Late Morula Stage Research Articles

Effect of post-activation culture conditions on the development of parthenogenetic embryos in cattle

Purpose: to conduct a comparative analysis of the effect of commercial media BO-IVC and СR1aa at the stage of the activation and subsequent culture of artificially activated oocytes on the formation and quality of parthenogenetic bovine embryos.Materials and methods. 3 groups of disemeters of 50 goals in each were formed. In the first experimental group, the disemeted was in a meticulous manner with a ram-industrialist (artificial kriproporchid), in the second experimental-with a penEexctomed ram-industrialist. In the third (control) group, a producer ram was used. In the first experimental group of a ram-industrialist (artificial kriproporchid) with attached taps were released into a group of sheep twice a day for 1.5-2 hours. In the second experimental group of a penEctomed ram, it was placed in the corral to the disemetery in the morning for 3 hours. In the third group, the lamb producer was constantly with the disemets for two weeks, then he was changed on a new ram i.e. Used the methodology used in the farm. During the experiment, they observed the behavior of animals of all groups. In the experimental groups, after the detection of disemeters in the hunt, their natural insemination of the manufacturer was carried out. Based on the results of the subsequent oster, the effectiveness of the reproduction of sheep was evaluated.Results. The cleavage rate did not differ between the experimental groups, varying from 73,0 to 76,5%. Also, there was not found a significant effect of the conditions for post-activation culture of oocytes on their development before late morula and late blastocyst stage, which was for the CR1aa/CR1aa, CR1aa/BO-IVC and BOIVC/ BO-IVC groups 28,9±1,7, 40,4±7,5 and 36,0±6.4%, respectively. Meanwhile, we found out the effect of tested culture conditions on the ability of parthenogenetic embryos to overcome the 8-16 cell block and their quality on the late stages of embryo development. The rate of embryos with less than 16 nuclei was the highest in the CR1aa/CR1aa group (56,8±2,1 %). The replacement of CR1aa medium to BO-IVC medium (BO-IVC/BO-IVC group) significantly reduced this level (p<0,05). The positive effect was enhanced when CR1aa medium was used at the stage of culture in the presence of 6-DMAP and cycloheximide, and subsequent embryo development was in BO-IVC medium (CR1aa/BO-IVC group) (p<0.001). Furthermore, when we used the mixed variant of culture, the total cell number in parthenogenetic morula and blastocyst stage embryos increased (p<0.05).Conclusion. Thus, the BO-IVC medium at the stages of post-activation and subsequent development of artificially activated bovine oocytes is comparable to the CR1aa medium in terms of the efficiency of obtaining parthenogenetic embryos at the blastocyst stage. Nevertheless, its replacement at the post-activation stage with CR1aa medium makes it possible to improve the quality of parthenogenetic embryos.

MEIOTIC AND MITOTIC ANEUPLOIDIES DRIVE ARREST OF IN VITRO FERTILIZED HUMAN PREIMPLANTATION EMBRYOS

The high incidence of chromosome aneuploidy in early human development is the primary cause of pregnancy loss, miscarriage, and still birth following natural conception as well as in vitro fertilization (IVF). Preimplantation genetic testing for aneuploidy (PGT-A) of IVF embryos at the blastocyst stage has confirmed the prevalence of aneuploidies of putative meiotic and postzygotic mitotic origins. However, only about half of normally fertilized embryos develop to the blastocyst stage in vitro, while the others arrest at cleavage to late morula or early blastocyst stages.

Multiple ovulation and embryo transfer in sheep: Effects of embryo developmental stage and quality on viability in vivo under farm conditions.

Multiple ovulation and embryo transfer (MOET) technologies are integral to genetic improvement programs in the sheep industries. Despite the protocols being well established, previous findings regarding the effects of embryo properties on MOET success remain contradictory. The objective of this study was to determine the effects of embryo developmental stage and quality on embryo viability following transfer to recipient ewes. Data including details of 377 embryos collected from 45 Merino donor ewes were obtained from MOET trials conducted on three separate farms on day 6 after laparoscopic artificial insemination (AI). A total of 270 embryos were classified as being of transferrable grade (grade 1: n = 233; grade 2: n = 37). One or two transferrable grade embryos were transferred to each of 256 synchronised recipient ewes and pregnancy diagnosis was performed on day 36 after embryo transfer. Embryos at the hatched blastocyst stage tended to have greater viability in vivo compared to embryos at the late morula stage (59.0 ± 10.6% vs. 36.2 ± 9.7%; P = 0.083). The viability of grade 1 embryos was greater than that of grade 2 embryos (53.6 ± 7.8% vs. 35.9 ± 10.2%; P < 0.05). The results suggest that the success of the MOET trials was influenced by the transfer of embryos at the late morula stage, almost half of which were classified as grade 2 embryos. These findings highlight the importance of following strict embryo quality grading criteria to inform the most economical management of recipient ewes and maximize pregnancy outcomes.

O-099 TEAD4 regulates trophectoderm differentiation upstream of CDX2 in human preimplantation embryos

Abstract Study question What is the main pathway regulating trophectoderm (TE) differentiation during pre-implantation development in mouse versus human embryos? Summary answer TEAD4 is acting upstream of CDX2 and is involved in TE differentiation, as TEAD4-null human embryos exhibit compromised TE lineage differentiation. What is known already TEAD4 is the earliest transcription factor during early embryo development, required for the expression of TE-associated genes leading to successful TE differentiation and subsequent blastocoel formation in mouse. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination have shown that Tead4-null embryos do not express TE specific genes, including Caudal-Type Homeobox Protein 2 (Cdx2) and GATA Binding Protein 3 (Gata3), but expression of inner cell mass (ICM)-specific genes, remains unaffected. Furthermore, ablation of Tead4 compromises embryonic development and subsequent blastocoel formation in mouse. The role of TEAD4, during human pre-implantation development has not been functionally characterized yet. Study design, size, duration CRISPR-Cas9 was introduced in mouse zygotes and editing efficiency was evaluated by next-generation sequencing (NGS) on 4.5dpc embryos (n = 55). Developmental kinetics were monitored in CRISPR-Cas9 targeted (n = 83), sham-injected (n = 26) and non-injected media-control (n = 51) mouse embryos. Immunofluorescence analysis was performed in Tead4 targeted (n = 57) and non-injected media-control embryos (n = 94). The same methodology was applied in human donated in vitro matured (IVM) metaphase-II (MII) oocytes, which were CRISPR-Cas9 targeted (n = 74) during ICSI or used as media-Control (n = 33). Participants/materials, setting, methods A gRNA-Cas9 mixture targeting exon 2 of Tead4/TEAD4 was microinjected in respectively mouse 2PN (pronuclear) stage zygotes, or human IVM MII oocytes along with the sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. Embryonic development and morphology were assessed daily, followed by a detailed scoring at the late blastocyst stage. Successful targeting following CRISPR-Cas9 introduction was assessed by immunostaining and NGS analysis of the targeted locus. Main results and the role of chance In mouse, we confirmed previous findings, as the developmental capacity of Tead4 targeted embryos was significantly reduced starting from the morula stage and blastocyst formation rates were 8.97% in the targeted group, compared to 87.23% in the control and 87.50% in the sham group, respectively. Immunofluorescence analysis of late morula and blastocyst stage embryos confirmed the absence of Tead4, Cdx2 and Gata3, resulting from the successful interruption of the Tead4 locus (n = 57). Exon 2 of TEAD4 was successfully targeted in human. In total, 21 embryos from various developmental stages were successfully NGS analyzed and 90,48% (19 out of 21) of the embryos carried genetic modifications as a result of CRISPR-Cas9 genome editing and seven blastocysts were identified carrying exclusively frameshift mutations. In contrast to mouse, the developmental capacity of human targeted embryos (25%) did not differ significantly from the control group (23%). However, the blastocyst morphology and quality were compromised in the targeted group showing mostly grade C TE scores, containing very few cells. Immunofluorescence analysis of targeted blastocysts (n = 6) confirmed successful editing by complete absence of TEAD4 and its downstream TE marker CDX2. Limitations, reasons for caution CRISPR-Cas9 germline genome editing results in multiple editing outcomes with variable phenotypic penetrance, the mosaic nature of which complicates the phenotypic analysis and developmental behaviour of the injected embryos. Wider implications of the findings Elucidation of the evolutionary conserved molecular mechanisms that regulate self-renewal of the trophoblast lineage can give us fundamental insights on early implantation failure. Trial registration number Not Applicable

Developmental stages and viability of Toxocara canis eggs outside the host

Toxocariasis is a soil-transmitted zoonotic disease caused mainly by ingestion of larvated eggs of Toxocara canis. To study the morphology of the intraovular developmental stages of Toxocara canis in culture, characterize non-viable eggs and the sequences of larval molting and compare the viability of eggs at the early stages of division and at reaching full maturation. Observation of developing embryos and characterization of non-viable eggs were done using light microscope. The proportions of viable eggs during embryonation were compared to the proportions of viable mature eggs. Cell division commenced after 24 hours of cultivation. Early stages were found to be present over a period of 3-5 days. The developmental stages identified were eggs with: One cell, two cells, three cells, four cells, early morula, late morula, blastula, gastrula, tadpole, pre-larva, first, second and third stage larva. Two larval molts occurred. Non-viable eggs had degenerated cytoplasm, thin or collapsed shell and the larvae did not move after exposure to light. No significant differences were found between the proportions of viable eggs from day five to day 21 as compared to viability of fully mature eggs (30 days). Developing embryos in the environment may be considered as a potential threat to the public health. The precise identification of developmental stages and the clear differentiation of viable and non-viable eggs can help in determining an accurate baseline rate of development that could be used in studies of ovicidal compounds.

Role of chemokine (C-C motif) ligand 24 in spatial arrangement of the inner cell mass of the bovine embryo.

The process of spatial rearrangement of cells of the inner cell mass (ICM) that are destined to become hypoblast is not well understood. The observation that the chemokine (C-C motif) ligand 24 (CCL24) and several other genes involved in chemokine signaling are expressed more in the ICM than in the trophectoderm of the bovine embryo resulted in the hypothesis that CCL24 participates in spatial organization of the ICM. Temporally, expression of CCL24 in the bovine embryo occurs coincidently with blastocyst formation: transcript abundance was low until the late morula stage, peaked in the blastocyst at Day 7 of development and declined by Day 9. Treatment of embryos with two separate antagonists of C-C motif chemokine receptor 3 (the prototypical receptor for CCL24) decreased the percent of GATA6+ cells (hypoblast precursors) that were located in the outside of the ICM. Similarly, injection of zygotes with a CCL24-specific morpholino decreased the percent of GATA6+ cells in the outside of the ICM. In conclusion, CCL24 assists in spatial arrangement of the ICM in the bovine embryo. This experiment points to new functions of chemokine signaling in the bovine embryo and is consistent with the idea that cell migration is involved in the spatial organization of hypoblast cells in the blastocyst.

Pluripotency Network in Porcine Embryos and Derived Cell Lines

Huge amounts of work have been dedicated to the establishment of embryonic stem cell lines from farm animal species since the successful isolation of embryonic stem cells from the mouse and from the human. However, no conclusive results have been obtained so far, and validated lines have yet to be established in domestic animals. Many limiting factors have been suggested and need to be studied further to isolate truly pluripotent cell lines from livestock. In this review, we will discuss the difficulties in deriving and maintaining embryonic stem cell lines from farm animal embryos and how can this lack of success be explained. We will summarize results obtained in our laboratory regarding derivation of pluripotent cells in the pigs. Problems related to the identification of standard methods for derivation, maintenance and characterization of cell lines will also be examined. We will focus our attention on the need for appropriate stemness-related marker molecules that can be used to reliably investigate pluripotency in domestic species. Finally, we will review data presently available on functional key pluripotency-maintaining pathways in farm animals.

Morphological Changes of Ascaris spp. Eggs During Their Development Outside the Host

Information on the infective stage of Ascaris lumbricoides and the pathology caused by the parasite is widely available in the literature. However, information about early embryonic development of A. lumbricoides and its life cycle outside the host is limited. The purpose of this study was to describe the morphological changes within the developing embryo during incubation in vitro at 28 C, as well as to explore differences in egg viability during incubation. Ascaris suum eggs (4,000 eggs/ml), used as a model for A. lumbricoides , were placed for incubation in 0.1N H(2)SO(4) at 28 C in the dark for 21 days. Every day, sub-samples of approximately 100 A. suum eggs were taken from the incubation solution for microscopic evaluation. Development, morphological changes, and viability of the first 40 eggs were observed and documented with photos. During this study, 12 stages were identified in the developing embryo by standard microscopy, 2 of which had not been previously reported. By the end of the first wk, most developing embryos observed were in the late-morula stage (72.5%). On day 14 of incubation, 90% had developed to larva-1 stage, and by day 21, 100% had developed to larva-2 stage. No significant differences were found in the viability recorded in a continuum from day 5 to day 21 of incubation (chi-square, P > 0.05). The result of this study complements and expands the stages of development of Ascaris spp. outside the host previously reported in the literature. It also suggests the potential use of early stages of development of the nematode to determine viability and safety of sewage sludge, wastewater, or compost after treatment recommended by USEPA.

Chimérisme tétragamétique : à propos d’un cas

We report the third case of spontaneous monochorionic dizygous pregnancy, discovered on foetal sex discordance. Blood group testing on the female twin revealed a hematopoietic chimera. The mechanism of monochorionic dizygous formation could be the fusion of two independent zygotes at a late morula stage. A single placental mass with vascular anastomosis then develops. Stem cells exchanged during early foetal life can thus lead to chimeras, in similar conditions to stem cell transfusion in adults. Immaturity of the foetal immune system allows cell graft in the other twin's marrow. Assisted reproductive procedures are believed to promote such pregnancies.

Cloning and expression of sheep DNA methyltransferase 1 and its development‐specific isoform

Unlike the mouse embryo, where loss of DNA methylation in the embryonic nucleus leaves cleavage stage embryos globally hypomethylated, sheep preimplantation embryos retain high levels of methylation until the blastocyst stage. We have cloned and sequenced sheep Dnmt1 and found it to be highly conserved with both the human and mouse homologues. Furthermore, we observed that the transcript normally expressed in adult somatic tissues is highly abundant in sheep oocytes. Throughout sheep preimplantation development the protein is retained in the cytoplasm whereas Dnmt1 transcript production declines after the embryonic genome activation at the 8-16 cell stage. Attempts to clone oocyte-specific 5' regions of Dnmt1, known to be present in the mouse and human gene, were unsuccessful. However, a novel ovine Dnmt1 exon, theoretically encoding 13 amino acids, was found to be expressed in sheep oocytes, preimplantation embryos and early fetal lineages, but not in the adult tissue. RNAi-mediated knockdown of this novel transcript resulted in embryonic developmental arrest at the late morula stage, suggesting an essential role for this isoform in sheep blastocyst formation.

DNA methylation pattern in pig in vivo produced embryos

DNA methylation/demethylation pattern, determined by 5-methylcytosine (5-MeC) immunostaining, was evaluated in porcine "in vivo" produced embryos from zygote up to the blastocyst stage. In one-cell stage embryos, only the maternal pronucleus showed a positive labeling whilst the paternal pronucleus showed almost no labeling. The intensity of labeling is high until the late morula stage. Blastocysts containing less than 100 cells showed the same intensity of labeling in both the inner cell mass (ICM) nuclei and the trophectodermal (TE) cell nuclei. Interestingly, with further cell multiplication, cells of the ICM became more intensively labeled when compared to TE cells. This distinct methylation pattern is even more profound in blastocysts containing about 200-300 cells and is not caused by the difference in the cell volume of ICM and TE cells.

97 ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS FOLLOWING VITRIFICATION BY CVM OR SLOW FREEZING WITH ETHYLENE GLYCOL AND TRIPLE TRANSFER

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P &lt; 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P &lt; 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).

80 EFFECT OF DNA METHYLATION ON SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT IN RHESUS MONKEY

Up to now, no primate animals have been successfully cloned with somatic cell nuclear transfer (SCNT) and little is known about molecular events occurring in SCNT embryos. DNA methylation reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals. Epigenetic characteristics of donor cell nuclei and their epigenetic reprogramming in oocyte cytoplasm have been supposed as major factors influencing the development of SCNT embryos. In Experiment 1, on donor cells used in a previous SCNT at our laboratory, global DNA methylation and histone 3 lysine 9 acetylation (H3K9ac) of three cell lines (S11, S1-04, and S1-03) derived from ear skin were examined after serum starvation by immunofluorescence with monoclonal antibody to 5-methyl cytosine (Oncogene, Science, Inc., Cambridge, MA, USA) and anti-acetyl-Histone H3 (Lys 9) (Upstate Jingmei Biotech, Ltd., Shenzhen, China). In the results, two cells lines, S11 and S1-04, supporting higher blastocyst development (about 20%) than that (7.8%) of S1-03, showed a higher level of H3K9ac than the S1-03 cell line. Global DNA methylation levels in the three cell lines were decreased after serum starvation, but no obvious correlation between the level and SCNT embryo developmental potential was found among the three cell lines. In Experiment 2, on SCNT and IVF embryos, global DNA methylation reprogramming during pre-implantation development was investigated with immunofluorescence and laser scanning microscopy techniques. In IVF embryos, active demethylation of paternal genome occurred soon after fertilization; subsequently, passive demethylation resulted in remarkably reduced global methylation level at the 8-cell stage and the morula stage. Thereafter, genomewide remethylation started at the late morula stage and an asymmetric methylation pattern was formed in blastocysts, with higher methylated trophectoderm than inner cell mass (ICM). Compared with IVF embryos, most SCNT 2-cell embryos and ICM in blastocysts showed higher methylation levels, and the asymmetric methylation pattern was not as evident as that in IVF blastocysts. Some SCNT 8-cell embryos showed higher methylation, but others were slightly stained, even lower than IVF embryos. In conclusion, the higher global H3K9 acetylation level of donor cells may benefit chromatin remolding and development of SCNT embryos. Abnormal methylation reprogramming in most SCNT embryos, especially in ICM of blastocysts, may be one main obstacle for primate cloning, although relatively high blastocyst development rate was obtained. DNA methylation reprogramming in rhesus monkey pre-implantation embryos, on the whole, was as conservative as that reported in other mammals.

152 IN VITRO AND IN VIVO DEVELOPMENT OF BOVINE IVP EMBRYOS FOLLOWING SINGLE-CELL BIOPSY ON DAY 4

Maximum advantage can be gained from gene discovery programs, by screening embryos carrying the desired genes(s) prior to immediate transfer. This requires an efficient and reliable genotyping system and a method for biopsy preparation that does not compromise subsequent embryo or fetal development. The present study examined the effect of removing a single-cell from the developing 8–16 cell embryo on its subsequent ability to continue development to at least the late morula stage in vitro and then survive following triple transfer to recipients. Abattoir-sourced ovaries were obtained and subjected to IVP as previously described (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). Briefly, oocytes were matured in TCM199 +10% FCS, 10 μg/mL FSH, 10 μg/mL LH, 1 μg/mL estradiol, and 100 μM cysteamine under 5% CO2 in air at 38.5°C for 24 h. Percoll-separated sperm (1 × 106/mL) were then co-incubated with the matured oocytes (Day 0) for 24 h with the presumptive zygotes further cultured in mSOF medium under 5%CO2, 7% O2, 88% N2. On Day 4 embryos with a minimum of 8 cells were selected and held at 38.5°C in HEPES-buffered SOF (HSOF) until biopsy at ambient temperature. Embryo biopsy was performed in HSOF medium + 5 μg/mL cytochalasin B. A single cell was removed using a 30 μm biopsy pipette. Both biopsied and control embryos were then further cultured in mSOF in individual wells prepared in a 1% agarose matrix (Peura TT 2003 Cloning Stem Cells 5, 13–24). Embryos were scored for grade and stage of development reached on Day 7, and Grades 1 and 2 blastocysts and expanded blastocysts were transferred to synchronized recipients (three embryos of the same stage and grade to each recipient; n = 50). Fetal number was determined on Day 35 and 62 of gestation. A model for embryo survival was fitted to the data (McMillan WH et al. 1998 Theriogenology 50, 1053–1070) in order to estimate embryo (“e”) and recipient (“r”) contributions to embryo survival. Values were then compared to those determined for control embryos, produced using identical IVP methods (van Wagtendonk-De Leeuw AM et al. 2004 Reprod. Fert. Dev. 16, 214 abst). A total of 358 control and 561 biopsied embryos were cultured. Removal of a single cell did not significantly affect in vitro development (60.1% vs. 56.0%; control vs. biopsy). Day 35 survival of biopsied embryos was 44.7% with calculated “e” and “r” values of 0.48 and 0.94, respectively, which did not differ from control values (44.1%; 0.50 and 0.89). However, by Day 62 fetal survival had significantly decreased with a concomitant drop in “e” but not “r” (30.0%; 0.32 and 0.94, respectively; control “e” and “r” were unchanged). In conclusion, single-cell biopsy of the 8–16-cell embryo does not affect in vitro development or embryo survival to Day 35. However, significant fetal loss occurs by Day 62 that may limit commercial application. Further work is required to elucidate the cause of and overcome fetal loss.

118 EXPRESSION PROFILES OF CIRCADIAN CLOCK GENES IN MOUSE OOCYTES AND PRE-IMPLANTATION EMBRYOS

Matsuo et al. reported that circadian clock genes regulate the timing of cell division in mouse regenerating liver cells (2003). Their results suggested the importance of circadian clock genes for organs or tissues for which functions are characterized by cell division, such as pre-implantation embryos. To obtain basic information on the molecular functions of circadian clock genes in pre-implantation embryos, we investigated the expression profiles of transcripts and proteins of some circadian clock genes, clock, bmal1, cry1, and per2, in mouse germinal vesicle oocytes (GV), MII oocytes (MII), and pre-implantation embryos using real-time PCR and immunocytochemistry (ICC). Germinal vesicle oocytes were collected from ICR females at 48 h after PMSG priming. The mouse at 48 h after PMSG priming was primed with hCG, and MII were collected at 15 h after hCG priming. The pre-implantation embryos were collected at 6, 12, 24, 36, 48, 60, 72, 84, and 96 h after insemination, and they corresponded to early 1-cell, late 1-cell, early 2-cell, late 2-cell, 4-cell, 8-cell, early morula, late morula, and blastocyst stages, respectively. cDNA was produced by mRNA isolated from 20 oocytes or embryos using oligo dT and was subjected to real-time PCR using a TaqMan Probe system (ABI). Three sets of 20 oocytes or embryos at each developmental stage were applied to mRNA extraction and real-time PCR analysis to ensure equal mRNA extraction efficiency between samples. The level of mRNA of each clock gene contained in 3 samples from each developmental stage was almost the same. Statistical analysis of the transcripts of each gene were done by ANOVA. Germinal vesicles, MII and embryos collected at each time point were subjected to ICC using antibodies of CLOCK, BMAL1, CRY1, and PER2. The oocytes or embryos treated with only secondary antibody did not produce any signal. All of the examined genes except per2 were expressed in oocytes and pre-implantation embryos. The transcript level of clock, bmal1, and cry1 in MII were significantly lower than those in GV (P &lt; 0.05). After fertilization, transcript levels of clock, bmal1, and cry1 significantly decreased from early 1-cell stage to late 2-cell stage (P &lt; 0.05). These decreased transcript levels were maintained until the blastocyst stage after the late 2-cell stage. Immunocytochemistry analysis showed the nuclear localization of CLOCK and BMAL1 in early and late 2-cell embryos and of CRY1 in early 2-cell embryos but no signals of PER2 in oocytes or pre-implantation embryos. Because mouse oocytes and 1- to 2-cell embryos are transcriptionally inert, the abundant transcripts of clock, bmal1, and cry1 in these stages seemed to indicate that they were synthesized and stored during the oocyte growth phase. Moreover, the nuclear localization of CLOCK, BMAL1, and CRY1 in the oocytes and 1- to 2-cell stage embryos suggested that some clock genes were translated and worked for oocyte maturation and early embryogenesis. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

102 DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) OOCYTES VITRIFIED AT DIFFERENT STAGES OF MATURATION IN VITRO

Cryopreservation of unfertilized oocytes at very low temperature (-196�C) is carried out to ensure their continuous availability during different assisted reproductive techniques. However, various problems associated with the freezing of oocytes influence their developmental competence, resulting in suboptimal embryo production. The present study was planned to assess the developmental competence of buffalo oocytes vitrified at different meiotic stages of maturation. Expression profile of developmentally important genes, viz, heat shock protein 70 (HSP 70) and glucose transporter 1 (Glut1), was verified in these vitrified warmed oocytes. Buffalo cumulus-oocyte complexes were collected from slaughterhouse ovaries and divided into six groups: control (no vitrification); 0 h group (vitrified before maturation), and 6-, 12-, 18-, and 24-h groups [vitrified respectively at 6, 12, 18, and 24 h post-in vitro maturation (IVM)]. Vitrification solution consisted of propylene glycol (40% w/v), and trehalose (0.25 mol/L) in PBS + BSA (4% w/v) and vitrification was carried out by directly plunging 0.25-mL French mini-straws into liquid nitrogen. After a minimum storage period of 7 days, the straws were thawed at 37�C for 30 sec. In all groups, the oocytes completed 24-h of maturation. After 24 h maturation, a few oocytes from each of the six groups were stained with ethidium bromide to reveal their nuclear status. The remaining oocytes were co-incubated with frozen thawed buffalo semen in fertilization TALP with 6 mg/mL fatty acid free BSA and 10 �g/mL heparin sodium salt for 18 h. Presumptive zygotes were cultured in mSOF for 8 days. Vitrified warmed oocytes were subjected to total RNA isolation and RT-PCR for detection of mRNA transcripts of HSP 70 and Glut1 genes. Data were analyzed by one-way ANOVA and F-test analysis. Differences of P &lt; 0.05 were considered significant. The percentage of oocytes recovered from all five vitrification groups varied from 89 to 92 out of which 84-91% of oocytes were morphologically normal. A higher proportion of nonvitrified control oocytes (72.8%; 40/55) reached the metaphase II stage than for the oocytes vitrified at 24 (60%; 36/60), 18 (54.4%; 31/57), 12 (42.3%; 22/52), 6 (33.3%; 20/60), and 0 (31.7%; 19/60) h of IVM. The cleavage rate of nonvitrified control oocytes was higher (36.8%) than that of oocytes vitrified at 0 (1.6%), 6 (2.0%), 12 (3.2%), 18 (5.3%), and 24 (5.2%) h of IVM. With regard to subsequent development, 0- and 6-h oocytes were blocked at 8 cells, whereas in other groups development reached the late morula (4.8%) and blastocyst (3.5%) stages, confirming that the stage of maturation at which oocytes are vitrified influenced the nuclear maturation and developmental competence. Total RNA content was 2.24 � 0.40 ng/oocyte in the control group and 2.11 � 0.22 ng/oocyte in the group vitrified after 24 h of IVM. The expression pattern of HSP 70 and Glut1 was identical in control and vitrified groups, indicating that the vitrification protocol did not alter the expression pattern of these genes.

Erratum: Inositol transport in preimplantation rabbit embryos: effects of embryo stage, sodium, osmolality and metabolic inhibitors

The preimplantation period in the rabbit consists of a 3 day cleavage stage during which the number of cells increases with little change in embryo size, followed by a 3-4 day blastocyst stage during which the inner cell mass, the blastocoel and the trophectodermal layer are formed and the embryo grows rapidly in size and protein content. This study used [3H]inositol to investigate the transport of inositol, an essential component of the phosphatidylinositol signal transduction system, over the 6 days of preimplantation development by rabbit embryos. In the presence of 15 micromol inositol-1 in the incubation medium, there was a small linear increase in inositol uptake from 0.07 pmol per embryo per h at the one-cell stage (day 1) to 0.135 pmol at the late morula (day 3) stage. Inositol uptake increased to 0.58 pmol per embryo per h for early blastocysts (day 4) and 23.7 pmol for late blastocysts (day 6). There was a significant linear relationship between inositol uptake and blastocyst diameter and surface area. Efflux of inositol from early morulae was minimal (about 1.25% of embryo content per h), whereas efflux from mid-blastocysts (day 5) was much greater (about 15.6% of embryo content per h). Efflux of inositol from both early morulae and mid-blastocysts was increased by decreasing the osmolality of the incubation medium. Varying the osmolality had no effect on inositol uptake up to 2 h. Inositol uptake was dependent on sodium in cleavage-stage embryos but independent of sodium in blastocyst stages. In early morulae, inositol uptake was inhibited by glucose and the sodium-dependent hexose transport inhibitor, phloridzin, but not by the facilitated transport inhibitor, phloretin. Inositol uptake in early morulae was saturable; estimates of 0.227 and 0.288 pmol per morula per h for V(max) and 0.045 and 0.038 mmol-1 [corrected] for Km were obtained for sodium-dependent transport in two separate experiments. All of these results are consistent with the hypothesis that transport in cleavage stages occurs via a sodium myo-inositol transporter (SMIT) protein. Uptake in blastocysts was non-saturable. Uptake into blastocysts appeared to take place by a transcellular rather than a paracellular route.

Stage-Specific Toxicity of Cypermethrin to Medaka (Oryzias latipes) Eggs and Embryos Using a Refined Methodology for an In Vitro Fertilization Bioassay

Using original artificial fertilization methods with medaka (Oryzias latipes), the effects of exposure to cypermethrin on gametes, fertilization, and embryonic development were investigated. The relative sensitivity was studied with 96-hour duration, 24-hour renewal exposures to six nominal concentrations of cypermethrin ranging from 3.1 100.0 microg cypermethrin/L. Tests were initiated at different developmental stages: unfertilized egg (stage 0), late morula (stage 9), eminence of swim bladder (stage 29), and maximum flexion of the atrioventricular region (stage 34). Cypermethrin did not affect the fertilization process. Predominant sublethal effects in embryos included transient visceral edemas intimately associated to the gall bladder with subsequent pericardial edemas. Other sublethal effects were observed in surviving larvae and included spastic movements with or without ability to respond to stimulus (>/=6.3 microg cypermethrin/L), spinal curvatures, and delayed or absence of swim bladder inflation (>/=12.5 microg cypermethrin/L). The exposure of the gametes (stage 0) or animals during earlier embryonic development (stages 9 or 29) was not a critical window for cypermethrin exposure. Although the incidence of edemas in embryos occurred mainly during exposure of these early developmental stages, embryo and larva lethality and the incidence of transient sublethal effects in hatchlings showed that the later exposure window (stage 34) was the most sensitive. The stage 34 group involved advanced organogenetic stages in which the chorion partially degraded before hatching. Our studies reinforced the idea that a combination of morphologic and functional impairment evaluation is a more sensitive response to developmental toxicants than morphologic defects alone.

Sexing of murine and bovine embryos by developmental arrest induced by high-titer H-Y antisera

Murine and bovine embryos at the late morula stage were cultured in medium containing high-titer rat H-Y antisera. After 12 h of incubation, embryos blocked at the late morulae stage were classified as males and those at the blastocyst stage were classified as females. Sexing of murine embryos by PCR and cytogenetics revealed that 83% of the embryos classified as males and 82% of those classified as females had their sex correctly predicted ( P < 0.05). Bovine embryos were transferred to recipient females. Pregnancy rates were 71.4% (10/14) for embryos classified as males and 68.8% (11/16) for embryos classified as females. The sex was correctly predicted for 80% (8/10) of the embryos classified as males and for 81.8% (9/11) of those classified as females (overall accuracy, 80.9%, P < 0.05). Therefore, the induction of developmental arrest by high-titer male-specific antisera was an efficient strategy for non-invasive embryo sexing. The procedure was straightforward and has considerable commercial potential for sexing bovine embryos.

Inositol transport in preimplantation rabbit embryos: effects of embryo stage, sodium, osmolality and metabolic inhibitors

The preimplantation period in the rabbit consists of a 3 day cleavage stage during which the number of cells increases with little change in embryo size, followed by a 3-4 day blastocyst stage during which the inner cell mass, the blastocoel and the trophectodermal layer are formed and the embryo grows rapidly in size and protein content. This study used [(3)H]inositol to investigate the transport of inositol, an essential component of the phosphatidylinositol signal transduction system, over the 6 days of preimplantation development by rabbit embryos. In the presence of 15 micromol inositol l(-1) in the incubation medium, there was a small linear increase in inositol uptake from 0.07 pmol per embryo per h at the one-cell stage (day 1) to 0.135 pmol at the late morula (day 3) stage. Inositol uptake increased to 0.58 pmol per embryo per h for early blastocysts (day 4) and 23.7 pmol for late blastocysts (day 6). There was a significant linear relationship between inositol uptake and blastocyst diameter and surface area. Efflux of inositol from early morulae was minimal (about 1.25% of embryo content per h), whereas efflux from mid-blastocysts (day 5) was much greater (about 15.6% of embryo content per h). Efflux of inositol from both early morulae and mid-blastocysts was increased by decreasing the osmolality of the incubation medium. Varying the osmolality had no effect on inositol uptake up to 2 h. Inositol uptake was dependent on sodium in cleavage-stage embryos but independent of sodium in blastocyst stages. In early morulae, inositol uptake was inhibited by glucose and the sodium-dependent hexose transport inhibitor, phloridzin, but not by the facilitated transport inhibitor, phloretin. Inositol uptake in early morulae was saturable; estimates of 0.227 and 0.288 pmol per morula per h for V(max) and 0.045 and 0.038 micromol l(-1) for K(m) were obtained for sodium-dependent transport in two separate experiments. All of these results are consistent with the hypothesis that transport in cleavage stages occurs via a sodium myo-inositol transporter (SMIT) protein. Uptake in blastocysts was non-saturable. Uptake into blastocysts appeared to take place by a transcellular rather than a paracellular route.