- Transcriptome analysis of the uterus of hens laying eggs differing in cuticle deposition. - The quantity of cuticle varies and the deposition of a good cuticle in the uterus (Shell-gland) prevents transmission of bacteria to the egg contents.. - This suggests an endogenous clock in the uterus may be a component of cuticle deposition control. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - Full list of author information is available at the end of the article. - The cuticle is a highly glycosylated pro- teinaceous layer that is specifically deposited on the out- side of avian eggs [6], filling the pores in the shell, so preventing aqueous access and bacterial contamination but still allowing gas exchange [7–9]. - The predominant cuticle proteins in the chicken appear to be the BPI fold containing family B member 3 (BPIFB3 also known as ovocalyxin-36), kunitz-like protease inhibitor, matrix extracellular phosphoglycoprotein (MEPE also known as ovocleidin-116), ovocleidin-17 (OC-17), ovocalyxin 25 (WAP), clusterin (CLU) and retinoic acid receptor re- sponder 1 (RARRES1 also known as ovocalyxin-32) [10].. - Despite these proteins having a not dissimilar con- stitution to the organic matrix of the shell [14], termin- ation of shell formation occurs before the deposition of the cuticle [6]. - Therefore, cuticle deposition is not an ex- tension of shell organic matrix production but is a spe- cific event that occurs in the uterus, often referred to as the shell gland pouch, very close to the time of ovipos- ition [6]. - This was elucidated by utilizing precise tem- poral manipulation of the hypothalamo-pituitary- gonadal-oviduct axis to manipulate the daily laying cycle and obtain eggs at the same time in the daily cycle which had cuticle deposition or did not have cuticle deposition [6]. - The question of what precise pathways are responsible for the secretion of the cuticle however, remains unresolved.. - Previously we speculated that there may even be in- trinsic timing events that control the succession of events in the uterus from mineralization through to cuticle deposition and oviposition [6]. - Namely, what genes or pathways are involved in the deposition of the cuticle and what genes contribute to the variation in cuticle deposition within a chicken breed? By generating uterus transcriptomes from hens that produced eggs with good or poor cuticle coverage and from hens that were manipulated to produce eggs with or without cuticle, our intention was to increase both our under- standing of the uterus transcriptome and to find new avenues to pursue in understanding the biology of this fascinating protective coating found on the surface of eggs.. - Experiment 1: comparison of the top and tail of the cuticle deposition distribution. - At post mortem, there was no difference in body weight between the hens sampled from the top (High) or the bottom (Low) of the distribution for cuticle quality g v g. - As it was the basis of selection of the samples, the cuticle (ΔAbs @640 nm) was more abundant in the High group versus the Low group as indicated by a larger difference in absorbance (High v Low ΔAbs @640 nm. - Nor was there a difference in the oviposited egg weight v. - F1,14 0.07) although the latter tended to be heavier in the AVT group. - Compar- ing the egg cuticle (ΔAbs @640 nm) in the two treat- ments there was little or no cuticle in the AVT group compared to the GNRH1 treated group eggs (GNRH v AVT ΔAbs @640 nm. - Pigmentation of the egg (Abs @ 640 nm) was also affected with a lower absorbance in the AVT treated groups eggs compared to the GNRH1 group which equates with a less pigmented egg (GNRH v AVT Abs @ 640 nm. - Eight biological replicate samples of chicken uterus from each of the extremes of the distribution of cuticle depos- ition, i.e. - Of a total of 18,346 genes present in the chicken genome, our sequencing reads were assembled onto 12,253 genes (RPKM >. - Of a total of 18,346 genes present in the chicken genome, our sequencing reads were assembled onto 12,248 genes (RPKM >. - Expression profile of genes in the uterus close to oviposition in experiment 1 and 2. - In the uterus, the 30 genes with the highest expression are detailed in Table 1. - There logCPM values are between 11 and 15.3, where the genes in the data set as a whole had a mean expression of 4.4 and an upper quartile value of 6.1 (Table 1).. - GO functional annotation and pathway enrichment analysis of the DEGs in experiment 2. - In the Bio- logical process ontology, the highest enrichment of DEGs included signal transduction (26 genes), system development (19 genes) and cell differentiation (19 genes). - In the Molecular function ontology, only the gly- cosaminoglycan binding category (GO:0005539) satisfied the cut-off criteria (P <. - There were no over-represented categories in the cellular com- ponent ontology.. - PER2 showed differential expres- sion but neither of the CRY genes were differentially expressed in the RT-qPCR (Fig. - In order to study further the expression of genes that were apparently regulated between the situations were cuticle production was changed, their expression was measured in the uterus at different stages during egg for- mation including when there was no egg in the uterus (Fig. - the expression was significantly lower when there was no egg in the uterus and the expression at the late period of shell formation was also significantly lower than either mid or early shell formation (Fig. - Expression was very low when no egg was present in the uterus (Fig. - .001), with significantly higher expression when no egg was in the uterus but no differences between the stages when an egg was present in it (Fig. - .001) with a significant peak of expression at the end of shell formation, whilst the expression was low during the rest of the stages (Fig. - previous studies that compared when an egg was in the uterus and when there was not [22–24]. - We present the analysis of the expression profile of genes in the uterus, close to oviposition. - There are 32 in- dividuals sampled at this stage of egg formation from 2 experiments, so we have a highly-replicated data set to identify those genes most highly expressed in the uterus at oviposition. - When the 30 genes with the Table 1 Profile of the thirty top expressed genes (log CPM >. - highest expression in the uterus were annotated as might be expected, several of the genes with extremely high ex- pression at oviposition either encode proteins that are known to be expressed and secreted to form the cuticle [10, 25] or the organic matrix of the eggshell [14] e.g.. - A number of these highly-expressed genes were also differentially expressed in the GNRH1 versus AVT experiment described in this study (Experiment 2);. - IGLL1, for example, has previously been reported in the uterus in a micro- array experiment [27]. - This protein was found in the protein component of the deeper layers of the shell [28]. - It is one of the most abundant proteins in the cuticle . - Overall, the most abundant genes in the uterus re- ported here are broadly in agreement with the 5 top expressed genes quoted in a recent NGS study that also featured the uterus with the unidentified transcript ENSGALG likely being homologous to ENSGALG NADH-ubiquinone oxidore- ductase chain 5 in this study. - Although not in the list of genes with the most significant differences in expression, we also found significant differences between the AVT and GNRH1 treated hens in the expression of PER2 (P CRY2 (ENSGALG00000008436. - These differences com- bined with the high level of expression of PER and CRY genes, confirmed by RT-qPCR, suggests that Clock genes may be important in driving the cyclic expression of genes in the uterus. - When examined over the egg laying cycle the expression of PER2 showed strong cyclicity re- lated to the time in the egg formation cycle. - Cyclicity of the clock related genes has previously been observed in the oviduct of mice which could be entrained by external. - Such a phase shift could be critical in the timing of cuticle deposition in the hen’s uterus or other key events during shell for- mation and therefore provides a testable hypothesis.. - A further aspect of this study was to compare the uterus transcriptome from the extremes of the popula- tion distribution for the trait. - A photograph of what the extremes of the cuticle deposition distribution look like is available in the supplemental material of a recent paper along with the associated the genetic parameters of the trait [18]. - One explanation could be that the genetic effects that re- sult in large phenotypic differences in this trait may not be expressed in the uterus, but may reside in neural struc- tures that control release of the cuticle proteins in the uterus or in a timing mechanism related to ovarian-uterus interaction or other unknown mechanisms. - Given that in the endo- crinological intervention using AVT/GNRH we observed clear effects on expression, it seems likely that if there. - Given the prominence of clock genes in the. - The y-axis scale therefore varies depending on the relative level of expression of the genes in the tissue, the larger the number the higher the relative expression of the gene. - Early, mid- and late describes the stage of shell formation in the uterus and indicates that the egg was not in the magnum when the samples was taken. - Pause represents no egg in the uterus and magnum is when the egg is in that region of the oviduct. - AVT/GNRH experiment the possibility that variation in their protein expression rather than gene expression is a possibility, indeed in many clock mechanisms there are observations of differences in the profile of the different components of the clock [37]. - These differences might be genetic differences in, for example, the half-life of clock proteins that would not be apparent in the tran- scriptome [37].. - For those genes tested, all were confirmed to have a lower level of expression in the GNRH1 group who had completed a normal albeit advanced egg formation cycle than the AVT group by RT-qPCR. - longer an egg in the oviduct and no requirement for events associated with glycosylation of matrix or cuticle proteins.. - A further category of genes identified from experiment 2 was a group of genes that had both relatively high ex- pression and a large significant difference in expression, where expression in the GNRH1 group was lower than in the AVT group. - This seems plausible because in the natural cycle (experiment 3) ex- pression was lower in the late phase of shell formation.. - In the chick oviduct JUN and FOS have also been shown to be responsive to the steroid environment, increasing with oestrogen and decreasing in response to progester- one [40–42]. - although arguably the timing effect of the previous surge could still be critical.. - For these genes, TNFSF10 and GKN2 (Ovoca- lyxin 21), expression was lower in the AVT group which produced eggs without cuticles versus the GNRH group that did have cuticles on the eggs produced. - OPG is present in the uterus and is highly upregulated as the steroid envir- onment changes with sexual maturity [45]. - This observation was confirmed by a transcriptome study comparing the uterus during and before egg shell calcification where TNFSF10 was higher when no egg was in the uterus [22].. - Using a microarray approach, Brionne [34] profiled the expres- sion of genes in the uterus in the presence of a hard shelled egg and identified a number of genes some of which are common to both studies. - categories of genes that may be responsible for control- ling the production of the cuticle. - Hens from the top and tail of the distribu- tion were collected from a Lohmann Tierzucht flock with permission. - Hens were killed immediately after oviposition as specified in Schedule 1 of the UK Animals (Scientific Procedures) Act (1986). - Hens were killed immediately after oviposition as specified in Schedule 1 of the UK Animals (Scientific Procedures) Act (1986) using an intra-venous injection of 200 mg/kg of Pentobarbitone Sodium (Euthatal, Merial Animal Health Limited, Harlow, Essex, UK).. - Briefly, uterus tissue was col- lected either when the egg was in the magnum where the albumen is secreted (n = 7), in the uterus (shell gland) and recorded as early (n = 9), mid- (n = 9), and late (n = 9) cal- cification based on electron microscopy of the shell or during a pause day (n = 11) when there was no ovulation and therefore, no egg in the oviduct.. - A small incision was made in the centre of the pouch and the uterine fluid removed. - Expression levels of the transcripts were quantified as RPKM (reads per kilo- base per million). - 1 in at least two samples were not included in the analysis. - Concentrations were normalized using a geometric mean of the expression of two ‘ housekeeping genes. - 1 resulting from the comparison of the top and tail of the cuticle deposition distribution.. - The authors would like to thank the staff in the National Avian Research Facility at Roslin for bird care.. - ICD, WI, ACJ, MMB constructed the hypothesis tested in the paper and sought the funding to execute the study. - For comparison of the top and tail of the distribution no work involved experimentation on living animals. - Material was collected at the end of the breeding cycle after euthanasia using an overdose of barbiturates.. - Diversity of the cuticle layer of avian eggshells. - Understanding avian egg cuticle formation in the oviduct: a study of its origin and deposition. - Proteomic analysis of the acid-soluble organic matrix of the chicken calcified eggshell layer. - Estrogen action: revitalization of the chick oviduct model. - Hen uterine gene expression profiling during eggshell formation reveals putative proteins involved in the supply of minerals or in the shell mineralization process.. - Mechanism of the circadian clock in physiology. - Circadian clock genes are rhythmically expressed in specific segments of the hen oviduct. - Involvement of the luteinizing hormone surge in the regulation of ovary and oviduct clock gene expression in mice.. - Molecular components of the mammalian circadian clock. - Involvement of osteopontin in egg shell formation in the laying chicken. - Rapid inhibition of the c-JUN protooncogene expression in avian oviduct by estrogen.. - Progesterone inhibits the estrogen- induced expression of c-FOS messenger-ribonucleic-acid in the uterus.. - Rapid down- regulation of c-Jun protooncogene transcription by progesterone in the avian oviduct. - accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. - An eQTL in the cystathionine beta synthase gene is linked to osteoporosis in laying hens
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