the spindle formed from the metamorphosed germinal vesicle projecting into
a protoplasmic prominence of the surface of the egg. Picric acid
preparation (copied from Fol).]
[Illustration: FIG. 7.--Portion of the ovum of Asterias glacialis at the
moment of the detachment of the first polar body and the withdrawal of the
remaining part of the spindle within the ovum. Picric acid preparation
(copied from Fol).]
[Illustration: FIG. 8.--Portion of the ovum of Asterias glacialis, with the
first polar body as it appears when living (copied from Fol).]
[Illustration: FIG. 9.--Portion of the ovum of Asterias glacialis
immediately after the formation of the second polar body. Picric acid
preparation (copied from Fol).]
The spindle, however it be formed, has up to this time been situated with
its axis parallel to the surface of the egg, but not long after the stage
last described a spindle is found with one end projecting into a
protoplasmic prominence which makes its appearance on the surface of the
egg (Fig. 6). Hertwig believes that the spindle simply travels towards the
surface, and while doing so changes the direction of its axis. Fol finds,
however, that this is not the case, but that between the two conditions of
the spindle an intermediate one is found in which a spindle can no longer
be seen in the egg, but its place is taken by a compact rounded body. He
has not been able to arrive at a conclusion as to what meaning is to be
attached to this occurrence. In any case the spindle which projects into
the prominence on the surface of the egg divides it into two parts, one in
the prominence and one in the egg (Fig. 7). The prominence itself with the
enclosed portion of the spindle becomes partially constricted off from the
egg as the first polar body (Fig. 8). The part of the spindle which remains
in the egg becomes directly converted into a second spindle by the
elongation of its fibres without passing through a typical nuclear
condition. A second polar cell next becomes formed in the same manner as
the first (Fig. 9), and the portion of the spindle remaining in the egg
becomes converted into two or three clear vesicles (Fig. 10) which soon
unite to form a single nucleus, the female pronucleus (Fig. 11). The two
polar cells appear to be situated between two membranes, the outer of which
is very delicate and only distinct where it covers the polar cells, while
the inner one is thicker and becomes, after impregnation, more distinct and
then forms what Fol speaks of as the vitelline membrane. It is clear, as
Hertwig has pointed out, that the polar bodies originate by a regular cell
division and have the value of cells.
[Illustration: FIG. 10.--Portion of the ovum of Asterias glacialis after
the formation of the second polar cell, shewing the part of the spindle
remaining in the ovum becoming converted into two clear vesicles. Picric
acid preparation (copied from Fol).]
[Illustration: FIG. 11.--Ovum of Asterias glacialis with the two polar
bodies and the female pronucleus surrounded by radial striæ, as seen in the
living egg (copied from Fol).]
_General conclusions._
Considering how few ova have been adequately investigated with reference to
the behaviour of the germinal vesicle any general conclusions which may at
present be formed are to be regarded as provisional, and I trust that this
will be borne in mind by the reader in perusing the following paragraphs.
There is abundant evidence that at the time of maturation of the egg the
germinal vesicle undergoes peculiar changes, which are, in part at least,
of a retrogressive character. These changes may begin considerably before
the egg has reached the period of maturity, or may not take place till
after it has been laid. They consist in appearance of irregularity and
obscurity in the outline of the germinal vesicle, the absorption of its
membrane, the partial absorption of its contents in the yolk, and the
breaking up and disappearance of the germinal spot. The exact fate of the
single germinal spot, or the numerous spots where they are present, is
still obscure; and the observations of Oellacher on the trout, and to a
certain extent my own on the skate, tend to shew that the membrane of the
germinal vesicle may in some cases be ejected from the egg, but this
conclusion cannot be accepted without further confirmation.
The retrogressive metamorphosis of the germinal vesicle is followed in a
large number of instances by the conversion of what remains into a striated
spindle similar in character to a nucleus previous to division. This
spindle travels to the surface and undergoes division to form the polar
cell or cells in the manner above described. The part which remains in the
egg forms eventually the female pronucleus.
The germinal vesicle has up to the present time only been observed to
undergo the above series of changes in a certain number of instances,
which, however, include examples from several divisions of the
Coelenterata, the Echinodermata, and the Mollusca, and also some of the
Vermes (Nematodes, Hirudinea, Sagitta). It is very possible, not to say
probable, that it is universal in the animal kingdom, but the present state
of our knowledge does not justify us in saying so. It may be that in the
case of the rabbit, and many Nematodes as described by van Beneden and by
Bütschli, we have instances of a different mode of formation of the polar
cells.
The case of Amphibians, as described by Bambeke (2) and Hertwig (12) cannot
so far be brought into conformity with our type, though observations are so
difficult to make with such opaque eggs that not much reliance can be
placed upon the existing statements. In both of these types of possible
exceptions it is fairly clear that, whatever may be the case with reference
to the formation of the polar cells, part of the germinal vesicle remains
behind as the female pronucleus.
There are a large number of types, including the whole of the Rotifera[365]
and Arthropoda, with a few doubtful exceptions, in which the polar cells
cannot as yet be said to have been satisfactorily observed.
Footnote 365: Flemming (6) finds that, in the summer and
probably parthenogenetic eggs of _Lacinularia socialis_, the
germinal vesicle approaches the surface and becomes invisible,
and that subsequently a slight indentation in the outline of
the egg marks the point of its disappearance. In the hollow of
the indentation Flemming believes a polar cell to be situated,
though he has not definitely seen one.
Whatever may be the eventual result of more extended investigation, it is
clear that the formation of polar cells according to our type is a very
constant occurrence. Its importance is also very greatly increased by the
discovery by Strasburger of the existence of an analogous process amongst
plants. Two questions about it obviously present themselves for solution:
(1) What are the conditions of its occurrence with reference to
impregnation? (2) What meaning has it in the development of the ovum or the
embryo?
The answer to the first of these questions is not difficult to find. The
formation of the polar bodies is independent of impregnation, and is the
final act of the normal growth of the ovum. In a few types the polar cells
are formed while the ovum is still in the ovary, as, for instance, in some
species of Echini, Hydra, &c., but, according to our present knowledge, far
more usually after the ovum has been laid. In some of the instances the
budding off of the polar cells precedes, and in others follows
impregnation; but there is no evidence to shew that in the later cases the
process is influenced by the contact with the male element. In Asterias, as
has been shewn by O. Hertwig, the formation of the polar cells may
indifferently either precede or follow impregnation--a fact which affords a
clear demonstration of the independence of the two occurrences.
To the second of the two questions it does not unfortunately seem possible
at present to give an answer which can be regarded as satisfactory.
The retrogressive changes in the membrane of the germinal vesicle which
usher in the formation of the polar bodies may very probably be viewed as a
prelude to a renewed activity of the contents of the vesicle; and are
perhaps rendered the more necessary from the thickness of the membrane
which results from a protracted period of passive growth. This suggestion
does not, however, help us to explain the formation of polar cells by a
process identical with cell division. The ejection of part of the germinal
vesicle in the formation of the polar cells may probably be paralleled by
the ejection of part or the whole of the original nucleus which, if we may
trust the beautiful researches of Bütschli, takes place during conjugation
in Infusoria as a preliminary to the formation of a fresh nucleus. This
comparison is due to Bütschli, and according to it the formation of the
polar bodies would have to be regarded as assisting, in some as yet unknown
way, the process of regeneration of the germinal vesicle. Views analogous
to this are held by Strasburger and Hertwig, who regard the formation of
the polar bodies in the light of a process of excretion or removal of
useless material. Such hypotheses do not unfortunately carry us very far.
I would suggest that in the formation of the polar cells part of the
constituents of the germinal vesicle which are requisite for its functions
as a complete and independent nucleus are removed to make room for the
supply of the necessary parts to it again by the spermatic nucleus (vide p.
541). More light on this, as on other points, may probably be thrown by
further investigations on parthenogenesis and the presence or absence of a
polar cell in eggs which develop parthenogenetically. Curiously enough the
two groups in which parthenogenesis most frequently occurs in the ordinary
course of development (_Arthropoda_ and _Rotifera_) are also those in which
polar cells, with the possible exception mentioned above, of the
parthenogenetic eggs of Lacenularia, are stated to be absent. This curious
coincidence, should it be confirmed, may perhaps be explained on the
hypothesis, I have just suggested, viz. _that a more or less essential part
of the nucleus is removed in the formation of the polar cells; so that in
cases, e.g. Arthropoda and Rotifera, where polar cells are not formed, and
an essential part of the nucleus not therefore removed, parthenogenesis can
much more easily occur than when polar cells are formed_.
That the part removed in the formation of the polar cells is not absolutely
essential, seems at first sight to follow from the fact of parthenogenesis
being possible in instances where impregnation is the normal occurrence.
The genuineness of all the observations on this head is too long a subject
to enter into here[366], but after admitting, as we probably must, that
there are genuine cases of parthenogenesis, it cannot be taken for granted
without more extended observation that the occurrence of development in
these rare instances may not be due to the polar cells not having been
formed as usual, and that when the polar cells are formed the development
without impregnation is less possible.
Footnote 366: The instances quoted by Siebold from Hensen and
Oellacher are not quite satisfactory. In Hensen's case
impregnation would have been possible if we can suppose the
spermatozoa to be capable of passing into the body-cavity
through the open end of the uninjured oviduct; and though
Oellacher's instances are more valuable, yet sufficient care
seems hardly to have been taken, especially when it is not
certain for what length of time spermatozoa may be able to live
in the oviduct. For Oellacher's precautions, vide _Zeit. für
wiss. Zool._ Bd. XXII. p. 202.
The remarkable observations of Professor Greeff (19) on the parthenogenetic
development of the eggs of _Asterias rubens_ tell, however, very strongly
against this explanation. Greeff has found that under normal circumstances
the eggs of this species of starfish will develop without impregnation in
simple sea water. The development is quite regular and normal though much
slower than in the case of impregnated eggs. It is not definitely stated
that polar cells are formed, but there can be no doubt that this is
implied. Professor Greeff's account is so precise and circumstantial that
it is not easy to believe that any error can have crept in; but neither
Hertwig nor Fol have been able to repeat his experiments, and we may be
permitted to wait for further confirmation before absolutely accepting
them.
It is possible that the removal of part of the protoplasm of the egg in the
formation of the polar cells may be a secondary process due to an
attractive influence of the nucleus on the cell protoplasm, such as is
ordinarily observed in cell division.
_Impregnation of the Ovum._
A far greater amount of certainty appears to me to have been attained as to
the effects of impregnation than as to the changes of the germinal vesicle
which precede this, and there appears, moreover, to be a greater uniformity
in the series of resulting phenomena. For convenience I propose to reverse
the order hitherto adopted and to reserve the history of the literature and
my discussion of disputed points till after my general account. Fol's paper
on _Asterias glacialis_, is again my source of information. The part of the
germinal vesicle which remains in the egg, after the formation of the
second polar cell, becomes converted into a number of small vesicles
(Fig. 10), which aggregate themselves into a single clear nucleus which
gradually travels toward the centre of the egg and around which as a centre
the protoplasm becomes radiately striated (Fig. 11). This nucleus is known
as the _female pronucleus_[367]. In _Asterias glacialis_ the most
favourable period for fecundation is about an hour after the formation of
the female pronucleus. If at this time the spermatozoa are allowed to come
in contact with the egg, their heads soon become enveloped in the investing
mucilaginous coat. A prominence, pointing towards the nearest spermatozoon,
now arises from the superficial layer of protoplasm of the egg and grows
till it comes in contact with the spermatozoon (Figs. 12 and 13), Under
normal circumstances the spermatozoon, which meets the prominence, is the
only one concerned in the fertilisation, and it makes its way into the egg
by passing through the prominence. The tail of the spermatozoa, no longer
motile, remains visible for some time after the head has bored its way in,
but its place is soon taken by a pale conical body which is, however,
probably in part a product of the metamorphosis of the tail itself
(Fig. 14). This body vanishes in its turn.
Footnote 367: According to Hertwig's most recent statement a
nucleolus is present in this nucleus.
[Illustration: FIGS. 12 AND 13.--Small portion of the ovum of Asterias
glacialis. The spermatozoa are shewn enveloped in the mucilaginous coat. In
Fig. 12 a prominence is rising from the surface of the egg towards the
nearest spermatozoon; and in Fig. 13 the spermatozoon and prominence have
met. From living ovum (copied from Fol).]
At the moment of contact between the spermatozoon and the egg the outermost
layer of the protoplasm of the latter raises itself as distinct membrane,
which separates from the egg and prevents the entrance of any more
spermatozoa. At the point where the spermatozoon entered a crater-like
opening is left in the membrane (Fig. 14).
[Illustration: FIG. 14.--Portion of the ovum of Asterias glacialis after
the entrance of a spermatozoon into the ovum. It shows the prominence of
the ovum through which the spermatozoon has entered. A vitelline membrane
with a crater-like opening has become distinctly formed. From living ovum
(copied from Fol).]
The head of the spermatozoon when in the egg forms a nucleus for which the
name _male pronucleus_ may be conveniently adopted. It grows in size by
absorbing, it is said, material from the ovum, though this may be doubted,
and around it is formed a clear space free from yolk-spherules. Shortly
after its formation the protoplasm in its neighbourhood assumes a radiate
arrangement (Fig. 15). At whatever point of the egg the spermatozoon may
have entered, it gradually travels towards the female pronucleus. This
latter, around which the protoplasm no longer has a radial arrangement,
remains motionless till it comes in contact with the rays of the male
pronucleus, after which its condition of repose is exchanged for one of
activity, and it rapidly approaches the male pronucleus, and eventually
fuses with it (Fig. 16).
[Illustration: FIG. 15.--Ovum of Asterias glacialis, with male and female
pronucleus and a radial striation of the protoplasm around the former. From
living ovum (copied from Fol).]
[Illustration: FIG. 16.--Three successive stages in the coalescence of the
male and female pronucleus in Asterias glacialis. From the living ovum
(copied from Fol).]
The product of this fusion forms the first segmentation nucleus (Fig. 17),
which soon, however, divides into the two nuclei of the two first
segmentation spheres. While the two pronuclei are approaching one another
the protoplasm of the egg exhibits amoeboid movements.
Of the earlier observations on this subject there need perhaps only be
cited one of E. van Beneden, on the rabbit's ovum, shewing the presence of
two nuclei before the commencement of segmentation. Bütschli was the
earliest to state from observations on _Rhabditis dolichura_ that the first
segmentation nucleus arose from the fusion of two nuclei, and this was
subsequently shewn with greater detail for _Ascaris nigrovenosa_, by
Auerbach (1). Neither of these authors gave at first the correct
interpretation of their results. At a later period Bütschli (5) arrived at
the conclusion that in a large number of instances (_Lymnæus_, _Nephelis_,
_Cucullanus_, &c.), the nucleus in question was formed by the fusion of two
or more nuclei, and Strasburger at first made a similar statement for
_Phallusia_, though he has since withdrawn it. Though Bütschli's statements
depend, as it seems, upon a false interpretation of appearances, he
nevertheless arrived at a correct view with reference to what occurs in
impregnation. Van Beneden (3) described in the rabbit the formation of the
original segmentation nucleus from two nuclei, one peripheral and the other
central, and he gave it as his hypothetical view that the peripheral
nucleus was derived from the spermatic element. It was reserved for Oscar
Hertwig (11) to describe in _Echinus lividus_ the entrance of a
spermatozoon into the egg and the formation from it of the male pronucleus.
[Illustration: FIG. 17.--Ovum of Asterias glacialis, after the coalescence
of the male and female pronucleus (copied from Fol).]
Though there is a general agreement between the most recent observers,
Hertwig, Fol, Selenka, Strasburger, &c., as to the main facts connected
with the entrance of one spermatozoon into the egg, the formation of the
male pronucleus, and its fusion with the female pronucleus, there still
exist differences of detail in the different descriptions which partly, no
doubt, depend upon the difficulties of observation, but partly also upon
the observations not having all been made upon the same species. Hertwig
does not enter into details with reference to the actual entrance of the
spermatozoon into the egg, but in his latest paper points out that
considerable differences may be observed in occurrences which succeed
impregnation, according to the relative period at which this takes place.
When, in Asterias, the impregnation is effected about an hour after the egg
is laid and previously to the formation of the polar cells, the male
pronucleus appears at first to exert but little influence on the
protoplasm, but after the formation of the second polar cell, the radial
striæ around it become very marked, and the pronucleus rapidly grows in
size. When it finally unites with the female pronucleus it is equal in size
to the latter. In the case when the impregnation is deferred for four hours
the male pronucleus never becomes so large as the female pronucleus. With
reference to the effect of the time at which impregnation takes place,
Asterias would seem to serve as a type. Thus in _Hirudinea_, _Mollusca_,
and _Nematodes_ impregnation normally takes place before the formation of
the polar bodies is completed, and the male pronucleus is accordingly as
large as the female. In _Echinus_, on the other hand, where the polar
bodies are formed in the ovary, the male pronucleus is always small.
Selenka, who has investigated the formation of the male pronucleus in
_Toxopneustes variegatus_, differs in certain points from Fol. He finds
that usually, though not always, a single spermatozoon enters the egg, and
that though the entrance may be effected at any part of the surface, it
generally occurs at the point marked by a small prominence where the polar
cell was formed. The spermatozoon first makes its way through the mucous
envelope of the egg, within which it swims about, and then bores with its
head into the polar prominence. The head of the spermatozoon on entering
the egg becomes enveloped by the superficial protoplasm, and travels inward
with its envelope, while the tail remains outside. As Fol has described, a
delicate membrane becomes formed shortly after the entrance of the
spermatozoon. The head continues to make its way by means of rapid
oscillations, till it has traversed about one eighth of the diameter of the
egg, and then suddenly becomes still. The tail in the meantime vanishes,
while the neck swells up and forms the male pronucleus. The junction of the
male and female pronucleus is described by Fol and Selenka in nearly the
same manner.
Giard gives an account of impregnation which is not easily brought into
harmony with that of the other investigators. His observations were made on
_Psammechinus miliaris_. At one point is situated a polar body and usually
at the pole opposite to it a corresponding prominence. The spermatozoa on
gaining access to the egg attach themselves to it and give it a rotatory
movement, but according to Giard none of them penetrate the vitelline
membrane which, though formed at an earlier period, now retires from the
surface of the egg.
Giard believes that the prominence opposite the polar cells serves for the
entrance of the spermatic material, which probably passes in by a process
of diffusion. Thus, though he regards the male pronucleus as a product of
impregnation, he does not believe it to be the head of a spermatozoon.
Both Hertwig and Fol have made observations on the result of the entrance
into the egg of several spermatozoa. Fol finds that when the impregnation
has been too long delayed the vitelline membrane is formed with comparative
slowness and several spermatozoa are thus enabled to penetrate. Each
spermatozoon forms a separate pronucleus with a surrounding sun; and
several male pronuclei usually fuse with the female pronucleus. Each male
pronucleus appears to exercise a repulsive influence on other male
pronuclei, but to be attracted by the female pronucleus. When there are
several male pronuclei the segmentation is irregular and the resulting
larva a monstrosity. These statements of Fol and Hertwig are at first sight
in contradiction with the more recent results of Selenka. In _Toxopneustes
variegatus_ Selenka finds that though impregnation is usually effected by a
single spermatozoon yet that several may be concerned in the act. The
development continues, however, to be normal if three or even four
spermatozoa enter the egg almost simultaneously. Under such circumstances
each spermatozoon forms a separate pronucleus and sun.
It may be noticed that, while the observations of Fol and Hertwig were
admittedly made upon eggs in which the impregnation was delayed till they
no longer displayed their pristine activity, Selenka's were made upon quite
fresh eggs; and it seems not impossible that the pathological symptoms in
the embryos reared by the two former authors may have been due to the
imperfection of the egg and not to the entrance of more than one
spermatozoon. This, of course, is merely a suggestion which requires to be
tested by fresh observations. We have not as yet a sufficient body of
observations to enable us to decide whether impregnation is usually
effected by a single spermatozoon, though in spite of certain conflicting
evidence the balance would seem to incline towards the side of a single
spermatozoon[368].
Footnote 368: The recent researches of Calberla on the
impregnation of the ovum of _Petromyzon Planeri_ support this
conclusion.
The discovery of Hertwig as to the formation of the male pronucleus throws
a flood of light upon impregnation.
The act of impregnation is seen essentially to consist in the fusion of a
male and female nucleus; not only does this appear in the actual fusion of
the two pronuclei, but it is brought into still greater prominence by the
fact that the female pronucleus is a product of the nucleus of a primitive
ovum, and the male pronucleus is the metamorphosed _head_ of the
spermatozoon which is itself developed from the nucleus of a spermatic
cell[369]. The spermatic cells originate from cells (in the case of
Vertebrates at least) identical with the primitive ova, so that the fusion
which takes place is the fusion of morphologically similar parts in the two
sexes.
Footnote 369: This seems the most probable view with reference
to the nature of the head of the spermatozoon, though the point
is not perhaps yet definitely decided.
It must not, however, be forgotten, as Strasburger has pointed out, that
part of the protoplasm of the generative cells of the two sexes also fuse,
viz. the tail of the spermatozoon with the protoplasm of the egg. But there
is no evidence that the former is of importance for the act of
impregnation. The fact that impregnation mainly consists in the union of
two nuclei gives an importance to the nucleus which would probably not have
been accorded to it on other grounds.
Hertwig's discovery is in no way opposed to Mr Darwin's theory of
pangenesis and other similar theories, but does not afford any definite
proof of their accuracy, nor does it in the meantime supply any explanation
of the origin of two sexes or of the reasons for an embryo becoming male or
female.
_Summary._
In what may probably be regarded as a normal case the following series of
events accompanies the maturation and impregnation of an egg:--
(1) Transportation of the germinal vesicle to the surface of the egg.
(2) Absorption of the membrane of the germinal vesicle and metamorphosis of
the germinal spot.
(3) Assumption of a spindle character by the remains of germinal vesicle,
these remains being probably largely formed from the germinal spot.
(4) Entrance of one end of the spindle into a protoplasmic prominence at
the surface of the egg.
(5) Division of the spindle into two halves, one remaining in the egg, the
other in the prominence. The prominence becomes at the same time nearly
constricted off from the egg as a polar cell.
(6) Formation of a second polar cell in same manner as first, part of the
spindle still remaining in the egg.
(7) Conversion of the part of the spindle remaining in the egg after the
formation of the second polar cell into a nucleus--the female pronucleus.
(8) Transportation of the female pronucleus towards the centre of the egg.
(9) Entrance of one spermatozoon into the egg.
(10) Conversion of the head of the spermatozoon into a nucleus--the male
pronucleus.
(11) Appearance of radial striæ round the male pronucleus which gradually
travels towards the female pronucleus.
(12) Fusion of male and female pronuclei to form the first segmentation
nucleus.
_List of important recent Publications on the Maturation and
Impregnation of the Ovum._
1. Auerbach. _Organologische Studien_, Heft 2.
2. Bambeke. "Recherches s. Embryologie des Batraciens." _Bull. de l'Acad.
royale de Belgique_, 2me sér., t. LXI. 1876.
3. E. Van Beneden. "La Maturation de l'OEuf des Mammifères." _Bull. de
l'Acad. royale de Belgique_, 2me sér., t. XL, no. 12, 1875.
4. E. Van Beneden. "Contributions à l'Histoire de la Vésicule Germinative,
&c." _Bull. de l'Acad. royale de Belgique_, 2me sér., t. XLI, no. 1, 1876.
5. Bütschli. _Eizelle, Zelltheilung, und Conjugation der Infusorien._
6. Flemming. "Studien in d. Entwicklungsgeschichte der Najaden." _Sitz. d.
k. Akad. Wien_, B. LXXI. 1875.
7. Fol. "Die erste Entwicklung des Geryonideneies." _Jenaische
Zeitschrift_, Vol. VII.
8. Fol. "Sur le Développement des Pteropodes." _Archives de Zoologie
Expérimentale et Générale_, Vols. IV and V.
9. Fol. "Sur le Commencement de l'Hénogénie." _Archives des Sciences
Physiques et Naturelles_. Genève, 1877.
10. Giard. _Note sur les premiers phénomènes du développement de l'Oursin._
1877.
11. Hertwig, Oscar. "Beit. z. Kenntniss d. Bildung, &c., d. thier. Eies."
_Morphologisches Jahrbuch_, Bd. I.
12. Hertwig, Oscar. Ibid. _Morphologisches Jahrbuch_, Bd. III, Heft. 1.
13. Hertwig, Oscar. "Weitere Beiträge, &c." _Morphologisches Jahrbuch_,
Bd. III, Heft 3.
14. Kleinenberg. _Hydra_. Leipzig, 1872.
15. Oellacher, J. "Beiträge zur Geschichte des Keimbläschens im
Wirbelthiereie." _Archiv f. micr. Anat._, Bd. VIII.
16. Selenka. _Befruchtung u. Theilung des Eies von Toxopneustes variegatus_
(Vorläufige Mittheilung). Erlangen, 1877.
17. Strasburger. _Ueber Zellbildung u. Zelltheilung._ Jena, 1876.
18. Strasburger. _Ueber Befruchtung u. Zelltheilung._ Jena, 1878.
19. R. Greeff. "Ueb. d. Bau u. d. Entwicklung d. Echinodermen." _Sitzun.
der Gesellschaft z. Beförderung d. gesammten Naturwiss. z. Marburg_, No. 5.
1876.
_Postscript_.--Two important memoirs have appeared since this paper was in
type. One of these by Hertwig, _Morphologisches Jahrbuch_, Bd. IV, contains
a full account with illustrations of what was briefly narrated in his
previous paper (13); the other by Calberla, "Der Befruchtungsvorgang beim
Ei von _Petromyzon Planeri_," _Zeit. für wiss. Zool._, Bd. XXX, shews that
the superficial layer of the egg is formed by a coating of protoplasm free
from yolk-spheres, which at one part is continued inwards as a column, and
contains the germinal vesicle. The surface of this column is in contact
with a micropyle in the egg-membrane. Impregnation is effected by the
entrance of the head of a single spermatozoon (the tail remaining outside)
through the micropyle, and then along the column of clear protoplasm to the
female pronucleus.
XII. ON THE STRUCTURE AND DEVELOPMENT OF THE VERTEBRATE OVARY[370].
Footnote 370: From the _Quarterly Journal of Microscopical
Science_, Vol. 18, 1878.
(With Plates 24, 25, 26.)
The present paper records observations on the ovaries of but two types,
viz., Mammalia and Elasmobranchii. The main points dealt with are
three:--1. The relation of the germinal epithelium to the stroma. 2. The
connection between _primitive ova_ in Waldeyer's sense and the permanent
ova. 3. The homologies of the egg membranes.
The second of these points seems to call for special attention after
Semper's discovery that the primitive ova ought really to be regarded as
_primitive sexual cells_, in that they give rise to the generative elements
of both sexes.
THE DEVELOPMENT OF THE ELASMOBRANCH OVARY.
The development of the Elasmobranch ovary has recently formed the subject
of three investigations. The earliest of them, by H. Ludwig, is contained
in his important work, on the 'Formation of the Ovum in the Animal
Kingdom[371].' Ludwig arrives at the conclusion that the ovum and the
follicular epithelium are both derived from the germinal epithelium, and
enters into some detail as to their formation. Schultz[372], without
apparently being acquainted with Ludwig's observations, has come to very
similar results for Torpedo.
Footnote 371: _Arbeiten a. d. zool.-zoot. Institut Würzburg_,
Bd. I.
Footnote 372: _Archiv f. micr. Anat._ Vol. XI.
Semper[373], in his elaborate memoir on the urogenital system of
Elasmobranchii, has added very greatly to our knowledge on this subject. In
a general way he confirms Ludwig's statements, though he shews that the
formation of the ova is somewhat more complicated than Ludwig had imagined.
He more especially lays stress on the existence of nests of ova
(Ureiernester), derived from the division of a single primitive ovum, and
of certain peculiarly modified nuclei, which he compares to spindle nuclei
in the act of division.
Footnote 373: _Arbeiten a. d. zool.-zoot. Institut Würzburg_,
Bd. II.
My own results agree with those of previous investigators, in attributing
to the germinal epithelium the origin both of the follicular epithelium and
ova, but include a number of points which I believe to be new, and,
perhaps, of some little interest; they differ, moreover, in many important
particulars, both as to the structure and development of the ovary, from
the accounts of my predecessors.
The history of the female generative organs may conveniently be treated
under two heads, viz. (1) the history of the ovarian ridge itself, and (2)
the history of the ova situated in it. I propose dealing in the first place
with the ovarian ridge.
_The Ovarian ridge in Scyllium._--At the stage spoken of in my monograph on
Elasmobranch Fishes as stage L, the ovarian ridge has a very small
development, and its maximum height is about 0.1 mm. It exhibits in section
a somewhat rounded form, and is slightly constricted along the line of
attachment. It presents two surfaces, which are respectively outer and
inner, and is formed of a layer of somewhat thickened germinal epithelium
separated by a basement membrane from a central core of stroma. The
epithelium is far thicker on the outer surface than on the inner, and the
primitive ova are entirely confined to the former. The cells of the
germinal epithelium are irregularly scattered around the primitive ova, and
have not the definite arrangement usually characteristic of epithelial
cells. Each of them has a large nucleus, with a deeply staining small
nucleolus, and a very scanty protoplasm. In stage N the ovarian ridge has a
pointed edge and narrower attachment than in stage L. Its greatest height
is about 0.17 mm. There is more stroma, and the basement membrane is more
distinct than before; in other respects no changes worth recording have
taken place. By stage P a distinction is observable between the right and
left ovarian ridges; the right one has, in fact, grown more rapidly than
the left, and the difference in size between the two ridges becomes more
and more conspicuous during the succeeding stages, till the left one ceases
to grow any larger, though it remains for a great part of life as a small
rudiment.
The right ovarian ridge, which will henceforth alone engage our attention,
has grown very considerably. Its height is now about 0.4 mm. It has in
section (vide Pl. 24, fig. 1) a triangular form with constricted base, and
is covered by a flat epithelium, except for an area on the outer surface,
in length co-extensive with the ovarian ridge, and with a maximum breadth
of about 0.25 mm. This area will be spoken of as the ovarian area or
region, since the primitive ova are confined to it. The epithelium covering
it has a maximum thickness of about 0.05 mm., and thins off rather rapidly
on both borders, to become continuous with the general epithelium of the
ovarian ridge. Its cells have the same character as before, and are several
layers deep. Scattered irregularly amongst them are the primitive ova. The
germinal epithelium in the ovarian region is separated by a basement
membrane from the adjacent stroma.
In succeeding stages, till the embryo reaches a length of 7 centimetres, no
very important changes take place. The ovarian region grows somewhat in
breadth, though in this respect different embryos vary considerably. In two
embryos of nearly the same age, the breadth of the ovarian epithelium was
0.3 mm. in the one and 0.35 mm. in the other. In the former of these
embryos, the thickness of the epithelium was slightly greater than in the
latter, viz. 0.09 mm. as compared with 0.08. In both the epithelium was
sharply separated from the subjacent stroma. There were relatively more
epithelial cells in proportion to primitive ova than at the earlier date,
and the individual cells exhibited great variations in shape, some being
oval, some angular, others very elongated, and many of them applied to part
of an ovum and accommodating themselves to its shape. In some of the more
elongated cells very deeply stained nuclei were present, which (in a
favourable light and with high powers) exhibited the spindle modification
of Strasburger with great clearness, and must therefore be regarded as
undergoing division. The ovarian region is at this stage bounded on each
side by a groove.
In an embryo of seven centimetres (Pl. 24, fig. 2) the breadth of the
ovarian epithelium was 0.5, but its height only 0.06 mm. It was still
sharply separated from the subjacent stroma, though a membrane could only
be demonstrated in certain parts. The amount of stroma in the ovarian ridge
varies greatly in different individuals, and no reliance can be placed on
its amount as a test of the age of the embryo. In the base of the ovarian
ridge the cells were closely packed, elsewhere they were still embryonic.
My next stage (Pl. 24, fig. 3, and fig. 4), shortly before the time of the
hatching of the embryo, exhibits in many respects an advance on the
previous one. It is the stage during which a follicular covering derived
from the germinal epithelium is first distinctly formed round the ova, in a
manner which will be more particularly spoken of in the section devoted to
the development of the ovum itself. The breadth of the ovarian region is
0.56 mm., and its greatest height close to the central border, 0.12 mm.--a
great advance on the previous stage, mainly, however, due to the larger
size of the ova.
The ovarian epithelium is still in part separated from the subjacent stroma
by a membrane close to its dorsal and ventral borders, but elsewhere the
separation is not so distinct, it being occasionally difficult within a
cell or so to be sure of the boundary of the epithelium. The want of a
clear line between the stroma and the epithelium is rendered more obvious
by the fact that the surface of the latter is somewhat irregular, owing to
projections formed by specially large ova, into the bays between which are
processes of the stroma. In an ovary about this stage, hardened in osmic
acid, the epithelium stains very differently from the subjacent stroma, and
the line of separation between the two is quite sharp. A figure of the
whole ovarian ridge, shewing the relation between the two parts, is
represented on Pl. 24, fig. 5.
The layer of stroma in immediate contact with the epithelium is very
different from the remainder, and appears to be destined to accompany the
vascular growths into the epithelium, which will appear in the next stage.
The protoplasm of the cells composing it forms a loose reticulum with a
fair number of oval or rounded nuclei, with their long axis for the most
part parallel to the lower surface of the epithelium. It contains, even at
this stage, fully developed vascular channels.
The remainder of the stroma of the ovarian ridge has now acquired a
definite structure, which remains constant through life, and is eminently
characteristic of the genital ridge of both sexes. The bulk of it (Pl. 24,
fig. 3, _str_) consists of closely packed polygonal cells, of about
0.014 mm. with large nuclei of about 0.009. These cells appear to be
supported by a delicate reticulum. The whole tissue is highly vascular,
with the numerous capillaries; the nuclei in the walls of which stand out
in some preparations with great clearness.
In the next oldest ovary, of which I have sections, the breadth of the
ovarian epithelium is 0.7 mm. and its thickness 0.096. The ovary of this
age was preserved in osmic acid, which is the most favourable reagent, so
far as I have seen, for observing the relation of the stroma and
epithelium. On Pl. 24, fig. 6, is represented a transverse section through
the whole breadth of the ovary, slightly magnified to shew the general
relations of the parts, and on Pl. 24, fig. 7, a small portion of a section
more highly magnified. The inner surface of the ovarian epithelium is more
irregular than in the previous stage, and it may be observed that the
subjacent stroma is growing in amongst the ova. From the relation of the
two tissues it is fairly clear that the growth which is taking place is a
definite growth of the stroma into the epithelium, and not a mutual
intergrowth of the two tissues. The ingrowths of the stroma are, moreover,
directed towards individual ova, around which, outside the follicular
epithelium, they form a special vascular investment in the succeeding
stages. They are formed of a reticular tissue with comparatively few
nuclei.
By the next stage, in my series of ovaries of _Scy. canicula_, important
changes have taken place in the constitution of ovarian epithelium. Fig. 8,
Pl. 24, represents a portion of the ovarian epithelium, on the same scale
as figs. 1, 2, 3, &c., and fig. 9 a section through the whole ovarian ridge
slightly magnified. Its breadth is now 1.3 mm., and its thickness 0.3 mm.
The ova have grown very greatly, and it appears to me to be mainly owing to
their growth that the greater thickness of the epithelium is due, as well
as the irregularity of its inner surface (vide fig. 9).
The general relation of the epithelium to the surrounding parts is much the
same as in the earlier stage, but two new features have appeared--(1) The
outermost cells of the ovarian region have more or less clearly arranged
themselves as a kind of epithelial covering for the organ; and (2) the
stroma ingrowths of the previous stage have become definitely vascular, and
have penetrated through all parts of the epithelium.
The external layer of epithelium is by no means a very marked structure,
the character of its cells varies greatly in different regions, and it is
very imperfectly separated from the subjacent layer. I shall speak of it
for convenience as _pseudo-epithelium_.
The greater part of the germinal epithelium forms anastomosing columns,
separated by very thin tracts of stroma. The columns are, in the majority
of instances, continuous with the pseudo-epithelium at the surface, and
contain ova in all stages of development. Many of the cells composing them
naturally form the follicular epithelium for the separate ova; but the
majority have no such relation. They have in many instances assumed an
appearance somewhat different from that which they presented in the last
stage, mainly owing to the individual nuclei being more widely separated. A
careful examination with a high power shews that this is owing to an
increase in the amount of protoplasm of the individual cells, and it may be
noted that a similar increase in the size of the bodies of the cells has
taken place in the pseudo-epithelium and in the follicular epithelium of
the individual ova.
The stroma ingrowths form the most important feature of the stage. In most
instances they are very thin and delicate, and might easily be overlooked,
especially as many of the cells in them are hardly to be distinguished,
taken separately, from those of the germinal epithelium. These features
render the investigation of the exact relation of the stroma and epithelium
a matter of some difficulty. I have, however, been greatly assisted by the
investigation of the ovary of a young example of _Scyllium stellare_,
16-1/2 centimètres in length, a section of which is represented in Pl. 25,
fig. 26. In this ovary, although no other abnormalities were observable,
the stroma ingrowths were exceptionally wide; indeed, quite without a
parallel in my series of ovaries in this respect. The stroma most clearly
divides up the epithelium of the ovary into separate masses, or more
probably anastomosing columns, the equivalents of the egg-tubes of Pflüger.
These columns are formed of normal cells of the germinal epithelium, which
enclose ovarian nests and ova in all stages of development. A comparison of
the section I have represented, with those from previous stages, appears to
me to demonstrate that the relation of the epithelium and stroma has been
caused by an ingrowth or penetration of the stroma into the epithelium, and
not by a mutual intergrowth of the two tissues. Although the ovary, of
which fig. 26 represents a section was from _Scy. stellare_, and the
previous ovaries have been from _Scy. canicula_, yet the thickness of the
epithelium may still be appealed to in confirmation of this view. In the
previous stage the thickness was about 0.096 mm., in the present one it is
about 0.16 mm., a difference of thickness which can be easily accounted for
by the growth of the individual ova and the additional tracts of stroma. A
pseudo-epithelium is more or less clearly formed, but it is continuous with
the columns of epithelium. In the stroma many isolated cells are present,
which appear to me, from a careful comparison of a series of sections, to
belong to the germinal epithelium.
The thickness of the follicular epithelium on the inner side of the larger
ova deserves to be noted. Its meaning is discussed on p. 567.
Quite a different interpretation to that which I have given has been put by
Ludwig and Semper upon the parts of the ovary at this stage. My
_pseudo-epithelium_ is regarded by them as forming, together with the
_follicular epithelium_ of the ova, the sole remnant of the original
germinal epithelium; and the masses of cells below the pseudo-epithelium,
which I have attempted to shew are derived from the original germinal
epithelium, are regarded as parts of the ingrowths of the adjacent stroma.
Ludwig has assumed this interpretation without having had an opportunity of
working out the development of the parts, but Semper attempts to bring
forward embryological proofs in support of this position.
If the series of ovaries which I have represented be examined, it will not,
I think, be denied that the general appearances are very much in favour of
my view. The thickened patch of ovarian epithelium can apparently be traced
through the whole series of sections, and no indications of its sudden
reduction to the thin pseudo-epithelium are apparent. The most careful
examination that I have been able to make brings to light nothing tending
to shew that the general appearances are delusive. The important difference
between us refers to _our views of the nature of the tissue subjacent to
the pseudo-epithelium_. If my results be accepted, it is clear that the
whole ovarian region is an epithelium interpenetrated by connective tissue
ingrowths, so that the region below the pseudo-epithelium is a kind of
honeycomb or trabecular net-work of germinal epithelium, developing ova of
all stages and sizes, and composed of cells capable of forming follicular
epithelium for developing ova. Ludwig figures what he regards as the
formation of the follicular epithelium round primitive ova during their
passage into the stroma. It is quite clear to me, that his figures of the
later stages, 33 and 34, represent fully formed permanent ova surrounded by
a follicular epithelium, and that their situation in contact with the
pseudo-epithelium is, so to speak, an accident, and it is quite possible
that his figures 31 and 32 also represent fully formed ova; but I have
little hesitation in asserting that he has not understood the mode of
formation of the follicular epithelium, and that, though his statement that
it is derived from the germinal epithelium is quite correct, his account of
the process is completely misleading. The same criticism does not exactly
apply to Semper's statements. Semper has really observed the formation of
the follicular epithelium round young ova; but, nevertheless, he appears to
me to give an entirely wrong account of the relation of the stroma to the
germinal epithelium. The extent of the difference between Semper's and my
view may perhaps best be shewn by a quotation from Semper, _loc. cit._,
465:--"In females the nests of primitive ova sink in groups into the
stroma. In these groups one cell enlarges till it becomes the ovum, the
neighbouring cells increase and arrange themselves around the ova as
follicle cells."
Although the histological changes which take place in the succeeding stages
are not inconsiderable, they do not involve any fundamental change in the
constitution of the ovarian region, and may be described with greater
brevity than has been so far possible.
In a half-grown female, with an ovarian region of 3mm. in breadth, and
0.8mm. in thickness, the stroma of the ovarian region has assumed a far
more formed aspect than before. It consists (Pl. 24, fig. 10) of a basis in
most parts fibrous, but in some nearly homogeneous, with a fair number of
scattered cells. Immediately below the pseudo-epithelium, there is an
imperfectly developed fibrous layer, forming a kind of tunic, in which are
imbedded the relatively reduced epithelial trabeculæ of the previous
stages. They appear in sections as columns, either continuous with or
independent of the pseudo-epithelium, formed of normal cells of the
germinal epithelium, nests of ova, and permanent ova in various stages of
development. Below this there comes a layer of larger ova which are very
closely packed. A not inconsiderable number of the larger ova have,
however, a superficial situation, and lie in immediate contact with the
pseudo-epithelium. Some of the younger ova, enclosed amongst epithelial
cells continuous with the pseudo-epithelium, are very similar to those
figured by Ludwig. It is scarcely necessary to insist that this fact does
not afford any argument in favour of his interpretations. The ovarian
region is honeycombed by large vascular channels with distinct walls, and
other channels which are perhaps lymphatic.
The surface of the ovarian region is somewhat irregular and especially
marked by deep oblique transverse furrows. It is covered by a distinct,
though still irregular pseudo-epithelium, which is fairly columnar in the
furrows but flattened along the ridges. The cells of the pseudo-epithelium
have one peculiarity very unlike that of ordinary epithelial cells. Their
inner extremities (vide fig. 10) are prolonged into fibrous processes which
enter the subjacent tissue, and bending nearly parallel to the surface of
the ovary, assist in forming the tunic spoken of above. This peculiarity of
the pseudo-epithelial cells seems to indicate that they do not essentially
differ from cells which have the character of undoubted connective tissue
cells, and renders it possible that the greater part of the tunic, which
has apparently the structure of ordinary connective tissue, is in reality
derived from the original germinal epithelium, a view which tallies with
the fact that in some instances the cells of the tunic appear as if about
to assist in forming the follicular epithelium of some of the developing
ova. In Raja, the similarity of the pseudo-epithelium to the subjacent
tissue is very much more marked than in Scyllium. The pseudo-epithelium
appears merely as the superficial layer of the ovarian tunic somewhat
modified by its position on the surface. It is formed of columnar cells
with vertically arranged fibres which pass into the subjacent layers, and
chiefly differ from the ordinary fibres in that they still form parts of
the cell-protoplasm enclosing the nucleus. In Pl. 25, fig. 34, an attempt
is made to represent the relations of the pseudo-epithelium to the
subjacent tissue in Raja. Ludwig's figures of the pseudo-epithelium of the
ovary, in the regular form of its constituent cells, and its sharp
separation by a basement membrane from the tissue below, are quite unlike
anything which I have met with in my sections either of Raja or Scyllium.
Close to the dorsal border of the ovary the epithelial cells of the
non-ovarian region have very conspicuous tails, extending into a more or
less homogeneous substance below, which constitutes a peculiar form of
tunic for this part of the ovarian ridge.
In the full-grown female the stroma of the ovarian region is denser and has
a more fibrous aspect than in the younger animal. Below the
pseudo-epithelium it is arranged in two or three more or less definite
layers, in which the fibres run at right angles. It forms a definite
ovarian tunic. The pseudo-epithelium is much more distinct, and the tails
of its cells, so conspicuous in previous stages, can no longer be made out.
_Formation of the permanent ova and the follicular epithelium._--In my
monograph on the development of Elasmobranch Fishes an account was given of
the earliest stages in the development of the primitive ova, and I now take
up their development from the point at which it was left off in that work.
From their first formation till the stage spoken of in my monograph as P,
their size remains fairly constant. The larger examples have a diameter of
about 0.035 mm., and the medium-sized examples of about 0.03 mm. The larger
nuclei have a diameter of about 0.16 mm., but their variations in size are
considerable. If the above figures be compared with those on page 350 of my
monograph on Elasmobranch Fishes, it will be seen that the size of the
primitive ova during these stages is not greater than it was at the period
of their very first appearance.
The ova (Pl. 24, fig. 1) are usually aggregated in masses, which might have
resulted from division of a single ovum. The outlines of the individual ova
_are always distinct_. Their protoplasm is clear, and their nuclei, which
are somewhat passive towards staining reagents, are granular, with one to
three nucleoli. I have noticed, up to stage P, the occasional presence of
highly refractive spherules in the protoplasm of the primitive ova already
described in my monograph (pp. 353, 354, Pl. 12, fig. 15). They seem to
occur up to a later period than I at first imagined. Their want of
constancy probably indicates that they have no special importance.
Professor Semper has described similar appearances in the male primitive
ova of a later period.
As to the distribution of the primitive ova in the germinal epithelium,
Professor Semper's statement that the larger primitive ova are found in
masses in the centre, and that the smaller ova are more peripherally
situated is on the whole true, though I do not find this distribution
sufficiently constant to lay so much stress on it as he does.
The passive condition of the primitive ova becomes suddenly broken during
stage Q, and is succeeded by a period of remarkable changes. It has only
been by the expenditure of much care and trouble that I have been able to
elucidate to my own satisfaction what takes place, and there are still
points which I do not understand.
Very shortly after stage Q, in addition to primitive ova with a perfectly
normal nucleus, others may be seen in which the nucleus is apparently
replaced by a deeply stained irregular body, smaller than the ordinary
nuclei (Pl. 24, fig. 11, _d.n._). This body, by the use of high objectives,
is seen to be composed of a number of deeply stained granules, and around
it may be noticed a clear space, bounded by a very delicate membrane. The
granular body usually lies close to one side of this membrane, and
occasionally sends a few fine processes to the opposite side.
The whole body, _i.e._ all within the delicate membrane is, according to my
view, a modified nucleus; as appears to me very clearly to be shewn by the
fact that it occupies the normal position of a nucleus within a cell body.
Semper, on the other hand, regards the contained granular body as the
nucleus, which he compares with the spindles of Bütschli, Auerbach,
&c.[374]. This interpretation appears to me, however, to be negatived by
the position of these bodies. The manner in which Semper may, perhaps, have
been led to his views will be obvious when the later changes of the
primitive ova are described. The formation of these nuclei would seem to be
due to a segregation of the constituents of the original nuclei; the solid
parts becoming separated from the more fluid. As a rule, the modified
nuclei are slightly larger than the original ones. In stage Q the following
two tables shew the dimensions of the parts of three unmodified and of
three modified nuclei taken at random.
Footnote 374: _Loc. cit._ p. 361.
_Primitive ova with unmodified nuclei_--
Nuclei.
0.014 mm.
0.012 mm.
0.01 mm.
_Primitive ova with modified nuclei_--
Granular
Nuclei. Bodies in nuclei.
0.018 mm. 0.006 mm.
0.018 mm. 0.006 mm.
0.012 mm. 0.009 mm.
For a slightly older stage than Q, the two annexed tables also shew the
comparative size of the modified and unmodified nuclei:
_Unmodified nuclei of normal primitive ova--_
0.014 mm.
0.016 mm.
0.014 mm.
0.016 mm.
0.016 mm.
_Nuclei of primitive ova with modified nuclei--_
Granular
Nuclei. Bodies in Nuclei.
0.018 mm. 0.008 mm.
0.016 mm. 0.008 mm.
0.016 mm. 0.01 mm.
0.016 mm.
0.018 mm.
These figures bring out with clearness the following points: (1) that the
modified nuclei are slightly but decidedly larger on the average than the
unmodified nuclei; (2) that the contained granular bodies _are very
considerably_ smaller than ordinary nuclei.
Soon after the appearance of the modified nuclei, remarkable changes take
place in the cells containing them. Up to the time such nuclei first make
their appearance the outlines of the individual ova are very clearly
defined, but subsequently, although numerous ova with but slightly modified
nuclei are still to be seen, yet on the whole the outlines of all the
primitive ova are much less distinct than before; and this is especially
the case with the primitive ova containing modified nuclei.
From cases in which three or four ova are found in a mass with modified
nuclei, but in which the outline of each ovum is fairly distinct, it is
possible to pass by insensible gradations to other cases in which two or
three or more modified nuclei are found embedded in a mass of protoplasm in
which no division into separate cells can be made out (fig. 14). For these
masses I propose to employ the term nests. They correspond in part with the
_Ureiernester_ of Professor Semper.
Frequently they are found in hardened specimens to be enclosed in a
membrane-like tunic which appears to be of the nature of coagulated fluid.
These membranes closely resemble and sometimes are even continuous with
trabeculæ which traverse the germinal epithelium. Ovaries differ
considerably as to the time and completeness of the disappearance of the
outlines marking the separate cells, and although, so far as can be
gathered from my specimens, the rule is that the outlines of the primitive
ova with modified nuclei soon become indistinct, yet in one of my best
preserved ovaries very large nests with modified nuclei are present in
which the outline of each ovum is as distinct as during the period before
the nuclei undergo these peculiar changes (Pl. 24, fig. 12). In the same
ovary other nests are present in which the outlines of the individual ova
are no longer visible. The section represented on Pl. 24, fig. 2, is fairly
average as to the disappearance of the outlines of the individual ova.
It is clear from the above statements, that in the first instance the nests
are produced by the coalescence of several primitive ova into a single mass
or syncytium; though of course, the several separate ova of a nest may
originally, as Semper believes, have arisen from the division of a single
ovum. In any case there can be no doubt that the nests of separate ova
increase in size as development proceeds; a phenomenon which is more
reasonably explained on the view that the ova divide, than on the view that
they continue to be freshly formed. The same holds true for the nests of
nuclei and this, as well as other facts, appears to me to render it
probable that the nests grow by division of the nuclei without
corresponding division of the protoplasmic matrix. I cannot, however,
definitely prove this point owing to my having found nests, with distinct
outlines to the ova, as large as any without such outlines.
The nests are situated for the most part near the surface of the germinal
epithelium. The smaller ones are frequently spherical, but the larger are
irregular in form. The former are about 0.05 mm. in diameter; the latter
reach 0.1 mm. Scattered generally, and especially in the deeper layers, and
at the edges of the germinal epithelium, are still unmodified or only
slightly modified primitive ova. These unmodified primitive ova are
aggregated in masses, but in these masses the outlines of each ovum, though
perhaps less clear than in the earlier period, are still distinct.
When the embryo reaches a length of seven centimètres, and even in still
younger embryos, further changes are observable. In the first place many of
the modified nuclei acquire fresh characters, and it becomes necessary to
divide the modified nuclei into two categories. In both of these the outer
boundary of the nucleus is formed by a very delicate membrane, the space
within which is perfectly clear except for the granular body. In the
variety which now appears in considerable numbers the granular body has an
irregular star-like form. The rays of the star are formed of fibres
frequently knobbed at their extremities, and the centre of the star usually
occupies an eccentric position. Typical examples of this form of modified
nucleus, which may be spoken of as the stellate variety, are represented on
Pl. 25, fig. 17; between it and the older granular variety there is an
infinite series of gradations, many of which are represented on Pl. 24,
figs. 12, 14, 15, 16. Certain of the stellate nuclei exhibit two centres
instead of one, and in some cases, like that represented on Pl. 25,
fig. 19, the stellate body of two nuclei is found united. Both of these
forms are possibly modifications of the spindle-like form assumed by nuclei
in the act of dividing, and may be used in proving that the nests increase
in size by the division of the contained nuclei. In addition to the normal
primitive ova, a few of which are still present, there are to be found,
chiefly in the deeper layers of the germinal epithelium, larger ova
differing considerably from the primitive ova. They form the permanent ova
(Pl. 24, fig. 3, _o_). Their average diameter is 0.04 mm., compared with
0.03 mm., the diameter of original primitive ova. The protoplasm of which
they are composed is granular, but at first a membrane can hardly be
distinguished around them; their nucleus is relatively large, 0.02 -
0.027 mm. in diameter. It presents the characters ascribed by Eimer[375],
and many other recent authors[376], to typical nuclei (vide Pl. 24, fig. 3,
and Pl. 24, 25, figs. 13, 14, 15, 16, 17, 18). It is bounded by a distinct
membrane, within which is a more or less central nucleolus from which a
number of radial fibres which stain very deeply pass to the surface; here
they form immediately internal to the membrane a network with granules at
the nodal points. In some instances the regularity of the arrangement of
these fibres is very great, in other instances two central nucleoli are
present, in which case the regularity is considerably interfered with. The
points in which the youngest permanent ova differ from the primitive may be
summed up as follows:--
(1) The permanent ova are larger, the smallest of them being larger than
the average primitive ova in the proportion of four to three. (2) They have
less protoplasm as compared to the size of the nucleus. (3) Their
protoplasm is granular instead of being clear. (4) Their nucleus is clear
with exception of a network of fibres instead of being granular as in the
primitive ova. It thus appears that the primitive ova and permanent ova are
very different in constitution, though genetically related in a way to be
directly narrated.
Footnote 375: _Archiv f. micr. Anat._ Vol. XIV.
Footnote 376: Vide especially Klein, _Quart. Journ. of Mic.
Sci._ July 1878.
The formation of permanent ova is at its height in embryos of about seven
centimètres or slightly larger. The nests at this stage are for the most
part of a very considerable size and contain a large number of nuclei,
which have probably, as before insisted, originated from a division of the
smaller number of nuclei present in the nests at an earlier stage. Figs.
14-18 are representations of nests at this period. The diameter of the
nuclei is, on the whole, slightly greater than at an earlier stage. A
series of measurements gave the following results:--
0.016 mm.
0.016 mm.
0.018 mm.
0.02 mm.
0.02 mm.
Both varieties of modified nuclei are common enough, though the stellate
variety predominates. The nuclei are sometimes in very close contact, and
sometimes separated by protoplasm, which in many instances is very slightly
granular. In a large number of the nests nothing further is apparent than
what has just been described, but in a very considerable number one or more
nuclei are present, which exhibit a transitional character between the
ordinary stellate nuclei of my second category, and the nuclei of permanent
ova as above described; and in these nests the formation of permanent ova
is taking place. Permanent ova in the act of development are indicated in
my figures by the letters _do_. Many of the intermediate nuclei are more
definitely surrounded by granular protoplasm than the other nuclei of the
nests, and accordingly have their outlines more sharply defined. Between
nuclei of this kind, and others as large as those of the permanent ova,
there are numerous transitional forms. The larger ones frequently lie in a
mass of granular protoplasm projecting from the nest, and only united with
it by a neck (Pl. 24, figs. 14 and 16). For prominences of this kind to
become independent ova, it is only necessary for the neck to become broken
through. Nests in which such changes are taking place present various
characters. In some cases several nuclei belonging to a nest appear to be
undergoing conversion into permanent ova at the same time. Such a case is
figured on Pl. 25, figs. 17 and 18. In these cases the amount of granular
protoplasm in the nest and around each freshly formed ovum is small. In the
more usual cases only one or two permanent ova at the utmost are formed at
the same time, and in these instances a considerable amount of granular
protoplasm is present around the nucleus of the developing permanent ovum.
In such instances it frequently happens several of the nuclei not
undergoing conversion appear to be in the process of absorption, and give
to the part of the nest in which they are contained a very hazy and
indistinct aspect (Pl. 24, fig. 15). Their appearance leads me to adopt the
view _that while some of the nuclei of each nest are converted into the
nuclei of the permanent ova, others break down and are used as the pabulum,
at the expense of which the protoplasm of the young ovum grows_.
It should, however, be stated, that after the outlines of the permanent ova
have become definitely established, I have only observed in a single
instance the inclusion of a nucleus within an ovum (Pl. 25, fig. 24). In
many instances normal nuclei of the germinal epithelium may be so observed
within the ovum.
The nuclei which are becoming converted into the nuclei of permanent ova
gradually increase in size. The following table gives the diameter of four
such nuclei:--
0.022 mm.
0.022 mm.
0.024 mm.
0.032 mm.
These figures should be compared with those of the table on page 564.
The ova when first formed are situated either at the surface or in the
deeper layers of the germinal epithelium. Though to a great extent
surrounded by the ordinary cells of the germinal epithelium, they are not
at first enclosed in a definite follicular epithelium. The follicle is,
however, very early formed.
My observations lead me then to the conclusion that in a general way the
permanent ova are formed by the increase of protoplasm round some of the
nuclei of a nest, and the subsequent separation of the nuclei with their
protoplasm from the nest as distinct cells--a mode of formation exactly
comparable with that which so often takes place in invertebrate egg tubes.
Besides the mode of formation of permanent ova just described, a second one
also seems probably to occur. In ovaries just younger than those in which
permanent ova are distinctly formed, there are present primitive ova, with
modified nuclei of the stellate variety, or nuclei sometimes even
approaching in character those of permanent ova, which are quite isolated
and not enclosed in a definite nest. The body of these ova is formed of
granular protoplasm, but their outlines are very indistinct. Such ova are
considerably larger than the normal primitive ova. They may measure
0.04 mm. In a slightly later stage, when fully formed permanent ova are
present, isolated ones are not infrequent, and it seems natural to conclude
that these isolated ova are the direct descendants of the primitive ova of
the earlier stage. It seems a fair deduction that in some cases primitive
ova undergo a direct metamorphosis into permanent ova by a modification of
their nucleus, and the assumption of a granular character in their
protoplasm, without ever forming the constituent part of a nest.
It is not quite clear to me that in all nests the coalescence of the
protoplasm of the ova necessarily takes place, since some nests are to be
found at all stages in which the ova are distinct. Nevertheless, I am
inclined to believe that the fusion of the ova is the normal occurrence.
The mode of formation of the permanent ova may then, according to my
observations, take place in two ways:--1. By the formation of granular
protoplasm round the nucleus in a nest, and the separation of the nucleus
with its protoplasm as a distinct ovum. 2. By the direct metamorphosis of
an isolated primitive ovum into a permanent ovum. The difference between
these two modes of formation does not, from a morphological point of view,
appear to be of great importance.
The above results appear clearly to shew that _the primitive ova in the
female are not to be regarded as true ova, but as the parent sexual cells
which give rise to the ova_: a conclusion which completely fits in with the
fact that cells exactly similar to the primitive ova in the female give
rise to the spermatic cells in the male.
Slightly after the period of their first formation the permanent ova become
invested by a very distinct and well-marked, somewhat flattened, follicular
epithelium (Pl. 24, fig. 3). Where the ova lie in the deeper layers of the
germinal epithelium, the follicular epithelium soon becomes far more
columnar on the side turned inwards, than on that towards the surface,
especially when the inner side is in contact with the stroma (Pl. 24,
fig. 7, and Pl. 25, figs. 24 and 26). This is probably a special provision
for the growth and nutrition of the ovum.
There cannot be the smallest doubt that the follicular epithelium is
derived from the general cells of the germinal epithelium--a point on which
my results fully bear out the conclusions of Ludwig and Semper.
The larger ova themselves have a diameter of about 0.06 mm., and their
nucleus of about 0.04 mm. The vitellus is granular, and provided with a
distinct, though delicate membrane, which has every appearance of being a
product of the ovum itself rather than of the follicular epithelium. The
membrane would seem indeed to be formed in some instances even before the
ovum has a definite investment of follicle cells. The vitellus is
frequently vacuolated, but occasionally the vacuoles appear to be caused by
a shrinking due to the hardening reagent. The nucleus has the same peculiar
reticulate character as at first. Its large size, as compared with the
ovum, is very noticeable.
With this stage the embryonic development of the ova comes to a close,
though the formation of fresh ova continues till comparatively late in
life. I have, however, two series of sections of ovaries preserved in osmic
acid, from slightly larger embryos than the one last described, about which
it may be well to say a few words before proceeding to the further
development of the permanent ova.
The younger of these ovaries was from a Scyllium embryo 10 centimètres
long, preserved in osmic acid.
A considerable number of nests were present (Pl. 24, fig. 13), exhibiting,
on the whole, similar characters to those just described.
A series of measurements of the nuclei in them were made, leading to the
following results:--
0.014 mm.
0.014 mm.
0.016 mm.
0.016 mm.
0.018 mm.
0.018 mm.
Thus, if anything, the nuclei were slightly smaller than in the younger
embryo. It is very difficult in the osmic specimens to make out clearly the
exact outlines of the various structures, the nuclei in many instances
being hardly more deeply stained than in the protoplasm around them. The
network in the nuclei is also far less obvious than after treatment with
picric acid. The permanent ova were hardly so numerous as in the younger
ovary before described. A number of these were measured with the following
results:--
Ovum. Nucleus.
0.03 mm. 0.014 mm.
0.034 mm. 0.018 mm.
0.028 mm. 0.016 mm.
0.03 mm. 0.02 mm.
0.04 mm. 0.02 mm.
0.04 mm. 0.02 mm.
0.048 mm. 0.02 mm.
These figures shew that the nuclei of the permanent ova are smaller than in
the younger embryo, and it may therefore be safely concluded that, in spite
of the greater size of the embryo from which it is taken, the ovary now
being described is in a more embryonic condition than the one last dealt
with.
Though the permanent ova appeared to be formed from the nests in the manner
already described, it was fairly clear from the sections of this ovary that
many of the original primitive ova, after a metamorphosis of the nucleus
and without coalescing with other primitive ova to form nests, become
converted directly into the permanent ova. Many large masses of primitive
ova, or at least of ova with the individual outlines of each ovum distinct,
were present. The average size of ova composing these was however small,
the body measuring about 0.016 mm., and the nucleus 0.012 mm. Isolated ova
with metamorphosed nuclei could also be found measuring 0.022, and their
nuclei about 0.014 mm.
The second of the two ovaries, hardened in osmic acid, was somewhat more
advanced than the ovary in which the formation of permanent ova was at its
height. Fewer permanent ova were in the act of being formed, and many of
these present had reached a considerable size, measuring as much as
0.07 mm. Nests of the typical forms were present as before, but the nuclei
in them were more granular than at the earlier period, and on the average
slightly smaller. A series measured had the following diameters:--
0.01 mm.
0.012 mm.
0.014 mm.
0.016 mm.
One of these nests is represented on Pl. 25, fig. 20. Many nests with the
outlines of the individual ova distinct were also present.
On the whole it appeared to me, that the second mode of formation of
permanent ova, viz. that in which the nest does not come into the cycle of
development, preponderated to a greater extent than in the earlier
embryonic period.
POST-EMBRYONIC DEVELOPMENT OF THE OVA.--My investigations upon the
post-embryonic growth and development of the ova, have for the most part
been conducted upon preserved ova, and it has been impossible for me, on
this account, to work out, as completely as I should have wished, certain
points, more especially those connected with the development of the yolk.
Although my ovaries have been carefully preserved in a large number of
reagents, including osmic acid, picric acid, chromic acid, spirit,
bichromate of potash, and Müller's fluid, none of these have proved
universally successful, and bichromate of potash and Müller's fluid are
useless. Great difficulties have been experienced in distinguishing the
artificial products of these reagents. My investigations have led me to the
result, that in the gradual growth of the ova with the age of the
individual the changes are not quite identical with those during the rapid
growth which takes place at periods of sexual activity, after the adult
condition has been reached--a result to which His has also arrived, with
reference to the ova of Osseous Fish. I propose dealing separately with the
several constituents of the egg-follicle.
_Egg membranes._--A vitelline membrane has been described by Leydig[377] in
Raja, and an albuminous layer of the nature of a chorion[378] by
Gegenbaur[379] in Acanthias--the membranes described in these two ways
being no doubt equivalent.
Footnote 377: _Rochen u. Haie._
Footnote 378: By _chorion_ I mean, following E. van Beneden's
nomenclature, a membrane formed by the follicular epithelium,
and, by _vitelline membrane_, one formed by the vitellus or
body of the ovum.
Footnote 379: "Bau und Entwicklung d. Wirbelthiereier," &c.,
_Müll. Archiv_, 1861.
Dr Alex. Schultz[380] has more recently investigated a considerable variety
of genera and finds three conditions of the egg membranes. (1) In Torpedo,
a homogeneous membrane, which is of the nature of a chorion. (2) In Raja, a
homogeneous membrane which is, however, perforated. (3) In Squalidæ, a
thick homogeneous membrane, internal to which is a thinner perforated
membrane. He apparently regards the perforated inner membrane as a
specialised part of the simple membrane found in Torpedo, and states that
this membrane is of the nature of a chorion.
Footnote 380: "Zur Entwicklungsgeschichte d. Selachier,"
_Arch. f. mikr. Anat._ Vol. XI.
My own investigations have led me to the conclusion that though the
egg-membranes can probably be reduced to single type for Elasmobranchii,
yet that they vary with the stage of development of the ovum. Scyllium
(stellare and canicula) and Raja have formed the objects of my
investigation. I commence with the two former.
It has already been stated that in Scyllium, even before the follicular
epithelium becomes formed, a delicate membrane round the ovum can be
demonstrated, which appears to me to be derived from the vitellus or body
of the ovum, and is therefore of the nature of a vitelline membrane. It
becomes the vitelline membrane of Leydig, the albuminous membrane of
Gegenbaur, and homogeneous membrane of Schultz.
In a young fish (not long hatched) with ova of not more than 0.12 mm., this
membrane, though considerably thicker than in the embryo, is not thick
enough to be accurately measured. In ova of 0.5 mm. from a young female
(Pl. 25, fig. 21) the vitelline membrane has a thickness of 0.002 mm. and
is quite homogeneous[381]. Internally to it may be observed very faint
indications of the differentiation of the outermost layer of the vitellus
into the perforated or radially striated membrane of Schultz, which will be
spoken of as _zona radiata_.
Footnote 381: The apparent structure in the vitelline membrane
in my figure is merely intended to represent the dark colour
assumed by it on being stained. The zona radiata has been made
rather too thick by the artist.
In an ovum of 1 mm. from the nearly full grown though not sexually mature
female, the zona radiata has increased in thickness and definiteness, and
may measure as much as 0.004 mm. It is always very sharply separated from
the vitelline membrane, but appears to be more or less continuous on its
inner border with the body of the ovum, at the expense of which it no doubt
grows in thickness.
In ova above 1 mm. in diameter, both vitelline membrane and zona radiata,
but especially the latter, increase in thickness. The zona becomes marked
off from the yolk, and its radial striæ become easy to see even with
comparatively low powers. In many specimens it appears to be formed of a
number of small columns, as described by Gegenbaur and others. The stage of
about the greatest development of both the vitelline membrane and zona
radiata is represented on Pl. 25, fig. 22.
At this time the vitelline membrane appears frequently to exhibit a
distinct stratification, dividing it into two or more successive layers. It
is not, however, acted on in the same manner by all reagents, and with
absolute alcohol appears at times longitudinally striated.
From this stage onwards, both vitelline membrane and zona gradually
atrophy, simultaneously with a series of remarkable changes which take
place in the follicular epithelium. The zona is the first to disappear, and
the vitelline membrane next becomes gradually thinner. Finally, when the
egg is nearly ripe, the follicular epithelium is separated from the yolk by
an immeasurably thin membrane--the remnant of the vitelline membrane--only
visible in the most favourable sections (Pl. 25, fig. 23, _vt._). When the
egg becomes detached from the ovary even this membrane is no longer to be
seen.
Both the vitelline membrane and the zona radiata are found in Raja, but in
a much less developed condition than in Scyllium. The vitelline membrane is
for a long time the only membrane present, but is never very thick (Pl. 25,
fig. 31). The zona is not formed till a relatively much later period than
in Scyllium, and is always delicate and difficult to see (Pl. 25, fig. 32).
Both membranes atrophy before the egg is quite ripe; and an apparently
fluid layer between the follicular epithelium and the vitellus, which
coagulates in hardened specimens, is probably the last remnant of the
vitelline membrane. It is, however, much thicker than the corresponding
remnant in Scyllium.
Though I find the same membranes in Scyllium as Alexander Schultz did in
other Squalidæ, my results do not agree with his as to Raja. Torpedo I have
not investigated.
It appears to me probable that the ova in all Elasmobranch Fishes have at
some period of their development the two membranes described at length for
Scyllium. Of these the inner one, or zona radiata, will probably be
admitted on all hands to be a product of the peripheral protoplasm of the
egg.
The outer one corresponds with the membrane usually regarded in other
Vertebrates as a chorion or product of the follicular epithelium, but, by
tracing it back to its first origin, I have been led to reject this view of
its nature.
_The follicular epithelium._--The follicular epithelium in the eggs of Raja
and Acanthias has been described by Gegenbaur[382]. He finds it flat in
young eggs, but in the larger eggs of Acanthias more columnar, and with the
cells wedged in so as to form a double layer. These observations are
confirmed by Ludwig[383].
Footnote 382: _Loc. cit._
Footnote 383: _Loc. cit._
Alexander Schultz[384] states that in Torpedo, the eggs are at first
enclosed in a simple epithelium, but that in follicles of .008 mm. there
appear between the original large cells of the follicle (which he describes
as granulosa cells and derives from the germinal epithelium) a number of
peculiar small cells. He states that these are of the same nature as the
general stroma cells of the ovary, and believes that they originate in the
stroma. When the eggs have reached 0.1 - 0.15 mm., he finds that the small
and large cells have a very regular alternating arrangement.
Footnote 384: _Loc. cit._
Semper records but few observations on the follicular epithelium, but
describes in Raja the presence of a certain number of large cells amongst
smaller cells. He believes that they may develop into ova, and considers
them identical with the larger cells described by Schultz, whose
interpretations he does not, however, accept.
My own results accord to a great extent with those of Dr Schultz, as far as
the structure of the follicular epithelium is concerned, but I am at one
with Semper in rejecting Schultz's interpretations.
In Scyllium, as has already been mentioned, the follicular epithelium is at
first flat and formed of a single layer of uniform cells, each with a
considerable amount of clear protoplasm and a granular nucleus. It is
bounded externally by a delicate membrane--the membrana propria folliculi
of Waldeyer--and internally by the vitelline membrane. In the ovaries of
very young animals the cells of the follicular epithelium are more columnar
on the side towards the stroma than on the opposite side, but this
irregularity soon ceases to exist.
In many cases the nuclei of the cells of the follicular epithelium exhibit
a spindle modification, which shews that the growth of the follicular
epithelium takes place by the division of its cells. No changes of
importance are observable in the follicular epithelium till the egg has
reached a diameter of more than 1 mm.
It should here be stated that I have some doubts respecting the
completeness of the history of the epithelium recorded in the sequel.
Difficulties have been met with in completely elucidating the chronological
order of the occurrences, and it is possible that some points have escaped
my observation.
The first important change is the assumption of a palisade-like character
by the follicle cells, each cell becoming very narrow and columnar and the
nucleus oval (Pl. 25, fig. 28). In this condition the thickness of the
epithelium is about 0.025 mm. The epithelium does not, however, become
uniformly thick over the whole ovum, but in the neighbourhood of the
germinal vesicle it is very flat and formed of granular cells with
indistinct outlines, rather like the hypodermis cells of many Annelida.
Coincidently with this change in the follicular epithelium the commencement
of the atrophy of the membranes of the ovum, described in the last section,
becomes apparent.
The original membrana propria folliculi is still present round the
follicular epithelium, but is closely associated with a fibrous layer with
elongated nuclei. Outside this there is now a layer of cells, very much
like an ordinary epithelial layer, which may possibly be formed of cells of
the true germinal epithelium (fig. 28, _fe´_). This layer, which will be
spoken of as the secondary follicle layer, might easily be mistaken for the
follicular epithelium, and it is possible that it has actually been so
mistaken by Eimer, Clark, and Klebs, in Reptilia, and that the true
follicular epithelium (in a flattened condition) has been then spoken of as
the _Binnenepithel_.
In slightly older eggs the epithelial cells are no longer uniform or
arranged as a single layer. The general arrangement of these cells is shewn
in Pl. 25, fig. 29. A considerable number of them are more or less
flask-shaped, with bulky protoplasm prolonged into a thin stem directed
towards the vitelline membrane, with which, in many instances if not all,
it comes in contact. These larger cells are arranged in several tiers.
Intercalated between them are a number of elongated small cells with scanty
protoplasm and a deeply staining nucleus, not very dissimilar to, though
somewhat smaller than, the columnar cells of the previous stage. There is
present a complete series of cells intermediate between the larger cells
and those with a deeply stained nucleus, and were it not for the condition
of the epithelium in Raja, to be spoken of directly, I should not sharply
divide the cells into two categories. In surface views of the epithelium
the division into two kinds of cells would not be suspected. There can, it
appears to me, be no question that both varieties of cell are derived from
the primitive uniform follicle cells.
The fibrous layer bounding the membrana propria folliculi is thicker than
in the last stage, and the epithelial-like layer (_fe´_) which bounds it
externally is more conspicuous than before. Immediately adjoining it are
vascular and lymph sinuses. The thickness of the follicular epithelium at
this stage may reach as much as 0.04 mm., though I have found it sometimes
considerably flatter. The cells composing it are, however, so delicate that
it is not easy to feel certain that the peculiarities of any individual
ovum are not due to handling. The absence of the peculiar columnar
epithelium on the part of the surface adjoining the germinal vesicle is as
marked a feature as in the earlier stage. When the egg is nearly ripe, and
the vitelline membrane has been reduced to a mere remnant, the follicular
epithelium is still very columnar (Pl. 25, fig. 23). The thickness is
greater than in the last stage, being now about 0.045 mm., but the cells
appear only to form a single definite layer. From the character of their
nuclei, I feel inclined to regard them as belonging to the category of the
smaller cells of the previous stage, and feel confirmed in this view by
finding certain bodies in the epithelium, which have the appearance of
degenerating cells with granular nuclei, which I take to be the
flask-shaped cells which were present in the earlier stage.
I have not investigated the character of the follicular epithelium in the
perfectly ripe ovum ready to become detached from the ovary. Nor can I
state for the last-described stage anything about the character of the
follicular epithelium in the neighbourhood of the germinal vesicle.
As to the relation of the follicular epithelium to the vitelline membrane,
and the possible processes of its cells continued into the yolk, I can say
very little. I find in specimens teased out after treatment with osmic
acid, that the cells of the follicular epithelium are occasionally provided
with short processes, which might possibly have perforated the vitelline
membrane, but have met with nothing so clear as the teased out specimens
figured by Eimer. Nothing resembling the cells within the vitelline
membrane, as described by His[385] in Osseous Fish, and Lindgren in
Mammalia, has been met with[386].
Footnote 385: _Das Ei bei Knochenfischen._
Footnote 386: _Arch. f. Anat. Phys._ 1877.
My observations in Raja are not so full as those upon Scyllium, but they
serve to complete and reconcile the observations of Semper and Schultz, and
also to shew that the general mode of growth of the follicular epithelium
is fundamentally the same in my representatives of the two divisions of the
Elasmobranchii. In very young eggs, in conformity with the results of all
previous observers, I find the follicular epithelium approximately uniform.
The cells are flat, but extended so as to appear of an unexpected size in
views of the surface of the follicle. This condition does not, however,
last very long. A certain number of the cells enlarge considerably, others
remaining smaller and flat. The differences between the larger and the
smaller cells are more conspicuous in sections than in surface views, and
though the distribution of the cells is somewhat irregular, it may still be
predicted as an almost invariable rule that the smaller cells of the
follicle will line that part of the surface of the ovum, near to which the
germinal vesicle is situated. On Pl. 25, fig. 30, is shewn in section a
fairly average arrangement of the follicle cells. Semper considers the
larger cells of such a follicle to be probably primitive ova destined to
become permanent ova. This view I cannot accept: firstly, because these
cells only agree with primitive ova in being exceptionally large--the
character of their nucleus, with its large nucleolus, being not very like
that of a primitive ovum. Secondly, because they shade into ordinary cells
of the follicle; and thirdly, because no evidence of their becoming ova has
come before me, but rather the reverse, in that it seems probable that they
have a definite function connected with the nutrition of the egg. To this
point I shall return.
In the next stage the small cells have become still smaller. They are
columnar, and are wedged in between the larger ones. No great regularity in
distribution is as yet attained (Pl. 25, fig. 31). Such a regularity
appears in a later stage (Pl. 25, fig. 32), which clearly corresponds with
fig. 8 on Pl. 34 of Schultz's paper, and also with the stage of Scyllium in
Pl. 25, fig. 29, though the distinction between the two kinds of cells is
here far better marked than in Scyllium. The big cells have now become
flask-shaped like those in Scyllium, and send a process down to the
vitelline membrane. The smaller cells are arranged in two or three tiers,
but the larger cells in a single layer. The distribution of the larger and
smaller cells is in some instances very regular, as shewn in the surface
view on Pl. 25, fig. 33. There can, it appears to me, be no doubt that
Schultz's view of the smaller cells being lymph-cells which have migrated
into the follicle cannot be maintained.
The thickness of the epithelium at this stage is about 0.04 mm. In the
succeeding stages, during which the egg is rapidly growing to the colossal
size which it eventually attains, the follicular epithelium does not to any
great extent alter in constitution. It grows thicker on the whole, and as
the vitelline membrane gradually atrophies, its lower surface becomes
irregular, exhibiting somewhat flattened prominences, which project into
the yolk. At the greatest height of the prominences the epithelium may
reach a thickness of 0.06 mm., or even more. The arrangement of the tissues
external to the follicular epithelium is the same in Raja as in Scyllium.
The most interesting point connected with the follicle, both in Scyllium
and Raja and presumably in other Elasmobranchii is that its epithelium at
the time when the egg is rapidly approaching maturity is composed with more
or less of distinctness of two forms of cells. One of these is large
flask-shaped and rich in protoplasm, the other is small, consisting of a
mere film of protoplasm round a nucleus. Considering that the larger cells
appear at the time of rapid growth, it is natural to interpret their
presence as connected with the nutrition of the ovum. This view is
supported by the observations of Eimer and Braun, on the development of
Reptilian ova. In many Reptilian ova it appears from Eimer's[387]
observations, that the follicular epithelium becomes several layers thick,
and that a differentiation of the cells, similar to that in Elasmobranchii,
takes place. The flask-shaped cells eventually undergo peculiar changes,
becoming converted into a kind of beaker-cell, with prolongations through
the egg membranes, which take the place of canals leading to the interior
of the egg. Braun also expresses himself strongly in favour of the
flask-shaped cells functioning in the nutrition of the egg[388]. That these
cells in the Reptilian ova really correspond with those in Elasmobranchii
appears to me clear from Eimer's figures, but I have not myself studied any
Reptilian ovum. My reasons for dissenting from both Semper's and Schultz's
views on the nature of the two forms of follicular cells have already been
stated.
Footnote 387: _Archiv f. mikr. Anat._ Vol. VIII.
Footnote 388: Braun, "Urogenitalsystem d. Amphibien,"
_Arbeiten a. d. zool.-zoot. Institut Würzburg_, Bd. IV. He
says, in reference to the flask-shaped cell, p. 166, "Höchstens
würde ich die Funktion der grossen Follikelzellen als
_einzellige Drüsen_ mehr betonen."
_The Vitellus and the development of the yolk spherules._--Leydig,
Gegenbaur, and Schultz, have recorded important observations on this head.
Leydig[389] chiefly describes the peculiar characters of the yolk
spherules.
Footnote 389: _Loc. cit._
Gegenbaur[390] finds in the youngest eggs fine granules, which subsequently
develop into vesicles, in the interior of which the solid oval spheres, so
characteristic of Elasmobranchii, are developed.
Footnote 390: _Loc. cit._
Schultz describes in the youngest ova of Torpedo the minute yolk spherules
arranged in a semilunar form around the eccentric germinal vesicle. In
older ova they spread through the whole. He also gives a description of
their arrangement in the ripe ovum. Dr Schultz further finds in the body of
the ovum peculiar protoplastic striæ, arranged as a series of pyramids,
with the bases directed outwards. In the periphery of the ovum a
protoplastic network is also present, which is continuous with the
above-mentioned pyramidal structures.
My observations do not very greatly extend those of Gegenbaur and Schultz
with reference to the development of the yolk, and closely agree with what
Gegenbaur has given in the paper above quoted more fully for Aves and
Reptilia than for Elasmobranchii.
In very young ova the body of the ovum is simply granular, but when it has
reached about 0.5 mm. the granules are seen to be arranged in a kind of
network, or sponge-work (Pl. 25, fig. 21), already spoken of in my
monograph on Elasmobranch Fishes.
This network becomes more distinct in succeeding stages, especially in
chromic acid specimens (Pl. 25, fig. 22), probably in part owing to a
granular precipitation of the protoplasm. In the late stages, when the yolk
spherules are fully developed, it is difficult to observe this network,
but, as has been shewn in my monograph above quoted, it is still present
after the commencement of embryonic development. An arrangement of the
protoplasmic striæ like that described by Schultz has not come under my
notice.
The development of the yolk appears to me to present special difficulties,
owing to the fact pointed out by His[391] that the conditions of
development vary greatly according to whether the ovary is in a state of
repose or of active development. I do not feel satisfied with my results on
this subject, but believe there is still much to be made out. Observations
on the yolk spherules may be made either in living ova, in ova hardened in
osmic acid, or in ova hardened in picric or chromic acids. The two latter
reagents, as well as alcohol, are however unfavourable for the purpose of
this study, since by their action the yolk spherules appear frequently to
be broken up and otherwise altered. This has to some extent occurred in
Pl. 25, fig. 21, and the peculiar appearance of the yolk of this ovum is in
part due to the action of the reagent. On the whole I have found osmic acid
the most suitable reagent for the study of the yolk, since without breaking
up the developing spherules, it stains them of a deep black colour. The
yolk spherules commence to be formed in ova, of not more than 0.06 mm. in
the ovaries of moderately old females. In young females they are apparently
not formed in such small ova. They arise as extremely minute, highly
refracting particles, in a stratum of protoplasm _some little way below the
surface, and are always most numerous at the pole opposite the germinal
vesicle_. Their general arrangement is very much that figured and described
by Allen Thomson in Gasterosteus[392], and by Gegenbaur and Eimer in young
Reptilian ova. In section they naturally appear as a ring, their general
mode of distribution being fairly typically represented on Pl. 25, fig. 27.
The ovum represented in fig. 27 was 0.5 mm. in diameter, and the yolk
spherules were already largely developed; in smaller ova they are far less
numerous, though arranged in a similar fashion. The developing yolk
spherules are not uniformly distributed but are collected in peculiar
little masses or aggregations (Pl. 25, fig. 21). These resemble the
granular masses, figured by His (_loc. cit._ Pl. 4, fig. 33) in the Salmon,
and may be compared with the aggregations figured by Götte in his monograph
on _Bombinator igneus_ (Pl. 1, fig. 9). It deserves to be especially noted,
that when the yolk spherules are first formed, the _peripheral layer of the
ovum_ is entirely free from them, a feature which is however apt to be lost
in ova hardened in picric acid (Pl. 25, fig. 21). Two points about the
spherules appear clearly to point to their being developed in the
protoplasm of the ovum, and not in the follicular epithelium. (1) That they
do not make their appearance in the superficial stratum of the ovum. (2)
That no yolk spherules are present in the cells of the follicular
epithelium, in which they could not fail to be detected, owing to the deep
colour they assume on being treated with osmic acid.
Footnote 391: _Das Ei bei Knochenfischen._
Footnote 392: "Ovum" in Todd's _Encyclopædia_, fig. 69.
It need scarcely be said that the yolk spherules at this stage are not
cells, and have indeed no resemblance to cells. They would probably be
regarded by His as spherules of fatty material, unrelated to the true food
yolk.
As the ova become larger the granules of the peripheral layer before
mentioned gradually assume the character of the yolk spheres of the adult,
and at the same time spread towards the centre of the egg. Not having
worked at fresh specimens, I cannot give a full account of the growth of
the spherules; but am of opinion that Gegenbaur's account is probably
correct, according to which the spheres at first present gradually grow and
develop into vesicles, in the interior of which solid bodies (nuclei of
His?) appear and form the permanent yolk spheres. When the yolk spheres are
still very small they have the typical oblong form[393] of the ripe ovum,
and this form is acquired while the centre of the ovum is still free from
them.
Footnote 393: The peculiar oval, or at times slightly
rectangular and striated yolk spherules of Elasmobranchii are
mentioned by Leydig and Gegenbaur (Pl. 11, fig. 20), and
myself, _Preliminary Account of Development of Elasmobranch
Fishes_, and by Filippi and His in _Osseous Fishes_.
The growth of the yolk appears mainly due to the increase in size and
number of the individual yolk spheres. Even when the ovum is quite filled
with large yolk spheres, the granular protoplastic network of the earlier
stages is still present, and serves to hold together the constituents of
the yolk. In the cortical layer of nearly ripe ova, the yolk has a somewhat
different character to that which it exhibits in the deeper layers, chiefly
owing to the presence of certain delicate granular (in hardened specimens)
bodies, whose nature I do not understand, and to special yolk spheres
rather larger than the ordinary, provided with numerous smaller spherules
in their interior, which are probably destined in the course of time to
become free and to form ordinary yolk spheres.
The mode of formation of the yolk spheres above described appears to me to
be the normal, and possibly the only one. Certain peculiar structures have,
however, come under my notice, which may perhaps be connected with the
formation of the yolk. One of these resembles the bodies described by
Eimer[394] as "Dotterschorfe." I have only met these bodies in a single
instance in ova of 0.6 mm., from the ovary (in active growth) of a specimen
of _Scy. canicula_ 23 inches in length. In this instance they consisted of
homogeneous clear bodies (not bounded by any membrane) of somewhat
irregular shape, though usually more or less oval, and rarely more than
0.02 mm. in their longest diameter. They were very numerous in the
peripheral layer of the ovum, but quite absent in the centre, and also not
found outside the ovum (as they appear to be in Reptilia). Yolk granules
formed in the normal way, and staining deeply by osmic acid, were present,
but the "Dotterschorfe" presented a marked contrast to the remainder of the
ovum, in being absolutely unstained by osmic acid, and indeed they appeared
more like a modified form of vacuole than any definite body. Their general
appearance in Scyllium may be gathered from Eimer's figure 8, Pl. 11,
though they were much more numerous than represented in that figure, and
confined to the periphery of the ovum.
Footnote 394: "Untersuchung über die Eier d. Reptilien,"
_Archiv f. mikros. Anat._ Vol. VIII.
Dr Eimer describes a much earlier condition of these structures, in which
they form a clear shell enclosing a central dark nucleus. This stage I have
not met with, nor can I see any grounds for connecting these bodies with
the formation of the yolk, and the fact of their not staining with osmic
acid is strongly opposed to this view of their function. Dr Eimer does not
appear to me to bring forward any satisfactory proof that they are in any
way related to the formation of the yolk, but wishes to connect them with
the peculiar body, well known as the yolk nucleus, which is found in the
Amphibian ovum[395].
Footnote 395: Vide Allen Thomson, article "Ovum," Todd's
_Encyclopædia_, p. 95.
Another peculiar body found in the ova may be mentioned here, though it
more probably belongs to the germinal vesicle than to the yolk. It has only
been met with in the vitellus of some of the medium sized ova of a young
female. Examples of this body are represented on Pl. 25, fig. 25A, _x_. As
a rule there is only one in each of the ova in which they are present, but
there may be as many as four. They consist of small vesicles with a very
thick doubly contoured membrane, which are filled with numerous deeply
staining spherical granules. At times they contain a vacuole. Some of the
larger of them are not very much smaller than the germinal vesicle of their
ovum, while the smallest of them present a striking resemblance to the
nucleoli (fig. 25B), which makes me think that they may possibly be
nucleoli which have made their way out of the germinal vesicle. I have not
found them in the late stages or large ova.
The following measurements shew the size of some of these bodies in
relation to the germinal vesicle and ovum:--
Diameter of Germinal Diameter of Body in
Diameter of Ovum. Vesicle. Vitellus.
0.096 mm. 0.03 mm. 0.009 mm.
0.064 mm. 0.025 mm. 0.012 mm.
{0.019 mm.
0.096 mm. 0.03 mm {0.003 mm.
_Germinal vesicle._--Gegenbaur[396] finds the germinal vesicle completely
homogeneous and without the trace of a germinal spot. In Raja granules or
vesicles may appear as artificial products, and in Acanthias even in the
fresh condition isolated vesicles or masses of such may be present. To
these structures he attributes no importance.
Footnote 396: _Loc. cit._
Alexander Schultz[397] states that there is nothing remarkable in the
germinal vesicle of the Torpedo egg, but that till the egg reaches 0.5 mm.,
a single germinal spot is always present (measuring about 0.01 mm.), which
is absent in larger ova.
Footnote 397: _Loc. cit._
The bodies described by Gegenbaur are now generally recognised as germinal
spots, and will be described as such in the sequel. I have very rarely met
with the condition with the single nucleolus described by Schultz in
Torpedo.
My own observations are confined to Scyllium. In very young females, with
ova not larger than 0.09 mm., the germinal vesicle has the same characters
as during the embryonic periods. The contents are clear but traversed by a
very distinct and deeply staining reticulum of fibres connected with the
several nucleoli which are usually present and situated close to the
membrane.
In a somewhat older female in the largest ova of about 0.12 mm., the
germinal vesicle measures about 0.06 mm., and usually occupies an eccentric
position. It is provided with a distinct though delicate membrane. The
network, so conspicuous during the embryonic period, is not so clear as it
was, and has the appearance of being formed of lines of granules rather
than of fibres. The fluid contents of the nucleus remain as a rule, even in
the hardened specimens, perfectly clear, though they become in some
instances slightly granular. There are usually two, three, or more nucleoli
generally situated, as described by Eimer, close to the membrane of the
vesicle, the largest of which may measure as much as 0.006 mm. They are
highly refracting bodies, containing in most instances a vacuole, and very
frequently a smaller spherical body of a similar nature to themselves[398].
Granules are sometimes also present in the germinal vesicle, but are
probably only extremely minute nucleoli.
Footnote 398: Compare, with reference to several points, the
germinal vesicle at this stage with the germinal vesicle of the
frog's ovum figured by O. Hertwig, _Morphologisches Jahrbuch_,
Vol. III. pl. 4, fig. 1.
In ova of 0.5 mm. the germinal vesicle has a diameter of 0.12 mm. (Pl. 25,
fig. 21). It is usually shrunk in hardened specimens though nearly
spherical in the living ovum. Its contents are rendered granular by
reagents though quite clear when fresh, and the reticulum of the earlier
stages is sometimes with difficulty to be made out, though in other
instances fairly clear. In all cases the fibres composing it are very
granular. The membrane is thick. Peculiar highly refracting nucleoli,
usually enclosing a large vacuole, are present in considerable numbers, and
are either arranged in a circle round the periphery, or sometimes
aggregated towards one side of the vesicle; and in addition, numerous
deeply staining smaller granular aggregations, probably belonging to the
same category as the nucleoli (from which in the living ovum they can only
be distinguished by their size), are scattered close to the inner side of
the membrane over the whole or only a part of the surface of the germinal
vesicle. In a fair number of instances bodies like that figured on Pl. 25,
fig. 27, are to be found in the germinal vesicle. They appear to be
nucleoli in which a number of smaller nucleoli are originating by a process
of endogenous growth, analogous perhaps to endogenous cell-formation. The
nucleoli thus formed are, no doubt, destined to become free. The above mode
of increase for the nucleoli appears to be exceptional. The ordinary mode
is, no doubt, that by simple division into two, as was long ago shewn by
Auerbach.
Of the later stages of the germinal vesicle and its final fate, I can give
no account beyond the very fragmentary statements which have already
appeared in my monograph on Elasmobranch Fishes.
_Formation of fresh ova and ovarian nests in the post-embryonic
stages._--Ludwig[399] was the first to describe the formation of ova in the
post-embryonic periods. His views will be best explained by quoting the
following passage:--