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.

Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development.

X-chromosome inactivation (XCI) in female mammals allows dosage compensation for X-linked gene products between the sexes. The developmental regulation of this process has been extensively investigated in mice, where the X chromosome of paternal origin (Xp) is silenced during early embryogenesis owing to imprinted expression of the regulatory RNA, Xist (X-inactive specific transcript). Paternal XCI is reversed in the inner cell mass of the blastocyst and random XCI subsequently occurs in epiblast cells. Here we show that other eutherian mammals have very different strategies for initiating XCI. In rabbits and humans, the Xist homologue is not subject to imprinting and XCI begins later than in mice. Furthermore, Xist is upregulated on both X chromosomes in a high proportion of rabbit and human embryo cells, even in the inner cell mass. In rabbits, this triggers XCI on both X chromosomes in some cells. In humans, chromosome-wide XCI has not initiated even by the blastocyst stage, despite the upregulation of XIST. The choice of which X chromosome will finally become inactive thus occurs downstream of Xist upregulation in both rabbits and humans, unlike in mice. Our study demonstrates the remarkable diversity in XCI regulation and highlights differences between mammals in their requirement for dosage compensation during early embryogenesis.

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.

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.

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.

Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development.

To investigate the embryonic genome organization upon fertilization and somatic cell nuclear transfer (SCNT), we tracked HP1ß and CENP, two well-characterized protein markers of pericentric and centromeric compartments respectively, in four types of embryos produced by rabbit in vivo fertilization, rabbit parthenogenesis, rabbit-to-rabbit, and bovine-to-rabbit SCNT. In the interphase nuclei of rabbit cultured fibroblasts, centromeres and associated pericentric heterochromatin are usually isolated. Clustering into higher-order chromatin structures, such as the chromocenters seen in mouse and bovine somatic cells, could not be observed in rabbit fibroblasts. After fertilization, centromeres and associated pericentric heterochromatin are quite dispersed in rabbit embryos. The somatic-like organization is progressively established and completed only by the 8/16-cell stage, a stage that corresponds to major embryonic genome activation in this species. In SCNT embryos, pericentric heterochromatin distribution typical for rabbit and bovine somatic cells was incompletely reverted into the 1-cell embryonic form with remnants of heterochromatin clusters in 100% of bovine-to-rabbit embryos. Subsequently, the donor cell nuclear organization was rapidly re-established by the 4-cell stage. Remarkably, the incomplete remodeling of bovine-to-rabbit 1-cell embryos was associated with delayed transcriptional activation compared with rabbit-to-rabbit embryos. Together, the results confirm that pericentric heterochromatin spatio-temporal reorganization is an important step of embryonic genome reprogramming. It also appears that genome reorganization in SCNT embryos is mainly dependent on the nuclear characteristics of the donor cells, not on the recipient cytoplasm.

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.

Comparison of In Vitro Corneal Permeation and In Vivo Ocular Bioavailability in Rabbits of Three Marketed Latanoprost Formulations.

BACKGROUND AND OBJECTIVE: All latanoprost formulations currently available for the treatment of glaucoma or ocular hypertension contain the same concentration of latanoprost (0.005%) but differ in excipients, which may affect corneal drug permeability or stability. This study aimed at comparing corneal penetration of three marketed latanoprost solutions with different excipient formulations in in vitro and in vivo drug permeability studies. METHODS: Three latanoprost formulations were tested under good laboratory practice conditions: a formulation containing benzalkonium chloride (BAK) but no surfactant (Preserved latanoprost); the same formulation except preservative-free (PF) without BAK or surfactant (SF) (PF SF latanoprost); and a different formulation without BAK but containing a non-ionic surfactant (MGHS 40 at 5%) combined with thickening agents (Carbomer 974P, Macrogol 4000) (PF latanoprost). Corneal permeation of latanoprost acid (LAT) was first determined in vitro using a reconstructed human corneal epithelium tissue. Then, in vivo pharmacokinetic studies were performed on pigmented rabbits, for which LAT concentration was measured in the aqueous humour (AH) and iris-ciliary body (ICB). RESULTS: In vitro, the cumulative transport of LAT was linear between 1 h and 4 h for preserved latanoprost and PF SF latanoprost, and LAT concentrations matched exactly at each timepoint. By contrast, the permeation of PF latanoprost was linear between 2 h and 12 h and was significantly lower than that of preserved latanoprost and PF SF latanoprost at 4 and 8 h (p < 0.001). In rabbits, the concentrations of LAT in AH and ICB were not statistically different between preserved latanoprost and PF SF latanoprost at each timepoint, except at 1 h in ICB (p = 0.005). By comparison, the LAT concentration of PF latanoprost was statistically (p < 0.05) lower than that of preserved latanoprost and PF SF latanoprost in AH and ICB from 0.5 to 3 h. CONCLUSION: BAK did not influence the corneal penetration of latanoprost in in vitro and in vivo studies. The formulation containing a non-ionic surfactant resulted in lower and slower ocular penetration compared with preserved or PF SF formulations. This raises questions about the relevance of BAK and some surfactants in enhancing corneal penetration of ocular formulations.

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.

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.

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.

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.

Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos.

Efficient reprograming of the donor cell genome in nuclear transfer (NT) embryos is linked to the ability of the embryos to sustain full-term development. As the nuclear architecture has recently emerged as a key factor in the regulation of gene expression, we questioned whether early bovine embryos obtained from transfer of cultured fibroblasts into enucleated oocytes would adopt an embryo-like nuclear organization. We studied the dynamics of constitutive heterochromatin in the stages prior to embryonic genome activation by distribution analysis of heterochromatin protein CBX1 (HP1), centromeric proteins CENPA and CENPB, and histone H3 three-methylated at lysine 9. Then we applied descriptive, quantitative, and co-localization analyses. A dramatic reorganization of heterochromatic blocks of somatic donor cells was first observed in the late one-cell stage NT embryos. Then at two- and four-cell stages, we found two types of NT embryos: one displaying noncondensed heterochromatin patches similar to IVF embryos, whereas the second type displayed condensed heterochromatin blocks, normally observed in IVF embryos only after the eight-cell stage. These analyses discriminate for the first time two contrasted types of nuclear organization in NT embryos, which may correspond to different functional states of the nuclei. The relationship with the somatic nucleus reprograming efficiency is discussed.

First-line latanoprost therapy in ocular hypertension or open-angle glaucoma patients: a 3-month efficacy analysis stratified by initial intraocular pressure.

BACKGROUND: Prospective, multicenter, randomized, double-masked trials have shown latanoprost instilled once daily to be at least as effective as and generally superior to timolol administered twice daily and to be as effective as other frequently prescribed prostaglandin analogues. This study prospectively assessed the efficacy of latanoprost monotherapy in a large cohort of treatment-naive patients with a broad range of baseline intraocular pressure (IOP) levels treated in actual clinical practice settings. METHODS: This prospective, open-label, multicenter, uncontrolled, phase IV study included treatment-naive ocular hypertension or open-angle glaucoma subjects initiating latanoprost once daily (evening). IOP levels were measured at baseline and after 1 and 3 months. The primary efficacy outcome was mean change in IOP from baseline to month 3. Analyses were stratified by baseline IOP: > or = 20 and <24 mmHg vs > or = 24 mmHg. RESULTS: Efficacy analyses (intent to treat) included 572 subjects: 20 to <24 mmHg group, N = 252; > or = 24 mmHg group, N = 320. Mean baseline IOP levels were 22.2 +/- 0.9 mmHg and 26.7 +/- 2.8 mmHg, respectively. At month 3, significant IOP reductions were seen in both groups (p < 0.0001, within-group differences); reductions were smaller in the 20 to <24 mmHg group (-6.3 +/- 2.4 vs -9.2 +/- 3.7 mmHg, respectively; -28.0 +/- 10.6% vs -34.1 +/- 11.9%, respectively). An IOP reduction of > or = 30% from baseline to month 3 was noted in 48.4% and 65.6% of subjects, respectively (p < 0.0001). At month 3, targets IOPs of < or = 18 mmHg were achieved by > or = 70% of subjects in both groups. Latanoprost was well tolerated with an adverse event profile similar to that reported in the literature. CONCLUSIONS: This "real world" study found once-daily latanoprost to be effective and safe in treatment-naive ocular hypertension or open-angle glaucoma patients. Patients with baseline IOP levels of 20 to <24 mmHg as well as > or = 24 mmHg benefitted from initial latanoprost therapy. TRIAL REGISTRATION NUMBER: NCT00647101.

Predictors of additional intraocular pressure reduction in patients changed to latanoprost/timolol fixed combination.

BACKGROUND: Given the growing number of ocular hypotensive medications available, it is important to be able to predict a positive response to therapy. The purpose of the present study was to identify predictors of an additional 10% intraocular pressure (IOP) reduction after 12 weeks of treatment with latanoprost/timolol fixed combination (FC) in patients requiring a change in their previous ocular hypotensive medication. METHODS: This multicenter, open-label, prospective, phase IIIb study included subjects >or=18 years of age with open-angle glaucoma (OAG) or ocular hypertension (OHT). Eligible subjects had baseline IOP >or=21 mmHg and insufficient response to current beta-blocker monotherapy. The primary efficacy analysis (logistic regression) identified predictors of a positive response after 12 weeks of latanoprost/timolol FC. RESULTS: The intent-to-treat (ITT) population included 383 subjects treated with >or=1 drop of FC and having >or=1 follow-up IOP assessment. Mean IOP was 22.19 +/- 2.16 mmHg at baseline and was reduced by 5.42 +/- 2.71 mmHg at study end. In all, 325 (84.9%) subjects had a positive response to latanoprost/timolol FC; the response rate was similar across groups: OAG (n = 208; 82.7%); OHT (n = 161; 87.6%); OAG+OHT (n = 14; 85.7%). Higher baseline IOP (odds ratio: 1.284; 95% confidence interval [CI]: 1.101, 1.497; p = 0.0014) and absence of adverse events (odds ratio: 0.318; 95% CI: 0.161, 0.629; p = 0.0010) were significant predictors of positive response. Age, gender, ethnic origin, diagnosis, family history of OAG/OHT, corneal thickness, and concomitant systemic beta-blocker were not significant predictors of a positive response in the ITT analysis. The FC was well tolerated. The most common adverse events were related to the eye and were consistent with known adverse events associated with latanoprost and timolol. CONCLUSIONS: These results support the use of latanoprost/timolol FC in patients whose IOP is insufficiently controlled on beta-blocker monotherapy. Patients with higher baseline IOP levels and who do not experience adverse events while on therapy are most likely to achieve a positive response to latanoprost/timolol FC.

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.

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.

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.

Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture.

In vitro production (IVP) has been shown to affect embryonic gene expression and often result in large offspring syndrome (LOS) in cattle and sheep. To dissect the effects of in vitro maturation, fertilization and culture on bovine embryos, we compared the expression profiles of single blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF); (2) in vivo maturation, fertilization and in vitro culture (IVD); and (3) in vivo maturation, fertilization and development (AI). To conduct expression profiling, total RNA was isolated from individual embryos, linearly amplified and hybridized to a custom bovine cDNA microarray containing approximately 6,300 unique genes. There were 306, 367, and 200 genes differentially expressed between the AI and IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and IVD embryos, making these potential candidates for LOS. There were 60 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology category "RNA processing" was over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on the ability to transcribe maternal RNA stores. A culture effect on the expression of genes involved in translation was also observed by the comparison of AI with IVD embryos.

Embryonic gene expression profiling using microarray analysis.

Microarray technology enables the interrogation of thousands of genes at one time and therefore a systems level of analysis. Recent advances in the amplification of RNA, genome sequencing and annotation, and the lower cost of developing microarrays or purchasing them commercially, have facilitated the analysis of single preimplantation embryos. The present review discusses the components of embryonic expression profiling and examines current research that has used microarrays to study the effects of in vitro production and nuclear transfer.