Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72–97% of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32–49% of the embryos. In summary, we established the simple and practical method of mouse oocyte vitrification with high survivability and developmental ability and the IVF using the vitrified-warmed oocytes with fresh, cold-stored or cryopreserved sperm with high fertility.     

Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. However, freezing the sperm of C57BL/6 mice, the most commonly used genetic background for genetically engineered mice, considerably reduces its fertility. We previously reported that methyl-beta-cyclodextrin dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. Recently, it was reported that exposing sperm to reduced glutathione may alleviate oxidative stress in frozen/thawed mouse sperm, thereby enhancing in vitro fertilization (IVF); however, the mechanism underlying this effect is poorly understood. In the present study, we examined the combined effects of methyl-beta-cyclodextrin and reduced glutathione on the fertilization rate of IVF with frozen/thawed C57BL/6 mouse sperm and the characteristic changes in the zona pellucida induced by reduced glutathione. Adding reduced glutathione to the fertilization medium increased the fertilization rate. Methyl-beta-cyclodextrin and reduced glutathione independently increased fertilization rates, and their combination produced the strongest effect. We found that reduced glutathione increased the amount of free thiols in the zona pellucida and promoted zona pellucida enlargement. Finally, 2-cell embryos produced by IVF with the addition of reduced glutathione developed normally and produced live offspring. In summary, we have established a novel IVF method using methyl-beta-cyclodextrin during sperm preincubation and reduced glutathione during the IVF procedure to enhance fertility of frozen/thawed C57BL/6 mouse sperm.     

Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs)

Superovulation, in vitro fertilization, embryo cryopreservation, and embryo transfer are assisted reproductive technologies (ARTs) widely used in laboratory mice. Inbred strains of mice have inherent genetic differences that cause them to respond differently to these technologies. Knowing how common inbred strains will perform when used for ARTs will ensure the most efficient use of mice, time, and resources. In this study, we characterized the ability of 10 inbred strains: 129S1/SvImJ, A/J, BALB/cJ, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, FVB/NJ, NOD/LtJ, and SJL/J to superovulate, fertilize in vitro, and produce live pups subsequent to embryo transfer. Three-week-old female mice were superovulated using eCG (5.0 IU) and hCG (5.0 IU). The resulting oocytes were fertilized in vitro in human tubal fluid medium with spermatozoa of the same strain. The following day, two-cell embryos were either transferred into pseudopregnant recipient females or cryopreserved. The cryopreserved embryos were later thawed and transferred into pseudopregnant recipient females. Differences in response to superovulation, fertilization, and number of live born produced after embryo transfer were observed between strains, substantiating the influence of genetic variability on ARTs. The response to the superovulation treatment varied among strains and ranged from 5+/-1(A/J) to 40+/-3 (129S1/SvImJ) normal oocytes per female. The average proportion of oocytes that fertilized ranged among strains from 24% (129S1/SvImJ) to 93% (DBA/2J and A/J). The average proportion of two-cell embryos that were transferred into recipient females and subsequently developed into live pups varied from 5% (A/J) to 53% (C57BL/6J) for fresh embryos and from 18% (BALB/cByJ) to 45% (129S1/SvImJ) for thawed embryos.     

Generation of Live Offspring from Vitrified Mouse Oocytes of C57BL/6J Strain

In mammals, unfertilized oocytes are one of the most available stages for cryopreservation because the cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has generally been reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. C57BL/6J mice, an inbred strain, are used extensively for the production of transgenic and knockout mice. If the oocytes from C57BL/6J mice can be successfully cryopreserved, the cryopreservation protocol used will contribute to the high-speed production of not only gene-modified mice but also hybrid mice. Very recently, we succeeded in the vitrification of mouse oocytes derived from ICR (outbred) mice. However, our protocol can be applied to the vitrification of oocytes from an inbred strain. The aim of the present study was to establish the vitrification of oocytes from C57BL/6J mice. First, the effect of cumulus cells on the ability of C57BL/6J mouse oocytes to fertilize and develop in vitro was examined. The fertility and developmental ability of oocyte-removed cumulus cells (i.e., denuded oocytes, or DOs) after IVF were reduced compared to cumulus oocyte complexes (COCs) in both fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop into the 2-cell and blastocyst stages compared to the vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate, equivalent to the rate obtained with IVF using fresh COCs. Taken together, our results demonstrate that we succeeded for the first time in the vitrification of mouse oocytes from C57BL/6J mice. Our findings will also contribute to the improvement of oocyte vitrification not only in animals but also in clinical applications for human infertility.     

昆明小鼠精子冷冻的研究（简报）

胚胎工程技术是动物品种、品系培育,种质资源保存及转基因动物制备、保种的重要手段。配子的冷冻保存技术目前广泛应用于胚胎工程。和胚胎冷冻相比小鼠精子冷冻技术方便、高效尤其适用于转基因及突变系小鼠的保种。成功的精子冷冻要求复苏后通过体外受精(IVF)获得胚胎,再移植入受     

Effects of a GnRH agonist on oocyte number and maturation in mice superovulated with eCG and hCG

The objective was to investigate the effects of a gonadotropin-releasing hormone agonist (GnRH) on ovulation rate and the number and maturation of oocytes in mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Thirty 3-month-old BALB/C female mice (weight: 25-30 g) were assigned to three experimental groups: control, superovulated, and superovulated with GnRH pretreatment (n=10 per group). Control mice received an i.p. injection of 0.1 ml physiological saline solution. Superovulation was induced with 5 IU eCG (i.p.) and 5 IU hCG 48 h later. Mice in the superovulated with GnRH pretreatment group were given GnRH (20 mg/kg Fertirelin, i.m.), 24 h before superovulation. Thirteen hours after hCG administration, mice were sacrificed by cervical dislocation and blood samples were collected to determine serum progesterone concentration (by radioimmunoassay). Ovaries and oviducts were also harvested to enumerate corpora lutea and cumulus-enclosed oocytes. Progesterone concentrations were not significantly different among groups. The oocyte number and the maturation, ovulation rate, and the number of corpora lutea were higher in GnRH-treated mice than both controls and superovulated mice. In conclusion, GnRH given 24 h before superovulation with eCG-hCG increased the number and maturation of oocytes and the rate of ovulation in mice.     

Treatment of sperm with extracellular adenosine 5'-triphosphate improves the in vitro fertility rate of inbred and genetically modified mice with low fertility

In vitro fertilization (IVF) is one of the most important techniques used for assisted reproduction in mouse colony management. As with natural mating, where mice have varying fertility indices, fertility rates of genetically modified (GM) [transgenic (Tg), knock out (KO) and congenic (Cg)] mice are influenced by their genetic background. Lines of GM mice that have poor fertility have a concomitant poor IVF outcome. Treatment of mouse sperm with extracellular adenosine 5'-triphosphate (ATPe) enhanced in vitro fertilization rates in outbred and hybrid mice. The objective of this study was to analyze the effects of using extracellular adenosine 5'-triphosphate-treated sperm for IVF of inbred wild type, and genetically modified mouse lines, for which standard IVF did not work well. The IVF was performed using the GM mice on C57BL/10SnJ, C57BL/6J, BALB/cJ and NFS/N background strains and wild type (WT) mice such as C57BL/6N, BALB/cAnN, and B6129SF1 strains. Oocytes from superovulated females were fertilized in vitro with sperm from the same background strain, and either treated or not treated with ATPe. The ATPe treatment enhanced IVF outcome in most of the GM and some WT strains, as indicated by the percentage of embryos that progressed to the two-cell stage. There was no marked difference between ATPe treated and control groups for the development rate of two-cell embryos to blastocysts in culture, or in the number of pups born after transfer of two-cell embryos into recipient females. The observed improvement of the IVF results following ATPe treatment of transgenic and KO mouse sperm were a potential solution for improving the outcome of assisted reproduction techniques used for rederivation or for gamete banking.     

Transplantation of male pronucleus derived from in vitro fertilization of enucleated oocyte into parthenogenetically activated oocyte results in live offspring in mouse

In this study, inter-strain reconstructed embryos were produced by combining the female pronucleus of Kunming mouse (white) with male pronucleus of C57BL/6 strain (black). Metaphase II (MII) oocytes of Kunming mouse were enucleated and the zona pellucida was removed. Then, the enucleated oocytes were inseminated by capacitated sperm of C57BL/6 mouse in vitro. At the same time, MII oocytes of Kunming mouse were artificially activated using strontium chloride solution, which did not contain cytochalasin B. Finally, we removed the male pronucleus derived from C57BL/6 sperm and injected it into a parthenogenetically activated one-pronucleus oocyte by micromanipulation. The reconstructed 2-cell embryos were transplanted into the oviducts of 22 foster mother mice, each receiving about 20 embryos. In the end, seven healthy and live pups were born from one recipient.     

Effect of partial incision of the zona pellucida by piezo-micromanipulator for in vitro fertilization using frozen-thawed mouse spermatozoa on the developmental rate of embryos transferred at the 2-cell stage.

Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.     

Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells

Sperm cryopreservation provides an economical means of storing genetically engineered mouse strains in resource facilities. In general, relatively high fertilization rates are obtained for frozen/thawed sperm of the CBA/JN, DBA/2N, and C3H inbred strains and some F1 hybrid strains. However, the fertilization rate for frozen/thawed sperm of C57BL/6, which is the main strain of genetically engineered mice, remains very low. Therefore, it is necessary to establish an in vitro fertilization (IVF) method for cryopreserved C57BL/6 sperm that can obtain a high rate of fertilization after thawing. In the present study, we focused on the effects of methyl-beta-cyclodextrin (MBCD) on the fertilizing ability of frozen/thawed C57BL/6 sperm. Our results have shown that the highest fertilization rate for frozen/thawed sperm was obtained with MBCD at 1.0 mM for 30 min (63.7% +/- 11.0%), but the effects were attenuated by long-term incubation for 120 min at 1.0 or 2.0 mM. The embryos with frozen/thawed sperm showed good developmental potential, and the offspring had normal fertility. The efflux of cholesterol from frozen/thawed sperm was increased by MBCD in a dose-dependent manner and occurred much earlier and to a greater extent than bovine serum albumin. The localization of cholesterol labeled by filipin in the sperm plasma membrane was drastically decreased by MBCD. In summary, we suggest that MBCD is useful for developing an IVF method for frozen/thawed C57BL/6 mouse sperm achieving a high fertilization rate, being involved in the capacity to sequester cholesterol from sperm membrane.     

Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm

Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%-12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%-88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%-63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.     

Cryopreservation of unfertilized mouse oocytes from inbred strains by ultrarapid freezing

Unfertilized mouse oocytes from inbred strains (BALB/c, C3H/He and C57BL/6) were frozen ultrarapidly by direct plunging into liquid nitrogen, immediately after exposure to a highly-concentrated solution (DAP 213: 2 M dimethyl sulphoxide, 1 M acetamide, and 3 M propylene glycol in PB 1), and were later thawed in a 37 degrees C waterbath. After thawing, 76.8-90.9% of recovered oocytes were morphologically normal. Following fertilization in vitro of cryopreserved oocytes, the proportion of 2-cell embryos 24 h after insemination ranged from 70.7% to 83.4%. Nearly all 2-cell embryos obtained from cryopreserved oocytes were transferred to the oviducts of pseudopregnant recipients and 31.0-43.0% of 2-cell embryos developed into normal young.     

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.g., bovine and human. In this study, we examined the efficiency of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6J, commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2F1. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh, 89 vs. 55%; frozen, 56 vs. 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89 vs. 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90 and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of two-cell embryos to the blastocyst stage was lower for C57BL/6J than B6D2F1 (42-61% and 84-98%) in all treatments but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%), indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More important, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.     

Successful cryopreservation of mouse ovaries by vitrification

We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-day-old green fluorescent protein (GFP)-transgenic mice (+/+). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P < 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 +/- 0.3 for the experimental group versus 2.0 +/- 0.7 for the control group, mean +/- SEM), the number of collected oocytes by superovulation per mouse (7.0 +/- 1.7 for the experimental group versus 22.7 +/- 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.     

系统低温生物学技术在实验小鼠资源保存中的建立与应用

目的建立小鼠胚胎与配子冷冻库,以安全、有效地保存小鼠资源。方法选择不同遗传背景（近交系、远交群、免疫缺陷、疾病模型和基因改变等）的实验小鼠,系统进行了超数排卵、体外受精（IVF）率、胚胎与精子的冷冻复苏效果、卵巢冷冻与移植、辅助体外受精等比较研究。结果①小鼠年龄和遗传背景的不同,其超数排卵的结果也不同（P〈0．01）。三个日龄段中,28日龄最好,其次为112日龄,56日龄最差;不同遗传背景小鼠的超数排卵结果显示,封闭群和大部分近交系小鼠优于转基因小鼠（P〈0．05）,自发性疾病小鼠和基因剔除小鼠的结果最差;②不同品系小鼠的新鲜精子和冻融精子的体外受精率差异有显著性（P〈0．05）,特别是C57BL／6J小鼠冻融精子的IVF率（10．3±4．2％）与新鲜精子（89．8±4.8％）相比,差异极显著（P〈0．01）;③不同品系小鼠的胚胎复苏率,除MRL／mp小鼠的复苏率略低外,其他小鼠品系均有较高的冷冻胚胎复苏率（58．2％～83．9％）,表明,不同遗传背景小鼠之间差异有显著性（P〈0．05）,但均可以达到有效保存小鼠资源的目的。④小鼠的遗传背景、年龄等对小鼠精子的冷冻效果都有影响,采用改良的FERTIUP冷冻保护剂和细胞质内单精注射（ICSI）技术可有效提高以C57BL／6J为背景的基因改变小鼠的精子冷冻复苏率。⑤卵巢冷冻保存可以改善雌性小鼠的繁殖困难或不孕。结论小鼠资源的安全保存,除了长期连续繁殖保种外,最好的或最保险的方法是低温保存。通过将胚胎、配子、卵巢等长期保存在液氮（-196℃）中避免遗传性状的改变,并在将来复苏后获得正常的小鼠后代,以用于生物学和医学等研究。     

The superovulation of synchronous adult rats using follicle-stimulating hormone delivered by continuous infusion

The estrous cycles of adult female rats were synchronized with an LHRH agonist on the morning of Day -4 (Day 0 = day of mating). On Day -2, animals received s.c. implants of continuous-infusion osmotic minipumps containing different doses of an FSH preparation (Folltropin) in combination with hCG at various ratios of hCG:FSH or were given single injections of eCG in doses ranging from 15 IU to 60 IU. Rats infused with the optimal dose (3.4 U/day) of FSH ovulated 44.1 +/- 5.4 oocytes/rat while rats treated with the most effective dose (60 IU) of eCG ovulated only 20.5 +/- 4.3 oocytes/rat on the morning of Day 1. The inclusion of hCG in pumps at ratios from 0.188:1 to 0.75:1 (hCG:FSH) had no significant effect on ovulation rate. The importance of synchronization of estrus in successful superovulation was demonstrated by the finding that only 70% of the unsynchronized animals ovulated (29.1 +/- 4.8 oocytes/rat) whereas 95% of the synchronized animals ovulated (51.0 +/- 3.6 oocytes/rat). Oocyte viabilities were assessed by determining fertilization rates and embryonic development in vivo following mating with fertile males. In rats superovulated by use of the FSH regimen, 92% (39.0 +/- 4.1) of the recovered embryos were 1-cell zygotes on Day 1, 89% (36.3 +/- 5.6) were at the 2-cell embryo stage of development on Day 2, and 88% (28.8 +/- 2.2) were at the morula and blastocyst stages on Day 5 following mating on Day 0. The high ovulation rates and oocyte viability in rats receiving infusions of Folltropin following estrus synchronization offer a reliable method for superovulation of adult rats.     

Efficient collection and cryopreservation of embryos in F344 strain inbred rats

In rats, it is now possible to produce genetically engineered strains, not only as transgenic animals but also using gene knockout techniques. Reproductive technologies have been used as indispensable tools to produce and maintain these novel valuable strains. Although studies for collecting and cryopreserving embryos have been reported using outbred rats, efficient methods have not been established in inbred strains. The F344 inbred strain is important in rat breeding and has been used for the production of transgenic/knockout strains and for genome sequencing. Here we studied the optimal conditions for oocyte collection by induction of superovulation, and the development of embryos after cryopreservation in F344 rats. The response to pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was examined by injection of 150 IU/kg PMSG + 75 IU/kg hCG or 300 IU/kg PMSG + 300 IU/kg hCG. Superovulation was achieved at high efficiency by an injection of 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, superovulation in this strain showed similar high response as Wistar rats. Of 2-cell embryos cryopreserved by vitrification in a solution containing 10% propylene glycol, 30% ethylene glycol, 20% Percoll and 0.3 M sucrose, more than 90% survived after warming and 32% developed to offspring. However, the freezability of pronuclear stage embryos was extremely low. This study demonstrated that sufficient unfertilized oocytes and embryos can be collected from F344 rats by the induction of superovulation with 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, cryopreservation of 2-cell embryos using this vitrification protocol can now be applied to maintaining valuable rat strains derived from the F344 inbred strain as genetic resources.     

Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice

We examined in vitro fertilizability and development of 10 inbred mouse strains (C57BL/6J, C57BL/10, C57BL/10.D2/newSn, C57BL/10-Thy1.1, C57BL/10.Br/Sn, C3H/He, RFM/Ms, STS/A, BALB/c-nu and C.B-17/Icr), and the viability of frozen-thawed in vitro fertilized (IVF) embryos after embryo transfer (ET). In seven strains, fertilizability was significantly greater in modified human tubal fluid (mHTF) compared with modified Krebs-Ringer's bicarbonate solution (TYH medium). The TYH medium supported almost no fertilization in four strains. More than 80% of IVF embryos developed to the blastocyst stage by 120 h in potassium-enhanced simplex optimization medium (KSOM). Reciprocal fertilization between C57BL/6J and BALB/c-nu gametes in TYH medium yielded poor fertilization o f BALB/c-nu due to spermatozoal deficiencies. Increased concentrations of bovine serum albumin and spermatozoa during capacitation and Percoll washing did not drastically affect fertilization. The mHTF, but not TYH medium, supported BALB/c-nu spermatozoa penetration into the zona pellucida irrespective of capacitation media. In vitro fertilized embryos frozen-thawed rapidly were transferred to surrogate mothers at the two-cell stage. Compared with that of unfrozen controls, rapid freezing had no significant effect on fetus development except in C57BL/10.D2/newSn mice. These results suggest that mHTF medium is superior with respect to IVF of inbred mice, and that KSOM adequately supports in vitro fertilized embryo development in inbred mice. The data also indicate that rapid freezing of pronucleate embryos following IVF is suitable for cryopreservation and embryo banking of inbred mice and for the production of genetically modified mice.     

Inhibition of In Vitro Fertilizing Capacity of Cryopreserved Mouse Sperm by Factors Released by Damaged Sperm,and Stimulation by Glutathione

Background

In vitro fertilization (IVF) of eggs by frozen and thawed C57BL/6J mouse sperm is inhibited by dead sperm and enhanced by preincubation of the sperm in calcium-free medium. In other species, the presence of sperm killed by freezing and thawing has been associated with the generation of hydrogen peroxide.

Methodology/Principal Findings

The proportion of eggs fertilized by cryopreserved C57BL/6J mouse sperm was increased significantly by increasing the volume of fertilization medium in which sperm and eggs were coincubated. Enhanced fertilization occurred even though the concentration of potentially fertile sperm was decreased fivefold. This suggested that if a putative soluble factor was inhibiting fertilization, dilution of that factor, but not the sperm, should increase the fertilization rate. This was achieved by coincubation of the gametes in cell culture inserts (Transwells^®) that during incubation were transferred progressively to wells containing fresh fertilization medium. Fertilization rates using inserts were high (66.6±2.4% versus 27.3%±2.8% in wells alone). On the assumption that the soluble factor could be H₂O₂, reduced glutathione was added to the fertilization medium. This enhanced fertilization rate significantly (76.6%±2.0% versus 21.2%±1.9%), while addition of oxidized glutathione did not (82.7%±6.5% with reduced glutathione; 44.5±8.8% with oxidized glutathione; 47.8%±12.1% with no glutathione). Positive effects of reduced glutathione on IVF were also seen with frozen 129S1, FVB, and C3H sperm, and sperm from two lines of genetically modified C57BL/6J mice.

Conclusions/Significance

IVF in cell culture inserts and addition of glutathione to fertilization medium significantly increased the proportion of eggs fertilized by cryopreserved mouse sperm from four inbred strains, suggesting that reactive oxygen species generated during fertilization inhibit fertilization. The modified IVF techniques developed here enhance the feasibility and efficiency of using cryopreserved sperm from genetically modified lines of inbred mice.     

Predictive variables of in vitro fertilization and pre-implantation embryo development in the mouse

The present study aims to analyze the cause-effect relationships among several in-vitro fertilization and pre-implantation embryo development variables in the mouse. Superovulation of hybrid (C57Bl/6JIco female X CBA/JIco male) female mice of 4-6 weeks of age was induced by a priming injection of pregnant mare's serum gonadotropin at the estrus stage of the estrous cycle followed after a 48-hr interval by human chrorionic gonadotropin. Ovulated cumulus-enclosed oocytes were inseminated with sperm from hybrid males of 12-16 weeks of age. The multiple linear regression analyses performed indicated that (a) total number of ovulated oocytes is a good predictor of both fertilization frequency and total number of cells in day-5 blastocysts; (b) fertilization frequency predicts percentage of day-5 blastocysts; (c) total number of cells in day-5 blastocysts is predicted by percentage of day-5 blastocysts; and (d) total number of cells in day-5 blastocysts predicts percentage of apoptotic cells, number of inner cell mass (ICM) and trophectoderm (TE) cells, and ICM/TE ratio in day-5 blastocysts. Mitotic index in day-5 blastocysts was positively correlated with total number of ovulated oocytes, percentage of ovulated cumulus-enclosed oocytes, fertilization frequency, percentage of day-5 blastocysts and total number of cells in day-5 blastocysts. On the contrary, it was negatively correlated with percentage of apoptotic cells in day-5 blastocysts.