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PLoS Biology Jun 2024In the highly regulative embryo of the sea urchin Paracentrotus lividus, establishment of the dorsal-ventral (D/V) axis critically depends on the zygotic expression of...
In the highly regulative embryo of the sea urchin Paracentrotus lividus, establishment of the dorsal-ventral (D/V) axis critically depends on the zygotic expression of the TGF-β nodal in the ventral ectoderm. nodal expression is first induced ubiquitously in the 32-cell embryo and becomes progressively restricted to the presumptive ventral ectoderm by the early blastula stage. This early spatial restriction of nodal expression is independent of Lefty, and instead relies on the activity of Panda, a maternally expressed TGF-β ligand related to Lefty and Inhibins, which is required maternally for D/V axis specification. However, the mechanism by which Panda restricts the early nodal expression has remained enigmatic and it is not known if Panda works like a BMP ligand by opposing Nodal and antagonizing Smad2/3 signaling, or if it works like Lefty by sequestering an essential component of the Nodal signaling pathway. In this study, we report that Panda functions as an antagonist of the TGF-β type II receptor ACVRII (Activin receptor type II), which is the only type II receptor for Nodal signaling in the sea urchin and is also a type II receptor for BMP ligands. Inhibiting translation of acvrII mRNA disrupted D/V patterning across all 3 germ layers and caused acvrII morphants to develop with a typical Nodal loss-of-function phenotype. In contrast, embryos overexpressing acvrII displayed strong ectopic Smad1/5/8 signaling at blastula stages and developed as dorsalized larvae, a phenotype very similar to that caused by over activation of BMP signaling. Remarkably, embryos co-injected with acvrII mRNA and panda mRNA did not show ectopic Smad1/5/8 signaling and developed with a largely normal dorsal-ventral polarity. Furthermore, using an axis induction assay, we found that Panda blocks the ability of ACVRII to orient the D/V axis when overexpressed locally. Using co-immunoprecipitation, we showed that Panda physically interacts with ACVRII, as well as with the Nodal co-receptor Cripto, and with TBR3 (Betaglycan), which is a non-signaling receptor for Inhibins in mammals. At the molecular level, we have traced back the antagonistic activity of Panda to the presence of a single proline residue, conserved with all the Lefty factors, in the ACVRII binding motif of Panda, instead of a serine as in most of TGF-β ligands. Conversion of this proline to a serine converted Panda from an antagonist that opposed Nodal signaling and promoted dorsalization to an agonist that promoted Nodal signaling and triggered ventralization when overexpressed. Finally, using phylogenomics, we analyzed the emergence of the agonist and antagonist form of Panda in the course of evolution. Our data are consistent with the idea that the presence of a serine at that position, like in most TGF-β, was the ancestral condition and that the initial function of Panda was possibly in promoting and not in antagonizing Nodal signaling. These results highlight the existence of key functional and structural elements conserved between Panda and Lefty, allow to draw an intriguing parallel between sea urchin Panda and mammalian Inhibin α and raise the unexpected possibility that the original function of Panda may have been in activation of the Nodal pathway rather than in its inhibition.
PubMed: 38913712
DOI: 10.1371/journal.pbio.3002701 -
Genomics & Informatics May 2024Small auxin-up RNA (SAUR) proteins were known as a large family that supposedly participated in various biological processes in higher plant species. However, the SAUR...
Genome-wide identification, characterization, and expression analysis of the small auxin-up RNA gene family during zygotic and somatic embryo maturation of the cacao tree (Theobroma cacao).
Small auxin-up RNA (SAUR) proteins were known as a large family that supposedly participated in various biological processes in higher plant species. However, the SAUR family has been still not explored in cacao (Theobroma cacao L.), one of the most important industrial trees. The present work, as an in silico study, revealed comprehensive aspects of the structure, phylogeny, and expression of TcSAUR gene family in cacao. A total of 90 members of the TcSAUR gene family have been identified and annotated in the cacao genome. According to the physic-chemical features analysis, all TcSAUR proteins exhibited slightly similar characteristics. Phylogenetic analysis showed that these TcSAUR proteins could be categorized into seven distinct groups, with 10 sub-groups. Our results suggested that tandemly duplication events, segmental duplication events, and whole genome duplication events might be important in the growth of the TcSAUR gene family in cacao. By re-analyzing the available transcriptome databases, we found that a number of TcSAUR genes were exclusively expressed during the zygotic embryogenesis and somatic embryogenesis. Taken together, our study will be valuable to further functional characterizations of candidate TcSAUR genes for the genetic engineering of cacao.
PubMed: 38907330
DOI: 10.1186/s44342-024-00003-6 -
The EMBO Journal Jun 2024Cell polarity networks are defined by quantitative features of their constituent feedback circuits, which must be tuned to enable robust and stable polarization, while...
Cell polarity networks are defined by quantitative features of their constituent feedback circuits, which must be tuned to enable robust and stable polarization, while also ensuring that networks remain responsive to dynamically changing cellular states and/or spatial cues during development. Using the PAR polarity network as a model, we demonstrate that these features are enabled by the dimerization of the polarity protein PAR-2 via its N-terminal RING domain. Combining theory and experiment, we show that dimer affinity is optimized to achieve dynamic, selective, and cooperative binding of PAR-2 to the plasma membrane during polarization. Reducing dimerization compromises positive feedback and robustness of polarization. Conversely, enhanced dimerization renders the network less responsive due to kinetic trapping of PAR-2 on internal membranes and reduced sensitivity of PAR-2 to the anterior polarity kinase, aPKC/PKC-3. Thus, our data reveal a key role for a dynamically oligomeric RING domain in optimizing interaction affinities to support a robust and responsive cell polarity network, and highlight how optimization of oligomerization kinetics can serve as a strategy for dynamic and cooperative intracellular targeting.
PubMed: 38907033
DOI: 10.1038/s44318-024-00123-3 -
Frontiers in Endocrinology 2024Polycystic ovary syndrome with insulin resistance (PCOS-IR) is the most common endocrine and metabolic disease in women of reproductive age, and low fertility in PCOS...
High coverage of targeted lipidomics revealed lipid changes in the follicular fluid of patients with insulin-resistant polycystic ovary syndrome and a positive correlation between plasmalogens and oocyte quality.
BACKGROUND
Polycystic ovary syndrome with insulin resistance (PCOS-IR) is the most common endocrine and metabolic disease in women of reproductive age, and low fertility in PCOS patients may be associated with oocyte quality; however, the molecular mechanism through which PCOS-IR affects oocyte quality remains unknown.
METHODS
A total of 22 women with PCOS-IR and 23 women without polycystic ovary syndrome (control) who underwent fertilization and embryo transfer were recruited, and clinical information pertaining to oocyte quality was analyzed. Lipid components of follicular fluid (FF) were detected using high-coverage targeted lipidomics, which identified 344 lipid species belonging to 19 lipid classes. The exact lipid species associated with oocyte quality were identified.
RESULTS
The number (rate) of two pronuclear (2PN) zygotes, the number (rate) of 2PN cleaved embryos, and the number of high-quality embryos were significantly lower in the PCOS-IR group. A total of 19 individual lipid classes and 344 lipid species were identified and quantified. The concentrations of the 19 lipid species in the normal follicular fluid (control) ranged between 10 mol/L and 10 mol/L. In addition, 39 lipid species were significantly reduced in the PCOS-IR group, among which plasmalogens were positively correlated with oocyte quality.
CONCLUSIONS
This study measured the levels of various lipids in follicular fluid, identified a significantly altered lipid profile in the FF of PCOS-IR patients, and established a correlation between poor oocyte quality and plasmalogens in PCOS-IR patients. These findings have contributed to the development of plasmalogen replacement therapy to enhance oocyte quality and have improved culture medium formulations for oocyte maturation (IVM).
Topics: Humans; Female; Polycystic Ovary Syndrome; Follicular Fluid; Oocytes; Adult; Lipidomics; Insulin Resistance; Plasmalogens; Fertilization in Vitro; Lipids; Infertility, Female; Lipid Metabolism; Embryo Transfer; Case-Control Studies
PubMed: 38904043
DOI: 10.3389/fendo.2024.1414289 -
International Journal of Molecular... May 2024Global methylation levels differ in in vitro- and in vivo-developed embryos. Follicular fluid (FF) contains extracellular vesicles (EVs) containing miRNAs that affect...
Global methylation levels differ in in vitro- and in vivo-developed embryos. Follicular fluid (FF) contains extracellular vesicles (EVs) containing miRNAs that affect embryonic development. Here, we examined our hypothesis that components in FF affect global DNA methylation and embryonic development. Oocytes and FF were collected from bovine ovaries. Treatment of zygotes with a low concentration of FF induced global DNA demethylation, improved embryonic development, and reduced DNMT1/3A levels. We show that embryos take up EVs containing labeled miRNA secreted from granulosa cells and the treatment of zygotes with EVs derived from FF reduces global DNA methylation in embryos. Furthermore, the methylation levels of in vitro-developed blastocysts were higher than those of in their vivo counterparts. Based on small RNA-sequencing and in silico analysis, we predicted miR-29b, -199a-3p, and -148a to target DNMTs and to induce DNA demethylation, thereby improving embryonic development. Moreover, among FF from 30 cows, FF with a high content of these miRNAs demethylated more DNA in the embryos than FF with a lower miRNA content. Thus, miRNAs in FF play a role in early embryonic development.
Topics: Animals; Female; MicroRNAs; Cattle; Follicular Fluid; Extracellular Vesicles; Embryonic Development; DNA Methylation; DNA Demethylation; Oocytes; Blastocyst; Embryo, Mammalian; Gene Expression Regulation, Developmental; Zygote
PubMed: 38892059
DOI: 10.3390/ijms25115872 -
International Journal of Molecular... May 2024In recent years, the awareness that pesticides can have other effects apart from generic toxicity is growing. In particular, several pieces of evidence highlight their...
Prediction of Pesticide Interactions with Proteins Involved in Human Reproduction by Using a Virtual Screening Approach: A Case Study of Famoxadone Binding CRBP-III and Izumo.
In recent years, the awareness that pesticides can have other effects apart from generic toxicity is growing. In particular, several pieces of evidence highlight their influence on human fertility. In this study, we investigated, by a virtual screening approach, the binding between pesticides and proteins present in human gametes or associated with reproduction, in order to identify new interactions that could affect human fertility. To this aim, we prepared ligand (pesticides) and receptor (proteins) 3D structure datasets from online structural databases (such as PubChem and RCSB), and performed a virtual screening analysis using Autodock Vina. In the comparison of the predicted interactions, we found that famoxadone was predicted to bind Cellular Retinol Binding Protein-III in the retinol-binding site with a better minimum energy value of -10.4 Kcal/mol and an RMSD of 3.77 with respect to retinol (-7.1 Kcal/mol). In addition to a similar network of interactions, famoxadone binding is more stabilized by additional hydrophobic patches including L20, V29, A33, F57, L117, and L118 amino acid residues and hydrogen bonds with Y19 and K40. These results support a possible competitive effect of famoxadone on retinol binding with impacts on the ability of developing the cardiac tissue, in accordance with the literature data on zebrafish embryos. Moreover, famoxadone binds, with a minimum energy value between -8.3 and -8.0 Kcal/mol, to the IZUMO Sperm-Egg Fusion Protein, interacting with a network of polar and hydrophobic amino acid residues in the cavity between the 4HB and Ig-like domains. This binding is more stabilized by a predicted hydrogen bond with the N185 residue of the protein. A hindrance in this position can probably affect the conformational change for JUNO binding, avoiding the gamete membrane fusion to form the zygote. This work opens new interesting perspectives of study on the effects of pesticides on fertility, extending the knowledge to other typologies of interaction which can affect different steps of the reproductive process.
Topics: Humans; Molecular Docking Simulation; Protein Binding; Pesticides; Retinol-Binding Proteins, Cellular; Binding Sites; Reproduction; Animals; Hydrogen Bonding; Ligands
PubMed: 38891976
DOI: 10.3390/ijms25115790 -
International Journal of Molecular... May 2024Species of the genus have served as favorite models in speciation studies; however, genetic factors of interspecific reproductive incompatibility are...
Species of the genus have served as favorite models in speciation studies; however, genetic factors of interspecific reproductive incompatibility are under-investigated. Here, we performed an analysis of hybrid female sterility by crossing females and males. Using transcriptomic data analysis and molecular, cellular, and genetic approaches, we analyzed differential gene expression, transposable element (TE) activity, piRNA biogenesis, and functional defects of oogenesis in hybrids. Premature germline stem cell loss was the most prominent defect of oogenesis in hybrid ovaries. Because of the differential expression of genes encoding piRNA pathway components, and , the functional RDC complex in hybrid ovaries was not assembled. However, the activity of the RDC complex was maintained in hybrids independent of the genomic origin of piRNA clusters. Despite the identification of a cohort of overexpressed TEs in hybrid ovaries, we found no evidence that their activity can be considered the main cause of hybrid sterility. We revealed a complicated pattern of Vasa protein expression in the hybrid germline, including partial piRNA targeting of the allele and a significant zygotic delay in expression. We arrived at the conclusion that the hybrid sterility phenotype was caused by intricate multi-locus differences between the species.
Topics: Animals; Female; Drosophila melanogaster; Male; Drosophila simulans; Drosophila Proteins; RNA, Small Interfering; DNA Transposable Elements; Ovary; Hybridization, Genetic; Oogenesis; Infertility; Crosses, Genetic; DEAD-box RNA Helicases
PubMed: 38891872
DOI: 10.3390/ijms25115681 -
International Journal of Molecular... May 2024The testis-specific double sex and mab-3-related transcription factor 1 () has long been recognized as a crucial player in sex determination across vertebrates, and its...
The testis-specific double sex and mab-3-related transcription factor 1 () has long been recognized as a crucial player in sex determination across vertebrates, and its essential role in gonadal development and the regulation of spermatogenesis is well established. Here, we report the cloning of the key spermatogenesis-related cDNA, named , from the gonads of (), with a molecular weight of 41.93 kDa and an isoelectric point of 7.83 (pI). Our hypothesis is that machinery governs spermatogenesis and regulates gonadogenesis. RNAi-mediated knockdown revealed its critical role in hindering spermatogenesis and reducing expression levels in boring giant clams. A histological analysis showed structural changes, with normal sperm cell counts in the control group (ds-EGFP) but significantly lower concentrations of sperm cells in the experimental group (). transcripts during embryogenesis exhibited a significantly high expression pattern ( < 0.05) during the early zygote stage, and whole-embryo in-situ hybridization confirmed its expression pattern throughout embryogenesis. A qRT-PCR analysis of various reproductive stages revealed an abundant expression of in the gonads during the male reproductive stage. In-situ hybridization showed tissue-specific expression of , with a positive signal detected in male-stage gonadal tissues comprising sperm cells, while no signal was detected in other stages. Our study findings provide an initial understanding of the molecular machinery controlling spermatogenesis and its specificity in male-stage gonads of the key bivalve species, , and suggest that predominantly functions as a key regulator of spermatogenesis in giant clams.
Topics: Animals; Spermatogenesis; Transcription Factors; Male; Testis; Bivalvia; Gene Expression Regulation, Developmental; Gonads; Hermaphroditic Organisms; Cloning, Molecular; Phylogeny; Amino Acid Sequence
PubMed: 38891762
DOI: 10.3390/ijms25115574 -
Frontiers in Cell and Developmental... 2024What is the utilization rate of embryos that exert inadequate zygote cleavage into three daughter cells?
RESEARCH QUESTION
What is the utilization rate of embryos that exert inadequate zygote cleavage into three daughter cells?
DESIGN
This study used a retrospective dataset from a single IVF Unit. A total of 3,060 embryos from 1,811 fresh IVF cycles were analyzed. The cleavage pattern, morphokinetics, and outcome were recorded. Only 2pn embryos, fertilized by ejaculated sperm, and cultured in a time-lapse system for at least 5 days were included. We generated three study groups according to the embryo's cleavage pattern: (I) Control, normal cleavage ( = 551); (II) fast cleavage, zygote to three cells within 5 h ( = 1,587); and (III) instant direct tripolar cleavage (IDC) from zygote to three cells ( = 922).
RESULTS
The rate of usable fast cleavage blastocysts was 108/1,587 (6.81%) and usable control blastocysts was 180/551 (32.67%). The time of PN fading and from fading to first cleavage differed significantly between the three groups. Although the pregnancy rate of control and fast cleavage blastocysts were comparable (40.35% and 42.55%, respectively), the amount of instant direct cleavage embryos that reached blastocyst stage was neglectable (only four embryos out of 922 analyzed IDC embryos) and unsuitable for statistical comparison of pregnancy rates.
CONCLUSION
Our results indicate the need to culture instant direct cleavage embryos for 5 days, up to the blastocyst stage, and avoid transfer of embryos that are fated to arrest even when their morphological grade on day 3 is acceptable, whereas fast cleavage embryos could be transferred on day 3 when there is no alternative.
PubMed: 38887513
DOI: 10.3389/fcell.2024.1398684 -
Research Square Jun 2024NLRP2 belongs to the subcortical maternal complex (SCMC) of mammalian oocytes and preimplantation embryos. This multiprotein complex, encoded by maternal-effect genes,...
BACKGROUND
NLRP2 belongs to the subcortical maternal complex (SCMC) of mammalian oocytes and preimplantation embryos. This multiprotein complex, encoded by maternal-effect genes, plays a pivotal role in the zygote-to-embryo transition, early embryogenesis, and epigenetic (re)programming. The maternal inactivation of genes encoding SCMC proteins has been linked to infertility and subfertility in mice and humans. However, the underlying molecular mechanisms for the diverse functions of the SCMC, particularly how this cytoplasmic structure influences DNA methylation, which is a nuclear process, are not fully understood.
RESULTS
We undertook joint transcriptome and DNA methylome profiling of pre-ovulatory germinal-vesicle oocytes from -null, heterozygous (Het), and wild-type (WT) female mice. We identified numerous differentially expressed genes (DEGs) in Het and -null when compared to WT oocytes. The genes for several crucial factors involved in oocyte transcriptome modulation and epigenetic reprogramming, such as DNMT1, UHRF1, KDM1B and ZFP57 were overexpressed in Het and -null oocytes. Absence or reduction of , did not alter the distinctive global DNA methylation landscape of oocytes, including the bimodal pattern of the oocyte methylome. Additionally, although the methylation profile of germline differentially methylated regions (gDMRs) of imprinted genes was preserved in oocytes of Het and -null mice, we found altered methylation in oocytes of both genotypes at a small percentage of the oocyte-characteristic hyper- and hypomethylated domains. Through a tiling approach, we identified specific DNA methylation differences between the genotypes, with approximately 1.3% of examined tiles exhibiting differential methylation in Het and -null compared to WT oocytes.
CONCLUSIONS
Surprisingly, considering the well-known correlation between transcription and DNA methylation in developing oocytes, we observed no correlation between gene expression differences and gene-body DNA methylation differences in -null versus WT oocytes or Het versus WT oocytes. We therefore conclude that post-transcriptional changes in the stability of transcripts rather than altered transcription is primarily responsible for transcriptome differences in -null and Het oocytes.
PubMed: 38883732
DOI: 10.21203/rs.3.rs-4457414/v1