Adverse eff ects of polymeric nanoparticle poly(ethylene glycol)- block-polylactide methyl ether (PEG-b-PLA) on steroid hormone secretion by porcine granulosa cells.

OBJECTIVES: Development of nanoparticles (NPs) for biomedical applications, including medical imaging and drug delivery, is currently undergoing a dramatic expansion. Diverse effects of different type NPs relating to mammalian reproductive tissues have been demonstrated. Th e objective of this study was to explore the in vitro effects of polymeric nanoparticle poly(ethylene glycol)-blockpolylactide methyl ether (PEG-b-PLA NPs) on functional state and viability of ovarian granulosa cells (GCs), which play an important role in maintaining ovarian function and female fertility. METHODS: The GCs isolated from porcine ovarian follicles were incubated with the different concentrations of PEG-b-PLA NPs (PEG average Mn=350 g/mol and PLA average Mn=1000 g/mol; 0.2-100 µg/ml) or poly(ethylene glycol) with an average molecular weight of 300 (PEG-300; 0.2- 40 mg/ml) in the presence or absence of stimulators, follicle-stimulating hormone (FSH; 1 µg/ml), androstenedione (100 nM), forskolin (10 µM) or 8Br-cAMP (100 µM), for different time periods (24, 48, 72 h). At the end of the incubation, progesterone and estradiol levels produced by GCs were measured in the culture media by radioimmunoassay. Th e viability of GCs was determined by the method using a colorimetric assay with MTT. RESULTS: Treatment of GCs with PEG-b-PLA NPs induced a significant decrease in basal as well as FSH-stimulated progesterone secretion above the concentration of 20 and 4 µg/ml, respectively. Moreover, PEG-b-PLA NPs reduced forskolin-stimulated, but not cAMP-stimulated progesterone production by GCs. A dose-dependent inhibition of androstenedione-stimulated estradiol release by GCs was found by the action of PEG-b-PLA NPs. Incubation of GCs with PEG-300 significantly inhibited basal as well as FSH-stimulated progesterone secretion above the concentration of 40 mg/ml. PEG-b-PLA NPs and PEG-300 significantly reduced the viability of GCs at the highest tested concentrations (100 µg/ml and 40 mg/ml, respectively). CONCLUSIONS: The obtained results indicate that polymeric NPs PEG-b-PLA might induce alterations in steroid hormone production by ovarian GCs and thereby could modify reproductive functions.

Effect of bone morphogenetic protein-15 on gonadotropin-stimulated synthesis of hyaluronan and progesterone in porcine ovarian follicle.

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.

Titanium dioxide nanoparticles: some aspects of toxicity/focus on the development.

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.

Impact of endocrine disruptors on ovarian steroidogenesis.

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.

Effects of RU486 and indomethacin on meiotic maturation, formation of extracellular matrix, and progesterone production by porcine oocyte-cumulus complexes.

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.

Bisphenol A alone or in combination with estradiol modulates cell cycle- and apoptosis-related proteins and genes in MCF7 cells.

OBJECTIVE: Bisphenol A (BPA) with its estrogenic properties is intensively studied since its presence in the environment and human body. Besides other adverse effects, the compound is suspected of contributing to hormone-related cancers. The present study was aimed to investigate short time (24 h) effects of BPA on the important genes/proteins involved in apoptosis and the cell cycle progression in the breast cancer cells MCF7. The experimental design covered cell treatment with a broad BPA concentration scale: 10-12M corresponding to ubiquitous exposure, 10-9M relevant to human levels, and 10-6M as experimentally usual. We further investigated the combined effects of low BPA dose (10-12M) with physiological concentration of estradiol (E2) (10-9M). METHODS: The expression of particular proteins and genes was studied by Western blotting and real time RT-PCR, respectively. RESULTS: Estrogenic effect of BPA was confirmed in the following checkpoints: mRNA expression of estrogen receptor α, expression of cyclin D1 and A2 proteins and CCNA2 gene, Bax and Bcl2 protein levels. For both cyclins protein levels, the maximum stimulation was present at 10-9M BPA and the effects resembled the "inverted U"-shape, a nonmonotonic dose-response curve reported for the action of xenoestrogens. The combined effect of low BPA dose with physiological E2 concentration differ from those of individual compounds, the character of stimulatory response is neither additive nor synergistic. CONCLUSIONS: The results obtained strongly support the evidence of BPA and BPA+E2 proliferation-promoting effects in human breast carcinoma cells, even after short time exposure, partially via reduced rate of apoptosis by the action of BPA+E2.

Effect of 1α,25-dihydroxyvitamin D3 on progesterone secretion by porcine ovarian granulosa cells.

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.

Role of interleukins in the regulation of ovarian functions.

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.

Inhibition of proteasomal proteolysis affects expression of extracellular matrix components and steroidogenesis in porcine oocyte-cumulus complexes.

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.

Polymeric nanoparticles - targeted drug delivery systems for treatment of CNS disorders and their possible endocrine disrupting activities.

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.

Inhibitory effect of cadmium and tobacco alkaloids on expansion of porcine oocyte-cumulus complexes.

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.

Components of cigarette smoke inhibit expansion of oocyte-cumulus complexes from porcine follicles.

The role of alkaloids in cigarette smoke was investigated in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral porcine follicles. Suppression of the cumulus expansion stimulated by FSH was observed in the presence of different concentration of cadmium, anabasine and nicotine but not its metabolite cotinine. There were comparable inhibitory effects of cadmium and nicotine on the synthesis and accumulation of hyaluronic acid in the cell/matrix compartment of OCC. The inhibitory effect of tested compounds on the cumulus expansion was accompanied by decreased progesterone synthesis by cumulus cells during 42 h incubation of OCC with FSH. The results suggest that cigarette smoking may affect intrafollicular processes, which are responsible for normal ovulation and fertilization.