Expression of Adiponectin and its Receptors in the Porcine Hypothalamus During the Oestrous Cycle T Kaminski, N Smolinska, A Maleszka, M Kiezun, K Dobrzyn, J Czerwinska, K Szeszko and A Nitkiewicz Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland Contents Adiponectin is a hormonal link between obesity and repro- duction, and its actions are mediated by two types of receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). This study compares the expression levels of adiponectin and adiponectin receptor mRNAs and proteins in selected areas of the porcine hypothalamus responsible for GnRH production and secretion: the medio- basal hypothalamus (MBH), pre-optic area (POA) and stalk median eminence (SME). The tissue samples were harvested on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Adiponectin mRNA expression in MBH was significantly lower on days 14–16, whereas in SME, the most pronounced gene expression was found on days 2–3 of the cycle (p < 0.05). Adiponectin protein in MBH was most abundant on days 17–19 and in POA on days 2–3 (p < 0.05). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.05) on days 14–16. AdipoR1 gene expression in POA was potentiated on days 2–3 and 10–12 of the oestrous cycle (p < 0.05). In SME, the highest AdipoR1 mRNA expression was noted on days 2–3 (p < 0.05). The concentrations of the AdipoR1 protein in POA were similar throughout the luteal phase (days 2–14 of the cycle), and they decreased on days 17–19 (p < 0.05). In SME, AdipoR1 protein expression peak occurred on days 2–3 (p < 0.05). The expression patterns of the AdipoR2 gene in MBH, POA and SME revealed the highest mRNA levels on days 2–3 of the cycle (p < 0.05). The highest content of AdipoR2 protein in MBH was reported on days 2–3 (p < 0.05), while in POA on days 17–19 and in SME on days 10–12 and 14–16 (p < 0.05). This study demonstrated that adiponectin and adiponectin receptor mRNAs and proteins are present in the porcine hypothalamus and that their expression levels are determined by the pig’s endocrine status related to the oestrous cycle. Introduction Adiponectin is one of the adipocytokines highly expressed in the adipose tissue and is one of the most abundant plasma proteins (2–25 lg/ml) in mammals (Nishizawa et al. 2002; B€ ottner et al. 2004). Adiponectin circulates in the serum in different multimeric forms, including trimers, hexamers and high-molecular-weight multimers, and as the globular fraction (globular adiponectin) generated by the proteolytic cleavage of multimers. Adiponectin controls energy homeostasis and insulin sensitivity, and it affects lipid synthesis, vasodilatation and atherogenic activity (Yamauchi et al. 2002; Trujillo and Scherer 2005). Plasma adiponectin concentrations are inversely correlated with the adipose tissue reservoir (Arita et al. 1999). Adiponectin is also identified in cerebrospinal fluid (Kos et al. 2007), and the hormone levels increase after its intravenous injec- tion (Qi et al. 2004). Adiponectin actions are mediated by two types of receptors: AdipoR1 and AdipoR2. Unlike G-coupled protein receptors, AdipoR1 and AdipoR2 are seven- transmembrane domain receptors with an extracellular carboxyl terminus and an intracellular amino terminus. AdipoR1 shows high affinity for the globular form of adiponectin, and AdipoR2 has an intermediate binding affinity for both full-length and globular species. The receptors are highly related and share 66.7% sequence identity in mice (Yamauchi et al. 2003). AdipoR1 and AdipoR2 are widely expressed in humans and rodents, which suggests that adiponectin has pleiotropic effects. The highest levels of AdipoR1 expression are observed in skeletal muscles, whereas AdipoR2 is most abundantly expressed in the liver (Yamauchi et al. 2003; Lord et al. 2005). The presence of the adiponectin receptors was observed in human and rodent hypothalami, including in the paraventricular nucleus (Kos et al. 2007; Guillod-Maximin et al. 2009), in periventricular areas and the pituitary (Yamauchi et al. 2003; Rodriguez- Pacheco et al. 2007; Psilopanagioti et al. 2009). Both types of receptors were also expressed in GT1-7 hypotha- lamic GnRH neuron cells (Wen et al. 2008). The expres- sion of adiponectin and its receptors in species other than humans and rodents remains largely unexplored. Adiponectin seems to be a hormonal link between obesity and reproduction. It was described as a factor affecting ovarian steroidogenesis, oocyte maturation, embryo implantation and development (Palin et al. 2012). Several lines of evidence also suggest that adiponectin influences the reproductive system by exerting central effects on the highest branch of the hypothalamic–pituitary–ovarian axis, inhibiting GnRH (Wen et al. 2008) and GnRH-induced LH secretion (Rodriguez-Pacheco et al. 2007; Lu et al. 2008). To date, there has been no research investigating the gene and protein expression patterns of adiponectin and its receptors in porcine hypothalamic structures respon- sible for GnRH production and secretion, including the mediobasal hypothalamus (MBH), pre-optic area (POA) and stalk median eminence (SME), or the effect of the oestrous cycle on transcript and protein levels in animals. The objective of this study was to compare the expression levels of adiponectin, AdipoR1 and AdipoR2 mRNAs and proteins in selected areas of the porcine hypothalamus during the oestrous cycle. Materials and Methods Animals and tissue collection This study was carried out in accordance with ethical standards of the Animal Ethics Committee at the © 2014 Blackwell Verlag GmbH Reprod Dom Anim 49, 378–386 (2014); doi: 10.1111/rda.12282 ISSN 0936–6768