15.9. 1975
Speeialia
were q u a n t i t a t i v e l y reproducible. L o w e r c o n c e n t r a t i o n s of VCN are ineffective, p e r h a p s because of t h e i r i n a b i l i t y to release t h e o p t i m u m a m o u n t of sialic acid to b r i n g a b o u t cellular deformabiliy. T h u s it a p p e a r s t h a t t h e o b s e r v e d effect as d e p i c t e d in t h e Figure is due to t h e action of VCN on t h e cell m e m b r a n e s t r u c t u r e of a m o e b a e . T h e e x a c t m e c h a n i s m as t o h o w VCN leads to t h e d i s i n t e g r a t i o n of t h e m e m b r a n e s t r u c t u r e followed b y t h e lysis of t h e cell b o d y of a m o e b a is n o t k n o w n . R e m o v a l of sialic acid m i g h t be associated w i t h t h e loss of t h e r i g i d i t y of t h e cell m e m b r a n e s t r u c t u r e s leading t o d e f o r m a t i o n 6 of t h e surface s t r u c t u r e of t h e cell. T h e s e p h e n o m e n a m i g h t c h a n g e t h e m o d e s of n o r m a l t r a n s p o r t across cell m e m b r a n e s 13-15, t h u s c r e a t i n g an i m b a l a n c e in t h e o s m o t i c p o t e n t i a l inside a n d outside t h e m e m b r a n e . I n t h i s situation, a t some p o i n t s , a n y t w o p a r t s of t h e m e m b r a n e s t r u c t u r e m a y fall a p a r t leading to t h e c o n s e q u e n c e d e t a i l e d above. I t m i g h t be r e l e v a n t to p o i n t o u t t h a t t h i s could be an isolated e x a m p l e of t h e o b s e r v e d effects because of t h e u n i q u e surface c o n f i g u r a t i o n of a m o e b a . I n t h e case of m a m m a l i a n cells, this kind of s i t u a t i o n is n o t n o r m a l l y
1077
o b s e r v e d in vitro, e v e n if i n c u b a t i o n is e x t e n d e d for longer periods a n d h i g h e r c o n c e n t r a t i o n s of V C N are used 10,11
S u m m a r y . R e m o v a l of cell surface sialic acid w i t h n e u r a m i n i d a s e b r i n g s a b o u t cell d e f o r m a t i o n in a m o e b a . The m e m b r a n e s of t h e s e d e f o r m e d cells are e v e n t u a l l y r u p t u r e d leading to t h e liberation of t h e cell mass. P. K. RAY a n d S. CHATTERJEE 16
Biomedical Group, M o d u l a r Laboratories, Bhabha A t o m i c Research Centre, Trombay, B o m b a y - 4 0 0 085 (India), 3 M a y 1974. 13 L. WEISS and C. J. LEVINSON, J. Cell Physiol. 73, 31 (1969). 14 D. H. BROWNand A. F. MICHAEL,Proc. Soc. exp. Biol. Med. 731, 568 (1969). 15 A. VAHERI, E. RUOSHATRIand S. NORDLING, Nature New Biol. 238, 211 (1972). 16 We thank Dr. E. W. RAJASEKHARfor his valuable help in preparing the manuscript.
T w o T y p e s of Bipolar Cells in the Chick Retinal D e v e l o p m e n t 1 B i p o l a r cells c o m p r i s e t h e bulk of t h e i n n e r n u c l e a r layer o f t h e retina. T h e s e ceils are i m p o r t a n t interm e d i a t e s of t h e r e t i n a l p a t h w a y s b e c a u s e t h e y f o r m synaptic contacts with both photoreceptor terminals, processes of a m a c r i n e cells a n d ganglion cells 2-5. E x t e n s i v e studies b y RAM6N u CAJAL6-s i n d i c a t e d t h a t , for e x a m p l e in t h e chicken, t h e r e are 2 t y p e s of b i p o l a r cells. H o w e v e r , t h e r e a p p e a r s to be little e x i s t i n g d a t a on w h e n t h e s e t w o t y p e s of bipolar cells arise a n d w h a t t h e i r m o r p h o l o g i c a l
Fig. 1. Type I and II bipolar cells are distinguished at stage 40 of chick embryonic retina. Note a centriole (Ce) in one of the type If cells and external projections (arrows) in the other. • 28,800.
c h a r a c t e r i s t i c s are. This s t u d y is an a t t e m p t t o s h e d s o m e i n s i g h t on t h i s p r o b l e m . Materials and method. Fertile w h i t e l e g h o r n chicken eggs were i n c u b a t e d in s t a n d a r d c o n d i t i o n s a n d s t a g e d a c c o r d i n g to HAMBURGER a n d HAMILTON ~ Chick emb r y o n i c eyes f r o m stages 36 to h a t c h i n g were t a k e n o u t a n d b i s e c t e d into halves. T h e n t h e p o s t e r i o r h a l v e s were p r e f i x e d w i t h 6% b u f f e r e d g l u t a r a l d e h y d e and p o s t f i x e d w i t h 1% b u f f e r e d o s m i u m t e t r o x i d e , d e h y d r a t e d in e t h a n o l , cleared in p r o p y l e n e oxide a n d e m b e d d e d in E p o n . Sections were cut w i t h a P o r t e r - B l u m MT 2 m i c r o t o m e a n d s t a i n e d w i t h u r a n y l a c e t a t e a n d lead citrate, a n d t h e n e x a m i n e d w i t h a Zeiss E M 9S2 electronmicroscope. Results. Bipolar cells c a n n o t be d i s t i n g u i s h e d u n t i l s t a g e 36 w h e n b o t h p l e x i f o r m layers are f o r m e d a n d t h e p r e s u m p t i v e b i p o l a r nuclei b e c o m e d i s t i n g u i s h a b l e b y t h e i r ellipsoidal shape. The c y t o p l a s m of t h e s e cells a t stage 36 h a v e a few m i t o c h o n d r i a , s o m e r o u g h e n d o p l a s m i c r e t i c u l u m a n d few ribosomes. S o m e b i p o l a r cells o f t e n lie close t o g e t h e r r e s e m b l i n g a t e l o p h a s e p h e n o m e non, i n d i c a t i n g t h a t t h e y h a v e j u s t c o m p l e t e d a m i t o t i c division. A t s t a g e 40, d e n d r i t i c p r o j e c t i o n s c a n be seen arizing f r o m t h e b i p o l a r cell bodies t o w a r d s t h e o u t e r p l e x i f o r m layer (Figure 1) a n d a t t h i s t i m e 2 t y p e s of bipolar cells can be distinguished. T h e u l t r a s t r u c t u r a l details of t h e s e 2 t y p e s of bipolars are o u t l i n e d in t h e
1 Supported by NIH grant No. EY00477 to DBM. 2 j. 1~. DOWLING, Proc. R. Soc., Lond. 170, 205 (1968). 3 j. E. DOWLINC.,Invest. Ophthal. 9, 655 (1970). 4 j. E. DOWLING and B. B. BOYCOTT, Proe. R. Soe. Lond. 766, 80 (1966). 5 J. E. DOWLING and B. B. BOYCOTT, Cold Spring Habor Syrup. quant. Biol. 30, 392 (1965). S. RAM6N Y CAJAL, Anat. Anz. d, 111 (1889). v S. RAM6N Y CAJAL, Cellule 9, 119 (1892). s S. RAMONY CAJAL, translated by S. A. THORPEand M. GLICKSTEIN, The Structure o] the Retina: A Complete Work (Charles C. Thomas, Springfield 1972). s U. HAMBURGERand H. L. HAMILTON, J. Morph. 88, 47 (1951).
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Comparison of the two bipolar cell types at stage 40
Nuclear position Nuclear morphology
Cytoplasm Cytoplasmic organelles,
Morphology of dendritic processes
Fig. 2. Type I and II bipolar cells at the same stage. The nucleus of the type I bipolar is large and located more externally. Note the presence of some rough endoplasmic reticulum near the nucleus. On the other hand, the long dendritic trunk in the type II bipolar contains a golgi apparatus, mitochondria and neurotubules. • 28,800.
T a b l e (Figures 1 a n d 2). I n r a r e instances, a c e n t r i o l e m a y b e p r e s e n t in t h e m a i n d e n d r i t e of t h e t y p e I I b i p o l a r cells (Figure 2) a t t h i s stage. Discussion. B i p o l a r cells are d e r i v e d e m b r y o l o g i c a l l y f r o m t h e o u t e r n e u r o b l a s t i c l a y e r 1~ n a n d i n i t i a l l y t h e y a p p e a r s p i n d l e - s h a p e d a n d c a n n o t b e easily d i f f e r e n t i a t e d f r o m t h e o t h e r r e t i n a l ceils. B y stage 36, w h e n t h e o u t e r plexiform layer appears and separates the photoreceptor ceils f r o m t h e i n n e r n u c l e a r layer, b i p o l a r cells c a n b e clearly identified. O u r e l e c t r o n m i c r o s c o p i c d a t a on t h e s e 2 t y p e s of c h i c k b i p o l a r cells a p p e a r t o c o n f i r m RAM6~ v CAJAL'S studies. T y p e I b i p o l a r cells (RAM65! Y CAJAL'S o u t e r bipolars) w i t h large nuclei a n d s h o r t dendritic branches can be distinguished from the type II b i p o l a r cells (RAM6N Y CAJAL'S i n n e r bipolars) e a c h of
Ovulation in an Echinoderm
Type I
Type II
Close to outer plexiform layer Large (2 [xln wide differ from horizontal cell nuclei which are spherical) Scanty Mitochondria, more rough endoplasmic reticulum, free ribosomes, some neurotubules
Close to inner layers Smaller (1.5 [xm wide)
Short branches of dendritic processes from cell body
Scanty Long mitochondria less rough endoplasmic reticulum free ribosomes, prominent long golgi, more neurotnbules Long main dendrite leading from body, then giving out branches
t h e m h a s a s m a l l e r n u c l e u s a n d a single long m a i n dend r i t i c t r u n k . F u r t h e r m o r e , o u r results also i n d i c a t e t h a t t h e r e are differences b e t w e e n c y t o p l a s m i c organelles in t h e 2 t y p e s of b i p o l a r cells. I t is h o p e d t h a t , b a s e d o n t h e u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s of t h e s e 2 t y p e s of c h i c k b i p o l a r cells, a d d i t i o n a l u n d e r s t a n d i n g of t h e c o m p l e x r e l a t i o n s h i p s b e t w e e n t h e b i p o l a r s a n d t h e d i f f e r e n t r e t i n a l cell t y p e s c a n t h e n b e obtained.
Summary. T w o t y p e s of b i p o l a r cells are identified in the chick embryonic retina. They can be distinguished by t h e i r c y t o p l a s m i c organelles. D. T. YEW a n d D. B. MEYER
Department o/Biology, University Science Center, Chinese University o[ Hong Kong, Shatin, N. T. (Hong Kong), and Department o/ A natomy, Wayne State University, Detrot (Michigan, USA), 2 April 1975. 10 I. MANN, Br. J. Ophthal. I2, 449 (1928). :t I. MANN,The Developmento! the Human Eye (Grune and Stratton Inc., New York 1950).
(Comanthus japonica)
O v u l a t i o n in several i n v e r t e b r a t e g r o u p s m a y be defined as t h e p a s s a g e of oocytes t h r o u g h a l a y e r of e p i t h e l i a l cells. T h i s p h e n o m e n o n h a s b e e n o b s e r v e d d i r e c t l y for c t e n o p h o r e s ~ a n d h a s b e e n i n f e r r e d f r o m c o m p a r i s o n s of o v a r i a n h i s t o l o g y before a n d a f t e r o v u l a t i o n for c o e l e n t e r a t e s ~, p r i a p u l i d s 3, p e n t a s t o m i d s 4 a n d c e p h a l o c h o r d a t e s 6 . T h e p r e s e n t r e p o r t describes t h e foregoing t y p e of o v u l a t i o n 6 for t h e first t i m e in a n e c h i n o d e r m ; m o r e o v e r , t h e o o c y t e s are i l l u s t r a t e d d u r i n g t h e i r t r a n s e p i t h e l i a l passage. T h e e c h i n o d e r m s t u d i e d was Comanthus japonica, a c r i n o i d for w h i c h t h e s p a w n i n g d a t e is p r e d i c t a b l e f r o m tile l u n a r c a l e n d a r ~. On t h e p r e d i c t e d day, a l m o s t all t h e
oocytes in all t h e females b e g i n m a t u r a t i o n w i t h g e r m i n a l vesicle b r e a k d o w n s h o r t l y before noon, a n d t h e m a t u r e o v a are s p a w n e d l a t e r t h a t afternoonS, 9. To d e m o n s t r a t e o v u l a t i o n in Comanthus, we r e m o v e d ovaries e v e r y 15 mill t h r o u g h o u t tile m o r n i n g a n d a f t e r n o o n of t h e s p a w n ing day. These ovaries were fixed b y p r e v i o u s l y p u b l i s h e d m e t h o d s 1 ~ for light- a n d e l e c t r o n microscopy. U p t h r o u g h 11.30 h, e v e r y o o c y t e h a d a g e r m i n a l vesicle c o n t a i n i n g a single nucleolus. E a c h o o c y t e l a y m a i n l y in t h e i n t e r m e d i a t e l a y e r of t h e ovary11; h o w e v e r , t h e e n d of t h e o 0 c y t e n e a r e s t t h e o v a r i a n l u m e n was closely assoc i a t e d w i t h a p l a q u e of c u b o i d a l s o m a t i c cells b e l o n g i n g to t h e o t h e r w i s e s q u a m o u s e p i t h e l i u m lining t h e ovary.
Speeialia
were q u a n t i t a t i v e l y reproducible. L o w e r c o n c e n t r a t i o n s of VCN are ineffective, p e r h a p s because of t h e i r i n a b i l i t y to release t h e o p t i m u m a m o u n t of sialic acid to b r i n g a b o u t cellular deformabiliy. T h u s it a p p e a r s t h a t t h e o b s e r v e d effect as d e p i c t e d in t h e Figure is due to t h e action of VCN on t h e cell m e m b r a n e s t r u c t u r e of a m o e b a e . T h e e x a c t m e c h a n i s m as t o h o w VCN leads to t h e d i s i n t e g r a t i o n of t h e m e m b r a n e s t r u c t u r e followed b y t h e lysis of t h e cell b o d y of a m o e b a is n o t k n o w n . R e m o v a l of sialic acid m i g h t be associated w i t h t h e loss of t h e r i g i d i t y of t h e cell m e m b r a n e s t r u c t u r e s leading t o d e f o r m a t i o n 6 of t h e surface s t r u c t u r e of t h e cell. T h e s e p h e n o m e n a m i g h t c h a n g e t h e m o d e s of n o r m a l t r a n s p o r t across cell m e m b r a n e s 13-15, t h u s c r e a t i n g an i m b a l a n c e in t h e o s m o t i c p o t e n t i a l inside a n d outside t h e m e m b r a n e . I n t h i s situation, a t some p o i n t s , a n y t w o p a r t s of t h e m e m b r a n e s t r u c t u r e m a y fall a p a r t leading to t h e c o n s e q u e n c e d e t a i l e d above. I t m i g h t be r e l e v a n t to p o i n t o u t t h a t t h i s could be an isolated e x a m p l e of t h e o b s e r v e d effects because of t h e u n i q u e surface c o n f i g u r a t i o n of a m o e b a . I n t h e case of m a m m a l i a n cells, this kind of s i t u a t i o n is n o t n o r m a l l y
1077
o b s e r v e d in vitro, e v e n if i n c u b a t i o n is e x t e n d e d for longer periods a n d h i g h e r c o n c e n t r a t i o n s of V C N are used 10,11
S u m m a r y . R e m o v a l of cell surface sialic acid w i t h n e u r a m i n i d a s e b r i n g s a b o u t cell d e f o r m a t i o n in a m o e b a . The m e m b r a n e s of t h e s e d e f o r m e d cells are e v e n t u a l l y r u p t u r e d leading to t h e liberation of t h e cell mass. P. K. RAY a n d S. CHATTERJEE 16
Biomedical Group, M o d u l a r Laboratories, Bhabha A t o m i c Research Centre, Trombay, B o m b a y - 4 0 0 085 (India), 3 M a y 1974. 13 L. WEISS and C. J. LEVINSON, J. Cell Physiol. 73, 31 (1969). 14 D. H. BROWNand A. F. MICHAEL,Proc. Soc. exp. Biol. Med. 731, 568 (1969). 15 A. VAHERI, E. RUOSHATRIand S. NORDLING, Nature New Biol. 238, 211 (1972). 16 We thank Dr. E. W. RAJASEKHARfor his valuable help in preparing the manuscript.
T w o T y p e s of Bipolar Cells in the Chick Retinal D e v e l o p m e n t 1 B i p o l a r cells c o m p r i s e t h e bulk of t h e i n n e r n u c l e a r layer o f t h e retina. T h e s e ceils are i m p o r t a n t interm e d i a t e s of t h e r e t i n a l p a t h w a y s b e c a u s e t h e y f o r m synaptic contacts with both photoreceptor terminals, processes of a m a c r i n e cells a n d ganglion cells 2-5. E x t e n s i v e studies b y RAM6N u CAJAL6-s i n d i c a t e d t h a t , for e x a m p l e in t h e chicken, t h e r e are 2 t y p e s of b i p o l a r cells. H o w e v e r , t h e r e a p p e a r s to be little e x i s t i n g d a t a on w h e n t h e s e t w o t y p e s of bipolar cells arise a n d w h a t t h e i r m o r p h o l o g i c a l
Fig. 1. Type I and II bipolar cells are distinguished at stage 40 of chick embryonic retina. Note a centriole (Ce) in one of the type If cells and external projections (arrows) in the other. • 28,800.
c h a r a c t e r i s t i c s are. This s t u d y is an a t t e m p t t o s h e d s o m e i n s i g h t on t h i s p r o b l e m . Materials and method. Fertile w h i t e l e g h o r n chicken eggs were i n c u b a t e d in s t a n d a r d c o n d i t i o n s a n d s t a g e d a c c o r d i n g to HAMBURGER a n d HAMILTON ~ Chick emb r y o n i c eyes f r o m stages 36 to h a t c h i n g were t a k e n o u t a n d b i s e c t e d into halves. T h e n t h e p o s t e r i o r h a l v e s were p r e f i x e d w i t h 6% b u f f e r e d g l u t a r a l d e h y d e and p o s t f i x e d w i t h 1% b u f f e r e d o s m i u m t e t r o x i d e , d e h y d r a t e d in e t h a n o l , cleared in p r o p y l e n e oxide a n d e m b e d d e d in E p o n . Sections were cut w i t h a P o r t e r - B l u m MT 2 m i c r o t o m e a n d s t a i n e d w i t h u r a n y l a c e t a t e a n d lead citrate, a n d t h e n e x a m i n e d w i t h a Zeiss E M 9S2 electronmicroscope. Results. Bipolar cells c a n n o t be d i s t i n g u i s h e d u n t i l s t a g e 36 w h e n b o t h p l e x i f o r m layers are f o r m e d a n d t h e p r e s u m p t i v e b i p o l a r nuclei b e c o m e d i s t i n g u i s h a b l e b y t h e i r ellipsoidal shape. The c y t o p l a s m of t h e s e cells a t stage 36 h a v e a few m i t o c h o n d r i a , s o m e r o u g h e n d o p l a s m i c r e t i c u l u m a n d few ribosomes. S o m e b i p o l a r cells o f t e n lie close t o g e t h e r r e s e m b l i n g a t e l o p h a s e p h e n o m e non, i n d i c a t i n g t h a t t h e y h a v e j u s t c o m p l e t e d a m i t o t i c division. A t s t a g e 40, d e n d r i t i c p r o j e c t i o n s c a n be seen arizing f r o m t h e b i p o l a r cell bodies t o w a r d s t h e o u t e r p l e x i f o r m layer (Figure 1) a n d a t t h i s t i m e 2 t y p e s of bipolar cells can be distinguished. T h e u l t r a s t r u c t u r a l details of t h e s e 2 t y p e s of bipolars are o u t l i n e d in t h e
1 Supported by NIH grant No. EY00477 to DBM. 2 j. 1~. DOWLING, Proc. R. Soc., Lond. 170, 205 (1968). 3 j. E. DOWLINC.,Invest. Ophthal. 9, 655 (1970). 4 j. E. DOWLING and B. B. BOYCOTT, Proe. R. Soe. Lond. 766, 80 (1966). 5 J. E. DOWLING and B. B. BOYCOTT, Cold Spring Habor Syrup. quant. Biol. 30, 392 (1965). S. RAM6N Y CAJAL, Anat. Anz. d, 111 (1889). v S. RAM6N Y CAJAL, Cellule 9, 119 (1892). s S. RAMONY CAJAL, translated by S. A. THORPEand M. GLICKSTEIN, The Structure o] the Retina: A Complete Work (Charles C. Thomas, Springfield 1972). s U. HAMBURGERand H. L. HAMILTON, J. Morph. 88, 47 (1951).
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Comparison of the two bipolar cell types at stage 40
Nuclear position Nuclear morphology
Cytoplasm Cytoplasmic organelles,
Morphology of dendritic processes
Fig. 2. Type I and II bipolar cells at the same stage. The nucleus of the type I bipolar is large and located more externally. Note the presence of some rough endoplasmic reticulum near the nucleus. On the other hand, the long dendritic trunk in the type II bipolar contains a golgi apparatus, mitochondria and neurotubules. • 28,800.
T a b l e (Figures 1 a n d 2). I n r a r e instances, a c e n t r i o l e m a y b e p r e s e n t in t h e m a i n d e n d r i t e of t h e t y p e I I b i p o l a r cells (Figure 2) a t t h i s stage. Discussion. B i p o l a r cells are d e r i v e d e m b r y o l o g i c a l l y f r o m t h e o u t e r n e u r o b l a s t i c l a y e r 1~ n a n d i n i t i a l l y t h e y a p p e a r s p i n d l e - s h a p e d a n d c a n n o t b e easily d i f f e r e n t i a t e d f r o m t h e o t h e r r e t i n a l ceils. B y stage 36, w h e n t h e o u t e r plexiform layer appears and separates the photoreceptor ceils f r o m t h e i n n e r n u c l e a r layer, b i p o l a r cells c a n b e clearly identified. O u r e l e c t r o n m i c r o s c o p i c d a t a on t h e s e 2 t y p e s of c h i c k b i p o l a r cells a p p e a r t o c o n f i r m RAM6~ v CAJAL'S studies. T y p e I b i p o l a r cells (RAM65! Y CAJAL'S o u t e r bipolars) w i t h large nuclei a n d s h o r t dendritic branches can be distinguished from the type II b i p o l a r cells (RAM6N Y CAJAL'S i n n e r bipolars) e a c h of
Ovulation in an Echinoderm
Type I
Type II
Close to outer plexiform layer Large (2 [xln wide differ from horizontal cell nuclei which are spherical) Scanty Mitochondria, more rough endoplasmic reticulum, free ribosomes, some neurotubules
Close to inner layers Smaller (1.5 [xm wide)
Short branches of dendritic processes from cell body
Scanty Long mitochondria less rough endoplasmic reticulum free ribosomes, prominent long golgi, more neurotnbules Long main dendrite leading from body, then giving out branches
t h e m h a s a s m a l l e r n u c l e u s a n d a single long m a i n dend r i t i c t r u n k . F u r t h e r m o r e , o u r results also i n d i c a t e t h a t t h e r e are differences b e t w e e n c y t o p l a s m i c organelles in t h e 2 t y p e s of b i p o l a r cells. I t is h o p e d t h a t , b a s e d o n t h e u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s of t h e s e 2 t y p e s of c h i c k b i p o l a r cells, a d d i t i o n a l u n d e r s t a n d i n g of t h e c o m p l e x r e l a t i o n s h i p s b e t w e e n t h e b i p o l a r s a n d t h e d i f f e r e n t r e t i n a l cell t y p e s c a n t h e n b e obtained.
Summary. T w o t y p e s of b i p o l a r cells are identified in the chick embryonic retina. They can be distinguished by t h e i r c y t o p l a s m i c organelles. D. T. YEW a n d D. B. MEYER
Department o/Biology, University Science Center, Chinese University o[ Hong Kong, Shatin, N. T. (Hong Kong), and Department o/ A natomy, Wayne State University, Detrot (Michigan, USA), 2 April 1975. 10 I. MANN, Br. J. Ophthal. I2, 449 (1928). :t I. MANN,The Developmento! the Human Eye (Grune and Stratton Inc., New York 1950).
(Comanthus japonica)
O v u l a t i o n in several i n v e r t e b r a t e g r o u p s m a y be defined as t h e p a s s a g e of oocytes t h r o u g h a l a y e r of e p i t h e l i a l cells. T h i s p h e n o m e n o n h a s b e e n o b s e r v e d d i r e c t l y for c t e n o p h o r e s ~ a n d h a s b e e n i n f e r r e d f r o m c o m p a r i s o n s of o v a r i a n h i s t o l o g y before a n d a f t e r o v u l a t i o n for c o e l e n t e r a t e s ~, p r i a p u l i d s 3, p e n t a s t o m i d s 4 a n d c e p h a l o c h o r d a t e s 6 . T h e p r e s e n t r e p o r t describes t h e foregoing t y p e of o v u l a t i o n 6 for t h e first t i m e in a n e c h i n o d e r m ; m o r e o v e r , t h e o o c y t e s are i l l u s t r a t e d d u r i n g t h e i r t r a n s e p i t h e l i a l passage. T h e e c h i n o d e r m s t u d i e d was Comanthus japonica, a c r i n o i d for w h i c h t h e s p a w n i n g d a t e is p r e d i c t a b l e f r o m tile l u n a r c a l e n d a r ~. On t h e p r e d i c t e d day, a l m o s t all t h e
oocytes in all t h e females b e g i n m a t u r a t i o n w i t h g e r m i n a l vesicle b r e a k d o w n s h o r t l y before noon, a n d t h e m a t u r e o v a are s p a w n e d l a t e r t h a t afternoonS, 9. To d e m o n s t r a t e o v u l a t i o n in Comanthus, we r e m o v e d ovaries e v e r y 15 mill t h r o u g h o u t tile m o r n i n g a n d a f t e r n o o n of t h e s p a w n ing day. These ovaries were fixed b y p r e v i o u s l y p u b l i s h e d m e t h o d s 1 ~ for light- a n d e l e c t r o n microscopy. U p t h r o u g h 11.30 h, e v e r y o o c y t e h a d a g e r m i n a l vesicle c o n t a i n i n g a single nucleolus. E a c h o o c y t e l a y m a i n l y in t h e i n t e r m e d i a t e l a y e r of t h e ovary11; h o w e v e r , t h e e n d of t h e o 0 c y t e n e a r e s t t h e o v a r i a n l u m e n was closely assoc i a t e d w i t h a p l a q u e of c u b o i d a l s o m a t i c cells b e l o n g i n g to t h e o t h e r w i s e s q u a m o u s e p i t h e l i u m lining t h e ovary.
