Ultrastructural and Cytochemical Studies on Sperm-Egg Interactions of the Horseshoe Crab, Limulus Polyphemus L " (1974)

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1974 Ultrastructural and cytochemical studies on sperm- egg interactions of the horseshoe crab, Limulus polyphemus L Jerry Bennett Iowa State University

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BENNETT, Jerry, 1942- ULTRASTRUCTURAL AND CYTOCHEMICAL STUDIES ON SPERM-EGG INTERACTIONS OF THE HORSESHOE CRAB, LIMCJLUS POLYPHEMUS L. • Iowa State University, Ph.D., 1974 Zoology

Xerox University iviicrofilms, Ann Arbor, Michigan 4810D

JERRY BENNETT

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. Ultrastructural and cytochemical studies on

sperm-egg interactions of the horseshoe

crab, Limulus polyphemus L.

Jerry Bennett

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of

The Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department: Zoology and Entomology Major: Cell Biology

Approved:

Signature was redacted for privacy. In Ch^ge of Major

Signature was redacted for privacy. For the Major Department

TABLE OF CONTENTS

Page

INTRODUCTION 1

REVIEW OF LITERATURE 3

Sperm Acrosome 3

Ultrastructure of Invertebrate Sperm Acrosome 5

Coelelenterata (Cnidaria) 5 Platyhelminthes 7 Nematoda 8 Annelida 9 Mollusca 11 Echinodermata 14 Arthropoda 17 Symphyla and Pauropoda 23

Ultrastructure of Vertebrate Sperm Acrosomes 32

Agnatha 33 Chondrichthyes 34 Osteichthyes 35 Amphibia 35 Reptilia 37 Aves 37 Mammalia 38

Chemical Content of the Sperm Acrosome 40

Sperm-Egg Interactions 47

Sperm-egg attachment 4 8 The acrosome reaction 49 Egg activation 52

Egg Envelopes 54

Invertebrate egg envelopes 55 Prochordate and vertebrate egg 60 Vertebrates 61

Cytochemistry of Egg Envelopes 65

Invertebrate egg envelopes 66 Vertebrate egg envelopes 74 îii

Page

Limulus Fertilization 78

Limulus sperm 78 Limulus egg 80 Sperm-egg interaction in Limulus 81

Approaches to Fertilization in Limulus 82

MATERIALS AND METHODS 84

Source and Maintenance of Animals 84

Collection and Preparation of Gametes 84

Preparation of Spermatozoa for Cytochemistry 85

Acid phosphatase 85 Acridine orange 86 Acrosome proteinase 87 Trypsin-like acrosome enzyme 87 Nonspecific esterase 88

Preparation of Sperm for Ultrastructure 89

Separation of Sperm Acrosome 90

Preparation of Eggs and Egg Sections for Cytochemistry 91

Uninseminated egg sections and eggs 91 Inseminated egg sections 92 Culture of fertilized eggs 100

Enzymatic Treatment of Egg Envelopes 101

Determination of the Types of Chemical Linkages Maintaining the Structural Integrity of Limulus Egg Envelopes 102

Amino Acid Analysis of Egg Envelopes 102

Preparation of Egg Envelopes for Ultrastructure 104

Uninseminated egg envelopes 104 Inseminated egg envelopes 105 iv

Page

Microscopic Examination and Photographing of Cytochemical Reactions and Developing Limulus Embryos 105

RESULTS 107

Limulus as a Laboratory Animal 107

Cytochemistry of the Sperm Acrosome 108

Acid phosphatase 108 Acridine orange 109 Acrosome proteinase 109 Trypsin-like acrosome enzyme 110 Nonspecific esterase 111

Ultrastructure of the Sperm Acrosome Complex 111

Separation of Sperm Acrosome 112

Cytochemistry of Uninseminated Egg Sections and Eggs 113

Carbohydrates 114 Proteins and nucleic acids 120 Lipids 122 Hydrolytic enzymes 123

Cytochemistry of Inseminated Egg Sections and Developing Eggs 124

Enzymatic Treatment of Uninseminated Egg Sections 125

Physiochemical Treatment of Egg Sections 127

Amino Acid Analysis of Isolated Uninseminated Egg Envelopes 127

Amino Acid Analysis of Isolated 72 h Egg Envelopes 130

Observations on the Embryology of Limulus polyphemus L, 130 V

Page

Ultrastructure of Sperm-Egg Interaction in Limulus polyphemus L. 134

DISCUSSION 136

Cytochemistry of Limulus Sperm Acrosome 136

Ultrastructure of the Sperm Acrosome Complex 139

Separation of the Sperm Acrosome 141

Cytochemistry of Uninseminated Egg Sections and Eggs 142

Carbohydrates 142 Proteins and nucleic acids 150 Lipids 154 Hydrolytic enzymes 158

Cytochemistry of Inseminated Egg Sections and Eggs 160

Physiochemical Treatment of Limulus Egg Sections 162

Amino Acid Composition of Limulus Egg Envelopes 167

Limulus polyphemus L Embryology 173

Ultrastructure of Uninseminated and Inseminated Eggs 177

CONCLUSIONS 179a

SUMMARY 180

BIBLIOGRAPHY 182

ACKNOWLEDGMENTS 219

APPENDIX A. SOURCES OF REAGENTS 220

APPENDIX B. FIGURES AND ABBREVIATIONS 223

Abbreviations 224 1

INTRODUCTION

"As the egg may be protected by follicle cells, jelly coat, vitelline membrane and perhaps other protective envelopes, it can be regarded as a treasure hidden by many seals, locked by many locks. The spermatozoon has to perform the unlocking. It must possess the proper keys and these may be immunological, enzymatic or mechanical" (Afzelius, 1970).

The spermatozoa of Limulus possess one and possibly two of the keys mentioned above. The presence and participation of sperm antigens during the sperm-egg interaction have been indicated by Mowbray, Brown and Metz (1970). Their observa tions suggest the occurrence of an interaction between specific sites on both the spermatozoon and the egg.

Observations by Shoger and Brown (1970) and Brown and

Humphreys (1971) that the thick egg envelope of the Limulus egg is penetrated by a 50y long acrosome filament during sperm-egg interactions suggest that a mechanical key is

involved in unlocking the treasures of the Limulus egg. Since

the normal sperm acrosome reaction with the egg envelope in

volves extrusion of an acrosome filament through two egg

layers and recent evidence suggests the filaments pass through

pre-existing pores in the outer layer (no such pores exist in

the inner layer of the egg), penetration of these layers is

either mechanical or enzymatic. 2

One approach to ascertaining whether the enzymatic key of the spermatozoon is involved in unlocking the treasures of the Limulus egg is to examine the chemical nature of enzymes in the sperm acrosome and to correlate the finding with the chemical makeup of the egg envelopes. Cytochemical methods along with several other methods are used in this study to investigate the cytochemistry of the sperm acrosome and egg envelopes. In addition, the feasibility of using Limulus polyphemus as a laboratory animal in embryological studies will be explored. The experimental procedures and observations are preceded by the following review on sperm acrosomes, egg envelopes, sperm-egg interactions and Limulus fertilization. 3

REVIEW OF LITERATURE

Sperm Acrosome

A vast amount of basic information on sperm ultrastructure has accumulated within the past fifteen years. The majority of the work contributing to our knowledge of sperm ultrastructure was performed on invertebrate organisms during the 1960's. Since many invertebrates, particularly marine invertebrates are easy to obtain and gametes can be collected in large quantities, they are ideally suited for such studies.

The typical primitive type metazoan spermatozoon consists of a round head, middle piece, and a tail (see Franzen, 1970 for review). Many electron microscopists studying mature spermatozoa have given detailed descriptions on the morphology and development of the head region. Here, in addition to the nucleus, is commonly found a vesicular structure termed the acrosome, a term which was probably first introduced by

Lenhossek (1898) and is now used to describe what has been referred to as the "acrosome cap", "head cap" or "galea capitus" (Austin, 1968). In its classical position, the acrosome is located apically or around the anterior portion of the sperm nucleus. However, as Bowen (1924) and others have observed, the acrosome in various species can occupy any position with respect to the sperm nucleus. For example, the arthropods represent a group of invertebrates where 4 spermatozoa of selected species have either no acrosome (some insects, phillips, 1970) or an acrosome almost completely surrounded by the sperm nucleus (some decapods, Langreth,

1965).

Diversity in size, shape, and substructure of the acrosome is not restricted to invertebrates. Vertebrates also display considerable variation from the common cap-type acrosome structures found in many amphibians, reptiles, aves, and mammals (Nicander, 1970).

Recently the acrosome has become the focal point for the localization and extraction of enzymes involved in sperm pene tration (for review see Williams, 1972). Investigations on acrosome enzymes has primarily been confined to mammalian spermatozoa. In the rabbit, for example, Srivastava, Adams and Hartree (1965a) have demonstrated the presence of a corona penetrating enzyme capable of removing the corona radiata of the ova while Stambaugh and Buckley (1969) have demonstrated a trypsin-like enzyme activity that dissolves the zona pellucida. Studies along these lines have created added interest in the sperm acrosome of mammals and most likely there will be renewed interest in the cytochemistry and bio chemistry of acrosomes in other organisms. 5

Ultrastructure of Invertebrate Sperm Acrosome

The following section on invertebrate sperm acrosome ultrastructure is a survey of the general acrosome types found among the invertebrates. Some groups will be discussed in more detail than others since spermatozoa of some phyla have received more investigative attention.

Coelenterata (Cnidaria)

A detailed report on the fine structure of the spermatozoa of coelenterates was lacking until a study by Summers (1970).

Even though Burnett, Davis and Ruffing (1966) conducted an ultrastructural study on "the germinal differentiation of interstitial cells from gland cells in Hydra viridus," they failed to include a description of the spermatozoa. A year later Schencariol, Habowsky and Winner (1967) published a report on spermatogenesis in Hydra fusca demonstrating a mature spermatozoon but with no description of the head region. Finally, Summers' report (1970) on the fine structure of the gymnoblastic hydroid Pennaria tiarella represented the first published ultrastructural description of the sperm head of any coelenterate spermatozoon.

Pennaria spermatozoa are conical and contain heads that range in length from 3.0y to 3.5%. Four spherical mitochondria lie at the base of the nucleus and within the fossa formed by the mitochondria are the distal and proximal centrioies. The 6 tails display the typical 9 + 2 flagelliam pattern and range in length from 30y to 40p. In Pennaria, the classical acrosome region is occupied by 30-40 small Golgi derived vesicles and are in striking resemblance to similar vesicles occupying the residual cytoplasmic droplets in bull and ram spermatozoa, which incidentally have been found to contain lysosomal • enzymes. Even though the vesicles of Pennaria occupy the region of the classical acrosome and may contain lysosomal type enzymes, they are not termed an acrosome by Summers

(1970). Such a conclusion will undoubtedly await the elucida tion of the role these vesicles play in fertilization.

Vesicles in the anterior borders of the sperm head seem to be a common feature among the Hydrozoa. Afzelius (1971) reported membrane bound vesicles of about 0.05u in width and

O.ly long in the conical sperm head of Tubularia. Even though these vesicles are located at the anterior borders and not the tip of the sperm head, their numbers (30) and description are similar to those found in Pennaria (Summers, 1970).

The vesicles observed in the acrosome region of a primitive form of Scyphozoa are more complicated in mor phology than those found in the Hydrozoa. In Nausithoe, a jellyfish, no evidence of an acrosome cap exists (Afzelius and

Franzen, 1971). Instead, the acrosome area is occupied by a cytoplasmic layer containing moderately electron dense granular cytoplasm and is not surrounded by a membrane of its 7 own. This area contains several vesicles, each having a lighter core. Both the core and vesicles are limited by a true (triple-layered) membrane. The total number of such vesicles may approach 20 in any given spermatozoon.

A comparative study of the spermatozoa of Tubularia,

Hydractinia, Metridium, Cyanea and Aurelia by Hinsch and Clark

(1970) helps complete the general picture of the acrosome in coelenterate sperm. Since no discrete acrosome has been described and only numerous membrane-bound vesicles occupy the presumed acrosome area of the cell, the presence of a classical acrosome remains to be determined by functional studies of the vesicles in fertilization.

Platyhelminthes

Electron microscopic studies on developing and mature

Platyhelminthes spermatozoa have failed to show or identify an acrosome (Christensen, 19 61; Gresson, 1962; Bonsdorff and

Telkka, 1965; Hershenov, Tulloch and Johnson, 1966;

Hendelberg, 1967; Tulloch and Hershenov, 1967; Bedini and

Papi, 1970 and Hendelberg, 1970). Morphologically, the mature spermatozoa of many different groups of platyhelminthes have been found in general to be thread-shaped. Investigators studying sperm morphology of the flatworms are primarily I interested in the unusual flagella and microtubular systems of the sperm. 8

Neir.atoda

There are relatively few studies concerning the ultrastructure of nematode spermatozoa. Those studies available have demonstrated little uniformity in spermatozoa of the nematodes. The only consistent features are the lack of a

flagellum and the lack of a well-defined acrosome (Poor,

1970). Although the acrosome is a common feature of the

flagellated sperm, its presence in the nonflagellated sperm of nematodes is a controversial issue (Clark, Moretti and

Thomson, 1967; Poor, 1970). Bowen (1925) suggested that the

refringent cone of the nematode sperm might correspond to the

acrosome of the typical animal sperm. The refringent cone is

a common feature in the posterior cytoplasm of ascarid

spermatozoa (Poor, 1970), but is lacking in the spermatozoa

of the stongyloids (Jamuar, 1966) and oxyuroids (Lee and Anya,

1967). The identification of the refringent bodies with

acrosomes was probably due to the claim of their Golgi origin

and superficial resemblance to other sperm acrosomes. Such

ideas were dispelled when Favard (1961) showed the refringent

cone did not originate from the Golgi complex as had been

demonstrated with true acrosomes during spermiogenesis. In

addition, the refringent cone does not give the positive

reaction for mucopolysaccharides which is now an accepted

criterion for identification of an acrosome for many species

(Clermont and Leblond, 1955). 9

Favard's (1961) description of a layer of proacrosomal vesicles located between the refringent cone and plasma membrane in the mature sperm of Ascaris mégalocephala has led to an interesting revelation. These vesicles were found to arise from the Golgi complex and similar vesicles have recently been demonstrated to originate from the Golgi apparatus by Beams and Sekhon (19 70) in the male gametes of

Rhabditis pellio. Clark, Moretti and Thompson (1967) have also shown that vesicles, similar to those observed in

A. megalocephala and R. pellio, fuse with the plasmalemma of mature spermatozoa in Acaris lumbricoides var. suum, undergo dehiscence, and release their contents into the lumen of the female genital tract. In a later study the same investigators

(19 72) have shown these vesicles in the sperm of A. lumbricoides var. suum to be PAS positive and contain acid phosphatase activity. These facts, along with other character istics of these vesicles, suggest an acrosomal function in the sperm of Ascaris.

Annelida

Studies on annelid spermatozoa revealed acrosomes of a very complex nature, both during spermiogenesis (Cameron and

Fogal, 1963; Anderson and Ellis, 1968) and as mature spermatozoa (Colwin and Colwin, 1961a; Austin, 1968; Shay,

1972). In the mature sperm of Lumbricus (Cameron and Fogal,

1963; Anderson and Ellis, 1968; Shay, 1972), the acrosome is 10

approximately 5-7% in length and apically located to the

elongated nucleus which is followed by a short middle piece

and a long flagellum. The sperm plasma membrane covering the

apical end of the acrosome is raised into numerous papillae,

the tips of which are differentiated into a thin membranous

dome. The acrosome vesicle, which is bounded by a membrane,

is indented at its base and contains an axial rod that extends

about ly into its body. This rod also extends posteriorly

into the dense granular matrix located between the base of the

acrosome and the nucleus. An electron-dense material of

finely granular structure is found throughout the length of

the acrosome vesicle. Anderson and Ellis C1968) suggest that

the intricate compartmentalization of the Lumbricus acrosome

reflects physiological and functional specialization involved in sperm attachment and penetration.

One of the most detailed descriptions of sperm-egg inter

action in any organism has been conducted on thé annelid

Hydroides by Colwin and Colwin (19 61a; 1961b; 1961c). The

acrosome of Hydroides, like that of Lunû^ricus, is a complex structure. The acrosome vesicle, about ly in diameter, is

positioned over the anterior quarter of the nucleus and bounded by a single continuous membrane. This membrane is

divided into three zones by the Colwins (1961a). The inner

zone lies against the apex of the nucleus; the intermediate zone encircles the sides and narrowed apical part of the 11 nucleus and the outer zone curves away from the nucleus and is closely invested by the plasma membrane. The inner acrosome membrane contains several short tubules representing the priinordia of the acrosome filaments (Austin, 1968). Centrally located on the acrosome outer zone is an apical vesicle that constitutes a "lid." According to the Colwins (1961a), the rim of this lid presents a natural "fracture line" or "rim of dehiscence" during the acrosome reaction.

In the polychetes, Nereis japonica and Nereis limbota, spermatozoa have acrosomes in the form of a double walled hollow cone. Contained within an enclosed cavity of the cone is the anterior half of an axial rod (Austin, 1968). The posterior half of the rod is located within a deep invagina tion of the nucleus. Acrosomes of N. japonica differ from those of N. limbota in size and shape. The acrosome of

N. japonica is a large triangular structure, whereas that of

N. limbota is relatively small and conical in shape. The complexity of the acrosome in Nereis fits the general pattern of acrosome complexity found among other members of the

Phylum Annelida.

Mollusea

Since sperm structure of gastropods, pelecypods, and

cephalopods has received considerable investigative attention

among mollusks, sperm acrosome morphology of these classes will

be considered in the following section. 12

The spermatozoa of gastropods are usually long and thread-like with an acrosome situated at the anterior tip.

The acrosome of the mature sperm of Nucella is a complex two cone type acrosome (Walker and Macgregor, 1968) and is in many respects similar to. the acrosome of the cricket. Acheta domestica described by Kaye (1962). The principle component, the acrosome cone is about lu long and consists of a membrane enclosing a ring of longitudinally arranged tubules. At its base the cone widens to form an invagination. Within this invagination are five rods embedded in a matrix. In mature ' testicular spermatozoa, a row of microtubules occupies a thin layer of cytoplasm separating the acrosome from the cell membrane. These microtubules, which extend longitudinally from near the tip of the acrosome to about 3/4 the length of the sperm head, are not present in mature spermatozoa from the testicular duct (Walker, 1969).'

Electron microscopic examination of different orders of pelecypod spermatozoa presents a similar design in the acrosome structure (Dan, 1967). The acrosome vesicle is in the form of a hollow cone due to invagination of the posterior end. The lumen of the invagination extends into the anterior end of the nucleus and is found to contain a fibrous material embedded in a matrix. This general plan of acrosome morphology is observed in Mytilus (Niijima and Dan, 1965a), Crassostrea virginica (Galtsoff and Philpott, 1960) and Barnea cardida 13

(Pasteels and de Harven, 1962). The sperm acrosome of the mussel, Mytilus, is a conical vesicle with a deeply invaginated base forming a sheath around an axial rod, which extends through the anterior part of the nucleus by means of a nuclear canal. This rod is solid at its anterior end and loosely fibrillar in nature within the nuclear indentation. The acrosome vesicle contains a minimum of two different sub stances on the basis of differences in appearance. A cup-like conical acrosome vesicle of highly osmiophilic substance is found in the oyster, Crassostrea virginica,and is separated from the nucleus by a very thin acrosome membrane. As in

Mytilus, an axial rod traverses the sperm from the posterior invagination of the acrosome to almost the base of the nucleus and is composed of a fibrous material. The acrosome vesicle of Barnea Candida is shaped like a doubled walled hollow cone.

The cavity of the acrosome vesicle contains a broad axial rod limited by a membrane. The contents of this vesicle appear as a sparse coarse granular material.

In a recent ultrastructural study on the spermatozoa of the cephalopod. Octopus bimaculatus, the structure designated as the acrosome is spiral-like in form (Longo and Anderson,

1970), approximately 7y long, highly electron dense, and periodically displays dense striations perpendicular to the long axis of the spermatozoa. 14

Acrosome morphology among the classes of Mollusca Is diverse and complex. The acrosomes are generally cone-shaped and usually contain some form of an axial rod associated with the acrosome. As with other groups of organisms, the basic form displays many variations.

Echinodermata

A great deal of the information concerning the morpho logical aspects of fertilization comes from studies on gametes of echinoderms. Four of the five classes of echinoderms studied have spermatozoa with spheroidal heads (Dan, 1967).

Only the echinoids have spermatozoa with conical heads

(Afzelius, 1955; Bernstein, 1962). However, the spermatozoa of all classes represent the so-called typical sperm as previously described.

One of the pioneer workers on the ultrastructure of the sea urchin sperm and the acrosomes was Afzelius (1955).

Afzelius (1955), and later Afzelius and Murray (1957) described the acrosome region of several species of sea urchins. Their description depicted the acrosome as a round structure approximately 0.24% in diameter occupying the mouth of an invagination in the anterior end of the nucleus. Echin- ocardium cordaturn deviated from this general pattern of acrosomal form by having the round acrosome held in the tip of a 2vi long projection. Bernstein's (1962) study on Arbacia punctulata revealed the acrosome to be similar in form to 15 those species of sea urchins previously described by Afzelius

(1955) and Afzelius and Murray (1957) in all but two respects.

First, he noted the acrosome granule was bounded by a membrane that was distinct from the cell membrane and secondarily, he was unable to identify thé'hyaline component underlying the acrosome granule.

Studies on Pseudocentrotus depressus and Heliocidaris crossispina by Dan, Ohori and Kushida (1964) described in more detail the structures formerly observed by earlier workers.

In addition, they described a material located in a wide band around the sperm head below or beneath the plasma membrane.

This material encircled the distal part of the nucleus and adjacent sides of the acrosome granule and corresponds with the periacrosome and subacrosome material described by Colwin and Colwin (1961a; 1963a) in the annelid, Hydroides hexagonus and Saccoglossus kowaleuskii (Austin, 1968). In this investi gation, Dan, Ohori and Kushida (1964) were unable to identify a membrane enclosing the acrosome components.

Franklin's (1965) observations on the spermatozoa of

Arbacia punctulata and Lytechinus variegatus confirmed pre vious descriptions of sea urchin acrosomes and presented some undiscovered features. He observed an apical vesicle at the anterior end of the acrosome granule and tubular invaginations of the inner acrosome membrane. Similar structures had been described earlier by Colwin and Colwin (1961a) in Hydroides 16 hexaqonus sperm, but until now, no such structures had ever been observed in sea urchin sperm.

The general form of the aerosome region found in the starfish has been revealed in studies by Dan (1960), Dan and

Hagiwara (1967) and Hagiwara, Dan and Saito (1967). Dan

(1960) described the acrosome of Asterias forbesi as being set into a depression in the nucleus. The depression con stitutes a bowl-shaped region lined with a dense nuclear membrane. At the base of the bowl-shaped region is found a sac-shaped depression. The acrosome consists of a granule of dense material completely surrounded by a material of low electron density which in turn is laterally surrounded by a thick band of a third, dense substance. The anterior tip of the acrosome displays a characteristic crater-like structure in its center. Fundamentally, the acrosomes of Asterias amurensis and Asterina pectinifera are similar to the acro somes of Asterias forbesi (Hagiwara, Dan and Saito, 1967).

The proportions of the components found in Asterina pec tinifera differ somewhat from those found in the two species of Asterias. Electron microscopic studies on starfish acrosomes demonstrated an important feature of these acro somes was the absence of a surrounding membrane. Using fixation methods that gave better preservation of structural details, Hagiwara, Dan and Saito, (1967) and Dan and Hagiwara (1967) confirmed this fact for Asterias amurensis and Asterina 17 pectinifera and they concluded that the membrane-bound acrosome vesicle found to be characteristic of other taxonomic groups is lacking in starfish spermatozoa.

Arthropoda

In respects to environmental adaptability, the members of Phylum Arthropoda have had extreme success in adapting to many diverse environmental niches. As a result, many species of Arthropoda have some economical importance to man and having achieved such status, the arthropods have become the subjects of a wide range of scientific investigations, including spermatology.

Arachnida In general, the spermatozoa of Arachnida are filiform (Aranea, Pseudoscorpiones) or tubuliform

(Ixodoidea) and in many instances encysted or nonmotile (see

Suleiman, 1973 for review). Sperm acrosomes described for species from the class Arachnida display diverse morphology.

The pseudoscorpions have an elongated aerosome that coils around the nucleus (Boissin and Manier, 1966, 1967; Tuzet,

Manier and Boissin, 1966), while the mature acrosome found in spider spermatozoa consists of a small pointed tip vesicular- like organelle occupying a depression at the anterior end of the nucleus. Traversing the central longitudinal axis in the spider acrosome is a small rod of moderately osmiophilic material. This rod is found to continue a short distance into the sperm nucleus. The sperm acrosome of the liphistid 18 spiders, the most primitive order of Aranaea, resembles the acrosome of the Mytilus sperm (Niijima and Dan, 1965a). It is deeply invaginated at its posterior surface and contains an acrosome fiber within the invagination. This fiber, which is surrounded by a membraneous sheath of nuclear envelope origin, forms a spiral around the nucleus. In the Acarina, the most advanced evolutionary order of Arachnida, a U-shaped acrosome is found (Reger, 1963; Breucker and Horstmann, 1968). The U- shaped acrosome of Amblyomma dessimili is located in the tail of the sperm where it occupies a deep invagination adjacent to the small nucleus (Reger, 1963).

Crustacea The majority of ultrastructural studies on crustacean spermatozoa has been centered on species of the Sub class Malacostraca, namely decapods (Moses, 1961; Chevallier and Mallet, 1965; Langreth, 1965; Brown, 1966; Pochon-Masson,

1968a, b) and isopods (Reger, 1964a, b, 1966; Hollande and

Fain, 1964, 1965; Fain-Maurel, 1970; Reger and Fain-Maurel,

1973). Ultrastructural investigations on the sperm morphology of Cephalocarida (Brown and Metz, 1967), Branchiopoda (Brown,

1970), Ostracoda (Reger and Florendo, 1969a, b; Reger, 1970a),

Mystacocarida (Brown and Metz, 1967), Copepoda (Brown, 1970) and Cirripedia (Munn and Barnes, 1970) have also received considerable attention.

Ultrastructural studies on mature spermatozoa of species

from some subclasses of Crustacea have failed to demonstrate a 19 defined acrosome. Artemia salina (Branchiopoda, Brown, 1970),

Calanus hyperboreus (Copepoda, Brown, 1970), Cypridopsis sp.

(Ostracoda, Reger, 1969a, b; 1970a), Argulus sp. (Branchiura,

Brown, 1970) and Argulus foliaceus (Branchiura, Wingstand,

1972) are classified as crustacea lacking definite acrosome structures. One may take exception to Argulus sp., Argulus foliaceus and Cypridopsis sp. since they display questionable morphology in the head region of their spermatozoa. A trough- shaped structure that extends the entire length of the sperm might be considered an acrosome in Argulus sp. (Brown, 1970) while certain Golgi derived membraneous organelles in

Cypridopsis sp. may be considered as modified acrosomes. The spermatids of Argulus foliaceus develop an acrosome vesicle and acrosome filament but these disappear in the mature fili

form spermatozoa. Only an insignificant vestige, the pseudo- acrosome, remains in the mature spermatozoa of Argulus foliaceus (Wingstrand, 1972).

Acrosome structures have been described in species from

the subclasses Cephalocarida, Mystacocarida and Malacostraca.

Brown and Metz (1967) described the acrosome region in a

cephalocarid, Hutchinsoniella macracantha, as a two part disc

shaped structure. The major surfaces of the distal part of

the acrosome lay in the plane of the long axis of the sperm

while the button-shaped proximal part lay perpendicular to the

long axis. In the mystacocarid, Derocheilocaris typicus, the 20 acrosome is trough-shaped (Brown and Metz, 1967). The entire structure is electron dense and slightly overlaps the flagellum and nucleus, which both arise at the base of the acrosome.

Mature sperm ultrastructure of the isopods, members of the subclass Malacostraca, have been studied by Reger (1964a),

Kutish and Hinsch (1970) and Reger and Fain-Maurel (1973). In addition, the developing spermatozoa of the isopods have been examined by Reger (1964b, 1966) and Fain-Maurel (1966). Ultra- structural descriptions of the morphology of the sperm head

(Reger, 1964a, b, 1966; Fain-Maurel, 1966; Hollande and Fain-

Maurel, 1964, 1965) as well as ultracytochemical investiga tions of this region (Kutish and Hinsch, 1970) have not settled the controversy concerning the existence of an acrosome.

Whereas the vesicular-like structure covering the nuclear-tail junction is referred to as an acrosome by Reger (1964b, 1966),

Hollande and Fain (1964, 1965) and Fain-Maurel (1966) refer to it as a "spermatic vesicle." This structure is not of

Golgi origin and according to Hollande and Fain (1964, 1965) and Fain-Maurel (1966) has no known function. The picture is further complicated by unsuccessful attempts to definitely localize acrosomal-type enzymes within this region (Kutish and

Hinsch, 1970).

Decapod spermatozoa are morphologically diverse and complex. All are characteristically nonmotile and generally 21 lacking typical mitochondria and flagella. They can be tack- shaped (Pochon-Masson, 1969) or stellate in form (Moses,

1961; Chevaillier and Maillet, 1965; Reger, 1970b; Hinsch,

19 69, 19 71; Brown, 1966; Langreth, 1969). Typically, the acrosome is spherical and covered by an apical cap which sur rounds an acrosome tubule. This entire complex is deeply embedded or surrounded by the nucleus of the spermatozoon.

Brown's (1966) description of the acrosome in Callinectes sapidus provides an excellent example as to the complexity of the decapod acrosome. The body of the acrosome, which is covered by an apical cap, is composed of several adjacent layers surrounding a central canal. Inside the central canal is found the acrosome tubule, a structure consisting of microtubules distally and a granular material proximally.

Diplopoda Horstmann (1970) and Horstmann and Breucker

(1969) have studied the sperm of the diplopod, Spirostreptus sp. This acrosome constitutes a complex structure composed of . five different regions. The overall shape of the acrosome complex is that of an "M", which constitutes an electron-dense granular material. The "M" shaped body is covered with an envelope of fibrous material and located in the center of the

"M" is a nail-shaped, membrane bounded, acrosome vacuole. . At the enlarged distal end of the vacuole is a collette of neatly arranged material that is surrounded basally and laterally by a crystalloid-like material. In complexity, this acrosome 22 rivals that of the previously described decapod, Callinectes sapidus (Brown, 1966).

In contrast to the complex acrosome found in

Spirostreptus sp., the absence of a morphologically identified acrosome in the mature sperm has been reported for the diplopod, Polydesmus sp. (Reger and Cooper, 1968). According to these authors "acrosome vesicles" coalesce with the chromatin and appear as electron-lucent areas in the mature sperm.

Chilopoda Very little information exists on the ultrastructure of Chilopoda spermatozoa. Sperm ultrastructure in two species has been described and the acrosomes in the spermatozoa of these species display gross morphological dif ferences. In Geophilus linearis, the sperm acrosome is a rod like structure with delicate longitudinal striations

(Horstmann, 1968) and is approximately 7y long and O.Sjj wide.

A membrane found surrounding the acrosome is separated from the sperm plasma membrane by a small amount of granular material. On the other hand, the sperm acrosome of Lithobius forficatus L. is conical-shaped (Descamps, 1972), 4vi long, and increases in width in an apical-basal direction. Like the acrosome of G. linearis sperm, the acrosome in L. forficatus sperm is also surrounded by a membrane. Instead of a granular material between the acrosome membrane and sperm plasma membrane, the acrosome of L. forficatus sperm contain a 23 fibrous material in this region.

Symphyla and Pauropoda

Like Chilopoda, very little information is available on the ultrastructure of spermatozoa in Symphyla and Pauropoda.

In a recent study on one species of Symphyla and two species of Pauropoda, Rosati, Baccetti and Dallai (1970) reported the absence of typical acrosomal-like structures in the sperm of each species. The authors did observe a system of Golgi derived proteinaceous concentric membranes in the sperm head of Symphylslla vulgaris. These structures, suggest the

authors, are analogous to the typical acrosome of other sperm

atozoa.

Insecta The ultrastructure of the sperm acrosome has

been described in a large number of insects. Acrosomes in

these creatures display many variations in size, shape and

form and are highly structured and complex as in the water-

strider, Gerris, or completely lacking, as in the mealy bug,

Pseudococcus (Ross and Robison, 1969).

Apterygota Of the four orders in the subclass

Apterygota listed by Clark and Panchen (1971), the ultra-

structure of the acrosome has been described in two; Thysanura

and Collembola. The acrosome complex in Thermobia, a member

of the order Thysanura, is conical in shape (Bawa, 1964). The

acrosome complex extends over one side of the nucleus and 24 occupies a position within the sperm that is posterior to the nucleus. Structurally, the Thermobia acrosome consists of a homogeneous electron dense granular material. In contrast, the morphology of the acrosomes in species of the order

Collembola is more complex. Dallai (1970), in a study on spermiogenesis in certain Collembola, described the acrosome as a cylindrical structure with a tapering top. He found the center of the acrosome to contain a cylinder or.rod-like structure composed of an amorphous material. This cylinder not only traversed the acrosome, but the nucleus as well.

Pterygota Fine structure details of the sperm acro some have been described for many of the orders listed under the subclass Pterygota. When one speaks of the diversity of acrosome structures found in insect spermatozoa it is with reference to the species of this subclass. To date, only two orders of the Pterygota have been found to possess sperm atozoa that characteristically lack an acrosome. These are orders Neuroptera (Baccetti, Dallai and Rosati, 1969b) and

Trichoptera (Phill:.ps, 1970; Baccetti, Dallai and Rosati, Î 1970). The absence of the sperm acrosome in an insect sperm is not a characteristic restricted to these two orders. A homopteran (Ross and Robison, 1969) and a coleopteran

(Furieri, 1963) have also been reported to lack a sperm

acrosome. 25

Recently, Baccetti, Dallai and Giusti {1969a) described

the sperm acrosome of Chloëon. This insect is classified as

a member of the order Ephemeroptera, the most primitive of the subclass Pterygota. These authors found the acrosome in

this species to be apically located and very small. The

contents of the acrosome were homogeneous and encased in a

membrane. In respect to smallness in size and homogeneity,

the acrosome of Chloëon resembles that of the dipteran Sciara

(Phillips, 1966).

The complexity of the orthopteran acrosome has been

demonstrated in a study by Kaye (1962) on acrosome morpho

genesis in the house cricket. Acheta domestica. This acrosome

is a bipartite structure consisting of two concentric hollow

cones. The outer cone, whose boundaries are marked by

membranes, contains a deep invagination. The inner cone,

which is not bounded by a membrane, extends the length of the

space created by the invagination. The base of the acrosome

is solid and bulges past the lower extremities of the inner

cone,

Morphogenesis of the acrosome in the cave cricket,

Ceuthophilus secretus, is similar to that in Acheta domestica

(Shay and Biesele, 1968). The mature acrosome is also

similar to that of Acheta and is composed of two distinct

areas. The developing spermatozoa of other orthopterans have

also been investigated (Kessel, 1967; Dass and Ris, 1958) but 26 details of the mature acrosome in these studies were not given.

Fine structure studies on dictyopteran spermatozoa has revealed at least three basic acrosome forms. Blaberus craniifer has an acrosome that consists of a single compact electron opaque layer (Rosati, 1967). The acrosome of the

American cockroach, Periplaneta, has a duck bill shape

(Eddleman, Breland and Biesele, 1970). Like that of Blaberus, the acrosome is homogeneous and is composed of an electron opaque material. The Periplaneta acrosome also contains rod like structures. An acrosome rod is also present in the cock roach, Pycnoscelus indicus, and occupies the central space found in the conical-shaped acrosome (Shahaney, Fisk and

Parrish, 1972). This rod passes to the nucleus and occupies an anterior invagination. The authors contend the acrosome rod of the cockroach corresponds to the "inner cone" of the cricket acrosome described by Kaye (1962).

The spermatozoa of species representing the order

Mallophaga have only been recently described (Baccetti, Dallai and Rosati, 1969b) and are biflagellate, an unusual character istic which is also found in the order Anoplura (Ito, 1966).

In the mallophagan, Menopon gallinae, the acrosome is spindle-shaped and consists of an electron-dense homogeneous material. Otherwise, very little information concerning the fine structure of the mallophagan sperm has been published. Filmed as received without page(s)

UNIVERSITY MICROFILMS. 28 osmiophilic in the center than at the periphery, and is surrounded by a double layer of membranes. Located between these membranes and the sperm plasma membrane are small bundles of microtubules. In a similar study on a species representing the order Neuroptera, the authors were unable to locate an acrosome. The species from the Order Mecoptera also have bundles of longitudinal microtubules associated with the mature acrosome (Baccetti, Dallai, and Rosati, 1969a). These microtubules were found to encase the conical-shaped elongated acrosomes of Panorpa annexa and Panorpa germanica.

From studies made to date, -the conical-shaped acrosome is characteristic of mecopterans. Recently, Gassner, Breland and

Biesele (1972) described the acrosome of another mecopteran,

Panorpa nuptialis, as consisting of an acrosome core, cone, and anterior space, but did not observe the presence of microtubules in association with the sperm acrosome.

Lepidopterans normally produce two types of spermatozoa.

One type has a nucleus (eupyrene) and the other type is

anucleate (apyrene). The following descriptions apply only

to eupyrene spermatozoa since apyrene spermatozoa lack an

acrosome. Testicular spermatozoa of several species of

lepidopterans have been examined at the ultrastructural level.

Andre (1959; 1961) has described spermiogenesis in Pieris and

Macroglossum. Observations on testicular spermatozoa have

also been made on various lepidopterans by Yasuzumi and Oura 29

(1964), Phillips (1970; 1972) and Riemann (1970). A general description of the acrosome found in lepidopterans was pre sented by Phillips (1972) in a summary of observations on the testis, vas deferens, and female tract of a large number of species. The acrosomes of all species examined are very small and composed of homogeneous, moderately electron-dense material. They usually take the form of a cup-shaped cap and cover the anterior tip of the nucleus. Some species have rod shaped acrosomes that are adjacent to the anterior portion of the nucleus.

Ultrastructural examination of the acrosomes of dipteran species usually display a rather common feature of the acro some occupying a nuclear groove or invagination (Bairati and

Perotti, 1970; Phillips, 1966; Gassner, Klemetson and Richard,

1972). The complexity of the acrosome varies in form and structure. Sciara copophila has a small homogeneous acrosome

(Phillips, 1966), while that of Drosophila is dagger-like in shape and contains zones of different densities within its fine granular matrix (Shoup, 1967; Bairati and Perotti, 1970).

In addition, the Sciara acrosome is not membrane bounded like

the acrosome of Drosophila.

Recently, acrosome structures in spermatids of the

blowfly (Warner, 1971) and the housefly (Gassner, Klemetson

and Richard, 1972) have been examined. The mature acrosome

of the blowfly consists of a membrane bound striated filament 30 enclosed at its tip by a series of concentric lamellar elements. The spermatids of the housefly, on the other hand, has a slipper-shaped acrosome that fits along the apex of the nucleus and into a nuclear invagination which receives the base of the acrosome.

Early ultrastructural studies on spermatozoa of the order

Hymenoptera (Rothschild, 1955; Wilkes and Lee, 1965) failed to mention an acrosome. In 1967, Thompson and Blum gave a brief description of the acrosome in the fire ant, Solenopsis

laevissima. This acrosome is described as a rod-shaped structure that originates deep within the head of the sperm.

Hoage and Kessel (1968) subsequently described the spermatid

acrosomes of Apis mellifera L., a honey bee, as a hollow

conical-shaped structure with an electron-dense granular peg.

The peg was found to extend the length of the acrosome. These

authors also observed adjacent to the acrosome in the

spermatid microtubules which disappear following spermio-

genesis. Microtubules adjacent to the acrosome are also

observed in the spermatids and mature spermatozoa of

Chryptothrips (Baccetti, Dallai and Rosati, 1969b), Panorpa

annexa, and Panorpa germanica (Baccetti, Dallai and Rosati,

1969a). Recently, de Cruz-Hofling, de Cruz-Landim and Kitajima

(1970) gave a more detailed description of the Apis mellifera

acrosome. 31

In conclusion, the acrosomes of dipteran species are quite variable in form and structure and commonly occupy a nuclear invagination within the sperm.

Species from the order Homoptera have thread-like or filamentous spermatozoa. One group of homopterans, the coccid insects, possess spermatozoa with no distinguishable head, middle piece, or tail (Robison, 1966; 1970; Ross and Robison,

1969) and no obvious acrosomes. However, not all homopterans have been found to lack acrosomes. For example, Peregrinus maidis has a fibrous' cap-like acrosome at the anterior end of the nucleus (Herold and Munz, 1967). A large part and several small parts of this acrosome extend posteriorly alongside the nucleus and the texture of. the acrosome is fibrous. In general, this acrosome is similar to the perforatorium ob served in the toad (Burgos and Fawcett, 1956). Several other homopteran spermatozoa have been examined and have acrosomes.

In most species studied by Folliot and Mailett (1970), the acrosome was not located at the tip of the spermatozoa.

Instead, the tip was occupied by a cytoplasmic bleb that was as long as the acrosome. The acrosome in these species is described as a fibrous structure containing a hollow cavity along one face.

In summary, the acrosomes of invertebrate spermatozoa

are extremely diverse in morphology and complexity. Some

invertebrates have spermatozoa which contain no acrosomes 32

(Platyhelminthes) while spermatozoa of other invertebrates contain only vesicles in the classical region of the acrosoiae

(coelenterates, nematodes). The sperm acrosome of many in vertebrates also contain an accessory structure, the acrosome rod which is either pre-formed as in the spermatozoa of mollusks or formed during the acrosome reaction as in sperm atozoa of echinoderms. In considering the many species of invertebrates examined, the acrosome position in the sperm is extremely varied. However, in most phyla the acrosome is located at the anterior end of the sperm. In Acarina the acrosome resides in the tail of the sperm and in Decapoda

Crustacea the acrosome may be partially surrounded by the nucleus. Thus, not only are no clear cut patterns in sperm morphology present from phylum to phylum but no recognizable patterns in the morphology and the position of an acrosome within a phylum are observed.

Ultrastructure of Vertebrate Sperm Acrosomes

Most of the attention devoted to the ultrastructure of vertebrate spermatozoa has been centered on mammalian species with relatively little on the lower chordates. The acrosome region of chordates generally comprises an acrosome and some subacrosome material. The typical morphological shape of the

vertebrate acrosome is that of a cap (Nicander, 1970) and is

represented in Dipnoi (Jespersen, 1971), Urodela (Picheral, 33

1967), Anura (Burgos and Fawcett, 1956; Poirier and Spink,

1971), Reptilia (Furieri, 1970), Aves (Nagano, 1962) and

Mammalia (Fawcett and Phillips, 1970). Only one major group, the teleost fish, are characterized by the absence of any acrosome structures (Stanley, 1969).

Agnatha

Electron micrographs of whole lamprey spermatozoa were published by Afzelius and Murray (1957) and Kille (1960).

Follenicus (1965) and Stanley (1967) later published detailed accounts on the ultrastructure of sectioned spermatozoa. The acrosome of Lampetra planeri is described as a vesicular structure located at the anterior end of the nucleus (Stanley,

1967). It is memb'rane-bound and composed of finely granular material. In appearance this acrosome vesicle resembles that of the hemichordate, Saccoglossus (Colwin and Colwin, 1963a).

Follenicus' (1955) description of the L. planeri acrosome is somewhat different from the descriptions by Stanley (1967).

He observed a number of clear vesicles in the membrane-bound cytoplasmic mass that he called the "apical vesicle"

(acrosome). Both Follenicus (1965) and Stanley (1967) described a subacrosome ring, and a long subacrosome fibre running through the nucleus. This rod extends to form a true

acrosome tubule during the acrosome reaction (Nicander and

Sjoden, 1968). 34

In Myxine glutinosa, a hagfish, the acrosome is more typical of vertebrates. It is cap-shaped and located at the tip of the nucleus (Nicander, 1970). Positioned between the nucleus and the acrosome is a granular subacrosome material.

In conclusion, sperm acrosomes of Agnatha are basically of two types; those resembling certain species in invertebrates in which the acrosome is vesicular and those resembling the typical cap-shaped vertebrate acrosome.

Chondrichthyes

Fine structure studies on elasmobranch spermatozoa have been few in number. In the first fine structure reports on the sperm of the spiny dogfish, Squalus suckleyi, no descrip tion of the acrosome was given (Stanley, 1964; 1965). Later, a more detailed description of spermatogenesis in £. acanthian

(Holstein, 1969) and spermiogenesis in S. suckleyi (Stanley,

1971a; 1971b) appeared in the literature. These studies showed the acrosome of the mature sperm of Squalus to be cap- shaped. In addition, the acrosome has a deep posterior invagination which is positioned over the pointed nuclear tip and within this invagination is an acrosome rod. Posteriorly, the edge of the acrosome is asymmetrical with one side more posterior than the other. Recently, Mattei (1970) published a comparative sperm study from a number of elasmobranchs. The author demonstrated that the skates and other elasmobranchs have the typical cap-shaped acrosomes of the vertebrates. 35

Os teichthyes

Ultrastructural studies on the spermatozoa of teleost fish demonstrated the absence of an acrosome in these organisms (Stanley, 1969; Mattel, 1970; Nicander, 1970;

Billard, 1970). The Dipnoi, on the other hand, display the typical cap-shaped acrosome. Information on the acrosome structures is known for two species; the African lungfish,

Protopterus annectens, (Mattel, 1970) and the Australian lungfish, Neoceratodus forsteri, (Jespersen, 1971). Both have similar acrosomes except for the presence of two rod-shaped structures that penetrate the acrosome and the nucleus in

Neoceratodus.

Amphibia

The first ultrastructural study of anuran sperm struc

ture was published by Burgos and Fawcett (1956) in a study on

spermatogenesis in the toad, Bufo areharum. Again the cap-

type acrosome is observed and takes the form of small rods

above and alongside the nuclear apex. These authors also

describe a perforatorium in the sperm head located posterior

to the acrosome. Similar morphology was observed in the

mature sperm of Bufo bufo as described by Furieri (1961).

A description of acrosome formation and the occurrence of

polysaccharides in Discoglossus has been reported by Sandoz

(1970a; 1970b). The acrosome complex of this species consist 36 of a cap-shape acrosome and some dense subacrosome material.

More recently, a study on the comparative fine structure of testicular spermatozoa in two species of Rana has been reported (Poirier and Spink, 1971). The acrosome in the sperm of Rana clamitans is described as a cap-shaped homogeneous structure surrounding many finger-like projections at the anterior portion of the nucleus. Since this acrosome is homogeneous and overlaps the nucleus, it resembles the acro some found in mammalian spermatozoa (Fawcett, 1970) more so than those of other anurans. The acrosome of Rana pipiens, on the other hand, is located at the tip of the nucleus and is not cap-shaped. In R. clamitans no inner and outer acrosome membranes are present. Only i±ie sperm plasma membrane bounds the acrosome. In addition, certain granules filled with small particles found in R. pipiens acrosomes have not been observed in R. clamitans acrosomes.

Ultrastructural observations on the sperm acrosomes of salamanders reveal the typical cap-shaped acrosome structure characteristic of the vertebrates (Baker and Biesele, 1967).

Posterior to the cap in the acrosome complex is found a long electron-dense subacrosome rod. This rod penetrates deeply into the nucleus in Pleurodeles (Ficherai, 1967) and has been found to display an internal honeycomb like appearance in

Triturus (Nicander, 1970). Among the sperm acrosomes observed in the urodeles, Pleurodeles (Picheral, 1967) and Notophtholmus 37

(Fawcett, 1970) have two of the most complex. The acrosome cap in Pleurodeles, which is similar to Notophtholmus viridescens has a blunt terminal knob and a hook at its tip.

In Notophtholmus, this cap is drawn out to a slender point and the acrosome has a projecting barb-shaped structure giving the intact acrosome the appearance of a harpoon. The contents of the acrosome appear homogeneous. Posterior to the cap exists a subacrosome space containing a long rod or perforatorium.

From a morphological standpoint, the acrosomes of these two urodeles resemble those found in certain mammals (Fawcett and

Phillips, 1970)1

Reptilia

Furieri (1970) has recently reviewed sperm morphology in two orders of reptiles, the Chelonia and Squamata. The sperm acrosome in Chelonia consists of two coaxial caps. The internal lesser opaque cap is composed of finely granular material while the outer more opaque cap is homogeneous. The acrosomes of Squamata differ from those of Chelonia by dis playing a paracrystalline structure in the internal cap. In general, sperm acrosomes of reptilia consist of two coaxial caps of variable morphology and internal composition.

Aves

Nagano (1962) investigated the; developing sperm in the domestic chicken. He found the acrosome in the late spermatid 38

stage to consist of a cap-shaped vesicular structure which was

located anterior to a mass of subacrosome material occupying

invaginations in both the acrosome and nucleus. This sub

acrosome material constitutes the axial rod or "perforatorium"

that is characteristically found in urodele spermatozoa.

Recently Lake, Smith and Young (1968) described this structure

in the acrosome of ejaculated fowl spermatozoa and found the

acrosome spine or subacrosome material to be composed of

densely-packed, longitudinally-arranged, layered material.

The structure of the spine is in contrast to the acrosome

proper which is homogeneous in composition.

Mammalia

Since the first review of mammalian sperm ultrastructure

by Fawcett (1958), numerous reviews (Hancock, 1966; Fawcett

and Phillips, 1970; Fawcett, 1970) and accounts of mammalian sperm ultrastructure have appeared in the literature (Nicander

and Bane, 1962; Pedersen, 1970). In general, the mammalian

acrosome has been considered simpler than those of non-

mammals because the substance of the acrosome appears to be

more homogeneous and the acrosome reaction does not involve

the formation of an acrosome filament. Recently biochemical

evidence has suggested that the acrosome contents of

mammalian acrosomes display a higher degree of organization.

Investigations by Phillips (1972) suggest the acrosome enzymes

are highly ordered and arranged within specific regions of the 39

acrosome. If this is the case, the mammalian aerosome may no

longer be considered of greater simplicity than the inverte

brate acrosome.

Morphologically, the mammalian acrosome is not simple,

particularly in shape and volume since extreme variability

exists (Fawcett, 1970). As a rule, the acrosome is not con

fined to the tip of the nucleus and is found to extend

posteriorly over a large portion of the nuclear surface area.

For convenience of description, Fawcett C1970) has divided the acrosome into apical, main, and equatorial segments. The

apical segment projects beyond the anterior margin of the nucleus; the main segment extends posteriorly over the anterior portion of the nucleus and the equatorial segment comprises the caudal end of the cap.

The apical segment of the acrosome is small and extends a short distance beyond the tip of the nucleus in the monkey

[Bedford, 1967), man (Pedersen, 1970), boar (Meander and

Bane, 1962), bull (Blom and Birch-Andersen, 1965), rabbit

(Bedford, 1968) and bat (Fawcett and Ito, 1965). In contrast,

the apical segment in the guinea pig (Fawcett, 1965),

chinchilla (Fawcett, 1970) and ground squirrel are large and

extremely elaborate in morphology.

Since the early I960's a number of investigators have

found structures in the subacrosome space of the rabbit (Hadek,

1963), bull (Blom and Birch-Andersen, 1965), boar, ram, dog 40 and stallion (Bane and Nicander, 1963) and many have suggested that theste structures are homologous to the perforatorium found in tiie spermatozoa of birds (Nagano, 1962) and toads

(Burgos and Fawcett, 1956). This suggestion is somewhat controversial since other investigators deny the existence of any such structures in mammalian sperm (Fawcett and Phillips,

1969). However, both camps do agree that a subacrosome space exists in mammalian spermatozoa but whether it contains a preformed component or just amorphous material has not been settled.

Even though vertebrate sperm acrosomes are most often cap-shaped, the acrosome may take the shape of an apical body as observed in some frogs and the river lamprey. In addition; an acrosome filament is associated with sperm acrosomes of the domestic fowl, urodele amphibians, and the river lamprey.

In other species, namely teleost fish, the sperm acrosome is completely lacking. Thus, considerable variation from the typical cap-shaped sperm acrosome is observed among vertebrate species.

Chemical Content of the Sperm Acrosome

Very little significance was attached to the chemistry and staining nature of the sperm acrosome until Wislocki

(1949) observed the PAS staining reaction to be highly specific for acrosome material. Since then it has been well 41

documented that acrosome material gives a positive PAS

reaction for polysaccharides (Leblond, 1950; Leuchtenberger

and Schrader, 1950; Clermont and Leblond, 1955; Moriber, 1956).

In addition to containing polysaccharides, acrosomes of cer

tain insects (Schrader and Leuchtenberger, 1951; Moriber,

1956), crustaceans (Chevaillier, 1967) and the marine worm,

Urechis caupo (Das, Micou-Eastwood, and Alfert, 1967) have

been found to contain a class of basic proteins different

from those characteristically located in the nucleus.

For some time the chemical nature of the acrosome has

been suspected as being similar to that of lysosomes. Bishop

and Smiles (1957, 1963) found that vital staining of rat and

guinea-pig spermatozoa produced a red fluorescence with

acridine orange in the acrosome. Confirmation of these

observations have been made in several other species of

mammals (Allison and Hartree, 1970) and in the horseshoe crab,

Limulus polyphemus (Bennett and Austin, 1970). The red

fluorescence observed in the acrosome is similar to that ob

served in lysosomes of other cells (Allison and Young, 1964).

Originally, the red fluorescence was thought to be specific

for RNA (Armstrong, 1956), but it is now believed that

staining living cells with aminoacridines is characteristic

of lysosomes (Allison and Young, 1964). According to Allison

and Hartree (1970), the lysosomal uptake of aminoacridines is

%;^ue to binding by a specific glycolipid component. This 42 component, which can be isolated by disc electrophoresis

(Barnett and Dingle, 1967), is probably also responsible for the PAS reaction observed in lysosomes and acrosomes. This, and the employment of biochemical methods to demonstrate a large number of lysosomal enzymes in the acrosome of the ram

(Allison and Hartree, 1970), further supports the thesis that acrosomes are in fact specialized lysosomes.

The majority of histochemical studies on sperm acro somes have been concerned with the localization of acid phosphatase. This enzyme is present in the acrosomes of the frog (Novikoff, 1961), salamander (Buongiorno-Nardelli and

Bertolini, 1967), guinea-pig (Ruffili, 1960), horseshoe crab

(Bennett and Austin, 1970), sea urchin (Anderson, 1968), and the nematode Ascaris (Clark, Moretti and Thomson, 1972).

Localization of acid phosphatase activity has also been reported in the subacrosome spaces of rabbit and bull spermatozoa (Teichman and Bernstein, 1971).

The histochemical localizations of other hydrolyases have also been studied. The localization of nonspecific esterases in guinea-pic acrosomes has been reported by Birns

and Masek (1961) and Allison and Hartree (1970). Meizel

(1970) also found nonspecific esterase activity in the bull sperm. In these spermatozoa, enzyme activity is found to be

associated with the head, middle-piece and main tail-piece.

More recently, the histochemical localization of arylsulphatase 43 activity in acrosomes of the rat has been reported (Seiguer and Castro, 1972).

Within the past few years, the cytochemical demonstration of proteolytic activity in acrosomes has gained considerable attention. Even though gelatin had been used for many years to demonstrate proteolytic activity in various mammalian tissues, Gaddum and Blandau (19 70) most recently adopted this procedure to visualize proteolytic activity in sperm acro somes. By preparing gelatin membranes impregnated with India ink., they were able to provide contrast for observing the proteolytic reactions in the acrosome regions of sperm from a number of mammals. These reactions take place when sperma tozoa are applied to gelatin membranes that have been fixed in glutaraldehyde. Penn, Gledhill and Darzynkiewicz (1972) claimed to have improved the reproducibility and reliability of the technique by using Kodak AR-10 autoradiographic plates as a source for the gelatin substrate. Their modified technique has been used to give the first direct demonstration of proteolytic activity in the acrosome region of an amphibian sperm.

Although the gelatin membrane technique for localizing acrosome proteolytic activity is receiving considerable attention, the cytochemical approach has not been overlooked.

Recently, Yanagimachi and Teichman (1971) used a cytochemical method to demonstrate sites of proteinase activity in the 44 ' sperm acrosome caps of several mammals and an avian.

The biochemical characterization of proteolytic activity

found in the acrosomes of spermatozoa- is also receiving a

great deal of attention. Since Yamane (1935a, b) first

reported the presence of proteolytic activity in mammalian

spermatozoa, interest in demonstrating proteolytic activity

associated with vertebrate spermatozoa has been steadily

increasing. Even though the majority of studies in this area

have been concerned with mammalian species, the proteolytic

activity in the spermatozoa of other vertebrates has not been

overlooked. The presence of a trypsin-like enzyme activity

in avian spermatozoa is well documented. Buruiona (1956)

reported that avian spermatozoa had a high level of trypsin-

like enzyme activity. More recently, multiple forms of

trypsin-like enzyme activity have been reported in the

spermatozoa of the domestic fowl (Ho and Meizel, 1970).

In the early biochemical studies on spermatozoa the

assignment of enzyme activity to any specific part of sperm

other than the head or tail fraction was extremely difficult.

In order to circumvent these difficulties, numerous attempts

were made to remove or separate the acrosome from the sperm

which would then allow a more precise localization of enzymes

determined through biochemical methods. Among the procedures

used to remove 'acrosomes have been those employing NaOH

(Clermont, Clegg and Leblond, 1955), cetyl-trimethylammonium 45 bromide (Hathaway and Hartree, 1963), barbiturate, Triton

X-100 (Teichman and Bernstein, 1969), sodium hypochlorite

(Bennett and Austin, 1970) and sonification (Stambaugh and

Buckley, 1969). The most widely accepted method for removing acrosomes in several species of mammals is a procedure introduced by Hartree and Srivastava (1965) utilizing the detergent hyamine. This procedure consisted of treating whole spermatozoa with hyamine followed by centrifugation to remove nuclei, midpieces and tails. Use of this procedure by

Hartree and Srivastava (1965) and Srivastava, Adams and

Hartree (1965a, b) was the first step in preparing a lipoglycoprotein acrosome extract from ram spermatozoa. They found the extract to be capable of removing the zona pellucida from the rabbit ova vitro. In addition, treatment of eggs with the extract released several amino acids. Stambaugh and

Buckley (1968, 1969) slightly modified the technique of

Hartree and Srivastava (1965) for obtaining acrosome extracts and were able to obtain an extract that removed the zona pellucida using epididymal rabbit spermatozoa. Also, the

extracts prepared by Stambaugh and Buckley (1968, 1969) were

found to contain trypsin-like enzyme activity.

Zaneveld, Srivastava and Williams (1969) further con

firmed the presence of trypsin-like enzyme activity in the

acrosome extracts of ejaculated rabbit spermatozoa. This

enzyme, termed acrosin, is presently considered essential for 46 fertilization in mammals (Williams, 1972). Similar trypsin- like enzyme activity has been demonstrated in the acrosomes of bull spermatozoa (Multamaki and Niemi, 1969) and human spermatozoa (Pedersen, 1972). Recently, Zaneveld, Polakoski and Williams (1972) and Polakoski, Zaneveld and Williams

(1972) obtained a 250-fold purification of acrosin from rabbit sperm acrosomes and found the enzyme to be a dimer of about 55,000 mol. wt. They suggested that this enzyme acts on the mucoprotein of the egg zona pellucida.

Until these biochemical characterizations of the mammalian sperm acrosome contents, the sperm penetration of the zona pellucida was thought to be due mainly to the action of hyaluronidase (Austin, 1951). In fact, hyaluronidase might still play some role in zona dissolution since the crude extracts prepared by Stambaugh and Buckley (1969; 1970) demonstrated the trypsin-like enzyme responsible for dis persing the zona is a high molecular weight complex of protein ase and hyaluronidase. In addition to hyaluronidase and trypsin-like enzyme activity, biochemical analysis of acro some contents has revealed the presence of other lysosomal enzymes. Among these are proteases (Teichman and Bernstein,

1969) a corona-penetrating enzyme (Zaneveld and Williams,

1970) and a neuraminidase-like factor (Srivastava, Zaneveld and Williams, 1970; Srivastava and Goetsch, 1972). 47

The roles of the various acrosome enzymes in fertiliza tion have recently been thoroughly reviewed by Williams (1972) and Stambaugh (1972). In analyzing these reviews, the majority of the work on the chemical content of sperm acro somes has been performed primarily on mammalian spermatozoa.

With the development of procedures to remove the acrosomes from the spermatozoa and subsequently to analyze the chemical content of the acrosome, a number of enzymes have been identified, purified, and their roles in sperm-egg interactions have been superficially investigated.

Sperm-Egg Interactions

Except for those instances in which development of an organism begins parthenogenetically, the beginning of develop ment of any organism involves the interaction of male and female gametes; namely the sperm and the egg. Approximity of the gametes to insure gamete encounter involves many kinds of special devices, which Austin (1965) has grouped into three major categories; (1) those involving chemical agents to attract gametes, (2) mechanical juxta-position of gametes, and

(3) synhronous production and release of the gametes. The reproductive cycles of most organisms involves at least one of these categories for insuring initial contact of the gametes. Once initial contact of the sperm and egg occurs the sequence of events leading to production of a new organism 48 proceeds. Among those first events in the scheme of develop ment are sperm-egg attachment, the acrosome reaction, and egg activation.

Sperm-egg attachment

Sperm-egg interactions leading to sperm-egg attachment most certainly involve molecular components found on both the egg surface (Metz, 1967; Mowbray, Brown and Metz, 1970;

Mowbray, 1972) and the sperm surface (Cooper and Brown, 1972). The concept of interacting gamete surface components is by no means new. Lillie (1912; 1913; 1919) first introduced the subject in his studies on the effects of sea urchin egg water on sperm agglutination. He found that the egg jelly or egg investments contained a sea water soluble substance (fertilizin) capable of reacting with a component on the sperm

(antifertilizin) and resulting in sperm agglutination. Today little doubt exists that this fertilizin-antifertilizin system functions in adhesion of the sperm to the egg (Metz, 1967).

Since Lillie's observations on sea urchins, soluble egg surface components have been demonstrated in annelids (Lillie,

1919), Molluska (Tyler, 1940), ascidians (Minganti, 1951), cyclostomes (Schartau and Montalenti, 1941), fish (Hartmann, 1944) and amphibians (Glasner, 1914). This wide spread! occurrence of this phenomenon within the animal kingdom, further supports the notion that gamete surface components are

involved in sperm-egg attachment. 49

The observations that sperm-egg attachment is highly species specific suggest the occurrence of an interaction between specific sites on the participating gamete surfaces.

This is borne out by Mowbray, Brown, and Metz (1970) in an in vitro study on sperm-egg interactions in several decapod crustaceans and in Limulus polyphemus. They found sperm-egg attachment to be largely species specific and to involve one or more sperm surface antigens. The presence and involvement of specific sites for attachment of sperm to egg may also explain the rare phenomenon of cross-fertilization. Rothschild

(1956) suggests a certain "rubberiness" of fit exist between gamete surface components. Such a condition would allow for sufficient Van der Waals forces to develop between the gamete surfaces, and. consequently result in gamete adhesion. Al though all the mechanics of sperm-egg attachment have not been worked out, the participation in this process of complementary binding molecules on the surfaces of the gametes is clearly indicated (Mowbray, 1972).

The acrosome reaction

Experimental evidence strongly indicates that initiation of the acrosome reaction, like sperm-egg attachment, involves the interaction of gamete surface components (Dan, 1967).

Popa (1927) was the first to report that egg water causes a material to be released from the acrosomes of sea urchin 50 spermatozoa. Since then initiation of the acrosome reaction by egg water has been reported for annelids, Molluska and other echinoderms (Wada, Collier and Dan, 1956); Colwin and

Colwin, 1955; Metz and Morrill, 1955).

The concept of the acrosome reaction was developed by

Dan (1952; 1956; 1967). She and her co-workers found the reaction to be calcium-dependent in marine invertebrates and to occur under normal conditions in calcium containing sea water when spermatozoa come into contact with eggs of the same species. Also noted was the fact that a normal appearing

reaction could be elicited with alkaline sea water and by

contact of spermatozoa to a glass surface (Dan, 1956; 1967).

Utilizing light microscopy, she observed in several species that the acrosome reaction involves a morphological change at the sperm apex and results in some sort of projection or

apical filament that may vary in length from one species of

organism to another. Fine structural studies have demonstrated

the events of the acrosome reaction in greater detail. The

first changes in the acrosome involve those leading to

dehiscence of the acrosome vesicle. As dehiscence proceeds

the acrosome membrane and the sperm plasma membrane fuse. At

the same time fusion is occurring, the acrosome material

(subacrosomal, periacrosomal and acrosomal membranes) begins

to form a filament that subsequently participates in penetra

tion of the egg envelopes. Excellent descriptions of the 51 stages that constitute the acrosome reaction as it occurs in spermatozoa exposed to eggs or egg jelly have been provided for annelids (Colwin and Colwin, 1961a, b ), hemichordates

(Colwin and Colwin, 1963 a, b ), echinoderms (Dan, Ohori and

Kushida, 1964; Hagiwara, Dan, and Saito, 1967; Dan and

Hagiwara, 1967) and Molluska (Niijima and Dan, 1965a, 1965b).

In addition, descriptions of the acrosome reactions under conditions other than natural has been described for Barnea (Pasteels and de Harven, 1962), Callinectes (Brown, 1966),

Xiphosura (Andre, 1963) and Nereis (Austin, 1968).

Also electron microscopic studies of the acrosome reaction has been described for mammalian spermatozoa.

Changes in the morphology of the acrosome region observed in the rat (Piko and Tyler, 1964), the rabbit and the hamster (Barros, Bedford, Franklin and Austin, 19 67;

Bedford, 1970). The morphological changes in the acrosome region involves a progressive breakdown and fusion between the outer acrosome membrane and the sperm plasma membrane

(Bedford, 1970). This results in the formation of numerous vesicles and openings which presumably allow the loss of the acrosome contents.

The evidence supporting thé actual site of the acrosome reaction during fertilization in mammals is quite contro versial. Zamboni (1971) and Yanagimachi and Noda (1972) are currently engaged in a literary feud as to when and where the 52 sperm loses its acrosome during fertilization. Zamboni (1971) is thoroughly convinced that the "fertilizing" sperm lacks an acrosome before contact with the egg; Yanagimachi and Noda

(19 72), on the other hand, cite their ^ vitro studies on mammalian fertilization as the main support for acrosome loss as the sperm progresses through the egg envelope. One thing is clear, the acrosome undergoes changes in morphology during fertilization in mammals and these changes are considered the acrosome reaction, but the exact stage of any morphological change awaits further clarification.

Egg activation

As the sperm reacts under normal conditions for fertiliza tion, the egg also reacts to contact and penetration of the sperm. Egg reaction, or egg activation as the process is commonly called, may take many forms. The egg may undergo changes in shape, initiate or resume maturation, undergo changes in metabolism, elevate a fertilization membrane, lose the capacity to form attachments with spermatozoa, show the breakdown of cortical granules, or display various other phenomena (Austin, 1968).

Just how the sperm is able to cause such changes in the egg still remains a mystery. This problem is further compli cated by the ability of certain eggs to become activated through means other than those supplied by the spermatozoa.

Heat, cold, detergents and mechanical stimuli are listed among 53 the things known to initiate egg activation (Austin, 1965).

This, and the fact that the egg development of many animals is

parthenogenic, makes egg activation one of the more perplexing

problems of fertilization.

One of the events of egg activation that has received

considerable attention is cortical granule structure and

cortical granule breakdown. As long ago as 1911 Harvey

recognized that certain refractile granules just under the

surface of the unfertilized sea urchin egg disappeared

immediately after sperm penetration. Since that time,

cortical granules have been described in mature unfertilized

eggs of various invertebrates (Afzelius, 1956; Humphreys,

1967; Austin, 1968) and vertebrates (Kemp and Istock, 1967),

including mammals (Sozollosi, 1962; Austin, 1965; Zamboni,

Mishell, Bell, and Baca, 1966). Cytochemical studies of

these cortical granules have demonstrated proteins, glyco

proteins and sulphated mucopolysaccharides (Monne and Harde,

1951; Aketa, 1962; Bal, 1970; Kelley, Berger and Schuel, 1970).

Schuel, Wilson, Bressler, Kelley and Wilson (1972) recently

isolated and purified cortical granules from unfertilized sea

urchin eggs. They demonstrated the presence of the enzyme

1-3 gluconase in the granules, that the total egg protein was

approximately 6.7% and identified sulfated acid mucopoly

saccharides as a component of isolated cortical granules.

Even though egg activation remains very much an unsolved 54 problem, the biochemical characterization of cortical granules in vitro should lead to a better understanding of their roles in the process of fertilization.

Egg Envelopes

In general the egg is a single cell surrounded by a plasma membrane. In addition to the plasma membrane, a number of surrounding layers may be present. The "vitelline membrane", a membrane just external to the plasma membrane, constitutes one such layer. Other layers found surrounding eggs may be composed of chorions, shells, jellies or follicle cells.

During oogenesis or egg maturation, egg envelopes may arise from the ovary or the oviduct. Those membranes having their origin from the oocyte are classified as primary membranes, those arising from the follicle cells are secondary membranes and those produced by the oviducts are tertiary membranes. Classifications of egg envelopes in one or the other of these three categories is not always an easy task.

Some eggs, like those of humans, have envelopes that arise from a combination of two of the above structures involved in membrane secretion (Wartenberg and Stegner, 1960).

In the following discussion no attempt will be made to exhaust the literature on the comparative aspects of egg envelope morphology. Instead, selected examples from various 55 invertebrate and vertebrate phyla will be presented as a means of illustrating the tremendous morphological diversity observed in the structures surrounding mature eggs.

Invertebrate egg envelopes

Coelenterata Eggs or egg cells of Hydrozoa are among the least complex as far as egg envelopes go. The hydrozoan egg is surrounded only by a delicate oolemma until after fertilization when the egg secretes a multilayered embryonic cover (Mergner, 19 71).

In Anthozoa, eggs undergo partial development inside the female gastric cavity, and are surrounded by a delicate oolemma similiar to the type covering hydrozoan eggs. Only those eggs that are shed at an early stage from the female

(Actinia, Urticina) are different. They possess a thick

prickly integument (Mergner, 1971).

Ctenophora Ctenophores, unlike the Hydrozoa, have

eggs possessing jelly envelopes. The ctenophore, Beroe ovata,

has a rather large egg (1mm in diameter) inside of which

three distinguishable concentric regions are displayed. The

surrounding egg envelope consists of a jelly coat and a thin

membrane which thickens after fertilization (Reverberi, 1971c).

Platyhelminthes In contrast to Hydrozoa and

Ctenophora, much more investigative attention has been

directed toward egg envelope morphology and chemistry of egg

envelopes among the representatives of phylum Platyhelminthes, 56 mainly since a large number of the members of this phylum are parasites of economic importance.

The "eggs" of trematodes consist of an ovum and a number of vitelline cells surrounded by a protective capsule or shell

(Smyth and Clegg, 1959). Basically this type of egg is common to most trematodes although differences occur in the capsules. The Monogenea usually have capsules with long polar filaments at both ends whereas the Digenea have no polar filaments. Most Digenea have capsules that character istically contain an operculum.

A wider variety in the structure of the egg is seen in the cestodes than in the trematodes. The eggs of cestodes that reach maturity inside the uterus generally have thin non- operculate capsules. Those that mature in the water have thick operculate capsules (Smyth and Clegg, 1959).

Annelida The eggs of annelids, notably the polychetes, have been used extensively in embryological research.

These eggs have the advantage of being easily obtained, are fertilizible ^ vitro and have the ability to undergo develop ment to advanced stages in the laboratory. Consequently, the egg envelopes in some of these organisms have been described in detail (Lillie, 1912; Austin, 1968; Costello, 1949).

The eggs of the marine polychete. Nereis japonica and

Nereis limbata, are surrounded by a chorion. From electron microscopy studies the chorion is revealed to be composed of 57 three layers and to be penetrated by microvilli. In addition, the cortex displays the presence of numerous large alveoli containing fibrous material which is presumed to be precursor jelly involved in the formation of the jelly coat (Fallon and

Austin, 1967; PasteeIs, 1966).

The vitelline membrane of Hydroides is composed of a homogeneous material (Colwin and Colwin, 1961a). Its outer border is covered with a layer of vesicles and microvilli are found to project from the surface of the egg into the vitel line membrane. In this respect, the vitelline membrane of

Hydroides is similar to the chorion of Nereis.

The eggs of Sabellaria affords an example of one of the more complex polychete eggs in terms of surrounding invest ments. The eggs are surrounded by a thick compound vitelline membrane, a layer of vesicles and an external layer of jelly

(Pasteels, 1965).

Echinodermata In general echinoderm eggs are covered by a thin vitelline membrane and a very thick jelly layer respectively. The jelly of the sea urchin egg lacks any definite structure in electron micrographs (Austin, 1968).

On the other hand, the jelly layer in the starfish. Asterias forbesi, is a loose fibrous material.

Molluska The egg envelopes of mollusks have been described in a number of investigations. Many mollusks, both marine (Clement, 1971) and freshwater (Hess, 1971), lay eggs 58 that are enclosed within a capsule. Removal of the capsule and jelly mass from around the eggs of Ilyanassa reveals eggs surrounded only by a plasma membrane (Clemenc, 1971).

Freshwater gastropods like Limnaea produce eggs enclosed in a vitelline membrane, which originates from the cell cortex of the oocyte, and a chorion that is formed by follicular cells. The chorion is positioned to the outside of the vitelline membrane and encloses a large capsular space filled with a nutritive liquid. A third envelope or jelly coat formed by cells of the oviduct surrounds the entire complex.

An aggregation of two or more eggs surrounded by this latter jelly coat constitutes an egg batch which is typically produced by freshwater pulmonates like Limnaea (Hess, 1971).

Eggs of the bivalve mollusk, I4ytilus, are shed spontane ously from the female (Reverberi, 1971c). The mature eggs, when examined with the electron microscope, are surrounded by

a thick vitelline membrane and a jelly layer (Reverberi and

Mancuso, 1961; Dan, 1962; Humphreys, 1962; 1967). Like the

eggs of the annelids, the vitelline membrane of Mytilus is

traversed by numerous microvilli. Eggs of octopods are without accessory jelly layers. The

eggs are covered only by a chorion which displays a special

modification. It is drawn into a filament on the side of the

egg opposite the micropyle, and in some species of octopods

this filament is woven into an egg mass. A correlation exists 59 between the size of an egg and whether or not the egg will be incorporated into an egg mass. Large eggs are laid singly, whereas small eggs like those of Octopus vulgaris, are incor porated into masses with the chorion filaments woven and cemented into a common strand (Arnold, 1971).

Squids, unlike the octopods, have jelly layers sur rounding the egg. The jelly layers may be thin, as in Sepia or of considerable thickness, as in Loligo (Arnold, 1971). The jelly layers of the squids are classified as tertiary mem branes since they are placed around the egg in its passage through the oviduct.

Arthropoda The eggs of arthropods show tremendous variation in size, shape and color. Variation in size tends to reflect the environmental niche of the animal. Terrestrial

Crustacea, for example, have larger eggs than aquatic

Crustacea (Green, 1971). In general, the eggs of arthropods are invested by two envelopes; the inner vitelline membrane and an outer hard chorion or shell. The shells or chorions of some arthropods display interesting chemical and structural

characteristics. For example, the shell or outer layer of

resting Artemia eggs often contain haematin (Gilchrist and

Green, 1960) and the chorions of Drosophila are ornamented with hexagonal and pentagonal figures (Sonnenblick, 1965). 60

The fairy shrimp, Chirocephalopsis bundyi, like Drosophila, has an outer shell ornamented with polygonal patterns (Linder,

1960) and consisting of two distinct layers. The outer layer is perforated by numerous pores that penetrate to an inner spongy layer. The morphology and relationship of the outer egg layer to the inner spongy layer in the chorion of C. bundyi is similar to the relationship of the two envelopes surrounding the Limulus egg, where numerous pores penetrate an outer layer termed the basement lamina and end at an inner layer called the vitelline envelope (Brown and Humphreys,

1971). In addition to a multilayered outer shell, the eggs of

Chirocephalopsis are surrounded by a chitin containing envelope located inside the outer shell. In conclusion, egg envelopes of invertebrate species display great diversity in morphology.

The least complex egg envelopes are found among hydrozoans

(unfertilized eggs) while the most complex are found in species of annelids (Sabellaria) and arthropods (Chiro cephalopsis).

Prochordate and vertebrate egg

Prochordate Mature ascidian eggs are surrounded by several envelopes, the outermost of which is the chorion. The

chorion of the ascidian egg contains a large number of

vacuolated cells called follicular cells or chorion cells

(Ursprung and Schabtach, 1964). In the eggs of Ciona the

follicular cells of the chorion are cone shaped whereas those 61 of Ascidia nigra are vesicular and sponge-like in structure.

Positioned inside the chorion is an additional envelope sur rounding the ascidian egg composed of pigmented vacuolated cells known as test cells. The test cells of Phallusia are pushed against the surface of the chorion by a transparent, structureless envelope located over the surface of the egg plasma membrane. In other ascidians the test cells float freely in a liquid contained within a space between the egg plasma membrane and the chorion (Reverberi, 1971b).

Electron microscopy of Ascidia nigra eggs reveals the chorion to be a fibrous or multilayered structure with an electron dense core (Ursprung and Schabtach, 1964). In

addition, an envelope unfortunately termed the "plasma membrane" is found to lie directly on the surface of the egg.

The cephalochordate, Amphioxus, has a less complex egg in terms of surrounding investments. The "mature" eggs when

released from the animal are surrounded by a thin layer of

follicular cells and a jelly layer (Reverberi, 1971a). Even

though egg envelopes of cephalochordates are not complex

morphologically, prochordate egg envelopes in general are

complex in morphology.

Vertebrates

Pisces The teleostean egg is invested by a thick two

layered chorion (Yamamoto, 1961) which at one end is 62 perforated by a small hole called the micropyle. In Oryzial latipes the chorion contains a number of projecting filaments which function in the formation of egg clusters and the attachment of the egg clusters to the vent of the female.

Hurley and Fisher (1966) termed the chorion around Salvelinus fontinalis eggs as the zona radiata. They noted the chorion was porous and external to a 0.02y thick vitelline membrane.

At maturity this zona radiata is a SOu thick layer and becomes hard upon exposure to water.

Amphibia The yolk of amphibian eggs are surrounded by a vitelline membrane and a jelly layer. The jelly layer is secreted as the eggs pass down the oviducts (Balinsky, 1965).

In Xenopus an individual jelly coat or capsule surrounds each egg. In other frogs a common occurrence is a continuous mass or string of jelly surrounding the eggs (Deuchar, 1966). The jelly coat of the amphibian egg serves as a protective barrier for the egg and provides a means of making the eggs adhere to one another. When amphibian eggs are deposited in water, the jelly layer swells tremendously as a result of water absorp tion»

Reptilia The invasion of terrestrial environs by vertebrates and the evolution of reproductive mechanisms not necessitating an aquatic environment resulted in the appearance of eggs wizh relatively complex envelopes. This trend is especially evident in organisms exhibiting oviparity. The typical reptilian egg is enclosed in a calcareous shell with an underlying shell membrane surrounding the egg albumin or white. The ovum in turn is surrounded by the vitelline membrane (Fisk and Tribe, 1949). Unlike the avian egg, the reptilian egg normally lacks an air space and the presence of chalazae.

Aves The most complicated egg envelopes are found among the avians, since no less than five envelopes may be distinguished (Balinsky, 1965). The innermost envelope is termed the vitelline membrane and is found surrounding the egg plasma membrane. In avians the vitelline membrane has a dual origin. The inner layer originates in the ovary and the outer layer originates in the fallopian tube (Balinsky, 1965). Both layers of the vitelline membrane are fibrous in texture.

The white of the egg is the next envelope and is mostly water and a mixture of proteins, particularly albumin.

Adjacent to the egg white are two layers of shell membranes consisting of fibers of keratin. The outermost envelope of the avian egg is the porous shell which is composed chiefly of calcium carbonate.

The envelopes of the avian egg serve in the capacity of protection and nourishment of the developing embryo and differs from egg envelopes found in many species in which protection of the embryo is the primary function. 64

Mammals In mammalian eggs the egg plasma membrane is often called the vitelline membrane (Austin, 1961) and has essentially the same structure and functions as the plasma membrane of other cells. The envelope surrounding the plasma membrane and which is similar in nature to the chorion of tunicates and the vitelline membrane of insects, Molluska, amphibians, and avians is called the zona pellucida (Balinsky,

1965). The zona pellucida is a transparent envelope best developed in eggs of placental mammals. External to this envelope is the cumulus oophorus composed of a large number of follicle cells embedded in a transparent jelly-like matrix

(Austin, 1961). The structural integrity of the cumulus breaks up readily before sperm penetration in sheep, the cow and the horse. In rodents, rabbits and the pig it disintegrates during the period of sperm penetration. This layer remains partially intact until after the first cleavage division in the eggs of the cat and dog. An additional envelope described as a mucin coat or albumin envelope is found surrounding the fertilized rabbit egg after the cumulus disintegrates and the egg enters the lower regions of the oviduct (Austin, 1961).

Eggs of marsupials, like Didelphis and Setonix, acquire a jelly-like coat with passage through the fallopian tube.

Didelphis acquires, in addition, a noncalcareous leathery shell membrane. The eggs of the monotremes, Ornithorhynchus and

Tachyglossus, are more similar to those of birds and reptiles 65 than mammals due to the presence of a thick albumin layer, a shell membrane and a leathery shell (Austin, 1961).

Cytochemistry of Egg Envelopes

Although the earliest use of biological stains were botanical, modern day cytochemical techniques were developed on zoological materials (Lillie, 1969). Many specialized staining methods used today are capable of yielding chemical information as to* the nature of the cell components. This has been in part due to the development of rapid freezing methods which results in materials that are unaltered chemically and consequently more representative of tissues in their natural states. Equally important has been the development and use of the Schiff reagents in the identification of polysaccha rides and nucleic acids. Similar developments in other areas of histochemistry have allowed accurate identification of a number of amino acids, lipids and enzymes in cells and tissues.

Much of the accumulated literature concerning the histo- , chemistry of female gametes has been concerned with vitellogenesis. The chemical composition of compound yolk and the nature of lipids formed and stored during oogenesis has been investigated in numerous invertebrates and vertebrates (Nath,

1968). Egg envelopes have also received attention in terms of their chemical composition. Echinoderm and amphibian egg 66

jellies have been of special interest to many biologists

(Monroy, 1965) and organisms of economic importance such as

parasitic members of the phyla Aschelminthes and Platyhelmin-

thes have also generated interest as to the chemical nature of

their egg investments (Monne, 1969; Smyth and Clegg, 1959).

Perhaps the most thoroughly investigated egg in terms of the

chemical and biochemical nature of its envelopes is the avian

egg (Simkiss and Tyler, 1957; Robinson and King, 1968). In

considering this wide range of studies some insight into the

chemical nature of invertebrate and vertebrate egg envelopes will be presented.

Invertebrate egg envelopes

Aschelminthes The eggs of nematode families (e.g.

Trichostrongylidae, Strongylidae) which hatch outside the body

of the host are covered with thin envelopes. These envelopes

contain a polysaccharide which is stainable by means of the

PAS method (Monne, 1959). Since acetylated polysaccharides

like chitin cannot be stained using this method, the poly

saccharide in the envelopes is not chitin but is probably a

nonacetylated chitin precursor (Monne, 1959). Egg envelopes

of Dictyocaulus viviparus and D. fiaria contain a PAS-positive

polysaccharide of similar properties and are considered to

represent the most primitive type of egg envelopes among the

parasitic nematodes (Monne, 1959). 67

The eggs of the ascarids are provided with thick envelopes even though these eggs characteristically do not hatch outside the body of the host- The egg envelopes of

Ascaris suum. Parascaris equorun and Ascaridia galli consist of chitin and are not stainable by means of the PAS method.

In contrast, the fully developed thick envelopes of Toxocara cati, a related genus, stains intensely following the PAS procedure (Monne, 1959).

The proteins in the egg envelopes of nematode species whose eggs hatch outside the body of the host are associated with polysaccharides (Monne, 19 59). The protein is gram- positive and subject to polyphenol-quinone tanning. Egg shells of nematode species whose eggs hatch inside the body of the host (Ascaris, Parascaris) are covered by a thick envelope that also contains a polyphenol-quinone-tanned protein. The envelopes of Ascaridia and Toxascaris and the eggshell of Toxocara cati also contains this protein. Thus, polyphenol-quinone-tanning of egg envelopes are a general phenomenon among nematodes.

Platyhelminthes , Reports of carbohydrates in platyhelminthes egg shells have been scant. Most reports on egg envelope chemistry deal with proteins. In a recent report on the chemical composition of egg envelopes of Hymenolepis diminuta, Lethbridge (19 71) found the shell and subshell

"membrane" to be PAS and alcian blue negative. These results 68 are in contrast with observations by Pence (1970) that the outer envelope on the mature eggs of Hymenolepis diminuta is

PAS positive. The cytoplasmic layer is also Alcian blue negative but PAS positive before and following amylase treat ment (Lethbridge, 1971). The nonglycogen polysaccharide nature of the cytoplasmic layer has also been reported by

Hedrick and Daugherty (1957) and by Cheng and Dyckman (1964).

Histochemical evidence demonstrates the presence of a quinone-tanned protein or sclerotin in the egg shell of most trematodes (Smyth and Clegg, 1959; Stephenson, 1947).

Wilson's (1967) data suggest that the eggshells of Fasciola hepatica consisted of a fibrous protein resembling vertebrate collagen and Madhavi (1966; 1968; 1971) has shown the egg shell of the amphistomes to contain a keratin type protein instead of the common quinone-tanned protein. The isolated shell protein of Schistosoma mansoni is mainly a glycoprotein

(Clegg, 1963).

A sclerotin type protein has also been demonstrated in the egg shells of pseudophyllidean cestodes (Smyth, 1956).

Lethbridge (1971) found the egg shell of the cestode

Hymenolepis diminuta to consist entirely of protein. Amino acid analysis revealed a marked preponderance of heterocyclic and aromatic acids. The only lipids found histochemically were those associated with the subshell "membrane". Con trasting results by Pence (1970) suggested that the outer coat 69 of a mature egg from H. diminuta contains mucopolysaccharides.

Echinodermata The chemical nature of the jelly enve lopes^ encasing echinoderm eggs have been examined by numerous investigators (see Monroy, 1965 for review). The"jelly envelope is strongly acidic as a result of a high content of sulfate groups. Variations in the carbohydrate component of the jelly envelope is found among echinoderms in different genera. The sea urchin egg jelly envelope is a glycoprotein

(Monroy, 1965). Ishihara (1968) reported the jelly envelopes of Arbacia punctulata contained hexose, hexosamine, sulfate, inorganic cations and seventeen different amino acids. In addition, Minganti and Vasseur (1953) reported 20 per cent of the dry weight of the jelly layers surrounding Paracentrotus lividus eggs is protein. Thus the egg envelopes of echino derms contain ample amounts of carbohydrates and proteins.

Molluska Considerable literature exists on the reproductive tracts of mollusks. Most reports are concerned with morphology and those of histochemistry are mainly con cerned with the reproductive tract and not its products, the sperm and the egg. The bulk of information on the histo chemistry of the molluscan egg has come from studies on gastropod egg capsules (Rangarao, 1963a, b; Bayne, 1968; Hunt,

1966), mature eggs (Smith, 1965), centrifuged eggs

(Sathananthan, 1972), and isolated egg membranes (Haino and

Kigawa, 1966). 70

The presence of carbohydrates in molluscan egg envelopes is well documented. Rangarao (1963a) reported galactogen in the capsular fluid and acid mucopolysaccharides in the jelly of Ariophanta ligulata egg capsules. The capsular "membrane" of this species also contains acid mucopolysaccharides. Hunt

(1966) later reported the presence of six monosaccharides in the egg capsules of another species of gastropod, Buccinum undantum L. Bayne (1968) examined the egg capsules from eight species of gastropods and found the envelopes of all except

Nucella lapillus to contain acid mucopolysaccarides. Nucella lipillus capsules were found to contain neutral poly saccharides instead of acid mucopolysaccharides. The nutritive supplies within the egg capsules of all the gastropod species examined by Bayne (1968) contained mostly neutral poly saccharides.

Histochemical tests on the egg of Arion ater revealed the outer shell and inner membranes of the egg as PAS positive

(Smith, 1965). The inner membranes were Alcian blue positive and biochemical analysis of isolated egg membranes from

Tegula pfeifferi revealed the presence of mucopolysaccharides

(Haino and Kigawa, 1966). Thus the presence of neutral or acid polysaccharides have been demonstrated to be a common feature of the molluscan egg envelopes.

There has been some controversy as to the nature of the proteins comprising the capsular envelopes of molluks. 71

Rangarao (1963a) found the concentration of tyrosine- containing proteins to be high in the capsular envelope of

Ariophanta ligulata and low in the capsular fluid. In con trast amino acid analysis of capsular material from Buccinum undatum L. demonstrated the absence of the amino acid tyrosine

(Hunt, 1966). These results may be explained on the basis that Hunt (1966) used post-hatched material in his study. ,

Examination of egg cases from a closely related species,

Nucella lapillus, showed tyrosine to be present only in the capsular plug and inner homogeneous layer (Bayne, 1968).

Negative results for tyrosine would be obtained in an amino acid analysis of post-hatched material since both the capsular plug and inner homogenous layer is absent from such material.

The total protein of egg capsules is reported to be sparse in supporting layers of stylommatophoran (terrestrial) eggs and abundant in those of Limnaea stagnalis (freshwater) and marine species (Bayne, 1968). Protein is a major component in the capsular wall of Nucella lapillus and is associated with polysaccharides (Bayne, 1968).

Studies on the isolated'egg envelope of the marine gastropod, Tegula pfeifferi, demonstrated a chemical composi tion similar to molluscan egg capsules studied by other investigators. For instance, envelopes were found to consist chiefly of mucopolysaccharides and proteins (Haino and Kigawa,

1966). The protein moiety of the envelope contains tyrosine. 72

Because of its solubility characteristics and amino acid

composition the protein moiety of Tegula pfeifferi egg

envelopes resemble some structural proteins (collagen) found

among members of the animal kingdom (Haino and Kigawa, 1966).

The presence of lipids in molluscan egg investments

varies from species to species. Free lipids have been

reported to be on the surface of Arion ater eggs (Bayne, 1968).

In contrast to Bayne's work is a report by Jura and George

(1958), who found lipids in all layers of Succinea putris and

Planorbis corneus eggs. Haino and Kigawa (1966) and Rangaroa

(1963a) reported no lipids were present in isolated envelopes of Teglua pfeifferi and egg capsules of Ariophanta ligulata respectively.

Arthropoda Arthropod eggs have been the subject of numerous investigations concerned with development and physiology. Although insect eggs have constituted the bulk of these investigations, some studies related to the chemical composition of egg envelopes have been performed.

The first appearance of a study on the chemical nature of the insect chorion or egg shell was an investigation by Tomita

(1921) in which the amino acid composition of the chorion sur

rounding the silkworm egg was determined. Since then the chorion or shell of the insect egg has been consistently found

to be composed of several layers of proteins (Beament, 1946;

Furneaux, 1970). These proteins are thought to be cross- 73 linked by quinones and disulfide bonds (Wilson, 1960b;

McFarlane, 1962). This view is somewhat controversial since cystine, a sulfur containing amino acid, is not commonly found in insect egg shells (Jaskoski and Butler, 1971). Another point of controversy has been the presence of chitin in insect egg shells. Most investigators agreed a few years ago that chitin was not present in insect egg shells (Richards,

1951). However, chitin has since been demonstrated in

Drosophila (Wilson, 1960b) and Acheta (McFarlane, 1962) egg shells. Most recently in another arthropod taxon, chitin has been demonstrated in the egg shells of the tick, Rhipicephalus sanguineus (Jaskoski and Butler, 1971).

In addition to proteins, stabilized lipids combined with proteins have been identified as components of the insect chorion (Beament, 1946; Davies, 1948; Wigglesworth, 1970).

According to Wigglesworth (1970), the lipid serves to stiffen the cuticle (egg shell) before sclerotization occurs. The role these lipids play in the chorion is apparently different from waxes found on the outer layer of some insect eggs (Lees,

1961). Recent evidence of a phosphoprotein has been reported in the chorion of freshly laid eggs of the cricket. Acheta domestieus (Furneaux, 1970). Such a protein is effective as a cation-exchange polymer and is capable of retaining in organic cations at high acidities. These properties may be of importance in maintaining the integrity of the chorion in an 74 environment of divalent cations.

In conclusion, most arthropod egg envelopes that have been investigated are composed of proteins or protein-lipid complexes.

Vertebrate egg envelopes

The following section on the vertebrates will primarily concern accumulated data on the chemical nature of amphibian and avian egg envelopes. A brief reference to mammalian egg envelopes will also be included.

Egg envelopes of Pisces and Reptilia have received very little attention in terms of their chemical composition.

Studies conducted on the female gametes of Pisces concentrated on the physiological (See Yamamoto, 1961 for review) and structural aspects of the egg (Hagstrom and Lonning, 1968;

Lonning, 1972). Those studies concerned with the chemistry of the egg have focused on the cortical alveoli (Yamamoto,

1961) which contain mucoproteins (Kusa, 1953; 1954). Hamano

(1949) also reported the inner layer of the chorion of the salmon egg to be composed mainly of glycoprotein or mucoid.

A study on the amino acid composition of the protein envelope of the egg of the tortoise demonstrated the same amino acids as contained in the egg of the chicken (Zusman,

1965). The tortoise egg envelopes contain considerably less amounts of all amino acids determined except tyrosine and phenylalanine. This data indicates that chemically reptilian 75 egg envelopes resemble avian membranes.

Amphibia Among vertebrates the egg envelopes of amphibians have received considerable interest in terms of their functional significance. Only recently has there been interest in the detailed chemical nature of the envelopes.

The jelly envelopes of numerous amphibians have been demon strated to be glycoprotein in nature (see Monroy, 1965 for review). Utilizing several cytochemical techniques, Salthe

(1963) studied the comparative morphology of the egg envelopes of 72 species of amphibians. The jelly layers of all of these species were found to be composed of acid muco polysaccharides. A recent study on the jelly envelopes of

Rana pipiens has provided even more insight into the chemical organization of the jelly envelopes of amphibians (Steinke and Benson, 1970). Cytochemical tests on R. pipiens egg envelopes demonstrated five discrete regions classified as Ml through M5 from the inner to the outermost region. All five regions contain neutral mucopolysaccharides. In addition. Ml and M3 contain sulphated mucopolysaccharides, M2 and M4 nonsulphated acid mucopolysaccharides, and M5 contains both sulphated and nonsulphated acid mucopolysaccharides (Steinke and Benson? 1970). A recent study by Gusseck and Hedrick

(1971) found disulfide bonds to be responsible for maintaining the structural integrity of the jelly coat surrounding Xenopus laevis eggs. 76

A chemical analysis of egg envelopes obtained from blastula- and gastrula-stage embryos of Bufo vulgaris has been conducted by Uchiyama, Ishihara and Ishida (1971), and has shown the envelopes to be composed mainly of amino acids, lipids and hexose. In addition, small amounts of hexosamine fucose and sulfate were also present. The authors noted the chemical composition of the egg envelopes of blastula and gastrula- stage embryos of B. vulgaris resembled the fertili zation membrane of the sea urchin egg.

From these studies on mature amphibian egg envelopes the major chemical constituent is glycoprotein. Following • fertilization and subsequent development, the jelly layer surrounding the embryo is primarily protein.

Aves Because of the nutritional value and importance of the domestic hen egg to man, more is known about the chemistry of its envelopes than other avians. The shell of the domestic hen egg is composed of five essential parts; the shell membrane, the adjacent crystalline mammillary layer, the spongy layer or main part of the shell, the outer cuticle and the pores which traverse the shell from the membranes to its outer surface (Robison and King, 1968). The mammillary layer is attached to the outer shell membrane by structures known as mammillary cores. The organic portion of the mammillary cores

and the main portion of the shell was reported by Simkiss and

Tyler (1957) to be basically similar in chemical composition. 77

They contained a protein-acidic polysaccharide complex.

Robison and King (1968) have recently found the mammillary cones to contain neutral mucin surrounded by a weakly acidic substance and have suggested that disulphide and noncovalent bonds were responsible for maintaining the integrity of the cores.

Bellairs, Harkness and Harkness (1963) have studied the chemical nature of the vitelline membrane of the hen egg. The separated inner and outer layers of the vitelline membrane were subjected to histochemical tests and chemical analysis.

Both were found to consist mainly of protein. The protein of the inner layer resembles materials found in connective tissue accompanying collagen and the outer layer resembled some of the proteins found in egg white (Bellairs, Harkness and

Harkness, 1963).

Mammalia The zona pellucida is the only envelope found in all mammalian eggs other than the egg plasma membrane

(Raven, 1961). The zona pellucida has a double origin. Its outer layer is secreted by follicle cells and consists of acid mucopolysaccharides. The inner zone is produced by the oocyte and consists of neutral mucopolysaccharides (Wartenberg and

Stegner, 1960). The vertebrate egg varies from species to species as to the chemical composition of its envelopes. An examination of the literature on histochemical methods employed on vertebrate egg envelopes reveals one family of 78 components that consistently appears in the envelopes of most species; the mucoproteins.

Limulus Fertilization

Limulus sperm

Several investigators have presented ultrastructural descriptions of the mature spermatozoa of Limulus polyphemus

(Philpott and Shaw, 1959; Philpott, 1960; André, 1963) and on the sperm-egg interaction (Shoger and Brown, 1970; and Brown and Humphreys, 1971). The most recent ultrastructural study to appear in the literature is one describing spermiogenesis in Limulus (Fahrenbach, 1973).

The sperm of Limulus is similar in form to the primitive metazoan types described by Franzen (1970). The sperm has a rather large cap-shaped acrosome, a spherical nucleus and a simple flagellar complex. The Limulus sperm differs from primitive metazoan spermatozoa in one major respect. Present within the subacrosome space and extending through the nucleus is an acrosome filament. Posterior to the nucleus the fila ment assumes a coiled configuration and occupies a circum- nuclear cisternae. According to Fahrenbach (1973), the filament has its origin from a thick bundle of fibrils at the tip of the subacrosome space. The functional role the acro some filament plays in the process of fertilization is unclear.

During sperm-egg interactions the 50y long filament extends 79 through the anterior end of the acrosome where it penetrates

two egg envelopes surrounding the Limulus egg (Shoger and

Brownf 1970; Brown and Humphreys, 1971). Recent evidence

suggests the filament passes through preexisting pores in the outer egg envelope (Brown, 1973). This is of considerable

interest since such pores are absent from the inner egg

envelope. The fact that mechanical or chemical treatment will

also cause extrusion of the acrosome filament suggests this

phenomenon is not entirely dependent on contact with the egg

(André, 1963). Structurally, the acrosome filament appears as

a bundle of microfibres sheathed in a membrane. The membrane,

contends Fahrenbach (1973), is an extension of the outer

nuclear membrane that forms during early spermiogenesis.

The contents of the acrosome vesicle constitute a

heterogeneous matrix in electron micrographs. Cross sections

through the acrosome vesicle reveal a doughnut-shaped area

in the anterior portion. This area, which is kidney-shaped

in longitudinal sections, has a greater electron opacity than

the surrounding region.

The subacrosome space contains a flocculent material

that extends to the lateral edges of the cap-shaped poster

iorly indented acrosome vesicle. Encasing the remaining

anterior portions of the vesicle are periacrosome filaments.

The filaments are located between the acrosome membrane and

the sperm plasma membrane, which surrounds the entire sperm. 80

The flagellvun is of the 9+2 pattern commonly found among annelids and mollusks. Other than the end-to-end association of the centrioles, which has been reported in only one other animal, Pholcus, the features at the posterior end of the sperm are similar to those found in primitive sperm (Fahrenbach,

1973).

Limulus egg

Limulus eggs have been adequately described in the scientific literature. Two extensive accounts on oogenesis in Limulus by Munson (1898) and Gardiner (1927) appeared some years ago and more recently, Dumont and Anderson (1967) con ducted an ultrastructural study on oocyte maturation in

Limulus. In addition, transmission and scanning electron micrographs showing the structure of the egg envelopes have been presented in studies on sperm-egg interactions (Shoger and Brown, 1970; Brown and Humphreys, 1971).

The mature egg is approximately 2.5mm in diameter (Brown and Humphreys, 1971). The yolk, which comprises the bulk of the egg, is enclosed by two membranes. The inner layer, termed the vitelline envelope, is approximately 40y thick and homogeneous in nature. The outer layer is approximately

5y thick and perforated by a large number of randomly spaced pores. This porous layer is called the basement lamina

(Dumont and Anderson, 1967). Both layers uniformly sur

round the egg. 81

Sperm-egg interaction in Limulus

The study of fertilization in Limulus has been enhanced by the ability of this organism to survive in the laboratory and to produce viable gametes on a year around basis. Since inseminated eggs develop to hatching, artificially collected gametes are used for ^ vitro fertilization studies and for developmental studies. Consequently, significant studies on the ultrastructure of the initial sperm-egg interaction

(Shoger and Brown, 1970; Brown and Humphreys, 1971) and on the immunological events associated with these interactions

(Cooper and Brown, 1972; Mowbray and Brown, 1974) have appeared recently.

The sperm-egg interaction in Limulus initially involves sperm motility and the sperm acrosome reaction. Under normal conditions Limulus spermatozoa are not motile until in the proximity of the egg surface. Upon contact with the egg surface, the sperm acrosome undergoes an acrosome reaction in which a dehiscence of the acrosome vesicle and an extrusion of a long acrosome filament occurs. The acrosome filament subsequently penetrates the egg envelopes.

Studies on sperm-egg interactions involving Limulus gametes have revealed the striking observation that the surfaces of both whole eggs and egg sections (Mowbray and

Brown, 1974; Brown and Humphreys, 1971) form attachment with large numbers of spermatozoa. Recent observations by Brown 82 and Knouse (1973) indicate certain variables, such as sperm concentration and sperm aging influence ^ vitro fertilization in Limulus. High sperm concentration (1,000,000 spermatozoa/ egg) and the aging of sperm were found to enhance a higher per cent of development.

In conclusion, the horseshoe crab, Limulus polyphemus has been found to be an excellent species in which studies on in vitro fertilization and development may be pursued.

Approaches to Fertilization in Limulus

Recent approaches to the study of gametes and the sperm- egg interaction in Limulus have been on the ultrastructural and immunological level. Ultrastructural studies on sperm atozoa (André, 1963; Fahrenbach, 19 73), oocyte maturation

(Dumont and Anderson, 1967) and the sperm-egg interaction

(Brown and Humphreys, 1971) have provided most of the present knowledge of fertilization in Limulus. In addition, immulogi- cal studies have given some insight into the participation of molecular components on both the spermatozoa and egg in sperm- egg attachment (Mowbray, Brown and Metz, 1970; Cooper and

Brown, 1972). An area receiving less attention is that con cerning the chemistry of the sperm acrosome and the chemistry of the egg. In this study the cytochemistry of the sperm acrosome, the egg and the sperm-egg interaction will be investigated. Also the ultrastructure of the sperm acrosome. 83 egg envelopes of the mature egg and certain biochemical and physiochemical properties of the egg envelopes will be examined in an effort to make more complete the existing information on fertilization in Limulus. 84

MATERIA S AND METHODS

Source and Maintenance of Animals

Specimens of male and female Limulus polyphemus L. were obtained from the Florida Marine Biological Specimen Company,

Panama City, Florida and from the Supply Department, Marine

Biological Laboratory, Woods Hole, Massachusetts. The animals were kept in Model 250 Instant Ocean Culture Systems at a temperature of 15°C for periods up to 6 months before dis carding. Approximately 90 animals were used during the course of this study.

Collection and Preparation of Gametes

Spermatozoa and eggs were collected by electrical stimulation with an Adjust-A-Volt electrical stimulator. A setting of 30 volts on the stimulator was maintained at all times. A transformer was positioned between the electrodes and the stimulator in order to obtain a potential of 3.8 volts.

The procedure of collecting spermatozoa and eggs involved placing the animal on its dorsal surface, lifting the genital operculum while depressing the gill opercula, and applying the electrodes to the gonopore region (Figures 1,2,3).

Spermatozoa were collected with disposable Pasteur pipets and subsequently transferred to graduated centrifuge tubes for use in insemination studies. Spermatozoa to be used 85 for morphological, cytochemical and biochemical studies were transfered to 10x75mm culture tubes. Routinely 10% sperm Q suspensions containing approximately 10 spermatozoa/ml were prepared with cold (0°-5*C) filtered Instant Ocean synthetic sea water or Marine Biological Laboratory (MBL) synthetic sea water (Cavanaugh, 1956) for use in insemination studies. Both diluted and undiluted spermatozoa were stored on ice or refrigerated at 5°C for periods up to 12 h.

Since freshly spawned eggs adhere to glass or metal, special care was taken during handling. Eggs were collected from the gonopore region of the female with a Parafilm "M" covered tablespoon. Eggs to be used in insemination experi ments were placed in Parafilm "M" lined petri dishes or in plastic petri dishes containing cold (0*-5°C) sea water.

Eggs to be used in cytochemical and biochemical experi ments were washed from the tablespoon with cold filtered sea water into a plastic test tube. All subsequent manipulations of fresh eggs were carried out with wood applicator sticks.

Preparation of Spermatozoa for Cytochemistry

Spermatozoa used in cytochemical studies were collected as described in the previous section. Microscope slides for cytochemical studies were prepared with spermatozoa from sperm suspensions that were centrifuged and washed two times in cold sea water and allowed to air dry. Centrifugation of sperm 86 suspensions were performed for 10 min at 2000 rpm (480 G) in a Sorvoll Superspeed RC-2-B refrigerated centrifuge. The centrifugation runs were maintained at a temperature of 0°C.

Other microslides were prepared with unwashed 10% suspensions of spermatozoa.

Acid phosphatase

The histochemical method of Barka and Anderson (1962) was used to demonstrate acid phosphatase. Sperm smears were prepared on glass microscope slides with one drop of a 10% sperm suspension and allowed to air dry. Air dried smears were fixed in formal-calcium (4°-5°C) for 2 h. Incubation was carried out at room temperature (23°C) for 10-30 min in a medium (pH 2.0; 6.0) containing a-naththyl phosphate (Sigma) as a substrate and hexazonium pararosanilin (Sigma) as a coupling agent. Control smears were incubated in media identical to that of experimentals but without a-naphthyl phosphate as the substrate. Following incubation, smears were counterstained with 1% chloroform-extracted methyl green for

30 sec, rinsed in distilled water (dHgO) and quickly dif ferentiated in a graded series of ethyl alcohols. Microslides were cleared in xylene and mounted in Kleenmount.

Acridine orange

Microscope slides of 10% sperm suspensions in sea water

were prepared and washed in dHgO and subsequently treated with 87 a dilute solution of acridine orange (Fisher, C.I. 4600s). A stock solution of acridine orange was made at a 1.6x10 ^ M

(1/2000) concentration in dHgO. For use this stock solution was diluted to a final concentration of 1/20,000 (pH 7.5) with a physiological saline solution (West, 1969).

Acrosome proteinase

The method of Yanagimachi and Teichman (1972) was employed to demonstrate an acrosome proteinase. Sperm smears prepared from undiluted and 10% sperm suspensions were allowed to air dry. Before incubation the smears were fixed in 100% ethyl alcohol (-20°C) for 30 min or in formal-calcium (0°-

5®C) at pH 7.0-7.2 for 2 h. Other sperm preparations were processed without prior fixation. The incubation medium

(adjusted to pH 6.5, 7.2 and 7.4) contained 1% mild silver protein (Argyrol) as the reaction substrate. Silver nitrate was substituted for mild silver protein in the media of control smears.

Trypsin-like acrosome enzyme

Glutaraldehyde-fixed gelatin membranes were prepared according to Owers and Blandau (19 71) and Gaddum and

Blandau (1970). Washed (10%) and unwashed sperm suspensions in Instant Ocean synthetic sea water (pH 6.5; 8.0) were placed on the glutaraldehyde fixed gelatin membranes and prepared as sperm smears. The smear preparations were covered with 88 microcover glasses and subsequently sealed with melted vaseline to prevent evaporation. In addition to this experi ment, reacted spermatozoa were also used. To induce a "true" acrosome reaction (André, 1963), some sperm suspensions were treated with 0.34M CaClg (three vol of CaCl2/one vol of sperm suspension). Aliquots (0.1 ml) of the 0.34M CaClg treated spermatozoa suspension were placed on glutaraldehyde fixed gelatin membranes and prepared as smears. All slides were incubated at room temperature (23°C) or 37°C for periods up to 24 h.

Nonspecific esterase

Sperm smears were prepared from 10% sperm suspensions according to the previously described method. Smears were treated unfixed or after fixation for 1 h in cold (0°-5°C) formol-calcium adjusted to pH 7.2. Each preparation was incubated in a medium that was prepared in the following manner:

(1) 0.8 ml of 4% NaNOg was mixed with 0.8 ml of 4%

pararosanilin HCl (Sigma) in 2N HCl and allowed

to stand at room temperature (23°C) for 30 min

(Bryan and Unnithan, 1972).

(2) 0.75 ml of 1% 1-naphthyl acetate (Sigma) dissolved

in absolute acetone was added dropwise with

stirring to 20 ml of a pH 7.1 0.15M phosphate

buffer (Bancroft, 1967). 89

(3) The naphthyl acetate/phosphate buffer solution

was added to the NaNOg/pararosanilin-HCl solution

and allowed to stand 20 itiin at room temperature

(23°C) following pH adjustments to 6.2 and 7.2.

Incubations were carried out at 23°C and 37°C for periods up

to 48 h. Controls were incubated in media in. which 1-naphthyl

acetate was omitted.

Preparation of Sperm for Ultrastructure

Spermatozoa were collected, suspended in sea water,

centrifuged, and washed twice before fixation as a pellet.

Centrifugation of sperm suspensions (10 min. at 0°C) was

carried out in a Sorvall RC-2-B centrifuge.

Sperm pellets were fixed 1 h in one of the following

fixatives:

(1) Cold (0°-5°C) 2.5% glutaraldehyde buffered with

0.2M sodium cacodylate buffer (pH 7.4) containing

3% NaCl and 3.5% sucrose.

(2) Cold C0°-5°C) 3% glutaraldehyde buffered with 0.2M

phosphate buffer (pH 7.4) containing 7% sucrose

or 7% NaCl.

After 10 min in the fixative the pellet was broken into

smaller fragments to facilitate fixative penetration. The

fragments of the pellet were subsequently washed in the buffer

used with the fixative for a total of 3 h. Post fixation in 90

1% OsO^ (prepared from a 2% aqueous solution with the buffer used in the glutaraldehyde fixative or with dHgO) for 1 h at

0°C or room temperature (23°C) was followed by a 1 h wash in three changes of the buffer used with the OsO^ or in dHgO.

Rapid dehydration in a graded series of ethyl alcohols or acetone followed osmication. Sperm pellet fragments were embedded in epon-araldite (Anderson and Ellis, 1965) or

Spurr's (1969) low viscosity resin, sectioned with glass knives on a Porter-Blum MT II Ultramicrotome, mounted on uncoated 300 mesh grids or carbon stabilized, parlodion coated

200 mesh grids, and stained with 5% uranyl acetate in 50% ethyl alcohol followed by lead citrate (Venable and Coggeshall,

1965). The grids were examined on a Hitachi HU llE-1 electron microscope.

Separation of Sperm Acrosome

Spermatozoa were collected and washed as described by

Bennett and Austin (1970). Acrosomes were separated by treating 5 ml of sperm suspension in an ice bath with 1 ml of

0.0025% sodium hypochlorite for 1-2 min followed by dilution to 25 ml with filtered cold MBL sea water. In addition to effecting release of sperm acrosomes, this treatment also damages the sperm nuclei resulting in a release of DNA. With in 5 min of treatment with sodium hypochlorite, a gel forms trapping the separated acrosomes (Figure 18). Treatment of 91 the gel with 0.02% DNAase I (Sigma) releases the acrosomes which accumulate in the supernatant. The remaining gel was resuspended and gently sonicated in an ice bath to liberate additional acrosomes. Free acrosomes were stained with acridine orange and tested for acid phosphatase . Gels con taining free acrosomes and acrosomes in various stages of separation were prepared for electron microscopy.

Preparation of Eggs and Egg Sections for Cytochemistry

Uninseminated egg sections and eggs

Following collection and washing, Limulus eggs were immediately frozen on a specimen holder at -20°C in an Inter

national Cryostat CT-1, sectioned at 12ii and collected on

cold glass microscope slides or 22mm sg. cover glasses, fixed

at 0°C in 10% neutral buffered formalin (Thompson, 1966) for

1-12 h and washed in running tap water for 1 h. Microslides

containing egg sections not immediately being used were stored

in plastic slide boxes at -25®C. These stored egg sections

were normally used within a week. Other eggs were collected

and placed directly into 10% neutral buffered formalin

(Thompson, 1966) at room temperature (23°C). To ensure fixa

tion, the eggs were punctured with fine point forceps and a

dissecting needle after 15 min in the fixative. Punctured

eggs were placed in fresh fixative for a period of 48 h and

then washed in running tap water overnight. The eggs were 92 subsequently dehydrated in a graded series of ethyl alcohols, cleared in benzene or xylene, embedded in Paraplast (56°-

57°C), sectioned at 8-lOy on an American Optical Model 820 microtome, and prepared for cytochemical methods.

The distribution and types of carbohydrates, proteins, amino acids, lipids, nucleic acids and phosphatases were determined on uninseminated Limulus egg sections. A summary of the cytochemical methods employed are listed in Table 1.

Inseminated egg sections

Limulus polyphemus L. spermatozoa will attach to and undergo the aerosome reaction with cryostat sections of

Limulus eggs. Twelve hours before insemination spermatozoa were collected and stored at 0° to 5°C as a 10% suspension in sea water. Egg sections (12%) were prepared as described in the previous section. They were subsequently treated with the spermatozoa suspension for periods of 5 min and 1 h.

Excess spermatozoa were washed away with sea water and the egg sections were subsequently fixed for 1 h in 10% neutral buffered formalin (Thompson, 1966). Following a 30 min wash in running tap water, the inseminated egg sections were stained with Alcian blue (pH 2.5) and nuclear fast red as a counterstain for the demonstration of mucosubstances

(Humason, 1972). Table 1. Cytochemical methods employed for visualizing chemical constituents of Limulus egg envelopes

Cytochemical method Specific result Reference(s) for procedure

(1) Periodic-acid Oxidation of VIC-hydroxyl Thompson, 1966 (Lillie, Schiff (PAS)- groups followed by the 1947; McManus, 1946; metanil yellow formation of deep Hotchkiss, 1948) counterstain purplish-red colored complexes with Schiff reagent

(2) Hydroxylamine Block aldehydes Bancroft, 1967 treatment (30 min to 2 h)-subsequently stained with PAS

(3) Potassium per- 1, 2-glycols- (Pale Thompson, 1966 mangenate Schiff red-purplish red) (PPS)

(4) Chromic acid-Schiff 1, 2-glycols- (Pale red- Thompson, 1966 purplish red)

(5) Congo red Glycoprotein- (Pale pink- Thompson, red)

(6) Alcian blue Sulphated mucosubstances- Pearse, 1968 pH 1.0 (1% in blue O.IN HCl)

(7) Alcian blue pH 2.5 Weakly acidic sulphated Humason, 1972; Thompson, (Counterstained with mucosubstances, 1966 nuclear fast red) sialomucins, hyaluronic acids- dark blue (8) Mild methylation Alcianophilia of non- Spicer, 1960 (0.1% HCl for 4 h sulphated mucins is at 37°C)-subsequently inhibited stained with Alcian blue (pH 2.5)

(9) Methylene blue Distinguishes between Dempsey and Singer, 1946 - extinction (0.1% glycosaminoglycans and solution in veronal- glycoproteins acetate buffers, pH 2.6-8.2)

(10) Toluidine blue T-metachromasia- red Pearse, 1968 (Aqueous) 3-metachromasia- purple

(11) Azure A(pH 1.0; 3.6; Sulphated mucosubstances Pearse, 1968 4.0), 30 min -24 h below pH 2.0, sialomucins above pH 3.0- blue

(12) Periodic acid- Neutral mucopolysacchar Pearse, 1968 (Spicer, 1965) paradiamine (PAD) ides- brown, periodate- reactive polymers- purple or gray brown, periodate unreactive mucosubstances- black

(13) Silver method Ascorbic acid - black Pearse, 1968 silver granules

(14) Mercuric bromphenol Proteins- blue Humason, 1972 (Mazia, blue Brewer and Alfert, 1953) Table 1 (Continued)

Cytochemical method Specific result Reference(s) for procedure

(15) Van Slyke's reagent Blocks amino groups Humason, 1972 (Lillie, (45 min -6 days)- (Deamination) 1954) subsequently stained with mercuric bromphenol blue

(16) Performic acid- Bisulphides- blue Bancroft, 1967 Alcian blue

(17) Ninhydrin-Schiff Amino groups- pink to red Bancroft, 1967

(18) Sakaguchi method Arginine- orange red Pearse, 1968

(19) Naphthol-yellow Basic proteins- yellow Pearse, 1968 S (Aqueous, pH 2.7)

(20) Oil red O Neutral fats-red Pearse, 1968

(21) Bromination (6h 25°C)- Block ethylene groups Pearse, 1968 subsequently stained with Oil red 0

(22) Okamoto's method Cholesterol- bluegreen Pearse, 196 8

(23) U-V Schiff method Unsaturated lipids- Pearse, 1968 (Irradiated 4 h) (Magenta- red)

(24) Holczinger's Fatty acids- (Greenish- Pearse, 1968 technique black (25) IN HCl treatment, Extracts lipids Elleder and Lojda, 1972 acetone extraction (1 h 23°C) - sub sequently stained using Holczinger's technique

(26) Methanol/Chloroform Extracts lipids Pearse, 1968 extraction (1:1; 1 h 50°C)- sub sequently stained using Holczinger's technique (27) Fischler's method Patty acids- dark blue Pearse, 1968 (Controls extracted 24 h in 3 changes of 100 ETOH/Ether; 1:1 37°C)

(28) Copper Phthalocyanin Phospholipids- blue Pearse, 1968 method (Counter Lipoprotein- blue stained with 1% neutral red)

(29) Methanol/Chloroform extraction (1:1, 5-24 h at 60°C)- subsequently stained with copper phthalocyanin method (30) Controlled chromation Phospholipids- blue Pearse, 1968 (Elftman, (18 h at 57°C, 60*C, Lipoprotein- blue pH 3.5)- subsequently stained with copper phthalocyanin method Table 1 (Continued)

Cytochemical method Specific result Reference(s) for procedure

(31) Sudan black B Phospholipids- black Pearse, 1968

(32) Modified Molisch Sugar-containing lipids- Pearse, 1968 reaction (Violet- red)

(33) Feulgen method DNA- (Reddish- purple) Pearse, 1968

(34) DNase I treatment Extracts DNA Pearse, 1968; Bancroft, ' (0.2 m Tris buffer, 1967 pH 7.6; 5 h at 37°C)- subsequently stained with Feulgen method (35) IN-HCl (3 h at 37°C) Extracts DNA and RNA Pearse, 1968

(36) Modified Gomori method Alkaline phosphatase- Pearse, 1968 for alkaline phos black phatase (Incubated for 30 min - 4 h at 37*C)

(37) Azo dye coupling Alkaline phosphatase- Bancroft, 1967 method (Incubated for (Reddish- brown) 10-60 min at room temp.)- counterstained with 2% chloroform extracted methyl green

(38) Naphthyl acetate Nonspecific esterase- Bancroft, 1967 method (Incubated for (Reddish- brown) 20 min at 37°C)- counterstained with 1% chloroform extracted methyl green (39) Azo dye coupling Acid phosphatase- red Bancroft, 1967 method (Incubated for 15-16 min at 37°C)- counterstained with 1% chloroform extracted methyl green

(40) Lead method for G-6-PO^- (Brownish- black Bancroft, 1967 glucose-6-phosphatase (G-6-PO4) (Incubated for 20 min at 37°C)

(41) Nitro-blue tetrazolium Succinic dehydrogenase- Ishida and Chang, 1965 method for succinic blue dehydrogenase (Incubated at 37°C for 1 h, pH 7.6)

(42) Diastase treatment Inhibits subsequent Thompson, 1966; Humason (10 min to 17 h at staining of glycogen 1972; Pearse, 1968 37°C)- subsequently with PAS stained with Alcian blue (pH 2.5) PAS, and Congo red

(43) Hyaluronidase Hydrolyses glycosamin- Thompson, 1966; Humason treatment (1-3 h oglycans 1972 at 37°C, pH 5.0)- subsequently stained with Alcian blue (pH 2.5) Table 1 (Continued)

Cytochemical method Specific result Reference(s) for procedure

(44) Trypsin treatment Hydrolyses proteins with Thompson, 1966; Humason, (45 min - 26 h at free epsilon-amino or 1972 37°C, pH 7.8)- delta-guanido groups subsequently stained with Congo red and mercuric bromphenol blue

(45) Neuraminidase (24- Inhibits alcianophilia Spicer and Duvenci, 1964; 48 h at 37°C)- sub of sialic-acid containing Humason, 1972 sequently stained with proteins Alcian blue (pH 2,5)

(46) Glucuronidase Hydrolyses conjugated Thompson, 1966 (1 h, 2 h, and 8 h glucuronidases at 37°C, pH 6.8)- subsequently stained with PAS and Alcian blue (pH 2.5) 100

Culture of fertilized eggs

Limulus polyphemus L. eggs may be cultured in the

laboratory following artificial insemination. Eggs were col

lected and placed in plastic petri dishes. Just enough sea water to form a pool around each batch of eggs was added to prevent desiccation. A 10% spermatozoa suspension was

immediately added and the sperm egg mixture was allowed to

sit for periods of 30 min to 1 h. The fertilized eggs were

then washed three times with sea water and placed in a

Percival PT-80 or Sherer RI-LTP Series environmental chamber

at 25°C. Throughout the culture period the sea water was

changed daily. Inseminated eggs were removed at 5 min, 1 h

and 72 h, frozen and sectioned at 12y, attached to glass

microslides or 22 mm sq cover glasses and immediately fixed

in cold (0®-5°C) neutral buffered formalin (Thompson, 1966)

for 1 h. At the end of a 30 min running tap water wash, the

fertilized egg sections were stained for mucosubstances with

Alcian blue (pH 2.5)/nuclear fast red (Humason, 1972). Some

egg sections were not fixed following sectioning. Instead

they were immediately treated for glucose-6-phosphatase

(Bancroft, 1967; Pearse, 1968) and succinic dehydrogenase

activity (Kachlas, Tsou, Souza, Cheng and Seligman, 1957).

Glucose-6-phosphatase controls were incubated without the

reaction substrate (glucose-6-phosphate) and with complete

media and neutral formalin in a 1:1 ratio. Succinic 101 dehydrogenase controls were incubated in media in which sodium succinate was omitted. Other embryos were allowed to develop up through the trilobite larval stage vrtiere they were used in studies and observations on the embryological development of

Limulus polyphemus L.

Enzymatic Treatment of Egg Envelopes

To obtain additional information on the nature of the egg envelopes. Cryostat (12y) sections of eggs were fixed in neutral buffered 10% formalin (1 h), washed in running tap water (1 h) and subsequently treated for 10 min to 8 h at

37°C with enzyme preparations of malt diastase, hyaluronidase? trypsin, neuraminidase and g-glucuronidase (Thompson, 1966;

Spicer and Duvenci, 1964; Lillie, 1947).

Malt diastase (Nutritional Biochemicals) was prepared in neutral phosphate buffered saline (Img/ml), hyaluronidase

(Bovine testes. Type I, Sigma) in O.IM acetate buffer (pH

5.0, 0.5mg/ml), trypsin (4X U.S.P. pancreatin. Nutritional

Biochemicals) in M/15 Sorensen's phosphate buffer (pH 7.8,

Img/ml), neuraminidase (CI. perfringens, Type V, Sigma) in

O.IM acetate buffer (at pH 5.0 in final enzyme: buffer conc. of 1:500 and 1:10,000) and ^-glucuronidase (Sigma) in 0.004M phosphate buffer (pH 6.8, 100 units/ml). A summary of enzyme treatments and subsequent cytochemical methods has been tabulated in Table 1 (Experiments 42,43,44,45,46). 102

Determination of the Types of Chemical Linkages Maintaining the Structural Integrity of Limulus Egg Envelopes

A number of solutions known to have specific effects on the linkages between proteins (Brown, 1950) and on egg envelopes (Rothschild, 1956; Austin, 1961) were employed in an attempt to obtain additional information on how the structural integrity of Limulus egg envelopes is maintained. Unfixed

Cryostat egg sections (12vi) were treated for periods up to

24 h at room temperature or 37®C. A summary of the methods employed is presented in Table 2.

Amino Acid Analysis of Egg Envelopes

Limulus eggs were collected and egg envelopes separated from the yolky components in filtered Instant Ocean synthetic sea water. The separations were accomplished by crushing the eggs with a spatula in a Syracuse watch glass. Both the spatula and watch glass were covered with Parafilm "M" to prevent egg envelopes from adhering to the glass. To remove all traces of yolk, the egg envelopes were washed in three 20 ml vol of filtered sea water in a 50 ml plastic beaker with stirring on a Fisher Flexa-Mix magnetic stirrer. Washed eggs were allowed to dry on glassine powder paper (Eli Lilly

Company, Indianapolis) before being prepared for amino acid determination. Table 2. Physiochemical methods employed on Limulus egg sections

Method or reagent Effect Reference

0.2 N HCl (pH 1.2) Breaks electrovalent bonds Brown, 1950

NaOH (0.2N, .05N, Breaks electrovalent bonds Brown, 1950 .005N, pH 12.5)

Urea (4%, 6M) Breaks electrovalent bonds Brown, 1950

Sodium sulphide (5%) Breaks covalent bonds, Brown, 1950 •-S-S-bonds

Sodium hypochlorite (10%) Breaks non-S-S-bonds Brown, 1950

Hydrogen peroxide 10% Oxidizing agent Austin, 1961

Ca^^ free sea H^O Dissolves hyaline layer of Rothschild, 1956 sea urchin eggs

Hyamine 1622 in Cationic emulsifier MBL sea HgO (0.1%)

Trypsin (O.OOlg/ml, Attacks bonds between carboxyl McFarlane, 1962 pH 7.8)-10 min-16 h group of lysine or arginine and amino group of another amino acid

Pepsin (O.OOlg/ml, Attacks peptide bonds with McFarlane, 1962 pH 0.7)-10 min-16 h aromatic residues on either or both sides of the bond 104

Each sample prepared for amino acid analysis (3mg, 6-12 eggs) was placed in a test tube with 1 ml of 6N-HC1 (Moore and

Stein, 1960). The test tube was evacuated and sealed under vacuum. The contents were hydrolyzed at 110®C for 22 h. At the end of the hydrolysis, the HCl was evaporated and replaced with 3 ml of 0.2N sodium citrate buffer (pH 2.2). The amino acid composition of the sample was determined on a Beckman 120

B Amino Acid Analyzer.

Preparation of Egg Envelopes for Ultrastructure

Uninseminated egg envelopes

Eggs were collected and placed in plastic petri dishes containing cold (0*-5°C) filtered Instant Ocean synthetic sea water. The eggs were punctured with fine point Dumont stain less steel tweezers and the yolk was removed. Egg envelopes were placed in 17 X 60 mm glass vials containing cold (0°-5°C)

2.5% glutaraldehyde in a buffer comprised of 0.2 M sodium cacodylate (pH 7.4) 3% NaCl and 3.5% sucrose. Total time of the tissue in the fixative was 1 h. At the end of the fixa tion period the tissues were washed for 1 h in four changes of

0.2 M sodium cacodylate buffer (pH 7.4) containing 3% NaCl and

3.5% sucrose. Post fixation for 1 h with cold (0®-5°C) 1%

OsO^ in 0.2 M sodium cacodylate buffer (pH 7.4) containing 3%

NaCl and 3.5% sucrose was followed by a 1 h wash in the cacodylate buffer (3 changes). The tissues were dehydrated in 105 a graded series of acetone, embedded in Spurr's (1969) low viscosity resin and sectioned on a Porter-Blum MT II ultramicrotome with glass knives. Sections were mounted on uncoated

300 mesh grids and stained with 5% uranyl acetate (in 50% ethyl alcohol) followed by lead citrate (Venable and

Coggeshall, 1965). The stained sections were examined on a

Hitachi HU llE-1 or a Philips EM201 electron microscope.

Inseminated egg envelopes

Egg envelopes were prepared as described in the section on uninseminated egg envelopes. The envelopes were treated with a 10% dilution of fresh unwashed spermatozoa for 10 min and subsequently prepared for electron microscopy in the manner previously described.

Microscopic Examination and Photographing of Cytochemical Reactions and Developing;Limulus Embryos

Microscopic slides of sperm smears and egg sections were examined and photographed on the Nikon S-Ke-II microscope with the PFM 35 mm photomicroscopy system, the Zeiss Standard

Universal microscope with the Olympus Auto PM-IO-A 35 mm photomicroscopy system and the Wild M 20 microscope with the

MKa 5 automatic 35 mm photomicroscopy system. Developing

Limulus embryos were photographed with a Wild M5 stereo- microscope and a Nikon AFM automatic 35 mm photomicroscopy system. Black and white images were recorded on Kodak 106

Panatomic-X film which was subsequently developed in Kodak

Microdol-X at 68°C and on Kodak Tri-X developed in Kodak D-76

(1:1 developer/water) at 68®C. Prints from black and white negatives were processed in Kodak Dektol (1:2 Dektol/water) at

68°C. Color images were recorded on Kodak Kodachrome II

(Daylight), Kodachrome II (Type A), High Speed Ektachrome

(Daylight), High Speed Ektachrome (Tungsten) and Photomicro

graphy color film 2483 (Formerly S0-456). Slides and prints were processed at a regional Eastman Kodak Laboratory. 107

RESULTS

The phenomenon of fertilization in this study has been investigated along three major lines of inquiry; morphological, cytochemical and molecular. Although most of the investiga tions dealt with morphology, many are concerned with the cyto

chemical and molecular aspects of fertilization.

Limulus polyphemus L. is an excellent organism for

conducting studies in all three categories. This is readily

evident from recent studies on the morphological (Brown and

Humphreys, 1971) and immunological (Cooper and Brown, 1972)

aspects of fertilization in this primitive arthropod. The

desirable features of Limulus as a useful laboratory animal in

fertilization studies have only been recently appreciated, a

fact which probably accounts for the absence of information on

the cytochemistry and biochemistry of fertilization in this

species.

Limulus as a Laboratory Animal

The increased interest in Limulus as a laboratory animal

stems from its availability and usefulness under laboratory conditions. Limulus can be maintained with little attention

in marine aquaria for several months at 15°C and produce

viable gametes even when not fed. Thus, investigators who are

located inland and not near a marine habitat are not deprived

from studying this species. 108

The significant breakthrough in terms of investigating the developmental biology of Limulus came following the discovery that gametes could be obtained through electrical stimulation (Shrank, Shoger, Schechtman and Bishop, 1967).

Limulus, like many aquatic species displaying external fertilization, releases large numbers of gametes during spawn ing. Through the use of electrical stimulation this potential to release large quantities of gametes is very useful in the laboratory, particularly in cytochemical and biochemical studies where large quantities of material are needed for investigation. Another important aspect in the developmental biology of Limulus is the collected gametes can be mixed and the resulting inseminated eggs can be cultured Vitro through the trilobite larval stage. Thus, not only can the varied problems of fertilization be investigated in Limulus, but a detailed study of the embryological development of this species is also possible in the laboratory. In summary, the life history of Limulus can be followed from the union of sperm and egg through the hatching of the trilobite larvae.

Cytochemistry of the Sperm Acrosome

Acid phosphatase

Staining of the acrosome vesicle and acrosome membrane was observed in sperm smears on experimental slides incubated in media containing a-naphthyl phosphate as the substrate and 109 hexazonium pararosanilin as the coupling agent. The sperm smears counterstained with 1% chloroform extracted methyl green exhibit green staining of the nucleus while the acrosome vesicle and acrosome membrane display a reddish hue (Figure 4).

Control smears incubated without the substrate show no staining of the acrosome vesicle or membranes. Only the nucleus is visible following staining with 1% chloroform extracted methyl green (Figure 5). Identical results were obtained with experi mental and control media at pH 2.0 and pH 6.0.

Acridine orange

Subsequent to acridine orange treatment and examination with ultraviolet light, the acrosome vesicle of each Liittulus sperm exhibits a brilliant red fluorescence. The nucleus on the other hand, fluoresced bright green. The results were the same for unwashed spermatozoa and for spermatozoa following a

10 min centrifugation (2000 rpm; 480 G) at 0°C. Examination following a second centrifugation under identical conditions reveals a bright orange fluorescence in the nucleus in con trast to the reddish-orange fluorescence exhibited by the acrosome.

Acrosome proteinase

Preservation of sperm morphology in unfixed sperm smears and smears fixed in 100% ETOH at -20°C was poor. In addition, an intense positive reaction was observed in both fixed and 110 unfixed experimental sperm smears. Consequently sperm smears prepared from undiluted spermatozoa, unfixed spermatozoa and spermatozoa fixed in 100% ETOH were not usable.

Preparation of sperm smears from 10% suspensions and sub sequent fixation in formal-calcium at pH 7.0-7.2 (0®-5®C) for

2 h gave excellent sperm morphology preservation. Incubation in experimental media at pH 7.2-7.4 gives a positive reaction in the acrosome region (Figure 6) while control preparations show no reaction in the acrosome (Figure 7). The reaction product is brownish in color and appears in the acrosome as granules (Figure 6).

Trypsin-likie acrosome enzyme

Glutaraldehyde fixed (0.05% in veronal acetate buffer at pH 7.0) gelatin membranes (some impregnated with India ink) were tested for stability and sensitivity and were found to be stable in dHgO at 60®C for 30 min and sensitive to trypsin

(1 mg/ml in 0.1 M Tris buffer, pH 8.0) treatment. In experi mental treatments spermatozoa suspended in sea water (pH 6.5,

8.0) and placed on these membranes failed to react with the membrane (Figure 8). Reacted spermatozoa treated with 0.34 M

CaClg (pH 6.0) also had no effect in digesting the gelatin membrane. Ill

Nonspecific esterase

Unfixed sind fixed sperm smears (formol-calcium, pH 7.2 for 1 h at 0°-5®C) were tested for nonspecific esterases in media at pH 6.2 and 7.2. No positive reactions were observed following incubation at 23®C and 37®C for periods up to 48 h

(Figure 9).

Ultrastructure of the Spem Acrosome Complex

The acrosome complex of Limulus consists of a large acrosome vesicle and a multifibrillar rod. The multifibrillar rod extends from a posterior indentation of the acrosome vesicle, through the nucleus, and into a posterior perinuclear cisterna where it assumes a coiled configuration

(Figures 10, 11, 12, 13). The acrosome vesicle is surrounded by an acrosome membrane and the sperm plasma membrane.

Located between these two membranes is the periacrosome mate

rial encompassing both the acrosome vesicle and the anterior

lateral portions of the nucleus (Figure 4). Occupying the

posterior acrosome indentation region is a subacrosome mate

rial. This material differs in texture from the periacrosome

material (Figure 11).

The multifibrillar rod, which is 50 y long, may assume

two positions in relation to the sperm following an acrosome

reaction. The "true" acrosome reaction involves rupture of

the acrosome vesicle and extrusion of the acrosome filament 112

anteriorly. This occurs normally when the sperm come in

contact with the basement lamina of the egg (Figures 14, 15)

and artifically when spermatozoa are treated with 0.34 M CaClg

(Figure 16). With the latter conditions the acrosome fila

ment appears as a long stiff rod. The other position is the

"false" acrosome reaction in which the acrosome rod protrudes

posteriorly from the perinuclear cisternae. The acrosome

vesicle always remains intact. The "false" reaction is

observed after spermatozoa are suspended in 2x MBL sea water

(Figure 17). This reaction also occurs in spermatozoa that

have been centrifuged (10 min at 480 G and 0°C) and allowed to

stand at room temperature (30 min) as a wet mount preparation.

In either case no "true" acrosome reactions are observed in

the population of spermatozoa.

Separation of Sperm Acrosome

Treatment of Limulus sperm suspensions with cold 0.0025%

sodium hypochlorite for 1-2 min will release the intact acro

some from the spermatozoa. This treatment also causes dis

integration of the nucleus and subsequent release of the DNA which forms a gel trapping the freed acrosomes (Figure 18).

Treatment of the viscous gel with 0.02% DNAase I dissolves it

and releases the acrosomes (Figure 19). Electron micrographs

taken of gels formed within 5 min after sodium hypochlorite

treatment show the sperm plasma membrane and nuclear membrane 113 are first affected (Figure 20). The dissolution of the nucleus continues until only the aerosome remains (Figures 21,

22). The subacrosome plate, which is found posterior to the subacrosome material, is found to persist to later stages of nuclear disintegration. This structure finally disintegrates along with the remaining nuclear material. The sperm plasma membrane surrounding the acrosome disintegrates along with the periacrosome material surrounding the anterior and peripheral regions of the acrosome (Figure 23).

Cytochemistry of Uninseminated Egg Sections and Eggs

The Limulus egg is surrounded by two clearly recognizable layers in sectioned material; the basement lamina and the vitelline envelope. The outer basement lamina is 5 y thick and transversed by numerous pores (Figure 14). These pores vary in diameter and terminate at the surface of a 35 y thick layer called the vitelline envelope. At the inner border of the vitelline envelope is a cytochemically identifiable cor tical region or zone. This region is approximately 5 y across in uninseminated eggs (Figure 29). The remainder and bulk of the egg consists of densely packed yolk.

Cryostat sections of Limulus eggs are easily and quickly prepared for cytochemistry. These sections are attached to cold glass microscope slides or cover glasses without the use of an adhesive. The egg layers adhere to glass strongly and 114

consequently survive many procedures that would ordinarily

result in loss of sections. A summary of cytochemical reac- ' • i tions observed in egg layers, the cortical region and yolk of

uninseminated egg sections and eggs is presented in Table 3.

Carbohydrates

The vitelline envelope, cortical region and yolk of the

eggs are PAS positive. The cortical region and yolk stain

red and the vitelline envelope stains pink after the PAS

reaction (Figure 24). Treatment to block aldehydes with

hydroxylamine eliminate the PAS positive reaction of the egg

components.

The yolk and cortical region stain brown following the

PAD method for characterizing neutral mucopolysaccharides,

periodate-reactive polymers and periodate-unreactive muco-

substances, indicating the presence of neutral mucopolysac

charides in the cortical region and yolk. On the other hand,

the vitelline envelope stains gray-brown indicating the

presence of periodate-reactive polymers. No staining reaction

is observed in the basement lamina following the PAD method.

The presence of 1, 2-glycols in the egg is indicated by

the CAS and PPS procedures. Whereas all components of the

egg give a positive reaction following the PPS method, only

the cortical region and yolk give positive reactions with the

CAS method. The reaction results in a light pink to light Table 3, Cytochemical reactions of uninseminated Limulus eggs^ Reaction Stain or test Basement Vitelline Cortical Yolk lamina envelope region

PAS +++ ++++ ++++ Hydroxylamine/PAS Potassium +++ ++++ ++++ Permanganate Schiff (PPS) Chromic Acid Schiff + + Congo red ++ +++ +++ Alcian blue +++ (pH 1.0) Alcian blue +++ (pH 2.5) Toluidine blue 0 ++ Azure A PAD ++ ++ ++ (gray-brown) (brown) (brown) Silver method Mild methylation/ Alcian blue (pH 2.5) Methylene blue extinction pH 2.6 pH 3.6 pH 4.3 + pH 5.3 + + + pH 6.7 ++ ++ ++ ++ pH 7.6 ++ +++ +++ +++ pH 8.1 ++ ++++ ++++ +++ Mercuric bromphenol blue ++++ ++++ ++++ ++++

Van Slyke's reagent/ — ++ ++ mercuric bromphenol blue (144 h) Performic acid-Alcian blue ++ + Ninhydrin-Schiff ++++ +++ ++ ++ Ninhydrin-Schiff/ ++++ +++ ++ + Hydroxylamine (3 h)

Ninhydrin-SChiff/ H M Van Slyke's reagent m (10 h) Sakaguchi method + + + Naphthol-yellow S +++ +++ +++ +++ Feulgen method ++ ++ IN-HCL/Feulgen ++ ++ DNase I/Feulgen ++ ++ Oil red O +++ +++ Bromination/ +++ +++ Oil red O Okamoto's method

^0 = Undecided; + = weak reaction moderate reaction; +++ = strong reaction; ++++ = very intense reaction no reaction. Table 3 (Continued)

Reaction Stain or test Basement Vitelline Cortical Yolk lamina envelope region

U-V Schiff method 0 + ++ ++ U-V Schiff control Sudan black B +++ (chromated) Holczinger's technique + + ++ ++ Methanol/chlorform IN HCl, Holczinger's technique IN HCL, acetone, Holczinger's technique Fischler's method 0 ++ + + 100% ETOH/Ether Copper phthalocyanin + - - Methanol-chloroform/ + — — Copper phthalocyanin Chromâtion/Copper + ++ ++ phthalocyanin Molisch Reaction Gomori's method ++ ++ (Alkaline phosphatase) Azo dye method - +• + (Alkaline phosphatase) Naphthyl acetate method (Esterases) Azo dye method (Acid phosphatase) Lead method +++ {Glucose-6-phosphatase) Succinic dehydrogenase ++++ 119 red color with, the cortical region and yolk having the same staining characteristics. Following the PPS procedure the vitelline envelope, cortical region and yolk stain intensely.

Only a weak positive reaction is observed in the basement lamina. The most striking observation concerning the PPS procedure is the deposition of dark stain deposits in the canals of the pores, thus demonstrating the vast pore network of the basement lamina (Figure 26).

The Congo red method for glycoprotein gives a strong positive reaction in the cortical region and the yolk. The reaction is much less intense in the basement lamina and vitelline envelope (Figure 25). A positive reaction for sulphated mucosubstances and weakly acidic sulphated mucosub stances is observed only in the cortical region of the egg after staining with Alcian blue at pH 1.0 and pH 2,5 respec tively (Figures 27, 29). Similar results are also observed with mild methylation and subsequent staining with Alcian blue

(pH 2.5) to prohibit staining of nonsulphated mucins. The use of toluidine blue O for the demonstration of metachromasia also gives a positive reaction that is restricted to the corti cal region. The color reaction following treatment is purple, an indication of 6-metachromasia.

Methylene blue extinction of the basement lamina is between pH 5.3 and 6.7 (Figures 34, 35), whereas that of the vitelline envelope, the cortical region and yolk is between 120 pH 2.6 and pH 4.3 (Figures 31, 33).. At higher pH values

(Figures 36, 37) the vitelline envelope displays a strong affinity for methylene blue (pH 7.6-pH 8.1) while the basement lamina, cortical region and yolk show no increase in affinity for the stain above pH 6.7 (Figure 35).

Azure A at pH 1.0, 3.6 and 4.0 produces no staining reactions in the egg. A similar situation results when the silver method is used to demonstrate ascorbic acid.

Proteins and nucleic acids

The entire egg reacts strongly to the mercuric- bromphenol blue method for proteins. The basement lamina stains blue and the vitelline envelope, cortical region and yolk stain blue-purple (Figu.re 38). Deamination (Van Slyke's reagent) immediately blocks the positive reaction in the base ment lamina (Figure 39), but only extended deamination of six days is successful in blocking the positive reaction of the vitelline envelope (Figure 40). Even with this extended deamination, a weak positive reaction remains in certain yolk

bodies. Two other general protein methods, ninhydrin-Schiff

and naphthol-yellow S produce strong positive reactions in the

egg components. The ninhydrin-Schiff reaction leaves the

basement lamina and the vitelline envelope rose colored with

the basement lamina darker in color than the vitelline envelope.

The yolk, which is not distinguishable from the cortical

region, displays a slightly weaker reaction than the vitelline 121

envelope (Figures 41, 42). Hydroxylamine treatment of egg sections prior to employment of the ninhydrin-Schiff method had no effect on their staining characteristics. Deamination with Van Slyke's reagent (Lillie, 1954) blocks the reaction in the yolk and cortical region. Both the basement lamina

and the vitelline envelope fail to stain red following this

procedure (Figure 43).

Following the Sakaguchi reaction the vitelline envelope

and the yolk of egg sections display a weak reaction for the

basic amino acid arginine by staining light pink. The base

ment lamina and vitelline envelope do not stain following the

performic acid-Alcian blue technique for disulphides. This

is in contrast to the cortical region and the yolk vdiich

stains blue with the cortical region displaying a stronger

affinity for the stain than the yolk.

The Feulgen reaction is positive in the yolk and the

cortical region, although these two regions are not dis

tinguishable from each other on the basis of staining character

istics since both regions stain red. DNase I failed to alter

the staining properties after 5 h at 37®C (sperm smear pre

parations of Limulus spermatozoa were prevented from staining

in the nucleus after 2 h at 37°C). Extraction with 1 N-HCl at

37°C for 3 h slightly reduces staining intensity. 122

Lipids

The basement lamina and the vitelline envelope are un stained following the Oil red 0 method for neutral fats (Figure

44), although certain yolk bodies are observed to stain red.

The cortical region remains indistinguishable from the yolk in staining characteristics. Similar results are observed following staining with Sudan black B dissolved in 60% tri- ethylphosphate (Figure 46). Controlled chromation and sub sequent staining with Sudan black B produces no change in the staining characteristics of the egg yolk. Haematoxylin staining used in conjunction with the Sudan black B procedure stains all yolk bodies as well as the basement lamina and vitelline envelope (Figure 47).

Okamoto's method (Pearse, 19 68) for the demonstration of cholesterol class lipids fails to give a positive reaction in

Limulus eggs. The only reaction observed is the swelling of the basement lamina to approximately two times its original thickness.

Unsaturated lipids are demonstrated in the vitelline envelope, and the yolk with the U-V Schiff method.. The cortical region displays light purple staining identical with that of the yolk. The reaction in the vitelline envelope is weaker than that of the yolk (Figure 48). No staining was observed in controls (Figure 49). 123

Fischler's method for fatty acids gives a dark blue positive reaction in the vitelline envelope and a lighter reaction in some yolk bodies. No other egg components react.

Holczinger's fatty acid technique gives a greenish-black positive reaction in all egg components. The strongest reaction is observed in the cortical region and yolk. The staining between these two regions is indistinguishable.

Neither methanol/chloroform extraction at 60°C nor 95% ethanol extraction at room temperature has any effect on the staining characteristics. On the other hand, treatment with IN HCl or

IN HCl followed by acetone treatment abolishes staining in all egg components.

A weak positive reaction for lipoproteins is observed in the vitelline envelope following the copper phthalocyanin method. The characteristic blue staining of this procedure is absent from the basement lamina, cortical region and yolk

CFigure 45). Methanol/chloroform extraction at 60°C for 24 h does not prevent staining. Controlled chromation at 57°C and

60°C over a period of 24 h produces no change in the staining characteristics of the egg envelopes. The cortical region and yolk elicit a moderate positive reaction.

Hydrolytic enzymes

Two methods for identification of alkaline phosphatase,

Gomori's and the AZO dye give positive results in the cortical

region of the egg and in the yolk. With Gomori's method the 124 activity is a brownish color of equal intensity in the corti cal region and the yolk. With the AZO dye method the positive reaction in these two regions is considerably weaker.

Tests for acid phosphatase were negative for the entire egg. The yolk gave a positive reaction for nonspecific esterases and the cortical region is positive for glucose-6- phosphatase and succinic dehydrogenase. Both glucose-6- phosphatase and succinic dehydrogenase procedures give intense positive reactions in the cortical region. No other part of the egg reacts (Figures 50, 54).

Cytochemistry of Inseminated Egg Sections and Developing Eggs

Egg sections inseminated with 10% sperm suspensions for

5 min and stained with Alcian blue (pH 2.5)/nuclear fast red give a positive reaction in the cortical region. The acrosomes of the spermatozoa are also Alcian blue positive (Figure

30J. Egg sections allowed to react with spermatozoa for a period of 1 h and subsequently stained with Alcian blue/ nuclear fast red are identical in staining characteristics to egg sections stained after 5 min of sperm-egg interaction.

Inseminated eggs allowed to develop before sectioning

are also Alcian blue positive in the cortical region of the

egg. After 4 h of development the Alcian blue positive region

begins spreading away from the cortical region into the yolk.

At 72 h the cortical region is no longer Alcian blue positive. 125

A few yolk bodies adjacent to and in the cortical region of

72 h eggs are Alcian blue positive (Figure 28).

Glucose-6-phosphatase activity, like the positive Alcian blue reaction, is found only in the cortical region of fresh and early developing eggs (Figures 50, 51). At 72 h the glucose-6-phosphatase activity has spread to the yolk and is indistinguishable in intensity from that of the cortical region (Figure 51).

Succinic dehydrogenase activity in the Limulus egg is also restricted to the cortical region (Figures 53, 54). Un like glucose-6-phosphatase activity, succinic dehydrogenase activity remains in the cortical region at 72 h of development

(Figure 55).

Enzymatic Treatment of Uninseminated Egg Sections

A summary of the cytochemical reactions of uninseminated egg sections following enzymatic treatment is presented in

Table 4. The enzymatic treatments had no effect on the staining characteristics of the egg sections in all cases except one when compared with untreated sections (compare

Table 3 with Table 4). 3-Glucuronidase treatment for a period of 8 h was the only enzymatic treatment to produce a decrease in staining intensity which is observed in the vitelline envelope, cortical region and yolk of uninseminated egg sections. Table 4. Enzymatic treatment and staining of uninseminated egg sections^

Enzyme treatment Stain or Results test Basement Vitelline Cortical Yolk lamina envelope region

Malt diastase Congo red ++ + +++ +++

PAS - +++ ++++ ++++

Alcian blue — — +++ — (pH 2.5)

Hyaluronidase Alcian blue - - +++ - Trypsin Congo red ++ + +++ +++ Mercuric ++++ ++++ ++++ ++++ bromphenol blue (Figure 41)

Neuraminidase Alcian blue - - +++ -

3-Glucuronidase Alcian blue - - +++ -

PAS (8 h)b - ++ +++ +++

^0 = Undecided; + = weak reaction; ++ = moderate reaction; +++ = strong reaction; ++++ = very intense reaction; - = no reaction.

^Decrease in staining intensity as compared with untreated sections. 127

Treatments with trypsin, malt diastase, or hyaluronidase cause most of the yolk to become suspended in the enzyme solutions. Coating sections with celloidin (Thompson, 1966) affords a small degree of protection if the microslides are handled gently. The enzyme solutions do not digest either the basement lamina or the vitelline envelope.

Physiochemical Treatment of Egg Sections

A number of reagents were employed to determine the types of bonds responsible for maintaining the structural integrity of the Limulus egg envelopes (Table 2). The results of these treatments for periods up to 24 h at room temperature are recorded in Table 5, while Figures 56, 57, 58, 59, 60, 61, 62, and 63 show the effects of various treatment on the egg sections.

Amino Acid Analysis of Isolated Uninseminated Egg Envelopes

Sixteen amino acids are found in the egg envelopes.

Amino acids containing sulfur as a sulfhydryl group (cysteine) and sulfur in a thioether linkage (Methionine) are absent or in low concentrations. Also absent are the amide forms of aspartic and glutamic acid. The amino acid composition of

Limulus egg envelopes has been tabulated in Table 6. Table 5. Effects of physiochemical reagents on egg sections Effect on egg layers Method or reagent Basement lamina Vitelline envelope 0.2 N HCl (pH 1.2) Expansion and increase No change (Figure 56) in size NaOH Expands on contact and Dissolves in some in- 0.2 N (Figure 61) dissolves immediately stances before basement lamina 0.5 N (Figures 62, 63) Dissolves in approximately Dissolves in approximately 14 min 14 min 0.005 N (Figure 60) Swelling No change Urea 4% No change No change 6M (Figures 57, 58) Expansion and point separa No change tions from the vitelline envelope Sodium sulphide (5%) No change No change Sodium hypochlorite (10%) Dissolves Dissolves Hydrogen peroxide (10%) No change No change free sea HgO No change No change Hyamine 1622 in MBL No change No change sea H^O (0.1%) Trypsin (0.001 g/ml, No change No change pH 7.8)-10 min-16 h (Figure 59) Pepsin (0.001 g/ml, Vesiculation No change pH 0.7)-10 min-16 h (Figures 64, 65, 66, 67) 129

Table 6. Amino acid composition of Limulus egg investments^

Amino acid y Moles/mg li Moles/egg

Lysine .415 .097

Histidine .117 .028

Ammonia .494 .119

Arginine .126 .031

Aspartic acid .501 .126

Threonine .288 .295

Serine .365 .090

Glutamic acid .465 .114

Proline .357 .087

Glycine .332 .081

Alanine .180 .044

Valine .406 .100

Methionine .048 .011

Isoleucine .373 .092

Leucine .431 .106

Tyrosine .527 .129

Phenylalanine .236 .058

^Mean of 3 determinations. 130

Amino Acid Analysis of Isolated 72 h Egg Envelopes

Seventeen amino acids are found in the egg envelopes at

72 h of development. The amino acid cystine, which is not

present in uninseminated egg envelopes, is present in 72 h

egg envelopes. Each amino acid found in the uninseminated egg envelopes shows a significant percentage of increase over the amount found in 72 h egg envelopes (Table 7). The average amount of increase for all amino acids found in uninseminated egg envelopes as compared with 72 h egg envelopes is 71.5%.

Observations on the Embryology of Limulus polyphemus L.

The feasibility of using Limulus polyphemus as a laboratory animal in studies on embryological development is enhanced since horseshoe crabs are easily maintained under laboratory conditions, provide thousands of gametes annually, and complete early developmental stages in culture conditions.

These desirable characteristics makes this species attractive for use as a representative of a primitive arthropod in an embryology course.

Freshly spawned eggs obtained through electrical stimula tion are green in color and highly refractive. The eggs typically contain one or two large indentations immediately following spawning. In addition, the surface of the yolk is perforated by numerous pits which persist for 2-3 h (Figures 131

Table 7. Amino acid composition of inseminated (72 h) Limulus. eggB envelopes

Amino acid y Moles/mg y Moles/egg % of increase at .72 h level

Lysine .753 .188 81.4

Histidine .266 .066 93.1

Ammonia .883 .221 78.4

Arginine .232 .058 84.1

Aspartic acid .878 .200 75.2

Threonine .484 .121 68.0

Serine .590 .147 61.6

Glutamic acid .814 .203 75.0

Proline .623 .156 74.5

Glycine .590 .148 77.7

Alanine .317 .079 76.1 _b Half Cystine .317 .079

Valine .742 .186 82.7

Methionine .072 .018 50.0

Isoleucine .669 .168 79.3

Leucine .736 .184 70.7

Tyrosine .712 .178 35.1

Phenylalanine .362 , .090 53.3

"^Mean of 3 determinations.

^Not present in uninseminated egg investments. 132

68, 69, 70). Within a span of 2-6 h following fertilization, the surface of the yolk becomes smooth and the eggs character istically enter a cycle of development during which the yolk fluctuates between a smooth and granular appearance (Figure

71). This cycle of events continues for the next 18-20 h of development. Between 20-27 h following fertilization the embryos enter a segmentation stage during which the yolk of the eggs is divided into several large unequal biastomeres

(Figures 72, 73). At 28-48 h of development nucleated blastomeres are observed between and on the surface of the large biastomeres (Figure 73).

During the next 24 h the embryos pass through several developmental changes, namely the continuous smoothing of the embryo surface, invasion of the embryo surface by nucleated biastomeres and the appearance of a blastopore. As the large biastomeres lose their boundaries and become smooth in appearance, the nucleated blastomeres take over the surface of the embryo until the entire surface of the embryo is covered with nucleated blastomeres (Figures 74, 75). Subsequent

division of the blastomeres and appearance of the blastopore

completes the developmental events of the first 72 h of the

Limulus embryo (Figures 76, 77).

Developmental changes observed in fertilized eggs up to

the blastopore stage parallel those observed in unfertilized eggs with the exception of one event; nucleated blastomeres do 133 not appear in unfertilized eggs. Beyond 48 h following spawn ing the yolk in unfertilized eggs looses its close apposition to the egg envelopes and maintains a granular appearance for the duration of the culture period.

The successive topographical stages of development involves division of the blastomeres until these units become incorporated into a smooth shiny layer surrounding the embryo.

Any additional observable features of continued development are not evident following fertilization until 6-8 days when four paired grooves are visible on the surface of the embryo

(Figure 78). Twenty-four hours later five paired grooves or depressions and the stomodeum have became visible (Figure 79).

As development proceeds the five paired grooves take on the appearance of small buds and the embryo, after further develop ment, is observed to have six paired limb buds instead of five

(Figure 80). Concurrent with development of the limb buds is the appearance of the embryonic disc as a separate structure from the yolk (Figure 80).

At 14 days of development the appendages show movement, the segmentation of the céphalothorax is evident and the divi sion between the caphalothorax and abdomen is well defined

(Figure 81). Shortly before the embryos emerge from the origi nal egg envelopes, indications of an embryonic molt are evident

(Figure 82). Immediately after the embryonic envelope swells from the imbibition of sea water and the embryos are released 134

from their original egg envelopes, the first embryonic molt

occurs (Figures 83, 84). All further development of the

- embryos up to the trilobite hatching stage takes place inside

the embryonic egg envelope where they are free to swim about

(Figure 84). With the exception of possessing a telson, the

swimming embryos are morphologically similar to the adult.

The embryos hatch from the embryonic egg envelope shortly

after the second embryonic molt in approximately 23 days after

fertilization (Figure 85). The first post-hatch molt occurs

approximately 25 days later (Figures 86, 87).

Ultrastructure of Sperm-Egg Interaction in Limulus polyphemus L.

Ultrastructural studies on the egg envelopes of Limulus

have revealed the basement lamina to be covered with a layer

of electron dense granules which cover the insides of the

pores in the basement lamina as well as the surface of the

vitelline envelope (Figures 88, 89). These observations are

in agreement with those of Shoger and Brown (1970), who also

noted the presence of granules associated with the basement

lamina in their electron micrographs. Aside from the

appearance of the granules in the basement lamina, both the

basement lamina and the vitelline envelope are homogeneous in

substructure.

The observations that Limulus sperm fail to undergo a

"complete" acrosome reaction upon contact with the Limulus 135 egg envelopes was noted in several instances (Figure 90). The previous assumption had been the acrosome reaction of Limulus sperm involved simultaneous dehiscence of the acrosome vesicle and extrusion of the acrosome filament. The observations made in this study reveals the acrosome vesicle of Limulus sperm may react with the basement lamina of the egg by undergoing dehiscence and depositing its enzyme content on the surface of the egg without the uncoiling and penetration of the egg envelopes by the acrosome filament. 136

DISCUSSION

Cytochemistry of Limulus Sperm Acrosome

The sperm acrosome is commonly viewed as a specialized lysosome which evolved in spermatozoa to facilitate fertiliza tion in multicellular organisms (Allison and Hartree, 1970).

In support of this view several investigators have searched for and located lysosomal-type enzymes in the sperm acrosomes of a number of different species. Among the lysosomal-type enzymes known to reside in acrosomes are acid phosphatases, aryl sulphatases, esterases and various proteases.

The cytochemical localization of acid phosphatase in the acrosomes of Limulus sperm adds this organism to the growing list of species with sperm acrosomes in which acid phosphatase has been identified. Among these are the frog (Novikoff,

1961), salamander (Buongiomo, Nardelli and Bertolini, 1967), guinea-pig (Ruffili, 1960) and Ascaris (Clark, Moretti, and

Thomson, 1972). Acid phosphatase activity in the Limulus sperm acrosome is demonstrable following use of a technique developed by Barka and Anderson (1962).

Enzyme activity is evenly distributed throughout the

acrosome vesicle and along the acrosome membrane. Since no

difference is found in staining intensity at pH 2.0 and pH

6.0, the acid phosphatase of the Limulus sperm acrosome has a 137a

comparable pH activity range to acid phosphatases demonstrated in various animal tissues (Humason, 1972).

Vital staining with acridine orange has been used to provide additional evidence for the lysosomal nature of sperm acrosomes. The similarity of staining between lysosomes and sperm acrosomes following vital staining with acridine orange has led to the opinion that lysosomes and sperm acrosomes are similar in their chemical makeup. When high concentrations (1 in 10^) of the acridine orange dye are taken up by lyso somes and nuclei of living cells, the lysosomes fluoresce orange-red and the nuclei fluoresce green (Allison and Young,

1964; Allison and Hartree, 1970).

Sperm acrosomes of several different animal species fluoresce orange-red while the nucleus fluoresces green following vital staining with acridine orange (Allison and

Hartree, 1970). Limulus sperm acrosomes fit the general pattern observed in other sperm acrosomes. A bright red staining is observed in the sperm aerosome while the nucleus stains green following vital staining with acridine orange and subsequent examination with ultraviolet light. Unlike the weak fluorescence observed in sperm acrosomes of man, ram, bull

and the boar (Allison and Hartree, 1970), the fluorescence in

the Limulus acrosome is intense. Since a glycolipid is 137b

believed to be involved in binding the acridine dye in the sperm acrosome (Barnett and Dingle, 1967), the LimuTus acrosome undoubtedly contains a high concentration of this glyco-

lipid.

In view of the fact that proteases are considered char acteristic of lysosomeë, the use of a cytochemical method to demonstrate an acrosome proteinase in the sperm acrosome of

Limulus sperm provides additional evidence that this acrosome is chemically similar to the lysosome. In addition,pro teinase activity in the LiffiUlus sperm acrosome was found to be more intense in an acid pH range (pH 6.5) than in alkaline

pH range (pH 7.2-7.4). Even though these observations are in

agreement with those of Yanagimachi and Teichman (1972) on sperm acrosomes of several species (mouse, rat, guinea pig, rabbit, dog, cock, and human), they are not in agreement with

reports on the pH optimum of a trypsin-like enzyme demonstrated

in sperm acrosomes of several species (rat, rabbit, guinea pig

and human) using a gelatin membrane technique (Gaddum and

Blandau, 1970). The fact that Limulus sperm acrosomes fail to

give a positive reaction with the gelatin membrane technique

at acidic (pH 6.0) or alkaline pH 8.0) pH strongly indicates

the proteinase demonstrated in Lintulus sperm acrosomes is

different from the proteinase demonstrated in the acrosomes

of the rat, rabbit, guinea pig, and human. Also, since no 138

reaction takes place with the gelatin membrane, the proteinase of Limulus sperm acrosomes cannot be considered trypsin-like.

Thus, the proteinase of Limulus spesrm acrosomes is an acidic, nontrypsin-like enzyme. Even though data in this study clearly indicates the presence of an acrosome proteinase with in the acrosome vesicle of Limulus sperm, its role in fertilization is an enigma. Lytic action of this proteinase on the protein containing egg envelopes of the Limulus egg has not been observed in light or electron micrographs of sperm-egg interactions.

The failure to demonstrate cytochemically nonspecific esterase activity in Limulus sperm acrosomes with formal- calcium fixed sperm smears is not too surprising since Bryan and Unnithan (1973) demonstrated this reagent abolishes non specific esterase activity in murine sperm acrosomes. The fact that unfixed sperm smear preparations also fail to demonstrate nonspecific esterase activity almost certainly rules out the presence of these enzymes in Limulus sperm acro somes since the method used in this study (Bryan and Unnithan,

19 73) is capable of detecting very low levels of nonspecific esterase activity.

In summary, these findings on the Limulus sperm acrosome have given additional support to the concept the acrosome is a modified lysosome. Limulus sperm acrosomes have been found 139

to contain acid phosphatase and a protease, enzymes which are characteristically associated with lysosomes. In addition,

Limulus sperm acrosomes and lysosomes display similar staining behavior following exposure to the fluorescent stain, acridine orange.

Ultrastructure of the Sperm Aerosome Complex

Even though the spermatozoa of Limulus are classified among the "primitive types" by Franzen (1970), the acrosome is quite complex. It consists of an acrosome vesicle and a

50 y long axial filament. The presence of a rigid preformed acrosome filament is not unique among invertebrate species

(Nereis, Mytilis), but the manner in which the filament coils posterior to the nucleus is unique to Limulus.

The acrosome vesicle is not a homogeneous structure in material prepared for electron microscopy. Instead, it con sists of two zones of electron opacity. In addition, the material surrounding the anterior protion of the acrosome vesicle (between the acrosome membrane and sperm plasma membrane) differs in texture from material at the base of the acrosome. The appearance of zones of different electron densities within the Limulus acrosome is no doubt correlated 140 with compartmentalization of acrosome enzymes within the acrosome complex. The idea that acrosome enzymes may occupy specific regions of the acrosome has been suggested from

ultrastructural studies conducted by Phillips (1972) on sperm

acrosomes of several species of mammals. These acrosomes, which were previously believed to be homogeneous from studies of sectioned material, display an ordered substructure in

certain regions of the acrosome. In addition, the plasm

membrane overlying the acrosome of the Chinese hamster and

mouse were found to contain vesicles and microtubules in an

area where the sperm heads tend to have a "sticky" quality.

The material between the plasma membrane and acrosome membrane

surrounding the anterior portion of Limulus sperm acrosomes

also contains microfibers or microtubules.

Examination of sperm smears prepared for the demonstration

of acid phosphatase activity reveals the region where the

microfibers and material surrounding the lateral portions of

the acrosome stain more intensely than the central region of

the acrosome vesicle. The same is true at alkaline pH (7.2-

7.4) for the localization of silver grains following procedures

to demonstrate an acrosome proteinase. The silver grains tend

to outline the anterior and lateral portions of the acrosome

rather than localize in the central region of the acrosome

vesicle. 141

In conclusion, ultrastructural investigations reveal the acrosome complex of Limulus sperm to be quite complex. The correlation of different densities observed within sperm acrosomes of Limulus with cytochemical evidence on the sperm acrosomes indicates compartmentalization of the acrosome enzymes within these spermatozoa.

Separation of the Sperm Acrosome

Removal of sperm acrosomes has been accomplished through the use of several reagents. Among these are NaOH (Clermont,

Clegg and Leblond, 1955), Triton X-100 (Teichman and Bern stein, 1969), and cetyl-trimethylammonium bromide (Hathaway and Hartree, 1963). The most widely used reagent has been hyamine (Hartree and Srivastava, 1965), which is employed in removal of sperm acrosomes from several mammalian species.

Physical methods such as sonic vibration have also been used to cleave sperm into heads and tails (Stambaugh and Buckley,

1969).

The use of sodium hypochlorite and mild sonic vibration to separate sperm acrosomes from Limulus spermatozoa leaves the acrosome vesicle intact. The fact the sperm plasma

membrane and the nuclear membrane of Limulus spermatozoa are

readily susceptible to sodium hypochlorite treatment indicates

these membranes are different in some manner or property from

the acrosome membrane. Since sodium hypochlorite disrupts non- 142

S-S-covalent linkages between protein chains (Brown, 1950),

it is suggested such linkages play only a minor role as stabilizing forces in the acrosome membrane of the Limulus

sperm.

The ability to demonstrate fluorescence following stain ing with acridine orange in separated Limulus sperm acrosomes strongly indicates the enzyme complement of the sperm acro some remains intact following the drastic chemical means of acrosome separation. This fact is further supported by the

demonstration of acid phosphatase in separated sperm acrosomes.

In conclusion, the sodium hypochlorite/sonication method of removing Limulus sperm acrosomes is a sufficient technique to use in preparing these acrosomes for subsequent biochemical

analysis of their enzyme content.

Cytochemistry of Uninseminated Egg Sections and Eggs

Carbohydrates

The PAS procedure is a histochemical technique that

stains tissue components containing 1, 2-glycols. According

to Hotchkiss (1948), any tissue that gives an oxidation

product which is not diffusible and contains 1, 2-glycol

groupings or an equivalent amino or alkyl-amino derivative

should give positive results. The intensity of the reaction

is dependent upon the concentration of the adjacent glycol or

aminohydroxyl groupings in the tissue (Thompson, 1966). 143

Observations that the vitelline envelope, cortical region and yolk is strongly PAS positive is indicative of the presence of a high concentration of nondiffusible 1, 2-glycol groups in these components of the Limulus egg. Such findings are further substantiated by the ability of an aldehyde blocking reagent (hydroxylamine) to eliminate completely the

PAS positive reaction of the vitelline envelope, cortical region and yolk.

Additional evidence supporting the presence of 1, 2-glycol groups in the Limulus egg comes from positive results following the CAS and PPS procedures, both of which indicate the presence of 1, 2-glycol groups. In general, the CAS reaction is con sidered to stain only those tissue components relatively rich in 1, 2-glycols or 1, 2-aminohydro3^ groups. Even though these are the same groups demonstrated by the PAS procedure, many

PAS positive components are not CAS positive (Thompson, 1966).

Such disparity may be explained by observations that chromic acid oxidation not only produces aldehydes, but destroys the aldehydes which have been produced (Lillie, 1951). Con sequently, those tissue components which are PAS positive but

CAS negative contain relatively few reactive 1, 2 glycol groups. Observations that the vitelline envelope of the

Limulus egg is PAS positive but CAS negative indicates the vitelline envelope contains relatively few reactive 1, 2- glycol groupings per molecule as conç>ared with the cortical region and yolk of the egg, which are PAS and CAS positive. 144

Generally tissue components stained by the CAS procedure

are also stained with the PPS procedure. However, according

to Thompson (1966), some substances may be CAS positive and

PPS negative or CAS negative and PPS positive. Such is the

case with the Limulus egg. The vitelline envelope is CAS

negative following the CAS procedure and PPS positive following

the PPS procedure. Since potassium permanganate has similar oxidation characteristics to chromic acid in producing and

destroying 1, 2-glycols, the positive PPS reaction with Limulus

egg components suggests potassium permanganate reacts more like

periodic acid by not destroying the 1, 2-glycols than like

chromic acid, which destroys these groups.

Egg sections digested in malt diastase and subsequently stained with the PAS procedure show no change in intensity of the PAS positive reaction, thus indicating the positive results are not due to a reaction with the abundant 1, 2-glycols of glycogen. In tissue sections treated with ^-glucuronidase, the PAS reaction of most tissue components which are normally

PAS positive remain unchanged (Thompson, 1966). This is not the case with Limulus egg sections. Treatment with 8- glucuronidase slightly decreases the staining intensity of the

PAS positive components of the egg (vitelline envelope, corti cal region and yolk), thus indicating the observed PAS positive reaction in the egg components is in part dependent on the presence of conjugated glucuronides; compounds which are 145 hydrolyzed by 3-glucuronidase. An example of a tissue component which exhibits similar staining characteristics following ^-glucuronidase digestion is cartilage matrix

(Thompson, 1966).

The presence of neutral mucopolysaccharides in the corti cal region and yolk is strongly indicated by the brown stain ing reaction following the PAD method. In addition, these components of the egg are PAS-reactive and diastase-resistant, which according to Spicer (1963) are also criteria for deter mining neutral mucopolysaccharides. While there are no positive indications of any periodate unreactive mucosubstances in the egg sections, the PAD method does give a posi tive staining reaction for the localization of periodate- reactive polymers in the vitelline envelope.

The localization of acid mucopolysaccharides is restricted to the cortical region of the Limulus egg. The employment of

Alcian blue at pH 1.0 gives a strong positive reaction in the cortical region. According to Lev and Spicer (1964), staining with Alcian blue at pH 1.0 is selective for sulphated muco polysaccharides. In a comparison of mucopolysaccharide staining by Alcian blue and toluidine blue 0, Vialli (1951) found the two methods gave similar results. The fact that metachromasia is considered characteristic of sulfated acid mucopolysaccharides (Thompson, 1966) and the cortical region of the Limulus egg exhibits metachromasia following toluidine 146 blue 0 staining could be viewed as additional evidence sup porting the presence of sulfated mucopolysaccharides in this region of the egg. This is not likely the case since the metachromasia exhibited following toluidine blue 0 staining is

g-metachromasia, which is indicative of the presence of weak acidic groups (Pearse, 1968). Pearse (1968) contends that from a theoretical point of view, metachromasia signifies only the presence of free electronegative surface charges of a minimum density, not specific chemical groups. Since the intensity of the toluidine blue O reaction in the cortical region is not a strong reaction, the presence of weak acidic groups or electronegative surface charges in this region are not considered to be concentrated.

A stronger case supporting the results of Alcian blue staining for th.e presence of sulphated mucopolysaccharides in the cortical region of Limulus egg sections may be taken from the observation that mild methylation has no effect on the

alcianophilia of this region. Acid mucopolysaccharides con

taining sulfuric acid groups are unaltered by mild methylation while those lacking sulphate groups lose their basophilia and

Alcian blue affinity following such treatment [Thompson,

1966). In addition, Spicer (1963) found some rodent muco

polysaccharides failed to display an affinity for azure A, but

were positive for Alcian blue. Many of the Alcian blue

positive-azure A negative acid mucopolysaccharides were found 147 to contain sulfuric acid groups. A similar staining situation is exhibited by the cortical region of the Limulus egg.

According to Spicer (1963), the Alcian blue staining of such sulfated azure A negative mucopolysaccharides results from a combination of the Alcian blue with the carboxyls of muco polysaccharides containing occluded sulfate groups. In con clusion, evidence from staining with Alcian blue strongly supports the presence of sulfated acid mucopolysaccharides in the cortical region of the egg.

The use of Alcian blue at high pH (2.5-3.0) stains weakly acidic sulphated mucosubstances, hyaluronic acid and sialomucins (Pearse, 1968). Like the reaction observed follow ing Alcian blue at pH 1.0, the positive Alcian blue reaction at pH 2.5 is localized only in the cortical region of the

Limulus egg. Exposure to neuraminidase digestion and sub sequent failure to eliminate alcianophilia suggests sialic acid is not responsible for the acidic nature of the substances within this region. In addition, hyaluronidase treatment failed to demonstrate an effect on the alcianophilia of the cortical region of the Limulus egg, indicating uronic acids

are not responsible for the observed staining characteristics.

The inability to localize either sialic acids or uronic acid, two important constituents of glycosaminoglycans, indicates the protein-polysaccharide substances in the cortical region of the Limulus egg are glycoproteins in nature. According to

Thompson (1966), Congo red, in addition to staining ait^loid. 148 also stains glycoprotein. Even though components of Limulus egg sections exhibit different degrees of Congo red staining, it is of interest that the cortical region exhibits a strong affinity for Congo red. Treatment of egg sections with malt diastase or trypsin has no effect on observed staining characteristics indicating the sensitive sites of the carbo hydrate and protein molecules are masked from the hydrolytic effects of these enzymes.

The observations that the cortical region of Limulus egg sections contain sulfated mucosubstances and glycoproteins is of interest since cytochemical studies have demonstrated these substances in cortical granules of other species (Monné and Harde, 1951; Szollosl, 1967; Schuel, Wilson, Bressler,

Kelley and Wilson, 1972). Cortical granule breakdown promotes a number of biochemical changes at fertilization which involve initiation of egg activation and the prevention of polyspermy.

The fact the cortical region of the Limulus egg is chemically similar to cortical granules in other species suggests this region in the Limulus egg may have a similar role in fertiliza tion.

In addition to using basic dyes in histochemistry to demonstrate the localization of mucopolysaccharides in tissues, dye binding at various pH levels has been used to indicate the dissociation characteristics of acid groups in tissues (French and Benditt, 1953). The methylene blue 149 extinction method as used by Pearse (1968) makes possible a comparative estimation of the degree of basophilia observed in tissue components. The pH at which an anionic tissue component fails to bind the dye is the methylene blue extinction of that tissue component and an indication of the dissociation characteristics of the anionic groups in the basophilic com ponents of the tissue (Dempsey, Bunting, Singer and Wislocki,

1947). In the Limulus egg the methylene blue extinction of the cortical region is pH 2.6, while that of the vitelline envelope and yolk is pH 3.6 and the basement lamina is pH 5.3.

Of interest are observations by Dempsey, Bunting, Singer and

Wislocki (1947) that sulfated mucopolysaccharides have lower

"pH signatures" or extinction than nonsulfated mucopolysac charides. The fact that the cortical region h.as the lowest extinction pH of the Limulus egg provides more evidence for the presence of sulfated-mucosubstances in this region.

Ascorbic acid is considered a polysaccharide since it is a derivative of a B-ketolactone (Thompson, 1966). Histo- chemical demonstration of this polysaccharide is restricted to silver reduction techniques. Even though ascorbic acid appears in many tissues (liver, ovary, kidney), it is not demonstrated in Lim:ulus egg sections with the silver nitrate method. The fact that this procedure gave negative results does not mean ascorbic acid is not present since many tissues such as liver contain ascorbic acid but fail to demonstrate its presence by 150 reducing silver nitrate.

In conclusion, the vitelline envelope, cortical region and yolk of the Limulus egg are rich in 1, 2-glycol groups with the vitelline envelope containing fewer reactive 1, 2-glycols per molecule than the other components of the egg. The PAS positive reaction of these components is in part due to con jugated glcuronides. The cortical region and yolk also contain neutral mucopolysaccharides. Only the cortical region of the egg is found to contain sulfated mucosubstances which are in part glycoprotein in nature.

Proteins and nucleic acids

The fact that considerable protein is found in the base ment lamina, vitelline envelope, cortical region and yolk of the Limulus egg is demonstrated by the staining affinity of these components following mercuric-bromphenol blue, naphthol- yellow S and the ninhydrin-Schiff methods. Treatment of egg sections with Van Slyke's reagent, a deaminating reagent, progressively blocks the staining of Limulus egg sections subsequently stained with mercuric-bromphenol blue (Figures

39, 40). Since this reagent most rapidly blocks a-amine groups and staining with mercuric-bromphenol blue follows the

Beer-Lambert laws (Mazia, Brewer and Alfert, 1953), it can be concluded that the basement lamina of the Limulus egg contains the least number of a-amino groups in the egg since this region is the first part of the egg that fails to stain with 151 mercuric-bromphenol blue following deamination. Observations that extended deamination will not abolish all staining in the cortical region and the yolk indicates either an abundance of a-amine groups in this region of the egg or that mercuric- bromphenol blue is staining materials other than proteins.

Baker (1958) and Kanwar (1960) have expressed some doubts as to the specificity of bromphenol blue as a protein stain. The opinion is advanced that bromphenol blue is a strong acid dye

capable of making links with basic groups in tissue components

and with certain acid groups through mercury. Thus, sub stances stained with mercuric-bromphenol blue may not always contain proteins and substances not stained are not necessarily devoid of proteins. The staining reaction observed in the basement lamina and vitelline envelope following mercuric bromphenol blue is undoubtedly due to proteins since deamina tion completely inhibits staining of these regions. Some of

the stain in the cortical region and yolk is probably due to

substances other than proteins since deamination does not

completely inhibit staining in these regions of the egg.

Failure to observe a reduction in staining intensity when the

mercuric bromphenol blue method is preceded by trypsin treat

ment indicates the sensitive sites of the proteins stained by

this method are masked.

The ninhydrin-Schiff reaction is specific for a-amino

groups (Burnstone, 1955). In the Limulus egg, the basement 152 lamina gives the most intense reaction thus indicating an abundance of such groups in this region. These conclusions would appear to contradict evidence obtained by deamination and subsequent mercuric-bromphenol blue staining. This discrepancy can be explained by examining the probable mode of action of the ninhydrin-Schiff reaction. Ninhydrin is a reagent that oxidizes «-amino acids to carbon dioxide, ammonia cind aldehyde (Thompson, 1966). The aldehydes resulting from oxidative deamination of a-amino acids are localized at the sites of their formation and during the ninhydrin-Schiff reaction are stained with the Schiff reagent. Since an oxida tive step is involved in this reaction, the possibility of additional reactive sites being unmasked is greater than in a reaction such as the mercuric-bromphenol blue reaction where oxidation before staining is not involved. Consequently, mercuric-bromphenol blue would tend to stain only those sites that are available and not the additional sites that would be

revealed by unmasking.

Protein determination in tissue sections is also based on

determination of some chemical group or groups characteristic

of proteins. Such determination on Limulus egg sections

contributes additional evidence on the presence of proteins in

the various egg components. A highly specific and reliable

reaction for arginine (Thompson, 1966), the Sakaguchi reac

tion, produces a very weak reaction in the vitelline envelope. 153 cortical region and yolk. Another method that has a high specificity but low sensitivity is the performic acid-alcian blue method for cystine (Pearse, 1968). Limulus egg sections stain only in the cortical region and the yolk following use of this method. Data from both methods correspond with amino acid analysis of the egg envelopes which reveals arginine to be present and cystine absent from the egg envelopes.

The localization of DNA in the cortical region and the yolk of Limulus egg sections was revealed following the Feulgen reaction. The extreme resistance of this DNA to extractive methods is indicated following DNase I treatment and 1 N HCL extraction. Even though DNase I was found to prevent Feulgen staining in Limulus sperm nuclei, it had no effect on the I cortical region and yolk of Limulus egg sections. Since the

Feulgen method is specific for DNA, it is concluded the DNase

I sensitive sites in Limulus sperm nuclei are more accessible to hydrolytic action than those of the Lintulus egg cortical region and yolk.

DNA is known to be associated with the cytoplasm of sea urchin eggs (Baltus, Quertier, Ficq and Brachet, 1965) and with mitochondria and yolk platelets in amphibian eggs

(Brachet, 1967). The physical and chemical properties of cytoplasmic DNA is similar to nuclear DNA in sea urchin and

Xenopus eggs (Brachet and Malpoix, 1971). The present study demonstrates a large amount of extranuclear DNA in the Limulus egg is also associated with yolk (yolk bodies). The role of 154 yolk DNA in the Limulus egg at present is speculative. One possible role could involve participation in yolk body break down during embryogenesis as is suggested in studies on amphibian eggs (Brachet and Malpoix, 1971). Another function of yolk DNA could be to supply nucleotides for nuclear DNA replication during development of the Limulus embryo.

Lipids

The various classes of lipids demonstrated in Limulus egg sections are primarily localized in the yolk. The demonstration of neutral fats following the Oil red 0 method reveals that not all the yolk bodies of the yolk are colored by this procedure. Neutral fats are restricted to certain yolk bodies. This is also true of phospholipids demonstrated with Sudan black B staining in conjunction with controlled

chromation and hematoxylin staining. According to Pearse

(1968), "nonchromated lipids are stained by Sudan black B only and nonlipids by hematoxylin only." Structures stained by both methods are considered phospholipids. Since hematoxylin

stains all yolk bodies in Limulus agg sections and Sudan black

B following chromation stains only certain yolk bodies, it is

concluded that not all the yolk bodies contain phospholipid.

Limulus egg sections give a weak positive reaction in the

vitelline envelope following use of the copper phthalocyanin

method. Even though this method is considered to demonstrate

phospholipids, especially myelin lipids, it has been 155 impossible to ascribe any specificity within the broad groups of lipids stained by this method (Pearse, 1968). Based on his view of the staining mechanism of copper phthalocyanin,

Pearse (1968) believes lipoproteins rather than lipids are responsible for the staining reaction in fixed tissues. If this interpretation is correct, the staining of the vitelline envelope in Limulus egg sections by copper phthalocyanin may be attributed to lipoproteins since formalin fixed sections were used in this study. On the other hand, observations, that controlled chromation followed by the copper phthalocyanin

method produces staining of the yolk, suggests this method

also stains phospholipids in Limulus egg sections. Phospho lipids combine readily with chromium salts and in the process are made insoluble as well as mordanted. This is not the case with formalin containing fixatives. Although the specificity of the controlled chromation method has not been established, it is clearly capable of demonstrating a number of phospho lipids in tissue sections (Pearse, 1968). Thus, the staining

of the yolk in Limulus egg sections following controlled

chromation by the copper phthalocyanin method is not likely

due to the presence of phospholipids. Since the vitelline

envelope also stains with copper phthalocyanin following con

trolled chromation, but not with Sudan black B, which is

specific for phospholipids when used in conjunction with

controlled chromation, the observed staining is prpbably not 156

the result of the presence of phospholipid.

The localization of unsaturated lipids in the vitelline

envelope, cortical region and yolk of Limulus egg sections was demonstrated with the U-V Schiff reaction. Even though

proof of specificity for this reaction is difficult to obtain

(Pearse, 1968), considerable evidence exists supporting the

demonstration of double bonds by this reaction (Belt and

Hayes, 1956). In Limulus egg sections the most intense reac

tion for unsaturated lipids is in the cortical region.

Staining of Limulus egg sections for fatty acids with

both Fischler's method and Holczinger method leaves some doubt

as to cytochemical demonstration of fatty acids in the basement

lamina. On the other hand, the vitelline envelope, cortical

region and yolk exhibits a weak to moderate staining reaction

following the use of these two methods. Of the two methods,

Holczinger's had been considered to be absolutely specific for

free fatty acids (Adams, 1965), but recent studies by Elleder

and Lojoda, (1972) cast some doubt as to the specificity of

Holczinger's method. Since these authors found the method

stained phospholipids, lipopigment, calcium deposits, acidic

groups in proteins and acid mucopolysaccharides, they sug

gested only materials giving a positive reaction in sections

pretreated with IN HCl and a negative reaction when pretreated

with IN HCl followed by acetone extraction could be considered

as fatty acids. The observations that Limulus egg sections 157 treated in the above manner display negative results in both instances may be related to the extractive effects of HCl on

Limulus egg sections. Treatment with 0.2 N HCl was found to affect the basement lamina of the egg (Figure 56) and higher concentrations of the acid may also extract other egg com ponents. In any event, the localization of fatty acids in the basement lamina, vitelline envelope and yolk of the Limulus egg is questionable and must await physical separation and subsequent biochemical analysis of these regions.

A modified Molisch reaction (Diezel, 1954) for sugar con taining lipids (gangliosides) and Okamoto's method for cholesterol produced no staining reactions in Limulus egg sections. The swelling of the basement lamina with Okamoto's cholesterol method is initiated by the sulphuric acid in the staining reagent. Similar results are observed in the basement lamina \dien HCL is used to determine types of linkages between protein chains in the egg envelopes. Swelling results when a high proportion of basic amino acid residues are present

(Brown, 1950).

Various extraction techniques were used in conjunction with many of the lipid procedures above with one general out

come; they failed to extract the lipids. Pearse (1968) sug

gests there is no reason to discontinue the use of extraction

techniques but there is a need to use great caution in

accepting their effects as being similar to those obtained 158 in vitro. Interpretations based on the effects of extraction techniques should be held in reserve until parallel chemical studies are conducted on each technique (Pearse, 1968). The failure of lipid extraction techniques to affect the staining characteristics of Limulus egg sections is not surprising since extraction and digestion techniques for carbohydrates and proteins also have no effect on staining characteristics.

The macromolecules of the Limulus egg are apparently well protected from the degradative effect of a number of enzymes and extractive techniques. This might be the key to the survival of this primitive organism. Even early investigators

(Kingsley, 1892) noted the hardiness of the Limulus egg.

In conclusion, the yolk of the Limulus egg contains neutral lipids, phospholipids, unsaturated lipids and free fatty acids, while neutral lipids, lipoproteins and un saturated lipids are localized in the vitelline envelope. The cortical region contains neutral lipids, phospholipids, un saturated and possibly free fatty acids.

Hydrolytic enzymes

Both Gomori's method and the Azo dye method give a weak to moderate positive reaction for alkaline phosphatase in the cortical region and yolk of Limulus egg sections. No acid phosphatase nor esterases are localized in egg sections. Two enzymes, glucose-6-phosphatase and succinic dehydrogenase are localized only in the cortical region of the egg sections. 159

The use of negative controls with both methods for alkaline phosphatase give results similar to those observed in experimentals. Interpretation of such results indicate that endogenous enzyme substrate is sufficient in the Limulus egg to elicit a reaction since experimentals give more intense reactions than negative controls.

The presence of glucose-6-phosphatase in the cortical region of Limulus egg sections is an indication that glyco genolytic pathways play a role in the metabolism of the Limulus egg. , According to Pearse (1968), glucose-6-phosphatase is often used as a marker for microsomes since it is predominantly found in microsomal fractions of tissue homogenates. This fact suggests the presence of microsomes in the cortical region of uninseminated Limulus eggs.

Succinic dehydrogenase, an enzyme closely related to the cytochrome system has been demonstrated in sea urchin eggs

(Maggio and Ghiretti-Magaldi, 1958) and recently in rabbit and hamster eggs (Ishida and Chang, 1965). The localization of succinic dehydrogenase in the cortical region of LimiuTus egg sections indicates the tricarboxylic acid cycle is functional in the uninseminated egg. Evidence from various studies show formation of the formazan granules produced by Nitro-blue tetrazolium during the demonstration of succinic dehydro genase is localized in the mitochondria (Sedar, Rosa and Tsou, 160

1962). In addition, intact mitochondria are known to contain a number of the tricarboxylic acid cycle enzymes (Brachet and

Mirsky, 1961). The inference of these finding is that the location and concentration of formazan granules in tissues are a good indication of the presence of mitochondria (Ishida and

Chang, 1965). If this is indeed the case, mitochondria of the

Limulus egg are located only in the cortical region.

In conclusion, alkaline phosphatases were demonstrated in the cortical region and yolk of Limulus egg sections while tests for acid phosphatases were negative. Two enzymes associated with glycolytic metabolic pathways, glucose-6- phosphatase and succinic dehydrogenase, were localized only in the cortical region of egg sections.

Cytochemistry of Inseminated Egg Sections and Eggs

The Alcian blue positive reaction observed in uninseminated egg sections is not present in sections of inseminated and developing eggs beyond 72 h of development. The dis sipation of this cytochemically defined region in inseminated eggs is probably analogous to the dissipation of cortical granule material following rupture of the cortical granules in other species (Austin, 1965, 1968). Since there is no change in the location of the material in the cortical region of the

Limulus egg immediately following insemination, the macromolecules of this region are probably not involved in the 161 production of a barrier against polyspermy. This notion is further supported by the observation that this chemically defined region appears in both uninseminated and inseminated eggs. Instead, the macromolecules of this region most likely participate in the formation of the embryonic egg envelope of the Limulus embryo. This is suggested from observations that the dissipation of the sulfated mucosubstances of the cortical region at 72 h of development coincides with the first appearance of half cystine in amino acid hydrolysates of the original egg envelopes; the layers that give rise to the embryonic egg envelope.

Glucose-6-phosphatase activity, which is restricted to the cortical region of uninseminated egg sections, is localized in both the cortical region and yolk of sections from in seminated Limulus eggs. An increase in glucose-6-phosphatase following insemination has also been noted in oyster eggs

(Kobayashi, 1969). The first appearance of this enzyme in the yolk of the Limulus egg during the early stages of development indicates glycogenolytic pathways are functional in this region only during development.

Succinic dehydrogenase activity, unlike glucose-6-

phosphatase activity, remains in the cortical region of the

Limulus egg following insemination. These observations indi

cate the tricarboxylic acid cycle during early development is

functional primarily in the cortical region of the egg, a 162 situation similar to that of the uninseminated egg.

In conclusion, sulfur containing macromolecules of the cortical region in uninseminated Limulus eggs are most likely involved in the formation of the embryonic egg envelope during development. The appearance of glucose-6-phosphate activity in the yolk during early development, while succinic dehydro genase activity remains in the cortical region, suggests compartmentalization of glycogenolytic and tricarboxylic acid metabolic pathways within specific region of the Limulus egg.

Physiochemical Treatment of Limulus Egg Sections

Basically, there are three types of bonds or linkages in volved in holding protein chains together; Van der Waals forces, electrovalent linkages and covalent linkages (Brown,

1950). Disruption of bonds between protein chains may be accomplished by employing various chemical reagents (Table 5) which break some or all the linkages of fibrous proteins causing subsequent swelling or dissolution as a result of i water molecules from the medium penetrating between the pro tein chains. The degree of response to any reagent depends on the relative proportions of the different types of bonds present in the protein.

Of the various reagents tested on LiMUlus egg sections, only those treatments specific for disrupting electrovalent bonds (HCl, NaOH, urea), covalent bonds other than those formed 163 by disulphide linkages (sodium hypochlorite), and pepsin had any noticeable effect on the egg envelopes. The use of acids and alkalis on fibrous protein have demonstrated that acids swell proteins containing high proportions of basic amino acid residues and alkalis swell proteins containing high propor tions of acidic amino acids (Brown, 1950). The fact that 0.2

N HCl readily swells the basement lamina indicates this por tion of the egg envelope contains a high proportion of basic amino acid residues. Since no change is observed in the vitelline envelope following HCl treatment, it is concluded basic amino acid residues do not constitute a significant part of this layer. On the other hand the vitelline envelope from its reaction following NaOH treatment is found to contain a high proportion of acidic amino acid residues. Similar con clusions may be reached concerning the basement lamina since it also expands and dissolves following NaOH treatment. Thus, the basement lamina of the Limulus egg most likely contains large proportions of acidic and basic amino acid residues, whereas the vitelline envelope contains only acidic residues.

Any reagent causing an increase in the dielectric constant of the medium in which a protein is present will decrease the attractive forces between the polar groups of the protein and subsequently cause swelling or solution of electrovalent- linked proteins. Urea in solution has a high dielectric constant and causes swelling but not solution of the basement 164 lamina of the liimulus egg. Since the ability of a reagent to cause swelling of a fibrous protein depends on the number and strength of the linkages present as well as the spatial dimensions of the protein, it can be concluded that the vitel line envelope has a greater number or stronger linked, closely arranged electrovalent linkages than the basement lamina. Such a conclusion is feasible since urea treatment has no effect on this layer but data from alkali treatment indicate the integrity of this layer is maintained by electrovalent link ages.

Interpretation of results demonstrating involvement of electrovalent linkages in maintaining the structural integrity of the Limulus egg envelope presents some difficulty when covalent linkages are also involved. To break covalent link ages, specifically the -S-S- bond, sodium sulphite solution is employed. The negative results obtained from this procedure demonstrated the absence of -S-S- linkages in the Limulus egg envelopes. Apparently in these egg envelopes, unlike those of some species (Gusseck and Hedrick, 1971), disulphide bonds do not play an integral part in maintaining structural integrity. On the other hand, covalent linkages other than

-S-S- linkages are important in holding the protein chains together in both the, basement lamina and vitelline envelope of th.e Limulus egg since sodium hypochlorite treatment dissolves these two layers. 165

Treatment of Limulus egg envelopes with a strong oxidizing agent (hydrogen peroxide), an emulsifier (hyamine), and Ca^^-free sea water failed to affect the structural integrity of the basement lamina and the vitelline envelope.

Strong oxidizing reagents and Ca^^-free sea water have been found to affect the structural integrity of egg envelopes of some species. Hydrogen peroxide removes the zona pellucida of rat and rabbit eggs (Austin, 1961) and Ca^^-free sea water has been found to dissolve the hyaline layer of sea urchin

eggs (Rothschild, 1956). Chemically, the zona pellucida is composed of weakly acid mucoproteins and the hyaline layer, which arises from the contents of the cortical granules, contains mucopolysaccharides. Failure of either the basement lamina or the vitelline envelope of the Limulus to be affected by a strong oxidizing reagent or Ca^^-free sea water is under standable since cytochemical tests reveal no acid mucoproteins or mucopolysaccharides are present in these layers.

Lipids play no major role in maintaining the structural

integrity of Limulus egg envelopes since neither hyamine nor

lipase has any effect on these layers. In addition, cyto

chemical tests fail to demonstrate the presence of neutral

lipids in these layers. Enzymatic treatment of Limulus egg envelopes with trypsin,

an enzyme that specifically attacks bonds between the carboxyl

group of arginine or lysine and the amino group of other amino 166 acids (or alcohol hydro:^1 groups), fails to have any effect on Limulus egg envelopes. The failure of trypsin to attack the envelopes may be due to the close packing of the protein chains in the egg envelopes. McFarlane (1962) found trypsin had no effect on the egg shells of freshly laid eggs of Acheta domesticus but did attack swollen eggs (eggs that had completed water absorption), which indicates the protein chains after being stretched exposed the peptide bonds to attack. Since amino gcid analysis data reveal small amounts of lysine and arginine to be present in Limulus egg envelopes, another pos sible explanation of the inability of trypsin to attack the egg envelopes is tjie location of these residues at carboxyl ends of the protein chain.

Pepsin, which preferentially attacks peptide bonds with aromatic residues on one or both sides of a bond, has a minor effect on the basement lamina of the egg and no effect on the vitelline envelope. These data indicate the amino acids phenylalanine and tyrosine are found only in the basement lamina of the Limulus egg since amino acid analysis confirms the presence of these two residues in the egg envelopes.

In conclusion, structural integrity of Limulus egg envelopes is maintained by electrovalent linkages and covalent linkages not involving the -S-S- bond. The basement lamina contains a high proportion of acidic and basic amino acid residues whereas the vitelline envelope contains a high 167 proportion of acidic amino acid residues. In addition, the protein chains responsible for maintaining the structural integrity of Limulus egg envelopes are closely packed and con sequently resistant to attack by proteolytic enzymes.

Amino Acid Composition of Limulus Egg Envelopes

Sixteen amino acids have been identified from the acid hydrolysate of uninseminated Limulus egg envelopes and 17 residues have been identified from similar hydrolysates of inseminated egg envelopes (Tables 6, 7). Comparison of the amino acid composition of uninseminated Limulus egg envelopes with egg envelopes (e.g. jellies, shells, membranes, chorions, capsules) from other animal species reveals the Limulus envelopes are not identical in amino acid composition with any of the nine species listed (Table 8). Differences center around the presence or absence of one or two amino acids. The amino acid composition of uninseminated egg envelopes strongly suggests the protein or group of proteins in the egg envelopes are primarily acidic. The most abundant amino acids are tyrosine, aspartic acid and glutamic acid. Of particular interest is the relatively large amount of tyrosine found in

Limulus egg envelopes. Relatively large amounts of tyrosine are also found in the egg shells of some insects, namely the fruit fly (Wilson, 1960a) and the silkworm (Tomita, 1921). It is well established that polyphenols and their guinone Table 8, Amino acids in acid hydrolysates of egg envelopes Amino acid Limulus Arbacia^ Bufo Acheta^ (jelly) (egg membrane) (egg shell) (egg shell) glycine X X X X X alanine X X X X X serine X X X X X threonine X X X X X valine X X X X X leucine X X X X X isoleucine X X X X X half cysteine - - X X X cystine - X - - X methionine X X X X - aspartic acid X X X X X tryptophan - - - - - glutamic acid X X X X X proline X X X X X hydroxyproline - - — - X histidine X X X X X arginine X X X X X lysine X X X X X phenylalanine X X X X - tyrosine X X X X X cystic acid — X — — — X amino acid present amino acid absent

^Ishihara (1968).

^Uchiyama, Ishihara and Ishida (1971).

°Fumeaux (1970).

^Wilson (1960a). Table 8 (Continued)

Amino acid Rhipicephalus^ Tegula Hymenolepis^ Buccinum (egg shell) (egg membrane) (egg membrane) (egg capsule) glycine X X X X alanine X X X X serine X X X X threonine X X X X valine X X X X leucine X X X X isoleucine X X X X half cysteine - - - - cystine - • — - - methionine - X - X aspartic acid X X X X tryptophan — X — — glutamic acid X X X X proline - X X X hydroxyproline X — - histidine - X X - arginine X X X X lysine X X X X phenylalanine - X X X tyrosine X X X — cysteic acid — X X —

®Jaskoski and Butler (1971)

^Haino and Kigawa (1966).

^Lethbridge (1971).

^Hunt (1966). Table 8 (Continued) i iç 1 Amino acid Hen Fundulus^ Salmo Testudo (vitelline membrane) (chorion) (chorion) (protein membrane) glycine X X X X alanine X X X X serine X X X X threonine X X X X valine X X X X leucine X X X X isoleucine X X X — half cysteine - X X - cystine X - - — methionine X X X X aspartic acid X X - X tryptophan - - X - glutamic acid X X X X proline X X X - hydroxyproline X - - - histidine X X X X arginine X X X X lysine X X X X phenylalanine X X X X tyrosine X X X X cysteic acid

^Bellairs, Harkness and Harkness (1963).

^Kaighn (1964).

^Young and Smith (1956).

^Zusman (1965). 171 derivatives play an important role in the darkening and hardening of the insect cuticle. The polyphenols are derived from tyrosine which is oxidized by tyrosinase (Richards, 1951).

In the insect cuticle this enzyme system is widely distributed so that hardening and darkening of the cuticle is controlled by the distribution of the substrate, tyrosine. From the amino acid data on Limulus egg envelopes it could be postulated that the observed toughness of these egg envelopes is due to a similar mechanism involving tyrosine, but that darkening of the envelopes is not as pronounced as in the insect cuticle.

Of equal interest is the absence of the amino acid cys teine in uninseminated Limulus egg envelopes. Recently, Gus- seck and Hedrick (1971) postulated on the importance of sulfhydryl groups in maintaining the integrity of egg envelopes

(jelly) in amphibians and in the process of fertilization

(membrane fusion). According to these authors, the structural integrity of jelly envelopes of Xenopus laevis as well as egg envelopes of other species displaying susceptibility to solubilization by disulfide bond breaking reagents are maintained by disulphide bonds. In addition, sperm-egg membrane fusion is also thought to be a direct result of the formation and interchange of sulfhydryl-disulfide bonds.

Neither hypothesis involving sulfhydryl-disulfide bond forma tion would appear to be applicable to gametes of Limulus.

Physiochemical tests reveal disulfide bond breaking reagents 172 have no solxibilizing effect on egg envelopes of Limulus and amino acid analysis reveals cysteine to be absent from Limulus egg envelopes.

Amino acid analysis of inseminated Limulus egg envelopes

(72 h development) demonstrates the presence of half-cysteine.

In addition, all other amino acids found in uninseminated egg envelopes show significant increases in amounts in inseminated egg envelopes. The presence of half-cysteine in Limulus egg envelopes makes these envelopes identical in amino acid compo sition with the egg membranes obtained from blastula- and gastrula-stage embryos of Bufo vulgaris (Uchiyama, Ishihara and Ishida, 1971), the egg shells of Acheta domesticus

(Furneaux, 1970) and with the chorions of Fundulus heteroclitus

(Kaighn/ 1964) and Salmo solar (Young and Smith, 1956). Egg envelopes of these species, like those of Limulus, also con tain relatively large amounts of acidic amino acid residues.

The appearance of half-cysteine and the incorporation or increase in amount of amino acids in the egg envelopes at 72 h following insemination probably has significance in events that will take place during subsequent embryonic developement.

The production or synthesis of the embryonic envelope which surrounds the embryo at the imbibition-swelling stage is one possible explanation for the tremendous increase in amount of amino acids present in the egg envelopes. The amino acids for the embryonic envelope, which develops inside the original 173 egg envelopes, may begin accumulation as early as 72 h follow ing insemination.

Limulus polyphemus L« Embryology

Since considerable literature exists on the embryology of

Limulus (Kingsley, 1885, 1892; Packard, 1870, 1871, 1873, 1885;

Iwanoff, 1932, Sekiguchi, 1970), it will not be necessary to enter into a detailed discussion on the various aspects of the embryology of this organism. Instead, some of the features of embryological development that have not been described in the literature for Limulus polyphemus will be discussed in the following section.

Various accounts on developmental stages of Limulus polyphemus immediately following fertilization have failed to describe the pits seen in the yolk. These pits, which vary in size and number, are prevalent over the entire surface of the yolk. The fact that the pits are seen in inseminated and freshly collected uninseminated eggs suggest they are not the result of the egg response to reacting spermatozoa. The dis appearance of the pits shortly after insemination is probably analogous to the cortical reaction observed in other species

(Austin, 1965, 1968) which is considered as one indication of egg activation. The observation that the pits also disappear in uninseminated eggs is of little consequence since unin seminated eggs are apparently activated and indistinguishable 174 in terms of developmental stages from inseminated eggs up to the blastopore stage. The observations that the pits do not appear in fresh cryostat sectioned material leads to the speculation that they are responsible for the acid mucopoly saccharide positive material observed only in the cortical region of the Limulus egg since chemical analysis of materials from cortical granules of other species have revealed the presence of sulfated mucopolysaccharides (Aketa, 1962; Bal,

1970; Schuel, Wilson, Bressler, Kelley and Wilson, 1972).

According to Iwanoff (1932), the embryos of Limulus moluccanus undergo three moults before hatching as trilobite larvae. Sekiguchi (1970), who investigated embryonic moulting in the Japanese species of horseshoe cr^ (Tachypleus tridentatus), contends the "primare Cuticula" observed by

Iwanoff is not a true embryonic molt since it is produced by the blastoderm layer before the embryonic disc is formed.

Thus Limulus moluccanus embryos undergo two embryonic moults before hatching, not three. Observation on the embryonic development of Tachypleus tridentatus reveals four moults take place before the embryo hatches as a trilobite larva

(Sekiguchi, 1970). The first molt occurs before the embryo breaks out of the original egg envelopes, the second at about the time the embryo breaks out of the original egg envelopes

(imbibition-swelling stage) and the third and fourth while the embryo is still inside the embryonic egg envelopes. From 175 topographical observations, Limulus polyphemus embryos are observed to undergo two embryonic moults before hatching. The first coincides with the rupture of the original egg envelopes

(imbibition-swelling stage) and the second occurs a few days

(1-5) before hatching.

The participation of a hatching enzyme similar to that observed in the teleost (Yamagami, 1970; Kaighn, 1964) has been considered in the emergence of the Limulus polyphemus embryo from the original egg envelopes. Since these envelopes constitute a thick, tough, protective covering for the embryo, it is reasonable to suspect the involvement of an enzyme in digestion these envelopes. Microscopic examination of ruptured Limulus egg envelopes fails to reveal any indications of enzymatic digestion. The emergence of the embryo from the ' original egg envelopes is apparently a mechanical process involving fracture of the egg envelope resulting from an increase of water inside the embryonic membrane. In addition, enzymes which attack protein chains and carbohydrates have no effect on these egg envelopes which is further indication that a hatching enzyme (s) is not involved in the emergence of the

Limulus embryo from its original egg envelopes. Hatching of the trilobite larvae from the embryonic egg envelope is also a mechanical process. It simply involves each larva rupturing its embryonic egg envelope to free itself. 176

Perhaps the most important aspect concerning the develop mental biology of Limulus polyphemus is the ease with which this organism may be reared and studied under laboratory condi tions. The ability to collect large quantities of viable gametes on a year around basis and to induce in-vitro fertiliza tion facilitates studies on the biochemical and immunological events of fertilization. From a practical standpoint, eggs may be collected, inseminated and used in embryology courses as a means of studying the developmental biology of a primi- • tive arthropod. In view of the recent development and use of

Limulus lysate, an extract of Limulus blood used to detect endotoxins, understanding the developmental biology of Limulus takes on added importance. Limulus, an organism that in the past has been of little economical importance to mankind, is suddenly on the verge of becoming an important tool in bio medical research (Schofield and Watson, 1974). It is quite possible the Limulus lysate test may be adopted as a standard test for detecting endotoxins. In the event this happens, and in all likelyhood it will, understanding the developmental biology of this organism will take on added importance. 177

Ultrastructure of Uninseminated and Inseminated Eggs

The initial events of sperm-egg interaction in Limulus polyphemus have been described and discussed by Shoger and

Brown (1970) and Brown and Humphreys (1971). These authors also described the Limulus egg envelopes" as seen with both the transmission (Shoger and Brown, 1970) and scanning (Brown and

Hiaiiç)hreys/ 1971) electron microscope. The findings in this study coincides with those of previous studies with the exception of one event involving the sperm-egg interaction.

Spermatozoa of speciés which involve the participation of an acrosome filament during sperm-egg interactions may be divided into two groups: those in which the unreacted sperm contains a preformed filament (Mytilus, Dan, 1967) and those in which the filament is: formed during the acrosome reaction

(starfish, Dan, Ohori and Kushida, 1964). The spermatozoa of

Limulus may be included in the first group. The stages of the acrosome reaction in Limulus during sperm-egg interactions are similar to acrosome reactions observed in other nonmammalian species where an acrosome filament is involved (for review see

Colwin and Colwin, 1967; Franklin, 1970). The initial reactions with the egg surface include the dehiscence of the acrosome vesicle, fusion of the sperm plasma membrane with the acrosome membrane and extrusion of the acrosome filament. The assumption has been that all Limulus spermatozoa (> 100,000 178

per egg) attached to the egg envelope had experienced an acro

some reaction in which the acrosome filament penetrated both

egg envelopes. The observation that the acrosome vesicle of

Limulus spermatozoa may undergo dehiscence and deposit its

contents on the surface of the egg without the extrusion of

the acrosome filament is an interesting revelation. In

Limulus, an organism in which large numbers of spermatozoa attach to each egg, it is quite possible that a significant

number of the attaching spermatozoa do not undergo a complete

acrosome reaction, but simply attach to the egg and deposit

the contents of the acrosome vesicle upon its surface. Granted, it will be virtually impossible to ascertain a clear indication of the number of spermatozoa participating in this "abbreviated" acrosome reaction since a sample for electron microscopic examination represents only a minute area in relation to the entire egg surface. But the fact this phenomenon takes place calls for additional examinations on the role of the acrosome filament and the acrosome vesicle in the process of fertiliza tion in Limulus.

In conclusion, the acrosome reaction in Limulus polyphemus L. may not always involve the entire acrosome complex. In some cases the acrosome vesicle undergoes dehiscence without extrusion of the acrosome filament. 179a

CONCLUSIONS

I Cytochemical and ultrastructural studies with Lintulus sperm as reported in this study present evidence that lysosomal-type enzymes are constituents of the Limulus sperm acrosome and that these enzymes are compartmentalized within the sperm acrosome.

Cytochemical studies on uninseminated Limulus egg sections demonstrate the presence of carbohydrates, proteins, and lipids in the egg envelopes. From these studies the structural integrity of the egg envelopes was determined to be maintained by electrovalent linkages and by non -S-S- covalent linkages between protein chains. In addition, a cytochemically defined zone, the cortical region, was demonstrated to be the only region of the egg containing sulfated mucosubstances. This region also contains enzymes associated with glycogenolytic pathways and the tricarboxylic acid cycle.

The positive reaction demonstrating sulfated muco substances in the cortical region of sections taken from in seminated eggs disappears at 72 h of development. Cytochemical studies on sections from inseminated eggs for glucose-6- phosphatase, an enzyme associated with glycogenolytic pathways,, demonstrate increased enzyme activity at 72 h of development while succinic dehydrogenase, an enzyme of the tricarboxylic cycle, shows no increase in activity during the same period of time. 179b

Developmental studies on Limulus polyphemus reveal this species is an excellent organism for laboratory studies in general embryology. Embryos of Limulus polyphemus L. were observed to undergo two moults before hatching when cultured at 25®C. 180

SUMMARY

Ultrastructural, cytochemical and biochemical studies were

conducted on electrically spawned male and female gametes

of Limulus Polyphemus L. The morphology of mature sperm

atozoa and eggs (cryostat sections) were examined by light

microscopy and transmission electron microscopy. Cyto-

chemical studies were conducted on sperm smear prepara

tions and cytochemical and biochemical studies were con

ducted on cryostat sections of mature eggs. Developing embryos were examined by light microscopy following mixing of electrically spawned gametes.

Cytochemical tests on Limulus sperm demonstrate the presence of acid phosphatase, glycolipid and a protease in the sperm acrosomes. The protease, which has an acid pH optimum, is not a trypsin-like enzyme.

Ultrastructural studies on the sperm reveal the contents of the acrosome vesicle are not homogeneous in appearance.

The vesicle consists of two regions of different electron densities.

A method of separating acrosomes from Limulus spermatozoa is introduced. This method, which involves the use of sodium hypochlorite and mild sonication, allows the cyto chemical identification of enzymes in the separated acro somes. 181a

5. Cytochemical tests for carbohydrates on egg sections

demonstrate the presence of periodate-reactive polymers,

which includes 1,2 glycols and conjugated glucuronides.

In addition, the cortical region of the egg is the only

region of the egg that is demonstrated to contain sulfated

mucosubstances.

6. Enzyme digestion techniques followed by cytochemical tests

for carbohydrates and proteins demonstrate glycoproteins

in the egg cortical region.

7. Cytochemical tests for proteins and DNA reveal protein in

all egg components and DNA in the yolk and cortical region.

Both the proteins and DNA are highly resistant to the

effects of enzymatic digestion.

8. Neutral lipids, unsaturated lipids, phospholipids, and

fatty acids are demonstrated with cytochemical methods in

the yolk of Limulus eggs while lipoproteins, unsaturated

lipids, and fatty acids are demonstrated in the egg

envelopes.

9. The enzymes glucose-6-phosphatase and succinic dehydro

genase are demonstrated by cytochemical tests to occur

only in the cortical region of uninseminated egg sections.

10. Physiochemical treatments of egg sections reveal the

structural integrity of Limulus egg envelopes is main

tained by electrovalent and non -S-S- covalent bonds

between protein chains. 181b

-1. Sixteen amino acid residues are identified in acid hydro-

lysates of uninseminated egg envelopes and seventeen amino

acids are identified in egg envelopes from developing eggs.

12. Limulus embryos are observed to undergo two moults before

hatching at approximately 47 days when cultured at 25°C.

13. The Limulus sperm acrosome vesicle is observed to rupture,

following contact with the basement lamina of the egg,

without extruding the acrosome rod. 182

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ACKNOWLEDGMENTS

I wish to thank Dr. George Gordon Brown, who suggested this study, for his help, encouragement, and assistance in I preparing this manuscript. I also wish to thank Dr. James

R. Redmond for co-chairing my committee. Many thanks are also due the members of my committee: Dr. Donald.Beitz, Dr.

Charles C. Bowen, Dr. John A. Mutchmor, and Dr. Joseph M.

Viles. I am grateful for the advice and assistance offered by G. Kutish, R. C. Mowbray, and S. Suleiman. Finally I wish to thank my patient and understanding wifp. Myrtle and daughter, Cheri, who encouraged and supported my study.

This study was supported in part by a Southern Fellow ship Fund Fellowship. Electron microscope facilities were furnished through a gift from the Houston Endowment Co., Inc.,

Houston, Texas. 220

APPENDIX A. SOURCES OF REAGENTS 221

Acridine orange (C.I.46005). Fisher Scientific Company, Fair Lawn, N.J.

Alcian Blue 8GX(C.I.74240). Allied Chemical, Morristown, N.J.

-Naphthyl acetate. Sigma Chemical Company, St. Louis, Mo.

-Naphthyl phosphate (Sodium salt). Sigma Chemical Company, St. Louis, Mo.

Azure A. Fisher Scientific Company, Fair Lawn, N.J.

Basic fuchsin (NB 300). Matheson Coleman and Bell, Norwood, Ohio.

Glucuronidase. Type II, Bacterial. Sigma Chemical Company, St. Louis, Mo.

Glycerophosphate (disodium salt). Sigma Chemical Company, St. Louis, Mo.

Bromaphenol Blue (Sodium salt). J. T. Baker Chemical Company, Phillipsberg, N.J.

Chlorantine Fast Red 5B (C.I.28160). Matheson Coleman and Bell, Norwood, Ohio.

Congo Red (C.I.370). National Aniline Division, New York, N.Y.

DeoxyribonuclecLse I. Ix crystallized, from bovine pancreas. Sigma Chemical Company, St. Louis, Mo.

Fast Garnet G.B.C. Salt. Practical Grade, Sigma Chemical Company, St. Louis, Mo.

Fast Red TR (diazonium salt). Sigma Chemical Company, St. Louis, Mo.

Glucose-6-phosphate (dipotassium salt). Sigma Chemical Company, St. Louis, Mo.

Hematoxylin (C.I.75290). Allied Chemical, Morristown, N.J.

Hyaluronidase. Type I, from bovine testes. Sigma Chemical Company, St. Louis, Mo.

Hyamine 1622 (Benzethonium Chloride). Sigma Chemical Company, St. Louis, Mo. "Luxol" fast Blue MBSN. J.T. Baker Chemical Company, Phillipsberg, N.J. 222

Malt Diastase. Nutritional Biochemicals Corporation, Cleveland, Ohio.

Metanil yellow (C.I.13065). Matheson Coleman and Bell, Norwood, Ohio.

Methyl Green (C.I.42590). Matheson Coleman and Bell, Norwood, Ohio. Methylene Blue (C.I.52015). Matheson Coleman and Bell, Norwood, Ohio. Mild Silver protein (Argyrol). Tilden-Yates Laboratories, Inc., Wayne, N.J. Naphthol yellow S (C.I.10316). Allied Chemical Morristown, N.J.

Neuraminidase. Type V from C^. Perfringens. Sigma Chemical Company, St. Louis, Mo.

Nitro Blue Tetrazolium. Grade III, Sigma Chemical Company, St. Louis, Mo.

N,N-Dimethyl-M^phenylenediamine dihydrochloride. Eastman Kodak Company, Rochester, N.Y.

N,N-Dimethyl-p-phenyleneamine monohydrochloride. J.T. Baker Chemical Company, Phillipsberg, N.J.

Nuclear fast Red (B 1367). Matheson Coleman and Bell, Norwood, Ohio.

Oil Red O (C.I.26125). Allied Chemical, Morristown, N.J.

Pararosanilin hydrochloride. Sigma Chemical Company, St. Louis-, Mo.

Pepsin. Grade B, 3X crystallized, from porcrine stomach mucosa. Calbiochem, Los Angeles, California.

Rubeanic Acid (Dithiooxamide). Fisher Scientific Company, Fair Lawn, N.J.

Sudan Black B. C.I.26150. Fisher Scientific Company, Fair Lawn, N.J.

Toluidine Blue O (C.I.52040). J.T. Baker Chemical Company, Phillipsberg, N.J.

Trypsin (4-X U.S.P. Pancreatin). Nutritional Biochemicals Corporation, Cleveland, Ohio. 223

APPENDIX B. FIGURES AND ABBREVIATIONS 224

Abbreviations

Ab Abdomen

Ac Acrosome

Af Acrosome filament

AR Acrosome rod

AV Acrosome vesicle

BB Basal bodies

BL Basement lamina

Ce Céphalothorax

CR Cortical region

Eg Eggs

El Electrodes

F1 Flagellum

GO Genital operculum

Gp Gonopores

J Gel

Nu Nucleus

PAI Posterior acrosome indentation

PNS Posterior nuclear cisternae

Po Pore

SAM Subacrosome material

SAP Subacrosome plate Sp Spermatozoa

VE Vitelline envelope

YB Yolk body

Yo Yolk Plate 1. Gamete collection

Figure 1. A ventral view of a female specimen of Limulus polyphemus L. showing the location of the genital operculum (GO)

Figure 2. Limulus gonopores (Gp) are located on the underside of the genital operculum (GO). Spawning is induced artificially by placing the electrodes (El) of an A. C. electrical stimulator (3.8 volts) at the base of the gonopores (Gp)

Figure 3. A female specimen of Limulus polyphemus L. has been induced to begin egg laying through the use of electrical stimulation. The eggs (Eg) may be collected and fertilized with spermatozoa obtained in a similar manner 226 Plate II. Cytochemistry of sperm acrosome

Figure 4. Mature spermatozoa collected through electrical stimulation and subsequently stained for acid phosphatase. The acrosome (Ac) gives a positive reaction for acid phosphotase. The nucleus (Nu) is stained with Methyl green. X2500

Figure 5, Acid phosphatase control spermatozoa, Note only the nucleus (Nu) stains. X2500

Figure 6, Mature spermatozoa treated for acrosome proteinase. The reaction product appears as grains in the acrosome (Ac) of the spermatozoa (Arrow). X2500

Figure 7. Acrosome proteinase control sperm. No reaction product appears in the acrosome. Only the nucleus (Nu) is visible. X2500

Figure 8. Mature spermatozoa on a glutaraldehyde fixed gelatin membrane impregnated with India ink. No reaction is observed with the membrane. Ac = acrosome. X2500

Figure 9, Mature spermatozoa stained for nonspecific esterase. No positive reaction is observed. The stain allows visualization of morphological features not ordinarily seen on light microscopy level. Note the posterior nuclear cisternae (PNS) and the space of the posterior acrosome indentation (PAI). X2500 228 Plate III. Ultrastructure of Limulus sperm

Figure 10. The Limulus sperm. Note the acrosome vesicle (AV), nucleus (Nu)/ basal bodies (BB) mitochondria (Mi), acrosome rod (AR), flagellum (Fl) and subacrosome plate (arrow). X23,750

Figure 11. The Limulus sperm. The acrosome vesicle (AV) of the sperm contains an indentation within which the acrosome rod (AR) and subacrosome material (SAM) is located. The material at the base of the acrosome differs in appearance from the material located at the anterior and lateral portions of the acrosome vesicle (AV). X65,000 230 Plate IV. Ultrastructure of Limulus sperm

Figure 12. The flagellum region of the Limulus sperm. Note the exit of the aerosome rod (AR) from the nuclear canal to the coil within the posterior nuclear cisternae (PNS). Mitochondria (Mi) are visible within the cytoplasm of the collar (Co) surround ing part of the flagellum (Fl). X35,000

Figure 13. A transverse section of a Limulus sperm through the posterior nuclear cisternae Tpns). The acrosome rod (AR) is visible in both transverse and longitudinal views. Also note mitochondia (Mi) and basal body (BB). X42,500

Plate V. Aerosorne reactions

Figure 14. Nomarski interference-contrast photomicrograph of a cryostat section (12 y) of a Limulus egg inseminated with Limulus spermatozoa (Sp). Spermatozoa are observed attached to the basement lamina (BL). Also visible are several laminated regions characteristically seen in the vitelline envelope (VE) of cryostat egg sections. The yolk (Yo), which comprises the bulk of the egg, is uniformly surrounded by the basement lamina and vitelline envelope. XlOOO

Figure 15. Normarski interference-contrast photomicrograph of Limulus spermatozoa following a "true" aerosome reaction at the basement lamina (BL) of a cryostat (12 y) egg section. Note the presence of numerous pores (Po) within the basement lamina and the absence of such pores in the vitelline envelope (VE). X2500

Figure 16. Phase contrast photomicrograph of a Limulus sperm (Sp) following treatment with 0.34 M CaCl2 to produce a "true" acrosome reaction. The acrosome filament (AF) appears as a long rod extending from the sperm. X2500

Figure 17. Phase contrast photomicrograph of a Limulus sperm following exposure to 2X MBL sea water. The sperm has experienced a "false" acrosome reaction in which the acrosome filament (AF) extends from the posterior nuclear cisternae (PNC). Note the acrosome vesicle (AV), nucleus (Nu) and flagellum (Fl). X2500 234 Plate VI. Acrosome separation

Figure 18. Gel formation following treatment of Liniulus spermatozoa with 0.0025% sodium hypochlorite. 1-2 min treatment with sodium hypochlorite releases acrosomes and causes disintegration of the sperm nucleus. Subsequent formation of a nucleic acid gel (J) traps the freed acrosomes (Ac). X2500

Figure 19. Separated acrosome (Ac) following treatment of the gel/acrosome complex with 0.02% DNAase I. X2500 236 Plate VII. Acrosome separation

Figure 20. A section of a Limtilus sperm showing the effects of 0.0025% sodium hypochlorite. The sperm plasma membrane has dissolved (arrows) leaving the nucleus (Nu) and acrosome (Ac) intact. X14,250

Figure 21. An electron micrograph of spermatozoa showing dissolution of the nucleus (Nu). The acrosome (Ac) and the subacrosome plate (SAP) persists after the nucleus begins to disintergrate. X14,250

Figure 22. Free aerosomes (Ac) appear in the nucleic acid gel following 0.0025% sodium hypochlorite treatment (1-2 min). X14,250

Figure 23. A separated sperm acrosome. The acrosome is intact even though the 0.0025% sodium hypochlorite removes some of the peri-acrosome material (arrows). X34,200 238 Plate VIII. Carbohydrate cytochemistry of uninseminated and inseminated egg sections

Figure 24. Cryostat section (12 y) of an egg. PAS stain with Metanil yellow counterstain. Note basement lamina (BL), vitelline envelope (VE) and yolk bodies (YB). X70 0

Figure 25. Cryostat section (12 u) of an egg. Congo red stain. Note intense staining of the cortical region (arrows). X700

Figure 26. Cryostat section (12 y) of an egg. Potassium permanganate Schiff method. Note the dark deposits at the base of the pores (arrows) in the basement lamina (BL). X700

Figure 27. Cryostat section (12 y) of an egg. Alcicin blue CpH 2.5)/nuclear fast red stain. The cortical region is the only component of the egg to give a positive Alcian blue reaction Carrows). X700

Figure 28. Cryostat section (12 y) of an egg following in semination and 72 h of development. Alcian blue (pH 2.5)/nuclear fast red stain. Note disappear ance of Alcian blue positive region seen in Figures 27 and 29. X200

Figure 29. Cryostat section (12 y) of an egg. Alcian blue stain (pH 1.0). The cortical region is the only Alcian blue positive region in the egg (arrow). X200

Figure 30. Inseminated cryostat egg section (12 y). Alcian blue (pH 2.5/nuclear fast red stain. Note positive Alcian blue reaction in the reacted sperm acrosome (arrow). XIOOO 240 Plate IX. Methylene blue extinction of Limulus egg

Figure 31. Cryostat section (12 y) of a Limulus egg. Methylene blue extinction method CpH 2.6). BL = basement lamina, VE = vitelline envelope, CR = cortical region. X150

Figure 32. Same method as Figure 31 but at pH 3.6. X150

Figure 33. Same method as Figure 31 but at pH 4.3. Note extinction of the vitelline envelope (VE), cortical region (arrows), and yolk (Yo) is between pH 2.6 (Figure 31) and pH 4.3 (Figure 32). X150

Figure 34. Same method as Figure 31 but at pH 5.3. X150

Figure 35. Same method as Figure 31. Note staining of all egg components at pH 6,7. BL = basement lamina, VE = vitelline envelope, CR = cortical region, Yo = yolk. X150

Figure 36, Same method as Figure 31. Note staining of base ment lamina (BL) and vitelline envelope (VE) at pH 7.6. X150

Figure 37. Same method as Figure 31, but at pH 8.1. The staining of basement lamina (BL), cortical region (arrows), and yolk (Yo) display little change in staining characteristics beyond pH 6.7) Figure 35). X150 242

• ^ Plate X. Protein cytochemistry of Limulus egg

Figure 38. Cryostat section (12 y) of an egg. Mercuric bromphenol blue stain. The basement lamina (BL) and vitelline envelope (VE) are not distinguishable from each other in terms of staining characteristics. Yo = yolk. X300

Figure 39. Cryostat section (12 u) of an egg deaminated with Van Slyke's reagent (45 min) and subsequently stained with mercuric bromphenol blue. Note blockage of staining reaction in the basement lamina (arrow). X750

Figure 40. Cryostat section (12 u) of an egg deaminated with Van Slyke's reagent (6 days) and subsequently stained with mercuric bromphenol blue. Both the basement lamina (BL) and vitelline envelope (VE) fail to stain. Some yolk bodies (YB) retain the stain after 6 days. X750

Figure 41. Cryostat section (12 u) of an egg treated with trypsin (1 mg/ml) for 30 rain at 37®C. Mercuric bromphenol blue stain. X300

Figure 42. Cryostat section (12 y) of an egg. Ninhydrin- Schiff method. Note darker staining of the basement lamina (arrows). X300

Figure 43. Cryostat section (12 u) of an egg deaminated with Van Slyke's reagent and subsequently stained with the Ninhydrin-Schiff method. The characteristic staining reaction of the basement lamina (BL), vitelline envelope (VE) and yolk (Yo) is blocked by this procedure. X300 244 mm Plate XI. Lipid cytochemistry of Limulus egg

Figure 44. Cryostat section (12 y) of an egg. Oil red 0 stain. Note only certain yolk bodies (YB) have an affinity for Oil red 0. BL = basement lamina, VE = vitelline envelope. X300

Figure 45. Cryostat section (12 y) of an egg. Copper phthalocyanin method (counterstained with 1% neutral red). Note positive reaction in the vitelline envelope (VE) and negative reaction of the base ment lamina (BL), cortical region (CR) and yolk (Yo). X300

Fi(_ .re 46. Cryostat section (12 y) of an egg. Controlled chromation followed by Sudan black B stain. Results are similar to those observed in Figure 44 with only certain yolk bodies (YB) staining. XlOO

Figure 47. Cryostat section (12 y) of an egg. Controlled chromation followed by Mayer's haematoxylin and stain. Structures stained by haematoxylin Sudan black B (Figure 46) are considered to be phospholipids. XlOO

Figure 48. Cryostat section (12 y) of an egg. U-V Schiff method for unsaturated lipids. Note staining of cortical region (arrows) and yolk (Yo). X300

Figure 49. Cryostat section (12 y) of an egg not exposed to U-V light as slide in Figure 48. No staining is observed in any part of the egg. X300 246 Plate XII. Enzyme cytochemistry of Limulus egg

Figure 50. Glucose-6-phosphatase in a cryostat section (12 li) of an egg. Note activity in the cortical region (arrows). X250

Figure 51. Glucose-6-phosphatase in a cryostat section (12 y) of an egg at 72 h development. X250

Figure 52. Control egg section (cryostat, 12 y). Note absence of reaction product in the cortical region. X250 248

SBBa Plate XIII. Enzyme cytochemistry of Limulus egg

Figure 53. Cryostat section (12 y) of Limulus egg. Sections stained for succinic dehydrogenase without substrate sodium succinate (control). BL = basement lamina, VE = vitelline envelope, CR = cortical region and Yo = yolk. X625

Figure 54. Succinic dehydrogenase in a cryostat section (12 li) of an egg. Note positive reaction in the cortical region (CR). Yo = yolk, X625

Figure 55. Succinic dehydrognease in a cryostat section (12 y) of an egg at 72 h development. An intense positive reaction is observed in the cortical region (CR) of the egg. Yo = yolk. X625 250 Plate XIV. Physiochemical treatment of Limulus egg sections

Figure 56. Cryostat section (12 u) of an egg. 0.2 N (pH 1.2) HCl treatment at room temperature for 24 h. Note expansion and increase in size of basement lamina (BL). VE = vitelline envelope, Yo = yolk. XIOOO

Figure 57. Cryostat section (12 p) an egg. 6 M urea treatment for 8 h at room température. BL = basement lamina, VE = vitelline envelope, Yo = yolk. X500

Figure 58. Cryostat section (12 u) of an egg. 6 M urea treatment for 24 h at room temperature. Note basement lamina (BL).i VE = vitelline envelope, Yo = yolk. X500

Figure 59. Cryostat section (12 y) of an egg. Trypsin (0.001 g/ml) treatment for 16 h at 37*C. BL = basement lamina, VE = vitelline envelope. XIOOO 252 Plate XV. Physiochemical treatment of Limulus egg sections

Figure 60. Cryostat section (12 y) of a Limulus egg. 0.005 N NaOH (pH 12.5) treatment for 1 h at room temperature. Note swelling of the basement lamina (BL). VE = vitelline envelope. XIOOO

Figure 61. Cryostat section (12 li) of an egg. 0.2 N NaOH treatment (2 min) at room temperature. Note basement lamina (BL) and vitelline envelope (VE). Yo = yolk. X500

Figure 62. Cryostat section (12 p) of eggs. 0.5 N NaOH (pH 12.5) treatment at room temperature for 3 min. The photomicrograph shoTfs two egg sections (ai and 32). Note basement lamina CBL) and yolk (Yo). The vitelline envelope has dissolved. X250

Figure 63. Same slide as Figure 62 at 13 min. Only the yolk (Yo) remains. X250 254 Plate XVI. Pepsin treatment of egg sections

Figure 64. Cryostat section (12 Vi) of Limulus egg. Pepsin (0.001 g/ml) treatment for 5 min at 37®C. BL = basement lamina, VE = vitelline envelope, Yo = yolk. XIOOO

Figure 65. Same conditions as Figure 64. Pepsin treatment for 1 h. Note vesiculation of basement lamina (BL). VE = vitelline envelope, Yo = yolk. XIOOO

Figure 66. Same conditions as Figure 64. Pepsin treatment for 2 Î1. BL = basement lamina, VE = vitelline envelope. Yo = yolk. XIOOO

Figure 67. Same conditions as Figure 64. Pepsin treatment for 16 h. BL = basement lamina, VE = vitelline envelope. XIOOO 256 Plate XVII. Liinulus development (unfertilized to 6 h after . fertilization)

Figure 68. Fresh unfertilized Liinulus eggs. Note the indentation characteristically found in freshly spawned eggs (arrow). The color of freshly spawned eggs varies from pale pink to green. X300

Figure 69. Limulus eggs 1 h following fertilization. Both fertilized and unfertilized eggs contain "pits" in the surface of the yolk (arrow) that persist up to 2 h following egg collection. X300

Figure 70. Same as Figure 69 but at a higher magnification. Note "pits" in the surface of the yolk (arrows). X625

Figure 71. Limulus eggs 6 h following fertilization. The yolk fluctuates between smooth, and granular in appearance. X300 258 Plate XVIII. Limulus development (segmentation stage to nucleated blastomere stage)

Figure 72. Limulus eggs appear in the segmentation stage (a) or granular stage (b) 24-27 hours following fertilization. The granular stage will eventually resemble the segmentation stage. X300

Figure 73. Eggs in segmentation stage. Note appearance of nucleated blastomeres (arrow). X300

Figure 74. Limulus eggs in early nucleated blastomere stage. Note nucleated blastomeres (arrows) have not covered entire surface of the yolk. X625

Figure 75. A later stage of Figure 74 at which nucleated blastomeres (arrow) have covered the surface of the yolk. X625 260 Plate XIX. Limulus development (blastopore stage to five limb bud stage)

Figure 76. Limulus eggs at 48 h of development. A blastopore appears in each embryo (arrow). X300

Figure 77. Five day eggs showing blastopore on the surface of the embryo (arrow). X300

Figure 78. Limulus embryo at 4- groove stage. The earliest sign of development in live developing eggs following the blastopore stage is appearance of paired grooves on the surface of the embryo. X300

Figure 79. Limulus embryo at 9 days development in five limb bud stage. Note stomodaeum which will become the mouth (arrow). X300 262 Plate XX. Limulus development (6 limb stage to imbibition- swelling stage)

Figure 80. 12 day Limulus embryo at advanced 6 limb stage= Note limbs 1-6 and embryonic disc on the surface of the egg (arrow). X300

Figure 81. 14 day Limulus embryo showing well developed appendages (arrow), segmented céphalothorax (Ce) and abdomen (Ab). X300

Figure 82. Ventral surface of Limulus embryo just before imbibition-swelling stage. A remnant of the first embryonic molt is visible at this stage (arrow). X300

Figure 83. Imbibition-swelling stage. A Limulus embryo emerging from original egg envelopes (arrow 1) inside the embryonic envelope formed during the previous developmental stages. Note remnant of first embryonic molt (arrow 2). X300

Plate XXI. Limulus development (imbibition-swelling stags to first post-hatch stage)

Figure 84. Imbibition-swelling stage Limulus embryo (27 days) swimming inside embryonic egg envelope. Some embryos still have original egg envelopes attached to embryonic envelope (arrow 1). Note remnant of first molt inside embryonic envelope (arrow 2). X150

Figure 85. Freshly hatched Limulus trilobite larva. The telson at this stage is only a small protrusion (arrow). X300

Figure 86. Ventral view of Limulus trilobite larva following first post-hatch molt [47 days). Note telson (arrow). X150

Figure 87. Dorsal view of Limulus trilobite larva shown in Figure 86. X150 266 Plate XXII, Sperm-egg interaction

Figure 88. Longitudinal section of reacted sperm attached to the basement lamina (BL) 10 min following insemination. The material of the acrosome vesicle (arrows) has been deposited on the surface of the egg but the acrosome rod (AR) has not reacted. VE = vitelline envelope. X18,750

Figure 89. Cross section of basement lamina (BL) demonstrating pores (Po) and electron dense granules along the surface of the basement lamina (BL) and the pores. X18,750

Figure 90. A higher magnification of a pore (Po) in the basement lamina demonstrating the electron dense granules on the pore surface. VE = vitelline envelope. X20,250 268 k