Zearalenone induces chromosome aberrations in mouse bone marrow: preventive effect of 17β-estradiol, progesterone and Vitamin E

The cytogenetic effect of zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, was evaluated in vivo, in mouse bone marrow cells, by assessing the percentage of cells bearing different chromosome aberrations. The studies included different conditions for animal treatment, as follows: (1) single intraperitoneal (ip) injection, (2) repeated ip injections, (3) pre-treatment for 24 h with Vitamin E (Vit E), and (4) pre-treatment for 4 h with 17β-estradiol (17β-Est) or progesterone (Prog). ZEN induced different types of chromosome aberrations, which was concentration-dependent (2–20 mg/kg bw). These doses corresponded to 0.4–4% of the LD₅₀ in the mouse. Interestingly, when the dose of ZEN (40 mg/kg) was fractionated into four equivalent doses (4 × 10 mg/kg bw), into three doses (15 + 10 + 15 mg/kg bw), or into two equivalent doses (2 × 20 mg/kg bw), given every 24 h, the percentage of chromosome aberrations increased significantly. This finding suggests that ZEN proceeds by reversible binding on receptors that could become saturated, and that it damages the chromosomes in a ‘hit and go’ manner. Furthermore, pre-treatment of animals with 17β-estradiol or progesterone significantly decreased the percentage of chromosome aberrations, suggesting that (i) these hormones bind to the same cytoplasmic receptors transported into the nucleus to elicit DNA damage, (ii) they may play a role in preventing chromosome aberrations induced by ZEN. Similarly, Vit E prevented these chromosome aberrations indicating that Vit E, previously reported to prevent most of the toxic effects induced by ZEN, may also bind to the same receptors.     

In vitro binding to and in vivo effect on the cytosol and nuclear progesterone receptors of various progestins,and their relationship to synthesis of uteroglobin in rabbit uterus

In vitro binding affinities of various progestins to cytosol and nuclear progesterone receptors of rabbit uterus were determined and correlated with the biological potency of these steroids. In addition, cytosol and nuclear progesterone receptor levels were measured after a 5-day administration of different progestins (0.5 mg/kg daily) with variable biologic activites. The receptor levels were compared with the bilological response; the induction of uteroglobin synthesis. Cytosol and nuclear progesterone receptors had identical steroid binding properties (r = 0.98). The correlation between the in vitro binding affinity (cytosol or nuclear) and the in vivo biologic activity of the steroids was good (r = 0.73). After a 5-day treatment with progestins, the nuclear receptor concentration correlated in an inverse manner (r = ?0.84) with the uterine fluid unteroglobin concentration. A similar, but slightly weaker correlation (r = ?0.81) was also found for the cytosol receptor content and uteroglobin secretion. These data indicate that not only nuclear, but also cytosol progesterone receptor levels decrease in the rabbit uterus during chronic hormone action. Decline in the nuclear progesterone receptor content seemed to occur during treatment with all progestational steroids, while onlyi progestins with high biological potency were capable of decreasing the cytosol receptor content.     

Sensitivity of progesterone receptors to aurintricarboxylic acid: Inhibition of nuclear uptake,ATP and DNA binding

When hen oviduct cytosol samples containing progesterone receptor complexed to [³H]progesterone were included with isolated nuclei in presence of 0.2 mM aurintricarboxylic acid, more than 50% inhibition occurred in the uptake of progesterone receptor by the nuclei. The activated form of progesterone receptor appeared to be more sensitive to the presence of aurintricarboxylic acid since pretreatment of non-activated progesterone receptor with the inhibitor and the subsequent removal of the latter prior to activation did not result in the inhibition of receptor uptake by the nuclei. Also, the binding of progesterone receptor to columns of DNA-cellulose or ATP-Sepharose was abolished under simmilar conditions. When nuclei, ATP-Sepharose or DNA-cellulose were preincubated with the inhibitor prior to the addition of receptor preparations, no such inhibition resulted indicating that the inhibitor may be interacting with the receptor protein and not complexing to ATP, DNA or sites in the nuclei. The steroid binding properties of progesterone receptor, however, remained intact under these conditions. Both A and B forms of progesterone receptor are equally sensitive to aurintricarboxylic acid presence when tested for their nuclear uptake. Aurintricarboxylic acid was also found to be very effective at low concentrations (0.25 mM) in eluting the receptor complexes off ATP-Sepharose columns without disrupting the steroid binding properties of progesterone receptor. Our results suggest that auintricarboxylic acid is an effective inhibitor of progesterone receptor and that it may be acting by interfering with a site(s) on progesterone receptor which may be exposed upon activation and are involved in such processes as ATP binding, nuclear uptake and DNA binding. These observations suggest the use of aurintricarboxylic acid as a chemical probe for the analysis of progesterone receptor.     

Antiluteogenic effects of serial prostaglandin F2α administration in cycling mares

A single dose of PGF2α does not consistently induce luteolysis in the equine CL until at least 5 days after ovulation, leading to the erroneous assumption that the early CL is refractory to the luteolytic effects of PGF2α. We hypothesized that serial administration of PGF2α in early diestrus would induce a return to estrus similar to mares treated with a single injection in mid-diestrus, and fertility of the induced estrus would not differ. The objectives of the study were to evaluate the effects of the 2 approaches as reflected by: (1) concentrations of plasma progesterone; (2) interovulatory and treatment-to-ovulation intervals; (3) the proportion of mares pregnant after artificial insemination. The study consisted of a balanced crossover design in which 10 reproductively normal Quarter Horse Mares were exposed to 2 treatments on 2 consecutive reproductive cycles. At detected ovulation (Day 0), mares were randomly allotted to 1 of 2 treatment groups: I, mid-diestrus treatment, administration of a single 10-mg dose of dinoprost tromethamine (PGF2α) im on Day 10; II, early diestrus treatment, administration of 10-mg PGF2α im twice daily on Days 0, 1, and 2 and once daily on Days 3 and 4. Mares in estrus and with a follicle 35 mm or greater in diameter were artificially inseminated with at least 2 billion motile sperm from a fertile stallion. Pregnancy was defined as detection of a growing embryonic vesicle on 2 consecutive examinations approximately 14 days after ovulation. Serial plasma samples were collected throughout the study period, and concentration of plasma progesterone was determined by RIA. A mixed-model ANOVA for repeated measures was used to analyze hormonal data. Interovulatory and treatment-to-ovulation intervals were compared by a paired t test and fertility by a McNemar chi-square analysis. All mares in group I underwent luteolysis after PGF2α administration denoted by mean (±SD) concentration of plasma progesterone of 0.25 ± 0.21 ng/mL detected 2 days after treatment. In group II, mean concentration of plasma progesterone remained below 1.0 ng/mL during treatment and until the onset of the next estrus. The mean interovulatory interval in group I was 18.5 ± 2.0 days compared with 13.1 ± 3.7 days in group II (P < 0.01). Treatment-to-ovulation intervals were 8.5 ± 2.0 days and 13.1 ± 3.7 days for groups I and II, respectively (P < 0.05). In both groups, 9 of 10 mares were pregnant (P = 1.0). Serial PGF2α administration beginning at ovulation consistently prevented luteal function in 10 of 10 mares in the present study without adversely affecting pregnancy rate of post-treatment cycles.     

Dissecting the signaling pathways involved in the function of sperm flagellum

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility.     

Hormone Rhythms and Breast Cancer Chronoepidemiology: Salivary Progesterone Concentrations in Pre- and Post-Menarchal Girls and in Normal Premenopausal Women

Orcadian and circatrigintan time series of salivary progesterone levels in premenarchal and adolescent girls and healthy mature premenopausal women have been investigated as a possible determinant for breast cancer risk. Circadian variations in progesterone appear to be more random than systematic and estimates of total daily progesterone output are better represented by samples pooled from several 2-hr specimens. Different patterns of circatrigintan progesterone secretion in girls are recognised and relate to those experienced in infertile and fertile women, though their relation to chronological or menarchal age is as yet uncertain. These data suggest that the measurement of salivary progesterone at premenarche, adolescence and maturity is a feasible, though statistically difficult, study for prospective identification of individuals at risk for breast cancer.     

The effect of progesterone on the localization of progesterone receptors in the nuclei of chick oviduct cells

Summary The location of occupied and unoccupied progesterone receptors (PR) in chick oviduct cells was studied by immuno-electron microscopy with the use of a highly specific polyclonal anti-PR antibody and pre-embedding modifications of the peroxidase-anti-peroxidase-(PAP-) or immunogold-silver methods. Both methods revealed a nuclear localization of the PRs. The location of the PR in the nucleus was studied in detail by means of the immunogold-silver method. The most intense labelling for unoccupied PRs was in the condensed chromatin. After occupation of PRs with progesterone (P), decondensation or dispersion of chromatin was observed. At the same time, the labelling in the border area of condensed and dispersed chromatin, and in the dispersed chromatin, increased. The changes were statistically significant. The results can be explained by conformational changes of the PR-containing chromatin rather than by translocation of PRs from one site to another.     

Progesterone and a phospholipase inhibitor increase the endosomal bis(monoacylglycero)phosphate content and block HIV viral particle intercellular transmission

Progesterone, the cationic amphiphile U18666A and a phospholipase inhibitor (Methyl Arachidonyl Fluoro Phosphonate, MAFP) inhibited by 70%–90% HIV production in viral reservoir cells, i.e. human THP-1 monocytes and monocyte-derived macrophages (MDM). These compounds triggered an inhibition of fluid phase endocytosis (macropinocytosis) and modified cellular lipid homeostasis since endosomes accumulated filipin-stained sterols and Bis(Monoacylglycero)Phosphate (BMP). BMP was quantified using a new cytometry procedure and was increased by 1.25 times with MAFP, 1.7 times with U18666A and 2.5 times with progesterone. MAFP but not progesterone or U18666A inhibited the hydrolysis of BMP by the Pancreatic Lipase Related Protein 2 (PLRP2) as shown by in-vitro experiments. The possible role of sterol transporters in steroid-mediated BMP increase is discussed.     

Follicular 17β-estradiol and progesterone concentrations and degree of cumulus cell expansion as predictors of in vivo-matured oocyte developmental competence in superstimulated heifers

The quality of an oocyte is crucial for successful generation of offspring, but few selection parameters have been identified that reliably predict oocyte developmental competence. The objective of the present study was to determine whether the developmental competence of in vivo-matured oocytes derived from superstimulated heifers could be predicted by 17β-estradiol and progesterone concentrations in follicular fluid, degree of cumulus cell expansion, and follicular diameter. Cumulus oocyte complexes were individually collected from follicles ≥8 mm 22 hours after an induced LH peak and individually fertilized and cultured in vitro. Only oocytes that originated from follicles with 17β-estradiol ≤0.25 μM and progesterone ≥0.26 μM developed into blastocysts. When a combination of these cutoff values was evaluated as a predictor of oocyte competence, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 75%, 49%, and 100%, respectively. Hormone concentrations in follicular fluid were also associated with the degree of cumulus cell expansion and only cumulus oocyte complexes with full expansion developed into blastocysts; sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 71%, 45%, and 100%, respectively, when full expansion was used as the predictive criterion for blastocyst production. Follicular diameter was not a good predictor of oocyte competence. In conclusion, concentrations of 17β-estradiol and progesterone in the preovulatory follicle and the degree of cumulus cell expansion are predictors of blastocyst production in superstimulated heifers and can be used as selection markers for oocyte developmental competency.     

Assessment of HER-2 gene overexpression in Isfahan province breast cancer patients using Real Time RT-PCR and immunohistochemistry

Purpose

Overexpression of proto-oncogene HER-2 is one of the main molecular markers of breast cancer involved in prognosis and diagnosis and also in trastuzumab therapy. Thus, a request for the evaluation of HER-2 status in breast cancer has been increasing. The aim of our study was assessment of HER-2 overexpression in malignant and benign breast cancer specimens by Real Time RT-PCR technique and comparison of its results with IHC outcomes.

Methods

Twenty benign and sixty malignant breast cancers in addition to fifteen normal breast tissue specimens were analyzed by Real Time RT-PCR method. Fresh tissue samples were disrupted by mortar and pestle. A syringe and a needle were used for complete homogenization of the tissues. The RNA was then isolated from the samples and converted to cDNA. A standard curve was initially plotted using BioEasy SYBR Green I and then all 95 specimens were studied by Real Time RT-PCR using 2^− ΔΔCt method.

Results

23.3% of 60 malignant specimens showed HER-2 overexpression, while all of the benign samples represented the normal expression level of HER-2 gene. The concordance rate between the results of Real Time RT-PCR and IHC was 86.6%.

Conclusion

Real Time RT-PCR method is an almost reliable technique and at least can be used as a complementary method for confirming IHC results. This is emanated from relatively high rate of concordance between outcomes of IHC test, as a routine method of detecting the HER-2 gene expression status, and Real Time RT-PCR technique.