Human induced pluripotent stem cells (BIONi010-C) generate tight cell monolayers with blood-brain barrier traits and functional expression of large neutral amino acid transporter 1 (SLC7A5).

The barrier properties of the brain capillary endothelium, the blood-brain barrier (BBB) restricts uptake of most small and all large molecule drug compounds to the CNS. There is a need for predictive human in vitro models of the BBB to enable studies of brain drug delivery. Here, we investigated whether human induced pluripotent stem cell (hiPSC) line (BIONi010-C) could be differentiated to brain capillary endothelial- like cells (BCEC) and evaluated their potential use in drug delivery studies. BIONi010-C hIPSCs were differentiated according to established protocols. BCEC monolayers displayed transendothelial electrical resistance (TEER) values of 5,829±354 Ω∙cm2, a Papp,mannitol of 1.09±0.15 ∙ 10-6 cm∙s-1 and a Papp,diazepam of 85.7 ± 5.9 ∙ 10-6 cm ∙s-1. The Pdiazepam/Pmannitol ratio of ~80, indicated a large dynamic passive permeability range. Monolayers maintained their integrity after medium exchange. Claudin-5, Occludin, Zonulae Occludens 1 and VE-Cadherin were expressed at the cell-cell contact zones. Efflux transporters were present at the mRNA level, but functional efflux of substrates was not detected. Transferrin-receptor (TFR), Low density lipoprotein receptor-related protein 1 (LRP1) and Basigin receptors were expressed at the mRNA-level. The presence and localization of TFR and LRP1 were verified at the protein level. A wide range of BBB-expressed solute carriers (SLC's) were detected at the mRNA level. The presence and localization of SLC transporters GLUT1 and LAT1 was verified at the protein level. Functional studies revealed transport of the LAT1 substrate [3H]-L-Leucine and the LRP1 substrate angiopep-2. In conclusion, we have demonstrated that BIONi010-C-derived BCEC monolayers exhibited, BBB properties including barrier tightness and integrity, a high dynamic range, expression of some of the BBB receptor and transporter expression, as well as functional transport of LAT1 and LRP1 substrates. This suggests that BIONi010-C-derived BCEC monolayers may be useful for studying the roles of LAT-1 and LRP1 in brain drug delivery.

Review: Recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods.

In vitro production (IVP) of embryos and associated technologies in cattle have shown significant progress in recent years, in part driven by a better understanding of the full potential of these tools by end users. The combination of IVP with sexed semen (SS) and genomic selection (GS) is being successfully and widely used in North America, South America and Europe. The main advantages offered by these technologies include a higher number of embryos and pregnancies per unit of time, and a wider range of potential female donors from which to retrieve oocytes (including open cyclic females and ones up to 3 months pregnant), including high index genomic calves, a reduced number of sperm required to produce embryos and increased chances of obtaining the desired sex of offspring. However, there are still unresolved aspects of IVP of embryos that limit a wider implementation of the technology, including potentially reduced fertility from the use of SS, reduced oocyte quality after in vitro oocyte maturation and lower embryo cryotolerance, resulting in reduced pregnancy rates compared to in vivo-produced embryos. Nevertheless, promising research results have been reported, and work is in progress to address current deficiencies. The combination of GS, IVP and SS has proven successful in the commercial field in several countries assisting practitioners and cattle producers to improve reproductive performance, efficiency and genetic gain.

Organelle reorganization in bovine oocytes during dominant follicle growth and regression.

BACKGROUND: We tested the hypothesis that organelles in bovine oocytes undergo changes in number and spatial distribution in a manner specific for phase of follicle development. METHODS: Cumulus-oocyte-complexes were collected from Hereford heifers by ultrasound-guided follicle aspiration from dominant follicles in the growing phase (n = 5; Day 0 = ovulation), static phase (n = 5), regressing phase (n = 7) of Wave 1 and from preovulatory follicles (n = 5). Oocytes were processed and transmission electron micrographs of ooplasm representing peripheral, perinuclear and central regions were evaluated using standard stereological methods. RESULTS: The number of mitochondria and volume occupied by lipid droplets was higher (P < 0.03) in oocytes from regressing follicles (193.0 ± 10.4/1000 µm(3) and 3.5 ± 0.7 %) than growing and preovulatory stages (118.7 ± 14.4/1000 µm(3) and 1.1 ± 0.3 %; 150.5 ± 28.7/1000 µm(3) and 1.6 ± 0.2 %, respectively). Oocytes from growing, static and preovulatory follicles had >70 % mitochondria in the peripheral regions whereas oocytes from regressing follicles had an even distribution. Oocytes from growing follicles had more lipid droplets in peripheral region than in central region (86.9 vs. 13.1 %). Percent surface area of mitochondria in contact with lipid droplets increased from growing (2.3 %) to static, regressing or preovulatory follicle stage (8.9, 6.1 and 6.2 %). The amount, size and distribution of other organelles did not differ among phases (P > 0.11). CONCLUSIONS: Our hypothesis was supported in that mitochondrial number increased and translocation occurred from a peripheral to an even distribution as follicles entered the regressing phase. In addition, lipid droplets underwent spatial reorganization from a peripheral to an even distribution during the growing phase and mitochondria-lipid contact area increased with follicle maturation.

Ultrastructural myocardial changes in seven cats with spontaneous hypertrophic cardiomyopathy.

OBJECTIVES: Hypertrophic cardiomyopathy (HCM) is the most common heart disease in cats and shares clinical and pathological characteristics with human HCM. Little is known about the pathogenic mechanisms underlying development of spontaneous feline HCM. ANIMALS: The study population consisted of seven cats diagnosed with HCM and eight age-matched cats with no evidence of cardiac disease. METHODS: Fresh myocardial biopsies taken from the middle of the left ventricular posterior free wall were obtained and examined with transmission electron microscopy. RESULTS: Electron microscopic examination showed ultrastructural aberrations of the myocardial cytoarchitecture and of the interstitium in the seven cats with HCM. In the most severely affected cats the myofibrils were disorganized and subsarcolemmal mitochondria were depleted. In control cats, contraction band artifacts were commonly seen. CONCLUSIONS: In this preliminary study we show that ultrastructural changes of the myocardium in seven cats with HCM involve the cytoskeleton and mitochondria. We suggest that our findings are important for future research aiming at elucidating the pathogenic mechanisms underlying the phenotypic expression of feline HCM. The results of this study prompt for a larger scale study, including quantitative measurements of mitochondrial distribution and cytoskeletal derangements in feline HCM.

Ultrastructural visualization of the Mesenchymal-to-Epithelial Transition during reprogramming of human fibroblasts to induced pluripotent stem cells.

The Mesenchymal-to-Epithelial Transition (MET) has been recognized as a crucial step for successful reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs). Thus, it has been demonstrated, that the efficiency of reprogramming can be enhanced by promoting an epithelial expression program in cells, with a concomitant repression of key mesenchymal genes. However, a detailed characterization of the epithelial transition associated with the acquisition of a pluripotent phenotype is still lacking to this date. Here, we integrate a panel of morphological approaches with gene expression analyses to visualize the dynamics of episomal reprogramming of human fibroblasts to iPSCs. We provide the first ultrastructural analysis of human fibroblasts at various stages of episomal iPSC reprogramming, as well as the first real-time live cell visualization of a MET occurring during reprogramming. The results indicate that the MET manifests itself approximately 6-12days after electroporation, in synchrony with the upregulation of early pluripotency markers, and resembles a reversal of the Epithelial-to-Mesenchymal Transition (EMT) which takes place during mammalian gastrulation.

Selection of pre- versus postpubertal pig oocytes for parthenogenetic activation and somatic cell nuclear transfer.

Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter ('small' ≤110µm; 'medium' >110µm; 'large' ≥120µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase.

Porcine melanotic cutaneous lesions and lymph nodes: immunohistochemical differentiation of melanocytes and melanophages.

Porcine melanomas have proven interesting in a wider biological perspective due to a common phenomenon of spontaneous regression, which is characterized by infiltration of macrophages, among others. Separation of neoplastic melanocytes from pigment-laden macrophages may, however, be challenging as the morphology of melanocytes varies considerably and sometimes resembles macrophages. The aim of this study was correspondingly to characterize and differentiate the cells in 20 porcine melanocytomas and regional lymph nodes by histologic examination and immunohistochemistry for melan A, PNL2, S100, lysozyme, alpha-1-antitrypsin, and ionized calcium binding adaptor molecule 1 (Iba1). Grossly, the melanocytomas were divided into 2 distinct types: pigmented maculae (n = 7) and raised tumors (n = 13). In the maculae, the pigmented cells were mainly melanocytes reactive for melan A, PNL2 and S100. In contrast, the majority of the cells in the raised tumors were melanophages, which expressed Iba1, alpha-1-antitrypsin, and lysozyme. Yet, cells histomorphologically indistinguishable from the melanophages expressed melan A and PNL2. These cells were Iba1 and S100 negative, and ultrastructurally, they were devoid of lysosomal bodies and filled with stage III and IV melanosomes. In the regional lymph nodes, melanocytes were present in the trabecular sinuses. In focally or diffusely black lymph nodes, pigmentation was, however, mainly due to aggregates of melanophages, which were confined to the trabeculae, deep cortex, and peripheral lymphoreticular tissue. Normal and neoplastic porcine melanocytes express melan A and PNL2, and immunohistochemical staining for melan A, PNL2, and Iba1 was found useful to identify and distinguish melanocytes and melanophages in porcine melanotic lesions.

Early embryonic development, assisted reproductive technologies, and pluripotent stem cell biology in domestic mammals.

Over many decades assisted reproductive technologies, including artificial insemination, embryo transfer, in vitro production (IVP) of embryos, cloning by somatic cell nuclear transfer (SCNT), and stem cell culture, have been developed with the aim of refining breeding strategies for improved production and health in animal husbandry. More recently, biomedical applications of these technologies, in particular, SCNT and stem cell culture, have been pursued in domestic mammals in order to create models for human disease and therapy. The following review focuses on presenting important aspects of pre-implantation development in cattle, pigs, horses, and dogs. Biological aspects and impact of assisted reproductive technologies including IVP, SCNT, and culture of pluripotent stem cells are also addressed.

Directed differentiation of porcine epiblast-derived neural progenitor cells into neurons and glia.

Neural progenitor cells (NPCs) are promising candidates for cell-based therapy of neurodegenerative diseases; however, safety concerns must be addressed through transplantation studies in large animal models, such as the pig. The aim of this study was to derive NPCs from porcine blastocysts and evaluate their in-vitro differentiation potential. Epiblasts were manually isolated from expanded hatched blastocysts and cultured on MEF feeder cells. Outgrowth colonies were passaged to MS5 cells and rosettes were further passaged to Matrigel-coated dishes containing bFGF and EGF. Three NPC lines were established which showed expression of SOX2, NESTIN and VIMENTIN. One line was characterised in more detail, retaining a normal karyotype and proliferating for more than three months in culture. Following differentiation, TUJI was significantly up-regulated in protocol 2 (RA and SHH; 58% positive cells) as were NF and TH. In contrast, MBP was significantly up-regulated in protocol 3 (FGF8 and SHH; 63% positive cells), whereas, GFAP was significantly up-regulated in protocols 1-4 (33%, 25%, 43% and 22%). The present study provides the first report of a porcine blastocyst-derived NPC line capable of differentiating into both neurons and glia, which may be of paramount importance for future transplantation studies in large animal models of neurodegenerative diseases.

OCT4 expression in outgrowth colonies derived from porcine inner cell masses and epiblasts.

The present study was conducted to test different methods for porcine inner cell mass (ICM) and epiblast isolation and to evaluate the morphology and expression of pluripotency genes in ICM- and epiblast-derived outgrowth colonies (OCs) and passages thereof with particular attention on the relationship between OCT4 expression and embryonic stem cell (ESC)-like morphology. A total of 104 zona pellucida-enclosed and 101 hatched blastocysts were subjected to four different methods of ICM and epiblast isolation, respectively: Manual isolation, immunosurgery, immunosurgery with manual cleaning, or whole blastocyst culture. OCs were established on mouse embryonic fibroblast (MEF) cells and categorized according to morphology and OCT4 staining. Although all isolation methods resulted in ESC-like OCs, immunosurgery with manual cleaning yielded significantly higher rates of ICM/epiblast attachment and subsequent ESC-like morphology, whereas no significant difference was found between ICM and epiblasts with respect to these characteristics. All ESC-like OCs showed nuclear OCT4 staining and expression of OCT4, NANOG and SOX2 as evaluated by RT-PCR. Upon initial passages, the expression of pluripotency markers was, however, gradually lost in spite of maintained ESC-like morphology. In conclusion, we have established a robust system for derivation of ESC-like OCs from porcine ICM and epiblasts and we have shown that localization of OCT4 is associated with an ESC-like morphology although this relationship is lost during early passages.

Immunohistochemistry of pancreatic development in cattle and pig.

The aim of this study was to characterize bovine and porcine pancreatic development by immunohistochemistry. In the pig, staining for both glucagon and insulin was noted at day 19. In cattle, glucagon staining was observed at day 25 and insulin staining from day 26. In both species, glucagon-stained cells were abundant initially, but later insulin-stained cells became most abundant. A few cells displayed co-localization of glucagon and insulin staining during initial development in both species. Initially, most of the cells of the pancreatic primordia and the duodenal epithelium displayed Pdx-1-staining. All insulin-stained cells displayed Pdx-1-stained nuclei, whereas no glucagon-stained cells did so. Many Pdx-1-stained cells lacked insulin staining, but with development, the relative number of these cells diminished. Nkx6.1-staining was initially seen in a pattern similar to that for Pdx-1, but was lacking duodenal staining. Subsequently, the number of Nkx6.1-stained cells diminished, but increased again to a level where practically all insulin-stained cells also presented Nkx6.1-staining. Glucagon-stained cells, on the other hand, never had Nkx6.1 staining. In conclusion, the localization of the two transcription factors, Pdx-1 and Nkx6.1, demonstrated that pancreas development appears to be controlled by mechanisms comparable with those operating in humans.

From zygote to implantation: morphological and molecular dynamics during embryo development in the pig.

The increasing focus on the pig as a biomedical model calls for studies which investigate morphological and molecular mechanisms during initial embryonic development in this species. In the pig, the paternal genome is actively demethylated in the zygote, whereas the maternal genome remains methylated. The major genome activation occurs at the four-cell stage, when prominent ribosome-synthesizing nucleoli develop in the blastomeres, allowing for trophectoderm and inner cell mass (ICM) differentiation. Unlike in mice, the pluripotency gene OCT4 is initially expressed in both compartments. The ICM differentiates into epiblast and hypoblast approximately at the time of hatching from the zona pellucida, and subsequently the loss of the Rauber's layer results in an uncovered epiblast establishing the embryonic disc again in contrast to mice. This particular and protracted ICM/epiblast biology may contribute to the lack of success in culturing porcine embryonic stem cells. The embryonic disc subsequently becomes polarized by a posterior thickening, which includes ingression of the first extra-embryonic mesoderm. Thereafter, the primitive streak forms and gastrulation results in formation of the somatic germ layers and germline, i.e. the primordial germ cells. The latter remain pluripotent for a period and may be isolated and cultured as embryonic germ cells in vitro.

Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines.

Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer of mouse embryonic fibroblast (MEF) and mouse HM1 embryonic stem cells (HM1), were processed for autoradiography following (3)H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF and nucleophosmin, B23). All early 2-cell embryos showed transcriptional activity only in nucleoplasm, not over nucleolar precursor bodies (NPBs). UBF was diffusely localized to cytoplasm and B23 to cytoplasm and nucleoplasm. Late 2-cell IVF and PG embryos displayed transcription over nucleoplasm and NPBs. Ultrastructurally, the latter were developing into functional nucleoli. NT-MEF and NT-HM1 embryos displayed transcription over nucleoplasm, but not over NPBs. Development of NPBs into nucleoli was lacking. UBF was in both groups localized to nucleoplasm or distinctly to presumptive NPBs. B23 was distinctly localized to NPBs. All 4-cell embryos presented nucleoplasmic transcription and developing fibrillo-granular nucleoli. UBF and B23 were distinctly localized to nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli were found in IVF and PG embryos, NT-MEF and -HM1 embryos displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both of these processes were delayed.

High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos.

Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim of our present study was to establish the optimal TSA treatment in order to improve the development of handmade cloned (HMC) porcine embryos and examine the effect of TSA on their development. The blastocyst percentage of HMC embryos treated with 37.5 nM TSA for 22-24 h after activation increased up to 80% (control group-54%; P<0.05). TSA mediated increase in histone acetylation was proved by immunofluorescence analysis of acH3K9 and acH4K16. 2-cell stage embryos derived from TSA treatment displayed significant increase in histone acetylation compared to control embryos, whereas no significant differences were observed at blastocyst stage. During time-lapse monitoring, no difference was observed in the kinetics of 2-cell stage embryos. Compact morula (CM) stage was reached 15 h later in TSA treated embryos compared to the control. Blastocysts (Day 5 and 6) from HMC embryos treated with TSA were transferred to 2 recipients resulting in one pregnancy and birth of one live and five dead piglets. Our data demonstrate that TSA treatment after HMC in pigs may affect reprogramming of the somatic genome resulting in higher in vitro embryo development, and enable full-term in vivo development.

Endoscopic recovery of early preimplantation bovine embryos: effect of hormonal stimulation, embryo kinetics and repeated collection.

The collection of extra numbers of bovine embryos by superstimulation of donors underlies variation concerning yield of morulae and blastocysts. Our study aimed at establishing a correlation between hormonal treatment and embryo development during oviductal passage including repeated flushing. A transvaginal endoscopic procedure was used to flush the oviducts at six different time intervals (beginning at 24 h until 105 h) after artificial insemination. In total, 119 animals were superovulated using either FSH or eCG. The hormonal treatment resulted in the stimulation of 2076 follicles of which 77% (1590 CL) ovulated. The bilateral flushing resulted in the collection of 1411 complexes (collection rate: 89%), of which 78% (1098) were assessed as viable embryos. The use of FSH resulted in significantly more stimulated follicles and ovulation sites compared with eCG (p < 0.001). Generally, the embryo kinetics were similar among the FSH and eCG treated animals. However, the embryo cleavage of the eCG treated animals was ahead of that of the FSH group comparing the different collection time points. The overall proportions of non-viable embryos in both groups were similar. Regarding the embryo collection intervals in the eCG group, this proportion significantly increased during 51-105 h compared to 24-50 h (p < 0.05), whereas FSH delivered constant results. It was shown that the repeated endoscopic collection of oviductal stage embryos had no negative influence on the collection parameters. It is concluded that the introduced transvaginal endoscopic technique could have main impact on further studies focusing on early embryo development.

The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos.

The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition, AD 0.2 microg/ml; total transcriptional inhibition, AD 2.0 microg/ml). Late four-cell embryos were cultured to late eight-cell stage in 0.2 microg/ml AD (MTA prevention, ADLT (long-term total transcriptional inhibition group). Embryos were processed for autoradiography, transmission electron microscopy, fluorescent in situ hybridization (ribosomal RNA, rRNA), silver staining (nucleolar proteins), and immunofluorescence (RPI). Control embryos displayed extranucleolar and nucleolar transcription, functional nucleoli, and distinct RPI localization. Nuclei (97%) showed large rRNA clusters, in 94.1% co-localized with nucleolar proteins deposits. In AD 0.2 group, only extranucleolar transcription was detected. Segregated dense-fibrillar and granular components, but no fibrillar centers, were observed. RPI was dispersed. Nuclei (55%) presented rRNA clusters, in 38.8% co-localized with silver-stained deposits. AD 2.0 and ADLT groups displayed no transcription and disintegrating nucleolar precursors. AD 2.0 (34%) and 14% (ADLT) of nuclei presented clusters of maternally inherited rRNA. In AD 2.0 group, RPI was dispersed, but 17.2% of nuclei showed colocalization of rRNA with nucleolar proteins. In ADLT group, RPI was lacking and clustering of nucleolar proteins was hampered. In conclusion, rDNA transcription is not required for targeting of rRNA processing proteins, rRNA is maternally inherited and target to rDNA independent of transcription, and de novo transcription is required for proper nucleologenesis in cattle.

Ribosomal RNA and nucleolar proteins from the oocyte are to some degree used for embryonic nucleolar formation in cattle and pig.

The nucleolus is the site of ribosomal RNA (rRNA) and ribosome production. In the bovine primordial follicle oocyte, this organelle is inactive, but in the secondary follicle an active fibrillo-granular nucleolus develops and proteins involved in rDNA transcription (topoisomerase I, RNA polymerase I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) localize to it. At the end of the oocyte growth phase, the nucleolus is inactivated again and transforms into a solid remnant. The nucleolar remnant is dissolved when meiosis is resumed. Upon fertilization, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities are engaged in the re-establishment of fibrillo-granular nucleoli at the major activation of the embryonic genome. This nucleolar formation can be classified into two different modes: one where nucleolus development occurs inside NPBs (internal; e.g. cattle) and the other where it occurs on the surface of NPBs (external; e.g. pig). Oocyte derived proteins engaged in late rRNA processing (nucleolin and nucleophosmin) may to some degree be re-used for nucleolar formation in the embryo, while the other nucleolar proteins require de novo embryonic transcription in order to be allocated to the developing nucleoli. Moreover, unprocessed rRNA inherited from the oocyte targets to the developing embryonic nucleoli. In conclusion, the nucleolus is important for the development of oocytes and embryos and may serve as a marker for the completion of oocyte growth and the normality of activation of the embryonic genome.

Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos.

The processes of cellular differentiation were studied in somatic cell nuclear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients on day 6. All embryos were processed for examination by light and transmission electron microscopy or immunohistochemical labelling for alpha-1-fetoprotein and vimentin. Overall, morphological development of in vivo embryos was superior to IVC and SCNT embryos. Day 7 and particularly day 9 IVC and SCNT embryos had impaired hypoblast development, some lacking identifiable inner cell masses. On day 11, only in vivo and IVC embryos had developed an embryonic disc, and gastrulation was evident in half of in vivo embryos and one IVC embryo. By day 13, all in vivo embryos had completed gastrulation whereas IVC and SCNT embryos remained retarded. On days 17 and 19, in vivo embryos had significantly more somites and a more developed allantois than IVC and SCNT embryos. We conclude that IVC and particularly SCNT procedures cause a retardation of embryo development and cell differentiation at days 7-19 of gestation.

Chronological appearance of apoptosis in bovine embryos reconstructed by somatic cell nuclear transfer from quiescent granulosa cells.

Efficiency of cloning has remained low and in spite of attempts to improve this technology, many reconstructed embryos do not implant or are lost during early pregnancy. Chromosomal aberrations, deviant gene expression patterns and abnormal regulation of cell death may be involved in this increased early embryonic loss. Here, we investigate the chronological onset of both apoptotic changes in nuclear morphology and DNA degradation [detected by transferase-mediated dUTP nick-end labelling (TUNEL) reaction] in bovine two-cell- to blastocyst-stage embryos. Such embryos were generated either by reconstruction with nuclear transfer from quiescent granulosa cells or by regular in vitro embryo production. Nuclear condensation was observed from the two-cell stage and TUNEL labelling was observed from the six-cell stage in reconstructed embryos, whereas nuclear condensation was evident from the eight-cell stage and TUNEL labelling from the 13-cell stage in embryos derived in vitro. Furthermore, reconstructed embryos displayed elevated ratios of embryos containing apoptotic nuclei at pre-compaction stages and higher indices of apoptotic nuclei in morula and blastocyst stages when compared with in vitro-produced embryos.