Arch. histol. jap., Vol. 42, No. 5 (1979) p. 543-550
An Unusual Architecture of Occluding Junctions between Surface Cells in Teleost Ovarian Follicles (Plecoglossus Kiyotaka
altivelis)*
TOSHIMORI and Chikayoshi
OURA
Department of Anatomy (Prof. C. OURA),Medical College of Miyazaki, Kiyotake-cho, Japan Received February 19, 1979
Summary. An unusual architecture of occluding junctions between surface cells in teleost ovarian follicles was observed by electron microscopy, by using the freeze-fracture technique. On the P-face, double strands of intramembraneous particles are observed. A narrow furrow-like gap is recognized between two rows of particles in such double strands. On the E-face, two types of grooves can be distinguished. Type I groove consists of a wide furrow. Type II groove, which appears to be sequent to type I groove, consists of a pair of "sub-grooves" running parallel. Further, a row of particles appears to be located in the bottom of type I groove or between two "sub-grooves" of type II groove. These observations suggest that the double strands on the P-face are registered with the grooves (type I or type II) on the complementary E-face and that a row of particles on the E-face is registered with a furrow-like region between two rows in the double strands on the P-face. In other words, a single unit of the juncture of the present occluding junction is thought to consist of triplicated junctional strands.
Unusual architectures of occluding junctions have been reported in previous studies. On the P-face, not only grooves but also double linear arrangements of intramembraneous particles have been shown by CLAUDE and GOODENOUGH(1973) and STAEHELIN(1973), and by NAGANOet al. (1977). On the other hand, the strand preferentially located on the E-face has been described by several investigators (SIMIONESCU et al., 1975, 1976; GILULA et al., 1976; NAGANO and SUZUKI, 1976a, 1976b; NAGANOet al., 1977). However, there seem to be no reports concerning the particular area which displays simultaneously both double strands of intramembraneous particles on the P-face and single strands at the bottom of a wide groove or between two narrow grooves on the complementary E-face. This paper will describe such an unusual architecture of occluding junctions between surface cells in teleost ovarian follicles by using the freeze-fracture technique.
* This investigation was supported in part by Grants to the second author (C. O.) from the Ministry of Education of Japan (No. 348083). 543
544
K. TOSHIMORI and C. OURA:
MATERIAL
AND
METHOD
About thirty fish of Plecoglossus altivelis, an annual water river fish, were collected in August or September at the Aya Fisheries Experiment Station (Miyazaki Prefecture). Ovaries dissected from the fish were placed in 2.5% glutaraldehyde buffered by cacodylate buffer (pH 7.2) for 2-4hrs for the freeze-fracture method. The materials fixed by glutaraldehyde were then immersed in 30% glycerine overnight in a refrigerator. The materials were mounted on specimen holders and frozen in liquid Freon
22
cooled
with
liquid
nitrogen.
Specimens
were
cleaved
at
-110℃
and
the
fractured surface contours were replicated by platinum-palladium followed by carbon in 10-6 Torr in a freeze-fracture device (HFZ-1, Hitachi company). Organic substances on the replica were digested in both disodium hypochlorite (bleach) and chromic acid mixture, and then the replicas were washed in distilled water. The replicas on a 300-400 mesh copper grid were observed with a JEOL 100B and a Hitachi H-300 electron microscopes. Some ovaries dissected from the fish were used for the conventional thinsectioning method. The pre-fixed ovaries with 2.5% glutaraldehyde buffered by cacodylate buffer (pH 7.2) were placed in 2% osmium tetroxide in the same buffer for 2hrs. They were then dehydrated in a series of ethanol followed by 2 changes in propylen oxide. The specimens were embedded in Epon 812. Thin sections cut on a Porter-Blum MT-2B ultratome with a diamond knife were stained by uranyl acetate and lead citrate and then examined with the same electron microscope mentioned above.
RESULTS
Fig.1.
A
part
oocyte layer
of
a
(O),
follicle
(BL),
surface
cells
closely
apposed
surface
including
chorion
(A),
lamina
the
follicle
thecal (SC).
cells
(C), cells
attaching (FC),
cells
basal
(TC)
Arrows
and
indicate
membranes (SC).
the
between ×12,000
The outermost covering cells in teleost ovarian follicles are conveniently termed "surface cells" i n this paper, since the cells have not been suitably named so far. The surface cells are clearly identified by the particular location and the squamous contour (Fig. 1). The so-called strands and
An Unusual
Fig.
2.
Architecture
of Occluding Junctions
545
Low power electron micrograph showing part of surface cells. Double strands of particles (asterisk) on the P-face (P) and single strands in grooves (arrows) on the E-face (E). ×20,000
grooves are displayed simultaneously on the P-face and the E-face of the plasma membrane of the surface cells (Fig. 2). On the P-face: Double strands of intramembraneous particles are frequently observed (Fig. 2-5). Such strands consist of two rows of rather irregular-shaped, low particles (Fig. 3-5). Double strands are less anastomosed and have simple architectures such as a snaky line (Fig. 3), a simple linear one (Fig. 4) and a parallel one of double strands (Fig. 5). A particle-free, furrow-like region can be observed between two rows of low particles in each of the double strands (Fig. 3-5). Tall particles are haphazardly scattered in some areas in such strands. The double strands have a considerable width, being rather narrower than the width of triple particles (Fig. 3-5). Discrete short strands consisting of irregular-shaped particles sometimes lie on one or the other side directly adjacent to a linear strand of regular-shaped, tall particles (Fig. 6). On the E-face: Less developed anastomosing networks consisting of rows of intramembraneous particles are frequently observed (Fig. 7-9). In some areas, a row of particles appears to be located in the bottom of a wide groove, or runs along linear
546
Fig.
K. TOSHIMORI and C. OURA:
3.
Snaky between
Fig.
4.
double strands two
rows
of
Linear double strands
of low particles particles
on the P-face
is visible.
on the P-face
(P).
A linear
particle-free region
×78,000
(P).
A linear
particle-free
region
is also visible.
×75,000
Fig.
5.
Parallel
double
strands
on
the
P-face
(P).
×75,000
Fig. 6. A single lineararrangement of tallparticles (arrows) and a short double strand of low particles (asterisk), both on the P-face (P). Discrete short strands of low particles run close
to
a single
strand
of
tall
particles
(double
arrows).
×75,000
An Unusual
Architecture
of Occluding
Junctions
547
furrow-like regions (Fig. 7, 8). Moreover, two types of grooves were discriminated on the basis of the morphology. Type I groove consists of a wide furrow, which is apparently wider than the width of a single particle, whereas it is rather narrower than the width of triple particles (Fig. 7, 8). Type II groove consists of a pair of
Fig.
Fig.
7.
S.
Strands bottom
and grooves on the E-face (E). of a wide groove (arrowheads).
grooves
(arrows).
A row of particles appears to be located at the A few rows of particles run along narrow
×67,000
Wide type I grooves (Ig) on the E-face (E) and a double strand (asterisk) on the P-face (P). A double strand appears to be sequent to a wide groove in both sides of the fracture (double arrow). Rows of particles are located at the bottom of some grooves (arrowheads).
×78,000
548
Fig.
K. TOSHIMORI
9.
and
Type
II grooves
Single
rows
of
C. OURA:
(IIg)
particles
on the E-face appear
to
be
(E). located
Two between
"sub-grooves"
run
two "sub-grooves."
parallel
(arrows).
×90,000
narrow furrows (Fig. 9). For convenience we term each furrow of a pair of narrow ones a "sub-groove" in this paper, in contrast to a wide groove (type I groove). Such two "sub-grooves" run parallel to each other, being divided by a gap about the width of a single particle. A strand of regular-shaped particles appears to be located between such two "sub-grooves" in some areas. The total width of a pair of "sub-grooves" is roughly equal to that of type I grooves, while each "sub-groove" is rather narrower than the width of a single particle. Further, the width of type I or type II grooves is roughly equal to the double strands on the E-face.
DISCUSSION The present findings provide new information about the architecture of occluding junctions. A particle-free region between two rows of particles in the double strands in this study seems to be much wider than that reported by NAGANOet al. (1977). In contrast to the usual grooves in previous investigations (WADE and KARNOVSKY, 1974; SIMIONESCU et al., 1975, 1976; GILULA et al., 1976; NAGANOand SUZUKI,1976a, 1976b; NAGANO et al., 1977), the structures reported in the present study deserve attention in two points: First, the width of type I or type II grooves in this study appears to be wider, and second, type II groove shown in Figure 8 is clearly distinguished into two "sub-grooves" and in some areas a strand of particles appears to be located between them. These morphological characteristics seem to have been reported by none of the previous authors. The observations in this study suggest that double strands on the P-face are registered with a wide groove (type I) or a pair of "sub-grooves" (type II) on the E-face; each row of the double strands is registered respectively with each "subgroove" of type II groove, and that a strand of particles in the bottom of the wide type I groove or between two "sub-grooves" of type II groove on the E-face is registered with a furrow-like region between two rows of particles of the double strand on the P-face; a furrow-like region may be homologous to a groove itself. Therefore, a single unit of the juncture of the present occluding junction is supposed to consist
An Unusual Architecture of Occluding Junctions
549
of triplets of the junctional strands. Type II groove may be a well clarified feature of such an occluding junction, while type I groove may be an unclarified one probably due to technical problems in the process of making replicas. In the point of adhering or sealing capacity, a unit consisting of triplicated junctional strands may be stronger than a usual unit consisting of a paired junctional strand, because of its sealing capacity of lanthanum tracer in spite of their less developed anastomosis (TOSHIMORI,YASUZUMIand OURA,unpublished data). On the other hand, a question is now raised as to why several short strands of irregular-shaped, low particles lie in close relation to a strand of regular-shaped, tall particles on the P-face as shown in Figure 5. The answer is still unknown. Acknowledgment.
We thank Professor Robert J. ADAMS for his advice on English usage.
硬 骨 魚 類 (ア ユ) 卵 巣 の卵 胞 を お お う表 層 細 胞 間 の 癒 着帯 の構 築 年 森 清 隆 と 大 浦 親 善
凍 結 割 断法 を用 い て観 察 した, 硬 骨 魚 類 卵 巣 内 の卵 胞 をお お う表 層 細 胞 間 に存 在 す る癒 着 帯 の構 築 は, 通 常 み られ な い珍 し い もの で あ った. 膜 内粒 子 の線 条 が み られ, そ し て 認 めた. 一 方E面
上 に は,
P面 上 には 対 を な し て配 列 す る2本 の
これ らの線 条 の間 に,
2種 類 の 形 の 溝 が 区 別 で きた. す な わ ちI型
し, II型 は 平 行 に 走 る1対 の さ らに 細 い溝 か らな り, た.
これ ら両 型 の溝 の 中央 には
P面 上 の2列 の線 条 は, 内粒 子 は,
明 らか に溝 の よ うな 狭 い 間 隙 を
この もの はI型
は広い溝を構成 の溝 に連 絡 して い
1列 に 配 列 す る膜 内 粒 子 が 存 在 す るの を 認 め た. 従 って
E面 上 のI型 ま た は 豆型 の溝 に符 合 し, また
E而 上 の1列 の 膜
P面 上 の2列 の線 条 の間 の狭 い 部 分 に符 合 す る こ とを 示 唆 した. 本 論 文 は, 硬
骨 魚 類 卵 巣 に お け る卵 胞 をお お う表 層 細 胞 間 にみ られ た 癒 着 帯 の結 び 目の 最 小 単 位 が3本 一 組 の線 条 か ら構 成 され てい る こ とを 示 唆 した.
REFERENCES Claude,
P. and D. “leaky”epithelia.
A.
Goodenough: J. Cell Biol.
58:
Fracture faces 390-400 (1973).
of
zonulae
occludentes
from“tight”and
Gilula,
N. B., D. W. Fawcett and A. Aoki: The Sertoli cell occluding junctions in mature and developing mammalian testis. Devel. Biol. 50: 142-168 (1976). Nagano, T. and F. Suzuki: Freeze-fracture observations on the intercellular junctions of Sertoli cells and of Leydig cells in the human testis. Cell Tiss. Res. 166: 37-48 (1976a). - -: The postnatal development of the junctional complexes of the mouse Sertoli
cells as revealed by freeze-fracture. Anat. Rec. 185: 403-418 (1976b). Nagano, T., F. Suzuki, Y. Kitayama and K. Katsumoto: Sertoli cell junctions in the germ cell-free testis of the congenic mouse. Lab. Invest. 36: 8-17 (1977). Simionescu, M., N. Simionescu and G. E. Palade: Segmental differentiations of cell junctions in the vascular endothelium. J. Cell Biol. 67: 863-885 (1975).
550
K. TOSHIMORIand C. OURA
Simionescu, M., N. Simionescu and G. E. Palade: Segmental differentiations of cell junctions in the vascular endothelium: arteries and veins. J. Cell Biol. 68: 705-723 (1976). Staehelin, L. A.: Further observations on the fine structure of freeze-cleaved tight junctions. J. Cell Sci. 13: 763-786 (1973). Wade, J. B. and M. J. Karnovsky: The structure of the zonula occludens. A single fibril model based on freeze-fracture. J. Cell Biol. 60: 168-180 (1974).
年 森 清 隆 〒889-16宮 崎 郡 清 武 町 木 原5200 宮崎医科大学 第 一 解 剖学 教 室
Dr. Kiyotaka TOSHIMORI Department of Anatomy Medical College of Miyazaki 5200 Kihara, Kiyotake-cho Miyazaki-gun, Miyazaki-ken 889-16 Japan
An Unusual Architecture of Occluding Junctions between Surface Cells in Teleost Ovarian Follicles (Plecoglossus Kiyotaka
altivelis)*
TOSHIMORI and Chikayoshi
OURA
Department of Anatomy (Prof. C. OURA),Medical College of Miyazaki, Kiyotake-cho, Japan Received February 19, 1979
Summary. An unusual architecture of occluding junctions between surface cells in teleost ovarian follicles was observed by electron microscopy, by using the freeze-fracture technique. On the P-face, double strands of intramembraneous particles are observed. A narrow furrow-like gap is recognized between two rows of particles in such double strands. On the E-face, two types of grooves can be distinguished. Type I groove consists of a wide furrow. Type II groove, which appears to be sequent to type I groove, consists of a pair of "sub-grooves" running parallel. Further, a row of particles appears to be located in the bottom of type I groove or between two "sub-grooves" of type II groove. These observations suggest that the double strands on the P-face are registered with the grooves (type I or type II) on the complementary E-face and that a row of particles on the E-face is registered with a furrow-like region between two rows in the double strands on the P-face. In other words, a single unit of the juncture of the present occluding junction is thought to consist of triplicated junctional strands.
Unusual architectures of occluding junctions have been reported in previous studies. On the P-face, not only grooves but also double linear arrangements of intramembraneous particles have been shown by CLAUDE and GOODENOUGH(1973) and STAEHELIN(1973), and by NAGANOet al. (1977). On the other hand, the strand preferentially located on the E-face has been described by several investigators (SIMIONESCU et al., 1975, 1976; GILULA et al., 1976; NAGANO and SUZUKI, 1976a, 1976b; NAGANOet al., 1977). However, there seem to be no reports concerning the particular area which displays simultaneously both double strands of intramembraneous particles on the P-face and single strands at the bottom of a wide groove or between two narrow grooves on the complementary E-face. This paper will describe such an unusual architecture of occluding junctions between surface cells in teleost ovarian follicles by using the freeze-fracture technique.
* This investigation was supported in part by Grants to the second author (C. O.) from the Ministry of Education of Japan (No. 348083). 543
544
K. TOSHIMORI and C. OURA:
MATERIAL
AND
METHOD
About thirty fish of Plecoglossus altivelis, an annual water river fish, were collected in August or September at the Aya Fisheries Experiment Station (Miyazaki Prefecture). Ovaries dissected from the fish were placed in 2.5% glutaraldehyde buffered by cacodylate buffer (pH 7.2) for 2-4hrs for the freeze-fracture method. The materials fixed by glutaraldehyde were then immersed in 30% glycerine overnight in a refrigerator. The materials were mounted on specimen holders and frozen in liquid Freon
22
cooled
with
liquid
nitrogen.
Specimens
were
cleaved
at
-110℃
and
the
fractured surface contours were replicated by platinum-palladium followed by carbon in 10-6 Torr in a freeze-fracture device (HFZ-1, Hitachi company). Organic substances on the replica were digested in both disodium hypochlorite (bleach) and chromic acid mixture, and then the replicas were washed in distilled water. The replicas on a 300-400 mesh copper grid were observed with a JEOL 100B and a Hitachi H-300 electron microscopes. Some ovaries dissected from the fish were used for the conventional thinsectioning method. The pre-fixed ovaries with 2.5% glutaraldehyde buffered by cacodylate buffer (pH 7.2) were placed in 2% osmium tetroxide in the same buffer for 2hrs. They were then dehydrated in a series of ethanol followed by 2 changes in propylen oxide. The specimens were embedded in Epon 812. Thin sections cut on a Porter-Blum MT-2B ultratome with a diamond knife were stained by uranyl acetate and lead citrate and then examined with the same electron microscope mentioned above.
RESULTS
Fig.1.
A
part
oocyte layer
of
a
(O),
follicle
(BL),
surface
cells
closely
apposed
surface
including
chorion
(A),
lamina
the
follicle
thecal (SC).
cells
(C), cells
attaching (FC),
cells
basal
(TC)
Arrows
and
indicate
membranes (SC).
the
between ×12,000
The outermost covering cells in teleost ovarian follicles are conveniently termed "surface cells" i n this paper, since the cells have not been suitably named so far. The surface cells are clearly identified by the particular location and the squamous contour (Fig. 1). The so-called strands and
An Unusual
Fig.
2.
Architecture
of Occluding Junctions
545
Low power electron micrograph showing part of surface cells. Double strands of particles (asterisk) on the P-face (P) and single strands in grooves (arrows) on the E-face (E). ×20,000
grooves are displayed simultaneously on the P-face and the E-face of the plasma membrane of the surface cells (Fig. 2). On the P-face: Double strands of intramembraneous particles are frequently observed (Fig. 2-5). Such strands consist of two rows of rather irregular-shaped, low particles (Fig. 3-5). Double strands are less anastomosed and have simple architectures such as a snaky line (Fig. 3), a simple linear one (Fig. 4) and a parallel one of double strands (Fig. 5). A particle-free, furrow-like region can be observed between two rows of low particles in each of the double strands (Fig. 3-5). Tall particles are haphazardly scattered in some areas in such strands. The double strands have a considerable width, being rather narrower than the width of triple particles (Fig. 3-5). Discrete short strands consisting of irregular-shaped particles sometimes lie on one or the other side directly adjacent to a linear strand of regular-shaped, tall particles (Fig. 6). On the E-face: Less developed anastomosing networks consisting of rows of intramembraneous particles are frequently observed (Fig. 7-9). In some areas, a row of particles appears to be located in the bottom of a wide groove, or runs along linear
546
Fig.
K. TOSHIMORI and C. OURA:
3.
Snaky between
Fig.
4.
double strands two
rows
of
Linear double strands
of low particles particles
on the P-face
is visible.
on the P-face
(P).
A linear
particle-free region
×78,000
(P).
A linear
particle-free
region
is also visible.
×75,000
Fig.
5.
Parallel
double
strands
on
the
P-face
(P).
×75,000
Fig. 6. A single lineararrangement of tallparticles (arrows) and a short double strand of low particles (asterisk), both on the P-face (P). Discrete short strands of low particles run close
to
a single
strand
of
tall
particles
(double
arrows).
×75,000
An Unusual
Architecture
of Occluding
Junctions
547
furrow-like regions (Fig. 7, 8). Moreover, two types of grooves were discriminated on the basis of the morphology. Type I groove consists of a wide furrow, which is apparently wider than the width of a single particle, whereas it is rather narrower than the width of triple particles (Fig. 7, 8). Type II groove consists of a pair of
Fig.
Fig.
7.
S.
Strands bottom
and grooves on the E-face (E). of a wide groove (arrowheads).
grooves
(arrows).
A row of particles appears to be located at the A few rows of particles run along narrow
×67,000
Wide type I grooves (Ig) on the E-face (E) and a double strand (asterisk) on the P-face (P). A double strand appears to be sequent to a wide groove in both sides of the fracture (double arrow). Rows of particles are located at the bottom of some grooves (arrowheads).
×78,000
548
Fig.
K. TOSHIMORI
9.
and
Type
II grooves
Single
rows
of
C. OURA:
(IIg)
particles
on the E-face appear
to
be
(E). located
Two between
"sub-grooves"
run
two "sub-grooves."
parallel
(arrows).
×90,000
narrow furrows (Fig. 9). For convenience we term each furrow of a pair of narrow ones a "sub-groove" in this paper, in contrast to a wide groove (type I groove). Such two "sub-grooves" run parallel to each other, being divided by a gap about the width of a single particle. A strand of regular-shaped particles appears to be located between such two "sub-grooves" in some areas. The total width of a pair of "sub-grooves" is roughly equal to that of type I grooves, while each "sub-groove" is rather narrower than the width of a single particle. Further, the width of type I or type II grooves is roughly equal to the double strands on the E-face.
DISCUSSION The present findings provide new information about the architecture of occluding junctions. A particle-free region between two rows of particles in the double strands in this study seems to be much wider than that reported by NAGANOet al. (1977). In contrast to the usual grooves in previous investigations (WADE and KARNOVSKY, 1974; SIMIONESCU et al., 1975, 1976; GILULA et al., 1976; NAGANOand SUZUKI,1976a, 1976b; NAGANO et al., 1977), the structures reported in the present study deserve attention in two points: First, the width of type I or type II grooves in this study appears to be wider, and second, type II groove shown in Figure 8 is clearly distinguished into two "sub-grooves" and in some areas a strand of particles appears to be located between them. These morphological characteristics seem to have been reported by none of the previous authors. The observations in this study suggest that double strands on the P-face are registered with a wide groove (type I) or a pair of "sub-grooves" (type II) on the E-face; each row of the double strands is registered respectively with each "subgroove" of type II groove, and that a strand of particles in the bottom of the wide type I groove or between two "sub-grooves" of type II groove on the E-face is registered with a furrow-like region between two rows of particles of the double strand on the P-face; a furrow-like region may be homologous to a groove itself. Therefore, a single unit of the juncture of the present occluding junction is supposed to consist
An Unusual Architecture of Occluding Junctions
549
of triplets of the junctional strands. Type II groove may be a well clarified feature of such an occluding junction, while type I groove may be an unclarified one probably due to technical problems in the process of making replicas. In the point of adhering or sealing capacity, a unit consisting of triplicated junctional strands may be stronger than a usual unit consisting of a paired junctional strand, because of its sealing capacity of lanthanum tracer in spite of their less developed anastomosis (TOSHIMORI,YASUZUMIand OURA,unpublished data). On the other hand, a question is now raised as to why several short strands of irregular-shaped, low particles lie in close relation to a strand of regular-shaped, tall particles on the P-face as shown in Figure 5. The answer is still unknown. Acknowledgment.
We thank Professor Robert J. ADAMS for his advice on English usage.
硬 骨 魚 類 (ア ユ) 卵 巣 の卵 胞 を お お う表 層 細 胞 間 の 癒 着帯 の構 築 年 森 清 隆 と 大 浦 親 善
凍 結 割 断法 を用 い て観 察 した, 硬 骨 魚 類 卵 巣 内 の卵 胞 をお お う表 層 細 胞 間 に存 在 す る癒 着 帯 の構 築 は, 通 常 み られ な い珍 し い もの で あ った. 膜 内粒 子 の線 条 が み られ, そ し て 認 めた. 一 方E面
上 に は,
P面 上 には 対 を な し て配 列 す る2本 の
これ らの線 条 の間 に,
2種 類 の 形 の 溝 が 区 別 で きた. す な わ ちI型
し, II型 は 平 行 に 走 る1対 の さ らに 細 い溝 か らな り, た.
これ ら両 型 の溝 の 中央 には
P面 上 の2列 の線 条 は, 内粒 子 は,
明 らか に溝 の よ うな 狭 い 間 隙 を
この もの はI型
は広い溝を構成 の溝 に連 絡 して い
1列 に 配 列 す る膜 内 粒 子 が 存 在 す るの を 認 め た. 従 って
E面 上 のI型 ま た は 豆型 の溝 に符 合 し, また
E而 上 の1列 の 膜
P面 上 の2列 の線 条 の間 の狭 い 部 分 に符 合 す る こ とを 示 唆 した. 本 論 文 は, 硬
骨 魚 類 卵 巣 に お け る卵 胞 をお お う表 層 細 胞 間 にみ られ た 癒 着 帯 の結 び 目の 最 小 単 位 が3本 一 組 の線 条 か ら構 成 され てい る こ とを 示 唆 した.
REFERENCES Claude,
P. and D. “leaky”epithelia.
A.
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年 森 清 隆 〒889-16宮 崎 郡 清 武 町 木 原5200 宮崎医科大学 第 一 解 剖学 教 室
Dr. Kiyotaka TOSHIMORI Department of Anatomy Medical College of Miyazaki 5200 Kihara, Kiyotake-cho Miyazaki-gun, Miyazaki-ken 889-16 Japan
