OBSERVATIONS ON OVULATION IN THE RABBIT - BY A. WALTON, PH.D., AND J. HAMMOND, M.A.
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190
OBSERVATIONS ON OVULATION IN THE RABBIT
BY A. WALTON, P H . D . , AND J. HAMMOND, M.A.
School of Agriculture, Cambridge.
{Received 2nd September t 1928.)
(With Plate V, and One Text-figure.)
Coloured Plate supplied by Author.
INTRODUCTION.
THE rabbit is particularly suitable material for the study of ovulation in that
ovulation is not spontaneous but occurs normally only after coitus and there is a
fairly definite interval of about 10 hr. between mating and actual rupture of the
follicle. Our observations fall under the following headings and approach the
problem from different angles.
(a) Histological study of the follicle previous to rupture.
(b) Observations with binocular dissecting microscope of the actual process of
ovulation in the anaesthetised animal together with subsequent histological study
of the individual follicles observed.
(c) Experimental rupture and ablation of ripe follicles.
The rabbits used were mainly of our own breeding from inbred strains which
were fairly uniform as regards size and fecundity. In the protocols the different
strains are designated by letters: C = Flemish strain, X = Fx of the cross Polish (F)
and Belgian (E), A, B = animals bought in open market and of unknown ancestry.
(a) HISTOLOGICAL STUDY OF THE FOLLICLE PREVIOUS TO RUPTURE.
Series of does were killed at o, 3, 6 and 9 hr. after coitus. The follicles were
observed fresh and the ovary then fixed in 10 per cent, formalin. After fixation
some mature follicles were sectioned freehand with a razor and the sections showing
the maximum surface area picked out for measurement with a micrometer eyepiece.
From each doe two follicles were chosen and from each follicle two sections were
selected and two diameters at right angles (the breadth and depth) measured. The
average of the resulting eight measurements was taken as representing the follicular
diameter characteristic of the doe. Assuming the follicle to be approximately
spherical the volume has been computed and the results are shown in Text-fig. 1.
It is seen that the follicles vary considerably in size and that there is a definite
increase in volume of the follicle after coitus. There was some indication from the
measurements that the follicle becomes rather deeper in proportion to its breadth
but the method of measurement is relatively crude and there is considerable
variation in the shape of the follicle depending upon how crowded they are on theObservations on Ovulation in the Rabbit 191
surface of the ovary. It is important to note that blood follicles (i.e. follicles which
do not ultimately rupture but in which vessels in the theca break down and allow
blood to escape into the cavity of the follicle) may attain a greater volume than the
normal follicle about to rupture.
Other follicles were embedded in paraffin and complete serial sections cut and
stained with haematoxylin and eosin. No detailed histological description need be
given since the subject has been very fully investigated by Sabotta (1897), Honore
(1900) and Corner (1919). Only points of special interest will be mentioned in
a brief summary. The observable changes in the follicle are relatively slight and
there is considerable variation from follicle to follicle. It would be impossible
5-0
1 4-0 - 0
a 0
0 #
1 3-0 - »
0
o t'
v m
B 2 0
t
I 0
*
1-0
i i i i
0 3 6
Hours after coitus
Fig. 1. Growth of Follicle after Coitus. Volume calculated from average follicular
diameter of each rabbit.
from observation alone to state with any certainty the exact stage which an isolated
follicle represented. If, however, large numbers of follicles in each series are
carefully compared more or less definite changes are detectable.
At coitus the mature follicle is of considerable size in comparison with those which
are immature and not destined to rupture. It is relatively flat and has a bluish tinge;
no macula pellucida is apparent. The discus proligerus is dense; the granulosa is about
4~8 cells in depth round the follicle and shows slight columnar arrangement in the layers
next the membrana propria. " Bodies of Call and Exner " are present in most sections and
more frequently in the discus. The membrana propria appears as a thin line. The "luteal
cells " of the theca interna are small. The liquor folliculi stains uniformly a pale magenta.
At 3 hr. after-coitus. Little or no change is apparent.192 A. WALTON and J. HAMMOND
At 6 hr. after coitus. The discus appears to be broken up by clear non-staining fluid
(secondary liquor folliculi of Robinson (1918)?). The cells of the thecainterna are possibly
slightly larger and follicular measurements show a slight change in the shape of the
follicle which protrudes more from the surface of the ovary.
At 9 hr. after coitus. The vascularity of the theca is increased. Secondary liquor
folliculi is more abundant. The granulosa is about 3-5 cells deep round the follicle. There
is some slight hypertrophy of the luteal cells of the theca interna.
The method of fixation did not allow a critical study to be made of the maturation
changes of the ovum but it has been shown by Heape (1905) and Sabotta (1897) that this
takes place during this time.
(b) OBSERVATIONS ON OVULATION IN THE ANAESTHETISED ANIMAL.
The process of ovulation was watched with a binocular dissecting microscope
through an abdominal incision in the middle line. The rabbit ovulates about 10 hr.
after coitus and observations were begun just prior to this time and continued for
as long as changes were detectable. Timed drawings and notes were made of the
appearance of the follicles (examples are shown in Table I). At the close of the
observations the animal was killed and the ovaries fixed in formalin. Serial sections
were cut of the follicles which had been under observation, each follicle being
identified from the drawings previously made.
Systematic research on the optimum conditions for the experiment was not
Table I. Observations on the follicles during
Appearance
Time Time ovaries
Doe copulated first observed
A B C D
(a.m.) (p.m.) (p.m.) (p.m.) (p.m.) (p-m.)
F336 8.15 6.20 Ruptured before 6.30. Congested First seen at 6.35. Rounded
22. ii. 26 (10 hr. 5 min. 6.20 with pale pro- 7.25. Had just 7.55. Congested
after coitus) 7.0. Red patch at truding pimple ruptured. Dif- but not ruptured
summit marked in centre fuse redandclear
but edges much 6.40. Ruptured jelly running
paler 6.45. Smaller, red from centre
all over, blood
and fluid run-
ning downovary,
blood on former
clear pimple and
periphery clearer
6.55. Dull diffuse
colour. No col-
lapse
F357 8.40 6.45 6.45. With clear 6.55. Red, not 6.55. Stringy mass First seen 7.5.
23.11. 26 (10 far. 5 min. pimple, not rup- ruptured but did running from Pimpleatsummit
after coitus) tured so shortly after clear central pim- 7.15. Clear pim-
7.10. Ruptured. 7.15. Blood round ple; blood extra- ple at summit;
Stringy mass pimple. Col- vasation at edge rest of follicle
running down lapsed of pimple with blood ves-
ovary from pim- 7.15. Collapsed; sels congested
ple blood round
edge of pimpleObservations on Ovulation in the Rabbit
carried out but after some failures the technique ultimately adopted with success
was as follows: The operation was begun only just before the normal time of
ovulation. The room was kept warm and moist. The animal was laid on a warm
tank on the operating table. Ether alone was used as anaesthetic. The intestines
were gently moved to one side and kept wrapped in warm moist cloths. The ovary
remained in its normal position or was propped slightly with swabs of cotton-wool
moistened with warm Ringer's solution. Warm Ringer's solution was used to keep
all exposed tissues moist. The ovary was only momentarily exposed for the purpose
of observation and special precaution was taken to avoid scorching with the light
bulb used for illumination. When not actually under observation the ovary was
carefully covered with the intestines or moist swabs.
The actual times after coitus at which the follicles ruptured under observation
varied from 9f-i3i hr. (see Table II). Occasionally some follicles had ruptured
before observation began. In any one animal not more than two or three ruptured
at exactly the same time although the majority appeared to follow each other at
fairly short intervals. It may have been that the rate of ovulation was influenced
by the anaesthetic and operation, in fact we attribute failure to observe ovulation
in A 8, A 4, Table II, to the early period (9 hr. 10 min., 8 hr. 55 min. after coitus)
at which operative interference began. There can be no doubt, however, that all
follicles of the same batch do not rupture at exactly the same time, for in several
cases where observations began about 10 hr. after coitus one or more follicles had
already ruptured and others ruptured subsequently under observation (A 9,
the time of rupture. (Examples of notes made.)
of follicles
Time doe
killed
E F G H 1
(p.m.) (p.m.) (p.m.) (p.m.) (p.m.) (p.m.)
6.33. Red spot in 7.2. Clear central 7.40. Ruptured. 6.20. Clear raised Clear, no change 8.0
centre patch, rest con- Clear thick fluid pimple with con- (11 hr. 45 min.
6.50. More pro- gested streaming from gested spot at after coitus)
minent 7.20. Blood ves- clear central edge
6.55. Central sels more marked patch 7.40. Ruptured.
pimple dark red 7.55. Congested Stringy fluid
under surface as all over but not streaming from
though blood yet ruptured central pimple
vessels broken
down
7.10. Blood at
surface of cen-
tral pimple
7-5O. More con-
gested
6.50. Ruptured, 6.50 and 7.0. Clear 6.50. Clear pim-
blood in centre pimple at sum- ple in centre (10 hr. 35 mm.
7-o. Smaller, col- mit 7.15. Clear cen- after coitus)
lapsed; red in 7.15. Ruptured. tre. Had rup-
centre Stringy mass tured but not
7.15. Centre clear, coming from completely col-
ring of extra- clear pimple; lapsed
vasated blood much smaller
round it. Much
smaller
BJEB'Vlii194 A. WALTON and J. HAMMOND
E 208, etc.). Several of the large follicles which had not ruptured when the animal
was killed would probably not have done so normally. Sections showed that they
were probably atrophic (F 359, G and H).
Plate V, figs. 1-6, illustrates the external changes of the follicle in plan and in
elevation. The ripe follicle, 9 hr. after coitus, has a convex surface with a fairly wide
base and is covered with a fine network of small blood vessels which can be easily
seen through the very transparent surface cells (Fig. 1). The first sign of approaching
ovulation is the gradual formation of the macula pellucida at the apex, formed
possibly by increase in internal pressure, cutting off the blood supply in this area.
The whole follicle protrudes more markedly from the ovarian surface (Fig. 2).
This is followed by further enlargement of the follicle and an increase in the
congestion of the blood vessels is distinctly seen (Fig. 3). In the next stage the
macula pellucida somewhat rapidly blows out as a pimple (Fig. 4). The capillaries
and small vessels at the base of the pimple are ruptured before the actual surface
and there may be some extravasation of blood into the follicle round the line of
rupture. It is obvious, however, that rupture of the theca precedes the extra-
vasation of blood and is in no way causally dependent upon it. Rupture of the
pimple to the surface rapidly follows and liquor folliculi, frequently stained with
blood, streams down the side of the follicle (Fig. 5). The whole process of ovulation,
the congestion and swelling of the follicle, the formation of a pimple at the surface
and the gradual rupture and extrusion of contents strongly resembles the formation
and rupture of a boil and, not unlikely, somewhat similar mechanisms are involved.
The ovum has not actually been observed coming through the orifice but it has
been picked up with a capillary pipette from the flow just after rupture.
The liquor folliculi does not squirt from the follicle but flows steadily and
continuously for some little time. During this process the follicle walls may
gradually collapse but in many cases there is not a very marked change in the
volume of the follicle. The congestion of the blood vessels diminishes. Gradually
the liquor folliculi ceases to flow and appears to clot and forms, in the orifice, a plug
which protrudes a little from the surface. The plug is stained with blood and is
a bright translucent red (Fig. 6).
Later the former pimple is filled by a growth of luteal tissue which forms a
characteristic projection on the surface of the newly formed corpus luteum and its
presence is a certain indication that ovulation and actual rupture have taken place.
A similar projection has been observed on the corpus luteum of the cow (Kiipfer,
1920; Hammond, 1927), sow (Kiipfer, 1920 b; Corner, 1915), rabbit (Colin, 1888,
Fig. 220) and rat (observations of the authors) and is probably a feature common
to other mammals.
After rupture the base of the follicle becomes much narrower owing probably
to elastic readjustment of the ovarian surface and the whole follicle protrudes more
abruptly from the surface of the ovary.
Subsequent histological examination of the follicles which had been under
observation extends the description already given of the stages previous to actual
ovulation. Shaw (1927) states that in the human follicle the cumulus which isTable II. Time of rupture of follicles under observation.
Letters before the bracket A [ denote follicles which ruptured before operation began. Letters between the brackets [A] denote follicles which ruptured
under observation. Those enclosed by small brackets (A) had ruptured unobserved just previously. Letters after the bracket ] A denote follicles which
were unruptured when doe was killed.
Time after coitus
Hr. 8 9 10 11 12 13
Min. 30 45 0 15 3O 45 0 15 30 45 0 is 30 45 0 15 30 45 0 15 30
Animals under an-
aesthetic :
A "1 I
Doe No. A 9 B H D
F r J y
L ~\AB
AS No follicles ruptured
G d uring observation CD
[ JEF
E154 BC r No follicles ruptured d uring observation ] A
DE L I AB
A4 (F)* CD
1 C
L C
E208 B A [ Q ) D
F336 A B (Q G DF
H El
E B AF1 nu
F357 C G}
AB C H
F359 D ] G
A C
F391 B E
l ]D
Summary of ob-
served ruptures 1+ 3+ 6 3 2 3 1 1 1
Animals killed:
Doe No. C 339 x f 11 ruptured
1 s unruptured
£425 112 ruptured
M
\ 1 unruptured196 A. WALTON and J. HAMMOND
originally directed towards the medulla gradually rotates until just before ovulation
it lies against the spot which is to become the stigma, but according to Thompson
(1919 a) the evidence is very conflicting. Examination of the follicles on the point
Table I I I . Histological appearance of follicles which had been under observation.
Fol- Ruptured Diameter Description
Doe licle nun.
F336 D No 1-25 Ovum present, small congested blood vessels in theca
but no extravasation of blood. Granulosa about
5 cells thick on side of cumulus which was lateral, 2—3
cells thick on other side. Theca lutein cells in thin
layer
H Yes, about 0-98 No ovum or cumulus present, liquor folliculi almost
20 min. all extruded. Extravasated blood in walls round
aperture and little in central cavity. Granulosa cells
about 12 thick all round follicle. Walls collapsed
and slightly wavy, basement membrane creased.
Theca layer thick
F359 No 1-29 Ovum present but degenerate: granulosa only present
on cumulus side of follicle, broken up and detached
on other side: little congestion of thecal vessels.
Theca only well developed on side -where granulosa
present
H No Similar to G
A Yes, about 1 hr. 0-78 Similar to F 336 (H); blood seen passing from
vessels below granulosa into cavity of follicle
B Yes, about 1 hr. 1 02 Similar to A
E Yes, 25 min. I'OO Similar to A but still containing much liquor folliculi
C Yes, 38 min. 0-96 Similar to A but no blood in central cavity
D Yes, about 1 hr. Similar to A but no blood in central cavity-
0-78
F391 A Yes, ii- hr. No ovum or cumulus present; very little liquor
0-80 folliculi. Granulosa about 35 cells thick. Walls
showing slight wrinkling and irregular folding of
theca. Little blood extravasated into theca at sides
of aperture. Theca interna layer thick
E Yes, 1 hr. 0-98 Similar to A but rather more liquor. Granulosa 23
cells thick. Extravasation of blood into theca at base
of follicle
A4 A,B, No Ovum present; granulosa 6 cells thick near cumulus
C,D and about 3 cells thick on other side. Theca luteal
cells small to moderate size, blood vessels of theca
congested. Wall in region of macula pellucida
wrinkled in outline
E No Atrophic: granulosa detached and cells scattered
throughout liquor folliculi
F Yes No ovum or cumulus present. Granulosa about
(in fixation?) 12 cells thick all round except near point of rupture,
where 3—4 cells thick. Much liquor folliculi present.
Theca wavy and luteal cells of theca large to
moderate size: blood vessels few and not very
congested
of rupture shows that in the rabbit the cumulus and ovum do not occupy any
definite position with respect to the point of rupture. The continuous nature of the
flow and gradual contraction of the follicle after rupture render it unnecessary to
postulate any particular position of the ovum with respect to the orifice as a factor
aiding its extrusion from the follicle. Since two follicles (F 359, G and H) which
had not ruptured at 10 hr. 50 min. after coitus showed the granulosa heaped upObservations onOvulation in the Rabbit 197
round the ovum and for the most part detached from the theca interna it was
thought that this might represent a stage in the process of extrusion but the ovum
appeared abnormal and it seems more probable that the foUicles were in process of
becoming atrophic and would not have ruptured normally.
The remains of the liquor folliculi in the centre of the follicle show stream lines
of clear non-staining fluid (secondary liquor folliculi) often marked by a few red
blood corpuscles extravasated from an occasional ruptured vessel in the theca or
membrana propria. The "bodies of Call and Exner," which are seen in almost all
follicles previous to rupture and particularly in those at a standstill in development,
are absent from the recently ruptured follicle. These bodies are in all probability
small accumulations of liquor folliculi which during slow development do not
coalesce, as they do later when active secretion is taking place in the ovulating
follicle.
After rupture the diameter of the follicle diminishes (see Table III) and as a
consequence the granulosa, which in the follicle before rupture is extended and
only about 6 cells thick near the cumulus and 2-3 cells thick elsewhere, becomes
packed together and forms a layer some 12—30 cells in depth, distributed fairly
evenly round the perimeter of the follicle; near the point of rupture, however, it
remains about 4 cells in depth. It seems probable that near the orifice the walls
of the follicle are not subject to so much lateral pressure from surrounding tissues
and the contraction of the walls is consequently less. The walls of the follicle have
a slightly wavy outline and the theca interna is well marked and thicker—the results
probably of collapse. The luteal cells of the interna are especially prominent and
appear hypertrophied. The membrana propria is now seen to consist largely of a
network of well-filled capillaries.
Subsequent changes in the follicle are associated with the formation of the
corpus luteum and have been recently described by Marshall (1925).
(c) EXPERIMENTAL RUPTURE AND ABLATION OF RIPE FOLLICLES.
Bouin and Ancel (1910) claimed that by pricking the ripe follicle in the rabbit
corpora lutea were occasionally formed but that more often the pricked follicles
filled with blood and became atretic. They also stated that if the follicles in one
ovary were artificially ruptured, the follicles in the other ovary might rupture
spontaneously. O'Donoghue (1913) confirmed their main findings but found that
corpora lutea did not invariably form after the operation. We have attempted to repeat
these experiments. In two rabbits the operation of artificial rupture of the follicles
in one ovary was performed through an incision in the flank and the uterus and
cervix left completely undisturbed so that all possibility of reflex action from
handling the uterus or cervix might be eliminated. In two other rabbits the
incision was medial and the uterus and cervix deliberately handled in the course
of the operation. Handling produced an intense vascular congestion of the cervix
and uterus. The animals were killed three or four days after the operation. The
results are shown in Table IV, Series I—III. In no case did the pricked folliclesvO
GO
Table IV. Artificial rupture and ablation of mature follicles.
Appearance at operation Appearance after killing
Days
after
Series Doe operation
Date Operated ovary killed Operated ovary Other ovary
I. Rupture. Uterus not C693 15. vii. 27 All large follicles (4) pricked with 4 No corpora lutea formed. 9 small No corpora lutea formed. 4 reddish
handled points of fine scissors and z large blood follicles and 6 large clear ripe but unruptured follicles. 3 old blood
blood follicles also pricked follicles (newly formed after opera- follicles >
tion)
E779 15. vii. 27 All large and moderate sized follicles 4 No corpora lutea formed. 5 blood No corpora lutea formed. 8 large but
(12) pricked with points of fine follicles (from pricked follicles) and unruptured follicles 3
scissors 7 large clear follicles (newly formed)
II. Rupture. Uterus and X841 26. vii. 27 All large and small follicles (8) pricked 3 No corpora lutea formed. 6 blood No corpora lutea formed. 8 large but
cervix handled with points of fine scissors follicles (red) or blood clots where unruptured follicles. 1 small old
follicles pricked. 2 moderately large blood follicle (black) o
follicles (newly formed)
E836 26. vii. 27 All large and moderate sized follicles 3 No corpora lutea formed. 2 large and No corpora lutea formed. 6 large ripe as
(8) pricked with needle 4 small blood follicles (from pricked unruptured follicles
follicles) and 5 large ripe follicles
(newly formed) §
III. Rupture. 6 hr. after C892 2. viii. 27 9 a.m. Copulated with bucks 3 5 fresh corpora lutea (formed from 6 fresh (reddish) corpora lutea
coitus. Uterus not handled 3 p.m. (6 hr. after coitus). AH large pricked follicles) and 1 moderate
and moderate sized follicles (8) sized follicles
pricked with needle H-4
C894 2. viii. 27 9.5 a.m. Copulated with bucks 2 large and 4 small blood follicles No corpora lutea formed. 9 large H-4
3
3.20 p.m. (6J hr. after coitus). All (formed from pricked follicles) and follicles
large and moderate sized follicles (8) 3 large follicles (newly formed)
pricked with needle
IV. Ablation. Uterus not C618 15. vii. 27 All the large and moderate sized 4 3 scars on surface of ovary where No fresh corpora lutea. 7 large un-
handled follicles (3) cauterised follicles cauterised. 3 large follicles ruptured follicles o
(newly formed)
C6is 15. vii. 27 All the fairly large follicles (8) 4 8 scars on surface of ovary where No fresh corpora lutea. 9 large un-
cauterised follicles cauterised. 3 large clear ruptured follicles o
follicles and a few small ones (newly
formed)
V. Ablation. Uterus and A9 26. vii. 27 All the large follicles (6) and blood 3 7 scars on surface of ovary where No fresh corpora lutea. 9 large un-
cervix handled follicles (4) cauterised follicles cauterised. 4 large follicles ruptured follicles and 1 small blood
(newly formed) follicle
VI. Hemi-castration. E833 36. vii. 27 Ovary completely removed; contained 3 No fresh corpora lutea. 2 very large
Uterus and cervix handled 9 large follicles, 2 large and 3 small (double normal size) follicles. 7 ripe
blood follicles follicles and 1 small blood follicleObservations on Ovulation in the Rabbit 199
form corpora lutea nor did the ripe follicles in the intact ovary rupture spon-
taneously. Whether the uterus was handled or not made no difference. It is therefore
concluded that mere rupture of the follicle, whether accompanied by stimulation
and congestion of the cervix and uterus or not, is insufficient to cause intact
follicles to rupture, or initiate the formation of corpora lutea in those ruptured.
It is possible that at the time their experiments were made Bouin and Ancel, and
O'Donoghue, did not realise that does might ovulate after jumping one another
without actual coitus with the male and there is no statement in their papers that the
rabbits were kept apart. Perhaps either this or chance spontaneous ovulations such as
may occur at the height of the breeding season were responsible for their findings.
Eight of the pricked follicles were cut in serial sections. In no case could any
trace of the ovum or granulosa cells be found. The follicles were lined by the
theca interna and filled with red blood corpuscles and some pigment granules
formed from their disintegration as in an ordinary blood follicle. It might therefore
be objected that failure to form a corpus luteum in the artificially ruptured follicle
was due to operative trauma. It is feasible to postulate that in the sudden rupture
due to pricking, as distinct from the normally gradual process, the granulosa cells
became detached and were carried out with the liquor folliculi and the flow of
blood. The absence of the granulosa might explain the failure to form a corpus
luteum since Marshall and others have shown that the granulosa plays an integral
part in this formation. However in two other rabbits (Series III) the ripe
follicles were artificially ruptured 6 hr. after coitus (i.e. 4 hr. before ovulation or
any marked morphological change in the follicle had taken place) and in one of
these normal corpora lutea were found. It seems therefore that failure to form a
corpus luteum after artificial rupture is not due to operative trauma but to the
absence of certain changes which take place during the 6 hr. following coitus and
which either ensure the retention of the granulosa or enable it to undergo those
changes which precede the formation of luteal tissue. In the second rabbit in which
the follicles were pricked after coitus no corpora lutea were formed. Presumably
the follicles were immature or the stimulus of coitus had been insufficient to initiate
ovulation.
Since artificial rupture of the follicle by pricking did not cause ovulation or
formation of corpora lutea, a further series of experiments was carried out in which
the ripe follicles in one ovary were completely destroyed by means of an electric
cautery, and in one rabbit the whole of one ovary was completely removed. It has
been shown by Asdell (1924) that removal of one ovary causes double the usual
number of follicles to ripen in the ovary which remains intact. One of us (Ham-
mond, 1925) has postulated that the number of follicles which ripen depends upon
a limiting amount of some nutritive substratum in the blood supply (Heape's
"generative ferment") rather than on any inherent potentiality of the ovary itself.
It was therefore thought that if a number of ripe follicles were destroyed the excess
supply of "generative ferment" if available for the remaining follicles might cause
them to ovulate spontaneously. The results are shown in Table IV, Series IV-VI.
The complete destruction of the follicles was verified by histological examination200 A. W A L T O N and J. HAMMOND
afterwards; scar tissue, polymorphonuclear leucocytes and extravasated blood only
were found at the site of the operation. No corpora lutea were found in the intact
ovary; instead, the operation caused a number of small follicles to ripen immediately
in one or other of the ovaries until the total number of follicles again reached the
normal for the strain to which the doe belonged. For example, in C 618 (Series IV)
all the large follicles (numbering 3) in one ovary were cauterised and when killed
four days later three scars of the destroyed follicles were visible and there were again
three large follicles in the ovary, bringing the total number of follicles in both
ovaries up to ten which is a normal number for rabbits of this strain. In other
cases where a larger number of follicles were destroyed a certain proportion of the
follicles which ripened subsequently, appeared in the intact ovary, but in every case
the total for the two ovaries was about normal for the animal. It is to be noted
that a similar compensatory growth of new follicles occurred in those does in which
follicles had been pricked although in this case blood follicles were present in the
ovary. It is apparent therefore that blood follicles do not inhibit the growth of
fresh follicles. Compensatory growth of follicles also occurred in the doe (Series VI)
in which one ovary was completely removed.
Incidentally it was noticed that two of the ripened follicles were twice the
normal diameter but had not ruptured. Occasionally such follicles have been
observed in normal ovaries and perhaps represent an early stage in cyst formation.
Sections showed that these large follicles were, except for their size, normal in
appearance but that the granulosa was very attenuated, being only about 1—2 cells
in depth. A little further expansion of the follicle would result in further attenuation
of the granulosa and if this were accompanied by loss of adhesion to the theca and
consequent degeneration it is possible that cyst formation might be brought about.
The significance of these double-sized follicles and their bearing on the problem
of ovulation will be noted in the discussion which follows.
DISCUSSION.
Various theories have been advanced to account for ovulation. Thompson
(1919 a) gives a very exhaustive review to which reference may be made. For the
most part the theories have been advanced from purely histological study of the
follicle at various stages in the oestrous cycle but without accurate reference to the
exact time of ovulation. It is only in an animal such as the rabbit which does not
ovulate spontaneously that it is possible to establish an exact time relationship.
Our observations have been timed from coitus onwards and provide a consecutive
and dynamic picture of the whole process. In the majority of animals ovulation
is spontaneous, the follicle ripens, reaches maturity and ruptures. Rupture is
followed by the formation of a corpus luteum which undergoes growth and then
more or less gradual regression. The regression of the corpus luteum is followed
by the ripening of a further batch of follicles and the cycle continues automatically.
In the rabbit, ferret and possibly the cat the cycle is arrested at the stage when
ripe follicles are present in the ovary and coitus is required for ovulation and theObservations on Ovulation in the Rabbit 201
reinitiation of the cycle. Bouin and Ancel (loc. cit.) have suggested that in those
animals which do not ovulate spontaneously inhibition of the cycle is due to the
presence of considerable interstitial tissue in the ovarian stroma, which they
consider to be physiologically equivalent to the corpus luteum and to have a similar
though not so pronounced inhibitory effect on follicular growth.
Similarly, we have postulated above that inhibition might be due to insufficiency
of "generative ferment" or the nutritive substratum which is essential for the
growth of the follicle. Our experiments have shown that destruction of some
follicles is followed by immediate rapid growth of new follicles, demonstrating the
constancy in the supply of " generative ferment" and this rapid growth of follicles
takes place in the presence of the interstitial tissue. It would seem therefore that
if the inhibition be due to the action of the interstitial tissue or to inadequacy in
the supply of growth-promoting substances the inhibition only becomes effective
in the later stages of follicular development or alternatively that the immature
follicles have a marked priority over the supply of such substances.
On the other hand, it has perhaps been too readily assumed that ovulation is but
a stage in the growth of the follicle and occurs automatically when the follicle
reaches a certain size. Although limited to the mature follicle, ovulation is inde-
pendent of the actual size attained. A small follicle may rupture simultaneously
with or before a larger one and, as previously noted, a follicle may attain double the
normal size without rupturing (cf. also the case of blood follicles). In the absence
of ovulation the follicle continues to grow although very gradually and it has been
shown how this gradual growth may lead to cyst formation. In the rabbit cyst
formation is transitory, the follicle tends rather to break down internally with the
formation of a blood follicle and this occurs frequently when coitus and ovulation
have been in abeyance for some time. In this connection it is suggestive to find
that cyst formation from follicles occurs frequently in ovarian grafts and in small
remnants of ovary left after incomplete ovariotomy (Lipschiitz, 1925, 1927). In
such cases cyst formation might be attributed to the growth of fibrous coverings,
but we have observed that even when the ovary of the rabbit was covered with 2
thick adhesion of connective tissue, as occurs frequently as a result of tubal infection,
ovulation can take place normally under this fibrous coat and occasionally through
it when it is very closely adherent to the ovarian surface. We are inclined to attribute
cyst formation to the absence of ovulation rather than to the presence of adhesive
tissues. Cyst formation is not uncommon in young growing animals and in heavily
milking cows where the follicle ripens slowly.
Ovulation is not then merely the invariable consequence of follicular growth,
and the changes in the follicle which follow coitus are not merely a continuation
of the normal growth processes. There is evidence that the follicle about to rupture
under the stimulus of coitus enters a very definite phase of secretory activity. There
is a copious formation of secondary liquor folliculi with distension of the follicular
wall to breaking point and a continuous flow of the liquor after rupture which is
sufficient to carry away the ovum and many attached granulosa cells. The rate of
secretion and the consequent increase in internal pressure is probably of primary202 A. W A L T O N and J. HAMMOND
importance in causing rupture, the cells and tissues of the follicular wall being
unable to accommodate themselves to the sudden change. When distension is
continuous and gradual, as in the normal growth of the follicle, the walls have time
to accommodate themselves to the change and do not rupture. Schochet (1916)
suggested that rupture might be due to enzymic disintegration of the follicular
walls, and found evidence of proteolytic activity of the liquor folliculi in vitro.
There is, however, no evidence of any such action in vivo, although possibly the
long interval between coitus and ovulation might be claimed as giving some
support to the hypothesis.
It is also possible that as a result of pressure cutting off the blood supply to
the macula pellucida slight necrosis may aid in the separation of the cells. The
Guttmachers (1921) found evidence of plain muscle fibres in the theca of the sow
and suggested that rupture of the follicle might be caused or at least aided by
muscular contraction. We have found no evidence of any appreciable quantity of
musclefibresin the follicle of the rabbit, nor did the injection of posterior-pituitrin in
sufficient amount to cause intense contraction of the uterus and death of the foetus
in the 21-day-pregnant animal (see Knaus, 1926) have any influence in causing
ovulation in a doe in which ripe follicles were present in the ovary. The long
interval between coitus and ovulation and the marked distension of the follicular
wall at the time of rupture, and the fact that the follicles do not all rupture simul-
taneously are in favour of secretory distension rather than muscular contraction as
the main cause of ovulation.
It has been suggested that ovulation is brought about by vasodilation and
distension of erectile tissues (Heape, 1905), but our observations do not support
this hypothesis. It is true that marked congestion of the vessels accompanies the
process of ovulation but the mechanical influence of the distended capillaries in
the walls must be relatively negligible as a force distending the follicle at the time
of rupture. Vasodilation, however, at the time of coitus may possibly initiate the
secretory activity within the follicle.
It has also been advanced that rupture is brought about by a breaking down of
blood vessels into the follicle and consequent distension and rupture. Our obser-
vations show, however, that any rupture of the capillaries is secondary to the
rupture and collapse of the follicle.
The internal changes in the follicle which accompany ovulation are highly
suggestive of a new phase of secretory activity or a change in the orientation of
metabolic processes. In its slight staining capacity the secondary liquor folliculi
can be distinguished from the primary. The ovum undergoes maturation with
formation of the polar bodies. The luteal cells of the theca interna hypertrophy
and after rupture the granulosa proceeds to the formation of the corpus luteum.
Our experiments have shown that this formation of the corpus luteum is dependent
upon the processes which accompany the early stages of ovulation (within 6 hr. of
coitus) and does not occur with artificial rupture alone, Marshall and Runciman
(1914) artificially ruptured the ripe follicles in the ovaries of a bitch and found
that they became converted into structures resembling corpora lutea but withObservations on Ovulation in the Rabbit 203
abnormally large cavities. In this animal, however, ovulation occurs spontaneously
and it is likely that this implies not merely spontaneous rupture but also spontaneous
release of the necessary stimulus to luteal formation. It is probable, therefore, that
this may have occurred independently of the operation and before the complete
disintegration of the follicles. In the rabbit crescents of luteai tissue are exceptionally
found in blood follicles; possibly the inrush of blood detaches the granulosa cells
from the theca and no luteal tissue is formed, but where the inrush of blood is
gradual and one sided some of the granulosa cells may remain attached to the
theca, their nutritional base, and form luteal cells. The influence of coitus on the
formation of the corpus luteum as demonstrated by these experiments, is not without
parallel in the rat. Long and Evans (1922) have shown that coitus or simply stimula-
tion of the cervix with a glass rod is followed by prolongation of the normal luteal
phase of the oestrous cycle and it is not improbable that this is due to a similar
influence on the spontaneously rupturing follicle.
Our experiments throw little light on the question of how coitus initiates the
secretory activity which leads to ovulation. Some component of the orgasm which
accompanies coitus rather than the mechanical stimulus of the penis or the presence
of semen in the vagina would appear to be involved since ovulation in the rabbit
can sometimes be induced by allowing does to jump each other, and since artificial
insemination only rarely causes ovulation. Yamane and Egashira (1925) found
8*3 per cent, of cases of ovulation following artificial insemination and Hammond
and Asdell (1926) 3-6 per cent., which percentage was, however, raised to 13*3 per
cent, when the doe was tried with the male but did not actually copulate. It is not
unreasonable, however, to suppose that an orgasm is occasionally induced by this
means. There are several possible channels through which the stimulus of coitus
might be transferred to the ovary, but the experimental evidence is very fragmentary
and it appears premature to operf a discussion of this field until it has been further
explored. The following research bears upon this problem and may be suggestive
of further enquiry. Heape (loc. cit.) states that ovulation does not follow copulation
if the blood supply is interfered with, and Macomber (1926) found in rabbits that
disturbance with the normal nerve and blood supply prevented the normal functioning
of the ovary and sometimes caused cystic follicles. He has also shown that in the
rat which ovulates spontaneously the nerve supply to the ovary is not necessary
for normal reproduction. Whether ovulation occurs spontaneously or can only be
induced by coitus in grafted ovaries in the rabbit is a subject for further enquiry
but would give valuable information as to whether the stimulus to the ovary was
hormonic or nervous.
SUMMARY AND CONCLUSIONS.
Ovulation has been directly observed in the anaesthetised rabbit. The follicle
ruptures, on the average, about 10 hr. after coitus but there is some variation and
all follicles do not rupture simultaneously. Ovulation is not the invariable conse-
quence of the normal growth of the follicles and, although confined to those which
are mature, is independent of the actual size attained. Ovulation is accompanied by204 A. WALTON and J. HAMMOND
increased vascularity of the follicle. The principal cause of rupture is the rapid
distension of the follicle by marked secretory activity. The ovum is carried out in
the flow of this secretion. Ovulation in the rabbit is initiated by some component
of the orgasm which accompanies coitus rather than by the mechanical stimulus
of the penis or the presence of semen in the vagina. Corpora lutea are not formed
if the follicle is ruptured artificially and this effect is not due to operative trauma
but to the absence of some effect of coitus necessary for the formation of the luteal
tissue. Artificial rupture or ablation of ripe follicles is followed by an immediate
compensatory growth of new follicles. Blood follicles formed from those artificially
ruptured do not inhibit this compensatory growth.
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