Quinton A. Winger

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.

Development Rates of Male Bovine Nuclear Transfer Embryos Derived from Adult and Fetal Cells

Abstract This study compared the nuclear transfer (NT) embryo development rates of adult and fetal cells within the same genotype. The adult fibroblast cells were obtained from a 21-yr-old Brahman bull. The fetal cells were derived from a Day 40 NT fetus previously cloned using cells from the Brahman bull. Overall, similar numbers of blastocysts developed from both adult (53 of 190; 28%) and fetal (39 of 140; 28%) donor cells. Improved blastocyst development rates were observed when fetal cells were serum-starved (serum-fed 12% vs. serum-starved 43%; P < 0.01) whereas there was no similar benefit when adult cells were serum-starved (both serum-fed and serum-starved 28%). Day 30 pregnancy rates were similar for blastocysts derived from adult (6 of 26; 23%) or fetal (5 of 32; 16%) cells. Day 90 pregnancy rates were 3 of 26 for adult and 0 of 32 for the fetal cell lines. One viable bull calf derived from a 21-yr-old serum-starved adult skin fibroblast was born in August 1999. In summary, somatic NT embryo development rates were similar whether adult or fetal cells, from the same genotype, were used as donor cells. Serum starvation of these adult donor cells did not improve development rates of NT embryos to blastocyst, but when fetal cells were serum-starved, there was a significant increase in development to blastocyst.

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.

Reprogramming of Fibroblast Nuclei after Transfer into Bovine Oocytes

Recent landmark achievements in animal cloning have demonstrated that the events of cell differentiation can, in principle, be reversed. This reversal necessarily requires large-scale genetic reprogramming, of which little is known. In the present study we characterized the extent to which blastocyst stage-specific mRNA expression would be conserved in bovine embryos produced by nuclear transfer (NT) using fetal fibroblasts as nuclei donors (FF NT). The mRNA pool of FF NT embryos was compared with that of NT embryos reconstructed from embryonic blastomeres (Emb NT), with embryos produced under in vivo or in vitro conditions, and finally with fibroblast cells. Embryo/cell-specific mRNA pools were contrasted using differential display methodology. Random oligonucleotide primer pair combinations were used to subfractionate mRNA populations and represent individual mRNAs as copy DNA (cDNA) bands ranging in size from 100 to 800 base pairs. Regardless of whether bovine blastocysts developed in vivo or in vitro, or were derived after nuclear transplantation with embryonic blastomeres or fetal fibroblasts, their mRNA profile was highly conserved and distinct from that of fetal fibroblast cells. There was approximately 95% conservation in cDNA banding patterns between FF NT, Emb NT, and in vivo derived blastocysts, when compared with in vitro derived blastocysts. In contrast, the cDNA banding in fibroblasts was only 67% conserved with in vitro derived blastocysts (p < 0.0001), indicating that dramatic changes in gene transcription are induced by nuclear transplantation. After nuclear transplantation, gene expression in fetal fibroblasts is reprogrammed so to mimic that of preimplantation embryo development. Future characterization of these changes will be invaluable for the identification of suitable cell types to serve as nuclear donors for embryo reconstruction and provide information that can be used to improve the efficiency of cloning animals by nuclear transplantation.

Bovine nuclear transfer embryo development using cells derived from a cloned fetus

Many different cell types have been used to generate nuclear transfer embryos and fetuses. However, little is known about the potential of fibroblasts derived from a nuclear transfer fetus as donor cells for nuclear transfer. The ability of cloned fetuses or animals to be cloned themselves is of great interest in determining whether successive generations of clones remain normal or accumulate genetic or phenotypic abnormalities. We generated a bovine fibroblast cell line from a cloned fetus, that continued to divide beyond 120 days (94 doublings,18 passages) in continuous culture. As long-term survival of cells in culture is a desirable characteristic for use in transgenic cell production, passage 2 and 18 cells were compared as donor cells for nuclear transfer (NT). When cells from passage 2 (2 weeks in culture) and passage 18 (4 months in culture) were used for nuclear transfer, there was no significant difference in development rate to blastocyst (35.4 versus 44.6%, P=0.07). A greater proportion of late passage cells were in G0/G1 whether under serum-fed (64 versus 56%, P<0.01) or serum-starved (95 versus 88%, P<0.01) culture conditions. Following embryo transfer, equivalent day 30 pregnancy rates were observed for each group (P 2: 2/19 versus P 18: 2/13). A slightly retarded fetus was surgically removed at day 56 and the remaining three fetuses died in utero by day 60 of gestation. Our results show that fibroblast cells derived from regenerated cloned fetuses are capable of both in vitro and in vivo development. The longevity of this regenerated cell line would allow more time for genetic manipulations and then to identify stable transfected cells prior to their use as NT donor cells. Although no live fetuses were produced in this study the results provide encouraging data to show that a cloned fetus can itself be recloned to produce another identical cloned fetus. Further studies on this and other recloned fetuses are necessary to determine whether the failure to produce live offspring was a result of inadequate sample size or due to the cell type selected.

High LIN28A Expressing Ovarian Cancer Cells Secrete Exosomes That Induce Invasion and Migration in HEK293 Cells

Epithelial ovarian cancer is the most aggressive and deadly form of ovarian cancer and is the most lethal gynecological malignancy worldwide; therefore, efforts to elucidate the molecular factors that lead to epithelial ovarian cancer are essential to better understand this disease. Recent studies reveal that tumor cells release cell-secreted vesicles called exosomes and these exosomes can transfer RNAs and miRNAs to distant sites, leading to cell transformation and tumor development. The RNA-binding protein LIN28 is a known marker of stem cells and when expressed in cancer, it is associated with poor tumor outcome. We hypothesized that high LIN28 expressing ovarian cancer cells secrete exosomes that can be taken up by nontumor cells and cause changes in gene expression and cell behavior associated with tumor development. IGROV1 cells were found to contain high LIN28A and secrete exosomes that were taken up by HEK293 cells. Moreover, exposure to these IGROV1 secreted exosomes led to significant increases in genes involved in Epithelial-to-Mesenchymal Transition (EMT), induced HEK293 cell invasion and migration. These changes were not observed with exosomes secreted by OV420 cells, which contain no detectable amounts of LIN28A or LIN28B. No evidence was found of LIN28A transfer from IGROV1 exosomes to HEK293 cells.

Characterization of a bovine cDNA encoding citrate synthase, and presence of citrate synthase mRNA during bovine pre-attachment development

Citrate synthase is a key regulatory metabolic enzyme that catalyzes the first step in the tricarboxylic acid (TCA) cycle, the synthesis of citrate from acetyl coenzyme A and oxaloacetate. Aerobic metabolism via the TCA cycle is high in bovine embryos at the 4‐cell stage then decreases until the compact morula stage before increasing at the expanded blastocyst stage. This study characterizes the presence of citrate synthase mRNA in bovine pre‐attachment embryos to determine if a variation in mRNA transcript expression patterns is associated with previous reports of the patterns of TCA cycle activity. The reverse transcription–polymerase chain reaction (RT‐PCR) method was used to detect citrate synthase mRNA from the 1‐cell to blastocyst stage of bovine embryo development, and in embryos cultured under either an atmosphere of 5% CO2 in air or 5% CO2/5% O2/90%N2. The nucleotide sequence encoding citrate synthase was determined from bovine heart cDNA by the rapid amplification of cDNA ends (RACE) technique. This 1455‐bp nucleotide fragment contained an open reading frame that encoded a deduced protein of 466 amino acids. The bovine nucleotide sequence was 92.1% and 93.8% identical to the human and porcine coding sequence, respectively. The amino acid sequence predicted from the bovine sequence is 95.1% identical to the human sequence and 96.3% identical to the porcine sequence. The porcine sequence contains a stop codon that results in a peptide truncated by 2 amino acids. The detection of citrate synthase transcripts from the 1‐cell to blastocyst stage demonstrates that the decrease in TCA cycle activity observed following the 4‐cell stage is not associated with an absence of citrate synthase mRNA. Mol. Reprod. Dev. 55:14–19, 2000.