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 , 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 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 (Yu et al.,1980,1981),the liver Lampetra reissneri,were collected from the Ohno produces a 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 seen to be disintegrated in structure. and after metamorphosis when compared with In these hepatocytes,the rough endoplasmic those of ammocoetes except for the disappearance

―319― 魚 類 学 雑 誌Japan.J.Ichthyol.32(3),1985

4

5

Fig.4.Electron-micrograph of hepatocytes of an adult female.G,Golgi apparatus;M,mitochondrion; RER,rough endoplasmic reticulum.8,250. Fig.5.Electron-micrograph of hepatocytes of a male lamprey at the young adult stage.G,Golgi ap- paratus;M,mitochondrion;N,nucleus;RER,rough endoplasmic reticulum.8,480. of the intracellular bile canaliculus and glycogen Discussion deposition at later stages of metamorphosis(Fig. In the Ohno River,southern Hokkaido,sand 5).The cells were mostly degenerative in adult lamprey,Lampetra reissneri,in the process of males as well as adult females during the spawn- metamorphosis could be collected only from mid- ing period. June to late September,some eight to eleven months before spawning.In this species of

―320― Fukayama:Ultrastructural Changes in Lamprey Liver lampreys,conspicuous growth of ovarian oocytes with reduced distribution in the cytoplasm.They starts and proceeds with the advance of vitello- also reported that the Golgi apparatus were a genesis during the metamorphic period(Fukayama conspicuous component of hepatocytes in lam- and Takahashi,1983 ;Tsuneki and Ouji,1984a,b). preys throughout metamorphosis.They assumed One of the most characteristic changes found in that the changes in the rough endoplasmic re- hepatocytes of female sand lampreys during ticulum of hepatocytes during metamorphosis metamorphosis was a conspicuous development might be a form of cellular adaptation to changes of the rough endoplasmic reticulum and the Golgi associated with intracellular bile canaliculus atresia. apparatus.In ammocoetes the rough endo- Although degeneration of the intracellular bile plasmic reticulum of hepatocytes consisted of canaliculus system was certainly caused during short flattened cisternae in both sexes,while in metamorphosis in the sand lamprey,disarrange- females at the metamorphic stages 4-5,when ment of the rough endoplasmic reticulum in ovarian oocytes were undergoing active vitello- hepatocytes was in no case observed to occur genesis,it was composed of long parallel cisternae. during that period.The discrepancy between The cisternae of the rough endoplasmic reticulum the two observations may be explained by the came to be further developed to make massive stacks fact that active vitellogenesis begins in adult surrounding the nucleus in hepatocytes of female females of the sea lamprey a few years after meta- lampreys after metamorphosis.The Golgi appara- morphosis(Lewis and McMillan,1965)in sharp tus were scarcely developed in hepatocytes of am- contrast to the sand lamprey which initiates ac- mocoetes,while they were active in those of females tive vitellogenesis during metamorphosis. at the metamorphic stages 4-5.At the young Some experimental studies,including sex steroid adult and the adult stages,the Golgi apparatus in treatment,that have been carried out in hepatocytes of female lampreys were invariably indicate that the development of the rough highly active,with many small vesicles containing endoplasmic reticulum and the Golgi apparatus electron-dense contents.By contrast,the rough in hepatocytes is closely related to enhanced endoplasmic reticulum and the Golgi apparatus activity of vitellogenin synthesis in these cells(cf. were generally inactive in hepatocytes of male Wiegand,1982).For instance,Van Bohemen lampreys during and after metamorphosis.Kitada et al.(1981)revealed that,in hepatocytes of the (1970)reported that,in hepatocytes of the sand rainbow trout,Salmo gairdneri,the rough endo- lamprey,the rough endoplasmic reticulum was plasmic reticulum formed long parallel stacks more stratified in adults than in ammocoetes,but and the Golgi apparatus were increased in size he did not give any consideration to sexual dif- with many electron-dense vesicles at the period of ferences in the ultrastructure of the cells.The extensive vitellogenesis when plasma vitellogenin work of Bertolini(1965)on hepatocytes of am- level reached a maximum.These conspicuous mocoetes and adult females of a nonparasitic features of hepatocytes in vitellogenic female brook lamprey,Lampetra zanandreai,in which strongly resemble those in vitellogenic vitellogenesis occurred in ovarian oocytes during female sand lampreys examined in the present metamorphosis,showed that the rough endo- study.Immunological detection of vitellogenin plasmic reticulum in adults was subdivided into in the blood of the sand lamprey revealed that vesicles,while in ammocoetes it was generally vitellogenin was increased in amount rapidly during formed by series of flat cisternae.He also re- metamorphosis and reached its maximal level ported that the Golgi apparatus were evident in at the young adult stage(Fukayama et al.,un- hepatocytes of adult lampreys.In the sea lamprey, published).Thus it is highly likely that the Petromyzon marinas,vitellogenesis takes place in characteristic changes of both the rough endo- ovarian oocytes in the parasitic phase fairly late plasmic reticulum and the Golgi apparatus in to metamorphosis.Sidon and Youson(1983b) hepatocytes of female sand lampreys may be im- reported that,in the sea lamprey,hepatocytes of plicated in promoted vitellogenin synthesis. ammocoetes showed extensive arrays of regularly The significance of dark hepatocytes appearing arranged rough endoplasmic reticulum,and that in female sand lampreys from the metamorphic those of lampreys at the metamorphic stages 4 to stage 7 to the young adult stage is unclear at 5 showed disarrayed rough endoplasmic reticulum present.The cells were characterized by having

―321― 魚i類 学 雑 誌Japan.J.Ichthyol.32(3),1985 the nucleus of irregular shapes and a large number crinol., 35: 197-204. of dilated vesicular cisternae of the rough endo- Lewis, J. C. and D. E. McMillan. 1965. The devel- plasmic reticulum packing the cytoplasm.Sidon opment of the ovary of the sea lamprey(Petromyzon and Youson(1983a,b)also showed that some marinus L.). J. Morphol., 117: 425-466. hepatocytes of the sea lamprey,Petromyzon Pickering, A. D. 1976. Effects of gonadectomy, oestradiol and testosterone on the migrating river marinus,during metamorphosis had highly elec- lamprey, Lampetra fluviatilis(L.). Gen. Comp. tron-dense cytoplasmic matrices,dilated rough Endocrinol., 28: 473-480. endoplasmic reticulum,and mitochondria with Sidon, E. W. and J. H. Youson. 1983a. Morphologi- electron-dense matrices.They considered the cal changes in the liver of the sea lamprey, Petro- "dark"cells to be hepatocytes in a stage of de - myzon marinus L., during metamorphosis: I. Atresia generation.This might be true for the dark of the bile ducts. J. Morphol., 177: 109-124. hepatocytes observed in the sand lamprey at the Sidon, E. W. and J. H. Youson. 1983b. Morphological metamorphic stages 4-5.However,the dark changes in the liver of the sea lamprey, Petromyzon hepatocytes appearing in the late metamorphic marinus L., during metamorphosis: II. Canalicular and young adult stages were a common occurrence degeneration and transformation of the hepatocytes. in the liver of young adult males of the sand J. Morphol., 178: 225-246. lamprey which were induced to undergo vitello- Tsuneki, K. and M. Ouji. 1984a. Morphometric changes during growth of the brook lamprey genin synthesis by administration of estradiol-17ƒÀ Lampetra reissneri. Japan. J. Ichthyol., 31: 38-46. (Fukayama et al.,unpublished).This result may Tsuneki, K. and M. Ouji. 1984b. Histological suggest that these dark hepatocytes synthesize changes of several organs during growth of the vitellogenin more actively than common hepato- brook lamprey Lampetra reissneri. Japan. J. cytes at those metamorphic stages. Ichthyol., 31: 167-180. Wallace, R. A. 1978. Oocyte growth in nonmam- Acknowledgment malian vertebrates. Pages 469-502 in R. E. Jones, ed. The vertebrate ovary. Plenum Press, New York The author wishes to express his sincere grat- and London. itude to Prof.H.Takahashi,Faculty of Fisheries, Wiegand, M. D. 1982. Vitellogenesis in fishes. Pages Hokkaido University,for his kind guidance and 136-146 in C. J. J. Richter and H. J. Th. Goos, eds. critical readings of the manuscript. Reproductive physiology of . Pudoc, Wagenin- gen. Youson, J. H. and I. C. Potter. 1979. A descrip- Literature cited tion of the stages in the metamorphosis of the Bertolini, B. 1965. The structure of the liver cells anadromous sea lamprey, Petromyzon marinus L. during the life cycle of a brook-lamprey(Lampetra Can. J. Zool., 57: 1808-1817. zanandreai). Zeit. Zell. Microsk. Anat., 67: 297- Youson, J. H., P. A. Sargent and E. W. Sidon. 1983. 318. Iron loading in the livers of metamorphosing lam- Bohemen, Ch. G. van, J. G. D. Lambert and J. Peute. preys, Petromyzon marinus L. Cell Tissue Res., 1981. Annual changes in plasma and liver in rela- 234: 109-124. tion to vitellogenesis in the female rainbow trout, Yu, J. Y. L., W. W. Dickhoff, Y. Inui and A. Gorb- Salmo gairdneri. Gen. Comp. Endocrinol., 44: 94- man. 1980. Sexual patterns of protein metabolism 107. in liver and plasma of , Eptatretus stouti with Fukayama, S. and H. Takahashi. 1983. Sex dif- special reference to vitellogenesis. Comp. Biochem. ferentiation and development of the gonad in the Physiol., 65B: 111-117. sand lamprey, Lampetra reissneri. Bull. Fac. Fish. Yu, J. Y. L., Dickhoff, W. W., Swanson, P. and Hokkaido Univ., 34: 279-290. Gorbman, A. 1981. Vitellogenesis and its hor- Kitada, J. 1970. The fine structure of the liver cell monal regulation in the Pacific hagfish, Eptatretus in larval and adult brook-lamprey, Entosphenus stouti L. Gen. Comp. Endocrinol., 43: 492-502. reissneri, observed by electron microscope. Japan. J. Ichthyol., 17: 161-165.(In Japanese). (Department of Biology,Faculty of Fisheries,Hok- Larsen, L. O. 1978. Subtotal hepatectomy in intact kaido University,Hakodate 041,Hokkaido,Japan; or hypophysectomized river lampreys(Lampetra Present address,Department of Biological Science, fluviatilis L.): Effects on regeneration, blood glucose Tokyo Institute,Kaken Pharmaceutical Co.,Ltd.,2- regulation, and vitellogenesis. Gen. Comp. Endo- 28-8,Honkomagome, Bunkyo-ku,Tokyo 113,Japan)

―322― Fukayama:Ultrastructural Changes in Lamprey Liver

性成熟に伴 うスナヤツメ肝臓の微細構造変化 胞 状 を呈 す る場 合 も あ った.ま た 細胞 質 中 に は ゴ ル ジ体 深 山昭 一 も頻 繁 に観 察 され,そ の付 近 には 例外 な く電 子 密 度 の高 性成 熟 に 伴 うスナ ヤ ツ メ肝 細 胞 の 形 態 変 化 を電 顕的 に い 小 胞 ない し顆 粒 が 認 め られ た .一 方,雄 の細 胞 質 中 に 観 察 した.ア ンモ シー テ ス の 肝 細 胞 の 組 織 像 に は雌 雄 差 は,す べ て の成 熟 段 階 を通 じ て,こ の よ うな粗 面 小 胞 体 は認 め られ ず,細 胞 質 中 に は毛 細 胆 管 ,グ リ コーゲ ン顆 や ゴル ジ体 の 著 しい 発達 は認 め られ な か っ た. 粒,油 球 の存 在 が 一般 的 に認 め られ た 。 一 方,粗 面 小 胞 以 上 の 結 果 よ り,変 態 期 間 中 に始 ま る雌 肝 細 胞 中 の 粗 体 の発 達 は顕 著 で は な く,扁 平 な 層 板 の小 塊 よ り成 って 面小 胞 体 お よび ゴ ル ジ体 の発 達 は,肝 細 胞 の 卵 黄 蛋 白前 い る にす ぎ な か っ た、 変 態 の 進 行 に伴 い雌 雄 と も に肝 細 駆 物 質(ビ テ ロゲ ニ ン)の 合 成 活 性 と密 接 な 関 連 を もつ 胞 質 中 の毛 細 胆 管,グ リコー ゲ ン顆 粒,油 球 は消 失 した . こ とが示 唆 され た . し か し,雌 の肝 細胞 中 で は,卵 黄 形成 が急 速 に進 行 す る 変 態 中 期 以 降,粗 面 小 胞体 や ゴ ル ジ体 の発 達 が極 め て 顕 1041函 館 市 港 町3-1-1北 海 道 大 学水 産 学 部 淡 水 増 殖 学 著 に認 め られ る よ うにな っ た.変 態 直 後 の卵 黄 形成 途上 講 座;現 住 所,113東 京 都 文 京 区 本駒 込2-28-8科 研製 の 幼 若 雌 の 肝 細 胞 中 の 粗 面小 胞 体 は核 を と りま く厚 い 層 薬 株 式 会 社 東 京 研 究 所 生 物 科 学 研究 部 生 理 活 性 研 究 室) 板 集 塊 の 発 達 を みせ,と き と して それ が著 し く拡 張 して

―323―