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.

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.

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.

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.

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.

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.

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.

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.

Chicken oviduct-the target tissue for growth hormone action: effect on cell proliferation and apoptosis and on the gene expression of some oviduct-specific proteins.

The aim of this study was to examine the in vivo effect of growth hormone (GH) on cell proliferation and apoptosis and on the gene expression of selected proteins in the chicken oviduct before sexual maturity (first oviposition). Ten-week-old Hy-Line Brown chickens were injected three times a week with 200 µg · kg(-1) body weight of recombinant chicken GH (cGH) until 16 weeks of age. Control hens received 0.9 % NaCl with 0.05 % bovine serum albumin as a vehicle. Treatment with cGH increased (P < 0.05) oviduct weight at 16 weeks of age, i.e. 1-2 weeks before onset of egg laying. The highest number of proliferating (determined by proliferating cell nuclear antigen [PCNA] immunocytochemistry) and apoptotic (determined by TUNEL assay) cells in the oviduct was found in the mucosal epithelium, and the lowest in the stroma. Administration of cGH did not increase (P > 0.05) the number of PCNA-positive cells but it decreased (P < 0.01) the number of TUNEL-positive cells, thus increasing the proliferating-to-apoptotic cell ratio in the oviduct. Gene expression (determined by real-time polymerase chain reaction) of apoptosis-related caspase-2 in the magnum and caspase-3 in the magnum and isthmus and their activity (determined by fluorometric assay) in the magnum were attenuated (P < 0.05) in cGH-treated hens. The gene expression of the magnum-specific ovalbumin and the shell-gland-specific ovocalyxins 32 and 36 was increased (P < 0.05) in cGH-treated chickens. In contrast, the expression of Bcl-2 and of caspases 8 and 9 was not affected by cGH in any of the oviductal segments. The results suggest that GH, via the orchestration of apoptosis and expression of some oviduct-specific proteins, participates in the development and activity of the chicken oviduct prior to the onset of egg laying.

Effect of growth hormone on basal and LH-stimulated steroid secretion by chicken yellow ovarian follicles. An in vitro study.

The aim of the present study was to determine the effect of growth hormone (GH) on basal and LH-regulated steroid secretion by yellow hierarchical follicles before and after maturation, and the granulosa and theca layers of the largest preovulatory follicles during the ovulatory cycle in the chicken. In the first experiment, whole yellow follicles (8-12 mm, 12-18 mm, 18-24 mm and 24-30 mm) isolated from 15 and 17-18 week-old chickens were used. In the second experiment, the granulosa and theca layers of the 3 largest yellow preovulatory follicles (F3

The role of thyroid hormones in regulation of chicken ovarian steroidogenesis.

In all vertebrates, including birds, the normal development of the ovary and ovarian follicles is under the regulatory influence of hormones produced by the reproductive axis. In recent years, it has become clear that in birds an adequate level of thyroid hormones (THs), i.e. thyroxine (T4) and triiodothyronine (T3), in blood circulation is of primary importance for normal female reproductive functions. In avian species, characterized by seasonal reproduction, THs are involved in the photoperiodic regulation of reproduction acting at the mediobasal hypothalamus. In domestic fowl, where the seasonality of reproduction has been eliminated, the role of THs in ovarian function is not fully elucidated. Recent studies have revealed that ovarian follicles of the laying hen express mRNAs of TH nuclear receptors (TRα and TRß0) as well as integrin (αVß3) plasma membrane receptors, indicating genomic and nongenomic action of THs in the chicken ovary. In vivo experiments carried out on laying hens have showed that the bolus injection of T3 decreases levels of luteinizing hormone (LH) and estradiol (E2) in blood, and a hyperthyroid state evoked by administration of T3 for few days diminishes LH, E2 and progesterone (P4) levels, reduces the weight of the ovary, induces atresia of preovulatory follicles and eventually causes stoppage of egg laying. In vitro studies have demonstrated that T3 decreases E2 secretion from white nonhierarchical follicles and the theca layer of yellow preovulatory follicles, while on the other hand, it elevates P4 production from the granulosa layer of these follicles. These effects have been associated with steroidogenic enzyme expression and cyclic AMP synthesis. This review summarizes the current knowledge concerning the role of THs in regulation of steroidogenesis in chicken ovarian follicles.

Effect of weak electromagnetic field on cardiac work, concentration of thyroid hormones and blood aminotransferase level in the chick embryo.

The aim of the study was to determine the effect of alternating electromagnetic field (EMF; 50 Hz frequency, 50 and 100 µT induction) on cardiac work of the chick embryo. Eggs from the experimental groups were exposed to EMF throughout incubation. During the experiment, heart rate (ballistocardiographic method), thyroxine (T4) and triiodothyronine (T3) concentrations, heart weight, ventricle wall thickness, and levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. The results show, for the first time, that the exposure of chick embryos to EMF augments the heart rate, especially from 17 days of incubation. The increased heart rate in the embryos exposed to EMF was associated with considerable increases in plasma T4 and T3 concentrations, which were recorded during the final stage of embryogenesis. The significant effect of the 100-µT field on heart weight and blood AST levels in the embryos suggests that EMF has a direct effect on the physiological function of cardiac muscle.

In vitro effects of TCDD, PCB126 and PCB153 on estrogen receptors, caspases and metalloproteinase-2 mRNA expression in the chicken shell gland.

Among the environmental chemicals which disturb endocrine functions, dioxins and polychlorinated biphenyls (PCBs) are known as the most toxic. Numerous studies in mammals revealed that dioxins and PCBs disrupt functions of the uterus, delay implantation and increase embryo loss. The direct effect of these chemicals on the avian oviduct is not known. Therefore, in the study chicken shell gland tissues were used to examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coplanar PCB126 and non-coplanar PCB153 on estrogen receptors (ERs), initiator caspase-1, executioner caspase-3 and metalloproteinase-2 (MMP-2) mRNA expression. Fragments of shell gland tissue isolated from the laying chicken were incubated for 24h with TCDD (100nM), PCB126 (100nM) or PCB153 (100 microM). Quantitative PCR analysis showed that: (1) TCDD increased ER beta (ERbeta) mRNA expression, (2) PCB126 increased ER alpha (ERalpha), ERbeta and caspase-1, and decreased MMP-2 mRNA expression, (3) PCB153 elevated the ERbeta and caspase-1 expression levels and (4) expression of caspase-3 was not altered by any investigated xenobiotics. The results obtained using the shell gland explants model indicate that dioxins and PCBs have a direct effect on the chicken oviduct, especially the shell gland, by affecting the expression of genes involved in the function of this oviductal segment. It is suggested that coplanar PCBs such as PCB126, by changing cellular and extracellular regulators gene expression, may lead to disruption of shell gland activity and impair egg components formed in this organ.

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.

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.

Independent, non-IGF-I mediated, GH action on estradiol secretion by prehierarchical ovarian follicles in chicken. In vitro study.

Information concerning the role of growth hormone (GH) in the local regulation of ovarian activity in birds is limited. Therefore, the aim of the present study was to determine whether in the domestic hen GH influences in vitro estradiol secretion by prehierarchical ovarian follicles. Moreover, the interaction between GH and IGF-I on estradiol secretion was examined. Small white (1-4 mm), large white (4-6 mm) and yellowish (6-8 mm) ovarian follicles were isolated at the stage of 2 h after ovulation. In the first experiment (n = 8 hens), whole follicles (small white, n = 6/dose/ovary; large white, n = 1/dose/ovary and yellowish, n = 1/dose/ovary) were ipcubated for 24 h at 38 degrees C in a medium supplemented with 0 (control), 1, 10 or 100 ng/ml of chicken GH (cGH). In the second experiment (n = 6 hens), follicles were incubated in the same way in a medium with 0 (control), 10 ng/ml cGH, 25 ng/ml human IGF-I or cGH+hIGF-I (10 ng/ml+25 ng/ml). Following incubation the estradiol concentration was determined in media (RIA) and protein in the tissues of the follicular wall (Lowry). The secretion of estradiol was expressed per milligram of protein. The experiments revealed that both cGH and hIGF-I stimulated estradiol secretion by examined chicken ovarian follicles. The simultaneous addition of cGH and hIGF-I increased estradiol secretion by ovarian follicles as compare to the control. These hormones added together did not have an additive effect when compared to their separate actions. The results obtained suggest that both GH and IGF-I are important stimulators of estradiol production in chicken nonhierarchical ovarian follicles. We propose independent, non-IGF-I-mediated GH action on estradiol secretion.

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.

Effect of growth hormone on steroid content, proliferation and apoptosis in the chicken ovary during sexual maturation.

The present study was undertaken to examine in vivo the effect of growth hormone (GH) on progesterone and estradiol levels and on cell proliferation and apoptosis in the chicken ovary during sexual maturation. Hy-Line chickens (10 weeks old) were injected three times a week with 200 µg recombinant chicken GH (cGH) per kilogram body weight until sexual maturity. GH treatment significantly increased ovarian weight at 16 weeks of age, i.e., ~1 week before onset of egg laying. The progesterone content in the ovary just before and at the time of sexual maturity and the estradiol content before onset of egg laying were also elevated after cGH injections. The highest number of proliferating (positive for proliferating cell nuclear antigen) and apoptotic (positive for terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labeling) cells was found in the ovarian stroma and white follicles (>1-4 mm diameter), whereas the lowest number of these cells was detected in yellow (>8-30 mm) follicles. Administration of cGH significantly stimulated cell proliferation and inhibited cell apoptosis in the ovarian stroma and small ovarian follicles. The number of ovarian follicles and the weight of the ovary prior to the first oviposition were also higher in cGH-injected hens. Thus, prior to and after the onset of egg laying, GH participates in the growth, maturation and hormonal activity of ovarian follicles in the chicken, via the regulation of steroidogenesis, proliferation and apoptosis processes.

Structure and steroidogenic activity of the granulosa layer of F1 preovulatory ovarian follicles of the hen (Gallus domesticus).

The study was performed to determine the structure and steroidogenic activity of granulosa cells derived from the germinal disc region, proximal region and distal region of the largest preovulatory ovarian follicle (F1) of the hen. The study was carried out on 34 Hy-Line Brown egg-laying hens aged 40 weeks. Morphology of the granulosa cells was studied by histological assessment and scanning electron microscopy. Moreover, the level of P4, histochemical activity of 3beta-HSD and expression of 3beta-HSD gene mRNA in granulosa cells of F1 follicle were determined. The findings indicate that the morphology and steroidogenic activity of the granulosa layer in F1 preovulatory ovarian follicle are associated with the region of the follicle. This is consistent with earlier studies. In the germinal disc region the granulosa cells form a multilayer while in the proximal and distal regions granulosa cells form a single layer. Analysis of P4 concentration revealed that its level in granulosa cells was markedly reduced closer to the germinal disc. Moreover, our study demonstrates for the first time the lower histochemical activity of 3beta-HSD and expression of 3beta-HSD mRNA in granulosa cells from the germinal disc region compared with the proximal and distal region.