Human Chorionic Gonadotropin receptors by [D-Trp6]-Luteinizing Hormone Releasing Hormone in hypophysectomized rats

Vol. 90, No. 3, 1979 October

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

12, 1979

Pages 687-693

REDUCTION OF TESTICULAR LUTEINIZING HORMONE/HUMAN CHORIONIC GONADOTROPIN RECEPTORS BY [D-T&l-LuTEINI~ING HORMONE RELEASING HORMONE IN HYPOPHYSECTOMIZED RATS A. Arimura,

P. Serafini,

S. Talbot

and A.V.

Schally

Departments of Medicine and Anatomy, Tulane University and Endocrine and Polypeptide School of Medicine, Laboratories, Veterans Administration Hsopital, New Orleans, LA 70112

Received

June

27,L979 SUMMARY

Adult and immature male rats were hypophysectomized and injected daily with saline or 0.2 or 2 pg of superactive Luteinizing Hormone Releasing Hormone (LHRH) agonist, [D-Trp']-LHRH subcutaneously for seven days - with, or without, concomitant treatment of 1 IU Human Chorionic Gonadotropin(hCG) or The administration of [D-Trp6]-LHRH reduced 50 IU Pregnant Mare Serum. Luteinizing Hormone/Human Chorionic Gonadotropin receptors in all cases. The magnitude of this reduction was dose-related. As small a dose as 0.2 ng of the peptide resulted in approximately a 72% reduction of the receptors. The results suggest a direct action of [D-T~~~]-LHRH on the testis. It also indicated that reduction of testicular Luteinizing Hormone/Human Chorionic Gonadotropin receptors by the peptide is not necessarily due to the overstimulation of Luteinizing Hormone (LH) release from the pituitary through a "down regulation" mechanism. Although release

Luteinizing

of both

Luteinizing

(FSH)

from

doses

of LHRH or potent

gonadal the

involution

testicular receptors

--in vivo

in animals

ova

These

(8);

Hormone/Human paradoxical

(LHRH) stimulates

and Follicle

- for

Stimulating

Even in fairly

(6);

effects,

interruption

effects

were

results

doses

in

some of

such as prevention of gestation

and reduction Chorionic

of large

period small

the Hormone

the administration a prolonged

paradoxical

of spermatogenesis

(10).

Hormone

and in vitro,

(l-5).

induce

of fertilized

Luteinizing

(LH)

LHRH agonists

agonists

of implantation

Releasing

Hormone

the pituitary

superactive

suppression

Hormone

of ovarian Gonadotropin believed

Abbreviations: LH, Luteinizing Hormone; FSH, Follicle LHRH, Luteinizing hCG, Human Chorionic Gonadotropin; Hormone; PMS, Pregnant Mare Serum; SC, subcutaneously; binding.

(l-7); (9)

and

(LH/hCG) to be due to

Stimulating Hormone; Hormone Releasing NSB, non-specific

0006-291X/79/190687-07$01.00/0

687

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@ 1979 by Academic Press, Inc. in unyform reserved.

of reproduction

Vol. 90, No. 3, 1979

"down

BIOCHEMICAL

regulation",

of LH release

In a previous gestation

was distinctively

ova,

as well

other

hand,

the wall none

paradoxical

effect

some fetal

fetuses

study

LHRH agonist

the

full

Yoshinaga

be reproduced

testicular

addresses

in the rat

treated term.

of LHRH agonists

of LH. not

swelling

of the uterus

survived

effect

The present

effect

swelling

is

if

in the

Is reduction

by lldown

of LHRH on gonadal

(7)

to investigate

question:

induced

These

regulation",

animal.

implantation

of fertilized

uterus.

- along

with

a large

On the thickening

suggest

be fully

accounted

reported

that of large

treatment absence

with

that for

the

paradoxical

doses

of LH.

LHRH agonist gland.

LH/hCG receptors it

caused

the

by

of the pituitary

of gonadal or is

of

dose of hCG, but

findings

by administration

LH/hCG receptors

- treated

of the

with

cannot

el.al.

[D-Trp']-LHRH

the

on

blocked

of the hCG-treated

prevented

and fetal

eventually

of the

that

from

and hCG effects

substances

from

completely

as ballooning

could

reduces

different

treatment

oversecretion

both

resulting

(9,10,18).

[D-Trp6]-LHRH

of the uterus

- was observed

of the

by an LHRH agonist

Although

(6).

RESEARCH COMMUNICATIONS

of LH/hCG receptors

we compared

the appearance

[D-Trp6]-LHRU

It

study

in rats

gestation, rat

reduction

i.e.,

overstimulation

AND BIOPHYSICAL

by

by a direct

receptors? METHODS

Animals: Adult or immature male rats of CD strain from Charles River were housed in animal quarters which were equipped with controlled lighting (light: 0500 hrs. to 1900 hrs.) and controlled temperature (24 + ZOOC). Animals were fed on Purina Chow rat diets and water ad libitum. Experimental rats were hypophysectomized through the auditory canal under Nembutal (Abbott Lab., North Chicago,IL) anesthesia supplemented with ether. were into

Experiment 1: Young adult male rats weighing 180-200g were all hypophysectomized through the external auditory canal, the following four groups: Group

A:

Received 0.2 ml 0.9% saline, subcutaneously( 7 days, starting on the day when the animal tomized.

Group

B:

Similar to Group (human chorionic New York, NY) SC hCG was given on

A, except that the animals gonadotropin for injection, every 2 days for 7 days. the day of hypophysectomy.

688

They used. and divided daily for was hypophysec-

received U.S.P. The first

1 IU hCG Ayerst Lab., injection of

Vol. 90, No. 3, 1979

Group

tion

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

C:

Received daily SC injection of 2 pg [D-Trp6]-LHRH/lOOg B.W. (Ayerst Lab., AY 25650) dissolved in 0.5ml 0.9% saline for 7 on the day of hypophysectomy. days, starting Group D: Similar to Group C, except that in addition to [D-Trp6]-LHRH, the animals also received 1 IU hCG in a manner similar to that of Group B. Seven days after hypophysectomy, all animals were sacrificed by decapitaand testicular tissue was assayed for LH/hCG receptors.

Experiment 2: Immature male rats of 26 days of age were divided into two groups, A and B. The rats of Group A were injected with 50 IU Pregnant Mare Serum (PMS) (Ayerst Lab., Montreal, Canada) SC and hypophysectomized 67 hrs later. Group B did not receive any treatment before hypophysectomy. Each group was subdivided into three groups which were composed of Al,A2,A3,Bl,BZ,B3 and designated as follows: Al and Bl: Injected with 0.2ml 0.9% saline SC daily at 0900 hr for 7 days, beginning on the day of hypophysectomy. A2 and B2: Injected with 0.2ml 0.9% saline containing 0.2 ug [D-Trp']LHRH SC. A3 and B3: Injected with 2 pg [D-Trp6]-LHRH SC daily for 7 days, beginning on the day of hypophysectomy. All animals were sacrificed 7 days after hypophysectomy, i.e., one day after the last injection of saline or [D-Trp6]-LHRH. LH/hCG receptor assay: The testicular LH/hCG receptor assay reported by After decapitation Catt et.al. (11) was modified for use in the present studies. of each animal, one testis was dissected, decapsulated and weighed. It was then homogenized in 2ml ice-cold 0.25 M sucrose, 5 mM MgC12 0.1 M Tris-HCl buffer, pH 7.4 using 15 strokes in a Teflon-glass Patter homogenizer. The homogenate was decanted into 25ml of the sucrose-containing Tris-HCl buffer and centrifuged at 13,000 rpm for 15 minutes. The pellet thus obtained was resuspended in 0.1% bovine serum albumin, 5mM MgC12, Tris-HCl buffer, pH 7.4 to obtain a concentration of 1Omg tissue per 100 ~1. The assay was performed using 1Omg equivalents of tissue per tube and l-2 ng 1251-hCG in a total volume of 0.5 ml. Non-specific binding was assessed by addition of 2 pg bovine LH (NIH-~-lo), which a preliminary determination indicated was sufficient to saturate receptor sites. By this means, non-specific binding (NSB) ranged from 0.1 to 0.5% of total bound radioactivity. Incubation was carried out in duplicate Reaction was stopped by addition of 1 ml at room temperature for 16-20 hours. ice-cold bovine serum albumin(BSA) - containing Tris-HCl buffer. The preThe incubate was then centrifuged at 3000 rpm at 4oC for 30 min. cipitate was washed and recentrifuged, then counted for radioactivity by a Searle Autoganna Counter, Model 1285 with an efficiency of 70%. The specific binding was calculated by subtracting non-specific from the total bound radioexpressed in cpm per mg of tissue. for each homogenate, activli2tz I - hCG was prepared by radioiodinating 5 pg hCG (Serono Labs, Boston, Mass. 15,000 IU/mg) with 1 mCi Na125 I, using a lactoperoxidase method as described by Miyachi et.al. (12). The reaction mixture was purified on a Sephadex G-100 column, 1 X 50 cm, using 0.1 M Tris-HCl buffer, pH 7.4 as eluent. The specific activity of 1251-hCG r anged from 40-80 lKZi/pg as calculated from the elution pattern. RESULTS As shown

on Table

hypophysectomized of the

testis,

adult but

1, daily rats

did not

injection

significantly affect

the

of 2 pg [D-Trp6]-LHRH (pcO.01) testicular

reduced wieght.

for

7 days

LH/hCG binding

Administration

in sites

of 1 IU

Vol. 90, No. 3, 1979

TABLE 1:

Group

BlOCHEMlCAL

RESEARCH COMMUNICATIONS

Effect of treatment with hCG and [D-Trp6]-LHRH receptor in hypophysectomised adult rats.

Mean Body Wt. + SE (g)

Treatment

A

Saline'

B

Saline

C

[D-Trp6]-LHRH

D

[D-Trp6]-LHRH + hCG

1

AND BIOPHYSICAL

on testicular

Testicular LH/hCG receptor5 (cpm I 10~4

Mean Testicular wt.4 + SE 6%)

170 * 11.84 (4)3

LH/hCG

628

f 10.7 (4)

(4)

901

f 40.5

163 f 12.6

(4)

656

f 40.8 (4)

443 2 103.5

(4)

161 f

(4)

872

f 38.2 (4)

213 f 100.2

(4)

+ hCG2 155 + 6.5

3.7

(4)

Saline or 2 ug [D-Trp']LHRH / 100 g B.W. was injected on the day of hypophysectomy. days, starting

2 1 IU hCG was injected of hypophysectomy.

1264 + 275.2

801 f 127.0 (4)

SC daily

SC every 2 days for seven days, starting

All animals were sacrificed on the day following 3 Number in parenthesis indicates number of rats. 4 Testicular weight: Avs. B p < 0.01 C vs. D p < 0.01 A vs. C Not significant B vs. D Not significant

(4)

for seven on the day

that of the last

injection.

5 LH/hCG receptors: Avs. B Not significant Avs. C p < 0.01 Avs. D p < 0.01 B vs. D p < 0.05

hCG every

2 days

decreased

mean LH/hCG binding

Daily

administration

binding

sites.

(p < 0.05) reduced value

the (Table

(Table

seven

of

that

number

sites;

[D-Trp6]-LHRH

the testicular

but

the

latter

concomitant

of LH/hCG binding

in the

saline-hCG

of LH/hCG binding

was not

hCG further

in this

The treatment fo l/3

(p
difference with

sites

group. sites

weight

to l/4

last

and significant.

reduced group

with of the

LH/hCG

was smaller

the peptide respective

alone control

1). treatment

in hypophysectomized 2).

days increased

The number

than

Similarly, sites

for

The larger

with

[D-Trp6]-LHRH

immature

rats

- whether

dose of the LHRH agonist

690

decreased

testicular

pretreated induced

LH/hCG binding with

a greater

PMS, or not reduction

of

BIOCHEMICAL

Vol. 90, No. 3, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 2:

Effect of PMS and different doses of [D-Trp6]-LHRH on testicular LH/hCG receptor in hypophysectomized immature rats. _____--__-Treatment Mean Body Wt. Mean Testicular Testicular Wt. _+ SE (m8) LH/hCG receptor4 f SE (g) (cpm / 10mg)

Group

Al

PMS' + Saline'

90 + 2.8 (6)3

360 f 25.0 (6)

34677

2 1385.8

(3)

A2

PMS + 0.2 ug [D-Trp6]-LHRH

89 t 1.5 (8)

286 t 20.4 (8)

9571

f 1066.4

(8)

A3

PMS +2ug [D-Trp6]-LHRH

86k2.2

(7)

325 * 18.4 (6)

4999

+ 448.6

(5)

Bl

Saline

84 + 3.2

(5)

169 f 14.8 (5)

22792

f 1330.8

(5)

B2

0.2 ug [D-Trp']LHRH

79 f 4.0

(5)

186 f 41.9 (5)

10070

f 1317.5

(5)

B3

2 ug [D-Trp6]LHRH

80 f 2.5

(6)

193 f 10.1 (5)

6567

+ 666.2

(5)

1

Saline or [D-Trp6]-LHRH was injected SC daily day when the animal was hypophysectomized.

2 50 IU PMS was injected 3 Number 4 LH/hCG Alvs.AZ Al vs. A2 vs.

in parenthesis

sites,

groups. the

: :

hypophysectomy.

number of rats

per group.

p < 0.01 p < 0.01

but

Injection

testicular

in Table

indicates

on the

receptors: : p < 0.01 A3 : p < 0.01 A3 : p < 0.01

Blvs.BZ Bl vs. B3

binding

67 hours before

for seven days, starting

the difference

50 IU PMS 67 hours weight

2).

did

as well

However,

not

reach

before

as an increase

PMS did

not

affect

a significant

level

in the B

hypophysectomy

caused

an increase

in LH/hCG binding

sites

(Al

the LH/hCG receptor-reducing

vs. effect

[D-Trp6]-LHHH. DISCUSSION The present LHRH results the pituitary by LHRH agonist

results

clearly

in a reduction This

gland. is

not

indicate

that

of testicular means that

necessarily

the administration

LH/hCG receptors the

caused

691

decrease

of

in the

absence

in LH/hCG receptors

by over-stimulation

[D-Trp6]-of

induced

of LH release

in Bl of

BIOCHEMICAL

Vol. 90, No. 3, 1979

from

the pituitary This

gland

reduction

related.

especially

PMS, a 72% reduction

in

thereby

resulted

from

lacking.

exogenously ular

effect

by the

[D-Trp6]-LHRH processes

which

LHRH and its

lead

agonists

adrenal

possible

steroidogenesis,

the other

testicular

evidence

was taken by prior

competitive

organs

receptor,

testicular

LH/hCG

(13),

It

is

is

in the

study

ovarian

by the

that testic-

administration inhibition

was

the binding

of

that

intracellular However,

and ovarian

still

revealed

possible

initiating

of LH/hCG receptors.

of the remains

such

hypogonadism gonadal

agonist

to be specific.

to a reduction

of adrenal that

in

likely

the

significance

The presence also

with

not

LH/hCG receptors

confirmatory

Since

than

of LH/hCG receptors

suppressed

agonist.

less

with

LH release.

reduction

[D-Trp6]-LHRR

decrease

pretreated

tertiary

autoradiographic

was competitively

but

doses

reduce

LIIRH or its

Our recent

is

testicular

testis,

125 I-labeled

testis

binds

physiological

is

(13).

reduce

that

for

rats

that

directly

dose-

a dramatic

likely

of enhanced

suggests

of receptors

in the pituitary,

tracer

induced

to treat

could

on the

dose of unlabeled

observed the

a direct

The uptake

of a large

doses the effect

strongly

administered

tissue.

small

the data

was reported

is

(9,19,18).

was also

immature

The attempt

nullifying

The presence

tissues

It

to significantly

in fairly

Although

by the LHRH agonist

observed.

of the pituitary.

by LHRH agonist receptors,

being

RESEARCH COMMUNICATIONS

mechanism.

in hypophysectomized

be sufficient

the absence

regulation"

a dose as 0.2 Wg [D-Trp6]-LHRH

receptors,

0.2 ug would

a "down

of LH/hCG receptors

As small

of the

through

AND BIOPHYSICAL

receptors

the for

unknown. LHRH receptors

the agonist

directly

and thereby

has also affects

influencing

Acknowledgements. This work was supported in part AM 09094, AM 07467, and Veterans Administration.

been

reported

the adrenal, testicular

(23).

It

altering LH/hCG receptors.

by USPHS research

grants

During the preparation of this manuscript, Hsueh and Erickson reported Addendum. that LHRH and its agonist inhibited FSH-induced increase of estrogen and proThey also stated gesterone porduction by rat ovarian granulosa cells. --in vitro that the agonists inhibited FSH-induced changes in ovarian function in hypophysectomized rats --in vivo. (Hsueh, A.J.W. and Erickson, G.F., Science -204: 854-855, 1979). These findings are compatible with the results of this present study.

692

Vol. 90, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11.

12. 13. 14.

Corbin, A., Beattie, C.W., Rees, R., Yardely, J., Foell, T-H., Chai, S.Y., McGregor, H., Garsky, V., Sarantakis, D., McKinley, W.A. (1977) Fertil. Steril., 28, 471. Johnson, E.S., Gendrich, R.L., White, W.F. (1976) Fertil. Steril., 27, 853. Lin, Y.C. and Yoshinaga, K. (1976) Progr. 58th Annual Meeting of the Endocrine Sot., 143. Rippel, R.H. and Johnson, E.S. (1976) Proc. Sot. Exp. Biol. Med. 152, 432. Corbin, A., Beattie, C.W., Tracy, J., Jones, R., Foell, T.J., Yardley, J ., Rees, R.W.A. (1978) Int. J. Fertility, 23, 81. Arimura, A., Pedroza, E., Vilchez-Martinez, J.A., Schally, A.V. (1977) Endo. Res. Commun., 4, 357. Yoshinaga, K. (1978) in Follicular and Corpus Lutem Function of the J. Marsh, W.A. Sadler, eds.), Plenum Mammalian Ovary (C.P. Channing, Publishing Corp., New York, in press. Pelletier, G., Cusan, L., Auclair, C., Kelly, P.A., Desy, L., and Labrie, F., (1978) Endocrinology, 103, 641. Kledzik, G.S., Cusan, L., Auclair, C., Kelly, P.A., Labrie, F. (1978) Fertil, Steril., 29, 560. Auclair, C., Kelly, P.A., Labrie, F., Coy, D.H., Schally, A.V. (1977) Biochem. Biophys. Res. Commun., 76, 855. Catt, K.J., Baukal, A.J., Davies, T.F., Dufau, M.L. (1979) Endocrinology, 104, 17. Miyachi, Y., Vaitukaitis, J-L., Nieschlag, E., Lipsett, M.B. (1972) J. Clin. Endocr. Metab., 34, 23. Mayar, M.Q., Tarnavsky, G.K., Reeves, J.J. (1977) Proc. West Section, Am. Sot. Anim. Science, 28, 182. Bernardo, L.A., Petrali, J.P., Weiss, L.P., Sternberger, L.A. (1978) J. Histochem. Cytochem., 26, 613.

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