General and Comparative Endocrinology 190 (2013) 88–95

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General and Comparative Endocrinology

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Review

Expression of adiponectin and its receptors in avian species

0016-6480/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ygcen.2013.05.004

⇑ Corresponding author. Fax: +1 814 865 5691.E-mail address: RameshR@psu.edu (R. Ramachandran).

Ramesh Ramachandran ⇑, Sreenivasa Maddineni, Olga Ocón-Grove, Gilbert Hendricks III,Regina Vasilatos-Younken, Jill A. HadleyDepartment of Animal Science, The Pennsylvania State University, University Park, PA 16802, USA

a r t i c l e i n f o a b s t r a c t

Article history:Available online 21 May 2013

Keywords:AdipoR1AdipoR2Post-translational modificationsTestisEnzyme immunoassayHMW adiponectin

Adipose tissue is a dynamic endocrine organ secreting a variety of hormones that affect physiologicalfunctions within the central nervous system, cardiovascular system, reproductive, and immune systems.The endocrine role of avian adipose tissue remains enigmatic as many of the classical hormones found inmammalian adipose tissue have not been found in avians. This mini-review summarizes our currentknowledge on avian adiponectin, one of the most abundant adipose tissue hormones, and its receptors.We cloned the genes encoding chicken adiponectin and its receptors, AdipoR1 and AdipoR2. Using anti-chicken adiponectin antibody, we found that chicken adipose tissue and plasma predominantly contain aunique polymer of adiponectin with a mass greater than 669 kDa, unlike mammalian adiponectin whichis found as three distinct oligomers. Mass spectrometric analyses of chicken adiponectin revealed certainpost-translational modifications that are likely to favor the unique multimerization of adiponectin inchickens. Unlike adiponectin, the nucleotide sequences of chicken AdipoR1- and AdipoR2 cDNA arehighly similar to that of mammalian adiponectin receptors. Both adiponectin and adiponectin receptorsare widely expressed in several tissues in the chicken. Herein, we review the unique biochemistry ofadiponectin as well as expression of adiponectin and its receptors in the chicken. Future studies shouldfocus on elucidating the role of adiponectin, AdipoR1, and AdipoR2 on metabolism, steroidogenesis, andadipose tissue remodeling during growth and reproduction in birds.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Adipose tissue is an organ that stores lipids and secretes a vari-ety of hormones that influence physiological functions of other or-gans related to growth, immunity, and reproduction. Following thediscovery of leptin nearly two decades ago (Frederich et al., 1995;Halaas et al., 1995), secretion and biological functions of severaladipose tissue hormones such as adiponectin, resistin and chemer-in, and several cytokines have been characterized in humans androdent models (Beutler et al., 1985; Goralski et al., 2007; Kernet al., 2001; Steppan et al., 2001). Despite the recent developmentsin sequencing of the chicken genome, adipose hormones remainenigmatic as many classical adipose hormones such as leptin andresistin have not been identified in the chicken genome (Fried-man-Einat et al., 1999; Sharp et al., 2008). Avian adipose tissue isan interesting organ as it undergoes constant remodeling to meetthe metabolic demands of growth, reproduction, and migration.In addition, avian adipose tissue is constantly exposed to a glucoselevel exceeding 17.5 mM that is considered hyperglycemic tomammalian adipocytes. A thorough knowledge of adipose tissueis therefore critical for understanding the uniqueness of metabolic

conditions encountered in avian species. This mini-review summa-rizes our understanding of the unique biochemical properties andfunctions of the most abundant adipose tissue hormone, adiponec-tin, and its receptors, AdipoR1 and AdipoR2.

2. Adiponectin in mammalian and avian Species

Adiponectin (also called Acrp30, apM1, GBP28, or adipoQ) is a30 kDa adipocytokine hormone exclusively secreted from the adi-pose tissue in mammals (Hu et al., 1996; Maeda et al., 1996; Nak-ano et al., 1996; Scherer et al., 1995). The adiponectin gene ishighly conserved among several mammalian species and codesfor a 244-amino acid protein that belongs to a family of C1q glob-ular domain proteins (Kishore and Reid, 1999, 2000). Adiponectinis the most abundant protein in human adipose tissue, and alsothe most abundant hormone in human plasma (1.9–17 lg/ml),accounting for 0.01% of plasma proteins (Arita et al., 1999). Adipo-nectin circulates as a trimer, hexamer, heavy molecular weightforms, and also as small proteolytic cleavage products in mouseand human plasma (Fruebis et al., 2001; Kishida et al., 2003).Adiponectin has multiple beneficial effects on glucose utilizationand insulin sensitivity thereby aiding in the prevention of type 2diabetes and cardiovascular diseases in humans (reviewed inWhitehead et al. (2006)). Decreased plasma adiponectin levels

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are associated with the development of insulin resistance, type 2diabetes, and cardiovascular diseases in humans and rodent mod-els. Although secreted from adipose tissue, adiponectin levels inthe circulation were found to be lower, paradoxically, in obese hu-man subjects (Arita et al., 1999). Despite its potential importancein avian species, adiponectin has not been thoroughly investigated.Therefore, we began our investigations by characterizing the bio-chemistry and expression of chicken adiponectin, as describedbelow.

3. Expression of adiponectin mRNA in chickens

We first sought to determine the nucleotide sequence of thechicken adiponectin cDNA. The open reading frame of the chickenadiponectin cDNA consists of 735 nucleotides that was 65–68%homologous to various mammalian adiponectin cDNAs (Maddine-ni et al., 2005; Yuan et al., 2006). In contrast, chicken adiponectinmRNA sequence shares a high degree of identity with other birdssuch as Meleagris gallopavo (93%; Accession# XR_118340.1), Anasplatyrhynchos (86%; Accession# GU236315.1), Anser anser (89%;

A B

C D

Fig. 1. (A and B) Non-reducing and non-heat-denaturing Western blot analysis of chickeprotein extracts of adipose tissue (Ad; 20 lg), skeletal muscle (SM; 20 lg) or liver (L; 4detected using anti-chicken adiponectin antibody (A, �Blocking peptide). Specificity ochicken adiponectin antibody with chicken adiponectin peptide (A; +Blocking peptide). Ptwo different ages (4- and 8-week-old) was separated under non-reducing and non-heat-D) Western blot analysis of adiponectin under reducing and denaturing conditions. Chicwith (+) or without (�) reducing agent, heat denatured (70 or 100 C) or not heat denaturwas detected by immunostaining using a rabbit anti-chicken adiponectin antibody (C; �Bpre-adsorption of the anti-chicken adiponectin antibody with chicken adiponectin peptid(2009; �2009, The Endocrine Society).

EU370686.1), and Taeniopygia guttata (82%; XM_002188735.1).Using RT-PCR and northern analysis, we detected chicken adipo-nectin mRNA transcript in adipose tissue, liver, anterior pituitarygland, diencephalon, skeletal muscle, liver, kidney, ovary, andspleen, but not in blood (Maddineni et al., 2005). In support ofour data, adiponectin gene and/or protein expression has been de-tected in skeletal muscle (Ding et al., 2004; Punyadeera et al.,2005), pituitary gland (Rodriguez-Pacheco et al., 2007), and testis(Caminos et al., 2008) in mammals. The concentration of adiponec-tin mRNA was found to be the highest in adipose tissue followed byliver, anterior pituitary, diencephalon, kidney and skeletal muscle.We also found that adiponectin mRNA quantity was significantlydecreased following a 48 h food deprivation in adipose tissue, liver,and anterior pituitary gland but not in diencephalon (Maddineniet al., 2005).

4. Biochemical properties of adiponectin

Structurally, adiponectin consists of four distinct domains thatinclude a signal peptide at the N-terminus followed by a short

n adiponectin under native conditions. Male broiler chicken plasma (P; 0.5 ll) and0 lg) were separated by electrophoresis under native conditions. Adiponectin wasf the adiponectin immunostaining was determined by pre-adsorption of the anti-lasma (0.5 ll) from two chickens belonging to two genetic lines (leghorn, broiler) atdenaturing conditions and adiponectin was detected by immunostaining (B). (C andken plasma (P; 0.5 ll) and adipose tissue protein extracts (Ad; 20 lg) were treateded (�), separated by electrophoresis and blotted onto PVDF membrane. Adiponectinlocking peptide). Specificity of the adiponectin immunostaining was determined by

e (D; +Blocking peptide). Figure reproduced with permission from Hendricks III et al.

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variable region, a collagenous domain, and a C-terminal globulardomain (Shapiro and Scherer, 1998). During biosynthesis, the30 kDa adiponectin monomers are assembled into oligomers andmultimers and secreted as three distinct isoforms denoted as low(LMW), medium (MMW), and heavy molecular weight (HMW) iso-forms in mammals (Tsao et al., 2002, 2003; Waki et al., 2003). TheLMW adiponectin isoform represents a homotrimer of three adipo-nectin monomers, while the MMW isoform is a hexamer composedof two homotrimers and the HMW adiponectin isoform is likely tocontain 18 or more adiponectin monomers (Tsao et al., 2003). Webegan to characterize the biochemical properties of chicken adipo-nectin using anti-chicken adiponectin antibodies developed in ourlaboratory, as our attempts to utilize anti-mammalian adiponectinantibody to detect chicken adiponectin failed, possibly due to poorhomology of chicken and mammalian adiponectin. Our studies re-vealed that under non-reducing and non-heat-denaturing condi-tions, adiponectin exists predominantly as a polymer with amolecular mass of approximately 720 kDa in chicken plasma, adi-pose tissue, and skeletal muscle (Fig. 1A). Traces of an oligomericadiponectin isoform of approximately 242 kDa was detected in leg-horn chickens or 4-week-old broiler chickens (Fig. 1B). Gel filtra-tion of chicken plasma or adipose tissue protein extract followedby Western analysis of the fractions under native conditions con-firmed that chicken adiponectin is predominantly a HMW isoformof mass equal to or greater than 669 kDa. (Hendricks et al., 2009).Chicken plasma and adipose tissue extracts were then subjected toelectrophoresis under reducing conditions with or without reduc-ing agent and/or heating. Omission of heat and reducing agent re-sulted in a major immunoreactive band that is >191 kDa, which isrepresentative of the HMW isoform, as well as a less intense64 kDa oligomeric adiponectin isoform in both plasma and adiposetissue extracts (Fig. 1C). Treatment with a reducing agent, however,led to a complete disappearance of the HMW isoform, which is re-duced primarily to a 64 kDa oligomer in plasma, and to a 64 kDaoligomer as well as a 30 kDa monomer in the adipose tissue extract(Fig. 1C). In contrast to chicken, human and mouse adiponectin ex-ists as three different molecular mass species (67, 136, and>300 kDa) termed LMW, MMW, and HMW isoforms, respectively(Waki et al., 2003). The presence of HMW adiponectin isoformsin chicken plasma and tissues, without significant amounts ofLMW and MMW oligomeric forms is enigmatic. The mechanism(s)that aid in the assembly of this single, major HMW multimeric iso-form in the chicken is currently unknown. However, the aminoacid sequence as well as post-translational modifications of chick-en adiponectin combined with physiological factor(s) unique tochickens may favor such unique multimerization.

To determine the post-translation modifications in chickenadiponectin, we isolated adiponectin from chicken adipose tissueby immunoprecipitation. Chicken adiponectin was then subjectedto tryptic digestion and UPLC/MS/MS. The collagenous domain ofchicken adiponectin was found to possess certain amino acids thatare likely to aid in multimerization (Fig. 2). For instance, there weretwice as many lysine residues (8 versus 4) compared to human ormouse adiponectin in the collagenous domain. Mutations in someor all of the lysine residues in the collagenous domain of mouseadiponectin leads to a progressive loss of multimerization (Rich-ards et al., 2006; Wang et al., 2006), suggesting that a greater num-ber of lysine residues are likely to favor multimerization. Inaddition, five lysine residues (three conserved and two non-con-served; Fig. 2) contained a glucosylgalactosyl hydroxyl moiety, apost-translational modification that is likely to aid in multimeriza-tion of chicken adiponectin. Similar to chicken adiponectin, all fourlysine residues in the collagenous domain of mammalian adipo-nectin are modified as glucosylgalactosyl hydroxylysine, and suchpost-translational modifications were found to be essential forimproving stability of the HMW adiponectin isoform and for con-

ferring certain biological activities (Peake et al., 2007; Richardset al., 2006; Wang et al., 2002, 2004, 2006). Further studies are re-quired to determine how the oligomers of chicken adiponectin areassembled into multimers and held stably in the circulation as apredominant HMW isoform.

5. Plasma adiponectin levels in chickens

Following production and verification of an anti-chicken adipo-nectin antibody, we developed a chicken-specific enzyme-linkedimmunosorbent assay (EIA) and sought to determine the levels ofadiponectin in chicken plasma (Hendricks et al., 2009). We foundthat the adiponectin levels are in the range of 4–10 lg/ml in broilerchicken plasma. Such high levels of adiponectin (1.9–17 lg/ml) havesimilarly been reported in human serum (Arita et al., 1999). Wefound that a 48 h fast did not affect plasma adiponectin levels inchickens, suggesting that circulating adiponectin levels are resistantto metabolic changes in response to fasting. Plasma adiponectin lev-els were lower in 8-week-old chickens when their body weight andabdominal fat pad mass increased by 2- and 1.5-fold, respectively,compared to 4-week-old broiler chickens (Hendricks et al., 2009).Changes associated with age or rapid growth may have also led tothe lower levels of circulating adiponectin in the older chickens.

6. Adiponectin receptors

Adiponectin signals through two receptors, designated as Adi-poR1 and AdipoR2 (Yamauchi et al., 2003). We cloned the genesencoding chicken AdipoR1 and AdipoR2 (GenBank Accession Nos.DQ072275, DQ072276, (Ramachandran et al., 2007). The chickenAdipoR1 cDNA and AdipoR2 cDNA sequences were only 68%homologous to each other, similar to the homology of mammalianAdipoR1 and AdipoR2 cDNA sequences (Yamauchi et al., 2003). Thechicken AdipoR1 cDNA, however, was found to be 80–83% homol-ogous to human, mouse, rat, or pig AdipoR1 cDNA, while the de-duced protein sequence was 91% similar to mammalian AdipoR1.Similarly, the chicken AdipoR2 cDNA was 76–78% homologous tohuman, mouse, or pig AdipoR2 cDNA, while the deduced proteinsequence was 82% similar to mammalian AdipoR2 (Ramachandranet al., 2007). A significant homology between avian and mamma-lian AdipoR1 and AdipoR2 sequences may suggest that both genesare evolutionarily conserved and may have important biologicalfunctions. Using a hydrophobicity plot analysis, the amino acid se-quence of chicken AdipoR1 and AdipoR2 have been predicted toform seven transmembrane domains (Ramachandran et al.,2007). AdipoR1 has been reported to have greater binding affinityto the globular domain of adiponectin while AdipoR2 binds to boththe globular and full length adiponectin with intermediate affinity(Yamauchi et al., 2003). While chicken adiponectin contains puta-tive globular and collagenous domains (Hendricks et al., 2009;Maddineni et al., 2005), the relative affinities of the chicken Adi-poR1 and AdipoR2 to adiponectin remain to be determined.

Our studies suggest that AdipoR1 and AdipoR2 genes are ex-pressed in multiple tissues in the chicken wherein both receptorsare likely to mediate the physiological effects of adiponectin. Wedetermined the relative expression of AdipoR1 and AdipoR2 mRNAin various tissues and found that skeletal muscle, adipose tissueand diencephalon were the principal organs where AdipoR1 genewas maximally expressed, while AdipoR2 mRNA expression wasthe highest in adipose tissue. In contrast to our findings in thechicken, mouse AdipoR1 and AdipoR2 mRNAs have been found tobe maximally expressed in the skeletal muscle and liver, respec-tively (Yamauchi et al., 2003). This may represent species variationin the gene expression of AdipoR1 and AdipoR2. The quantities ofAdipoR2 mRNA were found to be significantly higher in liver, skel-etal muscle, and ovary than in diencephalon, anterior pituitary

A

B

Fig. 2. (A and B) Mass spectrometric analysis of chicken adiponectin. (A) Schematic of the collagenous domain of chicken adiponectin and the amino acid sequence ofcollagenous domain showing hydroxylation (Hx) of proline residues (42, 45, 51, 69, and 93) and glycosylation of hydroxylysine residues (66, 75, 81, 99, and 102) derived fromMS/MS analysis. A short peptide sequence DGKDGKDGQK (boxed) deduced from the chicken adiponectin cDNA sequence [GenBank Accession No. NM_206991; (Maddineniet al., 2005)] within the collagenous domain could not be detected in the LC/MS/MS analysis. (B) Identification of peptide fragments and post-translational modifications ofamino acids in the chicken adiponectin protein by peptide mass fingerprinting and MS/MS ion analysis. Peptide A demonstrates hydroxylation of three proline residues (42,45, and 51) that account for a difference of 47.2611 Da representing three hydroxyl groups. Peptide B was found in four different masses each representing variouscombinations of hydroxylation of proline residue (69) as well as hydroxylation and/or glycosylation of lysine residues (66,75, and 81). Peptide C was found in two differentmasses with various combinations of hydroxylation (P93 and K102) and/or glycosylation (K99 and K102). Peptides (D–F) represent fragments from the N-terminal end of theglobular domain of chicken adiponectin wherein the proline and lysine residues were not hydroxylated or glycosylated. Amino acids that are underlined in Peptides (A–C)denote modified residues. Figure reproduced with permission from Hendricks III et al. (2009; �2009, The Endocrine Society).

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gland, kidney, and spleen. AdipoR2 mRNA expressed in the liver islikely to be involved in lipid metabolism, glucose utilization, andgluconeogenesis. AdipoR1 and AdipoR2 expressed in skeletal mus-cle may be involved in glucose utilization as in mammals. Expres-sion of AdipoR1 and AdipoR2 in the chicken pituitary glandsuggests that adiponectin may regulate pituitary hormone secre-tion. Prolactin and growth hormone have been found to affect Adi-poR1 and AdipoR2 gene expression in adipose tissue (Nilsson et al.,2005), and plasma growth hormone concentrations are inverselyrelated to adiponectin concentrations in mice (Berryman et al.,2004) and in humans (Mantzoros et al., 2004).

7. Expression of adiponectin and its receptors in testis andovary

We have characterized the expression of adiponectin and itsreceptors in the testes of broiler breeder chickens (Ocon-Groveet al., 2008). Using RT-PCR and immunohistochemistry, we found

that adiponectin, AdipoR1 and AdipoR2 genes are expressed inthe chicken testis. We developed chicken-specific antibodiesagainst adiponectin, AdipoR1 and AdipoR2 and localized adiponec-tin-, AdipoR1-, and AdipoR2-immunoreactive (ir) cells in the chick-en testis (Figs. 3 and 4). Both adiponectin- (Fig. 3) and AdipoR1-ir(Ocon-Grove et al., 2008) cells were localized to the peritubularcells and Leydig cells surrounding the seminiferous tubules inthe chicken testis (Fig. 3). Based on distinguishable flattened cellmorphology of the peritubular cells, adiponectin and AdipoR1 ap-pear to be expressed in peritubular myoid cells. AdipoR2-immuno-reactivity was predominantly observed in the Leydig cells as wellas in the adluminal and luminal compartments of the chicken sem-iniferous tubules (Fig. 4). AdipoR2 immunostaining was observedin the Sertoli cell syncytia suggesting a potential role of adiponec-tin in regulating Sertoli cell function. In addition, AdipoR2 immu-nostaining was noticed in the round spermatids, elongatingspermatids, and spermatozoa. Based on such widespread AdipoR2immunostaining throughout the adluminal and luminal compart-

Fig. 3. Representative photomicrographs of testicular tissue sections from adult chickens showing adiponectin-immunostained cells. Paraformaldehyde-fixed tissue sectionswere immunostained using anti-chicken adiponectin antisera (A–E) or anti-chicken adiponectin IgG (G–K). (A–C) Confocal images of testicular tissue section showingadiponectin immunostaining (green; A and C) and nuclear staining (red; B and C). Note adiponectin immunostaining in the peritubular area and in the interstitium around theseminiferous tubule (ST). (D–E) Confocal images of seminiferous tubules (ST) depicting adiponectin-immunostaining (green) in the peritubular cells (PC) or interstitial cells(IC) and nuclear (red) staining. (F) Confocal image of testicular tissue sections immunostained with pre-immune rabbit serum as negative control. (G) Adiponectinimmunostaining (arrows) around the seminiferous tubules in the peritubular area and in the interstitium. (H) Diffuse adiponectin immunostaining throughout theinterstitium (IC) and within the Leydig cells (arrows) around seminiferous tubule (ST). (I) Photomicrograph of a testicular tissue section that is counterstained followingimmunostaining with anti-chicken adiponectin IgG pre-adsorbed with the immunogen peptide. (J) Adiponectin immunostaining in flattened peritubular cells (arrows). Notethe absence of adiponectin immunostaining in spermatogonial cells (Sg) within the seminiferous tubule (ST). (K) Diffuse adiponectin staining in the interstitium, Leydig cells(arrows) and peritubular cells (arrow heads) outside of seminiferous tubules (ST). Figure reproduced with permission from Ocón-Grove et al. (2008; �2008, BioScientifica).

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ments of the seminiferous tubule, it is likely that adiponectin af-fects maturation and differentiation of spermatocytes and there-fore potentially influences spermatogenesis. Using quantitativereal-time PCR, testicular AdipoR1 and AdipoR2 mRNA quantitieswere 8.3- and 9-fold higher in adult broiler breeder chickens thanprepubertal chickens, respectively, suggesting that sexual matura-tion is associated with up- regulation of AdipoR1 and AdipoR2 geneexpression. A significant elevation of AdipoR1 and AdipoR2 geneexpression in sexually mature chickens is likely to be a result ofhigher metabolic activity related to spermatogenesis, testicularsteroid hormone production, and transport of spermatozoa and

testicular fluid. In this regard, plasma adiponectin levels werefound to be 4-fold higher in sexually mature versus sexually imma-ture mice (Combs et al., 2003).

Consistent with our findings on expression of adiponectin in thechicken testis, rat testis has been found to express adiponectin inthe interstitial Leydig cells (Caminos et al., 2008). Rat testicularadiponectin mRNA levels were modulated by gonadotropins, glu-cocorticoids, thyroxine, and peroxisome proliferator-activatedreceptor-c (Caminos et al., 2008). Furthermore, AdipoR1 werefound to be predominantly expressed in the rat seminiferous tu-bules while recombinant adiponectin treatment significantly

Fig. 4. Representative photomicrographs of testicular tissue sections from adult chickens showing AdipoR2-immunostained cells. Paraformaldehyde-fixed tissue sectionswere immunostained using anti-chicken AdipoR2 antiserum (A–I). (A) Intense AdipoR2 immunostaining in the adluminal and luminal compartments (bold arrows) of theseminiferous tubule (ST) and in the interstitium (open arrow). (B) AdipoR2-immunoreactive (ir) Leydig cells in the interstitium (box, IC; magnified in D) showing granularAdipoR2 staining in the cytoplasm and on (the) cell membrane (arrows in D). (C) AdipoR2 immunostaining in the seminiferous tubular (ST) adluminal and luminalcompartments (box; magnified in E). Round spermatids (Rs) and elongating spermatids (Es) show AdipoR2 immunostaining around the cell membrane in Panel E.Spermatozoa in the seminiferous tubular lumen (STL) show intense AdipoR2 immunostaining (arrow in Panel E). (F) Intense AdipoR2 immunostaining was also observed inthe Sertoli cell (S) syncytia (box; magnified in G; arrows). Peritubular area (PT) is marked to distinguish the two seminiferous tubules (ST). (H and I) Sertoli cells (S; arrows)and elongating spermatids (Es) towards seminiferous tubular lumen (STL) show intense AdipoR2 immunostaining. Peritubular area (PT) is marked to orient the seminiferoustubular cross-section in the photomicrographs. (J) Photomicrograph of testis tissue section immunostained with pre-immune serum in place of anti-chicken AdipoR2antiserum as negative control and counterstained. Figure reproduced with permission from Ocón-Grove et al. (2008; �2008, BioScientifica).

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inhibited testosterone secretion ex vivo (Caminos et al., 2008). Ta-ken together, adiponectin is likely to influence Leydig cellularmetabolism and steroidogenesis, possibly through activation ofAMP-activated protein kinase and peroxisome proliferator acti-vated receptors that have been previously identified in mammaliantestis tissues (Cheung et al., 2000; Froment et al., 2006). Further-more, expression of AdipoR2 appears to be critical for testicularfunction in mice as AdipoR2-deficient knockout mice exhibit re-duced testis weight characterized by atrophy of the seminiferoustubules and aspermia, while plasma testosterone levels remainedunaffected (Bjursell et al., 2007). Testicular adiponectin may likelyact as a paracrine/autocrine factor thereby supplementing blood-borne adiponectin to influence interstitial Leydig cells and seminif-

erous tubular cells in the chicken. Future studies should focus onelucidating the role of adiponectin signaling on steroidogenesis.

In chicken ovary, the adiponectin gene was found to be mainlyexpressed in theca cells and is suggested to exert a paracrine orautocrine effect on ovarian steroidogenesis (Chabrolle et al.,2007). A limited number of studies have revealed the effect ofadiponectin on ovarian steroidogenesis or its association withpolycystic ovarian syndrome (PCOS) in women. In porcine follicu-lar cells, adiponectin was found to increase steroidogenic acuteregulatory protein (StAR) transcript abundance but reduces cyto-chrome P450 aromatase expression (Ledoux et al., 2006). Similarly,adiponectin is suggested to exert an inhibitory effect on bovinetheca cell steroidogenesis (Lagaly et al., 2008). Adiponectin de-

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creased insulin-induced steroidogenesis and increased insulin-likegrowth factor-1-induced proliferation of cultured bovine granulosacells through a potential involvement of mitogen-activated proteinkinase (MAPK)-Erk1/2 pathway (Maillard et al., 2010). AdiponectinHMW isoform is selectively reduced in women with PCOS, inde-pendent of body mass index and insulin resistance (Aroda et al.,2008; O’Connor et al., 2010). Overall, adiponectin and its receptorsappear to be involved in steroidogenesis in the mammalian ovary.

8. Summary and conclusions

Our studies revealed that the chicken adiponectin mRNA se-quence shares only 65–68% homology to mammalian adiponectincDNA, although the deduced amino acid sequence of chickenadiponectin contained 22 glycine-X-Y repeats (where X and Y rep-resent any amino acid) at the N-terminal end as found in mamma-lian adiponectin. We also found that adiponectin is expressed in avariety of tissues in the chicken with the greatest expression inadipose tissue. Using gel filtration column chromatography andWestern blot analysis, adiponectin in chicken plasma and adiposetissue was found to be predominantly a multimeric HMW isoformthat is larger than 669 kDa mass. Mass spectrometric analysis ofchicken adiponectin revealed the presence of several post-transla-tional modifications to the lysine and proline residues in the col-lagenous domain. An enzyme immunoassay developed andvalidated for quantifying plasma adiponectin in chickens revealedthat plasma adiponectin levels ranged between 4 and 10 lg/ml inbroiler chickens. Chicken adiponectin receptors, AdipoR1 and Adi-poR2 appear to have seven distinct hydrophobic regions represent-ing seven transmembrane domains while their cDNA sequenceswere 76–83% identical to the respective mammalian sequences.Both AdipoR1 and AdipoR2 mRNA were found to be expressed inadipose tissue, liver, anterior pituitary gland, diencephalon, skele-tal muscle, kidney, spleen, ovary, and blood. Using anti-chickenadiponectin, AdipoR1, or AdipoR2 antibodies, adiponectin-, Adi-poR1-, AdipoR2-ir cells were localized in the broiler breeder chick-en testis. Furthermore, testicular AdipoR1 and AdipoR2 mRNAabundance were found to be significantly higher in adult chickenscompared to prepubertal chickens, suggesting that sexual matura-tion is likely associated with an up-regulation of testicular AdipoR1and AdipoR2 gene expression. We are currently elucidating thebiological functions of avian adiponectin and its importance toavian growth and reproduction.

Acknowledgments

This project was supported in part by National Research Initia-tive Competitive Grant No. 2007-35206-17905 from the USDACooperative State Research, Education, and Extension Service andby Agriculture and Food Research Initiative Competitive GrantNo. 2012-67015-30193 from the USDA National Institute of Foodand Agriculture.

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