JP2009005651A - Method for preserving spermatid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for conveniently and efficiently preserving a spermatid. <P>SOLUTION: Provided is a method for preserving a spermatid comprising steps of making the slices of orchis including the spermatid of animals, and freezing the prepared slices, wherein a spermatid is provided that is functional upon injection into an ovum by microinsemination through thawing the freezed slices. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a simple and efficient method for storing sperm cells.

  Analysis of gene function by creating genetically modified animals (eg, mice) has contributed greatly to the development of basic biology and medical fields. Many animals are expected to be produced in the future. This requires the development of an efficient system preservation method.

  The most widely used method for preserving sperm cells is a method in which sperm is filled in a straw-shaped container together with a cryoprotectant and stored in liquid nitrogen (Non-patent Document 1). In this method, since the sperm after thawing retains motility, progeny can be produced by microinsemination by IVF (in-vitro fertilization) in which sperm is co-cultured with unfertilized eggs. it can. According to the above method, for example, about 10 samples can be prepared from one male mouse.

  As a similar method, a technique has been reported in which sperm is filled in a straw-like container and stored in liquid nitrogen without using a cryoprotectant (Non-patent Document 2). In this method, since the sperm after thawing has no motor ability, it is necessary to produce offspring by a micro insemination method called ICSI (intracytoplasmic sperm injection) in which sperm is directly injected into an unfertilized egg with a micromanipulator. According to the above method, for example, it is reported that about 100 sperm samples can be prepared from one male mouse.

  In both of the above methods, since the sperm is filled in a straw-shaped container, the freezing operation is complicated. Moreover, liquid nitrogen is required to maintain the storage state.

  As a method developed in recent years, a method of storing testis in a freezing tube at −30 ° C. or −80 ° C. has been reported (Non-patent Document 3). Although it is recommended to moderate the decrease in temperature by using a specific container called a freezing container during freezing, the operation is easy because the sperm cells contained therein can be preserved simply by freezing the testis. Moreover, the use of a cryoprotectant or liquid nitrogen is unnecessary. However, since the testis itself is frozen, the number of samples that can be prepared from one animal is very limited.

  By any of the above methods, it is possible to preserve inseminated spermatozoa by microinsemination, etc., but a large number of male mice are required to prepare a sufficient amount of frozen samples, and the operation is complicated However, there are problems such as requiring considerable storage space for storing a large number of systems.

Nakagata N. Cryopreservation of mouse spermatozoa. Mammalian genome, 11: 572-576 (2000). Ward MA et al. Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotectoin. Biology of Reproduction, 69: 2100-2108 (2003). Ogonuki N et al. Spermatozoa and spermatids retrieved from frozen reproductive organs or frozen whole bodies of male mice can produce normal offspring.Proc Natl Acad Sci USA, 103: 13098-13103. (2006).

  An object of the present invention is to provide a simple and efficient method for storing sperm cells.

  In order to improve the above problems, the present inventors have intensively studied and found that progeny can be produced by microinsemination from frozen testis frozen sections. By this method, it is possible to prepare theoretically several hundred sperm samples from one male mouse, for example. Moreover, since the thickness per testis tissue is several tens of μm, a large amount of samples can be stored in a space-saving manner.

The present invention
[1] (i) preparing a section of an animal testis containing sperm cells; and (ii) freezing and storing the prepared section;
A method of storing sperm cells, comprising thawing a cryopreserved section to obtain functional sperm cells upon injection into an egg by microinsemination;
[2] The method according to [1], wherein the section is prepared using a cryostat;
[3] The method of [1] or [2], wherein the thickness of the section is 10 to 25 μm;
[4] The method according to any one of [1] to [3], wherein the frozen storage is performed for a period of 1 day to 1 month;
[5] The method according to any one of [1] to [4], wherein the frozen storage is performed at −30 ° C .;
[6] The method according to any one of [1] to [5], wherein the prepared section is attached to a support;
[7] A method for producing a section for storing sperm cells, comprising a step of preparing a section of an animal testis containing sperm cells, wherein the prepared section is frozen and then thawed, and then the egg is subjected to microinsemination. A method in which functional sperm cells are obtained upon injection into the cell;
[8] The method of [7], wherein the section is prepared using a cryostat;
[9] The method according to [7] or [8], wherein the thickness of the section is 10 to 25 μm;
[10] The method according to any one of [7] to [9], wherein the frozen storage is performed for a period of 1 day to 1 month;
[11] The method according to any one of [7] to [10], wherein the frozen storage is performed at −30 ° C .;
[12] The method according to any one of [7] to [11], wherein the prepared section is attached to a support;
[13] A composition for preserving sperm cells, including a testicular slice of an animal, containing sperm cells, wherein the sperm cells functional in injection into an egg by microinsemination are obtained by thawing the sections after cryopreservation. The resulting composition;
[14] The composition of [13], wherein the section is prepared using a cryostat;
[15] The composition of [13] or [14], wherein the section has a thickness of 10 to 25 μm;
[16] The composition according to any one of [13] to [15], wherein the frozen storage is performed for a period of 1 day to 1 month;
[17] The composition according to any one of [13] to [16], wherein the frozen storage is performed at −30 ° C .;
[18] The composition according to any one of [13] to [17], wherein the section is attached to a support.

  The present invention provides a simple and efficient method for preserving sperm cells.

  The method for preserving sperm cells of the present invention includes (i) a step of preparing a testis section of an animal containing sperm cells; and (ii) a step of cryopreserving the prepared section. By thawing, sperm cells that are functional in injection into the egg by microinsemination are obtained. Further, the method for producing a slice for sperm cell storage according to the present invention includes a step of preparing a testis slice of an animal containing sperm cells, and thawing the prepared slice by freezing and then thawing the egg by microinsemination. Functional sperm cells are obtained upon injection into Furthermore, the composition for preserving sperm cells of the present invention comprises a section of an animal testis containing sperm cells, and thawing the sections after cryopreservation so that functional sperm cells in injection into an egg by microinsemination can be obtained. can get.

  In this specification, the term “sperm cells that are functional in injection into an egg by microinsemination” means a sperm cell that has the ability to cause insemination and development when injected into an egg using microinsemination technology. Say. The ability to cause insemination / development is confirmed by, for example, formation of a pronucleus in the obtained inseminated egg, development into a 2-cell stage embryo, generation of offspring after transplantation of the 2-cell embryo into an animal, and the like.

  The term “microinsemination” in the present specification refers to a technique for artificially insemination under a microscope. As techniques for microinsemination, IVF (in-vitro fertilization, also called in vitro fertilization), ICSI (also called intracytoplasmic sperm injection, sperm injection in an egg cell), and the like are known. In the former IVF, fertilization is performed by co-culturing sperm with an unfertilized egg. This technique has the advantage of being easy to operate and capable of producing a large number of fertilized eggs at one time, but has the limitation that the sperm must retain motility. In the latter ICSI, sperm is directly injected into an unfertilized egg with a micromanipulator. This technique requires a certain period of training for proficiency, and the number of fertilized eggs that can be produced at one time is smaller than that of IVF. However, ICSI has an advantage that a fertilized egg can be obtained even if the sperm does not have a motor ability. Sperm cells obtained by the present invention are preferably used for microinsemination by ICSI.

  The animal used in the present invention is not limited, and any animal having a testis is used. In one embodiment, the animal is a mammal. Examples of mammals include humans, monkeys, cows, horses, sheep, goats, pigs, dogs, cats, rabbits, rats, and mice. The testis is an organ that performs spermatogenesis. Therefore, sperm cells are contained in the testis.

  In the present specification, the “sperm cell” is a cell contained in the testis, and there is no limitation as long as it has a function in injection into an egg by microinsemination. That is, sperm cells include mature sperm and more immature male germ cells (eg, spermatocytes). If the sperm cells used do not have the ability to activate the egg, the egg is artificially activated. As long as it has the above properties, sperm cells do not need to exhibit motility and do not need to be alive. The survival of sperm cells can be confirmed, for example, by propidium iodide (PI) staining. When using mature sperm, since only the head is generally injected into the egg, it may be usable even if the tail is cut.

  In the present invention, there is no limitation on the means for preparing a testis slice of an animal as long as functional sperm cells can be obtained by injection into an egg by microinsemination. In one embodiment, the section is made using a cryostat (also referred to as a cryostat). Cryostat uses a microscope to keep the morphology of cells and tissues under the microscope in order to know tissue lesions, localization of proteins and sugar chains inside cells, and the presence of DNA and RNA in pathological diagnosis. It is an apparatus used for observation purposes (for example, for tissue diagnosis performed during surgery, that is, rapid diagnosis during surgery). Specifically, a biological sample (such as an organ) to be sliced is frozen by wrapping it in a frozen tissue embedding agent (for example, OCT compound), and using a cryostat (generally −10 to −40 ° C., For example, a section is prepared by a microtome at −20 to −25 ° C., and then the prepared section is attached to a glass slide. The obtained sections are generally subjected to staining and in situ hybridization and observed (for example, Makoto Tsujiyasu, Bio View, 39, 38-41 (2002), Mitsuru Kajikawa et al., Medical Technology, 31 Vol. 10, pages 1064-1068 (2003)). Therefore, this technique has not been used for the purpose of preserving functional cells. Here, the above OCT compound is a typical frozen tissue embedding agent, and is a mixture of polynyl alcohol, polyethylene glycol, and an inactive substance. Although no harmful effects on the human body have been confirmed, the effect on sperm cells in frozen sections is unknown.

  The section is preferably thicker in order to avoid damage to sperm cells, but is preferably thinner in order to obtain a larger number of sections. One skilled in the art can determine the appropriate section thickness based on the present disclosure. In the present invention, the thickness of the slice is, for example, 2 to 100 μm, preferably 5 to 50 μm, more preferably 10 to 25 μm.

  As long as the function of sperm cells is maintained during the storage period, there is no limitation on the temperature at which the prepared sections are stored frozen. The temperature is, for example, -80 ° C to -5 ° C, preferably -60 ° C to -10 ° C, more preferably -40 ° C to -20 ° C, and further preferably -30 ° C. There is no limitation on the method for lowering the temperature, and the sample to be stored frozen may be directly put in a freezer, or the temperature may be lowered gently using a freezing container or a programmed cooling device. In the present invention, it is not necessary to use a cryoprotectant which is generally used for sperm storage. There is also no limitation on the storage period, and the period is, for example, 1 hour or more, preferably 5 hours or more, more preferably 1 day or more, for example, 10 years or less, preferably 1 year or less, more preferably 1 month or less.

  In one embodiment, the section is attached to the support. As the support, any material (glass, plastic, etc.) and form (tube, plate, etc.) can be used. Preferably, the support is a glass slide. By using a slide glass as a support, sperm cells obtained by thawing a section can be used immediately for microinsemination.

  Thaw frozen sections to obtain sperm cells for use in microinsemination. Specifically, the thawed section is suspended in an appropriate medium, and the separated sperm cells are collected.

  It was unexpected that the functional sperm cells were obtained in the separated state according to the present invention. This is in contrast to the traditional method of freezing sperm using cryoprotectants and the method of using sections compared to the case where the whole testis is frozen and the organ itself can act as a substitute for the protectant. This is because the following bad conditions were predicted. For example, when a cryostat is used, the testis is once frozen, and the prepared frozen section is thawed when attached to the slide glass, and the section attached to the slide glass is stored frozen and stored frozen for microinsemination. Thawed sections are thawed. That is, in this case, sperm cells are subjected to microinsemination after undergoing two freeze-thaw cycles. Here, it is generally known that freeze-thaw causes damage to sperm cells (Wakayama, T. et al., Journal of Reproduction and Fertility, 112: 11-17 (1998)). In addition, sperm cells themselves may be cut when preparing a section. In addition to these physical effects, the OCT compound used to prepare sections and oxidative stress from sectioning and exposing sperm cells to the open air (Twigg, JP et al., Human Reproduction, 13: 1864 -1871 (1998)) could have an adverse effect. Nevertheless, normal progeny could be efficiently obtained using sperm cells obtained from testicular sections preserved according to the present invention.

  The present invention is useful for strain preservation of animals useful as, for example, laboratory animals or livestock. The present invention can also be used for the treatment of male infertility (ie, male infertility due to impaired spermatogenesis) when no sperm is found in the injected semen but sperm is present in the testicular tissue. Furthermore, although human immunodeficiency virus (HIV) infects cells of the immune system, the lumen of the testicular seminiferous tubule is isolated from the immune system by the testis-blood vessel barrier, so according to the present invention, HIV infection Sperm cells without HIV can be obtained from the lumen of the male seminiferous tubule.

  A further advantage of being able to store sperm as a tissue section is that it does not require significant storage space. Tissue sections are very thin and may be stored in sheet form (or “book” form). For example, several hundred samples can theoretically be prepared from one mouse. Furthermore, it is expected that a frozen sample can be sent in a letter-like state, which is expected to be simpler than the conventional method (packed in a foamed polystyrene container).

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

Preparation of testicular slices and cryopreservation The mice used were B6D2F1 (C57BL / 6 × DBA / 2; hereinafter referred to as BDF1) adult male individuals (10 weeks old). It was. Specifically, testes were collected from BDF1 male mice and embedded in OCT compound (Sakura Finetechnical Co., Ltd.). After freezing in a freezer (−30 ° C.), frozen sections having a thickness of 10 μm or 25 μm were prepared using a cryostat (MICROM). The prepared section was attached to a glass slide and stored at −30 ° C.

Recovery of sperm cells from testicular frozen sections and generation of offspring by microinsemination Attempts were made to recover spermatozoa from frozen sections 24 hours (1 day) or 1 month after storage. First, in order to collect spermatozoa from the testis slices attached to the slide glass, the slide glass was removed from the NIM solution (Kuretake S. et al. Fertilization and development of mouse oocytes injected with isolated sperm heads. Biology of Reproduction, 55: 789-795 (1996)) (4 ° C., 2 minutes). Thereafter, the testis section was detached from the slide glass using a micropipette and transferred to a 1.5 ml microtube. It was well suspended using a micropipette and centrifuged at 2300 g for 2 minutes. After discarding the supernatant, it was resuspended in a NIM solution and stored at 4 ° C. until microinsemination.

Progeny generation by microinsemination was performed according to the method of Kimura and Yanagimachi (Kimura Y and Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biology of Reproduction, 52: 709-720. (1995)). Specifically, first, superovulation treatment (equine chorionic gonadotropin (eCG) (5 IU) is administered to female mice of BDF1 and human placental sexual stimulating hormone (hCG) (5 IU) is administered 48 hours later) The egg was collected from the oviduct. The cumulus cells around the ovum dissociate using hyaluronidase, and the resulting ovum is a CZB medium (Chatot CL et al. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. Journal of Reproduction and Fertility, 86: 679-688. (1989)) and cultured at 37 ° C. and 5% CO ₂ .

  The previously prepared sperm suspension was suspended in a 12% polyvinylpyrrolidone (PVP) solution, and only the sperm head was collected by piezopulse. Microinsemination was performed by injecting these sperm heads into unfertilized eggs. The inseminated ova were cultured in CZB medium for 24 hours, and embryos that developed at the 2-cell stage were transplanted into pseudopregnant mice. These mice were cesarean section at 18.5 days after transplantation to confirm the presence or absence of offspring.

Results Testicular sections were preserved as glass slides (FIG. 1a), and sperm collection was attempted. When the section before sperm collection was observed with a microscope, formation of sperm could be confirmed as shown in FIGS. After detaching the section with a micropipette, a cell suspension was prepared (FIG. 1d). As a result, a cell suspension that was capable of microinsemination although the tail was cut (FIG. 1e-g) was successfully prepared. Further, when sperm was stained by propidium iodide (PI) staining, it was revealed that these sperm were dead as cells (FIG. 1h).

  Next, the insemination / development ability of these sperm cells was confirmed by microinsemination ICSI. When 10 μm and 25 μm testis sections were stored at −30 ° C. for 24 hours (1 day) and ICSI was performed, 90% or more of the embryos formed pronuclei, of which about 60 to 70% of embryos were 2 cell stage embryos (Table 1). When these 2-cell embryos were transplanted into pseudopregnant mice, 10-20% of the transplanted embryos developed into normal offspring (Table 1, FIG. 1i). From the above results, it was revealed that spermatozoa prepared from testis slices have normal insemination / development ability.

  Furthermore, when spermatozoa were prepared from testis sections stored for 1 month and subjected to ICSI, they succeeded in producing offspring with the same results as sperms stored for 24 hours (Table 1). In addition, when the mice obtained by this method were observed, no abnormalities were observed in growth or fertility, and it is considered that normal mice were obtained. From these results, it became clear that functional sperm cells can be stored as testicular frozen sections.

  In all experimental sections, sperm prepared from 25 μm sections had a higher success rate than sperm prepared from 10 μm sections (Table 1). This suggests that the spermatozoa are damaged to some extent by sectioning.

  The present invention provides a simple and efficient method for preserving sperm cells.

FIG. 2 shows the collection of sperm from a frozen section and the production of its progeny: a testis section cryopreserved on a glass slide. FIG. 2 shows the collection of sperm from a frozen section and the production of its progeny: a preserved testis section (where spermatogenesis can be confirmed). FIG. 2 shows the collection of sperm from a frozen section and the production of its progeny: a preserved testis section (where spermatogenesis can be confirmed). FIG. 2 shows the collection of sperm from frozen sections and the production of their progeny: sperm cells prepared from testis sections. It is a figure which shows collection | recovery of the spermatozoon from a frozen section, and production of the offspring: Sperm (sperm cell near normal) It is a figure which shows collection | recovery of the sperm from a frozen section, and production of the offspring: Sperm (sperm cell by which the tail part was cut | disconnected) It is a figure which shows collection | recovery of the sperm from a frozen section, and production of the offspring: Sperm (sperm cell by which the tail part was cut | disconnected) FIG. 1 shows sperm recovery from frozen sections and production of their progeny: PI-stained sperm cells. FIG. 2 shows the collection of sperm from a frozen section and the production of its progeny: a mouse produced by microinsemination.

Claims (18)

(I) producing a section of an animal testis containing sperm cells; and (ii) cryopreserving the produced section.
A method of preserving sperm cells, comprising thawing a cryopreserved section to obtain functional sperm cells upon injection into an egg by microinsemination.   The method of claim 1, wherein the section is made using a cryostat.   The method according to claim 1 or 2, wherein the thickness of the section is 10 to 25 µm.   The method according to any one of claims 1 to 3, wherein the frozen storage is performed for a period of 1 day to 1 month.   The method according to any one of claims 1 to 4, wherein the cryopreservation is performed at -30 ° C.   The method according to claim 1, wherein the prepared section is attached to a support.   A method for producing a section for storing sperm cells, comprising a step of preparing a section of an animal testis containing sperm cells, and injecting the prepared section into an egg by microinsemination by thawing after cryopreservation In which functional sperm cells are obtained.   8. The method of claim 7, wherein the section is made using a cryostat.   The method according to claim 7 or 8, wherein the thickness of the section is 10 to 25 µm.   The method according to any one of claims 7 to 9, wherein the frozen storage is performed for a period of 1 day to 1 month.   The method according to any one of claims 7 to 10, wherein the cryopreservation is performed at -30 ° C.   The method according to any one of claims 7 to 11, wherein the prepared section is attached to a support.   A composition for preserving sperm cells, including a section of an animal testis, containing sperm cells, and thawing the sections after cryopreservation to obtain functional sperm cells in injection into an egg by microinsemination, Composition.   14. The composition of claim 13, wherein the section is made using a cryostat.   The composition according to claim 13 or 14, wherein the section has a thickness of 10 to 25 µm.   The composition according to any one of claims 13 to 15, wherein the frozen storage is performed for a period of 1 day to 1 month.   The composition according to any one of claims 13 to 16, wherein the cryopreservation is performed at -30 ° C.   18. A composition according to any one of claims 13 to 17, wherein the section is attached to a support.

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