Cortical Changes in Acipenserid Eggs during Fertilization and Artificial Activation

The work presented aims at describing cortical structures and their changes on fertilization and artificial activation in Acipenserid eggs; and at elucidating the relationship between fertilization impulse, cortical granule expulsion, perivitelline space formation, change in the properties of the egg membranes, and the stimulation of the nucleus. Special attention was given to the study of acid mucopolysaccharides and their alteration in the activation process, as well as to the effect of treatment of living eggs with Na-periodate.

Some of the data presented in this work have already been published elsewhere (Dettlaff & Ginsburg, 1954; Dettlaff, 1957).

Investigations were carried out on the eggs of sturgeon—Acipenser güldenstadti colchicus V. Marti, of sevruga—A. stellatus Pall., and of white sturgeon— Huso huso L. The material was collected during 1955-9 in Rogozhkino sturgeon hatchery (Lower Don). The eggs were procured from females which had been given a hypophyseal injection. Living observations on the changes in fertilized eggs and the study of fixed eggs in section (in 6 lots of sturgeon eggs, 2 of sevruga eggs, and one of white sturgeon) were made in parallel; comparison of the dynamics of the changes in fertilized eggs and in eggs activated by pricking was carried out on the eggs of two sturgeon and one white sturgeon female. The eggs were pricked with a glass needle at the border of the animal region, or in the vegetative pole. Activation by pricking allows precise recording of the time of stimulation (which coincides with the moment of pricking) and hence precise study of the dynamics of the egg-changes (see also Ginsburg, 1961).

Living observations were carried out under a binocular dissecting microscope with incident light, and in a vertical chamber (Ancel & Vintemberger, 1948) in transmitted light with the aid of a compound microscope. To study the eggs in sections, precisely timed fixations were made: during the first 10 seconds after insemination (or pricking), every 1–2 seconds; later, up to 30 seconds, every 5 seconds; during the next 4 minutes, at intervals of 15 and 30 seconds; up to the onset of the first division at intervals of 5 and 15 minutes; and then at the stages of 2, 4, 8, and many blastomeres. The eggs were fixed in Sanfelice and Bouin fluids, and in 4 per cent, formalin, and embedded in paraffin; 5–10 eggs were cut at each stage. Sections 7μ thick were stained by the azan and Schiff’s iodine methods, with Heidenhain’s iron haematoxylin, and by Feulgen counterstained with light green. In order to study acid polysaccharides of the cortical layer and their changes during activation, sturgeon eggs were fixed at successive stages with Shabadash (1949), Bouin-Allen, and Carnoy’s fluids, and with lead acetate and formalin by the Sylven method. We employed Schiff’s iodine method, pretreating sections with (a) diastase prepared from germinated barley seeds by Ivanov’s technique (1946), (b) crystalline ribonuclease, (c) hyaluronidase from bovine testis (put at my disposal by L. G. Smirnova), and (d) an unpurified aqueous extract of an acetonized homogenate of sturgeon testis¹ possessing hyaluronidase activity; staining was carried out with an old aqueous solution of pyronin and 0·5 per cent, aqueous solution of toluidine blue. In order to detect proteins, the eggs were stained with sublimate-phenol-blue (according to Mazia, Brower, & Alfert, 1953).

Particular methods employed in individual experiments are described in the text.

Acipenserid eggs are covered with an external jelly membrane and with two layers of vitelline membrane (zona radiata interna and externa). The membrane layers can be distinguished only under high magnifications. The membranes closely adjoin the egg surface. The inner border of the zona radiata interna in the living egg is invisible. Over the region of the polar spot where the spindle of the 2nd maturation division lies, micropylar canals are located (Plate 1, fig. 1).

The cortical layer of the unfertilized egg is formed by a layer of cytoplasm free of yolk and pigment granules¹ and by a deeper-lying layer of cortical granules (Dettlaff, 1957) which merges into a layer of pigment granules (Plate 1, figs. 6a, 7a). The cortical layer possesses this structure even in the oocyte, at the stage before nuclear membrane dissolution (Plate 1, fig. 2).

The animal portion of the egg is rich in cytoplasm; it contains small-granular yolk and yolk-free lacunae, some substances of which are involved in the formation of the perivitelline space colloid (Dettlaff & Ginsburg, 1954).

Cortical granules are oval or spherical and form a continuous layer over the whole egg surface, including the funnel of maturation. The largest cortical granules in sturgeon and sevruga reach 2·2 × 3·8 μ. Egg membranes are stained similarly with aniline blue and light green. A positive Schiff-iodine reaction of granules and egg membranes was preserved after 3- and 20-hour incubation of sections at 37–38° C. in 1 percent, solutions of diastase and testicular hyaluronidases, while in control preparations of liver and umbilical cord the staining specific to glycogen and hyaluronic acid completely disappeared during this time; glycogen present in large amount in the animal portion of the egg also ceased to be detected.

After staining sections with an old pyronin solution for 2–5 days, the cortical granules were equally intensively pink-coloured, whether or not the sections were pretreated with ribonuclease solution (1 mg. per 1 ml. borate buffer at pH 7–5 during 3–20 hours at 37° C.). On keeping the preparations over 24 hours in 0·5 per cent, aqueous toluidine blue, the granules showed γ-metachromasia. The latter, however, as in the case of cortical granules of sea urchins (Monné & Harde, 1951a), was not always distinctly revealed.

The Schiff-positive reaction, and the metachromatic staining with toluidine blue and pyronin, point to the presence of acid polysaccharides in the cortical granules. Since their specific staining is preserved after the action of hyaluronidases (including the species-specific one) which split both hyaluronic and chondroitinsulfuric acids (Pearse, 1953) they can be assumed to contain acid mucopolysaccharides of heparin type. (Employing both the histochemical and the clot methods, we failed to find the species-specificity in the action of testicular hyaluronidases noticed by Takashima, Mori, and Kawano (1955), when comparing hyaluronidase activity of the extracts from bovine and sturgeon testes.)

Unlike cortical granules, egg membranes contain neutral mucopolysaccharides: they did not show a clear metachromasia with toluidine blue, and the zona radiata was not at all stained with pyronin, while the jelly membrane was stained light violet.

Polysaccharides in granules, as well as in membranes, appear to be firmly linked to proteins, since they were preserved when fixed with non-specific fixatives and when sections were heated. A small amount of protein was revealed in them on treatment of sections with sublimate-bromine-phenol-blue. Trypsin diminished the firmness of the membranes (G. M. Ignatieva, unpublished), and this also suggests the presence of proteins in them.

No lipid membrane could be detected in the cortical granules of sturgeons. It can be seen in preparations made by Ginsburg (1956), who studied lipids in sturgeon oocytes and eggs (fixation with formol-calcium, embedding in gelatine, cutting with freezing microtome and staining with Sudan black B), that the cortical layer of the cytoplasm contains diffusely dispersed lipids, while cortical granules lack these. Because of this, the granules are clearly seen. Under maximal magnifications of the light microscope no lipid concentration can be seen on their surface.

Diffuse acid mucopolysaccharides cannot be revealed in the cortical layer of sturgeon eggs by means of the above methods, while the Hale (1946) method employed for this purpose in sea urchins (Monné & Slautterback, 1950; Immers, 1956; Runnström & Immers, 1956) gave in my experiments a nonspecific coloration; mucopolysaccharides of the cortical granules are not revealed at all by this method.

Treatment of living sturgeon and sevruga eggs with 0·01 N solution of Na periodate for 1·5–15 minutes brought about a depolymerization of the membrane polysaccharides, but it did not affect the polysaccharides of the cortical granules.

In sections through such eggs treated with Schiff’s solution without preliminary immersion of the sections in NaIO₄ solution, cortical granules remained unstained at all time intervals (Plate 1, fig. 6b), while the coloration intensity of the membranes progressively increased. In control sections of the same eggs pretreated with NaIO₄ solution before Schiff’s reagent, the granules showed a typical PAS-positive reaction (Plate 1, fig. 6a). Diminishing the concentration of NaIO₄ solution with which control sections were treated gave preparations in which the coloration intensity of the membranes hardly differed from that in the experimental sections, but cortical granules, nevertheless, were stained with the same intensity as the membranes. Since a 15-minute exposure to the action of 0·01 N Na periodate solution was close to an injurious one, the conclusion must be drawn that the cortical layer in the living sturgeon egg appears to be impermeable to Na periodate. In this connexion it is of interest that Na periodate does not activate sturgeon eggs (l·10⁻³ and 1·10⁻⁴ M solutions neither damage the eggs nor activate them even with a 6-hour exposure; 1–10⁻¹ and 1·10⁻² M solutions damage the eggs after a 30-minute exposure without activating them, while with a 5-minute exposure they neither damage nor activate the eggs).

A network of lacunae of varying size was described in the cytoplasm of the animal region of the egg of A. ruthenus by Salensky (1881). This network arises during the maturation period as a result of the dissolution of the nuclear membrane and the consequent passage of the karyoplasm into the cytoplasm (Plate 1, figs. 2, 3). Like the oocyte nucleus, these lacunae are stained with aniline blue. The staining is more successful after formalin fixation of the eggs. After fixation with Sanfelice fluid, round globules of a hydrophilic colloid may be seen in these karyoplasmic areas of the animal region (Plate 1, fig. 4). In nonactivated eggs kept for some time in water, these globules, after fixation and passing through the alcohols, shrink more strongly than the rest of the cytoplasm, so that they often appear to lie in larger vacuoles (Plate 1, fig 5). These globules will be called ‘the globules of the hydrophylic colloid’ until their composition has been finally elucidated.

After insemination or pricking the egg surface rapidly undergoes a change: the coloration alters in shade, and the membranes become transparent. The membrane layers can now be easily distinguished, and the inner border of the zona radiata interna appears. In fertilized eggs these changes start in the micropylar region and spread in a wave-like fashion over the egg surface, reaching the opposite pole in between 2 and 4 minutes, depending on the temperature. In the eggs activated by pricking, the changes start from the pricked area and spread towards the animal pole at a higher velocity than towards the vegetative pole (cf. also Ginsburg, 1960, 1961). The spread of these changes can be clearly followed only in certain darker-pigmented lots of eggs.

The brightening of the membranes is followed by their rapid swelling, connected with their active water uptake from the external medium (Zotin, 1955; Dettlaff & Ginsburg, 1954) (Text-fig. 3). The jelly-membrane acquires an alveolar structure and becomes sticky.

After the appearance of stickiness the eggs begin to turn within the membranes with the animal pole upwards. In some lots the rotation of the eggs is completed by 20–25 minutes, in others somewhat later.

When examining the eggs laterally under the microscope in a vertical chamber, the inner border of zona radiata interna can be seen, but the space between it and the egg surface is still lacking. Only at the termination of the egg rotation, 15–20 minutes after insemination (17·8–18·0° C.), the animal region of the egg begins to flatten and between this region and the internal layer of the zona radiata a narrow perivitelline space becomes visible for the first time and rapidly widens during the subsequent 20–25 minutes (see also Dettlaff & Ginsburg, 1954).

Observation of a living egg under a dissecting binocular shows that the turbid, somewhat opalescent colloid is secreted from the animal portion of the egg into the perivitelline space. In sturgeons the secreted substance is slightly violet and can be clearly seen.

Whether eggs are fertilized or activated by pricking, the perivitelline space is formed simultaneously and in the same manner. Independently of the site of pricking (in the animal or vegetative region of the egg), it arises over the animal region.

Concomitantly with the rotation of the egg and with the formation of the perivitelline space, a hardening of the egg membranes takes place. By the onset of the first division their toughness reaches its maximum (Zotin, 1953).

The first changes in the cortical layer of sturgeon eggs were observed 3 seconds after insemination (17·8—18·0° C.); small-sized, optically empty vacuoles appeared near the micropylar canals, in the cortical layer, among the granules; the contours of the granules were no longer clearly seen. During some seconds subsequently the size of the vacuoles somewhat increased and, at the same time, the process of vacuolization spread laterally (Plate. 1, fig. 7). The percentage of eggs in which the early stages of the cortical changes could be detected rapidly increased during the first 8 seconds after insemination and by 11–15 seconds reached its maximum (Textfig. 1); i.e. it underwent the same changes as the fertilization percentage in experiments on the insemination rate (Ginsburg, 1957), or in experiments on chelating Ca-ions with versene solution at different intervals after the addition of sperm (Dettlaff, 1958). In the eggs showing the first signs of activation, spermatozoa were found at the entrance of the terminal canal where, according to Ginsburg’s (1957) data, an effective contact of gametes is achieved. Thus, the cytoplasm of the cortical layer responds to the encounter with a spermatozoon by the formation of vacuoles during the first 1-2 seconds.

The cytoplasmic areas with their granule material, enclosed between vacuoles, acquire a columnar form. At this time the whole material of the columns is stained by aniline blue and shows a PAS-positive reaction. In those places where individual granules are preserved within the columns, the latter can be clearly seen to consist not only of the granule material but of the cytoplasm as well. With the later enlargement of the vacuoles the columns stretch, get thinner, and separate from the egg (Plate 1, fig 7e). In the eggs of some females, apparently after over-ripening of the eggs, as well as in non-fertilized eggs which have remained in water for a long time, small vacuoles can also be found some times in the cytoplasm of the cortical layer, but they are more often located under the granular layer and not among the granules. The appearance of vacuoles in this case does not lead to a rapid spreading of the cortical reaction and to the detachment of granules. It seems that this requires some other changes in the properties of the surface layer.

The bulk of the columnar substance with the protoplasmic membrane uniting them passes then to the internal surface of the zona radiata interna (Plate 1, fig. 7e). Fragments of cytoplasmic filaments are left on the egg surface, and individual unchanged granules can be found. In some eggs the separation of the columnar material could be observed in the centre of the animal region 5 seconds after insemination or pricking. Whether the detachment of the columns described above is a result of their separation, or whether the latter is accelerated in the process of histological treatment, can hardly be decided.

Having once started in the animal portion of the egg, the changes described spread in all directions and gradually embrace the whole egg surface. After 60 seconds the contact between the egg and the membranes in the animal region is preserved only in the area of the micropylar canals (Ginsburg, 1957; Dettlaff, 1958). In sections of eggs fixed 20–40 seconds after insemination all intermediate stages from separated to completely unchanged granules can be seen (Plate 1, figs. 7a-e).

The velocity of spread of the cortical changes gradually decreases from the animal towards the vegetative pole (Text-fig. 2). When eggs are activated by pricking, the cortical layer undergoes the same changes as after fertilization but they start from the site of pricking. The time of termination of the separation of cortical granules is similar to that after fertilization. Comparison of the spread of the appreciable colour change in the living egg during the first 2–3 minutes after insemination or pricking with the changes in the layer of cortical granules as seen in sections shows that they agree.

It is interesting to note that one of the first manifestations of the inadequacy of eggs is distortion of the cortical reaction. After a prolonged exposure of eggs to the body fluid, or at an unfavourable temperature, the uniformity in the spread of the cortical reaction is lost. The velocity of cortical changes concomitantly decreases, or, in parts of the egg surface, they do not proceed at all. This latter abnormality occurs particularly often in white sturgeon eggs at unfavourable temperatures. In that part of the egg where no extrusion of cortical granules has taken place, the membranes are not elevated over the egg surface and do not undergo changes. Those parts of the egg which have not undergone cortical changes do not subsequently undergo cleavage, and eggs with a partial discoidal cleavage thus arise.

After the termination of the extrusion of cortical granules, the external surface of the egg has a relatively even outer contour (Plate 3, fig la) and sometimes contains individual unexpelled cortical granules.

Cytoplasmic columns containing the material of the cortical granules, which were separated from the egg in the process of the cortical reaction, adjoin from within the zona radiata interna and form on its inner surface a kind of fringe. More or less isolated columns fuse with each other. From this moment the vitelline membrane with the underlying layer containing the substances of the cortical granules corresponds to the so-called fertilization membrane of sea urchins (Motomura, 1941; Runnström, 1948; Endo, 1952).

Mucopolysaccharides of the cortical granules do not undergo chemical change in the process of egg activation: the fringe on the inner surface of the vitelline membrane, which contains mucopolysaccharides, shows the same histochemical reactions as the cortical granules : the positive Schiff iodine reaction is preserved after the treatment of sections with diastase and hyaluronidases (Plate 2, figs. 3a, b).

The extrusion of cortical granules and the formation of the fertilization membrane completes the first phase of the cortical changes. Because of them the egg is released from close contact with the membranes and begins to rotate within them with its animal pole upward. The egg membranes swell and become sticky.

The first isolation of the egg from the egg membranes takes place as a result of the extrusion of cortical granules and of the formation of a slightly swollen granular layer (internal portion of the fertilization membrane) on the inner surface of the vitelline membrane. The formation of the extended perivitelline space over the animal region of the egg takes place later and is connected with the secretion of a hydrophilic colloid from the egg.

Globules of hydrophilic colloid which were described in an unfertilized egg and which separate within the lacunae of the karyoplasm do not fuse with the rest of the cytoplasm and, after activation, gradually rise to the surface cytoplasmic layer of the animal region. Because of this the surface layer of the animal region becomes enriched with a light hydrophilic colloid (Plate 2, figs, 1a-e; Plate 3, figs, 1a-d). At first globules form isolated islets in the surface layer, while later on a continuous light layer appears in the animal portion of the egg (Plate 2, fig. le, Plate 3, figs. b-d). At the egg surface the shape of the globules changes (cf. Plate 2, fig. 2 a, b) while their content acquires a somewhat increased water content and undergoes vacuolization. The swelling of the colloid is particularly clearly seen in formol-fixed eggs (Plate 3, fig. 2a). This effect may, however, be increased in the case of formol fixation by post-mortem swelling of the colloid.

The colloid appears in the perivitelline space (after formol fixation) 10–15 minutes after activation; during the subsequent 30–40 minutes its volume rapidly increases on account both of extrusion of new colloid from the egg (Plate 3, fig. 2b) and of water uptake from the external medium (Zotin, 1955) (Text-fig. 3). At this time the animal region flattens considerably. In sections the pigment turns out to be dispersed at these stages in the thicker cytoplasm layer; its individual granules are carried away with the colloid to the perivitelline space. The colloid extruded is sometimes very vacuolated at the egg surface (Plate 3, fig. 2d). In some cases the light layer at the egg surface and the colloid of the perivitelline space pass imperceptibly one into the other (Plate 3, fig. 2e). In other cases (Plate 3, fig. 2c) they are distinguishable, but there exists a direct connexion between the light surface layer and the colloid of the perivitelline space. In this picture it is difficult to determine the border between the egg surface and the colloid of the perivitelline space. By 45–50 minutes (18° C.) the volume of the perivitelline space in the sturgeon reaches almost its maximal size; by this time the extrusion of the colloid from the egg ceases as well.

Simultaneously with the rise of hydrophilic colloid globules to the surface layer, the endoplasm of the animal region acquires a more uniform structure (Plate 2, figs. 1d, e); karyoplasmic lacunae which pierce it disappear. By the stages of the first and second cleavage divisions the hydrophilic colloid also disappears from the surface layer of the egg (Plate 2, fig. 1f; Plate 3, fig. le). The egg surface acquires a smooth appearance. In the process of cleavage hydrophilic substances that were secreted at previous stages into the peri vitelline space are seen in the crevices between the blastomeres and, later on, in the blastocoel (see also Zotin, 1961). The volume of the perivitelline space over the animal portion of the embryo diminishes at this time, as can be seen both in section and by vital observation in the vertical chamber. Some of the aniline blue stained substance does not, however, pass to the perivitelline space, and instead submerges together with the pronuclei from the egg surface inside the animal part of the egg.

During the period of cleavage a small amount of substance stained by aniline blue is also found in the pathways of the presumptive cleavage furrows. Prior to the appearance of a furrow on the egg surface its pathway is prepared in the cytoplasm of the animal region. This pathway consists of a chain of optically empty vacuoles surrounded by single pigment granules (Plate 4, fig. 1) and terminates in a colloid aggregation stained by aniline blue (Plate 4, fig. 2). Later on. the surface egg layer with pigment granules deepens in the pathway prepared by the vacuoles and forms adjacent surfaces. The vacuoles in the pathways of the presumptive cleavage furrow in sturgeon eggs were described for the first time by Peltzam (1886) and then by Ginsburg (1959).

The hydrophilic colloid secreted from the egg into the perivitelline space differs from the material of the cortical granules not only in its origin but also in its chemical composition. Unlike the cortical granules, it does not contain mucopolysaccharides: the PAS-positive reaction of the light layer (Plate 2, fig. 3d) is due to the presence of glycogen granules and can be eliminated by a preliminary diastase treatment of the section (Plate 2, fig. 3b). Thanks to this, it can be distinguished from the substances of discharged cortical granules. Apart from glycogen, traces of RNA and protein can be revealed in the hydrophilic colloid. Elective staining with aniline blue facilitates their detection but it does not reveal their chemical nature, though there are some indications that aminosaccharides can be revealed by this method (Monné & Harde, 1951a; Kusa, 1956). When stained with aniline blue, the hydrophilic colloid of the light layer shows a different shade of coloration from the cortical granule material, which also facilitates their distinction when outside the egg (Plate 2, fig. 3c).

Despite the differences described in cortical granules and the hydrophilic colloid, their secretion from the egg represents consequent and interdependent processes: the cortical reaction, accompanied by the extrusion of cortical granules, stimulates the rise of hydrophilic colloid globules from the endoplasm to the egg surface. If the cortical reaction is defective and is not accompanied by the extrusion of granules, the hydrophilic colloid cannot pass to the perivitelline space, though it accumulates in the surface layer of the egg under the layer of cortical granules (Plate 1, fig. 8). (A small amount of the colloid under the membrane, as well as the separation of the membranes from the egg surface in this case (Plate 1, fig. 8), seem to be artefacts.) It can be clearly seen in this figure that the cortical granule material and hydrophilic colloid of the light surface layer exist simultaneously and independently of each other.

Comparison of the results of microscopic investigation with the data of vital observation in a vertical chamber shows that, while the rise of hydrophilic colloid globules to the egg surface starts in the first few minutes after activation and proceeds for 30–40 minutes, the formation of the perivitelline space visible under the microscope can be observed only after 15–20 minutes; thereafter during the subsequent 20–25 minutes it rapidly extends. Water entry into the egg (leaving apart the water taken up by the membranes), according to Zotin’s data (1955), starts also some minutes after activation (in the experiment shown in Text-fig. 3 after 12 minutes, i.e. during the period of onset of the formation of a widened perivitelline space or somewhat earlier). The hydrophilic colloid accumulating in the surface egg layer seems to increase somewhat in water content and to pass to the perivitelline space, where it swells considerably.

The change in egg-membrane properties starting soon after the expulsion of cortical granules seems, however, not to be causally related to the latter.

In experiments carried out in collaboration with A. I. Zotin (see also Zotin, 1961), sevruga eggs were placed in water with the sperm for 10, 60, and 120 seconds and thereafter kept in 0-1 N NaCl. (Some of the eggs were kept in water during the whole experiment up to the 4-cell stage, while others were placed in water after varying periods of time.)

The membranes of the eggs kept in water with the sperm for 60 seconds and more swelled normally in NaCl solution, became sticky, and hardened. Those of the eggs placed in 0·1 N NaCl solution 10 seconds after insemination did not change, but, after replacement in water, they swelled, became sticky, and hardened even at the stages of 2 and 4 blastomeres. Thus, 0·1 N NaCl solution does not prevent changes in the membranes themselves, but it blocks some process in the eggs which proceeds over the period from 10 to 60 seconds after insemination, and is necessary for the membrane stickiness and hardening. In order to elucidate the relation of this action of NaCl solution to the extrusion of cortical granules, the eggs were fixed at the moment of placing them in NaCl solution and later, when they were taken from NaCl solution, with both unchanged and normally hardened membranes. It turned out that in the eggs placed into NaCl solution 10 seconds after their insemination in water, the membranes of which did not become sticky and did not harden, the cortical reaction proceeded normally, i.e. cortical granules were extruded, the fringe on the inner surface of the membrane was formed, and the hydrophilic colloid secreted. Since the eggs placed in 0·1 N NaCl solution 60 seconds after insemination hardened under the same conditions, it can be assumed that the stickiness and hardness of membranes in sturgeons was related to substances other than cortical granules and hydrophilic colloid, the expulsion of which from the egg followed that of cortical granules and could be reversibly blocked by 0·1 N NaCl solution.

The transition of the nucleus to the active state follows the extrusion of cortical granules. Judging by the analogy with sea urchins and teleost fishes, it can be expected that the latter is, in its turn, preceded by invisible cortical changes, the ‘fertilization wave’ or ‘fertilization impulse’ (Yamamoto, 1944, 1956; Sugiyama, 1956; Runnström, 1956; Rothschild, 1956; Allen, 1958).

Since egg activation is possible without an extrusion of cortical granules (Motomura, 1934, 1941; Thomopoulos, 1953a; Kusa, 1953a; Ishikawa, 1954; and many others—see also Rothschild, 1958; Allen, 1958) there are grounds for believing that stimulation of the nucleus is due to the action of the fertilization impulse and, therefore, that the transition of the nucleus to the active state can be used as a criterion for a study of the fertilization impulse.

On fertilization in Acipenserid fishes a spermatozoon enters the egg through the micropylar canal in the direct vicinity of the female nucleus (Plate 1, fig. 1) and it is just here that the first visible changes in the layer of cortical granules arise in the first seconds after the fertilization impulse. In order to judge the velocity of spread of the fertilization impulse over the egg surface and its relation to the velocity of spread of visible cortical changes (extrusion of cortical granules), experimental conditions must be created in which the fertilization impulse arises at a different distance from the nucleus. With this aim the eggs of two sturgeons and one white sturgeon were activated by pricking with a fine glass needle: some of them in the border of the animal region, others in the vegetative pole. Since the expulsion of cortical granules, having started from the site of pricking, spreads over the cortical egg layer during some minutes, on pricking in the vegetative pole the process of cortical granule discharge reaches the region of the nucleus later than on pricking in the animal region. As to the fertilization impulse, if it spreads in sturgeon eggs with a greater velocity than the cortical granule discharge (as is suggested by Rothschild & Swann, 1949, for sea urchins), the difference in the pricking site would not bring about a considerable difference in the time when the impulse reaches the zone of location of the nucleus.

The eggs of two sturgeons and of one white sturgeon pricked in the animal or the vegetative regions were kept at an even temperature (sturgeon eggs at 14·9—15·2° and 16·3–16·5° C., white sturgeon eggs at 21·5– 21·6° C.). In order to follow the changes in the nuclei in detail, the eggs of one sturgeon were fixed each minute for 10 minutes and then after 15 minutes; the eggs of another sturgeon were fixed each minute during the period from 8 to 30 minutes after pricking; the eggs of the white sturgeon were fixed each minute for 20 minutes. Ten or 4 sturgeon eggs and 5 white sturgeon eggs were pricked at each time. Sections were stained by iron haematoxylin. Two-hundred and sixty nuclear figures were investigated. Mitotic patterns were drawn under the microscope by means of a camera lucida; distances between sister chromosomes and the length of spindles were measured in the drawings.

Apart from this, additional fixations were carried out to find the time required for the cortical reaction to reach the site of the nucleus after pricking in various areas. Sections were stained by the Heidenhain azan method.

The curves presented in Text-figs. 4 and 5 show changes in the distance between sister chromosomes (1) and in the spindle length of the second maturation division (2) in sturgeon (Text-fig. 4) and white sturgeon (Text-fig. 5) eggs pricked at the site of the animal (a) and vegetative (v) pole during 30 (sturgeon) and 20 (white sturgeon) minutes after pricking. Each point represents an average for 3– 4 eggs.

The dynamics of the process of nuclear activation and of the completion of the second maturation division is the same on pricking the eggs either in the animal or in the vegetative region. The prolonged period is occupied by a very slow separation of sister chromosomes, then follows a period during which the chromosomes rapidly diverge to the spindle poles and, simultaneously, the spindle itself lengthens. After reaching its maximal length the spindle shortens, and then begins the separation of the polar body.

When comparing the stages of nuclear division in white sturgeon and sturgeon eggs pricked at different places and fixed at the same time interval after pricking, it turns out that at any time interval, the eggs pricked in the vegetative pole lag behind the eggs pricked in the animal region (Plate 4, fig 3, A, V). The differences are most demonstrative in the period of rapid chromosome divergence, when the spindle considerably lengthens during a minute, while the chromosomes cover a great distance (Text-fig. 4, Plate 4, fig. 3). The time of retardation of nuclei in the eggs activated by pricking in the vegetative region is close to the time required for the cortical reaction to spread from the vegetative egg pole to the border of the animal region. In sturgeon it took minutes, in white sturgeon minutes. In some white sturgeon eggs the delay in nuclear division on pricking in the vegetative region was much greater,¹ but it corresponded to a slow spread of the cortical reaction in some eggs. No considerable differences could be found in the stages of nuclear development in white sturgeon eggs at the same time after pricking in the animal region.

A retardation in the stage of nuclear division on pricking in the vegetative region was also found in the experiment on the second sturgeon, but here it was less demonstrative, the eggs being fixed early.

According to Allen’s (1958) classification Acipenserid fishes have to be included in the group of animals with a labile cortex. The first phase of visible cortical change in Acipenserid eggs is related to cortical granule expulsion and has many features in common with cortical changes occurring in the eggs of sea urchins, Saccoglossus, teleost fishes, amphibians, and others. The cortical granules of Acipenserid fishes contain acid mucopolysaccharides, as do the cortical granules of sea urchins (Runnström, Monné, & Wicklund, 1946; Monné & Slautterback, 1950, Monné & Harde, 1951a; Nakano & Ohashi, 1954), lamprey (Kusa, 1957b), the cortical alveoli of many teleost species (Kusa, 1953,a, b, 1954, 1956, 1958b; Thomopoulos, 1953,a, b;Devillers, Thomopoulos, & Colas, 1953; Aketa, 1954; K. Yamamoto, 1956; Nakano, 1956; Sakun, 1960; also see Rothschild, 1958), and the granules of amphibians (Wartenberg, 1956; Rosenbaum, 1958). As in sea urchins (Vasseur, 1948; Vasseur & Immers, 1949; Monné & Härde, 1951a; Nakano & Ohashi, 1954), they are, in their chemical composition, similar to egg membranes. No morphologically manifested lipid membrane of cortical granules can be revealed in Acipenserids by histological methods; such a membrane is described in sea urchins (Runnström, 1956). In most teleost species a lipid membrane is not found (see Kusa, 1956), though their alveoli undergo destruction under the influence of lipoid solvents (T. Yamamoto, 1951; Kusa, 1958b).

Unlike the case of sea urchins (Allen & Griffin, 1958), changes in the layer of cortical granules in Acipenserids begin in the first seconds after the establishment of the contact between the spermatozoon and the egg cytoplasm (or after pricking), that is, after a very short latent period (cf. also Ginsburg, 1961).

The mode of extrusion of the cortical granular material in Acipenserids differs from the mode of alveolar breakdown in teleosts (Kusa, 1953a, 1956, 1958b) and from that of cortical granule discharge in sea urchins described by Endo (1952). It can be suggested, however, that changes in the teleost alveoli and in the ectoplasm of the cortical granule layer of Acipenserids are based on common processes. In this connexion it is interesting to note that narcotics inhibit cortical changes in the eggs of sea urchins (Hagstrom & Allen, 1956; Sugiyama, 1956), teleosts (T. Yamamoto, 1956), and Acipenserid fishes (Ginsburg, 1961) in the same way.

After extrusion, the material of the cortical granules adheres to the inner yolk membrane as in sea urchins (Motomura, 1941; Runnström, 1948; Endo, 1952), Saccoglossus (Colwin & Colwin, 1954), and amphibians (Motomura, 1952; Kemp, 1956). As in other animals, the egg of the Acipenserids, owing to the cortical granule discharge, is freed within the membranes and turns with its animal pole upwards under the action of gravity (Dettlaff & Ginsburg, 1954).

Changes in sturgeon eggs following the extrusion of cortical granules essentially differ from those described in echinoderm and teleost eggs. While in teleost fishes and sea urchins the destruction of cortical alveoli and granules is followed by the formation of the quickly widening perivitelline space (T. Yamamoto, 1939; Motomura, 1941; Runnström, 1948, 1952; Devillers, Thomopoulos, & Colas, 1953; Kanoh, 1953; Kusa, 1953; and others—see also Runnström, 1952; Allen, 1958; Kusa, 1956,1958; Rothschild, 1958), in Acipenserids the formation of the latter starts considerably later and is related to the secretion of the hydrophilic colloid globules not described in teleosts and sea urchins. The movement of these globules towards the egg surface seems, however, to represent a result of the same activating effect of the cortical reaction on the endoplasm as does the pigment migration in sea-urchin eggs (Allen & Rowe, 1958; cf. Allen, 1958). It is of interest that in echinoderms (Runnström, 1928; Monné & Harde, 1951b), as well as in Acipenserids homogeneity of the cytoplasm increases in the process of egg activation. In the cytoplasm of unripe sea-urchin eggs vacuoles arising in the process of maturation have been described; they stain with basic dyes. These vacuoles disappear in the course of fertilization as a result of changes in the physiological state of the cytoplasm (Monné & Härde, 1951b); their expulsion into the perivitelline space was not observed.

There is no general agreement in the literature about the problem of the origin of the colloid involved in the formation of the perivitelline space (cf. Rothschild, 1956; Allen, 1958). It seems that in different animals it appears in a different manner. In teleost fishes it is secreted after the destruction of cortical alveoli (T. Yamamoto, 1939); most of the workers believe this colloid to be contained in the alveoli themselves, which show a high osmotic activity upon isolation (Kusa, 1957a, 1958a). As to sea urchins, some authors (Runnström, Monné, & Wicklund, 1946; Runnström, 1952; Moore, 1951; Endo, 1952) think that the granules cannot form an osmotically active colloid, while others (Parpart & Laris, 1955; Afzelius, 1956; Parpart & Cagle, 1957) assume that a portion of the extruded material of the granules, while polymerizing, forms the hyaline layer and the colloid of the perivitelline space. Their opinion is shared by Allen (1958).

At the same time data are available in the literature on the extrusion from the egg of other substances, along with cortical granules, during the process of activation: in sea urchins a luminous substance (Moore, 1951) and a transparent surface layer (Hiramoto, 1954); in Saccoglossus transparent granules and a jelly-like mass (Colwin & Colwin, 1954,¹); in the brook lamprey fine particles (Kusa & Ootake, 1959). There are some old (O. Hertwig, 1877; Bialaszewicz, 1912) and recent (Wintrebert, 1933) data on the excretion of a colloid from the animal portion of amphibian eggs. In amphibians, as well as in Acipenserids, the release of the egg and its rotation within the membranes precedes the formation of the perivitelline space (Ancel & Vintemberger, 1948). According to unpublished data of the author, in Rana temporaria and Ambystoma mexicanum the rotation of the egg is connected with the extrusion of cortical granules (described in amphibians by Motomura (1952) and Kemp (1956)), while the formation of the widened perivitelline space, as in Acipenserids, is related to the colloid expulsion from the animal egg region.

Formations very similar to the globules of the hydrophilic colloid in sturgeon eggs were described in the eggs of Parascaris equorum (van Beneden, 1883; Fauré-Fremiet, Ebel, & Colas, 1954) under the name ‘sphères hyalines’. Like the hydrophilic globules, they are electively stained with aniline blue, arise during oocyte maturation in the perinuclear zone, and migrate to the egg surface after fertilization. Here they fuse and form a continuous layer which widens and disappears in the perivitelline space thus formed. Hyaline spheres contain proteins and lack polysaccharides; they are in a peculiar physical state, do not mix with the cytoplasm, and are soluble in water. The data presented thus show that the extrusion of the globules of hydrophilic colloid in egg activation is not confined to sturgeons.

The synthesis of the hydrophilic colloid (whose chemical nature is not clear) appears to proceed in sturgeons in the oocyte nucleus. Since this nucleus shows considerable hydrophily, though it is likely to proceed in the lacunae of the karyoplasm after it has passed into the cytoplasm (this problem requires further investigation). In the process of activation the hydrophilic colloid moves towards the membranes and takes part in the formation of the perivitelline space, coming to lie between blastomeres at cleavage stages and, it seems, participating in the formation of the blastocoel (cf. also Zotin, 1961) as well as in that of other cavities of the embryo, since, according to the data of Zotin & Krumin (1959), the colloid from the blastocoel moves during gastrulation to the gastrocoel and, later, into the lumen of the gut.

The passage of some of the substances staining like the karyoplasm with aniline blue along the pathways of the presumptive cleavage furrow, and the presence of these substances at the termination of a chain of vacuoles preparing the plane of blastomere separation, suggests that the hydrophilic colloid may play the same separating role in the process of egg cleavage as it plays in the formation of the perivitelline space and the cavities.

Apart from cortical granules and the hydrophilic colloid, there is a substance (or substances) extruded from the eggs following the cortical granule discharge. They bring about swelling, appearance of stickiness, and hardening of the egg membranes (cf. also Zotin, 1961) and differ both from the substance of granules and from the hydrophilic colloid. It seems that they correspond to the hardening factor in sea-urchin eggs (Motomura, 1950, 1954, Runnström, 1952) and to the hardening enzyme in the eggs of Salmonid fishes (Zotin, 1958).

As to the data on the stimulation of the female nucleus and the velocity of the spread of the fertilization impulse inducing this stimulation in sturgeon eggs, there are no similar data for other animals in the literature. The fact that the delay of nuclear division of eggs pricked in the vegetative egg region, as compared with eggs pricked in the animal region, is close to the time required for the spread of the cortical reaction from one site of pricking to the other, speaks against the suggestion of Rothschild & Swann (1949) that there are considerable differences in the velocity of the spread of the fertilization impulse and the extrusion of cortical granules. Ginsburg (1960, 1961) failed to find in sturgeon eggs a rapidly spreading fertilization impulse even when judging by the sign of its hypothetical action (Rothschild & Swann, 1949, 1952) on the block to polyspermy. It is reasonable to conclude that the impulse bringing about the transition of the nucleus to the active state spreads in the cortical egg layer at a rate similar to the velocity of the spread of changes in cortical granules.

The action of cortical changes on the egg-cleavage process is not exhausted by the stimulation of the resting female nucleus to develop; in sturgeons, as in sea urchins (Allen, 1954, Allen & Hagström, 1955), the cortical reaction causes an effect upon the endoplasm without which normal movement of the cleavage nuclei is impossible: a portion of the egg in which cortical changes are lacking does not undergo cleavage either in the sturgeon or in the sea urchin.¹

The last question to be considered deals with the behaviour of acid mucopolysaccharides of the cortical structures in sturgeon eggs in the process of activation. This problem is of some interest in connexion with the widely known theory of Runnström (1949, 1952) on the inhibitory function of acid mucopolysaccharides in unfertilized sea-urchin eggs. If the facts underlying this theory could be demonstrated on another object, it would be of great interest.

The PAS-iodine method does not reveal the presence of diffusely scattered acid mucopolysaccharides in the cortical layer of ripe unfertilized eggs of sturgeons. After activation, during the extrusion of cortical granules, a diffuse staining of the cytoplasmic columns appears, as in sea urchins (Monné & Slautterback, 1950; Immers, 1956; Runnström & Immers, 1956), but in sturgeons it is due first of all to the disruption of cortical granules inside the cortical layer. The protoplasmic mass containing these polysaccharides passes later to the membranes. The surface light layer, which arises in sturgeon eggs after extrusion of cortical granules, either does not contain acid mucopolysaccharides at all, or contains but a very small amount of them. Thus, the localization and changes in acid mucopolysaccharides of the cortical layer revealed by the PAS-iodine method in sea-urchin and sturgeon eggs are not identical. The results of vital Na-periodate treatment of the Acipenserid are also different from those in sea urchins (Runnström & Kriszat, 1950): it does not activate sturgeon eggs, causing only a depolymerization of the membranes, and it seems not to penetrate into the egg itself. Differences in the permeability of sea-urchin and sturgeon eggs are revealed by the action of other substances too. (For example 1 μ urea solution, though it does activate sturgeon eggs, does not dissolve the material of cortical granules; neutral ATP solution in river-water does not accelerate cortical reaction, and heparin solution does not affect mitosis.) Thus the data obtained on sturgeons do not provide additional convincing facts in favour of this theory, though they do not contradict it.

To compare changes upon fertilization and artificial activation of sturgeon eggs with those in sea-urchin eggs, the scheme put forward by Allen (1958) for echinoderms (Text-fig. 6) and the same scheme modified for sturgeons (Textfig. 7) are presented here. Along with some similar features, essential differences can be seen in them which have been discussed above. Further investigations must show which of these differences are essential and which are a result of the incompleteness of our present knowledge.

1. The paper describes the cortical structures of the Acipenserid egg and their changes during fertilization and artificial activation by pricking, and the relationship between fertilization impulse, cortical granule expulsion, formation of perivitelline space, membrane changes, and nuclear stimulation. The eggs of Acipenser güldenstadti colchicus V. Marti, A. stellatus Pall., and Huso huso L. were studied.

2. The fertilization impulse bringing about nuclear stimulation and the disintegration and discharge of cortical granules spreads in the cortical layer at a velocity close to that of the spread of visible changes in the cortical granule layer.

3. Changes in the layer of cortical granules begin 1-2 seconds after the origin of the fertilization impulse and spread in a wave-like fashion over the egg surface, from the place of pricking (or, in the case of fertilization, from the micropylar canal region) to the opposite egg pole (in sturgeon this process takes some 3 minutes at 18° C.).

4. The extrusion of cortical granules determines the first separation and rotation of the egg within its membranes and makes hydrophilic colloid globules rise to the egg surface and become extruded; at the time of extrusion of the cortical granules a substance (or substances?) is released which affects the swelling, stickiness, and hardening of the egg membranes.

5. Formation of the widened perivitelline space in sturgeons, unlike teleosts and sea urchins, does not directly follow the extrusion of cortical granules, and is related to the swelling of the colloid excreted from the animal region of the egg which differs from the cortical granule material. This colloid, unlike the cortical granules, does not contain sulphated acid mucopolysaccharides, being related in its origin to the lacunae of the karyoplasm arising in the oocyte cytoplasm during the maturation period. At cleavage stages this colloid enters the spaces between the blastomeres and participates in blastocoel formation.

6. The localization and fate of sulphated acid polysaccharides of the egg cortical layer during the process of activation in sturgeons differs from that in sea urchins. The problem is discussed in relation to Runnström’s theory of the inhibiting action of acid mucopolysaccharides in unfertilized eggs.

7. The dynamics of the process of completion of the second maturation division is established : a prolonged period is occupied by very slow separation of sister chromosomes, then follows a period of the same duration in which the chromosomes rapidly diverge to the spindle poles and, simultaneously, the spindle itself lengthens. After reaching its maximal length, the spindle shortens and then the separation of the polar body begins.

The author is greatly indebted to Drs. A. S. Ginsburg and A. I. Zotin, whose close contact was a great help in the study of various problems of Acipenserid fertilization. Grateful acknowledgement is made to Dr. G. M. Ignatieva and Prof. G. V. Lopashov for critical discussion of the manuscript and to Dr. R. I. Tatarskaya for consultation about and participation in hyaluronidase preparation, as well as to Miss S. E. Golossovskaya, Mrs. L. A. Filatova, and Miss R. V. Pagnaeva for their technical assistance.

Key: C.G., cortical granules; F.M., funnel of maturation; G.H.C., globules of hydrophilic colloid; G.M., granular material extruded from the egg (inner portion of the ‘fertilization membrane’); J.M., jelly membrane; L.C. lacunae of caryoplasm; L.L., light layer; M.C., micropylar canal; N, female nucleus; P.C., protoplasmic columns; P.G., pigment granules; P.S.C., perivitelline space colloid; v, vacuoles in the cortical layer; Z.R.I., zona radiata interna; Z.R.E. zona radiata externa.