Mark E. Westhusin

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.

Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies)

The neonatal abnormalities, treatments and outcomes in a group of 13 cloned transgenic calves and fetuses that progressed into the third trimester of pregnancy are described. From these 13 fetuses, 8 calves were born live, 4 stillborn fetuses were recovered from 3 cows that died 7 d to 2 mo before term, and 1 aborted fetus was recovered at 8 mo gestation. All fetuses and calves were derived from the same male fetal Holstein fibroblast cell line transfected with a beta-galactosidase marker gene. Six calves were delivered by Cesarian section and two by vaginal delivery between 278 and 288 d of gestation. Birth weights ranged from 44 to 58.6 kg. Five of the 8 live born calves were judged to be normal within 4 h of birth based on clinical signs and blood gas measurements. One of these 5 calves died at 6 wk of age from a suspected dilated cardiomyopathy. Three of the 8 calves were diagnosed with neonatal respiratory distress immediately following birth, one of which died (at 4 d of age) as a result of pulmonary surfactant deficiency coupled with pulmonary hypertension and elevated systemic venous pressures. Similar findings of chronic pulmonary hypertension were also observed in 2 of 5 fetuses. Placental edema was present in both calves that later died and in the 2 fetuses with cardiopulmonary abnormalities. Hydrallantois occurred with or without placental edema in 6 cows, and only 1 calf from this group survived. The 6 cows without hydrallantois or placental edema produced 5 live calves and 1 aborted fetus. The cardiopulmonary abnormalities observed in the calves and fetuses occurred in utero in conjunction with placental abnormalities, and it is likely that the cloning technique and/or in vitro embryo culture conditions contributed to these abnormalities, although the mechanism remains to be determined.

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.

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.

Impact of Bovine Oocyte Maturation Media on Oocyte Transcript Levels, Blastocyst Development, Cell Number, and Apoptosis

Abstract The objectives were 1) to investigate the effects of oocyte maturation in serum-free and amino acid-supplemented defined media on oocyte transcript levels, blastocyst cell number, and apoptosis; 2) to investigate the influence of oocyte maturation culture atmosphere on blastocyst development, total cell number, and apoptosis; and 3) to examine the influence of epidermal growth factor (EGF) during oocyte maturation on blastocyst cell number and apoptosis. The results demonstrate that blastocysts derived from in vitro maturation, fertilization, and embryo culture protocols undergo apoptosis but that apoptotic levels are not greatly influenced by the oocyte maturation environment. Amino acid supplementation of oocyte maturation media was associated with enhanced developmental frequencies, increased blastocyst cell number, and elevated oocyte maternal mRNA levels. Oocyte maturation with supplemented synthetic oviduct fluid medium (cSOFMaa) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199 + newborn calf serum. Blastocyst development was reduced following oocyte maturation under a 5% CO2, 7% O2, 88% N2 culture atmosphere. EGF supplementation of oocyte maturation medium resulted in a concentration-dependent increase in blastocyst development but did not influence blastocyst total cell number or apoptosis. Our findings indicate that cSOFMaa medium is an effective base medium for bovine oocyte maturation.

Temporal patterns of embryonic gene expression and their dependence on oogenetic factors

Successful development of a fertilized egg beyond early cleavage divisions requires the de novo initiation and subsequent regulation of embryonic transcription. The egg provides the specialized environment within which the newly formed zygotic nucleus initiates its developmental program and as a result plays an obligatory role in its regulation. Although the precise timing of the onset of embryonic transcription in mammals varies during early cleavage divisions, several common elements exist. In the present essay we review the current literature on the timing and control of embryonic gene expression in mammals, and discuss recent findings from our laboratory on gene expression patterns in bovine embryos and their relation to other species, and zygotic gene activation (ZGA). Lastly, we discuss the putative role of maternally inherited factors in conferring developmental competence to the blastocyst stage, and a method to identify such factors present in oocytes as mRNA.

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.

Gene expression regulating blastocyst formation.

Development of embryos to the blastocyst stage is a critical event in the early lives of all eutherian mammalian species. Blastocyst formation is essential for implantation and is the principal morphological determinant of embryo quality prior to embryo transfer. The physiological events and roles of specific gene families that regulate blastocyst formation are subjects of intense research Recent findings have demonstrated that bovine embryos express multiple members of the Na/K-ATPase ion transporter gene family. Two members of this family have been co-localized to bovine trophectoderm, but each becomes largely confined to opposing cell membrane margins. Bovine blastocysts display a greater sensitivity to ouabain (potent inhibitor of the Na/K-ATPase) than murine blastocysts, and enzyme activity (ouabain sensitive 86Rb+ uptake) undergoes a 9-fold increase from the bovine morula to the blastocyst stage. Disruption of Na/K-ATPase gene expression by antisense oligodeoxynucleotide inhibition abolishes blastocyst formation. These results have implicated the Na/K-ATPase as a key regulator of bovine blastocyst formation and have provided insights necessary for the production of healthy bovine embryos by the application of in vitro maturation, in vitro fertilization and in vitro culture methods.

Analysis of variation in relative mRNA abundance for specific gene transcripts in single bovine oocytes and early embryos.

Variation in the abundance of a specific gene transcript was assessed in single bovine oocytes and in vitro–derived blastocysts. Transcripts encoding the Na+,K+‐ATPase α1 subunit were detected by reverse‐transcription polymerase chain reaction (RT‐PCR) and quantified relative to an exogenously supplied rabbit α‐globin mRNA using laser‐induced fluorescence capillary electrophoresis (LIF‐CE). The precision of this relative abundance (RA) calculation was predicted and shown to resolve 2‐fold differences in transcript abundance between individual blastocysts and predicted in oocytes to resolve 3‐fold differences. The RA of the α1 subunit transcript differed by 2‐ to 3‐fold among blastocysts, and 3‐ to 6‐fold among oocytes. Comparison of a general population of oocytes with blastocysts revealed little overlap in RA values between the two groups, with a 8‐ to 14‐fold increase in the mean RA for each group with development observed in two successive experiments (P ≤ 0.05). In contrast, oocytes selected for their developmental competence on the basis of morphologic criteria exhibited only a 1.6‐ to 1.7‐fold developmental increase when the assay was performed on cDNA generated from either embryo pools (n = 6 versus 6) or individuals (n = 7 versus 7), respectively. These results provide the first characterization of the degree of heterogeneity in the abundance of a specific mRNA transcript among individual mammalian oocytes and preimplantation embryos and demonstrate that transcript relative abundance can be correlated with bovine oocyte morphology. Mol. Reprod. Dev. 49:119–130, 1998.