Endocrine Regulation of Fetal Adipose Tissue Metabolism in the Pig: Role of Hydrocortisone

Glucocorticoids have been shown to be essential for the excessive fat deposition and development of obesity in several animal models. This study was performed to characterize the role of glucocorticoids in the developmental regulation of adipose tissue metabolism. On day 70 of gestation, pig fetuses were hypophysectomized by micro-cauterization. Hypophysectomized fetuses were implanted subcutaneously with hydrocortisone pellets or received no hormone replacement. Fetuses were removed by laparotomy on day 90 of gestation. Additional fetuses were hypophysectomized on day 70, implanted with hydrocortisone pellets on day 90 and removed on day 105 of gestation. Several intact fetuses were also implanted subcutaneously with hydrocortisone pellets during this later gestational period. Serum cortisol concentrations were reduced in hypophysectomized pigs at both fetal ages and were restored to intact levels by hydrocortisone treatment. Hydrocortisone supplementation enhanced lipolytic response to isoproterenol in intact fetuses but failed to restore lipolytic response to isoproterenol in hypophysectomized animals at either fetal age. Hydrocortisone induced a slight increase in lipogenesis in hypophysectomized fetuses when administered from 70 to 90 days of gestation and a more dramatic increase when administered from days 90 to 105 of gestation. However, hydrocortisone had no effect on basal or insulin stimulated lipogenesis in intact fetuses when administered from days 90 to 105 of gestation. These results indicate that hydrocortisone may have a primary influence on adipose tissue metabolism during late fetal development only in the absence of inhibition from counterregulatory hormones of pituitary origin.     

Nonresponsiveness of the thyroid gland to goitrogen in the early neonatal period in the rat: light- and electron-microscopic observations.

The thyroid response of fetal and neonatal rats to propylthiouracil (PTU) as a goitrogen was studied with observation of the thyroid glands by light and electron microscopy. On day 19 of gestation and on days 1, 3, 5 and 8 after birth, fetal and neonatal rats were given a subcutaneous injection of PTU and were autopsied 2 days later. PTU induced conspicuous goiters in fetal rats but did not in neonatal rats aged up to day 5 after birth. Beyond that age, PTU again induced goiters. Histologically, the follicular cell height in goitrous thyroid glands was significantly increased. Ultrastructurally, follicular cells in goitrous thyroid glands often had colloid droplets and lysosomes. It seems that nonresponsiveness of the thyroid glands in early neonatal rats to goitrogen is due to a temporary decline of the pituitary activity of thyrotropin secretion. About 5 days or more after birth, the pituitary-thyroid system begins to operate again in response to goitrogen.     

The role of hypothalamic input on corticotroph maturation in fetal sheep

Corticotropin-releasing hormone receptor type 1 (CRH-R1) expression and vasopressin type 1b (V1b) receptor protein decrease in late-gestation fetal sheep. Because hypothalamo-pituitary disconnection (HPD) has been demonstrated to prevent the morphological maturation of corticotrophs, we hypothesized that hypothalamic input is necessary for the maturational changes in CRH-R1 and V1b receptor levels. We measured CRH-R1 and V1b receptor expression in the anterior pituitaries of fetuses at 140 days gestational age (dGA) that underwent HPD or sham surgery at 120 dGA. CRH-R1 mRNA decreased similarly in HPD and sham-operated fetuses compared with 120 dGA naive fetuses. However, CRH-R1 protein levels were elevated in HPD fetuses compared with sham and were not different from 120 dGA values. V1b protein levels decreased similarly in HPD and sham-operated fetuses compared with 120 dGA naive fetuses. We conclude that hypothalamic input to the pituitary is necessary for the decrease in CRH-R1 receptor protein levels in late-gestation fetal sheep. However, hypothalamic input is not necessary for the decrease in V1b receptor expression seen in late gestation.     

Circulating insulin-like growth factor-I and -II concentrations are not dependent on pituitary influences in the midgestation fetal sheep

Studies were performed to investigate the possible role of pituitary factors on the regulation of circulating concentrations of insulin-like growth factor-I and -II in the midgestation sheep fetus. Four fetuses were decapitated at 59-64 days of gestation and fetal serum obtained at sacrifice at 90-102 days of gestation. Insulin-like growth factor-I and -II concentrations were similar in these samples to those from 6 control fetuses (83-102 days). A further 4 fetuses were studied following electrolytic destruction of the median eminence of the hypothalamus at 108-110 days of gestation. Four sham operated controls were also studied. Circulating growth hormone concentrations were markedly reduced (P less than 0.01) by destruction of the median eminence. However neither insulin-like growth factor-I nor -II levels differed from those of sham operated fetuses. We conclude that, in the midgestation fetal sheep, growth hormone is not essential for the maintenance of circulating concentrations of insulin-like growth factor-I or -II.     

Pro-opiomelanocortin peptides in the human fetal pituitary

Levels of immunoreactive pro-opiomelanocortin (POMC) peptides (N- and C-terminal ACTH, N- and C-terminal LPH and α-MSH) have been measured in pituitary extracts from human fetuses of 12–22 weeks gestation. The levels of ACTH were 30–200 times higher than α-MSH in all fetuses studied. Sephadex G-75 and G-25 chromatography of 8 extracts showed peaks of 34 kilodaltons (K) POMC, 22K ACTH, β-LPH, γ-LPH, β-endorphin, approximately 8K ACTH, 1–39 ACTH, α-MSH and CLIP. The 8K and 22K forms of ACTH are both partly glycosylated.In vitro culture of pituitaries from 2 fetuses (22 and 26 weeks gestation) gave a detectable basal output of ACTH but not of α-MSH. Stimulation of these pituitary cells with human fetal and rat hypothalamic extracts and with synthetic ovine CRF-41 produced a significant increase in ACTH release, and either small or undetectable amounts of α-MSH.These results demonstrate the presence of POMC-related peptides in early gestation human fetal pituitaries and suggest that ACTH, and not α-MSH, is the major corticotrophic hormone at this stage of gestation.     

Effect of intravenous 5-hydroxytryptophan on hypothalamic concentration of norepinephrine, dopamine, serotonin and hydroxyindole acetic acid in the fetal lamb

To investigate the neurochemical mechanism of the response of growth hormone to 5-hydroxytryptophan (5-HTP), we administered 5-HTP (20 mg/kg) to 10 ovine fetuses (110 or 130 days old; term gestation 147 days). Ninety minutes after 5-HTP administration, and following increases in plasma growth hormone concentrations, the fetus was delivered by hysterotomy. After local anesthesia of the fetus and sacrifice by cervical spinal cord transection the hypothalamus rapidly dissected, and stored at -80 degrees C for later analysis of norepinephrine, dopamine, serotonin and hydroxyindole acetic acid. Compared to the administration of saline, 5-HTP caused a significant increase in the hypothalamic content of serotonin, and norepinephrine, at both gestational ages. 5-hydroxyindole acetic acid increased significantly only in the older fetuses. These results indicate that serotonin may not be the only neurotransmitter active in the growth hormone response to 5-HTP.     

Thyroid hormone modulates renin and ANG II receptor expression in fetal sheep

Fetal renin-angiotensin system (RAS) activity is developmentally regulated, increasing in late gestation toward term. At the same time, fetal hemodynamic parameters change, with blood pressure increasing and heart rate decreasing. During this period, fetal plasma thyroid hormone concentrations also increase significantly. In this study we utilized the technique of thyroidectomy (TX), which removes thyroid hormone from the circulation, to investigate the importance of thyroid hormone on the developmental changes in the RAS (in plasma, kidney, heart, and lung) and hemodynamic regulation in fetal sheep. TX was performed at 120 days of gestational age (dGA), and control fetuses were sham operated. Immediately before necropsy ( approximately 137 dGA), fetuses were infused with isoproterenol and the hemodynamic responses were noted. TX significantly decreased plasma thyroid hormone concentrations and renal renin mRNA and renal active renin levels but did not change fetal plasma active renin levels. TX decreased both angiotensin II receptor subtype 1 (AT1) mRNA and protein levels in kidney and lung but not in the left ventricle. TX also was associated with increased ANG II receptor subtype 2 (AT2) mRNA and protein at the 44-kDa band in kidney, whereas AT2 protein was decreased at the 78-kDa level in kidney and lung tissue only. TX fetuses had significantly lower basal mean arterial blood pressures (MAP) and heart rates than controls. Isoproterenol infusion decreased MAP in TX fetuses. These findings support the hypothesis that thyroid hormone is important in modulating maturation of RAS and cardiovascular function in the late-gestation fetal sheep.     

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.     

Studies on the growth of the fetal guinea pig. Changes in plasma hormone concentration during normal and abnormal growth

The endocrine changes associated with fetal growth retardation caused by unilateral uterine artery ligation of guinea pigs at day 30 of pregnancy were studied. Plasma hormone levels in fetuses that, about 20 or 30 days later, were 35-50% of normal size were measured by radioimmunoassay. The small fetuses were severely hypoglycaemic and hypoinsulinaemic; both showing close correlation and relationship to the degree of growth retardation. Plasma thyroid and cortisol and concentrations were much lower than normal and that glucagon and androstenedione were much higher. Plasma growth hormone level appeared to be unaffected by growth retardation. The developmental changes in glucagon and thyroid hormone concentrations were consistent with a delay in the timing of prenatal events in growth-retarded fetuses. However the late cortisol rise, although somewhat blunted, still occurred at 58-60 in the small fetal guinea pigs.     

Effects of simulated high altitude (3800 m) on reproductive function in the pregnant rat.

Ovarian appearance, uterine contents (including numbers of fetuses, size and weight), and levels of gonadotropic hormones were studied in pregnant rats exposed to simulated altitude (3800 m). Comparisons were made at 11, 20 and 21 days of gestation between sea level controls rats bred at sea level and placed at reduced barometric pressure on confirmation of pregnancy (SHA). Although fewer fetuses were present in SHA rats on day 11 of gestation, litter size at days 20 and 21 was similar to controls. Body weight and length of SHA fetuses were significantly reduced by day 21 of gestation while placental weights remained similar to controls. The follicle-stimulating hormone (FSH) content in the pituitaries of SHA rats was significantly reduced at days 11 and 21 of pregnancy as compared with controls; luteinizing hormone (LH) content was lower at day 20 than at day 11 among SHA rats. Plasma gonadotropin levels were similar between SHA and control rats all all three stages of gestation. It is suggested that, despite lack of placental adaptation and altered endocrine functions, exposure of rats to hypoxic conditions only during gestation permits better maintenance of pregnancy than reported in rats with longer exposure to high altitude.     

Light and electron microscopic localization of ACTH and pro-opiomelanocortin-derived peptides in human developmental and neoplastic cells

Oncofetal aspects of ACTH and pro-opiomelanocortin (POMC)-derived peptides were studied immunohistochemically at the light and electron microscopic level in human fetal pituitary glands, pituitary adenomas, and small-cell carcinoma of the lung. ACTH, beta-endorphin, and gamma-MSH were localized in the same cells of both fetal and adult pituitary, as well as in the above-mentioned neoplastic tissues. However, alpha-MSH was observed only in the early fetal pituitary, its concentration decreasing with advancing gestational age. The adult pituitary contained only a few alpha-MSH-positive cells. By immunoelectron microscopy, ACTH in the adult pituitary was localized exclusively in the secretory granules. In fetal pituitary at 9 weeks' gestation, ACTH was localized in the perinuclear spaces (PNS), cisternae of rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules. The staining pattern of ACTH in these organelles varied from cell to cell. In fetal pituitaries of greater gestational ages, ACTH was localized in secretory granules. The pituitary adenomas mimicked the staining characteristics of the adult pituitary, i.e., negative or only very occasional alpha-MSH staining and localization of ACTH in the secretory granules. The ectopic ACTH-producing tumors showed a staining pattern similar to that of the early fetal pituitary, i.e., positive staining for alpha-MSH and the presence of ACTH in PNS and cisternae of RER.     

Inhibitory effects of a luteinizing-hormone-releasing hormone agonist implant on ovine fetal gonadotrophin secretion and pituitary sensitivity to luteinizing-hormone-releasing hormone.

Sheep fetuses at day 70 of gestation (term = 145 days) were implanted subcutaneously with a biodegradable implant containing a luteinizing-hormone-releasing hormone (LHRH) agonist (buserelin) to investigate whether treatment with LHRH agonist would induce a state of desensitization of the fetal gonadotrophs and thus influence fetal gonadal development. Treatment with the LHRH agonist for 35-40 days caused a significant reduction in mean fetal plasma concentrations of LH and follicle-stimulating hormone (FSH) compared with control fetuses. LH pulses were evident in control fetuses but were completely abolished by buserelin treatment. Furthermore, the pituitary content of LH and FSH was significantly depleted in fetuses implanted with LHRH agonist. A bolus intravenous injection of 500 ng LHRH given to control fetuses caused a rapid and significant increase in plasma LH and FSH concentrations which was sustained for at least 60 min after injection. Pretreatment with buserelin completely abolished the LH and FSH responses to a bolus injection of LHRH. There were no differences between the sexes in fetal gonadotrophin concentrations or pituitary sensitivity to LHRH in control or agonist-treated fetuses. Furthermore, buserelin treatment for 35-40 days had no effect on the morphological appearance of the fetal gonads when compared with control fetuses, at least to day 110 of pregnancy. These results provide evidence for the induction of a state of desensitization of the LHRH receptors of the fetal pituitary gonadotrophs following long-term treatment with an LHRH agonist, but provide no evidence for a role for gonadotrophin secretion in gonadal development at this stage in fetal life.     

Measurement of messenger RNA for luteinizing hormone beta-subunit and alpha-subunit during gestation and the postpartum period in ewes

Pituitary content of luteinizing hormone (LH) and mRNAs for LH beta-subunit (LH beta), alpha-subunit, prolactin, and growth hormone were measured in ewes on Days 50 and 140 of gestation and on Days 2, 13, 22, and 35 postpartum. Content of LH in dissociated anterior pituitary cells declined (P less than 0.05) between Days 50 and 140 of gestation and remained low at 2 days postpartum. By 22 days postpartum, pituitary concentrations of LH were comparable to concentrations in normally cycling ewes. During gestation concentrations of mRNA for LH beta and alpha-subunit paralleled changes in cellular content of LH, reaching minimal levels on Day 140. By Day 2 postpartum, pituitary concentrations of mRNAs for LH beta and alpha-subunit began to increase; they reached maximum levels by Day 13 postpartum. There appeared to be a gradual linear increase in mRNA for prolactin through gestation and the postpartum period. No changes in mRNA for growth hormone were noted during the prepartum or postpartum periods. These data suggest that the decline in pituitary concentrations of LH during gestation is due to a decrease in cellular mRNA for LH beta and alpha-subunit. The increase in mRNA for LH beta and alpha-subunit appears to precede an increase in cellular content of LH in the postpartum ewe by several days.     

Pulmonary feedback and gestational age-dependent regulation of fetal breathing movements

Prenatal lung development requires fetal breathing movements (FBM). To investigate the dependence of FBM on feedback originating from the lung, we hypothesized that pneumonectomy stimulates FBM. Time-dated fetal sheep underwent bilateral pneumonectomy, unilateral pneumonectomy, or sham surgery at 125-130 days gestation. The incidence of FBM decreased in sham-operated fetuses at 142 days versus 130 days (p = 0.013), but was unchanged across all gestational ages in bilaterally pneumonectomized fetuses (p > or = 0.52). In unilaterally pneumonectomized fetuses, the incidence of FBM remained unchanged until 139 days and was higher than that of the bilaterally pneumonectomized fetuses at 130-136 days gestation (p < or = 0.03). The amplitude of integrated diaphragmatic electromyographic activity (integralEMG(di)) and total respiratory output (frequency of breathing x integralEMG(di)) were lower in pneumonectomized fetuses versus sham-operated fetuses at later gestational ages (p < 0.05). These decreases in integralEMG(di) and total respiratory output were most pronounced at 142 days in bilaterally pneumonectomized fetuses versus sham-operated fetuses (p = 0.006 and 0.016, respectively). Low-voltage electrocortical activity (ECoG) increased, and high-voltage ECoG decreased, in unilaterally pneumonectomized fetuses compared with sham-operated fetuses (p = 0.04). In conclusion, we provide new evidence that feedback from the fetal lung modulates the incidence and various components of phrenic nerve output, suggesting a positive feedback mechanism between FBM and lung development.     

Nutritional control of respiratory and other muscular activities in relation to plasma prostaglandin E in the fetal sheep

The effects of nutrient availability on fetal plasma prostaglandin E (PGE) concentrations, on fetal breathing movements and electromyographic (EMG) activities of fetal nuchal and forelimb muscles were investigated in pregnant ewes by varying dietary intake and by manipulation of fetal plasma glucose concentration. The incidence of fetal breathing movements (06.00-10.00 h) decreased with increasing gestational age while fetal arterial concentrations of plasma PGE increased significantly over the same period of gestation. Maternal fasting for 48 h reduced the incidence of fetal breathing movements and the amount of nuchal EMG activity (06.00-10.00 h) in animals older than 130 days but had no effect earlier in gestation. No changes in forelimb EMG activity were observed during fasting at any gestational age. Plasma PGE levels increased significantly during fasts begun both before and after 130 days of gestation. When data from fed and fasted states were combined for all fetuses, irrespective of gestational age, there was a significant inverse correlation between fetal breathing movements incidence and plasma PGE concentration in utero. This relationship was even more pronounced when the fetuses were considered individually. Insulin infusions induced hypoglycaemia, an increase in fetal plasma PGE concentration and a significant reduction in the incidence of fetal breathing movements at all ages. Glucose infusions of fetal breathing movements only after 130 days and had no effect on plasma PGE levels in utero at any gestational age. Neither insulin nor glucose infusions altered the EMG activities of the nuchal and forelimb muscles. The results show that glucose availability is an important factor in determining the incidence of fetal breathing movements in utero and indicate that nutritionally induced changes in fetal breathing movements are mediated in part by PGE. They also suggest that PGE is a physiological regulator of fetal breathing movements in the sheep during late gestation.     

Maturation of the pituitary-thyroid axis during the perinatal period.

To clarify the maturation process of the pituitary-thyroid axis during the perinatal period, thyrotropin (TSH) response to thyrotropin releasing hormone (TRH) and serum thyroid hormone levels were examined in 26 healthy infants of 30 to 40 weeks gestation. A TRH stimulation test was performed on 10 to 20 postnatal days. Basal concentrations of serum thyroxine (T4), free thyroxine (free T4) and triiodothyronine (T3) were positively correlated to gestational age and birth weight (p less than 0.001-0.01). Seven infants of 30 to 35 gestational weeks demonstrated an exaggerated TSH response to TRH (49.7 +/- 6.7 microU/ml versus 22.1 +/- 4.8 microU/ml, p less than 0.001), which was gradually reduced with gestational age and normalized after 37 weeks gestation. A similar decrease in TSH responsiveness to TRH was also observed longitudinally in all of 5 high responders repeatedly examined. There was a negative correlation between basal or peak TSH concentrations and postconceptional age in high responders (r = -0.59 p less than 0.05, r = -0.66 p less than 0.01), whereas in the normal responders TSH response, remained at a constant level during 31 to 43 postconceptional weeks. On the other hand, there was no correlation between basal or peak TSH levels and serum thyroid hormones. These results indicate that (1) maturation of the pituitary-thyroid axis is intrinsically controlled by gestational age rather than by serum thyroid hormone levels, (2) hypersecretion of TSH in preterm infants induces a progressive increase in serum thyroid hormones, and (3) although there is individual variation in the maturation process, the feedback regulation of the pituitary-thyroid axis matures by approximately the 37th gestational week.     

Fine structure of, and ACTH production by, human fetal pituitaries taken at different periods of gestation. An in vitro study

Pituitaries were taken from human fetuses between the 6th and 30th weeks of gestation. Organ and monolayer cultures were prepared. The fine structure of the cultures was examined by electron microscopy and their basal and stimulated ACTH release were studied by radioimmunoassay as a function of time in vitro. It was shown that pituitaries taken from the first trimester of gestation have a capacity of self-differentiation; i.e. there was an increase in the number of cells filled with secretory granules and there was an increase in the number of granules per cell. In contrast, pituitaries taken from older embryos have been losing their secretory granules during the cultivation. We failed to demonstrate any corticotropin responsiveness in pituitary cultures prepared from the 6 to 7-week-old embryos. ACTH release could be stimulated from the 10th week of gestation but in slight measure. Pituitary taken from 16-week-old fetuses revealed an adult-like responsiveness to corticotropin.     

Sensitivity of the fetal rat pituitary-adrenal system to corticotropin-releasing factor in organ culture.

Six groups of adrenal glands from 17-day fetal rats were explanted to organ culture for 2 days. In one group, adrenal gland was cultured alone, and in the remaining five groups adrenal gland was cultured with pituitaries from fetuses ranging in age from 14 to 18 days. In each of the groups, half of the cultures had corticotropin-releasing factor (CRF) added to the medium. A histometric parameter utilized the size of adrenocortical cells as an indicator of sensitivity of the pituitary-adrenal system. When 17-day adrenal gland was cultured alone, addition of CRF did not cause any enlargement of cortical cells. When the adrenal gland was cultured with two 14-day pituitaries, cortical cells were enlarged. Addition of CRF to this culture induced no further change. With two 15-day pituitaries in the presence of CRF, cortical cells were slightly larger than those in the absence of CRF. With 16- to 18-day pituitaries, a marked hypertrophy of cortical cells was induced, and the addition of CRF caused further acceleration in their enlargement. These results suggest that, in organ culture, 14-day pituitary can release some adrenocorticotropic hormone (ACTH) with or without additional CRF. Older pituitaries (16- to 18-day) can apparently release an amount of ACTH in the presence of CRF that is greater than their own spontaneous ACTH secretion.     

Ontogeny of pituitary responsiveness to corticotropin-releasing hormone in rat

The ontogeny of the pituitary's responsiveness to synthetic rat corticotropin-releasing hormone (CRH) in the late prenatal and early postnatal periods of rats was studied by a superfusion system using whole pituitaries. A significant increase of immunoreactive beta-endorphin (IR-beta-Ep) secretion in response to 10(-10) M CRH but not to 10(-11) M CRH was observed in pituitaries from the 15th day of gestation, the earliest day that we tested, whereas 10(-11) M CRH stimulated IR-beta-Ep release from the pituitaries of 17.5-day-old fetuses. Dose-related IR-beta-Ep secretions induced by 10(-12) M to 10(-10) M CRH were observed in pituitaries of 19.5- and 21.5-day-old fetuses, and 1-, 3- and 9-day-old newborn pups. CRH stimulated not only IR-beta-Ep and IR-adrenocorticotropic hormone (ACTH) but also IR-alpha-melanocyte-stimulating hormone (IR-alpha-MSH) secretions from fetal pituitaries. The content of IR-CRH in the hypothalamic extract from 15-day-old fetus was 6.6 +/- 3.6 pg/hypothalamus (mean +/- S.E.M.) and it gradually increased to reach 212.7 +/- 20.3 pg/hypothalamus on the 21.5th day of gestation. However, the content of IR-CRH in the hypothalamus dramatically decreased just after birth and then rapidly increased again from the 5th day after birth. These data indicate that the responsiveness of corticotrophs to CRH is already present on the 15th day of gestation, when the content of IR-CRH in the hypothalamus is extremely low and that the amount of hypothalamic IR-CRH dramatically dropped for several days just after birth in rats.