Quantitative evaluation of reference genes for real-time PCR during in vitro maturation of ovine oocytes.

Quantitative reverse transcription PCR (RT-qPCR) is a powerful molecular technique that enables gene expression studies to be performed on extremely small samples such as oocytes and pre-implantation embryos. However, the high sensitivity of this technique requires that to prevent bias in expression data interpretation, the reference genes used for normalization are fully validated before use. Difficulties in choosing a reference gene are further compounded by the variable RNA content of the maturing oocyte. In the present study, we evaluated eight commonly used reference genes such as ACTB, GAPDH, H2AFZ, HPRT1, PPIA, SDHA, TUBB and YWHAZ and for sheep oocyte RT-qPCR before and after in vitro maturation. We have also compared different cDNA priming strategies using random hexamers or oligo-dT. GeNorm analysis of the results identified the most reliable genes for normalization to be SDHA, TUBB and PPIA when oocyte cDNA was made with random hexamers, and YWHAZ, TUBB and SDHA when oligo-dT primers were used (H2AFZ and HPRT1 were excluded from the geNorm analysis). Interestingly, the analysis revealed that the least stable genes were ACTB and GAPDH, which are the conventional 'housekeeping' genes used in many studies. We recommend the use of three reference genes to calculate a normalization factor to accurately quantify transcript abundance in sheep oocytes and these vary with the cDNA priming strategy employed.

Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep.

Eukaryotic cells contain two distinct genomes. One is located in the nucleus (nDNA) and is transmitted in a mendelian fashion, whereas the other is located in mitochondria (mtDNA) and is transmitted by maternal inheritance. Cloning of mammals typically has been achieved via nuclear transfer, in which a donor somatic cell is fused by electoporation with a recipient enucleated oocyte. During this whole-cell electrofusion, nDNA as well as mtDNA ought to be transferred to the oocyte. Thus, the cloned progeny should harbour mtDNAs from both the donor and recipient cytoplasms, resulting in heteroplasmy. Although the confirmation of nuclear transfer has been established using somatic cell-specific nDNA markers, no similar analysis of the mtDNA genotype has been reported. We report here the origin of the mtDNA in Dolly, the first animal cloned from an established adult somatic cell line, and in nine other nuclear transfer-derived sheep generated from fetal cells. The mtDNA of each of the ten nuclear-transfer sheep was derived exclusively from recipient enucleated oocytes, with no detectable contribution from the respective somatic donor cells. Thus, although these ten sheep are authentic nuclear clones, they are in fact genetic chimaeras, containing somatic cell-derived nuclear DNA but oocyte-derived mtDNA.

Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture.

Manipulation or non-physiological embryo culture environments can lead to defective fetal programming in livestock. Our demonstration of reduced fetal methylation and expression of ovine IGF2R suggests pre-implantation embryo procedures may be vulnerable to epigenetic alterations in imprinted genes. This highlights the potential benefits of epigenetic diagnostic screening in developing embryo procedures.

Induced pluripotent stem cells: epigenetic memories and practical implications.

Induced pluripotent stem cells (iPSCs) may be obtained by direct reprogramming of different somatic cells to a pluripotent state by forced expression of a handful of transcription factors. It was generally assumed that iPSCs are functionally equivalent to their embryonic stem cell (ESC) counterparts. Recently, a number of research groups have demonstrated that this is not the case, showing that iPSCs retain 'epigenetic memory' of the donor tissue from which they were derived and display skewed differentiation potential. This raises the question whether such cells are fit for experimental, diagnostic or therapeutic purpose. A brief survey of the literature illustrates that differences at both epigenetic and transcriptome level are observed between various pluripotent stem cell populations. Interestingly, iPSC populations with perceived 'anomalies' can be coaxed to a more ESC-like cellular state either by continuous passaging--which attenuates these epigenetic differences--or treatment with small molecules that target the machinery responsible for remodelling the genome. This suggests that the establishment of an epigenetic status approximating an ESC counterpart is largely a passive process. The mechanisms responsible remain to be established. Meanwhile, other areas of reprogramming are rapidly evolving such as, trans-differentiation of one somatic cell type to another by the forced expression of key transcription factors. When it comes to assessing their practical usefulness, the same question will also apply.

Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts.

Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.

Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep.

Nuclear transfer offers a cell-based route for producing precise genetic modifications in a range of animal species. Using sheep, we report reproducible targeted gene deletion at two independent loci in fetal fibro-blasts. Vital regions were deleted from the alpha(1,3)galactosyl transferase (GGTA1) gene, which may account for the hyperacute rejection of xenografted organs, and from the prion protein (PrP) gene, which is directly associated with spongiform encephalopathies in humans and animals. Reconstructed embryos were prepared using cultures of targeted or nontargeted donor cells. Eight pregnancies were maintained to term and four PrP-/+ lambs were born. Although three of these perished soon after birth, one survived for 12 days. These data show that lambs carrying targeted gene deletions can be generated by nuclear transfer.

Ovine fetal development is more sensitive to perturbation by the presence of serum in embryo culture before rather than after compaction.

The effects on subsequent fetal development of the presence or absence of serum at different times during IVC of ovine zygotes were studied. Zygotes, recovered from superovulated ewes 36h after intrauterine AI using semen from a single sire, were cultured for 5 days in synthetic oviductal fluid (SOF) media supplemented with either BSA and amino acids (SOF-) or with 10% (v/v) steer serum (SOF+). Serum was present or absent during the first two and last 2 days of IVC giving four treatments (SOF-/SOF-; SOF-/SOF+;SOF+/SOF- and SOF+/SOF+). In total, 224 embryos, including 26 in vivo controls, were transferred singly at day 6 post-AI to synchronous recipients and the products of conception recovered at day 125 of gestation. Presence of serum during IVC had a biphasic effect on embryo development. The inclusion of serum during the first 2 days of IVC retarded early embryo development while the inclusion of serum during the last 2 days of IVC produced more blastocysts by day 6. These effects were independent of each other. The presence of serum during the first 2 days of IVC resulted in increased weights of gravid uterus, placenta, fetus, fetal heart and liver. The incidence of fetuses whose total or organ weights were greater than three standard deviations above the corresponding mean weights of control fetuses was also greater when serum was present during the first 2 days of IVC. However, even when serum was absent throughout IVC there was still an infrequent incidence of fetal weights greater than three standard deviations above the mean for control fetuses. These observations provide evidence that it is the early pre-compaction stages of embryo development that are particularly sensitive to perturbations leading to abnormal fetal development.

Modifications to improve the efficiency of zona-free mouse nuclear transfer.

In the present study, some modifications were made to the zona-free nuclear transfer technique in the mouse in order to achieve greater efficiency. Firstly, a 1-h interval was allowed between cumulus removal and zona pellucida digestion. Secondly, acid Tyrode's was selected for zona pellucida removal, because contrary to pronase, it allows embryo survival during parthenogenic activation in the absence of calcium. Even when the exposure time to pronase was reduced to as little as 1 min or washed with fetal calf serum to inhibit the enzyme, the percentage of lysis during activation in the absence of calcium was still very high. Thirdly, electrofusion was performed at room temperature (21 degrees C), instead of 30 degrees C as in our previous experiments. Finally, embryos were cultured in groups of 12-15, instead of individually, using a "well of the wells" system during activation and culture. When compared, parthenogenic activated control embryos showed an increase in the development to blastocyst when cultured in pairs instead of individually. By the end of the experiments and using embryonic stem (ES) cells, there was a significant increase in fusion rate (1.5-fold increase) and in development to morula/blastocyst from cleaved reconstructed embryos (1.5-fold increase) when compared with the results before the modifications. A 2.4-fold increase in overall efficiency was achieved from the oocyte to morula/blastocyst stages.

Zygote donor nitrogen metabolism and in vitro embryo culture perturbs in utero development and IGF2R expression in ovine fetal tissues.

Tests were made of the effects of altering nitrogen metabolism in zygote donor ewes on fetal development and expression of the gene encoding the type II insulin-like growth factor receptor (IGF2R) following the transfer of ovine embryos cultured from these zygotes, either in the absence or presence of serum. Zygotes, recovered from superovulated ewes (32 on a urea supplemented (30 g urea/kg) diet (high N) and 32 on a control diet (low N)) 36 h after intrauterine AI using semen from a single sire, were cultured for 5 days in synthetic oviductal fluid (SOF) media either with BSA and amino acids (SOF-) or with 10% (v/v) steer serum (SOF+). In total, 166 embryos, including 30 in vivo controls, were transferred singly at day 6 post-AI to synchronous recipients and the products of conception recovered at day 125 of gestation. Elevated plasma urea concentrations in zygote donors were associated with accelerated early embryo development, low pregnancy rates (16%) for embryos from the high N, SOF+ treatment, and significantly influenced fetal development and the expression of IGF2R in the fetal heart at day 125 of gestation. Importantly, the culture of sheep zygotes under serum-free conditions led to a high incidence of aberrant conceptus development and IGF2R expression. Consequently, maternal nitrogen metabolism prior to zygote recovery and in vitro culture can influence fetal development and the expression of an imprinted gene following embryo transfer, and these data support the notion that environmental effects on the follicle-enclosed oocyte may contribute to the etiology of the Large Offspring Syndrome.

Development of a zona-free method of nuclear transfer in the mouse.

In the present study, a zona-free nuclear transfer (NT) technique, which had been originally developed in cattle, was modified for the mouse. Steps involved in this approach include removing the zona pellucida and enucleating without a holding pipette; sticking donor cells to the cytoplast before electric pulses are applied to fuse them and culturing reconstructed embryos individually in single droplets, to prevent aggregation. Control zona-free and zona-intact embryos from mated donors showed no significant difference in development to blastocyst, but did show reduced development to term. Removal of the zona pellucida affected the response to activation by strontium in the absence of calcium as a significant proportion of zona-free control oocytes and embryos reconstructed by NT lysed during this treatment. A comparison between cumulus and ES cells as donor cells revealed significant differences in fusion efficiency (58.1 +/- 4.0%, n = 573 vs. 42.9 +/- 2.2%, n = 2064, respectively, p < 0.001), cleavage (77.2 +/- 3.4%, n = 334 vs. 40.8 +/- 2.7%, n = 903, respectively, p < 0.001) but not for development to morula/blastocyst (8.7 +/- 2.1%, n = 334 vs. 13.9 +/- 1.8%, n = 903, respectively, p < 0.1). The stage at which embryo development arrested was also affected by donor cell type. A majority of embryos reconstructed from cumulus cells arrested at two-cell stage, usually with two nuclei, whereas those reconstructed from ES cells arrested at one-cell stage, usually with two pseudo-pronuclei. After transfer of ES cell-derived NT embryos, a viable cloned mouse was produced (3.0% of transferred embryos developed to term). These observations establish that a zona-free cloning approach is possible in the mouse, although further research is required to increase the efficiency.

Effect of an inhibitor of 3 beta-hydroxysteroid dehydrogenase on progesterone concentrations and embryo survival in sheep.

The effect of an inhibitor of 3 beta-hydroxysteroid dehydrogenase on peripheral progesterone concentration during the luteal phase of the oestrous cycle and on embryo survival was determined in sheep. Following administration of 10, 50, 100 or 250 mg epostane (4,5-epoxy-17-hydroxy-4,17,dimethyl-3-oxo-androstane-2-carbonitrile) progesterone concentrations were significantly lower than control levels 4 h after injection, from 2.5 to 22 h, 1.5 to 24 h and 1 to 24 h after injection respectively. There appeared to be no effect on peripheral oestradiol concentrations. Adrenal progesterone production was small and not influenced by epostane treatment. Epostane was administered on day 9 of the oestrous cycle to cause a reduction in progesterone concentrations for approximately 12-18 h on day 9 only (group 1, 250 mg epostane on day 9), or a series of such reductions on 3 consecutive days (group 2, 50 mg epostane on days 9, 10 and 11) or a continuous reduction for 3 days (group 3, 250 mg epostane on days 9, 10 and 11). The proportion of ewes that were pregnant was significantly (P less than 0.05) lower in ewes treated to give a continuously low progesterone concentration for 3 days than in either controls or ewes in which progesterone concentration was reduced for less than 24 h (in controls and groups 1, 2 and 3 the proportion was 85, 92, 54 and 18% of ewes treated respectively). Embryo survival was not affected by administration of 250 mg epostane on days 9, 10 and 11 if luteal phase levels of progesterone were maintained by insertion of a silicone elastomer implant of the steroid.(ABSTRACT TRUNCATED AT 250 WORDS)

Strategies for production of pharmaceutical proteins in milk.

Although it is only a decade since the use of transgenic livestock for the production of pharmaceutical proteins in milk was first suggested, great progress has been made. There is every reason to expect that, as methods for the transfer of genomic clones are applied with efficient regulatory elements, many proteins will be produced at high concentrations in the milk of livestock species. In most cases, these proteins will be biologically active, however, it remains to be shown whether they will be identical in structure and function to the natural product.

Embryonic and somatic cell cloning.

Revolutionary opportunities in biology, medicine and agriculture arise from the observation that offspring are obtained after nuclear transfer if somatic donor cells are induced to become quiescent. Exploitation of many of these opportunities will depend upon optimizing procedures for nuclear transfer. This may come about through an understanding of the means by which factors in the oocyte cytoplasm act upon the DNA of the transferred nucleus to regulate gene expression. Similarly, research will extend the procedure to other species. This technology may be used for embryo production, the introduction of genetic change and the derivation of cells needed to treat human diseases. Groups of genetically identical animals will be used in research to control genetic variation and to allow transfer of cells between individuals. In agriculture, production of a small number of clones will separate genetic and environmental effects, whereas production of larger numbers of offspring will disseminate genetic improvement from nucleus herds. Precise genetic modification will be achieved by site specific recombination in the donor cells before nuclear transfer. In all mammals it will become possible to define the role of any gene product and to analyse the mechanisms that regulate gene expression. Medical uses of these techniques will include the production of proteins needed to treat disease and the supply of organs such as hearts, livers and kidneys from pigs. As genome mapping projects identify loci associated with traits of commercial importance in agriculture then gene targeting will be used to study this effect. Finally, cells capable of differentiation into any of the tissues of a patient will provide treatment for diseases reflecting damage to a specific cell population that neither repairs nor replaces itself.

Fetal growth and development following temporary exposure of day 3 ovine embryos to an advanced uterine environment.

The effect of exposing Day 3 ovine embryos to an advanced uterine environment for a period of 3 days on subsequent fetal growth and development between Day 35 and Day 135 of gestation was studied. Day 3 embryos were recovered from superovulated donor ewes and transferred to synchronous final or asynchronous temporary recipients for 3 days. Embryos were recovered from these temporary recipients and transferred to Day 6 final recipients. Gravid uteri were recovered, weighed and dissected on Days 35, 45, 60, 90, 110, 125 and 135 of gestation. Fetal weight and length data were analysed by fitting non-linear Gompertz models of the form log(e) y = a - be(-ct), where y is fetal size and t is time from conception. Various terms including treatment, gestational age, embryo stage at transfer and fetal sex were fitted to this model. Fetal development was assessed by relating organ weight to fetal bodyweight using the linear allometric equation log(e) y = log(e) a + b log(e) x, where y is organ weight and x is fetal weight. Temporary exposure of Day 3 embryos to an advanced uterine environment did not increase the rate of embryo development and had no effect on fetal growth and development between Days 35 and 135 of gestation in this study. A single Gompertz model (log(e) y = 10.134 - 17.047e(-0.1733t)) explained 99.8% of the variation in fetal weight. Of terms fitted to this model only gestational age and fetal sex influenced fetal weight, with male fetuses being 5% heavier (P<0.05) than female fetuses. Fetal development was also unaffected by experimental treatment in this study. Allometric coefficients established for various fetal components agreed well with those from previously published studies.

New opportunities in animal breeding and production - an introductory remark.

New opportunities in animal breeding and production will be offered by methods for embryo production and the introduction of precise genetic changes in livestock species. A review of current procedures shows that they have been improved during the last 4 years, but still have significant limitations. In the light of experience over the past 50 years it seems likely that these methods will contribute to animal health and productivity, but at some unknown time in the future and in ways that cannot be predicted precisely. When looking to the future, delegates to this meeting should take pride in what science continues to contribute.

Survival of ovine embryos stored at 4 degrees C for 24 hours.

This study was conducted to ascertain if sheep embryos collected for transfer can be stored for short periods without freezing to allow for international transport. Of twelve Finnish Landrace ewes treated with equine follicle stimulating hormone (FSH), eleven ewes ovulated with a mean of 9.1 +/- 4.3 (SD) corpora lutea. Recovery rate from the nine ewes with normal corpora lutea was 68 +/- 27%, providing 61 morulae which were then cooled to 4 C and stored for 24 h while transporting them from Scotland to France. Romanov recipients received either 4 (n = 14) or 5 (n = 1) of these morulae. Fourteen of the recipients lambed, with a mean lambing rate of 2.1 +/- 0.8, representing 48.3% of embryos transferred. Cooling of embryos to 4 C and storing them in ovum culture medium for 24 h at 4 C may be a valuable technique for the handling and short-term storage of embryos.

Pretreatment with recombinant bovine somatotropin enhances the superovulatory response to FSH in heifers.

One of the primary limiting factors to superovulation and embryo transfer in cattle has been the large variability in response, both between and within animals. It appears that the primary source of this problem is the variability in the population of gonadotropin-responsive follicles present in ovaries at the time of stimulation. We have shown that treatment of heifers with recombinant bovine somatotropin (rbGH) increases the number of small antral follicles (2 to 5 mm) and, therefore, enhances the subsequent superovulatory response to eCG. To investigate further the potential of using this approach to improve superovulatory regimens in cattle, the effect of rbGH pretreatment on the response to pituitary FSH was studied. The estrous cycles of 16 heifers were synchronized using PGF2alpha. On Day 7 of the synchronized cycle, half of the animals were injected with 320 mg sustained-release formulated rbGH, while the other half received 10 ml saline. Five days later, all heifers were given a decreasing-dose regimen of twice daily injections of oFSH for 4 d, incorporating an injection of PGF2alpha with the fifth FSH treatment, to induce superovulation. All animals were artificially inseminated twice with semen from the same bull during estrus. Ova/embryos were recovered nonsurgically on Days 6 to 8 of the following estrous cycle, and the ovulation rate assessed on Day 9 by laparoscopy. Using the same animals as described above, the experiment was repeated twice, 3 and 6 mo later, with no laparoscopy in the third experiment. The animals were randomized both between experiments and for the day of ova/embryo collection. Pretreatment of heifers with rbGH significantly (P < 0.01) increased the number of ovulations, total number of ova/embryos recovered and the number of transferable embryos. The percentage of transferable embryos was significantly (P < 0.05) increased by rbGH pretreatment. In addition, the incidence (2/16) of follicular cysts with a poor ovulatory response (< 6 ovulations) for the rbGH-pretreated heifers was significantly lower (P < 0.05) when compared with the incidence (7/16) in the control animals. It is concluded that pretreatment with rbGH may provide a useful approach for improving superovulatory response in cattle.

Embryo cloning in sheep: work in progress.

We summarize here the procedures for nuclear transfer using S-phase cytoplasts and describe a new method for avoiding loss of reconstructed embryos from the oviducts during in vivo culture. We obtained 2 clones of 5 genetically identical animals following the transfer of blastomeres from 16-cell embryos into enucleated preactivated cytoplasts. Metaphase II oocytes and embryos were surgically collected from superovulated Sarda breed ewes 54 and 120 h after sponge removal, respectively. Oocytes were exposed for 15 min to 5 mug/ml of Hoechst 33342 and were micromanipulated at room temperature. Efficiency in embryo reconstruction was 100% for enucleation and 98% for fusion. Embryos were embedded in agar as separate clones and transferred into the oviducts of temporary recipients. The fimbriae were closed with glass-nylon made filters. Embryo recovery from the temporary recipients was 97.3%, with a cleavage rate of 81.4%; development to morula-blastocyst stage was 70.6%. A total of 29 Grade 1 blastocysts corresponding to 5 clones were transferred into 13 naturally synchronous ewes, and scanning was performed at 30 and 90 d. Ten ewes were pregnant at the first scanning and nine at the second for a final pregnancy rate of 71.4%; the survival rate at term was 48%. Overall, we obtained 4 clones of identical lambs: two sets of 5 (one male set and one female set) and two sets of twins (both sets male). Pregnancy length in recipients carrying clones was longer than the standard period in Sarda breed (153 vs 150 d, respectively). Weight at birth was higher for male lambs obtained from nuclear transfer than for normal males (4.1 vs 3.6 kg), while the weight for females was normal.