RETENTION OF PLASMA SOMATOMEDIN ACTIVITY IN THE
FOETAL RABBIT FOLLOWING DECAPITATION IN UTERO R. D. G. MILNER of Paediatrics, University of Sheffield Children's Hospital, Sheffield, S10 2TH
D. J. HILL, P. DAVIDSON
Department
AND
(Received 25 July 1978) SUMMARY
One rabbit foetus within each litter was decapitated in utero on day 24 of gestation. Plasma somatomedin activity and costal cartilage metabolism were studied 5 days later in the experimental foetuses and control litter-mates. Somatomedin was assayed by the uptake of [35S]sulphate in vitro into costal cartilage from intact foetuses. Uptake was proportional to logarithmic increases in the concentration of both foetal and maternal rabbit plasma. The mean (\m=+-\1 s.d.) somatomedin activity of four plasma pools, each pool being derived from the intact foetuses within each of four litters, was 1\m=.\3\m=+-\0\m=.\3compared with a potency of unity for the reference pool of maternal plasma. The plasma somatomedin activity of decapitated foetuses did not differ significantly from that of control litter-mates when analysed by rank test, but the costal cartilage of decapitated foetuses took up less [35S]\x=req-\ sulphate in basal medium when compared with that of intact litter-mates. The headless body weight of the decapitated foetuses did not rank in a position significantly different from the one expected. The concentration of plasma growth hormone in the decapitated foetuses was less than 5 ng/ml and that of the intact foetuses was more than 157 ng/ml. It is concluded that plasma somatomedin activity in the rabbit foetus is not dependent on foetal growth hormone. INTRODUCTION
After birth,
longitudinal
skeletal
growth
in mammals is
thought
to be mediated
by
the
somatomedins, which are largely dependent on growth hormone (Van Wyk & Underwood,
1975). The control of foetal skeletal growth may be different because in several species growth has been shown to progress normally after decapitation or hypophysectomy in utero (Jost, 1954; Eguchi, 1961; Chez, Hutchinson, Salazer & Mintz, 1970). Somatomedin activity has been detected in the circulation of the foetal pig, man and sheep (D'Ercole,
Foushee & Underwood, 1976; Gluckman & Brinsmead, 1976; Falconer, Forbes, Hart, Robinson & Thorburn, 1977) and thymidine uptake into human foetal chondrocytes is stimulated by somatomedin (Ashton & Francis, 1978); both facts suggested that somato¬ medin may be involved in foetal growth. Bone length and skeletal maturation are similar in decapitated and control rabbit foetuses (Jack & Milner, 1975). The purpose of the present study was to determine whether normal skeletal growth after foetal decapitation was dependent on somatomedin and growth hormone. Foetal somatomedin activity was assayed using foetal cartilage as this is the
appropriate physiological target.
MATERIALS AND METHODS
Assays Irvine, Ayr)
were
Chemicals performed using Waymouth's medium supplemented with sodium bicarbonate
MB752/1 (Flow Laboratories Ltd, (840 mg/1), penicillin (100 units/ml),
streptomycin (100 µg/ml) and JV-2-hydroxyethyl piperazine-iV'-ethane sulphonic acid (HEPES) buffer (4-76 mg/ml), and adjusted to pH 7-4 with NaOH. Medium was sterilized by passage through Millipore filters (0·45 µ ). [^SJSulphate was obtained from the Radiochemical Centre, Amersham, Bucks.
Animals Pregnant Dutch rabbits were obtained from the Joint Animal Breeding Unit, University of Nottingham, and were used for all studies. They were maintained on rabbit pellets (Glovers Animal Feeds Ltd, Sheffield) and water was given ad libitum. Foetal decapitation rabbits anaesthetized on day 24 of gestation by intravenous injection of 0-8 were Pregnant ml sodium pentobarbitone (Sagatal ; May & Baker, London) and uterine contractions were reduced to a minimum by supplying the rabbit with a mixture of halothane (Fluothane; ICI, Macclesfield) and air (3 litres/min) through a small face mask. One foetal rabbit within each litter was decapitated by the technique of Beam (1968). Following laparotomy, a foetus within the uterus was chosen at random and gently removed from the abdominal cavity. A small incision was made within a purse string suture applied to the uterine wall, taking care at this stage not to rupture the foetal membranes. The foetal head and surrounding membranes were then delivered through the incision and a catgut ligature was tightened around the neck to occlude the blood vessels and close the foetal membranes around the neck. Decapitation was performed above the ligature and the resulting loss in amniotic fluid was confined to the small quantity contained between the head and the portion of the foetal membranes removed with it. The headless foetus was returned to the uterus and the uterine and abdominal incisions were closed. On day 29 of gestation the doe was killed by an intravenous injection of Sagatal and all the foetuses were delivered by Caesarean section. Approximately 1 ml blood, taken by aortal puncture from each foetus, was placed in heparinized tubes and the plasma separated by centrifugation. Samples were stored at —20 °C until assay. Intact foetuses were decapitated and weighed as well as the experimentally decapitated foetus.
Somatomedin assay Plasma somatomedin activity was assayed by the uptake of [^SJsulphate into foetal rabbit costal cartilage in vitro. The method has been described previously by Hill, Francis & Milner (1977) for rat costal cartilage. Cartilage for the assay was derived from litters containing five or more intact foetuses, to obtain sufficient assay material. Consequently plasma samples from litters containing less than five intact foetuses (see Fig. 3) were stored and later assayed using cartilage from a litter of appropriate size. The rib cages were removed from five intact foetuses at delivery, and maintained in physiological saline until and during dissection. Cartilage was stored in closed dishes at 4 °C where possible before incubation, the time elapsing between the death of the doe and the start of incubation being approximately 2 h. Incorporation of isotope into cartilage was measured by liquid scintillation counting and the results are expressed as counts min-1 mg wet cartilage-1. Estimation of cartilage metabolism Sections of costal cartilage were prepared from each foetus within a litter as described above. Five sections from each individual foetus were added to each assay tube which contained 3 ml Waymouth's medium. After the addition of [^Sjsulphate (3 µ ) the tubes were incubated and the cartilage was treated as previously described.
Assay ofgrowth hormone Plasma growth hormone was measured by radioimmunoassay using reagents kindly donated by Dr A. F. Parlow through the rat pituitary hormone distribution programme of the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland, U.S.A. Rabbit growth hormone (AFP-684-C) was used as the standard and also as the tracer after labelling with 125iodine by the chloramine method (Greenwood, Hunter & Glover, 1963). The primary antibody was NIAMDD anti-rat growth hormone serum and a second antibody separation was employed using goat anti-human serum (Miles Laboratories Ltd, Stoke Poges, Slough). The procedure recommended by the NIAMDD was followed. Statistical analysis Assay results were evaluated by analysis of variance and acceptable assays were required to possess variance ratios showing significant regression ( 005). The 95% fiducial limits were calculated by Fieller's theorem according to Finney (1952). Comparison of parameters of growth involving the decapitated foetus and intact litter-mates could not be made between assays as the information was discontinuous and unsuitable for the use of direct parametric methods, the mean value for a litter being dependent on such factors as the state of nutrition. Such comparisons have been made by assigning each foetus a rank within the litter, the highest value ranking one. If the experimental procedure had no effect on the parameter being studied, it would be expected that the mean rank would not differ significantly from half the mean litter size. Using this hypothesis the differences between the observed and expected rank were com¬ pared by Student's paired f-test. RESULTS
Response offoetal cartilage to somatomedin An increased uptake of [^SJsulphate into the assay cartilage was observed with increasing concentrations of test plasma ; a logarithmic dose transformation resulted in a linear response in isotope uptake. The actual incorporation of isotope into cartilage from a particular litter varied greatly between litters which made direct comparison between assays difficult. To determine whether the assay was responding to plasma somatomedin activity the cartilage was exposed to increasing concentrations (2-5-10%) of pooled plasma from normal male rats, from six hypophysectomized rats and from three hypophysectomized rats given replacement injections of human growth hormone (Fig. 1). A decreased but parallel uptake of isotope was observed in the presence of plasma from hypophysectomized animals compared with the uptake in the presence of plasma from normal animals and hypo¬ physectomized rats treated with growth hormone. Exposure to increasing concentrations of rat growth hormone (5-500 ng/ml, NIAMDD-Rat GH-B5) failed to stimulate isotope uptake above the levels seen in medium alone. and maternal rabbit plasma Comparison was made between various pools of foetal plasma and one reference pool of maternal plasma. The foetal plasma for an assay was taken from a pool derived from the intact foetuses within a particular litter. The maternal plasma belonged to a pool taken from five animals when killed on day 29 of gestation. Because of the limited amount of foetal costal cartilage available in each assay individual maternal plasma samples could not be included within the assay at several doses, but the maternal pool plasma was included in every assay as a standard plasma. A parallel response in [35S]sulphate uptake was seen between foetal and maternal rabbit plasma (Fig. 2). A linear uptake of isotope occurred between plasma concentrations of 2·5
Response
to foetal
¡
6000,
4000
o
2000 u
JX "3 2-5 5
5
50
10 (rat plasma %) 500 (ng rat growth hormone/ml)
Fig. 1. Incorporation of [^SJsulphate into foetal rabbit cartilage in the presence of normal adult plasma (·), hypophysectomized rat plasma (A), plasma from hypophysectomized rats treated with growth hormone ( ) and rat growth hormone (o). Duplicate determinations are shown. rat
f
12 000-
(a)
)
8000 c o
o
a Ih O a
4000
CU
"3
L· 1-25 2-5 5
10 20
1-25 2-5 5
10 20
Plasma concentration (%) 2. Dose-response of foetal rabbit cartilage to pooled maternal rabbit plasma (broken lines) pooled foetal rabbit plasma taken from the intact foetuses within a litter (solid lines) in two assays. Duplicate determinations are shown and slopes were obtained by least squares analysis. Pooled foetal plasma in each assay was derived from a separate litter but both assays contained the same maternal plasma pool. Somatomedin potency for foetal plasma against the maternal plasma standard (a) 1-7 units/ml, (¿>) 0-9 units/ml.
Fig.
and
20% but linearity was sometimes lost at a concentration of 1-25% plasma (Fig. 2b). At 2-5% concentration the incorporation of [^Sjsulphate was usually two to three times greater than in the absence of plasma, and increased another two to five times between plasma concentrations of 2-5 and 10%. As insufficient foetal plasma was available to include and a
Table 1. Somatomedin potencies ofplasma pools from intact rabbit foetuses within four litters Potency* (units somatomedin/ml)
Index of precision
()
95% fiducial limits
A
1-7 0-9
C D Mean ± s.d.
1-3 1-3
0-18 0-36 019
2-4-1-3 1-1-0-7 1-6-1-0 2-0-0-9
Assay
0-39
1-3 ±0-3 *
Compared with standard of maternal plasma.
20% concentration assay point the maternal and foetal plasma were compared at concen¬ trations of 2-5, 5 and 10%. All six assays were found to be statistically valid but in two assays the index of precision ( ) exceeded 0-4 and these were discounted. Of the remaining four assays the mean potency ( ± s.d.) of the pools of foetal plasma was 1·3 ± 0·3 units somato¬ medin/ml (Table 1), similar to the maternal plasma potency of unity. A pool of foetal plasma taken from a rabbit not subjected to surgery possessed somatomedin activity of 1·0 unit/ml (seven foetuses, 95% fiducial limits 0-70-1-64). Four decapitated foetuses were required to provide sufficient plasma for assay at concen¬ trations of 2-5, 5 and 10%. A parallel dose-response to the maternal plasma standard was seen over this range of concentration. However, because of the impracticality of collecting adequate volumes of plasma from decapitated foetuses this rigorous test of potency was not repeated and a meaningful relative potency of plasma somatomedin activity in the decapi¬ tated foetus could not be calculated. a
Comparison ofplasma somatomedin from decapitated and intact foetuses To compare plasma somatomedin activity in the decapitated foetus and intact litter-mates,
plasma was collected from individual foetuses in a series of eight litters. Insufficient plasma could be taken from single foetuses for a potency estimation and therefore somatomedin activity was assessed by using a single 5% concentration of plasma which was in the centre of the linear dose-response. The incorporation of [^Sjsulphate in the presence of plasma from individual foetuses is shown in Fig. 3. There was considerable variation in the range of somatomedin activity between litters. This could have been due to differences in plasma somatomedin concentration or in the foetal cartilage used for bioassay. The variation in [35S]sulphate uptake by cartilage in response to the maternal plasma pool indicated that the latter explanation was partly responsible for the observed results. The range of foetal plasma somatomedin activities varied considerably from the maternal reference plasma pool indicating that there were important differences in foetal plasma somatomedin concentration between litters. There was no apparent relationship between litter size and the foetal/maternal plasma activity ratio. Within each litter the rank of plasma somatomedin activity of the decapitated foetus did not differ significantly from that expected (Table 2). Growth hormone assay Plasma growth hormone for five decapitated foetuses and for their intact litter-mates was estimated by radioimmunoassay. None of the decapitated foetuses possessed detectable growth hormone (> 5 ng/ml) whereas intact foetuses had levels of plasma growth hormone ranging from 157 to more than 400 ng/ml. Costal cartilage metabolism shows 4 the Figure incorporation of [^Sjsulphate into costal cartilage from individual foetuses during incubation in plasma-free medium. A large intralitter and interlitter variation was apparent but the mean observed rank of the decapitated foetus within the litter was
M
20 000
7
e
S
12000-
T _
o
S
4000
S
"3g, 3 4 5 6 7 Litter number Fig. 3. Incorporation of [^SJsulphate into foetal rabbit cartilage after incubation with plasma from individual rabbit foetuses (5% concentration) in eight litters. Each point represents the mean of duplicate determinations for plasma from decapitated foetuses (O) or from intact foetuses(e). Plasma samples from each litter were measured in a separate assay which included a 5% concentra¬ tion of pooled maternal plasma (lines). Litters one, three and eight each contained one foetus additional to the number represented in the Figure. Plasma samples from these foetuses were either inadequate for assay or accidentally destroyed. 12
L
Table 2. Mean ( ± s.d.) ranked positions of the decapitatedfoetus within the litters for plasma somatomedin activity (see Fig. 3), incorporation of [^S^ulphate into costal cartilage in plasma-free medium (see Fig. 4) and headless body weight Plasma somatomedin activity
Isotope uptake in cartilage
8
9
2-69 ±0-75
2-89 ±0-70
9 2-94 ±0-63
2-88 ±1-73
4-67 ±2-29*
3-67 ±2-00
Number of litters Mean expected position of decapitated foetus in litter Mean observed position of decapitated foetus in litter
Student's i-test for *
Mean
paired results.
expected position
v. mean
observed
Headless
body weight
position: P
FOETAL RABBIT FOLLOWING DECAPITATION IN UTERO R. D. G. MILNER of Paediatrics, University of Sheffield Children's Hospital, Sheffield, S10 2TH
D. J. HILL, P. DAVIDSON
Department
AND
(Received 25 July 1978) SUMMARY
One rabbit foetus within each litter was decapitated in utero on day 24 of gestation. Plasma somatomedin activity and costal cartilage metabolism were studied 5 days later in the experimental foetuses and control litter-mates. Somatomedin was assayed by the uptake of [35S]sulphate in vitro into costal cartilage from intact foetuses. Uptake was proportional to logarithmic increases in the concentration of both foetal and maternal rabbit plasma. The mean (\m=+-\1 s.d.) somatomedin activity of four plasma pools, each pool being derived from the intact foetuses within each of four litters, was 1\m=.\3\m=+-\0\m=.\3compared with a potency of unity for the reference pool of maternal plasma. The plasma somatomedin activity of decapitated foetuses did not differ significantly from that of control litter-mates when analysed by rank test, but the costal cartilage of decapitated foetuses took up less [35S]\x=req-\ sulphate in basal medium when compared with that of intact litter-mates. The headless body weight of the decapitated foetuses did not rank in a position significantly different from the one expected. The concentration of plasma growth hormone in the decapitated foetuses was less than 5 ng/ml and that of the intact foetuses was more than 157 ng/ml. It is concluded that plasma somatomedin activity in the rabbit foetus is not dependent on foetal growth hormone. INTRODUCTION
After birth,
longitudinal
skeletal
growth
in mammals is
thought
to be mediated
by
the
somatomedins, which are largely dependent on growth hormone (Van Wyk & Underwood,
1975). The control of foetal skeletal growth may be different because in several species growth has been shown to progress normally after decapitation or hypophysectomy in utero (Jost, 1954; Eguchi, 1961; Chez, Hutchinson, Salazer & Mintz, 1970). Somatomedin activity has been detected in the circulation of the foetal pig, man and sheep (D'Ercole,
Foushee & Underwood, 1976; Gluckman & Brinsmead, 1976; Falconer, Forbes, Hart, Robinson & Thorburn, 1977) and thymidine uptake into human foetal chondrocytes is stimulated by somatomedin (Ashton & Francis, 1978); both facts suggested that somato¬ medin may be involved in foetal growth. Bone length and skeletal maturation are similar in decapitated and control rabbit foetuses (Jack & Milner, 1975). The purpose of the present study was to determine whether normal skeletal growth after foetal decapitation was dependent on somatomedin and growth hormone. Foetal somatomedin activity was assayed using foetal cartilage as this is the
appropriate physiological target.
MATERIALS AND METHODS
Assays Irvine, Ayr)
were
Chemicals performed using Waymouth's medium supplemented with sodium bicarbonate
MB752/1 (Flow Laboratories Ltd, (840 mg/1), penicillin (100 units/ml),
streptomycin (100 µg/ml) and JV-2-hydroxyethyl piperazine-iV'-ethane sulphonic acid (HEPES) buffer (4-76 mg/ml), and adjusted to pH 7-4 with NaOH. Medium was sterilized by passage through Millipore filters (0·45 µ ). [^SJSulphate was obtained from the Radiochemical Centre, Amersham, Bucks.
Animals Pregnant Dutch rabbits were obtained from the Joint Animal Breeding Unit, University of Nottingham, and were used for all studies. They were maintained on rabbit pellets (Glovers Animal Feeds Ltd, Sheffield) and water was given ad libitum. Foetal decapitation rabbits anaesthetized on day 24 of gestation by intravenous injection of 0-8 were Pregnant ml sodium pentobarbitone (Sagatal ; May & Baker, London) and uterine contractions were reduced to a minimum by supplying the rabbit with a mixture of halothane (Fluothane; ICI, Macclesfield) and air (3 litres/min) through a small face mask. One foetal rabbit within each litter was decapitated by the technique of Beam (1968). Following laparotomy, a foetus within the uterus was chosen at random and gently removed from the abdominal cavity. A small incision was made within a purse string suture applied to the uterine wall, taking care at this stage not to rupture the foetal membranes. The foetal head and surrounding membranes were then delivered through the incision and a catgut ligature was tightened around the neck to occlude the blood vessels and close the foetal membranes around the neck. Decapitation was performed above the ligature and the resulting loss in amniotic fluid was confined to the small quantity contained between the head and the portion of the foetal membranes removed with it. The headless foetus was returned to the uterus and the uterine and abdominal incisions were closed. On day 29 of gestation the doe was killed by an intravenous injection of Sagatal and all the foetuses were delivered by Caesarean section. Approximately 1 ml blood, taken by aortal puncture from each foetus, was placed in heparinized tubes and the plasma separated by centrifugation. Samples were stored at —20 °C until assay. Intact foetuses were decapitated and weighed as well as the experimentally decapitated foetus.
Somatomedin assay Plasma somatomedin activity was assayed by the uptake of [^SJsulphate into foetal rabbit costal cartilage in vitro. The method has been described previously by Hill, Francis & Milner (1977) for rat costal cartilage. Cartilage for the assay was derived from litters containing five or more intact foetuses, to obtain sufficient assay material. Consequently plasma samples from litters containing less than five intact foetuses (see Fig. 3) were stored and later assayed using cartilage from a litter of appropriate size. The rib cages were removed from five intact foetuses at delivery, and maintained in physiological saline until and during dissection. Cartilage was stored in closed dishes at 4 °C where possible before incubation, the time elapsing between the death of the doe and the start of incubation being approximately 2 h. Incorporation of isotope into cartilage was measured by liquid scintillation counting and the results are expressed as counts min-1 mg wet cartilage-1. Estimation of cartilage metabolism Sections of costal cartilage were prepared from each foetus within a litter as described above. Five sections from each individual foetus were added to each assay tube which contained 3 ml Waymouth's medium. After the addition of [^Sjsulphate (3 µ ) the tubes were incubated and the cartilage was treated as previously described.
Assay ofgrowth hormone Plasma growth hormone was measured by radioimmunoassay using reagents kindly donated by Dr A. F. Parlow through the rat pituitary hormone distribution programme of the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland, U.S.A. Rabbit growth hormone (AFP-684-C) was used as the standard and also as the tracer after labelling with 125iodine by the chloramine method (Greenwood, Hunter & Glover, 1963). The primary antibody was NIAMDD anti-rat growth hormone serum and a second antibody separation was employed using goat anti-human serum (Miles Laboratories Ltd, Stoke Poges, Slough). The procedure recommended by the NIAMDD was followed. Statistical analysis Assay results were evaluated by analysis of variance and acceptable assays were required to possess variance ratios showing significant regression ( 005). The 95% fiducial limits were calculated by Fieller's theorem according to Finney (1952). Comparison of parameters of growth involving the decapitated foetus and intact litter-mates could not be made between assays as the information was discontinuous and unsuitable for the use of direct parametric methods, the mean value for a litter being dependent on such factors as the state of nutrition. Such comparisons have been made by assigning each foetus a rank within the litter, the highest value ranking one. If the experimental procedure had no effect on the parameter being studied, it would be expected that the mean rank would not differ significantly from half the mean litter size. Using this hypothesis the differences between the observed and expected rank were com¬ pared by Student's paired f-test. RESULTS
Response offoetal cartilage to somatomedin An increased uptake of [^SJsulphate into the assay cartilage was observed with increasing concentrations of test plasma ; a logarithmic dose transformation resulted in a linear response in isotope uptake. The actual incorporation of isotope into cartilage from a particular litter varied greatly between litters which made direct comparison between assays difficult. To determine whether the assay was responding to plasma somatomedin activity the cartilage was exposed to increasing concentrations (2-5-10%) of pooled plasma from normal male rats, from six hypophysectomized rats and from three hypophysectomized rats given replacement injections of human growth hormone (Fig. 1). A decreased but parallel uptake of isotope was observed in the presence of plasma from hypophysectomized animals compared with the uptake in the presence of plasma from normal animals and hypo¬ physectomized rats treated with growth hormone. Exposure to increasing concentrations of rat growth hormone (5-500 ng/ml, NIAMDD-Rat GH-B5) failed to stimulate isotope uptake above the levels seen in medium alone. and maternal rabbit plasma Comparison was made between various pools of foetal plasma and one reference pool of maternal plasma. The foetal plasma for an assay was taken from a pool derived from the intact foetuses within a particular litter. The maternal plasma belonged to a pool taken from five animals when killed on day 29 of gestation. Because of the limited amount of foetal costal cartilage available in each assay individual maternal plasma samples could not be included within the assay at several doses, but the maternal pool plasma was included in every assay as a standard plasma. A parallel response in [35S]sulphate uptake was seen between foetal and maternal rabbit plasma (Fig. 2). A linear uptake of isotope occurred between plasma concentrations of 2·5
Response
to foetal
¡
6000,
4000
o
2000 u
JX "3 2-5 5
5
50
10 (rat plasma %) 500 (ng rat growth hormone/ml)
Fig. 1. Incorporation of [^SJsulphate into foetal rabbit cartilage in the presence of normal adult plasma (·), hypophysectomized rat plasma (A), plasma from hypophysectomized rats treated with growth hormone ( ) and rat growth hormone (o). Duplicate determinations are shown. rat
f
12 000-
(a)
)
8000 c o
o
a Ih O a
4000
CU
"3
L· 1-25 2-5 5
10 20
1-25 2-5 5
10 20
Plasma concentration (%) 2. Dose-response of foetal rabbit cartilage to pooled maternal rabbit plasma (broken lines) pooled foetal rabbit plasma taken from the intact foetuses within a litter (solid lines) in two assays. Duplicate determinations are shown and slopes were obtained by least squares analysis. Pooled foetal plasma in each assay was derived from a separate litter but both assays contained the same maternal plasma pool. Somatomedin potency for foetal plasma against the maternal plasma standard (a) 1-7 units/ml, (¿>) 0-9 units/ml.
Fig.
and
20% but linearity was sometimes lost at a concentration of 1-25% plasma (Fig. 2b). At 2-5% concentration the incorporation of [^Sjsulphate was usually two to three times greater than in the absence of plasma, and increased another two to five times between plasma concentrations of 2-5 and 10%. As insufficient foetal plasma was available to include and a
Table 1. Somatomedin potencies ofplasma pools from intact rabbit foetuses within four litters Potency* (units somatomedin/ml)
Index of precision
()
95% fiducial limits
A
1-7 0-9
C D Mean ± s.d.
1-3 1-3
0-18 0-36 019
2-4-1-3 1-1-0-7 1-6-1-0 2-0-0-9
Assay
0-39
1-3 ±0-3 *
Compared with standard of maternal plasma.
20% concentration assay point the maternal and foetal plasma were compared at concen¬ trations of 2-5, 5 and 10%. All six assays were found to be statistically valid but in two assays the index of precision ( ) exceeded 0-4 and these were discounted. Of the remaining four assays the mean potency ( ± s.d.) of the pools of foetal plasma was 1·3 ± 0·3 units somato¬ medin/ml (Table 1), similar to the maternal plasma potency of unity. A pool of foetal plasma taken from a rabbit not subjected to surgery possessed somatomedin activity of 1·0 unit/ml (seven foetuses, 95% fiducial limits 0-70-1-64). Four decapitated foetuses were required to provide sufficient plasma for assay at concen¬ trations of 2-5, 5 and 10%. A parallel dose-response to the maternal plasma standard was seen over this range of concentration. However, because of the impracticality of collecting adequate volumes of plasma from decapitated foetuses this rigorous test of potency was not repeated and a meaningful relative potency of plasma somatomedin activity in the decapi¬ tated foetus could not be calculated. a
Comparison ofplasma somatomedin from decapitated and intact foetuses To compare plasma somatomedin activity in the decapitated foetus and intact litter-mates,
plasma was collected from individual foetuses in a series of eight litters. Insufficient plasma could be taken from single foetuses for a potency estimation and therefore somatomedin activity was assessed by using a single 5% concentration of plasma which was in the centre of the linear dose-response. The incorporation of [^Sjsulphate in the presence of plasma from individual foetuses is shown in Fig. 3. There was considerable variation in the range of somatomedin activity between litters. This could have been due to differences in plasma somatomedin concentration or in the foetal cartilage used for bioassay. The variation in [35S]sulphate uptake by cartilage in response to the maternal plasma pool indicated that the latter explanation was partly responsible for the observed results. The range of foetal plasma somatomedin activities varied considerably from the maternal reference plasma pool indicating that there were important differences in foetal plasma somatomedin concentration between litters. There was no apparent relationship between litter size and the foetal/maternal plasma activity ratio. Within each litter the rank of plasma somatomedin activity of the decapitated foetus did not differ significantly from that expected (Table 2). Growth hormone assay Plasma growth hormone for five decapitated foetuses and for their intact litter-mates was estimated by radioimmunoassay. None of the decapitated foetuses possessed detectable growth hormone (> 5 ng/ml) whereas intact foetuses had levels of plasma growth hormone ranging from 157 to more than 400 ng/ml. Costal cartilage metabolism shows 4 the Figure incorporation of [^Sjsulphate into costal cartilage from individual foetuses during incubation in plasma-free medium. A large intralitter and interlitter variation was apparent but the mean observed rank of the decapitated foetus within the litter was
M
20 000
7
e
S
12000-
T _
o
S
4000
S
"3g, 3 4 5 6 7 Litter number Fig. 3. Incorporation of [^SJsulphate into foetal rabbit cartilage after incubation with plasma from individual rabbit foetuses (5% concentration) in eight litters. Each point represents the mean of duplicate determinations for plasma from decapitated foetuses (O) or from intact foetuses(e). Plasma samples from each litter were measured in a separate assay which included a 5% concentra¬ tion of pooled maternal plasma (lines). Litters one, three and eight each contained one foetus additional to the number represented in the Figure. Plasma samples from these foetuses were either inadequate for assay or accidentally destroyed. 12
L
Table 2. Mean ( ± s.d.) ranked positions of the decapitatedfoetus within the litters for plasma somatomedin activity (see Fig. 3), incorporation of [^S^ulphate into costal cartilage in plasma-free medium (see Fig. 4) and headless body weight Plasma somatomedin activity
Isotope uptake in cartilage
8
9
2-69 ±0-75
2-89 ±0-70
9 2-94 ±0-63
2-88 ±1-73
4-67 ±2-29*
3-67 ±2-00
Number of litters Mean expected position of decapitated foetus in litter Mean observed position of decapitated foetus in litter
Student's i-test for *
Mean
paired results.
expected position
v. mean
observed
Headless
body weight
position: P
