CN101048496B

CN101048496B - Process for enriching a population of sperm cells

Info

Publication number: CN101048496B
Application number: CN2005800303094A
Authority: CN
Inventors: J·A·格雷厄姆
Original assignee: Monsanto Technology LLC
Current assignee: Monsanto Technology LLC
Priority date: 2004-07-22
Filing date: 2005-07-22
Publication date: 2012-07-04
Anticipated expiration: 2025-07-22
Also published as: ZA200700862B; CN101048496A

Abstract

Processes for selectively enriching a population of viable sperm cells with respect to a characteristic without physically sorting the cells are disclosed. The cells contained in such an enriched population benefit from the advantage of not being subjected to a sorting process. Processes of inseminating a female mammal and processes of forming a sperm dispersion utilizing the processes of selectively enriching a population of viable sperm cells are also disclosed.

Description

The method of enrichment population of sperm cells

Technical field

The present invention relates generally to the enrichment population of sperm cells.Particularly, the present invention relates generally to physical separation cell not and to the enrichment of the population of sperm cells of living.

Background of invention

Through artificial insemination (AI) make animal fertilization and in vitro fertilization after embryo transfer be a sophisticated operation.In the Domestic Animal industry, can exert one's influence and make thereafter generation have one or more desired characters remarkable advantages is arranged the breeding result.For example, it is female to guarantee the output of milk cow to select the offspring to help in the milk-product industry in advance, then can bring economic benefit.Sperm is separated into the cell mass that has X and Y chromosome of enrichment, and gender enriched sperm that promptly is called or gender enriched seminal fluid are one of methods that realizes the preliminary election offspring.

People such as Johnson (U.S. Patent number 5,135,759) have described use flow cytometer/cell sorter and have been separated into the cell mass that has X and the enrichment of Y chromosome sperm according to the sperm group that the DNA content will have complete X and Y chromosome.As said, sperm and DNA selected marker thing are mixed 1 hour (39 ℃) to 1.5 hours (30 ℃) under 30 ℃ to 39 ℃ temperature.Then use flow cytometer to measure the fluorescence volume that sends during through laser beam when sperm.Contain more DNA (being about 3% to 5% by the species decision) owing to have the sperm of X chromosome than the sperm that has Y chromosome, the sperm that has X chromosome produces bigger fluorescence light intensity than the sperm that has Y chromosome.Make on the drop band of the single sperm that contains predetermined fluorescent intensity electric charge and make its electrostatic deflection to collection container.Then the sperm group with the gender enriched of collecting is used for microinjection or artificial insemination.Obviously, this method needs the physical separation spermoblast to obtain the sperm group of gender enriched.Physical separation according to Johnson is time-consuming and expensive.

Summary of the invention

One of many-side of the present invention is the preparation method about a kind of sperm dispersion of characteristic enrichment (being sometimes referred to as suspension-s).In one embodiment, for example, method of the present invention is used to prepare the sperm dispersion about X or the enrichment of Y chromosome sperm.

In brief, therefore, the present invention relates to the ability that subgroup that selectivity reduces spermoblast in the sperm cell dispersion fertilizes an egg.This method is included in the dispersion-s that forms the spermoblast of mark in the liquid; Said liquid comprise induce sperm not mobility chemical agent or have and induce the not temperature of mobility of sperm, wherein with the existence of spermoblast bonded mark, lack or quantity indication dispersion-s in heredity, proteomics, structure or the functional character of subpopulation of sperm cells.This method comprises that also the optical detection dispersion-s is to identify the individual sperm cells as the subgroup member; Confirm the position of dispersion-s Central Asia group members; Reduce the ability that the subgroup member fertilizes an egg with different positions delivery of energy dosage in dispersion-s with selectivity and do not influence the spermoblast of other position in dispersion-s similarly.

The invention still further relates to the method for the population of sperm cells insemination female mammal that uses enrichment.This method is included in the dispersion-s that forms the spermoblast of mark in the liquid; Said liquid comprise induce sperm not mobility chemical agent or have and induce the not temperature of mobility of sperm, wherein with the existence of spermoblast bonded mark, lack or quantity indication dispersion-s in the characteristic of heredity, proteomics, structure or function of subpopulation of sperm cells.This method comprises that also the optical detection dispersion-s is to identify the individual sperm cells as the subgroup member; Confirm the position of dispersion-s Central Asia group members; Different positions sends that certain energy reduces the ability that the subgroup member fertilizes an egg with selectivity and the spermoblast that do not influence other position in dispersion-s similarly in dispersion-s; Use this dispersion-s or derivatives thereof insemination female mammal thereafter.

The invention still further relates to method in vitro fertilization.This method is included in the dispersion-s that forms the spermoblast of mark in the liquid; Said liquid comprise induce sperm not mobility chemical agent or have and induce the not temperature of mobility of sperm, wherein with the existence of spermoblast bonded mark, lack or quantity indication dispersion-s in the characteristic of heredity, proteomics, structure or function of subpopulation of sperm cells.This method comprises that also the optical detection dispersion-s is to identify the individual sperm cells as the subgroup member; Confirm the position of dispersion-s Central Asia group members, different positions delivery of energy dosage reduces the ability that the subgroup member fertilizes an egg with selectivity and influences the spermoblast of other position in dispersion-s similarly in dispersion-s; Use said dispersion-s or derivatives thereof to make egg's external fertilization thereafter.Can be in the uterus that thereafter zygote is imported female mammal.

The invention still further relates to the method that forms the refrigerated sperm dispersion.This method is included in the sperm cell dispersion that forms mark in the liquid; Said liquid comprise induce sperm not mobility chemical agent or have and induce the not temperature of mobility of sperm, wherein with the existence of spermoblast bonded mark, lack or quantity indication dispersion-s in the characteristic of heredity, proteomics, structure or function of subpopulation of sperm cells.This method comprises that also the optical detection dispersion-s is to identify the individual sperm cells as the subgroup member; Confirm the position of dispersion-s Central Asia group members; Different positions delivery of energy dosage reduces the ability that the subgroup member fertilizes an egg with selectivity and does not influence the spermoblast of other position in dispersion-s similarly in dispersion-s; Thereafter this dispersion-s of freezing.

Others of the present invention and characteristic are conspicuous, and part will be explained in hereinafter.

Detailed Description Of The Invention

Advantageously, the population of sperm cells that does not use the physical separation cell to live about a kind of characteristic enrichment according to the present invention.This characteristic can be that for example, spermoblast has X or Y chromosome.Alternatively, this characteristic can be another hereditary feature, the for example existence of SNP (" SNP "), and the animal fertility (for example, the milk yield of raising) that said polymorphum coding improves, or coding improves the lipid of the refrigeration of selected cell.This characteristic can also be the protein group characteristic, for example improves the protein of sperm performance, for example will be through improving the protein that useful acrosome characteristic improves performance in uterus.This characteristic can also be constitutional features (a for example acrosomal integnity), or functional character (for example propulsion property).

Can realize the enrichment of population of sperm cells about heredity, protein group, structure or functional character; It passes through; The spermoblast that for example has (or, alternatively, lack) this characteristic in the tagging populations; Make spermoblast motionless basically and optionally apply certain energy with the viability that reduces the cell used or reduce the ability (that is, after the insemination) that the cell used fertilizes an egg at least in external or body to motionless spermoblast.Because the spermoblast in the dispersion-s (being sometimes referred to as suspension-s) does not move and being selected property mark basically, so can the specific position that energy-beam is delivered in the dispersion-s to use to individual sperm cells; Through repeating this method steps, the spermoblast that promptly individually in dispersion-s, does not move on the different positions is used, and has the subpopulation of sperm cells of desired character in can effectively enrichment dispersion-s, for example with regard to the per-cent of cell subset with desired characteristic; With regard to owing to the percentage ratio of the filial generation with certain heredity or protein group characteristic that produces with spermoblast fertilization is discussed or effective enrichment with regard to the two.

The cell that in any event, can have a desired character without physical sepn is the specific subgroup of enrichment population of sperm cells with the cell (that is, need not separate cell of using and the cell of not using) that lacks desired character.Randomly, can be according to the following method subgroup of opening with cellular constituent that do not use that use through physical sepn, additionally the said cell of purifying is to obtain the further enrichment of cell.

Sperm cell dispersion

Spermoblast density

Generally speaking, can have with wide region spermoblast density preparation can be with the sperm cell dispersion of the colony of certain characteristic enrichment.Yet usually spermoblast density is at least about 1 * 10 ³Individual sperm/ml, and be no more than about 5 * 10 usually ¹⁰Individual sperm/ml, and more preferably be no more than about 5 * 10 ⁸Individual sperm/ml dispersion-s.For example, in one embodiment, can contain the sperm of " low relatively " density in the dispersion-s, promptly sperm concentration is less than about 1 * 10 ⁷Individual sperm/ml preferably is less than about 1 * 10 ⁶Individual sperm/ml, more preferably from about 1 * 10 ³To about 5 * 10 ⁶Individual sperm/ml, also more preferably from about 1 * 10 ³To about 1 * 10 ⁶Individual sperm/ml, even more preferably from about 1 * 10 ⁴To about 1 * 10 ⁵Individual sperm/ml and most preferably from about 1 * 10 ⁵Individual sperm/ml dispersion-s.In alternate embodiment, can contain the sperm of " centre " density in the dispersion-s, promptly density is about 1 * 10 ⁷To about 1 * 10 ⁸Individual sperm/ml dispersion-s.In a further embodiment, can contain the sperm of " high relatively " density in the dispersion-s, promptly sperm concentration is at least about 1 * 10 ⁸Individual sperm/ml, preferred about 1 * 10 ⁸To about 5 * 10 ¹⁰Individual sperm/ml, more preferably from about 1.5 * 10 ⁸To about 2 * 10 ¹⁰Individual sperm/ml, even more preferably from about 1.5 * 10 ⁸To about 2 * 10 ⁸Individual sperm/ml, also more preferably from about 1.5 * 10 ⁸Individual sperm/ml dispersion-s.Therefore, for example, in one embodiment, can contain in the dispersion-s at least about 0.04 * 10 ⁶, in another embodiment at least about 1 * 10 ⁶, in another embodiment at least about 1.5 * 10 ⁶, in another embodiment at least about 2 * 10 ⁶, in another embodiment at least about 3 * 10 ⁶, in another embodiment at least about 0.5 * 10 ⁷, in another embodiment at least about 1 * 10 ⁷, in another embodiment at least about 1.25 * 10 ⁷, in another embodiment at least about 2 * 10 ⁷, in another embodiment at least about 3 * 10 ⁷, in another embodiment at least about 4 * 10 ⁷, in another embodiment at least about 5 * 10 ⁷, in another embodiment at least about 6 * 10 ⁷, in another embodiment at least about 7.0 * 10 ⁷, in another embodiment at least about 8 * 10 ⁷, in another embodiment at least about 9 * 10 ⁷, in another embodiment at least about 10 * 10 ⁷, in another embodiment at least about 11 * 10 ⁷, in another embodiment at least about 12 * 10 ⁷, in another embodiment at least about 1.0 * 10 ⁸, in another embodiment at least about 1.25 * 10 ⁸, in another embodiment at least about 1.5 * 10 ⁸, in another embodiment at least about 1.75 * 10 ⁸, in another embodiment at least about 2.0 * 10 ⁸, in another embodiment at least about 2.25 * 10 ⁸, in another embodiment at least about 2.5 * 10 ⁸, in another embodiment at least about 2.75 * 10 ⁸, in another embodiment at least about 3 * 10 ⁸, in another embodiment at least about 5 * 10 ⁸, in another embodiment at least about 7.0 * 10 ⁸, or even at least about 8 * 10 ⁸Individual sperm/ml dispersion-s.In alternate embodiment, dispersion-s can contain and be less than about 9 * 10 ⁵, be less than about 7 * 10 ⁵, be less than about 5 * 10 ⁵, be less than about 2 * 10 ⁵, be less than about 1 * 10 ⁵, be less than about 1 * 10 ⁴, or even less than about 1 * 10 ³Individual sperm/ml dispersion-s.

The density of sperm can change based on many factors, and said factor comprises, for example the difference among the difference between the Mammals different plant species, the single species of Mammals with in addition different single Mammals, the difference in (once penetrating) seminal fluid.For example, Niu Jingzi can high-density, but smaller volume is present in the dispersion-s usually, for example at about 0.5ml in the volume of about 25ml 0.5 * 10 ⁶Individual sperm/ml is to about 8 * 10 ⁷Individual sperm/ml.Yet the pig sperm can be than low density, but bigger volume is present in the dispersion-s usually, for example at about 50ml in the volume of about 250ml 0.04 * 10 ⁶Individual sperm/ml is to about 1 * 10 ⁷Individual sperm/ml.

Sperm concentration in the sperm dispersion depends on that also spermoblast is subsequently by the method for enrichment or sorting.For example, can be as at the said use selected by flow cytometry apoptosis spermoblast of U.S. Patent Application Publication US 2005/0112541 (its content is incorporated herein by reference by complete at this).Under this type situation, dispersion-s can be generally the sperm of " centre " or " high relatively " density.The more low-density sperm of applying marking thing (dyestuff for example as herein described and affinity tag) mark is as low relatively " sperm of density is applicable to like hereinafter other sorting or beneficiation technologies in greater detail.

Sperm concentration in all right artificially operation sperm dispersion is to obtain the dispersion-s of specific spermatozoa density.The operation of (for example be included in insemination suction pipe in) sperm concentration can be based on following factor in the sperm dispersion, as the storable temperature of dispersion-s, storage time length, sperm in sperm dispersion be sorting or unsorted, from its collect the boar of sperm species, collect the mammiferous fertility of sperm and the species of the female mammal that will be inseminated from it.

Can also be through concentrating the density that sperm (for example through centrifugal) influences sperm in the sperm dispersion simply.Under this type situation, said dispersion-s will be divided into (being commonly referred to) deposition (cell mass that contains small amount of liquid) and supernatant (soluble liquid fraction) basically.Then can not destroy deposition ground and outwell supernatant, produce the fine and close relatively deposition of spermoblast that contains a small amount of suppressor factor thus, this effect minimizing volume of dispersion and the component of dispersion-s of not changing.As a result, sedimentary spermoblast remains on not kinestate.

The not mobility of spermoblast

Sperm cell dispersion contains the spermoblast that reduces mobility on the quite big degree.Minimizing in the sperm cell dispersion on the quite big degree of spermoblast mobility can obtain in several ways; It for example comprises, through spermoblast is contacted with the mobility suppressor factor, temperature through reducing immediate environment (being sperm dispersion) around spermoblast or the spermoblast or through both combination.In preferred embodiments; Spermoblast shows the behavior of epididymal sperm characteristic mode in some aspects in the sperm dispersion of the present invention, and for example, the spermoblast in the colony does not move basically; And/or for the sperm of washing or ejaculation just, its endogenous respiration speed is lower.Advantageously; The spermoblast that does not move (being sometimes referred to as static spermoblast) is when separating from suppressor factor or be exposed to the temperature of rising; Its mobility can show the characteristic behavior of the sperm of ejaculation; And in one embodiment, its mobility all can show the characteristic behavior of penetrating sperm with breathing.

In one embodiment; For example; Analyze (Hamilton-Thorne HTM-IVOS area of computer aided sperm analysis system through the HTM-IVOS sperm; Hamilton-Thorne Research; Beverly MA) measure, path velocity, forward speed or both that path velocity (being sometimes referred to as mobility or path mobility), forward speed (being sometimes referred to as propulsion property) or both that the reduction of suppressor factor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least about 50% simultaneously.Preferably through HTM-IVOS sperm analysis to measure; Path velocity, forward speed or both that path velocity, forward speed or both that the reduction of depressomotor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least about 60% simultaneously.More preferably through HTM-IVOS sperm analysis to measure; Path velocity, forward speed or both that path velocity, forward speed or both that the reduction of depressomotor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least about 70% simultaneously.Also more preferably through HTM-IVOS sperm analysis to measure; Path velocity, forward speed or both that path velocity, forward speed or both that the reduction of depressomotor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least about 80% simultaneously.Even more preferably through HTM-IVOS sperm analysis to measure; Path velocity, forward speed or both that path velocity, forward speed or both that the reduction of depressomotor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least about 90% simultaneously.Even more preferably through HTM-IVOS sperm analysis to measure; Path velocity, forward speed or both that path velocity, forward speed or both that the reduction of depressomotor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least about 95% simultaneously.Most preferably through HTM-IVOS sperm analysis to measure; Path velocity, forward speed or both that path velocity, forward speed or both that depressomotor can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least about 99% simultaneously.

Can use depressomotor to reduce the mobility of spermoblast in the sperm cell dispersion to a considerable extent.Suppressor factor can be in the inhibited a series of compsns of sperm motility property any one.This based composition comprises like the sodium channel suppressor factor, like Strophanthin G; The compsn that comprises potassium ion; And the compsn that comprises potassium ion and sodium ion.For example, the potassium ion of relative high density can suppress sperm motility property in the dispersion-s.Therefore generally speaking, the potassium concn that preferred dispersions comprises in potassium ion source and the dispersion-s is at least about 0.05mol/L.More preferably potassium concentration is at least about 0.05mol/L to about 0.5mol/L.Also more preferably potassium concentration is at least about 0.1mol/L to about 0.3mol/L.Most preferably potassium concentration is about 0.173mol/L.This type of dispersion-s (but nonessential) usually also comprises source of sodium ions.When having sodium, the mol ratio of potassium and sodium generally is equal to or greater than 1: 1 respectively, but mol ratio generally is no more than 8: 1.The mol ratio of preferred potassium and sodium is at least about 1.25: 1.Also more preferably the mol ratio of potassium and sodium is at least about 1.5: 1.Also more preferably the mol ratio of potassium and sodium is at least about 1.75: 1.Also more preferably the mol ratio of potassium and sodium is at least about 1.78: 1.In a specific embodiments, the mol ratio of potassium and sodium is at least about 2: 1.And in another embodiment, the mol ratio of potassium and sodium is at least about 3: 1.In another embodiment, the mol ratio of potassium and sodium is at least about 4: 1.In another embodiment, the mol ratio of potassium and sodium is at least about 5: 1.In another embodiment, the mol ratio of potassium and sodium is at least about 6: 1.In another embodiment, the mol ratio of potassium and sodium is at least about 7: 1.In another embodiment, the mol ratio of potassium and sodium is at least about 8: 1.

Also can contain the ion or the carbon dioxide source that can promote the mobility downward modulation in the sperm dispersion in addition.In this embodiment, the source of carbonic acid gas can do, one or more carbonate for example.In a present preferred embodiment, sperm dispersion contains NaHCO ₃And KHCO ₃(potassium and source of sodium ions are provided thus) and the partial pressure of carbon dioxide (with respect to ambient atmosphere) that increases.For example, in a present preferred embodiment, sperm dispersion contains NaHCO ₃And KHCO ₃The aqueous solution, preferred NaHCO ₃, KHCO ₃And C ₆H ₈O ₇H ₂The aqueous solution of O; Generally speaking, KHCO in the dispersion system ₃Concentration can be at least about 0.05mol/L.More preferably KHCO ₃Concentration be at least about 0.05mol/L to about 0.5mol/L.More preferably KHCO also ₃Concentration be at least about 0.1mol/L to about 0.3mol/L.In particularly preferred embodiments, like Salisbury & Graves, J.Reprod.Fertil., disclosed that kind among the 6:351-359 (1963) is used to contain 0.097mol/LNaHCO ₃, 0.173mol/L KHCO ₃, 0.090mol/L C ₆H ₈O ₇H ₂The depressomotor of the aqueous solution of O forms dispersion-s.As long as spermoblast is exposed to depressomotor, they generally all can keep static.

In dispersion-s, contain C ₆H ₈O ₇H ₂During O, KHCO ₃With NaHCO ₃Between mol ratio can be as stated.KHCO ₃With C ₆H ₈O ₇H ₂Mol ratio between O generally is equal to or greater than 1: 1 respectively, but generally can not surpass mol ratio 8: 1.Preferred KHCO ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 1.25: 1.More preferably KHCO also ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 1.5: 1.More preferably KHCO also ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 1.75: 1.In a specific embodiments, KHCO ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 1.78: 1.In another embodiment, KHCO ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 2: 1.In an embodiment again, KHCO ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 3: 1.Again in another embodiment, KHCO ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 4: 1.Again in another embodiment, KHCO ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 5: 1.Again in another embodiment, KHCO ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 6: 1.Again in another embodiment, KHCO ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 7: 1.Again in another embodiment, KHCO ₃With C ₆H ₈O ₇H ₂Mol ratio between O is at least about 8: 1.In particularly preferred embodiments, like Salisbury & Graves, J.Reprod.Fertil., disclosed that kind among the 6:351-359 (1963) is used to contain 0.097mol/L NaHCO ₃, 0.173mol/L KHCO ₃, 0.090mol/L C ₆H ₈O ₇H ₂The inhibition damping fluid of the aqueous solution of O forms dispersion-s.As long as spermoblast is exposed to depressomotor, they generally all can keep static.

Experimental evidence is up to now also pointed out, if sperm cell dispersion is kept at reduction or prevents that oxygen from the atmosphere of this dispersion-s diffusion, can improve the holistic health and other key characters of spermoblast.Through replace the atmosphere of sperm dispersion top with the gas that increases like carbonic acid gas, nitrogen or other rare gas element intrinsic standoff ratio ambient air, can reach this purpose.In concrete embodiment, dispersion-s maintains under the atmosphere with partial pressure of carbon dioxide higher than ambient air.In preferred embodiments, the partial pressure of carbon dioxide of dispersion-s top air is at least about 0.0001atm, but is generally less than about 5atm normal atmosphere.In one embodiment, partial pressure of carbon dioxide is that about 0.5atm is to about 2atm normal atmosphere; In another embodiment, partial pressure of carbon dioxide is that about 0.9atm is to about 2atm normal atmosphere; In another embodiment, partial pressure of carbon dioxide is that about 0.95atm is to about 2atm normal atmosphere.In particularly preferred embodiments, the atmospheric partial pressure of carbon dioxide in dispersion-s top is 0.9atm at least; More preferably at least about 0.95atm.

Except that using depressomotor or alternative be that the temperature that can change spermoblast or dispersion-s is not moved to cause that spermoblast becomes.Induce sperm not the temperature of mobility can be induced, for example the temperature through reducing spermoblast or dispersion-s is to about 0 ℃ to about 15 ℃, preferably from about 1 ℃ to about 10 ℃; More preferably from about 2 ℃ to about 8 ℃; Also more preferably from about 3 ℃ to about 6 ℃; Even more preferably about 4 ℃ to about 5 ℃; More preferably from about 5 ℃.Yet, preferably spermoblast is not exposed to and can the pair cell viability causes under the temperature of essence detrimentally affect or combination of remarkably influenced spermoblast or picked-up affinity tag ability.

The temperature that in another embodiment, can change spermoblast or sperm dispersion make its between about 4 ℃ to about 50 ℃ of scopes; Preferably from about 7 ℃ to about 43 ℃; More preferably from about 10 ℃ to about 39 ℃; Also more preferably from about 15 ℃ to about 30 ℃; And most preferably from about 17 ℃ to about 25 ℃ temperature.In particularly preferred embodiments, temperature of dispersion-s is about 4 ℃ around spermoblast or its.

In a single day Mammals obtains spermoblast from the source at any time, thereby the temperature that can be exposed to reduction is not moved it basically.For example; Can be from source Mammals collecting cell the time, when cell is mixed with damping fluid, when forming the mark mixture (before comprising labeling process, during or afterwards) or when forming the dispersion-s of labeled cell, reduce the temperature of spermoblast, cause not mobility of sperm thus.Yet, before prior to the optical detection of dispersion-s, pass through to reduce not mobility of temperature-induced sperm usually.

For example, can after labeled cell, reduce spermoblast temperature (that is, inducing not mobility of sperm), thereby carry out mark under the preferred temperature that allows to discuss hereinafter.In preferred embodiments, can be behind mark and before the optical detection cell, reduce the spermoblast or the temperature of dispersion-s on every side.

The temperature or both combinations that spermoblast are exposed to suppressor factor, reduction induce spermoblast not move.In one embodiment; For example; Mobility, propulsion property or both that mobility, propulsion property or both that the reduction of depressomotor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least 60% simultaneously.Preferably; Mobility, propulsion property or both that mobility, propulsion property or both that the reduction of depressomotor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least 70% simultaneously.More preferably; Mobility, propulsion property or both that mobility, propulsion property or both that the reduction of depressomotor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least 80% simultaneously.Preferably; Mobility, propulsion property or both that mobility, propulsion property or both that the reduction of depressomotor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least 90% simultaneously.Preferably; Mobility, propulsion property or both that mobility, propulsion property or both that the reduction of depressomotor, temperature or both combinations can make spermoblast in the dispersion-s have just penetrated spermoblast in the seminal fluid with respect to same species simultaneously reduce at least 99% simultaneously.

No matter employed method preferably makes cell not move, thereby allows adequate time to carry out the optical detection of dispersion-s, confirms the position of subgroup member cell and energy source is applied to member's cell of subgroup.If expectation physically separates the cell of being used and the cell of not using, can also preferably in this process steps, keep spermoblast in kinestate not.Likewise, if the refrigeration spermoblast, can make in the step it maintain not kinestate (cell of no matter before refrigeration, using whether with the cellular segregation of not using) in refrigeration.In preferred embodiments, the refrigeration step process remains cell and does not move.

Can be through cell be separated with the mobility suppressor factor; Be exposed to air; The temperature of rising cell or cell dispersion-s (preferably to the representative temperature that just penetrates seminal fluid); Through using saline water people such as (, 1963) Salisbury or such as the damping fluid dilution of TCA damping fluid or PBS, or through above combination (it depends on the not used method of mobility of inducing) arbitrarily not motor cell return to active condition (behavioural characteristic of promptly firm ejaculation seminal fluid).Generally speaking; According to HTM-IVOS sperm analysis to measure; At least about 20%, preferably at least about 50%, more preferably at least about 60%, also more preferably at least about 70% in addition more preferably at least about 80% in addition more preferably at least about 90%, also more preferably at least about 95%, most preferably at least about 99% recover active condition cell (being the reactivate cell) have such path velocity, preceding tropism's speed or both simultaneously: promptly its for the mobility inhibitor mixed before spermoblast (spermoblast that has promptly just penetrated) path velocity, preceding tropism's speed or both whiles at least about 50%; Preferably at least about 60%; More preferably at least about 70%, also more preferably at least about 80%, even more preferably at least about 90%; Even more preferably at least about 95%, most preferably at least about 99%.

From the Mammals collecting cell

The method of known multiple collection sperm alive.These class methods comprise, for example, hand grip, use artificial vagina and electro-ejaculation.

When collecting; Or thereafter; Can be with any one mixing in the many different damping fluids of the seminal fluid of collecting and suitable sperm; Said damping fluid does, such as TCA, HEPES, PBS or in U.S. Patent Application Publication No. US 2005/0003472 (its content intactly is incorporated herein by reference herein) disclosed any other damping fluid.For example, can contain the 500000000 ox seminal fluid samples to about 10,000,000,000 spermoblasts of having an appointment with common every milliliter directly is collected into the container that contains damping fluid to form sperm suspensions from the source Mammals.Alternatively, can and within several minutes after the collection to several hours, make it immediately seminal fluid sample collection to empty container to contact to form sperm suspensions with damping fluid.

Alternatively, can collect spermoblast and make it with replace damping fluid or damping fluid outside depressomotor contact, thereby form sperm dispersion.Can spermoblast directly be collected into the container that contains depressomotor to form sperm dispersion from animal; Or alternatively, be collected in the empty receptacle and within several minutes (or even several hours) of collecting, make it immediately contact with the formation sperm dispersion with depressomotor.

Sperm dispersion can also contain a series of other additives to improve sperm viability.The additive of exemplary comprises protein source, microbiotic, growth factor and regulates in the cell and/or the compsn of extracellular oxidation/reduction reaction.The various instances of these additives are well-known in the art, as, for example prove in U. S. application series number 60/557,407 and 11/092,313 open (its content here be introduced into as a reference) separately.

The mark of cell

Can use any one label sperm cells of multiple different affinity tags; Said affinity tag comprises affinity tag (for example fluorescently-labeled antibody) and cross-cell membrane that is bonded to outside and the affinity tag (for example, fluorescent DNA selective dye) that is bonded to entocyte.Generally speaking; Marking method is included under the temperature or pH that allows quick and effective combination or picked-up affinity tag; Spermoblast is contacted with certain density affinity tag (thereby form the mark mixture; Be sometimes referred to as dye mixture), make the time sufficiently long with the mark degree that obtains expectation the viability of non-materially affect cell.

Sperm can be pure seminal fluid form, or alternatively, contain sperm semen derivative (its through centrifugal or use other method separate seminal fluid divide as level obtain).Then spermoblast is contacted with affinity tag or additionally mixed with formation mark mixture; Randomly, this affinity tag can be the form of solid or solution.Yet generally speaking, affinity tag, spermoblast or both are at medium simultaneously, for example in the damping fluid.

In one embodiment, spermoblast is mixed with damping fluid to form sperm suspensions.Can use any of many different damping fluids of being fit to sperm, such as TCA, HEPES, PBS or disclosed damping fluid in U.S. Patent Application Publication No. US 2005/0003472.In case the formation sperm suspensions can mix it to form the mark mixture with source of label; Randomly, said affinity tag can be solid or liquid form and, as further selection, can additionally comprise any above-mentioned damping fluid.

In another embodiment, affinity tag can form mark suspension-s with the buffering liquid-phase mixing and this mark suspension-s mix formation mark mixture with sperm cell source, and said sperm cell source is pure seminal fluid, contain the semen derivative of sperm or the form of sperm suspensions.

In preferred embodiments, use the damping fluid that contains the mobility suppressor factor to form the mark mixture.For example, the mobility suppressor factor can be included in the damping fluid that is used to form sperm suspensions (it then mixes with affinity tag) or mark suspension-s (it then mixes with sperm cell source) to form the mark mixture.In any one event, produce the sperm dispersion that contains mobility suppressor factor and affinity tag.

Can be through using any of multiple affinity tag; As in the past at U.S. Patent number 5; 135,759 with (all quoting as a reference herein) described in the WO 02/41906, one or more can form the mark mixture through the DNA of UV or excited by visible light selective dye.The light activated DNA selective dye of the UV of exemplary comprises Hoechst 33342 and Hoechst 33258, and the two all can (St.Louis MO) be purchased from Sigma-Aldrich.The dyestuff of the excited by visible light of exemplary comprises can be from Molecular Probe; Inc. (Eugene; OR) SYBR-14 that is purchased; And the two benzimide-BODIPY described in the WO 02/41906

conjugate 6-{ [3-((2Z)-2-{ [1-(boron difluoride alkyl)-3; 5-dimethyl--1H-pyrroles-2-yl] methylene radical }-2H-pyrroles-5-yl) propionyl group] amino }-N-[3-(methyl { 3-[({ 4-[6-(4-N-METHYL PIPERAZINE-1-yl)-1H; 3 ' H-2,5 '-bisbenzimidazole-2 '-yl] phenoxy } ethanoyl) amino] propyl group } amino) propyl group] hexanamide (" BBC ").Use can used or make up in these dyestuffs each separately; Alternatively; Also can use separately other cell permeability UV and excited by visible light dyestuff or itself and aforementioned dye combinations used, as long as can not produce extremely unacceptable degree of detrimentally affect when said dyestuff uses under the concentration that can carry out the said sorting in other places to the viability of spermoblast.

Alternatively, can use fluorescent polyamide to form the mark mixture, more specifically be conjugated with the polymeric amide of fluorescent marker or reporter molecule.This type of affinity tag can send fluorescence when being incorporated into nucleic acid.The instance that is connected with the polymeric amide of fluorescent marker or reporter molecule comprises, like people such as Best, and Proc.Natl.Acad.Sci.USA, 100 (21): 12063-12068 (2003); People such as Gygi, Nucleic Acids Res., 30 (13): 2790-2799 (2002); U.S. Patent number 5,998,140; U.S. Patent number 6,143,901 and U.S. Patent number 6,090,947 in those disclosed, wherein the content of each piece all is incorporated herein by reference herein.

Fluorescent nucleotide sequences also can be used for label sperm cells.This type of nucleotides sequence is listed in and can sends fluorescence when containing the nucleic acid hybridization of target sequence or complementary sequence, but when non-hybridization state, then can not send fluorescence.This type of Nucleotide openly is shown in like U.S. Patent Application Publication 2003/0113765 (being incorporated herein by reference) here.

Sex specific antibodies also can be used for the spermoblast in the mark mark mixture.For example, in this embodiment, sex specific antibodies can be puted together fluorescence part (or equivalent reporter molecule).Since antibodies only be present in have X chromosome or, alternatively, have the antigen on the cell of Y chromosome, can optionally identify this type of cell according to its fluorescence (with respect to the no fluorescence of unlabeled cells).In addition, can use more than one sex specific antibodies simultaneously, every kind of antibody connects different fluorescence parts.Thereby can they be distinguished according to the intercellular different fluorescence that have X chromosome or Y chromosome.

Also can use the spermoblast in the luminous color selectivity nano crystalline mark mark mixture.These particles are also referred to as quantum dot, and as U.S. Patent number 6,322,901 are (all quoting as a reference herein) well known in the art with U.S. Patent number 6,576,291 is said.These nanocrystals are puted together in multiple biomaterial, comprise like peptide, antibody, nucleic acid, Streptavidin and polysaccharide (seeing like U.S. Patent number 6,207 392,6,423; 551,5,990,479 and 6,326; 144, all quote as a reference herein), and be used for the detection of biological target and (see like U.S. Patent number 6,207; 392 and 6,247,323, both all quote as a reference herein).

The preferred concentration of affinity tag is the function of a series of variablees in the mark mixture, and said variable comprises, for example, whether whether affinity tag is incorporated into outside or it must cross-cell membrane; Cell is for the passing through property (if the necessary cross-cell membrane of affinity tag) of selected affinity tag; The temperature of mark mixture; Carry out the time quantum of mark and the selectivity degree of expectation.Generally speaking, preferred marker concentrations is enough to reach in the short time in appropriateness the desired mark degree of cell, and the detrimentally affect that can not produce essence to sperm viability.For example, Hoechst 33342, Hoechst 33258, SYBR-14 or the concentration of BBC in the mark mixture generally at about 0.1 μ M between about 1.0M, be preferably from about 0.1 μ M to about 700 μ M, more preferably about 100 μ M are about 200 μ M extremely.In particularly preferred embodiments, Hoechst 33342, Hoechst 33258, SYBR-14 or the concentration of BBC in dye mixture generally at about 400 μ M to about 500 μ M, most preferably be about 450 μ M.Therefore, be provided with down in a kind of flag condition, the concentration of Hoechst 33342 is preferably about 100 μ M.Be provided with down in another kind of flag condition, the concentration of Hoechst 33342 is about 150 μ M.Be provided with down in another kind of flag condition again, concentration is preferably about 200 μ M.And be provided with down in another kind of flag condition, the concentration of Hoechst33342 most preferably is about 450 μ M.

Again for example, the concentration of fluorescent polyamide (for example those described in the U. S. application publication number 2001/0002314) is generally about 0.1 μ M between about 1mM, preferably from about 1 μ M to about 1mM, and 5 μ M about 100 μ M extremely more preferably from about, even 10 μ M more preferably from about.

In a single day the mark mixture forms, and can under the arbitrary temp in the series of temperature scope, preserve; For example, use Hoechst 33342 or Hoechst 33258 marks to carry out to about 50 ℃ of scopes at about 4 ℃ usually.For example, can the mark mixture be stored under the temperature of " low relatively ", promptly temperature is about 4 ℃ to about 30 ℃; In this embodiment, temperature more preferably from about 25 ℃ to about 30 ℃, most preferably is about 28 ℃ preferably from about 20 ℃ to about 30 ℃.Alternatively, can the mark mixture be stored in " centre " TR, promptly temperature is about 30 ℃ to about 39 ℃; In this embodiment, temperature is preferably at about 34 ℃ to about 39 ℃, more preferably about 37 ℃.In addition, also can the mark mixture be stored in the TR of " high relatively ", promptly temperature is about 40 ℃ to about 50 ℃; In this embodiment, temperature more preferably from about 40 ℃ to about 43 ℃, most preferably is about 41 ℃ preferably from about 40 ℃ to about 45 ℃.A series of variablees are generally depended in selection for preferred temperature, comprise whether be incorporated into outside or it like affinity tag whether must cross-cell membrane, cell is for the selectivity degree of passing through concentration, the time quantum that carries out mark and the expectation of affinity tag in property (if affinity tag must cross-cell membrane), the mark mixture of selected affinity tag.

The pH of mark mixture can remain in any pH value scope.For example, use Hoechst33342 or Hoechst 33258 marks generally to carry out in about 5.0 to about 9.0 pH scope.For example, the mark mixture is remained under " subacidity " pH, promptly from about 5.0 to about 7.0.In this embodiment, preferred pH is from about 6.0 to about 7.0, more preferably from about 6.0 to about 6.5, most preferably is about 6.2.Alternatively, the mark mixture is remained under " slight alkalinity " pH, promptly from about 7.0 to about 9.0.In this embodiment, preferred pH is from about 7.0 to about 8.0, more preferably from about 7.0 to about 7.5, and most preferably is about 7.3.Yet, generally speaking,,, just the mark mixture is adjusted to about 7.0 pH in case accomplish the needed time of mark that is enough to carry out expected degree if form mark at the pH except about 7.0.

Randomly, the mark mixture also contains additive to improve sperm viability.The additive of exemplary comprises that as above literary composition is collected microbiotic, the growth factor of discussing or regulated in the cell and/or the compsn of extracellular oxidation/reduction reaction for cell sample.Can in view of the above these additives be added the mark mixture.

Affinity tag in spermoblast picked-up or the bonding mark mixture can continue sufficiently long for some time, with the dispersion-s of the spermoblast that obtains desired degree mark.The said time generally is enough to make affinity tag to be incorporated into the DNA of spermoblast or spermoblast, thus the subgroup member of ability identification of cell and definite its position in dispersion-s.A series of variablees are depended in the selection of preferred time usually, and whether must cross-cell membrane, cell is for concentration, the temperature of mark mixture and the selectivity degree of expectation of passing through affinity tag in property (if affinity tag must cross-cell membrane), the mark mixture of selected affinity tag if comprising whether affinity tag for example is incorporated into outside or it.For example, the said time can be the time of the spermoblast DNA that is enough to that the fluorescent DNA selective dye is incorporated into and has X and Y chromosome, thereby can both sortings be come according to different and measurable fluorescence intensity between the two.For example, when using Hoechst 33342 or Hoechst 33258 marks, generally speaking, this mark time will be no more than about 160 minutes, preferably be no more than about 90 minutes, also more preferably no more than about 60 minutes, most preferably from about 5 minutes to about 40 minutes.

Some affinity tag, and some dyestuff particularly can permeate spermoblast and specificity and combine DNA and need not additionally intervene to increase the property of passing through of cell.Yet, use other affinity tag can expect can reduce viability or mobility acceptably to increase infiltration rate prior to mark pre-treatment spermoblast.Can use any suitable method that well known to a person skilled in the art.These class methods comprise electroporation, use the solution (like the tensio-active agent of gentleness) or the chemical shock that promote Premeabilisation of cells.When wherein use other or stricter technology was desired or favourable, this type processing can comprise used the perhaps many those skilled in the art of liposome to use the technology that staining agent, dyestuff, gene or carrier is imported viable cell.These methods comprise; But (used (Proc.Natl.Acad.ScI.USA1 77 (12): 7380-4 (1980)) like people such as Gordon to be not limited to microinjection; And extend to rabbit, sheep, ox and pig since then), the transfer of DEAE-VISOSE mediation, use co-precipitation and other technology of calcium phosphate, these technology are well known to those skilled in the art.Under another situation; Can expect centrifugal sperm and in another medium resuspension centrifugal sperm (although being based on identical or essentially identical buffer solution system), but some component (it possibly formerly be added into sperm dispersion) to remove interfere later stage operation steps.

Increasing spermoblast is well-known optoinjection (optoinjection) method to the special preferable methods of passing through property of affinity tag, and as at U.S. Patent number 6,753,161 is disclosed, and its content is quoted as a reference herein.Generally speaking, the optoinjection method is through using the pulses of radiation exposing cell instantaneously to change the method for cell thoroughly.Detection based on cell illumination is illuminated, is identified and locate cell, and uses the pulses of radiation irradiating cell that is enough to instantaneousization cell.For example, applied like the present invention, thus the affinity tag (for example, being bonded to the affinity tag of DNA or RNA) that can use instantaneousization of optoinjection spermoblast to make to be bonded to entocyte gets into cell more easily and effectively.Therefore, for example, can use optoinjection to reduce and use fluorescent DNA selective dye (like Hoechst 33342, Hoechst 33258) or use the abundant label sperm cells required time of fluorescent polyamide.

Optoinjection also is used in labeled cell under the temperature of reduction.Before, spermoblast had generally used, for example the fluorescent DNA selective dye surpass 30 ℃ with in addition 40 ℃ temperature under mark (because higher temperature helps to increase the dyestuff picked-up).That yes is feasible for mark under these temperature, and (time that is particularly prolonging) is useful under the higher temperature yet avoid spermoblast is exposed to.Therefore, optoinjection can being used for spermoblast, allow at a lower temperature labeled cell thus and still keep or surpass usually with higher temperature under relevant dyeing efficient and the speed of mark.

Therefore, in one embodiment, form the mark mixture that contains spermoblast, mobility suppressor factor and concentration dyestuff, and this dye mixture is kept for some time in about 41 ℃ temperature from about 100 μ M to about 200 μ M.In another embodiment, the mobility suppressor factor contains 0.204g NaHCO in every 25ml purified water ₃, 0.433g KHCO ₃With 0.473g C ₆H ₈O ₇H ₂O (NaHCO ₃0.097mol/L, KHCO ₃0.173mol/L, C ₆H ₈O ₇H ₂The aqueous solution of O 0.090mol/L).

In another embodiment, form the mark mixture that contains spermoblast, mobility suppressor factor and concentration dyestuff, and this dye mixture is kept for some time in about 41 ℃ temperature from about 400 μ M to about 500 μ M.In another embodiment, dye strength is 450 μ M.In another embodiment, the mobility suppressor factor contains 0.204g NaHCO in every 25ml purified water ₃, 0.433g KHCO ₃With 0.473g C ₆H ₈O ₇H ₂O (NaHCO ₃0.097mol/L, KHCO ₃0.173mol/L, C ₆H ₈O ₇H ₂The aqueous solution of O 0.090mol/L).

In another embodiment, form and contain spermoblast, mobility suppressor factor and concentration mark mixture, and this dye mixture is kept for some time in about 28 ℃ temperature from about 100 μ M to about 200 μ M dyestuffs.In another embodiment, the mobility suppressor factor contains 0.204g NaHCO in every 25ml purified water ₃, 0.433g KHCO ₃With 0.473g C ₆H ₈O ₇H ₂O (NaHCO ₃0.097mol/L, KHCO ₃0.173mol/L, C ₆H ₈O ₇H ₂The aqueous solution of O 0.090mol/L).

In a further embodiment, form the mark mixture that contains spermoblast, mobility suppressor factor and concentration dyestuff, and this dye mixture is kept for some time under about 28 ℃ temperature from about 400 μ M to about 500 μ M.In another embodiment, dye strength is 450 μ M.In another embodiment, the mobility suppressor factor contains 0.204g NaHCO in every 25ml purified water ₃, 0.433g KHCO ₃With 0.473g C ₆H ₈O ₇H ₂O (NaHCO ₃0.097mol/L, KHCO ₃0.173mol/L, C ₆H ₈O ₇H ₂The aqueous solution of O 0.090mol/L).

The formation of labeled cell dispersion-s

In case form the mark mixture, this mark mixture is used to form the dispersion-s of labeled cell, its to be detected immediately with use.This type dispersion-s comprises the spermoblast of mark and the chemical agent of inducing sperm not move.Alternatively, or additionally, dispersion-s can also comprise liquid except that the spermoblast of mark, for example aforesaid damping fluid, and wherein the temperature-induced sperm of cell or liquid does not move.

The spermoblast of mark can be any one of various ways.For example, the cell of mark can also be the part of mark mixture.In such cases, labeled cell also can be in unnecessary or unconjugated affinity tag.Perhaps, the cell of mark can be separates with any unnecessary or unlabelled affinity tag, for example through washed cell or through rotating cell (for example through centrifugal), and then cell is separated with unconjugated affinity tag.Under this type situation, labeled cell usually with collection like the preceding text cell sample in the damping fluid discussed mix.In the event in office, thus the spermoblast in the dispersion-s be labeled with one or more spermoblast bonded affinity tags lack or its quantity makes heredity, protein group, structure or the functional character that can identify subpopulation of sperm cells in the dispersion-s.This spermoblast can maintain and cause or increase sperm not under the temperature of mobility.

The dispersion-s of the cell of mark can also contain induces the not chemical agent of mobility of sperm, for example, and the mobility suppressor factor of as above discussing.Can this chemical agent be added mark mixture or labeled cell any time before the optical detection dispersion-s (for example, before the spermoblast mark, or afterwards).Can chemical agent be mixed with labeled cell, labeled cell is any one (that is, still in the mark mixture or from it, shifting out) of the various ways of above discussion.In concrete embodiment, the mark mixture of formation comprises spermoblast and affinity tag, and be about to the mark mixture with induce sperm not the chemical agent of mobility mix.With respect to chemical agent alternatively, or outside this, the temperature of the reduction mark mixture that can as above discuss is to cause or to increase not mobility of sperm.

The detection of cell, confirm and use

In case form the dispersion-s of labeled cell; This dispersion-s of optical detection is to identify the individual sperm cells as the subgroup member; Confirm the subgroup member's in dispersion-s position and energy-beam be passed to disperse intravital different positions optionally the energy being applied to the subgroup member, thus reduce by the viability of dosed cells or at least they the ability that fertilizes an egg and do not influence the spermoblast of other position in the dispersion-s simultaneously.

These steps are usually through equipment with commercial LEAP

(analysis of laser active and the processing) technology platform (Cyntellect that is called; Inc.; San Diego, method CA) is carried out.Generally speaking, this method needs applying marking substance markers cell to identify and to be positioned at the cell mixture or the individual cells of jumpbogroup inner cell subgroup more.Then illuminate cell mass, make the position of the individual cells that can identify subgroup.The localization process laser apparatus that continues makes it that the change of energy-beam with the identification of cell of inducing subgroup can take place.This inductive change is generally necrocytosis.These method and apparatus are at U.S. Patent number 6,534, are further described in 308,6,514,722,6,753,161 and 6,642,018, all quote as a reference herein.

The energy that uses among the present invention can be any source; When it is used for spermoblast with certain dosage; Can reduce by the viability of dosed cells, or their abilities of fertilizing an egg at least, and the spermoblast of other position of dispersion-s kind is had minimum or do not have similar influence.Usually, the energy is the form of energy-beam.The instance of the suitable energy comprises laser, the parallel or non-laser light that focuses on, RF energy (RF energy), the particle that quickens, ultrasonic energy, electron beam or other radiation beam of focusing.Yet preferred energy source is a laser, because laser has the HS energy and the relatively effective advantage of energy utilization and minimum heat production is provided in compact volume, thereby permission is used and disadvantageous effect peripheral cell indistinctively to individual cells.

Can cell be placed any surface that pair cell carries out optical detection and uses that is suitable for.Generally speaking, this type surface has the surface (top, bottom, or both whiles) of level, and it is permeable on the optics for the energy that is used for the optical detection cell with being used for to the energy that the subgroup member uses.The surface that this type is suitable comprises; For example glass, plastics or other relevant polymkeric substance; And Pyrex

, and can be the form of slide glass, petridish, single hole flat board or the porous plate of putting down.Instance is for example coming into question in the U.S. Patent number 6,534,308 and 6,514,722.

Can the sample of spermoblast be divided into several littler individual sample, for example be used for porous plate through being divided into many individual sample.Various samples (for example, in each hole) that can the enrichment same characteristic features produce thus and respectively are freed from a kind of characteristic and by a plurality of samples of enrichment.Yet, various samples that preferably can the enrichment different characteristics.For example, can sample of sperm cells be divided into littler individual sample, and each individual sample is placed a dull and stereotyped hole of 96 holes.Then can be with the independent sample in the every hole of a kind of characteristic enrichment different with other every hole sample, produce separately 96 individuals samples with the different character enrichment.

In case use the energy to use to member's cell of subgroup, can be through the further enrichment of cell crowd of the cell (spermoblast that does not promptly use energy to use) that purifying is not used.Can produce and contain subgroup through removing by dosed cells from dispersion-s or not carried out the not purifying of dosed cells by dosed cells by the not dosed cells of special characteristic enrichment.For example, if this special characteristic is the spermoblast that has Y chromosome, thereby the cell of can purifying not using its contain the spermoblast that has a Y chromosome at least about 85%; Preferably at least about 90% have a Y chromosome spermoblast; More preferably at least about 95% have a Y chromosome spermoblast; Even more preferably at least about 97% have a Y chromosome spermoblast; Most preferably at least about 99% have a Y chromosome spermoblast.Alternatively, if this specific desired character is the spermoblast that has X chromosome, thereby the cell of can purifying not using its contain the spermoblast that has an X chromosome at least about 85%; Preferably at least about 90% have an X chromosome spermoblast; More preferably at least about 95% have an X chromosome spermoblast; Even more preferably at least about 97% have an X chromosome spermoblast; Most preferably at least about 99% have an X chromosome spermoblast.

Any one of known several different methods realized removing dosed cells or dosed cells not from the dispersion-s used (promptly from comprising the dosed cells and the bigger population of sperm cells of dosed cells not) by one of skill in the art.These class methods comprise, rotation whole dispersion-s (for example through centrifugal) down for example, and then remove or remove the supernatant that contains dosed cells through wick effect.Another method comprises to dispersion-s and adds highdensity medium.The high-density medium that can add dispersion-s comprises, for example Percoll

and Isolate

.Generally speaking; In high-density separation; Viable cell (being the application's not dosed cells) can move about to the upper strata of high-density medium and can skimmed from the medium upper strata thereafter; And damaged cell or dead cell (being dosed cells) are dispersed in maintenance in the high-density medium, usually among the body phase.The method of using this type medium is well-known in the art.

Preferably the dispersion-s of labeled cell can contain the cell subsets that uses different affinity tag marks.Every kind of affinity tag can be identified different heredity, protein group, structure or the functional character of subpopulation of sperm cells in the dispersion-s.In addition, when being incorporated into spermoblast, every kind of affinity tag can be detected respectively, promptly different affinity tags can be detected respectively.For example, affinity tag can send fluorescence separately at different wave length.

Can add the different markers thing to the dispersion-s of mark mixture or labeled cell.Alternatively, can after arbitrary step of detection, mensuration or the step of applying of cell, add the different markers thing.Yet, preferably after the using of dispersion-s, add the different markers thing.For example; In case the subgroup member to spermoblast uses; Can be by the dosed dispersion (only comprising not dosed cells) of the dispersion-s used (comprise dosed cells and not dosed cells) or purifying by mark once more; But use the different markers thing, and usually disclosed like preceding text, can based on the lacking or the detection of quantity repetitive cell, measure and use of the different affinity tags of spermoblast bonded.

Generally speaking; Can use different affinity tags to identify not other heredity, protein group, structure or the functional character of dosed cells, said characteristic can be different from previous evaluation to the used characteristic of the subgroup member of its delivery of energy dosage (promptly be different from before confirmed to use or not the used characteristic of dosed cells).This provides the further enrichment approach of the cell mass of enrichment.

For example, use the fluorescent DNA selective dye can form the dispersion-s of labeled cell.Can follow this dispersion-s of optical detection has X chromosome with evaluation individual sperm cells.Confirming of can continuing has the position of the spermoblast of X chromosome, and then energy dose is delivered to one or more cell that has X chromosome more, obtains the viable cell crowd who has Y chromosome of enrichment thus.Thereafter; Can use dispersion-s that another affinity tag mark of showing acrosomal integnity uses ((the having Y chromosome) cell that comprises (having X chromosome) of using and do not use) or purifying by dosed dispersion (only comprising not dosed cells); The PNA (PE-PNA) puted together of phycoerythrin for example; When its when having the reaction or the cells contacting of impaired acrosome, the fluorescence of inducing cell fluorescence, particularly acrosome.Can then carry out the step of optical identification and definite PE-PNA fluorescence generation cell position, and impose energy to those cells.Generation have Y chromosome with have unreacted with damage (being complete) acrosome not by the subgroup of dosed cells.See people such as Nagy for example, Biol Reprod, 68:1828-1835 (2003).

The freezing prolongation (cryoextension) of cell

In case use the energy after member's cell of subgroup is used, whole population of sperm cells (by use and cell that do not used) or this crowd's subclass (cell of only not used) can be cooled or freezing (for example in the fertilization operation) for future use.In this case, adding freezing prolongation agent to minimize cooling or freezing influence to the viability or the back mobility of thawing, is useful to the spermoblast of not used.

Generally speaking, freezing prolongation agent can comprise protein source, cryoprotectant and mobility suppressor factor.If contain protein source, can it be added so that the cell support to be provided.Protein source can be any not dosed sperm cells viability and protein source compatible with the mobility suppressor factor of can not disturbing.The instance of common protein sources comprises breast (comprising hot homogenate and skimming milk), newborn extract, yolk, yolk extract, Sunlover 10 and soybean protein extract.This type of proteinic working concentration can be about 10% (v/v) to about 30% (v/v), and preferred about 10% (v/v) is to about 20% (v/v), more preferably from about 20% (v/v).

Preferably contain cryoprotectant in the freezing prolongation agent, to alleviate or to avoid freezing shock or the fertility of the spermoblast that keeps not used.Numerous cryoprotectant known in the art.Be suitable for multiple choices being arranged, depend on the species of treating the frozen sperm source with the common cryoprotectant that uses of given prolongation agent.The instance of suitable cryoprotectant comprise as, glycerine, DMSO 99.8MIN., terepthaloyl moietie, Ucar 35, trehalose, Triladyl and their combination.If contain cryoprotectant, then its amount in freezing prolongation agent is generally about 1% (v/v) to about 15% (v/v), and preferably its amount is extremely about 10% (v/v) of about 5% (v/v), and more preferably its amount is about 7% (v/v), and most preferred amount is about 6% (v/v).

In addition, freezing prolongation agent can contain the mobility suppressor factor of discussing as in the collection of preceding text cell sample.Can add the mobility suppressor factor to freezing prolongation agent according to it.

In a specific embodiments, freezing prolongation agent comprises mobility suppressor factor, water, Triladyl

, yolk and pyruvic acid.And in another embodiment, freezing prolongation agent comprises 0.097mol/LNaHCO ₃, 0.173mol/L KHCO ₃, 0.090mol/L C ₆H ₈O ₇H ₂The aqueous solution of O and in every 75ml water, contain 25g Triladyl , 25g yolk and 10mM pyruvic acid.

In another embodiment, freezing prolongation agent comprises mobility suppressor factor, water, Triladyl

and yolk.And in another embodiment, freezing prolongation agent comprises 0.097mol/L NaHCO ₃, 0.173mol/L KHCO ₃, 0.090mol/L C ₆H ₈O ₇H ₂The aqueous solution of O and in every 75ml water, contain 25g Triladyl

With 25g yolk.

Also can randomly contain in the microbiotic discussed in collecting like the preceding text cell sample or the adjusting cell in the freezing prolongation agent and/or the compsn of extracellular oxidation/reduction reaction.Can in view of the above every kind of additive be added in the freezing prolongation agent.

The refrigeration of the whole sperm group refrigeration of dosed dispersion (promptly through) makes and forms the frozen dispersion with two subgroups, and each subgroup differs from one another in fact.Yet each subgroup is made up of the cell of basic homogeneity.That is to say that each subgroup is made up of cell, each somatocyte of single subgroup have with identical subgroup in each other cell common characteristic.In preferred embodiments, through purifying dispersion-s further enrichment dispersion-s before refrigeration, said purifying is to carry out based on existing or lacking the common characteristic according to aforesaid method.

Therefore; For example; Can use present method to form the frozen sperm dispersion-s; Said dispersion-s comprises cell subsets of being used (whole cells of the subgroup of wherein using are the cells that has X chromosome) and the cell subsets of not using (whole cells of the subgroup of wherein not using are the cells that has Y chromosome).According to embodiment of the present invention, the cell (that is the cell that has Y chromosome of non-dosed) of not accepting energy dose comprises the spermoblast that has Y chromosome at least about 85%; Preferably at least about 90% have a Y chromosome spermoblast; More preferably at least about 95% have a Y chromosome spermoblast; Even more preferably at least about 97% have a Y chromosome spermoblast; Most preferably at least about 99% have a Y chromosome spermoblast.

Alternatively; Can use method of the present invention to form the frozen sperm dispersion-s; Said dispersion-s comprises through cell subsets of using (whole cells of the subgroup of wherein using are the cells that has Y chromosome) and the cell subsets (whole cells of the subgroup of wherein not using are the cells that has X chromosome) do not used.According to embodiment of the present invention, the cell of not using that has X chromosome can comprise the spermoblast that has X chromosome at least about 85%; Preferably at least about 90% have an X chromosome spermoblast; More preferably at least about 95% have an X chromosome spermoblast; Even more preferably at least about 97% have an X chromosome spermoblast; Most preferably at least about 99% have an X chromosome spermoblast.

Fertilization

The present invention also provides and fertilizes an egg or the novel method of the female mammal of inseminating, and uses this novel method selectivity to be reduced in the viability of subpopulation of sperm cells in the aforesaid cell dispersion-s usually.

In case the dispersion-s to labeled cell is used, can be with being used to make the female mammal fertilization through the dispersion-s used (comprise through that use with cell non-dosed).Can be fertilized according in the several different methods well known to those skilled in the art any one.These methods comprise, for example, and microinjection, artificial insemination and other method well known to those skilled in the art.For example, can with comprise use with dosed cells not through dosed dispersion, only comprise the dispersion-s of the purifying of dosed cells not or any one the verivate female mammal (for example through artificial insemination) that is used to inseminate among both.

Alternatively, in case accomplish using of labeled cell dispersion-s, can use this dispersion-s to fertilize an egg, more specifically be external ovum.Then can be through in multiple those skilled in the art's well-known process any one, zygote is imported the intrauterine of female mammal, for example through embryo transfer.For example, can with the dispersion-s of the dispersion-s of using, purifying or among both any one verivate be used for the external feritilization of ovum.Then can zygote be imported the intrauterine of female mammal.

Can be after accomplishing the using of dispersion-s soon; For example within about 7 days; Preferably within about 5 days, more preferably within about 3 days, also more preferably within about 2 days; With in concrete embodiment, carry out the in vitro fertilization of female mammal fertilization or ovum within about 1 day using of completion dispersion-s.Under this type situation, this dispersion-s can be without freezing (that is, this dispersion-s be fresh or comprises fresh spermoblast) before fertilization female mammal or ivf zygote usually; Instead it can remain in the mobility suppressor factor and/or refrigerate in about 2 ℃ to about 7 ℃, and more preferably from about 3 ℃ to about 5 ℃ and 4 ℃ temperature most preferably from about.Alternatively, can the freezing dispersion-s and before fertilization female mammal or ivf zygote, thawing of continuing (that is, this dispersion-s be frozen/thaw or comprise freeze/thaw spermoblast).Generally speaking, under this type situation, can be in the dispersion-s of thawing at once before and refrigerating in vitro fertilization of female mammal fertilization or ovum.

Preceding text have been described the present invention in detail, and it is obvious that, in not breaking away from the invention scope of accompanying claims definition, can modify and change it.

Claims

1. selectivity reduces the method for the ability that subpopulation of sperm cells fertilizes an egg in the sperm cell dispersion, and this method comprises:

A) with the spermoblast in the DNA selective dye label sperm cells sample in liquid, to form dispersion-s through the spermoblast of mark; This liquid comprise induce sperm not mobility chemical agent and/or have and induce the not temperature of mobility of sperm; The heredity, protein group, structure or the functional character that wherein show subpopulation of sperm cells in the dispersion-s with the quantity of spermoblast bonded affinity tag, and wherein in the dispersion-s density of sperm be 1 * 10 ³To 5 * 10 ¹⁰Between individual sperm/ml;

B) the optical detection dispersion-s is to identify that individual sperm cells is the subgroup member;

C) confirm the position of dispersion-s Central Asia group members; With

D) different positions reduces the ability that the subgroup member fertilizes an egg with selectivity in delivery of energy dosage to the dispersion-s, and does not influence the spermoblast of other position in the dispersion-s similarly.

2. method in vitro fertilization, this method comprises: in liquid, to form the dispersion-s through the spermoblast of mark, this liquid comprises with DNA selective dye label sperm cells sample

A) induce sperm not mobility chemical agent and/or have and induce the not temperature of mobility of sperm; The heredity, protein group, structure or the functional character that wherein show subpopulation of sperm cells in the dispersion-s with the quantity of spermoblast bonded affinity tag, and wherein in the dispersion-s density of sperm be 1 * 10 ³To 5 * 10 ¹⁰Between individual sperm/ml;

B) the optical detection dispersion-s is to identify that individual sperm cells is the subgroup member; With

C) confirm the position of dispersion-s Central Asia group members;

D) different positions reduces the ability that the subgroup member fertilizes an egg with selectivity in delivery of energy dosage to the dispersion-s, and does not influence the spermoblast of other position in the dispersion-s similarly; With

E) use said dispersion-s or derivatives thereof to make egg's external fertilization thereafter.

3. form the method for frozen sperm dispersion-s, this method comprises:

A) with DNA selective dye label sperm cells sample in liquid, to form dispersion-s through the spermoblast of mark, this liquid comprises

B) induce sperm not mobility chemical agent and/or have and induce the not temperature of mobility of sperm; The heredity, protein group, structure or the functional character that wherein show subpopulation of sperm cells in the dispersion-s with the quantity of spermoblast bonded affinity tag, and wherein in the dispersion-s density of sperm be 1 * 10 ³To 5 * 10 ¹⁰Between individual sperm/ml;

C) the optical detection dispersion-s is to identify that individual sperm cells is the subgroup member;

D) confirm the position of dispersion-s Central Asia group members;

E) different positions reduces the ability that the subgroup member fertilizes an egg with selectivity in delivery of energy dosage to the dispersion-s, and does not influence the spermoblast of other position in the dispersion-s similarly; With

F) refrigerate dispersion-s afterwards.

4. each method among the claim 1-3, wherein the quantity with spermoblast bonded affinity tag shows that spermoblast is the spermoblast that has X chromosome.

5. each method among the claim 1-3, wherein the quantity with spermoblast bonded affinity tag shows that spermoblast is the spermoblast that has Y chromosome.

6. each method among the claim 1-3, wherein said DNA selective dye is that UV or visible light can excite.

7. the method for claim 6, wherein said dyestuff is a DNA selectivity optical dye.

8. the method for claim 7, wherein said dyestuff is Hoechst 33342, Hoechst 33258 or SYBR-14.

9. each method among the claim 1-3, wherein energy dose is selected from radiation beam, laser beam, parallel non-laser light, the non-laser light of focusing and the ultrasonic energy of focusing.

10. each method among the claim 1-3, wherein said method comprise that also purifying do not accept the spermoblast of energy dose, the cell of promptly not used.

11. the method for claim 10, wherein the cell do not used of purifying comprises centrifugal dispersion-s and removes the cell of being used.

12. the method for claim 10, wherein the cell do not used of purifying comprises dispersion-s is contacted with high-density medium.

13. each method among the claim 1-3, wherein optical detection dispersion-s is to identify that individual sperm cells is that the subgroup member comprises optical detection captured cell image.

14. each method among the claim 1-3 wherein was distributed in dispersion-s on the porous plate before the optical detection dispersion-s.

15. the method for claim 14, wherein said porous plate are 96 or 384 orifice plates.

16. each method among the claim 1-3 is wherein compared with the spermoblast of not accepting energy dose, said energy dose is enough to reduce subgroup member's viability.

17. each method among the claim 1-3, wherein said energy dose is enough to cause subgroup member's death.

18. each method among the claim 1-3, wherein said method refrigerate dispersion-s after also being included in delivery of energy dosage.

19. the method for claim 2, wherein the in vitro fertilization of ovum carried out within 7 days, 5 days, 3 days, 2 days or 1 day after accomplishing the using of cell.

20. the method for claim 2, the dispersion-s of wherein said enrichment did not refrigerate before the fertilization of ovum.

21. the method for claim 2, the dispersion-s of wherein said enrichment refrigerated before the fertilization of ovum.

22. the method for claim 3, the spermoblast of wherein not accepting energy dose comprises at least 85% the spermoblast that has X chromosome.

23. the method for claim 3, the spermoblast of wherein not accepting energy dose comprises at least 85% the spermoblast that has Y chromosome.

24. the method for claim 3, the spermoblast of wherein not accepting energy dose comprises at least 90% the spermoblast that has X chromosome.

25. the method for claim 3, the spermoblast of wherein not accepting energy dose comprises at least 90% the spermoblast that has Y chromosome.

26. the method for claim 3, the spermoblast of wherein not accepting energy dose comprises at least 95% the spermoblast that has X chromosome.

27. the method for claim 3, the spermoblast of wherein not accepting energy dose comprises at least 95% the spermoblast that has Y chromosome.

28. the method for claim 3, the spermoblast of wherein not accepting energy dose comprises at least 97% the spermoblast that has X chromosome.

29. the method for claim 3, the spermoblast of wherein not accepting energy dose comprises at least 97% the spermoblast that has Y chromosome.

30. the method for claim 3, the dispersion-s that wherein will be used placed suction pipe before freezing.

31. each method of claim 1-3, said method also comprises:

The dispersion-s of using other affinity tag mark to be used, wherein with the existence of the said other affinity tag of spermoblast bonded, lack or quantity shows heredity, protein group, structure or the functional character of subpopulation of sperm cells in the dispersion-s of being used;

The dispersion-s that optical detection is used is to identify that individual sperm cells is the subgroup member;

The position of the dispersion-s Central Asia group members that definite quilt is used; With

Positions different in delivery of energy dosage to the dispersion-s reduce the ability that the subgroup member fertilizes an egg with selectivity, and do not influence the spermoblast of other position in the dispersion-s similarly.

32. each method among the claim 1-3, wherein the temperature through the spermoblast of mark is 0 ℃ to 15 ℃.

33. each method among the claim 1-3, wherein the temperature through the spermoblast of mark is 1 ℃ to 10 ℃.

34. each method among the claim 1-3, wherein the temperature through the spermoblast of mark is 2 ℃ to 8 ℃.

35. each method among the claim 1-3, wherein the temperature through the spermoblast of mark is 3 ℃ to 6 ℃.

36. each method among the claim 1-3, wherein the temperature through the spermoblast of mark is 4 ℃ to 5 ℃.

37. each method among the claim 1-3, wherein the temperature through the spermoblast of mark is 5 ℃.

38. each method among the claim 1-3, wherein the temperature through the spermoblast of mark is 4 ℃.