Relationship between Differential Expression of Estrogen Receptor and Follicle Stimulating Hormone Receptor Genes in Ovary and Heterosis of Egg Number Traits in Chickens*

: In order to understand the molecular mechanism of heterosis of reproduction traits in chickens, we used the quantitative real-time reverse transcriptional polymerase chain reaction (Quantitative real-time RT-PCR) technique to investigate the differential expression of estrogen receptor (ESR) and follicle stimulating hormone receptor (FSHR) genes in 32-week-old ovaries of inbred chickens and their hybrid offspring in 4 x 4 diallel crosses, which involved White Plymouth Rock (E), CAU Brown (D), Silkies (C) and White Leghorn (A). We found that there were significant differences in mRNA expression of ESR and FSHR genes not only between hybrids and their parental lines (p<0.01), but also among different crosses (p<0.01). Furthermore, positive correlations between differential expression of both ESR and FHSR in hybrids and heterosis percentages of 32-week-old and 42-week-old egg number traits were significant at p<0.05. Our results suggested that differential expression of ESR and FSHR genes in the ovaries of inbred chickens and their hybrids could play roles in the formation of heterosis of egg number traits to some extent. ( Key Words : ESR, FHSR, Heterosis, Chicken, Gene Expression)


INTRODUCTION
Heterosis has been exploited in domestic animal production with significant contributions to the world's egg and meat supplies, and study on its genetic basis has been one of the most attractive and difficult studies of life science over the past years.Three hypotheses exist for explaining heterosis: dominance (Davenport, 1908), over dominance (Shull, 1908) and epistasis (Wright, 1951).More recently, some achievements on the molecular mechanism of heterosis have been obtained.With the mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR) technique, many studies demonstrated that there was significant differential gene expression between F1 hybrids and their parents in rice (Cheng et al., 1996;1997), wheat (Sun et al., 1999), maize (Cheng et al., 1997) and chickens (Wang et al., 2005), which was classified into various patterns by comparing gene expression of hybrids to their parents.Furthermore, patterns of differential gene expression were found to be correlated with heterosis percentages of some traits of rice (Xiong et al., 1998), wheat (Xie et al., 2003), maize (Tian et al., 2002;2003) and chicken (Wang et al., 2004;Sun et al., 2005;Sun et al., 2005).However, all of these reports focused only on qualitative differential gene expression of hybrids and cannot completely explain the genetic basis of heterosis, because those traits responsible for significant heterosis appear to be complex quantitative traits that are controlled by multiple minor-effect genes coupled with environmental effects.It is therefore necessary to investigate quantitative differential gene expression between hybrids and their parents and its relationship with heterosis.
Our previous study showed that gene expression altered significantly between inbred chickens and their hybrid offspring in 32-week-old ovaries and classified it into seven patterns of differential gene expression (T1-T7) using the DDRT-PCR method (Wang et al., 2005); the patterns of T3 (over-expression) and T7 (hybrid-specific expressed) were correlated with the heterosis percentages of egg number of 32-week-old and 42-week-old chickens (p<0.01,p<0.01).
Based on these results, we proposed to investigate the relationships between the differentially expressed important candidate genes and heterosis of reproduction traits in chickens.
Steroid hormones perform many essential roles in vertebrates during embryonic development, reproduction, growth, water balance and responses to stress (Sutherland et al., 1998), of which estrogen and follicle stimulating hormone are the most important hormones relating to reproduction.Estrogen plays a key role in regulation of the neuroendocrine system and control of reproductive behavior, and it is essential for normal reproductive activity and has direct actions during sex determination in some vertebrates (Maxwell et al., 1987;Griffin et al., 2001) through activation and binding to its estrogen receptor (ESR) (Griffin et al., 1999;Katsu et al., 2004).So the ESR gene was considered as one of the candidates for litter size and determined to be associated with litter size in pigs (Rothschild et al., 1996;1997).Follicle stimulating hormone (FSH) accelerates the maturation of germ cells and ovulation through stimulating and binding to the FSH receptor (FSHR) (Dankbar, 1995).Also, previous study has indicated that the FSHR gene is related to litter size in pigs (Chen et al., 2002).
In the present study, the quantitative real-time reverse transcriptional polymerase chain reaction (Quantitative real time RT-PCR) technique was used to investigate the differential mRNA expression of ESR and FSHR genes in 32-week-old ovaries between chicken hybrids and their parents in 4x4 diallel crosses, which involved White Plymouth Rock (E), CAU Brown (D), Silkies (C) and White Leghorn (A), and the correlations between differential expression of these two genes and heterosis percentages of 32-week-old and 42-week-old egg number traits were further analyzed.

Anim 지 s
A 4x4 diallel cross was developed using four chicken breeds which were White Leghorn (A), Silkies (C), CAU Brown (D) and White Plymouth Rock (E).Sixteen crosses including a total of 3084 female individuals were obtained, namely, AA, CC, DD, EE, AC, CA, AD, DA, AE, EA, CD, DC, CE, EC, DE and ED, with about 200 (more than 192) females included in each cross.Individual 32-week-old and 42-week-old egg numbers were measured.To ensure that traits of hybrids were measured under the same condition as their parents and to eliminate systematic environmental error, phenotypic values for 32-week-old and 42-week-old egg number traits of four parents (A, C, D and E) were measured using about 200 female offspring from the four respective purebred crosses (AA, CC, DD and EE).The heterosis percentage of egg number traits for the 12 hybrids was calculated as: x 100 Fresh ovaries of eight healthy females were randomly collected from 12 F1 hybrids and four purebred cross at 32 weeks old and stored in liquid nitrogen.

Total RNA extraction and synthesis of cDNA
Total RNA was extracted from each sample using Trizol reagent (Gibco-BRL, Gaithersburg, MD, USA).Minor DNA amongst the total RNA was digested using DNase (Promega, Madison, WI, USA) (Liang and Pardee, 1992).
In the total volume of 20 jil, the PCR mixture contained 12 il the SYBR Green Master Mix (ABI), containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl B2, 20 mM dNTPs and 1 U Taq DNA polymerase, 200 pM of each primer and 4 il cDNA.The PCR mixture was programmed at 50°C for 2 min and then at 95°C for 10 min for the first cycle followed by 40 cycles of 92°C for 15 s, 59°C for 50 s, and 72°C for 50 s, and then extension at 72°C for 7 min.GAPDH was used as housekeeping gene for generation of a

Statistical analysis
Using SAS (v.8.02) software, Duncan's Multiple Range Test was performed to test the differences of mRNA expression of ER and FSHR among the 16 crosses.In addition, correlations were calculated betwen the mRNA level of ESR or FSHR gene of each hybrid, as X variable, and heterosis percentage of each trait of each cross, as Y variable.

Heterosis percentage of egg number traits
The heterosis percentages of 32-week-old and 42-weekold egg number traits of 12 F1 hybrids were calculated and are listed in Table 1.Results indicated that each hybrid showed significant heterosis for 32-week-old and 42-weekold egg number traits, suggesting the experimental chickens employed in this study were suitable for investigation of the genetic basis of heterosis for reproduction traits.

Differenti이 expression of ESR and FSHR between crosses
To determine if there were differences in mRNA expression of ESR and FSHR between F1 hybrids and their parents, quantitative RT-PCR analysis was employed (Figures 1 and 2).After normalization with the corresponding GAPDH mRNA level of each sample followed by Duncan's Multiple Range Test, as expected, we found there were significant differences of ESR expression between hybrids and their parents (DC, CD vs. DD, CC; EC, CE vs. EE, CC; AE, EA vs. AA, EE; CA, AC vs. CC, AA; AD, DA vs. AA, DD; DE, ED vs. DD, EE), eg., the mRNA level of ESR in DC of 0.948 was higher than that of its parents DD and CC at 0.931 and 0.88 (p<0.01),respectively.Also, significant differences of ESR mRNA expression were observed among some crosses.The mRNA level of ESR in combinations DD, DA and EE was significantly different from all other hybrids (p<0.01).There was no difference in ESR expression in combinations DD, DE and AA (p>0.05), but these were significantly different from crosses DC, CE,CA, AD, AC, EA, ED, CD, CC, DA and EE (p<0.01).
Table 2 shows all the comparisons of mRNA levels of ESR in various crosses.
Similarly, the FSHR gene was significantly differentially expressed not only between hybrids and their parents (p<0.01),but also among some crosses (p<0.01,Table 2).
These findings could be related to the different degree of heterosis for 32-week-old and 42-week-old egg number traits among different hybrids.

Correlations between differential expression of ESR and FSHR and heterosis percentages
Based on the results of mRNA expression levels of ESR and FSHR, the differential expression of these two genes between each hybrid and their parents was calculated through subtracting the mean expression level of parent from that of the hybrid.Having obtained the differential expression of ESR and FSHR genes of 12 F1 hybrids, then correlations were analyzed between the differential expression of ESR gene of hybrids and heterosis percentages of 32 week-old and 42 week-old egg number traits as well as between the differential expression of FSHR gene and heterosis percentages of these two reproduction traits.Correlation coefficients are presented in Table 3. Statistical analysis of the results showed that positive correlations between the differential expression of both ESR and FSHR genes and heterosis percentages of 32-week and 42-week old egg number traits were significant at p<0.05.

DISCUSSION
In the present study, we first demonstrated that ESR and FSHR were differentially expressed in the ovary between chicken hybrids and their parents using the quantitative real-time RT-PCR technique (p<0.01).The results of statistical tests showed that positive correlations between the differential expression of both ESR and FSHR genes and heterosis percentages of 32-week and 42-week old egg number traits were significant at p<0.05.Our finding is coincident with previous reports in rice, wheat and chickens, which showed that there was differential gene expression between F1 hybrids and their parents (Cheng et al., 1996;1997;Sun et al., 1999;Xiong et al., 1998;Tian et al., 2002;2003;Xie et al., 2003;Wang et al., 2004;Sun et al., 2005;Wang et al., 2005).Taken together, it may be concluded that it is the differences of gene expression that result in the heterosis of hybrids.Although all the genes in hybrid F1 were derived from their parental lines, the hybrid's genetic performance is not simply the additive product of genetic material from both parents, but the result of variations in quantitative and qualitative expression of two sets of genes within the hybrid by interactions resulting in the occurrence of heterosis.
The FSH receptor belongs to the family of G proteincoupled receptors, complex transmembrane proteins characterized by seven hydrophobic hrlices inserted in the plasmalemma and by intracellular and extracellular domains of variable dimensions depending on the type of ligand.The intracellular portion of the FSH receptor is coupled to a Gs protein and, upon receptor activation by the hormonal interaction with the extracellular domain, initiates the cascade of events that ultimately leads to the specific biological effects of the gonadotropin.
Correlation analysis showed that there was significant correlation between the differential expression of ESR and FSHR and heterosis percentages of 32-week and 42-week old egg number traits (p<0.05).It is suggested that the differential expression of ESR and FSHR genes between hybrids and their parents is related to the formation of heterosis of egg number traits, at least partly explaining its genetic mechanism.This could be due to the important roles of ESR and FSH in the regulation of ovulation rate and follicle development through activation and binding to their receptor, ESR and FSHR, respectively.In addition, our finding indicated that ESR and FSHR genes were over expressed in some hybrids than in their parents, which was consistent with our previous research indicating the gene differential expression patterns of T3 (over-expression) was correlated with the heterosis percentages of egg number of 32-week and 42-week old chickens at p<0.01 and p<0.01, respectively (Wang et al., 2005).

Table 3 .
Correlation coefficients between differential expression of ESR and FSHR and heterosis percentages of egg number traits * p<0.05.

Table 2 .
mRNA expression of ESR and FSHR in various crosses (Concentration of cDNA: copy numbers/ml)