Rita S.F. Lee

Cloned Cattle Fetuses with the Same Nuclear Genetics Are More Variable Than Contemporary Half-Siblings Resulting from Artificial Insemination and Exhibit Fetal and Placental Growth Deregulation Even in the First Trimester

Abstract The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome), and fetal overgrowth. Fetal and placental development was investigated at Day 50, during placentome formation; at Day 100, when placentation was completed; and at Day 150, when the hydrops syndrome frequently develops. The NT fetuses were compared with contemporary half-siblings generated from in vitro-produced embryos or by artificial insemination (AI). Fetal cotyledon formation and vascularization of the chorioallantoic membranes was initiated normally in NT conceptuses, but fewer cotyledons successfully formed placentomes. By Day 100, the mean number of placentomes was significantly lower in surviving NT fetuses. Only those with normal placentome numbers were represented in surviving NT pregnancies at Day 150. The mean total caruncle tissue weight of the placentomes was significantly higher in the surviving NT groups at Days 100 and 150, irrespective of the placentome numbers, indicating that increased NT placental weight was caused by excessive uterine tissue growth. By Day 100, NT fetuses exhibited growth deregulation, and those that survived to Day 150 were 17% heavier than contemporary AI controls. Placentome, liver, and kidney overgrowth accompanied the hydrops syndrome at Day 150. The NT fetal overgrowth was not a consequence of in vitro embryo culture and showed no correlation with placental overgrowth. However, in vitro culture and incomplete reprogramming of the donor genome are epigenetic effects that may override genetic traits and contribute to the greater variability in placental and fetal development in the NT group compared with AI half-siblings.

Review: Placental perturbations induce the developmental abnormalities often observed in bovine somatic cell nuclear transfer

Since the first success in cloning sheep, the production of viable animals by somatic cell nuclear transfer (SCNT) has developed significantly. Cattle are by far the most successfully cloned species but, despite this, the technique is still associated with a high incidence of pregnancy failure and accompanying placental and fetal pathologies. Pre- and early post-implantation losses can affect up to 70% of the pregnancies. In the surviving pregnancies, placentomegaly and fetal overgrowth are commonly observed, but the incidence varies widely, depending on the genotype of the nuclear donor cell and differences in SCNT procedures. In all cases, the placenta is central to the onset of the pathologies. Although cellular organisation of the SCNT placenta appears normal, placental vascularisation is modified and fetal-to-maternal tissue ratios are slightly increased in the SCNT placentomes. In terms of functionality, steroidogenesis is perturbed and abnormal estrogen production and metabolism probably play an important part in the increased gestation length and lack of preparation for parturition observed in SCNT recipients. Maternal plasma concentrations of pregnancy-associated glycoproteins are increased, mostly due to a reduction in turnover rate rather than increased placental production. Placental glucose transport and fructose synthesis appear to be modified and hyperfructosemia has been observed in neonatal SCNT calves. Gene expression analyses of the bovine SCNT placenta show that multiple pathways and functions are affected. Abnormal epigenetic re-programming appears to be a key component of the observed pathologies, as shown by studies on the expression of imprinted genes in SCNT placenta.

In vitro and early in vivo development of sheep gynogenones and putative androgenones.

Genomic imprinting, where only one of the two parental genes is expressed, occurs in many phyla. In mammals, however, this phenomenon has been primarily studied in mice, and to a lesser extent, in humans. To understand how genomic imprinting may affect development in other species, particularly those with a different mode of placental development from mice and humans, 339 sheep zygotes were micromanipulated to contain either 2 large (presumptive male) or 2 small (presumptive female) pronuclei. One hundred and twenty‐seven of these embryos and 86 manipulated and nonmanipulated control embryos were transferred to recipient ewes over 3 breeding seasons. Twenty‐one control and 7 experimental conceptuses were recovered on day 21. Four of these conceptuses derived from zygotes with 2 small pronuclei were identified by karyotyping to be gynogenones (maternal‐derived genome). While the gross morphology of the embryos appeared no different to those of normal controls, the extra‐embryonic tissue from the conceptuses showed some hypertrophy and hypervascularization. Preliminary Northern blots of mRNA from allantoic and trophoblast tissue showed an overexpression of H19 and an underexpression of IGF2. Although the sheep gynogenetic phenotype contrasts with that seen in mice, these two genes appear to be similarly differentially expressed. Mol. Reprod. Dev. 50:154–162, 1998.

Insulin-Like Growth Factor-I and Binding Proteins 1, 2, and 3 in Bovine Nuclear Transfer Pregnancies

Abstract In cloned pregnancies, placental deficiencies, including increased placentome size, reduced placentome number, and increased accumulation of allantoic fluid, have been associated with low cloning efficiency. To assess differences in paracrine and endocrine growth regulation in cloned versus normal bovine placentomes and pregnancies, we have examined the expression of insulin-like growth factor (IGF)-I and -II and their binding proteins (IGFBP)-1 through -3 in placentomes of artificially inseminated (AI), in vitro-produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation. Fetal, maternal, and binucleate cell counts in representative placentomes were performed on Days 50–150 of gestation in all three groups. Increased numbers of fetal, maternal, and binucleate cells were present in NT placentomes at all stages of gestation examined. Immunolocalization studies showed that spatial and temporal patterns of expression of IGFBP-2 and -3 were markedly altered in the placentomes of NT pregnancies compared to AI/IVP controls. Concentrations of IGF-I in fetal plasma, as determined by RIA, were significantly higher (P = 0.001) in NT pregnancies (mean ± SEM, 30.3 ± 2.3 ng/ml) compared with AI (19.1 ± 5.5 ng/ml) or IVP (24.2 ± 2.5 ng/ml) pregnancies on Day 150 of gestation. Allantoic fluid levels of IGFBP-1 were also increased in NT pregnancies. These findings suggest that endocrine and paracrine perturbations of the IGF axis may modulate placental dysfunction in NT pregnancies. Furthermore, increased cell numbers in NT placentomes likely have significant implications for fetomaternal communication and may contribute to the placental overgrowth observed in the NT placentomes.

Perturbations in the Biochemical Composition of Fetal Fluids Are Apparent in Surviving Bovine Somatic Cell Nuclear Transfer Pregnancies in the First Half of Gestation

Abstract Amniotic and allantoic fluid volumes and composition change dynamically throughout gestation. Cattle that are pregnant with somatic cell nuclear transfer (NT) fetuses show a high incidence of abnormal fluid accumulation (particularly hydrallantois) and fetal mortality from approximately midgestation. To investigate fetal fluid homeostasis in these pregnancies, Na, K, Cl, urea, creatinine, Ca, Mg, total PO4, glucose, fructose, lactate, total protein, and osmolalities were measured in amniotic and allantoic fluids collected at Days 50, 100, and 150 of gestation from NT pregnancies and those generated by the transfer of in vitro-produced embryos or by artificial insemination. Deviations in fetal fluid composition between NT and control pregnancies were apparent after placental and fetal organ development, even when no gross morphological abnormalities were observed. Individual NT fetuses were affected to varying degrees. Elevated allantoic Na was associated with lower K and increased allantoic fluid volume or edema of the fetal membranes. Total PO4 levels in NT allantoic and amniotic fluid were elevated at Days 100 and 150. This was not accompanied by hypophosphatemia at Day 150, suggesting that PO4 acquisition by NT fetuses was adequate but that its readsorption by the kidneys may be impaired. Excessive NT placental weight was associated with low allantoic glucose and fructose as well as high lactate levels. However, the fructogenic ability of the NT placenta appeared to be normal. The osmolality of the fetal fluids was maintained within a narrow range, suggesting that the regulation of fluid composition, but not osmolality, was impaired in NT pregnancies.

Altered Placental Lactogen and Leptin Expression in Placentomes from Bovine Nuclear Transfer Pregnancies

Abstract Appropriate growth, development, and function of the placenta is central to the success of nutrient partitioning between the mother, placenta, and fetus. Hormones such as placental lactogen (PL) and leptin are produced in the bovine placenta and play an important role in nutrient partitioning and regulation of placental and fetal growth. Nuclear transfer pregnancies are associated with a number of fetal and placental abnormalities, including increased placental growth and macrosomia, and hence represent a unique situation to gain insight into fetoplacental growth regulation. We have examined the expression of bovine PL (bPL) and leptin in placentomes of artificially inseminated (AI), in vitro produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation in the cow. Immunolocalization studies showed that spatial and temporal patterns of expression of bPL and leptin were markedly altered in the placentomes of NT pregnancies compared with AI or IVP controls. Concentrations of bPL in allantoic fluid, as determined by radioimmunoassay (RIA), were significantly higher (P ≤ 0.001) in NT pregnancies (17.9 ± 3.2 ng/ml; mean ± SD) compared with AI (2.03 ± 1.5 ng/ml), but not IVP (23.4 ± 12.8 ng/ ml) pregnancies on Day 150 of gestation. In contrast, amniotic fluid levels of bPL were significantly decreased in NT pregnancies at Day 150 gestation. Leptin mRNA expression, as determined by real-time reverse transcription-PCR, was increased 2.4- to 3.0-fold in NT placentomes compared with AI controls at all gestational ages examined. We speculate that the observed dysregulation of expression of bPL and leptin in NT placentomes could contribute to aberrations in cell migration and invasion and subsequently to alterations in placental metabolism and transfer of nutrients to the fetus, thus leading to increased placental and fetal macrosomia in NT pregnancies.

Development of a formula for estimating plasma free cortisol concentration from a measured total cortisol concentration when elastase-cleaved and intact corticosteroid binding globulin coexist.

Cortisol bound to corticosteroid binding globulin (CBG) contributes up to 90% of the total cortisol concentration in circulation. Therefore, changes in the binding kinetics of cortisol to CBG can potentially impact on the concentration of free cortisol, the only form that is responsible for the physiological function of the hormone. When CBG is cleaved into elastase-cleaved CBG (eCBG) by the activity of neutrophil elastase, its affinity for cortisol is reduced. Therefore, when eCBG coexists with intact CBG (iCBG) in plasma, the calculation of free cortisol concentration based on the formulae that considers only one CBG pool with the same affinity for cortisol may be inappropriate. In this study, we developed in vivo and in vitro models of cortisol partitioning which considers two CBG pools, iCBG and eCBG, with different affinities for cortisol, and deduce a new formula for calculating plasma free cortisol concentration. The formula provides better estimates of free cortisol concentration than previously used formulae when measurements of the concentrations of the two CBG forms are available. The model can also be used to estimate the affinity of CBG and albumin for cortisol in different clinical groups. We found no significant difference in the estimated affinity of CBG and albumin for cortisol in normal, sepsis and septic shock groups, although free cortisol was higher in sepsis and septic shock groups. The in vivo model also demonstrated that the concentration of interstitial free cortisol is increased locally at a site of inflammation where iCBG is cleaved to form eCBG by the activity of elastase released by neutrophils. This supports the argument that the cleavage of iCBG at sites of inflammation leads to more lower-affinity eCBG and may be a mechanism that permits the local concentration of free cortisol to increase at these sites, while allowing basal free cortisol concentrations at other sites to remain unaffected.

DNA methylation patterns are appropriately established in the sperm of bulls generated by somatic cell nuclear transfer.

The cloning of animals by somatic cell nuclear transfer (SCNT) has the potential to allow rapid dissemination of desirable traits from elite animals. However, concern has been expressed that aberrant epigenetic marks in SCNT-derived animals may be passed onto the next generation, even though the offspring of clones appear to be mainly normal. Here, we compared the DNA methylation patterns at 10 genomic regions in sperm from SCNT bulls with that from normal, naturally conceived bulls and with the nuclear donor somatic cells. Eight of the 10 genomic regions were differentially methylated in sperm compared with the donor cell DNA. All three satellite sequences examined here were less methylated in sperm than in the donor cells, contradicting the belief that the sperm genome is always highly methylated. The DNA methylation patterns at all 10 regions were almost identical between SCNT and control sperm, with only one out of the 175 CpG sites/groups of sites examined showing significant difference. These results provide the first molecular evidence that the donor cell genome is correctly reprogrammed upon passage through the germ line in males, and that any epigenetic aberrations harbored by SCNT bulls are unlikely to be passed onto their offspring.

Dynamic Regulation of Expression of Colony-Stimulating Factor 1 in the Reproductive Tract of Cattle During the Estrous Cycle and in Pregnancy

Abstract Colony-stimulating factor 1 (CSF-1) is a hematopoetic cytokine that also plays an important role in placental physiology. We report here the molecular cloning of two alternative splice variants of the bovine gene coding for a putative secreted and a membrane-bound form of the cytokine and the dynamic regulation of expression in the reproductive tract of cattle during the estrous cycle and pregnancy. Bovine CSF-1 was expressed mainly as the 3- and 4-kilobase (kb) transcripts, but 1.4- and 0.8-kb mRNAs were also detected in Day 50–70 pregnant uterine tissue. During the estrous cycle, both the 4- and 3-kb mRNAs were present, but the 3-kb putative membrane-bound form was more abundant than the 4-kb secreted form during diestrus. This pattern of expression was reversed in pregnancy, so that the exponential increase in CSF-1 expression seen during pregnancy was due predominantly to increased abundance of the 4-kb transcript. The change in the 4-kb:3-kb ratio was detected between Day 14 and Day 17, approximately the time of maternal recognition of pregnancy. Thus, CSF-1 was identified as one gene whose expression in the uterus might be altered early in response to the presence of the conceptus. CSF-1 was also expressed in the extraembryonic membranes of the conceptus and in the trophoblastic cells of the fetal cotyledons after the formation of the placentomes. The high level of CSF-1 expression during bovine pregnancy in uteroplacental tissues is consistent with its proposed role in placental physiology.

Influence of pathogenic bacteria species present in the postpartum bovine uterus on proteome profiles

In the first 2-3 weeks after parturition >90% of dairy cows will have some form of uterine infection. Uterine contamination with pathogens, such as Trueperella (formerly Arcanobacterium) pyogenes increases the risk of developing more severe endometritis, which can reduce conception rates. In this study, we compared the uterine proteome of cows infected with Trueperella pyogenes with that of uninfected cows, using 2D gel electrophoresis, and identified annexins A1 and A2 (ANXA1 and ANXA2), apolipoprotein A-1, calprotectin (S100A9), cathelicidin, enolase 1 (ENO1), peptidoglycan recognition protein 1 (PGLYRP1), phosphoglycerate mutase 1 (PGAM1), serine dehydratase (SDS) and serine protease inhibitors (SERPIN) B1, B3 and B4 proteins as differing in abundance in endometritis. Subsequently, levels of ten of these proteins were monitored in uterine samples collected from a herd of lactating, dairy cows at 15 and 42 days post-partum (DPP). The levels were compared with the cytology scores of the samples and the bacterial species isolated from the uterus. Cathelicidin, PGLYRP1, SERPINB1 and S100A9 levels at 15DPP showed strong positive correlations (r=0.78, 0.80, 0.79, and 0.68 respectively; P<0.001) with % of polymorphonuclear neutrophils (PMN). When compared with other bacterial pathogens identified, Streptococcus agalactiae and Truperella pyogenes induced increased expression of the indicator proteins, suggesting that these organisms may adversely affect the subsequent ability of the cow to conceive. Interestingly, there was no difference in the proportion of cows pregnant at 6 and 17 weeks after start of mating between the cows with high or low %PMN.