observations on ovulation in the rabbit · 2 0 1-0--t ' t 0 * i 0 0 # » 0 m i i i 0 0 3 6...

190

OBSERVATIONS ON OVULATION IN THE RABBIT

BY A. WALTON, PH.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 thatovulation is not spontaneous but occurs normally only after coitus and there is afairly definite interval of about 10 hr. between mating and actual rupture of thefollicle. Our observations fall under the following headings and approach theproblem 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 studyof 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 differentstrains 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 wereobserved fresh and the ovary then fixed in 10 per cent, formalin. After fixationsome mature follicles were sectioned freehand with a razor and the sections showingthe maximum surface area picked out for measurement with a micrometer eyepiece.From each doe two follicles were chosen and from each follicle two sections wereselected and two diameters at right angles (the breadth and depth) measured. Theaverage of the resulting eight measurements was taken as representing the folliculardiameter characteristic of the doe. Assuming the follicle to be approximatelyspherical 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 definiteincrease in volume of the follicle after coitus. There was some indication from themeasurements that the follicle becomes rather deeper in proportion to its breadthbut the method of measurement is relatively crude and there is considerablevariation in the shape of the follicle depending upon how crowded they are on the

Observations on Ovulation in the Rabbit 191

surface of the ovary. It is important to note that blood follicles (i.e. follicles whichdo not ultimately rupture but in which vessels in the theca break down and allowblood to escape into the cavity of the follicle) may attain a greater volume than thenormal follicle about to rupture.

Other follicles were embedded in paraffin and complete serial sections cut andstained with haematoxylin and eosin. No detailed histological description need begiven since the subject has been very fully investigated by Sabotta (1897), Honore(1900) and Corner (1919). Only points of special interest will be mentioned ina brief summary. The observable changes in the follicle are relatively slight andthere is considerable variation from follicle to follicle. It would be impossible

1

a1

ovB

I

5-0

4-0

3-0

2 0

1-0

-

-

t '

t

0

*

i

0

0 #

»

0

m

i i i

0

0 3 6Hours after coitus

Fig. 1. Growth of Follicle after Coitus. Volume calculated from average folliculardiameter of each rabbit.

from observation alone to state with any certainty the exact stage which an isolatedfollicle represented. If, however, large numbers of follicles in each series arecarefully compared more or less definite changes are detectable.

At coitus the mature follicle is of considerable size in comparison with those whichare 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 about4~8 cells in depth round the follicle and shows slight columnar arrangement in the layersnext the membrana propria. " Bodies of Call and Exner " are present in most sections andmore frequently in the discus. The membrana propria appears as a thin line. The "lutealcells " 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 possiblyslightly larger and follicular measurements show a slight change in the shape of thefollicle which protrudes more from the surface of the ovary.

At 9 hr. after coitus. The vascularity of the theca is increased. Secondary liquorfolliculi is more abundant. The granulosa is about 3-5 cells deep round the follicle. Thereis 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 maturationchanges of the ovum but it has been shown by Heape (1905) and Sabotta (1897) that thistakes place during this time.

(b) OBSERVATIONS ON OVULATION IN THE ANAESTHETISED ANIMAL.

The process of ovulation was watched with a binocular dissecting microscopethrough 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 foras long as changes were detectable. Timed drawings and notes were made of theappearance of the follicles (examples are shown in Table I). At the close of theobservations the animal was killed and the ovaries fixed in formalin. Serial sectionswere cut of the follicles which had been under observation, each follicle beingidentified from the drawings previously made.

Systematic research on the optimum conditions for the experiment was not

Table I. Observations on the follicles during

Doe

F336

F357

Timecopulated

(a.m.)8.15

22. ii. 26

8.4023.11. 26

Time ovariesfirst observed

(p.m.)6.20

(10 hr. 5 min.after coitus)

6.45(10 far. 5 min.after coitus)

A

(p.m.)Ruptured before

6.207.0. Red patch atsummit markedbut edges muchpaler

6.45. With clearpimple, not rup-tured

7.10. Ruptured.Stringy massrunning downovary from pim-ple

B

(p.m.)6.30. Congestedwith pale pro-truding pimplein centre

6.40. Ruptured6.45. Smaller, redall over, bloodand fluid run-ning downovary,blood on formerclear pimple andperiphery clearer

6.55. Dull diffusecolour. No col-lapse

6.55. Red, notruptured but didso shortly after

7.15. Blood roundpimple. Col-lapsed

C

(p.m.)First seen at7.25. Had justruptured. Dif-fuse redandclearjelly runningfrom centre

6.55. Stringy massrunning fromclear central pim-ple; blood extra-vasation at edgeof pimple

7.15. Collapsed;blood roundedge of pimple

Appearance

D

(p-m.)6.35. Rounded7.55. Congested

but not ruptured

First seen 7.5.Pimpleatsummit

7.15. Clear pim-ple at summit;rest of folliclewith blood ves-sels congested

Observations on Ovulation in the Rabbit

carried out but after some failures the technique ultimately adopted with successwas as follows: The operation was begun only just before the normal time ofovulation. The room was kept warm and moist. The animal was laid on a warmtank on the operating table. Ether alone was used as anaesthetic. The intestineswere gently moved to one side and kept wrapped in warm moist cloths. The ovaryremained in its normal position or was propped slightly with swabs of cotton-woolmoistened with warm Ringer's solution. Warm Ringer's solution was used to keepall exposed tissues moist. The ovary was only momentarily exposed for the purposeof observation and special precaution was taken to avoid scorching with the lightbulb used for illumination. When not actually under observation the ovary wascarefully covered with the intestines or moist swabs.

The actual times after coitus at which the follicles ruptured under observationvaried from 9f-i3i hr. (see Table II). Occasionally some follicles had rupturedbefore observation began. In any one animal not more than two or three rupturedat exactly the same time although the majority appeared to follow each other atfairly short intervals. It may have been that the rate of ovulation was influencedby the anaesthetic and operation, in fact we attribute failure to observe ovulationin 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 allfollicles of the same batch do not rupture at exactly the same time, for in severalcases where observations began about 10 hr. after coitus one or more follicles hadalready ruptured and others ruptured subsequently under observation (A 9,

the time of rupture. (Examples of notes made.)

of follicles

E

(p.m.)6.33. Red spot incentre

6.50. More pro-minent

6.55. Centralpimple dark redunder surface asthough bloodvessels brokendown

7.10. Blood atsurface of cen-tral pimple

7-5O. More con-gested

6.50. Ruptured,blood in centre

7-o. Smaller, col-lapsed; red incentre

7.15. Centre clear,ring of extra-vasated bloodround it. Muchsmaller

F

(p.m.)7.2. Clear centralpatch, rest con-gested

7.20. Blood ves-sels more marked

7.55. Congestedall over but notyet ruptured

6.50 and 7.0. Clearpimple at sum-mit

7.15. Ruptured.Stringy masscoming fromclear pimple;much smaller

G

(p.m.)7.40. Ruptured.

Clear thick fluidstreaming fromclear centralpatch

6.50. Clear pim-ple in centre

7.15. Clear cen-tre. Had rup-tured but notcompletely col-lapsed

H

(p.m.)6.20. Clear raisedpimple with con-gested spot atedge

7.40. Ruptured.Stringy fluidstreaming fromcentral pimple

1

(p.m.)Clear, no change

Time doekilled

(p.m.)8.0

(11 hr. 45 min.after coitus)

(10 hr. 35 mm.after coitus)

BJEB'Vlii


E 208, etc.). Several of the large follicles which had not ruptured when the animalwas killed would probably not have done so normally. Sections showed that theywere probably atrophic (F 359, G and H).

Plate V, figs. 1-6, illustrates the external changes of the follicle in plan and inelevation. The ripe follicle, 9 hr. after coitus, has a convex surface with a fairly widebase and is covered with a fine network of small blood vessels which can be easilyseen through the very transparent surface cells (Fig. 1). The first sign of approachingovulation is the gradual formation of the macula pellucida at the apex, formedpossibly 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 thecongestion of the blood vessels is distinctly seen (Fig. 3). In the next stage themacula pellucida somewhat rapidly blows out as a pimple (Fig. 4). The capillariesand small vessels at the base of the pimple are ruptured before the actual surfaceand there may be some extravasation of blood into the follicle round the line ofrupture. 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 thepimple to the surface rapidly follows and liquor folliculi, frequently stained withblood, 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 surfaceand the gradual rupture and extrusion of contents strongly resembles the formationand 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 hasbeen picked up with a capillary pipette from the flow just after rupture.

The liquor folliculi does not squirt from the follicle but flows steadily andcontinuously for some little time. During this process the follicle walls maygradually collapse but in many cases there is not a very marked change in thevolume of the follicle. The congestion of the blood vessels diminishes. Graduallythe liquor folliculi ceases to flow and appears to clot and forms, in the orifice, a plugwhich protrudes a little from the surface. The plug is stained with blood and isa bright translucent red (Fig. 6).

Later the former pimple is filled by a growth of luteal tissue which forms acharacteristic projection on the surface of the newly formed corpus luteum and itspresence 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 commonto other mammals.

After rupture the base of the follicle becomes much narrower owing probablyto elastic readjustment of the ovarian surface and the whole follicle protrudes moreabruptly from the surface of the ovary.

Subsequent histological examination of the follicles which had been underobservation extends the description already given of the stages previous to actualovulation. Shaw (1927) states that in the human follicle the cumulus which is

Tab

le I

I.

Tim

e of

rup

ture

of f

olli

cles

und

er o

bser

vati

on.

Let

ters

bef

ore

the

brac

ket A

[ d

enot

e fo

llicl

es w

hich

rup

ture

d be

fore

ope

rati

on b

egan

. L

ette

rs b

etw

een

the

brac

kets

[A

] de

note

folli

cles

whi

ch r

uptu

red

unde

r ob

serv

atio

n. T

hose

enc

lose

d by

sm

all

brac

kets

(A

) ha

d ru

ptur

ed u

nobs

erve

d ju

st p

revi

ousl

y.

Let

ters

aft

er t

he b

rack

et ]

A d

enot

e fo

llicl

es w

hich

wer

e un

rupt

ured

whe

n do

e w

as k

ille

d.

M <*»

&

Tim

e af

ter

coitu

sH

r.M

in.

Ani

mal

s un

der

an-

aest

heti

c :

Doe

No.

A 9

AS

E15

4

A4

E208

F336

F357

F359

F391

Sum

mar

y of

ob

-se

rved

rup

ture

s

Ani

mal

s ki

lled

:D

oe N

o. C

339

£4

25

A 7

A i

s

8 30

45 L

9 0 [

15

3O

45

BC

r

DE

L

B A AB D lA 1 +

x al

l un

rupt

ured

10 0 A B F No

No E C B 3 +

15

30

45

r L G

folli

cles

rup

ture

d d

folli

cles

rup

ture

d d

I A

B(F

)*

CD

1 C

B B

AF

1 n

C

G}

u

]C E 6

32

112

rupt

ured

\ 1

unru

ptur

ed

X 1

1 al

l ru

ptur

ed

11 0

urin

g

urin

g

(Q H G

is

30 H

obse

rvat

ion

obse

rvat

ion

A

[ Q

)G H

] D

3 1

45 D

]

1

12 0

15

A C D DF

El

30

45

~\A

B CD

JEF

13 0 15

30

"1

I

J y

1

x f 1

1 ru

ptur

ed1

s un

rupt

ured

* R

uptu

re f

irst

not

iced

in

sect

ione

d m

ater

ial.


originally directed towards the medulla gradually rotates until just before ovulationit 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.

Doe

F336

F359

F391

A 4

Fol-licle

D

H

HABECDA

E

A,B,C,D

E

F

Ruptured

No

Yes, about20 min.

No

NoYes, about 1 hr.

Yes, about 1 hr.Yes, 25 min.Yes, 38 min.Yes, about 1 hr.Yes, ii- hr.

Yes, 1 hr.

No

No

Yes(in fixation?)

Diameternun.

1-25

0-98

1-29

0-78

1 02I'OO0-960-78

0-80

0-98

Description

Ovum present, small congested blood vessels in thecabut no extravasation of blood. Granulosa about5 cells thick on side of cumulus which was lateral, 2—3cells thick on other side. Theca lutein cells in thinlayer

No ovum or cumulus present, liquor folliculi almostall extruded. Extravasated blood in walls roundaperture and little in central cavity. Granulosa cellsabout 12 thick all round follicle. Walls collapsedand slightly wavy, basement membrane creased.Theca layer thick

Ovum present but degenerate: granulosa only presenton cumulus side of follicle, broken up and detachedon other side: little congestion of thecal vessels.Theca only well developed on side -where granulosapresent

Similar to GSimilar to F 336 (H); blood seen passing fromvessels below granulosa into cavity of follicle

Similar to ASimilar to A but still containing much liquor folliculiSimilar to A but no blood in central cavitySimilar to A but no blood in central cavity-No ovum or cumulus present; very little liquorfolliculi. Granulosa about 35 cells thick. Wallsshowing slight wrinkling and irregular folding oftheca. Little blood extravasated into theca at sidesof aperture. Theca interna layer thick

Similar to A but rather more liquor. Granulosa 23cells thick. Extravasation of blood into theca at baseof follicle

Ovum present; granulosa 6 cells thick near cumulusand about 3 cells thick on other side. Theca lutealcells small to moderate size, blood vessels of thecacongested. Wall in region of macula pellucidawrinkled in outline

Atrophic: granulosa detached and cells scatteredthroughout liquor folliculi

No ovum or cumulus present. Granulosa about12 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 tomoderate size: blood vessels few and not verycongested

of rupture shows that in the rabbit the cumulus and ovum do not occupy anydefinite position with respect to the point of rupture. The continuous nature of theflow and gradual contraction of the follicle after rupture render it unnecessary topostulate any particular position of the ovum with respect to the orifice as a factoraiding its extrusion from the follicle. Since two follicles (F 359, G and H) whichhad not ruptured at 10 hr. 50 min. after coitus showed the granulosa heaped up

Observations onOvulation in the Rabbit 197

round the ovum and for the most part detached from the theca interna it wasthought that this might represent a stage in the process of extrusion but the ovumappeared abnormal and it seems more probable that the foUicles were in process ofbecoming atrophic and would not have ruptured normally.

The remains of the liquor folliculi in the centre of the follicle show stream linesof clear non-staining fluid (secondary liquor folliculi) often marked by a few redblood corpuscles extravasated from an occasional ruptured vessel in the theca ormembrana propria. The "bodies of Call and Exner," which are seen in almost allfollicles previous to rupture and particularly in those at a standstill in development,are absent from the recently ruptured follicle. These bodies are in all probabilitysmall accumulations of liquor folliculi which during slow development do notcoalesce, as they do later when active secretion is taking place in the ovulatingfollicle.

After rupture the diameter of the follicle diminishes (see Table III) and as aconsequence the granulosa, which in the follicle before rupture is extended andonly about 6 cells thick near the cumulus and 2-3 cells thick elsewhere, becomespacked together and forms a layer some 12—30 cells in depth, distributed fairlyevenly round the perimeter of the follicle; near the point of rupture, however, itremains about 4 cells in depth. It seems probable that near the orifice the wallsof the follicle are not subject to so much lateral pressure from surrounding tissuesand the contraction of the walls is consequently less. The walls of the follicle havea slightly wavy outline and the theca interna is well marked and thicker—the resultsprobably of collapse. The luteal cells of the interna are especially prominent andappear hypertrophied. The membrana propria is now seen to consist largely of anetwork of well-filled capillaries.

Subsequent changes in the follicle are associated with the formation of thecorpus 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 rabbitcorpora lutea were occasionally formed but that more often the pricked folliclesfilled with blood and became atretic. They also stated that if the follicles in oneovary were artificially ruptured, the follicles in the other ovary might rupturespontaneously. O'Donoghue (1913) confirmed their main findings but found thatcorpora lutea did not invariably form after the operation. We have attempted to repeatthese experiments. In two rabbits the operation of artificial rupture of the folliclesin one ovary was performed through an incision in the flank and the uterus andcervix left completely undisturbed so that all possibility of reflex action fromhandling the uterus or cervix might be eliminated. In two other rabbits theincision was medial and the uterus and cervix deliberately handled in the courseof the operation. Handling produced an intense vascular congestion of the cervixand uterus. The animals were killed three or four days after the operation. Theresults are shown in Table IV, Series I—III. In no case did the pricked follicles

Tab

le I

V.

Art

ific

ial

rupt

ure

and

abla

tion

of m

atur

e fo

llic

les.

vO GO

Seri

es

I.

Rup

ture

. U

teru

s no

tha

ndle

d

II.

Rup

ture

. U

teru

s an

dce

rvix

han

dled

III.

R

uptu

re.

6 hr

. af

ter

coit

us.

Ute

rus

not h

andl

ed

IV.

Abl

atio

n.

Ute

rus

not

hand

led

V.

Abl

atio

n.

Ute

rus

and

cerv

ix h

andl

ed

VI.

H

emi-

cast

rati

on.

Ute

rus

and

cerv

ix h

andl

ed

Doe

C69

3

E77

9

X84

1

E83

6

C89

2

C89

4

C61

8

C6

is

A9

E83

3

App

eara

nce

at o

pera

tion

Dat

e

15. v

ii. 2

7

15. v

ii. 2

7

26. v

ii. 2

7

26. v

ii. 2

7

2. v

iii. 2

7

2. v

iii. 2

7

15. v

ii. 2

7

15. v

ii. 2

7

26. v

ii. 2

7

36. v

ii. 2

7

Ope

rate

d ov

ary

All

larg

e fo

llicl

es

(4)

pric

ked

with

poin

ts o

f fin

e sc

isso

rs a

nd

z la

rge

bloo

d fo

llicl

es a

lso

pric

ked

All

larg

e an

d m

oder

ate

size

d fo

llicl

es(1

2)

pric

ked

with

po

ints

of

fi

nesc

isso

rs

All

larg

e an

d sm

all f

ollic

les

(8) p

rick

edw

ith p

oint

s of

fin

e sc

isso

rs

All

larg

e an

d m

oder

ate

size

d fo

llicl

es(8

) pr

icke

d w

ith n

eedl

e

9 a.

m.

Cop

ulat

ed w

ith b

ucks

3 p.

m.

(6 h

r. a

fter

coitu

s).

AH

lar

gean

d m

oder

ate

size

d fo

llicl

es

(8)

pric

ked

with

nee

dle

9.5

a.m

. C

opul

ated

with

buc

ks3.

20 p

.m.

(6J

hr.

afte

r co

itus

).

All

larg

e an

d m

oder

ate

size

d fo

llicl

es (

8)pr

icke

d w

ith n

eedl

e

All

the

larg

e an

d m

oder

ate

size

dfo

llicl

es (

3) c

aute

rise

d

All

the

fairl

y la

rge

folli

cles

(8

)ca

uter

ised

All

the

larg

e fo

llicl

es

(6)

and

bloo

dfo

llicl

es (

4) c

aute

rise

d

Ova

ry c

ompl

etel

y re

mov

ed; c

onta

ined

9 la

rge

folli

cles

, 2

larg

e an

d 3

smal

lbl

ood

folli

cles

Day

saf

ter

oper

atio

nki

lled

4 4 3 3 3 3 4 4 3 3

App

eara

nce

afte

r ki

lling

Ope

rate

d ov

ary

No

corp

ora

lute

a fo

rmed

. 9

smal

lbl

ood

folli

cles

and

6 la

rge

clea

r ri

pefo

llicl

es (

new

ly f

orm

ed a

fter

oper

a-ti

on)

No

corp

ora

lute

a fo

rmed

. 5

bloo

dfo

llicl

es (

from

pri

cked

fol

licle

s) a

nd7

larg

e cl

ear

folli

cles

(ne

wly

for

med

)

No

corp

ora

lute

a fo

rmed

. 6

bloo

dfo

llicl

es

(red

) or

blo

od c

lots

whe

refo

llicl

es p

rick

ed.

2 m

oder

atel

y la

rge

folli

cles

(ne

wly

for

med

)N

o co

rpor

a lu

tea

form

ed.

2 la

rge

and

4 sm

all

bloo

d fo

llicl

es (

from

pri

cked

folli

cles

) an

d 5

larg

e ri

pe

folli

cles

(new

ly f

orm

ed)

5 fr

esh

corp

ora

lute

a (f

orm

ed

from

pric

ked

folli

cles

) an

d 1

mod

erat

esi

zed

folli

cles

2 la

rge

and

4 sm

all

bloo

d fo

llicl

es(f

orm

ed f

rom

pri

cked

fol

licle

s) a

nd3

larg

e fo

llicl

es (

new

ly f

orm

ed)

3 sc

ars

on

surf

ace

of

ovar

y w

here

folli

cles

cau

teri

sed.

3

larg

e fo

llicl

es(n

ewly

for

med

)8

scar

s on

su

rfac

e of

ov

ary

whe

refo

llicl

es

caut

eris

ed.

3 la

rge

clea

rfo

llicl

es a

nd a

few

sm

all o

nes

(new

lyfo

rmed

)

7 sc

ars

on

surf

ace

of

ovar

y w

here

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No

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No

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rupt

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fo

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all

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rupt

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No

fres

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No

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No

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ollic

les

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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 thereforeconcluded that mere rupture of the follicle, whether accompanied by stimulationand congestion of the cervix and uterus or not, is insufficient to cause intactfollicles 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, andO'Donoghue, did not realise that does might ovulate after jumping one anotherwithout actual coitus with the male and there is no statement in their papers that therabbits were kept apart. Perhaps either this or chance spontaneous ovulations such asmay 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 anytrace of the ovum or granulosa cells be found. The follicles were lined by thetheca interna and filled with red blood corpuscles and some pigment granulesformed from their disintegration as in an ordinary blood follicle. It might thereforebe objected that failure to form a corpus luteum in the artificially ruptured folliclewas due to operative trauma. It is feasible to postulate that in the sudden rupturedue to pricking, as distinct from the normally gradual process, the granulosa cellsbecame detached and were carried out with the liquor folliculi and the flow ofblood. The absence of the granulosa might explain the failure to form a corpusluteum since Marshall and others have shown that the granulosa plays an integralpart in this formation. However in two other rabbits (Series III) the ripefollicles were artificially ruptured 6 hr. after coitus (i.e. 4 hr. before ovulation orany marked morphological change in the follicle had taken place) and in one ofthese normal corpora lutea were found. It seems therefore that failure to form acorpus luteum after artificial rupture is not due to operative trauma but to theabsence of certain changes which take place during the 6 hr. following coitus andwhich either ensure the retention of the granulosa or enable it to undergo thosechanges which precede the formation of luteal tissue. In the second rabbit in whichthe follicles were pricked after coitus no corpora lutea were formed. Presumablythe follicles were immature or the stimulus of coitus had been insufficient to initiateovulation.

Since artificial rupture of the follicle by pricking did not cause ovulation orformation of corpora lutea, a further series of experiments was carried out in whichthe ripe follicles in one ovary were completely destroyed by means of an electriccautery, and in one rabbit the whole of one ovary was completely removed. It hasbeen shown by Asdell (1924) that removal of one ovary causes double the usualnumber 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 upona 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 excesssupply of "generative ferment" if available for the remaining follicles might causethem to ovulate spontaneously. The results are shown in Table IV, Series IV-VI.The complete destruction of the follicles was verified by histological examination

200 A. W A L T O N and J . H A M M O N D

afterwards; scar tissue, polymorphonuclear leucocytes and extravasated blood onlywere found at the site of the operation. No corpora lutea were found in the intactovary; instead, the operation caused a number of small follicles to ripen immediatelyin one or other of the ovaries until the total number of follicles again reached thenormal 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 killedfour days later three scars of the destroyed follicles were visible and there were againthree large follicles in the ovary, bringing the total number of follicles in bothovaries up to ten which is a normal number for rabbits of this strain. In othercases where a larger number of follicles were destroyed a certain proportion of thefollicles which ripened subsequently, appeared in the intact ovary, but in every casethe total for the two ovaries was about normal for the animal. It is to be notedthat a similar compensatory growth of new follicles occurred in those does in whichfollicles had been pricked although in this case blood follicles were present in theovary. It is apparent therefore that blood follicles do not inhibit the growth offresh 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 thenormal diameter but had not ruptured. Occasionally such follicles have beenobserved in normal ovaries and perhaps represent an early stage in cyst formation.Sections showed that these large follicles were, except for their size, normal inappearance but that the granulosa was very attenuated, being only about 1—2 cellsin depth. A little further expansion of the follicle would result in further attenuationof the granulosa and if this were accompanied by loss of adhesion to the theca andconsequent degeneration it is possible that cyst formation might be brought about.The significance of these double-sized follicles and their bearing on the problemof 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 themost part the theories have been advanced from purely histological study of thefollicle at various stages in the oestrous cycle but without accurate reference to theexact time of ovulation. It is only in an animal such as the rabbit which does notovulate spontaneously that it is possible to establish an exact time relationship.Our observations have been timed from coitus onwards and provide a consecutiveand dynamic picture of the whole process. In the majority of animals ovulationis spontaneous, the follicle ripens, reaches maturity and ruptures. Rupture isfollowed by the formation of a corpus luteum which undergoes growth and thenmore or less gradual regression. The regression of the corpus luteum is followedby 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 whenripe follicles are present in the ovary and coitus is required for ovulation and the


reinitiation of the cycle. Bouin and Ancel (loc. cit.) have suggested that in thoseanimals which do not ovulate spontaneously inhibition of the cycle is due to thepresence of considerable interstitial tissue in the ovarian stroma, which theyconsider to be physiologically equivalent to the corpus luteum and to have a similarthough not so pronounced inhibitory effect on follicular growth.

Similarly, we have postulated above that inhibition might be due to insufficiencyof "generative ferment" or the nutritive substratum which is essential for thegrowth of the follicle. Our experiments have shown that destruction of somefollicles is followed by immediate rapid growth of new follicles, demonstrating theconstancy in the supply of " generative ferment" and this rapid growth of folliclestakes place in the presence of the interstitial tissue. It would seem therefore thatif the inhibition be due to the action of the interstitial tissue or to inadequacy inthe supply of growth-promoting substances the inhibition only becomes effectivein the later stages of follicular development or alternatively that the immaturefollicles have a marked priority over the supply of such substances.

On the other hand, it has perhaps been too readily assumed that ovulation is buta stage in the growth of the follicle and occurs automatically when the folliclereaches a certain size. Although limited to the mature follicle, ovulation is inde-pendent of the actual size attained. A small follicle may rupture simultaneouslywith or before a larger one and, as previously noted, a follicle may attain double thenormal size without rupturing (cf. also the case of blood follicles). In the absenceof ovulation the follicle continues to grow although very gradually and it has beenshown how this gradual growth may lead to cyst formation. In the rabbit cystformation is transitory, the follicle tends rather to break down internally with theformation of a blood follicle and this occurs frequently when coitus and ovulationhave been in abeyance for some time. In this connection it is suggestive to findthat cyst formation from follicles occurs frequently in ovarian grafts and in smallremnants of ovary left after incomplete ovariotomy (Lipschiitz, 1925, 1927). Insuch 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 2thick adhesion of connective tissue, as occurs frequently as a result of tubal infection,ovulation can take place normally under this fibrous coat and occasionally throughit when it is very closely adherent to the ovarian surface. We are inclined to attributecyst formation to the absence of ovulation rather than to the presence of adhesivetissues. Cyst formation is not uncommon in young growing animals and in heavilymilking 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 continuationof the normal growth processes. There is evidence that the follicle about to ruptureunder the stimulus of coitus enters a very definite phase of secretory activity. Thereis a copious formation of secondary liquor folliculi with distension of the follicularwall to breaking point and a continuous flow of the liquor after rupture which issufficient to carry away the ovum and many attached granulosa cells. The rate ofsecretion and the consequent increase in internal pressure is probably of primary

202 A. W A L T O N and J. H A M M O N D

importance in causing rupture, the cells and tissues of the follicular wall beingunable to accommodate themselves to the sudden change. When distension iscontinuous and gradual, as in the normal growth of the follicle, the walls have timeto accommodate themselves to the change and do not rupture. Schochet (1916)suggested that rupture might be due to enzymic disintegration of the follicularwalls, 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 thelong interval between coitus and ovulation might be claimed as giving somesupport to the hypothesis.

It is also possible that as a result of pressure cutting off the blood supply tothe macula pellucida slight necrosis may aid in the separation of the cells. TheGuttmachers (1921) found evidence of plain muscle fibres in the theca of the sowand suggested that rupture of the follicle might be caused or at least aided bymuscular contraction. We have found no evidence of any appreciable quantity ofmuscle fibres in the follicle of the rabbit, nor did the injection of posterior-pituitrin insufficient amount to cause intense contraction of the uterus and death of the foetusin the 21-day-pregnant animal (see Knaus, 1926) have any influence in causingovulation in a doe in which ripe follicles were present in the ovary. The longinterval between coitus and ovulation and the marked distension of the follicularwall 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 asthe main cause of ovulation.

It has been suggested that ovulation is brought about by vasodilation anddistension of erectile tissues (Heape, 1905), but our observations do not supportthis hypothesis. It is true that marked congestion of the vessels accompanies theprocess of ovulation but the mechanical influence of the distended capillaries inthe walls must be relatively negligible as a force distending the follicle at the timeof rupture. Vasodilation, however, at the time of coitus may possibly initiate thesecretory activity within the follicle.

It has also been advanced that rupture is brought about by a breaking down ofblood vessels into the follicle and consequent distension and rupture. Our obser-vations show, however, that any rupture of the capillaries is secondary to therupture and collapse of the follicle.

The internal changes in the follicle which accompany ovulation are highlysuggestive of a new phase of secretory activity or a change in the orientation ofmetabolic processes. In its slight staining capacity the secondary liquor folliculican be distinguished from the primary. The ovum undergoes maturation withformation of the polar bodies. The luteal cells of the theca interna hypertrophyand after rupture the granulosa proceeds to the formation of the corpus luteum.Our experiments have shown that this formation of the corpus luteum is dependentupon the processes which accompany the early stages of ovulation (within 6 hr. ofcoitus) and does not occur with artificial rupture alone, Marshall and Runciman(1914) artificially ruptured the ripe follicles in the ovaries of a bitch and foundthat they became converted into structures resembling corpora lutea but with


abnormally large cavities. In this animal, however, ovulation occurs spontaneouslyand it is likely that this implies not merely spontaneous rupture but also spontaneousrelease of the necessary stimulus to luteal formation. It is probable, therefore, thatthis may have occurred independently of the operation and before the completedisintegration of the follicles. In the rabbit crescents of luteai tissue are exceptionallyfound in blood follicles; possibly the inrush of blood detaches the granulosa cellsfrom the theca and no luteal tissue is formed, but where the inrush of blood isgradual and one sided some of the granulosa cells may remain attached to thetheca, their nutritional base, and form luteal cells. The influence of coitus on theformation of the corpus luteum as demonstrated by these experiments, is not withoutparallel 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 lutealphase of the oestrous cycle and it is not improbable that this is due to a similarinfluence on the spontaneously rupturing follicle.

Our experiments throw little light on the question of how coitus initiates thesecretory activity which leads to ovulation. Some component of the orgasm whichaccompanies coitus rather than the mechanical stimulus of the penis or the presenceof semen in the vagina would appear to be involved since ovulation in the rabbitcan sometimes be induced by allowing does to jump each other, and since artificialinsemination only rarely causes ovulation. Yamane and Egashira (1925) found8*3 per cent, of cases of ovulation following artificial insemination and Hammondand Asdell (1926) 3-6 per cent., which percentage was, however, raised to 13*3 percent, when the doe was tried with the male but did not actually copulate. It is notunreasonable, however, to suppose that an orgasm is occasionally induced by thismeans. There are several possible channels through which the stimulus of coitusmight be transferred to the ovary, but the experimental evidence is very fragmentaryand it appears premature to operf a discussion of this field until it has been furtherexplored. The following research bears upon this problem and may be suggestiveof further enquiry. Heape (loc. cit.) states that ovulation does not follow copulationif the blood supply is interfered with, and Macomber (1926) found in rabbits thatdisturbance with the normal nerve and blood supply prevented the normal functioningof the ovary and sometimes caused cystic follicles. He has also shown that in therat which ovulates spontaneously the nerve supply to the ovary is not necessaryfor normal reproduction. Whether ovulation occurs spontaneously or can only beinduced by coitus in grafted ovaries in the rabbit is a subject for further enquirybut would give valuable information as to whether the stimulus to the ovary washormonic or nervous.

SUMMARY AND CONCLUSIONS.

Ovulation has been directly observed in the anaesthetised rabbit. The follicleruptures, on the average, about 10 hr. after coitus but there is some variation andall follicles do not rupture simultaneously. Ovulation is not the invariable conse-quence of the normal growth of the follicles and, although confined to those whichare mature, is independent of the actual size attained. Ovulation is accompanied by


increased vascularity of the follicle. The principal cause of rupture is the rapiddistension of the follicle by marked secretory activity. The ovum is carried out inthe flow of this secretion. Ovulation in the rabbit is initiated by some componentof the orgasm which accompanies coitus rather than by the mechanical stimulusof the penis or the presence of semen in the vagina. Corpora lutea are not formedif the follicle is ruptured artificially and this effect is not due to operative traumabut to the absence of some effect of coitus necessary for the formation of the lutealtissue. Artificial rupture or ablation of ripe follicles is followed by an immediatecompensatory growth of new follicles. Blood follicles formed from those artificiallyruptured do not inhibit this compensatory growth.

REFERENCES.ASDELL (1924). Brit. Journ. Exp. Biol. 1, 473.BOUIN and ANCEL (1910). Journ. de Phys. et de Path. Gen. 12, 1.COLIN (1888). Traite de Phys. Compar. des Animaiix, Vol. 11, 895. Paris.CORNER (1915). Contrib. to Embryology, Carnegie Instit. Wash. 2, 71.

(1919). Amer.Joiirn.Anat.2B, 117.GUTTMACHER, M. S. and A. F. (1921). Johns Hopkins Hosp. Bull. 32, 394.HAMMOND and MARSHALL (1925). Reproduction in the Rabbit, 78. Edinburgh.HAMMOND and ASDELL (1926). Brit. Journ. Exp. Biol. 4, 155.HAMMOND (1927). Reproduction in the Coza, 35. Cambridge.HEAPE (1905). Proc. Roy. Soc. B. 76, 260.HONORE (1900). Arch, de Biol. T . 16, 563.KNAUS (1926). Journ. Physiol. 61, 383.KiiPFER (1920). Denkschr. d. Schzoeiz. Naturfor. Gesell. 56, Plate VI.

(1920 b). Vierteljahrsch. d. Natur. Gesel. in Zurich, 65, 377.LIFSCHUTZ (1925). Brit. Journ. Exp. Biol. 2, 331.

(1927). Biol. Reviezvs, 2, 263.LONG and EVANS (1922). Mem. Univ. California, 6.MACOMBER (1926). Boston Med. and Surg. Journ. 194, 529.

(1927). Boston Med. and Surg. Journ. 196, 21.MARSHALL and RUNCIMAN (1914). Journ. Physiol. 49, 17.MARSHALL (1925). See HAMMOND and MARSHALL (1925), 55.O'DONOGHUE (1913). "Proc. Phys. Soc." Jour, of Phys. 46, vi.ROBINSON (1918). Trans. Roy. Soc. Edin. 5, 52.SABOTTA (1897). Anat. Hefte, 8, 469.SCHOCHET (1916). Anat. Rec. 10, 447.SHAW (1927). Journ. Obst. and Gynaec. Brit. Emp. 34, No. 3, 1.THOMPSON (1919). Journ. of Anat. 3rd ser. 14, 172.

(1919 «)• Journ. of Anat. 3rd ser. 15, 1.YAMANE and EGASHIRA (1925). Journ. Jap. Soc. Vet. Sci. 4, 101.

JOURNAL OF EXPERIMENTAL BIOLOGY VOL. VI, PLATE V.

iWALTON AND HAMMOND—OBSERVATIONS ON OVULATION IN THE RABBIT.

observations on ovulation in the rabbit · 2 0 1-0--t ' t 0 * i 0 0 # » 0 m i i i 0 0 3 6...

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