4°3
CHANGES AT THE SURFACE OF NEREIS LIMBAT A
EGGS AFTER INSEMINATION
BY ALEX B. NOVIKOFF
Biology Department, Brooklyn College,1 Department of Zoology,
Columbia University, and the Marine Biological Laboratory,
Wood's Hole
(Received 13 December 1938)
(With Two Text-figures)
ONE of the classic descriptions of the insemination process is that of F. R. Lillie
(1911) in the egg of the annelid, Nereis. According to Lillie, there exists beneath
the vitelline membrane of the unfertilized Nereis egg a wide cortical layer. He
describes it as "a coarsely alveolar layer with homogeneous alveolar contents. The
walls of the alveoli are continuous internally with the protoplasm of egg, and unite
externally to form a protoplasmic layer applied to the vitelline membrane." Within
2 or 3 min. after the attachment of the spermatozoon to the egg, an outpouring of
jelly from the cortical alveoli occurs, so that in about 15 min. this layer is represented only by the perivitelline space and the delicate walls of the original alveoli
crossing this space to the vitelline membrane. A fertilization cone rises from the egg
surface, moves gradually across the perivitelline space, and comes in contact with
the membrane beneath the sperm. After the retraction of the cone, the sperm head
passes through the membrane into the egg. There is no discussion of the nature of
the alveolar walls, nor is there any account of how these walls disappear even though
they are not visible in many of the figures.
Chambers (1933) has offered an alternative interpretation of the cortical structure
of the unfertilized Nereis egg and, consequently, another interpretation of the
surface changes at insemination. He suggested that the jelly is not within the egg
as described by Lillie, but rather it is extra-protoplasmic. It lies among numerous
protoplasmic strands or filaments which extend the egg surface to the vitelline
membrane. At fertilization the spermatozoon becomes attached to one or several
of these strands. This is soon followed by the extrusion of the jelly and a withdrawal of all protoplasmic strands except those connected with the sperm. The
latter strands broaden and coalesce to form the entrance cone. The cone is then
withdrawn and the head of the sperm is pulled slowly through the vitelline (fertilization) membrane into the egg interior.
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jEB-xviiv
Contribution no. 26.
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404
ALEX B. NOVIKOFF
There are certain similarities between Chambers's description of the unfertilized egg of Nereis and the description of the unfertilized Sabellaria egg by the
writer (Novikoff, 1939). In both eggs, the vitelline membrane is separated from the
egg surface by a considerable distance—about 7/1 in Nereis and about 12 \i in
Sabellaria. The distance is traversed by numerous radiating filaments extending
from egg to membrane. In both cases, a jelly is present among these filaments. A
striking similarity exists between Chambers's account of the formation of the
entrance cone through a fusion of several filaments and the subsequent withdrawal
of the remaining filaments, and the writer's description of the same process in
Sabellaria eggs, if fertilized immediately after shedding.
In the course of an investigation of the suggested similarities in the surface
changes in the two eggs, the following observations were recorded.1 All observations were made on living eggs of Nereis limbata, obtained in the manner described
by Just (1928). The temperature of the sea water varied from 22 to 280 C.
THE UNFERTILIZED OVUM
Examination with high powers (440 x , 950 x) fails to show any constant structure of the cortical region between the vitelline membrane and the yolk granules.
This region contains a great many granules (Fig. 2 A). The granules are sometimes
arranged clearly in close radiating lines, which appear like the radiating lines in
Chambers's diagram (1933, p. 138). But they are quite unlike the protoplasmic
filaments of Sabellaria or of fertilized Nereis eggs.
With the use of an isotonic NaCl solution, brought to pH 9-6 by the addition
of NajCOa, the vitelline membrane may be removed from unfertilized Nereis eggs.
The eggs are washed quickly in the solution and then transferred to fresh solution.
The membrane swells from the surface and, with gentle agitation, it breaks at one
point and the naked egg rolls out. A great many eggs do not survive this treatment,
but those that do generally show no change in the structure of the cortical layer;
the granular arrangement is preserved. Rolling several eggs together or immersing
them in a suspension of China ink shows that there is no jelly surrounding the egg
surface. When sperm are added to such denuded eggs an outpouring of jelly occurs
(Fig. iB). It is evident, therefore, that the removal of the vitelline membrane
without insemination causes no exudation of jelly. Such an exudation of jelly
would be expected if, as Chambers suggests, the jelly were already extra-ovular,
and were lying packed between-radiating strands of cytoplasm beneath the vitelline
membrane. It is, therefore, more plausible to assume that the precursor of the
jelly resides in the cortical zone of cytoplasm and is released from the egg during
insemination.
One chance observation on an unfertilized egg is of importance for the present
discussion. In this egg, the germinal vesicle had been pushed from its usual
position to one side of the egg where it forced the vitelline membrane from the egg
1
The writer wishes to express his gratitude to Profs. L. G. Barth and Robert Chambers for their
generous help in the execution of the investigation and in the preparation of the manuscript.
Changes at Surface of Eggs after Insemination
405
surface. Fig. iA shows the numerous short protoplasmic strands, apparently torn
at their ends, which project from the cortical zone towards the membrane. This
observation indicates that among the granules of the normal unfertilized ovum there
is a hyaline protoplasm which adheres in spots to the vitelline membrane. This
would account for the development of protoplasmic strands when the egg surface
recedes from the membrane on fertilization.
v.m.
I.
Fig. 1. A. Camera lucida outline drawing of a portion of an unfertilized ovum of Nereis limbata. g.v.
germinal vesicle; c.l. cortical layer; v.m. vitelline membrane. B. Photograph of a denuded ovum of
Nereis limbata following insemination. The jelly surrounding the egg is indicated by the absence of
spermatozoa'.
INSEMINATION
Immediately after being mixed with the eggs,.the spermatozoa attach themselves
to the vitelline membrane. Within 2 min. the jelly pours from the egg. This is
followed by a decrease in the width of the cortical layer and its separation from the
vitelline membrane with the formation of the perivitelline space (Fig. 2B, C). The
fate of the cortical granules as the cortical zone disappears, could not be ascertained.
The formation of the perivitelline space is due to a retraction of the egg surface
from the vitelline membrane, although a slight elevation of the membrane itself
cannot be ruled out as a factor. As the separation occurs, numerous granule-free
strands or filaments are pulled out from the cortical protoplasm. As these filaments
lengthen, through the further withdrawal of the egg, the egg surface becomes
irregular, extending out at the base of each filament. The surface assumes an even
contour only when the ovum withdraws further and when the granular cortical
layer becomes much reduced and the filaments longer (Fig. 2D). The reduction in
the width of the granular region leaves the yolk granules close to the periphery so
that for the first time they are clearly visible from the surface,
27-2
406
ALEX B. NOVIKOFF
These changes occur over the entire surface except in the neighbourhood of the
attached spermatozoon. There the withdrawal of the egg surface may begin but it
never proceeds very far. At most, there are produced short, stubby protoplasmic
strands, and even these soon lose their identity in the substance of the forming
entrance cone which rises slightly to come in contact with the vitelline membrane
(Fig. 2B-D). The entrance cone becomes relatively larger when the egg surface
adjacent to it withdraws from the membrane. As the surface withdraws, the long
filaments are pulled out. But the egg in the region of the cone remains close to the
vitelline membrane and no such filaments are produced there. Within 10 or 15 min.
A
Fig. 2. Portion of the egg surface of Nereis limbata, highly magnified. A, unfertilized ovum; B-H,
after insemination.
after insemination, the entrance cone begins to flatten out, drawing with it the head
of the spermatozoon. Simultaneously with the flattening of the cone, all of the
remaining egg surface rises to approach the vitelline membrane. The behaviour of
the protoplasmic strands at this time is striking. As the perivitelline space decreases
in width, the filaments shorten and become coarser (Fig. 2E-F). When the perivitelline space has been entirely obliterated by the approximation of egg and membrane, both of which usually become irregular at this time, the filaments are no
longer visible (Fig. 2G). In several minutes the egg surface separates again from
the vitelline membrane. This movement is not as uniform as the initial separation
following the exudation of the jelly; extensive areas of the egg flatten out and usually
Changes at Surface of Eggs after Insemination
407
become straight. The perivitelline space is much widened in these regions (Fig. 2H).
As the flattening begins, one can distinguish again the granule-free, hyaline protoplasm. But now, in most cases, it is not seen in the form of filaments, but as stubby
stumps on the egg surface, unattached to the vitelline membrane. In a few minutes,
these stumps are withdrawn into the egg and the ovum possesses the even contour
which it maintains through later development. The flattened regions may soon
become rounded while others flatten out. This movement of the egg surface
everywhere except in. the vicinity of the sperm continues for some time. (See
Hoadley (1934) for a description of the deformations of the egg as a whole.) That
the separation of the filaments from the membrane may be independent of the
movement of the egg towards the membrane is indicated by what occurs in an
occasional egg. When the egg flattens out, new protoplasmic filaments are drawn
out and then these filaments may be seen to lose their contacts with the membrane
and flatten on to the egg surface, while the perivitelline space remains wide.
According to these observations, we must conceive of the strands radiating
across the perivitelline space of the fertilized egg, not as the walls of the emptied
alveoli, as Lillie suggested, but as the deeply indented surface of the protoplasm.
Moreover, it is evident that the strands, developing from the indentation of the
surface, are not identical with the variable cortical radiations in the unfertilized egg.
The cortical protoplasmic strands in the fertilized egg are well anchored to the
vitelline membrane. This can be shown simply by exerting pressure on the fertilized
egg, when the strands have formed, sufficient toflattenthe egg so that the perivitelline
space is entirely obliterated, and by then releasing the pressure. The egg springs back
to its normal position—with intact filaments. This may be repeated time and again on
the same egg, with the same result. That the filaments are capable of further
extension is indicated by what occurs in fertilized eggs which happen to be at the
edge of a cover slip where the sea water is evaporating. The perivitelline space may
be considerably wider than usual and the filaments may be pulled to three or four
times their normal length. They are then much finer and appear very much like the
filaments of the Sabellaria egg.
That the filaments do not come into existence merely through an outpouring
of jelly from cortical alveoli, is shown by adding sperm to denuded eggs. Such eggs
will not form jelly unless they are inseminated. Although a considerable amount of
jelly leaves the egg when inseminated, no signs of filaments are visible (Fig. 1B).
Thus, without the vitelline membrane, filaments cannot form. These filaments-are
produced as the egg surface withdraws from the membrane, only if the contact
between egg and membrane is maintained. This is further borne out by the behaviour,
upon insemination, of the egg drawn in Fig. iA, in which the vitelline membrane had been pushed away from a portion of egg cytoplasm by the displaced
germinal vesicle. After sperm contact, long radiating filaments were formed over
all of the egg surface except where the membrane and protoplasm had been
separated.
408
ALEX B. NOVIKOFF
CONCLUSION
The details of insemination in Nereis limbata which are presented in this paper
have not been described by Lillie or Chambers. The chief feature omitted from these
earlier descriptions is the movement of the egg surface away from the vitelline
membrane when the jelly leaves the cortical layer. The egg protoplasm adheres at
points to the membrane so that numerous protoplasmic filaments are formed which
maintain the connexion between the membrane and egg across the perivitelline
space. These filaments remain for about 15 min. after insemination.
Whereas in Nereis the separation of the egg surface from the vitelline membrane
occurs after insemination, in Sabellaria the separation occurs without insemination,
when the egg comes in contact with sea water. Thus, the protoplasmic filaments are
found in fertilized Nereis eggs but in unfertilized eggs of Sabellaria.
REFERENCES
CHAMBERS, R. (1933). J. exp. Biol. 10, 130.
HOADLEY, L. (1934). Biol. Bull. Woods Hole, 67, 484.
JUST, E. E. (1928). Coll. Net, 3, No. 3, 9.
LILLIE, F. R. (1911). J. Morph. 22, 361.
NOVIKOFF, A. B. (1939). (In the Press.)