Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors.

In this paper, we investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities on ovarian follicles of adult pigs. Piglets were injected with MXC (20 µg/kg body weight) or corn oil (controls) from postnatal Day 1 to Day 10 (n = 5 per group). Then, mRNA expression, protein abundance and immunolocalization of growth and differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), anti-Müllerian hormone (AMH) and cognate receptors (ACVR1, BMPR1A, BMPR1B, TGFBR1, BMPR2, and AMHR2), as well as FSH receptor (FSHR) were examined in preantral and small antral ovarian follicles of sexually mature gilts. The plasma AMH and FSH levels were also assessed. In preantral follicles, neonatal exposure to MXC increased GDF9, BMPR1B, TGFBR1, and BMPR2 mRNAs, while the levels of AMH and BMP15 mRNAs decreased. In addition, MXC also decreased BMP15 and BMPR1B protein abundance. Regarding small antral follicles, neonatal exposure to MXC upregulated mRNAs for BMPR1B, BMPR2, and AMHR2 and downregulated mRNAs for AMH, BMPR1A, and FSHR. MXC decreased the protein abundance of AMH, and all examined receptors in small antral follicles. GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles of control and treated ovaries. All analyzed receptors were detected in the oocytes and granulosa cells of preantral follicles, and in the granulosa and theca cells of small antral follicles. The exception, however, was FSHR, which was detected only in the granulosa cells of small antral follicles. In addition, MXC decreased the plasma AMH and FSH concentrations. In conclusion, the present study may indicate long-term effects of neonatal MXC exposure on GDF9, BMP15, AMH, and FSH signaling in ovaries of adult pigs. However, the MXC effects varied at different stages of follicular development. It seems that neonatal MXC exposure may result in accelerated initial recruitment of ovarian follicles and impaired cyclic recruitment of antral follicles.

Effects of neonatal exposure to methoxychlor on corpus luteum in gilts: A transcriptomic analysis.

This study investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities, on luteal function in pigs. Piglets were injected subcutaneously with MXC (20 µg/kg body weight) or corn oil (control) between postnatal Days 1 and 10 (N = 5/group). Corpora lutea from sexually mature gilts were examined for luteal steroid and prostaglandin concentrations and processed for total RNA isolation and subsequent RNA sequencing. Intra-luteal concentrations of androstenedione and prostaglandin E2 were greater, while that of estrone was lower when compared to control. Fifty-three differentially expressed (DE) microRNAS (miRNAs) (p-adjusted <.05 and log2(fold change) ≥.5) and 359 DE genes (p-adjusted <.05 and log2(fold change) ≥1) were identified in luteal tissue in response to neonatal MXC treatment. MXC was found to affect the expression of genes related to lipogenesis, steroidogenesis, membrane transport, immune response, cell signaling and adhesion. These results suggest an earlier onset of structural luteolysis in pigs caused by MXC actions in neonates. Since negative correlation analysis showed the potential interactions of miRNAs with specific messenger RNAs, we propose that these miRNAs are potential mediators of the long-term MXC effect on the CL function in pigs.

Altered circadian dynamics of Per2 after cystathionine-ß-synthase and/or cystathionine-γ-lyase pharmacological inhibition in serum-shocked NIH-3T3 cells.

Circadian clock genes are found in almost every cell that has a nucleus; they regulate the rhythmic nature of all processes that are cyclical. Among the genes controlled by the circadian clock, there are numerous factors that regulate key processes in the functioning of the cell. Disturbances in the functioning of the circadian clock are associated with numerous disorders. A recent study has shown the key role of H2S in regulating circadian rhythm. In this study, we investigated the in vitro effect of pharmacological inhibition of cystathionine-ß-synthase (CBS) and/or cystathionine-γ-lyase (CSE) on the circadian dynamics of Per2 expression in serum-shocked NIH-3T3 cells. Alternatively, Cbs and Cse were knocked down by transfection with siRNA. The 48-h treatment of serum-shocked NIH-3T3 cells with 1 mM dl-propargylglycine (PAG), a specific CSE inhibitor, significantly decreased the amplitude and baseline expression of Per2. During exposure to an effective CBS and CSE inhibitor (aminooxyacetic acid [AOAA]), the amplitude of oscillation and baseline expression of Per2 significantly increased. Incubation of NIH-3T3 cells with both inhibitors also significantly increased the amplitude and baseline expression of Per2 messenger RNA (mRNA). siCbs or siCse knockdowan significantly reduced the baseline and amplitude of oscillation of Per2. In conclusion, we showed that CBS/CSE/H2S pathway participates in the regulation of the circadian clock system. PAG and AOAA, change the general expression and dynamics of Per2 genes, but the increase of amplitude and overall Per2 mRNA level due to exposure to AOAA is probably caused by factors other than CBS and CSE activity.

Risk of wild fungi treatment failure: Phallus impudicus-induced telomere damage triggers p21/p53 and p16-dependent cell cycle arrest and may contribute to male fertility reduction in vitro.

The multifunctional characteristics of Phallus impudicus extract encourage to conduct research for its potential use in medical applications. Well, science is constantly seeking new evidence for the biological activity of extracts of natural origin. Drugs of natural origin should not cause any side effects on the physiological functions of the human body; however, this is not always successful. In this study, we used in vitro approach to evaluate the toxicity of alcohol Phallus impudicus extract on spermatogenic cells. We show, for the first time, cytotoxic properties of Phallus impudicus treatment associated with a decrease in cellular metabolic activity, dysregulation of redox homeostasis and impairment of selected antioxidant cell protection systems. As a consequence, p53/p21- and p16-mediated cell cycle arrest followed by p27 activation is initiated. The observed changes were associated with telomere shortening and numerous DNA damage at the chromosome ends (altered expression pattern of TRF1 and TRF2 proteins), as well as upregulation of cleaved caspase-3 with a decrease in Bcl-2 expression, suggesting induction of apoptotic death. Therefore, these results provide molecular evidence for mechanistic pathways and novel adverse outcomes linked to the Phallus impudicus treatment towards men's health and fertility reduction.

Molecular Consequences of Depression Treatment: A Potential In Vitro Mechanism for Antidepressants-Induced Reprotoxic Side Effects.

The incidence of depression among humans is growing worldwide, and so is the use of antidepressants. However, our fundamental understanding regarding the mechanisms by which these drugs function and their off-target effects against human sexuality remains poorly defined. The present study aimed to determine their differential toxicity on mouse spermatogenic cells and provide mechanistic data of cell-specific response to antidepressant and neuroleptic drug treatment. To directly test reprotoxicity, the spermatogenic cells (GC-1 spg and GC-2 spd cells) were incubated for 48 and 96 h with amitriptyline (hydrochloride) (AMI), escitalopram (ESC), fluoxetine (hydrochloride) (FLU), imipramine (hydrochloride) (IMI), mirtazapine (MIR), olanzapine (OLZ), reboxetine (mesylate) (REB), and venlafaxine (hydrochloride) (VEN), and several cellular and biochemical features were assessed. Obtained results reveal that all investigated substances showed considerable reprotoxic potency leading to micronuclei formation, which, in turn, resulted in upregulation of telomeric binding factor (TRF1/TRF2) protein expression. The TRF-based response was strictly dependent on p53/p21 signaling and was followed by irreversible G2/M cell cycle arrest and finally initiation of apoptotic cell death. In conclusion, our findings suggest that antidepressants promote a telomere-focused DNA damage response in germ cell lines, which broadens the established view of antidepressants' and neuroleptic drugs' toxicity and points to the need for further research in this topic with the use of in vivo models and human samples.

Klotho-mediated changes in the expression of Atg13 alter formation of ULK1 complex and thus initiation of ER- and Golgi-stress response mediated autophagy.

In the previous paper of our group, we have demonstrated that one of the crucial factors involved in the crosstalk between autophagy and apoptosis is klotho protein. We have shown that klotho silencing in normal human fibroblasts intensifies lipopolysaccharide (LPS)-induced p-eIF2a-mediated stress of endoplasmic reticulum and thus leads to retardation of prosurvival autophagy and induction of apoptotic cell death. In this study, we have performed a detailed step-by-step analysis of autophagy flux-related genes' expression and endoplasmic reticulum and Golgi stress related pathways in order to determine the exact mechanistic event when autophagy is inhibited in klotho-deficient cells on account of apoptosis initiation. We provide evidence that klotho-silencing in LPS-treated cells results in differential course of ER- and Golgi-mediated stress response. Further, we show that in klotho-deficient cells formation of ULK1 complex is inhibited and thus autophagy initiation is blocked on the account of apoptosis activation, while in the control cells cytoprotective autophagy is activated. Finally, in klotho-deficient cells formation of ULK1 complex is prevented by downregulated expression of Atg13. Thus, this study suggests a novel targeting pathway for efficient elimination of autophagy-deficient cells.

Autophagy as a consequence of seasonal functions of testis and epididymis in adult male European bison (Bison bonasus, Linnaeus 1758).

The European bison is still an animal endangered with extinction, so by learning factors that regulate its reproduction, we can contribute to the survival of this species. On the other hand, autophagy is a dynamic, lisosomal, and evolutionary conserved process which is essential for animal cell survival, homeostasis, and differentiation. This process was demonstrated in many species and in many organs; however, information on the metabolic course of autophagy in the male reproductive system in seasonally reproducing species is lacking. Therefore, in this study, we examined for the first time several autophagy-related factors (mTOR, ULK1, Atg13, PI3K, beclin1, beclin2, Atg14, Atg5, Atg16L, LC3) in testicular and epididymal tissues obtained from adult male individuals of the European bison. We compared the level of gene expression, protein synthesis, and localization of autophagy-related factors between June, September, and December (before, during, and after reproductive activity, respectively). We confirmed that the induction of autophagy was at the highest level in the period after reproductive activity, i.e., in December, when a significant increase in the gene and protein expression was observed for the majority of these factors, probably to ensure cellular protection. However, autophagy was also clearly marked in September, during the intense spermatogenesis, and this may indicate a great demand for autophagy-related proteins required for the normal development of reproductive cells. Obtained results seem to confirm that autophagy pathway, as a consequence of seasonal reproduction, may control the normal course of spermatogenesis in the male European bison.

Altered dynamics in the circadian oscillation of clock genes in serum-shocked NIH-3T3 cells by the treatment of GYY4137 or AOAA.

BACKGROUND AND PURPOSE: Several members of the core clock mechanism are equipped with a Per-Arnt-Sim (PAS) domain through which they can bind haem [Fe(II)]. Haem is a ligand for the orphan receptors REV-ERBα/ß (NR1D1/2), which regulate circadian rhythm and metabolism. The ability to bind haem sensitises these clock components to the action of small molecule gases, including NO, CO and H2S. Studies conducted with European hamsters revealed that during winter sleep, key clock genes stop oscillating. At the same time, H2S, when administered at subtoxic concentrations, can induce a hypometabolic state in the cell. We suppose that core clock components, including the nuclear receptors REV-ERBs, neuronal PAS domain protein 2 (nPAS2) and PER2, can be H2S targets. The general objective of this study was to investigate the effect of the H2S system on the expression profile of the core clock genes in cells in vitro. EXPERIMENTAL APPROACH: We analysed the expression of Per1, Per2, Per3, Bmal1, Cry1, Cry2, Nr1d1, Nfil-3 and Dbp messenger RNA (mRNA) in serum-shocked NIH-3T3 cells treated with a slow-releasing H2S donor (GYY4137) or the cystathionine beta-synthase (CBS) inhibitor (AOAA) cultured under constant darkness and collected during 3 days in 3 h interval. KEY RESULTS AND CONCLUSIONS AND IMPLICATIONS: We found that pharmacological CBS inhibition increased the general expression and dynamics of several clock genes. On the other hand, increased H2S decreased Per2 expression. These data suggest that CBS can affect circadian clock and effect on clock-controlled transcription output.

The seasonal changes of the heme oxygenase in the retina pig.

The eye is a very important organ in the human body which is affected by various external factors. One of these factors is the sunlight which can cause the visual impairment and as well as the increase in the oxidative stress. The heme oxygenase I (HO-1) plays a very important role in the fight against the oxidative stress. The HO enzyme catalyses the degradation of the heme to the ferrous iron, the biliverdin and the carbon monoxide (CO). The HO-2 is the isoform HO-1 and is mainly constitutively expressed. We have studied the changes in the HO-1 and the HO-2 in the retina on the level of the RNA and the protein in the summer and in the winter season (the biggest difference is in the length of the day light). The retina of the eye was obtained from the breeding pigs in concern (Sus scrofa f. domestica) posthumously. The expression of the HO-1 genes in the retina cells is higher in the winter and the amount of protein decreases. However, the HO enzyme concentration definitely increases in the summer, when the production of the free radicals (the oxidative stress) related to the exposition to the sunlight is greater. The obtained results suggest that various factors have the influence on the protein synthesis. One of the factors, can be the miRNA which blocks the synthesis of the HO. Another factors, influencing the HO are the biological clock, the sunlight and the UV radiation associated with it.

Klotho modulates ER-mediated signaling crosstalk between prosurvival autophagy and apoptotic cell death during LPS challenge.

Bacterial endotoxins have been shown to induce prosurvival autophagy or apoptosis in fibroblasts and thus impair the wound healing process. Endoplasmic reticulum has been proposed as a molecular switch between these processes and klotho protein possessing pleiotropic characteristics seems to be involved in both processes, however the exact molecular mechanism is unknown. In this study, we have evaluated the effect of klotho silencing on human fibroblasts exposed to a non-toxic dose of lipopolysaccharide in terms of in vitro wound healing ability. We show for the first time, that klotho silencing in fibroblasts intensified lipopolysaccharide-induced oxidative stress and inflammatory response, what resulted in genomic instability, p-eIF2a-mediated ER stress, retardation of prosurvival autophagy, induction of apoptotic cell death and finally in impaired wound closure. Therefore, our data suggest that klotho serves as a part of cellular defense mechanism engaged in providing protection against bacterial infections during wound healing by modulating ER-signaling crosstalk between autophagy and apoptosis.

Neuronal life or death linked to depression treatment: the interplay between drugs and their stress-related outcomes relate to single or combined drug therapies.

Depression is a serious medical condition, typically treated by antidepressants. Conventional monotherapy can be effective only in 60-80% of patients, thus modern psychiatry deals with the challenge of new methods development. At the same moment, interactions between antidepressants and the occurrence of potential side effects raise serious concerns, which are even more exacerbated by the lack of relevant data on exact molecular mechanisms. Therefore, the aims of the study were to provide up-to-date information on the relative mechanisms of action of single antidepressants and their combinations. In this study, we evaluated the effect of single and combined antidepressants administration on mouse hippocampal neurons after 48 and 96 h in terms of cellular and biochemical features in vitro. We show for the first time that co-treatment with amitriptyline/imipramine + fluoxetine initiates in cells adaptation mechanisms which allow cells to adjust to stress and finally exerts less toxic events than in cells treated with single antidepressants. Antidepressants treatment induces in neuronal cells oxidative and nitrosative stress, which leads to micronuclei and double-strand DNA brakes formation. At this point, two different mechanistic events are initiated in cells treated with single and combined antidepressants. Single antidepressants (amitriptyline, imipramine or fluoxetine) activate cell cycle arrest resulting in proliferation inhibition. On the other hand, treatment with combined antidepressants (amitriptyline/imipramine + fluoxetine) initiates p16-dependent cell cycle arrest, overexpression of telomere maintenance proteins and finally restoration of proliferation. In conclusion, our findings may pave the way to better understanding of the stress-related effects on neurons associated with mono- and combined therapy with antidepressants.

Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes.

Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells.

Aloe arborescens juice prevents EMF-induced oxidative stress and thus protects from pathophysiology in the male reproductive system in vitro.

More and more studies suggest that prolonged exposure to EMF may cause adverse biological effects and point directly to a significantly negative correlation between EMF and human health, especially men fertility. In our previous study, we reported that this could be related to the EMF-induced reactive oxygen species formation, followed by DNA damage, cell cycle arrest and apoptosis induction. In this study, we decided to expand our research by the search for substances which would prevent EMF-induced damage in spermatogenic cells. Such an agent seems to be Aloe arborescens Mill. juice, which was shown to possess a wide range of protective properties. The administration of aloe extract helps among others to prevent the formation of free radicals by various biochemical pathways. Therefore, the main aim of our study was to provide a significant knowledge concerning the mechanism involved in the multi-pathway cytoprotective response of aloe juice against EMF. The study was carried out in an in vitro mouse spermatogenesis pathway cell lines (GC-1 spg and GC-2 spd). Our results suggest that the aloe juice has many positive effects, especially for the cellular antioxidant systems by reducing the intracellular reactive oxygen species pool induced by EMF. In consequence, aloe juice prevents DNA damage, cell cycle arrest and therefore the viability and metabolic activity of both cell line tested are preserved. In conclusion, our study provides new insight into the underlying mechanisms through which aloe juice prevents spermatogenic cells from cytotoxic and genotoxic events.

Vascular endothelial growth factor (VEGF-A) and fibroblast growth factor (FGF-2) as potential regulators of seasonal reproductive processes in male European bison (Bison bonasus, Linnaeus 1758).

Growth factors: vascular endothelial growth factor A (VEGF-A) and fibroblast growth factor (FGF-2) were reported to affect normal physiological reproductive processes in human, domestic and free living animals. Moreover, some reports suggest that VEGF-A and FGF-2 may be directly involved in the control of the annual reproductive cycle of seasonally breeding animals but detailed knowledge is still missing. Our study aimed to demonstrate the expression of mRNA and protein for both factors in the tissues of testis and epididymis (caput, corpus, cauda) at different periods of the year (March, June, November, December) in European bison as a model of seasonally breeding animal. Results suggest, that VEGF-A expression was more pronounced in testis than in epididymis and the highest expression was noted in December and June. Surprisingly, the highest protein accumulation was observed in June at the same level in all tissues analyzed. On the other hand, the highest FGF-2 mRNA expression was noted in testis in June and in epididymis in March. However, no differences in protein expression of FGF-2 were found between analyzed groups. The results indicate that both factors are necessary for proper functioning of the reproductive system and their levels differ seasonally. Perhaps, it is linked to increased need of these factors in the testis as well as epididymis during preparation for the reproductive functions. Moreover, VEGF-A and FGF-2 not only may regulate reproductive functions by affecting vascularization and cell nutrition, but it also may be possible that they possess protective functions by stabilizing the reproductive cells. Therefore, obtained results provide new insight into mechanisms underlying seasonal breeding of the male European bison.

Extremely low frequency variable electromagnetic fields affect cancer and noncancerous cells in vitro differently: Preliminary study.

The exposure to extremely low frequency electromagnetic field (ELF-EMF) may result in various changes at the cellular level. To identify the effect of ELF-EMF exposure on viability of cells, cancer cells (U87-MG; 143B) and noncancerous cells (BJ; HEK) in exponential growth phase were exposed or sham-exposed to different values of frequency (2, 20, 30, 50 and 60 Hz), different shapes (sinusoidal, square and triangular) and time of exposure (0.5, 1, 2, 3 h) to electromagnetic field. After exposure, viability of cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We found a different effect of exposition of cancer and noncancerous cells to ELF-EMF on viability of cells. This preliminary study revealed that electro magentic field(EMF) might serve as a potential tool for manipulating viability of cells.

Impact of endocrine-active compounds on adrenal androgen production in pigs during neonatal period.

This study investigated the impact of neonatal exposure to endocrine-active compounds (EACs): flutamide (antiandrogen), 4-tert-octylphenol (an estrogenic compound), and methoxychlor (an organochlorine insecticide exhibiting estrogenic, antiestrogenic and antiandrogenic activities) on androgen production within porcine adrenal glands. The expression of genes related to androgen synthesis and the level of androgen production were analyzed (i) in the adrenal glands of piglets exposed to EACs during the first 10 days of life (in vivo study), and (ii) in adrenal explants from sow-fed or formula-fed 10-day-old piglets incubated with EACs (ex vivo study). EACs affected the expression of genes linked to adrenal androgen biosynthesis. The prominent effect of methoxychlor on downregulation of StAR, CYP11A1 and HSD3B and upregulation of CYP17A1 and SULT2A1 were demonstrated. Furthermore, our study revealed divergent response to EACs between sow-fed and formula-fed piglets, suggesting that natural feeding may provide protection against adverse EACs effects, particularly those interfering with estrogens action.

Antiandrogen flutamide affects folliculogenesis during fetal development in pigs.

Androgen deficiency during prenatal development may affect the expression of genes involved in the folliculogenesis regulation. In order to study the effect of antiandrogen on fetal ovarian development, pregnant gilts were injected with flutamide (for 7 days, 50 mg/kg bodyweight per day) or corn oil (control groups) starting on gestation days 43 (GD50), 83 (GD90), or 101 (GD108). The obtained fetal ovaries were fixed for histology and immunohistochemistry or frozen for real-time PCR. Morphological evaluation, TUNEL assay, and expression of selected factors (Ki-67, GATA binding transcription factor 4 (GATA4), E-Cadherin and tumor necrosis factor a (TNFa)) were performed. On GD90 and GD108, ovaries following flutamide administration showed a higher number of egg nests and lower number off ollicles than those in respective control groups. An increased mRNA and protein expression of Ki-67 was observed in flutamide-treated groups compared with controls on GD50 and GD108 but decreased expression was found on GD90. In comparison to control groups a higher percentage of TUNEL-positive cells was shown after flutamide exposure on GD50 and GD90 and a lower percentage of apoptotic cells was observed on GD108. These data were consistent with changes in TNF (TNFa) mRNA expression, which increased on GD90 and decreased on GD108. E-cadherin mRNA and protein expression was upregulated on GD50 and downregulated on GD90 and GD108. In conclusion diminished androgen action in porcine fetal ovaries during mid- and late gestation leads to changes in the expression of genes crucial for follicle formation. Consequently, delayed folliculogenesis was observed on GD90 and GD108. It seems however that androgens exhibit diverse biological effects depending on the gestational period.

Senescence and autophagy relation with the expressional status of non-canonical estrogen receptors in testes and adrenals of roe deer (Capreolus capreolus) during the pre-rut period.

The roe deer bucks represent a spontaneous model to study the synchronized testicular involution and recrudescence cycles. However, cellular processes and hormonal control of steroidogenic glands are scarcely known. For the present study testes and adrenal glands obtained from roe deer during the pre-rut season were used. We aimed to determine (i) senescence and autophagy involvement in testis atrophy (immunohistochemical analysis for tumor suppressor protein encoded by the cyclin-dependent kinase inhibitor 2A; p16 and microtubule-associated protein 1A/1B-light chain 3; LC3, respectively), (ii) the size of the adrenal cortex and medulla (morphometric analysis), (iii) G-protein coupled estrogen receptor (GPER) and estrogen-related receptors (ERRs; type α, ß, and Y) distribution and expression (qRT-PCR and immunohistochemical analyses) and (iv) serum testosterone and estradiol levels (immunoassay ELISA). This study revealed pre-rut characteristics of testis structure with the presence of both senescence and autophagy-positive cells and higher involvement of senescence, especially in spermatogenic cells (P < 0.05). In the adrenal cortex, groups of cells exhibiting shrinkage were observed. The presence of ERRs in cells of the seminiferous epithelium and interstitial Leydig cells and GPER presence distinctly in Leydig cells was revealed. In adrenals, these receptors were localized in groups of normal-looking cells and those with shrinkage. Morphometric analysis showed differences in cortex width which was smaller (P < 0.05) than that of the medulla. A weak immunohistochemical signal was observed for ERRß when compared to ERRα and ERRγ. The mRNA expression level of ERRα and ERRγ was lower (P < 0.001 and P < 0.05, respectively) while ERRß was higher (P < 0.001) in adrenals when compared to testes. mRNA GPER expression was similar in both glands. In the pre-rut season, the testosterone level was 4.89 ng/ml while the estradiol level was 0.234 ng/ml. We postulate that: (i) senescence and autophagy may be involved in both reinitiation of testis function and/or induction of abnormal processes, (ii) hormonal modulation of testis inactivity may affect adrenal cortex causing cell shrinkage, (iii) ERRs and GPER localization in spermatogenic cells and interstitial cells, as well as cortex cells, may maintain and control the morpho-functional status of both glands, and (iv) androgens and estrogens (via ERRs and GPER) drive cellular processes in the testis and adrenal pre-rut physiology.

Cadmium-induced changes in genomic DNA-methylation status increase aneuploidy events in a pig Robertsonian translocation model.

Although cadmium is a well-established human carcinogen, the mechanisms by which it induces cancer are poorly understood. It is suggested that cadmium-mediated carcinogenesis may include the modulation of gene expression and signal-transduction pathways, interference with antioxidant enzymes, inhibition of DNA repair and DNA methylation, and induction of apoptosis. Nevertheless, no predominant mechanism playing a role in metal-induced carcinogenesis has been reported. In the present study, we used a pig Robertsonian translocation model, which is a cross between a wild boar and domestic pig resulting in Robertsonian translocation (37,XX,der15;17 or 37,XY,der15;17), to determine the role of cadmium sulfate in the modulation of genomic DNA-methylation status and the induction of aneuploidy. We found a cadmium-mediated increase in aneuploidy within chromosome group A and C, but not within chromosome group D containing the translocated chromosome der15,17 which indicates that translocated chromosome is not more prone to chromosomal aberrations than are other chromosomes. We suggest that cadmium-induced aneuploidy (up to 5-µM concentration) may be mediated by global DNA hypermethylation as monitored with HPLC and 5-mdC immunostaining. In addition, the cyto- and genotoxic potential of cadmium was evaluated. Cadmium sulfate was able to induce apoptosis, inhibit cell-proliferative status and expression of nucleolar organizer regions (NORs), and increase oxidative DNA damage (8-oxoG content).

Cancer on-target: Selective enhancement of 3-bromopyruvate action by an electromagnetic field in vitro.

Cancer is one of the leading causes of death in the modern world. Nowadays, most often treatment methods used in clinical oncology are drug therapies applied as monotherapy or combined therapy. Additionally, recent studies focus on developing approaches with the use of a drug in combination with other factors, not only chemical, to improve the probability and magnitude of therapeutic responses and reduce the possibility of chemoresistance. Such a promising factor seems to be an electromagnetic field (EMF) application. Here, we tested the effect of continuous or pulsed EMF on human cancer cells of different origin treated or not with 3-bromopyruvate, a small and powerful alkylating agent with a broad spectrum of anticancer activities. We provide strong evidence suggesting that ELF-EMF potentiates the anti-cancer activity of 3BP in human cancer cells through inhibition of TNFα secretion leading to irreversible p21/p27-dependent G2/M cell cycle arrest and finally cancer cell death. Our findings suggest a novel approach combining pharmacotherapy with ELF-EMF. In conclusion, electromagnetic field seems to be a potential modulator of anti-cancer efficacy of 3BP while combined therapy offers off-target activity. These features contribute to the development of innovative therapeutic strategies for cancer treatment.