Original article Metabolic state defines the response of rabbit ovarian cells to leptin Abdel Halim Harrath a , Olga Østrup b , Jan Rafay d,e , Iveta Koni9 cková (Florkovi9 cová) d , Jozef Laurincik c , Alexander V. Sirotkin a,c,d, * a Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia b Center for Genomic Medicine, Copenhagen University Hospital, Copenhagen, Denmark c Department of Zoology and Anthropology, Constantine the Philosopher University, 949 74 Nitra, Slovakia d Department of Genetics and Reproduction, Research Institute of Animal Production, 949 59 Lužianky, Slovakia e Department of Biotechnology, University of SS. Cyril and Methodius, 917 01 Trnava, Slovakia A R T I C L E I N F O Article history: Received 11 October 2016 Received in revised form 12 November 2016 Accepted 18 November 2016 Available online xxx Keywords: Caloric restriction Leptin Ovary Proliferation Apoptosis Hormones Rabbit A B S T R A C T Leptin is a hormone that mediates the effect of the metabolic state on several biological functions, including reproduction. Leptin affects reproductive functions via alterations in the release of hormonal regulators. However, the extent to which caloric restriction (CR) can affect the complex processes of reproduction by other mechanisms, such as altering ovarian functions via direct binding/response to leptin, is unknown. Therefore, the aim of the present study was to show basic ovarian cell functions and CR on the response of ovarian cells to leptin. Female rabbits were subjected to 50% CR restriction for 10 days before ovulation. On the day of ovulation, both control and CR animals were sacrificed. Isolated granulosa cells were cultured for 2 days with and without leptin (100 ng/ml), and the accumulation of various markers was evaluated using immunocytochemistry; i.e., cell proliferation (PCNA and cyclin B1), apoptosis (bax), MAP/ERK1,2 kinase (MAPK), protein kinase A (PKA), and IGF-I. In addition, the release of IGF-I and estradiol (E 2 ) by cells cultured with and without leptin (1, 10, 100, 1000, or 10,000 ng/ml) was assessed by radioimmunoassay (RIA). In the granulosa cells of control animals, leptin promoted cyclin B1, MAPK, and PKA accumulation, but not that of PCNA, and reduced bax and IGF-I accumulation. These cells responded to leptin by increased IGF-I, but not E 2 release. In cells of CR animals, leptin increased cyclin B1 accumulation, but decreased PCNA, MAPK, and IGF-I expression. Bax and PKA were not affected. Leptin resulted in a decrease in IGF-I release. CR modulated the influence of leptin on E 2 release dose dependently, i.e., E 2 increased at 10 and decreased at 10,000 ng/ml. Therefore, CR modified the influence of leptin on PCNA, E 2 , bax, PKA, MAPK, and IGF-I release, but it did not change the effect of leptin on cyclin B1 and IGF-I accumulation within the cells. Our data showed that leptin directly affected proliferation, apoptosis, and hormone release by ovarian cells, probably via PKA- and MAPK-dependent pathways. Furthermore, it was demonstrated that nutrition could influence reproduction by affecting the response of ovarian cells to leptin. ã 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved. 1. Introduction Leptin (L) is a metabolic hormone that plays an important role in the control of reproduction. It can affect reproduction at different regulatory levels including the hypothalamus, pituitary, and reproductive organs [2,20,35]. Metabolic state affects repro- duction and synchronizes reproduction (an energy demanding process) with optimal nutritional conditions, and defines livestock reproductive efficiency. Caloric restriction (CR) affects metabolism and modulates reproductive processes by altering the release of metabolic hormones including L, and subsequently suppresses the hypothalamo-hypophysial-gonadal axis and disturbs reproductive processes, such as puberty and fertility [2,17,23,26,8]. In rabbits, CR suppresses the release of reproductive hormones and embryo development [3,10,18,22], and promotes oocyte maturation, fertility, and kindling rates [11,9,25,1,6,7]. CR inhibits L, a promoter of reproductive processes in various mammalian species [2,20,23,26,8]. L was able to affect steroid and IGF-I release by cultured rabbit ovarian cells, whilst L injections to rabbit does reduced the level of these hormones in plasma and promoted * Corresponding author at: Department of Zoology and Anthropology, Con- stantine the Philosopher University, 949 74 Nitra, Slovakia. E-mail address: asirotkin@ukf.sk (A.V. Sirotkin). http://dx.doi.org/10.1016/j.repbio.2016.11.002 1642-431X/ã 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved. Reproductive Biology xxx (2016) xxx–xxx G Model REPBIO 211 No. of Pages 6 Please cite this article in press as: A.H. Harrath, et al., Metabolic state defines the response of rabbit ovarian cells to leptin, Reprod Biol (2016), http://dx.doi.org/10.1016/j.repbio.2016.11.002 Contents lists available at ScienceDirect Reproductive Biology journal homepage: www.elsevier.com/locate/repbio