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The ovary of the lobster, Homarus americanus
JOURNAL OF ULTRASTRUCTURE RESEARCH
76, 235-248 (1981)
The Ovary of the Lobster, Homarus americanus I. Architecture of the Mature Ovary PRUDENCE TALBOT 1
Department of Biology, University of California, Riverside, California 92521, and Bodega Marine Laboratory, Bodega Bay, California 94923 Received April 7, 1981 We have examined the architecture of the mature lobster (Homarus americanus) ovary using microscopical techniques. The ovary is comprised of five tubular lobes which are organized in an H-shaped pattern. Each lobe contains four morphological components which are: (i) the ovarian wall, (ii) the epithelium, (iii) the follicles, and (iv) the extensions of the ovarian wall. The wall is made up of blood vessels and sinuses, blood cells, and nonstriated muscle cells which may undergo spontaneous contraction when the ovary is cut. The epithelial cells are flat and rest on a thick basal lamina. They form a thin seet on the luminal surface of the ovary. Portions of this sheet fold outward toward the ovarian wall. Epithelial cells are continuous with and may give rise to follicle cells around oocytes. The mature ovary contains large (mature, 1.2-1.6 mm)-, intermediate (0.10.4 ram)-, and small (0.07-0.1 mm)-sized follicles. Small follicles and some intermediate follicles are found embedded in the epithelium lining the lumen. Most intermediate follicles form chains linking the luminal epithelium to mature follicles. The mature follicles are stacked in tiers three to five follicle layers deep between the ovarian wall and the lumen. The extensions of the ovarian wall consist of both blood vessels and muscle cells; these project into the follicular region and insert on follicle walls and on the epithelium. This description of the mature lobster ovary extends our knowledge on the organization of the decapod ovary and provides a basis for future detailed structural and physiological analyses of this organ.
Apart from several histological examinations (2, 9, 10), and two recent ultrastructural studies of vitellogenesis (13, 19), very little work has been done on the structure of ovaries in the lobster, Homarus americanus. In order to understand ovarian processes, such as o o c y t e development', ovulation, and spawning, it is necessary to have a complete knowledge of the structure of this organ. We have therefore used light and electron microscopy to examine the morphology of lobster ovaries. The purpose of this report is to present an overview of the architecture of mature lobster (Homarus americanus) ovaries at the ultrastructural level. This will provide a framework for subsequent, more detailed descriptions of various components of the lobster ovary.
1 Present address: Department of Biology, University of California, Riverside, Calif. 92521.
MATERIALS AND METHODS Female lobsters (Homarus arnericanus) were obtained between July and September and candled (8) to estimate the degree of ovarian maturity. Ten females Were found to have mature ovaries which were close to natural ovulation. While it is not yet possible to predict precisely when lobsters will ovulate, these ovaries appeared to be in late stage 5 or stage 6 (ripe) of development according to the classification scheme proposed by Aiken and Waddy (1). These females were used in this and the following study. Lobsters were sacrificed and dissected to observe the anatomical features of the mature ovary. Pieces of ovary were then removed for study with light and electron microscopy. General features of ovarian organization were assessed using both a stereoscopic and a phase contrast microscope. For more detailed cytological analysis, tissue was fixed for 3 hr in a solution containing 3% glutaraldehyde, 12% glucose, and 0.1 M sodium cacodylate (pH 7.4). Following several rinses in 0.1 M cacodylate with 24% sucrose, tissue was postfixed for 1-2 hr in 1% OsO4 in 0.1 M cacodylate with 24% sucrose. Tissue was then washed in a 0.1 M cacodylate series containing 24, 12, or 6% sucrose, and dehydrated in a graded series of acetone. For light microscopy and transmission electron microscopy, 235 0022-5320/81/090235-14502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tissue was infiltrated overnight in Spurr's plastic/acetone (1:1), e m b e d d e d in Spurr's plastic (23), sectioned on a P o r t e r - B l u m MT-2B ultramicrotome, and stained with toluidine blue (thick sections) or lead and uraniu m salts (thin sections). Thick sections were examined with a Zeiss Standard light microscope; thin sections were viewed using a Hitachi H-500 transmission electron microscope. For scanning electron microscopy, pieces of ovary were fixed and dehydrated as described above. Specimens in 100% acetone were critically point dried in a Samdri PVT-3 apparatus, m o u n t e d on a l u m i n u m pedestals, coated with gold and platinum in a Technics H u m m e r II, and studied with a Jeol JSM-35C scanning electron microscope. In s o m e cases, tissue which had been e x a m i n e d with the scanning electron microscope was placed in 100% a c e t o n e , infiltrated and e m b e d d e d in plastic, sectioned, stained, and e x a m i n e d with the Hitachi H-500 electron microscope as previously described.
RESULTS
General Features of the Mature Ovary Homarus ovaries are comprised of five lobes which are united in an H-shaped pattern. The ovaries are positioned dorsally and extend from the anterior-most region of the cephalothorax to the third, fourth, or fifth abdominal segment (Fig. 1). The lobe forming the cross segment of the H lies ventral to the heart, and it is at this region that the oviducts leave the ovary and extend ventrally to the gonopores at the base of the third walking leg. Each lobe of the ovary is tubular. The lumen of the tube is demonstrated in Fig. 4 which shows the tip of a pair of forceps inserted into the center of a transected lobe. The ovary is dark green in color and usually contains a small number of yellow or orange spots, presumed to be either atretic or degenerating follicles. T h e s e are scattered r a n d o m l y throughout the ovary, and in one female were quite numerous. Lobes of ovaries were cut open to form flat strips (Figs. 2 and 3) and were examined by light and electron microscopy. These observations revealed that lobster ovaries are comprised of four distinct components: (i) the ovarian wall, (ii) the epithelium, (iii) the follicles (mature and immature), and
(iv) the extensions of the ovarian wall. Each of these components and their relationships to each other will next be considered in greater detail.
The Ovarian Wall In living ovaries, the ovarian wall is a pale white, translucent layer which is best seen after the ovary has been cut into strips (Figs. 3 and 5). The tissue of the wall is densely packed (Figs. 6-8) and is composed of long thin muscle cells, blood sinuses (Fig. 8), blood vessels, and blood cells (not shown). The muscle cells have centrally positioned nuclei and are surrounded by a t h i c k basal l a m i n a (Fig. 8). A l t h o u g h p a c k e d with microtubules and microfilaments (Fig. 8, insets), these cells do not show a striated pattern characteristic of most decapod muscle. Individual muscle cells are organized together to form flat bundles which branch (Figs. 6, 7). The peripheral bundles are oriented longitudinally (Fig. 6), while bundles which are more proximal to the lumen have a mixed pattern of organization (Fig. 7), suggesting that a wide range of contractile movements are possible. In scanning electron micrographs, the surfaces of many muscle bundles had a rough, pleated contour characteristic of contracted cells (Figs. 6 and 7). Similarly, the thin sectioned cell in Fig. 8 has a folded nucleus and pleated plasma membrane indicating it was contracting when fixed. To facilitate optimum fixation, the ovary had to be cut, and this seemed to induce some contraction in most of the ovarian walls we examined. In fact, several females had ovarian walls with such high contractile activity that small strips of cut ovary turned inside out and rounded up into a ball when placed in seawater. The ovarian wall is permeated by blood sinuses (Fig. 8) and vessels. Blood vessels in the wall are not shown but resemble those of the wall extensions depicted in Figs. 18-20. The vessels, but not the sinuses, generally are filled with a fine granular material which is fixed h e m o l y m p h (see
LOBSTER OVARY ARCHITECTURE Figs. 18, 19). B l o o d cells are p r e s e n t in b o t h the b l o o d v e s s e l s (Fig. 19) a n d s i n u s e s (Fig. 18) a n d g e n e r a l l y r e s e m b l e the cell t ~ r m e d a c o a g u l o c y t e in crayfish h e m o l y m p h (25). W h e n e v e r b l o o d cells w e r e o b s e r v e d outside of a b l o o d v e s s e l , t h e y were a t t a c h e d to a b a s a l l a m i n a (also see (26)).
The Epithelium T h e l u m i n a l s u r f a c e of the m a t u r e o v a r y is c o v e r e d b y a s h e e t of epithelial cells. W h e n e x a m i n e d with a d i s s e c t i n g micros c o p e , the e p i t h e l i u m is t r a n s l u c e n t , b a r e l y visible, a n d c o n t a i n s small white cells (imm a t u r e o o c y t e s ) s u s p e n d e d in it (Fig. 2). T h e e p i t h e l i u m is b e t t e r v i s u a l i z e d in scanning electron micrographs which reveal that it is a t h i n l a y e r that folds o u t w a r d tow a r d the p e r i p h e r a l m a r g i n s of the o v a r y (Fig. 9). T h e epithelial cells are flat, a n d a d j a c e n t cells h a v e h i g h l y i n t e r d i g i t a t e d m a r g i n s (Fig. 10). T h e cells c o n t a i n a large c e n t r a l l y
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l o c a t e d n u c l e u s , m i c r o t u b u l e s , microfilaments, mitochondria, and occasionally 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 Golgi b o d i e s ( F i g . 10; n o t all o r g a n e l l e s a r e s h o w n ) . A " n a k e d " p l a s m a m e m b r a n e is p r e s e n t o n the cell s u r f a c e facing the lum e n , while the o p p o s i t e s u r f a c e is c o v e r e d b y t h i c k b a s a l l a m i n a . W e k n o w the " n a k e d " m e m b r a n e faces l u m e n w a r d as it is c o a t e d b y g o l d / p l a t i n u m in t i s s u e p r e p a r e d for S E M (see Fig. 10). T h e p r o m i n e n t basal l a m i n a is c o m p o s e d of a n u m b e r of l a m e l l a e s t a c k e d o n top of e a c h o t h e r (Fig. 10). T h e epithelial cells are c o n t i n u o u s with the follicle cells s u r r o u n d i n g i m m a t u r e a n d mat u r e follicles a n d h a v e a s t r o n g s t r u c t u r a l r e s e m b l a n c e to follicle cells, in p a r t i c u l a r t h o s e s u r r o u n d i n g i m m a t u r e o o c y t e s (Fig. 11).
The Follicles T h e m a t u r e l o b s t e r o v a r y c o n t a i n s large (mature)-, intermediate-, and small-sized
FIG. 1. A female lobster dissected to show the mature (arrows) ovary. The lobes extend anteriorly to the front of the cephalothorax and posteriorly to the fourth abdominal segment. Beneath the heart (H) the four lobes join and at this region the oviducts (not shown) leave the ovary. Mature oocytes are visible within the ovary. FIG. 2. The luminal surface of a lobe cut open and viewed with a stereoscopic microscope. Large mature follicles (MF), which in life are green, constitute the bulk of the ovary. Many intermediate and small follicles (arrows) are also evident. These are supported in the thin translucent ovarian epithelium which is best visualized with the scanning electron microscope. In this micrograph, the existence of the epithelium may be inferred by the position of some small follicles (small arrows) which appear to float in space, x 10. FIG. 3. The outer surface of a mature lobe which was cut into a strip and photographed with a stereoscopic microscope. The cut edge of the thin, translucent wall (W) is easily seen (arrows). This strip is still relatively flat, but in some instances strips turned completely inside out due to contraction of the wall, x 10. FIG. 4. A portion of a mature lobe after removal from the lobster. One tong of the forceps has been inserted into the lumen of the ovary. × 1. FIG. 5. Scanning electron micrograph showing ovarian wall (W) and its extension after the follicles were removed. Sinuses can be seen in several regions of the wall (arrow). About × 20. Fic. 6. Scanning electron micrograph showing ovary viewed from outside looking inward. The surface of the muscle cells (wall, W) is seen in the lower left. These muscles are composed of fibers bound into long flat bands. Several mature follicles (MF) with wall extension (arrow) inserting on their surface are also visible. x 60. F~G. 7. Scanning electron micrograph of the ovarian wall viewed from inside looking outward. Muscle (M) tissue here is branched and probably in part contracted. × 60. FIG. 8. Transmission electron micrograph showing muscle cells of the ovarian wall. Each muscle cell contains microtubules and microfilaments (difficult to resolve at this low magnification, but see insets) and is surrounded by a discrete basal lamina (small arrows). These cells were partially contracted when fixed (note indentations of the nucleus at large arrows and convolutions of the cell surface). Inset a: microtubules; inset b: microfilaments. × 9000.
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follicles. The small follicles are clustered together in the epithelium lining the lumen, while mature and intermediate-sized follicles are located between this epithelium and the ovarian wall (Figs. 2, 3, 6, 9, 12, and 13). The mature follicles are dark green in color and measure 1.2-1.6 mm in diameter. The green color is due to the presence of a pigment, ovoverdin, in the mature oocyte (see (3, 24, 28)). Only the mature oocytes would be ovulated during this round of maturation, and following spawning the ovaries would be white. In cross sections through the ovary, three to five layers of mature follicles extend between the lumen and ovarian wall. Each follicle consists of an oocyte, surrounded by a " c h o r i o n " or investing coat, a single layer of follicle cells, and most peripherally a basal lamina. A detailed description of the mature follicle will be presented in the following paper (26). The intermediate-sized, immature follicles are white and range in size from 0.1 to 0.4 mm in diameter. The largest of the intermediate-sized follicles abut the surface of a mature follicle (Fig. 13). A chain or series of progressively smaller follicles extends away from the mature follicle. These follicles are interconnected by epithelial cells, and the smallest follicles (0.05-0.1 ram) of the chain join with the flat sheet of epithelium adjacent to the lumen. Both the intermediate- and small-sized follicles consist of a centrally positioned oocyte which is surrounded by thin, flat follicle cells and a thick basal lamina (Fig. 11). These follicle cells, unlike those around mature oocytes, are undifferentiated. Microtubules and mitochondria are the most conspicuous organelles of their cytoplasm; evidence of synthetic activity was not seen. Their cell boundaries are highly interdigitated, which may permit easy expansion of the follicle wall as the oocyte later enlarges. A chorion is not present around any intermediate or small follicles. The oocytes in intermediatesized follicles closest to mature follicles show evidence of primary vitellogenesis (Fig. ll).
Extensions of the Ovarian Wall A major component of the mature lobster ovary is "thread-like" extensions observed in the follicular zone of the ovary (Figs. 5, 6, 12, 14-17). The abundance and distribution of these extensions is readily appreciated by observing ovaries with the scanning electron microscope (Figs. 14-17). The extensions originate in the ovarian wall (Figs. 5, 14, 16). Figure 15 shows a major e x t e n s i o n of the wall with n u m e r o u s branching and anastomosing fibers projecting off of it. These extensions can be traced to the surface of follicles and into the epithelium where they insert (Figs. 6, 9, 12, 17). Analysis of thin sections revealed that the wall extensions are both muscle cells (Fig. 18) and blood vessels (Figs. 18-20). Hemocytes were also present both within (Fig. 19) and outside (Fig. 18) the vessels. The muscle cells appear identical to those found in the wall. They are packed with filaments and microtubules, and each cell is surrounded by a thick basal lamina (Fig. 18). Muscle cells and blood vessels may extend off the wall together (Fig. 18), or may leave the ovarian wall separately (Figs. 19 and 20 show isolated vessels; isolated muscle cells are not shown). In the blood vessel in Fig. 18, hemolymph is contained by a single layer of flat endothelial cells. These cells are interconnected by desmosome-like junctions. The blood vessels in Figs. 19 and 20 are s u r r o u n d e d by highly i n t e r d i g i t a t e d endothelial-like cells, but muscle cells similar to the one shown in Fig. 18 are not present. The walls of these blood vessels may nevertheless be contractile. The vessel in Fig. 20 has a contoured surface and the lumen has been shut down apparently by contraction. In contrast, the vessel in Fig. 19 is smooth surfaced and the vessel lumen distended. The cell labeled " H " in Fig. 19 is a hemocyte which has completely distended an adjacent arterial lumen; note that a small amount of hemolymph is visible at the periphery of the hemocyte. We often observed more than one lumen per vessel.
LOBSTER OVARY ARCHITECTURE E a c h o f t h e s e v e s s e l s ( F i g s . 19, 20) w a s f i r s t e x a m i n e d b y S E M a n d t h u s is c o a t e d w i t h an electron-dense layer of gold/platinum. DISCUSSION We have described the organization of t h e m a t u r e o v a r y o f t h e l o b s t e r , Homarus americanus. O u r m a i n o b s e r v a t i o n s a r e s u m m a r i z e d s c h e m a t i c a l l y i n F i g . 21. T h i s o r g a n i z a t i o n a l p a t t e r n is o b s e r v e d t h r o u g h out the length of the ovary. In general, these results agree with and extend those o f o t h e r s o n Homarus (2, 9, 10, 13, 19).
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M o r e o v e r , t h e Hornarus o v a r y is s i m i l a r t o t h a t o f o t h e r l o b s t e r s (Jasus, ( 7 ) ; Ne-
phrops, (6). A s is t r u e f o r m o s t c r u s t a c e a n s , t h e l o b s t e r o v a r y is r e l a t i v e l y s i m p l e i n d e s i g n . I t is c o m p l e t e l y s u r r o u n d e d by a muscular w a l l a n d c o n t a i n s a c e n t r a l l u m e n . T h e foll i c l e s lie b e t w e e n t h e m u s c l e a n d l u m e n . The fully mature follicles constitute most of the mass of mature ovaries. The musculature of the ovarian wall has several unusual features. Unlike most decapod muscle, including involuntary muscle
FIG. 9. Scanning electron micrograph showing epithelium and follicles of various size. The epithelium (E) is viewed from the lumen looking outward. Nests of small follicles are indicated by small arrows. The epithelium can be seen to invaginate (large arrows) and connect intermediate-sized follicles (IF). Mature follicles (MF) and thread-like extensions of the ovarian wall are also evident, x 36. FIG. 10. Transmission electron micrograph through the ovarian epithelium. The luminal surface of the cell layer is coated by a dense layer of gold/platinum (arrow) deposited for examination with the scanning electron microscope. The opposite surface is supported on a thick basal lamina (BL). The cells appear undifferentiated and have highly interdigitated boundaries, x 8000. FIG. 11. Transmission electron micrograph through follicle cells (FC) surrounding an immature oocyte (IO). These follicle cells appear similar to undifferentiated epithelium. Their outer surface is covered by a thick basal lamina (BL) which in this micrograph has a gold/platinum coating as the tissue was first examined with a scanning electron microscope. Adjacent cell boundaries are highly interdigitated, x 12 000. FIG. 12. Scanning electron micrograph showing ovarian epithelium, follicles of various size, and "threadlike" extensions of the ovarian wall. A nest of very small follicles is held in the epithelium (E) at arrow. Intermediate (IF) and mature (MF) follicles are also present. A vast network of thread-like projections can be observed in the vicinity of the mature follicles. The projections originate in the ovarian wall and attach to the epithelium and follicles of all sizes, x 58. Fx6. 13. A "chain" of follicles. A mature follicle (MF) appears adjacent to the ovarian wall (W), barely visible in the right of the micrograph. Two follicles of intermediate size (IF) attach to the mature follicle. Presumably one of these IF will mature in the next round of ovarian development. Progressively smaller follicles are linked together forming a "chain" between the mature follicle and epithelium lining the lumen (not shown). Portions of epithelium have invaginated from the luminal surface and connect the follicles of the chain together (see arrows), x 48. FIGS. 14-17. Scanning electron micrographs showing extensions of the ovarian wall. In Fig. 14 the follicles were removed; the view is from the lumen looking outward. Muscl~ cells (MC) of the wall and numerous extensions are evident. Figure 15 shows one major wall extension from which numerous smaller extensions branch and anastomose. Figure 16 is a high magnification showing junctions between the ovarian wall (W) and wall extensions (WE). These particular projections appear to be flat bands of muscle. The small spheres are yolk which fell out of oocytes during preparation. Figure 17 shows the surface of a mature follicle with attachment of numerous wall extensions. Here both flat bands of tissue and one strand which appears contracted (arrow) rest on the surface of the follicle. Fig. 14, x 100; Fig. 15, x 125; Fig. 16, × 600; Fig. 17, x 650. FIG. 18. Transmission micrograph of an extension from the ovarian wall. This section was taken close to the wall. Here two muscle cells (MC) each surrounded by a thick basal lamina (arrows) are seen packed with microfilaments and microtubules. A hemocyte (H) appears between the muscle cell and lining of a blood vessel (BV). Hemolymph (HE) is evident with the vessel, z 10 000. FrGs. 19 AND 20. Cross sections through two extensions of the ovarian wall; both are blood vessels. In Fig. 19, two lumens are present (this was commonly seen). One is empty, while the other contains a hemocyte (H); a bit of hemolymph is visible at the lower fight (arrow) of the hemocyte. Figure 20 is a blood vessel which appears contracted. Its surface is folded and the lumen (arrow) has been occluded. Fig. 19, x 4000; Fig. 20, x 8000.
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(5, 11), it is not striated. Upon initial inspection, it appears to resemble smooth muscle of mammals (22); however, it differs markedly from smooth muscle in having an abundance of cytoplasmic microtubules. It is likely that the contraction of ovarian muscles which we observed in vitro in living cells in our micrographs of fixed cells was due at least in part to these microtubules. To the best of our knowledge, this muscle is unique, inasmuch as it has such a high concentration of microtubules and in that these microtubules appear to function in muscular contraction. Further investigation of this point would be useful in understanding both microtubule function and muscle physiology. It is likely, as Herrick (10) suggested, that such muscle contraction moves ovulated oocytes through the lumen of the ovary and into the oviduct. Thus one function of this muscle layer would be to promote spawning. We also suggest that cont r a c t i o n o f t h e s e m u s c l e cells m a y be important in circulating hemolymph through ovarian blood vessels and sinuses. In particular, blood which collects in the sinuses could be forced along to the pericardium by continual, gentle contraction of the ovarian wall musculature. A new finding of this study is that muscle cells branch off the ovarian wall and form a thread-like network in the follicular region of the ovary. Structurally, these extensions resemble the muscles of the ovarian wall. In thin sections, we have seen muscle cells atop the walls of mature follicles where they insert. The presence of muscle insertion into the follicles may be significant in o v u l a t i o n which in o t h e r species involves contraction of cells surrounding follicles (e.g., (12, 14, 16, 20)). The blood vessels and sinuses of the ovarian wall were reported previously (2, 10), but the vast collection of vessels in the follicular zone has not been completely recognized previously. Herrick (10) has reported that blood leaves the vessels of the wall and permeates into the follicular por-
tion of the ovary via sinuses. H o w e v e r , the many vessels in the follicular zone of the ovary and the presence of coagulated hemolymph in these vessels indicates that blood flow within the follicular zone of the lobster ovary is at least partially intravascular. Considering the importance of hemolymph in crustacean vitellogenesis (reviewed by (17)), it would be useful in future investigations to determine the precise path of h e m o l y m p h flow in the c r u s t a c e a n ovary. It is also noteworthy that blood vessels in the follicular zone appeared contractile. The contractility could be important in movement of hemolymph through vessels or may restrict hemolymph flow to follicles in the process of rupturing. Blood cells were observed both in and out of vessels. When outside of vessels, they were always attached to a basal lamina. It is possible t h e s e e x t r a v a s c u l a r h e m o c y t e s are involved in repair or formation of the extensive basal lamina system within the ovary, especially since some ovarian cells (muscle) do not appear well equipped for this activity. The extensions of the ovarian wall (both the vessels and muscles) are best visualized with the scanning e l e c t r o n m i c r o s c o p e . H o w e v e r , since we are not able to distinguish between blood vessels and muscles with the technique, we cannot yet precisely catalog the distribution of each extension in the follicular region. Thus, the representation of the extensions presented in Fig. 21 is intended to give a tentative, not necessarily a complete, idea of their organization. The mature follicles abut the ovarian wall and extend in three to five tiers to the lumen. Intermediate follicles are found adjacent to mature follicles, and the smallest follicles of the ovary are near the lumen. This type of arrangement has been noted in several crustaceans (e.g. (15, 27)) and is simpler than the design usually seen among insects (5, 21). A detailed discussion of the mature follicle is given in the following paper.
LOBSTER OVARY ARCHITECTURE H
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( "\ Flo. 21. Schematic diagram of mature lobster ovary. The relationship between the ovarian wall (W), mature follicles (MF), epithelium (E), and extension of the wall (WE) is shown. The extensions of the wall consist of both blood vessels and muscle cells. We do not yet know the exact distribution of each of these. This diagram therefore represents a tentative scheme for blood vessel and muscle distribution in the follicular zone. m = muscle cells; IF = intermediate-sized follicles; SF = small-sized follicles; H = hemocytes; and BV = blood vessels.
The epithelial cells form the innermost lining of the ovary. Little is known about their origin or function. Herrick (10) has reported glandular epithelium near the wall of less mature ovaries. We did not observe such epithelium, but it is possible that ovarian epithelium, like the follicular epithelium (see (26)), goes through cyclic activity. In mature ovaries, it appears relatively quiescent. The epithelial cells also bear a strong structural resemblance to follicle cells, especially those surrounding immature follicles. Thus, they may give rise to follicle cells. Herrick (10) reported that immature oocytes also originate from epithelial cells, although we have no evidence to either support or contradict his observation. The mature lobster ovary is remarkable for its lack of extracellular connective tissue elements. The well-developed basal lamina which subtends the epithelium and surrounds the follicles and muscle cells appears to be the main connective tissue component of the ovary. Its primary functions are probably to support the various tissues
and to maintain structural organization within the o v a r y . W e l l - d e v e l o p e d basal lamina similar to this have been previously reported in other crustacean (18) and insect (21) ovaries. Our purpose has been to present an overview of the organization of mature ovaries from the lobster. To the best of our knowledge, this is the first complete ultrastructural analysis of ovaries from any decapod. Considering the current interest in decapod reproductive p h y s i o l o g y (see reviews by (1, 4, 17)), we hope this structural analysis will be helpful to many investigators working with crustaceans. Our descriptions will also provide a basis for subsequent detailed analysis of each region of the lobster ovary and for the interpretation of ovarian processes such as ovulation. In the following paper, the structure of the mature follicle is analyzed in detail. Supported by a Sea grant from NOAA, an Intercampus Travel grant from the University of California, and a Career Development Award from NIH.
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1. AIKEN, D. E., AND WADDY, S. (1980) in The Biology and Management of Lobsters, Vol. 1, p. 215, Academic Press, New York. 2. BuMPUS, H. C. (1891) J. Morphol. 5, 215. 3. CECCALDI, H. J., CHEESEMAN, D. F., AND ZAGALSKY, P. F. (1966) C. R. Soc. Biol. 160, 587. 4. CrtARNIAux-CoTTON, H. (1978) Arch. Zool. Exp. Gen. 119, 365. 5. CLARKE, K. U. (1963) Biology of the Arthropoda, Clowes, London. 6. FARMER, A. S. D. (1974) J. Zool. Lond. 174, 161. 7. FIELDER, D. R. (1964) Austr. J. Mar. Freshw. Res. 15, 133. 8. HEDGECOCK, D., MOFFET, W. L., BORGESON, W., AND NELSON, K. (1978) Proceedings of the 9th Annual Meeting of the World Mariculture Society, p. 497. 9. HERRICK, F. H. (1895) Bull. U.S. Fish. Commission, p. 1. U.S. Govt. Printing Office, Washington, D.C. 10. HERRICK, F. H. (1911) Bull. Fish. 29, 149. 11. HUXLEY, T. H. (1880) The Crayfish, Appleton, New York. 12. JAEBERT, B., AND SZOLLOZI, D. (1975) Prostaglandins 9, 765. 13. KESSEL, R. G. (1968) Z. Zellforsch. 89, 17.
14. LARSEN, J. H., SCHROEDER, P. C., AND WALDO, A. E. (1977) Cell Tissue Res. 181, 505. 15. LINDER, H. J. (1959) J. Morphol. 104, I. 16. MARTIN, G. G., AND TALBOT, P. (1981) J. Exp. Zool. 216, 469. 17. MEUSY, J. J. (1980) Reprod. Nutr. Develop. 20, 1. 18. RATEAU, J., AND ZERBIB, C. (1978) C. R. Acad. Sci. 286, 65. 19. SCHADE, M. L., AND SHIVERS, R. R. (1980) J. MorphoL 163, 13. 20. SCHROEDER, P. (1971) Naturinissenschaften 58, 270. 21. SMITH, D. S. (1968) Insect Cells, p. 337, Oliver & Boyd, Edinburgh. 22. SOMLYO, A. P., AND SOMLYO, A. V. (1977) Vol. 3. Plenum, New York. 23. SPURR, A. R. (1969) J. Ultrastruct. Res. 26, 31. 24. STERN, K. G., AND SALOMAN, K. (1937) Science 86, 310. 25. STERNSTEIN, D. J., AND BURTON, P. R. (1980) J. Morphol. 165, 67. 26. TALBOT, P. (1981) J. Ultrastruct. Res. 76, 249. 27. WORSMANN,T. U., BARCELLOS, S. R., AND A. G. FERRI (1976) Bolm Inst. Oceanogr. S. Paulo p. 43. 28. WYCKOFE, R. W. G. (1937) Science 86, 311.
76, 235-248 (1981)
The Ovary of the Lobster, Homarus americanus I. Architecture of the Mature Ovary PRUDENCE TALBOT 1
Department of Biology, University of California, Riverside, California 92521, and Bodega Marine Laboratory, Bodega Bay, California 94923 Received April 7, 1981 We have examined the architecture of the mature lobster (Homarus americanus) ovary using microscopical techniques. The ovary is comprised of five tubular lobes which are organized in an H-shaped pattern. Each lobe contains four morphological components which are: (i) the ovarian wall, (ii) the epithelium, (iii) the follicles, and (iv) the extensions of the ovarian wall. The wall is made up of blood vessels and sinuses, blood cells, and nonstriated muscle cells which may undergo spontaneous contraction when the ovary is cut. The epithelial cells are flat and rest on a thick basal lamina. They form a thin seet on the luminal surface of the ovary. Portions of this sheet fold outward toward the ovarian wall. Epithelial cells are continuous with and may give rise to follicle cells around oocytes. The mature ovary contains large (mature, 1.2-1.6 mm)-, intermediate (0.10.4 ram)-, and small (0.07-0.1 mm)-sized follicles. Small follicles and some intermediate follicles are found embedded in the epithelium lining the lumen. Most intermediate follicles form chains linking the luminal epithelium to mature follicles. The mature follicles are stacked in tiers three to five follicle layers deep between the ovarian wall and the lumen. The extensions of the ovarian wall consist of both blood vessels and muscle cells; these project into the follicular region and insert on follicle walls and on the epithelium. This description of the mature lobster ovary extends our knowledge on the organization of the decapod ovary and provides a basis for future detailed structural and physiological analyses of this organ.
Apart from several histological examinations (2, 9, 10), and two recent ultrastructural studies of vitellogenesis (13, 19), very little work has been done on the structure of ovaries in the lobster, Homarus americanus. In order to understand ovarian processes, such as o o c y t e development', ovulation, and spawning, it is necessary to have a complete knowledge of the structure of this organ. We have therefore used light and electron microscopy to examine the morphology of lobster ovaries. The purpose of this report is to present an overview of the architecture of mature lobster (Homarus americanus) ovaries at the ultrastructural level. This will provide a framework for subsequent, more detailed descriptions of various components of the lobster ovary.
1 Present address: Department of Biology, University of California, Riverside, Calif. 92521.
MATERIALS AND METHODS Female lobsters (Homarus arnericanus) were obtained between July and September and candled (8) to estimate the degree of ovarian maturity. Ten females Were found to have mature ovaries which were close to natural ovulation. While it is not yet possible to predict precisely when lobsters will ovulate, these ovaries appeared to be in late stage 5 or stage 6 (ripe) of development according to the classification scheme proposed by Aiken and Waddy (1). These females were used in this and the following study. Lobsters were sacrificed and dissected to observe the anatomical features of the mature ovary. Pieces of ovary were then removed for study with light and electron microscopy. General features of ovarian organization were assessed using both a stereoscopic and a phase contrast microscope. For more detailed cytological analysis, tissue was fixed for 3 hr in a solution containing 3% glutaraldehyde, 12% glucose, and 0.1 M sodium cacodylate (pH 7.4). Following several rinses in 0.1 M cacodylate with 24% sucrose, tissue was postfixed for 1-2 hr in 1% OsO4 in 0.1 M cacodylate with 24% sucrose. Tissue was then washed in a 0.1 M cacodylate series containing 24, 12, or 6% sucrose, and dehydrated in a graded series of acetone. For light microscopy and transmission electron microscopy, 235 0022-5320/81/090235-14502.00/0 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
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tissue was infiltrated overnight in Spurr's plastic/acetone (1:1), e m b e d d e d in Spurr's plastic (23), sectioned on a P o r t e r - B l u m MT-2B ultramicrotome, and stained with toluidine blue (thick sections) or lead and uraniu m salts (thin sections). Thick sections were examined with a Zeiss Standard light microscope; thin sections were viewed using a Hitachi H-500 transmission electron microscope. For scanning electron microscopy, pieces of ovary were fixed and dehydrated as described above. Specimens in 100% acetone were critically point dried in a Samdri PVT-3 apparatus, m o u n t e d on a l u m i n u m pedestals, coated with gold and platinum in a Technics H u m m e r II, and studied with a Jeol JSM-35C scanning electron microscope. In s o m e cases, tissue which had been e x a m i n e d with the scanning electron microscope was placed in 100% a c e t o n e , infiltrated and e m b e d d e d in plastic, sectioned, stained, and e x a m i n e d with the Hitachi H-500 electron microscope as previously described.
RESULTS
General Features of the Mature Ovary Homarus ovaries are comprised of five lobes which are united in an H-shaped pattern. The ovaries are positioned dorsally and extend from the anterior-most region of the cephalothorax to the third, fourth, or fifth abdominal segment (Fig. 1). The lobe forming the cross segment of the H lies ventral to the heart, and it is at this region that the oviducts leave the ovary and extend ventrally to the gonopores at the base of the third walking leg. Each lobe of the ovary is tubular. The lumen of the tube is demonstrated in Fig. 4 which shows the tip of a pair of forceps inserted into the center of a transected lobe. The ovary is dark green in color and usually contains a small number of yellow or orange spots, presumed to be either atretic or degenerating follicles. T h e s e are scattered r a n d o m l y throughout the ovary, and in one female were quite numerous. Lobes of ovaries were cut open to form flat strips (Figs. 2 and 3) and were examined by light and electron microscopy. These observations revealed that lobster ovaries are comprised of four distinct components: (i) the ovarian wall, (ii) the epithelium, (iii) the follicles (mature and immature), and
(iv) the extensions of the ovarian wall. Each of these components and their relationships to each other will next be considered in greater detail.
The Ovarian Wall In living ovaries, the ovarian wall is a pale white, translucent layer which is best seen after the ovary has been cut into strips (Figs. 3 and 5). The tissue of the wall is densely packed (Figs. 6-8) and is composed of long thin muscle cells, blood sinuses (Fig. 8), blood vessels, and blood cells (not shown). The muscle cells have centrally positioned nuclei and are surrounded by a t h i c k basal l a m i n a (Fig. 8). A l t h o u g h p a c k e d with microtubules and microfilaments (Fig. 8, insets), these cells do not show a striated pattern characteristic of most decapod muscle. Individual muscle cells are organized together to form flat bundles which branch (Figs. 6, 7). The peripheral bundles are oriented longitudinally (Fig. 6), while bundles which are more proximal to the lumen have a mixed pattern of organization (Fig. 7), suggesting that a wide range of contractile movements are possible. In scanning electron micrographs, the surfaces of many muscle bundles had a rough, pleated contour characteristic of contracted cells (Figs. 6 and 7). Similarly, the thin sectioned cell in Fig. 8 has a folded nucleus and pleated plasma membrane indicating it was contracting when fixed. To facilitate optimum fixation, the ovary had to be cut, and this seemed to induce some contraction in most of the ovarian walls we examined. In fact, several females had ovarian walls with such high contractile activity that small strips of cut ovary turned inside out and rounded up into a ball when placed in seawater. The ovarian wall is permeated by blood sinuses (Fig. 8) and vessels. Blood vessels in the wall are not shown but resemble those of the wall extensions depicted in Figs. 18-20. The vessels, but not the sinuses, generally are filled with a fine granular material which is fixed h e m o l y m p h (see
LOBSTER OVARY ARCHITECTURE Figs. 18, 19). B l o o d cells are p r e s e n t in b o t h the b l o o d v e s s e l s (Fig. 19) a n d s i n u s e s (Fig. 18) a n d g e n e r a l l y r e s e m b l e the cell t ~ r m e d a c o a g u l o c y t e in crayfish h e m o l y m p h (25). W h e n e v e r b l o o d cells w e r e o b s e r v e d outside of a b l o o d v e s s e l , t h e y were a t t a c h e d to a b a s a l l a m i n a (also see (26)).
The Epithelium T h e l u m i n a l s u r f a c e of the m a t u r e o v a r y is c o v e r e d b y a s h e e t of epithelial cells. W h e n e x a m i n e d with a d i s s e c t i n g micros c o p e , the e p i t h e l i u m is t r a n s l u c e n t , b a r e l y visible, a n d c o n t a i n s small white cells (imm a t u r e o o c y t e s ) s u s p e n d e d in it (Fig. 2). T h e e p i t h e l i u m is b e t t e r v i s u a l i z e d in scanning electron micrographs which reveal that it is a t h i n l a y e r that folds o u t w a r d tow a r d the p e r i p h e r a l m a r g i n s of the o v a r y (Fig. 9). T h e epithelial cells are flat, a n d a d j a c e n t cells h a v e h i g h l y i n t e r d i g i t a t e d m a r g i n s (Fig. 10). T h e cells c o n t a i n a large c e n t r a l l y
237
l o c a t e d n u c l e u s , m i c r o t u b u l e s , microfilaments, mitochondria, and occasionally 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 Golgi b o d i e s ( F i g . 10; n o t all o r g a n e l l e s a r e s h o w n ) . A " n a k e d " p l a s m a m e m b r a n e is p r e s e n t o n the cell s u r f a c e facing the lum e n , while the o p p o s i t e s u r f a c e is c o v e r e d b y t h i c k b a s a l l a m i n a . W e k n o w the " n a k e d " m e m b r a n e faces l u m e n w a r d as it is c o a t e d b y g o l d / p l a t i n u m in t i s s u e p r e p a r e d for S E M (see Fig. 10). T h e p r o m i n e n t basal l a m i n a is c o m p o s e d of a n u m b e r of l a m e l l a e s t a c k e d o n top of e a c h o t h e r (Fig. 10). T h e epithelial cells are c o n t i n u o u s with the follicle cells s u r r o u n d i n g i m m a t u r e a n d mat u r e follicles a n d h a v e a s t r o n g s t r u c t u r a l r e s e m b l a n c e to follicle cells, in p a r t i c u l a r t h o s e s u r r o u n d i n g i m m a t u r e o o c y t e s (Fig. 11).
The Follicles T h e m a t u r e l o b s t e r o v a r y c o n t a i n s large (mature)-, intermediate-, and small-sized
FIG. 1. A female lobster dissected to show the mature (arrows) ovary. The lobes extend anteriorly to the front of the cephalothorax and posteriorly to the fourth abdominal segment. Beneath the heart (H) the four lobes join and at this region the oviducts (not shown) leave the ovary. Mature oocytes are visible within the ovary. FIG. 2. The luminal surface of a lobe cut open and viewed with a stereoscopic microscope. Large mature follicles (MF), which in life are green, constitute the bulk of the ovary. Many intermediate and small follicles (arrows) are also evident. These are supported in the thin translucent ovarian epithelium which is best visualized with the scanning electron microscope. In this micrograph, the existence of the epithelium may be inferred by the position of some small follicles (small arrows) which appear to float in space, x 10. FIG. 3. The outer surface of a mature lobe which was cut into a strip and photographed with a stereoscopic microscope. The cut edge of the thin, translucent wall (W) is easily seen (arrows). This strip is still relatively flat, but in some instances strips turned completely inside out due to contraction of the wall, x 10. FIG. 4. A portion of a mature lobe after removal from the lobster. One tong of the forceps has been inserted into the lumen of the ovary. × 1. FIG. 5. Scanning electron micrograph showing ovarian wall (W) and its extension after the follicles were removed. Sinuses can be seen in several regions of the wall (arrow). About × 20. Fic. 6. Scanning electron micrograph showing ovary viewed from outside looking inward. The surface of the muscle cells (wall, W) is seen in the lower left. These muscles are composed of fibers bound into long flat bands. Several mature follicles (MF) with wall extension (arrow) inserting on their surface are also visible. x 60. F~G. 7. Scanning electron micrograph of the ovarian wall viewed from inside looking outward. Muscle (M) tissue here is branched and probably in part contracted. × 60. FIG. 8. Transmission electron micrograph showing muscle cells of the ovarian wall. Each muscle cell contains microtubules and microfilaments (difficult to resolve at this low magnification, but see insets) and is surrounded by a discrete basal lamina (small arrows). These cells were partially contracted when fixed (note indentations of the nucleus at large arrows and convolutions of the cell surface). Inset a: microtubules; inset b: microfilaments. × 9000.
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follicles. The small follicles are clustered together in the epithelium lining the lumen, while mature and intermediate-sized follicles are located between this epithelium and the ovarian wall (Figs. 2, 3, 6, 9, 12, and 13). The mature follicles are dark green in color and measure 1.2-1.6 mm in diameter. The green color is due to the presence of a pigment, ovoverdin, in the mature oocyte (see (3, 24, 28)). Only the mature oocytes would be ovulated during this round of maturation, and following spawning the ovaries would be white. In cross sections through the ovary, three to five layers of mature follicles extend between the lumen and ovarian wall. Each follicle consists of an oocyte, surrounded by a " c h o r i o n " or investing coat, a single layer of follicle cells, and most peripherally a basal lamina. A detailed description of the mature follicle will be presented in the following paper (26). The intermediate-sized, immature follicles are white and range in size from 0.1 to 0.4 mm in diameter. The largest of the intermediate-sized follicles abut the surface of a mature follicle (Fig. 13). A chain or series of progressively smaller follicles extends away from the mature follicle. These follicles are interconnected by epithelial cells, and the smallest follicles (0.05-0.1 ram) of the chain join with the flat sheet of epithelium adjacent to the lumen. Both the intermediate- and small-sized follicles consist of a centrally positioned oocyte which is surrounded by thin, flat follicle cells and a thick basal lamina (Fig. 11). These follicle cells, unlike those around mature oocytes, are undifferentiated. Microtubules and mitochondria are the most conspicuous organelles of their cytoplasm; evidence of synthetic activity was not seen. Their cell boundaries are highly interdigitated, which may permit easy expansion of the follicle wall as the oocyte later enlarges. A chorion is not present around any intermediate or small follicles. The oocytes in intermediatesized follicles closest to mature follicles show evidence of primary vitellogenesis (Fig. ll).
Extensions of the Ovarian Wall A major component of the mature lobster ovary is "thread-like" extensions observed in the follicular zone of the ovary (Figs. 5, 6, 12, 14-17). The abundance and distribution of these extensions is readily appreciated by observing ovaries with the scanning electron microscope (Figs. 14-17). The extensions originate in the ovarian wall (Figs. 5, 14, 16). Figure 15 shows a major e x t e n s i o n of the wall with n u m e r o u s branching and anastomosing fibers projecting off of it. These extensions can be traced to the surface of follicles and into the epithelium where they insert (Figs. 6, 9, 12, 17). Analysis of thin sections revealed that the wall extensions are both muscle cells (Fig. 18) and blood vessels (Figs. 18-20). Hemocytes were also present both within (Fig. 19) and outside (Fig. 18) the vessels. The muscle cells appear identical to those found in the wall. They are packed with filaments and microtubules, and each cell is surrounded by a thick basal lamina (Fig. 18). Muscle cells and blood vessels may extend off the wall together (Fig. 18), or may leave the ovarian wall separately (Figs. 19 and 20 show isolated vessels; isolated muscle cells are not shown). In the blood vessel in Fig. 18, hemolymph is contained by a single layer of flat endothelial cells. These cells are interconnected by desmosome-like junctions. The blood vessels in Figs. 19 and 20 are s u r r o u n d e d by highly i n t e r d i g i t a t e d endothelial-like cells, but muscle cells similar to the one shown in Fig. 18 are not present. The walls of these blood vessels may nevertheless be contractile. The vessel in Fig. 20 has a contoured surface and the lumen has been shut down apparently by contraction. In contrast, the vessel in Fig. 19 is smooth surfaced and the vessel lumen distended. The cell labeled " H " in Fig. 19 is a hemocyte which has completely distended an adjacent arterial lumen; note that a small amount of hemolymph is visible at the periphery of the hemocyte. We often observed more than one lumen per vessel.
LOBSTER OVARY ARCHITECTURE E a c h o f t h e s e v e s s e l s ( F i g s . 19, 20) w a s f i r s t e x a m i n e d b y S E M a n d t h u s is c o a t e d w i t h an electron-dense layer of gold/platinum. DISCUSSION We have described the organization of t h e m a t u r e o v a r y o f t h e l o b s t e r , Homarus americanus. O u r m a i n o b s e r v a t i o n s a r e s u m m a r i z e d s c h e m a t i c a l l y i n F i g . 21. T h i s o r g a n i z a t i o n a l p a t t e r n is o b s e r v e d t h r o u g h out the length of the ovary. In general, these results agree with and extend those o f o t h e r s o n Homarus (2, 9, 10, 13, 19).
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M o r e o v e r , t h e Hornarus o v a r y is s i m i l a r t o t h a t o f o t h e r l o b s t e r s (Jasus, ( 7 ) ; Ne-
phrops, (6). A s is t r u e f o r m o s t c r u s t a c e a n s , t h e l o b s t e r o v a r y is r e l a t i v e l y s i m p l e i n d e s i g n . I t is c o m p l e t e l y s u r r o u n d e d by a muscular w a l l a n d c o n t a i n s a c e n t r a l l u m e n . T h e foll i c l e s lie b e t w e e n t h e m u s c l e a n d l u m e n . The fully mature follicles constitute most of the mass of mature ovaries. The musculature of the ovarian wall has several unusual features. Unlike most decapod muscle, including involuntary muscle
FIG. 9. Scanning electron micrograph showing epithelium and follicles of various size. The epithelium (E) is viewed from the lumen looking outward. Nests of small follicles are indicated by small arrows. The epithelium can be seen to invaginate (large arrows) and connect intermediate-sized follicles (IF). Mature follicles (MF) and thread-like extensions of the ovarian wall are also evident, x 36. FIG. 10. Transmission electron micrograph through the ovarian epithelium. The luminal surface of the cell layer is coated by a dense layer of gold/platinum (arrow) deposited for examination with the scanning electron microscope. The opposite surface is supported on a thick basal lamina (BL). The cells appear undifferentiated and have highly interdigitated boundaries, x 8000. FIG. 11. Transmission electron micrograph through follicle cells (FC) surrounding an immature oocyte (IO). These follicle cells appear similar to undifferentiated epithelium. Their outer surface is covered by a thick basal lamina (BL) which in this micrograph has a gold/platinum coating as the tissue was first examined with a scanning electron microscope. Adjacent cell boundaries are highly interdigitated, x 12 000. FIG. 12. Scanning electron micrograph showing ovarian epithelium, follicles of various size, and "threadlike" extensions of the ovarian wall. A nest of very small follicles is held in the epithelium (E) at arrow. Intermediate (IF) and mature (MF) follicles are also present. A vast network of thread-like projections can be observed in the vicinity of the mature follicles. The projections originate in the ovarian wall and attach to the epithelium and follicles of all sizes, x 58. Fx6. 13. A "chain" of follicles. A mature follicle (MF) appears adjacent to the ovarian wall (W), barely visible in the right of the micrograph. Two follicles of intermediate size (IF) attach to the mature follicle. Presumably one of these IF will mature in the next round of ovarian development. Progressively smaller follicles are linked together forming a "chain" between the mature follicle and epithelium lining the lumen (not shown). Portions of epithelium have invaginated from the luminal surface and connect the follicles of the chain together (see arrows), x 48. FIGS. 14-17. Scanning electron micrographs showing extensions of the ovarian wall. In Fig. 14 the follicles were removed; the view is from the lumen looking outward. Muscl~ cells (MC) of the wall and numerous extensions are evident. Figure 15 shows one major wall extension from which numerous smaller extensions branch and anastomose. Figure 16 is a high magnification showing junctions between the ovarian wall (W) and wall extensions (WE). These particular projections appear to be flat bands of muscle. The small spheres are yolk which fell out of oocytes during preparation. Figure 17 shows the surface of a mature follicle with attachment of numerous wall extensions. Here both flat bands of tissue and one strand which appears contracted (arrow) rest on the surface of the follicle. Fig. 14, x 100; Fig. 15, x 125; Fig. 16, × 600; Fig. 17, x 650. FIG. 18. Transmission micrograph of an extension from the ovarian wall. This section was taken close to the wall. Here two muscle cells (MC) each surrounded by a thick basal lamina (arrows) are seen packed with microfilaments and microtubules. A hemocyte (H) appears between the muscle cell and lining of a blood vessel (BV). Hemolymph (HE) is evident with the vessel, z 10 000. FrGs. 19 AND 20. Cross sections through two extensions of the ovarian wall; both are blood vessels. In Fig. 19, two lumens are present (this was commonly seen). One is empty, while the other contains a hemocyte (H); a bit of hemolymph is visible at the lower fight (arrow) of the hemocyte. Figure 20 is a blood vessel which appears contracted. Its surface is folded and the lumen (arrow) has been occluded. Fig. 19, x 4000; Fig. 20, x 8000.
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(5, 11), it is not striated. Upon initial inspection, it appears to resemble smooth muscle of mammals (22); however, it differs markedly from smooth muscle in having an abundance of cytoplasmic microtubules. It is likely that the contraction of ovarian muscles which we observed in vitro in living cells in our micrographs of fixed cells was due at least in part to these microtubules. To the best of our knowledge, this muscle is unique, inasmuch as it has such a high concentration of microtubules and in that these microtubules appear to function in muscular contraction. Further investigation of this point would be useful in understanding both microtubule function and muscle physiology. It is likely, as Herrick (10) suggested, that such muscle contraction moves ovulated oocytes through the lumen of the ovary and into the oviduct. Thus one function of this muscle layer would be to promote spawning. We also suggest that cont r a c t i o n o f t h e s e m u s c l e cells m a y be important in circulating hemolymph through ovarian blood vessels and sinuses. In particular, blood which collects in the sinuses could be forced along to the pericardium by continual, gentle contraction of the ovarian wall musculature. A new finding of this study is that muscle cells branch off the ovarian wall and form a thread-like network in the follicular region of the ovary. Structurally, these extensions resemble the muscles of the ovarian wall. In thin sections, we have seen muscle cells atop the walls of mature follicles where they insert. The presence of muscle insertion into the follicles may be significant in o v u l a t i o n which in o t h e r species involves contraction of cells surrounding follicles (e.g., (12, 14, 16, 20)). The blood vessels and sinuses of the ovarian wall were reported previously (2, 10), but the vast collection of vessels in the follicular zone has not been completely recognized previously. Herrick (10) has reported that blood leaves the vessels of the wall and permeates into the follicular por-
tion of the ovary via sinuses. H o w e v e r , the many vessels in the follicular zone of the ovary and the presence of coagulated hemolymph in these vessels indicates that blood flow within the follicular zone of the lobster ovary is at least partially intravascular. Considering the importance of hemolymph in crustacean vitellogenesis (reviewed by (17)), it would be useful in future investigations to determine the precise path of h e m o l y m p h flow in the c r u s t a c e a n ovary. It is also noteworthy that blood vessels in the follicular zone appeared contractile. The contractility could be important in movement of hemolymph through vessels or may restrict hemolymph flow to follicles in the process of rupturing. Blood cells were observed both in and out of vessels. When outside of vessels, they were always attached to a basal lamina. It is possible t h e s e e x t r a v a s c u l a r h e m o c y t e s are involved in repair or formation of the extensive basal lamina system within the ovary, especially since some ovarian cells (muscle) do not appear well equipped for this activity. The extensions of the ovarian wall (both the vessels and muscles) are best visualized with the scanning e l e c t r o n m i c r o s c o p e . H o w e v e r , since we are not able to distinguish between blood vessels and muscles with the technique, we cannot yet precisely catalog the distribution of each extension in the follicular region. Thus, the representation of the extensions presented in Fig. 21 is intended to give a tentative, not necessarily a complete, idea of their organization. The mature follicles abut the ovarian wall and extend in three to five tiers to the lumen. Intermediate follicles are found adjacent to mature follicles, and the smallest follicles of the ovary are near the lumen. This type of arrangement has been noted in several crustaceans (e.g. (15, 27)) and is simpler than the design usually seen among insects (5, 21). A detailed discussion of the mature follicle is given in the following paper.
LOBSTER OVARY ARCHITECTURE H
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( "\ Flo. 21. Schematic diagram of mature lobster ovary. The relationship between the ovarian wall (W), mature follicles (MF), epithelium (E), and extension of the wall (WE) is shown. The extensions of the wall consist of both blood vessels and muscle cells. We do not yet know the exact distribution of each of these. This diagram therefore represents a tentative scheme for blood vessel and muscle distribution in the follicular zone. m = muscle cells; IF = intermediate-sized follicles; SF = small-sized follicles; H = hemocytes; and BV = blood vessels.
The epithelial cells form the innermost lining of the ovary. Little is known about their origin or function. Herrick (10) has reported glandular epithelium near the wall of less mature ovaries. We did not observe such epithelium, but it is possible that ovarian epithelium, like the follicular epithelium (see (26)), goes through cyclic activity. In mature ovaries, it appears relatively quiescent. The epithelial cells also bear a strong structural resemblance to follicle cells, especially those surrounding immature follicles. Thus, they may give rise to follicle cells. Herrick (10) reported that immature oocytes also originate from epithelial cells, although we have no evidence to either support or contradict his observation. The mature lobster ovary is remarkable for its lack of extracellular connective tissue elements. The well-developed basal lamina which subtends the epithelium and surrounds the follicles and muscle cells appears to be the main connective tissue component of the ovary. Its primary functions are probably to support the various tissues
and to maintain structural organization within the o v a r y . W e l l - d e v e l o p e d basal lamina similar to this have been previously reported in other crustacean (18) and insect (21) ovaries. Our purpose has been to present an overview of the organization of mature ovaries from the lobster. To the best of our knowledge, this is the first complete ultrastructural analysis of ovaries from any decapod. Considering the current interest in decapod reproductive p h y s i o l o g y (see reviews by (1, 4, 17)), we hope this structural analysis will be helpful to many investigators working with crustaceans. Our descriptions will also provide a basis for subsequent detailed analysis of each region of the lobster ovary and for the interpretation of ovarian processes such as ovulation. In the following paper, the structure of the mature follicle is analyzed in detail. Supported by a Sea grant from NOAA, an Intercampus Travel grant from the University of California, and a Career Development Award from NIH.
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PRUDENCE TALBOT REFERENCES
1. AIKEN, D. E., AND WADDY, S. (1980) in The Biology and Management of Lobsters, Vol. 1, p. 215, Academic Press, New York. 2. BuMPUS, H. C. (1891) J. Morphol. 5, 215. 3. CECCALDI, H. J., CHEESEMAN, D. F., AND ZAGALSKY, P. F. (1966) C. R. Soc. Biol. 160, 587. 4. CrtARNIAux-CoTTON, H. (1978) Arch. Zool. Exp. Gen. 119, 365. 5. CLARKE, K. U. (1963) Biology of the Arthropoda, Clowes, London. 6. FARMER, A. S. D. (1974) J. Zool. Lond. 174, 161. 7. FIELDER, D. R. (1964) Austr. J. Mar. Freshw. Res. 15, 133. 8. HEDGECOCK, D., MOFFET, W. L., BORGESON, W., AND NELSON, K. (1978) Proceedings of the 9th Annual Meeting of the World Mariculture Society, p. 497. 9. HERRICK, F. H. (1895) Bull. U.S. Fish. Commission, p. 1. U.S. Govt. Printing Office, Washington, D.C. 10. HERRICK, F. H. (1911) Bull. Fish. 29, 149. 11. HUXLEY, T. H. (1880) The Crayfish, Appleton, New York. 12. JAEBERT, B., AND SZOLLOZI, D. (1975) Prostaglandins 9, 765. 13. KESSEL, R. G. (1968) Z. Zellforsch. 89, 17.
14. LARSEN, J. H., SCHROEDER, P. C., AND WALDO, A. E. (1977) Cell Tissue Res. 181, 505. 15. LINDER, H. J. (1959) J. Morphol. 104, I. 16. MARTIN, G. G., AND TALBOT, P. (1981) J. Exp. Zool. 216, 469. 17. MEUSY, J. J. (1980) Reprod. Nutr. Develop. 20, 1. 18. RATEAU, J., AND ZERBIB, C. (1978) C. R. Acad. Sci. 286, 65. 19. SCHADE, M. L., AND SHIVERS, R. R. (1980) J. MorphoL 163, 13. 20. SCHROEDER, P. (1971) Naturinissenschaften 58, 270. 21. SMITH, D. S. (1968) Insect Cells, p. 337, Oliver & Boyd, Edinburgh. 22. SOMLYO, A. P., AND SOMLYO, A. V. (1977) Vol. 3. Plenum, New York. 23. SPURR, A. R. (1969) J. Ultrastruct. Res. 26, 31. 24. STERN, K. G., AND SALOMAN, K. (1937) Science 86, 310. 25. STERNSTEIN, D. J., AND BURTON, P. R. (1980) J. Morphol. 165, 67. 26. TALBOT, P. (1981) J. Ultrastruct. Res. 76, 249. 27. WORSMANN,T. U., BARCELLOS, S. R., AND A. G. FERRI (1976) Bolm Inst. Oceanogr. S. Paulo p. 43. 28. WYCKOFE, R. W. G. (1937) Science 86, 311.