Search

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. Indeed, in XX PIS–/– gonads the deletion of PIS leads to the transcriptional extinction of at least 3 neighboring genes, FOXL2, PFOXic and PISRT1. Among them, only FOXL2 is a ‘classical’ gene, encoding a highly conserved transcription factor. On the other hand, knock-out of Foxl2 in mice results in an early blocking of follicle formation without sex-reversal. This phenotype discrepancy leads to two hypotheses, either FOXL2 is responsible for XX sex-reversal in goat assuming distinct functions of its protein during ovarian differentiation in different mammals, or other PIS-regulated genes are involved. To assess the second possibility, PISRT1 expression was constitutively restored in XX PIS–/– gonads. Six transgenic fetuses were obtained by nuclear transfer and studied at 2 developmental stages, 41 and 46 days post-reconstruction. The gonads of these fetuses appear phenotypically identical to those of cloned non-transgenic controls. Conclusively, this result argues for FOXL2 being responsible for the PIS gonad-associated phenotype. Its invalidation in goat will help to better understand this complex syndrome.

Chapitre 2; International audience; 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.

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. The persistence 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 microtubular 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.

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%).

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. Whereas the GJ decreased in diameter after 24 h of culture, their number was still substantially great at that time. The Cx43-positive GJ, localized between corona radiata cell projections and oolemma, disappeared after 6 h of culture. Concomitantly, the OCC lost the ability to transfer LY from cumulus to oocyte, and connexin32 (Cx32) became detectable in the oocytes. Both the changes in corona-oocyte coupling and cumulus expansion were preceded by the redistribution of F-actin in cytoplasm of CC. These data indicate that functional GJ linked the CC until the second meiotic arrest. However, the removal of Cx43-positive GJ interconnecting cytoplasmic projections of corona radiata cells with the oocyte was temporally correlated with germinal vesicle breakdown. The present results suggest the pivotal role of the cytoskeleton (F-actin) in cumulus expansion.

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. Indeed, in XX PIS(-/-) gonads the deletion of PIS leads to the transcriptional extinction of at least 3 neighboring genes, FOXL2, PFOXic and PISRT1. Among them, only FOXL2 is a 'classical' gene, encoding a highly conserved transcription factor. On the other hand, knock-out of Foxl2 in mice results in an early blocking of follicle formation without sex-reversal. This phenotype discrepancy leads to two hypotheses, either FOXL2 is responsible for XX sex-reversal in goat assuming distinct functions of its protein during ovarian differentiation in different mammals, or other PIS-regulated genes are involved. To assess the second possibility, PISRT1 expression was constitutively restored in XX PIS(-/-) gonads. Six transgenic fetuses were obtained by nuclear transfer and studied at 2 developmental stages, 41 and 46 days post-reconstruction. The gonads of these fetuses appear phenotypically identical to those of cloned non-transgenic controls. Conclusively, this result argues for FOXL2 being responsible for the PIS gonad-associated phenotype. Its invalidation in goat will help to better understand this complex syndrome.

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.

International audience; 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. In other mammalian species, however, this transition occurs later after several cell cycles, and little is known concerning the regulation of maternal information during this period. To address this question, we have used rabbit pre-implantation embryos to analyze the translational activation and stability of two maternal mRNAs, mm 41 and mm61. During the cleavage period, these mRNAs exhibit distinct kinetics for both their translational activation and degradation. In addition, these mRNAs both undergo cytoplasmic polyadenylation but with different efficiencies. This polyadenylation was functionally correlated with the translational activation of these mRNAs; inhibiting polyadenylation prevented translational activation. The differential efficiency of cytoplasmic polyadenylation, driven by cis-elements in the 3' untranslated region of these mRNAs, was also observed in Xenopus laevis embryos, which emphasizes the high conservation of this mechanism between species.

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. © 2000 Wiley-Liss, Inc.

Summary 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.

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. In a second experiment, sex was determined from a single cell and the other cells were used for nuclear transfer. The effect of sex on in vitro development was studied in 386 male and 314 female reconstructed embryos derived from 19 male and 14 female parent embryos, respectively. Developmental competence in vitro of male and female constructs over 7 days was not statistically different (25.2 and 23.1% blastocysts on day 7, respectively; P > 0.05). After the transfer of predetermined male (n = 30) and female (n = 27) cloned embryos into recipient heifers, no effect of sex was observed on pregnancy rates at day 21, 35 and 90, or on calving rates (P > 0.05). These rates did not differ between single and twin transfer (P > 0.05). The sex of the calves born always corresponded to that determined from a single blastomere. These results show that sex can be determined accurately when using a single blastomere before nuclear transfer and that the sex of the parent embryo does not affect in vitro development or in vivo survival rates of cloned embryos.

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. Embryonic nuclear transfer has improved and is associated with sexing to generate sets of clones despite a great variability of results between parent embryos. The factors of progress are reviewed here. Recently, somatic cells used as a source of nuclei in bovine nuclear transfer has been demonstrated. Here we present the results of the developmental potential of nuclei from skin and muscle 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 mid-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.

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. Changes in nucleolar ultrastructure after the nuclear transfer reflected the inhibition and subsequent reactivation of rRNA transcription. Nucleoli of 32-cell embryos had a typical structure of active nucleoli; many fibrillar centers surrounded and interconnected by threads of the dense fibrillar component and embedded in the granular component. Six hours following nuclear transplantation, these nucleoli underwent drastic changes including loss of granular material, collapse of nucleolar structure, and segregation of nucleolar components. Following the first cleavage, segregated fibrillar components of nucleoli manifested a complete inhibition of nucleolar transcription. Ribosomal RNA transcription was restored at the eight-cell stage and the sequence of ultrastructural changes was similar to that of the normal development. However, at the 32-cell stage, excessive extrusion of pre-ribosomal particles in the cytoplasm occurred, suggesting a possible alteration in regulating mechanisms of ribosome delivery. These results show that after fusion with enucleated metaphase II cytoplasm and subsequent activation, transcription is inhibited in donor embryonic nuclei and progressively increases again during cleavage; almost as in normal embryos. Migration of ribosomes into cytoplasm appears more intense in 32-cell stage reconstituted embryos but this does not seem to inhibit blastocyst building.

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. However, after a further 4-day culture of the cleaved embryos, the blastocyst formation rate was four times higher in those made with male (16%) than with female (4%) gonial cells. No implantation sites were detected following transfer of reconstituted embryos into recipient females. These results show that the nuclei of male and female rabbit diploid germ cells differ in their capability to be “reprogrammed” and bring about development to the blastocyst stage following nuclear transfer. The origin of this difference, which is evidenced long before the onset of meiosis, is discussed.

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. For this purpose the phosphorylation profiles of four groups of in vitro-matured bovine oocytes (aged oocytes, aged-cooled oocytes, enucleated-aged oocytes and enucleated-aged-cooled oocytes (recipient cytoplasts)) were analyzed. These recipient cytoplasts exhibited a phosphorylation profile similar to that of activated oocytes. Maturation promoting factor (MPF) activity, which was high in young metaphase II oocytes, in aged oocytes, in enucleated-aged oocytes and in aged-cooled oocytes, dropped to the basal level in enucleated-aged-cooled oocytes (recipient cytoplasts), while mitogen-activated protein kinase (MAPK) activity remained elevated. The combination of enucleation, ageing and cooling following oocyte in vitro maturation resulted in an interphase-like stage cytoplasm having a phosphorylation profile and low MPF activity similar to activated oocytes, but exhibiting high MAPK activity.

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.

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. This number was not significantly different from that of control IVF embryos cultured under the same conditions during the same period (mean = 88.89 +/- 7.53). Semi-thin sections revealed that most of the blastocysts had an inner cell mass (10/12) and a blastocoele. Under transmission electron microscopy, the trophectoderm appeared well differentiated as a polarized epithelium with apical microvilli and lateral junctions including desmosomes with bound intermediate filaments. The cytoplasm sometimes contained immature mitochondria or a large number of residual bodies. About half of the blastocysts examined had a large amount of cellular debris in the perivitelline space or inside the blastocoele cavity. The cloned blastocysts were also able to hatch in vitro by day 8 and SEM indicated a normal morphology of the trophectoderm cells with numerous apical microvilli. The high number of excluded or degenerating cells found in some embryos may partially explain early embryonic mortality that follows transfer. However, these observations do not give a clear explanation for the high incidence of fetal losses.

International audience

International audience

International audience

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. Whereas the GJ decreased in diameter after 24 h of culture, their number was still substantially great at that time. The Cx43-positive GJ, localized between corona radiata cell projections and oolemma, disappeared after 6 h of culture. Concomitantly, the OCC lost the ability to transfer LY from cumulus to oocyte, and connexin32 (Cx32) became detectable in the oocytes. Both the changes in corona-oocyte coupling and cumulus expansion were preceded by the redistribution of F-actin in cytoplasm of CC. These data indicate that functional GJ linked the CC until the second meiotic arrest. However, the removal of Cx43-positive GJ interconnecting cytoplasmic projections of corona radiata cells with the oocyte was temporally correlated with germinal vesicle breakdown. The present results suggest the pivotal role of the cytoskeleton (F-actin) in cumulus expansion.