金色中仓鼠胚胎植入小鼠子宫后PTEN、FAK、Shc信号分子的异常表达

除了马属动物外,种间妊娠的胚胎总在妊娠的一定时期流产。以往的研究多集中在宏观结构和解剖学差别的研究上,对于这种现象产生的分子机制却少有报道。为了从信号分子角度阐明种间妊娠维持失败的可能原因,我们通过免疫组化和明胶酶谱法比较了科间和同种妊娠部位整合素αvβ3、FAK、p-ERK、IGFⅡ、IGFBP-1、Shc、PTEN和MMP-2、-9表达的差异。科间妊娠是将金色中仓鼠(Mesocricetus auratus)的胚胎移植入假孕小鼠(Mus musculus)子宫并取妊娠D4、D8、D12的子宫为实验材料,相应时间同种小鼠胚胎移植小鼠妊娠后的子宫和假孕小鼠子宫分别为阳性和阴性对照。免疫组化结果表明D8时,科间妊娠孕体integrinαvβ3、IGF-Ⅱ、IGFBP-1、PTEN、Shc、FAK的表达强度均与正常同种妊娠有差异。而妊娠D8时同种、科间妊娠子宫p-ERK的表达部位与表达量无明显差异。明胶酶谱显示,科间妊娠时MMP-2,-9的分泌模式与同种妊娠明显不同。这些结果表明,上述信号通路分子的差异表达有可能是造成科间妊娠时母胎界面异常的组织学表现的一个原因。     

Viability and development of 'tube-locked' mouse embryos

'Tube-locked' morulae and blastocysts were recovered from the ampulla of the oviduct of centchroman-treated mice between Days 4 and 12 post coitum and transferred to the uteri of pseudopregnant female mice. Pregnancy and implantation rates were lower and the post-implantation resorption rate was higher in the treated than in the control group. There was little difference in the pregnancy or implantation rates between embryos recovered on Days 4 or 12 post coitum, but the resorption rate increased with increasing duration of embryos in the oviducts and was 100% for the Day-12 embryos. The resorption rate was similar even when these embryos were transferred to the sterile uterine horn of unilaterally pregnant mice. Centchroman did not produce any deleterious effect on embryos which survived until Day 19 of pregnancy in foster mothers. The average fetal weight was also comparable to those of control fetuses.     

Requirement for progesterone priming and its long-term effects on implantation in the mouse

At least 48 hr of progesterone (P4) priming has been documented to be essential for P4 and estrogen to initiate implantation in the rat. However, the length of this P4 priming requirement for implantation in the mouse has not been experimentally defined. Therefore, our first objective was to determine the length of P4-priming requirement for implantation in the mouse. Day 4 blastocysts were transferred into the uteri of Day 5 or Day 6 pseudopregnant mice that were ovariectomized on Day 1 (= vaginal plug) and treated with a single injection of P4 and 17 beta-estradiol (E2) only on Day 5, or a single injection of P4 on Day 5 followed by a second injection of P4 plus E2 on Day 6, respectively. Although none of the transferred blastocysts implanted in the uteri of P4-unprimed recipients, 46% of the transferred blastocysts implanted into the uteri of all recipients that were first primed with P4 24 hour prior to a second injection of P4 and E2. These results suggest that in contrast to the rat, the mouse uterus requires at most 24 hr of P4 priming before P4 and estrogen can initiate implantation. Our second objective was to determine whether P4 priming has a long-term effect on implantation in the mouse. Our present results and those of others suggest that the mouse uterus is exposed to rising P4 levels for 24 hr prior to implantation on Day 4 of pregnancy. Therefore, in the present investigation, induction of implantation by an injection of P4 and E2 following 5 days of ovariectomy performed on Day 4 of pregnancy clearly suggests that once exposed to P4 for 24 hr, the mouse uterus retains a long-term effect, i.e., following P4 withdrawal for several days, 24 hr of initial P4 priming is no longer required for P4 and estrogen to initiate implantation. Our next objective was to explore whether this long-term effect of P4 priming on implantation can be prolonged and potentiated by increasing the length of initial P4 priming. Thus, when the mice were ovariectomized on Day 4 of pregnancy and treated with P4 beginning on Day 5 for 4 days, the long-term effect on implantation was prolonged (8 days vs 5 days following P4 withdrawal) and potentiated (94% vs 0% mice with implantation following 8 days of P4 withdrawal) as compared with those with no P4 priming after ovariectomy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of centchroman on tubal transport and preimplantation embryonic development in rats

A single oral administration of centchroman (1.25 mg/kg) to adult female rats within 24 h of mating induced slight acceleration in the rate of transport of embryos through the oviducts. The compound did not seem to produce any deleterious effect on preimplantation embryonic development since well organized and apparently normal embryos were collected from the genital tract up to Day 12 of pregnancy. The recovery rate of embryos from centchroman-treated rats was, however, significantly reduced after Day 4 of pregnancy. There was some stimulation in the rate of cleavage of embryos and morula to blastocyst transformation, but retardation in the shedding of the zona pellucida. The rate of blastocyst formation was not altered when 6-8-cell embryos collected from the oviducts of control rats were transferred to the uteri of control or centchroman-treated females. A delay in zona shedding was observed in the centchroman-treated recipients.     

Effects of E-cadherin on mouse embryo implantation and expression of matrix metalloproteinase-2 and -9

E-cadherin is a cell surface glycoprotein, which is responsible for adhesion between epithelial cells. Whether it is involved in embryo implantation is still unknown. In a mouse intrauterine horn injection model, one uterine horn in each mouse was injected with different doses of E-cadherin antibody on day 3 of pregnancy. The results showed that embryo implantation was significantly inhibited in the mice injected with 3 microg E-cadherin antibody. The mouse uteri in this group were collected on days 5, 6, and 7 of pregnancy and expressions of MMP-2 and -9 were studied. In situ hybridization and RT-PCR results showed that the expression of MMP-2 and -9 mRNAs in uteri of E-cadherin antibody treated group was increased on days 5-7. The results of gelatin zymography of MMPs showed that the activities of pro-MMP-2, MMP-2, and pro-MMP-9 were increased significantly on days 5 and 6, and pro-MMP-9 activity was increased on day 7. The present study suggested that E-cadherin was involved in embryo implantation through decreasing the expressions and activities of MMP-2 and -9.     

Ultrastructural studies on maternal-embryonic cell interaction during experimentally induced implantation of rat blastocysts to the endometrium of the mouse.

Rat blastocysts collected around noon on Day 5 of pregnancy were transferred to the uterus of the mouse on Day 3 of pseudopregnancy. Out of 430 rat blastocysts transferred, a total of 37 were recovered as xenogeneic implants from the recipient mice killed 36, 48, 52, 58, 72, 96, and 120 hr after transfer. None of the transferred blastocysts was found surviving in the host uterus beyond 96 hr after transfer. Electron microscopic examination of the recovered implants revealed that rat blastocysts can successfully undergo the stages of ovum implantation in the mouse uterus from the early attachment to the initial phase of the trophoblastic invasion of the endometrium. During the late attachment stage, desmosomes (maculae adhaerentes), intermediate junctions (zonulae adhaerentes), and gap junctions (nexuses) were formed xenogeneically between the foreign trophoblast and the uterine epithelial cells of the host. Trophoblast cells of xenogeneic implants were destroyed shortly after the penetration of the basement membrane of the luminal epithelium of the host endometrium, leading to the degeneration and sloughing off of the transferred embryos.     

Factors affecting the efficiency of embryo cryopreservation and rederivation of rat and mouse models

The efficiency of embryo banking for rat and mouse models of human disease and normal biological processes depends on the ease of obtaining embryos. Authors report on the effect of genotype on embryo production and rederivation. In an effort to establish banks of cryopreserved embryos, they provide two databases for comparing banking efficiency: one that contains the embryo collection results from approximately 11,000 rat embryo donors (111 models) and another that contains the embryo collection results from 4,023 mouse embryo donors (57 induced mutant models). The genotype of donor females affected the efficiency of embryo collection in two ways. First, the proportion of females yielding embryos varied markedly among genotypes (rats: 16-100 %, mean =71 %; mice: 24-95 %, mean =65 %). Second, the mean number of embryos recovered from females yielding embryos varied considerably (rats: 4-10.6, mean =7.8; mice 5.3-32.2, mean =13.7). Genotype also affected the efficiency of rederivation of banked rat and mouse embryos models by embryo transfer. For rats, thawed embryos (n =684) from 33 genotypes were transferred into 66 recipient females (pregnancy rate, 78 %). The average rate of developing live newborns for individual rat genotypes was 30 % with a range of 10 to 58 %. For mice, thawed embryos (n =2,064) from 59 genotypes were transferred into 119 pseudopregnant females (pregnancy rate: 76 %). The average rate of development of individual mouse genotypes was 33 % with a range of 11 to 53 %. This analysis demonstrates that genotype is an important consideration when planning embryo banking programs.     

Beneficial effect of agar for the frozen storage of bisected embryos

The effect of agar embedding on the viability of intact and bisected goat embryos during freezing and thawing was examined. Blastocysts or hatched blatocysts were bisected into haves by micromanipulation. After embedding with or without agar, they were stored by deep freezing. After thawing, undamaged and some partially damaged embryos were transferred into uteri of recipients. Of 22 demi-embryos embedded with agar, only one was undamaged, but of 58 demi-embryos embedded with agar, 29 were undamaged. Although one set of monozygotic twins was obtained after the transfer of 15 sets of frozen-thawed bisected embryos, the pregnancy rate (47%) and the proportion of young (27%) were lower than those obtained by transfer of frozen-thawed, intact embryos (67 to 80% for pregnancy rate, 45 to 53% for the young).     

Viability of frozen-thawed bovine embryos bisected in sucrose: A preliminary report

A method for producing identical twin calves is described in which Day 7 frozen-thawed bovine embryos in 12.5% sucrose solution were bisected using a fine microsurgical blade. The resulting bisected embryos were transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/-1 d), two bisected embryos per recipient. The pregnancy rate when both halves remained in the same zona pellucida was 50% (5 10 ); the pregnancy rate was 1 5 for morulae and 4 5 for blastocysts. The pregnancy rate for unfrozen morulae bisected in PBS and transferred without zona pellucida was 27% (4 15 ). The in vitro survival rate of embryos bisected in 12.5% sucrose when both halves remained in the original zona pellucida was 82% (18 22 ), which was higher than when embryos were bisected in PBS (53%, 9 17 ).     

A Globosus amorphus from an in vitro fertilized embryo transferred to a Japanese black cow

A Globosus amorphus along with a living calf was encountered following the transfer of blastocysts obtained by in vitro fertilization of in vitro-matured follicular oocytes in Japanese black cattle. Two embryos obtained 9 days after in vitro fertilization developed into either a hatched blastocyst with distinct inner cell mass or an expanded blastocyst with indistinct inner cell mass. The embryos were loaded into a 0.25-ml plastic straw and were nonsurgically transferred to the uterus of a heifer on Day 8 (Day 0 = estrus). On Day 75, a twin pregnancy was ultrasonically diagnosed in the right uterine horn, in which a live fetus with distinct limbs and a concomitant ovoid mass were detected. On Day 287, the dam developed parturient paralysis with dropsy of the fetal membranes. By palpation per rectum an ovoid mass was detected in the body of the uterus [corpus uteri] and a larger live fetus was in the uterine horn. A cesarean section was performed to extract a live fetus and a Globosus amorphus. The live fetus was female with the 60, XX female complements.     

The ovine uterus as a host for in vitro-produced bovine embryos

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.     

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to −6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), −1 (68%), −2 (63%), −3 (66%), −4 (76%), −5 (61%), and −6 (27%). The −6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to −5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.     

Expression of matrix metalloproteinase -2, -9 and tissue inhibitors of metalloproteinase -1, -2, -3 mRNAs in rat uterus during early pregnancy

Zymography and in situ hybridizition were used to investigate matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and expression of mRNAs for MMP-2, -9 and tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3) in the rat uterus during early pregnancy (day 1-7). The zymography results showed two forms of MMP-2 (64 and 67 kDa) in the rat uteri during early pregnancy. The 64-kDa MMP-2 activity was the highest on day 2 (P < 0.01) and higher on day 5 and 6 (P < 0.05). The 67-kDa MMP-2 activity reached the highest on day 5 and 6 (P < 0.01). The 64-kDa MMP-2 activity at the implantation sites was higher than those at interimplantation sites (P < 0.05). Furthermore, the 67 kDa MMP-2 can be converted to 64 kDa forms by incubation with p-aminophenylmercuric acetate (APMA) and trypsin in vitro. The 92-kDa MMP-9 activity was only detected on day 5 and 6 of pregnancy (P < 0.01). In situ hybridization showed that on day 1-4 of pregnancy, both MMP-2 and TIMP-2 mRNAs were evidently localized in the basal stromal cells. On day 5, MMP-2 mRNA signals were decreased in the basal stromal cells and mRNA for TIMP-2 was expressed in the epithelial cells and subepithelial stromal cells. The mRNAs for MMP-9, TIMP-1, and -3 were mainly expressed in epithelial cells on day 1-5. At the implantation site on day 6, the mRNAs for MMP-2, -9, TIMP-1, -2, and -3 were highly expressed in the primary decidual zone surrounding the implanting embryo, and in the whole decidualized stromal cells (the primary and secondary decidual zones) at the implantation site on day 7. The intensities of mRNAs for the TIMPs in decidualized stromal cells at the implantation site on day 6 and 7 were stronger than those for the MMPs. The weak mRNAs for MMP-2, -9, TIMP-1, and -3 but not TIMP-2 were also observed in the ectoplacental cone/trophoblastic cells of the implanting embryos. However, at the interimplantation sites on day 6 and 7, MMP-2, -9, TIMP-1, -2, and -3 mRNAs were weakly expressed in the epithelial cells, subepithelial stromal cells, and myometrium. The results suggested that the implanting rat embryo strongly induced MMP-2 and -9 proteins and gene expression for decidulization and embryo invasion, which were strictly controlled and balanced by the simultaneous expression of TIMP-1, -2 and -3.     

Invasiveness of mouse embryos to human ovarian cancer cells HO8910PM and the role of MMP-9

ABSTRACT: Background Our previous work found that mouse embryos could invade malignant cancer cells. In the process of implantation, embryo trophoblast cells express matrix metalloproteinases and the invasive ability of trophoblast cells is proportional to matrix metalloproteinase-9 protein expression. So the purpose of this study is to observe the effects of mouse embryos on human ovarian cancer cells in the co-culture environment in vitro and explore the possible mechanism of matrix metalloproteinase-9. Methods Several groups of human ovarian cancer cells HO8910PM were co-cultured with mouse embryos for different time duration, after which the effects of mouse embryos on morphology and growth behavior of HO8910PM were observed under the light microscope real-time or by H.E staining. Apoptosis was detected under laser confocal microscope by Annexin V-EGFP/PI staining in situ. Invasion ability of tumor cells was studied by transwell experiments. After matrix metalloproteinase 9 (MMP -9) activity was inhibited by MMP-9 Inhibitor I, the interaction between mouse embryos and human ovarian cancer cells HO8910PM was observed. Results Mouse embryos were able to invade co-cultured human ovarian cancer cell layer which extended in the bottom of the culture dish, and gradually pushed away tumor cells to form their own growth space. The number of apoptosis tumor cells surrounding the embryo increased under laser confocal microscope. After co-cultured with mouse embryos, tumor cells invasive ability was lowered compared with the control group. After MMP-9 activity was inhibited, the interaction between mouse embryos and HO8910PM cells had no significant difference compared with the normal MMP-9 activity group. Conclusion Mouse embryos were able to invade human ovarian cancer cells in vitro and form their own growth space, promote apoptosis of human ovarian cancer cells and lower their invasive ability. The mouse embryo was still able to invade human ovarian cancer cells after MMP-9 activity was inhibited.     

Production of mouse by inter-strain inner cell mass replacement

The developmental ability of reconstructed blastocysts from C57BL/6 strain mouse inner cell masses (ICMs) and Kunming strain mouse trophoblasts was assessed. The procedure of ICM replacement was as follows: C57BL/6 ICMs were separated from the blastocysts using immunosurgery. A slit was made in the zona pellucida of a Kunming blastocyst to allow its ICM to extrude. The C57BL/6 ICM was injected into the Kunming blastocoele, and the extruded Kunming ICM was cut off. The reconstructed blastocysts were able to re-expand (77%) and hatch (27.3%) in vitro. A total of 64 reconstructed blastocysts and 124 Kunming blastocysts were co-transferred into the uteri of 11 pseudopregnant Kunming mice, and an ICM replacement offspring was born. The results indicate that reconstructed embryos obtained by inter-strain ICM replacement have the ability to develop to term. This technique may provide a method to solve the pregnancy failure in interspecific cloning.     

The synthetic surfactants AS and LAS interrupt pregnancy in mice

Alcohol sulfate (AS) interrupted pregnancy when applied to the dorsothoracic area (2 × 3 cm) of pregnant mice from Day 1 to 10 of gestation. This effect was due to interference with development of the embryos during the cleavage stage, because when pregnant mice were treated with AS in the same way before implantation (from Day 0 to 3), significant numbers of embryos (29.1%) collected from the uteri and oviducts were deformed (dead or dying). Similar results were obtained with a linear alkylbenzene sulfonate (LAS). AS had no detectable teratogenicity in the offspring when applied to the mothers similarly from Day 1 to 17 or carcinogenicity when applied from Day 12 to 17. The offspring of mice treated with AS from Day 12 to 17 showed growth retardation, but this disappeared after weaning.     

Production of identical twins by separating two-cell rat embryos

Rat identical twins were produced from two-cell embryos. In the presence of cytochalasin B, rat two-cell embryos could be separated efficiently into two blastomeres by micromanipulation. Isolated blastomeres, embedded in agar cylinders and cultivated in ligated rat oviducts for 3 days, developed to the morula or blastocyst stage. After removing the agar, pairs of developed one-half embryos were transferred into Day 1 oviducts or Day 4 uteri of pseudopregnant rats. The percentage of embryos, separated either in the presence or absence of cytochalasin B, that developed into live fetuses was higher in cases of uterine transfer than in cases of oviduct transfer (38% vs. 18%, 31% vs. 15%, respectively). Throughout the present experiment, nine pairs of identical twins were successfully produced. This is the first report of the production of identical rat twins by separating two-cell embryos.     

Effect of various procedures on the viability of mouse embryos containing half the normal number of blastomeres

Half embryos produced from 8-cell or compacted stages were cultured in vitro for 1-2 days and transferred to oviducts or uteri of recipients at different stages of pseudopregnancy. The proportion of live fetuses was low (8-12%), except for one group (27%) in which half embryos were cultured in vitro for 1 day and transferred into oviducts on the 1st day of pregnancy. Monozygotic twin production rate, however, was low (1 out of 10) even in this group. Fetal weight on the 18th day of gestation was significantly lower after transfer of half embryos than after transfer of similarly treated but undivided embryos. Half embryos produced from the 2-cell stage were inserted into empty zonae, embedded in agar, cultured in ligated mouse oviducts for 2-4 days and transferred to oviducts of recipient females on the 1st day of pregnancy or pseudopregnancy. When twin embryos cultured for 2-3 days were transferred to pseudopregnant recipients together with control embryos, 4 sets of monozygotic twins and 5 singletons out of 10 sets of twin embryos were obtained on Days 18-19 of gestation, giving a survival rate of 65%.     

Embryos of different ages transferred to the rat oviduct enter the uterus at different times

Indirect evidence of embryo signalling to the oviduct was sought in rats by examining the transport of embryos of different ages. One-cell or four-cell embryos were transferred to the oviducts of recipient rats on Day 1 of pregnancy, and the number, condition, and location of native and transferred embryos was assessed on Day 4. To control for the effect of the presence of foreign embryos and excess number of eggs and the transfer procedure upon the fate of native embryos, other groups of rats were sham-operated or left undisturbed. Recipients had a mean number of ova significantly higher than controls. In controls and recipients of 1-cell embryos, the majority of eggs reached the morula stage and all of them were located in the oviducts. In those animals receiving 4-cell embryos, half of the eggs had reached the blastocyst stage and 28% were in the uteri (p less than 0.005). These results support the idea that advanced embryos can influence the timing of their entrance to the uterus in rats.