Composition of marsupial zona pellucida: a molecular and phylogenetic approach Carla Moros-Nicola ´s A,G , Pascale Chevret B , Marı ´a Jose ´ Izquierdo-Rico A , William V. Holt C , Daniela Esteban-Dı ´az A , Manel Lo ´ pez-Be ´jar D , Eva Martı ´nez-Nevado E , Maria A. Nilsson F , Jose ´ Ballesta A and Manuel Avile ´s A,G A Department of Cell Biology and Histology, Faculty of Medicine, University of Murcia, Campus Mare Nostrum and IMIB, Murcia 30100, Spain. B Laboratoire de Biome ´trie et Biologie Evolutive, UMR5558, CNRS, Universite ´ de Lyon, Universite ´ Claude Bernard Lyon 1, Villeurbanne 69622, France. C Institute of Zoology, Regent’s Park, London NW1 4RY, UK. D Department of Animal Health and Anatomy, Universitat Auto ` noma de Barcelona, Barcelona 08193, Spain. E Veterinary Department, Zoo-Aquarium Madrid, Casa de Campo s/n., Madrid 28011, Spain. F Senckenberg Biodiversity and Climate Research Centre, Senckenberg Gesellschaft fu ¨r Naturforschung, Senckenberganlage 25, Frankfurt am Main D-60325, Germany. G Corresponding authors. Emails: carla.moros@um.es; maviles@um.es Abstract. The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennett’s wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species. Additional keywords: evolution, fertilisation, oocyte, sperm–egg interaction. Received 23 December 2016, accepted 20 September 2017, published online 22 November 2017 Introduction During fertilisation, female and male gametes are involved in a series of complex events that allow the formation of a zygote. In marsupials, preimplantation embryos are enclosed by three acellular envelopes: the zona pellucida (ZP), a mucoid coat and an outer shell coat (Selwood 2000). Furthermore, in some marsupials there is a sub-zonal, extra-cellular matrix coat (Selwood 2000). In eutherian mammals, the structure and composition of the ZP has been studied in detail, but this is not the case in most marsupials. The ZP plays a key role in species- specific binding between gametes in most species, as well as in the induction of the acrosome reaction, polyspermy block and protection of the preimplantation embryo (Yanagimachi 1994; Dean 2004, 2007; Wassarman and Litscher 2009). Eutherians can be classified into three categories according to their ZP composition: (1) species with a ZP formed from ZP1, ZP2 and ZP3, in which ZP4 is a pseudogene (to date, only the house mouse; Bleil and Wassarman 1980; Lefie `vre et al. 2004; Evsikov et al. 2006; Goudet et al. 2008), (2) species with three proteins, in which ZP1 is a pseudogene (pig, cow, dog, common marmoset, dolphin, tarsier, Antarctic fur seal, Weddell seal and fox; Hedrick and Wardrip 1987; Noguchi et al. 1994; Goudet CSIRO PUBLISHING Reproduction, Fertility and Development, 2018, 30, 721–733 https://doi.org/10.1071/RD16519 Journal compilation Ó CSIRO 2018 www.publish.csiro.au/journals/rfd