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Cortical reaction in inseminated eggs of the horseshoe crab, Limulus polyphemus L
DEVELOPMENTAL
BIOLOGY
76,410-417
(1980)
Cortical Reaction in Inseminated Eggs of the Horseshoe Limulus polyphemus L.
Crab,
GEORGE GORDON BROWN’ AND DAVID L. CLAPPERS Department of Zoology, Iowa State University, Ames, Iowa 50011 Received July 20, 1979; accepted in revised form November 28, 1979 The egg cortical reaction in Limuluspolyphemus is described in four events. Approximately 10 min after insemination, small “pits” are visible in the cortex of the inseminated Limulus egg. These pits progressively enlarge, eventually coalesce, and finally disappear, leaving a smoothappearing surface approximately 60-90 min after insemination. Based on these visible changes, the following four events comprise the cortical reaction: (I) uninseminated egg-smooth surface, (II) inseminated egg-appearance and growth of pits, (III) inseminated egg-coalescence of pits, and (IV) inseminated egg-appearance of smooth surface. Preparation of these events for SEM studies demonstrated morphological characteristics and sequential development of the pits. Also numerous microvilli are found on the surface of uninseminated and inseminated eggs during the cortical reaction. An increase in their diameter during the reaction is particularly unique. INTRODUCTION
The Limulus egg cortical reaction has not previously been described, although extensive investigations have been performed on later embryonic stages (Lockwood, 1870; Packard, 1885; Kingsley, 1892,1893; Scholl, 1977) and on the closely related species, Tachypleus gigas (Miiller) (Iwanoff, 1933; Roonwal, 1944) and Tachypleus tridentatus Leach (Kishinouye, 1892; Sekiguchi, 1960, 1973). The bulk of this literature considers the developmental stages occurring after blastulation, more specifically, organogenesis. Technical problems have contributed to this void of information on early development. The main problems have been the lack of a suitable in vitro fertilization system which produces a high percentage of development and the lack of adequate fixation techniques for the early stages of development. Sekiguchi (1973) has adequately described 21 stages for the development of Tachypleus tridentatus. However, due to difficulties in following early development, ’ All communications should be with this author. ’ Present address: Hopkins Marine Laboratory, Pacific Grove, California 93950. 410 0012-1606/80/060410-08$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
he placed all events before the appearance of cleavage nuclei on the surface into Stage 1. Thus, this present study not only describes an unique cortical reaction but also provides needed information on early development in xiphosurids. Although the egg cortical reaction has been described in numerous species, sea urchin eggs have received the most attention due to availability of gametes. However, the Limulus egg is an excellent system, since the eggs are relatively large (1.9 mm in diameter), large quantities of gametes are available year around (Brown, 1976), in vitro fertilization is now routine, and the events of the cortical reaction as demonstrated in this paper are easily observed. The following paper is concerned with elucidating mechanisms by which the cortical reaction takes place (Bannon and Brown, 1980). MATERIALS
AND
METHODS
Source of Animals Specimens of Limulus polyphemus L. were obtained from Florida Marine Biological Specimen Co., Inc., Panama City, Florida, maintained at 15°C in 150-gal Instant
BROWN
AND CLAPPER
Ocean aquaria (Instant Ocean artificial seawater (ASW), Aquarium Systems, Inc., Eastlake, Ohio), and were exposed to a light cycle of 14-10 LD. Seventeen females and six males were used in this study.
Gamete Collection and Insemination Gametes were collected by brief electrical stimulation (1.5 V, 0.5-1.0 mA, ac) of the gonoducts proximal to the genital pores. Semen was diluted with ASW to produce a 10% sperm suspension (10’ spermatozoa/ ml) which was used immediately or stored at 5°C for several hours before using. Approximately 15-20 eggs were spawned and immediately transferred with wooden applicators to a lo-cm plastic petri dish containing 35 ml of ASW. One or two drops of the sperm suspension was added, and the dish was swirled to ensure adequate mixing of gametes. All cultures were maintained at 18.5”C.
Events of the Cortical Reaction Inseminated eggs were observed with the aid of a dissecting microscope and continued at 5-min intervals until the cortical reaction was completed. “Events” were designated when obvious structural changes occurred in the cortex. Variations in the duration of these events were frequently noticed when egg batches from different females were used. Aliquots of all egg batches were maintained for 2 weeks to ensure normal development was occurring.
Scanning Electron Microscopy An aliquot of inseminated eggs at each event of the cortical reaction was fixed in a 2.5% glutaraldehyde solution (cacodylate buffer 0°C). To expedite fixative penetration, a small break was made immediately in each egg envelope by pinching with watchmaker forceps. After lo-20 min of fixation, each egg envelope was removed mechanically with the forceps. The inseminated eggs were fixed for 12-24 hr, washed, postfixed in 0~0~ for 2 hr, and stored in ddH,O for l-5 days. Specimens were then
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dehydrated in ethanol and cleared into Freon 113 in which the critical-point technique was applied using liquid carbon dioxide. Uninseminated eggs were prepared in a like manner. Specimens were mounted on stubs with silver paint, carbon and gold coated, and examined on a JEOL JSM-35 scanning electron microscope. RESULTS
Events of the Cortical Reaction Event I, uninseminated egg-smooth surface. Freshly spawned eggs frequently exhibit one or more large indentations (Fig. la) which are caused by oviductal packing. The egg (as observed with a dissecting microscope) has a smooth-appearing surface. No SEM of the whole uninseminated egg was obtained since the egg envelope is very difficult to remove without destroying the egg. However, the egg surface can be observed when small pieces of the egg envelope are pinched off with watchmaker forceps (Fig. lg). The surface topography is dominated by a mat of microvilli (Figs. lh and i). No other surface structures or features are observed.
Event II, inseminated egg-appearance and growth of pits. Pits become obvious approximately 10 min after insemination and gradually increase in size for the next 15-20 min. These pits (Figs. lb and 2) are randomly distributed over the entire inseminated egg surface. The pits are hemispherical, with irregular edges, and are approximately 45-80 pm in diameter. The basins of the pits are homogeneous in appearance. The interpit regions consist of randomly aligned ridges interspersed with irregularly shaped grooves (Figs. 2a and b). Microvilli coat the entire surface of the inseminated egg including the ridges (Figs. 2c and d).
Event III, inseminated egg-coalescing ofpits. Approximately 35 min after insemination, the continuing enlargement of the pits results in their coalescence (Figs. lc and 3a). This event is represented by a highly pitted surface with irregularly shaped pits containing noticeable ridges in
412,
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their basins (Fig. 3b). The interpit region, although greatly reduced, still contains irregularly shaped ridges and grooves. The pits continue to enlarge (Fig. Id) and eventually form several large crater-like structures (Fig. le), approximately 55 min after insemination. Event IV, inseminated egg-appearance of smooth surface. By 90 min after insemination, the surface has a smooth appearance (Fig. If) which resembles that of the uninseminated egg (Figs. la and 3~). Microvilli are now longer in length and doubled in diameter as compared to microvilli in earlier events (Fig. 3d). This stage was very sensitive to SEM preparations and good mounts were difficult to achieve (Fig. 3~). Variations
of the Cortical
Reaction
In a batch of inseminated eggs from one female, variations found in the duration of the cortical reaction are minimal (usually 5%). However, when comparing egg batches between different females, two distinct rates of the cortical reaction have been observed: (1) a fast rate which is completed in 60-90 min and (2) a slow rate which lasts for between 125 and 150 min. The latter was found in approximately 20% of the fe-
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males tested. In the slow rate the pits become more pronounced and deeper, and persist longer, thus, causing an extension of Event III. In the fast rate, the pits form at the same time as in the slow rate, but coalesce and disappear more rapidly. Occasionally a female was found in which the egg batches did not form pits, but instead wrinkles or large shallow depressions were observed. However, regardless of the variations found in the cortical reaction, a high percentage (>90%) of development was always obtained. DISCUSSION
The egg reaction in Limulus is described in four events based on morphological structures of the egg surface before and after insemination. The uninseminated egg is representative of Event I and is characterized by a smooth-appearing surface liberally coated with microvilli. In approximately 10 min after insemination pits which are visible with the aid of a dissecting microscope form on the surface. As pits reach a maximum size (completion of Event II) coalescing or an extensive overlapping of the pits occurs (Event III), causing a characteristic ridging of the pit basins. Even-
FIG. 1. Cortical reaction in Limbs polyphemus at 18.5”C in 100% seawater: (a) Event I, uninseminated egg-smooth surface; (b) Event II, inseminated egg-appearance and growth of pits; (c-e) Event III, inseminated egg-coalescing of pits; (f) Event IV, inseminated egg-appearance of smooth surface. (a) Although this particular egg was photographed 4 min after insemination, no structural differences are observed at this magnification when compared to an uninseminated egg. The surface of the vitellus has a smooth appearance and no unique surface structures are observed. The indentations noted in the whole egg are caused by the packing of eggs in the oviduct. The transparent egg envelope is observed at the edges. x 22. (b) Twenty-five minutes after insemination. The pits in the cortex are obvious and are increasing in size. The distribution of these pits is quite uniform and the diameters are fairly constant. Pits in this inseminated egg were present at lo-15 min after insemination, but were difficult to record photographically. x 22. (c) Thirty-five minutes after insemination. As pits continue to increase in size, an overlapping or coalescing occurs with adjacent pits. Numerous pits are observed in an oblong shape which represents two or more pits coalescing. x 22. (d) Fortyfive minutes after insemination. The coalescing pit areas are continuing to increase in size. x 22. (e) Fifty-five minutes after insemination. Extensive coalescing has led to obvious crater-like structures separated by ridges. (fj Ninety minutes after insemination. The surface of the inseminated egg again has a smooth appearance, which remains thus until approximately 3 hr after insemination when a granular appearance occurs. x 22. (g) Event I, uninseminated egg-smooth surface. Since in uninseminated eggs the egg envelope is difficult to remove without disrupting the vitellus, this micrograph demonstrates a place where the egg envelope (EE) has been partially removed by watchmakers forceps and the surface of the vitellus is exposed. Note the absence of pits or ridges on the vitellus. x 5200. (h) Higher magnification of g demonstrating numerous microvilli covering the surface of the vitellus. x 3090. (i) The microvilli in Event I are very numerous and extensively packed, and have an approximate diameter of 0.1 pm. Compare with Fig. 3d. x 13,500.
F IG. 2. Event II, inseminated egg-appearance and growth of pits. (a) Twenty minutes after inseminat ion. The pits have reached their maximum size just prior to coalescing. The pits and the numerous ridges in the intei spit region are quite obvious. x 64. (b) In the first of a series of higher magnifications, the surface is obsei *ved to ccx&in true pits and numerous irregular ridges. The basins of the pits contain no ridges. x 340. (cl Hi1;her mag nification of a pit rim and surrounding area reveals numerous connecting ridges covered with micro villi. The microvilli are found over the entire surface including the basins of the pits. X 1250. (d) The microvill 1 are obse wed attached to the plasma membrane (arrows). x 3650. 414
FIG. 3. (a) Event III, inseminated egg-coalescing of pits; 35 min after insemination. Pits are enlarging and coalescing. x 65. (b) During the coalescing of pits, noticeable ridges are developing in the basins of the pits. x 190. (c) Event IV, inseminated egg-appearance of smooth surface; SOmin after insemination. The inseminated egg demonstrating a surface lacking pits. The ridge-like structures observed are coating material. This event was very difficult to prepare properly, thus explaining the artifacts (cracks) present in this micrograph. x 59. (d) Microvilli have become longer and have nearly doubled in diameter (0.2 pm) since the egg was observed 415
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tually, the pits and ridges disappear and the surface resumes a smooth appearance (Event IV). An unusual phenomenon is the doubling of the microvilli diameter during the cortical reaction. An advantage of the Limulus system is the duration of the cortical reaction. The mechanisms in cortical reactions of numerous species are difficult to examine since the duration (fusion of cortical vesicles and smoothing of disrupted surface) is quite rapid. For example, in sea urchins, this reaction is completed in 4-9 min (Schroeder, 1979); in the teleost, Oryzias, 5 min (Iwamatsu and Keino, 1978); in the frog, Xenopus, 5 min (Wolf, 1974); and in the peneid shrimp, Penaeus, less than 5 min (Clark and Lynn, 1977). In Limulus, the cortical reaction ranges in duration from 60 to 90 min. Nereis limbata exhibits a cortical reaction similar to that of Limulus. However, the cortical reaction is completed in 15-20 min (Fallon and Austin, 1967). In mammals, the duration is 15 and 60 min in mouse and hamster eggs, respectively (Nicosia et al., 1977; Fukuda and Chang, 1978). The longer duration in Limulus allows the identification of morphological changes and the possible separation of chemical activities. Pit-like structures have been observed with SEM during the cortical reaction of other species and designated by various terms: depressions, pits, alveoli, pockets, and crypts. In the sea urchin, Strongylocentrotus purpuratus, small pits (1.0 pm in diameter) are observed in the surface approximately 30 set after insemination and are the results of the perigranular membrane fusing with the plasmalemma (Eddy and Shapiro, 1976; Tegner and Epel, 1976). In the teleost, Oryzias Zatipes, cortical alveoli observed before insemination fuse with the plasma membrane after insemination, forming cortical alveolus pockets (Iwamatsu and Keino, 1978). These pockets are initially 2-3 pm in diameter, but undergo reduction within 3 min as incorporation into the plasmalemma occurs. In the peneid shrimp, Penaeus aztecus and P. se-
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tiferus, a rather unusual situation exists in that preformed crypts are present in the cortex and contain rods (10 pm in diameter x 40 pm in length) which are external to the plasmalemma (Clark and Lynn, 1977). Upon egg activation the rods are extruded to form an investment layer around the egg. Presumably, these crypts disappear by 5 min after activation. In all cases involving the aforementioned species, material is released from the “pits” and may contribute as a polyspermy prevention substance or to initial formation of an embryonic protective layer. The appearance of pits during the cortical reaction is used as a criterion for Limulus early development. The duration of the presence of pits varies between egg batches and has led to the distinction of fast (60-90 min) and slow (120-150 min) rates of cortical reactions. Occasionally (~5%) a female is obtained whose egg batches produce a ruffled appearance or a highly ridged surface in response to insemination. Since normal development occurs in these inseminated egg batches, this phenomenon is not abnormal but demonstrates flexibility of the cortical reaction. Also, initial studies in our laboratory have demonstrated that changes in pH and salinity have significant effects on the cortical reaction. Normal and induced variations of the cortical reaction need to be examined critically. This research was supported in part by Iowa State University Graduate College and an Iowa State University Research Grant. We are grateful to Dr. Harry T. Horner of ISU for the use of scanning electron microscope facilities. REFERENCES G. A., and BROWN, G. G. (1980). Vesicle involvement in the egg cortical reaction of the horseshoe crab, Linulus polyphemus L. Develop. Biol. 76, 418-427. BROWN, G. G. (1976). Scanning electron-microscopical and other observations of sperm fertilization reactions in Limuluspolyphemus L. (Merostomata: Xiphosura). J. Cell Sci. 22,547~562. CLARK, W. H., JR., and LYNN, J. W. (1977). A Mg”
BANNON,
BROWN AND CLAPPER dependent cortical reaction in the eggs of penaeid shrimp. J. Exp. 2001. 200, 177-183. EDDY, E. M., and SHAPIRO, B. M. (1976). Changes in the topography of the sea urchin egg after fertilization. J. Cell Biol. ‘71, 35-48. FALLON, J. F., and AUSTIN, C. R. (1967). Fine structure of gametes of Nereis limbata (Annelida) before and after interaction. J. Exp. 2001. 166, 225-242. FUKUDA, Y., and CHANG, M. C. (1978). The time of cortical granule breakdown and sperm penetration in mouse and hamster eggs inseminated in uitro. Biol. Reprod. 19,261-266. IWAMATSU, T., and KEINO, H. (1978). Scanning electron microscopic study on the surface change of eggs of the teleost, Oryzias latipes, at the time of fertilization. Develop. Growth Different. 20, 237-250. IWANOFF, P. P. (1933). Die embryonale Entwicklung von Limulus molluccanus. Zool. Jb. Anat. Ont. 56, 163-348. KINGSLEY, J. S. (1892). The embryology of Limulus. J. Morphol. 7, 35-68. KINGSLEY, J. S. (1893). The embryology of Limulus, II. J. Morphol. 8, 195-268. KISHINOUYE, K. (1892). On the development of Limulus longispina. J. Cell. Sci. Imp. Univ. Tokyo 5, 53-100. LOCKWOOD, S. (1870). The horse-foot crab. Amer. Natur. 4, 257-274. NICOSIA, S. V., WOLF, D. P., and INOUE, M. (1977).
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Cortical granule distribution and cell surface characteristics in mouse eggs. Develop. Biol. 57, 56-74. PACKARD, A. S. (1885). On the embryology of Limulus polyphemus III. Amer. Natur. 19, 722-727. ROONWAL, M. L. (1944). Some observations on the breeding biology, and on the swelling, weight, watercontent and embryonic movements in the developing eggs of the moluccan king crab, Tachypleus gigas (Muller) (Arthropoda, Xiphosurida). Proc. Ind. Acad. Sci. Sect. B 20, 115-129. SCHOLL, G. (1977). Beritrage zur Embryonalentwicklung von Limulus polyphemus L. (Chelicerata, Xiphosura). Zoomorphologie 86,99-154. SCHROEDER, T. E. (1979). Surface area change at fertilization: Resorption of the mosaic membrane. Develop. Biol. 70,306-326. SEKIGUCHI, K. (1960). Embryonic development of the horse-shoe crab studied by vital staining. Bull. Mar. Biol. Sta. Asamuchi Tohuku Univ. 10, 161-164. SEKIGUCHI, K. (1973). A normal plate of the development of the Japanese horse-shoe crab, Tachypleus tridentatus. Sci. Rep. Tokyo Kyoiku Daigaku B 15, 153-162. TEGNER, M. J., and EPEL, D. (1976). Scanning electron microscope studies of sea urchin fertilization. J. Exp. Zool. 197, 31-58. WOLF, D. P. (1974). The cortical granule reaction in living eggs of the toad, Xenopus laevis. Develop. Biol. 36,62-71.
BIOLOGY
76,410-417
(1980)
Cortical Reaction in Inseminated Eggs of the Horseshoe Limulus polyphemus L.
Crab,
GEORGE GORDON BROWN’ AND DAVID L. CLAPPERS Department of Zoology, Iowa State University, Ames, Iowa 50011 Received July 20, 1979; accepted in revised form November 28, 1979 The egg cortical reaction in Limuluspolyphemus is described in four events. Approximately 10 min after insemination, small “pits” are visible in the cortex of the inseminated Limulus egg. These pits progressively enlarge, eventually coalesce, and finally disappear, leaving a smoothappearing surface approximately 60-90 min after insemination. Based on these visible changes, the following four events comprise the cortical reaction: (I) uninseminated egg-smooth surface, (II) inseminated egg-appearance and growth of pits, (III) inseminated egg-coalescence of pits, and (IV) inseminated egg-appearance of smooth surface. Preparation of these events for SEM studies demonstrated morphological characteristics and sequential development of the pits. Also numerous microvilli are found on the surface of uninseminated and inseminated eggs during the cortical reaction. An increase in their diameter during the reaction is particularly unique. INTRODUCTION
The Limulus egg cortical reaction has not previously been described, although extensive investigations have been performed on later embryonic stages (Lockwood, 1870; Packard, 1885; Kingsley, 1892,1893; Scholl, 1977) and on the closely related species, Tachypleus gigas (Miiller) (Iwanoff, 1933; Roonwal, 1944) and Tachypleus tridentatus Leach (Kishinouye, 1892; Sekiguchi, 1960, 1973). The bulk of this literature considers the developmental stages occurring after blastulation, more specifically, organogenesis. Technical problems have contributed to this void of information on early development. The main problems have been the lack of a suitable in vitro fertilization system which produces a high percentage of development and the lack of adequate fixation techniques for the early stages of development. Sekiguchi (1973) has adequately described 21 stages for the development of Tachypleus tridentatus. However, due to difficulties in following early development, ’ All communications should be with this author. ’ Present address: Hopkins Marine Laboratory, Pacific Grove, California 93950. 410 0012-1606/80/060410-08$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
he placed all events before the appearance of cleavage nuclei on the surface into Stage 1. Thus, this present study not only describes an unique cortical reaction but also provides needed information on early development in xiphosurids. Although the egg cortical reaction has been described in numerous species, sea urchin eggs have received the most attention due to availability of gametes. However, the Limulus egg is an excellent system, since the eggs are relatively large (1.9 mm in diameter), large quantities of gametes are available year around (Brown, 1976), in vitro fertilization is now routine, and the events of the cortical reaction as demonstrated in this paper are easily observed. The following paper is concerned with elucidating mechanisms by which the cortical reaction takes place (Bannon and Brown, 1980). MATERIALS
AND
METHODS
Source of Animals Specimens of Limulus polyphemus L. were obtained from Florida Marine Biological Specimen Co., Inc., Panama City, Florida, maintained at 15°C in 150-gal Instant
BROWN
AND CLAPPER
Ocean aquaria (Instant Ocean artificial seawater (ASW), Aquarium Systems, Inc., Eastlake, Ohio), and were exposed to a light cycle of 14-10 LD. Seventeen females and six males were used in this study.
Gamete Collection and Insemination Gametes were collected by brief electrical stimulation (1.5 V, 0.5-1.0 mA, ac) of the gonoducts proximal to the genital pores. Semen was diluted with ASW to produce a 10% sperm suspension (10’ spermatozoa/ ml) which was used immediately or stored at 5°C for several hours before using. Approximately 15-20 eggs were spawned and immediately transferred with wooden applicators to a lo-cm plastic petri dish containing 35 ml of ASW. One or two drops of the sperm suspension was added, and the dish was swirled to ensure adequate mixing of gametes. All cultures were maintained at 18.5”C.
Events of the Cortical Reaction Inseminated eggs were observed with the aid of a dissecting microscope and continued at 5-min intervals until the cortical reaction was completed. “Events” were designated when obvious structural changes occurred in the cortex. Variations in the duration of these events were frequently noticed when egg batches from different females were used. Aliquots of all egg batches were maintained for 2 weeks to ensure normal development was occurring.
Scanning Electron Microscopy An aliquot of inseminated eggs at each event of the cortical reaction was fixed in a 2.5% glutaraldehyde solution (cacodylate buffer 0°C). To expedite fixative penetration, a small break was made immediately in each egg envelope by pinching with watchmaker forceps. After lo-20 min of fixation, each egg envelope was removed mechanically with the forceps. The inseminated eggs were fixed for 12-24 hr, washed, postfixed in 0~0~ for 2 hr, and stored in ddH,O for l-5 days. Specimens were then
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dehydrated in ethanol and cleared into Freon 113 in which the critical-point technique was applied using liquid carbon dioxide. Uninseminated eggs were prepared in a like manner. Specimens were mounted on stubs with silver paint, carbon and gold coated, and examined on a JEOL JSM-35 scanning electron microscope. RESULTS
Events of the Cortical Reaction Event I, uninseminated egg-smooth surface. Freshly spawned eggs frequently exhibit one or more large indentations (Fig. la) which are caused by oviductal packing. The egg (as observed with a dissecting microscope) has a smooth-appearing surface. No SEM of the whole uninseminated egg was obtained since the egg envelope is very difficult to remove without destroying the egg. However, the egg surface can be observed when small pieces of the egg envelope are pinched off with watchmaker forceps (Fig. lg). The surface topography is dominated by a mat of microvilli (Figs. lh and i). No other surface structures or features are observed.
Event II, inseminated egg-appearance and growth of pits. Pits become obvious approximately 10 min after insemination and gradually increase in size for the next 15-20 min. These pits (Figs. lb and 2) are randomly distributed over the entire inseminated egg surface. The pits are hemispherical, with irregular edges, and are approximately 45-80 pm in diameter. The basins of the pits are homogeneous in appearance. The interpit regions consist of randomly aligned ridges interspersed with irregularly shaped grooves (Figs. 2a and b). Microvilli coat the entire surface of the inseminated egg including the ridges (Figs. 2c and d).
Event III, inseminated egg-coalescing ofpits. Approximately 35 min after insemination, the continuing enlargement of the pits results in their coalescence (Figs. lc and 3a). This event is represented by a highly pitted surface with irregularly shaped pits containing noticeable ridges in
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their basins (Fig. 3b). The interpit region, although greatly reduced, still contains irregularly shaped ridges and grooves. The pits continue to enlarge (Fig. Id) and eventually form several large crater-like structures (Fig. le), approximately 55 min after insemination. Event IV, inseminated egg-appearance of smooth surface. By 90 min after insemination, the surface has a smooth appearance (Fig. If) which resembles that of the uninseminated egg (Figs. la and 3~). Microvilli are now longer in length and doubled in diameter as compared to microvilli in earlier events (Fig. 3d). This stage was very sensitive to SEM preparations and good mounts were difficult to achieve (Fig. 3~). Variations
of the Cortical
Reaction
In a batch of inseminated eggs from one female, variations found in the duration of the cortical reaction are minimal (usually 5%). However, when comparing egg batches between different females, two distinct rates of the cortical reaction have been observed: (1) a fast rate which is completed in 60-90 min and (2) a slow rate which lasts for between 125 and 150 min. The latter was found in approximately 20% of the fe-
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413
males tested. In the slow rate the pits become more pronounced and deeper, and persist longer, thus, causing an extension of Event III. In the fast rate, the pits form at the same time as in the slow rate, but coalesce and disappear more rapidly. Occasionally a female was found in which the egg batches did not form pits, but instead wrinkles or large shallow depressions were observed. However, regardless of the variations found in the cortical reaction, a high percentage (>90%) of development was always obtained. DISCUSSION
The egg reaction in Limulus is described in four events based on morphological structures of the egg surface before and after insemination. The uninseminated egg is representative of Event I and is characterized by a smooth-appearing surface liberally coated with microvilli. In approximately 10 min after insemination pits which are visible with the aid of a dissecting microscope form on the surface. As pits reach a maximum size (completion of Event II) coalescing or an extensive overlapping of the pits occurs (Event III), causing a characteristic ridging of the pit basins. Even-
FIG. 1. Cortical reaction in Limbs polyphemus at 18.5”C in 100% seawater: (a) Event I, uninseminated egg-smooth surface; (b) Event II, inseminated egg-appearance and growth of pits; (c-e) Event III, inseminated egg-coalescing of pits; (f) Event IV, inseminated egg-appearance of smooth surface. (a) Although this particular egg was photographed 4 min after insemination, no structural differences are observed at this magnification when compared to an uninseminated egg. The surface of the vitellus has a smooth appearance and no unique surface structures are observed. The indentations noted in the whole egg are caused by the packing of eggs in the oviduct. The transparent egg envelope is observed at the edges. x 22. (b) Twenty-five minutes after insemination. The pits in the cortex are obvious and are increasing in size. The distribution of these pits is quite uniform and the diameters are fairly constant. Pits in this inseminated egg were present at lo-15 min after insemination, but were difficult to record photographically. x 22. (c) Thirty-five minutes after insemination. As pits continue to increase in size, an overlapping or coalescing occurs with adjacent pits. Numerous pits are observed in an oblong shape which represents two or more pits coalescing. x 22. (d) Fortyfive minutes after insemination. The coalescing pit areas are continuing to increase in size. x 22. (e) Fifty-five minutes after insemination. Extensive coalescing has led to obvious crater-like structures separated by ridges. (fj Ninety minutes after insemination. The surface of the inseminated egg again has a smooth appearance, which remains thus until approximately 3 hr after insemination when a granular appearance occurs. x 22. (g) Event I, uninseminated egg-smooth surface. Since in uninseminated eggs the egg envelope is difficult to remove without disrupting the vitellus, this micrograph demonstrates a place where the egg envelope (EE) has been partially removed by watchmakers forceps and the surface of the vitellus is exposed. Note the absence of pits or ridges on the vitellus. x 5200. (h) Higher magnification of g demonstrating numerous microvilli covering the surface of the vitellus. x 3090. (i) The microvilli in Event I are very numerous and extensively packed, and have an approximate diameter of 0.1 pm. Compare with Fig. 3d. x 13,500.
F IG. 2. Event II, inseminated egg-appearance and growth of pits. (a) Twenty minutes after inseminat ion. The pits have reached their maximum size just prior to coalescing. The pits and the numerous ridges in the intei spit region are quite obvious. x 64. (b) In the first of a series of higher magnifications, the surface is obsei *ved to ccx&in true pits and numerous irregular ridges. The basins of the pits contain no ridges. x 340. (cl Hi1;her mag nification of a pit rim and surrounding area reveals numerous connecting ridges covered with micro villi. The microvilli are found over the entire surface including the basins of the pits. X 1250. (d) The microvill 1 are obse wed attached to the plasma membrane (arrows). x 3650. 414
FIG. 3. (a) Event III, inseminated egg-coalescing of pits; 35 min after insemination. Pits are enlarging and coalescing. x 65. (b) During the coalescing of pits, noticeable ridges are developing in the basins of the pits. x 190. (c) Event IV, inseminated egg-appearance of smooth surface; SOmin after insemination. The inseminated egg demonstrating a surface lacking pits. The ridge-like structures observed are coating material. This event was very difficult to prepare properly, thus explaining the artifacts (cracks) present in this micrograph. x 59. (d) Microvilli have become longer and have nearly doubled in diameter (0.2 pm) since the egg was observed 415
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DEVELOPMENTAL
BIOLOGY
tually, the pits and ridges disappear and the surface resumes a smooth appearance (Event IV). An unusual phenomenon is the doubling of the microvilli diameter during the cortical reaction. An advantage of the Limulus system is the duration of the cortical reaction. The mechanisms in cortical reactions of numerous species are difficult to examine since the duration (fusion of cortical vesicles and smoothing of disrupted surface) is quite rapid. For example, in sea urchins, this reaction is completed in 4-9 min (Schroeder, 1979); in the teleost, Oryzias, 5 min (Iwamatsu and Keino, 1978); in the frog, Xenopus, 5 min (Wolf, 1974); and in the peneid shrimp, Penaeus, less than 5 min (Clark and Lynn, 1977). In Limulus, the cortical reaction ranges in duration from 60 to 90 min. Nereis limbata exhibits a cortical reaction similar to that of Limulus. However, the cortical reaction is completed in 15-20 min (Fallon and Austin, 1967). In mammals, the duration is 15 and 60 min in mouse and hamster eggs, respectively (Nicosia et al., 1977; Fukuda and Chang, 1978). The longer duration in Limulus allows the identification of morphological changes and the possible separation of chemical activities. Pit-like structures have been observed with SEM during the cortical reaction of other species and designated by various terms: depressions, pits, alveoli, pockets, and crypts. In the sea urchin, Strongylocentrotus purpuratus, small pits (1.0 pm in diameter) are observed in the surface approximately 30 set after insemination and are the results of the perigranular membrane fusing with the plasmalemma (Eddy and Shapiro, 1976; Tegner and Epel, 1976). In the teleost, Oryzias Zatipes, cortical alveoli observed before insemination fuse with the plasma membrane after insemination, forming cortical alveolus pockets (Iwamatsu and Keino, 1978). These pockets are initially 2-3 pm in diameter, but undergo reduction within 3 min as incorporation into the plasmalemma occurs. In the peneid shrimp, Penaeus aztecus and P. se-
VOLUME
76, 1980
tiferus, a rather unusual situation exists in that preformed crypts are present in the cortex and contain rods (10 pm in diameter x 40 pm in length) which are external to the plasmalemma (Clark and Lynn, 1977). Upon egg activation the rods are extruded to form an investment layer around the egg. Presumably, these crypts disappear by 5 min after activation. In all cases involving the aforementioned species, material is released from the “pits” and may contribute as a polyspermy prevention substance or to initial formation of an embryonic protective layer. The appearance of pits during the cortical reaction is used as a criterion for Limulus early development. The duration of the presence of pits varies between egg batches and has led to the distinction of fast (60-90 min) and slow (120-150 min) rates of cortical reactions. Occasionally (~5%) a female is obtained whose egg batches produce a ruffled appearance or a highly ridged surface in response to insemination. Since normal development occurs in these inseminated egg batches, this phenomenon is not abnormal but demonstrates flexibility of the cortical reaction. Also, initial studies in our laboratory have demonstrated that changes in pH and salinity have significant effects on the cortical reaction. Normal and induced variations of the cortical reaction need to be examined critically. This research was supported in part by Iowa State University Graduate College and an Iowa State University Research Grant. We are grateful to Dr. Harry T. Horner of ISU for the use of scanning electron microscope facilities. REFERENCES G. A., and BROWN, G. G. (1980). Vesicle involvement in the egg cortical reaction of the horseshoe crab, Linulus polyphemus L. Develop. Biol. 76, 418-427. BROWN, G. G. (1976). Scanning electron-microscopical and other observations of sperm fertilization reactions in Limuluspolyphemus L. (Merostomata: Xiphosura). J. Cell Sci. 22,547~562. CLARK, W. H., JR., and LYNN, J. W. (1977). A Mg”
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BROWN AND CLAPPER dependent cortical reaction in the eggs of penaeid shrimp. J. Exp. 2001. 200, 177-183. EDDY, E. M., and SHAPIRO, B. M. (1976). Changes in the topography of the sea urchin egg after fertilization. J. Cell Biol. ‘71, 35-48. FALLON, J. F., and AUSTIN, C. R. (1967). Fine structure of gametes of Nereis limbata (Annelida) before and after interaction. J. Exp. 2001. 166, 225-242. FUKUDA, Y., and CHANG, M. C. (1978). The time of cortical granule breakdown and sperm penetration in mouse and hamster eggs inseminated in uitro. Biol. Reprod. 19,261-266. IWAMATSU, T., and KEINO, H. (1978). Scanning electron microscopic study on the surface change of eggs of the teleost, Oryzias latipes, at the time of fertilization. Develop. Growth Different. 20, 237-250. IWANOFF, P. P. (1933). Die embryonale Entwicklung von Limulus molluccanus. Zool. Jb. Anat. Ont. 56, 163-348. KINGSLEY, J. S. (1892). The embryology of Limulus. J. Morphol. 7, 35-68. KINGSLEY, J. S. (1893). The embryology of Limulus, II. J. Morphol. 8, 195-268. KISHINOUYE, K. (1892). On the development of Limulus longispina. J. Cell. Sci. Imp. Univ. Tokyo 5, 53-100. LOCKWOOD, S. (1870). The horse-foot crab. Amer. Natur. 4, 257-274. NICOSIA, S. V., WOLF, D. P., and INOUE, M. (1977).
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Cortical granule distribution and cell surface characteristics in mouse eggs. Develop. Biol. 57, 56-74. PACKARD, A. S. (1885). On the embryology of Limulus polyphemus III. Amer. Natur. 19, 722-727. ROONWAL, M. L. (1944). Some observations on the breeding biology, and on the swelling, weight, watercontent and embryonic movements in the developing eggs of the moluccan king crab, Tachypleus gigas (Muller) (Arthropoda, Xiphosurida). Proc. Ind. Acad. Sci. Sect. B 20, 115-129. SCHOLL, G. (1977). Beritrage zur Embryonalentwicklung von Limulus polyphemus L. (Chelicerata, Xiphosura). Zoomorphologie 86,99-154. SCHROEDER, T. E. (1979). Surface area change at fertilization: Resorption of the mosaic membrane. Develop. Biol. 70,306-326. SEKIGUCHI, K. (1960). Embryonic development of the horse-shoe crab studied by vital staining. Bull. Mar. Biol. Sta. Asamuchi Tohuku Univ. 10, 161-164. SEKIGUCHI, K. (1973). A normal plate of the development of the Japanese horse-shoe crab, Tachypleus tridentatus. Sci. Rep. Tokyo Kyoiku Daigaku B 15, 153-162. TEGNER, M. J., and EPEL, D. (1976). Scanning electron microscope studies of sea urchin fertilization. J. Exp. Zool. 197, 31-58. WOLF, D. P. (1974). The cortical granule reaction in living eggs of the toad, Xenopus laevis. Develop. Biol. 36,62-71.