Nuclear transfer in rabbit.

Since 2002, our INRA laboratory (Biologie du Développement et de la Reproduction) has developed a method to produce live somatic clones in rabbit, one of the mammalian species considered as difficult to clone. This chapter presents the technical protocol used nowadays to achieve the goal to obtain cloned embryos able to develop to term using fresh somatic cumulus cells.

First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning.

Two experiments have been performed to clone the bucardo, an extinct wild goat. The karyoplasts were thawed fibroblasts derived from skin biopsies, obtained and cryopreserved in 1999 from the last living specimen, a female, which died in 2000. Cytoplasts were mature oocytes collected from the oviducts of superovulated domestic goats.

Attempt to rescue sex-reversal by transgenic expression of the PISRT1 gene in XX PIS-/- goats.

The Polled Intersex Syndrome (PIS mutation) in goats leads to an absence of horn and to an early sex-reversal of the XX gonads. This mutation is a deletion of an 11.7-kb DNA fragment showing a tissue-specific regulatory activity.

In utero characterisation of fetal growth by ultrasound scanning in the rabbit.

Fetal development is an important factor influencing the susceptibility of adults to metabolic diseases. In order to study the influence of fetal growth on further development in animal models like the rabbit, methods of measurement of fetal and placental size and viability must be established and validated. In this study, 42 New Zealand does bred naturally (N=12) or transferred with in vivo produced embryos (2, 4 or 6 embryos/doe) have been scanned every 2-3 days with a 7.

Cloned rabbits produced by nuclear transfer from adult somatic cells.

We have developed a method to produce live somatic clones in the rabbit, one of the mammalian species considered up to now as difficult to clone. To do so, we have modified current cloning protocols proven successful in other species by taking into account both the rapid kinetics of the cell cycle of rabbit embryos and the narrow window of time for their implantation after transfer into foster recipients. Although our method still has a low level of efficiency, it has produced several clones now proven to be fertile.

Transcription-dependent nucleocytoplasmic distribution of hnRNP A1 protein in early mouse embryos.

A unique feature of certain members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins is that they shuttle continuously between nucleus and cytoplasm and their accumulation in the nucleus is transcription-dependent. An extensively characterised protein of this group is hnRNP A1. To date, most studies addressing the transcription-dependent transport of hnRNP A1 have been performed on cultured cell lines treated with transcription inhibitors.

Centrosome inheritance in sheep zygotes: centrioles are contributed by the sperm.

The inheritance and duplication of the sperm centriole in the sheep zygote was studied by transmission electron microscopy. We found two centrioles at one pole and a single centriole at the opposite pole of the first mitotic spindle, in monospermic eggs, 20-21 hours postinsemination. This indicated both duplication and relocation of centrioles to opposite spindle poles during fertilization.

Differential regulation of the translation and the stability of two maternal transcripts in preimplantation rabbit embryos.

In most species, transcription is essentially silent during the first mitotic cell cycles that follow fertilization. This means that the regulation of gene expression in early embryos heavily relies on the translational activation or inactivation of maternal mRNAs. In mammals, the mechanisms that control the translation of maternal mRNAs have been mainly studied in the mouse when maternal to zygotic transition occurs after the first mitotic division.

Lymphoid hypoplasia and somatic cloning.

Background: Adult somatic cloning by nuclear transfer is associated with high rate of perinatal mortality but there is still no evidence that nuclear transfer itself is responsible for these failures. We report on a longlasting defect linked to somatic cloning.

Methods: Skin cells grown from an ear biopsy specimen from a 15-day-old calf were used as a source of nuclei.

Cloning in cattle: from embryo splitting to somatic nuclear transfer.

The ability to obtain genetically identical offspring in cattle (clones) is useful for research and for potential applications to breeding schemes. Experimental possibilities for generating such animals have evolved considerably in the last two decades. Embryo splitting has become a relatively simple technique but is limited to twinning.

Nuclear transfer from sexed parent embryos in cattle: efficiency and birth of offspring.

The objectives of this study were to evaluate the efficiency and reliability of embryo sexing from isolated single blastomeres, and after nuclear transfer to examine the influence of the sex of donor embryos on development in vitro and in vivo up to calving. The sex of the donor embryo was determined by revealing a specific Y DNA sequence by PCR and electrophoresis after isolation of one, two, three, or more than five cells. The efficiency of sex determination was over 90% and reliability was 100% independent of the number of blastomeres used.

Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells.

Muscle and skin biopsies taken from bovine fetuses and young calves have been used as a source of donor nuclei for cloning experiments. After culture, cells were individually fused to enucleated matured oocytes and the resulting blastocysts obtained after 7 d of culture (3-8% depending on the cell type) were transferred to foster recipient heifers. Two calves, a female and a male, both originating from muscle cells were born, and four additional pregnancies have surpassed mild-term gestation.

In vivo aging of oocytes influences the behavior of nuclei transferred to enucleated rabbit oocytes.

The present study examined nuclear remodeling in rabbit nuclear transfer (NT) embryos formed from metaphase II (MII) oocytes aged in vivo until 19 hr postcoitum (hpc), enucleated, and fused at 22-26 hpc with 32-cell morula blastomeres by means of electric fields, which also induced recipient oocyte activation. Post-activation events observed during the first hour following the fusion/activation pulse were studied in terms of chromatin, lamins, and microtubules, and revealed that transferred nuclei underwent premature chromosomes condensation (PCC) in only one-third of NT embryos and remained in interphase in others. Recipient oocytes were mostly not activated by manipulations performed before the fusion/activation pulse.

Bovine embryo cloning: Characterization of the recipient cytoplasts by phosphorylation patterns and kinase activities.

When in vitro-matured oocytes were enucleated, aged and kept at 10°C before reconstitution, the in vitro development of nuclear transfer embryos to the blastocyst stage did not differ from that obtained with in vitro fertilization. This suggests that these recipient cytoplasts constitute a suitable environment for the development of the nuclear transplant. The aim of the present study was to investigate, at the biochemical level, the result of the preparation of recipient oocytes, including enucleation, ageing and cooling.

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer.

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post coïtum (d.p.

Differential ability of male and female rabbit fetal germ cell nuclei to be reprogrammed by nuclear transfer.

The pluri- or totipotency of gonial cells, isolated from rabbit fetuses at 18-20 days of pregnancy, has been investigated by transferring their nuclei into enucleated oocytes and following the development of the resulting reconstituted embryos both in vitro (in a total of 726 embryos) and in vivo (in 135 embryos). The gonial cells exhibited pseudopodial activity like that of primordial germ cells and ultrastructural studies confirmed that neither male nor female cells had entered meiosis. When the gonial cells were used immediately after isolation, about 37% of the reconstituted embryos of both sexes cleaved, with no significant difference according to sex.

Transcriptional activity and nucleolar ultrastructure of embryonic rabbit nuclei after transplantation to enucleated oocytes.

Changes in the level of transcriptional activity in 32-cell stage morula nuclei were studied after blastomere electrofusion to enucleated oocytes. Nuclear transplant recipients were pulse labelled with 3H-uridine during cultivation in vitro, embryos were then fixed and processed for autoradiography and electron microscopy. Transcriptional activity substantially decreased after 4.

Cellular evaluation of bovine nuclear transfer embryos developed in vitro.

Cloned blastocysts developed in vitro for 7 d had a mean number of cells (82.86 +/- 5.35) as evaluated by nuclei counting in serial optical sections using confocal microscopy, after staining with propidium iodide.

Developmental ability of bovine embryos after nuclear transfer based on the nuclear source: in vivo versus in vitro.

Bovine nuclear transfer embryos reconsitituted from in vitro-matured recipient oocyte cytoplasm and different sources of donor nuclei (in vivo, in vitro-produced or frozen-thawed) were evaluated for their ability to develop in vitro. Their cleavage rate and blastocyst formation are compared with those of control IVF embryos derived from the same batches of in vitro-matured oocytes that were used for nuclear transfer and were co-cultured under the same conditions on bovine oviducal epithelial cell monolayers for 7 d. Using fresh donor morulae as the source of nuclei resulted in 30.

Dynamic changes of gap junctions and cytoskeleton during in vitro culture of cattle oocyte cumulus complexes.

Changes in cell-to-cell contact and distribution of cytoskeletal components were investigated during in vitro culture of cattle oocyte cumulus complexes (OCC). Freeze-fracture analysis (FF), microinjections of the fluorescent dye Lucifer Yellow (LY), immunofluorescence, and ultrastructural immunocytochemistry were used. The cumulus cells (CC) remained in close contact via gap junctions (GJ) constituted of connexin43 (Cx43) during the entire culture time.

Nuclear transfer in cattle: birth of cloned calves and estimation of blastomere totipotency in morulae used as a source of nuclei.

The birth of a clone of five bull calves, each produced by fusing a cell taken from the same young embryo with an oocyte whose own nucleus has been removed, opens the way to the use of these new animal models for research and animal selection. The embryonic cells came from a 31-cell morula. Our results indicate that most of the nuclei at this stage are totipotent which seems to no longer be the case for those taken from the cells of the inner cell mass at the blastocyst stage.

Lamb production using superovulation, embryo bisection, and transfer.

In this study, 39 embryos from donor ewes superovulated with follicle stimulating hormone-pituitary (FSH-P) were bisected to produce pairs of monozygotic twin lambs for experimentation. Each pair obtained by bisecting 8-, 9- or 10-day-old embryos was immediately transferred surgically into a recipient ewe at the same physiological stage. Of the 39 recipients which received a pair of half-embryos by transfer into the uterine horn ipsilateral to the corpus luteum, 28 (72%) lambed.

Improvement of survival rate of frozen cattle blastocysts after transfer with trophoblastic vesicles.

An experiment was conducted to determine if the loss of viability due to deep freezing could be overcome by addition of trophoblastic tissue to the embryo at transfer time. Forty-nine recipient heifers in a cotransfer group each received one frozen blastocyst + two frozen trophoblastic vesicles. The confirmed pregnancy rates by Day 45, 60, and 90 were 73, 61, and 57%, respectively.