Japanese Journal of Ichthyology 魚 類 学 雑 誌 32巻3号1985年 Vol.32,No.3 1985 Ultrastructural Changes in the Liver of the Sand Lamprey, Lampetra reissneri,during Sexual Maturation Shoichi Fukayama (Received March 5,1985) Abstract Ultrastructural changes of hepatocytes were examined in the sand lamprey,Lampetra reissneri,during various phases of the life cycle.In hepatocytes of ammocoetes,the rough endo- plasmic reticulum was composed of short cisternae and the Golgi apparatus were scarcely de- veloped,showing no sexual differences at this stage of life cycle.In hepatocytes of female lampreys at the metamorphic stages 4 to 5,the rough endoplasmic reticulum was developed to form long parallel cisternae and the Golgi apparatus were well-developed.The rough endoplasmic reticulum developed further to form stacks of long parallel cisternae extending over the cytoplasm in hepato- cytes of females at the young adult stage,and became composed of both long parallel and vesicular cisternae in the cells of females at the adult stage.The Golgi apparatus were invariably well- developed in hepatocytes of young adult and adult females.No consipcuous development was observed in profiles of the rough endoplasmic reticulum and the Golgi apparatus in hepatocytes of males during and after metamorphosis.The ultrastructural changes of the rough endoplasmic reticulum and the Golgi apparatus observed in hepatocytes of female sand lampreys are considered to have an intimate relation to the activity of vitellogenin synthesis in the liver,and it is suggested that the hepatocytes begin to rapidly synthesize vitellogenin in the sand lamprey at the metamorphic stages 4 to 5. In oviparous nonmammalian vertebrates in- Material and methods cluding lampreys(Pickering,1976;Larsen,1978) Larval,metamorphosing,and adult lampreys, and hagfishes(Yu et al.,1980,1981),the liver Lampetra reissneri,were collected from the Ohno produces a yolk precursor protein(vitellogenin River,southern Hokkaido,in 1983 and 1984 which is released into circulation and is finally incorporated into ovarian oocytes(cf.Wallace, (Table 1).For electron microscopic studies, 1978,and Wiegand,1982).In the sand lamprey, pieces of the liver taken from anesthetized animals Lampetra reissneri,a conspicuous growth of were fixed in Karnovsky's glutaraldehyde-para- formaldehyde mixture in 0.2 M cacodylate buffe ovarian oocytes accompanying active vitellogenesis starts and proceeds during the metamorphic period (pH 7.4)for 4 to 5 hours at room temperature, followed by postfixation in 1% osmium tetroxide (Fukayama and Takahashi,1983;Tsuneki and in the same buffer for 2hours at 4•Ž,and then Ouji,1984a,b).Only a few reports have dealt embedded in Epon.Ultrathin sections stained with some ultrastructural changes occurring in the double with uranyl acetate and lead citrate were hepatocytes of lampreys during metamorphosis observed with a Hitachi H-300 electron micro- (Sidon and Youson,1983a,b;Youson et al., scope.The livers from 37 lampreys in total were 1983),mainly with changes relating to degenera- tion of the intracellular bile canaliculus system in examined in the present study. Stages of metamorphosis of the sand lamprey the liver.So far as the present writer knows,no were determined according to the criteria proposed information has been given thus far about ultra- by Youson and Potter(1979). structural changes of the hepatocytes of lampreys during the period of active vitellogenesis.In the present study,ultrastructural changes of the Results hepatocytes of the sand lamprey were observed during various periods in the life cycle in order to Sand lampreys in the process of metamorphosis, know their particular relationship to the develop- ranging from 136 to 222mm in total length,were ment of vitellogenesis. collected during a period from mid-June to late ―316― Fukayama:Ultrastructural Changes in Lamprey Liver September(Table 1).During metamorphosis, Round,oval or irregularly elongated mito- initiation and rapid progress of vitellogenesis were chondria were distributed sporadically in the cyto- found in ovarian oocytes of female lampreys, plasm.The rough endoplasmic reticula,which resulting in a remarkable increase in diameter of were distributed concentrically around the nucleus the oocytes.By contrast,ovarian oocytes never in many cases,were few in number and generally showed vitellogenic changes in female lampreys consisted of short parallel cisternae.The Golgi which were larger than 140mm in total length apparatus was seldom conspicuous at this stage. but still did not show any signs of metamorphosis. Glycogen deposition occurred in some places and The growth of vitellogenic oocytes was particularly lipid droplets were seen distributed throughout prominent in later stages of metamorphosis. the cytoplasm.Furthermore,a large number of Relative weight of the liver(HSI;liver weight electron-dense myelin figures of various sizes 100/body weight)in female lampreys was•~ 0.45 and shapes,measuring up to about 5 ,um in at the ammocoetes stage while it was 0.84 at the maximum diameter,were found dispersed in the young adult stage just after metamorphosis,in- cytoplasm. dicating a clear increase during metamorphosis. At the metamorphic stages 4-5,female lampreys There was no noticeable difference in HSI values had ovaries with oocytes undergoing active vitel- between the sexes in ammocoetes,but HSI value logenesis(Table 1).In hepatocytes of these was higher in metamorphosing females than in females,moderately dilated,parallel cisternae of males at comparable metamorphic stages. the rough endoplasmic reticulum extended through In ammocoetes there were no notable differences the cytoplasm,encompassing a centrally located between the sexes in hepatocyte ultrastructure.A nucleus(Fig.1).The Golgi apparatus was well- frequent existence of intracellular bile canaliculi developed,without showing any regional differences was noticed in hepatocytes of ammocoetes. in their distribution,and was often attended by Table 1.Gross characteristics of lampreys examined. ―317― 魚 類 学 雑 誌Japan.J.ichthyol.32(3),1985 Fig.1.Electron-micrograph of hepatocytes of a female sand lamprey at the metamorphic stage 4.A dark hepatocyte containing lipid droplets(LD)in the cytoplasm is also shown(arrow).DB,dense body;G,Golgi apparatus;M,mitochondrion;N,nucleus;RER,rough endoplasmic reticulum•~ 5,500. small vesicles with electron-dense contents.Elec- roundish vesicles of the rough endoplasmic re- tron-dense bodies of irregular shapes,as seen in ticulum containing moderately electron-dense, hepatocytes of ammocoetes,still existed in al- granular and fibrillar material(Fig.3).Innumera- most all hepatocytes,and multivesicular bodies ble free ribosomes were also observed to be scat- measuring about 0.5 in diameter were occa- tered throughout the cytoplasm.Smooth endo- sionally observed.Intracellular bile canaliculi plasmic reticulum was scarcely observed,and were no longer present in hepatocytes of lampreys well-developed Golgi apparatus existed sporad- examined at later stages of metamorphosis.In ically in these dark hepatocytes.Furthermore, some cases,atypical dark hepatic cells with elec- these dark cells had mitochondria with somewhat tron-dense nuclei were found in the liver(Fig.1). electron-lucent matrices,in which neither tubular During the period from the metamorphic stage nor parallel cristae were obvious. 7 to the young adult stage,female lampreys had Adult female lampreys sampled in November ovaries with oocytes showing advanced vitello- had ovaries packed with maturing oocytes of genesis(Table 1).Their hepatocytes were pro- about 600-800 µm in diameter(Table 1).In vided with the rough endoplasmic reticulum in hepatocytes of these females,remarkably regular the cytoplasm which had further developed to arrays of parallel cisternae of the rough endo- form stacks of long parallel cisternae near the plasmic reticulum were extended throughout the nucleus(Fig.2).Well-developed Golgi apparatus cytoplasm,though some of the cisternae were with minute electron-dense vesicles also existed. still dilated to form roundish vesicles with mod- In the liver of females examined at that period, erately electron-dense contents(Fig.4).The some dark hepatocytes,which were characterized Golgi apparatus showed highly active configura- by having electron-dense nucleus and cytoplasmic tions with minute electron-dense vesicles.Mito- matrix,also occurred interspersed among the chondria of various shapes were scattered through common hepatocytes.The nucleus of these cells the cytoplasm.The smooth endoplasmic reti- was small in size and irregular in shape.The culum could scarcely be observed in the hepato- cytoplasm was filled with a large number of dilated cytes. ―318― Fukuyama:Ultrastructural Changes in Lamprey Liver 2 3 Figs.2 and 3.Electron-micrographs of hepatocytes of female lampreys at the young adult stage.Dark hepatocytes with roundish cisternae of the rough endoplasmic reticulum(RER)are shown in Fig.3 DB,dense body;G,Golgi apparatus;N,nucleus.Fig.2,v 7 ,690; Fig.3,:•~8,300. Hepatocytes of adult females during the spawn- reticulum was composed of short flattened cisternae ing period were quite different in ultrastructure distributed irregularly in the cytoplasm.The from those of maturing females sampled in Novem- Golgi apparatus was inconspicuous in these cells. ber.Their hepatocytes were indistinct in contour. Hepatocytes of male lampreys showed no and some of them with pycnotic nuclei of irregular significant changes in their ultrastructure during shapes were