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Variation in sequences and mRNA expression levels of growth hormone (GH), insulin-like growth factor I (IGF-I) and II (IGF-II) genes between prolific Lezhi black goat and non-prolific Tibetan goat (Capra hircus)
General and Comparative Endocrinology 187 (2013) 1–5
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Short Communication
Variation in sequences and mRNA expression levels of growth hormone (GH), insulin-like growth factor I (IGF-I) and II (IGF-II) genes between prolific Lezhi black goat and non-prolific Tibetan goat (Capra hircus) q Xiang-Dong Zi a,⇑, Xiao-Kun Mu a,b, Yong Wang a a Sichuan Provincial Key-Laboratory of Protection and Utilization of Animal Genetic Resources in Qinghai-Tibet Plateau, Southwest University for Nationalities, Chengdu 610041, PR China b Zhaoqing University, Zhaoqing 526061, PR China
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Article history: Received 3 December 2012 Revised 27 February 2013 Accepted 20 March 2013 Available online 8 April 2013 Keywords: Growth hormone IGF-I IGF-II Cloning Gene expression Goat
a b s t r a c t Growth hormone (GH), insulin-like growth factor-I (IGF-I), and II (IGF-II) play a key role in the development of preantral to preovulatory follicles in some species. To better understand the role of these genes in controlling follicular development and fecundity in goats, in the present study, we first cloned the cDNA encoding GH, IGF-I and IGF-II from prolific Lezhi black goat and non-prolific Tibetan goat (Capra hircus), and their mRNA expression between the two breeds were compared. By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length 688-bp GH, 493-bp IGF-I, and 566-bp IGF-II cDNAs, encoding for 217 amino acid (aa) GH, 154 aa IGF-I, and 179 aa IGF-II putative proteins. Analysis of their nucleotide and amino acid sequences revealed a high degree of identity between the two breeds, although one base change in GH resulted in one amino acid substitution in the translated proteins. However, two base changes in IGF-I and IGF-II did not lead to any amino acid changes. Real-time PCR analyses revealed that in the middle of estrus, GH, IGF-I and IGF-II genes were expressed, albeit at different levels, in all three tissues (anterior pituitary, endometrium and ovary) examined. GH was most highly expressed in ovary (P < 0.01) and its expression was greater in all three tissues examined in Lezhi black goat than in Tibetan goat (P < 0.05). IGF-I and IGF-II genes were expressed at a higher (P < 0.05) level in anterior pituitary of Lezhi black goat than that in Tibetan goat, but they had a similar expression pattern in endometrium and ovary. These results provide the foundation of information required for future studies of these gene effects on goat fecundity. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Reproductive rate, especially fecundity, is one of the most economically important traits in animal production and is regulated by genetic and environmental factors. Ovulation rate can be a major determinant of reproductive efficiency. In cattle, single ovulations occur most frequently, and in sheep and goats the number of ova released can range from one to many depending upon the breed, whilst the pig is polyovular. The processes of recruitment and selection determine the number of ovulatory follicles in all these species with FSH and subsequently LH playing major roles
q The goat GH, IGF-I, and IGF-II cDNA sequences reported in this paper have been deposited in the GenBank database as the following accession numbers: JF813118, JF896275, and JF896277 for Lezhi black goat, and JF896274, JF896276, and JF896278 for Tibetan goat respectively ⇑ Corresponding author. Address: College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, PR China. Fax: +86 28 85522310. E-mail address: [email protected] (X.-D. Zi).
0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.03.023
(Hunter et al., 2004; Silva et al., 2009). However, primordial to early antral follicle development has generally been considered to be largely gonadotrophin-independent and mechanisms governing the initiation of growth of the primordial follicles are not completely known. There is a growing body of evidence that growth hormone (GH) and members of the insulin-like growth factors (IGF-I and IGF-II) family system (Baker et al., 1996; Echternkamp et al., 2004; Hastie and Haresign, 2006; Hunter et al., 2004; Li et al., 2011; Lucy, 2011; Reinecke, 2010; Shimizu et al., 2008; Silva et al., 2009; Zhao et al., 2001; Zhao et al., 2002; Zhou et al., 1996) play a key role both in the development of preantral to preovulatory follicles and in the process of follicular atresia. Growth hormone exerts direct and/or indirect effects on virtually every organ in the body, with IGF-I mediating its indirect actions (Butler and Le Roith, 2001; Duan et al., 2010), whereas placental-specific IGF-II is a major modulator of placental and fetal growth (Constância et al., 2002; DeChiara et al., 1990; Velazquez et al., 2008). Growth hormone, a single-chain protein belongs to the structurally and functionally related prolactin, somatolactin, and placental
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lactogen family and is synthesized predominantly by the somatotrophs of the anterior pituitary (Ayuk and Sheppard, 2006; Filby and Tyler, 2007), but also by a number of extrapituitary sites (Abir et al., 2008; Carlsson et al., 1993; Le Roith et al., 2001). IGF-I and IGF-II are single-chain polypeptides with structural homology to proinsulin. Mature IGF-I and IGF-II consist of B, C, A, and D domains (Filby and Tyler, 2007; Funes et al., 2006; Jones and Clemmons, 1995). IGF-I is produced in organs of reproductive significance such as hypothalamus, anterior pituitary, ovaries, oviducts, and uterus (Adam et al., 2000; Bach and Bondy, 1992; Constância et al., 2002; Eppler et al., 2007; Filby and Tyler, 2007; Funes et al., 2006; Gonzalez-Parra et al., 2001; Hastie and Haresign, 2006; Hunter et al., 2004; Iida, 2005; Irwin and Van Der Kraak, 2011; Jevdjovic et al., 2007; Mikawa et al., 1995b; Olchovsky et al., 1993; Silva et al., 2008; Velazquez et al., 2008). However, most of the IGF-I measured in blood is produced by the liver (Funes et al., 2006; Yakar et al., 1999). IGF-II mRNAs were also detected in liver and in variety of extrahepatic tissues, as was IGF-I mRNA (Funes et al., 2006; Silva et al., 2009). Recently, the IGF system has garnered more attentions for its potential role in ovarian development with the discovery of the gonad specific IGF-III, a third form of IGF (Berishvilia et al., 2010; Irwin and Van Der Kraak, 2011; Reinecke, 2010). However, there is little information about GH and IGF system in goats (Mikawa et al., 1995a,b; Silva et al., 2008; Yamano et al., 1988; Yato et al., 1988). The present study, for the first time, investigated the nucleotide sequences and mRNA expression levels of GH, IGF-I and IGF-II genes in the Lezhi black goats, a local Chinese breed famous for its high fecundity (producing 3–5 kids per kidding), and Tibetan goats (Capra hircus), a single-birth breed characterized by adapting to cold, hypoxic ecological conditions in the Qinghai-Tibet Plateau. This intends to examine whether base mutations or expression levels of mRNA of these genes could be associated with the reproductive difference in goats. 2. Materials and methods 2.1. Animals and sample collection All experimental procedures were performed according to the guide for animal care and use of laboratory animals of the Institutional Animal Care and Use Committee of Southwest University for Nationalities. Lezhi black goats were supplied by the Lezhi Black Goat Breeding Farm in Lezhi County (30°300 N, 105°020 E and 596.3 m altitude), China, and Tibetan goats were purchased from Li County (31°420 N, 103°160 E and 2800 m altitude) of Qinghai-Tibet Plateau in China during the breeding season (October). Lezhi black goats were kept in shelters, and Tibetan goats were grazed on the pasture. Animals aged 3–5 year, with a history of multiple births (twin or triplet births) for Lezhi black goats (n = 6) and single birth for Tibetan goats (n = 6) were selected to investigate the nucleotide sequences and mRNA expression levels of GH, IGF-I, and IGF-II genes. Estrus was detected twice a day, and a doe was considered in estrus only when she allowed the male to mount. Goats were slaughtered at 12–24 h after onset of estrus (in the middle of estrus) for collection of anterior pituitaries, ovaries, and endometria. Part of the removed tissue samples was snap-frozen in liquid nitrogen and then stored at 80 °C to be used for RNA extraction. 2.2. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from the tissue samples (pituitaries, ovaries, and endometria) using RNAprep pure Tissue Kit (Tiangen Biotech, Beijing), following the manufacture’s instructions. The
RNA samples were spectrophotometrically quantified at A260 nm and A280 nm, and then analyzed for their integrities on agarose gel (1.2%). Reverse transcription was performed using RNA PCR kit (AMV) (TaKaRa, Dalian, China) in a volume of 10 lL reaction mixture (Zi et al., 2012). The generated cDNA was then amplified using gene specific primers (GH: 50 -CAATGGGAAAAATCAGCA GTCT-30 /50 GAGGGGTAGTAACAACAGATGG-30 ; IGF-I: 50 -CAATGGGA AAAATC AGCAGTCT-30 /50 -ATT CTTCGCTCTTTAGGAAGGGC-30 ; GF-II: 50 -GCAGAGACATCAATGGG GATC-30 /50 -ACTTTGGCTCACT TCTAATCGC-30 , sense and antisense, respectively) designed from regions of GH, IGF-I, and IGF-II cDNAs conserved between sheep, goat and yak available in the NCBI GenBank database. PCR amplification was carried out in a volume of 25 lL of reaction mixture (Zi et al., 2012). The purified PCR products were ligated into a pMD19-T vector and transformed into DH5a ( Escherichia coli) using standard techniques (Sambrook et al., 1989). The DNA sequence was determined with an ABI PRISM 3700 automated DNA sequencer by Shanghai Invitrogen Biotechnology (Shanghai, China). 2.3. Sequence analysis The sequences of goat GH, IGFI and IGFII cDNA were subjected to BLAST analysis to verify that the sequence was of GH, IGFI and IGFII. The nucleotide and deduce amino acid sequence identity was performed using the Clustal option in MegAlign (Lasergene Software, DNASTAR). 2.4. Quantitative real-time PCR (qPCR) GAPDH gene was chosen as reference gene for normalizing expression levels of target genes. Primers specific for target goat genes were designed with Beacon Designer 7.0 software (Premier Biosoft International, Palo Alto, CA USA) according to manufacturers guidelines (GH: 50 -CTGAAGGACCTGGAGGAA-30 /50 -GGA GAGCA GACCGTAGTT-30 ; IGF-I: 50 -TGCTCTCCAGTTCGTGTG-3/5-CATCTCCA GCCTCCTCAG-30 ; IGF- II: 50 -CACCCTCCAGTTTGTCTG-30 /50 -GGCACAG TAAGTCTCCAG-30 ; GAPDH: 50 -AGTTCCACGGCACA GTCAAG30 /50 -ACTCAGCACCAGCATCACC-30 ). Total RNA of different tissues was extracted, and reverse transcribed as above. Real-time PCR was performed using on an iCycler iQ5 Real-Time Detection System (Bio-Rad, CA, USA) with the SsoFast™ EvaGreen Supermix (Bio-Rad, CA, USA) in a volume of 10 lL. The cycle parameters were 3 min at 95 °C, followed by 45 cycles of 30 s at 95 °C and 5 s at Tm, followed by a melting curve analysis. The efficiency of each primer pair and mean Ct (threshold cycles) values were calculated and used for determination of target gene RNA transcript levels (Pfaffl, 2001). Each sample was tested in triplicate. 2.5. Statistical analysis All data were expressed as mean values ± SEM. Statistical differences were assessed by Student’s t-test or one-way ANOVA followed by Dunn’s multiple pairwise comparison test. 3. Results 3.1. Cloning and molecular characterization of cDNAs for GH, IGF-I, and IGF-II genes As expected, the goat GH, IGF-I, and IGF-II cDNA sequences were found to consist of 688, 493, and 566 bp, respectively. They were respectively submitted to NCBI GenBank Accession Nos. JF813118, JF896275, and JF896277 for Lezhi black goat, and JF896274, JF896276, and JF896278 for Tibetan goat respectively. The coding region (nucleotides 6–656) of GH cDNA encoded a
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putative GH peptide of 217-amino acids (aa) and the predicted cleavage of the signal peptide (residues 1–26) would free a mature GH of 191 aa (residues 27–217) (Fig. 1A). Four cysteine residues (underlined) which may participate in the construction of structural features present in goat GH at the same positions as those for other vertebrate GHs (Kawauchi and Yasuda, 1989). The coding region of IGF-I cDNA encoded a putative IGF-I of 154 aa. Predicted cleavage of the 49 aa signal peptide (residues 1–49) and the 35 aa C-terminal extension (E) – peptide (residues 119–154) would free a mature IGF-I of 70 aa. The four domains, characteristic of mature IGF-I, were identified as follows: residues 50–75 (B-domain), 76– 90 (C-domain), 91–111 (A-domain), and 112–119 (D-domain). The six characteristic cysteine residues involved in the formation of the disulfide bounds were conserved (B6, B18, A6, A7, A11, and A20) (Fig. 1B). For goat IGF-II, the signal peptide consisted of 24 aa. A total of 32, 8, 21, 6, and 88 aa were detected for B-, C-, A-, D-, and E- domains, respectively. The sequence exhibited the six characteristic cysteine residues (B9, B21, A6, A7, A11, and A20) (Fig. 1C).
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Comparison of the nucleotide sequences between the two breeds showed that the sequence identities of GH, IGF-I, and IGFII were respectively 99.85, 99.59, and 99.65%. There was one base changes in GH (C to T from Lezhi black to Tibetan goat at bases 372) resulting in one amino acid substitution (Val 123 Phe). There were two base changes in IGF-I (G to A and T to C at bases 146 and 177), and two base changes in IGF-II (C to T and T to C at bases 267 and 315), but both of these changes do not result in amino acid substitution. 3.2. Tissue expression of GH, IGF-I, and IGF-II genes In the middle of estrus, GH, IGF-I and IGF-II genes were expressed, albeit at different levels, in all three tissues examined in both prolific Lezhi black goat and non-prolific Tibetan goat (Fig. 2A–C). In the same breed, GH was highly expressed in ovary (P < 0.01) than in anterior pituitary and endometrium. Comparison of the two breeds, GH mRNA expression was higher (P < 0.05) in all three tissues of Lezhi black goats than that in Tibetan goats
Fig. 1. Nucleotide and deduced amino acid sequences of Tibetan goat (Capra hircus) GH (A), preproIGF-I (B) and preproIGF-II (C). Open reading frames are in uppercase. Signal peptides are shaded. IGF-I and IGF-II mature proteins consisting of B, C, A, and D domains are in boldfaced letters. The six conserved cysteine residues are underlined. Nucleotides and amino acids different from those of Lezhi black goat are indicated as bold double-underlined. Numbering to the left denotes nucleotide number (regular font) and translated amino acid number (bold italic font).
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Fig. 2. GH, IGF-I, and IGF-II mRNA levels in different tissues of Tibetan goat (Capra hircus) and Lezhi black goat. Transcripts were quantitated using total RNA from tissues of six does at 12–24 h after onset of estrus for each breed. The experiments were carried out in three replicates. The expression of these mRNAs was normalized to the expression of GAPDH measured in the same RNA preparation. Results were expressed as the mean ± SEM. Different letters in the same tissue indicate the difference between breeds (P < 0.05).
(Fig. 2A). IGF-I gene was expressed at a significantly (P < 0.05) higher level in anterior pituitary of Lezhi black goat than that in Tibetan goat, but it had a similar expression pattern in ovary and endometrium of the two breeds (Fig. 2B and C). In both of these breeds, IGF-II was highly expressed in ovary and endometrium than in anterior pituitary (P < 0.01). IGF-II mRNA expression was higher (P < 0.05) in anterior pituitary of Lezhi black goat than that in Tibetan goat, but there was no difference in ovary and endometrium between the two breeds (P > 0.05). 4. Discussion In this study, we isolated cDNA clones encoding GH, IGF-I and IGF-II molecules of the prolific Lezhi black goat and non-prolific Tibetan goat (C. hircus). Analysis of their nucleotide and amino acid sequences revealed a high degree of identity between the two goat breeds, although one base change in GH resulted in one amino acid
substitution in the translated proteins (Val 123 Phe). Comparing the nucleotide and amino acid sequences from other goat breeds (Yamano et al., 1988; Yato et al., 1988), it appears that this mutation in GH only occurs in Lezhi black goat. Mutations in GH have been found to dramatically change its biological activity; when glycine at 120 of GH is replaced by arginine, lysine, or a variety of amino acids, GH is converted from a growth enhancer to a growth suppressor or a GH antagonist (Chen et al., 1991). However, two base changes in IGF-I and IGF-II did not lead to any amino acid changes. Therefore, the amino acid substitution in GH may be an important molecular mechanism that regulates the differential fecundity of these goat breeds, but the base changes in IGF-I and IGF-II may be attributed to the evolutional divergence. During the last decades, involvement of GH, IGFs and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied. In vitro studies and knockout experiments demonstrated in most mammalian species, GH and IGFs do not appear to be required for primordial to primary follicles transition, but they promote secondary follicle growth and antrum formation. In that regard, GH enhances the development of small antral follicles to the gonadotrophin-dependent stages and stimulates oocyte maturation, whereas IGFs increase granulose cell proliferation, steroidogenesis and oocyte growth in most mammalian species (Hastie and Haresign, 2006; Hunter et al., 2004; Silva et al., 2009; Velazquez et al., 2008). However, there is little information about them in goats (Mikawa et al., 1995a,b; Silva et al., 2008), especially, no clear differences between breeds with different fecundity have been reported. Therefore, to enable investigations of GH, IGF-I and IGF-II genes signaling at expression levels on fecundity, the differences in tissue (anterior pituitary, ovary, and endometrium) mRNA expression levels of these genes in the middle of estrus were compared between the prolific Lezhi black goats and non-prolific Tibetan goats in the present study. GH is synthesized and stored by somatotroph cells within the anterior pituitary gland (Ayuk and Sheppard, 2006; Filby and Tyler, 2007). GH mRNA is highly localized to the pituitary, and low-level expression in gonad and in some other tissues (Abir et al., 2008; Biga et al., 2004; Filby and Tyler, 2007). Extra-pituitary GH is not believed to be released into systematic circulation since plasma GH is undetectable in hypophysectomized animals (Lazarus and Scanes, 1988). It is, therefore, likely to reflect local paracrine/autocrine effects of GH in goat gonad, distinct from (and in additional to) GH’s systemic effects. In the present study, the finding that goat GH mRNA is highly expressed to the pituitary is in agreement with most findings (Abir et al., 2008; Biga et al., 2004; Filby and Tyler, 2007). In contrast, GH mRNA is also highly expressed in ovary and endometrium. In addition, GH mRNA was significantly greater in all tissues examined in prolific Lezhi black goat than in non-prolific Tibetan goat, indicating that GH may play an important role in the development of preovulatory follicles and the number of ovulatory follicles, and hence, ovulation rate in goat. However, future studies are needed to confirm whether GH can really highly expressed in ovary and endometrium. In agreement with previous studies (Bach and Bondy, 1992; Filby and Tyler, 2007; Mikawa et al., 1995b), IGF-I and IGF-II mRNAs were also found to be expressed in all three tissues examined, and they had a similar expression pattern in ovary and endometrium of these two breeds. Interestingly, both of IGF-I and IGF-II mRNAs were expressed at a significantly higher level in anterior pituitary of prolific Lezhi black goat than in non-prolific Tibetan goat. The IGF-I and IGF-II synthesized by anterior pituitary cells regulate pituitary function, including release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Adam et al., 2000; Gonzalez-Parra et al., 2001; Li et al., 2011). Therefore, the differential fecundity of these goat breeds might be partly controlled by the difference in IGFs expression in their anterior pituitary.
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In summary, the cDNA encoding GH, IGF-I and IGF-II were cloned, sequenced and characterized in prolific Lezhi goat and non-prolific Tibetan goat. They prove to be identical to each other except for one base change resulting in one amino acid substitution in GH, two base changes in IGF-I, and two base changes in IGF-II between the two breeds. Using real-time PCR techniques, we have demonstrated that GH mRNA expression was greater in anterior pituitary, ovary, and endometrium of Lezhi black goat than in Tibetan goat, but IGF-I and IGF-II expressions were only greater in anterior pituitary of Lezhi black goat. These results provide the foundation of information required for future studies of these gene effects on goat fecundity. Acknowledgments This work was supported by National Key Technology R&D Program (No. 2012BAD13B06 and No. 2008BADC3B01) and Southwest University for Nationalities (12NZYTH07; 2011XWD-S0905). References Abir, R., Garor, R., Felz, C., Nitke, S., Krissi, H., Fisch, B., 2008. Growth hormone and its receptor in human ovaries from fetuses and adults. Fertil. Steril. 90, 1333– 1339. Adam, C.L., Gadd, T.S., Findlay, P.A., Wathes, D.C., 2000. IGF-I stimulation of luteinizing hormone secretion, IGF-binding proteins (IGFBPs) and expression of mRNAs for IGFs, IGF receptors and IGFBPs in the ovine pituitary gland. J. Endocrinol. 166, 247–254. Ayuk, J., Sheppard, M.C., 2006. Growth hormone and its disorders. Postgrad. Med. J. 82, 24–30. Bach, M.A., Bondy, C.A., 1992. Anatomy of the pituitary insulin-like growth factor system. Endocrinology 131, 2588–2594. Baker, J., Hardy, M.P., Zhou, J., Bondy, C., Lupu, F., Bellve, A.R., Efstratiadis, A., 1996. Effects of an IGF-I gene null mutation on mouse reproduction. Mol. Endocrinol. 10, 903–918. Berishvilia, G., Baroiller, J.F., Eppler, E., Reinecke, M., 2010. Insulin-like growth factor-3 (IGF-3) in male and female gonads of the tilapia: development and regulation of gene expression by growth hormone (GH) and 17aethinylestradiol (EE2). Gen. Comp. Endocrinol. 167, 128–134. Biga, P.R., Schelling, G.T., Hardy, R.W., Cain, K.D., Overturf, K., Ott, T.L., 2004. The effects of recombinant bovine somatotropin (rbST) on tissue IGF-I, IGF-I receptor, and GH mRNA levels in rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 135, 324–333. Butler, A.A., Le Roith, D., 2001. Control of growth by the somatotrophic axis: growth hormone and the insulin-like growth factors have related and independent roles. Annu. Rev. Physiol. 63, 141–164. Carlsson, B., Nilsson, A., Isaksson, O.G.P., Billig, H., 1993. Growth hormone-receptor messenger RNA in the rat ovary: regulation andlocalization. Mol. Cell. Endocrinol. 95, 59–66. Chen, W.Y., Wight, D.C., Mehta, B.V., Wagner, T.E., Kopchick, J.J., 1991. Glycine 119 of bovine growth hormone is critical for growth promoting activity. Mol. Endocrinol. 5, 1845–1852. Constância, M., Hemberger, M., Hughes, J., Dean, W., Ferguson-Smith, A., Fundele, R., Stewart, F., Kelsey, G., Fowden, A., Sibley, C., Reik, W., 2002. Placental-specific IGF-II is a major modulator of placental and fetal growth. Nature 417, 945–948. DeChiara, T.M., Efstratiadis, A., Robertson, E.J., 1990. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 345, 78–80. Duan, C.M., Ren, H.X., Gao, S., 2010. Insulin-like growth factors (IGFs), IGF receptors, and IGF-binding proteins: Roles in skeletal muscle growth and differentiation. Gen. Comp. Endocrinol. 167, 344–351. Echternkamp, S.E., Roberts, A.J., Lunstra, D.D., Wise, T., Spicer, L.J., 2004. Ovarian follicular development in cattle selected for twin ovulations and births. J. Anim. Sci. 82, 459–471. Eppler, E., Jevdjovic, T., Maake, C., Reinecke, M., 2007. Insulin-like growth factor I (IGF-I) and its receptor (IGF-1R) in the rat anterior pituitary. Eur. J. Neurosci. 25, 191–200. Filby, A.L., Tyler, C.R., 2007. Cloning and characterization of cDNAs for hormones and/or receptors of growth hormone, insulin-like growth factor-I, thyroid hormone, and corticosteroid and the gender-, tissue-, and developmentalspecific expression of their mRNA transcripts in fathead minnow (Pimephales promelas). Gen. Comp. Endocrinol. 150, 151–163. Funes, V., Asensio, E., Ponce, M., Infante, C., Canavate, J.P., Manchado, M., 2006. Insulin-like growth factors I and II in the sole Solea senegalensis: cDNA cloning and quantitation of gene expression in tissues and during larval development. Gen. Comp. Endocrinol. 149, 166–172. Gonzalez-Parra, S., Argente, J., Chowen, J.A., Van Kleffens, M., van Neck, J.W., Lindenbeigh-Kortleve, D.J., Drop, S.L., 2001. Gene expression of the insulin-like growth factor system during postnatal development of the rat pituitary gland. J. Neuroendocrinol. 13, 86–93.
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Hastie, P.M., Haresign, W., 2006. Expression of mRNAs encoding insulin-like growth factor (IGF) ligands, IGF receptors and IGF binding proteins during follicular growth and atresia in the ovine ovary throughout the oestrous cycle. Anim. Reprod. Sci. 92, 284–299. Hunter, M.G., Robinson, R.S., Mann, G.E., Webb, R., 2004. Endocrine and paracrine control of follicular development and ovulation rate in farm species. Anim. Reprod. Sci. 82–83, 461–477. Iida, K., Rosen1, C.J., Ackert-Bicknell, C., Thorner, M.O., 2005. Genetic differences in the IGF-I gene among inbred strains of mice with different serum IGF-I levels. J. Endocrinol. 186, 481–489. D.A. Irwin, G.J. Van Der Kraak, Regulation of the novel insulin-like growth factor III ligand in the ovary of zebrafish, Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology doi: 10.3389/conf.fendo.2011.04.00144. Jevdjovic, T., Bernays, R.L., Eppler, E., 2007. Insulin-like growth factor-I mRNA and peptide in the human anterior pituitary. J. Neuroendocrinol. 19, 335–341. Jones, J.I., Clemmons, D.R., 1995. Insulin-like growth factors and their binding proteins: biological actions. Endocr. Rev. 16, 3–34. Kawauchi, H., Yasuda, A., 1989. Evolutionary aspects of growth hormones from nonmammalian species. In: Muller, E.E., Cocchi, D., Locatelli, V. (Eds.), Advances in Growth Hormones and Growth Factor Research. Pythagora Press/Springer, Roma-Milano/Berlin, pp. 51–68. Lazarus, D.D., Scanes, C.G., 1988. Acute effects of hypophysectomy and administration of pancreatic and thyroid hormones on circulating concentrations of somatomedin C in young chickens: relationships between growth hormone and somatomedin C. Domest. Anim. Endocrinol. 5, 283–289. Le Roith, D., Bondy, C., Yakar, S., Liu, J.L., Butler, A., 2001. The somatomedin hypothesis. Endocr. Rev. 22 (2001), 53–74. Li, L., Fu, Y.C., Xu, J.J., Chen, X.C., Lin, X.H., Luo, L.L., 2011. Caloric restriction promotes the reproductive capacity of female rats via modulating the level of insulin-like growth factor-1 (IGF-1). Gen. Comp. Endocrinol. 174, 232–237. Lucy, M.C., 2011. Growth hormone regulation of follicular growth. Reprod. Fertil. Dev. 24, 19–28. Mikawa, S., Yoshikawa, G.-I., Aoki, H., Yamano, Y., Sakai, H., Komano, T., 1995a. Dynamic aspects in the expression of the goat insulin-like growth factor-I (IGFI) gene: diversity in transcription and post-transcription. Biosci. Biotechnol. Biochem. 59, 87–92. Mikawa, S., Yoshikawa, G.-I., Yamano, Y., Sakai, H., Komano, T., Hosoi, Y., Utsumi, K., 1995b. Tissue- and development-specific expression of goat insulin-like growth factor-I (IGF-I) mRNAs. Biosci. Biotechnol. Biochem. 59, 759–761. Olchovsky, D., Song, J., Gelato, M.C., Sherwood, J., Spatola, E., Bruno, J.F., Berelowitz, M., 1993. Pituitary and hypothalamic insulin-like growth factor-I (IGF-I) and IGF-I receptor expression in food-deprived rats. Mol. Cell. Endocrinol. 93, 193– 198. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Res. 29, 2002–2007. Reinecke, M., 2010. Insulin-like growth factors and fish reproduction. Biol. Reprod. 82, 656–661. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbour Library Press. Shimizu, T., Murayama, C., Sudo, N., Kawashima, C., Tetsuka, M., Miyamoto, A., 2008. Involvement of insulin and growth hormone (GH) during follicular development in the bovine ovary. Anim. Reprod. Sci. 106, 143–152. Silva, J.R.V., Brito, I.R., Leitao, C.C.F., Silva, A.W.B., Passos, M.J., Vasconcelos, G.L., 2008. Protein and messenger RNA quantification of insulin-like growth factor-1 (IGF-1) in goat ovarian follicles. Acta Sci. Vet. 36 (Suppl. 2), 466. Silva, J.R.V., Figueiredo, J.R., van den Hurk, R., 2009. Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis. Theriogenology 71, 1193–1208. Velazquez, M.A., Spicer, L.J., Wathes, D.C., 2008. The role of endocrine insulin-like growth factor-I (IGF-I) in female bovine reproduction. Domest. Anim. Endocrinol. 35, 325–342. Yakar, S., Liu, J.-L., Stannard, B., Butler, A., Accili, D., Saur, B., LeRoith, D., 1999. Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc. Nat. Acad. Sci. USA 96, 7324–7329. Yamano, Y., Oyabayashi, K., Okuno, M., Yato, M., Kioka, N., Manabe, E., Hashi, H., Sakai, H., Komano, T., Utsumi, K., Iritani, A., 1988. Cloning and sequencing of cDNA that encodes goat growth hormone. FEBS Lett. 228, 301–304. Yato, M., Yamano, Y., Oyabayashi, K., Okuno, M., Kioka, N., Manabe, E., Hashi, H., Sakai, H., Komano, T., Utsumi, K., Iritani, A., 1988. Nucleotide sequence of the growth hormone gene cDNA from goat Capra hircus L. (Tokara). Nucleic Acids Res. 16, 3578. Zhao, J., Taverne, M.A., van de Weijden, G.C., Bevers, M.M., van den Hurk, R., 2001. Insulin-like growth factor-I (IGF-I) stimulates the development of cultured rat pre-antral follicles. Mol. Reprod. Dev. 58, 287–296. Zhao, J., Taverne, M.A., van de Weijden, G.C., Bevers, M.M., van den Hurk, R., 2002. Immunohistochemical localisation of growth hormone (GH), GH receptor (GHR), insulin-like growth factor I (IGF-I) and type I IGF-I receptor, and gene expression of GH and GHR in rat pre-antral follicles. Zygote 10, 85–94. Zhou, J., Adesanya, O.O., Vatzias, G., Hammond, J.M., Bondy, C.A., 1996. Selective expression of insulin-like growth factor system components during porcine ovary follicular selection. Endocrinology 137, 4893–4901. Zi, X.D., Chen, D.W., Wang, H.M., 2012. Molecular characterization, mRNA expression of prolactin receptor (PRLR) gene during pregnancy, nonpregnancy in the yak (Bos grunniens). Gen. Comp. Endocrinol. 175, 384–388.
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Short Communication
Variation in sequences and mRNA expression levels of growth hormone (GH), insulin-like growth factor I (IGF-I) and II (IGF-II) genes between prolific Lezhi black goat and non-prolific Tibetan goat (Capra hircus) q Xiang-Dong Zi a,⇑, Xiao-Kun Mu a,b, Yong Wang a a Sichuan Provincial Key-Laboratory of Protection and Utilization of Animal Genetic Resources in Qinghai-Tibet Plateau, Southwest University for Nationalities, Chengdu 610041, PR China b Zhaoqing University, Zhaoqing 526061, PR China
a r t i c l e
i n f o
Article history: Received 3 December 2012 Revised 27 February 2013 Accepted 20 March 2013 Available online 8 April 2013 Keywords: Growth hormone IGF-I IGF-II Cloning Gene expression Goat
a b s t r a c t Growth hormone (GH), insulin-like growth factor-I (IGF-I), and II (IGF-II) play a key role in the development of preantral to preovulatory follicles in some species. To better understand the role of these genes in controlling follicular development and fecundity in goats, in the present study, we first cloned the cDNA encoding GH, IGF-I and IGF-II from prolific Lezhi black goat and non-prolific Tibetan goat (Capra hircus), and their mRNA expression between the two breeds were compared. By reverse transcriptase-polymerase chain reaction (RT-PCR) strategy, we obtained full-length 688-bp GH, 493-bp IGF-I, and 566-bp IGF-II cDNAs, encoding for 217 amino acid (aa) GH, 154 aa IGF-I, and 179 aa IGF-II putative proteins. Analysis of their nucleotide and amino acid sequences revealed a high degree of identity between the two breeds, although one base change in GH resulted in one amino acid substitution in the translated proteins. However, two base changes in IGF-I and IGF-II did not lead to any amino acid changes. Real-time PCR analyses revealed that in the middle of estrus, GH, IGF-I and IGF-II genes were expressed, albeit at different levels, in all three tissues (anterior pituitary, endometrium and ovary) examined. GH was most highly expressed in ovary (P < 0.01) and its expression was greater in all three tissues examined in Lezhi black goat than in Tibetan goat (P < 0.05). IGF-I and IGF-II genes were expressed at a higher (P < 0.05) level in anterior pituitary of Lezhi black goat than that in Tibetan goat, but they had a similar expression pattern in endometrium and ovary. These results provide the foundation of information required for future studies of these gene effects on goat fecundity. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Reproductive rate, especially fecundity, is one of the most economically important traits in animal production and is regulated by genetic and environmental factors. Ovulation rate can be a major determinant of reproductive efficiency. In cattle, single ovulations occur most frequently, and in sheep and goats the number of ova released can range from one to many depending upon the breed, whilst the pig is polyovular. The processes of recruitment and selection determine the number of ovulatory follicles in all these species with FSH and subsequently LH playing major roles
q The goat GH, IGF-I, and IGF-II cDNA sequences reported in this paper have been deposited in the GenBank database as the following accession numbers: JF813118, JF896275, and JF896277 for Lezhi black goat, and JF896274, JF896276, and JF896278 for Tibetan goat respectively ⇑ Corresponding author. Address: College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, PR China. Fax: +86 28 85522310. E-mail address: [email protected] (X.-D. Zi).
0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.03.023
(Hunter et al., 2004; Silva et al., 2009). However, primordial to early antral follicle development has generally been considered to be largely gonadotrophin-independent and mechanisms governing the initiation of growth of the primordial follicles are not completely known. There is a growing body of evidence that growth hormone (GH) and members of the insulin-like growth factors (IGF-I and IGF-II) family system (Baker et al., 1996; Echternkamp et al., 2004; Hastie and Haresign, 2006; Hunter et al., 2004; Li et al., 2011; Lucy, 2011; Reinecke, 2010; Shimizu et al., 2008; Silva et al., 2009; Zhao et al., 2001; Zhao et al., 2002; Zhou et al., 1996) play a key role both in the development of preantral to preovulatory follicles and in the process of follicular atresia. Growth hormone exerts direct and/or indirect effects on virtually every organ in the body, with IGF-I mediating its indirect actions (Butler and Le Roith, 2001; Duan et al., 2010), whereas placental-specific IGF-II is a major modulator of placental and fetal growth (Constância et al., 2002; DeChiara et al., 1990; Velazquez et al., 2008). Growth hormone, a single-chain protein belongs to the structurally and functionally related prolactin, somatolactin, and placental
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lactogen family and is synthesized predominantly by the somatotrophs of the anterior pituitary (Ayuk and Sheppard, 2006; Filby and Tyler, 2007), but also by a number of extrapituitary sites (Abir et al., 2008; Carlsson et al., 1993; Le Roith et al., 2001). IGF-I and IGF-II are single-chain polypeptides with structural homology to proinsulin. Mature IGF-I and IGF-II consist of B, C, A, and D domains (Filby and Tyler, 2007; Funes et al., 2006; Jones and Clemmons, 1995). IGF-I is produced in organs of reproductive significance such as hypothalamus, anterior pituitary, ovaries, oviducts, and uterus (Adam et al., 2000; Bach and Bondy, 1992; Constância et al., 2002; Eppler et al., 2007; Filby and Tyler, 2007; Funes et al., 2006; Gonzalez-Parra et al., 2001; Hastie and Haresign, 2006; Hunter et al., 2004; Iida, 2005; Irwin and Van Der Kraak, 2011; Jevdjovic et al., 2007; Mikawa et al., 1995b; Olchovsky et al., 1993; Silva et al., 2008; Velazquez et al., 2008). However, most of the IGF-I measured in blood is produced by the liver (Funes et al., 2006; Yakar et al., 1999). IGF-II mRNAs were also detected in liver and in variety of extrahepatic tissues, as was IGF-I mRNA (Funes et al., 2006; Silva et al., 2009). Recently, the IGF system has garnered more attentions for its potential role in ovarian development with the discovery of the gonad specific IGF-III, a third form of IGF (Berishvilia et al., 2010; Irwin and Van Der Kraak, 2011; Reinecke, 2010). However, there is little information about GH and IGF system in goats (Mikawa et al., 1995a,b; Silva et al., 2008; Yamano et al., 1988; Yato et al., 1988). The present study, for the first time, investigated the nucleotide sequences and mRNA expression levels of GH, IGF-I and IGF-II genes in the Lezhi black goats, a local Chinese breed famous for its high fecundity (producing 3–5 kids per kidding), and Tibetan goats (Capra hircus), a single-birth breed characterized by adapting to cold, hypoxic ecological conditions in the Qinghai-Tibet Plateau. This intends to examine whether base mutations or expression levels of mRNA of these genes could be associated with the reproductive difference in goats. 2. Materials and methods 2.1. Animals and sample collection All experimental procedures were performed according to the guide for animal care and use of laboratory animals of the Institutional Animal Care and Use Committee of Southwest University for Nationalities. Lezhi black goats were supplied by the Lezhi Black Goat Breeding Farm in Lezhi County (30°300 N, 105°020 E and 596.3 m altitude), China, and Tibetan goats were purchased from Li County (31°420 N, 103°160 E and 2800 m altitude) of Qinghai-Tibet Plateau in China during the breeding season (October). Lezhi black goats were kept in shelters, and Tibetan goats were grazed on the pasture. Animals aged 3–5 year, with a history of multiple births (twin or triplet births) for Lezhi black goats (n = 6) and single birth for Tibetan goats (n = 6) were selected to investigate the nucleotide sequences and mRNA expression levels of GH, IGF-I, and IGF-II genes. Estrus was detected twice a day, and a doe was considered in estrus only when she allowed the male to mount. Goats were slaughtered at 12–24 h after onset of estrus (in the middle of estrus) for collection of anterior pituitaries, ovaries, and endometria. Part of the removed tissue samples was snap-frozen in liquid nitrogen and then stored at 80 °C to be used for RNA extraction. 2.2. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from the tissue samples (pituitaries, ovaries, and endometria) using RNAprep pure Tissue Kit (Tiangen Biotech, Beijing), following the manufacture’s instructions. The
RNA samples were spectrophotometrically quantified at A260 nm and A280 nm, and then analyzed for their integrities on agarose gel (1.2%). Reverse transcription was performed using RNA PCR kit (AMV) (TaKaRa, Dalian, China) in a volume of 10 lL reaction mixture (Zi et al., 2012). The generated cDNA was then amplified using gene specific primers (GH: 50 -CAATGGGAAAAATCAGCA GTCT-30 /50 GAGGGGTAGTAACAACAGATGG-30 ; IGF-I: 50 -CAATGGGA AAAATC AGCAGTCT-30 /50 -ATT CTTCGCTCTTTAGGAAGGGC-30 ; GF-II: 50 -GCAGAGACATCAATGGG GATC-30 /50 -ACTTTGGCTCACT TCTAATCGC-30 , sense and antisense, respectively) designed from regions of GH, IGF-I, and IGF-II cDNAs conserved between sheep, goat and yak available in the NCBI GenBank database. PCR amplification was carried out in a volume of 25 lL of reaction mixture (Zi et al., 2012). The purified PCR products were ligated into a pMD19-T vector and transformed into DH5a ( Escherichia coli) using standard techniques (Sambrook et al., 1989). The DNA sequence was determined with an ABI PRISM 3700 automated DNA sequencer by Shanghai Invitrogen Biotechnology (Shanghai, China). 2.3. Sequence analysis The sequences of goat GH, IGFI and IGFII cDNA were subjected to BLAST analysis to verify that the sequence was of GH, IGFI and IGFII. The nucleotide and deduce amino acid sequence identity was performed using the Clustal option in MegAlign (Lasergene Software, DNASTAR). 2.4. Quantitative real-time PCR (qPCR) GAPDH gene was chosen as reference gene for normalizing expression levels of target genes. Primers specific for target goat genes were designed with Beacon Designer 7.0 software (Premier Biosoft International, Palo Alto, CA USA) according to manufacturers guidelines (GH: 50 -CTGAAGGACCTGGAGGAA-30 /50 -GGA GAGCA GACCGTAGTT-30 ; IGF-I: 50 -TGCTCTCCAGTTCGTGTG-3/5-CATCTCCA GCCTCCTCAG-30 ; IGF- II: 50 -CACCCTCCAGTTTGTCTG-30 /50 -GGCACAG TAAGTCTCCAG-30 ; GAPDH: 50 -AGTTCCACGGCACA GTCAAG30 /50 -ACTCAGCACCAGCATCACC-30 ). Total RNA of different tissues was extracted, and reverse transcribed as above. Real-time PCR was performed using on an iCycler iQ5 Real-Time Detection System (Bio-Rad, CA, USA) with the SsoFast™ EvaGreen Supermix (Bio-Rad, CA, USA) in a volume of 10 lL. The cycle parameters were 3 min at 95 °C, followed by 45 cycles of 30 s at 95 °C and 5 s at Tm, followed by a melting curve analysis. The efficiency of each primer pair and mean Ct (threshold cycles) values were calculated and used for determination of target gene RNA transcript levels (Pfaffl, 2001). Each sample was tested in triplicate. 2.5. Statistical analysis All data were expressed as mean values ± SEM. Statistical differences were assessed by Student’s t-test or one-way ANOVA followed by Dunn’s multiple pairwise comparison test. 3. Results 3.1. Cloning and molecular characterization of cDNAs for GH, IGF-I, and IGF-II genes As expected, the goat GH, IGF-I, and IGF-II cDNA sequences were found to consist of 688, 493, and 566 bp, respectively. They were respectively submitted to NCBI GenBank Accession Nos. JF813118, JF896275, and JF896277 for Lezhi black goat, and JF896274, JF896276, and JF896278 for Tibetan goat respectively. The coding region (nucleotides 6–656) of GH cDNA encoded a
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putative GH peptide of 217-amino acids (aa) and the predicted cleavage of the signal peptide (residues 1–26) would free a mature GH of 191 aa (residues 27–217) (Fig. 1A). Four cysteine residues (underlined) which may participate in the construction of structural features present in goat GH at the same positions as those for other vertebrate GHs (Kawauchi and Yasuda, 1989). The coding region of IGF-I cDNA encoded a putative IGF-I of 154 aa. Predicted cleavage of the 49 aa signal peptide (residues 1–49) and the 35 aa C-terminal extension (E) – peptide (residues 119–154) would free a mature IGF-I of 70 aa. The four domains, characteristic of mature IGF-I, were identified as follows: residues 50–75 (B-domain), 76– 90 (C-domain), 91–111 (A-domain), and 112–119 (D-domain). The six characteristic cysteine residues involved in the formation of the disulfide bounds were conserved (B6, B18, A6, A7, A11, and A20) (Fig. 1B). For goat IGF-II, the signal peptide consisted of 24 aa. A total of 32, 8, 21, 6, and 88 aa were detected for B-, C-, A-, D-, and E- domains, respectively. The sequence exhibited the six characteristic cysteine residues (B9, B21, A6, A7, A11, and A20) (Fig. 1C).
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Comparison of the nucleotide sequences between the two breeds showed that the sequence identities of GH, IGF-I, and IGFII were respectively 99.85, 99.59, and 99.65%. There was one base changes in GH (C to T from Lezhi black to Tibetan goat at bases 372) resulting in one amino acid substitution (Val 123 Phe). There were two base changes in IGF-I (G to A and T to C at bases 146 and 177), and two base changes in IGF-II (C to T and T to C at bases 267 and 315), but both of these changes do not result in amino acid substitution. 3.2. Tissue expression of GH, IGF-I, and IGF-II genes In the middle of estrus, GH, IGF-I and IGF-II genes were expressed, albeit at different levels, in all three tissues examined in both prolific Lezhi black goat and non-prolific Tibetan goat (Fig. 2A–C). In the same breed, GH was highly expressed in ovary (P < 0.01) than in anterior pituitary and endometrium. Comparison of the two breeds, GH mRNA expression was higher (P < 0.05) in all three tissues of Lezhi black goats than that in Tibetan goats
Fig. 1. Nucleotide and deduced amino acid sequences of Tibetan goat (Capra hircus) GH (A), preproIGF-I (B) and preproIGF-II (C). Open reading frames are in uppercase. Signal peptides are shaded. IGF-I and IGF-II mature proteins consisting of B, C, A, and D domains are in boldfaced letters. The six conserved cysteine residues are underlined. Nucleotides and amino acids different from those of Lezhi black goat are indicated as bold double-underlined. Numbering to the left denotes nucleotide number (regular font) and translated amino acid number (bold italic font).
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Fig. 2. GH, IGF-I, and IGF-II mRNA levels in different tissues of Tibetan goat (Capra hircus) and Lezhi black goat. Transcripts were quantitated using total RNA from tissues of six does at 12–24 h after onset of estrus for each breed. The experiments were carried out in three replicates. The expression of these mRNAs was normalized to the expression of GAPDH measured in the same RNA preparation. Results were expressed as the mean ± SEM. Different letters in the same tissue indicate the difference between breeds (P < 0.05).
(Fig. 2A). IGF-I gene was expressed at a significantly (P < 0.05) higher level in anterior pituitary of Lezhi black goat than that in Tibetan goat, but it had a similar expression pattern in ovary and endometrium of the two breeds (Fig. 2B and C). In both of these breeds, IGF-II was highly expressed in ovary and endometrium than in anterior pituitary (P < 0.01). IGF-II mRNA expression was higher (P < 0.05) in anterior pituitary of Lezhi black goat than that in Tibetan goat, but there was no difference in ovary and endometrium between the two breeds (P > 0.05). 4. Discussion In this study, we isolated cDNA clones encoding GH, IGF-I and IGF-II molecules of the prolific Lezhi black goat and non-prolific Tibetan goat (C. hircus). Analysis of their nucleotide and amino acid sequences revealed a high degree of identity between the two goat breeds, although one base change in GH resulted in one amino acid
substitution in the translated proteins (Val 123 Phe). Comparing the nucleotide and amino acid sequences from other goat breeds (Yamano et al., 1988; Yato et al., 1988), it appears that this mutation in GH only occurs in Lezhi black goat. Mutations in GH have been found to dramatically change its biological activity; when glycine at 120 of GH is replaced by arginine, lysine, or a variety of amino acids, GH is converted from a growth enhancer to a growth suppressor or a GH antagonist (Chen et al., 1991). However, two base changes in IGF-I and IGF-II did not lead to any amino acid changes. Therefore, the amino acid substitution in GH may be an important molecular mechanism that regulates the differential fecundity of these goat breeds, but the base changes in IGF-I and IGF-II may be attributed to the evolutional divergence. During the last decades, involvement of GH, IGFs and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied. In vitro studies and knockout experiments demonstrated in most mammalian species, GH and IGFs do not appear to be required for primordial to primary follicles transition, but they promote secondary follicle growth and antrum formation. In that regard, GH enhances the development of small antral follicles to the gonadotrophin-dependent stages and stimulates oocyte maturation, whereas IGFs increase granulose cell proliferation, steroidogenesis and oocyte growth in most mammalian species (Hastie and Haresign, 2006; Hunter et al., 2004; Silva et al., 2009; Velazquez et al., 2008). However, there is little information about them in goats (Mikawa et al., 1995a,b; Silva et al., 2008), especially, no clear differences between breeds with different fecundity have been reported. Therefore, to enable investigations of GH, IGF-I and IGF-II genes signaling at expression levels on fecundity, the differences in tissue (anterior pituitary, ovary, and endometrium) mRNA expression levels of these genes in the middle of estrus were compared between the prolific Lezhi black goats and non-prolific Tibetan goats in the present study. GH is synthesized and stored by somatotroph cells within the anterior pituitary gland (Ayuk and Sheppard, 2006; Filby and Tyler, 2007). GH mRNA is highly localized to the pituitary, and low-level expression in gonad and in some other tissues (Abir et al., 2008; Biga et al., 2004; Filby and Tyler, 2007). Extra-pituitary GH is not believed to be released into systematic circulation since plasma GH is undetectable in hypophysectomized animals (Lazarus and Scanes, 1988). It is, therefore, likely to reflect local paracrine/autocrine effects of GH in goat gonad, distinct from (and in additional to) GH’s systemic effects. In the present study, the finding that goat GH mRNA is highly expressed to the pituitary is in agreement with most findings (Abir et al., 2008; Biga et al., 2004; Filby and Tyler, 2007). In contrast, GH mRNA is also highly expressed in ovary and endometrium. In addition, GH mRNA was significantly greater in all tissues examined in prolific Lezhi black goat than in non-prolific Tibetan goat, indicating that GH may play an important role in the development of preovulatory follicles and the number of ovulatory follicles, and hence, ovulation rate in goat. However, future studies are needed to confirm whether GH can really highly expressed in ovary and endometrium. In agreement with previous studies (Bach and Bondy, 1992; Filby and Tyler, 2007; Mikawa et al., 1995b), IGF-I and IGF-II mRNAs were also found to be expressed in all three tissues examined, and they had a similar expression pattern in ovary and endometrium of these two breeds. Interestingly, both of IGF-I and IGF-II mRNAs were expressed at a significantly higher level in anterior pituitary of prolific Lezhi black goat than in non-prolific Tibetan goat. The IGF-I and IGF-II synthesized by anterior pituitary cells regulate pituitary function, including release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Adam et al., 2000; Gonzalez-Parra et al., 2001; Li et al., 2011). Therefore, the differential fecundity of these goat breeds might be partly controlled by the difference in IGFs expression in their anterior pituitary.
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In summary, the cDNA encoding GH, IGF-I and IGF-II were cloned, sequenced and characterized in prolific Lezhi goat and non-prolific Tibetan goat. They prove to be identical to each other except for one base change resulting in one amino acid substitution in GH, two base changes in IGF-I, and two base changes in IGF-II between the two breeds. Using real-time PCR techniques, we have demonstrated that GH mRNA expression was greater in anterior pituitary, ovary, and endometrium of Lezhi black goat than in Tibetan goat, but IGF-I and IGF-II expressions were only greater in anterior pituitary of Lezhi black goat. These results provide the foundation of information required for future studies of these gene effects on goat fecundity. Acknowledgments This work was supported by National Key Technology R&D Program (No. 2012BAD13B06 and No. 2008BADC3B01) and Southwest University for Nationalities (12NZYTH07; 2011XWD-S0905). References Abir, R., Garor, R., Felz, C., Nitke, S., Krissi, H., Fisch, B., 2008. Growth hormone and its receptor in human ovaries from fetuses and adults. Fertil. Steril. 90, 1333– 1339. Adam, C.L., Gadd, T.S., Findlay, P.A., Wathes, D.C., 2000. IGF-I stimulation of luteinizing hormone secretion, IGF-binding proteins (IGFBPs) and expression of mRNAs for IGFs, IGF receptors and IGFBPs in the ovine pituitary gland. J. Endocrinol. 166, 247–254. Ayuk, J., Sheppard, M.C., 2006. Growth hormone and its disorders. Postgrad. Med. J. 82, 24–30. Bach, M.A., Bondy, C.A., 1992. Anatomy of the pituitary insulin-like growth factor system. Endocrinology 131, 2588–2594. Baker, J., Hardy, M.P., Zhou, J., Bondy, C., Lupu, F., Bellve, A.R., Efstratiadis, A., 1996. Effects of an IGF-I gene null mutation on mouse reproduction. Mol. Endocrinol. 10, 903–918. Berishvilia, G., Baroiller, J.F., Eppler, E., Reinecke, M., 2010. Insulin-like growth factor-3 (IGF-3) in male and female gonads of the tilapia: development and regulation of gene expression by growth hormone (GH) and 17aethinylestradiol (EE2). Gen. Comp. Endocrinol. 167, 128–134. Biga, P.R., Schelling, G.T., Hardy, R.W., Cain, K.D., Overturf, K., Ott, T.L., 2004. The effects of recombinant bovine somatotropin (rbST) on tissue IGF-I, IGF-I receptor, and GH mRNA levels in rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 135, 324–333. Butler, A.A., Le Roith, D., 2001. Control of growth by the somatotrophic axis: growth hormone and the insulin-like growth factors have related and independent roles. Annu. Rev. Physiol. 63, 141–164. Carlsson, B., Nilsson, A., Isaksson, O.G.P., Billig, H., 1993. Growth hormone-receptor messenger RNA in the rat ovary: regulation andlocalization. Mol. Cell. Endocrinol. 95, 59–66. Chen, W.Y., Wight, D.C., Mehta, B.V., Wagner, T.E., Kopchick, J.J., 1991. Glycine 119 of bovine growth hormone is critical for growth promoting activity. Mol. Endocrinol. 5, 1845–1852. Constância, M., Hemberger, M., Hughes, J., Dean, W., Ferguson-Smith, A., Fundele, R., Stewart, F., Kelsey, G., Fowden, A., Sibley, C., Reik, W., 2002. Placental-specific IGF-II is a major modulator of placental and fetal growth. Nature 417, 945–948. DeChiara, T.M., Efstratiadis, A., Robertson, E.J., 1990. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 345, 78–80. Duan, C.M., Ren, H.X., Gao, S., 2010. Insulin-like growth factors (IGFs), IGF receptors, and IGF-binding proteins: Roles in skeletal muscle growth and differentiation. Gen. Comp. Endocrinol. 167, 344–351. Echternkamp, S.E., Roberts, A.J., Lunstra, D.D., Wise, T., Spicer, L.J., 2004. Ovarian follicular development in cattle selected for twin ovulations and births. J. Anim. Sci. 82, 459–471. Eppler, E., Jevdjovic, T., Maake, C., Reinecke, M., 2007. Insulin-like growth factor I (IGF-I) and its receptor (IGF-1R) in the rat anterior pituitary. Eur. J. Neurosci. 25, 191–200. Filby, A.L., Tyler, C.R., 2007. Cloning and characterization of cDNAs for hormones and/or receptors of growth hormone, insulin-like growth factor-I, thyroid hormone, and corticosteroid and the gender-, tissue-, and developmentalspecific expression of their mRNA transcripts in fathead minnow (Pimephales promelas). Gen. Comp. Endocrinol. 150, 151–163. Funes, V., Asensio, E., Ponce, M., Infante, C., Canavate, J.P., Manchado, M., 2006. Insulin-like growth factors I and II in the sole Solea senegalensis: cDNA cloning and quantitation of gene expression in tissues and during larval development. Gen. Comp. Endocrinol. 149, 166–172. Gonzalez-Parra, S., Argente, J., Chowen, J.A., Van Kleffens, M., van Neck, J.W., Lindenbeigh-Kortleve, D.J., Drop, S.L., 2001. Gene expression of the insulin-like growth factor system during postnatal development of the rat pituitary gland. J. Neuroendocrinol. 13, 86–93.
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Hastie, P.M., Haresign, W., 2006. Expression of mRNAs encoding insulin-like growth factor (IGF) ligands, IGF receptors and IGF binding proteins during follicular growth and atresia in the ovine ovary throughout the oestrous cycle. Anim. Reprod. Sci. 92, 284–299. Hunter, M.G., Robinson, R.S., Mann, G.E., Webb, R., 2004. Endocrine and paracrine control of follicular development and ovulation rate in farm species. Anim. Reprod. Sci. 82–83, 461–477. Iida, K., Rosen1, C.J., Ackert-Bicknell, C., Thorner, M.O., 2005. Genetic differences in the IGF-I gene among inbred strains of mice with different serum IGF-I levels. J. Endocrinol. 186, 481–489. D.A. Irwin, G.J. Van Der Kraak, Regulation of the novel insulin-like growth factor III ligand in the ovary of zebrafish, Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology doi: 10.3389/conf.fendo.2011.04.00144. Jevdjovic, T., Bernays, R.L., Eppler, E., 2007. Insulin-like growth factor-I mRNA and peptide in the human anterior pituitary. J. Neuroendocrinol. 19, 335–341. Jones, J.I., Clemmons, D.R., 1995. Insulin-like growth factors and their binding proteins: biological actions. Endocr. Rev. 16, 3–34. Kawauchi, H., Yasuda, A., 1989. Evolutionary aspects of growth hormones from nonmammalian species. In: Muller, E.E., Cocchi, D., Locatelli, V. (Eds.), Advances in Growth Hormones and Growth Factor Research. Pythagora Press/Springer, Roma-Milano/Berlin, pp. 51–68. Lazarus, D.D., Scanes, C.G., 1988. Acute effects of hypophysectomy and administration of pancreatic and thyroid hormones on circulating concentrations of somatomedin C in young chickens: relationships between growth hormone and somatomedin C. Domest. Anim. Endocrinol. 5, 283–289. Le Roith, D., Bondy, C., Yakar, S., Liu, J.L., Butler, A., 2001. The somatomedin hypothesis. Endocr. Rev. 22 (2001), 53–74. Li, L., Fu, Y.C., Xu, J.J., Chen, X.C., Lin, X.H., Luo, L.L., 2011. Caloric restriction promotes the reproductive capacity of female rats via modulating the level of insulin-like growth factor-1 (IGF-1). Gen. Comp. Endocrinol. 174, 232–237. Lucy, M.C., 2011. Growth hormone regulation of follicular growth. Reprod. Fertil. Dev. 24, 19–28. Mikawa, S., Yoshikawa, G.-I., Aoki, H., Yamano, Y., Sakai, H., Komano, T., 1995a. Dynamic aspects in the expression of the goat insulin-like growth factor-I (IGFI) gene: diversity in transcription and post-transcription. Biosci. Biotechnol. Biochem. 59, 87–92. Mikawa, S., Yoshikawa, G.-I., Yamano, Y., Sakai, H., Komano, T., Hosoi, Y., Utsumi, K., 1995b. Tissue- and development-specific expression of goat insulin-like growth factor-I (IGF-I) mRNAs. Biosci. Biotechnol. Biochem. 59, 759–761. Olchovsky, D., Song, J., Gelato, M.C., Sherwood, J., Spatola, E., Bruno, J.F., Berelowitz, M., 1993. Pituitary and hypothalamic insulin-like growth factor-I (IGF-I) and IGF-I receptor expression in food-deprived rats. Mol. Cell. Endocrinol. 93, 193– 198. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Res. 29, 2002–2007. Reinecke, M., 2010. Insulin-like growth factors and fish reproduction. Biol. Reprod. 82, 656–661. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbour Library Press. Shimizu, T., Murayama, C., Sudo, N., Kawashima, C., Tetsuka, M., Miyamoto, A., 2008. Involvement of insulin and growth hormone (GH) during follicular development in the bovine ovary. Anim. Reprod. Sci. 106, 143–152. Silva, J.R.V., Brito, I.R., Leitao, C.C.F., Silva, A.W.B., Passos, M.J., Vasconcelos, G.L., 2008. Protein and messenger RNA quantification of insulin-like growth factor-1 (IGF-1) in goat ovarian follicles. Acta Sci. Vet. 36 (Suppl. 2), 466. Silva, J.R.V., Figueiredo, J.R., van den Hurk, R., 2009. Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis. Theriogenology 71, 1193–1208. Velazquez, M.A., Spicer, L.J., Wathes, D.C., 2008. The role of endocrine insulin-like growth factor-I (IGF-I) in female bovine reproduction. Domest. Anim. Endocrinol. 35, 325–342. Yakar, S., Liu, J.-L., Stannard, B., Butler, A., Accili, D., Saur, B., LeRoith, D., 1999. Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc. Nat. Acad. Sci. USA 96, 7324–7329. Yamano, Y., Oyabayashi, K., Okuno, M., Yato, M., Kioka, N., Manabe, E., Hashi, H., Sakai, H., Komano, T., Utsumi, K., Iritani, A., 1988. Cloning and sequencing of cDNA that encodes goat growth hormone. FEBS Lett. 228, 301–304. Yato, M., Yamano, Y., Oyabayashi, K., Okuno, M., Kioka, N., Manabe, E., Hashi, H., Sakai, H., Komano, T., Utsumi, K., Iritani, A., 1988. Nucleotide sequence of the growth hormone gene cDNA from goat Capra hircus L. (Tokara). Nucleic Acids Res. 16, 3578. Zhao, J., Taverne, M.A., van de Weijden, G.C., Bevers, M.M., van den Hurk, R., 2001. Insulin-like growth factor-I (IGF-I) stimulates the development of cultured rat pre-antral follicles. Mol. Reprod. Dev. 58, 287–296. Zhao, J., Taverne, M.A., van de Weijden, G.C., Bevers, M.M., van den Hurk, R., 2002. Immunohistochemical localisation of growth hormone (GH), GH receptor (GHR), insulin-like growth factor I (IGF-I) and type I IGF-I receptor, and gene expression of GH and GHR in rat pre-antral follicles. Zygote 10, 85–94. Zhou, J., Adesanya, O.O., Vatzias, G., Hammond, J.M., Bondy, C.A., 1996. Selective expression of insulin-like growth factor system components during porcine ovary follicular selection. Endocrinology 137, 4893–4901. Zi, X.D., Chen, D.W., Wang, H.M., 2012. Molecular characterization, mRNA expression of prolactin receptor (PRLR) gene during pregnancy, nonpregnancy in the yak (Bos grunniens). Gen. Comp. Endocrinol. 175, 384–388.