The origin and structure of the tertiary envelope in thick-shelled eggs of the brine shrimp, Artemia

© 1970 by Academic Press, Inc.

J. ULTRASTRUCTURERESEARCH32, 497-525 (1970)

497

The Origin and Structure of the Tertiary Envelope in Thick-Shelled Eggs of the Brine Shrimp, Arternia EVERETTANDERSON, J. H. LOCHHEAD,M. S. LOCHHEAD,AND ERWlN HUEBNER Department of Zoology, The University of Massachusetts, Amherst, Massachusetts 01002, The University of Vermont, Burlington, Vermont 05401, and The Marine Biological Laboratory, Woods Hole, Massachusetts 02543 Received February 6, 1970 The shell glands of the brine shrimp Artemia are composed of clusters of shell gland units. Each unit usually consists of two rather oblong cells separated by a lumen. Each lumen communicates with the uterus by a duct, formed by a neck cell and a small number of duct cells. Often, two or more ducts join before entering the uterus. The plasma rnembrane facing the lumen between gland cells is thrown into short microvilli. Elsewhere, the plasmalemma lacks such specializations. The two gland cells are joined together by maculae adhaerentes and septate desmosomes. The nuclei of these cells are large and contain abundant chromatin, arranged in a reticular pattern. The cytoplasmic matrix contains glycogen, lipids, mitochondria, a few microtubules, Golgi complexes, and large amounts of rough endoplasmic reticulum. When Artemia produces thick-shell eggs, the material for the outer portion of the shell (the tertiary envelope) is synthesized in the shell glands. During this synthesis numerous membrane-bounded secretory granules are formed that contain lipoprotein. Prior to their liberation the granules show a decrease in density and their membranes fuse with the plasmalemma adjacent to the lumen between the gland cells. Other granules fuse with those already being liberated, thereby enlarging the lumen of the shell gland unit. The tertiary envelope that is deposited around an embryo is at first homogeneous. Later it becomes filled with alveolus-like structures, the surfaces of which are composed of hexagonal porous units, through which the alveoli interconnect. Still later, in the gastrula stage, these interconnections are lost, and a more homogeneous outer layer is added. The brine shrimp, Artemia, is a crustacean about 12 m m long that lives in extremely saline lakes and pools. Zoologists have long been attracted to this animal because of m a n y unusual features in its a n a t o m y and physiology and because it is easy to obtain and to rear in the laboratory. A m o n g the problems that have excited the curiosity of n u m e r o u s cell biologists about Artemia, are those having to do with its early

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development. Especially challenging among these problems is the remarkable capacity shown by Artemia embryos for withstanding prolonged desiccation (13, 14, 17, 21, 33). The particular type of dormancy experienced by Artemia embryos has been referred to by Keilin (29) as cryptobiosis. He defines cryptobiosis as "the state of art organism when it shows no visible signs of life and when its metabolic activity becomes hardly measurable or comes reversibly to a standstill." Some races of Artemia are parthenogenetic, others are bisexual. In either case, successive batches of eggs enter the uterus at intervals of about 4 days. While in the uterus, some batches of eggs acquire thin, membranous shells and develop into nauplii that are born ovoviviparously, whereas other batches of eggs acquire thick shells and develop only to a late gastrula stage (17)before being laid. It has been suggested that the fate of any particular batch of eggs may depend entirely on the nature and amount of the secretion discharged into the uterus from the shell glands

(34). The present paper deals with the origin and structure of the outer layer, or tertiary envelope, of the thick egg shells of Artemia. The topic is one of special interest, since the ability of an embryo to enter cryptobiosis probably depends on the nature of the surrounding shell (34). Accordingly, we have felt it worthwhile to extend the elegant observations of Linder (31, 32), who studied the shell glands and egg shells of the freshwater Anostracan Chirocephalopsis, a close relative of Artemia. With regard to the ultrastructure of the shell, some results have been published by Morris and Afzelius (40), but these authors studied only fully formed egg shells that had been dried. The main points to which we shall direct attention are: (a) the anatomy of the shell gland and associated structures; (b) the origin and release from these glands of the material needed to form the tertiary envelope; (c) the architecture of the tertiary envelope in successive stages of its deposition on the surface of an egg. MATERIALS AND METHODS

Artemia for this study came from a culture maintained by two of us (J. H. L. and M. S. L.) at Burlington, Vermont, during the academic year and at Woods Hole, Massachusetts, during the months of June, July, and August. The culture was started from eggs derived from salterns on San Francisco Bay, California, where the animals are diploid and bisexual. The species involved cannot be named with any certainty. Recent work has shown that in some localities two species are present that are unable to interbreed (23). There is some indication that the animals from San Francisco may belong to a third species (9). Even after further work, it will be difficult to decide which species should be called A. salina (L.), since the specimens seen by Linnaeus are not available for cross-breeding experiments. To obtain desired stages in the secretory cycle of the shell glands and in the development of the tertiary envelope, living females that were estimated to be in appropriate stages of

TERTIARY ENVELOPEIN EGGS OF BRINE SHRIMP, Artemia

49?

their reproductive cycle (see below) were selected for both light and electron microscopy Only specimens producing a brown shell gland secretion were used, since specimens with white shell glands reproduce ovoviviparously. From the selected females uterus-shell gland complexes were dissected out and immediately put into a fixative. For light microscope analysis, the fixatives used were aqueous Bouin's, 10 % formalin, formol calcium, and Carnoy's (25). The fixed material was dehydrated, embedded in paraffin or in Piccolyte-paraffin (15), and sectioned at ~ 10 ~. The sections were treated with the following stains: (a) Heidenhain's iron hematoxylin and Mallory's triple stain, for the general histological picture; (b) Best's carmine, with and without prior c~-amylase digestion, for the detection of glycogen; (c) periodic acid-Schiff staining, also with and without prior eamylase digestion, for the detection of polysaccharides or oxidized lipids; (d) mercuric bromophenol blue for the detection of proteins; (e) gallocyanine, for basophilia; (f) Millon's reagent, for tyrosine in proteins; (g) Feulgen and methyl green-pyronin Y staining (25), with and without prior RNase and DNase digestion, ~ for nucleic acids (22); (h) Berenbaum's Sudan Black B method "C" for masked lipids (12). Some of the formalin-fixed material was embedded in gelatin, frozen, and sectioned with a Harris International cryostat. Some tissue was fixed in 3 % paraformaldehyde, embedded in agar, and sectioned with the Smith/Farquhar tissue sectioner. The frozen sections or those from the tissue sectioner were stained for general lipids with Sudan Black B and for phospholipids by the acid hematin test of Baker (3, 44). The specificity of the acid hematin test was verified with pyridine extraction. Some Epon-embedded material (see below) was sectioned at 1 ~ on a Porter-Blum MT-1 ultramicrotome and stained according to the recommendation of Richardson et al. (46) or Ito and Winchester (27). For electron microscopy, material was prefixed for 2 hours in 3 % glutaraldehyde buffered with phosphate buffer (48), washed for 2 hours, and postfixed in a 1.33 % s-collidinebuffered osmium tetroxide solution (7). The tissue was rapidly dehydrated in a graded series of ethanol solutions, then infiltrated and embedded in Epon (36). Thin sections were made with a Porter-Blum MT-2 ultramicrotome and stained with uranyl acetate (56) followed by the lead citrate stain of Venable and Coggeshall (55). Examination was with a Philips 200 electron microscope. OBSERVATIONS Female Artemia reproduces about once every 4 days. Some of the major events occurring during the 4-day reproductive cycle are indicated in Table I.

General morphology and light microscopic observations Artemia has three pairs of shell glands, located posterolateral dorsomedial, and anterolateral to the uterus (sometimes referred to as an ovisac). Each gland resembles clusters of grapes, and each opens by way of ducts into the uterus. A l t h o u g h the histology of the shell gland is reasonably well k n o w n for the freshwater anostracan 1 Ribonuclease and deoxyribonuclease were obtained from the Worthington Biochemical Corporation, Freehold, New Jersey.

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ANDERSON ET AL. TABLE I MAJOR EVENTS DURING REPRODUCTIVE CYCLE IN Artemia

stages (approximate times after molt) 0-4 Hours

Uterus

Empty of eggs. Sperm are deposited

Shell Glands Full of secretion

into the distal part 4-25 Hours

Eggs enter from oviducts. Sperms penetrate eggs. Polar bodies )arm (18). Cleavage commences. Shell

Secretion gradually released into the uterus, surrounding the uterine eggs

glands' secretion surrounds eggs, and shells begin to form 25-30 Hours

Shell continue to form around eggs

Very scanty secretion, or empty

30-36 Hours

Tertiary envelope around eggs completed during this period

Secretion starts to accumulate in cytoplasm

36 H o u r s - 4 days

Shelled eggs develop to prenauplius stage (17). Ovoviviparous eggs develop to nauplius stage

Secretion increasingly accumulates in cytoplasm

Last 2 hours of day 4

Eggs or nauplii leave. Uterine

Cytoplasm filled with secretion

cuticular lining shed when animal molts At 0 hour, cycle starts over again

Chirocephalopsis (31, 32), some photomicrographs are included to aid in the interpretation and orientation of the electron micrographs. The shell glands are composed of numerous shell gland units each of which consists of two large cells each containing a large nucleus with numerous nucleoli (Figs. 1 and 3). The large amount of chromatin and the many nucleoli have been thought to indicate a high degree of polyploidy (4) such as has been reported for gland cells in other organisms (16). The reticulated nature of the chromatin is reminiscent of such material in the cells comprising the spinning gland of some insects (5). The cells of a shell gland unit are partly separated from each other by a slit-like lumen (Fig. 1). Usually only two cells form each shell gland unit; occasionally, however, one finds four cells. That portion of the cell facing the lumen will hereinafter be referred to as the lumenal border and the portions facing in other directions as peripheral. The cytoplasm of these cells at about 25-30 hours within the cycle is FIGS. 1-3. Photomicrographs of cells of the shell gland (GC) unit filled with secretory granules (SG). D, duct; N, nucleus of shell gland unit; NU, nucleolus; DN, nucleus of duct cell. Epon embedded, toluidine blue-stained. × 400. FIG. 4. Photomicrograph of ceils of the shell gland unit (GC) almost devoid of secretory granules. N, nucleus; D, duct. Epon embedded, toluidine blue-stained, x 400.

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TABLE II CHART OF RESULTS OF STAINING PROCEDURES a Stain

Nucleus

Acid hematin Sudan Black B for masked lipids Sudan Black B on frozen sections Best's carmine PAS before c~-amylasedigestion PAS after c~-amylasedigestion Millon's reagent Mercuric bromophenol blue + Feulgen + + + Gallocyanine + Methyl green-pyronin, DNA + ++ Methyl green-pyronin, RNA + +

Cytoplasm

Granules

+ + + + ++ + ++ +

+ + + + + + + ++ + ++ -

a Symbols: +, + +, + + +, increasing degrees of positive staining intensity; - , negative reaction.

intensely basophilic. D u r i n g the periods of 0 to 9 hours a n d 72 hours to 4 days, the cytoplasm is filled with n u m e r o u s intensely staining secretory granules of various sizes. D u r i n g the period when secretory granules are being released (8 to 25 hours) there is no alteration in their staining properties. Figure 4 illustrates the m o r p h o l o g y of cells devoid of secretory granules. Table II summarizes the results of staining a n d histochemical procedures used in a n effort to characterize the secretory granules. Each pair of g l a n d cells is connected to a duct (Fig. 2, D). Some ducts e m p t y directly into the uterus, while others j o i n first with one or more n e i g h b o r i n g ducts. The cells that comprise a duct consist of a single neck cell, in contact with the gland cells, a n d a n average of a b o u t three duct cells, each one of which extends a r o u n d the l u m e n of the duct. I n b o t h the neck a n d the duct cells the c h r o m a t i n in the nuclei displays a reticular p a t t e r n (Fig. 2, D N ) a n d the cytoplasm is moderately basophilic. This basophilia is abolished b y prior digestion with RNase. Also present in the cytoplasm of these cells are a few clumps of glycogen, indicated by staining with Best's carmine, and, i n the duct cells, small PAS-positive bodies, the staining of which is n o t abolished by prior digestion with c~-amylase. F~G. 5. An area between cells of the shell gland unit depicting septate desmosomes (SD). x 105,000. FIG. 6. A region of two cells of a shell gland unit facing each other. MV, microvilli; SG, secretory granules, x 43,000. FIGS. 7 and 8. Sections through the nucleus of a shell gland unit. NE, nuclear envelope; NU, nucleolus; CH, chromatin. Fig. 7, x 40,000; Fig. 8, × 70,000. FIGS. 9 and 10. Sections through shell gland unit depicting Golgi complexes (GS). ER, endoplasmic reticnlum; ER1, endoplasmic reticulum adjacent the Golgi complex; SG, secretory granules; GLY, glycogen. Figs. 9 and 10, x 60,000.

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When the lumen of a duct is not filled with secretion from the shell gland cells. it can be seen to contain a secretion from the duct cells, either as one or more filamentous strands or as a lining adhering to the cells. When a lining is present, it has the appearance of a cuticle (Fig. 19, CL), except that between it and the cells there may be a relatively thick layer of less dense material. Where the shell gland ducts open into the uterus, no unusual cytological features have been observed. The wall of the uterus is comprised of striated muscle cells and epithelial cells, both of which contain glycogen. The cytoplasm of the epithelial cells is basophilic and secretes a cuticular lining. Both this cuticle and the material secreted by the duct cells are colored blue by Mallory's triple stain and an intense red by the PAS technique. These staining reactions may indicate the presence of chitin, but this suspicion requires confirmation. ELECTRONMICROSCOPY

Shell-gland unit PlasmaIemma and junctional complex. The lumenal plasmalemma of the secretory cell possesses rather short microvilli (Fig. 6, MV). The peripheral portions of the plasmalemma are morphologically unspecialized, display an irregular contour, and rest on a basement lamina. The cells constituting the shell gland unit are joined together by two kinds of junctional complexes. Maculae adhaerentes are located nearest the lumen and septate desmosomes (Fig. 5, SD) are found more peripherally. Nucleus. Figure 7 is a low magnification electron micrograph depicting the reticular pattern of the chromatin. Here the chromatin appears to be composed of tightly packed filaments. At the periphery of each mass of chromatin are clumps of particles. The nucleus also contains numerous nucleoli each of which is composed of both filaments and dense particles (Figs. 7 and 8, NU). The chromatin and nucleoli are suspended in a granular nucleoplasm limited by a nuclear envelope perforated at intervals by pores (Figs. 7 and 8, NE). The outer lamina of the nuclear envelope is studded with ribosomes. The inner lamina of the nuclear envelope is devoid of a "fibrous lamina" (20). The nuclear structure is similar during all phases of the secretory cycle. Mitochondria, glycogen, and microtubules. The mitochondria are rather large and contain cristae of varied orientations. Some cristae are transverse to the long axis; FIG. 11. A small portion of the cytoplasm of the shell gland unit showing Golgi complex GS, endoplasmic reticulum ER, mitochondria M, and secretory granules SG. x 40,000. FIGS. 12-14. Section of secretory granules showing a granular cortex (SG1) and a granular cortex with striations (SG2). Figs. 12-14. x 75,000.

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some are parallel to the long axis, while others are oriented irregularly (Figs. 11, 15, and 16). During the initial phase of the secretory cycle, glycogen appears as large aggregations of particles among the organelles (Fig. 10, GLY). During the production of the secretory granules (see below) the rather large glycogen clumps are no longer seen, but one observes smaller ones. Mingled with these various components are a few randomly distributed microtubules. Endoplasmic reticulum. At the time when the cells of the shell gland unit contain few or no secretory granules (25 to 30 hours), there is an abundance of rough endoplasmic reticulum, appearing as long, slender, tortuous cisternae (Fig. 9, ER) that contain some flocculent material. This organelle is presumably responsible for the intense basophilia of the cytoplasm. Unattached ribosomes are interspersed between the cisternae of the endoplasmic reticulum. The cisternae of the endoplasmic reticulum adjacent to the Golgi complex is devoid of ribosomes and displays some evaginations (Figs. 9 and 10, ER). Often the cisternae of the endoplasmic reticulum appear continuous with the saccules of the Golgi complex. When the cell obtains a full complement of secretory granules (see below) one finds only a few cisternae of the endoplasmic reticulum among the granules, but many of them still within the remaining, i.e., peripheral, areas of the cytoplasmic matrix (Figs. 12 and 15, ER). Golgi complex. The Golgi complexes of the gland cells are numerous and randomly dispersed. Just prior to the initiation and during the early phases of the secretory cycle, the Golgi complexes appear like those in Figs. 9, 10, and 11. Each Golgi complex occupies a rather large area within the cell and each consists of a number of saccules and varied sized vesicles, some of which have a clear interior while others possess an electron opaque substance (Figs. 10 and 11, SG). These opaque structures are identified as the secretory granules that are utilized during the formation of the tertiary coat (see below). In many instances it is difficult to identify whether the saccules are part of the Golgi complex or are smooth regions of the endoplasmic reticulum (Fig. 9). It may be that what one is observing is the production of Golgi saccules by cisternae of the endoplasmic reticulum where the latter organelle becomes devoid of ribosomal particles. Secretory granule. As noted above, secretory granules first appear in the vicinity of the Golgi complex. The secretory granules are membrane bounded; they vary in size and internal configuration. The variability in size may be a consequence of their

FIG. 15. A small portion of the cytoplasmof a shell gland unit. SG, SG~,SG~;secretory granules; M, mitochondrion, x 40,000.

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ANDERSON ET AL.

"maturation," for profiles similar to those depicted in Fig. 15 (SGz) seem to show that two secretory granules may fuse with each other. Some secretory granules possess a dense homogeneous interior (Fig. 6. SG); some are divided into large and small portions, often eccentrically placed, which may or may not be similar in structure; still others possess a dense medulla with a less dense cortex. At times the cortical area of the secretory granule possesses a reticular pattern (Fig. 12, SG1). Occasionally we have found some secretory granules whose cortical region possesses rod-like structures (Figs. 13 and 14, SG2). The rod-like structures appear as striations oriented normal or obliquely to the medullary portion. When the production of all secretory granules is completed, the cytoplasm between the nucleus and the lumen becomes filled with these granules. The release of the secretory granules is a gradual process, occurring during approximately the 8th to 25th hours of the cycle. During this period, while secretion is being discharged into the uterus, one sees some morphological changes within the secretory granules. Just prior to the release of the secretion, one notices that numerous vesicles appear like those shown in Figs. 15 and 16. Each of these vesicles is filled with a low density material. The granule labeled SGz shows that the substance comprising a portion of its cortex and medulla is organized in a reticular pattern; the one labeled SG4 (Figs. 15 and 16) illustrates some rather clear flocculent areas among a dense peripheral component. Frequently, one sees large areas within the cytoplasm that contain the reticular substance. These areas may be thought of as a compound granule resulting from the fusion of two or more single ones. When a granule is actually being secreted, there are further changes in its internal configuration. The dense bi- or tripartite structure becomes reticular (Fig. 16, SM). Then the membrane surrounding the reticular substance becomes confluent with the plasmalemma, thereby discharging its contents (Fig. 16, arrows). Often one notes a deep incursion within the cells that is continuous with the lumen, suggesting that many granules have fused with each other in a sort of chain reaction, thereby enlarging the lumen. It should be pointed out that the thickness of the plasmalemma of the new lumenal surface is approximately the same as that in a nonsecreting shell gland unit. It is interesting to note that secretory material within the lumen and in

FIG. 16. Portion of two cells of the shell gland unit during the secretory phase. M, mitochondrion;

SG, SGa; secretory granules; SM, secretory material; SM1 secretory material within the lumen;

arrows, break in plasma membrane after fusion, x 20,000. Fins. 17, 18, and Inset. Section through shell gland unit showing secretory material within the lumen (SM1, Fig. 17) and within the duct (SM1, inset and Fig. 18). CL, cuticle lining the duct. Fig. 17, × 15,000; Fig. 18, x 40,000. Photomicrograph (inset). Epon embedded, toluidine blue-stained. × 400. FIG. 19. Section through duct cell of shell gland unit. DN, duct cell nucleus; M', mitochondria; ER', endoplasmic reticulum; MTS, microtubules (inset); CL, cuticular lining; SD', septate desmosomes; BL, basement lamina. Fig. 19, x 18,000; inset, ×40,000.

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the various ducts en route to the uterus is reticular (Figs. 16, 17, inset, 18), however, once within the uterus, the secretion no longer appears reticular but becomes electron opaque and homogeneous. This material coats the surface of cleaving eggs (Fig. 26,

ST). When the release of the secretory product has terminated, the gland cells once again display organized cisternae of the endoplasmic reticulum and Golgi complexes with only a few secretory granules of varied sizes.

Duct cells of shell gland unit The duct cells have nuclei that resemble those of the gland cells (Fig. 19, DN). Each duct cell is squamous in shape and wraps around the central lumen. Where the two borders of the cell meet, on the side opposite to the nucleus, their adjacent cell membranes display both maculae adhaerentes and septate desmosomes (Fig. 19, SD). In the cytoplasm, there is an extensive array of rough endoplasmic reticulum, a host of ribosomes, mitochondria with the cristae mainly transversely oriented (Fig. 19, M'), and an abundance of microtubules (Fig. 19, inset, MTS). Most of the microtubules are oriented parallel to the long axis of the duct. A Golgi complex lies close to the nucleus consisting of a stack of saccules and vesicles, some of which contain an electron dense material. Dispersed within the rest of the cytoplasm are vesicles containing a dense material similar to that seen in some of the Golgi vesicles. The forementioned dense substance may be a precursor of the cuticular material in the duct. Both the lumenal and the external plasma membranes of the duct cells sometimes produce cytoplasmic projections of varying lengths. The external surface rests on a basement lamina (Fig. 19, BL). The lumen that is surrounded by the duct cells contains a cuticular material, separated from the plasmalemma by a space of varying width. This space is filled with a sparse, reticular material, together with a few dense bodies of variable size. The dense cuticular layer may be of even thickness, forming a regular, very wavy cuticle that entirely surrounds the lumen (Fig. 19, CL). Or, the cuticular layer may be much more uneven (Fig. 18), reminiscent of the filamentous strands sometimes seen with the light microscope. Uterine epithelial and muscle cells. The cuticular lining of the ducts is continuous with the cuticular lining of the uterus, as already reported by Linder for ChirocephaFIG. 20. Section through the surface of a newly laid egg showing the vitelline envelope (VE). x 18,000. FIG. 21. Section through a fertilized egg showing the possible first appearance of the tertiary envelope (TC). × 40,000. F~G. 22. Section through two-cell stage of the embryo showing a small portion of the newly deposited tertiary envelope, TC', Epon embedded, toluidine blue-stained, x 400. FI~. 23. Section showing the dense tertiary envelope (TC'), vermiform area (TV), superficial matt region (MT), and glycogen (GLY) within the cytoplasm of a blastomere, x 40,000.

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ANDERSONET AL.

lopsis (31). The only striking feature of the uterine epithelial cells (Fig. 26, UE) is an abundance of glycogen. Microtubules appear to be absent. The nucleus is rather large and morphologically similar to that described for the duct cells. The cytoplasm contains endoplasmic reticulum of the rough variety, a complement of ribosomes, and some rather large mitochondria. The Golgi complex lacks dense cored vesicles such as are seen in duct cells. The epithelial cells are joined to one another by septate desmosomes. Muscle. The contractions of the uterus are presumably accomplished by the striated muscle cells. The fine structure of this muscle will not be dealt with in this report.

Tertiary envelope The tertiary envelope is formed from the secretion fabricated by the shell gland units. Fig. 20 is a section of an uncleaved egg possessing the vitelline envelope (VE). From the oolemma outward, this envelope is composed of two light, and two dense, layers that alternate with each other. After about 1 hour in utero, patches of electron opaque material appear on the surface of the vitelline envelope. This might represent the initial deposition of the tertiary coat, but it also could result from adhesion of seminal fluid or oviduct secretion. By the time the first cleavage occurs, which is about 4-5 hours subsequent to the arrival of the egg in the uterus, the tertiary envelope is an electron opaque layer of irregular thickness (Fig. 22, TC'). Later, the material of the tertiary envelope becomes evenly distributed over the surface of the eggs, presumably as a result of the constant churning movements of the uterus. Approximately 7 hours after liberation of the egg into the uterus the tertiary coat is a relatively thick homogeneous structure (Fig. 23, TC'). In the region of the coat immediately adjacent to the surface of the blastomeres one notices some electron lucid vermiform areas (Fig. 23, TV). Later (about 8 hours), the vermiform regions appear to anastomose with each other (Fig. 24, TV) forming larger areas that eventually become spherical (Figs. 24 and 25, A V). The inner surfaces of the spherical regions, which we shall hereinafter call alveoli, have dense structures that alternate with electron lucid areas. When seen in tangential section, the surfaces of the alveoli display a hexagonal pattern like that shown in Fig. 25. The alveoli gradually spread throughout the entire electron opaque layer, first forming adjacent to the embryo and then moving outward. Eventually (15-20 hours) the coat appears to be composed of numerous alveolus-like structures of varied sizes (Figs. 26 and 27). These structures are embedded in the dense granular matrix. The surface of each alveolus is composed FIGS. 24 and 25. Sections of the tertiary coat (TC). GLY, glycogen within the cytoplasm of blasto= meres; TV, vermiform region; A V, alveoli; TP, pores within the surface of alveoli; MT, superficial matt region. Fig. 24, × 18,000; Fig. 25, x 60,000.

518

ANDERSON ET AL.

of the hexagonal units which appear to interconnect alveoli (Fig. 28, inset). The interior of each alveolus contains a flocculent material. The surface of the whole envelope is overlaid with what appears as a matt of fine filaments (Figs. 21, 23, 24, 25, 27, and 28). The embryo encompassed by the tertiary envelope is a blastula. Alterations are seen in this envelope by the time the gastrula stage is reached (Fig. 29). There is now more electron opaque material between the alveoli, so that neighboring hexagonal pores are no longer in contact with each other. The hexagonal interconnections have been lost, and the tubular interconnections are fewer. External to the alveoli there is now a thick electron opaque outer surface layer, perforated by pores or aeropyles (Figs. 29 and 30). On the outer surface of the tertiary envelope, the matt of fine filaments has been replaced by a dense stratum. In general, the structure now resembles that reported for dried egg shells by Morris and Afzelius (40). While this study is not concerned with the embryology of Artemia, we think it worthwhile to point out two interesting facts: (a) the peripheral cytoplasm of the blastomeres adjacent to the forming tertiary coat is rich in glycogen (Figs. 23-25, GLY); accmulation of this cortical glycogen in the cleavage furrows has been reported by Fautrez-Firlefyn and Fautrez (•9); (b) after the tertiary envelope has been completed, the developing embryo synthesizes a layer as indicated at EC in Fig. 31. This layer consists of chitin, impregnated with protein (33). DISCUSSION According to Ludwig (35) an envelope produced around an egg by nonovarian tissue is a tertiary envelope (1, 37, 45). The covering of the egg of Arternia is produced by the secretion from the shell glands and is thus a tertiary envelope. Data gathered during this study indicate that the material comprising this tertiary envelope is fabricated in the shell glands through the conjoined efforts of primarily the abundant rough endoplasmic reticulum and the Golgi complex. We also noted that the surface of the cisternae of the endoplasmic reticulum facing the Golgi complex was devoid of ribosomes and that the interior of the cisternae of the endoplasmic reticulum was sometimes confluent with the region of the Golgi complex. We assume that the endoplasmic reticulum synthesizes the protein component of the secretory granule and that this protein then "flows" to the Golgi complex, where it is packaged and concentrated. Once concentrated, the granules enlarge by fusing with one another, As noted earlier, our cytochemical findings suggest that the secretory granule is primarily FIG. 26. A photomicrograph of a blastula within the uterus (UE). TC, tertiary envelope; ST, coagulated secretory material. Epon embedded, toluidine blue-stained, x 400. FIG. 27. An electron micrograph of the tertiary envelope of the blastula MT, outer matt region; A V, alveoli, x 9000.

27 3zl--701829 J . Ultrastructure Research

TERTIARY ENVELOPE IN EGGS OF BRINE SHRIMP,

Artemia

521

Fios. 29 and 30. Sections of the tertiary envelope surrounding the gastrula. A V1, alveoli; AP, aeropyles. Fig. 29, x 27,000; Fig. 30, x 40,000. F~G. 31. Section through the embryo illustrating the embryonic cuticle (EC). Paraffin embedded, Mallory's triple-stained, x 800. lipoprotein. Some carbohydrate may also be present, as indicated by PAS staining, but such staining could result from the presence of lipoprotein complexes (44). Extensive evidence implicates the Golgi in the synthesis of carbohydrates in a variety of cell types (24, 41, 42, 47), but much less is known concerning sites for lipid synthesis. It does not seem unreasonable to us that the biochemical machinery of the Golgi complex in the shell gland cells of Artemia, and presumably in other cases, is directed toward the concentration of proteins, or of some complex, without synthesizing a moiety to be added to that which it packages and concentrates. Our results provide no evidence as to where in the cell the lipid is added to the protein. Linder (32) inFIG. 28. Section of the tertiary envelope of the blastula, MT, matte region; A V, alveoli; TP, pores between alveoli (also see inset). Fig. 28, × 20,000; inset, x 105,000.

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ferred from his results that the secretory granules consist at first of lipoprotein, and that additional lipid is added to them later, The literature indicates that cells, whose secretory products are mainly protein, such as those comprising the ampullate gland of the spider, Araneus sericatus (6), may be deficient in Golgi complexes. Kramer and Poort (30), investigating the pancreas, state: "... the proteins synthesized in the ergastoplasm can follow two pathways: they can either be condensed in the Golgi complex and stored in a granular form, or be secreted directly without a substantial condensation." In some respects, the condensation of secretory material in the shell gland of Artemia is similar to that of the acinar cells of the pancreas (26, 28, 43) and to that of the secretory cells of the mammalian oviduct (10). Just before being released, the secretory granules become somewhat granular or reticular. The membrane surrounding the granule then fuses with the plasma membrane and thereby releases its contents into the lumen. A little later the secretion can be seen as a finely granular suspension in the duct enroute to the uterus. Once this material enters the uterus, it becomes deposited around each embryo as a dense homogeneous layer within which there develops an architecturally complex pattern. Just how this organization is achieved, or how the material hardens is unknown. The nature of the vermiform areas at the surface of the blastomeres (Figs. 23 and 24) is enigmatic. In some respects they resemble the folded lamellate structures seen at the surface of two-celled embryos by Anteunis et al. (2). However, we believe the vermiform structures represent the initial beginnings of the architecturally complex pattern. We have confirmed, for Artemia, the observations of Linder (32) on Chirocephalopsis, that the shell gland secretion contains a lipoprotein rich in tyrosine. Although he was unable to find any polyphenol oxidase or diphenols, Linder supposed that during egg shell formation the lipoprotein is sclerotized, presumably by a process involving spontaneous oxidation of the tyrosine residues to quinones. Sclerotization certainly seems likely, but its mechanism needs further clarification, especially in light of the recent work by Sekeris (49) and others (39). Biochemical mechanisms eventually proposed will have to take into account certain morphological findings, such as the separation of the material in the tertiary coat into dense and alveolar components, and the conversion into long fibers of the material that is being deposited during the later stages (Fig. 28). It is tempting to compare the situation in Artemia with some of the findings reported for the colleterial glands of lepidopterans and orthopterans among the insects (8, 11, 38, 53, 57). These glands secrete the material for the tertiary envelope in Lepidoptera and for the ootheca in some Orthoptera. In Lepidoptera, the two glands are thought to secrete identical material (8), but in Orthoptera the left gland produces the neces-

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sary substrates while the right gland produces one of the enzymes needed for sclerotization (11). Since there are three pairs of shell glands in Artemia, their secretions might differ, possibly in the same respects as in orthopterans. However, our studies to date give no hint of any such differences. As already noted, some batches of eggs in Arternia develop while in the uterus into free-swimming nauplii, and other batches acquire thick hard shells and are laid when the embryos are in a late gastrula stage. The suggestion has been made that the fate of any given batch of eggs may depend entirely on the nature and amount of the secretion from the shell glands (17, 34, 54). Among the hard-shelled eggs, not all are destined to enter a state of cryptobiosis. Some of them may hatch only a few days after being laid, others may not hatch for months, and others may be dried and then remain dormant for years. Casual observations suggest that the eggs in any one batch are Mike in their behavior, those in some batches hatching almost immediately, while those in other batches enter a more or less prolonged period of cryptobiosis. It is certainly tempting to suppose that cryptobiosis of an Artemia embryo is dependent on some degree of impermeability of the egg shell. The controlling factor may be only the total thickness of the shell, as is suggested in the three references just cited (17, 34, 54). Or, more complex factors may be involved, such as the thickness, density, microstructure, or chemical composition of particular layers in the shell. What role the tertiary coat may play in the cryptobiotic state of Artemia embryos remains obscure at present. Perhaps the situation will be found to be comparable to that in the grasshopper, in which it has been shown that diapause of the embryo depends on waterproofing of a special portion of the egg shell termed the hydropyle (50-52). Further discussion of the topic of cryptobiosis in Artemia will be found in papers by Dutrieu and by Morris and Afzelius (17, 40). Morris and Afzelius (40) suggest that the alveoli within the tertiary coat may contain a gas and thus may serve for flotation. However, it must be pointed out that newly laid eggs of Artemia float only in rather strong brines, in which most other organic materials also float. Even dried eggs of Artemia sink in brines of about twice the salinity of seawater, as soon as wetting of their shell surfaces is complete. To elucidate the role of the tertiary envelope in the embryogenesis of Artemia, further studies will be needed on the biochemistry and permeability of the different layers of the tertiary coat. Such studies will also have to be extended to the inner chitinous coat, alluded to only briefly in the present report. This investigation was supported by a grant (HD 04924-09) from the National Institutes of Child Health and Human Development, United States Public Health Service. The authors wish to thank Mrs Gloria S. Lee, and Mr and Mrs L. Musante for their technical assistance.

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REFERENCES 1. ANDERSON,E., J. Cell Biol. 35, 193 (•967).

2. ANTEUNIS,A., FAUTREZ-FmLEFYN,N. and FAUTREZ,J., Exp. Cell Res. 25, 463 (1961). 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

BARKA, T. and ANDERSON, P. J., Histochemistry. Harper & Row, New York, 1963. BARIGOZZI,C., Chromosoma 2, 251 (1942). BEAMS,H. W. and Wu, C. F., J. MorphoL 47, 261 (1929). BELL, A. L. and PEAKALL,D. B., d. Cell BioL 42, 284 (1969). BENNETT,H. S. and LUFT, J. H., J. Biophys. Biochem. Cytol. 6, 113 (1959). BERRY, S. J., J. Morphol. 125, 259 (1968). BOWEN, S. T., Genetics 52, 695 (1965). BROWER, L. K. and ANDERSON, E., Biol. Reprod. 1, 130 (1969). BRUNET, P. C. J. and KENT, P. W., Proc. Roy. Soc. Ser. B144, 259 (1955). CLARA, M., in GRAUMANN, W. and NEUMANN, K. (Eds.), Methoden der Lipidhistochemie, Bd. V, Erster Tell, p. 54. Fischer, Stuttgart, 1965. CLEGG,J. S., Comp. Biochem. Physiol. 20, 801 (1967). CLEGG,J. S. and GOLUB, A. L., Develop. Biol. 19, 178 (1969). CLONEY,R. A., Amer. Zool. 1, 67 (1961). DACRuz-LANDIM,C. and MELLO, M. L. S., Y. Exp. Zool. 170, 149 (1969). DUTRIEU,J., Arch. ZooL Exptl. Gen. 99, 3 (1960). FAUTREZ,J. and FAUTREZ-FYRLEFYN,N., Arch. Biol. 72, 611 (1961). FAUTREZ-FIRLEFYN,N. and FAUTREZ, J., Biol. Jaarboek, Gent 30, 81 (1962). FAWCETT,n . W., Am. J. Anat. 119, 129 (1966). FINAMORE,F. J. and CLEGG, J. S., in PADILLA, G. M., WHITSON, G. L. and CAMERON, I. L. (Eds.), The Cell Cycle. Academic Press, New York, 1969. GLICK, D., Techniques of Histo- and Cytochemistry. Wiley (Interscience), New York, 1949. HALFER-CERvINI,A. M., PICCINELLI,M., PROSDOCIMI,T. and BARATELLI-ZAMBRUN/,L., Evolution 22, 373 (1968). HORWlTZ,A. L. and DORFMAN, A., J. Ceil Biol. 38, 358 (1968). HUMASON,G. L., Animal Tissue Techniques. Freeman, San Francisco, 1962. ICmKAWA,A., J. Cell Biol. 24, 369 (1965). ITO, S. and WINCHESTER,R. J., d. Cell Biol. 16, 541 (1963). JAMIESON,J. D. and PALADE, G. E., d. Cell Biol. 34, 577 (1967). KEILIN, D., Proe. Roy. Soc. Ser. B 150, 149 (1959). KRAMER,M. F. and POORT, C., Z. Zellforsch. Mikroskop. Anat. 86, 475 (1968). LINDER,H. J., J. Morphol. 104, 1 (1959). -ibid. 107, 259 (1960). LOCHHEAD,J. H., Turtox News 19, 41 (1941). LOCHHEAD,J. H. and LOCHI-mAD,M. S., Anat. Rec. 78, Suppl., p. 75 (1940). LUDWIG,H., Wurzb. Zool. Inst. Arb. 1, 287 (1874). LUFT, J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). MAWSON, M. L. and YONGE, C. M., Quart. d. Microsc. Sci. 80, 553 (1938). MERCER, E. H. and BRUNET, P. C. J., Y. Biophys. Biochem. Cytol. 5, 257 (1959). MILLS, R. R., LAKE, C. R. and ALWORTH,W. L., J. Insect Physiol. 13, 1539 (1967). MORRIS, J. E. and AFZELIUS,B., d. Ultrastruct. Res. 20, 244 (1967).

TERTIARY ENVELOPE IN EGGS OF BRINE SHRIMP, Artemia

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41. NEUTRA, M. and LEBLOND,C. P., J. Cell Biol. 30, 119 (1966). 42. - ibid. 30, 137 (1966). 43. PALADE, G. E., SIEKEVITZ, P. and CARO, L. G., Exoerine Pancreas Norm. Abnorm. Functions Ciba Found. Syrup. 1961.

44. 45. 46. 47. 48. 49. 50. 51. 52.

53. 54. 55. 56. 57.

PEARSE,A., Histochemistry, Theoretical and Applied. Little, Brown, Boston, 1961. QUATTROPANI,S. L. and ANDERSON,E., Z. Zellforsch. Mikroskop. Anat. 95, 495 (1969). RICHARDSON,K. C., JARETT, L. and FINKE, E. H., Stain Technol. 35, 313 (1960). Ross, R. and BENDITT,E. P., J. Cell Biol. 27, 83 (1965). SABATINI,D. D., BENSCH, K. and BARRNETT,R. J., J. Cell Biol. 17, 19 (1963). SEKERIS,C. E., in KARLSON,P. (Ed.), Mechanisms of Hormone Action, p. 149. Thieme, Stuttgart, 1965. SLIFER,E. H., Quart. J. Microsc. Sci. 80, 437 (1938). -J. Morphol. 87, 239 (1950). -Y. Exp. Zool. 138, 259 (1958). STAY, B. and ROTH, L. M., Ann. Entomol. Soc. Amer. 55, 124 (1962). STEFANI,R., Riv. Biol. 54, 457 (1961). VENABLE,J. H. and COGGESHALL,R., J. Cell Biol. 25, 407 (1965). WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958). WILLIS, J. H. and BRUNET, P. C. J., J. Exp. Biol. 44, 363 (1966).