Hypophysectomized Fetuses Research Articles

The Influence of Human Growth Hormone (GH) and Thyroxine (T4) on the Differentiation of Adipose Tissue in the Fetus

Late term fetuses from genetically obese dams have slightly larger fat cells, greater adipose tissue lipoprotein lipase (LPL) activities, elevated levels of thyroid hormones, and depressed growth hormone (GH) levels when compared to fetuses from lean dams. We have investigated the influence of thyroid hormone and GH status per se on these and other adipose tissue traits by chronically treating hypophysectomized (hypox) fetuses (day 70) between day 90 and 105 of gestation with either thyroxine (T4) or human GH. Treatment with T4 decreased body weights (P<.05), increased serum T4 levels (P<.05), and enhanced skin and hair development (P<.05). Quantitative analysis of sections of perirenal and subcutaneous adipose tissue indicated that T4 increased LPL activity (P<.05), slightly increased fat cell size, and more than doubled (P<.05) lipid accretion. A hypox induced deficit in fat cell cluster number in the outer layer of subcutaneous tissue was normalized by T4 (P<.05). Conversely, human GH (hGH) treatment had no influence on body weight, increased serum hGH levels, decreased fat cell size (P<.05) and LPL activity (P<.05) but had no influence on lipid accretion. Quantitative analysis of adipose tissue sections provided direct and indirect evidence of a "critical" or "sensitive" period between 90 and 105 days, since fetal hypox at day 70 severely impeded preadipocyte recruitment/replication during this period. Furthermore, T4 but not GH effectively normalized this hypox-induced deficiency in preadipocyte development. Therefore, T4 may have a major role in preadipocyte recruitment/replication during late fetal life.

The influence of thyroxine on the differentiation of adipose tissue and skin during fetal development.

Preobese fetuses have elevated thyroid hormone levels and depressed growth hormone levels relative to lean fetuses. Therefore, we are studying various experimental fetal pig models to explore the relationship between endocrine status and onset of obesity. In the present study, intact and hypophysectomized (d 70) fetuses were implanted with thyroxine (T4) pellets on d 70 of gestation, and blood, adipose tissue, and skin samples were obtained upon removal of d 90 of gestation. Body weights were similar for all groups and T4 treatment reversed myxedema in hypophysectomized fetuses. Serum T4 levels were elevated (p less than 0.05) and skin and hair development were enhanced (p less than 0.05) to a similar degree by T4 treatment in intact and hypophysectomized fetuses. However, T4 did not influence adipose tissue development in intact fetuses, but markedly enhanced development in hypophysectomized fetuses. For instance, fat cell size and lipogenic enzyme activities in hypophysectomized fetuses were increased (p less than 0.05) by 5-mg and 15-mg T4 treatments, with a marked increase (p less than 0.05) in apparent fat cell number with the 15-mg T4 treatment. In contrast, there was no effect of T4 (15 mg) on these parameters in intact fetuses. Therefore, fetal obesity may be directly associated with elevated thyroid hormone levels and suppressed growth hormone levels, but not with elevated T4 levels alone.

Hypophysectomy and Porcine Fetal Lung Development

The effect of hypophysectomy on the development of the lung parenchyma and maturation of pulmonary alveolar type II cells was examined in the fetal pig. In fetuses from four different gilts, hypophysectomy was performed on gestational day 69 or 70. Littermates from the same gilts served as controls. Fetuses were delivered by caesarean section at term (113 +/- 1 days of gestation), and the lungs were fixed by intratracheal instillation. Plasma cortisol and thyroxine concentrations in the umbilical artery were markedly lower for hypophysectomized fetuses compared with control fetuses. Body weight was similar for both groups of fetuses. Total lung volume was 53% smaller in hypophysectomized fetuses compared with control fetuses. However, alveolar septal tissue and capillary luminal volumes were similar in both groups. Total lung alveolar surface area was twice as great in control animals compared with hypophysectomized animals. The volumes of epithelium, interstitium, and endothelium of centriacinar alveolar septa per unit surface area of epithelial basal lamina were 3.7, 4.8, and 2.4 times greater in hypophysectomized fetuses compared with control fetuses. Alveolar type II cell composition also differed significantly between groups. The volume fraction of glycogen in type II cells was 51% for hypophysectomized fetuses and 12% for control fetuses, while lamellar body volume fraction was 8% in hypophysectomized fetuses and 23% in control fetuses. The frequency of alveolar type II cell contact with mesenchymal interstitial cells via foot processes was 5 times greater in the lungs of control animals compared with hypophysectomized animals. These findings demonstrate significant effects of hypophysectomy on the morphogenetic and cytodifferentiation activities of all major tissue compartments of the pulmonary gas exchange area during the final trimester of fetal development.

EFFECT OF HYPOPHYSECTOMY ON BODY AND ORGAN WEIGHTS AND SUBSEQUENT DEVELOPMENT IN THE FETAL PIG

The effect of hypophysectomy on fetal growth and development was studied in 45 fetuses in which the pituitary had been destroyed by electrocoagulation between 68 and 84 d of gestation, and in 35 control littermates. Hypophysectomized fetuses were significantly heavier than their intact litter mates (1203 g vs. 1057 g; P &lt; 0.05), were more edematous and had visible body fat stores. Fetuses hypophysectomized before 70 d of gestation were hairless and myxedematous, whereas in those hypophysectomized after day 80 the body surface either was similar to that of intact litter mates or had mild hypotrichosis. Hypophysectomy resulted in significant reductions in the weights of adrenal and thyroid glands and the testes, but did not influence fetal ovarian weight. In fetuses hypophysectomized after 80 d of gestation, no alteration of thyroid weight was noted. The spleen and kidneys were significantly heavier in hypophysectomized fetuses, whereas liver and heart weights were lighter. It is concluded that fetal hypophysectomy may increase fetal growth in the pig, and selectively alters the growth and maturation of individual organs. Key words: Hypophysectomy, body weight, organ weight, fetus

INFLUENCE OF FETAL NUMBERS AND HYPOPHYSECTOMY ON THE LENGTH OF GESTATION IN THE PIG

Parturition was not delayed in gilts carrying intact and hypophysectomized fetuses when four or more intact fetuses were present but its timing varied when fewer were present. In normal farrowings, gestation periods were longer for litters of two or three but not for litters of four. It is suggested that four fetuses are required for consistent delivery at the expected time. Key words: Gestation length, litter size, hypophysectomy

Effect of hypophysectomy with and without thyroxine replacement on growth and circulating concentrations of insulin-like growth factors I and II in the fetal lamb.

The role of the pituitary gland in the regulation of skeletal growth and plasma insulin-like growth factor (IGF)-I and IGF-II concentrations in the late gestation sheep fetus has been studied. Singleton fetuses were either hypophysectomized (n = 14) or sham operated (n = 8) between days 110 and 125. Fetal and maternal blood samples were collected three times weekly through the remainder of gestation. In some hypophysectomized fetuses (HXT4 group, n = 4), T4 was administered (100 micrograms L-T4/day) to overcome the hypothyroidism caused by hypophysectomy. The other hypophysectomized fetuses received no replacement therapy (HXNR group, n = 10). Six HXNR fetuses were allowed to remain in utero post term and were killed at day 163 of gestation. All other animals were killed at day 147. All values are group means +/- SE. Hypophysectomized fetuses had significantly shorter limbs and long bones and delayed osseous maturation at term compared to sham controls. Plasma free T4 concentrations in HXT4 fetuses were not significantly different from those measured in sham fetuses (P greater than 0.05). Bone maturation at term was normal in HXT4 fetuses although there was no improvement in limb or bone elongation. Retention of hypophysectomized fetuses in utero until 16 days past term yielded fetuses which were heavier than controls but whose limb and bone lengths were no greater than hypophysectomized fetuses killed at term. Osseous maturation was appropriate for term in five of the six postterm hypophysectomized fetuses. The plasma IGF-I and IGF-II concentrations were not significantly affected by hypophysectomy, hypophysectomy with T4 replacement, gestational age, or prolonged gestation. The plasma IGF-I concentrations in the sham, HXNR, and HXT4 fetuses were 35.4 +/- 6.6, 28.2 +/- 3.0, and 34.4 +/- 1.7 ngeq human (h)IGF-I/ml, and the IGF-II concentrations were 656.3 +/- 59.2, 635.3 +/- 56.3, and 645.5 +/- 71.9 ngeq hIGF-II/ml, respectively, and remained within these ranges throughout the experiment. Fetal IGF-I concentrations were significantly lower than mean maternal IGF-I concentrations (88.0 +/- 6.8 ngeq hIGF-I/ml, P less than 0.05), and fetal IGF-II concentrations were significantly higher than mean maternal IGF-II concentrations (362.4 +/- 24.0 ngeq hIGF-II/ml, P less than 0.05). We conclude that in the late gestation fetal sheep, elongation of the appendicular skeleton is under some direct pituitary control whereas appendicular maturation exhibits some dependence on circulating T4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of the fetal pituitary-adrenal axis on fetal and maternal progesterone and unconjugated oestrogen concentrations in the pig.

Fetal and maternal plasma progesterone and unconjugated oestrone and oestradiol-17 beta concentrations were measured in intact pig fetuses and those in which the pituitary had been destroyed. Progesterone concentrations were significantly higher (P less than 0.05) and oestrogen concentrations significantly lower (P less than 0.01) in hypophysectomized fetuses than in intact fetuses. When fetuses in one uterine horn only were hypophysectomized, oestrogen concentrations in the uterine vein draining this horn were lower than those from the contralateral vein. The results indicate that both fetal and maternal oestrogen concentrations are influenced by the fetal pituitary. When dexamethasone was infused (at 27 micrograms/h for 96 h) into 5 chronically-catheterized hypophysectomized fetuses no changes in peripheral fetal progesterone or oestrone were observed.

Control of binucleate cell migration in the placenta of sheep and goats.

In the ruminant placenta 15-20% of the trophectodermal epithelium consists of granulated binucleate cells (BNC). In the sheep the granules contain ovine placental lactogen (oPL). These cells migrate from the trophectoderm to form fetomaternal hybrid tissue from implantation to term. The number of BNC, their percentage migration and the potential secretory activity of the syncytium they form were estimated by semiquantitative transmission electron-microscopical techniques after several surgical techniques and hormone or drug infusions. BNC numbers decrease normally just before parturition, and this fall could be eliminated by fetal hypophysectomy or induced early by administration of tetracosactrin to intact or hypophysectomized fetuses. If only one twin was treated with tetracosactrin the placenta of the untreated twin did not show the fall in BNC numbers found in the other unless it died in utero some time before sampling. This indicates fetal control of BNC number and migration. However, fetal catheterization, hypophysectomy, stalk section, adrenalectomy, infusion of mouse epidermal growth factor or bromocriptine had little or no effect on binucleate cell numbers or migration percentages. Maternal carunclectomy, ovariectomy, or epostane or bromocriptine administration also had no consistent significant effect. Previous reports of degeneration of BNC structure plus a decrease in their number (with bromocriptine) or an increase in migration frequency (after adrenalectomy or stalk section) have not been confirmed by this study. The BNC migration delivers the oPL-containing BNC granules close to the maternal circulation but the variation in migration seems only loosely correlated with the reported maternal oPL concentrations. The results indicate that BNC migration is independent of the hormonal milieu, but that BNC production is greatly modified by the hormonal changes just before parturition, with cortisol production by the fetus a possible primary cause.

In vitro spontaneous and adrenocorticotropin-dependent maturation of the steroidogenic pathway of ovine fetal adrenal cells.

Adrenal cells from intact or hypophysectomized fetuses and newborn lambs cultured for 6 days under several conditions were tested for their ability to metabolize [14C]pregnenolone (P5) (5 X 10(-5) M) during a 2-h period. Also, their ability to produce P5 was investigated. At the beginning of the culture, the metabolism of P5 by adrenal cells from hypophysectomized fetuses was lower than that of cells from intact fetuses which in turn was lower than that of cells from newborns. After 6 days in culture in the absence of ACTH-(1-24), the metabolism of P5 by cells from both intact and hypophysectomized fetuses increase, whereas that of cells from newborns slightly decreases. Continuous ACTH-(1-24) (10(-8) M) treatment produced a striking increase in the capacity to metabolize P5 by cells from both intact and hypophysectomized fetuses and the maintainance of this capacity of cells from newborns was demonstrated. At the beginning of the culture the main product of [14C]P5 was corticosterone with cells from intact and hypophysectomized fetuses, and cortisol with newborn cells. After 6 days in culture in ACTH-free medium more than 50% of P5 added appeared as progesterone in all cases. Addition of ACTH-(1-24) to the culture medium induced within 5 days, a striking increase in the activation of 3 beta-hydroxysteroid dehydrogenase-isomerase, 17 alpha-, 21, and 11 beta-hydroxylases of adrenal cells from fetuses. However, the activity of 17 alpha-hydroxylase in adrenal cells from hypophysectomized fetuses was lower than that of cells from intact fetuses. ACTH-(1-24) treatment maintained the enzymatic activity of cultured newborn adrenal cells, except for 11 beta-hydroxylase which declined to about 50% of its activity on day 0. The capacity of fetal adrenal cells to produce P5 after acute ACTH-(1-24) stimulation increased 30-fold when cells were cultured in the absence of ACTH and up to 110-fold when cells were treated with ACTH-(1-24) from day 1 on. These results, together with those reported previously, show that the spontaneous in vitro maturation of fetal adrenal cell steroidogenic pathway is related to an increased activity of the 3 beta-hydroxylase dehydrogenase-isomerase and an enhanced capacity to produce P5. Both steps were stimulated further by ACTH. In addition, the hormone is an absolute requirement for the development and/or the maintenance of 17 alpha-hydroxylase activity.

Ovine placental lactogen concentrations during premature delivery induced by infusion of dexamethasone or adrenocorticotrophin to the fetus.

Premature delivery was induced by intrafetal infusion of dexamethasone or (1-24)ACTH, starting between days 124 and 129 of pregnancy in twelve ewes carrying intact (n = 7) or hypophysectomized (n = 5) single fetuses. Concentrations of ovine placental lactogen (oPL) in maternal plasma were measured by specific radioimmunoassay before and during premature delivery. There was no significant change in oPL concentrations before delivery in ewes carrying either intact or hypophysectomized fetuses. The results suggest that a prepartum decline in oPL is not an essential prerequisite for delivery, and indicate that fetal glucocorticoids are not directly responsible for the prepartum fall in oPL concentrations observed at spontaneous delivery.

Adrenal cortex of fetal lamb: changes after hypophysectomy and effects of Synacthen on cytoarchitecture and secretory activity.

The effects of hypophysectomy on the cell population of the adrenal cortex has been examined with light and electron microscopy. The sensitivity of the adrenal cortical cells to exogenous ACTH in both normal and hypophysectomized fetuses has been investigated by comparing plasma cortisol concentrations with structural changes in the adrenal cortex. Hypophysectomy was carried out at about 100 d and the subsequent analysis was made at about 135 d on fetuses catheterized at least 6 d earlier. The zona fasciculata of intact fetuses contained about 25% mature and 75% immature cells at 130-136 d. After hypophysectomy this zone contained a uniform population of immature cells and no mature cells; there was a significant reduction in both adrenal weight and cortical thickness. The zona glomerulosa was unaffected by hypophysectomy. The infusion of Synacthen 0 . 25 mg/d I.V. for 48 h did not affect the zona glomerulosa but resulted in a 2-fold increase in the thickness of the zona fasciculata in hypophysectomized and intact fetuses. This cortical growth was due to both hypertrophy and hyperplasia. All the cells in the zona fasciculata were mature following Synacthen treatment in both groups. The most striking difference between the secretory response of the adrenal cortex to Synacthen in the two groups was found within 3-6 h of the onset of infusion. Within this period there was a significant increment in the plasma cortisol concentration in the intact fetuses while no response was observed in the hypophysectomized animals. After 12 h infusion by contrast, a steadily rising response was observed in both groups. There was a significant correlation between the final plasma cortisol concentration and both the adrenal weight and cortical thickness after 48 h treatment. The increased sensitivity of the adrenal cortex to exogenous ACTH as term approaches in the sheep is considered to be dependent upon the increasing proportion of mature zona fasciculata cells within the adrenal cortex.

Effects of glucocorticosteroids and glucagon on argininosuccinate synthetase, argninosuccinase, and arginase in fetal rat liver.

In the present study we examined the in vivo effects of glucocorticosteroids and glucagon on the developmental formation of the individual urea cycle enzymes argininosuccinate synthetase, argininosuccinase, and arginase during the late fetal period. In particular, addition of exogenous glucagon caused a rise in argininosuccinase and arginase activities in the livers of rat fetuses at term but not at earlier stages. Glucagon produced a rise in argininosuccinase activity earlier if fetuses were previously treated with cortisol. When fetuses were deprived of corticosteroid (hypophysectomy in utero), glucagon no longer promoted the argninosuccinase activity, indicating that adrenal glucocorticoids are required for normal enhancement of the enzyme activity by glucagon. Dibutyryl cAMP was still effective in hypophysectomized fetuses. Results obtained by injected combinations of inducers indicated that a glucocorticosteroid-glucagon interaction might be involved in the regulation of argininosuccinate synthetase and argininosuccinase. No synergistic action was found on arginase activity in vivo.

Induction of labour in sheep after fetal hypophysectomy: an investigation of the possible involvement of a fetal pituitary secretion in the activation of placental enzymes by fetal cortisol

Activities of 17 alpha-hydroxylase, C-17, 20 lyase and aromatase were measured in placentae of intact and hypophysectomized fetuses after premature induction of parturition by fetal administration of Synacthen. Activities of these enzymes were compared with concentrations of steroids produced by the placenta, which were measured in maternal plasma before and during parturition. Hypophysectomy was assessed by determining fetal plasma concentrations of LH before and after administering LH-RH, and histologically post partum. Concentrations of progesterone, which decreased before parturition, and of 17 alpha, 20 alpha-dihydroxypregn-4-en-3-one and oestradiol-17 beta, which increased, were not affected by fetal hypophysectomy. However, fetal hypophysectomy reduced the normal rise in oestrone sulphate which precedes parturition and abolished the normal rise in androstenedione. Although placental activities of C-17, 20 lyase and aromatase (but not 17 alpha-hydroxylase) tended to be reduced by fetal hypophysectomy, these effects were not statistically significant. The data support an earlier suggestion that an unidentified product of the fetal pituitary may be involved in the activation of C-17, 20 lyase by fetal cortisol before parturition. However, the results of the present study show that the action of such a factor is more likely to be permissive than obligatory.

Maternal peripheral concentrations of estradiol, estrone, cortisol, and progesterone during late pregnancy in rhesus monkeys (Macaca mulatta) and after experimental fetal anencephaly and fetal death

Estradiol, estrone, cortisol, and progesterone were measured serially in maternal venous plasma during late pregnancy in four intact rhesus monkeys (Macsca mulatta) and in six monkeys with experimental anencephaHc fetuses (functionally hypophysectomized fetuses). Estradiol and estrone increased gradually and significantly (P <0.05) during the 2 weeks preceding spontaneous vaginal delivery in normal monkeys. Progesterone did not decrease prior to parturition. Cortisol increased sharply on the day of delivery, whereas estradiol, estrone, and progesterone decreased precipitously. In mothers with live, anencephalic fetuses, estradiol was significantly lower than in normal monkeys during late gestation (P <0.05); however, estrone, cortisol, and progesterone values were not different from those in control monkeys. Fetal death occurred in three monkeys at various times after experimental fetal anencephaly and resulted in substantial decreases in maternal estradiol and estrone. Cortisol and progesterone were not affected. Spontaneous vaginal delivery occurred in two animals; pregnancy was substantially prolonged in four. Vaginal delivery was induced by administering estradiol benzoate to a monkey with prolonged pregnancy and a dead fetus. These data suggest that estrogens are involved in the onset of parturition in primates and that a live fetus with an intact hypothalamic-pituitary-adrenal axis is essential for normal concentrations of maternal estrogens but not cortisol or progesterone.

The sheep fetus at parturition

Spontaneous parturition in sheep requires activation of the fetal pituitary-adrenal axis. Before parturition there is a rise in the concentration of adrenocorticotrophin (ACTH) but this occurs after the increase in cortisol concentrations. Radioimmunoassayable ACTH is present in fetal plasma in a number of molecular forms. The factors influencing ACTH concentrations in the sheep fetus include hypoxia, hypoglycaemia, haemorrhage, vasopressin and catecholamine infusions, and infusion of prostaglandin E 2. Comparison of the endocrine changes in the fetus and ewe during infusion of Synactrien or dexamethasone to intact or hypophysectomized fetuses, suggests that some pituitary factor(s) other than ACTH 1–24 is required to mimic the endocrine changes characteristic of spontaneous parturition, in ewes carrying hypophysectomized fetuses.

Prostaglandins in fetal tracheal and amniotic fluid from late pregnant sheep

Concentrations of prostaglandin E (PGE), prostaglandin F (PGF) and 13,14-dihydro-15-keto-prostaglandin F (PGFM) have been measured in fetal tracheal and amniotic fluid from chronically catherized sheep during late pregnancy. Amniotic fluid contained significantly greater concentrations of these prostaglandins than tracheal fluid (p<0.01); there was no correlation between the level of prostaglandins found in each fluid. In tracheal fluid concentrations of PGE and PGFM exceeded those of PGF (P<0.01) whereas no significant differences were found in amniotic fluid. The levels of prostaglandins in these fluids were similar in ewes bearing hypophysectomized fetuses.