On an Ovum of Ornithorhynchus exhibiting
Polar Bodies and Polyspermy.
By
Professor J. Bronte Gatenby,
Dublin University,
and
Professor J. P. Hill, F.R.S.,
University College, London.
With Plate 10 and 1 Text-figure.
AMONGST the Monotreme material in the possession of one
of us are two large slides of sections of a uterine egg of Ornithorhynchus labelled c, diameter G-75 mm. (No. 1, August 19,
1901). This particular egg was cut on the sliding microtome,
after embedding in Stepanow's clove-oil celloidin, at the timo
Wilson and Hill were working up the material for their paper
on the development of Ornithorhynchus (1) in the hope that
it would exhibit an early phase of cleavage. In this respect
the egg proved a disappointment, being unsegmented, and it
was put on one side, though it was noted at the time that it
was possibly at the stage of polar body formation. We have
recently had occasion to re-examine the sections, and though
we have had considerable difficulty in interpreting the structural appearances in the region of the germinal disc and have
been unable to distinguish either the male and female pronuclei or the cleavage nucleus, we think that the egg is actually
at the stage just following the formation of the polar bodies.
It is therefore unique and worthy of a brief description. Moreover, the sections, considering the refractory nature of the
material, are quite good, the sectional plane coinciding almost
perfectly with the vertical or polar axis, and afford a very
clear picture of the general structure of the egg and mor
230
J. BRONTE GATBNBY AND J. I'. HILL
especially of the latebra and its relations to the germinal disc,
of which no detailed account exists.
The diameter of the intact egg (c) enveloped by the albumen
and shell is 6-75 mm. ; after removal of the envelopes the
ovum proper measures (in sections) 3-8 x 3-7 mm. in diameter.
The twin egg (cc) had the same diameter as (c). .The ovum,
measured in spirit, has a diameter of about 4-5 mm. Wilson
and Hill's eggs A and AA (in the eight-celled cleavage stage)
measured in the intact state 5-5 mm. in diameter, after removal
of shell and albumen 4 mm.; their egg NN in a later cleavage
stage measured 5 mm. Their egg o, in an early phase of
germ-layer formation, measured in the intact condition
4-75 mm., and after removal of shell and albumen 4 mm.,
whilst their egg a, in the early bilaminar blastocyst phase,
measured in diameter 6-5 x 6 mm. We have three other
records of early uterine eggs which may be mentioned here :
JJ, September 1905, single egg in an early phase of cleavage,
intact 6 mm., after removal of shell and albumen 4-5 mm. ;
No. 2, September 9, 1905, twin eggs, intact 6-5 mm. and
6-75 mm., after removal of shell and albumen 4-5 mm. in
diameter; No. 3, September 9,1905, twin eggs, intact 6-75 mm.
and 7 mm., ovum proper 5 x4-5 mm. and 4-5 mm. respectively.
The largest ovarian ovum we have observed measures (in
section) 4-4 x 4-16 mm. in diameter. There is evidently some
variation in the size of the ovarian ovum, but on the average
it may be said to measure from 4 to 4-5 in diameter. Our
records are thus at variance with those of Caldwell (2), who
states that the ripe ovarian ovum measures in Echidna 3 mm.
in diameter and in Platypus 2-5 mm. On the other hand, he
records the diameter of segmenting uterine eggs of Echidna
and Ornithorhynchus as in both cases 6 mm.
The variations in size of early intra-uterine eggs (prior to
the formation of a complete enclosing layer of ectoderm)
may be attributed partly to variations in the size of the ovum
itself, partly to variations in the thickness of the albumen,
the latter variation being probably due, mainly at least, to
a variable amount of absorption of fluid from the uterine lumen.
OVUM OP ORNITHORHYNCHU3
231
GENERAL DESCRIPTION OF THE OVUM.
The general structure of the Monotreme ovum has been
dealt with by one of us (Gatenby) in a paper on the gametogenesis of Ornithorhynchus (3), to which the reader is referred
for references to the papers of Caldwell and Sernon.
PI. 10, fig. 1, is a drawing of a median vertical section through
the entire ovum. The main bulk of the ovum, it may be recalled,
is formed of yolk-spheres, of a yellow tint in the fresh condition, which stain deeply and uniformly with haematoxylin
and which vary in size from fine to coarse, the latter attaining
a diameter up to 0-036 mm. They are disposed in two zones,
viz. a peripheral closely packed outer yolk-zone (OZY) and
a central less closely packed zone, the inner yolk-zone (IRY).
Towards the periphery of the ovum the yolk-spheres of the
outer zone become smaller, and so form a thin rather illdefined peripheral zone (PZ) which extends round to become
continuous with the margin of the germinal disc occupying
the upper pole.
The germinal disc (GD) appears in the intact ovum as a
circular whitish spot with a slightly raised margin, its colour
rendering it conspicuous by contrast with the yellow tint
of the yolk-spheres of the rest of the ovum.
The surface diameter of the disc is just over 0-05 mm. and
its thickness down to the clear vacuolated zone is about
0-025 mm.
In section the disc is seen to merge peripherally into the
outer yolk-zone, whilst below it passes into continuity with
a clear vacuolated mass (cz in PI. 10, fig. 1) in the position
of the so-called nucleus of Pander of the bird's ovum, which is
continued down as the slender neck of the latebra. This
latter penetrates into the central yolk-zone and, gradually
widening, constitutes the club-like latebra (LZL) occupying
the central region of the egg.
The germinal disc is formed of alveolar cytoplasm presenting
a dense granular appearance owing to the presence in it of
numerous yolk-spheres. The spheres in the central region
OVUM OV ORNITHORHYNCHUS
233
mass (PI. 10, fig. 1, cz) which occupies the position of the
so-called nucleus of Pander of the bird's ovum, and which
apparently represents the upper portion of the latebra. From
this mass the neck of the latebra extends down for some
distance as a thin elongated cord, and then gradually expands
to form an elongated club-like mass (LZL), occupying the
central region of the egg, the neck and this latter mass having
a combined length of 1-8 mm. The neck of the latebra is
enclosed in a tube-like investment of closely packed large
yellow yolk-spheres (PI. 10, fig. 1, UAL), prolonged up from
the central yolk-zone, outside which is a second investment of
less coarse spheres prolonged down from the peripheral region
of the germinal disc. At the level of the upper part of the
latebral neck, these two investments are separated for a short
distance by a downward prolongation of the clear zone (PL 10,
fig. 1, IAV).
The latebra has essentially the same structure as the clear
zone, i.e. it consists of a cytoplasmic basis containing very
fine yolk-spheres. In its deepest part (LZL) it presents a hollow
appearance, owing to the' presence of large, irregular vacuolar
spaces, whilst in its neck region similar vacuolar spaces are
present, only they are spherical and separated by thin intervening cross-strands, with the result that the neck presents
a ladder-like appearance in section (PI. 10, fig. 1).
POLAR BODIES AND NUCLEAR STRUCTURES IN THE
REGION OF THE GERMINAL DISC.
Careful examination of the germinal disc in the sections
has shown that in addition to the structures which we regard
as in all probability the first and second polar bodies, there
are present in its substance a number of free nuclei. In PI. 10,
fig. 1, the positions of the polar bodies and of certain of these
nuclei have been indicated diagrammatically as if they had
all been cut in one plane ; they are marked by the numerals
1, 2,. 3, 4.
The number 4 indicates the position of two bodies, situated
234
J. BRONTE GATBNBY AND J. P. HILL
outside the region of the germinal disc, which we regard as the
products of division of the first polar body and which are
represented at a much higher magnification in PI. 10, fig. 5.
The two structures in question lie in relation to a shallow
depression on the surface of the yolk, situated about 1-34 mm.
from the centre of the germinal disc. They have the form of
ovoidal masses, lying in contact with each other and with the
floor of the depression, but quite distinct and separate from
the yolk. They are of unequal size ; the one nearest the disc
is the smaller of the two and measures 0-038 x 0-031 mm. in
diameter, the other, the larger, measures 0-05 x 0-031 mm.
They have stained very lightly, appear definitely contoured,
and consist of alveolar cytoplasm in the framework of which
are present fine yolk-spheres similar to those of the disc.
No chromosomes are recognizable, but certain granules in the
cytoplasm which stain more deeply than the yolk-spheres are
probably of chromatinic nature. We have little hesitation
in identifying these two structures as the products of division
of the first polar body. If our identification is correct, then
we must assume that their present position, outside the limits
of the germinal disc, is the result of secondary displacement.
The numeral 3 in PI. 10, fig. 1, indicates the position of the
structure which we regard as in all probability the second
polar body. It is represented under higher magnification in
PI. 10, fig. 3. Present in four adjoining sections, the structure
in question appears as a flattened ovoidal body situated in
a surface depression of the disc, a little to one side of the centre
of the latter and measuring 0-036x0-026 mm. in diameter.
Structurally it resembles the first polar bodies in consisting
of alveolar cytoplasm in which there are present fine yolkspheres, but it differs from them in that it is not free but is
still continuous below with the cytoplasm of the germinal
disc. In other words, the second polar body is not yet completely constricted off from the oocyte. No definite chromosomes are distinguishable in it, though the altered remains of
such are probably represented by certain minute bodies readily
distinguishable by their characters from the yolk-spheres.
OVUM OF OENITHOBHYNCHUS
235
One such body recalling a sperm-head and consisting of darker
and lighter-staining segments is shown at xx in PI. 10, fig. 3.
Immediately adjacent to the second polar body is a small
clear area of the disc (PI. 10, fig. 3) in which the cleavage
nucleus or alternatively the two pronuclei might have been
expected to occur, since such clear areas are present around
other nuclei in the disc and indeed are of general occurrence
round the nuclei in yolk-laden eggs during fertilization and
cleavage ; but we have failed to make out any nuclear structures
in relation to this particular area.
Turning now to the nuclei which we have actually found
present in the disc, the numeral 1 in PI. 10, fig. 1, indicates
the site of a nucleus which is shown, along with the immediately surrounding area, in PL 10, fig. 4. This nucleus is
situated well towards the margin of the disc, surrounded by
a clear zone of cytoplasm, and measures 0-016 x 0-014 mm. in
diameter. It lies in close contact with the membrane-like
layer (egg-membrane) investing the surface of the disc, is
diffusely staining and vesicular in • character, and possesses
a faint reticulum and a darkly staining nucleolus with a
characteristic vacuolated appearance. To one side of it is
a vacuole in the cytoplasm of the disc, so that it has evidently
caused a certain amount of disturbance in the latter. Four
other nuclei showing essentially the same characters as that
just described are present in other parts of the marginal region
of the disc ; two of them, having diameters of 0-019 x 0-016 mm.
and 0-02 x 0-019 mm. respectively, lie in contact in one section.
From their marginal position we think there is little doubt
that all these five nuclei are accessory sperm-nuclei. If that
interpretation is correct, the Monotrerne ovum must be held
to be polyspermic like the similarly meroblastic ova of the
Elasmobranchs, Birds, and Reptiles.
There remains for mention a curious group of nuclei, the
position of which is indicated by the numeral 2 in PI. 10,
fig. 1, and which is shown under higher magnification in PI. 10,
fig. 2 (combined from two sections). This group consists of
five nuclei, two occurring in one section, three in the next,
NO. 270
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236
J. BRONTE GATENBY AND J. P. HILL
situated some nine sections to one side of the centre of the
disc and accordingly not so definitely marginal in position
as the nuclei described above. The five nuclei lie massed
together in a clear area of the disc, and, with the exception
of one which is situated below the others, are in close contact
with the egg-membrane, investing the free surface of the disc.
Structurally the nuclei are all similar, and, so far as we can see,
they differ in no essential particular from the accessory spermnuclei described above, being pale staining and vesicular
and containing each a single deeply staining and vacuolated
nucleolus. The largest nucleus in the group has a diameter
of about 0-02 x 0-019 mm.
On account of their more central position we were at first
inclined to think that these nuclei might have some relationship to the cleavage nucleus, but the facts that their number
is an odd one, that no one of them is sufficiently large to be
accounted for by belated division, and that they are structurally identical with the marginal nuclei are all against that
idea, and favour the view that they are, like the latter, accessory
sperm-nuclei, though it must be admitted that the clumping
together of accessory sperm-nuclei to form a regular nest appears
to be unknown amongst the polyspermic eggs of lower forms.
It is singularly unfortunate that we have been unable to
recognize either the cleavage nucleus or the male and female
pronuclei. This may be due to a defect in the series or to the
fact that in some of the sections the surface of the disc has
suffered slight abrasion. Apart from our inability to find
these nuclei, we have no reason to regard this ovum as other
than normal.
CONCLUSIONS.
So far as our observations go, and they obviously require
confirmation and extension, they would appear to indicate
(1) that the two polar bodies in Ornithorhynchus are of
unequal size, the first being larger than the second and undergoing division before the latter is completely separated off, and
(2) that the ovum is polyspermatic.
OVUM OF ORNITHORHYISICHUS
237
We are not acquainted with any detailed account of polar
body formation in the Reptilia, but amongst Birds Harper (4)
has described the formation of the polar bodies and the process
of fertilization in the ovum of the pigeon. He states that the
polar bodies are given off whilst the ovum is situated in the
proximal region of the glandular segment of the oviduct.
They lie below the egg-membrane, in a depression in the
cytoplasm. Comparison of Harper's fig. 23 (showing the
first polar body separated off and the second polar spindle)
with his fig. 23 (showing the second polar body not yet completely separated and the clumped chromosomes of the female
pronucleus) demonstrates that the first polar body is much
larger than the second. The second polar body is said to
degenerate before the first. No mention is made of any
division of the latter. Harper's fig. 25 rather strikingly
recalls our fig. 3, PI. 10.
The polyspermatic condition of the ovum of Ornithorhynchus
is of interest in view of the general occurrence of polysperniy
in other meroblastic eggs, and more especially in the Eeptiles
and Birds. In these forms numerous accessory sperm-nuclei
are present in the marginal region of the blastodisc, and
condition the appearance of a transitory accessory cleavage
around the periphery of the blastodisc proper. In the Ornithorhynchus ovum the number of these accessory nuclei is relatively
quite small, and it seems likely that they very soon degenerate
and disappear. The reader is referred to the papers of
Nicolas (5) and Harper (4) for detailed accounts of the
phenomena associated with polysperniy in a reptile (Anguis)
and a bird (Columba) respectively.
BlBLIOGEAPHY.
1. Wilson, J. T., and Hill, J. P.—" Observations on the Development of
Ornithorhynchus", ' Phil. Trans. Boy. Soc.', vol. 199 B, 1907.
2. Caldwell, W. H.—" The Embryology of the Monotremata and Marsupialia", ibid., vol. 178B, 1887.
3. Gatenby, J. Bronte.—" Some Notes on the Gametogenesis of Ornithorhynchns paradoxus ", ' Quart. Journ. Micr. Sci.', vol. 66, 1922.
R2
238
J. BRONTE GATENBY AND J. P. HILL
4. Harper, E. H.—" The Fertilization and early Development of the
Pigeon's Egg ", ' Amer. Journ. of Anat.', vol. 3, 1904.
5. Nicolas, A.—" Recherohes sur l'embryologie des Reptiles. 4. I.a
segmentation chez 1/Orvet (Anguis fragilis)", ' Archiv. de Biol.',
torn, xx, 1904.
EXPLANATION OB1 PLATE 10
LETTERING.
cz, clear area beneath germinal disc (in site of nucleus of Pander) ;
GD, germinal disc; IEY, inner yolk-zone; LW, downward continuation
of cz ; LZL, lower portion of latebra; N, nucleus, OZY, outer yolk-zone ;
rz, peripheral zone of yolk; UAL, inner yolk-investment of neck of latebra ;
xx, possible chromatin body in second polar body; z, zona (vitelline
membrane).
Fig. 1.—Ovum of Ornithorhynchus in vertical median section, showing
germinal disc and latebra. The numerals 1, 2, 3, 4 mark the positions of
the polar bodies and nuclear structures. At the left upper corner (inside
a square) are three spermatozoa drawn to the same scale as the ovum.
Fig. 2.—Composite figure drawn from two adjoining sections, showing
group of five nuclei (the position of which is indicated by the numeral 2
in fig. 1). The significance (if any) of the minute projection at the surface
of the disc is not clear.
Fig. 3.—High-power view of area indicated by the numeral 3 in fig. 1,
showing second polar body.
Fig. 4.—Accessory sperm-nucleus and adjoining area of the disc, from
the marginal region of the latter (numeral 1 in fig. 1).
Fig. 5.—High-power view of the area indicated by the numeral 4 in
fig. 1, showing what are regarded as the products of division of the first
polar body.
Fig. 0.—The spermatozoa of Ornithorhynchus, drawn to the same scale
as figs. 2-5.
Quart. Journ. Micr. Sci.
Vol. 68, N. S., PL 10