The presence of metabolites of 3H-testosterone in the lumen of the cauda epididymidis of the rat

413

THE PRESENCE OF METABOLITES OF 3H-TESTOSTERONE

IN THE

LUMEN OF THE CAUDA EPIDIDYMIDIS OF THE RAT

D. J. Back Department of pharmacology and Unit of Reproductive Biology, Liverpool L.69 3BX. England. University of Liverpool,

Received: I/22/75 ABSTRACT '1 Following the administration of 'H-testosterone to adult male rats radioactivity appeared in epididymal fluid and at 60 minutes was in excess of the level in tissue of the cauda epididymidis and blood plasma. Ligation of the arterial blood supply to this region caused a significant decline in the radioactive content of epididymal fluid and cauda tissue. It is concluded that direct transfer occurs from the systemic circulation into the cells of the cauda epididymidis and thence into the lumen of the duct. The major radiometabolite of 'H-testosterone identified in chloroform extracts of epididymal tissue (60.6%) and epididymal fluid (72.8%) was 17B-hydroxy-5a-androstan-3-one, INTRODUCTION Many compounds, including steroids

(1) and alkanesulphonic

esters

(2) have been shown to pass the 'blood-testis barrier' and have been identified in rete testis fluid.

There exists, however, a gap in our

knowledge of the entrance of exogenously administered compounds into the cauda epididymidis.

It is clearly of interest to know if exogenously

administered compounds can pass directly from the circulation into the lumen of the epididymal duct either in an unchanged form or as metabolites. It has previously been shown (3, 4) that the rat epididymis metabolizes 'H-testosterone are qualitatively conditions.

in vivo and in vitro.

and quantitatively

The metabolites formed

the same under both experimental

These experiments clearly show that the rat epididymis

contains the necessary enzymes for the metabolism of testosterone.

VoZwne 25, Number 3

S

T6ROfD6

March, 1975

S

414

In the of the

present

cauda

been

made

both

in the

work

epididymidis

to identify tissue

the

TIIEOIDm

of the

rat

quantitatively

of the

of 3 H-testosterone

passage

cauda

has

been

studied

the

most

important

epididymidis

MATERIALS

into

AND

and

and

the

lumen

an attempt

has

metabolites

in epididymal

fluid.

METHODS

Radioactive materials. Testosterone-1,2,6,7-H3 (87 Ci/mmol) wasobtained from the Radiochemical Centre, Amersham, England. After evaporation of the organic vehicle under nitrogen the steroid was redissolved in a mixture of 0.9% saline:methanol (9:l v/v). Tests for purity were made by thin layer chromatography (TLC) in solvent system A. Experimental procedure in rats. In initial experiments, mature male rats (300~&OO g) of the Wistar strain were anaesthetixed with urethane (14% w/v in water; 10.0 ml/kg, i.p.), tracheotomized and polyethylene catheters inserted into the carotid artery and femoral vein. The testes were exposed through a scrotal incision. In some experiments the corpus epididymidis was ligatured; in others, the vasal and inferior epididymal arteries were ligatured. For collection of epididymal fluid, a 23 gauge needle attached to a length of polyethylene tubing was inserted retrogradely into the ductus deferens, the other end of the tubing being The contents of the cauda epididymidis attached to a needle and syringe. were collected by aspiration from both ductuli deferentes aided by mild electrical stimulation (0.5 to 5.0 V) of the smooth muscle of the cauda. 3

H-testosterone (100 pCi/kg) was injected into the femoral vein Carotid arterial blood was collected at 60 over a one minute period. minutes and the radioactivity in the plasma determined. Epididymal fluid was dispensed directly into preweighed scintillation vials. NCS 1.0 ml) was added to the samples tissue solubilixer (Amersham/Searle; and the vials placed in a preheated water bath (50°C) for 5 hours, to ensure complete digestion of the fluid. To avoid problems of chemoluminescence in liquid scintillation counting, glacial acetic acid (50 ~1) was added to each sample to bring the pH to near neutrality. After collection of epididymal contents, animals were killed, and the radioactivity in caudae epididymides epididymides removed, determined after solubilization. 3 Epididymal fluid was Extraction of metabolites of H-testosterone. collected from six mature male Wistar rats (300-400 g) 60 minutes after the intravenous administration of 3H-testosterone. The caudae epididymides were also removed and frozen prior to homogenization in ice Steroids were extracted using the method cold sucrose solution (O.25M). The final chloroform-methanol phase was of Bruchovsky and Wilson (5). One aliquot taken to dryness and resuspended in chloroform (50 ul). (2 ul) was analysed for total radioactivity and the rest analysed for radiometabplites by TLC. Thin

layer

chromatography.

Thin

layer

chromatograms

(TLC aluminium

S

TRROID13

415

sheets Silica gel 60/Kieselguhr F254; Aluminium oxide F254 neutral; 20 x 20 cm; Merck; Darmstadt) were activated at 1OOoC for 60 minutes. The aluminium oxide plates were treated with an alcoholic solution of The TLC acetic acid for 5 minutes to ensure a pH of approximately 7. systems used were: A. B. C. D. E.

Methylene chloride:diethyl ether . . . Methylene chloride:diethyl ether . . . Methylene chloride:ethyl acetate ... Methylene chloride:ethyl acetate .. . Benzene:ethanol . .. . . . .. . . . .. . . .. . .

(80:20 v/v) on silica (95:5 (95:5 (98.2 (97.3

" II

gel 1, II

v/v) v/v)

' 11

v/v) v/v)

" 'I " on aluminium oxide

Radioactivity on the TLC plates Detection of radioactive metabolites. was detected by scraping horizontal bands (0.5 cm width) individually adding scintillation fluid and counting. into scintillation vials, Authentic standards were run in each solvent system and were detected by spraying with an anisaldehyde-sulphuric acid reagent (3). Acetate formation. Acetylation was performed pyridine-acetic anhydride solution (2:1, v/v) which were left at 60Oc for 2 hours.

by adding 0.15 ml of dry to the eluted metabolites

Measurement of radioactivity. Liquid scintillation fluid of the following composition (per litre): 2,5-diphenyl oxazole (6 g), Triton X-100 (333 ml), toluene (666 ml), and water (80 ml per litre) was routinely used. Radioactivity was counted at an efficiency of approximately 35% in a Unilux Nuclear Chicago liquid scintillation spectroCounting efficiency was determined by the channels ratio method meter, using a i33Ba external standard. The bulk of the chloroform extracts were Identification of steroids. subjected to thin layer chromatography in system A and four radioactive Peak I (polar compounds at the origin) was not peaks were located. Peak II (Rf=O.31) was eluted from the TLC plate and examined further. rechromatographed in system E. Two distinct peaks of radioactivity were found with the same mobility as 5a-androstane-3a,l7B-diol (3a-diol, 'Ihe radio~f=0.16) and 5a-androstane-3B,17B-diol (3S-diol, Rf=O.ll). active substance behaving like 3a-diol was eluted, acetylated and rechromatographed in system B. 'Ihe mobility of the peak was the same as Peak III from system A was eluted authentic 3a-diol acetate (Rf=O.73). and divided into two parts. One part was rechromatographed in system C and two distinct peaks were seen with a mobility like authentic 3ahydroxy-5a-androstan-3-one (androsterone, Hf=O.24) and DHT (Hf=O.32). 'lhe radioactive substance behaving like DHT was eluted and crystallized to constant specific activity. The second part was acetylated, divided into two equal parts and rechromatographed in solvent systems C and D. In both systems distinct peaks were seen corresponding to the authentic acetates of androsterone and DHT (system C - androsterone acetate, l?f=O.64, DHT acetate, Hf-0.59; system D - androsterone acetate, Rf=O.54, DHT acetate, of-0.48). Peak IV from system A was distinct and corresponded to 5cc-androstane-3,17-dione (Rf=O.74). This peak was not examined further.

S

416

TElEOIDl

RESULTS Testosterone tissue the

uptake.

of the

cauda

intravenous

epididymidis

rapidly

of the

level

in blood

Table

1.

concentration and

appeared

level

of radioactivity epididymal

in epididymal

in epididymal

60 minutes

is shown

fluid

tissue

in blood

fluid,

of 3 H-testosterone

administration

Radioactivity in excess

lhe

and

in Table

at 60 minutes

and

greatly

in excess

of radioactivity epididymal

Control ligation vessel

ligation

*Significantly Ligation

or

epididymal

midis

corpus

37.9 2 5.6

96.5 2 1.3

39.0 + 2.0

*27.6 2 5.3

fluid

after of the

tissue

and

from

control

epididymidis

(dpm/mg)

did

in tissue

*43.8 +‘ 2.1

significantly

of the

cauda

of the

labelled

arterial

supply

to the

decline

epididymal

of metabolites

of

blood in the fluid

radioactive

from

this

3 H-testosterone.

2 17.3

102.0

P(O.01

value, not

96.4 2 13.9

administration

a significant

Identification

64.1 2 5.3

of radioactivity

ligation

caused

epididymal

of the

fluid

38.1 + 5.4

different

of the

concentration

contrast,

was

epididymal

tissue

the

1.

'Ihe concentration of radioactivity (dpm/mg) in blood plasma, epididymal tissue and epididymal fluid of rats 60 minutes after the intravenous administration of 3H-testosterone. Also shown is the effect of ligation of the corpus epididymidis and ligation of the inferior epididymal artery and vasal artery on the level of radioactivity. Bach value is the mean + SE of four experiments.

Concentration

Blood

after

plasma.

plasma

Corpus

plasma,

decrease

epididymidis steroid.

cauda

epididy-

content

region

In

of the

Radioactivity

of both duct.

extracted

after

partitioned When

four

the

distinct

II and

and Table

found.

peaks

Table

2.

epididymal

intravenous

relative

2 shows

the

by the

to TLC

found

the

procedure

constituents

of the

of metabolites

of epididymal

tissue

peaks of

and

The relative amount of metabolites of 3H-testosterone in the chloroform phase of cauda epididymal tissue and epididymal fluid 60 minutes after the intravenous administration of labelled steroid.

% of total

radioactivity

chloroform epididymal

Polar

**

3cL-diol

23.5

*

gp-diol

2.2

**

Androsterone

11.0

$

DHT

60.6

*

Androstanedione

*

tentatively

epididymal

14.2 0

8.5 72.8 4.5

1.2

identified in several

by

chromatography

only.

chromatographic

systems

chromatographic

systems,

and

by

chemical

transformation. identified

in several

transformation

and

by recrystallization.

fluid

0

1.5

metabolites

identified

in the

extract

tissue

*

4

A, Peaks

(see Methods).

identification

extracts

in system

fluid.

Metabolite

**

of 'H-testosterone

identification

of

tentative

in chloroform

subjected

were

percentage

of anaesthetised

water.

were

of radioactivity resolved

fluid

administration

and

extracts

further

the

and

chloroform

chloroform

H-testosterone

epididymal

tissue

the

between

III were

described

3

epididymal

60 minutes

rats was

from

by

chemical

S

418

The major metabolite

TDEOXDI

in both tissue of the cauda epididymidis

and epididymal fluid (72.8% was DHT. recrystallization

(60.6%)

This was further identified by

to constant specific activity

(Table 3).

There was

no evidence under the experimental conditions described of unmetabolized testosterone. Table 3.

Determination by crystallization of the identity of 3Hdihydrotestosterone isolated by TLC from extracts of cauda epididymal tissue and epididymal fluid.

Crystallization

Specific activity epididymal tissue

(dpm/mg)

epididymal fluid

1.

Methanol

712

122

2.

Ethyl acetate-n-hexane

664

143

3.

Benzene-n-hexane

660

136

4.

Cyclohexane-ethyl

689

142

acetate

The separation ability of system A was as follows:

The radio-

activity in peak II of epididymal tissue extracts consisted of 91% 3a-diol and 9% 3P-diol, whilst peak III consisted of 85% DHT and 15% androsterone.

In epididymal fluid extracts peak II consisted of 100%

3a-diol and peak III 90% DHT and 10% androsterone. DISCUSSION It has been shown in hamsters

(6,7) and guinea pigs (8) that

circulating testosterone is of major importance for the maintenance of fertilizing capacity.

In addition, Jones and Glover (9) have

demonstrated in the rabbit that in the presence of androgen, the epididymal cells actively maintain a constant milieu in the lumen of the duct by their capacity for absorption and secretion.

There are,

S clear

however,

indications

5cc-reductase

activity

affinity

DHT

for

metabolite of

has

shown

exogenously

the

epididymal into

that

DHT

and

the

lumen

initiation

cells

and

lumen

in rat been

main

identified The

in epididymal

fluid

would

indicate,

DHT

epididymidis.

to

main

maintenance

preferentially

ratio

tissue

of DHT

than

since

fluid passes

of in vivo.

in epididymal

(5.1:1)

in epididymal

to 3a-diol

in epididymal

the

total

exceeded from

fluid

that

in

epididymal

cells

duct.

therefore,

seem

of

intranuclear

certain

in addition,

the

metabolites

epididymal

higher

of the

of the

the

cauda

that

as the

(3), the present

et al

of the

tissue,

for

high

of spermatozoa.

testosterone

tend

with

DHT,

importance

are

have

protein

13) that

12,

of Djoseland

3a-diol

metabolites

of nucleus-associated

binding

(11,

maturation

of radioactivity

It would,

the

and

(2.6:1) and this

concentration

androgen

is of primary

results

lumen

419

presence

epididymis

the

these

the

and

with

considerably

tissue

the

in rat

administered

In addition,

was

(lo),

capacity

Consistent

within

from

of testosterone,

fertilizing

study

TElEOIDb

its

that

possible

epididymal

duct

DHT

is undoubtedly events

role

needs

within

important the

as an active

for

epididymal

androgen

within

investigation.

ACKNOWLEDGEMENTS The work was supported in part by a research grant from the Ford Foundation. I am indebted to Dr. T. D. Glover, Director of the Unit of Reproductive Biology, Liverpool University, for initiating this study. I also wish to thank Mrs. J. A. Broughton for technical assistance and Miss V. Chapman for typing the manuscript. REFERENCES 1.

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T. G. and

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G. M.

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508,

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S. J.,

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O.,

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