Exp. Clin. Endocrinol. Vol. 98, No. 2, 1991, pp. 131 139
J. A. Barth, Leipzig
Institute of Experimental Endocrinology (Head: Prof. Dr. Sc. med. Dr. h. c. G. Dörner), Humboldt University Medical School (Charité), Berlin/Germany
The Influence of Fetal Adrenals on the Androgen Levels during Brain Differentiation in Human Subjects and Rats
With 8 Figures Summary: Measurements of plasma total and free testosterone (T) levels in human subjects from fetal to postpubertal life showed about twofold higher total T and 15-fold higher free T levels in female fetuses than in female adults. The ratios between the sexes were only moderate in fetal life. Between the 17th and 31st week of pregnancy the ratios (male : female) of total T were found to be 6.6 in week 17, 1.5 in week 22, 2.3 in week 28 and 1.2 in week 31 of pregnancy compared to 16.2 in adulthood. The corresponding ratios of free T were calculated to be 5.6 in week 17, 1.4 in week 22, 0.9 in week 28 and 0.7 in week 31 of pregnancy compared to 34 in adulthood. In amniotic fluids, we measured even an overlapping of T values between the two sexes. The reason for the observed striking difference of T levels between the sexes in fetal and postpubertal life may be the high adrenal activity and secretion rate in fetal life during brain differentiation. In rats, the contribution of adrenals to plasma T levels is only moder.ate and much smaller than
in human beings. As measured in adult female rats, the portion was found to be about 20% only, contrary to about 60% in women. The main sources of T in female rats appear to be the gonads. The mainly gonadal secretion may be the reason that exposure of pregnant rats to stress diminished the T levels in male fetuses, but did not significantly elevate the T levels in females. The contribution of adrenals to androstenedione (A), however, appears to be similar in both species (about 40%). Therefore, we found a significant increase of A levels in female rats after stress, both in mothers, their fetuses and non-pregnant females. In adult males, however, A levels decreased significantly after stress. The same effect has been also observed after surgical stress in men. Contrary to female rats, surgical stress increased significantly T levels in women, whereas in males a significant decrease in both species has been observed.
Key words: Testosterone - Androstenedione - Fetal adrenals - Stress - Human subjects Rats
Introduction
It is evident from the structural and biochemical differences seen between fetal, neonatal and adult adrenal glands that the regulation of growth of the adrenal and steroid secretion in prenatal life is unique. In particular, the control mechanism must be able to maintain an enlarged gland, it must allow the growth of the fetal zone which occupies 80% of the mass at term and it must regulate the secretion of 100 200 mg steroid per day, an amount at least five times of that normally
Downloaded by: University of Pittsburgh. Copyrighted material.
F. STAHL, FRANZISKA GÖTZ and G. DÖRNER
132
Exp. Clin. Endocrinol. 98 (1991) 2
produced by the adult gland (Makin, 1984). This high fetal adrenal activity and secretion rate may influence fetal plasma testosterone (T) levels, especially in females. Therefore, we measured plasma T levels from fetal to postpubertal life in human subjects and in rats. It is well-known that stress changes the androgen levels either by stimulation of the adrenals and/or by inhibition of the testes (Nahashima et al., 1975; Stahl et al.; 1978, 1987; Parker, 1989). In order to investigate the influence of stress on androgen levels, we determined plasma T and androstenedione (A) levels before and after stress in adult men and women, in male and femal rats and in pregnant rats and their fetuses.
Human fetuses were obtained by induced abortion because of different medical indication between week 17 and 31 of pregnancy. Blood samples were drawn from patients before and after thyroid
surgery. In all animal experiments adult Wistar rats were used being in full reproductive age (Males: 3-4 months; 370 ± 60 g; Females: 3-4 months; 220 ± 20 g). The rats were stressed by immobilization in small plastic boxes with additional illumination three times a day for 45 min over a period of 4 days. For pregnant rats the period of stress lasted from day 15 to day 21 of gestation. The offspring were either delivered by Caesarian section on day 22 of pregnancy or sacrificed immediately, 2 12 hrs, 12-24 hrs, 3 days or 5 days after spontaneous delivery. Determination of plasma T and A levels were performed by RIA. An appropriate volume of plasma was diluted to I ml with aqua bidest and extracted once with 4 ml ether. The ether phase was evaporated to dryness and the residue re-dissolved in an antiserum dilution. The used antibodies were raised against testosterone-3- or androstenedione-3-(carboxymethyl)-oxim-BSA, respectively. Free T was determined by a dialysis method described by Stahl et al. (1975).
Results
Plasma levels under normal condition
As demonstrated in Fig. 1, plasma total T levels in human female fetuses were found to be about twofold higher (p < 0.001) than in cyclic women and the ratios between the sexes were very much smaller in fetal than in postpubertal life. The mean total T from week 17 to week 31 of fetal life was calculated to 2.1 ± 0.35 nmol/l (n = 4) for females compared to 1.2 ± 0.3 nmol/l (n = 25) in cyclic women. The ratios (male:female) between the sexes were 6.6 in week 17, 1.5 in week 22,
2.3 in week 28 and 1.2 in week 31 of pregnancy compared to a ratio of 16.2 in adulthood. As shown in Fig. 2, in free T levels these differences between fetal and adult life are still stronger. Free T levels in female fetuses were even 15-fold higher than in female adults. The mean free T level from week 17 to week 31 of fetal life was 142 ± 20 pmol/l (n = 4) for females compared to 13.5 ± 4.1 pmol/l (n = 25)
in cyclic women. The ratios between the sexes were 5.6 in week 17, 1.4 in week 22, 0.9 in week 28 and 0.7 in week 31 of pregnancy compared to a ratio of 34 in adulthood. Measurements of unconjugated T levels in amniotic fluids revealed even an overlapping of T levels between the two sexes (Fig. 3). In rats, however, we have not found significant differences of total T levels between female fetuses and female adults. We determined a mean value of 0.6 ± 0.18 nmol/l (n = 10) on day 22 of pregnancy and 0.83 ± 0.38 nmol/l(n = 19) in adult females at the age of 3 months.
Downloaded by: University of Pittsburgh. Copyrighted material.
Material and Methods
F. STAHL et al., Fetal Adrenal Androgens in Men and Rats
133
-I
Birth
25 20 15
1
L
10
I
i
5 o I
idIIiIIIiiIlPr,,..,!
Tt!lhilî !IIIIIIL1
17 22 28 31
10 20 50 75 95 145 20 45 90 120240 4
5
ours
Weeks
8
25
Years
Days
Females
Males
Fig. 1 Plasma total T levels (mean ± SD) from fetal life in weeks of pregnancy (single values) to postpubertal life in hours, days and years after birth in human subjects (n = 7-12 in postnatal and n = 25-28 in postpubertal life)
Birth
pmol/l
800
600
I --
r___________________________
400 I
200
o
1II.
17
22
[hi 28 31
1 _I
lillílí rl 11M.UL. I1 1
5
10
20
50 75
95 145 20
45
90 120 240
ays D Males
4
8
25
e a rs
Females
Fig. 2 Plasma free T levels (mean ± SD) from fetal life in weeks of pregnancy (single values) to postpubertal life in hours, days and years after birth in human subjects (n = 7-12 in postnatal
and n = 25-28 in postpubertal life)
Downloaded by: University of Pittsburgh. Copyrighted material.
nmol/l
Exp. Clin. Endocrinol. 98 (1991) 2
134 nmol/I 1.5 s
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13-15
16
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26-3 1
32-37
38-40
Weeks of Pregnancy Fig. 3 Unconjugated T levels in amniotic fluids of human subjects from week 13 to 40 of pregnancy. Shaded area represents mean ± SD. Points: males; asterisks: females
The difference is not significant but the tendency is rather higher in adult than in fetal life. The corresponding values in males were 2.3 ± 0.9 nmol/l (n = 11) on day 22 of pregnancy and 11.7 ± 6.1 nmol/l (n = 11) in adult life (Fig. 4). Plasma levels under stress conditions We studied the responses of plasma T and A levels in patients undergoing thyroid surgery. During surgical stress there was a clear-cut difference between men and women. After surgery, plasma T levels decreased in men. However, significant
decreases have been measured not till the first and second postoperative day (Fig. 5), whereas plasma T in women increased significantly on the day of surgery and was unchanged afterwards. Plasma A, on the other hand, increased significantly both in men and in women on the operative day. Contrary to women, A values in men decreased postoperatively, parallel to the decrease of T levels. On the second postoperative day the examined A level was significantly lower than the initial level (Fig. 6). Contrary to the results under stress conditions in human subjects, we found only a significant decrease of T levels in male rats but in females the values remained unchanged. As demonstrated in Fig. 7, plasma T levels decreased significantly in
male fetuses, in male newborns and in male adults. An exception was only the group sacrificed immediately after birth. In females, however, no significant increase has been observed neither in fetuses, newborns or in adult females. Re-
Downloaded by: University of Pittsburgh. Copyrighted material.
s
F. STAHL et al., Fetal Adrenal Androgens in Men and Rats
135
nmolfl
20
15
22 Days
2-12 Hrs
Birth
12-24 Hrs
5 Days
3 Days
3 Months
Age
U Males
Females
Fig. 4 Plasma T levels (mean ± SD) from fetal life (day 22 of pregnancy; n = 10 pubertal life in hours, days and months after birth (n = 13 23) in rats
* p
J. A. Barth, Leipzig
Institute of Experimental Endocrinology (Head: Prof. Dr. Sc. med. Dr. h. c. G. Dörner), Humboldt University Medical School (Charité), Berlin/Germany
The Influence of Fetal Adrenals on the Androgen Levels during Brain Differentiation in Human Subjects and Rats
With 8 Figures Summary: Measurements of plasma total and free testosterone (T) levels in human subjects from fetal to postpubertal life showed about twofold higher total T and 15-fold higher free T levels in female fetuses than in female adults. The ratios between the sexes were only moderate in fetal life. Between the 17th and 31st week of pregnancy the ratios (male : female) of total T were found to be 6.6 in week 17, 1.5 in week 22, 2.3 in week 28 and 1.2 in week 31 of pregnancy compared to 16.2 in adulthood. The corresponding ratios of free T were calculated to be 5.6 in week 17, 1.4 in week 22, 0.9 in week 28 and 0.7 in week 31 of pregnancy compared to 34 in adulthood. In amniotic fluids, we measured even an overlapping of T values between the two sexes. The reason for the observed striking difference of T levels between the sexes in fetal and postpubertal life may be the high adrenal activity and secretion rate in fetal life during brain differentiation. In rats, the contribution of adrenals to plasma T levels is only moder.ate and much smaller than
in human beings. As measured in adult female rats, the portion was found to be about 20% only, contrary to about 60% in women. The main sources of T in female rats appear to be the gonads. The mainly gonadal secretion may be the reason that exposure of pregnant rats to stress diminished the T levels in male fetuses, but did not significantly elevate the T levels in females. The contribution of adrenals to androstenedione (A), however, appears to be similar in both species (about 40%). Therefore, we found a significant increase of A levels in female rats after stress, both in mothers, their fetuses and non-pregnant females. In adult males, however, A levels decreased significantly after stress. The same effect has been also observed after surgical stress in men. Contrary to female rats, surgical stress increased significantly T levels in women, whereas in males a significant decrease in both species has been observed.
Key words: Testosterone - Androstenedione - Fetal adrenals - Stress - Human subjects Rats
Introduction
It is evident from the structural and biochemical differences seen between fetal, neonatal and adult adrenal glands that the regulation of growth of the adrenal and steroid secretion in prenatal life is unique. In particular, the control mechanism must be able to maintain an enlarged gland, it must allow the growth of the fetal zone which occupies 80% of the mass at term and it must regulate the secretion of 100 200 mg steroid per day, an amount at least five times of that normally
Downloaded by: University of Pittsburgh. Copyrighted material.
F. STAHL, FRANZISKA GÖTZ and G. DÖRNER
132
Exp. Clin. Endocrinol. 98 (1991) 2
produced by the adult gland (Makin, 1984). This high fetal adrenal activity and secretion rate may influence fetal plasma testosterone (T) levels, especially in females. Therefore, we measured plasma T levels from fetal to postpubertal life in human subjects and in rats. It is well-known that stress changes the androgen levels either by stimulation of the adrenals and/or by inhibition of the testes (Nahashima et al., 1975; Stahl et al.; 1978, 1987; Parker, 1989). In order to investigate the influence of stress on androgen levels, we determined plasma T and androstenedione (A) levels before and after stress in adult men and women, in male and femal rats and in pregnant rats and their fetuses.
Human fetuses were obtained by induced abortion because of different medical indication between week 17 and 31 of pregnancy. Blood samples were drawn from patients before and after thyroid
surgery. In all animal experiments adult Wistar rats were used being in full reproductive age (Males: 3-4 months; 370 ± 60 g; Females: 3-4 months; 220 ± 20 g). The rats were stressed by immobilization in small plastic boxes with additional illumination three times a day for 45 min over a period of 4 days. For pregnant rats the period of stress lasted from day 15 to day 21 of gestation. The offspring were either delivered by Caesarian section on day 22 of pregnancy or sacrificed immediately, 2 12 hrs, 12-24 hrs, 3 days or 5 days after spontaneous delivery. Determination of plasma T and A levels were performed by RIA. An appropriate volume of plasma was diluted to I ml with aqua bidest and extracted once with 4 ml ether. The ether phase was evaporated to dryness and the residue re-dissolved in an antiserum dilution. The used antibodies were raised against testosterone-3- or androstenedione-3-(carboxymethyl)-oxim-BSA, respectively. Free T was determined by a dialysis method described by Stahl et al. (1975).
Results
Plasma levels under normal condition
As demonstrated in Fig. 1, plasma total T levels in human female fetuses were found to be about twofold higher (p < 0.001) than in cyclic women and the ratios between the sexes were very much smaller in fetal than in postpubertal life. The mean total T from week 17 to week 31 of fetal life was calculated to 2.1 ± 0.35 nmol/l (n = 4) for females compared to 1.2 ± 0.3 nmol/l (n = 25) in cyclic women. The ratios (male:female) between the sexes were 6.6 in week 17, 1.5 in week 22,
2.3 in week 28 and 1.2 in week 31 of pregnancy compared to a ratio of 16.2 in adulthood. As shown in Fig. 2, in free T levels these differences between fetal and adult life are still stronger. Free T levels in female fetuses were even 15-fold higher than in female adults. The mean free T level from week 17 to week 31 of fetal life was 142 ± 20 pmol/l (n = 4) for females compared to 13.5 ± 4.1 pmol/l (n = 25)
in cyclic women. The ratios between the sexes were 5.6 in week 17, 1.4 in week 22, 0.9 in week 28 and 0.7 in week 31 of pregnancy compared to a ratio of 34 in adulthood. Measurements of unconjugated T levels in amniotic fluids revealed even an overlapping of T levels between the two sexes (Fig. 3). In rats, however, we have not found significant differences of total T levels between female fetuses and female adults. We determined a mean value of 0.6 ± 0.18 nmol/l (n = 10) on day 22 of pregnancy and 0.83 ± 0.38 nmol/l(n = 19) in adult females at the age of 3 months.
Downloaded by: University of Pittsburgh. Copyrighted material.
Material and Methods
F. STAHL et al., Fetal Adrenal Androgens in Men and Rats
133
-I
Birth
25 20 15
1
L
10
I
i
5 o I
idIIiIIIiiIlPr,,..,!
Tt!lhilî !IIIIIIL1
17 22 28 31
10 20 50 75 95 145 20 45 90 120240 4
5
ours
Weeks
8
25
Years
Days
Females
Males
Fig. 1 Plasma total T levels (mean ± SD) from fetal life in weeks of pregnancy (single values) to postpubertal life in hours, days and years after birth in human subjects (n = 7-12 in postnatal and n = 25-28 in postpubertal life)
Birth
pmol/l
800
600
I --
r___________________________
400 I
200
o
1II.
17
22
[hi 28 31
1 _I
lillílí rl 11M.UL. I1 1
5
10
20
50 75
95 145 20
45
90 120 240
ays D Males
4
8
25
e a rs
Females
Fig. 2 Plasma free T levels (mean ± SD) from fetal life in weeks of pregnancy (single values) to postpubertal life in hours, days and years after birth in human subjects (n = 7-12 in postnatal
and n = 25-28 in postpubertal life)
Downloaded by: University of Pittsburgh. Copyrighted material.
nmol/l
Exp. Clin. Endocrinol. 98 (1991) 2
134 nmol/I 1.5 s
'
. sa
s.
.. s. SS
s
: I.
s s5
s.. 5
0.5
0 00
000 00
s.s a.
000
00
00 o
000
Iiis. s
so. s
s
s
s. I
s
I
s
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4
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s
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a
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00
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00 o
,'O .05
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0o
C
00
o
o
0000
s o.
Ola
o
o
13-15
16
17
18
19
20-23
26-3 1
32-37
38-40
Weeks of Pregnancy Fig. 3 Unconjugated T levels in amniotic fluids of human subjects from week 13 to 40 of pregnancy. Shaded area represents mean ± SD. Points: males; asterisks: females
The difference is not significant but the tendency is rather higher in adult than in fetal life. The corresponding values in males were 2.3 ± 0.9 nmol/l (n = 11) on day 22 of pregnancy and 11.7 ± 6.1 nmol/l (n = 11) in adult life (Fig. 4). Plasma levels under stress conditions We studied the responses of plasma T and A levels in patients undergoing thyroid surgery. During surgical stress there was a clear-cut difference between men and women. After surgery, plasma T levels decreased in men. However, significant
decreases have been measured not till the first and second postoperative day (Fig. 5), whereas plasma T in women increased significantly on the day of surgery and was unchanged afterwards. Plasma A, on the other hand, increased significantly both in men and in women on the operative day. Contrary to women, A values in men decreased postoperatively, parallel to the decrease of T levels. On the second postoperative day the examined A level was significantly lower than the initial level (Fig. 6). Contrary to the results under stress conditions in human subjects, we found only a significant decrease of T levels in male rats but in females the values remained unchanged. As demonstrated in Fig. 7, plasma T levels decreased significantly in
male fetuses, in male newborns and in male adults. An exception was only the group sacrificed immediately after birth. In females, however, no significant increase has been observed neither in fetuses, newborns or in adult females. Re-
Downloaded by: University of Pittsburgh. Copyrighted material.
s
F. STAHL et al., Fetal Adrenal Androgens in Men and Rats
135
nmolfl
20
15
22 Days
2-12 Hrs
Birth
12-24 Hrs
5 Days
3 Days
3 Months
Age
U Males
Females
Fig. 4 Plasma T levels (mean ± SD) from fetal life (day 22 of pregnancy; n = 10 pubertal life in hours, days and months after birth (n = 13 23) in rats
* p
