Anna Ajduk

Rhythmic actomyosin-driven contractions induced by sperm entry predict mammalian embryo viability

Fertilization-induced cytoplasmic flows are a conserved feature of eggs in many species. However, until now the importance of cytoplasmic flows for the development of mammalian embryos has been unknown. Here, by combining a rapid imaging of the freshly fertilized mouse egg with advanced image analysis based on particle image velocimetry, we show that fertilization induces rhythmical cytoplasmic movements that coincide with pulsations of the protrusion forming above the sperm head. We find that these movements are caused by contractions of the actomyosin cytoskeleton triggered by Ca2+ oscillations induced by fertilization. Most importantly, the relationship between the movements and the events of egg activation makes it possible to use the movements alone to predict developmental potential of the zygote. In conclusion, this method offers, thus far, the earliest and fastest, non-invasive way to predict the viability of eggs fertilized in vitro and therefore can potentially improve greatly the prospects for IVF treatment.

Cytoplasmic maturation of mammalian oocytes: development of a mechanism responsible for sperm-induced Ca2+ oscillations.

The oocytes of most mammalian species, including mouse and human, are fertilized in metaphase of the second meiotic division. A fertilizing spermatozoon introduces an oocyte-activating factor, phospholipase C zeta, triggering oscillations of the cytoplasmic concentration of free calcium ions ([Ca(2+)](i)) in the oocyte. [Ca(2+)](i) oscillations are essential for the activation of the embryonic development. They trigger processes such as resumption and completion of meiosis, establishment of the block to polyspermy and recruitment of maternal mRNAs necessary for the activation of the embryo genome. Moreover, it has been recently shown that [Ca(2+)](i) oscillations may also influence the development of the embryo. The ability to generate [Ca(2+)](i) oscillations develops in mammalian oocytes during meiotic maturation and requires several cytoplasmic changes, including: 1/ reorganization of endoplasmic reticulum, the main stockpile of calcium in the oocyte, 2/ increase in the number of 1,4,5-inositol triphosphate (IP(3)) receptors, 3/ changes in their biochemical properties (e.g.: sensitivity to IP3), and possibly both 4/ an increase in the concentration of Ca(2+) ions stored in endoplasmic reticulum (ER) and 5/ redistribution of Ca(2+)-binding ER proteins. The aim of this review is to present the state of current knowledge about these processes.

Quality control of embryo development.

Although in recent years we have seen an undeniable improvement in the field of reproductive biology and medicine, the efficiency of in vitro fertilization (IVF) procedures remains relatively low, ranging from 4% to 40% depending on the patients age. It is believed that this is in a large part caused by inaccurate assessment of embryo quality prior to transfer to mothers-to-be. Thus there is a strong need for further refinement of existing selection methods and development of novel, robust and, if only possible, non-invasive procedures to ensure that only embryos with the highest developmental potential are chosen for transfer. In the present review we compare various methods for assessing the quality of preimplantation embryos either currently used in IVF clinics or still to be tested. These methods include assessment of embryonic morphology, the genetic material, the transcriptomes of the oocyte and its accompanying follicular cells, and the embryos metabolism. We discuss what information these parameters actually provide about the processes occurring in the embryo itself. We also present novel methods for selecting healthy embryos based on most recent advanced time-lapse imaging techniques, which show great promise and are likely to lead to increased in vitro fertilization efficiency.

Advances in embryo selection methods.

Despite many recent advances in the field of reproductive biology and medicine, the efficiency of in vitro fertilization procedures remains relatively low. There is a need for a reliable and non-invasive method of embryo selection to ensure that only embryos with the highest developmental potential are chosen for transfer to mothers-to-be. Here, we compare various methods currently used for assessing embryonic viability, such as examination of embryonic morphology, quality of the genetic material, or metabolism. Additionally, we discuss novel procedures for embryonic assessment based on advanced time-lapse imaging techniques, which show great promise and may lead to increased in vitro fertilization efficiencies.

The basal position of nuclei is one pre-requisite for asymmetric cell divisions in the early mouse embryo

The early mouse embryo undertakes two types of cell division: symmetric that gives rise to the trophectoderm and then placenta or asymmetric that gives rise to inner cells that generate the embryo proper. Although cell division orientation is important, the mechanism regulating it has remained unclear. Here, we identify the relationship between the plane of cell division and the position of the nucleus and go towards identifying the mechanism behind it. We first find that as the 8-cell embryo progresses through the cell cycle, the nuclei of most – but not all – cells move from apical to more basal positions, in a microtubule- and kinesin-dependent manner. We then find that all asymmetric divisions happen when nuclei are located basally and, in contrast, all cells, in which nuclei remain apical, divide symmetrically. To understand the potential mechanism behind this, we determine the effects of modulating expression of Cdx2, a transcription factor key for trophectoderm formation and cell polarity. We find that increased expression of Cdx2 leads to an increase in a number of apical nuclei, whereas down-regulation of Cdx2 leads to more nuclei moving basally, which explains a previously identified relationship between Cdx2 and cell division orientation. Finally, we show that down-regulation of aPKC, involved in cell polarity, decreases the number of apical nuclei and doubles the number of asymmetric divisions. These results suggest a model in which the mutual interdependence of Cdx2 and cell polarity affects the cytoskeleton-dependent positioning of nuclei and, in consequence, the plane of cell division in the early mouse embryo.

Polarity and cell division orientation in the cleavage embryo: from worm to human

Cleavage is a period after fertilization, when a 1-cell embryo starts developing into a multicellular organism. Due to a series of mitotic divisions, the large volume of a fertilized egg is divided into numerous smaller, nucleated cells—blastomeres. Embryos of different phyla divide according to different patterns, but molecular mechanism of these early divisions remains surprisingly conserved. In the present paper, we describe how polarity cues, cytoskeleton and cell-to-cell communication interact with each other to regulate orientation of the early embryonic division planes in model animals such as Caenorhabditis elegans, Drosophila and mouse. We focus particularly on the Par pathway and the actin-driven cytoplasmic flows that accompany it. We also describe a unique interplay between Par proteins and the Hippo pathway in cleavage mammalian embryos. Moreover, we discuss the potential meaning of polarity, cytoplasmic dynamics and cell-to-cell communication as quality biomarkers of human embryos.

Mouse oocytes fertilised by ICSI during in vitro maturation retain the ability to be activated after refertilisation in metaphase II and can generate Ca2+ oscillations

BackgroundAt fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca2+ ([Ca2+]i). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca2+]i oscillations through the generation of inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release by binding to IP3 receptors located on the endoplasmic reticulum (ER) of the oocyte. Ability to respond to this activating stimulus develops during meiotic maturation of the oocyte. Here we examine how the development of this ability is perturbed when a single spermatozoon is introduced into the oocyte prematurely, i.e. during oocyte maturation.ResultsMouse oocytes during maturation in vitro were fertilised by ICSI (intracytoplasmic sperm injection) 1 – 4 h after germinal vesicle break-down (GVBD) and were subsequently cultured until they reached metaphase II (MII) stage. At MII stage they were fertilised in vitro for the second time (refertilisation). We observed that refertilised oocytes underwent activation with similar frequency as control oocytes, which also went through maturation in vitro, but were fertilised only once at MII stage (87% and 93%, respectively). Refertilised MII oocytes were able to develop [Ca2+]i oscillations in response to penetration by spermatozoa. We found however, that they generated a lower number of transients than control oocytes. We also showed that the oocytes, which were fertilised during maturation had a similar level of MPF activity as control oocytes, which were not subjected to ICSI during maturation, but had reduced level of IP3 receptors.ConclusionMouse oocytes, which were experimentally fertilised during maturation retain the ability to generate repetitive [Ca2+]i transients, and to be activated after completion of maturation.

Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.

Optical coherence microscopy as a novel, non-invasive method for the 4D live imaging of early mammalian embryos

Imaging of living cells based on traditional fluorescence and confocal laser scanning microscopy has delivered an enormous amount of information critical for understanding biological processes in single cells. However, the requirement for a high numerical aperture and fluorescent markers still limits researchers’ ability to visualize the cellular architecture without causing short- and long-term photodamage. Optical coherence microscopy (OCM) is a promising alternative that circumvents the technical limitations of fluorescence imaging techniques and provides unique access to fundamental aspects of early embryonic development, without the requirement for sample pre-processing or labeling. In the present paper, we utilized the internal motion of cytoplasm, as well as custom scanning and signal processing protocols, to effectively reduce the speckle noise typical for standard OCM and enable high-resolution intracellular time-lapse imaging. To test our imaging system we used mouse and pig oocytes and embryos and visualized them through fertilization and the first embryonic division, as well as at selected stages of oogenesis and preimplantation development. Because all morphological and morphokinetic properties recorded by OCM are believed to be biomarkers of oocyte/embryo quality, OCM may represent a new chapter in imaging-based preimplantation embryo diagnostics.

Time-lapse imaging of cleavage divisions in embryo quality assessment

In vitro fertilization (IVF) is one of the most important procedures for treating infertility. As several embryos are usually produced in a single IVF cycle, it is crucial to select only the most viable ones for transfer to the patient. Morphokinetics, i.e. analysis of the dynamics of cleavage divisions and processes such as compaction and cavitation, has provided both biologists and clinicians with a new set of data regarding embryonic behaviour during preimplantation development and its association with embryo quality. In the current review, we focus on biological significance of morphokinetic parameters and show how they can be used to predict a reproductive outcome. We also explain the statistics behind the predictive algorithms and discuss the future perspectives of morphokinetics.