Effect of mono-(2-ethylhexyl) phthalate on human and mouse fetal testis: In vitro and in vivo approaches.

The present study was conducted to determine whether exposure to the mono-(2-ethylhexyl) phthalate (MEHP) represents a genuine threat to male human reproductive function. To this aim, we investigated the effects on human male fetal germ cells of a 10⁻5 M exposure. This dose is slightly above the mean concentrations found in human fetal cord blood samples by biomonitoring studies. The in vitro experimental approach was further validated for phthalate toxicity assessment by comparing the effects of in vitro and in vivo exposure in mouse testes. Human fetal testes were recovered during the first trimester (7-12 weeks) of gestation and cultured in the presence or not of 10⁻5 M MEHP for three days. Apoptosis was quantified by measuring the percentage of Caspase-3 positive germ cells. The concentration of phthalate reaching the fetal gonads was determined by radioactivity measurements, after incubations with ¹4C-MEHP. A 10⁻5 M exposure significantly increased the rate of apoptosis in human male fetal germ cells. The intratesticular MEHP concentration measured corresponded to the concentration added in vitro to the culture medium. Furthermore, a comparable effect on germ cell apoptosis in mouse fetal testes was induced both in vitro and in vivo. This study suggests that this 10⁻5 M exposure is sufficient to induce changes to the in vivo development of the human fetal male germ cells.

[Effects of estrogens on the development of the testis during fetal and neonatal life]. / Effets des estrogènes sur le développement du testicule pendant la vie foetale et néonatale.

Estrogens are classically known to play a major role in female reproduction but there is now compelling evidence that they may also be involved in the regulation of male reproductive function. In humans, a decrease in sperm count and an increase in the incidences of testicular cancer, cryptorchidism and hypospadia have been observed in many countries over the last 50 years. Male reproductive alterations were also observed in wildlife. Such male reproductive disorders have been attributed to the increase in concentration of xenobiotics, and of xenoestrogens in particular, in the environment and in food. Epidemiological, clinical and experimental studies have suggested that excessive exposure to estrogens during fetal/neonatal life can lead to reproductive disorders in adulthood. Using an in vitro model we showed that estrogens directly affected the development of the fetal testis and we evidenced the existence of periods of sensitivity throughout development. Lastly, we clearly demonstrated that the fetal and neonatal testis is very sensitive to estrogens since the invalidation of estrogen receptor alpha leads to an increase of steroidogenesis and the invalidation of estrogen receptor beta enhances the development of the germ cell lineage in the male.

Development of the foetal and neonatal testis.

The foetal testis originates from a proliferation of the mesonephric and the coelomic epithelia which are colonized by the primordial germ cells. In the foetal testis, the development and functions of the three main cell type precursors (Leydig, Sertoli and germ cells) do not depend upon gonadotropins. Numerous intra- and extra-testicular factors are candidates for the control of its development and functions. To study the potential involvement of these factors, we developed an organotypic culture system. In absence of any growth factors or hormone, this system allows a development of the three main cell types which mimics that observed in vivo. The effects of different regulators (gonadotropin-releasing hormone, follicle-stimulating hormone, transforming growth factor-beta, insulin-like growth factor-I, anti-Mullerian hormone, retinoic acid, oestrogens) were tested in this system. Whether or not some of the effects observed in vitro have a physiological relevance was evaluated using appropriate transgenic mice. It is concluded that the foetal testis cannot be considered as an adult mini-testis since it has a specific physiology which largely differs from that of the immature or adult testis.

Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: immunolocalization and implication in the control of the number of gonocytes.

Retinoids have pleiotropic effects on embryonic development and are essential for spermatogenesis in the adult, where they act via nuclear retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We used immunohistochemistry to examine the cellular localization of RARs and RXRs in the rat testis from Day 13.5 postconception (13.5 dpc) until Day 8 postpartum (8 dpp), and these findings were compared with those for immature and adult testes. RARalpha and RARbeta were detected in the interstitial tissue from 14.5 dpc, with intense staining in the gonocytes from 20. 5 dpc to 8 dpp. The nuclei of all cell types stained faintly for RARgamma from 8 dpp. Immunoreactivity for RXRalpha was intense in the gonocytes from 13.5 dpc and in the Leydig cells from 16.5 dpc, and persisted throughout the period studied. RXRbeta was always detected in the Leydig cells and during a short neonatal period in the gonocytes. RXRgamma gave a faint reaction in the nuclei of all cell types from 20.5 dpc. Unexpectedly, immunostaining for all the receptors tested, except RARgamma and RXRgamma, was detected in the cytoplasmic compartment of the cells of fetal and neonatal testes, while it was found in the nuclei in immature and adult testes. In cultures of dispersed testicular cells from 3 dpp pups, retinoic acid had a dose-dependent deleterious effect on the survival of the gonocytes and, to a lesser extent, of the somatic cells. These results suggest that retinoids act on the testicular development, especially on germ cells, via RARs and/or RXRs.

Apoptosis and mitosis in gonocytes of the rat testis during foetal and neonatal development.

This study was undertaken to determine the extent of apoptosis and mitosis in the various testicular cell types throughout rat development from foetal day 14.5 to postnatal days 9-10. Apoptotic activity was studied by detecting DNA fragmentation (TUNEL method) in situ. A TUNEL-positive reaction was detected in gonocytes, while none of the other testicular cells were labelled. The morphology of the TUNEL-positive gonocytes was characteristic of apoptotic cells and was different from that observed in experimentally induced necrosis. The percentage of stained gonocytes peaked on day 15.5-16.5 post-conception (dpc), decreased thereafter and no TUNEL-positive gonocytes were found from foetal day 18.5 onwards. On postnatal day 2, apoptosis resumed and increased to reach a maximum on day 7. Mitosis in the gonocytes, as evaluated by the immunodetection of 5-bromo-2'-deoxyuridine (BrdU) incorporation, was present during the same developmental periods but the ratio of BrdU-positive/TUNEL-positive gonocytes was much greater in the foetal period than in the neonatal period. In an organotypic culture system, the changes in the apoptotic and mitotic activities of the gonocytes in testicular explants from foetuses on days 18.5 and 20.5 or from neonates on day 3, cultured for two days were similar to those observed in vivo. Addition of LH or FSH did not influence either apoptosis or mitosis in the germ cells. These results suggest that both apoptosis and mitosis of gonocytes are independent of gonadotrophins and are mainly controlled by intratesticular factors.

Expression and regulation of transforming growth factor beta1 mRNA and protein in rat fetal testis in vitro.

The expression and secretion of Transforming Growth Factor beta1 (TGFbeta1) by cultured testes of day 20.5 rat fetuses were investigated. The testes were found to express two TGFbeta1 mRNA transcripts of 2.5 and 1.8 kb. By using mink lung epithelial cell bioassay based on the measurement of the inhibition of tritiated thymidine incorporation in response to TGFbeta1 immunoreactive material, the fetal testes were shown to secrete TGFbeta1 protein in organ culture. This secretion was positively regulated by dibutyryl cyclic AMP or by LH and FSH together, but not by LH alone and very slightly by FSH alone, which suggests interactions between Leydig and Sertoli cells for the control of TGFbeta1 production. These regulations probably take place at a posttranscriptional step since no concomitant increase of TGFbeta1 mRNA levels was observed. Such a positive regulation of TGFbeta1 secretion by gonadotropins could be a characteristic of the rat fetal testis.

Transforming growth factor beta1 inhibits steroidogenesis in dispersed fetal testicular cells in culture.

TGF beta1 has been detected by immunohistochemistry in the rat fetal testis. Therefore, we attempted to determine whether this factor can act as a local regulator of Leydig cell function during fetal development. An inhibitory effect of TGF beta1 on basal and luteinizing hormone (LH)-stimulated testosterone secretion by fetal testes in vitro was observed only with testes from 13.5 day-old fetuses and not with testes from older stages. The lack of effect of exogenous TGF beta1 in organ culture after day 13.5 might be related to an elevated intratesticular concentration that would already exert maximal biological effect. On the contrary, in a model of dispersed testicular cells in culture, TGF beta1 was able to inhibit LH-stimulated testosterone production by fetal Leydig cells from 16.5 and 20.5 day-old fetuses. This inhibition of LH-stimulated testosterone production was dose- and time-dependent and was maximal after 48 h of treatment with 1 ng/ml TGF beta1, with testosterone secretion being reduced to 25% of control values. Inhibition of testosterone secretion was also observed in basal and dbcAMP-stimulated conditions, suggesting that one site of action of TGF beta1 is located after the production of cAMP. However, TGF beta1 was also able to inhibit LH-induced cAMP production. As demonstrated by the transformation of steroidogenic precursors into testosterone, TGF beta1 did not significantly alter 3beta-hydroxysteroid dehydrogenase (3beta HSD) activity but induced a strong inhibition of cytochrome P450 17alpha-hydroxylase/C17-20 lyase (P450C17) activity which was associated with a marked diminution of cytochrome P450C17 mRNA levels (26% of control values) but not of cytochrome P450scc mRNA. In addition to its effect on steroidogenesis, TGF beta1 exhibited morphogenic actions on the fetal testicular cells, inducing spreading when the cells were adherent and aggregation when the cells were cultured in conditions of lesser adherence and without any significant effect on either total cell number or 3beta HSD positive cells. Taken together these results suggest that TGF beta1 likely plays a morphogenic and physiological role very early in the fetal testis via paracrine/autocrine mechanisms.

The immunohistochemical localization of transforming growth factor-beta 2 in the fetal and neonatal rat testis.

The localization of transforming growth factor beta-2 (TGF beta 2) in the fetal and neonatal testis (from day 13.5 of fetal life to postnatal day 9) was investigated by an immunohistochemical staining method employing a specific polyclonal antibody. Immunostaining appeared on fetal day 13.5 in primitive Sertoli cells as they begin to come in contact with each other and surround the germ cells to form the seminiferous cords. Staining in Sertoli cells was still clearly observed until fetal day 16.5 and became faint or undetectable from fetal day 18.5 onwards. In fetal-type Leydig cells, a positive reaction for TGF beta 2 appeared on day 16.5 and became very intense from day 18.5 onwards. In the germ cells, immunoreactivity for TGF beta 2 appeared on fetal day 20.5, rose to a maximum on postnatal day 4 and decreased thereafter. On postnatal day 9, staining was still present in type A spermatogonia and absent in type B spermatogonia. No immunoreactivity was detected in peritubular cells on any day studied. In conclusion, our results are in favour of an autocrine/paracrine role of TGF beta 2 in the differentiation of the testis during the perinatal period. It may be involved in the organization of the seminiferous cords, the regulation of testosterone production and the regulation of the number of germ cells. When compared with the immunolocalization of TGF beta 1 that we have previously reported [1], the present study suggests that the roles of TGF beta 2 in the developing rat testis can be specific but also overlap from those of TGF beta 1.

Immunohistochemical localization of transforming growth factor beta 1 and beta 2 in the fetal and neonatal rat ovary.

The localization of transforming growth factor beta (TGF beta) in the differentiating ovary (from fetal day 13.5 to postnatal day 14) was investigated immunohistochemically using polyclonal antibodies for TGF beta 1 and TGF beta 2. Immunostaining was undetectable in the gonadal primordium on fetal day 13.5. From fetal day 14.5 and throughout fetal life, there was intense immunostaining for TGF beta 1 and faint staining for TGF beta 2 in some ovarian somatic cells which were identified as epithelial cells at the end of fetal life. This pattern of staining was also found in the presumptive granulosa cells present between the oocytes on postnatal day 1. The staining for TGF beta 2 persisted while the staining for TGF beta 1 decreased in the granulosa cells of primordial and primary follicles of older rats. In the subsequent stages of follicular development, the staining for TGF beta 1 disappeared while faint staining for TGF beta 2 persisted in the granulosa cells of secondary and small antral (postnatal day 14) follicles. On day 14, the newly functional thecal/interstitial cells were moderately stained for TGF beta 2 and intensely stained for TGF beta 1. These results plus our previous immunolocalization of TGF beta 1 in the fetal testis [13] show that, in both sexes: (1) TGF beta 1 is detectable in the gonads on the same fetal age (day 14.5); (2) TGF beta 1 is present in the somatic cells which are the precursors of Sertoli and granulosa cells, at the time of their organization in seminiferous tubules or in primordial follicles; and (3) TGF beta 1 is largely present in cells synthesizing androgens, from the onset of their steroidogenic capacity (fetal day 16.5 for Leydig cells and postnatal day 14 for thecal/interstitial cells.

Immunohistochemical localization of transforming growth factor-beta 1 in the fetal and neonatal rat testis.

The localization of transforming growth factor-beta 1 in the fetal and neonatal rat testis (from day 13.5 of fetal life to postnatal day 20) was investigated by an immunohistochemical staining method employing a polyclonal anti-TGF-beta 1 antibody that does not cross react with either TGF-beta 2 or TGF-beta 3. In testis and mesonephros tissue, immunostaining for TGF-beta 1 was undetectable on fetal day 13.5 and appeared exclusively in the primordial Sertoli cells on fetal day 14.5. Staining in Sertoli cells was still clearly observed on days 15.5 and 16.5 of fetal life and became faint from fetal day 18.5 onwards. In fetal Leydig cells, a positive reaction for TGF-beta 1 appeared on day 16.5 and became very intense during late fetal life. After birth, fetal-type Leydig cells, which were still observed on postnatal days 4 and 20, also exhibited a very strong immunostaining for TGF-beta 1, whereas adult-type Leydig cells, observed on day 20, showed a slight staining. No immunoreactivity for TGF-beta 1 was found in germ cells and peritubular cells on any day studied. In conclusion, TGF-beta 1 is present very early in the fetal rat testis and its prevailing localization shows age-related changes, which suggests that this factor plays an autocrine/paracrine role in the regulation of testicular function and differentiation, during early development.

Stimulatory effect of follicle-stimulating hormone on basal and luteinizing hormone-stimulated testosterone secretions by the fetal rat testis in vitro.

The in vitro effect of FSH on testosterone secretion by the fetal rat testis was studied. Testes were cultured in the presence or absence of either commercial human (h) FSH (Metrodine; 200 mIU/ml) or recombinant hFSH (200 mIU/ml) for 3 days and with 100 ng/ml ovine LH during the last 4 h of culture. To avoid a stimulatory effect by the 0.4% LH that contaminates Metrodine, the cultures were performed in the presence of a monoclonal anti-hLH beta antibody and with a concentration of Metrodine that had no short term stimulatory effect on testosterone production by the fetal testes in vitro. Metrodine treatment had a positive long term effect on both basal and LH-stimulated testosterone secretion by fetal testes explanted on days 18.5, 20.5, and 22.5 postconception, which was abolished by the addition of a monoclonal anti-hFSH beta antibody. LH-free recombinant FSH also augmented basal and LH-stimulated testosterone secretion of testes explanted on days 13.5, 14.5, and 18.5 postconception. The positive effect of recombinant hFSH appeared during the second day of treatment with day 14.5 and 18.5 testes and on the third day of treatment with day 13.5 testes. As it is widely accepted that FSH receptors are exclusively localized on Sertoli cells, these results suggest that on or before day 15.5 of fetal life, 1) Sertoli cells are able to respond to FSH, 2) Sertoli cells can produce factors that are able to act on Leydig cell function, and 3) Leydig cells are sensitive to FSH-induced Sertoli cell factors. In conclusion, this study points out a potential paracrine control of fetal Leydig cell function and/or differentiation by fetal Sertoli cells as soon as fetal Leydig cells differentiate.

Glucose utilization and insulin binding in discrete brain areas of obese rats.

The present study was carried out to determine whether genetically obese Zucker rats present changes in brain glucose utilization and/or insulin binding when compared to their lean counterparts. Glucose utilization in the whole brain, determined by measurement of 2-deoxy(1-3H)glucose-6-phosphate, was significantly lower in obese than in lean Zucker rats. In order to precise the structure involved, we then used quantitative autoradiography methods after either (1-14C) 2-deoxyglucose injection or 125I-insulin incubation. In obese rats, local cerebral glucose utilization (LCGU) was significantly decreased in the external plexiform layer (-37%, p < 0.05), in the lateral hypothalamus (-23%, p < 0.05), and in the basolateral amygdaloid nucleus (-30%, p < 0.05). In contrast, no difference in specific insulin binding was found between the two genotypes in any of the areas studied. These results are consistent with some data showing a decrease of LCGU in hyperinsulinemic rats. All together, these data show perturbations of glucose utilization, particularly in structures linked to the regulation of body weight and food intake in obese Zucker rats.

Developmental changes in testosterone production by the rat testis in vitro during late fetal life.

The age-related evolution of the in vitro capacity of the rat testis to produce testosterone and to respond to luteinizing hormone (LH) during 3 and 24 h incubation was studied from day 18.5 to day 21.5 of fetal life. Basal testosterone production by testes from 18.5-day-old fetuses was significantly higher than production by testes from 20.5- and 21.5-day-old fetuses when secretion was expressed as either per testis or per microgram of testicular protein. When maximal LH-stimulated testosterone secretion was expressed on a per testis basis, it was significantly lower for day 18.5 testes than for day 20.5 and 21.5 testes. However, when it was expressed on the basis of testicular protein content, it decreased significantly between days 18.5 and 20.5. Basal and LH-stimulated secretions displayed the same time-related decrease throughout 24 h of incubation for the three ages studied. The dose-response curves for LH showed that the sensitivity was similar for day 18.5 and 20.5 testes (ED50 = 10 vs. 14 ng/mL, respectively). These results showed an age-related decrease in testicular steroidogenic capacity without a change in the coupling efficiency of LH receptor to testosterone production during late fetal life in rats.

Fatty acid synthetizing enzymes in adipocytes and stromavascular fraction of hyperthyroid rat adipose tissue.

In the rat, hyperthyroidism induced from birth has been shown to increase both adipose cell recruitment and de novo lipogenesis at 6 weeks of age. Since preadipose cells are included into the stromavascular fraction (SVF) of adipose tissue, fatty acid synthetizing enzyme activities were estimated separately in adipocytes and SVF cells of adipose tissue of 6 week-old hyperthyroid rats. Fatty acid synthetase (FAS), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities were generally increased in adipocytes and in SVF cells when compared to age-matched controls. Among the enzymes studied, ME activity displayed the highest stimulation in SVF cells and was much more stimulated in these cells than in adipocytes (214% and 73% increase, respectively, above control values, P less than 0.001). These results show that, in vivo, SVF cells and adipocytes can respond differently to an hormonal stimulation and raise the question of the role of ME in the adipose conversion.

Acceleration by triiodothyronine of adipose conversion of rat preadipocytes from two adipose localizations.

Since we have previously reported that hyperthyroidism induces adipose tissue hyperplasia in the young rat, the effect of thyroid hormones on growth and differentiation of preadipocytes from retroperitoneal (RPAT) and epididymal (EAT) adipose tissue was studied in a primary culture system which allows a precocious cell differentiation. In this culture system, preadipocytes from RPAT exhibited a greater potentially to differentiate than cells from EAT. Chronic exposure to triiodothyronine (T3) induced an acceleration of the differentiating process as shown by a transient increase of the number of differentiated cells without alteration of cell multiplication. This effect was more important in cultures of cells from RPAT than from EAT. T3 was ineffective on lipoprotein-lipase activity but induced a stimulation of the esterification pathway which was durable and could likely be related to an increased lipid turn-over. T3 induced also a stimulation of fatty acid biosynthesis, only on the first stages of morphological differentiation which suggests that this effect could be specifically in relation with the stimulation of adipose conversion.

Hepatic and adipose tissue lipogenesis as related to age and thyroid status in the rat.

We have previously reported that, in the rat, chronic thyroxine (T4) treatment induced a transient adipose tissue hyperplasia and that, in preadipocytes cultures, lipogenesis as well as adipose conversion were enhanced by triiodothyronine. Therefore we looked for the possibility of a relationship between in vivo stimulation of adipose tissue lipogenesis and the stimulation of fat cell recruitment by thyroid hormones. Hepatic and adipose tissue de novo lipogenesis were estimated by the incorporation of 3H2O into lipids in rats of various ages made slightly hyperthyroid by daily injections of T4 (0.2 microgram/g/day) from birth. Hepatic and adipose tissue lipogenesis were increased at 3 and 6 weeks of age, no stimulation being observed when animals get older. 21 week-old animals were therefore acutely treated with 0.2 or 2 micrograms T4/g/day. In this case, only the high T4 dose was able to induce a consistent lipogenesis stimulation in liver and in retroperitoneal adipose tissue and failed to induce it in epididymal adipose tissue. These results pointed out that thyroid hormones can stimulate lipogenesis both in liver and adipose tissue. However, there is an age related fall in the sensitivity to thyroid hormones for lipogenesis stimulation, not only in the liver, but also and more pronounced in adipose tissue, in parallel to that observed in vivo for adipose differentiation; moreover, this decreased sensitivity seems to be accelerated by a long lasting hyperthyroidal state.

Lipogenesis as related to endocrine control of adipose cellularity in the rat.

The incorporation rate of 3H from 3H2O in the total lipids of rat retroperitoneal adipose tissue (RPAT) according to age was followed in slightly hyperthyroid rats, in type-2 diabetic rats and in control rats. Until 12 weeks of age, the RPAT of the hyperthyroid animals exhibited hyperplasia and an increase in lipogenesis per g of wet weight or per whole fat pad. Fat cel number and lipogenesis per g of wet weight or per whole fat pad were reduced in adult diabetic rats. The concomitant changes in fat cell number and lipid synthesis in the RPAT of the two groups of treated rats have been discussed.

Effects of thyroid hormones on adipose tissue development in Sherman and Zucker rats.

It has been reported that mild hyperthyroidism in the young Sherman rat induces adipose tissue hyperplasia, concomitant with cell size reduction, and that hypothyroidism induces opposite effects. The present experiments were designed to study the evolution of cellularity in retroperitoneal and epididymal adipose tissue during a long term thyroxine (T4) treatment or in T4-treated rats, after the treatment had been stopped. In both cases, hyperplasia was transient with the observation that the adipocyte number observed in 3-mo-old control rats was reached earlier in T4-treated rats. In hypothyroid rats, hypoplasia was also transient, because once the treatment was stopped, the cell number overtook that of controls. Zucker rats were also treated with T4, because hypoplasia has been observed in young obese (fa/fa) rats and these rats are reported to be hypothyroid. T4 treatment increased their adipocyte number up to the level of nonobese (Fa/fa) untreated rats, while hypertrophy, although reduced, was persistent. In Sherman and Zucker rats, adipose tissue lipoprotein lipase activity was decreased by T4 treatment in parallel with and perhaps because of adipocyte size reduction. We suggest that hyperplasia induced by thyroid hormones results from a precocious differentiation of preadipocytes and does not necessarily imply an increased preadipocytes multiplication.

Effect of early and chronic hypoinsulinism on adipose tissue cellularity in the rat.

The effect of chronic hypoinsulinism on the development of retroperitoneal adipose tissue was studied in rats injected with streptozotocin at birth. The streptozotocin injection induced an acute neonatal diabetes which regressed spontaneously after one week and led to a chronic state of chemical diabetes in the young and in the adult rat. Growth of chemically diabetic rats was normal although the retroperitoneal adipose tissue showed a relative hypoplasia which appeared at two months and evolved with age so that at 10 months the number of adipose cells in the retroperitoneal adipose tissue was largely decreased with respect to control animals (1.34 +/- 0.12 x 10(6) versus 2.23 +/- 0.11 x 10(6)). This relative hypoplasia was still present at 20 months. Whereas the hypoplasia associated with the chemical diabetes was highly reproducible, the mean adipocyte size was modified in a variable manner but was never significantly decreased in chemically diabetic rats. These findings indicate that insulin is involved in the control of retroperitoneal adipose tissue cellularity and suggest that the effect of hypoinsulinism on adipocyte number does not depend on a decrease of the mean adipocyte volume.

Thyroid hormones and adipose tissue development.

The effect of thyroid hormones on the cellularity of the retroperitoneal adipose tissue (R.P.A.T.) was investigated in rats that were 3, 6 and 12 weeks old. Two groups of rats were respectively made hypothyroid by the antithyroid compound propylthiouracil, or hyperthyroid by thyroxine. The number of adipocytes was less in the hypothyroid rats than in the controls; it was higher in the hyperthyroid rats without any concomitant increase in the weight of their R.P.A.T. Moreover, there was no significant correlation between adipose cell number and adipose tissue weight within any group of T4 or control rats. In all groups of rats, the number of adipose cells in the R.P.A.T. was larger in males than in females; the difference was highly significant in 12 week old control rats.