Immunolocalization and activity of aromatase in the bank vole testes.

A direct approach to identify the cellular source of P450 aromatase in the bank vole testes (seasonally breeding rodents) is the use of immunohistochemistry with a specific antibody that recognizes this enzyme. To confirm the presence of functional aromatase, its activity was measured in microsomal preparations of whole testes and of seminiferous tubules by means of biochemical assay with tritiated androstenedione. The assay was validated using increasing concentrations of both microsomal preparations. Immunoreactive aromatase was found in Leydig cells, Sertoli cells, and germ cells, especially in spermatocytes and spermatids. The aromatase activity was present in microsomal fractions of whole testis and seminiferous tubules. The immunolocalization of P450 aromatase and aromatase activity have been found as photoperiod-dependent.     

Aromatization and antioxidant capacity in the testis of seasonally breeding bank voles: Effects of LH, PRL and IGF-I

Aromatization and antioxidant strategies in the male gonads are important processes, which are involved in control of normal fertility. The objective of this study was to show whether luteinizing hormone (LH), prolactin (PRL), and insulin-like growth factor-I (IGF-I) as well as the length of photoperiod are able to exert an effect on aromatase expression, steroid hormone levels, and antioxidant concentrations in testes of bank voles, seasonally breeding rodents. Mature bank voles that were kept under short light cycles or long light cycles served as the animal model. Testicular sections were used for immunohistochemical visualization of aromatase expression, whereas testicular homogenates were used for radioimmunological measurement of steroids, biochemical determination of superoxide dismutase activity, total antioxidant status (TAS) and protein content. In the testes of hormone-treated voles a stronger immunostaining for aromatase was concurrent with the increase in testosterone and estradiol levels, and total antioxidant status compared with the controls. In contrast, there was a decrease in superoxide dismutase (SOD) activity. The strongest effect on aromatase immunoexpression and steroid hormone levels was detected after combined doses of LH and IGF-I, indicating a stimulatory role of these hormones on estrogen synthesis in the bank vole. An increase in total antioxidant status in testes of hormone-treated bank voles suggests the presence of testicular defense, whereas a decrease in superoxide dismutase activity indicates a protective role of administered hormones against toxic oxygen radicals. The present study also demonstrates a significant, photoperiod-dependent relationship between aromatization and antioxidant capacity in the testis of the bank vole.     

Detection of aromatase, androgen, and estrogen receptors in bank vole spermatozoa

Spermatozoa are highly specialized cells which transport a single-copy haploid genome to the site of fertilization. Before this, spermatozoa undergo a series of biochemical and functional modifications. In recent years, the crucial role of androgens and estrogens in proper germ cell differentiation during spermatogenesis has been demonstrated. However, their implication in the biology of mature male gametes is still to be defined. Our study provides evidence for the first time that aromatase, the androgen receptor (AR), as well as the estrogen receptors α and β (ERα and ERβ), are present in bank vole spermatozoa. We demonstrated the region-specific localization of these proteins in bank vole spermatozoa using confocal microscopy. Immunoreactive aromatase was observed in the proximal head region and in both the proximal and distal tail regions, whereas steroid hormone receptors were found only in the proximal region of the sperm head. Protein expression in sperm lysates was detected by Western blot analysis. Immunohistochemical results were analyzed quantitatively. Our results show that bank vole spermatozoa are both a source of estrogens and a target for steroid hormone action. Moreover, the presence of aromatase and steroid hormone receptors in the bank vole spermatozoa indicates a potential function of these proteins during capacitation and/or the acrosome reaction.     

Hormonal regulation of oestrogen formation by Leydig cells in vitro: immunocytochemical approach

The ability of the testis to convert androgens into oestrogens is related to the presence of a microsomal enzyme, aromatase, in testicular cells. The aim of this study was to show whether the supplementation of culture media with LH or an aromatase inhibitor could affect the process of aromatisation in Leydig cells of the bank vole in vitro. This was investigated by means of immunocytochemistry and radioimmunological assays. In control cultures of Leydig cells, both steroid hormones secretion as well as immunoreactivities for aromatase and oestrogen receptor were weaker than in those treated with LH. On the contrary, the addition of aromatase inhibitor into the culture medium resulted in a decreased intensity of immunocytochemical stainings in comparison with the control. Concomitantly, the androgen level was slightly higher, whereas that of oestrogen significantly lower than in the control cultures. Additionally, to check whether steroid hormones are able to regulate aromatase or oestrogen receptor immunoexpressions, some of the Leydig cell cultures were enriched with testosterone or oestradiol, respectively. Strong immunoreactivities for both aromatase and oestrogen receptor were observed. This suggests that Leydig cells in vitro are able to regulate directly the secretion of oestrogens by active aromatase. Finally, it is concluded that oestrogen formation in bank vole Leydig cells in vitro can be influenced by various factors. It should be stressed, however, that the effect of hormone stimulation or aromatase inhibitor action appeared to be dependent on the length of light cycles that bank voles were exposed prior to the isolation of Leydig cells.     

The site of aromatization in the mouse cryptorchid testis

Using the mouse cryptorchid model, degenerations of germ cells were observed as well as a reduced size of seminiferous tubules, while the area of the interstitial tissue increased. Aromatase, the enzyme responsible for the conversion of androgens into oestrogens, was immunolocalized in Leydig cells and in germ cells from both scrotal and abdominal testes, and in Sertoli cells only in a control testis. In the cryptorchid testis, aromatase was strongly expressed in a few tubules, including those spermatids that were still present. Other cells inside the tubules were negative for aromatase. In both testes, oestrogen receptors alpha were expressed only in Leydig cells. Strong aromatase expression in germ cells indicates an additional source of oestrogens in the testis besides the interstitial tissue.     

Effect of photoperiod on cultured granulosa cells of the bank vole, Clethrionomys glareolus

Gonadal function of the bank vole females depends on the photoperiod. This experiment was to show whether photoperiod applied on the whole animal in vivo would affect the function of ovarian cells in vitro. Granulosa cells from large ovarian follicles of bank vole reared in long or short photoperiod were cultured as monolayers in control or luteinizing hormone supplemented media. Formation of cell colonies, activity of delta5, 3beta-hydroxy steroid dehydrogenase and progesterone secretion were investigated. First colonies of long day cells were formed already on day 1. On day 2 they enlarged and became abundant. Short day cells formed colonies only on day 2. Colonies of similar size to 2 day colonies of long day cells appeared only on day 6. There were also differences in steroid dehydrogenase activity and in progesterone secretion between long and short day control and hormone treated cultures. We conclude that photoperiod applied in vivo affects ovarian cell function in vitro.     

Activité aromatase dans les cellules germinales mâles: quelle signification fonctionnelle?

The ability of the male gonad to convert androgens into estrogens is well known. According to age, aromatase activity has been already measured in immature and mature rat Leydig cells as well as in Sertoli cells. Recently, in different studies, a cytochrome P450_arom has even been immunolocalized not only in Leydig cells but also in germ cells of mouse, brown bear and rooster whereas in pig, ram and human the aromatase is mainly present in Leydig cells. Our purpose was to investigate the testicular cell distribution of cytochrome P450_arom mRNA in adult rat using RT-PCR. With 2 highly specific primers located on exons 8 and 9, we have been able to amplify a 289 bp aromatase fragment not only in Leydig cells and Sertoli cells but more importantly in highlyenriched preparations of pachytene spermatocytes, round spermatids and testicular spermatozoa. These amplified products showed 100% homology with the corresponding fragment of the rat ovary cDNA. In parallel, using an anti-human cytochrome P450_arom antibody we have demonstrated the presence of a 55 kDa protein in seminiferous tubules and crude germ cell (pachytene spermatocytes and round spermatids) preparation of the mature rat. After incubation with tritiated androstenedione, the aromatase activities in the microsomal fractions were 3.12±0.19 pmoles/mg/h in the testis, 1.25±0.13 in the seminiferous tubules and 1.53±0.15 in the crude germ cells. In purified testicular spermatozoa the aromatase activity was 2.96±0.69 pmoles/mg/h and found to be 5-fold higher when compared to that of either purified pachytene spermatocytes or round spermatids. Using a quantitative RT-PCR method with a standard cDNA 29 bp shorter, we have compared the amount of cytochrome P450_arom mRNA in mature rat Leydig cells and Sertoli cells. In purified Leydig cells from 90 day-old rats the P450_arom mRNA level was: 36.2±3.4×10^?3 amoles/μg RNA whereas in Sertoli cells the mRNA level was 10 fold lower. In pachytene spermatocytes, round spermatids and testicular spermatozoa the P450_arom mRNA levels were re pectively 367.2±76.6, 117.6±22.0 and <1×10^?3 amole/μg RNA. In conclusion we have demonstrated that the P450 aromatase is present not only in Sertoli cells and Leydig cells from mature rat testis but a biologically active aromatase exists also in germ cells (pachytene spermatocytes, round spermatids and spermatozoa). The existence of an additional source of estrogens within the genital tract of the male is now well documented and that suggests a putative role for these hormones during the male germ cell development.     

Genetic efficacy of low doses of ionizing radiation in chronically-irradiated small mammals

Earlier we have established the genetic effects of low dose chronic irradiation in bank vole (somatic and germ cells, embryos), in pond carp (fertilized eggs, embryos, fry) and in laboratory mice (somatic and germ cells) in the range of doses from near-background to 10 cGy. These low dose effects observed in mammals and fish are not expected from extrapolation of high dose experiments. For understanding reasons this discrepancy the comparative analysis of genetic efficiency of low dose chronic irradiation and the higher doses of acute irradiation was carried out with natural populations of bank vole which inhabited the two sites differing in ground of radionuclide deposition. For comparing efficiency the linear regression model of dose-effect curve was used. Dose-effect equations were obtained for animals from two chronically irradiated bank vole populations. The mean population absorbed doses were in the range 0.04-0.68 cGy, the main part of absorbed doses consisted of external radiation of animals exposed to 137Cs gamma-rays. Dose-effect equations for acute irradiation to 137Cs gamma-rays (10-100 cGy) were determined for the same populations. Comparison of genetic efficiency was made by extrapolation, using regression coefficient beta and doubling dose estimation. For chronic exposure the doubling doses calculated from low-dose experiments are 0.1-2 cGy and the doubling doses determined from high-dose experiments are in the range of 5-20 cGy. Our hypothesis that the doubling dose estimate is calculated in higher-dose ionizing radiation experiments should be much higher than the deduced from the low dose line regression equation was verified. The doubling dose estimates for somatic cells of bank vole and those for germ cells of laboratory mice are in close agreement. The radiosensitivity of bank vole chromosomes were shown is practically the same as that for human lymphocytes since doubling dose estimates for acute irradiation close to each other. For low LET radiation a higher genetic efficiency of chronic low doses in comparison with the higher doses of acute gamma-irradiation (137Cs source) was proved by three methods.     

Immunolocalization of PTHrP in the sublingual glands of three mammals

The present study deals with immunohistochemical localization of PTHrP in sublingual glands of white mouse, bank vole, and common vole. PTHrP immunoreactivity was observed in epithelial cells of striated, interlobular and main excretory ducts of the salivary glands in all the three animal species tested. However, we found no positive reaction for PTHrP in epithelial cells of the intercalated ducts. In striated duct cells, the reaction intensity was species-dependent. In bank vole and common vole, the reaction was very strong, while in white mouse very weak. In the remaining segments of excretory ducts (interlobular and main excretory duct) we found no species-related differences in the reaction intensity or character. Myoepithelial cells surrounding ducts and mucous tubules with serous demilunes in sublingual glands were also PTHrP-negative in all the three animal species tested.     

Immunolocalization of cytochrome P450 aromatase in rat testis during postnatal development

Aromatization of androgens into estrogens in rat testis is catalyzed by the microsomal enzyme cytochrome P450 aromatase. In this work, aromatase cellular site was investigated in prepuberal, peripuberal and postpuberal testis, from 10-, 21- and 60-day-old rats respectively. Paraffin-embedded testis sections were processed for P450arom immunostaining using a rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase. Next, biotinylated anti-rabbit IgG was applied, followed by ABC/HRP/complex amplification with diaminobenzidine as chromogen. Prepuberal testis sections showed a strong immunoreactivity of aromatase in Sertoli cell cytoplasm while interstitial cells were immunonegative. In peripuberal testis sections, cytoplasmic immunoreaction was weak in Sertoli cells, but it was strong in spermatocytes and sporadic in Leydig cells. Postpuberal testis sections displayed a moderate aromatase immunoexpression in spermatocytes while a strong immunostaining was observed in round and elongated spermatids, as well as in Leydig cells. These results indicate a different age-dependence of aromatase localization in rat testicular cells during gonadal development. In particular, inside the seminiferous tubules, the aromatization site moves from Sertoli cells to late germ cells, suggesting a proliferative role of aromatase in prepuberal testis and its subsequent involvement in meiotic and post-meiotic germ cell maturation.     

Direct interference or indirect exploitation? An experimental study of fitness costs of interspecific competition in voles

Studies on competing mammalian species in the past have focused mainly on the competitive exclusion of one species from the preferred habitat of the other. Investigations on effects of competition and coexistence on individual fitness are rare . In this study we were able to measure effects of interspecific competition on major fitness components, using a system with two vole species in asymmetric competition. Survival, reproduction and space use of bank vole Clethrionomys glareolus females were monitored in 32 enclosed populations over four replicates of eight parallel run enclosures. Into half of the enclosures we introduced an additional number of field voles Microtus agrestis , a dominant competitor.
Survival of bank vole females was lower under competitive conditions. Total number of breeding females was lower in populations coexisting with competitors. Territory size of bank vole females decreased. Females body weight and litter size bank vole litters conceived during the experiment were not affected by interspecific competition. These characteristics should respond to differences in food resources, and territory size should increase if food was scarce, thus we found no indication of direct exploitation competition between the two species. Space use was overlapping between the species, but individuals of both species were never caught together in the same trap, indicating avoidance behaviour.
We conclude that adult bank vole females do suffer fitness consequences through interference competition with field voles, probably basing on increased number of aggressive encounters in the presence of the dominant species. Our results suggest, that direct interference rather than indirect exploitation competition may be the cause for observed fitness decrease in bank vole females.