Maternal Growth Hormone and Growth and Function VICKI R. SARA L. LAZARUS Garvan Institute of Medical Research, St. Vincent S Hospital Sydney, N.S. W., Australia
Factors regulating somatic growth postnatally have been proposed to affect brain growth and subsequent function when applied prior to birth. This study pertains to the role of growth hormone administered to pregnant rats and the subsequent growth of the progeny. The results showed a significant increase in brain weight and cortical neurone number as determined by the incorporation of labelled thymidine into DNA and subsequent autoradiography. At maturity, learning performance on a series of conditional discrimination tasks was found to be enhanced.
Physiological theories of learning imply that performance is dependent upon cortical neurone interaction, a function of the number of neurones and the extent of their axo-dendritic processes (Sholl, 1956). Mathematical models of cortical organization predict that increments in neuronal interaction result in a mechanism whch processes a large amount of information rapidly (Cragg & Temperly, 1954; Sholl & Uttley, 1953) with a decrease in the probability of error (Winograd & Cowan, 1963). Learning ability varies with axo-dendritic development (Eayrs, 1961) but, although phylogenetic evidence is supportive, a similar relationship to neuronal number has not been investigated. Neuronal proliferation in most species including the rat (Berry & Rogers, 1965) and man (Dobbing & Sands, 1970) is complete prior to birth and, thus, alterations in neuronal number must occur in utero. We therefore proposed that growth-promoting factors during prenatal development would stimulate the proliferation of cortical neurones and thereby enhance learning ability. Winick (1971) clearly demonstrated that the effect of any growth factor is a function o f the time it is active during the sequence of growth. Consequently, factors regulating somatic growth postnatally should influence growth of the brain, particularly cortical neurone proliferation, when applied prior to birth. As pituitary growth hormone is one of the primary regulators of postnatal growth (Tanner,1972) and pharmacological doses during pregnancy stimulate brain growth (Zamenhof, 1941, 1942; Zamenhof, Mosley, & Schuller, 1966), its action prior to birth was investigated. The relation between dose o f hormone administered to pregnant rats and the response in progeny was examined.
Received for publication 11 July 1974 Revised for publication 20 December 1974 Developmental Psychobiology, 8 (6): 489-502 (1975) 0 1975 by John Wiley & Sons, Inc.
489
490
SARA AND LAZARUS
Method Wistar rats (Rattusnowegicus), obtained from Prince Henry Hospital Animal Breeding Station (Sydney, Australia), were maintained on a 12-hr dark/l2-hr light cycle, the light cycle beginning at 0600 hours. Females were 100 days old and weighed approximately 200 g prior to mating. Time of conception was determined by vaginal smears taken each morning and the date of finding sperm was called Day 1 of gestation. Pregnant rats were randomly allocated to control and experimental conditions and body weights recorded daily. Experimental rats were randomly allocated to 4 dose-level groups. Each pregnant female rat was injected subcutaneously with 10 pg, 100 pg, 1 mg, or 3 mg of porcine growth hormone dissolved in .5 ml of normal saline per day. Porcine growth hormone (pGH), with an activity of .6 International Unit per mg, was obtained in pure crystalline form from Sigma Chemical Co., U.S.A., and stored under dessication at 4°C. Control animals received the same volume of vehicle alone. Injections were given daily at 0900 hours from Day 7 to Day 20 of gestation, a period from implantation in the uterine wall and formation of the neural tube on Day 7 until labelling of cortical neuroblasts on Day 20 of gestation. Neuronal number in the cerebral cortex was specifically determined by the selective labelling procedure (see Brain Examination). On Day 20 of gestation, at 0900 hours, each pregnant rat was injected intraperitoneally with 500 pCi of tritiated thymidine (3H-TdR) dissolved in .5 ~nl of sterile saline. The 3H-TdR labelled on the methyl group, with a specific activity of 6.7 Ci/mmole, was obtained in sterile aqueous solution (1 mCi/ml) from the New England Nuclear Corporation, U.S.A. On Day 7 of postnatal life each litter was randomly split and reduced to 4-5 animals which were allowed to grow to maturity. (The remaining pups were sacrificed by decapitation and the brains examined.) On Day 28, the animals were weaned, weighed, and housed in wire mesh cages with usually 3 of the same sex per cage. Littermates were housed in separate cages. They were then left undisturbed with food and water ad libitum until Day 90 when they were again weighed and placed on a 23-hr food deprivation schedule. Animals were fed wet mash for 30 min daily in individual feeding boxes. During a 3-week adaption period animals were gentled by 30 min of daily free exploration and handling, and on Day 130 were tested on the 1st behavioral task. Testing on the 2nd task began on Day 150. In the initial stages of the experiment 3 litters died, one 3-mg and 2 Control litters. The final distribution of litters was: Control, 6; lO-pg, 3; 100-pg, 4; I-mg, 4; and 3-mg, 4.
Brain Examination Cortical neurone proliferation was determined by a selective labelling procedure. Administration of 3H-TdR at the time of neuroblast origin on day 20 of gestation produces a permanent and selective label of supragranular neurones. Site of label incorporation was determined by autoradiography. The brains of 75 rats were examined after formation of cortical laminations on Day 7. After weighmg, they were decapitated and the brains immediately removed minus olfactory lobes and cerebellum. Brains were weighed on a Stanton Unimatic single-pan balance accurate to lod5g. Brain removal and all subsequent steps were carried out at
ACTION OF MATERNAL GROWTH HORMONE
491
4°C to avoid degradation of the deoxyribonucleic acid (DNA). The DNA was extracted from individual brains by the method of Munro and Fleck (1966) with additional hot acid separation of DNA from protein by the method of Santen and Agranoff (1963). A maximum of 5 min elapsed between sacrifice and homogenization. From each DNA fraction .2-ml aliquots were taken and mixed with 10 ml Bray’s solution (675 ml Dioxan, 675 ml 2-methoxy ethanol, 9 g PPO, and 112.5 g napthalene [scintillation grade] in 900 ml toluene; chemicals obtained from May & Baker and Laboratory Supply, Sydney, Australia). Each sample was counted in a Nuclear Chicago Scintillation Spectrometer for 80 min. The 3H-beta emission was recorded between .001 and .007 MeV with an efficiency of 555 x lo3 c/min/pCi. Duplicate background measurements were determined by counting .2-ml aliquots of extracted “cold” DNA in I 0 ml of Bray’s solution at the beginning and end of each set of samples. Background count and error of the counling rate was found to be 53 k 1.6 cpm for 95% confidence limits. Quench corrections were made by external standardization and the total radioactivity in each sample was determined by correcting for volume. One brain from each litter was left intact and examined by autoradiography. Following removal, the brain was fixed in 10%neutral formalin and double-embedded in paraffin. Coronal sections were cut at 4-7 pm in thickness through the frontal cortex, hippocampus, and cerebellum. Following deparaffinization, slides were coated with Kodak NTB-3 nuclear bulk emulsion by the dripping technique in a dark room. The emulsion was diluted with distilled water (1: 1) and melted in a constant temperature water bath at 43°C. Slides were prewarmed to 43°C and dipped into a cylinder containing the emulsion. Excess emulsion was removed and, following drying, slides were stored in boxes containing a dessicant. The boxes were sealed and stored in a light- and air-tight container at 4°C for 4 months. Development was carried out by emersion in Kodak D 19-B developer at 18°C for 6 miii and washing in 1% acetic acid for 30 sec, Amfix high speed fixer for 5 min, tap water for 10 min, and distilled water for 1 0 min. Slides were stained with Cresyl violet and examined microscopically.
Behavioral Examination The subjects were 42 male and 31 female Wistar rats, 110 days old at the beginning of the evaluation. The apparatus used for both discrimination tasks was a single-unit T-maze with removable arms of varying brightness and an insertable floor of different textures. These stimuli served as discriminative cues. The stem and start box had neutral grey walls and floor so designed that a neutral grey, rough wire mesh floor could be inserted over the smooth aluminum floor. Each of the 4 removable arms had a goal box; 2 had black walls and floor, and 2 had white. The stem and boxes were covered with hinged, clear Perspex lids. Clear Perspex guillotine doors were located at the entry to each alley and box. When the door of the start box was raised, a microswitch was opened which started a Standard Electric Timer (10 msec resolution). Each arm was fitted to hold a light source and a photoelectric cell mounted on opposite sides of the alley at a distance of 1 3 cm from the goal box. Regardless of the arm the subject entered, interruption of the light beam stopped the timer. Food pellets, each weiglvng approximately 66 mg, were used as reinforcement and were placed in aluminum food cups attached to the end of each god box.
492
SARA AND LAZARUS
Experimental Procedure. All animals were run at 23-hr food deprivation. On edch trial for both tasks the rat was removed from an individual cage and placed in the start box. After the subject oriented towards the starting door, the door was raised. A noncorrection technique was employed. After entering the correct arm and eating the reinforcement (2 food pellets), the animal was removed. The animal was removed from the incorrect arni after a 15-sec delay, equivalent to the average time taken to consume the reward. When a rat entered a goal box, the door to that box was closed. Criterion of response choice was crossing the light beam and stopping the timer. Following removal from the apparatus the rat was returned to the cage. The intertrial interval was no less than 3 min. All subjects reccived 16 trials per day, consisting of 4 blocks of 4 trials each, with each block consisting of a free choice trial first and the remainder forced to equate number of reinforced and nonreinforced trials in each block. The stimulus configuration was the same for each block of 4 trials. On forced trials the arin was blockcd by closing the guillotine door at its entrance. The response on the free trial and latencies on all trials were recorded. The learning criterion was 1 0 correct responses on the free trial in 10 consecutive 4-trial blocks. Task 1. Both of the maze arms were either black or white on any given block of 4 trials. The schedule of presentation was randomized such that each day on half the trials both arms were black and on the other half they were white. The right arm contained the food reinforcement when both arms were black, and the left arm contained reinforcement when both arms were white. Brightness stimuli were presented simultaneously, one of the arms being Task 2. black and the other w h t e on any given trial. The spatial orientation of the arms altered according to a fixed schedule to eliminate position bias. The floor of the alley arm was made either rough or smooth by the insertion of the wire mesh floor. The trial schedule was randomi7ed such that the presentation of stimuli was balanced and without any fixed order. On half the trials, the black arm was on the left and on half it was on the right. Similarly, the alley floor was rough on half the trials. For example, 1 daily trial schedule was Black-Left-Rough; Black-Left-Smooth; Black-Right-Rough; Black-Right-Smooth. Four trials were given for each stimulus configuration, consisting of 1 free choice trial and 3 forced trials. The black arm contained the food reinforcement when the floor of the alley was rough and the white arm contained the reinforcement when the floor was smooth. The forced trial procedure was terminated after 75 4-trial blocks, i.e., 300 trials. The rat then received 25 additional free choice trials in daily 4-trial blocks providing a total of 100 free choice trials. Running was terminated at this stage due to limitations on the time for experimentation. If the animal did not reach criterion, a score of 100 was assigned and included for analysis.
Statistical Analysis Sexual differentitation wils not made at Day 7 of postnatal life and measurements obtained at this time were analyzed by a 1-way analysis of variance for unequal group sizes. Other variables were analyzed using a 2-way analysis of variance for unequal group sizes (Snedecor, 1938, p, 235). Following a significant F value, post-hoc comparisons between groups were performed according to the method of Scheffd (1959).
ACTION OF MATERNAL GROWTH HORMONE
*
"1
"1
*
*
**
493
*
*
340
300 v7
>
m
260 0,
220
Fig. 1. Mean body weight (8) taken at days 7, 28, and 90 of postnatal life. Error b a n represent __ standard deviation and * refers to a significant difference as compared to controls @J < .05). @ -pooled male and female; @-male; 0 -female.
Results Prenatal growth hormone administration produced no significant effect (p > .05) on weightgainduringgestation ( F = .69, df = 4/16), gestation period ( F = 2.04, df= 4/14), or litter size ( F = 1.49, df= 4/16). The body weights of offspring are given in Figure 1 . At Day 7, weights were recorded prior to sacrifice, whereas measurements on Day 28 and 90 were from animals examined behaviorally. A significant treatment effect was obtained at Day 7 (F = 25.71, df = 4/70, p < .Ol), Day 28 ( F = 12.62, df= 4/63, p < .Ol), and Day
494
SARA AND LAZARUS
90 ( F = 3.47, df = 4/63, p
Factors regulating somatic growth postnatally have been proposed to affect brain growth and subsequent function when applied prior to birth. This study pertains to the role of growth hormone administered to pregnant rats and the subsequent growth of the progeny. The results showed a significant increase in brain weight and cortical neurone number as determined by the incorporation of labelled thymidine into DNA and subsequent autoradiography. At maturity, learning performance on a series of conditional discrimination tasks was found to be enhanced.
Physiological theories of learning imply that performance is dependent upon cortical neurone interaction, a function of the number of neurones and the extent of their axo-dendritic processes (Sholl, 1956). Mathematical models of cortical organization predict that increments in neuronal interaction result in a mechanism whch processes a large amount of information rapidly (Cragg & Temperly, 1954; Sholl & Uttley, 1953) with a decrease in the probability of error (Winograd & Cowan, 1963). Learning ability varies with axo-dendritic development (Eayrs, 1961) but, although phylogenetic evidence is supportive, a similar relationship to neuronal number has not been investigated. Neuronal proliferation in most species including the rat (Berry & Rogers, 1965) and man (Dobbing & Sands, 1970) is complete prior to birth and, thus, alterations in neuronal number must occur in utero. We therefore proposed that growth-promoting factors during prenatal development would stimulate the proliferation of cortical neurones and thereby enhance learning ability. Winick (1971) clearly demonstrated that the effect of any growth factor is a function o f the time it is active during the sequence of growth. Consequently, factors regulating somatic growth postnatally should influence growth of the brain, particularly cortical neurone proliferation, when applied prior to birth. As pituitary growth hormone is one of the primary regulators of postnatal growth (Tanner,1972) and pharmacological doses during pregnancy stimulate brain growth (Zamenhof, 1941, 1942; Zamenhof, Mosley, & Schuller, 1966), its action prior to birth was investigated. The relation between dose o f hormone administered to pregnant rats and the response in progeny was examined.
Received for publication 11 July 1974 Revised for publication 20 December 1974 Developmental Psychobiology, 8 (6): 489-502 (1975) 0 1975 by John Wiley & Sons, Inc.
489
490
SARA AND LAZARUS
Method Wistar rats (Rattusnowegicus), obtained from Prince Henry Hospital Animal Breeding Station (Sydney, Australia), were maintained on a 12-hr dark/l2-hr light cycle, the light cycle beginning at 0600 hours. Females were 100 days old and weighed approximately 200 g prior to mating. Time of conception was determined by vaginal smears taken each morning and the date of finding sperm was called Day 1 of gestation. Pregnant rats were randomly allocated to control and experimental conditions and body weights recorded daily. Experimental rats were randomly allocated to 4 dose-level groups. Each pregnant female rat was injected subcutaneously with 10 pg, 100 pg, 1 mg, or 3 mg of porcine growth hormone dissolved in .5 ml of normal saline per day. Porcine growth hormone (pGH), with an activity of .6 International Unit per mg, was obtained in pure crystalline form from Sigma Chemical Co., U.S.A., and stored under dessication at 4°C. Control animals received the same volume of vehicle alone. Injections were given daily at 0900 hours from Day 7 to Day 20 of gestation, a period from implantation in the uterine wall and formation of the neural tube on Day 7 until labelling of cortical neuroblasts on Day 20 of gestation. Neuronal number in the cerebral cortex was specifically determined by the selective labelling procedure (see Brain Examination). On Day 20 of gestation, at 0900 hours, each pregnant rat was injected intraperitoneally with 500 pCi of tritiated thymidine (3H-TdR) dissolved in .5 ~nl of sterile saline. The 3H-TdR labelled on the methyl group, with a specific activity of 6.7 Ci/mmole, was obtained in sterile aqueous solution (1 mCi/ml) from the New England Nuclear Corporation, U.S.A. On Day 7 of postnatal life each litter was randomly split and reduced to 4-5 animals which were allowed to grow to maturity. (The remaining pups were sacrificed by decapitation and the brains examined.) On Day 28, the animals were weaned, weighed, and housed in wire mesh cages with usually 3 of the same sex per cage. Littermates were housed in separate cages. They were then left undisturbed with food and water ad libitum until Day 90 when they were again weighed and placed on a 23-hr food deprivation schedule. Animals were fed wet mash for 30 min daily in individual feeding boxes. During a 3-week adaption period animals were gentled by 30 min of daily free exploration and handling, and on Day 130 were tested on the 1st behavioral task. Testing on the 2nd task began on Day 150. In the initial stages of the experiment 3 litters died, one 3-mg and 2 Control litters. The final distribution of litters was: Control, 6; lO-pg, 3; 100-pg, 4; I-mg, 4; and 3-mg, 4.
Brain Examination Cortical neurone proliferation was determined by a selective labelling procedure. Administration of 3H-TdR at the time of neuroblast origin on day 20 of gestation produces a permanent and selective label of supragranular neurones. Site of label incorporation was determined by autoradiography. The brains of 75 rats were examined after formation of cortical laminations on Day 7. After weighmg, they were decapitated and the brains immediately removed minus olfactory lobes and cerebellum. Brains were weighed on a Stanton Unimatic single-pan balance accurate to lod5g. Brain removal and all subsequent steps were carried out at
ACTION OF MATERNAL GROWTH HORMONE
491
4°C to avoid degradation of the deoxyribonucleic acid (DNA). The DNA was extracted from individual brains by the method of Munro and Fleck (1966) with additional hot acid separation of DNA from protein by the method of Santen and Agranoff (1963). A maximum of 5 min elapsed between sacrifice and homogenization. From each DNA fraction .2-ml aliquots were taken and mixed with 10 ml Bray’s solution (675 ml Dioxan, 675 ml 2-methoxy ethanol, 9 g PPO, and 112.5 g napthalene [scintillation grade] in 900 ml toluene; chemicals obtained from May & Baker and Laboratory Supply, Sydney, Australia). Each sample was counted in a Nuclear Chicago Scintillation Spectrometer for 80 min. The 3H-beta emission was recorded between .001 and .007 MeV with an efficiency of 555 x lo3 c/min/pCi. Duplicate background measurements were determined by counting .2-ml aliquots of extracted “cold” DNA in I 0 ml of Bray’s solution at the beginning and end of each set of samples. Background count and error of the counling rate was found to be 53 k 1.6 cpm for 95% confidence limits. Quench corrections were made by external standardization and the total radioactivity in each sample was determined by correcting for volume. One brain from each litter was left intact and examined by autoradiography. Following removal, the brain was fixed in 10%neutral formalin and double-embedded in paraffin. Coronal sections were cut at 4-7 pm in thickness through the frontal cortex, hippocampus, and cerebellum. Following deparaffinization, slides were coated with Kodak NTB-3 nuclear bulk emulsion by the dripping technique in a dark room. The emulsion was diluted with distilled water (1: 1) and melted in a constant temperature water bath at 43°C. Slides were prewarmed to 43°C and dipped into a cylinder containing the emulsion. Excess emulsion was removed and, following drying, slides were stored in boxes containing a dessicant. The boxes were sealed and stored in a light- and air-tight container at 4°C for 4 months. Development was carried out by emersion in Kodak D 19-B developer at 18°C for 6 miii and washing in 1% acetic acid for 30 sec, Amfix high speed fixer for 5 min, tap water for 10 min, and distilled water for 1 0 min. Slides were stained with Cresyl violet and examined microscopically.
Behavioral Examination The subjects were 42 male and 31 female Wistar rats, 110 days old at the beginning of the evaluation. The apparatus used for both discrimination tasks was a single-unit T-maze with removable arms of varying brightness and an insertable floor of different textures. These stimuli served as discriminative cues. The stem and start box had neutral grey walls and floor so designed that a neutral grey, rough wire mesh floor could be inserted over the smooth aluminum floor. Each of the 4 removable arms had a goal box; 2 had black walls and floor, and 2 had white. The stem and boxes were covered with hinged, clear Perspex lids. Clear Perspex guillotine doors were located at the entry to each alley and box. When the door of the start box was raised, a microswitch was opened which started a Standard Electric Timer (10 msec resolution). Each arm was fitted to hold a light source and a photoelectric cell mounted on opposite sides of the alley at a distance of 1 3 cm from the goal box. Regardless of the arm the subject entered, interruption of the light beam stopped the timer. Food pellets, each weiglvng approximately 66 mg, were used as reinforcement and were placed in aluminum food cups attached to the end of each god box.
492
SARA AND LAZARUS
Experimental Procedure. All animals were run at 23-hr food deprivation. On edch trial for both tasks the rat was removed from an individual cage and placed in the start box. After the subject oriented towards the starting door, the door was raised. A noncorrection technique was employed. After entering the correct arm and eating the reinforcement (2 food pellets), the animal was removed. The animal was removed from the incorrect arni after a 15-sec delay, equivalent to the average time taken to consume the reward. When a rat entered a goal box, the door to that box was closed. Criterion of response choice was crossing the light beam and stopping the timer. Following removal from the apparatus the rat was returned to the cage. The intertrial interval was no less than 3 min. All subjects reccived 16 trials per day, consisting of 4 blocks of 4 trials each, with each block consisting of a free choice trial first and the remainder forced to equate number of reinforced and nonreinforced trials in each block. The stimulus configuration was the same for each block of 4 trials. On forced trials the arin was blockcd by closing the guillotine door at its entrance. The response on the free trial and latencies on all trials were recorded. The learning criterion was 1 0 correct responses on the free trial in 10 consecutive 4-trial blocks. Task 1. Both of the maze arms were either black or white on any given block of 4 trials. The schedule of presentation was randomized such that each day on half the trials both arms were black and on the other half they were white. The right arm contained the food reinforcement when both arms were black, and the left arm contained reinforcement when both arms were white. Brightness stimuli were presented simultaneously, one of the arms being Task 2. black and the other w h t e on any given trial. The spatial orientation of the arms altered according to a fixed schedule to eliminate position bias. The floor of the alley arm was made either rough or smooth by the insertion of the wire mesh floor. The trial schedule was randomi7ed such that the presentation of stimuli was balanced and without any fixed order. On half the trials, the black arm was on the left and on half it was on the right. Similarly, the alley floor was rough on half the trials. For example, 1 daily trial schedule was Black-Left-Rough; Black-Left-Smooth; Black-Right-Rough; Black-Right-Smooth. Four trials were given for each stimulus configuration, consisting of 1 free choice trial and 3 forced trials. The black arm contained the food reinforcement when the floor of the alley was rough and the white arm contained the reinforcement when the floor was smooth. The forced trial procedure was terminated after 75 4-trial blocks, i.e., 300 trials. The rat then received 25 additional free choice trials in daily 4-trial blocks providing a total of 100 free choice trials. Running was terminated at this stage due to limitations on the time for experimentation. If the animal did not reach criterion, a score of 100 was assigned and included for analysis.
Statistical Analysis Sexual differentitation wils not made at Day 7 of postnatal life and measurements obtained at this time were analyzed by a 1-way analysis of variance for unequal group sizes. Other variables were analyzed using a 2-way analysis of variance for unequal group sizes (Snedecor, 1938, p, 235). Following a significant F value, post-hoc comparisons between groups were performed according to the method of Scheffd (1959).
ACTION OF MATERNAL GROWTH HORMONE
*
"1
"1
*
*
**
493
*
*
340
300 v7
>
m
260 0,
220
Fig. 1. Mean body weight (8) taken at days 7, 28, and 90 of postnatal life. Error b a n represent __ standard deviation and * refers to a significant difference as compared to controls @J < .05). @ -pooled male and female; @-male; 0 -female.
Results Prenatal growth hormone administration produced no significant effect (p > .05) on weightgainduringgestation ( F = .69, df = 4/16), gestation period ( F = 2.04, df= 4/14), or litter size ( F = 1.49, df= 4/16). The body weights of offspring are given in Figure 1 . At Day 7, weights were recorded prior to sacrifice, whereas measurements on Day 28 and 90 were from animals examined behaviorally. A significant treatment effect was obtained at Day 7 (F = 25.71, df = 4/70, p < .Ol), Day 28 ( F = 12.62, df= 4/63, p < .Ol), and Day
494
SARA AND LAZARUS
90 ( F = 3.47, df = 4/63, p
