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.

Response of the hen ovary to eCG treatment: Insight into morphology and expression of genes related to steroidogenesis and vitellogenesis.

The present study aimed to examine the effect of equine chorionic gonadotropin (eCG) treatment on the chicken ovarian folliculogenesis and steroidogenesis. The expression of vitellogenesis-related genes in the liver was also investigated. Laying hens were injected with 75 I.U./kg of body weight/0.2 mL of eCG, once a day for 7 successive days. On day 7 of the experiment hens, including control hens which were receiving vehicle, were euthanized. The liver and ovarian follicles were harvested. Blood was collected daily through the whole experiment. The eCG treatment resulted in the cessation of egg laying after 3 or 4 days. The eCG-treated hens had heavier ovaries with a higher number of yellowish and yellow follicles arranged in a non-hierarchical way in contrast to ovaries of control hens. Moreover, these birds had elevated plasma estradiol (E2) and testosterone (T) concentrations. The molar ratios of E2:progesterone (P4) and T:P4 were increased in chickens injected with eCG. Real-time polymerase chain reaction revealed changes in mRNA abundances of steroidogenesis-associated genes (StAR, CYP11A1, HSD3ß, and CYP19A1) in ovarian follicles: white, yellowish, small yellow, and the largest yellow preovulatory (F3-F1) as well as VTG2, apoVLDL II, and gonadotropin receptors in the liver. In general, the abundances of gene transcripts were higher in eCG-treated hens than in control hens. Western blot analyses showed an elevated abundance of aromatase protein in the prehierarchical and small yellow follicles of eCG-treated hens. Unexpectedly, there was presence of both FSHR and LHCGR mRNA in the liver and the level of expression was shifted in eCG-treated hens. In summary, eCG treatment leads to disruption of the ovarian hierarchy with accompanying changes in circulating steroids and ovarian steroidogenesis.

Non­viral transfection methods optimized for miRNA delivery to human dermal fibroblasts.

Fibroblasts are beneficial model cells for in vitro studies and are frequently used in tissue engineering. A number of transfection reagents have been employed to deliver microRNAs (miRNAs/miRs) into cells for genetic manipulation. The present study aimed to establish an effective method of transient miRNA mimic transfection into human dermal fibroblasts. The experimental conditions included three different methods: Physical/mechanical nucleofection, and two lipid­based methods, Viromer® Blue and INTERFERin®. To evaluate the impact of these methods, cell viability and cytotoxicity assays were performed. The silencing effect of miR­302b­3p was revealed to alter the expression levels of its target gene carnitine O­octanoyltransferase (CROT) by reverse transcription­quantitative PCR. The present study showed that all selected non­viral transient transfection systems exhibited good efficiency. It was also confirmed that nucleofection, for which a 21.4­fold decrease in the expression of the CROT gene was observed 4 h after 50 nM hsa­miR­302b­3p transfection, was the most effective method. However, these results indicated that lipid­based reagents can maintain the silencing effect of miRNAs up to 72 h after transfection. In summary, these results indicated that nucleofection may be the optimal method for the transport of small miRNA mimics. However, lipid­based methods allow for the use of lower concentrations of miRNA and maintain longer­lasting effects.

An Overview of the Current Known and Unknown Roles of Vitamin D3 in the Female Reproductive System: Lessons from Farm Animals, Birds, and Fish.

Recent studies have clearly shown that vitamin D3 is a crucial regulator of the female reproductive process in humans and animals. Knowledge of the expression of vitamin D3 receptors and related molecules in the female reproductive organs such as ovaries, uterus, oviduct, or placenta under physiological and pathological conditions highlights its contribution to the proper function of the reproductive system in females. Furthermore, vitamin D3 deficiency leads to serious reproductive disturbances and pathologies including ovarian cysts. Although the influence of vitamin D3 on the reproductive processes of humans and rodents has been extensively described, the association between vitamin D3 and female reproductive function in farm animals, birds, and fish has rarely been summarized. In this review, we provide an overview of the role of vitamin D3 in the reproductive system of those animals, with special attention paid to the expression of vitamin D3 receptors and its metabolic molecules. This updated information could be essential for better understanding animal physiology and overcoming the incidence of infertility, which is crucial for optimizing reproductive outcomes in female livestock.

Alterations in connexin 43 gene and protein expression in the chicken oviduct following tamoxifen treatment.

Connexins (Cxs) are a group of gap junction proteins involved in the direct exchange of small molecules between neighboring cells. Information concerning the expression and regulation of Cxs in the chicken oviduct is lacking, but likely has potential implications for functioning of the oviduct and the quality of the egg laid by commercially used hens. The present study was designed to examine whether selected Cxs are present in the chicken oviduct and, if so, whether expression of the most abundant Cx changes following tamoxifen (TMX; estrogen receptor modulator) treatment. Hy-Line Brown laying hens were injected (s.c.) daily with a vehicle (n = 6) or with TMX (n = 6), at a dose of 6 mg/kg of body weight for 7 days until complete cessation of egg laying by TMX-treated hens. All oviductal segments (infundibulum, magnum, isthmus, shell gland, and vagina) were collected from hens on day 8 of the experiment. First, the gene expression of GJA1 (i.e. Cx43 protein), GJA4 (Cx39), GJB1 (Cx32), and GJD2 (Cx36) was investigated by real-time PCR in tissues of control birds. The results demonstrated gene- and oviductal segment-dependent expression of GJB1, GJD2, GJA4, and GJA1 mRNA. Since the GJA1 transcript was the most abundant in all oviductal parts, subsequently, the Cx43 expression and localization were examined in the oviduct of all hens. The relative expression of GJA1 mRNA in control hens was highest in the infundibulum and vagina and lowest in the magnum. The pattern of Cx43 protein abundance evaluated by Western blot was similar to that of mRNA. Treatment of hens with TMX decreased the GJA1 mRNA levels in the magnum and isthmus, and Cx43 protein abundances were reduced in the isthmus and vagina. Immunofluorescence demonstrated cell- and segment-dependent localization of Cx43 protein in the oviductal wall; the most intense immunoreactivity was observed in the muscle cells of the shell gland and vagina. In TMX-treated hens, the immunoreactivity for Cx43 in all oviductal segments was slightly reduced and had a different signal pattern compared with control chickens. These results suggest that Cx43 likely takes part in the regulation of oviduct functioning, especially in the coordination of muscle contraction required for egg transport and oviposition. In addition, the results suggest a contribution of estrogen in the regulation of Cx43 expression and/or fates in the chicken oviduct. New insights into the expression and regulation of Cxs in the hen oviduct, indicating their potential involvement in the mechanisms of egg formation and transport that may affect poultry production, were obtained in this study.

In vitro effects of polychlorinated biphenyls and their hydroxylated metabolites on the synthesis and metabolism of iodothyronines in the chicken (Gallus domesticus) thyroid gland.

To assess the effect of polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) on thyroid hormone [TH: thyroxine (T4) and triiodothyronine (T3)] secretion, the concentrations of iodothyronine deiodinases (DIO1, DIO2, DIO3), and mRNA expression of genes involved in TH synthesis (TSHR, NIS, TPO, TG), metabolism (DIO1, DIO2, DIO3), and transport (OATP1C1, MCT8, MCT10, LAT1), chicken thyroid explants were incubated in medium supplemented with TSH (250 mU/ml), PCB118, PCB153, 4-OH-PCB107, and 3-OH-PCB153 (0.5 × 10-8 M), and TSH together with each PCB and OH-PCB. The results of the in vitro experiment revealed that, except for 4-OH-PCB107, all applied PCBs and OH-PCBs inhibited basal and TSH-stimulated T4 secretion. Moreover, they increased basal and reduced TSH-stimulated T3 secretion. PCBs and OH-PCBs decreased the TSH-stimulated TSHR expression. Following PCB and OH-PCB exposure, significant changes in mRNA expression of NIS, TPO, and TG were observed. PCBs and OH-PCBs affected DIO1 and DIO3 transcript levels and protein abundances of each DIO. Furthermore, PCB-dependent effects on OATP1C1, MCT8, and MCT10 mRNA expression were found. In conclusion, both PCB118 and PCB153 and their OH-PCBs affect TH synthesis and deiodination processes in the chicken thyroid gland and influence TH transport across the thyrocyte membrane. In addition, the effects of PCBs and OH-PCBs depended mainly on the type of PCB congener and the exposure time. These results indicate that not only parental PCBs but also OH-PCBs are hazardous for the thyroid gland and may disrupt its endocrine function. Further studies are necessary to explain a mechanism of PCB and OH-PCB action in the avian thyroid gland.

Effects of Silver Nanoparticles on Proliferation and Apoptosis in Granulosa Cells of Chicken Preovulatory Follicles: An In Vitro Study.

The continuous development of poultry production related to the growing demand for eggs and chicken meat makes it necessary to use modern technologies. An answer to this demand may be the use of nanotechnology in poultry farming. One of the promising nanomaterials in this field are silver nanoparticles (AgNPs), which are used as disinfectants, reducing microbial pollution and the amounts of greenhouse gases released. This study aimed to evaluate the effect of AgNPs on the proliferation and apoptosis process in the granulosa cells of chicken preovulatory follicles. The in vitro culture experiment revealed that both 13 nm and 50 nm AgNPs inhibited the proliferation of the granulosa cells. However, a faster action was observed in 50 nm AgNPs than in 13 nm ones. A size-dependent effect of AgNP was also demonstrated for the caspase-3 activity. AgNPs 13 nm in size increased the caspase-3 activity in granulosa cells, while 50 nm AgNPs did not exert an effect, which may indicate the induction of distinct cell death pathways by AgNPs. In conclusion, our study reveals that AgNPs in vitro inhibit granulosa cell proliferation and stimulate their apoptosis. These results suggest that AgNPs may disrupt the final stage of preovulatory follicle maturation and ovulation.

Sodium Fluoride In Vitro Treatment Affects the Expression of Gonadotropin and Steroid Hormone Receptors in Chicken Embryonic Gonads.

Sodium fluoride (NaF), in addition to preventing dental decay may negatively affect the body. The aim of this study was to examine the effect of a 6 h in vitro treatment of gonads isolated from 14-day-old chicken embryos with NaF at doses of 1.7 (D1), 3.5 (D2), 7.1 (D3), and 14.2 mM (D4). The mRNA expression of luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), estrogen receptors (ESR1 and ESR2), progesterone receptor (PGR), and the immunolocalization of progesterone receptors were examined in the tissue. In the ovary, the expression of FSHR and LHR increased following the NaF treatment. In the case of FSHR the highest stimulatory effect was noticed in the D2 group, while the expression of LHR increased in a dose-dependent manner. A gradual increase in ESR1 and PGR mRNA levels was also observed in the ovary following the NaF treatment, but only up to the D3 dose of NaF. The highest ESR2 level was also found in the D3 group. In the testes, the lowest dose of NaF significantly decreased the expression of FSHR, ESR1, ESR2, and PGR. On the other hand, an increase in PGR expression was observed in the D3 group. The expression of LHR in the testes was not affected by the NaF treatment. Immunohistochemical analysis showed that NaF exposure increased progesterone receptor expression in the ovarian cortex, while it decreased its expression in the testes. These results reveal that NaF may disturb the chicken embryonic development and different mechanisms of this toxicant action exist within the females and males.

Response of the matrix metalloproteinase system of the chicken ovary to prolactin treatment.

The expression and activity of several matrix metalloproteinases (MMPs) has been demonstrated in the chicken ovary during various physiological states; these data indicate that MMPs are involved in the remodeling of the extracellular matrix (ECM) during follicle development, ovulation, atresia, and regression. The regulation of MMPs in the avian ovary, however, remains largely unknown. The present study aimed to examine the effect of recombinant chicken prolactin (chPRL) treatment on the expression of selected MMPs and their tissue inhibitors (TIMPs), as well as MMP-2 and MMP-9 activity in the hen ovary. Real-time polymerase chain reaction revealed changes in the mRNA expression of MMP-2, MMP-7, MMP-9, MMP-10, MMP-13, TIMP-2, and TIMP-3 in the following ovarian follicles: white, yellowish, small yellow, and the largest yellow preovulatory (F3-F1). Western blot analysis showed alterations in the abundance of latent and active forms of the MMP-2 protein, as well as the abundance of the MMP-9 protein. Moreover, minor changes in MMP-2 and MMP-9 total activities were found in ovarian follicles of chPRL-treated hens. The response to chPRL treatment depended upon the stage of follicle development, the layer of follicular wall, and the type of MMPs or TIMPs studied. In general, the results indicate that chPRL, is a positive regulator of MMP expression in the yellow preovulatory follicles. Our findings suggest that PRL participates in the mechanisms orchestrating ECM turnover during ovarian follicular development in the hen ovary via regulating the transcription, translation, and/or activity of some constituents of the MMP system.

Effect of eCG treatment on gene expression of selected matrix metalloproteinases (MMP-2, MMP-7, MMP-9, MMP-10, and MMP-13) and the tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3) in the chicken ovary.

Several metalloproteinases (MMPs) are present and functional in the chicken ovary and regulate the extracellular matrix (ECM) during follicle development, ovulation, atresia, and regression. The regulation of the abundance of MMPs in avian ovarian follicles, however, is largely unknown. The aim of the present study was to examine effects of equine chorionic gonadotropin (eCG) on abundance of selected MMPs and relevant tissue inhibitors of MMPs (TIMPs) in the hen ovary. The MMP-2 and MMP-9 activity was also determined. Results indicated there were effects of eCG on abundances of MMP-2, MMP-7, MMP-9, MMP-10, MMP-13, TIMP-2, and TIMP-3 mRNA transcript and/or protein relative abundances in white, yellowish, small yellow, and the largest yellow preovulatory (F3-F1) ovarian follicles. The response to eCG depended on the stage of follicle development, layer of follicular wall, and the type of MMPs or TIMPs affected by eCG. Furthermore, there was a pause in egg laying when eCG was administered and there were morphological changes in the ovary following eCG treatment that were associated with alterations in MMP-2 and MMP-9 activity. In general, the results indicate that eCG, which has primarily follicle stimulating hormone (FSH)-like bioactivities, is a negative regulator of MMP abundance and activity in the largest yellow preovulatory follicles. Results from the present study indicate the gonadotropins, especially FSH, by the regulation of transcription, translation, and/or activity of proteins of the MMP system have effects on the mechanisms that underlie ECM remodeling and cell function throughout ovarian follicle development in the chicken ovary.

Administration of silver nanoparticles affects ovarian steroidogenesis and may influence thyroid hormone metabolism in hens (Gallus domesticus).

This study aimed to determine the in vivo effect of silver nanoparticles (AgNPs) on the concentration of sex steroids (progesterone - P4, estradiol - E2, testosterone - T) and thyroid hormones (thyroxine - T4, triiodothyronine - T3) in the blood plasma as well as the messenger ribonucleic acid (mRNA) and protein expression of HSD3ß, CYP17A1 and CYP19A1 enzymes and steroid hormone concentrations in chicken ovarian follicles. AgNPs did not affect serum steroid hormone levels, but increased T3 levels depending on the size and concentration of AgNPs. At the level of ovarian tissues, AgNPs: (i) affected the levels of E2 and T in prehierachical follicles; (ii) reduced the expression of CYP19A1 mRNA and protein and consequently diminished E2 concentration in small white follicles; and (iii) increased the expression of CYP17A1 mRNA in large white follicles, without changing its protein expression. The results indicate that AgNPs affect chicken ovarian steroidogenesis. The effects of AgNPs depend on exposure time, the type of follicle and the degree of its development and are associated with the modulation of steroidogenic gene expression and E2 and T synthesis. Prehierachical follicles seem to be more susceptible to AgNPs than preovulatory ones. In conclusion, AgNPs by targeting the chicken ovary may indirectly influence the selection processes of prehierarchical follicles to the pre-ovulatory hierarchy and disturb the ovarian steroidogenesis. Furthermore, AgNPs may affect thyroid hormone metabolism in different ways by size which in turn may influence energy homeostasis of the target cells.

Altered vitamin D3 metabolism in the ovary and periovarian adipose tissue of rats with letrozole-induced PCOS.

Vitamin D3 (VD3) plays an important role in the ovary and its deficiency is associated with ovarian pathologies, including polycystic ovary syndrome (PCOS). However, there is no data related to VD3 metabolism in the ovary during PCOS. Herein, we investigated differences in the expression of VD3 receptor (VDR) and key VD3 metabolic enzymes, 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1), in the ovary and periovarian adipose tissue (POAT) of control (proestrus and diestrus) and PCOS induced by letrozole rats. Vdr, Cyp27b1 and Cyp24a1 mRNA expression was determined, their protein abundance was examined and immunolocalized. Furthermore, VD3 metabolite concentrations in plasma (25OHD) and tissues (ovary and POAT; 1,25(OH)2D3), and plasma calcium level were determined. 25OHD concentration decreased markedly in letrozole-treated rats in comparison with controls, whereas calcium concentration did not vary among the examined groups. The amount of 1,25(OH)2D3 decreased in both ovary and POAT of PCOS rats. In the ovary, we found decreased Cyp27b1 and increased Vdr mRNA expression in letrozole-treated and diestrus control group. Corresponding protein abundances were down-regulated and up-regulated, respectively but only following letrozole treatment. In POAT, only Cyp27b1 transcript level and CYP27B1 protein abundance were decreased in letrozole-treated rats. VDR was immunolocalized in healthy and cystic follicles, while CYP27B1 and CYP24A1 were found exclusively in healthy ones. Concluding, our results provide the first evidence of disrupted VD3 metabolism in the ovary and POAT of PCOS rats. The reduced 1,25(OH)2D3 concentration in those tissues suggests their contribution to VD3 deficiency observed in PCOS and might implicate in PCOS pathogenesis.

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.

Aquaporin 4 in the chicken oviduct during a pause in laying induced by food deprivation.

In the present study we hypothesize that aquaporin 4 (AQP4) expression in the chicken oviduct would change during a pause in egg laying that was induced by fasting. Accordingly, the aim of this investigation was to examine the AQP4 mRNA and protein expression, and immunolocalization in the chicken oviduct during the course of regression. The experiment was carried out on laying hens subjected to a pause in laying that was induced by food deprivation for 5 days. Control hens were fed ad libitum. The birds were sacrificed on day 6 of the experiment and all segments of the oviduct were isolated, including the infundibulum, magnum, isthmus, shell gland, and vagina. Subsequently, the gene and protein expressions of AQP4 in the tissues were tested by real-time PCR and Western blot, respectively. The relative mRNA expression of AQP4 was the highest in the infundibulum and vagina and the lowest, and least detectable, in the magnum. The level of AQP4 protein was the highest in the infundibulum and the lowest in the magnum. Fasting resulted in a decrease of the AQP4 mRNA expression (P<0.001) in the infundibulum, a decrease in protein abundance (P<0.01) in the shell gland, and an increase in protein level (P<0.001) in the vagina. Immunohistochemistry demonstrated tissue- and cell-dependent localization of AQP4 protein in the oviductal wall. The intensity of staining was as follows: the infundibulum > shell gland > vagina ≥ isthmus ≫ magnum. In the control hens, the immunoreactivity for AQP4 in the vagina was similar, whereas in other oviductal segments, the immunoreactivity was stronger when compared with the chickens subjected to a pause in laying. In summary, these findings suggest that the AQP4 is an essential protein involved in the regulation of water transport required to create a proper microenvironment for fertilization and egg formation in the hen oviduct.

Dans la présente étude, nous posons l'hypothèse que l'expression de l'aquaporine 4 (AQP4) dans l'oviducte de poule changerait pendant une pause lors de la ponte induite par un jeûne. Ainsi, le but de notre expérimentation était de déterminer l'expression de l'ARNm et de la protéine AQP4 ainsi que son immunolocalisation dans l'oviducte de poule au cours de la régression. L'expérience a été réalisée sur des poules pondeuses soumises à une pause de ponte induite par une privation alimentaire pendant 5 jours. Les poules témoins ont été nourries ad libitum. Les oiseaux ont été sacrifiés au jour 6 de l'expérience et tous les segments de l'oviducte ont été isolés, à savoir l'infundibulum, le magnum, l'isthme, la glande coquillière, et le vagin. Les expressions géniques et protéiques d'AQP4 dans ces tissus ont été testées respectivement par PCR en temps réel et Western blot. L'expression relative d'ARNm d'AQP4 était la plus élevée dans l'infundibulum et le vagin et la plus faible et la moins détectable dans le magnum. Le niveau de la protéine AQP4 était le plus élevé dans l'infundibulum et le plus bas dans le magnum. Le jeûne a entraîné une diminution de l'expression de l'ARNm AQP4 (P<0,001) dans l'infundibulum, une diminution de l'abondance des protéines (P<0,01) dans la glande coquillière et une augmentation du niveau de protéines (P<0,001) dans le vagin. L'immunohistochimie a démontré une localisation dépendante des tissus et des cellules de la protéine AQP4 dans la paroi oviductale. L'intensité de la coloration était la suivante : infundibulum > glande coquillière > vagin ≥ isthme ≫ magnum. Chez les poules témoins, l'immunoréactivité de l'AQP4 dans le vagin était similaire, tandis que dans d'autres segments oviductaux, l'immunoréactivité était plus forte par rapport aux poulets soumis à une pause de ponte. En résumé, ces résultats suggèrent que l'AQP4 est une protéine essentielle impliquée dans la régulation du transport de l'eau nécessaire pour créer un micro-environnement approprié pour la fécondation et la formation d'œufs dans l'oviducte de poule.

Expression of gelatinases (MMP-2 and MMP-9) and tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3) in the chicken ovary in relation to follicle development and atresia.

Matrix metalloproteinases (MMPs) are a family of peptidases that possess the ability to break down extracellular matrix macromolecules associated with tissue turnover in various physiological and pathological conditions. Their activity is largely regulated by specific tissue inhibitors of MMPs (TIMPs). Information concerning the role of MMPs in the chicken ovary is very limited. The aim of the present study was to determine the expression and localization of selected members of the MMP system in different compartments of the laying hen ovary and to investigate whether their expression changes at different stages of the ovulatory cycle. MMP-2 and -9 activity was also examined. Expression of MMP-2, -9 and tissue inhibitors of MMPs (TIMP-2 and -3) in the ovarian follicles was examined 22 h and 3 h before F1 ovulation. Real-time polymerase chain reaction and western blot revealed differential mRNA and protein expression of MMP-2, MMP-9, TIMP-2, and TIMP-3 in the ovarian follicles: white, yellowish, small yellow, the largest preovulatory (F3-F1), and white atretic. Within the ovary, the relative expression of MMP and TIMP mRNA depended on follicle development, the layer of follicular wall, and ovulation stage. The relatively higher expression of MMP-2 and MMP-9 mRNA in the ovarian follicles 3 h compared to 22 h before ovulation was found. As follicle development progressed toward ovulation, elevated MMP-2 and -9 activity was noted. Atresia of white follicles was accompanied by an increase in gelatinase activities. Immunohistochemistry demonstrated tissue- and follicle-dependent immunoreactivity of the examined MMPs and TIMPs. In summary, the results show tissue- and stage of the ovulatory cycle-dependent differences in MMP and TIMP expression, as well as MMP-2 and -9 activity. Findings that suggest these molecules might significantly participate in the complex remodeling of extracellular matrix required for follicle development, ovulation, and atresia in the chicken ovary.

Expression of aquaporin 4 in the chicken oviduct following tamoxifen treatment.

This study was designed to examine whether aquaporin 4 (AQP4) is present in the chicken oviduct, and if so, whether its expression changes during pause in laying induced by tamoxifen (TMX; oestrogen receptor modulator) treatment. The control chickens were injected with a vehicle (ethanol) and the experimental ones with TMX at a dose of 6 mg/kg of body weight. Birds were treated daily until complete cessation of egg laying. The oviductal parts, that is the infundibulum, magnum, isthmus, shell gland and vagina were isolated from hens on day 8 of the experiment, and subsequently, the gene and protein expressions of AQP4 in tissues were examined by real-time PCR and Western blot, respectively. Immunohistochemical localization of AQP4 in the wall of the chicken oviduct was also investigated. Both mRNA and protein of AQP4 were found in all segments of the chicken oviduct. The relative expression [RQ] of AQP4 was the highest in the infundibulum and the vagina and the lowest, less detectable, in the magnum and isthmus. The pattern of AQP4 protein expression was similar to that of mRNA. Treatment of hens with TMX decreased the mRNA and protein levels of AQP4 in the oviduct. Immunohistochemistry demonstrated tissue and cell-dependent localization of AQP4 protein in the oviductal wall. The intensity of the immunopositive reaction was as follows: the infundibulum > vagina > shell gland ≥ isthmus >˃ magnum. In the control chickens, the immunoreactivity for AQP4 in all oviductal segments was stronger compared with the TMX-treated hens. The results obtained indicate that AQP4 takes part in the regulation of water transport required for the formation of egg in the chicken oviduct. Moreover, a relationship between oestrogen action and AQP4 gene and protein expression is suggested.

Involvement of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the regression of chicken postovulatory follicles.

The study was undertaken to examine mRNA expression and localization of selected matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), and the activity of MMPs in chicken postovulatory follicles (POFs) during their apoptotic regression. Apoptotic cells and apoptosis-related caspase expression and activity were examined as well. Chickens were sacrificed 2 h and 21 h after ovulation, and five POFs (POF1 to POF5) were isolated from the ovaries. It was found that the number of apoptotic cells (TUNEL-positive) increased along with follicle regression. The relative expression (RQ) of caspase-2, -3, -8 and -9 mRNA increased (P < 0.05) in POF5, while the activity of all examined caspases elevated gradually (approximately 80-150%) reaching the highest level in POF3, and then slowly decreased to the value noted in POF1 (P < 0.05 - P < 0.001). Real-time polymerase chain reaction revealed different expression of MMP-2, -7, -9 and TIMP-2 and -3 on mRNA levels, and activity assay showed the changes in activity of MMP-2 and -9 in the POFs. Regression of the follicles was accompanied predominantly by an increase in the relative expression of MMP-2, and a decrease in TIMP-2 and -3 mRNAs (P < 0.05 - P < 0.001). The activity levels of MMP-2 and -9 showed pronounced changes during the examined period. During follicle regression elevated activity of MMP-2 and -9 was found (P < 0.05 - P < 0.001). Immunohistochemistry demonstrated tissue- and follicle-dependent immunoreactivity of the examined members of the MMP system. In summary, the results showing the apoptotic regression-related changes as well as tissue-dependent differences in the expression of selected MMPs and TIMPs, and activity of MMP-2 and MMP-9, point to the significance that these molecules might participate in the complex orchestration of chicken POF regression.

Selection of reference genes for quantitative real-time PCR analysis in chicken ovary following silver nanoparticle treatment.

Reliable results of quantitative real time PCR (qPCR) analysis require normalization of target gene expression level using reference genes (RGs). However housekeeping genes expression may vary under experimental conditions, so selection of the proper RGs is a crucial step in a qPCR analysis. Several algorithms have been developed to address this problem: geNorm, NormFinder and BestKeeper. In this study, we have used these three tools to evaluate the stability of RGs in the ovarian tissues of hens treated with silver nanoparticles. Eight genes were selected for the validation: HPRT, HMBS, VIM, SDHA, TBP, RPL13, GAPDH and 18S rRNA. According to geNorm the best combination of reference genes is SDHA and TPP. NormFinder also selected SDHA as the most suitable gene, but in combination with RPL13. Analysis in BestKeeper showed that SDHA, RPL13 might be the best choice in gene expression studies using the chicken ovary. In conclusion, the results obtained depend on the algorithm used and it arises from the diverse calculation strategies used in these programs. The outcome from the NormFinder is considered to be the most trustworthy and used in further qPCR analysis.

Effects of PCB 126 and PCB 153 on secretion of steroid hormones and mRNA expression of steroidogenic genes (STAR, HSD3B, CYP19A1) and estrogen receptors (ERα, ERß) in prehierarchical chicken ovarian follicles.

The objective of this study was to assess the in vitro effects of dioxin-like PCB 126 and non-dioxin-like PCB 153 on basal and ovine LH (oLH)-stimulated testosterone (T) and estradiol (E2) secretion and expression of steroidogenic genes (STAR, HSD3B and CYP19A1) and estrogen receptors α (ERα) and ß (ERß) in white (WF) and yellowish (YF) prehierarchical follicles of the hen ovary. Steroid concentrations in a medium and gene expression in follicles following 6h of exposition were determined by RIA and real-time qPCR, respectively. Both PCBs increased basal and oLH-stimulated T secretion by the WF follicles. PCB 126 reduced basal E2 secretion by the WF follicles. PCB 153 elevated but PCB 126 reduced oLH-stimulated E2 secretion by the prehierarchical follicles. PCB 126 increased basal STAR and HSD3B and reduced CYP19A1 mRNA expression in these follicles. PCB 153 increased basal expression of STAR and HSD3B in YF follicles, but diminished HSD3B mRNA levels in the WF. The studied PCBs had an opposite effect on basal and oLH-stimulated CYP19A1 mRNA expression in prehierarchical follicles. Both PCBs modulated basal and inhibited oLH-stimulated ERα and ERß gene expression in the prehierarchical follicles. In conclusion, data of the current study demonstrate the congener-specific effects of PCBs on sex steroid secretion by prehierarchical follicles of the chicken ovary, which are at least partly related to STAR, HSD3B and CYP19A1 gene expression. It is suggested that PCBs, by influencing follicular steroidogenesis and expression of estrogen receptors, may impair development and selection of yellowish follicles to the preovulatory hierarchy.

Changes in proliferating and apoptotic markers in the oviductal magnum of chickens during sexual maturation.

The avian oviduct is characterized by dynamic hormonal, biochemical, and cellular changes during its development. To better understand the molecular mechanisms regulating proper development of this organ in birds, the rate of cell proliferation and apoptosis as well as these processes-related gene expressions in the chicken oviduct during the sexual maturation were examined. The oviducts were isolated from Hy-Line Brown chickens at 2-week intervals from 10 to 16 weeks of age, and at 17 weeks, i.e. just after the onset of egg laying. In the tissue from the middle part of the oviduct (the magnum) the following parameters were tested: (1) proliferating (proliferating cell nuclear antigen [PCNA]-positive) and apoptotic (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive) cells, (2) mRNA expression of bcl-2, caspases 2, 3, 8, and 9, PCNA, survivin-142, and ovalbumin by quantitative real-time polymerase chain reaction, (3) protein expression of Bcl-2, PCNA, and caspases 3 and 9 by Western blot, (4) activity of caspases 2, 3, 8, and 9 by fluorometric method, and (5) localization of Bcl-2 and caspases by immunohistochemistry. It was found that the number of proliferating cells per unit area did not change during the examined period. The number of apoptotic cells in the oviductal wall remained on the same level until 14 weeks of age followed by a gradual decrease, reaching the lowest number at 17 weeks. The mRNA expression of all caspases and Bcl-2 gradually decreased during maturation, and PCNA decreased after 14 weeks of age. Survivin-142 mRNA level increased in 14-week-old chickens and then diminished, whereas ovalbumin expression was dramatically elevated in birds 16 weeks old and older. Patterns of protein expression of Bcl-2, PCNA, and caspases and activity of caspases were similar to mRNA, although not as pronounced. In the wall of the magnum the apoptotic cells and examined proteins were localized predominantly in the mucosa (surface epithelium and tubular glands). In summary, the results obtained provide some evidence of changes in selected proliferation- and apoptosis-related gene expression, alterations in activity of multiple apoptotic markers, and differences in the frequency of proliferating and apoptotic markers between mucosa and stroma in the oviductal magnum during the sexual maturation. Concluding, we suggest that Bcl-2, PCNA, survivin-142, and some caspases may cooperatively orchestrate a cascade of events mainly related to the cell proliferation, apoptosis, and differentiation in the chicken oviduct over the course of its development.