Exencephalic Fetuses Research Articles

Cell necrosis, intrinsic apoptosis and senescence contribute to the progression of exencephaly to anencephaly in a mice model of congenital chranioschisis

Exencephaly/anencephaly is one of the leading causes of neonatal mortality and the most extreme open neural tube defect with no current treatments and limited mechanistic understanding. We hypothesized that exencephaly leads to a local neurodegenerative process in the brain exposed to the amniotic fluid as well as diffuse degeneration in other encephalic areas and the spinal cord. To evaluate the consequences of in utero neural tissue exposure, brain and spinal cord samples from E17 exencephalic murine fetuses (maternal intraperitoneal administration of valproic acid at E8) were analyzed and compared to controls and saline-injected shams (n = 11/group). Expression of apoptosis and senescence genes (p53, p21, p16, Rbl2, Casp3, Casp9) was determined by qRT-PCR and protein expression analyzed by western blot. Apoptosis was measured by TUNEL assay and PI/AV flow cytometry. Valproic acid at E8 induced exencephaly in 22% of fetuses. At E17 the fetuses exhibited the characteristic absence of cranial bones. The brain structures from exencephalic fetuses demonstrated a loss of layers in cortical regions and a complete loss of structural organization in the olfactory bulb, hippocampus, dental gyrus and septal cortex. E17 fetuses had reduced expression of NeuN, GFAP and Oligodendrocytes in the brain with primed microglia. Intrinsic apoptotic activation (p53, Caspase9 and 3) was upregulated and active Caspase3 localized to the layer of brain exposed to the amniotic fluid. Senescence via p21-Rbl2 was increased in the brain and in the spinal cord at the lamina I-II of the somatosensory dorsal horn. The current study characterizes CNS alterations in murine exencephaly and demonstrates that degeneration due to intrinsic apoptosis and senescence occurs in the directly exposed brain but also remotely in the spinal cord.

Teratogenicity of valproic acid and its constitutional isomer, amide derivative valnoctamide in mice.

The anticonvulsant valproic acid (VPA) has a known teratogenic effect capable of inducing major congenital malformations and developmental disorders. A comparative teratogenicity study of VPA and its analog valnoctamide (VCD), which is a new generation candidate antiepileptic drug, was carried out using Swiss Vancouver (SWV) mice. Pregnant SWV dams were treated with either a single intraperitoneal injection of VPA (1.8 and 2.7 mmol/kg), VCD (1.8 and 2.7 mmol/kg), or vehicle on E8:12 (gestational day:hour). The numbers of implantation and resorption, viable and dead fetuses, and the presence of gross fetal visceral and skeletal abnormalities were determined (E18). Real-time Polymerase chain reaction (RT-PCR) arrays were used to analyze the expression of 84 genes related to the processes of neurogenesis and neural stem cell differentiation. Significant decreases in pregnancy weight gain and the number of live fetuses were observed when VPA was administered at the high dose, whereas the percentage of exencephalic fetuses was significantly increased in VPA treated compared with an equivalent VCD dosage group. There was a dose-related increase in visceral defects in the VPA-exposed fetuses. Missing skull bones and fused vertebrae in fetuses occurred at the high dose of VPA. Three genes (Mtap2, Bmp8b, and Stat3) were significantly upregulated and one (Heyl) was downregulated in samples from VPA-treated dams. The study demonstrates that the teratogenicity of VPA was significantly greater than that of an equimolar dose of VCD. Four genes (Mtap2, Bmp8b, Stat3, and Heyl) represent candidate target genes for the underlying teratogenic mechanism responsible for VPA-induced malformations.

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.

Exencephaly-anencephaly sequence: proof by ultrasound imaging and amniotic fluid cytology.

We present and discuss major current theories about the developmental natural history of the anencephalic human fetus. We confirm previous observations made using transvaginal ultrasonography of exencephalic fetuses which were later imaged and/or delivered as anencephalic fetuses. We explore the possibility of proving the theory of the slowly rubbed-off exposed brain tissue by cytologic examination and special staining of aspirated cells in amniotic fluid. Three fetuses with a typical sonographic picture of exencephaly at 13-15 postmenstrual weeks underwent amniocentesis. The aspirated fluid contained pathognomonic neural cells. The same fetuses later showed the characteristic sonographic and postabortion picture of anencephaly. Our results support the theory that exencephaly is the forerunner of anencephaly.

Neuronal overmaturation in dysraphism: ontogenic expression of neuropeptides in the fetal brain and developmental anomalies in exencephaly

Starting from knowledge obtained in our previous studies of experimental dysraphism in chick embryos, the entity of neuronal overgrowth observed in exencephaly was further investigated. The ontogenic expression of neuron-specific enolase (NSE), vasoactive intestinal polypeptide (VIP), and somatostatin was analyzed both in chick exencephaly of the natural product and in normal chick fetuses by carrying out immunohistochemical studies. In normal fetuses, immunostained elements positive for NSE first appeared in the spinal gray matter on postincubation day 16 and increased in intensity during the fetal period. By postnatal day 2, the cerebral peduncle, brain stem neurofibers, molecular layer of the cerebellum, corpus striatum, and piriform cortex became immunoreactive. No immunohistochemical reaction to VIP was observed during these stages. Somatostatin-positive elements were not identified during the fetal period, except in limited regions, such as the corpus striatum, which appeared to have weakly positive staining on day 21. The exencephalic fetuses, however, demonstrated extremely advanced neuronal maturation, with intense immunoreactivity already being manifest in various regions, including the corpus striatum, piriform cortex, spinal gray matter, and brain stem nuclei, on day 16 of the fetal period. Somatostatin-positive elements also appeared at this stage in chick exencephaly, but such immunoreactivity was localized, particularly in the overgrown foci. The present study showed that the neuronal maturation process in some neurons of exencephalic brain and spinal cord was definitely further advanced than that in normal controls. A possible clinical application of NSE and somatostatin measurement as markers for dysraphic states in the fetus is suggested.

Effect of pre-treatment with aspirin on alcohol-induced neural tube defects in the TO mouse fetuses

This study was planned to investigate the effects of ethanol on the genetic susceptibility of the TO mouse to neural tube malformations and to determine the ameliorative effects, if any, of aspirin a potent prostaglandin inhibitor. The TO mouse exhibits a spontaneous incidence of 3.6% exencephaly. The mice were exposed to single (i.p.) dose of 0.03 ml/gm body weight of a solution of (25%v/v) of absolute alcohol in physiological saline on day 7 or 8 of gestation. Subteratogenic doses (150 or 200 mg/kg) of aspirin were administered (i.p.) an hour before ethanol exposure. Fetuses were collected on day 18 and compared with those of the untreated, and saline treated pair-fed pair-watered controls as well as with those of the aspirin alone treatment group. A total of 175 litters were studied. Alcohol caused a three-fold increase against the background incidence of exencephaly. Several craniofacial anomalies and growth retardation were also observed. Alizarin red-S stained skeletal preparations revealed extensive malformations of the craniofacial skeleton in the exencephalic fetuses. Both doses of aspirin administered prior to alcohol treatment significantly accentuated the alcohol-induced prenatal mortality. The rescue effect of aspirin on alcohol-induced intrauterine growth retardation was also significant although fetal weight was not restored to levels comparable to those of the controls. Pre-treatment with aspirin (both 150 and 200 mg/kg) on day 8 of gestation resulted in a numerical, though not statistically significant increase in alcohol-induced exencephaly. On the other hand pre-administration of the lower dose on day 7 of gestation caused a significant reduction while the higher dose gave rise to a significant increase in the incidence of this malformation. Aspirin also reduced the frequency of alcohol-induced arched palate and the baseline exencephaly. These data provide evidence for the possible interaction of alcohol with the genetic susceptibility to exencephaly in this strain of mice. The lack of a clear dose-dependent antagonistic effect of aspirin on alcohol-induced exencephaly suggests that the production of this malformation is probably not mediated by prostaglandin as it was shown for limb and renal abnormalities (Randall, C.L., Anton, R.F. and Becker, H.C. (1991). Asprin dose dependently reduces alcohol-induced birth defects and prostaglandin E levels in mice. Teratology 44, 521–529).

Is Exencephaly the Forerunner of Anencephaly?

Exencephaly is said to precede anencephaly resulting from failure of the rostral neuropore closure. In order to verify if the exencephaly induced after neural tube closure would also lead to anencephaly, exencephaly was induced in rat fetuses by maternal administration of a single dose (15 mg/kg) of cyclophosphamide on day 12 of gestation and pregnancy was prolonged by uterine ligation until postconception (PC) day 24. Fetal death was found to increase with prolongation of gestation and no sign of recovery from growth retardation was observed. Alizarin red-S-stained skeletal preparations substantiated the persistence of skull malformations in the exencephalic fetuses. Histological observations of the mesenchyme and the brain indicated degenerative changes that were intensifying with time. The ventricular system expanded progressively; the ependyma was denuded and neural mass lay free in the ventricle. The choroid plexus appeared to be elaborate and extensive. The haemorrhagic capillary network around the brain tissue was highly proliferative and appeared to penetrate the former from the exterior. Tissue necrosis seemed to progress unabated. Cystic spaces appeared beneath the base of the brain and became progressively large and it appeared as if the brain was being pushed out of the shallow cranial fossae. By PC day 24, most of the brain tissue had degenerated, thus giving clearly the appearance of the anencephalic condition.

Evidence for embryotoxicity of gossypol in mice and chicks with no evidence of mutagenic activity in the ames test

Effects of gossypol treatment were studied in pregnant mice and chick embryos. Pregnant Balb C mice were treated orally with 60 or 120 mg/kg of gossypol acetic acid on days 6–13 of pregnancy and killed on day 18. The uteri were removed, the number of resorptions and late fetal deaths were recorded, and the fetuses were weighed and assessed for malformations. Fertilized hen eggs were injected with 0.25 mg gossypol/egg at 24, 48, 72, or 96 h of incubation. The embryos were examined at day 9 of incubation. The mutagenic potential of gossypol was determined by the Ames test. Treatment of mice with gossypol produced significant adverse effects on the dam and offspring including decreased pregnancy weight gain of the dam and growth retardation of the offspring. There were increased resorptions and late fetal deaths in mice and high mortality in chick embryos. Exencephalic fetuses were observed in one of four litters exposed to the higher dose of gossypol, micromelia was observed in one of 26 chick embryos treated at 24 h, and gastroschisis was observed in one of 21 chick embryos treated at 72 h. No malformations were observed in the controls. The number of revertants per plate in the gossypol treatment group (100 or 500 mg gossypol/plate) did not differ significantly from that of control. This study provides evidence that gossypol has embryotoxic and possibly teratogenic activity in mouse and chick embryos but no mutagenic activity according to the Ames assay.

Common hierarchies of susceptibility to the induction of neural tube defects in mouse embryos by valproic acid and its 4-propyl-4-pentenoic acid metabolite.

The teratogenic effects of valproic acid and its 4-propyl-4-pentenoic acid (4-en) metabolite were investigated in three inbred mouse strains that were known to possess differing sensitivity to heat-induced neural tube defects. In the heat-resistant DBA/2J strain, administration of either valproic acid or the metabolite during the critical period of neural tube development failed to produce any abnormal offspring. Similar treatment in the moderately heat-sensitive LM/Bc strain resulted in up to 19.8% exencephalic fetuses. The highly heat-sensitive SWV strain was also very susceptible to the induction of neural tube defects by either valproic acid or its 4-en metabolite. When administered on gestational day 8 plus 12 hours, the parent compound produced 35% exencephalic fetuses, while the metabolite had a response frequency of 32.4%. Thus, the hierarchy of susceptibility for the induction of neural tube defects in these inbred mouse strains was exactly the same whether the teratogen was a physical agent such as hyperthermia or a chemical compound such as valproic acid. If such diverse agents as these should interact to produce malformations, then it is possible that a wide variety of other agents might interact in a similar manner to produce neural tube defects.

Amniotic fluid cholinesterase of valproate-induced exencephaly in the mouse: an animal model for prenatal diagnosis of neural tube defects

After a single administration of the antiepileptic drug valproic acid (VPA; i.p.:600 mg/kg) on day 8 of gestation in the mouse embryotoxicity and amniotic fluid (AF) cholinesterase (ChE) were evaluated on day 16 of gestation. VPA treatment induced an increase in embryolethality, neural tube defects (exencephaly), cleft palate, deformed vertebrae, open eyes, and a reduction in fetal weight. In VPA-exposed fetuses AF total ChE (TChE) activity of exencephalic fetuses was higher than that of normal fetuses. However, in 3 out of 110 normal fetuses of the control group TChE activity was found in the AF. There was no correlation between blood contamination of AF and its TChE activity, either in non-exencephalic control or treated embryos. Using ethopropazine as a "pseudo"-ChE inhibitor in vitro, the percentage of acetyl-ChE in blood-contaminated AF was similar to that of fetal rather than maternal serum, indicating that AF was contaminated with fetal and not with maternal blood. VPA-induced exencephaly in mice may provide an animal model to further investigate biochemical markers for prenatal diagnosis of neural tube defects.

Hyperthermia-induced exencephaly in mice: effect of multiple exposures.

Pregnant LM/Bc female mice were given a 10-minute hyperthermic exposure in a 43 degrees C waterbath during the period of neural tube closure. On day 15.5 of gestation, the females were killed, and the fetuses were examined for exencephaly. Following a single treatment on day 8.0, 8.5, 8.75, or 9.0 of gestation 1.7, 13.6, 2.9, and 0.8% of the respective fetuses displayed exencephaly. With two treatments, one on each of gestational days 8.5 and 8.75, or three treatments, one on each of gestational days 8.5, 8.75, and 9.0, the percentage of exencephalic fetuses increased to 28.3 and 59.3%, respectively. The increased response to multiple treatments was not due to an increase in the number of susceptible embryos but rather was due to the increased number of treatments. The results of this study suggest that with increasing numbers of treatments, the embryo's ability to recover from the hyperthermic exposure is lessened, resulting in an increase in exencephaly.

Abnormalities of the ear associated with exencephaly in mouse fetuses induced by maternal exposure to cadmium.

Exencephaly was induced in mouse fetuses by maternal injection of cadmium chloride (CdCl2) on day 7 of gestation. The heads of exencephalic, nonexencephalic experimental, and control fetuses were embedded in paraffin and sections were stained with hematoxylin and eosin. Compared to those of controls, the ears of the exencephalic fetuses were smaller (microtia) and low set. The meatal plug representing the external auditory canal was thick, variously branched, and often directed inferiorly. Usually, there were just two ossicles. The stapedial artery, facial nerve, and stapedius muscle were hypoplastic; the tensor tympani was small or absent. There were 1.0 to 2.0 turns of the cochlea in contrast to 2.5 turns in the controls. The organ of Corti was underdifferentiated; the spiral ganglion had fewer cells. In the control, the long axes of the anterior and posterior semicircular ducts were at right angles to each other and in vertical planes, but in the exencephalics, they tended more laterally towards the horizontal plane. The differentiation of the cristae ampullares and maculae was also severely affected. In several specimens, the entire membranous labyrinth had been distended; these labyrinths also had unusual epithelial infoldings. In cadmium-treated nonexencephalic fetuses, the external ears were normal and appropriate to the body size; five of them were examined histologically; in all, the five middle ear contents were hypoplastic; in three, the cochlea had a maximum of two turns and the organ of Corti, crista ampullaris, and macula were hypoplastic. By an analogy to abnormalities of mutants with neural tube defects, it is suggested that the exencephaly induced by cadmium might affect the differentiation of the ear. Partial involvement of the ear in nonexencephalic experimental embryos may be the result of direct action of cadmium during critical stages of development.

Exencephaly in human fetuses.

In some anencephalic fetuses exposed neural tissue mass of varied size can be demonstrated. This is known as exencephaly. The authors diagnosed by ultrasound 10 typical exencephalic cases prenatally between 14 and 21 weeks of gestation. Nine singular pregnancies were terminated and in the twin pregnancy a selective feticide of the exencephalic co-twin was carried out. The pregnancy continued to term and a healthy newborn infant and a fetus papyraceus were born. The mummified co-twin was anencephalic and showed only the remnants of the exposed brain. Authors suggest that, as in experiments with animals, the exencephaly in humans, by the degeneration of the exposed neural tissue converts to anencephaly and in this process the macrophages in fetal circulation and in the amniotic fluid may play a significant role. The large number of these actively phagocytic macrophages can be demonstrated in the amniotic fluid samples from exencephalic fetuses.

Exencephaly and axial skeletal dysmorphogenesis induced by acute doses of ethanol in mouse fetuses.

Administration of single doses (0.02 ml and 0.03 ml/g body wt.) of a solution of absolute alcohol (25%, v/v) in saline to MF1 mice on day 8 of gestation resulted in significant fetal mortality, growth retardation and gross malformations of the fetuses at term. The malformations included cranioschisis with exencephaly, maxillary hypoplasia, exophthalmia and various digital anomalies. The exencephalic fetuses, cleared in KOH and stained with alizarin red revealed the absence of the skull vault and hypoplasia of the facial bones. The skull base indicated under ossification and overcrowding of the constituent bones resulting in severe reduction in the cranial volume and asymmetry of cranial base. These cranial skeletal malformations were accompanied by abnormalities of the vertebral bodies, arches, ribs and sternum. It is concluded that acute doses of alcohol during critical stages of neural tube development is deleterious to both the central nervous system (CNS) and axial skeleton in the mouse.

Malformations of the axial skeleton in cranioschisis aperta and exencephaly in rat fetuses induced after neural tube closure.

Single doses of cyclophosphamide were administered (IP) to groups of Wistar rats, on different days of gestation after neural tube closure (days 12-15) and fetuses were collected on day 20. A very large number of the fetuses treated during days 12-14 exhibited cranioschisis and exencephaly. Ethanol fixed, alizarin red stained specimens were observed for axial skeletal abnormalities. The exencephalic ones lacked ossified skulls. The basicranial bones were either under ossified or had undergone extensive fusion resulting in reduction in cranial volume. The basicraniovertebral angle was obtuse. The vertebral bodies and arches showed varying degrees of hypoplasia, fusion and/or agenesis. The development of ribs and sternebrae was also extensively affected. Treatment on day 15 did not induce exencephaly but the axial skeleton was hypoplastic. Wavy ribs were a remarkable feature of these fetuses. All exencephalic fetuses had subcutaneous haemorrhages; many of them were obviously oedematous. These data indicate that the susceptible period for induction of these anomalies does not stop at neuropore closure.

The teratogenicity of cytochalasin D and its inhibition by drug metabolism.

Pregnant rats received intraperitoneal injections of cytochalasin D (CD) on gestational days 7-11. Doses of 400 micrograms/kg were only minimally and nonsignificantly teratogenic, leading to 2 exencephalic fetuses among the 111 fetuses delivered. By contrast, embryos exposed to CD in vitro on day 10 showed significant frequencies of neural tube abnormalities when exposed to CD concentrations at or above 3.1 ng/ml. These embryos also exhibited significantly decreased protein, somite counts, and crown-rump lengths. In order to understand this apparent discrepancy between the teratogenicity of CD in vivo and in vitro we performed experiments to determine whether drug metabolism could inhibit the teratogenicity in vitro. Male rats were pretreated with a mixture of polychlorinated biphenyls as cytochrome P-450 inducers, and their livers were used to prepare a microsome-rich fraction (S-9). This S-9 fraction, plus a source of reducing equivalents (NADPH), significantly inhibited the teratogenicity of CD. The teratogenicity was restored by the omission of NADPH, and was partially restored with the addition of carbon monoxide. These results led us to conclude that the teratogenic effect of CD can be inhibited by drug metabolism in vitro; additionally, it is likely that some or all of this drug metabolism may depend on cytochrome P-450. We further speculate that CD may be inactivated in vivo by these same systems, explaining the discrepancy between the teratogenicity of CD in vivo and in vitro.

Alpha-fetoprotein and albumin in experimentally-induced exencephaly in the rat.

Alpha-fetoprotein and albumin were quantified in the sera and amniotic fluids from control, Vitamin A-treated non-exencephalic and Vitamin A-treated exencephalic rat fetuses. Exencephaly was associated with amniotic fluid alpha-fetoprotein concentrations which were significantly elevated over those of Vitamin A-treated non-exencephalic and of untreated fetuses. Amniotic fluid albumin concentrations also were higher in the exencephalic fetuses than in the non-exencephalic fetuses. Serum alpha-fetoprotein and albumin concentrations were lower in the exencephalic than in the non-exencephalic fetuses. The results are cosistent with simple diffusion across a defective barrier as the cause of elevated amniotic fluid alpha-fetoprotein concentrations in the presence of open neural tube defects. This experimental model of neural tube defects result in changes in amniotic fluid alpha-fetoprotein similar to those changes found in human amniotic fluid alpha-fetoprotein concentrations in the presence of neural tube defects.

Skullbase malformations in rat fetuses with hypervitaminosis A-induced exencephaly.

AbstractExencephalic fetuses (16th–20th day), offspring of female Wistar rats treated with vitamin A palmitate orally during pregnancy, were studied in serial sections. The median part of the skullbase of the rat is formed by the basioccipital, basisphenoid, and presphenoid. In exencephalic fetuses the basioccipital had a normal shape; the hypophyseal fossa in the basisphenoid was too deep; the presphenoid was malformed. The angle between the cervical vertebral column and basioccipital was increased and those between the basioccipital and basisphenoid, and the basisphenoid and presphenoid were reduced. As a result the longitudinal axis of the skullbase was shortened. In all exencephalic fetuses the pontine and cervical flexures were less well marked than in controls. No anomalies of the rostral part of the notochord were observed. It is concluded that there is a relation between abnormal flexures of the neural tube and the malformation of the skullbase. The resemblance of the skullbase in human anencephaly and rat exencephaly is emphasized.

AN EXPERIMENTAL STUDY ON TERATOGENIC EFFECT OF CADMIUM

Administration of 5 mg per kilogram of cadmium chloride to pregnant mice on 7 days of gestation produced a variety of malformations in the surviving fetuses.Exencephaly was found most among the fetuses and such exencephalic fetuses also often exhibited open eye.Clefts palate and lack of tail or rachischisis were also found in some other fetuses.Skeletal malformations were observed in the skull region, vertebral parts, ribs and tails.The total rate of malformation appearance exceeded about 80%.The less the dosis of cadmium chloride, the less the rate of malformation appear-ance: 19% for 2.5 mg/kg 1.0% or less for 0.63 mg/kg and none for 0.33 mg/kg.The quantities of cadmium in the liver, the kidneys and the placentas of the mother animals and fetuses were measured by atomic absorption spectrophotometry.The cadmium content in the liver of cadmium chloride injected mother animal was 800 times greater than the control animal, that in the kidneys was 450 times and that in the placenta 10 times, but measurable amount of cadmium was not de-tected among fetuses of cadmium chloride injected mice.Above mentioned fact expresses that cadmium chloride in the late pregnancy does not transfer through the placenta and stays in the mother's side.

Lack of hypertrophy of the adrenal in exencephalic fetal rats subjected to hypervitaminosis A and then to maternal adrenalectomy.

Mother rats were treated daily with 30,000 USP units of vitamin A on the 8th, 9th and 10th days of gestation. Subsequently, some of them were adrenalectomized on the 20th day of gestation or were subjected to laparotomy for the injection of 1 IU of ACTH to each exencephalic fetus on the 21st day of gestation. Autopsy was performed on the 22nd day of gestation. Maternal hypervitaminosis A induced exencephaly in some fetuses in which the growth of the adrenal was retarded despite the presence of the anterior pituitary. Maternal adrenalectomy did not cause a significant hypertrophy of the fetal adrenal in exencephalics but did in non-exencephalics in the same litter. The adrenals of exencephalics given ACTH were enlarged significantly. The observations suggest that the fetal brain-pituitary region plays an essential role in the mechanism of hypertrophy of the fetal adrenal following maternal adrenalectomy. (Endocrinology 88: 1526, 1971) T ADRENALS of anencephalic or hydrocephalic human fetuses are invariably small, regardless of the presence or absence of the anterior pituitary (1). Similar retardation of the adrenal growth has been observed in fetal rats under various experimental conditions such as complete removal of the hypothalamus by decerebration of a fetus (2), destruction of the fetal hypothalamic area with an electrocoagulator, compression of the fetal brain by intracranial paraffin injection, and induction of fetal exencephaly by hypervitaminosis A (3). These observations suggest that the brain, including the hypothalamus, is necessary for the growth of the adrenal during prenatal life. However, such observations still do not necessarily rule out the possibilities of partial autonomy of the fetal pituitary-adrenal axis or some influence of maternal CRF on the axis. The present studies were carried out to compare the extent of retardation of the adrenal growth in the absence of the pituitary with that which occurs when the brain is defective and to examine to what extent the adrenal of a fetus in which the brain was destroyed could enlarge following maternal adrenalectomy. The defect of the fetal brain was induced by maternal hypervitaminosis A. Materials and Methods Rats of the Wistar strain were used. They were given a commercial diet (Oriental pellets NMF) and water, both ad lib. Days of gestation were counted from the day following an overnight matReceived June 2, 1970. ing, which was counted as the 1st day of gestation. The experimental groups and designs were: Group 1. Mother rats were subjected to midventral laparotomy under ether anesthesia on the 20th day of gestation in order to decapitate surgically 3 or 4 fetuses in utero in each litter by the method of Domm and Leroy (4) for the purpose of hypophysectomy. Two days later, at autopsy, these decapitated fetuses were matched at random with intact litter-mate fetuses of the same sex. Group 2. Mother rats were adrenalectomized on the 20th day of gestation via the dorsolumbar approach. Subsequently, on the same day, they were subjected to laparotomy and 2 or 3 fetuses in each litter were decapitated. Two days later, autopsy was performed. Group 3. Mother rats were treated daily with 30,000 USP units of vitamin A palmitate (Sigma Chemical Co.) suspended in 0.6 ml of saline by a stomach tube on the 8th, 9th and 10th days of gestation. Autopsy was performed on the 22nd day of gestation. Litters in which all or none of the fetuses showed exencephaly were discarded. In each litter used, exencephalic fetuses were matched at random with non-exencephalic fetuses of the same sex. The remaining odd fetuses were discarded. Group 4. Mother rats were treated with vitamin A as in group 3. They were subjected to adrenalectomy on the 20th day of gestation. Exencephalic fetuses were paired with litter-mate non-exencephalic fetuses in the same way as in group 3. Group 5. Mother rats were treated with vitamin A as in group 3. They were subjected to laparotomy on the 21st day of gestation for the injection of