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.     

Corticotropin releasing hormone concentrations in umbilical cord blood of preterm fetuses.

Corticotrophin releasing hormone (CRH), dehydroepiandrosterone sulfate (DHEAS) and cortisol were measured in umbilical cord plasma obtained from 90 preterm and 98 term fetuses. Maternal plasma was obtained from 23 women who delivered preterm and from 23 women matched for gestational age who ultimately delivered term infants. Mean umbilical cord plasma CRH concentration was significantly higher in the preterm fetuses (n = 69, 538 +/- 63 pg/ml) compared to the term fetuses (n = 98, 280 +/- 22 pg/ml, P < 0.01). Mean DHEAS level in the preterm fetuses was 208 +/- 22 mg/dl (n = 56), cortisol level was 7 +/- 1 mg/dl (n = 58). Umbilical plasma CRH concentrations (808 +/- 170 pg/ml) were significantly higher at 24-27 weeks than at 28-31 or 31-34 weeks gestation. Cortisol levels (12 +/- 3 micrograms/dl) were highest at 24-27 weeks. Mode of delivery and the presence of labor did not affect fetal CRH levels. The highest fetal CRH levels were measured in the pregnancies complicated by hypertension as well as prematurity; however, fetal CRH levels remained higher in the preterm group compared to the term group when hypertensive pregnancies were excluded. Maternal plasma CRH levels were significantly higher in the group that delivered preterm compared to women who delivered at term matched for gestational age (1058 +/- 184 pg/ml compared to 456 +/- 71 pg/ml, P < 0.00).(ABSTRACT TRUNCATED AT 250 WORDS)     

Luteinizing hormone-releasing hormone in thymus and hypothalamus of rat fetuses: suppressing effect of antagonist and of antibodies on concanavalin A-induced proliferation of thymocytes

The effect of endogenous luteinizing hormone-releasing hormone (LHRH) on the proliferation induced by concanavalin A (Con A) in rat fetal thymocytes was studied. A selective antagonist (2 microg per fetus) or antibodies to LHRH (20 microl per fetus) were injected in utero into 20-day-old rat fetuses, and this resulted in a two- or fivefold decrease in the Con A-induced proliferation of thymocytes, respectively. In combined culture of the antagonist (10-5-10-6 M) with fetal thymocytes, the proliferative response was not decreased. The concentration of LHRH was determined by radioimmunoassay in tissues of immunocompetent organs and in blood serum of 18- and 21-day-old fetuses, and the hormone was found in the hypothalamus, thymus, and peripheral blood. The initially low level of LHRH in the thymus increased by 65 and 40%, respectively, on the first day after birth and became similar to the level in the hypothalamus. In the fetal blood serum, the LHRH level was significantly higher than in the thymus and hypothalamus of fetuses of the same age. The hormone concentration was greatest in the 18-day-old fetuses, and it decreased twofold by the 21st day. The findings indicate that LHRH is involved in regulation of T-cell immunity even during prenatal ontogenesis.     

Corticotrophin-releasing factors in the hypothalamus of the developing fetal sheep.

Corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from the hypothalamic median eminence to elicit the secretion of ACTH from the pituitary corticotrophs. During fetal development there is progressive maturation of the hypothalamic-pituitary-adrenal axis, manifest as increasing plasma ACTH and cortisol concentrations, which in species such as sheep culminates in the onset of birth. However, the precise nature of the hypothalamic signal controlling fetal pituitary ACTH secretion remains poorly understood. To investigate the ontogeny of this hypothalamic signal, the present study examined immunoreactive and bioactive ACTH-releasing factors in the developing fetal sheep hypothalamus. Immunoreactive CRH and AVP were measured by radioimmunoassay in extracts of hypothalami taken at day 70, day 100, and day 130 gestation (term = 145 days). There was a progressive and significant (P < 0.01) increase in hypothalamic CRH and AVP concentrations which was particularly marked between d100 and d130 gestation. AVP was always present in higher concentrations that CRH, although this difference was significantly reduced by day 130 gestation as the result of a large increase in the content of CRH relative to AVP. Sephadex G50 chromatography revealed that immunoreactive CRH and AVP in hypothalamic extracts existed as single molecular forms corresponding to synthetic peptides at each gestational age. In addition, these immunoreactive forms of CRH and AVP possessed significant ACTH-releasing bioactivity as measured in primary cultures of adult sheep anterior pituitary cells. Furthermore, significant bioactivity was present in high and low molecular weight fractions eluted after chromatography which did not contain any CRH or AVP immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ghrelin is released from rat hypothalamic explants and stimulates corticotrophin-releasing hormone and arginine-vasopressin.

Ghrelin and synthetic growth hormone secretagogues have diverse effects on the hypothalamus including effects on appetite and the growth hormone axis as well as on the hypothalamus-pituitary-adrenal (HPA) axis. We previously studied the effect of synthetic growth hormone secretagogues on CRH and AVP release from rat hypothalami in vitro, and now report on the effects of ghrelin on CRH and AVP release. The ghrelin protein content and ghrelin output from rat hypothalamic explants was measured using a specific novel ghrelin enzyme immunoassay. The effect of 10(-8) M to 10(-6) M ghrelin on CRH and AVP release was studied in the rat hypothalamic explants, where stimulation with des-octanoyl ghrelin was used as control. The presence of both ghrelin mRNA and protein could be shown in the rat hypothalamus. Ghrelin output was detected in the incubation fluid of rat hypothalamic explants and could be stimulated with high potassium concentrations. Our data also demonstrated a dose-dependent effect of ghrelin on both CRH and AVP release, while des-octanoylated ghrelin showed no effect on either peptide. In summary, the current data suggest that ghrelin is expressed in the hypothalamus both at RNA and the protein levels. Ghrelin stimulates the HPA axis in the rat via stimulation of both CRH, and particularly, AVP release from the hypothalamus. The local autocrine/paracrine and endocrine effects of ghrelin in the hypothalamus could influence all the hormonal systems involved in ghrelin effects, including growth hormone release, the HPA axis and appetite.     

Changes in testosterone levels in rabbit embryo testes after removal of the hypothalamus (encephalectomy)

Brain hypothalamus region was removed by encephalectomy in utero in 21-23-day-old rabbit fetuses to find out when hypothalamic control over testicular androgenous function was set up during rabbit prenatal development. Testosterone concentration in testicular tissue was measured by radioimmunoassay at different terms after the operation. Encephalectomy resulted in a reliable decrease of testosterone concentration in 29- and 25-day-old fetuses, however, no significant changes were observed in 23-day-old fetuses. The greatest reduction in hormone gland concentration was noted in 25-day-old fetuses, encephalectomized on day 23 of their development. Introduction of LH-RH to 25-day-old encephalectomized fetuses 30 min before fixation significantly increased androgen concentration in the gland. The data obtained indicate that hypothalamic control over testicular androgenous function is set up on days 23-25 of prenatal development.     

Pituitary and hypothalamic hormones in normal and neoplastic adrenal medullae: biologically active corticotropin-releasing hormone and corticotropin

Six normal and 8 neoplastic adrenal medullae were assayed for several immunoreactive (IR) proopiomelanocortin (POMC) and hypothalamic peptides. IR-POMC peptides were found in normal and tumor tissue in concentrations ranging from 0.0003 to 0.1% of those in pituitary. Their molecular sizes resembled those of pituitary intermediate lobe POMC peptides. No intact POMC was found. One pheochromocytoma contained fully bioactive IR-adrenocorticotropic hormone (IR-ACTH; Mr approximately 4,500) and an intermediate-sized (Mr approximately 10,000) IR-ACTH with approximately 69% bioactivity. Normal and tumorous medullae contained IR-corticotropin-releasing hormone (CRH) in concentrations ranging from 0.6 to 4% of those in hypothalamus except for one pheochromocytoma that contained 40 times that amount of IR-CRH, which was chromatographically indistinguishable from hypothalamic CRH and fully bioactive. IR-somatostatin and IR-growth hormone-releasing hormone were found in both tissue types, but IR-gonadotropin-releasing hormone and IR-thyrotropin-releasing hormone (TRH) were not, although IR-histidyl-proline diketopiperazine, a putative TRH metabolite, was found. IR-arginine vasopressin was found in two normal medullae, but not in pheochromocytomas.     

Influence of endogenous growth hormone-releasing factor (GRF) on the secretion of GH during the perinatal period in the rat

Passive immunization of pregnant rats with a specific antiserum to rat GRF (GRF-AS) is followed by a decrease in fetal serum GH on the 19th day of gestation. A significant reduction in serum GH is still observed in older fetuses and newborn rats. Pituitary GH content increases in 19- and 20-day-old fetuses after GRF-AS administration to their mothers. These results suggest that endogenous fetal hypothalamic GRF (or placenta GRF) play a physiological role in the secretion of pituitary GH as early as the 19th day of fetal life and may be responsible for the peak of GH release that occurs in fetuses at the end of gestation.     

Radioimmunoassay of testosterone in late fetal and newborn rabbit plasma, gonads and adrenal glands]

A radioimmunoassay was used for measuring testosterone in the plasma, gonads and adrenals of 28, 29, 30 and 31-day-old rabbit fetuses of both sexes and newborns. A marked sex difference was shown in the concentrations of testosterone in plasma and in gonads whereas in adrenals the levels of testosterone were low in both sexes (34 to 147 pg/10 mg). In male fetuses, plasma testosterone levels increased from the 28th (133 +/- 20 pg/ml) to the 31st day (361 +/- 119 pg/ml) of intrauterine life, reaching then the values observed in the newborns (387 +/- 73 pg/ml). Plasma from males, on the other hand contained, at all stages studied, significantly more testosterone than plasma from female fetuses (21 +/- 6 to 41 +/- 11 pg/ml) and female newborns (42 +/- 6 pg/ml). In the same way, fetal testicular testosterone concentrations varying from 1 382 +/- 218 to 2 317 +/- 333 pg/10 mg were similar to those measured in the newborns (1 940 +/- 304 pg/10 mg) and significantly higher than fetal (13 to 34 pg/10 mg) or neonatal (44 pg/10 mg) ovarian concentrations. These results showed at evidence the endocrine activity of the fetal testis during this period.     

Hypothalamic influence on differentiation of the immunoreactive ACTH beta-LPH, alpha- and beta-endorphin containing cells in adenohypophysial primordia of rat fetus in organ culture (author's transl)]

Adenohypophysial primordia were isolated in rat fetuses from day 12.5 to day 15.5 of gestation. The organ culture employed for maintenance of the primordia was made up according to Watanabe et al. (1973). The fixation of primordia in Bouin Hollande's solution was performed after 9, 8, 7 or 6 days of culture when the normal duration of pregnancy was achieved. The cultivated primordia were immunologically studied using different antisera: anti-alpha(17-39)ACTH, anti-beta(1-24)ACTH, anti-beta-LPH, anti-alpha and anti-beta-endorphins, with immunoperoxidase or immunofluorescence techniques, including control experiments of the specificity of the antisera. A similar study was performed on pituitaries removed from normal rat fetuses from day 16.5 of gestation and each day up to birth, and fixated immediately. In vivo the first cells reacting with all the antisera used in this study were observed on day 16.5 of gestation; their number increased during gestation (Fig. 1 A, B and C). Immunoreactive cells with the different antisera could be detected in primordia isolated on day 12.5 of gestation after 9 days of culture. Numerous groups of cells were observed in primordia of older fetuses (Fig. 2 A and B). These data indicate that the corticotropic cells in rat fetuses could start to be differentiated without stimuli from the hypothalamus since primordia were isolated before the appearance of this cell type in normal rat fetuses and before the differentiation of the hypothalamus. The presence of ACTH and other peptides such as beta-LPH or beta-endorphin would support the hypothesis of a common precursor in this cell type existing early in gestation. Similar results were obtained in human fetuses.     

Ontogeny of androgen receptors in fetal guinea pig brain

Sexual differentiation of the guinea pig brain is androgen dependent. To understand the cellular mechanisms of androgen action, we studied the ontogeny of cytosolic (ARc) and nuclear (ARn) androgen receptors in the brains and anterior pituitaries of fetal, neonatal, and adult guinea pigs. Using cytosol from the hypothalamus-preoptic area-amygdala-septum of 60- to 65-day fetuses and nuclear preparations from 6-day-old neonates treated with testosterone propionate, validation studies revealed an AR with an apparent Kd of 1.9 +/- 1.1 (mean +/- SEM, n = 3) x 10(-10) M (ARc) and 3.4 +/- 3.2 (n = 3) x 10(-10) M (ARn). The cytosolic receptors were highly specific for androgens. After assay validation, AR content was determined from specific brain regions of fetuses obtained on Days 30, 40, 50, and 59 of gestation and on Days 6 and 120 postpartum. ARc differed significantly (p less than 0.05) between brain regions and times of gestation, but no sex differences were apparent. In contrast, ARn showed little difference between tissues or with gestational age, but there were significant differences between males and females, especially in late gestation and early postnatal life, with males having greater ARn binding (p less than 0.05). These data demonstrate the presence of ARc and ARn in the fetal brain and pituitary gland during the critical period of sexual differentiation (Days 30-37 of gestation), thus establishing the identity of cellular structures involved in androgen action.     

The role of monoamines in female puberty

The estradiol positive feedback mechanism appears to become mature between days 10 and 20 after birth. Rising serum prolactin levels between day 20 after birth and puberty are correlated with high hypothalamic norepinephrine turnover. High prolactin levels stimulate hypothalamic dopamine (DA) turnover, which may actively inhibit hypothalamic luteinizing hormone-releasing hormone (LHRH) release. Hypothalamic DNA receptor sensitivity is high in 10- to 20-day-old rats and gradually decreases between day 20 after birth and puberty. The reason for this desensitization may be the high hypothalamic DA turnover. This may result in a less strong inhibition of LHRH release allowing the positive feedback action of estradiol to elicit the first preovulatory luteinizing hormone (LH) surge initiating puberty.     

Haematological values during normal reproduction of the maternal and the fetal rabbit

1. Haematological values of non-pregnant/non-lactating, pregnant as well as lactating rabbits and 28-day-old fetuses were measured. 2. The haemoglobin content in does decreased during the observed periods from 122 +/- 8 g/l to 100 +/- 11 g/l. In 28-day-old fetuses it was 85 +/- 0 g/l. 3. The erythrocyte count in 28-day-old fetuses was 2.4 X 10(12)/l. In the does, the erythrocyte count was 5.2 X 10(12)/l in week 4 of gestation. The erythrocyte volume in fetuses was about 45% higher than that of the doe. 4. In fetuses the leucocyte count was approximately one ninth that of the mother in week 4 of gestation (0.41 +/- 0.08 X 10(9)/l vs 3.8 +/- 0.4 X 10(9)/l).     

Reduced weight in mice offspring after in utero exposure to 2450-MHz (CW) microwaves

Time-bred CD-1 mice (100) were sham-irradiated or irradiated with 2450-MHz (CW) microwaves at 28 mW/cm² for 100 minutes daily from the 6th through 17th day of gestation. The offspring were examined either as fetuses after hysterotomy on the 18th day of gestation or as naturally born neonates on the 1st and 7th day of age. Fetuses of half of the dams were examined on the 18th day of gestation. The incidence of pregnancy and the numbers of live, dead, resorbed, and total fetuses were similar in both groups. The mean weight was significantly lower (10%) in live microwave-irradiated fetuses, and ossification of sternal centers was significantly delayed. In the offspring that were born naturally, the mean weight of microwave-irradiated 7-day-old suckling mice was significantly lower (10%) than that of the sham-irradiated group. Survival rates of neonates in these two groups were not different. These data demonstrate that the decreased fetal weight seen in microwave-irradiated mice is retained at least 7 days after birth. Evidence from other published studies is presented to show that the retarded growth is persistent and might be interpreted as permanent stunting.     

Effects of restricting uteroplacental blood flow on concentrations of corticotrophin-releasing hormone, adrenocorticotrophin, cortisol, and prostaglandin E2 in the sheep fetus during late pregnancy.

We have examined the effects of reduced uterine blood flow and prolonged fetal hypoxemia on the temporal relationship between changes in hormones associated with the activity of the pituitary-adrenal axis (corticotrophin-releasing hormone (CRH), adrenocorticotrophin (ACTH), cortisol, and prostaglandin E2 (PGE2) in the ovine fetus at 120-125 days of pregnancy, and we sought evidence for placental secretion of CRH and ACTH during prolonged hypoxemia. Uterine blood flow was reduced by placing an adjustable Teflon clamp around the maternal common internal iliac artery to decrease fetal arterial oxygen saturation from mean values of 59.1 +/- 3.3 to 25.7 +/- 4.6% (+/- SEM, n = 10). There was a transient peak in immunoreactive (IR-) CRH at 1-2 h after reducing uterine blood flow. IR-ACTH rose to peak values at +2 h, then gradually decreased to control level by +12 h. Fetal plasma cortisol and PGE2 concentrations were elevated significantly by +2 and +4 h, respectively, and at 20-24 h. The identity of IR-CRH in fetal plasma and in ovine placental extracts was confirmed by HPLC, but there was no consistent umbilical vein--femoral arterial concentration difference for either IR-CRH or IR-ACTH during normoxemia or hypoxemia. We conclude that a sequence of endocrine changes involving CRH, ACTH, PGE2, and cortisol occurs in the fetus during a prolonged reduction in uterine blood flow. However, we did not obtain evidence, for placental secretion of either CRH or ACTH in response to this manipulation.     

Ontogeny of neuropeptidergic systems: luteinizing hormone releasing hormone (LHRH); somatostatin (SRIF) and neurophysin (NF) in the hypothalamus of the domestic pig by immunocytochemistry

The ontogenic development of some hypothalamic neuropeptides: luteinizing hormone releasing hormone (LHRH); somatostatin (SRIF) and neurophysin (NF) and their localization in the hypothalamus of fetuses in different stages of the fetal life were studied by immunoperoxidase method. It was found that differentiation of the neurons which produce the examined hormones begins in the midstage of pregnancy. LHRH is stored in the nerve terminals of the median eminence (ME) and organum vasculosum of the lamina terminalis (OVLT) since 72 day of gestation and its amount gradually increases with the development of the embryo. In this stage a few immunoreactive (ir) LHRH perikarya appear but they are most numerous in the last days of pregnancy (110 day). They are localized in the most anterior periventricular parts of the hypothalamus, area preoptica, diagonal band of Broca and very rare in the medial-basal hypothalamus. Somatostatin is produced in the separate neuronal system and appears in the last days of fetal life. Neurophysin is present in both magnocellular nuclei in 72 day-old fetuses, but at the end of gestation it is seen also in some preoptico-septal region.     

The corticotropin releasing hormone gene is expressed in human placenta

Maternal plasma immunoreactive corticotropin-releasing hormone (IR-CRH) increases progressively with pregnancy. This elevated plasma IR-CRH is presumably secreted by the placenta. To investigate further this hypothesis, we searched for the CRH mRNA and its peptide product in full term human placentae. Using a radiolabelled 48-mer oligonucleotide probe complementary to a portion of human CRH mRNA, we identified a 1300 nucleotide RNA from human placenta and rat hypothalami. We next examined the chromatographic characteristics of the placental IR-CRH. The bulk of the IR-CRH extracted from placenta and the IR-CRH secreted in vitro by placental fragments had the same chromatographic profiles as synthetic CRH. These findings indicate that the CRH gene is expressed in human placenta and imply that this organ is a site of CRH biosynthesis 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.     

Development of gastric sensitivity to histamine in the rat

Sensitivity of the developing rat stomach to histamine (HA) was examined on isolated gastric mucosae of rats of various ages from the fetal to adult periods. Spontaneous acid secretion in mu eq/h.cm2 occurred at all the ages studied, at a basal rate of 0.45 +/- 0.07 in fetuses to 0.22 +/- 0.03 (day 5), 0.11 +/- 0.04 (day 10), 0.12 +/- 0.04 (day 12), 0.22 +/- 0.08 (day 16) and 0.33 +/- 0.04 (adults). In the fetal rats as in the adults, marked responses to respectively 10(-5) and 10(-4) M HA were demonstrated. The H2-receptor antagonist cimetidine diminished HA-induced secretion by 66 and 57% in fetuses and adults respectively. Between these two stages (from days 5 to 12), basal secretion and the response to HA dropped significantly. On day 21 of gestation, as well as on the critical days 5 and 12 after parturition, db-cAMP (10(-4) M) caused maximal stimulation of acid secretion. These results indicate that the development of responsiveness to HA in the rat is biphasic. They suggest that after birth, the H2-receptor adenylate cyclase system undergoes major modifications which might lead to the complete lack of responsiveness to HA by day 12.     

Angiotensin II vascular receptors in fetal and neonatal rats

Specific binding sites for angiotensin II in aorta and renal arteries have been studied in rat fetuses (18th day of pregnancy) and 1-day-old newborn rats by binding studies in arterial membranes using [125I] ileu-5-angiotensin II. One type of angiotensin receptor was found both in fetuses and in the newborns; the capacity of this (RT) decreased immediately after birth (from 0.06 +/- 0.01 nM to 0.02 +/- 0.005 nM; +/- SEM) and the affinity (Kd) increased at birth (from 3.5 +/- 0.6 nM to 19.5 +/- 1.2 nM; +/- SEM). Localization of the specific binding sites was studied by autoradiography on arteries from fetal and newborn rats either perfused with iodinated angiotensin II by cannulation of the aorta or in vitro on cryostat sections incubated with the radioactive angiotensin II. Both in fetuses and in the newborn the binding sites were located in the tunica media of the arteries.