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.

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.

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.

Alternations in the expression of selected matrix metalloproteinases (MMP-2, -9, -10, and -13) and their tissue inhibitors (TIMP-2 and -3) and MMP-2 and -9 activity in the chicken ovary during pause in laying induced by fasting.

Matrix metalloproteinases (MMPs) are a large group of proteolytic enzymes involved in extracellular matrix turnover in the ovary. Under physiological conditions, the activity of MMPs is controlled by specific tissue inhibitors of MMPs (TIMPs). Information concerning the role and regulation of MMPs in the chicken ovary is scarce. This study was undertaken to examine the expression of selected MMPs and their TIMPs in the chicken ovary during a pause in egg laying induced by feed deprivation. The activities of MMP-2 and MMP-9 were investigated as well. Real-time polymerase chain reaction and Western blot analyses showed changes in the expression of gelatinases (MMP-2, MMP-9), stromelysin (MMP-10), collagenase (MMP-13), TIMP-2, and TIMP-3 on mRNA and/or protein levels in the prehierarchical white (WFs) and yellowish (YFs) follicles, as well as in the largest yellow preovulatory (F3-F1) follicles. In feed-deprived hens, the occurrence of ovarian regression was accompanied by (1) a pronounced decrease in mRNA expression of the examined MMPs and TIMP-3 in all tissues except the YFs where the expression of MMP-13 was higher than in the control hen ovary; (2) an increase in the transcript abundance of TIMP-2 in the yellow atretic follicles; (3) a decrease or no changes in MMP-2 and MMP-9 protein expression in all tissues; (4) an increase in the total activity of gelatinases in the YFs and theca layer of F3; and (5) a decrease in the activity of MMP-2 in F3-F1 follicles and MMP-9 in the theca of F3. In summary, the results of the current study suggest that the selected MMPs and TIMPs may not be involved in the regulation of the advanced stages of atresia of the largest yellow preovulatory follicles in the chicken ovary. This event may require different cell signaling pathways.

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.

Tamoxifen-induced alterations in the expression of selected matrix metalloproteinases (MMP-2, -9, -10, and -13) and their tissue inhibitors (TIMP-2 and -3) in the chicken ovary.

Matrix metalloproteinases (MMPs) are a family of peptidases that disintegrate extracellular matrix (ECM) molecules associated with tissue remodeling, including reproductive tissues. Their actions are largely controlled by specific tissue inhibitors of MMPs (TIMPs). The role and regulation of MMPs in the chicken ovary is largely unknown. The aim of the present study was to examine the effect of tamoxifen (TMX; estrogen receptor modulator) treatment on the expression of selected members of the MMP system in the laying hen ovary. The activity of MMP-2 and -9 was also examined. Real-time polymerase chain reaction and western blot analyses revealed changes in mRNA and/or protein expression of MMP-2, -9, -10, -13, TIMP-2, and TIMP-3 in the following ovarian follicles after TMX treatment: white (WF), yellowish (YF), small yellow (SYF), and the largest yellow preovulatory (F3-F1). The response to TMX depended on the stage of follicle development and the layer of follicular wall. Moreover, ovarian regression following TMX treatment was accompanied by both an increase in total activity of MMP-2 in the theca layer of F3-F2 and granulosa layer of F2, and a decrease in total activity of MMP-2 in the WF, YF, and SYF, and MMP-9 in theca of F3-F1. In conclusion, the TMX-induced changes in MMP-2, -9, -10, and -13, and TIMP-2 and -3 mRNA expression, as well as MMP-2 and -9 activity, were dependent on tissue and the stage of follicular maturation. Our findings strongly suggests a role for estrogen in regulating the transcription, translation, and/or posttranslational activity of members of the MMP system. Further, these components may be involved in the orchestration of ECM turnover and cellular functions during ovary regression, which occur under conditions of reduced estrogenic activity.

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.