PLASMA AND ADRENAL CORTISOL CONCENTRATIONS IN FOETAL, NEWBORN AND MOTHER GUINEA-PIGS DURING
THE PERINATAL PERIOD M. DALLE AND P. DELOST Laboratoire de Physiologie Animale et ERA CNRS d'Endocrinologie du Développement, Université de Clermont-Ferrand, Ensemble Scientifique des Cézeaux, B.P.45, 63170 Aubière, France
(Received 14 October 1975) SUMMARY
Concentrations of cortisol, corticosterone and cortisone in the plasma and adrenal glands, liver glycogen and plasma glucose of foetal, newborn and mother guinea-pigs were estimated during the last 6 days of pregnancy and throughout the first 24 h post partum. At the same time progesterone was measured in the plasma of the mother. During the prepartum rise in foetal plasma cortisol levels and liver glycogen, no significant change in the foetal adrenal cortisol content was observed. The plasma and adrenal cortisol concentrations of the mother were much higher than those observed in the foetus and increased significantly before parturition. In the mother as in the foetus, cortisone and corticosterone represent only a small percentage of corticosteroids compared with cortisol. These results indicate that the autonomous capacity of foetal adrenals, inhibited by maternal secretions before term, appears suddenly at birth.
INTRODUCTION
The very high concentrations of plasma cortisol associated with a decrease in adrenal cortisol levels observed in the newborn guinea-pig (Dalle & Delost, 1974) indicate that in this species the adrenals of the newborn are able to respond to the stress of parturition. The study of adrenal and plasma corticosteroids in the foetus and mother before birth may give further important contributions to this problem. It has been observed in cows (Comline, Hall, Lavelle, Nathanielsz & Silver, 1974) and ewes (Bassett & Thorburn, 1969; Drost, Kumaga'i & Guzman, 1973) that the rise in foetal plasma cortisol occurring during the last days of pregnancy is not associated with a similar increase in maternal plasma cortisol levels, which are always lower than those in the foetus. In these species an increase of foetal adrenal glucocorticoid synthesis appears just before parturition. The adrenal glands of the foetal rat are able to synthesize and release corticosteroids before birth and placental transfer of corticosterone from the foetus to the mother has been demonstrated (Dupouy, Coffigny & Magre, 1975). These findings have not been confirmed in all other species. In the rabbit during late pregnancy, plasma corticosteroid levels are higher in the mother than in the foetus (Mulay, Giannopoulos & Solomon, 1973); the rise in foetal plasma corticosteroids before parturition may be due to a transfer of these hormones from the mother to the foetus. It is difficult to draw conclusions from similar studies performed in guinea-pigs since investigations have not been carried out at precise times around parturition (Illingworth,
Challis, Ackland, Burton, Heap & Perry, 1974; Jones, 1974). Thus we have made a system¬
atic
study of the changes in the values of plasma and adrenal corticosteroids in the mother and foetus from 6 days prepartum to 24 h post partum. MATERIALS AND METHODS
Animals about 70 pregnant
were carried out on guinea-pigs and 180 male and female foetal and newborn guinea-pigs (Dunkin-Hartley strain). When the vagina was entirely open a female guinea-pig was caged with a male for 24 h. Thus, the time of fertilization was known to within + 12 h. The day of mating was designated day 0 of pregnancy. In our colony, parturition occurs at 68 days of pregnancy. Pregnant guinea-pigs were isolated 3 weeks before parturition in individual cages, in a quiet room. The animal room was entered only once a day when one pregnant guinea-pig or one mother and its offspring were
Experiments
sampled.
Pregnant guinea-pigs
by decapitation between
09.00 and 11.00 h on days 62, collected immediately by Caesarean section and decapitated. Newborn pups and mothers were decapitated at 0, 12 and 24 h after parturition. The blood was taken from the neck, centrifuged and deep frozen at 35 °C, as were the adrenals and liver. Samples were not pooled. The groups comprised ten mothers and 8-16 foetuses or newborn pups of each sex. For each age, animals were of the same weight and were kept in litters of 2-4 animals, if more than four pups were born both mother and offspring were excluded from the experiments. were
killed
64, 66 and 67 of pregnancy and their foetuses
were
—
Assays Plasma and adrenal cortisol, corticosterone and cortisone were measured in each animal according to the protein-binding procedure of Murphy (1967): hormones were extracted with chloroform and separated by paper chromatography in Bush (1952) system no. 5. They were eluted with methanol and separated into two parts. One part was used to deter¬ mine the percentage recovery of [4-14C]corticosteroids (New England Nuclear Corporation) added to the glands and plasma extracts; in our hands the percentage recovery was about 80%. The other part was measured by competition against a protein-binding solution of 2-5% de-steroided dog plasma bound to [l,2-3H]cortisol (New England Nuclear Corpora¬ tion). Plasma (0-5 ml) and both homogenized adrenals of each animal were assayed separ¬ ately and in duplicate. The sensitivity of the method was 0-5 ng for cortisol and 1 ng for cortisone and corti¬ costerone. The precision was 10 % between 2 and 15ng for the three hormones. Progesterone was also measured in the same individual plasma samples from the pregnant mothers. Before extraction of the other hormones with chloroform, plasma was treated with 0-1 MNaOH to destroy the high affinity progesterone-binding protein, and progesterone was extracted with light petroleum (b.p. 40-60 °C). The hormone was estimated by a proteinbinding procedure (Attal & Engels, 1971). In each animal, the plasma glucose and liver glycogen were estimated by colorimetry according to Trinder (1969) and Darlympe &Hamm (1973) respectively. Levels are expressed as /ig/100 ml plasma and as /¿g/100 mg fresh weight of adrenals for steroids, as mg/100 ml plasma for glucose and as mg/g wet weight of liver for glycogen. Means ± s.e.m. are given; probability and significance were calculated by Student's /-test.
RESULTS
Changes in adrenal weight In foetuses and newborn guinea-pigs of both sexes the absolute adrenal weight did not increase significantly between 6 days prepartum and 24 h post partum. The relative adrenal weight (mg/100 g body wt) remained stable from day 62 of pregnancy to parturition. Then, it rose between 0 and 24 h by 20 % (P < 0-001) in both sexes, due to a decrease in body weight. A sharp increase in absolute adrenal weight (45 %, < 0-001) and relative adrenal weight (50 %, < 0-001) in pregnant guinea-pigs occurred between day 62 of pregnancy and parturition.
Changes in the plasma concentration of cortisol (Fig. 1), cortisone and corticosterone In the foetus, plasma cortisol concentration increased progressively by 770 % (P < 0-001) in the male and 580 % (P < 0-001) in the female between days 62 and 67 with little further variation between day 67 and 24h after birth. No sexual dimorphism was observed. In the mother, plasma cortisol levels were already very high at 62 days (274 ±30/ig/100 ml) and increased between days 62 and 67 by 86 % (P < 001) and then decreased quickly after birth by 47 % (P < 0001) between day 67 and 24 h after birth. The plasma cortisol ratio of mother : foetus or newborn pup decreased regularly in the perinatal period: 19 on day 62, 7-5 on day 64, 5 on day 66, 4-6 on day 67, 3-5 at 0 h and 2-8 at 24 h. Plasma corticosterone concentration increased very little from day 62 to birth in male and female foetuses; values remained low (between 2 and 12/ig/100 ml) and represented only 20 and 10 % of the plasma cortisol levels on day 62 and at 0 h respectively. During the same period, the plasma cortisone concentration varied very little (about 10/ig/100 ml). During the last 6 days of pregnancy, there was a parallelism between the changes in corticosterone and cortisone and those in cortisol in the mothers ; values of corticosterone and cortisone were only 4 and 10 % that of cortisol. Changes in the concentrations of cortisol (Fig. 2), cortisone and corticosterone in the adrenals
In the foetus the relative concentrations of cortisol did not change between days 62 and 67 (about 0-60/ig/lOO mg). At the time of birth, the concentration increased sharply by 178 % in the male (P < 0-001) and 93 % in the female (P < 0-001). A progressive significant increase (400 %,P< 0-001) in the maternal adrenal cortisol concentration occurred between days 62 and 67, followed by a decrease after parturition. In foetal and neonatal guineapigs, the relative concentrations of corticosterone were parallel to that of adrenal cortisol and represented respectively 20 and 50 % of cortisol values during gestation and at birth. At the same time the relative concentration of cortisone remained stable (about 0-20 µg/ 100 mg). In the mother the relative concentration of corticosterone and cortisone varied very little throughout the perinatal period (about 0-07/ig/100 mg for corticosterone and 0-03 /ig/100 mg for cortisone).
Changes in the concentrations of liver glycogen and plasma glucose (Fig. 3) Three phases can be seen in the changes in liver glycogen content during the perinatal period in the foetus and newborn guinea-pigs of both sexes: (i) a regular increase by 187 % (P < 0-001) in the male and 193 % (P < 0-001) in the female between days 62 and 67; (ii) a marked rise by 35 % (P < 0-001) in the male and 53 % (P < 0001) in the female between day 67 and birth and (iii) a decrease of 80 % in both sexes (P < 0-001) from 0 to 24 h after birth. Maternal liver glycogen remained stable from 62 to 67 days, then a significant
12
24 Hours
Fig. 1. Maternal plasma progesterone and cortisol levels and foetal plasma cortisol levels in the perinatal period in the guinea-pig. Mother: A, progesterone; V, cortisol. Foetuses: O, male;
·, female. The vertical lines indicate
+
s.e.m.,
=
8-12.
0-80 -
2 0-60 ¡= 0-40 -
12 24 Hours
Fig. 2. Maternal and foetal adrenal cortisol levels in the perinatal period in the guinea-pig. Mother: . Foetuses: O, male; ·. female. The vertical lines indicate + s.e.m., 8-16. =
decrease (47 %, < 0-001) occurred between day 67 and parturition. Foetal plasma glucose lower than 100 mg/100 ml between days 62 and 67. It increased between day 67 and 0 h (96 % in the male, < 0-001 ; 53 % in the female, < 0001), then decreased quickly by 50 % (P < 0-001) in both sexes between 0 and 24 h after birth. During the perinatal period, maternal glucose was stable (125 mg/100 ml). was
Changes in maternal plasma concentrations of progesterone (Fig. 1) plasma progesterone concentrations remained very high until the last day of pregnancy; values were about 17 pg/100 ml between days 62 and 66 and increased by 52 % at day 67 (P > 0-05). A fall of 72 % (P < 0-001) was observed between day 67 and parturition. Plasma levels of progesterone reached 1 /ig/100 ml 24 h after birth. Maternal
o
150
Fig. 3. Plasma glucose concentrations (a) and liver glycogen content (b) in mothers and foetuses of guinea-pigs in the perinatal period. Mother: A. Foetuses: O, male; ·. female. The vertical 8-16. lines indicate + s.e.m., =
DISCUSSION
An
analysis of our results adds to the knowledge about the adrenal capacity of the guineapig to secrete corticosteroids in the foetus just before parturition and in the newborn pup. An increase in foetal adrenal capacity might be indicated by several observations during late pregnancy: (i) the rise of foetal plasma cortisol concentration, (ii) the decrease of plasma cortisol ratio mother:foetus, and (iii) the increase in liver glycogen. This increase in foetal liver glycogen during the last days of pregnancy confirms results already reported in foetal guinea-pigs and other mammals (Shelley, 1961). It is known that the presence of an intact pituitary-adrenal axis is necessary for the appearance of raised liver glycogen levels in late pregnancy (Jost & Jacquot, 1955). There is also a rise in aldosterone concentrations in foetal guinea-pig plasma and adrenals before birth (Giry & Delost, 1974). In addition,
glands of foetal guinea-pigs are capable of responding to adrenocorticotrophin early as 60 days of pregnancy (Donovan & Peddie, 1974). All these observations suggest that the hypothalamo-hypophysial-adrenal axis may be active before birth in the foetal guinea-pig as in the foetal rat (Dupouy, 1971; Dupouy et al. 1975). Nevertheless several observations are not consistent with this hypothesis: indeed foetal adrenal weight and foetal adrenal cortisol concentrations do not increase between days 62 and 67 of pregnancy adrenal as
and it is unlikely that there is any increase in the rate of cortisol secretion from the gland. The rise in foetal plasma cortisol levels might be due either to transfer from the mother, or to an increase in foetal corticosteroid-binding globulin (CBG) levels, which would increase plasma cortisol levels in the absence of placental transfer from the mother or increased secretion from the foetal adrenal. Seal 8c Doe (1967) have shown the CBG levels in the foetal guinea-pig near term are around eight times higher than those of the non-pregnant adult and are sufficient to account for the high foetal plasma cortisol levels. In the species looked at so far, the guinea-pig is the only one with such high foetal CBG levels. Plasma free cortisol levels decrease in the mother and increase in the foetus in the last part of pregnancy (Jones, 1974). This may suggest a placental transfer of cortisol from the mother to the foetus and in part would also explain the rise in foetal plasma cortisol during the same period. The significant increase of maternal adrenal weight and adrenal and plasma cortisol concentrations, which are higher than those of the foetus, suggest that there is marked adrenal stimulation of the mother before parturition. The very high maternal plasma cortisol concentrations are in accordance with the high binding capacity of CBG observed in late pregnancy in the mother guinea-pig (Diamond, Rust & Westphal, 1969). In the guinea-pig, foetal adrenal activity may be partly inhibited by maternal steroid secretion during the last 6 days of pregnancy. Nevertheless plasma corticosterone and cortisone levels remained constant up to the time of birth, whereas in the mother the plasma levels of these two hormones rose progressively at this time. Assuming all three cortico¬ steroids cross the placenta at similar rates, if the increasing cortisol levels of the foetus were due to transfer from the mother then one would expect an increase in foetal levels of corticosterone and cortisone to occur, which is not the case. Maternal adrenalectomy might answer this question. The autonomous capacity of the adrenals of the newborn pup clearly appears at birth, since plasma cortisol concentrations remain high in the newborn guinea-pig while they decrease sharply in the mother after parturition, and adrenal cortisol and corticosterone, and liver glycogen rise in the last 24 h of intrauterine life. The sustained high plasma cortisol levels in newborn pups could also be due to a lower capacity of the neonatal liver to metabolize corticosteroids transferred from the mother before birth (Schapiro, Percin & Kotichas, 1971). The increase of plasma glucose in the newborn guinea-pig, probably due to hypoxia (Hard, Reynolds & Winbury, 1944; Shelley & Neligan, 1966), might also be related to the high cortisol levels and to the important increase in adrenal adrenaline observed at 0 h in the same animals (Girvès & Delost, 1975). Nevertheless, it is difficult to resolve the problem of adrenal capacity in the foetal and newborn guinea-pig throughout the perinatal period without knowing the placental transfer, metabolic clearance and binding capacity for cortisol. We are at present studying these factors. Lastly, regarding the influence of foetal corticosteroids in the induction of parturition, well known in sheep and cattle (see Jöchle, 1973), our results suggest that foetal corticosteroids cannot play the same role in the guinea-pig, as demonstrated by Donovan & Peddie (1974). Indeed, plasma cortisol concentrations are much lower in the foetus than in the mother in late pregnancy, confirming the observations made by Illingworth et al. (1974). Furthermore, the very high maternal plasma progesterone levels observed at day 67 indicate that there is probably a
sharp drop in plasma progesterone levels in the last 12 h before parturition. The levels found at 0 h were low compared with day 67, and bearing in mind the longer half-life of progesterone in late pregnancy (Illingworth, Heap & Perry, 1970) it seems likely that a sharp decrease in secretion rate must occur some hours before parturition. We wish to thank the Laboratory of I.N.R.A. (Clermont-Ferrand, France) for its advice on the estimation of the plasma glucose and the liver glycogen.
helpful
REFERENCES J. A. A (1971). simple technique for the assay of progesterone in pregnancy. Journal of Attal, Engels, Endocrinology 50, 703-704. Bassett, J. M. & Thorburn, G D. (1969). Foetal plasma corticosteroids and the initiation of parturition in sheep. Journal of Endocrinology 44, 285-286. Bush, I. E. (1952). Methods of paper chromatography of steroids applicable to the study of steroids in mammalian blood and tissue. Biochemical Journal SO, 370-375. Comline, R. S., Hall, L. W., Lavelle, R. B., Nathanielsz, P. W. & Silver, M. (1974). Parturition in cow: endocrine changes in animals with chronically implanted catheters in the foetal and maternal circulations. Journal of Endocrinology 63, 451-472. Dalle, M. & Delost, P. (1974). Changes in the concentrations of cortisol and corticosterone in the plasma and adrenal glands of the guinea-pig from birth to weaning. Journal of Endocrinology 63, 483-488. Darlympe, R. H. & Hamm, R. (1973). A method for the extraction of glycogen and metabolites from a single muscle sample. Journal of Food Technology 8, 439-444. Diamond, M., Rust, N. & Westphal, U. (1969). High affinity binding of progesterone, testosterone and cortisol in normal and androgen treated guinea-pigs during various reproductive stages: relationship to masculinisation. Endocrinology 84, 1129-1133. Donovan, B. T. & Peddie, M. J. (1974). Adrenal function, oestrogen and the control of parturition in the guinea-pig. Progress in Brain Research 41, 281-288. Drost, M., Kumagaï, L. F. & Guzman, M. (1973). Sequential foetal-maternal plasma cortisol levels in sheep. Journal of Endocrinology 56, 483-492. Dupouy, J. P. (1971). Réponse du complexe hypothalamo-hypophysaire du foetus de rat à un blocage de la biosynthèse de corticostéroïdes par la metopirone. Influence du cortisol. Comptes Rendus Hebdoma¬ daires des Séances de Académie des Sciences 273, 962-965. Dupouy, J. P., Coffigny, H. & Magre, S. (1975). Maternal and foetal corticosterone levels during pregnancy in rats. Journal of Endocrinology 65, 347-352. Girvès, . & Delost, P. (1975). Les catecholamines surrénaliennes chez la mère, le foetus et le nouveau-né de Cobaye. Journal de Physiologie 71, 286A-287A. Giry, J. & Delost, P. (1974). Evolution de l'aldostéronémie du foetus et du nouveau-né de Cobaye au cours de la période périnatale. Comptes Rendus Hebdomadaires des Séances de l'Académie des Sciences 279,
J. &
2087-2090.
Hard, W. L., Reynolds, O. E. & Winbury, M. (1944). Carbohydrate, fat and moisture relationships in the pregnant, foetal and newborn guinea-pig. Journal of Experimental Zoology 96, 189-199. Illingworth, D. V, Challis, J. R. G, Ackland, N., Burton, A. M., Heap, R. B. & Perry, J. S. (1974). Parturition in the guinea-pig; plasma levels of steroid hormones, steroid-binding proteins and oxytocin, and the effect of corticosteroids, prostaglandins and adrenocorticotrophin. Journal of Endocrinology 63, 557-570.
Illingworth, D. V., Heap, R. B. & Perry, J. S. (1970). Changes in the metabolic clearance rate of progesterone in the guinea-pig. Journal of Endocrinology 48, 409—417. Jöchle, W. (1973). Corticosteroid induced parturition in domestic animals. Annual Review of Pharmacology 13, 33-35. Jones, C. T. (1974). Corticosteroid concentrations in the plasma of fetal and maternal guinea-pig during gestation. Endocrinology 95, 1129-1133. Jost, A. & Jacquot, R. (1955). Recherche sur les facteurs endocriniens de la charge en glycogène du foie foetal chez le lapin (avec des indications sur le glycogène placentaire). Annales d'Endocrinologie 16, 849-872.
Mulay, S., Giannopoulos, G. & Solomon, S. (1973). Corticosteroid levels in the mother and fetus of the rabbit during gestation. Endocrinology 93, 1342-1348. Murphy, B. E. P. (1967). Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurements of various steroids in body fluids by competitive protein-binding radioassay. Journal of Clinical Endocrinology and Metabolism 27, 973-990. Schapiro, S., Percin, C. S. & Kotichas, F. J. (1971). Half life of plasma corticosterone during development. Endocrinology 89, 284-286.
U. S. & Doe, R. P. (1967). The role of corticosteroid-binding globulin in mammalian pregnancy. In Proceedings of the Second International Congress on Hormonal Steroids, Milan, May 1966. I.C.S. No. 132,
Seal,
pp. 697-706. Eds L. Martini, F. Fraschini & M. Motta.'Amsterdam: Excerpta Medica Foundation. Shelley, H. J. (1961). Glycogen reserves and their changes at birth and in anoxia. British Medical Bulletin 17, 137-143. Shelley, H. J. & Neligan, G. A. (1966). Neonatal hypoglycaemia. British Medical Bulletin 22, 34-39. Trinder, H. J. (1969). Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry 6, 24-27.
THE PERINATAL PERIOD M. DALLE AND P. DELOST Laboratoire de Physiologie Animale et ERA CNRS d'Endocrinologie du Développement, Université de Clermont-Ferrand, Ensemble Scientifique des Cézeaux, B.P.45, 63170 Aubière, France
(Received 14 October 1975) SUMMARY
Concentrations of cortisol, corticosterone and cortisone in the plasma and adrenal glands, liver glycogen and plasma glucose of foetal, newborn and mother guinea-pigs were estimated during the last 6 days of pregnancy and throughout the first 24 h post partum. At the same time progesterone was measured in the plasma of the mother. During the prepartum rise in foetal plasma cortisol levels and liver glycogen, no significant change in the foetal adrenal cortisol content was observed. The plasma and adrenal cortisol concentrations of the mother were much higher than those observed in the foetus and increased significantly before parturition. In the mother as in the foetus, cortisone and corticosterone represent only a small percentage of corticosteroids compared with cortisol. These results indicate that the autonomous capacity of foetal adrenals, inhibited by maternal secretions before term, appears suddenly at birth.
INTRODUCTION
The very high concentrations of plasma cortisol associated with a decrease in adrenal cortisol levels observed in the newborn guinea-pig (Dalle & Delost, 1974) indicate that in this species the adrenals of the newborn are able to respond to the stress of parturition. The study of adrenal and plasma corticosteroids in the foetus and mother before birth may give further important contributions to this problem. It has been observed in cows (Comline, Hall, Lavelle, Nathanielsz & Silver, 1974) and ewes (Bassett & Thorburn, 1969; Drost, Kumaga'i & Guzman, 1973) that the rise in foetal plasma cortisol occurring during the last days of pregnancy is not associated with a similar increase in maternal plasma cortisol levels, which are always lower than those in the foetus. In these species an increase of foetal adrenal glucocorticoid synthesis appears just before parturition. The adrenal glands of the foetal rat are able to synthesize and release corticosteroids before birth and placental transfer of corticosterone from the foetus to the mother has been demonstrated (Dupouy, Coffigny & Magre, 1975). These findings have not been confirmed in all other species. In the rabbit during late pregnancy, plasma corticosteroid levels are higher in the mother than in the foetus (Mulay, Giannopoulos & Solomon, 1973); the rise in foetal plasma corticosteroids before parturition may be due to a transfer of these hormones from the mother to the foetus. It is difficult to draw conclusions from similar studies performed in guinea-pigs since investigations have not been carried out at precise times around parturition (Illingworth,
Challis, Ackland, Burton, Heap & Perry, 1974; Jones, 1974). Thus we have made a system¬
atic
study of the changes in the values of plasma and adrenal corticosteroids in the mother and foetus from 6 days prepartum to 24 h post partum. MATERIALS AND METHODS
Animals about 70 pregnant
were carried out on guinea-pigs and 180 male and female foetal and newborn guinea-pigs (Dunkin-Hartley strain). When the vagina was entirely open a female guinea-pig was caged with a male for 24 h. Thus, the time of fertilization was known to within + 12 h. The day of mating was designated day 0 of pregnancy. In our colony, parturition occurs at 68 days of pregnancy. Pregnant guinea-pigs were isolated 3 weeks before parturition in individual cages, in a quiet room. The animal room was entered only once a day when one pregnant guinea-pig or one mother and its offspring were
Experiments
sampled.
Pregnant guinea-pigs
by decapitation between
09.00 and 11.00 h on days 62, collected immediately by Caesarean section and decapitated. Newborn pups and mothers were decapitated at 0, 12 and 24 h after parturition. The blood was taken from the neck, centrifuged and deep frozen at 35 °C, as were the adrenals and liver. Samples were not pooled. The groups comprised ten mothers and 8-16 foetuses or newborn pups of each sex. For each age, animals were of the same weight and were kept in litters of 2-4 animals, if more than four pups were born both mother and offspring were excluded from the experiments. were
killed
64, 66 and 67 of pregnancy and their foetuses
were
—
Assays Plasma and adrenal cortisol, corticosterone and cortisone were measured in each animal according to the protein-binding procedure of Murphy (1967): hormones were extracted with chloroform and separated by paper chromatography in Bush (1952) system no. 5. They were eluted with methanol and separated into two parts. One part was used to deter¬ mine the percentage recovery of [4-14C]corticosteroids (New England Nuclear Corporation) added to the glands and plasma extracts; in our hands the percentage recovery was about 80%. The other part was measured by competition against a protein-binding solution of 2-5% de-steroided dog plasma bound to [l,2-3H]cortisol (New England Nuclear Corpora¬ tion). Plasma (0-5 ml) and both homogenized adrenals of each animal were assayed separ¬ ately and in duplicate. The sensitivity of the method was 0-5 ng for cortisol and 1 ng for cortisone and corti¬ costerone. The precision was 10 % between 2 and 15ng for the three hormones. Progesterone was also measured in the same individual plasma samples from the pregnant mothers. Before extraction of the other hormones with chloroform, plasma was treated with 0-1 MNaOH to destroy the high affinity progesterone-binding protein, and progesterone was extracted with light petroleum (b.p. 40-60 °C). The hormone was estimated by a proteinbinding procedure (Attal & Engels, 1971). In each animal, the plasma glucose and liver glycogen were estimated by colorimetry according to Trinder (1969) and Darlympe &Hamm (1973) respectively. Levels are expressed as /ig/100 ml plasma and as /¿g/100 mg fresh weight of adrenals for steroids, as mg/100 ml plasma for glucose and as mg/g wet weight of liver for glycogen. Means ± s.e.m. are given; probability and significance were calculated by Student's /-test.
RESULTS
Changes in adrenal weight In foetuses and newborn guinea-pigs of both sexes the absolute adrenal weight did not increase significantly between 6 days prepartum and 24 h post partum. The relative adrenal weight (mg/100 g body wt) remained stable from day 62 of pregnancy to parturition. Then, it rose between 0 and 24 h by 20 % (P < 0-001) in both sexes, due to a decrease in body weight. A sharp increase in absolute adrenal weight (45 %, < 0-001) and relative adrenal weight (50 %, < 0-001) in pregnant guinea-pigs occurred between day 62 of pregnancy and parturition.
Changes in the plasma concentration of cortisol (Fig. 1), cortisone and corticosterone In the foetus, plasma cortisol concentration increased progressively by 770 % (P < 0-001) in the male and 580 % (P < 0-001) in the female between days 62 and 67 with little further variation between day 67 and 24h after birth. No sexual dimorphism was observed. In the mother, plasma cortisol levels were already very high at 62 days (274 ±30/ig/100 ml) and increased between days 62 and 67 by 86 % (P < 001) and then decreased quickly after birth by 47 % (P < 0001) between day 67 and 24 h after birth. The plasma cortisol ratio of mother : foetus or newborn pup decreased regularly in the perinatal period: 19 on day 62, 7-5 on day 64, 5 on day 66, 4-6 on day 67, 3-5 at 0 h and 2-8 at 24 h. Plasma corticosterone concentration increased very little from day 62 to birth in male and female foetuses; values remained low (between 2 and 12/ig/100 ml) and represented only 20 and 10 % of the plasma cortisol levels on day 62 and at 0 h respectively. During the same period, the plasma cortisone concentration varied very little (about 10/ig/100 ml). During the last 6 days of pregnancy, there was a parallelism between the changes in corticosterone and cortisone and those in cortisol in the mothers ; values of corticosterone and cortisone were only 4 and 10 % that of cortisol. Changes in the concentrations of cortisol (Fig. 2), cortisone and corticosterone in the adrenals
In the foetus the relative concentrations of cortisol did not change between days 62 and 67 (about 0-60/ig/lOO mg). At the time of birth, the concentration increased sharply by 178 % in the male (P < 0-001) and 93 % in the female (P < 0-001). A progressive significant increase (400 %,P< 0-001) in the maternal adrenal cortisol concentration occurred between days 62 and 67, followed by a decrease after parturition. In foetal and neonatal guineapigs, the relative concentrations of corticosterone were parallel to that of adrenal cortisol and represented respectively 20 and 50 % of cortisol values during gestation and at birth. At the same time the relative concentration of cortisone remained stable (about 0-20 µg/ 100 mg). In the mother the relative concentration of corticosterone and cortisone varied very little throughout the perinatal period (about 0-07/ig/100 mg for corticosterone and 0-03 /ig/100 mg for cortisone).
Changes in the concentrations of liver glycogen and plasma glucose (Fig. 3) Three phases can be seen in the changes in liver glycogen content during the perinatal period in the foetus and newborn guinea-pigs of both sexes: (i) a regular increase by 187 % (P < 0-001) in the male and 193 % (P < 0-001) in the female between days 62 and 67; (ii) a marked rise by 35 % (P < 0-001) in the male and 53 % (P < 0001) in the female between day 67 and birth and (iii) a decrease of 80 % in both sexes (P < 0-001) from 0 to 24 h after birth. Maternal liver glycogen remained stable from 62 to 67 days, then a significant
12
24 Hours
Fig. 1. Maternal plasma progesterone and cortisol levels and foetal plasma cortisol levels in the perinatal period in the guinea-pig. Mother: A, progesterone; V, cortisol. Foetuses: O, male;
·, female. The vertical lines indicate
+
s.e.m.,
=
8-12.
0-80 -
2 0-60 ¡= 0-40 -
12 24 Hours
Fig. 2. Maternal and foetal adrenal cortisol levels in the perinatal period in the guinea-pig. Mother: . Foetuses: O, male; ·. female. The vertical lines indicate + s.e.m., 8-16. =
decrease (47 %, < 0-001) occurred between day 67 and parturition. Foetal plasma glucose lower than 100 mg/100 ml between days 62 and 67. It increased between day 67 and 0 h (96 % in the male, < 0-001 ; 53 % in the female, < 0001), then decreased quickly by 50 % (P < 0-001) in both sexes between 0 and 24 h after birth. During the perinatal period, maternal glucose was stable (125 mg/100 ml). was
Changes in maternal plasma concentrations of progesterone (Fig. 1) plasma progesterone concentrations remained very high until the last day of pregnancy; values were about 17 pg/100 ml between days 62 and 66 and increased by 52 % at day 67 (P > 0-05). A fall of 72 % (P < 0-001) was observed between day 67 and parturition. Plasma levels of progesterone reached 1 /ig/100 ml 24 h after birth. Maternal
o
150
Fig. 3. Plasma glucose concentrations (a) and liver glycogen content (b) in mothers and foetuses of guinea-pigs in the perinatal period. Mother: A. Foetuses: O, male; ·. female. The vertical 8-16. lines indicate + s.e.m., =
DISCUSSION
An
analysis of our results adds to the knowledge about the adrenal capacity of the guineapig to secrete corticosteroids in the foetus just before parturition and in the newborn pup. An increase in foetal adrenal capacity might be indicated by several observations during late pregnancy: (i) the rise of foetal plasma cortisol concentration, (ii) the decrease of plasma cortisol ratio mother:foetus, and (iii) the increase in liver glycogen. This increase in foetal liver glycogen during the last days of pregnancy confirms results already reported in foetal guinea-pigs and other mammals (Shelley, 1961). It is known that the presence of an intact pituitary-adrenal axis is necessary for the appearance of raised liver glycogen levels in late pregnancy (Jost & Jacquot, 1955). There is also a rise in aldosterone concentrations in foetal guinea-pig plasma and adrenals before birth (Giry & Delost, 1974). In addition,
glands of foetal guinea-pigs are capable of responding to adrenocorticotrophin early as 60 days of pregnancy (Donovan & Peddie, 1974). All these observations suggest that the hypothalamo-hypophysial-adrenal axis may be active before birth in the foetal guinea-pig as in the foetal rat (Dupouy, 1971; Dupouy et al. 1975). Nevertheless several observations are not consistent with this hypothesis: indeed foetal adrenal weight and foetal adrenal cortisol concentrations do not increase between days 62 and 67 of pregnancy adrenal as
and it is unlikely that there is any increase in the rate of cortisol secretion from the gland. The rise in foetal plasma cortisol levels might be due either to transfer from the mother, or to an increase in foetal corticosteroid-binding globulin (CBG) levels, which would increase plasma cortisol levels in the absence of placental transfer from the mother or increased secretion from the foetal adrenal. Seal 8c Doe (1967) have shown the CBG levels in the foetal guinea-pig near term are around eight times higher than those of the non-pregnant adult and are sufficient to account for the high foetal plasma cortisol levels. In the species looked at so far, the guinea-pig is the only one with such high foetal CBG levels. Plasma free cortisol levels decrease in the mother and increase in the foetus in the last part of pregnancy (Jones, 1974). This may suggest a placental transfer of cortisol from the mother to the foetus and in part would also explain the rise in foetal plasma cortisol during the same period. The significant increase of maternal adrenal weight and adrenal and plasma cortisol concentrations, which are higher than those of the foetus, suggest that there is marked adrenal stimulation of the mother before parturition. The very high maternal plasma cortisol concentrations are in accordance with the high binding capacity of CBG observed in late pregnancy in the mother guinea-pig (Diamond, Rust & Westphal, 1969). In the guinea-pig, foetal adrenal activity may be partly inhibited by maternal steroid secretion during the last 6 days of pregnancy. Nevertheless plasma corticosterone and cortisone levels remained constant up to the time of birth, whereas in the mother the plasma levels of these two hormones rose progressively at this time. Assuming all three cortico¬ steroids cross the placenta at similar rates, if the increasing cortisol levels of the foetus were due to transfer from the mother then one would expect an increase in foetal levels of corticosterone and cortisone to occur, which is not the case. Maternal adrenalectomy might answer this question. The autonomous capacity of the adrenals of the newborn pup clearly appears at birth, since plasma cortisol concentrations remain high in the newborn guinea-pig while they decrease sharply in the mother after parturition, and adrenal cortisol and corticosterone, and liver glycogen rise in the last 24 h of intrauterine life. The sustained high plasma cortisol levels in newborn pups could also be due to a lower capacity of the neonatal liver to metabolize corticosteroids transferred from the mother before birth (Schapiro, Percin & Kotichas, 1971). The increase of plasma glucose in the newborn guinea-pig, probably due to hypoxia (Hard, Reynolds & Winbury, 1944; Shelley & Neligan, 1966), might also be related to the high cortisol levels and to the important increase in adrenal adrenaline observed at 0 h in the same animals (Girvès & Delost, 1975). Nevertheless, it is difficult to resolve the problem of adrenal capacity in the foetal and newborn guinea-pig throughout the perinatal period without knowing the placental transfer, metabolic clearance and binding capacity for cortisol. We are at present studying these factors. Lastly, regarding the influence of foetal corticosteroids in the induction of parturition, well known in sheep and cattle (see Jöchle, 1973), our results suggest that foetal corticosteroids cannot play the same role in the guinea-pig, as demonstrated by Donovan & Peddie (1974). Indeed, plasma cortisol concentrations are much lower in the foetus than in the mother in late pregnancy, confirming the observations made by Illingworth et al. (1974). Furthermore, the very high maternal plasma progesterone levels observed at day 67 indicate that there is probably a
sharp drop in plasma progesterone levels in the last 12 h before parturition. The levels found at 0 h were low compared with day 67, and bearing in mind the longer half-life of progesterone in late pregnancy (Illingworth, Heap & Perry, 1970) it seems likely that a sharp decrease in secretion rate must occur some hours before parturition. We wish to thank the Laboratory of I.N.R.A. (Clermont-Ferrand, France) for its advice on the estimation of the plasma glucose and the liver glycogen.
helpful
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