Levels of Testosterone in the Plasma, Gonads, and Adrenals During Fetal Development of the Rabbit GEORGES VEYSSIERE, MICHEL BERGER, CHRISTIANE JEAN-FAUCHER, MARC D E TURCKHEIM, AND CLAUDE JEAN Physiologie Comparee et Endocrinologie, Universite de Clermont, Complexe Scientifique des C&zeaux, B.P. 45-63170 Aubiere, France ABSTRACT. Testosterone (T) concentrations in the plasma, gonads, and adrenals were measured by radioimmunoassay in 188 male and 160 female rabbit fetuses. Determinations were performed daily from the 20th to the 31st day of gestation and were correlated with the maternal plasma concentration of T. The T content of both testes remained relatively constant from the 20th (3040 pg) to the 26th day (3940 pg), and subsequently decreased until the 31st day (1630 pg). The concentration of testicular T fluctuated only slightly from the 20th (1040 pg/mg) to the 23rd day (783 pg/ mg), and thereafter decreased until the 31st day
(138 pg/mg). The T levels in plasma of males (132-361 pg/ml) were significantly higher than those of females (21-116 pg/ml). Plasma T levels in males were relatively constant and did not exhibit any rise, which is similar to observations of the testis during differentiation of the genital tract. Testosterone concentrations were low in the adrenals (3.5-12.3 pg/mg) of both sexes and in the ovaries (1.5-20.4 pg/mg) of fetuses. These data provide the first evidence for testicular T secretion at the time of genital differentiation in the rabbit (Endocrinology 99: 1263, 1976)
^T^HE influence of the mammalian fetal Materials and Methods \. testis on sexual differentiation has been thoroughly investigated in the rat (1), rabbit Animals (2), and mouse (3). In these species, the fetal Female rabbits of the New Zealand strain were testis plays a vital role in directing the raised in the laboratory under identical condidifferentiation of the male genital tract tions of temperature (20 C), lighting (daylight), and also induces Miillerian duct regression. and nutrition (complete pelleted chow and water The means by which the testis effects given freely). The time of gestation was conthese changes are not known but, according sidered to be 0 at the hour of copulation. Pregto Jost (4), the differentiation of the Wolffian nant females were sacrificed daily from the 20th duct, urogenital sinus, and genital tubercle to the 31st day after copulation (parturition could be caused by androgens secreted by occurs at 32 days). Mothers were killed by section the jugular and carotid vessels, and fetuses the fetal testis. Testosterone has been of were delivered by hysterectomy. Fetal blood quantified in the fetal testis of sheep (5), was obtained by decapitation (from 27 to 31 monkey (6), man (7,8), rat (9,10), and more days) or by cardiac puncture (20-26 days). recently in the rabbit (11). Testosterone has Blood samples were collected with (27-31 days) been also measured in the fetal blood of or without (20-26 days) anticoagulant (anticlot: some species, such as the monkey (12), Laboratoire Delagrange, Paris). Differentiation cattle (13), pig (14), sheep (15), and man of the gonad in the rabbit fetus occurs at the (16-18). The purpose of our study was to 14th day of intrauterine life (20). On days 20 and determine the quantity of testosterone 21 one gonad of each fetus was used for the present in the plasma, gonads, and adrenals histologic determination of sex. For the older sex identification could be determined of rabbit fetuses of both sexes at different fetuses, using such morphological criteria as the position, stages of intrauterine life. Preliminary re- form, weight, and vascularization of the gonad. sults, concerning late pregnancy, have Testosterone was measured in one or both previously been reported (19). gonads of each male fetus at all stages. Plasma, Received February 26, 1976.
ovaries, and adrenals were pooled as indicated in Table 1. Testosterone was also measured in 1263
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Endo i 1976 Vol 99 . No 5
VEYSSIERE ETAL.
1264
the plasma of pregnant mothers. For these studies, 58 pregnant animals and their 348 embryos (188 males, 160 females) were used. Radioimmunoassay of testosterone Testosterone was measured by the method of Mahoudeau and Bricaire (21). All samples of plasma (0.5 to 3.0 ml), gonads and adrenals were extracted with 6 ml of ethyl acetate-isooctane (3/7, vol/vol) after adding 2,000 dpm of [3H]testosterone (43 Ci/mmol). The extracts were washed with distilled water and the organic layer was dried in an airstream and redissolved in 2 ml of heptane-benzene (98/2, vol/vol) for transfer to celite columns. Each column, containing 0.5 g of celite-0.25 ml of formamide was washed and saturated with heptane. Dihydrotestosterone was eluted with 10 ml of heptane-benzene (98/2, vol/vol) and the testosterone was then eluted with 2.2 ml of benzene. This eluate was dried and redissolved in 1 ml of ethanol. A 0.3 ml aliquot was taken for determination of recovery. Two additional aliquots of 0.5 ml and 0.1 ml were taken from testicular extracts; only a single 0.5 ml aliquot was taken from extracts of plasma, ovaries, and adrenals and 15,000 dpm of [3H]testosterone was added to each sample tube. Testosterone standards, ranging from 0.02 to 1 ng were pipetted in duplicate into identical tubes and 15,000 dpm of tritiated testosterone were added to each tube. After evaporation, 300 /x\ of testosterone—antiserum diluted to 1:45,000 were added to each sample and standard tube. After incubation overnight at 4 C, antibodybound and free hormone were separated using dextran-charcoal. The tubes were centrifuged
and the supernatant, containing the bound steroid, was decanted into counting vials and counted for 4 min in the following scintillation fluids:for non-aqueous samples, PPO-POPOP solution (4 g PPO, 40 mg POPOP, 1 liter toluene); for aqueous samples, a solution of dioxane (700 ml), toluene (300 ml), naphthalene (20 g), POPOP (0.1 g), and PPO (4 g). Distilled water blanks were included in each assay and these blanks generally did not exceed 10 pg. Accuracy was determined by adding known amounts (50, 100, 200, 500, 1000, 2000 pg) of testosterone to distilled water. The estimates (mean ± SD) were:64 ± 12, 111 ± 15, 196 ± 26, 517 ± 32, 947 ± 55, and 1887 ± 100, respectively. If known amounts (50 to 2000 pg) of testosterone were added to the plasma of adult male rabbits, initial values were found with a mean variation of 6.2%. A highly significant correlation (r = 0.998) was found between assay value and volume of plasma extracted. The intra- and inter-assay variations, determined by repeated measurements of the same adult male rabbit plasma, were 5.6% and 9.5%, respectively. The lower limit of the assay was 50 pg. The antibody was prepared in female rabbits by simultaneous multiple intradermal injections (70-80) of testosterone-3-0-carboxymethyl oxime conjugated to bovine albumin (8 to 10 mol of testosterone were bound to each mol of BSA). The rabbits were killed from 43 to 46 days after immunization, the plasma was diluted to 1:100 in 0.05M phosphate buffer, pH 7.4, containing 0.1% (wt/vol) gelatin and was stored in 0.4 ml aliquots at — 25 C. A 0.3 ml aliquot of this antiserum diluted to 1:45,000 and incubated with 15,000 dpm of tritiated testosterone bound 7,500 dpm [3H]testos-
TABLE 1. Summary of the experimental conditions (M = male; F = female); from 23 to 31 days; determinations performed daily in the same experimental conditions
Fetal age (days)
Gonads
Adrenals
Number of samples pooled
Volume (ml)
Number of pools measured
Number of gonads pooled
Number of pools measured
20
M F
9 12
0.8-1.4
2 1
1 12
18 1
21
M F
4 4
0.6-0.9
4 3
22
M F
0.55-1.35
3 4
23-31
M F
4 4 2 2
1 8-9 2 11-12
0.5-3.0
7-9 5-9
2 8-12
16 2 12 2 14-18 2-3
Number of adrenals pooled
Number of pools measured
OO OO
Plasma
2 1
4-8 4-8
3-6 3-6
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1265
TESTOSTERONE IN THE FETAL RABBIT
relatively steady from the 20th (3040 pg) to the 26th day (3940 pg), and thereafter decreased until the 31st day (1630 pg). The only significant reduction was observed between the 26th and 27th day (0.0KP < 0.02). At the end of gestation, testicular testosterone content was reduced by half compared to that of the 20th day, while the testis weight was four times higher. The concentration of testicular testosterone during fetal life was consistently higher than that measured in adult rabbits (95 pg/ mg). The highest value was observed at 20 days (1040 pg/mg). It subsequently exhibited a 30% decline from the 23rd to the 24th day (0.02 < P < 0.05), remained constant until the 26th day, and then decreased Results until the 31st day (138 pg/mg). In male fetuses, the plasma testosterone In male fetuses, testosterone could be concentration fluctuated between 132 ± 22 measured in one or both testes of each pg/ml and 361 ±119 pg/ml. (Table 3). These animal at every stage (Table 2). The testosvalues are very much smaller than those terone content of both testes remained
terone. The major steroids reacting in this system were testosterone (100%) and dihydrotestosterone (69%). The two were separated on a celite column. Cross-reactions by the 17 other steroids studied were low:5a-androstane-3a, 17/3-diol (7.8%), 5a-androstane-3/3, 17/3-diol (7.8%), androst-5-ene-3/3, 17/3-diol (2%), androstenedione (1.8%), estradiol-17/3 (0.5%) and androsterone (0.4%). Etiocholanolone, dehydroepiandrosterone, progesterone, pregnenolone, estrone, cortisol, cortisone, 17a-hydroxyandrost-4-en-3one and 5a-androstane-3, 17-dione were all less than 0.4%. The significance of differences between means was calculated using Student's t test. Differences between means were considered significant when P < 0.05.
TABLE 2. Testosterone levels in the testes and adrenals of fetal male rabbits Testicular testosterone Fetal age (days)
Body weight
20 (18)§ 21 (16) 22 (12) 23 (16) 24 (16) 25 (16) 26 (16)
3.0 ± 0.1
27
(18) 28 (18) 29 (14) 30 (14) 31 (14)
pg/2 testes
Pg/mg testis
2.8 ± 0.1
3,040 ± 530
1,040 ± 198
4.9 ± 0.2
3.0 ± 0.1
2,400 ± 450
768 ± 110
6.7 ± 0.2
3.5 ± 0.2
3,120 ±510
925 ± 161
9.9 ± 0.5
4.0 ± 0.2
3,120 ± 350
783 ± 84
11.7 ± 0.6
4.5 ± 0.2
2,370 ± 190
548 ± 45*
15.3 ± 1.0
5.3 ± 0.3
3,230 ± 300*
639 ± 63
20.2 ± 1.3
6.4 ± 0.2
3,940 ± 500
615 ± 70
25.1 ± 1.0
6.4 ± 0.3
2,510 ± 250f
405 ± 39t
38.7 ± 0.7
8.7 ± 0.3
2,230 ± 350
232 ± 3 3 |
41.8 ± 2.9
8.9 ± 0.6
1,700 ± 330
183 ± 33
45.3 ± 1.0
9.6 ± 0.3
1,540 ± 370
159 ± 36
57.6 ± 2.0
11.1 ± 0.6
1,630 ± 330
138 ± 22
(g)
weight (mg)
Testosterone in adrenals pg/10 adrenals
pg/mg adrenal
74
12.3
75
6.9
122
10.0
165
10.4
156
7.1
104
3.5
§ Numbers in parentheses indicate number of fetuses. * Significantly different from the preceding stage (* 0.02 < P < 0.05; t 0.01 < P < 0.02; t 0.001 < P < 0.01). Values are means ± SE.
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1266
VEYSSIERE ETAL.
Endo • 1976 Vol 99 • No 5
At every stage, testosterone could be measured in pools containing ovaries from 4 to 6 animals (Table 4). Ovarian testosterone Comparison concentration was very low compared to that Fetal between male of the testis. It was relatively steady from Pregnant Male Female and female age mothers fetuses fetuses fetuses (days) 20 to 27 days and subsequently began to decline. Testosterone levels of the adrenals 20 63 234 ± 59 44 ± 10 (175-293) of female fetuses were identical to those of 174 ± 41 21 34 ± 8 65 ± 15 male fetuses of the same age and to those (28-38) (80-258) measured in fetal ovaries. 74 ±21 74 ±24 146 ± 27 22 Plasma testosterone levels of female (95-188) (38-133) fetuses assayed at each stage varied little 23 0.1 < P < 0 . 2 64 ± 12 55 ± 8 136 ± 40 (Table 3). Two significant variations were (29-113) (48-269) observed, however: a 123% rise from the 24 0.02
(138 pg/mg). The T levels in plasma of males (132-361 pg/ml) were significantly higher than those of females (21-116 pg/ml). Plasma T levels in males were relatively constant and did not exhibit any rise, which is similar to observations of the testis during differentiation of the genital tract. Testosterone concentrations were low in the adrenals (3.5-12.3 pg/mg) of both sexes and in the ovaries (1.5-20.4 pg/mg) of fetuses. These data provide the first evidence for testicular T secretion at the time of genital differentiation in the rabbit (Endocrinology 99: 1263, 1976)
^T^HE influence of the mammalian fetal Materials and Methods \. testis on sexual differentiation has been thoroughly investigated in the rat (1), rabbit Animals (2), and mouse (3). In these species, the fetal Female rabbits of the New Zealand strain were testis plays a vital role in directing the raised in the laboratory under identical condidifferentiation of the male genital tract tions of temperature (20 C), lighting (daylight), and also induces Miillerian duct regression. and nutrition (complete pelleted chow and water The means by which the testis effects given freely). The time of gestation was conthese changes are not known but, according sidered to be 0 at the hour of copulation. Pregto Jost (4), the differentiation of the Wolffian nant females were sacrificed daily from the 20th duct, urogenital sinus, and genital tubercle to the 31st day after copulation (parturition could be caused by androgens secreted by occurs at 32 days). Mothers were killed by section the jugular and carotid vessels, and fetuses the fetal testis. Testosterone has been of were delivered by hysterectomy. Fetal blood quantified in the fetal testis of sheep (5), was obtained by decapitation (from 27 to 31 monkey (6), man (7,8), rat (9,10), and more days) or by cardiac puncture (20-26 days). recently in the rabbit (11). Testosterone has Blood samples were collected with (27-31 days) been also measured in the fetal blood of or without (20-26 days) anticoagulant (anticlot: some species, such as the monkey (12), Laboratoire Delagrange, Paris). Differentiation cattle (13), pig (14), sheep (15), and man of the gonad in the rabbit fetus occurs at the (16-18). The purpose of our study was to 14th day of intrauterine life (20). On days 20 and determine the quantity of testosterone 21 one gonad of each fetus was used for the present in the plasma, gonads, and adrenals histologic determination of sex. For the older sex identification could be determined of rabbit fetuses of both sexes at different fetuses, using such morphological criteria as the position, stages of intrauterine life. Preliminary re- form, weight, and vascularization of the gonad. sults, concerning late pregnancy, have Testosterone was measured in one or both previously been reported (19). gonads of each male fetus at all stages. Plasma, Received February 26, 1976.
ovaries, and adrenals were pooled as indicated in Table 1. Testosterone was also measured in 1263
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Endo i 1976 Vol 99 . No 5
VEYSSIERE ETAL.
1264
the plasma of pregnant mothers. For these studies, 58 pregnant animals and their 348 embryos (188 males, 160 females) were used. Radioimmunoassay of testosterone Testosterone was measured by the method of Mahoudeau and Bricaire (21). All samples of plasma (0.5 to 3.0 ml), gonads and adrenals were extracted with 6 ml of ethyl acetate-isooctane (3/7, vol/vol) after adding 2,000 dpm of [3H]testosterone (43 Ci/mmol). The extracts were washed with distilled water and the organic layer was dried in an airstream and redissolved in 2 ml of heptane-benzene (98/2, vol/vol) for transfer to celite columns. Each column, containing 0.5 g of celite-0.25 ml of formamide was washed and saturated with heptane. Dihydrotestosterone was eluted with 10 ml of heptane-benzene (98/2, vol/vol) and the testosterone was then eluted with 2.2 ml of benzene. This eluate was dried and redissolved in 1 ml of ethanol. A 0.3 ml aliquot was taken for determination of recovery. Two additional aliquots of 0.5 ml and 0.1 ml were taken from testicular extracts; only a single 0.5 ml aliquot was taken from extracts of plasma, ovaries, and adrenals and 15,000 dpm of [3H]testosterone was added to each sample tube. Testosterone standards, ranging from 0.02 to 1 ng were pipetted in duplicate into identical tubes and 15,000 dpm of tritiated testosterone were added to each tube. After evaporation, 300 /x\ of testosterone—antiserum diluted to 1:45,000 were added to each sample and standard tube. After incubation overnight at 4 C, antibodybound and free hormone were separated using dextran-charcoal. The tubes were centrifuged
and the supernatant, containing the bound steroid, was decanted into counting vials and counted for 4 min in the following scintillation fluids:for non-aqueous samples, PPO-POPOP solution (4 g PPO, 40 mg POPOP, 1 liter toluene); for aqueous samples, a solution of dioxane (700 ml), toluene (300 ml), naphthalene (20 g), POPOP (0.1 g), and PPO (4 g). Distilled water blanks were included in each assay and these blanks generally did not exceed 10 pg. Accuracy was determined by adding known amounts (50, 100, 200, 500, 1000, 2000 pg) of testosterone to distilled water. The estimates (mean ± SD) were:64 ± 12, 111 ± 15, 196 ± 26, 517 ± 32, 947 ± 55, and 1887 ± 100, respectively. If known amounts (50 to 2000 pg) of testosterone were added to the plasma of adult male rabbits, initial values were found with a mean variation of 6.2%. A highly significant correlation (r = 0.998) was found between assay value and volume of plasma extracted. The intra- and inter-assay variations, determined by repeated measurements of the same adult male rabbit plasma, were 5.6% and 9.5%, respectively. The lower limit of the assay was 50 pg. The antibody was prepared in female rabbits by simultaneous multiple intradermal injections (70-80) of testosterone-3-0-carboxymethyl oxime conjugated to bovine albumin (8 to 10 mol of testosterone were bound to each mol of BSA). The rabbits were killed from 43 to 46 days after immunization, the plasma was diluted to 1:100 in 0.05M phosphate buffer, pH 7.4, containing 0.1% (wt/vol) gelatin and was stored in 0.4 ml aliquots at — 25 C. A 0.3 ml aliquot of this antiserum diluted to 1:45,000 and incubated with 15,000 dpm of tritiated testosterone bound 7,500 dpm [3H]testos-
TABLE 1. Summary of the experimental conditions (M = male; F = female); from 23 to 31 days; determinations performed daily in the same experimental conditions
Fetal age (days)
Gonads
Adrenals
Number of samples pooled
Volume (ml)
Number of pools measured
Number of gonads pooled
Number of pools measured
20
M F
9 12
0.8-1.4
2 1
1 12
18 1
21
M F
4 4
0.6-0.9
4 3
22
M F
0.55-1.35
3 4
23-31
M F
4 4 2 2
1 8-9 2 11-12
0.5-3.0
7-9 5-9
2 8-12
16 2 12 2 14-18 2-3
Number of adrenals pooled
Number of pools measured
OO OO
Plasma
2 1
4-8 4-8
3-6 3-6
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1265
TESTOSTERONE IN THE FETAL RABBIT
relatively steady from the 20th (3040 pg) to the 26th day (3940 pg), and thereafter decreased until the 31st day (1630 pg). The only significant reduction was observed between the 26th and 27th day (0.0KP < 0.02). At the end of gestation, testicular testosterone content was reduced by half compared to that of the 20th day, while the testis weight was four times higher. The concentration of testicular testosterone during fetal life was consistently higher than that measured in adult rabbits (95 pg/ mg). The highest value was observed at 20 days (1040 pg/mg). It subsequently exhibited a 30% decline from the 23rd to the 24th day (0.02 < P < 0.05), remained constant until the 26th day, and then decreased Results until the 31st day (138 pg/mg). In male fetuses, the plasma testosterone In male fetuses, testosterone could be concentration fluctuated between 132 ± 22 measured in one or both testes of each pg/ml and 361 ±119 pg/ml. (Table 3). These animal at every stage (Table 2). The testosvalues are very much smaller than those terone content of both testes remained
terone. The major steroids reacting in this system were testosterone (100%) and dihydrotestosterone (69%). The two were separated on a celite column. Cross-reactions by the 17 other steroids studied were low:5a-androstane-3a, 17/3-diol (7.8%), 5a-androstane-3/3, 17/3-diol (7.8%), androst-5-ene-3/3, 17/3-diol (2%), androstenedione (1.8%), estradiol-17/3 (0.5%) and androsterone (0.4%). Etiocholanolone, dehydroepiandrosterone, progesterone, pregnenolone, estrone, cortisol, cortisone, 17a-hydroxyandrost-4-en-3one and 5a-androstane-3, 17-dione were all less than 0.4%. The significance of differences between means was calculated using Student's t test. Differences between means were considered significant when P < 0.05.
TABLE 2. Testosterone levels in the testes and adrenals of fetal male rabbits Testicular testosterone Fetal age (days)
Body weight
20 (18)§ 21 (16) 22 (12) 23 (16) 24 (16) 25 (16) 26 (16)
3.0 ± 0.1
27
(18) 28 (18) 29 (14) 30 (14) 31 (14)
pg/2 testes
Pg/mg testis
2.8 ± 0.1
3,040 ± 530
1,040 ± 198
4.9 ± 0.2
3.0 ± 0.1
2,400 ± 450
768 ± 110
6.7 ± 0.2
3.5 ± 0.2
3,120 ±510
925 ± 161
9.9 ± 0.5
4.0 ± 0.2
3,120 ± 350
783 ± 84
11.7 ± 0.6
4.5 ± 0.2
2,370 ± 190
548 ± 45*
15.3 ± 1.0
5.3 ± 0.3
3,230 ± 300*
639 ± 63
20.2 ± 1.3
6.4 ± 0.2
3,940 ± 500
615 ± 70
25.1 ± 1.0
6.4 ± 0.3
2,510 ± 250f
405 ± 39t
38.7 ± 0.7
8.7 ± 0.3
2,230 ± 350
232 ± 3 3 |
41.8 ± 2.9
8.9 ± 0.6
1,700 ± 330
183 ± 33
45.3 ± 1.0
9.6 ± 0.3
1,540 ± 370
159 ± 36
57.6 ± 2.0
11.1 ± 0.6
1,630 ± 330
138 ± 22
(g)
weight (mg)
Testosterone in adrenals pg/10 adrenals
pg/mg adrenal
74
12.3
75
6.9
122
10.0
165
10.4
156
7.1
104
3.5
§ Numbers in parentheses indicate number of fetuses. * Significantly different from the preceding stage (* 0.02 < P < 0.05; t 0.01 < P < 0.02; t 0.001 < P < 0.01). Values are means ± SE.
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1266
VEYSSIERE ETAL.
Endo • 1976 Vol 99 • No 5
At every stage, testosterone could be measured in pools containing ovaries from 4 to 6 animals (Table 4). Ovarian testosterone Comparison concentration was very low compared to that Fetal between male of the testis. It was relatively steady from Pregnant Male Female and female age mothers fetuses fetuses fetuses (days) 20 to 27 days and subsequently began to decline. Testosterone levels of the adrenals 20 63 234 ± 59 44 ± 10 (175-293) of female fetuses were identical to those of 174 ± 41 21 34 ± 8 65 ± 15 male fetuses of the same age and to those (28-38) (80-258) measured in fetal ovaries. 74 ±21 74 ±24 146 ± 27 22 Plasma testosterone levels of female (95-188) (38-133) fetuses assayed at each stage varied little 23 0.1 < P < 0 . 2 64 ± 12 55 ± 8 136 ± 40 (Table 3). Two significant variations were (29-113) (48-269) observed, however: a 123% rise from the 24 0.02
