Effect of prenatal gamma irradiation during the late fetal period on the postnatal development of the mouse

Pregnant Swiss albino mice were exposed to 0.3, 0.5, 1.0, or 1.5 Gy of gamma radiation on day 17 of gestation. Sham-exposed controls were examined for comparison. Exposed mice as well as controls were left to complete gestation and parturition. Pups were observed up to age 6 weeks; appearance of physiological markers (pinna detachment, eye opening, fur development, vaginal opening, and testes descent), postnatal mortality, body weight, body length, head length, head width, and tail length were recorded. A significant delay in fur development was observed at 0.3 Gy and in other physiological markers at doses above 0.3 Gy, while a significant increase in mortality and growth retardation occurred only at 1.0 and 1.5 Gy. Although congenital anomalies such as syndactyly and bent tail were observed at doses of 0.5-1.5 Gy, only syndactyly showed a statistically significant increase in frequency. A statistically significant lower body weight was observed during the first week of postnatal life, but body weights increased to normal levels by the second week in animals exposed to doses less than 1.0 Gy. At higher doses, low body weight persisted throughout the postnatal period. Head length and tail length showed a significant decrease from controls at 0.5-1.5 Gy, and the effect was evident from birth to age 6 weeks. But a similar effect on body length and head width was noticed only at 1.0 and 1.5 Gy. These studies indicate that even in the absence of any major morphological changes, normal development of physiological landmarks and postnatal growth can be impaired by fetal irradiation at 17 days p.c. (post coitus). Morphological changes appear to have a threshold between 0.3-0.5 Gy, while physiological marker effects may occur with a lower threshold.     

Air pollutant effects on fetal and early postnatal development

Numerical research on the health effects of air pollution has been published in the last decade. Epidemiological studies have shown that children's exposure to air pollutants during fetal development and early postnatal life is associated with many types of health problems including abnormal development (low birth weight [LBW], very low birth weight [VLBW], preterm birth [PTB], intrauterine growth restriction [IUGR], congenital defects, and intrauterine and infant mortality), decreased lung growth, increased rates of respiratory tract infections, childhood asthma, behavioral problems, and neurocognitive decrements. This review focuses on the health effects of major outdoor air pollutants including particulates, carbon monoxide (CO), sulfur and nitrogen oxides (SO(2), NOx), ozone, and one common indoor air pollutant, environmental tobacco smoke (ETS). Animal data is presented that demonstrate perinatal windows of susceptibility to sidestream smoke, a surrogate for ETS, resulting in altered airway sensitivity and cell type frequency. A study of neonatal monkeys exposed to sidestream smoke during the perinatal period and/or early postnatal period that resulted in an altered balance of Th1-/Th2-cytokine secretion, skewing the immune response toward the allergy-associated Th2 cytokine phenotype, is also discussed.     

Cell proliferation and differentiation in the fetal and early postnatal mouse thymus

The relationships between cell proliferation and cell differentiation during thymus ontogeny were studied by labeling DNA-synthesizing thymocytes with bromodeoxyuridine and staining with antibodies against CD4, CD8, J11d, phagocytic glycoprotein 1, TCR V beta 8 chain, Thy-1, and IL-2R surface proteins. The development of the thymus was discontinuous, with two well defined growth periods from 13 days to 18 days of fetal life and from 3 days to 6 days after birth, and more progressive growth from day 8 to 2 wk. Cell proliferation started on fetal day 12, 1 day after the arrival of hemopoietic stem cells in the third branchial pouch. These cells were phagocytic glycoprotein 1-positive but IL-2R and Thy-1 negative. Thus, cell proliferation preceded IL-2R expression. Until day 15, CD4-8- thymocytes expanded without differentiation. Then CD4-8+ and CD4+8+ cells appeared; this induction was proliferation dependent and occurred on cells which had already lost IL-2R, but just after maximum expression of this receptor. During several days, the thymus remained of constant size (around 10(7) cells) and behaved like the steady state thymus. On day 3 after birth, expansion started again and was correlated with an increase in CD4-8- proliferation index and IL-2R expression. At the same time, the thymic subset capable of expansion without differentiation was again, transiently, detectable. These results suggest that the inflow of precursor cells into the thymus is permanent but transiently increased at several times during ontogeny. Moreover, the behavior of fetal CD4-8- cells does not appear radically different from that of adult precursors, but the actual difference resides in the variation of the relative proportion of CD4-8- cells at different maturation stages, as revealed by striking variations of IL-2R expression by cycling cells.     

Immunohistochemical localization of gonadotropin-releasing hormone (GnRH) in the fetal and early postnatal mouse brain.

The objectives were to (a) determine the age in development when GnRH is first detectable in the brain and (b) observe the distribution of GnRH throughout the fetal and early postnatal period. GnRH was localized immunohistochemically in fetal (15, 16, 17 and 19 days of gestation) and early postnatal (1- and 7-day-old) mice with the peroxidase-antiperoxidase (PAP) method of Sternberger. In the organum vasculosum of the lamina terminalis (OVLT) and in the median eminence of the fetus, GnRH was first detected at 17 days of gestation. In the OVLT, GnRH was found ventral to the preoptic recess of the third ventricle near the ventral surface of the brain. In addition, GnRH was located adjacent to the superficial portal capillaries near the surface of the median eminence. At 19 days of gestation, the distribution of GnRH was similar to that observed at 17 days and there was a marked increase in amount. In the newborn mouse, GnRH was undetectable in the OVLT and its content in the median eminence was decreased as compared to that observed in the fetus. By the seventh postnatal day, a considerable accumulation of GnRH had occurred in the OVLT and median eminence. In the OVLT, it was associated with capillaries ventral to the preoptic recess, and its distribution in the median eminence was similar to that in the adult mouse. In both the OVLT and median eminence of the fetal and early postnatal mouse GnRH appeared to be stored in axons and axon endings, but was not detectable in nerve cell bodies or ependymal cells. These observations suggest that the potential for neuroendocrine control of gonadotropin secretion exists in the fetal mouse early as 17 days of gestation.     

Effect of early postnatal long-term fasting on the development of peptide hydrolysis in chicks.

1. Early development of peptide hydrolysis in the digestive tract was investigated in experiments with fasted and fed ad lib. chicks during the first decade of postnatal period. 2. Pancreatic carboxypeptidase A (CPA) activity was maximal at the moment of hatch. On the second day CPA activity considerably diminished in starved and fed animal groups; further starvation (3-4 days) led to the significant increase of CPA total and specific activity, whereas the amount of enzyme in pancreas of fed chicks was rather low. 3. Aminopeptidase (AP) activity of the small intestinal surface was less sensitive to starvation. The increase of activity in all intestinal parts was observed only on the 4th day of fasting. The most sensitive to starvation were dipeptidases. Changes in their activity (2-fold increase) were detected after 24 hr of starvation. 4. The formation of specific physiological proximo-distal gradient of intestinal exopeptidase activities began only after the moment of the first feeding. 5. This gives evidence that the development of peptide hydrolysis depends not only on the age of the animal but also on the normal physiological beginning of the process of exogenous nutrition.     

Impact of postnatal glucocorticoids on early lung development.

Inhaled glucocorticoid treatment during the first 2 yr of life is controversial because this is a period of major structural remodeling of the lung. Rabbits received aerosolized budesonide (Bud; 250 microg/ml) or injected dexamethasone (Dex; 0.05 mg.ml(-1).kg(-1)) between 1 and 5 wk of age. Treatment with Bud caused specific growth retardation of the lung. Dex but not Bud affected the mechanical properties of the lung parenchyma, when corrected for lung volume. Small peripheral airway walls in both glucocorticoid groups were thinner and had fewer alveolar attachment points with greater distance between attachments than controls, but collagen content was not affected by glucocorticoids. Dex led to reduced body weight, lung volume, alveolar number, and surface area. The alveolar size and number and elastin content, when related to lung volume, was not affected by Bud, suggesting normal structural development but inhibition of total growth. Arterial wall thickness and diameter were affected by Bud. This study demonstrates that developing lungs are sensitive to inhaled glucocorticoids. As such, the use of glucocorticoids in young infants and children should be monitored with caution and only the lowest doses that yield significant clinical improvement should be used.     

Effect of whole-body irradiation of females on pregnancy and postnatal development of the offspring

In 218 primigravida rats of Wistar strain with body mass 200-250 g radiosensitivity during the whole period of pregnancy has been studied after a single total gamma-radiation with 60Co given in LD50 2.0 Gy. The multifactor analysis includes: dynamics of the body mass of the pregnant rats, ability to implantation and pregnancy, duration of pregnancy, number of births that failed, body mass loss during delivery, death of the embryos during the antenatal period, number of viable offsprings, dynamics of body mass in the offsprings and their viability during 30 days after birth, time for covering with fur and eyes opening, developmental anomalies etc. Quantitative and qualitative postradiative disorders of the intrauterine and postnatal development of the offsprings are described. The most sensitive days of pregnancy are revealed (the 1st--3d and the 9th--11th days).     

Effect of long-term selection for early postnatal growth rate on survival and prenatal development of transferred mouse embryos

Reciprocal embryo transfer procedures were performed among mouse selection lines to examine prenatal maternal effects on survival and development of transferred embryos. Mice were from generations 28 and 29 of an experiment to select for (i) increased body weight again from 0 to 10 days (E+); (ii) decreased body weight gain from 0 to 10 days (E-); or (iii) a randomly bred control line (C). A total of 118 embryo transfer procedures performed 12 h after conception resulted in 983 progeny born to 89 litters. There was a 39% overall embryo survival rate and 75% overall pregnancy success rate. Response to superovulation and oestrous synchronization was significantly lower (P < 0.01) in the E+ line. E+ individuals that did superovulate produced an average of 37 oocytes per flush, which was significantly higher than in the control line mice (29 oocytes per flush; P < 0.01). The ability to complete pregnancy successfully was not influenced by uterine environment or embryo-uterine interaction. In contrast, embryo survival in successful pregnancies was significantly affected by uterine environment. There were large maternal effects for body weight and tail length at birth; E+ recipients produced pups that were significantly larger than E- recipient pups (P < 0.01), which in turn were significantly larger than pups gestated by control recipients (P < 0.01).     

Cytokeratin expression during mouse embryonic and early postnatal mammary gland development

Cytokeratins are intermediate filament proteins found in most epithelial cells including the mammary epithelium. Specific cytokeratin expression has been found to mark different epithelial cell lineages and also to associate with putative mammary stem/progenitor cells. However, a comparative analysis of the expression of cytokaratins during embryonic and postnatal mammary development is currently lacking. Moreover, it is not clear whether the different classes of putative mammary stem/progenitor cells exist during embryonic development. Here, we use double/triple-label immunofluorescence and immunohistochemistry to systematically compare the expression of cytokeratin 5 (K5), cytokeratin 6 (K6), cytokeratin 8 (K8), cytokeratin 14 (K14) and cytokeratin 19 (K19) in embryonic and early postnatal mouse mammary glands. We show that K6⁺ and K8⁺/K14⁺ putative mammary progenitor cells arise during embryogenesis with distinct temporal and spatial distributions. Moreover, we describe a transient disconnection of the expression of K5 and K14, two cytokeratins that are often co-expressed, during the first postnatal weeks of mammary development. Finally, we report that cytokeratin expression in cultured primary mammary epithelial cells mimics that during the early stages of postnatal mammary development. These studies demonstrate an embryonic origin of putative mammary stem/progenitor cells. Moreover, they provide additional insights into the use of specific cytokeratins as markers of mammary epithelial differentiation, or the use of their promoters to direct gene overexpression or ablation in genetic studies of mouse mammary development.     

Effect of low extracellular calcium and ryanodine on muscle contraction of the mouse during postnatal development.

We have examined the effects of low Ca2+ solutions, Co2+, and ryanodine on the isometric tension and contraction speed of isolated, developing mouse EDL muscles. Twitch responses of young muscles (7-14 days postnatal) were more sensitive to lowered [Ca2+]o than those of more fully developed muscles (22-35 days postnatal). Responses of EDL muscles from a middle-aged group (15-21 days postnatal) were intermediate between the two other groups. Overall, the time course of contraction in a single twitch was accelerated by low [Ca2+]o. Ca(2+)-free solution induced a 7.95 and 9.25 mV depolarization in young and "old" muscle fibres, respectively. The presence of cobalt ions (5 mM) in the Krebs solution had a similar effect as Ca(2+)-free Krebs in terms of reduction of the isometric twitch and tetanic tensions of EDL muscles from the various age groups. In contrast, the shortening of the contraction time seen with Ca(2+)-free solution did not take place following exposure to Co(2+)-containing solutions. Finally, young (7-14 days postnatal) muscles were less sensitive to the inhibitory action of ryanodine on the twitch compared with more fully developed muscles (22-35 days postnatal). Taken together, our results indicate that from birth to maturity, there is a gradual change in the spectrum of calcium utilization for the contractile process.     

Long-term effects of diagnostic ultrasound during fetal period on postnatal development and adult behavior of mouse

Pregnant Swiss albino mice were exposed to diagnostic levels of ultrasound (3.5 MHz, intensity 65 mW, I(SPTP) = 1 W/cm(2), I(SATA) = 240 W/cm(2)) for 10, 20 and 30 minutes on day 14 of gestation. Sham exposed controls were maintained for comparison. Fifteen pregnant mice were exposed for each group. Exposed as well as control animals were left to complete gestation and parturition. Ultrasound induced changes in maternal vaginal temperature was recorded. The changes in the physiological reflexes and postnatal mortality up to 6 weeks of age were recorded. The litters were subjected to behavioral tests for locomotor activity, learning and memory at 4 month and 1 year of age. Neither the physiological reflexes nor the postnatal mortality was affected by ultrasound exposure. However, there was a noticeable impairment in both locomotor and learning behavior even after a 10 min exposure, which further increased with increases in exposure time. Thus the present study demonstrates the neurotoxicity of diagnostic ultrasound and the high susceptibility of early fetal brain to induction of lasting detrimental changes by ultrasound exposure.     

Consequences of fetal irradiation on follicle histogenesis and early follicle development in rat ovaries

Follicle histogenesis, in which follicles arise from fragmenting ovigerous cords, is a poorly understood mechanism that is strictly dependent upon the presence of germ cells. Our previous studies have shown that severely germ cell-depleted rat ovaries after fetal gamma-irradiation display modifications of follicular endowment and dynamics during the immature period. The primordial follicle stock was absent and the follicles with primary appearance remained quiescent longer than in control ovaries during the neonatal period. The aim of the present work was to analyze the initial steps of follicle histogenesis, and to investigate the etiology of the alterations observed in the development of irradiated ovaries. Just after birth, we observed, in addition to sterile ovigerous cords, the emergence of the first follicles which exhibited several abnormal features as compared to those of control ovaries. Most of the follicles appeared as primary follicles, as they were composed of a layer of cuboidal-shaped granulosa cells surrounding an enlarged oocyte. Interestingly, the granulosa cells of these primary-like follicles did not proliferate and did not express the genes for anti-Müllerian hormone (Amh) or bone morphogenetic protein receptor type II (Bmpr2), both of which are normally expressed from the primary stage onwards. In contrast, the oocytes strongly expressed the gene for growth and differentiation factor 9 (Gdf9), which is normally upregulated from the primary follicle stage onwards, which suggests an uncoupling of granulosa cell development from oocyte development. In addition, irradiated ovaries displayed a higher frequency of follicles that contained 2 or 3 oocytes, which are also referred to as multi-oocyte follicles (MOFs). Examination at the time of follicle histogenesis indicated that MOFs arise from incomplete ovigerous cord breakdown. Taken together, the results of this study indicate that severe perturbations of follicular histogenesis take place following irradiation and massive germ cell depletion during fetal life. In addition to the classically described sterile cords, we have pointed out the differentiation of MOFs and primary-like quiescent follicles, which finally evolve into growing follicles and participate in ovarian function. We propose that these phenotypes are closely correlated to the proportion of granulosa cells to oocytes at the time of neonatal follicle histogenesis.     

Effect of substrate stiffness on early mouse embryo development

It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68±15% vs. 59±18%), blastocyst (64±9.1% vs. 50±18%) and hatching blastocyst stages (54±25% vs. 21±16%) and an increase in the number of trophectodermal cell (TE,65±13 vs. 49±12 cells) compared to control embryos cultured in PD (mean±S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.     

Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads

Fluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCA_I) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEA_I) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only). Distinct developmental-related changes were observed when female germ cells were labeled with fluorescein-conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled. I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.     

Thymocyte subpopulations during early fetal development in the BALB/c mouse

Phenotypic analysis of thymocytes during murine fetal development may be of use in determining the pathways of thymocyte differentiation. The expression of the functionally significant molecules Lyt-2 (CD8), L3T4 (CD4), and the TCR has already been described. However, mAb specific for several other murine lymphocyte surface markers are now available and, although these have been used to characterize adult thymocytes, a detailed analysis of fetal thymocytes with these antibodies has not previously been undertaken. In this study, we have used mAb specific for Thy-1, J11d, Pgp-1, and the IL-2R, in addition to those for Lyt-2 and L3T4, to identify subpopulations of early fetal thymocytes. By using two-color flow cytometric analysis of cells obtained from fetal thymuses on sequential days of gestation, we have been able to follow the development of various subpopulations through early fetal ontogeny. Our data indicate that the earlier thymocytes are found in the J11d+/Pgp-1+ subset which is abundant at fetal day 14 but constitute a numerical minority by day 16.