Taeyoung Shin

Evidence for Placental Abnormality as the Major Cause of Mortality in First-Trimester Somatic Cell Cloned Bovine Fetuses

Abstract The production of cloned animals is, at present, an inefficient process. This study focused on the fetal losses that occur between Days 30–90 of gestation. Fetal and placental characteristics were studied from Days 30–90 of gestation using transrectal ultrasonography, maternal pregnancy specific protein b (PSPb) levels, and postslaughter collection of fetal tissue. Pregnancy rates at Day 30 were similar for recipient cows carrying nuclear transfer (NT) and control embryos (45% [54/120] vs. 58% [11/19]), although multiple NT embryos were often transferred into recipients. From Days 30–90, 82% of NT fetuses died, whereas all control pregnancies remained viable. Crown-rump (CR) length was less in those fetuses that were destined to die before Day 90, but no significant difference was found between the CR lengths of NT and control fetuses that survived to Day 90. Maternal PSPb levels at Days 30 and 50 of gestation were not predictive of fetal survival to Day 90. The placentas of six cloned and four control (in vivo or in vitro fertilized) bovine pregnancies were compared between Days 35 and 60 of gestation. Two cloned placentas showed rudimentary development, as indicated by flat, cuboidal trophoblastic epithelium and reduced vascularization, whereas two others possessed a reduced number of barely discernable cotyledonary areas. The remaining two cloned placentas were similar to the controls, although one contained hemorrhagic cotyledons. Poor viability of cloned fetuses during Days 35–60 was associated with either rudimentary or marginal chorioallantoic development. Our findings suggest that future research should focus on factors that promote placental and vascular growth and on fetomaternal interactions that promote placental attachment and villous formation.

A cat cloned by nuclear transplantation.

Sheep, mice, cattle, goats and pigs have all been cloned by transfer of a donor cell nucleus into an enucleated ovum, and now we add the successful cloning of a cat (Felis domesticus) to this list. However, this cloning technology may not be readily extendable to other mammalian species if our understanding of their reproductive processes is limited or if there are species-specific obstacles.

Cell biology: A cat cloned by nuclear transplantation

Sheep, mice, cattle, goats and pigs have all been cloned by transfer of a donor cell nucleus into an enucleated ovum, and now we add the successful cloning of a cat (Felis domesticus) to this list. However, this cloning technology may not be readily extendable to other mammalian species if our understanding of their reproductive processes is limited or if there are species-specific obstacles.

A Highly Efficient Method for Porcine Cloning by Nuclear Transfer Using In Vitro-Matured Oocytes

To date, the efficiency of pig cloning by nuclear transfer of somatic cell nuclei has been extremely low, with less than 1% of transferred embryos surviving to term. Even the utilization of complex procedures such as two rounds of nuclear transfer has not resulted in greater overall efficiencies. As a result, the applicability of the technology for the generation of transgenic and cloned animals has not moved forward rapidly. We report here a simple nuclear transfer protocol, utilizing commercially available in vitro-matured oocytes, that results in greater than 5% overall cloning efficiency. Of five recipients receiving nuclear transfer embryos produced with a fetal fibroblast cell line as nuclear donor, all five established pregnancies by day 28 (100%), and 4/5 (80%) went to term. Efficiencies for each transfer were 7% (9 piglets/128 doublets transferred), 5% (5/100), 12% (7/59), and 6.6% (7/106). The overall efficiency in all recipients was 5.5% and in pregnant recipients 7.7%, with a total of 28 cloned piglets produced. With the average fusion rate being 58%, the percentage of fused doublets producing a live piglet approached 12%. The method described here can be undertaken by a single micromanipulator at a reasonable cost, and should facilitate the broad utilization of porcine cloning technology in transgenic and nontransgenic applications.

Cloning to reproduce desired genotypes

Cloned sheep, cattle, goats, pigs and mice have now been produced using somatic cells for nuclear transplantation. Animal cloning is still very inefficient with on average less than 10% of the cloned embryos transferred resulting in a live offspring. However successful cloning of a variety of different species and by a number of different laboratory groups has generated tremendous interest in reproducing desired genotypes. Some of these specific genotypes represent animal cell lines that have been genetically modified. In other cases there is a significant demand for cloning animals characterized by their inherent genetic value, for example prize livestock, household pets and rare or endangered species. A number of different variables may influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, and the techniques employed for nuclear transfer. At present, there is no solid evidence that suggests cloning will be limited to only a few specific animals, and in fact, most data collected to date suggests cloning will be applicable to a wide variety of different animals. The ability to reproduce any desired genotype by cloning will ultimately depend on the amount of time and resources invested in research.

Development of porcine oocytes from preovulatory follicles of different sizes after maturation in media supplemented with follicular fluids

The development of porcine oocytes from large (3.1-8.0 mm in diameter) or small (<3.1 mm) follicles was examined after maturation culture in medium containing porcine follicular fluid (pFF). Large follicles yielded larger (256 microm v. 221 microm; P<0.05) cumulus-oocyte complexes and more (22 v. 14%) morphologically normal oocytes than small follicles (Experiment 1). In Experiments 2-4, maturation media supplemented with mixed pFF (10%) from small and large follicles was used. More oocytes from large follicles matured (58% v. 91%), formed pronuclei (81% v. 90%) and developed to the blastocyst stage (2% v. 10%) than oocytes from small follicles. In Experiments 5-7, the effects of pFF collected from either small or large follicles on oocyte development were examined. Regardless of the source of oocytes, large-follicle-derived pFF more significantly enhanced preimplantation development than did small-follicle-derived pFF. The highest rate of blastocyst formation (16%) was found when oocytes from large follicles were cultured in maturation medium containing large-follicle-derived pFF. These results suggest that oocytes from large follicles have greater developmental potential than oocytes from small follicles, and that the origin of pFF, which is added to the maturation media, might be an important factor for improving in vitro development of porcine oocytes.

Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei.

Adult animal cloning has progressed to allow the production of offspring cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT‐PCR was used to evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1‐cell, 2‐cell, 6–8 cell, morula, and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK‐L, PFK‐M, and PFK‐P. PFK‐L and PFK‐P were not detected in bovine oocytes. PFK subunits were not detected in 6–8 cell embryos but were detected in blastocysts. Results from NT embryo RT‐PCR demonstrated that PFK was not detected in 8‐cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst stage in both bovine IVF and NT embryos. LDH‐A and LDH‐B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to the blastocyst stage. A third subunit, LDH‐C was not detected at the blastocyst stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH‐C mRNA was detected in gonad isolated from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer. Mol. Reprod. Dev. 56:458–464, 2000.

Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A

Summary.  Background: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. Objectives: To re‐establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. Patients/methods: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. Results and conclusions: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non‐existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame‐shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

Regenerated bovine fetal fibroblasts support high blastocyst development following nuclear transfer.

Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.

Post‐activation treatment with demecolcine improves development of somatic cell nuclear transfer embryos in pigs by modifying the remodeling of donor nuclei

The objective of this study was to examine the effect of cytochalasin B (CB) and/or demecolcine (Dc) on the remodeling of donor nuclei, nuclear ploidy, and development of somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) pig embryos. SCNT and PA oocytes were either untreated (control), or treated with CB, Dc, or both CB and Dc after electric activation, and then cultured or transferred to surrogates. In SCNT, blastocyst formation was higher after treatment with CB and/or Dc (26–28%) than in the controls (16%). The number of oocytes that formed a single pronucleus (PN) was higher after treatment with Dc (86%) and CB + Dc (86%) than under control conditions (44%) or after treatment with CB (63%). In PA, blastocyst formation was higher after CB treatment (47%) than under control conditions (28%), while the formation of a single PN was higher after treatment with Dc (88%) and CB + Dc (84%) compared to controls (34%). The rate of formation of diploid embryos was higher after treatment with Dc and CB + Dc than under control conditions. Dc treatment resulted in a farrowing rate of 50% with 1.1% production efficiency, while controls showed a farrowing rate of 37.5% and a production efficiency of 0.7%. The results of our study demonstrate that post‐activation treatment with Dc improves preimplantation development and supports normal in vivo development of SCNT pig embryos, probably because Dc induces formation of a single PN and this leads to normal nuclear ploidy. Mol. Reprod. Dev. 76: 611–619, 2009.