METHODS AND COMPOSITIONS FOR ENHANCING DEVELOPMENTAL POTENTIAL OF OOCYTES AND PREIMPLANTATION EMBRYOS

RELATED APPLICATION

This application claims the priority benefit to US Provisional Patent Application No. 61/505,780 filed July 8, 2011.

FIELD OF THE INVENTION

The invention relates to compositions and methods for enhancing the developmental potential of oocytes and preimplantation embryos.

BACKGROUND OF THE INVENTION

According to the Centre for Disease Control, one in every eight North American couples seeks medical treatment for infertility. Embryo quality remains a strong determining factor for predicting the outcome of assisted reproductive technology (ART) [1]. Molecular defects responsible for failed preimplantation development are frequently attributed to poor oocyte quality of unknown etiology. Mathematical modeling of death rates in human preimplantation embryos has suggested that the factors predisposing an embryo to arrest are determined at or even before the zygote stage [2,3]. The ability of the conceptus to pass through the transition from maternal to zygotic control in vitro has been proposed to be a function of the cytoplasmic components of the oocyte with minimal impact of the newly formed zygotic genome [4]. Thus, oocytes must possess cytoplasmic components which accumulate during oogenesis and support development through the blocking stage [5], and these components are lacking or non-functional in those embryos that arrest.

Ooplasm transfer experiments demonstrated that an unidentified ooplasmic factor(s) can prevent embryo arrest [6]. This pioneering work led to controversial clinical attempts to rescue human embryos with poor developmental potential by transferring 'healthy' donor ooplasm into recipient oocytes prone to abnormal development [7]. Such ooplasm transfers in humans were performed for patients with increased maternal age, repeated embryonic developmental failure or poor ovarian reserve, and have resulted in the birth of at least thirty children worldwide [8]. Unfortunately, transfer of ooplasm results in offspring carrying mitochondria from both the donor and recipient, thus creating mitochondrial heteroplasmy [9]. While benefits of this mitochondrial enrichment are clearly evident during thei early developmental stages, mitochondrial heteroplasmy can have late physiological consequences [10]. Therefore, it is desirable to identify molecules responsible for suboptimal oocyte quality and devise molecular strategies for treatment options. SUMMARY

The present invention relates to a method for enhancing developmental potential of oocytes and preimplantation embryos comprising introducing or administering an effective amount of a Bcl-XL modified protein. A method of the invention may additionally comprise fertilizing the oocytes to obtain a zygote with increased levels of a Bcl-XL modified protein.

The invention further relates to a method for decreasing, inhibiting or reversing mitochondrial defects, and/or regulating reactive oxygen species (ROS) production in oocytes or preimplantation embryos comprising administering to the oocytes or preimplantation embryos a Bcl-XL modified protein.

The invention also relates to a method for enhancing developmental potential of preimplantation embryos comprising introducing a Bcl-XL modified protein into the preimplantation embryo. In an embodiment, the preimplantation embryo is a zygote and a Bcl- XL modified protein is introduced in the zygote. In a particular embodiment, the proteins are introduced into the pronucleus or cytoplasm.

The invention further relates to an oocyte or a preimplantation embryo comprising a

Bcl-XL modified protein in the oocyte or preimplantation embryo. In an aspect, an oocyte or a preimplantation embryo obtained from a method of the invention is provided wherein the oocyte or preimplantation embryo comprises increased levels of a Bcl-XL modified protein.

In a further aspect the invention relates to a composition comprising a Bcl-XL modified protein in a form or effective amount for enhancing developmental potential of oocytes or preimplantation embryos. In an embodiment a composition of the invention comprises a pharmaceutically acceptable carrier, excipient or vehicle. In a particular embodiment, a composition of the invention comprises a Bcl-XL modified protein with a terminal half-life of less than about 24, 20, 15, 10, 9, 8, 7, 6, or 5 hours.

The invention relates to the use of a Bcl-XL modified protein in the manufacture of a medicament for use in improving embryo development after in vitro fertilization or embryo transfer in a female mammal.

The invention also relates to the use of a Bcl-XL modified protein in the manufacture of a medicament for use in reducing, inhibiting or decreasing mitochondrial defects, and/or regulating ROS production in oocytes or preimplantation embryos.

In another aspect, the invention provides a method for fertilizing oocytes comprising removing oocytes from a follicle of an ovary, introducing a Bcl-XL modified protein in the oocytes, and fertilizing the resulting oocytes with spermatozoa. The introduction of the protein and the spermatozoa can be carried out simultaneously, sequentially, or separately. In an embodiment, the protein and spermatozoa are simultaneously injected. In a still further aspect the invention provides a method for storing and then enhancing the developmental potential of oocytes comprising cryopreserving immature oocytes, thawing the cryopreserved oocytes, and introducing a Bcl-XL modified protein into the oocytes.

The methods and compositions of the invention can improve the quality of the oocytes that are being fertilized and the quality of preimplantation embryos to increase the rate of success in embryo development and ongoing pregnancy.

In an aspect, the invention provides a method for improving embryo development after in vitro fertilization or embryo transfer in a female mammal comprising implanting into the female mammal an embryo derived from an ooctye or preimplantation embryo (e.g., zygote) comprising a Bcl-XL modified protein. The invention provides methods of improving the success of in vitro fertilization, gamete intrafallopian transfer, or zygote intrafallopian transfer comprising introducing a a Bcl-XL modified protein in oocytes or preimplantation embryos employed therein.

In an aspect, the invention provides a method for improving the success of in vitro fertilization in a female subject comprising:

(a) removing oocytes from the subject;

(b) introducing a Bcl-XL modified protein in the oocytes;

(c) fertilizing the oocytes with spermatozoa; and

(d) transferring fertilized oocytes from step (c) into the uterus of the subject.

In another aspect, the invention provides a method for improving the success of zygote intrafallopian transfer in a female subject comprising:

(a) removing oocytes from the subject;

(b) introducing a Bcl-XL modified protein in the oocytes;

(c) fertilizing the oocytes with spermatozoa; and

(d) transferring fertilized oocytes from step (c) into a fallopian tube of the subject.

In a further aspect, the invention provides a method for improving the success of gamete intrafallopian transfer in a female subject comprising:

(a) removing oocytes from the subject;

(b) introducing a Bcl-XL modified protein in the oocytes;

(c) combining the oocytes with spermatozoa; and

(d) immediately introducing the oocytes and spermatozoa from step (c) into a fallopian tube of the subject.

An oocyte may be a recipient oocyte in a nuclear transfer method. Thus, the invention relates to a method for enhancing developmental potential of recipient oocytes in a nuclear transfer method comprising introducing a Bcl-XL modified protein in the oocytes. The invention also contemplates recipient oocytes comprising an exogenous (e.g., isolated or recombinant) Bcl-XL modified protein, and preimplantation embryos, blastocyts, embryos, and non-human animals formed from a nuclear transfer method of the invention. In conventional nuclear transfer methods, the donor nucleus is placed in an enucleated oocyte obtained from a different individual. The invention by introducing a Bcl-XL modified protein into recipient oocytes enhances the developmental potential of the recipient oocytes. This is expected to increase the live birth rate in nuclear transfer methods.

In an embodiment, the invention provides a method of cloning a non-human mammalian embryo by nuclear transfer comprising:

(a) introducing a donor cell nucleus derived from a donor cell of a non-human mammal and an exogenous Bcl-XL modified protein into an enucleated recipient oocyte of the same species as the donor cell to form a nuclear transfer unit; and

(b) culturing the nuclear transfer unit to form an embryo.

The method may further comprise permitting the embryo to develop into a cloned mammal.

The invention also provides a method of cloning a non-human mammal by nuclear transfer comprising:

(a) introducing a donor cell nucleus derived from a donor cell of a non-human mammal and an exogenous Bcl-XL modified protein into a non-human mammalian enucleated recipient oocyte of the same species as the donor cell to form a nuclear transfer unit;

(b) culturing the nuclear transfer unit to form an embryo;

(c) implanting the embryo into the uterus of a surrogate mother of said species; and

(d) permitting the embryo to develop into the cloned mammal.

In yet another embodiment, a method of cloning a non-human mammalian fetus by nuclear transfer is provided comprising the following steps:

(a) introducing a donor cell nucleus from a donor cell of a non-human mammal and a Bcl-XL modified protein into an enucleated recipient oocyte of the same species as the donor cell to form a nuclear transfer unit;

(b) culturing the nuclear transfer unit until greater than the 2-cell developmental stage; and

(c) transferring the cultured nuclear transfer unit to a host non-human mammal of the same species such that the nuclear transfer unit develops into a fetus.

The method may also comprise developing the fetus into an offspring.

In embodiments of methods of the invention, the Bcl-XL modified protein is from the same species as the donor cell, preferably from the same species and cell type as the donor cell, more preferably from the non-human mammal from which the donor cell nucleus is derived.

In a further aspect the invention provides a recipient oocyte comprising a perivitelline space and a donor cell nucleus and a Bcl-XL modified protein, preferably from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, most preferably from the same individual from which the donor cell nucleus is derived, deposited in the perivitelline space.

The invention also includes kits and articles-of-manufacture for conducting the methods of the invention.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which:

Figure 1 shows the automated robotic microinjection of mouse zygotes. (A) The robotic system employs a glass micro device to immobilize a large number of mouse zygotes into a regular pattern via fine vacuum and micrometer-sized through holes underneath cells. Based on precise position control and microscopy vision feedback, a three-degrees-of- freedom (3-DOF) micromanipulator, a motorized X-Y stage, and an in-house developed rotational stage are automatically controlled by a host computer to control an injection micropipette and position/orient the zygotes, respectively. An inverted microscope mounted with a digital camera is used to provide visual feedback and therefore, guide motions ^, of the micropipette and zygotes to achieve automated microinjection. (B) A mouse zygote with the tip of a micropipette at the cytoplasmic center after material deposition. A droplet of mineral oil, which is easy to observe under a non-fluorescent microscope, was injected for visualization to verify the success of material deposition into the cytoplasmic center. (C) Schematic of the glass micro device for zygote immobilization. Vacuum is applied to each zygote for immobilization via micrometer-sized through holes. (D) Mouse zygotes robotically injected with PBS buffer are developed into blastocysts. (E) Calibration data of deposition volumes as a function of deposition time and pressure. Micropipettes with an opening of 1.2 μτη were used, as shown in the inlet. (F) Automated robotic microinjection induced significantly lower lysis rates than manual injection (n=400 for robotic protein and buffer injection; n=229 for manual injection). Bars indicate mean ± s.e.m. Kruskal Wallis test followed by Dunn's post test was used for statistical analysis. Figure 2 shows the impact of culture medium on developmental competence and BCL-X protein expression of mouse embryos. (A) HTF culture medium induces 2-cell arrest in a subset of embryos and compromises preimplantation embryo development when compared to KSOM medium (n=273 embryos/medium). Rates of blastocyst formation at day 4.5 (-96 hours in culture) as well as total cell number (TCN) are dramatically reduced, while cell death index (CDI) is elevated (n=54 embryos for KSOM and n=36 embryos for HTF). Bars indicate mean ± s.e.m. Mann Whitney U-test was used for pairwise comparison. (B) Poor quality of embryos is also reflected by nuclear staining (DAPI), showing smaller blastocysts with multiple apoptotic cells (arrows). (C) Expression of BCL-X protein is decreased in 2-cell embryos cultured for 24 hours in HTF medium. Significant reduction in fluorescent intensity (RFU), generated after immunocytochemical analysis for BCL-X was detected in embryos cultured in HTF (n=10), when compared to KSOM cultured embryos (n=9). Control embryos, exposed to no-specific IgG (n=6), exhibited only very small amount of fluorescence, which was subtracted from the intensity generated by BCL-X antibody. Bars indicate mean ± s.e.m. Student's t-test was used for calculating significance of difference between KSOM and HTF groups.

Figure 3 shows the impact of recBCL-XL (ΔΤΜ) microinjection on early embryo development. (A) Ability of mouse zygotes to progress through the development and form blastocysts in suboptimal HTF medium were significantly increased upon microinjection of recBCL-XL (ΔΤΜ) protein (n=424) when compared to buffer injected embryos (n=414). In addition, total cell number (TCN) per embryo was significantly increased and cell death index (CDI) was decreased (n=71 for buffer injection; n=1 10 for protein injection). Nuclear counterstaining (DAPI) images of blastocysts at day 4.5 reflect differences in embryo quality. Mann-Whitney U-test was used for pairwise comparison. (B) Reactive oxygen species (ROS) accumulation, determined by fluorescent measurement of DCHFDA probe fluoresce at 2-cell stage was determined 24 hours after microinjection of either buffer (n=15) or recBCL-XL (ΔΤΜ) protein (n=15) and relative fluorescence units (RFU) were used to express fluorescent signal. Injection of recBCL-XL (ΔΤΜ) significantly reduced the accumulation of ROS (student's t-test). (C) Immunocytochemical analysis of total p66SHC or phosphorylated p66SHC on Ser36 was decreased in embryos injected with recBcl-xL (ΔΤΜ) (n=15/antibody), when compared to buffer injected embryos (n=15/antibody). In addition, Ser10 p66SHC (green) localized to the mitochondria (Mitotracker red), with preferential clustering in subcortical and peri-nuclear regions (yellow overlap; arrows), but this was greatly reduced in recBCL-XL (ΔΤΜ) microinjected embryos. Bars indicate mean ± s.e.m.

Figure 4 shows the impact of recBCL-XL (ΔΤΜ) microinjection of embryo metabolism and mitochondrial distribution at 2-cell stage. (A) Mitochondrial distribution (Mitotracker Red) at 2-cell stage was evaluated by computerized image analysis approach (extracted features) and compared among cultured conditions. RecBCL-XL (ΔΤΜ) protein maintained diffuse mitochondrial pattern (n=30 embryos), while buffer (n=30; similar to HTF culture alone), caused preferential clustering of these organelles to subcortical and perinuclear regions (arrows) of 2-cell embryos maintained in culture for 24 hours. (B) Microinjection of recBCL-XL (ΔΤΜ) protein stabilized redox state of 2-cell stage embryos reduced (NAD(P)H and oxidized FAD autofluorescence signal expressed in RFU; n=15 embryos per condition) and improved Krebs cycle outcome (Citrate/ATP ratio n=15 embryos per condition). Student's t-test was used for statistical analysis. Bars indicate mean ± s.e.m.

Figure 5 shows the expression of BCL-X in human oocytes. (A) Distribution of human oocytes obtained form 43 patients based on their BCL-X expression in either germinal vesicle stage (GV - left) or meiosis I stage (Ml - right). Arrows point to groups of oocytes with insufficient endowment of BCL-X transcript. (B) Visualization of protein interaction network that connects BCL-X (BC2L1 ) with other targets known to be deregulated in arrested human embryos. Node shape, represented by triangles, indicates trends of expression. Shape of triangles pointing up corresponds to genes up-regulated and triangles pointing down correspond to genes down-regulated in arrested human embryos; circles represent direct interacting partners that link BCL-X (BCL2L1 ) to up- and down-regulated targets. Red highlight on nodes represents the set of cross-linked proteins. Node color is based on gene ontology as per legend. To reduce network complexity, all other nodes and edges are made partially transparent.

Figure 6 shows zygote immobilization using a glass-based cell holding device. (A) A completed glass cell holding device. (B) A zoomed-in picture of the through holes. (C) A 5*5 array of immobilized mouse zygotes using the cell holding device.

Figure 7 shows the overall flow of microrobotic mouse embryo injection. (A) Contact between micropipette tip and cell holding cavity is detected using a vision-based algorithm [15]. (B) The micropipette tip is elevated to a home position H, and the first embryo is brought into the field of view, recognized and centered. If the polar body faces the penetration site, the embryo is properly rotated through automatic orientation control. (C) Micropipette is moved to a switch point, S. (D) The micropipette penetrates the embryo and deposits materials to the target destination. (E) The micropipette is retracted out of the embryo. (F) Micropipette is moved to the home position. Simultaneously, the next embryo is brought into the field of view. This injection process is repeated until all the embryos in the batch are injected.

Figure 8 shows mouse zygote orientation. (A) Side view and (B) top view of the zygote and injection micropipette before orientation. When the polar body appears in the space of quadrant II, there are risks of either direct polar body penetration or large stress induced polar body damage. The desired target orientation is either 12 o'clock or 6 o'clock. (C) Top view of the embryo after orientation. Polar body is now at the 12 o'clock position. Figure 9 shows the impact of injection modes of delivery (manual vs. automated injection) on rates of blastocyst formation and embryo quality. Both modes of delivery significantly improved developmental potential of embryos injected with recBCL-XL (ΔΤΜ) protein (manual injection: n= 107 for buffer and n=122 for protein; automated injection: n=307 for buffer and n=302 for protein). No significant difference (p=0.359 for buffer injection; p=0.762 for protein injection) was found between rates of blastocyst formation if protein was delivered into either cytoplasm or pronucleus. Microinjection of recBCL-XL (ΔΤΜ) protein also significantly enhanced the embryo quality (manual injection: n=39 for buffer and n=65 for protein; automated injection: n=32 for buffer and n=44 for protein). Bars indicate mean ± s.e.m. Student's t-test was used for pairwise comparison.

Figure 10 shows the impact of culture medium on reactive oxygen species (ROS) levels. Assessment of the relative amounts of ROS measured by DCHFDA probe in 2-cell embryos cultured for 24 hours in KSOM (n=15), HTF (n=17), HTF with 15 ng/μΙ of BH4 peptide (n=15). Bars indicate mean ± s.e.m. Kruskal Wallis test followed by Dunn's post test was used for statistical analysis.

Figure 11 shows computationally quantitated mitochondrial distributions at 2-cell stage in (A) un-injected and (B) injected embryos. Using Euler number computation, mitochondrial distribution in un-injected embryos is significantly altered by culture medium (n=19 for HTF; n=20 for KSOM), which can be corrected by microinjection of recBCL-XL (ΔΤΜ) protein (n=22 for each condition). Bars indicate mean ± s.e.m. Mann-Whitney U-test was used for pairwise comparison.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See for example, Sambrook, Fritsch, & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); and B. Perbal, A Practical Guide to Molecular Cloning (1984).

Unless defined otherwise, 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 invention belongs.

Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." The term "about" means plus or minus 0.1 to 50%, 5-50%, or 10-40%, preferably 10- 20%, more preferably 10% or 15%, of the number to which reference is being made. Further, it is to be understood that "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

A "Bcl-XL modified protein" refers to a modified native sequence Bcl-XL polypeptide or isolated or substantially pure Bcl-XL polypeptide, oligopeptide, peptide or isoform thereof, or pharmaceutically acceptable salts thereof. In aspects of the invention, a Bcl-XL modified protein is an analogue of a Bcl-XL polypeptide. In other aspects a Bcl-XL modified protein is a derivative or variant of a Bcl-XL polypeptide or analogue thereof. In aspects of the invention, a Bcl-XL modified protein is a mimetic or part of a chimeric polypeptide. A Bcl-XL modified protein includes amino acid sequences from humans and from any source whether natural, synthetic, semi-synthetic, or recombinant. The structure of the Bcl-XL polypeptide comprises eight a-helices connected by loops of varying length with the two central helices (a5 and a6) forming the core of the polypeptide. A unique element of a Bcl-XL polypeptide is the presence of a long loop between the a1 and a2 helices (Petros et al, 2004, Biochimica et Biophysica Acta 1644:83-94).

A "native sequence" polypeptide comprises a polypeptide having the same amino acid sequence of a polypeptide derived from nature. Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. Amino acid sequences for Bcl-XL polypeptides (and nucleic acids encoding the polypeptides) are available in public databases such as the NCBI database (see for example, UniProt Q07817, NCBI NP_001182, and SEQ ID NO. 1 ).

SEQ ID NO. 1 :

MSQSNRELVV DFLSYKLSQK GYSWSQFSDV EENRTEAPEG TESEMETPSA INGNPSWHLA

DSPAVNGATG HSSSLDAREV IPMAAVKQAL REAGDEFELR YRRAFSDLTS QLHITPGTAY QSFEQWNEL FRDGVNWGRI VAFFSFGGAL CVESVDKEMQ VLVSRIAAWM ATYLNDHLEP

WIQENGGWDT FVELYGNNAA AESRKGQERF NRWFLTG TV AGWLLGSLF SRK

The terms "substantially pure" or "isolated," refer to a protein that is separated as desired from RNA, DNA, proteins or other contaminants with which they are naturally associated. For example, when referring to proteins and polypeptides, a protein or polypeptide is considered substantially pure when that protein makes up greater than about 50% of the total protein content of the composition containing that protein, and typically, greater than about 60% of the total protein content. More typically, a substantially pure or isolated protein or polypeptide will make up at least about 75%, at least about 80%, at least about 85%, more preferably, at least about 90%, at least about 95% of the total protein. Preferably, the protein will make up greater than about 90%, and more preferably, greater than about 95% of the total protein in the composition. An "isoform" refers to a polypeptide that contains the same number and kinds of amino acids as a native sequence polypeptide but the isoform has a different molecular structure. Isoforms preferably have the same properties (e.g., biological and/or immunological activity) as a native sequence polypeptide.

An "analogue" includes a polypeptide wherein one or more amino acid residues of a native sequence polypeptide have been substituted by another amino acid residue, one or more amino acid residues of a native polypeptide have been inverted, one or more amino acid residues of the native polypeptide have been deleted, and/or one or more amino acid residues have been added to the native polypeptide. Such an addition, substitution, deletion, and/or inversion may be at either of the N-terminal or C-terminal end or within the native polypeptide, or a combination thereof.

A "derivative" includes a polypeptide in which one or more of the amino acid residues of a polypeptide have been chemically modified. A chemical modification includes adding chemical moieties, creating new bonds, and removing chemical moieties. In particular, a chemical modification can include internal linkers (e.g. spacing or structure-inducing) or appended molecules, such as molecular weight enhancing molecules (e.g., polyethylene glycol, polyamino acid moieties, etc.,), or tissue targeting molecules. A polypeptide may be chemically modified, for example, by alkylation, acylation, glycosylation, pegylation, ester formation, deamidation, or amide formation.

A "variant" refers to a polypeptide having at least about 60%, 65%, 70%, 75%, 80%,

85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity, particularly at least about 70-80%, more particularly at least about 85%, still more particularly at least about 90%, most particularly at least about 95% amino acid sequence identity with a native-sequence polypeptide. Such variants include for instance polypeptides wherein one or more amino acid residues are added to, or deleted from the N- or C-terminus of the full-length or mature sequences of the polypeptide, including variants from other species. A naturally occurring allelic variant may contain conservative amino acid substitutions from the native polypeptide sequence or it may contain a substitution of an amino acid from a corresponding position in a polypeptide homolog, for example, a murine polypeptide.

"Identity" as known in the art and used herein, is a relationship between two or more amino acid sequences as determined by comparing the sequences. It also refers to the degree of sequence relatedness between amino acid sequences as determined by the match between strings of such sequences. Identity and similarity are well known terms to skilled artisans and they can be calculated by conventional methods (for example, see Computational Molecular Biology, Lesk, A.M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W. ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G. eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G. Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J. eds. M. Stockton Press, New York, 1991 , Carillo, H. and Lipman, D., SIAM J. Applied Math. 48:1073, 1988). Methods which are designed to give the largest match between the sequences are generally preferred. Methods to determine identity and similarity are codified in publicly available computer programs including the GCG program package (Devereux J. et al., Nucleic Acids Research 12(1 ): 387, 1984); BLASTP, BLASTN, and FASTA (Atschul, S.F. et al. J. Molec. Biol. 215: 403-410, 1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al. NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al. J. Mol. Biol. 215: 403-410, 1990).

Mutations may be introduced into a polypeptide by standard methods, such as site- directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions can be made at one or more predicted non-essential amino acid residues. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue with a similar side chain. Amino acids with similar side chains are known in the art and include amino acids with basic side chains (e.g. Lys, Arg, His), acidic side chains (e.g. Asp, Glu), uncharged polar side chains (e.g. Gly, Asp, Glu, Ser, Thr, Tyr and Cys), nonpolar side chains (e.g. Ala, Val, Leu, Iso, Pro, Trp), beta-branched side chains (e.g. Thr, Val, Iso), and aromatic side chains (e.g. Tyr, Phe, Trp, His). Mutations can also be introduced randomly along part or all of the native sequence, for example, by saturation mutagenesis. Computer programs, for example DNASTAR, may be used to determine which amino acid residues may be substituted, inserted, or deleted without abolishing biological and/or immunological activity.

In aspects of the invention, the Bcl-XL modified protein is a Bcl-XL polypeptide lacking the carboxy-terminal transmembrane domain region or a portion thereof. In an embodiment, the Bcl-XL modified protein is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 missing the last 21 amino acids (i.e., amino acid residues 213 to 233 of SEQ ID NO. 1 ). In a particular embodiments, the Bcl-XL modified protein is a recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 1 missing the last 21 amino acid residues [also referred to herein as recBCL-XL (ΔΤΜ)].

"Mimetic" refers to a synthetic chemical compound that has substantially thes same structural and/or functional characteristics of a Bcl-XL modified protein. A mimetic can be composed entirely of synthetic, non-natural analogues of amino acids, or, is a chimeric polypeptide of partly natural peptide amino acids and partly non-natural analogues of amino acids. A polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, N.Y.). Mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a motif or peptide. A particular mimetic refers to a molecule, the structure of which is developed based on the structure of a Bcl-XL modified protein or portions thereof, and is able to effect some of the actions of chemically or structurally related molecules.

A "chimeric polypeptide" comprises all or part (preferably biologically active) of a Bcl- XL modified protein operably linked to a heterologous polypeptide (i.e., a polypeptide other than the same Bcl-XL modified protein). Within the chimeric polypeptide, the term "operably linked" is intended to indicate that the Bcl-XL modified protein and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the N-terminus or C-terminus of the Bcl-XL modified protein or analogue thereof. A useful chimeric polypeptide is a GST fusion protein in which a Bcl-XL modified protein is fused to the C-terminus of GST sequences. Another example of a chimeric polypeptide is an immunoglobulin fusion protein in which all or part of a Bcl-XL modified protein is fused to sequences derived from a member of the immunoglobulin protein family. Chimeric polypeptides can be produced by standard recombinant DNA techniques.

A Bcl-XL modified protein can be prepared by a variety of methods known in the art such as solid-phase synthesis, purification of the proteins from natural sources, recombinant technology, or a combination of these methods. See for example, United States Patent Nos. 5,188,666, 5,120,712, 5,523,549, 5,512,549, 5,977,071 , 6,191 ,102, Dugas and Penney 1981 , Merrifield, 1962, Stewart and Young 1969, and the references cited herein. Derivatives can be produced by appropriate derivatization of an appropriate backbone produced, for example, by recombinant DNA technology or peptide synthesis (e.g. Merrifield-type solid phase synthesis) using methods known in the art of peptide synthesis and peptide chemistry. In aspects of the invention a Bcl-XL modified protein is a recombinant protein or a synthesized protein.

The term "oocytes" refers to the gamete from the follicle of a female animal, whether vertebrate or invertebrate. The animal is preferably a mammal, including a human, non- human primate, a bovine, equine, porcine, ovine, caprine, buffalo, guinea pig, hamster, rabbit, mice, rat, dog, cat, or a human. Suitable oocytes for use in the invention include immature oocytes, and mature oocytes from ovaries stimulated by administering to the oocyte donor, in vitro or in vivo, a fertility agent(s) or fertility enhancing agent(s) (e.g. inhibin, inhibin and activin, clomiphene citrate, human menopausal gonadotropins including FSH, or a mixture of FSH and LH, and/or human chorionic gonadotropins). In some embodiments of the invention, the oocytes are aged (e.g. from humans 40 years +, or from animals past their reproductive prime). Methods for isolating oocytes are known in the art.

In the nuclear transfer embodiments of the invention oocytes are used as recipient cells (such cells are referred to herein as "recipient oocytes"). The recipient ooctyes are obtained from mammals, especially non-human mammals, in particular domestic, sports, zoo, and pet animals including but not limited to bovine, ovine, porcine, equine, caprine, buffalo, and guinea pigs, rabbits, mice, hamsters, rats, primates, etc.

"Preimplantation embryo" refers to the very early free-floating embryo of an animal, from the time the oocyte is fertilized (zygote), until the beginning of implantation (in humans, a period of about 6 days). The term also includes embryos resulting from nuclear transfer, in all the development stages through the blastocyst stage. A preimplantation embryo may be from a vertebrate or an invertebrate, preferably a mammal, more preferably a human, a non-human primate, a bovine, equine, porcine, ovine, caprine, buffalo, guinea pig, hamster, rabbit, mice, rat, dog, or cat.

The term "zygote" refers to a fertilized oocyte prior to the first cleavage division.

The expression "enhancing the developmental potential of oocytes" refers to increasing the quality of the oocyte so that it will be more capable of being fertilized and/or enhancing mitochondrial function or activity in the oocyte for subsequent development and reproduction. Increasing the quality of the oocyte, and thus the fertilized oocyte (e.g. zygote), preferably results in enhanced development of the oocyte into an embryo and its ability to be implanted and form a healthy pregnancy. The expression "enhancing the developmental potential of preimplantation embryos" refers to increasing the quality of the preimplantation embryos and/or enhancing mitochondrial function or activity in the preimplantation embryos for subsequent development and reproduction. Increasing the quality of the preimplantation embryos, preferably results in enhanced development of the preimplantation embryos into an embryo and their ability to be implanted and form a healthy pregnancy. Quality can be assessed by the appearance of the developing embryo by visual means and by the IVF or nuclear transfer success rate. Criteria to judge quality of the developing embryo by visual means include, for example, their shape, rate of cell division, fragmentation, appearance of cytoplasm, and other means recognized in the art of IVF and nuclear transfer.

"Spermatozoa" refers to male gametes that can be used to fertilize oocytes.

The term "pharmaceutically acceptable carrier, excipient, or vehicle" refers to a medium which does not interfere with the effectiveness or activity of an active ingredient and which is not toxic to the hosts to which it is administered. A carrier, excipient, or vehicle includes diluents, binders, adhesives, lubricants, disintegrates, bulking agents, wetting or emulsifying agents, pH buffering agents, and miscellaneous materials such as absorbants that may be needed in order to prepare a particular composition. Examples of carriers etc. include but are not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The use of such media and agents for an active substance is well known in the art. Description of Embodiments of the Invention

The present invention generally involves the use of Bcl-XL modified proteins to enhance the developmental potential of animal oocytes and preimplantation embryos, especially mammals, including sports, zoo, pet, and farm animals, in particular dogs, cats, cattle, pigs, horses, goats, buffalo, rodents (e.g. mice, rats, guinea pigs), monkeys, sheep, and humans, especially humans. In the nuclear transfer methods, Bcl-XL modified proteins are used to enhance the developmental potential of recipient oocytes, especially non-human recipient oocytes.

Methods of the invention involve removing the oocytes from follicles in the ovary. This can be accomplished by conventional methods for example, using the natural cycle, during surgical intervention such as oophorohysterectomy, during hyperstimulation protocols in an IVF program, or by necropsy. Oocyte removal and recovery can be suitably performed using transvaginal ultrasonically guided follicular aspiration.

In a method of the invention for enhancing developmental potential of oocytes, a Bcl- XL modified protein is introduced into the oocytes, or the oocytes can be cryopreserved for storage in a gamete or cell bank. If the oocytes are not cryopreserved the oocytes can be treated in accordance with the method of the invention preferably within 48 hours after aspiration. If the oocytes are frozen, they can be thawed when it is desired to use them and treated in accordance with a method of the invention.

A Bcl-XL modified protein may be introduced into the oocytes (or zygotes) by conventional microinjection techniques, electroporation, methods using viral fusion proteins or cationic lipids, and methods devised by a person skilled in the art (see for example, Protein Delivery: Physical Systems, Sanders and Hendren (eds) (Plenum Press, 1997). The proteins may be introduced into the cytoplasm, the pronucleus of an oocyte, or the pronucleus of a zygote (in particular the male pronucleus).

Automated microinjection systems using robotic technologies [11 ,12,13,14,15,16,17,18] may be utilized to introduce a Bcl-XL protein into oocytes/embryos. In an embodiment, a robotic system that leverages motion control, computer vision microscopy, and micro device technology is employed to achieve automated microinjection with high speed, reproducibility, and post-injection survival rate. This system employs microfabricated cell holding devices and vision-position-based control of multiple micropositioning devices to achieve easy sample immobilization, rapid cell orientation, and fast injection of mouse embryos. Technical aspects of the system are described by Liu and Sun, 2009 [19,20].

Bcl-XL modified proteins may be formulated as pharmaceutical compositions which can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions. Suitable pharmaceutically acceptable carriers, excipients and vehicles are described, for example, in Remington's Pharmaceutical Sciences, 19 Edition (Mack Publishing Company, Easton, Pa., USA 1995). On this basis, the compositions include, albeit not exclusively, solutions of the proteins in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. A Bcl-XL modified protein may be formulated in a pharmaceutically acceptable delivery composition that can be used in the form or a solid, a solution, an emulsion, a dispersion, a micelle, a liposome, and the like, in admixture with an organic or inorganic carrier or excipient suitable for administration to oocytes or preimplantation embryos. The Bcl-XL modified protein may be a concentrate including lyophilized compositions which may be diluted prior to use.

A Bcl-XL modified protein may be in the form of a kit, in particular a kit or article-of manufacture including a Bcl-XL modified protein either as a concentrate (including lyophilized compositions), which may be further diluted prior to use or at the concentration of use, where the vials may include one or more dosages.

In an aspect, the invention provides an article-of-manufacture comprising packaging material and a pharmaceutical composition identified for improving embryo development after in vitro fertilization or embryo transfer contained within the packaging material, the pharmaceutical composition including as an active ingredient, a Bcl-XL modified protein, and a pharmaceutically acceptable carrier, excipient, or vehicle.

After introduction, simultaneously with, or prior to the introduction of the Bcl-XL modified protein, the oocytes are fertilized with suitable spermatozoa from the same species. The fertilization can be carried out by known techniques including sperm injection, in particular intracytoplasmic sperm injection (ICSI). In ICSI, sperm is injected directly into an oocyte with a microscopic needle.

In an embodiment, the oocytes are simultaneously injected with a Bcl-XL modified protein and sperm. In another embodiment, oocytes are fertilized with sperm followed by introduction of the protein into the fertilized oocytes (zygotes).

The fertilized oocytes (zygotes) can be cultured or immediately transferred to the subject. Suitable human in vitro fertilization and embryo transfer procedures that can be used include in vitro fertilization (IVF) (Trounson et al. Med J Aust. 1993 Jun 21 ;158(12):853-7, Trouson and Leeton, in Edwards and Purdy, eds., Human Conception in Vitro, New York:Academic Press, 1982, Trounson, in Crosignani and Rubin eds., In Vitro Fertilization and Embryo Transfer, p. 315, New York: Academic Press, 1983); intracytoplasmic sperm injection (ICSI) (Casper et al., Fertil Steril. 1996 May;65(5):972-6); in vitro fertilization and embryo transfer (IVF-ET)(Quigly et al, Fert. Steril., 38: 678, 1982); gamete intrafallopian transfer (GIFT) (Molloy et al, Fertil. Steril. 47: 289, 1987); and pronuclear stage tubal transfer (PROST) (Yovich et al., Fertil. Steril. 45: 851 , 1987). Generally, in IVF methods the fertilized oocytes are introduced into the uterus of the subject while in other methods such as GIFT and ZIFT the fertilized oocytes are transferred to a fallopian tube.

The invention also contemplates improved nuclear transfer methods using Bcl-XL modified proteins. Nuclear transfer methods or nuclear transplantation methods are known in the literature and are described in for example, Campbell et al, Theriogenology, 43:181 (1995); Collas et al, Mol. Report Dev., 38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935- 939 (1994); Sims et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, WO 90/03432, U.S. Pat. Nos. 4,944,384 and 5,057,420.

Methods for isolation of recipient oocytes suitable for nuclear transfer methods are well known in the art. Generally, the recipient oocytes are surgically removed from the ovaries or reproductive tract of a mammal, e.g., a bovine. Once the oocytes are isolated they are rinsed and stored in a preparation medium well known to those skilled in the art, for example buffered salt solutions.

Recipient oocytes must generally be matured in vitro before they may be used as recipient cells for nuclear transfer. This process generally requires collecting immature (prophase I) oocytes from mammalian ovaries, and maturing the oocytes in a maturation medium prior to fertilization or enucleation until the oocyte attains the metaphase II stage. Metaphase II stage oocytes, which have been matured in vivo, may also be used in nuclear transfer techniques.

Enucleation of the recipient oocytes may be carried out by known methods, such as described in U.S. Pat. No.4,994,384. For example, metaphase II oocytes may be placed in HECM, optionally containing cytochalasin B, for immediate enucleation, or they may be placed in a suitable medium, (e.g. an embryo culture medium), and then enucleated later, preferably not more than 24 hours later. Enucleation may be achieved microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm (McGrath and Solter, Science, 220:1300, 1983), or using functional enucleation (see U.S. 5,952,222). The recipient oocytes may be screened to identify those which have been successfully enucleated.

The recipient oocytes may be activated on, or after nuclear transfer using methods known to a person skilled in the art. Suitable methods include culturing at sub-physiological temperatures, applying known activation agents (e.g. penetration by sperm, electrical and chemical shock), increasing levels of divalent cations, or reducing phosphorylation of cellular proteins (see U.S. 5, 496,720).

A nucleus of a donor cell, preferably of the same species as the enucleated oocyte, is introduced into the enucleated recipient oocyte. The donor cell nucleus may be obtained from any mammalian cells. Donor cells may be differentiated mammalian cells derived from mesoderm, endoderm, or ectoderm. In particular, the donor cell nucleus may be obtained from epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, and muscle cells. Suitable mammalian cells may be obtained from any cell or organ of the body. The mammalian cells may be obtained from different organs including skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organ, bladder, kidney and urethra.

The nucleus of the donor cell is preferably membrane-bounded. A donor cell nucleus may consist of an entire blastomere or it may consist of a karyoplast. A karyoplast is an aspirated cellular subset including a nucleus and a small amount of cytoplasm bounded by a plasma membrane. (See Methods and Success of Nuclear Transplantation in Mammals, A. McLaren, Nature, Volume 109, June 21 , 194 for methods for preparing karyoplasts).

Bcl-XL modified proteins are introduced into the enucleated recipient oocyte. The proteins are preferably derived from the same species as the donor cell, more preferably from the same species and cell type as the donor cell, and most preferably from the same individual from which the donor cell nucleus is derived. Methods for preparing the proteins are known to a person skilled in the art.

Donor cells may be propagated, genetically modified, and selected in vitro prior to extracting the nucleus.

The nucleus of a donor cell may be introduced into an enucleated recipient oocyte using micromanipulation or micro-surgical techniques known in the art or disclosed herein (see McGrath and Solter, supra). For example, the nucleus of a donor cell may be transferred to the enucleated recipient oocyte by depositing an aspirated blastomere or karyoplast under the zona pellucida so that its membrane abutts the plasma membrane of the recipient oocyte. This may be accomplished using a transfer pipette.

Fusion of the donor nucleus and the enucleated oocyte may be accomplished according to methods known in the art. For example, fusion may be aided or induced with viral agents, chemical agents, or electro-induced. Electrofusion involves providing a pulse of electricity sufficient to cause a transient breakdown of the plasma membrane. (See U.S. 4, 994,384). In some cases (e.g. with small donor nuclei) it may be preferable to inject the nucleus directly into the oocyte rather than using electroporation fusion. Such techniques are disclosed in Collas and Barnes, Mol. Reprod. Dev., 38:264-267 (1994).

The clones produced using the nuclear transfer methods as described herein may be cultured either in vivo (e.g. in sheep oviducts) or in vitro (e.g. in suitable culture medium) to the morula or blastula stage. The resulting embryos may then be transplanted into the uteri of a suitable animal at a suitable stage of estrus using methods known to those skilled in the art. A percentage of the transplants will initiate pregnancies in the surrogate animals. The offspring will be genetically identical where the donor cells are from a single embryo or a clone of the embryo. Zygote injection may also be employed in some aspects of the invention (see for example, the description in U.S. Pat. No. 4,736,866). This method involves allowing a fertilized egg or zygote comprising a Bcl-XL modified protein to develop in a pseudo-pregnant female.

The following non-limiting examples are illustrative of the present invention:

Example 1

AUTOMATED MICROINJECTION OF RECOMBINANT BCL-X INTO MOUSE ZYGOTES ENHANCES EMBRYO DEVELOPMENT

The following materials and methods were used in the study described in this example:

Ethics Statement: All mouse experiments were performed in accordance with Canadian Council on Animal Care (CCAC) guidelines for Use of Animals in Research and Laboratory Animal Care under protocols (permit or protocol #:AUP0015) approved by the animal care committees at Mount Sinai Hospital (MSH), Toronto and Toronto Centre for Phenogenomics (TCP). Single immature human oocytes at germinal vesicle or metaphase I stage were donated to research after obtaining patient consent approval in writing, which was approved by the Research Ethics Board at Mount Sinai Hospital, Toronto.

Manual and Robotic Microinjection: The workstation for manual microinjection consisted of an inverted microscope equipped with differential interference contrast (DIC) optics (Leica Microsystems, Wetzlar, Germany). Microinjection pipettes were backloaded and connected to a microinjector (FemtoJet; Eppendorf, Hamburg Germany). Holding pipettes (100 /vm O.D., 30 ji m I.D.) were prepared on a microforge (DeFonbrune) from 1.0mm O.D. * 0.75mm I.D. borosilicate glass capillaries (FHC 27-30-0) and connected to a manually controlled oil-based holding syringe system (Narishige, Japan). Mouse zygotes with visible pronuclei were selected for microinjection. All the zygotes were injected within the time window of 1-3 hr post- collection.

Measurement of Reactive Oxygen Species (ROS) Content: In the experiments, ROS content of the injected and un-injected embryos was measured at the 2-cell stage. The level of ROS content was quantified using the dichlorodihydrofluorescein diacetate (DCHFDA, Molecular Probes, Invitrogen, Carlsbad, CA, USA) method as previously described [49]. Live imaging and quantitation were conducted on a deconvolution microscope (Olympus IX70, Applied Precision Inc. Issaquah, WA, USA) using an image analysis program (SoftwoRx, Applied Precision Inc., Issaquah, WA, USA).

Immunocytochemistry for pSHC and BCL-X: Embryos, fixed with 10% formalin, were incubated overnight with appropriate primary antibody, rabbit anti-SHC (BD Transduction laboratories), mouse anti-SHC/phospho S36 antibody (Abeam) or rabbit anti mouse BCL-X (Santa Cruz, CA). After washing, embryos were incubated with appropriate secondary antibodies. Following a 15 minute counterstain with DAPI, embryos were mounted and imaged on a deconvolution microscope (Olympus IX70; Applied Precision Inc., Issaquah, WA, USA), and relative fluorescence in each image was quantitated as described above.

Metabolic Assays and Mitochondrial Labeling (M;tofracJcer);Microanalytical metabolic assay for ATP and citrate levels were performed as previously described [50]. Live embryos were stained with Mitotracker Red (Molecular Probes, Invitrogen, Carlsbad, CA, USA) at a final concentration of 200 μΜ. Embryos were then imaged on a deconvolution microscope (Olympus IX70, Applied Precision Inc., Issaquah, WA, USA) under the TRITC filter. NAD(P)H and FAD autofluorescence of the embryos were also imaged under DAPI and FITC filters, respectively as previously described [51]. Quantitation of the mitochondrial DNA copy number and mitochondrial membrane potential (JC-1 , Molecular probes, Invitrogen, Carlsbad, CA, USA) labeling was performed as previously described [34] and distribution was evaluated as outlined below.

Network Analysis and Visualization: Genes reported to be differentially expressed in human arrested or fragmented embryos, were chosen based on previous publications [23,24,37,38,39]. These gene targets were mapped to proteins and used to assess connectivity to BCL-X using the known, physical protein-protein interactions. Network was generated by querying I2D database Version 1.95 [52,53]. Network visualization was performed in NAViGaTOR 2.2 ([54,55]).

Statistical Analysis: Data were presented as mean ± s.e.m. and analyzed using either student's t-test, Mann-Whitney U-test, or Kruskal Wallis test followed by Dunn's post test (SigmaStat 3.5, Systat Software Inc.), as appropriate. For patient data analysis, Pearson correlation was used for maternal age and Anova on Ranks for patient diagnosis, hormonal stimulation and pregnancy outcome. All the statistical analyses were performed using SigmaStat 3.5 (Systat Software Inc.).

Animal husbandry and Culture Conditions: Animals were maintained on 12 h light/dark cycle and provided with food and water ad libitum in open cages (MSH) or individually ventilated units (TCP). Female ICR mice (Harlan) at 6 weeks of age were stimulated with 5 IU of pregnant mare serum gonadotropin (PMSG) (National Hormone and Peptide Program, NIDDK, Bethesda MD) followed 48 hours later by 5 IU of human chorionic gonadotropin (hCG) (Calbiochem, Gibbstown, NJ, USA) by intraperitoneal injection and mated overnight with ICR males. Embryos were recovered from the oviducts at 0.5 day post coitum (dpc) at the zygote stage. Ampullae were opened in 0.3 mg/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) in modified HTF (mHTF; Irvine Scientific, Irvine, CA, USA) to release cumulus masses.

Before microinjection experiments, 20 μΙ droplets of either KSOM supplemented with amino acids (KSOM; Millipore, Billerica, MA, USA) or HTF medium Irvine Scientific, Irvine, CA, USA) supplemented with 0.1 % BSA (Sigma-Aldrich, St. Louis, MO, USA.) were set up in 35 mm Petri dishes, covered with mineral oil, and pre-equilibrated overnight in a humidified 37°C incubator with 5% C0 ₂ . Microinjection of mouse zygotes was conducted in mHTF. After injection with either recBcl-xL (ΔΤΜ) protein (1.5 μς/μΙ) diluted in EmbryoMax Injection buffer (Millipore, Billerica, MA, USA) or buffer alone, embryos were cultured in appropriate medium with 10-20 embryos per group. For some experiments, TAT-BH4 peptide (Calbiochem, Gibbstown, NJ, USA) was supplemented in HTF culture medium (15 ng/μΙ). In addition, uninjected embryos were also cultured in HTF and KSOM droplets as control groups. Developmental progression was recorded daily. At day 4.5 (-96 hours in culture) embryos were fixed and total cell number and cell death index were determined as previously described [56].

Mitochondrial distribution analysis: Computer-based morphometry was used to quantitate differences in mitochondrial distribution, as previously described [23,24]. Briefly, regions of the stained mitochondria were identified using Mitotracker red staining (Figure 4A), and mitochondrial distribution was quantitated by calculating an Euler number. The Euler number measures the topology of an image and is defined as the number of objects in the image minus the number of holes in those objects. A lower Euler number indicates more clustered mitochondria, while higher number is indication of higher fragmentation of mitochondria.

Real time RT-PCR: Obtained single human oocytes, free of cumulus cells were loaded into guanidine iso-thio cyanate (GITC) solution and total nucleic acid was precipitated as previously described [23,24]. Upon treatment with DNase (Sigma-Aldrich, St.Louis, MO, USA), reverse transcription was performed on whole sample using Fermentas Revert Aid kit (Fermentas, Burlington, Ontario, Canada) and oligo-dT primers. 1/40 of the cDNA product was used in a real time PCR reaction using LightCycler ^® 480 SYBR Green I Master (Roche Applied Science, Indianapolis, IN, USA) and LightCycler ^® 480 (Roche, Mannheim, Germany). Primer sequences were located in the 3'UTR corresponding to β-ACTIN (Forward: 5'- CACAGGGGAGGTGATAGCAT-3' [SEQ ID NO: 2]; Reverse: 5'- CACGAAGGCTCATCATTCAA-3' [S EQ ID NO: 3) or BCL-X (Forward: 5'- GAGCTGTTTATGGCCTCAGC-3' [SEQ ID NO: 4] Reverse: 5'- GCTCCCATAGCTGTTCCTGA-3' [SEQ ID NO: 4]) and the samples were run simultaneously in duplicates for each gene. Amplification profile included a pre-incubation step at 95 °C for 5 minutes, followed by denaturation at 95 °C for 20 s, annealing at 62 °C for 15 s and extension at 72 °C for 15 s. Initially, all oocyte were pre-screened for βΑΟΤΙΝ expression and the samples that had a crossing point (C _P ) value less than 35 were used for further analysis. The target gene concentration for the oocyte samples was extrapolated utilizing the standard curve for BCL-XL and the data were expressed as relative ratio C _P value of β-ACTIN. Robotic Mouse Embryo Injection System: The technical development of the robotic mouse embryo injection system was partially described previously [19,20]. To facilitate the discussion of the testing results of system performance, the key features of the robotic system are briefly summarized as follows.

Glass cell holding device for cell immobilization: Vacuum-based glass cell holding devices (Figure 1A and Figure 6A) were constructed via standard microfabrication for rapidly immobilizing an array of mouse embryos [19]. Briefly, micrometer-sized through-holes (37 μηη±0.5 μπι in diameter) are formed on a standard cover slip (-170 μΐη thick) using two-side hydrofluoric acid (HF) wet etching. The device consists of a top glass layer with an array of through-holes, a bottom glass layer, and a PDMS spacer for forming a vacuum chamber. Devices with arrays of 3*3 and 5*5 through-holes were used for immobilizing mouse zygotes (-98 pm). Low pressures of 1.6 kPa-2.2 kPa were experimentally determined to be effective for holding the cells in place with sufficient forces during micropipette penetration. The complete cell immobilization process including the removal of extra cells typically takes 31 s for devices with an array of 5*5 through holes.

Injection micropipette fabrication and volume control: The injection micropipette is pulled using a programmable micropipette puller (P-97, Sutter), and the tip of pulled micropipette is then abraded using a micropipette beveler (BV-10, Sutter) to form a sharp tip and a 1.2 μπι opening (inlet picture in Figure 1 E). This micropipette fabrication process is highly repeatable, providing precisely controlled shape and size of the micropipette tip.

The deposition volume as a function of the pressure level and deposition time was accurately calibrated by measuring the size of spherical droplet blown out of the micropipette tip under an inverted microscope. Figure 1 E shows a calibration data example of the deposition volume using deionized water. The resolution of material deposition volume was 1fL

Micropipettes with an inner diameter (I.D.) of 1.2 pm and a sharp angle of 10° were used in experiments for evaluating the system performance. Due to protein viscosity, micropipettes with an I.D. of 3 pm and a sharp angle of 20° were used for injecting the BCL- XL (ΔΤΜ) protein and TE buffer.

Injection control sequence: A batch of mouse embryos are transferred and immobilized on the embryo holding device placed on the motorized rotational stage. Control flow of the injection process, shown in Figure 7, starts with vision-based contact detection [15] to vertically align the injection micropipette tip and the bottom surface of the cell holding cavity. The injection pipette tip is then raised to a home position, H (Figure 7B).

Simultaneously, the first embryo is brought into the field of view. The cytoplasm center and the polar body center are identified visually by a human operator and input to the system through computer mouse clicking. The embryo is then brought to the center of the field of view by the X-Y translational stage. If the polar body faces the penetration site, it is rotated away via visually servoed embryo orientation control.

Once the embryo is oriented, if needed, the microrobot controls the micropipette to a switch point, S. After the switch point, the micropipette diagonally penetrates the embryo and deposits materials at the injection destination (i.e., cytoplasm center). Upon retracting out of the embryo (Figure 7E), the micropipette is moved back to the home position, H. The next embryo is then brought into the field of view (Figure 7F). This injection process is repeated until all the embryos in the batch are injected.

Throughout the process, motions of the X-Y stage and 3-DOF micro-robot are controlled via proportional-integral-derivative (PID) control law. Transformations among the multiple coordinate frames are achieved during the operation of the system without requiring an off-line process.

Embryo structure identification: The determination of the injection destination (cytoplasm center) and the polar body center for orientation control is conducted by a human operator and input to the robotic system via computer mouse clicking in the graphical user interface of the control software. The system remains this minimal human-machine intervention to guarantee reliability. When the polar body of an embryo is within the depth of field, the human operator directly selects image coordinates of the cytoplasm and polar body centers. When the polar body is not visible (out of focus), the embryo is focally scanned by the z-motor on the microscope. During this scanning process, the human operator identifies and selects the polar body center. The control software accepts the image coordinates of the cytoplasm and polar body centers from user input, which are subsequently converted into Cartesian frame coordinates via online coordinate transformations. In the experiments, it was found that only 8.2% of the injected embryos appeared in the space of quadrant II (Figure 8A), which required automated re-orientation.

Vision-based cell orientation control: Since an embryo within a batch is rarely coincident along the rotational axis of the rotational stage, coupled translational motions during rotation cause the embryo to move out of the field of view. Thus, the system conducts 3-DOF cooperative control of the X-Y translational stage and the rotational stage to bring the embryo back into the field of view after orientation [57]. This control method permits high-speed orientation of cells.

Coordinate transformation between the frames of the rotational stage and X-Y translational stage is calibrated by image-based visual servoing [58] of the X-Y stage for always keeping the embryo inside the field of view during cell orientation. This calibration procedure is only required/conducted on the first embryo that requires re-orientation for an entire batch of immobilized embryos, since pitches (300/ym) between adjacent embryos are accurately known. The results of the study are discussed below.

Automated Microinjection of Mouse Zygotes: In contrast to conventional manual injection systems, the robotic system (Figure 1A) uses a microfabricated glass cell holding device (Figures 1C and 6A) to immobilize many mouse zygotes into a regular pattern (Figure 6C, ). Switching from one cell to another for injection was greatly simplified and automatically performed via precise position control (Figures 7 and 8), dramatically enhancing the injection speed. A vision-based cell orientation control technique as well as an in-house developed motorized rotational stage (Figure 1A) was integrated into the robotic system for fast and automated cell orientation. A motorized micromanipulator (i.e., injection microrobot) was automatically controlled to inject mouse zygotes (Figure 1 B) in a high-speed manner with high repeatability.

During system development, 306 mouse zygotes were injected with PBS buffer by the robotic system. Through these trials, an injection speed of 200 pm/s and a retraction speed of 500 pm/s were experimentally determined to be optimal in terms of minimizing injection- induced cell lysis. In order to ensure viability of injected zygotes and investigate dose effects of the injected materials, deposition volume was accurately controlled (Figure 1 E), achieved by fabricating injection micropipettes with a high consistency and precisely regulating the pressure unit output. The resolution of material deposition volume was 1 femtoliter (fl_).

To quantify system performance, the robotic system injected an additional 240 mouse zygotes with PBS buffer, demonstrating an average injection speed of 12 zygotes/min (vs. ~2 zygotes/min in manual injection by highly skilled technicians, data provided by microinjection operators at the Toronto Center for Phenogenomics). Based on visual inspection right after injection, the robotic system achieved a low cell lysis rate (1.1 %, Table 1 ). Developmental competence of microinjected embryos assessed by the rate of blastocyst formation (Table 1 ) in vitro after 96 hours in culture was 89±1.3% (mean ± s.e.m.), indicating minimal detrimental effect of robotic injection on embryo quality.

Table 1. Statistics of non-lysis and blastocyst formation rates of mouse embryos with PBS injection using the robotic system. The injected embryos were cultured in KSOM medium.

Due to the higher viscosity of the recombinant proteins (vs. PBS buffer), the need for increased size of microinjection pipettes was determined next. This modification increased the embryo lysis rates (20.3%; Figure 1 F); however, the robotic system still provided significantly lower lysis rates than manual injection (37.7%; Figure 1 F). There was no significant difference between the lysis rates of protein injection or buffer injection produced by robotic and manual microinjection. Thus, the robotic system has better performance in terms of cellular damage caused by microinjection. Automation enables users to operate the system without the long training needed for microinjection while achieving high injection consistency.

Culture induced developmental arrest: To use the system to address interesting questions in developmental biology, strategies for overcoming early embryo arrest were devised. Outbred colonies of mice often exhibit compromised preimplantation embryo development in suboptimal culture conditions [21]. First it was determined that human tubal fluid (HTF) medium, often used by in-vitro fertilization (IVF) clinics in the past, delays mouse preimplantation embryo development in vitro (Figures 2A and 2B). The HTF culturing model well recapitulates developmental arrest and was used to study the impact of ooplasmic transfer in the mouse model [22]. It has been previously reported that embryos of suboptimal quality often exhibit altered expression levels of genes known to regulate cell death [23,24]. In addition, females lacking Bcl-x (officially called Bcl2L1) in their oocytes exhibit decreased breeding performance that could not be attributed to a defect in ovarian reserve [25]. In order to determine whether altered levels of Bcl-2 family members accompany embryo arrest in the mouse, the possibility of a change in BCL-X protein levels in 2-cell stage mouse embryos due to culture in HTF medium, was explored.

Immunocytochemistry revealed -25% reduction of BCL-X protein expression in the embryos that were cultured for 24 hours in HTF in comparison to potassium simplex optimization medium (KSOM) (Figure 2C), most commonly used for in vitro culture of murine embryos. These results suggest that the depletion of BCL-X may contribute to poor embryo survival in the suboptimal culture conditions and may be one of the cytoplasmic factors responsible for improved embryo development observed during ooplasmic rescue.

Enhancing embryo development by automated microinjection of recBCL-XL (ATM): Thus, next an attempt was made to transiently supplement BCL-X levels by microinjecting recBCL-XL (ΔΤΜ) [26] into zygotes and to examine their in vitro developmental potential under an adverse culture condition (HTF). Injection of recBCL-XL (ΔΤΜ) protein significantly improved preimplantation embryo development, when compared to buffer-injected, HTF- cultured embryos (p<0.001 ; Figure 3A). Rates of blastocyst formation, total cell number (TCN) and cell death index (CDI), which all reflect embryo quality, were restored by recBCL-XL (ΔΤΜ) microinjection to levels comparable with embryos cultured in KSOM medium (Figure 3A and Figure 2). As a negative control, zygotes were also injected with BSA dissolved in microinjection buffer, and this did not significantly improve developmental rates (47%; n=66) or embryo quality (TCN: 64±5.5%, CDI: 4.2±0.6; n=13). These results show that microinjection of the recBCL-XL (ΔΤΜ) protein is capable of restoring developmental competence and improving quality of embryos facing conditions of stress. Furthermore, there was no significant difference in studied outcomes if protein was delivered into the cytoplasm or pronucleus (Figure 9). As robotic microinjection resulted in lower lysis rates and a higher degree of consistency, all experiments described below were performed with robotic recBCL- XL (ΔΤΜ) delivery into the cytoplasm.

Gametes and early mammalian embryos are susceptible to damage caused by excessive reactive oxygen species (reviewed in [27]). While reactive oxygen species (ROS) are key signaling molecules mediating basic cellular functions such as proliferation, differentiation and programmed death, excessive ROS production has been implicated in DNA damage, ATP depletion and permanent embryo arrest similar to that of cellular senescence [28]. As BCL-X has been previously shown to have antioxidant activities [29], the possibility of BCL-X increasing developmental competence via preventing ROS accumulation was explored.

HTF medium triggered excessive production/accumulation of ROS when compared to KSOM. Embryos microinjected with recBCL-XL (ΔΤΜ) protein and maintained in HTF medium had significantly reduced ROS levels (Figure 3B). Supplementing HTF medium with BCL-X derived BH4 domain TAT-synthetic peptide was sufficient to maintain a physiological ROS profile (Figure 10). However, developmental competence of embryos maintained in medium supplemented with BH4 peptide was not restored. These results indicate that the BH4 domain can alleviate excessive ROS, but only full length BCL-X protein, albeit lacking the transmembrane region (BCL-XL(ATM)), is capable of effectively restoring the developmental quality of embryos.

Previous work in bovine embryos identified the adaptor protein p66SHC as a mediator of permanent embryo arrest. Developmentally compromised bovine embryos have been reported to exhibit higher p66SHC expression accompanied by elevated ROS levels, and knockdown of p66Shc significantly reduced the occurrence of permanent embryo arrest in the bovine model [30,31]. Immunocytochemistry revealed that recBCL-XL (ΔΤΜ) microinjection decreased expression of both total and activated (pSer-36) p66SHC (Figure 3C) protein. As the P-p66SHC isoform has been shown to enter mitochondria and contribute to hydrogen peroxide release into the cytosol, decreased expression of p66SHC/Ser-36, particularly in the mitochondria, was accompanied by lower ROS levels in recBCL-XL (ΔΤΜ) microinjected embryos.

Suboptimal culture conditions may result in both excessive ROS production and the alteration of embryo metabolism. BCL-X, in addition to its anti-apoptotic role, has also been implicated in mitochondrial biogenesis [32] and the regulation of mitochondrial metabolism [33]. Therefore, whether mitochondrial distribution, an indicator of embryo health [34,35], can be altered by culture conditions, was next explored. As anticipated, embryos cultured in HTF medium exhibit altered sub-cellular distribution of mitochondria in comparison to KSOM (Figure 1 1 ). Furthermore, this distribution was corrected by recBCL-XL (ΔΤΜ) microinjection (Figure 4A), indicating that this Bcl-2 family member is capable of maintaining the correct cellular mitochondrial network in developing embryos.

Mitochondrial copy number (mtDNA content), mitochondrial activity (Mitotracker intensity) as well as mitochondrial membrane potential (JC-1 ratio), were also explored. However, none of these parameters was affected by culture conditions or by BCL-X microinjection (data not shown). Finally, whether recBCL-XL (ΔΤΜ) microinjection can affect the metabolism of preimplantation embryos, was investigated. Early embryos are not capable of glycolysis and rely on oxidative phosphorylation to sustain their energy demands. With development, they progressively gain the ability to utilize glucose [36]. Autofluorescent signals, reflecting cellular content of reduced NAD(P)H and oxidized FAD were significantly decreased in embryos upon microinjection of recBCL-XL(ATM) protein with concomitant increase in the citrate/ATP ratio (Figure 4B). Thus, BCL-X at the 2-cell stage modulates mitochondrial output, with outcomes indicating more efficient Krebs cycle metabolism and carbohydrate utilization.

Expression of BCL-X in human oocytes: While experiments described above dealt with in vitro induced phenotype, they have clinical relevance to human IVF, where embryos are always maintained in culture for at least 3 days. In addition, it is also possible that some oocytes may lack sufficient storage of maternally derived Bci-x gene products. In order to determine if variability in the endowment of maternally accumulated BCL-X transcripts could contribute to poor quality of human embryos in patients undergoing IVF, its expression in human oocytes was analyzed. As Bcl-x transcript expression peaks in germinal vesicle (GV) stage of mouse oocytes, followed by dramatic decline in metaphase II stage [23], only human GV and metaphase I (Ml) arrested oocytes were used. The GV and Ml oocytes were analyzed separately. Within the cohort of oocytes obtained from patients undergoing infertility treatment, 6/72 GV and 14/65 Ml oocytes failed to express detectable levels of BCL-X transcripts. An additional sub-group of oocytes (8 at GV stage) expressed reduced levels (less than mean) of BCL-X transcripts (Figure 5A). These data indicate that -20% of human growing oocytes obtained from infertile patients either completely lack or possess diminished endowment of BCL-X transcripts.

Next, a possible link between BCL-X and genes known to be significantly deregulated in human arrested or fragmented embryos by considering known physical protein-protein interactions from the I2D database, was analyzed. These targets included genes ZAR1 , YBX2, SYMPK, CPEB1 , TARBP2, DICER1 , DGCR8, MYLC2, ECT2, DIAPH1 , CFL1 , NELF, BTF3, IGFR2, YY1 , TERT, DNMT3B, CTNNB1 , HRK, BCL-XS, BCL2L10, P27KIP1 [23,24,37,38,39]. The resulting protein interaction network comprises 1 ,810 proteins and 20,562 interactions. Thick edges represent direct interactions among 22 up- and down- regulated genes/proteins. Thin, light grey edges link BCL-X (BCL2L1 ) with the up- and down- regulated genes, via 25 additional protein partners (small circles). On the left side are proteins that mostly link to down-regulated genes. EP300 is linked to both up- and down-regulated targets, while CASP8 is most linked to up-regulated genes (Figure 5B). This network highlights how lack of BCL-X may be connected with targets known to be differentially expressed in arrested embryos.

Enabled by the microfabricated embryo holding devices and vision-position based control of multiple motion control devices, the robotic mouse embryo injection system is capable of fast immobilization, switching, orientation, and injection of mouse embryos. The automated system requires minimal human involvement (i.e., a few computer mouse clicks), and is therefore, independent of operator skills, enabling the acquisition of large-scale molecule testing data with a high reproducibility. The robotic system performed microinjection of mouse zygotes at a speed of 12 zygotes/min, six times the speed of manual injection (~2 zygotes/min). High accuracy and consistency of the robotic system produced lower cell lysis rates and higher blastocyst formation rates than proficient microinjection technicians. Except the micro devices for immobilizing embryos and the motorized rotational stage for orienting cells, the automated system contains no other custom developed components and presents no difference in hardware compared to a conventional microinjection system, which is an advantage promising its use in biology laboratories and mouse core facilities. The robotic cell manipulation technology presented here can also be adapted with further technological modifications and applied to other cell micromanipulation tasks, for instance, intracytoplasmic sperm injection (ICSI) and nuclear transfer procedures.

The experimental results suggest that embryo arrest caused by suboptimal culture conditions is mostly driven by inefficient metabolism, in part due to decreased Bcl-x expression triggered by as yet unknown mechanisms. However, this altered cellular milieu can be restored by the microinjection of recombinant BCL-X protein, which repairs mitochondrial bioenergetics, prevents ROS accumulation, and facilitates the development of preimplantation mouse embryos. These effects start to become apparent at the 2-cell stage, and the consequences are still evident in the blastocyst stage (higher cell number and lower cell death rate; Figure 3A). While Bcl-x deletion causes midgestational embryonic lethality mostly due to defects in neuronal and hematopoietic lineages [40], its haplo-insufficiency triggers ovarian follicle loss, which became obvious with aging [41]. However, deletion of Bcl-x in follicles (oocyte and granulosa cells) did not compromise ovarian reserve in young age, but did result in decreased fertility of females [25].

It has been previously shown that Bcl-x is maternally deposited into the oocytes, but becomes upregulated, likely from embryonic genome, at the late 1 -cell stage [25]. In addition, some human as well as murine fragmenting early embryos alter splicing of Bcl-x gene, producing pro-apoptotic Bcl-xS isoform [24,25]. It has been recently shown that blastocysts do not require Bcl-xL for their survival, as downregulation of this isoform at the morula stage did not affect blastocyst quality. However, downregulation of Bcl-xL with concomitant induction of Bcl-xS had detrimental developmental consequences, as these blastocysts contain fewer cells and suffer from increased oxidative stress [42]. Data from the current study, however, point to a dependency on BCL-X during the critical window of transition from maternal to embryonic control, at the 2-cell stage.

The mechanism by which BCL-X promotes survival of 2-cell embryos does not appear to involve suppression of apoptosis, but rather points to the regulation of mitochondrial metabolism. These findings are consistent with previously published results in yeast and mammalian cell lines, which proposed that BCL-X can regulate a metabolic switch from glycolysis to oxidative phosphorylation [33], and can also maintain metabolite passage and activity of VDAC under conditions of stress [43,44]. BCL-X and other BCL-2 family members had also been shown to dynamically remodel the mitochondrial network (e.g., fission and fusion; reviewed in [45]). Thus, changes in mitochondrial distribution without the effect on mitochondrial DNA copy number observed in recBCL-XL microinjected embryos, are not surprising.

Intriguing is, however, the connection between phosphorylation of p66SHC and BCL- X protein levels. It is presently unknown how BCL-X influences the phosphorylation of p66SHC, besides regulating OS levels. Suppression of apoptosis by BCL-2 and BCL-XL can be attributed to protection against ROS and/or a shift of the cellular redox potential to a more reduced state [46]. Paradoxically, an elevated release of hydrogen peroxide was observed from BCL-XL overexpressing mitochondria, which led to an enhanced cellular antioxidant defense and superior protection against death [47]. It is also possible that dual crosstalk exists between BCL-X and p66SHC as previous work revealed that ablation of p66SHC increased expression of BCL-XL [48]. However, our data point to an additional role of BCL-X besides ROS safeguarding. While suboptimal culture clearly triggered oxidative stress, it is unlikely that ROS is the sole reason behind early embryo arrest, as BH4 peptide could alleviate the ROS accumulation, but could not support further embryo development. Likely, a combined role in mitochondrial metabolism with physiological ROS maintenance facilitates the viability of early embryos. An attempt was also made to explore if microinjection of recBCL-XL protein could have a clinical relevance. Expression screen of oocytes from infertile patients revealed variability in the BCL-X endowment. Within the cohort of freshly collected immature human oocytes, one-fifth either lacked or expressed lower amounts of BCL-X transcripts. However, the level of BCL-X did not correlate with clinical parameters such as patient infertility diagnosis, maternal age, stimulation protocol as well as pregnancy outcome (data not shown). It was observed that oocytes from the same patient would be variably affected, indicating that not all oocytes are created equal and likely would not equally well support preimplantation embryo development. Speculatively, embryos conceived from oocytes lacking BCL-X would be incapable of progression through preimplantation development and would arrest during in vitro culture. As expression of BCL-X in the transferred embryos was not assessed, this may explain the lack of BCL-X correlation with pregnancy outcome. Nonetheless, microinjection of recBCL-XL could have potential use during IVF treatment, particularly for cases where patients experience repeated IVF failure due to poor embryo quality.

Microinjection of recombinant mitochondrial proteins is capable of improving the developmental competence of embryos without genetic alterations in the offspring, a problematic factor of ooplasmic transfer. In contrast to mitochondrial heteroplasmy that results from ooplasmic transfer, the recombinant form of the BCL-X protein has a terminal half-life. Therefore, its addition to the embryos is aimed at providing transient support during the time that embryos are most susceptible to demise without resulting in permanent genetic modifications of the offspring. Further screening of additional protein targets using the automated microinjection technique could lead to the selection of the most efficacious proteins for improving embryo survival.

The present invention is not to be limited in scope by the specific embodiments described herein, since such embodiments are intended as but single illustrations of one aspect of the invention and any functionally equivalent embodiments are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. All publications, patents and patent applications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the methodologies etc. which are reported therein which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Full Citations for References Referred to in the Specification

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