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

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

Haploid maternal genome derived from early diplotene oocytes can substitute for the female pronucleus in preimplantation mouse development

We describe the preimplantation development of mouse embryos that have received the haploid maternal genome derived from early diplotene nuclei of primordial oocytes (PO). Two generations of recipient egg-cells were used. Induction of two meiotic divisions of the PO nucleus and the reduction of the number of chromosomes to the haploid level were achieved in preovulatory oocytes (primary recipients). The developmental potential of the obtained haploid genome was examined in zygotes (secondary recipients). The nuclei of PO obtained from newborn mice were transferred by cell electrofusion to in vitro maturing (IVM) and enucleated preovulatory mouse oocytes. The reconstructed oocytes which had completed maturation, i.e., reached metaphase II, were artificially activated (8% ethanol + CHX). Activated oocytes were used as donors of haploid pronuclei of PO origin which were transferred (by karyoplast fusion) to partially enucleated zygotes containing only the male pronucleus. Thus, reconstituted zygotes were transplanted to the ligated oviducts of the cycling mice and 27% of them developed to the blastocyst stage. Our experiments demonstrate that 1) the nucleus of PO can be induced to premature meiotic divisions in an IVM enucleated preovulatory oocyte; 2) in the presence of a normal male pronucleus, the haploid pronucleus of PO origin can substitute for a female pronucleus during preimplantation development. Mol. Reprod. Dev. 48:488–495, 1997. © 1997 Wiley-Liss, Inc.     

Developmental arrest of fertilized eggs from the B6.YDOM sex-reversed female mouse

When the Y chromosome of a Mus musculus domesticus mouse strain is placed onto the C57BL/6J (B6) inbred background, the XY progeny develop ovaries or ovotestes but never normal testes during fetal life. While some of the hermaphroditic males become fertile, none of the XY females produces litters. Here, we examined the fertility and development of oocytes derived from the XY female mouse. With or without preceding injection of gonadotropins, female mice were mated with normal B6 males, and their embryos were recovered at various developmental stages. In vitro fertilization was performed with the eggs recovered from the oviduct after treatment with go-nadotropins. Development of embryos was examined by both light and electron microscopy. The results indicate that the oocytes released from the B6.Y^DOM ovary were efficiently fertilized and often initiated the first cell cleavage, but all embryos died during early preimplantation periods. Even when oocytes were fertilized in vitro, minimizing their exposure to the XY oviduct/uterus environment, most embryos died at the 1- or 2-cell stage. A few exceptional embryos reached the 4- or 8-cell stage, but abnormalities were evident in both nuclear and cytoplasmic structures of all embryos. After cleavage, neighbouring blastomeres were only loosely associated, and microvilli were abundant at the intercellular interfaces. We postulate that oocytes of the B.6.Y^DOM female mouse become defective during XY ovarian differentiation, and, hence, fail to proceed through normal embryonic development. © 1994 Wiley-Liss, Inc.     

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 μM 2',3'-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.     

Effect of ATM and HDAC Inhibition on Etoposide-Induced DNA Damage in Porcine Early Preimplantation Embryos

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.     

Presence and possible role of C-ras and nuclear (c-fos and c-jun) proto-oncogene products in preimplantation embryonic development in mice

The presence and possible role of products of nuclear (c-fos and c-jun) and c-ras proto-oncogenes were investigated in preimplantation embryonic development in mice. Polyclonal antibodies to c-fos or c-jun proto-oncogene products did not affect development of in vitro-cultured embryos from two-cell to morula or from morula to late blastocyst stages. However, v-H-ras monoclonal antibody (mAb) to c-ras protein (p²¹), although it did not inhibit the development of in vitro-cultured embryos from two-cell to morula stages, it significantly (P < .001–.005) inhibited the development of morula to late blastocyst stages in a dose-dependent manner. The effects of v-H-ras mAb were specific, since immunoabsorption with synthetic ras peptide completely blocked inhibitory effects of v-H-ras mAb. Neither c-fos nor c-jun antibodies reacted with specific proteins corresponding to c-fos (62 kDa) and c-jun (39 kDa) products on the Western blots of various murine ova/embryos extracts. However, the c-fos and c-jun antibodies reacted with 62 and 39 kDa protein bands, respectively, on the blot of NIH 3T3 cells extract. The v-H-ras mAb specifically identified 21 ± 3 kDa protein corresponding to c-ras p²¹ on the blots of early as well as late blastocyst extracts. The rat control ascites IgG₁ did not react with any protein band on the blots of various ova/embryo extracts. The reactions of v-H-ras mAb on the Western blots of blastocyst extracts were specific, since immunoabsorbed antibody was unable to react with any specific band on blots of early or late blastocyst extract. These results were further confirmed by immunoprecipitation procedure utilizing v-H-ras mAb. Again, the v-H-ras mAb immunoprecipitated a 21 kDa band from early as well as late blastocyst extracts. The rat control ascites IgG₁ did not react with any band corresponding to p ²¹ in the immunoprecipitation procedure. These results suggest that the specific products of nuclear proto-oncogenes, the c-fos and c-jun, are not detected in murine ova and preimplantation embryos, and the respective antibodies do not inhibit embryogenesis, indicating that they may not play a major role in early embryonic development. On the other hand, the product of c-ras proto-oncogene is specifically expressed in the blastocyst-stage embryos and may have a possible role in preimplantation embryonic development in mice. © 1993 Wiley-Liss, Inc.     

Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo

Investigations were conducted to quantitate polyadenylic acid and estimate the synthesis of polyadenylated RNA in mouse embryos at several stages of preimplantation development. Poly(A) was assayed by molecular hybridization of total embryonic RNA with [³H]polyuridylic acid. The mean values of poly(A) in the ovulated oocytes and in the one-cell, two-cell, and blastocyst stages of the embryo were 1.9, 1.6, 0.68, and 3.8 pg, respectively. Synthesis of polyadenylated RNA was estimated by affinity chromatography of [³H]uridine-labeled embryo RNA on oligo(dT)-cellulose. The proportions of newly synthesized RNA bound by oligo(dT)-cellulose at the 2-cell, 8- to 16-cell, and blastocyst stages were 6.7, 3.5, and 3.3%, respectively. These results suggest that significant quantities of maternal mRNA are present during early development of the mouse, but that polyadenylation of RNA transcribed from the embryonic genome occurs as early as the two-cell stage.     

Promotion of Human Early Embryonic Development and Blastocyst Outgrowth In Vitro Using Autocrine/Paracrine Growth Factors

Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6–8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner’s criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.     

Effect of the presence and location of corpus luteum on competence of bovine cumulus-oocyte complexes

This study aimed to determine the effect of presence of the corpus luteum (CL) and its influence on cumulus–oocyte complexes (COCs) obtained from the ipsilateral or contralateral ovary in bovine on the recovery and capacity of the oocytes to sustain mono-spermic fertilization, undergo preimplantation development, and develop to the blastocyst stage. Ovaries were collected at a local slaughterhouse and kept in pairs corresponding to the same animal. In the first experiment the variables evaluated were compared between cows with (CCL⁺) and without (CCL^-) CL, and for the second experiment, comparisons were made between ovaries with an ipsilateral (CL⁺), contralateral (CL⁻), and no (NCL). The recovery rate of COCs was higher in ovaries from CCL⁻ cows, and a higher proportion of grade 1 COCs were recovered from this group. A higher proportion of metaphase I oocytes at 7 h of maturation, and a higher rate of cleavage were observed in the CCL⁺ group; however, a higher proportion of embryos were obtained from the CCL⁻ group. Besides, COCs from the CL⁺ group had a lower proportion of grades 1 and 2 morphological qualities, lower rate of metaphase II oocytes at 22 h of maturation, and lower rate of formation of two pronuclei, whereas a higher proportion of unfertilized oocytes after in vitro fertilization. On the other hand, the COCs from the CL⁻ group displayed a lower proportion of oocytes with more than two pronuclei, higher cleavage rate, and higher final blastocyst production were obtained when compared to CL+. Thus, the effects of CL on the competence of bovine COCs are different depending on the anatomical proximity of their location in the animal, negatively affecting the quality of COCs located in the same ovary, but not having negative effects on the competence of COCs in the ovaries contralateral to their location.     

Molecular differentiation of the rabbit ovum. II. During the preimplantation development of in vivo and in vitro matured oocytes

The developmental capacity of in vitro matured rabbit oocytes was assessed after transfer to inseminated, ovariectomized recipients such that fertilization and preimplantation development occurred in vivo. The results demonstrate that of the total number of transferred oocytes (1) 75% were fertilized, (2) 50% underwent cleavage, and (3) 13% developed into expanded blastocysts. By light microscopic criteria, embryos recovered at representative stages of preimplantation development were morphologically indistinguishable from embryos recovered at comparable stages from normally mated animals. Autoradiographs produced by high resolution, two-dimensional polyacrylamide gel electrophoresis demonstrated that changes in the pattern of polypeptide synthesis during the preimplantation stages were directly and entirely comparable for embryos derived either from normally mated animals or from in vivo or in vitro matured and transferred oocytes. Up to approximately the eight-cell stage, the translational patterns indicate the progressive disappearance of numerous oocyte-characteristic polypeptides from the autoradiographs as well as the appearance of some new species of polypeptides. Between the eight-cell and early blastocyst period, extensive and complex changes (qualitative and quantitative) occur in the patterns, whereas, in contrast, the phase of blastocyst growth and expansion that occurs during the latter portion of the preimplantation period is characterized by a fairly uniform and constant translational pattern.     

Clonal lines of transgenic fibroblast cells derived from the same fetus result in different development when used for nuclear transfer in pigs

Different factors are believed to influence the outcome of nuclear transfer (NT) experiments. Besides the cell cycle stage of both recipient cytoplast and donor karyoplast, the origin of the donor cells (embryonic, fetal, and adult) is of interest. We compared in vitro development of NT embryos derived from small serum-starved (G0) or small cycling (G1) porcine fetal fibroblast cells. Serum starvation did not have a positive effect on cleavage rate or the percentage of embryos that developed to the morula and blastocyst stages. Next, we investigated the development of porcine NT embryos derived from different transgenic clonal cell lines that had originated from the same fetus. When different clonal lines of fetal fibroblasts were fused to enucleated metaphase II oocytes, differences in fusion rates as well as in development to the morula and blastocyst stages were observed (P < 0.05). When oocytes derived from sow ovaries were used as recipient cytoplasts, significantly better cleavage (P = 0.03) and blastocyst formation (P < 0.014) was obtained when compared with oocytes derived from gilts. Our data indicate that not only different cell lines, but also different clones derived from one primary cell line, result in different development when used for NT. In addition, the use of sow oocytes as a cytoplast source also improves the efficiency of NT experiments.     

Nuclear transfer in sheep embryos: The effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes

The developmental ability of nuclear transplant sheep embryos derived from in vitro matured oocytes was studied by controlling cell-cycle coordination of donor embryonic nuclei and recipient cytoplasts. Oocytes were recovered from nonatretic antral follicles of adult sheep ovaries and cocultured with follicle shells in M199-based medium supplemented with gonadotrophins in a nonstatic system. Effective activation of IVM oocytes was obtained by applying two pulses of 1.0 kv/cm 22 min apart in inositol-based electroporation medium to oocytes matured in vitro for 27 hr. Synthesis of DNA (S-phase) was assessed by BrdU incorporation and was found to initiate around 5 hpa (hours postactivation) and to persist until 18 hpa. Mitotic blastomeres were induced by treating embryos with 6.6 μM nocodazole for 14–17 hr. Three types of transfers were compared directly: “S → S,” early embryonic nuclei (mostly in S-phase) were transferred to presumptive S-phase cytoplasts; “M → MII,” nocodazole-treated embryonic nuclei (most in M-phase) were transferred to MII-phase cytoplasts; and control (S → MII), conventional nuclear transfer of fusion and activation simultaneously. The results showed that fusion and recovery rates did not differ among the three groups. However, after 6 days of in vivo culture, the morula and blastocyst formation rate was significantly higher for the M → MII combination than for the control (28.3% vs. 8.1%, P < 0.05), while no significant differences in developmental rate were observed between S → S and M → MII, and between S → S and control, though developmental rate was also increased for S → S compared to control (20.9% vs. 8.1%, P > 0.05). Transfer of blastocysts derived from M → MII or S → S nuclear cytoplasmic reconstitution to synchronized recipient ewes resulted in the birth of lambs. These data suggest that in vitro matured oocytes can support full-term development of nuclear transplant sheep embryos when the cell cycle of nucleus and cytoplasm is coordinated, and that M → MII nuclear transfer might be an efficient and simple way to improve the developmental competence of the reconstituted embryos. Mol. Reprod. Dev. 47:255–264, 1997. © 1997 Wiley-Liss, Inc.     

BMP4 plays a role in apoptosis during human preimplantation development

Comprehensive understanding of lineage differentiation and apoptosis processes is important to increase our knowledge of human preimplantation development in vitro. We know that BMP signaling is important for different processes during mammalian development. In mouse preimplantation embryos, BMP signaling has been shown to play a role in the differentiation into extra‐embryonic trophectoderm (TE) and primitive endoderm (PE). In this study, we aimed to investigate the effect of bone morphogenetic protein 4 (BMP4) supplementation on human preimplantation embryos cultured in vitro. The BMP4 treatment impaired human blastocyst formation. No differences in the expression of the early lineage markers NANOG, CDX2, GATA3, and GATA6 were found between BMP4‐treated embryos and controls. Instead, BMP4 supplementation triggered apoptosis in the human blastocyst. We focused on P53, which is known to play a major role in the apoptosis. In BMP4‐treated embryos, the P53 responsive gene expression was not altered; however, the P53 deacetylase SIRT1 was downregulated and acetylated P53 was increased in mitochondria. Altogether, our findings suggest that BMP4 plays a role in the apoptosis during human preimplantation development.     

Dynamics of zonula occludens-2 expression during preimplantation embryonic development in the hamster

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster.