A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

The effect of maternal HMB supplementation on bone mechanical and geometrical properties, as well as histomorphometry and immunolocalization of VEGF, TIMP2, MMP13, BMP2 in the bone and cartilage tissue of the humerus of their newborn piglets.

The presented experiment focuses on assessing the impact of HMB (hydroxy-ß-methobutyrate) supplementation of mothers during pregnancy on the development of the skeletal system of their offspring. For this purpose, an experiment was carried out on 12 clinically healthy sows of the Great White Poland breed, which were divided randomly into two groups the control and the HMB group. All animals were kept under standard conditions and received the same feed for pregnant females. In contrast, females from the HMB group between 70 and 90 days were supplemented with 3-hydroxy-3-methylbutyle in the amount of 0.2g/kg b.w/day. Immediately after birth, the piglets were also divided into groups based on: sex, and presence or lack HMB supplementation, and subsequently were euthanized and humerus bones from all piglets were collected. Mother's HMB supplementation during pregnancy affected the multiple index of their offspring. The higher humerus mass and length was observed with the greater effect in males. Maternal supplementation also influenced on the geometrical and mechanical properties of the humerus as in the case of mass, this effect was higher in males. Also, the collagen structure of the compacted and trabecular bone changed under the HMB addition. Maternal supplementation also affected the expression of selected proteins in growth cartilage and trabecular bone. The obtained results show that the administration to the mother during pregnancy by the HMB significantly affects the development of the humerus in many ways. The obtained results also confirm the utility of such experiments in understanding of the importance of the pregnancy diet as an develop and adaptable factor of offspring organisms and are the base for further research in that area as well as in the protein markers expression area.

Fetal echocardiographic assessment of cardiovascular impact of prolonged support on EXTrauterine Environment for Neonatal Development (EXTEND) system.

OBJECTIVE: EXTrauterine Environment for Neonatal Development (EXTEND) is a system to support ongoing fetal growth and organ development in an extrauterine environment, utilizing a pumpless low-resistance oxygenator circuit. The aim of this study was to evaluate hemodynamics and cardiac function in fetal sheep sustained on the EXTEND system. METHODS: This was a prospective study of fetal sheep supported for a minimum of 3 weeks on EXTEND. Hemodynamic parameters were assessed weekly and included heart rate, mean arterial pressure (MAP), Doppler-echocardiography-derived cardiac output (CO), pulsatility indices (PIs) of the fetal middle cerebral artery (MCA), umbilical artery (UA) and ductus venosus and cardiac function, as assessed by speckle-tracking-derived global longitudinal strain and strain rate in the right (RV) and left (LV) ventricles. Parameters were compared at 0 days and 1, 2 and 3 weeks following placement on EXTEND. RESULTS: Of 10 fetal sheep enrolled, seven survived for 3 weeks and were included in the analysis. Median gestational age at cannulation was 107 (range, 95-109) days. Heart rate decreased and MAP increased significantly, but within acceptable ranges, during the study period. The quantities and relative ratios of right and left CO remained stable within the anticipated physiological range throughout the study period. Vascular tracings and PIs appeared to be similar to those seen normally in the natural in-utero state, with MCA-PI being higher than UA-PI. UA tracings demonstrated maintained abundant diastolic flow despite the absence of placental circulation. In both the RV and LV, strain decreased significantly at 1 and 2 weeks relative to baseline but returned to baseline values by week 3. CONCLUSIONS: The EXTEND mechanical support system replicates natural physiology and creates a stable and sustainable cardiovascular construct that supports growth over a 3-week period. However, there is a period of depressed contractility within the first week with subsequent improvement by week 3. This may reflect a period of physiological accommodation that warrants further investigation. This study lays the foundation for further exploration as the EXTEND system moves towards human application. © 2019 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Intrauterine exposure to oestradiol promotes sex-specific differential effects on the prostatic development of neonate gerbils.

The effects of intrauterine exposure to 17ß-oestradiol (E2) are well studied for the male prostate and there are accumulating evidences that the exposure to high dosages leads to a hypomorphic development. However, there is a lack of information about the effects of intrauterine exposure to E2 in the prostate of rodent females, and such research becomes relevant in view of the presence of functional prostate in a proportion of women, and the morphophysiological similarities between the prostate of female rodents and the prostate of women. This study uses histochemical, immunohistochemical, immunofluorescence and three-dimensional (3D) reconstruction techniques to evaluate the effects of intrauterine exposure to E2 (500 BW/d) on neonatal prostate development in both male and female gerbils. It was verified that intrauterine exposure to E2 promotes epithelial proliferation and growth of prostatic budding in females, whereas in males the prostatic budding shows hypomorphic growth in the VMP (Ventral Mesenchymal Pad) as well as reduced epithelial proliferation. Together, the data demonstrate that intrauterine exposure to E2 causes different effects on male and female prostates of the gerbil even at the early postnatal development of the gland.

Altered immune response in mallard ducklings exposed to lead through maternal transfer in the wild.

Lead (Pb) poisoning has caused significant mortality in waterfowl populations worldwide. In spite of having been banned since 2003, prevalence of Pb shot ingestion in mallards (Anas platyrhynchos) from the Ebro delta was still 15.5% in 2011-12. We collected mallard eggs from this area to study the effects of maternally transferred Pb on eggshell properties and on immune response and oxidative balance of ducklings. Eggshell Pb levels were positively correlated with Pb levels in the blood of ducklings. Ducklings with blood Pb levels above 180 ng mL(-1) showed reduced body mass and died during the first week post hatching. Blood Pb levels positively correlated with humoral immune response, endogenous antioxidants and oxidative stress biomarkers, and negatively correlated with cellular immune response. Pb shot ingestion in birds can result in maternal transfer to the offspring that can affect their developing immune system and reduce their survival in early life stages.

A structure-based extracellular matrix expansion mechanism of fibrous tissue growth.

Embryonic growth occurs predominately by an increase in cell number; little is known about growth mechanisms later in development when fibrous tissues account for the bulk of adult vertebrate mass. We present a model for fibrous tissue growth based on 3D-electron microscopy of mouse tendon. We show that the number of collagen fibrils increases during embryonic development and then remains constant during postnatal growth. Embryonic growth was explained predominately by increases in fibril number and length. Postnatal growth arose predominately from increases in fibril length and diameter. A helical crimp structure was established in embryogenesis, and persisted postnatally. The data support a model where the shape and size of tendon is determined by the number and position of embryonic fibroblasts. The collagen fibrils that these cells synthesise provide a template for postnatal growth by structure-based matrix expansion. The model has important implications for growth of other fibrous tissues and fibrosis.

Acceleration of reflex maturation and physical development in suckling rats: effects of a maternal diet containing lipids from goat milk.

OBJECTIVE: To investigate the effects of lipids from goat milk containing conjugated linoleic acids on body weight and reflex ontogeny of neonatal rats treated during the prenatal and suckling periods. METHODS: Three groups were studied: soybean oil (S), coconut oil (C), and goat milk lipids (GM). Reflex maturation (palm grasp, righting reflex, cliff avoidance, vibrissae placing, negative geotaxis, auditory startle, and free-fall righting) as well as body weight evolution were recorded during lactation. RESULTS: Data demonstrated that the lipids from goat milk accelerated body weight evolution as well as all the reflex maturation investigated (P < 0.05). DISCUSSION: The supply of goat's milk offered to Wistar rats during pregnancy and lactation provided a variety of fatty acids necessary to accelerate the development of offspring.

The effect of albumen removal before incubation (embryonic protein under-nutrition) on the post-hatch performance, regulators of protein translation activation and proteolysis in neonatal broilers.

Albumen was removed from broiler eggs before the start of incubation to induce prenatal protein under-nutrition in chicken embryos. With this method, the direct effect of protein deficiency was investigated, differing from mammalian models manipulating the maternal diet where indirect, hormonal effects can interfere. Based on the estimated albumen/egg weight ratio, 10 % of albumen was removed with an 18G needle, after making a hole at the sharp end of the egg with another 18G needle. Eggs were taped thereafter. The sham group underwent the same procedure, except that no albumen was removed. Control eggs did not receive any treatment. The removal of albumen decreased both embryonic and post-hatch body weight up to day 7 compared with the control group. On embryonic day 18, embryos from the albumen-deprived group had higher plasma uric acid levels compared with the sham (P= 0·016) and control (P= 0·009) groups. Moreover, a lower plasma amino acid concentration was observed at hatch compared with the sham (P= 0·038) and control (P= 0·152) groups. These findings indicate an altered protein metabolism. At hatch, a higher mRNA expression of muscle ring finger-1 (MuRF1), a gene related to proteolysis, was observed in albumen-deprived chicks compared with the control and sham chicks, together with an up-regulated expression of atrogin-1 (another atrogene) at this time point in the male protein-deficient chicks. These findings suggest that muscle proteolysis is transiently increased by the removal of albumen before the start of incubation. No evidence was found for altered protein synthesis capacity and translational efficiency in albumen-deprived chicks.

RA-GEF-1 (Rapgef2) is essential for proper development of the midline commissures.

The cerebral hemispheres are directly connected by three major interhemispheric fibers: the corpus callosum, the anterior commissure, and the hippocampal commissure. RA-GEF-1 (also termed Rapgef2) is a guanine nucleotide exchange factor responsible for sustained activation of Rap1. We previously reported anatomical defects of the major forebrain commissures in the adult dorsal telencephalon-specific RA-GEF-1 conditional knockout (cKO) mice. In this study, we use neuroanatomical tracing and immunohistochemistry to study the formation of the commissural fibers during early postnatal development. DiI anterograde tracing reveals the inability of the callosal axons to cross the midline in cKO mice, thereby forming Probst bundles on the ipsilateral side, which is associated with the absence of the indusium griseum glia and the glial sling at the cortical midline. Wheat germ agglutinin-conjugated horseradish peroxidase retrograde tracing verifies the agenesis of the anterior commissure in cKO mice, and DiI anterograde tracing confirms the deviation of the fibers from their original tract. As for the hippocampal commissure, agenesis and hypoplasia are observed in its dorsal and ventral parts, respectively. These results indicate the essential role of RA-GEF-1 in the proper formation of the cerebral midline commissures.

Why are hatching and emergence success low? Mercury and selenium concentrations in nesting leatherback sea turtles (Dermochelys coriacea) and their young in Florida.

Leatherback sea turtles (Dermochelys coriacea) have low hatching and emergence success compared to other sea turtle species. Postmortem examinations of hatchlings showed degeneration of heart and skeletal muscle that was similar to that found in other neonates with selenium deficient mothers. Selenium deficiency can result from elevated concentrations of bodily mercury. Ingested mercury is detoxified by the liver through mercury-selenium compound formation. In animals persistently exposed to mercury, the liver's ability to detoxify this element may decrease, especially if dietary selenium is insufficient. We measured mercury and selenium concentrations in nesting female leatherbacks and their hatchlings from Florida and compared the levels to hatching and emergence success. Both liver selenium and the liver selenium-to-mercury ratio positively correlated with leatherback hatching and emergence success. This study provides the first evidence for the roles of mercury and selenium in explaining low reproductive success in a globally imperiled species, the leatherback sea turtle.

TNF-α and the development of the neonatal immune system: implications for inhibitor use in pregnancy.

Inflammatory bowel diseases, Crohn's disease, and ulcerative colitis have an unpredictable course during and after pregnancy (1). There is a great deal of interest in treating moderate to severe active inflammatory bowel disease with anti-tumor necrosis factor (anti-TNF) biologics in pregnant women (2). We lack definitive information about the effects of these agents on the development of the immune system of the human fetus and the newborn baby. Anti-TNF agents fall within US Food and Drug Administration's (FDA) category B regarding fetal risk, indicating that no adequate and well-controlled studies have been conducted in pregnant or nursing women. Here, we review animal studies (of both mice and nonhuman primates) that examine the role of TNF and its inhibitors in the normal development of the immune system.

Foetal and neonatal exposure to chlorpyrifos: biochemical and metabolic alterations in the mouse liver at different developmental stages.

The mechanisms implicated in the age-related toxicity, including its neurobehavioral effects after subtoxic developmental exposure to chlorpyrifos (CPF), a widely used insecticide, have not been fully elucidated yet. With the aim of investigating whether metabolic differences during ontogeny could account for the age-related susceptibility to CPF, we examined the developmental time-course of hepatic metabolizing enzymes and CPF metabolism in a cohort of mice exposed either prenatally (gestational day 15-18) and/or postnatally (postnatal day (PND) 11-14) to CPF at doses which were previously reported to induce neurobehavioural alterations, in the absence of brain acetyl-cholinesterase inhibition. Testosterone hydroxylase activity, CPF ex vivo biotransformation, glutathione content, as well as aromatase activity were determined in the liver of control and treated male and female mice at PND0, 9, 15 and 150. In control mice most Cyp activities were detectable and progressively increased up to PND15. In newborn control mice CPF bioactivation was much higher than the Cyp-catalysed detoxication, negligible at birth, indicating a possible increased susceptibility to CPF-induced effects in newborn mice. Detoxication rapidly increased with age, so that Cyp-related metabolic features cannot explain the higher susceptibility of juvenile mice. The observed age-dependent metabolic picture was partially altered by CPF prenatal treatment. Following in utero exposure CPF detoxifying capability was enhanced at birth and reduced at PND15, when CPF-oxon formation was slightly increased. No effects were evident at adulthood. Prenatal dosing was more effective in causing metabolic alterations than CPF postnatal treatment; no potentiation was observed in mice experiencing pre- plus post-natal CPF administration. Both in utero and postnatal CPF exposure decreased aromatase activity by 50% at PND9 and 15; this effect together with the presence of higher levels of the sex-specific Cyp2c activity at adulthood in male mice may suggest the occurrence of long-lasting impairment in the expression of hepatic Cyps under hormonal regulation. Altogether, the alterations in CPF Cyp-mediated biotransformation caused by perinatal CPF exposure seem not sufficient per se to explain the reported vulnerability of developing central nervous system to this insecticide, which can be due also to the parent compound itself or to the activation of different toxicological pathways. The hypothesis that observed effects on aromatase and sex-specific Cyp activity may be associated with a possible interference with the long-term alterations in sex-specific behavioural pattern deserves further investigation.

Isolator and other neonatal piglet models in developmental immunology and identification of virulence factors.

The postnatal period is a 'critical window', a time when innate and passive immunity protect the newborn mammal while its own adaptive immune system is developing. Neonatal piglets, especially those reared in isolators, provide valuable tools for studying immunological development during this period, since environmental factors that cause ambiguity in studies with conventional animals are controlled by the experimenter. However, these models have limited value unless the swine immune system is first characterized and the necessary immunological reagents developed. Characterization has revealed numerous features of the swine immune system that did not fit mouse paradigms but may be more generally true for most mammals. These include fetal class switch recombination that is uncoupled from somatic hypermutation, the relative importance of the molecular mechanisms used to develop the antibody repertoire, the role of gut lymphoid tissue in that process, and the limited heavy chain repertoire but diverse IgG subclass repertoire. Knowledge gained from studies of adaptive immunity in isolator-reared neonatal pigs suggests that isolator piglets can be valuable in identification of virulence factors that are often masked in studies using conventional animals.

Is the fetal lung a mineralocorticoid receptor target organ? Induction of cortisol-regulated genes in the ovine fetal lung, kidney and small intestine.

BACKGROUND: Lung, kidney and small intestine are involved in fetal volume regulation and amniotic fluid secretion and play a pivotal role in the transition from intrauterine to extrauterine life. OBJECTIVE: This study was performed to determine the ontogeny of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR), and of MR- and GR-regulated genes and proteins, serum and glucocorticoid-induced kinase (Sgk-1), epithelial sodium channel (ENaC alpha), and Na,K-ATPase alpha1. METHODS: Lung, renal cortex and medulla, and small intestine were collected from fetuses at 80, 100, 120, 130 and 145 days' gestation and from day 1 and 7 neonatal lambs. Real-time PCR was performed to determine mRNA concentration for MR, GR, the 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD1 and 2), Sgk-1, ENaC alpha, and Na,K-ATPase alpha1. Protein expression of ENaC alpha and Na,K-ATPase alpha1 in whole cell and membrane fractions was determined by immunoblotting. RESULTS: Expression of corticosteroid-induced genes in renal cortex increases at term; in small intestine the induction occurs postnatally. In contrast, in lung expression of MR and GR mRNAs were greater at 100 days to term than postnatally and 11 beta-HSD1 peaked at 145 days; the corticosteroid-induced genes also increased prenatally: Sgk-1 and ENaC alpha increased by 120 days, peaking at 145 days, and Na,K-ATPase alpha1 was greatest at 130 days. CONCLUSIONS: The expression of high levels of MR and 11 beta-HSD1 in preterm fetal lung suggest low endogenous fetal cortisol may exert actions at the high affinity MR in vivo, leading to increases in expression of sodium channels important in the regulation of lung liquid secretion and reabsorption.

Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse.

Congenitally hypomyelinated shiverer mice fail to generate compact myelin and die by 18-21 weeks of age. Using multifocal anterior and posterior fossa delivery of sorted fetal human glial progenitor cells into neonatal shiverer x rag2(-/-) mice, we achieved whole neuraxis myelination of the engrafted hosts, which in a significant fraction of cases rescued this otherwise lethal phenotype. The transplanted mice exhibited greatly prolonged survival with progressive resolution of their neurological deficits. Substantial myelination in multiple regions was accompanied by the acquisition of normal nodes of Ranvier and transcallosal conduction velocities, ultrastructurally normal and complete myelination of most axons, and a restoration of a substantially normal neurological phenotype. Notably, the resultant mice were cerebral chimeras, with murine gray matter but a predominantly human white matter glial composition. These data demonstrate that the neonatal transplantation of human glial progenitor cells can effectively treat disorders of congenital and perinatal hypomyelination.

Krox-20 gene expression: influencing hindbrain-craniofacial developmental interactions.

Krox-20 is a C(2)H(2)-type zinc-finger transcription factor that plays an essential role in hindbrain development. The Krox-20 null mutation results in hindbrain anomalies that result in neonatal death due to respiratory and feeding deficits. Here we review our studies of how the Krox- 20 null mutation impacts the development of motor and sensory systems critical for the production of consummatory behaviors (suckling/chewing). First, we demonstrated that Krox-20 null mutants suffer a selective loss of primary jaw-opening muscles during prenatal development. In vivo and in vitro studies are reviewed that highlight intrinsic defects in mutant jaw-opener muscles that contribute to muscle degeneration. Next we focus on the impact of the mutation on proprioceptive neurons activated during consummatory behaviors. Mesencephalic trigeminal (Me5) neurons are primary sensory neurons that relay jaw proprioception to the central nervous system. These cells are unique because their cell bodies are located in the central as opposed to the peripheral nervous system. Data are reviewed that demonstrate the impact of the mutation on Me5 neurons, a cell group traditionally thought to emerge from the mesencephalon. We show that Krox-20 null mutants have twice as many Me5 neurons relative to wildtypes at E15, but by birth have half the number of Me5 cells as wildtypes. TUNEL assays performed in each set of studies reveal that Krox-20 expression acts to protect both muscle and mesencephalic trigeminal neurons against apoptosis, suggesting that Krox-20, in addition to its role in hindbrain patterning, has a broader, long-lasting role in development.

Prenatal glucocorticoids and long-term programming.

Epidemiological evidence suggests that low birth weight is associated with an increased risk of cardiovascular, metabolic and neuroendocrine disorders in adult life. Glucocorticoid administration during pregnancy reduces offspring birth weight and alters the maturation of the lung and other organs. We hypothesised that prenatal exposure to excess glucocorticoids or stress might represent a mechanism linking foetal growth with adult pathophysiology. In rats, birth weight is reduced following prenatal exposure to the synthetic steroid dexamethasone, which readily crosses the placenta, or to carbenoxolone, which inhibits 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the physiological feto-placental 'barrier' to maternal glucocorticoids. As adults, the offspring exhibit permanent hypertension, hyperglycaemic, increased hypothalamic-pituitary-adrenal (HPA) axis activity and behaviour reminiscent of anxiety. Physiological variations in placental 11beta-HSD2 activity correlate directly with foetal weight. In humans, 11beta-HSD2 gene mutations cause low birth weight. Moreover, low-birth-weight babies have higher plasma cortisol levels throughout adult life, indicating HPA axis programming. The molecular mechanisms may reflect permanent changes in the expression of specific transcription factors, key among which is the glucocorticoid receptor (GR) itself. The differential programming of the GR in different tissues reflects effects upon one or more of the multiple tissue-specific alternate first exons/promoters of the GR gene. Overall, the data suggest that both pharmacological and physiological exposure prenatally to excess glucocorticoids programmes cardiovascular, metabolic and neuroendocrine disorders in adult life.

The endocannabinoid system: function in survival of the embryo, the newborn and the neuron.

Since the identification and cloning of the first cannabinoid (CB1) receptor and the subsequent discovery of the endogenous cannabinoid ligands (endocannabinoids), anandamide, 2-arachidonoyl glycerol (2-AG) and noladin ether, a intensive search for their function in health and disease has been launched. The endocannabinoids in the central nervous system bind Gi/o coupled CB1 receptors that modulate adenylyl cyclase, ion channels and extracellular signal-regulated kinases. The present review discusses the nature of endocannabinoid (anandamide and 2-AG) neurotransmission, the activity of cannabinoids and the possibility that some of these activities are mediated via a receptor, yet to be discovered, which is distinct from the brain specific CB1 receptor. Three physiological functions in which the endocannabinoids play a critical role are also discussed: embryonal implantation, feeding and appetite, and neuroprotection.

Differential expression of alpha 3 and alpha 6 integrins in the developing mouse inner ear.

The development of the mammalian inner ear involves a complex series of cell-cell and cell-extracellular matrix interactions. These interactions are likely to be mediated by families of adhesion molecules, including the integrins. We have studied the expression of three integrin subunits known to be expressed on epithelia in a number of tissues (namely, alpha3, alpha6, and beta4) during the development of the murine inner ear. At E10.5, both alpha3 and alpha6 were expressed in the epithelial layers of the otocyst. The expression of alpha6 was concentrated in an anterioventral region of the epithelium and in a proportion of the cells forming the cochlear-vestibular and facial ganglia. By E12.5, alpha6 showed a more restricted expression, confined mainly to the pro-sensory epithelia and the neural processes from the cochlear-vestibular ganglion. In contrast, alpha3 was expressed in epithelia adjacent to the pro-sensory areas. This reciprocal expression pattern was maintained until birth. Between birth and P6, a switch in expression occurred such that alpha3 was upregulated and alpha6 was downregulated in the sensory epithelia of both the auditory and vestibular systems. At this stage, alpha3 was expressed in all the epithelia lining the scala media, thus defining the endolymph compartment. The expression of beta4 was restricted to epithelial/mesenchymal borders throughout the developmental stages studied, suggesting that alpha6 expression observed within the epithelium and neuronal tissue was alpha6beta1. The early expression and changing pattern of alpha3 and alpha6 integrins during development of the mammalian inner ear suggests that they may be involved in the molecular processes that define epithelial boundaries and guide sensory innervation.

Chronic exposure to the opioid growth factor, [Met5]-enkephalin, during pregnancy: maternal and preweaning effects.

The opioid peptide, [Met(5)]-enkephalin (termed opioid growth factor, OGF), is an autocrine growth factor that serves as a constitutively active inhibitory agent. OGF crosses the placenta and depresses DNA synthesis in the fetus. The role of OGF in pregnancy and parturition, and the influence exerted on prenatal and neonatal features of the offspring, were studied in rats. Females received daily injections of 10 mg/kg OGF throughout gestation; all offspring were cross-fostered to lactating noninjected dams at birth. No effects on the length of gestation, course of pregnancy, behavior of the pregnant dam, maternal weight gain, or food and water intake throughout gestation were recorded in OGF-treated mothers. Moreover, nociceptive response in these females was not altered by chronic OGF exposure, and no signs of physical dependence or withdrawal could be observed following a challenge by the opioid antagonist naloxone. Litter size and the number of live births per litter of OGF-treated mothers were reduced by 25% from control subjects and a fourfold increase in stillborns was noted for mothers receiving OGF compared to control levels. Histopathologic analysis confirmed the stillborns to have died in utero. OGF-exposed neonates were normal in body weight and crown-to-rump length, but these pups were observed to be lethargic and cyanotic, and had subnormal weights of many organs. Body weights of 10-, 15-, and 21-day-old OGF-exposed rats were reduced 11-27% from control levels. Wet and dry organ weights of the rats maternally subjected to OGF were decreased from control values in six of the eight organs evaluated at 10 days. At weaning, some organs were subnormal in weight. These data lead us to hypothesize that a native opioid peptide-OGF-is integral to certain aspects of maternal, neonatal, and postnatal well-being, and that disruptions in this opioid peptide have serious repercussions on the course of pregnancy and fetal outcome.