CN111876376A - Perilla polysaccharide composition capable of enhancing maturation of oocyte and embryo - Google Patents
Perilla polysaccharide composition capable of enhancing maturation of oocyte and embryo Download PDFInfo
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- 235000004347 Perilla Nutrition 0.000 title claims abstract description 48
- 241000229722 Perilla <angiosperm> Species 0.000 title claims abstract description 48
- 210000000287 oocyte Anatomy 0.000 title claims abstract description 46
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- 230000035800 maturation Effects 0.000 title claims abstract description 33
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- 230000002708 enhancing effect Effects 0.000 title claims abstract description 20
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 26
- 238000000338 in vitro Methods 0.000 claims description 15
- 210000003101 oviduct Anatomy 0.000 claims description 14
- 210000002257 embryonic structure Anatomy 0.000 claims description 12
- 238000010255 intramuscular injection Methods 0.000 claims description 12
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- 108010010803 Gelatin Proteins 0.000 claims description 7
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- 235000011852 gelatine desserts Nutrition 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 7
- 241000699670 Mus sp. Species 0.000 claims description 6
- 210000000625 blastula Anatomy 0.000 claims description 6
- 230000004720 fertilization Effects 0.000 claims description 5
- 230000008182 oocyte development Effects 0.000 claims description 5
- 210000000918 epididymis Anatomy 0.000 claims description 4
- 201000010063 epididymitis Diseases 0.000 claims description 4
- 210000004392 genitalia Anatomy 0.000 claims description 4
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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- C12N2501/90—Polysaccharides
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Abstract
One or more embodiments of the present disclosure provide a perilla polysaccharide composition for enhancing maturation of oocyte and embryo, wherein perilla is a traditional Chinese medicine entering lung meridian and spleen meridian, has the functions of promoting qi circulation, regulating the middle warmer, guiding qi downward, preventing miscarriage, and the like, and can be used for treating fetal irritability, miscarriage, and the like. The composition can be combined with some proper polysaccharides to improve the embryo blastocyst rate, and has the characteristics of small dosage of used medicines and simple and easy preparation operation, thereby becoming the prerequisite condition for popularization and application. The fertilized egg or the activated parthenogenetic embryo is treated by using the composition of perilla polysaccharide, red date polysaccharide and the like with certain concentration, so that the embryo quality can be obviously improved, the blastocyst rate of the embryo is increased, and the production efficiency of the embryo is greatly improved.
Description
Technical Field
One or more embodiments of the present disclosure relate to the technical field of polysaccharide compositions for enhancing maturation of oocytes and embryos, and in particular, to a perilla polysaccharide composition for enhancing maturation of oocytes and embryos.
Background
The life science, especially the embryo development technology, has made great efforts for human beings since the 21 st century. However, the in vitro culture conditions of the embryo obtained by researchers or the embryo after the 'transformation' often have a series of problems such as low development rate, embryo fragmentation, embryo hypoplasia, low quality grade of the embryo and the like, and the problems cause that the number of the obtained embryo is too low, the quality is poor, the heredity is unstable, and abortion, fetal death and fetal weakness occur in the uterus of the transplanted animal.
Disclosure of Invention
In view of the above, one or more embodiments of the present disclosure are directed to a perilla polysaccharide composition for enhancing maturation of oocytes and embryos, so as to solve the problems of the prior art, such as low number, poor quality, unstable inheritance, abortion, fetal death, and weak fetus in uterus of transplanted animals.
In view of the above, one or more embodiments of the present disclosure provide a perilla polysaccharide composition for enhancing maturation of oocyte and embryo development, which is characterized by comprising perilla polysaccharide and a polysaccharide component a, wherein the polysaccharide component a comprises any one of red date polysaccharide, dried orange peel polysaccharide, donkey-hide gelatin polysaccharide and peach gum polysaccharide.
Optionally, the concentration ratio of the perilla polysaccharide to the polysaccharide component A is 2.5-10: 2-10 mg/mL.
Optionally, the concentration ratio of the perilla polysaccharide to the polysaccharide component A is 4-7.5: 2.5-5 mg/mL.
An operation method for enhancing oocyte and embryo development and maturation by adopting a perilla polysaccharide composition comprises the following steps:
intramuscular injection: selecting adult female mice in estrus, and performing intramuscular injection for 10 days by adopting perilla polysaccharide and polysaccharide component A with different concentrations respectively;
obtaining an oocyte: killing the intramuscular injection treated female mouse by a cervical dislocation method, splitting the abdomen of the female mouse after the abdomen is disinfected, and exposing a reproductive system; lifting the oviduct, cutting off fat and uterine mesentery, cutting off the joint of the oviduct and ovary and uterine horn, and putting the oviduct into a vessel containing operating liquid;
obtaining an embryo: killing sexually mature male mouse by dislocation, splitting open the external genitalia after disinfecting, exposing reproductive organs, collecting mature sperms of the male mouse at the epididymis, directly adding the obtained oocytes, performing in vitro fertilization incubation, and detecting the number of secondary cells, quaternary cells, morulae embryos and blastulas in 48h, 56h, 72h and 96h respectively.
Optionally, the incubation environmental parameter is 5% CO at 37 deg.C in normal embryo culture solution2And (5) incubation in an incubator. .
From the above, it can be seen that according to the perilla polysaccharide composition for enhancing maturation of oocyte and embryo provided by one or more embodiments of the present specification, traditional Chinese medicine conditioning can improve ovarian function and quality of ova, stimulate growth of endometrium by tonifying kidney, make endometrium favorable for accepting fertilized egg planting, and also has functions of conditioning qi and blood and promoting reproduction. The fertilized eggs or the parthenogenetic embryos after activation can be treated by using the perilla polysaccharide and the polysaccharide component A with certain concentrations to obviously improve the embryo quality, and the blastocyst rate of the embryos is increased, so that the acquisition of high-quality embryos becomes possible. The method has the characteristics of small dosage of used medicines and simpler operation and easy preparation, and becomes a prerequisite condition for popularization and application. The method is convenient and quick, has low medicine price, obtains a large number of blastula, and is suitable for popularization and application.
Drawings
In order to more clearly illustrate one or more embodiments or prior art solutions of the present specification, the drawings that are needed in the description of the embodiments or prior art will be briefly described below, and it is obvious that the drawings in the following description are only one or more embodiments of the present specification, and that other drawings may be obtained by those skilled in the art without inventive effort from these drawings.
FIG. 1 is a schematic diagram illustrating the method of operation of one or more embodiments of the present disclosure.
Detailed Description
To make the objects, technical solutions and advantages of the present disclosure more apparent, the present disclosure is further described in detail below with reference to specific embodiments.
The invention provides a perilla polysaccharide composition capable of enhancing the maturation of oocytes and embryos, which is characterized by comprising perilla polysaccharide and a polysaccharide component A, wherein the polysaccharide component A comprises any one of red date polysaccharide, dried orange peel polysaccharide, donkey-hide gelatin polysaccharide and peach gum polysaccharide. Meanwhile, the operation method for enhancing the maturation of the oocyte and the embryo by adopting the perilla polysaccharide composition comprises the following steps:
intramuscular injection: selecting adult female mice in estrus, and performing intramuscular injection for 10 days by adopting perilla polysaccharide and polysaccharide component A with different concentrations respectively;
obtaining an oocyte: killing the intramuscular injection treated female mouse by a cervical dislocation method, splitting the abdomen of the female mouse after the abdomen is disinfected, and exposing a reproductive system; lifting the oviduct, cutting off fat and uterine mesentery, cutting off the joint of the oviduct and ovary and uterine horn, and putting the oviduct into a vessel containing operating liquid;
obtaining an embryo: killing sexually mature male mouse by dislocation, splitting open the external genitalia after disinfecting, exposing reproductive organs, collecting mature sperms of the male mouse at the epididymis, directly adding the obtained oocytes, performing in vitro fertilization incubation, and detecting the number of secondary cells, quaternary cells, morulae embryos and blastulas in 48h, 56h, 72h and 96h respectively.
Specifically, for example, the perilla polysaccharide composition for enhancing the maturation of oocytes and embryos provided in embodiment 1 of the invention includes perilla polysaccharide and red date polysaccharide. The concentration ratio of the perilla polysaccharide to the red date polysaccharide is 2.5-10: 2-10 mg/mL.
The specific operation steps are as follows:
1. perilla polysaccharide concentration gradient experiment
Setting 5 concentration gradients for carrying out a concentration gradient experiment, wherein the concentrations are 0mg/mL, 1mg/mL, 2mg/mL, 3mg/mL and 4mg/mL respectively, taking eggs after continuous intramuscular injection for 10 days, counting the maturation rate of the oocytes, and determining that the concentration with the highest maturation rate of the oocytes is 4 mg/mL.
2. Red date polysaccharide concentration gradient experiment
Setting 5 concentration gradients for carrying out a concentration gradient experiment, wherein the concentrations are 0mg/mL, 1mg/mL, 2mg/mL, 3mg/mL and 4mg/mL respectively, taking eggs after continuous intramuscular injection for 10 days, counting the maturation rate of the oocytes, and determining that the concentration with the highest maturation rate of the oocytes is 3 mg/mL.
3. Mouse oocyte and fertilized egg retrieval
(1) Separated oviduct
The mother mouse treated by polysaccharide intramuscular injection is killed by neck dislocation, and the abdomen of the mother mouse is cut open by scissors after being disinfected by alcohol spraying, so as to expose the reproductive system. Accurately finding out the position of the oviduct along the uterus, lifting the oviduct with forceps, cutting off fat and uterine mesentery with scissors, cutting at the joint of the oviduct, the ovary part and the uterine horn, and placing the oviduct into a small dish of 35mm containing operating fluid.
(2) Embryo production by in vitro fertilization
Killing sexually mature male mouse by dislocation, spraying alcohol to the external genitalia for disinfection, splitting with scissors, exposing reproductive organs, collecting mature sperm of male mouse in epididymis, directly adding oocyte obtained in the concentration screening process, and incubating for in vitro fertilization. In normal embryo culture, 5% CO at 37 deg.C2And (4) incubating in an incubator, detecting the number of two cells, four cells, morula and blastula in 48h, 56h, 72h and 96h respectively, and culturing the blastula development rate in vitro.
Three tests were performed for each polysaccharide fraction and the results of the measurements are shown in tables 1 and 2.
TABLE 1 Effect of Perilla frutescens polysaccharide and Red jujube polysaccharide on mouse oocyte maturation
TABLE 2 Effect of Perilla frutescens polysaccharide and Red jujube polysaccharide on mouse in vitro embryo development
After 4mg/mL perilla polysaccharide and 3mg/mL red date polysaccharide are used for treating a mother mouse, the number of oocytes and the in-vitro sperm embryo receiving time can be remarkably increased by 24 hours.
Specifically, for example, the perilla polysaccharide composition for enhancing maturation of oocyte and embryo provided in example 2 includes perilla polysaccharide and tangerine peel polysaccharide. The concentration ratio of the perilla polysaccharide to the tangerine peel polysaccharide is 2.5-10: 2-10 mg/mL. The concentration of perilla polysaccharide determining the highest oocyte maturation rate is 5mg/mL, the concentration of tangerine peel polysaccharide determining the highest oocyte maturation rate is 5mg/mL, the other steps are the same as example 1, and the performance tests are shown in tables 3 and 4.
TABLE 3 Effect of Perilla frutescens polysaccharide and Citrus reticulata polysaccharide on mouse oocyte maturation
TABLE 4 Effect of Perilla frutescens polysaccharide and Citrus reticulata polysaccharide on in vitro embryonic development in mice
After the female mouse is treated by using 5mg/mL perillaseed polysaccharide and 5mg/mL tangerine peel polysaccharide, the number of oocytes and the in-vitro sperm embryo receiving time can be remarkably increased by 24 hours.
Specifically, for example, the perilla polysaccharide composition for enhancing maturation of oocyte and embryo provided in example 3 includes perilla polysaccharide and donkey-hide gelatin polysaccharide. The concentration ratio of the perilla polysaccharide to the donkey-hide gelatin polysaccharide is 2.5-10: 2-10 mg/mL. The concentration of perilla polysaccharide for determining the highest oocyte maturation rate is 7.5mg/mL, the concentration of donkey-hide gelatin polysaccharide for determining the highest oocyte maturation rate is 2.5mg/mL, other steps are the same as example 1, and performance tests are shown in tables 5 and 6.
TABLE 5 Effect of Perilla frutescens polysaccharide and colla Corii Asini polysaccharide on mouse oocyte maturation
TABLE 6 influence of Perilla frutescens polysaccharide and colla Corii Asini polysaccharide on in vitro embryonic development of mice
After the female mouse is treated by using 7.5mg/mL perilla polysaccharide and 2.5mg/mL donkey-hide gelatin polysaccharide, the number of oocytes and the in-vitro sperm embryo receiving time can be remarkably increased by 24 hours.
Specifically, for example, the perilla polysaccharide composition for enhancing the maturation of oocytes and embryos provided in example 4 comprises perilla polysaccharide and peach gum polysaccharide. The concentration ratio of the perilla polysaccharide to the peach gum polysaccharide is 2.5-10: 2-10 mg/mL. The concentration of perilla polysaccharide determining the highest oocyte maturation rate is 7.5mg/mL, the concentration of peach gum polysaccharide determining the highest oocyte maturation rate is 5mg/mL, the other steps are the same as example 1, and the performance tests are shown in tables 7 and 8.
TABLE 7 Effect of Perilla frutescens polysaccharide and peach gum polysaccharide on mouse oocyte maturation
TABLE 8 Effect of Perilla frutescens polysaccharide and peach Gum polysaccharide on in vitro embryonic development in mice
After the female mouse is treated by using 7.5mg/mL perilla polysaccharide and 5mg/mL peach gum polysaccharide, the number of oocytes and the blastocyst rate can be obviously improved by receiving sperms in vitro for 24 hours.
The Perillae herba is a Chinese medicinal preparation for lung and spleen meridians, has effects in promoting qi circulation, regulating stomach, guiding qi downward, and preventing miscarriage, and can be used for treating threatened abortion, pernicious vomiting, etc. The composition can be combined with some proper polysaccharides to improve the embryo blastocyst rate, and has the characteristics of small dosage of used medicines and simple and easy preparation operation, thereby becoming the prerequisite condition for popularization and application.
The fertilized egg or the activated parthenogenetic embryo is treated by using the composition of perilla polysaccharide, red date polysaccharide and the like with certain concentration, so that the embryo quality can be obviously improved, the blastocyst rate of the embryo is increased, and the production efficiency of the embryo is greatly improved.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the spirit of the present disclosure, features from the above embodiments or from different embodiments may also be combined, steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present description as described above, which are not provided in detail for the sake of brevity.
It is intended that the one or more embodiments of the present specification embrace all such alternatives, modifications and variations as fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements, and the like that may be made without departing from the spirit and principles of one or more embodiments of the present disclosure are intended to be included within the scope of the present disclosure.
Claims (5)
1. The perilla polysaccharide composition capable of enhancing oocyte and embryo development maturity is characterized by comprising perilla polysaccharide and a polysaccharide component A, wherein the polysaccharide component A comprises any one of red date polysaccharide, dried orange peel polysaccharide, donkey-hide gelatin polysaccharide and peach gum polysaccharide.
2. The perilla polysaccharide composition for enhancing maturation of oocyte and embryo according to claim 1, wherein the concentration ratio of the perilla polysaccharide to the polysaccharide component A is 2.5-10: 2-10 mg/mL.
3. The perilla polysaccharide composition for enhancing maturation of oocyte and embryo according to claim 1, wherein the concentration ratio of the perilla polysaccharide to the polysaccharide component A is 4-7.5: 2.5-5 mg/mL.
4. An operation method for enhancing oocyte and embryo development and maturation by adopting a perilla polysaccharide composition is characterized by comprising the following steps:
intramuscular injection: selecting adult female mice in estrus, and performing intramuscular injection for 10 days by adopting perilla polysaccharide and polysaccharide component A with different concentrations respectively;
obtaining an oocyte: killing the intramuscular injection treated female mouse by a cervical dislocation method, splitting the abdomen of the female mouse after the abdomen is disinfected, and exposing a reproductive system; lifting the oviduct, cutting off fat and uterine mesentery, cutting off the joint of the oviduct and ovary and uterine horn, and putting the oviduct into a vessel containing operating liquid;
obtaining an embryo: killing sexually mature male mouse by dislocation, splitting open the external genitalia after disinfecting, exposing reproductive organs, collecting mature sperms of the male mouse at the epididymis, directly adding the obtained oocytes, performing in vitro fertilization incubation, and detecting the number of secondary cells, quaternary cells, morulae embryos and blastulas in 48h, 56h, 72h and 96h respectively.
5. The manipulation method of claim 4 for enhancing oocyte and embryo development and maturation by using perilla polysaccharide composition, wherein the incubation is performed in a normal embryo culture medium at 37 ℃ and 5% CO2And (5) incubation in an incubator.
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|---|---|---|---|---|
| CN105533705A (en) * | 2015-12-27 | 2016-05-04 | 劲膳美生物科技股份有限公司 | Medical formula food for recuperating women during second trimester |
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| US20190151390A1 (en) * | 2016-06-28 | 2019-05-23 | Inqpharm Group Sdn Bhd | Perilla extract composition |
| US20190382722A1 (en) * | 2016-12-09 | 2019-12-19 | The University Of Adelaide | Compositions and methods for maturation of oocytes in vitro |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105533705A (en) * | 2015-12-27 | 2016-05-04 | 劲膳美生物科技股份有限公司 | Medical formula food for recuperating women during second trimester |
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| US20190151390A1 (en) * | 2016-06-28 | 2019-05-23 | Inqpharm Group Sdn Bhd | Perilla extract composition |
| US20190382722A1 (en) * | 2016-12-09 | 2019-12-19 | The University Of Adelaide | Compositions and methods for maturation of oocytes in vitro |
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