CN111616830A - Novel ICR mouse superovulation method - Google Patents
Novel ICR mouse superovulation method Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D19/00—Instruments or methods for reproduction or fertilisation
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Reproductive Health (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a novel mouse superovulation method, which is characterized in that 15nmol of humanized Kisspeptin54 is injected into an ICR mouse twice at intervals of 48h, and 14h after the injection is finished, the mouse can obtain more than 16 oocytes which are far higher than the ovulation number (average 6-8 oocytes per mouse in primary ovulation) of the ICR mouse under normal physiological conditions. Meanwhile, Kisspeptin54 is a green novel polypeptide hormone compared with the traditional hormone. The invention can replace the traditional hormone for mouse superovulation and lays a good foundation for the application research of Kisspeptin in superovulation on large animals and human assisted reproduction.
Description
Technical Field
The invention relates to the technical field of superovulation, in particular to a novel superovulation method for mice.
Background
Superovulation is a technical support and guarantee for successfully researching animal genetic engineering, is a key link for obtaining a large number of embryo sources so as to realize embryo transplantation, and is an effective method for improving the fertility of excellent dams and excavating the breeding potential of the dams. Currently, superovulation continues to be treated with conventional exogenous hormones. However, with the pursuit of green consumption, the problem of residual traditional hormones in animal products and the problem of "desensitization" of the hormones caused by long-term, high-volume use have become increasingly prominent, and new green technologies are urgently needed to replace traditional super-exclusion technologies.
Kisspeptin (KP), a pharmaceutical compositionKiss1The product of the gene, which is currently believed to be the most important factor in regulating GnRH release,can indirectly or directly promote the release of pituitary gonadotropin.Kiss1The gene encodes 145 amino acids, which are further processed and cleaved in vivo into 4 bioactive peptides of different lengths (Kisspeptin 54, Kisspeptin14, Kisspeptin 13, Kisspeptin 10), of which Kisspeptin54 is most active. Researches show that Kisspeptin is a main factor for regulating and controlling the GnRH release of hypothalamus, can directly act on the periphery of the hypothalamus median bulge and promote the GnRH release at the end of GnRH nerve synapse, and can also directly act on the anterior pituitary to stimulate the release of pituitary gonadotropins. The Kisspeptin injection in vitro not only can obviously improve the FSH and LH level in the animal breeding season, but also can ensure that the animal in the non-breeding season is oestrous and ovulated. Caraty et al (2007) continued treatment of the seasonally stringy sheep with Kisspeptin10 allowed oestrus and ovulation. Redmond et al (2011) treated lambs before sexual maturation with Kisspeptin found that Kisspeptin could activate their axis of reproduction early and enter early stages early. Therefore, exogenous Kisspeptin injections can trigger the synthesis and release of endogenous gonadotropins in animals, and application to animal superovulation is theoretically possible.
However, the application of Kisspeptin to superovulation of animals has not been reported. Therefore, Kisspeptin was first explored on ICR mice for its application to superovulation, and a novel superovulation method different from the past was successfully obtained. The method not only can achieve the effect of superovulation, but also effectively avoids the defects of the traditional superovulation hormone, and is a pollution-free and green superovulation method. The novel superovulation method not only realizes the successful substitution of the traditional superovulation hormone on mice, but also lays a good foundation for the application of Kisspeptin in the assisted reproduction of large animals and even human beings.
Disclosure of Invention
According to a large number of experimental researches, the feasibility of carrying out superovulation on a mouse by using novel polypeptide humanized Kisspeptin54 instead of traditional hormones is discussed, the optimal dosage of Kisspeptin54 applied to superovulation of the mouse is optimized, different procedures of Kisspeptin54 applied to superovulation of the mouse are compared, and finally the novel mouse superovulation method is obtained.
In the investigation of the injection of different types of Kisspeptin, the existence of different degrees of promotion of the release of GnRH of the hypothalamus of a mouse is found, so that the gonadotropin level in the body is improved, and the ovarian function is activated. In Kisspeptin10 injections, oestrus and ovulation can be promoted. When Kisspeptin14 is injected, the reproductive axis of the Kisspeptin can be activated in advance, so that the Kisspeptin can be promoted to enter the initial stage in advance. The invention discovers that the Kisspeptin54 injection has obvious superovulation effect on mice, is obviously higher than the ovulation effect under normal physiological (non-superovulation condition) condition, has good ovulation quality, and opens up a new way for the deep research of animal superovulation and even human assisted reproduction. The exogenous injection Kisspeptin54 can indirectly and directly promote the release of endogenous gonadotropin in an animal body by directly acting on the median eminence of the hypothalamus or directly acting on the pituitary, and promote the mature and ovulation of a plurality of follicles, thereby achieving the effect of superovulation of the animal. Therefore, Kisspeptin54 was feasible in the application of animal superovulation. The invention firstly uses Kisspeptin54 injection to replace the hormone hCG used in the traditional super-excretion; secondly, the search was for a replacement of the traditional creosomal PMSG with a certain amount of Kisspeptin54 and further to define the optimal superseding procedure of Kisspeptin54+ Kisspeptin 54. The invention relates to application of Kisspeptin54 in improving total number of embryos/blastula of animals, the number of available embryos, the quality of mature eggs or the quality of embryos after fertilization. Superovulation occurs after injection of Kisspeptin54 into mice; kisspeptin54 replaced the traditional super-excretion hormones PMSG and hCG. 15Noml of Kisspeptin54 was injected and the superovulation procedure of Kisspeptin54 injected at the same dose was repeated after 48h intervals.
Notably, the present invention is a supersort procedure with injections of Kisspeptin54+ Kisspeptin54, time interval 48 h; the injected doses were all 15nom of Kisspeptin 54.
The superovulation method of the invention comprises the following steps:
A) on the first day, mice were first injected intraperitoneally with 150 μ L of Kisspeptin54 at a concentration of 15 nmol;
B) on the third day, mice were injected a second time with 150 μ L of Kisspeptin54 at a concentration of 15 nmol;
C) 14h after the second injection of Kisspeptin54, the ampulla of the oviduct of the white rat was removed aseptically, and the number of oocytes ejected by the first polar body was counted under a stereomicroscope after removal of cumulus cells. The ovulation number of a single white mouse is 17-30. The invention stimulates the thalamus and the pituitary by Kisspeptin54, promotes the release of endogenous gonadotropin in a mouse, and promotes multiple follicles to mature and ovulate.
Furthermore, the invention compares the super-excretion effect of the traditional hormone. The optimal Kisspeptin54 concentration for replacing the conventional super-excretion hormone hCG, namely PMSG + Kisspeptin54 combination with different concentrations, is screened to carry out super-excretion on ICR mice, and the best super-excretion effect of replacing hCG by 15nmol of Kisspeptin54 is determined.
Further, the optimal concentration of Kisspeptin54 for replacing the traditional PMSG is screened, namely Kisspeptin54+15nmol Kisspeptin54 combinations with different concentrations are selected for carrying out superovulation on ICR mice, and finally the best superovulation effect of the PMSG replaced by 15nmol Kisspeptin54 is determined.
Further, comparing the superovulation with 15nmol Kisspeptin54+15nmol Kisspeptin54 using different superovulation procedures, it was finally determined that the best superovulation was obtained with the injection procedure of Kisspeptin54 injecting the same dose of Kisspeptin 48h after the injection of 15nmol Kisspeptin 54. The optimal 15nmol Kisspeptin54+15nmol Kisspeptin54, mouse superovulation program with interval of 48h injection was finally determined.
Furthermore, animal experiments prove that the method can remarkably improve the total number of embryos/blastula washed embryos and available embryos after the animal is subjected to insemination, and can improve the quality of mature ova and the embryos after fertilization.
Through a large number of tests, the feasibility of replacing the traditional excretion hormone with the novel medicine Kisspeptin54 is determined, the optimal dosage and injection procedure are preferably selected, and the novel mouse excretion method is finally created. The invention discloses a novel super-drainage method which is close to green, effective and novel. Superovulation of white mice was performed using the novel polypeptide Kisspeptin54 instead of the traditional hormone.
The invention also has the beneficial effects that the Kisspeptin54 drives the release of endogenous gonadotropin, and avoids the large amount of hormone residues in the animal body caused by the long-term and continuous use of large dose of exogenous hormone (such as FSH, PMSG, hCG and the like) in the traditional super-excretion method. Furthermore, Kisspeptin54 acts directly or indirectly on multiple sites of the hypothalamus or pituitary gland, reducing the occurrence of "desensitization" of the hormone resulting from the large dose, prolonged use of conventional hormones, and causing failure of the superovulation. By using the novel superovulation method, more than 16 oocytes can be obtained from each mouse, and the superovulation quantity is lower than that of the conventional hormone but far higher than that of ICR mice under normal physiological conditions (the average number of primary ovulations is 6-8 oocytes per mouse). Therefore, Kisspeptin54 is a novel, green method of mouse superovulation compared to the traditional hormones.
The specific process of the super-row effect comparison and selection of the invention is shown in tables 1, 2 and 3.
TABLE 1 PMSG + Effect of Kisspeptin54 combinations at different concentrations on ICR mice superovulation
Note: the representation in the table is significantly different between completely different letters in the same vertical row,p＜0.05。
preferably, the optimal concentration of Kisspeptin54 of PMSG replacing traditional super-excretion hormone, namely, the combination of Kisspeptin54+15nmol Kisspeptin54 with different concentrations carries out super-excretion on ICR mice, the specific experimental data are shown in Table 2, and finally the best super-excretion effect of PMSG replacing 15nmol Kisspeptin54 is determined.
TABLE 2 Effect of different concentrations of Kisspeptin54+15nmol Kisspeptin54 on ICR mice superovulation
Note: the difference in the representation between different letters in the same vertical row in the table is significant,p＜0.05。
table 315 nmolKisspeptin54+15nmolKisspeptin54 screens for the ICR mouse best super-exclusion program in place of PMSG + hCG.
Preferably, the best results of the third superranking procedure were finally determined by comparing superranking with 15nmol Kisspeptin54+15nmol Kisspeptin54 of the different injection procedures, and the specific experimental data are shown in table 3.
Table 315 nmol kisspeptin54+15nmol kisspeptin54 optimal supersequencing screening of ICR mice replacing PMSG + hCG.
Note: the representation differences between the different letters in the table are significant,p＜0.05。
preferably, the ICR white mouse is healthy for 4 weeks, has a weight of about 18-20 g, and ensures diet, drinking water and indoor temperature during treatment.
In conclusion, Kisspeptin54 responds to the superscalar, which is simple and economically feasible.
Detailed Description
The following will explain the specific implementation method of the present invention in detail by combining examples.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
ICR white mouse superovulation
1. Medicine preparation: PMSG, hCG, human Kisspeptin54
2. Selection of healthy animals of appropriate age
The ICR white mice are selected to be healthy for 4 weeks, the weight of the ICR white mice is about 18-20 g, and normal diet, drinking water and indoor temperature are guaranteed during treatment.
3. Superovulation procedure
On day 1, ICR mice were injected intraperitoneally with Kisspeptin54 at a concentration of 15nmol at a dose of 150 μ L.
On day 3, ICR mice were again given a 150 μ L dose of intraperitoneal injection of Kisspeptin54 at 15nmol concentration at the same time.
And (3) killing an ICR mouse 14 hours after 15nmol of Kisspeptin54 is injected for the second time, taking out the ampulla of the oviduct aseptically, scratching the ampulla with a disposable aseptic 1 mL syringe under a body type microscope, extruding a cumulus-oocyte complex, putting the extruded cumulus-oocyte complex into a 35mm flat dish containing 300 IU/mL hyaluronidase DPBS, acting for 1-2 min at 37.5 ℃, completely removing cumulus cells by using a self-made oral suction tube, and counting the number of oocytes discharged by a first polar body under the body type microscope.
4. Super-drainage effect
The novel superovulation method can obtain 16 oocytes per mouse on average, and is lower than the traditional superovulation method (PMSG + hCG, which can obtain 25 oocytes per mouse on average) but much higher than the number of ovulation in ICR white mice under normal physiological conditions (the average number of primary ovulation is 6-8/mouse). Meanwhile, Kisspeptin54 is a novel green polypeptide that drives the release of endogenous excretion hormones compared to conventional hormones. Therefore, it is a novel and green method for superovulation of mice.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Claims (3)
1. A novel ICR mouse superovulation method is characterized in that: the Kisspeptin54 is applied to the aspects of improving the total number of embryos/blastula of animals, the number of available embryos, the quality of mature ova or the quality of embryos after fertilization;
it is characterized in that after Kisspeptin54 is injected into a mouse, superovulation occurs;
the preparation is characterized in that Kisspeptin54 replaces traditional super excretion hormones PMSG and hCG;
characterized in that the injection is a super-exclusion procedure of Kisspeptin54+ Kisspeptin54 with a time interval of 48 h; it is characterized in that the injection dosage is 15nom Kisspeptin 54.
2. The novel method of superovulation in ICR mice as claimed in claim 1, wherein said superovulation method comprises the steps of:
A) on the first day, mice were injected intraperitoneally with a first 150 μ L dose of Kisspeptin54 at a concentration of 15 nmol;
B) on the third day, mice were injected a second time with 150 μ L of a dose of 15nmol i.p. with Kisspeptin 54;
C) 14h after the second injection of Kisspeptin54, the ampulla of the oviduct of the mouse was removed aseptically, and the number of oocytes ejected by the first polar body was counted under a stereomicroscope after removal of cumulus cells.
3. The novel method of claim 1, wherein said method comprises stimulating the thalamus and pituitary gland with Kisspeptin54 to promote the release of endogenous gonadotropins in mice, thereby promoting the maturation and ovulation of multiple follicles.
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