KR102293384B1 - Preparation of recipient model for donor testicular germ cell - Google Patents

Abstract

The present invention relates to a method for preparing a donor animal for spermatogonial cell transplantation, and by dividingly injecting an alkylating agent into a spermatogonial cell recipient animal, endogenous germ cells are depleted and, in particular, a donor animal can be effectively prepared for a stallion.

Description

Preparation of recipient animal for spermatogonial cell transplantation {Preparation of recipient model for donor testicular germ cell}

The present invention relates to a method for preparing a recipient animal for spermatocyte transplantation.

In general, genetically modified animals have been produced by micro-injection of fertilized eggs. Heterologous or homologous DNA or hybrid DNA is micro-injected into the pronucleus of the fertilized egg. Next, a micro-injected fertilized egg is transferred to the animal's genitalia to produce a genetically modified animal.

However, the production of such genetically modified animals requires a large amount of fertilized eggs, and the loss rate of fertilized eggs is high during micro-injection. Moreover, engineered embryos are less likely to implant and survive in the uterus. Therefore, the production of these genetically modified animals is mainly used in mice with high fertility due to low efficiency. Small animals such as mice have been proven to be suitable models for specific diseases, but their value is limited.

Most male animals produce at least 100 million sperm per ejaculation, as opposed to 10 to 20 eggs in mice after treatment with an ovulation inducer. For this reason, sperm, which are male reproductive cells, can be more efficient after simple natural mating.

Spermatogonial stem cells are the cells that are the foundation for the spermatogenesis process, and are stem cells that have the ability to self-renew and differentiate, like other types of adult stem cells, from which the final differentiation is completed. phosphorus sperm is produced. Sperm stem cells are the only adult stem cells that have the characteristics of a germ cell that can transmit genetic material to the next generation. Because of these advantages, they are perfect cells for transformation, and worldwide attention is focused on technology development in the field of transgenic animal production using garden stem cells.

Sperm cell transplantation technology is known to produce donor-derived sperm from a variety of livestock, including goats. In particular, in 1994, Brinster's research team showed that donor-derived progeny were produced when spermatogonial stem cells from donor mice were transplanted into infertile mice. By transplanting spermatogonial cells to a recipient animal, the existence of spermatogonial stem cells in the testis of a mammal and the possibility of transplantation of spermatogonial stem cells were demonstrated. Since then, the transplantation technique of spermatogonial stem cells into the testis of a recipient animal has been used as an essential and most essential technique in the identification of biological characteristics of garden stem cells and the development of utilization methods.

Until recently, methods for improving the purity of garden stem cells, genetic manipulation techniques, and in vitro culture techniques have been developed through research, and the utilization of garden stem cells is increasing. In addition, as the technology for producing pluripotent cells from garden stem cells has been developed, the use of garden stem cells has been proposed as a resource for the development of cell therapy products.

In order to apply the intratestis transplantation technique, which is essential for the analysis of the biological activity of spermatogonial stem cells and the production of transgenic animals, efficient preparation of the recipient animal must be preceded. Efficient testis of a recipient animal for transplantation should provide an environment in which the transplanted spermatogonial stem cells are located in the seminiferous tubules of the testes of the recipient animal, and then perform functions such as self-renewal and production of differentiated cells without any abnormality. To this end, first, the testis state in which the spermatogonial stem cells and differentiated germ cells of various stages have been removed in the testis of the recipient animal should be maintained. In the case of experimental mice, the intraperitoneal injection of Busulpan, a kind of anticancer drug, is commonly used to prepare the recipient animals. In addition, various treatments have been applied, including topical irradiation, treatment of low temperature ischemia, hyperthermia, and injection of an alkylating agent such as glycerin. However, glycerin therapy can be toxic in livestock such as pigs and dogs, and can be fatal in rodents by suppressing hematopoiesis due to severe bone marrow depression. Accordingly, the dosage of the alkylating agent and the appropriate time for use as a recipient animal after treatment show significant differences between recipient animal species.

In the case of horses, there is an urgent need to develop garden stem cell technology as a technology that can produce progeny of castrated horses with excellent genetic ability. However, as a recipient animal, the technology to remove the testis cells in the testis of horses has not yet been developed.

Therefore, the present inventors evaluated the effect on the removal of endogenous germ cells through intravenous injection of the alkylating agent, spermatogenesis, testicular characteristics, sperm motility and the target of the present invention for an ideal treatment method for an alkylating agent for horses. was completed.

(Patent Document 1) KR10-2013-0075228 A

Accordingly, it is an object of the present invention to provide a safe and effective method for preparing a horse garden stem cell recipient animal for transplantation of spermatogonial stem cells.

The method for preparing a recipient animal according to the present invention for achieving the above object is characterized in that an alkylating agent is dividedly injected into a subject of a spermatogonial recipient animal.

Here, the alkylating agent may be one of busulfan, chlorambucil, glycerin, cyclophosphamide, melphalan, or ethylsulfonic acid.

Here, the recipient animal may be a mammal selected from non-human primates, horses, dogs, rats, hamsters, rabbits, sheep, cattle, cats, or pigs, or birds selected from ducks, geese, turkeys, chickens, ostriches, pigeons or quails. have.

Also, the total injection amount of the alkylating agent may be 15 mg/kg.

In addition, the alkylating agent may be injected intravenously in the recipient animal subject.

In addition, the alkylating agent can split equally or progressively escalate.

In addition, the divided dose of the alkylating agent may be a single dose divided by the time period until conferable.

Further, the alkylating agent may be a powdered alkylating agent having a dose of 15 mg/kg of the body mass of the recipient animal to be treated diluted in 50 ml of a solvent.

According to the present invention, intravenous injection of an alkylating agent including busulpan depletes endogenous germ cells, thereby providing an effective method for preparing a recipient animal, particularly for horses.

In addition, according to the present invention, death due to the toxicity of the alkylating agent in the recipient animal can be prevented, and spermatogonial cells can be efficiently removed from the testis. In addition, it is possible to protect the sperm production ability of the recipient animal by minimizing damage or destruction of the testes.

The effects according to the present invention in the above are not limited to the above, and other effects of the present invention can be clearly identified by those of ordinary skill in the art through the examples and claims to be described later. can be understood

1 shows the survival rate of stallions in the intravenous injection type using the busulpan formulation.
Figure 2 shows the testes weight (g) of the Buseulpan-treated group and the control group.
Figure 3 shows the type of spermatogenesis in semi-castrated testicular seminiferous tubules.
Figure 4 shows the ratio (%) of spermatogenesis in the cross section of semi-castration after 11 weeks.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for preparing a recipient animal for spermatocyte transplantation.

A method for preparing a recipient animal according to a preferred embodiment of the present invention comprises injecting an alkylating agent into a spermatogonial recipient animal by divided injection.

Here, the recipient animal is a mammal selected from non-human primates, horses, dogs, rats, hamsters, rabbits, sheep, cattle, cats, and pigs, or birds selected from ducks, geese, turkeys, chickens, ostriches, pigeons, or quails. can be

In addition, the alkylating agent may use one of busulfan, chlorambucil, glycerin, cyclophosphamide, melphalan or ethylsulfonic acid, preferably For example, busulfan can be used.

In the present invention, the alkylating agent is injected into the recipient animal subject in a total injection amount of 15 mg/kg of body mass.

In addition, the alkylating agent may be injected intravenously in the recipient animal subject.

According to the present invention, genetic manipulation may or may not be performed prior to transplantation into the testis of a recipient animal so that the germ cells isolated from the testis living body can be resettled in the testis.

According to the present invention, it is possible to produce by naturally mating a recipient animal transplanted with germ cells or by selecting progeny having a desired trait with the help of reproductive assistance technology. In addition, it is applicable to the production of genetically modified animals and the modification of the genome of animal models by adding or altering genes.

The present invention aims to improve the existing methods for genetic manipulation of animal germ cells and the production of transgenic animals, and the existing methods include direct injection of DNA produced in vitro or in vivo, or stem cells derived from undifferentiated embryonic cells. It is known to produce chimeric embryos using Thereafter, the treated embryo is transplanted into the uterus or fallopian tube, and this method is time consuming and costly, and the production of transformed progeny is difficult to predict because it can be produced with a very low probability.

Through the present invention, a fast and efficient method at a lower cost to obtain a genetically modified animal or an animal of a desired trait was identified.

Transplantation of endogenous male germ cells should not destroy or seriously damage the precise structure of the testes. If the process of vasculogenesis is disrupted, exogenous spermatogonial cells may become unable to reproduce in the testis. In addition, if the vas deferens collapse and the semen transport is impaired, reproduction may become impossible, so damage or destruction of small testicles should not be caused. to reduce the risk.

The alkylating agent may be one of busulfan, chlorambucil, glycerin, cyclophosphamide, melphalan, or ethylsulfonic acid.

In particular, busulfan (1,4-butanediol dimethane sulfonate; Myleran) is known to be administered to eliminate male germ cells and spermatogonial cells in the testes of recipient animals. In addition, various alkylating agents are known to kill spermatogonial cells in the testis. It is known that the alkylating agent is administered in an amount sufficient for the death of spermatogonial cells, but an appropriate concentration and amount has been established because the death of the recipient animal due to cytotoxicity has become a problem.

A preferred dose of the alkylating agent in the present invention is 10 to 20 mg per 1 kg of body weight, more preferably 15 mg per 1 kg of body weight.

Administration of the alkylating agent in the present invention may include intraperitoneal injection, intramuscular injection, transdermal delivery, or oral administration, but for uniform administration at a constant rate, the intravenous injection method is most preferable, and the intravenous injection method using a catheter is more preferable.

According to the present invention, death due to the toxicity of the alkylating agent in recipient animals can be prevented, and spermatogonial cells can be efficiently removed from the testes. In addition, it is possible to protect the sperm production ability of the recipient animal by minimizing damage or destruction of the testes.

Therefore, according to the present invention, it is possible to provide a safe and efficient recipient animal for spermatocyte transplantation.

According to the method of dividing the alkylating agent in the present invention, the total injection amount of the alkylating agent can be divided equally or divided so that the injection amount is gradually increased. This is to effectively remove spermatogonial cells in the testis while improving the survival rate of the recipient animal.

Specifically, the divided injection amount of the alkylating agent is a single injection amount, but the total injection amount can be divided by the period until the spermatogonial cells can be conferred.

In the present invention, an alkylating agent solution obtained by diluting a powdery alkylating agent having a dose of 15 mg/kg of the recipient animal's body mass in 50 ml of a solvent may be used as the alkylating agent in the present invention.

Preferably, the solvent in the present invention includes a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, and a suppository for preparing a busulpan solution as a solvent for injection of a recipient animal subject. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Preferably, dimethyl for easy drug delivery during intravenous injection. Dimethyl Sulfoxide (DMSO) may be used.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are only for specifying the contents of the present invention, and the present invention will not be limited thereby.

<Example 1>

1-1 Animal Preparation

The present invention was carried out in a research facility for livestock in Gyeongbuk, and was approved by the Animal Experimentation and Use Committee of Kyungpook National University (2017-0030). A total of 9 Thoroughbred stallions were used, and the average age of these stallions was 6.16±2.38 years, and they were between 3 and 18 years of age.

All stallions were housed in a 3m x 4.5m individual space and a 20m x 30m revolving paddock. The nine stallions were divided into three groups of three each, and one group was used as a control group. In the present invention, testis samples were collected through general in situ castration. Hay and water were provided as feed at 5% of body weight per stallion.

1-2 experimental design

Nine thoroughbred stallions were divided into a treatment group and a control group, and the busulpan was injected at 15 mg/kg of body weight (BM) to three stallions in group 1.

Phosphate buffersaline (PBS) was injected to the stallions of group 3 as a control group, in the same amount as the total amount of sulphate in groups 1 and 2.

The injection period, number, and dose are shown in Table 1 below.

1 injection
Blastpan (15 mg/kg BW) in split injection
Blastpan (15 mg/kg BW) control
PBS (15 mg/kg BW) type T1 T2 T3 Mean ±
SEM T1 T2 T3 Mean ±
SEM C1 C2 C3 Mean ±
SEM Germ cells (%) 0 Dead Dead 67.4 4.8 15.8 21 13.8±4.7 98.2 100 97.6 98.8±0.9 30.9 85.2 71.2 41 65.8±13 0 0 0 0 ehrtoli cells only, (%) 99.4 Damaged (%) 0.6 1.5 10 13 38 20.3±8.8 1.8 0 2.4 1.4±0.7

In the present invention, all stallions were ejaculated on the straw model 3 times a week, and semen was collected for 3 to 6 weeks.

Semen was collected at 1, 4, and 8 weeks before and after crushing plate treatment. The testes of the stallion were hemi-castrated, and the testes were weighed 11 weeks after treatment.

1-3 Stallion slab treatment

The stallion was given an intravenous injection of the squirrel plate. Intravenous injection of bisulpan is a type of injecting 15 mg/kg per body weight (BM) once a week (group 1), dividing 15 mg/kg per body weight for 5 to 6 weeks, and injecting it once a week (group 2) divided by

For the intravenous injection method, Buseulpan was dissolved in 50ml of DMSO (dimethyl sulfoxide) and filtered through a 2 μm syringe filter (Chromdisc, Daegu, Gyeongsanbuk, Korea).

Then, after fixing with a 16 gauge IV catheter (1.7 x 45 mm, Becton Dickinson, NJ, USA) to the calf of the stallion, 50 ml of the dissolved and filtered busulfan solution was injected at 5 ml / min with a melt contact injector (Model Fusion 720, Chemyx) Inc, Stafford, TX).

In the present invention, bisulpan (246.30 g / mol, Sigma, St, Louis, MO, USA) is dissolved in 50 ml of DMSO (dimethyl sulfoxide, 99.7%, Sigma), and 0.22 um PVDF membrane is used to make it sterile. was filtered.

Thereafter, after fixing the catheter to the male vein, 50 ml of the busulpan solution was injected at a rate of 5 cc/min using an infusion pump.

1-4 Quantification of Spermatogenesis Patterns

After hemi-castration of the stallion of the example, the testes were transferred to the laboratory in a 4 °C icebox. The removed testicular tissue was cut to a size of 1 cm and immersed in 4% paraformaldehyde at room temperature for at least 24 hours.

The method of determining the amount of sperm from the testis was stained according to the Hematoxylin & Eosin (H & E) staining procedure in the section of the lavage tube in which the testis testis tissue was sectioned.

Morphological states of spermatogenesis were classified into three different patterns. Sertoli cells were then counted in sperm obtained from seminiferous tubules using a Leica DMIL LED microscope (Leica, Wet-zlar, Germany).

1-5 Semen Collection and Assessment

Stallion semen was collected using a horse model CSU (Animal Reproduction System, Chino, CA) 1 week before and 8 weeks after the treatment of the stilts.

The ejaculated semen was filtered using a disposable nylon mesh gel filter provided on the top of the collection bottle.

After collection, the semen was preheated in a 37°C water bath (JS Research, Gongju-si, Chungcheong-si, Korea), and the semen was diluted (1:20) with INRA96 (IMV technology, L'Aigle, France) extender.

Diluted semen was assessed for motility using a computer-assisted movement analysis system (MICROPTIC, Sperm Class Analyzer 5.4, Barcelona, Spain) and an optical microscope (Nicon, E200, Konan, Minato-ku, Tokyo).

The total/progressive movement of sperm was investigated with a phase-contrast microscope (Nikon) at 100 magnification and photographed with a BASLER camera (BASLER, Ahrensburg, Germany).

To calculate sperm concentration, ejaculated semen was diluted 1:20, fixed with 4% paraformaldehyde (Formalin 10 Equine semen dilution, Animal Reproduction Systems), and cytometer (MARIENFELD, Lauda-konigshofen; Germany) was used to count (Nicon).

1-6 Sexual Impulsivity (Libido)

When endogenous germ cells are removed, it is important to maintain the sex drive of the donor embryos and the recipient. All stallions were monitored and counted the time from breeding area to erection (minutes), time from washing to ejaculation (minutes), time from surface to ejaculation (minutes), and time from mounting on the model to ejaculation. . The time of each sexual activity was measured with an electronic timer (Electronics Tomorrow Limited, Cheung Street, Hung Hom, Hong Kong). In order to test the sexual behavior of the stallion, a sexual behavioral environment was provided by moving a model such as teasing a mare with the same cycling.

<Example 2>

2-1 Stallion survival rate (%) after treatment

The survival rate of the stallion was monitored as a result of the single-injection and split-injection of the bustle plate, and the results are shown in FIG. 1 .

As a result, in group 1, 2 of 3 stallions died after treatment with 15 mg/kg of body weight busulpan (survival rate = 33%).

However, in group 2, three stallions injected with 15 mg/kg of body weight divided and injected survived (survival rate = 100%).

2-2 Testicular volume

The weight of the testicles of the castrated stallion was measured at 11 weeks after the treatment with the beesulhwan, and the weight of the testicles is shown in FIG. 2 .

The average weight (g) of testicles (224 ± 22) of the horseshoe plate treated stallions was not significantly different from the average weight (g) of testes (203 ± 9.4) of the control stallions treated with PSB.

2-3 Effect of shatterboard treatment on spermatogenesis

Hemi-castration of the stallions was performed 11 weeks after treatment.

H & E staining was performed to quantify the morphological state of spermatogenesis in the seminiferous tubule cross-section by the processing of the sesame plate, and the results are shown in FIGS. 3 and 4 . As shown, the state of spermatogenesis appeared normal spermatogenesis, only Sertoli cells, and a disrupted spermatogenesis pattern.

The average percentage (%) of the cross section in which normal sperm was formed was 13.8 ± 4.7, that of Sertoli cells was 65.8 ± 13, and that of destroyed was 20.3 ± 8.8. The mean percentages of Sertoli cells and disrupted spermatogenesis were significantly higher (p <.05) than that of the control group (0, 1.4 ± 0.7).

2-4 Evaluation of semen motility

The semen of the stallions was collected for analysis of total/progressive sperm motility 1 week before and 4 and 8 weeks after the treatment with the stilt.

The total motility of ejaculated sperm of stallions was not significantly different between 1 week, 2 weeks, and 4 weeks after treatment with saphenous and PBS (P> .05).

However, the total sperm count in the group 8 weeks after the treatment was significantly decreased compared to the one week before the treatment (P> .05).

There was no significant difference in total sperm count before and after PBS treatment in the control group (P > .05).

The total/progressive motility of sperm collected in the saline treatment group was not different from that of the control group (P > .05).

In group 1, no sperm data or statistical analysis could be performed, because one injection of 15 mg of fusulpan per kg of body weight resulted in the death of two of the three stallions.

2-5. sexual impulsivity (Libido)

In this experiment, the libido of the stallion did not differ significantly between the PBS-treated group and the Bu-seulpan-treated group in the erection time in the breeding area before ejaculation, the ejaculation time after the washing step, and the number of seizures before ejaculation, and decreased libido. was found not to appear.

<Example 3>

3-1 Discussion

The present inventors confirmed the possibility of removing endogenous spermatogonial cells by intravenous injection by dividing the spleen plate to prepare a recipient horse for transplantation of spermatogonial stem cells in a stallion.

In the present invention, it was confirmed that the split injection of the saphenous pancreas can be safely performed from hematopoiesis or toxicity and suppression of donor sperm development.

As a result, it was shown that the survival rate of the stallions injected with the buseulpan intravenously was higher than the single injection administration and the divided injection administration. Two of the three stallions in the group injected intravenously with a single injection of mussel plate died, but all the stallions of the group that were divided and injected intravenously survived. This indicates that, if the concentration of musulpan is excessive during one injection, the mortality rate of the stallion increases in intravenous administration, and the mortality rate can be reduced through divided administration.

As described above, specific embodiments of the present invention have been described in detail for the method for preparing a recipient animal for spermatogonial cell transplantation according to the present invention. Through addition, change, deletion, etc., other degenerative inventions or other embodiments included within the scope of the spirit of the present invention may be easily proposed. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the claims described later rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. should be interpreted as

Claims (9)

A method for preparing a recipient animal other than a human, comprising the step of dividingly injecting an alkylating agent into a spermatogonial cell recipient animal,
The recipient animal is characterized in that the horse,
The alkylating agent is characterized in that busulfan (busulfan),
The busulfan is characterized in that it is injected into the vein of the recipient animal,
The divided injection is to divide the total injection amount of busulfan equally and inject it once a week at intervals of 1 week for the period until it is conferable,
The equal division is to divide the total injection amount of busulfan by the period until awardable,
The total injection amount of the busulfan is characterized in that 15 mg / kg,
The method for producing a recipient animal other than a human, characterized in that the period until the award is 5 to 6 weeks. delete delete delete delete delete delete According to claim 1,
The buseulpan is a method for preparing a recipient animal other than a human, wherein a powdered buseulpan having a dose of 15 mg/kg of the body mass of the recipient animal to be treated is diluted in 50 ml of a solvent. A recipient animal, other than a human, for spermatogonial cell transplantation prepared by the method of claim 1 or 8.

Images