Evaluate mechanical method, enzymatic method and enzymatic hydrolysis? The effects of mechanical combination and other separation methods on the development and reproductive potential of isolated follicles from transplanted ovaries in mice were studied, and a better technical scheme for follicular separation was established.
Methods After ovarian transplantation in B6D2F 1 newborn mice, mechanical method, long-term enzymolysis, short-term enzymolysis or enzymolysis were used respectively. Preantral follicles in ovarian tissue are separated by mechanical adhesion. The recovered follicles were cultured in vitro for 65438±02d days, treated with hCG for 26 h, and the cumulus complex was harvested for in vitro fertilization.
Results The recovery rate, survival rate and ovulation rate of mechanical method were higher than those of enzymatic method, and the ovulation rate of short-term enzymatic method was higher than that of long-term enzymatic method. The average follicular recovery rate, average ovulation rate and oocyte maturation rate of enzymatic hydrolysis combined with mechanical method were higher than those of other groups.
Conclusion Mechanical combined with enzymatic hydrolysis is a good technical scheme for separating mouse follicles.
Follicular oocyte culture technology embryo tissue transplantation
Artificial induction of primordial follicles in embryonic ovary can provide sufficient sources of oocytes for fertilization mechanism research and medical cloning. It is one of the key steps to artificially induce follicular development to separate the growing follicles from the ovaries that have developed to a certain extent after allogeneic transplantation [1? [3] So far, two follicular separation technologies have been developed, including enzymatic separation and mechanical separation. These two methods have their own shortcomings, which make the follicular recovery rate and maturity rate of each ovary stay at a low level. In this study, the effects of enzymatic hydrolysis, mechanical method and enzymatic hydrolysis, mechanical combination and other separation methods on follicular development and reproductive ability in transplanted ovaries of mice were discussed in order to establish a better technical scheme for follicular separation. 1. 1 animal C57BL/6(B6) female mice and DBA/2(D2) male mice were purchased from Shanghai slack Experimental Animal Co., Ltd. [Certificate: SCXK (Shanghai) 2007? B6D 1 (F 1) mice are formed by mating B6 female mice with D2 male mice. Mice were kept in a cage with independent ventilation system (IVC? Type ⅱ, Suzhou Shi Feng Experimental Animal Equipment Co., Ltd.), bright 14 h, dark 10 h, free to eat and drink.
1.2 reagent α? MEM？ Hpss (42360? 032),α? MEM(3257 1？ 036), collagenase I (17100017), deoxyribonuclease (DNaseI, 18047? 0 19) and its (insulin? Transferrin? Selenium mixture) are all products of Gibco company in the United States; RFSH (recombinant human follicle stimulating hormone for injection, Serono, Switzerland); LH (luteinizing hormone, Sigma, USA); HCG (Livzon Pharmaceutical Factory of China Livzon Group); FCS (fetal bovine serum), penicillin/streptomycin (penicillin/streptomycin) and KSOM+AA(MR? 106? D) All products are products of Chemicon Company of the United States.
1.3 Ovary transplantation B6 female rats (8 ~ 10 weeks old) and D2 male rats (10 weeks old) were caged to breed F 1 generation at the ratio of 4: 1, and the female rats were recorded as E0.5d the next day; Take 20 days as the pregnant period, and check whether pregnant rats give birth every morning and evening 3 days before the expected date of delivery; The newborn F 1 mice within 12 h were decapitated and their bilateral ovaries were taken out for transplantation. Adult (8 ~ 10 weeks) F 1 female rats (transplant recipients) were anesthetized with pentobarbital (15 mg/mL). Sterilize the back of mice, cut longitudinally into abdominal cavity at the corner of costal ridge, remove bilateral ovaries, then implant two newborn mice ovaries under the capsule of left kidney, and sew the wounds layer by layer. The recipient mouse wakes up and sends it back to the mouse house.
1.4 After ovarian transplantation in newborn mice 14 d, the recipient mice were decapitated and the transplanted ovaries were taken out from under the renal capsule. The size of the ovarian graft is about 2 mm, and abundant reconstructed blood vessels can be seen on the surface. Follicles in transplanted ovaries can be clearly observed under stereomicroscope. Each ovary was cut into two pieces with the needle tip of a 1 mL syringe (3203 10, BD Company, USA), and four pieces of ovarian tissues were randomly divided after mixing.
1.4. 1 A group (mechanical method only), ovarian tissue was transferred to follicular fluid (α? MEM？ HEPES, 10%FCS, 100 IU/mL penicillin, 100 mg/mL streptomycin, without collagenase and dnase), in a hot plate at a constant temperature of 37℃ (MATS? U55SZX2A, Olympus), using fine-pointed tweezers (straight 5#, T 1? 18 1, China Huai 'an Teshen Medical Equipment Co., Ltd.) to exfoliate the follicles in the ovary. Every 5 minutes, the intact follicles that have been exfoliated are transferred to cells containing 1 mL (α? MEM, 5% fetal bovine serum, 0. 1 iu/ml follicle stimulating hormone, 0.0 1 iu/ml luteinizing hormone, 1% luteinizing hormone. * * * Separation for 4 times, with a total time of about 20 minutes.
1.4.2 B group (long-term enzymolysis) cut ovarian tissue into three small pieces with the needle tip of 1 mL syringe, and put them in 500 μL follicular enzymolysis solution (α? MEM？ HEPES (collagenase I, 3 mg/mL, 65438+20 min/mL DNase I) was digested in a CO2 incubator with a volume fraction of 0.05 at 37℃ for 20 min, then taken out of the incubator at the 20th minute, and the digestive juice was sucked 30 times with a 200 μL pipette. The intact free follicles were transferred to a 35 mm Petri dish pre-filled with 1 mL follicular culture medium and observed under a stereomicroscope.
1.4.3 C group (short-term enzymolysis), the ovarian tissue was cut into three pieces with the needle tip of 1 mL syringe, put into 500 μL follicular enzymolysis solution, digested in a CO2 incubator with a volume fraction of 0.05 at 37℃, and the tissue was taken out of the incubator every 5 minutes, and the digestive juice was sucked 30 times with a 200 μL pipette. * * * Digest for 20 minutes.
1.4.4 D group (combined method) cut ovarian tissue into three pieces with the needle tip of 1 mL syringe, transferred to 500 μL follicular enzymolysis solution, and digested in a CO2 incubator with a volume fraction of 0.05 at 37℃ for 5 min. Then the follicles were separated from the enzymatic hydrolysate of follicles with fine-pointed tweezers under the stereomicroscope of 37℃ constant temperature hot plate. Every 5 minutes, the separated intact follicles were transferred to a 35 mm Petri dish pre-loaded with 1 mL follicular medium. * * * Separation for 4 times, 20 min.
1.5 double fluorescent staining: some separated follicles were randomly selected from each group, and calcein AM(4 mmol/L, Sigma, C 1359) and ethidium homodimer were added. I( 10 mmol/L, Sigma, E 1903) was cultured in a CO2 incubator with a volume fraction of 0.05 at 37℃ for 45 min. Inverted fluorescence microscope was used to observe follicular suspension. Under the excitation of blue light, living follicles emit green light and dead follicles emit red light, and the numbers of dead follicles and live follicles are counted respectively.
1.6 follicles cultured in vitro 12 days. On the 0 th day, the isolated follicles were cultured in a 35 mm Petri dish with 1 mL medium. On 1 day, add 1 mL fresh culture medium; After that, change the liquid once every half day; On day 12, the whole amount was replaced with follicular maturation medium (α? MEM，5%FCS，0. 1 IU/mL FSH，0.0 1 IU/mL LH， 1% ITS， 1.5 IU/mL hCG)； On day 13, the discharged cumulus complexes (OGCs) were collected 26 hours after adding follicular maturation medium.
1.7 in vitro fertilization All OGC groups were fertilized in vitro. After 4 hours of fertilization culture, the oocytes were washed out from the semen droplets and transferred to the pre-balanced KSOM+AA droplets for culture. The maturation rate of oocytes (expressed as the percentage of oocytes containing the first polar body) and fertilization rate (expressed as the percentage of oocytes with double pronuclear oocytes occupying the first polar body) were observed.
SPSS 1 1.5 was used for statistical analysis. The survival rate of follicles, maturation rate of cultured follicles and ovulation rate of the four groups were analyzed by one-way ANOVA, and the mean values of each group were compared and the minimum significant difference test was made. The maturation rate and fertilization rate of oocytes were tested by χ2. 2. 1 After the separation operation, a large number of preantral follicles can be seen under the microscope. Most follicles are round or oval with clear boundaries. Some follicles are more or less surrounded by ovarian interstitial tissue. The oocyte is round and located in the center of the follicle, and the zona pellucida is clearly visible (figure 1A). Follicles with clear and smooth follicular boundaries were recovered under stereomicroscope, and the number of follicles recovered in each group was counted (table 1). Table 1 Comparison of follicular recovery in different groups
2.2 Double fluorescent staining results After double fluorescent staining, under the excitation of blue light, living cells showed green fluorescence and dead cells showed red fluorescence. Oocytes with green color and follicular cells ≥70% green color are considered as living follicles (Figure 1B). The percentage of live follicles in each group is shown in Table 2. Table 2 Comparison of follicular survival rate in each group
2.3 Follicle culture results In vitro, the follicles of each group were cultured under the same conditions for 65438 02d, and the survival rate of follicles was counted for 5 days and 65438 02d. After adding HCG hCG 26 h, the ovulation rate was counted. The results of each group of indicators are shown in Table 3. Table 3 Comparison of in vitro culture results of follicles in each group
2.4 In vitro fertilization results The OGCs harvested in each group were fertilized separately, and the density of capacitated sperm was controlled at 5× 105 mL? 1。 Early embryos were cultured in KSOM+AA, and the density of eggs was 15 20μ. The counting results of maturity rate and fertilization rate are shown in Table 4. The maturity rate of group D was higher than that of the other three groups, and there was no significant difference among the other three groups. There was no difference in fertilization rate among the four groups. Table 4 Comparison of in vitro maturation and fertilization results of each group In recent years, great progress has been made in the technical system of follicular induction and development in many mammals such as mice, rabbits, pigs and humans [4? 7]。 In these systems, most of them tend to isolate follicles in ovaries and then culture them. It has been reported that isolated follicles can be successfully cultured in vitro only if their basement membrane is not damaged and there are a few interstitial cells and immature round oocytes in the center [8]. Up to now, there are two commonly used methods to separate follicles. One is to digest ovarian connective tissue with hydrolytic enzymes (such as pronase, collagenase and deoxyribonuclease) to obtain free follicles, and the other is to mechanically separate follicles with sharp instruments [6,9? 10]。 Judging from the current data, the two methods have their own advantages and disadvantages. Enzymatic hydrolysis is a common method to separate cells from various tissues and organs in cell biology research, and it is widely used because of its simple and effective operation. When separating follicles by enzymatic hydrolysis, collagenase and deoxyribonuclease are usually used to prepare a separation solution. This separation liquid digests collagen in ovarian interstitial tissue by enzyme and releases follicles from ovarian tissue, so it is expected to harvest more free follicles in a limited time. It is reported that oocytes separated from follicles by enzymatic hydrolysis tend to have higher maturation rate and cleavage rate [1 1]. However, when the separated liquid is soaked in ovarian tissue for a long time, it may obviously cause damage to the basement membrane of follicles, which makes follicles more prone to degeneration in subsequent culture and reduces the survival rate of follicular culture. Therefore, the concentration and time of enzyme action is a key issue.
Mechanical separation of follicles, if properly handled, may maintain the integrity of follicular basement membrane, thus making it easier to maintain the three-dimensional structure of follicular culture in vitro and ensuring a higher survival rate. Carrell et al. and Park et al. showed that the survival rate of mechanically separated follicles was significantly higher than that of enzymatically separated follicles [12? 13]; Shen et al. further cultured mature oocytes from mouse follicles obtained by mechanical method and successfully prepared IVF? ET mice [4]. However, it has also been reported that the ovarian sections after transplantation show that the oocytes of some preantral follicles are weakly connected with the surrounding granulosa cells [14]. When separating follicles from dense ovarian interstitial tissue by mechanical means, it is often difficult to avoid pulling follicles too hard, which is easy to destroy the connection between oocytes and granulosa cells, while abnormal gap connection between oocytes and granulosa cells will affect the development of oocytes and lead to meiosis obstruction [15? 16]。 Therefore, whether the mechanical method can ensure the survival rate of follicles depends largely on the operation skills, and it is difficult to improve the recovery rate of follicles within a limited separation operation time. According to the current report, the number of follicles obtained from each ovary by mechanical method is very limited, with an average of only 12.55438+0 and 1 1.27 [4, 14] per ovary.
The results of this study show that the recovery rate of follicles, the survival rate of isolated follicles and the ovulation rate by enzymatic method are all lower than those by mechanical method (table 1 3), but the viability of follicles just separated is higher than that by mechanical method (table 2). This result is inconsistent with Demeestere's report, but similar to Carrell's and Park's report [1 1? 13]。 It shows that enzymatic hydrolysis has little direct damage to follicles, but it is not good for the long-term survival of follicles. It is worth noting that in this study, when separating follicles by enzymolysis, group C was set up, that is, short-time enzymolysis, and 1 follicle was harvested every 5 minutes during the enzymolysis process. The ovulation rate of this group was significantly higher than that of the conventional 20-minute enzymolysis treatment group, which proved that it was valuable to reduce the action time of the enzyme.
In order to improve the recovery rate of isolated follicles and ensure the high culture survival rate, ovulation rate and reproductive potential of follicles, a technical scheme of separating preantral follicles and ovarian grafts from newborn mice by enzymatic hydrolysis combined with mechanical method was established. Combined method (Group D): The ovarian tissue blocks were digested at 37℃ for 5 min in the separation solution containing collagenase and deoxyribonuclease, and then peeled off by the same operation method as the simple mechanical method, and the peeled follicles were immediately transferred to the culture solution without enzyme. This method makes the connective tissue around the follicle digested by enzyme, and the ovarian tissue becomes loose, thus reducing the pulling injury to the follicle during mechanical stripping, and at the same time making the free follicle leave the separation solution in time, reducing the action time of hydrolase on the follicle, which is more conducive to maintaining the three-dimensional structure of the follicle than simple enzymatic hydrolysis or simple mechanical method. The experimental results showed that the survival rate of isolated follicles in Group D was not different from that in the simple enzymatic hydrolysis method (Group B and Group C), but higher than that in the simple mechanical method (Group A), while the follicular recovery rate, average ovulation number and oocyte maturation rate of each ovary were higher than those in other groups, indicating that the combined method was absolutely superior to the two methods alone. At the same time, 97. 13 follicles (equivalent to 194 follicles/ovary) were obtained from each ovarian transplantation block by combination method, of which 40.66% were ovulated, which was significantly higher than the corresponding data reported in domestic and foreign literatures. It is worth noting that in this study, oocytes cultured by four separation methods were fertilized in vitro, and there was no significant difference in fertilization rate among groups, indicating that separation technology only affected the development and maturation of follicles in subsequent culture, but had no significant effect on the reproductive potential of those mature oocytes.
The experimental results show that the enzymatic hydrolysis established in this study? Mechanical combination method is a good technique for follicular separation, which has practical application value for the technical progress of ovarian tissue engineering.
[1] picton H M, Harris S E, Muruwe, et al. Growth and maturation of follicles in vitro [J]. Replication, 2008, 136(6):703? 7 15.
Min Xu, Ban Kai, et al. Growth of secondary follicles and maturation of oocytes in alginate gel after cryopreservation of ovaries or single follicles [J]. Biotechnology Bioenergy, 2009, 103(2):378? 386.
Adriaenssens T, Mazoyer C, Segues I, et al. Difference of collagen expression in cumulus cells after exposure to high-purity gonadotropins or recombinant follicles? Stimulating hormone in mouse follicular culture mode [J]. Biological reproduction, 2009,21:21.
Shen Wei, Zhang Dan, Qing Tao, et al. Living offspring produced by oocytes derived from mouse embryonic germ cells in the early and middle stages [J]. Biological Reproduction, 2006,75 (4): 615? 623.
[5] Eppig J J, O 'Brien M J. In vitro development of mouse primordial follicle oocytes. Biological reproduction. 1996; 54: 197? 207.
Hirao Y, Gaina T, Kubo M, et al. In vitro growth and maturation of porcine oocytes [J].J Reprod Fertil, 1994, 100:333? 339.
Abir R, Roizman P, Fisch B, et al. Preliminary study on isolated early human follicles cultured in collagen gel for 24 hours [J]. Humreprod, 1999, 14: 1299? 130 1.
Xu M, Kreeger P K, Shea L D et al. Organization? Engineering follicles produce living and fertile offspring. Tissue Engineering, 2006, 12( 10):2739? 2746.
[9] Hartshorne. In vitro culture of follicles [J].Rev Reprod, 1997, 2:94? 104.
[10] Jin Jianhui, Park Guosheng, Song Haibo, et al. A simple method for isolating mouse preantral follicles [J].Kor J Fertil Steril, 2000, 27:235? 243.
[1 1] Demeestere I, Delbaere A, Gervy C, et al. Effects of antral follicle separation on follicular growth, oocyte maturation and in vitro fertilization in mice [J]. Humreprod, 2000,15 (Abst. Book1): 89? 90.
[12] Liu Liang, Yi Huang, et al. Comparison of oocyte maturation, meiosis and chromosome aneuploidy in vitro culture of mouse secondary follicles [J].j Assisted Reproductive Genetics, 2005,22 (9? 10): 347? 354.
[13] Park K S, Lee T H, Park Y K, et al. Separation methods (mechanical or enzymatic methods) for pre? Mouse antral follicles [J].j Assisted Reproductive Genetics, 2005,22 (9? 10):355? 359.
[14] Liu Jun, Vanderster, vandenbroucke, et al. Maturity study of mouse primordial follicle transplantation combined with in vitro culture [J]. Bioreproduction, 2000, 62: 12 18? 1223.
[15] EPPIG J. Control of oocytes on the development and function of mammalian ovarian follicles [J]. Reproduction, 200 1, 122(6):829? 838.
[16] Klinger F G, de Felici M. In vitro development of embryonic mouse oocytes: stage? Specific regulation of stem cell factors and granulosa cells [J]. Developmental Biology, 2002,244 (1): 85? 95.