CN103525759A - Application of cordycepin in in-vitro maturation culture of oocytes of small pig follicles - Google Patents
Application of cordycepin in in-vitro maturation culture of oocytes of small pig follicles Download PDFInfo
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Abstract
The invention relates to application of cordycepin in in-vitro maturation culture of oocytes of small pig follicles, which belongs to the field of auxology of animals. Degrees of polyadenylation of the maternal genes consisting of cyclinB1, C-mos, GDF9, BMP15 and Cdc2 in oocytes determine expression levels of the genes, which has certain promotion effects on the recruitment process where small follicles become dominant follicles and on maturation of oocytes. The expression levels of the genes consisting of C-mos, GDF9 and BMP15 play a leading role in maturation of the oocytes of small follicles. Addition of a 3'-dA inhibitor can improve the expression levels of the genes consisting of C-mos, GDF9 and BMP15, so the maturation rate of the small follicles is increased. The technology of in-vitro maturation culture of oocytes is expected to be popularized and applied in research fields like transgenic technology, nuclear transplantation technology and embryonic stem cell technology.
Description
Technical field
The invention belongs to animal development and learn field, relate to Cordyceps militaris (L.) Link. element to the application in pig ovule bubble In vitro maturation, specifically Cordyceps militaris (L.) Link. element is to the little follicular oocyte of pig ripe GVBD, M I, M II period in vitro, restraining effect is in various degree carried out in adenosine acidifying to source of parents gene, thereby causes the difference of part source of parents gene expression difference and the little Oocytes in Vitro Maturation rate of pig.
Background technology
Along with going deep into of transgenic technology, nuclear transplantation technology, embryonic stem cell technical study, further promoted developing rapidly of technology in vitro fertilization.Yet ovocyte quantity is limited, only have a small amount of ovocyte to be applied to embryo transfer technology research, greatly limited the application of breeding new technology.The maturation in vitro technology of ovocyte not only can be reproduced internal fertilization process, and is conducive to the research of mechanism of fertilization.The maturation in vitro technology of ovocyte can provide the embryo of a large amount of cheapnesss for external embryo transfer technology, be conducive to promote the development of the technology such as nuclear transplantation, transgenosis.
The size of ovarian follicle in ovary is, very close (the Liu et al.2002 of the maturation in vitro relation of the diameter of ovocyte and ovocyte; Lucas et al.2002; Griffin et al.2006).Many results of study show, in non-atretic follicle, along with the increase of ovarian follicle volume, the maturing rate of its ovocyte, rate of fertilization and blastocyst rate all improve thereupon.The result of study of Melanie A. etc. (2007) shows, from the ovocyte blastocyst rate of 5-8mm ovarian follicle, is almost < three times (55% to 17%) of the little follicular oocyte of 3mm.Little follicular oocyte is as providing huge resource follicular oocyte more and more to receive in recent years the concern of Chinese scholars.After entering the nineties, the Vitro Culture Techniques of little follicular oocyte has had further development.Numerous scholar both domestic and external, by research, set up the Vitro Culture Techniques of a lot of little follicular oocyte, yet these technology are set up mostly on the basis of follicular oocyte extracorporeal culturing method.Foundation to the little follicular oocyte culture technique of pig is more late, until Tsafriri A in 1975 etc. get follicle size first, at the ovocyte of 1-2mm, cultivates, and the maturing rate of cultivation only has 15%～25%.
Cordyceps militaris (L.) Link. element (cordycepin) claims again cordycepin, 3'-Deoxyadenosine (3 '-dA), is (especially ucleosides) main active ingredient in Cordyceps militaris (L.) Link., is also first nucleoside antibiotics of separating from fungi.In normal Oocyte Maturation Process, genetic expression control is mainly post-transcriptional level; it is mainly the impact that is subject to cytoplasmic Polyadenylation; this process is not degraded and is excited them to translate to play an important role (Bettegowda and Smith, 2007 for protection mRNA; Zhang et al., 2009).3 '-dA can cause by deadenylation the degraded (reviewed in Bettegowda and Smith, 2007) of mRNA.Thereby affect stability and the translation efficiency of mRNA in the process of oocyte maturation, genome can affect the sex change of mRNA before or after activating during fetal development in early days.Krischek and Meinecke (2002) study and show, bovine oocyte in vitro maturation needs the Polyadenylation of source of parents gene.Therefore in large follicle ovocyte culturing process, the restraining effect of Polyadenylation can cause the decline of maturing rate and ripe quality.
Summary of the invention
The invention provides the application of Cordyceps militaris (L.) Link. element in pig ovule bubble In vitro maturation.
The technical scheme that the present invention takes is: the application of Cordyceps militaris (L.) Link. element in pig ovule bubble In vitro maturation.
The collection method of the little follicular oocyte of pig of the present invention is: ovary is adopted in just and butchered the ovary of sow, puts into 37 ℃ of physiological saline containing 75mg/ml penicillin and 50mg/ml Streptomycin sulphate, and with physiological saline, cleans 2～3 times within 2 hours; The female ovarian cumulus complex body of ovum that extracts the little ovarian follicle of diameter < 3mm at the syringe of No. 18 syringe needles of the indoor use of aseptic technique, picking has 3～5 layers of granulosa cell parcel under the microscope, and tenuigenin is even, ovarian cumulus ovocyte (COCs) complex body that zona pellucida is complete.
The method of one boar ovule bubble In vitro maturation, comprises the following steps:
(1) collection of the female ovarian cumulus complex body of pig ovule bubble ovum
Ovary is adopted in just and is butchered the ovary of sow, puts into 37 ℃ of physiological saline containing 75mg/ml penicillin and 50mg/ml Streptomycin sulphate, and with physiological saline, cleans 2～3 times within 2 hours; At the syringe of No. 18 syringe needles of the indoor use of aseptic technique, extract diameter and be < the female ovarian cumulus complex body of ovum of 3mm ovarian follicle, picking has 3～5 layers of granulosa cell to wrap up under the microscope, and tenuigenin is even, the ovarian cumulus ovocyte complex body that zona pellucida is complete;
(2) preparation and the cultivation of pig ovule bubble oocyte in vitro maturation culture solution
Preparation nutrient solution, basic medium is TCM-199, adds 0.1%PVA (w/v), 3.05mM D-Glucose before balance, 1mM Sodium.alpha.-ketopropionate, 0.57mM halfcystine, 10ng/ml EGF, 10IU/ml PMSG, 10IU/ml hCG, 75mg/ml penicillin, 50mg/ml Streptomycin sulphate, finally adding Cordyceps militaris (L.) Link. element final concentration is 2 μ g/ml;
First with Hepes-TL-PVA, wash after 2 times, nutrient solution more than use balance 2h 2 times, culture condition is 5%CO
2, 39 ℃ of temperature and maximal phase are to saturated humidity; Cultivate 44 hours during to M II, ovocyte is discharged first polar body and is maturation; Collect the female ovarian cumulus complex body of ovum, with the Unidasa of 0.1% concentration, process 2min.
The effect of part source of parents gene in oocyte maturation that the present invention is clear and definite, provides theoretical foundation for improving the ovule bubble oocyte maturation quality of diameter < 3mm.The present invention is the impact on the maturation in vitro of little ovarian follicle by research Polyadenylation, the mechanism of action of illustrating source of parents gene pairs oocyte maturation that can be more deep; Can excavate to a greater extent the breeding potential of jenny, for the research of animal cloning, transgenic technology provides high-quality ovum source.
The invention has the advantages that: source of parents gene cyclinB1, C-mos, GDF9, BMP15, the Cdc2 Polyadenylation degree in ovocyte determines the expression amount of these genes, ovule bubble is become to the process of raising of dominant follicle and the maturation of ovocyte has certain pushing effect.Wherein the expression amount of C-mos, GDF9 and BMP15 gene plays a major role to the maturation of little follicular oocyte.Add 3 '-dA inhibitor and can improve the expression amount of C-mos, GDF9 and BMP15 gene, thereby increase the maturing rate of little ovarian follicle.To being widely applied in research fields such as transgenic technology, nuclear transplantation technology, embryonic stem cell technology.
Accompanying drawing explanation
Fig. 1 is the maturation in vitro rate comparison diagram for the treatment of group and the little follicular oocyte of untreated fish group pig;
Fig. 2 is the expression amount variation diagram that Real time PCR detects the medium and small follicular oocyte different times of in-vitro maturity of porcine oocytes process CyclinB1 gene;
Fig. 3 is the expression amount variation diagram that Real time PCR detects the medium and small follicular oocyte different times of in-vitro maturity of porcine oocytes process C-mos gene;
Fig. 4 is the expression amount variation diagram that Real time PCR detects the medium and small follicular oocyte different times of in-vitro maturity of porcine oocytes process GDF9 gene;
Fig. 5 is the expression amount variation diagram that Real time PCR detects the medium and small follicular oocyte different times of in-vitro maturity of porcine oocytes process BMP15 gene;
Fig. 6 is the expression amount variation diagram that Real time PCR detects the medium and small follicular oocyte different times of in-vitro maturity of porcine oocytes process Cdc2 gene;
Fig. 7 is that PAT method detects cyclinB1, c-MOS, GDF9, BMP-15, Cdc2 gene at the little follicular oocyte different times of pig GVBD, M I and M II poly(A in period) depth map of tail.
Embodiment
TCM-199 is purchased from GIBCO company; Hepes, PVA, PMSG, EGF, hCG, D-Glucose, ketone acid sodium, the medicines such as halfcystine are purchased from sigma company.
The method of pig ovule bubble In vitro maturation of the present invention is as follows:
(1) collection of the female ovarian cumulus complex body of pig ovule bubble ovum
Ovary is adopted in just and is butchered the ovary of sow, puts into 37 ℃ of physiological saline containing 75mg/ml penicillin and 50mg/ml Streptomycin sulphate, and with physiological saline, cleans 2～3 times within 2 hours; At the syringe of No. 18 syringe needles of the indoor use of aseptic technique, extract diameter and be < the female ovarian cumulus complex body of ovum of 3mm ovarian follicle, picking has 3～5 layers of granulosa cell to wrap up under the microscope, and tenuigenin is even, ovarian cumulus ovocyte (COCs) complex body that zona pellucida is complete;
(2) preparation and the cultivation of pig ovule bubble oocyte in vitro maturation culture solution
Preparation nutrient solution, basic medium is TCM-199, adds 0.1%PVA (w/v), 3.05mM D-Glucose before balance, 1mM Sodium.alpha.-ketopropionate, 0.57mM halfcystine, 10ng/ml EGF, 10IU/ml PMSG, 10IU/ml hCG, 75mg/ml penicillin, 50mg/ml Streptomycin sulphate, finally adding Cordyceps militaris (L.) Link. element final concentration is 2 μ g/ml; Be little follicular oocyte nutrient solution;
First with Hepes-TL-PVA, wash after 2 times, nutrient solution more than use balance 2h 2 times, culture condition is 5%CO
2, 39 ℃ of temperature and maximal phase are to saturated humidity; Cultivate 44 hours during to M II, ovocyte is discharged first polar body and is maturation, collects the female ovarian cumulus complex body of ovum, with the Unidasa of 0.1% concentration, processes 2min, filters out under the microscope ripe ovocyte and records maturing rate.
Below by the contrast experiment who adds Cordyceps militaris (L.) Link. element or do not add Cordyceps militaris (L.) Link. element, further illustrate effect of the present invention.
One, the maturation in vitro of the little follicular oocyte of pig
1. the collection of the female ovarian cumulus complex body of pig ovule bubble ovum
Ovary is adopted in just and is butchered the ovary of sow, puts into 37 ℃ of physiological saline containing 75mg/ml penicillin and 50mg/ml Streptomycin sulphate, and with physiological saline, cleans 2～3 times within 2 hours; At the syringe of No. 18 syringe needles of the indoor use of aseptic technique, extract diameter and be < the ovum mother ovarian cumulus complex body of 3mm ovarian follicle, picking has 3～5 layers of granulosa cell parcel under the microscope, tenuigenin is even, and ovarian cumulus ovocyte (COCs) complex body that zona pellucida is complete, washes 2 times with Hepes-TL-PVA.
2. the maturation in vitro controlled trial of the little follicular oocyte of pig
Preparation nutrient solution, basic medium is TCM-199, adds 0.1%PVA (w/v) before balance, 3.05mM D-Glucose, 1mM Sodium.alpha.-ketopropionate, 0.57mM halfcystine, 10ng/ml EGF, 10IU/ml PMSG, 10IU/ml hCG, 75mg/ml penicillin, 50mg/ml Streptomycin sulphate, be untreated fish group nutrient solution, it is 2 μ g/ml that treatment group is added Cordyceps militaris (L.) Link. element final concentration again.
First with Hepes-TL-PVA, wash respectively after 2 times, untreated fish group, treatment group are washed 2 times with nutrient solution separately more than balance 2h respectively, and culture condition is 5%CO
2, 39 ℃ of temperature and maximal phase are to saturated humidity; When GVBD, M I, M II, collect respectively the female ovarian cumulus complex body of ovum, with the Unidasa of 0.1% concentration, process 2min.And record oocyte maturation rate on the same group not.Exposed ovocyte is collected and is stored in liquid nitrogen, and for further gene expression analysis, every 40 of ovocyte is divided into one group.
Two, the expression amount of source of parents gene different times detects
From the porcine oocytes in three periods of GVBD, M I, M II, extract RNA, dilution RNA control of the concentration is 10ng/uL, carries out reverse transcription, and reverse transcription test kit is from TOYOBO company.The primer of fluorescent quantitation is to carry out design of primers (table 1) with primer5.0, what fluorescence dye was selected is TOYOBO company green fluorescence dyestuff, fluorescent quantitation step is as follows: 95 ℃ of denaturations 3 minutes, 40 circulations comprise 30 seconds 95 ℃ of times, 60 ℃ of 30 seconds times, 72 ℃ of 30 seconds times, last 4 ℃ of preservations.Sample carries out in triplicate technology repetition.Each group all arranges the internal reference of contemporaneity, and internal reference is used β-actin.Fluorescent quantitation data acquisition is analyzed by the method for 2-Δ Δ CT.
Table 1 source of parents gene cyclin B1, cdc2, C-mos, GDF9, BMP15 fluorescent quantitation primer
| Genes | Primer?sequence(5’–3’) |
| cyclin?B1-F | TTGACTGGCTAGTGCAGGTTC |
| cyclin?B1-R | CTGGAGGGTACATTTCTTCATA |
| cdc2-F | AAGACTCCACGCTTCCATT |
| cdc2-R | GTCCACCTACTTCCAGAACAACCT |
| C-mos-F | TGGGAAGAAACTGGAGGACA |
| C-mos-R | TTCGGGTCAGCCCAGTTCA |
| GDF9-F | GAGCTCAGGACACTGTAAGCT |
| GDF9-R | CTTCTCGTGGATGATGTTCTG |
| BMP15-F | CCCTCGGGTACTACACTATG |
| BMP15-R | GGCTGGGCAATCATATCCT |
| β-Action-F | CACGCCATCCTGCGTCTGGA |
| β-Action-R | AGCACCGTGTTGGCGTAGAG |
Three, the poly(A) mensuration of the length of tail
By PCR method, determine ploy(A) length of tail carries out according to following method: article one reverse transcription primer be Oligo (dT) (5 '-GCGAGCTCCGCGGCC-GCGT12-3 ' Salles and Strickland, 1999). it is downstream primer that ensuing PCR be take Oligo (dT), upstream primer is shown in (table 2). in reaction tubes, add Taq enzyme 1.25 μ l, DEPC water 9.5 μ l, cDNA template 1 μ l, 1 μ l concentration is 10nM reverse primer, the anchor primer that 1 μ l concentration is 10nM, mixes and adds in each reaction tubes after mixing and all.Then sample is carried out to following PCR program: first under 93 ℃ of conditions, carry out 5min, then be the process of 35 circulations, at 93 ℃, move 30S, at 60 ℃, carry out 1min, after moving 3min under 72 ℃ of conditions, stop 35 circulations, under 72 ℃ of conditions, move 7min, then remain on 4 ℃ (Eppendorf company, Germany).Then sample is carried out to 1% agarose gel electrophoresis, under gel image analyser, observe and retain result.
Table 2 source of parents gene cyclin B1, cdc2, C-mos, GDF9, BMP15PAT length amplimer
| Genes | GenBank?accession?number | Primer?sequence(5’–3’) |
| C-mos | NM_001113219 | GCTGAACTGGGCTGACCCGAAAC |
| Cyclin?B1 | L48205 | TCTTGATAATGGTGAATGGACACCA |
| GDF9 | AY626786 | CTGCGTACCTGCCAAGTACAGCC |
| BMP15 | NM_001005155 | CCCTCGGGTACTACACTATG |
| Cdc2 | AB045783 | CTGTTAACTCTGCTTTTGTCTTGTGT |
Conclusion:
1. the impact of Polyadenylation on ovule bubble oocyte maturation rate
The maturing rate of little follicular oocyte treatment group after adding 3 '-dA is 61.7%, and the maturing rate of the little follicular oocyte of untreated fish group is 48.4%.Two groups of maturing rate comparing differences remarkable (p < 0.05).Little follicular oocyte process and untreated rear relatively mortality ratio and prematurity rate difference remarkable (p > 0.05) (Fig. 1).
2. the impact of Polyadenylation on the genetic expression of ovarian follicle source of parents
In untreated fish group group, CyclinB1 has higher expression amount in M I period, and expression amount interim when ovocyte GVBD and M II is lower, and CyclinB1 has reached the maximum in these 3 periods at the expression amount in the M of ovocyte I period.Comparatively speaking, in treatment group, the expression amount of CyclinB1 in little follicular oocyte all declined than untreated fish group group to some extent three periods, and interim expression amount is slightly improved (Fig. 2) than the expression amount in the little follicular oocyte M II of untreated fish group period when little follicular oocyte M II.
C-mos is GVBD, M I and the stepped downward trend of expression amount in M II period in the little follicular oocyte of untreated fish group.Yet in treatment group, C-mos at the expression amount in middle GVBD, M I and M II period apparently higher than untreated fish group.C-mos expression amount in the treatment group M II little follicular oocyte in period is these three periods the highest (Fig. 3).
GDF9 expression amount in M II period in the little follicular oocyte of untreated fish group is much lower period compared with GVBD and M I, and the expression amount in GVBD, M I and M II period presents downward trend gradually.The expression amount of GDF9 in treatment group M II ovocyte in period is high compared with the expression amount in M I and GVBD period.And exceed more than 3 times than the expression amount in untreated fish group group M II large follicle in period ovocyte.Expression amount in treatment group M I little follicular oocyte in period slightly declines compared with the expression amount in GVBD period.(Fig. 4)
BMP15 GVBD, M I and expression amount in M II period in the little follicular oocyte of untreated fish group present downward trend gradually.Yet after interpolation inhibitor, BMP15 GVBD, M I and expression amount in M II period in the little follicular oocyte for the treatment of group presents the trend rising gradually.(Fig. 5).
Cdc2 GVBD, M I and expression amount in M II period in the little follicular oocyte of untreated fish group present downward trend gradually.After suppressing, the expression amount of experimental group Cdc2 in GVBD, M I and M II size in period follicular oocyte is too and gone out gradually downward trend.(Fig. 6)
3.poly(A) the mensuration of the length of tail
Adopt PAT method to detect the poly(A of normal pig ovocyte GVBD, M I and M II source of parents in period gene cyclinB1, C-mos, GDF9, BMP15, Cdc2) tail length.Wherein, cyclinB1 compares and differs not obvious with control group after adding 3 '-dA inhibitor.C-mos gene is after adding 3 '-dA inhibitor, and the M I in the little follicular oocyte of pig and M II period be poly(A) length of tail diminishes.M I and M II poly(A in period in the untreated fish group of GDF9) length of tail diminishes.After adding 3 '-dA inhibitor, M I and M II poly(A in period in the little follicular oocyte of pig) length of tail increases.BMP15 after adding 3 '-dA inhibitor, M II poly(A in period) length of tail obviously diminishes.The untreated fish group of Cdc2 is at GVBD, M I and M II poly(A in period) length of tail do not change, after 3 '-dA inhibitor, M I band in period diminish (Fig. 7).
Result of study shows, source of parents gene cyclinB1, C-mos, GDF9, BMP15, the Cdc2 Polyadenylation degree in ovocyte determines the expression amount of these these genes, and ovule bubble is become to raising in process of dominant follicle and play a role, and oocyte maturation is played to key pushing effect.Wherein the expression amount of C-mos, GDF9, BMP15 height plays Main Function for the maturation of little follicular oocyte.Poly(A) length of tail shows, the interpolation of 3 '-dA does not make GDF9 adenosine turn into being affected, can also maintain stability and the translation efficiency of its mRNA, as can be seen here, the expression amount of C-mos, GDF9, BMP15 gene can increase ovule bubble and become the possibility of dominant follicle, thereby increases the maturing rate of little ovarian follicle.To being widely applied in research fields such as transgenic technology, nuclear transplantation technology, embryonic stem cell technology.
Claims (2)
1. the application of Cordyceps militaris (L.) Link. element in pig ovule bubble In vitro maturation.
2. the method for a boar ovule bubble In vitro maturation, comprises the following steps:
(1) collection of the female ovarian cumulus complex body of pig ovule bubble ovum
Ovary is adopted in just and is butchered the ovary of sow, puts into 37 ℃ of physiological saline containing 75mg/ml penicillin and 50mg/ml Streptomycin sulphate, and with physiological saline, cleans 2～3 times within 2 hours; At the syringe of No. 18 syringe needles of the indoor use of aseptic technique, extract diameter and be < the female ovarian cumulus complex body of ovum of 3mm ovarian follicle, picking has 3～5 layers of granulosa cell to wrap up under the microscope, and tenuigenin is even, the ovarian cumulus ovocyte complex body that zona pellucida is complete;
(2) preparation and the cultivation of pig ovule bubble oocyte in vitro maturation culture solution
Preparation nutrient solution, basic medium is TCM-199, adds 0.1%PVA (w/v), 3.05mM D-Glucose before balance, 1mM Sodium.alpha.-ketopropionate, 0.57mM halfcystine, 10ng/ml EGF, 10IU/ml PMSG, 10IU/ml hCG, 75mg/ml penicillin, 50mg/ml Streptomycin sulphate, finally adding Cordyceps militaris (L.) Link. element final concentration is 2 μ g/ml;
First with Hepes-TL-PVA, wash after 2 times, nutrient solution more than use balance 2h 2 times, culture condition is 5%CO
2, 39 ℃ of temperature and maximal phase are to saturated humidity; Cultivate 44 hours during to M II, ovocyte is discharged first polar body and is maturation; Collect the female ovarian cumulus complex body of ovum, with the Unidasa of 0.1% concentration, process 2min.
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| CN104140952A (en) * | 2014-08-08 | 2014-11-12 | 山东威高新生医疗器械有限公司 | Hyaluronidase and preparation method thereof |
| CN109112100A (en) * | 2018-09-04 | 2019-01-01 | 安徽农业大学 | The collection method of the small cumulus oocyte complex for having chamber of one boar |
| CN109554484A (en) * | 2018-12-19 | 2019-04-02 | 安徽农业大学 | A kind of method of quick detection Pig embryos full-length genome transcriptional activity |
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| DING-XIAO ZHANG ET AL: "Involvement of Polyadenylation Status on Maternal Gene Expression During In Vitro Maturation of Porcine Oocytes", 《MOLECULAR REPRODUCTION & DEVELOPMENT》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104140952A (en) * | 2014-08-08 | 2014-11-12 | 山东威高新生医疗器械有限公司 | Hyaluronidase and preparation method thereof |
| CN104140952B (en) * | 2014-08-08 | 2018-03-16 | 山东威高新生医疗器械有限公司 | hyaluronidase and preparation method thereof |
| CN109112100A (en) * | 2018-09-04 | 2019-01-01 | 安徽农业大学 | The collection method of the small cumulus oocyte complex for having chamber of one boar |
| CN109554484A (en) * | 2018-12-19 | 2019-04-02 | 安徽农业大学 | A kind of method of quick detection Pig embryos full-length genome transcriptional activity |
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