Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.

Genetic compensation induced by deleterious mutations but not gene knockdowns

Fertilization calcium waves are introduced, and the evidence from which we can infer general mechanisms of these waves is presented. The two main classes of hypotheses put forward to explain the ge...

/pdf/calcium-at-fertilization-and-in-early-development-4f8xabt5ha.pdf

Calcium at fertilization and in early development.

BACKGROUND: Mammalian oocytes are activated by intracellular calcium (Ca(2+)) oscillations following gamete fusion. Recent evidence implicates a sperm-specific phospholipase C zeta, PLCζ, which is introduced into the oocyte following membrane fusion, as the responsible factor. This review summarizes the current understanding of human oocyte activation failure and describes recent discoveries linking certain cases of male infertility with defects in PLCζ expression and activity. How these latest findings may influence future diagnosis and treatment options are also discussed. METHODS: Systematic literature searches were performed using PubMed, ISI-Web of Knowledge and The Cochrane Library. We also scrutinized material from the United Nations and World Health Organization databases (UNWHO) and the Human Fertilization and Embryology Authority (HFEA). RESULTS AND CONCLUSIONS: Although ICSI results in average fertilization rates of 70%, complete or virtually complete fertilization failure still occurs in 1-5% of ICSI cycles. While oocyte activation failure can, in some cases, be overcome by artificial oocyte activators such as calcium ionophores, a more physiological oocyte activation agent might release Ca(2+) within the oocyte in a more efficient and controlled manner. As PLCζ is now widely considered to be the physiological agent responsible for activating mammalian oocytes, it represents both a novel diagnostic biomarker of oocyte activation capability and a possible mode of treatment for certain types of male infertility.

/pdf/oocyte-activation-phospholipase-c-zeta-and-human-infertility-26p9acbddm.pdf

Oocyte activation, phospholipase C zeta and human infertility

PLCζ(zeta): A sperm protein that triggers Ca2+ oscillations and egg activation in mammals

Background The complexity of fertilization failure during assisted reproductive technologies (ART) is often under-appreciated, as this failure can occur at any number of essential mechanistic and cellular events. Importantly, successful fertilization is heavily dependent upon inherent qualities of the oocytes, and thus reliant upon fidelity of oocyte maturation. Methods Pubmed and medline were searched up to April 2008 for papers on oocyte fertilization and its mechanistic components. References to clinical/human studies were selected wherever possible. Results Successful oocyte maturation cannot simply be determined via visual assessment of polar body extrusion, but rather entails coordination of numerous cytoplasmic processes not readily observed. Proper regulation of intra-oocyte signaling cascades is crucial for sufficient production and storage of carbohydrates and proteins, successful relocation of organelles and regulation of metabolic pathways required for an apparently mature metaphase II oocyte to complete subsequent fertilization events; such as cumulus penetration, sperm/oocyte binding, fusion, oocyte activation, sperm processing and pronuclear (PN) formation. Regulation of oocyte maturation begins during oocyte growth and is intimately connected with events influencing folliculogenesis. Therefore, the oocyte is subject to a multitude of potential effector impacting fertilization potential and developmental competence long before encountering the artificial environment of the IVF laboratory. Conclusions Although meticulous care and continued research is essential for future improvement, failure to fertilize and properly form PN following clinical ART is likely to be dependent on historical events in oocyte maturation, not easily explained or prevented through simple modification of contemporary laboratory protocols.

ART failure: oocyte contributions to unsuccessful fertilization

In mammals, the sperm triggers a series of cytosolic Ca(2+) oscillations that continue for approximately 4 hours, stopping close to the time of pronucleus formation. Ca(2+) transients are also seen in fertilized embryos during the first mitotic division. The mechanism that controls this pattern of sperm-induced Ca(2+) signalling is not known. Previous studies suggest two possible mechanisms: first, regulation of Ca(2+) oscillations by M-phase kinases; and second, regulation by the presence or absence of an intact nucleus. We describe experiments in mouse oocytes that differentiate between these mechanisms. We find that Ca(2+) oscillations continue after Cdk1-cyclin B1 activity falls at the time of polar body extrusion and after MAP kinase has been inhibited with UO126. This suggests that M-phase kinases are not necessary for continued Ca(2+) oscillations. A role for pronucleus formation in regulating Ca(2+) signalling is demonstrated in experiments where pronucleus formation is inhibited by microinjection of a lectin, WGA, without affecting the normal inactivation of the M-phase kinases. In oocytes with no pronuclei but with low M-phase kinase activity, sperm-induced Ca(2+) oscillations persist for nearly 10 hours. Furthermore, a dominant negative importin beta that inhibits nuclear transport, also prevents pronucleus formation and causes Ca(2+) oscillations that continue for nearly 12 hours. During mitosis, fluorescent tracers that mark nuclear envelope breakdown and the subsequent reformation of nuclei in the newly formed two-cell embryo establish that Ca(2+) oscillations are generated only in the absence of a patent nuclear membrane. We conclude by suggesting a model where nuclear sequestration and release of a Ca(2+)-releasing activity contributes to the temporal organization of Ca(2+) transients in meiosis and mitosis in mice.

Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei.

Segregating Chromosomes in the Mammalian Oocyte.

Chromosome segregation defects in cancer cells lead to encapsulation of chromosomes in micronuclei (MN), small nucleus-like structures within which dangerous DNA rearrangements termed chromothripsis can occur. Here we uncover a strikingly different consequence of MN formation in preimplantation development. We find that chromosomes from within MN become damaged and fail to support a functional kinetochore. MN are therefore not segregated, but are instead inherited by one of the two daughter cells. We find that the same MN can be inherited several times without rejoining the principal nucleus and without altering the kinetics of cell divisions. MN motion is passive, resulting in an even distribution of MN across the first two cell lineages. We propose that perpetual unilateral MN inheritance constitutes an unexpected mode of chromosome missegregation, which could contribute to the high frequency of aneuploid cells in mammalian embryos, but simultaneously may serve to insulate the early embryonic genome from chromothripsis.

https://www.pnas.org/content/pnas/113/3/626.full.pdf

Greg FitzHarris

Papers

Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei.

Segregating Chromosomes in the Mammalian Oocyte.

Micronucleus formation causes perpetual unilateral chromosome inheritance in mouse embryos

Mammalian Oocytes Locally Remodel Follicular Architecture to Provide the Foundation for Germline-Soma Communication

Intrinsically Defective Microtubule Dynamics Contribute to Age-Related Chromosome Segregation Errors in Mouse Oocyte Meiosis-I