We studied the relationship between shape, size, and developmental time in the embryonic ontogeny of 15 species of the frog genus Physalaemus. As in other anuran exotrophic embryos, shape changes are correlated with size increase and mainly concern tail elongation, decrease in body height, and increase in fin height. Size ranges and developmental times vary interspecifically. Embryos of the P. signifer Clade and the P. gracilis Group are among the largest, are slightly peramorphic, and develop fast regarding congeneric species. Embryos of P. cicada combine the smallest sizes with fast development and the most peramorphic shapes. The paedomorphic shapes of embryos of P. biligonigerus and P. henselii groups are correlated with fast vs. slow developmental times respectively. Trajectories in the P. cuvieri Group are diverse and in general differ in size and developmental time. The embryos of P. cristinae and from the Argentinean lineage of P. cuvieri stand out with the longest development. Sequences of developmental events are overall conserved in the genus, and main differences concern mouthpart ontogeny. This study constitutes the first attempt to evaluate morphological, allometric, and heterochronic parameters of the early ontogeny of anurans and how these can vary and contribute to diversification in taxonomic groups.

Ultrasonography is widely used to monitor pregnancy in viviparous species, but it is underutilized as a tool to characterize embryonic development in oviparous species. Currently, a multi-institutional effort is underway to re-wild the endangered zebra shark (Stegostoma tigrinum) to locations where this species was previously extirpated by leveraging the reproductive efforts of aquarium sharks as a source of brood stock. Zebra sharks are oviparous and fecund, but a large percentage of their yolked eggs do not result in hatchlings. Therefore, ultrasonography represents a potential tool for distinguishing fertile eggs with developing embryos from degrading eggs, and to diagnose changes in early embryonic development predictive of poor outcomes. The objectives of the current study were to use ultrasonography to assess egg fertility, monitor early embryonic development, and identify morphological indicators that may be predictive of early embryonic mortality. Freshly laid eggs from four female zebra sharks were collected and inventoried daily at Aquarium of the Pacific. Eggs were incubated undisturbed for 2 to 4 weeks and subsequently examined weekly via ultrasound to assess fertility and monitor embryo development. Among 120 fertile eggs, embryos were identified as early as 8 days post-oviposition, with average (±SD) time to first observation at 30 ± 7 days. Morphological and behavioral abnormalities were observed for most embryos (n = 84, 70%) as early as 16 days and up to 95 days post-oviposition. Common abnormalities included: bent or curled tails, vesicle(s) at the base of the yolk stalk, and slow or weak movement. Only one embryo survived to hatch during the study and was genetically-confirmed parthenogenetic, suggesting hatching success for parthenotes is low (<1%). Ultrasonography was demonstrated to be an effective and non-invasive method to determine egg fertility, identify embryos with developmental abnormalities, and monitor embryo growth.

Monobutyl phthalate (MBP) is the primary active metabolite of dibutyl phthalate (DBP), the key plasticizer component. A substantial body of evidence from studies conducted on both animals and humans indicates that MBP exposure could result in harmful impacts on toxicity pathways. In addition, it can seriously affect human and animal reproductive health. In our present study, we showed that exposure to MBP causes abnormal epigenetic modifications in porcine oocytes and failure of early embryonic development. However, glycine (Gly) can protect oocytes and early embryos from damage caused by MBP. Our study indicated a significant decrease in the percentage of porcine oocytes that reached the metaphase II (MII) phase when exposed to MBP. SET-domain-containing 2(SETD2)-mediated H3K36me3 histone methylation was detected, and the results showed that MBP significantly decreased the protein expression of H3K36me3 and SETD2. Moreover, the expression of the DNA break markers γH2AX and the mRNA expression of Asf1a, and Asf1b increased in the MBP group. The detection of DNA methylation marker proteins showed that MBP significantly increased the fluorescence intensity of 5-methylcytosine (5mC). The results from our RT-qPCR analysis demonstrated a significant decrease in the mRNA expression of the DNA methylation-related genes Dnmt1 and Dnmt3a, as well as the embryonic developmental potential-related genes Oct4 and Nanog, in porcine oocytes following exposure to MBP. Additionally, the mRNA expression of p53 significantly increased. Subsequently, the effects of MBP on early embryonic development were examined via parthenogenesis activation (PA) and in vitro fertilization (IVF). Exposure to MBP significantly impacted the development of embryos in both PA and IVF processes. The TUNEL staining data showed that MBP significantly increased embryonic apoptosis. However, Gly can ameliorate MBP-induced defects in oocyte epigenetic modifications and early embryonic development.

Ageing populations, mass \"baby-free\" policies and children born to mothers at the age at which they are biologically expected to become grandmothers are growing problems in most developed societies. Therefore, any opportunity to improve the quality of infertility treatments seems important for the survival of societies. The possibility of indirectly studying the quality of developing oocytes by examining their follicular fluids (hFFs) offers new opportunities for progress in our understanding the processes of final oocyte maturation and, consequently, for predicting the quality of the resulting embryos and personalising their culture. Using mass spectrometry, we studied follicular fluids collected individually during in vitro fertilisation and compared their composition with the quality of the resulting embryos. We analysed 110 follicular fluids from 50 oocyte donors, from which we obtained 44 high-quality, 39 medium-quality, and 27 low-quality embryos. We identified 2182 proteins by Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) using a TripleTOF 5600+ hybrid mass spectrometer, of which 484 were suitable for quantification. We were able to identify several proteins whose concentrations varied between the follicular fluids of different oocytes from the same patient and between patients. Among them, the most important appear to be immunoglobulin heavy constant alpha 1 (IgA1hc) and dickkopf-related protein 3. The first one is found at higher concentrations in hFFs from which oocytes develop into poor-quality embryos, the other one exhibits the opposite pattern. None of these have, so far, had any specific links to fertility disorders. In light of these findings, these proteins should be considered a primary target for research aimed at developing a diagnostic tool for oocyte quality control and pre-fertilisation screening. This is particularly important in cases where the fertilisation of each egg is not an option for ethical or other reasons, or in countries where it is prohibited by law.

The Dlk1-Dio3 domain is important for normal embryonic growth and development. The heart is the earliest developing and functioning organ of the embryo. In this study, we constructed a transcriptional termination model by inserting termination sequences and clarified that the lack of long non-coding RNA (lncRNA) expression in the Dlk1-Dio3 domain caused the death of maternal insertion mutant (MKI) and homozygous mutant (HOMO) mice starting from E13.5. Parental insertion mutants (PKI) can be born and grow normally. Macroscopically, dying MKI and HOMO embryos showed phenomena such as embryonic edema and reduced heart rate. Hematoxylin and eosin (H.E.) staining showed thinning of the myocardium in MKI and HOMO embryos. In situ hybridization (IHC) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) showed downregulation of lncGtl2, Rian, and Mirg expression in MKI and HOMO hearts. The results of single-cell RNA sequencing (scRNA-Seq) analysis indicated that the lack of lncRNA expression in the Dlk1-Dio3 domain led to reduced proliferation of epicardial cells and may be an important cause of cardiac dysplasia. In conclusion, this study demonstrates that Dlk1-Dio3 domain lncRNAs play an integral role in ventricular development.

Cell fate specification occurs along invariant species-specific trajectories that define the animal body plan. This process is controlled by gene regulatory networks that regulate the expression of the limited set of transcription factors encoded in animal genomes. Here we globally assess the spatial expression of ~90% of expressed transcription factors during sea urchin development from embryo to larva to determine the activity of gene regulatory networks and their regulatory states during cell fate specification. We show that >200 embryonically expressed transcription factors together define >70 cell fates that recapitulate the morphological and functional organization of this organism. Most cell fate-specific regulatory states consist of ~15-40 transcription factors with similarity particularly among functionally related cell types regardless of developmental origin. Temporally, regulatory states change continuously during development, indicating that progressive changes in regulatory circuit activity determine cell fate specification. We conclude that the combinatorial expression of transcription factors provides molecular definitions that suffice for the unique specification of cell states in time and space during embryogenesis.

BACKGROUND: Appropriate regulation of genes expressed in oocytes and embryos is essential for acquisition of developmental competence in mammals. Here, we hypothesized that several genes expressed in oocytes and pre-implantation embryos remain unknown. Our goal was to reconstruct the transcriptome of oocytes (germinal vesicle and metaphase II) and pre-implantation cattle embryos (blastocysts) using short-read and long-read sequences to identify putative new genes.
RESULTS: We identified 274,342 transcript sequences and 3,033 of those loci do not match a gene present in official annotations and thus are potential new genes. Notably, 63.67% (1,931/3,033) of potential novel genes exhibited coding potential. Also noteworthy, 97.92% of the putative novel genes overlapped annotation with transposable elements. Comparative analysis of transcript abundance identified that 1,840 novel genes (recently added to the annotation) or potential new genes were differentially expressed between developmental stages (FDR < 0.01). We also determined that 522 novel or potential new genes (448 and 34, respectively) were upregulated at eight-cell embryos compared to oocytes (FDR < 0.01). In eight-cell embryos, 102 novel or putative new genes were co-expressed (|r|> 0.85, P < 1 × 10-8) with several genes annotated with gene ontology biological processes related to pluripotency maintenance and embryo development. CRISPR-Cas9 genome editing confirmed that the disruption of one of the novel genes highly expressed in eight-cell embryos reduced blastocyst development (ENSBTAG00000068261, P = 1.55 × 10-7).
CONCLUSIONS: Our results revealed several putative new genes that need careful annotation. Many of the putative new genes have dynamic regulation during pre-implantation development and are important components of gene regulatory networks involved in pluripotency and blastocyst formation.

Growing evidence suggests that metabolic regulation directly influences cellular function and development and thus may be more dynamic than previously expected. In vivo and in real-time analysis of metabolite activities during development is crucial to test this idea directly. In this study, we employ two metabolic biosensors to track the dynamics of pyruvate and oxidative phosphorylation (Oxphos) during the early embryogenesis of the sea urchin. A pyruvate sensor, PyronicSF, shows the signal enrichment on the mitotic apparatus, which is consistent with the localization patterns of the corresponding enzyme, pyruvate kinase (PKM). The addition of pyruvate increases the PyronicSF signal, while PKM knockdown decreases its signal, responding to the pyruvate level in the cell. Similarly, a ratio-metric sensor, Grx-roGFP, that reads the redox potential of the cell responds to DTT and H2O2, the known reducer and inducer of Oxphos. These observations suggest that these metabolic biosensors faithfully reflect the metabolic status in the cell during embryogenesis. The time-lapse imaging of these biosensors suggests that pyruvate and Oxphos levels change both spatially and temporarily during embryonic development. Pyruvate level is increased first in micromeres compared to other blastomeres at the 16-cell stage and remains high in ectoderm while decreasing in endomesoderm during gastrulation. In contrast, the Oxphos signal first decreases in micromeres at the 16-cell stage, while it increases in the endomesoderm during gastrulation, showing the opposite trend of the pyruvate signal. These results suggest that metabolic regulation is indeed both temporally and spatially dynamic during embryogenesis, and these biosensors are a valuable tool to monitor metabolic activities in real-time in developing embryos.

SETD2 is the only enzyme responsible for transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3). Mutations in SETD2 cause human diseases including cancer and developmental defects. In mice, Setd2 is essential for embryonic vascular remodeling. Given that many epigenetic modifiers have recently been found to possess noncatalytic functions, it is unknown whether the major function(s) of Setd2 is dependent on its catalytic activity or not. Here, we established a site-specific knockin mouse model harboring a cancer patient-derived catalytically dead Setd2 (Setd2-CD). We found that the essentiality of Setd2 in mouse development is dependent on its methyltransferase activity, as the Setd2CD/CD and Setd2-/- mice showed similar embryonic lethal phenotypes and largely comparable gene expression patterns. However, compared with Setd2-/-, the Setd2CD/CD mice showed less severe defects in allantois development, and single-cell RNA-seq analysis revealed differentially regulated allantois-specific 5\' Hoxa cluster genes in these two models. Collectively, this study clarifies the importance of Setd2 catalytic activity in mouse development and provides a new model for comparative study of previously unrecognized Setd2 functions.

BACKGROUND: Monoamine oxidases (MAOs) is an enzyme that catalyzes the deamination of monoamines. The current research on this enzyme is focused on its role in neuropsychiatric, neurodevelopmental, and neurodegenerative diseases. Indeed, MAOs with two isoforms, namely, A and B, are located on the outer mitochondrial membrane and are widely distributed in the central nervous system and peripheral tissues. Several reports have described periodic changes in the levels of this enzyme in the human endometrial tissue.
RESULTS: The novel role of MAOs in endometrial receptivity establishment and embryonic development by maintaining monoamine homeostasis was investigated in this study. MAOs activity was observed to be enhanced during the first trimester in both humans and mice under normal conditions. However, under pathological conditions, MAOs activity was reduced and was linked to early pregnancy failure. During the secretory phase, the endometrial stromal cells differentiated into decidual cells with a stronger metabolism of monoamines by MAOs. Excessive monoamine levels cause monoamine imbalance in decidual cells, which results in the activation of the AKT signal, decreased FOXO1 expression, and decidual dysfunction.
CONCLUSIONS: The findings suggest that endometrial receptivity depends on the maintenance of monoamine homeostasis via MAOs activity and that this enzyme participates in embryo implantation and development.