Normal Pups Research Articles

Effects of gestational intermittent hypoxia on the autoresuscitation response following anoxia-induced apneas in neonatal mice

Introduction: Sudden infant death syndrome (SIDS) is the leading cause of death in infants from one month to one year of age. Smoking is a significant risk factor in SIDS, and smoking probably causes intermittent hypoxia in the fetus. Therefore, we modeled the effects of intermittent hypoxia during gestation in mice to determine if gestational intermittent hypoxia would adversely affect the capacity of neonatal mice to autoresuscitate effectively after exposure to anoxic stimuli. A degraded capacity to autoresuscitate could explain, in part, why infants born to mothers who smoke have an increased risk of dying from of SIDS. The primary objective of this study was to investigate the effects of intermittent gestational hypoxia on the autoresuscitation responses to anoxia-induced apneas in neonatal mice. Our hypothesis was that pups born to intermittently hypoxic dams would struggle more to recover from anoxic apneas — would have impaired cardiorespiratory responses to anoxic stress compared to the control mice born to dams exposed to room air during gestation. Methods: We studied C57Bl/6 dams who previously had one litter to prove their capability to breed and rear pups. Dams were placed in either an hypoxic or room air chamber at approximately embryonic day 5 (E5; fifth day of pregnancy). The fractional inspired oxygen concentration (FIO2) was reduced to 11% for 10 minutes, three times per hour, with 10 minute periods of 21% oxygen between them. Each hour of intermittent hypoxia was followed by an hour breathing 21% FiO2. The room air control chamber was maintained at a constant FIO2 of 21% with inflows of compressed air that mimicked the noise and flow of nitrogen in the hypoxic chamber. The dams spent the entirety of their pregnancies in the environmental chambers. Once the pups were delivered, the cages were placed in room air, housing environments. The pups were allowed to stay with their mothers until approximately post-natal day 8 (P8). On P8, each pup was fitted into a head out plethysmograph to measure respiratory flow using a pneumotachograph. We recorded respiratory activity, ECG activity, and body movements (using a piezo sensitive film). The pups were maintained at ~34 degrees C in the pneumotachographic chamber. We measured the highest respiratory rate, highest heart rate, longest apnea, gasp latency, mean heart rate recovery, and mean respiratory rate recovery. Results: The mean heart rate recovery and mean longest apnea (in seconds) doubled in the mice exposed to gestational intermittent hypoxia compared to pups born to dams exposed to room air during gestation. The mean heart rate recovery and mean longest apnea in gestationally hypoxic pups was 41.0 +/- 13.7 seconds and 22.05 +/- 12.0 seconds, respectively. The mean heart rate recovery and mean longest apnea in normal, room air-exposed pups was 22.4 +/- 11.1 seconds and 10.3 +/- 7.0 seconds, respectively. The longest apnea in gestational intermittent hypoxic pups was significantly longer than in control pups (53.0 +/- 13.7 seconds and 19.1 +/- 15.0 seconds). Pups exposed to gestational intermittent hypoxia also had increased mean respiratory rate recovery times and gasp latency times (start of apnea until the first gasp). The average body weight of hypoxic pups was 4.3 grams while the average body weight for the normal pups was 4.9 grams. Conclusion: Gestational intermittent hypoxia was associated with delayed and prolonged the autoresuscitation responses in neonatal mice following acute anoxic exposure. We hypothesize that this is due to changes in the brain’s serotonin system. We are investigating how intrauterine hypoxia may decrease serotonin and serotonergic signaling pathways as changes in serotonergic function may degrade the capacity of mice to autoresuscitate following anoxic gas exposure. NIH/NICHD. HD100823 and NIH/NICHD. HD 107060. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prenatal treatment with corticosterone via maternal injection induces learning and memory impairments via delaying postsynaptic development in hippocampal CA1 neurons of rats.

Previously, we reported that prenatal exposure to high corticosterone induced attention-deficit hyperactivity disorder (ADHD)-like behaviors with cognitive deficits after weaning. In the present study, cellular mechanisms underlying cortisol-induced cognitive dysfunction were investigated using rat pups (Corti.Pups) born from rat mothers that were repetitively injected with corticosterone during pregnancy. In results, Corti.Pups exhibited the failure of behavioral memory formation in the Morris water maze (MWM) test and the incomplete long-term potentiation (LTP) of hippocampal CA1 neurons. Additionally, glutamatergic excitatory postsynaptic currents (EPSCs) were remarkably suppressed in Corti.Pups compared to normal rat pups. Incomplete LTP and weaker EPSCs in Corti.Pups were attributed to the delayed postsynaptic development of CA1 neurons, showing a higher expression of NR2B subunits and lower expression of PSD-95 and BDNF. These results indicated that the prenatal treatment with corticosterone to elevate cortisol level might potently downregulate the BDNF-mediated signaling critical for the synaptic development of hippocampal CA1 neurons during brain development, and subsequently, induce learning and memory impairment. Our findings suggest a possibility that the prenatal dysregulation of cortisol triggers the epigenetic pathogenesis of neurodevelopmental psychiatric disorders, such as ADHD and autism.

Influence of maternal cholesterol-enriched diet on chemical composition of teeth enamel in offspring of mice.

Aim: To determine the chemical composition of the tooth enamel of two-day-old mice from hypercholesterolemic mothers by energy dispersive X-ray spectroscopy. Materials and Methods: Forty mature female mice were randomly assigned (n = 20/group) to either a standard chow vivarium diet (control group) or a cholesterol-enriched chow diet (experimental group). After fertilization, pregnancy and birth, on postnatal day 2, the incisor segments of 6 pups form each group were used for energy dispersive X-ray spectroscopy. Results: Influence of maternal hypercholesterolemic diet on tooth development and mineralization was examined, which revealed changes in enamel chemical composition. First, the results indicate the presence of seven elements (Na, Cl, Ca, P, Mg, S, Fe) in the enamel of both the hypercholesterolemic and normal offspring, but the content of element Ca2+ decreased, the content of elements P5+, Na+, Cl- tended to increase in pups from hypercholesterolemic mice. Second, the initial level of mineralization according to the atomic (%) Ca / P in hypercholesterolemic pups ratio was 1.26, comparing with normal pups where level of mineralization was 1.34. Taking into account that irreversible changes in the structure of the enamel were observed when the Ca / P ratio was below 1.33, we can suggest that the eruption of teeth with an imperfect structure could be because of maternal hypercholesterolemic diet. Conclusions: Results of this study suggest that hypercholesterolemic diet during gestation and lactation leads to altered enamel mineralization in mice because of changes in chemical composition and may link to the early childhood caries.

Role for Nongenomic Estrogen Signaling in Male Fertility.

Estrogen actions are mediated by both nuclear (n) and membrane (m) localized estrogen receptor 1 (ESR1). Male Esr1 knockout (Esr1KO) mice lacking functional Esr1 are infertile, with reproductive tract abnormalities. Male mice expressing nESR1 but lacking mESR1 (nuclear-only estrogen receptor 1 mice) are progressively infertile due to testicular, rete testis, and efferent ductule abnormalities similar to Esr1KO males, indicating a role for mESR1 in male reproduction. The H2NES mouse expresses only mESR1 but lacks nESR1. The goal of this study was to identify the functions of mESR1 alone in mice where nESR1 was absent. Breeding trials showed that H2NES males are fertile, with decreased litter numbers but normal pup numbers/litter. In contrast to Esr1KO mice, H2NES testicular, and epididymal weights were not reduced, and seminiferous tubule abnormalities were less pronounced. However, Esr1KO and H2NES males both had decreased sperm motility and a high incidence of abnormal sperm morphology. Seminiferous tubule and rete testis dilation and decreased efferent ductule epithelial height characteristic of Esr1KO males were reduced in H2NES. Consistent with this, expression of genes involved in fluid transport and ion movement that were reduced in Esr1KO (Aqp1, Car2, Car14, Cftr) were partially or fully restored to wild-type levels in H2NES. In summary, in contrast to Esr1KO males, H2NES males are fertile and have reduced phenotypic and functional abnormalities in the testis and efferent ductules. Thus, mESR1 alone, in the absence of nESR1, can partially regulate male reproductive tract structure and function, emphasizing its importance for overall estrogen action.

Maternal obesity driven changes in collagen linearity of breast extracellular matrix induces invasive mammary epithelial cell phenotype

Obesity has been linked with numerous health issues as well as an increased risk of breast cancer. Although effects of direct obesity in patient outcomes is widely studied, effects of exposure to obesity-related systemic influences in utero have been overlooked. In this study, we investigated the effect of multigenerational obesity on epithelial cell migration and invasion using decellularized breast tissues explanted from normal female mouse pups from a diet induced multigenerational obesity mouse model. We first studied the effect of multigenerational diet on the mechanical properties, adipocyte size, and collagen structure of these mouse breast tissues, and then, examined the migration and invasion behavior of normal (KTB-21) and cancerous (MDA-MB-231) human mammary epithelial cells on the decellularized matrices from each diet group. Breast tissues of mice whose dams had been fed with high-fat diet exhibited larger adipocytes and thicker and curvier collagen fibers, but only slightly elevated elastic modulus and inflammatory cytokine levels. MDA-MB-231 cancer cell motility and invasion were significantly greater on the decellularized matrices from mice whose dams were fed with high-fat diet. A similar trend was observed with normal KTB-21 cells. Our results showed that the collagen curvature was the dominating factor on this enhanced motility and stretching the matrices to equalize the collagen fiber linearity of the matrices ameliorated the observed increase in cell migration and invasion in the mice that were exposed to a high-fat diet in utero. Previous studies indicated an increase in serum leptin concentration for those children born to an obese mother. We generated extracellular matrices using primary fibroblasts exposed to various concentrations of leptin. This produced curvier ECM and increased breast cancer cell motility for cells seeded on the decellularized ECM generated with increasing leptin concentration. Our study shows that exposure to obesity in utero is influential in determining the extracellular matrix structure, and that the resultant change in collagen curvature is a critical factor in regulating the migration and invasion of breast cancer cells.

Surfactant Treatment Shows Higher Correlation Between Ventilator and EIT Tidal Volumes in an RDS Animal Model.

Neonatal respiratory distress syndrome (RDS) is a condition of pulmonary surfactant insufficiency in the premature newborn. As such, artificial pulmonary surfactant administration is a key treatment. Despite continued improvement in the clinical outcomes of RDS, there are currently no established bedside tools to monitor whether pulmonary surfactant is effectively instilled throughout the lungs. Electrical impedance tomography (EIT) is an emerging technique in which physiological functions are monitored on the basis of temporal changes in conductivity of different tissues in the body. In this preliminary study, we aimed to assess how EIT tidal volumes correlate with ventilator tidal volumes in an RDS animal model, namely untreated, surfactant-treated, and normal control rabbit pups. Tidal volumes were measured simultaneously on an EIT system and a mechanical ventilator and compared at different peak inspiratory pressures. The linear correlation between tidal volumes measured by EIT and by ventilator had an R 2 of 0.71, 0.76 and 0.86 in the untreated, surfactant-treated, and normal control groups, respectively. Bland–Altman analysis showed a good correlation between the measurements obtained with these two modalities. The intraclass correlation coefficients (ICC) between ventilator tidal volume and EIT tidal volume were 0.83, 0.87, and 0.93 (all p < 0.001) in the untreated, surfactant-treated, and normal control groups, respectively, indicating that the higher ICC value, the better inflated status of the lung. In conclusion, we demonstrated that EIT tidal volume correlated with ventilator tidal volume. ICC was higher in the surfactant treated group.

Hepatic cytochrome P450 function is reduced by life-long Western diet consumption in guinea pig independent of birth weight

IntroductionNon-alcoholic fatty liver disease (NAFLD) is characterised by accumulation of triglycerides and cholesterol within the liver and dysregulation of specific hepatic cytochrome P450 (CYPs) activity. CYPs are involved in the metabolism of endogenous and exogenous chemicals. Hepatic CYP activity is dysregulated in human studies and animal models of a Western diet (WD) or low birth weight (LBW) independently, but the additive effects of LBW and postnatal WD consumption are unknown. As such, the aim of this study was to determine the independent and combined effect of birthweight and postnatal diet on hepatic CYP activity in a guinea pig model. MethodsLBW was generated via uterine artery ablation at mid gestation (term = 70 days gestation). Normal birthweight (NBW) and LBW pups were allocated either a control diet (CD) or WD at weaning. After 4 months of dietary intervention, guinea pigs were humanely killed, and liver tissue collected for biochemical and functional hepatic CYP activity analyses. ResultsIndependent of birthweight, functional activity of CYP3A was significantly reduced in female and male WD compared to CD animals (female, P < 0.0001; male, P = 0.004). Likewise, CYP1A2 activity was significantly reduced in male WD compared to CD animals (P = 0.020) but this same reduction was not observed in females. ConclusionDiet, but not birthweight, significantly altered hepatic CYP activity in both sexes, and the effect of diet appeared to be greater in males. These findings may have clinical implications for the management of NAFLD and associated co-morbidities between the sexes.

The postnatal leptin surge in mice is variable in both time and intensity and reflects nutritional status

Background/objectivesThe murine postnatal leptin surge occurs within the first 4 weeks of life and is critical for neuronal projection development within hypothalamic feeding circuits. Here we describe the influence of nutritional status on the timing and magnitude of the postnatal leptin surge in mice.MethodsPlasma leptin concentrations were measured 1–3 times per week for the first 4 weeks of life in C57BL/6J pups reared in litters adjusted to 3 (small), 7–8 (normal), or 11–12 (large) pups per dam fed breeder chow or raised in litters of 7–8 by dams fed high-fat diet (HFD) ad libitum starting either prior to conception or at parturition.ResultsMice raised in small litters become fatter than pups raised in either normal or large litters. The leptin surge in small litter pups starts earlier, lasts longer, and is dramatically larger in magnitude compared to normal litter pups, even when leptin concentrations are normalized to fat mass. In mice reared in large litters, weight gain is diminished and the surge is both significantly delayed and lower in magnitude compared to control pups. Pups reared by HFD-fed dams (starting preconception or at parturition) are fatter and have augmented leptin surge magnitude compared to pups suckled by chow-fed dams. Surge timing varies depending upon nutritional status of the pup; the source of the surge is primarily subcutaneous adipose tissue. At peak leptin surge, within each group, fat mass and plasma leptin are uncorrelated; in comparison with adults, pups overproduce leptin relative to fat mass. Plasma leptin elevation persists longer than previously described; at postnatal day 27 mice continue overproducing leptin relative to fat mass.ConclusionsIn mice, small litter size and maternal HFD feeding during the perinatal period augment the plasma leptin surge whereas large litter size is associated with a delayed surge of reduced magnitude.

Platelet FV Deficiency Restores Survival of TFPI Null Mice Independently of Plasma FV

Introduction: Platelets are essential for the hemostatic process, releasing “just in time” prothrombotic molecules, such as factor V (FV) and prothrombin to sites of vascular injury. Paradoxically, platelets also carry potent antithrombotic molecules, including tissue factor pathway inhibitor (TFPI), which may prevent hemostatic platelet plugs from expanding into vascular occluding thrombi. The TFPI gene undergoes alternative splicing to produce several isoforms, including TFPIα and TFPIβ. Platelets are the primary location of TFPIα in the murine circulation. TFPIα has a basic 9 amino acid sequence in its C-terminal region nearly identical to a basic sequence in the FV B domain. The TFPIα basic region tightly binds to an acidic region of the FV B domain. This binding takes place only when the FV basic region is absent, as occurs following proteolysis by factor Xa (FXa), in some forms of FV naturally present in platelet α-granules, or in genetically altered forms of FV, such as FV-short (associated with East Texas bleeding disorder). This interaction provides an essential exosite for TFPIα inhibition of the prothrombinase complex (FXa-FVa) assembled with forms of FVa retaining the B domain acidic region. Since prothrombinase assembles on the surface of activated platelets, we hypothesized that inhibition of prothrombinase via the platelet TFPIα-platelet FV interaction is a physiologically important biochemical mechanism for the inhibiting early stages of coagulation. We probed this hypothesis using TFPI K1 deleted (TFPI-/-), FV deleted (F5-/-) and FV transgenic (F5Tg) mice to uncover whether specifically reducing or removing platelet FV would rescue TFPI-/- mice from embryonic lethality.Methods/Results: We performed crossbreeding experiments using F5-/- mice carrying liver- or platelet-specific transgenes. F5LiverTg produces 50% plasma FV with no FV in platelets, whereas F5PltTg produces 3% platelet FV with no FV in plasma. F5-/- F5LiverTgand F5-/- F5PltTgmice were bred into TFPI+/- mice. Offspring from this cross were bred to determine the effects of the liver or platelet transgene on the survival of mice lacking TFPI.F5-/- is embryonic lethal in mice. F5-/- mice are rescued by transgenes expressing either liver-specific FV or platelet specific FV. When we bred F5+/- TFPI+/- x F5+/- F5LiverTg TFPI+/-, neither F5-/- TFPI-/- nor F5+/- TFPI-/- mice without the liver transgene were viable. Liver transgene expression did not rescue F5+/- TFPI-/- mice, however, it produced grossly normal pups lacking TFPI (F5-/- F5LiverTg TFPI-/-) that lived to adulthood with no overt phenotypes. We further characterized this phenotype by crossbreeding F5+/- TFPI+/- x F5-/- F5LiverTg TFPI-/-. In this cross, F5-/- F5LiverTg TFPI-/- pups survived with ~80% penetrance (45 of 234 total mice, p<5.0 x10-12). Next, we bred F5+/- TFPI+/- x F5+/- F5PltTgTFPI+/-. Platelet transgene expression also produced grossly normal pups lacking TFPI (F5-/- F5PltTg TFPI-/-) that lived to adulthood with no overt phenotypes. Further crossbreeding, as described for the liver transgene, revealed a similar ~80% survival of F5-/- F5PltTg TFPI-/- pups (56 of 291 total mice, p<9.0 x10-18). Both male and female platelet and liver specific F5-/- F5Tg TFPI-/- were fertile, and mothers supported pups until weaning.Conclusions: Neither total loss of FV nor concerted 50% reduction in plasma and platelet FV rescued TFPI-/- embryonic lethality, indicating that TFPI anticoagulant activity is not simply counterbalanced by FV procoagulant activity (or vice versa) during embryogenesis. Remarkably, either the total lack of platelet FV with 50% normal plasma FV, or the total lack of plasma FV with 3% normal platelet FV, resulted in the nearly complete rescue of F5-/- TFPI-/- mice with pups surviving to adulthood without grossly observable pathology. These observations indicate that severe reduction of platelet FV, independent of the plasma FV concentration, produces this extraordinary rescue of TFPI-/- embryonic lethality. Collectively, these findings expose the critical role for platelet TFPIα in regulating procoagulant activity of prothrombinase assembled with platelet FV. This platelet FV-platelet TFPIα interaction highlights the important biological activity of pro and anticoagulant proteins located within platelets in regulating hemostatic balance during murine development and into adulthood. DisclosuresMast:Novo Nordisk: Research Funding.

Intergenerational trauma transmission is associated with brain metabotranscriptome remodeling and mitochondrial dysfunction

Intergenerational trauma increases lifetime susceptibility to depression and other psychiatric disorders. Whether intergenerational trauma transmission is a consequence of in-utero neurodevelopmental disruptions versus early-life mother–infant interaction is unknown. Here, we demonstrate that trauma exposure during pregnancy induces in mouse offspring social deficits and depressive-like behavior. Normal pups raised by traumatized mothers exhibited similar behavioral deficits to those induced in pups raised by their biological traumatized mothers. Good caregiving by normal mothers did not reverse prenatal trauma-induced behaviors, indicating a two-hit stress mechanism comprising both in-utero abnormalities and early-life poor parenting. The behavioral deficits were associated with profound changes in the brain metabotranscriptome. Striking increases in the mitochondrial hypoxia marker and epigenetic modifier 2-hydroxyglutaric acid in the brains of neonates and adults exposed prenatally to trauma indicated mitochondrial dysfunction and epigenetic mechanisms. Bioinformatic analyses revealed stress- and hypoxia-response metabolic pathways in the neonates, which produced long-lasting alterations in mitochondrial energy metabolism and epigenetic processes (DNA and chromatin modifications). Most strikingly, early pharmacological interventions with acetyl-L-carnitine (ALCAR) supplementation produced long-lasting protection against intergenerational trauma-induced depression.

Prenatal Exposure to High Cortisol Induces ADHD-like Behaviors with Delay in Spatial Cognitive Functions during the Post-weaning Period in Rats.

High levels of cortisol in blood are frequently observed in patients with major depressive disorders and increased cortisol level induces depressivelike symptoms in animal models. However, it is still unclear whether maternal cortisol level during pregnancy is a critical factor resulting in neuropsychiatric disorders in offspring. In this study, we increased cortisol level in rats by repetitively injecting corticosterone subcutaneously (Corti. Mom, 20 mg/kg/day) during pregnancy and evaluated the behavioral patterns of their pups (Corti.Pups) via forced swimming (FS), open field (OF), elevated plus maze (EPM) and Morris water maze (MWM) tests during the immediate post-weaning period (postnatal day 21 to 25). In results, corticosterone significantly increased plasma cortisol levels in both Corti.Moms and Corti.Pups. Unlike depressive animal models, Corti.Pups showed higher hyperactive behaviors in the FS and OF tests than normal pups (Nor.Pups) born from rats (Nor.Moms) treated with saline. Furthermore, Corti.Pups spent more time and traveled longer distance in the open arms of EPM test, exhibiting higher extremity. These patterns were consistent with behavioral symptoms observed in animal models of attention deficit hyperactivity disorder (ADHD), which is characterized by hyperactivity, impulsivity, and inattention. Additionally, Corti.Pups swam longer and farther to escape in MWM test, showing cognitive declines associated with attention deficit. Our findings provide evidence that maternal cortisol level during pregnancy may affect the neuroendocrine regulation and the brain development of offspring, resulting in heterogeneous developmental brain disorders such as ADHD.

Demetylation of the sex-determining region Y gene promoter and incidence of disorder of sex development in cloned dog males.

Canine cloning is occasionally accompanied by abnormal sexual development. Some male donor cells produce cloned pups with female external genitalia and complete male gonadal dysgenesis, which is classified as an XY disorder of sex development (XY DSD). In this study, we examine the potential of 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to reduce the phenotypic abnormality XY DSD in somatic cell nuclear transfer (SCNT)- derived pups. We used a 9-year-old normal male German Shepherd dog as a cell donor. Donor cells were treated with 10 nM 5-aza-dC for 4 days before being used for SCNT. At the same stage of cell development, significantly lower levels of DNA methylation of the sex-determining region Y (SRY) promoter was observed in the treated donor cells compared to that in the untreated cells (95.2% versus 53.3% on day 4 for the control and treated groups, respectively). No significant differences were observed in the control or treatment groups concerning fusion rate, pregnancy rate (30 days or entire period), the number of pups, or the incidence of XY DSD. However, more XY DSD dogs were observed in the control group (31.25%) than in the treatment group (14.29%). Hypermethylation of the SRY promoter was observed in the XY DSD cloned pups in both the treatment (84.8%) and control groups (91.1 ± 1.4%) compared to the methylation level in the phenotypically normal male pups of the treatment (23.2 ± 20.9%) and control groups (39.1 ± 20.1%). These results suggest that 5-aza-dC treatment of donor cells can reduce the methylation level of the SRY promoter in donor cells, and thus, 5-aza-dC is advantageous for reducing the incidence of XY DSD in canine cloning.

CXCL5-CXCR2 signaling is a senescence-associated secretory phenotype in preimplantation embryos.

Pregnancy rate of women decreases with age due to declining quality of oocytes and embryos. However, there is no established method to improve pregnancy rate in aging women. In this study, we identified a senescence‐associated secretory phenotype (SASP) factor partially responsible for the decline in embryo implantation potential. Based on microarray analysis using young and aging human embryos at the same morphological grade, 702 genes showed >fivefold increases in aging human blastocysts. Among these genes, C‐X‐C motif chemokine 5 (CXCL5) showed 7.7‐fold increases in aging human blastocysts. However, no‐age‐dependent changes in expression of the CXCR2, the cognate receptor for CXCL5, were found. In aging mice, Cxcl5 transcript levels were also increased in oocytes and embryos. Treatment of young mouse embryos with CXCL5 decreased implantation rates, together with increased expression of aging markers (P53, P21, Pai‐1, and Il‐6). Moreover, CXCL5 treatment suppressed trophoblast outgrowth in young mouse blastocysts. Conversely, suppression of CXCL5‐CXCR2 signaling in aging mouse embryos using neutralizing antibodies and a receptor antagonist improved the implantation rate, leading to increases in pregnancy and delivery of normal pups. The gene expression pattern of these embryos was comparable to that in young mouse embryos showing enriched cell proliferation‐related pathways. In conclusion, we identified CXCL5 as a SASP factor in human and mouse embryos and suppression of CXCL5‐CXCR2 signaling during embryo culture improved pregnancy success in aging mice. Future analysis on CXCL5‐CXCR2 signaling suppression in human embryos could be the basis to improve embryo development and pregnancy outcome in middle‐aged infertile patients.

Maternal diesel particle exposure promotes offspring asthma through NK cell-derived granzyme B.

Mothers living near high-traffic roads before or during pregnancy are more likely to have children with asthma. Mechanisms are unknown. Using a mouse model, here we showed that maternal exposure to diesel exhaust particles (DEP) predisposed offspring to allergic airway disease (AAD, murine counterpart of human asthma) through programming of their NK cells; predisposition to AAD did not develop in DEP pups that lacked NK cells and was induced in normal pups receiving NK cells from WT DEP pups. DEP NK cells expressed GATA3 and cosecreted IL-13 and the killer protease granzyme B in response to allergen challenge. Extracellular granzyme B did not kill, but instead stimulated protease-activated receptor 2 (PAR2) to cooperate with IL-13 in the induction of IL-25 in airway epithelial cells. Through loss-of-function and reconstitution experiments in pups, we showed that NK cells and granzyme B were required for IL-25 induction and activation of the type 2 immune response and that IL-25 mediated NK cell effects on type 2 response and AAD. Finally, experiments using human cord blood and airway epithelial cells suggested that DEP might induce an identical pathway in humans. Collectively, we describe an NK cell-dependent endotype of AAD that emerged in early life as a result of maternal exposure to DEP.

Sealed culture system for supporting mouse preimplantation embryo development in vitro.

This study investigated the possibility of a sealed culture system in polymerase chain reaction (PCR) tubes to maintain embryo development. The embryo density that could support the development of 2-cell stage mouse embryos to the hatching stage was determined. At an embryo density of 1:2 (100 embryos cultured in 200μL CZB medium that had been pretreated with a reference gas containing 5% O2), the developmental rate was higher and fewer embryos exhibited reactive oxygen species- or hypoxia-induced injury compared with other sealed culture groups. Expression of growth factors (insulin-like growth factor (IGF) 1, IGF2, epidermal growth factor and transforming growth factor-α) and their receptors was evaluated, with similar expression patterns seen for embryos in sealed culture (5% O2, embryo density of 1:2) compared with the control group (embryos cultured in microdrops and placed in a 37°C, 5% CO2 water-jacketed incubator; P>0.05). After transfer of blastocysts generated by the sealed culture into recipients, there were no obvious differences in the rate of normal live pups births between the sealed culture and control groups (P>0.05). Thus, the sealed embryo culture system in PCR tubes is feasible for use in situations which cannot use a traditional incubator, such as in space and during the transport of embryos.

N-acetyl cysteine restores the fertility of vitrified-warmed mouse oocytes derived through ultrasuperovulation.

Oocyte cryopreservation is useful for preserving fertility and storing genetic resources. However, the small number of oocytes acquired using conventional treatment to induce superovulation and the reduction of fertility due to cryopreservation represent significant problems. Herein, we vitrified the oocytes derived through high-yield superovulation using inhibin antiserum and equine chorionic gonadotropin (IAS + eCG: IASe) and examined the yield of cryopreserved oocytes and survival rates relative to those of vitrified–warmed mouse oocytes derived through conventional superovulation using equine chorionic gonadotropin (eCG). Furthermore, we investigated the effects of N-acetyl cysteine on the fertility and developmental potential of vitrified–warmed oocytes derived using IASe. Compared with eCG, IASe increased the yield of cryopreserved oocytes and achieved equivalent survival rates. N-acetyl cysteine (0.5 mM) increased the fertilization rate of vitrified–warmed oocytes derived using IASe. Vitrification decreased thiol levels in the zona pellucida (ZP), while warming followed by N-acetyl cysteine treatment increased free thiol levels in ZP. Moreover, N-acetyl cysteine treatment recovered zona hardening by cleaving disulfide bonds and promoting the expansion of ZP. Two-cell embryos derived via in vitro fertilization using N-acetyl cysteine developed into normal pups through embryo transfer. Therefore, we developed an efficient technique for the production of cryopreserved oocytes using IASe through superovulation and found that N-acetyl cysteine improves the fertility of vitrified–warmed oocytes by cleaving the disulfide bonds and promoting the expansion of ZP.

Novel mRNA-Based Therapy Reduces Toxic Galactose Metabolites and Overcomes Galactose Sensitivity in a Mouse Model of Classic Galactosemia.

Classic galactosemia (CG) is a potentially lethal inborn error of galactose metabolism that results from deleterious mutations in the human galactose-1 phosphate uridylyltransferase (GALT) gene. Previously, we constructed a GalT-/- (GalT-deficient) mouse model that exhibits galactose sensitivity in the newborn mutant pups, reduced fertility in adult females, impaired motor functions, and growth restriction in both sexes. In this study, we tested whether restoration of hepatic GALT activity alone could decrease galactose-1 phosphate (gal-1P) and plasma galactose in the mouse model. The administration of different doses of mouse GalT (mGalT) mRNA resulted in a dose-dependent increase in mGalT protein expression and enzyme activity in the liver of GalT-deficient mice. Single intravenous (i.v.) dose of human GALT (hGALT) mRNA decreased gal-1P in mutant mouse liver and red blood cells (RBCs) within 24h with low levels maintained for over a week. Repeated i.v. injections increased hepatic GalT expression, nearly normalized gal-1P levels in liver, and decreased gal-1P levels in RBCs and peripheral tissues throughout all doses. Moreover, repeated dosing reduced plasma galactose by 60% or more throughout all four doses. Additionally, a single intraperitoneal dose of hGALT mRNA overcame the galactose sensitivity and promoted the growth in a GalT-/- newborn pup.

CoQ10 alleviate preeclampsia symptoms by enhancing the function of mitochondria in the placenta of pregnant rats with preeclampsia

ABSTRACTObjective: To assess whether supplementation with Coenzyme Q10 (CoQ10) during pregnancy alleviate preeclampsia symptoms and the underlying mechanism in the rats with preeclampsia.Methods: Forty-five pregnant Wistar rats were equally divided into three groups randomly and received subcutaneous saline injection (control group, n = 15) or 200 mg/kg L-NAME injection to induce preeclampsia symptoms (PE group, n = 30). The PE rats were treated by distilled water (PE+DW group, n = 15) and CoQ10 (PE+CoQ10 group, n = 15) on day 15 to 21 of gestation randomly. Physiological characteristics such as urine volume, total urine protein, blood pressure, number and weight of pups were recorded. Fluorescent dye was used to detect mitochondrial membrane potential in placenta. Real-time fluorescent quantitative PCR was used to detect the expression of mitochondrial DNA(mtDNA) in placenta.Results: There was no statistic difference among all the three groups on day 10 of gestation in SBP and 24-h proteinuria (P > 0.05). Whereas, SBP and 24-h proteinuria were significantly higher in PE group than control group on day 15 and 21 of gestation (P < 0.05). SBP and 24-h proteinuria were significantly lower in PE+CoQ10 group than PE+DW group on day 21 of gestation (P < 0.05). The number and weight of normal pups were significantly lower in PE group than the control group (P < 0.05), which were most notably in distilled water group, and the number and weight of normal pups were markedly bigger in PE+CoQ10 group rats compared to PE+DW (P < 0.05). The PE+CoQ10 group showed a significantly higher in level of mitochondrial membrane potential than PE+DW group. The expression of mtDNA was significantly higher in the PE+CoQ10 group compared with PE+DW group (P < 0.05).Conclusions: CoQ10 can alleviate preeclampsia symptoms and enhance the function of mitochondria in the placenta.

Retinoic acid: A potential therapeutic agent for cryptorchidism infertility based on investigation of flutamide-induced cryptorchid rats in vivo and in vitro

Cryptorchidism is a common disorder in children and may cause infertility in adults. The BTB is essential for maintaining the microenvironment necessary for normal spermatogenesis. This study investigated whether retinoic acid (RA) may regulate the proteins that are essential for integrity of the BTB in cryptorchidism. Female Sprague-Dawley rats were administrated flutamide during late pregnancy to induce a model of cryptorchidism in male offspring. The concentrations of RA and BTB tight and gap junction protein levels were significantly lower in untreated cryptorchid pups compared with normal pups, but almost normal in cryptorchid pups given RA. Studies in vitro corroborated these findings. The sperm quality of RA-treated model pups was better compared with the untreated model. RA treatment may have therapeutic potential to restore retinoic acid and proteins associated with integrity of the BTB in cryptorchid testis.

2022-P: Neonatal IL-4 Administration Decreases Body Fat Content and Weight and Improves Glucose Tolerance in Adulthood

Intrauterine growth restriction (IUGR) due to uteroplacental insufficiency is a common complication of pregnancy and leads to an increased risk for type 2 diabetes (T2D) in adulthood. We model IUGR by performing bilateral uterine artery ligation in pregnant rats at d18 of gestation (term 22 days). Offspring are growth restricted and newborn males, but not females have decreased ß-cell proliferation and impaired insulin secretion which is associated with inflammation and increased IL-4 levels. IUGR males develop obesity and insulin resistance in adulthood. Neutralizing IL-4 antibody given on postnatal days 1-6 normalizes hyperglycemia in adult IUGR animals. The aim of this study was to determine whether IL-4 is sufficient to recapitulate the IUGR phenotype. IL-4 (50ng or 100ng) or PBS was injected subcutaneously to normal newborn rat pups on postnatal days 1-6. IL-4 had no adverse effects on newborn rat pups and weights did not differ between the IL-4 and PBS injected pups (n=15). IL-4 injection had no effect on insulin secretion as measured by both static incubation experiments and perifusion square wave studies (n=6). Surprisingly, at P14, animals treated with IL-4 in the newborn period had decreased body fat content (p=0.0173) compared to controls (n=6). Further, at 7 weeks of age IL-4 injected rats weighed less than controls (n=7; p=0.00017) and the weight difference increased with age. At 10 weeks, IL-4 injected rats had decreased fasting blood glucose levels (n=6-7; p=0.0055) and improved glucose tolerance and the area under the curve (AUC) was less than controls (n=3; p=0.0513). These results suggest that IL-4 is not sufficient to recapitulate the IUGR metabolic phenotype. However, IL-4 administered transiently during the neonatal period has lasting effects in adulthood, decreasing fat content and improving glucose tolerance. Disclosure T. Ying: None. R.A. Simmons: None. Funding National Institutes of Health