Cell Differentiation, 5 ( 1 9 7 6 ) 1 9 9 - - 2 0 6 199 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press, A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
INTERCELLULAR
B R I D G E S IN L I Z A R D O O G E N E S I S *
S. F I L O S A a n d C. T A D D E I
Institute of Histology and Embryology, Laboratory of Comparative Anatomy, University of Naples, Italy A c c e p t e d 2 May 1 9 7 6
S t u d y o f t h e g e r m i n a l e p i t h e l i u m in t h e a d u l t lizard s h o w s t h a t t h e germ cells constit u t e clusters o f s y n c h r o n i z e d cells i n t e r c o n n e c t e d b y i n t e r c e l l u l a r bridges. S u c h bridges i n t e r c o n n e c t o o g o n i a as well as early m e i o t i c p r o p h a s e o o c y t e s ( z y g o - p a c h i t e n e ) . Besides t r u e i n t e r c e l l u l a r bridges in o o c y t e s , t h e r e are p l a s m a m e m b r a n e i n t e r r u p t i o n s f o r m i n g large z o n e s t h a t e n s u r e c y t o p l a s m i c c o n t i n u i t y b e t w e e n a d j a c e n t cells. In early d i p l o t e n e , germ cells are isolated. Later, d u r i n g a u x o c y t o s i s , w h e n t h e p o l y m o r p h i c follicular e p i t h e l i u m a r o u n d t h e o o c y t e starts d i f f e r e n t i a t i n g , i n t e r c e l l u l a r bridges a p p e a r b e t w e e n t h e follicle cells a n d o o c y t e . No r e l a t i o n s h i p is o b s e r v e d b e t w e e n t h e i n t e r c e l l u l a r bridges f o u n d in t h e g e r m i n a l epit h e l i u m a n d t h o s e f o u n d b e t w e e n t h e follicle cells a n d o o c y t e .
Intercellular bridges connecting differentiating germ cells were first observed by Fawcett et al. (1959) in spermatogenesis, and interpreted as being correlated to the synchronous differentiation of germ cell clusters. Also reported in the literature are intercellular bridges that connect oogonia as well as early meiotic prophase o o c y t e s during embryonic development of the ovary in different vertebrates (Zamboni et al., 1968; R u b y et al., 1969, 1970; Skalko et al., 1972}. Beside the role of synchronizing the developing germ cells, these bridges have been considered as having a function in transferring cytoplasmic materials from one cell to another. In the vertebrates studied so far with the only exception being the lizard, intercellular bridges disappear at the onset of diplotene, when auxocytosis begins. In the lizard, true intercellular bridges connecting follicle cells (pyriform cells) and o o c y t e (Ghiara et al., 1968; Hubert, 1971; Neaves, 1971; Taddei, 1972b) have been observed throughout auxocytosis. It was consequently thought that it would be interesting to extend our investigations to the early stages of oogenesis in order to find o u t whether intercellular bridges also interconnect germ cells during the early stages of * Part o f t h e s e results was p r e s e n t e d at t h e II I n t e r n a t i o n a l C o n f e r e n c e o f D i f f e r e n t i a t i o n , Copenhagen, 8--12 September, 1975. This w o r k was s u p p o r t e d b y C.N.R. G r a n t No. 7 3 / 0 5 9 1 a n d carried o u t at t h e E l e c t r o n M i c r o s c o p y C e n t e r o f t h e F a c u l t y o f Sciences, University o f Naples, Naples, Italy.
200 lizard oogenesis. In addition, we have investigated at which stage the intercellular bridges between the pyriform cells and oocyte appear for the first time in order to clarify whether such bridges originate following possible interaction between the cells constituting the germinal epithelium, or by a fusion of the follicle cells with the growing oocyte. L a c e r t a sicula Raf. was chosen since oogenesis here, as in most reptiles, is an uninterrupted process lasting t h r o u g h o u t the reproductive life of the adult animal (Franchi et al., 1962). MATERIALS AND METHODS Adult, sexually mature females of the lizard L a c e r t a s. sicula Raf. (6--7 cm in snout-vent length) were collected in the neighbourhood of Naples. The ovaries were exposed and the germinal epithelia isolated under a dissecting microscope. The whole germinal epithelium (about 1 mm in diameter) was fixed in phosphate buffered formaldehyde~glutaraldehyde pH 7.4 (Karnovsky, 1965) post-fixed with phosphate buffered 2% osmium, dehydrated with alcohol and embedded in Epon. Ultrathin sections, obtained with an LKB ult r o t o m e and stained with uranyl acetate and lead citrate {Reynolds, 1963), were observed under a Siemens Elmiskop IA electron microscope. RESULTS The germinal epithelium is a small area (1 mm wide) close to the ovarian hilum. It contains oogonia, early meiotic prophase oocytes and stroma cells. Germ cells in the same differentiation stage appear to be organized in groups, each bounded by stroma cells and composed of several closely-connected cells {Fig. 1). The germ cells are easily identified by their large size, transparent cytoplasm and round-shaped nucleus. They can be easily distinguished from the electron-denser stroma cells scattered inside the germinal epithelium, which have an elongated body, a small irregularly shaped nucleus containing masses of very condensed chromatin and a well developed rough endoplasmic reticulum. Oogonia are characterized by the organization of their chromatin in scattered and marginal clumps of moderate density. A cytoplasmic continuity between these cells is assured by true intercellular bridges; these stand out clearly because of a 400 /~ thick band of dense material lying close to the plasma membrane lining the bridge {Fig. 2). The fine structure of the cytoplasmic matrix of the bridge is identical to that of the two contiguous cells; it contains ribosomes, mitochondria and vesicles. Not u n c o m m o n are bundles of microtubules, probably the remnant of a mitotic spindle, crossing the bridges from one cell to another (Fig. 3). During early meiotic prophase (up to zygo-pachitene), the germ cell increases in size. The condensed chromatin inside the nuclei disappears and synaptonemal complexes take place. The organization of the germ cell clus-
Fig. 1. A cluster o f oogonia (OG) s u r r o u n d e d by s t r o m a cells (SC). The a r r o w p o i n t s to an intercellular bridge m o r e e v i d e n t in Fig. 2. Fig. 2. A h i g h e r m a g n i f i c a t i o n o f the bridge. N o t e the c o n t i n u i t y o f t h e plasma m e m b r a n e o f t h e t w o cells at t h e level o f t h e bridge and the b o u n d a r y m a d e up o f d e n s e material b e l o w the plasma m e m b r a n e . Fig. 3. A b u n d l e o f m i c r o t u b u l e s crossing t h e bridge b e t w e e n t w o oogonia. N o t e the material o f the residual m i d - b o d y .
Fig. 4, T w o o o c y t e s at z y g o - p a c h y t e n e c o n n e c t e d b y an i n t e r c e l l u l a r bridge. Inside th~ nucleus, s y n a p t o n e m a l c o m p l e x e s are e v i d e n t (arrow). Fig. 5. A h i g h e r m a g n i f i c a t i o n o f t h e intercellular c o n n e c t i o n s h o w n in Fig. 4. I n t e r r u p t i o n s of t h e p l a s m a m e m b r a n e are also e v i d e n t (arrows). Fig. 6. C o m p l e t e a b s e n c e o f p l a s m a m e m b r a n e s b e t w e e n t w o cells at z y g o - p a e h y t e n e .
203
ters is maintained during this stage;easily detectable are intercellular bridges similar in structure to those connecting oogonia (Fig. 4), as well as wide gaps in the plasma membrane between adjacent germ cells of the cluster (Fig. 5). Such gaps seem to originate from the fusion of the plasma membrane of two adjacent cells; however, unlike true intercellular bridges, they do not exhibit the thickening of the plasma membrane. The plasma membrane between adjacent cells often completely disappears (Fig. 6). At early diplotene, germ cells are isolated and surrounded by stroma cells in such a way that the cluster organization of the germ cells is no longer detectable. The follicular epithelium is not yet organized, and intercellular bridges do not yet exist. At this stage, the cells are characterized by a remarkable enlargement of the cytoplasm, which is denser than during the previous stages, and by an abundance of both electron-transparent vesicles and ribosomal bodies (Taddei, 1972a). The nucleus is irregularly shaped and contains dispersed, finely granular chromatin (Fig. 7).
Fig. 7. O o c y t e at early d i p l o t e n e . T h e follicular e p i t h e l i u m is n o t y e t o r g a n i z e d a r o u n d the o o c y t e . No i n t e r c e l l u l a r bridges are d e t e c t a b l e at this stage. T h e o o c y t e c y t o p l a s m is enlarged a n d m o r e c o m p a c t . It s h o w s large n u m b e r s of vacuoles a n d t w o r i b o s o m a l bodies
(arrows).
Fig. 8. A m o n o l a y e r e d follicular e p i t h e l i u m ( F E ) s u r r o u n d i n g an o o c y t e ( O o ) of 100 p m in d i a m e t e r . T h e p l a s m a m e m b r a n e s of o o c y t e a n d follicular cells are in c o n t a c t and num e r o u s m i c r o v i l l o u s d i g i t a t i o n s ( a r r o w s ) are e v i d e n t . Fig. 9. I n t e r c e l l u l a r bridges (IB) c o n n e c t i n g t h r o u g h t h e ' z o n a p e l l u c i d a ' (ZP), a different i a t i n g p y r i f o r m cell ( P C ) a n d an o o c y t e ( O o ) of 300 p m in d i a m e t e r .
205 A monolayered follicular epithelium forms around the o o c y t e when the latter reaches 100 pm in diameter. Follicle cells and o o c y t e are in close contact via a number of intertwining microvillous digitations (Fig. 8), b u t their cytoplasms are not in continuity. When the o o c y t e is 200--300 pm in diameter, and the multilayered, polymorphic follicular epithelium begins to differentiate, cytoplasmic bridges, identical to those observed in larger follicles between differentiated pyriform cells and o o c y t e (Taddei, 1972b), are detectable between follicle cells and o o c y t e via the 'zona pellucida', which, at this stage, makes its first appearance (Fig. 9). No structural differentiation of the plasma membrane is observed around these bridges. DISCUSSION This study shows that in the adult lizard, during the early differentiation stages sister germ cells are interconnected by intercellular bridges thus giving rise to a syncytial organization. The bridges are structurally similar to those described during the embryonic development of the ovary in other vertebrates (Zamboni et al., 1968; R u b y et al., 1969, 1970; Skalko et al., 1972) and represent a true cytoplasmic continuity between germ cells during early oogenesis. The finding of microtubule bundles only inside the bridges that connect oogonia b u t never inside those that connect oocytes, suggests that these microtubules are a remnant of the mitotic spindle, and supports the hypothesis that intercellular bridges are formed by incomplete cytodieresis. True intercellular bridges represent the only cytoplasmic continuity between oogonia; whereas between o o c y t e s (at zygo-pachitene), in addition to intercellular bridges, more extensive communications are established by the disappearance of part or all of their plasma membrane. At early diplotene, the syncytial organization of the clustered germ cells disappears, leading to isolated oocytes. Later, when the multilayered follicular epithelium around the o o c y t e begins to differentiate, cytoplasmic bridges are detectable between o o c y t e and follicle cells (pyriform cells). Hence, this observation discounts any correlation between the intercellular bridges connecting sister germ cells and those connecting pyriform cells and the growing oocyte. In conclusion, during o o c y t e differentiation and growth in the lizard two different types of cell connections are established. The first type is present between sister germ cells: it originates at the beginning of differentiation and determines the syncytial organization either of oogonia or of early meiotic prophase oocytes. This situation is similar to that found in other vertebrates. The second t y p e is established between pyriform cells and oocyte: this is characteristic of lizard oogenesis since intercellular bridges between follicle cells and o o c y t e have never been described in other vertebrates.
206 T h e biological significance o f this s y n c y t i a l o r g a n i z a t i o n is obscure. One m i g h t speculate t h a t the c o n n e c t i o n s b e t w e e n sister germ cells and b e t w e e n follicle cells and o o c y t e results in genetic c o o p e r a t i o n b e t w e e n the cells involved. T h e r e is n o d i r e c t evidence as y e t t h a t such a g e n o m e c o o p e r a t i o n does in f a c t exist; the presence o f c y t o p l a s m i c material inside the intercellular bridges suggests e x c h a n g e o f c y t o p l a s m i c c o m p o n e n t s t h a t m a y include i n f o r m a t i o n a l m a c r o m o l e c u l e s . In the case o f the c o n n e c t i o n s b e t w e e n pyrif o r m cells and o o c y t e , t h e r e is evidence o f c o o p e r a t i o n b e t w e e n the t w o t y p e s o f cells: t h e r e is indeed evidence o f a transfer o f r i b o s o m e s f r o m the p y r i f o r m cells into the o o c y t e c y t o p l a s m via intercellular bridges (Taddei, 1972b). ACKNOWLEDGEMENT We are very grateful to Professors G. Ghiara and A. Monroy for helpful discussions during the preparation of this manuscript.
REFERENCES Fawcett, D.W., S. Ito and D. Slautterback: J. Biophys. Biochem. Cytol. 5, 453--460 (1959). Franchi, L.L., A.M. Mandl and S. Zuckerman: The Ovary, ed.;S. Zuckerman (Academic Press, New York) Vol. 1, 1--88 (1962). Ghiara, G., E. Limatola and S. Filosa: Conference on Electron Microscopy {Tip. Poliglotta, Roma) 2, 331 (1968). Hubert, J.: Z. Zellforsch. 116, 240 (1971). Karnovsky, M.J.: J. Cell Biol. 27,137 A (1965). Neaves, W.B.: Anat. Rec. 170,285--302 (1971). Reynolds, E.S.: J. Cell Biol. 17, 208--212 (1963). Ruby, J.R., R.F. Dyer and R.G. Skalko: J. Morph. 127,307--340 (1969). Ruby, J.R., R.F. Dyer, R.G. Skalko and E.P. Volpe: Anat. Rec. 167, 1--10 (1970). Skalko, R.G., J.M. Kerrigan, J.R. Ruby and R.F. Dyer: Z. Zellforsch. 128, 31--41 (1972). Taddei, C.: Exp. Cell Res. 70, 285--292 (1972a). Taddei, C.: Exp. Cell Res. 72, 562--566 (1972b). Zamboni, L. and B. Gondos: J. Cell Biol. 36, 276--282 (1968).
INTERCELLULAR
B R I D G E S IN L I Z A R D O O G E N E S I S *
S. F I L O S A a n d C. T A D D E I
Institute of Histology and Embryology, Laboratory of Comparative Anatomy, University of Naples, Italy A c c e p t e d 2 May 1 9 7 6
S t u d y o f t h e g e r m i n a l e p i t h e l i u m in t h e a d u l t lizard s h o w s t h a t t h e germ cells constit u t e clusters o f s y n c h r o n i z e d cells i n t e r c o n n e c t e d b y i n t e r c e l l u l a r bridges. S u c h bridges i n t e r c o n n e c t o o g o n i a as well as early m e i o t i c p r o p h a s e o o c y t e s ( z y g o - p a c h i t e n e ) . Besides t r u e i n t e r c e l l u l a r bridges in o o c y t e s , t h e r e are p l a s m a m e m b r a n e i n t e r r u p t i o n s f o r m i n g large z o n e s t h a t e n s u r e c y t o p l a s m i c c o n t i n u i t y b e t w e e n a d j a c e n t cells. In early d i p l o t e n e , germ cells are isolated. Later, d u r i n g a u x o c y t o s i s , w h e n t h e p o l y m o r p h i c follicular e p i t h e l i u m a r o u n d t h e o o c y t e starts d i f f e r e n t i a t i n g , i n t e r c e l l u l a r bridges a p p e a r b e t w e e n t h e follicle cells a n d o o c y t e . No r e l a t i o n s h i p is o b s e r v e d b e t w e e n t h e i n t e r c e l l u l a r bridges f o u n d in t h e g e r m i n a l epit h e l i u m a n d t h o s e f o u n d b e t w e e n t h e follicle cells a n d o o c y t e .
Intercellular bridges connecting differentiating germ cells were first observed by Fawcett et al. (1959) in spermatogenesis, and interpreted as being correlated to the synchronous differentiation of germ cell clusters. Also reported in the literature are intercellular bridges that connect oogonia as well as early meiotic prophase o o c y t e s during embryonic development of the ovary in different vertebrates (Zamboni et al., 1968; R u b y et al., 1969, 1970; Skalko et al., 1972}. Beside the role of synchronizing the developing germ cells, these bridges have been considered as having a function in transferring cytoplasmic materials from one cell to another. In the vertebrates studied so far with the only exception being the lizard, intercellular bridges disappear at the onset of diplotene, when auxocytosis begins. In the lizard, true intercellular bridges connecting follicle cells (pyriform cells) and o o c y t e (Ghiara et al., 1968; Hubert, 1971; Neaves, 1971; Taddei, 1972b) have been observed throughout auxocytosis. It was consequently thought that it would be interesting to extend our investigations to the early stages of oogenesis in order to find o u t whether intercellular bridges also interconnect germ cells during the early stages of * Part o f t h e s e results was p r e s e n t e d at t h e II I n t e r n a t i o n a l C o n f e r e n c e o f D i f f e r e n t i a t i o n , Copenhagen, 8--12 September, 1975. This w o r k was s u p p o r t e d b y C.N.R. G r a n t No. 7 3 / 0 5 9 1 a n d carried o u t at t h e E l e c t r o n M i c r o s c o p y C e n t e r o f t h e F a c u l t y o f Sciences, University o f Naples, Naples, Italy.
200 lizard oogenesis. In addition, we have investigated at which stage the intercellular bridges between the pyriform cells and oocyte appear for the first time in order to clarify whether such bridges originate following possible interaction between the cells constituting the germinal epithelium, or by a fusion of the follicle cells with the growing oocyte. L a c e r t a sicula Raf. was chosen since oogenesis here, as in most reptiles, is an uninterrupted process lasting t h r o u g h o u t the reproductive life of the adult animal (Franchi et al., 1962). MATERIALS AND METHODS Adult, sexually mature females of the lizard L a c e r t a s. sicula Raf. (6--7 cm in snout-vent length) were collected in the neighbourhood of Naples. The ovaries were exposed and the germinal epithelia isolated under a dissecting microscope. The whole germinal epithelium (about 1 mm in diameter) was fixed in phosphate buffered formaldehyde~glutaraldehyde pH 7.4 (Karnovsky, 1965) post-fixed with phosphate buffered 2% osmium, dehydrated with alcohol and embedded in Epon. Ultrathin sections, obtained with an LKB ult r o t o m e and stained with uranyl acetate and lead citrate {Reynolds, 1963), were observed under a Siemens Elmiskop IA electron microscope. RESULTS The germinal epithelium is a small area (1 mm wide) close to the ovarian hilum. It contains oogonia, early meiotic prophase oocytes and stroma cells. Germ cells in the same differentiation stage appear to be organized in groups, each bounded by stroma cells and composed of several closely-connected cells {Fig. 1). The germ cells are easily identified by their large size, transparent cytoplasm and round-shaped nucleus. They can be easily distinguished from the electron-denser stroma cells scattered inside the germinal epithelium, which have an elongated body, a small irregularly shaped nucleus containing masses of very condensed chromatin and a well developed rough endoplasmic reticulum. Oogonia are characterized by the organization of their chromatin in scattered and marginal clumps of moderate density. A cytoplasmic continuity between these cells is assured by true intercellular bridges; these stand out clearly because of a 400 /~ thick band of dense material lying close to the plasma membrane lining the bridge {Fig. 2). The fine structure of the cytoplasmic matrix of the bridge is identical to that of the two contiguous cells; it contains ribosomes, mitochondria and vesicles. Not u n c o m m o n are bundles of microtubules, probably the remnant of a mitotic spindle, crossing the bridges from one cell to another (Fig. 3). During early meiotic prophase (up to zygo-pachitene), the germ cell increases in size. The condensed chromatin inside the nuclei disappears and synaptonemal complexes take place. The organization of the germ cell clus-
Fig. 1. A cluster o f oogonia (OG) s u r r o u n d e d by s t r o m a cells (SC). The a r r o w p o i n t s to an intercellular bridge m o r e e v i d e n t in Fig. 2. Fig. 2. A h i g h e r m a g n i f i c a t i o n o f the bridge. N o t e the c o n t i n u i t y o f t h e plasma m e m b r a n e o f t h e t w o cells at t h e level o f t h e bridge and the b o u n d a r y m a d e up o f d e n s e material b e l o w the plasma m e m b r a n e . Fig. 3. A b u n d l e o f m i c r o t u b u l e s crossing t h e bridge b e t w e e n t w o oogonia. N o t e the material o f the residual m i d - b o d y .
Fig. 4, T w o o o c y t e s at z y g o - p a c h y t e n e c o n n e c t e d b y an i n t e r c e l l u l a r bridge. Inside th~ nucleus, s y n a p t o n e m a l c o m p l e x e s are e v i d e n t (arrow). Fig. 5. A h i g h e r m a g n i f i c a t i o n o f t h e intercellular c o n n e c t i o n s h o w n in Fig. 4. I n t e r r u p t i o n s of t h e p l a s m a m e m b r a n e are also e v i d e n t (arrows). Fig. 6. C o m p l e t e a b s e n c e o f p l a s m a m e m b r a n e s b e t w e e n t w o cells at z y g o - p a e h y t e n e .
203
ters is maintained during this stage;easily detectable are intercellular bridges similar in structure to those connecting oogonia (Fig. 4), as well as wide gaps in the plasma membrane between adjacent germ cells of the cluster (Fig. 5). Such gaps seem to originate from the fusion of the plasma membrane of two adjacent cells; however, unlike true intercellular bridges, they do not exhibit the thickening of the plasma membrane. The plasma membrane between adjacent cells often completely disappears (Fig. 6). At early diplotene, germ cells are isolated and surrounded by stroma cells in such a way that the cluster organization of the germ cells is no longer detectable. The follicular epithelium is not yet organized, and intercellular bridges do not yet exist. At this stage, the cells are characterized by a remarkable enlargement of the cytoplasm, which is denser than during the previous stages, and by an abundance of both electron-transparent vesicles and ribosomal bodies (Taddei, 1972a). The nucleus is irregularly shaped and contains dispersed, finely granular chromatin (Fig. 7).
Fig. 7. O o c y t e at early d i p l o t e n e . T h e follicular e p i t h e l i u m is n o t y e t o r g a n i z e d a r o u n d the o o c y t e . No i n t e r c e l l u l a r bridges are d e t e c t a b l e at this stage. T h e o o c y t e c y t o p l a s m is enlarged a n d m o r e c o m p a c t . It s h o w s large n u m b e r s of vacuoles a n d t w o r i b o s o m a l bodies
(arrows).
Fig. 8. A m o n o l a y e r e d follicular e p i t h e l i u m ( F E ) s u r r o u n d i n g an o o c y t e ( O o ) of 100 p m in d i a m e t e r . T h e p l a s m a m e m b r a n e s of o o c y t e a n d follicular cells are in c o n t a c t and num e r o u s m i c r o v i l l o u s d i g i t a t i o n s ( a r r o w s ) are e v i d e n t . Fig. 9. I n t e r c e l l u l a r bridges (IB) c o n n e c t i n g t h r o u g h t h e ' z o n a p e l l u c i d a ' (ZP), a different i a t i n g p y r i f o r m cell ( P C ) a n d an o o c y t e ( O o ) of 300 p m in d i a m e t e r .
205 A monolayered follicular epithelium forms around the o o c y t e when the latter reaches 100 pm in diameter. Follicle cells and o o c y t e are in close contact via a number of intertwining microvillous digitations (Fig. 8), b u t their cytoplasms are not in continuity. When the o o c y t e is 200--300 pm in diameter, and the multilayered, polymorphic follicular epithelium begins to differentiate, cytoplasmic bridges, identical to those observed in larger follicles between differentiated pyriform cells and o o c y t e (Taddei, 1972b), are detectable between follicle cells and o o c y t e via the 'zona pellucida', which, at this stage, makes its first appearance (Fig. 9). No structural differentiation of the plasma membrane is observed around these bridges. DISCUSSION This study shows that in the adult lizard, during the early differentiation stages sister germ cells are interconnected by intercellular bridges thus giving rise to a syncytial organization. The bridges are structurally similar to those described during the embryonic development of the ovary in other vertebrates (Zamboni et al., 1968; R u b y et al., 1969, 1970; Skalko et al., 1972) and represent a true cytoplasmic continuity between germ cells during early oogenesis. The finding of microtubule bundles only inside the bridges that connect oogonia b u t never inside those that connect oocytes, suggests that these microtubules are a remnant of the mitotic spindle, and supports the hypothesis that intercellular bridges are formed by incomplete cytodieresis. True intercellular bridges represent the only cytoplasmic continuity between oogonia; whereas between o o c y t e s (at zygo-pachitene), in addition to intercellular bridges, more extensive communications are established by the disappearance of part or all of their plasma membrane. At early diplotene, the syncytial organization of the clustered germ cells disappears, leading to isolated oocytes. Later, when the multilayered follicular epithelium around the o o c y t e begins to differentiate, cytoplasmic bridges are detectable between o o c y t e and follicle cells (pyriform cells). Hence, this observation discounts any correlation between the intercellular bridges connecting sister germ cells and those connecting pyriform cells and the growing oocyte. In conclusion, during o o c y t e differentiation and growth in the lizard two different types of cell connections are established. The first type is present between sister germ cells: it originates at the beginning of differentiation and determines the syncytial organization either of oogonia or of early meiotic prophase oocytes. This situation is similar to that found in other vertebrates. The second t y p e is established between pyriform cells and oocyte: this is characteristic of lizard oogenesis since intercellular bridges between follicle cells and o o c y t e have never been described in other vertebrates.
206 T h e biological significance o f this s y n c y t i a l o r g a n i z a t i o n is obscure. One m i g h t speculate t h a t the c o n n e c t i o n s b e t w e e n sister germ cells and b e t w e e n follicle cells and o o c y t e results in genetic c o o p e r a t i o n b e t w e e n the cells involved. T h e r e is n o d i r e c t evidence as y e t t h a t such a g e n o m e c o o p e r a t i o n does in f a c t exist; the presence o f c y t o p l a s m i c material inside the intercellular bridges suggests e x c h a n g e o f c y t o p l a s m i c c o m p o n e n t s t h a t m a y include i n f o r m a t i o n a l m a c r o m o l e c u l e s . In the case o f the c o n n e c t i o n s b e t w e e n pyrif o r m cells and o o c y t e , t h e r e is evidence o f c o o p e r a t i o n b e t w e e n the t w o t y p e s o f cells: t h e r e is indeed evidence o f a transfer o f r i b o s o m e s f r o m the p y r i f o r m cells into the o o c y t e c y t o p l a s m via intercellular bridges (Taddei, 1972b). ACKNOWLEDGEMENT We are very grateful to Professors G. Ghiara and A. Monroy for helpful discussions during the preparation of this manuscript.
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