Cl. JEAN. Physiologie. Compar#{233}e et Endocrin ologie,. Universit#{233} de ... and sexual behavior. (Pfeiffer,. 1936;. Harris,. 1964). Testicu- lar androgens are.
BIOLOGY
OF
REPRODUCTION
15,
Developmental
M.
561-564
(1976)
Patterns of Plasma and Testicular Testosterone Rabbits from Birth to 90 Days of Age J.
BERGER,
CHAZAUD,
Ch.
G.
JEAN-FAUCIIER,
VEYSSIERE
Physiologie
and
M. Cl.
DE
in
TURCKHEIM,
JEAN
Compar#{233}e et Endocrin
ologie,
Universit#{233} de Clerm out, Complexe Scientifique des C#{233}zeaux., B.P. 45-63l7OAubiere-France ABSTRACT every 10 days in plasma and testes of male rabbits, from birth to 90 varied little from birth (3.8 ng/10 ml) to 40 days (4.6 ng/10 ml) thereafter until the 60th day (40.1 ngIlO ml). This level, characteristic of puberty, unchanged until the 90th day and decreased thereafter. Testicular testosterone content a similar pattern. It remained steady from the 1st to the 20th day. From the 20th day to day it rose slowly. Between the 50th and 60th day it increased rapidly and reached its at the 60th day (339.8 ng). It remained at the same level until the 90th day. Testicular concentration was steady from 1 to 20 days, then increased quickly, reaching a at 60 days (154.8 ng/100 mg). It then decreased until the 90th day (48.4 ng/100 mg).
Testosterone days of age. and increased
remained followed the 50th maximum testosterone maximum
was measured Plasma testosterone
INTRODUCTION
Testicular
androgens,
terone,
play
opment
at
natal
an
different
life.
tion
and of
fetus,
the
nervous
and
pubertal
understanding plete knowledge, of the testis, to adulthood. concerning al.,
1975)
neonatal,
genital species,
neonatal the
from We the
involved
and
we
To
the
sexual Testicufor the
psychological puberty.
obtain
a
In of neogood
these periods for a species,
requires of the
its embryonic have already
differentiation reported results
period
report
here
and
MATERIALS One hundred New-Zealand strain,
in and
critical periods during embryonic,
embryonic
prepubertal
tract like
testicular differentia-
Harris, 1964). responsible
life.
of
post-
is responsible
physiological and which characterize of ontogenesis, activity occur
devel-
and
secretion
behavior (Pfeiffer, 1936; lar androgens are also
natal
testis
structures
of gonadotropin
morphological, modifications
fetal
of the male short-gestation
In
testos-
in sexual
of
particularly rats, influence irreversibly
regulation
the course testicular
especially
role
stages
In the
for differentiation (Jost, 1947). rodents secretions
and
important
AND
same conditions of temperature (20#{176}C ± 1), lighting (daylight) and feeding (complete pelleted diet and water given freely), were used. Animals were sacrificed every 10 days from birth to 90 days. Nine males, sexually mature, were sacrificed at 180 days. All rabbits were killed by section of the jugular and carotid vessels. The blood plasma and both testes were removed and frozen at -25#{176}C until assayed. This study was first undertaken with the older animals (180-30 days) by the technique of Attal (1969), which was used at this time by our laboratory, using GLC with electron capture detection. This method requires large blood volumes and, for this reason, it could not be applied to the younger animals (1-20 days). A more sensitive method was therefore necessary to adequately determine the endogenous level of testosterone in individual samples. The radioimmunoassay method was chosen and used for these animals (Veyssiere et a!., 1975). Plasma testosterone was also measured, by RIA, in females sacrificed at 1, 30 and 60 days.
data
pubertal
Gas Cbromatoraphy
a comfunction
Plasma with
et
referring
to
METHODS the the
found
with
July March
testosterone
a mean
reproducibility Accepted Received
testicular
The
androgens was converted
were extracted then isolated by to testosterone-
17(3-heptafluorobutyrate, purified and injected into a Packard chromatograph equipped with an electron detector (63 Ni). The specificity of the method has been previously proven (Attal, 1969; Jean et a!., 1975). The lowest amount of testosterone measurable in biological samples was 1 ng. The precision and accuracy of the method was determined by recovering known amounts of testosterone (1, 5, 10, 20 and 100 ng) added to 5 ml of distilled water. Initial values were
periods.
of under
and
thin-layer-chromatography,
(Veyssiere
and thirty nine rabbits raised in the laboratory
ether.
measuring
20, 1976. 9, 1976.
male
561
rabbits
of
the
same plasma)
precision the
method
of
10
percent.
The
was evaluated by sample (3 or 6 ml of 90 day old three times. The values measured
BERGERETAL.
562
were: 13.5; 13.9; 10.5 ng/3 ml (mean 1.9) and 24.8; 27.8; 22.8 ng/6 ml (mean
SD: SD:
± ±
12.6 25.2
± ±
0
be
“'
‘fl’’S
0 0, V
Radiozrnmunoassay
0
o The androgens were extracted with ethyl acetateisooctane. Dihydrotestosterone was eliminated by chromatography on a celite column. Testosterone, tritiated testosterone and the antibody diluted to 1/45000 were incubated overnight at +4#{176}C. Antibody-bound and free hormone were separated using dextran coated charcoal. Accuracy was determined by adding known amounts of testosterone to distilled water. The amounts of testosterone added were 50, 100, 200, 500, 1000, 2000 pg and the estimates (mean ± SD) were 64 ± 12; 111 ± 15; 196 ± 26; 947 ± 55; 1887 ± 100 respectively. The assay was sensitive to 50 pg, with intraand inter-assay coefficients of variation of 5.6 percent and 9.5 percent respectively. The antibody was prepared in rabbits by simultaneous multiple intradermal injections of testosterone-3-0carboxymethyl oxime bovine albumin. The major
cross-reacting
steroid
in this system,
considered
not significant when
+1
0’
05
00 ‘-‘‘0
-‘
*
-‘
‘fl
N
N
N
‘f N
‘fi ‘fi
OS N
0
00 00
N
N
+1
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+1
+1
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N
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50
Os N
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+1
+1
N
Os
*
N N .
N N
000s
_
* “5
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-‘
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N
-
-,
fl
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iS
00 ‘0
N *
+1
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+1
+1
+1
00
*
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0
0-.
I-
000
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0
*
+1
+1
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0
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00
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+1
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‘000’0
N**000’*00’5ws
be 0
#{149}
‘Oils
5N’SNN #{149}
V
di
00’W5IfiNs0N’0 +1
*1
+1
+1
+1
+1
+1
+1
+1
+1
+1
* *
.0
‘ON
0
56
-
+1
> -‘0 560 0 .0
a >5
a
P>0.05.
+1
*
-,
V
5o-dihydrotestos-
terone (69 percent) was eliminated by chromatography. Among the 17 other steroids studied, the cross reaction of 5cs-androstane-3j3, 1 7!3-diol and 5o-androstane-3o, 17-diol was 7.8 percent. For the others, it was less than 2 percent. Simultaneous measurements of various samples, by RIA and GLC, showed an average variation of 12 percent, with a highly significant coefficient of correlation (r = 0,87) which permitted the comparison of the results obtained by both methods. The significance of differences between means was calculated using the Student’s : test. Differences between means are
c.
‘0
‘0
N
00
0
0
0
+1
+1
+1
*1
‘-‘
US
N
,-‘
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+1
4-I
+1
0
00” N
US
+1
+1
+1
0#{149} 0 a
0”0 -
-
“S 4
*
“S
-
-
OSSO N
N
*50”5
L’S
*
*
*
‘0 0
0’
RESULTS
Plasma
testosterone
relatively
constant
40
of
days
concentrations from
age
0
birth
(4.6
ng/10
ng/10
ml).
ml)
Then
.0* 0
.0
remain
(3.8
.0 56 V
to
it
rises
50
days
.0
E 0
V
,,04
a V
1-’
‘0
0
9-
and
steeply and
40.1
typical
reaches ng/10
of
from
60
ml
the to
adult 1).
stage
occur
at
60
It
only
In
one levels measured at remain relatively constant 0.06;
0.46
plasma
± 0.03; from
lower
than
0.66
in plasma
of
and
plasma
*
‘IS
N’O
+1
*1
*1
0”'
*4
*4
(10
*1
CCL’S
N
*
‘0
“‘0
Os’-’
0 -
N
‘0
* *
L5
‘000 N
N
‘0
00.-’
N
O5.-4 -
*
VS Os
,n
V C 0 V
days 60
a 0
days
the
*1
in
same
N
a 0
-
“S N’0
00 N *
N “5
be
V
significantly of
“‘‘00’
a
testoster-
Testosterone
always males
0
0
1, 30 and 60 days (in ng/10 ml: 0.42 ± is
-4-I
ng/10
10
50
± 0.05).
females
*1
6
in
variations
and
females,
+1
NNN’00’NO©
a
between
significant
between
+1
V
1
and
17.2
analysis
that
(0.001
