JPS60114137A - Concentrating separation of x, y sperms of mammal - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the extraction of spermatozoa having an X-chromosome (hereinafter abbreviated as "xx sheath") and spermatozoa having a Y-chromosome (hereinafter "Y-sperm sperm tract") from mammalian semen. The present invention relates to a method for concentrating and separating X and X spermatozoa by concentrating and separating them, respectively.Specifically, the present invention uses a discontinuous density gradient medium and uses a new principle that utilizes the difference in the passing speed of X and X sperm at a density interface. The present invention relates to a method of roughly separating the two based on the following.

Although it is extremely difficult to completely separate mammalian semen into This is important in that it makes it possible to statistically select the gender of the person.

In 1270, the F-body method for human sperm was discovered as a method for determining x and
It has become possible to apply this method to mammalian spermatozoa, and it has become relatively easy to consider separation techniques.

Conventionally, methods for concentrating and separating mammalian X and Y spermatozoa include:
Several methods are known, but none of them
lko and Y'f? This method is based on the premise that there are differences in cell properties such as density (specific gravity) and size among I cells.

For example, Tokko Akira! ;-X proposed by "Method for controlling the sex of mammalian offspring" described in No. 362-7
Similar to the present invention, the method for preparing Y spermatozoa uses a density gradient medium formed in a bottomed tube, but it is clear that
It is based on the use of the density difference between Y spermatozoa.

That is, the separation method described in the above patent publication prepares λ density media each having a predetermined density in each container, suspends these containers from a pulley, and when one container is raised, the other container is the same. the density medium in each container is simultaneously introduced into the mixing chamber by its static pressure;
X by successively liquefying the mixing proportion of each density medium using a device configured to introduce the density medium from the mixing chamber into a bottomed tube.

This is a method in which a density gradient medium that continuously changes within the range of the density difference between Y spermatozoa is formed, semen is introduced into the center of the density gradient medium, and the two are separated by buoyancy due to the density difference between x and Yf77 offspring. .

However, using a continuous density gradient medium, X.

In the above-mentioned known method based on the utilization of the density difference between Y spermatozoa, the X and Y spermatozoa must be in an immobile state, and therefore, the method described in the above-mentioned patent publication also applies; It requires cooling the density gradient medium and the mucus introduced into it to a low temperature at which the sperm become immobile.

However, in such cooling treatment, the resulting temperature shock has a detrimental effect on sperm viability and reproductive ability. Of course, the cooling process is carried out as late and as carefully as possible, but it is still difficult to completely eliminate temperature shock. Furthermore, performing such cooling treatment each time makes the separation operation complicated.

Furthermore, in the method described in the above patent publication, the iF7 liquid is mixed in advance with an appropriate nutrient medium in order to equalize the density of the semen introduced into the center point of the density gradient medium. However, such treatment is also inconvenient in terms of separation operation.

The present invention has a PH of X and Y sperm that eliminates the above-mentioned drawbacks.
In order to provide the I method, we have intensively studied the 7'c improvement method using a density gradient medium, and we have developed a discontinuous density gradient method.
We obtained the fundamental finding that there is a relatively large difference in the speed at which the X+Y spermatozoa pass through the density interface between the lifting platform and the density interface of the X+Y spermatozoa in the body.

The present invention was completed as a result of further studies based on the above knowledge, and its purpose is to create a concentrated fraction n' of X and Y sperm based on the new hλ theory, which does not require cooling of semen. [
The purpose is to provide a method.

Therefore, according to the present invention, the density difference of each layered medium as a density gradient medium is o, o o sS'/
Using a discontinuous density gradient medium of m1 or more, Q, and consisting of 5 or more layers, semen containing mucus with an This can easily be achieved by layering the semen in layers, applying a centrifugal force of 100 G or more to concentrate and distribute the semen containing the X-chromosome below the enrichment gradient medium, and then separating both appropriately. .

The present invention will be explained in detail below.

Various conventionally known density media can be used as the density medium used in the present invention. - In other words, as a density medium, in accordance with the knowledge in the field, in order not to have a detrimental effect on the viability and fertility of spermatozoa,
t,o-g,.

Any material having a pH of 2 tO to 300 mosM and an osmotic pressure of 2 tO to 300 mosM may be used. Of course, it is also necessary that the density medium has a suitable viscosity. Specifically, Tokko Akira! ! -3
Various liquid media described in Publication No. 6.29/ and colloidal dispersion media described below can be used. for example,
Milk is a typical liquid medium, but aqueous solutions of egg yolk, textrose, coconut cream, tomato juice, glucose, fructose, lecithin, and amino acids may also be used. Furthermore, as the colloid dispersion medium, it is possible to use various colloid dispersion liquid media that have the above-mentioned physical properties and are used for the separation of various substances related to living organisms, but in particular, colloid dispersion media such as those from FARMA JAPAN A colloidal dispersion medium commercially available under the tradename "PerCOll" is suitable.

This media is coated with polyvinylpyrrolidone
It is a dispersion of colloidal phosphorus with a diameter of ~30 nm,
It has an extremely low viscosity (1~/1 cp) and is one of the most suitable density media for the separation method of the present invention.

In the method of the present invention, the density media as described above are layered in a bottomed tube in order from the heavy medium, and the density difference of each MN medium is o,
A discontinuous density gradient medium having ootφ1 or more and consisting of five or more layers is formed.

The density range from the top layer to the bottom layer of a discontinuous density gradient medium is
Since the separation method of the present invention does not utilize the density difference between X and X sperm, it is not particularly limited in principle, but in general, the density is similar to that of the mammal to be separated. range. From the point of view of centrifugation processing and collection processing, which will be described later, the fineness of the top layer should be such that when semen is layered thereon, X and X spermatozoa do not settle under normal conditions, and
The density of the lowest layer is preferably such that X spermatozoa normally settle. Specifically, /, 0 /~/,
Or? For bovine semen in the /ml range, the density of the top layer is approximately 0.3 ml, and the density of the bottom medium is approximately 0.03 ml.

Is the density difference between each multilayer medium 0.00 j? /frL1 or higher is required, and this is it! In the case of 1/J1, the situation is similar to that of a continuous density gradient medium, and it is difficult to substantially separate spermatozoa with <X, , and On the other hand, if the density difference between each multilayer medium is too large, it may not be possible to secure the necessary number of multilayer stages, which will be described later, when the entire range of the discontinuous density gradient medium is set to the above-mentioned preferred range. Taking this into consideration, it is usually set to 0.0/f/ml or less. The preferred density difference between each layered medium is 0.007 for cow semen.
~θ,001? /rul range.

Since the separation method of the present invention utilizes the density interface, the thickness of each multilayer medium does not need to be particularly large.In fact, if it is too large, the thickness x
The yvI particles may be remixed on the way to the next density interface. Therefore, the thickness of each multilayer medium is determined to be 10 mrr, preferably 3 to tra, considering the ease of multilayer operation.
selected from a range of n.

The number of stages of the multilayer media forming the discontinuous density gradient medium must be 4 or more, and if the number of stages is less than this,
It is not possible to form a sufficiently concentrated distribution of X,X sperm. In other words, it is not possible to separate X spermatozoa with high purity. The higher the number of layers in the layered medium, the higher the purity of the sorted sperm, but on the other hand, the recovery rate decreases, so it is usually
The range is 5 or less, preferably A-12.

Layering of density media can be done by the glaze method, but it is easier to layer media of various densities prepared in advance by using a pipette or a pipette 2 and passing them through the tube wall of a bottomed tube of ++ m order. It is.

Various types of bottomed pipes can be used, including those with a valve structure at the bottom, but in general, pipes with an appropriate diameter,
For example, a test tube with a diameter of 1010-20 is used.

In the method of the present invention, semen is layered on the top layer of a specific discontinuous density gradient medium formed as described above, and a predetermined centrifugal force is applied to reduce the amount of X in the semen.

This is to concentrate and separate X sperm.

Centrifugal force is expressed as mW21, where the angular velocity is (W), the mass is (m), and the distance from the rotation axis is (1).
In the present invention, (1) is the center point 1 of the bottomed tube from the rotation axis.
means the distance in

Therefore, in the method of the present invention, the centrifugal force applied to the discontinuous density gradient medium is within a range of values that allows X, Y sperm to pass through the density interface and maintain the concentrated distribution state formed according to the difference in passing speed. selected from. Specifically, a centrifugal force of at least 10 OG is required, and the upper limit varies depending on the number of stages of the discontinuous density gradient medium, the density difference between each layered medium, and the duration of action of the centrifugal force, but it is usually less than 10 OG. be done. The preferred centrifugal force is 200~
This is the range of JOOG. Also, the action time of centrifugal force is
200~300G, which is selected as appropriate by the above-mentioned Token
In the case of centrifugal force of usually 1O-Il, 0 minutes, preferably,
It is said to be in the range of 20 to 30 minutes.

Furthermore, the separation method of the present invention is completely different from the method that utilizes the density difference between X and Y spermatozoa, and there is no need to cool down either the discontinuous density gradient medium or the semen layered thereon. The separation operation is carried out at room temperature. Incidentally, semen to be subjected to separation with a high viscosity is preferably diluted to a viscosity equal to or lower than that of the density medium used.

X in the semen layered on top of the discontinuous density gradient medium,
Y sperm passes through the density interface due to centrifugal force, but at that time, X:! Since the passing speed of J sperm is relatively thicker than that of Y sperm, X
Sperm are concentrated and distributed, and x+ YH offspring are roughly separated from each other. The thus concentrated and separated spermatozoa are collected from the top or bottom of the bottomed tube by appropriate means using a pipette or syringe.

When fractionating X and Y spermatozoa, whether fractionation is performed using a discontinuous density gradient medium has a direct effect on the purity and recovery rate of the target spermatozoa, but based on the data of preliminary tests conducted under the same conditions, It can be arbitrarily selected based on the

The separation method of the present invention can be applied to semen of all mammals, and specific examples include semen of cows, pigs, sheep, rabbits, dogs, foxes, and the like.

According to the uninvented method described above, there is no need for cooling treatment of semen and density gradient medium, which is essential in conventional methods, so the separation operation is not only simple, but also does not impair the viability or reproductive ability of sperm. However, the present invention will greatly contribute to the field of dairy farming and the like.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples unless the gist thereof is exceeded.

Example [Formation of discontinuous density medium] tPθrco11” (commercially available from Pharmacia Japan)
Percoll, trade name) stock solution (density: i, tJo±0.0
03 P/shoulder j) was mixed with 1 volume of 10 times concentrated culture solution, pH 7, ta, 70% of osmotic pressure 3θOmosM (
V/V) A Percoll solution was prepared. This was further diluted with culture solution by changing the magnification, i, oz~/, IO1/r
Percoll solution density media with a series of densities in the TTL range were created.

As a culture solution, use 20mMHF! PKS, / OmM
Glucose, +2mM Na2HPO4, 2mM Mg
O12,,2mMQac12.2. jytl N-K
OH and 7. jm Aqueous solution containing/containing N-NaOH (μ
(7, ψ, λ = O~300 mOsM) was used.

Each of the above density media o, 7! Sequentially starting with the heavy medium using a pipette/! In a test tube with a minor diameter? The layers were layered to form a microdiscontinuous density gradient medium with the following characteristics:

Thickness of each multilayer density medium: Approximately 2cm Density difference:
0.007t ~ Number of layers of multilayer medium: 5D Density of top layer: Approx. 0 J Ot/Mg Density of bottom layer: Approx. 100 (Separation of X, Y sperm) Immediately after being collected from a dairy bull Approximately 0.3*1 semen was collected with a pipette and gently layered on the top layer of each using the discontinuous density gradient medium moss described above.

Insert this test tube into the holder of the centrifuge, set the other two test tubes in the holder, balance them, and 2! The centrifugal force of OG was applied for 2j minutes.

Next, from the test tube layered with semen, each density medium was removed from the top with an 11th order pipette, X and Y spermatozoa were identified by quinacrine fluorescent staining, and the percentages of X and Y spermatozoa in each density medium were determined. The results shown in the table below were obtained.

(Note) The number of columns in the table is the number from the top layer to the bottom layer.

The spermatozoa (%) in the table is a value with the spermatozoa identified in each stage number as ioo.

The sperm identification method using the quinacrine fluorescent staining method employed in the above examples was carried out in the following manner.

(1) Removal of Percoll Percoll is removed to facilitate observation of F-body.

To remove Percoll, after diluting it several times with each fractional culture solution, centrifugal force was applied for 10 minutes under the conditions of GOOa.
Next, the precipitate is collected and resuspended in culture solution/d to be used as a sample.

(2) Quinacrine fluorescently stained sample was mixed with ≠000 times diluted papain solution and a small amount of anionic surfactant, reacted at 37°C for 5 minutes, then immediately stopped the reaction, and then placed on a non-fluorescent slide glass. After drying, fix using Carnoy's fixative.

This is immersed in O1θOj% (vv) quinataline mustard solution and dyed for 20 minutes. Next, Mac Engineering 1
The cells are washed three times with vaine buffer (pH 7,77), sealed with a cover glass using the internal solution, and then observed and measured using a fluorescence microscope.

(3) Judgment This staining method shows that the flu is located approximately in the center of the sperm head.
It is determined that the sperm is a YB7 child with an orescent body (F-body) observed.

per sample! Create specimens, observe over 200 sperm in each specimen, and calculate the ratio of x and y sperm (
For the X sperm, subtract the Y sperm from the observed mucus.)
.

Applicant: Hideo (name: name) Agent: Patent attorney Hase - (7 others)

Claims (1)

[Claims] (X) A method of concentrating and separating mammalian X and X sperm using a density gradient medium obtained by sequentially layering an electric medium in a bottomed tube. /rn1 or more and using a discontinuous density gradient medium consisting of 5 or more layers, and after layering semen containing spermatozoa having an X-chromosome and spermatozoa having a Y-chromosome on the top layer of the density gradient medium. / Concentrated separation of mammalian X, X sperm, characterized by applying a centrifugal force of 00 G or more to concentrate and distribute semen containing the X chromosome below the density gradient medium, and then separating both appropriately. Method.