Large-scale production method of rat ova based on novel superovulation induction treatment

Abstract

The invention aims to provide a superovulation inducing method for rats. According to the present invention, it is possible to provide a method for inducing superovulation, characterized in that an anti-statin antibody and a horse chorionic gonadotropin (eCG) are simultaneously administered to a female rat less than 6 weeks old, followed by a human chorionic gonadotropin (hCG). In addition, according to the present invention, an in vitro fertilization method using an ovum produced by the method of the present invention can be provided. The method can be used for rats of various strains.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for inducing superovulation in female rats. BACKGROUND

[0002] Experimental animals are essential for research for in-depth understanding of healthy or pathological states, and evaluation of effectiveness of candidate therapies intended to be applied to human diseases. Comprehensive databases / platforms on experimental rats such as the Japanese National Bioresource Project "Rat", the Rat Genome Database in the United States have been developed. Rats have contributed greatly to the development of therapeutic drugs, treatment regimens in the form of human disease models, but the use of rats in genetic modification research has not reached the level of mice because of the difficulty in implementing basic embryonic engineering techniques such as superovulation, in vitro fertilization (IVF), etc.

[0003] The number of strains of rats is much smaller than that of mice. Rats have the following characteristics: they are larger in size than mice, so it is easy to take a larger amount of blood, urine, and the like over time than mice, and in addition, the size of organs and tissues is sufficient, with the advantage that they can also be used for surgical operations. In addition, rats are docile and easy to tame, so they are also suitable for experiments such as behavioral observation.

[0004] Sexual maturation of female rats is at the age of 50 to 80 days. The vaginal opening of females is closed by skin before about 20 days, but opens with growth and vigorous ovarian function. The appropriate breeding period is 60 to 80 days in females, and 12 to 15 eggs are discharged in a 4-day cycle.

[0005] Superovulation is one of the most important techniques for efficient production of transgenic rats. For this purpose, superovulation methods using pregnant mare serum gonadotropin [pregnant mare serum gonadotropin: PMSG; also known as equine chorionic gonadotropin (eCG)], human chorionic gonadotropin (hCG) have been reported, but there are strains that do not respond to the above superovulation methods. It has been reported that in superovulation using PMSG and hCG, the number of eggs per rat is 17.0 in the Wistar strain, 31.0 in the F344 strain, and 2.2 in the BN strain (Non-Patent Literature 1).

[0006] Therefore, various methods have been proposed. For example, a method has been proposed in which oocyte-granulosa cell complexes (OGC) are recovered after culturing ovaries taken from females after administration of PMSG in vitro, and it has been reported that the number of OGC recovered per rat is increased to 27.7 in Wistar strain, 34.7 in F344 strain, and 24.7 in BN strain (Non-Patent Literature 1). In addition, by administering anti-inhibin serum (AIS) to rats at the same time as PMSG and hCG, 19.9 oocytes per rat can be recovered in BN strain (Non-Patent Literature 2). It has been reported that by administering PMSG and hCG after synchronization of estrus cycle using luteinizing hormone-releasing hormone (LH-RH), 22.3 oocytes can be recovered in BN strain rats, and further, by administering PMSG and hCG after synchronization of estrus cycle using LHRH and AIS, 42.0 oocytes per rat can be recovered in BN strain rats, 42.5 oocytes in Wistar strain, 33.3 oocytes in F344 strain, 45.5 oocytes in LE strain, and 53.9 oocytes in SD strain (Non-Patent Literature 2).

[0007] Prior Art Documents

[0008] Non-Patent Literature

[0009] Non-Patent Literature 1: H. Taketsuru and T. Kaneko, Journal of Reproduction and Development, Vol. 62, No 5, 2016

[0010] Non-Patent Literature 2: A. Honda et al., Scientific Reports (2019) 9:11571

[0011] Non-Patent Literature 3: T. Aoto et al., Transgenic Res. (2011) 20:1245-12526

[0012] Non-Patent Literature 4: N. Nakagata et al., Sci Rep. 2020 Jan 9;10(1):93. doi:10.1038 / s41598-019-57090-7

[0013] Non-patent literature 5: K. Yamaga et al., Sci Rep. 2021; 11: 22641. doi: 10.1038 / s41598-021-02166-6

[0014] Non-patent literature 6: H. Kishi et al., The Journal of endocrinology 151(1): 65-75, 1996

[0015] Non-patent literature 7: Nakagata, Exp. Anim. 41(3), 387-388 (1992) SUMMARY

[0016] As the superovulation treatment technique for rats, as described above, there are known methods of administering pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and methods of further using luteinizing hormone-releasing hormone (LHRH), anti-steroidin serum (AIS), but so far, a method in which about 50 eggs can be recovered per rat in multiple strains of rats has only been reported by Honda et al. (Non-patent literature 2) using LHRH, AIS, PMSG, and hCG. Since LHRH is expensive and cannot be stored for a long period of time, a method using a hormone reagent that can be stored for a long period of time and utilizing a simple method to recover sufficient eggs in various strains of rats is desired.

[0017] To solve the above problems, the present inventors conducted intensive studies, as a result of which it was found that the effects of hormones administered in superovulation treatment differ depending on the age of rats, and thus the present invention was completed. Specifically, it was found that sufficient superovulation can be induced by the combination of the hormone used and the age of the rats to which the hormone is administered.

[0018] In addition, the present inventors confirmed the quality of the eggs produced by the superovulation treatment of the present invention by in vitro fertilization (IVF) with sperm, as a result of which it was also found to be good. Furthermore, the development ability of the embryos obtained was confirmed by embryo transfer. As a result, the development potential was also good.

[0019] The present invention includes the following.

[0020] [1] A method of inducing superovulation, characterized by simultaneously administering an anti-steroidin antibody (preferably, anti-steroidin serum) and equine chorionic gonadotropin (eCG) to a female rat less than 6 weeks old (preferably, 3 weeks old or older and less than 6 weeks old), and then administering human chorionic gonadotropin (hCG).

[0021] [2] The method according to the above [1], wherein the anti-inhibin antibody is administered in the form of anti-inhibin serum.

[0022] [3] The method according to the above [1] or [2], wherein the female rats are 3 to 5 weeks old.

[0023] [4] The method according to any one of the above [1] to [3], wherein the female rats are not administered with exogenous luteinizing hormone-releasing hormone (LH-RH) before administration of the equine chorionic gonadotropin.

[0024] [5] The method according to any one of the above [1] to [3], wherein the female rats are not administered with exogenous gonadotropin-releasing hormone before administration of the equine chorionic gonadotropin.

[0025] [6] The method according to any one of the above [2] to [5], wherein the anti-inhibin serum administered is 0.3 mL or less per rat.

[0026] [7] The method according to the above [6], wherein the anti-inhibin serum administered is 0.05 mL to 0.2 mL per rat.

[0027] [8] The method according to any one of the above [1] to [7], wherein the equine chorionic gonadotropin administered is 5 IU to 40 IU (preferably 10 IU to 30 IU) per rat.

[0028] [9] The method according to any one of the above [1] to [8], wherein the female rats are of a strain selected from the group consisting of CD (SD), Wistar, Long-Evans, F344, BN and LEW.

[0029]

[10] An in vitro fertilization method comprising the following steps:

[0030] Step a. preparing oocytes using the method according to any one of the above [1] to [9], and

[0031] Step b. fertilizing the oocytes prepared in the above step with sperm (preferably sperm of a genetically modified male rat) of a male rat.

[0032]

[11] An in vitro fertilization method comprising the following steps:

[0033] Step (1) administering an anti-inhibin antibody and equine chorionic gonadotropin (eCG) to a female rat less than 6 weeks old (preferably more than 3 weeks old and less than 6 weeks old) at the same time, followed by administration of human chorionic gonadotropin (hCG) to induce superovulation,

[0034] Step (2) involves recovering oocytes from female rats that have undergone superovulation, and

[0035] Step (3) involves fertilizing the eggs recovered in the above steps with sperm from male rats (preferably sperm from genetically modified male rats).

[0036]

[12] According to the in vitro fertilization method described in

[11] above, the above-mentioned female rats are sperm derived from male rats selected from the strains of CD (SD), Wistar, Long-Evans, F344, BN and LEW.

[0037]

[13] The method described in

[11] or

[12] above, wherein the anti-inhibitor antibody is given in the form of anti-inhibitor serum.

[0038]

[14] The method according to any one of

[11] to

[14] above, wherein the above-mentioned female rat is a female rat aged 3 to 5 weeks.

[0039]

[15] The method according to any one of

[11] to

[15] above, wherein the above-mentioned female rat is a female rat that has not been given exogenous gonadotropin-releasing hormone before the administration of equine chorionic gonadotropin.

[0040]

[16] According to the method described in

[11] to

[15] above, wherein the above-mentioned female rats are female rats that have not been given exogenous luteinizing hormone-releasing hormone (LH-RH) before the administration of equine chorionic gonadotropin.

[0041]

[17] The method according to any one of

[11] to

[16] above, wherein the amount of antiinhibin serum given is less than 0.3 mL.

[0042]

[18] According to the method described in

[17] above, the anti-inhibin serum administered is 0.05 mL to 0.2 mL.

[0043]

[19] The method according to any one of

[11] to

[18] above, wherein the equine chorionic gonadotropin given above is 5 IU to 40 IU (preferably 10 IU to 30 IU).

[0044] The novel superovulation technique of this invention can efficiently produce a large number of eggs from a single female rat compared to previous methods. Attached Figure Description

[0045] Figure 1Results obtained by applying the method of the present application to 5-week-old Crl:CD (SD) female rats are shown. Data represent mean ± SEM, dots represent individual subjects (rats). * indicates P<0.05.

[0046] Figure 2 A photograph of a superovulation-induced zygote obtained by in vitro fertilization between the eggs obtained by inducing superovulation by the method of the present application and fresh sperm of the same strain of Crl:CD (SD) is shown.

[0047] Figure 3 A photograph of a pup born by implanting the zygote produced by in vitro fertilization between the eggs obtained by inducing superovulation by the method of the present application and fresh sperm into the oviduct of a surrogate female mouse is shown.

[0048] Figure 4 A photograph of a pup born by implanting the zygote produced by in vitro fertilization between the eggs obtained by inducing superovulation by the method of the present application and frozen EGFP-transgenic rat sperm into the oviduct of a surrogate female mouse is shown.

[0049] Figure 5 Results obtained by applying the method of the present application to 5-week-old Wistar female rats are shown. Data represent mean ± SEM, dots represent individual subjects (rats) (the same applies to the following figures, unless otherwise specified). * indicates P<0.05, n.s. indicates no significant difference (the same applies to the following figures, unless otherwise specified).

[0050] Figure 6 Results obtained by applying the method of the present application to 5-week-old LE female rats are shown.

[0051] Figure 7 Results obtained by applying the method of the present application to 5-week-old F344 female rats are shown.

[0052] Figure 8 Results obtained by applying the method of the present application to 5-week-old BN female rats are shown.

[0053] Figure 9 Results obtained by applying the method of the present application to 5-week-old LEW female rats are shown.

[0054] Figure 10 Results obtained by applying the method of the present application to 3-week-old Crl:CD (SD) female rats are shown.

[0055] Figure 11 Results obtained by applying the method of the present application to 6-week-old Crl:CD (SD) female rats are shown.

[0056] Figure 12Results of applying the method of the present application to 7-week-old Crl:CD (SD) female rats are shown. DETAILED DESCRIPTION

[0057] The present application will be described with respect to exemplary embodiments as examples only and as illustrative of the preferred methods and materials that can be used in the practice of the application. It is to be understood that throughout the description and claims of this application, all relative terms are to be construed in the manner typical to one of ordinary skill in the art in the field of the application. In addition, any materials and methods similar or equivalent to those described herein can be used in the practice of the present application. Also, the practice of the present application will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, genetics, immunology, and pharmacology, within the skill of the art; such methods are explained fully in the literature.

[0058] In the present specification, the description of "A to B" indicating a numerical range means a numerical range including A and B as end points. Also the same applies to "A to B". Also, in the present specification, "about" is used in the sense of allowing ±10%.

[0059] In the present specification, pregnant mare serum gonadotropin (PMSG) and equine chorionic gonadotropin (eCG) mean the same substance and are used as terms that can be replaced with each other. Also, in the present specification, anti-inhibin serum (AIS) and anti-inhibin serum (IAS) mean the same substance and are used as terms that can be replaced with each other.

[0060] The present inventors have found that when anti-inhibin serum and equine chorionic gonadotropin (eCG) are simultaneously administered to a female rat less than 6 weeks old, followed by administration of human chorionic gonadotropin (hCG), superovulation can be induced efficiently. One embodiment of the present application is a method of inducing superovulation, characterized by simultaneously administering anti-inhibin serum and equine chorionic gonadotropin (eCG) to a female rat less than 6 weeks old, preferably more than 3 weeks old and less than 6 weeks old, followed by administration of human chorionic gonadotropin (hCG).

[0061] Another embodiment of the present application is an in vitro fertilization method (IVF) comprising: a step (i) of inducing superovulation by simultaneously administering anti-inhibin serum and equine chorionic gonadotropin (eCG) to a female rat less than 6 weeks old, preferably more than 3 weeks old and less than 6 weeks old, followed by administration of human chorionic gonadotropin (hCG); a step (ii) of recovering oocytes from the female rat in which superovulation has been induced; and a step (iii) of fertilizing the recovered oocytes with sperm of a male rat, preferably sperm of a genetically modified male rat.

[0062] The female rats used in the method of the present application can be any strain of female rats. For example, female rats of strains of Jackson Laboratory, female rats of strains of SLC, Japan can be mentioned. The female rat strain is not limited thereto, and Crl:CD (SD), Crlj:WI, Crlj:LE, F344 / DuCrlCrlj, LEW / CrlCrlj, BN / CrlCrlj, WKY / NCrlCrlj, Slc:SD, Slc:Wistar, Slc:Wistar / ST, lar:Long-Evans (LE), F344 / NSlc, DA / Slc, LEW / SsNSlc, BN / ScNSlc, ACI / NSlc, in particular, the commonly used SD strain, Wistar strain, F344 strain, LEW strain, LE strain, BN strain are preferable. Genetically modified rats (transgenic rats), various disease model rats made using these strains can also be used in the method of the present application. As disease model rats, PCK / CrlCrlj-Pkhd1 pck / CrlCrlj, ZDF-Lepr fa / CrlCrlj, GK / Slc, Slc:Zucker-fa / fa, Hos:ZFDm-Lepr fa , SHR / lzm, SHRSP / lzm, DIS / EisSlc, DIR / EisSlc, SHRAP5 / Dmcr, HWY / Slc. In addition, female rats of new strains to be established in the future, genetically modified rats, and disease model rats can also be used in the method of the present application.

[0063] The female rats are sexually mature at 50 days to 80 days, and rats less than this are referred to as immature rats. Therefore, rats less than 7 weeks of age are immature rats, and the method of the present application is characterized by using younger female rats less than 6 weeks of age. The female rats used in the present application are 3 weeks of age or older and less than 6 weeks of age, preferably 3 to 5 weeks of age, and more preferably 4 to 5 weeks of age. In the present specification, 3 weeks of age means 21 days to 27 days after birth, 4 weeks of age means 28 days to 34 days after birth, and 5 weeks of age means 35 days to 41 days after birth. Therefore, female rats 3 weeks of age or older and less than 6 weeks of age mean female rats 21 days to 41 days after birth.

[0064] When the age is mentioned in the present application, it means the age at the time of administration of PMSG (eCG), IAS, or hCG, and the age is preferably the age at the time of administration of PMSG.

[0065] The anti-inhibin antibody used in the present application is only required to be an antibody having an activity of neutralizing endogenous inhibin in rats. For example, anti-inhibin serum containing an anti-inhibin antibody can be obtained by immunizing a castrated goat with purified 32 kDa porcine, mouse or rat inhibin as an antigen. The anti-inhibin activity can be determined by a conventional method. For example, the immune titer can be determined by an immunoassay. In the present application, the anti-inhibin antibody can be in the form of anti-inhibin serum or a purified (including crude purification) antibody.

[0066] When the anti-inhibin serum is used, for example, 0.05 mL to 0.3 mL, preferably 0.05 mL to 0.2 mL, more preferably 0.05 mL to 0.15 mL, further preferably about 0.1 mL of the prepared antiserum is administered to the rat, but is not limited thereto.

[0067] The amount of equine chorionic gonadotropin (eCG) administered to the female rat in the superovulation induction method of the present application uses an amount generally used in the superovulation treatment of experimental rats. For example, generally 2 to 50 IU, preferably 5 to 40 IU, more preferably 10 to 30 IU, further preferably 15 to 25 IU per rat. By using 5 to 40 IU, preferably 10 to 30 IU of eCG in combination with 0.05 to 0.3 mL, preferably 0.05 to 0.2 mL of IAS per rat for a female rat of, for example, more than 3 weeks old and less than 6 weeks old, more preferably 4 to 5 weeks old, excellent superovulation induction can be achieved.

[0068] The amount of human chorionic gonadotropin (hCG) administered to the female rat in the superovulation induction method of the present application uses an amount generally used in the superovulation treatment of experimental rats. For example, 2 to 50 IU, preferably 5 to 40 IU, more preferably 10 to 30 IU, further preferably 15 to 25 IU per rat.

[0069] The superovulation induction method of the present application is characterized in that an anti-inhibin antibody (for example, anti-inhibin serum) and equine chorionic gonadotropin (eCG) are simultaneously administered to a female rat of less than 6 weeks old, and then human chorionic gonadotropin (hCG) is administered. The "simultaneous administration" of the anti-inhibin antibody and eCG means that they can be administered separately at the same time or administered together as a mixture, and in the case of separate administration, they can be administered at approximately the same time. The administration is performed, for example, by injection into the abdominal cavity, but is not limited thereto. Then, after a certain period of time, for example, after about 2 days to about 3 days, preferably about 2 days, hCG is administered. The administration of hCG is performed, for example, by injection into the abdominal cavity, but is not limited thereto.

[0070] The superovulation induction method of the present application can obtain significantly more oocytes than the number of oocytes obtained by the method reported in the past by administering an anti-inhibin antibody (preferably, anti-inhibin serum) and equine chorionic gonadotropin (eCG) to a female rat as an object of less than 6 weeks old, particularly 3 weeks old or more and less than 6 weeks old, and then administering human chorionic gonadotropin (hCG).

[0071] The oocyte obtained by the present application can be fertilized with sperm of a male rat. The sperm can be taken from an adult male rat using a conventional method, and can be fertilized with the oocyte produced in the present application in a fresh state or after cold storage or cryopreservation. The male rat that provides the sperm is preferably a rat of the same strain as the female rat used for fertilization. Fertilization can be performed according to a conventional method. For example, it can be performed according to the methods reported in the following documents (Non-Patent Documents 3 to 5: T. Aoto et al., Transgenic Res. (2011) 20: 1245-1252, N. Nakagata et al., Sci Rep. 2020 Jan 9; 10(1): 93. doi: 10.1038 / s41598-019-57090-7, K. Yamaga et al., Sci Rep. 2021; 11: 22641. doi: 10.1038 / s41598-021-02166-6, which are incorporated herein by reference). The zygote obtained by the oocyte produced by the present application can be developed into a cleavage embryo. The method of producing a cleavage embryo in this way is also included in the present application. Furthermore, the cleavage embryo thus obtained can be transplanted into a recipient by embryo transfer to develop into a pup. Embryo transfer can be performed by a conventional method. The method of producing a rat in this way is also included in the present application.

[0072] Therefore, one mode of the present application is also an in vitro fertilization method of in vitro fertilizing the oocyte taken from a female rat using the method of inducing superovulation of the present application with sperm of a male rat (preferably, a genetically modified rat).

[0073] Another mode of the present application is also a method of producing a cleavage embryo by culturing the zygote obtained by the in vitro fertilization method of the present application.

[0074] Still another mode of the present application is also a method of developing a pup by transplanting the cleavage embryo obtained by the method of producing a cleavage embryo of the present application into a recipient by embryo transfer.

[0075] In the present specification, "exogenous" in the gonadotropin-releasing hormone or luteinizing hormone-releasing hormone refers to a hormone that is not produced in the body of a living rat but is administered to the rat from the outside. The source of the exogenous gonadotropin-releasing hormone or luteinizing hormone-releasing hormone can be either a rat or another species (e.g., pig, mouse).

[0076] Examples

[0077] Hereinafter, the present application will be specifically described by way of examples, but the present application is not limited to the following examples.

[0078] 1. Materials and methods

[0079] (1-1) Animals

[0080] Female and male rats of each strain were purchased from Jackson Laboratory Japan (formerly Charles River Japan). Female rats were used as egg donors using female rats of each recorded week of age, and male rats were used as sperm donors at 12 to 14 weeks of age. All animals were allowed to freely take in feed and water, and were reared at 22°C ± 1°C with a 12-hour light-dark cycle (lighting time: 7:00 to 19:00). Animal experiments were performed in accordance with the protocols approved by the Kumamoto University Animal Experiment Committee.

[0081] (1-2) Culture medium

[0082] The pre-culture medium for sperm and the in vitro fertilization culture medium used human tubal fluid (mHTF) with increased calcium. Rat embryo culture medium (R1 ECM) was used for the manipulation of two-cell stage embryos or the culture of embryos until the blastocyst stage.

[0083] (1-3) Ovulation and ovum collection

[0084] Female rats were administered PMSG (Meiji Seika Kaisha, Ltd.) alone, a combination of IAS and PMSG, or a combination of LH-RH (Peptide Institute, Inc.) and IAS and PMSG. IAS was prepared by using mouse inhibin peptide according to the method described in the paper by Kishi et al. (Non-patent document 6: H. Kishi et al., The Journal of endocrinology 151(1): 65-75, 1996, which is incorporated herein by reference) and the prepared antiserum was used directly. The prepared IAS was subjected to determination of its antibody activity by ELISA method and used after being standardized as necessary. Specifically, mouse inhibin peptide at a concentration of 5 μg / mL was adsorbed to an enzyme plate for ELISA, followed by washing and blocking. Anti-inhibin serum diluted 1000-fold to 128,000-fold was subjected to treatment, and an anti-goat IgG antibody labeled with horseradish peroxidase was used as a secondary antibody to perform ELISA, and it was confirmed that inhibin antibody could be detected sufficiently even at the maximum dilution ratio, and used in the following experiments.

[0085] In the experimental group administered with LH-RH, LH-RH was administered about 2 days before PMSG was administered. In the experimental group administered with IAS, IAS was administered at the same time as PMSG.

[0086] (1-4) In vitro fertilization

[0087] Sperm was taken from genetically modified rats of SD, Wistar, or LE strain background as follows. After male rats were euthanized by cervical dislocation, the cauda epididymidis was taken and transferred to a petri dish for pre-culture of sperm covered with paraffin oil. Sperm clumps were recovered from the cauda epididymidis using a glass rod and transferred to a 400-μL droplet of mHTF medium, followed by secondary dilution in a 200-μL droplet of mHTF medium at a final sperm concentration of 500 sperm / μL. Ova were administered with hCG (Meiji Seika Kaisha, Ltd.) to rats about 2 days after PMSG administration. One day after hCG administration, rats were euthanized by cervical dislocation, and the oviducts of the rats were quickly removed, and cumulus-oocyte complexes (COCs) were taken from the oviducts under a microscope and introduced into a 200-μL droplet of mHTF medium containing sperm (insemination). Seven hours after insemination, ova were washed in four droplets (80 μL) of mHTF. After ova were washed, the number of ova was counted. After 22 to 24 hours of fertilization, the total number of pronuclear stage fertilized ova and two-cell stage embryos was divided by the total number of ova and multiplied by 100, thereby calculating the fertilization rate.

[0088] (1-6) Embryo transfer

[0089] Embryo transfer was performed according to the procedure previously reported by the present inventors (Non-patent Literature 7, Nakagata, N. Embryo transfer through the wall of the fallopian tube in mice. Exp. Anim. 41(3), 387-388 (1992), which is incorporated herein by reference). Fertilized eggs and two-cell stage embryos obtained by in vitro fertilization were transferred into the oviduct of CD (SD) female rats on the day of vaginal plug appearance (day 1 of pseudo-pregnancy) (10 embryos / oviduct). Embryos were transferred through the oviduct wall. The number of pups was recorded 22 days later.

[0090] (1-7) Statistical processing

[0091] Statistical analysis was performed using Prism 5.0 (GraphPad). Results are expressed as mean ± standard deviation. Results of groups were compared using analysis of variance after the inverse sine conversion of percentages. p < 0.05 was determined to be statistically significant.

[0092] (Example 1)

[0093] PMSG (eCG), IAS, and hCG were administered to 4-week-old or 5-week-old Crl:CD (SD) female rats under the following four conditions (1 to 4) to obtain oocytes.

[0094] • Condition 1: hCG (300 IU / kg) was administered 48 to 50 hours after administration of PMSG (300 IU / kg).

[0095] • Condition 2: PMSG (300 IU / kg) was administered 52 to 53 hours after administration of LH-RH (0.04 mg dissolved in 0.2 mL of physiological saline), and hCG (300 IU / kg) was further administered 48 to 50 hours after administration of PMSG.

[0096] • Condition 3: hCG (300 IU / kg) was administered 48 to 50 hours after administration of PMSG (300 IU / kg) and IAS (0.1 mL).

[0097] • Condition 4: PMSG (300 IU / kg) and IAS (0.1 mL) were administered 52 to 53 hours after administration of LH-RH (0.04 mg dissolved in 200 μL of physiological saline), and hCG (300 IU / kg) was further administered 48 to 50 hours after administration of PMSG.

[0098] Four female rats in each group were used as oocyte donors. In vitro fertilization was performed between the obtained oocytes and the sperm of genetically modified rats.

[0099] The results are shown in Table 1 below.

[0100] [Table 1]

[0101]

[0102] It was shown that superovulation was significantly induced by administering IAS and PMSG to female rats simultaneously, followed by administration of hCG. In addition, according to the comparison between Condition 1 and Condition 2 and the comparison between Condition 3 and Condition 4, it was shown that there was no effect of LH-RH when rats of the CD (SD) strain at the age of 4 weeks and 5 weeks were used. In particular, in the rats at the age of 5 weeks, it was shown that administration of LH-RH inhibited the induction of superovulation by administration of IAS and PMSG. It should be noted that the fertilization rate showed a good fertilization rate in any case.

[0103] (Example 2)

[0104] The same operation as in Example 1 was performed using 5-week-old Crl:CD (SD) female rats to confirm the effects of LH-RH, IAS, and PMSG under the following three conditions (experimental zones). The amounts of LH-RH, IAS, PMSG, and hCG administered were 0.04 mg, 0.1 mL, 20 IU, and 20 IU per rat, respectively. The administration conditions are described below.

[0105] • Experimental Zone LH-RH: IAS and PMSG were administered 2 days after administration of LH-RH, and hCG was further administered 2 days after that.

[0106] • Experimental Zone IAS e: IAS and PMSG were administered, and hCG was administered 2 days after that.

[0107] • Experimental Zone PMSG: PMSG was administered, and hCG was administered 2 days after that.

[0108] The same operation as in Example 1 was performed using the obtained eggs to perform in vitro fertilization, and further, the fertilized eggs were transplanted into recipients to develop into pups.

[0109] The results of the number of ovulations and the fertilization rate are shown in Tables 2 and 3 below. Figure 1 .

[0110] [Table 2]

[0111]

[0112] It was shown that superovulation was significantly induced by simultaneous administration of IAS and PMSG, followed by administration of hCG, to 5-week-old Crl:CD (SD) female rats. In addition, it was shown that the effect of superovulation induction was in the order of IAS > LH-RH > PMSG, and that administration of LH-RH effectively suppressed superovulation induced by administration of IAS and PMSG. It should be noted that the fertilization rate exhibited good fertilization rates in any case.

[0113] Using the oocytes taken from the female rats in which superovulation was induced, in vitro fertilization was performed with fresh sperm of Crl:CD (SD). Photographs of the resulting fertilized eggs are shown in Figure 2 . In all of the oocytes, pronuclei and sperm tails were seen in the cytoplasm of the oocytes. In addition, photographs of the offspring born by implanting the prepared fertilized eggs into the oviducts of surrogate female mice are shown in Figure 3 .

[0114] In addition, in vitro fertilization was performed between the oocytes taken from the female rats in which superovulation was induced and frozen-preserved EGFP (Enhanced Green Fluorescent Protein) transgenic rat sperm, and the prepared fertilized eggs were implanted into the oviducts of surrogate female mice to produce offspring. The results are shown in Figure 4 . The EGFP-derived gene was expressed throughout the body, resulting in green offspring. All of the offspring obtained expressed EGFP.

[0115] (Example 3)

[0116] The effect of combined administration of IAS and PMSG to various strains of rats was investigated. Using 5-week-old female rats of various strains (Wistar, LE, F344, BN, and LEW), the induction of superovulation by LH-RH, IAS, and PMSG was investigated, in the same manner as in Example 2.

[0117] The results for Wistar female rats are shown in the following table and Figure 5 .

[0118] [Table 3]

[0119]

[0120] It was shown that superovulation was significantly induced by simultaneous administration of IAS and PMSG, followed by administration of hCG, to 5-week-old Wistar female rats. In addition, it was shown that the effect of superovulation induction was in the order of IAS > LH-RH > PMSG, and that administration of LH-RH effectively suppressed superovulation induced by administration of IAS and PMSG. It should be noted that the fertilization rate exhibited good fertilization rates in any case.

[0121] Results for LE female rats are shown in the following table and Figure 6 .

[0122] [Table 4]

[0123]

[0124] indicate that superovulation was significantly induced by the simultaneous administration of IAS and PMSG, followed by hCG, to 5-week-old F344 female rats. It is also indicated that the effect of superovulation induction was in the order of IAS > LH-RH > PMSG, and that the administration of LH-RH effectively suppressed superovulation induced by the administration of IAS and PMSG. It should be noted that the fertilization rate exhibited a good fertilization rate in any case.

[0125] Results for F344 female rats are shown in the following table and Figure 7 .

[0126] [Table 5]

[0127]

[0128] indicate that superovulation was significantly induced by the simultaneous administration of IAS and PMSG, followed by hCG, to 5-week-old F344 female rats. It is also indicated that the effect of superovulation induction was in the order of IAS > LH-RH > PMSG, and that the administration of LH-RH effectively suppressed superovulation induced by the administration of IAS and PMSG. It should be noted that the fertilization rate exhibited a good fertilization rate in any case.

[0129] Results for BN female rats are shown in the following table and Figure 8 .

[0130] [Table 6]

[0131]

[0132] indicate that superovulation was significantly induced by the simultaneous administration of IAS and PMSG, followed by hCG, to 5-week-old F344 female rats. It is also indicated that the effect of superovulation induction was in the order of IAS > LH-RH > PMSG, and that the administration of LH-RH effectively suppressed superovulation induced by the administration of IAS and PMSG. It should be noted that the fertilization rate exhibited a good fertilization rate in any case.

[0133] Results for LEW female rats are shown in the following table and Figure 9 .

[0134] [Table 7]

[0135]

[0136] It was shown that superovulation was significantly induced by simultaneous administration of IAS and PMSG, followed by administration of hCG to 5-week-old LE female rats. In addition, it was shown that the superovulation-inducing effect was in the order of IAS > LH-RH > PMSG, and that administration of LH-RH effectively suppressed superovulation induced by administration of IAS and PMSG. It should be noted that the fertilization rate showed a good fertilization rate in any case.

[0137] (Example 4)

[0138] The effect of IAS, PMSG was confirmed using 3-week-old Crl:CD (SD) female rats, in the same manner as in Example 2. The administration amounts of IAS, PMSG, hCG were 0.1 mL, 20 IU, 20 IU per one rat, respectively. The administration condition was administration of IAS and PMSG, followed by administration of hCG two days later. The obtained oocytes were used to perform in vitro fertilization, in the same manner as in Example 2. The results are shown in the following table and Figure 10 The ovulation effect almost equivalent to that of 4-week-old, 5-week-old rats was confirmed even for 3-week-old rats.

[0139] [Table 8]

[0140]

[0141] (Comparative Example) Effect of LH-RH on superovulation induction by IAS + PMSG

[0142] According to the report by Honda et al. (Non-patent literature 2), it was reported that administration of IAS and PMSG, followed by administration of hCG after administration of LH-RH to immature BN rats significantly induced ovulation compared to the case where LH-RH was not administered. In addition, it was shown that the same tendency was observed in other strains of rats (Wistar, F344, LE, and SD).

[0143] However, as shown in Examples 1 to 3, it was confirmed that in all strains of rats, although administration of IAS and PMSG significantly induced superovulation, the superovulation-inducing effect by administration of IAS and PMSG was suppressed by administration of LH-RH. In addition, the suppressing effect was more significant in 5-week-old than in 4-week-old Crl:CD (SD) female rats.

[0144] In order to confirm the difference, the effect of LH-RH, IAS, and PMSG on superovulation induction was confirmed using 6-week-old and 7-week-old female rats as immature rats.

[0145] The effects of LH-RH, IAS, and PMSG were confirmed in the same manner as in Example 2, using 6- and 7-week-old Crl:CD (SD) female rats, under the following three conditions (experimental zones). The amounts of LH-RH, IAS, PMSG, and hCG administered were 0.04 mg, 0.1 mL, 20 IU, and 20 IU per rat, respectively. The administration conditions are described below.

[0146] • Experimental zone LH-RH: After 2 days of administration of LH-RH, IAS and PMSG were administered, and then 2 days later, hCG was administered.

[0147] • Experimental zone IAS e: IAS and PMSG were administered, and 2 days later, hCG was administered.

[0148] • Experimental zone PMSG: PMSG was administered, and 2 hours later, hCG was administered.

[0149] In vitro fertilization was performed using the obtained eggs in the same manner as in Example 1.

[0150] The results of the number of ovulations and the fertilization rate are shown in the following tables and figures. Table 9 and Figure 11 Table 10 and Figure 12 show the results for 7-week-old rats.

[0151] [Table 9]

[0152]

[0153] When IAS and PMSG were simultaneously administered to 6-week-old Crl:CD (SD) female rats, followed by administration of hCG, a tendency toward superovulation was observed, but statistically significant effects relative to PMSG alone could not be confirmed. However, by administering LH-RH, a significant superovulation-inducing effect could be confirmed. The results were consistent with the report by Honda et al.

[0154] [Table 10]

[0155]

[0156] When IAS and PMSG were simultaneously administered to 7-week-old Crl:CD (SD) female rats, followed by administration of hCG, a tendency toward superovulation was observed, but statistically significant effects relative to PMSG alone could not be confirmed. However, by administering LH-RH, a significant superovulation-inducing effect could be confirmed. The results were consistent with the report by Honda et al.

[0157] The above results show that the effect of administration of LH-RH to immature rats differs between 6-week-old and 7-week-old rats and younger 4-week-old and 5-week-old rats. Specifically, the superovulation induction effect by administration of IAS and PMSG is confirmed but not so clear in 6-week-old and 7-week-old rats, whereas it shows a clear effect in 4-week-old and 5-week-old rats. In addition, the superovulation induction effect by administration of IAS and PMSG is significantly enhanced by administration of LH-RH in 6-week-old and 7-week-old rats, whereas it is suppressed in 4-week-old and 5-week-old rats. Thus, the superovulation induction effect by administration of IAS and PMSG and the effect of LH-RH thereon differ depending on the age of rats as the target.

[0158] The above detailed description merely illustrates the object and the target of the present application and does not limit the scope of the appended patent claims. Various modifications and substitutions of the described embodiments according to the teachings described in the present specification will be apparent to those skilled in the art without departing from the scope of the appended patent claims.

[0159] Industrial applicability

[0160] The present application provides a superovulation induction method for rats. The method of the present application can reduce the number of female rats as an egg donor, and is extremely useful for the realization of the 3Rs principle in animal experiments, the rat resource bank for research using genetically modified rats, and the research efficiency of research institutes around the world.

[0161] This application is based on Japanese Patent Application No. 2023-070207 (Filing Date: April 21, 2023) filed in Japan, and the content thereof is incorporated herein by reference in its entirety.

Claims

1. A method for inducing superovulation, characterized in that, Female rats under 6 weeks of age were simultaneously given antiinhibin antibody and equine chorionic gonadotropin (eCG), followed by human chorionic gonadotropin (hCG).

2. The method according to claim 1, wherein, The antiinhibin antibody was administered in the form of antiinhibin serum.

3. The method according to claim 2, wherein, The female rats were female rats older than 3 weeks.

4. The method according to claim 2, wherein, The female rats were 3 to 5 weeks old.

5. The method according to claim 2, wherein, The female rats were female rats that had not been given exogenous luteinizing hormone-releasing hormone (LH-RH) before administration of equine chorionic gonadotropin.

6. The method according to claim 2, wherein, The female rats were female rats that had not been given exogenous gonadotropin-releasing hormone prior to administration of equine chorionic gonadotropin.

7. The method according to any one of claims 2 to 6, wherein, The amount of antiinhibin serum administered was less than 0.3 mL per rat.

8. The method according to claim 7, wherein, The anti-inhibin serum administered was 0.05 mL to 0.2 mL per rat.

9. The method according to claim 8, wherein, The amount of equine chorionic gonadotropin administered was 5 IU to 40 IU per rat.

10. The method according to any one of claims 1 to 6, wherein, The female rats were selected from the CD (SD), Wistar, Long-Evans, F344, BN and LEW strains.

11. An in vitro fertilization method, comprising the following steps: Step a, producing an egg using the method described in any one of claims 1 to 6, and Step b involves fertilizing the eggs produced in the aforementioned steps with sperm from a male rat.

12. An in vitro fertilization method, comprising the following steps: Procedure (1): Female rats under 6 weeks of age were simultaneously given antiinhibin antibody and equine chorionic gonadotropin (eCG), followed by human chorionic gonadotropin (hCG) to induce superovulation. Step (2) involves recovering oocytes from female rats that have undergone superovulation, and Step (3) involves fertilizing the eggs recovered in the previous steps with sperm from male rats.

13. The method according to claim 12, wherein, The antiinhibin antibody was administered in the form of antiinhibin serum.

14. The in vitro fertilization method according to claim 13, wherein, The female rats used were derived from sperm from male rats selected from the strains of CD (SD), Wistar, Long-Evans, F344, BN, and LEW.

15. The method according to claim 14, wherein, The female rats were 3 to 5 weeks old.

16. The method of claim 14, wherein, The female rats were female rats that had not been given exogenous luteinizing hormone-releasing hormone (LH-RH) before administration of equine chorionic gonadotropin.

17. The method according to any one of claims 14 to 16, wherein, The amount of anti-inhibin serum administered was less than 0.3 mL.

18. The method according to claim 17, wherein, The amount of anti-inhibin serum administered was 0.05 mL to 0.2 mL.

19. The method of claim 17, wherein, The amount of equine chorionic gonadotropin administered was 5 IU to 40 IU.

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