Maternal exposure to di(2-ethylhexyl)phthalate (DEHP) promotes the transgenerational inheritance of adult-onset reproductive dysfunctions through the female germline in mice.

Endocrine disruptors (EDs) are compounds known to promote transgenerational inheritance of adult-onset disease in subsequent generations after maternal exposure during fetal gonadal development. This study was designed to establish whether gestational and lactational exposure to the plasticizer di(2-ethylhexyl)phthalate (DEHP) at environmental doses promotes transgenerational effects on reproductive health in female offspring, as adults, over three generations in the mouse. Gestating F0 mouse dams were exposed to 0, 0.05, 5mg/kg/day DEHP in the diet from gestational day 0.5 until the end of lactation. The incidence of adult-onset disease in reproductive function was recorded in F1, F2 and F3 female offspring. In adult F1 females, DEHP exposure induced reproductive adverse effects with: i) altered ovarian follicular dynamics with reduced primordial follicular reserve and a larger growing pre-antral follicle population, suggesting accelerated follicular recruitment; ii) reduced oocyte quality and embryonic developmental competence; iii) dysregulation of the expression profile of a panel of selected ovarian and pre-implantation embryonic genes. F2 and F3 female offspring displayed the same altered reproductive morphological phenotype and gene expression profiles as F1, thus showing transgenerational transmission of reproductive adverse effects along the female lineage. These findings indicate that in mice exposure to DEHP at doses relevant to human exposure during gonadal sex determination significantly perturbs the reproductive indices of female adult offspring and subsequent generations. Evidence of transgenerational transmission has important implications for the reproductive health and fertility of animals and humans, significantly increasing the potential biohazards of this toxicant.

Maternal exposure to a mixture of di(2-ethylhexyl) phthalate (DEHP) and polychlorinated biphenyls (PCBs) causes reproductive dysfunction in adult male mouse offspring.

We investigated the effects of maternal exposure to the plasticizer di(2-ethylhexyl) phthalate (DEHP) and the organic industrial compounds polychlorinated biphenyls (PCBs), singly and combined, on the reproductive function of male mouse offspring. Mice dams were exposed throughout pregnancy and lactation to 1µg PCBs (101+118)/kg/day, 50µg DEHP/kg/day, or the DEHP/PCB mixture in the diet. The mixture induced permanent alterations in adult F1 males' reproductive health in a way, differently from the single compounds. Depending on the endpoint, we observed: (1) synergy in altering the gross and histological morphology of the testis; (2) antagonism on the expression levels of genes involved in pituitary-gonadal cross-talk; (3) non-interactions on sperm parameters and testosterone production. This study illustrates the complex action of a DEHP/PCB mixture, leading to a unique panel of effects on the male reproductive system, indicating the need for research on the reproductive hazards of combined endocrine disruptors.

Effects of polychlorinated biphenyls in CD-1 mice: reproductive toxicity and intergenerational transmission.

Several studies indicate that in utero and perinatal exposure to polychlorinated biphenyls (PCBs) induces adverse reproductive effects, but it remains unclear whether such effects may be transmitted to subsequent generations. We therefore investigated the association between maternal exposure to PCBs and reproductive health in male and female offspring over three generations. Mouse dams were fed 0, 1, 10, and 100 µg/kg/day of a PCB mixture (101 + 118) during pregnancy and lactation. PCB levels were measured in the tissues of both dams and offspring. PCB concentrations at all doses investigated were greater in the offspring than in the dams (p ≤ 0.0001) confirming that the progeny were exposed as a result of maternal exposure. In F1 offspring, exposure to PCBs resulted in reductions in (1) testis weight (p ≤ 0.05) and seminiferous tubule diameter (p ≤ 0.05), (2) sperm viability (p ≤ 0.0001) and developmental capacity (p ≤ 0.05), (3) ovary weight (p ≤ 0.05), (4) oocyte developmental capacity (p ≤ 0.05), and (5) increased follicular atresia (p ≤ 0.0001). In females, adverse effects were observed only in the F1 animals. In contrast, male offspring exhibited reduced sperm viability and altered seminiferous tubule distribution up to the third generation, showing intergenerational transmission. In summary, our data indicate that exposure to PCBs at the time of gonadal sex determination perturbed, significantly, the reproductive physiology of male and female offspring in adulthood. Furthermore, male reproductive deficiencies may be observed in at least two further generations. These findings have significant implications for reproductive health and fertility of animals and humans.

Exposure to di(2-ethyl-hexyl) phthalate (DEHP) in utero and during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring.

The present study examined the effects in mice of exposure to di(2-ethyl-hexyl) phthalate (DEHP) throughout pregnancy and lactation on the development and function of the pituitary-gonadal axis in male and female offspring once they have attained adulthood. Groups of two to three dams were exposed with the diet from gestational d 0.5 until the end of lactation, at 0, 0.05, 5, and 500 mg DEHP/kg · d. The experiment was repeated three times (total: seven to 10 dams per treatment). The 500-mg dose caused complete pregnancy failure, whereas exposure to doses of 0.05 and 5 mg did not affect pregnancy and litter size. In total, about 30 male and 30 female offspring per group were analyzed. Offspring of the DEHP-treated groups, compared with controls, at sexual maturity showed: 1) lower body weight (decrease 20-25%, P < 0.001); 2) altered gonad weight (testes were ∼13% lighter and ovaries ∼40% heavier; P < 0.001); 3) poor germ cell quality (semen was ∼50% less concentrated and 20% less viable, and ∼10% fewer oocytes reached MII stage, P < 0.001); 4) significant lower expression of steroidogenesis and gonadotropin-receptor genes in the gonads; and 5) up-regulated gonadotropin subunit gene expression in the pituitary. In conclusion, our findings suggest that, in maternally exposed male and female mice, DEHP acts on multiple pathways involved in maintaining steroid homeostasis. Specifically, in utero and lactational DEHP exposure may alter estrogen synthesis in both sexes. This, in turn, induces dysregulation of pituitary-gonadal feedback and alters the reproductive performance of exposed animals.

The role of teat skin contamination in the epidemiology of Staphylococcus aureus intramammary infections.

Knowledge of the epidemiological pattern and the potential sources of infections is important to control Staphylococcus aureus in dairy herds. This paper reports the results of a study applying both pulse field gel electrophoresis (PGFE) and the assessment of a selected number of virulence genes to investigate the role of teat skin on Staph. aureus transmission among cows and on the contamination of milk. Overall 61 isolates were considered, 23 from teat skin, 33 from milk samples and 5 from curd samples. Teat swabs were taken in five herds, but in only three of them could Staph. aureus be isolated. Curd was sampled in three herds, but Staph. aureus could be isolated in only two herds. The distribution of isolates among herds confirmed the presence of herd-specific Staph. aureus strain in most of the herds. The same pattern was observed in teat skin samples, in quarter milk samples, and in the curd samples. Our findings are consistent with other studies showing the role of teat skin as a potential reservoir. Moreover, Staph. aureus was isolated from teat skin of confirmed Staph. aureus-negative cows that were segregated from infected ones. Our findings also suggest that some strains have higher chances to survive on teat skin and therefore to increase the risk for contamination of milk and milk products due to the persistence of intramammary infections.

Mink growth hormone structural-functional relationships: effects of renaturing and storage conditions.

Fourier-transform infrared spectroscopy, in vitro bioassay and enzyme-linked immunoassay were used to study the structural-functional relationships of recombinant mink growth hormone (mGH), refolded and stored under different conditions. Porcine GH (pGH) was synthesized and used as an example. These two hormones, when refolded and stored the same way, had the same secondary structures, biological and immunological efficacy, and biological potency. Only the immunological potency differed, mGH being significantly less potent than pGH. Renaturation pH and storing frozen or at 4 degrees C in 5% glycerol did not affect either the secondary structure or the activity. However, freeze-drying raised the content of buried alpha-helices and lowered that of solvated alpha-helices and of unordered structures. These conformational changes were associated with a reduction of immunological and biological potency of mGH and of immunological potency of pGH. These findings provide original information on the secondary structure of mGH, and show that conformational changes induced by lyophilization adversely affect its activity.

Differentiation of five tuna species by a multiplex primer-extension assay.

A novel methodology based on analysis of mtDNA-cytb diagnostic sites was performed to discriminate four closely related species of Thunnus (Thunnus alalunga, Thunnus albacares, Thunnus obesus and Thunnus thynnus) and one species of Euthynnus (Katsuwonus pelamis) genus in raw and canned tuna. The primers used in the preliminary PCR designed in well conserved region upstream and downstream of the diagnosis sites successfully amplified a 132bp region from the cytb gene of all the species taken into consideration. The sites of diagnosis have been interrogate simultaneously using a multiplex primer-extension assay (PER) and the results were confirmed by fragment sequencing. The applicability of the multiplex PER assay to commercial canned tuna samples was also demonstrated. The proposed test could be useful for detection of fraud and for seafood traceability.

Production of recombinant mink growth hormone in E. coli.

Escherichia coli cells expressing mink (Mustela vison) growth hormone were grown in a batch fermentation process. The expression level was estimated to be 27% of the total cellular protein after 3 h of induction with 1 mM isopropyl beta-D-thiogalactoside (IPTG). If the expression of mink growth hormone (mGH) was induced with 0.2 mM IPTG, the concentration of target protein was slightly lower and was found to be 23% at the same time after induction. mGH expressed as inclusion bodies was solubilized in 8 M urea and renatured by dilution protocol at a protein concentration of 1.4-2.1 mg/ml in the presence of glutathione pair in a final concentration of 11.3 mM. [GSH]/[GSSG] ratio equal to 2/1 was used. Two-step purification process comprising of ion-exchange chromatography on Q-Sepharose and hydrophobic chromatography on Phenyl-Sepharose was developed. Some 25-30 mg of highly purified and biologically active mGH was obtained from 4 g of biomass. The method presented in this study allows producing large quantities of mGH and considering initiation of scientific investigation on mGH effect on mink in vivo and availability in fur industry.

Isoelectric focusing identification of four freshwater fish commercially labeled "perch".

Isoelectric focusing (IEF) was used to distinguish 4 freshwater fish species that are sold in the European Union under the generic label of "perch": Perca fluviatilis (European perch), Lates niloticus (Nile perch), Stizostedion lucioperca (European pikeperch), and Morone chrysops x saxatilis (Sunshine bass). These species have different commercial values but are easily interchangeable because they are sold already filleted, in view of the numerous bones of the whole fish. IEF of the water-soluble proteins extracted from fish muscle resulted in species-specific patterns. None of the bands was common to all 4 species. Intraspecies polymorphism was low and did not concern the bands identified as characteristic of the species.

Characterization of biologically active bovine pituitary FSH purified by immunoaffinity chromatography using a monoclonal antibody.

A substantial amount of highly purified, biologically active bovine FSH was isolated from pituitary extracts by immunoaffinity chromatography based on a novel anti-bovine FSH beta-subunit monoclonal antibody. The biological activity was assessed in vitro using a steroidogenic granulosa cell line constitutively expressing the FSH receptor. Amino acid analysis, N-terminal amino acid sequencing, and peptide mass mapping demonstrated that primary structure modifications do not contribute to the heterogeneity of bovine FSH. The monosaccharide composition of the N-linked oligosaccharides was quantified and remarkably two distinct forms of sialic acids, N-acetyl- and N-glycolyl-neuraminic acids were found. In conclusion, we showed that isoform differences in bovine FSH is likely due only to sugar chain heterogeneity, and we give the first evidence that two substituted sialic acids contribute to the diversity of mammalian glycoprotein hormone isoforms.

Monoclonal antibodies as a probe for the unfolding of porcine growth hormone.

Monoclonal antibodies (mAbs) were generated against pituitary porcine growth hormone (pGH). Ten mAbs were selected for their specificity and affinity for pGH. These mAbs were of the immunoglobulin G (IgG)(1) kappa subclass, with dissociation constants (K(d)) between 7.42 and 0.26 nM, and recognised seven non-overlapping epitopes. We measured whether the mAbs detected alterations of the pGH three-dimensional structure by comparing the antibody reactivity to native pGH and to pGH experimentally unfolded by heating at 50 degrees C, 75 degrees C and 100 degrees C or by reduction and S-carboxymethylation. The antibody-antigen interactions were studied with two enzyme-linked immunosorbent assays (ELISA), based either on a direct binding or inhibition format. The results show that: 1) one mAb, mAb D12, is a conformation-sensitive antibody that recognises an epitope present only in the native pGH. Because the intact three-dimensional structure is essential for the expression of biological activity, mAb D12 could be used to detect altered pGH molecules in biological samples (blood, pituitary extracts or material produced with recombinant technology), and for the one-step purification of biologically active pGH by immunoaffinity chromatography; 2) one mAb, mAb I4, binds to a linear epitope that is not significantly modified in the denatured hormone. This mAb was able to detect the hormone in assays where protein conformation is usually strongly altered, i.e. immunoblotting and immunohistochemistry; 3) the performances of the other eight mAbs differed significantly in the competitive and non-competitive ELISA.