11beta hydroxysteroid dehydrogenase type 2 in the adrenal gland by testosterone withdrawal of adult rats.

AIM: 11beta hydroxysteroid dehydrogenase (11beta HSD) catalyzes the interconversion of glucocorticoids to inert metabolites in man and rodents and plays a crucial role in regulating corticosteroid hormone action. The physiological role and regulation of 11beta HSD type 2 in the adrenal gland remains obscure. Therefore, the aim of the present study was to establish the pattern of 11beta HSD type 2 expression in rat adrenal gland under conditions of testosterone withdrawal. MATERIAL AND METHODS: We performed immunohistochemical analyses of adrenal gland sections of ethane dimethanesulphonate (EDS)-treated adult rats. RESULTS: In controls, strong positive 11beta HSD type 2 signals were detected in the adrenal cortex cells, but not in the medulla. We observed the lowest 11beta HSD type 2 expression intensity 7 days after initial treatment with ethane dimethanesulphonate (EDS) followed by progressive increase in the immunoreactivity toward days 14 and 21. Maximal staining intensity of 11beta HSD type 2 in the adrenocorticocytes was found by day 35 after EDS treatment. CONCLUSIONS: By using the EDS model the present study provides new data about 11beta HSD type 2 expression in the adrenal gland under conditions of testosterone withdrawal of adult rats. Our results elucidate further the functional significance of 11beta HSD system in rat adrenal gland and the regulatory role of testosterone in its activity.

Sex-specific differences in fetal germ cell apoptosis induced by ionizing radiation.

BACKGROUND: We have previously shown that male human fetal germ cells are highly radiosensitive and that their death depends on p53 activation. Male germ cell apoptosis was initiated with doses as low as 0.1 Gy and was prevented by pifithrin alpha, a p53 inhibitor. In this study, we investigated the radiosensitivity of early female and male fetal proliferating germ cells. METHODS AND RESULTS: Both male and female fetal germ cells displayed a similar number of gamma H2AX foci in response to ionizing radiation (IR). In organ culture of human fetal ovaries, the germ cells underwent apoptosis only when exposed to high doses of IR (1.5 Gy and above). Accumulation of p53 was detected in irradiated male human fetal germ cells but not in female ones. Inhibition of p53 with pifithrin alpha did not affect oogonia apoptosis following irradiation. IR induced apoptosis similarly in mouse fetal ovaries in organ culture and in vivo during oogonial proliferation. Germ cell survival in testes from p53 knockout or p63 knockout mice exposed to IR was better than wild-type, whereas female germ cell survival was unaffected by p53 or p63 knockout. CONCLUSIONS: These findings show that pre-meiotic male and female fetal germ cells behave differently in response to a genotoxic stress--irradiation--with oogonia being less sensitive and undergoing p53-independent apoptosis.

c-erbAalpha and c-erbaAbeta in the Leydig cell repopulation by ethane-1,2-dimethanesulphonate model of mature rats.

INTRODUCTION: The regulatory effect of thyroid hormones on the proliferation and maturation of the Leydig cells (LC) in testis is still poorly understood. To date, it remains obscure whether the thyroid hormones have direct effect on the LC, as far as in rat testis the thyroid hormones receptors are localized predominantly in the Sertoli cells. A single intraperitoneal dose of cytotoxin ethane-1,2-dimethanesulphonate (EDS) injected into mature rats caused a rapid, selective elimination of the adult LC associated with temporary impairment of fertility. Regeneration of the LC population by EDS model is a result of the differentiation of LC progenitors as well as of the proliferation of the newly formed LC whereas the process is similar with the development of adult LC in the prepubertal testis. AIM: The present study aimed to establish the immunohistochemical expression of high affinity triiodothyronine nuclear receptors c-erbAalpha and c-erbAbeta in the regenerating LC after treatment with EDS of mature rats. MATERIAL AND METHODS: Mature male Wistar rats were divided into two groups: (1) a group of rats receiving a single intraperitoneal (i.p.) injection of EDS (75 mg/kg body weight) and (2) a group of control animals. The animals were killed 24 hours, 7, 14, 21 and 35 days after treatment. Testicular fragments were prepared for routine histological and immunohistochemical examinations. RESULTS: The immunohistochemical analysis revealed similar changes in the immunoreactivity for both c-erbAalpha and c-erbAbeta after EDS administration. On day 1 after EDS treatment, the intensity of the immune reactions for c-erbAalpha and c-erbAbeta in the LCs decreased simultaneously with their number. Seven days after EDS administration there was neither LCs nor c-erbAalpha nor c-erbAbeta-immunoreactivity. The first positive stained LCs were found 14 days after EDS when LCs progenitors were detected. The most prominent c-erbAalpha- and c-erbAbeta-immunostaining in the regenerating LCs was evident 21 days after EDS; this coincided with the increased number of LCs progenitors and their transformation into adult LCs population. Thirty-five days after EDS c-erbAalpha and c-erbAbeta-positive LCs were abundant their number and localization in the testicular interstitium being very similar to that in the control rats. CONCLUSION: The observed change in the intensity of the immune reactions for c-erbAalpha and c-erbAbeta in LC repopulation after EDS treatment corresponds to the process of differentiation of progenitors into mature LC. The results obtained support the idea about the regulatory role of thyroid hormones in the differentiation of LC in prepubertal rat testis.

INSLF3-LGR8 ligand-receptor system in testes of mature rats after exposure to ethane dimethanesulphonate (short communication).

The cytotoxic agent ethane-1,2-dimethanesulphonate (EDS) specifically destroys the Leydig cells (LC) in the adult testis, followed by a complete regeneration. The process of LC renewal after exposure to EDS shows homology to the development of the adult-type LC population in prepubertal testis. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a peptide hormone, a novel member of the insulin/relaxin family, and seems to be localized predominantly in the gonadal tissues. INSL3 mRNA is expressed in the LC in a constitutive fashion and INSL3 thus seems to be a useful marker of LC differentiation status. The present study was aimed at establishing the chronology and dynamic of expression of INSL3 and its specific receptor LGR8 in the LC repopulation after exposure to mature rats to EDS. As material, testes of mature Wistar rats that received single intraperitoneal injection of EDS (75 mg/kg body weight) were used. The animals were killed 1, 7, 14, 21 and 35 days after the initial treatment. The pattern of INSL3-LGR8 expression in newly formed LC after EDS administration was established using a high sensitive immunohistochemical polymer detection kit. After treatment with EDS, the immunoreactivity for INSL3 and LGR8 disappeared from the testis and reappeared again at the time of regeneration of the first LC, 14 days after EDS. The INSL3-LGR8 positive cells grew in number concomitantly with the increase of the LC repopulation. Thirty-five days after EDS destruction a larger number of immunopositive LC were seen in form of clusters corresponding with the regeneration of adult type LC population. The present findings support the hypothesis that EDS-treated rats can serve as a model for studying the LC development in the prepubertal testis and indicate a specific role of hormonal factors like INSL3 in this process.

Loss and recovery of androgen receptor protein expression in the adult rat testis following androgen withdrawal by ethane dimethanesulfonate.

Androgens are especially important for the maintenance of spermatogenesis in adulthood and the experimental withdrawal of testosterone (T) production by ethane dimenthanesulfonate (EDS) is a valuable tool for studying androgen-dependent events of spermatogenesis. The aim of the present study was to investigate the specific changes in immunoexpression of androgen receptor (AR) in the testis in relation to degeneration and regeneration of Leydig cell (LC) population and seminiferous epithelium. Immunohistochemistry for AR and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) as well as TUNEL assay for apoptosis were performed on testicular sections of control and EDS-treated rats. Serum LH and T levels were measured by RIA. Our results revealed a total loss of AR immunoexpression from the nuclei of Sertoli (SCs), LCs and peritubular cells during the first week after EDS administration and that coincided with severe drop in T levels. Two weeks after EDS administration, the AR expression was recovered in these cells but normal stage-specificity in SCs was replaced by uniform intensity of AR immunostaining at all the stages of the spermatogenic cycle. The stage-specific pattern of androgen expression in SCs with a maximum at stages VII-VIII appeared 5 weeks after treatment. LC immunoreactivity for 3beta-HSD at different time points after EDS administration correlated with values of T concentration. The maximal germ cell apoptosis on day 7 was followed by total loss of elongated spermatids 2 weeks after EDS treatment. Regeneration of seminiferous epithelium 3 weeks after EDS administration and onwards occurred in tandem with the development of new LC population indicated by the appearance of 3beta-HSD-positive cells and gradual increase in T production. The specific changes in AR after EDS including their loss and recovery in Sertoli cells paralleled with degenerative and regenerative events in Leydig and germ cell populations, confirming close functional relationship between Sertoli, Leydig and germ cells.

Age-related changes in the expression of 11beta-hydroxysteroid dehydrogenase type 2 in rat Leydig cells.

Previous studies in rats have shown that the ability of Leydig cells (LCs) to produce testosterone significantly declines with age. To address the possible mechanisms by which aging LCs lose their steroidogenic function, we determined the effect of aging on the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2. The enzyme plays a protective role in blunting the suppressive effects of glucocorticoids on LCs steroidogenesis. Our immunohistochemical analysis revealed progressive decline in 11beta-HDS type 2 expression in LCs of the 18 months of age rats and the most significant reduction in 11beta-HSD2 immunoreactivity was evident in the testicular interstitium of 24- month-old rats. The decrease in the 11beta-HDS type 2 immunostaining in LCs during aging coincided with decline in insulin-like 3/relaxin-like factor (INSL3/RLF) expression, an independent marker for LCs differentiation status. Concomitant with the age-related decrease of 11beta-HDS type 2 immunoreactivity in the LCs population, the immunoexpression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), marker for LCs steroidogenic activity, was greatly reduced at 24 months compared to 3-month-old control. Similar pattern of expression exhibited also androgen receptor (AR) which is localized in the nuclei of Sertoli cells (SCs), LCs, and peritubular cells. During ages we observed progressive decrease in the immunoreactivity for AR in the testicular types and there was a loss of stage specificity in SCs at age of 24 months. It now seems evident that a variety of factors are likely to be involved in age-related decreases in LCs steroidogenesis, including 11beta-HSD type 2. The observed reduction in 11beta-HSD type 2 expression in aging LCs reflects the decline in their protection ability, opposing the suppressive effect of glucocorticoids on testosterone production.

Ontogenesis of testicular function in humans.

The two major functions of the testis, steroidogenesis and gametogenesis, take place during fetal life. These two functions have been extensively studied in rodents and adult humans. However, their onset during fetal life is poorly documented in humans. In the first part of this work we presented both our experimental data and some data of literature concerning the development of the human fetal testis. In the second part of this article, using the organ culture system we previously developed, we have investigated the regulations or perturbations of fetal testis development both in rodent and human models. Our findings provide important insight into the potential role of exposure to environmental pollutants (physical factors, in particular ionizing radiation, cadmium and endocrine disruptors such as phthalates) during fetal testicular development and their potential deleterious effects on male fertility in adulthood. Our results highlight the specificity of the human model compared with rodent models.

11beta-hydroxysteroid dehydrogenase type 2 expression in the newly formed Leydig cells after ethane dimethanesulphonate treatment of adult rats.

The enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible conversion of physiologically active corticosterone to the biologically inert 11beta-dehydrocorticosterone in rat testis and protect the Leydig cells (LCs) against the suppressive effect of glucocorticoids. The developmental pathway of the adult LCs population is accompanied with an increase in the 11beta-HDS activity. Thus, 11beta-HDS together with its role in controlling the toxicological effect of glucocorticoids on LCs can be used as a marker for their functional maturity. Ethane 1,2-dimethanesulphonate (EDS) treatment of adult rats become unique appropriate model, which enable to answer many questions related to the differentiation of adult LCs in the prepubertal rat testis. The aim of the present study was to investigate the specific changes in the 11beta-HDS type 2 immunoreactivity in tandem with the expression of androgen receptor (AR) during renewal of LCs population after EDS treatment. In the present study, we observed the first appearance of immunostaining for 11beta-HSD2 in new LCs population on day 14 after EDS administration when the progenitor LCs were detected. Our immunohistochemical analysis revealed progressive increases in the 11beta-HSD2 reaction intensity on 21 days after EDS treatment and reached a maximum on day 35. AR immunoexpression was found in new LCs on day 14 and 21 after EDS injection with an increasing curve of intensity. The most prominent AR immunostaining in new population LCs was evident by 35 days after EDS and that coincided with the increased number of LCs and restoration of adult LCs population. Our results demonstrated similar pattern of immunoreactivity for 11beta-HSD2 and AR in new LCs population after EDS treatment and suggested that the changes in 11beta-HSD2 expression can be used for evaluation of adult LCs differentiation in rat testis.

Catecholamine-synthesizing enzymes in the adult and prenatal human testis.

Catecholamines play functional roles in the mature and developing mammalian testis but the cell types responsible for their local synthesis are still controversially discussed. Here, we demonstrate that four enzymes involved in the biosynthesis of catecholamines, namely, tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DBH) and phenylethanolamine- N-methyltransferase (PNMT), are expressed in Leydig cells of the human testis. Tyrosine hydroxylase, the key enzyme of the biosynthesis of catecholamines, was localized to Leydig cells both at the transcript level (by RT-PCR analyses and by in situ hybridization assays) and at the protein level (by immunoblotting and by immunohistochemistry). The other enzymes were also demonstrated in Leydig cells by RT-PCR and immunohistochemical analyses. The presence of TH, AADC, DBH, and PNMT in human Leydig cells was found, in addition, by immunohistochemical approaches carried out on sections from prenatal human testes. Thus, the present study identifies the Leydig cells as the presumed sites of catecholamine production in both the mature and fetal human testes and further supports the previously recognized neuroendocrine characteristics of this cell type.

Induction of male germ cell apoptosis by testosterone withdrawal after ethane dimethanesulfonate treatment in adult rats.

OBJECTIVE: To carry out a detailed quantitative analysis of male germ cell apoptosis in seminiferous epithelium in a long period after EDS administration. METHODS: The apoptosis in adult rat testes was induced by a single i.p. injection of ethane dimethanesulphonate (EDS) in a dose of 75 mg/kg body weight. The TUNEL assay for in situ detection of apoptosis and quantitation of apoptotic germ were performed in testicular sections days 1, 3, 7, 14, 21 and 35 after EDS treatment. Plasma levels of testosterone (T) and luteinizing hormone (LH) were measured by RIA. RESULTS: First signs of seminiferous epithelium regression were manifested by a marked increase in the number of apoptotic cells on 3rd day after EDS treatment. The maximal value of germ cell apoptosis was established on 7th day post EDS that coincided with lowest T levels. Later, until the end of investigated period, the elevated values of all investigated parameters for quantification of germ cell apoptosis decreased, but remained still higher as compared to control and, in addition, also T concentrations returned to normal range and their mean values were lower than these in controls. The pachytene spermatocytes and spermatids were the predominant cell types that underwent apoptosis after EDS treatment. CONCLUSIONS: Quantitative patterns of germ cell death after testosterone deprivation reveal in advance the kinetic of germ cell depletion and regeneration in a long period after EDS. These new findings bring additional support to the concept that germ cell apoptosis is a hormonally regulated process. Induction of germ cell apoptosis by EDS could be considered as a result of differential alterations occurring in the main testicular cell types, more than one pathway being probably involved in that physiological cell death in the testis.