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Microarray analysis of genes involved with shell strength in layer shell gland at the early stage of active calcification.

INTRODUCTION

The chicken eggshell is a porous bioceramic container which protects the egg against physical damage and microbial contamination. Avian eggshell consists of the innermost bilayered membranes, a calcified layer composed of a mamillary and pallisade layer, and the outermost cuticle. The calcified layer consists of both inorganic minerals and extracellular matrix. It is well known that the shell mineral amount (thickness or density) is the main factor contributing to the mechanical properties of the eggshell (Ahmed et al., 2005). However, the organic matrix, although its content in the calcified layer is only 2 to 3.5%, is of great importance to the deposition of bicarbonate and calcium ions, and to eggshell strength by controlling calcite crystal nucleation, growth, size and orientation (Greenfield et al., 1984).

The organic matrix in the calcified layer is comprised of a complex suite of components. In the acid soluble part of chicken eggshell matrix, 520 proteins have been identified (Mann et al., 2006), including several abundant proteins such as ovalbumin (Hincke, 1995), ovotransferrin (Gautron et al., 2001b), lysozyme (Hincke et al., 2000), osteopontin (Pines et al., 1995), sialoprotein (Solomon, 1999), clusterin (Mann et al., 2003), ovocleidin-17 (Hincke et al., 1995), ovocleidin-23 (Mann, 1999), ovocleidin-116 (Carrino et al., 1997), ovocalyxin-32 (Gautron et al., 2001a) and ovocalyxin-36 (Gautron et al., 2007). Many of the above components have been reported to undergo various post-translation modifications,which allow them to be effective chelators for interacting with the inorganic materials (Veis, 1989; Reyes-Grajeda et al., 2004; Mann et al., 2007), or to mediate protein-protein interactions to facilitate the assembly of the organic matrix (Lakshminarayanan et al., 2002; Ney et al., 2006).

It has been demonstrated that some genes in hen oviduct are associated with eggshell formation, whose expression is dependent on mechanical strain (Pines et al., 1995; Lavelin et al., 1998; Lavelin et al., 2002). It is proposed that some genes may function as crucial modulators for eggshell quality through regulating signal transduction, ion transportation, expression or modification of organic components, and many other processes. However, despite the importance of eggshell strength in the poultry industry, very few transcriptome-wide studies regarding this trait have been published to date (Yang et al., 2007; Dunn et al., 2009; Jonchere et al., 2010).

It is well documented that various parts of the avian eggshell are formed in specific regions of the oviduct as the egg passes through them. During the laying sequence, about 4 h after previous oviposition, the next egg arrives at and will take about 1h to pass through the white isthmus, in which the bilayered shell membranes are built around the egg. Then the egg enters the initial part of the shell gland, the red isthmus (tubular shell gland), and stays there for about 5 h to form mammillary knobs (Reyes-Grajeda et al., 2004). Finally the egg reaches the uterus (the main part of shell gland) and stays for an additional 15 h to form the palisade layer (Creger et al., 1976). It is known that the mamillary layer is the base of calcite crystal nucleation and crystal growth, and the palisade layer is the main part of the calcified shell, both of which affect global eggshell quality (Reyes-Grajeda et al., 2004; Jonchere et al., 2010).

In this study, we focused on the shell gland (uterus tissue near the red isthmus) at about 9 h post oviposition (corresponding to the early stage of active calcification, or to the transition stage from mammillary knob formation to construction of the palisade layer), and identified differentially expressed genes (DE-genes) in the layers with high shell strength compared to those with weak eggshell. Our results provide insight into the candidate genes involved in the mamillary layer formation and calcification that is crucial to the mechanical properties of avian eggshells.

MATERIALS AND METHODS

Animal treatments

Ninety purebred Xianju hens (a widely-bred Chinese indigenous chicken breed) of 28 weeks old were individually housed in laying cages. Birds were maintained under a cycle of 16 h light and 8 h dark. All birds were fed ad libitum with water and a mash layer diet (165 g protein, 35 g Ca, 11.29 MJ ME/kg, as recommended by NRC of China, 2004).

After 10 d of adaptation for hens, the oviposition time of each egg was initiated to be observed and recorded, then egg weight and shape index (length/width) were measured immediately. Following strength testing, the egg content was discarded and the shell was washed, dried at room temperature and weighed. Shell thickness without membranes was measured with a digital micrometer. Shell index (g/100 [cm.sup.2]) (Sauveur, 1988) was calculated as I = (C/S)x100, in which C is the weight of shell with membranes, S is the shell surface ([cm.sup.2]) with S = 4.68x[P.sup.2/3] where P = egg weight (g). All above measurements were consecutively carried out daily for 16 d.

Finally, 2 groups of 2 hens with consistent high or low shell breakage strength were found. The differences between the eggshell properties of the selected 4 hens were analyzed by One-way ANOVA variance analysis in SPSS statistic software.

The four hens of interest were humanely sacrificed about 9 h after the previous oviposition. It is of note that all of the sacrificed hens had eggs in their uteruses (Figure 1A). The fat was removed from the uterus tissues near red isthmus and the tissues were then frozen in liquid nitrogen immediately and stored at -80[degrees]C. The animal treatments were approved by the Commission for Animal Welfare of Zhejiang A&F University.

Measurement of eggshell strength

After egg weight and shape index measurements, the uncracked fresh eggs were individually placed lengthways with its blunt end upward in the FHK testing machine (Fujihara Co., Tokyo, Japan), and the vertical pressure was increasingly loaded upon the eggshell until the eggshell cracked and the eggshell strength was recorded as the maximum load (kgf).

RNA preparation

About 500 mg of the tissue of the uterus near red isthmus, including the mucosa, muscularis and outer serosa was powdered under liquid nitrogen. The total RNA was extracted using the RNAiso Plus Mini Kit (TaKaRa, Dalian, P.R. China) according to the manufacturer's instructions. RNA concentration and purity were measured by a NanoDrop spectrophotometer (NanoDrop[R] ND-1000, NanoDrop Technologies, DE).

Microarray hybridization and image acquisition

Microarray analysis was performed by the Bioassay Laboratory of CapitalBio Corporation (CapitalBio Co., Beijing, China). Briefly, the RNA integrity was firstly assessed using a Bioanalyzer (Agilent Technologies, Cheshire, UK), then 2 [micro]g of total RNA was used for reverse transcription and biotin-labeled cRNA synthesis according to the manufactures' instructions, and finally subjected to microarray hybridization. The Affymetrix GeneChip[R] Chicken Genome Array (Affymetrix, Santa Clara, CA, USA) was used in this study, which contains 38,535 probesets corresponding to >28,000 chicken genes. Following 16 h of hybridization, the arrays were immediately washed, stained and scanned using Affymetrix[R] GeneChip[R] scanner 3000 (Affymetrix, Santa Clara, CA, USA), and the image files were processed into raw CEL intensity files using GeneChip Operating Software (GCOS version 1.2).

Pre-processing and normalization of microarray data

The raw intensity files generated by GCOS were imported and processed by R with Bioconductor packages. The total RNA quality was firstly verified statistically again by plotting the 5'-3' hybridization signal trends across all target transcripts. Then the microarray intensity was processed into transcript expression by the Affymetrix MAS5.0 method implemented in the R package, a procedure including background normalization, PM/MM probe correction, expression summarization and constant normalization on probeset level.

Identification of DE-transcripts

To identify DE-transcripts, the 4 array samples were first grouped into two pairs of high vs. low eggshell strength according to eggshell property differences of the hens (see results). According to Cheuk and Cheng (2011), Affymetrix platform is relatively precise and sensitive in detecting signals, the DE-transcripts were identified as those with fold-change >= 2 in either of the two pairs of comparison and a statistical significant difference between high strength and low strength samples (p<0.05, Welch t-test). It is of note that the log-odds values (Lods) of expression fold-change were used in the analysis; therefore, the DE-transcripts always have an absolute Lods value no less than 1 ([absolute value of Lods] [greater than or equal to] 1).

Gene ontology enrichment analysis

Gene ontology enrichment analysis for DE-transcripts was performed using the web-based GOEAST (Zheng and Wang, 2008) Affymetrix analysis tool, with FDR cut-off of 0.05 using Yekutieli's FDR adjustment method.

Validation of differential expression by qRT-PCR experiments

Twenty-one DE-transcripts, with a fold-change ranging from low to high, were selected for further validation with qRT-PCR experiments; and all two groups of microarray samples were tested. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference in all the PCR experiments. The primer sequences for qRT-PCR experiments can be found in Table 1.

To begin, total RNA was individually reverse transcribed with the SYBR[R] PrimeScript[TM] RT-PCR kit II (TaKaRa, Dalian, China) according to the manufacturer's instructions. Then above RT-PCR kit was further used for fluorescence detection on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, USA). All samples were analyzed in triplicates.

Dissociation curve analysis was conducted to ensure that a single PCR product with appropriate size was amplified in each reaction. On the other hand, the examination of PCR efficiency was performed based on LinRegPCR program (12.X) (Ramakers et al., 2003; Ruijter et al., 2009) to ensure internal and target transcript primers were amplified with similar efficiency.

The differential expression levels (Log2 units) were calculated using the equation Log2 [units.sub.(high versus low)] = -[DELTA][DELTA]Ct, where [DELTA][DELTA]Ct = ([Ct.sup.th] - [Ct.sup.ih]) - ([Ct.sup.tl] - [Ct.sup.il]). Ct is the threshold cycle number when the amount of amplified product reaches a stable threshold. [Ct.sup.th] and [Ct.sup.ih] represented the Ct of target transcript and internal reference transcript of "high eggshell strength sample", respectively. Correspondingly, [Ct.sup.tl] and [Ct.sup.il] represented the Ct of target transcript and inner-reference transcript of "low eggshell strength sample", respectively.

RESULTS

Eggshell quality of hens under study

Among 90 tested hens, only 6 of them laid eggs at a similar laying rate with consistent high eggshell strength (defined as [greater than or equal to] 4.5 kgf) or low strength (defined as [less than or equal to] 3.5 kgf). Two of these 6 hens were sacrificed at about 11.5 to 12 h post oviposition, but the eggshells collected from the shell glands showed more calcification extent than expected (Figure 1A and 1B). To focus on the initial stage of active calcification, we decided to use the uterus tissues near red isthmus from the remaining 4 hens, namely #19, #35, #40 and #80, at about 9 hr post oviposition. The eggs and eggshells harvested from these 4 hens at the moment of tissue sampling are shown in Figure 1A and Figure 1B. The eggshell breaking strength is also shown in Table 2 for these 4 hens, with consistent high (#19 and #40) or low (#35 and #80) eggshell quality.

To eggs from #19 and #35 hens, the differences of both shell strength and shell weight were very significant (p<0.01, t-test), but there was no significant difference (p>0.05) for other eggshell quality metrics, such as shell thickness or shell index (Table 2). On the other hand, the differences of all of above eggshell metrics between eggs from #40 and #80 hens were very significant (p<0.01) (Table 2). To get rigorous microarray data, therefore, we grouped the #19 with #35 hens due to the similarity of some of eggshell mechanical properties of the paired individuals; while #40 and #80 hens were also grouped as another pair.

Differentially expressed transcripts

The expression level of all probesets in 4 array samples were analyzed, and 1,195 DE-transcripts between uterus samples with high shell strength and low shell strength were identified. These DE-transcripts correspond to 941 unique genes. Among them, 407 genes were up-regulated in high strength samples comparing to low strength samples, and the other 534 genes were down-regulated. The expression profile of all 1,195 DE-transcripts is shown in the heatmap in Figure 2. As shown in the heatmap, samples #19 with #40 and #35 with #80 were grouped as clusters among different samples, consistent with the similarity of their eggshell quality.

According to gene ontology annotations, the DE-transcripts are involved in a variety of biological processes. The most prominent DE-transcripts were found related to the following processes: signal transduction (88 DE-transcripts), ion transport and extracellular matrix organization (77 DE-transcripts), carbohydrate metabolism and protein modification (26 DE-transcripts) (Table 3).

Furthermore, avian calcified eggshell is a biomaterial composed of calcium salt and special ECM. The ECM is mainly comprised of collagens, glycoproteins and proteoglycans. Among the DE-transcripts, COL8A2, COL12A1, COL13A1, LOC424798, LAMA2, LAMA4, LAMB4, and LAMC1 may be related to extracellular matrix formation; while CHST3, GALNTL1, NDST4, LARGE, POFUT2, RCJMB04_28l23, and MAN1A2 are all localized in the endoplasmic reticulum or Golgi apparatus, and likely mediate the processes of carbohydrate metabolism, or posttranslation glycosylation modification.

Gene ontology (GO) term enrichment of DE-transcripts

It is of note that although many DE-transcripts were found related to various biological processes according to their ontology annotations, they are not necessarily correlated to the eggshell quality, due to random noise or other non-specific confounding factors commonly existing in microarray or other high-throughput experiments. Therefore, using web-based GOEAST (Zheng and Wang, 2008) we further identified significantly enriched GO terms among all the DE-transcripts. According to biology processes or molecular functions, the enriched GO terms can be roughly classified into several groups (Tables 4 and 5).

A group of processes are involved in reproductive hormone regulation, which contain Somatotropin secreting cell differentiation (GO:0060126), adenohypophysis development (GO:0021984), and response to estradiol stimulus (GO:0032355) (Table 4).

As shown in Table 4 and Table 5, many DE-transcripts are involved in signal transduction, such as GO terms purinergic nucleotide receptor activity (GO:0001614), nucleotide receptor activity (GO:0016502), purinergic receptor activity (GO:0035586), transmembrane signaling receptor activity (GO:0004888), and negative regulation of BMP signaling pathway (GO:0030514). Among them, GO:0004888 dominantly contains 33 transcripts encoding signal receptors, and these receptors could be further classified into several subgroups: OXTR, LOC431251 and SSTR3 belong to reproductive hormone receptors; CHRM2, ADRA2B, P2RX4, P2RY2, EDNRB2, GABRB2, GABRG2, LOC428961 and NPFFR2 function as receptors mediating neurotransmitters or neuropeptide; GRIN2B and GRIN3A could modulate the efficiency of synaptic transmission; NTRK1 and NTRK2 belong to the receptor tyrosine kinase (RTK) family, and are involved with neurotrophin (GO:0005030--neurotrophin receptor activity; and GO:0043121--neurotrophin binding) (Table 5).

Besides various enriched molecular function shown above, many biophysical processes are also found to be enriched among the DE-transcripts, including a series of processes and subgroups (Tables 4 and 5). GO:0003951 (NAD+ kinase activity) modulate the metabolism or redox in cell (Table 5). Enrichment of GO:0009409 (response to cold) may reflect the fact the rearing condition of experimental hens was in the winter at room temperature about 2 to 10[degrees]C. GO:0046209 (nitric oxide metabolic process) may regulate vascular or smooth muscle relaxation or other functions. GO:0002028 is involved in ion transportation. While the subgroup processes of muscular development and activity include skeletal muscle fiber development (enrichments of GO:0048741, GO:0048747 and GO:0055002) and striated muscle contraction regulation (enrichments of GO:0055117 and GO:0006942). It is of note that there is almost no striated muscle in avian uterus except smooth muscle. However, the chicken genome project was completed in 2004, and the functional gene database of G. gallus remains incomplete, some ontology annotations of DE-genes may refer to mammalian homologs, which may account for our results. The genes related to muscular cell contraction are likely to modulate the mobility of uterus to facilitate egg rotation and calcification (Johnson, 1986; Jonchere et al., 2010). Similarly, there is no digestion process in the uterus, three genes in GO:0007586 (digestion process), PGA5 (an aspartic acid protease, which is involved in ovulation (Peluffo et al., 2011)), PRSS2 and LOC396365 (preprogastrin), are likely to promote the maturation of secretary extracellular proteins or regulate the secretion of uterus glands and mobility of uterus.

The final group of reproductive biophysical processes also includes several subgroups of processes (Table 4). Epithelial tube morphogenesis (GO:0060562) may regulate the development of uterus glands (tubular epithelial glands). Oocyte development subgroup contains oocyte differentiation (GO:0009994) and oocyte development (GO:0048599). Female pregnancy subgroup contains enrichments of GO:0060135, GO:0060745, GO:0060748, GO:0060444 and GO:0060603.

Overall, laying is an avian reproductive behavior, and eggshell calcification is regulated by relative reproductive hormones and neurotransmitters, which may finally affect eggshell quality through a complex suite of biophysical reactions.

Confirmation of DE-transcripts by qRT-PCR

21 DE-transcripts (9 up-regulated and 12 down-regulated) were chosen for validation using qRT-RCR experiments, and the four microarray samples were tested in pairs for #19 vs #35 and #40 vs #80, respectively. As shown in Figure 3, 16 out of the 21 tested transcripts (76%) were confirmed by qRT-PCR experiments, though the absolute fold-change values are slightly different. The remaining transcripts, CRYBB1, EXOC6B, LOC416916, MAN1A2 and CHST3, showed inconsistent differential expression between qRT-PCR and microarray experiments.

GAPDH, CHST3, GALNTL1, NDST4, LARGE, SP1, RHOBTB2, and WDR72 were selected to examine the PCR efficiency. The results showed the PCR efficiency of these genes ranged from 86.8% to 94.2%, and the PCR efficiency of inner reference (GAPDH) and other genes seemed nearly similar.

DISCUSSION

Laying is regarded as avian reproductive behavior, which is regulated by reproductive hormones and neurotransmitters. The chicken oviduct has been extensively used as a model to study hormonal induction of protein synthesis (Khuong and Jeong. 2011). Under the control of steroid hormones or neurotransmitters, the tubular gland epithelial cells synthesize and secrete a great variety of proteins to form egg white and eggshell when egg passes through the oviduct (Mann et al., 2006). In this paper, 1,195 DE-transcripts have been identified to be related with eggshell strength. GOEAST analysis further identify some significantly enriched GO terms, and the enriched GO terms suggest that some DE-transcripts mediate reproductive hormones or neurotransmitters to affect eggshell quality (Tables 4 and 5).

Both terms GO:0060126 and GO:0021984 are involved in reproductive hormone regulation, and share two genes, OTX2 and WNT4.

Otx2 is a paired-like homeodomain transcription factor, which can mediate GnRH (gonadotropin releasing hormone) signaling (Kelley et al., 2000). Functional studies revealed that Otx2 is required as early as gastrulation for neural induction, and even for brain development (Rhinn et al., 1998). However, Otx2 is also of importance for neurogenesis and cellular proliferation in multiple other tissues (Layman et al., 2011).

As a member of the WNT family, Wnt4 is a secreted glycoprotein signaling molecule and involved in paracrine signaling (Diaz et al., 2011). Wnt4 is critical for female sex determination and differentiation (Chen et al., 2011). In the female, Wnt4 is positively involved in ovarian development; while in the male mutated WNT4 will result in aberrant testis development (Diaz et al., 2011; Barrionuevo et al., 2012). On the other hand, Wnt4 is also potent to regulate the development of the female reproductive tract (Franco et al., 2011). Furthermore, WNT4 is expressed postnatally in ovarian follicles and corpora lutea, and its expression increases in response to gonadotropin (Hsieh et al., 2002). Wnt4 mediates follicle development and fertility by regulating the expression of genes involved in steroidogenesis, prostaglandin biosynthesis, tissue remodeling, and angiogenesis (Hsieh et al., 2002; Boyer et al., 2010). Moreover, excluding its reproductive contributions, WNT4 is also tightly associated with bone strength (Zmuda et al., 2011).

Our results also show that some DE-transcripts are involved in signal transduction (Tables 4 and 5), among which, NTRK1, NTRK2, P2RX4, and P2RY2 are overlapped in multiple enriched GO terms (Table 5).

Ntrk1, also named TrkA, and Ntrk2 TrkB, are two members of the neurotrophic tyrosine kinase receptor (NTKR) family. These kinases are membrane-bound receptors mediating various functions of neurotrophins, such as cell survival, migration, outgrowth of axons and dendrites, synaptogenesis, remodeling of synapses, and synaptic transmission (Ohira1 and Hayashi, 2009). So far, several neurotrophins have been well studied, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and NT-4/5. NTKRs are high affinity receptors of neurotrophins. TrkA mediates the biological response of NGF, while BDNF and NT-4/5 are the preferred ligands for TrkB (Ohira1 and Hayashi, 2009). Additionally, NTKRs also play roles in some biomaterials. NT-4 may modulate proliferation and differentiation of the dental epithelium and promote production of the enamel matrix via the TrkB-MAPK pathway (Yoshizaki et al., 2008).

Both P2RX4 and P2RY2 are purinergic receptors. Purinergic receptors are subdivided into metabotropic P2Y receptors and ionotropic P2X receptors. P2Y receptors are coupled to G-protein and trigger inositol 1,4,5-triphosphate (IP3)-induced intracellular [Ca.sup.2+] release following activation of phospholipase C, while P2X receptors are ligand-gated ion channels. P2RX4 will be discussed later, while P2RY2 performs a dominant role in calcium signaling during osteoblast differentiation (Nishii et al., 2009). It is known that extracellular ATP, UTP, and PPi can strongly block the mineralization of matrix nodules, while this potent inhibition of bone formation is mediated by P2RY2 (Orriss et al., 2007). Furthermore, P2RY2 is also involved in inhibition of intercellular communication between osteoblasts (Hoebertz et al., 2003).

At present, there are at least three cDNA microarray studies globally investigating the gene expression in chicken shell gland (Yang et al., 2007; Dunn et al., 2009; Jonchere et al., 2010), but the overlap among the DE-genes from these studies is not plentiful. Different animals, tissue samples or treatment methods may partially account for this problem.

Yang et al. (2007) harvested uterus tissues at 2 h post oviposition, and screened out 34 known genes in the shell glands between hens with low and high egg productive rates. This study and our data share a single gene CALD1 (caldesmon 1) (Figure 4). CALD1 is a ubiquitous actin and calmodulin binding protein, and functions as a substrate for mitogen-activated protein kinase (Childs et al., 1992) or as serine and threonine kinases (Sutherland et al., 1994).

Dunn et al. (2009) identified 266 DE-genes in shell glands from 25-week old mature hens comparing to 12-week old juveniles from high and low bone quality lines, respectively. The tissues sampled when eggs passed through the oviducts but not in shell glands. Three DE-genes are also found in our data: NADK (NAD kinase), LOC422993 (Similar to interferon-induced membrane protein Leu-13/9-27), and LAMP3 (lysosomal-associated membrane protein 3) (Figure 4), suggesting potential crucial function of these genes in not only early stage of eggshell calcification but also other stages of eggshell formation.

Jonchere et al. (2010) used the 40-week old hens at 18 h post oviposition (corresponding to the rapid phase of calcification), and identified 469 DE-known genes in uterus versus both white isthmus, and magnum. There are 7 genes consistently identified in their study and our data, such as P2RX4 (purinergic receptor P2X, ligand-gated ion channel, 4), FSTL1 (follistatin-like 1), TUBGCP4 (Tubulin, gamma complex associated protein 4), WDR77 (WD repeat domain 77), RCJMB04_6g16 (microtubule-associated protein 1 light chain 3 beta), PWP1 (PWP1 homolog in S. cerevisiae) and SGK1 (serum/glucocorticoid regulated kinase 1) (Figure 4).

On the other hand, three additional DE-genes in our data were previously found in the acid soluble part of chicken eggshell organic matrix (Mann et al., 2006). These three genes, FSTL1 (follistatin-like 1), CAMK2D (calcium/ calmodulin-dependent protein kinase (CaM kinase) II delta) and KRT75 (keratin 75) (Figure 4), could reflect potential interaction of eggshell calcification and organic matrix formation.

Among these overlapping DE-genes, both P2RX4 (Jonchere et al., 2010) and NADK (Yang et al., 2007) are also present in our enriched GO terms (Tables 4 and 5), and FSTL1 (Mann et al., 2006; Jonchere et al., 2010) occurs in more than three relative studies.

P2RX4 is one member of the P2X receptors (P2RX). P2RX are ionotropic ATP-gated ion channels conducting [Ca.sup.2+] inflow (Fodor et al., 2009), with high capability of [Ca.sup.2+] permeabilities corresponding to at least 100-fold those of [Na.sup.+] (Burnashev, 1998). In chondrogenic mesenchymal cells, P2X4 receptors could conduct [Ca.sup.2+] inflow to elevate intracellular [Ca.sup.2+] levels, and finally promoting extracellular matrix production (Fodor et al., 2009). Eggshell calcification requires considerable ion transportation, especially [Ca.sup.2+], and various extracellular matrix synthesis and secretion, whether and how P2RX4 channels regulate these processes requires further studies.

NAD kinases (NADKs) are a family of enzymes transferring a phosphate group from ATP to NAD to generate and maintain the cellular NADP pool (Pollak et al., 2007). It is reported, that during development of placenta, the expression level of NADK appears drastically elevated (Lerner et al., 2001).

Fstl1 is a secreted glycoprotein belonging to the BM-40/SPARC/osteonectin family containing both calciumbinding domain and Follistatin-like domain (Hambrock et al., 2004). As a mesenchymal factor, Fstl1 is critical for oviduct development, and determines the differentiation of secretary epithelial cells and ciliated epithelial cells in the oviduct (Umezu et al., 2010). This means Fstl1 may modulate chicken endometrium development during eggshell formation. However, Fstl1 is also present in the organic part of eggshells (Mann et al., 2006), and Jonchere et al. (2010) propose it may be a uterine antiprotease

IMPLICATIONS

Above all, using Affymetrix Chicken Array, 1,195 DE-transcripts were identified in the shell gland between "high shell strength" and "low shell strength" hens, which represent 941 unique known genes. According to gene ontology annotations, these transcripts are involved in a wide range of biological processes; the most prominent DE-transcripts relate to signal transduction, metabolism, extracellular matrix, or ion transport and homeostasis, and so on. Furthermore, Gene Ontology (GO) term enrichment of DE-transcripts suggests that avian eggshell calcification is likely to be regulated by relative reproductive hormones and neurotransmitters, which may finally affect eggshell quality through a complex suite of biophysical processes.

http://dx.doi.org/ 10.5713/ajas.2012.12398

ACKNOWLEDGEMENTS

This work was supported by National Natural Science Foundation of China (grant no. 30700567) and Zhejiang Provincial Natural Science Foundation of China (grant no. LY12C17002).

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Zhangguo Liu1 (1,2,a), Qi Zheng (3,a), Xueyu Zhang (4) and Lizhi Lu (5) *

* Corresponding Author: Lizhi Lu. Tel: +86-571-86406682, Fax: +86-571-86406682, E-mail: Lulizhibox@163.com

(1) The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Lin'an, Zhejiang, 311300, China

(2) College of Animal Science, Zhejiang A. & F. University, Lin'an, Zhejiang, 311300, China.

(3) Department of Biology, 104 Lynch Laboratory, University of Pennsylvania, Philadelphia, PA 19104, USA.

(4) Institute of Poultry Science, Chinese Academy of Agricultural Science, Yangzhou, Jiangsu, 225003, China.

(5) Institute of Animal Husbandry and Veterinary Science, Zhejiang Provincial Academy of Agricultural Science, Hangzhou, Zhejiang, 310021, China.

(a) These authors contributed equally to this work.

Submitted Jul. 18, 2012; Accepted Sept. 15, 2012; Revised Oct. 8, 2012

Table 1. Descriptions of specific primers used for
real-time RT-PCR

Gene symbol     Accession      Forward        Reverse        Amplicon
                no.            primer         primer         (bp)
                               (5'-3')        (5'-3')

ACYP2           XM_419292      CGGCTCGC-      GGCC-          152
                               TCAAGTC-       CTGAAC-
                               GGTGG          TTGGCCCGTC
AMDHD1          XM_416158      GCACTGG-       TCTTCC-        175
                               GAAGT-         GTGGC-
                               GCGTA-         CTTCCT-
                               TTGCCA         GGTGT
ATP6V1A         NM_204974      TGCAAC-        TGCCA-         187
                               ATGGC-         GGCCC-
                               AGGTGCTGCT     CAGTTCCACT
CA5B            XM_414195      CAGCT-         ACACG-         175
                               TGGCCA-        TCGCT-
                               CCTGCACTCC     GGGTCGTAGCT
CHST3           NM_205121      TGATGGCCAC-    CTGCAGCACG-    170
                               CACACGCACC     TCGCGGTACA
COL12A1         NM_205021      AGGCGAGTCT-    GCGCTGTCCT-    171
                               TCCCCGACGG     CATGTCTGCCC
CRABP1          NM_001030539   CGCCCCGCCA-    AACTGGTCCC-    161
                               TGCCTAACTT     CGTCCTGGCG
CRYBB1          NM_204180      ACCTGGCGGA-    CGGTAGCTGC-    151
                               CTGCGGGTT      TGGACCAGGTG
EXOC6B          XM_420892      AACCCCACCA-    TGGCTGTTGA-    149
                               CAGCCCTCGT     TGAGGCCGCG
FGB             XM_420369.2    GCTGCTCCTG-    GTGCCACGGG-    155
                               CTGCTCCTGC     CCTGAGTGTG
GAS2L3          XM_416172      GGAGTAGTGC-    CCTGGGCCGT-    193
                               TGGCAGTCCTGC   GTCTGGGAGT
GIT2            NM_204206      TCGCTTGCCA-    GCAACGTGGA-    168
                               TGCCGTGAGG     GCGGGGTGTT
MAN1A2          XM_416490      ACGTGGACACC-   TCCTTTGCCTC-   148
                               AGCAAGGGGG     TTCCAGG-
                                              GCCTTT
NDST4           XM_420638      CGAGCA-        TGCCCA-        156
                               GCTTCC-        GGGGC-
                               CTCATCCCCAA    TTGACGTAA
NPY             NM_204587      GAGGACGCTCC-   TCGAAGGGTCT-   175
                               CGCAGAGGA      TCAAACCGGGA
OC416916        XM_415207      TGGAGG-        CCACCGA-       200
                               TGGAGC-        GCACA-
                               ACAAACATCTGC   CAGCCAGAAA
PLCXD1          NM_001128637   CCTGG-         AGCCA-         137
                               CCTGCAG-       CGCTGC-
                               GAATTTTGATGG   CACATGGTC
RCJMB04_34k20   NM_001031112   GGACA-         TGGTGG-        126
                               GGCGGG-        TAACAC-
                               CGAGAGAGT      GCACGCTGA
SLC8A1          XM_415002      CGTGTTTGTGG-   ATGGCCGCGAT-   159
                               CACTGGGGACA    GGACCAAGC
TBXAS1          XM_416334      TGTGT-         ATACAGC-       188
                               GGTGCT-        CACGG-
                               GGGACAGCGT     GGTCCTGCT
WDR72           XM_425069      GGCTG-         GCACAC-        161
                               TTATC-         GCAGCA-
                               AGGGGG-        CACTACGC
                               CCAGGA
GAPDH           NM_204305      GGGCTG-        TCAGGG-        177
                               CTAAG-         GCCCAT-
                               GCTGTGGGG      CAGCAGCA

Table 2. Parameters related to eggshell quality of hens in
this study

Hen     shell strength (kgf)     shell thickness (mm)

#40   5.17 [+ or -] 0.40 (A)   0.367 [+ or -] 0.016 (A)
#19   4.75 [+ or -] 0.21 (A)   0.328 [+ or -] 0.012 (B)
#35   2.99 [+ or -] 0.71 (B)   0.324 [+ or -] 0.023 (B)
#80   2.54 [+ or -] 0.69 (B)   0.272 [+ or -] 0.022 (C)

Hen   shell index (g/100 [cm.sup.2])     shell weight (g)

#40   8.07 [+ or -] 0.28 (A)             4.297 [+ or -] 0.144 (a)
#19   7.18 [+ or -] 0.25 (B)             3.968 [+ or -] 0.195 (b)
#35   6.97 [+ or -] 0.68 (B)             4.363 [+ or -] 0.426 (a)
#80   6.25 [+ or -] 0.71 (C)             3.451 [+ or -] 0.417 (c)

Hen        egg weight (g)             shape index

#40   38.46 [+ or -] 1.52 (C)    1.274 [+ or -] 0.024 (B)
#19   40.72 [+ or -] 1.26 (B)    1.322 [+ or -] 0.030 (A)
#35   48.60 [+ or -] 1.38 (A)    1.278 [+ or -] 0.032 (AB)
#80   40.58 [+ or -] 1.06 (B)    1.313 [+ or -] 0.052 (AB)

Values are from eggs laid by each hen of interest during the
period of observation. Distinct capital letters in the same
column indicate parameters between
hens with a significant difference (p< 0.01), and distinct
small letters indicate the significant difference is at level
p<0.05.

Table 3. i) DE-transcripts related with signaling,
ion transportation, extracellular matrix protein, and
carbohydrate metabolism or post-translation
glycosylation modification

Gene symbol   Transcript ID     Log2 units       p-value   Category
                              (strong VS weak)

LOC771699     XM_001234946         3.853         0.0004    signaling
SH3PXD2A        XM_421741          3.410         0.0397    signaling
LOC429955       XM_427511          3.296         0.0034    signaling
PDCL2           XM_420702          3.100         0.0115    signaling
LOC430487       XM_428042          3.055         0.0074    signaling
SEMA3G          XM_414289          2.933         0.0213    signaling
RHOBTB2       XM_001232709         2.905         0.0069    signaling
RXFP1           XM_420385          2.795         0.0205    signaling
PIK3C2B         XM_417956          2.777         0.0194    signaling
OR10A7          XM_425093          2.768         0.0067    signaling
NPY             NM_205473          2.401         0.0329    signaling
PDE8B           XM_425218          2.401         0.0498    signaling
GREM2           XM_419552          2.284         0.0423    signaling

Table 3. ii) DE-transcripts related with signaling, ion
transportation, extracellular matrix protein, and carbohydrate
metabolism or post-translation glycosylation
modification (Continued)

Gene symbol     Transcript ID   Log2 units    p-value   Category
                                (strong VS
                                   weak)

SPAG9             XM_420098        2.215      0.0423    signaling
LOC396365         NM_205400        2.166      0.0009    signaling
MAPKBP1           XR_026772        2.157      0.0187    signaling
OXTR            NM_001031569       2.128      0.0134    signaling
STC2              XM_414534        2.120      0.0135    signaling
MPP3              XM_418108        1.921      0.0491    signaling
GREM1             NM_204978        1.903      0.0220    signaling
C20orf32          XM_417499        1.889      0.0117    signaling
CRABP1          NM_001030539       1.778      0.0115    signaling
C1orf107        NM_001031051       1.770      0.0411    signaling
RGS9              XM_415685        1.657      0.0177    signaling
ARHGEF12          XM_417890        1.437      0.0014    signaling
LTBP3             XM_426444        1.433      0.0409    signaling
NGEF            NM_001010841       1.390      0.0307    signaling
PDE1A             XM_421969        1.363      0.0127    signaling
CRHBP             XM_424801        1.355      0.0159    signaling
SRGAP1          NM_001080101       1.335      0.0426    signaling
PDE9A             XM_416748        1.196      0.0475    signaling
NPFFR2          NM_001034825       1.124      0.0283    signaling
WNT4              NM_204783        1.114      0.0180    signaling
FGD4              XM_416365        1.099      0.0121    signaling
SOCS2             NM_204540        1.033      0.0377    signaling
TOB1            NM_001001467       1.015      0.0347    signaling
RND3              XM_422158        0.887      0.0094    signaling
ARHGAP28          XM_419140       -4.485      0.0231    signaling
WNT3              NM_204675       -3.902      0.0422    signaling
LOC428961       NM_001142671      -3.469      0.0058    signaling
VAV2              NM_204142       -3.397      0.0232    signaling
TBC1D20         XM_001235014      -3.154      0.0389    signaling
HTT               XM_420822       -3.086      0.0065    signaling
P2RY2             XM_425667       -3.079      0.0294    signaling
GRAP2           XM_001234081      -2.967      0.0402    signaling
INPP4A            XM_416886       -2.749      0.0130    signaling
ITSN1             XM_416715       -2.744      0.0139    signaling
EDNRB2            NM_204120       -2.644      0.0317    signaling
FGB               XM_420369       -2.631      0.0194    signaling
RXFP3             XM_429217       -2.616      0.0048    signaling
LOC420403         XM_418509       -2.531      0.0049    signaling
SSTR3           NM_001024583      -2.501      0.0203    signaling
GPR39           NM_001080105      -2.376      0.0087    signaling
GPR97             XM_413998       -2.356      0.0230    signaling
FGF14             NM_204777       -2.322      0.0175    signaling
SFRP4             XM_418831       -2.204      0.0162    signaling
LOC430333       XM_001235474      -1.880      0.0153    signaling
GARNL1            XM_421244       -1.850      0.0477    signaling
RAPH1             XM_421961       -1.842      0.0035    signaling
C14orf138         XM_421460       -1.756      0.0012    signaling
LOC421876         XM_419893       -1.750      0.0333    signaling
NLE1              XM_415857       -1.735      0.0463    signaling
CHRM2           NM_001030765      -1.721      0.0310    signaling
LOC768958       XM_001232128      -1.622      0.0278    signaling
RASL10B         XM_001233673      -1.546      0.0473    signaling
SIPA1L2           XM_419564       -1.545      0.0020    signaling
LOC769317       XM_001231944      -1.531      0.0287    signaling
PLXDC2            XM_418613       -1.503      0.0155    signaling
SPOCK1            XM_414622       -1.491      0.0375    signaling
CSF2RB          XM_001234608      -1.468      0.0498    signaling
LOC429163         XM_426718       -1.434      0.0220    signaling
PLXNC1            XM_416143       -1.398      0.0103    signaling
RCJMB04_19g9      XM_419989       -1.339      0.0019    signaling
PLXNA1            XM_414370       -1.305      0.0467    signaling
TSPAN5            XM_420654       -1.277      0.0142    signaling
LOC431251       NM_001127171      -1.226      0.0414    signaling
ANXA10          XM_001233661      -1.176      0.0441    signaling
RCJMB04_18c11   NM_001012909      -1.153      0.0436    signaling
SPRED2            XM_419341       -1.124      0.0146    signaling
MOBKL1A           XM_420601       -1.116      0.0026    signaling
ALS2CL            XR_026875       -1.038      0.0119    signaling
MPP1            NM_001007917      -1.036      0.0424    signaling
FGF12             NM_204888       -1.016      0.0006    signaling
GNA13             XM_415686       -1.007      0.0195    signaling
ARL10             XM_414552       -0.975      0.0492    signaling
ADRA2B            XM_425203       -0.885      0.0415    signaling
CCKAR           NM_001081501      -0.868      0.0352    signaling
RCJMB04_3n15    NM_001030902      -0.815      0.0373    signaling
TBC1D24         XM_001232296       4.629      0.0023       IT
SCN9A             XM_422021        3.745      0.0047       IT
KCNT2             XM_426614        3.077      0.0015       IT
LOC395893                          3.030      0.0392       IT
NIPAL4            XM_414566        2.796      0.0163       IT
ATP6V0A4        NM_001080102       2.790      0.0397       IT
GRIN2B            XM_416204        2.781      0.0291       IT
KCNJ1             XM_425795        2.363      0.0011       IT
POR               XM_415768        2.294      0.0047       IT
KCNK2           XM_001234269       2.270      0.0447       IT
KIRREL3           XR_026874        2.143      0.0193       IT
GABRG2            NM_205345        1.985      0.0176       IT
SLC4A1            NM_205522        1.794      0.0041       IT
NDUFA7            XM_418185        0.995      0.0464       IT
JPH3              XM_414192        0.925      0.0193       IT
CACNA2D1        XM_001231265       0.852      0.0429       IT
EFCAB5            XM_415833       -3.593      0.0342       IT
SPATA22         XM_001235167      -3.590      0.0176       IT
RCJMB04_1f1     NM_001031133      -3.197      0.0328       IT
LOC428404         XM_425965       -3.177      0.0442       IT
ATP13A3           XM_422709       -3.059      0.0065       IT
GABRB2          XM_001232377      -2.963      0.0472       IT
SLC8A1          NM_001079473      -2.694      0.0342       IT
CACNA2D3          XM_414338       -2.622      0.0100       IT
SERINC5           XM_424762       -2.615      0.0129       IT
LOC421866         XR_027148       -2.613      0.0307       IT
LOC425295         XM_423073       -2.586      0.0212       IT
LOC772391       XM_001235535      -2.573      0.0163       IT
KCNK17            XM_419477       -2.506      0.0047       IT
KCTD16            XM_425217       -2.363      0.0076       IT
CNNM1             XM_421703       -2.309      0.0446       IT
KCNJ5             XM_417864       -2.138      0.0324       IT
RCJMB04_11e10   NM_001030630      -2.102      0.0226       IT
GRIN3A          XM_001232181      -1.680      0.0355       IT
ATP6V1A           NM_204974       -1.202      0.0065       IT
RCJMB04_16a12   NM_001031305      -1.030      0.0261       IT
CNGA3             NM_205221       -0.962      0.0479       IT
P2RX4             NM_204291       -0.882      0.0252       IT
MEGF10            XM_424719        3.910      0.0398       EM
FAT2              XM_414584        3.892      0.0052       EM
SDK2              NM_204538        2.905      0.0097       EM
NRXN3             XM_421297        2.643      0.0139       EM
LAMA4             XM_419780        2.569      0.0322       EM
NTNG1           XM_001231446       2.004      0.0328       EM
CRTAC1          NM_001080211       1.930      0.0132       EM
LAMC1             NM_204166        1.680      0.0257       EM
LAMB4           XM_001232877       1.642      0.0161       EM
PPFIA1            XM_421074        1.583      0.0166       EM
CLDN20          XM_001232002       1.438      0.0101       EM
CDH9            XM_001231501       1.296      0.0311       EM
CHAD              XM_416236        1.268      0.0240       EM
LOC396026         NM_205128        1.239      0.0403       EM
PCDH21          NM_001001759       1.174      0.0187       EM
EPDR1             XM_418830        1.158      0.0469       EM
CPNE8           XM_001231388       1.121      0.0219       EM
COL12A1           NM_205021        0.996      0.0003       EM
PKP2              XM_416362        0.983      0.0447       EM
CD72              NM_205052        0.855      0.0263       EM
NINJ2             XM_416382       -4.039      0.0409       EM
OTOF              XM_420015       -3.980      0.0016       EM
COL13A1         XM_001232218      -3.260      0.0006       EM
SVEP1             XM_424917       -2.967      0.0250       EM
OTOP1             XM_420790       -2.830      0.0263       EM
GPNMB             XM_425991       -2.771      0.0327       EM
PKP1              XM_419240       -2.349      0.0324       EM
LAMA2             XM_419746       -2.336      0.0000       EM
COL8A2            XM_425780       -2.295      0.0170       EM
FNBP4             XM_424260       -2.279      0.0486       EM
EGFL6             XM_416835       -2.268      0.0392       EM
CDH18             XM_426046       -1.955      0.0090       EM
CLDN8             XM_425544       -1.598      0.0288       EM
RCJMB04_34k20   NM_001031112      -1.214      0.0085       EM
SRPX              XM_416781       -1.123      0.0244       EM
DLG1              XM_422701       -1.084      0.0171       EM
FBLN1             NM_204165       -0.981      0.0206       EM
F13A1             NM_204685       -0.949      0.0234       EM
FREM1             XM_424932       -0.910      0.0057       EM
MEGF10            XM_424719        3.910      0.0398       EM
MGAT4C            XM_425447        3.007      0.0497    GM or CM
CHST3             NM_205121        2.839      0.0372    GM or CM
EDEM3             XM_422293        2.539      0.0179    GM or CM
LARGE           NM_001004404       2.071      0.0429    GM or CM
GFPT2             XM_424573        1.919      0.0173    GM or CM
GALNTL1         XM_001231964       1.895      0.0452    GM or CM
WDR77           NM_001030916       1.805      0.0445    GM or CM
NDST3             XM_426325        1.403      0.0121    GM or CM
B3GALT1           XM_426584        1.254      0.0483    GM or CM
OGDHL             XM_421503        1.144      0.0022    GM or CM
MAN1A2            XM_416490       -4.700      0.0087    GM or CM
POFUT2            XM_421892       -2.804      0.0156    GM or CM
LOC772154       XM_001235329      -2.798      0.0006    GM or CM
KLB               XM_423224       -2.335      0.0380    GM or CM
TRIM7.2         NM_001099354      -1.986      0.0041    GM or CM
LOC771361       XM_001234647      -1.433      0.0369    GM or CM
RCJMB04_28l23   NM_001039316      -1.324      0.0034    GM or CM
NDST4             XM_420638       -1.239      0.0261    GM or CM
PFKM              NM_204223       -1.170      0.0460    GM or CM
NUP153            XM_418937       -1.109      0.0378    GM or CM
GPD1L             XM_418763       -0.947      0.0383    GM or CM
PHKA2             XM_416811       -0.926      0.0326    GM or CM
B3GNTL1           XM_415599       -0.883      0.0109    GM or CM
PMM1              XM_416228       -0.787      0.0490    GM or CM
MMP11           XM_001232776       2.209      0.0391    GM or CM
ST3GAL4           XM_417860       -1.043      0.0094    GM or CM

IT represents ion/proton transporter, EM represents extracellular
matrix, GM represents post-translation glycosylation modification,
and CM represents carbohydrate metabolism.

Table 4. Enriched gene ontology (GO) terms revealed from
identified DE-transcripts according to biological_process
ontology

Group                 GOID              Term             p

Reproductive       GO:0060126   Somatotropin           0.013
  hormone                       secreting cell
  synthesis                     differentiation
  and regulation   GO:0021984   Adenohypophysis        0.044
                                development
                   GO:0032355   Response to            0.044
                                estradiol stimulus
Signal             GO:0030514   Negative regulation    0.030
  transduction                  of BMP signaling
                                pathway
Biophysical        GO:0048741   Skeletal muscle        0.012
  processes                     fiber development
                   GO:0015074   DNA integration        0.012

                   GO:0055117   Regulation of          0.013
                                cardiac muscle
                                contraction
                   GO:0009409   Response to cold       0.018
                   GO:0048747   Muscle fiber           0.022
                                development
                   GO:0046209   Nitric oxide           0.024
                                metabolic process
                   GO:0007586   Digestion              0.030

                   GO:0015849   Organic acid           0.033
                                transport

                   GO:0046942   Carboxylic acid        0.033
                                transport

                   GO:0055002   Striated muscle        0.034
                                cell development
                   GO:0006942   Regulation of          0.037
                                striated muscle
                                contraction
                   GO:0002028   Regulation of          0.044
                                sodium ion
                                transport
Reproductive       GO:0060748   Tertiary               0.009
                                branching
  biophysical                   involved in
                                mammary
  processes                     gland duct
                                morphogenesis
                   GO:0060745   Mammary gland          0.013
                                branching
                                involved in
                                pregnancy
                   GO:0060562   Epithelial             0.019
                                tube
                                morphogenesis

                   GO:0060444   Branching              0.020
                                involved in
                                mammary gland
                                duct
                                morphogenesis
                   GO:0009994   Oocyte                 0.024
                                differentiation
                   GO:0048599   Oocyte                 0.024
                                development
                   GO:0060603   Mammary                0.033
                                gland duct
                                morphogenesis
                   GO:0060135   Maternal               0.037
                                process
                                involved in
                                female pregnancy

Group                 GOID         Gene symbol
                                or representative
                                    public ID

Reproductive       GO:0060126   OTX2, WNT4
  hormone
  synthesis
  and regulation   GO:0021984   OTX2, WNT4

                   GO:0032355   SOCS2, AREGB

Signal             GO:0030514   TOB1, GREM1
  transduction

Biophysical        GO:0048741   SLC23A2, CHAT
  processes
                   GO:0015074   LOC770294,
                                LOC770705, ENS-3
                   GO:0055117   P2RX4, NKX2-5

                   GO:0009409   IL4, SLC27A1
                   GO:0048747   SLC23A2, CHAT

                   GO:0046209   P2RX4, CPS1

                   GO:0007586   PGA5, PRSS2,
                                LOC396365
                   GO:0015849   SLC23A2, OCA2,
                                LOC770309,
                                SLC7A14, SLC27A1
                   GO:0046942   SLC23A2, OCA2,
                                LOC770309,
                                SLC7A14, SLC27A1
                   GO:0055002   SLC23A2, CHAT,
                                TTN, NKX2-5
                   GO:0006942   P2RX4, NKX2-5

                   GO:0002028   NKX2-5, NEDD4L

Reproductive       GO:0060748   WNT4, AR

  biophysical

  processes

                   GO:0060745   WNT4, AR

                   GO:0060562   DEAF1, WNT3,
                                GREM1, WNT4,
                                NKX2-5, HOXA11,
                                AR, AREGB
                   GO:0060444   WNT4, AR, AREGB

                   GO:0009994   WNT4, GDF9

                   GO:0048599   WNT4, GDF9

                   GO:0060603   WNT4, AR,
                                AREGB

                   GO:0060135   WNT4, AR

GOID represents the identifiers, and Term represents term
definitions for Gene Ontology term entities. p: p-value of
significance (Welch t-test).

Table 5. Enriched gene ontology (GO) terms revealed from
identified DE-transcripts according to molecular function
ontology

Group            GOID         Term             p

Signal           GO:0005030   Neurotrophin     0.013
  transduction                receptor
                              activity
                 GO:0001614   Purinergic       0.017
                              nucleotide
                              receptor
                              activity
                 GO:0016502   Nucleotide       0.017
                              receptor
                              activity
                 GO:0043121   Neurotrophin     0.024
                              binding
                 GO:0035586   Purinergic       0.026
                              receptor
                              activity
                 GO:0004888   Transmembrane    0.049
                              signaling
                              receptor
                              activity

Biophysical      GO:0003951   NAD+             0.013
  processes                   kinase
                              activity
                 GO:0005319   Lipid            0.049
                              transporter
                              activity

Group            GOID         Gene symbol or
                              representative
                              public ID

Signal           GO:0005030   NTRK1, NTRK2
  transduction

                 GO:0001614   P2RX4, P2RY2,
                              ENSGALG00000012080

                 GO:0016502   P2RX4, P2RY2,
                              ENSGALG00000012080

                 GO:0043121   NTRK1, NTRK2

                 GO:0035586   P2RX4, P2RY2,
                              ENSGALG00000012080

                 GO:0004888   OXTR, LOC431251,
                              SSTR3, CHRM2, ADRA2B,
                              P2RX4, P2RY2, EDNRB2,
                              GABRB2, GABRG2,
                              LOC428961,
                              NPFFR2, GRIN2B, GRIN3A,
                              NTRK1, NTRK2,
                              EPHB6, DDR2,
                              TMPRSS6, PCSK5, CCKAR,
                              IFNAR2, CSF1R,
                              TLR5, OR10A7,
                              LOC768958, LOC769317,
                              LOC777484, GPR39, GPR97,
                              ENSGALG00000017405,
                              ENSGALG00000017093,
                              ENSGALG00000012080.
Biophysical      GO:0003951   C5orf33, NADK
  processes

                 GO:0005319   ATP11C, ATP8A2,
                              ATP8B3, APOB,
                              LOC769564, SLC27A1

GOID represents the identifiers, and Term represents term
definitions for gene ontology term entities. p: p-value of
significance (Welch t-test).
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Article Details
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Title Annotation:chicken eggshells
Author:Liu, Zhangguo; Zheng, Qi; Zhang, Xueyu; Lu, Lizhi
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Geographic Code:9CHIN
Date:May 1, 2013
Words:8991
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