Ethane Dimethane Sulphonate Research Articles

Neurotrophin-3 stimulates stem Leydig cell proliferation during regeneration in rats.

Neurotrophin‐3 (NT‐3) acts as an important growth factor to stimulate and control tissue development. The NT‐3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT‐3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT‐3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT‐3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle‐stimulating hormone levels. NT‐3 increased CYP11A1‐positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT‐3 significantly down‐regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT‐3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT‐3 dose‐dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT‐3 antagonist Celitinib can antagonize NT‐3 action in vitro. In conclusion, the present study demonstrates that NT‐3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.

Disruption of androgen signaling during puberty affects Notch pathway in rat seminiferous epithelium

BackgroundOnset of spermatogenesis at puberty is critically dependent on the activity of hypothalamic-pituitary-gonadal axis and testosterone production by Leydig cells. The aim of this study was to examine whether activation of Notch receptors and expression of Notch ligands and effector genes in rat seminiferous epithelium are controlled by androgen signaling during puberty.MethodsPeripubertal (5-week-old) Wistar rats received injections of flutamide (50 mg/kg bw) daily for 7 days to reduce androgen receptor (AR) signaling or a single injection of ethanedimethane sulphonate (EDS; 75 mg/kg bw) to reduce testosterone production. Gene and protein expressions were analyzed by real-time RT-PCR and western blotting, respectively, protein distribution by immunohistochemistry, and steroid hormone concentrations by enzyme-linked immunosorbent assay. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test or by Kruskal-Wallis test, followed by Dunn’s test.ResultsIn both experimental models changes of a similar nature in the expression of Notch pathway components were found. Androgen deprivation caused the reduction of mRNA and protein expression of DLL4 ligand, activated forms of Notch1 and Notch2 receptors and HES1 and HEY1 effector genes (p < 0.05, p < 0.01, p < 0.001). In contrast, DLL1, JAG1 and HES5 expressions increased in seminiferous epithelium of both flutamide and EDS-treated rats (p < 0.05, p < 0.01, p < 0.001).ConclusionsAndrogens and androgen receptor signaling may be considered as factors regulating Notch pathway activity and the expression of Hes and Hey genes in rat seminiferous epithelium during pubertal development. Further studies should focus on functional significance of androgen-Notch signaling cross-talk in the initiation and maintenance of spermatogenesis.

Establishment of a stem Leydig cell line capable of 11-ketotestosterone production.

The deficiency or insufficiency of androgen can trigger a range of reproductive diseases as well as other symptoms. Stem Leydig cells (SLCs) are critical for the formation and maintenance of a functional androgen-producing cell (Leydig cell, LC) population throughout adult male life. However, to date, our knowledge about SLCs is poor. Here we report the derivation and characterisation of a clonal stem LC line (designated as TSL) capable of 11- ketotestosterone (11-KT) production from a 3-month-old Nile tilapia (Oreochromis niloticus) testis. The cells retained stable proliferation after 77 generations with normal karyotype and growth factor dependency. They expressed platelet-derived growth factor receptor-α (pdgfrα), nestin and chicken ovalbumin upstream promoter transcription factor II (coup-tfIIa), which are characteristic of SLCs. Upon induction in defined medium, TSLs could undergo differentiation into steroidogenically active LCs and produce 11-KT. When implanted into recipient Nile tilapia testes from which endogenous LCs had been eliminated by ethane dimethanesulphonate (EDS) treatment, the PKH26-labelled TSLs could colonise the interstitium, subsequently express steroidogenic genes and restore 11-KT production. Taken together, our data suggest that TSLs possess the ability of continuous proliferation and potential of differentiation into functional LCs invitro and invivo. To the best of our knowledge TSL might represent the first stem LC line capable of 11-KT production to date. Our study may offer new opportunities for investigating the self-renewal of SLCs and steroidogenesis invitro, and provide an invaluable invitro model for investigating endocrine disruptors.

Fibroblast growth factor homologous factor 1 stimulates Leydig cell regeneration from stem cells in male rats.

Fibroblast growth factor homologous factor 1 (FHF1) is an intracellular protein that does not bind to cell surface fibroblast growth factor receptor. Here, we report that FHF1 is abundantly present in Leydig cells with up‐regulation during its development. Adult male Sprague Dawley rats were intraperitoneally injected with 75 mg/kg ethane dimethane sulphonate (EDS) to ablate Leydig cells to initiate their regeneration. Then, rats daily received intratesticular injection of FHF1 (0, 10 and 100 ng/testis) from post‐EDS day 14 for 14 days. FHF1 increased serum testosterone levels without affecting the levels of luteinizing hormone and follicle‐stimulating hormone. FHF1 increased the cell number staining with HSD11B1, a biomarker for Leydig cells at the advanced stage, without affecting the cell number staining with CYP11A1, a biomarker for all Leydig cells. FHF1 did not affect PCNA‐labelling index in Leydig cells. FHF1 increased Leydig cell mRNA (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3, Nr5a1 and Hsd11b1) and their protein levels in vivo. FHF1 increased preadipocyte biomarker Dlk1 mRNA level and decreased fully differentiated adipocyte biomarker (Fabp4 and Lpl) mRNA and their protein levels. In conclusion, FHF1 promotes Leydig cell regeneration from stem cells while inhibiting the differentiation of preadipocyte/stem cells into adipocytes in EDS‐treated testis.

Fibroblast growth factor 16 stimulates proliferation but blocks differentiation of rat stem Leydig cells during regeneration.

ObjectivesWe aim to investigate the effects of fibroblast growth factor 16 (FGF16) on Leydig cell regeneration in ethane dimethane sulphonate (EDS)‐treated rat testis.MethodsWe intraperitoneally inject 75 mg/kg EDS to adult male Sprague Dawley rats and then intratesticularly inject FGF16 (0, 10 and 100 ng/testis/day) from post‐EDS day 14 for 14 days. We investigate serum hormone levels, Leydig cell number, gene and protein expression in vivo. We also explore the effects of FGF16 treatment on stem Leydig cell proliferation in vitro.ResultsFGF16 lowers serum testosterone levels (21.6% of the control at a dose of 100 ng/testis) without affecting the levels of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) on post‐EDS day 28 in vivo. FGF16 increases Leydig cell number at doses of 10 and 100 ng/mg without affecting Sertoli cell number, increases the percentage of PCNA‐positive Leydig cells, and down‐regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) and Sertoli cell genes (Fshr, Dhh and Sox9) and their proteins in vivo. FGF16 increases phosphorylation of AKT1 and AKT2 as well as EKR1/2 in vivo, indicating that it possibly acts via AKT1/ATK2 and ERK1/2 pathways. FGF16 also lowers medium testosterone levels and down‐regulates the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1 and Hsd17b3) but increases EdU incorporation into stem Leydig cells in vitro.ConclusionsThese data suggest that FGF16 stimulates stem and progenitor Leydig cell proliferation but blocks their differentiation, thus lowering testosterone biosynthesis.

Identification of Leydig cell-specific mRNA transcripts in the adult rat testis

The adult population of Leydig cells acts to secrete testosterone which is essential for reproductive health and fertility in the adult male. However, other physiological functions of these cells are uncertain, and to address this issue a cell ablation model has been used to identify Leydig cell-specific mRNA transcripts. Ethane dimethane sulphonate (EDS) was synthesised by a novel process and was used to ablate Leydig cells in adult male rats previously treated with butane dimethane sulphonate (busulphan) to delete the germ cell population. Levels of mRNA transcripts were measured in the testis using microarrays 1, 3, 5, 8 and 12 days after EDS injection. During this period, there was a significant change in the levels of 2200 different transcripts with a marked decline in the levels of canonical Leydig cell transcripts, such as Cyp11a1, Cyp17a1 and Insl3. A total of 95 transcripts showed a similar decline in expression after EDS treatment, suggesting that they have a Leydig cell-specific origin. Analysis of selected transcripts confirmed that they were expressed specifically in Leydig cells and showed that most had a late onset of expression during adult Leydig cell development. Apart from transcripts encoding components of the steroidogenic apparatus, the most common predicted function of translated proteins was endogenous and xenotoxicant metabolism. In addition, a number of transcripts encode acute-phase proteins involved in reduction of oxidative stress. Results show that, in addition to androgen secretion, Leydig cells may have a critical role to play in protecting the testis from damage caused by toxicants or stress.

The endocrine disruptors dibutyl phthalate (DBP) and diethylstilbestrol (DES) influence Leydig cell regeneration following ethane dimethane sulphonate treatment of adult male rats

The manner by which endocrine-disrupting xenobiotics, such as phthalates, can induce changes in the development of the male reproductive system still remains largely unknown. Herein, we have explored the application of ethane dimethane sulphonate (EDS) to eliminate adult-type Leydig cells in the mature rat testis, leading to their regeneration from resident stem cells, as a novel system to investigate the effects of dibutyl phthalate (DBP) and diethylstilbestrol (DES) on adult-type Leydig cell differentiation. The advantage of this model is that one can study adult-type Leydig cell differentiation in vivo divorced from the concomitant endocrine development of puberty. In these preliminary studies, we show that both DBP and/or DES, given for 2 or 4 days following EDS application, indeed affect Leydig cell differentiation in the adult testis, largely by increasing early Leydig cell proliferation and possibly thereby delaying early differentiation. In particular, on day 27 post-EDS, a time-point when the differentiation trajectory appears to be most discriminating, we observe that both DBP and/or DES cause a fourfold increase in Leydig cell density, and a significant increase in the expression of the Leydig cell-specific marker transcripts INSL3, LH receptor, Cyp17a1 and Cyp 11a1. In conclusion, both DBP and DES are able to affect adult-type Leydig cells during their differentiation to cause a significant perturbation in their ultimate functional capacity.

Distinct ageing- and androgen-dependent effects on cGMP signalling proteins in three male rat urogenital organs (bladder, prostate, epididymis)

Background Aging of the male reproductive system leads to changes in endocrine signalling and is frequently associated with the emergence of bothersome conditions owing to prostate hyperplasia and bladder dysfunctions. Recent findings suggest that urogenital organs are promising targets for therapeutic interventions with inhibitors of the cGMPdegrading phosphodiesterase 5 (PDE5) [1]. However, the cGMP signalling system in these tissues and possible agerelated alterations are only partially characterized. This study investigated key proteins of cGMP pathways in bladder, prostate and epididymis of young (3 months) and old (23-24 months) Wistar rats. To address androgen-dependent effects, we (i) used an experimental model, where injection of ethane dimethane sulphonate (EDS) into adult rats evokes a temporary elimination of the testosterone-producing Leydig cells [2] and (ii) examined effects induced by castration and testosterone supplementation.

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

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. We performed immunohistochemical analyses of adrenal gland sections of ethane dimethanesulphonate (EDS)-treated adult rats. 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. 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.

Response of testicular macrophages to EDS-induced Leydig cell death.

The response of testicular macrophages to massive Leydig cell death was studied by the administration of the specific Leydig cell cytotoxic ethylene dimethane sulphonate (EDS) to sham-operated (SO), short-term (STHX), and long-term (LTHX) hypophysectomized rats. EDS-killed Leydig cells showed the morphological features of the programmed cell death or apoptosis. A 2-fold increase in the number of macrophages was found on days 1-2 after treatment in both SO and STHX rats, and dead Leydig cells were completely eliminated by day 3 after treatment. Otherwise, in LTHX rats, there was a delay in the increase in the number of macrophages, and EDS-killed Leydig cells remained in the testicular interstitium for several days. These results indicate that the phagocytic capacity of the macrophage population was diminished in hypophysectomized rats, and particularly after long-term hypophysectomy.

Gonadotrophin Levels in Male Rats Following Impairment of Leydig Cell Function by Ethylene Dimethanesulphonate

The effects of the antispermatogenic compound, ethylene dimethanesulphonate on anterior pituitary and serum FSH, LH and prolactin levels in male rats were investigated. A rapid and sustained rise in serum LH, reaching castrate levels, indictated a normal pituitary response to androgen deprivation. Whilst no change in prolactin was observed, serum FSH showed a biphasic increase which is discussed in relation to the spermatogenic process.

Superoxide dismutase activity along rat seminiferous epithelial wave: effects of ethane dimethanesulphonate and 3.0 Gy of X-irradiation.

Changes in generation of reactive oxygen species and antioxidant enzyme activities are associated with differentiation processes. The authors have studied the activity of superoxide dismutase (SOD) in sequentially cut stage-defined segments of rat seminiferous tubules. Great variation was observed in SOD activity along the seminiferous epithelial wave. At its highest, four-fold increases were observed in individual tubules. However, these changes showed no clear correlation to the stages of the cycle. To determine the effect of testosterone withdrawal, rats treated with ethane dimethanesulphonate (EDS) were studied. This treatment had no effect on the pattern of SOD activity along the seminiferous epithelial wave. Testes of other rats were exposed to local 3.0 Gy X-irradiation to cause selective loss of germ-cell populations. SOD activity in the seminiferous epithelium was not affected at 30 min or 7 d after X-irradiation. On day 31 post-irradiation, SOD activity increased at stages XIV-VI, peaking at stage III (P < 0.01 for comparison of stages XIV-VI with the other stages). The data presented here suggest that the activity of SOD in seminiferous epithelium is regulated over a wide range during spermatogenesis. Testosterone plays no major role in the control of seminiferous tubule SOD activity. The loss of spermatocytes and early spermatids by day 31 after X-irradiation revealed a stage-specific increase in SOD activity, which may be associated with the differentiation of elongated spermatids.

Connexin 43 expression in the testis of the frogRana esculenta

Testicular cell-to-cell interactions play a key role in the regulation of spermatogenesis. In the testis, cell contacts are mediated through several mechanisms, including paracrine and direct contacts depending on gap junctional pathways. Gap junctions require connexin (Cx) channels and connexin-43 (Cx43) represent the most abundant Cx found in mammalian testis. Little is known about Cx expression in non-mammalian testis. Here we report the partial cloning of a Cx43 transcript of 381 bp from Rana esculenta testis. We also demonstrate that, in the frog testis, Cx43 transcript and protein show a parallel temporal and spatial pattern of expression throughout the reproductive annual cycle, with higher levels from September to January (when spermatogenesis is at a maximum level). In situ hybridization, carried out on testis collected in October, indicated that Leydig cells (LC) and Sertoli cells express Cx43 transcript, while the hybridization signal was less intense in germ cells. To obtain more information on Cx43 expression in the frog testis, we have used ethane-dimethane sulphonate (EDS), a toxin that specifically destroys LC. RT-PCR analysis shows a progressive decrease in Cx43 expression in EDS-treated testis from day 1 to day 4 after the injection, associated with LC destruction. Moreover, Cx43 expression returns to normal on day 28, when a new population of LC reappear in the interstitium, indicating that Cx43 is mainly expressed by LC. Taken together our data provide evidence that Cx43 is present in the frog testis with an important role in spermatogenesis.

Single Subcutaneous Administration of Chorionic Gonadotropin to Rats Induces a Rapid and Transient Increase in Testicular Expression of Pro-Inflammatory Cytokines

hCG has been reported to cause an inflammation-like effect in the testis, although the background and consequences of this phenomenon remain to be understood. This investigation reveals that a single injection of hCG (100 U) induces a transient surge in pro-inflammatory cytokine expression in the adult rat testis. Reverse transcriptase PCR analysis demonstrated onset of testicular expression of IL-1beta and IL-6 mRNA and increases in the levels of mRNA encoding the constitutively expressed cytokines IL-1alpha, IL-1 receptor antagonist, and tumor necrosis factor-alpha 4 h after hCG injection and a maximal response after 8-12 h. These increases were accompanied by a transient increase in testicular IL-1 bioactive protein. Twenty-four hours after administration of hCG, the levels of all cytokine mRNA had decreased, although most were still elevated above control. Immunohistochemical staining revealed that the IL-1beta protein was undetectable in normal testes but was seen to be localized to interstitial macrophages but not Leydig cells after hCG treatment. Testes devoid of Leydig cells after pretreatment with ethane dimethane sulphonate exhibited normal staining for interstitial macrophages but failed to respond to hCG with increases in IL-1beta mRNA and protein expression. We conclude that hCG induces testicular inflammation via local activation by Leydig cells of the production of pro-inflammatory cytokines by resident macrophages. It remains to be investigated whether the high-dose hCG regimens used for treatment of boys with cryptorchidism could induce similar increases of pro-inflammatory cytokines in the human testis and if such treatments could adversely affect future testicular function.

Morphometric changes in the adrenal medulla of intact and ethane dimethanesulphonate (EDS)-injected rats subjected to chronic treatment with isoproterenol or propranolol

Morphometric characteristics of adrenal medulla were analysed stereologically in adult male rats injected with a single dose of ethane dimethanesulphonate (EDS), an agent that causes atrophy of the inner adrenocortical zone, or vehicle, and subjected to isoproterenol (ISO) or propranolol (PROP) treatment over the following 15 days. Compared with dimethylsulphoxide (DMSO) vehicle-injected controls, in EDS-administered rats the volume of chromaffin cell nuclei was decreased. ISO treatment in these rats increased the volumes of chromaffin cells and their nuclei. Furthermore, in both EDS- and vehicle-injected rats ISO significantly enlarged the total volume of medullary blood vessels, suggesting a vasodilatatory effect of β -adrenoceptor stimulation. However, unexpectedly, in EDS-injected rats PROP treatment also caused an increase in the volumes of chromaffin cells and their nuclei. This finding, most likely, may be related to a non- β -adrenoceptor-related action of PROP. Collectively, the present results suggest that the response of adrenomedullary chromaffin cells to chronic ISO-induced β -adrenoceptor stimulation is dependent on the functional status of adrenal cortex, so that the stereologically detectable changes were found only in rats previously exposed to the destructive action of EDS on the adrenal gland cortical cells. Moreover, they indicate that ISO treatment exerts a reversing effect on the morphometrical changes of chromaffine cells induced by EDS administration.

Testicular Activity of Mos in the Frog, Rana esculenta: A New Role in Spermatogonial Proliferation1

Mos is a MAPK kinase kinase with an expression that is highly restricted to the gonads. Its function is mainly associated to the meiotic metaphase II arrest occurring during female gametogenesis, whereas to our knowledge, its role during spermatogenesis has not yet clarified. In the present paper, we report the isolation of c-mos cDNA and the identification of a 60-kDa Mos protein from the testis of the anuran amphibian, Rana esculenta. Both the transcript and the protein are always present at low levels in the testis during the frog annual sexual cycle, with single significant peaks of expression in March and May, respectively. Mos is mainly localized in the cytoplasm of primary and secondary spermatogonia (SPG). Therefore, we have used treatments with ethane-dimethane sulphonate (EDS), which blocks spermatogonial mitosis in frogs. Four days after a single EDS injection, Mos expression in SPG highly increases concomitantly with the temporary arrest of mitosis. From 8 to 28 days after the injection, the normal proliferative activity of SPG is restored, and Mos expression gradually decreases to control levels. These results strongly indicate that the c-mos proto-oncogene exerts a new role associated to the regulation of spermatogonial proliferation.

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.

Expression of fractalkine in the rat testis: molecular cloning of a novel alternative transcript of its gene that is differentially regulated by pro-inflammatory cytokines.

The testis is a very complex organ in which cellular communications and interactions are central to spermatogenesis and where inflammatory processes can lead to sterility. Fractalkine (CX3CL1) is a chemokine involved in cell-cell interactions and in leukocyte chemoattraction. It has been reported to be expressed in testis, but its cellular expression and function in this organ has not been described. In this study we report constitutive expression of fractalkine in the testis. Expression is higher in Leydig cells than Sertoli cells, spermatogonia, pachytene spermatocytes and elongated spermatids. In both, Sertoli cells stimulated by interleukin-1beta and tumour necrosis factor alpha, and in Leydig cells, two forms of fractalkine mRNA were observed: the previously described transcript of 3.7 kb and a novel transcript of 4.2 kb. The 4.2 kb transcript has a 5' elongation and is differentially regulated. To investigate fractalkine function in testis, we abolished Leydig cell expression of fractalkine by specific destruction of this cell type using ethylene dimethane sulphonate. The absence of fractalkine expression in Leydig cells did not seem to affect the fractalkine expression by other testicular cells. In addition, the destruction of testicular macrophages by Cl2MDP (chlodronate) did not seem to affect Leydig cell expression of fractalkine. We conclude that Leydig cell expression of fractalkine could be preferentially involved in inflammation in interstitial space whereas fractalkine expressed by germ cells may participate in the cellular interactions between germ cells and other seminiferous tubule cell types.

Ethane 1,2-dimethane sulphonate is a useful tool for studying cell-to-cell interactions in the testis of the frog, Rana esculenta

Ethane 1,2-dimethane sulphonate (EDS), a toxin which specifically destroys Leydig cells (LC), has been used to study cellular interactions in the testis of the frog Rana esculenta. Animals received three consecutive EDS injections and were sacrificed on day 4, 8, and 28 from the first injection. No significant morphological differences were observed between present observation and that obtained, in a previous experiment, after four consecutive EDS injections. In fact, on day 4, in the germinal tubules adjacent to apparently normal LC, Sertoli cells surrounding primary spermatogonia (I SPG) show heterochromatic nuclei and loss of cellular adhesion. Interestingly, I SPG surrounded by the heterochromatic Sertoli cells present grossly swollen mitochondria with ballooned cristae. On day 8, sometimes in the interstitium many LC appear strongly damaged and the germinal tubules appear disorganized; the only cell type still distinguishable is the I SPG. On day 28 from the first EDS injection a new population of LC reappear in the interstitium and spermatogenesis normalizes. These data confirm the close relationship between the interstitial and the geminal compartments. Immunocytochemical data obtained using a polyclonal antibody anticonnexin-43 (Cx-43, the most abundant Cx found in mammalian testis) demonstrate the presence of Cx-43 in the frog testis. In particular, Cx-43 is present between LC in the interstitium, between Sertoli and germ cells in the cysts and between Sertoli cells and I SPG. Cx-43 immunopositivity sharply decreases on day 4 from the first EDS injection simultaneously with the loss of cellular adhesion between Sertoli and germ cells. On day 8 and 28 from the first EDS injection Cx-43, immunopositivity is restored and, this data is also supported by Western blot analysis. Our data provide, for the first time, evidence that Cx-43 protein is present in the frog testis and confirm that EDS is a useful tool for studying cellular communication at the paracrine pathway or through direct contact depending on the gap junctional pathway in R. esculenta testis

The effects of acth, isoproterenol and Dexamethasone on the rat adrenal gland response to ethane dimethanesulphonate (Eds): A stereological study

Ethane dimethanesulphonate (EDS), an alkylating agent, caused marked atrophy of the adrenal cortex of adult male rats, in addition to its toxic effect on testicular Leydig cells. The aim of this work was to examine whether a 9-day treatment with ACTH (40 IU/kg/d), isoproterenol (120 mg/kg/d) or dexamethasone (0.25 mg/kg/d), which started 4 days prior to intraperitoneal administration of a single dose of EDS (75 mg/kg), affected the morphological changes in the adrenal cortex evoked by EDS alone. The animals were killed 15 days after EDS injection. Stereological analysis revealed that both ACTH and isoproterenol almost completely prevented cortical atrophy induced by EDS. They also considerably stimulated corticosterone secretion in EDS-injected animals. By contrast, in dexamethasone-suppressed rats, the deleterious effect of EDS on adrenocortical cells was augmented. The volume and cellularity of all cortical zones were reduced, but the remaining cells of the zona reticularis displayed considerable hypertrophy which was probably responsible for the maintenance of corticosterone secretion. These results clearly demonstrate that both ACTH and b adrenoceptor stimulation have protective action against the toxic effects of EDS on rat adrenal cortex, whereas dexamethasone exerts an opposite influence.