Patent Application
20200080052
N.A.
The present disclosure relates to a method of reducing chromosome segregation error in cells of the early embryo. More specifically, the present disclosure is concerned with a method of reducing chromosome segregation error in cells of the early embryo using an inhibitor that directly binds to APC/C.
N.A.
The goal of cell division is to accurately share replicated chromosomes between newly forming daughter cells. Errors result in cells with erroneous numbers of chromosomes, termed aneuploidy. The early mammalian embryo is a notable scenario where aneuploidy is common. As a result of segregation errors during the first several mitoses after fertilisation, preimplantation embryos are often mosaic, with both euploid and aneuploid cells, which reduces the potential of a successful pregnancy Vanneste et al., 2009, Vázquez-Diez et al., 2018). However, the underlying cellular cause for the inherent tendency of embryonic blastomeres to mis-segregate chromosomes is unknown.
The major cellular safeguard preventing segregation errors is the spindle assembly checkpoint (SAC), a near-ubiquitous signalling pathway that delays anaphase onset until chromosomes are correctly aligned with kinetochores correctly attached to spindle microtubules. Mis-attached kinetochores catalyse generation of Mitotic Checkpoint Complex (MCC), a diffusible signal that inhibits the anaphase promoting complex (APC/C), delaying anaphase until correct attachment is achieved (Lara-Gonzalez et al., 2012; Musacchio et al., 2015). Whilst embryo aneuploidy can be exacerbated by SAC inhibition (Bolton et al., 2016; Wei et al., 2011), a detailed examination of SAC function, or an explanation as to why segregation errors are normally common in the early embryo, is yet to be presented. SAC function has been well studied in mammalian oocytes, where it is relatively inefficient in preventing segregation errors (Gui et al., 2012; Lane et al., 2017; Kolano et al., 2012; Nagaoka et al., 2012). This is likely due in part to the need for limited amounts of diffusible MCC to inhibit APC/C throughout the cytoplasm in an extraordinarily large cell (Kyogoku et al., 2017; Lane et al., 2017). Consistently, in C. elegans embryos the SAC is ineffective in early embryos and becomes more effective as cells reduce in size during early development (Galli et al., 2016; Gerhold et al., 2018).
In the present disclosure, the inventors used live imaging, micromanipulation, gene knockdown, and pharmacological approaches in mouse embryos to appraise SAC function in in the early mammalian embryo. The inventors show that SAC signalling operates in embryos and limits the number of segregation errors but that cells frequently enter anaphase despite severely misaligned chromosomes with SAC-active kinetochores. While the inventors tested the hypothesis that large cytoplasmic volume hampers the checkpoint in the very earliest stages of development until smaller cell sizes are attained, they unexpectedly discovered that frequent segregation error in the mouse embryo and failure to delay anaphase is not attributable to the large size of early embryo cell but is permitted by a failure of SAC-active kinetochores to prevent anaphase, that is not related to cytoplasmic volume. The present disclosure shows that mildly inhibiting the APC/C can prolong mitosis and thereby increase the probability of correct alignment being achieved before the completion of cell division, and thus lower the number of segregation errors. The present disclosure shows that modulation of the SAC-APC/C axis increases the likelihood of faithful chromosome segregation in cultured embryos.
More specifically, in accordance with the present disclosure, there is provided the following items:
Item 1. Method for reducing chromosome segregation errors in cultured cells of the early embryo by contacting the cells with an effective dose of an inhibitor of the anaphase promoting complex (APC/C) that directly binds to the APC/C.
Item 2. The method of item 1, wherein the inhibitor is proTAME or TAME.
Item 3. The method of item 1 or 2, wherein the effective dose is a nanomolar amount.
Item 4. The method of item 1 or 2, wherein the effective dose is about 0.001 nM to about 500 nM.
Item 5. The method of any one of items 1 to 3, wherein the effective dose is about 0.01 nM to about 100 nM.
Item 6. The method of any one of items 1 to 4, wherein the cells are at the 2-cell stage to blastocyst stage.
Item 7. The method of any one of items 1 to 5, wherein the inhibitor increases the duration of mitosis, reduces the number of micronucleis per cell, reduces the incidence misaligned chromosomes at anaphase onset or a combination thereof.
Item 8. The method of any one of items 1 to 7, wherein the inhibitor does not influence blastocyst cell number as compared to cells of the early embryo not contacting with the inhibitor.
Item 9. The method of any one of items 1 to 8, wherein the cells are mammalian cells.
Item 10. The method of item 9, wherein the cells are human cells.
Item 11. Cells of the early embryo obtained by the method defined in any one of items 1 to 10.
Item 12. Composition comprising the cells of the early embryo defined in item 11.
Item 13. A kit for reducing chromosome segregation errors in cultured cells of the early embryo comprising (a) an inhibitor of the anaphase promoting complex (APC/C) that directly binds to the APC/C; and (b) (i) instructions to use the inhibitor for reducing chromosome segregation errors in cultured cells of the early embryo; (ii) cells of the early embryo; and (iii) a combination of (i) and (ii).
The present disclosure provides a method for reducing chromosome segregation errors in cultured cells of the early embryo by contacting the cells with an effective dose of an inhibitor of the anaphase promoting complex (APC/C), wherein the inhibitor binds directly to the APC/C.
The present invention therefore relates to a method that can result in an embryo with reduced aneuploidy (as compared to when no inhibitor of the present invention is used) and improve embryo development and survival. This method can be used to improve the success of assisted reproduction, in vitro fertilization and in the generation of transgenic animals.
The present invention encompasses APC/C inhibitors which exercise their inhibiting activity by directly binding to APC/C. In a specific embodiment, by such binding, the inhibitor prevents the APC/C activators such as Cdc20 and Cdh1 to interact with and/or activate APC/C. Without being so limited, such inhibitors include proTAME, and TAME.
As used herein, the term “reduces” or “reducing” (in the context of e.g., reducing chromosome segregation errors such as but not limited to reducing the number of micronucleis per cell; reducing the incidence misaligned chromosomes at anaphase onset; reducing kinetochore-microtubule misattachment; reducing chromosome non-disjunction; reducing chromosome breakage; and/or reducing chromosome bridges) refers to a reduction of at least 10% as compared to a control (e.g., control embryotic cells not treated with an inhibitor of the present disclosure), in an embodiment of at least 20% lower, in a further embodiment of at least 30% lower, in a further embodiment of at least 40% lower, in a further embodiment of at least 50% lower, in a further embodiment of at least 60% lower, in a further embodiment of at least 70% lower, in a further embodiment of at least 80% lower, in a further embodiment of at least 90% lower, in a further embodiment of 100% (complete inhibition).
Similarly, as used herein, the term “increases” or “increasing” (in the context of e.g., increasing the duration of mitosis) refers to an increase of at least 10% as compared to a control (e.g., control embryotic cells not treated with an inhibitor of the present disclosure), in an embodiment of at least 20% higher, in a further embodiment of at least 30% higher, in a further embodiment of at least 40% higher, in a further embodiment of at least 50% higher, in a further embodiment of at least 60% higher, in a further embodiment of at least 70% higher, in a further embodiment of at least 80% higher, in a further embodiment of at least 90% higher, in a further embodiment of 100% higher, in a further embodiment of 200% higher, etc.
As used herein, the term “cultured” in the context of “cultured cells of the early embryo” refers to cells of the early embryo grown in conditions suitable to maintain their function and viability. Without being so limited, such culture conditions include ibidi micro-insert wells mounted on a glass-bottom dish with distilled water providing humidified CO2 gas supply and previously described in extensive detail (Vázquez-Diez et al., 2018) or any standard mammalian embryo culture system. For example, an artificial culture medium or in an autologous endometrial coculture (on top of a layer of cells from the woran's own uterine lining) can be used. With artificial culture medium, there can either be the same culture medium throughout the period, or a sequential system can be used, in which the embryo is sequentially placed in different media.
For example, when culturing to the blastocyst stage, one medium may be used for culture to day 3, and a second medium is used for culture thereafter. Single or sequential medium are equally effective for the culture of human embryos to the blastocyst stage. Artificial embryo culture media basically contain glucose, pyruvate, and energy-providing components, and optionally amino acids, nucleotides, vitamins, and cholesterol. Methods to permit dynamic embryo culture with fluid flow and embryo movement are also available. (see also Mantikou et al., 2013).
As used herein the term “cells of the early embryo” refers to the one-cell stage embryo (i.e. fertilized oocyte) up until the blastocyst stage of the embryo. In a specific embodiment, it refers to the two-cells stage up until the blastocyst stage (including all stages in between including the four-cell stage, eight-cell stage, morula stage (e.g., sixteen to thirty-two cell stage) and blastocyst stage (e.g., 64-128 cell stage).
As used herein the term effective dose refers to an amount that effectively reduces chromosome segregation errors without inducing significant toxicity. In a specific embodiment, it refers to a nanomolar dose (i.e. about 0.001 nM to about 900 nM). In another specific embodiment, it refers to a dose of about 0.01 nM to about 500 nM. In another specific embodiment, it refers to a dose of about 0.01 nM to about 100 nM.
The inhibitor may be applied to the cells of the early embryo with at least one carrier and/or other agents able to improve cells of the early embryo function and/or survival. Carriers for use in such context optimally can dissolve the inhibitor (e.g. proTAME). Without being so limited such carriers include DMSO. Without being so limited, such agents include APCin.
The present disclosure also encompasses cells of the early embryo produced using the method of the present disclosure. Such cells advantageously contain less aneuploidy than cells generated in vitro without using an inhibitor of the instant disclosure.
Kits
The present disclosure also relates to a kit for reducing chromosome segregation errors in cultured cells of the early embryo comprising an inhibitor of the anaphase promoting complex (APC/C), wherein the inhibitor binds directly to the APC/C in accordance with the present disclosure (e.g., proTAME); and (i) cultured cells of the early embryo; and/or (ii) instructions to use the kit to reduce chromosome segregation errors in cultured cells of the early embryo. Such kits may further comprise at least one other active agent able to prevent or treat an osteocalcin associated disease or condition, or symptom thereof. In addition, a compartmentalized kit in accordance with the present disclosure includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.
No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.
Herein, the term “about” has its ordinary meaning. In embodiments, it may mean plus or minus 10% of the numerical value qualified.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
The present disclosure is illustrated in further details by the following non-limiting examples.
Embryo collection, culture and chemical treatments. All experiments were approved by the Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM) Comité Institutionnel de Protection des Animaux (CIPA). Two-cell embryos were obtained from superovulated 2-3-month-old CD-1 female mice mated with CD-1 males. Embryos were collected in M2 media and cultured in KSOM media drops covered in mineral oil at 37° C. in 5% CO2. For chemical treatments, media was supplemented with the following compounds: AZ3146 (20 μM), GSK923295 (various concentrations), nocodazole (various concentrations), Cytochalasin B (5 μg/ml), MG132 (25 μM), proTAME (various concentrations). All drug stocks are dissolved in DMSO. For experiments involving proTAME, embryos were cultured without oil in 500 μL of media in 4-well plates.
Cytoplasmic Removal and Microinjection.
All micromanipulations were performed on a Leica™ DM14000 inverted microscope fitted with Narashige micromanipulators. Cytoplasmic aspiration and removal were achieved with hydraulic-controlled glass pipettes with paraffin liquid hydraulic control mounted on a piezo-electric drill, to perforate the zona pellucida, in two-cell embryos treated with 10 μM nocodazole, an anti-mitotic agent, and 5 μg/mL Cytochalasin B, known to block the formation of microfilaments. Embryos where thoroughly washed in over a dozen drops of M2 media prior to transfer and incubation in equilibrated KSOM medium for at least 2 hours prior to imaging to allow them to recover from the cytoplasmic removal procedure. Microinjections were performed as described previously (Nakagawa and FitzHarris, 2016; Vázquez-Diez and FitzHarris, 2018; Vázquez-Diez et al., 2016). cRNA was synthesized using mMessenger Machine kit (Ambion) as previously (Nakagawa et al., 2017; Vázquez-Diez et al., 2018), from the following plasmids; pRN4-H2B:RFP, psC2-GAP43:GFP, MajSatTALE:mClover (pTALYM3B15)(Miyanari et al., 2013).
Live Cell Imaging and Immunofluorescence.
All imaging was performed on a Leica™ SP8 confocal microscope with HyD detectors. For live imaging embryos were placed in a heated top-stage incubator with CO2 supply and imaged with a 20×0.75 NA objective as previously described (Vázquez-Diez et al., 2016). All imaging was performed in ˜2 μL drops under mineral oil, for experiments involving proTAME, embryos were cultured in Ibidi micro-insert wells mounted on a glass-bottom dish with distilled water and the inventors' setup was modified to provide humidified CO2 gas supply, previously described in extensive detail (Vázquez-Diez et al., 2018).
Z-stack images of embryos (˜50 μm) with step size 2 μm were obtained at a time interval of 30 sec for experiments concerning H2BRFP only, 2 min for H2B:RFP-GAP43:GFP, and 3 min H2B:RFP-Mad1:EGFP experiments. For live-cell experiments at two-cells, eight cell, morula and blastocyst, imaging was started at ˜48 h, 72 h, 84 h and ˜96 h post-hCG administration and mating, respectively. For immunofluorescence embryos were fixed in 2% PFA in PBS (20 min) and permeabilised in 0.25% Triton™ X-100 in PBS (10 min), for Mad2 immunofluorescence embryos were fixed for 15 min in PHEM buffer containing 2% PFA and 0.05% Triton™ X-100, and permeabilised in 0.05% Triton™ X in PHEM buffer for 15 min. Blocking was performed in 3% bovine serum albumin in PBS, for 1 hr at 37° C. or overnight at 4° C. Primary antibodies used were rabbit anti-Mad2 (1:300), mouse anti-alpha-Tubulin (1:1000), anti-rabbit and anti-mouse AlexaFluor secondaries were used at 1:1000 dilution. Alexa-555-Phalloidin (1:300) and Hoechst (1:1000) were used to visualize F-actin and DNA, respectively. Briefly, immunofluorescence confocal imaging was performed on a Leica™ SP8 microscope using a 63×1.4 NA oil objective lens, using a 1.5 μm optical section and acquiring Z-stacks with a step size of 1.5 μm, described in more detail in (Vázquez-Diez et al., 2018). In fixed-cell experiments involving GSK923295 and nocodazole embryos were exposed from the 8-cell stage, ˜72 h post-hCG and mating for ˜16 h and immediately fixed. Blastocyst immunofluorescence analysis was performed at late blastocyst stage, ˜120 h post-hCG.
Image Analysis and Statistics.
All image analysis was performed using Fiji software. Time-lapse Z-stacks were examined to determine mitosis duration and identify chromosome alignment and segregation errors. Spindle length measurements were performed measuring the distance between spindle poles, using Pythagoras' theorem where spindle poles were located on different Z-slices. Mad2 Immunofluorescence was quantified by subtracting nuclear envelope and background maximum grey values. To calculate cell volume for each cell at metaphase, areas delineated by the cell membrane (GAP43:GFP signal) were manually traced and measured at each Z-slice, the sum of the areas was multiplied by the step size (2 μm). All data analysis was performed using GraphPad™ Prism, statistical test used were accordingly noted in figures, α=0.05.
Mouse embryos progress from a fertilised 1-cell embryo to a 16-32 cell stage morula and 64-128 cell blastocyst in 4-5 days, blastomeres approximately halving in size at each division. The inventors first examined chromosome segregation in live H2B:RFP-expressing embryos. Classically in somatic cells, a single misaligned chromosome is sufficient to sustain robust SAC signalling and prevent anaphase (Rieder et al., 1995). In morula-stage embryos, chromosomes were fully aligned at the time of anaphase onset in 76% of divisions, anaphase occurring without any obvious defect. However, a significant subset of divisions progressed into anaphase despite the presence of mildly (18%) or severely (6%) misaligned chromosomes (
The inventors next tested the ability of the SAC to respond to spindle defects. Treating embryos with the spindle poison nocodazole caused a pronounced mitotic arrest (mitotic index 40% and 95% in 100 nM and 1 μM nocodazole, respectively) (
The inventors wondered whether the kinetochores of misaligned chromosomes mount a SAC signal. Fixed-cell analysis showed that Mad2 immunolocalised on kinetochores of misaligned, but not aligned chromosomes in prometaphase (
Recent work in mouse oocytes and C. elegans embryos has revived the longstanding notion that SAC efficiency could be affected by cell size (Chen et al., 2016; Hara et al., 1980; Minshull et al., 1994; Zhang et al., 2015). Specifically, it is thought that diffusible MCC may fail to sufficiently inhibit APC/C throughout the cytoplasm in very large cells (Kyogoku et al., 2017; Lane et al., 2017; Galli et al., 2016). Preimplantation mitotic divisions are unaccompanied by cell growth, causing cells to decrease in size ˜40-fold from ˜200 pL 1-cell stage, similar to the oocyte, to ˜5 pL at blastocyst stage (Courtois et al., 2012; Tsichlaki et al., 2016). The inventors tested whether failure of SAC to arrest mitosis may relate to the cytoplasmic volume of early preimplantation blastomeres, and that SAC efficiency might strengthen as blastomeres decrease in size during development. They tested this hypothesis with two series of experiments.
Firstly, the inventors examined SAC strength throughout preimplantation development. They performed live imaging of H2B:RFP-expressing embryos in the presence and absence of a mild spindle challenge (10 nM nocodazole) and used the increase in mitotic duration as a quantitative measure of SAC strength (Lane et al., 2017; Galli et al., 2016). Strikingly, nocodazole prolonged mitosis appreciably at the two-cell stage and in blastocysts (˜32-64 cell stage,
Secondly, the inventors used embryo micromanipulation to directly alter cell size (
The inventors tested whether low concentrations of proTAME would improve chromosome segregation outcomes in normally-cultured embryos.
ProTAME is a is a cell permeable prodrug that is converted to TAME (Tosyl-L-Arginine Methyl Ester) by intracellular esterases. TAME structurally mimics the IR-tail of the co-activators and therefore binds to APC/C, blocking the interaction of Cdc20 or Cdh1 with the APC/C.
Embryos were cultured in various concentrations of proTAME from the 2-cell stage onwards. Micromolar concentrations (1-10 μM) of proTAME prevented embryo development to blastocyst (
The scope of the claims should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.
This application claims benefit, under 35 U.S.C. § 119(e), of U.S. provisional application Ser. No. 62/729,090, filed on Sep. 10, 2019. The above-document is incorporated herein in its entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62729090 | Sep 2018 | US |
