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A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

University of Pavia

Mouse antral oocytes can be classified in two different types termed SN or NSN oocytes, depending on the presence or absence, respectively, of a ring of Hoechst 33342-positive chromatin surrounding the nucleolus. The aim of the present study was to test the developmental competence to blastocyst of the two types of oocytes. Here we show that following isolation, classification and culture of cumulus-free antral oocytes, 14.7% and 74.5% of NSN and SN oocytes, respectively, reached the metaphase II stage. When fertilised and further cultured none of the metaphase II NSN oocytes developed beyond the 2-cell stage whilst 47.4% of the metaphase II SN oocytes reached the 4-cell stage and 18.4% developed to blastocyst. The findings reported in this paper may contribute to improved procedures of female gamete selection for in vitro fertilisation of humans and farm animals. Furthermore, the selection of oocytes with better developmental potential may be of interest for studies on nuclear/cytoplasm interaction, particularly in nuclear-transfer experiments.

The analysis of chromatin organisation allows selection of mouse antral oocytes competent for development to blastocyst.

The imprinted mouse Xist (X-inactive specific transcript) gene is involved in the initiation of X-chromosome inactivation. Only the paternal Xist is expressed in preimplantation development beginning from the 4-cell stage, preceding and in correlation with paternal X-inactivation in the extraembryonic lineage of the blastocyst. To better understand the mechanisms regulating Xist expression in early development, we investigated the precise timing of its onset. We set up a single-cell RT-PCR for the simultaneous analysis on single embryos of Xist and Hprt (internal control) cDNAs and a Y-chromosome specific DNA sequence, Zfy (for embryo sexing). Applying this procedure, we demonstrate that Xist expression begins at the G2-phase of 2-cell female embryos, earlier than previously reported and at the same time of the major wave of zygotic genome activation (ZGA). We then examined, if Xist expression at the 2-cell stage is dependent on the lapse of time spent since fertilization, as previously reported for zygotic genes. One-cell embryos at the G2-phase of the first cell-cycle were cultured with cytochalasin D (inhibitor of cytokinesis but not of DNA synthesis or nuclear progression) for a time equivalent to the 4-cell stage in control, untreated embryos. We show that Xist activation occurs at a scheduled time following fertilization despite the embryos being blocked at the 1-cell stage, suggesting the existence of a zygotic clock involved in the regulation of the transcription of this imprinted gene.

Mouse Xist expression begins at zygotic genome activation and is timed by a zygotic clock.

Although the female gamete is blocked at the dictyate stage of the first meiotic prophase during the whole folliculogenesis, many important epigenetic changes occur to organise the genome to attend early embryonic development. In this paper, we will describe the results of a number of studies aimed to improve our understanding of the nuclear organization of the mouse oocyte during folliculogenesis. Using silver methods that stain NOR, centromeres and heterochromatin, as well as, the use of specific antibodies for the demonstration of centromeres, we have described the changes to the chromatin organisation and to the spatial localisation of chromocenters and centromeres during oocyte growth; these changes have been correlated to the developmental competence of the resulting antral and metaphase II (MII) oocyte.

Chromatin organisation and nuclear architecture in growing mouse oocytes.

The maternal contribution of transcripts and proteins supplied to the zygote is crucial for the progression from a gametic to an embryonic control of preimplantation development. Here we compared the transcriptional profiles of two types of mouse MII oocytes, one which is developmentally competent (MIISN oocyte), the other that ceases development at the 2-cell stage (MIINSN oocyte), with the aim of identifying genes and gene expression networks whose misregulated expression would contribute to a reduced developmental competence.

Maternal Oct-4 is a potential key regulator of the developmental competence of mouse oocytes.

Regenerative medicine deals with the possible use of stem cells to repair tissues damaged by aging and related diseases, including amyloidoses. In the latter case, the toxicity of the amyloid deposits can, in principle, question the possibility to graft specific tissues by undifferentiated cells. To assess whether stem cells are vulnerable to amyloid toxicity, we exposed, in culture, murine embryonic stem (ES) cells and haematopoietic progenitor (HP) cells to oligomers of the amyloidogenic peptide Aβ42 at concentrations previously shown to be cytotoxic to several other cell types. These stem cells did not display any sign of apoptosis and their survival, proliferation and differentiation were not affected by the oligomers although the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay revealed that ES, but not HP, cells displayed some impaired ability to reduce the tetrazole salts possibly as a result of transient oxidative stress. Our results support a remarkable resistance of the investigated stem cells against amyloids and hence their potential use in cell therapy of Alzheimer's disease and, possibly, other amyloid diseases.

Embryonic stem and haematopoietic progenitor cells resist to Aβ oligomer toxicity and maintain the differentiation potency in culture.

Bioinformatics tools have been recently applied to study the differentiation of the mammalian oocyte during folliculogenesis. In this review, we will summarize our knowledge of 1) the use of biological databases for the extraction of relevant information, 2) bioinformatics methods for knowledge extraction and representation, 3) the application of these methods to the study of mammalian oocyte differentiation and 4) state-of the-art prediction approaches for the assessment and estimation of the cell differentiation status.

Knowledge-based bioinformatics for the study of mammalian oocytes.

Within species, populations differing by chromosomal rearrangements ("chromosomal races") may become reproductively isolated in association with reduced hybrid fertility due to meiotic aberrations. Speciation is also possible if hybridizing chromosomal races accumulate genetic differences because of reduced meiotic recombination in the heterozygous configuration in hybrids. Here, we examine recombination in pure races and hybrids within a model system for chromosomal speciation: the hybridization of the Poschiavo (CHPO) and Upper Valtellina (IUVA) chromosomal races of house mouse in Upper Valtellina, Italy. These races differ by Robertsonian fusions/whole-arm reciprocal translocations, such that hybrids produce a pentavalent meiotic configuration. We determined the number and position of the recombination points (using an antibody against the MutL homolog 1 [MLH1] protein) on synaptonemal complexes at pachytene in laboratory-reared CHPO, IUVA, and hybrid males, analyzing at least 112 spermatocytes per karyotypic group, up to a total of 534 spermatocytes. The mean ± standard deviation numbers of MLH1 foci per spermatocyte were 22.2 ± 3.2, 20.1 ± 2.9, 20.7 ± 2.3, and 21.9 ± 2.9 for CHPO, IUVA, CHPO × IUVA, and IUVA × CHPO, respectively. Altogether, 10,146 chromosome arms were examined, allowing multiple comparisons. Overall, recombination events were more frequently distal than proximal or interstitial. The average number of proximal MLH1 foci per chromosome arm decreased going from telocentric to metacentric bivalents to pentavalents (when present), which (together with other factors) influenced the average number of MLH1 foci per cell between CHPO, IUVA, and hybrid mice. The low frequency of proximal recombination in pentavalents of CHPO-IUVA hybrids may promote reproductive isolation between the CHPO and IUVA races, when coupled with reduced hybrid unfitness.

Chromosomal speciation in mice: a cytogenetic analysis of recombination.

Ovarian follicle encapsulation in synthetic or natural matrixes based on biopolymers is potentially a promising approach to in vitro maturation (IVM) process, since it maintains follicle 3D organisation by preventing its flattening and consequent disruption of gap junctions, preserving the functional relationship between oocyte and companion follicle cells. The aim of the work was to optimise physico-chemical parameters of alginate microcapsules for perspective IVM under 3D environments. On this purpose alginate and cross-linking agent concentrations were investigated. Alginate concentration between 0.75% and 0.125% w/w and Mg(2+), Ba(2+), Ca(2+ )at concentration between 100 and 20 mM were tested. Follicle encapsulation was obtained by on purpose modified diffusion setting gelation technique, and evaluated together with beads, chemical and mechanical stability in standard and stressing conditions. Beads permeability was tested towards albumin, fetuin, pyruvate, glucose, pullulan. Results demonstrated that 0.25% alginate cross-linked in 100 mM CaCl2 beads is suitable to follicle encapsulation.

Formulation and stability evaluation of 3D alginate beads potentially useful for cumulus-oocyte complexes culture.

Full-grown mouse antral oocytes are classified as surrounding nucleolus (SN) or not-surrounding nucleolus (NSN), depending on the respective presence or absence of a ring of Hoechst-positive chromatin surrounding the nucleolus. In culture, both types of oocytes resume meiosis and reach the metaphase II (MII) stage, but following insemination, NSN oocytes arrest at the two-cell stage whereas SN oocytes may develop to term. By coupling time-lapse bright-field microscopy with image analysis based on particle image velocimetry, we provide the first systematic measure of the changes to the cytoplasmic movement velocity (CMV) occurring during the germinal vesicle-to-MII (GV-to-MII) transition of these two types of oocytes. Compared to SN oocytes, NSN oocytes display a delayed GV-to-MII transition, which can be mostly explained by retarded germinal vesicle break down and first polar body extrusion. SN and NSN oocytes also exhibit significantly different CMV profiles at four main time-lapse intervals, although this difference was not predictive of SN or NSN oocyte origin because of the high variability in CMV. When CMV profile was analyzed through a trained artificial neural network, however, each single SN or NSN oocyte was blindly identified with a probability of 92.2% and 88.7%, respectively. Thus, the CMV profile recorded during meiotic resumption may be exploited as a cytological signature for the non-invasive assessment of the oocyte developmental potential, and could be informative for the analysis of the GV-to-MII transition of oocytes of other species.

Maurizio Zuccotti

Laboratorio di Biologia dello Sviluppo,
Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani",
Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani"

University of Pavia

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Maurizio Zuccotti

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratorio di Biologia dello Sviluppo,Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani",Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani"

University of Pavia

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Laboratorio di Biologia dello Sviluppo,
Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani",
Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia e Biotecnologie "Lazzaro Spallanzani"