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Observations on ovulation in the rabbit.

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Department of Anatomy, Xtanford University
There are two prevailing theories in regard to the mechanism of development of intrafollicular pressure in ovulation :
one explains the rupture by action of the smooth muscle in the
stroma of the ovary either by direct compression of the follicle
or by producing a rise in ovarian blood pressure (Rouget,
1858; Pfliiger, 1857; Aeby, 1859 ; Winiwarter and Sainmont,
'09; Thomson, '19 ; Guttmacher and Guttmacher, '21) ; the
other states that a gradual secretion of follicular liquor increases the intrafollicular pressure suficiently to cause rupture (Kiwasch, 1851; Brandt, 1877; Long and Mark, '11;
Robinson, '18 ; Strassmann, '23 ; Hartman, '32). Regardless
of what transpires within the ovary in preparation for rupture, it is a fact that it occurs on the surface of the ovary in
a region known as the rupture point, where, just preceding
rupture in the rabbit, there is a projection (the stigma) from
the surface of the ovary. It is a matter of interest to know
whether the intrafollicular pressure must be built up to a level
at which it can produce rupture through a normal tunica
albuginea and germinal epithelium, or whether there is a
weakening in the ovarian wall coincident with the rise in intrafollicular pressure. Kehrer (1865) spoke of a well-recognized
stigma, a thinner region relatively free of vessels but surrounded by a rosette of small vessels. Wester ('21) believed
This investigation was conducted with the aid of a grant from the Sex Division
of the National Research Council and of the Rockefeller Foundation Grant for
Fluid Research in the Medical Sciences at 6tanford University.
that the thinning of the tunica albuginea was the result of a
pressure necrosis produced by the dome-like bulging of the
follicle. Long and Nark ('11)described an actual disappearance of the theca folliculi and the tunica albuginea in the
mouse so that the follicular liquor came in direct contact with
the germinal epithelium, which was stretched out into a thin
membrane with widely scattered nuclei. Walton and Hammond ('28) figured and described a white avascular spot which
developed at the point of future rupture on the surface of the
follicle as ovulation impended in the rabbit. Kelly ( '31) and
Smith ('34) confirmed their observations. At the present
time, it is quite generally believed that there is a change in
the tunica albuginea and germinal epithelium preceding actual
The material from which these observations were made consisted of 50 pairs of rabbit ovaries: 16 pairs from animals
with transected mid-brain (the plane of section extending from
the rostra1 border of the superior colliculi to the exit of the
oculomotor nerves), 14 from large and 6 from small mid-brainhypophysectomized animals (Hinsey and Markee, '33), 10
from animals in which the ovaries had been partially or totally
denervated (Hinsey and Markee, '32)' and 4 from normal
animals. Ovulation was induced in all of these animals by the
injection of 10 cc. of human pregnancy urine. Each ovary was
fixed in Bouin's solution and cut into a complete series of 10 p
sections which were stained either with hematoxylin and eosin
or with the Mallory-azan technique.
Reconstructions were made from 24 of these animals (9
mid-brain, 8 mid-brain-hypophysectomized,5 denervated and
2 normal controls). Three types of reconstruction were used :
1) the contents within the antrum of ninety ruptured follicles
by the three-dimensional blotting paper technique, 2) the area
of each of these rupture points by the graphic method, and
3) the theca, granulosa and the contents of the antrum of five
of these follicles by the three-dimensional blotting paper
We have been able to confirm the observations of Walton
and Hammond ('28) and Kelly ('31) on the events that take
place at ovulation, by means of observations made on transplants of the ovary into the anterior chamber of the eye,
through a glass window placed into the lateral abdominal wall
and on the ovary in situ. On the surface of a well-vascularized
follicle, an avascular spot appears which is surrounded by dilated vessels. This avascular spot develops into a papilla, the
tip of which opens, and the contents of the follicle flow slowly
out of this opening. It may be emphasized that the avascular
spot appears before the papilla protrudes noticeably above the
surface. It is difficult to say definitely whether the avascular
spot is the result of a local pressure upon the vessels in it or
whether there is a localized constriction of the vessels in this
region. An emmination of figure 1-a section through a
mature follicle (judged by the maturation of the nucleus and
loosening of the cumulus)-shows the presence of a relatively
avascular area surrounded by dilated vessels before there is
either formation of a papilla or any considerable stretching
of the superficial ovarian tissue. This is typical of a number
of other ovaries removed from rabbits just before the occurrence of rupture in the follicles.
From a study of the serial sections of ruptured follicles,
oval openings were found in eighty-eight out of ninety cases.
I n the other two cases the openings were shaped like a figure
eight with a bridge of tissue between two circular openings.
The bridge consisted of stretched germinal epithelium, tunica
albuginea, and theca, the granulosa being absent. Uniformly,
around the oval openings, there were dilated vessels which
seemed to be dilated more than the vessels around the rest
of the follicle, although this difference in caliber of blood
vessels has not been noted in our experience in the stages just
preceding rupture. The edges of the rupture points were not
serrated, there was no evidence of tearing, and, although we
looked carefully for it, there was no indication of any retraction unless the protrusion of granulosa cells into the ruptured
point be regarded as such. However, we believe that this
represents a stage in the formation of the 'bouchon epithelial.'
Ordinarily, there was a relatively small amount of hemorrhage
into the antrum of the follicle, although there was some variation in this. This coincides with the observations of Clark
(1896) and Thomson ('19) in the cat and of Sobotta (1896)
in the mouse.
As f a r as our histological observations go, we were unable
to find any variations from normal in the ruptured follicles of
animals which had been hypophysectomized (mid-brain animals) two hours before the injection of pregnancy urine
(Hinsey and Markee, '33) and in those in which the ovaries
had been denervated (Hinsey and Markee, '32). I n the latter
group, the vagi had been cut bilaterally and the spinal cord
removed from 4 or 5 T through 2 or 3 L and from 6 L through
4 S. Here the ovaries had been completely severed from any
possible connection with the central nervous system. The
presence of the ring of dilated vessels about the rupture point
in these ovaries (fig. 2) demonstrates that the vascular
changes in ovulation are local phenomena independent at least
of the central nervous system.
I n order to check our observations in regard to the shape
of the rupture points, we have made graphic reconstructions
of ninety of these. In each case the area (in square millimeters by planimeter) and the greatest and least axes (in
millimeters) were measured. An examination of these figures
will show that in eighty-eight out of ninety cases the rupture
points are oval in shape. The reconstructions themselves
showed no serrations (fig. 3). There was considerable variation in the size of the openings. The mean of their areas was
0.062, the greatest axes, 0.3169 mm., and the least,
0.215 mm. The mean areas of 11 rupture points in normal
controls was 0.065, of 36 mid-brain ones wclr, of 29 in mid-brain-hypophysectomized animals was
0.053 and of 10 in denervated ovaries was 0.038
Although there are not enough reconstructions in each group
to submit these measurements to a statistical analysis, the
denervated animals show a tendency toward small rupture
points I n lieu of more complete data, we attach no particular
significance to this variation in size.2
In an attempt to find whether there is any correlation between the size of the rupture point and the residual fluid in
the itntra, three-dimensional reconstructions of the latter were
made in the ninety cases where graphic reconstructions of the
rupture points were done. It was found that the correlation
of the size of the rupture points with the amount of residual
fluid in the antra gave a ratio of 0.377 with a probable error
0.061. This would indicate that there was a tendency
for the follicles with larger rupture points to contain more
residual fluid, but an examination of the data will show that
this may not always hold.
Figure 4 illustrates reconstructions of the theca, granulosa
and follicular liquor of a mature follicle in which the first polar
body had been formed. This shows the regularity of outline
of all components and is to be contrasted with the description
of Evans and Cole ( '31) of folding of the granulosa preceding
rupture in the dog. Figure 5 is a similar one showing the inside of these layers in another follicle where the cumulus had
been nearly freed by vacuolization. The attachment is rather
wide (over 400 1.1) but it is only 40 1.1thick.
Figure 6 shows a reconstruction of the theca (A) and the
granulosa (€3) of a follicle which had ruptured. Its shows the
irregular outline and the fact that one side of the follicle had
collapsed much more than the other. This resulted in the
liquor streaming from the collapsed side more than from the
other. Figure 7 is a section through this follicle, which contains an ovum that had failed to escape at the time of rupture.
This sort of retention of an ovum with subsequent fertilization
might very well explain the phenomenon of ovarian pregnancy
of the primary type.
One ovary was examined with a hand lens before fixation
and was found to contain four ripe follicles with well-developed
stigmata. Within 30 seconds after it was placed in Bouin's
* Tablea covering this
data may be obtained by writing the authors.
solution for fixation, it was observed that there were four
thread-like projections (about 4 to 6 m. in length) from the
surface, which in our judgment represented ruptures of the
ripe follicles. Histological examination confirmed this. While
we realize that these ruptures were probably due to the action
of the fixative upon the ovary, we feel that a description is
justified in the light of the fact that it may represent to some
degree what takes place in normal ovulation. Figure 8 is a
reconstruction of one of the follicles, showing the theca (A),
granulosa (B), contents of the antrum (C) and the precipitated follicular liquor outside the follicle (D). On the very
tip, this thread-like projection contained groups of cells from
the germinal epithelium which had been carried out by the
fluid as it made its exit. These cells were probably derived
from the portion of the stigma within the vascular ring. Since
they were found only at the tip, it seems that they formed a
plug which was, in effect, blown out a t the start of the rupture.
The ovum was found within 0.7 mm. of the end of the thread,
which was 6.8 mm. long. This would indicate that it was
situated near the stigma of the unruptured follicle and was
expelled with the first portion of the escaping follicular liquor.
Figure 9 shows a cross section of this ovum through the
nucleus, which was in spindle-stage. It is near the center of
the thread and is surrounded by approximately the normal
number of corona radiata cells. When we first saw these
threads they were straight, but the rolling of the ovary in the
bottle of fixative produced a bending which is demonstrated
in the reconstruction. This jet-like projection of the follicular
contents under an increase of pressure produced by the action
of the fixative is different from the slow flow which is seen
normally under direct observation. This may have its explanation in a difference in the state of the stigma within the
vascular ring. In our case, a greater pressure of the follicular
liquor may have been built up before the tip of the stigma gave
way, thus explaining the jet-like projection of the follicular
contents, Gerlach (1890, two cases in mouse), Sobotta (1895,
mouse), Longley ( '11, cat) and Hartman ( '32, opossum) have
recorded cases of recovery of ova in exit from the follicle.
These observations from reconstruction showing an absence
of serrations in the rupture-points are in line with the histological ones of Brandt (1877, frog), Long and Mark ('11,
mouse), Strassmann ( '23, dog) and Evans and Cole ( '31, dog).
I n the presence of oval-shaped rupture points, it is difficult to
explain rupture by an increase in the intrafollicular pressure
alone. Such an increase in pressure must be accompanied by
some local change in the wall of the ovary in such a manner
that a portion of it is removed at the time of rupture. One
would expect that the rupture due to tearing would not cause
the formation of an oval opening. It is impossible to say that
the rupture point is oval-shaped at the time of rupture because
a subsequent change in the state of the ovary might alter the
shape. However, we do know that in the u n k e d ovary
which is examined with a dissecting or a Leitz ultra-opaque
microscope, the rupture points are oval-shaped and free from
serrations. The effect of the action of the fixative is recognized, but it must be encountered to obtain quantitative observations such as we have made.
The phenomenon of ovulation has been followed in the living animals, and histological observations have been made
on fifty pairs of serially-sectioned rabbit ovaries in which
ovulation had occurred. Attention was focussed upon the
changes in the vascular bed about the follicle and the histology
of the rupture point. A ring-shaped area of dilated vessels
appeared about a relatively avascular area before any marked
stigma was present. As the stigma developed, these vessels
were definitely dilated in this ring as well as about the follicle
as a whole, with the exception of the central portion of the
stigma. These vascular changes as well as the other events in
ovulation showed no observable variation in ovaries that had
been completely severed from their connection with the central
nervous system or in ones in which the hypophysis had been
removed 2 to 3 hours before the injection of pregnancy urine.
Graphic reconstructions made from ninety rupture points in
twenty-four ovaries showed the rupture points to be ovalshaped, with a mean area of 0.062, and the mean of the
greatest axes 0.3169 and the least 0.215. No evidences of serrations which might indicate a slit-like tearing of the ovarian
wall were present. These observations indicate that a portion
of the ovarian wall bounding the stigma is actually removed
at the time rupture starts.
Three-dimensional reconstructions of the theca, granulosa
and contents of the antrum are described for mature follicles
before and after rupture. One of these is modelled from an
ovary that had ruptured in fixative and shows the thread-like
projection of follicular liquor with the occurrence of germinal
epithelial cells at the tip and the ovum near the tip. Another
reconstruction shows a ruptured follicle in which the ovum
failed to leave the antrum of the follicle. This observation is
significant in an explanation of primary ovarian pregnancy.
Amy, C. 1859 Ueber glatten Mushelfasern im Ovarium und Mesovarium von
Wirbelthieren. Arch. f. Anat., Physiol. u. wissensch. Med., S. 675-676.
1877 Fragmenterische Bemerkungen iiber daa Ovarium des
Froches. Zeitschr. f. wissensch. Zool., Bd. 28, S. 575-586.
CLARK,J. G. 1898 The origin, growth and fate of the corpus luteum a8 observed in the ovary of the pig and man. Johns Hopkins Hosp. Rep.,
V O ~ . 7, pp. 181-222.
EVANS,H. M., rn €
I.COLE 1931 An introduction to the study of the
oestroue cycle in the dog. Mem. Univ. Calif., vol. 9, pp. 65-119.
GEWCH, L. 1890 Beitrage zur Morphologie und Physiologie des Ovulationsvorganges der Saugethiere. Sitzungsber. d. phys.-med. Soe. zu Erlang.
Munchen, Bd. 22, Hft. 43 (not seen).
1921 Morphological and physiological studies on the musculature of the mature Graafian follicle of the
sow. Johns Hopkins Eosp. BulI., vol. 32, pp. 394-399.
C. (2. 1932 Ovulation and the transport and viability of ova in the
female genital tract. Chapter 14 in ‘Sex and internal secretions.’
Baltimore, Williams and Wilkins.
HINSEY,J. C., AND J. E. MARKEE1932 A search for neurological mechanisms in
ovulation. €’roc. SOC.Exp. Biol. and Med., vol. 30, pp. 136-138.
1933 Studies on prolan-indueed ovulation in midbrain and midbrainhypophysectqfaized rabbits. Am. J. Physiol., vol. 106, pp. 48-54.
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Mechanismus der Einwanderung des Ovulum in der Fransentrichter.
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G. I;. 1931 Direction observation of rupture of graafian follicle i n
mammal. J. Flx. Xctl. ~Issoc.,vol. 17, 1). 422.
KIWASCH,F. A . 1851 Uir Qehrtskuiide. Erlarigcn, F. Enke.
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Cariiegie Inst. Wash., Pulil. no. 142; also Fontrili. 110. 216, Zoul. Lab.
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cat. Am. J. Anat., vol. 12, pp. 139-172; also Science, 1910, 11.9. vol.
31, pp. 468466.
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Arch. f . Anat. u.
Physiol. u. wissensch. Mrtl., 8. 30-32.
ROBINSON,A. 1918 The forniwtion, rupture, and closure of o\arian follicles in
ferrets and ferret-polecat hybrids and some associated phenomena.
Trans. RIJS. Soc. Edinburgh, vol. 52, pp. 303-362.
ROTJGET,C. 1858 T. Rcchcrches sur les organe 6rectiles de la femme et sui1 'appareil niusculaire tubo ovarien, dans lcurs rapports avee 1'ovulation
et la menstruation. J. de la Physiol., T. 1, pp. 320-343.
1934 Some ohscrvations on the rupture of Graafian follicles in
rabbits. h i . J. Obst. and G p e c . , vol. 27, pp. 729-731.
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mikr. Anat., Bd. 4.5, S. 15-93.
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mikr. Anat., Bd. 47, S. 261-308.
N. 1923 Waruni platzt der FolltkelB Arch. f . Gynak., Bd. 119,
THOMSON,A. 1919 The ripe liuinan Graafian follicle together with soinr suggestions as to its mode nf rupture. .T. Anat., vol. 54, pp. 1 40.
WALTON, A., AND a. H A M X O V D 1928 Obsertations on ovulation in the rabbit.
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1 Photoiiiicrograph of n section through a ripe graafian follicle showing a
central relatively amscular area surronndcd by dilated 1 essels. X 130.
2 fhotoniicrograph showing dilated vessels around rupture point of a follicle in
a deiiervated ovary (both vagi c u t ; spinal cord rtxmovrd from 5T-2L and below 5L).
x 130.
3 Graphic reconstruction of rupture points to show shape and size of three
typical oncs and one atypical one. (a)A rupturc point rcpreseiitativc of the
ciglit)-eight oval oncs. (E) Oiie of thc two rupture points shaped like a figure
eight. X 85.
4 Reconstruction of ( A ) thcca, ( E ) graiiulosa and ( C ) follicular liquor, showing snioot,h contour and lack of folding i r i a mature uiiruptured follicle. Volurrics:
them, 2.435 cu.mni.; granulosx 2.943 cumm.; fluid within follicuiar aiitruin,
1.218 X 38.
5 Reconstructions of ( A ) thraa, (B) granulosa, and (C) follicular liquor of a
ripe unruptured follicle, illustrating smooth contour and wide although thin at
txchment of nearly loosened cumulus. This shows one-half of each reconstruction,
viewed f r o m within. Volumes : theca, 1.630 cu.rnin. ; granulosn, 2.105; fluid
within antrum of follicle, 1.982 cu.mii. X 38.
G Xeconstruction of ( A ) tlieca nnrl ( U ) granulosa of a ruptured follicle from
which ovum did not escape. The follicle had collapsed marlicdlv on one side only.
Yolumcs : t h a , 1.753 ; grauulosa, 2.173 cu.ni111.; fluid within folliclr, 0.3965
c u m m . X 38.
7 A section through t h e folhclt., the rcvonstructinn of which i s illustrated in
figure 6. Thc oruni is hhoivn on one side of the follicle and the streaiii of fluid
froiii the other (collapsed) side of the €ollicle passes out through the rupture point.
8 Fieeonstructioli of a follicle that ruptured after being placed iii Bouin’s
fixative. ( A ) thcca, (B) granulosa, ( C ) fluid vithin antruni of follicle and (U)
follicular fluid outside the ovary. (The bending aiid constrictions appeared after
fixation.) The position o f the ovmi is indicated b) the ball superimposerl on t h e
reeoiistruetioii of t h e fluid 1). Volumes: tlieca, 1.081 eimini. ; granulosa, 1.637
eu.Inm.; fluid within follicle, 0.3042; fluid outside folliclc, 1.095
9 Section through the nucleus of the o w m . T h e sertion WRS 0.7 iiiiii. f r o m the
end of D in figure 8. X 240.
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rabbits, observations, ovulation
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