Aromatase immunolocalization in male goat reproduction tract and accessory glands

In recent years ample evidence has been accumulated that estrogens are essential for normal male fertility and that they likewise may also interfere with fertility (6). The opinion that estrogens are essential for male fertility, however, was delayed until a knockout mouse (ko) and rat lacking a functional estrogen receptorα(ERα) gen (ERαko) was developed (5). Mice lacking a functional  aromatase gene (Arko) are also infertile (8). Therefore, it is now accepted that estrogens play a physiological role in male reproduction (2,7). cytochrome P450 aromatase is a terminal enzyme catalyzing the conversion of androgens to estrogens, and its expression in male reproductive tract tissues suggests local estrogen biosynthesis (1,9). In several mammalian species (mouse, rat, boar, cattle, ram, stallion, dog, raccoon and human) aromatase has been reveled in leydig cells, sertoli cells and germ cells, suggesting estrogen involving in gonadal development and in sperm maturation (1, 3).

The aim of the present study was to investigate localization of aromatase P450 in testis, proximal epididymis ductus, prostate and vesicle seminal glands tissues of native male goat.

Testicular tissues and accessory glands were obtained from five native adult male goat (2 and 4 years old). The testis and accessory glands were excised immediately from animals after slaughter and fixed in Bouin solution and dehydrated in a series of ethanol concentration s and paraffin-embedded. The sections cut (5 µm thick) and mounted on slides coated with a suitable tissue adhesive deparaffinized and rehydrated (4-5 serial sections for each sample). Morphological analysis was carried out by haematoxylin and eosin staining. Immunohistochemistry was performed after heat-treated for antigen retrieval using citrate buffer(10 mM, PH6) for break of protein cross- links. Peroxides block (5 min) was used to neutralize endogenous peroxides. Protein block (5 min) was used to block non-specific binding sites. P450 _arom immunodetection was carried out using a mouse polyclonal aromatase antibody as primary antibody and polyclonal anti-mouse IgG antibody (HRP) as secondary antibody (ABBiotec). They were then incubated overnight with primary antibody diluted  in PBS in a moist chamber at 4?. The section were incubated with post primary block (30 min), then developed peroxides activity with DAB working solution (5min), and section were counterstained with haematoxilin, dehydrated in alcohol, and mounted with cytology glue. In controls? normal mouse serum or PBS alone was applied instead of primary antibody. 

Aromatase was immunolocalized in stages of spermatogenesis, leydig, sertoli cells, proximal ductus of epididymis, prostate and vesicle seminal glands. prostate cells were immunostained very much more other cells.( Figures 1 a-f). Control sections were all immunonegative. (Figures 2 a-e )

Figure1.Immunolocalization of P450arom in adult male goat reproductive tract and accessory glands. P450arom was identified in seminiferous tubules (a), Leydig cells (b) of testis, proximal ductus of epididymis (c), prostate (d) and vesicle seminal glands(e, f).

No immunostaining was detected in control seminiferous tubules (a), Leydig cells (b) of testis, proximal ductus of epididymis (c), vesicle seminal (d) and prostate glands (e) in wich normal mouse serum was used instead of primary antibody.

These data show, aromatase is expressed in seminiferous tubules, leydig, sertoli cells, proximal ductus of epididymis, prostate and vesicle seminal glands. This suggesting that estrogen is biosynthesis in these parts of the genital tract. It is likely that local aromatase production in stages of spermatogenesis, leydig, sertoli cells in male goat, would be necessary for spermatogenesis. In several species, a functional role of estrogens on epididymal tissue has been indicated by estrogen receptor expression (4). The primary function of the ductuli efferents is the transport of sperm from the rerte testis to the epididymal duct and reabsorbing of the luminal fluid (90 %) to increase concentration. In the ductuli efferentes of rat and mouse, it has been demonstrated that estrogens, via estrogen receptors, can regulate Na⁺ reabsorbing and passive water transport, and are also responsible for maintenance of the apical cytoarchitecture in epithelial cells (10).

On the basis of observation present in this study and in previous studies, we conclude that leydig, sertoli cells proximal ductus of epididymis, prostate and vesicle seminal glands can synthesize estrogen and that production of estrogen is possibly important to spermatogenesis, sperm maturation, and epididymal function.

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