Statin drugs markedly inhibit testosterone production by rat Leydig cells in vitro: implications for men.

Statin drugs lower blood cholesterol by inhibiting hepatic 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase. Statins are known to inhibit sterol production in the testis, but effect of statins on testosterone production has not been studied critically in vitro and clinical data are controversial. We measured 18-h testosterone production in vitro, using highly purified rat Leydig cells exposed to atorvastatin, mevastatin, or simvastatin and also determined if statin-induced inhibition of testosterone production could be bypassed with substrate distal to cholesterol. Statins had no effect on testosterone production during culture without LH. However, with 10ng/mL LH, testosterone production was ≥12-fold higher and markedly inhibited (-40%) by ≥0.3µM statin. Leydig cells provided sub-saturating pregnenolone or progesterone to bypass the site of statin action, maintained LH-stimulated testosterone production at or above amounts observed with LH stimulation and no statin. Pregnenolone resulted in greater testosterone production, but LH responsiveness was lost. With progesterone, LH responsiveness was maintained.

Placental cadmium and progesterone concentrations in cigarette smokers.

Cadmium and progesterone concentrations were evaluated in term placentas collected from 56 healthy parturients in the city of Zagreb. Concentrations of lead, iron, zinc, and copper in placentas were analyzed. Data collected by questionnaire identified 29 nonsmoking and 27 smoking women. From each placenta, three samples from different locations were taken. Metals were measured by atomic absorption spectrometry. Progesterone was determined by specific radioimmunoassay in homogenized and lyophilized tissue samples after steroid extraction with ethanol. No effect of sample location was found. In placentas of smoking women an increase in cadmium, reduced progesterone and a decrease in iron concentrations were found. Placental copper and zinc concentrations were not altered. In conclusion, the results present new evidence that maternal smoking reduces placental progesterone content and support the established association of smoking with placental cadmium.

Effects of in vitro cadmium exposure on ovarian steroidogenesis in rats.

The purpose of this study was to evaluate the direct effect(s) of in vitro cadmium (Cd) exposure on steroidogenesis in rat ovaries during different reproductive states. Sprague-Dawley rats were killed on the day of proestrus, or on gestation day 6 or 16. Ovaries were removed, placed in medium and minced. Culture from each ovary was incubated with Cd2+ ions in concentrations of 0, 100, 500, 1000, 1500, or 2000 microM. One-hour whole-ovary production of progesterone (P4), testosterone and estradiol (E2) in culture medium was evaluated in the absence and presence of human chorionic gonadotropin (hCG) or hCG plus pregnenolone by specific radioimmunoassay. Under in vitro Cd exposure the most affected were productions of P4 and testosterone in proestrus rats and less in pregnant dams, whereas E2 was not affected at all. Cadmium appears to interfere with the ovarian steroidogenic pathway in rats at more than one site.

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.

Biomarkers of heavy metal reproductive effects and interaction with essential elements in experimental studies on female rats.

Experimental studies in laboratories in Croatia and U.S.A. were conducted on female rats exposed to lead or cadmium to evaluate effects on the female reproductive integrity. The health condition of the offspring and relationship with essential elements were also evaluated. By using simple biomarkers of reproductive effects it was found that subchronic oral exposure to lead (1500-5500 ppm) or cadmium (50 ppm) during pregnancy and lactation decreased pup body weight, and that lead also decreased pup viability. Acute exposure to cadmium (3 or 5 mg/kg body weight s.c.) in vivo suppressed serum concentrations of progesterone and estradiol depending on the reproductive stage. Organ accumulations of lead or cadmium were accompanied by changes in the concentrations of iron and zinc in both mother and pups. Future research should focus on the effects of metals on endocrine disruption in the ovary and placenta, and on concomitant interaction of toxic and essential metals in mother and offspring.

Therapy methodology. Riding the bus: teaching an adult with a brain injury to use a transit system to travel independently to and from work.

A man, 8 years post-injury, who was still experiencing poor impulse control and poor directional orientation, was taught to follow directions and to take city buses to and from his vocational placement. This was a new skill for the subject as he was from a rural area and thus had no premorbid experience using a transit system. In-vivo functional training was conducted, similar to the method used by Sowers et al. [1] to train a severely retarded adult to ride buses to and from work. In-vivo training was supplemented by daily planning sessions in which the subject would review the route and instructions prior to each ride. The subject was able to learn the bus route from the treatment centre to the job site in 1 week. Upon discharge to a transitional living centre in the same city the subject was able, again in 1 week, to learn new bus routes, and to take the buses back and forth between his new residence and his vocational placement.

The use of cultured ovarian fragments to assess toxicant alterations in steroidogenesis in the Sprague-Dawley rat.

This study was conducted to determine the utility of using steroid production by cultured ovarian fragments to assess toxicant-induced alterations in ovarian steroidogenesis in Sprague-Dawley rats. To this end, serum steroid concentration and steroid production (progesterone (P4), testosterone (T), estradiol (E2)) by cultured ovarian fragments is described during a normal 4-day estrous cycle. This culture system was then used to profile the effects of aminoglutethimide shown to have two sites of steroidogenic inhibition, side chain cleavage enzyme and aromatase. LH, FSH, P4, and E2 concentrations in serum during the 4-day estrous cycle confirmed that described in the literature for untreated rats. All of the steroids measured had peak production levels during proestrus. The patterns of P4 and E2 production by the ovaries in an unstimulated culture mimics that seen in serum. Stimulation with hCG (100 mIU/mL) after the initial 1 h culture tends to even out the production of P4, while T production rises faster and peaks earlier. The pattern and levels of estradiol production in hCG-stimulated cultures are very similar to those in the unstimulated culture, both in pattern and in production levels. When cultured ovarian fragments from proestrous rats were treated in vitro with aminoglutethimide (1 to 16 microM), the pattern of steroid production that characterized the inhibitory effects were similar to those reported in the literature using isolated cell culture procedures. This pattern showed a rapid decrease in E2 production (IC50 of 2.43 microM), a concurrent rise in T production, and a decrease in P4 production (IC50 of 15.5 microM). This culture system is an appropriate system to rapidly assess toxicant effects on ovarian steroidogenesis following in vivo or in vitro exposure.

Acute cadmium exposure and ovarian steroidogenesis in cycling and pregnant rats.

The purpose of this study was to evaluate the effect(s) of acute in vivo cadmium (Cd) exposure on steroidogenesis in rat ovaries during different reproductive states. Sprague-Dawley rats were injected subcutaneously on the day of diestrus, or on day 7 or 16 of gestation with a single dose of 0, 3, or 5 mg Cd/kg bw, and evaluated 24 h later. Serum progesterone and estradiol concentrations were determined. Whole-ovary culture was used to evaluate Cd effects on the production of progesterone, testosterone, and estradiol. Liver, kidney, spleen, ovary, placenta, and blood were analyzed for Cd and iron (Fe) concentrations. No general toxic effects, no disruption of estrous cyclicity, and no change in fetal viability were seen. Histologic evaluation revealed moderate Cd-related thecal congestion in ovaries of pregnant rats. The highest Cd concentrations, except for liver, were found in the fetal portion of the placenta. Interestingly, Cd-related decreases in Fe concentration were found in several tissues from rats in proestrus and on gestation day 8, and in fetal placenta from rats on gestation day 17. Cadmium appears to interfere with normal steroidogenesis at a number of sites in the biosynthetic pathway with serum estradiol concentration and ovarian estradiol production the most affected. Acute Cd effects on steroidogenesis are most severe in rats evaluated in proestrus or in early pregnancy, while in late pregnancy steroidogenesis is relatively unaffected.

The ethane dimethanesulfonate-induced decrease in the fertilizing ability of cauda epididymal sperm is independent of the testis.

Several decades ago it was reported that when adult male rats were exposed to a single injection of 50 mg/kg body weight ethane dimethanesulfonate (EDS) and mated with untreated females, average litter size was significantly reduced as early as 2 weeks later. Recently, we demonstrated that EDS exerts multiple effects in the epididymis of adult rats. Some of these effects were independent of reduced serum testosterone (T) levels. Later we found that EDS has direct effects on epididymal epithelial cells in vitro. Herein, we sought to determine whether EDS perturbs the fertilizing ability of cauda epididymal sperm. Four days after exposure to 50 mg/kg EDS, sperm from the proximal cauda epididymidis were inseminated into adult receptive females in utero; on the next day the percentage of fertilized eggs was determined. Exogenous T administration and castration were used to determine what role, if any, androgen deprivation and the testis had on the fertilizing ability of proximal cauda epididymal sperm. Sperm motion parameters, serum T, T in the caput/corpus epididymidis, and detergent-extracted sperm protein were evaluated and correlated with fertilizing ability. We found that both castration and EDS exposure significantly compromised the fertilizing ability of sperm in proximal cauda epididymidis 4 days after exposure. Exogenous T, sufficient to maintain serum T, completely restored the fertilizing ability of sperm following castration, but not after EDS exposure. Moreover, exogenous T failed to restore fertilizing ability when castrated animals were exposed to EDS. Thus, the effects that EDS exerts on sperm maturation in vivo are independent of the testis. Finally, the only endpoint that was well correlated with fertilizing ability was the relative amount of an acidic 18-kDa sperm protein.

Chloroethylmethanesulfonate-induced effects on the epididymis seem unrelated to altered Leydig cell function.

Decades ago it was reported that when male rats were exposed to chloroethylmethanesulfonate (CEMS) for 5 days prior to weekly matings with untreated females, the second mating resulted in reduced litter size. Since fertility was not assessed at earlier time points, it was not possible to determine whether CEMS exerted any effects on sperm in the epididymis. In this study, we used a 4-day exposure and assessed multiple reproductive endpoints on Day 5 to characterize effects of CEMS exposure (6.25-25 mg/kg) on Leydig cells and the epididymis. Exposure to CEMS caused a dose-related decline in serum testosterone (T) levels. This occurred at a dose lower than that required to decrease T production in vitro by testicular parenchyma. The in vitro decline was not attributed to a decrease in maximal hCG-stimulated T production, but to a decrease in unstimulated T production. CEMS was 5-fold less sensitive than ethane dimethanesulfonate (EDS) in reducing maximal hCG-stimulated T production. To control for alterations in the epididymis resulting from decreased serum T alone, T was implanted in CEMS-treated animals to maintain serum T at a concentration similar to that found in normal rats. This exogenous T failed to prevent the CEMS-induced decrease in the weight of the caput/corpus epididymidis but did prevent the CEMS-induced decrease in seminal vesicle weight. Implantation of T failed to prevent the CEMS-induced reduction in sperm reserves in the cauda epididymidis, and it failed to prevent the CEMS-induced alterations in the histology of both the corpus and proximal cauda epididymidis. The height of the epithelium in both of these regions was increased, and clear cells disappeared from the proximal cauda epididymidis. These results demonstrate that CEMS might alter the ability of the Leydig cell to respond to LH stimulation in vivo, and that alterations in the structure and function of the epididymis occur even when the serum concentration of T is maintained.

Effects of ethane dimethanesulfonate (EDS) on adult and immature rabbit Leydig cells: comparison with EDS-treated rat Leydig cells.

Ethane-dimethanesulfonate (EDS) has been shown to selectively kill Leydig cells and depress testosterone production in adult rats. A recent study has shown that immature rat Leydig cells are less sensitive to EDS exposure. There is evidence that the rabbit metabolizes EDS to methane sulfonic acid more rapidly than does the rat, reducing exposure to the parent compound. In the study reported here, we examined the effects of EDS on the Leydig cells in both adult and immature rabbits and compared the effects found with those previously reported in the rat. In vivo, EDS exposure demonstrated that Leydig cells from adult rabbits were affected, with both serum and interstitial testosterone production depressed. EDS effects in adult rabbits and rats were compared by exposing explants of testicular parenchyma to EDS in vitro and evaluating testosterone production. With this procedure, the rabbit testis was less sensitive to EDS treatment than the rat, with a 50% reduction rate (EC50) achieved with 2026 microM EDS for the rabbit and with 336 microM EDS for the rat. Perfusion of adult and immature rabbit testis with 430 microM EDS demonstrated the insensitivity of the immature testis to EDS exposure: adult testosterone production was reduced 50% in 3.5 h, whereas no diminution was found in the immature rabbit. EDS exposure of interstitial cell preparations further demonstrated the insensitivity of immature rabbit Leydig cells, with an EC50 of 4397 microM compared to an EC50 of 1137 microM EDS in adult preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of [14C]ethane dimethanesulfonate in immature and adult male rats following an acute exposure.

In the adult rat, ethane dimethanesulfonate (EDS) reduces testosterone (T) production by killing Leydig cells. Studies have also shown that acute EDS administration produces transient infertility and epididymal effects. Although these later effects were believed to be indirect results of the reduced Leydig cell T production, it was recently found that the epididymal effects were partially a direct result of in vivo EDS treatment. In contrast to the Leydig cells of the adult rat, immature Leydig cells are affected by EDS only at doses four- to sixfold higher than those that affect mature Leydig cells. In fact, the Leydig cells of the adult rat seem to be uniquely susceptible to the cytotoxic effects of EDS. Steroidogenesis in other organs, like the adrenal and ovary, are unaffected in vivo at doses that eliminate T production in males. In addition, studies have shown that doses of EDS that kill Leydig cells in vitro, isolated from the testes of adult rats, have no effect on similarly exposed hepatocytes. Hence, it was the objective of this study to describe the distribution and temporal fate of EDS in target (testes and epididymides) and nontarget tissues in immature and adult male rats and to determine if this information would explain either the age- or tissue-related susceptibility to EDS. We have concluded from this study that tissue distribution, integrated in vivo EDS dose, and differences in EDS metabolism are not the only factors contributing to the difference in sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered steroidogenesis in whole-ovary and adrenal culture in cycling rats.

Cultures of minced, whole-ovary (whole-ovary culture) were used to determine if three selected chemicals altered steroidogenic profiles. First, phenolsulfonthalein (PST), when used in culture medium, was tested for its influence on in vitro steroidogenesis. Next, aminoglutethimide (AGTP; 0 or 150 mg/kg once) and di(2-ethylhexyl)phthalate (DEHP; 0 or 1500 mg/kg/day for 10 days) were administered in vivo to young adult cycling rats, and the ovaries and adrenals were removed and cultured for 1 h. Ovarian steroidogenic profiles of progesterone (P), testosterone (T), and estradiol (E) release into the medium were measured using radioimmunoassay techniques. PST in medium significantly decreased ovarian P production and altered T and E production so that the T/E ratio was significantly altered. Therefore, PST was excluded in the later studies. DEHP altered steroid profiles so that proestrus appeared to be delayed. AGTP decreased P and E production significantly, and T production was increased slightly in proestrus ovaries. These AGTP alterations in T and E resulted in a highly significant increase in the T/E ratio. Adrenals from the DEHP and AGTP experiments were also cultured for 1 h, and P was assayed in the medium. AGTP, but not DEHP, significantly increased the production of P in adrenals. Whole-ovary culture is recommended as an in vitro test for chemicals suspected of interfering with steroidogenesis in vivo. This test model should be placed strategically between in vivo studies of reproductive toxicity and complex in vitro mechanistic studies.

Steroidogenic assessment using ovary culture in cycling rats: effects of bis(2-diethylhexyl)phthalate on ovarian steroid production.

In vitro ovary culture in rats was used to characterize ovarian steroidogenesis and to evaluate changes produced by in vivo exposure to bis(2-diethylhexyl)phthalate (DEHP). Steroid profiles [progesterone (P4), estradiol (E2), and testosterone (T)] from cultures of minced ovary were obtained in untreated immature and mature rats, and from mature rats treated with DEHP. A 1-h incubation without human chorionic gonadotropin (hCG) was used to produce an initial steroidogenic profile. Three 1-h incubations with hCG were used to produce a stimulated steroid profile. A combination of initial and stimulated ovarian steroid profiles was shown to correctly identify the stage of the cycle in all untreated rats, using multivariate statistical analysis. Separately, initial or stimulated ovarian steroid profiles correctly identified the stage of the cycle in more than 90% of the rats. The statistical analysis using a combination of variables (multivariate) indicated that DEHP-treated rats were significantly different (P < 0.001) from sham-treated rats. In fact, the alteration caused by DEHP in the in vitro ovarian steroidogenic profile was most apparent in rats during diestrus and estrus. In DEHP-treated rats in diestrus, ovarian steroidogenesis appeared to shift to the production of more T and more E2 than in untreated rats in diestrus. The change seen in steroid profiles in DEHP-treated rats in estrus is to decreased E2 production. The steroid profile from ovary culture in conjunction with vaginal cytology was very useful in correctly identifying in vivo DEHP-treated rats, and will be a useful in vitro technique in the evaluation of ovarian toxicants in cycling females.

Use of the fungicide carbendazim as a model compound to determine the impact of acute chemical exposure during oocyte maturation and fertilization on pregnancy outcome in the hamster.

Early pregnancy loss due to acute chemical exposure is difficult to detect and essentially impossible to characterize in humans. Here we use a hamster animal model to identify early pregnancy loss due to an acute chemical exposure to the female during the perifertilization interval. The fungicide carbendazim (methyl 1H-benzimidazole-2-carbamate), a microtubule poison with antimitotic activity, was selected as a model compound because it would be expected to perturb microtubule-dependent events occurring in the oocyte during meiotic maturation and fertilization. Such effects would likely lead to aneuploidy in the zygote with subsequent early pregnancy loss. Female hamsters were given a single oral dose of carbendazim during meiosis I (the afternoon of proestrus) prior to breeding, or during meiosis II (the morning of estrus) following overnight breeding. Pregnancy outcome was assessed on Day 15 (the afternoon before parturition). When given during during meiosis I, carbendazim treatment (750 or 1000 mg/kg body weight) significantly reduced the percentage of pregnant hamsters. In those animals that became pregnant, the average number of live pups was significantly lower at all dosages of carbendazim used (250, 500, 750, and 1000 mg/kg), an effect attributable to both preimplantation and early postimplantation losses. When given early on the morning of estrus, shortly before and during fertilization (0500 or 0600 hr), carbendazim treatment (1000 mg/kg) produced a similar decrease in litter size. This effect disappeared when carbendazim was administered at a slightly later time (0800 or 0900 hr), after the microtubule-dependent events of fertilization have occurred. These results demonstrate that a single exposure to a microtubule poison such as carbendazim at critical times, coincident with microtubule-dependent meiotic events, can result in very early pregnancy loss. Such loss was readily measurable in this animal model and serves as the basis for further mechanistic studies which would be impossible to conduct in humans.

Immunotoxicity of 2-methoxyethanol following oral administration in Fischer 344 rats.

The immunotoxicity of the glycol ether 2-methoxyethanol (ME) was evaluated in adult Fischer 344 rats using a variety of in vitro and in vivo immune function assays. In the first phase of this study, male rats were dosed by oral gavage with ME in water, at dosages ranging from 50 to 200 mg/kg/day, for 10 consecutive days. Decreases in thymus weights were observed at dosages of 50-200 mg/kg/day in the absence of decreased body weights. Lymphoproliferative (LP) responses to concanavalin A and phytohemagglutinin were reduced at 50-200 mg/kg/day while pokeweed mitogen and Salmonella typhimurium mitogen responses were reduced at 200 mg/kg/day. No alterations were observed in natural killer cell activity, mixed lymphocyte reaction, or cytotoxic T lymphocyte responses. The frequency of W3/25-positive splenocytes was reduced in rats dosed at 200 mg/kg/day. Interleukin-2 production was reduced in splenocytes from rats exposed to all dosages of ME. The plaque-forming cell (PFC) response to sheep red blood cells was enhanced in rats dosed at 50 mg/kg/day. However, the PFC response to trinitrophenyl-lipopolysaccharide (TNP-LPS) was suppressed at all dosages. Similarly, the PFC response to TNP-LPS was suppressed in adult female rats dosed with ME. A reduction in the expulsion of adult worms was observed in rats dosed at 200 mg/kg/day that were infected with Trichinella spiralis. A number of male reproductive parameters were also evaluated in rats dosed with ME over 10 days. A significant reduction in testicular weight was observed in rats dosed at 200 mg/kg/day. In the second phase of this study, the PFC response to TNP-LPS was employed to assess the role that metabolism of ME to 2-methoxyacetic acid (MAA) plays in the immunotoxicity of this glycol ether. Ten-day oral dosing with MAA resulted in the inhibition of the PFC response to TNP-LPS at dosages of 50-200 mg/kg/day. Concomitant exposure of rats to ME and the alcohol dehydrogenase inhibitor 4-methylpyrazole blocked ME-induced suppression of this PFC response. Attempts to ameliorate ME-induced suppression of the PFC response with serine, which has been shown to reverse ME-induced developmental and reproductive toxicity, were unsuccessful. These results suggest that the immune system may be more sensitive than the reproductive system to the toxic effects of ME. Furthermore, it appears that MAA is the proximate toxicant for ME-induced alterations in the immune system, as has been demonstrated for ME-induced reproductive and developmental toxicity.

Effect of cadmium and other metal cations on in vitro Leydig cell testosterone production.

In vivo assessment of toxicant action on Leydig cell function is subject to homeostatic mechanisms which make it difficult to determine whether any changes seen in serum testosterone (T) concentration are due to extragonadal endocrine alterations or to a direct effect on the Leydig cell. For example, metal cations administered in vivo have been shown to depress serum T concentration and alter serum concentrations of pituitary hormones in laboratory animals. The studies reported here use a testicular cell culture technique to evaluate Leydig cell testosterone biosynthesis in the presence of several metal cations. To determine the site of toxic action, the Leydig cells were stimulated to produce testosterone by using human chorionic gonadotrophin (hCG), dibutyl cyclic adenosine monophosphate (db-cAMP), or several substrates required for the biosynthesis of testosterone. hCG was chosen because resultant T production requires an intact membrane receptor and db-cAMP was used to test for post LH receptor defects caused by the metals. The other substrates were chosen to isolate the effect of metals on enzymatic pathways. Collagenase dispersed testicular cells (15% Leydig cells) were incubated with metal cations (1 to 5000 microM) for 3 hr in the absence and presence of maximally stimulating concentrations of hCG, db-cAMP, 20 alpha-hydroxycholesterol (HCHOL), or pregnenolone (PREG), and T concentration was determined by radioimmunoassay. In one separate experiment we also tested the effect of the substrates progesterone, 17 alpha-hydroxy-progesterone, and androstenedione on Cd2(+)-treated Leydig cells. The results show no change in Leydig cell viability with any metal cation treatment during the 3-hr incubation. Ca2+, Cr3+, Fe3+, Mg2+, Na+, or Pb2+ had no effect on stimulated testosterone. Dose-response depression in both hCG- and db-cAMP-stimulated T production were seen with Cd2+, Co2+, Cu2+, Hg2+, Ni2+, and Zn2+ treatment. Surprisingly, Cd2+, Co2+, Ni2+, and Zn2+, which caused a depression in hCG- and db-cAMP-stimulated T production, caused significant increases in HCHOL- and PREG-stimulated T production over untreated and similarly stimulated cultures. This indicates that these cations may act at multiple sites within the Leydig cell.

In vitro/in vivo effects of ethane dimethanesulfonate on Leydig cells of adult rats.

Although ethane dimethanesulfonate (EDS) is well recognized as a Leydig cell toxicant, the dose responsiveness of Leydig cells to EDS, both in vitro and in vivo, is not well established. In addition, the cellular site of action of EDS during Leydig cell toxicity and the status of Leydig cell viability during the affected period remain controversial. We determined the in vitro EC50 (370 microM) and in vivo ED50 (60 mg/kg) for human chorionic gonadotropin (hCG)-stimulated testosterone (T) production using both highly purified (98%) and interstitial (14%) Leydig cell preparations, respectively. Leydig cells were recovered in approximately equal numbers following all in vivo and in vitro EDS exposures. The Leydig cells in these preparations were viable and steroidogenically active (3 beta-HSD positive) subsequent to all exposures, both before and after incubations to stimulate T biosynthesis. When hCG-stimulated T production was decreased 50% following in vivo or in vitro exposures, the morphological integrity of the Leydig cells appeared normal, with no discernible lesion at either the light or the electron microscope level. We used stimulants of various reactions in the pathway of T biosynthesis (20 alpha-hydroxycholesterol and pregnenolone) to determine the site of action impaired when T biosynthesis was decreased. Our results indicate that when Leydig cells are exposed to EDS either in vitro or in vivo, the biosynthesis of T is compromised between the cyclic adenosine monophosphate activation of protein kinase and the cholesterol side chain cleavage enzyme.

Multiple effects of ethane dimethanesulfonate on the epididymis of adult rats.

Ethane dimethanesulfonate (EDS), a Leydig cell toxicant which results in transient infertility, was used in a 4 day postexposure experimental protocol designed to identify any effects this compound might exert on the epididymis. The techniques of efferent duct ligation and testosterone (T) implantation were used to negate the role of testicular effects on the epididymal parameters. Numerous evaluations were performed including light and electron microscopy, computer assisted sperm motion analyses, and electrophoresis of sperm membrane proteins. EDS was shown to affect the epididymis in a dose-dependent fashion. The action of EDS on the epididymis is in part due to Leydig cell cytotoxicity and the resulting decrease in circulating androgen since T implantation prevented some of the changes in sperm proteins and motility. However, neither efferent duct ligation nor T implantation prevented the formation of sperm granulomas in the caput epididymidis, the distinct morphological alterations of the corpus epididymidis, the modification of certain sperm membrane proteins, or the decrease in the progressive motility and velocity of sperm following EDS treatment. Although we cannot prove these effects of EDS are due to a direct action on the epididymis, it is now clear that EDS has a distinct action on the epididymis which is unrelated to circulating T or testicular fluid.

Serum chemistries of Coturnix coturnix japonica given dietary manganese oxide (Mn3O4).

1. Plasma creatinine and inorganic phosphorus were increased in manganese oxide (Mn3O4)-treated adult male Coturnix quail, but BUN, BUN/creatinine ratio, uric acid, and total calcium were decreased. 2. Serum enzymes (alkaline phosphatase, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and lactic dehydrogenase) were elevated in Mn3O4-treated adult male Coturnix quail, but creatine phosphokinase was not affected. 3. Dietary Mn3O4 at 5000 ppm did not produce overt signs of toxicosis.