CN212426029U - Device for separating nucleated sperms and non-nucleated sperms of silkworms - Google Patents

Abstract

The utility model belongs to the technical field of the separator of lepidoptera insect second type sperm cell, concretely relates to device of separation silkworm nucleated sperm and no nuclear sperm. The utility model discloses a device of separation silkworm nucleated sperm and anucleate sperm, be in including cell sieve and setting cell sieve below, the sample collection device who is used for accepting the sample, sample collection device one end opening just the cell sieve with sample collection device's open end links to each other, the aperture of the screen cloth of cell sieve is 70-100 mu m. The device for separating the nucleated sperms and the anucleated sperms of the silkworms of the utility model can conveniently and efficiently realize the separation of the nucleated sperms and the anucleated sperms of the silkworms.

Description

Device for separating nucleated sperms and non-nucleated sperms of silkworms

Technical Field

The utility model belongs to the technical field of the separator of lepidoptera insect second type sperm cell, concretely relates to device of separation silkworm nucleated sperm and no nuclear sperm.

Background

Silkworms belong to one of the lepidopteran insects, and the sperms thereof have a dual type, i.e., nucleated sperms and anucleated sperms, which can fertilize eggs and develop into individuals; the anucleated sperm cannot fertilize the egg, and the nucleated sperm enters the mated female moth body before playing an auxiliary role in the process of fertilization of the nucleated sperm. Type II spermatozoa have significant differences in subcellular morphological structure and formation stage, but it is not clear at present what mechanism leads to differentiation of silkworm nucleated and non-nucleated spermatozoa.

The spermary of silkworm is the place where the sperm is formed by the internal reproductive organ of male silkworm. The surface of the silkworm spermary is distributed with a plurality of organ clusters. The outermost layer of the anatomy of the testis is a thin membrane called the adventitia. The inner surface is an inner membrane consisting of a layer of flattened cells, which is derivatized inward into three septa that divide the testis into four compartments, where sperm generation, maturation and formation occur.

In order to reveal the differentiation mechanism of the sperm type II, it is necessary to separately study nucleated sperm and non-nucleated sperm. In 1992 Minoru Osanai, Liangzheng et al used Percoll density gradient centrifugation to separate nucleated and non-nucleated sperm from male silkworm moth semen. The method needs density gradient centrifugation, the number of the obtained sperms is small, and the separation effect is not good, so the method is not widely applied. In 2015 Timothy l.karr et al elutriated nucleated and non-nucleated sperm of adult dipterus americanus but this method was not ideal in silkworms.

SUMMERY OF THE UTILITY MODEL

The utility model provides a device for separating nucleated sperms and anucleated sperms of silkworms, which solves the technical problem that the nucleated sperms and the anucleated sperms of silkworms can not be effectively separated in the prior art.

The utility model discloses a device of separation silkworm nucleated sperm and anucleated sperm adopts following technical scheme: the device for separating the nucleated sperms and the anucleated sperms of the silkworms is characterized by comprising a cell sieve and a sample collection device which is arranged below the cell sieve and used for receiving a sample, wherein one end of the sample collection device is open, the cell sieve is connected with the open end of the sample collection device, and the aperture of a screen mesh of the cell sieve is 70-100 mu m.

Preferably, the sample collection device contains TC-100 complete culture medium; the TC-100 complete culture medium also comprises FBS with the mass concentration of 10%.

Preferably, the mesh has a pore size of 70 μm.

Preferably, the number of the cell sieves is 2 or more.

Preferably, the cell screen is inverted over the sample collection device such that the sample trapped on the cell screen can be transferred from the cell screen by dipping the cell screen in a solution. By inverting the cell screen over the sample collection device, nucleated sperm passing through the screen of the cell screen and trapped on the screen can be transferred from the screen by simply dipping the cell screen in a solution (e.g., TC-100 medium). If the cell sieve is placed right, the nucleated sperms on the upper layer need to be washed from the bottom by the culture medium if the nucleated sperms are dissolved, so that a large amount of solution is consumed, the washing is not easy, the concentration of the sperms is low, the sperms need to be concentrated in a large volume, and the subsequent steps are inconvenient. Normally, during filtration, the screen is located at the bottom end (or near the bottom end) of the cell sieve as a whole; the term "upside down" as used herein means that when the cell sieve is used for filtration, the mesh of the cell sieve is located at the top of the cell sieve, and when the cell sieve is turned 180 degrees so that the cell sieve is upright, the sample on the mesh is more easily transferred, and when the cell sieve is immersed in a container containing TC-100 complete medium (liquid containing 10wt% FBS), the cell sieve is more easily contacted with the liquid, so that the sample can be transferred easily, without consuming a large amount of solvent and helping to maintain the concentration of the sample.

Preferably, the cell sieve is further provided with a handle.

The utility model has the advantages that: the device for separating the nucleated sperms and the anucleated sperms of the silkworms of the utility model adopts

The silkworm spermary cells are separated by the cell sieve of 70-100 mu m, so that nucleated sperms can be enriched above the screen mesh of the cell sieve, and the proportion of the nucleated sperms in samples on the screen mesh or the proportion of the collected non-nucleated sperms in the filtrate can be increased.

The cell sieve with the aperture of 70 mu m is adopted to separate the silkworm spermary cells, and the separation effect is good. When 2 layers of screens (cell screens) with the aperture of 70 mu m are arranged, a sample with the anucleate sperm ratio of 95 percent can be obtained, and the separation effect is good.

Through placing the cell sieve upside down in sample collection device top, the transfer of the sample that can be convenient realization is held back on the cell sieve screen cloth helps sparingly being used for washing the solution of screen cloth, also can effectively avoid the sample to be diluted.

The TC-100 complete culture medium contained in the sample collection device is helpful for maintaining the activity of nucleated sperms and anucleated sperms in the sample and avoiding damaging the sample.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.

FIG. 1 is a schematic structural view showing an exploded view of one embodiment of the apparatus for separating nucleated and non-nucleated spermatozoa of silkworms according to the present invention;

FIG. 2 is a plan view of a cell sieve according to one embodiment of the apparatus for separating nucleated and non-nucleated spermatozoa of silkworms of the present invention;

FIG. 3 is a schematic cross-sectional view of one embodiment of the apparatus for separating nucleated and non-nucleated spermatozoa of silkworms according to the present invention;

in the figure: 1-cell sieve, 11-upper surface of cell sieve, 12-lower surface of cell sieve, 13-screen, 14-handle and 21-convex edge.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative work belong to the protection scope of the present invention.

Example 1

As shown in fig. 1-2, the device for separating nucleated sperm and anucleated sperm of silkworms of the present invention comprises a cell sieve 1 and a sample collection device 2 disposed below the cell sieve 1 and used for receiving a sample, wherein one end of the sample collection device 2 is open, the cell sieve 1 is connected with the open end of the sample collection device 2, and the aperture of the screen 13 of the cell sieve 1 is 70-100 μm.

The cell sieve is provided with a screen 13 on both the bottom and side surfaces thereof to improve the efficiency of sample separation (or may be provided with a screen only on the bottom of the cell sieve 1).

In practical use, a person skilled in the art can select a culture dish (for example, a small 35mm culture dish) or a centrifuge tube as the sample collection device 2 as required, so that the size of the sample collection device 2 can be matched with the cell sieve 1 (based on the fact that the cell sieve can be fixed above the sample collection device 2, the cell sieve can also be fixed by means of a bracket or a fixed connection structure which is common in the art, and details are not described here). In order to facilitate taking, grasping and the like of the cell sieve 1, a handle 12 may be further provided on the cell sieve 1. In addition, in order to improve the tightness of the connection between the cell sieve and the sample collection device, a fastening tape, a clip (a fastening member such as a fastening tape or a clip may be a commercially available member), or the like may be used to reinforce the connection.

The utility model discloses a separation silkworm nucleated sperm and anucleated sperm's device's theory of use: taking a sample obtained by dissecting from a silkworm spermary (cells including nucleated sperms and non-nucleated sperms are dispersed in a solution, wherein TC-100 complete culture medium is selected to disperse silkworm spermatids), pouring the sample into a cell sieve 1, filtering the sample by the cell sieve 1, transferring the sample intercepted on a screen 13 of the cell sieve 1 into the TC-100 culture medium, centrifuging, discarding redundant supernatant, leaving 50-100 mu L, lightly blowing and uniformly spotting on a glass slide by using a sheared gun head, drying in the air, fixing for 5min by using 4% paraformaldehyde PBS solution, staining for 1 min by 10 mu g/mL DAPI aqueous solution, covering a cover glass, sealing with glycerol, and taking a picture by using a fluorescence microscope to observe the separation effect of the nucleated sperms and the non-nucleated sperms in the sample.

Similarly, the sample in the sample collection device 2 is centrifuged, the excess supernatant is discarded, 50-100 μ L of the sample is left to be lightly blown and evenly spotted on a glass slide by a cut gun head, the sample is fixed for 5min by using 4% paraformaldehyde PBS solution after being dried, the sample is stained for 1 min by using 10 μ g/mL DAPI aqueous solution, a cover glass is covered, glycerol is sealed, and the separation condition of the nucleated sperm and the non-nucleated sperm in the filtrate can be obtained by taking pictures by a positive fluorescence microscope.

The experimental results show that: when the aperture of the screen 13 of the selected cell sieve 1 is 70 μm, 78% of the nucleated sperm are contained in the sample remaining above the screen 13; when the aperture of the screen mesh 11 of the selected cell sieve 1 is 100 μm, the filtrate collected in the sample collection device 2 contains 85% of the anucleated sperms (before the device for separating the nucleated sperms and the anucleated sperms of silkworms of the utility model is used for separation, the ratio of the nucleated sperms and the anucleated sperms in the sperms of silkworms is close to 1: 1).

Example 2

On the basis of embodiment 1, the device for separating nucleated sperms and anucleated sperms of silkworms of the utility model comprises 2 cell sieves 1 and a sample collection device 2 arranged below the cell sieves 1 and used for receiving samples, wherein one end of the sample collection device 2 is open, the cell sieves 1 are connected with the open end of the sample collection device 2, and the aperture of the screen mesh of the cell sieves 1 is 70-100 μm.

The experimental result shows that when the 2-layer cell sieve 1 is adopted, if the aperture of the screen 13 of the cell sieve 1 is 70 μm (the aperture of the screen of the 2-layer cell sieve is 70 μm), the proportion of the anucleated sperms in the filtrate collected in the sample collection device 2 can reach 95%; if the aperture of the screen 13 of the cell sieve 1 is 100 μm (the apertures of the screens of the 2-layer cell sieves are all 100 μm), the sample remaining on the screen 11 contains only 66% of nucleated sperm; when the device for separating the nucleated sperms and the anucleated sperms of the silkworms of the utility model is provided with 2 layers of cell sieves 1 with the screening aperture of 70 mu m, the sample with the anucleated sperms proportion of 95 percent can be obtained through separation, and the separation effect is good.

Example 3

Based on the embodiment 1 or 2, as shown in the attached figure 3 of the specification, the cell sieve 1 can be placed upside down above the sample collecting device 2 (the upper surface 11 of the cell sieve 1 is in contact with the upper surface of the sample collecting device 2; then the lower surface 12 of the cell sieve is positioned above the whole device), after the sample is processed by the device for separating the nucleated sperms and the anucleated sperms of the silkworms of the utility model, the cell sieve 1 is turned over 180 degrees, the lower surface 12 of the cell sieve is immersed into the complete culture medium containing TC-100, and the sample is slightly dipped or shaken in the liquid, so that the sample trapped at the position of the screen 13 can conveniently enter the complete culture medium containing 10% of FBS, no large amount of liquid (for example, the complete culture medium containing 10% of TC-100) is needed to be washed, and the dilution of the sample caused by the FBS can be effectively avoided, increasing the difficulty of subsequent operations.

As shown in FIG. 3, to enhance the stability of the connection between the cell sieve 1 and the sample collection device 2, the sample collection device 2 may be selected to have an outwardly protruding ledge 21 to increase the contact area between the upper surface 11 of the cell sieve 1 and the upper surface of the sample collection device 2, thereby enhancing the stability.

The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention, and any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (6)

1. The device for separating the nucleated sperms and the anucleated sperms of the silkworms is characterized by comprising a cell sieve and a sample collection device which is arranged below the cell sieve and used for receiving a sample, wherein one end of the sample collection device is open, the cell sieve is connected with the open end of the sample collection device, and the aperture of a screen mesh of the cell sieve is 70-100 mu m.

2. The device for separating nucleated and anucleated bombyx mori sperm of claim 1, wherein said sample collection device contains TC-100 complete medium; the TC-100 complete culture medium also comprises FBS with the mass concentration of 10%.

3. The apparatus for separating bombyx mori nucleated sperm and anucleated sperm according to claim 1, wherein the mesh has a pore size of 70 μm.

4. The apparatus for separating nucleated spermatozoa of silkworms and anucleated spermatozoa according to any one of claims 1 to 3, wherein the number of said cell sieves is 2 or more.

5. The device for separating nucleated spermatozoa from anucleated spermatozoa of silkworms according to claim 4, wherein the cell sieve is inverted over the sample collection device such that the sample trapped on the cell sieve screen can be transferred from the cell sieve by dipping the cell sieve in a solution.

6. The apparatus for separating bombyx mori nucleated sperm and anucleated sperm according to claim 5, wherein said cell sieve further comprises a handle.

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