BIOLOGY

OF

12,

REPRODUCTION

Studies

194.211

(1975)

on Reproduction

with

Antigonadotropins

and H.

Department

of Animal

Emphasis

Science,

H.

Award

with

humility

Universily

and

would

be remiss

inspiring ates, Dr.

if I did

influence H. M.

Though

their

January, Evans

acknowledge

personalities

cycle

differed

until

bitch

with

Dr.

but I saw very September. When

and

I the

Evans

first

in the

cow

and

and

tant

used

account.

awesome

knowl-

with

him

was

was for

by no racing

Dr.

Evans

of biology.

had

My

exhilarating experience, means diminished by with in

delivery

wagons

Berkeley

control East

and

lights

an

contact

which passion

his down

for

on our

way

descripcolorful

Telegraph

running

kennel

of the Berkeley.

Hart

was

at this

Acting

Chairman

time

Division of Veterinary He apparently received

report

of my

because Degree

before I left at the University

me that he had the Department

work

in Dr.

Evans’

© 1975 by The of reproduction

Society in any

for form

the Study reserved.

were

we had

preceded of

a

concenduring by the Hart-

G. me,

Dr.

I shall

Hartman.

An

of Dr. Hartman cogent contributions

was to

at

the

annual

Association

audience.

meetings

of

His

of

Anatomists.

discuss my

an

the His

with

modest

and

Dr.

with Dr. Hartman was Duke University in 1936 meetings of the American It

inspire a neophyte

him

wisdom

his colleagues.

the

was

his

un-

confidence which in biology, to

significance

of some

of

findings.

indirect

findings

and

with

Anatomists. to

contact, actually order, with Dr. one

of correspondence

of at

of Reproduction.

of

My second chronological

laboratory

animation

to conversation

canny ability prompted me,

194 Copyright All rights

6 months

have

characteristic to make

Association in

Science at a favorable

chairmanship Husbandry

of the

zeal

gonadotropic of the mare

eulogies

My first conversation in a washroom at while attending the

to work for my Ph.D. of Minnesota he told

accepted the of Animal

within

blood and

of papers on a wide variety of He was a conspicuous and an impor-

extended

Oakland.

Dr.

in the

enthusiasm

of the blood

member

the an

traffic

to the dog

then

pregnant He joined activity,

remarks on papers presented were sprinkled with witticisms which were always a delight to

Avenue

yellow

their

American

my drab a more

edge

to

impressive his ability

slides

graphs. Finally, he couched tion of our findings into

of

who

microphoto-

be

and His and

Award

for

vaginal

to

blood

man

smears

the

and

the precedent established recipients of the Carl

discussions subjects.

and

urine,

mare.

in stud-

in Davis, I inof gonadotropic

Following distinguished

An artist reproduc-

selected

of my

pregnancy.

in

racy of every aspect of the was brought in for drawings tract

urine

inspiring

add

were

me a position

completion

after arriving of the finding

in the

from a 5-month European trip, I had made significant progress. For the next 4 months he worked with me daily checking on the accu-

tive

offered

upon

fair knowledge tration in the

little of Dr. he returned

work. of the

tentatively

95616

women by Aschheim and Zondek. me in the search for gonadotropic

most in

California

Department

activity

widely

Davis,

ies. Shortly formed him

both had a dramatic I began work on the

of the

1926

Davis

of two of my early associEvans and Dr. G. H. Hart.

many respects, they influence on my life. estrous

not

of California,

the

deep

appreciation. This honor, more appropriately. belongs to my colleagues and graduate students; I accept the honor in their behalf.

Progonadotropins

COLE

Upon considering the large number of outstanding scientists in the field of reproductive physiology, I accept this Sixth Carl G. Hartman

on Gonadotropins,

between

Hartman. to

stemming

him

Dr. which

Evans

the first in Hartman was

from

an

Dr.

H. had

suggested

exchange M.

Evans

related

our

that

the

STUDIES

unfertilized ble

ovum

in

the

1924)

that

the

more

than

24 hours.

ovum

and

study

by

Smith

view, based on the in the mouse and on opossum (Hartman,

cates

that

the

days.

remained

viable

the beginning

of the obtained

Phemister

Simpson

extends

Dr.

remained

several

for

not

of estrus,

the first

male. Comparable by Bischoff (1845), (1971)

(1973).

and

Hartman

Andersen (1932)

it teleologically unreasonable, long estrus in the dog, for

results HoIst and

considered

so early. To

the length of the viability of ovum, 2 dogs were tested

daily for estrus with active was deferred until the 6th

in the uterine and female therefore, ble for

7th or whelped.

that late

9th

day

the basis Amoroso

tion and

may that

the

found

remain fertilizaovulation. This with

statements

Phemister bred either

estrus

(1971) on the

conceived

and

of indirect evidence, Griffiths (1939) concluded that ovula-

by follicle,

and

may

nonetheless,

a viability

of

may result and also with

occur as late as the 6th ovulation is probably

concluded, Our (1845) indicate

conclusion

of not

day of estrus successive,

take

that less

than

3 days.

They

dog’s

ovum

the

48 hours.

own studies and those of Bischoff and of HoIst and Phemister (1971) that the ova are closely grouped in

oviduct

making

it unlikely

quite

on

ovum

in that

mies The

to determine report of

we

the did

that

ovulation

indi-

bitch

correctly

occurs

not

of

the

perform

the exact Phemister

dog’s

laparoto-

time of ovulation. el a!. (1973) that

ovulation may occur in Beagle bitches where from the second day preceding 7th day

to our viability

of estrus

conclusion of at least that

the

must

dog

adds

some

authors

uncertainty

found

ovulated by the evidence

ovum

is viable

reluctantly

anyestrus

that the dog ovum has a 4 days. It should be noted,

these

32 bitches had estrus. Although

this oc-

criticized

viability

that

21 of

the 3rd day of favors the view

of

fertilization in estrus

of

in estrus. (1932)

A recent

acceptance and that ovulation

on the 6th day

may after

of HoIst and that 5 bitches

and

has

the egg 4 days

in the

on the viability of the mammalian evidence suggests that the ovum

fortunately

is in conformity

conclusions who found

follicle

bred were

surge

of

end of the oviduct showing male pronuclei. Our studies indicate,

breeders mating

On

ova

LH

a single set of apparently

sacrificed beginning

In the dog

that at least

conclusion dog from

early Hartman

I).

(1974)

still

dogs showed corpora lutea

age.

(Fig.

McDonald

on the 8th and of estrus, re-

spectively. Both well developed fertilized

days

and

that

were the

uniform

several

males but mating and 11th days of

estrus; the dogs 13th days after

estrus,

curs

however,

determine unfertilized

the

over

to the

in view of the ovulation to occur

195

about one day before first provides further evidence

our

in the dog (Evans and Cole, that ovulation occurs within

after

acceptance have been

via-

bitch

for

Hartman held a contrary studies of Long (1912) his own studies in the

24 hours

REPRODUCTION

of the

oviduct

Our studies 1931) indicated

ON

for several

admit

that

the

has

reviewed

days,

I

evidence

is

inconclusive. Blandau

less

than

hamster, man but the

(1962)

24

hours

in the

the

literature

ovum. is viable

opossum,

mouse,

rat, rabbit, sheep, cow, monkey and 30 hours in the ferret and possibly in

guinea

pig.

Thus, Hartman’s (1924) view “that cannot wait long for the spermatozoa-these must be on hand when ovulation takes has

wide

application

fertilizable tion,

for

this

Possibly division mammals, tion

and

several

days

for the

the

that

fact

in the dog and the is not completed

fox, until

Stricht, has

1923;

some

period of viability of the if indeed this conclusion PITUITARY

I had biology upon

intended of the

rereading

on mammalian occasion two

place,” is

ovulaovum.

maturation unlike after

most ovula-

Pearson

bearing

egg

ovum

mammalian first

der

dog

the

following

the

1943)

THE

if the

is unique

(Van

Enders,

The for

and

on the

ovum in the is correct.

long

oviduct,

GONADOTROPINS to discuss pituitary Roy

Greep’s

at some length gonadotropins excellent

gonadotropins years ago, I have

the but

lecture

on a similar decided that

196

COLE

I. Five

FIG.

close and

fertilized

grouping Cole.

my

of ova

comments

would

(Greep,

comments

to

remarks

constitute

1973). a

relating

in the

uterine

end and

to the

plaguing

biology

Zondek

dualistic

theory

a major I will limit and

some

and

the

Thus,

confession

concerning

gonadotropins. Aschheim posed

ova

is characterisitc

mone

of a bitch

that

the

of the (1927)

to

a

re-

LH

my

mone

few

sented

Dr.

people

might

inconsistpituitary first

pro-

(LH) (FSH).

and According

follicle

stimulating to a reliable

was

on the

interval

horsource,

not

first

of release

named

because

he wished

the Hisaw’s

day

of metestrus.

of ova

is short

probably

follicle

luteinizing

contraction,

FLH,

initials

interpret

his name

I at one time validity of the tary

of gonadotropins

and evidence was soon obtained by Fevold et a!. (1931) for the separation of two gonadotropins from pituitary tissue. Hisaw and his group named the two hormones luteinizing hormone

of oviduct

indicates

This (Evans

1931).

dundancy

encies

segmenting in the oviduct

this

he feared

as indicating

attached

to the

had some doubts dualistic concept

gonadotropins. due

and

This in

part

to

horreprethat that

hormone.

about the of the pitui-

skepticism the

was

fact

that

Dr.

Harold Goss and I were unable to separate the follicle stimulating and luteinizing activities of pregnant mare serum precipitation procedures. My doubts on the dualistic

by

fractional

concepi

were

STUDIES

fortified by with pituitary

studies, activity:

two criteria were used to identify LH I) the ability ofan extract to increase

and

when

2)

the

augment

the

extracts. substances

It was would

response

ranging

albumen

given

ability ovarian

weight

1934;

found that the age had a pronounced

question mone.

Cole,

always ovarian

agreement, ascorbic

low

(1961)

and

contained

of

biological

ventral

mg

weight

than

100 weight.

one.

A number

the biological rations can reduction these

only

of

of

referred

Originally

studies

units

3%

of

A total purified prostates mg

for

as

much

LH

as

cite evidence concerning the

ventral

prostate for

presented

and LH.

evidence

will augment the secretion have

the

Johnson which

reported

that

of gonadotropic prepawithout a concurrent

immunological In my

activities view,

the

ultimate

prouteri fe-

considered

for early secretion of eta!.

that follicular estrogen.

(1970)

support opinion inducing

growth.

Greenwald

Interestingly,

(1974)

release

during

initiating

assign

proestrus

follicular

successive

to

Bast a burst

and

of FSH

a prominent

role

development

for

in the

estrus.

For many the ovulating

years, LH was considered hormone but Lostroh and

to be John-

son

reported

either

(1966)

purified

FSH

of FSH that

that

or LH

and the

I g

of LH.

FSH

of this repair

statement they the interstitial

than

female

The

which

and

rats. reported

con-

in support not of

On the

other

that

when

they

FSH

was negligithe finding of (Schwartz, 1969: Schwartz el a!.,

report

that

in

antiserum did not LI-I antiserum did. inconsistencies

by no means detract of the remarkable

knowledge the biology

state

preparation is neutralized “the ovulation inducing

rats FSH whereas seeming

authors

note that 180 zg did tissue of the ovaries

capacity of this LH-free ble”. This study supports Schwartz and co-workers Schwartz and Ely, 1970; in

in

feof 3 zg

employed

1% LH

et a!. (1974)

the LH in an FSH with LH antiserum

female ovulation

of

effective

These

preparation

more

Lipner

zg

equally

in hypophysectomized with 3 daily doses

not

hand,

20

were

tained

tioned cance of

of their

this view but a more widely accepted is that LH and FSH synergize in

1973)

the action of of testoster-

Sher-

II g to above,

it was

hypophysectomized

this FSH and co-

whereas

that

weighing 53 mg and mg in hypophysectomized

inducing ovulation male rats pretreated

NIH-LH-Sl.

authors views

of workers

in the

is

between of Par-

to 10.1

in assaying

activity be reduced

preparations.

this

indicated that by Sherwood

NIH-FSH-S4. These that there are conflicting

(1971)

there

assay

of dry

dose

Radioimmunoassay preparation prepared

indicated that FSH LH in stimulating

Ac-

Sherwood et a!. (1970) frozen sheep pituitaries

12.7 mg as compared

Ewing

(1966)

prostate

weighing

and

assays.

dry weight of produced ventral

the

us to hor-

for example, depletion assay

21

of

led

rats.

follicular

more

response

(Saun-

facts

a luteinizing

dose of preparation

OAAD

extracts

These

et a!.

per

specificity

preparation, ovaries 113

female rats. alone has no

weight

of Sherwood

which

contained

purified duced weighing

concerns the ovarian weight

FSH

found

The

et a!. (1941). FSH from

workers

ovarian (1970)

responsible and the

the

dry

on et a!.

wood

FSH was development

by FSH

be its biologi-

conflict the

in hypophysectomized (1962) state that

male

Greep prepared

zg

response Li et a!.

on

we

inconsistency concerning lack of conformity of with biological assays and

not the

a 40

Cole,

must

of

rat of

Hashimoto

g

and

point FSH

of

female extent

between

NIH-FSH-S4

or egg

hormone

of the immature effect on the

1936a).

inconsistencies

Saunders

Another influence

influence

to FSI-I

to casein

for a given activity.

Next

One disturbing LH is the apparent radioimmunoassay to

to

test cal

1938).

the existence We were wrong!

cording

response

Cole,

induced

and

extracts;

fractions

ZnSo4

and

luteinization

FSH

pituitary

from

Saunders

ders

which we In the early

found that a wide variety of augment the ovarian weight

(Maxwell,

1936;

with

of

197

REPRODUCTION

further obtained

luteinization

some results fractions.

ON

of the of the

molecular gonadotropins

I have from the advances

mature block mensignifiin the

structure and eloquently

I 98

COLE

described by Greep ery of Schally and a!., 1972) substance and

LH

(1973). The his associates

that brings

one hypothalmic about release

is another

isolation

of

(Reichert

the

and

milestone a

Ward,

as is the

HUMAN

LH

to whether

that

there

has

of HCG property

preparations of the HCG

were due molecule

was

to contaminating

due

Butt

(1959)

excreted

reported

amounts

from less equivalents

2. (a) Ovary

FIG.

of rat

found

to inhibit

uninjected

800

250 lU.

controls

Crooke

(Cole

receiving lU.

of

of HCG. and

responses

erties

of the

second

depended HCG

HCG

rise female

in ovarrats

concluded

that

as

Further support does HCG bind

of this to LH

FSH

of

follicle

receptors

intrinsic (Fig.

antiserum the response

we

a!.,

upon

molecule

intrinsic

and

the

rat

prop-

2). I might augmented to FSH.

HCG

stimulating

that

molecule properties.

view, is that not only receptors but also to testes

et

(Rabinowitz

1974).

and PREGNANT

In serum,

lU.

of HCG

pIus

0.1

ml

size of the largest 1967).

MARE

SERUM

GONADOTROPIN

ranging

800 The

the immature

has

per day much of

HCG

Bigelow,

these

woman

in the urine

of rat

both

in intact

Thus

to an intrinsic or whether it

up to 13 mg 20A. However

receiving

inhibited

from

process. We (Cole and found that HCG anti-

of

properties

a pregnant

of FSH

many that

a question

FSH. that

than 1.8 of HMG

Ovary

has to

been

stimulating

separated

add that this HCG rather than depressed

HCG

follicle

be

follicular development in hypophysectomized rats in a manner anticipated on the basis

OF

comparable

but the

of hFSH

(HCG)

properties

would

ian weight

CHORIONIC

is agreement

pituitary

serum

recent

EFFECTS

FSH

during the purification Bigelow, 1967) have

1974).

GONADOTROPIN There

this

releasing of both FSH

$ subunits

and

PHYSIOLOGICAL

biological

signal discov(Redding et

alone. of HCG follicles

(PMSG)

1935, we reported after treatment

Note

the marked

antiserum. has been

This reduced

that with

pregnant H3S,

mare became

follicular

development.

(b)

amount

of antiserum

was

to approximately

that

of

STUDIES

completely the capacity pituitary ders

ineffective by to augment gonadotropic

and

Cole,

itself the

ON

but retained response to

preparations

1935).

We

(Saun-

erroneously

con-

from this finding that pregnant mare contained two gonadotropins-one was effective by itself and another

which

synergized

pituitary

with

extracts.

In the

ever, tation

we correctly of pituitary

pended

upon

the

proteins

in the

pregnant

tion

to slow

the

effects

of

pregnant the

capacity

basis

estrous

cycle,

gonadotropin the

mares

we

was

of

depending

cycle,

noted

the

were serum

studies

(Cole 2 to

collected cycle. lents in

upon in the of

the

intact

non-

stages

of

serum

quantitative

at best, and

gonadotropin(s) mares.

proximately

present

rat.

of the

effects

of

that and

biological pregnant

25

mg

to

an

action

the the

ysectomized PMSG old

allowed However

studies on purimare serum indi-

vided lates and

a single

gonadotropin

together

with

our

was studies

non-pregnant that only

onadotropin was secreted by the mare that this hormone might appropriately called equine gonadotropin. Though the clusion that the gonadotropic activity mare serum depends upon has been verified, we were

on one and be conof

a single in error

superovulation low mating, Albaugh PMSG

the and

ovulation 1937)

1933)

age

males

in

and

28-day

if mating

will result (Fig. produces ovulation rats

(Cole

(1966)

is 3). in

et a!.,

have

pro-

PMSG stimugonadotropins

ovulation

in

the

intact

alone on the will produce

and superfecundity response is erratic

Cole,

375

prostate

in the hypoph-

PMSG given cycle in cows

may fol(Wagnon.

unpublished).

produced

of the gonads a!., 1931) and

about

immature

female

facilitates

in-

hypophyvalue of

of the

evidence suggesting that the release of pituitary thus

daily)

of

Bogdanove

animal. Although 17th day of the

et a!.,

intact

(Cole,

and

(ap-

controls of the same the strong “luteinizing”

superfecundity PMSG rarely Gay

the

days

I.U.

is confirmed

hypophysectomized

of non-pregnant

this,

rats

20

that

animal. will produce

female

male”. referred

20

weight

in

1932)

for

to

exceeded

reported eta!.,

(Cole

obvious,

of

weight

the been

of immature from a control

average

of uninjected intact about 4 times, Thus

nature

to

hormone,

daily

equivalent

the

define

secured

in

stimulating

were

is

was

frequently

R.U.

serum

crudely

activity in us to suggest

follicle of 20

throughout

only

the gonadotropic mare serum led

pregnant substance

a

mg-this

tedious studies 50 samples of

qualitative

as

injection

these

It

words, in spite of attained in the

evidence

has

creased the prostates sectomized male rats

In

unet a!.

Pencharz

development

in

stage

to

1940).

In any instance, fied extracts of cated

PMSG

no

blood

in the serum

tissue

Although

that

1939).

only

germinal

found

the

biolog-

the testis, and a second growth in the female and

to that

that our rather the extraction of could,

tissue of follicular

the

biological

had

from

serum.

no

non-

and

approx-

solids

“In other of purification

of

7 days

immature

mare

extracts,

Leydig causing

It usually took 100 to 400 ml equivaof serum to produce an ovarian response

therefore, involving

pregnant

Pen-

indicated

indistinguishable

that

Goss,

3 liters 4 to

of

1940;

1940)

containing total

serum

mares

and

every

serum

the

similar

treated

Cole,

et a!.,

I.U./mg

properties

serum.

support the view that mare serum contains 2 distinct hormones, one specifically affecting the intersititial tissue of the ovary and the

biological

concluded in

pregnant

differences,

gonad-

at several

present

serum

of pituitary

the

ical

secreted identical to

mare

and

Cole

preparations

7000

present

serum prepara-

the

from

purified

inert

studies

collected

(Goss 1940;

(1940) concluded, the high degree

their biologi1936).

extracts

studies

the augmenextracts de-

serum

of

in pregnant

et a!.,

imately

how-

to enhance and Cole, of

Our that

in

year

of

mare

the absorption

mares

pregnant

following

gonadotropin

charz

gonadotropin

concluded that gonadotropic

otropins and, thus, cal effects (Saunders On

a

in concluding that the gonadotropins by the pituitary of the mare are the

cluded serum which

199

REPRODUCTION

dramatic

increases

in size

of the pregnant mare (Cole et in the gonads of the fetus (Cole but

the

teleological

significance

200

COLE

FIG.

day

3. Uterus

of rat given

of pregnancy

of these

(Cole.

changes

16 lU.

of PMSG

on 28th

awaits

clarification

(Fig.

4).

Catchpole et a!. (1935) found that the half-life of exogenous PMSG in the gelding was six days as compared to 26 hours in the rabbit.

Parlow

and

following

half-lives

in

rat:

the

HCG-4.9 half-life was

reported apparently metabolism amounts

at the found earlier

(1961)

of several murine

terectomized tion

Ward

be

no

of PM SG. of PMSG

are

hours. in mares

The hys-

of hormonal

secre-

comparable

in the gelding.

plays

the

minutes,

PMSG-26 PMSG peak

to

reported gonadotropins

LH-I7

hours and of endogenous

day

of age with

25 fetuses.

Necropsy

on the

12th

1937).

to

Thus

the ovary

significant

role

Further,

though

excreted

that

in the

in

the

and

milk

amounts rate the

of

pregnant

are

of disappearance

interest. On concentration pituitary and Lyons Goss,

of this

the basis of of gonadotropic the chorion,

(1934)

concluded

by 1943)

in

et a!., 1967). formation of

blood (Cole The site of

produced

lactating

insignificant

the found

little

but evidence

activity of the allantochorion been carefully rinsed with

small

adhering

urine

found

endometrial tremendous

cup amounts

the the

hormone

from

PMSG studies activity Catchpole

that

chorion

mares explaining

is

PMSG we

of

on the in the and

(Cole

was and

of hormonal

if this tissue had saline to remove secretion. of the hormone

We in

STUDIES

4.

FIG.

U nstained

Crown-rump fetus, 22 cm. gonads

are

gonads

have

length

(CR.) Four now

many

greatly

times

creted

larger

regressed.

which

(M)

the

(Cole

et al..

were

epithelial

cells

glands

in the

lining

that

unique

the

studies, provided

a!., se-

uterus

endometrial

hormone

decidual-like

cups which have migrate into the

in the

their origin endometrium eta!.,

1973). These chorionic to secrete hormone days

(Fig.

rionic cells cup secrete finding remarkable

that

6).

The

incorporated PMSG the

is

stages

length blood

of gestation.

(cor). (b) CR. of fetus. 50cm. of fetus.

when

the

reported

when

the

Clegg

el

that was

a!.

(1962)

blood.

Clegg

found

the

PMSG individual

secretion variations

present could (1962)

the

that

change

the excretory

rate

estrogen.

fetus amount

has

a of

PMSG formed (Bielanski et a!., 1956; Clegg et a!., 1962). Based on the relatively insensitive rabbit ovulation test, Bielanski et a!.

foal.

At 2 days

present

significantly,

after

to 7).

(1934) showed at mid-pregnancy

and Cole (1934) showed that present in the urine of the mare foaling and in the first voided

endometrial with the

due (Fig.

Cole mare

of

More

in

then to a levels of

that in

cho-

significantly

4 mares

evidences suggests by the fetal placenta

not

and the

that

but also that be detected in

simply mares

did

in

but

only

bred

been over

Hart

present fetus

a stallion and the different were not between

Parenthetically, estrogen is secreted

not

not

cells have in vitro for

the

fetal C.R.

a mule

the horse. ovariectomy

of the

The

attains

was

carrying

eta!.

years to show that

by

PMSG

mare

successive jack to

endometrial

that

(a)

length of The fetal

85 cm. fetus

girdle area (Allen 1973; Hamilton

in the consistent upon

C.R.

and evi-

in cells which from the cho-

conclusion

genotype

influence

at various

endometrial cups were small amounts of PMSG

cup

R. Allen convincing

is produced cells

in the allantochorionic Moor, 1972; Allen

el a!., shown

W.

(d) maternal

(1956)

et was

the

the

the

dence

(F) gonads

1933).

designated

hormone

uterine

In some elegant his associates have

150

fetal

ovaries. from

area.

non and

and

maternal

disappears

5). Clegg

cups (Fig. that the

by the the

than

PMSG

30 cm.

structures

as endometrial (1954) suggested

of maternal

201

REPRODUCTION

length of fetus. 7 cm. The maternal ovary contains 2 corpora nearly solid corpora are present in this section. (c) C. R. length

of approximately

specialized

and

cross-sections

ON

foaling,

in recognizable

Catchpole estrogen the day urine

estrogen

amounts

was before of the was

by the

not tests

employed the foal.

in the urine of either the mother or The precipitous drop in urinary

estrogens

of both

the dam

and

the

foal

is most

202

FIG. the cup constitute

COLE

5. (a) area

Endometrial are

many

cup times

an allantochorionic

is an autolysis

of all

tissues

from larger pouch.

in center

mare than x7.

sacrificed outside

on

105th

day

of pregnancy.

the cup

area.

The

cup

(b) Endometrial

of cup.

x 14. (Cole

cup and

from Goss.

and

mare 1943).

At this

its secretion

sacrificed

stage, and

on 63rd

the uterine

the allantochorion day

of pregnancy.

glands

in now

There

STUDIES

ON

REPRODUCTION

203

500

400

.2

300

4,

200 4, 4,

100

C, 0

0 20

0

40

80

60 Age

6.

FIG.

PMSG

production

by four

cultures

100 of cultures

of

horse

120

140

160

180

(days)

allantochorionic

girdle

cells.

(Allen

and

Moor.

1972).

easily explained by assuming that the fetal placenta which is separated from both the dam and the foal at birth is the source of the hormone.

pituitary given

gonadotropic

and further stimulate the Because

GONADOTROPIC PROGONADOTROPIC SERA

OF

ANIMALS

WITH It is now

AND ACTIVITIES IMMUNIZED

GONADOTROPINS widely

accepted

injection

of gonadotropins

formation activity

of antibodies of the gonadotropin.

there

have

same

type

presence

been

The

views

treatment

term

posed

by

enhanced

or

Thus refer

in

serum

progonadotropic

response

activity” (1938)

to

to an

(1937)

and of a horse a gonadotropic

pro-

refers

to

a gonadotropin

appropriate

pituitaries

would

found

the when

with a with the

test

that

treated for preparation

enhance

the

the with

activity.

“progonadotropic Rowlands

this

animal.

the term is not used, as is proinsulin, to a precursor of a hormone.

Thompson dogs with

in the

result

in the

serum of an animal treated chronically gonadotropin is given concurrently gonadotropin

chronic

as to why

may

of a substance(s)

gonadotropic

the result

which inhibit the On the contrary,

divergent

of

that may

the 20-80 from

response

sera

to

when

immature

female

that the serum ovaries of these

the serum

response IN

preparation

concurrently

did not

rats

alone would intact animals.

produce

an ovarian

in hypophysectomized

female

rats,

Thompson not depend the serum

concluded that these activities did upon a gonadotropin present in but, rather, that the chronic treat-

ment

resulted

had

in the

formation

of anti-

bodies to the pituitary antagonist a!., 1936; Leonard, 1934). Several authors (Rowlands, Collip, et

1937;

a!.,

Katzman

1947)

sponse given

to sheep concurrently

stergaard

progonadotropic

only

against

animal

progonadotropic tained by

of

gonadotropin

days sheep

antigen

of the

of

sera

of

concluded

that

sheep

Later

response injecting the than

et a!., (1938) that

itself

which

found

the

one

the

that

can easily antiserum

injection

used

with of

a

be obwith a as an

1968).

concurred the

generally

with it was

gorewhen rats.

asserts

gonadotropin

other

Thompson

that

response

the

(Snook

Rowlands

Katzman

pituitary gonadotropins to immature female

is treated.

to

1938;

1939;

with sheep pituitary would augment the

(1942)

the

1937,

et

el a!.,

reported

treated chronically nadotropic extract

(Evans

the

view

pituitary

204

COLE

200

180

160

140

120 E a,

100

80

60

40

20

0 20

0

40

60

80 of pregnancy

Days 7. PMSG

FIG.

stallion jack;

extracts

x x, #{149}--

concentration mare

-#{149}.

resulted

mare

in

in the serum

814 bred 860

the

bred

to stallion:

of mares

x

-

-

-

that

partial provide

inhibition a better

activity with the gonadal

the

antiserum of LH balance

when

x. mare

bred

to a jack

860 bred

as compared

to stallion;

to breeding mare

#{149}-,

to a

814 bred

to

to jack.

formation

of

1937)

anti-

might activity of FSH

induce and and

a consequent augmentation response. Collip (1934,

suggested

that

was under

normally certain

substance which

bodies against the pituitary antagonist thus resulting in a progonadotropic or gonadotropic response. Later Rowlands (1939) suggested

120

100

an

antigonadotropic

present in the circumstances

blood was

masked. a

thus LH of 1935,

Katzman

et a!.

progonadotropic antiserum evidence a!. (1950)

(1939) activity

but

they

in support suggested

postulated is due

provided

that to LH

no

of this view. that a small

the

in the

convincing Deutsch amount

et of

STUDIES

the

antibody

body

formed

complex

a soluble

resulting

antibody nadotropin

resulted with

authors the

also

may

a second contain

response,

the

tracts. other that

J

Q

#{212} #{212} CAJU

0

U

‘

z I

I

gonadotropic,

and

with

Our

experience

I

anactivicrude

with

HCG

preparations

is like

and in HCG

that

of

investigators working in this many unexplained inconsistencies In some

to give several weeks duration

-

00

LII

encountered.

a progonadotropic

-

the

and progonadotropic of mares treated

sera

crude

that

response.

9

studied

containing about 2300 I.U./mg sera of ewes treated with similar

univalent antian antigonado-

latter

have

FSH,

biva-

postulate

precipitating

lent and non-precipitating bodies; the former producing tropic

the

in precipitation of the goresulting inhibition. These

proposed

antiserum

We

tigonadotropic ties in the

elimina-

given concurrently; amounts of

larger

205

REPRODUCTION

antigen-anti-

in a slower

tion of the gonadotropin on the other hand,

ON

u Q

QQ

Q

00

I

I

instances

series each

o

Q00OC

many field

in are

it is necessary

of treatments before any

)

-

the ex-

of about gonadotropic

6

O(j

CsJ It)

OO#{224}#{212}OO

00

lUll

II

I!

(I, 4.

150

0

-

,\‘

0

.v

0C.)

In’.

I“I’

I\\ ,ix\

U, 4-

100

/\

\

-

\ Ix

I I

a)’. 4-9-

0 4-

o

llI-

U,

toe, 00

50

-

/\

x

II 9-

4-.-

C

-D

a). >

0’

III’

-

,

-D 4,.-

#{149},__ I,’I,

\T

.% ‘5

/X

a)’. >.

0

-50

x

-

-

,0

-S

#{149}o’

-

o_.o

tt,

-

a, Cl)

I

-100 0

I

10

20

30

I

I

40

50

60

Weeks 8.

FIG.

Responses

subsequent

to the

hours

the initial

after

third

of

intact

series injection.

of the seminal vesicle weights period of injection. X. ventral

immature of injections. The

figures

from those prostate:

male The

rats rats

at the top

to

30 ml

were

injected

of the graph

of 6 uninjected controls. vesicles: 0.

#{149}, seminal

of

FSH twice

indicate The testes.

antiserum daily

collected

for 3 days

the significance

with

during

and

necropsy

96

of the difference

bars along the abscissa (Snook et al.. 1968).

indicate

the

206

COLE

activity

or progonadotropic

instances the serum

series. Gonadotropic in

responses

intact

immature

increases

or

decreases

mare

treated

with

effect whereas

the

anti-FSH increased

vesicles

and

prostates

activity

in the

the

of

crude

on endogenous weights of the are

indicative

gonadotropins

for the activities

of evidence

that

seminal vesicles do not support period

after cause and

latent

not

sible

responses

suggest

that

extract

for

used

the

LII

are

gonadotestes of the

shown such

in Fig. a view.

treatment

with

increases seminal

in the vesicles

contaminating

LH

as an antigen

activity

in

is respon-

of the

FSH

anti-

serum. The ventral hypophysectom from mg

8.7

prostates of ized males

mg

of untreated

in animals

antisera. responses,

the

the

contain LH-S8

about (Snook

ovarian

ascorbic

serum

was

animals

receiving

On

basis

sera

not

of the

acid greater

ventral

were

estimated

equivalents of 1968). However, depletion than

of the that

depend

solely

upon

increased

to

HCG

obtained

and

HCG

FSH rat,

tralizing

either

HCG

ventral

as well

alone.

is given

with

mized mized

females males,

the did of LH

when

When FSH

the to

FSH

Evidence

in

the for

the HCG

antianti-

hypophysecto-

or with LH to hypophysectothe progonadotropic responses

are presumably due to complexing neutralizing HCG antibodies with nous

LII

we postulated,

response

is given

as the

In the hypophof non-neuwith

accounts,

serum

we the FSH

of the intact immafor the gonado-

antibodies

serum

gonadoThus

between and

antiserum,

prostate

mMg/ml small to

its

complex antibodies

and LH accounts

antiserum

by radioim-

activity.

a soluble HCG

the

responses the amounts of

10 to 91 were too

for

postulated that non-neutralizing

which

in both

antiserum

progonadotropic

endogenous ture female

the The

depressed

involved

progonadotropic However,

in the

LI-I in the

also

was

was

responsible

or

depressed antiserum.

LH

ranging from of NIH-LH-S1

solely

tropic

rats

evidence LH

gonadotropic and to HCG antiserum. found

produced

response obtained when was given in conjunction to hypophysectomized fe-

Thus that

munoassay, equivalents

FSI-I

of the ovine

to

of control

levels

female activity

antibody

rats.

crude

of

injection

antiserum with NIH-LH in hypophysectomized male of rabbit antibody to ovine with the HCG antiserum in

the progonadotropic the HCG antiserum with NIH-FSH-S7 male

gonado-

concurrent

with

immature

suggested

the

response in hypophysectorats as did the concurrent

NIHthe anti-

fraction

responses

The

antiserum

intact

the

prostate

sera. This provided the first clue that ventral prostate response of the antiserum not

antisera.

injection of the Sprague-Dawley rats. The injection LH concurrently

HCG

FSH

in the

studied

tropic response in this animal. ysectomized male, complexing

up to 38.0

8 ml of certain

studied

25 g et a!.,

Sprague-Dawley were increased

found

(1972)

a progonadotropic mized female

be

same

gonadotropic

progonadotropic

HCG

LH

best

precipitation.

a!.

and

rabbit

antigens

result of residual exogenous in the sera. The decreased and the increased weights

FSH

LU

and progoMany lines

these

FSH before the sera weights of the prostates the

as

gonadotropic of the sera.

indicate

prostates and 8, for example,

does

of

in this

the

were

ethanol et

gonadotropic

with

responsible nadotropic

long

FSI-I seminal

concern has been to determine in the sera of animals treated

chronically

The

antigen

the deinhibiting

sera.

Our main substance(s)

not the tropins weights

as an

and

Ahern ovine

a

that

responses

of HCG

of

Furthermore,

found

the greatest antigonadotropic activity the serum was subjected to ammonium

tropic

of intact

Presumably, reflect the

hormone

in

of the

serum

FSH

Fig. 8. weights

it was

sulfate

but not percentage

vesicles

receiving

antiserum.

study having when

be obtained

weights

seminal

rats

in the

hypophysectomized

in the

and

male

are shown in pressed testes

in other evident in to a single

intact females females. The

prostates

immature

be

may

or

males, in immature hypophysectomized testes,

is found;

these activities may of animals subsequent

the

of nonexoge-

or LH. that

HCG

antibodies

complex

STUDIES

ON

207

REPRODUCTION

V0 (Blue

Dextran)

160 HCG antiserum plus 1311-OLH

Normal plus

serum 1311-OLH

120

I I $

I I I I

‘7 0 I’ U,

I-

z

0

I

0

I I I I

0.9

1.0

1.1

1.2

1.3

1.4

1.6

1.5

Ve Ftc. “1ILH (1.5 pH void

9.

The

influence

was incubated x 81 cm) 8.5, vol

hormone

of HCG with

of Sephadex

containing

0.153

superfine

M NaCI.

The

Ewe

9-437

was

also

incubated

on

0.4 ml of serum G-I0O

(V0).

column: an arrow denotes 90, 1619-1632 (1972).

antiserum

was

treated

with the position

a rabbit

V0 migration

of iodinated

at 4#{176} for approximately equilibrated

column with

the

HCG

as an antigen;

antiserum

of the peak

24 hr. and

and developed

was calibrated to ovine

of radioactivity

LH

with LH

then

placed

at 22#{176} with

0.025

dextran,

in order

Blue ewe

on a Sephadex

9-388

was

(RAOLH)

emerging

on the column

M barbital

separately

the column

buffer.

to determine

untreated. and

from

column.

The

the

iodinated ran

on the

Endocrinologt

208

COLE

with

ovine

LH

has

on a Sephadex “I-LU and HCG

been

G-l00 column either normal

antiserum.

radioactivity incubated

As can

with as

the

antibodies

to form

being

due

be seen

in Fig.

when serum

to

the

with

a larger

by filtration

of incubates sheep serum

emerges earlier the immune

interpret HCG

obtained

of or 9 the

“I-LH which

is we

complexing

the

LII

SUBSTANCES

IN

BY

CHROMATOGRAPHY

indicated

HCG

above,

ANTISERUM

we

have

successful in separating the and gonadotropic activities chemical We now

bodies whereas

the

are bound at least

not

free or one

hypothesis

that

neutralizing

with

the

than

HCG

antigenic

sites

on

antigen

one

which the

molecule,

HCG

binds

type

the

of anti-

antibody

pends

upon Snook

is likely

column.

When

this

by to to

effluent

or female combines

rats, with

respectively, the these gonadotropins

form

a larger

molecule,

their

half-lives

and

producing

above

is only

thereby

than

gamma

gamma

we

have activity

globulin. (1968)

not de-

As

showed

of HCG globulin

stated that

antiserum fraction

gonadotropic

and

the was and it

progonad-

due in part at least to However, the gamma

fraction, contain

I look

having

been

reproductive present

upon

obtained substances

by

back

with

date.

great

a participant

I am

from

astounded

which

has

pleasure

in research

physiology

ble progress

1926

in

to

embarrassing for

this

that

most

the

committee

honor,

but

of us can

made

in this

time

and our

thus

who rather

expect

area

rather

some of

nominated

to emphasize

to be wrong

it is better

errors

the

by the rem arka-

been

of biology. I have made it a point to cite of my errors, not with the intention me

salt other

globulins.

In closing, to

that

depends

to

than

part

forthrightly

to

attempt

to

them. I hope my exposition has that a host of problems remain one to concentrate concerning the

of

made to be

on our mechanism

lack of

hormonal action, the gaps of our knowledge would appear much greater. I will end my

the

discourse

with

pass

Herbert

con-

E. C. Amoroso have

antito

increasing a progonado-

a hypothesis,

or

the

is injected concurrently into hypophysectomized

male body

LH

postulated

antibodies, progonadotropic

the

solved. Were of knowledge

hormone the

(1972)

et a!.

that

and

that

the

enhancing

pituitary

activity

a

conceal it clear

is free

of

eta!.

the

that one of against only

molecule

this

this

taining this antibody with LU or FSH

tropic response. Though the

on

from combining with by a masking of some the

Ahern

above,

admit

of the

activity

progonadotropic

the

is prevented the column

through

Though the

more

antibody HCG on

Thus

anti-

because

of markedly

FSH.

to combine or react with exogenous FSH or LH. At can suggest, as a working

a portion

Sepharose

biological

and proantiserum

importance

capable

globulin enriched precipitation may

for the progonadothrough the column. substance(s) which

is formed against HCG, types of antibody is formed

column.

the

are

otropic activities are the same substance.

to the HCG on the column some of the non-neutralizing

combine

column is endogenous this stage, body these

been

through a column coupled to acti-

HCG

substance(s) responsible tropic response pass Thus the non-neutralizing does

not

methods of protein fractionation. have unpublished results indicating

Sepharose,

of practical

gonadotropic activity present in the gamma

antigonadotropic by conventional

that if the serum is passed in which HCG is covalently vated

be

probodies

non-neutralizing proven that

SEPARATION OF NEUTRALIZING AND NON-NEUTRALIZING

As

may

of

radioactive

molecule.

AFFINITY

ability to separate antigonadotropic gonadotropic activities in HCG

the

M. cited

“There

a

favorite

Evans and

in his may

quotation

from

Euripedes

of which

George

W. Corner

biographical

memoir:

be many

And many things God Past hope or fear.

Dr. Dr.

(1972)

shapes

of mystery,

makes

to be

And the end men looked for cometh not, And a path is there where no man thought, So hath it follow here.”

STUDIES

ON

209

REPRODUCTION

tion

REFERENCES

as

an

intrinsic

gonadotropin. C., COLE,

AHERN.

(1972).

H. H., GESCHWIND,

Physiological

gonadotropic of ewes

studies

I. I. AND

on

the

and progonadotropic immunized

against

ITzE,

L.

of

the

identity

substance(s) HCG.

in sera

Endocrinology

90,

R.,

W.

(1973). Invasion

D.

HAMILTON.

The

Rec.

origin

of

W.

equine

AND

endometrial

of the endometrium

R.

M.

cups.

II

Moox,

by the trophoblast.

W.

equine

R. AND

cal

R.

MOOR.

cups.

trophoblastic

cells.

E. C. AND

AMOROSO,

Memoirs

M.

The

I. Production J. Reprod.

Fellows

of

the

origin

of

Fert.

of

PMSG

by

29, 313-3

G. W. (1972).

CORNER,

of

(1972).

16.

Biographi-

A. and

Geron-X

C.

Royal

SIMPsoN, Cycle

Los

S. AND

ASCHHEIM,

Altos.

Society

18,

J.

BAST,

D.

profiles

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