Ethane Dimethane Sulphonate Research Articles

Replacement of Surgical Castration by GnRH-Inhibition or Leydig Cell Ablation in the Male Rat Hershberger Antiandrogen Assay

An obstacle to the widespread adoption of the Hershberger antiandrogen assay is the surgical castration procedure required to produce androgen deficiency in the test animals. Here we describe two chemical treatments that produce similar effects to surgical castration. The first is use of ethane dimethane sulphonate (EDS), a specific toxin to the testosterone-producing Leydig cells of the mature testes. The second class of compound is the decapeptide inhibitors of the gonadotrophin-releasing hormone (GnRH), compounds such as Antarelix and Antide. Administration of either EDS or the GnRH inhibitors results in loss of weight of the testes, epididymides, and sex-associated tissues. Co-administration of testosterone to these animals leads to reversal of the induced effects. The basic test protocol for both of these assay modifications is described. Flutamide was used as a representative potent antiandrogen, and DDE as an example of a weakly active antiandrogen. The 5α-reductase inhibitor finasteride was used to inhibit the transformation of testosterone to dihydrotestosterone. It is shown that the EDS assay is sensitive to the antiandrogen flutamide, but that it fails to detect the weaker antiandrogen DDE. In contrast, the Antarelix assay performs as well as does the classical castration assay, leading to the detection as antiandrogens of flutamide, DDE, and finasteride. It is concluded that the GnRH inhibition Hershberger assay is more convenient to conduct than the original surgical castration assay, and it involves less stress to the test animals.

Expression of Amphiphysin I in Sertoli Cells and Its Implication in Spermatogenesis

Amphiphysin I is a protein concentrated in nerve terminals and involved in the endocytosis of synaptic vesicle membrane. We show here that amphiphysin I is expressed in the rat testis, localized exclusively in the Sertoli cells. In the postnatal testicular development, expression of amphiphysin I was not evident at birth, but became significant at postnatal day 15 (P15), coinciding with the onset of spermatogenesis. The expression level of amphiphysin I increased 10-fold between P15 and P25 to reach the adult level. In adult testes reversibly damaged by ethane dimethane sulphonate administration, expression of amphiphysin I did not change following the damage, whereas the protein was transiently converted into its phosphorylated form. The increase in levels of phosphorylated amphiphysin I was closely associated with the severe histological damage to germ cells. The present findings suggest that amphiphysin I in Sertoli cells is involved in spermatogenesis, probably through endocytic processes.

Developmental and hormonal regulation of type II DNA topoisomerase in rat testis.

Type II DNA topoisomerase (topo II) is required for diverse biological functions including DNA replication, maintenance of genome stability, chromosome segregation and chromosome condensation. While the identity of topo II in rodent testis has been established, the regulation of topo II expression during the development of the postnatal testis and gametogenesis is unclear. Here, we report that rat testis topo II is developmentally and hormonally regulated. Topo IIalpha mRNA levels peaked prior to the onset of puberty, declined sharply thereafter and stabilized in adult testis. In contrast, the topo II enzyme content was lower in prepubertal testis but increased after the onset of puberty. Topo II was expressed in a cell-specific manner within germ cells, being detected only in pachytene spermatocytes. While testosterone markedly increased topo IIalpha mRNA levels in prepubertal testis, continued treatment failed to enhance topo IIalpha mRNA above postpubertal control levels. The extent of topo II activity remained steady regardless of the testosterone-induced increase in topo IIalpha mRNA levels. Inhibition of testosterone function in postpubertal animals by ethanedimethane sulphonate (EDS) and flutamide resulted in a significant decrease in topo IIalpha gene expression and topo II activity. The administration of exogenous testosterone (T) to EDS- and flutamide-treated rats restored topo IIalpha mRNA levels and topo II activity similar to the levels seen in the testis of age-matched control animals. Histochemical analyses of testes indicated that the effect of T on spermatogenesis was separable from its effect on topo IIalpha expression. Our results reveal that testosterone acts as a positive regulator of topo IIalpha gene expression and is required for the maintenance of topo IIalpha expression during the development of the postnatal testis and spermatogenesis.

Apoptosis related gene products in differentiated and tumorigenic rat Leydig cells and following regression induced by the cytotoxin ethane dimethanesulphonate.

Androgen secreting Leydig cells in the adult are differentiated with a very low turnover, however, Leydig cell tumours can arise spontaneously or after treatment with toxins. This study in the rat investigated whether changes in components of programmed cell death could be involved. In contrast to their absence in differentiated Leydig cells, antiapoptotic Bcl-2 and proapoptotic Bax were expressed in tumours. Bak and Bcl-xl were found in both tumour and normal Leydig cells. Apoptosis was induced in subcutaneous implants of Leydig cell tumour by ethane dimethanesulphonate (EDS) which is known to kill differentiated Leydig cells. The marked regression of the tumour following EDS treatment was transient and re-growth occurred between 6 and 14 days later. Tumour regression and growth was associated with a similar weight pattern in the seminal vesicles caused by changes in serum testosterone. During tumour regression, clusterin and Bax proteins were elevated but Bak, Bcl-xl and Bcl-2 were unchanged. Fas-R, Fas-L and Bax were upregulated after tumour regression had taken place. These data show that Leydig cell tumours possess many of the apoptosis related gene products and can die by apoptosis, however, regulation is clearly different in differentiated and mitotic Leydig cells.

Opposite effects of dexamethasone and ACTHon the adrenal cortex response to ethane dimethanesulphonate (EDS)

Recently we have reported that ethane dimethane-sulphonate (EDS), the Leydig cell cytotoxin, caused marked atrophy of the adrenal cortex of adult male rats. The aim of this work was to examine whether a 9-day treatment with dexamethasone (0.25 mg/kg/d) or ACTH (40 IU/kg/d), which started 4 days prior to administration of a single dose of EDS (75 mg/kg), influenced the response of the inner adrenocortical zones to the toxin. On day 15 after administration of EDS, adrenal weight was significantly decreased in saline treated rats, but glandular and serum corticosterone levels were not altered. In dexamethasone-suppressed rats, the effect of EDS was augmented; an additional decrease in adrenal weight was accompanied by reduced adrenal and serum corticosterone levels. In ACTH-treated animals EDS was ineffective. These results demonstrate that the deleterious effects of EDS on rat adrenal cortex can be prevented by ACTH and potentiated by dexamethasone.

Effects of thyroid hormone on Leydig cell regeneration in the adult rat following ethane dimethane sulphonate treatment.

We tested the effects of thyroid hormone on Leydig cell (LC) regeneration in the adult rat testis after ethane dimethyl sulphonate (EDS) treatment. Ninety-day-old, thyroid-intact (n = 96) and thyroidectomized (n = 5) male Sprague-Dawley rats were injected intraperitoneally (single injection) with EDS (75 mg/kg) to destroy LC. Thyroid-intact, EDS-treated rats were equally divided into three groups (n = 32 per group) and treated as follows: control (saline-injected), hypothyroid (provided 0.1% propyl thiouracil in drinking water), and hyperthyroid (received daily subcutaneous injections of tri-iodothyronine, 100 microg/kg). Testing was done at Days 2, 7, 14, and 21 for thyroid-intact rats and at Day 21 for thyroidectomized rats after the EDS treatment. Leydig cells were absent in control and hyperthyroid rats at Days 2, 7, and 14; in hypothyroid rats at all ages; and in thyroidectomized rats at Day 21. The LC number per testis in hyperthyroid rats was twice as those of controls at Day 21. 3beta-Hydroxysteroid dehydrogenase (LC marker) immunocytochemistry results agreed with these findings. Mesenchymal cell number per testis was similar in the three treatment groups of thyroid-intact rats on Days 2 and 7, but it was different on Days 14 and 21. The highest number was in the hypothyroid rats, and the lowest was in the hyperthyroid rats. Serum testosterone levels could be measured in control rats only on Day 21, were undetectable in hypothyroid rats at all stages, and were detected in hyperthyroid rats on Days 14 and 21. These levels in hyperthyroid rats were twofold greater than those of controls on Day 21. Serum androstenedione levels could be measured only in the hyperthyroid rats on Day 21. Testosterone and androstenedione levels in the incubation media showed similar patterns to those in serum, but with larger values. These findings indicate that hypothyroidism inhibits LC regeneration and hyperthyroidism results in accelerated differentiation of more mesenchymal cells into LC following the EDS treatment. The observations of the EDS-treated, thyroidectomized rats confirmed that the findings in hypothyroid rats were, indeed, due to the deficiency of thyroid hormone.

Effects of finasteride and bicalutamide on prostatic blood flow in the rat.

To determine whether finasteride and bicalutamide, both currently used in the clinical management of patients with prostate diseases because they have anti-androgenic properties, have any effects on prostatic blood flow in a rat prostate model, as androgens are known to be involved in the regulation of prostatic blood flow and angiogenesis. Both finasteride and bicalutamide were supplied as oral suspensions in water and given daily to rats for 7 days by tube feeding. Blood flows to the ventral and dorsal prostates, and to the kidneys, were measured using the radioactive microsphere technique. In the bicalutamide experiments, some rats were treated with the Leydig cell toxin ethane dimethane sulphonate (EDS), to obtain a castration-like effect, and one group of these rats received testosterone. Finasteride induced a clear decrease in blood flow to the ventral and dorsal prostates after 7 days of treatment, with no significant changes in blood pressure or kidney blood flow. Bicalutamide inhibited the testosterone-induced increment of prostatic blood flow observed in EDS-treated animals. Finasteride, a blocker of 5alpha-reductase, decreases prostate blood flow after 7 days of administration. The response was slower than that after castration, but was of similar magnitude. Blood flow was also decreased after treatment with the androgen-receptor inhibitor bicalutamide. These observations suggest that prostatic blood flow is increased by dihydrotestosterone, and that the androgen receptor is responsible for mediating this effect.

Effects of multiple injections of ethane 1,2-dimethane sulphonate (EDS) on the frog,Rana esculenta, testicular activity

Ethane 1,2-dimethane sulphonate (EDS) is an alkylating agent, which has a selective cytotoxic effect on Leydig cells in some mammalian species. Similarly, in the frog, Rana esculenta, Leydig cells are destroyed after a single EDS injection and regenerate after 28 days. Regeneration of Leydig cells in frogs appears to be independent of the pituitary. The present experiments in R. esculenta were carried out: a) to investigate Leydig cell responsiveness to gonadotropin stimulation during 58 days after a single EDS injection; and b) to assess whether four consecutive EDS injections induce additional effects on the testicular cell population. Our results show that androgen stimulation after gonadotropin injections is restored after 44 days from a single EDS injection. Since the interstitial compartment appears to be normal at least 28 days after EDS treatment, it is likely that new Leydig cells lack gonadotropin receptors. With respect to multiple-EDS injections, Leydig cells completely disappear in several areas and the adjacent germinal compartment is disorganised. In some cases damaged germinal compartment is still surrounded by intact Leydig cells. Surprisingly, testicular and plasma androgens strongly increase in EDS-treated animals. Therefore, Sertoli cells may produce substances inhibiting androgen production in Leydig cells. J. Exp. Zool. 287:384-393, 2000.

Mitosis of resident macrophages in the adult rat testis.

Resident macrophages are maintained at a comparatively high, yet stable, tissue concentration in the adult rat testis. After destruction of Leydig cells by ethane dimethane sulphonate treatment, the number of resident macrophages increases briefly and then decreases to below normal values, but returns to normal after the reappearance of Leydig cells. The mechanisms by which the adult testicular macrophage population is maintained, either by monocyte recruitment or by mitosis of the resident macrophages, have not been examined. An immunohistochemical dual labelling approach using a specific monoclonal antibody for resident macrophages, ED2, and markers of mitotic activity (bromodeoxyuridine incorporation and expression of the proliferating cell nuclear antigen) was used to investigate resident macrophage proliferation in Bouin's-fixed paraffin wax-embedded adult rat testes. Detection of the normally fixation sensitive antigen recognized by ED2 was achieved by using a decreased fixation time and antigen retrieval. Peaks of resident macrophage mitotic activity were observed during the phases of macrophage proliferation immediately after ethane dimethane sulphonate treatment and during the recovery phase associated with Leydig cell restoration. These data demonstrate that resident macrophages have the capacity to proliferate within the adult rat testis and, thus, this population of resident macrophages is maintained, at least in part, by mitotic division in situ.

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.

Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat.

Antiandrogenic chemicals alter sexual differentiation by a variety of mechanisms, and as a consequence, they induce different profiles of effects. For example, in utero treatment with the androgen receptor (AR) antagonist, flutamide, produces ventral prostate agenesis and testicular nondescent, while in contrast, finasteride, an inhibitor of 5 alpha-dihydrotestosterone (DHT) synthesis, rarely, if ever, induces such malformations. In this regard, it was recently proposed that dibutyl phthalate (DBP) alters reproductive development by a different mechanism of action than flutamide or vinclozolin (V), which are AR antagonists, because the male offsprings display an unusually high incidence of testicular and epididymal alterations--effects rarely seen after in utero flutamide or V treatment. In this study, we present original data describing the reproductive effects of 10 known or suspected anti-androgens, including a Leydig cell toxicant ethane dimethane sulphonate (EDS, 50 mg kg-1 day-1), linuron (L, 100 mg kg-1 day-1), p,p'-DDE (100 mg kg-1 day-1), ketoconazole (12-50 mg kg-1 day-1), procymidone (P, 100 mg kg-1 day-1), chlozolinate (100 mg kg-1 day-1), iprodione (100 mg kg-1 day-1), DBP (500 mg kg-1 day-1), diethylhexyl phthalate (DEHP, 750 mg kg-1 day-1), and polychlorinated biphenyl (PCB) congener no. 169 (single dose of 1.8 mg kg-1). Our analysis indicates that the chemicals discussed here can be clustered into three or four separate groups, based on the resulting profiles of reproductive effects. Vinclozolin, P, and DDE, known AR ligands, produce similar profiles of toxicity. However, p,p'-DDE is less potent in this regard. DBP and DEHP produce a profile distinct from the above AR ligands. Male offsprings display a higher incidence of epididymal and testicular lesions than generally seen with flutamide, P, or V even at high dosage levels. Linuron treatment induced a level of external effects consistent with its low affinity for AR [reduced anogenital distance (AGD), retained nipples, and a low incidence of hypospadias]. However, L treatment also induced an unanticipated degree of malformed epididymides and testis atrophy. In fact, the profile of effects induced by L was similar to that seen with DBP. These results suggest that L may display several mechanisms of endocrine toxicity, one of which involves AR binding. Chlozolinate and iprodione did not produce any signs of maternal or fetal endocrine toxicity at 100 mg kg-1 day-1. EDS produced severe maternal toxicity and a 45% reduction in size at birth, which resulted in the death of all neonates by 5 days of age. However, EDS only reduced AGD in male pups by 15%. Ketoconazole did not demasculinize or feminize males but rather displayed anti-hormonal activities, apparently by inhibiting ovarian hormone synthesis, which resulted in delayed delivery and whole litter loss. In summary, the above in vivo data suggest that the chemicals we studied alter male sexual differentiation via different mechanisms. The anti-androgens V, P, and p,p'-DDE produce flutamide-like profiles that are distinct from those seen with DBP, DEHP, and L. The effects of PCB 169 bear little resemblance to those of any known anti-androgen. Only in depth in vitro studies will reveal the degree to which one can rely upon in vivo studies, like those presented here, to predict the cellular and molecular mechanisms of developmental toxicity.

Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p′-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat

Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p′-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat

Leydig cell apoptosis in the rat testes after administration of the cytotoxin ethane dimethanesulphonate: role of the Bcl-2 family members.

Ethane dimethanesulphonate (EDS) is cytotoxic to Leydig cells in the adult rat. To investigate the role and regulation of apoptosis in the Leydig cell, EDS (100 mg/kg i.p.) was administered to adult male rats and the testes examined 6, 12, 18, 24, 48 and 72 h later. Numbers of Leydig cells, identified by 3 beta-hydroxysteroid dehydrogenase immuno-histochemistry started to fall by 12 h after EDS injection and were almost undetectable by 72 h. Apoptotic cells in the interstitium, visualised by in situ end labelling of DNA, increased in number to reach a maximum 24 h after injection of EDS, and were undetectable by 72 h. In many tissues the apoptosis-related gene products act in cohort: Bcl-2 and Bcl-xl promoting survival of a cell, whilst Bax promotes cell death often positively regulated by the tumour-suppressor gene p53. Western blot analysis showed that: (1) Bcl-2 and p53 were absent from interstitial Leydig cells but were expressed in the seminiferous tubules. (2) Bax protein although expressed in the interstitium was not present in the Leydig cells. (3) Bcl-xl in Leydig cells was transiently increased after EDS. In conclusion, EDS kills Leydig cells by apoptosis; however the control of Leydig cell death does not involve p53 or the Bcl-2 family members but may require other gene products yet to be identified.

Role of the testis in the response of the pituitary-testicular axis to nitric oxide-related agents.

Nitric oxide (NO) is generated from the guanidine group of L-arginine by NO synthases (NOS) in a wide variety of tissues, including endocrine organs. In order to discriminate between central and local effects of NO-related agents on the pituitary-testicular axis, adult rats were injected intraperitoneally with 1 g/kg body weight (bw) L-arginine methyl ester (L-AME, an exogenous substrate of NOS), 0.5 mg/kg bw sodium nitroprusside (SNP, an NO donor) or vehicle (0.9% NaCl) or intratesticularly with 2 mg/testis L-AME, 2 micrograms/testis SNP or 25 microliters vehicle, and killed at 60 or 120 min after treatment. Both intraperitoneal and intratesticular administration of L-AME had the same effects: a decrease in the serum concentrations of LH and testosterone and in those of testosterone in the testicular interstitial fluid. However, treatment with SNP was more effective when given intratesticularly, inducing a decrease in serum and interstitial fluid testosterone concentrations, without significant changes in LH concentrations. Furthermore, when rats were injected intraperitoneally with 4 mg L-AME (the same dose as that given intratesticularly), serum LH concentrations were not changed. In addition, L-AME administration was not effective in modifying serum LH concentrations in castrated rats. To test the possible role of Leydig cells, the effects of systemic administration of L-AME were studied in rats depleted of Leydig cells by treatment with ethylene dimethane sulphonate. In these animals L-AME significantly decreased serum LH concentrations. To study the role of macrophages in this system, rats depleted of testicular macrophages by the liposome-suicide approach were injected intraperitoneally (1 g/kg bw) or intratesticularly (2 mg/testis) with L-AME or vehicle, 10 days after macrophage depletion, and killed at 120 min after treatment. The effects of L-AME on serum LH concentrations were blocked when the drug was administered intratesticularly.

Investigation of the potential role of the germ cell complement in control of the expression of transferrin mRNA in the prepubertal and adult rat testis.

Iron is required for the normal development of germ cells during spermatogenesis. Because these cells have no direct access to systemic iron, there exists a shuttle system involving production and secretion of the iron-transporting protein transferrin by the Sertoli cells. Previous reports using cultures of immature Sertoli cells exposed to adult germ cells, or in vivo studies involving germ cell depleted adult rat testes, concluded that production of transferrin by Sertoli cells is modulated by germ cell complement. In the present study we have used in situ hybridisation with cRNA probes directed against the 5' and 3' ends of transferrin mRNA to examine the pattern of expression of transferrin in the immature and adult rat testis. Adult rats were treated with ethane dimethane sulphonate or methoxyacetic acid (MAA) to manipulate their testosterone levels or germ cell complement respectively. Initial findings obtained using the 3' probe showed a decrease in transferrin mRNA associated with round spermatid depletion. However, these data were not confirmed by in situ hybridisation when the 5' probe was used. The specificity of the probes was examined using Northern blotting and the 3' probe was found to hybridise to the germ cell transcript for hemiferrin even under conditions of high stringency. Examination of immature and pubertal rat testes by in situ hybridisation using the 5' transferrin-specific probe found that as early as 14 days of age the level of expression of transferrin mRNA was clearly different between tubules, and the mRNA appeared to be expressed in Leydig cells on and after day 31. In the adult rat testis, maximal expression of transferrin mRNA was found at stages VIII-XIV, calling into question the interpretation of the results of some previous studies showing expression of transferrin mRNA at all stages of the spermatogenic cycle. This stage-specific pattern of expression was not altered by acute germ cell depletion using MAA. However, Northern blot analysis showed a statistically significant increase in transferrin mRNA expression at 7 days after MAA treatment when pachytene spermatocytes were depleted from tubules at all stages of the spermatogenic cycle at which transferrin is normally expressed. In conclusion, we found that transferrin mRNA expression was not modulated by round spermatids as has been reported previously but that meiotic germ cells may influence expression of transferrin at specific stages of the spermatogenic cycle.

Thymic regression and apoptosis in the rat after treatment with the Leydig cell cytotoxin ethylene dimethanesulphonate (EDS)

Ethylene dimethanesulphonate (EDS) is an alkylating agent which is widely assumed to specifically kill Leydig cells leaving other biological systems intact. However, after EDS treatment of the male rat the thymus reversibly involutes and the gonadal regional lymph nodes are activated. In the present experiments we have demonstrated that EDS has a direct action upon the thymus both in vivo and in vitro. EDS treatment of the intact and castrated male rat and the intact female rat caused regression of the thymus by up to 50% 3 days later. Total cellularity decreased while the proliferative index increased suggesting a compensatory mechanism. Thymocytes were exposed to EDS in vitro and the response compared to the glucocorticoid methylprednisolone (P), a well characterised thymic apoptotic stimulant. EDS and P increased apoptosis in the thymocyte as characterised by the appearance of cells containing nuclei with apoptotic morphology and with DNA fragmentation visualised by a characteristic ladder after agarose gel electrophoresis. The effects of both EDS and P were time and dose dependent but, in contrast to the effects in Leydig cells, P was the most effective apoptotic stimulus (for instance 100%-P compared to 30%-EDS or 7% control/DMSO after 24 h incubation). The immunological responses of the gonadal lymph nodes were not associated with testicular regression as it was seen in the castrated rat but may be related to a direct action upon the epididymis. In conclusion, tissue specificity of the Leydig cell cytotoxin needs to be extended to the thymus and epididymis. The mode of cell death in Leydig cells and thymocytes after both glucocorticoids and EDS is apoptosis which suggests that they possess some common mechanism(s) which is responsible for the toxicity of these diverse compounds.

Leydig cell apoptosis in response to ethane dimethanesulphonate after both in vivo and in vitro treatment.

The biological effects of ethane dimethanesulphonate (EDS) are unique since cytotoxicity in the adult rat is almost exclusively confined to the Leydig cells. For this reason, EDS has been used extensively to investigate the physiological role of the Leydig cell and its products. Experiments were conducted to determine whether the Leydig cell will undergo apoptosis in response to EDS or methylprednisolone (MP), a glucocorticoid known to cause apoptosis in a number of other cell types. Percoll-purified Leydig cells were incubated for 24 hours with EDS (750 micrograms/ml), at which time the cells attached to the culture plate became rounded up while control cells were flattened and polyhedral. Following incubation with EDS or MP (10 microM), cells that became detached from the plate were characteristically apoptotic when stained with the fluorescent DNA dye, acridine orange. These cells had shrunk and the nuclear chromatin had become condensed, which is an early characteristic of apoptosis in other cells; eventually, apoptotic bodies formed, reflecting a later apoptotic stage. Electrophoresis of DNA extracted from the treated Leydig cells exhibited the characteristic ladder of the apoptotic process. Increasing the concentration of EDS or MP resulted in a dose-dependent increase in the incidence of apoptosis that reached a maximum of 25% (EDS) or 12% (MP) of detached cells. Administration of EDS in vivo caused a 20-fold increase in the number of apoptotic cells observed in interstitial cell preparations. In conclusion, the data indicates that programmed cell death, apoptosis, can occur in the Leydig cell and that this is the likely mechanism by which EDS kills the cells in vivo and in vitro.

Discriminant analysis indicates a single sperm protein (SP22) is predictive of fertility following exposure to epididymal toxicants.

In a previous study, we found that ethane dimethanesulphonate (EDS) compromised the fertilizing ability of proximal cauda epididymal sperm from the rat within 4 days of exposure, an effect that persisted in castrated, testosterone (T)-implanted animals, establishing direct action on the epididymis. This EDS-induced reduction in fertilizing ability was highly correlated with a quantitative decrease in specific sperm protein. Here we sought to determine whether the fertility of proximal cauda epididymal sperm recovered from animals exposed to a variety of male reproductive toxicants could be predicted by assessing quantitative changes in specific sperm protein(s), or whether more common endpoints (e.g., sperm motility, sperm morphology, serum and epididymal tissue T, cauda epididymal sperm reserves) also are required to predict fertility. Intact adult male rats were dosed with EDS (25 or 50 mg/kg), chloroethylmethanesulphonate (CEMS; 12.5 or 18.75 mg/kg), or epichlorohydrin (EPI; 3 or 6 mg/kg) daily for 4 days. Castrated, T-implanted rats were dosed with hydroxyflutamide (HFLUT; 12.5 or 25 mg/kg) daily for 5 days. On day 5, proximal cauda epididymal sperm were inseminated in utero into receptive, cervically stimulated adult females, and on day 9, fertility (implants/corpora lutea) was assessed. Fertility-was decreased by the higher dose of each toxicant (P < 0.05) and also by the lower dose of EPI and HFLUT. Likewise, an acidic 22 kDa sperm protein (SP22) was decreased quantitatively (P < 0.05) in silver-stained two-dimensional gels by the higher dose of each toxicant as well as by the lower dose of EPI and HFLUT. Although sperm motility and serum T were altered by specific exposures, these endpoints were not useful in predicting fertility. In contrast, SP22 was highly correlated (P < 0.0001; r2 = 0.83) with fertility. Indeed, the amount of SP22 correctly predicted 90% and 94% of the fertile (> 50% fertility) and subfertile (< 50 fertility) animals, respectively, when discriminant analysis was performed. Thus, the amount of SP22 in a cauda epididymal sperm sample may be a useful predictor of fertility in toxicant-treated animals.

Leydig cells secrete factors which increase vascular permeability and endothelial cell proliferation

Adult intact control rats, and animals treated with human chorionic gonadotrophin (hCG) or with ethane dimethane sulphonate (EDS) to deplete Leydig cells, were injected with bromodeoxyuridine (BrdU) to label proliferating cells. Apoptotic cells were visualized by in-situ end labelling (ISEL) of fragmented DNA. Three per cent of testicular endothelial cells were labelled with BrdU and few were apoptotic in intact testes. The BrdU endothelial cell labelling index was increased by hCG-treatment and decreased in Leydig cell-depleted testes. Immunohistochemical staining showed that Leydig cells and testicular macrophages contain immunoreactive vascular endothelial growth factor (irVEGF). The ability of testicular cells to stimulate angiogenesis was studied further by transplanting interstitial cells or seminiferous tubule segments under the kidney capsule. A prominent vascular network was observed around interstitial cell grafts, but not around tubule grafts. Treatment of transplanted rats with human chorionic gonadotrophin (hCG, 50 i.u.) resulted in an accumulation of PMN-leukocytes and an increase in vascular permeability in the remaining testis and in interstitial cell grafts. Interstitial cells from Leydig cell-depleted (EDS-treated) testes were also transplanted under the kidney capsule. This type of graft caused only a discrete stimulation of angiogenesis, and there was no increase in vascular permeability around the graft after hCG treatment. It is suggested that Leydig cells secrete angiogenic factors and that they are the source of the inflammation mediator(s) produced in the testis after hCG treatment. The high proliferation rate in endothelial cells suggests continuous remodelling of the testicular microvasculature, but the functional significance of this remains unknown.

Inhibin-related proteins in rat prostate.

Inhibin and activin are members of the transforming growth factor beta (TGF beta) family which can regulate cell proliferation in a number of tissues. The presence of inhibins and the related proteins, activins, in the prostate has been implicated by the detection of activin type II receptors. The aim of this study was to determine whether or not immunoactive (ir) inhibin and ir-activin are present in the rat prostate and to study the acute regulation by androgens. The results showed that mRNAs for the alpha and beta inhibin subunits were detected in rat prostate by reverse transcription-PCR together with ir-inhibin and ir-activin in prostate cytosols. The levels of ir-activin in the prostate (223 +/- 44 ng/gland) were greater than the levels of ir-inhibin (6.89 ng/gland), and activin immunoreactivity was localised to the epithelial cells. The presence of these proteins and the subunit mRNAs suggests that these proteins are produced in the prostate and may have a role in prostate function. The study of the effect of androgen withdrawal on the levels of ir-activin and ir-inhibin in these tissues showed no change in the content of ir-inhibin or ir-activin (ng/g tissue) after 3 days of castration or following the administration of the cytotoxic drug ethane dimethane sulphonate (EDS), although there was a significant (P < 0.01) decline in prostate weight. Fourteen days after EDS treatment, as the prostate weight fell significantly lower, the amount of ir-inhibin and ir-activin per prostate gland was significantly (P < 0.01) reduced although the concentration was unaffected. These data demonstrate, for the first time, that inhibin alpha and beta subunit mRNA and ir-inhibin and ir-activin are present in the prostate; the role of these proteins in prostate function remains to be established.