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C Callies, T G Cooper and C H Yeung

The nature of the membrane channels mediating water transport in murine spermatozoa adjusting to anisotonic conditions was investigated. The volume of spermatozoa subjected to physiologically relevant hypotonic conditions either simultaneously, or after isotonic pre-incubation, with putative water transport inhibitors was monitored. Experiments in which quinine prevented osmolyte efflux, and thus regulatory volume decrease (RVD), revealed whether water influx or efflux was being inhibited. There was no evidence that sodium-dependent solute transporters or facilitative glucose transporters were involved in water transport during RVD of murine spermatozoa since phloretin, cytochalasin B and phloridzin had no effect on volume regulation. However, there was evidence that Hg2 +- and Ag+-sensitive channels were involved in water transport and the possibility that they include aquaporin 8 is discussed. Toxic effects of these heavy metals were ruled out by evidence that mitochondrial poisons had no such effect on volume regulation.

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TAPANI VANHA-PERTTULA

Summary.

Aminoacyl naphthylamidases or aminopeptidases of the rat testicular tissue were fractionated by DEAE-cellulose chromatography and their subcellular site was evaluated by continuous sucrose gradient centrifugation. Four different enzymes were separated with distinct enzymatic properties.

Enzyme I preferentially hydrolysed methionyl-β-NA followed by valyl-, isoleucyl-, leucyl-, phenylalanyl- and alanyl-β-NA at pH 7·0. A slight activation with cysteine and EDTA and a marked inhibition with heavy metal ions was observed. This enzyme was connected to the particles of the mitochondrial—lysosomal fraction. Its main site of function is presumably within the testicular interstitial tissue.

Enzyme II readily hydrolysed a wide variety of substrates with preference for lysyl- and arginyl-β-NA at pH 6·8. It was markedly activated by chelating agents and sensitive to heavy metal ions as well as to some divalent metal ions. This enzyme was tentatively localized in membranous structures and exclusively in the testicular interstitial tissue.

Enzyme III showed the typical characteristics of aminopeptidase B with a marked activation by halide ions. It hydrolysed only arginyl- and lysyl-β-NA optimally at pH 6·5. It is a soluble enzyme and mainly located within the seminiferous tubules.

Enzyme IV showed a large substrate spectrum with optimum at pH 8·0. It was an SH-dependent soluble enzyme. Its main site was within the seminiferous tubules.

None of the four enzymes appear specific for testicular tissue since similar enzymes have been reported in other tissues. Their physiological significance can be evaluated with differential quantification during experimental conditions.

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E Gómez, E Correia-Álvarez, J N Caamaño, C Díez, S Carrocera, N Peynot, D Martín, C Giraud-Delville, V Duranthon, O Sandra and M Muñoz

Early in cow embryo development, hepatoma-derived growth factor (HDGF) is detectable in uterine fluid. The origin of HDGF in maternal tissues is unknown, as is the effect of the induction on developing embryos. Herein, we analyze HDGF expression in day 8 endometrium exposed to embryos, as well as the effects of recombinant HDGF (rHDGF) on embryo growth. Exposure to embryos did not alter endometrial levels of HDGF mRNA or protein. HDGF protein localized to cell nuclei in the luminal epithelium and superficial glands and to the apical cytoplasm in deep glands. After uterine passage, levels of embryonic HDGF mRNA decreased and HDGF protein was detected only in the trophectoderm. In fetal fibroblast cultures, addition of rHDGF promoted cell proliferation. In experiments with group cultures of morulae in protein-free medium containing polyvinyl alcohol, adding rHDGF inhibited blastocyst development and did not affect cell counts when the morulae were early (day 5), whereas it enhanced blastocyst development and increased cell counts when the morulae were compact (day 6). In cultures of individual day 6 morulae, adding rHDGF promoted blastocyst development and increased cell counts. Our experiments with rHDGF indicate that the growth factor stimulates embryonic development and cell proliferation. HDGF is synthesized similarly by the endometrium and embryo, and it may exert embryotropic effects by autocrine and/or paracrine mechanisms.

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J. A. THOMAS, M. MAWHINNEY and G. WENGER

Summary.

Prostate gland fructose was measured enzymatically in normal and castrate mice. Regardless of protein precipitation procedure, castration resulted in a loss of prostatic fructose.

Prostate gland fructose levels were higher, using the resorcinol method, only when tricholoroacetic acid (TCA) was used to precipitate tissue proteins. Similar procedures employing supernatants obtained from barium hydroxide-zinc sulphate precipitated organs were comparable to enzymatically measured fructose.

The use of radio-active fructose and fructose-1,6-diphosphate established that heavy metal precipitation resulted in over 90% of the labelled fructose residing in the supernatant and a similar amount of phosphate ester found in the precipitate. The use of TCA to precipitate prostate gland proteins revealed that both fructose and fructose-1,6-diphosphate were predominantly found in the supernatant (93 and 95% respectively). Very little radio-activity was found in the precipitate fraction, indicating that TCA was unable to separate free hexose adequately from phosphate esters.

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M. Kantola, M. Saaranen and T. Vanha-Perttula

Summary. High levels of selenium and glutathione peroxidase (GSH-Px) were found in bull seminal plasma but low concentrations in human seminal plasma. In man the seminal plasma selenium was associated with two macromolecules separable by gel filtration, but no GSH-Px was found in the same fractions. Selenium in bull seminal plasma was associated with two proteins, which could be separated by gel filtration and anion exchange chromatography. Both macromolecules coeluted with GSH-Px activity and had identical optima at pH 7·0. Their responses to thermal treatment, however, differed. Seminal vesicle secretory fluid in the bull contained both these proteins, while the larger molecule was also found in fractionations of ampulla, prostate and Cowper's glands. The larger enzyme form is evidently a tetramer of the smaller one. Both enzyme forms were extremely sensitive to heavy metals and some divalent metal ions. GSH caused an activation while other reducing agents were suppressive. Triton X-100 had no effect, while sodium deoxycholate was inhibitory. These properties are typical for a phospholipid hydroperoxide GSH-Px. It is concluded that this selenium-dependent enzyme may be important in the protection of bovine spermatozoa against damage caused by oxygen radicals, while in man such a mechanism is not functional.

Keywords: selenium; glutathione peroxidase; seminal plasma; human; bull

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T Leahy, JP Rickard, RJ Aitken and SP de Graaf

Abstract

Head-to-head agglutination of ram spermatozoa is induced by dilution in the Tyrode’s capacitation medium with albumin, lactate and pyruvate (TALP) and ameliorated by the addition of the thiol d-penicillamine (PEN). To better understand the association and disassociation of ram spermatozoa, we investigated the mechanism of action of PEN in perturbing sperm agglutination. PEN acts as a chelator of heavy metals, an antioxidant and a reducing agent. Chelation is not the main mechanism of action, as the broad-spectrum chelator ethylenediaminetetraacetic acid and the copper-specific chelator bathocuproinedisulfonic acid were inferior anti-agglutination agents compared with PEN. Oxidative stress is also an unlikely mechanism of sperm association, as PEN was significantly more effective in ameliorating agglutination than the antioxidants superoxide dismutase, ascorbic acid, α-tocopherol and catalase. Only the reducing agents cysteine and dl-dithiothreitol displayed similar levels of non-agglutinated spermatozoa at 0 h compared with PEN but were less effective after 3 h of incubation (37 °C). The addition of 10 µM Cu2+ to 250 µM PEN + TALP caused a rapid reversion of the motile sperm population from a non-agglutinated state to an agglutinated state. Other heavy metals (cobalt, iron, manganese and zinc) did not provoke such a strong response. Together, these results indicate that PEN prevents sperm association by the reduction of disulphide bonds on a sperm membrane protein that binds copper. ADAM proteins are possible candidates, as targeted inhibition of the metalloproteinase domain significantly increased the percentage of motile, non-agglutinated spermatozoa (52.0% ± 7.8) compared with TALP alone (10.6% ± 6.1).

Reproduction (2016) 151 1–10

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D. Tsatas, M. S. Baker and G. E. Rice

A number of tightly regulated proteolytic enzyme systems, including the plasminogen activation cascade and matrix metalloproteases, play integral roles in the remodelling of extracellular matrices during pregnancy and parturition. This study assessed these labour-associated changes in protease activity in human gestational tissues. Amnion, choriodecidua and placenta collected from women before (at caesarean section, not in labour), during (at caesarean section, in labour) and after (spontaneous-onset labour, normal vaginal delivery) labour were examined on gelatin-substrate SDS-PAGE zymography. All tissues displayed major 55 kDa plasminogen-dependent activity that was abolished by the serine protease inhibitors (10 mmol phenylmethyl-sulphonylfluoride l−1, 100 mmol epsilon aminocaproic acid l−1, 1 mmol Glu-Gly-Arg chloromethylketone l−1). The enzymic activity was identified as urokinase plasminogen activator on the basis of its co-migration with reference standard and western blot analysis, and did not vary with labour status. An additional protease with an apparent molecular mass of approximately 90 kDa was detected in all tissues. Densitometric measurement of these tissues showed a significant (P < 0.05) increase in this enzyme activity with labour onset. Heavy metal chelators (1 mmol 1, 10 phenanthroline l−1 and 10 mmol EDTA l−1) selectively blocked the 90 kDa activity, consistent with the proposal that it is a metalloprotease. Co-migration with reference standard and western blot analysis confirmed the identity of this protease as the matrix metalloprotease 9 (MMP-9). Immunoreactive MMP-9 protein was also significantly (P < 0.05) increased during and after labour compared with before labour in all tissues examined. It is proposed that the upregulated expression of MMP-9 is involved in fetal membrane rupture and placental separation during and after labour onset, respectively. In conclusion, the regulated repertoire of protease activities expressed by human gestational tissues implies an important role for matrix-degrading enzymes during human parturition.

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Fátima Nogales, M Luisa Ojeda, María Fenutría, M Luisa Murillo and Olimpia Carreras

Selenium (Se), an essential trace metal, is important in both growth and reproduction and is the constituent of different selenoproteins. The glutathione peroxidase (GPx) family is the most studied as it prevents oxidative stress. Liver oxidation is considered as another mechanism involved in low birth weight. Therefore, in order to ascertain whether GPx is related to the effects of Se on growth during gestation and lactation, three groups of rat pups were used: control, Se deficient (SD), and Se supplemented (SS). Morphological parameters and reproductive indices were evaluated. Hepatic Se levels were measured by graphite furnace atomic absorption while spectrophotometry was used for activity of antioxidant enzymes and oxidative stress markers in liver and western blotting for expression of hepatic GPx1 and GPx4. The SD diet increased mortality at birth; decreased viability and survival indices; and stunted growth, length, and liver development in offspring, thus decreasing hepatic Se levels, GPx, glutathione reductase, and catalase activities, while increasing superoxide dismutase activity and protein oxidation. The SS diet counteracted all the above results. GPx1 expression was heavily regulated by Se dietary intake; however, although Se dietary deficiency reduced GPx4 expression, this decrease was not as pronounced. Therefore, it can be concluded that Se dietary intake is intimately related to growth, length, and directly regulating GPx activity primarily via GPx1 and secondly to GPx4, thus affecting liver oxidation and development. These results suggest that if risk of uterine growth retardation is suspected, or if a neonate with low birth weight presents with signs of liver oxidation, it may be beneficial to know about Se status.

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J. M. Goldman, T. E. Stoker, R. L. Cooper, W. K. McElroy and M. B. Parrish

The presence of noradrenergic neuronal innervation in the ovaries and cyclic alterations in ovarian noradrenaline suggest a role for such innervation in oocyte release. The current experiments evaluated the relationship between ovulation and alterations in ovarian concentrations of noradrenaline induced by unilateral, intrabursal administration of the specific noradrenergic neurotoxin DSP4. Intrabursal injections of DSP4 (0–10 μmoles per ovary) given at 19:00 h at pro-oestrus induced a prompt, dose-related reduction in ovarian noradrenaline on the injected and non-injected sides. Although this result suggests that injected material was reaching the contralateral ovary, ovulation was suppressed only on the injected side. This suppression was persistent, and lasted through at least the next two cycles following either unilateral or bilateral treatment. The reductions in noradrenaline could be mostly, if not entirely, attenuated by prior administration of desipramine which blocks re-uptake of noradrenaline, while the ipsilateral ovulatory effects remained unchanged. Although it has been reported that DSP4 binds the opiate receptor, intrabursal co-administration of the antagonist naloxone was ineffective in altering ovulatory suppression. These results suggest that while decreases in ovarian noradrenaline in response to local exposure to a noradrenergic neurotoxin may accompany a reduction in oocyte release or a block in ovulation, the anti-ovulatory effect of DSP4 is independent of the changes in noradrenaline concentrations and may be due to some other ovarian response.

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Wendy N Jefferson, Heather B Patisaul and Carmen J Williams

Phytoestrogens, estrogenic compounds derived from plants, are ubiquitous in human and animal diets. These chemicals are generally much less potent than estradiol but act via similar mechanisms. The most common source of phytoestrogen exposure to humans is soybean-derived foods that are rich in the isoflavones genistein and daidzein. These isoflavones are also found at relatively high levels in soy-based infant formulas. Phytoestrogens have been promoted as healthy alternatives to synthetic estrogens and are found in many dietary supplements. The aim of this review is to examine the evidence that phytoestrogen exposure, particularly in the developmentally sensitive periods of life, has consequences for future reproductive health.