Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles†.

In mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein Gs to elevate cAMP, but both of these hormones can also elevate Ca2+ under some conditions. Here, we investigate FSH- and LH-induced Ca2+ signaling in intact follicles of mice expressing genetically encoded Ca2+ sensors, Twitch-2B and GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca2+ within the granulosa cells of preantral and antral follicles. The Ca2+ rise begins several minutes after FSH application, peaks at ∼10 min, remains above baseline for another ∼10 min, and depends on extracellular Ca2+. However, suppression of the FSH-induced Ca2+ increase by reducing extracellular Ca2+ does not inhibit FSH-induced phosphorylation of MAP kinase, estradiol production, or the acquisition of LH responsiveness. Like FSH, LH also increases Ca2+, when applied to preovulatory follicles. At a physiological concentration (10 nM), LH elicits Ca2+ oscillations in a subset of cells in the outer mural granulosa layer. These oscillations continue for at least 6 h and depend on the activity of Gq family G-proteins. Suppression of the oscillations by Gq inhibition does not inhibit meiotic resumption, but does delay the time to 50% ovulation by about 3 h. In summary, both FSH and LH increase Ca2+ in the granulosa cells of intact follicles, but the functions of these Ca2+ rises are only starting to be identified.

Regulator of G-protein signaling 2 (RGS2) suppresses premature calcium release in mouse eggs.

During oocyte maturation, capacity and sensitivity of Ca(2+) signaling machinery increases dramatically, preparing the metaphase II (MII)-arrested egg for fertilization. Upon sperm-egg fusion, Ca(2+) release from IP3-sensitive endoplasmic reticulum stores results in cytoplasmic Ca(2+) oscillations that drive egg activation and initiate early embryo development. Premature Ca(2+) release can cause parthenogenetic activation prior to fertilization; thus, preventing inappropriate Ca(2+) signaling is crucial for ensuring robust MII arrest. Here, we show that regulator of G-protein signaling 2 (RGS2) suppresses Ca(2+) release in MII eggs. Rgs2 mRNA was recruited for translation during oocyte maturation, resulting in ∼ 20-fold more RGS2 protein in MII eggs than in fully grown immature oocytes. Rgs2-siRNA-injected oocytes matured to MII; however, they had increased sensitivity to low pH and acetylcholine (ACh), which caused inappropriate Ca(2+) release and premature egg activation. When matured in vitro, RGS2-depleted eggs underwent spontaneous Ca(2+) increases that were sufficient to cause premature zona pellucida conversion. Rgs2(-/-) females had reduced litter sizes, and their eggs had increased sensitivity to low pH and ACh. Rgs2(-/-) eggs also underwent premature zona pellucida conversion in vivo. These findings indicate that RGS2 functions as a brake to suppress premature Ca(2+) release in eggs that are poised on the brink of development.

Friend or Foe: Epigenetic Regulation of Retrotransposons in Mammalian Oogenesis and Early Development.

Epigenetics is the study of phenotypic variation arising from developmental and environmental factors regulating gene transcription at molecular, cellular, and physiological levels. A naturally occurring biological process driven by epigenetics is the egg-to-embryo developmental transition when two fully differentiated adult cells - egg and sperm - revert to an early stem cell type with totipotency but subsequently differentiates into pluripotent embryonic stem cells that give rise to any cell type. Transposable elements (TEs) are active in mammalian oocytes and early embryos, and this activity, albeit counterintuitive because TEs can lead to genomic instability in somatic cells, correlates to successful development. TEs bridge genetic and epigenetic landscapes because TEs are genetic elements whose silencing and de-repression are regulated by epigenetic mechanisms that are sensitive to environmental factors. Ultimately, transposition events can change size, content, and function of mammalian genomes. Thus, TEs act beyond mutagenic agents reshuffling the genomes, and epigenetic regulation of TEs may act as a proximate mechanism by which evolutionary forces increase a species' hidden reserve of epigenetic and phenotypic variability facilitating the adaptation of genomes to their environment.

The Protein Ontology: a structured representation of protein forms and complexes.

The Protein Ontology (PRO) provides a formal, logically-based classification of specific protein classes including structured representations of protein isoforms, variants and modified forms. Initially focused on proteins found in human, mouse and Escherichia coli, PRO now includes representations of protein complexes. The PRO Consortium works in concert with the developers of other biomedical ontologies and protein knowledge bases to provide the ability to formally organize and integrate representations of precise protein forms so as to enhance accessibility to results of protein research. PRO (http://pir.georgetown.edu/pro) is part of the Open Biomedical Ontology Foundry.

Novel Alu retrotransposon insertion leading to Alström syndrome.

Alström syndrome is a clinically complex disorder characterized by childhood retinal degeneration leading to blindness, sensorineural hearing loss, obesity, type 2 diabetes mellitus, cardiomyopathy, systemic fibrosis, and pulmonary, hepatic, and renal failure. Alström syndrome is caused by recessively inherited mutations in the ALMS1 gene, which codes for a putative ciliary protein. Alström syndrome is characterized by extensive allelic heterogeneity, however, founder effects have been observed in some populations. To date, more than 100 causative ALMS1 mutations have been identified, mostly frameshift and non-sense alterations resulting in termination signals in ALMS1. Here, we report a complex Turkish kindred in which sequence analysis uncovered an insertion of a novel 333 basepair Alu Ya5 SINE retrotransposon in the ALMS1 coding sequence, a previously unrecognized mechanism underlying the mutations causing Alström syndrome. It is extraordinarily rare to encounter the insertion of an Alu retrotransposon in the coding sequence of a gene. The high frequency of the mutant ALMS1 allele in this isolated population suggests that this recent retrotransposition event spreads quickly, and may be used as a model to study the population dynamics of deleterious alleles in isolated communities.

Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence.

Voltage-sensitive phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid phosphatase specific for phosphoinositides, and for ascidian and zebrafish VSPs, the phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl-VSP1 and Xl-VSP2), whereas X. tropicalis has only one gene (Xt-VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl-VSP2 activates only at positive membrane potentials outside of the physiological range, Xl-VSP1 and Xt-VSP phosphatase activity is regulated in the voltage range that regulates sperm-egg fusion at fertilization.

Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos.

A comprehensive analysis of transposable element (TE) expression in mammalian full-grown oocytes reveals that LTR class III retrotransposons make an unexpectedly high contribution to the maternal mRNA pool, which persists in cleavage stage embryos. The most abundant transcripts in the mouse oocyte are from the mouse transcript (MT) retrotransposon family, and expression of this and other TE families is developmentally regulated. Furthermore, TEs act as alternative promoters and first exons for a subset of host genes, regulating their expression in full-grown oocytes and cleavage stage embryos. To our knowledge, this is the first example of TEs initiating synchronous, developmentally regulated expression of multiple genes in mammals. We propose that differential TE expression triggers sequential reprogramming of the embryonic genome during the oocyte to embryo transition and in preimplantation embryos.

Evolutionary origin and phylogenetic analysis of the novel oocyte-specific eukaryotic translation initiation factor 4E in Tetrapoda.

The transcriptionally active, growing oocyte accumulates mRNAs essential for early stages of development, the oocyte-to-embryo transition, in a stable, dormant form. Translational repression of mRNAs in eggs of various species is conferred by interactions, either direct or via intermediate proteins, of repressive factors bound to the 3'-untranslated regions with the proteins of the eukaryotic translation initiation factor 4E (eIF4E) family bound to the 5'-cap of the transcripts. Recently, a novel oocyte-specific eIF4E encoded by the Eif41b gene in mammals has been identified by our group. To further investigate this gene, the available cDNA libraries, as well as genome assemblies of nonmammalian vertebrates, were surveyed. This analysis revealed that the Eif4e1b gene arose in Tetrapoda as a result of the ancestral Eif4e locus duplication. Unlike other known proteins of three subfamilies comprising eIF4E family (eIF4E1, eIF4E2, and eIF4E3), cDNA library evidence suggests that Eif41b locus has an oocyte-restricted expression across all classes of Tetrapoda. To further understand the role of eIF4E1B during oocyte maturation, injections of antisense morpholino nucleotides in the X. tropicalis fully-grown stage VI oocytes were performed. The resulted ablation of eIF4E1B protein led to significant acceleration of oocyte maturation after progesterone induction; morpholino-injected oocytes formed the metaphase plate 30 min faster than the control groups. These results suggest that eIF4E1B protein acts as a repressor in translational regulation of maternal mRNAs activated during, and required for, oocyte maturation.

Gene expression during the oocyte-to-embryo transition in mammals.

The seminal question in modern developmental biology is the origins of new life arising from the unification of sperm and egg. The roots of this question begin from 19th to 20th century embryologists studying fertilization and embryogenesis. Although the revolution of molecular biology has yielded significant insight into the complexity of this process, the overall orchestration of genes, molecules, and cells is still not fully formed. Early mammalian development, specifically the oocyte-to-embryo transition, is essentially under "maternal command" from factors deposited in the cytoplasm during oocyte growth, independent of de novo transcription from the nascent embryo. Many of the advances in understanding this developmental period occurred in tandem with application of new methods and techniques from molecular biology, from protein electrophoresis to sequencing and assemblies of whole genomes. From this bed of knowledge, it appears that precise control of mRNA translation is a key regulator coordinating the molecular and cellular events occurring during oocyte-to-embryo transition. Notably, oocyte transcriptomes share, yet retain some uniqueness, common genetic motifs among all chordates. The common genetic motifs typically define fundamental processes critical for cellular maintenance, whereas the unique genetic features may be a source of variation and a substrate for sexual selection, genetic drift, or gene flow. One purpose for this complex interplay among genes, proteins, and cells may allow for evolution to transform and act upon the underlying processes, at molecular, structural and organismal levels, to increase diversity, which is the ultimate goal of sexual reproduction.

Aligning the Aligners: Comparison of RNA Sequencing Data Alignment and Gene Expression Quantification Tools for Clinical Breast Cancer Research.

The rapid expansion of transcriptomics and affordability of next-generation sequencing (NGS) technologies generate rocketing amounts of gene expression data across biology and medicine, including cancer research. Concomitantly, many bioinformatics tools were developed to streamline gene expression and quantification. We tested the concordance of NGS RNA sequencing (RNA-seq) analysis outcomes between two predominant programs for read alignment, HISAT2, and STAR, and two most popular programs for quantifying gene expression in NGS experiments, edgeR and DESeq2, using RNA-seq data from breast cancer progression series, which include histologically confirmed normal, early neoplasia, ductal carcinoma in situ and infiltrating ductal carcinoma samples microdissected from formalin fixed, paraffin embedded (FFPE) breast tissue blocks. We identified significant differences in aligners' performance: HISAT2 was prone to misalign reads to retrogene genomic loci, STAR generated more precise alignments, especially for early neoplasia samples. edgeR and DESeq2 produced similar lists of differentially expressed genes, with edgeR producing more conservative, though shorter, lists of genes. Gene Ontology (GO) enrichment analysis revealed no skewness in significant GO terms identified among differentially expressed genes by edgeR versus DESeq2. As transcriptomics of FFPE samples becomes a vanguard of precision medicine, choice of bioinformatics tools becomes critical for clinical research. Our results indicate that STAR and edgeR are well-suited tools for differential gene expression analysis from FFPE samples.

The Transcriptomic Toolbox: Resources for Interpreting Large Gene Expression Data within a Precision Medicine Context for Metabolic Disease Atherosclerosis.

As one of the most widespread metabolic diseases, atherosclerosis affects nearly everyone as they age; arteries gradually narrow from plaque accumulation over time reducing oxygenated blood flow to central and periphery causing heart disease, stroke, kidney problems, and even pulmonary disease. Personalized medicine promises to bring treatments based on individual genome sequencing that precisely target the molecular pathways underlying atherosclerosis and its symptoms, but to date only a few genotypes have been identified. A promising alternative to this genetic approach is the identification of pathways altered in atherosclerosis by transcriptome analysis of atherosclerotic tissues to target specific aspects of disease. Transcriptomics is a potentially useful tool for both diagnostics and discovery science, exposing novel cellular and molecular mechanisms in clinical and translational models, and depending on experimental design to identify and test novel therapeutics. The cost and time required for transcriptome analysis has been greatly reduced by the development of next generation sequencing. The goal of this resource article is to provide background and a guide to appropriate technologies and downstream analyses in transcriptomics experiments generating ever-increasing amounts of gene expression data.

Embryonic exposures of lithium and homocysteine and folate protection affect lipid metabolism during mouse cardiogenesis and placentation.

Embryonic exposures can increase the risk of congenital cardiac birth defects and adult disease. The present study identifies the predominant pathways modulated by an acute embryonic mouse exposure during gastrulation to lithium or homocysteine that induces cardiac defects. High dose periconceptional folate supplementation normalized development. Microarray bioinformatic analysis of gene expression demonstrated that primarily lipid metabolism is altered after the acute exposures. The lipid-related modulation demonstrated a gender bias with male embryos showing greater number of lipid-related Gene Ontology biological processes altered than in female embryos. RT-PCR analysis demonstrated significant change of the fatty acid oxidation gene Acadm with homocysteine exposure primarily in male embryos than in female. The perturbations resulting from the exposures resulted in growth-restricted placentas with disorganized cellular lipid droplet distribution indicating lipids have a critical role in cardiac-placental abnormal development. High folate supplementation protected normal heart-placental function, gene expression and lipid localization.

Metabolite profile of a mouse model of Charcot-Marie-Tooth type 2D neuropathy: implications for disease mechanisms and interventions.

Charcot-Marie-Tooth disease encompasses a genetically heterogeneous class of heritable polyneuropathies that result in axonal degeneration in the peripheral nervous system. Charcot-Marie-Tooth type 2D neuropathy (CMT2D) is caused by dominant mutations in glycyl tRNA synthetase (GARS). Mutations in the mouse Gars gene result in a genetically and phenotypically valid animal model of CMT2D. How mutations in GARS lead to peripheral neuropathy remains controversial. To identify putative disease mechanisms, we compared metabolites isolated from the spinal cord of Gars mutant mice and their littermate controls. A profile of altered metabolites that distinguish the affected and unaffected tissue was determined. Ascorbic acid was decreased fourfold in the spinal cord of CMT2D mice, but was not altered in serum. Carnitine and its derivatives were also significantly reduced in spinal cord tissue of mutant mice, whereas glycine was elevated. Dietary supplementation with acetyl-L-carnitine improved gross motor performance of CMT2D mice, but neither acetyl-L-carnitine nor glycine supplementation altered the parameters directly assessing neuropathy. Other metabolite changes suggestive of liver and kidney dysfunction in the CMT2D mice were validated using clinical blood chemistry. These effects were not secondary to the neuromuscular phenotype, as determined by comparison with another, genetically unrelated mouse strain with similar neuromuscular dysfunction. However, these changes do not seem to be causative or consistent metabolites of CMT2D, because they were not observed in a second mouse Gars allele or in serum samples from CMT2D patients. Therefore, the metabolite 'fingerprint' we have identified for CMT2D improves our understanding of cellular biochemical changes associated with GARS mutations, but identification of efficacious treatment strategies and elucidation of the disease mechanism will require additional studies.

Micro axial tomography: a miniaturized, versatile stage device to overcome resolution anisotropy in fluorescence light microscopy.

With the development of novel fluorescence techniques, high resolution light microscopy has become a challenging technique for investigations of the three-dimensional (3D) micro-cosmos in cells and sub-cellular components. So far, all fluorescence microscopes applied for 3D imaging in biosciences show a spatially anisotropic point spread function resulting in an anisotropic optical resolution or point localization precision. To overcome this shortcoming, micro axial tomography was suggested which allows object tilting on the microscopic stage and leads to an improvement in localization precision and spatial resolution. Here, we present a miniaturized device which can be implemented in a motor driven microscope stage. The footprint of this device corresponds to a standard microscope slide. A special glass fiber can manually be adjusted in the object space of the microscope lens. A stepwise fiber rotation can be controlled by a miniaturized stepping motor incorporated into the device. By means of a special mounting device, test particles were fixed onto glass fibers, optically localized with high precision, and automatically rotated to obtain views from different perspective angles under which distances of corresponding pairs of objects were determined. From these angle dependent distance values, the real 3D distance was calculated with a precision in the ten nanometer range (corresponding here to an optical resolution of 10-30 nm) using standard microscopic equipment. As a proof of concept, the spindle apparatus of a mature mouse oocyte was imaged during metaphase II meiotic arrest under different perspectives. Only very few images registered under different rotation angles are sufficient for full 3D reconstruction. The results indicate the principal advantage of the micro axial tomography approach for many microscopic setups therein and also those of improved resolutions as obtained by high precision localization determination.

MouseCyc: a curated biochemical pathways database for the laboratory mouse.

Linking biochemical genetic data to the reference genome for the laboratory mouse is important for comparative physiology and for developing mouse models of human biology and disease. We describe here a new database of curated metabolic pathways for the laboratory mouse called MouseCyc http://mousecyc.jax.org. MouseCyc has been integrated with genetic and genomic data for the laboratory mouse available from the Mouse Genome Informatics database and with pathway data from other organisms, including human.

Cracking the egg: molecular dynamics and evolutionary aspects of the transition from the fully grown oocyte to embryo.

Fully grown oocytes (FGOs) contain all the necessary transcripts to activate molecular pathways underlying the oocyte-to-embryo transition (OET). To elucidate this critical period of development, an extensive survey of the FGO transcriptome was performed by analyzing 19,000 expressed sequence tags of the Mus musculus FGO cDNA library. Expression of 5400 genes and transposable elements is reported. For a majority of genes expressed in mouse FGOs, homologs transcribed in eggs of Xenopus laevis or Ciona intestinalis were found, pinpointing evolutionary conservation of most regulatory cascades underlying the OET in chordates. A large proportion of identified genes belongs to several gene families with oocyte-restricted expression, a likely result of lineage-specific genomic duplications. Gene loss by mutation and expression in female germline of retrotransposed genes specific to M. musculus is documented. These findings indicate rapid diversification of genes involved in female reproduction. Comparison of the FGO and two-cell embryo transcriptomes demarcated the processes important for oogenesis from those involved in OET and identified novel motifs in maternal mRNAs associated with transcript stability. Discovery of oocyte-specific eukaryotic translation initiation factor 4E distinguishes a novel system of translational regulation. These results implicate conserved pathways underlying transition from oogenesis to initiation of development and illustrate how genes acquire and lose reproductive functions during evolution, a potential mechanism for reproductive isolation.

Molecular control of the oocyte to embryo transition.

The elucidation of the molecular control of the initiation of mammalian embryogenesis is possible now that the transcriptomes of the full-grown oocyte and two-cell stage embryo have been prepared and analysed. Functional annotation of the transcriptomes using gene ontology vocabularies, allows comparison of the oocyte and two-cell stage embryo between themselves, and with all known mouse genes in the Mouse Genome Database. Using this methodology one can outline the general distinguishing features of the oocyte and the two-cell stage embryo. This, when combined with oocyte-specific targeted deletion of genes, allows us to dissect the molecular networks at play as the differentiated oocyte and sperm transit into blastomeres with unlimited developmental potential.

The Gs-linked receptor GPR3 maintains meiotic arrest in mammalian oocytes.

Mammalian oocytes are held in prophase arrest by an unknown signal from the surrounding somatic cells. Here we show that the orphan Gs-linked receptor GPR3, which is localized in the oocyte, maintains this arrest. Oocytes from Gpr3 knockout mice resume meiosis within antral follicles, independently of an increase in luteinizing hormone, and this phenotype can be reversed by injection of Gpr3 RNA into the oocytes. Thus, the GPR3 receptor is a link in communication between the somatic cells and oocyte of the ovarian follicle and is crucial for the regulation of meiosis.

Maternal beta-catenin and E-cadherin in mouse development.

The oocyte to embryo transition in metazoans depends on maternal proteins and transcripts to ensure the successful initiation of development, and the correct and timely activation of the embryonic genome. We conditionally eliminated the maternal gene encoding the cell adhesion molecule E-cadherin and partially eliminated the beta-catenin gene from the mouse oocyte. Oocytes lacking E-cadherin, or expressing a truncated allele of beta-catenin without the N-terminal part of the protein, give rise to embryos whose blastomeres do not adhere. Blastomere adhesion is restored after translation of protein from the wild-type paternal alleles: at the morula stage in embryos lacking maternal E-cadherin, and at the late four-cell stage in embryos expressing truncated beta-catenin. This suggests that adhesion per se is not essential in the early cleavage stage embryos, that embryos develop normally if compaction does not occur until the morula stage, and that the zona pellucida suffices to maintain blastomere proximity. Although maternal E-cadherin is not essential for the completion of the oocyte-to-embryo transition, absence of wild-type beta-catenin in oocytes does statistically compromise developmental success rates. This developmental deficit is alleviated by the simultaneous absence of maternal E-cadherin, suggesting that E-cadherin regulates nuclear beta-catenin availability during embryonic genome activation.