Insights into in vivo follicle formation: a review of in vitro systems

In vitro systems capable of reconstituting the process of mouse oogenesis are now being established to help develop further understanding of the mechanisms underlying oocyte/follicle development and differentiation. These systems could also help increase the production of useful livestock or genetically modified animals, and aid in identifying the causes of infertility in humans. Recently, we revealed, using an in vitro system for recapitulating oogenesis, that the activation of the estrogen signaling pathway induces abnormal follicle formation, that blocking estrogen-induced expression of anti-Müllerian hormone is crucial for normal follicle formation, and that the production of α-fetoprotein in fetal liver tissue is involved in normal in vivo follicle formation. In mouse fetuses, follicle formation is not carried out by factors within the ovaries but is instead orchestrated by distal endocrine factors. This review outlines findings from genetics, endocrinology, and in vitro studies regarding the factors that can affect the formation of primordial follicles in mammals.

Optimized nickase- and nuclease-based prime editing in human and mouse cells

Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. Currently, the delivery of PE components to mammalian cell lines requires multiple plasmid vectors. To overcome this limitation, we generated all-in-one prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease generated intended edits with efficiencies that were similar, and in some cases exceeded, the PE-nickase system. We also show that the nuclease prime editor can generate intended modifications in mouse fetuses with up to 100% efficiency.

Assessments of Heart Rate and Sympathetic and Parasympathetic Nervous Activities of Normal Mouse Fetuses at Different Stages of Fetal Development Using Fetal Electrocardiography

Heart rate is controlled by the activity of the autonomic nervous system: the sympathetic and parasympathetic nervous systems increase and suppress heart rate, respectively. To evaluate the activity of the autonomic nervous system, it is possible to determine heart rate variability using electrocardiography (ECG). During the fetal period, the heart and autonomic nerves develop in coordination; however, physiological changes, including autonomic nervous activities that occur during the fetal stage, remain largely unknown. Therefore, in this study, we measured ECG signals of mouse fetuses using our established method to evaluate the development of heart rate and autonomic nervous activity at different fetal developmental stages. We found that heart rate was significantly increased in fetal mice at embryonic day (E) 18.5 compared with that at E13.5, E15.5, and E17.5, indicating that fetal heart rate increases only at the stage immediately prior to birth. Interestingly, fetal parasympathetic nervous activity was reduced at E17.5 and E18.5 compared with that at E13.5, whereas fetal sympathetic nervous activity remained unchanged, at least from E13.5 to E18.5. These results indicate that parasympathetic activity rather than sympathetic activity affects fetal heart rate and that the decrease in parasympathetic activity toward the end of pregnancy could result in the observed increase in fetal heart rate.

Transcriptome and DNA Methylation Profiles of Mouse Fetus and Placenta Generated by Round Spermatid Injection

Low birth efficiency and developmental abnormalities in embryos derived using round spermatid injection (ROSI) limit the clinical application of this method. Further, the underlying molecular mechanisms remain elusive and warrant further in-depth study. In this study, the embryonic day (E) 11.5 mouse fetuses and corresponding placentas derived upon using ROSI, intracytoplasmic sperm injection (ICSI), and naturalin vivofertilized (control) embryos were collected. Transcriptome and DNA methylation profiles were analyzed and compared using RNA-sequencing (RNA-seq) and whole-genome bisulfite sequencing, respectively. RNA-seq results revealed similar gene expression profiles in the ROSI, ICSI, and control fetuses and placentas. Compared with the other two groups, seven differentially expressed genes (DEGs) were identified in ROSI fetuses, and ten DEGs were identified in the corresponding placentas. However, no differences in CpG methylation were observed in fetuses and placentas from the three groups. Imprinting control region methylation and imprinted gene expression were the same between the three fetus and placenta groups. Although 49 repetitive DNA sequences (RS) were abnormally activated in ROSI fetuses, RS DNA methylation did not differ between the three groups. Interestingly, abnormal hypermethylation in promoter regions and low expression ofFggyandRec8were correlated with a crown-rump length less than 6 mm in one ROSI fetus. Our study demonstrates that the transcriptome and DNA methylation in ROSI-derived E11.5 mouse fetuses and placentas were comparable with those in the other two groups. However, some abnormally expressed genes in the ROSI fetus and placenta warrant further investigation to elucidate their effect on the development of ROSI-derived embryos.

Ionizable lipid nanoparticles for in utero mRNA delivery

Clinical advances enable the prenatal diagnosis of genetic diseases that are candidates for gene and enzyme therapies such as messenger RNA (mRNA)–mediated protein replacement. Prenatal mRNA therapies can treat disease before the onset of irreversible pathology with high therapeutic efficacy and safety due to the small fetal size, immature immune system, and abundance of progenitor cells. However, the development of nonviral platforms for prenatal delivery is nascent. We developed a library of ionizable lipid nanoparticles (LNPs) for in utero mRNA delivery to mouse fetuses. We screened LNPs for luciferase mRNA delivery and identified formulations that accumulate within fetal livers, lungs, and intestines with higher efficiency and safety compared to benchmark delivery systems, DLin-MC3-DMA and jetPEI. We demonstrate that LNPs can deliver mRNAs to induce hepatic production of therapeutic secreted proteins. These LNPs may provide a platform for in utero mRNA delivery for protein replacement and gene editing.

Increased RNA editing in maternal immune activation model of neurodevelopmental disease

The etiology of major neurodevelopmental disorders such as schizophrenia and autism is unclear, with evidence supporting a combination of genetic factors and environmental insults, including viral infection during pregnancy. Here we utilized a mouse model of maternal immune activation (MIA) with the viral mimic PolyI:C infection during early gestation. We investigated the transcriptional changes in the brains of mouse fetuses following MIA during the prenatal period, and evaluated the behavioral and biochemical changes in the adult brain. The results reveal an increase in RNA editing levels and dysregulation in brain development-related gene pathways in the fetal brains of MIA mice. These MIA-induced brain editing changes are not observed in adulthood, although MIA-induced behavioral deficits are observed. Taken together, our findings suggest that MIA induces transient dysregulation of RNA editing at a critical time in brain development.

Two sides of a coin: a Zika virus mutation selected in pregnant rhesus macaques promotes fetal infection in mice but at a cost of reduced fitness in nonpregnant macaques and diminished transmissibility by vectors

ABSTRACTAlthough fetal death is now understood to be a severe outcome of congenital Zika syndrome, the role of viral genetics is still unclear. We sequenced Zika virus (ZIKV) from a rhesus macaque fetus that died after inoculation and identified a single intra-host mutation, M1404I, in the ZIKV polyprotein, located in NS2B. Targeted sequencing flanking position 1404 in 9 additional macaque mothers and their fetuses identified M1404I at sub-consensus frequency in the majority (5 of 9, 56%) of animals and some of their fetuses. Despite its repeated presence in pregnant macaques, M1404I occurs rarely in humans since 2015. Since the primary ZIKV transmission cycle is human-mosquito-human, mutations in one host must be retained in the alternate host to be perpetuated. We hypothesized that ZIKV I1404 increases fitness in non-pregnant macaques and pregnant mice but is less efficiently transmitted by vectors, explaining its low frequency in humans during outbreaks. By examining competitive fitness relative to M1404, we observed that I1404 produced lower viremias in non-pregnant macaques and was a weaker competitor in tissues. In pregnant wildtype mice ZIKV I1404 increased the magnitude and rate of placental infection and conferred fetal infection, contrasting with M1404, which was not detected in fetuses. Although infection and dissemination rates were not different, Ae. aegypti transmitted ZIKV I1404 more poorly than M1404. Our data highlight the complexity of arbovirus mutation-fitness dynamics, and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics.IMPORTANCEAlthough Zika virus infection of pregnant women can result in congenital Zika syndrome, the factors that cause the syndrome in some but not all infected mothers are still unclear. We identified a mutation that was present in some ZIKV genomes in experimentally inoculated pregnant rhesus macaques and their fetuses. Although we did not find an association between the presence of the mutation and fetal death, we performed additional studies with it in non-pregnant macaques, pregnant mice, and mosquitoes. We observed that the mutation increased the ability of the virus to infect mouse fetuses but decreased its capacity to produce high levels of virus in the blood of non-pregnant macaques and to be transmitted by mosquitoes. This study shows that mutations in mosquito-borne viruses like ZIKV that increase fitness in pregnant vertebrates may not spread in outbreaks when they compromise transmission via mosquitoes and fitness in non-pregnant hosts.

Obstetric Ultrasonography to Detect Fetal Abnormalities in a Mouse Model for Zika Virus Infection

In 2015 Zika virus (ZIKV) emerged for the first time in South America. The following ZIKV epidemic resulted in the appearance of a clinical phenotype with microcephaly and other severe malformations in newborns. So far, mechanisms of ZIKV induced damage to the fetus are not completely understood. Previous data suggest that ZIKV may bypass the placenta to reach the fetus. Thus, animal models for ZIKV infection are important to facilitate studies about ZIKV infection during pregnancy. Here, we used ultrasound based imaging (USI) to characterize ZIKV induced pathogenesis in the pregnant Type I interferon receptor-deficient (IFNAR-/-) mouse model. Based on USI we suggest the placenta to be a primary target organ of ZIKV infection enabling ZIKV spreading to the fetus. Moreover, in addition to direct infection of the fetus, the placental ZIKV infection may cause an indirect damage to the fetus through reduced uteroplacental perfusion leading to intrauterine growth retardation (IUGR) and fetal complications as early as embryonic day (ED) 12.5. Our data confirmed the capability of USI to characterize ZIKV induced modifications in mouse fetuses. Data from further studies using USI to monitor ZIKV infections will contribute to a better understanding of ZIKV infection in pregnant IFNAR-/- mice.