Somatic cell nuclear transfer in the sheep induces placental defects that likely precede fetal demise.

The efficiency of cloning by somatic cell nuclear transfer (SCNT) is poor in livestock with approximately 5% of transferred cloned embryos developing to term. SCNT is associated with gross placental structural abnormalities. We aimed to identify defects in placental histology and gene expression in failing ovine cloned pregnancies to better understand why so many clones generated by SCNT die in utero. Placentomes from SCNT pregnancies (n = 9) and age matched, naturally mated controls (n = 20) were collected at two gestational age ranges (105-134 days and 135-154 days; term = 147 days). There was no effect of cloning on total placental weight. However, cloning reduced the number of placentomes at both gestational ages (105-134 days: control 55.0 +/- 4.2, clone 44.7 +/- 8.0 and 135-154 days: control 72.2 +/- 5.1, clone 36.6 +/- 5.1; P < 0.001) and increased the mean individual placentome weight (105-134 days: control 10.6 +/- 1.3 g, clone 18.6 +/- 2.8 g and 135-154 days: control 6.6 +/- 0.6 g, clone 7.0 +/- 2.0 g; P < 0.02). Placentomes from cloned pregnancies had a significant volume of shed trophoblast and fetal villous hemorrhage, absent in controls, at both gestational age ranges (P < 0.001) that was shown to be apoptotic by activated caspase-3 immunoreactivity. Consequently, the volume of intact trophoblast was reduced and the arithmetic mean barrier thickness of trophoblast through which exchange occurs was altered (P < 0.001) at both gestational age ranges in clones. In addition, cloning reduced placental expression of key genes in placental differentiation and function. Thus, cloning by SCNT results in both gross and microscopic placental abnormalities. We speculate that trophoblast apoptosis, shedding, and hemorrhage may be causal in fetal death in ovine clones.

Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos.

The processes of cellular differentiation were studied in somatic cell nuclear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients on day 6. All embryos were processed for examination by light and transmission electron microscopy or immunohistochemical labelling for alpha-1-fetoprotein and vimentin. Overall, morphological development of in vivo embryos was superior to IVC and SCNT embryos. Day 7 and particularly day 9 IVC and SCNT embryos had impaired hypoblast development, some lacking identifiable inner cell masses. On day 11, only in vivo and IVC embryos had developed an embryonic disc, and gastrulation was evident in half of in vivo embryos and one IVC embryo. By day 13, all in vivo embryos had completed gastrulation whereas IVC and SCNT embryos remained retarded. On days 17 and 19, in vivo embryos had significantly more somites and a more developed allantois than IVC and SCNT embryos. We conclude that IVC and particularly SCNT procedures cause a retardation of embryo development and cell differentiation at days 7-19 of gestation.

The effect of peri-conception nutrition on embryo quality in the superovulated ewe.

Evidence indicates that oocyte/embryo quality in the sheep is affected by nutrient status during the cycle of conception. This study aimed to determine, in the superovulated ewe, if there are stages during the peri-conception period (-18 days to +6 days relative to the day of ovulation [Day 0]) when quality is more likely to be influenced by nutrition. In Experiment 1, ewes were provided with either a 0.5 x maintenance (L), 1.0 x maintenance (M) or 1.5 x maintenance (H) diet (in terms of daily energy requirements) during the peri-conception period. Diet did not affect the mean ovulation rate (range: 15.4+/-1.47 to 16.1+/-1.55) nor the mean number of embryos collected per ewe (range: 10.9+/-2.05 to 12.4+/-1.82) but there was an increase (P<0.05) in the mean number of cells per blastocyst in the L diet (74.7+/-1.45) compared with either the M (66.4+/-1.29) or H (62.0+/-0.84) diets. This increase was due to an increase in the number of trophectoderm (Tr) cells, resulting in a shift (P<0.05) in the Tr:inner cell mass (ICM) cell ratio (range 0.69+/-0.03 to 0.73+/-0.04). In Experiment 2, six diets (HHH, MHH, MHL, MLH, MLL and LLL) were imposed during three 6-day periods commencing 12 days before and continuing until 6 days after ovulation. Although diet had minimal effect on the superovulatory response, both the mean number of cells per blastocyst and the Tr:ICM ratio were increased (P<0.05) when the L diet was provided after Day 0 (diets MHL, MLL and LLL). It is concluded that the ewe is able to respond to acute changes in nutrition imposed immediately after ovulation, resulting in changes in embryo development including cell lineage differentiation. The significance of these findings, in terms of fetal development, embryo-maternal signalling and the nutritional management of the ewe is discussed.

No differences in sheep somatic cell nuclear transfer outcomes using serum-starved or actively growing donor granulosa cells.

The aim of this study was to compare serum-starved and non-starved donor cells in sheep nuclear transfer with a special emphasis on cloning outcomes. Sheep oocytes, derived either in vivo or in vitro, were fused with cultured serum-starved or actively growing adult granulosa cells. Resulting blastocysts were transferred to recipients fresh or after vitrification, and subsequent pregnancies followed to term. Donor cell treatment did not significantly affect preimplantation development, pregnancy rates, fetal loss or neonate survival rates. Of 22 lambs born, ten survived the immediate perinatal period but all succumbed at various timepoints within the first few weeks of life. The results of the study suggest that the use of serum-starved cells offers no advantages or disadvantages to cloning outcomes. Neither were significant differences in outcomes observed when using either in vivo- or in vitro-derived oocytes or embryos transferred fresh or after vitrification. Yet, these results continue to highlight problems associated with somatic cell cloning as indicated by offspring mortality. It remains unclear whether the high offspring mortality in the current study was related to species, associated with the cell lines used or the result of other causes.

Fetoplacental growth in sheep administered progesterone during the first three days of pregnancy.

We have previously shown that administration of progesterone during early pregnancy in sheep enhances fetal weight and crown-rump length. The present study examined the effect of this treatment on individual fetal organ weights and on placental growth and structure. Embryos that had been exposed to either a normal or a high concentration of progesterone on days 1-3 in initial recipient ewes were transferred at random to final recipient ewes that had or had not been treated with progesterone on days 1-3. Embryos in an additional group of ewes were exposed to progesterone on days 1-3 with oviducts of the ewes ligated. An increase in fetal weight was observed in the final recipient group that had been treated with progesterone (P< 0.01) but not in the initial group treated with progesterone. Fetal weight was increased (P< 0.05) in the initial recipients treated with progesterone plus ligation. Placental weight did not differ between any of the treatments in either initial or final recipients, while placental volumes of chorionic membrane and maternal crypts were increased by progesterone, with and without ligation, in initial recipients (P< 0.05). The responses of fetal weight in final recipients were associated with increases in the weight and linear dimensions of specific fetal components (e.g. brain, kidney, heart, spleen, total gut, head width, thorax circumference). Proportionate increases were observed for most parameters with the exception of brain, heart and M tibialis anterior weight; adjusted least squares means indicated disproportionate increases in these of 5 per cent, 32 per cent and 26 per cent respectively. Enhanced fetal weight in the progesterone plus ligation group was associated with increased (P< 0.05) heart weight; a disproportionate increase of 39 per cent was recorded. Increased fetal weight and fetal heart, skeletal muscle and brain weight were correlated with increased volumes and surface area of the fetal trophectoderm and maternal fetomaternal syncytium in the final recipients treated with progesterone. It is concluded that alteration of the embryo's environment during the first few days of development enhances fetal growth disproportionately, in close association with increased abundance of the exchange epithelia in the placenta.

Long-term effects on offspring of exposure of oocytes and embryos to chemical and physical agents.

Central to this review is the knowledge that, in some livestock species, the environment in which fertilization and embryo development occurs influences not only preimplantation embryo development but also the phenotype of resulting offspring. This knowledge is based on in-vitro studies where the induced changes in the embryo can result in an array of developmental abnormalities after transfer including fetal overgrowth. Whilst such findings are of immediate relevance to assisted reproduction in the human, they also raise another equally important but less obvious issue. Can the in-vivo environments in which fertilization and embryo development normally occur be influenced by exogenous factors (either physical or chemical) in such a way that long-term development is adversely affected? In a global environment of increased use of synthetic chemicals and increased production of pollutants, it is an issue of growing relevance. This review examines technical information that is pertinent to these issues together with a brief assessment of some possible molecular mechanisms responsible for aberrant development. The review concludes with an assessment of the clinical significance of the findings.

Early influences on embryonic and placental growth.

Growth of the placenta is influenced by events before and during early pregnancy. Some of these events set the growth trajectory of the placenta and the fetus for the remainder of the pregnancy. Maternal size and nutrition, and the local metabolic, cytokine and hormonal environment of the embryo all affect growth of the placenta.