Genome reprogramming is the ability of a nucleus to modify its epigenetic characteristics and gene expression pattern when placed in a new environment. Low efficiency of mammalian cloning is attributed to the incomplete and aberrant nature of genome reprogramming after somatic cell nuclear transfer (SCNT) in oocytes. To date, the aspects of genome reprogramming critical for full-term development after SCNT remain poorly understood. To identify the key elements of this process, changes in gene expression during maternal-to-embryonic transition in normal bovine embryos and changes in gene expression between donor cells and SCNT embryos were compared using a new cDNA array dedicated to embryonic genome transcriptional activation in the bovine. Three groups of transcripts were mostly affected during somatic reprogramming: endogenous terminal repeat (LTR) retrotransposons and mitochondrial transcripts were up-regulated, while genes encoding ribosomal proteins were downregulated. These unexpected data demonstrate specific categories of transcripts most sensitive to somatic reprogramming and likely affecting viability of SCNT embryos. Importantly, massive transcriptional activation of LTR retrotransposons resulted in similar levels of their transcripts in SCNT and fertilized embryos. Taken together, these results open a new avenue in the quest to understand nuclear reprogramming driven by oocyte cytoplasm.

Background Preimplantation mammalian embryo development is characterized by fundamental changes in nuclear function as genomes of gametes, egg and sperm unite to give rise to a totipotent embryonic genome. This totipotency is however a transient property since the first events of differentiation occur after few cell cycles, giving rise to the trophectoderm cells that co-exist with the pluripotent and no more totipotent cells of the inner cell mass at the blastocyst stage. Concomitantly to these cellular events, the newly formed embryonic genome becomes progressively transcriptionally active. We analyzed embryonic gene expression over this period in both the rabbit [1] and the bovine species. We have chosen these species as embryonic models in preference to the mouse since contrarily to this later species, embryonic genome activation (EGA) spans over several cell cycles and is preceded by progressive epigenetic modifications.

Early mammalian development is characterized by extensive changes in nuclear functions that result from epigenetic modifications of the newly formed embryonic genome. While the first embryonic cells are totipotent, this status spans only a few cell cycles. At the blastocyst stage, the embryo already contains differentiated trophectoderm cells and pluripotent inner cell mass cells. Concomitantly, the embryonic genome becomes progressively transcriptionally active. During this unique period of development, the gene expression pattern has been mainly characterized in the mouse, in which embryonic genome activation (EGA) spans a single cell cycle after abrupt epigenetic modifications. To further characterize this period, we chose to analyze it in the rabbit, in which, as in most mammals, EGA is more progressive and occurs closer to the first cell differentiation events. In this species, for which no transcriptomic arrays were available, we focused on genes expressed at EGA and first differentiation and established a 2,000-gene dedicated cDNA array. Screening this with pre-EGA, early post-EGA, and blastocyst embryos divided genes into seven clusters of expression according to their regulation during this period and revealed their dynamics of expression during EGA and first differentiation. Our results point to transient properties of embryo transcriptome at EGA, due not only to the transition between maternal and embryonic transcripts but also to the transient expression of a subset of embryonic genes whose functions remained largely uncharacterized. They also provide a first view of the functional consequences of the changes in gene expression program.

The Saola (Pseudoryx nghetinhensis), a new large mammal species, was discovered to science in 1993 and listed as critical endangered. Its systematic position within the family Bovidae, however, remains confused since previous studies have led to several conflicting hypotheses. In the present study, an analysis of microsatellite data was performed to determinate the taxonomic status of this enigmatic species. Cross species amplification of 19 cattle microsatellite loci was tested on a panel of 56 individuals representing 9 different species of the Bovidae. Phylogenetic analyses were carried out on matrices from the Dps and Dc genetic distances using the neighbour-joining method. The results obtained from separate and combined analyses strongly support a grouping of Pseudoryx with subtribe Bubalina, which contains all species of Bubalus and Syncerus. (Resume d'auteur)

BACKGROUND:The wild gaur (Bos gaurus) is an endangered wild cattle species. In Vietnam, the total number of wild gaurs is estimated at a maximum of 500 individuals. Inbreeding and genetic drift are current relevant threats to this small population size. Therefore, information about the genetic status of the Vietnamese wild gaur population is essential to develop strategies for conservation and effective long-term management for this species. In the present study, we performed cross-species amplification of 130 bovine microsatellite markers, in order to evaluate the applicability and conservation of cattle microsatellite loci in the wild gaur genome. The genetic diversity of Vietnamese wild gaur was also investigated, based on data collected from the 117 successfully amplified loci.RESULTS:One hundred-thirty cattle microsatellite markers were tested on a panel of 11 animals. Efficient amplifications were observed for 117 markers (90%) with a total of 264 alleles, and of these, 68 (58.1%) gave polymorphic band patterns. The number of alleles per locus among the polymorphic markers ranged from two to six. Thirteen loci (BM1314, BM2304, BM6017, BMC2228, BMS332, BMS911, CSSM023, ETH123, HAUT14, HEL11, HEL5, ILSTS005 and INRA189) distributed on nine different cattle chromosomes failed to amplify wild gaur genomic DNA. Three cattle Y-chromosome specific microsatellite markers (INRA124, INRA126 and BM861) were also highly specific in wild gaur, only displaying an amplification product in the males. Genotype data collected from the 117 successfully amplified microsatellites were used to assess the genetic diversity of this species in Vietnam. Polymorphic Information Content (PIC) values varied between 0.083 and 0.767 with a mean of 0.252 while observed heterozygosities (Ho) ranged from 0.091 to 0.909 (mean of 0.269). Nei's unbiased mean heterozygosity and the mean allele number across loci were 0.298 and 2.2, respectively.CONCLUSION:Extensive conservation of cattle microsatellite loci in the wild gaur genome, as shown by our results, indicated a high applicability of bovine microsatellites for genetic characterization and population genetic studies of this species. Moreover, the low genetic diversity observed in Vietnamese wild gaur further underlines the necessity of specific strategies and appropriate management plans to preserve this endangered species from extinction.

Reproductive activity in swamp buffalo is characterized by a clearly demonstrated anestrus season. The aim of the present study was to evaluate season effect on the oocyte collection, in vitro maturation, and somatic cell nuclear transfer. The ovaries collected from a slaughterhouse were divided into 3 groups according to the collection period: (1) G1: from January to April; G2: from May to August, which is characterized by higher climate temperature and low reproductive activity; and G3: from September to December. Cumulus–oocyte complexes (COCs) were aspirated from follicles 2-6 mm in diameter using an 18-gauge needle, washed in HEPES-buffered TCM-199 (Sigma-Aldrich, St Louis, MO, USA), and classified following 3 different quality levels: A (with 4–6 layers of cumulus cells), B (with 2–3 layers of cumulus cells), and C (few or without cumulus cells). The oocytes of A and B categories were used for IVM in maturation media currently used in cattle (TCM-199 medium + 10% fetal bovine serum) with an increase of FSH concentration (30 g mL-1) and estradiol-17β (3 g mL-1). Maturation was carried out at 39C in a water-saturated incubator, under 5% CO2 for 22 h. The oocytes were observed for the cumulus expanding and the presence of polar body (PB). The oocytes with PB were used for further enucleation and cell nuclear transfer using buffalo quiescent fibroblast cells and the technique described previously (Nguyen et al. 2000 Theriogenology 53, 235). The percentages of intact and fused oocytes as well as reconstructed embryos developed to blastocyst stage were compared for the oocytes from G1 and G2. The results indicated that the average number of good quality COCs collected per ovary for the G1, G2, and G3 period were 6.00 4.08 (n = 426), 2.93 2.55 (n = 346), and 4.78 1.05 (n = 445), respectively. The percentages of A and B oocytes were 62.4% (1.58 0.51 vs. 2.17 1.54), 63.2% (0.90 0.32 vs. 0.95 0.50), and 54.7% (1.12 0.25 vs. 1.49 0.53), respectively; the maturation rate was 55.08%, 56.28%, and 52.16%, respectively. There were no significant differences between G1 and G2 in the percentage of intact and fused oocytes (93.7% and 59% for G1; 100% and 60% for G2, respectively), but the rate of embryos developed to blastocyst stage was higher for oocytes from G1 (18.5% vs. 10.2%). In conclusion, in swamp buffalo, the hot season affected significantly the number of oocytes collected per animal and the subsequent results of somatic cell nuclear transfer. The optimal period for working with buffalo oocyte is from January to April. This work was aupported by a grant from the Vietnam-Italy 3AB3 Project.

Production of cloned nonhuman primate embryos has been reported using mature oocytes obtained from donors treated in vivo with a high dose of recombinant human FSH (r-hFSH, 35 IU per day for 10 days). The disadvantages of this approach are the high cost of hormones and the need to use the oocytes shortly after collection. Our study aimed to investigate the possibility of using initial in vivo treatment with a reduced FSH dose followed by in vitro culture for long periods of up to 3 days to produce mature monkey oocytes as host ooplasts for somatic cell nuclear transfer (SCNT). Adult female long-tailed Macaque (Macaca fascicularis) monkeys were treated with r-hFSH (Serono, Aubonne, Switzerland, 35 IU per day, i.m.) either for 10 days with an injection of hCG (1000 IU, i/m) 34 h before oocyte collection (G.I) or with only r-hFSH for 7 days (G.II). Cumulus oocyte complexes (COCs) were collected by follicular aspiration and then cultured in TCM-199 medium (GIBCO) supplemented with estradiol-17β, FSH, LH, and 10% FCS at 39°C in an incubator with 5% CO2 in air. The maturation rate based on the level of cumulus expansion and the presence of the first polar body was recorded at the moment of collection and during 24 h, 48 h, and 72 h of in vitro maturation (IVM). For SCNT, the mature Metaphase II oocytes were separated from cumulus cells and selected for enucleation in the presence of cytochalasin B (Sigma, St. Louis, MO, USA). Skin fibroblasts obtained from adult monkeys were cultured in DMEM+ 10% FCS and induced to quiescence in DMEM 0% FCS 2 days before use. A single cell was transferred under the zona of each enucleated oocyte. Couplets were fused with two direct current (DC) pulses of 220 V/mm for 25 μs in Zimmerman medium. Fused oocytes were cultured in medium containing cyclohexamide for 6 h before placing them into monkey culture medium (Cook, Brisbane, Australia). The average number of oocytes collected per animal were 21.2 (n = 18) and 18.6 (n = 12) for the G.I and G.II treatments, respectively. For G.I, the rate of COCs with fully expanded cumulus was 42% at collection and was maximal (80%) at Day 1 of IVM. For G.II, fully expanded cumulus was not observed at the time of collection and during the first 2 days of IVM, but 75% of COCs had full cumulus expansion by Day 3 of IVM. The rates of intact and fused oocytes were 50.3% for G.I and 55.4% for G.II. From the fused oocytes, 67.8% and 64.4% developed to the 4- to 8-cell stages at Days 2–3 after nuclear transfer for G.I and G.II, respectively. From these data, it can be concluded that this approach can be applied to optimize production of mature oocytes for non-human primate SCNT and ART (assisted reproductive technologies) programs. This work was supported by AIRE-Development.

Background SSH has emerged as a widely used technology to identify genes that are differentially regulated between two biological situations. Because it includes a normalisation step, it is used for preference to clone low abundance differentially expressed transcripts. It does not require previous sequence knowledge and may start from PCR amplified cDNAs. It is thus particularly well suited to biological situations where specific genes are expressed and tiny amounts of RNA are available. This is the case during early mammalian embryo development. In this field, few differentially expressed genes have been characterized from SSH libraries, but an overall assessment of the quality of SSH libraries is still required. Because we are interested in the more systematic establishment of SSH libraries from early embryos, we have developed a simple and reliable strategy based on reporter transcript follow-up to check SSH library quality and repeatability when starting with small amounts of RNA. Results Four independent subtracted libraries were constructed. They aimed to analyze key events in the preimplantation development of rabbit and bovine embryos. The performance of the SSH procedure was assessed through the large-scale screening of thousands of clones from each library for exogenous reporter transcripts mimicking either tester specific or tester/driver common transcripts. Our results show that abundant transcripts escape normalisation which is only efficient for rare and moderately abundant transcripts. Sequencing 1600 clones from one of the libraries confirmed and extended our results to endogenous transcripts and demonstrated that some very abundant transcripts common to tester and driver escaped subtraction. Nonetheless, the four libraries were greatly enriched in clones encoding for very rare (0.0005% of mRNAs) tester-specific transcripts. Conclusion The close agreement between our hybridization and sequencing results shows that the addition and follow-up of exogenous reporter transcripts provides an easy and reliable means to check SSH performance. Despite some cases of irregular normalisation and subtraction failure, we have shown that SSH repeatedly enriches the libraries in very rare, tester-specific transcripts, and can thus be considered as a powerful tool to investigate situations where small amounts of biological material are available, such as during early mammalian development.

Early embryonic development is initially dependent on mRNAs that have been transcribed during oocyte growth (maternal transcripts). Newly formed zygotic transcripts then become required during what is called the maternal-to-zygotic transition. In cattle, this transition initiates at the 8-cell stage and ends before the morula stage. Because of its decisive role in the further development of the embryo we are interested in characterizing the regulative functions of those cattle zygotic transcripts that are differentially expressed at the end of this transition. For that reason a subtracted cDNA library corresponding to the first zygotic transcripts was established at the early compacted morula stage using suppressive subtractive hybridization (SSH; Clontech, LePont de Claix, France). Morula derived cDNAs were used as Tester and 4-cell stage cDNAs as Driver materials. Cattle embryos were obtained from slaughterhouse-derived ovaries using standard in vitro maturation and fertilization techniques. Since, in cattle, early cleaving (2-cell-stage) zygotes are more likely to develop to the blastocyst stage than their later-cleaving counterparts, all embryos used to establish the cDNA library were selected from zygotes that were already at the 2-cell stage 32 h post-in vitro fertilization. Total RNA was extracted from batches of 140 (morula) and 200 (4-cell-stage) embryos and the amount of PolyA+ RNAs was estimated according to Duranthon and Renard (in Biology and Pathology of the Oocyte, Trounson and Gosden eds, Cambridge Univ. Press, 2003, p. 96). Double-stranded cDNAs were synthesized with the SMART cDNA amplification kit (Clontech) before SSH was undertaken. Upon RNA extraction, exogenous transcripts obtained from Arabidopsis thaliana (Stratagene, La Jolla, CA, USA) were added either to the Tester only (at three concentrations: 10−3, 5 × 10−3, 5 × 10−2) or to both the Tester and the Driver materials (at two concentrations: 5 × 10−3, 5 × 10−2). These transcripts allowed us to report on the efficiency of our subtraction procedure and on the quality of the bacterial library in terms of tester-specific transcript enrichment. We found the library to be enriched in specific transcripts of the Tester (morula stage) by a factor of 300. Normalization of the library, as determined from the proportion of exogenous transcripts after bacterial transformation, was effective for those added initially at low (10−3) or moderate (5 × 10−3) concentrations but not for abundant ones (5 × 10−2). These conditions are thus beneficial for the isolation of rare zygotic transcripts present at an initial concentration of only 10−3 of the messengers. Ongoing study using various differential screening of this cattle library with morula- and 4-cell-stage probes will now allow us to identify zygotic transcripts specifically expressed at the onset of genome activation and not present in the pool of maternal transcripts up to the 4-cell stage. This work was supported by an INRA CIRAD grant (BioDiva) to LCB.

Increasing the diary population and milk production is a goal of many tropical developing countries. We report in this paper an attempt to develop a system of intercontinental shipping for transfer of fresh crossbred Bos taurus×Bos indicus IVF embryos into local Laisind (Bos indicus) recipients as a way to produce tropical dairy calves with highly improved milk productivity. The production of embryos was done at BOMED, Inc (Madison, WI, USA). Oocytes collected from ovaries of Holstein (Bos taurus) at slaughter and semen from milking Gir (Bos indicus) were used for IVP. Cleaved embryos were selected for air shipping in portable incubators at Day 4 (Group 1), Day 3 (Group 2) or Day 2 (Group 3) after IVF. The duration of shipping varied from 60 to 65h. Embryo transfer was done in Vietnam. Laisind cows (Yellow cattle×Red Sindhi) with body weight more then 280kg and normal reproductive activity were selected for treatment of estrous synchronization with double 11-day interval injection of PG2α (Intervet, Boxmeer, The Netherlands) and single injection of eCG (SABC Vietnam) two days before the second injection of PG2α. Timing of injections was calculated according to the IVF schedule. Embryos collected from portable incubators were transferred to a CO2 incubator for further culture at 39°C. Two experiments were carried out: (1) transfer of embryos without sexing;; (2) transfer of embryos after biopsy and sex determination by PCR. In experiment 2, compact morulae or morula-blastocysts were selected for sex determination. Four to five blastomeres were aspirated from each embryo using a cutting pipette and an aspiration pipette of 30-μm diameter. PCR was done as previously described (Uoc et al., 1999 J. Biology). After biopsy, embryos were kept in culture for one day to observe the development in vitro. Embryos developed to morula-blastocyst or hatching blastocyst at Day 7 or Day 8 after IVF were transferred nonsurgically to recipients with estrus detected in the period from 0 to 12h before or after the starting IVF. Pregnancy was confirmed by rectal palpation 3 months after embryo transfer. The average rate of embryos developed into morula-blastocyst was more then 50% (Table 1) and there were no significant differences among different shipping groups. For experiment 2, more then 87 % of embryos biopsied developed in vitro to expanding and hatching embryos. The average rate of female embryos was 56.3%. The pregnancy rate at 3 months was more then 44% (n=188). The first group of calves was born without unusual birthing problems. In conclusion, the system of embryo transfer using intercontinental shipping of fresh IVF embryos and local Bos indicus recipients can be applied for production of dairy calves. Supported by grant from the AIRE-Development agency. Table 1 Development in vitro of IVP embryos