Comparative genomics of hormonal signaling in the chorioallantoic membrane of oviparous and viviparous amniotes
Graphical abstract
Introduction
Reproduction is fundamental for organisms because it is the only mechanism that allows genes to be transmitted through generations. The ancestral reproductive mode for amniote vertebrates (reptiles, birds, and mammals) is egg-laying (oviparity), but live birth (viviparity) has evolved independently multiple times in reptiles, and once in mammals (Blackburn, 2014, Griffith et al., 2015). Embryonic development requires a suitable supply of respiratory gases and water. In viviparous species where the embryo is not exposed to the external environment, adequate structures are required to facilitate the exchange of materials between mother and offspring. In oviparous species, the chorioallantoic membrane (CAM, Fig. 1) is an embryonic tissue that lines the majority of the internal surface of the eggshell late in incubation, where it is the primary gas exchange organ between the embryo and the external environment (Piiper et al., 1980).
In contrast, in viviparous amniotes transfer of materials between mother and offspring occurs in a placenta composed of both maternal (uterine) and embryonic (chorioallantoic or yolk sac membranes) tissues (Blackburn, 2006, Van Dyke et al., 2014a, Wake, 1992). In eutherian mammals, the definitive placenta is produced by the uterus and the embryonic CAM. Chorioallantoic placentae are also found in all viviparous squamate reptiles, with the placental region in some species having morphological and physiological specializations for gas exchange and nutrient transport (Adams et al., 2005, Biazik et al., 2010, Blackburn, 1993, Griffith et al., 2013a, Van Dyke et al., 2015). Viviparous amniote placentae are extremely diverse in structure and function, with some placentae transporting respiratory gases, water, and small amounts of other molecules, and others transporting all classes of nutrients required for embryonic development (placentotrophy) (Ferner and Mess, 2011, Thompson and Speake, 2002, Thompson et al., 2002). Placentotrophy has evolved relatively few times in amniotes, and exists along a continuum, with complete reliance on egg yolk resources (lecithotrophy, in most squamates) at one end and complete reliance on placental nutrient transfer on the other end (as in eutherian mammals and some lizards). There are also species with intermediate reliance on both placental transfer and ovulated yolk resources, such as the lizard Pseudemoia entrecasteauxii (Ferner and Mess, 2011, Thompson et al., 2000, Van Dyke et al., 2014b).
To understand the evolution of viviparity, it is essential to understand the processes that regulate key pregnancy functions and how these processes have been modified during evolutionary transitions (Thompson and Speake, 2006). Hormones play an essential role in almost every step of amniote reproduction. Hormonal signals are responsible for egg production, vitellogenesis, ovulation, maintenance of the egg in utero, and oviposition/parturition (Callard et al., 1992, Custodia-Lora and Callard, 2002, Jones, 2017, Licht, 1979). Furthermore, hormones are required to induce tissue-level changes that facilitate pregnancy, including, for example, cellular changes in the uterus for nutrient transport to offspring. Finally, hormones facilitate communication between maternal and embryonic tissue throughout gestation, which is important for coordination of developmental features such as apposition of vascular beds within the placenta (Murphy et al., 2011).
The fundamental difference between oviparous and viviparous species is that in viviparous species eggs are retained in utero until development is complete. As embryonic development in ectotherms is temperature rather than time-dependent, viviparous mothers cannot precisely determine the developmental state of offspring without relying on embryonic cues. Hormone production by embryos therefore represents one possible way for eggs to be retained in utero and deposited at the correct developmental stage which would ultimately give rise to viviparity. For example, progesterone is a key hormonal regulator of pregnancy/gravidity in both mammals and reptiles (Custodia-Lora and Callard, 2002). Genes involved in progesterone biosynthesis have been identified in the chorioallantoic tissue of the chicken, suggesting that this tissue may ancestrally produce progesterone (Albergotti et al., 2009). Production of progesterone by the chorioallantoic placenta could play a significant role in the evolution of viviparity, because egg retention could be achieved in part by simply increasing the transfer of progesterone from embryo to mother by, for example, reducing eggshell thickness.
As well as facilitating maternal-offspring communication, embryonic production of hormones allows embryos to manipulate maternal physiology, which can facilitate parent-offspring conflict. Parent-offspring conflict is a major driver of the evolution of viviparity and placental functions in vertebrates (Blackburn, 2015, Crespi and Semeniuk, 2004, Zeh and Zeh, 2008). Conflict arises when offspring demand more resources than is optimal for the lifetime reproductive success of the parent (Trivers, 1974). For maternal-offspring conflict to occur during pregnancy, embryos must be able to manipulate maternal reproductive physiology, such as nutrient transport across the placenta. This manipulation can be achieved either by gaining direct access to maternal resources through invasive placentation, or through embryonic secretions that change the physiology of maternal tissue. In species with non-invasive epitheliochorial placentation, such as those examined in this study, nutrients must be actively transported across maternal tissue to the embryo, restricting embryonic control. However, embryonic control of transport could still occur in these species (Fowden et al., 2006): In the horse, which has non-invasive epitheliochorial placentation, embryos actively manipulate placental nutrient supply, likely by the production of hormones such as insulin like growth factor 2 (Allen et al., 2002).
In species with non-invasive placentation, if offspring are able to manipulate the activity of maternal tissues by the production and transport of hormones to maternal tissues, conflict may occur. Extensive embryonic production of growth factors occurs in the embryonic trophoblast of mammals (Fowden and Forhead, 2009, Fowden et al., 2006). Understanding how the production of growth factors correlates with different reproductive modes and the evolution of placentotrophy will allow us to understand if embryonic manipulation, through growth factor production, is a novel function of the chorioallantois in placentotrophic lineages. Alternatively, the production of growth factors may be the result of modifications to processes that occurred ancestrally in chorioallantoic tissue, with growth factors expressed in the chorioallantois of oviparous species.
We measured gene expression in the embryonic chorioallantoic tissue of species across the amniote phylogeny. To characterize the hormonal activity of the chorioallantoic membrane in amniotes we quantified the expression of genes which encode peptide hormones, proteins responsible for hormone synthesis, and hormone receptors. From these data we addressed two specific questions: 1) Is hormone production by the chorioallantoic placenta widespread in viviparous amniotes and 2) Has placental hormonal signaling evolved following the recruitment of hormone related genes in viviparous lineages, or is expression of hormone-related genes a result of coopting ancestral gene expression patterns.
Section snippets
Species selection
Recent advances in next-generation sequencing technology make it possible to simultaneously measure the expression of most genes in a given tissue (transcriptome). We evaluated the expression of hormone-related genes in the CAM of multiple species using newly-generated and published transcriptome data for the chorioallantoic membrane (Fig. 2). We sampled two oviparous taxa including the chicken (Gallus gallus) and an oviparous population of the skink Lerista bougainvillii (which is a bimodally
CAM hormone gene expression
The CAM is a hormone-producing organ in amniotes: of the 423 hormone-related genes examined, 321 genes are expressed in the CAM of at least one of the studied species. Expression of hormone-related genes is not confined to viviparous taxa, which confirms findings previously reported in oviparous birds, crocodiles, and tortoises (Albergotti et al., 2009, Cruze et al., 2013, Cruze et al., 2012). Ninety-three genes show conserved expression across all studied species, suggesting they may perform
Conclusions and future research
The conserved expression of hormone producing genes observed here suggests that the chorioallantoic membrane is ancestrally an endocrine organ. Ancestral expression of several hormones in all studied amniotes, including the angiogenic factors VEGFA and PGF, suggests that some placental signaling function has evolved by utilizing genes expressed in the CAM of the ancestral amniote. An important evolutionary question follows from our study: if many hormones important for placental functions in
Acknowledgments
Lizards were collected under New South Wales National Parks and Wildlife licence to MBT (SL100401). All animal work was conducted with University of Sydney Animal Ethics approval. We thank James Van Dyke, Fran van den Berg, Melanie Laird, and Jessica Dudley for comments on the manuscript. We thank Intersect Australia Ltd. for supercomputing resources and Bosch MBF for laboratory resources. Project supported by Gaylord Donnelley Environmental Postdoctoral Fellowship Grant to OWG, the Australian
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