Hen Ovary Research Articles

Regulation of egg quality and lipids metabolism by Zinc Oxide Nanoparticles

This investigation was designed to explore the effects of Zinc Oxide Nanoparticles (ZnO NP) on egg quality and the mechanism of decreasing of yolk lipids. Different concentration of ZnO NP and ZnSO4 were used to treat hens for 24 weeks. The body weight and egg laying frequency were recorded and analyzed. Albumen height, Haugh unit, and yolk color score were analyzed by an Egg Multi Tester. Breaking strength was determined by an Egg Force Reader. Egg shell thickness was measured using an Egg Shell Thickness Gouge. Shell color was detected by a spectrophotometer. Egg shape index was measured by Egg Form Coefficient Measuring Instrument. Albumen and yolk protein was determined by the Kjeldahl method. Amino acids were determined by an amino acids analyzer. Trace elements Zn, Fe, Cu, and P (mg/kg wet mass) were determined in digested solutions using Inductively Coupled Plasma-Optical Emission Spectrometry. TC and TG were measured using commercial analytical kits. Yolk triglyceride, total cholesterol, pancreatic lipase, and phospholipids were determined by appropriate kits. β-carotene was determined by spectrophotometry. Lipid metabolism was also investigated with liver, plasma, and ovary samples. ZnO NP did not change the body weight of hens during the treatment period. ZnO NP slowed down egg laying frequency at the beginning of egg laying period but not at later time. ZnO NP did not affect egg protein or water contents, slightly decreased egg physical parameters (12 to 30%) and trace elements (20 to 35%) after 24 weeks treatment. However, yolk lipids content were significantly decreased by ZnO NP (20 to 35%). The mechanism of Zinc oxide nanoparticles decreasing yolk lipids was that they decreased the synthesis of lipids and increased lipid digestion. These data suggested ZnO NP affected egg quality and specifically regulated lipids metabolism in hens through altering the function of hen's ovary and liver.

Alteration of gene expression by zinc oxide nanoparticles or zinc sulfate in vivo and comparison with in vitro data: A harmonious case

Granulosa cells (GCs) are those somatic cells closest to the female germ cell. GCs play a vital role in oocyte growth and development, and the oocyte is necessary for multiplication of a species. Zinc oxide (ZnO) nanoparticles (NPs) readily cross biologic barriers to be absorbed into biologic systems that make them promising candidates as food additives. The objective of the present investigation was to explore the impact of intact NPs on gene expression and the functional classification of altered genes in hen GCs in vivo, to compare the data from in vivo and in vitro studies, and finally to point out the adverse effects of ZnO NPs on the reproductive system. After a 24-week treatment, hen GCs were isolated and gene expression was quantified. Intact NPs were found in the ovary and other organs. Zn levels were similar in ZnO-NP-100 mg/kg– and ZnSO4-100 mg/kg–treated hen ovaries. ZnO-NP-100 mg/kg and ZnSO4-100 mg/kg regulated the expression of the same sets of genes, and they also altered the expression of different sets of genes individually. The number of genes altered by the ZnO-NP-100 mg/kg and ZnSO4-100 mg/kg treatments was different. Gene Ontology (GO) functional analysis reported that different results for the two treatments and, in Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, 12 pathways (out of the top 20 pathways) in each treatment were different. These results suggested that intact NPs and Zn2+ had different effects on gene expression in GCs in vivo. In our recent publication, we noted that intact NPs and Zn2+ differentially altered gene expression in GCs in vitro. However, GO functional classification and KEGG pathway enrichment analyses revealed close similarities for the changed genes in vivo and in vitro after ZnO NP treatment. Furthermore, close similarities were observed for the changed genes after ZnSO4 treatments in vivo and in vitro by GO functional classification and KEGG pathway enrichment analyses. Therefore, the effects of ZnO NPs on gene expression in vitro might represent their effects on gene expression in vivo. The results from this study and our earlier studies support previous findings indicating ZnO NPs promote adverse effects on organisms. Therefore, precautions should be taken when ZnO NPs are used as diet additives for hens because they might cause reproductive issues.

Flaxseed and its components differentially affect estrogen targets in pre-neoplastic hen ovaries

Flaxseed has been studied for decades for its health benefits that include anti-cancer, cardio-protective, anti-diabetic, anti-inflammatory properties. The biologically active components that mediate these effects are the omega-3 fatty acids and the lignan, secoisolariciresinol diglucoside. We have previously shown that whole flaxseed supplemented diet decreases the severity and incidence of ovarian cancer while a 15% dose of flaxseed is most protective against inflammation and estrogen-induced chemical and genotoxicity. The objective of this study was to dissect the independent effects of the two flaxseed components on estrogen signaling and metabolism. Two and half year old hens were fed either a control diet, 15% whole flaxseed diet, defatted flax meal diet or 5% flax oil diet for 3 months after which the animals were sacrificed and blood and tissues were harvested. Whole flaxseed diet caused a decrease in expression of ERα. ERα target gene expression was assessed using RT2 profiler PCR array. Some targets involved in the IGF/insulin signaling pathway (IRS1, IGFBP4, IGFBP5) were downregulated by flaxseed and its components. Flaxseed diet also downregulated AKT expression. A number of targets related to NF-kB signaling were altered by flaxseed diet including a series of targets implicated in cancer. Whole flaxseed diet also affected E2 metabolism by increasing CYP1A1 expression with a corresponding increase in the onco-protective E2 metabolite, 2-methoxyestradiol.The weak anti-estrogens, enterolactone, enterodiol and 2-methoxyestradiol, might be working synergistically to generate a protective effect on the ovaries from hens on whole flaxseed diet by altering the estrogen signaling and metabolism.

Intracellular lipid dysregulation interferes with leukocyte function in the ovaries of meat-type hens under unrestricted feed intake

Meat-type Red-feather country hens fed ad libitum (AD-hens) exhibit obesity-associated morbidities and a number of ovarian irregularities. Leukocyte participations in ovarian activities are unstudied in AD-hens. In contrast to feed-restricted hens (R-hens), ovulatory process of the F1 follicle appeared delayed in AD-hens in association with reduced F1 follicle progesterone content, gelatinase A (MMP-2) and collagenase-3 (MMP-13) activities coincident with elevated IL-1β and no production (P<0.05), and increased leukocyte infiltration of inflamed necrotic follicle walls. Extracts of AD-hen F1 follicle walls induced greater leukocyte migration than extracts from F1 follicle wall extracts of R-hens (P<0.05). Co-cultures of granulosa cells with increasing numbers of leukocytes from either AD-hens or R-hens exhibited dose dependent reductions in progesterone production and increases in cell death. AD-hen leukocytes were less proapoptotic than their R counterparts (P<0.05). Granulosa MMP-13 and MMP-2 activities were also suppressed in the co-cultures with heterophils or monocytes in a dose-dependent manner (P<0.05). AD heterophils and R monocytes had a greater inhibitory effect on MMP activities in the co-cultures than their respective counterparts (P<0.05). Both basal and LPS-induced IL-1β secretion and MMP-22 or MMP-2 activities in freshly isolated AD-hen leukocytes were reduced (P<0.05). Exposure of AD or R leukocytes to 0.5mM palmitate impaired IL-1β secretion and MMP-22 or MMP-2 activity. Inhibition of ceramide synthesis with FB1 and ROS production with n-MPG scavenging rescued MMP activity and IL-1β production in palmitate treated heterophils, but exacerbated monocyte suppression. These latter findings suggest that intracellular lipid dysregulation in leukocytes contributes to ovarian dysfunction in AD-hens.

Estrogen Receptor Subtype Expression is Altered in the Hen Model of Ovarian Cancer

There is growing evidence that estrogens may promote tumor progression, including ovarian tumors. Estrogens exert their actions in tissues through two different receptor subtypes (ESR1 and ESR2). Studies have shown that hens develop ovarian cancer spontaneously, therefore providing a suitable animal model for the disease. Our aim was to determine the expression of mRNA and protein of the estrogen receptor subtypes in ovaries of normal hens and ovaries from hens with ovarian cancer. Ovarian tissue from normal hens and hens with ovarian cancer was collected for quantitative real-time PCR and immunofluorescence analysis. Quantitative real-time PCR results showed that the relative mRNA expression of ESR1 and the ratio of ESR1/ESR2 are significantly greater from hens with ovarian cancer when compared to normal ovarian tissue. Immunofluorescence analysis showed differential ESR1 and ESR2 protein expression in ovarian tissue sections from normal hens and hens with ovarian cancer, with results parallel to the mRNA data. There was no significant difference in plasma estradiol levels between normal hens and hens with ovarian cancer. These data suggest an increase in downstream estrogen-mediated actions in chicken ovarian tumors and, indeed, microarray analysis reveals a functionally significant estradiol-signaling pathway in chicken ovarian tumors. Interestingly, expression of a putative ovarian tumor suppressor, EPB41L3, is down regulated in this pathway. Taken together, these results suggest that, in the hen, ESR1 may be mediating a proliferative response in ovarian cancer cells. Although additional studies are required to define the role of ESR1 in tumor formation in the hen, these results support the utility of the hen for testing possible endocrine therapies.

Granulosa cell responsiveness to follicle stimulating hormone during early growth of hen ovarian follicles

In the laying hen ovary, the cyclic recruitment of a follicle represents a process in which a single follicle is selected to enter the rapid growth phase and undergo final maturation prior to ovulation. Published data support the proposal that final differentiation of the granulosa cell (GC) layer commences at the time of follicle selection. This process is characterized by the enhanced capacity for FSH-induced cell signaling via the protein kinase A/cyclic adenosine monophosphate (cAMP) pathway. One consequence of such signaling within the GC layer is the initial capacity for steroidogenesis (predominantly progesterone production) mediated by increased expression of mRNA encoding steroidogenic acute regulatory protein (STAR) and the cholesterol side-chain cleavage enzyme (CYP11A). Prior to selection, the GC layer remains minimally responsive to a 3 h challenge with FSH (10 ng/mL), in vitro, compared to that from the most recently selected 9- to 12-mm follicle. By comparison, when the duration of the cell culture prior to FSH challenge is increased to 18 h, GCs collected from 1- to 2-mm, 3- to 5-mm, and 6- to 8-mm follicles respond to a 3 h FSH challenge by increasing STAR expression and progesterone production, with the greatest response from GCs collected from 6- to 8-mm follicles. Culture with Bone Morphogenetic Protein 6 (BMP6) enhances both CYP11A expression and FSH responsiveness at each stage of development, with the greatest response again occurring in GCs from 6- to 8-mm follicles. Significantly, factors that activate mitogen activated protein kinase (MAPK) or protein kinase C (PKC) signaling prevent the ability of prolonged culture or culture with BMP6 to induce FSH-responsiveness and the initiation of GC differentiation at each stage of development. Collectively, these results provide further support for the hypothesis that prior to follicle selection, inhibitory cell signaling (e.g., MAPK, PKC) maintains the GC layer in an undifferentiated state in follicles of all sizes, even in the presence of a differentiation-promoting signal (BMP6). The process by which the GC layer from the single 6- to 8-mm follicle selected each ovulatory cycle ultimately escapes inhibitory signaling to initiate FSH-responsiveness remains to be established.

Characterization of annexin A2 in chicken follicle development: Evidence for its involvement in angiogenesis

Annexin A2 (ANXA2) is a calcium-dependent, phospholipid-binding protein found in various cells and tissues. ANXA2 plays multiple roles in regulating cellular functions and is often over-expressed in different types of tumors including ovarian cancer. Others and we previously found that ANXA2 was up-regulated in the ovaries of hens with higher laying rate, indicated that ANXA2 is involved in avian follicle development. In this study, we found that ANXA2 mRNA expression increased during chicken ovary maturation and follicle development. In the pre-ovulatory follicles, ANXA2 expression level was significantly higher in theca cells than granulosa cells. In theca cells, ANXA2 expression could be stimulated by follicle-stimulating hormone (FSH) and estrogen but not luteinizing hormone (LH) or progesterone. The core promoter regions control the basal and FSH-induced ANXA2 gene expression were identified. Forced expression of ANXA2 could induce the expression of angiogenic factors and receptors in theca cells. Furthermore, ANXA2 overexpression resulted increased vascular endothelial growth factor A (VEGFA) secretion and theca cell proliferation. Current study not only provides the first evidence of expression and regulation of ANXA2 in chicken ovary, but also suggests that ANXA2 is involved in follicular angiogenesis and contributes to successful follicle development and ovulation.

Expression and effect of NAMPT (visfatin) on progesterone secretion in hen granulosa cells

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is an adipokine produced by adipose tissue that is found in intracellular and extracellular compartments. The intracellular form of NAMPT is a nicotinamide phosphoribosyltransferase, whereas the extracellular form is considered an adipokine. In humans, NAMPT regulates energy metabolism and reproductive functions, such as ovarian steroidogenesis. To date, no study has investigated the role of NAMPT in hen ovaries. We investigated whether NAMPT is present in hen ovarian follicles and its role in granulosa cells. Using RT-PCR, western blotting and immunocytochemistry, we detected mRNA transcripts and proteins related to NAMPT in theca and granulosa cells from pre-ovulatory follicles. Using RT-PCR, we demonstrated that mRNA NAMPT levels were higher in granulosa cells than they were in theca cells and that during follicle development, theca cell levels decreased, whereas levels remained unchanged in granulosa cells. NAMPT protein quantities were significantly higher in theca cells than they were in granulosa cells, but they were unchanged during follicular development. Plasma NAMPT levels, as determined by ELISA and immunoblotting, were significantly lower in adult hens than they were in juveniles. In vitro, treatment with human recombinant NAMPT (100 ng/ml, 48 h) halved basal and IGF1-induced progesterone secretion, and this was associated with a reduction in STAR and HSD3B protein levels and MAPK3/1 phosphorylation levels in granulosa cells. These effects were abolished by the addition of FK866, a specific inhibitor of NAMPT enzymatic activity. Moreover, NAMPT had no effect on granulosa cell proliferation. In conclusion, NAMPT is present in hen ovarian cells and inhibits progesterone production in granulosa cells.

Flaxseed reduces the pro-carcinogenic micro-environment in the ovaries of normal hens by altering the PG and oestrogen pathways in a dose-dependent manner.

The objective of the present study was to find the optimum dose of flaxseed that would decrease PG and alter oestrogen pathway endpoints implicated in ovarian cancer. In the study, four groups of fifty 1.5-year-old chickens were fed different amounts of flaxseed (0, 5, 10 or 15% of their total diet) for 4 months and were then killed to collect blood and tissues. Levels of flaxseed lignan metabolites, Enterolactone (EL) and Enterodiol (ED) were measured in the serum, liver and ovaries by liquid chromatography-MS/MS, and n-3 and n-6 fatty acid (FA) levels were measured by GC. The effects of the varied flaxseed doses were assessed by measuring levels of PGE2 and oestrogen metabolites (16-hydroxyestrone (16-OHE1) and 2-hydroxyestrone (2-OHE1)) as well as by analysing the expression of the oestradiol metabolising enzymes CYP3A4 (cytochrome p450, family 3, subfamily A, polypeptide 4), CYP1B1 (cytochrome p450, family 1, subfamily B, polypeptide 1) and CYP1A1 (cytochrome p450, family 1, subfamily A, polypeptide 1) and that of oestrogen receptor α (ERα) in the ovaries. The ratio of n-3:n-FA increased with an increase in flaxseed supplementation and corresponded to a dose-dependent decrease in cyclo-oxygenase-2 protein and PGE2 levels. EL and ED increased in the serum, liver and ovaries with increased concentrations of flaxseed. Flaxseed decreased the expression of ERα in the ovaries. The ratio of 2-OHE1:16-OHE1 in the serum increased significantly in the 15% flaxseed diet, and there was a corresponding increase in CYP1A1 in the liver and decrease in CYP3A4 in the ovaries. CYP1B1 mRNA also decreased with flaxseed diet in the ovaries. The 15% flaxseed-supplemented diet significantly decreased inflammatory PGE2, ERα, CYP3A4, CYP1B1 and 16-OHE1, but it increased CYP1A1 and 2-OHE1, which thus reduced the inflammatory and pro-carcinogenic micro-environment of the ovaries.

Pivotal roles for hormonally regulated expression of the HEP21 gene in the reproductive tract of chickens for oviduct development and in ovarian carcinogenesis

Hen egg protein (HEP21) is a 21-kDa secreted protein and has a single copy of the Ly6/uPAR domain. Although HEP21 is expressed primarily in the chicken oviduct, its biological function(s) in the reproductive system of chickens is not known. Thus, in the present study, we investigated expression patterns of HEP21 with respect to hormonal regulation, oviduct development, changes in expression in laying hens undergoing induced molting, and in the development of ovarian carcinogenesis in laying hens. Results of present study indicated that HEP21 messenger RNA (mRNA) expression increased (P < 0.001) in the chicken oviduct in response to estrogen. In situ hybridization analyses revealed expression of HEP21 mRNA predominantly in glandular (GE) and luminal epithelia of the magnum of the chicken oviduct in response to estrogen. The expression of HEP21 mRNA decreased (P < 0.001) as the oviduct regressed during induced molting and increased (P < 0.001) with recrudescence of the oviduct following molting. HEP21 mRNA was most abundant in GE of the oviduct during recrudescence, but not during oviduct regression following induced molting. Moreover, we found abundant expression of HEP21 in GE of cancerous ovaries, but not in normal ovaries of hens. Collectively, results of present study suggest that HEP21 is an estrogen-responsive gene in the oviduct of hens that likely regulates development of the chicken oviduct, and egg production and formation. Furthermore, there is increased expression of HEP21 in epithelial-derived ovarian cancer suggesting that HEP21 could be used for diagnosis and monitoring carcinogenesis in laying hens and in women.

The avian ovary and follicle development: some comparative and practical insights

This review focuses on recent research related to the avian ovary and, in particular, cellular mechanisms mediating embryonic growth of the ovary, postembryonic follicular development, and the process of follicular selection. Attention is drawn to a variety of fundamental, yet unresolved, aspects of ovarian function, several of which are uniquely related to avian reproductive strategies. Some notable differences in ovarian function between birds and the most closely related extant group, the crocodilians, are illustrated. Moreover, because the sauropsid (reptilian and avian) and synapsid (mammalian) lineages diverged shortly after the emergence of amniotes some 340 million years ago, consideration is given to both derived and convergent characteristics of ovarian function in avian species versus those in eutherian mammals. Two examples of divergent characteristics are the differences in sex-determining mechanisms between birds and mammals and the requirement for progesterone synthesized within the follicular granulosa as a requirement for ovulation in virtually all species of birds, compared to the site of synthesis and role for estradiol in mammals. As an example of a convergent process, both birds and select mammals (e.g., humans, cattle, and horses) typically ovulate a single egg per reproductive cycle, yet the processes associated with follicular selection occur via unique mechanisms. Finally, reference is made to several practical outcomes from studies of the avian ovary, including applications within the poultry industry and use of the domestic hen ovary as a model for human ovarian cancer.

Anti-inflammatory effects of fish oil in ovaries of laying hens target prostaglandin pathways

BackgroundAn effective way to control cancer is by prevention. Ovarian cancer is the most lethal gynecological malignancy. Progress in the treatment and prevention of ovarian cancer has been hampered due to the lack of an appropriate animal model and absence of effective chemo-prevention strategies. The domestic hens spontaneously develop ovarian adenocarcinomas that share similar histological appearance and symptoms such as ascites and metastasis with humans. There is a link between chronic inflammation and cancer. Prostaglandin E2 (PGE2) is the most pro-inflammatory ecoisanoid and one of the downstream products of two isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. PGE2 exerts its effects on target cells by coupling to four subtypes of receptors which have been classified as EP1-4. Fish oil is a source of omega-3 fatty acids (OM-3FAs) which may be effective in prevention of ovarian cancer. Our objective was to assess the potential impact of fish oil on expression of COX enzymes, PGE2 concentration, apoptosis and proliferation in ovaries of laying hens.Methods48 white Leghorn hens were fed 50, 100, 175, 375 and 700 mg/kg fish oil for 21 days. The OM3-FAs and omega-6 fatty acids contents of egg yolks were determined by Gas Chromatography. Proliferation, apoptosis, COX-1, COX-2 and prostaglandin receptor subtype 4 (EP4) protein and mRNA expression and PGE2 concentration in ovaries were measured by PCNA, TUNEL, Western blot, quantitative real-time qPCR and ELISA, respectively.ResultsConsumption of fish oil increased the incorporation of OM-3FAs into yolks and decreased both COX-1 and COX-2 protein and mRNA expression. In correlation with COXs down-regulation, fish oil significantly reduced the concentrations of PGE2 in ovaries. EP4 protein and mRNA expression in ovaries of hens was not affected by fish oil treatment. A lower dose of fish oil increased the egg laying frequency. 175 and 700 mg/kg fish oil reduced proliferation and 700 mg/kg increased apoptosis in hen ovaries.ConclusionsOur findings suggest that the lower doses of fish oil reduce inflammatory PG and may be an effective approach in preventing ovarian carcinogenesis. These findings may provide the basis for clinical trials utilizing fish oil as a dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer.

Flaxseed enriched diet-mediated reduction in ovarian cancer severity is correlated to the reduction of prostaglandin E2 in laying hen ovaries

Prevention of ovarian cancer is the best approach for reducing the impact of this deadly disease. The laying hen is a robust model of spontaneous ovarian cancer that recapitulates the human disease. Dietary intervention with flaxseed, the richest vegetable source of omega-3 fatty acids (OM-3FAs) and phytoestrogen lignans, demonstrate the potential for effective prevention and amelioration of ovarian cancer by targeting inflammatory prostaglandin pathways. Prostaglandin E2 (PGE2) is the most pro-inflammatory ecoisanoid and one of the downstream products of two isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Our objective was to investigate the effect of flaxseed supplementation for one year on ovarian cancer and correlate its effects to expression of COX enzymes and concentrations of prostaglandins. White Leghorn hens were fed 10% flaxseed-enriched or standard diet for one year. The severity of ovarian cancer was determined by gross pathology and histology. COX-1 and COX-2 localization and protein and mRNA expression and PGE2 and PGE3 concentrations in ovaries were measured by IHC, western blot, quantitative real-time PCR and LC–MS–MS, respectively. The results demonstrated a significant reduction in late stage ovarian tumors in the flaxseed-fed hens compared with the control diet-fed hens. In correlation with decreased ovarian cancer severity, concentrations of PGE2 and expression of COX-2 were diminished in ovaries of flaxseed-fed hens. PGE3 concentrations were below the level of detection. The results demonstrated that in normal ovaries, COX-1 was localized to the granulosa cell layer surrounding the follicles and ovarian surface epithelium (OSE) whereas COX-2 protein was localized to the granulosa cell layer in the follicle. Extensive COX-1 and COX-2 protein expression was found throughout the ovarian carcinoma. Our findings suggest that the flaxseed-mediated reduction in the severity of ovarian cancer in hens is correlated to the reduction in PGE2 in the ovaries of flaxseed-fed hens. These findings may provide the basis for clinical trials of dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer.

Avian WNT4 in the female reproductive tracts: potential role of oviduct development and ovarian carcinogenesis.

The wingless-type MMTV integration site family of proteins (WNTs) is highly conserved secreted lipid-modified signaling molecules that play a variety of pivotal roles in developmental events such as embryogenesis, tissue homeostasis and cell polarity. Although, of these proteins, WNT4 is known to be involved in genital development in fetuses of mammalian species, its role is unknown in avian species. Therefore, in this study, we investigated expression profiles, as well as hormonal and post-transcriptional regulation of WNT4 expression in the reproductive tract of female chickens. Results of this study demonstrated that WNT4 is most abundant in the stromal and luminal epithelial cells of the isthmus and shell gland of the oviduct, respectively. WNT4 is also most abundant in the glandular epithelium of the shell gland of the oviduct of laying hens at 3 h post-ovulation during the laying cycle. In addition, treatment of young chicks with diethylstilbestrol (DES, a synthetic estrogen agonist) stimulated WNT4 only in the glandular epithelial cells of the isthmus and shell gland of the oviduct. Moreover, results of our study demonstrated that miR-1786 influences WNT4 expression via specific binding sites in its 3′-UTR. On the other hand, our results also indicate that WNT4 is expressed predominantly in the glandular epithelium of cancerous ovaries, but not in normal ovaries of hens. Collectively, these results indicate cell-specific expression of WNT4 in the reproductive tract of chickens and that it likely has crucial roles in development and function of oviduct as well as initiation of ovarian carcinogenesis in laying hens.

Long term consumption of flaxseed enriched diet decreased ovarian cancer incidence and prostaglandin E2 in hens

ObjectiveOvarian cancer is the most lethal gynecological malignancy. Prevention may be the best approach to reduce ovarian cancer. Flaxseed is the richest vegetable source of omega-3 fatty acids which may be effective in the prevention of ovarian cancer. Prostaglandin E2 (PGE2) is the most pro-inflammatory ecoisanoid and one of the downstream products of two isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Our objective was to determine if long-term consumption of a flaxseed enriched diet decreased ovarian cancer severity and incidence in the laying hen and to investigate its potential correlation with the expression of COX enzymes and PGE2 concentration. MethodsWhite Leghorn hens were fed 10% flaxseed-enriched or standard diet for 4years. The severity and incidence of ovarian cancer were determined by gross pathology and histology. COX-1 and COX-2 protein and mRNA expression and PGE2 concentrations in ovaries were measured by Western blot, quantitative real-time PCR and ELISA, respectively. ResultsThe results demonstrated that there was a reduction in ovarian cancer severity and incidence in hens fed flaxseed diet. In correlation with decreased ovarian cancer severity and incidence, concentration of PGE2 and expression of COX-2 were diminished in ovaries of hens fed flaxseed. ConclusionsOur findings suggest that the lower levels of COX-2 and PGE2 are the main contributing factors in the chemo-suppressive role of long-term flaxseed consumption in ovarian cancer in laying hens. These findings may provide the basis for clinical trials of dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer.

Bone Morphogenetic Protein 4 Supports the Initial Differentiation of Hen (Gallus gallus) Granulosa Cells

In the hen ovary, selection of a follicle into the preovulatory hierarchy occurs from a small cohort of prehierarchal (6-8 mm) follicles. Prior to follicle selection the granulosa layer remains in an undifferentiated state despite elevated follicle-stimulating hormone receptor (FSHR) expression. The present studies describe a role for bone morphogenetic protein 4 (BMP4) in supporting FSHR mRNA expression in granulosa cells from prehierarchal follicles and promoting differentiation at follicle selection. Culture of undifferentiated granulosa cells in culture medium alone resulted in a significant decline in levels of FSHR mRNA (by ~80% compared to freshly collected cells). By comparison, granulosa cultured with BMP4 (10-100 ng/ml) maintained FSHR and expression at approximately in vivo levels. Because both granulosa and theca tissues from prehierarchal follicles express BMP4, it is suggested that BMP4 acts in a paracrine and/or autocrine fashion to support elevated FSHR expression prior to follicle selection. Granulosa cells cultured with BMP4 for 24 h also initiated FSH-induced cAMP production and indirectly initiated anti-Mullerian hormone (AMH), CYP11A, and STAR expression plus progesterone production. However, pretreatment with the BMP antagonist NOGGIN or the mitogen-activated protein kinase (MAPK) agonist transforming growth factor alpha attenuated or blocked each action promoted by BMP4. We conclude that prior to and immediately after selection, BMP4 serves to support FSHR expression within the granulosa layer, yet prior to selection, multiple factors (including inhibitory MAPK signaling, AMH, and BMP antagonists) can modulate FSHR expression and suppress FSH-mediated cell signaling to prevent granulosa cell differentiation prior to follicle selection.

Age dependent increase in prostaglandin pathway coincides with onset of ovarian cancer in laying hens

Chronic inflammation has been linked to cancer. Prostaglandin E2 (PGE2) is the most pro-inflammatory lipid and one of the downstream products of 2 isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Ovarian cancer is the most lethal gynecological malignancy and mainly occurs in older women. The factors that contribute to the correlation of age and ovarian cancer are unknown. The purpose of this study was to examine the expression of COX enzymes and PGE2 levels in ovaries and correlate them to ovarian cancer and aging. White Leghorn hens aged 1, 1.5, 2, 2.5, 3 and 3.5 years were used. The incidence of ovarian cancer was determined by gross pathology and histology. COX-1 and COX-2 protein and mRNA expression and PGE2 concentrations in ovaries were measured using Western blot, quantitative real-time PCR and ELISA, respectively. Our results indicated an increase in ovarian cancer incidence and expression of both COX enzymes in ovaries of older hens. In correlation with ovarian cancer incidence and COX enzymes expression, PGE2 concentrations were elevated with age. Ovaries with tumor had elevated COX-1 expression and PGE2 concentration compared to normal ovaries. Our findings suggest that the up-regulation of COX enzymes with age is the main contributing factor in the age associated increase in PGE2. Furthermore, elevated PGE2 in ovaries of hens concomitant with age suggests its important role in early stages of ovarian carcinogenesis. These finding may provide the basis for clinical trials utilizing COX specific inhibitors or dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer.

Molecular characterization of prostaglandin F receptor (FP) and E receptor subtype 3 (EP3) in chickens

Prostaglandin E and F regulate diverse physiological functions including gastrointestinal motility, fever induction and reproduction. This multitude of biological effects is mediated via their four E receptor subtypes (EP1, EP2, EP3 and EP4) and F receptor (FP), respectively. Majority of these studies was performed in mammalian species, while investigations on their roles were impeded by inadequate information on their receptors in avian species. In present study, full-length cDNAs of chicken EP3 (cEP3) and two isoforms of FP – cFPa and cFPb – were cloned from adult hen ovary. The putative cEP3 and cFPa share high amino acid sequence identity with their respective orthologs, while the predicted cFPb is a novel middle-truncated splice variant which lacks 107 amino acids between transmembrane domains 4 and 6. RT-PCR showed that cEP3, cFPa and cFPb are widely expressed in adult tissues examined, including ovary and oviduct. Using a pGL3-CRE luciferase reporter system, cEP3-expressing DF1 cells inhibited forskolin-induced luciferase activity (EC50: <1.9pM) upon PGE2 treatment, suggesting that cEP3 may functionally couple to Gi protein. Upon PGF2α addition, cFPa was shown to potentially couple to intracellular Ca2+-signaling pathway by pGL3-NFAT-RE reporter assay (EC50: 2.9 nM), while cFPb showed no response. Using a pGL4-SRE reporter system, both cEP3 and cFPa exhibited potential MAPK activation by PGE2 and PGF2α at EC50 0.34 and 13nM, respectively. Molecular characterization of these receptors paved the road to the better understanding of PGE2 and PGF2α roles in avian physiology and comparative endocrinology studies.

Expression of Matrix Metalloproteinase-2 mRNA in the Chicken Ovary in Relation to Follicle Remodelling

In the mammalian ovary, matrix metalloproteinase-2 (MMP-2) is thought to be involved in the regulated turnover of the extracellular matrix during the process of follicle development, ovulation and regression. Information concerning the role of MMP-2 in the chicken ovary is scarce. The present investigation was undertaken to determine the distribution of MMP-2 mRNA within the laying hen ovary as well as to define whether the expression of MMP-2 mRNA changes during the ovulatory cycle. Expression of MMP-2 mRNA in ovarian compartments was determined by RT-PCR analysis at two stages of the ovulatory cycle, i.e. at 22 h and 3 h before ovulation. MMP-2 mRNA was detected in the ovarian stroma and follicles: white, yellowish, small yellow, the largest preovulatory (F3-F1) and postovulatory (P1-P5). In the wall of preovulatory follicles MMP-2 mRNA was found in the theca and granulosa layers. Within the ovary the relative expression of MMP-2 mRNA depended on follicular size, the layer of the follicular wall, the stage of the ovulatory cycle and the degree of regression in the postovulatory follicles. The results obtained indicate that in the chicken ovary MMP-2 may take part in the extracellular matrix remodelling required for follicle growth, development and regression.

Paradoxical expression of AHCYL1 affecting ovarian carcinogenesis between chickens and women

We investigated S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) gene expression in human epithelial ovarian cancer (EOC) using the chicken, which is the most relevant animal model. Ovarian cancer was detected in 10 of 136 laying hens (7.4%). Results of the present study indicated that AHCYL1 mRNA and protein are most abundant in the glandular epithelium of adenocarcinoma of cancerous, but not normal, ovaries of hens. In addition, bisulfite sequencing to examine methylation patterns in the promoter region of the AHCYL1 gene revealed that 30-38% of the three CpG sites were demethylated in ovarian cancer cells as compared with normal ovarian cells. Furthermore, in human ovarian cancer cells such as OVCAR-3, AHCYL1 protein was predominantly in the nucleus and had a similar expression pattern to that in chicken ovarian cancer cells. Thereafter, we examined the prognostic value of AHCYL1 expression in patients with EOC using multivariate linear logistic regression and Cox's proportional hazard analyses. In 109 human patients with EOC, 14 (12.8%), 41 (37.6%) and 54 (49.6%) patients showed weak, moderate and strong expression of AHCYL1 protein, respectively. However, intermediate or high expression of AHCYL1 protein was a favorable factor for overall responses (adjusted odds ratio, 7.23; 95% confidence interval [CI], 1.36-38.39), and for progression-free survival (adjusted hazard ratio, 0.20; 95% CI, 0.07-0.55). From these results, we conclude that AHCYL1 expression is associated with ovarian carcinogenesis as an oncogene in chickens, whereas it plays the role of tumor suppressor in human EOC, suggesting a paradoxical function of AHCYL1 in ovarian carcinogenesis.