Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.     

An approach for producing transgenic cloned cows by nuclear transfer of cells transfected with human alpha 1-antitrypsin gene

In an attempt to produce transgenic cloned cows secreting alpha 1-antitrypsin (alpha1-AT) protein into milk, bovine cumulus cells were transfected with a plasmid containing an alpha1-AT gene and green fluorescent protein (GFP) reporter gene using Fugene 6 as a lipid carrier. The GFP-expressing cells were selected and transferred into enucleated bovine oocytes. Couplets were fused, chemically activated and cultured. Developmental competence was monitored and the number of inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts were counted after differential staining. The percentage of blastocysts was lower (P < 0.05) in transgenic cloned embryos compared to non-transgenic cloned embryos (23% versus 35%). No difference in the numbers of ICM and TE cells between the two groups of embryos was observed. One or two GFP-expressing blastocysts were transferred into the uterus of each recipient cow. Out of 49 recipient cows, three pregnancies were detected by non-return estrus and rectal palpation. However, the pregnancies failed to maintain to term; two fetuses were aborted at Day 60 and 150, respectively, and one fetus at Day 240. The genomic DNA from the aborted fetus was amplified by polymerase chain reaction (PCR) to investigate integration of the transgene in the fetus. The expected PCR product was sequenced and was identical to the sequence of alpha1-AT transgene. In conclusion, the present study demonstrated that developmental competence of cloned embryos derived from transgenic donor cells was lower than embryos derived from non-transfected donor cells. Although we failed to obtain a viable transgenic cloned calf, integration of alpha1-AT gene into the fetus presents the possibility of producing transgenic cloned cows by somatic cell nuclear transfer.     

TGFalpha and EGFR in ovine preimplantation embryos and effects on development

The present study aimed to assess location and relative amounts of transforming growth factor alpha (TGFalpha) and its receptor (EGFR) in ovine oocytes and preimplantation embryos by using immunohistochemical technique that was graded on a relative scale of 0-3, with 0 representing absence of staining, and 3 exhibiting prominent staining, and to evaluate the effects of TGFalpha/EGF on in vitro development of preimplantation embryos by adding different concentrations of EGF and TGFalpha to culture medium. The results showed that EGFR was abundant in cell plasma membranes in immature and mature oocytes, cumulus cells of immature cumulus-oocyte complexes (COC), fertilized oocytes and at different stages of embryo development. However, the relative amounts in inner cell mass (ICM) (1+) was less than that in trophectoderm (TE) cells (2+) at the blastocysts stage. The staining pattern for TGFalpha was a similar to EGFR. However, the staining for TGFalpha slightly increased in the fertilized oocytes (1-2+) as compared to immature and mature oocytes (1+). TGFalpha was mainly detected in the cytoplasm close to the membrane in both ICM and trophectoderm (TE) cells. The developmental rate of 8-cell stage embryos cultured with 5 ng/ml TGFalpha was increased as compared to other treatments (P<0.05). There was no significant difference in the rate of development of blastocysts cultured with 5 ng/ml TGFalpha, 20 ng/ml EGF, 20 ng/ml EGF+5 ng/ml TGFalpha or the control treatment (P>0.05). In addition, there was no significant difference in the number of cells in blastocyst stage as compared with different treatments (P>0.05). However, TGFalpha alone enhanced cell survival rated (P<0.01) and reduced apoptosis. We concluded that TGFalpha can improve development of ovine preimplantation embryos at the 8-cell and blastocyst stages in vitro.     

Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL

Programmed cell death (apoptosis) characteristically affects the single cells of blastocysts whereas necrosis affects cluster of cells in both the inner cell mass (ICM) and the trophectoderm (TE). This study uses the trophectodermrminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) assay as a way of evaluating the proportion of apoptotic cells and, thus, bovine blastocyst quality during in vitro culture at Days 6,7, and 8. Furthermore, parthenogenetic blastocysts were compared to in vitro fertilized blastocysts at Day 7. Confocal microscopy was used to generate three-dimensional reconstructions of the blastocysts. Apoptosis was observed in both early (Day 6) and late (Day 8) developing blastocysts. The dead cell index (DCI, total number of apoptotic nuclei/total number of nuclei) tend to increase as the in vitro culture time increases, and apoptosis is proportionately higher in the ICM than in the TE. The ratio of ICM to TE cells remains relatively constant even as the blastocysts cell number increases (Day 6 = 11.9 +/- 2.2, Day 7 = 11.2 +/- 0.5, Day 8 = 11.7 +/- 0.4). The overall cell number is significantly reduced in parthenogenetic blastocysts compared to Day 7 in vitro produced blastocysts (P = 0.037). The parthenogenetic blastocysts also show an increase of apoptosis over Day 7 controls. The decrease in cell number in the parthenogenetic blastocysts may be due to the increase of apoptotic nuclei observed. Based on these results we found the TUNEL assay to be a useful method for evaluating in vitro culture conditions of pre-implantation bovine embryos.     

Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

Beneficial effects of dithiothreitol on relative levels of glutathione S-transferase activity and thiols in oocytes, and cell number, DNA fragmentation and allocation at the blastocyst stage in the mouse

We analyzed the effect of in vitro aging of mouse oocytes in the presence of dithiothreitol (DTT) on relative levels of glutathione S-transferase (GST) activity and thiols in oocytes, and cell number, DNA fragmentation and cellular allocation to the inner cell mass (ICM) and trophectoderm (TE) lineage at the blastocyst stage. Ovulated oocytes from gonadotropin primed hybrid female mice of 6-8 weeks of age were aged in vitro in the presence of 0, 5, 50, or 500 microM DTT for 6 hr prior to insemination. Relative levels of GST activity and thiols in oocytes were determined by confocal laser scanning microscopy, DNA fragmentation using a single-step TUNEL method, and cell allocation to the ICM and TE lineage by blastocyst staining with propidium iodide and Hoechst 33258. Non-aged oocytes exhibited higher relative levels of GST activity and thiols when compared to oocytes aged in the presence of 0, 5, and 50 microM DTT. Day 5 blastocysts from the 5, 50, and 500 microM DTT groups exhibited higher total number of cells, number of ICM cells, and ICM/TE ratio, but lower percentage of number of nuclei with DNA fragmentation/number of ICM cells than blastocyst from the 0 microM DTT group. These data show that DTT counteracts the negative effects of a post-ovulatory aging of mouse oocytes in vitro on relative levels of GST activity and thiols in oocytes, and percentage of number of nuclei with DNA fragmentation/number of ICM cells, total number of cells, number of ICM cells and ICM/TE ratio in Day 5 blastocysts.     

A comparative investigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocysts to produce chimeras

The ability of trophectoderm (TE) cells to produce chimeric mice (pluripotency) was compared with that of inner cell mass (ICM) cells. TE and ICM cells of blastocysts and hatching or hatched blastocysts derived from albino mice (CD-1, Gpi-1a/a) were aggregated with zona cut 8- to 16-cell stage embryos or injected into the blastocoele from non-albino mice (C57BL/6 x C3H/He, Gpi-1b/b). After transfer to pseudopregnant female mice, the contribution of the donor cells was examined by glucose phosphate isomerase (GPI) analysis of embryos, membrane and placenta at mid-gestation (Day 10.5 and 12.5) or by the coat color of newborn mice. In contrast to ICM cells, there was no contribution of TE cells in the conceptuses and no coat color chimeric young were obtained. After pre-labeling of TE cells with fluorescent latex microparticles, they were aggregated with embryos and the allocation of TE cells at the compacted morula and blastocyst stages was observed under a fluorescent microscope. Although the TE cells were observed attached onto the surface of the embryos at morula and blastocyst stages, unlike the ICM cells, they were not positively incorporated into the embryos. Thus, the pluripotency of TE cells from mouse blastocysts was not induced by the aggregation and injection methods.     

Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts

Abortions of nuclear transfer (NT) embryos are mainly due to insufficient placentation. We hypothesized that the primary cause might be the aberrant allocations of two different cell lineages of the blastocyst stage embryos, the inner cell mass (ICM) and the trophectoderm (TE) cells. The potential for development of NT embryos to blastocysts was similar to that for in vitro fertilized (IVF) embryos. No difference in the total cell number was detected between NT and IVF blastocysts, but both types of embryos had fewer total cells than did in vivo-derived embryos (P < 0.05). The NT blastocysts showed a higher ratio of ICM:total cells than did IVF or in vivo-derived embryos (P < 0.05). Individual blastocysts were assigned to four subgroups (I: <20%, II: 20-40%, III: 40-60%, IV: >60%) according to the ratio of ICM:total cells. Most NT blastocysts were placed in groups III and IV, whereas most IVF and in vivo-derived blastocysts were distributed in group II. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocations of NT embryos to the ICM and TE cells during early development.     

EGF and TGF-alpha supplementation enhances development of cloned mouse embryos

In this study, we sought to determine the extent to which mitogenic growth factors affect the survival and development of cloned mouse embryos in vitro. Cloned embryos derived by intracytoplasmic nuclear injection (ICNI) of cumulus cell nuclei into enucleated oocytes were incubated in culture media supplemented with EGF and/or TGF-alpha for 4 days. Compared to control, treatment with either growth factor significantly increased the blastocyst formation rate, the total number of cells per blastocyst, the cell ratio of the inner cell mass and the trophectoderm (ICM:TE ratio), and EGF-R protein expression in cloned embryos. In most instances these effects were enhanced in cloned embryos when EGF and TGF-alpha were combined. Although fewer blastocysts developed from cloned than from fertilized one-cell stage embryos, growth factor treatment appeared to have the greatest effect on cloned embryos. These results demonstrate that mitogenic growth factors significantly enhance survival and promote the preimplantation development of cloned mouse embryos.     

Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication

Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P < 0.05), respectively. Epifluorescence microscopy showed that the proportion of blastocysts with eGFP-positive cells in the ICM was higher in the PHA group than in the no-PHA group (40% vs. 16%; P < 0.05). Confocal laser scanning microscopy revealed that the total cell numbers of blastocysts from the PHA group of aggregation chimeras (n = 17; 207.8 ± 67.3 [mean ± standard deviation]) were higher (P < 0.05) than those of embryos without ZP and exposed to PHA (n = 30; 159.6 ± 42.2) and of handling control embryos (n = 19; 176.9 ± 53.3). The same was true for ICM cell counts (56.5 ± 22.0 vs. 37.7 ± 14.2 and 38.7 ± 12.4) and TE cell counts (151.2 ± 58.0 vs. 121.9 ± 37.4 and 138.3 ± 53.0), whereas the ICM/total cell number ratio was not different between the groups. Of the 17 chimeric blastocysts analyzed by confocal laser scanning microscopy, nine had eGFP-positive cells (three of them in the ICM, three in the TE, and three in both lineages). When integration in the ICM occurred, the number of eGFP-positive cells in this compartment was 8.3 ± 2.3 (mean ± standard error of the mean). We conclude that PHA is advantageous for the formation of aggregation chimeras, but the approach tested in the present study with only two donor blastomeres and two host embryos did not result in multiplication of genetically valuable donor embryos.     

Development to the blastocyst stage of immature pig oocytes arrested before the metaphase-II stage and fertilized in vitro

In vitro fertilization (IVF) and embryonic development of mature and meiotically arrested porcine oocytes were compared in the present study. After in vitro maturation (IVM) of cumulus-oocyte complexes for 48 h, 75.4% of them extruded a visible polar body (PB). Most of the oocytes with a first polar body (PB+ group) were at the metaphase-II (M-II) stage (91.4%). Most of the oocytes without a visible polar body (PB− group) appeared to be arrested at the germinal vesicle (GV) (41.6%) and metaphase-I (M-I) (34.0%) stages. After IVF of oocytes (day of IVF = Day 0), there was no difference between PB⁺ and PB⁻ groups in rates of sperm penetration, mono-spermy, however oocyte activation rate after penetration was greater in the PB+ than in the PB− group (P < 0.05). On Day 2, there was no difference between rates of embryos cleaved at the 2–4 cell stages in PB+ and PB− groups (42.1 ± 48.8% and 33.6 ± 2.1%, respectively). On Day 4, the rate of PB+ embryos developing beyond the 4-cell stage was greater than that of PB− embryos (P < 0.05, 31.7 ± 3.9% and 14.1 ± 1.5%, respectively), and PB+ embryos had more cells than the PB− embryos (P < 0.05, 8.3 ± 0.4 and 6.0 ± 0.8 cells, respectively). On Day 6, a greater proportion of PB+ embryos developed to the blastocyst stage than did PB− embryos (P < 0.05, 34.6 ± 2.4% and 20.7 ± 2.8%, respectively). However, when the GV oocytes of the PB− group were not included in recalculations, there was no difference in blastocyst rates between M-I arrested and M-II oocytes (35.3 and 34.6%, respectively). The number of blastomere nuclei in embryos obtained from the PB+ group (52.0 ± 2.5) was greater than that from the PB− group (P < 0.05, 29.1 ± 2.8). The proportion of degenerated parts in the blastocysts, as determined by morphological appearance, was the same in the PB+ and PB− groups. Although the quality of PB+ embryos was enhanced as compared with that of the PB− group, the proportion of inner cell mass and trophectoderm cells in PB+ and PB− blastocysts did not differ (1:1.9 and 1:2.2, respectively). Chromosome analysis revealed that PB+ blastocysts had more diploidy (P < 0.05, 69.7%) than did PB− blastocysts (44.0%), whereas PB− blastocysts had more triploid cells (P < 0.05, 34.0%) than did PB+ oocytes (8.4%). These results indicate that pig oocytes arrested before the M-II stage (M-I oocytes) undergo cytoplasmic maturation during maturation culture and have the same ability to develop to blastocysts after IVF as M-II oocytes, but some of them resulted in degeneration or delayed development with poor embryo quality.     

Cell division and death in the mouse blastocyst before implantation

Summary The numbers of cells in the trophectoderm (TE) and inner cell mass (ICM) of mouse blastocysts were counted by differentially labelling their nuclei with two polynucleotide-specific fluorochromes. Blastocysts recovered from the uterus at intervals between their formation early on Day 4 to the initial stages of implantation on day 5 were analysed. TE cell number increase was initially rapid, indicating some synchronisation of the sixth division, but slowed down progressively and plateaued on Day 5, possibly due to the onset of primary giant cell formation. ICM cell number increase was slower than the corresponding TE cells. As a result, TE cell number more than quadrupled, whereas ICM cell number only doubled over this period. Although the mitotic index of both populations of cells fell steadily, there was no significant difference between them. The decline in the proportion of ICM cells, therefore, is likely to be due to cell death, first detected in early blastocysts and predominantly located in the ICM. In addition, however, a contribution of ICM cells to the overlying polar TE cannot be excluded.     

A paucity of structural integrity in cloned porcine blastocysts produced in vitro

The structural integrity of blastocyst stage embryos, consisting of the inner cell mass (ICM) and trophectoderm (TE) cells, is a prerequisite for normal development after implantation in mammals. In this study, allocation of nuclear transfer (NT)-derived porcine blastocysts to the ICM and to the TE cells was examined and compared with IVF- and in vivo-derived embryos. NT-derived embryos had a lower developmental competence to the blastocyst stage than IVF-derived embryos (P < 0.05). Total cell number of NT-derived blastocysts was inferior to that of IVF-derived embryos (P < 0.05), although no difference was detected between the two groups in the ratio of ICM to total cells. However, in vivo-derived blastocysts had a higher proportion of ICM to total cells compared with in vitro-produced embryos (P < 0.01). To investigate what proportions of in vitro-produced porcine embryos represent normal structural integrity, differentially-stained blastocysts were individually classified into three presumptive groups (I: <20%; II: 20-40%; III: >40%) according to the ratio of ICM to total cells. Low proportions of NT- (12.5%, 7/56) and IVF-derived blastocysts (15.8%, 9/57) were assigned to Group II, presumptively having a normal range of structural integrity, whereas, almost all in vivo-derived embryos (97.5%, 39/40) were allocated to Group II. In conclusion, limited structural integrity may lead to the poor survival to term of NT- or IVF-derived porcine embryos produced in vitro.     

Transfer of inner cell mass cells derived from bovine nuclear transfer embryos into the trophoblast of bovine in vitro-produced embryos

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.     

Production of a healthy calf by somatic cell nuclear transfer without micromanipulators and carbon dioxide incubators using the Handmade Cloning (HMC) and the Submarine Incubation System (SIS)

The aim of this work was to investigate the minimum technical requirements for production of live offspring with somatic cell nuclear transfer. The experiment was performed in a field type laboratory without micromanipulators and carbon dioxide incubators. All long-term incubations were performed in the Submarine Incubation System (SIS) using various gas mixtures. The somatic cell culture was established from ear biopsy of a 9-year-old Holstein cow. Nuclear transfer was performed using the Handmade Cloning (HMC) technique. Zona-free oocytes were randomly bisected by hand with a disposable blade and a stereomicroscope. Cytoplast were selected using Hoechst staining and a fluorescent microscope. After a two-step fusion embryos were activated with calcium ionophore and dimethylaminopurine. Embryos were cultured in microwells (WOWs) in SOFaaci medium supplemented with 5% cattle serum. In two consecutive experiments, six blastocysts were produced from 52 reconstructed embryos. On Day 7, five blastocysts were transferred into synchronized recipients. All three recipients became pregnant but two pregnancies aborted at 6 and 7 months, respectively. A heifer calf weighing 27 kg was delivered at term by Caesarean section from the third pregnancy. The healthy 6-month-old heifer, the first cloned animal of Africa, is living evidence that nuclear transfer technology may be successfully used under basic laboratory conditions.     

Prepubertal bovine oocyte: A negative model for studying oocyte developmental competence

To identify potential markers of maturation quality, differences in developmental capacity between cow and calf oocytes were compared in parallel with their constitutive and neosynthetic protein profiles before and after in vitro maturation (IVM). A comparison was also made between the protein profiles of follicular fluid (FF) from calf and cow ovaries. The effect of epidermal growth factor (EGF) during IVM on the subsequent development of prepubertal calf oocytes was examined. The effect of the presence of fetal calf serum (FCS) during development of embryos originating from calf oocytes was also examined. No differences were noted between the constitutive proteins of cow and calf oocytes and only a minor modification was observed before IVM in the pattern of neosynthesized proteins (presence of a band of 37 kD and a slight increase in the intensity of band of 78 kD in cow as compared to calf oocytes). However, the comparison of constitutive protein profiles from calf and cow FF demonstrated quantitative (the bands of 34 and 45 kD were more intense for cow than for calf) differences. EGF receptors (EGF-R) were demonstrated on cumulus—oocytes complexes (COCs) by immunofluorescence. There was no difference in intensity between cow and calf COCs. Furthermore, the addition of EGF during IVM of calf oocytes dramatically stimulated cumulus expansion and significantly increased the cleavage rate at 72 h post-insemination (82% vs 67%), as well as the proportion of embryos at the 5- to 8-cell stage at this time (54% vs 43%). Also, blastocyst yields at day 6 (11% vs 5%) and at day 8 (17% vs 10%) were significantly higher in the presence of EGF P < 0.05). The addition of FCS to synthetic oviduct fluid droplets at day 2 of culture (48 hpi) had no effect on cleavage, blastocyst yield, or blastocyst cell number. In conclusion, differences in developmental ability between calf and cow oocytes would appear to be not solely linked to differences in oocyte protein patterns. It is likely that the FF, which constitutes the microenvironment in which the oocyte develops, plays a major modulating role in determining the fate of the oocyte/follicle. © 1996 Wiley-Liss, Inc.     

Preimplantational embryo development and incidence of blastomere apoptosis in bovine somatic cell nuclear transfer embryos reconstructed with long-term cultured donor cells

This study was performed to investigate whether types and/or age of donor cells affect preimplantational embryo development and the incidence of apoptosis in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine fetal or adult ear fibroblasts were isolated, cultured in vitro and categorized into fresh or long-term cultured cells in terms of population doublings (PD): in fetal fibroblasts, <16 being considered fresh and >50 being long-term cultured; in adult ear fibroblasts, <16 being considered fresh and >30 being long-term cultured. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199, enucleated and reconstructed by SCNT. The reconstructed oocytes were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39 degrees C in a humidified atmosphere of 5% CO(2) air for 7 days. The early development of SCNT embryos was monitored under a microscope and the quality of blastocysts was assessed by differential counting of inner cell mass (ICM) and trophectoderm (TE) cells and by apoptosis detection in blastomeres using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. As results, types and/or age of donor cells did not affect the rate of blastocyst formation and the number of ICM and TE cells. However, a significant increase in apoptotic blastomeres was observed in SCNT embryos reconstructed with long-term cultured fetal or adult ear fibroblasts compared to those in SCNT embryos derived from fresh fetal or adult ear fibroblasts. In conclusion, these results indicated that the long-term culture of donor cells caused increased the incidence of apoptosis in bovine SCNT embryos but did not affect the developmental competence and the cell number of blastocysts.