Abstract: The expression of IGF2 and IGFBP3 mRNA in the heart, liver, lung, kidney, spleen, intestine muscle and stomach of wuzhishan pigs from 30-150 days of age was investigated with fluorescent quantitative PCR assay. Results showed that IGF2 expression showed a significant reduction trend (p<0.05) in all of 8 tissues from the highest level at 30 days of age to the lowest at 150 days of age; IGFBP3 expression showed a similar reduction trend among 6 tissues (heart, lung, spleen, kidney, intestine muscle and stomach) from 30-150 days of age while a trend of increase during early development (until 90 days of age in liver or until 60 days in skeletal muscle) then decrease thereafter was seen with lowest at 150 days of age.

INTRODUCTION

Insulin-like growth factors (IGFs including IGF1 and IGF2) are multifunctional cell proliferation regulators named after their structural resemblance to insulin. Studies have shown that IGFs precisely regulate skeletal muscles development, myofibril generation and hypertrophy (Barton, 2006; Rosenblatt et al., 1994; Scandinavica, 1999; Florini et al., 1991a, b). IGF2 is composed of 67 amino acid residues. It plays an very important regulatory functions in fetal development, tumor proliferation and muscle growth (De Chiara et al., 1991; Gerrard et al., 1998; Florini et al., 1986).

In myocyte culture, IGF2 is required for the differentiation of satellite cells (Florini et al., 1991a, b). Because of its essential function, IGF2 has been a focus of research for a long time. However, the mechanism by which it functions is still unclear (Erbay et al., 2003). IGF2 functions by binding with IGF2 receptor (IGFR). Few IGFs exist unbound in plasma. Most IGFs (97%) are compounded with one of the 6 IGF binding proteins (IGFBP1-6). These IGFBPs serve as an IGF reservoir, increase its half life and regulate its activities (Neely and Rosenfeld, 1992). Among the 6 IGFBPs, IGFBP3 is the predominant binding protein.

The wuzhishan pig breed is endangered, precious and lightest pig breed in China. It has high tolerance, stable inheritance, low metabolic rate, thin skin and backfat. Because its anatomy, physiology and pathology are similar to humans, wuzhishan pigs have been used as a model in pharmacological, veterinary and comparative medicine studies (Haitao and Hong, 2007). Therefore, it is very important for the development of human society to understand the special gene expression and developmental changes. The ontogeny of IGF2 and IGFBP3 has investigated in 8 tissues of wuzhishan pigs with real time quantitative PCR method in order to provide new data for further research of the molecular mechanism of developmental regulation.

MATERIALS AND METHODS

Materials: Wuzhishan pigs were obtained from China Tropical Agriculture Research Institute. Ten dams which had delivered 2 or 3 L before were selected for pure breeding and their 3rd or 4th L was used in this study. The piglets were raised with conventional standard and weaned 30 days old. Males were castrated 5 day old. Three castrated males and three females were sacrificed at 30, 60, 90, 120 and 150 days of age. Heart, liver, lung, spleen, kidney, muscle, stomach and small intestine were collected and frozen briefly in liquid nitrogen and stored at -80°C.

Major equipment and reagent: Maxwell 16 total RNA purification kit and AMVRTase were purchased from Promega (USA). SYBR PremixEx Taq was purchased from Dalian Bao Biotech Co. Nucleic acid extraction device (Maxwell16) was from Promega. PCR equipment was from Biometra (Germany). Gel image system 120 was from Kodak.

Quantitative PCR
Total RNA extraction and reverse transcription: Total RNA was extracted according to the instruction of the kit manufacturer examined with agarose gel electrophoresis. The concentration was determined with ultraviolet spectrophotometer and diluted to 1 μg μL^-1.

Table 1:	Real time RT-PCR primer information
ACTB (β-actin) was the internal reference

Reverse transcription: Reaction mix containing 1 μL of total RNA, 1 μL of oligo (dT) 18 (50 μmol L^-1), 5 μL of dd H₂O (RNase free) was incubated at 70°C for 10 min chilled rapidly on ice. Then, 1 μL of 5X buffer (containing Mg²⁺), 5 μL of 10 mmol L^-1 dNTP, 1 μL of Rnase inhibitor (40 U μL^-1) 1 μL of M-MLV RTase (200 U μL^-1), 2 μL of dd H₂O (RNase-free) were added to the mix which was subsequently incubated at 42°C for 60 min followed by inactivation for 5 min at 99°C. Finally the reaction mix was chilled on ice and stored at -20°C until use.

Primer design: Primers were designed using Primer 3.0 (http://frodo.wi.mit.edu/) and synthesized by Dalian Bao Biotech Co. Relevant information was shown in Table 1 (Erkens et al., 2006; Tilley et al., 2007).

Real time PCR: Reactions were performed with Reaplex⁴ quantitative PCR equipment (Eppendorf). The reaction mix (25 μL) contained 12.5 μL of SYBR Premix ExTaq, 1 μL of primers (10 μmol L^-1), 2 μL of DNA template, 9.5 μL of ddH₂O. The thermal cycle parameter was as the following: preheat at 95°C for 30 sec; 45 cycles of denaturation at 95°C for 5 sec; annealing/extension 20 sec (Table 1) and extension at 72°C for 15 sec. During melting curve analysis, temperature was gradually raised from 60-95°C at a rate of 0.875°C/30 sec. Each time, internal reference and unknown samples were assessed with triplicates and one No-Template Control (NTC) was used.

Data analysis: Relative quantification method was used to measure mRNA quantity and ACTB served as an internal reference as described by Winer et al. (1999). The expression levels at 30 days were set as one unit of expression. Variance analysis was used to test significance of difference. Multiple comparisons were done with SAS 9.01 software.

RESULTS AND DISCUSSION

Expression character of IGF2 mRNA: As shown in Table 2, levels of IGF2 mRNA decreased in each tissue during animals developing. The highest mRNA levels were observed at 90 days of age in each tissue while the lowest were observed at 150 days of age. The differences were very significant (p<0.01). Levels of IGF2 mRNA in heart, liver, lung, spleen and kidney were higher at 30, 60 and 90 days of age than at 120 and 150 days of age (p<0.01). No significant differences were seen in IGF2 mRNA levels between 120 and 150 days of age (p>0.05). At 150 days of age, liver and lung mRNA levels were significantly reduced comparing with those at 120 days of age. In muscle and stomach, the levels at 30 and 60 days of age were significantly higher than those at 90, 120 and 150 days of age (p<0.01).

The developmental changes of IGF2 mRNA levels were shown in Fig. 1. Results showed that the patterns of IGF2 expression were similar among eight tissues. The mRNA levels were high during early development and gradually decreased. The tendency of reduction was the smallest in liver and greatest in spleen.

Expression character of IGFBP3 mRNA: Levels of IGFBP3 mRNA were shown in Table 3. Results showed that IGFBP3 mRNA levels showed a trend of reduction in heart, lung, spleen, kidney, intestine and stomach as the pigs grew older. The highest IGFBP3 mRNA levels were seen at 30 days of age and the lowest were seen at 150 days of age. The differences between the highest and lowest were statistically significant (p<0.01). In the liver, IGFBP3 mRNA level increased from 30-90 days of age then declined to the lowest at 150 days of age. In muscle, IGFBP3 mRNA level was highest at 60 days of age and lowest at 150 days of age. The difference was not statistically significant between 30 and 60 days of age but very significant among 90, 120 and 150 days of age (p<0.01). Developmental changes of IGFBP3 mRNA levels were shown in Fig. 2. Levels of IGFBP3 mRNA exhibited similar trend among heart, lung, spleen, kidney and stomach: high during early stage then decrease continuously.

The levels in muscle and liver were similarly increased during early stage until 60 or 90 days of age then started to decrease gradually to the lowest at 150 days of age. IGF2 is a typical paternal imprinting gene regulating animal growth. Studies have shown that loss of paternal IGF2 expression resulted in the reduction in body weight by 40% (De Chiara et al., 1991). Other reports showed that IGF2 is associated with growth rate and backfat thickness(Jeon et al., 1999; Knoll et al., 2000).

Table 2:	IGF2 mRNA levels in 8 tissues of wuzhishan pig

Table 3:	Levels of IGFBP3 mRNA in 7 tissues of wuzhishan pigs

Fig. 1:	Developmental changes of IGE3 mRNA in 8 tissues of wuzhishan pigs

Fig. 2:	Ontogeny of IGE3 mRNA in 7 pig tissues

Wuzhishan pig is the smallest breed among the medium sized pigs that can be used in medical studies (Haitao and Hong, 2007). Therefore, an understanding of the relationship between IGF2 expression and pig growth is important for veterinary and comparative medicine and human health. Tilley et al. (2007) studied IGF2 expression in muscle tissue of swine fetuses at 45, 65 and 100 days of pregnancy. They demonstrated that IGF2 mRNA level decreased from 65-100 days of pregnancy (Tilley et al., 2007). Gerrard et al. (1998) studied IGF2 expression in skeletal muscle in fetus (30, 44, 59, 68, 75, 89 and 109 days of pregnancy) and young pig (21 days and 6 months of age). They showed that fetal IGF2 mRNA reached a peak level at 59 days of pregnancy and decreased thereafter (Gerrard et al., 1998). Tang studied IGF2 mRNA levels in Tongcheng and large white fetuses at 30, 65 and 90 days of pregnancy. They found that skeletal muscle IGF2 mRNA levels were the highest at 65 days of pregnancy (Zhonglin et al., 2008). These studies demonstrated that IGF2 is a fetal growth factor participating in early physiological process.

IGF2 mRNA level reduces during postnatal development in other pig breeds. This trend is a similar in wuzhishan pigs. However, the prenatal IGF2 expression in wuzhishan pigs requires further study. Guo et al. (2008) determined IGF2 mRNA in large white and Taihu pigs of 30, 60, 90 120 and 150 days of age. They discovered that IGF2 mRNA level in large white was the highest at 30 days of age (p<0.01).

After that the level decreased, reaching the lowest at 120 days of age (Guo et al., 2008). Stinckens et al. (2007) investigated developmental changes of IGF2 mRNA in muscles tissue of Landrace at 4, 8, 16 and 26 weeks of age. Pigs of all genotype showed a decrease in IGF2 mRNA level (Stinckens et al., 2007). These reports are consistent with the results. The study has also shown the temporal reduction of IGF2 mRNA in heart, liver, lung, spleen, kidney, muscle, small intestine and stomach. The levels are different among different tissue. The liver IGF2 mRNA was most abundant all the time suggesting that liver is the major IGF2 synthesis organ. This result is similar to those of Peng et al. (1998).

CONCLUSION

IGFs exert their biological action through binding with their respective receptors. The biological availability of IGFs is modulated by IGFBPs. The expression of IGF2 and IGFBP3 were coordinated in most tissues of wuzhishan pig, suggesting that IGFBP3 expression affect development through an IGF2-dependent mechanism. IGFBP3 expression has similar tissue differences with the liver expressing the highest amount all the time. Other studies showed similar results (Bar et al., 1987; Dewu et al., 2008).

Different from IGF2 expression, hepatic IGFBP3 was the highest at 90 days of age while muscle IGFBP3 peaked at 60 days of age. Mohan and Baylink (2002) reported that IGFBP3 was abundant in infant peaked in adolescent and decreased thereafter (Mohan and Baylink, 2002). Guo et al. (2008) showed the highest level of IGFBP3 appeared in liver at 90 days of age in Lantang pigs. These studies indicate that IGFBP3 expression may peak after birth suggesting that IGFBP3 may have other functions in addition to binding with IGF2. Whether it is related to development awaits further study.

ACKNOWLEDGEMENTS

This research was supported by Basic Commonweal Research Fund for National Academic Institution of Chinese Academy of Tropical Agricultural (grant PZS-013) and Communion Platform Constructional Project for Livestock and Poultry Germplasm Resource (grant 2005DKA21101).