ARTICLE |
Correspondence to: Safa Moslemi, Laboratoire de Biochimie et Biologie Moléculaire, EA 2608, IBFA, Université de Caen, Esplanade de la Paix, 14032 Caen cedex, France. E-mail: bioch.bio.mol@ibba.unicaen.fr
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Summary |
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High levels of plasma estrogens constitute an endocrine peculiarity of the adult stallion. This is mostly due to testicular cytochrome P450 aromatase, the only irreversible enzyme responsible for the bioconversion of androgens into estrogens. To identify more precisely the testicular aromatase synthesis sites in the stallion, testes from nine horses (25 years) were obtained during winter or spring. Paraplast-embedded sections were processed using rabbit anti-equine aromatase, followed by biotinylated goat anti-rabbit antibodies, and amplified with a streptavidinperoxidase complex. Immunoreactivity was detected with diaminobenzidine. Immunofluorescence detection, using fluoroisothiocyanate-conjugated goat anti-rabbit antibodies, was also applied. Specific aromatase immunoreactivity was observed intensely in Leydig cells but also for the first time, to a lesser extent, in the cytoplasm surrounding germ cells at the junction with Sertoli cells. Interestingly, the immunoreactivity in Sertoli cells appears to vary with the spermatogenic stages in the basal compartment (with spermatogonia) as well as in the adluminal one (with spermatids). Relative staining intensity in Leydig and Sertoli cells and testicular microsomal aromatase activity increased with age. The present study in stallions indicates that in addition to Leydig cells, Sertoli cells also appear to participate in estrogen synthesis, and this could play a paracrine role in the regulation of spermatogenesis.
(J Histochem Cytochem 51:311318, 2003)
Key Words: aromatase, estrogens, immunohistochemistry, Leydig, Sertoli, testis, stallion, equine, horse, spermatogenesis
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Introduction |
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STALLION TESTES represent the major source of aromatase and therefore the major site for estrogen biosynthesis (
The stallion is commonly considered to be a seasonal breeder, with a breeding season extending in general from March to July in the northern hemisphere (April to July in Normandy, France). However, unlike other seasonal breeders that cease spermatozoa production during winter, stallions continue to produce spermatozoa throughout the year. Moreover, the stallion breeding season is characterized by an increase in testicular size and weight and in Leydig, Sertoli, and germ cell numbers (mainly type A and B spermatogonia). During this time, the daily production of sperm (
In addition to seasonal variations, both testicular and hormonal characteristics of stallions also change with age. Horse puberty begins at 11.5 years of age, depending on the breed, and spermatogenesis is completed by 2 years of age (
Aromatase is the only enzyme responsible for the irreversible bioconversion of androgens to estrogens. This enzyme is considered to be crucial and rate-limiting for the estrogen/androgen balance in the body, and thus important for estrogen-dependent processes such as bone maturation (
The aim of the present study was to better define the testicular distribution of aromatase in the stallion.
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Materials and Methods |
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Animals and Sampling
Testes from five postpubertal (2-year-old) and four adult (35-year-old) stallions were obtained from a veterinary clinic (Veterinary Clinic; Crèvecoeur-en-Auge, Normandy, France) after castration under general anesthesia. This was performed both during the breeding (April to June, n=6) and non-breeding (February and March, n=3) seasons. Tissues were immersed in NaCl 0.9% or in Eagle's minimum essential medium (EMEM) and transported to the laboratory on ice. In the laboratory, each testis was cut transversely into two equivalent pieces and either used immediately for immunohistolocalization or stored at 70C for microsomal preparation.
Chemicals and Tissue Processing
All chemicals and reagents were obtained from Sigma (St Quentin Fallavier, France) except when stated. Fragments of testicular parenchyma, 0.5 x 0.5 x 1 cm, were taken from the center and periphery of testes, fixed in Bouin's solution or 4% paraformaldehyde (PFA) in Dulbecco's PBS, pH 7.4, for 24 hr at 4C, dehydrated in ethanol, and embedded in paraplast. Tissue blocks were serially sectioned at 57 µm and attached to poly-L-lysine-coated slides. Slides were then dried at 37C for 48 hr before immunohistochemistry.
Immunohistolocalization
The preparation and specificity of primary rabbit anti-equine aromatase antibodies were reported previously (
Microsomal Preparations, Aromatase Activity Assays, and Data Analyses
Microsomes from each testis were prepared by successive ultracentrifugations as previously described (
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Results |
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Both Bouin and PFA fixatives induced similar immunoreactivity characteristics. However, Bouin's solution was better than PFA for conservation of cell morphology. Therefore, all results presented here were obtained from tissue fixed in Bouin's solution.
Aromatase was immunolocalized in both Leydig and Sertoli cells; Leydig cells were immunostained very much more strongly than Sertoli cells (Fig 1 Fig 2 Fig 3). In the latter, immunostaining appears heterogeneously in the cytoplasm surrounding germ cells. However, labeling in the peripheral membrane of germ cells is not excluded. In all testes examined, staining intensity was consistent between serial sections or between staining methods (Fig 1A1C). Higher magnification views indicate that staining was localized only in the cytoplasm of immunopositive cells; nuclei were negative (Fig 1B1D). All other cell types in the tubular, peritubular, and interstitial compartments were immunonegative.
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Cytoplasmic staining intensity in both Leydig and Sertoli cells was increased noticeably with stallion age. Leydig cells of 3- and 5-year-old stallions were stained more strongly than those of 2-year-old stallions (Fig 2A2B). Moreover, in aged testes almost all interstitial spaces were filled by clusters of Leydig cells; this phenomenon amplified the signal. Because of their immunoreactivity with the antiserum, two types of Leydig cells were observed; one type reacted less than the other. In 2-year-old stallions, the presence of the less immunoreactive cell type was observed more frequently than in 3- or 5-year-olds. In addition, the 2-year-old stallion testes contained small clusters of Leydig cells in the interstitial spaces, and individual Leydig cells were observed more frequently (Fig 2A). No differences in immunostaining were observed between winter (February) and spring (April to June) whatever the age.
Although immunoreactivity of Sertoli cells was seen in all spermatogenic stages, some differences across the seminiferous epithelium were observed (Fig 3). In stages III and VI, the intensity of the staining was close to the basal lamina and appeared higher at the junctions of Sertoli cells, i.e., around spermatogonia and zygotene spermatocytes. In contrast, in stages IV and VII the staining was higher in the adluminal section, i.e. around the diakinese spermatocytes and elongated spermatids. In all other stages, I, II, V, and VIII, the staining was distributed uniformly across the tubules within Sertoli cells (Fig 3). The intercellular spaces between germ cells appear to be positive where Sertoli cells support germ cells. There was no difference in both distribution and intensity of the staining between testes collected from April to June in 3-year-old stallions. About 150 tubules were observed per testis.
Testicular aromatase activity increased significantly with age between 2 and 35 years (Fig 4A; p< 0.001). However, no difference was observed between testes collected in February and April for 2-year-old stallions (Fig 4B).
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Discussion |
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We report here that aromatase is expressed not only in Leydig cells of stallion testis but that immunoreactivity is also present to a lesser extent in the seminiferous tubules, in the cytoplasm surrounding germ cells. Sertoli cells therefore appear positive. It can be considered that both cell types could contribute differently to estrogen synthesis in the stallion. However, these results are different from those of Eisenhauer et al. (1994), Almadhidi et al. (1995), and
Moreover, our study indicates that there are two types of Leydig cell populations in horse testes from 2-year-old postpubertal stallions that react differently with anti-equine aromatase. This is in agreement with results reported by Eisenhauer et al. (1994). Heterogeneity of Leydig cell populations, i.e., light and dark cell populations, was previously reported in stallion (
In the present study, the immunoreactivity of aromatase in Leydig cells was obviously very much stronger than that in Sertoli cells, indicating that Leydig cells are still the main sites of estrogen synthesis in stallion testes. This is a general phenomenon observed in most mammalian species (for review see
In the stallion the seminiferous epithelium cycle consists of eight stages with a helical pattern distribution in the tubule (
In this work, we show microsomal aromatase activity in stallion testes increases with age. It is significantly higher in 35-year-old than in 2-year-old stallions. This is in agreement with results previously reported by our group concerning enzymatic activity, proteins, and aromatase mRNA in the horse testis (
In conclusion, it is likely that local aromatase production in Sertoli cells, which seems age- and stage-related in the stallion, would be necessary for spermatogenesis, particularly for spermatogonia undergoing proliferation and meiosis, and for maturation of spermatozoa or spermiogenesis.
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Acknowledgments |
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Supported by Fonds Feder and CRAB (Comité de Recherche pour les Agrobioindustries, Calvados), and Conseil Général du Calvados. HS had a studentship from SFERE (Société Française d'Exportation des Ressources Educatives).
We wish to thank Dr J. Fresnel for excellent assistance with microphotography.
Received for publication June 12, 2002; accepted October 25, 2002.
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