Early Blastocyst Stage Research Articles

Completion of Meiosis I of preovulatory oocytes and facilitation of preimplantation embryo development by glial cell line-derived neurotrophic factor

Optimal maturation of oocytes and successful development of preimplantation embryos is essential for reproduction. We performed DNA microarray analyses of ovarian transcripts and identified glial cell line-derived neurotrophic factor (GDNF) secreted by cumulus, granulosa, and theca cells as an ovarian factor stimulated by the preovulatory LH/hCG surge. Treatment of cumulus–oocyte complexes with GDNF enhanced first polar body extrusion with increase in cyclin B1 synthesis and the GDNF actions are likely mediated by its receptor GDNF family receptor-alpha1 (GFRA1) and a co-receptor ret proto-oncogene (Ret), both expressed in oocytes. However, treatment with GDNF did not affect germinal vesicle breakdown and cytoplasmic maturation of oocytes. During the preimplantation stages, GDNF was expressed in pregnant oviducts and uteri, whereas GFRA1 and Ret were expressed in embryos throughout early development with an increase after the early blastocyst stage. In blastocysts, both GDNF and GFRA1 were exclusively localized in trophectoderm cells, whereas Ret was detected in both cell lineages. Treatment with GDNF promoted the development of two-cell-stage embryos into blastocysts showing increased cell proliferation and decreased apoptosis mainly in trophectoderm cells. Our findings suggest potential paracrine roles of GDNF in the promotion of completion of meiosis I and the development of early embryos.

Does IVF with PGD and blastocyst transfer affect offspring's sex-ratio?

Almost 30 years since the first IVF newborn, conception through ART should still be followed for unknown consequences. A shifting of male to female ratio following IVF and blastocyst transfer has been recently suggested. We used PGD results of gender to investigate whether the sex ratio was altered and if gender affects embryo development in vitro, implantation, and eventually delivery rate. Retrospective data analysis. 143 consecutive cycles of IVF with PGD for aneuploidy were analyzed. 48.2% (466/966) of the embryos were normal for 5–9 chromosomes and the gender was known. In vitro embryo development until day 5–6, implantation rate, miscarriage rate, and delivery rate by gender were evaluated. 46.8% of the embryos tested by PGD were diagnosed as males (n = 218) and 53.2% (n = 248) as females (P=NS). There were no significant differences in their potential development to early or advanced blastocyst stages. 111 male blastocysts and 129 female blastocysts were transferred. Implantation rates were comparable, 32.4% and 31%, respectively, and 78% of the implanted embryos in each group were delivered. Male and female embryos generated by IVF-PGD treatment have similar developmental, implantation and delivery potential. This is the first study to use PGD results to follow in vitro development and implantation of embryos by gender. Our data suggest that ART with extended blastocyst culture is not associated with sex-ratio imbalance.

Sequential co-culture of preimplantation embryos with oviductal epithelial cells first is more effective than that with endometrial epithelial cells first in human

To address the physiological relevance of the process that fertilized embryos gradually move from the oviduct to the uterine cavity during their development into the blastocyst. We studied preimplantation embryo development in vitro using two sequential co-culture conditions. One hundred and two surplus frozen embryos (G2 or G3 based on the Veeck's classification) in the IVF program were used for the co-culture. Such embryos at 4 cell stage were randomly allocated into two groups as follows: the oviductal (2 days)-endometrial (2 days) sequential co-culture group (OEG) and the endometrial (2 days)-oviductal (2 days) sequential co-culture group (EOG). SV40TAg-transfected oviductal or endometrial epithelial cell lines were used for co-culture. Cells were cultured in RPMI1640 supplemented with 10% FCS. The medium was replaced 24 h before co-culture was started. Only cells at 80% confluence in 6-well plates (Falcon) were used for embryo co-culture. Conditioned medium for 48 h cell culture was assayed for bFGF, HGF (hepatocyte growth factor), TGF-β1, IGF-I, IL-1β, IL-4, IL-6 and IL-8 with EIA (for bFGF, TGF-β1, IL-1β, IL-6) or IRMA (for IGF-I) or ELISA (for IL-4, IL-8 and HGF). Differences in embryonic development between OEG and EOG were compared with Fisher's exact test. Differences in concentrations between culture conditions were compared with two-tailed t-test. Of 46 and 43 frozen-thawed embryos with good quality (3 or 4 blastomeres were viable after thawing) in OEG and EOG, 31 (67.4%) and 26 (60.5%) embryos reached the morula stage (N.S.), 23 (50.0%) and 12 (27.9%) embryos reached the early blastocyst stage (P<0.05), and 8 (17.4%) and 6 (14.0%) reached the expanded blastocyst stage (N.S.), respectively. IL-1β and HGF concentrations were significantly higher in the conditioned medium of the oviductal epithelial cell culture. IL-6 and IL-8 concentrations were significantly higher in the conditioned medium of the endometrial epithelial cell culture. IGF-I and IL-4 were not detected in each assay. Although both oviduct and uterus are developed from the Müllerian tubercle, each tissue might play differentiated roles in the preimplantation embryo growth. We could not show clear difference in the development into expanded blastocyst stage possibly due to the limitation of our co-culture conditions that did not include hormonal regulation or stromal cells.

Regulation of preimplantation embryo development by brain-derived neurotrophic factor

Hormonal factors secreted by embryos and reproductive tracts are important for successful development of preimplantation embryos. We found expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) transcripts at its highest levels in the blastocyst stages. The transcripts for their receptor, TrkB, were detectable throughout the early embryonic stages with an increase after the early blastocyst stage. Both BDNF and TrkB are expressed in trophectoderm cells, whereas ligand-binding studies indicated specific binding of BDNF to trophectoderm cells. Furthermore, BDNF and NT-4/5 were produced in pregnant oviducts and uteri. Treatment with BDNF promoted the development of two-cell-stage embryos into blastocysts showing increased proliferation and decreased apoptosis. The effects of BDNF were blocked by the TrkB ectodomain or a Trk receptor inhibitor, K252a. Studies using specific inhibitors demonstrated the roles of the PI3K, but not the ERK, pathway in mediating BDNF actions. Under high-density embryo cultures, treatment with the TrkB ectodomain or K252a alone also inhibited embryonic development and survival, suggesting potential autocrine actions of BDNF produced by the embryo. In vivo experiments further demonstrated that K252a treatment suppressed early embryo development by inhibiting blastocyst cell numbers, and increasing blastocyst apoptosis. Our findings suggested that BDNF signaling plays important paracrine roles during blastocyst development by promoting the development of preimplantation embryos.

The effects of time of first cleavage, developmental stage, and delipidation of nuclear-transferred porcine blastocysts on survival following vitrification

The effect of removing cytoplasmic lipid droplets (delipidation) at the 2-cell and developmental stages on the survival of porcine somatic cell nuclear-transferred blastocysts developed from the enucleated oocytes receiving somatic cells from kidney of an adult female after cryopreservation was examined. Vitrification was performed using the Cryoloop method with a small volume of medium (0.5 μl). To select 2-cell embryos with a high potential to develop into blastocysts, the relationship between the timing of the first cleavage and the developmental potential was examined. The potential of nuclear-transferred oocytes to develop into blastocysts in the intermediate-cleavage group (20–24 h after activation, 25%) was slightly or significantly ( P < 0.05) higher than that in fast-cleavage (<20 h after activation, 13%) and slow-cleavage groups (>24 h after activation, 5%). Most non-delipidated blastocysts did not survive after thawing (0% for early-stage and 9% for advanced-stage blastocysts), but the survival rate of delipidated blastocysts 48 h after culture (54% and 72%, respectively) was not significantly different from that of non-vitrified blastocysts (80% and 92%, respectively). The survival rate of advanced-stage blastocysts after vitrification was slightly higher than that of early-stage blastocysts. The present study demonstrates that somatic cell nuclear-transferred porcine blastocysts developed from embryos selected at the 2-cell stage can be preserved by vitrification with a small volume of medium if the lipid droplets of the embryos are first removed.

Targeted disruption of the murine large nuclear KIAA1440/Ints1 protein causes growth arrest in early blastocyst stage embryos and eventual apoptotic cell death

The KIAA1440 protein contains no significant domains that allow for a prediction of its function, despite the fact that it is an extremely large protein comprising 2222 amino acids. In our current study, we show that the developing KIAA1440 −/− mouse embryo in a pure ICR background arrests its growth at the early blastocyst stage, whereas the majority of the KIAA1440 −/− embryos of mixed genetic backgrounds do not progress beyond the morula stage, approximately 0.5 days earlier. KIAA1440 −/− embryos exhibited no abnormal localization of E-cadherin or β-catenin and no obvious compaction abnormalities at the morula stage. In addition, E3.5 KIAA1440 −/− embryos are not viable even in in vitro cultures. Both TUNEL and FAM-caspase-3/7 assays performed on these embryos consistently showed that E3.5 KIAA1440 −/− embryos had activated caspase-3/7, which then induced an apoptotic response predominantly within the inner cell mass of the blastocyst. Moreover, qRT-PCR analysis showed that KIAA1440 −/− embryos had increased levels of the unprocessed, primary U2 snRNA transcript but decreased levels of the mature U2 snRNA transcript compared to heterozygotes. The impaired processing of U2 snRNA and the predominantly nuclear localization of KIAA1440 protein is also very consistent with recently reported data showing that it is the largest subunit of the integrator complex, which mediates U1 and U2 snRNA 3′-end processing. Large nuclear KIAA1440/Ints1 is thus suggested to play non-redundant roles in the cell such as the formation of a scaffold for the assembly of the integrator complex.

Effect of genistein on mouse blastocyst development in vitro

To examine the cytotoxic effects of genistein, an isoflavone compound, on early postimplantation embryonic development in vitro. Mouse blastocysts were incubated in medium with or without genistein (25 or 50 micromol/L) or daidzein (50 micromol/L) for 24 h. Cell proliferation and growth was investigated by dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and apoptotic or necrotic cells were visualized by Annexin-V and propidium iodide (PI) staining. Implantation and postimplantation development of embryos were measured by in vitro development analysis. TUNEL staining and Annexin-V/PI staining showed that genistein dose-dependently increased apoptosis in mouse blastocysts, while daidzein, another soy isoflavone, had no such effect. The pretreatment of the blastocysts with genistein caused fewer cells than the control group and this effect was primary in the inner cell mass. The genistein-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer genistein-pretreated embryos reached the later stages of embryonic development versus the controls, with many of the former embryos dying at relatively early stages of development. In addition, genistein treatment decreased the development of morulas into blastocysts, and dietary genistein was found to induce cell apoptosis and decrease cell proliferation in an animal assay model of embryogenesis. Our results collectively indicate that genistein treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death in vitro, while dietary genistein appears to negatively affect mouse embryonic development in vivo by inducing cell apoptosis and inhibiting cell proliferation. These novel findings provide important new insights into the effect of genistein on mouse blastocysts.

138 CELL INTEGRITY OF HOLSTEIN AND JERSEY EMBRYOS: COMPARISON OF TWO FREEZING METHODS

The aim of this study was to compare cell integrity of Holstein and Jersey embryos using the standard method of freezing and vitrification. Embryos were harvested and processed following standard procedures approved by IETS (International Embryo Transfer Society), and then underwent two methods of freezing. (1) Standard method: embryos were put into 1.5 m ethylene glycol (EG) for 5 min at room temperature, and then at –6.5�C for 10 min for stabilization. After seeding, the temperature was lowered by 0.5�C until it reached –32�C, and then straws containing embryos were plunged into liquid nitrogen. (2) Vitrification: embryos were put for 3 min into vitrification solutions V1: 5 m EG in EmCare (ICPbio, Ltd., Auckland, New Zealand) and V2: 7 m EG, 0.5 m galactose (Sigma Chemical Co., St. Louis, MO, USA), and 18% Ficoll70 (Sigma) in EmCare for 45 s. Embryos were loaded into straws and then plunged into liquid nitrogen. De-freezing was done as follows: 5 s at room temperature, and then 30 s in water at 25�C. Vitrified embryos were de-frozen for 10 s at room temperature followed by 30 s in water at 25�C. All embryos were subjected to a combined staining, Hoechst 33342 and propidium iodide (Sigma-Aldrich Canada, Toronto, Ontario, Canada). The combined staining with these dyes makes analysis of cell death possible. Propidium iodide is specific to dead cells, whereas Hoechst stains all of the cells. Embryos were put in a PBS solution containing 20 µg mL–1 of Hoechst 33342 and propidium iodide for 15 min at 37�C and placed between a slide and coverslip. Fluorescence microscopy was then used to assess blue nuclei (live cells) and red nuclei (dead cells) of the cow embryos. For this experiment, a total of 51 grade 1 embryos (IETS classification) at early blastocyst or blastocyst stages were used (25 Holstein embryos from 4 donor cows and 26 Jersey embryos from 3 donor cows). The standard method of freezing was performed on 26 embryos (13 from Holstein cows and 13 from Jersey cows) and vitrification was performed on 25 embryos (12 from Holstein cows and 13 from Jersey cows). Embryos from a given cow (Holstein or Jersey) were evenly allocated to the standard and vitrification methods. The GLM procedure of SAS (SAS statistical software version 8; SAS Institute, Inc., Cary, NC, USA) was used to compare numbers of dead and live cells between vitrification and the standard method of freezing in Holstein and Jersey embryos. The mean numbers (�SE) of live cells of Holstein embryos were, respectively, 87.92 � 8.48 and 85.5 � 6.46 for standard method and vitrification. For Jersey embryos, means (�SE) of live cells were 94.42 � 2.6 and 73.93 � 7.39, respectively, for the standard method and vitrification. Means (�SE) of dead cells of Holstein embryos were, respectively, 5.58 � 1.37 and 13.67 � 2.91 for the standard method and vitrification. For Jersey embryos, means (�SE) of dead cells were 8.08 � 1.01 and 27.6 � 7.06, respectively, for the standard method and vitrification. In conclusion, vitrification significantly increased (P ≤ 0.05) the number of dead cells of embryos over the standard method of freezing. This effect was more evident (P ≤ 0.05) in Jersey than in Holstein embryos. The authors thank Dr. Vincent Girard for his help with statistics.

The effect of hyperthermia in vitro on vitality of rabbit preimplantation embryos

The aim of our study was to test the influence of short exposure (6 h) of preimplantation rabbit embryos to elevated temperatures (41.5 degrees C or 42.5 degrees C) in vitro on their developmental capacity. Fertilized eggs recovered from female oviducts at the pronuclear stage (19 hpc) were cultured at standard temperature (37.5 degrees C) until the morula stage (72 hpc). Afterwards, the embryos were divided into two groups, cultured for 6 h either at hyperthermic (41.5 degrees C or 42.5 degrees C) or standard temperature (control 37.5 degrees C), post-incubated overnight (16-20 h) at 37.5 degrees C and then evaluated for developmental stages, apoptosis (TUNEL), proliferation (cell number), actin cytoskeleton and presence of heat-shock proteins Hsp70. It was observed that hyperthermia at 41.5 degrees C did not alter progression of embryos to higher preimplantation stages (expanded and hatching/hatched blastocysts), rate of apoptosis, total cell number of blastocysts and structure of actin filament compared to 37.5 degrees C. Western-blotting revealed the presence of heat stress-induced 72 kDa fraction of Hsp70 proteins in granulosa cells (exposed to 41 degrees C) and embryos (exposed to 41.5 degrees C). Following the elevation of temperature to 42.5 degrees C embryo development was dramatically compromised. The embryos were arrested at the morula or early blastocyst stage, showed an increased rate of apoptosis and decreased total cell number compared to control. The structure of actin filaments in most of blastomeres was damaged and such blastomeres often contained apoptotic nuclei. In this group a presence of heat-stress-induced fraction of Hsp70 proteins had not been confirmed. This is the first report demonstrating a threshold of thermotolerance of rabbit preimplantation embryos to hyperthermic exposure in vitro. A detrimental effect of higher temperature on the embryo is probably associated with the loss of their ability to produce Hsp70 de novo, which leads to cytoskeleton alterations and enhanced apoptosis.

Tumor Necrosis Factor Regulation of Apoptosis in Mouse Preimplantation Embryos and Its Antagonism by Transforming Growth Factor Alpha/Phosphatidylionsitol 3-Kinase Signaling System1

Survival and apoptosis of cells in preimplantation embryos are fundamental for successful pregnancy. Relevant to these processes, tumor necrosis factor (TNF) and transforming growth factor alpha (TGFA) are produced by mammalian oviducts and uteri. In early embryos, TNF induces apoptosis, whereas TGFA could act as a survival factor. Here we investigated the TNF regulation of apoptosis in early mouse embryos and its antagonism by TGFA. TNF receptor superfamily, member 1a mRNA was detectable throughout early embryonic stages, with an increase after the early blastocyst stage, whereas the expression of TNF receptor superfamily, member 1b transcripts were detected only at the expanded blastocyst stage. Although pregnant uteri produced TNF, physiologic levels were low during the preimplantation period. Treatment with TNF inhibited the development of two-cell stage embryos to blastocysts showing decreased proliferation and increased apoptosis both in vitro and in vivo. These detrimental effects of TNF on early embryo development and survival were blocked by a neutralizing anti-TNF antibody. In addition to the death receptor-mediated pathway, TNF-induced apoptosis was further mediated by disruption of mitochondrial functions, characterized by release of cytochrome c and activation of caspase 9. The proapoptotic effects of TNF in blastocysts were counteracted by cotreatment with TGFA. The antagonistic effect of TGFA on TNF-induced apoptosis was blocked by phosphatidylionsitol 3-kinase (PI3K) inhibitors. The present findings demonstrate the stage-selective susceptibility to the apoptosis-inducing effect of TNF in mouse preimplantation embryos and that the TGFA/PI3K signaling system has an important role in the control of TNF-induced apoptosis in blastocysts.

Mouse zona pellucida dynamically changes its elasticity during oocyte maturation, fertilization and early embryo development

A change in the elasticity of mouse zona pellucida was quantitatively evaluated during oocyte maturation, fertilization and early embryo development. Young's modulus of zona pellucida of germinal vesicle (GV), metaphase-II (MII), pronuclear (PN), 2cell, 4cell, 8cell, morulae (M) and early blastocyst (EB) stages was measured using a micro tactile sensor (MTS) and a chamber exclusively designed for the measurement. The MTS has very high sensitivity and a deformation of only 5 microm was sufficient to calculate the Young's modulus and the oocyte/embryo maintained its original spherical shape during the measurement. The Young's modulus of GV, MII, PN, 2cell, 4cell, 8cell, M and EB was 22.8+/-10.4 kPa (n=30), 8.26+/-5.22 kPa (n=74), 22.3+/-10.5 kPa (n=66), 13.8+/-3.54 kPa (n=41), 12.6+/-3.34 kPa (n=19), 5.97+/-4.97 kPa (n=6), 1.88+/-1.34 kPa (n=8) and 3.39+/-1.86 kPa (n=4), respectively. Experimental results clearly demonstrated that the mouse zona pellucida hardened following fertilization. Interestingly, once the zona pellucida hardened at the PN stage, it gradually softened as the embryo developed (i.e. it was found that the zona hardening is a transient phenomenon). Furthermore, the zona pellucida of the GV oocyte was as hard as that of the PN embryo and became soft as it matured to the MII stage. In addition, the safety of the MTS measurement for oocytes and embryos was discussed both theoretically and experimentally.

Batchwise assessment of porcine embryos for cryotolerance

The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze–thawing and in vitro culture varied depending on the batch and was lower (31.0 ± 10.2%, mean ± S.E.M.; P < 0.01) than that of unfrozen controls (96.8 ± 2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 μm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.

P-241: Removal of bacterial contamination from developing blastocysts in extended culture via micromanipulation in PVP

The presence of bacterial contamination in human embryos, resistant to antibiotics and sperm processing-purification, has been recently identified to occur after initiation of extended culture. Previous reports have identified Enterococcus faecalis, a Gram-positive bacteria that is resistant to many commonly used antibiotics, as the main cause of bacterial contamination. Removal of bacteria has been attempted by transferring the embryos via pipetting to fresh media every 24 hrs until the performance of ET. However, this approach represents a short-term solution because even minute amounts of the contaminated media may contain enough bacteria to colonize fresh media or affect the implantation and pregnancy In utero. Furthermore, another source for the bacteria may also be present in the zona pellucida (ZP). The objective of this study was to develop a method of containing or isolate the bacteria via micromanipulation in a medium of high viscosity before transferring the embryos to fresh media used for culture and ET. Case report. Bacterial contamination of culture media was detected on Day 4, following the transfer of embryos from cleavage media (Day 3) to blastocyst media for extended culture. Isolation of bacterial contamination was performed via micromanipulation of the embryos in PVP (7% (v/v) + 5 mg HSA/ml). The embryos were placed in a drop of PVP and a holding pipet was introduced next to the embryo. The selected embryo was moved to a separate area of the PVP drop and the holding pipet was raised over the plane of visibility in reference to the embryo at the 6 o’clock position. Negative pressure was applied to the holding pipet, which resulted in the formation of a vortex and a spinning motion on the embryo. The embryo was simultaneously moved in a zigzag route to aid in the removal and isolation of bacteria. The embryo was then placed in the opposite area of the PVP drop from where it was originally placed. The embryo was rinsed 3 times in blastocyst media and kept in culture until the time of ET or cryopreservation. The same treatment was applied to all embryos considered for ET or cryopreservation. All dishes where the embryos had contact with culture media or reagents were kept for examination on the next day. Most embryos were at the compact morula or early blastocyst stages when treated with PVP to isolate the bacteria. Culture drops in which the embryos resided before PVP treatment showed bacterial colonization at 24 hrs. The culture drops in which the embryos were cultured after PVP treatment were free of bacterial contamination at 24 hrs post-treatment. Embryos continued developing as expected for day 5 following PVP treatment. Two expanded blastocysts were transferred on day 5 resulting in an ongoing twin pregnancy. The remaining embryos were cryopreserved. Exposure and micromanipulation of embryos in PVP resulted in the isolation of bacteria as evident by the absence of colonization in fresh media 24 hrs after treatment. It is possible that the viscous properties of PVP serve to mechanically dislodge the bacteria from the ZP and also contain the bacteria and prevent re-colonization during the micromanipulation of the embryos. Treatment with PVP seems to be an adequate alternative to avoid transmitting bacteria once established in extended culture, and which can also represent risks for the establishment of implantation and pregnancy once the embryos are replaced in the uterus.

Ginkgolide B induces apoptosis and developmental injury in mouse embryonic stem cells and blastocysts

Ginkgolide B, the major active component of Ginkgo biloba extracts, can both stimulate and inhibit apoptotic signalling. We previously showed that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early post-implantation blastocyst development and increases early-stage blastocyst death. Here, we report more detailed examinations of the cytotoxic effects of ginkgolide B on mouse embryonic stem cells (ESCs) and blastocysts and their subsequent development in vitro and in vivo. Using cell culture assay model, we revealed in our results that ginkgolide B treatment of ESCs (ESC-B5) induced apoptosis via reactive oxygen species (ROS) generation, c-Jun N-terminal kinase (JNK) activation, loss of mitochondrial membrane potential (MMP) and the activation of caspase-3. Furthermore, an in vitro assay model showed that ginkgolide B treatment inhibited cell proliferation and growth in mouse blastocysts. Finally, an in vivo model showed that treatment with 10 microM ginkgolide B caused resorption of post-implantation blastocysts and fetal weight loss. Our results reveal for the first time that ginkgolide B retards the proliferation and development of mouse ESCs and blastocysts in vitro and causes developmental injury in vivo.

Effects of Glutamine on Blastocoele Formation and Hatching in Mouse Preimplantation Embryos

ABSTRACT The effects of glutamine supplementation on the blastocoele formation and hatching of mouse preimplantation embryos were investigated. When culturing the 1-cell or 2-cell stage embryos in glutamine-supplemented media, the incidence of development to each stage from the morula stage to the completely hatched blastocyst stage was higher than for culturing in media without glutamine supplementation. When culturing 4-cell stage, 8-cell stage or morula stage embryos in glutamine-supplemented media, percentage of development up to the expanded blastocyst stage was comparable to culturing in media without glutamine supplementation, but the hatching rate for the glutamine-supplemented media was higher or tended to be higher compared to culturing in media without glutamine supplementation. Further, culturing in glutamine-supplemented media from the early blastocyst stage, the blastocyst stage or the expanded blastocyst stage resulted in a higher hatching rate compared to culturing in media without glutami...

Shear Stress Induces Preimplantation Embryo Death That Is Delayed by the Zona Pellucida and Associated with Stress-Activated Protein Kinase-Mediated Apoptosis1

In this study, we discovered that embryos sense shear stress and sought to characterize the kinetics and the enzymatic mechanisms underlying induction of embryonic lethality by shear stress. Using a rotating wall vessel programmed to produce 1.2 dynes/cm2 shear stress, it was found that shear stress caused lethality within 12 h for E3.5 blastocysts. Embryos developed an approximate 100% increase in mitogen-activated protein kinase 8/9 (formerly known as stress-activated protein kinase/junC kinase 1/2) phosphorylation by 6 h of shear stress that further increased to approximately 350% by 12 h. Terminal deoxynucleotidyltransferase dUTP nick end labeling/apoptosis was at baseline levels at 6 h and increased to approximately 500% of baseline at 12 h, when irreversible commitment to death occurred. A mitogen-activated protein kinase 8/9 phosphorylation inhibitor, D-JNKI1, was able to inhibit over 50% of the apoptosis, suggesting a causal role for mitogen-activated protein kinase 8/9 phosphorylation in the shear stress-induced lethality. The E2.5 (compacted eight-cell/early morula stage) embryo was more sensitive to shear stress than the E3.5 (early blastocyst stage) embryo. Additionally, zona pellucida removal significantly accelerated shear stress-induced lethality while having no lethal effect on embryos in the static control. In conclusion, preimplantation embryos sense shear stress, chronic shear stress is lethal, and the zona pellucida lessens the lethal and sublethal effects of shear stress. Embryos in vivo would not experience as high a sustained velocity or shear stress as induced experimentally here. Lower shear stresses might induce sufficient mitogen-activated protein kinase 8/9 phosphorylation that would slow growth or cause premature differentiation if the zona pellucida were not intact.

159 LOCALIZATION OF CLAUDIN FAMILY PROTEINS IN PIG EMBRYOS DURING PRE-IMPLANTATION DEVELOPMENT

Tight junctions (TJ) are critical for blastocoel formation in mammalian pre-implantation embryos. Claudin family proteins, which are TJ proteins, are generally important for the barrier function of TJ. However, the expression and localization of claudin proteins are not clarified in mammalian pre-implantation embryos. The present study was designed to examine changes in the localization of claudin isoforms, claudin-1, -2, and -4, in pig parthenogenetic diploids through pre-implantation development. Oocyte–cumulus–granulosa cell complexes were collected from the follicles 4–6 mm in diameter, and then maturation-cultured. Only oocytes with a prominent first polar body were subjected to electro-stimulation after maturation, and they were treated with cytochalasin B to produce parthenogenetic diploids. Presumptive diploids were then cultured for 168 h and observed every 24 h. Embryos of each developmental stage from 2-cell to blastocyst were subjected to immunofluorescence staining of the anti-TJ proteins. Zona-free embryos were fixed and treated with rabbit anti-claudin-1 and -2 polyclonal antibodies or mouse anti-claudin-4 monoclonal antibody, followed by treatment with Alexa fluor 488-labeled goat anti-rabbit IgG antibody or goat anti-mouse IgG antibody. Some embryos were treated with rabbit anti-occludin antibody or anti-ZO-1 antibody for the detection of the TJ-net work. All embryos were counterstained with Hoechst 33342 and observed under an epifluorescence microscope after whole-mounting. The specific fluorescence for all TJ proteins examined was observed in embryos at all pre-implantation stages. Occludin and ZO-1 were detected in the cytoplasm before the morula stage (96 h), and localized toward the boundary region among cells from the morula and early blastocyst stages, reflecting the distribution of the tight junction in embryos. Claudin-1 and -2 localized in the cytoplasm at early-cleaving stages. They were detected in the nucleus after compaction, and the distribution of these proteins in the nucleus was dominant at the blastocyst stage. The distribution of the 2 claudin proteins in the nucleus was quite different; claudin-1 distributed rather homogeneously, but claudin-2 formed several bright spots in the nucleus. Claudin-4 also showed a unique distribution pattern in embryos; it was detected in the cytoplasm with strong fluorescence at the periphery of the nucleus of the 2- and 4-cell embryos. Claudin-4 changed its localization toward the boundary region of cells around the early blastocyst stage (120 h), and then the distribution of claudin-4 was restricted to the boundary region later in the blastocyst stage (144 h). These results indicate that claudin-4, but not claudin-1 and -2, is responsible for the formation of TJ in the pre-implantation embryos, although the function of these claudin proteins in the nucleus is unknown.

Developmentally retarded frozen blastocysts can be rescued by synchronizing culture prior to transfer

Asynchrony between embryo development and endometrial differentiation is the limiting step of successful pregnancy in assisted reproduction. The aim of this study was to investigate whether or not post-thaw synchronization culture of day 5–6 frozen embryos, prior to transfer, with endometrial differentiation resulted in pregnancy. A total of 142 cycles of 134 patients were transferred in three protocols. Blastocysts with cavities larger than half of the entire blastocyst volume were transferred without synchronizing culture on day 5 or 6 of progesterone commencement (P5/6) in hormone replacement treatment cycles (protocol 1). Blastocysts with cavitation below half of the entire blastocyst were cultured for 1 or 2 days after thawing prior to transfer on P5 or P6 (protocol 2). Morulae and very early stage blastocysts were thawed on the days corresponding to P5 and P6, and only the embryos that reached expanded or hatching blastocysts were transferred on P7 without synchronizing culture (protocol 3). Pregnancy rate in protocol 2 (32.0%) was comparable with that of protocol 1 (35.0%). It is concluded that developmentally retarded frozen embryos can be rescued with synchronizing culture prior to transfer by evading asynchrony.

120 SUCCESSFUL PRODUCTION OF PIGLETS DERIVED FROM VITRIFIED MORULAE AND EARLY BLASTOCYSTS WITHOUT DIRECT EXPOSURE TO LIQUID NITROGEN

Recently piglets have been produced from morulae and early blastocysts vitrified by using methods such as open pulled straw and microdroplet. However, a problem of pollution, such as contamination of embryos with pathological microorganisms from the liquid nitrogen (LN), to the embryos is possible, because embryos must be exposed directly to LN in these methods. Therefore, we designed a new device in which the embryo is vitrified on the stainless steel chip inserted into a 0.25-mL straw without direct contact with LN. To serve as a stainless steel chip, a part of the body of a 19 G needle was cut to reveal its inner surface as the loading site for embryos, and 2 to 15 embryos in a small amount of a vitrification solution were placed on the inner surface of the needle. Then the needle with the embryos was inserted into the 0.25-mL straw which was pre-cooled in LN. This study was conducted to determine the efficiency of vitrification using the new device for porcine embryos at early developmental stages. In Experiment 1, embryos at the compact morula to early blastocyst stages were collected from 7 gilts. The embryos were equilibrated in 20 mM HEPES-buffered PZM (Yoshioka et al. 2003 Biol. Reprod. 69, 2092–2099) (H-PZM) supplemented with 1.8 M ethylene glycol (EG), and then in 1.8 M EG, 0.3 M sucrose, and 2% (w/v) bovine serum albumin (BSA) for 5 min. After equilibration, the embryos were vitrified using H-PZM supplemented with 0.6 M sucrose, 2% (w/v) BSA, and 4 M, 5 M, 6 M, or 7 M EG (ESB). The vitrified embryos were thawed, diluted by directly plunging the needle of the device into H-PZM supplemented with 1.8 M EG and 0.6 M sucrose for 5 min at 38�C, and subsequently cultured in PZM supplemented with 10% fetal bovine serum for 48 h; fresh embryos were cultured as a control group. In Experiment 2, embryos were vitrified using 5 M EG ESB, and then 56 vitrified embryos (collected from 4 gilts) were transferred to 4 recipient gilts (12 to 15 embryos per recipient). In Experiment 1, the survival rates of vitrified embryos after 48 h of culture in 4 M, 5 M, 6 M, 7 M, and control groups were 72.7, 86.4, 90.0, 87.0, and 100% (n = 20 to 23), respectively. The 4 M group showed a significantly lower survival rate than the control group (P &amp;lt; 0.05; chi-square test). The expanding and hatching rates of the vitrified embryos in these groups after 48 h of culture were 68.2, 77.3, 70.0, 65.2, and 95.0%, respectively. The 7 M group had a significantly lower expanding and hatching rate than the control group (P &amp;lt; 0.05). In Experiment 2, 3 recipients showed estrus at 19, 29, and 44 days after their previous estrus. One recipient became pregnant and 6 normal live piglets were born. These results indicated that porcine embryos at the compact morula to early blastocyst stages can survive after vitrification using our new device, which can prevent embryos from direct exposure to LN, and that piglets can be obtained from vitrified embryos without fear of pollution from LN.

Ginkgolides induce apoptosis and decrease cell numbers in mouse blastocysts

In this report, we examine the cytotoxic effect of ginkgolides, the major components of Ginkgo biloba extracts, on the blastocyst stage of mouse embryos and on subsequent early postimplantation embryonic development in vitro. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed that blastocysts treated with 5 or 10 μM ginkgolide A or ginkgolide B showed increased apoptosis versus untreated controls. This could be correlated with the observation that ginkgolide-treated blastocysts showed a significant reduction in the average number of total cells in the blastocyst and trophectoderm/inner cell mass lineage versus controls. In addition, ginkgolide-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer embryos reached the later stages of embryonic development in the treatment groups versus the controls, instead dying at relatively early stages of development. Our results collectively indicate that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death. These novel findings provide important new insights into the effect of Ginkgo biloba extracts on mouse blastocysts.