Ovary-specific novel peroxisome proliferator activated receptors-gamma transcripts in buffalo Isha Sharma a , Rachna Monga a , Natwar Singh a , Tirtha Kumar Datta b , Dheer Singh a, ⁎ a Molecular Endocrinology Laboratory, Animal Biochemistry Division, National Dairy Research Institute, Karnal-132001, Haryana, India b Animal Biotechnology Centre, National Dairy Research Institute, Karnal-132001, Haryana, India abstract article info Article history: Accepted 30 April 2012 Available online 16 May 2012 Keywords: PPARγ Granulosa cells Ovary Buffalo RACE Tissue-specific transcripts In the present study, we describe the isolation and characterization of the transcripts encoding peroxisome proliferator-activated receptor gamma (PPARγ1 and PPARγ2) in buffalo ovary. 5′ RACE experiments and se- quence analysis showed that these transcripts (PPARγ1a, PPARγ1b and PPARγ2) were transcribed by the dif- ferent promoter usage and alternative splicing of terminal 5′-exon. The distribution of these isoforms of PPARγ transcripts in different tissues (ovary, mammary gland, spleen, liver, lung, adipose tissue) was inves- tigated using quantitative real time analysis. Tissue- and transcript-specific expression analyses showed that a transcript, transcribed from distal promoter, not only expressed preferentially in ovary but contributes predominantly to PPAR gamma expression in ovary. Western blot analysis of both, in vivo and in vitro, exper- iments also supported that PPARγ1 predominantly expressed in ovary. In buffalo granulosa cells culture, the isolated transcripts were found to be up-regulated by both natural (CLA) and synthetic (Rosiglitazone) li- gands and effect was reversed by PPARγ antagonist GW9662. In conclusion, the present study identified an ovary-specific novel transcript, transcribed by distal promoter, predominantly expressed in ovary which could have functional relevance in buffalo ovary. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Peroxisomes are cytoplasmic organelles which are important in mammals in modulation of lipid homeostasis, including the metabolism of long-chain fatty acids and conversion of cholesterol to bile salts. Receptors found on these organelles perform many cellular and meta- bolic roles, which are named initially on the basis of the stimulation of peroxisome proliferation in rodents (Lazarow and Fujiki, 1985; Nemali et al., 1989; Vamecq and Draye, 1989). The peroxisome proliferators- activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor super family (Issemann and Green, 1990). They share a high degree of structural homology with all members of the super family, particularly in the DNA-binding domain and Ligand and cofactor binding domain. PPARs heterodimerize with the retinoid X receptor (RXR) belong to the same receptor super family (Keller et al., 1993; Kliewer et al., 1992) and bind to specific DNA sequence peroxisome proliferator responsive elements (PPREs) located within the promoter of the respective gene (Issemann and Green, 1990; Keller et al., 1993; Perlmann and Jansson, 1995). PPARs are activated by a variety of chemical compounds and they are targeted by varied number of structurally diverse ligands (Dreyer et al., 1992; Issemann and Green, 1990). In the context of their diverse physiological and developmental functions, PPARs exhibit broad, but isotype and tissue-specific, expression patterns (Braissant et al., 1996; Kliewer et al., 1992). One of the PPAR isotypes i.e. PPARγ is expressed as two or more isoforms, of which PPARγ2 is found at high levels in the adipose tissues, whereas PPARγ1 has a broader expression pattern (Tontonoz et al., 1994; Zhu et al., 1995). The two most upstream exons (A1, A2) of mouse PPARγ1, comprise the 5′-untranslated region (UTR) and are spliced- to the six most 3′-proximal exons (1–6) which extends the common coding region shared by the two isoforms (Zhu et al., 1995). The single exon (B), located between exon A2 and exon A1 of PPARγ2, comprise the 5′-UTR and an additional 30 amino acid (Zhu et al., 1995). Like all nuclear receptor, PPARs are also highly conserved among species (Komar and Curry, 2002). PPARs have three major domains, one exon for the N terminal A/B domain, two exons for the Gene 504 (2012) 245–252 Abbreviation: PPARγ, peroxisome proliferative activated receptor-gamma; RACE, random amplified cDNA ends; RLM-RACE, RNA ligase mediated random amplified cDNA ends; cDNA, complementary DNA; UTR, untranslated regions; CLA, conjugated linoleic acid; RXR, retinoid X receptor; PPRE, peroxisome proliferator responsive elements; DBD, DNA-binding domain; LBD, ligand-binding domain; CIP, calf intestine alkaline phospha- tase; TAP, tobacco acid pyrophosphatase; Q-PCR, quantitative polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; TSS, transcription start site; SF, small follicle; LF, large follicle; CL, corpus luteum; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; TBST, tris-buffered saline and tween 20; PBS, phosphate‐buffered saline; DMEM, Dulbecco's modified eagle medium; FSH, follicle stimulating hormone; IGF-1, insulin like growth factor-1; BSA, bovine serum albumin; LHr, luteinizing hormone receptor; NCBI, National Center for Biotechnology Information. ⁎ Corresponding author at: Molecular Endocrinology Laboratory, Animal Biochemistry Division, National Dairy Research Institute (NDRI), Karnal-132001, Haryana, India. Tel.: +91 184 2259135; fax: +91 184 2250042. E-mail address: drdheer.singh@gmail.com (D. Singh). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.04.090 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene