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steroid biosynthesis in goose granulosa cells
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.
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Revised Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells
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Shenqiang Hu, Chao Gan, Rui Wen, Qihai Xiao, Hua Gou, Hehe Liu, Yingying Zhang, Liang Li, Jiwen Wang*
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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
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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
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significantly stimulated, while progesterone increased slightly and testosterone decreased dramatically after leptin treatment. In accordance with the data for steroids, expression of Lepr,
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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
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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
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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
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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
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(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
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on ovarian function in female chickens due to altered ovarian steroidogenesis and attenuated follicular apoptosis [13, 22].
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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
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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
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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
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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
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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
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(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
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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.
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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.
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synthesis kit (TaKaRa, JAPAN) according to the manufacturer’s instruction with 1 µg of total
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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
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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
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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
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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
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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
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color changes at a wavelength of 450 nm.
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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<0.05) in the pituitary than in other tissues with the exception of adrenal glands, followed by ovary, oviduct, brain, and hypothalamus (Fig. 1).
ACCEPTED MANUSCRIPT 3.2 Lepr mRNA expression during follicle development The mRNA levels of Lepr were determined in different sized follicles from laying geese by qRTPCR (Fig.2). Lepr mRNA was found in both theca and granulosa cells isolated from all follicles. In the 4-8 mm diameter size class and F5, transcripts of Lepr were significantly higher in granulosa cells than in theca cells (p<0.05). By contrast, Lepr gene expression was significantly higher in theca cells than in granulosa cells isolated from F4 and F2 (p<0.05). In addition,
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expression of Lepr in granulosa cells exhibited a tendency of decreasing gradually throughout follicle development, with fluctuating expression in F5 and F3. Noticeably, the peak of Lepr mRNA expression was found in granulosa cells from 4-6 mm follicles, which was significantly higher than any other follicles (p<0.05). As follicle development ensues, expression of Lepr
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mRNA in theca cells underwent a slight decrease from the 6-8 mm cohorts to F5, then exhibited a transient increase and declined later. The mRNA expression level of Lepr was higher in theca cells from F4 than that from other developing follicles (p<0.05).
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3.3 Effect of leptin treatment on steroid hormone release
Leptin-induced estradiol, progesterone, and testosterone production by goose granulosa cells from preovulatory follicles were determined in the media after 24 h culture. As shown in Fig.3, the 1 ng/ml dose of recombinant leptin significantly stimulated estradiol release (p<0.05) in comparison to the control group. However, no significant difference was observed at 10 or 100 ng/ml. Although the effect of recombinant leptin on progesterone levels was not significant at all doses
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(p>0.05), a slight increase was seen after leptin addition. In addition, testosterone release was not significantly altered by leptin treatment (p>0.05), but a strong decline was observed. 3.4 Effect of leptin treatment on the expression of Lepr mRNA
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Changes in Lepr mRNA levels in goose granulosa cells after leptin treatment were shown in Fig.4. A significant increase in the expression of Lepr mRNA was observed at 1 and 10 ng/ml (p<0.05). The addition of high concentrations of leptin (100ng/ml) significantly reduced Lepr mRNA
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expression (p<0.05) when compared with the lower doses, but no significant difference was found compared to controls (p>0.05). 3.5 Expression of sterol/steroidogenic genes induced by leptin treatment After exposure to different doses of recombinant leptin, all selected genes in the control of sterol/steroid biosynthesis were expressed in all cultured granulosa cell preparations but displayed different expression patterns (Fig.5). Among genes regulating sterol biosynthesis, sterol regulatory element binding protein 1 (Srebp1) and cytochrome P450 14α-sterol demethylases (Cyp51) were significantly up-regulated at leptin concentrations of 1 and 10 ng/ml (p<0.05), subsequently decreased at 100 ng/ml. No significant differences were found in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (Hmgcs1) and 3β-hydroxysterol-∆24 reductase (Dhcr24) in response to different concentrations of leptin (p>0.05), when compared with
ACCEPTED MANUSCRIPT those from controls. Within steroidogenic genes, comparison of transcripts of P450 side-chain cleavage enzyme (Cyp11a1) and 17α-hydroxylase (Cyp17) among different doses of leptin showed no significant differences compared to controls (p>0.05), but showed a slightly increasing tendency with elevated concentrations of leptin. The mRNA levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450 aromatase (Cyp19a1) were significantly higher at leptin concentrations of 1 and 10 ng/ml, and 10 ng/ml, respectively, when compared with controls
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(p<0.05). Notably, although there were no significant differences in 3β-hydroxysteroid dehydrogenase/∆5-∆4 isomerase (3β-hsd) or 17β-hydroxysteroid dehydrogenase (17β-hsd) gene expression responding to different doses of leptin when compared with controls (p>0.05), a strong increase was seen. In addition, the proposed pathways and key genes in the regulation of leptin-mediated steroid hormone release in goose preovulatory follicle granulosa cells are
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summarized in Fig.6.
Discussion
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Despite the controversy on the identity of the leptin gene in birds [29-31], increasing evidence suggests the existence of a leptin-like immunoreactive substance [32] and a functional leptin receptor [33, 34]. Furthermore, the effects of exogenous recombinant murine or chicken leptin on chicken lipid metabolism [35, 36] and ovarian follicle development [13, 22] have been demonstrated. In addition, a recent study reported that leptin in vivo administration could promote the recovery of regressed ovary in fasted ducks [37], and the full-length cDNA of goose Lepr has
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been characterized with a high identity to that of other birds [38]. Considering the similar tertiary structures of leptin and highly conserved binding sites between leptin and its receptors in vertebrates [30, 31], it is conceivable that recombinant chicken leptin could be utilized for researches in waterfowl. To the best of our knowledge, the present study was the first to evaluate ovarian expression of goose Lepr mRNA during follicle development and to reveal the role of
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leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells. Transcripts of Lepr were ubiquitous in all tissues examined, with pituitary and adrenal glands
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being the sites of relatively high gene expression. Such widespread expression of Lepr mRNA was coincident with the involvement of leptin in diverse physiological and pathological processes [7], indicating the central role of leptin in the control of avian reproductive biology and neuroendocrinology through hypothalamic-pituitary-gonadal/adrenal (HPG/HPA) axes. The presence of Lepr mRNA in goose ovary suggests a peripheral effect of leptin, which has also been shown in chicken [34] and turkey [39]. Moreover, the detectable mRNA levels of Lepr in the oviduct supported the possible participation of leptin in the process of egg white protein synthesis, eggshell formation, and final oviposition. Accordingly, similar to other birds, a leptin-like system in controlling female reproduction might also occur in geese. It is well known that avian ovarian follicles are arranged in a distinct follicular hierarchy and are committed to ovulation. Follicle maturation is a hormonally controlled process accompanied by
ACCEPTED MANUSCRIPT the transition from prehierarchical (undifferentiated) to preovulatory (differentiated) stage [40, 41]. In this study, the ubiquity of goose Lepr mRNA in both theca and granulosa cells isolated from all developing follicles further supported a direct action of leptin at the level of the ovary. Besides, the different expression patterns of Lepr in either theca or granulosa cells throughout follicle maturation indicated that leptin might be involved in maintaining the normal follicular hierarchy and regulating follicle development, which was in good accordance with other studies. In ad
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libitum-fed broiler chickens the dramatic up-regulation of ovarian expression of Lepr during follicle development was associated with the formation of abnormal follicular hierarchy and led to significant reproductive dysfunction [21]. During the re-feeding period of fasting hens, the continuation of leptin treatment considerably delayed the recovery of regressed ovary by reducing the entry of white follicles into the hierarchical follicles and inhibiting the growth of yellow
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follicles [13]. The walls of the ovarian follicles could synthesize three major sex steroids including estradiol, testosterone, and progesterone. Estradiol is mainly produced by theca externa cells and
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testosterone is synthesized by theca interna cells, while the granulosa cells are considered as the main resource for progesterone [42]. The steroidogenic activity of all types of cells differs at different stages of follicle development [42]. Therefore, the level of expression of Lepr mRNA in theca and granulosa cells during follicle maturation could be a factor in the regulation of ovarian steroidogenesis. In human granulosa cells, leptin inhibited the stimulatory effects of luteinizing hormone (LH) on estradiol production [8], and passive immunization against leptin in sheep contributed to an abrupt increase in ovarian estradiol secretion [20]. In cultured rat ovarian
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follicles, leptin suppressed the stimulatory effect of insulin-like growth factor I (IGF-I) on follicle stimulating hormone (FSH)-induced estradiol production [19]. The relatively higher-level expression of goose Lepr in granulosa cells from follicles of 4-8 mm in diameter than that from large follicles suggested a more potent inhibitory effect of leptin on insulin-stimulated aromatase
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activity of granulosa cells from small follicles [43, 44]. The significant increase of Lepr gene expression in granulosa cells from F5 follicle might facilitate follicle selection, during which the follicles undergo the transition from an undifferentiated to a differentiated state in association with
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a shift from FSH receptor (FSHR)-dominance to LH receptor (LHR)-dominance [40]. However, expression of Lepr decreased in both theca and granulosa cells between F4 and F1 follicles, which were in good accordance with previously published findings [10, 21], indicating less sensitivity of large follicles to the negative control of leptin as the follicles developed [43]. Notably, transcripts of Lepr were found to be constantly higher in theca cells than in granulosa cells from F4 to F1 follicle, implying a more active action of leptin in theca cells linked to the release of E2 and T. The results from our in vitro experiment provided novel evidence that leptin directly affected the production of steroid hormones independent of HPG influence and altered expression of several sterol/steroidogenic genes in goose granulosa cells. Concentrations of E2 were significantly stimulated at the 1 ng/ml dose of recombinant leptin, whereas no significant difference was observed at 10 or 100 ng/ml when compared with controls. Although there were no statistically
ACCEPTED MANUSCRIPT significant changes in the P4 and T concentrations among all granulosa cells exposed to different doses of leptin, a slight increase in P4 and a strong decline in T secretion were seen with the elevated leptin doses. Parallel observations were found in recent data from other studies. The up-regulated levels of E2 and P4, as well as the down-regulated levels of T induced by recombinant chicken leptin were found in cultured fragments of follicular wall isolated from chicken ovarian follicles [25]. Of particular note is that the difference in the release of P4 and T could result from
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the clearance of theca cells in the current study, because the significant changes observed in Sirotkin et al are not primary, but secondary [25], and may be associated with the granulosa-theca interaction in the secretion of sex steroids [45]. Among the key genes in regulating sterol biosynthesis, significantly elevated mRNA expression levels of Srebp1 and Cyp51 were shown at leptin concentrations of 1 and 10 ng/ml, while neither Hmgcs1 nor Dhcr24 gene expression were
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significantly different compared to controls despite showing a slight increasing tendency. Similarly, genes within biosynthetic pathways were regulated by FSH and two forkhead box
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transcription factor (FOXO1) mutants in cultured granulosa cells [46]. As for the steroidogenic genes, the mRNA levels of StAR and Cyp19a1 were significantly higher at leptin concentrations of 1 and 10 ng/ml, and 10 ng/ml, respectively, when compared with controls. No statistically significant changes but strong up-regulated levels were observed in the expression of 3β-hsd and 17β-hsd compared to the control group. Additionally, comparison of expression of 3β-hsd and 17β-hsd showed significantly higher levels at 1 ng/ml than at 100 ng/ml doses of leptin. Cyp11a1 and Cyp17 gene expression were not significantly altered by leptin treatment in granulosa cells,
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but a slight increase was seen. StAR is considered as the key gene in controlling the rate-limiting step of steroidogenesis by mediating transfer of cholesterol from the outer to the inner mitochondrial membrane where Cyp11a1 promotes the conversion of cholesterol to pregnenolone, which subsequently undergoes conversion to P4 by 3β-hsd [47]. Aromatase enzyme plays key
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roles in the conversion of T into E2 and thus regulates estrogen biosynthesis [48]. Our results showed that leptin significantly stimulated estradiol production through an increase in StAR and Cyp19a1 mRNA levels, and a subtle increasing tendency was observed in P4 secretion in
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association with the increase of StAR, Cyp11a1, and 3β-hsd gene expression. The down-regulated concentrations of T might be linked to the activation of aromatase enzyme (mainly Cyp19a1) by leptin, which in turn accelerated the conversion of T to E2. An alternative explanation is that no statistically significant differences in expression of Cyp17 and 17β-hsd were found in granulosa cells exposed to recombinant leptin in comparison with controls. Besides, since cholesterol is thought to be the precursor for steroid biosynthesis, the elevated mRNA levels of Srebp1 and Cyp51 could promote steroid hormone production in goose granulosa cells. A slight increase in both Hmgcs1 and Dhcr24 expression suggested that they might play a role in this process. Leptin exerts its effect by interacting with its receptors in both the hypothalamic-hypophysis system and the ovary to regulate follicular steroidogenesis [6]. Leptin is able to modulate the expression of its receptors in the rat hypothalamic-pituitary-ovarian axis [49]. A direct action of
ACCEPTED MANUSCRIPT leptin on the ovary in chicken has been previously described [13, 21], and the capability of chicken leptin receptor to bind with endogenous ligand as well as mammalian leptin has also been demonstrated in vitro [50]. Furthermore, the observation of presence of Lepr mRNA in goose ovary, theca, and granulosa cells throughout follicle development indicated the possibility of leptin’s direct effects on the ovary through its receptors. Consistent with changes in steroid hormone production and sterol/steroidogenic gene expression, Lepr mRNA was also up-regulated
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by leptin at its doses of 1 and 10 ng/ml. Thus, it could be inferred that leptin modulates secretion of steroid hormones by regulating cellular sterol/steroid biosynthesis via interaction with its receptor in goose granulosa cells (Summarized in Fig.6).
In conclusion, all these results clearly suggest that leptin is involved in the development of goose
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ovarian follicles. The effect of leptin on ovarian steroid hormone production could be due to altered sterol/steroidogenic gene expression via interaction with its receptor.
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Acknowledgements
The work was supported by the National Waterfowl Industrial Technology System, China (No. CARS-43-6).
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[23] Barkan D, Jia H, Dantes A, Vardimon L, Amsterdam A, Rubinstein M. Leptin modulates the glucocorticoid-induced ovarian steroidogenesis. Endocrinology. 1999;140:1731-8. [24] Ruiz-Cortes ZT, Martel-Kennes Y, Gevry NY, Downey BR, Palin MF, Murphy BD. Biphasic effects of leptin in porcine granulosa cells. Biol Reprod. 2003;68:789-96. [25] Sirotkin AV, Grossmann R. Leptin directly controls proliferation, apoptosis and secretory activity of cultured chicken ovarian cells. Comp Biochem Physiol A Mol Integr Physiol. 2007;148:422-9. [26] Kovacs J, Forgo V, Peczely P. The fine structure of the follicular cells in growing and atretic ovarian follicles of the domestic goose. Cell Tissue Res. 1992;267:561-9.
ACCEPTED MANUSCRIPT [27] Gilbert AB, Evans AJ, Perry MM, Davidson MH. A method for separating the granulosa cells, the basal lamina and the theca of the preovulatory ovarian follicle of the domestic fowl (Gallus domesticus). J Reprod Fertil. 1977;50:179-81. [28] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402-8. [29] Ohkubo T, Adachi H. leptin signaling and action in birds. The Journal of Poultry Science.
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2008;45:233-40.
[30] Denver RJ, Bonett RM, Boorse GC. Evolution of leptin structure and function. Neuroendocrinology. 2011;94:21-38.
[31] Prokop JW, Duff RJ, Ball HC, Copeland DL, Londraville RL. Leptin and leptin receptor: analysis of a structure to function relationship in interaction and evolution from humans to fish.
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Peptides. 2012;38:326-36.
[32] Neglia S, Arcamone N, Gargiulo G, de Girolamo P. Immunocytochemical detection of
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leptin-like immunoreactivity in the chicken gastroenteric tract. Gen Comp Endocrinol. 2008;155:432-7.
[33] Horev G, Einat P, Aharoni T, Eshdat Y, Friedman-Einat M. Molecular cloning and properties of the chicken leptin-receptor (CLEPR) gene. Mol Cell Endocrinol. 2000;162:95-106. [34] Ohkubo T, Tanaka M, Nakashima K. Structure and tissue distribution of chicken leptin receptor (cOb-R) mRNA. Biochim Biophys Acta. 2000;1491:303-8.
[35] Hu Y, Zhang R, Zhang Y, Li J, Grossmann R, Zhao R. In ovo leptin administration affects Biotechnol. 2012;3:16.
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hepatic lipid metabolism and microRNA expression in newly hatched broiler chickens. J Anim Sci [36] Denbow DM, Meade S, Robertson A, McMurtry JP, Richards M, Ashwell C. Leptin-induced decrease in food intake in chickens. Physiol Behav. 2000;69:359-62.
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[37] Song Y, Wang C, Wang C, Lv L, Chen Y, Zuo Z. Exogenous leptin promotes the recovery of regressed ovary in fasted ducks. Anim Reprod Sci. 2009;110:306-18. [38] Wang F, Lu L, Yuan H, Tian Y, Li J, Shen J, et al. Molecular cloning, expression, and
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regulation of goose leptin receptor gene in adipocytes. Mol Cell Biochem. 2011;353:267-74. [39] Richards MP, Poch SM. Molecular cloning and expression of the turkey leptin receptor gene. Comp Biochem Physiol B Biochem Mol Biol. 2003;136:833-47. [40] Johnson AL, Woods DC. Dynamics of avian ovarian follicle development: cellular mechanisms of granulosa cell differentiation. Gen Comp Endocrinol. 2009;163:12-7. [41] Johnson PA. Follicle selection in the avian ovary. Reprod Domest Anim. 2012;47 Suppl 4:283-7. [42] Porter TE, Hargis BM, Silsby JL, el Halawani ME. Differential steroid production between theca interna and theca externa cells: a three-cell model for follicular steroidogenesis in avian species. Endocrinology. 1989;125:109-16. [43] Spicer LJ, Francisco CC. The adipose obese gene product, leptin: evidence of a direct inhibitory role in ovarian function. Endocrinology. 1997;138:3374-9.
ACCEPTED MANUSCRIPT [44] Munoz-Gutierrez M, Findlay PA, Adam CL, Wax G, Campbell BK, Kendall NR, et al. The ovarian expression of mRNAs for aromatase, IGF-I receptor, IGF-binding protein-2, -4 and -5, leptin and leptin receptor in cycling ewes after three days of leptin infusion. Reproduction. 2005;130:869-81. [45] Kato M, Shimada K, Saito N, Noda K, Ohta M. Expression of P450 17 alpha-hydroxylase ovarian follicles during follicular growth. Biol Reprod. 1995;52:405-10.
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and P450aromatase genes in isolated granulosa, theca interna, and theca externa layers of chicken [46] Liu Z, Rudd MD, Hernandez-Gonzalez I, Gonzalez-Robayna I, Fan HY, Zeleznik AJ, et al. FSH and FOXO1 regulate genes in the sterol/steroid and lipid biosynthetic pathways in granulosa cells. Mol Endocrinol. 2009;23:649-61. and HSD3B. Exp Biol Med (Maywood). 2009;234:880-907.
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[47] Lavoie HA, King SR. Transcriptional regulation of steroidogenic genes: STARD1, CYP11A1 [48] Simpson ER, Davis SR. Minireview: aromatase and the regulation of estrogen
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biosynthesis--some new perspectives. Endocrinology. 2001;142:4589-94.
[49] Di Yorio MP, Bilbao MG, Pustovrh MC, Prestifilippo JP, Faletti AG. Leptin modulates the expression of its receptors in the hypothalamic-pituitary-ovarian axis in a differential way. J Endocrinol. 2008;198:355-66.
[50] Adachi H, Takemoto Y, Bungo T, Ohkubo T. Chicken leptin receptor is functional in
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activating JAK-STATpathway in vitro. J Endocrinol. 2008;197:335-42.
ACCEPTED MANUSCRIPT Table 1 Primer sequences used in this study
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Tm ( )
Size(bp)
60
159
60
92
58.2
91
56.7
104
56.4
83
58.6
87
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sequence (5’ to 3’) F AGCAGTTGCCCGTTGTAT Lepr R AAGGGAGGGTTGGTTTCA F CGAGTACATCCGCTTCCTGC Srebp1 R TGAGGGACTTGCTCTTCTGC F TTCCCTCTGCTTGACTGTG Hmgcs1 R TGTCTCCGTTCCAACTTCC F GCCCTTTCGGAACTTATCAT Cyp51 R TCCAAATCGGCGTAGAGC F CTGGTCCCGATGAAGAGC Dhcr24 R AAAGGGCATAACCAAAGAGG F AGAATCTTGACCTCTTTGACGCTG StAR R GAGACGGTGGTGGATAACGGA F AGGGAGAAGTTGGGTGTCTACGA Cyp11a1 R CGTAGGGCTTGTTGCGGTAGT F GCTCCCTCTGCTTCAACTCCT Cyp17 R CCTGACCTTGAGGCACTTCTTC F CTGGTCCTGGTCTCGTGCGTAT Cyp19a1 R GATGTGTCAAGCATGATCCGTCTC F GACCTGGGGTTTGGAATTGAG 3β-hsd R TAGGAGAAGGTGAATGGGGTGT F TCCTTGGGTGCTATTGTC 17β-hsd R TGCTCCCTTGAGACTCTATC F TTGGTGGAGCGATTTGTC 18s R ATCTCGGGTGGCTGAACG F CAACGAGCGGTTCAGGTGT β-actin R TGGAGTTGAAGGTGGTCTCGT F, sense primers; R, antisense primers
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Gene
61.2
89
60.5
179
59.2
139
60.3
170
59.4
169
53.9
129
59.6
92
ACCEPTED MANUSCRIPT Figure Legends Fig.1. Comparison of Lepr mRNA expression by semi-quantitative RT-PCR in various tissues of laying goose (n=3). 1, brain; 2, hypothalamus; 3, pituitary; 4, adrenal glands; 5, ovary; 6, oviduct; 7, adipose tissue as a positive control; 8, water as a negative control. (A) Gel electrophoresis of semi-quantitative RT-PCR products. (B) Analysis of Lepr mRNA relative to β-actin mRNA. Values represent mean and S.D., and bars with different lowercase letters are significantly
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different (p<0.05).
Fig.2. Expression of Lepr mRNA in goose ovarian theca and granulosa cells from developing follicles (n=3). Prehierarchical follicles were grouped by diameter (4-6 mm, 6-8 mm, and 8-10 mm). Hierarchical follicles were classified by their diameters with the largest yellow-yolk-filled
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follicle (next to ovulate) designated as the F1 follicle and followed by F2, F3, F4, and F5. β-actin and 18S rRNA were used as internal controls. Bars with different lowercase letters are significantly different between the same type of cell from different sized follicles (p<0.05). * and
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** indicate significant differences between theca and granulosa cell in the same size class (p<0.05 and p<0.01, respectively).
Fig.3. Leptin effects (0, 1, 10, and 100 ng/ml) on steroid hormone accumulation in goose granulosa cells. (A) estradiol, (B) progesterone, and (C) testosterone. Values (mean ± S.D.) with different lowercase letters are significantly different (p<0.05).
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Fig.4. Effect of different doses of leptin on the mRNA levels of Lepr by cultured granulosa cells. Values (mean ± S.D.) with different lowercase letters are significantly different (p<0.05). Fig.5. Effect of leptin on expression of selected genes in the sterol/steroid biosynthetic pathway by cultured granulosa cells. (A) Genes regulating sterol biosynthetic pathways, and (B) genes
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regulating steroid biosynthetic pathways. Values (mean ± S.D.) with different lowercase letters are significantly different (p<0.05).
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Fig.6. Exploration of mechanism by which leptin modulated steroid hormone production. indicates a significantly increase of target gene expression, indicates a slightly but not significantly increase, decrease.
indicates a significantly decrease.
indicates a slightly but not significantly
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Figure 1
ACCEPTED MANUSCRIPT Figure 2 a**
Theca cells Granulosa cells
8 6 4
b* ab
bc
cd de
bc** d
a** de
ab
cd
bc** e
2 0 4-6mm
6-8mm 8-10mm
F5
F4
F3
F2
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Follicular size
d de
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LEPR mRNA relative levels
10
F1
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Figure 3
(B)
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(A)
(C)
ACCEPTED MANUSCRIPT Figure 4
leptin-R
a
2
a
b
b
1.5
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Relative Expression
2.5
1 0.5 0
1 10 100 Concentration of leptin(ng/ml)
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0
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Figure 5
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Figure 6
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.
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Revised Role of leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells
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Shenqiang Hu, Chao Gan, Rui Wen, Qihai Xiao, Hua Gou, Hehe Liu, Yingying Zhang, Liang Li, Jiwen Wang*
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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
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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
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significantly stimulated, while progesterone increased slightly and testosterone decreased dramatically after leptin treatment. In accordance with the data for steroids, expression of Lepr,
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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
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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
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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
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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
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(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
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on ovarian function in female chickens due to altered ovarian steroidogenesis and attenuated follicular apoptosis [13, 22].
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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
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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
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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
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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
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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
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(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
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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.
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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.
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synthesis kit (TaKaRa, JAPAN) according to the manufacturer’s instruction with 1 µg of total
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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
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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
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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
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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
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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
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color changes at a wavelength of 450 nm.
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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<0.05) in the pituitary than in other tissues with the exception of adrenal glands, followed by ovary, oviduct, brain, and hypothalamus (Fig. 1).
ACCEPTED MANUSCRIPT 3.2 Lepr mRNA expression during follicle development The mRNA levels of Lepr were determined in different sized follicles from laying geese by qRTPCR (Fig.2). Lepr mRNA was found in both theca and granulosa cells isolated from all follicles. In the 4-8 mm diameter size class and F5, transcripts of Lepr were significantly higher in granulosa cells than in theca cells (p<0.05). By contrast, Lepr gene expression was significantly higher in theca cells than in granulosa cells isolated from F4 and F2 (p<0.05). In addition,
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expression of Lepr in granulosa cells exhibited a tendency of decreasing gradually throughout follicle development, with fluctuating expression in F5 and F3. Noticeably, the peak of Lepr mRNA expression was found in granulosa cells from 4-6 mm follicles, which was significantly higher than any other follicles (p<0.05). As follicle development ensues, expression of Lepr
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mRNA in theca cells underwent a slight decrease from the 6-8 mm cohorts to F5, then exhibited a transient increase and declined later. The mRNA expression level of Lepr was higher in theca cells from F4 than that from other developing follicles (p<0.05).
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3.3 Effect of leptin treatment on steroid hormone release
Leptin-induced estradiol, progesterone, and testosterone production by goose granulosa cells from preovulatory follicles were determined in the media after 24 h culture. As shown in Fig.3, the 1 ng/ml dose of recombinant leptin significantly stimulated estradiol release (p<0.05) in comparison to the control group. However, no significant difference was observed at 10 or 100 ng/ml. Although the effect of recombinant leptin on progesterone levels was not significant at all doses
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(p>0.05), a slight increase was seen after leptin addition. In addition, testosterone release was not significantly altered by leptin treatment (p>0.05), but a strong decline was observed. 3.4 Effect of leptin treatment on the expression of Lepr mRNA
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Changes in Lepr mRNA levels in goose granulosa cells after leptin treatment were shown in Fig.4. A significant increase in the expression of Lepr mRNA was observed at 1 and 10 ng/ml (p<0.05). The addition of high concentrations of leptin (100ng/ml) significantly reduced Lepr mRNA
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expression (p<0.05) when compared with the lower doses, but no significant difference was found compared to controls (p>0.05). 3.5 Expression of sterol/steroidogenic genes induced by leptin treatment After exposure to different doses of recombinant leptin, all selected genes in the control of sterol/steroid biosynthesis were expressed in all cultured granulosa cell preparations but displayed different expression patterns (Fig.5). Among genes regulating sterol biosynthesis, sterol regulatory element binding protein 1 (Srebp1) and cytochrome P450 14α-sterol demethylases (Cyp51) were significantly up-regulated at leptin concentrations of 1 and 10 ng/ml (p<0.05), subsequently decreased at 100 ng/ml. No significant differences were found in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (Hmgcs1) and 3β-hydroxysterol-∆24 reductase (Dhcr24) in response to different concentrations of leptin (p>0.05), when compared with
ACCEPTED MANUSCRIPT those from controls. Within steroidogenic genes, comparison of transcripts of P450 side-chain cleavage enzyme (Cyp11a1) and 17α-hydroxylase (Cyp17) among different doses of leptin showed no significant differences compared to controls (p>0.05), but showed a slightly increasing tendency with elevated concentrations of leptin. The mRNA levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450 aromatase (Cyp19a1) were significantly higher at leptin concentrations of 1 and 10 ng/ml, and 10 ng/ml, respectively, when compared with controls
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(p<0.05). Notably, although there were no significant differences in 3β-hydroxysteroid dehydrogenase/∆5-∆4 isomerase (3β-hsd) or 17β-hydroxysteroid dehydrogenase (17β-hsd) gene expression responding to different doses of leptin when compared with controls (p>0.05), a strong increase was seen. In addition, the proposed pathways and key genes in the regulation of leptin-mediated steroid hormone release in goose preovulatory follicle granulosa cells are
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summarized in Fig.6.
Discussion
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Despite the controversy on the identity of the leptin gene in birds [29-31], increasing evidence suggests the existence of a leptin-like immunoreactive substance [32] and a functional leptin receptor [33, 34]. Furthermore, the effects of exogenous recombinant murine or chicken leptin on chicken lipid metabolism [35, 36] and ovarian follicle development [13, 22] have been demonstrated. In addition, a recent study reported that leptin in vivo administration could promote the recovery of regressed ovary in fasted ducks [37], and the full-length cDNA of goose Lepr has
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been characterized with a high identity to that of other birds [38]. Considering the similar tertiary structures of leptin and highly conserved binding sites between leptin and its receptors in vertebrates [30, 31], it is conceivable that recombinant chicken leptin could be utilized for researches in waterfowl. To the best of our knowledge, the present study was the first to evaluate ovarian expression of goose Lepr mRNA during follicle development and to reveal the role of
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leptin in the regulation of sterol/steroid biosynthesis in goose granulosa cells. Transcripts of Lepr were ubiquitous in all tissues examined, with pituitary and adrenal glands
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being the sites of relatively high gene expression. Such widespread expression of Lepr mRNA was coincident with the involvement of leptin in diverse physiological and pathological processes [7], indicating the central role of leptin in the control of avian reproductive biology and neuroendocrinology through hypothalamic-pituitary-gonadal/adrenal (HPG/HPA) axes. The presence of Lepr mRNA in goose ovary suggests a peripheral effect of leptin, which has also been shown in chicken [34] and turkey [39]. Moreover, the detectable mRNA levels of Lepr in the oviduct supported the possible participation of leptin in the process of egg white protein synthesis, eggshell formation, and final oviposition. Accordingly, similar to other birds, a leptin-like system in controlling female reproduction might also occur in geese. It is well known that avian ovarian follicles are arranged in a distinct follicular hierarchy and are committed to ovulation. Follicle maturation is a hormonally controlled process accompanied by
ACCEPTED MANUSCRIPT the transition from prehierarchical (undifferentiated) to preovulatory (differentiated) stage [40, 41]. In this study, the ubiquity of goose Lepr mRNA in both theca and granulosa cells isolated from all developing follicles further supported a direct action of leptin at the level of the ovary. Besides, the different expression patterns of Lepr in either theca or granulosa cells throughout follicle maturation indicated that leptin might be involved in maintaining the normal follicular hierarchy and regulating follicle development, which was in good accordance with other studies. In ad
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libitum-fed broiler chickens the dramatic up-regulation of ovarian expression of Lepr during follicle development was associated with the formation of abnormal follicular hierarchy and led to significant reproductive dysfunction [21]. During the re-feeding period of fasting hens, the continuation of leptin treatment considerably delayed the recovery of regressed ovary by reducing the entry of white follicles into the hierarchical follicles and inhibiting the growth of yellow
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follicles [13]. The walls of the ovarian follicles could synthesize three major sex steroids including estradiol, testosterone, and progesterone. Estradiol is mainly produced by theca externa cells and
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testosterone is synthesized by theca interna cells, while the granulosa cells are considered as the main resource for progesterone [42]. The steroidogenic activity of all types of cells differs at different stages of follicle development [42]. Therefore, the level of expression of Lepr mRNA in theca and granulosa cells during follicle maturation could be a factor in the regulation of ovarian steroidogenesis. In human granulosa cells, leptin inhibited the stimulatory effects of luteinizing hormone (LH) on estradiol production [8], and passive immunization against leptin in sheep contributed to an abrupt increase in ovarian estradiol secretion [20]. In cultured rat ovarian
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follicles, leptin suppressed the stimulatory effect of insulin-like growth factor I (IGF-I) on follicle stimulating hormone (FSH)-induced estradiol production [19]. The relatively higher-level expression of goose Lepr in granulosa cells from follicles of 4-8 mm in diameter than that from large follicles suggested a more potent inhibitory effect of leptin on insulin-stimulated aromatase
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activity of granulosa cells from small follicles [43, 44]. The significant increase of Lepr gene expression in granulosa cells from F5 follicle might facilitate follicle selection, during which the follicles undergo the transition from an undifferentiated to a differentiated state in association with
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a shift from FSH receptor (FSHR)-dominance to LH receptor (LHR)-dominance [40]. However, expression of Lepr decreased in both theca and granulosa cells between F4 and F1 follicles, which were in good accordance with previously published findings [10, 21], indicating less sensitivity of large follicles to the negative control of leptin as the follicles developed [43]. Notably, transcripts of Lepr were found to be constantly higher in theca cells than in granulosa cells from F4 to F1 follicle, implying a more active action of leptin in theca cells linked to the release of E2 and T. The results from our in vitro experiment provided novel evidence that leptin directly affected the production of steroid hormones independent of HPG influence and altered expression of several sterol/steroidogenic genes in goose granulosa cells. Concentrations of E2 were significantly stimulated at the 1 ng/ml dose of recombinant leptin, whereas no significant difference was observed at 10 or 100 ng/ml when compared with controls. Although there were no statistically
ACCEPTED MANUSCRIPT significant changes in the P4 and T concentrations among all granulosa cells exposed to different doses of leptin, a slight increase in P4 and a strong decline in T secretion were seen with the elevated leptin doses. Parallel observations were found in recent data from other studies. The up-regulated levels of E2 and P4, as well as the down-regulated levels of T induced by recombinant chicken leptin were found in cultured fragments of follicular wall isolated from chicken ovarian follicles [25]. Of particular note is that the difference in the release of P4 and T could result from
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the clearance of theca cells in the current study, because the significant changes observed in Sirotkin et al are not primary, but secondary [25], and may be associated with the granulosa-theca interaction in the secretion of sex steroids [45]. Among the key genes in regulating sterol biosynthesis, significantly elevated mRNA expression levels of Srebp1 and Cyp51 were shown at leptin concentrations of 1 and 10 ng/ml, while neither Hmgcs1 nor Dhcr24 gene expression were
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significantly different compared to controls despite showing a slight increasing tendency. Similarly, genes within biosynthetic pathways were regulated by FSH and two forkhead box
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transcription factor (FOXO1) mutants in cultured granulosa cells [46]. As for the steroidogenic genes, the mRNA levels of StAR and Cyp19a1 were significantly higher at leptin concentrations of 1 and 10 ng/ml, and 10 ng/ml, respectively, when compared with controls. No statistically significant changes but strong up-regulated levels were observed in the expression of 3β-hsd and 17β-hsd compared to the control group. Additionally, comparison of expression of 3β-hsd and 17β-hsd showed significantly higher levels at 1 ng/ml than at 100 ng/ml doses of leptin. Cyp11a1 and Cyp17 gene expression were not significantly altered by leptin treatment in granulosa cells,
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but a slight increase was seen. StAR is considered as the key gene in controlling the rate-limiting step of steroidogenesis by mediating transfer of cholesterol from the outer to the inner mitochondrial membrane where Cyp11a1 promotes the conversion of cholesterol to pregnenolone, which subsequently undergoes conversion to P4 by 3β-hsd [47]. Aromatase enzyme plays key
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roles in the conversion of T into E2 and thus regulates estrogen biosynthesis [48]. Our results showed that leptin significantly stimulated estradiol production through an increase in StAR and Cyp19a1 mRNA levels, and a subtle increasing tendency was observed in P4 secretion in
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association with the increase of StAR, Cyp11a1, and 3β-hsd gene expression. The down-regulated concentrations of T might be linked to the activation of aromatase enzyme (mainly Cyp19a1) by leptin, which in turn accelerated the conversion of T to E2. An alternative explanation is that no statistically significant differences in expression of Cyp17 and 17β-hsd were found in granulosa cells exposed to recombinant leptin in comparison with controls. Besides, since cholesterol is thought to be the precursor for steroid biosynthesis, the elevated mRNA levels of Srebp1 and Cyp51 could promote steroid hormone production in goose granulosa cells. A slight increase in both Hmgcs1 and Dhcr24 expression suggested that they might play a role in this process. Leptin exerts its effect by interacting with its receptors in both the hypothalamic-hypophysis system and the ovary to regulate follicular steroidogenesis [6]. Leptin is able to modulate the expression of its receptors in the rat hypothalamic-pituitary-ovarian axis [49]. A direct action of
ACCEPTED MANUSCRIPT leptin on the ovary in chicken has been previously described [13, 21], and the capability of chicken leptin receptor to bind with endogenous ligand as well as mammalian leptin has also been demonstrated in vitro [50]. Furthermore, the observation of presence of Lepr mRNA in goose ovary, theca, and granulosa cells throughout follicle development indicated the possibility of leptin’s direct effects on the ovary through its receptors. Consistent with changes in steroid hormone production and sterol/steroidogenic gene expression, Lepr mRNA was also up-regulated
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by leptin at its doses of 1 and 10 ng/ml. Thus, it could be inferred that leptin modulates secretion of steroid hormones by regulating cellular sterol/steroid biosynthesis via interaction with its receptor in goose granulosa cells (Summarized in Fig.6).
In conclusion, all these results clearly suggest that leptin is involved in the development of goose
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ovarian follicles. The effect of leptin on ovarian steroid hormone production could be due to altered sterol/steroidogenic gene expression via interaction with its receptor.
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Acknowledgements
The work was supported by the National Waterfowl Industrial Technology System, China (No. CARS-43-6).
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ACCEPTED MANUSCRIPT Table 1 Primer sequences used in this study
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Tm ( )
Size(bp)
60
159
60
92
58.2
91
56.7
104
56.4
83
58.6
87
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sequence (5’ to 3’) F AGCAGTTGCCCGTTGTAT Lepr R AAGGGAGGGTTGGTTTCA F CGAGTACATCCGCTTCCTGC Srebp1 R TGAGGGACTTGCTCTTCTGC F TTCCCTCTGCTTGACTGTG Hmgcs1 R TGTCTCCGTTCCAACTTCC F GCCCTTTCGGAACTTATCAT Cyp51 R TCCAAATCGGCGTAGAGC F CTGGTCCCGATGAAGAGC Dhcr24 R AAAGGGCATAACCAAAGAGG F AGAATCTTGACCTCTTTGACGCTG StAR R GAGACGGTGGTGGATAACGGA F AGGGAGAAGTTGGGTGTCTACGA Cyp11a1 R CGTAGGGCTTGTTGCGGTAGT F GCTCCCTCTGCTTCAACTCCT Cyp17 R CCTGACCTTGAGGCACTTCTTC F CTGGTCCTGGTCTCGTGCGTAT Cyp19a1 R GATGTGTCAAGCATGATCCGTCTC F GACCTGGGGTTTGGAATTGAG 3β-hsd R TAGGAGAAGGTGAATGGGGTGT F TCCTTGGGTGCTATTGTC 17β-hsd R TGCTCCCTTGAGACTCTATC F TTGGTGGAGCGATTTGTC 18s R ATCTCGGGTGGCTGAACG F CAACGAGCGGTTCAGGTGT β-actin R TGGAGTTGAAGGTGGTCTCGT F, sense primers; R, antisense primers
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Gene
61.2
89
60.5
179
59.2
139
60.3
170
59.4
169
53.9
129
59.6
92
ACCEPTED MANUSCRIPT Figure Legends Fig.1. Comparison of Lepr mRNA expression by semi-quantitative RT-PCR in various tissues of laying goose (n=3). 1, brain; 2, hypothalamus; 3, pituitary; 4, adrenal glands; 5, ovary; 6, oviduct; 7, adipose tissue as a positive control; 8, water as a negative control. (A) Gel electrophoresis of semi-quantitative RT-PCR products. (B) Analysis of Lepr mRNA relative to β-actin mRNA. Values represent mean and S.D., and bars with different lowercase letters are significantly
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different (p<0.05).
Fig.2. Expression of Lepr mRNA in goose ovarian theca and granulosa cells from developing follicles (n=3). Prehierarchical follicles were grouped by diameter (4-6 mm, 6-8 mm, and 8-10 mm). Hierarchical follicles were classified by their diameters with the largest yellow-yolk-filled
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follicle (next to ovulate) designated as the F1 follicle and followed by F2, F3, F4, and F5. β-actin and 18S rRNA were used as internal controls. Bars with different lowercase letters are significantly different between the same type of cell from different sized follicles (p<0.05). * and
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** indicate significant differences between theca and granulosa cell in the same size class (p<0.05 and p<0.01, respectively).
Fig.3. Leptin effects (0, 1, 10, and 100 ng/ml) on steroid hormone accumulation in goose granulosa cells. (A) estradiol, (B) progesterone, and (C) testosterone. Values (mean ± S.D.) with different lowercase letters are significantly different (p<0.05).
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Fig.4. Effect of different doses of leptin on the mRNA levels of Lepr by cultured granulosa cells. Values (mean ± S.D.) with different lowercase letters are significantly different (p<0.05). Fig.5. Effect of leptin on expression of selected genes in the sterol/steroid biosynthetic pathway by cultured granulosa cells. (A) Genes regulating sterol biosynthetic pathways, and (B) genes
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regulating steroid biosynthetic pathways. Values (mean ± S.D.) with different lowercase letters are significantly different (p<0.05).
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Fig.6. Exploration of mechanism by which leptin modulated steroid hormone production. indicates a significantly increase of target gene expression, indicates a slightly but not significantly increase, decrease.
indicates a significantly decrease.
indicates a slightly but not significantly
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Figure 1
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Theca cells Granulosa cells
8 6 4
b* ab
bc
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bc** d
a** de
ab
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bc** e
2 0 4-6mm
6-8mm 8-10mm
F5
F4
F3
F2
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Follicular size
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LEPR mRNA relative levels
10
F1
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Figure 3
(B)
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(A)
(C)
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leptin-R
a
2
a
b
b
1.5
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Relative Expression
2.5
1 0.5 0
1 10 100 Concentration of leptin(ng/ml)
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0
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Figure 5
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Figure 6