Urinary estrogens II excretion of estrogens in rhesus monkeys stimulated with human gonadotropins

305

URINARY EXCRETION

OF ESTROGENS

STIMULATED

Meinert

ESTROGENS

II

IN RHESUS MONKEYS

WITH HUMAN GONADOTROPINS 1

Breckwoldt 2, Taras Murawec,

and

Joseph C. Touchstone

Steroid Laboratory, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Received 7/30/70 Revised 10/5/70 ABSTRACT

Spontaneously non-cycling rhesus monkeys were treated with gonadotropins extracted from human pituitaries and an HMG preparation. Treatment was carried out for i0 - ii days with 75 IU FSH daily followed by a single injection of 2000 IU LH. Daily urinary estrogen levels were determined by a method involving conjugate extraction, enzyme hydrolysis, and phenolic separation followed by two thin layer separations and gas chromatography. Estrone was found to be the major urinary estrogen. Estradiol-178 and estriol were detectable only in small quantities coinciding with high concentrations of estrone. Estrone levels as high as 56 ug/24 hours were found when multiple ovulations occurred. Ovulation time and ovarian size were determined by laparotomy.

INTRODUCTION

The many advantages

of using the rhesus monkey

306

ST ER O I DS

(macaca mulatta)

as a model

17:3

in studying

human reprod-

uction have been pointed

out by Balin and Wan

However,

is limited by the lack of

this usefulness

information

concerning

ovarian

(3).

steroid metabolism

(i0)

in this species. Estrogen monkeys, humans

excretion,

as studied

showed a different (8).

in pregnant

pattern

There are conflicting

from that of reports

sence of estriol

since Short and Eckstein

ted no estriol.

The evidence

urinary

estrogens

icting. Brown

Values (6) method

by Laumas

are contradictory

by Short and Eckstein

and during

tone appears and estradiol

monkey

of the is confl-

(9) using

the

to those reported

the quantitation

in the rhesus monkey stimulation

(ii) detec-

(ii).

This paper describes estrogens

on the pre-

for the nature

in the nonpregnant

reported

rhesus

in a control

of urinary period

with human gonadotropin.

to be the major estrogen rarely being

MATERIALS

Est-

with estriol

found.

AND METHODS

During the summer season some of the rhesus monkeys in our colony spontaneously develop amenorrhea. Those animals were chosen for this study. The animals had a history of a two to three month amenorrhea, and did not show any evidence of ovarian activity as indicated by monophasic atrophic vaginal cytology. All animals used were apparently of m i d - r e p r o d u c t i v e age. The animals were kept in metabolic cages for urine collection. Ovarian stimulation was achieved by treat-

March 1971

ST ER O I D S

307

ment with gonadotropins extracted from human pituitaries according to Bettendorf et al (4) and Breckwoldt (5). The following preparations were used: HHG (FSH activity of 92.0 IU/mg and LH activity 42.7 IU/mg, FSH/LH = 2.1), N4N69 (FSH activity 26.0 IU/mg and LH activity 160 IU/mg, FSH/LH = 0.16), and an HMG p r e p a r a t i o n (FSH 6.4 IU/mg and LH 1.3 IU/mg, FSH/LH = 5.0). The biological activities were determ i n e d by bioassays, Steelman and Pohley (12) for FSH, Parlow (13) for LH activity respectively. HMG - IRP II was used as reference. The treatment schedule was similar to clinical practice. Daily injections of 75 IU FSH for i0 to 12 days were given. On day ii or 12 a single injection of 2000 IU LH was administered. The ovarian response due to this treatment was followed by inspecting and m e a s u r i n g the ovarian size via laparotomy and by d e t e r m i n a t i o n of estrone (El), estradiol-178 (E 2) and estriol (E3) in the 24 hour urine. The estrogens were d e t e r m i n e d according to the method of Touchstone et al as described in the paper in this issue. This is outlined below. Scheme for Estrogen .

2.

Determination

Conjugate

extraction (ethyl acetate) ¢ Enzyme hydrolysis (8-glucuronidase)

4.

Free steroid e x t r a c t i o n (ether) + Phenolic separation (toluene - NaOH)

5.

I.

6.

II.

3.

.

TLC + TLC

(20% acetone - isopropyl ether) (12% methanol

- methylene chloride)

Gas chromatography

The phenolic fractions were subjected to two different thin layer chromatographic separations. The first d e v e l o p m e n t was in 20% acetone, 80% isopropyl ether. After eluting El, E2, and E 3 fractions separately, each fraction was r e c h r o m a t o g r a p h e d in a 12% methanol: 88% m e t h y l e n e chloride system. Finally, the samples were q u a n t i t a t e d by gas chromatography using a 10% QF-I plus 5% L45 column as described in the preceding paper. Recovery studies were carried out by adding purified labeled steroids.

308

ST ER O I D S

17:3

RESULTS

Figure ovarian

I illustrates

response

hour u r i n a r y ment period day

the t r e a t m e n t

in m o n k e y

estrogen

#143

levels.

m a x i m u m on day 6 w i t h

was continued, observed

on day

showed

ii.

56.0 ug/24

increased

flushing

i0 fold,

points.

the f a l l o p i a n

signs of o v u l a t i o n

by

while

After

This was

fol-

hours was

the left of the ovar-

injection occurred

3 ovulation

tube,

its

As the t r e a t m e n t

Neither

3 ovulations

as i n d i c a t e d

On

time the size of the

a 4 fold increase.

IU LH on day 12,

right ovary

estrogens.

hours.

hours.

At this

ies s h o w e d o v u l a t i o n 2000

the p r e t r e a t -

a s e c o n d peak of 43.0 ug/24

right o v a r y was ovary

During

to rise and r e a c h e d

lowed by a drop to I0 ug/24

and

in terms of the 24

there were no d e t e c t a b l e

3 the E 1 o u t p u t b e g a n

schedule

2 ova w e r e

of

on the

points.

On

recovered.

No

could be seen on the o p p o s i t e

ovary. The E 2 o u t p u t The El, E 2 ratio highest

followed

the E1 e x c r e t i o n

ranged between

a m o u n t of E2 found

ug on day ii of the cycle,

7:1 and 9:1.

pattern. The

in a 24 h o u r urine was coinciding

with

8.4

the s e c o n d

E1 peak. E 3 was d e t e c t e d maximally coincide

1.4 ~g/24 with

only hours.

in 2 s p e c i m e n s

in a m o u n t s

The E 3 p a t t e r n

the E1 and E2 e x c r e t i o n

did not

peaks.

of

March 1971

ST E R O I D S

309

ovulolion 3 CorporaLuteo

~

,o.~1

~"

IOjug-

FIGURE

I

FIGURE

II

1.2m-

l

I00-

2O0O

"

7/30 s;'l

el5

e)lo

m

eh5

e/zo,s9

BBMenses ovulotion ~L 1 Corpus Luteum

Monkey¢1:187

I0/~¢.

.~ ~% zorn~' osm.

- - - A

,oo

,u~.

. . . . . . . . . . . . . . . . .

L=o_.T

ttttttttttt 8;20

['u~"/ 8]25

8)30

9/o

9]10

,Menses

FIGURES

I AND

II

ESTROGEN CYCLE

EXCRETION

INDUCED

TREATMENT RHESUS

IN

BY

DURING

GONADOTROPIN

2 AMENORRHEIC

MONKEYS.

A

310

S T E R O I D S

17:3

The average estrogen output during the follicular development was much higher than during the luteal phase.

During the luteal phase the urinary

output of E 1 ranged between Ten days after ovulation

7.6 and 16.0 ug/24 hours.

the onset of m e n s t r u a t i o n

was observed. Figure

II shows the estrogen output during a

treatment cycle in monkey

#187,

except for an addi-

tional injection of 2000 IU of LH during the luteal phase of the treatment in monkey

#143.

schedule was the same as used

No estrogens were detectable

the p r e t r e a t m e n t period.

On day 3 of treatment a

marked increase of E 1 output was noted. follicular development

were

During the

3 major peaks of E 1 excretion

were observed with values of 9.8, 24 hours.

during

10.2 and 13.6 ug/

Coinciding with the E1 peaks,

found with values of 2.56,

3 E 2 peaks

2.4 and 1.8 ~g/24 hours.

The El/E2 ratio ranged between 4:1 and i0:i.

0.5 ug

E 3 per 24 hours was found in only one specimen, all other samples E 3 was not detected. the right ovary increased

in

The size of

7 fold during the treatment,

while the left ovary increased

4 fold.

After injec-

tion of 2000 IU LH, a single o v u l a t i o n occurred on the right side. On day 15 an additional was administered.

A slight,

injection of 2000 IU LH but not significant,

March 1971

ST ER O I D S

elevation of E 1 output was noted. cycle of m o n k e y

311

The treatment

#144 is shown in Figure

this case an HMG p r e p a r a t i o n was used, containing

64 IU FSH and 13 IU LH.

day was a d m i n i s t e r e d

for i0 days,

injection of 2000 IU LH was given ovulation.

III.

In

each ampule

One ampule per on day ii a single for induction of

The estrogen excretion p a t t e r n showed a

single peak one day prior to ovulation. output of estrone was found to be 28.0

The h i g h e s t Ug/24 hours.

Simultaneously,

4.2 ug of E2 could be detected in

this specimen.

Estriol was not detectable

out the whole

through-

cycle.

Only a minor

fluctuation

in the estrone e x c r e t i o n

was observed during the luteal phase ranging b e t w e e n 2.8 and 6.9 monkey

ug/24 hours.

The treatment

#203 is shown in Figure

IV.

cycle of

This animal rec-

eived the same treatment as animal #144 but did not respond.

Using another H M G - p r e p a r a t i o n

containing

IU FSH and 840 IU LH the ovarian response in Figure increase

IV was observed.

84

illustrated

On day 6 and 7 a slight

of the E1 excretion was found and continued

to rise reaching 24 hours.

a single peak on day i0 with 21.0

After the injection of 2000 IU HCG on day

i0, 3 ovulations

occurred,

as indicated by 2 corpora

lutea on the right ovary and 1 corpus left side.

ug/

By flushing the tubes

luteum on the

1 ovum could be

312

ST ER O I D S

17:3

|ovulation 30Jug.

M o n k e y :1:1:144

~t'

7 Corpora

Luteo

20,ug-

lOJug-

FIGURE

III

FIGURE

IV

L, ~ 5,~-

.

.

.

.

.

.

z

-

z

-

-

t

1

20~

100.

iU FSH t t t t t t t t t t 10115 •

-

-

~

.

.

.

.

.

.

.

.

==

1115

II/I

_-

lU LH

I 1/10

IIA5

Menses

ovulotion

Je

20 Jug- Monkey:t:1:203

3 Corporo Lutea

15~ugI O,Jg5~JgA

--

J v

T

~

IO0IU FSH r

9

HCG 2 0 0 0 lU

!

,/i

,>K~

a~zo

nl;3o

d/ao

,Menses

FIGURES

III A N D

IV

URINARY

ESTROGEN

RESULTING

FROM

STIMUI~TION RHESUS

LEVELS

GONADOTROPIN

IN 2 A M E N O R R H E I C

MONKEYS.

March 1971

ST ER O I D S

recovered.

313

S i m u l t a n e o u s l y with the E 1 peak 4 ug of

E 2 could be found in the 24 hour urine was undetectable

sample.

E3

throughout the whole treatment cycle.

14 days after the HCG injection the onset of menstruation was observed. Recovery studies with labeled and purified estrone resulted in constant values between 50 and 60%, with the average of 55.0 ± 3.2%.

DISCUSSION

The urinary estrogen output of the n o n p r e g n a n t rhesus m o n k e y has been investigated by Chatterjee and Anand (6). ween

(7) using a m o d i f i c a t i o n

of Brown's method

Daily excretion of total estrogens varied bet1 and 2 ug.

There appeared to be two major

peaks during the normal cycle. estrogens was done. (ii) estriol

According

to Short and Eckstein

seemed to be absent in the urine of the

normally cycling monkey. Laumas

No separation of the

Using the Brown m e t h o d

(9) was able to detect all 3 classical

in the urine of the nonpregnant monkey. estradiol

estrogens

Estrone,

and estriol were found in about the same

amount ranging between 1 and 3 ~g/24 hours. Using a highly ermination

specific m e t h o d for estrogen det-

in our experiments,

estrone was the major

314

ST E R O I D S

urinary estrogen.

17:3

In most instances

and estriol were not detectable.

estradiol-178

In a few instances

E 2 and E 3 were found in small quantities m o s t l y coinciding with very high levels of E1 during stimulation with human gonadotropins.

Our results

indicate

that

the estrogen excretion pattern in the m o n k e y regarding the El, E2, E 3 r e l a t i o n s h i p that of the human.

is different

from

This statement gets further sup-

port by the findings of Hopper and Tullner

(8) in the

pregnant monkey.

combined

Using gas chromatography

with TLC these authors abundant estrogen, small quantities during pregnancy.

to be the most

estriol was present in only very

and showed only a 4.5 fold increase In late p r e g n a n c y estriol was

found in concentration hours.

found estrone

ranging between

These distinct differences

1 and 2 ug/24

in urinary estro-

gen between the human and the rhesus monkey might be due to a different peripheral since there is evidence

estrogen m e t a b o l i s m

from in vitro

studies

(14)

that the secretory product of the monkey ovary is similar to that of the human. Comparing the estrogen output of the 4 monkeys, the patterns of monkey

#143 and #178 appear

similar

with 2 and 3 major E 1 peaks during follicular development.

The first peaks are noted on day 6 of the

March 1971

treatment.

ST ER O I D S

315

This may indicate maximal

follicular estrogen synthesis.

stimulation of

The subsequent

signi-

ficant drop of E 1 excretion may be due to follicular regression.

No ovulation occurred.

that the m o r p h o l o g i c a l

It can be supposed

development of the stimulated

follicles was not completed

for ovulation at this

time of the treatment cycle. As the treatment continued another ovarian response to the exogenous g o n a d o t r o p i n

is noted after a

two day period of relative refraction. day stimulation period,

After a i0

LH was a d m i n i s t e r e d and ovu-

lation occurred. The E1 excretion patterns of monkeys #203 are different

from the others

(Figures I and II)

since the E 1 output increased slowly,

reaching

m a x i m u m on day ii and i0 respectively. tions occurred after administration of LH or HCG.

preparation.

in E 1 excretion

Three ovula-

of a single dose

is due to the differ-

since these animals received an HMG However,

it can be concluded that in

these cases m o r p h o l o g i c a l ovary occurred

its

It is difficult to determine whether

this difference ent treatment,

#144 and

and endocrine

response of the

simultaneously.

The unusual high quantities

of E 1 found in the

urine indicate that certainly more than one follicle was responding

to the treatment.

The incidence of

316

S T E R O I D S

multiple reached

ovulations

proves

17:3

that more

than one follicle

full maturation.

REFERENCES

i.

,

3.

.

.

,

7.

.

o

Supported in part by the USPHS Grants AM-K-14013 and HDO 1199 and a grant from the Ford Foundation. Ford Foundation

Fellow

Balin, H. and Wan, 273, 1969.

in Reproductive

L. S.

J. Reprod.

Biology.

Med.

2,

Bettendorf, G., Apostolakis, M. A. and Voigt, K.D. Acta Endocr. 41, i, 1961. Breckwoldt, M. Hypophysares luteinisierungs Hormon (Habil. Schrift) Hamburg, 1969. Brown,

J. B.

Biochem J. 60,

Chatterjee, S. and Anand, Res. 55, 973, 1967.

B. K.

Hopper, B. R. and Tullner, 517, 1967. Laumas,

U. R.

85, 1955.

W. W.

J. Endocrinol.

J. D'A.

31,

J. Endocrinol.

Ind.

J. Med.

Steroids

297,

95,

1965.

i0.

Jeffery,

ii.

Short, R. V. and Eckstein, 22, 15, 1961.

12.

Steelman, S. L. and Pohley, 53, 604, 1953.

13.

Parlow, A. F. in Human Pituitary Gonadotropins, ed. A. Albert. Ch. C. Thomas, Springfield, Ill., 1961.

14.

Flickinger,

P. J.

F. M.

G. L. and Breckwoldt,

34, 387,

1966.

Endocrinol.

Endocrinology

M.

Unpublished.