Developmental potential of bovine androgenetic and parthenogenetic embryos: a comparative study

In this study, we compared the developmental capacity of bovine haploid and diploid androgenetic and parthenogenetic embryos obtained by different methods. Androgenetic embryos were produced by piezo-intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) of enucleated oocytes with or without subsequent pronuclear transfer from one haploid zygote to another. Parthenogenetic embryos were obtained by activation of matured oocytes by ionomycin combined with cycloheximide or 6-dimethylaminopurine (DMAP) treatment. Only few cleaved androgenetic haploid embryos were able to compact (2.7%) and to form blastocysts (1.8%), while significantly more haploid parthenogenotes underwent compaction (24-37%) and a minority developed to blastocysts at different rates, depending on the activation procedure (cycloheximide 3%, 6-DMAP 14.5%). By contrast, development to blastocyst of diploid androgenotes, cloned androgenetic embryos, and parthenogenotes (31%, 39%, and 43%, respectively) was similar to IVF control embryos (35%). Cell number on Day 7 was higher for IVF blastocysts and decreased in consecutive order in diploid androgenotes, diploid parthenogenotes, and haploid uniparental embryos. Following transfer of diploid androgenetic embryos, a pregnancy was established and maintained up to Day 28.     

Fate of haploid parthenogenetic cells in mouse chimeras during development

The developmental capability of haploid parthenogenetic cells was investigated by studies on haploid parthenogenetic in equilibrium fertilized mouse chimeras. Two chimeras were born. One female chimera was smaller at birth and grew slower than its littermates. The distribution of haploid-derived cells in the chimeras was analyzed 11 months after their birth. Cells derived from haploid embryos were found only in the brain, eyes, pigment cells in hair follicles, and spleen, in which they constituted 30%, 20%, 10%, and less than 5%, respectively, of the cells. The correlation between the parthenogenetic contribution to the brain and growth retardation is discussed. All of the cells examined in these chimeric organs (brain and eyes) contained a diploid amount of DNA, suggesting that diploidization of the haploid parthenogenetic cells occurred during development. Possibly, the haploid state is not sufficient for cell growth, even in chimeras with fertilized embryos.     

Cell allocation and chromosomal complement of parthenogenetic and IVF bovine embryos

Considerable concerns exist regarding the quality of parthenogenetically activated embryos in terms of sufficient numbers of cells comprising the inner cell mass (ICM) and trophectoderm (TE) and the ploidy. Therefore, these two parameters were used to assess the quality of embryos derived from parthenogenetic activation by using calcium ionophore A23187 (CaI) followed by either 6‐dimethylaminopurine (6‐DMAP, 3.5 hr or 6.5 hr) or cycloheximide (CHX) plus cytochalasin D (CD). The conventional in vitro (IVF) produced embryos served as a control. Double staining of the parthenogenetic blastocysts showed that the total cell number (TC) of embryos from the 6‐DMAP 3.5 hr (87.0 ± 5.3) and CHX+CD (79.0 ± 6.1) groups was not different (P > 0.05), but was lower than that of control embryos (116.0 ± 5.8, P < 0.001). The mean ratios of inner cell mass (ICM) and trophectoderm (TE) cells in the 6‐DMAP 3.5 hr group (0.57 ± 0.04) and the control IVF group (0.50 ± 0.02) did not differ significantly. Both were higher than those of the CHX+CD group (0.36 ± 0.02; P < 0.05). Further analysis of chromosomal compositions of developing stage embryos at day four after IVF or parthenogenetic activation demonstrated that prolonged treatment with 6‐DMAP for 6.5 hr resulted in a significantly lower percentage of diploid embryos and a significantly higher percentage of abnormal ploidy embryos compared to treatment with 6‐DMAP for 3.5 hr or with CHX and IVF. In conclusion, parthenogenetic activation of bovine oocytes with CaI followed by 6‐DMAP for 3.5 hr could produce better quality embryos in terms of total cell numbers, the number of cells allocated to the ICM, and the ploidy of embryos. Mol. Reprod. Dev. 54:57–62, 1999. © 1999 Wiley‐Liss, Inc.     

Temporal and spatial selection against parthenogenetic cells during development of fetal chimeras

The fate of parthenogenetic cells was investigated during development of fetal and early postnatal chimeras. On day 13 of embryonic development, considerable contribution of parthenogenetic cells was observed in all tissues of chimeric embryos, although selection against parthenogenetic cells seemed to start before day 13. Between days 13 and 15 of development, parthenogenetic cells came under severe selective pressure, which was most striking in tongue. The disappearance of parthenogenetic cells from tongue coincided with the beginning of myoblast fusion in this tissue. Severe selection against parthenogenetic cells was also observed in pancreas and liver, although in the latter, parthenogenetic cells were eliminated later than in skeletal muscle or pancreas. In other tissues, parthenogenetic cells may persist and participate to a considerable extent throughout the gestation period and beyond, although a significant decrease was observed in all tissues. Parthenogenetic in equilibrium fertilized chimeras were significantly smaller than their non-chimeric littermates at all developmental stages. These results suggest that the absence of paternal chromosomes is largely incompatible with the maintenance of specific differentiated cell types. Furthermore, paternally derived genes seem to be involved in the regulation of proliferation of all cell types, as indicated by the drastic growth decceleration of parthenogenetic in equilibrium fertilized chimeras and the overall decrease of parthenogenetic cells during fetal development. Chromosomal imprinting may have a role in maintaining a balance between cell growth and differentiation during embryonic development. The major exception to the selective elimination of parthenogenetic cells appear to be the germ cells; viable offspring derived from parthenogenetic oocytes were detected, sometimes at a high frequency in litters of female parthenogenetic in equilibrium fertilized chimeras.     

Developmental competence of porcine parthenogenetic embryos relative to embryonic chromosomal abnormalities

Parthenogenetically activated (PA) embryos exhibit delayed development, a lower blastocyst rate, and less successful development in vitro compared to in vitro fertilized (IVF) embryos. To investigate the possible mechanisms for unsuccessful parthenogenetic development, this study analyzed the chromosome abnormalities and developmental potential of porcine PA embryos. Mature oocytes were electrically activated and cultured in Porcine Zygote Medium-3 (PZM3) supplemented with 3 mg/ml BSA for 6, 7, or 8 days. The percentage of PA blastocysts was lower than that of IVF embryos on days 6 and 7 (16.4 +/- 7.4 vs. 28.7 +/- 3.7; 10.9 +/- 2.8 vs. 21.5 +/- 4.7, P < 0.05; respectively), and the PA blastocysts had significantly fewer nuclei than IVF blastocysts (23.2 +/- 1.8 vs. 29.7 +/- 0.8; 29.7 +/- 3.3 vs. 32.0 +/- 2.4, P < 0.05). The percentage of abnormal PA embryos (including embryos with condensed nuclei, arrested embryos and fragmented embryos) was higher than that of IVF embryos (PA: 52.9 +/- 12.8 vs. 16.4 +/- 7.4 on day 6), and increased with culture time (71.9 +/- 12.1 vs. 10.9 +/- 2.8. on day 7,and 75.0 +/- 22.6 vs. 12.1 +/- 2.3 on day 8, P < 0.05). The Day-6 PA blastocysts (n = 147) were divided into three classes according to the total number of nuclei (<20, 20-39, >40) and into three groups according to the morphological diameter (<150, 150-180, >180 microm). Of the haploid blastocysts, 56.1% had less than 20 nuclei, and 71.5% were less than 150 microm in diameter. Of all (114) blastocysts suitable for analysis, 55.5% displayed chromosomal abnormalities. Among chromosomal abnormalities in PA blastocysts, haploid blastocysts were most prevalent (43.6%), while polyploidy (4.4%) and mixoploidy (7.7%) embryos were less prevalent. Chromosomal abnormalities of porcine PA embryos might contribute to a higher rate of abnormal embryonic development. We suggest that a careful consideration should be given when using the blastocysts with smaller size, and establishing the optimum culture condition for PA embryos development in vitro.     

Chimeras between parthenogenetic or androgenetic blastomeres and normal embryos: allocation to the inner cell mass and trophectoderm

A series of chimeras was generated by injecting single normal, parthenogenetic, or androgenetic blastomeres carrying transgenic markers under the zona pellucida of nontransgenic eight-cell embryos. These chimeras were cultured to the blastocyst stage and sectioned, and the transgenic component was detected by in situ hybridization. No statistically significant difference was found among the normal, parthenogenetic, and androgenetic chimeras in the number of chimeric blastocysts with a transgenic contribution to the inner cell mass (ICM), the trophectoderm, or both the ICM and trophectoderm. Since androgenetic and parthenogenetic cells were present in chimeras at a high frequency in both the ICM and trophectoderm at the blastocyst stage, but not in similar chimeras at late gastrulation, these cells must not respond normally to developmental signals subsequent to blastocyst formation and prior to late gastrulation.     

Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes

Microinjection of isolated mitochondria into oocytes is an effective method to introduce exogenous mitochondrial DNA. In nuclear transfer procedures in which donor cell mitochondria are transferred with nuclei into recipient oocytes; development and survival rates of reconstructed embryos may be also directly influenced by mitochondrial viability. Mitochondrial viability is dramatically affected by cell culture conditions, such as serum starvation prior to nuclear transfer. This study was conducted to examine the influence of exogenous mitochondria using bovine and mouse parthenogenetic models. Mitochondria were isolated from primary cells at confluency and after serum starvation. The bovine oocytes injected with serum-starved mitochondria showed lower rates of morula and blastocyst formation when compared to uninjected controls (P < 0.05). However, the developmental rates between non-starved mitochondria injection and controls were not different (P > 0.05). The murine oocytes injected with serum-starved mitochondria showed lower rates of development when compared with non-starved mitochondria and controls (P < 0.01). In contrast to mitochondria transfer, ooplasm transfer did not affect murine or bovine parthenogenetic development (P > 0.05). The overall results showed that injection of serum-starved mitochondria influenced parthenogenetic development of both bovine and murine oocytes. Our results illustrate that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development; particularly when using serum-starved cells as donor cells.     

Influence of sequence duration and number of electrical pulses upon rabbit oocyte activation and parthenogenetic in vitro development

Electroactivation of in vivo mature young rabbit oocytes was investigated here. The effects of four or eight electrical pulse treatment over 90, 150 or 270 min upon oocyte activation frequency and type, and even upon their subsequent in vitro development, were studied. The lowest activation frequency was observed after applying four-pulses over 90 min (54%). However, extending four-pulse treatment duration over 150 or 270 min induced more oocytes to activate (from 84% to 100%), as did the eight pulsing treatments (from 91% to 97%). With eight pulses, extending treatment duration improved the normal activation rates (from 47% to 76%; P<0.05). Nevertheless, the haploid morulae and blastocyst rates decreased significantly with extended eight pulsing treatment duration (morulae: from 94% to 41% and blastocysts: from 31% to 0%).     

The effect of 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and nuclear transfer embryos

The effects of activation by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and SCNT embryos were investigated. The results revealed that the blastocyst development of parthenogenetic embryos was significantly higher (P < 0.05) in 6-DMAP activated oocytes, compared to those activated with CHX (21.0 +/- 0.9 vs. 14.9 +/- 0.5, respectively). In contrast, the blastocyst frequencies did not significantly differ (P > 0.05) between the two activation treatment groups for SCNT embryos. The 6-DMAP or CHX treatment did not result in any significant difference in the blastocyst total cell number in either parthenote or SCNT embryos. The chromosomal analysis revealed that all the parthenogenetic embryos (100.0%) derived from 6-DMAP treatment, were chromosomally abnormal whereas in CHX-treated embryos, it was significantly lowered (93.6%, P < 0.05). Conversely, the proportions of chromosomally abnormal SCNT embryos did not significantly differ (P > 0.05) among the 6-DMAP and CHX- treated embryo groups (60.0% vs. 56.2%, respectively). This study demonstrated that oocyte activation agents such as DMAP and CHX have differing effects on meiotic or mitotic nuclei. The study also highlighted the feasibility of using bovine X and Y chromosome specific painting probes in sheep embryos.     

Microinjection of cytoplasm or mitochondria derived from somatic cells affects parthenogenetic development of murine oocytes

Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.     

Relationship between plasmid and chromosomal hemolysin determinants of Escherichia coli

Plasmid hemolysin (hly) determinants have been shown previously to comprise three cistrons (hlyA, hlyB, hlyC), coding for the synthesis and transport of hemolysin. Using recombinant plasmids as specific probes for these cistrons, we were able to analyze the chromosomal hly determinants of nine Escherichia coli strains which belonged to serotypes O4, O6, O18, and O75 and were isolated from urinary tract infections and fecal flora. The chromosomal hly genes shared extensive sequence homology with the cloned plasmid hly determinant. Nevertheless, small differences were observed, and these were found to lie mainly within cistron A (hlyA), which has been shown to determine the hemolysin protein itself. These fine variations were not specific for the O-serotype.     

Effects of 5-azacytidine on the development of parthenogenetic mouse embryos

This study describes the effects of 5-azacytidine (5-azaC) on the development of diploid parthenogenetic embryos (PE) of CBA, C57BL/6 and (CBA × C57BL/6)F₁ mice in vitro at the 1-cell or the blastocyst stage or in vivo after implantation. Our findings indicate that genomic imprinting is modulated by genetic background. Non-fertilized C57BL/6 eggs form diploid parthenogenetic blastocysts at a much higher frequency than CBA eggs. Eggs from F₁ hybrid females form parthenogenetic blastocysts at an approximately intermediate level between these inbred strains of mice. C57BL/6 PE do not develop to the somite stages. In contrast, CBA PE and F₁ PE develop to various somite stages. Following administration of 5–azaC at 1.0 μmol/L in vitro at the 1- -cell stage, the number of implantations of C57BL/6 PE transferred to pseudopregnant females increased. In contrast, the number of implantations and somite F₁ PE did not significantly change following exposure to 5–azaC. However, administration of 5-azaC at the 1-cell stage stimulates development of somite F₁ PE. Administration of 5-azaC at 0.2 and 1.0 μmol/L in vitro at the blastocyst stage did not change the number of implantations of C57BL/6 PE. However, the number of implantations and somite CBA PE decreased. After injection of 5azaC at 0.24mg/kg in vivo at day 8 of gestation, some F₁ PE developed to 26–35 somites compared with a maximum of 25 somites in controls. The different effects of 5-azaC on the development of PE depend upon the mouse strain used and the stage of development.     

Anti-apoptotic effect of melatonin on preimplantation development of porcine parthenogenetic embryos

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.     

Unrestricted lineage differentiation of parthenogenetic ES cells

 The developmental potential of parthenogenetic embryonic stem (P-ES) cells was studied in teratomas and mouse chimaeras. Teratomas derived from P-ES cells contained a mixture of tissue types with variable proportions of specific tissues. Three of the eight P-ES cell lines analysed showed high proportions of striated muscle in teratomas, similar to teratomas from normal embryos or ES cell lines derived from fertilised embryos (F-ES cells). Our study also revealed that one P-ES cell line showed little lineage restriction in injection chimaeras. Descendants of the P-ES cells contributed to most tissues of chimaeric fetuses in patterns similar to F-ES cells. Normal colonisation of muscle, liver and pancreas was found in adult chimaeras. P-ES cells also showed similar haematopoietic differentiation and maturation as F-ES cells. However, extensive P-ES cell contribution was associated with a reduction in body size. These findings suggest that, while P-ES cells display more extensive developmental potential than the cells of parthenogenetic embryos from which they were derived, they only retained properties related to the presence of the maternal genome. To elucidate the molecular basis for the lack of lineage restriction during in vivo differentiation, the expression of four imprinted genes, H19, Igf2r, Igf2 and Snrpn was compared among five P-ES and two F-ES cell lines. Expression levels of these genes varied among the different ES cell lines, both in undifferentiated ES cells and in embryoid bodies.     

Metabolism and cell allocation during parthenogenetic preimplantation mouse development

Diploid parthenogenetic postimplantation mouse embryos, containing two maternal genomes, are characterized by poor development of extraembryonic membranes derived from the trophectoderm and primitive endoderm of the blastocyst. This is thought to be caused by a deficiency of expression of paternally derived imprinted genes. Here we have compared the inner cell mass, from which the primitive endoderm and fetal lineages are derived, and the trophectoderm, which forms a major component of the placenta, in parthenogenetic and fertilized preimplantation embryos. We have also studied the metabolism from the 1-cell to the blastocyst stage. Cell numbers were reduced in the ICM and TE of parthenogenetic blastocysts compared to fertilized blastocysts. This was thought to be due to the increased levels of cell death observed in these lineages. Pyruvate and glucose uptake by parthenogenetic embryos was similar to that by fertilized embryos throughout preimplantation development. However, at the expanded blastocyst stage glucose uptake by parthenogenetic embryos was significantly higher than by fertilized embryos. The implications of the actions of imprinted genes and of X-inactivation is discussed. © 1996 Wiley-Liss, Inc.     

Effects of activation methods and culture conditions on development of parthenogenetic porcine embryos

The effects of different activation methods and culture conditions on early development of porcine parthenotes were examined. Three different activation methods were tested: (1) electroporation; (2) electroporation followed by incubation in the presence of butyrolactone I, an inhibitor of cdc2 and cdk2 kinases; and (3) electroporation followed by a treatment with cycloheximide, a blocker of protein synthesis. The activated oocytes were cultured in two different media, NCSU-23 and PZM-3 under 5% CO2 in air. In a separate experiment, the effects of high (approximately 20%) or low (5%) O2 tension on early embryo development were also evaluated. The average pronuclear formation was less (p<0.05) in the electroporated oocytes (83.9+/-1.7%) compared with those activated by electroporation and butyrolactone I or electroporation plus cycloheximide (92.8+/-0.8 and 93.0+/-1.0%). In PZM-3 medium, the average frequencies of blastocyst formation (59.7+/-3.6%) and hatching (10.6+/-1.3%) were greater than those in NCSU-23 medium (39.9+/-3.1% blastocyst formation, p<0.05; and 0.2+/-0.2% hatching; p<0.001). Furthermore, the average nuclear number was also greater (p<0.001) in blastocysts developed in PZM-3 (50.2+/-1.3) than in those developed in NCSU-23 (35.3+/-1.1). Blastocyst formation was similar (p>0.10) among the three activation procedures when parthenotes were cultured in NCSU-23, while in PZM-3 more (p<0.05) parthenotes produced by electroporation plus butyrolactone or electroporation plus cycloheximide developed into blastocysts compared to electroporation alone (64.9+/-5.2 and 68.6+/-3.5% compared with 45.6+/-4.7%). Incidences of apoptotic nuclei were similar (p>0.10) among all treatments. No difference in development was found between parthenotes that developed under high versus low O2 tension (p>0.10). These results demonstrate that activation methods targeting the calcium signaling pathway at several points trigger embryonic development more efficiently than electroporation alone. The data also imply that the PZM-3 medium provides for enhanced culture conditions for the early development of parthenogenetic porcine embryos than NCSU-23.