Animal Reproduction Science 148 (2014) 53–62 Contents lists available at ScienceDirect Animal Reproduction Science jou rn al hom epage : w ww.elsevier.com/locate/anir eprosci Expression of orexins and their precursor in the porcine ovary and the influence of orexins on ovarian steroidogenesis in pigs Anna Nitkiewicz, Nina Smolinska, Anna Maleszka, Katarzyna Chojnowska, Tadeusz Kaminski ∗ Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, ul. Oczapowskiego 1A, 10-719 Olsztyn, Poland a r t i c l e i n f o Article history: Received 14 October 2013 Received in revised form 16 March 2014 Accepted 23 March 2014 Available online 9 May 2014 Keywords: Orexin Corpus luteum Granulosa cells Theca interna cells Steroid hormones Pig a b s t r a c t Orexins A and B are hypothalamic neuropeptides associated with homeostasis and the reproductive system. The aim of the study was to compare the expression of the prepro- orexin gene and the intensity of orexins immunoreactivity in the porcine ovary (corpora lutea, granulosa and theca interna cells) during four different stages of the oestrous cycle (days: 2–3, 10–12, 14–16 and 17–19) and to examine the in vitro effect of orexins on the secretion of steroid hormones by porcine luteal, granulosa and theca interna cells. The high- est expression of prepro-orexin mRNA was observed in theca interna cells on days 17–19 of the oestrous cycle. The highest content of immunoreactive orexin A was noted in corpora lutea on days 10–12 and the highest level of immunoreactive orexin B on days 14–16 of the cycle. Immunoreactive orexin A concentrations were higher in theca interna cells than in granulosa cells, whereas similar levels of immunoreactive orexin B were observed in both cell types. Under in vitro conditions, at the concentration of 10 nM, orexins A and B inhibited FSH-induced oestradiol secretion by granulosa cells. The obtained results suggest that the pattern of orexin peptide expression in the porcine ovary is related to the animals’ hormonal status. Our findings imply that orexins can affect porcine reproductive functions through modulation of ovarian steroidogenesis. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Orexins A and B, also known as hypocretin 1 and 2, are hypothalamic neuropeptides which were discovered simultaneously in 1998 by two independent laboratories (Sakurai et al., 1998; de Lecea et al., 1998). Both orexins are derived by proteolytic cleavage from a common 130-amino acid (aa) precursor molecule, prepro-orexin (PPO). Orexin A (OXA) is a 33-aa peptide, while orexin B (OXB) consists of 28 amino acids. The amino-acid sequence of orexin A is ∗ Corresponding author. Tel.: +48 89 523 39 04; fax: +48 89 523 39 37. E-mail address: tkam@uwm.edu.pl (T. Kaminski). highly conserved among mammalian species. The amino acid sequence of OXA is identical in pigs, humans, rats and cows, and the sequence of OXB in pigs and humans dif- fers by only one amino acid residue (Sakurai et al., 1998; Dyer et al., 1999). The biological action of hormones is mediated by binding two distinct, but structurally similar G protein-coupled receptors: orexin receptor type 1 (OX1R) and orexin receptor type 2 (OX2R). Orexin A is a more selec- tive ligand for OX1R, whereas OX2R binds both orexins with similar affinity (Sakurai et al., 1998; de Lecea et al., 1998). Orexins were originally discovered in the lateral hypothalamus, a structure involved in the control of food intake and energy balance (Sakurai et al., 1998; de Lecea http://dx.doi.org/10.1016/j.anireprosci.2014.03.015 0378-4320/© 2014 Elsevier B.V. All rights reserved.