Embryonic suckling and maternal specializations in the live-bearing teleost Zoarces viviparus Skov, Peter Vilhelm; Steffensen, John Fleng; Sorensen, Thomas Flarup; Qvortrup, Klaus Published in: Journal of Experimental Marine Biology and Ecology DOI: 10.1016/j.jembe.2010.08.024 Publication date: 2010 Document version Early version, also known as pre-print Citation for published version (APA): Skov, P. V., Steffensen, J. F., Sorensen, T. F., & Qvortrup, K. (2010). Embryonic suckling and maternal specializations in the live-bearing teleost Zoarces viviparus. Journal of Experimental Marine Biology and Ecology, 395(1-2), 120-127. https://doi.org/10.1016/j.jembe.2010.08.024 Download date: 07. apr.. 2020 Journal of Experimental Marine Biology and Ecology 395 (2010) 120–127 Contents lists available at ScienceDirect Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe Embryonic suckling and maternal specializations in the live-bearing teleost Zoarces viviparus Peter Vilhelm Skov a,⁎, John Fleng Steffensen a, Thomas Flarup Sørensen b, Klaus Qvortrup c a Marine Biological Laboratory, University of Copenhagen, DK-3000 Helsingør, Denmark b Department of Science, Systems and Models, Roskilde University, DK-4000 Roskilde, Denmark c Department of Biomedical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark article info abstract Article history: The European eelpout follows an aplacental viviparous reproductive strategy, in which gestation lasts 4– Received 25 March 2010 5 months. During the last months of development yolk reserves are depleted, and embryos depend on an Received in revised form 20 August 2010 external source of nutrients. Here we provide evidence for novel specialized physiological, morphological Accepted 23 August 2010 and behavioural adaptations, which we propose as the responsible mechanisms for the exchange of nutrients and gases between the maternal organism and her embryos. Ovarian follicles contain an internal glomerulus- Keywords: like structure within the distal tip of each follicle. Ultrastructural examination indicated a capacity for steroid Matrotrophy fl Nutrition synthesis and secretion. Gel electrophoresis demonstrated a protein size distribution in the follicular uid Oxygen different from that of the maternal serum, and that ovarian fluid is devoid of protein. From vascular casts and Suckling histological sections the follicle was reconstructed. The glomerulus has a central canal that is exteriorized at Viviparity the tip of the follicle, allowing passage of follicular fluid. Oxygen measurements across the ovary of near- term females showed a strongly hypoxic ovary lumen, yet ovarian fluid adjacent to follicles was oxygen saturated. As another novel observation, embryos were seen engaged in suckling on follicles. We hypothesize that embryos use the follicles on the ovarian wall as placental analogues and that they use their mobile jaw apparatus to attach themselves and apply suction. © 2010 Elsevier B.V. All rights reserved. 1. Introduction of near-term embryos, several hypothesis have been put forward as to how Z. viviparus provides nutrition for their young. Soin (1968) The course of embryonic development in the European eelpout, described the development of a mobile jaw apparatus early in the Zoarces viviparus (Linnaeus, 1758), has been described in detail (Soin, embryonic development, which for some time was considered a 1968; Kristoffersson et al., 1973; Korsgaard and Andersen, 1985; mechanism to facilitate ingestion of ovarian fluid, the uptake of which Rasmussen et al., 2006). In brief, the female reproductive cycle is is facilitated by a hypertrophied hind gut (Kristoffersson et al., 1973). initiated by the simultaneous maturation of 25–400 oocytes in the Nutritive substances in the ovarian fluid were thought to originate ovarian wall (Soin, 1968; Rasmussen et al., 2006). Each oocyte has its from a fatty mucous fluid secreted by the ovary wall (Stuhlmann, own vascular supply in the form of follicles that grow during 1887). However, as no nutritional components have been found in the maturation and project perpendicularly into the ovary lumen. During ovarian fluid (Kristoffersson et al., 1973; Korsgaard, 1983) this ovulation, a single mature oocyte is released by each follicle into the scenario appears unlikely. lumen of the ovary, and follicles do not undergo atresia during the In contrast, the fluid within the follicle has been shown to be rich subsequent development of embryos. in protein, free fatty acid and glucose, with levels identical to that of Gestation in the eelpout lasts 18–22 weeks. Eggs hatch 3 weeks maternal serum (Korsgaard, 1983). Because the follicle is a closed after fertilization and the developing embryos deplete their yolk sac structure, these nutritional substances are not readily available to reserves during the following 6 weeks (Rasmussen et al., 2006). For developing embryos, and various proposals have been made as to the remaining 9–13 weeks until parturition, embryos have a wet mass which mechanisms might facilitate transepithelial passage (Skov gain of more than 1500% (Kristoffersson et al., 1973; Korsgaard and et al., 2007). Andersen, 1985), and must depend on an external nutritional source. Further to uncertainties pertaining embryonic nutrition, there is As extra-vitelline sources must account for some 90% of the wet mass the question of oxygen delivery to the embryos. Embryos of Z. viviparus lie freely suspended in the ovarian fluid with no direct maternal connection or specialized structures to facilitate oxygen ⁎ Corresponding author. DTU Aqua, Technical University of Denmark, DK-9850 fl Hirtshals, Denmark. Tel.: +45 3588 3263; fax: +45 3588 3260. uptake. Oxygen content in the ovarian uid of Z. viviparus is low, with E-mail address: [email protected] (P.V. Skov). a PO2 of less than 5 kPa (Hartvig and Weber, 1984). Despite a high 0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2010.08.024 P.V. Skov et al. / Journal of Experimental Marine Biology and Ecology 395 (2010) 120–127 121 oxygen affinity in embryonic whole-blood, with a half-saturation incision was made to access the ovary at its widest point, the ovary tension of 1.2 kPa PO2 (Hartvig and Weber, 1984), embryonic wall was punctured and the oxygen sensor advanced to the opposing development in a hypoxic environment cannot be unproblematic. ovary wall. Using a micromanipulator, the oxygen sensor was Among viviparous fishes, embryos typically have specialized struc- retracted 1 mm at a time and the partial pressure of oxygen recorded. tures to facilitate maternal–embryonic exchange, in the form of Distances across the ovary varied from 32 to 38 mm. Measurement external portal networks, trophotaeniae (external outgrowths of the points were transformed to percentage of distance across the ovary gut) or vascularized enlarged fin folds (Balon, 1975; Turner, 1947; and binned to the nearest 5% interval. Subsequently oxygen pressures Webb and Brett, 1972). In some viviparous poeciliid species, maternal were measured adjacent to the tip of 3 follicles from each individual. specializations in the form of vascularized ovarian skin-folds, ‘grow Animals were killed by spinal transection, the embryos were into the opercular opening’ of the embryo (Turner, 1947). These removed, counted, and weighed. structures may be solely for gas transfer or gas and nutrient transfer (Jollie and Jollie, 1964), during what is a comparatively short gestation time (~3–4 weeks). 2.4. Gel electrophoresis The present work was spurred on by observations made during fl dissection of a fixed near-term pregnant female, in which several The size distribution of proteins in female serum, ovarian uid and fl embryos had an ovarian follicle within the oral cavity. The aim of the follicular uid from 2 late term pregnant females was analysed by study was to investigate any morphological and physiological SDS-PAGE using a NuPAGE system (Invitrogen, Denmark). Female fl adaptations to facilitate maternal–embryonic exchange of oxygen serum was obtained from the caudal vein, while ovarian uid was and nutrients to developing embryos in Z. viviparus. Using vascular obtained by Pasteur pipettes after the ovaries were dissected out. μ casts, histology and electron microscopy we reconstructed the Fluid from the follicles was obtained using 10 l capillary tubes and – internal follicular structure, to identify possible pathways for pooled into 0.2 ml Eppendorf tubes. A 10% BIS TRIS gel with MES nutritional transfer and ultra structural specializations. In order to running buffer was used (Invitrogen) with a Mark12 unstained – provide an estimate of the metabolic costs associated with pregnancy standard (2.5 200 kDa) (Invitrogen). Female serum and follicular fl we used respirometry to determine resting metabolic rate of non- uid were diluted 10-fold, while ovarian samples were diluted 2-fold μ reproductive males and near-term pregnant females. prior to loading. A 15 l sample was loaded onto individual lanes and the gel was run at fixed voltage (200 V) for 60 min before being 2. Materials and methods stained in Coomassie Blue R-250 for 45 min and destained overnight in 10% acetic acid / 30% methanol. 2.1. Animals 2.5. Vascular casting European eelpout, Z. viviparus were caught locally in set eel traps from October to November. Animals were kept in 450 l aerated Vascular casting was performed as described previously (Skov and holding tanks at the Marine Biological Laboratory, University of Bennett, 2004). In brief, animals were euthanized in an overdose of Copenhagen, according to Danish institutional guidelines for animal Benzocaine (0.15 g L−1), before the ventral aorta was cannulated research. Holding tanks were continuously supplied (8 l min−1) with normograde via the heart. The animal was cleared of blood with 40– 10 °C recirculating filtered seawater (30 ppt.) and kept under a 12 h 60 ml of 1.0% NaCl (pH 7.8) containing Heparin (20 IU ml−1) and light: 12 h dark regime. Fish were fed to apparent satiation twice sodium nitroprusside (10 μM). A polyurethane resin (vasQtec, Zurich, weekly with chopped mussels (Mytilus edulis).