Objectives: The aim of our study was to determine a possible relationship between the redox state of the organism and the exposure to TiO2 Nanoparticles (NPs). Design and methods: TiO2 NPs (1% or 10% LD50 TiO2) were intraperitoneally administered to female Wistar rats from the Postnatal Day 4 (PND4) to PND7. Rats were sacrificed on the PND15 (infantile rats) or after the PND220 (adult rats). In plasma, Antioxidant Capacity (TEAC), concentration of protein carbonyls and lipoperoxides were measured. Hemolysates were used to determine the changes in catalase, Glutathione Peroxidase (GPx) and Superoxide Dismutase (SOD) activities. Results: Young rats: 1% and 10% LD50 TiO2 induced the decrease in TEAC, lipoperoxide levels and Gluthione Peroxidase (GPx) activities and higher Superoxide Dismutase (SOD) activities in infantile rats, compared to controls. Adults: Significantly lower lipoperoxide levels, higher SOD and GPx activities were found in adults compared to young rats. 1% LD50 TiO2 significantly increased TEAC value and reduced SOD and GPX activities, but 10% LD50 TiO2 reduced TEAC compared to controls. Conclusion: Our results demonstrate that neonatal exposure to TiO2 NPs may modulate redox balance in female rats depending on the age, which might lead to alterations of their physiological functions.

Bone morphogenetic protein-15 (BMP-15), an oocyte-derived growth factor, has been shown to play integral roles in regulation of ovarian follicular function in mammals. Despite the recognition of the physiological importance of the BMP system in regulation of gonadotropin action in the ovary, molecular mechanisms of BMP-15 effect on oocyte and somatic follicular cell functions remain poorly understood. The objective of this study was to determine the effect of BMP-15 on the FSH/LH-stimulated synthesis of hyaluronan (HA) by oocyte cumulus complexes (OCC) and progesterone by OCC and granulosa cells (GC) in the presence or absence of serum using primary porcine cultures. In addition, the effect of BMP-15 on oocyte maturation- and steroidogenesis-related transcripts after 4, 8, 16, and 24 hours of cultivation was evaluated using real-time RT-PCR. We demonstrated that the FSH/LH-induced cumulus expansion was accompanied by a significant increase in CD44, PTGS2, CYP11A1 (at 4 h) and AREG, HAS2, TNFAIP6, STAR (at 8 h) mRNAs. While FSH/LH-stimulated total HA synthesis by OCC was not affected by BMP-15 in serum-supplemented medium, its retention within the complex was significantly increased after the action of BMP-15 in comparison to FSH/LH alone (P < 0.001; 65% versus 35%, respectively). Moreover, we detected a significant increase in the expression of AREG and TNFAIP6 (both at 16 h), and CYP11A1 (at 24 h) in FSH/LH-stimulated OCC due to the action of BMP-15 compared to complexes cultured only with FSH/LH. In the presence of serum, BMP-15 markedly increased FSH/LH-stimulated progesterone secretion by OCC (about 69%) and induced a significant decrease in FSH/LH-induced progesterone release by GC (about 35%) compared to FSH/LH alone. The present results indicate that the addition of BMP-15 to the gonadotropin-stimulated OCC cultured in serum-supplemented medium might improve oocyte-cumulus maturation.

Wildlife intrusion has been associated with pathogen contamination of produce. However, few studies have examined pathogen transfer from wildlife feces to pre-harvest produce. This study was performed to calculate transfer coefficients for Escherichia coli from simulated wildlife feces to field-grown lettuce during irrigation. Rabbit feces inoculated with a 3-strain cocktail of non-pathogenic E. coli were placed in a lettuce field 2.5–72 h before irrigation. Following irrigation, the E. coli concentration on the lettuce was determined. After exclusion of an outlier with high E. coli levels (Most Probable Number = 5.94*108), the average percent of E. coli in the feces that transferred to intact lettuce heads was 0.0267% (Standard Error [SE] = 0.0172). Log-linear regression showed that significantly more E. coli transferred to outer leaves compared to inner leaves (Effect = 1.3; 95% Confidence Interval = 0.4, 2.1). Additionally, the percent of E. coli that transferred from the feces to the lettuce decreased significantly with time after fecal placement, and as the distance between the lettuce and the feces, and the lettuce and the sprinklers increased. These findings provide key data that may be used in future quantitative risk assessments to identify potential intervention strategies for reducing food safety risks associated with fresh produce.

Nanosized titanium dioxide (TiO2) particles belong to the most widely manufactured nanoparticles (NPs) on a global scale because of their photocatalytic properties and the related surface effects. TiO2 NPs are in the top five NPs used in consumer products. Ultrafine TiO2 is widely used in the number of applications, including white pigment in paint, ceramics, food additive, food packaging material, sunscreens, cosmetic creams, and, component of surgical implants. Data evidencing rapid distribution, slow or ineffective elimination, and potential long-time tissue accumulation are especially important for the human risk assessment of ultrafine TiO2 and represent new challenges to more responsibly investigate potential adverse effects by the action of TiO2 NPs considering their ubiquitous exposure in various doses. Transport of ultrafine TiO2 particles in systemic circulation and further transition through barriers, especially the placental and blood-brain ones, are well documented. Therefore, from the developmental point of view, there is a raising concern in the exposure to TiO2 NPs during critical windows, in the pregnancy or the lactation period, and the fact that human mothers, women and men in fertile age and last but not least children may be exposed to high cumulative doses. In this review, toxicokinetics and particularly toxicity of TiO2 NPs in relation to the developing processes, oriented mainly on the development of the central nervous system, are discussed Keywords: nanoparticles, nanotoxicity, nanomaterials, titanium dioxide, reproductive toxicity, developmental toxicity, blood brain barrier, placental barrier.

This study was designed to determine whether inhibition of either cyclooxygenase-2 (COX-2) by indomethacin or progesterone receptor (PR) by PR antagonist, RU486, affects oocyte maturation, progesterone production, and covalent binding between hyaluronan (HA) and heavy chains of inter-α trypsin inhibitor, as well as expression of cumulus expansion-associated proteins (HA-binding protein, tumor necrosis factor α-induced protein 6, pentraxin 3) in oocyte-cumulus complexes (OCCs). The experiments were based on freshly isolated porcine OCC cultures in which the consequences of PR and COX-2 inhibition on the final processes of oocyte maturation were determined. Granulosa cells (GCs) and OCCs were cultured in medium supplemented with FSH/LH (both 100 ng/mL) in the presence/absence of RU486 or indomethacin. Western blot analysis, (3)H-glucosamine hydrochloride assay, immunofluorescence, and radioimmunoassay were performed. Only treatment with RU486 (25 μM) caused a decrease in the number of oocytes that reached germinal vesicle breakdown and metaphase II stage compared with indomethacin (100 μM) or FSH/LH treatment alone after 44 h. All treated OCCs synthesized an almost equal amount of HA. Heavy chains (of inter-α trypsin inhibitor)-HA covalent complexes were formed during in vitro FSH/LH-stimulated expansion in RU486- or indomethacin-treated OCCs. Follicle-stimulating hormone/LH-induced progesterone production by OCCs was increased in the presence of RU486 after 44 h. In contrast, a decrease of FSH/LH-stimulated progesterone production by GCs was detected in the presence of either RU486 or indomethacin after 72 h. We suggest that the PR-dependent pathway may be involved in the regulation of oocyte maturation. Both PR and COX-2 regulate FSH/LH-stimulated progesterone production by OCCs and GCs.

Production of steroid hormones by the ovary plays a key role in the female phenotype maintenance, as well as is critical for regular ovarian processes, including follicle growth, oocyte maturation and ovulation. Thus, optimal ovarian steroid synthesis is an indispensable requisite for the female reproductive health. In the past decades, along with an increased incidence of female reproductive disorders, an increasing concern for the potential reproductive impact of exogenous factors, particularly of environmental pollutants with endocrine disrupting properties, has risen. The scientific studies report that ovarian steroid hormone production is being recognized as an important target for the action of endocrine disrupting chemicals (EDCs). The fact that these chemicals have been detected in the biological samples of general population, and even directly in the follicular fluid of women, emphasizes the demands for testing the influence of EDCs on ovarian steroidogenesis. For these purposes, different methodological approaches have been employed, from in vivo studies on female rodents to in vitro experimental procedures using steroidogenically active follicular cells. In the present review, the effects of selected EDCs (pesticides, phthalate and phenol derivatives, and halogenated arylhydrocarbons) on the processes of ovarian steroidogenesis are summarized, and possible mechanisms of action of these agents are outlined.

The essential role of vitamin D (VD) in bone metabolism and mineral homeostasis is well established knowledge. Research indicates that classical and non-classical pathways of VD affect also cell proliferation and differentiation, the immune system, infection, and cancer. VD receptor (VDR) and VD metabolizing enzymes have been detected in female reproductive tissues, such as ovary, uterus and placenta. The presence of VD metabolites was demonstrated in follicular fluid (FF) in women undergoing in vitro fertilization and embryo transfer (IVF-ET). The recent studies show that VD regulates the expression of a large number of genes in reproductive tissues implicating a role for VD in female reproduction and pregnancy outcomes. There is increasing human and animal data suggesting that VD status may be associated with impaired fertility, endometriosis, polycystic ovary syndrome (PCOS), and ovarian cancer. The presence of VDR in both animal and human ovarian tissue has raised the question of a possible direct role for 1α,25-dihydroxyvitamin D [1α,25(OH)2D3] in the regulation of steroid hormone synthesis and secretion. Our recent data have demonstrated that 1α,25(OH)2D3 may affect in vitro insulin- and follicle-stimulating hormone (FSH)-induced progesterone secretion by porcine ovarian granulosa cells. The molecular mechanisms of this action should be further investigated.

UNLABELLED:Reproduction is a result of coordinated signaling network between gonads and pituitary/hypothalamus. Ovarian functions, such as follicular development, ovulation, luteinisation, luteolysis, and remodeling of the endometrium, are controlled by endocrine, paracrine and autocrine factors. Ovulation is a unique biological process by which the complex of a mature oocyte and surrounding somatic cumulus cells (CCs), named oocyte-cumulus complex (OCC), is released from the follicle into the oviduct for transport and fertilization. Recently, evidence has been accumulated that the immune system might represent an additional local regulator of the ovarian functions that are essentially modulated by gonadotropins. Moreover, the ovulation is similar to an inflammatory response: follicles become hyperemic, produce prostaglandins (PGs), and synthesize a hyaluronan-rich extracellular matrix. Cytokines are originally referred as numerous signaling substances secreted by certain cells of the immune system which influence the activity of other cells. A number of studies have shown that cytokines may modulate ovarian functions and play an important role in the ovulation. The most known cytokines related to the reproduction are interleukins (ILs). These molecules have been localized in the reproduction-related body fluids and various ovarian cell types, such as the oocytes, granulosa (GCs) and theca cells (TCs) in several mammalian species. Moreover, macrophages in the ovary have been shown to secrete cytokines, including ILs. The present review summarizes the current knowledge on ILs in regard of their role in the regulation of selected ovarian functions.KEYWORDS:cytokines, interleukins, ovary, steroidogenesis, ovulation, corpus luteum.

Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of TNFAIP6 and HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of TNFAIP6 and HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway-regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.

OBJECTIVE:The aim of the present study was to introduce a new approach of the light microscopic immunohistochemical triple-staining enabling to study the differences in the activity of at least two different phenotypes of neurons on the same histological section. For this purpose combination of Fos (a product of the immediate early gene) labeling with nickel intensified diaminobenzidine (DAB-Ni) and two neuropeptides labeled with Alexa488 and Alexa555 fluorescent dyes on cryo-processed 35-40 µm thick free-floating brain sections was selected.METHODS:The parallel occurrence of three antibodies studied, i.e. Fos, hypocretin (HCRT), and melanin-concentrating hormone (MCH), was studied by a new methodic approach utilizing combination of Fos immunolabeled with DAB-Ni and HCRT and MCH labeled with Alexa488 and Alexa555 fluorescent dyes, respectively. Fos stimulation was induced by a single immobilization (IM0) for 120 min. Then, the rats were sacrificed, the brains removed, soaked with 30% sucrose in 0.1 M phosphate buffer (PB), cryo-sectioned throughout the hypothalamus into 35-40 μm thick coronal sections, collected, and washed in the same buffer for 10-15 min. Fos was revealed by avidin-biotin-peroxidase (ABC) complex and visualized by diaminobenzidine chromogen containing nickel chloride salt. HCRT and MCH neurons were visualized by the above mentioned fluorescent dyes. Evaluation of the Fos and fluorescent staining was performed in the computerized Axo Imager Carl Zeiss microscope using light and fluorescent illuminations.RESULTS:All the antibodies used showed clear immunoreactive staining. Fos staining occurred in the form of black color located in the cell nuclei. HCRH and MCH neuropeptides showed clear green and red fluorescence in the cell perikarya, respectively. The final merged picture showed Fos protein in the activated green HCRT or red MCH neurons in the form of white nuclei.CONCLUSIONS:The present study clearly demonstrate that the combination of Fos labeling with DAB-Ni and neuropeptides labeled with Alexa488 and Alexa555 on cryo-processed 35-40 µm thick free-floating brain sections is an excellent approach providing further advantages for quick and reproducible triple immuno-staining enabling to compare the activity of at least two phenotypes of neurons on the same section.KEYWORDS:Alexa488 and Alexa555 fluorescent dyes, Fos, hypocretin, melanin-concentrating hormone, cryostat sections, triple labeling immunohistochemistry, rat.

Drug delivery to the central nervous system (CNS) represents one of the most priority challenges in research and development of pharmaceutical nanotechnology products. Among the various non-invasive approaches for CNS delivery, nanoparticle carriers and particularly polymeric nanoparticles (PNs) seem to be one of the most interesting. This review deals with PNs as CNS drug delivery systems and their potential endocrine disrupting properties. Possible interference with the development of neuroendocrine-reproductive system is considered. Special regard is being paid to potential mechanisms of PNs toxicity. Necessity to investigate the toxicity of nanomaterials and their impact on human health are discussed.

Effects of FSH, LH and follicular fluid isolated from preovulatory follicles on secretion of progesterone, cAMP a cGMP by cultured immature porcine granulosa cells were investigated. Exposure of granulosa cells to gonadotropins and follicular fluid resulted in increase of progesterone and cGMP formation. Follicular fluid had an inhibitory effect on cAMP synthesis. The findings suggest a possible intermediary role of cGMP for the stimulatory action of follicular fluid in the process of luteinization of granulosa cells.

Treatment of the rat ovarian membrane-bound and Triton X-100 solubilized LH/hCG receptor with the tryptophan-specific reagents N-bromosuccinimide (NBS) and 2-hydroxy-5-nitrobenzyl bromide (HNB-Br) resulted in inactivation of the receptor to bind hCG. Fluorescence quenching studies indicated that oxidation of tryptophan residues by NBS decreased the accessibility of fluorophores for acrylamide. Preceding binding of hCG to receptor sites was found to protect fluorophores from NBS action. Modification of tryptophan residues was associated with alteration in the rigidity of ovarian membranes and with destabilization of the LH/hCG receptor structure. The results suggest that tryptophan residue is essential for hCG binding to the receptor.

The time and dose dependent cytotoxic effects of phenol derivatives on human skin carcinoma cells A431 were investigated. The cells were cultured for 16, 24, 48 and 72 hours with 4-octylphenol (OP), 4-nonylphenol (NP), phenylphenol (PP), bisphenol A (BPA) and bisphenol A dimetacrylate (BPA-DM) in the dose range 0.1-100 ng/mL. The cultured media and compounds tested were changed for fresh ones every 24 h. The release of lactate dehydrogenase (LDH) was used as the marker of cytotoxicity on cell membrane level. The results express the effect of substance exceeding basal values of control cells. The highest effect was induced by acute (16 h) and chronic (72 h) treatment with 5 ng/mL BPA, while the maximal effect of BPA-DM was shifted to higher doses of 25-20 ng/mL. The dose response curves displayed inverted U shaped responses, i.e. higher doses did not induce an elevated effect. NP was less effective than OP. Chronic exposure to 20 ng/mL PP had the highest effect, with decreased efficiency present at higher doses. Maximal LDH cytotoxic activity for low doses of the environmental pollutants tested has important implication in hazard and risk assessment for human health.

Studies aimed at the influence of smoking on reproductive functions have found out fertility disorders in smokers occurring at any stage of reproductive processes. In our experiments the role of cadmium, nicotine and anabasine was investigated in the expansion of oocyte-cumulus complexes (OCC) isolated from large antral porcine follicles. Suppression of FSH-induced cumulus expansion and significant inhibition of synthesis and accumulation of hyaluronic acid in the cell/matrix compartment of the OCC was observed in the presence of different concentrations of tested compounds. The suppressive effect of cadmium and tobacco alkaloids on the cumulus expansion was accompanied by decreased progesterone production by cumulus cells during 42 h incubation of the OCC with FSH.