The PRAMOS Study: PRostaglandin Analogues Monotherapy-Awareness Survey on Ocular Surface Involvement.

INTRODUCTION: Even though the local tolerance of prostaglandin (PG) analogues has improved drastically since the introduction of preservative-free (PF) eye drops, prescription patterns still vary widely among practitioners and between countries and could have an impact on the ocular surface of treated patients and, in consequence, their adherence. The aim of this study is to explore the prescribing patterns of PG analogues monotherapy in France and to evaluate their impact on ocular surface status. METHODS: This was a national multicenter cross-sectional observational study that was conducted by 18 glaucoma experts in France. Patients over 18 years of age and receiving monotherapy with topical PG analogues for the treatment of ocular hypertension and/or glaucoma, with no history of prior glaucoma surgery, were consecutively selected from the glaucoma outpatient clinics of participating physicians and underwent an ocular surface examination. RESULTS: A total of 344 eyes of 344 patients were enrolled between November 2022 and November 2023. Prescribed PG monotherapy was PF in 271 (78.7%) patients. Clinical history and ocular surface evaluation indicated that 79.4% of the study population (n = 273) presented with at least one symptom or clinical sign of dry eye and that three patients out of four had an unstable tear film. Subgroup analysis comparing preserved and PF PG analogues showed a higher prevalence of conjunctival hyperemia and corneal staining in the preserved group. Multivariate analysis identified conjunctival hyperemia as consistently associated with preservative use (odds ratio = 7.654; p = 0.003 for moderate conjunctival hyperemia). CONCLUSIONS: This study highlights the growing trend toward PF PG analogue prescriptions by specialists in France. However, ocular surface issues remain prevalent, impacting patient adherence and treatment efficacy. Comprehensive ocular surface examinations are crucial in glaucoma management to enhance long-term tolerance, compliance, and overall treatment success.

Massive dysregulation of genes involved in cell signaling and placental development in cloned cattle conceptus and maternal endometrium.

A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients before or during the implantation window. We investigated this problem by using RNA sequencing (RNA-seq) to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at day (d) 18 (preimplantation) and d 34 (postimplantation) of gestation. In addition, endometrium was profiled to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality before or during the implantation window. At d 18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d 18, <0.4% of expressed genes were affected by the presence of a cloned conceptus, whereas at d 34, ∼36% and <0.7% of genes were differentially expressed in intercaruncular and caruncular tissues, respectively. Functional analysis of DEGs in placental and endometrial tissues suggests a major disruption of signaling between the cloned conceptus and the endometrium, particularly the intercaruncular tissue. Our results support a "bottleneck" model for cloned conceptus survival during the periimplantation period determined by gene expression levels in extraembryonic tissues and the endometrial response to altered signaling from clones.

Multicenter clinical trial of high-intensity focused ultrasound treatment in glaucoma patients without previous filtering surgery.

PURPOSE: To evaluate the efficacy and safety of the ultrasonic circular cyclocoagulation procedure in patients with open-angle glaucoma naïve of previous filtering surgery. METHODS: Prospective non-comparative interventional clinical study conducted in five French University Hospitals. Thirty eyes of 30 patients with open-angle glaucoma, intra-ocular pressure (IOP) > 21 mmHg and with no previous filtering glaucoma surgeries were sonicated with a probe comprising six piezoelectric transducers. The six transducers were activated with a 6-s exposure time. Complete ophthalmic examinations were performed before the procedure and at 1 day, 1 week, 1, 2, 3, 6 and 12 months after the procedure. Primary outcomes were qualified surgical success (defined as IOP reduction from baseline ≥20% and IOP > 5 mmHg with possible re-intervention and without hypotensive medication adjunction) and complete surgical success (defined as IOP reduction from baseline ≥20%, IOP > 5 mmHg and IOP < 21 mmHg with possible re-intervention and without hypotensive medication adjunction) at the last follow-up visit and vision-threatening complications. Secondary outcomes were mean IOP at each follow-up visit compared with baseline, medication use, complications and re-interventions. RESULTS: Intra-ocular pressure was significantly reduced (p < 0.05) from a mean pre-operative value of 28.2 ± 7.2 mmHg (n = 3.6 hypotensive medications) to 19.6 ± 7.9 mmHg at 12 months (n = 3.1 hypotensive medications and n = 1.1 procedures) (mean IOP reduction of 30%). Qualified success was achieved in 63% of eyes (19/30) (mean IOP reduction of 37% in these eyes) and complete success in 46.7% of eyes (14/30) (mean IOP reduction of 37% in these eyes) at the last follow-up. No major intra- or post-operative complications occurred. CONCLUSIONS: The UC(3) procedure seems to be an effective and well-tolerated method to reduce IOP in patients with open-angle glaucoma without previous filtering surgery.

Changes in WNT signaling-related gene expression associated with development and cloning in bovine extra-embryonic and endometrial tissues during the peri-implantation period.

We determined if somatic cell nuclear transfer (SCNT) cloning is associated with WNT-related gene expression in cattle development, and if the expression of genes in the WNT pathway changes during the peri-implantation period. Extra-embryonic and endometrial tissues were collected at gestation days 18 and 34 (d18, d34). WNT5A, FZD4, FZD5, LRP5, CTNNB1, GNAI2, KDM1A, BCL2L1, and SFRP1 transcripts were localized in extra-embryonic tissue, whereas SFRP1 and DKK1 were localized in the endometrium. There were no differences in the localization of these transcripts in extra-embryonic tissue or endometrium from SCNT or artificial insemination (AI) pregnancies. Expression levels of WNT5A were 11-fold greater in the allantois of SCNT than AI samples. In the trophoblast, expression of WNT5A, FZD5, CTNNB1, and DKK1 increased significantly from d18 to d34, whereas expression of KDM1A and SFRP1 decreased, indicating that implantation is associated with major changes in WNT signaling. SCNT was associated with altered WNT5A expression in trophoblasts, with levels increasing 2.3-fold more in AI than SCNT conceptuses from d18 to d34. In the allantois, expression of WNT5A increased 6.3-fold more in SCNT than AI conceptuses from d18 to d34. Endometrial tissue expression levels of the genes tested did not differ between AI or SCNT pregnancies, although expression of individual genes showed variation across developmental stages. Our results demonstrate that SCNT is associated with altered expression of specific WNT-related genes in extra-embryonic tissue in a time- and tissue-specific manner. The pattern of gene expression in the WNT pathway suggests that noncanonical WNT signal transduction is important for implantation of cattle conceptuses.

Nutritional, lifestyle and environmental factors in ocular hypertension and primary open-angle glaucoma: an exploratory case-control study.

PURPOSE: To evaluate known and potential risk factors, including nutritional, lifestyle and environmental factors, differentiating patients with high-tension primary open-angle glaucoma (POAG) from control subjects with ocular hypertension (OHT). METHODS: In 2006-2007, 111 French ophthalmologists prospectively enrolled 339 cases of POAG and 339 age-matched controls with OHT. After a clinical examination with assessment of ocular risk factors, the ophthalmologist filled, during face-to-face interview, a detailed questionnaire developed by nutritionists and epidemiologist on lifestyle and environmental risk factors, including socio-demographic variables, dietary habits related to omega-3 fatty acids intake, smoking and alcohol drinking and professional exposure to pesticides and other chemicals. Associations of POAG with risk factors were estimated using conditional logistic regression, with adjustment for age, gender and duration of disease. RESULTS: In the final multivariate model, by comparison with OHT, POAG was significantly associated with more frequent use of pesticides during the professional life [OR = 2.65, 95% confidence interval (CI): 1.04-6.78, p = 0.04] and with low consumption of fatty fish (OR = 2.14, 95% CI: 1.10-4.17, p = 0.02) and walnuts (OR = 2.02, 95% CI: 1.18-3.47, p = 0.01). POAG was also associated with higher frequency of heavy smoking (40 pack-years or more, OR = 3.93, 95% CI: 1.12-13.80, p = 0.03) but not with moderate (20-40 pack-years) and light smoking (<20 pack-years). CONCLUSIONS: These exploratory observations suggest a protective effect of omega-3 fatty acids and a deleterious effect of heavy smoking and professional exposure to pesticides in POAG. This will need to be confirmed in future studies.

Synergic reprogramming of mammalian cells by combined exposure to mitotic Xenopus egg extracts and transcription factors.

Transfer of somatic cell nuclei to enucleated eggs and ectopic expression of specific transcription factors are two different reprogramming strategies used to generate pluripotent cells from differentiated cells. However, these methods are poorly efficient, and other unknown factors might be required to increase their success rate. Here we show that Xenopus egg extracts at the metaphase stage (M phase) have a strong reprogramming activity on mouse embryonic fibroblasts (MEFs). First, they reset replication properties of MEF nuclei toward a replication profile characteristic of early development, and they erase several epigenetic marks, such as trimethylation of H3K9, H3K4, and H4K20. Second, when MEFs are reversibly permeabilized in the presence of M-phase Xenopus egg extracts, they show a transient increase in cell proliferation, form colonies, and start to express specific pluripotency markers. Finally, transient exposure of MEF nuclei to M-phase Xenopus egg extracts increases the success of nuclear transfer to enucleated mouse oocytes and strongly synergizes with the production of pluripotent stem cells by ectopic expression of transcription factors. The mitotic stage of the egg extract is crucial, because none of these effects is detected when using interphasic Xenopus egg extracts. Our data demonstrate that mitosis is essential to make mammalian somatic nuclei prone to reprogramming and that, surprisingly, the heterologous Xenopus system has features that are conserved enough to remodel mammalian nuclei.

A small set of extra-embryonic genes defines a new landmark for bovine embryo staging.

Axis specification in mouse is determined by a sequence of reciprocal interactions between embryonic and extra-embryonic tissues so that a few extra-embryonic genes appear as 'patterning' the embryo. Considering these interactions as essential, but lacking in most mammals the genetically driven approaches used in mouse and the corresponding patterning mutants, we examined whether a molecular signature originating from extra-embryonic tissues could relate to the developmental stage of the embryo proper and predict it. To this end, we have profiled bovine extra-embryonic tissues at peri-implantation stages, when gastrulation and early neurulation occur, and analysed the subsequent expression profiles through the use of predictive methods as previously reported for tumour classification. A set of six genes (CALM1, CPA3, CITED1, DLD, HNRNPDL, and TGFB3), half of which had not been previously associated with any extra-embryonic feature, appeared significantly discriminative and mainly dependent on embryonic tissues for its faithful expression. The predictive value of this set of genes for gastrulation and early neurulation stages, as assessed on naive samples, was remarkably high (93%). In silico connected to the bovine orthologues of the mouse patterning genes, this gene set is proposed as a new trait for embryo staging. As such, this will allow saving the bovine embryo proper for molecular or cellular studies. To us, it offers as well new perspectives for developmental phenotyping and modelling of embryonic/extra-embryonic co-differentiation.

Identification of noncompliant glaucoma patients using Bayesian networks and the Eye-Drop Satisfaction Questionnaire.

OBJECTIVE: To identify poorly compliant glaucoma patients, using the Eye-Drop Satisfaction Questionnaire (EDSQ). METHODS: This was an observational cross-sectional study with compliance data collected by an electronic monitoring device. Patients with primary open-angle glaucoma or ocular hypertension completed the EDSQ, a six-dimension self-reported questionnaire addressing "treatment concern", "disease concern", "patient-clinician relationship", "positive beliefs", "treatment convenience", and "self-declared compliance". A Bayesian network (BN) was applied to explore compliance associations with EDSQ. RESULTS: Among 169 patients who completed the EDSQ, 113 had valid Travalert® data, of whom 25 (22.1%) demonstrated low compliance. All six EDSQ dimensions were associated directly, or indirectly, with compliance. Two profiles exhibited low compliance, ie, patients aged younger than 77.5 years with a poor patient-physician relationship and self-declared poor compliance and patients aged older than 77.5 years with a poor patient-physician relationship and self-declared good compliance. The third profile showed high compliance, ie, patients aged younger than 77.5 years with a good patient-physician relationship and self-declared good compliance. CONCLUSION: Our results confirm a central role for the patient-physician relationship in the compliance process. Age, self-declared compliance, and patient satisfaction with the patient-physician relationship are all dimensions worth exploring before glaucoma medication is switched or proceeding to laser treatment or surgery.

Nuclear transfer-derived epiblast stem cells are transcriptionally and epigenetically distinguishable from their fertilized-derived counterparts.

Mouse embryonic pluripotent stem cells can be obtained from the inner cell mass at the blastocyst stage (embryonic stem cells, ESCs) or from the late epiblast of postimplantation embryos (epiblast stem cells, EpiSCs). During normal development, the transition between these two stages is marked by major epigenetic and transcriptional changes including DNA de novo methylation. These modifications represent an epigenetic mark conserved in ESCs and EpiSCs. Pluripotent ESCs derived from blastocysts generated by nuclear transfer (NT) have been shown to be correctly reprogrammed. However, NT embryos frequently undergo abnormal development. In the present study, we have examined whether pluripotent cells could be derived from the epiblast of postimplantation NT embryos and whether the reprogramming process would affect the epigenetic changes occurring at this stage, which could explain abnormal development of NT embryos. We showed that EpiSCs could be derived with the same efficiency from NT embryos and from their fertilized counterparts. However, gene expression profile analyses showed divergence between fertilized- and nuclear transfer-EpiSCs with a surprising bias in the distribution of the differentially expressed genes, 30% of them being localized on chromosome 11. A majority of these genes were downregulated in NT-EpiSCs and imprinted genes represented a significant fraction of them. Notably, analysis of the epigenetic status of a downregulated imprinted gene in NT-EpiSCs revealed complete methylation of the two alleles. Therefore, EpiSCs derived from NT embryos appear to be incorrectly reprogrammed, indicating that abnormal epigenetic marks are imposed on cells in NT embryos during the transition from early to late epiblast.

Faithful reprogramming to pluripotency in mammals - what does nuclear transfer teach us?

Nuclear reprogramming toward pluripotency has been now achieved either in vivo by somatic cell nuclear transfer into the ooplasm of an enucleated egg, or in vitro by three different approaches, namely cell fusion, treatment with cell extracts and more recently, forced expression of a reduced set of defined transcription factors. This last technique has expanded our view of genome plasticity with important applied perspectives in regenerative biomedicine. Because of their ease of generation, induced pluripotent stem cells represent a major hope in the field of regenerative medicine. However, the extent to which such an in vitro induced pluripotency can be considered to be equivalent to embryonic-derived pluripotency remains undetermined and also largely dependent on how pluripotency is assessed. Here, we provide an overwiew of the data published in the recent literature on the ability of each of the above techniques to reprogram somatic nuclei into pluripotent embryonic-like nuclei. These data support the view that even though nuclear transfer is technically demanding, it remains a fast and efficient means for a systematic derivation of bona fide embryonic stem cells from somatic donor cells. We conclude that nuclear transfer has still much to teach us about faithful nuclear reprogramming to pluripotency.

Gene expression profiles of bovine caruncular and intercaruncular endometrium at implantation.

At implantation the endometrium undergoes modifications necessary for its physical interactions with the trophoblast as well as the development of the conceptus. We aim to identify endometrial factors and pathways essential for a successful implantation in the caruncular (C) and the intercaruncular (IC) areas in cattle. Using a 13,257-element bovine oligonucleotide array, we established expression profiles at day 20 of the estrous cycle or pregnancy (implantation), revealing 446 and 1,295 differentially expressed genes (DEG) in C and IC areas, respectively (false discovery rate = 0.08). The impact of the conceptus was higher on the immune response function in C but more prominent on the regulation of metabolism function in IC. The C vs. IC direct comparison revealed 1,177 and 453 DEG in cyclic and pregnant animals respectively (false discovery rate = 0.05), with a major impact of the conceptus on metabolism and cell adhesion. We selected 15 genes including C11ORF34, CXCL12, CXCR4, PLAC8, SCARA5, and NPY and confirmed their differential expression by quantitative RT-PCR. The cellular localization was analyzed by in situ hybridization and, upon pregnancy, showed gene-specific patterns of cell distribution, including a high level of expression in the luminal epithelium for C11ORF34 and MX1. Using primary cultures of bovine endometrial cells, we identified PTN, PLAC8, and CXCL12 as interferon-tau (IFNT) target genes and MSX1 and CXCR7 as IFNT-regulated genes, whereas C11ORF34 was not an IFNT-regulated gene. Our transcriptomic data provide novel molecular insights accounting for the biological functions related to the C or IC endometrial areas and may contribute to the identification of potential biomarkers for normal and perturbed early pregnancy.

Early alteration of the self-renewal/differentiation threshold in trophoblast stem cells derived from mouse embryos after nuclear transfer.

Development after nuclear transfer (NT) is subjected to defects originating from both the epiblast and the trophoblast parts of the conceptus and is always accompanied by placentomegaly at term. Here we have investigated the origin of the reprogramming errors affecting the trophoblast lineage in mouse NT embryos. We show that trophoblast stem (TS) cells can be derived from NT embryos (ntTS cells) and used as an experimental in vitro model of trophoblast proliferation and differentiation. Strikingly, TS derivation is more efficient from NT embryos than from controls and ntTS cells exhibit a growth advantage over control TS cells under self-renewal conditions. While epiblast-produced growth factors Fgf4 and Activin exert a fine-tuned control on the balance between self-renewal and differentiation of control TS cells, ntTS cells exhibit a reduced dependency upon their micro-environment. Since the supply of growth factors is known do decrease at the onset of placental formation in vivo we propose that TS cells in NT embryos continue to self-renew during a longer period of time than in fertilized embryo. The resulting increased pool of progenitors could contribute to the enlarged extra-embryonic region observed in the early trophoblast of in vivo grown mouse NT blastocysts that results in placentomegaly.

The immune status of bovine somatic clones.

Agronomical applications of cloned livestock produced by somatic cell nuclear transfer (SCNT) have been authorized in the United States and the European Food Safety Authority published that there was no evidence of risks associated with the use of cloned animal in the breeding industry. Both assessments, however, underlined that complementary data are needed to update their conclusions. SCNT is associated with a high incidence of perinatal losses. After birth, cloned cattle appear to possibly present subtle immune defects, requiring extensive studies to be properly evidenced. Twenty-five cloned Holstein heifers from five distinct genotypes and their contemporary age- and sex-matched controls were compared. An extensive survey of leukocyte subsets was performed and the humoral and T-cell immune responses to exogenous antigens were studied. Cloned cattle presented a normal representation of leukocyte subsets. Functional immunity was not modified in cloned heifers, as they were able to raise an antibody response and to develop B and T cell-specific responses against the model antigen OVA (ovalbumin) and against a rotavirus vaccine as in controls. Thus, this extensive analysis supports previous data suggesting that cloned cattle have a normal immunity.

Endometrium as an early sensor of in vitro embryo manipulation technologies.

Implantation is crucial for placental development that will subsequently impact fetal growth and pregnancy success with consequences on postnatal health. We postulated that the pattern of genes expressed by the endometrium when the embryo becomes attached to the mother uterus could account for the final outcome of a pregnancy. As a model, we used the bovine species where the embryo becomes progressively and permanently attached to the endometrium from day 20 of gestation onwards. At that stage, we compared the endometrial genes profiles in the presence of an in vivo fertilized embryo (AI) with the endometrial patterns obtained in the presence of nuclear transfer (SCNT) or in vitro fertilized embryos (IVF), both displaying lower and different potentials for term development. Our data provide evidence that the endometrium can be considered as a biological sensor able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process. Compared with AI, numerous biological functions and several canonical pathways with a major impact on metabolism and immune function were found to be significantly altered in the endometrium of SCNT pregnancies at implantation, whereas the differences were less pronounced with IVF embryos. Determining the limits of the endometrial plasticity at the onset of implantation should bring new insights on the contribution of the maternal environment to the development of an embryo and the success of pregnancy.

Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term.

BACKGROUND: Genome reprogramming in early mouse embryos is associated with nuclear reorganization and particular features such as the peculiar distribution of centromeric and pericentric heterochromatin during the first developmental stage. This zygote-specific heterochromatin organization could be observed both in maternal and paternal pronuclei after natural fertilization as well as in embryonic stem (ES) cell nuclei after nuclear transfer suggesting that this particular type of nuclear organization was essential for embryonic reprogramming and subsequent development. RESULTS: Here, we show that remodeling into a zygotic-like organization also occurs after somatic cell nuclear transfer (SCNT), supporting the hypothesis that reorganization of constitutive heterochromatin occurs regardless of the source and differentiation state of the starting material. However, abnormal nuclear remodeling was frequently observed after SCNT, in association with low developmental efficiency. When transient treatment with the histone deacetylase inhibitor trichostatin A (TSA) was tested, we observed improved nuclear remodeling in 1-cell SCNT embryos that correlated with improved rates of embryonic development at subsequent stages. CONCLUSION: Together, the results suggest that proper organization of constitutive heterochromatin in early embryos is involved in the initial developmental steps and might have long term consequences, especially in cloning procedures.

Trophoblast stem cell derivation, cross-species comparison and use of nuclear transfer: new tools to study trophoblast growth and differentiation.

The trophoblast is a supportive tissue in mammals that plays key roles in embryonic patterning, foetal growth and nutrition. It shows an extensive growth up to the formation of the placenta. This growth is believed to be fed by trophoblast stem cells able to self-renew and to give rise to the differentiated derivatives present in the placenta. In this review, we summarize recent data on the molecular regulation of the trophoblast in vivo and in vitro. Most data have been obtained in the mouse, however, whenever relevant, we compare this model to other mammals. In ungulates, the growth of the trophoblast displays some striking features that make these species interesting alternative models for the study of trophoblast development. After the transfer of somatic nuclei into oocytes, studies in the mouse and the cow have both underlined that the trophoblast may be a direct target of reprogramming defects and that its growth seems specifically affected. We propose that the study of TS cells derived from nuclear transfer embryos may help to unravel some of the epigenetic abnormalities which occur therein.

Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning.

Therapeutic cloning, whereby somatic cell nuclear transfer (SCNT) is used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs), holds great promise for the treatment of many human diseases. ntESCs have been derived in mice and cattle, but thus far there are no credible reports of human ntESCs. Here we review the recent literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodeling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which ntESCs are derived, seems to be highly efficient. Defects in the extraembryonic lineage are probably the major cause of the low success rate of reproductive cloning but are not expected to affect the derivation of ntESCs. We remain optimistic that human therapeutic cloning is achievable and that the derivation of patient-specific ntESC lines will have great potential for regenerative medicine.

Cloned ferrets produced by somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.

Developmental abnormalities of NT mouse embryos appear early after implantation.

In mammals, cloning by nuclear transfer (NT) into an enucleated oocyte is a very inefficient process, even if it can generate healthy adults. We show that blastocysts derived from embryonic stem (ES) donor cells develop at a high rate, correctly express the pluripotential marker gene Oct4 in ICM cells and display normal growth in vitro. Moreover, the majority of them implant in the uterus of recipient females. We combine embryological studies, gene expression analysis during gastrulation and generation of chimaeric embryos to identify the developmental origin (stage and tissue affected) of NT embryo mortality. The majority died before mid-gestation from defects arising early, either at peri-implantation stages or during the gastrulation period. The first type of defect is a non-cell autonomous defect of the epiblast cells and is rescued by complementation of NT blastocysts with normal ES or ICM cells. The second type of defect affects growth regulation and the shape of the embryo but does not directly impair the initial establishment of the patterning of the embryo. Only chimaeras formed by the aggregation of NT and tetraploid embryos reveal no growth abnormalities at gastrulation. These studies indicate that the trophoblast cell lineage is the primary source of these defects. These embryological studies provide a solid basis for understanding reprogramming errors in NT embryos. In addition, they unveil new aspects of growth regulation while increasing our knowledge on the role of crosstalk between the extra-embryonic and the embryonic regions of the conceptus in the control of growth and morphogenesis.