Zika virus infection of pregnant Ifnar1-/- mice triggers strain-specific differences in fetal outcomes

Zika virus (ZIKV) is a flavivirus that causes a constellation of adverse fetal outcomes collectively termed Congenital Zika Syndrome (CZS). However, not all pregnancies exposed to ZIKV result in an infant with apparent defects. During the 2015-2016 American outbreak of ZIKV, CZS rates varied by geographic location. The underlying mechanisms responsible for this heterogeneity in outcomes have not been well defined. Therefore, we sought to characterize and compare the pathogenic potential of multiple Asian/American-lineage ZIKV strains in an established Ifnar1 pregnant mouse model. Here, we show significant differences in the rate of fetal demise following maternal inoculation with ZIKV strains from Puerto Rico, Panama, Mexico, Brazil, and Cambodia. Rates of fetal demise broadly correlated with maternal viremia but were independent of fetus and placenta virus titer, indicating that additional underlying factors contribute to fetus outcome. Our results, in concert with those from other studies, suggest that subtle differences in ZIKV strains may have important phenotypic impacts. With ZIKV now endemic in the Americas, greater emphasis needs to be placed on elucidating and understanding the underlying mechanisms that contribute to fetal outcome.

Identification of mundulone and mundulone acetate as natural products with tocolytic efficacy in mono- and combination-therapy with current tocolytics

Currently, there are a lack of FDA-approved tocolytics for the management of preterm labor. We previously observed that the isoflavones mundulone and mundulone acetate (MA) inhibit intracellular Ca2+-regulated myometrial contractility. Here, we further probed the potential of these natural products to be small molecule leads for discovery of novel tocolytics by: (1) examining uterine-selectivity by comparing concentration-response between human primary myometrial cells and a major off-target site, aortic vascular smooth muscle cells (VSMCs), (2) identifying synergistic combinations with current clinical tocolytics to increase efficacy or and reduce off-target side effects, (3) determining cytotoxic effects and (4) investigating the efficacy, potency and tissue-selectivity between myometrial contractility and constriction of fetal ductus arteriosus (DA), a major off-target of current tocolytics. Mundulone displayed significantly greater efficacy (Emax = 80.5% vs. 44.5%, p=0.0005) and potency (IC50 = 27 μM and 14 μM, p=0.007) compared to MA in the inhibition of intracellular-Ca2+ from myometrial cells. MA showed greater uterine-selectivity, compared to mundulone, based on greater differences in the IC50 (4.3 vs. 2.3 fold) and Emax (70% vs. 0%) between myometrial cells compared to aorta VSMCs. Moreover, MA demonstrated a favorable in vitro therapeutic index of 8.8, compared to TI = 0.8 of mundulone, due to its significantly (p<0.0005) smaller effect on the viability of myometrial (hTERT-HM), liver (HepG2) and kidney (RPTEC) cells. However, mundulone exhibited synergism with two current tocolytics (atosiban and nifedipine), while MA only displayed synergistic efficacy with only nifedipine. Of these synergistic combinations, only mundulone + atosiban demonstrated a favorable TI = 10 compared to TI=0.8 for mundulone alone. While only mundulone showed concentration-dependent inhibition of ex vivo mouse myometrial contractions, neither mundulone or MA affected mouse fetal DA vasoreactivity. The combination of mundulone and atosiban yielded greater tocolytic efficacy and potency on term pregnant mouse and human myometrial tissue compared to single-drugs. Collectively, these data highlight the difference in uterine‐selectivity of Ca2+‐mobilization, effects on cell viability and tocolytic efficacy between mundulone and MA. These natural products could benefit from medicinal chemistry efforts to study the structural activity relationship for further development into a promising single- and/or combination-tocolytic therapy for management of preterm labor.

The Circadian Clock Gene Bmal1 Modulates Myometrium Contractile Function in Pregnant Mice

Caesarian section should be avoided unless medically required. Caesarian section is often a result of failed labor enhancement. While most natural births occur at night, labor enhancement is often scheduled during the day. We propose that the disparity between the commonly used timing of labor enhancement in the clinic, from the timing of natural birth, could contribute to the high rate of labor induction failure. Oxytocin receptor agonists are the most used labor enhancing agents. In pregnant non-human primates, oxytocin enhances uterine contractions more efficiently at night than during the day, suggesting a daily change in uterine sensitivity to oxytocin in pregnancy. To identify the molecular mechanisms generating daily changes in uterine function, we here explore the role of the molecular clock gene, Bmal1 (Brain and muscle ARNTL1-like), in the pregnant mouse myometrium. BMAL1 is a transcription factor required to generated circadian rhythms at the cellular level. We hypothesize that Bmal1 in uterine myometrial cells generates circadian rhythms and establishes the daily change in uterine contractile response to oxytocin. To evaluate circadian rhythms ex vivo, we collected myometrium samples from the validated circadian Per2:luciferase reporter mice at gestation day 17-18. We found that the pregnant mouse myometrium possesses circadian rhythms, which are generated by the molecular clock, as triple transgenic Per2:luciferase mice with Bmal1 conditionally deleted in the myometrium (cKO) do not have rhythmic expression of the Per2:luciferase reporter. To determine if BMAL1 is required to establish uterine contractions, we used a myograph to measure ex vivo uterine contractions at gestation day 17-18. In controls, uterine contraction force was significantly higher at ZT15 (3h after lights OFF) versus ZT3 (3h after lights ON). Interestingly, our preliminary data show increased basal contractile force at ZT15 in cKO as compared to controls. In addition, the cKO uterus contracted stronger to oxytocin than controls. Our findings identify Bmal1 as a clock gene modulating basal contractions in the mouse uterus and indicate Bmal1 might be a regulator of uterine sensitivity to oxytocin. Future work will focus on identifying the molecular mechanisms driven by BMAL1 to regulate uterine function in pregnancy. This work has the potential to provide insights into how we can improve labor enhancing treatment strategies in the clinic in the future.

Placenta-derived IL-32β activates neutrophils to promote preeclampsia development

Immune activation at the maternal-fetal interface is a main pathogenic factor of preeclampsia (PE). Neutrophils (PMNs) are activated in PE patients, but the mechanism and consequences of PMN activation need to be further explored. Here, we demonstrated that interleukin-32 (IL-32) expression was significantly upregulated in syncytiotrophoblasts (STBs) and that IL-32β was the major isoform with increased expression in the placenta of severe PE (sPE) patients. Furthermore, the level of IL-32 expression in the placenta was correlated with its level in the serum of sPE patients, indicating that IL-32 in the serum is derived mainly from the placenta. Then, in vitro experiments showed that IL-32β could highly activate PMNs and that these IL-32β-activated PMNs were better able to adhere to endothelial cells (HUVECs) and enhance the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) in HUVECs, which could be reversed by preincubation with the NADPH oxidase inhibitor VAS 2870. In addition, we showed that IL-32β mainly activated PMNs by binding to proteinase 3. Finally, IL-32β administration induced a PE-like phenotype in a pregnant mouse model. This study provides evidence of the involvement of IL-32β in the pathogenesis of PE.

Exposure to sevoflurane results in changes of transcription factor occupancy in sperm and inheritance of autism

ABSTRACTOne in 54 children in the U.S. is diagnosed with Autism Spectrum Disorder (ASD). De novo germline and somatic mutations cannot account for all cases of ASD, suggesting that epigenetic alterations triggered by environmental exposures may be responsible for a subset of ASD cases. Human and animal studies have shown that exposure of the developing brain to general anesthetic (GA) agents can trigger neurodegeneration and neurobehavioral abnormalities but the effects of general anesthetics on the germ line have not been explored in detail. We exposed pregnant mice to sevoflurane during the time of embryonic development when the germ cells undergo epigenetic reprogramming and found that more than 38% of the directly exposed F1 animals exhibit impairments in anxiety and social interactions. Strikingly, 44-47% of the F2 and F3 animals, which were not directly exposed to sevoflurane, show the same behavioral problems. We performed ATAC-seq and identified more than 1,200 differentially accessible sites in the sperm of F1 animals, 69 of which are also present in the sperm of F2 animals. These sites are located in regulatory regions of genes strongly associated with ASD, including Arid1b, Ntrk2, and Stmn2. These findings suggest that epimutations caused by exposing germ cells to sevoflurane can lead to ASD in the offspring, and this effect can be transmitted through the male germline inter and trans-generationally.Summary sentencePregnant mouse F0 females exposed to sevoflurane give rise to F1 males with sociability and anxiety defects. These behaviors are transmitted to F2 and F3 males. Their sperm show changes in transcription factor occupancy in genes implicated in autism.

Combination of histochemical analyses and micro-MRI reveals regional changes of the murine cervix in preparation for labor

The cervix is responsible for maintaining pregnancy, and its timely remodeling is essential for the proper delivery of a baby. Cervical insufficiency, or “weakness”, may lead to preterm birth, which causes infant morbidities and mortalities worldwide. We used a mouse model of pregnancy and term labor, to examine the cervical structure by histology (Masson Trichome and Picrosirius Red staining), immunohistochemistry (Hyaluronic Acid Binding Protein/HABP), and ex-vivo MRI (T2-weighted and diffusion tensor imaging), focusing on two regions of the cervix (i.e., endocervix and ectocervix). Our results show that mouse endocervix has a higher proportion of smooth muscle cells and collagen fibers per area, with more compact tissue structure, than the ectocervix. With advanced gestation, endocervical changes, indicative of impending delivery, are manifested in fewer smooth muscle cells, expansion of the extracellular space, and lower presence of collagen fibers. MRI detected three distinctive zones in pregnant mouse endocervix: (1) inner collagenous layer, (2) middle circular muscular layer, and (3) outer longitudinal muscular layer. Diffusion MRI images detected changes in tissue organization as gestation progressed suggesting the potential application of this technique to non-invasively monitor cervical changes that precede the onset of labor in women at risk for preterm delivery.

Vaccination with Neospora GRA6 Interrupts the Vertical Transmission and Partially Protects Dams and Offspring against Neospora caninum Infection in Mice

Vaccination is the mainstay of preventative measures for numerous infectious diseases. Neospora caninum infection induces storms of abortion in pregnant cows and ewes, resulting in drastic economic losses because of fetal losses and culling of the dams. Herein, we evaluated the potential of recombinant protein of N. caninum dense granule protein 6 fused with glutathione-S-transferase (NcGRA6+GST) as a vaccine candidate against neosporosis in a pregnant mouse model. The protective efficacy was investigated by subcutaneous inoculation of BALB/c mice with recombinant NcGRA6+GST (25 pmol), and GST alone (25 pmol) or phosphate-buffered saline (PBS) as the controls. This study revealed the partial ability of NcGRA6+GST to protect the dams and offspring from N. caninum infection during the critical period of pregnancy. This ability was revealed by higher survival rate and lower parasite burden in brains of offspring of the NcGRA6+GST-immunized group in comparison with the control groups. In addition, mouse dams from NcGRA6+GST-immunized groups exhibited lower clinical score and minimum alteration in body weight in comparison with PBS or GST groups after challenge with N. caninum tachyzoites. Taken together, our results suggest the efficacy of recombinant NcGRA6 for interrupting the vertical transmission of N. caninum in mice by reducing the severity of infections in dams and offspring.

Transgenerational inheritance of BPA-induced obesity correlates with transmission of new CTCF sites in the Fto gene

The mechanisms by which epiphenotypes are transmitted transgenerationally through the parental germlines are poorly understood. Here we show that exposure of pregnant mouse F0 females during E7.5-E13.5 to bisphenol A results in obesity in the F2 progeny in the absence of additional exposure. This epiphenotype can be transmitted through the male and female germlines up to the F5 generation, decreases in F6, and disappears in F7. Analyses of chromatin changes in the sperm of the F1 generation reveal a widespread increase in chromatin accessibility at binding sites for CTCF and other transcription factors accompanied by alterations in 3D organization. Comparison of the transmission of obesity between F2 and F5 and its disappearance in F7 with alterations in the binding of these transcription factors points to the activation of two enhancers located in intronic and intergenic regions of the Fto gene as the cause of transgenerational inheritance. These enhancers form an autoregulatory feedback loop that, in combination with a decrease of m6A in sperm RNAs, may cause alterations of gene expression in the embryo after fertilization. Given the established involvement of SNPs in FTO in human obesity, the results suggest that both genetic and epigenetic alterations of the same gene can lead to the same phenotypic outcomes on human health.