BIOLOGY

OF

REPRODUCTION

Peripheral

18, 401 -408

Blood

(1978)

and Ovarian Cyclic

in the

Hamster and

K. SAIDAPUR’

SRINIVAS

Levels of Sex Steroids

GILBERT

S. GREENWALD2

Department of Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas

City,

Kansas

66103

ABSTRACT (P), testosterone (T), estrone (E, ) and estradiol-1 7j3 (E2) in blood, ovary, corpora lutea (CL) and in the nonluteal portion of the ovary (NLO) were the cyclic hamster by RIA. In addition, androstenedione (A) was measured, but only Serum P levels were the same on Days 1 (day of ovulation) and 2 at 0900 h (5 ng/ml), but markedly on Day 3 and on the morning of Day 4 (proestrus). After the LU surge on the Changes

of Day

4, serum

peripheral studied in

in serum. declined

afternoon

h to 11-18 ng/ml and and the ovary occurred T levels declined rapidly

and ovarian levels of P increased abruptly until 1900 slowly thereafter. Peak T levels in both serum (209 pg/mi) 1400-1500 h of Day 4, paralleling the rising P levels. However,

declined between

after

in progesterone

levels by 1900 h, but there was a second peak of T by 2300 h in by the ovary. Serum levels of E, and E2 began to rise on Day 2, reaching peak values by Day 4 between 1200 and 1400 h (41 pg/mi and 152 pg/mi, respectively). Peak ovarian concentrations of E, and E2 were reached on Day 4 at 0900 h. These high levels were maintained up to 1600 h of Day 4 and declined rapidly thereafter. The serum levels of E, were about one-third as high as E2 and were not as drastically affected on the evening of Day 4. 1600

h to

which

serum

Serum

undetectable

was matched

androstenedione

striking

as for

levels of secretion

from

steroids.

of 5 g

significant

synthesis

ng/ml, but changes throughout the cycle were not as increased levels of A were associated with decreased the circulating levels of A probably reflect altered

Therefore,

LH on the evening

increases

in

on the evening

preovulatory

1-2

However,

estrogens and vice-versa. rates of other steroids.

Administration caused

ranged

the other

P

of Day

and

T

but

4 cannot

not be

of Day 4 (when all steroid E2. Hence, the refractory

attributed

There

are a few

bevels

1970; Shaikh 1974;

of

isolated

progesterone

Lukaszewska

concentrations

reports

and

and Saksena, 1972; Ridley and Greenwald,

have

not

been

on

(Leavitt

gen (Shaikh, 1972; Baranczuk 1973) in the cyclic hamster. bevels of androstenedione (A) (T)

insufficient

period. INTRODUCTION

ing

to

levels are declining) period of estrogen levels of LH during the

one,

circulat-

and

ovary

Blaha,

Greenwald,

nonbuteab

1970;

toto

and

mentioned

the

‘Supported

by

an

International

Postdoctoral

FelDivi-

of In-

dia.

2Address Department cal Center,

reprint requests to: Dr. of Physiology, University Kansas City, Kansas 66103.

(P),

estradiol luteal, has

testoster-

(E2) as not

in

the

opposed to been docu-

steroids

was

in serum AND

and

ovary.

METHODS

Adult female golden hamsters (Alesocricetus auratus) weighing 80-120 g and maintained on a 14 h light:10 h dark schedule (lights on: 0500 h-1900 h, CST) were used after 3 or 4 consecutive 4 day cycles with 6-8 animals in each group. Day 1 is defined as the day of ovulation. Hamsters were killed by decapitation on Days 1-4 at 0900 h and on Day 4 animals were also used at 1200, 1400, 1500 (proestrus), 1600, 1700, 1800, 1900 and 2300 h (estrus). The trunk blood was allowed to clot at 4#{176}C for 24 h and serum was then collected and kept frozen until assayed. One

September 20, 1977. August 11, 1977.

lowship of the Population Council, Biomedical sion, New York. Permanent address: Department Zoology, Karnatak University, Dharwar-580003,

in

compartments

MATERIALS

Accepted Received

and

therefore designed to account of changes in sex steroids throughout the 4 day estrous cycle of the hamster by measuring all of the afore-

and Greenwabd, However, blood and testosterone Similarly,

in

progesterone

(E1)

mented. The present paper provide a comprehensive

Blaha and Leavitt, 1975) and estro-

reported.

of

estrone

G. S. Greenwald, of Kansas Medi-

401

SAIDAPUR

402

AND

ovary was weighed to the nearest 0.01 mg and stored at -20#{176} C in vials containing 0.4 cc 95% alcohol. From the other ovary, corpora lutea (CL) and the nonluteal

portion of the ovary (NLO) were separated, weighed and likewise stored in alcohol. However, due to the profound luteal regression on Day 4, it was not possible to dissect out CL at different times of Day 4 and therefore the entire ovary was used in animals killed from 1200 h and on. Steroid

Assays

The mination

radioimmunoassays (RIAS) used for deterof steroids in sera and tissues were similar to

procedures

described

previously

for

progesterone

by

Thorneycroft and Stone (1972), using specific antisera for progesterone (Surve et al., 1976), testosterone (Pang and Johnson, 1974), androstenedione (antiserum obtained from Dr. John Resko, Regional Primate Research Center, Beaverton, Oregon), estrone (Wright et al., 1973) and estradiol-1713 (Exley et al., 1971). The testosterone antiserum cross reacted 58% with 5o-dihydrotestosterone and 2% or less with androstenedione. In the cyclic hamster, the preovulatory surge of androgen represents testosterone exclusively and no detectable amounts of DHT are present (Connor and Greenwald, unpublished). Hormone lev-

els

determined

with

this

antiserum

are

therefore

GREENWALD

serum

Changes

On

counter,

Model

The

recovery

of

internal

standard

from

2 of

the

nadir

steroids

ranged

significant

rise

the

Fig.

During

the

the

cycle,

serum

by

the

from

of

by

Days

serum

P occurred

a slow

decline

3 and

h of

of

to

P was

decreased

0900

(afternoon

continued

Cycle

bevels

mornings

in

bevels

followed

(P)

reached

proestrus

Day

4),

time

first

1500

until

with

4.

the

at

rise

4,

Day

h

1800

h

(Table

1,

1). The

the

concentration

ovarian

serum

levels. P

at

There

1400

and

concentration

1900

h

served

(Table

1500

h but,

decrease

Day

on

the

sharp

3 and ovary

the

by

4.

The

in

the

values

reached

next

value

Day

on

day

ovary

the

signifi-

of

the

the

of

a

cycle.

progres-

with

morning

of

1 with

dropped

nonbuteab by

ob-

a precipitous highest

was

P concentration

sively

until was

P increased

followed

Days

Thereafter,

stable

decrease

of

2,

by

in

2).

nonluteab

drop

rise thereafter,

relatively

significant

2, Fig.

P paralleled

a significant

concentration

on

P in

a

of

was

was

when

Luteal

cantly

Day

lowest 4 (Table

3).

in Estradiol

Changes

On were

Day

1 of

barely

(12

(E2) the

above

pg/mI).

The

subsequently

showed

(when

serum

striking at

1700

values

The

values

were

progressive h of

Day

1, Fig. ovarian

levels of by

Day

found

on

The the

2 and highest

afternoon

at

increased).

in

serum

culminating the

E2

assay

increase

P abruptly

4,

of

the Day

3.

no appreciable decline

approximating

Cycle

serum

doubled by

4, but

the

sensitivity

bevels

E2

h

cycle,

tripled

of Day 1500

During

the

serum

h (Table percentage

all the

but

on

During

the

scintillation

1 and constant

the

For each steroid assay, quantities of sera, homogenized tissues and standards were diluted to 1.0cc with distilled water and extracted with 2.0 cc anhydrous diethyl ether. For RIA of progesterone (P), 20 MI of sera, 1-S il of homogenized tissue and 5 p1(5.0-400 pg) standard P in methanol were assayed. For RIA of androstenedione (A), 20 p1 of serum and 5 pI (4.0-1000 pg) standard A were used. For RIAs of testosterone (T), estrone (E, ) and estradiol (E2), 300

liquid

Days

with

ovarian

3003.

for

Progesterone

significantly

estradiol.

Tri-Carb

in

relatively

and

a Packard

as tissues

RESULTS

referred to as testosterone. The antiserum for androstenedione cross reacted 70% with 5c-androstane3,17-dione and about 4% with dehydroepiandrosterone (DHEA). Hormone levels measured by this antiserum are collectively referred to as androstenedione. The antiserum for estrone cross reacts only 1% with

p1 of serum, 50 pI of homogenized tissues and 5 p1 (2.0-300 pg) standard T, E, or E2 in methanol were assayed. After extraction with ether, each tube was dried using a vacuum evaporator. The dry residue was suspended in 0.2 cc phosphate buffered gelatin (PBSG, 0.1 M phosphate buffer, pH 7.0), stirred on a vortex and incubated in a 45#{176}Cwater bath for 20 mm. To each assay tube, 0.1 cc antibody (dilution: 1/3000 for T; 1/50,000 for Pand E2; 1/3500 for A;and 1/25,000 for E1) and 0.1 cc radiolabeled steroid in 0.1 M PBSG were added. The assay tubes were stirred on a vortex and incubated for 24 h at 4#{176}C.Free and bound steroids were separated at 4#{176}C by adding 0.8 cc PBSG containing 0.625% charcoal Norit A and 0.0625% dextran. The assay tubes were vortexed and incubated for 20 mm. The tubes were then centrifuged at 1500 X g for 20 mm. The supernatants were decanted into counting vials and 1.0 cc dioxane and 9.0 cc toluene counting solution (15 g omnifluor/gallon toluene) were then added. Each vial was counted for 10 mm in

as well

80-90%. The lower limit of sensitivity for different steroid assays was as follows: P, 5.0 pg; A, 4.0 pg; T, E, and E2, 2.0 pg. Serum from ovariectomized (6 weeks) and hypophysectomized (1 week before the autopsy) hamsters contained approximately 0.6 ng P/mI; 0.5 ng A/mi; 30 pg T/ml; 9.0 pg E, /ml and 12 pg E2/mI, respectively. The hormone levels for tissues are expressed in pg or ng/mg wet weight of tissue. More details of the assay procedures are given in a recent paper (Terranova and Greenwald, 1978). Data were analyzed using Duncan’s Multiple Range Test (Steel and Torrie, 1960) and where appropriate, Student’s t test was employed. Differences were adjudged significant if P‘

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HOUR

HOUR FIG.

peak

at

0900

h of

ues remained 4.

levels

in

hormones

3, at which

bevels

ovarian

until

time

1500

TABLE

rose

concentration

2. Ovarian

from

the

of

steroid

lowest

hormone

values

levels

OF

the

val-

E2

CYCLE hamster.

1 to

h of Day

1600 h of Day 4; 1 h before of E2 began to drop (Table 1, Fig. E2

DAY

in the cyclic

at

Luteal

Day

Day

at high

fall

The

evident

levels of steroid

1. Serum

AND

a peak

followed

in buteal

was

on next

E2

(Table

serum 1).

in the

nonluteal

with

increasingly

at

Comparison

Day

in the cyclic

the

shows high

the

of

E2

cycle.

This

was

by a 50% reduction concentration of E2

3). The

ovary

a different

levels

pattern Day 3. in the

until

concentration

hamster.

Progesterone

Estradiol

Testosterone

(ng/mg)

Day Day Day Day Day Day

3 of

morning

and

hour of the cycle

Day Day Day Day Day Day

Day

the

1 0900 2 0900 3 0900 40900 4 1200 4 1400 4 1500 4 1600 4 1700 4 1800 4 1900 42300

h h h h h h h h h h h h

P values were ap‘

-o

0 > 0

0

0’

-p

E

3 C”

‘0

Ui

0 ‘C 0

0

“C

Ui

I-

a, 0.

HOUR FIG.

2. Ovarian

nonbuteab

levels of steroid

ovary

concentration

(Table (Table

all

of

be

accounted

the

hormone

hormones

3) 2)

with

by

the

by

DAY

ovarian

that

almost

the

OF

in the cyclic

the

indicates

produced

for

AND

ovary

nonluteal

hamster.

pattern of E2, but E1 bevels were usually one-third to one-half the values for estradiob (Table 1, Fig. 1). The highest serum E1 bevels were reached on the afternoon of Day 4 at

can

compart-

1400 and significant

ment. Changes

in Estrone

Changes

TABLE

CYCLE

in

3. Steroid

(E1)

serum

During

E1

hormones

the

closely

Cycle

in corpora

lutea

the

E1

was

not

plummeting

and nonluteal

Day of the cycle (0900 h)

Tissue

in

resembled

1500 h. This was followed decrease at 1600 h, but the

ovary

Progesterone

E2

of

lutea

Nonluteal

P values ap