Oocyte Proteins Research Articles

Efficient mutagenesis by CRISPR/Cas system during meiotic maturation of porcine oocytes

Genome editing using the CRISPR/Cas system can induce mutations with high efficiency, and allows easier production of genome-modified animals than that offered by the conventional method where embryonic stem cells are used. However, studies using CRISPR/Cas systems have been mostly limited to proliferating somatic cells and pronuclear-stage fertilized eggs. In contrast, the efficiency of a CRISPR/Cas system in immature and maturing oocytes progressing through meiosis has not yet been assessed. In the present study, we evaluated the genome-modification efficiency of the CRISPR/Cas system during meiotic maturation of porcine oocytes. Additionally, the localization of the Cas9 protein in immature oocytes was analyzed in relation to nuclear transport and mutation induction. The results showed that CRISPR/Cas induced mutation with high efficiency even in maturing oocytes with condensed chromosomes, whereas mutations were not induced in GV-stage oocytes. The localization analysis of enhanced green fluorescent protein (EGFP)-tagged Cas9 (Cas9-EGFP) revealed that the nuclei contained lesser Cas9 than the cytoplasm in immature oocytes. Treatment with leptomycin B, a nuclear export inhibitor, increased the amount of nuclear Cas9 and enabled mutation induction in GV oocytes. Our results suggest that CRISPR/Cas systems can be applied to oocytes during meiotic maturation and be implemented in novel applications targeting female genomes.

SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways.

Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis.

Comparative Proteomic Analysis of Buffalo Oocytes Matured in vitro Using iTRAQ Technique.

To investigate the protein profiling of buffalo oocytes at the germinal vesicle (GV) stage and metaphase II (MII) stage, an iTRAQ-based strategy was applied. A total of 3,763 proteins were identified, which representing the largest buffalo oocytes proteome dataset to date. Among these proteins identified, 173 proteins were differentially expressed in GV oocytes and competent MII oocytes, and 146 proteins were differentially abundant in competent and incompetent matured oocytes. Functional and KEGG pathway analysis revealed that the up-regulated proteins in competent MII oocytes were related to chromosome segregation, microtubule-based process, protein transport, oxidation reduction, ribosome, and oxidative phosphorylation, etc., in comparison with GV and incompetent MII oocytes. This is the first proteomic report on buffalo oocytes from different maturation stages and developmental competent status. These data will provide valuable information for understanding the molecular mechanism underlying buffalo oocyte maturation, and these proteins may potentially act as markers to predict developmental competence of buffalo oocyte during in vitro maturation.

Good Egg or Bad Egg: Developing markers of oocyte competence for Assisted Reproductive Interventions.

The oocyte is the foundation of life. It develops from a single fertilized cell to a multicellular organism capable of an independent existence. Competence to achieve this maximum potential is acquired following the protracted, but, highly co-ordinated process of growth and subsequently, maturation. The environment in which all or part of these processes occur, can ultimately have long-term consequences for female fertility and the health of resulting offspring. The pressure to identify and select oocytes or embryos with the highest developmental potential has intensified as the number of patients and the range of options available to them have increased. In particular, as in vitro maturation and single embryo transfer become more routine in assisted reproductive technology, selection is critical to a successful outcome. Moreover, the identification of markers of oocyte health and quality is essential to monitor the impact of these technologies on gametes and embryos. Technologies, such as transcriptomics, proteomics and metabolomics, offer more sophisticated methods for oocyte and embryo selection, with the emphasis on the predictive value of non-invasive protocols which profile follicular fluid, follicle/ granulosa cells and cumulus cells, for assessment of oocyte quality. Using the bovine as a model, we have employed a range of approaches and identified many potential markers, such as oocyte and cumulus candidate proteins and transcripts and follicular fluid fatty acids and amino acids. The models, technologies, and future strategies will be discussed in detail.

Zfp207 is a Bub3 binding protein regulating meiotic chromosome alignment in mouse oocytes.

Zinc finger proteins are a massive, diverse family of proteins that serve a wide variety of biological functions. However, the roles of them during meiosis are not yet clearly defined. Here, we report that Zfp207 localizes at the kinetochores during mouse oocyte meiotic maturation. Depletion of Zfp207 leads to a significantly higher proportion of impaired spindle organization and misaligned chromosomes in oocytes. This is coupled with the defective kinetochore-microtubule attachments, and resultantly increasing incidence of aneuploid metaphase II eggs. The precocious polar body extrusion and escape of metaphase I arrest induced by nocodazole treatment in Zfp207-depleted oocytes indicates that Zfp207 is essential for activation of SAC (Spindle Assembly Checkpoint) activity. Notably, we find that Zfp207 binds to Bub3 to form a complex and maintains its protein level in oocytes, and that overexpression of Bub3 is able to partially rescue the occurrence of aneuploid eggs in Zfp207-depleted oocytes. Collectively, we identify Zfp207 as a novel Bub3 binding protein in oocytes which plays an important role in controlling meiotic chromosome alignment and SAC function.

Hypoxia Inducible Factor 1 Alpha Is Expressed in Germ Cells throughout the Murine Life Cycle

Pluripotent stem cells of the early embryo, and germ line cells, are essential to ensure uncompromised development to adulthood as well as species propagation, respectively. Recently, the transcription factor hypoxia inducible factor 1 alpha (Hif1α) has been shown to have important roles in embryonic stem cells; in particular, regulation of conversion to glycolytic metabolism and, as we have shown, maintenance of functional levels of telomerase. In the present study, we sought to assess whether Hif1α was also expressed in the primitive cells of the murine embryo. We observed expression of Hif1α in pre-implantation embryos, specifically the 2-cell stage, morula, and blastocyst. Robust Hif1α expression was also observed in male and female primordial germ cells. We subsequently assessed whether Hif1α was expressed in adult male and female germ cells. In the testis, Hif1α was robustly expressed in spermatogonial cells, in both juvenile (6-week old) and adult (3-month old) males. In the ovaries, Hif1α was expressed in mature oocytes from adult females, as assessed both in situ and in individual oocytes flushed from super-ovulated females. Analysis of Hif1α transcript levels indicates a mechanism of regulation during early development that involves stockpiling of Hif1α protein in mature oocytes, presumably to provide protection from hypoxic stress until the gene is re-activated at the blastocyst stage. Together, these observations show that Hif1α is expressed throughout the life-cycle, including both the male and female germ line, and point to an important role for Hif1α in early progenitor cells.

Effective protein inhibition in intact mouse oocytes through peptide nanoparticle-mediated antibody transfection.

Female meiosis is a fundamental area of study in reproductive medicine, and the mouse oocyte model of in vitro maturation (IVM) is most widely used to study female meiosis. To investigate the probable role(s) of an unknown protein in female meiosis, the method traditionally used involves microinjecting a specific antibody into mouse oocytes. Recently, in studies on somatic cells, peptide nanoparticle-mediated antibody transfection has become a popular tool because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, untill now no researchers have tried using this technique on mouse oocytes because the zona pellucida surrounding the oocyte membrane (vitelline membrane) is usually thought or proved to be a tough barrier to macromolecules such as antibodies and proteins. Therefore, we attempted to introduce an antibody into mouse oocytes using a peptide nanoparticle. Here we show for the first time that with our optimized method, an antibody can be effectively delivered into mouse oocytes and inhibit its target protein with high specificity. We obtained significant results using small GTPase Arl2 as a test subject protein. We propose peptide nanoparticle-mediated antibody transfection to be a superior alternative to antibody microinjection for preliminary functional studies of unknown proteins in mouse oocytes.

Purification of Human and Mammalian Membrane Proteins Expressed in Xenopus laevis Frog Oocytes for Structural Studies.

This protocol describes the isolation of recombinant human and mammalian membrane proteins expressed in Xenopus laevis frog oocytes for structural studies. The cDNA-derived cRNA of the desired genes is injected into several hundreds of oocytes, which are incubated for several days to allow protein expression. Recombinant proteins are then purified via affinity chromatography. The novelty of this method comes from the design of a plasmid that produces multi-tagged proteins and, most importantly, the development of a protocol for efficiently discarding lipids, phospholipids, and lipoproteins from the oocyte egg yolk, which represent the major contaminants in protein purifications. Thus, the high protein purity and good yield obtained from this method allows protein structure determination by transmission electron microscopy of single detergent-solubilized protein particles and of 2D crystals of membrane protein embedded in lipid bilayers. Additionally, a radiotracer assay for functional analysis of the expressed target proteins in oocytes is described. Overall, this method is a valuable option for structural studies of mammalian and particularly human proteins, for which other expression systems often fail.

Translation repression by maternal RNA binding protein Zar1 is essential for early oogenesis in zebrafish.

A large amount of maternal RNA is deposited in oocytes and is reserved for later development. Control of maternal RNA translation during oocyte maturation has been extensively investigated and its regulatory mechanisms are well documented. However, translational regulation of maternal RNA in early oogenesis is largely unexplored. In this study, we generated zebrafish zar1 mutants that result in early oocyte apoptosis and fully penetrant male development. Loss of p53 suppresses the apoptosis in zar1 mutants and restores oocyte development. zar1 immature ovaries show upregulation of proteins implicated in endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). More importantly, loss of Zar1 causes marked upregulation of zona pellucida (ZP) family proteins, while overexpression of ZP proteins in oocytes causes upregulation of stress-related activating transcription factor 3 (atf3), arguing that tightly controlled translation of ZP proteins is essential for ER homeostasis during early oogenesis. Furthermore, Zar1 binds to ZP gene mRNAs and represses their translation. Together, our results indicate that regulation of translational repression and de-repression are essential for precisely controlling protein expression during early oogenesis.

Tob1 is expressed in developing and adult gonads and is associated with the P-body marker, Dcp2.

Tob1 is a member of the BTG/TOB family of proteins with established antiproliferative function. In Danio rerio and Xenopus laevis, the Tob1 gene is expressed from the one-cell stage through to early gastrula stages, followed in later development by discrete expression in many tissues including the notochord and somites. In both mouse and human, Tob1 is expressed in many adult tissues including the testis and ovary; however, the specific cell types are unknown. We examine Tob1 gene expression in mouse in developing germ cells and in sorted male germ cells (gonocytes, spermatogonia, pachytene spermatocytes and round spermatids) by reverse transcription and droplet digital polymerase chain reaction (RT-ddPCR) and in adult ovary and testis by immunofluorescence with anti-Tob1 protein staining. By RT-ddPCR, Tob1 expression was low in developing male germ cells but was highly expressed in round spermatids. In developing female germ cells undergoing entry into meiosis, it increased 10-fold. Tob1 was also highly expressed in round spermatids and in oocytes in all stages of folliculogenesis. Notably, a marker for P-bodies, Dcp-2, was also highly expressed in round spermatids and all oocyte stages examined. The cytoplasmic presence of Tob1 protein in round spermatids and oocytes and the association of Tob1 protein with Dcp2 in both cell types suggest that Tob1 protein plays a role in post-transcriptional mechanisms.

FSH Regulates mRNA Translation in Mouse Oocytes and Promotes Developmental Competence.

A major challenge in assisted reproductive technology is to develop conditions for in vitro oocyte maturation yielding high-quality eggs. Efforts are underway to assess whether known hormonal and local factors play a role in oocyte developmental competence and to identify the molecular mechanism involved. Here we have tested the hypothesis that FSH improves oocyte developmental competence by regulating the translational program in the oocyte. Accumulation of oocyte proteins (targeting protein for the Xenopus kinesin xklp2 and IL-7) associated with improved oocyte quality is increased when cumulus-oocyte complexes are incubated with FSH. This increase is due to enhanced translation of the corresponding mRNAs, as indicated by microinjection of constructs in which the 3' untranslated region of the Tpx2 or Il7 transcripts is fused to the luciferase reporter. A transient activation of the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte preceded the increase in translation. When the epidermal growth factor (EGF) receptor is down-regulated in follicular cells, the FSH-induced rate of maternal mRNA translation and AKT activation were lost, demonstrating that the effects of FSH are indirect and require EGF receptor signaling in the somatic compartment. Using Pten(fl/fl):Zp3cre oocytes in which the AKT is constitutively activated, translation of reporters was increased and was no longer sensitive to FSH stimulation. More importantly, the oocytes lacking the phosphate and tensin homolog gene showed increased developmental competence, even when cultured in the absence of FSH or growth factors. Thus, we demonstrate that FSH intersects with the follicular EGF network to activate the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte to control translation and developmental competence. These findings provide a molecular rationale for the use of FSH to improve egg quality.

140 EXPRESSION OF PERILIPIN IN PORCINE OOCYTES AND CUMULUS CELLS AT DIFFERENT STAGES OF IN VITRO MATURATION

Perilipin, one of the perilipin adipophilin tail-interacting protein of 47 kDa (PAT) family, has been found to coat the surface of intracellular lipid droplets. It limits the interaction of lipases with intracellular lipid droplets and is involved in the formation and regulation of lipids in various kinds of cells. However, little is known about the effect of perilipin on porcine oocytes and cumulus cells. Therefore, this study aimed to detect the expression of perilipin1 (PLIN1), perilipin2 (PLIN2), perilipin3 (PLIN3), and perilipin4 (PLIN4) in porcine oocytes and cumulus cells at 4 stages of in vitro maturation (IVM) by quantitative real-time PCR (RT-qPCR). Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) stage were found to occur predominantly at 18 to 24 h, after 24 h, 30 to 36 h, and 36 to 44 h of IVM, respectively. Cumulus-oocyte complexes (COC) were cultured in IVM medium and oocytes and cumulus cells were isolated after different durations of IVM (20, 26, 32, and 44 h). The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, Sn Diego, CA, USA) and the threshold for statistical significance was set at P < 0.05. Messenger RNA expression of PLIN1 was not detected in either oocytes or cumulus cells during all periods of IVM. PLIN2, on the other hands, showed a significant lower expression in GVBD, MI, and MII oocytes compared with the GV oocytes, but showed no difference in cumulus cells. PLIN3 expression was significantly decreased in oocytes of MI stage, whereas PLIN3 from cumulus cells was expressed significantly lower in GVBD and MII stage compared with GV stage. Expression of PLIN4 was significantly decreased in only cumulus cells of GVBD and MI stage. These findings suggest that PLIN2 may have important roles in lipid metabolism during porcine oocyte maturation, whereas PLIN4 may play a major role in cumulus cells. PLIN3 can be hypothesised as a common lipid droplet-associated protein in both oocytes and cumulus cells. Further studies should be conducted to characterise the expression and distribution of PLIN1, PLIN2, PLIN3, and PLIN4 in porcine oocytes and cumulus cells. This study was supported by Ministry of Trade, Industry and Energy (#10048948), Korea IPET (#311011-05-4-SB010), Research Institute for Veterinary Science, and the BK21 plus program.

Postovulatory aging affects dynamics of mRNA, expression and localization of maternal effect proteins, spindle integrity and pericentromeric proteins in mouse oocytes.

Is the postovulatory aging-dependent differential decrease of mRNAs and polyadenylation of mRNAs coded by maternal effect genes associated with altered abundance and distribution of maternal effect and RNA-binding proteins (MSY2)? Postovulatory aging results in differential reduction in abundance of maternal effect proteins, loss of RNA-binding proteins from specific cytoplasmic domains and critical alterations of pericentromeric proteins without globally affecting protein abundance. Oocyte postovulatory aging is associated with differential alteration in polyadenylation and reduction in abundance of mRNAs coded by selected maternal effect genes. RNA-binding and -processing proteins are involved in storage, polyadenylation and degradation of mRNAs thus regulating stage-specific recruitment of maternal mRNAs, while chromosomal proteins that are stage-specifically expressed at pericentromeres, contribute to control of chromosome segregation and regulation of gene expression in the zygote. Germinal vesicle (GV) and metaphase II (MII) oocytes from sexually mature C57B1/6J female mice were investigated. Denuded in vivo or in vitro matured MII oocytes were postovulatory aged and analyzed by semiquantitative confocal microscopy for abundance and localization of polyadenylated RNAs, proteins of maternal effect genes (transcription activator BRG1 also known as ATP-dependent helicase SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) and NOD-like receptor family pyrin domain containing 5 (NLRP5) also known as MATER), RNA-binding proteins (MSY2 also known as germ cell-specific Y-box-binding protein, YBX2), and post-transcriptionally modified histones (trimethylated histone H3K9 and acetylated histone H4K12), as well as pericentromeric ATRX (alpha thalassemia/mental retardation syndrome X-linked, also termed ATP-dependent helicase ATRX or X-linked nuclear protein (XNP)). For proteome analysis five replicates of 30 mouse oocytes were analyzed by selected reaction monitoring (SRM). GV and MII oocytes were obtained from large antral follicles or ampullae of sexually mature mice, respectively. Denuded MII oocytes were aged for 24 h post ovulation. For analysis of distribution and abundance of polyadenylated RNAs fixed oocytes were in situ hybridized to Cy5 labeled oligo(dT)20 nucleotides. Absolute quantification of protein concentration per oocyte of selected proteins was done by SRM proteome analysis. Relative abundance of ATRX was assessed by confocal laser scanning microscopy (CLSM) of whole mount formaldehyde fixed oocytes or after removal of zona and spreading. MSY2 protein distribution and abundance was studied in MII oocytes prior to, during and after exposure to nocodazole, or after aging for 2 h in presence of H2O2 or for 24 h in presence of a glutathione donor, glutathione ethylester (GEE). The significant reduction in abundance of proteins (P < 0.001) translated from maternal mRNAs was independent of polyadenylation status, while their protein localization was not significantly changed by aging. Most of other proteins quantified by SRM analysis did not significantly change in abundance upon aging except MSY2 and GTSF1. MSY2 was enriched in the subcortical RNP domain (SCRD) and in the spindle chromosome complex (SCC) in a distinct pattern, right and left to the chromosomes. There was a significant loss of MSY2 from the SCRD (P < 0.001) and the spindle after postovulatory aging. Microtubule de- and repolymerization caused reversible loss of MSY2 spindle-association whereas H2O2 stress did not significantly decrease MSY2 abundance. Aging in presence of GEE decreased significantly (P < 0.05) the aging-related overall and cytoplasmic loss of MSY2. Postovulatory aging increased significantly spindle abnormalities, unaligned chromosomes, and abundance of acetylated histone H4K12, and decreased pericentromeric trimethylated histone H3K9 (all P < 0.001). Spreading revealed a highly significant increase in pericentromeric ATRX (P < 0.001) upon ageing. Thus, the significantly reduced abundance of MSY2 protein, especially at the SCRD and the spindle may disturb the spatial control and timely recruitment, deadenylation and degradation of developmentally important RNAs. An autonomous program of degradation appears to exist which transiently and specifically induces the loss and displacement of transcripts and specific maternal proteins independent of fertilization in aging oocytes and thereby can critically affect chromosome segregation and gene expression in the embryo after fertilization. We used the mouse oocyte to study processes associated with postovulatory aging, which may not entirely reflect processes in aging human oocytes. However, increases in spindle abnormalities, unaligned chromosomes and H4K12 acetylated histones, as well as in mRNA abundance and polyadenylation have been observed also in aged human oocytes suggesting conserved processes in aging. Postovulatory aging precociously induces alterations in expression and epigenetic modifications of chromatin by ATRX and in histone pattern in MII oocytes that normally occur after fertilization, possibly contributing to disturbances in the oocyte-to-embryo transition (OET) and the zygotic gene activation (ZGA). These observations in mouse oocytes are also relevant to explain disturbances and reduced developmental potential of aged human oocytes and caution to prevent oocyte aging in vivo and in vitro. The study has been supported by the German Research Foundation (DFG) (EI 199/7-1 | GR 1138/12-1 | HO 949/21-1 and FOR 1041). There is no competing interest.

Embryonic Poly(A)-Binding Protein (EPAB) Is Required for Granulosa Cell EGF Signaling and Cumulus Expansion in Female Mice.

Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos before zygotic genome activation. EPAB is required for translational activation of maternally stored mRNAs in the oocyte and Epab(-/-) female mice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aim of this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab(-/-) mice. Using a coculture system of oocytectomized cumulus oophorus complexes (OOXs) with denuded oocytes, we found that when wild-type OOXs were cocultured with Epab(-/-) oocytes, or when Epab(-/-) OOXs were cocultured with WT oocytes, cumulus expansion failed to occur in response to epidermal growth factor (EGF). This finding suggests that oocytes and cumulus cells (CCs) from Epab(-/-) mice fail to send and receive the necessary signals required for cumulus expansion. The abnormalities in Epab(-/-) CCs are not due to lower expression of the oocyte-derived factors growth differentiation factor 9 or bone morphogenetic protein 15, because Epab(-/-) oocytes express these proteins at comparable levels with WT. Epab(-/-) granulosa cells (GCs) exhibit decreased levels of phosphorylated MEK1/2, ERK1/2, and p90 ribosomal S6 kinase in response to lutenizing hormone and EGF treatment, as well as decreased phosphorylation of the EGF receptor. In conclusion, EPAB, which is oocyte specific, is required for the ability of CCs and GCs to become responsive to LH and EGF signaling. These results emphasize the importance of oocyte-somatic communication for GC and CC function.

High-resolution microscopy of active ribosomal genes and key members of the rRNA processing machinery inside nucleolus-like bodies of fully-grown mouse oocytes

Nucleolus-like bodies (NLBs) of fully-grown (germinal vesicle, GV) mammalian oocytes are traditionally considered as morphologically distinct entities, which, unlike normal nucleoli, contain transcribed ribosomal genes (rDNA) solely at their surface. In the current study, we for the first time showed that active ribosomal genes are present not only on the surface but also inside NLBs of the NSN-type oocytes. The “internal” rRNA synthesis was evidenced by cytoplasmic microinjections of BrUTP as precursor and by fluorescence in situ hybridization with a probe to the short-lived 5′ETS segment of the 47S pre-rRNA. We further showed that in the NLB mass of NSN-oocytes, distribution of active rDNA, RNA polymerase I (UBF) and rRNA processing (fibrillarin) protein factors, U3 snoRNA, pre-rRNAs and 18S/28S rRNAs is remarkably similar to that in somatic nucleoli capable to make pre-ribosomes. Overall, these observations support the occurrence of rDNA transcription, rRNA processing and pre-ribosome assembly in the NSN-type NLBs and so that their functional similarity to normal nucleoli. Unlike the NSN-type NLBs, the NLBs of more mature SN-oocytes do not contain transcribed rRNA genes, U3 snoRNA, pre-rRNAs, 18S and 28S rRNAs. These results favor the idea that in a process of transformation of NSN-oocytes to SN-oocytes, NLBs cease to produce pre-ribosomes and, moreover, lose their rRNAs. We also concluded that a denaturing fixative 70% ethanol used in the study to fix oocytes could be more appropriate for light microscopy analysis of nucleolar RNAs and proteins in mammalian fully-grown oocytes than a commonly used cross-linking aldehyde fixative, formalin.

A peculiar lamin in a peculiar mammal: Expression of lamin LIII in platypus (Ornithorhynchus anatinus)

Platypus (Ornithorhynchus anatinus) holds a unique phylogenetic position at the base of the mammalian lineage due to an amalgamation of mammalian and sauropsid-like features. Here we describe the set of four lamin genes for platypus. Lamins are major components of the nuclear lamina, which constitutes a main component of the nucleoskeleton and is involved in a wide range of nuclear functions. Vertebrate evolution was accompanied by an increase in the number of lamin genes from a single gene in their closest relatives, the tunicates and cephalochordates, to four genes in the vertebrate lineage. Of the four genes the LIII gene is characterized by the presence of two alternatively spliced CaaX-encoding exons. In amphibians and fish LIII is the major lamin protein in oocytes and early embryos. The LIII gene is conserved throughout the vertebrate lineage, with the notable exception of marsupials and placental mammals, which have lost the LIII gene. Here we show that platypus has retained an LIII gene, albeit with a significantly altered structure and with a radically different expression pattern. The platypus LIII gene contains only a single CaaX-encoding exon and the head domain together with coil 1a and part of coil1b of the platypus LIII protein is replaced by a novel short non-helical N-terminus. It is expressed exclusively in the testis. These features resemble those of male germ cell-specific lamins in placental mammals, in particular those of lamin C2. Our data suggest (i) that the specific functions of LIII, which it fulfills in all other vertebrates, is no longer required in mammals and (ii) once it had been freed from these functions has undergone structural alterations and has adopted a new functionality in monotremes.

Embryonic Poly(A)-Binding Protein Is Required During Early Stages of Mouse Oocyte Development for Chromatin Organization, Transcriptional Silencing, and Meiotic Competence.

During oocyte maturation, fertilization, and early embryo development until zygotic genome activation (ZGA), transcription is suppressed, and gene expression is dependent upon the timely activation of stored mRNAs. Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos and is important for regulating translational activation of maternally stored mRNAs. EPAB is critical for early development because Epab(-/-) female mice do not produce mature eggs and are infertile. In this study, we further characterize morphological and molecular aspects of Epab(-/-) oocytes. We demonstrated that Epab(-/-) oocytes are smaller in size, contain peripheral germinal vesicles, and are loosely associated with cumulus cells. The chromatin reorganization of the surrounded nucleolus (SN) configuration and transcriptional silencing that normally occurs during oocyte growth does not occur in Epab(-/-) oocytes. Interestingly, microinjection of Epab mRNA into Epab(-/-) preantral follicle-enclosed oocytes rescues reorganization of chromatin and oocyte maturation to metaphase II. Overall, these results demonstrate an important role for EPAB during oocyte growth and the acquisition of meiotic competence.

Monitoring channel activities of proteoliposomes with SecA and Cx26 gap junction in single oocytes

Establishing recordable channels in membranes of oocytes formed by expressing exogenous complementary DNA (cDNA) or messenger RNA (mRNA) has contributed greatly to understanding the molecular mechanisms of channel functions. Here, we report the extension of this semi-physiological system for monitoring the channel activity of preassembled membrane proteins in single cell oocytes by injecting reconstituted proteoliposomes along with substrates or regulatory molecules. We build on the observation that SecA from various bacteria forms active protein-conducting channels with injection of proteoliposomes, protein precursors, and ATP–Mg2+. Such activity was enhanced by reconstituted SecYEG–SecDF•YajC liposome complexes that could be monitored easily and efficiently, providing correlation of in vitro and intact cell functionality. In addition, inserting reconstituted gap junction Cx26 liposomes into the oocytes allowed the demonstration of intracellular/extracellular Ca2+-regulated hemi-channel activities. The channel activities can be detected rapidly after injection, can be monitored for various effectors, and are dependent on specific exogenous lipid compositions. This simple and effective functional system with low endogenous channel activity should have broad applications for monitoring the specific channel activities of complex interactions of purified membrane proteins with their effectors and regulatory molecules.

Actin-capping proteins play essential roles in the asymmetric division of maturing mouse oocytes

Actin polymerization is essential for various stages of mammalian oocyte maturation, including spindle migration, actin cap formation, polar body extrusion and cytokinesis. The heterodimeric actin-capping protein is an essential element of the actin cytoskeleton. It binds to the fast-growing (barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. However, the roles of capping protein in mammalian oocyte maturation are poorly understood. We investigated the roles of capping protein in mouse oocytes and found that it is essential for correct asymmetric spindle migration and polar body extrusion. Capping protein mainly localized in the cytoplasm during maturation. By knocking down or ectopically overexpressing this protein, we revealed that it is crucial for efficient spindle migration and maintenance of the cytoplasmic actin mesh density. Expression of the capping-protein-binding region of CARMIL (also known as LRRC16A) impaired spindle migration and polar body extrusion during oocyte maturation and decreased the density of the cytoplasmic actin mesh. Taken together, these findings show that capping protein is an essential component of the actin cytoskeleton machinery that plays crucial roles in oocyte maturation, presumably by controlling the cytoplasmic actin mesh density.

Differences in embryo quality are associated with differences in oocyte composition: a proteomic study in inbred mice.

Current models of early mouse development assign roles to stochastic processes and epigenetic regulation, which are considered to be as influential as the genetic differences that exist between strains of the species Mus musculus. The aim of this study was to test whether mouse oocytes vary from each other in the abundance of gene products that could influence, prime, or even predetermine developmental trajectories and features of derivative embryos. Using the paradigm of inbred mouse strains, we quantified 2010 protein groups (SILAC LC-MS/MS) and 15205 transcripts (RNA deep sequencing) present simultaneously in oocytes of four strains tested (129/Sv, C57Bl/6J, C3H/HeN, DBA/2J). Oocytes differed according to donor strain in the abundance of catalytic and regulatory proteins, as confirmed for a subset (bromodomain adjacent to zinc finger domain, 1B [BAZ1B], heme oxygenase 1 [HMOX1], estrogen related receptor, beta [ESRRB]) via immunofluorescence in situ. Given a Pearson's r correlation coefficient of 0.18-0.20, the abundance of oocytic proteins could not be predicted from that of cognate mRNAs. Our results document that a prerequisite to generate embryo diversity, namely the different abundances of maternal proteins in oocytes, can be studied in the model of inbred mouse strains. Thus, we highlight the importance of proteomic quantifications in modern embryology. All MS data have been deposited in the ProteomeXchange with identifier PXD001059 (http://proteomecentral.proteomexchange.org/dataset/PXD001059).