Nitrophenols disrupt the expression and activity of biotransformation enzymes (CYP3A and COMT) in chicken ovarian follicles in vivo and in vitro.

Nitrophenols are environmental pollutants and xenobiotics, the main sources of which are diesel exhaust fumes and pesticides. The biotransformation processes that take place in the liver are defence mechanisms against xenobiotics, such as nitrophenols. Our previous study showed that the chicken ovary is an additional xenobiotic detoxification place and that nitrophenols disrupt steroidogenesis in chicken ovarian follicles. Therefore, the present study aimed to determine the in vivo and in vitro effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on the expression and activity of phase I (CYP3A) and phase II (COMT) biotransformation enzymes in chicken ovary. In an in vivo study, hens were treated with a vehicle or 10 mg PNP or PNMC/kg b.wt. per day for 6 days. In an in vitro study, prehierarchical white and yellowish follicles, as well as the granulosa and theca layers of the three largest preovulatory follicles (F3, F2 and F1), were isolated and then incubated in a control medium or medium supplemented with PNP (10-6 M) or PNMC (10-6 M) for 24 or 48 h. Both in vivo and in vitro studies showed that nitrophenols exert tissue- and compound-dependent (PNP or PNMC) effects on CYP3A and COMT gene (real-time PCR) protein (Western blot) expression and their activity (colorimetric methods). The inhibitory effect of nitrophenols in vivo on the activity of biotransformation enzymes suggest that the ovary has the capacity to metabolise PNP and PNMC.

The potential effect of leptin co-administration on photodynamic damage using quail chorioallantoic membrane model.

BACKGROUND: The chorioallantoic membrane (CAM) of the Japanese quail is an excellent model for studying photodynamic therapy (PDT) due to its rich vascularization. PDT is used not only in oncological treatment but also in infectious diseases, or psoriasis, where it yields significant advantages. This treatment also has its limitations, such as burning, itching, erythema, redness, swelling, and delayed wound healing. The aim of this study was to analyse the potentially protective properties of the tissue hormone leptin during PDT. METHODS: Japanese quail embryos incubated ex ovo were used in this experiment. On the 9th day of embryonic development, leptin (5 µg) and photosensitiser hypericin (79 µM) were topically applied, followed by irradiation. The effect of leptin co-administration was evaluated from CAM images and histological structure analysis, histological samples, and qPCR, where the expression of genes involved in angiogenesis, apoptosis, and oxidative stress was monitored. RESULTS: We observed vascular damage in all experimental groups, the highest damage was found after the application of hypericin without leptin coadministration. Histological analysis confirmed the protective effect of leptin. qPCR analysis presented differences in FREK gene expression, but also in genes involved in oxidative stress like SOD, NRF-1, NRF-2, and GPX7. The application of leptin significantly reduced the expression of apoptosis regulatory proteins CASP3, cytochrome C, and APAF1. CONCLUSIONS: Our results in the CAM model suggest a possible protective effect of leptin to prevent PDT damage and aid in the subsequent regeneration of target tissues after antimicrobial PDT.

Nitrophenols are negative modulators of steroidogenesis in preovulatory follicles of the hen (Gallus domesticus) ovary: An in vitro and in vivo study.

This study assessed the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the granulosa layers (GLs) and theca layers (TLs) of chicken preovulatory follicles in vitro and in vivo. In the in vitro experiment, three of the largest yellow preovulatory follicles (F3 < F2 < F1) were exposed to PNP or PNMC (10-8-10-4 M), ovine luteinising hormone (oLH; 10 ng/mL), and combinations of oLH and PNP or PNMC (10-6 M). In the in vivo experiment, laying hens were treated for 6 days with PNP or PNMC (10 mg/kg). In vitro experiments revealed that PNP and PNMC decreased basal and oLH-stimulated P4 secretion from the GL as well as T and E2 secretion from the TLs of F3-F1 follicles. Treatment of laying hens with nitrophenols lowered plasma concentrations of luteinising hormone and all three steroids. The reduction of steroid secretion was associated with decrease in LHR, HSD3B1 and CYP19A1 mRNA expression in the GL and/or TLs of the preovulatory follicles, both in vitro and in vivo. Moreover, PNP decreased HSD3B protein expression in the GL of F2 follicles in vitro and in vivo, while PNMC diminished its expression in the GL of F1 follicles in vivo. In vitro, nitrophenols did not affect CYP19A1 protein expression; however, nitrophenols inhibited its expression in the TLs of F3 and F2 follicles in vivo. The results obtained clearly demonstrate that nitrophenols are negative modulators of steroidogenesis in chicken preovulatory follicles and, in consequence, may not only impair ovulation process, but also affect function of the hypothalamic-pituitary-ovarian axis.

Immunolocalization of Leptin Receptor and mRNA Expression of Leptin and Estrogen Receptors as well as Caspases in the Chorioallantoic Membrane (CAM) of the Chicken Embryo.

The chicken chorioallantoic membrane (CAM) is used as a model in tests of angiogenesis, the biocompatibility of materials as well as tumor invasive potential. To assess the properties of CAM tissue, the localization of leptin receptor in the CAM, and the mRNA expression of two leptin receptor isoforms, estrogen receptors (ERα and ERß) and caspases (-1 and -3) in the CAM on embryonic days 12 (E12), 15 (E15) and 18 (E18) were investigated. The leptin receptor was immunolocalized in each structure of the CAM (chorionic epithelium, allantoic epithelium, mesodermal layer and the walls of blood vessels) and did not change among analyzed stages of embryonic development (E12, E15 and E18) and between sexes. Expression of mRNA of genes encoding leptin and estrogen receptors as well as caspases was detected in the CAM of female and male chicken embryos at all three analysed stages of development. The relative mRNA expression of the long form of leptin receptor exceeded that of its short isoform. The mRNA expression of ERß was significantly higher than ERα as well as caspase-3 in comparison with caspase-1. There were no differences in mRNA expression of these genes between sexes and among analyzed developmental days. The results indicate that the CAM is a target tissue for leptin as well as for estrogens and that CAM development is partially regulated by caspase-1 and caspase-3 dependent cell death. These results should be taken into consideration in studies in which the CAM is used as an experimental model.

Expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1) and CYP1 family monooxygenase mRNAs and their activity in chicken ovarian follicles following in vitro exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

The aim of this in vitro study was to determine the effect of TCDD and luteinizing hormone (LH) on mRNA expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1), and the CYP1 family monooxygenases (CYP1A4, CYP1A5, CYP1B1), and to assess the basal and TCDD-induced activity of these enzymes in chicken ovarian follicles. White (WF) and yellowish (YF) prehierarchical follicles and fragments of the theca (TL) and granulosa (GL) layers of the 3 largest preovulatory follicles (F3-F1) were exposed to TCDD (10nM), ovine LH (oLH; 10ng/mL) or a combination of TCDD (10nM) and oLH (10ng/mL), and increasing doses of TCDD (0.01-100nM). AHR1 and ARNT1 mRNA transcripts were found in all examined follicles. The effect of TCDD and oLH on AHR1 and ARNT1 mRNA expression depended on the maturational state of the follicle. CYP1A4 was predominantly expressed in the GL of the F3-F1 follicles; in comparison with the WF, a higher level of CYP1A5 mRNA was found both in the GL and TL of F3-F1 follicles. Alternatively, the highest level of CYP1B1 mRNA was noticed in the WF follicles. In different developmental stages of the follicle TCDD and oLH induced a different CYP1 isoform. TCDD increased EROD and MROD activities in all the investigated ovarian follicles. In conclusion, AHR1 and ARNT1 mRNA expression indicate that the chicken ovary is a target tissue for dioxin and dioxin-like compounds. The expression of CYP1-family genes and TCDD-inducible EROD and MROD activities in ovarian follicles suggest the possibility of xenobiotic detoxification in the chicken ovary.

Localization of apoptotic and proliferating cells and mRNA expression of caspases and Bcl-2 in gonads of chicken embryos.

The aim of the present study was to analyze participation of apoptosis and proliferation in gonadal development in the chicken embryo by: (1) localization of apoptotic (TUNEL) and proliferating (PCNA immunoassay) cells in male and female gonads and (2) examination of mRNA expression (RT-PCR) of caspase-3, caspase-6 and Bcl-2 in the ovary and testis during the second half of embryogenesis and in newly hatched chickens. Apoptotic cells were found in gonads of both sexes. At E18 the percentage of apoptotic cells (the apoptotic index, AI) in the ovarian medulla and the testis was lower (p<0.05) than in the ovarian cortex. In the ovarian medulla, the AI at E18 was lower (p<0.05) than on E12. In the testis, the AI was significantly lower (p<0.05) at E18 than at E15 and 1D. The percentage of proliferating cells (the proliferation index: PI) within the ovary significantly increased from E15 to 1D in the cortex, while proliferating cells in the medulla were detected only at E15. In the testis, the PI gradually increased from E12 to 1D. The mRNA expression of caspase-3 and -6 as well as Bcl-2 was detected in male and female gonads at days 12 (E12), 15 (E15) and 18 (E18) of embryogenesis and the day after hatching (1D). The expression of all analyzed genes on E12 was significantly higher (p<0.05) in female than in male gonads. This difference was also observed at E15 and E18, but only for the caspase-6. The results obtained showed tissue- and sex-dependent differences in the number of apoptotic and proliferating cells as well as mRNA expression of caspase-3, -6 and Bcl-2 genes in the gonads of chicken embryos. Significant increase in the number of proliferating cells in the ovarian cortex and lack of these cells in the ovarian medulla (stages E12, E18, 1D) simultaneous with decrease in the intensity of apoptosis only in the medulla indicates that proliferation is the dominant process involved in the cortical development, which constitutes the majority of the functional structure of the fully developed ovary. No pronounced changes in the expression of apoptosis-related genes found during embryogenesis suggest that they cannot be considered as important indicators of gonad development. The molecular mechanisms of the regulation of balance between apoptosis and proliferation in developing avian gonads need to be further investigated.

Expression and localization of growth hormone receptor in the oviduct of the laying hen (Gallus domesticus).

The purpose of the present study was to examine growth hormone receptor (GHR) gene expression by real-time PCR and demonstrate immunocytochemically the localization of GHR in four chicken oviductal parts, i.e. infundibulum, magnum, isthmus and shell gland. Experiments were carried out on Hy-Line laying hens decapitated 2 h after oviposition. GHR mRNA was expressed in all examined oviductal segments with a significantly lower level in the infundibulum in comparison to other parts of the oviduct. Specific GHR immunoreactivity was also detected in the wall of the oviduct. The intensity of the staining was as follows: infundibulum < or = isthmus < or = shell gland < or = magnum. In all oviductal parts, a positive reaction for GHR was observed in the mucosa whereas a very weak or no reaction was observed in the stroma. Within the mucosa a strong reaction for GHR was observed in the epithelium of the infundibulum and in the tubular gland of the magnum, isthmus and shell gland. Immunoreactivity for GHR was very weak in the mucosal epithelium of the magnum, isthmus and shell gland. In conclusion, the results point to the possibility of an important role of GH in oviduct functions in domestic hens.

The expression of pituitary FSHbeta and LHbeta mRNA and gonadal FSH and LH receptor mRNA in the chicken embryo.

In avian species, synthesis of sex steroids by embryonic gonads is regulated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In order to elucidate the role of the two gonadotropins in gonadal axis development during the second half of chicken embryogenesis, pituitary expression of LH beta subunit (LHbeta) and FSH beta subunit (FSHbeta) mRNAs as well as gonadal expression of LH and FSH receptor (LHR and FSHR) mRNAs were determined on days 11 (E11) and 17 (E17) of embryonic development and after hatching (D1). In the pituitary of the female embryo, the gene expression of FSHbeta was the lowest on E11 and increased on E17. In the male pituitary, the expression of FSHbeta did not differ among the studied days. The FSHbeta mRNA expression on E11 was higher in the male than in the female pituitary gland. The expression of LHbeta mRNA in the female pituitary increased on D1 in comparison to E11. In the male pituitary gland, the expression of LHbeta gene was relatively constant. The expression of mRNA encoding FSHR in the ovary increased on E17, while in testes it did not differ among the studied days. There were no significant alterations in LHR gene expression in the ovary or in the testes in the examined period however, the gene expression on E17 was higher in the ovary than in the testes. We observed positive correlations between the pituitary FSHbeta mRNA expression and ovarian expression of FSHR mRNA (r = 0.63; p<0.01) as well as between LHbeta mRNA and LHR mRNA in the testes (r=0.65; p<0.01). The reported alterations in gene expression of FSHbeta, LHbeta and their receptors between sexes and among the stages of embryonic development indicate time- and sex-dependent action of gonadotropins in gonads of chicken embryos.

MRNA expression and immunocytochemical localization of leptin receptor in the oviduct of the laying hen (Gallus domesticus).

The role of leptin in female reproduction is fairly well established in mammals, whereas reports concerning leptin action in birds are scarce. The aim of the present study was to detect leptin receptor (LEP-R) mRNA and to localize the leptin receptor protein in the oviduct of laying hens 2h after ovulation by the RT-PCR method and immunocytochemical staining. The RT-PCR reaction demonstrated expression of the long form ofleptin receptor mRNA in all examined oviductal parts (infundibulum, magnum, isthmus and shell gland) and the weakest level was found in the isthmus. The expression of the short isoform was lower than the long form in all examined tissue samples and no differences between oviductal parts were observed. Immunostaining specific for leptin receptor was found in the walls of all examined oviductal parts. The intensity of the immunopositive reaction was the strongest in the epithelium of all examined parts of the oviduct and in the endothelium and muscles of blood vessels. The weakest immunopositive reaction was observed in tubular glands, the connective tissue layer and in circular and longitudinal muscles. The results obtained in this experiment suggest that the oviduct may be a target tissue for leptin, where this polypeptide hormon may participate in egg formation and/or its transport through the oviduct of the domestic hen.