Pigs are increasingly being recognized as good large-animal models for translational research, linking basic science to clinical applications in order to establish novel therapeutics. This article reviews the current status and future prospects of genetically modified and cloned pigs in translational studies. It also highlights pigs specially designed as disease models, for xenotransplantation or to carry cell marker genes. Finally, use of porcine somatic stem and progenitor cells in preclinical studies of cell transplantation therapy is also discussed.

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It has been shown previously that tumor necrosis factor alpha-induced protein 6 (TNFAIP6) is essential for formation of the cumulus extracellular matrix and female fertility. Therefore, we studied the expression of TNFAIP6 mRNA in porcine preovulatory follicles. In addition, we asked whether the expression of TNFAIP6 mRNA changes in mural granulosa cells (MGCs) during the periovulatory period or after culture of oocyte-cumulus complexes (OCCs) or MGCs in vitro. Mural granulosa cells obtained from follicles on day 12 (D 12) and day 15 (D 15) of the estrous cycle, eCG-stimulated follicles, follicles at 4-32 h after hCG stimulation and MGCs and OCCs obtained from immature gilts and cultured for 0-44 h in vitro with gonadotropins were used for extraction of total RNA and assessment of the relative abundance (RA) of TNFAIP6 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The levels of TNFAIP6 mRNA were low in the follicles on D 12 and D 15 of the estrous cycle and at 66 h after eCG stimulation but were significantly increased at 4 h after hCG. The high level of TNFAIP6 expression was maintained until 16 h after hCG stimulation and gradually decreased at 24 and 32 h after hCG. During in vitro culture, FSH/LH-induced TNFAIP6 mRNA was expressed in both OCCs and MGCs in a similar temporal pattern as seen in vivo. We conclude that TNFAIP6 expression in the pig, like other species, increases in preovulatory follicles following the LH (hCG) surge. The OCC and MGC display similar patterns of TNFAIP6 expression under both in vivo and in vitro conditions.

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Streptolysin O (SLO) is a known bacterial protein that forms very large pores in the plasma membranes of mammalian cells. SLO has been used in the delivery of proteins into living cells following permeabilization. The objective of this study was to investigate the effects of SLO treatment in donor cells that generate pores in the plasma membrane during in vitro development of porcine reconstructed embryos. In experiment 1, fetal fibroblast cells were trypsinized, treated with SLO for 0, 30, 50 and 70 min and then their fusion rates and developmental capabilities were tested following reconstruction. SLO treatment for 50 min produced a higher blastocyst formation rate compared with the other treatments. In experiment 2, fetal fibroblasts were treated with 200 ng/ml SLO for 50 min at the confluent or single-cell stages of the nuclear transfer protocol (i.e., before and after trypsinization, respectively), and the in vitro development of the resulting porcine reconstructed embryos was investigated over 6 days in culture. Oocytes receiving cells treated with SLO post-trypsinization showed higher fusion and blastocyst formation rates compared with those receiving untreated cells. Collectively, these findings show that SLO-mediated permeabilization of porcine fetal fibroblast cells appears to improve the fusion rates and in vitro development of porcine reconstructed embryos.

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To investigate molecular effects of anti-sperm autoantibodies on fertilization, we previously established anti-mouse sperm-head auto-monoclonal antibodies (mAbs). Among the mAbs established, one mAb (named Ts4) recognized the sugar moiety of TEX101, a germ cell-marker glycoprotein. In the present study, we examined the immunoreactivity of Ts4 in mouse spermatozoa and fertilized eggs during early embryogenesis to clarify the distribution of the Ts4-reactive antigen in the fertilization process. Similar to TES101 mAb (a specific probe for TEX101), immunopositive staining of Ts4 was observed on spermatocytes, spermatids and spermatozoa within the testis. In contrast to the results obtained with TES101 mAb, Ts4 reacted with the sperm acrosomal region within the cauda epididymis. A Western blot analysis of epididymal sperm extract revealed that Ts4 mainly detected two bands between 100 and 150 kDa, while Ts4 faintly detected a band corresponding to TEX101 at 38 kDa. In addition, Ts4-reactive molecules were observed in the growing early embryo after fertilization. Since Ts4-reactive antigen, potentially a carbohydrate chain, is only observed in reproduction-related areas such as the testis, epididymal sperm-head and early embryo, it is expected to have an effect on fertilization. Therefore, additional studies of this antigen may elucidate the molecular mechanisms underlying the reproductive process.

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Glucocorticoids have heterogeneous effects on reproductive function. We used a gonadotropin-primed, immature rat model to study the influence of dexamethasone (1 mg/kg), given during the latter stages of follicular development, on litter size, the number of oocytes released, and pituitary hormone levels. Dexamethasone-treated females released a larger number of oocytes at ovulation and gave birth to larger litters indicating the oocytes were viable. Survival to weaning age was not affected but average weight at weaning was lower for pups born to DEX-treated females. Serum FSH and LH were assayed at 12, 24 and 48 h following eCG and did not differ between dexamethasone-treated and control animals, but prolactin showed a prolonged pattern of elevation in DEX-treated females. Prolactin, which normally exhibits an elevation on proestrous, may modulate follicular development. Dexamethasone enhances fertility and fecundity possible through an effect of prolactin on follicle development, or by other direct effects on the ovary. These results may improve our understanding of the usefulness of DEX in assisted reproductive therapies for women.

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Satellite cells are skeletal muscle progenitor/stem cells that reside between the basal lamina and plasma membranes of skeletal muscle fibers in vivo and can give rise to both myogenic and adipogenic cells. The isolated skeletal muscle single fiber culture is an in vitro model that has the advantage of enabling the researcher to monitor the differentiation of satellite cells in a closer microenvironment found in living muscle in vivo. The aim of the present study was to establish single fiber culture of goat skeletal muscle and monitor satellite cell differentiation in this system. Fine single fibers were isolated from goat intercostal muscle by enzymatic treatment, and satellite cells on the fibers were analyzed immunocytochemically. Satellite cells on freshly isolated fibers were positive for Pax7, but negative for MyoD and myogenin, indicating that they are quiescent. As the culture period progressed, the satellite cells became positive for myogenic markers MyoD and myogenin. The satellite cells that detached from the fiber and migrated onto the culture dish were positive for both MyoD and myogenin, and some of them formed multinucleated myosin heavy chain-positive myotubes. In addition, hypercontraction of isolated fine fibers induced adipogenic differentiation of satellite cells. The present study is the first to describe establishment of a goat skeletal muscle single fiber culture that can be used as a model system for further studies on satellite cell behavior in ruminants.

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Ketone bodies are considered to act as a signal to suppress gonadotropin release during negative energy balance. The present study examined the effect of 48-h fasting on the mRNA expressions of monocarboxylate transporter 1 (MCT1) and MCT2, which are involved in ketone body transport, in several brain regions. Quantitative real-time RT-PCR analysis showed that the MCT2 mRNA levels were significantly increased by 48-h fasting in the area postrema-solitary tract nucleus (AP-NTS) region but not the arcuate nucleus-ventromedial hypothalamic nucleus (ARC-VMH) and central gray-supragenual nucleus around the 4th ventricle (CG-SGe) regions. Fasting did not significantly affect MCT1 mRNA expression in any of the brain areas examined. Luteinizing hormone (LH) pulse frequency significantly decreased and plasma concentrations of β-hydroxybutyric acid, a ketone body, significantly increased after 48-h fasting. The present results suggest that increased uptake of ketone bodies via MCT2 in the AP-NTS region is likely involved in the mechanism of fasting-induced suppression of LH secretion in rats.

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In the pig, the periimplantation period is critical for successful establishment of pregnancy. We studied the influence of embryos on oxytocin (OT) and progesterone (P₄) regulated endometrial and myometrial secretion of 1) luteotrophic prostaglandin E₂ (PGE₂) and 2) luteolytic prostaglandin F_2α and its metabolite (PGFM) on days 12-14 of pregnancy in pigs. We used unilaterally pregnant pigs created by a surgical procedure in which one uterine horn remained intact and the second horn was cut transversely so that part of the horn was detached from the uterine body. The animals were divided into two groups, inseminated gilts (days 12-14 of pregnancy, n=6) and uninseminated cyclic gilts, which were used as controls (days 12-14 of estrous cycle, n=5). Embryos developed only in the patent part of the uterus and not in the occluded horn. The abundance of OTR mRNA was increased in the endometrium and decreased in the myometrium of the gravid uterine horn in the pregnant pigs compared with the non-gravid uterine horn or either uterine horn in the cyclic pigs, indicative of a local effect of the conceptus. The presence of embryos in the uterine horn during the periimplantation period determines endometrial metabolism of PGF_2α and the local response of the endometrium to OT and P₄. OT stimulates PGF_2α secretion and PGFM accumulation in endometrial cultures only from the non-gravid uterine horn and controls PGE₂ secretion from the endometrium and myometrium in the pregnant gilts. The results indicate a more systemic affect of pregnancy on the uterine response to OT and a possibly the local effect of the conceptus in promoting progesterone's inhibition of OT-stimulated PGE₂ secretion and uterine metabolism of PGF_2α.

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We found frequent and spontaneous proliferation of glandular epithelium and dilated cysts in the uteri of interleukin-2 receptor (IL2R) β-chain transgenic (Tg2Rβ) mice. The aim of this study was to examine the involvement of IL2R β-chain in the pathogenesis of endometrial hyperplasia (EH). Mouse uteri and serum were collected from Tg2Rβ and normal littermates (NL), which were classified into three groups according to age. The incidence of EH increased in an age-dependent manner in both types of mice. However, in old age, Tg2Rβ mice showed more serious phenotypes of EH than NL. Immunohistochemical analysis revealed specific localization of IL2R β-chain in the glandular epithelial cells, with a correlation to the degree of EH, not only in the Tg2Rβ uteri but also in the NL uteri. Immunoreactions of CD3 and CD25 were detected in the uteri of Tg2Rβ but were weak in the uteri of NL, and CD25-positive cells were distributed in the endometrial stroma and myometrium in the Tg2Rβ mice. These findings suggest that the IL2R β-chain induces growth potential for glandular epithelial cells and an immune-privileged condition mediated by CD25+regulatory-T cells.

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We cloned a porcine ortholog of homeodomain transcription factor Msx1 from the porcine pituitary cDNA library. The amino acid sequence of Msx1 shows high conservation among mammalian species. RT-PCR for porcine fetal and postnatal pituitaries showed that Msx1 is already expressed at early fetal day 40, decreases to a low level before birth and then remarkably decreases after birth. On the other hand, Msx1 expression was observed in all pituitary-derived cell line tested, with most in a gonadotrope lineage LβT4. Transfection assay demonstrated that Msx1 markedly repressed the basal Cga and Fshb gene expression, while Lhb expression was affected slightly. Taken together, Msx1 may play a role in repressing gene expression in the fetal and postnatal periods.

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The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible tissues for transplantation.

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Agrin is the heparan sulfate proteoglycan (HSPG) that is well known as the molecule that aggregates acetylcholine receptor (AChR) through muscle specific kinase (MuSK) and rapsyn at neuromuscular junctions. HSPGs are spatiotemporally expressed in embryonic and maternal tissues during implantation. The present study examined the role of agrin in the mouse embryo using leukemia inhibitory factor (LIF)-deficient mice, which show complete sterility. Agrin was detected widely in the cytoplasm of uterine luminal epithelial cells at the third day of pregnancy (Day 3) and Day 4. At Day 5, agrin moved to the apical surface of the luminal epithelium. This migration was not found in LIF-deficient mice. AChR was also found in the apical surface of the uterine epithelium at Day 4 and Day 5 in normal mice. LIF-deficient mice did not show this pattern of expression. Only nAChR b1 subunit mRNA was increased at Day 5 in normal mice. Furthermore, acetylcholinesterase was active in the uterine stroma of normal mice throughout the implantation period and was exclusively active in the uterine epithelium at Day 4. Taken together, agrin signaling was activated in the uterus during embryo implantation in the mice. Here, we suggest that the agrin pathway is involved in closure of the uterine epithelium toward placentation.

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We evaluated the developmental competence of somatic cell nuclear transfer (SCNT) embryos using in vitro embryo culture systems. Embryos were cultured in NCSU-23, NCSU-23 supplemented with essential and non-essential amino acids (NCSU-23aa) or modified PZM-5 supplemented with BSA instead of PVA (mPZM-5). The rates of blastocyst formation were significantly higher in the mPZM-5 group than in the other groups, regardless of the method of embryo production (38.0 vs. 25.3 or 29.1% for IVF, 18.2 vs. 8.7 or 9.4% for SCNT, respectively). The mean cell numbers of IVF and SCNT blastocysts were also significantly higher in mPZM-5 than in the other groups (62.0 vs. 42.3 or 43.0 for IVF, 46.5 vs. 29.4 or 31.3 for SCNT, respectively). Next, the embryos were cultured in mPZM-5 from days 0 to 4 and then in mPZM-5 (P/P), NCSU-23 (P/N) or NCSU-23aa (P/Naa) until day 6. The rates of blastocyst formation were similar among the 3 two-step culture systems in both embryo groups (36.2, 34.2, and 33.6% for IVF, 20.8, 14.1, and 17.2% for SCNT, respectively). The mean cell number in the IVF and SCNT blastocysts was significantly lower in P/N than in P/P and P/Naa (46.5 vs. 63.5 and 68.7 for IVF, 29.3 vs. 45.5 and 39.7 for SCNT, respectively). Next, we examined the effect of media on apoptosis in IVF and SCNT blastocysts. The apoptosis indices in the blastocysts derived from either NCSU-23 or mPZM-5 were analyzed by TUNEL assay. The apoptosis index of the SCNT blastocysts was significantly lower in mPZM-5 than in NCSU-23 (8.8 vs. 13.6%), whereas no such difference was observed between groups in the IVF embryos (5.1 vs. 4.4%). These data suggested that SCNT embryos were more easily affected by culture environment compared with IVF embryos, offering the possibility to further enhance the developmental competence of SCNT embryos by developing more appropriate culture conditions in pigs.

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This study was conducted in bovine to investigate whether CD9 (a member of the tetraspanin superfamily of proteins) is present on oocytes and whether it functions in sperm-oocyte binding and fusion. First, the presence of CD9 in bovine matured oocytes was examined by immunofluorescence with the anti-CD9 monoclonal antibody (mAb) and fluorescein isothiocyanate-conjugated goat anti-mouse antibody, and the results showed that CD9 was expressed on the plasma membrane of matured oocytes. Sperm binding and fusion with oocytes was then examined by in vitro fertilization. When the zona pellucida-free matured oocytes were fertilized, both sperm binding to ooplasma and sperm penetrating into oocytes were significantly (P<0.01) reduced in anti-CD9 antibody-treated oocytes (6.3 ± 0.7 per oocyte and 41.6%, respectively) compared with untreated control oocytes (19.0 ± 0.7 per oocyte and 81.3%, respectively), indicating that the anti-CD9 mAb potentially inhibits sperm-oocyte binding and fusion. These results demonstrated that the CD9 present on bovine matured oocytes is involved in sperm-oocyte interaction during fertilization.

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The aim of this study was to examine the hypothesis that follicular and uterine perfusion as well as endometrial gene expression during the ovulatory period differs after induction of luteolysis during the 1^st follicular wave compared with the 2^nd wave or in intact cycle. Nine healthy non-lactating Holstein-Friesian cows were examined during three estrous cycles. A prostaglandin F_2α analogue (PGF) was administered randomly either on Day 7 (1^st wave cycle) or Day 11 (2^nd wave cycle) after detection of ovulation (=Day 1). No hormonal treatment was used in the intact cycle with spontaneous ovulation. Transrectal Doppler sonography was conducted daily after PGF injection and in the intact cycle beginning on Day 18 of the estrous cycle until ovulation. Follicular blood flow (FBF) was determined by measuring the maximum area of colour pixels on digitalized images of the follicles. Uterine blood flow was quantified by the time-averaged maximum velocity (UTAMV) and pulsatility index (PI) in both uterine arteries. Blood flow measurements were carried out on Days -1 and 0. Endometrial biopsy specimens were taken on Day 1 and analyzed for the gene expressions of estrogen, progesterone, oxytocin and VEGF receptors and eNOS and iNOS using RT-PCR. The interval from PGF injection to ovulation was shorter (P<0.05) in 1^st wave cycles than in 2^nd wave cycles. On Days 0 and -1, FBF was greater (P<0.05) in 1^st wave cycles than in intact and 2^nd wave cycles. On Day -1, UTAMV was greater (P<0.05) in 1^st wave cycles than in intact and 2^nd wave cycles. There were no differences (P>0.05) in FBF and UTAMV values between 2^nd wave and intact cycles. No differences (P>0.05) were detected in the gene expressions of endometrial receptors and enzymes between intact, 1^st and 2^nd wave cycles. The results show that follicular and uterine perfusion during the ovulatory phase are higher after induction of luteolysis during the 1^st follicular wave compared with the 2^nd wave or intact cycle. There were no effects on endometrial gene expression.

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Functional and structural changes in the mammalian ovary are coordinately regulated by the pituitary glycoprotein hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), leading to follicular development, ovulation and transformation of follicles into corpus lutea. To investigate protein profiles during these processes of the mouse ovarian cycle, we applied combined methods (two-dimensional gel electrophoresis [2-DE] for separation and visualization of proteins plus matrix laser desorption/ionization time-of-flight mass spectrometry [MALDI-TOF/MS] analysis for protein identification) for comparative proteomic analysis using immature mice at 3 weeks of age. Protein profiles were obtained from proteins extracted from intact ovaries that had been collected from pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed immature mice at 0 (no PMSG), 24 and 48 h post PMSG, as well as at 10 and 20 h post hCG. The results showed that 1028 common protein spots were found in representative gels that had been separated in the 3 to 11 pH range and the 15-200 kDa range, 253 protein spots (24.6%) of which were differentially expressed (p<0.05) during the mouse ovarian cycle. Of these 253 protein spots, 99 were identified by MALDI-TOF/MS. This comparative proteomic approach to identifying proteins that were potentially involved in the complex process of the ovarian cycle could contribute to our understanding of the molecular basis of functional and structural changes in the ovary in response to gonadotropins. Furthermore, the interesting ovarian proteins identified in this study may eventually serve as diagnostic biomarker candidates of ovarian function.

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Protein serine/threonine phosphorylation in mammalian sperm flagella has been considered to play important roles in regulation of motility. Protein phosphorylation state reflects balance of enzymatic activities between protein phosphatases and protein kinases [predominantly protein kinase A (PKA)]. The aims of this study were to disclose roles of protein phosphatases in the regulation of sperm motility and to provide evidence for suppression of PKA full activation by protein phosphatases in sperm flagella. Mouse epididymal spermatozoa were incubated with a cell-permeable protein phosphatase 1 (PP1)/protein phosphatase 2A (PP2A) inhibitor (calyculin A: 25-125 nM) at 37.5 C. After incubation, they were used for immunodetection of phosphorylated proteins, PKA and PP1 γ2, assessment for motility and co-immunoprecipitation of PP1γ2 with PKA. Incubation with calyculin A enhanced the phosphorylation states of several proteins (>250 kDa, 170 kDa, 155 kDa, 140 kDa and 42 kDa for serine/threonine phosphorylation and 70 kDa for tyrosine phosphorylation) and PKA catalytic subunits [at the autophosphorylation residue (Thr-197) for its full enzymatic activation] in the flagella. Coincidently, this incubation induced changes of sperm flagellar movement from the progressive type to the hyperactivation-like type. Indirect immunofluorescence and co-immunoprecipitation showed that PKA was co-localized with PP1 γ2 in the principal pieces of sperm flagella. These findings suggest that calyculin A-sensitive protein phosphatases (PP1/PP2A) suppress full activation of PKA as well as enhancement of the phosphorylation states of other flagellar proteins in sperm flagella in order to prevent precocious changes of flagellar movement from the progressive type to hyperactivation.

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Hysterosalpingo-contrast sonography is an accurate first-line method used to test for tubal patency in human gynecology. Tubal pathology occurs in dairy cattle and is a reason for infertility, but easy and accurate methods to test for tubal patency are not available in the living cow. In this study it was thus investigated if contrast sonography (CS) using Echovist^®-200 as the echo-contrast medium is a feasible procedure to test for tubal patency in dairy cattle. In eight repeat breeder Holstein Frisian cows, all of them being in behavioral estrus, Echovist^®-200 was given into the uterus and its exit into the abdominal cavity then imaged by transrectal conventional B-mode ultrasound, and used as indicator for tubal patency. Animals were slaughtered one day later and the genital tracts subjected to gross morphology and histology in order to confirm the results of CS. In two cows, CS was prematurely terminated after examination of one oviduct because of rectal bleeding, while CS completely failed in another cow because of poor image quality. In five cows, both oviducts could be examined by CS, respectively. A total of five oviducts were found patent by CS and confirmed by post mortem examination. Two out of five oviducts diagnosed as occluded by CS were morphologically intact and thus misdiagnosed. Of the three non-patent oviducts, two were occluded because of a hydrosalpings, respectively, while the third was inflamed. In conclusion, CS has been shown a feasible procedure to test for tubal patency in dairy cattle. Further studies with more animals are however recommended to warrant this result.

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This study investigated the effects of N, N-Dimethylglycine (DMG) on the development of in vitro produced (IVP) bovine embryos. IVP embryos were obtained by in vitro fertilization of in vitro matured oocytes for 6 h. In Experiment 1, IVP embryos were cultured in mSOFaa supplemented with bovine serum albumin but without glucose (SOF1) for 4 days, transferred to mSOFaa (with 5% fetal bovine serum and 1.5 mM glucose; SOF2) supplemented with 0 (control), 0.1,1 or 10 μM DMG and cultured for an additional 7 days (11 days in total) to assess their development in vitro. When cultured in the medium with 0.1 μM DMG, a significantly higher number of IVP embryos developed to the blastocyst and hatched blastocyst stages (40.3 and 40.8%, respectively) compared with the other groups (18.7-31.0% and 15.0-28.7%, respectively; P<0.05, analysis of variance). In Experiment 2, IVP embryos were cultured in SOF1 with or without 0.1 μM DMG for 4 days, transferred to SOF2 with or without 0.1 μM DMG and further cultured as in Experiment 1; DMG was added to either SOF1 or SOF2 and to both of them to assess its exposure effects on embryo development. When cultured continuously with DMG for 11 days, significantly higher rates of IVP embryos developed into blastocyst and hatched blastocyst stages (39.0 and 47.7%, respectively) compared with the other groups (31.0-32.2% and 29.5-31.0%, respectively; P<0.05). In Experiment 3, we examined developmental speed of IVP embryos cultured with or without addition of 0.1 μM DMG to IVC medium after 7 days of IVC. When DMG was added to IVC medium, the ratio of embryos developed to advanced developmental stages (No. of embryos developed to the blastocyst and expanded blastocyst stages/No. of embryos developed to the morula stage) was 28.7% (86/3) and 7 times higher than that of those cultured without DMG, 4.0% (52/13). These results suggest that addition of 0.1 μM DMG to mSOFaa during IVC of IVP bovine embryos has a promoting effect on their development.

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Mammalian preimplantation embryonic development is achieved by tightly coordinated regulation of a great variety of temporal and spatial changes. Therefore, it would be valuable to analyze these events three-dimensionally and dynamically. We have previously developed a live-cell imaging method based on the expression of fluorescent proteins, using mRNA injection and time-lapse florescence microscopy. However, with conventional fluorescent microscopy, three-dimensional images could not be obtained due to the thickness of the embryos and the optical problem in which `out-of focus blur' cannot be eliminated. Moreover, as the repeated exposure of intense excitation light to the cell yields phototoxicity, long-term observation was detrimental to embryonic development. Here, we improved our imaging system to enable six-dimensional live-cell imaging of mouse preimplantation embryos (x, y and z axes, time-lapse, multicolor and multisample). Importantly, by improving the imaging devices and optimizing the conditions for imaging, such as intensity of excitation and time intervals for image acquisition, the procedure itself was not detrimental to full-term development, although it is a prolonged imaging process. For example, live pups were obtained from embryos to which two different wavelengths of excitation (488 and 561 nm) were applied at 7.5-min intervals for about 70 h, and 51 images were acquired in the z axis at each time point; thus, a total of 56,814 fluorescent images were taken. All the pups were healthy, reproductively normal and not transgenic. Thus, this live-cell imaging technology is safe for full-term mouse development. This offers a novel approach for developmental and reproductive research in that it enables both retrospective and prospective analyses of development. It might also be applicable to assessment of embryo quality in fields such as human reproductive technology and production animal research.

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