Comparison of in vitro bioactivity of chicken prolactin and mammalian lactogenic hormones.

Recombinant chicken prolactin, expressed in Escherichia coli as an unfolded protein, was successfully refolded and purified to homogeneity as a monomeric protein. Its biological activity was evidenced by its ability to interact with rabbit prolactin receptor extracellular domain and stimulate prolactin receptor-mediated proliferation in three cell types possessing mammalian prolactin receptors. Chicken prolactin activity in those assays was 20-100-fold lower than that of mammalian lactogenic hormones, likely due to lower affinity for mammalian prolactin receptors and not to improper refolding, because in two homologous bioassays, chicken prolactin activity was equal to or higher than that of ovine prolactin and the CD spectra of chicken and human prolactin were almost identical. Our results using seven mammalian lactogenic hormones from five species in three bioassays revealed the major role of species specificity in testing biological activity in vitro. Heterologous bioassays may be misleading and homologous assays are strongly recommended for predicting the activity of species-specific lactogenic hormones in vivo.

Expression and localization of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct during pause in laying induced by tamoxifen.

Induced pause in egg laying simulates natural molting events in which the hen's reproductive organs regress and rejuvenate. Such processes require extracellular matrix remodeling that is maintained, at least in part, by the action of proteolytic enzymes known as matrix metalloproteinases (MMPs). Nevertheless, information concerning the expression and hormonal regulation of MMP system members in chickens is scarce. Therefore, MMP-2, -7, and -9 and their tissue inhibitors (TIMP-2, -3) expression and localization were investigated in all segments of the domestic hen oviduct (infundibulum, magnum, isthmus, shell gland, vagina) during a pause in egg laying induced by tamoxifen (TMX)-an estrogen receptor modulator. Hy-Line Brown hens were treated daily with TMX (n = 6) at a dose of 6 mg/kg of body weight or a vehicle (n = 6) until complete cessation of egg laying (for 7 days). Chickens were decapitated on Day 7 of the experiment. Real-time polymerase chain reaction and Western blotting revealed section-dependent expression of MMP-2, -7, -9 and TIMP-2 and -3. Immunohistochemistry found tissue and cell-dependent localization of examined proteins in the wall of the oviduct. The MMP-2, TIMP-2, and TIMP-3 were localized mainly in the luminal epithelium, MMP-7 in the luminal and glandular epithelium, whereas MMP-9 was detected only in the connective tissue. Treatment of chickens with TMX markedly elevated the relative expression of MMP-7 and MMP-9 mRNA in the oviduct, but did not affect MMP-2, TIMP-2, and TIMP-3 mRNA levels. However, TMX increased the MMP-2 protein level in the infundibulum, shell gland, and vagina as well as activity of MMP-2 evaluated by gelatin zymography. The results obtained indicate that MMP-2, MMP-7, and MMP-9 are involved in chicken oviduct regression. Moreover, changes in the expression and activity of chosen MMPs after TMX treatment may indicate a contribution of estrogen in the regulation of transcription, translation, and/or the activity of selected elements of the MMP system.

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

This study was undertaken to examine mRNA and protein expression, localization of selected MMPs and their tissue inhibitors (TIMPs), and the activity of MMPs in all segments of the chicken oviduct (infundibulum, magnum, isthmus, shell gland, vagina) during a pause in egg laying induced by food deprivation. Control chickens (n = 18) were fed ad libitum, while experimental birds (n = 18) were fasted for 5 days, followed by being fed every other day, and then fed daily from day 10 onwards. Chickens were decapitated on day 6 (when the oviduct was regressed), day 13 (during oviduct recrudescence), and days 17-20 (oviduct rejuvenation) of the experiment. A pause in laying occurred between days 6 and 13 and resumption of laying before days 17-20. Real-time polymerase chain reaction and western blot revealed different expression of MMP-2, -7, -9 and TIMP-2 and -3 on mRNA and protein levels, respectively, as well as activity of MMP-2 and -9 by activity assay in the oviduct parts. Immunohistochemistry showed cell- and tissue-dependent localization of the examined members of the MMP system. Regression of the oviduct was accompanied predominantly by an increase in the relative expression of MMP-2, -7 and -9, and TIMP-2 and -3 mRNAs. The protein expression levels and localization of MMPs and TIMPs did not show pronounced changes during the examined period. During oviduct regression and recrudescence, elevated activity of MMP-2 and -9 was found. In summary, the results showing the stage of reproductive cycle-dependent changes as well as cell- and 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 be the local regulators of remodeling and functions of the hen oviduct.

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.

Expression and localization of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct during maturation.

Although participation of matrix metalloproteinases (MMPs) in reproductive tract remodeling has been strongly suggested in mammalian species, the role of MMPs in the avian oviduct has received little attention. To gain a better understanding of the potential role of the MMP system in avian oviduct development, mRNA and protein expression, localization of selected MMPs and their tissue inhibitors (TIMPs), and gelatinolytic activity in the oviduct of growing chickens were examined. The oviducts were collected from Hy-Line Brown hens before (10, 12, 14 and 16 weeks of age) and after (week 17) the onset of egg laying. The MMP-2, -7, -9 and TIMP-2 and -3 genes were found to be differentially expressed in all examined oviductal sections: the infundibulum, magnum, isthmus and shell gland on both mRNA (by real time polymerase chain reaction) and protein (by western blotting and immunohistochemistry) levels. In the course of oviduct development, the relative expression of all genes decreased in most sections. Protein level of MMP-9 was diminished, while MMP-7 and TIMP-3 were elevated in the oviduct of growing birds. MMP-2 and TIMP-2 protein levels remained constant, with a slight increase in MMP-2 concentration just before reaching maturity. The relative activity of MMP-2 and -9 (assessed by gelatin zymography) was higher (P < 0.05, P < 0.01) in immature birds compared with adults. Immunohistochemistry demonstrated cell- and tissue-specific localization of MMPs and TIMPs in the wall of the chicken oviduct. We concluded that changes in the expression of examined MMPs and their inhibitors, as well as alterations in MMP activity occurring simultaneously with changes in the morphology of the chicken oviduct, suggest the involvement of the MMP system in the proper development and functioning of this organ. Mechanisms regulating the expression and activity of MMPs require further clarification.

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.