Vitaliano Borromeo

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.

Relationship between some Staphylococcus aureus pathogenic factors and growth rates and somatic cell counts

Staphylococcus aureus isolates produce several pathogenic factors. The combination of these products influences the pathogenic role of different isolates, but their specific effects are well known in the pathogenesis of udder infections. This study focused on the association of polymorphism of the coagulase gene, protein A gene, collagen-binding protein gene, and of fibrinogen-binding protein gene on somatic cell count (SCC) and on Staph. aureus growth rate. Fifty Staph. aureus isolates from 13 dairy cow herds, located in seven different provinces, were considered. The results showed a low frequency of cna gene, similar to the one observed in human isolates. Meanwhile, the high frequency of efb gene indirectly confirmed the role of this factor in bacterial pathogenesis, being associated with adhesion to epithelia. The association of these two single genes with SCC and growth rate showed to be not significant. The polymorphism of spa gene was confirmed to be significantly associated with inflammatory response and growth rate, albeit with a pattern different from the one suggested for human isolates. Sorting of isolates based on the clusters obtained by combining polymorphisms of spa and coa genes and the presence of cna and efb genes, showed that a single cluster (cluster V) was prevalent in the different herds and provinces, while the other six clusters identified were widely spread among the remaining 60% of the isolates. Results showed that clusters VI and VII had significantly higher growth rates at 3, 4, and 6 h in comparison with the other clusters. Meanwhile, quarters infected with these strains showed significantly lower SCC levels. The frequency of isolates from cluster V, suggested that they should possess pathogenic factors increasing their invasiveness, even if in the presence of a stronger inflammatory response. These results indirectly confirm previous findings on the different interactions between isolates and the udder immune system. They also suggest that isolates with higher growth rates and inducing a lower inflammatory response have better chances to spread among the herd. The relatively simple genomic method proposed in this study could be applied by an increasing number of diagnostic laboratories and could be useful in studying the epidemiology of Staph. aureus intra-mammary infections in dairy herds when collecting data from the field.

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.

Relationship between S. aureus gene pattern and dairy herd mastitis prevalence

The importance to evaluate Staphylococcus aureus virulence analysing the combination of virulence genes is largely recognised, and the recent availability of simplified microarray tools allows performing these analyses also in the dairy field. The combined availability of herd-specific S. aureus mastitis prevalence data, isolates from these herds, and microarray technology offered the opportunity to investigate the relationship between S. aureus genetic pattern and their prevalence among dairy herds. Eleven commercial dairy herds following a S. aureus control programme were enrolled in the study, and 33 S. aureus isolates were collected from these herds. Diagnostic DNA microarrays based on the array-tube platform were used for genotyping of staphylococcal DNA. The genetic analysis of the 157-genes microarray showed as only 19 genes were present in all the isolates considered, and among them the genes coding for the leukocidin subunits (LukF, LukS and LukY), haemolysins (hla, hld and an unnamed haemolysin) and enterotoxin X. Several genes considered in the arrays were absent in all the isolates, including the ones encoding the resistance to most of the antimicrobials, except for tetracycline. In our isolates, some agr alleles were never identified (B-III, C-III, D-III, C-IV and D-IV). The comparison of epidemiological data with the genetic pattern suggests that agr type II is associated to the most diffusive isolates, being recovered from the largest number of herds and with the highest frequency. Microarray technique showed to be a useful method to assess the characteristics of virulence of S. aureus isolated in dairy herds and to investigate the relationship with the prevalence of the microorganism. These results support previous evidence that specific gene patterns could be associated to S. aureus mastitis.

A collaboration of aquaporins handles water transport in relation to the estrous cycle in the bitch uterus

Fluid movement through uterine cell membranes is crucial, as it can modulate the tissue imbibition pattern in the different phases of the estrous cycle. To gain insight into the mechanisms underlying steroid-controlled water handling, the presence and distribution of aquaporins (AQPs), integral membrane channel proteins permitting rapid passive water movement, was explored in bitch uterine tissues. Immunohistochemistry and Western immunoblot analysis were used to study the presence of AQP1, AQP2, and AQP5 in the layers of the bitch uterine wall during the different estrous phases. Presence of endothelial nitric oxide-generating enzyme NO synthase (NOS3) was also investigated, as it is known that the vasodilator NOS3 might be involved in the development of uterine edema. The results demonstrated the following: (1) AQP1, AQP2, and AQP5 were present in the uterus of cycling bitches. (2) AQP1 was localized within uterine mesometrial, myometrial, and endometrial blood vessels and in the circular and longitudinal layers of myometrium. AQP1 localization and expression were unaffected by the estrous cycle. (3) The estrogenic milieu was probably at the basis of AQP2 expression in the glandular and luminal epithelium of the endometrium. (4) AQP5 water channels were present in the apical plasma membrane of uterine epithelial cells in coincidence with plasma progesterone increase. (5) NOS3 was localized in the myometrial and epithelial tissues as well as in blood vessels indicating a contribution of this vasoactive peptide to the uterine imbibition processes. Thus, we can hypothesize that a functional and distinctive collaboration exists among diverse AQPs in water handling during the different functional uterine phases.

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.

Growth hormone but not prolactin concentrations in the fluid of bovine ovarian cysts are related to the cystic stage of luteinization

Regulation of follicular growth and ovulation as well as steroid production by the ovary depends principally on gonadotropins. However nonsteroid systemic hormones and autocrine and paracrine factors contribute to the regulation of ovarian function. The objectives of the present work were 1) to asses the presence of growth hormone (GH) and prolactin (PRL) in fluid drawn from normal bovine ovarian follicles, cysts or cystic corpora lutea; 2) to relate the stage of luteinization of the cyst with the GH and PRL concentrations in fluids; and 3) to asses the feasibility of providing a defined nonsteroid hormone marker to distinguish between normal and pathological ovarian structures. Cysts were classified according to histological and morphological appearance as follicular or luteal. Concentrations of GH, PRL, estrogens (E2), progesterone (P4) and testosterone (T) were measured in follicular and cystic fluids. On the basis of the E2 to P4 ratio, ovarian formation classes were further divided into two subclasses (E2 dominant and P4 dominant). The results provide evidence of 1) the presence of immunoreactive GH and PRL in all the follicular and cystic fluids assayed, 2) an increasing concentration of GH correlated to the stage of luteinization of the cyst and a direct correlation between GH and P4 concentrations, 3) a significant variability of intraovarian fluid PRL concentration not related to the histological class of the cyst nor to the concentrations of steroid hormones examined, and 4) the possibility of distinguishing 6 different ovarian formation classes by merely measuring GH, P4, E2 and T concentrations in fluids. These data contribute to a better understanding of the endocrine milieu of bovine ovarian cystic degeneration.

Growth hormone stimulates the secretion of insulin-like growth factor binding protein-2 (IGFBP-2) by monolayer cultures of sheep costal growth plate chondrocytes

Using monolayer cultures of costal chondrocytes established from four week old Clun Forest lambs, we have demonstrated that, under serum free conditions the cells release three IGFBPs (32, 29 and 21 kDa) into the medium. The most abundant of these—the 32 kDa BP-was shown to be IGFBP-2 by Western blotting. Furthermore we demonstrate that the levels of IGFBP 2 in conditioned medium are acutely increased (6, 12 and 24 h time points) following treatment of cells with bovine GH (1–100 ng/ml).In a parallel set of experiments, using ovine fibroblasts (derived from dermis) we show that IGFBPs of Mr 32, 29 and 21 kDa are also secreted by this cell type. However the relative abundance of these BPs differed from that seen in the chondrocyte cultures, with the 21 kDa species now the most abundant. In addition, prolonged exposure of autoradiographs indicated that fibroblasts secreted a higher Mr IGFBP (most probably ovine BP-3) that was not detected in any of our chondrocyte cultures. Most significant however was the demonstration that bGH did not dramatically affect the levels of IGFBPs in fibroblast cell cultures. We conclude that GH stimulates BP-2 production from chondrocytes and this is a cell-type specific effect in as much as it is not replicated in cultures of dermal fibroblasts.

Circadian and circannual plasma secretory patterns of growth hormone and prolactin in fresian heifers: hormonal profiles and signal analysis

The circadian and circannual secretory patterns of growth hormone (GH) and prolactin (PRL) in Fresian heifers were analysed to identify the significant pulses, the shape and the periodic components of the pulsatile signal that may be relevant to intercellular communication. The possible influence of temperature and photoperiod over these hormonal secretions has also been investigated. In the bovine GH (bGH) secretory patterns the basal levels and number, magnitude and amplitude of significant secretory peaks seem to be independent and not affected by seasonal changes. All the bGH circadian patterns show two periodic components with distinct frequencies. The bovine PRL (bPRL) circadian secretion shows basal levels, number and magnitude of the secretory peaks greater in summer than in winter; the periodic components are only one in winter, but two in summer. There is a significant increase of plasma bPRL in the May-July period and a correlation of the circannual hormone profile with both temperature and daylength.

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.