Müllerian-inhibiting substance inhibits rat Leydig cell regeneration after ethylene dimethanesulphonate ablation

The postnatal development of Leydig cell precursors is postulated to be controlled by Sertoli cell secreted factors, which may have a determinative influence on Leydig cell number and function in sexually mature animals. One such hormone, Mullerian inhibiting substance (MIS), has been shown to inhibit DNA synthesis and steroidogenesis in primary Leydig cells and Leydig cell tumor lines. To further delineate the effects of MIS on Leydig cell proliferation and steroidogenesis, we employed the established ethylene dimethanesulphonate (EDS) model of Leydig cell regeneration. Following EDS ablation of differentiated Leydig cells in young adult rats, recombinant MIS or vehicle was delivered by intratesticular injection for 4 days (Days 11-14 after EDS). On Days 15 and 35 after EDS (1 and 21 days post-MIS injections), endocrine function was assessed and testes were collected for stereology, immunohistochemistry, and assessment of proliferation and steroidogenesis. Although serum testosterone and luteinizing hormone (LH) were no different, intratesticular testosterone was higher on Day 35 in MIS-treated animals. At both time points, intratesticular 5alpha-androstan-3alpha,17beta-diol concentrations were much higher than that of testosterone. MIS-treated animals had fewer mesenchymal precursors on Day 15 and fewer differentiated Leydig cells on Day 35 with decreased numbers of BrdU+ nuclei. Apoptotic interstitial cells were observed only in the MIS-treated testes, not in the vehicle-treated group on Day 15. These data suggest that MIS inhibits regeneration of Leydig cells in EDS-treated rats by enhancing apoptotic cell death as well as by decreasing proliferative capacity.     

Abnormal development of the testis after administration of the Leydig cell cytotoxic ethylene-1,2-dimethanesulphonate to the immature rat

Male rats were injected with 50 mg ethylene-1,2-dimethanesulphonate/kg from Day 5 to Day 16 after birth and control rats received injections of the same volume of vehicle. Testes were studied at various times from Day 6 to Day 108 using histochemistry, light and electron microscopy. Fine structural degenerative changes were observed in the Leydig cells and seminiferous tubules of EDS-treated animals as early as Day 6. By Day 11 no Leydig cells could be detected and the interstitia of EDS-treated testes contained large numbers of fibroblast-like cells which formed peritubular collars 3-5 cells thick; the tubules contained Sertoli cells with heterogeneous inclusions and large numbers of lipid droplets. A small number of Leydig cells was found at Day 14 and their numbers increased so that, in animals of 28 days and older, large clusters of Leydig cells were present between severely atrophic tubules. These tubules contained Sertoli cells with few organelles; germinal cells were not observed after 28 days in EDS-treated animals. These results show that EDS destroys the fetal population of Leydig cells postnatally and this mimics the well documented effect of EDS on adult Leydig cells. The seminiferous tubules were permanently damaged by EDS in the present experiments. Tubular damage could have been due to a direct cytotoxic effect of multiple injections of EDS on the tubule before the blood-testis barrier develops or due to withdrawal of androgen support secondary to Leydig cell destruction.     

Effects of thyroid and luteinizing hormones on the onset of precursor cell differentiation into leydig progenitor cells in the prepubertal rat testis

Leydig cells in the adult rat testis differentiate during the neonatal-prepubertal period. However, the stimulus for the initiation of their differentiation is still not clear. In the present study our objectives were to test the effects of thyroid hormone and LH on the initiation of precursor cell differentiation into Leydig cells in the prepubertal rat testis. Four groups of Sprague-Dawley rats were used. All treatments began at postnatal Day 1. Rats in groups I, II, and III received daily s.c. injections of saline (200 microl, controls), triiodothyronine (T(3), 50 microg/kg body weight, hyperthyroid), and LH (ovine LH 10 microg/rat/day), respectively. Rats in group IV were made hypothyroid from postnatal Day 1 by adding 0.1% propylthiouracil (PTU) to their mother's drinking water. Testes of rats were collected at 7, 8, 9, 10, 11, 12, 16, and 21 days of age, fixed in Bouin's solution, and embedded in paraffin for immunocytochemical studies. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and LH receptors (LHR) in testicular interstitial cells (other than the fetal Leydig cells) was observed using the avidin-biotin method. In control rats, out of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for 3beta-HSD, beginning from the postnatal Day 11. However, positive immunolabeling for LHR was first detected in these cells at Day 12, i.e., after acquiring the steroidogenic enzyme activity. In T(3)-treated rats 3beta-HSD positive spindle-shaped cells were first observed at Day 9 (i.e., 2 days earlier than controls), and LHR-positive cells were first observed on Day 11 (2 days later than obtaining 3beta-HSD immunoactivity); they were exclusively the peritubular mesenchymal cells. The 3beta-HSD- and LHR-positive spindle-shaped cells were absent in the testis interstitium of LH-injected rats from Days 7 through 12 but were present at postnatal Day 16. In addition, more fetal Leydig cell clusters and fetal Leydig cells in mitosis were present in LH-treated rats compared to rats in all other treatment groups. Following their first detection, the number of positive cells for each protein continued to increase at each subsequent age in controls, T(3)-, and LH-injected groups. In PTU rats, 3beta-HSD and LHR-positive spindle-shaped cells were absent throughout the experimental period. From these observations, it is possible to suggest the following regarding the developing rat testis interstitium. 1) The precursor cells for the adult generation of Leydig cells in the postnatal rat testis are the peritubular mesenchymal cells. 2) Luteinizing hormone does not initiate the onset of mesenchymal cell differentiation into Leydig cells, instead it delays this process. However, daily LH treatment causes mitosis in fetal Leydig cells and increase in fetal Leydig cell clusters. 3) Thyroid hormone is critical to initiate the onset of mesenchymal cell differentiation into adult Leydig cells.     

Effects of tri-iodothyronine on testicular interstitial cells and androgen secretory capacity of the prepubertal Rat

The main objective of the study was to investigate the effects of hyperthyroidism on the rat testis interstitium during prepuberty, which is not well understood at present. Male Sprague Dawley rats were injected subcutaneously daily with saline (controls) or tri-iodothyronine (T(3), 50 microg/kg body weight; hyperthyroids) from postnatal Day 1. Rats were killed at Days 5, 7, 9, 12, 16, and 21. One testis of each rat was used to determine LH-stimulated (100 ng/ml) testicular androgen secretory capacity in vitro. The other testis was used either for morphometric studies (n = 5) or for immunolocalization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) to identify steroidogenic cells (n = 3) and 11 beta-hydroxysteroid dehydrogenase 1 (11 beta-HSD1) to differentially identify adult Leydig cells. Daily T(3) injections resulted in significant reductions in body and testis weights. Morphometric analysis revealed that lower testis weights in rats treated with T(3) were mainly the result of reductions of total volume of seminiferous cords/tubules. The number of interstitial mesenchymal cells (MCs) was lower (P < 0.05) in T(3) rats compared with age-matched controls. The number of fetal Leydig cells (FLCs) was not different between the two groups; however, FLC hypotrophy was detected in T(3) rats at Day 16 in contrast to Day 21 in control rats. In both groups, morphologically identifiable adult Leydig cells (ALCs) were observed at Day 12 and thereafter; however, the ALC number per testis in T(3) rats was twice as much as those of controls. Positive immunolabeling for 3beta-HSD was first detected in MC/progenitor cells on Day 9 in rats in the T(3) group (cells were still spindle-shaped) and on Day 12 in rats in the control group. Testicular testosterone production in vitro was lower (P < 0.05) in T(3) rats compared with controls at each age tested and further reductions (<0.05) were observed in T(3) rats at Days 16 and 21. Testicular androstenedione production was also lower (P < 0.05) in T(3) rats at Days 5 and 7, but increased (P < 0.05) thereafter, than in control rats. These findings support that there are more newly formed ALCs in T(3) testes than in those of controls. Moreover, these results demonstrate that hyperthyroidism stimulates premature hypotrophy of FLCs and early differentiation of increased numbers of MCs to ALCs in the prepubertal rat testis, further supporting the view that thyroid hormone has a regulatory role in initiating MC differentiation into ALCs in the prepubertal rat testis.     

The effects of ethylene dimethane sulphonate (EDS) on rat Leydig cells: evidence to support a connective tissue origin of Leydig cells

Ethylene dimethane sulphonate (DS) administered to adult male rats in a single dose of 75 mg/kg body weight results in a rapid destruction of Leydig cells which, in turn, is associated with a marked decline in levels of serum testosterone. For 24-72 h after treatment with EDS (post-EDS) the Leydig cells undergo degenerative changes consisting of chromatin condensation and cytoplasmic vacuolation, and testicular macrophages progressively remove Leydig cells from the intertubular tissue by phagocytosis. This results in the total absence of Leydig cells on Days 7-14 and the absence of any detectable specific 125I-hCG binding to testis homogenates. Associated with the low levels of serum testosterone, levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum rise, LH to levels found in castrate rats. Morphometric and 125I-hCG binding studies indicate that a new generation of Leydig cells develop from Day 21 and reach control levels by Day 49. Morphologic observations suggest that the Leydig cells arise by differentiation from a pool of connective tissue cells that includes fibroblasts, lymphatic endothelial cells and pericytes. The new Leydig cells, which appear around Day 21 post-EDS, have the features of fetal Leydig cells. The latter appear to transform into Leydig cells typical of normal adult rats between 35-49 days post-EDS. The differentiation of new Leydig cells is associated with a reestablishment of normal levels of testosterone 21 days post-EDS. Serum LH and FSH return to normal at 28 days and 49 days respectively.     

Functional properties of developing rat Leydig cells after treatment with ethylene dimethanesulphonate (EDS)

The development of a new population of Leydig cells after specific elimination of existing Leydig cells in mature rats by ethylene dimethanesulphonate (EDS) was characterized by investigating the testicular activities of 5 alpha-reductase and non-specific esterase, the serum concentrations of 3 alpha-androstanediol and testosterone and the Leydig cell morphology. Plasma concentrations of both androgens were strongly reduced up to 15 days after administration of EDS. Thereafter, in contrast to the gradual and continuous increase of serum testosterone values, the changes in serum 3 alpha-androstanediol were transient, with the highest level on Day 35. The temporal pattern of testicular 5 alpha-reductase activity was almost similar to that of serum 3 alpha-androstanediol. The testicular esterase activity increased gradually from Day 25 until Day 76. The temporal changes in steroid concentrations and enzyme activities after EDS administration indicate that the development of the Leydig cells in EDS-treated rats occurs in a fashion similar to that in pubertal rats. However, the numerous lipid droplets and large nuclei in these Leydig cells indicate that these cells may also be classified as fetal cells. It is concluded that, after treatment with EDS, fetal and pubertal characteristics are present in Leydig cells. It is, however, unknown whether both characteristics are present in one or in two distinct cell populations.     

Stimulation of interstitial cell growth after selective destruction of foetal Leydig cells in the testis of postnatal rats

Summary Five-day-old male rats received a single treatment of ethane dimethanesulphonate (EDS), and the response of the testis on days 6–10 and 21 was examined by light microscopy and morphometry, supplemented by measurement of peripheral testosterone levels. One day after treatment, foetal Leydig cells degenerated, showing fragmentation, condensation and nuclear pyknosis. Macrophages phagocytosed the foetal Leydig cells resulting in their disappearance by day 7. Destruction of foetal Leydig cells was followed by an arrest of testicular growth in comparison to testes of intact age-matched control rats. In testes of EDS-treated rats, gonocytes and spermatogonia also degenerated, forming pyknotic bodies within the seminiferous cords. In contrast, interstitial fibroblasts and mesenchymal cells showed proliferative activity, which on days 4 and 5 after treatment resulted in peritubular hyperplasia surrounding each seminiferous cord. Thereafter, on day 21 after EDS administration, the previously depressed serum testosterone levels became markedly elevated coincident with the development of many immature-type Leydig cells, of which the total volume per testis was similar to that of Leydig cells in control testes, despite a four- to five-fold difference in testicular volumes. The results indicate that, although EDS destroys the foetal Leydig cells and impairs spermatogenesis, the interstitial tissue exhibits increased cell growth. The latter probably occurs in response to altered gonadotrophic stimulation and/or disturbances in the interaction between the seminiferous cords and the interstitial tissue.     

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.     

Regulation of androgen receptor mRNA and protein in the rat testis by testosterone.

Adult rats were treated with ethane dimethane sulphonate (EDS), an agent that destroys Leydig cells. Within 5 days after EDS treatment, the levels of testosterone (T) in the circulation and in the testis were decreased to very low values, which makes it possible to manipulate the testicular T concentration through administration of exogenous T. Spermatogenesis was not markedly affected within 5 days after EDS treatment, also not in the absence of T administration. In testes of EDS-treated rats, the androgen receptor mRNA (ARmRNA) level remained unaltered for 5 days. In ventral prostate, however, this treatment caused a pronounced upregulation of the level of ARmRNA, which could be counteracted by implantation of silastic T implants immediately after EDS treatment. In EDS-treated rats carrying a T implant and in untreated rats, the same number of specific [3H]R1881 binding sites was observed using a total testis nuclear fraction (Scatchard analysis). In testes from EDS-treated rats without T implants, androgen receptors (AR) did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals (measured by nuclear [3H]R1881 binding after receptor transformation through injection of a high dose of T, 2 h before killing the rats) remained unaltered. Immunoprecipitation and Western blotting using anti N-terminal antibodies seemed to indicate that the total testicular amount of AR protein in the EDS-treated rats was very low as compared to that in EDS-treated rats carrying T implants and in untreated rats. Even after receptor retransformation (by injection of a high dose of T) the receptors were not quantitatively detected by immunoprecipitation and Western blotting. This may point to a structural modification of the AR that occurs in the prolonged absence of androgens.     

Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity

Changes in the rat testis interstitium from birth to adulthood were studied using Sprague Dawley rats of 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Our objectives were 1) to understand the fate of the fetal Leydig cells (FLC) in the postnatal rat testis, 2) to determine the volume changes in testicular interstitial components and testicular steroidogenic capacity in vitro with age, 3) to differentially quantify FLC, adult Leydig cells (ALC), and different connective tissue cell types by number and average volume, and 4) to investigate the relationship between mesenchymal and ALC numbers during testicular development. FLC were present in rat testes from birth to 90 days, and they were the only steroidogenic cells in the testis interstitium at Days 1 and 7. Except for FLC, all other interstitial cell numbers and volumes increased from birth to 90 days. The average volume of an FLC and the absolute volume of FLC per testis were similar at all ages except at Day 21, when lower values were observed for both parameters. FLC number per testis remained constant from birth through 90 days. The observations suggested that the significance of FLC in the neonatal-prepubertal rat testis is to produce testosterone to activate the hypothalamo-hypophyseal-testicular axis for the continued development of the male reproductive system. ALC were the abundant Leydig cell type by number and absolute volume per testis from Day 14 onwards. The absolute numbers of ALC and mesenchymal cells per testis increased linearly from birth to 90 days, with a slope ratio of 2:1, respectively, indicating that the rate of production of Leydig cells is 2-fold greater than that of mesenchymal cells in the postnatal rat testis through 90 days. In addition, this study showed that the mesenchymal cells are an active cell population during testis development and that their numbers do not decrease but increase with Leydig cell differentiation and testicular growth up to sexual maturity (90 days).     

Identification of markers for precursor and leydig cell differentiation in the adult rat testis following ethane dimethyl sulphonate administration.

Administration of ethane dimethane sulphonate (EDS) to adult rats results in the destruction of all Leydig cells, followed by a complete regeneration. We investigated this regeneration process in more detail, using different markers for precursor and developing Leydig cells: the LH receptor, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), transforming growth factor alpha (TGFalpha), and a new marker for Leydig cell maturation, relaxin-like factor (RLF). LH receptor immunoreactivity was found in Leydig cell-depleted testes at 3 and 8 days after EDS administration. The positive (precursor) cells had a mesenchymal-like morphology. The number of LH receptor-positive cells 8 days after EDS administration was 15 +/- 4 per 500 Sertoli cell nuclei. Fifteen days after EDS administration, the first new Leydig cells could be observed. These cells stained positively with both the antibodies against the LH receptor and 3beta-HSD, while some cells also stained positively for TGFalpha. After EDS administration, RLF mRNA disappeared from the testis and reappeared again at the time of the appearance of the first Leydig cells. Concomitant with the increase in the number of Leydig cells, the number of RLF-expressing cells increased. The observations of the present study give further support to the hypothesis that Leydig cell development in the prepubertal testis, and in the adult testis following EDS administration, takes place along the same cell lineage and suggest, therefore, that the adult EDS-treated rat can serve as a model for studying the adult-type Leydig cell development that normally occurs in the prepubertal rat testis.     

Expression and regulation of lipocalin-type prostaglandin d synthase in rat testis and epididymis

Lipocalin-type prostaglandin D synthase (L-PGDS), a bifunctional protein, is expressed in the male reproductive organs of many species. However, the expression and regulation of L-PGDS in rat are still uncertain. The present study investigated the regionalization and regulation of L-PGDS expression in rat testis and epididymis by in situ hybridization and immunohistochemistry under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments. In sexually mature rats, L-PGDS mRNA was weakly expressed only in the testicular peritubular cells, whereas L-PGDS immunostaining was highly detected in the Leydig cells by Day 70 postpartum. During sexual maturation, L-PGDS mRNA expression was highly detected in the caput, corpus, and cauda of the epididymis 70 days after birth. Compared with normal L-PGDS expression in adult epididymis, both L-PGDS mRNA expression and protein immunostaining were significantly reduced in the caput, corpus, and cauda epididymis after castration. Testosterone propionate treatment induced a significant increase of L-PGDS expression in the epididymis of castrated rats. Compared with adult rat epididymis, L-PGDS mRNA and protein expression was down-regulated after EDS treatment. Testosterone propionate treatment could induce an increase of L-PGDS mRNA and protein expression in the epididymis of EDS-treated rats. In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.     

Rat testicular endogenous steroids and number of Leydig cells between the fetal period and sexual maturity

Endogenous androgens (androstenedione, testosterone, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol), and some of their C21 precursors (pregnenolone, progesterone and 17-hydroxyprogesterone) were measured in rat testes between Day 18.5 of pregnancy and Day 64 postpartum, and correlated with numerical densities of Leydig cells. The latter parameter showed an early maximum on Day 19.5 of the fetal period, a nadir on Day 15 postpartum, and a gradual increase thereafter. The two dominating androgens, testosterone and 5 alpha-androstane-3 alpha,17 beta-diol, had similar levels until 15 days of age, but the 5 alpha-diol predominated thereafter. The total steroid content per Leydig cell was highest on Day 18.5 of gestation (77 ng/10(6) cells). A decline started already in utero, and reached a nadir of 5 ng/10(6) cells on Day 29. Thereafter, a slight increase occurred with advancing age. It is concluded that: The fetal testis has highest Leydig cell and endogenous steroid concentrations. A nadir in these parameters is seen 2-4 wk postpartum. The Leydig cell concentration increases around puberty on Days 40-60, but only a slight concomitant increase occurs in steroids. A sharp decline in steroid content per Leydig cell occurs during the last fetal days, but the postnatal decline of testicular steroids is due to Leydig cell loss. The new Leydig cell generation after 15 days has a persistently low steroid concentration through puberty.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testicular blood flow and microcirculation in rats after treatment with ethane dimethyl sulfonate

The effects of ethane dimethyl sulfonate (EDS) on total testicular blood flow, microcirculation, and the testicular interstitial fluid volume (IFV) in rats were studied. In agreement with previous studies, treatment of control rats with human chorionic gonadotropin (hCG) induced an increase in IFV and total testicular blood flow as measured with radioactive microspheres. These effects of hCG were completely abolished in rats pretreated with EDS; in EDS-treated rats not receiving any hCG, there were decreases in IFV when compared with untreated control rats. Furthermore, the pulsatile pattern of testicular microcirculation registered with laser-Doppler flowmetry was abolished after EDS treatment, and this effect was not influenced by hCG treatment. The hCG-induced increase in IFV is associated with an increased accumulation of polymorphonuclear leukocytes locally in the testis, but this accumulation of leukocytes was not observed in rats pretreated with EDS. It was concluded from the present study that hCG-induced changes in total testicular blood flow and testicular microcirculation require functionally intact Leydig cells.     

Gene expression analysis of toxicological pathways in TM3 leydig cell lines treated with Ethane dimethanesulfonate

Ethane dimethanesulfonate (EDS), a well-known alkylating agent, selectively destroys Leydig cells. To clarify the molecular pathways underlying EDS action on Leydig cells, we analyzed gene expression profiles of an EDS-treated TM3 Leydig cell line. In this study, we analyzed the representative canonical pathways and toxicity pathways/gene lists using the Ingenuity Pathways Analysis program. In TM3 cells, 677 and 6756 genes were identified as being up- or downregulated after 3 and 24 h EDS treatments, respectively, (>1.3-fold changes, p < 0.05). Toxicological pathway analysis revealed that expression of genes related to Nrf2-mediated oxidative stress response showed remarkable changes in early or later stage of EDS-treated TM3 cells. Several genes related to steroidogenesis and apoptosis were also differentially expressed at 24 h in EDS-treated TM3 cells. Overall, toxicological pathway analysis using gene expression profiling showed that oxidative stress might be an important factor in cell death in TM3 cells affected by EDS treatment.     

Gestational exposure to ethane dimethanesulfonate permanently alters reproductive competence in the CD-1 mouse

Although the adult mouse Leydig cell (LC) has been considered refractory to cytotoxic destruction by ethane dimethanesulfonate (EDS), the potential consequences of exposure during reproductive development in this species are unknown. Herein pregnant CD-1 mice were treated with 160 mg/kg on Gestation Days 11-17, and reproductive development in male offspring was evaluated. Prenatal administration of EDS compromised fetal testosterone (T) levels, compared with controls. EDS-exposed pups recovered their steroidogenic capacities after birth because T production by hCG-stimulated testis parenchyma from prepubertal male offspring was unchanged. However, prepubertal testes from prenatally exposed males contained seminiferous tubules (STs) devoid of germ cells, indicating a delay in spermatogenesis. In adults, some STs in exposed males still contained incomplete germ cell associations corroborating observed reductions in epididymal sperm reserves, fertility ratios, and litter size. Morphometry revealed an EDS-induced increase in interstitial area and a concomitant decrease in ST area, but stereology revealed an unexpected decrease in the number and size of the LCs per testis in exposed males. Paradoxically, there was an increase in both serum LH and T production by adult testis parenchyma, indicating that the LCs were hyperstimulated. These data demonstrate permanent lesions in LC development and spermatogenesis caused by prenatal exposure in mice. Thus, although adult mouse LCs are insensitive to EDS, EDS appears to have direct action on fetal LCs, resulting in abnormal testis development.     

Leydig cell involvement in the paracrine regulation of mast cells in the testicular interstitium of the rat

The accumulation of mast cells in the rat testicular interstitium was studied under different experimental conditions in order to correlate this accumulation with the alterations of specific testicular tissue compartments or cell types. Estrogen treatment was effective in inducing mast cell proliferation when administered on Day 1 or at higher doses at 10 days of age. Estrogens were ineffective beyond 20 days of age. Postnatal treatment of neonatal-estrogen-treated rats with FSH and LH prevented the appearance of mast cells. In contrast, treatment with the Leydig cell cytotoxic ethylene dimethane sulphonate (EDS) was effective in inducing mast cell accumulation only when administered to adult rats, inducing small numbers of mast cells at 45 days of age; it was ineffective on 30-day-old rats. Hypophysectomy alone did not determine the appearance of mast cells. However, when atrophic Leydig cells were destroyed with EDS, high numbers of mast cells accumulated in the testis. These results support the existence of Leydig cell-related inhibitory factors for mast cells in the rat testicular interstitium.     

Thyroid hormone-induced haemoglobin changes and antioxidant enzymes response in erythrocytes

Thyroid hormones modulate haemoglobin and reactive oxygen species (ROS) production, leading to antioxidant changes. This study evaluated the antioxidant response to ROS in erythrocytes in hypothyroid and hyperthyroid rats. Wistar rats were divided into four groups: control; hyperthyroid (T4-12 mg 1(-1) in drinking water); sham operated (simulation of thyroidectomy); and hypothyroid (thyroidectomized). Four weeks after, blood was collected and haemoglobin and T(4) levels, lipid peroxidation (LPO), protein oxidation, superoxide dismutase (SOD), catalase (CAT) , glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities, and total radical antioxidant potential (TRAP) were measured. SOD, CAT and GST immunocontent was evaluated. Haemoglobin levels were increased in hyperthyroid erythrocytes. LPO and carbonyls were augmented (65% and 55%, respectively) in hyperthyroid and reduced (31% and 56%, respectively) in hypothyroid group. SOD and CAT activities have not changed, as well as CAT immunocontent. TRAP was diminished in both hyperthyroid and hypothyroid groups (36% and 37%, respectively). GST activity and immunocontent, as well as GPx activity, were increased in hyper and hypothyroid rats. The data suggest that thyroid hormone changes determine ROS concentration changes and decrease of some antioxidant defences that would lead to a compensatory answer of the GST and GPx enzymes, which could be consider as credible biomarkers.     

Influence of thyroid states on stress gastric ulcer formation

Thyroid hormones exert a critical developmental and regulatory role on the morphology and biochemistry of gastrointestinal mucosal cells. However, the relationship between thyroid function and stress gastric lesion formation remains undetermined. This study was designed to test the hypothesis that thyroid states may affect the acute development of gastric lesions induced by cold-restraint stress. Normal (euthyroid), hyperthyroid (200 micrograms of T4 i.p. x 7 days) and hypothyroid (thyroidectomized) rats were used. Gastric lesion incidence and severity was significantly (p less than 0.05) increased in hypothyroid rats, whereas in contrast hyperthyroid rats developed significantly less gastric lesions. As anticipated, plasma levels of thyroxin (T4) were significantly (p less than 0.01) elevated in hyperthyroid rats, and undetectable in hypothyroid rats. Acute pretreatment with i.p. cimetidine (100 mg/Kg), but not T4 (200 micrograms/Kg) 1 h prior to stress completely prevented gastric lesions formation in hypothyroid rats. Finally, binding of 3H-dihydroalprenolol to beta-adrenergic receptors on brain membranes prepared from frontal cortex was reduced by 20% in hypothyroid rats after 3 h of stress. These and other data contained herein suggest that thyroid hormones contribute to modulate the responsiveness of the gastric mucosa to stress. The increased rate of ulcerogenesis observed in hypothyroid rats appears to be mediated by gastric acid secretion. The central mechanism of this response may involve decreased brain nonadrenergic receptor function.