Autoradiographic studies on the distribution of labeled maternal RNA in early mouse embryos

Maternal RNA of mouse eggs and embryos was labeled by exposure of growing ovarian oocytes to 3H-uridine in vivo 8 to 16 days before ovulation and fertilization. Labeled embryos from the 1-cell stage to the blastocyst stage were collected, fixed, and autoradiographs of plastic sections prepared. The observed grain density was similar in the pronuclei and in the cytoplasm of 1-cell embryos. Knowing the volumes of nucleus and cytoplasm, it was determined that 3% of the maternal RNA was found in the pronuclei. It is suggested that some of this nuclear RNA may be stable small nuclear RNAs (e.g. U1 RNA) retained from the germinal vesicle stage through meiotic maturation. During the 2-cell stage and beyond, maternal RNA is degraded and labeled precursor is reincorporated into nuclear RNA, making it difficult to accurately quantitate the amount of nuclear maternal RNA. It is known that about one third of the total maternal RNA is lost between the 8-cell and blastocyst stages. It was found that cytoplasmic grain densities in inner and outer cells of the morula and blastocyst were not significantly different. Thus, the loss of maternal RNA does not proceed more rapidly in the differentiating trophoblast than in the inner cell mass.     

Influences of retrieval stages and glutathione addition on post-thaw viability of quick frozen mouse morula during in vitro culture

Effects of the embryo retrieval stages and addition of glutathione (GSH) on post-thaw development of mouse morula were evaluated in 2 consecutive experiments. In the first experiment, 1-, 2-, 3- to 4- and 5- to 8-cell stage embryos were collected and cultured to the morula stage in Whitten's medium containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). The development rate of 1-cell embryos to the morula stage was lower than that of the other stages (P<0.01). The post-thaw development rate of the morulae obtained from in vitro culture of 1-, 2-, 3- to 4-, and 5- to 8-cell embryos and from in vivo embryos (control) to the blastocyst stage was 55.5, 84.9, 87.4, 90.1 and 90.8%, respectively. The post-thaw development rate of morula obtained from in vitro produced 1-cell embryos was significantly lower than from the other stages or from the in vivo counterparts (P<0.0001). In Experiment 2, the impact of GSH supplementation of the culture medium in the presence or absence of EDTA was evaluated for embryo development to the morula stage and post-thaw survival, using in the 2 x 2 factorial design. Although EDTA supplementation increased development rates to the morulae (P<0.01) stage, GSH did not have an influence on morula development. However, the presence of either GSH or EDTA in the culture medium supported development to the blastocyst stage (P<0.01) of in vitro produced morulae. These data demonstrate that 1-cell embryos from a blocking-strain mouse cultured in vitro to the morula stage have a lower development rate following freezing and thawing than embryos collected at the 2-cell or later stages. Addition of EDTA or GSH, individually or in combination, to the culture medium may improve the development rate of morula to blastocyst stage following cryopreservation.     

Cyclooxygenases and prostaglandin E synthases in preimplantation mouse embryos

Prostaglandin E2 (PGE2) is shown to be essential for female reproduction. Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandin synthesis from arachidonic acid and exists in two isoforms: COX-1 and COX-2. Prostaglandin E synthase (PGES) is a terminal prostanoid synthase and can catalyse the isomerization of the COX product PGH2 to PGE2, including microsomal PGES-1 (mPGES-1), cytosolic PGES (cPGES) and mPGES-2. This study examined the protein expression of COX-1, COX-2, mPGES-1, cPGES and mPGES-2 in preimplantation mouse embryos by immunohistochemistry. Embryos at different stages collected from oviducts or uteri were transferred into a flushed oviduct of non-pregnant mice. The oviducts containing embryos were paraffin-embedded and processed for immunostaining. COX-1 immunostaining was at a basal level in zygotes and a low level at the 2-cell stage, reaching a high level from the 4-cell to blastocyst stage. COX-2 immunostaining was at a low level at the zygote stage and was maintained at a high level from the 2-cell to blastocyst stages. A low level of mPGES-1 immunostaining was observed from the zygote to 8-cell stages. The signal for mPGES-1 immunostaining became stronger at the morula stage and was strongly seen at the blastocyst stage. cPGES immunostaining was strongly observed in zygotes, 2-cell and 8-cell embryos. There was a slight decrease in cPGES immunostaining at the 4-cell, morula and blastocyst stages. mPGES-2 immunostaining was at a low level from the zygote to morula stages and at a high level at the blastocyst stage. We found that the COX-1, COX-2, mPGES-1, cPGES and mPGES-2 protein signals were all at a high level at the blastocyst stage. PGE2 produced during the preimplantation development may play roles during embryo transport and implantation.     

Effect of lipid polarization by centrifugation at different developmental stages on post-thaw survival of bovine in vitro produced 16-cell embryos

The developmental rate to the blastocyst stage of frozen-thawed bovine in vitro produced embryos at stages earlier than Day 6 morula is not sufficiently high for practical utilization. The present study was undertaken to determine the effect of polarization of lipid droplets in the cytoplasm of bovine in vitro produced embryos from zygotes to the 8-cell stage, by centrifugation without following micromanipulation, on the survival rate of Day 4 16-cell embryos. After centrifugation at 15,500 x g in medium containing cytochalasin D, embryos were cultured to the 16-cell stage, classified as either mostly or partially delipidated by degree of lipid droplet removal, and then frozen. Embryos centrifuged at the 2-cell stage developed to the 16-cell stage similarly to those centrifuged at the 8-cell stage. The developmental rate to blastocysts after freezing of the mostly delipidated 16-cell embryos centrifuged at the 2-cell stage was higher than that of those centrifuged at the zygote stage, those that were partially delipidated at the 2-cell stage, and those that were not centrifuged. The results demonstrate that polarization of lipid droplets at the 2-cell stage by centrifugation without micromanipulation improved the survival rate of mostly delipidated 16-cell embryos after freezing.     

Effect of egg composition on the developmental capacity of androgenetic mouse embryos.

Analysis of the developmental capacities of androgenetic and gynogenetic mouse embryos (bearing two paternal or two maternal pronuclei, respectively) revealed a defect in blastocyst formation of androgenetic, but not gynogenetic, embryos that was a function of the maternal genotype. Androgenetic embryos constructed using fertilized eggs from C57BL/6 or (B6D2)F1 mice developed to the blastocyst stage at frequencies similar to those previously reported, whereas androgenetic embryos constructed with fertilized eggs from DBA/2 mice developed poorly, the majority failing to progress beyond the 16-cell stage and unable to form a blastocoel-like cavity, regardless of whether the male pronuclei were of C57BL6 or DBA/2 origin. This impaired development was observed even in androgenetic embryos constructed by transplanting two male pronuclei from fertilized DBA/2 eggs to enucleated C57BL/6 eggs, indicating that the defect cannot be explained as the lack of some essential component in the DBA/2 cytoplasm that might otherwise compensate for androgeny. Rather, the DBA/2 egg cytoplasm apparently modifies the incoming male pronuclei differently than does C57BL/6 egg cytoplasm. Several specific alterations in the protein synthesis pattern of DBA/2 androgenones were observed that reflect a defect in the regulatory mechanisms that normally modulate the synthesis of these proteins between the 8-cell and blastocyst stages. These results are consistent with a model in which cytoplasmic factors present in the egg direct a strain-dependent modification of paternal genome function in response to epigenetic modifications (genomic imprinting) established during gametogenesis and indicate that preimplantation development can be affected by these modifications at both the morphological and biochemical levels.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Effects of oxygen tension and humidity on the preimplantation development of mouse embryos produced by in vitro fertilization: analysis using a non-humidifying incubator with time-lapse cinematography

To examine the effects of oxygen tension and humidity on early embryonic development, the preimplantation development of mouse embryos produced by in vitro fertilization was assessed by time-lapse cinematography to evaluate morphokinetic development with higher precision. Zygotes were produced from spermatozoa and oocytes from ICR mice and cultured in KSOM under low or high oxygen tension in a non-humidified incubator with time-lapse cinematography (CCM-iBIS). The developmental rates of embryos to the 4-cell and blastocyst stages under lower oxygen tension in CCM-iBIS were significantly higher than those under higher oxygen tension in CCM-iBIS. Ninety-six hours after insemination, a large number of embryos cultured under low oxygen tension developed to the hatching blastocyst stage. Embryonic development was more synchronized under lower oxygen tension. Non-humidified cultures did not affect embryonic development. On average, mouse embryos cultured at lower oxygen tension reached 2-cell at 18 h, 3-cell at 39 h, 4-cell at 40 h, initiation of compaction at 58 h, morula at 69 h, and blastocyst at 82 h after insemination. In conclusion, lower oxygen tension better supports preimplantation development of mouse embryos fertilized in vitro, and non-humidified culture conditions do not influence the embryonic development in vitro.     

Assessment of cytoplasmic effects on the development of mouse embryonic nuclei transferred to enucleated zygotes

In this study, cytoplasmic effects on the development of nuclear transplant embryos were examined. In addition, the production of offspring from nuclear transplant embryos was attempted. Nuclei from cleavage-stage embryos were transplanted to enucleated zygotes at different cell cycle stages and with different cytoplasmic volumes. A greater developmental rate to the blastocyst stage was observed in reconstituted late stage zygotes that received nuclei from late 2-cell stage embryos than in early stage zygotes (46.3% vs. 16.9%). A further increase in developmental rate to the blastocyst stage (85.5%) and in cell number was obtained in reconstituted late stage zygotes with reduced cytoplasmic volume. However, developmental potential of nuclei from 4- and 8-cell stage embryos was very limited, although they were transferred to enucleated late stage zygotes with reduced cytoplasm. After the transfer of blastocysts derived from nuclear transplant embryos to recipient females, live young were obtained from reconstituted embryos that received nuclei from late 2-cell stage embryos (28.6%). These results confirm that the development of nuclear transplant embryos can be affected by recipient cell cycle stage and cytoplasmic volume. Furthermore, the nuclei from late 2-cell stage embryos in which activation of the embryonic genome had occurred can be reprogrammed to a certain extent when transplanted into enucleated zygotes, especially late stage zygotes with reduced cytoplasmic content.     

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.     

The absence of direct effects of prostaglandin F2α on preimplantation mouse embryos in vitro

Preimplantation mouse embryos were cultured in vitro in concentrations of PGF_2α ranging from 5 nanograms/ml to 100 micrograms/ml. Embryos were cultured at the 2-cell stage, 4-cell stage, 8-cell stage and the morula stage and observed for normal development to the blastocyst stage. There was no significant difference (P < .1) between the number of blastocysts attained in the control group and those in the experimental groups. After reaching the blastocyst stage, the embryos were transferred to the uteri of pseudopregnant recipient females. Again, there was no significant difference (P < .3) between controls and PGF_2α treated embryos in the number developing to term. These data suggest that exposure to PGF_2α does not result in teratogenic effects or early death in preimplantation embryos.     

应用FRAP技术分析小鼠嵌合体和正常胚胎发育中细胞间隙连接介导通讯

应用激光扫描共聚焦显微镜的光漂白恢复(fluorescence redistribution after photobleaching,FRAP)技术分析小鼠嵌合体胚胎和正常胚胎的卵裂球之间细胞间隙连接介导通讯(gap junctional inter-cellular communica-tion,GJIC),结果发现:8-细胞期嵌合体胚胎的光漂白恢复率(24.3%)明显低于正常胚胎(64.2%),提示GJIC的降低可能是影响嵌合体胚胎发育率降低的因素之一;囊胚的光漂白恢复率也较低(22.7%),提示随着细胞分化,GJIC的水平有所降低。     

Development of sheep preimplantation embryos in media supplemented with glucose and acetate

Two experiments were conducted to examine the effect of supplemental glucose (G; 1.5 mM) and/or acetate (A; 0.5 mM) on the development of early sheep embryos to blastocysts when cultured in vitro in glucose-free synthetic oviductal fluid (SOF) + sheep serum or bovine serum albumin (BSA). In Experiment 1, 2- to 4-cell, 8- to 16-cell and >16-cell embryos were cultured in SOF, SOF+G, SOF+A or SOF+G+A. All media were supplemented with 10% sheep serum. In addition, embryos were cultured in either microdrops under polysiloxane oil or in multiwell dishes. Overall, development to the blastocyst stage was 3%, 30% and 68% for 2- to 4-cell, 8- to 16-cell and >16-cell stages, respectively, suggesting that an 8-cell developmental block existed under our culture conditions. Glucose supplementation had little effect on embryo development, and no overall effect was observed from the addition of acetate. In Experiment 2, 8- to 16-cell embryos were cultured in SOF or SOF+G, both supplemented with BSA. Development to the blastocyst stage was 25% and 18%, respectively. The results show that the presence of glucose or acetate did little to enhance embryonic development in our incubation systems. Further work is required to evaluate fully the energy requirements for development of the early sheep embryo.     

Culture of early stage ovine embryos to blastocyst enhances survival rate after cryopreservation

The current study assessed both the effects of in vitro culture and developmental stage of early stage in vivo produced ovine embryos on their ability to survive cryopreservation. Early stage embryos (n=226) were recovered from the oviduct, at different days of the early luteal phase, at three different developmental stages: 2- to 4-cell, 5- to 8-cell and 9- to 12-cell. For each stage, half of the embryos were cultured to the blastocyst stage and frozen thereafter (CF), while the remainder was frozen just after recovery (EF). A third experimental group (BF; n=43) included blastocysts obtained from the uterus and frozen immediately after recovery. Embryo viability post-thawing was determined by assessing their rate of development to the hatched blastocyst stage following in vitro culture. Culture negatively affected embryo viability, since survival rate was higher in blastocysts obtained from the uterus than in those from culture (83.7% versus 66.1%; P<0.05); also the cryosurvival of cultured embryos was lower when the timing of blastocyst formation was extended (P<0.01). However, survival following freezing-thawing of early developmental stages was significantly lower when compared to viability of their counterparts cultured to the blastocyst stage (23.1% versus 66.1%, P<0.0001). In conclusion, our results indicate that, despite the deleterious effects of culture per se, the culture of early in vivo produced ovine embryos to the blastocyst stage prior to be frozen improves their survival after thawing.     

In vitro culture of bovine IVM-IVF embryos: Cooperative interaction among embryos and the role of growth factors

The objective of this study was to determine whether there is a cooperative interaction among bovine embryos during in vitro culture. Furthermore, culture medium was supplemented with the growth factors, epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), to determine if these factors had a stimulatory effect on bovine embryo development similar to that seen in mouse development. In vitro matured - in vitro fertilized bovine embryos (2- to 8-cell) were cultured singly and in groups of five in 25 mul of medium (CR1 + amino acids + fatty acid-free bovine serum albumin) with or without EGF and TGF-beta1. Bovine embryos cultured in groups had a significantly higher rate of development to the blastocyst stage than embryos cultured singly. Neither EGF (10 ng/ml) nor TGF-beta1 (2 ng/ml) affected blastocyst development, hatching or the cell number of the embryos cultured in groups. Epidermal growth factor stimulated hatching of embryos cultured singly from the 8-cell stage, but did not significantly affect blastocyst development.     

Aberrant profile of gene expression in cloned mouse embryos derived from donor cumulus nuclei

Somatic cell nuclear transfer has successfully been used to clone several mammalian species including the mouse, albeit with extremely low efficiency. This study investigated gene expression in cloned mouse embryos derived from cumulus cell donor nuclei, in comparison with in vivo fertilized mouse embryos, at progressive developmental stages. Enucleation was carried out by the conventional puncture method rather than by the piezo-actuated technique, whereas nuclear transfer was achieved by direct cumulus nuclear injection. Embryonic development was monitored from chemically induced activation on day 0 until the blastocyst stage on day 4. Poor developmental competence of cloned embryos was observed, which was confirmed by lower cell counts in cloned blastocysts, compared with the in vivo fertilized controls. Subsequently, real-time polymerase chain reaction was used to analyze and compare embryonic gene expression at the 2-cell, 4-cell, and blastocyst stages, between the experimental and control groups. The results showed reduced expression of the candidate genes in cloned 2-cell stage embryos, as manifested by poor developmental competence, compared with expression in the in vivo fertilized controls. Cloned 4-cell embryos and blastocysts, which had overcome the developmental block at the 2-cell stage, also showed up-regulated and down-regulated expression of several genes, strongly suggesting incomplete nuclear reprogramming. We have therefore demonstrated that aberrant embryonic gene expression is associated with low developmental competence of cloned mouse embryos. To improve the efficiency of somatic cell nuclear transfer, strategies to rectify aberrant gene expression in cloned embryos should be investigated.This project was funded mainly by the National University of Singapore (grant number: R-174-000-065-112/303).