Morula-blastocyst Transition Research Articles

METTL3-mediated m6A methylation negatively modulates autophagy to support porcine blastocyst development‡.

N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Here, we show that METTL3-mediated m6A methylation sustains porcine blastocyst development via negatively modulating autophagy. We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 (a key regulator of autophagy) and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3'UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell qPCR revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Taken together, these results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.

Tripolar mitosis and partitioning of the genome arrests human preimplantation development in vitro

Following in vitro fertilisation (IVF), only about half of normally fertilised human embryos develop beyond cleavage and morula stages to form a blastocyst in vitro. Although many human embryos are aneuploid and genomically imbalanced, often as a result of meiotic errors inherited in the oocyte, these aneuploidies persist at the blastocyst stage and the reasons for the high incidence of developmental arrest remain unknown. Here we use genome-wide SNP genotyping and meiomapping of both polar bodies to identify maternal meiotic errors and karyomapping to fingerprint the parental chromosomes in single cells from disaggregated arrested embryos and excluded cells from blastocysts. Combined with time lapse imaging of development in culture, we demonstrate that tripolar mitoses in early cleavage cause chromosome dispersal to clones of cells with identical or closely related sub-diploid chromosome profiles resulting in intercellular partitioning of the genome. We hypothesise that following zygotic genome activation (ZGA), the combination of genomic imbalance and partial genome loss disrupts the normal pattern of embryonic gene expression blocking development at the morula-blastocyst transition. Failure to coordinate the cell cycle in early cleavage and regulate centrosome duplication is therefore a major cause of human preimplantation developmental arrest in vitro.

Cell death is involved in sexual dimorphism during preimplantation development

In bovine preimplantation development, female embryos progress at lower rates and originate smaller blastocysts than male counterparts. Although sex-specific gene expression patterns are reported, when and how sex dimorphism is established is not clear. Differences among female and male early development can be useful for human assisted reproductive medicine, when X-linked disorders risk is detected, and for genetic breeding programs, especially in dairy cattle, which requires female animals for milk production. The aim of this study was to characterize the development of female and male embryos, attempting to identify sex effects during preimplantation development and the role of cell death in this process. Using sex-sorted semen from three different bulls for fertilization, we compared kinetics of bovine sex-specific embryos in six time points, and cell death was assessed in viable embryos. For kinetics analysis, we detected an increased population of female embryos arrested at 48 and 120h.p.i., suggesting this time points as delicate stages of development for female embryos that should be considered for testing improvement strategies for assisted reproductive technologies. Assessing viable embryos quality, we found 144h.p.i. is the first time point when viable embryos are phenotypically distinct: cell number is decreased, and apoptosis and cell fragmentation are increased in female embryos at this stage. These new results lead us to propose that sex dimorphism in viable embryos is established during morula-blastocyst transition, and cell death is involved in this process.

36 RESTORATION OF TELOMERE LENGTH IN CLONED PIG EMBRYOS DURING EARLY EMBRYOGENESIS IS NOT DEPENDENT ON TELOMERE LENGTH AND TYPE OF DONOR CELLS

Analyses on telomere length in cloned animals have revealed diverse results depending on the donor cell types. In mice and cattle, telomere length is reset during morula-blastocyst transition and the restoration is thought to be a telomerase-dependent process. However, it is still unknown whether the pattern of telomere elongation during this transition is dependent on donor cell types. In the present study, we examined the changes of telomere length during morula-blastocyst transition in cloned porcine embryos using different types of donor cell. Embryonic stem-like cells (ES), cumulus cells (C), fibroblasts at passages 7 and 10 (F7 and F10, respectively) were used as donor cells to produce NT embryos (ES, C, F7, and F10 groups, respectively). Telomere lengths of ES (35.8 ± 1.5 kb), C (24.4 ± 0.5 kb), P7 (18.7 ± 0.6 kb), and P10 (17.2 ± 0.1 kb) cells were significantly different. In contrast, cloned morulae in ES, C, F7, and F10 groups did not have any significant differences in telomere length (18.2 ± 0.3, 17.8 ± 0.7, 18.5 ± 0.3, and 18.4 ± 0.4 kb, respectively). Likewise, cloned blastocysts in ES, C, F7, and F10 groups had similar telomere length (22.3 ± 1.5, 23.5 ± 2.6, 20.2 ± 1.0, and 20.9 ± 1.0 kb, respectively). However, the telomere of the blastocyst was significantly longer (P < 0.05) compared with the morula in the respective group. Furthermore, relative telomerase activities of cloned morulae in ES, C, F7, and F10 groups (4.2 ± 0.4, 4.0 ± 0.5, 5.1 ± 0.4, and 4.9 ± 0.4, respectively) were significantly lower (P < 0.01) than those of cloned blastocysts in the same groups (8.2 ± 1.1, 8.6 ± 0.6, 12.5 ± 2.9, and 8.3 ± 1.1, respectively). The proportions of blastocysts in cloned embryos for ES, C, F7, and F10 groups (10.0 ± 5.2, 17.3 ± 2.9, 13.5 ± 2.9, and 13.1 ± 3.6%, respectively) did not significantly differ. Total cell numbers in blastocysts for ES, C, F7, and F10 groups (28.3 ± 2.9, 32.6 ± 3.6, 30.4 ± 3.1, and 27.4 ± 2.2, respectively) were not significantly different as well. In the present study, we found that the telomere elongation in cloned pig embryos occurs during morula-blastocyst transition. This is consistent with the previous findings in in vivo and in vitro fertilization and cloned embryos in cattle and mice. We also revealed that although different types of cells (ES, C, and F) or the same type of cells with different telomere length (F7 and F10) were used for NT, their resultant morulae and blastocysts had similar telomere length. This suggests that the telomere restoration during morula-blastocyst transition is independent of telomere length and type of donor cells. An increase in telomerase activity during morula-blastocyst transition indicates that the elongation of telomere length was likely a telomerase-dependent process. In conclusion, restoration of telomere length in cloned porcine embryos during morula-blastocyst transition was independent of telomere length and type of donor cells, and likely a telomerase-dependent process.

91 SPERMATOZOA TELOMERE LENGTH DETERMINES EMBRYONIC TELOMERE LENGTH BEFORE EMBRYONIC GENOME ACTIVATION

A critical issue for species integrity is the existence of a telomere elongation program during embryogenesis that ensures sufficient telomere reserves in mammalian newborns. Two different mechanisms have been reported to act on telomere elongation during early embryogenesis: first, the telomerase, the ribonucleoprotein that adds telomeric repeats onto the chromosome ends, known to be responsible for the telomere lengthening at the morula-blastocyst transition in mice and bovine; second, in laboratory mice strains, mature oocytes increase the length of their relatively short telomeres between the 1-cell and 2-cell stages by a recombination or ALT-like pathway. In contrast, spermatozoa, the terminally differentiated male gametes, exhibit a very long telomere length (TL). The aim of this study was to clarify the potential role of the spermatozoa TL in the telomere lengthening occurring between oocyte and the 2-cell stage. For this purpose, we used 2 mouse species known to differ greatly in their TL [Mus musculus (hybrid C57CBAF1), long TL, and Mus spretus, short TL]. First, we compared relative TL in sperm samples from 5 age-matched males of each species by quantitative real-time PCR, with the numbers of telomere repeats being normalized, to the amount of DNA present in the sample (based on quantification of the Rn18S gene) by the comparative Ct method. Then, 1- and 2-cell embryos were produced by fertilizing Mus musculus oocytes with either Mus musculus or Mus spretus spermatozoa. The TL analysis in oocytes, zygotes, or 2-cell embryos was carried out by absolute quantification of telomere repeats by qPCR and normalized to the highest Ct observed value. Twenty to thirty samples per stage were analyzed, with each sample consisting in 2 matured oocytes, 2 zygotes, or one 2-cell embryo, to allow comparisons between stages. One-way ANOVA was used for statistical analysis. Mus spretus spermatozoa had significantly shorter telomeres than did Mus musculus (1.0 ± 0.1 v. 9.0 ± 1.5, respectively; P ≤ 0.01). The TL increased after fertilization from oocyte to zygote and 2-cell embryo stages in Mus musculus (1.0 ± 0.1, 1.5 ± 0.1, and 2.4 ± 0.2, respectively; P ≤ 0.01). In contrast, no differences were found in the TLs between the 3 stages in Mus spretus hybrids (oocyte: 1.0 ± 0.1; zygote: 1.0 ± 0.1; and 2-cell embryo: 1.0 ± 0.1), indicating that no elongation occurred after fertilization with spermatozoa with short telomeres. Herein, we demonstrated that before embryonic genome activation occurs, spermatozoa TL determines TL of the early embryo, suggesting that spermatozoon telomeres may act as recombination templates for early telomere lengthening right after syngamia.

Importance of culture conditions during the morula-to-blastocyst period on capacity of inner cell-mass cells of bovine blastocysts for establishment of self-renewing pluripotent cells

The hypothesis was tested that the pluripotency of the inner cell mass (ICM) of the bovine embryo is enhanced by the glycogen synthase kinase-3β inhibitor CHIR99021 and the MAPK1 and MAPK3 inhibitor PD032591. Treatment with the two inhibitors from Days 6 to 8 after insemination increased blastocyst steady state concentrations of mRNA for NANOG (P < 0.05) and SOX2 (P = 0.055) and tended to decrease (P = 0.09) expression of GATA6. To evaluate pluripotency, the inner cell mass was isolated by immunosurgery at Day 8, seeded on a feeder layer of bovine embryonic fibroblasts, and cultured in the presence of the inhibitors. Ten of 52 (19%) ICM from control embryos had primary outgrowth formation vs. 23 of 50 (46%) of the ICM from embryos cultured with inhibitors (P < 0.01). For ICM outgrowths from embryos cultured without inhibitors, colonies either did not persist through Passage 2 or became differentiated. In contrast, for the inhibitor group, four colonies survived beyond Passage 2, and one line persisted for 19 passages. This cell line possessed alkaline phosphatase activity, expressed several genes characteristically expressed in pluripotent cells, and differentiated into embryoid bodies when cultured in the absence of the signal transduction inhibitors and the feeder layer. Propagation of the cells was difficult due to slow growth and inefficiency in survival through each passage. In conclusion, exposure to inhibitors during the morula-blastocyst transition facilitated formation of self-renewing pluripotent cell lines from bovine blastocysts.

Exposure to High Glucose During the Pre-Compaction Period Impacts on Transcriptomic Profile and ATP Content at the Morula-Blastocyst Transition in Bovine Embryos.

Exposure to High Glucose During the Pre-Compaction Period Impacts on Transcriptomic Profile and ATP Content at the Morula-Blastocyst Transition in Bovine Embryos.

Somatic Cell Nuclear Transfer Cloning: Practical Applications and Current Legislation

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

In vivo oocyte IGF-1 priming increases inner cell mass proliferation of in vitro-formed bovine blastocysts

Studies addressing the effects of supraphysiological levels of IGF-1 on oocyte developmental competence are relevant for unravelling conditions resulting in high bioavailability of IGF-1, such as the polycystic ovary syndrome (PCOS). This study investigated the effects of supraphysiological levels of IGF-1 during in vivo folliculogenesis on the morula-blastocyst transition in bovine embryos. Compacted morulae were non-surgically collected and frozen for subsequent mRNA expression analysis (IGF1R, IGBP3, TP53, AKT1, SLC2A1, SLC2A3, and SLC2A8), or underwent confocal microscopy analysis for protein localization (IGF1R and TP53), or were cultured in vitro for 24 h. In vitro-formed blastocysts were subjected to differential cell staining. The mRNA expression of SLC2A8 was higher in morulae collected from cows treated with IGF-1. Both IGF1R and TP53 protein were present in the plasma membrane and cytoplasm. IGF-1 treatment did not affect protein localization of both IGF1R and TP53. In vitro-formed blastocysts derived from morulae recovered from IGF-1-treated cows displayed a higher number of cells in the inner cell mass (ICM). Total cell number (TCN) of in vitro-formed blastocysts was not affected. A higher mean ICM/TCN proportion was observed in in vitro-formed blastocysts derived from morulae collected from cows treated with IGF-1. The percentage of in vitro-formed blastocysts displaying a low ICM/TCN proportion was decreased by IGF-1 treatment. In vitro-formed blastocysts with a high ICM/TCN proportion were only detected in IGF-1 treated cows. Results show that even a short in vivo exposure of oocytes to a supraphysiological IGF-1 microenvironment can increase ICM cell proliferation in vitro during the morula to blastocyst transition.

MicroRNA regulation via DNA methylation during the morula to blastocyst transition in mice†

Epigenetic regulation is responsible for transcriptional silencing of genes and parental imprinting. This study addresses the question whether microRNAs (miRNAs) could be affected by DNA methylation during morula-blastocyst transition. Mouse embryos were treated with/without a DNA methyltransferase inhibitor (5-aza-2'-deoxycytidine, 5-aza-dC, 10 nM-5 μM). Changes of miRNAs were analyzed by quantitative real-time (Q-PCR)-based megaplex pre-amp microRNA assays. Development from morula to blastocyst in mice was inhibited by 5-aza-dC in a dose-dependent manner (10 nM-5 μM), with half of the embryos arrested at morula stage when treated with levels of 5-aza-dC as low as 50 nM. In total, 48 down-regulated microRNAs and 17 up-regulated microRNAs (≥5-fold changes) were identified after 5-aza-dC treatment, including let-7e, mir-20a, mir-21, mir-34b, mir-128b and mir-452. Their predicted targets were selected based on software analysis, published databases and further confirmed by Q-PCR. At least eight targets, including dnmt3a, jagged 1, sp1, edg2, abcg4, numa1, tmsb10 and csf1r were confirmed. In conclusion, 5-aza-dC-modified microRNA profiles and identification of the microRNA's targets during the morula to blastocyst stage in mice provide information that helps us to explore the relationship between fertility, microRNA regulation and epigenetic intervention.

The Unfolded Protein Response Contributes to Preimplantation Mouse Embryo Death in the DDK Syndrome1

DDK syndrome is the polar-lethal embryonic death that occurs at the morula-blastocyst transition when female mice of the DDK strain are mated with males from many other inbred strains (so-called alien males). Embryonic death is caused by incompatibility between a DDK oocyte factor and an alien male gene, both of which map to the Om locus on mouse chromosome 11. We compared global transcription patterns of DDK x DDK embryos (high viability) and DDK x C57BL/6 embryos (low viability) at the morula stage, approximately 24 h before any morphological manifestations of DDK syndrome are observed. Of the transcripts that are differentially more abundant in the DDK x C57BL/6 embryos, many are the products of genes induced by the "unfolded protein response." We confirmed by quantitative RT-PCR that a number of genes in this pathway are upregulated in the DDK x C57BL/6 embryos. Immunostaining of the endoplasmic reticulum (ER) marker BIP/GRP78 (immunoglobin-binding protein/glucose-regulated protein of 78 kDa), official symbol HSPA5, heat shock protein 5 revealed an accompanying abnormal HSPA5 accumulation and ER structure in the DDK x C57BL/6 embryos. Immunostaining for HERPUD1 (homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1) and ATF4 (activating transcription factor 4) also revealed accumulation of these stress-response products. Our results indicate that the unfolded protein response is induced in embryos destined to die of DDK syndrome and that the embryonic death observed is associated with inability to resolve the associated ER stress.

103 ECTOPIC EXPRESSION OF TELOMERASE IN BOVINE PREIMPLANTATION EMBRYOS

Recently, we have demonstrated a stage-specific increase of telomere length at morula-blastocyst transition in bovine and murine embryos (Schaetzlein et al. 2004 PNAS 101, 8034–8038). Telomeres are composed of repetitive hexanucleotide sequences (TTAGGG) encompassing several kilobasepairs and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage up to the morula stage, telomerase is not active or only found at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) was selected as the target molecule due to its 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from local abattoir followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. In preliminary tests, it was shown that co-injection of green fluorescent protein (GFP) and red fluorescent protein (dsRed) constructs resulted in the simultaneous expression of the 2 proteins. The onset of fluorescent protein expression was recorded 30 to 40 hours after injection by fluorescence microscopy. Because all cDNA were driven by the cytomegalovirus (CMV) promoter, it was assumed that hTERT is expressed in parallel. In the main experiment, 2 constructs encoding human TERT and GFP were co-injected to allow live separation of embryos. A total of 400 bovine embryos were injected, 209 (53%) of the treated embryos showed specific GFP fluorescence; out of a total of 104 blastocysts (26%), 55 showed GFP fluorescence (53%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic human TERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. Surprisingly, expression pattern of the endogenous TERT revealed a transient increase at the 2–4 cell stage and a major increase at the blastocyst stage. The mRNA level of the ectopic hTERT started to rise from 4 to 8 cell stage and remained high up to the morula stage. Currently, qFISH and TRAP techniques are being employed to assess enzymatic activity of hTERT and to quantitatively determine telomere length. In conclusion, this study demonstrates the ectopic expression of TERT and fluorescent proteins in early embryos; overexpression of TERT may facilitate the derivation of bovine ES cells. Supported by DFG and Goyaike S.A.A.C.I. Y.F.

Up-regulation of glucose metabolism during male pronucleus formation determines the early onset of the s phase in bovine zygotes.

After in vitro fertilization with spermatozoa from bulls with high in vitro fertility, a beneficial paternal effect is manifested during the G1 phase of the first cell cycle. This benefit determines an earlier onset of the first S phase, and then a successful morula-blastocyst transition 7 days later. We hypothesized that the origin of the paternal effect could be a shift of the metabolism of the fertilized oocyte, because in mice, sperm decondensation is responsible for a dramatic increase in glucose metabolism. In this study we investigated the interaction between both pronuclei and compared glycolysis and pentose phosphate pathway (PPP) activities in bovine oocytes fertilized with spermatozoa from bulls of high or low fertility. Here we demonstrate that male pronucleus formation is necessary for the onset of the S phase in the female pronucleus, and that the component promoting an early S phase in both pronuclei is metabolic and linked to an up-regulation of the PPP during the male pronucleus formation. This long-lasting paternal effect is more evidence of the important role of epigenetic control during early embryo development.

Preimplantation embryo morphology following early luteal phase anti-nidatory treatment with mifepristone (RU486) in the rhesus monkey.

The ultrastructural characteristics of peri-implantation stage embryos recovered on day 6 after ovulation from rhesus monkeys with or without mifepristone (RU486) treatment during the early luteal phase were examined in the present study. Monkeys were randomly allocated to two groups; group 1 animals were injected s.c. with 2 ml vehicle (1:4, benzyl benzoate: olive oil, v/v, n = 21) and group 2 animals received a single dose of mifepristone (2 mg/kg body weight, w/v, n = 30) in the same volume of vehicle on day 2 after ovulation in mated cycles. On day 6 after ovulation, female monkeys of both groups were laparotomized and their reproductive tracts were flushed to retrieve preimplantation stage embryos. Embryos that showed frank degeneration or desynchrony on gross microscopical examination were not included in the present study. Preimplantation embryo growth on day 6 after ovulation was significantly (P < 0.05) affected in the morula-blastocyst transition stage in mifepristone-treated monkeys compared with that in the control group of monkeys. Ultrastructurally, administration of mifepristone on day 2 after ovulation depressed preimplantation stage embryo development, characterized by loss of cell polarity, lack of mitochondrial maturity, and lack of differentiation in trophoblast cells. Furthermore, preimplantation embryos from mifepristone-treated animals displayed a higher occurrence of inter-blastomere space, intra-cytoplasmic vacuoles, myelinoid bodies, accumulation of lipid droplets, lysosomes, lipofuscins, autophagosomes and multivesicular bodies. Collectively, it appears that the developmental potential of preimplantation embryos was significantly compromised in mifepristone-treated cycles.

Beta-catenin is a major tyrosine-phosphorylated protein during mouse oocyte maturation and preimplantation development.

During mouse preimplantation development, the components of the E-cadherin-catenin complex are derived from both maternal and zygotic gene activity and the adhesion complex is increasingly accumulated and stored in a nonfunctional form, ready to be used for compaction and the formation of the trophectoderm cell layer (Ohsugi et al., Dev. Dyn. 206:391-402, 1996). Here, we show that beta-catenin is a major tyrosine-phosphorylated protein in oocytes and early cleavage-stage embryos and that the relative amount of phosphorylated beta-catenin is greatly reduced during the morula-blastocyst transition. Peptide-specific antibodies indicate that beta-catenin undergoes conformational changes and/or that the carboxy-terminal region of beta-catenin is blocked during preimplantation development. Moreover, the availability of a carboxy-terminal epitope seems to depend on the tyrosine phosphorylation state of beta-catenin and becomes unmasked when oocytes are treated with the tyrosine kinase inhibitor genistein. Our results suggest that tyrosine phosphorylation of beta-catenin represents a molecular mechanism to keep the accumulating E-cadherin adhesion complex in a nonfunctional form. Dev Dyn 1999;216:168-176.

Early luteal phase administration of mifepristone inhibits preimplantation embryo development and viability in the rhesus monkey.

It is generally believed that progesterone is essential for inducing the changes in oviduct and uterus necessary for embryo viability and implantation in a number of mammalian species. The aim of this study was, in the rhesus monkey, to examine in conception cycles with and without early luteal phase antiprogestin (mifepristone; RU 486) treatment: (i) the growth status of preimplantation embryos and (ii) the implantation ability of the preimplantation embryo after transfer to a synchronous-cycle surrogate recipient. A total of 43 proven fertile rhesus monkeys were randomly placed in the control (group 1, n = 18) and mifepristone (group 2, n = 25) groups. All monkeys cohabited with proven fertile male monkeys on cycle days 8-16 and were injected with vehicle alone [benzyl benzoate:olive oil, 1:4 (v/v), s.c.] for group 1 and with mifepristone (2 mg/kg body weight s.c.) for group 2, on day 2 after the presumed day of ovulation. A total of 12 preimplantation embryos [premorula (n = 1), morula (n = 2), zona-encased (n = 7) and zona-free (n = 1) blastocysts and degenerate embryos (n = 1)] were recovered from 17 ovulatory, mated cycles in group 1 on day 6 after ovulation. In group 2, of the 23 ovulated cycles, 12 preimplantation embryos [premorula (n = 2), morula (n = 7), zona-encased blastocyst (n = 1), and degenerate embryos (n = 2)] were retrieved. Despite no significant difference in the recovery rate between the two groups, early luteal phase RU 486 exposure induced delay (P < 0.01) in preimplantation embryo growth, primarily at the morula-blastocyst transition stage. Nine of the embryos from group 1 and seven of the embryos from group 2 recovered on day 6 were transferred to naturally synchronized, non-mated and untreated surrogate recipients. In group 1, five embryos implanted (55%) and, of these, three (60%) gave rise to live infants through natural delivery; implantation was assessed from extension of the cycle (i.e. no menstrual bleeding) and rise in concentrations of oestradiol and progesterone from day 10 of conception; rectal palpation was performed on cycle day 50 to confirm clinical pregnancy. In group 2, however, there was not a single case of establishment of pregnancy following transfer of embryos retrieved from mifepristone-exposed monkeys. Thus, preimplantation embryos recovered from RU 486-exposed monkeys failed to establish evolutive implantation and pregnancy, while significant (P < 0.02) success was observed in transfers of embryos from the control group. We postulate that progesterone-mediated actions are involved in mediating the growth and viability of preimplantation-stage embryos in the rhesus monkey.

In vitro co-culture of in vitro fertilized (IVF) bovine embryos with different cell monolayers

Studies addressing the effects of supraphysiological levels of IGF-1 on oocyte developmental competence are relevant for unravelling conditions resulting in high bioavailability of IGF-1, such as the polycystic ovary syndrome (PCOS). This study investigated the effects of supraphysiological levels of IGF-1 during in vivo folliculogenesis on the morula-blastocyst transition in bovine embryos. Compacted morulae were non-surgically collected and frozen for subsequent mRNA expression analysis (IGF1R, IGBP3, TP53, AKT1, SLC2A1, SLC2A3, and SLC2A8), or underwent confocal microscopy analysis for protein localization (IGF1R and TP53), or were cultured in vitro for 24 h. In vitro-formed blastocysts were subjected to differential cell staining. The mRNA expression of SLC2A8 was higher in morulae collected from cows treated with IGF-1. Both IGF1R and TP53 protein were present in the plasma membrane and cytoplasm. IGF-1 treatment did not affect protein localization of both IGF1R and TP53. In vitro-formed blastocysts derived from morulae recovered from IGF-1-treated cows displayed a higher number of cells in the inner cell mass (ICM). Total cell number (TCN) of in vitro-formed blastocysts was not affected. A higher mean ICM/TCN proportion was observed in in vitro-formed blastocysts derived from morulae collected from cows treated with IGF-1. The percentage of in vitro-formed blastocysts displaying a low ICM/TCN proportion was decreased by IGF-1 treatment. In vitro-formed blastocysts with a high ICM/TCN proportion were only detected in IGF-1 treated cows. Results show that even a short in vivo exposure of oocytes to a supraphysiological IGF-1 microenvironment can increase ICM cell proliferation in vitro during the morula to blastocyst transition.

The Effects of Plasminogen on in Vitro Ovine Embryo Development1

Plasminogen activator production by ovine embryos and the effects of plasminogen on ovine embryo development and zona pellucida integrity were evaluated. Eight-cell to sixteen-cell embryos were cultured in Whitten's medium containing 0, 60, or 120 micrograms/ml plasminogen. Plasmin and plasminogen activator concentrations in the medium were determined by a caseinolytic assay. More blastocysts hatched in medium containing 60 and 120 micrograms/ml plasminogen (33 and 21%, respectively) than 0 microgram/ml plasminogen (0%; p less than 0.05). Zona pellucida dissolution time in acidified phosphate-buffered saline was less after incubation in medium with 60 and 120 micrograms/ml plasminogen (7.2 and 5.9 min, respectively) than 0 microgram/ml plasminogen (9.4 min; p less than 0.05). Plasminogen activator production was low until the morula stage, increased during morula-blastocyst transition, and remained elevated through blastocoelic expansion and hatching. Zona pellucida solubility, plasminogen activator production, and plasminogen conversion to plasmin increased as embryonic stage advanced; however, plasminogen activator production and plasmin conversion to plasmin were poorly correlated with zona pellucida solubility. The results indicate that ovine embryos produce plasminogen activator, and plasmin can increase zona pellucida solubility; however, other factors may also be involved in altering zona pellucida integrity prior to hatching.

Blastocoel formation in half and double mouse embryos.

We have tested the alleged causal relationship between the peripheral sealing of the embryo and the formation of the blastocoel by observing with the electron microscope the diffusion of a colloidal lanthanum tracer into advanced morulae and early blastocysts grown from half and double mouse embryos. In these experimental conditions and in the controls, the peripheral sealing of the embryos always precedes the appearance of the blastocoel, supporting a causal relationship between both phenomena. By counting the number of cells present at the morula-blastocyst transition in half and double embryos, as well as in control embryos, we have observed that neither the total number of cells nor the number of cell cycles correlate well with the appearance of the blastocoel. Results suggest that blastocoel formation depends on time elapsed since fertilization and that the experimental manipulation diminishes the cleavage rate.