FUS driven circCNOT6L biogenesis in mouse and human spermatozoa supports zygote development

Circular RNA (circRNA) biogenesis requires a backsplicing reaction, promoted by inverted repeats in cis-flanking sequences and trans factors, such as RNA-binding proteins (RBPs). Among these, FUS plays a key role. During spermatogenesis and sperm maturation along the epididymis such a molecular mechanism has been poorly explored. With this in mind, we chose circCNOT6L as a study case and wild-type (WT) as well as cannabinoid receptor type-1 knock-out (Cb1−/−) male mice as animal models to analyze backsplicing mechanisms. Our results suggest that spermatozoa (SPZ) have an endogenous skill to circularize mRNAs, choosing FUS as modulator of backsplicing and under CB1 stimulation. A physical interaction between FUS and CNOT6L as well as a cooperation among FUS, RNA Polymerase II (RNApol2) and Quaking (QKI) take place in SPZ. Finally, to gain insight into FUS involvement in circCNOT6L biogenesis, FUS expression was reduced through RNA interference approach. Paternal transmission of FUS and CNOT6L to oocytes during fertilization was then assessed by using murine unfertilized oocytes (NF), one-cell zygotes (F) and murine oocytes undergoing parthenogenetic activation (PA) to exclude a maternal contribution. The role of circCNOT6L as an active regulator of zygote transition toward the 2-cell-like state was suggested using the Embryonic Stem Cell (ESC) system. Intriguingly, human SPZ exactly mirror murine SPZ.

High Genetic Diversity of Shorea acuminata Dyer in the Rehabilitated Area of a Degraded Lowland Dipterocarp Tropical Rainforest

The United Nation’s Decade on Ecosystem Restoration 2021–2030 aims to halt ecosystem degradation to achieve Sustainable Development Goals (SDGs) by 2030. In Malaysia, the concept of sustainable forest management (SFM) has been practiced since 1901. In this study, we evaluated the genetic diversity of the native dipterocarp timber tree Shorea acuminata in a rehabilitated area at Kenaboi Forest Reserve (Kenaboi FR). The rehabilitated area was formerly a degraded forest managed with the taungya restoration system for 50 years. All trees with diameter at breast height (DBH) of 5 cm and over were measured, tagged and identified in a one-hectare study plot. A total of 132 inner bark samples were collected for DNA extraction. Four SSR markers (Sle280, Sle392, Sle475 and Sle566) and two EST-SSR markers (SleE07 and SleE16) were used to analyse 95 good-quality DNA samples. Genetic diversity parameters including maternal contribution were determined for 75 samples. The genetic diversity of big trees (He = 0.656 ± 0.19) and small trees (He = 0.652 ± 0.17) were high and both were in genetic equilibrium, with Fis values of the big trees being 0.035 and small trees being 0.164. Clustering analysis based on Jaccard’s similarity values (at 95% confidence level) confirmed that big trees in the Kenaboi FR rehabilitated area had originated from genetically diverse seed trees of the Sungai Menyala Forest Reserve which were used as the planting stock for the taungya restoration system. Maternal contribution showed that the allele contribution of the small trees came from the planted S. acuminata trees within the study area. The high genetic diversity of small trees in this study provides strong evidence that the existing big trees would be suitable for a genetically diverse seed collection to rehabilitate other degraded forests. Sustainable forest management must emphasise genetic diversity in order to ensure the resilience of rehabilitated forest ecosystems.

Circulating Maternal sFLT1 (Soluble fms-Like Tyrosine Kinase-1) Is Sufficient to Impair Spiral Arterial Remodeling in a Preeclampsia Mouse Model

One driving factor for developing preeclampsia—a pregnancy disorder, often associated with poor spiral artery (SpA)-remodeling and fetal growth restriction—is the anti-angiogenic sFLT1 (soluble fms-like tyrosine kinase-1), which is found to be highly upregulated in preeclampsia patients. The sFLT1-mediated endothelial dysfunction is a common theory for the manifestation of maternal preeclampsia symptoms. However, the influence of sFLT1 on SpA-remodeling and the link between placental and maternal preeclampsia symptoms is less understood. To dissect the hsFLT1 (human sFLT1) effects on maternal and/or fetoplacental physiology in preeclampsia, sFLT1-transgenic mice with systemic hsFLT1 overexpression from midgestation onwards were used. SpA-remodeling was analyzed on histological and molecular level in placental/mesometrial triangle tissues. Maternal kidney and aorta morphology was investigated, combined with blood pressure measurements via telemetry. hsFLT1 overexpression resulted in maternal hypertension, aortic wall thickening, and elastin breakdown. Furthermore, maternal kidneys showed glomerular endotheliosis, podocyte damage, and proteinuria. preeclampsia symptoms were combined with fetal growth restriction already at the end of the second trimester and SpA-remodeling was strongly impaired as shown by persisted vascular smooth muscle cells. This phenotype was associated with shallow trophoblast invasion, delayed presence of uterine natural killer cells, and altered lymphatic angiogenesis. Overall, this study showed that circulating maternal hsFLT1 is sufficient to induce typical maternal preeclampsia-like symptoms in mice and impair the SpA-remodeling independent from the fetoplacental compartment, revealing new insights into the interaction between the placental and maternal contribution of preeclampsia.

Isoform-specific roles of the Drosophila filamin-type protein Jitterbug (Jbug) during development

Filamins are highly conserved actin-crosslinking proteins that regulate organization of the actin cytoskeleton. As key components of versatile signaling scaffolds, filamins are implicated in developmental anomalies and cancer. Multiple isoforms of filamins exist, raising the possibility of distinct functions for each isoform during development and in disease. Here, we provide an initial characterization of jitterbug (jbug), which encodes one of the two filamin-type proteins in Drosophila. We generate Jbug antiserum that recognizes all of the spliced forms and reveals differential expression of different Jbug isoforms during development, and a significant maternal contribution of Jbug protein. To reveal the function of Jbug isoforms, we create new genetic tools, including a null allele that deletes all isoforms, hypomorphic alleles that affect only a subset, and UAS lines for Gal4-driven expression of the major isoforms. Using these tools, we demonstrate that Jbug is required for viability and that specific isoforms are required in the formation of actin-rich protrusions including thoracic bristles in adults and ventral denticles in the embryo. We also show that specific isoforms of Jbug show differential localization within epithelia and that maternal and zygotic loss of jbug disrupts Crumbs (Crb) localization in several epithelial cell types.

Partitioning maternal and exogenous diet contributions to otolith 87Sr/86Sr in Kokanee Salmon (Oncorhynchus nerka)

Strontium isotopes (87Sr/86Sr) recorded in the otoliths of Pacific Salmon (Oncorhynchus spp.) are commonly used to identify natal origin. For species that migrate at or soon after emergence, the embryonic region of the otolith provides the only record of provenance. However, maternal contribution of Sr from the yolk can confound the isotopic signature of the natal site. We experimentally quantified maternal and exogenous diet contributions to otolith 87Sr/86Sr over embryonic development in Kokanee salmon (O. nerka). Eggs from two populations in isotopically distinct lakes were incubated and reared in a common water source. Timing of developmental events and proportional contribution from yolk to otolith 87Sr/86Sr differed significantly between the two populations. We suggest that the magnitude of difference in 87Sr/86Sr between yolk and water, the relative concentrations of Sr and Ca in these isotopic sources, and population-specific effects on otolith growth and composition contribute to this variation. Understanding how these factors affect otolith 87Sr/86Sr could extend the use of otolith geochemistry for determining provenance to species and populations in which natal site rearing is limited.

Maternal vgll4a promotes blastoderm cohesion enabling yap1-mediated mechano-transduction during zebrafish epiboly

Cellular cohesion provides tissue tension, which is then sensed by the cytoskeleton and decoded by the activity of mechano-transducers, such as the transcriptional cofactor Yap1, thereby enabling morphogenetic responses in multi-cellular organisms. How cell cohesion is regulated is nevertheless unclear. Here we show that, zebrafish epiboly progression, a prototypic morphogenetic event that depends on Yap activity, requires the maternal contribution of the proposed yap1 competitor vgll4a. In embryos lacking maternal/zygotic vgll4a (MZvgll4a), spreading epithelial cells are ruffled, blastopore closure is delayed and the expression of the yap1-mediator arhgap18 is decreased, impairing the actomyosin ring at the syncytial layer. Furthermore, rather than competing with Yap1, vgll4a coordinate the levels of the E-Cadherin/β-catenin adhesion complex components at the blastomere plasma membrane and hence their actin cortex distribution. Taking these results together, we propose that maternal vgll4a may act at epiboly initiation to coordinate blastomere adhesion/cohesion, which is a fundamental piece of the self-sustained bio-mechanical regulatory loop underlying morphogenetic rearrangements during gastrulation.

Isoform-specific roles of the Drosophila filamin-type protein Jitterbug (Jbug) during development

ABSTRACTFilamins are highly conserved actin-crosslinking proteins that regulate organization of the actin cytoskeleton. As key components of versatile signaling scaffolding complexes, filamins are implicated in developmental anomalies and cancer. Multiple isoforms of filamins exist, raising the possibility of distinct functions of each isoform during development and in diseases. Here, we provide an initial characterization of jitterbug (jbug), which encodes one of the two filamin-type genes in Drosophila. We generate Jbug antiserum that recognizes all of the spliced forms, which reveals differential expression of different Jbug isoforms during development with a significant maternal contribution of Jbug protein. To reveal the function of Jbug isoforms, we create new genetic tools, including a null allele that deletes all isoforms, hypomorphic alleles that affect only a subset, and UAS lines for expression of the major isoforms. Using these tools, we demonstrate that Jbug is required for viability and that specific isoforms of Jbug are required in the formation of actin-rich protrusions such as thoracic bristles in adults and ventral denticles in the embryo. We also provide evidence for trans-splicing in the jbug locus.

Parental contribution to trisomy in heterozygous androgenetic complete moles

Complete hydatidiform moles (CHMs) comprise a proliferative trophoblastic disorder and are known to be androgenetic and diploid. Androgenetic CHMs are classified as having monospermic and dispermic origins. Rarely, some CHMs have other genetic constitutions, such as biparental diploid or tetraploid. Previous studies have shown the possibility that androgenetic heterozygous CHMs have an additional chromosome with high frequency. This study aimed to comprehensively analyse the molecular karyotyping of androgenetic dispermic CHMs and the parental contribution of their additional chromosomes. Single-nucleotide polymorphism arrays were performed with the genomic DNA of CHMs and patients. The B allele frequency and selected B allele frequency plotting of CHM were visualised. Among the 31 dispermic CHMs, eight showed trisomy and one showed double trisomy; of the 10 additional chromosomes, seven were of maternal original and three were of paternal origin. In addition, three disomic chromosomes comprised one maternal and one paternal chromosome, although these should theoretically have had two paternal chromosomes in the case of androgenetic CHMs. The subclassification of heterozygous CHMs, with or without maternal contribution, is a new approach and could be a candidate indicator of gestational trophoblastic neoplasia risk.

Spontaneous embryo resorption in the mouse is triggered by embryonic apoptosis followed by rapid removal via maternal sterile purulent inflammation

Background In normal mammalian development a high percentage of implantations is lost by spontaneous resorption. This is a major problem in assisted reproduction and blastocyst transfer. Which embryo will be resorbed is unpredictable. Resorption is very fast, so that with conventional methods only final haemorrhagic stages are encountered. Here we describe the histology and immunohistochemistry of 23 spontaneous embryo resorptions between days 7 and 13 of murine development, which were identified by high-resolution ultrasound (US) in a previous study. Results In the early resorptions detected at day 7, the embryo proper was replaced by maternal haemorrhage and a suppurate focus of maternal neutrophils. In the decidua maternal macrophages transformed to foam cells and formed a second focus of tissue dissolution. In the late resorptions detected at day 9, the embryo underwent apoptosis without involvement of maternal cells. The apoptotic embryonic cells expressed caspase 3 and embryonic blood cells developed a macrophage like phenotype. Subsequently, the wall of the embryonic vesicle ruptured and the apoptotic embryo was aborted into the uterine lumen. Abortion was initiated by degeneration of the embryonic lacunar trophoblast and dissolution of the maternal decidua capsularis via sterile inflammation and accompanied by maternal haemorrhage, invasion of the apoptotic embryo by maternal neutrophils, and contraction rings of the uterine muscle layers. Conclusions We conclude that spontaneous resorption starts with endogenous apoptosis of the embryo without maternal contribution. After break down of the foetal-maternal border, the apoptotic embryo is invaded by maternal neutrophils, aborted into the uterine lumen, and rapidly resorbed. We assume that the innate maternal unspecific inflammation is elicited by disintegrating apoptotic embryonic cells. Graphical abstract

Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition

SUMMARYIn animal embryos the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. We show that the maternal proteome represents over half of the protein coding capacity of the Drosophila melanogaster genome and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). While the ubiquitin-proteasome system is necessary for clearance of all four repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL and ME31B for degradation early in the MZT; the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG makes the protein immune to degradation. We show that artificially persistent SMG downregulates the zygotic re-expression of mRNAs whose maternal contribution is cleared by SMG. Thus, clearance of SMG permits an orderly MZT.