Accepted Manuscript Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells Shenqiang Hu, Chao Gan, Rui Wen, Qihai Xiao, Hua Gou, Hehe Liu, Yingying Zhang, Liang Li, Jiwen Wang PII:
S0093-691X(14)00267-2
DOI:
10.1016/j.theriogenology.2014.05.025
Reference:
THE 12820
To appear in:
Theriogenology
Received Date: 21 February 2014 Revised Date:
19 May 2014
Accepted Date: 22 May 2014
Please cite this article as: Hu S, Gan C, Wen R, Xiao Q, Gou H, Liu H, Zhang Y, Li L, Wang J, Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells, Theriogenology (2014), doi: 10.1016/j.theriogenology.2014.05.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Revised Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells
RI PT
Shenqiang Hu, Chao Gan, Rui Wen, Qihai Xiao, Hua Gou, Hehe Liu, Yingying Zhang, Liang Li, Jiwen Wang*
AC C
EP
TE D
M AN U
SC
Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Ya’an, Sichuan 625014, P. R. China
*Corresponding author: Jiwen Wang Professor
College of Animal Science and Technology Sichuan Agricultural University, Ya’an, Sichuan 625014, P.R. China Tel.: +86-835-2885463 Fax: +86-835-2891889 E-mail: [email protected]
ACCEPTED MANUSCRIPT Abstract: Leptin is critical for reproductive endocrinology. The aim of this study is to assess the expression patterns of leptin receptor (Lepr) during ovarian follicle development and to reveal the mechanism by which leptin affects steroid hormone secretion in goose granulosa cells. Transcripts of Lepr were ubiquitous in all tested tissues with pituitary and adrenal glands being the predominant sites. Goose ovarian follicles were divided into several groups by diameter including prehierarchical (4-6 mm, 6-8 mm, 8-10 mm) and hierarchical (F5-F1) follicles. Lepr gene
RI PT
expression was significantly higher in granulosa cells than in theca cells from follicles of 4-8 mm in diameter. Expression of Lepr in granulosa cells decreased gradually as follicles developed, with fluctuating expression in F5 and F3 follicles. Lepr mRNA in theca cells underwent a slight decrease from the 6-8 mm cohorts to F5 follicle and then exhibited a transient increase and declined later. In vitro experiments in cultured goose granulosa cells showed estradiol release was
SC
significantly stimulated, while progesterone increased slightly and testosterone decreased dramatically after leptin treatment. In accordance with the data for steroids, expression of Lepr,
M AN U
Srebp1, Cyp51, StAR, and Cyp19a1 were induced by addition of leptin, and the concomitant changes in Hmgcs1, Dhcr24, Cyp11a1, 17β-hsd, Cyp17, and 3β-hsd gene expression were seen. These results suggested that leptin is involved in the development of goose ovarian follicles, and leptin’s effect on steroid hormone secretion could be due to altered sterol/steroidogenic gene expression via interaction with its receptor.
Keywords: goose; leptin; leptin receptor; ovary; steroid hormone Introduction
TE D
1.
Leptin, a member of the type I helical cytokine family, is an adipose-derived hormone that plays a critical role in relaying energetic status to the brain and thus is involved in the regulation of numerous biological activities, including metabolism, growth, development, and neuroendocrine
EP
and immune function [1]. Recent years have seen advances in revealing the peripheral actions of leptin on the reproductive axis from mutant mouse models. It was found that ob/ob mice, which have a mutated leptin gene, were infertile, but administration of exogenous leptin eliminated the
AC C
sterility [2, 3]. It has previously been indicated that circulating leptin levels could act as a metabolic signal that communicates information about energy reserves to the reproductive system and appears to be a permissive factor in the onset of puberty and in maintaining normal fertility [4, 5]. Leptin regulates reproductive functions by acting centrally on the hypothalamic-hypophysis axis and/or peripherally at the ovary [6]. Although abundant evidence demonstrates the central effect of leptin on the reproductive neuroendocrine axis, the mechanisms of leptin signaling in the ovary still remain to be determined. Leptin is encoded by the obese (ob or Lep) gene, and it exerts its effects by activating its specific cellular receptors. The wide distribution of leptin receptor (Lepr) is concordant with leptin’s complex physiological functions [7]. Lepr has been characterized in the ovary, theca, and granulosa cells of humans [8], mice [9], cows [10], pigs [11], rabbits [12], and chicken[13],
ACCEPTED MANUSCRIPT demonstrating the direct actions of leptin on the reproductive system at the level of the ovary. Leptin deficiency in mice leads to impaired folliculogenesis and increased granulosa cell apoptosis [14]. The abundance of Lepr mRNA in ovarian follicular cells during different physiological stages clearly supports the involvement of leptin in follicular maturation and oocyte development [15, 16]. In leptin-treated female rabbits, a significantly higher number of live newborns were found than in the control group, accompanied by reduced plasma progesterone (P4), testosterone
RI PT
(T), and estradiol (E2) levels [17]. Besides, through in vivo injections, the effects of leptin on ovarian function have been demonstrated to be associated with the secretion of steroid hormone by theca and granulosa cells in several species [18-20]. Furthermore, leptin was shown to be involved in the dysfunction of follicular hierarchy observed in ad libitum-feed breeder hens [21]. Exogenous leptin could advance the initiation of puberty and ameliorate the influences of fasting
SC
on ovarian function in female chickens due to altered ovarian steroidogenesis and attenuated follicular apoptosis [13, 22].
M AN U
Regarding the role of leptin in steroidogenesis of granulosa cells, divergent results from in vitro experiments were obtained in different species. In rat and human granulosa cells, leptin inhibited the glucocorticoid-induced steroidogenesis [23], whereas in the porcine cells leptin modulated steroidogenesis in a biphasic and dose-dependent manner [24]. In addition, in rabbit granulosa cells, leptin significantly decreased P4 secretion at lower doses, but increased P4, T, and E2 levels at higher doses [17]. When added to chicken granulosa cells in vitro, leptin stimulated P4 and E2 secretion but had an inhibitory effect on T secretion [25]. The mechanisms by which leptin
TE D
induces these changes and the steroidogenic genes regulated by leptin remain poorly understood. Taking geese (Anser cygnoides) as a model to study the interaction between genes and steroid hormones is advantageous with regard to the larger size of ovarian follicles and relatively low-laying performance. To date, nothing is known about the role of leptin in goose ovary. The
EP
objective of this study was to determine the expression patterns of Lepr mRNA in both theca and granulosa cells during follicle development in the laying geese. The direct effects of leptin on
AC C
steroid hormone secretion and expression of key genes in the sterol/steroid pathways in goose granulosa cells were also investigated. In addition, expression of Lepr mRNA was assessed in response to different doses of exogenous leptin.
2.
Materials and Methods
2.1. Animals and sample collection The healthy maternal line of Tianfu meat geese (Anser cygnoide), 35-45 weeks of age and laying in regular sequences of at least 2-3 eggs, were used in all studies described. The geese were kept under natural conditions of light and temperature at the Experimental Farm of Waterfowl Breeding of Sichuan Agricultural University (Sichuan, China), and were provided with free access to feed and water. Individual laying cycles were monitored for each goose throughout the laying sequence.
ACCEPTED MANUSCRIPT Geese were euthanized by cervical dislocation 7-9 h after the first or second oviposition in an egg sequence. Brain, hypothalamus, pituitary, adrenal glands, ovary, oviduct, adipose tissues, and ovarian theca and granulosa cells from 4-6 mm-, 6-8 mm-, 8-10 mm-diameter prehierarchical follicles, and F5-F1 (measuring F1>F2>F3>F4>F5 in diameter) hierarchical follicles were collected, rapidly frozen in liquid nitrogen, and finally stored at -80
until RNA extraction.
Morphologically normal follicles were characterized as previously described [26], and the theca
RI PT
and granulosa cells were separated according to the method introduced by Gilbert et al., [27]. All procedures in this study were approved by the Beijing Animal Welfare Committee. 2.2 Granulosa cell culture
The granulosa cells harvested from F3-F1 follicles were washed with phosphate buffer saline
SC
(PBS, pH 7.4) and dispersed with type collagenase (Sigma, St Louis, USA). Cell viability was assessed by the Trypan blue dye exclusion test. The cells were diluted with the media to a concentration of ~ 5×105 /mL and then added to 6-well culture plates and incubated at 38.5
M AN U
under 5% CO2 in humidified air to allow the cells to reach a confluence. The media consisted of Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture (DMEM/F12) containing 3% fetal bovine serum (Sigma, St Louis, USA). After 2 days of pre-culture, the medium was replaced by fresh medium without or with chicken recombinant leptin (purchased from ProSpec-Tany TechnoGene Ltd., Rehovot, Israel) at doses 0, 1, 10 or 100 ng/ml. After that, the cells were cultured for 24 h. Each group had three replicates, and the same treatment was repeated in triplicate.
TE D
2.3 RNA extraction and cDNA synthesis
Total RNA was extracted from all the samples using Trizol (Invitrogen, USA) according to the manufacturer’s instructions, and the quality of the resulting RNA was assessed by visualizing the ribosomal RNA bands via agarose gel electrophoresis. The cDNA was obtained using a cDNA RNA as a template.
EP
synthesis kit (TaKaRa, JAPAN) according to the manufacturer’s instruction with 1 µg of total
AC C
2.4 Semi-quantitative RT-PCR analysis Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the relative levels of Lepr mRNA in several goose tissues using β-actin as an internal reference. According to the concentration of total RNA samples, the smallest amount was selected to be the standard for sample dilution to ensure the consistent initial concentration. The normalized RNA from each sample was used as a template for reverse transcription. PCR was finally performed under the following conditions, 5 min denaturation at 94 sec and 72
, followed by 30 cycles (94
, 30 sec) for Lepr and 30 cycles (94
β-actin, ending with a 5 min extension at 72
, 30 sec, 59.6
, 30 sec, 60
, 30
, 30 sec and 72 , 45 sec) for
. PCR products were separated by electrophoresis
on Goldview (SBS Genetech, China) stained 2% agarose gels. The optical density (OD) value was analyzed by Quantity One software (Bio-Rad, USA). The abundance of Lepr mRNA was obtained
ACCEPTED MANUSCRIPT on the basis of the gray scale ratios between Lepr and β-actin. All of the primers were designed using Primer Premier 5 software (Primer Biosoft International, USA) and synthesized by Invitrogen Corporation (Applied Invitrogen, Shanghai, China). The RT-PCR primers are listed in Table 1. 2.5 Real-time PCR
RI PT
The mRNA expression levels of Lepr and selected sterol/steroid biosynthetic genes in goose ovarian follicles were assessed by quantitative real-time PCR (qRT-PCR). The qRT-PCR was performed in a 96-well IQTM5 System (Bio-Rad, USA) using a Takara ExTaq RT-PCR kit and SYBR Green as the detection dye (Takara, Dalian, China). The procedure included 1 cycle of 95 for 10 sec, followed by 40 cycles of 95
for 5 sec and primer-specific annealing temperature for
increasing by 0.5
SC
30 sec. An 80-cycle melting curve was performed, starting at a temperature of 55
and
every 10 sec to determine primer specificity. Only one product of the desired
size was idenfied and one single peak was observed in a melting curve for each primer. Each
M AN U
sample was repeated three times and the relative expression of all interested genes were normalized to β-actin and 18s rRNA using the 2-∆∆Ct method [28]. The primers designed for real-time PCR are also listed in Table 1. 2.6 Hormone concentration measurement
Progesterone, estradiol, and testosterone in media samples were quantified by Geese P4, E2, and T
TE D
ELISA Kit provided by Shanghai Yanhui Biotechnology Co. Ltd (Shanghai, P.R. China), respectively. Steroid contents were undetectable (P4 < 1 ng/ml, E2 < 20 ng/ml, and T < 10 ng/L). The standard curve for measurement of each hormone was established by the diluted standard samples, and the concentration of each hormone in all samples was determined by comparing the OD values of corresponding sample to the standard curve on the basis of the spectrophotometric
EP
color changes at a wavelength of 450 nm.
AC C
2.7 Statistical analysis
The data were subjected to analysis of variance (ANOVA), and the means were assessed for significant differences using Duncan's Multiple Range Test. The results were presented as the mean ± S.D., and p-values below 0.05 were considered statistically significant. All statistical analyses were performed using SAS 9.13 (SAS Institute., Cary, NC, USA).
3.
Results
3.1 Tissue distribution of Lepr mRNA Lepr mRNA expression was assessed in seven tissues from laying geese by semi-quantitative RT-PCR. Transcripts of Lepr were present in all tested tissues. The mRNA levels of Lepr were significantly higher (p
S0093-691X(14)00267-2
DOI:
10.1016/j.theriogenology.2014.05.025
Reference:
THE 12820
To appear in:
Theriogenology
Received Date: 21 February 2014 Revised Date:
19 May 2014
Accepted Date: 22 May 2014
Please cite this article as: Hu S, Gan C, Wen R, Xiao Q, Gou H, Liu H, Zhang Y, Li L, Wang J, Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells, Theriogenology (2014), doi: 10.1016/j.theriogenology.2014.05.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Revised Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells
RI PT
Shenqiang Hu, Chao Gan, Rui Wen, Qihai Xiao, Hua Gou, Hehe Liu, Yingying Zhang, Liang Li, Jiwen Wang*
AC C
EP
TE D
M AN U
SC
Institute of Animal Genetics and Breeding, Sichuan Agricultural University, Ya’an, Sichuan 625014, P. R. China
*Corresponding author: Jiwen Wang Professor
College of Animal Science and Technology Sichuan Agricultural University, Ya’an, Sichuan 625014, P.R. China Tel.: +86-835-2885463 Fax: +86-835-2891889 E-mail: [email protected]
ACCEPTED MANUSCRIPT Abstract: Leptin is critical for reproductive endocrinology. The aim of this study is to assess the expression patterns of leptin receptor (Lepr) during ovarian follicle development and to reveal the mechanism by which leptin affects steroid hormone secretion in goose granulosa cells. Transcripts of Lepr were ubiquitous in all tested tissues with pituitary and adrenal glands being the predominant sites. Goose ovarian follicles were divided into several groups by diameter including prehierarchical (4-6 mm, 6-8 mm, 8-10 mm) and hierarchical (F5-F1) follicles. Lepr gene
RI PT
expression was significantly higher in granulosa cells than in theca cells from follicles of 4-8 mm in diameter. Expression of Lepr in granulosa cells decreased gradually as follicles developed, with fluctuating expression in F5 and F3 follicles. Lepr mRNA in theca cells underwent a slight decrease from the 6-8 mm cohorts to F5 follicle and then exhibited a transient increase and declined later. In vitro experiments in cultured goose granulosa cells showed estradiol release was
SC
significantly stimulated, while progesterone increased slightly and testosterone decreased dramatically after leptin treatment. In accordance with the data for steroids, expression of Lepr,
M AN U
Srebp1, Cyp51, StAR, and Cyp19a1 were induced by addition of leptin, and the concomitant changes in Hmgcs1, Dhcr24, Cyp11a1, 17β-hsd, Cyp17, and 3β-hsd gene expression were seen. These results suggested that leptin is involved in the development of goose ovarian follicles, and leptin’s effect on steroid hormone secretion could be due to altered sterol/steroidogenic gene expression via interaction with its receptor.
Keywords: goose; leptin; leptin receptor; ovary; steroid hormone Introduction
TE D
1.
Leptin, a member of the type I helical cytokine family, is an adipose-derived hormone that plays a critical role in relaying energetic status to the brain and thus is involved in the regulation of numerous biological activities, including metabolism, growth, development, and neuroendocrine
EP
and immune function [1]. Recent years have seen advances in revealing the peripheral actions of leptin on the reproductive axis from mutant mouse models. It was found that ob/ob mice, which have a mutated leptin gene, were infertile, but administration of exogenous leptin eliminated the
AC C
sterility [2, 3]. It has previously been indicated that circulating leptin levels could act as a metabolic signal that communicates information about energy reserves to the reproductive system and appears to be a permissive factor in the onset of puberty and in maintaining normal fertility [4, 5]. Leptin regulates reproductive functions by acting centrally on the hypothalamic-hypophysis axis and/or peripherally at the ovary [6]. Although abundant evidence demonstrates the central effect of leptin on the reproductive neuroendocrine axis, the mechanisms of leptin signaling in the ovary still remain to be determined. Leptin is encoded by the obese (ob or Lep) gene, and it exerts its effects by activating its specific cellular receptors. The wide distribution of leptin receptor (Lepr) is concordant with leptin’s complex physiological functions [7]. Lepr has been characterized in the ovary, theca, and granulosa cells of humans [8], mice [9], cows [10], pigs [11], rabbits [12], and chicken[13],
ACCEPTED MANUSCRIPT demonstrating the direct actions of leptin on the reproductive system at the level of the ovary. Leptin deficiency in mice leads to impaired folliculogenesis and increased granulosa cell apoptosis [14]. The abundance of Lepr mRNA in ovarian follicular cells during different physiological stages clearly supports the involvement of leptin in follicular maturation and oocyte development [15, 16]. In leptin-treated female rabbits, a significantly higher number of live newborns were found than in the control group, accompanied by reduced plasma progesterone (P4), testosterone
RI PT
(T), and estradiol (E2) levels [17]. Besides, through in vivo injections, the effects of leptin on ovarian function have been demonstrated to be associated with the secretion of steroid hormone by theca and granulosa cells in several species [18-20]. Furthermore, leptin was shown to be involved in the dysfunction of follicular hierarchy observed in ad libitum-feed breeder hens [21]. Exogenous leptin could advance the initiation of puberty and ameliorate the influences of fasting
SC
on ovarian function in female chickens due to altered ovarian steroidogenesis and attenuated follicular apoptosis [13, 22].
M AN U
Regarding the role of leptin in steroidogenesis of granulosa cells, divergent results from in vitro experiments were obtained in different species. In rat and human granulosa cells, leptin inhibited the glucocorticoid-induced steroidogenesis [23], whereas in the porcine cells leptin modulated steroidogenesis in a biphasic and dose-dependent manner [24]. In addition, in rabbit granulosa cells, leptin significantly decreased P4 secretion at lower doses, but increased P4, T, and E2 levels at higher doses [17]. When added to chicken granulosa cells in vitro, leptin stimulated P4 and E2 secretion but had an inhibitory effect on T secretion [25]. The mechanisms by which leptin
TE D
induces these changes and the steroidogenic genes regulated by leptin remain poorly understood. Taking geese (Anser cygnoides) as a model to study the interaction between genes and steroid hormones is advantageous with regard to the larger size of ovarian follicles and relatively low-laying performance. To date, nothing is known about the role of leptin in goose ovary. The
EP
objective of this study was to determine the expression patterns of Lepr mRNA in both theca and granulosa cells during follicle development in the laying geese. The direct effects of leptin on
AC C
steroid hormone secretion and expression of key genes in the sterol/steroid pathways in goose granulosa cells were also investigated. In addition, expression of Lepr mRNA was assessed in response to different doses of exogenous leptin.
2.
Materials and Methods
2.1. Animals and sample collection The healthy maternal line of Tianfu meat geese (Anser cygnoide), 35-45 weeks of age and laying in regular sequences of at least 2-3 eggs, were used in all studies described. The geese were kept under natural conditions of light and temperature at the Experimental Farm of Waterfowl Breeding of Sichuan Agricultural University (Sichuan, China), and were provided with free access to feed and water. Individual laying cycles were monitored for each goose throughout the laying sequence.
ACCEPTED MANUSCRIPT Geese were euthanized by cervical dislocation 7-9 h after the first or second oviposition in an egg sequence. Brain, hypothalamus, pituitary, adrenal glands, ovary, oviduct, adipose tissues, and ovarian theca and granulosa cells from 4-6 mm-, 6-8 mm-, 8-10 mm-diameter prehierarchical follicles, and F5-F1 (measuring F1>F2>F3>F4>F5 in diameter) hierarchical follicles were collected, rapidly frozen in liquid nitrogen, and finally stored at -80
until RNA extraction.
Morphologically normal follicles were characterized as previously described [26], and the theca
RI PT
and granulosa cells were separated according to the method introduced by Gilbert et al., [27]. All procedures in this study were approved by the Beijing Animal Welfare Committee. 2.2 Granulosa cell culture
The granulosa cells harvested from F3-F1 follicles were washed with phosphate buffer saline
SC
(PBS, pH 7.4) and dispersed with type collagenase (Sigma, St Louis, USA). Cell viability was assessed by the Trypan blue dye exclusion test. The cells were diluted with the media to a concentration of ~ 5×105 /mL and then added to 6-well culture plates and incubated at 38.5
M AN U
under 5% CO2 in humidified air to allow the cells to reach a confluence. The media consisted of Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture (DMEM/F12) containing 3% fetal bovine serum (Sigma, St Louis, USA). After 2 days of pre-culture, the medium was replaced by fresh medium without or with chicken recombinant leptin (purchased from ProSpec-Tany TechnoGene Ltd., Rehovot, Israel) at doses 0, 1, 10 or 100 ng/ml. After that, the cells were cultured for 24 h. Each group had three replicates, and the same treatment was repeated in triplicate.
TE D
2.3 RNA extraction and cDNA synthesis
Total RNA was extracted from all the samples using Trizol (Invitrogen, USA) according to the manufacturer’s instructions, and the quality of the resulting RNA was assessed by visualizing the ribosomal RNA bands via agarose gel electrophoresis. The cDNA was obtained using a cDNA RNA as a template.
EP
synthesis kit (TaKaRa, JAPAN) according to the manufacturer’s instruction with 1 µg of total
AC C
2.4 Semi-quantitative RT-PCR analysis Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the relative levels of Lepr mRNA in several goose tissues using β-actin as an internal reference. According to the concentration of total RNA samples, the smallest amount was selected to be the standard for sample dilution to ensure the consistent initial concentration. The normalized RNA from each sample was used as a template for reverse transcription. PCR was finally performed under the following conditions, 5 min denaturation at 94 sec and 72
, followed by 30 cycles (94
, 30 sec) for Lepr and 30 cycles (94
β-actin, ending with a 5 min extension at 72
, 30 sec, 59.6
, 30 sec, 60
, 30
, 30 sec and 72 , 45 sec) for
. PCR products were separated by electrophoresis
on Goldview (SBS Genetech, China) stained 2% agarose gels. The optical density (OD) value was analyzed by Quantity One software (Bio-Rad, USA). The abundance of Lepr mRNA was obtained
ACCEPTED MANUSCRIPT on the basis of the gray scale ratios between Lepr and β-actin. All of the primers were designed using Primer Premier 5 software (Primer Biosoft International, USA) and synthesized by Invitrogen Corporation (Applied Invitrogen, Shanghai, China). The RT-PCR primers are listed in Table 1. 2.5 Real-time PCR
RI PT
The mRNA expression levels of Lepr and selected sterol/steroid biosynthetic genes in goose ovarian follicles were assessed by quantitative real-time PCR (qRT-PCR). The qRT-PCR was performed in a 96-well IQTM5 System (Bio-Rad, USA) using a Takara ExTaq RT-PCR kit and SYBR Green as the detection dye (Takara, Dalian, China). The procedure included 1 cycle of 95 for 10 sec, followed by 40 cycles of 95
for 5 sec and primer-specific annealing temperature for
increasing by 0.5
SC
30 sec. An 80-cycle melting curve was performed, starting at a temperature of 55
and
every 10 sec to determine primer specificity. Only one product of the desired
size was idenfied and one single peak was observed in a melting curve for each primer. Each
M AN U
sample was repeated three times and the relative expression of all interested genes were normalized to β-actin and 18s rRNA using the 2-∆∆Ct method [28]. The primers designed for real-time PCR are also listed in Table 1. 2.6 Hormone concentration measurement
Progesterone, estradiol, and testosterone in media samples were quantified by Geese P4, E2, and T
TE D
ELISA Kit provided by Shanghai Yanhui Biotechnology Co. Ltd (Shanghai, P.R. China), respectively. Steroid contents were undetectable (P4 < 1 ng/ml, E2 < 20 ng/ml, and T < 10 ng/L). The standard curve for measurement of each hormone was established by the diluted standard samples, and the concentration of each hormone in all samples was determined by comparing the OD values of corresponding sample to the standard curve on the basis of the spectrophotometric
EP
color changes at a wavelength of 450 nm.
AC C
2.7 Statistical analysis
The data were subjected to analysis of variance (ANOVA), and the means were assessed for significant differences using Duncan's Multiple Range Test. The results were presented as the mean ± S.D., and p-values below 0.05 were considered statistically significant. All statistical analyses were performed using SAS 9.13 (SAS Institute., Cary, NC, USA).
3.
Results
3.1 Tissue distribution of Lepr mRNA Lepr mRNA expression was assessed in seven tissues from laying geese by semi-quantitative RT-PCR. Transcripts of Lepr were present in all tested tissues. The mRNA levels of Lepr were significantly higher (p
