JP5334033B2 - How to save mammalian sperm - Google Patents

Description

  The present invention relates to a method for preserving mammalian sperm.

  With the advent of the post-genomic era, the production of genetically modified mice (transgenic / knockout mice) has increased dramatically as a tool for clarifying gene functions. Along with this, sperm cryopreservation has been carried out worldwide as preservation of these mouse strains (lines). Since cryopreservation of mouse spermatozoa was the first in the world by Tada et al. In 1990 using raffinose as a cryoprotectant, Nakagata et al. Improved the cryopreservation method using raffinose, and now It is widespread worldwide as 'Katagata' (Non-Patent Documents 1 and 2). In addition, there are increasing opportunities for transportation between research institutions such as universities in the state of frozen sperm.

However, in conventional cryopreservation, liquid nitrogen is mostly stored, and liquid nitrogen must be periodically replenished. Moreover, when transporting frozen spermatozoa, it must be carried in a special transport container such as a dry shipper, and liquid nitrogen needs to be replenished.
Furthermore, there are strain differences in the viability of mouse sperm after cryopreservation and fertilization rate after in vitro fertilization, and in particular, the survival rate after freezing-thawing of C57BL / 6J mice, which is a general genetic background of genetically modified mice. It has been revealed that the fertilization rate is extremely low (Non-patent Document 3).

On the other hand, a successful example of litter production by ICSI using freeze-dried mouse sperm has been reported by Wakayama et al. This lyophilization storage method is also attracting attention as a method that can replace the above cryopreservation method. However, a relatively expensive vacuum dryer is necessary, sperm DNA damage due to lyophilization is remarkable, and room temperature. There is a problem that long-term storage under is impossible (Non-Patent Document 5).
Tada N. et al. , J .; Reprod. Fert. 89, 511-516, 1990 Nakagata N. et al. & Takeshima T. , Exp. Anim. 42, 317-320, 1993 Thorton CE. et al. Mamm. Genome, 10, 987-992, 1999 Sztein JM. et al. Biol. Reprod. 63, 1774-1780, 2000 Wakayama T.W. & Yanagimachi R. Nat. Biotechnol. 16, 639-641, 1998

  An object of the present invention is to provide a new storage method capable of stably storing mammalian sperm for a long time at room temperature.

Sperm after storage can be fertilized by ICSI method (intracytoplasmic sperm injection: microcytosemination method) even if no motility is observed, and normal pups can be obtained. In storage, it is important to suppress chromosomal abnormalities and DNA damage in the sperm nucleus.
The present inventor conducted various studies on methods for preserving mammalian sperm while retaining fertilization ability, and after opening a hole in the cell membrane of sperm collected from the mammal by α-hemolysin treatment, disaccharide or trisaccharide was added. It was found that it can be stored for a long period of time even at room temperature without causing chromosomal abnormalities or DNA damage in sperm by allowing it to flow into cells, incubating it for a certain period of time, and then air drying. And the sperm preserve | saved by the said method was fertilized by carrying out micro insemination (ICSI) to an unfertilized egg, discovered that it could produce a litter, and completed this invention.

  That is, the present invention relates to preservation of mammalian sperm, characterized in that sperm collected from mammals is treated with α-hemolysin to allow disaccharides or trisaccharides to flow into the cells, followed by incubation for a certain period of time and then air drying. A method is provided.

  According to the present invention, stable long-term storage and transportation of mammalian sperm at room temperature is possible. Moreover, the method of the present invention does not require energy such as liquid nitrogen or dry ice and special containers for storage and transportation of mammalian spermatozoa, and is therefore inexpensive and simple.

  In the present invention, first, sperm is collected from a mammal. Here, the mammalian sperm to be subjected to the preservation method of the present invention may be any mammalian sperm, and examples of mammals include humans, monkeys, cows, mice, rats, and rabbits. Of these, mice are preferable, and genetically modified mice are particularly preferable.

As a method for collecting sperm from mammals, for example, the epididymis tail is collected from the testis of mammals, and this may be cut into pieces in a preservation solution. Thereby, the suspension in which the sperm is suspended in the preservation solution can be prepared. This sperm suspension is preferably sliced from the epididymal tail, allowed to stand at room temperature for about 5 minutes, and then removed from the suspension by centrifugation or the like.
The number of sperm in the suspension is preferably 1 to 5 million / ml, particularly 4 to 5 million / ml.

In order to prevent sperm chromosome abnormalities and DNA damage, the preservation solution is prepared by adding a chelating agent such as EGTA or EDTA to a buffer such as Tris-HCl buffer, Hepes buffer, or phosphate buffer; glucose, sucrose, lactose, It preferably contains saccharides such as trehalose and raffinose.
The concentration of the chelating agent in the preservation solution is preferably 50 to 100 mM. Moreover, it is preferable that the density | concentration of the saccharides in a preservation | save liquid is 0.5-1.0M.

  The preservation solution may further contain inorganic salts such as potassium salt, sodium salt, calcium salt and phosphate; organic acids such as Hepes and pyruvic acid; antibiotics such as penicillin and streptomycin; various amino acids and serum. . In particular, from the viewpoint of preventing oxidative stress of sperm during drying and storage, it is preferable to contain 100 to 200 mM of green tea-derived polyphenols such as epigallocatechin gallate, epicatechin, catechin, and epigallocatechin. Specifically, a cryopreservation solution such as Tris-HCl-EGTA, a culture solution such as NIM (nucleus isolation medium), mCZB-Hepes is used as the preservation solution.

  Next, 6 to 20 μg / ml, preferably 10 to 15 μg / ml of α-hemolysin solution is added to the sperm suspension, and then allowed to stand for 15 minutes to 1 hour in the dark at room temperature. This treatment opens micropores in the sperm cell membrane. It is preferable to use α-hemolysin after dissolving it in the above preservation solution from the viewpoint of preventing chromosomal abnormalities of sperm and DNA damage. After the treatment, it is preferable to remove the supernatant by centrifugation.

Subsequently, after adding disaccharide or trisaccharide, it incubates for a fixed period. As a result, these saccharides are taken into the cell membrane from the micropores opened in the sperm cell membrane. After the α-hemolysin treatment, these saccharides quickly flow into the cells, thereby suppressing chromosomal abnormalities and DNA damage in the sperm nucleus.
Examples of the disaccharide or trisaccharide used here include disaccharides such as sucrose, maltose, lactose and trehalose; and trisaccharides such as raffinose. These can be used alone or in combination of two or more. Of these, sucrose, trehalose, and raffinose are preferable, and trehalose is particularly preferable.

  These saccharides are preferably added to the suspension so as to be 0.2 to 1.5M, preferably 0.4 to 1.2M. In addition, it is preferable to use these saccharides by dissolving them in the above-mentioned storage solution from the viewpoint of preventing chromosomal abnormalities of sperm and DNA damage.

  The incubation period is preferably 0 ° C. to room temperature, preferably 4 ° C. to room temperature for 20 minutes to 10 days, preferably 30 minutes to 7 days from the viewpoint of preventing sperm chromosome abnormalities and DNA damage. .

  The sperm suspension is then dropped onto the plate and allowed to air dry. The plate is not particularly limited, and examples thereof include a culture plate such as a culture plate, a microplate, and a slide glass. The sperm suspension is preferably dropped on the plate in an amount of 20 to 50 μl.

The method of air-drying the sperm suspension is sufficient if water in the suspension is removed, and examples thereof include natural drying, heat drying, and vacuum drying. Especially, it is preferable to air-dry (air-dry) at 20-50 degreeC from the point which prevents damage to a sperm DNA, Preferably it is room temperature (20-25 degreeC).
The drying time is preferably about 30 minutes to 2 hours, although it depends on the drying method, the type of plate, and the amount of suspension on the plate.
The water content of the suspension after drying is not particularly limited, but is preferably 35% or less from the viewpoint of storage stability.

  The dried sperm suspension is preferably stored in a desiccator (room temperature, vacuum) or in a refrigerator (about 4 ° C.) in a low humidity and low oxygen atmosphere, for example, in an aluminum foil or gas barrier film.

  The sperm thus preserved retains fertilization ability and can be fertilized by microinsemination, preferably by the ICSI method, on unfertilized eggs after condensing at the time of use. The condensate is not particularly limited as long as it does not affect the fertilizing ability of sperm, and for example, pure water, the above-described preservation solution, or the like can be used.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1 [Dry storage of sperm]
(1) Collect the epididymis tail on both sides from 8-16 weeks old BDF1 mice, put it into 1.0 ml of each preservation solution shown in Table 1 in the culture dish, and collect spermatozoa. did. After standing at room temperature for 10 minutes, the epididymis tail was removed, and then the sperm suspension was transferred to a microtube.
(2) The microtube was centrifuged (10,000 rpm, 5 minutes, room temperature), and then the supernatant was discarded. Next, 500 ml of each stock solution was added to the sperm mass, and light pipetting was performed to prepare a sperm suspension.
(3) α-hemolysin (final concentration 12 μg / ml) dissolved in 500 ml of each stock solution was added to the tube at room temperature, protected from light, and then treated for 30 minutes. After the treatment, the supernatant was discarded by centrifugation (10,000 rpm, 5 minutes, room temperature).
(4) Next, 200 μl (0.5 to 1.0 M) of a trehalose solution dissolved in each preservation solution was added to the tube, and after light pipetting, the tube was allowed to stand at room temperature for 30 minutes. The tube was left on ice until dry.
(5) 50 μl of sperm suspension was dropped into each well of a culture plate (24 well, # 35-3847, manufactured by Falcon). Further, 20 μl each was dropped on a slide glass (micro slide glass, ring mark, 76 × 26 mm, Matsunami). After dropping, the culture plate was air-dried for 2 hours and the slide glass for 30 minutes in a safety cabinet at room temperature.
After air drying, the culture plate was wrapped with aluminum wheels and stored in a vacuum desiccator (room temperature). Also, slide glass covers the dried sperm suspension with a cover glass (HybriSlip Hybridization Cover, 22 x 22 mm, Funakoshi), sealed with parafilm, put in a polyethylene bag, put into a vacuum pack, and placed in a vacuum desiccator (room temperature ) For 7 days.

Comparative Example 1
In the same manner as in Example 1 (1) and (2), a sperm suspension was obtained using NIM, mCZB-Hepes or Tris-HCl-EGTA, respectively, and then α-hemolysin treatment and trehalose addition were not performed. In the same manner as in Example 1 (5), the culture plate was air-dried and stored in a vacuum desiccator (room temperature) for 7 days.

Test example 1
1. [Microinsemination of dry-preserved sperm]
Microinsemination was performed on the dried sperm after storage. That is, 100 μl or 1 ml of sterilized milli-Q water, NIM, mCZB-Hepes or Tris-HCl-EGTA is added to each well of a dried sperm suspension or a culture plate on a slide glass, and at room temperature for 5 minutes. Condensed. After the condensate, sperm suspensions were prepared by light pipetting and transferred to microtubes.
Subsequently, the supernatant was discarded by centrifugation (10,000 rpm, 5 minutes, room temperature), and then 100 μl of mCZB-Hepes was added to suspend the sperm. The tube was left on ice until ICSI.
ICSI was performed by a conventional method. That is, 8-16 weeks old BDF1 female mice treated with superovulation by intraperitoneal administration of 10 IU each of PMSG and hCG, which are gonadotropins, 48 hours apart, eggs about 16 hours after hCG administration. Unfertilized eggs wrapped in cumulus cells were collected from the enormous portion of the tube. These unfertilized egg and cumulus cell clumps separated unfertilized eggs and cumulus cells in a drop of 100 ml modified Whitten's medium (mW) containing 0.1% hyaluronidase. Thereafter, unfertilized eggs were cultured in mW until ICSI was performed. When performing ICSI, the micromanipulator (made by Leica) and the injector (made by Eppendorf) were adjusted. After the adjustment, a holding pipette and an injection pipette were connected to the tips of the instrument curlers connected to the left and right injectors, respectively. After that, under an inverted microscope (DM IRB, with Hoffman contrast, manufactured by Leica), insert the tip of the injection pipette into a mixture of 5 ml of mCZB-Hepes containing 12% PVP plus 2 ml of the suspension of dried sperm. The sperm head and tail were separated by hooking the sperm head to the tip and applying a pulse of Piezo Drive (Primetech). This operation was repeated several times to inhale several sperm heads into the pipette. Next, the stage of the microscope was moved to move to a drop of mCZB-Hepes containing unfertilized eggs, and the unfertilized eggs were sucked and fixed at the tip of the holding pipette. After that, the tip of the injection pipette is brought into close contact with the zona pellucida of the unfertilized egg, a hole is made in the zona pellucida by applying a piezo drive pulse, and then the cytoplasm of the unfertilized egg is similarly punctured with piezo, and the sperm head One was released into the egg cytoplasm. Unfertilized eggs including the sperm head were allowed to stand in the drop of mCZB-Hepes. This operation was performed several times within 30 minutes. Eggs after ICSI were washed once with mW and cultured in a 20 ml mW drop for 5 days. During this culture, fertility and blastocyst development were confirmed. In addition, some oocytes that were developed in 2-cell stage embryos 24 hours after ICSI were transplanted into the oviducts of recipient mice (ICR; 9-12 weeks of age) on day 0.5 of pseudopregnancy. . Thereafter, recipient tubes transplanted with fallopian tubes were euthanized 13.5 days after transplantation, and intrauterine fetuses were confirmed.
In addition, after preparing sperm suspension using mCZB-Hepes containing 1M trehalose, immediately performing ICSI without storage; after preparing sperm suspension using mCZB-Hepes, suspension treated with α-hemolysin Immediate ICSI was performed without storing the suspension; sperm suspension was prepared using mCZB-Hepes, and then ICSI was performed immediately without storage.

2. [result]
The results are shown in Table 1.
As is clear from Table 1, all sperm preserved by the method of the present invention had a high fertilization rate, and the incidence of morula embryos and blastocysts was good. This was particularly good when Tris-EGTA was used as the stock solution. When 1M trehalose and 0.5M trehalose were compared, there was no difference in the incidence of morula embryos, but 0.5M trehalose had a better incidence in blastocysts.
In addition, when the condensate used for condensate was examined, it was found that in the NIM group, a similar preservation solution had a better incidence of blastocysts after ICSI than sterilized milli-Q water. It was. On the other hand, in the Tris-EGTA group, there was no difference in fertilization rate and incidence after ICSI due to differences in condensate. Overall, the Tris-EGTA group containing 0.5 M trehalose was found to have the best incidence of blastocysts after ICSI.
Furthermore, when the Tris-EGTA group containing 0.5 M trehalose was compared with the group (* in Table 1) that had been subjected to ICSI immediately after preparation of a sperm suspension using mCZB-Hepes containing 1 M trehalose, without storage. Although the 0.5M trehalose / Tris-EGTA group showed a lower tendency in the blastocyst development rate, there was little difference between the two in terms of fertilization rate and development rate to the morula stage. It was. Thus, according to the method of the present invention, it was confirmed that it can be stored for a long time even at room temperature without causing chromosomal abnormalities or DNA damage in sperm.

On the other hand, in the dry sperm prepared in Comparative Example 1, 73.9% in the NIM group and 90% in the mCZB-Hepes group were fertilized. Among these fertilized eggs, the NIM group 82.4% and the mCZB-Hepes group 77.8% were 2- Developed into cell stage embryo. However, the incidence of morula embryos when cultured for 3 days after ICSI markedly decreased to 23.5% in the NIM group and 27.8% in the mCZB-Hepes group. Moreover, what developed to the blastocyst was not recognized by any preservation group.
In addition, in sperm preserved with Tris-EGTA alone, fertilization rate was 93.1%, 2-cell stage embryo development rate 96.3%, morula stage embryo development rate 37%, NIM group and mCZB-Hepes group Although a tendency toward a higher value was observed, the incidence of blastocysts after ICSI was as low as 7.4%. Thus, when α-hemolysin treatment and trehalose addition were not performed, air-drying with NIM, mCZB-Hepes and Tris-EGTA alone and storage of sperm for 7 days at room temperature resulted in relatively high fertilization in both groups. Although the rate was obtained, the incidence of blastocysts after ICSI was found to be low.
In addition, after preparing a sperm suspension with mCZB-Hepes alone, the group (** and *** in Table 1) that was immediately subjected to ICSI without storage was fertilized, regardless of the presence or absence of α-hemolysin treatment, The incidence to the morula stage and the incidence to the blastocyst were both high. This suggests that the addition of trehalose to the preservation solution has an effect on the development of the egg after ICSI after the morula stage when it is not stored at room temperature.

Test example 2
In vivo development after ICSI of mouse spermatozoa stored at room temperature for 7 days in Tris-EGTA containing 0.5 M trehalose was examined.
In the same manner as in Example 1, mouse sperm was stored for 7 days in Tris-EGTA containing 0.5 M trehalose. The dried sperm was condensed using Tris-EGTA or sterilized milli-Q water, and fertilized eggs were obtained by ICSI in the same manner as in Test Example 1. These fertilized eggs were cultured for 1 day to develop to 2-cell stage embryos, and then transplanted into the oviduct of recipient mice on the 0.5th day of pseudopregnancy (the day of plug confirmation was the 0.5th day of pseudopregnancy). On day 13.5 after transplantation, these mice were sacrificed to confirm fetuses in the uterus.
As a result, as shown in Table 2, even when Tris-EGTA or Milli-Q water was used as the condensate, normal fetuses were obtained, and no difference was observed in the incidence between the two. From these results, it was found that even a room temperature-preserved sperm develops to a normal fetus after ICSI.

It is a figure which shows the outline of this invention method.

Claims (4)

  A method for preserving mammalian sperm, comprising treating sperm collected from a mammal with α-hemolysin and allowing a disaccharide or trisaccharide to flow into the cell, followed by incubation for a certain period of time and then air drying.   The method for preserving mammalian sperm according to claim 1, wherein the disaccharide is trehalose and the trisaccharide is raffinose.   The method for preserving mammalian sperm according to claim 1 or 2, wherein the incubation is performed at 0 ° C to room temperature for 20 minutes to 10 days.   The method for preserving mammalian sperm according to any one of claims 1 to 3, wherein the mammal is a mouse.