Ontogeny of the response to growth hormone-releasing factor

Primary cell cultures were prepared from fetal, neonatal and adult rat pituitaries and evaluated for their ability to secrete growth hormone (GH) in response to growth hormone-releasing factor (GRF). Pituitary cells prepared from fetuses at days 19 and 21 of gestation, neonatal animals at the day of birth (day 0) or the following day (day 1) and peripubertal male rats showed full dose response curves to GRF with maximal GH release when stimulated with 1 X 10(-10) M rat GRF. At this concentration of GRF, the amount of GH released was not different from that elicited by activation of adenylate cyclase with 1 X 10(-5) M forskolin. In contradistinction, a preparation of cells from fetuses at day 18 of gestation did not show the same release of GH when challenged with 1 X 10(-10) M GRF and forskolin (0.057 +/- 0.001, compared to 0.076 +/- 0.003 micrograms/10(5) cells per 4.5 h), although the cells clearly responded to both secretagogues (basal levels of GH, 0.029 +/- 0.002 micrograms/10(5) cells per 4.5 h). While cells prepared from fetuses at day 21 of gestation or from animals after birth released 5-10% of their total cellular GH content, those prepared from 18- and 19-day fetuses released as much as 40% of their total GH suggesting there is a maturation of intracellular GH processing that occurs late in gestation. The results show that, in late pregnancy, the rat fetal pituitary is highly responsive to growth hormone-releasing factor and suggest that this peptide participates in regulating GH levels during the perinatal period.     

Soluble T lymphocyte antigen-specific molecules.

Growth hormone (GH) was measured in the sera of control, hypothyroid (thyroidectomized [Tx]) and GH-treated Tx rats and their fetuses on Days 19, 20, 21, and 22 of gestation and in their progenies on postnatal Days 1, 5, 30, and 75. Maternal endogenous serum GH increased dramatically between the 19th and 20th days of gestation and remained elevated through the 22nd day in control rats, but was depressed significantly in Tx and GH-treated Tx rats during this period. GH was not always detected in the sera of 19-day-old fetuses. On Day 20, GH was depressed in fetuses of Tx mothers as compared with those form controls or GH-treated Tx mothers. GH was elevated in sera of fetuses from GH-treated Tx rats over fetuses of control and Tx only rats on the 22nd day of gestation. In postnatal rats, those from GH-treated mothers continued to show elevated serum GH on Day 1 as compared with those from Tx only mothers. On postnatal Days 5 and 30, progenies of Tx mothers had significantly elevated GH as compared with progenies of control mothers. At 75 days of age, the GH levels of these progenies had normalized. We have shown previously that the hormonal secretions of the pituitary-thyroid axis are badly disrupted in the progenies of Tx and GH-treated Tx mothers and that even as adults these animals have tissue (brain and liver) deficits of active thyroid hormones. Although the onset of GH secretion is mildly delayed in fetuses of Tx but not GH-treated Tx mothers, the serum GH levels of both groups of progenies are elevated during most of the neonatal period through the time of puberty. It is, therefore, concluded that GH in the absence of adequate levels of thyroid hormones is ineffective in preventing many of the learning and memory deficits induced in the progenies of Tx mothers.     

Experimental production of myeloschisis associated with hindbrain crowding in the central nervous system of rat fetuses by a single intragastric administration of ethylenethiourea

66 pregnant rats were divided into 9 groups according to gestation day 11 to 19. These pregnant rats were subjected to a single intragastric administration of ethylenethiourea (ETU) and cesarian sectioned on day 20. No dam died following the ETU treatment, but the rate of fetal death was as high as 21.2% on day 11, followed by a gradual decrease in the fetal death rate thereafter. The rate of production of various types of externally visible malformations was 100% except in the fetuses of dams treated with ETU on gestation day 19. The important results were as follows. (I) Fetuses of dams treated with ETU from gestation day 11 were found to suffer from a high incidence of myeloschisis associated with hindbrain crowding. (II) Exencephaly and an abnormally enlarged head with occipital bossing due to herniation of the mesencephalic tectum, with and without dilatation of the mesencephalic and 4th ventricle, were induced among the fetuses of dams given ETU at gestation day 12 and 13. (III) Various degrees of hydranencephaly and dysgenic hydrocephalus were found among the fetuses of dams treated with ETU from gestation days 14 to 18. The above results suggest that ETU may be a useful agent for the production of congenital malformations in the rat.     

Pituitary-thyroid function of fetuses of hypothyroid and growth hormone treated hypothyroid rats.

Maternal hypothyroidism induced by surgical thyroidectomy (Tx) of the rat resulted in significantly higher fetal serum levels of thyroid stimulating hormone (TSH) and thyroxine (T4) on day 22 of gestation. Surprisingly, administration of growth hormone (GH) to hypothyroid mothers increased further the fetal serum T4 and TSH. The in vitro uptake of 131I-T4 by erythrocytes was elevated significantly when incubated with serum from fetuses of both hypothyroid and hypothyroid GH-treated mothers. Although the plasma protein levels of hypothyroid mothers and their fetuses are decreased significantly as compared to controls this is not true of hypothyroid GH-treated mothers and their fetuses. The T4 levels of both groups of Tx mothers were significantly below that of controls. However, as in the case of their fetuses, the serum T4 of GH-treated hypothyroid mothers was elevated from that of Tx only animals. It is concluded that the pituitary-thyroid system of fetuses of hypothyroid mothers is activated excessively during late gestation, that considerable T4 can be transported from the fetus to the mother during this period and that these fetuses are in fact born in a hyperthyroid state which is aggravated by maternal treatment with GH.     

Immunohistochemical study on the fetal rat pituitary in hyperthermia-induced exencephaly

Hyperthermia of fetal rats is known to cause malformations of various organs including brain. The present study was carried out to investigate the effect of the hyperthermia-induced brain damages on the development of the adenohypophysis. Mother rats of day 9.5 of pregnancy were anesthetized and immersed in hot water (43 degrees C) for 15 min. At day 21.5 of gestation, fetuses were removed by caesarian section and examined for exencephaly. Hyperthermal stress induced varying degrees of exencephaly in 36% of surviving fetal rats. In extreme cases a considerable part of head was lost. Even in those fetuses with severe brain deformities, the hypophysial stalk and neural lobe were present though they were markedly underdeveloped. In exencephalic fetuses, no immunoreactive vasopressin was detected in the neural lobe of the hypophysis. Immunohistochemical examination of the adenohypophysis showed that exencephaly caused a marked decrease in the number of growth hormone (GH)-producing cells. Other types of hormone-producing cells appeared to be unaffected by brain anomaly. The reason for a decreased population of GH cells in exencephalic fetuses is discussed in relation to their adrenocortical hypotrophy.     

Fetal lung development in streptozotocin-induced experimental diabetes: cytidylyl transferase activity, disaturated phosphatidyl choline and glycogen levels

The influence of streptozotocin-induced maternal diabetes on choline phosphate cytidylyltransferase activity (EC.2.7.7.15) glycogen content and disaturated phosphatidyl choline in fetal lung was studied between 19 and 21 days of gestation. In this experimental model, induction of maternal diabetes two days after mating, resulted in fetal hyperglycemia and hyperinsulinemia; the fetuses were neither macrosomic nor showed any evidence of fetal growth retardation. The glycogen content of lungs on days 19 and 20, but not on day 21 of gestation was significantly higher in fetuses of diabetic rats than in controls. The pulmonary cytosol cytidylyltransferase activity was similar in the two groups of fetuses on days 19 and 20. On day 21 of gestation the enzyme activity was significantly lower in fetuses of diabetic rats than in those of controls. On day 21 of gestation and in newborns of diabetic mothers, although there was no difference in the total pulmonary phospholipids, the levels of disaturated phosphatidyl cholines were significantly lower than in controls.     

Experimental study of umbilical cord length as a marker of fetal alcohol syndrome

Umbilical cord length has long been investigated as a potential marker of intrauterine events that may place the neonate at risk for future adverse developmental sequelae. Experimentally, significantly shortened cords have been reported in association with prenatal exposure to common drugs of abuse. This study in rats reports the time course of effects on umbilical cord length of a daily maternal ethanol gavage (3,200 mg/kg) from gestational day 6 through termination of pregnancy at either day 17, 18, 19, or 20. A total of 786 fetuses derived from 60 litters were examined. Control fetuses demonstrated a linear increase in umbilical cord length and body weight gain during late gestation, findings that support previous studies. The body weights of the ethanol-exposed fetuses were reduced significantly on all gestational days examined, indicating intrauterine growth retardation, a characteristic of fetal alcohol syndrome. Similarly, acute fetal akinesia as well as long-term sequelae stemming from impaired neurological development would result from the elevated blood ethanol levels achieved in this study. The umbilical cords of ethanol-exposed fetuses were significantly shorter on gestational days 19 and 20 in comparison to their controls, while cord lengths on days 17 and 18 were not shortened significantly. A stretch hypothesis has been proposed suggesting that the degree of fetal activity is the main determinant of umbilical cord length. In rats, there is a physiologic diminution of the volume of amniotic fluid (oligohydramnios) in late gestation (day 19 to term), which restricts fetal movements but does not appear to alter the linear relationships between gestational age and cord length in controls, thus arguing against the stretch hypothesis. However, cord lengths in the ethanol-exposed fetuses plateaued in late gestation, suggesting possible adherence to a stretch hypothesis. This dichotomy is discussed emphasizing fetal growth and activity as well as intrauterine space.     

Dynamic of the GRF-induced GH response in genetically obese Zucker rats: influence of central and peripheral factors

To determine the time onset of the growth hormone (GH) alteration in the genetically obese rat, we studied the in vivo and in vitro rat growth hormone releasing factor (rGRF(1-29)NH2)-induced GH secretion in 6- and 8-week-old lean and obese male Zucker rats. Under sodium pentobarbital anesthesia, rGRF(1-29)NH2 (GRF) was injected intravenously at two doses: 0.8 and 4.0 micrograms/kg b.w. Basal serum GH concentrations were similar in lean and obese age-matched animals. The GH response to both GRF doses tested was unchanged in 6-week-old obese rats as compared to their lean litter mates. In contrast, a significant decrease of the GH secretion in response to 4.0 micrograms/kg b.w. GRF was observed in the 8-week-old obese rats. The effect of GRF (1.56, 6.25 and 12.5 pM) was further studied in vitro, in a perifusion system of freshly dispersed anterior pituitary cells of lean and obese Zucker rats. Basal GH release was similar in the 6-week-old animal group. In contrast, it was significantly decreased in 8-week-old obese rats as compared to their lean litter mates. Stimulated GH response to 1.56 and 6.25 pM GRF was significantly greater in the 6-week-old obese group than in the age-matched control group. In contrast, the GH response to all GRF concentrations tested was significantly decreased in the 8-week-old obese rats as compared to their respective lean siblings. In 8-week-old obese rats, a decrease of GH pituitary content and an increase of hypothalamic somatostatin (SRIF) concentration were observed. Insulin and free fatty acid serum were significantly increased in 8-week-old obese rats. In contrast, lower insulin-like growth factor I serum levels were observed in the obese animals as compared to their lean litter mates. Finally, to further clarify the role of the periphery in the inhibition of GH secretion observed in the 8-week-old fatty rats, we exposed cultured pituitary cells of 8-week-old lean animals to 17% serum of their obese litter mates. A significant decrease of GRF-stimulated GH secretion of lean rat pituitary cells exposed to the obese serum was noted (P less than 0.05). This study demonstrates that, in the obese Zucker rat, an alteration of the GH response to GRF is evident by the 8th week of life. This defective GH secretion could be related to peripheral and central abnormalities.     

Growth hormone secretion during pregnancy: altered effects of growth hormone releasing factor and insulin-like growth factor-I in vitro

Growth hormone (GH) secretion is controlled by growth hormone releasing factor (GRF) but changes in the circulating level of this hormone are difficult to measure. Insulin-like growth factor (IGF-I) is a GH-dependent growth factor which significantly but slightly inhibits stimulated GH release in vitro. We have tested the effects of GRF and IGF-I on GH release in pregnancy, a state in which serum concentrations of GH are elevated and levels of IGF-I are lowered. We have found, in a system of acutely dispersed adenohypophysial cells prepared from pregnant (day 21-23) or control cycling female rats, that adenohypophysial cells from pregnant rats have an increased GH release with GRF. In contrast, IGF-I inhibition is similar but slightly smaller. These altered responses may result in elevated serum GH levels during pregnancy.     

Ultrastructural study on the hypothalamic-hypophysial-adrenal axis in fetal rats

Summary The adrenal glands of decapitated and encephalectomized fetal rats were investigated electron microscopically and compared to those of normal intact fetal rats. Although the adrenal cortices did not show three zones (zona glomerulosa, fasciculata, and reticularis) on the 16.5th day of gestation when the decapitation or encephalectomy was carried out in utero, the zonation was recognized in fetuses operated on the 21.5th day of gestation. The same was true for normal control fetuses. However, cytoplasmic characteristics suggesting steroidogenesis in the cortical cells were reduced to various degrees in the encephalectomized or decapitated fetuses, especially in the latter ones. The change in cytoplasmic appearance was more conspicuous in the inner portion of the cortex. This result suggests that for the maintenance of normal adrenocortical function the hypothalamus may be indispensable even during the prenatal life of rats.     

The effect of hadacidin, ultraviolet irradiation of blood (UVB) and thiamine on the prenatal development of Uje:WIST rats: correlation with the hepatic activity of gamma-glutamyltranspeptidase (GGT)]

The influence of hadacidin, a model substance for induction of cheilognathouranoschisis in rat fetuses (2,550 mg/kg b.m. at gestation day 12), ultraviolet irradiation (UVB) of blood (1 week before gestation) and thiamine (25 mg/kg b.m. from gestation days 12 to 15) on the prenatal development of rats at the 20th day of gestation was investigated. Using the body mass and the hepatic GGT-activity as parameters. Hadacidin caused a distinct retardation of the fetal somatic development. Partially, the embryotoxic effect was compensated by UVB or thiamine. The combination of both procedures was more effective. There is a good correlation between the maturation grade of fetuses at day 20 of gestation and the hepatic GGT activity.     

The development of beta-adrenergic mediated inhibition of growth hormone secretion in the ovine fetus

The ontogeny of the suppressive effect of the beta-adrenergic agonist, isoprenaline, on fetal growth hormone (GH) release was examined in 14 chronically-catheterized ovine fetuses. Isoprenaline was administered as an intravenous infusion over 1 h (200 micrograms/kg). In seven fetuses between 72 and 99 days of gestation, isoprenaline had no effect on fetal plasma GH concentrations. In seven older fetuses between 114 and 140 days of gestation, isoprenaline infusion suppressed (P less than 0.02) fetal GH release. No effect was observed in five saline-treated control fetuses (119-131 days). Propranolol (250 micrograms/kg i.v.) administered 5 min prior to the isoprenaline infusion to four fetuses (117-136 days) delayed (P less than 0.05) the onset of the suppressive effect of isoprenaline demonstrating that the action of isoprenaline was mediated by the beta-adrenergic receptor. Propranolol alone (n = 6) had no effect. These observations demonstrate that the potential for beta-adrenergic inhibition of fetal GH release differentiates after 100 days of gestation. Comparison with previous studies of the ontogenesis of the control of GH secretion suggests that the hypothalamic beta-adrenergic control of GH release differentiates with an intermediate time course compared to other potential neuroendocrine controls.     

Endocrine Regulation of Fetal Adipose Tissue Metabolism in the Pig: Interaction of Porcine Growth Hormone and Thyroxine

HAUSMAN, D.B., G.J. HAUSMAN, AND R.J. MARTIN. Endocrine regulation of fetal adipose tissue metabolism in the pig: interaction of porcine growth hormone and thyroxine. Obes Res. 1999;7:76–82. Objective : This study tested the hypothesis that combined treatment of thyroxine (T₄) and growth hormone (GH) could normalize cellular and metabolic aspects of adipose tissue development of hypophysectomized fetal pigs. Research Methods and Procedures : On day 70 of gestation, pig fetuses were hypophysectomized by microcauterization or remained intact. Hypophysectomized fetuses remained untreated or were treated from day 90 to day 105 of gestation with T₄, GH, or a combination of both hormones. Results : Body weights were unaffected by hypophysectomy or hormone treatment. De novo lipogenesis in subcutaneous adipose tissue was increased 10-fold by hypophysectomy, consistent with our previous results. This increase was abolished by GH treatment in the hypophysectomized fetuses. In contrast, T₄ treatment of the hypophysectomized fetuses resulted in a 12-fold further increase in adipose tissue lipogenesis, an effect that was negated by concomitant administration of GH. Lipolytic response to isoproterenol was decreased by hypophysectomy, unaffected by GH treatment, and restored to intact values by T₄ or by T₄+GH treatment in the hypophysectomized fetuses. Discussion : In contrast to T₄, GH does not influence serum insulin-like growth factor-I or adipose tissue lipolysis, but decreases lipogenesis in the fetal pig. However, replacing both T₄ and GH normalized hypophysectomized fetuses to a greater extent than either GH or T₄ alone. Thus, any influence of thyroid hormones on stimulating adipose tissue lipogenesis in the developing fetal pig may be normally counterregulated by pituitary-derived growth hormone.     

Iron deficiency in the rat: biochemical studies of fetal metabolism

The effects of dietary-induced iron deficiency on fetal and maternal metabolism were studied in the rat. Concentrations of phenylalanine, but not tyrosine, were significantly elevated in plasma from iron-deficient maternal and fetal rats at day 20 of gestation with individual fetal plasma levels of phenylalanine as high as 10 mg per 100 ml. Concentrations of total 5-hydroxyindole compounds were significantly decreased in brain tissue from iron-deficient fetuses (day 20 of gestation), suggesting that synthesis of the compounds may be inhibited by iron deficiency. Mitochondrial NADH oxidase activity was markedly decreased (60%) in homogenates of fetuses at day 14 of gestation and may account for the high fetal resorption rate and small fetal size observed in the rat in iron deficiency.     

Topographical and ontogenetic study of the neurons producing growth hormone-releasing factor in human hypothalamus

Neurons producing growth hormone-releasing factor have been characterized and analyzed by immunohistochemistry in the hypothalami of human fetuses, neonates, infants and adults, using two antibodies against human pancreatic GRF (hpGRF). One of the antibodies recognized both the hpGRF(1-40)OH and hpGRF(1-44)NH2 in the mid portion (between the 28th and 39th amino acid), the other one specifically recognized the C-terminal end of hpGRF(1-44)NH2. These two antibodies stain a single neuronal system with cell bodies mainly located in the infundibular (arcuate) nucleus, and in the ventromedial and lateralis tuber nuclei. These neurons project to the median eminence where they give numerous endings in contact with portal vessels. These neurons are distinct from those containing LH-RH, somatostatin, CRF or pro-opiocortin. In fetuses, neurons immunoreactive with hpGRF antibodies are first detected at the 29th week. They display a neuroblastic aspect which persists after birth. Immunoreactive fibers are detectable in the median eminence after the 31st week. These results demonstrate that the infundibular nucleus plays a major role in control of GH secretion in man and that secretion of GRF appears late during fetal life; this suggests that the first stages of differentiation and development of GH producing cells in the human fetus do not depend on hypothalamic GRF secretion.     

Influence of maternal dexamethasone treatment on morphometric characteristics of pituitary GH cells and body weight in near-term rat fetuses

Growth hormone (GH) and glucocorticoids have a powerful influence on controlling fetal growth, differentiation and maturation of numerous tissues. In the present study, the effect of maternal dexamethasone (Dx) treatment on GH cells and body weight in 19- and 21-day-old rat fetuses was investigated using immunocytochemical and morphometric methods. Pregnant female rats received daily injections of 1.0-0.5-0.5 mg Dx/kg b.w. on days 16-18 of pregnancy (experimental group), while the control group received an equal volume of saline. Dx treatment of pregnant rats enhanced immunostaining intensity and significantly increased (p<0.05) GH nuclear and cell volume, as well as volume density and number of GH cells per square millimeter in 19-day-old fetuses compared to the controls. In 21-day-old fetuses after maternal Dx administration, immunoreactivity, volume density and number of GH cells remained significantly increased (p<0.05). Dx treatment of pregnant rats resulted in marked body weight reduction of 21-day-old but not 19 days old fetuses in comparison with the corresponding controls. The presented results demonstrate that maternal Dx application has pronounced effect on morphometric parameters of GH cells of 19- and 21-day-old fetuses. Also, in near-term rat fetuses body weight was largely independent of pituitary GH cell activity.     

Fetal cells in the maternal circulation during first trimester in pregnancies

To investigate the presence of fetal cells in the maternal circulation during early pregnancy, the polymerase chain reaction was used to test the presence of human Y chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood specimens from 19 women carrying male fetuses and 12 women carrying female fetuses. The presence of fetal cells was suggested as early as 6 weeks gestation in 1 of the 19 women bearing male fetuses. Fetal cells were present in the maternal circulation of 15 of the 19 women by 9 weeks gestation, and in only 1 of the 19 were fetal cells not detected until the 12th week after conception. These results suggest that identification of fetal cells in the maternal circulation is possible with a properly designed and executed polymerase chain reaction. However, there was considerable variation with respect to when these fetal cells first became detectable during pregnancy. These fetal cells are potentially a valuable source of material for biochemical and genetic studies of the fetuses.     

Onset of the constrictive effect of indomethacin on the ductus arteriosus in fetal rats.

The time of onset of the constrictive effect of indomethacin on the ductus arteriosus (DA) in fetal rats was assessed by measurement of the caliber of the DA after maternal treatment with indomethacin on days 19-21 of gestation. The day following overnight mating was regarded as day 0 of gestation. Observation was performed by direct exposure of the DA by hand shaving of intact frozen fetuses. On days 20 and 21, the DA was significantly constricted 3 h after maternal treatment with 1 mg/kg of indomethacin. When the DA was examined at 19 1/2 and 19 2/3 days of gestation (3 h after indomethacin exposure), it was significantly constricted at 19 2/3 days but not at 19 1/2 days. Higher doses of indomethacin (10 and 100 mg/kg) induced a significant constriction of the DA at day 19 1/2, but not at the beginning of the same day (1.00 a.m.). These results suggest that the onset of the susceptibility of the DA to the constrictive effect of indomethacin occurs in the first half of day 19 of gestation.     

Effects of Anoectochilus formosanus Hayata extract and glucocorticoid on lung maturation in preterm rats.

We investigated the effects of maternal administration of Anoectochilus formosanus extract and dexamethasone on lung maturation in preterm rats. A. formosanus group mothers were tube-fed A. formosanus extract (300 mg/kg body wt./day) for 7 days from days 12-18 of gestation. Dexamethasone group mothers were injected intraperitoneally with dexamethasone (0.2 mg/kg body wt.) in saline on day 18 of gestation. Control group mothers were similarly injected with saline alone. On day 19 of gestation, fetuses were delivered by cesarean section. A. formosanus treatment significantly increased the fetal lung/body weight ratio, as compared to dexamethasone treatment. Saturated phosphatidylcholine levels in fetal lung tissue and growth hormone levels in maternal serum were significantly increased in the A. formosanus- and dexamethasone-treated groups as compared to controls. The histological appearance of preterm rat lungs revealed extensive branching of intermediate airways, denser mesenchyme, and more epithelial tubules in the dexamethasone and A. formosanus groups as compared with the control group. These results suggest that antenatal A. formosanus treatment may play a role in accelerating fetal rat lung maturation.