli cells. However, peritubular myoid cells were immunostained. Most epithelial cells of the efferent ducts were immunolabeled, whereas the epithelial cells of the rete testis (extratesticular segments), epididymis (caput, corpus, and cauda regions), and ductus deferens were unstained. However, most epithelial cells from the intratesticular segments of the rete were immunopositive. Some cells identified as intraepithelial macrophages and lymphocytes, apical cells, and narrow cells were intensely immunolabeled. Regional differences in the distribution of these cell types along the ducts studied were also noted. The possible functional roles for taurine in these cells are discussed. (J Histochem Cytochem 48:313320, 2000)
Key Words: 2-aminoethanesulfonic acid, ß-amino acid, testis, male reproductive tract, immunohistochemistry
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Introduction |
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Taurine (2-aminoethanesulfonic acid) is a free ß-amino acid that is present in high concentrations in several cell types. Two sources of taurine could account for the intracellular levels of this amino acid: a biosynthetic route from cysteine and/or the specific uptake from the extracellular space. Extracellular taurine comes from dietary sources or from some tissues in which it is synthesized and released into circulation, such as the liver (for review see
In the male reproductive system, taurine has been identified as the major free amino acid of sperm cells (
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Materials and Methods |
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Albino Wistar rats (250 g) were used for the present study. Animals were anesthetized with a ketamine (0.08 ml/100 g), xylocaine (0.04 ml/100 g), and atropine (0.02 ml/animal) mixture and perfused through the ascending aorta with 100 ml phosphate buffer (0.12 M, pH 7.4), followed by the fixative solutions. The compositions of the fixatives used were 4% paraformaldehyde, 0.5%, 1%, or 1.5% glutaraldehyde, and 4% paraformaldehyde (control specimens). Tissue samples were removed from the rats and stored in the same fixative solutions used during perfusion for 624 hr, washed in buffer, dehydrated, and embedded in paraffin according to conventional methods (
StreptavidinPeroxidase Method
Serial sections of the paraffin-embedded samples were obtained and the immunolabeling was performed in one section each 30 µm. Sections (5 µm thick) were mounted on silanized slides and allowed to dry overnight before immunohistochemical staining. After xylene deparaffinization, sections were hydrated and incubated for 20 min in 0.3% H2O2 followed by 0.03% H2O2 in methanol, washed in Tris-buffered saline (TBS), pH 7.5, and incubated for 10 min in a 0.1 M sodium borohydride solution in TBS, pH 7.5. Sections were then washed and incubated in 3% normal goat serum with 0.05% Triton X-100 in TBS, pH 7.5, at room temperature (RT) for 30 min, followed by overnight incubation at 4C with the rabbit anti-taurine antibody (Chemicon International; Temecula, CA) diluted from 1:500 to 1:1500 in the same buffer. Afterwards the sections were washed twice in TBS to remove unbound primary antibody and then incubated with the secondary antibody, biotinylated goat anti-rabbit IgG (Zymed Labs; San Francisco CA), for 1 hr at RT, and washed in TBS. Sections were incubated with the streptavidinperoxidase complex (Zymed) for 1030 min and washed in TBS, followed by Tris-HCl buffer, pH 7.6. The peroxidase activity was revealed using 3'-diaminobenzidine tetrahydrochloride (DAB) as chromogen (Sigma; St Louis, MO). The reaction product of DAB was intensified with nickel nitrate to obtain a dark black color of immunostained antigens [10 µl of nickel nitrate (8%) in 1 ml DABH2O2 solution]. Thereafter, the sections were dehydrated in ethanol and mounted in Eukitt (O. Kindler; Freiburg Germany) without being counterstained. To estimate the percentage of immunostained cells, 20 areas for each tissue region were chosen at random and at least 50 cells of each type were counted in each area. Only those cells with definitely recognizable nuclei were counted. Values obtained for each cell type were expressed as mean ± SD.
Control Experiments
The specificity of the immunohistochemical procedures was assessed by means of the negative controls performed: (a) omitting the primary antibody; (b) using nonimmune serum instead of the primary antibody; (c) incubating with an inappropriate secondary antibody after the incubation with the primary antibody at optimal titers; and (d) immunolabeling on 4% paraformaldehyde-fixed samples. The last control should not give a positive immunoreaction because the anti-taurine antibody used here was prepared using taurine conjugated by glutaraldehyde to poly-L-lysine as immunogen, and therefore it recognizes only haptenic taurine on glutaraldehyde-fixed samples (
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Results |
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The distribution of taurine in the testis (Fig 1a1g), rete testis (Fig 1h and Fig 1i), efferent ducts (Fig 1j and Fig 1k), epididymis (Fig 2), and ductus deferens (Fig 3a and Fig 3b) of the rat was studied. With the different paraformaldehydeglutaraldehyde solutions used, no differences in the pattern of taurine distribution were observed (Fig 1a1g). No staining was found in the negative controls performed (not shown). Positive controls (Fig 3k) showed a taurine distribution in Purkinje cells of rat cerebellar sections similar to that reported in other studies (
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In the testis (Fig 1a1g) taurine was detected in Leydig cells, vascular endothelial cells, and other interstitial cells. Most Leydig cells were stained (80.7 ± 4.1% of all Leydig cells). Leydig cells showed a variable degree of taurine immunostaining in their nuclei, being weak in some cells and as strong or stronger than in the cytoplasm in other cells. Moreover, taurine was homogeneously distributed in the cytoplasm of some Leydig cells, whereas in other cells it appeared to be located at discrete foci of the cytoplasm (Fig 1b1e). In the seminiferous tubules, germ cells at all spermatogenic stages and Sertoli cells were unstained. However, peritubular myoid cells showed cytoplasmic immunolabeling along their thin cell processes that surround the seminiferous tubules, and a nuclear pool of taurine (Fig 1a, Fig 1f, and Fig 1g). In the tunica albuginea, some fibroblasts and/or myoid cells were stained.
In the rete testis, most epithelial cells of the initial segments (tubuli recti and tunical part) were immunopositive for taurine (Fig 1h and Fig 3d). However, the epithelial cells from the extratesticular segment of the rete were immunonegative (Fig 1i). Moreover, taurine was also detected in some cells and blood vessels of the interstitial tissue located between the different segments of the rete testis (Fig 1h).
Most epithelial cells of the efferent ducts (Fig 1j and Fig 1k) were immunostained (86.2 ± 9.1% of all epithelial cells). Frequently, taurine appeared homogeneously distributed in the nucleus and cytoplasm of these cells. However, a minor population of these epithelial cells showed intensely stained cytoplasm and weakly stained nuclei. Moreover, some cells scattered in the efferent ducts, which showed many cytoplasmic processes, were more intensely immunolabeled than adjacent epithelial cells. These cells were identified by their size, morphology, and distribution as intraepithelial macrophages (Fig 1j, Fig 1k, and Fig 3g). These macrophages were also intensely stained in other ducts, such as the rete testis (Fig 1i) and the epididymis (Fig 2d and Fig 2j). In this cell type, taurine appeared mainly distributed in the perinuclear cytoplasm and in thin cytoplasmic processes, whereas the nucleus was usually weakly stained.
In the pseudostratified columnar epithelium of the epididymis, both main cell types, principal and basal cells, were immunonegative (Fig 2). Small round cells, which appeared isolated in the epididymal epithelium (probably intraepithelial lymphocytes), showed intense immunolabeling (Fig 2c and Fig 3e). These lymphocyte-like cells were found at specific segments of the epididymis, being scattered all along the length of the caput and corpus, whereas they were not localized in the cauda epididymidis.
Taurine was found in some scattered epididymal cells, identified by the position of their nuclei in the upper half of the cytoplasm. This minor population of epididymal epithelial cells have been termed apical mitochondria-rich cells (
In the ductus deferens, most epithelial cells were unstained. However, taurine appeared to be distributed in some scattered cells that were identified as intraepithelial lymphocytes (Fig 3a) and apical cells (Fig 3b and Fig 3j).
In the interstitial region among the different ducts studied, vascular endothelial cells and other interstitial cells (fibroblasts and/or periductal myoid cells) were also stained (Fig 1j, Fig 1k, and Fig 2). Moreover, a variable degree of taurine immunostaining in the endothelial cells of the capillaries was observed. Some capillaries were intensely immunoreactive, whereas others were not stained (Fig 1j).
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Discussion |
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Mammalian spermatozoa are immature when they leave the testis. Their development continues during their passage through the epididymis, where they acquire fertilizing ability and a mature motility pattern. This maturation process appears to be inherent to the sperm cells as they age and is also influenced by the epididymal epithelium, which modifies the luminal environment by secretion and reabsorption of several substances (for review see
Taurine/hypotaurine in sperm cells could originate not only via amino acid transport from the duct fluids but could also be synthesized from cysteine via the cysteine sulfinic acid decarboxylase (CSAD) pathway. Previous studies suggest a correlation between hypotaurine content and CSAD activity in the reproductive tissues of guinea pigs, being both higher in the epididymis and lower in the testis (
The rat ductus epididymis is lined by an epithelium consisting of two main cell types (principal and basal cells) and other less common cell types, such as apical, narrow, and halo cells. Halo cells are actually considered intraepithelial lymphocytes and macrophages (
Apical and narrow cells are a minor population of epididymal epithelial cells. Although their function is unknown, it has been suggested that they could modify the luminal environment in which spermatozoa undergo maturation by cooperation with principal cells in reabsorption of testicular fluid (
Leydig cells are the cellular source of androgen, i.e., testosterone, which is necessary for the regulation of spermatogenesis in mammals. Previous studies have demonstrated a relation between hypotaurine and testosterone. Hypotaurine almost disappears from the prostate and seminal vesicle of the guinea pig, rat, and mouse after castration, and its concentration is restored to almost normal values by administration of testosterone (
In neurons of the rat spinal cord, variable taurine immunostaining in their nuclei has been reported, being weak in some cells and as strong or stronger than in the cytoplasm in other cells (
This study is the first to demonstrate the localization of taurine in the male reproductive organs. Such a specific distribution implies that taurine may play important roles in the male reproductive system and highlights the need for further investigation into the functions of taurine in the reproductive organs.
Received for publication September 30, 1999; accepted November 4, 1999.
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