Effects of FGF10 on bovine oocyte meiosis progression, apoptosis, embryo development and relative abundance of developmentally important genes in vitro

Contents Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion-related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22h in TCM-199 supplemented with 0, 2.5, 10 or 50ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5ng/ml FGF10 increased (p<0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real-time RT-PCR showed a tendency of 50ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.

Molecular basis of maturation promoting factor

Maturation promoting factor (MPF), which is functionally defined by its ability to induce Xenopus oocyte maturation, is an M phase (meiosis and mitosis) specific activity that is present in all species tested. It was hypothesized that MPF is a universal trigger of the interphase to M phase transition during the cell cycle. The current model for the molecular basis of MPF is that MPF is a protein kinase having the cdc2 protein as its catalytic subunit and is identical to the M phase-specific histone H1 kinase. In the present study, I have shown that more than just cdc2 kinase contributes to MPF activity, and M phase-specific H1 kinase is composed of at least two entities, instead of just cdc2 kinase. Therefore, the simple model of MPF = cdc2 kinase = M phase-specific H1 kinase should be ruled out.^ My study began with the characterization of the mitosis-specific monoclonal antibody MPM-2. MPM-2 reacts specifically with M phase cells from different species by recognizing a discrete set of proteins once they are phosphorylated at the G$\sb2$/M transition. I found that phosphorylation of MPM-2 antigens coincided with the appearance of MPF activity during oocyte maturation stimulated by progesterone. If MPM-2 was injected into oocytes before the stimulation, MPF activity failed to appear, and the oocytes could not mature. Furthermore, MPM-2 was able to deplete MPF activity from M phase extracts. These results identified MPM-2 as a probe that recognizes either MPF itself or a regulator of MPF.^ Since M phase-specific H1 kinase was believed to be identical to cdc2 kinase and MPF, I proceeded to determine whether MPM-2 recognized the M phase-specific H1 kinase. I found that MPM-2 did recognize an M phase-specific H1 kinase. However, this kinase was not cdc2 kinase. This kinase (MPM-2 kinase) is present in a latent form in immature oocytes and is activated in tandem with the activation of MPF during oocyte maturation. It appears to accelerate progesterone-induced oocyte maturation. Therefore, MPM-2 kinase may be a novel positive regulator of MPF activation.^ MPM-2 depletes MPF activity, but not cdc2 kinase activity. This discrepancy caused me to question the equivalency of MPF with cdc2 kinase. I found that when a high percentage of MPF activity was recovered from gel filtration of mature oocyte extract, the recovered MPF activity was due to two factors, cdc2 kinase and a factor recognized by MPM-2. This factor might activate and stabilize cdc2 kinase. Identification of this factor in the present study may contribute to the understanding of the autoactivation of MPF. ^

Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I.

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: Possible role of the of developmental competence

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.

Sex steroid feedback regulation of pituitary gonadotropins during early secondary oocyte growth in coho salmon (Oncorhynchus kisutch) – a potential target of endocrine disruption.

Thesis (Ph.D.)--University of Washington, 2016-06

Examination of the role of FSH in periovulatory events in the hamster

he need for endogenous FSH in the periovulatory events such as oocyte maturation, ovulation, luteinization, maintenance of luteal function and follicular maturation was examined in the cyclic hamster. A specific antiserum to ovine FSH, shown to be free of antibodies to LH and to cross-react with FSH of the hamster, was used to neutralize endogenous FSH at various times. Administration of this antiserum during pro-oestrus did not affect oocyte maturation and ovulation, as judged by the normality of the ova to undergo fertilization and normal implantation. It also had no effect on the process of luteinization or on the maintenance of luteal function, as indicated by the normal levels of plasma and luteal progesterone during pro-oestrus and oestrus during the cycle and in pregnancy. All these processes were, however, disrupted by administration of an antiserum to ovine LH, thereby demonstrating their dependence on endogenous LH. Although FSH antiserum given at pro-oestrus did not prevent the imminent ovulation, it blocked the ovulation occurring at oestrus of the next cycle. This antiserum was effective in preventing the ensuing ovulation when given at any other time of the cycle until the morning of pro-oestrus. It is concluded that, in the hamster, high levels of FSH during pro-oestrus and oestrus are required for initiating maturation of a new set of follicles which are dependent on the trophic support of FSH throughout the cycle until the morning of pro-oestrus. Such follicles then appear to need only LH for subsequent ovulatory and associated processes.

Staging ovaries of Haddock (Melanogrammus aeglefinus): implications for maturity indices and field sampling practices

We build on recent efforts to standardize maturation staging methods through the development of a field-proof macroscopic ovarian maturity index for Haddock (Melanogrammus aeglefinus) for studies on diel spawning periodicity. A comparison of field and histological observations helped us to improve the field index and methods, and provided useful insight into the reproductive biology of Haddock and other boreal determinate fecundity species. We found reasonable agreement between field and histological methods, except for the regressing and regenerating stages (however, differentiation of these 2 stages is the least important distinction for determination of maturity or reproductive dynamics). The staging of developing ovaries was problematic for both methods partly because of asynchronous oocyte hydration during the early stage of oocyte maturation. Although staging on the basis of histology in a laboratory is generally more accurate than macroscopic staging methods in the field, we found that field observations can uncover errors in laboratory staging that result from bias in sampling unrepresentative portions of ovaries. For 2 specimens, immature ovaries observed during histological examination were incorrectly assigned as regenerating during macroscopic staging. This type of error can lead to miscalculation of length at maturity and of spawning stock biomass, metrics that are used to characterize the state of a fish population. The revised field index includes 3 new macroscopic stages that represent final oocyte maturation in a batch of oocytes and were found to be reliable for staging spawning readiness in the field. The index was found to be suitable for studies of diel spawning periodicity and conforms to recent standardization guidelines.

Spawning cycles and habitats for ballyhoo (Hemiramphus brasiliensis) and balao (H. balao) in south Florida

Two halfbeak species, ballyhoo (Hemiramphus brasiliensis) and balao (H. balao), are harvested as bait in south Florida waters, and recent changes in fishing effort and regulations prompted this investigation of the overlap of halfbeak fishing grounds and spawning grounds. Halfbeaks were sampled aboard commercial fishing vessels, and during fishery-independent trips, to determine spatial and temporal spawning patterns of both species. Cyclic patterns of gonadosomatic indices (GSIs) indicated that both species spawned during spring and summer months. Histological analysis demonstrated that specific stages of oocyte development can be predicted from GSI values; for example, female ballyhoo with GSIs >6.0 had hydrated oocytes that were 2.0−3.5 mm diameter. Diel changes in oocyte diameters and histological criteria demonstrated that final oocyte maturation occurred over a 30- to 36-hour period and that ballyhoo spawned at dusk. Hydration of oocytes began in the morning, and ovulation occurred at sunset of that same day; therefore females with hydrated oocytes were ready to spawn within hours. We compared maps of all locations where fish were collected to maps of locations where spawning females (i.e. females with GSIs >6.0) were collected to determine the degree of overlap of halfbeak fishing and spawning grounds. We also used geographic information system (GIS) data to describe the depth and bottom type of halfbeak spawning grounds. Ballyhoo spawned all along the coral reef tract of the Atlantic Ocean, inshore of the reef tract, and in association with bank habitats within Florida Bay. In the Atlantic Ocean, balao spawned along the reef tract and in deeper, more offshore waters than did ballyhoo; balao were not found inshore of the coral reef tract or in Florida Bay. Both halfbeak species, considered together, spawned throughout the fishing grounds of south Florida.

Age, growth, and reproduction of permit (Trachinotus falcatus) in Florida waters

We examined 536 permit (Trachinotus falcatus, 65–916 mm FL) collected from the waters of Florida Keys and from the Tampa Bay area on Florida’s Gulf coast to describe their growth and reproduction.Among permit that we sexed, females ranged from 266 to 916 mm in length (mean=617) and males ranged from 274 to 855 mm (mean=601). Ages of 297 permit ranging from 102 to 900 mm FL were estimated from thin-sectioned otoliths (sagittae). The large proportion of otoliths with an annulus on the margin and an otolith from an OTC-injected fish suggested that a single annulus was formed each year during late spring or early summer.Permit reach a maximum age of at least 23 years.Permit grew rapidly until an age of about five years, and then growth slowed considerably. Male and female von Bertalanffy growth models were not significantly different, and the sexes-combined growth model was FL=753.1(1–e –0.348(Age+0.585)). Gonad development was seasonal, and spawning occurred during late spring and summer over artificial and natural reefs at depths of 10–30 m. Ovaries that contained oocytes in the final stages of oocyte maturation or postovulatory follicles were found during May–July. We estimated that 50% of the females in the population had reached sexual maturity by 547 mm and an age of 3.1 years and that 50% of the males in the population had reached sexual maturity by 486 mm and an age of 2.3 years. Because Florida regulations restrict the maximum size of permit caught in recreational and commercial fisheries to 20-inch (508-mm), most fish harvested are sexually immature. With the current size selectivity of the fishery, the spawning stock biomass of permit could decrease quickly in response to moderate levels of fishing mortality; thus, the regulations in place in Florida to restrict harvest levels appear to be justified.

A comparative study on the induced breeding and in vitro fertilization performance by various inducing agents in catfish, Heteropneustes fossilis (Bloch)

Brood catfish, Heteropneustes fossilis were collected from Tamirabarani river basin of Tamil Nadu, India and kept in cement tanks. Three inducing hormones viz, Ovaprim, Ovatide and WOVA-FH were injected at the rate of 0.5, 1.0, 1.5 and 2.0 ml/kg body weight in order to induce oocyte maturation and ovulation. After 10-13h of injection at a water temperature of 27±-0.5°C, stripping of eggs and in vitro fertilization was done. Ovaprim gave maximum (94.67%) hatching rate followed by Ovatide (90.33%) and WOVA-FH (77.33%).

Histology and hormonal effect of Ghrelin on ovary of Benni (Barbus sharpeyi) in order to study sexual maturity, zygote and larval generation

Benni (Barbus sharpeyi) is valuable fish that Khuzastan fisheries office propagated it artificially in Susangerd Fish Propagation Center every year. Pituitary gland is used for this aim but female fish lost their fertilization power after 2-3 years, so in present research, new hormone, that is called Ghrelin. The aims of this research are histology, hormonal, zygote and larval generation studies and comparing the results with each other. Ghrelin is a multifunctional peptidyl hormone which increases GTH-II in fish, amphibian, and birds and mammalian so its effect on Benni sexual maturation was studied. Human Ghrelin (hGRL) was obtained from ANASPEC, Canada, with 28 amino acids. In the present study, three levels of ghrelin including 0 (sham treatments), 0.10 (treatment 1) and 0.15 μg/g (treatment 2) body wt and one level of pituitary gland 4000 μg/g (pituitary treatment) with two replications were used. 56 specimens were injected intraperitonealy and their ghrelin level was evaluated immediately after injection and after 24 h. Control fish(n=16) were just injected by physiological saline. For hormonal studies sham and experimental fish(n=40) were anesthetized with MS-222 at a concentration of 250 mg l-1, and blood samples were collected and kept at 4ْC, then spun to collect serum. Serum samples were stores at -20ْC until the RIA for CTH-II. For histology studies immediately after injection a piece of ovary was collected from control fish (Sham zero) after being anesthetized. The sampled ovaries were fixed in Buin solution and embedded in paraffin, and stained to Sections of 5–6 μm using haematoxylin and eosin. The ovarian samples were performed with a compound microscope. Histology and micrometry studies had done. The mature oocytes had given from mature fish, then weighted and the working fecundity were counted. The mature oocytes fertilized, the eggs were incubated and the percentage of fertilization was calculated. After 72h the eggs hatched and the percentage of hatch was counted. The percentage of hindrance was calculated after 6 days. Hormonal results indicate that ghrelin and pituitary increase significantly the GTH-II level in comparison to sham. Macroscopic observations (before taking ovary) showed that ovaries with green colored have couple oval structure located in the abdominal cavity. Microscopic studies of dissected ovaries indicated simultaneous growth of 127 oocytes with 6 stages. The type of the ovary is asynchronous. The results indicated that both of the ghrelin treatment increased the percentage of mature follicles followed by decrease of immature follicles. There were significant differences (P<0.05) between the number of mature and immature follicles. Average diameter of follicle in both of the ghrelin treatment was significantly (P<0.05) declined in the stages of the vitellogenesis when the result compared to the other treatment. Just treatment 1 and pituitary treatment can give mature oocytes. The fecundity of pituitary treatment significantly increase in comparision to ghrelin treatment (P<0.05). In food-restricted fish where endogenous ghrelin levels are known to be increased, a chronic administration of ghrelin induces overt negative effect in releasing mature oocytes. The percentage of fertilization was significantly increase (P<0.05) in ghrelin t. in comparison to pituitary t. and the percentage of hatch was significantly increase (P<0.05) in pituitary t. in comparison to ghrelin t. There was no significant difference (P>0.05) in terms of percentage of hindrance between treatments. In conclusion, the present study demonstrated that ghrelin has positive effect on the level of GTH-II, oocyte maturation, ovarian vitellogenesis and the number of mature follicles of Barbus sharpeyi ovary. Increasing of the mature follicles number reduces their average diameter, indicating stimulating effect of ghrelin in sexual maturation of Barbus sharpeyi.The ghrelin and pituitary treatment have equal chance in the post-stage of spawning.

The survey of sexual maturity in Mugil cephalus so measuring sex hormones and histologic studies

In this study the process of female gray mullet brooders was carried out by using histological study and masurment of sex steroids. Results of histological studies showed that oocyte of gray mullet brooders in Gomishan Rearing Center conditions of develop to the end of yolk globule stage. The results were observed with oocyte in chromatin nucleolar stage (first stage) with means of diameter of 20 p m, in August, perinucleolar stage (second stage) in September with mean diameter of 87 p m, yolk vesicle stage (third stage) in October with mean diameter 200 p m and yolk granules stage (forth stage) from October to November with average diameter of 180 — 650 p m. For the reason of stopping oocyte develop at the end of fourth stage, hormonal induction to final oocyte maturation and ovulation was used. For this purpose, carp pituitary , HCG and LRH-A2 with different combinations were used in two stages, second injection was used 24 hours after first injection. 15 females brooders were divided in 5 groups, different hormonal combinations were injected to four groups and to fifth group as control, only saline, was injected. The process of female brooder rippening in hormonal induction was studied via masurment of sex steroids including 17 a - hydroxy progestrone, estradio1-17)6 and testosterone. Blood samples were collected from caudal vein during first injection, 24, 30 and 48 hours after the first injection. At the same time, for distinguishing histological changes the sample has been attained from the gonads Sex stroid fluctuation patterns in different brooder groups that injected hormon were similar, however hormonal composition had similar effects. All brooder that their oocyte in the beginning of hormonal injection were At the end of fourth stage with oocyte diameter average of 600 p m received to final maturation and ovulation. The brooder that its oocytes were At the begining or mid-fourth stage did not show ovulation but hormonal induction caused oocyte develop at the beginning of fifth stage. Study of 17-hydroxy progestrone fluctuation showed that the maximum level of this steroid (0.347 ng/ml) measured 30 hours after the first injection and was significantly higher (p< 0.05) than those of control group. So, 17-hydroxy progestrone is probably precursor of maturation inducing steroid (MIS). However the maximum level of that observed was coincident with germinal vesicle breakdown, oil droplets coalescence and dissolution of yolk granuls The maximum levels of esteradiol— 17/0 and testosterone (3.778 and 16.801ng/ml,respectively) in spawned brooders,were observed 24 hours after the first injection. levels of those steroids were significantly higher (p<0.05) than control group. Maximum level of sex steroids in the brooders that did not spawn to the end of treatment was observed with more delay than those in spawned brooders. Therefor maximum level of 17a-hydroxy progestrone (0.264 ng/ml) in those brooders observed in fourth sampling time and the maximum levels of estradio1-17a and testosterone (2.944 and 18.993 ng/ml, respectivly)observed in third sampling time that was significantly higher (p<0.05) than those of control group. For the study of stress effect on brooders during the hormonal induction, level of cortisol was measured in every sampling time. level of cortisol had high fluctuation that showed handling level and stress effect on brooders. However maximum level of cortisol in majority of brooders was dominant in third sampling time that was coincident with final maturation.

Survey of reproductive physiology on Epinephelus coioides in Khozestan province, Persian Gulf

Epinephelus coioides (family serranidae) is protogynous. This species is one of the most important fishes in food chain of marine proteins of persian Gulf. Therefore knowing about the reproductive biology and physiology of this species is an important role on aquaculture procedures. Monthly samples of Epinephelus coioides were obtained in khozestan Bahrekan province from 2001 to 2002 for annual variation of base line of reproductive hormone. The hormones such as: 17-B estradiol, Testosteron, Progesterone, Gonadotropin I ,II GTHI, II) and cortisol have assayed and also different stages of gonads from the histological point of view were studied by light and electron microscope. Aditional to morphometric and fecundity measurements, the important factors such as : Gonadosomatic index (GSI) Hepatosomotic index (HSI) and Condition factor (KF) were also studied. Environmental factors such as temperature, salinity, photoperiod and pH were analyzed for the determination of effective factors responsible for the changes of reproductive cycles. The flactmation of estroid hormones and gonadotropines show a significant variation in different stages of maturation, e.g 17-B estradiol's concentration in the third stages, GTH II in fourth stages of sexual maturation or final oocyte maturation, plasma Testosteron in post ovulation and Progesterone during maturation indicates the highest levels of above mentioned hormones. The total calcium concentration was high in all year. calcium concentration was correlated with GTH II synthesis and increases with GTH II in June. 17-B estradiol concentration was also correlated with GSI. The high concentration of cortisol throughout the year was an index of stress and development of ovary maturational processes. This species was protogynous synchronous hermaphrodites , and belongs to annual spawning species, being monandric. The sexual transition was found to occure in individuals of 51.2- 105 cm in length. GSI and HSI level confirms the time of spawning period is in April- June. Electrone microscopic studies of gonad tissues showed some changes in mitochondria and endoplasmic reticulum in the post ovulation, maturation and post spawning periods. During the monthly sampling the biochemistry of tissues variations indicated decrease in protein and lipid content, but an increase in water content of spawning fishes which was correlated to the maturation of Epinephelus coioides . sex ratio indicative of higher frequences of females to males during monthly sampling periods. The females were smaller than males in sizes, therefore the females lived in 8-15m depth, but males were living in upper limits of depth. The results indicated that the temperature was the most effective parameter in reproductive cycle of Epinephelus coioides and the mean 24°c was a convenient temperature for spawning. Photoperiod was the second effective. factor on the reproductive cycle for this species. It seemed that the increase in the photoperiod between January to May caused a development of the oocyte. Regarding to the results of this research, it seems that the period of spawning in Epinephelus coioides is in May- June and the aquaculture procedure of Epinephelus coioides could be performed in the above mentioned periods.

Histone H2A Has a Novel Variant in Fish Oocytes

Histone variants and their modification have significant roles in many cellular processes. In this study, we identified and characterized the histone H2A variant h2af1o in fish and revealed its oocyte-specific expression pattern during oogenesis and embryogenesis. Moreover, posttranslational modification of H2af1o was observed that results from phosphorylation during oocyte maturation. To understand the binding dynamics of the novel core histone variant H2af1o in nucleosomes, we cloned ubiquitous gibel carp h2afx as a conventional histone control and investigated the dynamic exchange difference in chromatin by fluorescence recovery after photobleaching. H2af1o has significantly higher mobility in nucleosomes than ubiquitous H2afx. Compared with ubiquitous H2afx, H2af1o has a tightly binding C-terminal and a weakly binding N-terminal. These data indicate that fish oocytes have a novel H2A variant that destabilizes nucleosomes by protruding its N-terminal tail and stabilizes core particles by contracting its C-terminal tail. Our findings suggest that H2af1o may have intrinsic ability to modify chromatin properties during fish oogenesis, oocyte maturation, and early cleavage.

Identification of a C1q family member associated with cortical granules and follicular cell apoptosis in Carassius auratus gibelio

C1q family proteins with C1q domain have been reported in vertebrates, but their biological roles are currently unknown. In this study, a C1q-like factor, designated Carassius auratus gibelio ovary-specific C1q-like factor (CagOC1q-like), was identified as a cortical granules component. Immunofluorescence localization revealed that the C1q family member was specifically expressed in follicular epithelial cells, and associated with cortical granules in fully grown oocytes. Moreover, it was discharged to the perivitelline space and egg envelope upon fertilization. As it is the first identified C1q family member that is expressed in follicular cells that surround oocyte, CagOC1q-like was applied to detection of follicular cell apoptosis and deletion. The entire cytological process of follicular cell apoptosis and deletion was clearly seen from double visualizations of follicular cells with CagOC1q-like immunofluorescence and apoptotic follicular cells labeled by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) during oocyte maturation and ovulation. (C) 2008 Elsevier Ireland Ltd. All rights reserved.