Rabbit Blastocysts Research Articles

183 ADDITION OF DIPHENYLENEIODONIUM OR DEHYDROEPIANDROSTERONE TO CULTURE MEDIA INHIBITS REACTIVE OXYGEN SPECIES PRODUCTION DURING THE EARLY CLEAVAGE STAGES OF IN VITRO-PRODUCED PORCINE EMBRYOS

Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), an enzyme required to catalyze the oxidation of NADPH to NADP during the metabolism of glucose via the pentose phosphate pathway (PPP), was considered as contributing to intracellular reactive oxygen species (ROS) production. Production of superoxide anion and H2O2 via NADPH oxidase has been reported on a rabbit blastocyst surface (Manes and Lai 1995 J. Reprod. Fertil. 104, 69–75). The objective of this study was to examine the effects on in vitro development and intracellular ROS content after the addition of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, or dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), to culture medium during the early embryonic development of in vitro-produced (IVP) porcine embryos. To confirm that these inhibitors lead to reduction in NADPH concentration in the embryo and hence likely to be inhibiting the PPP, a brilliant cresyl blue (BCB) test was performed on Day 2 (the day of insemination = Day 0) of culture. Porcine cumulus–oocyte complexes were matured and fertilized in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Prezumptive zygotes were then cultured in NCSU-37 supplemented with 5.5 mM glucose and DPI at concentrations of 0.5 or 1 nM or DHEA at concentrations of 10 or 100 �M (DPI-0.5, DPI-1, DHEA-10 and DHEA-100 groups, respectively) from Day 0 to Day 2 of culture. All of the embryos were cultured subsequently until Day 6 in NCSU-37 supplemented with only 5.5 mM glucose. Data were analyzed by ANOVA. On Day 6, the development to the blastocyst stage of embryos in DPI-0.5, DPI-1, DHEA-10, and DHEA-100 groups were 16.1, 17.6, 16.1, and 19.5%, respectively, which were not significantly different from that of the control group (17.5%) (n d 165 per group, 5 replicates). However, the mean cell number in blastocysts derived from DPI-1, DHEA-10, and DHEA-100 groups (40.8 � 2.3, 39.3 � 1.7, and 42.5 � 2.7, respectively) was significantly higher (P < 0.01) than those in the control (33.4 � 1.6) and DPI-0.5 (32.7 � 1.6) groups. At 20 min after an exposure to BCB, the percentage of BCB+ embryos in DPI-1, DHEA-10, and DHEA-100 groups (73.8, 79.9, and 77.8%, respectively) were significantly higher (P < 0.01) than those in the control and DPI-0.5 groups (42% and 53.9%, respectively) (n = 81-92 per group, 6 replicates), indicating that these two inhibitors effectively induce the reduction of NADPH concentration in the embryos. Moreover, the addition of DPI at 1 nM or DHEA at 10 or 100 �M significantly decreased the H2O2 content of Day 2 embryos as compared with control embryos (n = 48-53 per group, 7 replicates). These results suggest that the addition of either DPI or DHEA to the medium during the first 2 days of culture did not impair the development of the embryos to the blastocyst stage. Decrease of cellular ROS production in Day 2 embryos in this study is interpreted as a result of inhibition of the NADPH oxidase by DPI or of the G6PDH by DHEA.

Lineage-specific effects of polychlorinated biphenyls (PCB) on gene expression in the rabbit blastocyst

The ubiquitous toxic environmental contaminants polychlorinated biphenyls (PCBs) change gene expression in preimplantation embryos. Cell lineage-specific effects of PCB are not known. Rabbit day 6 blastocysts were exposed in vitro to low (0.1 ng/congener/mL medium) and high (1 μg) PCB levels of coplanar (PCB 77, 126, and 169) or non-coplanar PCBs (PCB 28, 52, 101, 118, 138, 153, and 180). Embryoblast (ICM) and trophoblast cells (TE) were separated and analyzed for transcriptional changes of PCB-and implantation-associated genes by semiquantitative RT-PCR. PCBs increased CYP 1A1 mRNA only in the ICM. CYP 1B1, VEGFR2, and COX-2 transcript numbers were elevated in both ICM and TE. Transcripts for HIF-1α were decreased in the ICM. No obvious differences in gene expression following exposure to coplanar and non-coplanar PCBs were detected. Our results show that transcriptional responses to PCBs differ between the cell lineages of the rabbit blastocyst, indicating that PCBs can influence the highly sensitive process of early mammalian development.

Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts

The addition of insulin during in vitro culture has beneficial effects on rabbit preimplantation embryos leading to increased cell proliferation and reduced apoptosis. We have previously described the expression of the insulin receptor (IR) and the insulin-responsive glucose transporters (GLUT) 4 and 8 in rabbit preimplantation embryos. However, the effects of insulin on IR signaling and glucose metabolism have not been investigated in rabbit embryos. In the present study, the effects of 170 nM insulin on IR, GLUT4 and GLUT8 mRNA levels, Akt and Erk phosphorylation, GLUT4 translocation and methyl glucose transport were studied in cultured day 3 to day 6 rabbit embryos. Insulin stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) Erk1/2 and levels of IR and GLUT4 mRNA, but not phosphorylation of the phosphatidylinositol 3-kinase-dependent protein kinase, Akt, GLUT8 mRNA levels, glucose uptake or GLUT4 translocation. Activation of the MAPK signaling pathway in the absence of GLUT4 translocation and of a glucose transport response suggest that in the rabbit preimplantation embryo insulin is acting as a growth factor rather than a component of glucose homeostatic control.

Effects of transferring in vitro-cultured rabbit embryos to recipient oviducts on mucin coat deposition, implantation and development

A mucin coat is deposited on rabbit embryos during passage through the oviduct; rabbit blastocysts cultured from the 1-cell stage in vitro have no mucin coat. When cultured blastocysts are transferred to recipients, the lack of mucin coat might account in part for subsequent failure of pregnancy. We have investigated the possibility that mucin coat deposition is induced following transfer of in vitro 72 h-cultured blastocysts to oviducts of asynchronous or synchronous recipients. One-cell embryos were collected by flushing oviducts 19-20 h post-coitus and were cultured in vitro for 72 h until they reached the blastocyst stage. The blastocysts were transferred to the oviducts of recipients that were synchronized either with the donors (synchronous) or 1 day later than the donors (asynchronous). They were recovered after 24-48 h and the mucin coat thickness and embryo degeneration rate were measured. The degeneration rate of blastocysts recovered from uteri of synchronous recipients was higher than that from asynchronous recipients (72.2% vs 40.0%). The mucin coats around embryos recovered from oviducts of asynchronous recipients after 48 h were thicker than those from synchronous recipients. More asynchronous recipients were pregnant and gave birth to more pups than synchronous recipients. These results indicate that the oviducts of asynchronous recipients secreted more mucin around the transferred embryos, causing higher rates of implantation of the in vitro-cultured blastocysts.

Vitrification of zona-free rabbit expanded or hatching blastocysts: a possible model for human blastocysts.

The purpose of this study was to test the effectiveness of one two-step (A) and two one-step (B1 and B2) vitrification procedures on denuded expanded or hatching rabbit blastocysts held in standard sealed plastic straws as a possible model for human blastocysts. The effect of blastocyst size was also studied on the basis of three size categories (I: diameter <200 micro m; II: diameter 200-299 micro m; III: diameter >/==" BORDER="0">300 micro m). Rabbit expanded or hatching blastocysts were vitrified at day 4 or 5. Before vitrification, the zona pellucida was removed using acidic phosphate buffered saline. For the two-step procedure, prior to vitrification, blastocysts were pre- equilibrated in a solution containing 10% dimethyl sulphoxide (DMSO) and 10% ethylene glycol (EG) for 1 min. Different final vitrification solutions were compared: 20% DMSO and 20% EG with (A and B1) or without (B2) 0.5 mol/l sucrose. Of 198 vitrified blastocysts, 181 (91%) survived, regardless of the vitrification procedure applied. Vitrification procedure A showed significantly higher re-expansion (88%), attachment (86%) and trophectoderm outgrowth (80%) rates than the two one-step vitrification procedures, B1 and B2 (46 and 21%, 20 and 33%, and 18 and 23%, respectively). After warming, blastocysts of greater size (II and III) showed significantly higher attachment (54 and 64%) and trophectoderm outgrowth (44 and 58%) rates than smaller blastocysts (I, attachment: 29%; trophectoderm outgrowth: 25%). These result demonstrate that denuded expanded or hatching rabbit blastocysts of greater size can be satisfactorily vitrified by use of a two-step procedure. The similarity of vitrification solutions used in humans could make it feasible to test such a procedure on human denuded blastocysts of different sizes.

A Role for αvβ3 Integrin During Implantation in the Rabbit Model

The study of implantation has been facilitated by the identification of specific biomarkers that are associated with uterine receptivity. The avb3 integrin is a cell surface adhesion receptor, whose expression has been shown to be elevated in the endometrium at the time of implantation in both humans and other mammalian species; however, the distribution of avb3 in the rabbit model is unknown. The rabbit has been shown to be an excellent model for the study of implantation. As an obligate ovulator, the timing of pregnancy can be precisely established, and embryonic attachment occurs through specialized trophoblastendometrial structures known as trophoblastic knobs. In the present study, the expression of avb3 integrin subunit in the rabbit uterus was examined by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridization. Expression of the avb3 integrin was examined in Day 6.5 embryos, flushed from pregnant does. Immunofluorescence demonstrated strong immunostaining on the rabbit blastocyst (Day 6.5). RT-PCR analyses showed higher levels of mRNA for b3 subunit at the implantation site, with reduced expression in nonimplantation sites and in nonpregnant adult and immature endometrium. Immunohistochemistry demonstrated little, if any, b3 immunoreactivity on the endometrial epithelium. In contrast, in situ hybridization showed expression of the b3 integrin subunit mRNA in the uterine myometrium and on the trophoblast. To further determine the functional significance of avb3 integrin expression during implantation, pregnant female rabbits that underwent ventral laparotomy on the morning of Day 6 received intrauterine injection of the following into the right uterine horn: 1) the monoclonal avb3 neutralizing antibody (LM609), 2) arg-gly-asp (RGD) hexapeptides (GRGDSP), 3) non-RGD hexapeptides (GRGESP), and 4) IgG isotype matched control antibody. The left horn served as a control and received only saline injections. A significant reduction in the number of implantation sites was observed in the horns receiving anti-avb3 antibody (P , 0.001) and the RGD peptides (P 5 0.03). In the rabbit, the avb3 integrin is present on the embryo and trophoblast and appears to be involved in early embryo-maternal interaction. embryo, implantation, uterus

A role for alphavbeta3 integrin during implantation in the rabbit model.

The study of implantation has been facilitated by the identification of specific biomarkers that are associated with uterine receptivity. The alpha(v)beta(3) integrin is a cell surface adhesion receptor, whose expression has been shown to be elevated in the endometrium at the time of implantation in both humans and other mammalian species; however, the distribution of alpha(v)beta(3) in the rabbit model is unknown. The rabbit has been shown to be an excellent model for the study of implantation. As an obligate ovulator, the timing of pregnancy can be precisely established, and embryonic attachment occurs through specialized trophoblast-endometrial structures known as trophoblastic knobs. In the present study, the expression of alpha(v)beta(3) integrin subunit in the rabbit uterus was examined by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridization. Expression of the alpha(v)beta(3) integrin was examined in Day 6.5 embryos, flushed from pregnant does. Immunofluorescence demonstrated strong immunostaining on the rabbit blastocyst (Day 6.5). RT-PCR analyses showed higher levels of mRNA for beta(3) subunit at the implantation site, with reduced expression in nonimplantation sites and in nonpregnant adult and immature endometrium. Immunohistochemistry demonstrated little, if any, beta(3) immunoreactivity on the endometrial epithelium. In contrast, in situ hybridization showed expression of the beta(3) integrin subunit mRNA in the uterine myometrium and on the trophoblast. To further determine the functional significance of alpha(v)beta(3) integrin expression during implantation, pregnant female rabbits that underwent ventral laparotomy on the morning of Day 6 received intrauterine injection of the following into the right uterine horn: 1) the monoclonal alpha(v)beta(3) neutralizing antibody (LM609), 2) arg-gly-asp (RGD) hexapeptides (GRGDSP), 3) non-RGD hexapeptides (GRGESP), and 4) IgG isotype matched control antibody. The left horn served as a control and received only saline injections. A significant reduction in the number of implantation sites was observed in the horns receiving anti-alpha(v)beta(3) antibody (P < 0.001) and the RGD peptides (P = 0.03). In the rabbit, the alpha(v)beta(3) integrin is present on the embryo and trophoblast and appears to be involved in early embryo-maternal interaction.

Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure

The study was designed to evaluate the efficiency of a modified (sealed) open pulled straw (mOPS) method for cryopreserving rabbit embryos by vitrification or rapid freezing. An additional objective was to determine whether the mOPS method could cause the vitrification of a cryoprotectant solution generally used in rapid freezing procedures. Two consecutive experiments of in vitro and in vivo viability were performed. In Experiment 1, the in vitro viability of rabbit embryos at the morula, compacted morula, early blastocyst and blastocyst stages was assessed after exposure to a mixture of 25% glycerol and 25% ethylene glycol (25GLY:25EG: vitrification solution) or 4.5M (approximately 25% EG) ethylene glycol and 0.25M sucrose (25EG:SUC: rapid freezing solution). Embryos were loaded into standard straws or mOPS and plunged directly into liquid nitrogen. The mOPS consisted of standard straws that were heat-pulled, leaving a wide opening for the cotton plug and a narrow one for loading embryos by capillarity. The embryos were aspirated into the mOPS in a column positioned between two columns of cryoprotectant solution separated by air bubbles. The mOPS were then sealed with polyvinyl-alcohol (PVA) sealing powder. The vitrification 25GLY:25EG solution became vitrified both in standard straws and mOPS, whereas the rapid freezing 25EG:SUC solution crystallized in standard straws, but vitrified in mOPS. The total number of embryos cryopreserved was 1695. Embryos cryopreserved after exposure to each solution in mOPS showed higher rates (88.2%) of survival immediately after thawing and removal of the cryoprotectant than those cryopreserved in 0.25ml standard straws (78.8%; P<0.0001). After culture, the developmental stage of the cryopreserved embryos significantly affected the rates of development to the expanded blastocyst stage. Regardless of the cryoprotectant used, lower rates of in vitro development were obtained when the embryos were cryopreserved at the morula stage, and higher rates achieved using embryos at blastocyst stages. Based on the results of Experiment 1, the second experiment was performed on blastocysts using the mOPS method. Experiment 2 was designed to evaluate the in vivo viability of cryopreserved rabbit blastocysts loaded into mOPS after exposure to 25GLY:25EG or 25EG:SUC. Embryos cryopreserved in mOPS and 25GLY:25EG solution gave rise to rates of live offspring (51.7%) not significantly different to those achieved using fresh embryos (58.5%). In conclusion, the modified (sealed) OPS method allows vitrification of the cryoprotectant solution at a lower concentration of cryoprotectants than that generally used in vitrification procedures. Rabbit blastocysts cryopreserved using a 25GLY:25EG solution in mOPS showed a similar rate of in vivo development after thawing to that shown by fresh embryos.

Stage-specific expression of the intermediate filament protein cytokeratin 13 in luminal epithelial cells of secretory phase human endometrium and peri-implantation stage rabbit endometrium.

In preparation for blastocyst implantation, uterine luminal epithelial cells express new cell adhesion molecules on their apical plasma membrane. Since one mechanism epithelial cells employ to regulate membrane polarity is the establishment of specific membrane-cytoskeletal interactions, this study was undertaken to determine if new cytokeratin (CK) intermediate filament assemblies are expressed in endometrial epithelial cells during developmental stages related to blastocyst implantation. Type-specific CK antibodies were used for immunocytochemical and immunoblot analyses of 1) intermediate filament networks of the endometrial epithelium during embryo implantation in rabbits and 2) proliferative and secretory phases of the human menstrual cycle. CK18, a type I CK found in most simple epithelia, was expressed in all luminal and glandular epithelial cells of both the human and rabbit endometrium at all developmental stages analyzed; it was also strongly expressed in trophectoderm of the implanting rabbit blastocyst. In contrast, CK13, another type I cytokeratin, exhibited a regulated expression pattern in luminal, but not glandular, epithelial cells of secretory phase human and peri-implantation stage rabbit endometrium. Furthermore, in the rabbit implantation chambers, CK13 was predominantly localized at the cell apex of luminal epithelial cells, where it assembled into a dense filamentous network. These data suggest that the stage-specific expression of CK13 and a reorganization of the apical intermediate filament cytoskeleton of uterine luminal epithelial cells may play important functions in preparation for the implantation process.

Proteolipid protein 2 mRNA is expressed in the rabbit embryo during gastrulation.

Differential display technology applied to rabbit blastocysts identified an mRNA that encodes a motif similar to that of the proteolipid protein PLP2/A4 of man, mouse and sheep. The open reading frame (456bp) has 88% amino acid identity to human PLP2/A4. The gene is maximally expressed at the beginning of gastrulation: in situ hybridizations exhibited a sickle-shaped area of labelling at the posterior pole of day 7 post-coitum embryos, which appeared at day 6.5 and decreased in size up to day 8. Weaker labelling was found in the extraembryonic mesoderm, in the anterior part of the primitive streak and in the trophoblast. Time and site of gene expression coincide with emerging morphogenetic activities at the posterior pole of the embryo at the beginning of gastrulation.

Social Capital for Health and Human Development

Crystalline inclusion bodies (CIB) may be found as prominent ultrastructural components of the trophoblast cells of rabbit blastocysts and of progestational uterine endometrium. In the work reported here we have sought to describe developmental steps in crystal formation, to correlate these events with embryonic age and to determine if the uterus is essential (either as a source or an environment) for crystal formation in the embryo. CIB, which are of a size and periodicity to make them appear to be clusters or packages of microtubules, are first detectable in embryos 4 days 6 h post coitum and by 41/2 days are well established in significant numbers. Another structural component, granular vesicles, may be seen in embryos as early as 21/2 days post coitum, and decrease in number during the same time the CIB are increasing. We believe that the CIB originate from the pre-existing granular vesicles and present electron micrograph evidence of crystal formation progressing from such vesicles. CIB formation does not occur in 41/2- to 5-day-old embryos which have been locked in the oviduct by a suture around the utero-tubal junction. However, when such tube-locked embryos are transplanted into the uterus, they develop crystals within 36 h thereafter. We conclude that the uterus is essential for CIB formation to occur in the rabbit embryo.

Successful pregnancy after transfer of rabbit blastocysts grown in vitro from single-cell zygotes

To establish successful pregnancy in rabbits after the transfer of blastocysts cultured in vitro for 72 h, pregnancy rates were compared according to synchronization methods of recipient and embryo transfer sites. Also, the effect of RDH (1:1:1 mixture of RPMI, DMEM and Ham's F10) medium with additives such as BSA and taurine was evaluated for developmental capacity and cell number. Developmental capacity and cell number were considered important for implantation. When we evaluated the relative survival of rabbit one-cell embryos after culture in Ham's F10, in RD or in RDH for 72 h, embryos cultured in RDH and RD developed much better than in Ham's F10. When the effects of BSA and taurine in RDH medium were tested for rabbit embryo development, BSA or taurine promoted transition to the blastocyst stage and increased cell numbers of cultured embryos in RDH medium. The BSA and taurine together in RDH medium had a synergistic effect on embryo development. By transferring cultured blastocysts to the oviduct of the recipient doe synchronized one day behind the donor, live-born pups were obtained successfully. These results demonstrated that rabbit blastocysts can develop to normal pups after in vitro culture and embryo transfer.

Receptor-mediated effects of chlorinated hydrocarbons.

This short review summarizes some recent findings on arylhydrocarbon (dioxin) receptor expression during early pregnancy in mammals. The arylhydrocarbon receptor is a ligand-activated transcription factor and was originally described as a mediator of the toxic effects of dioxins and other chlorinated hydrocarbons such as polychlorinated biphenyls. Orally administered polychlorinated biphenyls accumulate in uterine secretions, in the pre-implantation blastocyst, in the foetus and in the placenta. Coplanar polychlorinated biphenyls are known ligands of the arylhydrocarbon receptor. Deletion experiments indicate physiological roles of the arylhydrocarbon receptor during development and for the function of various organs. During early pregnancy, the arylhydrocarbon receptor is specifically expressed in pre-implantation embryos, during blastocyst differentiation and implantation, in the endometrial epithelium and in the decidua cells of the placenta. Coplanar polychlorinated biphenyls were found to be embryotoxic in low doses, i.e. in doses found in tissues of not specifically exposed individuals originating from xenobiotic environmental background contamination. In initial experiments, low-dose exposure of rabbit blastocysts to coplanar polychlorinated biphenyls in vitro did not induce transcriptional changes of the so-called arylhydrocarbon receptor gene battery. The embryological and toxicological implications of the findings are discussed.

Oxygen stress increases prolyl cis/trans isomerase activity and expression of cyclophilin 18 in rabbit blastocysts.

The peptidyl-prolyl cis/trans isomerase (PPIase) activity and the expression of cyclophilins were studied in 6-day-old rabbit preimplantation embryos cultured under physiological and increased oxygen concentrations of 5% and 20% O(2), respectively. The PPIase activity was completely inhibited by cyclosporin A (CsA). The inhibitor of FK506-binding proteins, rapamycin, had no effect on the PPIase activity, indicating that the PPIase activity in rabbit blastocysts originates from cyclophilins. Using CsA affinity chromatography, only one cyclophilin with a molecular mass of about 17.8 kDa was separated. The cDNA of rabbit cyclophilin was cloned and sequenced. Analysis of the 682-base pair cDNA revealed an open reading frame coding for a polypeptide of 164 amino acid residues with a molecular weight of 17.83 kDa. Homologies of 90% and 96% for the cDNA and amino acid sequence, respectively, to the human CyP18 were found, suggesting that the novel rabbit cyclophilin is a member of the CyP18 family (rabCyP18). The transcription level of rabCyP18 mRNA was 8.3 +/- 0.6 pg in 100 ng total RNA in noncultured blastocysts. In vitro culture with moderate oxygen stress (20% O(2)) resulted in a 1.5-fold increase in rabCyP18 transcription and an increased PPIase activity compared to that of blastocysts cultured with 5% O(2). Increase in transcription rate and PPIase activity by oxygen stress suggests an involvement of CyP18 in oxygen defense in rabbit preimplantation embryos.

Insulin and insulin-like growth factor-I promote rabbit blastocyst development and prevent apoptosis.

Insulin as well as insulin-like growth factor-I (IGF-I) promote early embryo development, and IGF-I binds to the coats of preimplantation rabbit embryos. As the IGF-I receptor is expressed from the morula stage onwards, the embryos are capable of responding to insulin and IGF-I, which is present in the oviductal and uterine secretions that surround them. The embryonic coats were removed to exclude any influence by IGF-I bound to the coats. The in vitro development of such embryos under classical conditions appears to be retarded. Addition of IGF-I (68 pM-6.8 nM) or insulin (68 nM-6.8 microM), however, promotes blastocyst formation. Embryo development under such conditions is not significantly different from that of embryos cultured with intact coats. In contrast, coat-free embryos cultured without IGF-I or insulin supplementation show apoptosis. Because IGF-I stimulates cell proliferation and prevents apoptosis, we investigated whether insulin or IGF-I may act as "survival factors" in preimplantation development. Therefore, apoptosis was induced by slight UV irradiation (254 nm wave length; 11.8 W/m2). Compared to the untreated controls, embryos displaying retarded development or degeneration were increased by 22% and 14%, respectively. Addition of IGF-I or insulin to the culture medium of UV-irradiated embryos improved [3H]thymidine incorporation and blastocyst formation significantly. By immunohistochemistry we could show that addition of insulin (0.68-68 nM) decreased apoptosis and increased cell proliferation in a dose-dependent manner, supporting blastocyst development significantly.

Demi-embryo production from hatching of zona-drilled bovine and rabbit blastocysts

It is known that the pregnancy rate resulting after transfer of bisected embryos is lower than after transfer of whole embryos. The main reason is the reduced cell number in the demi-embryo which is less than 1 2 of that in the intact embryo, since a number of blastomeres is damaged as a result of the procedure used in conventional embryo splitting. The aim of our experiment was to develop a non-invasive procedure which would limit cell losses during microsurgery. The experiment was carried out on bovine IVM-IVF embryos at middle, late and expanded blastocyst stage and rabbit embryos at late blastocyst stage cultured in vitro from in vivo produced zygotes. The zona pellucida of these embryos was drilled on the line between the inner cell mass and the trophoblast using a glass microneedle (≤ 2 μm) or micropipette (≤ 30 μm). The procedure resulted in expulsion of the blastocyst through the perforation and formation of an incomplete demi-embryo configuration, connected by a very thin cell bridge (figure eight in shape). To separate the parts of the embryo, the cell bridge was cut using a glass microneedle. During the separation only a few cells were damaged. As a result of the procedure 4 20 (20.0%), 48 144 (33.3%) and 3 40 (7.5%) middle, late and expanded blastocysts hatched according to the pattern described. The developed procedure could be considered as a non-invasive alternative to conventional embryo splitting.

Morphology and/or hatching ability of in vitro produced bovine embryos is no reliable indicator of inner cell mass cell number

This study documents the spatial and temporal distribution of Oct-4, Cdx-2 and acetylated H4K5 (H4K5ac) by immunocytochemistry staining using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where the signal was similarly weak in both the ICM and TE cells. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts, and became evident in the TE cells exclusively afterwards. A consistently strong H4K5ac signal was observed in the TE cells in all blastocyst stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Double staining of H4K5ac with either Oct-4 or Cdx-2 on embryos at different blastocyst stages confirmed these findings. This work suggests that day 4 is a critical timing for lineage formation in rabbit embryos. A combination of Oct-4, Cdx-2 and H4K5ac can be used as biomarkers to identify different lineage cells in rabbit blastocysts.There are very limited studies on key transcription factors and epigenetic programming events in preimplantation-stage rabbit embryos. Previously we reported the spatial and temporal distribution of Oct-4 and acetylated H4K5 (H4K5ac) in-vitro-cultured rabbit embryos. In the present study, we report the similar distribution patterns of Oct-4 and H4K5ac using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where such signal was similarly weak in both the ICM and the TE cells. A consistent strong H4K5ac signal was observed in the TE cells in all stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Importantly, we also report, as far as is known for the first time, the distribution pattern of Cdx-2 in rabbit embryos. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts. It became evident in the TE cells exclusively afterwards. The present study provides novel information on key transcription factors and epigenetic events during rabbit embryo development, and demonstrates that a combination of Oct-4, Cdx-2 and H4K5ac could be used as biomarkers to identify different lineage cells in rabbit blastocysts.

Uterine fibroblast growth factor-2 and embryonic fibroblast growth factor receptor-1 at the beginning of gastrulation in the rabbit.

Fibroblast growth factor-2 (FGF-2) induces gastrulation of rabbit blastocysts in vitro and is present in the uterine secretion at day 6 after mating. The following study was made in order to show if changes in the uterine FGF-2 concentration or in the FGF receptor concentration of the embryonic tissues point to a regulation of this event. By the use of the ELISA technique and immunohistochemistry, FGF-2 concentration was determined in the endometrial tissue, uterine secretion and blastocyst between day 4 and day 8 of pregnancy, in the uterine secretion after induction of pseudopregnancy, in day 6 blastocysts after in vitro culture, and FGF immunoreactivity was localized in the endometrial tissue. FGF receptor-1 (FGFR-1) concentration was examined correspondingly in the blastocyst. Cross-linking experiments using 125I-FGF-2 were done to identify binding proteins in the blastocyst. In the uterine secretion, FGF-2 was constantly high up to day 6.5 but showed an increase thereafter. Similar values in pseudopregnant uterine secretions indicated that the growth factor was of uterine origin. It was probably synthesized by the uterine epithelium as shown by immunohistochemistry. Under culturing conditions, the blastocyst produced small amounts of FGF-2. In the blastocyst, FGFR-1 as well as binding of 125I-FGF-2 showed a dramatic increase from day 6.0 to day 6.5, coinciding with the onset of gastrulation. Receptor antigenicity was located in the embryonic disc at day 6.5 and day 7.0. Two binding proteins of about 200 and 130 kDa were found by cross-linking. The results indicate that a regulation of growth factor influence on embryonic differentiation is more probable via expression of the embryonic receptor than via differential release of the uterine growth factor.

Successful implantation of in vitro cultured rabbit embryos after uterine transfer: a role for mucin.

The functional role of the mucin layer for development of rabbit embryos was examined by uterine transfer of embryos with different thicknesses of mucin. Embryos collected at various intervals after human chorionic gonadotropin (hCG) injection were cultured until 90 hr post-coitum (p.c.) and transferred to the uterus of synchronized recipients. When embryos collected at 20 or 25 hr p.c. were used for transfer, no implantation occurred. By contrast, embryos collected at 35 or 40 hr p.c. developed to term at high rates (53 and 80%, respectively). The thickness of the mucin layer on the embryos was different between these two groups. Embryos collected before 25 hr p.c. have less than 11.2 +/- 0.2 microns of thickness of mucin and embryos collected after 35 hr p.c. have more than 34.3 +/- 5.5 microns. To examine whether mucin deposition is required for in vitro cultured rabbit blastocysts to continue development after uterine transfer, embryos were collected at 20 hr p.c., cultured for 60 or 70 hr in vitro, and then temporarily transferred to the oviducts of recipient does to add mucin. These embryos were recovered from the oviducts at 24 hr after transfer, classified according to the thickness of mucin deposition, and transferred again to the uterus of synchronized recipients. Twenty live young were obtained from 67 embryos with a 20-40 microns thick mucin layer. No live young were obtained from 57 embryos with less than a 20 microns thick mucin. The thickness of the mucin layer appears to be an important factor for successful implantation of rabbit embryos.

What is the relationship between the metabolism of preimplantation embryos and their developmental competence?

What is the relationship between the metabolism of preimplantation embryos and their developmental competence?