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1 KOREAN JOURNAL OF ICHTHYOLOGY, Vol. 26, No. 3, , September 2014 ISSN: (Print), (Online) Received: September 1, 2014 Revised: September 20, 2014 Accepted: September 25, 2014 멸종위기어류돌상어 Gobiobotia brevibarba 의난자형성과정과난막의형태 최웅선 김재구 박종영 * 전북대학교자연과학대학생명과학과, 전북대학교생물다양성연구소 Structure of Egg Envelope and Oogenesis of the Korean Endangered Fish Gobiobotia brevibarba (Pisces: Cyprinidae) by Wung Sun Choi, Jae Goo Kim and Jong Young Park* (Department of Biological Science and Institute for Biodiversity Research, College of Natural Sciences, Chonbuk National University, Jeonju , Korea) ABSTRACT A histological study on the egg envelope and oogenesis of Gobiobotia brevibarba (Pisces, Cyprinidae) was carried out by a light microscope and a scanning electron microscope. Various developmental cells appeared in the ovary caught during May 2014, spawning season. For the relative area of oocyte, the ovary consisted of mature stage (74.5%), a vitellogenic stage (yolk granule stage, 16.6% and yolk vesicle stage, 6.6%) and previtellogenic stage (perinucleolus stage 2.2%), which means its spawning season. The cytoplasm of the perinucleolus oocyte is acidic and many nucleoli are located at the inner side of the nuclear membrane. The yolk vesicles, an early vitellogenic stage, has a follicular layer and a zona radiata clearly. Numerous villi, called an egg envelope, begin to form on the zona radiata. The yolk granules, an another vitellogenic stage, proceeds and they show a strong eosinophilic nature. Such yolk granules appeared between the yolk vesicles occupying most cytoplasm, and as the stage proceeds, there are some yolk masses fused with each other. Egg envelope is covered with plenty of villi (2~3 μm in the length) over the entire egg surface. Key words : Egg envelope, villi, oogenesis, Gobiobotia brevibarba, endangered fish 서 론 *Corresponding author: Jong Young Park Fax: , park7877@jbnu.ac.kr 일반적으로어류의난모세포는여포세포층 (follicular layer) 과방사대 (zona radiata) 그리고난세포질 (oocyte plasma membrane) 등 3층으로구성되어있다. 특히여포세포층은 theca cell과 follicular cell (=granulosa cell) 로구성되는데 theca cell은방어기능을수행하는반면에여포세포는호흡, 난모세포와배발생에영양공급, 효소분비, 난황및 steroid 형성등많은기능을수행하고있다 (Kjesbu and Kryvi, 1989; Hirai, 1993; Riehl and Greven, 1993). 이러한다양한기능이외에도일부어류에서는여포세포층에서의난막구조변화 (egg membrane, adhesive material, adhesive membrane, egg envelope, vitelline envelope) 가나타나기도한다. 이러한난막구조는종의산란습성과관련된부착기능 (adhesive function), 수압조절, 배아보호기능뿐아니라분류학적목적으로도널리이용되어왔다 (Blaxter, 1969; Lagler et al., 1977; Laale, 1980; Groot and Alderdice, 1985; Erickson and Pikitch, 1993; Thiaw and Mattei, 1996; Park and Kim, 2001a, b; Kim and Park, 2013). 또한체외수정을선택한대부분의담수어류의정자머리에는첨체를보유하지않기때문에난자의동물극쪽에위치한난문 (micropyle) 을통해수정이이루어지는데이들의난문은종마다모양과크기가다양하게나타남으로써종특이성을가지고있다. 일반적으로어류에서난문은다른종의정자가침입하는것을방해하고다수정을방지하는역할을수행하는형태적특이성을가지고있어종을분류하는중요한특징으로이용되고있다 (Park, 1996; Yoon, 1999; Chen et al., 1999; Mori

2 180 최웅선 김재구 박종영 sawa, 1999; Debus et al., 2002). 돌상어는잉어목잉어과모래무지아과에속하는담수어류로물이깨끗하고유속이빠르며, 바닥에자갈이깔린곳에서식하고주로수서곤충을먹고산다. 우리나라의한강, 임진강, 금강에만서식하는한국고유종현재멸종위기야생생물 II급으로지정되어보호받고있다 (Kim and Park, 2002). 돌상어에관한연구는난발생및초기생활사 (Ko et al., 2011), 식성 (Choi et al., 2001b), 산란생태 (Choi et al., 2001a) 등은있었지만난자형성과정과난막의형태에관한연구는아직까지보고된바없고, 현재서식처교란으로인해개체수가급격히감소하고있는현실정에서생식세포형성과정에대한연구의필요성이절실하다. 따라서돌상어의난자형성과정과산란특성에따른부착구조를조사하여국내에출현하는 3종의꾸구리속 (Gobiobotia) 어류연구에대한기초자료를얻고자한다. 재료및방법 % PNS YVS YGS MS Relative area of oocyte in ovary Fig. 1. Cell-type distribution represented by relative area of four different types of oocytes in ovaries of Gobiobotia brevibarba in May MS, mature stage; PNS peri-nucleolus stage; YGS, yolk granule stage; YVS, yolk vesicle stage. 1. 실험재료돌상어포획은금강유역환경청의포획허가 ( 허가번호제 ) 를받은후2014년 5월에충남금산군제원면용화리일대에서족대 ( 망목 4 4 mm) 를이용하여 5개체 ( 전장 8.5~10.3 cm) 를채집하였다. 이후살아있는상태의돌상어를실험실로운반하여실험을진행하였다. 2. 실험방법 1) 광학현미경관찰채집된 5개체중 3개체의난소를적출하여 4 C 의 10% formalin에 12시간고정한후 12시간수세하였다. 일반적인 ethanol 탈수법으로탈수한다음 xylene으로치환하여 paraffin으로포매하였고 block을제작하였다. Block은 microtome (Leica-820, Germany) 으로 5 μm 간격으로절단하여 hematoxylin과 eosin으로이중염색하여표본을제작하였다. 광학현미경 (Carl zeiss, AX10, Germany) 을이용하여난소를관찰하였고난소내분화단계별세포의면적은 Axio vision (Carl zeiss, Germany) 으로 0.01 μm 2 까지측정하여백분율로환산하였다. 2) 주사전자현미경관찰 2014년 5월에채집한성체로부터직접압박법을통해얻은난을재료로 0.1 M phosphate buffer (ph 7.4) 로조정된 2.5% glutaraldehyde에 4 C 에서 4시간전고정후 1% osmium tetroxide (O S O 4 ) 로 90분간후고정하여 ethanol series를통해탈수시켰다. Tert-butyl alcohol (TBA) 을이용하여동결 건조시킨뒤 O S O 4 를증착시켜주사전자현미경 (JEOL JSM- 6400, Japan) 으로관찰하였다. 결과산란기의돌상어난소는체강의등쪽에낭상형으로배치되어있었으며, 길이는체강과비슷하였다. 제작된슬라이드를광학현미경으로관찰한결과, 난소내에는염색인기 (chromatin-nucleolus stage), 주변인기 (peri-nucleolus stage), 난황포기 (yolk vesicle stage), 난황구기 (yolk granule stage), 성숙기 (mature stage) 등다양한분화단계의생식세포들이존재하였다 (Fig. 2A). 이러한난세포들의상대면적은성숙란 (74.5%) 이가장많은부분을차지하고있었으며, 난황물질이형성되는난황구기 (16.6 %), 난황포기 (6.6%), 그리고주변인기 (2.3%) 순으로구성되었다 (Figs. 1, 2). 염색인기의난세포는주변인기로진행되었고핵막의안쪽에는여러개의인 (nucleolus) 들이배열하고있었으며, 세포질은 hematoxylin에강하게염색되었다. 난세포의외측에는단층의편평상피세포 (squamous epithelial cell) 로구성된여포세포층이뚜렷하게발달하기시작하였다. 주변인기의난세포는직경 98.9~215.4 μm( 평균 160.1±39.5 μm) 로다양한크기를보였다 (Fig. 2B). 난황포기의난세포에서는핵내에존재하는인들의크기가작아지면서핵막인근으로이동하였고, 세포질에는외측에서부터난황포 (yolk vesicle) 가형성되기시작하였다 (Fig. 2C). 난황구기의난세포에서는호산성의난황과립들이핵막주변의내질층으로부터피질

돌상어의난자형성과정과난막의형태 181 Fig. 2. Photos of a light microscopy (A-F) and a scanning electron microscopy (G and H). A-F, Transverse section with hematoxylin and eosin staining.

3 돌상어의난자형성과정과난막의형태 181 Fig. 2. Photos of a light microscopy (A-F) and a scanning electron microscopy (G and H). A-F, Transverse section with hematoxylin and eosin staining. A, There are various development stages in the full-grown ovary. B, Early perinucleolus stage with many nucleolus arranged along inner nuclear membrane. C, Late yolk vesicle stage during vitellogenesis. The formation of follicular layer (f), villus (v) on the zona radiata (z) begins rapidly. D, Early yolk granule stage. E, Mature stage. F, Follicular layer has theca cell (t) and granulosa cell (g). H, Lots of villi (v) cover the whole full grown oocyte (z). f, follicular layer; gv, germinal vesicle; mo, mature oocyte; mp, micropyle; n, nucleolus; op, ooplasm; v, villi; yg, yolk granule; yv, yolk vesicle; z, zona radiata. Scales indicate 200 μm (A), 50 μm (B), 10 μm (C), 20 μm (D), 200 μm (E), 10 μm (F), 67 μm (G), 7 μm (H), respectively.

4 182 최웅선 김재구 박종영 층으로확산되었다 (Fig. 2D). 발달과정이진행됨에따라성숙기의난세포는평균 ±64.7 (1052.2~1251.3) μm까지커졌으며이미형성된난황과립들은서로융합하면서세포질의대부분을차지하고있었다 (Fig. 2E). 난황구기에서성숙기로진행되면서여포세포와 theca cell로구성된 2 층의여포세포층이증가하였으며, 세포질과여포세포층사이에줄무늬모양 (striated appearance) 의방사대는두꺼워졌다 (Fig. 2D). 성숙란시기의방사대는약 10~15 μm 두께를가지며호산성을보였다 (Fig. 2D, F). 방사대와여포세포층사이에존재하는융모 (villi) 는난황포기의난세포에서출현하기시작하여성숙란에서는그길이가 2~3 μm까지길어졌다 (Fig. 2C, D, F, G, H). 주사전자현미경을이용하여관찰한결과, 성숙란의표면에는깔때기모양의난문이관찰되었으며, 그외경은약 13 μm였으나내경이점차작아지는특징을보였다 (Fig. 2G). 또한난문을제외한대부분의난표면에는 2~3 μm 길이의미세융모가일정하게분포하고있었다 (Fig. 2H). 고찰경골어류성어의난소는나출형과낭상형으로구분되는데 (Park,1996; Parenti and Grier, 2004; Koya and Munoz, 2007), 돌상어의난소는체강의앞쪽에서좌우한쌍으로나타났으며항문부근에서융합된낭상형으로체강의길이와비슷한크기로존재하고있었다. 이러한특징은모래무지아과어류인 Coreoleuciscus splendidus (Kim et al., 2010), Pseudorasbora parva (Kim et al., 2007) 의난소와유사한형태를보이고있었다. 이는여울부에서식하는저서성어류모래무지아과에서나타나는특징으로보인다. 부레를갖는 Rhynchocypris oxycephalus (Kim et al., 2009) 와해산어류인양볼락과어류 Scorpaena notata (Munoz et al., 2002) 의난소들은쌍으로구성되어있어돌상어와는차이를보였다. 성숙한돌상어의난소내에는핵안에인이산재한주변인기와세포질외측에난황포가형성되는난황포기, 난황포에난황구를축적하여난황물질이핵막주변의내질층으로부터피질로확산중인난황구기등다양한분화단계의생식세포들이관찰되었고성숙기난세포의평균크기는 (1052.2~1251.3) μm로이전의난발생연구 (Ko et al., 2011) 와거의일치하였다. 또한성숙기난세포의최외각에는편평한모양의여포세포가층을이루고있었으며약 10~15 μm 두께의방사대가관찰되었다. 일반적으로난막의두께는부성란이아닌침성란, 난태생어류보다난생어류, 친어가알을보호하지않는경우, 유속이더빠른곳에서식하는어류에서더두꺼운것으로알려져있다 (Guraya, 1986). 따라서두꺼운난막을가지는돌상어는난생의침성란으로유속 이빠른곳에서산란하는생태학적특성을잘반영한다고사료된다. 돌상어의난막표면을광학현미경과주사전자현미경으로관찰한결과, 많은수의융모가관찰되었는데이러한미세융모는난황포가형성되는시기에발달하기사작하여난황물질들이형성됨에따라더욱뚜렷해졌다. 이와같이난막에존재하는다양한구조들은부착기능, 수압조절, 배아보호기능등을수행하는것으로알려져있다 (Laale, 1980; Hiromi, 1984; Rizzo et al., 2002). 특히저서성어류인한국산미꾸리과어류는난막표면구조물을과립형 (granular form), 융모형 (villous form), 사상형 (filamental form), 거치형 (saw-shaped form), 소구형 (hillock-shaped form), 울타리형 (fence-shaped form) 그리고무구조형 (non-structural form) 등 7가지로분류하였고, 이러한구조는산란장소와깊은관계가있는것으로보고하였다 (Park 1996). 또한이러한난막의부착구조는서식환경과밀접한생태적연관성을보이고있으며일부분류군에서는분류학적특징으로이용되기도한다 (Groot and Alderdice, 1985; Hirai, 1993; Yoon, 1999; Park and Kim, 2001a, 2001b; Rizzo et al., 2002; Kim et al., 2011; Kim and Park, 2013). 돌상어의난막에서는외경이크고내경이작아지는깔때기모양의한개의난문이관찰되었는데이는종의특이성을보일뿐아니라다른종의정자가침입하는것을방어하고다수정방지기능을수행하는것으로알려져있다 (Grierson and Neville, 1981; Cameron and Hunter, 1984). 또한일부어류에서는난문의수가 2개이상나타나고있어중요한분류학적특징으로이용되고있다 (Chen et al., 1999; Morisawa, 1999; Debus et al., 2002). 이상과같이돌상어의난자형성과정과난막의미세구조에대한연구는국내의멸종위기야생생물로지정된꾸구리속어류3종의생태학적특성을파악할수있는기초자료와분류학적특성을제공할수있을것으로생각된다. 요약잉어과 (Pisces, Cyprinidae) 에속하는멸종위기어류인돌상어 Gobiobotia brevibarba의난막미세구조및난자형성과정을광학현미경과주사전자현미경을이용하여관찰하였다. 2014년 5월의돌상어난소내에는다양한발생단계의생식세포들이관찰되었다. 난모세포들의상대면적은성숙란 (74.5 %), 난황물질이형성되는난황구기 (16.6%), 난황포기 (6.6%), 그리고미숙한발달단계인주변인기 (2.3%) 순으로구성되어있었다. 주변인기단계의세포질은 hematoxylin에강하게염색되며, 핵막의안쪽에는다수의인들이존재하였다. 난황형성물질인난황포기와초기난황구기단계에서는여포층과방사대가뚜렷하게형성되기시작되었으며, 방사대

5 돌상어의난자형성과정과난막의형태 183 위에는미세융모의난막구조가더욱뚜렷해졌다. 난황구기및성숙란의단계에서는난황구일부가융합되면서강한호산성을보였다. 또한성숙란의표면에는깔때기모양의난문이관찰되었으며, 2~3 μm 길이의미세융모가일정하게분포하였다. 인용문헌 Blaxter, J.H.S Development: eggs and larvae. In: Hoar, W.S., D.J. Randall and E.M. Donalson (eds.), Fish physiology. Academic Press, New York, pp Cameron, I.L. and K.E. Hunter Regulation of the permeability of the medaka fish embryo chorion by exogeneous sodium and calciumions. J. Exp. Zool., 231: Chen, K.C., K.T. Shao and J.S. Yang Using micropylar ultrastructure for species identification and phylogenetic inference among four species of Sparidae. J. Fish Biol., 55: Choi, J.S., H.K. Byeon and O.K. Kwon. 2001a. Reproductive Ecology of Gobiobotia brevibarba (Cyprinidae). Korean J. Ichthyol., 13: (in Korean) Choi, J.S., O.K. Kwon, J.H. Park and H.K. Byeon. 2001b. Feeding Habit of Gobiobotia brevibarba (Cyprinidae) from the Hongcheon River, Korea. Korean J. Ichthyol., 13: (in Korean) Debus, L., M. Winkler and R. Billard Structure of micropyle surface on oocytes and caviar grains in Sturgeons. Internat. Rev. Hydrobiol., 87: Erickson, D.L. and E.K. Pikitch A histological description of shortspine thorny head, Sebastolobus alascanus, ovaries: structures associated with the production of gelatinous egg masses. Env. Biol. Fish., 36: Grierson, J.P. and A.C. Neville Helicoidal architecture of fish eggshell. Tiss. Cell, 13: Groot, E.P. and D.F. Alderdice Fine structure of the external egg membranes of five species of Pacific salmon and steelhead trout. Can. J. Zool., 63: Guraya, S.S Monographs in developmental biology, The cell and molecular biology of fish oogenesis. Karger, 18: Hirai, A Fine structure of the egg membrane in four species of Pleuronectinae. Japanese J. Ichthyol., 40: Hiromi, O Electron microscopic study on adhesive material of pacific herring (Clupea pallasi) eggs. Japanese J. Ichthyol., 30: Kim, C.H., J.G. Kim and J.Y. Park Structure of egg envelope and oogenesis of Kichulchoia multifasciata (Pisces, Cobitidae). Korean J. Electron Microscopy, 41: (in Korean) Kim, D.H., B.S. Chang, H.S. Jung, Y.C. Teng, S. Kim and K.J. Lee The oogenesis of Chinese minnow, Leuciscinae, Teleostei. Korean J. Electron Microscopy, 39: (in Korean) Kim, D.H., K.J. Lee, S. Kim and Y.K. Deung A study on the oogenesis of false dace (Pseudorasbora parva). Korean J. Electron Microscopy, 37: (in Korean) Kim, D.H., K.J. Lee, Y.C. Teng, W.J. Kim and S. Kim The oogenesis of Coreoleuciscus splendidus, Cyprinidae, Teleostei. Korean J. Electron Microscopy, 40: (in Korean) Kim, I.S. and J.Y. Park Freshwater fishes of Korea. Kyo-hak Publishing Co., Ltd., pp (in Korean) Kim, J.G. and J.Y. Park Reproductive Cycle of the Korean loach, Kichulchoia multifasciata (Pisces, Cobitidae). Korean J. Ichthyol., 25: (in Korean) Kjesbu, O.S. and H. Kryvi Oogenesis in cod, Gradus morhua L., studied by light and electron microscopy. J. Fish Biol., 34: Ko, M.H., S.Y. Park, I.R. Lee and I.C. Bang Egg development and early life history of the endanger species Gobiobotia brevibarba (Pisces, Cyprinidae). Korean J. Limnol., 44: (in Korean) Koya, Y. and M. Munoz Comparative study on ovarian structures in scorpaenids: possible evolutional process of reproductive mode. Ichthyol. Res., 54: Laale, H.W The perivitelline space and egg envelopes of bony fishes, a reviews. Copeia, Lagler, K.F., J.E. Bardach, R.R. Miller and D.R.M. Passono nd ed. Ichthyology. John Wiley & Sons. pp Morisawa, S Fine structure of micropylar region during late oogenesis in eggs of the hagfish Eptatretus burgeri (Agnatha). Develop. Growth Differ., 41: Munoz, M., M. Casadevall and S. Bonet The ovarian morphology of Scorpaena notata shows a specialized mode of oviparity. J. Fish Biol., 61: Parenti, L.R. and H.J. Grier Evolution and phylogeny of gonad morphology in bony fishes. Integr. Comp. Biol., 44: Park, J.Y A morphological study on the gonad of the species in the family Cobitidae (Pisces, Cypriniformes) from Korea. Dissertation, Chonbuk National Univ., pp (in Korean) Park, J.Y. and I.S. Kim, 2001a. Fine structure of oocyte envelopes of the related cobitid species in the genus Iksookimia (Cobitidae), Ichthyological Res., 48: Park, J.Y. and I.S. Kim, 2001b. Ultrastructure of the external egg envelopes in two Cobitid fishes (Cobitidae). Korean J. Ichthyol., 13: Riehl, R. and H. Greven Fine structure of egg envelopes in some viviparous goodeid fishes, with comments on the relation of envelope thinness to viviparity. Can. J. Zool., 71: Rizzo, E., Y. Sato, B.P. Barreto and H.P. Godinho Adhesiveness and surface patterns of eggs in neotropical freshwater teleosts. J. Fish Biol., 61:

6 184 최웅선 김재구 박종영 Thiaw, O.T. and X. Mattei Ultrastructure of the secondary egg envelope of Cyprinodontidae of the genus Epiplatys Gill, 1862 (Pisces, Teleostei). Acta. Zool., 77: Yoon, J.M Ultrastructure of egg micropyles and zona radiata in three aquacultural teleosts. Korean J. Ichthyol., 11:

04)한국산(칼라)

04)한국산(칼라) KOREAN JOURNAL OF ICHTHYOLOGY, Vol. 25, No. 4, 216-221, December 2013 ISSN: 1225-8598 (Print), 2288-3371 (Online) Received: December 4, 2013 Revised: December 15, 2013 Accepted: December 25, 2013 한국산수수미꾸리