Mariana Bakalska

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.

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 β-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.

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.

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.

Ontogenèse et régulations des fonctions testiculaires chez le fœtus humain

Two major functions are assumed by the testis: the production of male gametes (that is, spermatozoa) and the production of steroid hormones. Both two functions are established during fetal life and are essential to the adult fertility and the masculinization of the internal tract and genitalia. For many years, our laboratory has been interested in the ontogeny of those two functions in rodents and, since 2003, in collaboration with gynecology and obstetrics service of professor R. Frydman in Antoine-Béclère hospital, we have studied them in human. The first aim of this work was to improve the global knowledge of the human fetal testis development by using both our experimental data and the literature. Then, we focused on the different defects that can occur during the fetal testis development. Indeed, male reproductive abnormalities have been steadily increasing since the last decades and are thought to be related to the concomitant increase of the concentration of contaminants and particularly of endocrine disruptors in the environment. Thus, we decided to study the effect of endocrine disruptors on human fetal testis and, more particularly, the effect of phthalates, by using an organ culture system developed for human. In contrast to the data obtained in rat, mono (ethylhexyl)-phthalate (MEHP), an active metabolite of the most widespread phthalate in the environment, does not disturb the steroidogenic function. On the other hand, it has a negative effect on the male germ cells number. This study is the first experimental demonstration of a negative effect of phthalates directly on human fetal testis.

Treizièmes Journées nationales de la FFER (Paris, 17–19 septembre 2008)Ontogenèse et régulations des fonctions testiculaires chez le fœtus humainDevelopment and regulations of testicular functions in the human foetus

Two major functions are assumed by the testis: the production of male gametes (that is, spermatozoa) and the production of steroid hormones. Both two functions are established during fetal life and are essential to the adult fertility and the masculinization of the internal tract and genitalia. For many years, our laboratory has been interested in the ontogeny of those two functions in rodents and, since 2003, in collaboration with gynecology and obstetrics service of professor R. Frydman in Antoine-Béclère hospital, we have studied them in human. The first aim of this work was to improve the global knowledge of the human fetal testis development by using both our experimental data and the literature. Then, we focused on the different defects that can occur during the fetal testis development. Indeed, male reproductive abnormalities have been steadily increasing since the last decades and are thought to be related to the concomitant increase of the concentration of contaminants and particularly of endocrine disruptors in the environment. Thus, we decided to study the effect of endocrine disruptors on human fetal testis and, more particularly, the effect of phthalates, by using an organ culture system developed for human. In contrast to the data obtained in rat, mono (ethylhexyl)-phthalate (MEHP), an active metabolite of the most widespread phthalate in the environment, does not disturb the steroidogenic function. On the other hand, it has a negative effect on the male germ cells number. This study is the first experimental demonstration of a negative effect of phthalates directly on human fetal testis.

Ganglioside spinal cord changes in chronic relapsing experimental allergic encephalomyelitis induced in the Lewis rats.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in Lewis rats by inoculation with guinea-pig myelin and complete Freunds adjuvant followed by treatment with low-dose cyclosporin A. Rats were sacrified at different phases of the disease (just before the onset of clinical signs, during the first clinical episode of CREAE and during the first recovery). Gangliosides were extracted from the spinal cord, analysed after purification by two-dimensional chromatography and quantified densitometrically. An increase of GM1, the main rat myelin ganglioside, and a decrease of GT1b, suggested to play a role in mediating the interactions between oligodendroglia and axons, were observed during the development of the CREAE. These findings indicating significant ganglioside changes in CREAE give further support to the concept concerning the involvement of gangliosides in autoimmune demyelination.

11β Hydroxysteroid Dehydrogenase Type 2 in the Adrenal Gland by Testosterone Withdrawal of Adult Rats

11β Hydroxysteroid Dehydrogenase Type 2 in the Adrenal Gland by Testosterone Withdrawal of Adult Rats Aim: 11β hydroxysteroid dehydrogenase (11β 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 11β HSD type 2 in the adrenal gland remains obscure. Therefore, the aim of the present study was to establish the pattern of 11β 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 11β HSD type 2 signals were detected in the adrenal cortex cells, but not in the medulla. We observed the lowest 11β 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 11β 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 11β HSD type 2 expression in the adrenal gland under conditions of testosterone withdrawal of adult rats. Our results elucidate further the functional significance of 11β HSD system in rat adrenal gland and the regulatory role of testosterone in its activity. 11β Гидроксистероид Дегидрогена - 3А Типа 2 В Надпочечнойжелезе ЗрелыХ Крыс В УсловияХ Дефицита Тестостерона Цель: 11 β гидроксистероид дегидрогеназа (11 β ГСД)катализирует превращение глюкокортикостероидое в инертные метаболиты у человека и грызунов и играет важную роль в регуляции действия кортико-стероидныХ гормонов. Физиологическая роль и регу-лирование lift ГСД типа 2 в надпочечной железе все еще остаются неясными. В связи с этим настоящее исследование ставит себе целью установить модель экспрессии lift ГСД типа 2в надпочечной железе крыс в условияХ дефицита тестостерона. Материал и методы:Применен иммуногисто- Химический анализ срезов надпочечной железы зрелыХ крыс. Срезы обработаныэтан диметансульфонатом (ЭДС). Результаты: У крыс контрольной группы llβ ГСД типа 2сигналы сильной интенсивности обна-ружены в клеткаХ надпочечной коры, но не и в медулле. Самая слабо выраженная 11 β ГСД типа 2 экспрессия установлена 7 дней после обработки с помощью ЭДС; следует прогрессивное увеличивание интенситета иммунореактивности на 14-ый и 21-ый день. Максимальный интенситет окрашивания 11 р ГСД типа 2 в адренокортикоцитаХ обнаружен на 35-ый день после ЭДС воздействия. Выводы: Применяя ЭДС модель, настоящее иссле- дование дает новые данные насчет экспрессии ll β ГСД типа 2в надпочечнике зрелыХ крыс в условияХ дефицита тестостерона. Полученные результаты способствуют выяснению функционального значения 11 β ГСД системы в надпочечной железе, а также и выяснению регуляторной роли тестостерона в ее активности.

Morphological Changes in Rat Leydig Cells Reflecting the Decreased Testicular Steroidogenic Capacity during Aging

The Leydig cells situated in the interstitial compartment of the mammalian testis are responsible for most of the testosterone produced in males. Previous studies in rats have shown that the ability of Leydig cells to produce testosterone significantly declines with age. The present study was focused on the description of some morphological alterations in rat Leydig cells that could be associated with the decreased testicular steroidogenic capacity during aging. Ultrastructural study of aging rat testes revealed the presence of Leydig cells with intact morphology as well as Leydig cells with different degree of degeneration. The decrease of both smooth endoplasmic reticulum and mitochondria, together with an accumulation of lipid droplets and residual bodies in aging Leydig cells, were observed. Our electron microscopical analysis revealed that the majority of the mitochondria in aged Leydig cells (24-month-old) exhibited significant features of degeneration: increasing in size, swelling, lighten of the matrix, reduction of cristae number, and alterations in the structural integrity of the membranes. During aging progressive increase was established in the number of Leydig cells exhibiting nuclear changes such as chromatin compaction and fragmentation and nuclear shrinkage that could be a sign of elevated apoptotic tendency. The observed morphological alterations in rat Leydig cells reflect the decreased testicular steroidogenic capacity during aging, resulting in decrease of testosterone production.