P. Chesné

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. Our work indicates that cloning can probably be carried out successfully in any mammalian species by taking into account physiological features of their oocytes and embryos. Our results will contribute to extending the use of rabbit models for biomedical research.

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. The donor animal was a clone of three females obtained from embryonic cells. Clinical examination, haematological, and biochemical profiles, and echocardiography of the somatic clone were done from birth to death. FINDINGS After 6 weeks of normal development, the somatic cloned calf had a sudden and rapid fall in lymphocyte count and a decrease in haemoglobin. The calf died on day 51 from severe anaemia. Necropsy revealed no abnormality except thymic atrophy and lymphoid hypoplasia. INTERPRETATION Somatic cloning may be the cause of long-lasting deleterious effects. Our observation should be taken into account in debates on reproductive cloning in human beings.

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. The pregnancies include one fetus established from a transgenic nucleus from a fetal skin cell, and another one resulting from a skin biopsy performed on a female calf. Our data demonstrate that nuclei from cultured bovine somatic cells obtained from differentiated tissues can be made multipotent.

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. Oocytes were enucleated and coupled to bucardos fibroblasts by electrofusion. Reconstructed embryos were cultured for 36h or 7d and transferred to either Spanish ibex or hybrid (Spanish ibex malex domestic goat) synchronized recipients. Embryos were placed, according to their developmental stage, into the oviduct or into the uterine horn ipsilateral to an ovulated ovary. Pregnancy was monitored through their plasmatic PAG levels. In Experiment 1, 285 embryos were reconstructed and 30 of them were transferred at the 3- to 6-cells stage to 5 recipients. The remaining embryos were further cultured to day 7, and 24 of them transferred at compact morula/blastocyst stage to 8 recipients. In Experiment 2, 154 reconstructed embryos were transferred to 44 recipients at the 3- to 6-cells stage. Pregnancies were attained in 0/8 and 7/49 of the uterine and oviduct-transferred recipients, respectively. One recipient maintained pregnancy to term, displaying very high PAG levels. One morphologically normal bucardo female was obtained by caesarean section. The newborn died some minutes after birth due to physical defects in lungs. Nuclear DNA confirmed that the clone was genetically identical to the bucardos donor cells. To our knowledge, this is the first animal born from an extinct subspecies.

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 4.5 hr and was completely inhibited at last 15 hr after fusion. Transcription resumed thereafter in two‐cell stage embryos and could be detected in both nuclei from 70% of the embryos analyzed. Transcription activity rapidly increased at the eight 16‐cell stages, reaching the level typical for 32‐cell stage nuclei used for the transfer.

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 30.2% blastocyst formation (150 497 ), which was similar to that of control IVM-IVF embryos (33.8% blastocysts, 222 657 ). When IVF embryos were used as the source of nuclei for cloning, a slightly lower blastocyst formation rate (22.6%, 41 181 ) was obtained but not significantly different from that using fresh donor morulae. Nuclear transfer embryos derived from vitrified donor embryos showed poor development in vitro (7.1%, 11 154 ). No difference in morphology or cell number was observed after 7 d of co-culture between blastocysts derived from nuclear transfer or control IVF embryos. The viability of 34 in vitro-developed nuclear transfer blastocysts was tested in vivo and resulted in the birth of 11 live calves (32.3%).

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 micro‐tubules, 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. The persistance of transferred nuclei in interphase resulted from the rapid progression of recipient oocytes to interphase after activation, suggesting that the cytoplasmic state of MII oocytes aged in vivo was poised for the approach to interphase. Studying micro‐tubular organization in MII oocytes before nuclear transfer manipulations, we found that 19 hpc MII oocytes aged in vivo differed from 14 hpc MII oocytes (freshly ovulated) and from 19‐hpc MII oocytes aged in vitro (collected at 14 hpc and cultured for 5 hr), notably by the presence of microtubule asters and tubulin foci or only tubulin foci dispersed throughout the cytoplasm. When PCC was avoided, remodeling of the transferred nucleus was well advanced 1 hr after nuclear transfer, and NT embryos developed better to the blastocyst stage. Mol. Reprod. Dev. 46:325–336, 1997.

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.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.

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. The absence of centrioles in mature sheep oocytes was confirmed. Following activation by the calcium ionophore A 23187, mature oocytes entered mitosis and formed a bipolar spindle 18 hours later. Centrioles were not detected in the mitotic spindle of parthenogenotes. Androgenetic eggs were obtained by excision of the anaphase II/telophase II meiotic spindle of fertilized eggs. They were capable of undergoing mitosis and formed one or two bipolar spindle(s) in monospermic and dispermic eggs, respectively, 20–24 hours postinsemination. In two monospermic androgenetic eggs, two centrioles were found at one pole and a single centriole at the opposite pole of the first mitotic spindle. Three centrioles were also observed in another androgenetic egg in prometaphase of the first mitotic division, in close vicinity to the sperm neck‐piece. These data provide evidence that the sperm centriole do reproduce and occupy a pivotal position on opposite spindle poles at syngamy. Altogether, the present findings suggest that centrioles of sheep zygotes are paternally derived. Microsc. Res. Tech. 49:445–450, 2000.

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. Eighteen of 28 recipients lambing (64%) produced pairs, i.e., 7 male and 11 female pairs. Ten of 28 lambings produced a single lamb, i.e., six males and four females. Overall yield (the number of lambs produced in relation to the number of embryos used) was 118%. This percentage tended to increase, depending on the day of collection (Day 8, 100%; Day 9, 118%; and Day 10, 131%).