Gonadal sex steroid hormone secretion after exposure of male rats to estrogenic chemicals and their combinations.

Environmental chemicals can interfere with the endocrine axis hence they are classified as endocrine disrupting chemicals (EDCs). Bisphenol S (BPS) is used in the manufacture of consumer products because of its superior thermal stability and is thought to be a safe replacement chemical for its analog bisphenol A (BPA). However, the safety profile of these compounds alone or in the presence of other EDCs is yet to be fully investigated. Also, the estrogenic chemical 17α-ethinyl estradiol (EE2) and a constituent of female oral contraceptives for women, is present in water supplies. To simulate concurrent exposure of the population to chemical mixtures, we investigated the effects of BPA, BPS, EE2, and their combinations on sex steroid secretion in the growing male rat gonad. Prepubertal and pubertal male rats at 21 and 35 days of age were provided test chemicals in drinking water (parts per billion) for 14 days. At termination of exposure, some individual chemical effects were modified by exposure to chemical combinations. Single chemical exposures markedly decreased androgen secretion but their combination (e.g., BPA + BPS + EE2) caused the opposite effect, i.e., increased Leydig cell T secretion. Also, the test chemicals acting alone or in combination increased testicular and Leydig cell 17ß-estradiol (E2) secretion. Chemical-induced changes in T and E2 secretion were associated with altered testicular expression of the cholesterol side-chain cleavage (Cyp11a1) and 17ß-hydroxysteroid dehydrogenase (Hsd17ß) enzyme protein. Additional studies are warranted to understand the mechanisms by which single and chemical combinations impact function of testicular cells and disrupt their paracrine regulation.

Exposures of male rats to environmental chemicals [bisphenol A and di (2-ethylhexyl) phthalate] affected expression of several proteins in the developing epididymis.

Hormonally active agents are released into the environment from industrial and manufacturing activity. Evidence in the literature indicates that impaired reproductive capacity in wildlife and laboratory species is associated with chemical exposures. In particular, bisphenol A (BPA) and di (2-ethylhexyl) phthalate (DEHP) have generated public interest due to their presence in several consumer products. In this study, we determined that expression of steroid hormone receptors (estrogen and androgen receptors), Wnt4, and ß-catenin was greater (p < 0.05) in the rat epididymis at 35 days of age compared to 21 and 90 days. Second, timed-pregnant Long-Evans dams were exposed to the chemicals BPA and DEHP by gavage from gestational days 12-21. The caput epididymis was collected from cohorts of male offspring at 35 and 90 days of age and processed for Western blot analysis. Results showed that prenatal BPA and DEHP exposures affected (p < 0.05) expression of estrogen and androgen receptor, Wnt4, ß-catenin, MAPK, and HOXD4 protein in the epididymis. Data have implications for morphological development of the epididymis, a possibility that would be explored in future studies.

Effects of methoxychlor and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane on 3ß-hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase-3 activities in human and rat testes.

Human and rat testis microsomes were used to investigate direct inhibitory activities of methoxychlor (MXC) and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3). The 3ß-HSD and 17ß-HSD3 enzymes are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. The results demonstrated that MXC and HPTE inhibited human 3ß-HSD activity at a concentration of 10 nm. The half maximal inhibitory concentration (IC(50) ) for MXC inhibition of 3ß-HSD was 53.21 ± 15.52 µm (human) and 46.15 ± 17.94 µm (rat), and for HPTE, it was 8.29 ± 2.49 µm (human) and 13.82 ± 2.26 µm (rat). At the higher concentration of 100 µm, MXC did not affect human and rat 17ß-HSD3 activity. However, the IC(50) for HPTE inhibition of 17ß-HSD3 was 12.1 ± 1.9 µm (human) and 32 .0 ± 8.6 µm (rat). The mode of action of MXC and HPTE on 3ß-HSD activity was non-competitive with the substrate pregnenolone, but was competitive with the cofactor NAD(+) . The mode of HPTE inhibition of 17ß-HSD3 was non-competitive with the substrate androstenedione, but was competitive with the cofactor NADPH. In summary, our results showed that HPTE, which is the biologically active metabolite of MXC, has the capacity for direct inhibition of 3ß-HSD and 17ß-HSD3 enzyme activity. Inhibition of enzyme activity is presumably associated with suppression of steroidogenesis in gonadal tissues and has implications for testis function.

Oestrogenic and antiandrogenic chemicals in the environment: effects on male reproductive health.

Exposures of human populations to pesticides and industrial pollutants, and to synthetic chemicals present in foods, beverages, and plastics, have raised concern that these substances can interfere with endogenous sex hormone function. Interference with sex hormone action can, in turn, result in a variety of developmental and reproductive anomalies. Compounds in this class are thus referred to as endocrine disruptors (EDs). EDs that affect reproductive processes in vertebrates act primarily by altering oestrogenic and antiandrogenic activities. The recent cloning of a second oestrogen receptor (ER) subtype (ERbeta) and its widespread tissue distribution pattern indicates that the first ER to be cloned, ERalpha, may not be the only, or even the primary, mediator of oestrogen action. It is anticipated that this discovery will lead to development of antagonist compounds specific to either ER subtype, and help to determine the function of each receptor subtype in reproductive and other tissues. Growing evidence suggests that EDs interfere with reproductive function at low exposure levels and cause distinct effects at different concentrations within the same organ. Developing organisms have increased susceptibility to the actions of EDs because differentiating tissues are more vulnerable to changes in hormonal milieu. Thus, children are at greater risk of toxicant-related illnesses than adults. However, most data are collected from laboratory studies, and it remains to be determined that the levels of chemicals in the environment can impair human reproductive health. There is also significant genetic variability between human and animal species in their reactions to chemicals. The effects of low-dose, chronic, and multiple chemical exposures warrant further investigation in order to characterize the risk of environmental agents to humans. The aims of this review, which will focus on male reproduction, are to: 1) identify synthetic chemicals in the environment that fall into the ED class; 2) describe their mechanisms of toxicity in reproductive tissues; and, 3) outline the direction of future research efforts with respect to EDs.

Modulation of rat Leydig cell steroidogenic function by di(2-ethylhexyl)phthalate.

Exposure of rodents to phthalates is associated with developmental and reproductive anomalies, and there is concern that these compounds may be causing adverse effects on human reproductive health. Testosterone (T), secreted almost exclusively by Leydig cells in the testis, is the primary steroid hormone that maintains male fertility. Leydig cell T biosynthesis is regulated by the pituitary gonadotropin LH. Herein, experiments were conducted to investigate the ability of di(2-ethylhexyl)phthalate (DEHP) to affect Leydig cell androgen biosynthesis. Pregnant dams were gavaged with 100 mg(-1) kg(-1) day(-1) DEHP from Gestation Days 12 to 21. Serum T and LH levels were significantly reduced in male offspring, compared to control, at 21 and 35 days of age. However, these inhibitory effects were no longer apparent at 90 days. In a second set of experiments, prepubertal rats, from 21 or 35 days of age, were gavaged with 0, 1, 10, 100, or 200 mg(-1) kg(-1) day(-1) DEHP for 14 days. This exposure paradigm affected Leydig cell steroidogenesis. For example, exposure of rats to 200 mg(-1) kg(-1) day(-1) DEHP caused a 77% decrease in the activity of the steroidogenic enzyme 17beta-hydroxysteroid dehydrogenase, and reduced Leydig cell T production to 50% of control. Paradoxically, extending the period of DEHP exposure to 28 days (Postnatal Days 21-48) resulted in significant increases in Leydig cell T production capacity and in serum LH levels. The no-observed-effect-level and lowest-observed-effect-level were determined to be 1 mg(-1) kg(-1) day(-1) and 10 mg(-1) kg(-1) day(-1), respectively. In contrast to observations in prepubertal rats, exposure of young adult rats by gavage to 0, 1, 10, 100, or 200 mg(-1) kg(-1) day(-1) DEHP for 28 days (Postnatal Days 62-89) induced no detectable changes in androgen biosynthesis. In conclusion, data from this study show that DEHP effects on Leydig cell steroidogenesis are influenced by the stage of development at exposure and may occur through modulation of T-biosynthetic enzyme activity and serum LH levels.

Morphological changes in the gonads of the Sabi ram experimentally infected with Trypanosoma congolense.

Indigenous Sabi rams of Southern Africa were experimentally infected with Trypanosoma congolense for 8 and 16 weeks. Testes weights (g) were significantly (P<0.05) lower in the infected (249.7+/-26.4) compared to the control (372.63+/-19.4) animals. Histopathological and ultrastructural changes included seminiferous tubular atrophy and mononuclear infiltration in the testis, and lesions in the epithelium of the corpus epididymidis (middle segment) as well as spermatozoa in the cauda epididymidis. The gonadal lesions may have the capability to impair fertility in Sabi rams infected with Trypanosoma congolense.

A metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane, reduces testosterone biosynthesis in rat leydig cells through suppression of steady-state messenger ribonucleic acid levels of the cholesterol side-chain cleavage enzyme.

Postnatal development of Leydig cells involves transformation through three stages: progenitor, immature, and adult Leydig cells. The process of differentiation is accompanied by a progressive increase in the capacity of Leydig cells to produce testosterone (T). T promotes the male phenotype in the prepubertal period and maintains sexual function in adulthood; therefore, disruption of T biosynthesis in Leydig cells can adversely affect male fertility. The present study was designed to evaluate the ability of a xenoestrogen, methoxychlor (the methoxylated isomer of DDT [1,1, 1-trichloro-2,2-bis(p-chlorophenyl)ethane]), to alter Leydig cell steroidogenic function. Purified progenitor, immature, and adult Leydig cells were obtained from, respectively, 21-, 35-, and 90-day-old Sprague-Dawley rats treated with graded concentrations of the biologically active metabolite of methoxychlor, 2, 2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and assessed for T production. HPTE caused a dose-dependent inhibition of basal and LH-stimulated T production by Leydig cells. Compared to the control value, reduced T production by progenitor and immature Leydig cells was apparent after 10 h of HPTE treatment in culture; the equivalent time for adult Leydig cells was 18 h. The reversibility of HPTE-induced inhibition was evaluated by incubating Leydig cells for 3, 6, 10, 14, or 18 h and measuring T production after allowing time for recovery. After treatment with HPTE for 3 h, T production by immature and adult Leydig cells for the 18-h posttreatment period was similar to the control value, but that of progenitor Leydig cells was significantly lower. The onset of HPTE action and the reversibility of its effect showed that Leydig cells are more sensitive to this compound during pubertal differentiation than in adulthood. T production was comparable when control and HPTE-treated immature Leydig cells were incubated with pregnenolone, progesterone, and androstenedione, but HPTE-treated Leydig cells produced significantly reduced amounts of T when incubations were conducted with 22R-hydroxycholesterol (P < 0.01). This finding suggested that HPTE-induced inhibition of T production is related to a decrease in the activity of cytochrome P450 cholesterol side-chain cleavage enzyme (P450(scc)) and cholesterol utilization. The reduced steady-state mRNA level for P450(scc) in HPTE-treated Leydig cells was demonstrated by reverse transcription-polymerase chain reaction and densitometry. In conclusion, this study showed that HPTE causes a direct inhibition of T biosynthesis by Leydig cells at all stages of development. This effect suggests that reduced T production could be a contributory factor in male infertility associated with methoxychlor and, possibly, other DDT-related compounds.

Differential response of legumes and creep feeding on gut morphology and faecal composition in weanling pigs.

The effects of creep feeding and different levels of soybean meal (SBM) and cowpea meal on the intestinal morphology and faecal characteristics were investigated in weaners. Prior to the feeding trial, one group of piglets was creep-fed and the other noncreep-fed. The two groups of piglets were weaned at 28 days and randomly assigned to four different diets, the main protein sources of which were: T1--skimmed milk power (control); T2--31% soybean meal (high SBM), T3--15% soybean meal and 12% skimmed milk powder (low SBM), and T4--100% raw cowpea meal. Live weight gain was highest in the control group, and least in cowpea-fed piglets. At weaning, only the noncreep-fed weaners showed villus atrophy and crypt hyperplasia but at 7 days postweaning, these changes were evident in all groups except the control and were more severe in the noncreep SBM and cowpea-fed groups. At 21 days postweaning, only noncreep cowpea-fed pigs showed a reduced villus height when compared to the control group. A mild diarrhoea was generally observed in all noncreep-fed weaners, but its onset was more rapid (P < 0.01) and the duration much longer (P < 0.05) in the high SBM and cowpea-fed pigs than in low SBM and control groups. A lower faecal pH was observed in weaners that had diarrhoea when compared with a pH of 7.1 in pigs with normal faecal moisture. The observations of enteropathology and low growth performance in the cowpea group suggest that feeding raw cowpea to weaners may induce antigenicity in the intestinal mucosa, causing damage and a consequent decrease in productivity. However, the introduction of creep feeding before weaning appears to have some ameliorative effects.

The influence of protein malnutrition on the antifertility action of gossypol in the Trypanosoma brucei-infected rat: some ultrastructural observations from the testis.

The testes of adult male Wistar rats that were variously protein malnourished, gossypol treated, and/or trypanosome infected (Trypanosoma brucei) were evaluated ultrastructurally. The findings included several necrotic germ cells in the seminiferous epithelium and focal degeneration of Sertoli cells. Leydig cells showed a remarkable paucity of smooth endoplasmic reticulum, and aggregation of mitochondria. These observations were prevalent in those animals that were simultaneously protein-malnourished/gossypol treated/ trypanosome infected, and were either absent or markedly reduced in the other groups. These structural alterations are probably related to increased gossypol availability to the testis and/or represent the additive effects of gossypol and trypanosomes. Such tissue changes could compromise spermatogenesis in affected animals, and suggest that trypanosome infections may exacerbate testicular lesions in the gossypol-treated rat.

Chronic ethanol intake may delay the onset of gossypol-induced infertility in the male rat.

Parameters were obtained from the reproductive organs of ethanol-fed, gossypol-treated Sprague Dawley rats. The experimental animals were fed either on a normal (15.17%) or low protein (8.00%) diet. Measurements included reproductive organ weights, seminal characteristics, serum concentration of testosterone and histological, stereological and histomorphometric evaluation of the testis. The testis size, length and diameter of the seminiferous tubule had the least values in the protein-malnourished, gossypol-treated rats (3.01 +/- 0.26 g, 0.56 +/- 0.03 m, 281.34 +/- 11.30 microns), in comparison to corresponding animals which had received ethanol simultaneously with gossypol (3.40 +/- 0.25, 0.71 +/- 0.06 m, 314.42 +/- 11.61 microns). As gossypol and ethanol are both associated with reduced reproductive capacity, this unexpected but interesting finding lends support to the hypothesis that either a normal dietary protein or ethanol consumption may modify the action of gossypol on body tissues, including the testis. This effect, presumably mediated through changes caused to the bioavailability of gossypol, modifies its antifertility activity. The present observation further highlights the need to consider the concurrent administration of other drugs, such as alcohol, and the nutritional status in the evaluation of gossypol for various potential uses.

Ethanol intake may modify gossypol toxicosis in the rat.

Some aspects of the toxicity of gossypol were investigated in ethanol-fed, gossypol-treated Sprague-Dawley rats, with and without protein malnutrition. Serum creatine phosphokinase activity was depressed in all gossypol-treated rats and gossypol caused greater decreases than ethanol in those animals that were fed the normal diet. Even though the adrenal glands were enlarged in all gossypol-treated rats, this was accompanied by depressed serum cortisol levels. Histopathological findings, including vacuolar degeneration of hepatocytes and dilation of the sinusoids in the liver, atrophy and leucocytic infiltration of cardiac myofibres, vacuolation of the renal glomerulus and cellular depletion of the spleen and thymus, were most severe in the protein-malnourished, gossypol-treated rats; these were generally absent in the other groups. These findings indicated that either adequate levels of protein in the diet and/or ethanol consumption have the effect of reducing gossypol toxicity, probably by reducing the amount of gossypol available for tissue exposure through enhanced metabolism and/or clearance from the body. However, an evaluation of the metabolic pathways involved in gossypol metabolism in ethanol-fed individuals might provide a better understanding of these observations.

Gossypol toxicosis in the rat associated with protein malnutrition and experimental infection with Trypanosoma brucei.

The toxicity of gossypol, a compound occurring naturally in the cotton plant, was investigated in Trypanosoma brucei-infected, gossypol-treated rats, with and without protein malnutrition. The liver, heart, lungs, spleen and adrenal glands were enlarged in all gossypol-treated rats. Gossypol treatment or trypanosome infection, either alone or together, invariably caused significant reductions in the serum activity of creatine phosphokinase and amylase and in the serum concentration of cortisol. The serum biochemical changes, together with histopathological findings in various organs, indicated that the toxicity of gossypol and pathology of trypanosome infection, either alone or in concert, could be exacerbated by protein malnutrition. This finding suggests that the previously reported antiparasitic properties of gossypol may be of little ultimate benefit due to these serious side effects. The spleen in the protein-malnourished, trypanosome-infected and gossypol-treated animals exhibited only a slight decrease in the number of lymphatic nodules, but a marked cellular depletion, especially of cortical tissue, was observed in the thymus. These observations would seem to justify further study of the immune status of trypanosome-infected, gossypol-treated animals.

Infection with Trypanosoma brucei potentiates the antifertility effect of gossypol, especially in the protein-malnourished male rat.

Reproductive parameters were investigated in gossypol-treated male rats that had been infected experimentally with Trypanosoma brucei and then place on one of two diets of differing (low and normal) protein content. The endpoints assessed were reproductive organ weights, semen epididymal sperm counts, serum testosterone and histological, stereological and histomorphometric evaluation of the testis and accessory reproductive organs. Most of the parameters studied were lowest in the protein-malnourished, gossypol-treated, trypanosome-infected animals, when compared to those obtained from the corresponding animals that were only gossypol-treated or trypanosome-infected. Mean testicular size and the diameter and the total length of seminiferous tubules were especially reduced, indicating that the overall volume of the seminiferous epithelium in these animals was smaller. These findings suggest that reproductive capacity could be impaired in protein-malnourished, trypanosome-infected animals fed on gossypol-containing products, even when there are no obvious clinical signs of disease. This could translate into increased production costs in a farm enterprise. Screening for haemoparasitism would also seem to be worthwhile prior to evaluation and/or use of gossypol (or perhaps other potential contraceptives) in human subjects, especially in communities that are confronted with severe food shortages.

Effects of dietary soybean and cowpea on gut morphology and faecal composition in creep and noncreep-fed pigs.

The effects of creep feeding and different levels of soybean meal (SBM) and cowpea meal on the intestinal morphology and faecal characteristics were investigated in weaners. Prior to the feeding trial, one group of piglets was creep-fed and the other noncreep-fed. The two groups of piglets were weaned at 28 days and randomly assigned to four different diets whose main sources of protein were: T1-skimmed milk powder (control); T2-31 % soybean meal; t(3)-15% soybean meal and 12 % skimmed milk powder; and T(4)-100% raw cowpea meal. Live weight gain was highest in the T1 group, and least in the T(4) group. At weaning only the noncreep-fed weaners showed villus atrophy and crypt hyperplasia, but at 7 days postweaning these changes were evident in all groups except the control and were more severe in the noncreep (T2,T3) and cowpea-fed groups. At 21 days postweaning, only noncreep cowpea-fed pigs showed a reduced villus height when compared to the T1 group. A mild diarrhoea was generally observed in all noncreep-fed weaners but its onset was more rapid (P < 0.01) and the duration longer (P < 0.05) in the T2 and T4 pigs than in T3 and T1 groups. A lower faecal pH was observed in weaners that had diarrhoea when compared with a pH of 7.1 in pigs with normal moisture. The glucose content of the faeces was found to be significantly higher (P<0.05) in the T2 and T4 groups. The observations of enteropathology and low growth performance in the T4 group suggest that feeding raw cowpea to weaners is capable of inducing considerable antigenicity in the intestinal mucosa, causing damage and a consequent decrease in productivity. However, the introduction of creepfeeding before weaning appears to have some ameliorative effects.

The effects of protein malnutrition and experimental infection with Trypanosoma brucei on gossypol treatment in the rat: haematological and serum biochemical changes.

This study was designed to evaluate the interaction between protein malnutrition, gossypol treatment and blood parasitosis (Trypanosoma brucei) in the Wistar rat. Haematological and serum biochemical changes were evaluated in the rats, which were placed on two planes of nutrition--low protein (LP) and normal protein (NP)--and either treated with gossypol or infected with Trypanosoma brucei, or both. Higher parasitaemia occurred in gossypol-treated NP rats than in the corresponding LP group. Gossypol treatment and trypanosomal infection, either alone or in concert, caused an anaemia that was both macrocytic and hypochromic. Both treatments together also caused increases in serum alkaline phosphatase and alanine aminotransferase activities, which were accompanied by depressed serum albumin concentrations, suggestive of hepatic dysfunction in affected rats. These results suggest that, with adequate protein intake, the growth and infectivity of trypanosomes is not inhibited by gossypol but that protein malnutrition has a beneficial effect of reduced parasitaemia. Unfortunately, this beneficial effect is counteracted by gossypol enhancement of hepatic dysfunction caused by trypanosomes.

Evaluation of some reproductive parameters in the indigenous boar of Zimbabwe.

A number of reproductive parameters were evaluated in 12 adult indigenous boars. The seminal vesicles, prostate and bulbourethral glands had masses of 184.41 +/- 18.00 g, 16.69 +/- 2.42 g and 142.05 +/- 16.12 g, respectively, while the penile length measured 45.71 +/- 4.49 cm. The testes and epididymides had masses of 211.82 +/- 26.74 g and 108.81 +/- 11.49 g. The number of sperm averaged 2.78 +/- 0.59 x 10(9) in the testes and 11.76 +/- 2.11 x 10(9) in the epididymides. The daily sperm production per gram (DSPG) of testicular tissue and the daily sperm production rate (DSP) were calculated to be 2.98 +/- 0.31 x 10(6) and 0.62 +/- 0.14 x 10(9) spermatozoa, respectively. The serum level of testosterone measured 11.98 +/- 0.81 ng/ml. These values suggest a reduced reproductive capacity in these animals. However, appropriate selection techniques may be evolved to upgrade this indigenous stock while nutrition and management may be improved to increase body size, and hence, gonadal development.

Haematological and serum biochemical changes in the rat due to protein malnutrition and gossypol-ethanol interactions.

The effects of protein malnutrition on haematological and serum biochemical values were evaluated in gossypol-treated rats which were simultaneously fed with ethanol. Gossypol caused anaemia, leucopenia and thrombocytopenia in malnourished animals, suggesting a depression of bone marrow activity. Gossypol also caused a significant elevation of serum alkaline phosphatase and alanine aminotransferase activities and increases in the concentrations of Mg++ and Ca++ with reduced albumin, regardless of the nutritional status. These changes were more severe with malnutrition. Ethanol alone caused a thrombocytopenia but no other significant haematological changes. However, it appeared to cause derangement of lipid and protein metabolism as reflected in serum cholesterol and urea. The toxic effects seen in gossypol-treated rats were significantly reduced in animals simultaneously given ethanol. As the livers of gossypol-treated rats were significantly heavier than in these animals, it seems possible that ethanol consumption enhances the ability of the liver to metabolize gossypol, thereby reducing its accumulation and consequently its toxicity. However, further studies are needed to determine the mechanisms responsible.