Laboratory of Veterinary Physiology, Faculty of ...€¦ · adult pattern of testosterone-dominance, an androstenedione-dominant pattern was found in the immature rat. The change

Endocrinol. Japon. 1966, 13(2), 326～337

ANDROGENS IN TESTICULAR VENOUS BLOOD IN THE

RAT, WITH SPECIAL REFERENCE TO PUBERAL

CHANGE IN THE SECRETORY PATTERN

INORU HASHIMOTO AND YOSHISUKE SUZUKI

Laboratory of Veterinary Physiology, Faculty of Agriculture,University of Tokyo, Tokyo

SYNOPSIS

A puberal change in the secretory pattern of androgen was investigated in therat. Using immature and adult rats which received an acute human chorionic

gonadotrophin(HCG) stimulation, androstenedione and testosterone were measuredboth in the testicular venous blood(TVB) and in the testis by means of gaschromato-

graphy after preliminary separation by paperchromatography. In contrast to theadult pattern of testosterone-dominance, an androstenedione-dominant pattern wasfound in the immature rat. The change from immature to the adult pattern wasassumed to take place at around day 40, being coincident with the onset of pubertyin this species. The adult pattern of androgen secretion, however, could not beinduced precociously even in the immature rat pretreated with HCG for 2 weeks,up to 34 days of age.

The ratio of testosterone to androstenedione in TVB or in the testis of theintact adult animal was significantly increased by an acute stimulation of HCG.This finding is discussed in connection with the locus(ci) in the pathway of testo-sterone-biogenesis which HCG accelerates.

Testosterone has been characterized as the androgen predominantly secretedfrom the testis in many species of mammals; the dog(West et al., 1952; Brink-

Johnsen and Eik-Nes, 1957; Mason and Samuels, 1961; Ibayashi et al., 1965), thebull(Linder, 1959 and 1961a; Savard et al., 1961) the man(Hollander and Hollander,1958) and the ram, boar and stallion(Linder, 1961b). Suzuki and Eto(1962)demonstrated that in the adult rat also, testosterone is the major androgenelaborated and secreted from the testis, by analysis of the pure effluent blood ofthe testis.

Linder(1959 and 1961a) and Linder and Mann(1960) have found that in pre-

puberal bulls the testis secretes more androstenedione(androst-4-ene-3,17-dione)than testosterone, and this androstenedione-dominant pattern changes to the adulttestosterone-dominant pattern during the course of sexual maturation. They alsodemonstrated that this puberal change could not be induced precociously bymeans of treatment with several gonadotrophins. In other species(sheep andboar), however, an androstenedione-dominant pattern has not been found in the

prepuberal period(Linder, 1961b).Recently, through a slight modification in the original collection method

Received for publication July 4, 1966.

Vol.13, No.3 PUBERAL CHANGE OF ANDROGEN IN RAT 327

described by Suzuki and Eto(1962), the collection of pure effluent blood fromthe immature rat testis was made possible. In order to inquire if the puberalchange in the secretory pattern of androgen takes place in the rat, comparisonwas made between the adult and the immature rat by measuring concentrationsof androstenedione and testosterone both in the testicular venous blood(TVB)and in the testis. The effect of exogenous gonadotrophin stimulation on the ratioof the two steroids in the adult was also investigated.

MATERIALS AND METHODS

Immature and adult rats of Wistar strain bred in our laboratory were used. They were

divided into the following groups;

(1) IMM-S Group: Two hundred immature male rats were divided into 2 batches. The first

batch(IMM-S-I) consisted of 98 rats, average 35.7 days of age(35•`38 days), and the second

(IMM-S-II), 102 rats, average 43.5 days of age (39•`46 days). Each animal received a single

dose of 100 I. U. human chorionic gonadotrophin(HCG,"PUBEROGEN"Tomoda Pharm.

Co.), dissolved in 0.2ml of saline solution intravenously at 3 hrs.before the start of TVB

collection.

(2) IMM-L Group: Fifty, 21 day old male rats received daily injections of 5 I.U. HCG dissolved

in 0.2ml saline solution subcutaneously for 2 weeks; TVB collection was performed 1 day after

the last injection(on day 35). Two batches, each consisting of 25 animals, were used(IMM-L-I

and-II).

(3) AD-S Group: Seven adult male rats, about a year old, were pretreated with a single dose

of HCG in the same manner as in the IMM-S Group. They were distributed into four batches,

each consisting of 1 or 2 animals(AD-S-I•`-IV).

(4) AD-INT Group: Fifteen adult male rats, about a year old, were divided into 4 batches,

each consisting of 3 or 4 animals(AD-INT-I-I•`V). They received no pretreatment.

Collection of testicular venous blood

The collection method of TVB was similar to that of the previous paper(Suzuki and Eto,

1962), however instead of polyethylene tubing used in the original method, glass capillaries were

used for cannulae. They were approximately 10cm long with an inner-diameter of 0.4mm for

immature rats, and about 0.7mm for the adult. Cannulae were ground aslant at the tip and

carefully cleaned with water and suitable organic solvents before use.

Under pentobarbital sodium anesthesis(Nembutal sodium, 40mg per kg body weight), one

testis, usually the right, was withdrawn through the abdominal incision and placed on the

inguinal region of the abdomen. After an intravenous injection of 100 U.(0.1 ml) heparin

solution(Heparin Sodium, Takeda Pharm. Co.), a small puncture was made with the tip of a

hypodermic needle on the wall of a large branch of testicular veins through the albuginal

membrane, and a glass cannula inserted into the vein and fixed to the albuginal membrane with

adhesive(Eastman 910, Eastman Co.). After completion of cannulation, the free end of the

cannula was sheathed with a slightly larger glass tube filled with heparin solution, as shown

Figure 1, and fixed with adhesive. In this manner, coagulation of effluent blood at the free

tip was effectively prevented.

The effluent blood was allowed to flow freely into a graduated tube placed in an ice cold

bath. The collection was continued in a thermo-regulating box at 35•Ž for 1.5 to 2.0 hrs. in

the immature rat and for 1.0 to 1.5 hrs. in the adult rat. As seen in Figure 2, about 3ml of

TVB from an immature rat and about 10ml from an adult were usually obtainable without

any sign of abnormal congestion in the testis.

328 HASHIMOTO AND SUZUK1

Endocrinol. Japon.Sept. 1966

Fig.1. Showing Cannulation into a Branch of Testicular Veins, and

a Devise for Prevention of Blood Coagulation at the Tip of Cannula

・Adult

・lmmature

Fig.2. Relation between Volume of Testicular Venous Blood(TVB)

and the Inner-diameter of Cannula

Immediately after the blood collection both testes were removed for chemical analysis. Inimmature groups(IMM-S and IMM-L),one fifth of the animals in each batch were selected atrandom and their accessory reproductive organ weighed. In all animals the penis type describedby Suzuki(1954b) was observed as an indicator of sexual development.

Chemical ProcessesExtraction: In each animal, TVB was diluted five times with distilled water and the

decapsulated testis was homogenized in a glass homogenizer with distilled water to produce a20% homogenate. The diluted blood and the testicular homogenate were immediately extracted


five times with an equal volume of ethylacetate,and these extracts were stored in a deep-freezer

until further processing. The extracts of both TVB and testis from rats of the above mentioned

batches were respectively pooled and analyzed in batches. Further procedure for extraction was

identical to that of the previous paper. The final residues obtained were again partitioned

between n-heptane and 70% methanol and purified using the final steps of the extraction method

and submitted to paperchromatography.

Paperchromatography: The paper and systems of paperchromatography were the same as

those described in the previous paper. To estimate the location of steroids on the test lanes one

or two control lanes were always provided, on which 20ƒÊg each of authentic testosterone,

androstenedione, progesterone and testosterone acetate were applied. Separation of testosterone

from androstenedione was accomplished by the first chromatography using n-heptane: benzene

(2: 1 v/v)-formamide system. The areas of the test lane corresponding to androstenedione and

to testosterone were eluted with methanol, and were rechromatographed on n-heptane-formamide

for the former, and on n-heptane: benzene(2: 1 v/v)-formamide for the latter(2nd chromato.).

Eluate from the testosterone area was brought to dryness and acetylated with acetic anhydride

in pyridine, remaining in dark at room temperature over night, and again chromatographed on

n-heptane-formamide(3rd chromato.). Eluate from the androstenedione area was purified by an

additional paperchromatography on n-heptane-formamide system(3rd chromato.).

Quantitative estimation of testosterone and androstenedione: An aliquot of each sample,

obtained by elution of the area corresponding to androstendione or to testosterone acetate in the

last paperchromatograms, was quantitated by gas-liquid chromatography using SHIMAZU

Gaschromatography(Model GC 1-B, Shimazu Co.), equipped with a hydrogen ionizing detector

(Type HFD-1, Shimazu Co.) on Column SE-30(Shimazu Co., 2.26m long, 4mm in inner-

diameter). The peak areas on the gaschromatograms corresponding to the respective steroids

were measured, and amounts of each steroid was estimated by interpolating to the respective

standard area-dose curves. The standard curves were prepared by gaschromatography of graded

doses of authentic steroids under the same conditions.

RESULTS AND DISCUSSION

Results obtained in gaschromatographic measurement of androstenedione and

testosterone in TVB and in the testis are given in Tables 1 and 2. Relative

retention times to cholesterol(RRT) of several authentic steroids, as well as the

two questioned steroids, gaschromatographed under the same conditions are also

given in Table 3. RRT obtained in the biological samples were 0.43+0.013

(S.D.) for testosterone acetate and 0.31•}0.004 for androstenedione, and compared

well with those of the authentic steroids. For further support, in most batches a

small amount of each authentic steroid was added to an aliquot of the respective

sample and gaschromatograghy was made. The identity was proved in every

case by an increase in height of the questioned peak without disturbing its shape,

as seen in Figure 3.

Levels of androstenedione and testosterone expressed in concentration(ƒÊg/100ml

TVB or ƒÊg/100g testis), are compared and given in Figures 4 and 5. The sum

of androstenedione and testosterone was tentatively used as a measure of total

androgen level.

330 HASHIMOTO AND SUZUKI


Table 1. Lev

els of T

est

oster

one and Androstene

di

onｅ in Testicular Veno

us Blood and Te

st

isin Im

ma

ture.

Rat

s with pretreatm

ent w

ith Gonadotro

phin

, HCG

*1 IMM-S-I-and-I re

ceived 1

00 I. U. HCG intra

venously 3 hr

s.b

ef

ore the start of T

VB col

lection. Batch I co

nsisted of rats,aver

age 35.7 da

ys

o

ld, and bat

ch II, average 43.5 day s

old

. IMM-

Ia

nd-I receioed dai

ly injection

s of 51. U. HC

G sub

ecutaneously for tw

o weeks

u

p to a

days befo

re TVB c

ol

lection. B

oth batc

hes c

ons

isted of r

ats 35 days old.

*2 Relative re

te

ntio

n time to ch

olestero

l.

*

3 C

once

nt

ra

tion in μg/100ml or μg/100

g.

*4 Ratio o

f testosterone(c

on

c.) to an

drostenedion

e(

conc.

).

*5 TVB: Testicular v

en

ous bl

oo, T: T

esticular t

issu

e.


TAbl

e 2. Le

vels of Te

sto

stero

ne and A

ndrostened

ione in Testicular V

eno

us Blood and Te

st

is in Ad

ul

t Rats

wit

h and without Pre

drea

tment with gon

adotrop

hin, HCG

*1 AD-S-1 to

-IV: Adult rats a

bo

ut one year old re

ceiv

ed 100 I. U. HCG intr

avenously 3 hrs. befo

re th

e st

art of TVB collec

tion

. AD-INT-I to

-IV: Adult rats ab

out one ye

ar o

ld re

cei

ved no pret

reatm

ent.

*2 Relative r

et

enti

on time to c

holstero

l.

*3 Conce

nt

ra

tion in μg/100ml or μg 100

g.

*4 Ratio of testosterone(c

onc.) to androstenddio

ne (conc

.).

*5 TVB: T

es

ticula

r venou

s blood, T: T

esti

s.

332 HASHIMOTO AND SUZUKIEndocrinol. Japon.Sept. 1966

Table 3. Relative Retention Times of Several Steroids to Cholesterolon Column SE-30(RRT)

*Average of more than 4 determinations in each steroid .

Fig. 3. Gaschromatograms of Testosterone as its Acetate(top left) andAndrostenedione(top right), separated from TVB of Immature Rats.

In the bottom two graphs, a small amount of each authenticsteroid was added to the respective sample given above for identifi-cation. Cholesterol was added in every case for determination ofrelative retention times of the steroids(RRT).


Androgen in adult rats and effect of acute gonadotrophin stimulation

The secretion level of total androgen in the intact adult was rather low

(3ƒÊg/100ml TVB), however this was immediately increased about seven times by

exogenous HCG(Fig. 4). In the case of the testis, the intact level(16ƒÊg/100g

testis) was increased 3.5 times by acute HCG stimulation(Fig. 5). Both in TVB

and the testis, testosterone always predominated over androstenedione in the adult

period. These data were in good accord with those of the previous paper, except

that a little higher level of total androgen in the testis was obtained in the pre-

sent study.

In the previous paper, the ratio of testosterone to androstenedione(T/A ratio)

in the intact adult rat was not determined due to the shortage in androstenedione

level for measurement. By employing gaschromatography, levels of androstenedione

could be measured in every case in the present study, and T/A ratios in the intact

adult(AD-INT) were determined as 1.5•}0.31(S. D.) in TVB and 2.3•}0.12 in the

testis. It is noteworthy that these ratios were significantly increased by acute

stimulation of HCG and showed 4.0•}0.67 in TVB and 3.2•}0.76 in the testis in

AD-S Group(AD-INT versus AD-S: TVB, P<0.005; testis, P<0.05 in t test).

Suzuki et at.(1962) and Suzuki (1964) observed a similar change in ratio of the

two steroids converted from progesterone-4-14C in the adult rat testis in vivo with

an acute HCG treatment.

Pregnenolone(3 j3-hydroxypregn-5-en-20-one) and androstenedione have been

thought to be obligatory intermediates in the pathway of testosterone-biogenesis

of this species until quite recent years. In taking this point of view, the above

mentioned findings may strongly suggest that the accelerating action of HCG on

testosterone-biogenesis is also precipitated in the step between androstenedione and

testosterone. Recently, however, Slaunwhite and Burgetts(1965) using the rat

testis in vitro, have suggested a new alternative pathway whereby testosterone is

Levels of androgens in testicular venous blood

Fig.4. Levels of Androgens in Testicular Venous Blood(TVB) in the Rat.Explanation for group and batch is given in the note of Tables 1 and 2 .



Levels of androgens in lestieular lissue

Fig.5. Levels of Androgens in Testicular Tissue in the Rat.

Explanation for group and batch is given in the note of Tables 1 and 2.

produced directly from 17ƒ¿-hydroxypregnenolone(3ƒÀ, 17ƒ¿-dihydroxypregn-5-en-20-

one), probably via androstenediol (androst-5-ene-3ƒÀ, 17ƒÀ-diol), but not through

dehydroepiandrosterone(3ƒÀ-hydroxyandrost-5-en-17-one) and androstenedione.

Taking this into consideration, the increase in T/A ratio by HCG stimulation

could also be explained as a result of selective simulation on an alternative route

other than via-androstenedione route. Even so, it may still be considered that

the stimulation of HCG may take part also in the steps later than pregnenolone,

as far as this steroid is assumed to be an obligatory intermediate. However,

Brink-Johnsen and Eik-Nes(1957) and Mason and Samuels(1961) could not find

a definite change in T/A ratio in spermatic vein blood in the dog with an acute

HCG stimulation, and Hall(1966) could not demonstrate the stimulatory effect of

interstitial cell stimulating hormone(ICSH) upon conversion of pregnenolone-7ƒ¿-3H

to testosterone-3H in the rabbit testis in vitro. These workers, therefore, have

considered that the trophic action of HCG or ICSH should be precipitated only

in the earlier steps than pregnenolone.

Further studies would be needed to clarlify the locus(ci) which the gonado-

trophins exerted their stimulating effect in the pathway of testosterone-biogenesis.


Table 4. Weights of Reproductive Organs and the Penis Type in Immature Ra

t Groups

* E

xplan

at

ion f

or group and batc

h is given in N. B. of Table 1

.

** Weighed in batch and expressed in weight per anima

l.



Puberal Change in Secretory Pattern of Androgen in the RatPredicting a shortage in androgen for measurement in the immature intact

rat, androgen determination was performed only in the immature rats pretreatedwith a single injection of HCG(IMM-S), or with multiple long term injectionsof HCG(IMM-L).

The total androgen level in immature rats pretreated with a single HCGinjection(IMM-S), was very low both in TVB and in the testis, when comparedwith the adult group under similar conditions(AD-S). But it was not so muchless than those of the intact adult(AD-INT), especially in TVB(Figs. 4 and 5).

In contrast to the adult rat, the testis of immature rats of this group secretedandrostenedione in high proportion, and progressive increase in T/A ratio wasfound with advance of age, i.e. T/A ratios were 0.29 on day 36(IMM-S-I) and1.09 on day 44(IMM-S-II) in TVB. In the testis, only androstenedione wasdetected on day 36, and a T/A ratio of extraordinary high proportion in testo-sterone(7.66) was observed on day 44.

Thus, the change of secretory pattern from androstenedione-dominance to oneof testosterone-dominance was proved to exist in the rat as well as in the bull(Linder, 1959 and 1961a). The T/A ratio of unusually high value in the testisand relatively low value in TVB which was found on day 44, may indicate thatthis age is coincident with the transient phase of this change. Judging from theage, the weights of reproductive organs and their penis type(IMM-S in Table 4),these animals are assumed to be just in the advent of the sexual maturation

process, having just completed"Epoch I"(onset of puberty), but not yet"EpochII"(sexual maturity). (cf. Suzuki, 1952, 1954a and b). Therefore, this change insecretory pattern of androgen is assumed to be one of the puberal changes inthe rat.

In the immature rats pretreated with HCG daily for two weeks ending oneday fefore collection of TVB on day 35(IMM-L), manifestations of androgensecretion, such as increase in weight of accessory sexual glands and precociousappearance of"U"type penis, were quite evident as seen in Table 4. Thetotal androgen levels, however, were unexpectedly low, especially in TVB.Moreover, it is noteworthy that the adult pattern of testosterone-dominant androgensecretion could not be induced precociously by this treatment. This finding isalso in good accord with that obtained in the bull(Linder, 1959 and 1961a), andstrongly suggests that the puberal change in the secretory pattern of androgen cannot be accomplished by gonadotrophin alone.

REFERENCES

Brink-Johnsen, T. and K. Eik-Nes (1957). Endocrinology 61, 676.Hall, P. F.(1966). Ibid. 78, 690.Hollander, N. and V. P. Hollander (1958). J. Clin. Endocrinol. and Metablo. 18, 966.Ibayashi, H., M. Nakamura, T. Uchikawa, S. Murakawa, S. Yoshida, K. Nakao and S.

Okinaka (1965). Endocrinology 76, 347.Linder, H. R.(1959). Nature 183, 1605.

Vol.13, No.3 PUBERALCHANGEOF AND ROGEN IN RAT 337

Linder, H. R.(1961 a). J. Endocrinol. 23, 139.Linder, H. R.1961 b). Ibid. 23, 171.Linder, H. R. and T. Mann (1960). Ibid. 21, 341.Mason, N. R. and L. T. Samuels (1961). Endocrinology 68, 899.Savard, K. N. R. Mason, J. I. Ingram and F. X. Gassner (1961). Ibid. 69, 324.Slaunwhite, Jr. W. R. and M. J. Burgett (1965). Steroids 6, 721.Suzuki, Y.(1952). Japan. J. Vet. Sci. 14, 115.(in Japanese with Engl.)Suzuki, Y.(1954). Ibid. 16, 1.(in Japanese with Engl.)Suzuki, Y.(1954). Ibid. 16, 87.(in Japanese with Engl.)Suzuki, Y.(1964). Folia Endocrinol. Japan. 40, 1424.(in Japanese)Suzuki, Y. and T. Eto (1962). Endocrinol. Japon. 9, 277.Suzuki, Y. and T. Eto and H. Masuda (1962). Japan. J. Animal Reprod. 8, 41.(in Japanese

with Engl.)West, C. D., V. P. Hollander, K. T. K. Krichevsky and K. Dobriner (1952). J. Clin. Endocrinol.

and Metabol. 12, 915.

Laboratory of Veterinary Physiology, Faculty of ...€¦ · adult pattern of testosterone-dominance, an androstenedione-dominant pattern was found in the immature rat. The change

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