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.

Alterations in apoptotic markers and egg-specific protein gene expression in the chicken oviduct during pause in laying induced by tamoxifen.

The aim of this study was to examine the cell apoptosis, gene expression and activity of caspases 2, 3, 8 and 9, and the mRNA expression of selected egg-specific proteins in the chicken oviduct during pause in egg laying induced by tamoxifen (TMX) treatment. The experiment was carried out on Hy-Line Brown laying hens. The control birds were treated subcutaneously with vehicle (ethanol) and the experimental ones with TMX at a dose of 6 mg/kg of body weight. Hens were treated daily until a pause in egg laying occurred and sacrificed on Day 7 of the experiment. Within the oviductal wall, the highest number of apoptotic cells (TUNEL-positive) was found in the luminal epithelium and the lowest in the stroma. The administration of TMX increased the percentage of apoptotic cells in the magnum, isthmus, and shell gland as well as immunoreactivity for caspases 3 and 9. Real-time PCR analysis revealed the segment-dependent mRNA expression of caspases 2, 3, 8 and 9. Treatment of hens with TMX elevated the level of caspase-2 transcript in the infundibulum, caspases 2, 3 and 8 in the isthmus, and caspase-9 in the shell gland (P < 0.05 - P < 0.001). As shown by fluorometric method TMX caused an increase in the activity of caspases 3 and 8 in the magnum, isthmus and shell gland, and the activity of caspases 2 and 9 in the isthmus and shell gland (P < 0.05 - P < 0.01). The expression of ovalbumin, avidin and ovocleidin-116 mRNAs was decreased (P < 0.05 - P < 0.001), ovocalyxin-36 mRNA level tended to increase, and ovocalyxin-32 expression was not affected by TMX. The results obtained indicate that caspases are involved in the chicken oviduct regression during a pause in laying induced by TMX, and estrogen is involved in the regulation of examined caspase expression and activity. The changes in mRNA transcript levels of some examined egg-specific proteins after TMX treatment suggest that there is a relationship between estrogen action and the expression of these genes.