0736-5748/83 $3.00+0.00 Pergamon Press Ltd. ~) 1983 ISDN
Int. J. Devl. Neuroscience, Vol. 1, No. 3, pp. 187-190, 1983 Printed in Great Britain.
H Y P O T H A L A M I C T E S T O S T E R O N E I N C R E A S E IN T H E MALE RAT AT BIRTH J. RHODA,P. CORBIERand J. ROFFI
Laboratoire d'Endocrinologie, Centre d'Orsay, Universit6de Paris XI, F 91405 Orsay C6dex, France (Accepted 6 April 1983)
A b s t r a c t - - I n the male rat, a dramatic increase in serum testosterone occurs during the first 2 h of postnatal life. Since the hypothalamus is known to be an important site for sexual differentiation of the brain, this early testosterone surge was a good model to use to study the transfer of serum testosterone to the hypothalamus and cerebral cortex. E n d o g e n o u s testosterone was m e a s u r e d by radioimmunoassay in the h y p o t h a l a m u s and the cerebral cortex of the foetus and newborn rats during the first 6 h following birth. In the male, hypothalamic testosterone increased between 0 h in utero and 2 h; in the males gonadectomized at 0 h in utero and killed at the age of 2 h, the testosterone surge was abolished, clearly indicating the testicular origin of this h o r m o n e in the neonate. T h e small testosterone increase in the cerebral cortex compared with that in the hypothalamus reflects a preferential uptake of this h o r m o n e by the h y p o t h a l a m u s of the newborn. In the female, hypothalamic testosterone slightly decreased between 0 h in utero and 6 h. These results are in a g r e e m e n t with the view that hypothalamic modifications form the basis for some behavioral and physiological changes attributed to the effect of perinatal hormonal stimulation. Key words: Testosterone, Newborn rat, H y p o t h a l a m u s , Sexual differentation of the brain.
In the newborn male rat, the pituitary testicular axis is active at the time of birth. 6 A sudden increase in serum gonadotropins is followed by a transient elevation of serum testosterone level, between 0 and 2 h after birth. Evidence indicates that the masculinization of the central nervous system requires the presence of testicular androgens, probably testosterone, during the perinatal period,I° and at least part of this effect is probably exerted through action on the hypothalamus of the neonate. 3'15 In this case, hypothalamic tissue of the neonatal male should have a higher level of testosterone than hypothalamic tissue of the neonatal female. The main purpose of this study was to compare hypothalamic testosterone levels in male and female neonate rats within the 6 h following birth. The effects of perinatal gonadectomy at 0 h in utero on hypothalamic testosterone were also studied. MATERIALS AND METHODS Animals
Experiments were performed on laboratory bred Sherman rats. Animals were maintained on a 14:10 h light-dark cycle, with food and water ad libitum. Conditions of breeding and the timing of the pregnancies have been previously described. 5 The onset of gestation was defined as the estimated hour of ovulation (02.00 h on day 0), during the night of cohabitation. Parturition most often occurred during the day time of day 21 or 22 of gestation. Testosterone assays were performed on the following groups of" animals: Fetus 21 days 8 h: on day 21, between 09.00 and 11.00 h a.m., the mother was killed by cervical fracture and the fetuses were immediately removed from the uterine horns by caesarian section. Fetus 0 h in utero: at the moment of appearance of the first newborn, the mother was killed by cervical fracture and the fetuses were removed from the uterine horns. Newborns at 0 h ex utero: taken between 35 and 45 min after onset of parturition. Newborns at 2, 4 or 6 h after birth: the animals were left with their mothers until killed. Experimental procedure Dissection o f brain tissues. The fetuses and newborns of both sexes were killed by decapitation and the head immediately placed on ice. The entire hypothalamus was cut out as a block limited anteriorly by the optic chiasma, laterally by the hypothalamic fissures and posteriorly by the mammilary bodies; the block was approximately 2 mm deep from the basal surface of the hypothalamus.
187
| 88
J. Rhoda et al.
Cortical tissue was excised from parietal lobes of the brain. The tissues were freed from meninges and blotted with filter paper. For each tissue one determination involved a pool of 8-1(I pieces of tissue from several litters. All tissues were stored in 1 ml of distilled water at - 3 0 ° C until assay. G o n a d e c t o m y at 0 h in utero. Newborn male rats were gonadectomized or sham gonadectomized in the following manner: when the first newborn appeared, the mother was killed with a blow on the neck and the fetuses removed from the interne horns. Males were quickly placed in a freezer and gonadectomized or sham gonadectomized within 15 min after onset of delivery through a midline incision under cryoanesthesia. The incision was closed with a drop of collodion and the pups were rewarmed in a humidicrib at 34°C. They were killed by decapitation 2 h later and brain tissues were treated as above. H o r m o n e analyses. After decongelation, hypothalamic and cortical samples were homogenized by vigorously disrupting the tissues on a vortex mixer. Approximately 2500 dpm of tritiated testosterone (1,2,6,7[3H]testosterone, S.A. 109 Ci/mmol, A m e r s h a m , U . K . ) were added to the different tissue homogenates for recovery estimation. After a 30 rain incubation, at 37°C, the samples were extracted with twice 6 ml of diethyl ether. The pooled extracts were then dried under a stream of nitrogen and redissolved in 1 ml of 0.01 M sodium phosphate buffer p H 6.9 containing 0.1% gelatin, 0.1% sodium azide and 0.9% NaC1. This buffer will be referred to as PGSB. The extracts were stored at - 3 0 ° C until assay. In some cases, hypothalamic extracts were chromatographed on celite microcolumns according to Challis et al. 4 Testosterone was estimated in the extracts with a sensitive radioimmunoassay as previously described. 6 The antiserum (provided by M. Forest) used to measure testosterone was obtained by inoculating rabbits with testosterone 3carboxymethoxime conjugated with bovine serum albumin. The antibodies obtained exhibited a cross-reaction mainly with 17[3-hydroxy-5~-androstan 3-one (62.5%) and, to a lesser extent, with androstene 3[3.1713-diol (9.2%). Briefly, aliquots of the reconstituted extracts were incubated overnight at 4°C with 20,000 dpm of tritiated testosterone and antiserum in a final volume of 0.8 ml 0.01 M PGSB p H 6.9. Free from bound testosterone was separated by adding 1 ml of a cold solution of charcoal-dextran in PGSB (0.5% charcoal and 0.05% dextran T 70). After 20 min of incubation at 4°C, the samples were then centrifuged for 10 min at 4000 g in a refrigerated centrifuge. The radioactivity of the supernatant was counted in a LKB liquid scintillation spectrometer with 10 ml of scintillation liquid (toluene 1.5 1; triton X-100 0.75 1; scintimix 12.4 g). Using this procedure, water blanks were usually less than the assay sensitivity which was around 10 pg/tube. The intraassay and inter-assay coefficient of variation were, respectively, 9.7 and 12.6%. Statistics. Results were expressed as mean _+1 S.E.M. An Ftest was used for overall comparisons of the hormonal data and a t test for the intergroup differences. A probability of ~
Int. J. Devl. Neuroscience, Vol. 1, No. 3, pp. 187-190, 1983 Printed in Great Britain.
H Y P O T H A L A M I C T E S T O S T E R O N E I N C R E A S E IN T H E MALE RAT AT BIRTH J. RHODA,P. CORBIERand J. ROFFI
Laboratoire d'Endocrinologie, Centre d'Orsay, Universit6de Paris XI, F 91405 Orsay C6dex, France (Accepted 6 April 1983)
A b s t r a c t - - I n the male rat, a dramatic increase in serum testosterone occurs during the first 2 h of postnatal life. Since the hypothalamus is known to be an important site for sexual differentiation of the brain, this early testosterone surge was a good model to use to study the transfer of serum testosterone to the hypothalamus and cerebral cortex. E n d o g e n o u s testosterone was m e a s u r e d by radioimmunoassay in the h y p o t h a l a m u s and the cerebral cortex of the foetus and newborn rats during the first 6 h following birth. In the male, hypothalamic testosterone increased between 0 h in utero and 2 h; in the males gonadectomized at 0 h in utero and killed at the age of 2 h, the testosterone surge was abolished, clearly indicating the testicular origin of this h o r m o n e in the neonate. T h e small testosterone increase in the cerebral cortex compared with that in the hypothalamus reflects a preferential uptake of this h o r m o n e by the h y p o t h a l a m u s of the newborn. In the female, hypothalamic testosterone slightly decreased between 0 h in utero and 6 h. These results are in a g r e e m e n t with the view that hypothalamic modifications form the basis for some behavioral and physiological changes attributed to the effect of perinatal hormonal stimulation. Key words: Testosterone, Newborn rat, H y p o t h a l a m u s , Sexual differentation of the brain.
In the newborn male rat, the pituitary testicular axis is active at the time of birth. 6 A sudden increase in serum gonadotropins is followed by a transient elevation of serum testosterone level, between 0 and 2 h after birth. Evidence indicates that the masculinization of the central nervous system requires the presence of testicular androgens, probably testosterone, during the perinatal period,I° and at least part of this effect is probably exerted through action on the hypothalamus of the neonate. 3'15 In this case, hypothalamic tissue of the neonatal male should have a higher level of testosterone than hypothalamic tissue of the neonatal female. The main purpose of this study was to compare hypothalamic testosterone levels in male and female neonate rats within the 6 h following birth. The effects of perinatal gonadectomy at 0 h in utero on hypothalamic testosterone were also studied. MATERIALS AND METHODS Animals
Experiments were performed on laboratory bred Sherman rats. Animals were maintained on a 14:10 h light-dark cycle, with food and water ad libitum. Conditions of breeding and the timing of the pregnancies have been previously described. 5 The onset of gestation was defined as the estimated hour of ovulation (02.00 h on day 0), during the night of cohabitation. Parturition most often occurred during the day time of day 21 or 22 of gestation. Testosterone assays were performed on the following groups of" animals: Fetus 21 days 8 h: on day 21, between 09.00 and 11.00 h a.m., the mother was killed by cervical fracture and the fetuses were immediately removed from the uterine horns by caesarian section. Fetus 0 h in utero: at the moment of appearance of the first newborn, the mother was killed by cervical fracture and the fetuses were removed from the uterine horns. Newborns at 0 h ex utero: taken between 35 and 45 min after onset of parturition. Newborns at 2, 4 or 6 h after birth: the animals were left with their mothers until killed. Experimental procedure Dissection o f brain tissues. The fetuses and newborns of both sexes were killed by decapitation and the head immediately placed on ice. The entire hypothalamus was cut out as a block limited anteriorly by the optic chiasma, laterally by the hypothalamic fissures and posteriorly by the mammilary bodies; the block was approximately 2 mm deep from the basal surface of the hypothalamus.
187
| 88
J. Rhoda et al.
Cortical tissue was excised from parietal lobes of the brain. The tissues were freed from meninges and blotted with filter paper. For each tissue one determination involved a pool of 8-1(I pieces of tissue from several litters. All tissues were stored in 1 ml of distilled water at - 3 0 ° C until assay. G o n a d e c t o m y at 0 h in utero. Newborn male rats were gonadectomized or sham gonadectomized in the following manner: when the first newborn appeared, the mother was killed with a blow on the neck and the fetuses removed from the interne horns. Males were quickly placed in a freezer and gonadectomized or sham gonadectomized within 15 min after onset of delivery through a midline incision under cryoanesthesia. The incision was closed with a drop of collodion and the pups were rewarmed in a humidicrib at 34°C. They were killed by decapitation 2 h later and brain tissues were treated as above. H o r m o n e analyses. After decongelation, hypothalamic and cortical samples were homogenized by vigorously disrupting the tissues on a vortex mixer. Approximately 2500 dpm of tritiated testosterone (1,2,6,7[3H]testosterone, S.A. 109 Ci/mmol, A m e r s h a m , U . K . ) were added to the different tissue homogenates for recovery estimation. After a 30 rain incubation, at 37°C, the samples were extracted with twice 6 ml of diethyl ether. The pooled extracts were then dried under a stream of nitrogen and redissolved in 1 ml of 0.01 M sodium phosphate buffer p H 6.9 containing 0.1% gelatin, 0.1% sodium azide and 0.9% NaC1. This buffer will be referred to as PGSB. The extracts were stored at - 3 0 ° C until assay. In some cases, hypothalamic extracts were chromatographed on celite microcolumns according to Challis et al. 4 Testosterone was estimated in the extracts with a sensitive radioimmunoassay as previously described. 6 The antiserum (provided by M. Forest) used to measure testosterone was obtained by inoculating rabbits with testosterone 3carboxymethoxime conjugated with bovine serum albumin. The antibodies obtained exhibited a cross-reaction mainly with 17[3-hydroxy-5~-androstan 3-one (62.5%) and, to a lesser extent, with androstene 3[3.1713-diol (9.2%). Briefly, aliquots of the reconstituted extracts were incubated overnight at 4°C with 20,000 dpm of tritiated testosterone and antiserum in a final volume of 0.8 ml 0.01 M PGSB p H 6.9. Free from bound testosterone was separated by adding 1 ml of a cold solution of charcoal-dextran in PGSB (0.5% charcoal and 0.05% dextran T 70). After 20 min of incubation at 4°C, the samples were then centrifuged for 10 min at 4000 g in a refrigerated centrifuge. The radioactivity of the supernatant was counted in a LKB liquid scintillation spectrometer with 10 ml of scintillation liquid (toluene 1.5 1; triton X-100 0.75 1; scintimix 12.4 g). Using this procedure, water blanks were usually less than the assay sensitivity which was around 10 pg/tube. The intraassay and inter-assay coefficient of variation were, respectively, 9.7 and 12.6%. Statistics. Results were expressed as mean _+1 S.E.M. An Ftest was used for overall comparisons of the hormonal data and a t test for the intergroup differences. A probability of ~
