Journal of Neuroendocrinology, 2014, 26, 870–887 ORIGINAL ARTICLE © 2014 British Society for Neuroendocrinology Molecular Analysis of the Koala Reproductive Hormones and Their Receptors: Gonadotrophin-Releasing Hormone (GnRH), Follicle-Stimulating Hormone b and Luteinising Hormone b with Localisation of GnRH E. R. Busby*, S. Soeta†, N. M. Sherwood* and S. D. Johnston‡ *Department of Biology, University of Victoria, Victoria, B.C., Canada. †School of Veterinary Medicine, Nippon Veterinary and Life Science University, Tokyo, Japan. ‡School of Agriculture and Food Science, The University of Queensland, Gatton, Australia. Journal of Neuroendocrinology Correspondence to: Ellen R. Busby, Biology Department, University of Victoria, Petch Bldg, 3800 Finnerty Road, Victoria, BC V8W 3N5, Canada (e-mail: erbusby@uvic.ca). During evolution, reproductive hormones and their receptors in the brain-pituitary-gonadal axis have been altered by genetic mechanisms. To understand how the neuroendocrine control of reproduction evolved in mammals, it is important to examine marsupials, the closest group to placental mammals. We hypothesised that at least some of the hormones and receptors found in placental mammals would be present in koala, a marsupial. We examined the expression of koala mRNA for the reproductive molecules. Koala cDNAs were cloned from brain for gonado- trophin-releasing hormones (GnRH1 and GnRH2) or from pituitary for GnRH receptors, types I and II, follicle-stimulating hormone (FSH)b and luteinising hormone (LH)b, and from gonads for FSH and LH receptors. Deduced proteins were compared by sequence alignment and phyloge- netic analysis with those of other vertebrates. In conclusion, the koala expressed mRNA for these eight putative reproductive molecules, whereas at least one of these molecules is missing in some species in the amniote lineage, including humans. In addition, GnRH1 and 2 are shown by immunohistochemistry to be expressed as proteins in the brain. Key words: marsupials, gonadotrophin-releasing hormone, luteinising hormone, follicle- stimulating hormone, receptors, neuropeptides. doi: 10.1111/jne.12208 Although the marsupial brain differs from other mammals in having reduced development of the neocortex and lack of a corpus callo- sum between the hemispheres, the areas of the brain that control reproduction, such as the hypothalamus, appear similar to those described in eutherian species (1). Whereas both groups are vivipa- rous, their pattern of reproduction has fundamental differences, as might be expected because they last shared a common therian ancestor between 130 and 148 million years ago (2–4). Marsupial young are born at a very immature stage of development, which is followed by a long period of lactation within the pouch and pro- longed developmental stages compared to placental mammals. To understand the evolution of neuroendocrine control of reproduction in mammals, it is important to identify orthologous hormones and receptors in the brain-pituitary-gonadal axis in a marsupial such as the koala. Detailed knowledge of the koala reproductive system, including the structure of the reproductive hormones, is also essential for functional studies associated with the genetic and reproductive management of this species (5,6). One approach for studying the reproductive axis is the molecu- lar analysis of hormones and receptors in three tissues: (i) brain tissue for characterisation and histochemical location of gonado- trophin-releasing hormones (GnRHs); (ii) pituitary tissue for GnRH receptors (GnRHRs), follicle-stimulating hormone (FSH) and lutein- ising hormone (LH); and (iii) gonadal tissue for the LH receptor (LHR) and FSH receptor (FSHR). Divergence of these molecules in amniotes is useful for phylogenetic analysis to clarify the evolu- tionary path of mammalian reproduction. The origin of mammals has been traced to therapsids, which resembled modern lizards. This amniote lineage includes monotremes (e.g. platypus and echidna) that are lactating mammals with lizard-like reproductive tracts and egg laying. The monotremes, marsupials and placental mammals last shared an ancestor 166 million years ago, followed