β-Endorphin in male rat reproductive organs

BIOCHEMICAL

Vol. 95, No. 2, 1980

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 31, 1980

Pages 618-623

B-ENDORPHIN IN MALE RAT REPRODUCTIVE ORGANS Burt

Sharp,

Endocrine

A. Eugene

Research

Received

Pekary,

Nancy V. Meyer and Jerome

M. Hershman

Laboratory, Veterans Administration Wadsworth Los Angeles, California 90073

Medical

Center,

May 30,198O

SUMMARY 5-Endorphin and related peptides have been detected within the male rat reproductive system. The f3-endorphin immunoreactivity of testis exhibits parallelism with synthetic human B-endorphin on serial dilution and coelutes with ,6-endorphin on Sephadex G-50 and reverse phase high pressure liquid chroma tography. Sephadex G-50 chromatography also indicates the presence of B-lipotropin. @Endorphin concentrations in rat seminal vesicles and prostate were approximately 25% of that in testis. 6-Endorphin pituitary

opioid

and gastrointestinal

histochemistry opioid

and other

(l-9).

receptors

peptides 6-endorphin testis, MATERIALS

tract

This

widespread

rat

distribution

and seminal

(10)

prompted We report

immunoreactive

peptides

vesicles

from

in the

bioassay

sexually

mature

us to look here

of for

evidence

in extracts

brain,

and immuno-

and the presence

system.

reproductive

and 6-endorphin-like

have been identified

by radioimmunoassay,

in the mouse vas deferens

in the male

prostate

peptides

opioid for

both

of

rats.

AND METHODS

200 g male Sprague-Dawley rats given access ad libitum to water and standard rat chow were decapitated and the following reproductive organs were immediately removed: a single testis (decorticate), single caudate seminal vesicles and both vgs deferens. Each was epididymis, prostate, weighed and placed into 1.0 ml of 0.2 M HCl at 97 C for 15 minutes to Samples were homogenized with a Polytron destroy enzyme activity (3,ll). for 5-10 seconds. The residue on the homogenizer probe was recovered by rinsing it into the corresponding homogenate with 1.0 ml of HCl. Homogenates were centrifuged at 1,008 x g for 1 hour, and th&n the supernatants thawing at 26 C, the pH of each were aspirated and frozen at -20 C. After sample was adjusted to 7.5 with 1.0 N NaOH and then the sample was frozen at -2OOC. After thawing the sample, a final centrifugation at 1,000x g yielded a clear, colorless supernatant for chromatography and RIA. A pooled testis extract, prepared from equal volumes of the supernatant from 8 rats, was chromatographed on a 0.9x60 cm column of Sephadex G-50 precoated with 2% crystalline bovine serum albumin and eluted with Fractions of 0.62 ml were collected 0.2 M acetic acid, 0.01% Triton-X-100. at 0.15 ml/minute. Prior to radioimmunoassay (RIA), each fraction was raised to pH 7.5 with 1.0 N NaOH. 0006-291X/80/140618-06$01.00/0 Copyright :B 1980 by Academic Press, Inc. All rights of reproducfion in any form reserved.

618

Vol.

95, No.

2, 1980

8lOCHEMlCAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Reversed phase high pressure liquid chromatography was performed with an Altex system interfaced to a computerized gradient making accessory. A flow rate of 1.0 ml/min was maintained through an Altex ODS Ultrasphere of less than column, 4.6 mm x 25 cm, 5 urn particle size , with a backpressure 2,000 psi. Linear acetonitrile (Fisher Scientific Co.) gradients with stepwise increasing slopes were used (modified from Ref. 12). The sample and starting buffer consisted of 0.1 M phosphate, pH 2.1. Two and one-half min after sample injection a linear acetonitrile gradient was begun which rose to 40% at 37.5 min after injection. From this point the acetonitrile increased linearly to 60% at 45 min and to 100% at 55 min after sample injection. Retention times for methionine enkephalin, leucine enkephalin and human B-endorphin were measured by injecting 100 ug/ml of each of the purified peptides and detecting the ultraviolet absorbance at 225 nm. Fractions from the G-50 chromatography of the testis extract, which demonstrated B-endorphin immunoreactivity eluting in the region of synthetic human B-endorphin, were combined, lyophilized and reconstituted in starting buffer. A 250 ~1 sample was injected into the system, and fractions were collected at 1 min intervals. Fractions of interest were raised to pH 7.5, lyophilized and reconstituted in 0.45 ml of RIA buffer. B-Endorphin was quantified by an RIA (13) which showed equimolar cross-reactivity with purified B-lipotropin (NIAMDD) and no displacement by a and v-endorphin (Peninsula Lab), methionine and leucine enkephalin (Calbiochem.), or ACTH (l-24). The minimum detectable dose was 2.2 pg/tube (22 pg/ml of unknown) and the intra-assay coefficient of variation using human B-endorphin (obtained from NIAMDD) was 4%. 0.05 M phosphate, pH 7.7, containing 0.75% crystalline bovine serum albumin and 0.01% merthiolate was used as the RIA buffer. [1251]-B-endorphin with a specific activity of 125-175 pCi/ug was prepared usin sodium iodide (Amersham) according to the chloramine-T method (14). [ 9 Z51]-B-endorphin and [12511-iodide were separated using a 10 ml G-50 column eluted with 0.2 M acetic acid and 0.1% Triton-X-100. Unknown samples of 100 ~1 were added to 200 ~1 of rabbit anti-B-endorphin antibody (Bioflex gorp) diluted l/8000 in RIA buffer. After 40 hours of incubation at 4 C, [12511-B-endorphin was added, and separation was begun 14 hours later by adding normal rabbit serum and goat anti-rabbit u-globulin. The doses of the standard curve and unknown samples were prepared in triplicate.

RESULTS AND DISCUSSION Serial

dilution

B-endorphin

of pooled

immunoreactivity

testis which

(Fig

1) (statistical

analysis,

this

pooled

demonstrated

ing

to the

specimen regions

[1251]-fi-endorphin

where (Fig

B-lipotropin

was 6.1.

volume

could

which

extract was parallel

Ref. 15 ).

In addition

represent

The molar

the 31,000

and B-endorphin.

619

gel

showed

RIA standard chromatography

irmnunoreactivity

(detected

a small

8 rats

to the

Sephadex

B-endorphin

f3-lipotropin 2) elute.

from

and

of B-endorphin/

peak was detected

dalton

of

correspond-

by radioimmunoassay) ratio

curve

precursor

at the

to B-lipotropin

void

Vol.

95,

No.

BIOCHEMICAL

2, 1980

25

AND

50

130

250

p - ENDOf?PHIN

Fig.

High ing

1.

Serial dilution radioimmunoassay extract.

pressure

to the

liquid

B-endorphin

The broadening human B-endorphin

of

synthetic compared

with

elutes

500

ooo

profile

human serial

COMMUNICATIONS

2oQo

( pg/ml)

6-endorphin dilution

detected of rat

by testis

of the G-50 fractions

confirmed

elution

RESEARCH

IMMUNOREACTIVITY

chromatography region

of the

RIOPHYSICAL

the

presence

about

may represent

correspond-

of a-endorphin

the fraction

where

(Fig

synthetic

the micro-heterogeneity

of

B-endorphin

1'25-/3- EP 1

8-EP

IWWNOREACTIVITY ,60 (pg/

fraction) 120. 00-

40_ 20-IO,

-

+I),

o

Fig. 2.

I

8

Sephadex G-50 radioimmunoassay. centration of

g

I

IO

I

12

14

I8

20

ELUTION VOLUME

(ml)

chromatography of --indicates human B-endorphin.

620

16

rat the

22

3).

24

testis extract detected minimum detectable

26

by con-

BIOCHEMICAL

Vol. 95, No. 2, 1980

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FRACTIONS

Fig.

due

to

3.

High pressure obtained from chromatography. centration of

variation

traction

in

effects,

peak

with

the or

constant

native

both.

width

at

synthetic

6-endorphin

sidered

together,

the

evidence

peptides

such

reported

levels

between

200-400

(Table

1)

for as

the

in

the

Rat N

of

found

only

for

0.5

the

Data

for

rat

which

1,250

B-endorphin

1.25

to.15

Prostate

4

0.28

t .04

4

0.24

i .09

Epididymis

4

0.0

Vas

4

0.0

deferens

In

might

immunoreactivity

621

0.2

and other

in of

and

since rat

in

rat

the

serum

are

testis

due to blood

immunoreactivity

of

Con-

imnunoreactive

basal

occur

after

provide

addition,

pg/g

the

(pg/mg)

ex-

elution

M HCl.

profiles

testis.

7

Ves.

in

(16),

narrow

before

15 minutes

Testis

Seminal

a single,

minute

Table 1 Illiillunoreactivity in the Orqans Reproductive System (mean +SD) C-endorphin

endorphin

chromatographic

amount

that

testis extract Sephadex G-50 detectable con-

tissue

immunoreactivity

exceeds extract.

forms

B-endorphin

of

(17,18),

e-Endorphin Tissue

97'C

presence

of rat region of minimum

of

and

8-endorphin

pg/ml

the

height to

B-lipotropin for

of

half

we

parallelism

significantly

tamination

chemical However,

heating

strong

liquid chromatography [Ilz5]-B-endorphin - indicates human B-endorphin.

conin

the

Vol.

95, No.

2, 1980

testis

BIOCHEMICAL

was not corrected

85% following

the addition

of 4 testes

prior

and other

pooled

and concentrated dilutes

vesicles

(data

was not

parallel

for

those

in the

reactivity

at least

to the

was examined;

regulatory

1).

that

to be to each

of the

were

and other

in the

the

of organ

immunoreactivity

prostate the

the

increased

The tissue

approximately

may reflect

seminal

extract

curve.

to detect

were which

in the

may reflect

As an extra-reproductive

25% of

B-endorphin

imm,Jno-

very

amounts

small

control,

mesenteric

was not detected. is

present

by testicular

B-endorphin

each type

of the standard

vesicles

between

was found

this

The inability

immunoreactivity

functions

region

B-endorphin

binding

curve

standard,

and epididymis

extraction.

from

the response

and seminal

(Table

fat

suggests

Although

in vas deferens

with

was found

in weight

B-Endorphin

standard

B-endorphin

prostate

used for

consistent

COMMUNICATIONS

human fi-endorphin

differences

5-fold.

at the low dose

testis

B-Endorphin

which

2 extracts

to the

shown).

of tissue (N=4)

organs,

in parallel not

both

of the large

reproductive

variance

levels

RESEARCH

efficiency

of 1.0 ng synthetic

the effect

testis

serially

extraction

BIOPHYSICAL

to homogenization.

To diminish

response

for

AND

in testis

receptors opioid

reproductive

at levels

which

or de novo synthesis.

peptides

may have

are This

important

system.

ACKNOWLEDGEMENTS: Support for this research by Veterans Medical Research Funds and NIH Grant HD-7181 is gratefully We thank Florence Rosen for typing the manuscript.

Administration acknowledged.

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8.

Rossier, J., Vargo, T., Minnick, S., Ling, N.,Bloom, F.E., and Guillemin, R. (1977) Proc. Natl. Acad. Sci. USA 74, 5162-5165. Duka, T., Hollt, V., Przewlocki, R., and Wesche, D. (1978) Biochem, Biophys. Res. Commun. 85, 1119-1127. Liotta, A.S., Suda, T. and Krieger, D.T. (1978) Proc. Natl. Acad. Sci. USA 75, 2950-2954. Larsson, L-I, Childers, S., and Snyder, S.H. (1979) Nature 282, 407-410. Nilaver, G., Zimmerman, E.A., Defendini, R., Liotta, A.S., Krieger, D.T., and Brownstein, M.J. (1979) J. Cell Biology 81, 50-58. H.G. (1979) Life Sci. 24, 111-118. Salih, H., Panerai, A.E., and Friesen, E., Rossier, J., Ling, N., Leppaluoto, J., Bloom, F., Battenberg, Vargo, T.M., and Guillemin, R. (1977) Life Sci. 20, 43-48. Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., Morgan, B.A. and Morris, H.R. (1975) Nature 258, 577-579.

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BIOPHYSICAL

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Bradbury, A.F., Smyth, D.G., Snell, C.R., Birdsall, N.J.M., and Hulme, E.C (1976) Nature 260, 793-795. Hughes, J., Kosterlitz, H.W., and Leslie, F.M. (1975a) Br. J. Pharmacol. 53, 371-381. Rossier, J., Bayon, A., Vargo, T.M., Ling, N., Guillemin, R., and Bloom, F. (1977) Life Sci. 21, 847-852. Nice, E.C., and O'Hare, M.J. (1979) J. Chromatogr. 162, 401-407. Sharp, B., Melmed, S., Goldberg, R., Carlson, H.E., Refetoff, S., and Hershman, J.M. (1980), submitted. Hunter, W.M., and Greenwood, F.C. (1962) Nature 194, 495-496. Pekary, A.E. (1979) Comput. Biol. Med. 9, 355-362. Smyth, D.G., Massey, D.E., Zakarian, S., and Finnie, M.D.A. (1979) Nature 279, 252-254. Akil, H., Watson, S.J., Barchas, J.D., and Li, C.H. (1979) Life Sci. 24, 1659-1666. Hollt, V., Przewlocki, R., and Her-z, A. (1978) Naunyn-Schmiedeberg's Arch. Pharmacol. 303, 171-174.

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