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.

Vitamin D3 Receptors and Metabolic Enzymes in Hen Reproductive Tissues.

In recent years, vitamin D3 has been revealed as an important regulator of reproductive processes in humans and livestock; however, its role in the female reproductive system of poultry is poorly known. The aim of this study was to examine vitamin D3 receptor (VDR and PDIA3) and metabolic enzyme (1α-hydroxylase and 24-hydroxylase) mRNA transcript and protein abundances, and protein localization within the hen ovary, oviductal shell gland, pituitary, liver, and kidney. We demonstrated, for the first time, the patterns of the relative mRNA and protein abundances of examined molecules in the ovary, dependent on follicle development and the layer of follicle wall, as well as in other examined organs. Immunohistochemically, PDIA3, 1α-hydroxylase, and 24-hydroxylase are localized in follicular theca and granulosa layers, luminal epithelium and tubular glands of the shell gland, pituitary, liver, and kidney. These results indicate that reproductive tissues have both receptors, VDR, primarily involved in genomic action, and PDIA3, probably participating in the rapid, non-genomic effect of vitamin D3. The finding of 1α-hydroxylase and 24-hydroxylase expression indicates that the reproductive system of chickens has the potential for vitamin D3 synthesis and inactivation, and may suggest that locally produced vitamin D3 can be considered as a significant factor in the orchestration of ovarian and shell gland function in hens. These results provide a new insight into the potential mechanisms of vitamin D3 action and metabolism in the chicken ovary and oviduct.

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 gonadotropin, prolactin, androgen and estrogen receptor and steroidogenesis-associated gene expression in gander testes in relation to the annual period.

Physiological mechanisms of seasonal changes in testicular function in birds are not fully elucidated. The balance between androgens and estrogens and testis sensitivity for gonadotropin and gonadal steroids are still unclear. The aim of the study was to examine: (1) the changes in circulating and intra-testicular steroid hormone levels and their relationship; (2) the mRNA expression of testicular gonadotropin, prolactin (PRL), progesterone (P4), androgen, and estrogen receptors, and (3) key steroidogenesis processes-related genes with immunofluorescent localization of aromatase in gander testes during the annual period. Testes from ganders (n = 25) in the first reproduction season were obtained at five breeding stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under breeding conditions. It was found that plasma P4 levels decreased at the PoB and NB stages, whereas intra-testicular P4 was the highest in the NB stage. Intra-testicular estradiol (E2) levels were higher at the PoB and NB stages than the other stages, whereas testosterone (T) levels showed a nearly opposite pattern. The plasma estradiol-to-testosterone ratios were higher at the PrB, PoB and NB stages compared to other stages. The transcript abundances for luteinizing hormone receptor (LHR), PRL receptor (PRLR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERß) also change in testicular tissue during the annual period. Moreover, StAR mRNA expression was upregulated at the PoB and NB stages, and CYP11A1 transcript level was the highest at the PoB stage. Stage-dependent changes in the CYP19A1 mRNA and aromatase protein levels with higher abundances of transcript at PoB and NB stages and protein at the NB stage were observed. Localization and immunofluorescent signal intensity for aromatase also differed in relation to the examined stages. It may be suggested that differential E2 levels, as well as aromatase expression and localization across annual stages are responsible for the seasonal activation/inactivation stages of testis spermatogenesis in domestic ganders. These data strongly suggest a role of aromatase in the control of gander steroidogenesis as changes in this enzyme level are associated with alternation in gonadal steroid hormones. In addition, joint action with others hormones, like PRL and LH, seems to be important in the final effect of seasonal reproduction potential.

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 gene expression of selected matrix metalloproteinase system proteins in the broiler chicken gastrointestinal tract during post-hatch development and coccidia infection.

Matrix metalloproteinases (MMPs) are a family of proteases, that can process extracellular matrix (ECM) components and non-ECM molecules. MMPs can also function intracellularly in proteolytic and nonproteolytic functions. The participation of MMPs in the remodeling of the chicken gastrointestinal tract is largely unknown. The aim of the present study was to examine 1) the early neonatal developmental changes and effect of delayed access to feed immediately post-hatch (PH) and 2) the effect of Eimeria infection on mRNA expression of selected MMPs, their tissue inhibitors (TIMPs), and a disintegrin and metalloproteinase (ADAM) metallopeptidase with thrombospondin type 1 motif 8 (ADAMTS8) in the gastrointestinal tract of chicken. Protein localization of MMPs and TIMPs was also carried out in the normal ileal wall at -48, 24, and 336 h relative to hatch using immunofluorescence. In experiment 1, newly hatched Ross 708 chicks received feed and water immediately PH or were subjected to 48 h delayed access to feed. Chickens were sampled at -48, 0, 4, 24, 48, 72, 96, 144, 192, 240, 288, and 336 h PH. Ileum was collected for investigation of gene expression or fixed in paraformaldehyde for immunofluorescence. In experiments 2 and 3, Ross 708 male broilers were infected, at 21 d of age with Eimeria maxima or E. acervulina or sham-infected with water. Intestinal tissues were collected at 7 and 10 d postinfection for gene expression analysis. In general, mRNA expression patterns of all examined genes showed downregulation during the first 2 wk PH and were not affected by delay in feed access. These development-dependent changes in expression and tissue-dependent localization in the ileum of selected MMPs and TIMPs indicate that these molecules participate in the remodeling of chicken intestinal tissues during PH development. Increased expression of MMP-7 and MMP-9 transcripts in the intestine of Eimeria infected birds suggests an important role for these enzymes in the process of tissue remodeling and destruction in pathological conditions. The findings of this study are important for understanding the relationship between the expression of the MMP system and intestinal development, as well its role in gastrointestinal infection and subsequent recovery.

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.

The expression and localization of selected matrix metalloproteinases (MMP-2, -7 and -9) and their tissue inhibitors (TIMP-2 and -3) in follicular cysts of sows.

Matrix metalloproteinases (MMPs) are a family of enzymes that degrade extracellular matrix (ECM) molecules, playing a vital role in tissue remodeling under physiological and pathological conditions. Their expression and/or activity are regulated by specific tissue inhibitors of MMPs named TIMPs. Recently, an imbalance in the MMP/TIMP system has been found in human and bovine ovarian cysts, but its role in porcine cyst pathogenesis is unknown. This study examined mRNA expression, protein abundance and localization for selected members of the MMP/TIMP system in follicular cysts of sows. Based on histological analysis, we have assessed follicular (FC) and follicular lutein (FLC) cysts with preovulatory follicles (PF) used as a control. Regarding the pattern of MMP expression, increased MMP2, MMP7 and MMP9 mRNA levels were observed in FLC. Furthermore, both pro- and active forms of MMP-2 and MMP-9 proteins were more abundant in FLC. In FC, the abundance of latent and active forms of MMP-9 and the active form of MMP-2 were greater when compared with PF. In relation to TIMPs, TIMP-2 mRNA and protein expression were increased in FLC, whereas TIMP-3 was up-regulated in both FC and FLC only at the protein level. Using immunofluorescence, MMP-2, MMP-7, TIMP-2 and TIMP-3 were detected in granulosa and theca compartments of FC and within the entire luteinized wall of FLC. Notably, MMP-9 occurred weakly in the granulosa layer of FC, but abundantly in the theca compartment of FC and in the luteinized FLC. Taken together, our findings indicate altered expression of the MMP/TIMP system, suggestive of increased ECM degradation, in sow follicular cysts. These components may be involved in the pathogenesis of porcine ovarian cysts through the ECM remodeling.

Annual changes in cell proliferation and apoptosis and expression of connexin 43 in the testes of domestic seasonal breeding ganders.

Molecular mechanisms of seasonal changes in testicular morphology, histoarchitecture, and functions in birds have not been fully elucidated. The aim of the study was to examine the alternations in cell proliferation and apoptosis, these processes-related gene expressions, and gap junction protein (GJA1gene, connexin 43 protein; Cx43) expression and localization in gander testes during annual stages. Testes from domestic ganders (n = 28) in the first reproduction season were obtained at five stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under controlled breeding conditions. Testicular weight, morphometry, and histology (H&E staining) were evaluated, and the following parameters were tested: (1) number of proliferating (PCNA-positive) and apoptotic (TUNEL-positive) cells; (2) mRNA and protein abundances of PCNA, caspase-3, and Cx43 by qRT-PCR and Western blot, respectively; (3) activity of caspase-3 by fluorometric method; and (4) localization of Cx43 by immunofluoresence. Testicular weight was found to decrease by 4-fold at the NB stage with massive depletion of germ cells concomitantly with a reduction of seminiferous tubule (ST) diameter and ST lumen compared to the PR and OR stages. The number of proliferating germ cells was higher at PrB and PR than at the PoB stage, whereas the number of apoptotic cells was higher at PoB and NB compared to PrB and OR. Thus, proliferation-to-apoptosis ratios were lower in PoB and NB than other stages. Moreover, mRNA expression of PCNA exhibited down regulation in these stages compared to PrB and PR. Stage-dependent changes in the Cx43 mRNA level and in the localization pattern in the ST germinal epithelium were observed. Lower abundances of GJA1 transcript during NB and OR than at PoB the stage and irregular distribution of Cx43 protein located near the lumen of ST primarily at PrB and NB compared to the remaining stages were noted. These results suggested that during gander testis development, function, regression, and recrudescence, the interaction between processes of cell proliferation, apoptosis, and changes in the localization of Cx43 protein in the germinal epithelium occurred. The balance between these processes may determine the final functional activity or inactive stage of the testes connected with weight and histoarchitecture changes.

Matrix Metalloproteinases (MMPs) and Inhibitors of MMPs in the Avian Reproductive System: An Overview.

Many matrix metalloproteinases (MMPs) are produced in the mammalian reproductive system and participate in the regulation of its functions. In birds, the limited information available thus far indicates that MMPs are significant regulators of avian ovarian and oviductal functions, too. Some MMPs and inhibitors of MMPs are present in the hen reproductive tissues and their abundances and/or activities change according to the physiological state. The intraovarian role of MMPs likely includes the remodeling of the extracellular matrix (ECM) during folliculogenesis, follicle atresia, and postovulatory regression. In the oviduct, MMPs are also involved in ECM turnover during oviduct development and regression. This study provides a review of the current knowledge on the presence, activity, and regulation of MMPs in the female reproductive system of birds.

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.

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.

miRNA expression profile in chicken ovarian follicles throughout development and miRNA-mediated MMP expression.

Accumulating evidence has demonstrated the role of microRNAs (miRs) in the avian ovary. In this study, high-throughput transcriptome analyses were employed to study the differential miR expression profiles in the chicken ovary, aiming to reveal miR-targeting matrix metalloproteinase (MMP) expression during follicular growth, maturation, and atresia. Using tissues of chicken ovarian follicles at key steps of development (slow growing - white, the most recently recruited - small yellow, and preovulatory - F2) and regression (the third postovulatory), 14 small RNA (sRNA) libraries were constructed. The 25 most highly expressed known miRs were identified along with eight significantly differentially expressed (DE) miRs (gga-miR-let-7d, gga-miR-31-3p, gga-miR-138-1-3p, gga-miR-1552-5p, gga-miR-92-3p, gga-miR-31-5p, gga-miR-202-3p, and gga-miR-6648-3p) which were further examined by quantitative real time-PCR (qRT-PCR) in white, yellowish, small yellow, and atretic follicles as well as in the granulosa and theca layer of yellow preovulatory F3-F1 follicles (n = 6 hens). These miRs were mainly associated with four pathways: inhibition of MMPs, axonal guidance signaling, HIF1α signaling, and GP6 signaling. Four predicted target genes (i.e. MMP-16, ADAM10, COL4A2, and COL4A5) were examined by qRT-PCR and negatively correlated with DE miRs. The identified candidate miR:mRNA target pairs include gga-miR-31-5p or gga-miR-92-3p:MMP-16, gga-miR-31-5p or gga-miR-92-3p:ADAM10, let-7d:COL4A2, and gga-miR-138-1-3p:COL4A5 are potentially associated with MMP modulation in the hen ovary, mostly in the granulosa and theca cells of the largest preovulatory follicles. These results provide a novel insight to the role of miRs in follicle development by identifying a miR target network that is putatively engaged in remodeling of the extracellular matrix during ovarian follicle development in chickens.

Altered vitamin D metabolic system in follicular cysts of sows.

This study aimed to examine 25OHD3 concentration in the fluid of follicular and follicular lutein cysts of sows in comparison with preovulatory follicles as well as immunolocalize vitamin D metabolic enzymes (CYP27B1 and CYP24A1) and determine their protein abundances in the cyst wall. We have shown for the first time that 25OHD3 level in the fluid of both cyst types was significantly lower than in preovulatory follicles. Furthermore, we have demonstrated CYP27B1 and CYP24A1 protein immunolocalization and abundance in follicular and follicular lutein cysts. The abundance of protein for both metabolic enzymes was decreased in ovarian cysts when compared to preovulatory follicles. We propose that altered VD metabolism in ovarian cyst might associate with their formation in sows.

Effects of dietary supplementation with algae, sunflower oil or soybean oil on folliculogenesis in the rabbit ovary during sexual maturation.

This study aimed to examine the effects of algae (rich in omega-3 fatty acids), sunflower oil (rich in omega-6 fatty acids) and soybean oil (rich in omega-6 fatty acids) on the entire folliculogenesis in juvenile and sexually mature rabbits. After weaning, rabbits were randomly divided into four experimental groups of 14 animals each. Control animals received non-supplemented pellets, while in the other groups, the pellets contained 1% marine algae, 3% sunflower oil or 3% soybean oil. Animals from each group were slaughtered at 12 weeks of age (n = 7 per group) or at 18 weeks of age (n = 7 per group). The ovaries were harvested and fixed for hematoxylin-eosin staining, immunohistochemical localization of PCNA and TUNEL assay. Algae-enriched diet markedly decreased the number of primordial and primary follicles, while addition of sunflower oil reduced the number of primary follicles in 12-week-old rabbits. The number of antral follicles was higher following algae supplementation, but lower after addition of soybean oil in that age group. Proliferating index was decreased following supplementation with algae and soybean oil in juvenile rabbits, whereas it was increased after addition of algae and decreased following vegetable oils in mature ones. Dietary PUFAs did not impact apoptosis in the rabbit ovary of both age groups. The obtained results suggest that PUFA-enriched diet regulate either early folliculogenesis or antral follicle development in rabbits that might influence reproductive performance as a consequence. It appears that observed effects are attributed to sexual maturity.

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.

The effect of parachlorophenylalanine treatment on the activity of gonadal and lactotrophic axes in native Polish crested chickens stimulated to broodiness.

Brooding behavior, a common characteristic of native breeds of the domestic chicken, is marked by elevated prolactin (PRL) levels, which is necessary for incubation and connected with changes in hypothalamic-pituitary-gonadal axis activity. Evidence indicates the serotoninergic system is a potent modulator of PRL secretion. The objective of this study is to investigate whether blocking serotonin synthesis with parachlorophenylalanine (PCPA) prevents incubation behavior in native Polish crested chickens. In addition, we examined the effect of PCPA on the gene expression of the gonadal and lactotrophic axes. Birds were stimulated to broodiness by artificial eggs in nests. At 34 wk of age (April: spring period), the hens were divided into 2 groups (14 hens in each group): control and PCPA-treated (50 mg/kg BW) group. After 5 wk of treatment, the artificial eggs were removed from the nests. Egg production, incubation activity, and levels of plasma ovarian steroids progesterone (P4), testosterone (T), estradiol (E2), and PRL were examined. At the end of the experiment (45 wk of age, June: summer period), ovarian characteristics and mRNA gene expression of gonadal (gonadotropin-releasing hormone [GnRH] I, luteinizing hormone [LH] ß, follicle-stimulating hormone [FSH] ß) and lactotrophic (vasoactive intestinal peptide [VIP], PRL) axes were measured by quantitative real-time PCR. Incubation activity was observed in the hens of both groups but with lower frequency in PCPA-treated birds. Moreover, the PCPA group had a higher cumulative egg production than the controls. During the first six and 8 wk of the experiment, levels of P4 and E2, respectively, were similar in both groups, but all concentrations increased in the PCPA-treated hens after this period. In addition, increased GnRH-I, LHß, and FSHß and decreased VIP mRNA expression was observed in the PCPA group compared with the controls. There were no differences in PRL mRNA expression, the PRL level, and ovarian morphometry between the 2 groups. These results indicate that blockage of serotonin synthesis by PCPA does not effectively prevent incubation in native Polish crested chickens. However, treatment with PCPA increased gonadal axis activity and improved reproductive performance.

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.