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Effects of Daidzein on Messenger Ribonucleic Acid Expression of Gonadotropin Receptors in Chicken Ovarian Follicles

College of Animal Sciences, Zhejiang University, Hangzhou 310029, P.R. China

¹ Corresponding author: cqzhang{at}zju.edu.cn

	ABSTRACT

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Effects of daidzein on expression of mRNA of gonadotropin receptors [follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR)] were evaluated in ovarian follicles of ISA laying hens that were 13 mo old in the postpeak period of egg laying. The hens were randomly allocated as control and daidzein-treated groups, with daidzein supplemented to the basal diet at the level of 10 mg/kg for 7 wk. The granulosa layers of preovulatory follicles (F1, F2, F3, F4, F5) and follicular layers of the small yellow follicle (SYF), large white follicle (LWF), and atretic follicle were collected. The mRNA expression of related genes was measured by semiquantitative reverse transcription PCR. Results showed that daidzein significantly increased the egg-laying rate (P < 0.05) and the number of SYF and LWF (P < 0.05). The relative abundance of the FSHR mRNA decreased in the granulosa layers from F5 to F1, but LHR mRNA displayed the opposite trend in developmental changes. Treatment with daidzein resulted in increased expression of FSHR mRNA in LWF, SYF, and granulosa layers of F4 to F2 and LHR mRNA in granulosa layers of F4 and F1 (P < 0.05). These results indicated that dietary supplementation of daidzein upregulated mRNA expression of gonadotropin receptors to improve follicle development in chicken developing follicles and laying performance after the peak laying period.

Key Words: gonadotropin receptor • daidzein • follicle • chicken

	INTRODUCTION

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In birds, estrogen is proposed to be a pivotal factor involved in the development of sexual differentiation, female secondary sexual characteristics, and vitellogenesis. Phytoestrogens can bind to the estrogen receptor to induce estrogen-like effects in animals, humans, and cultured cells (Hong et al., 2004; Liu et al., 2006). Daidzein belongs to the most common group of phytoestrogens found in plants such as soybeans, clover, and bluegrass and has been studied extensively for possible beneficial biological activities, including estrogen-like and estrogen-independent effects (Payne et al., 2001; Liu et al., 2006). Feeding daidzein to Shaoxing ducks or white silky fowls significantly improved the laying performance (Zhao et al., 2004; Liu et al., 2007). However, the information about the influence of daidzein on reproductive performance of domestic animals is relatively scarce.

The growth and development of ovarian follicles undergo a series of complex biochemical and physiological changes, which include gonadotropin receptor expression, steroid biosynthesis, cell proliferation, and differentiation. Among these changes, the expression of gonadotropin receptors [follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR)] plays very important roles in inducing follicular development (Zhang et al., 1997). Our previous studies revealed that daidzein stimulated germ cell proliferation in embryonic chickens through estrogenic and antioxidant actions (Liu et al., 2006; Mi et al., 2007). The objective of this study was to investigate the effects of daidzein on laying performance, follicle development, and mRNA expression of gonadotropin receptors in laying hens.

	MATERIALS AND METHODS

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Birds

The ISA hens (13 mo old) in the postpeak period of egg laying were randomly allocated as control and daidzein (Xuanhua Chemical Plant, Zhangjiakou, China)-treated groups (n = 8), with daidzein supplemented to the basal diet at the level of 10 mg/kg continuously for a period of 7 wk. Hens were given free access to water and feed under natural light cycles. The egg-laying rate was determined as follows: egg-laying rate = total number of eggs/(total number of hens x experiment days).

Collection of Ovarian Follicles

The hens were killed by cervical bleeding postanesthesia (thiopental, 90 mg/kg i.m.). The granulosa layers of preovulatory follicles (PRF; F5 to F1, >10 mm) and atretic follicles were separated and snap-frozen in liquid N. Whole small yellow follicles (SYF; 6 to 8 mm) and large white follicles (LWF; 2 to 4 mm) were frozen after removal of the yolk.

Total RNA Isolation

Total RNA of granulosa layers of F5 to F1, the follicular layers of SYF and LWF, were extracted by Trizol reagent (Gibco BRL, Grand Island, NY) according to the instructions of the manufacturer. Ribonucleic acid concentration and purity were determined with a spectrophotometer by calculating the ratio of optical density at 260 and 280 nm. The ratios were from 1.8 to 2.0.

Reverse Transcription and PCR

Total RNA (1 µg) was denatured at 70°C for 5 min with 0.5 µg oligo (deoxythymidine)₁₈ primer (BioSynthesis, Lewisville, TX) and reverse-transcribed with 200 units of Moloney murine leukemia virus reverse transcriptase (Gibco BRL) in a 20-µL reaction. The reaction mix was incubated for 60 min at 42°C and inactivated by heating at 70°C for 15 min. Two microliters of the reverse-transcribed product was subsequently used for PCR amplification. For PCR, 0.5 µM specific primers for respective target genes and 0.5 units of Taq DNA polymerase (Promega, Madison, WI) in a total volume of 50 µL were used. For PCR amplifications, the following primer pairs were designed according to the chicken mRNA sequences (synthesized by Shanghai Sangon Co. Ltd., Shanghai, China). Primers for FSHR: 5'-AGAAGGCCAACAACCTCGTG-3' and 5'-ACAGCAATGGCTAGGATAGGT-3' (521 bp); LHR: 5'-CTCAGGCGGATACACAACGA-3' and 5'-TCA-GAACAGCTTCCAGCAGG-3' (193 bp); β-actin: 5'-ACGTCGCACTGGATTTCGAG-3' and 5'-TGTCAGCAATGCCAGGGTAC-3' (282 bp). Each target gene was coamplified with β-actin in the same reaction. By adjusting the ratio of β-actin primers to those of the target genes, the overall PCR amplification efficiency of β-actin can be adjusted to the level comparable to the target genes. All samples were included in the same run of RT-PCR with at least 3 repeats.

Quantitation of PCR Products

The PCR product (10 µL) was analyzed by electrophoresis on 1.2% agarose gel. The gel was stained with ethidium bromide. The net intensities of individual bands were measured using Image Master VDS Software (Pharmacia Biotech, Uppsala, Sweden). The ratios of the intensities of target genes to β-actin represented the relative mRNA levels of the target genes. The average level of 3 repeats was used for statistical analysis.

Statistical Analysis

All data were expressed as the means ± SD and analyzed by ANOVA and Duncan’s multiple range tests using the SAS 9.0 software (SAS Institute Inc., Cary, NC). A value of P < 0.05 was considered significantly different.

	RESULTS

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Effects of Daidzein on Egg-Laying Performance

As shown in Table 1, the egg-laying rate in the daidzein-treated group increased by 11.86% (P < 0.05) compared with the control over the experimental period of 7 wk. An increase trend in mean egg weight was observed, but there was no significant difference between the control and daidzein-treated hens.

There was no significant difference in the number of PRF between the control and daidzein-treated hens, but there was a marked increase in the number of SYF and LWF after daidzein treatment over the experimental period of 7 wk (Figure 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. Effects of daidzein (DAI) on follicle development in laying hens. Values are the mean ± SD. a–eBars with different letters are statistically different (P < 0.05; n = 8). LWF = large white follicle; SYF = small yellow follicle; PRF = preovulatory follicle; ATF = atretic follicle.

In the granulosa layers of the PRF, relative abundance of FSHR mRNA expression showed the highest level in the F5, decreased in the granulosa layers of F4 and F3, and remained low in F2 and F1. Meanwhile, the FSHR mRNA level of LWF was lower compared with that of SYF. After treatment with daidzein supplemented in the feedstuff, the FSHR mRNA manifested an increased expression in all follicles except the F5 and F1 follicles (Figures 2A and 3; P < 0.05).

View larger version (45K): [in this window] [in a new window]   Figure 2. Effects of daidzein (DAI) on expression of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) mRNA in ovarian follicles of laying hens. Panels A and B represent electrophoresis of reverse transcription-PCR products for FSHR and LHR vs. β-actin mRNA, respectively. LW = large white follicle; SY = small yellow follicle; F5 to F1 = granulosa layers of F5 to F1.

View larger version (11K): [in this window] [in a new window]   Figure 3. Effects of daidzein (DAI) on expression of follicle-stimulating hormone receptor (FSHR) mRNA in ovarian preovulatory (A) and prehierarchical follicles (B) of laying hens. The FSHR mRNA levels are expressed as arbitrary units relative to β-actin mRNA. Values are the mean SD. a–fBars with different letters are statistically different (P < 0.05; n = 4). LWF = large white follicle; SYF = small yellow follicle; F5 to F1 = granulosa layers of F5 to F1.

In contrast to FSHR mRNA expression in the granulosa layers of PRF, LHR mRNA abundance showed the lowest level in the granulosa layer of F5, increased in the granulosa layers of F4 to F2, and increased markedly in the granulosa layer of F1 to reach the highest expression. Meanwhile, the LHR mRNA abundance in LWF was lower compared with that of SYF. In the daidzein-treated group, LHR mRNA expression in the granulosa layers of F1 and F4 was significantly higher than that of the control group (P < 0.05). However, no obvious changes of LHR mRNA level were found in daidzein-treated SYF and LWF follicles, compared with the control (Figures 2B and Figure 4).

View larger version (12K): [in this window] [in a new window]   Figure 4. Effects of daidzein (DAI) on expression of luteinizing hormone receptor (LHR) mRNA in ovarian preovulatory (A) and prehierarchical follicles (B) follicles of laying hens. The LHR mRNA levels are expressed as arbitrary units relative to β-actin mRNA. Values are the mean ± SD. a–fBars with different superscripts are statistically different (P < 0.05; n = 4). LWF = large white follicle; SYF = small yellow follicle; F5 to F1 = granulosa layers of F5 to F1.

	DISCUSSION

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Gonadotropins are the primary regulators of follicular growth and ovulation. Follicle-stimulating hormone is responsible for follicular recruitment and growth of the smaller follicles. The primary target for luteinizing hormone is the granulosa layer of the larger PRF (Calvo and Bahr, 1983). Gonadotropins exert their actions by binding to specific G protein-coupled gonadotropin receptors. Many studies on FSHR and LHR gene expression have been conducted in mammals, but relatively less work in avian species has been reported. In the present study, we found that the highest expression of FSHR mRNA in the granulosa layer of F5, the intermediate expression in F4 to F3, and the lowest expression in F2 and F1. The FSHR mRNA level of SYF was higher than that of LWF. These results were consistent with the reports that the granulosa layer of SYF and F6 to F3 are the primary targets for FSH (Calvo and Bahr, 1983; Zhang et al., 1997). The marked expression of LHR mRNA in the granulosa layer of F1 to F3 and lower expression in the F4 to F5 may be important for LHR protein synthesis and subsequently increase progesterone production in F1 destined for ovulation. The LHR mRNA level of SYF was higher than that of LWF and the granulosa layer of F5. These results were in agreement with the reports that luteinizing hormone promotes progesterone secretion by the granulosa cells of F3 to F1 as the follicles approach ovulation (Hammond et al., 1981). Meanwhile, expression of FSHR and LHR mRNA in daidzein-treated groups was increased, which suggests that daidzein upregulated the expression of FSHR and LHR mRNA in the partial prehierarchical follicles and also the granulosa layer of PRF. Estrogen stimulates the proliferation of granulosa cells in follicles and the expression of gonadotropin and serves to facilitate the actions of them (Richards, 1980; Ing and Tornesi, 1997; Dickey and Swanson, 1998). Furthermore, estrogen synergizes with follicle-stimulating hormone and exogenous cyclic adenosine monophosphate to increase the number of FSHR (Richards, 1980). Moreover, our studies showed that daidzein increased the P450 aromatase expression in immature follicles before selection in the laying ISA hens (data not shown). According to these studies, the effect of daidzein on the upregulated expression of FSHR and LHR mRNA might be through the direct estrogenic actions.

In conclusion, this study revealed the developmental stage-related difference in mRNA expression of gonadotropin receptors in chicken ovarian follicles. Moreover, dietary supplementation of daidzein improved laying performance and the follicle development via stage-related upregulated mRNA expression of gonadotropin receptors. These results indicated that daidzein, as a dietary supplement, may promote laying performance and follicle development involving increased mRNA expression of gonadotropin receptor mRNA closely related with follicle development in laying hens after the peak laying period.

	ACKNOWLEDGMENTS

 
This study was supported by the National Natural Science Foundation of China (No. 30471245), Ministry of Education (NCET-05-0514), and Zhejiang Provincial Natural Science Foundation (No. R305035). We thank Ningying Xu and Weidong Zeng (Zhejiang University) for assistance during the experiments and Jie Chen (Nanjing Agricultural University, Nanjing, Jiangsu, China) for providing daidzein.

Received for publication July 5, 2007. Accepted for publication November 18, 2007.

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