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The Expression of Pituitary Gland Genes in Laying Geese¹

C. F. Yen^*, H. W. Lin^*, J. C. Hsu, C. Lin^*, T. F. Shen^* and S. T. Ding^*,2

^* Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan; and Department of Animal Science, National Chung Hsing University, Taichung 402, Taiwan

² Corresponding author: sding{at}ccms.ntu.edu.tw

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The purpose of this study was to detect differential expression of genes in the pituitary gland in laying geese by suppression subtractive hybridization (SSH). Pituitary glands from prelaying and laying geese were dissected for mRNA extraction. The cDNA from pituitary glands of prelaying geese was subtracted from the cDNA from the pituitary glands of laying geese (forward subtraction); the reverse subtraction was also performed. We screened 384 clones with possible differentially expressed gene fragments by differential screening. Sixty-five clones from the differential screening results were subjected to gene sequencing and further analysis. We found that at least 19 genes were highly expressed in the pituitary glands of laying geese compared with prelaying geese. Among these, 6 genes (including 4 novel genes) were confirmed by virtual Northern analysis. We found that prolactin and visinin-like protein were highly expressed in the pituitary glands of laying geese compared with prelaying geese (P < 0.05). Further investigation is needed to demonstrate specific functions of the novel genes discovered in the current study.

Key Words: laying geese • pituitary gland • prolactin

	INTRODUCTION

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The pituitary gland secretes several proteins involved in the function of egg laying in birds. For instance, luteinizing hormone and follicle-stimulating hormone are 2 of the most important hormones involved in regulating ovulation (Scanes et al., 1977; Johnson and van Tienhoven, 1980; Wilson et al., 1983). Prolactin is secreted from the anterior pituitary at a high level at the onset of egg laying in chickens and Japanese quail (Sharp et al., 1979; Goldsmith and Hall, 1980).

Pituitary gene expression during chicken embryonic development is well characterized by cDNA microarray (Ellestad et al., 2006). Growth hormone and thyroid-stimulating hormone ß-subunit mRNA increase as embryonic development proceeds (Ellestad et al., 2006). However, gene expression data for laying birds are lacking.

The goose requires a short lighting period for reproduction. With stimulation from a short lighting program, mature geese start ovulation and oviposition (Wang et al., 2005). The lighting program can be used to modulate the egg-laying period in geese (Wang et al., 2005). Although considerable information on the expression of genes in the pituitary of the chicken is available (Carre et al., 2006; Ellestad et al., 2006), the goose is not well studied. Understanding gene expression in the pituitary glands of laying geese is the first step toward improving the low laying performance in geese. Therefore, the purpose of this study was to detect differentially expressed genes in the pituitary gland of laying geese by suppression subtractive hybridization (SSH).

	MATERIALS AND METHODS

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Birds and Diets
The animal protocol used in the present experiment was approved by the Animal Care and Use Committee of the National Chung Hsing University. The geese (6 per group) were purchased from a commercial goose farm and were raised according to the standard program used at the farm. The prelaying geese were killed at 5 mo of age (average BW = 4.2 ± 0.6 kg). The laying geese were killed at 17 mo of age (average BW = 4.2 ± 0.4 kg). Geese were killed by electrical stunning followed by exsanguination. Tissue samples were rapidly removed, wrapped in foil, frozen in liquid nitrogen, and then stored at – 70 ° C until analysis.

SSH
The SSH procedure utilized the PCR Select Kit (Clontech, Palo Alto, CA), as previously described (Wang et al., 2006). In brief, tester DNA (cDNA from pituitary glands of laying geese) was divided into 2 groups and ligated with adaptor 1 (tester 1 DNA) or adaptor 2 (tester 2 DNA), respectively. The driver DNA (cDNA from pituitary glands of prelaying geese) was not ligated with any adaptor. Tester 1 or tester 2 DNA was denatured at 95 ° C for 10 min and hybridized with denatured driver DNA in separate tubes. After hybridization, any single-stranded DNA with adaptor 1 or adaptor 2 represented genes expressed specifically in pituitary glands of laying geese, but not in pituitary glands of prelaying geese, whereas the single-stranded DNA without adaptors represented genes expressed in pituitary glands of prelaying geese, but not in pituitary glands of laying geese. The resulting 2 populations were pooled for a second hybridization with fresh denatured drivers. The resulting molecules with both adaptor 1 and 2 represent gene sequences preferentially expressed in pituitary glands of laying geese. The differentially expressed gene fragments were then cloned into pGEM-T Easy TA cloning vector (Promega, Madison, WI). We selected 384 clones for further differential screening, sequencing using an ABI 3730 genetic analyzer (Applied Biosystems, Foster City, CA), and virtual Northern analysis to confirm the differential expression of genes between pituitary glands of laying geese and pituitary glands of pre-laying geese.

Differential Screening
The differential screening procedure followed the manufacturer’s instructions in the PCR-Select Differential Screening Kit user manual (Clontech). Details of the screening procedure were as described by Wang et al. (2006). This procedure was used to eliminate false-positive clones.

Transcript Analysis
Total RNA was extracted from the goose pituitary gland by the guanidinium-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987) with modifications (Wang et al., 2004). The virtual Northern analysis was performed for determining the concentrations of the transcripts of interest. The procedure followed the manufacturer’s instructions (Clontech). In short, the membrane was prehybridized at 42 ° C in UltraHyb (Ambion, Cambridgeshire, UK) for 1 h, and then the denatured cDNA probe (95 ° C for 5 min) was added at a concentration of 1 pmol to hybridize with the targeted genes overnight at 42 ° C. The ß-actin probe sequence was from a chicken gene fragment (GenBank Accession no. NM_205518 [GenBank] , nucleotides 522–672). The probe sequences of the other genes were generated from the current study (Table 1). Hybridization results were quantified by using a Typhoon 9200 Phosphorimager with ImageQuant software (GE Healthcare, Livingston, NJ) as previously described (Ding et al., 2004). The densitometric value for an individual transcript in a sample lane was normalized to the densitometric value for ß-actin in the same lane.

	RESULTS AND DISCUSSION

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Three hundred eighty-four clones of gene fragments were subjected to differential screening to reduce false-positive clones. After differential screening, 65 clones were confirmed to be differentially expressed between prelaying and laying geese. Sequences of these gene fragments showed that there were at least 19 genes highly expressed in the pituitary glands of laying geese compared with prelaying geese (Table 1). Six of these genes, including 4 novel genes, were confirmed by virtual Northern analysis (Figure 1). Limited genes were selected for further study due to limitation of resources (pituitary mRNA) and based on their importance in reproduction function.

View larger version (22K): [in this window] [in a new window]   Figure 1. Differential expression of genes in the pituitary gland in laying geese (Lay) compared with prelaying geese (Prelay). On the day of sampling, 6 geese from each group were killed and pituitary glands were dissected for RNA extraction. The mRNA concentrations of prolactin, visinin-like protein (VILIP), and 4 pituitary-expressed unknown genes (PEUG 1 to 4) were determined by virtual Northern analysis. The mRNA values were normalized to ß-actin content and expressed in arbitrary units (AU). Bars in panel A indicate means with SD. *Denotes a significant difference between the groups (P < 0.05).

Visinin-like protein (VILIP) was highly expressed in the pituitary glands of laying geese compared with prelaying geese (Figure 1; P < 0.05). This is the first report of VILIP expression in the pituitary of avian species. The partial sequence of the goose VILIP was highly homologous to that of chicken (97%, Lenz et al., 1992; GenBank accession no. NM_205255 [GenBank] ) and human (81%, GenBank accession no. NM_003385 [GenBank] ). The VILIP belongs to the superfamily of calcium sensor proteins. They are involved in modulation of the activity of the acetylcholine receptor (Lin et al., 2002), mitogen-activated protein kinase signaling pathway (Spilker et al., 2002), and cyclic adenosine monophosphate functions (Mahloogi et al., 2003; Gonzalez-Guerrico et al., 2005). Calcium metabolism is altered upon the change of lighting program in laying hens (Parsons and Combs, 1981). The calcium sensor protein VILIP may be involved in regulating such calcium flux through guanylate cyclase (Lambrecht and Koch, 1991). Therefore, high expression of VILIP in the laying goose pituitary may be involved in regulating functions in aforementioned pathways, or it may be a direct result of photostimulation in the laying goose.

The function of the novel genes (PEUG 1 to 4) is not known, but they were highly expressed in the pituitary gland of the laying goose, suggesting the possible involvement of these genes in goose reproduction. Further investigation is needed to demonstrate specific functions of the novel genes discovered in the current study.

In conclusion, we have demonstrated, using an SSH method, that there are a number of genes specifically expressed in the pituitary gland of the laying goose compared with the prelaying goose. We are the first to demonstrate the expression of goose prolactin, VILIP, and 4 novel genes in the laying goose pituitary. Further demonstration of the functions of genes discovered in the current study will add great value to the understanding of the reproductive biology of the goose.

	ACKNOWLEDGMENTS

 
The authors thank Harry Mersmann for his editorial assistance.

	FOOTNOTES

 
¹ This work was supported by the Council of Agriculture in Taiwan.

Received for publication June 7, 2006. Accepted for publication July 26, 2006.

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