The in vitro development of cloned sheep embryos treated with Scriptaid and Trichostatin (A).

Although, it has been success in the generation of animal clones from somatic cells in various animal species, the information related to nuclear reprogramming of cloned embryos is found to be limited. This study aims to compares the effect of both Scriptaid (SCR) and Trichostatin (A) treatments in improving cloning efficiency, and embryos developmental rate of cloned sheep embryos in vitro. Three groups were formed, i.e., one SCR group, second TSA group, with both treatment concentrations of 5 nM, 50 nM, and 500 nM, respectively, and third were control group with 0 nM. Methods: Ovaries of slaughtered sheep were collected and oocytes were recovered from antral follicles using aspiration method and in vitro maturation of oocytes were done. Then zona dissecting with micropipettes and oocyte enucleation were carried out under the micromanipulator. Later nuclear transfer, cell fusion and activation were done via cell fusion machine. Finally the embryo cultured in incubating chamber at the CO2 incubator up to 9 days. The result: In general the results showed that when the concentration increases the cleavage rate increased. The cleavage rates of the SCNT embryos treated with SCR at different concentrations are closely related to cleavage rate of embryos treated with TSA at same concentration; such as 39.47% for 500 nM TSA, 38.09% for 500 nM SCR; 18.6% for 50 nM TSA, 19.17% for 50 nM SCR, and 22.64% for 5 nM TSA, 17.18% for 5 nM SCR. As for the control group, the cleavage rate of the SCNT embryos cleavage ratewere27.47%., 30% and 30.85% respectively for bothtreatments. While there is a significant difference in TSA treatments at an eight-cell stage at the concentration (5 and 50 nM TSA) compared to the all other cleavage cell stages of (500 nM TSA and control). Also their were a differences between (50 nM of TSA) compared to the (50 nM SCR). Also there were a significant differences between the 16 cell stage at the (500 nM TSA) compared to other treatment (5 nM, 50 nM TSA and control). Regarding the SCR there were a significant difference at 8 cell stage between (5 nM SCR), compared to the other treatment (50 nM, 500 nM SCR and control). Also there were a significant difference at 16 cell stage between (50 nM, and 500 nM SCR), compared to the other treatment (5 nM SCR and control). While in the development of the embryos reach to blastocyst stage the SCR and the control group show a higher rate, in compered to TSA that did not show any development to blastocyst stage. The total SCR treatment showed (3/41 = 7.31%), and the total control showed (4/89 = 4.49%) blastula stage. It concludes that SCR improve the final development blastula stage compared to the TSA treatments that did not improved embryos reach to final developmental blastula stages may be due to spices differences or to the toxicity of TSA, especially at higher concentrations.

In Vitro Culture of Single Bovine Embryos with Microwell Plates Made of Poly(dimethylsiloxane) Cured under Low Pressure.

Single embryo culture is useful for assessing the developmental competence of an embryo in detail. Recently, a device made of poly(dimethylsiloxane) (PDMS), which is biocompatible and nontoxic, has been widely used for culture various types of cells. However, PDMS plates are porous, causing the serious osmolality increment of the medium (over 600 mOsm/kg from Day 4 to Day 7). Here, we report that curing the PDMS under low pressure (LP-PDMS) greatly reduced the porosity, resulting in a constant osmolality of the medium. The blastocyst rate of single bovine embryos cultured with LP-PDMS microwell (MW) plates was the same as that of group-cultured embryos (25 embryos/50 µl droplet; control, P>0.05). These results indicate that MWs on a plate made of PDMS cured under low pressure can be successfully used for individual embryo culture.

Early development of cloned bovine embryos produced from oocytes enucleated by fluorescence metaphase II imaging using a conventional halogen-lamp microscope.

Enucleation of a recipient oocyte is one of the key processes in the procedure of somatic cell nuclear transfer (SCNT). However, especially in bovine species, lipid droplets spreading in the ooplasm hamper identification and enucleation of metaphase II (MII) chromosomes, and thereby the success rate of the cloning remains low. In this study we used a new experimental system that enables fluorescent observation of chromosomes in living oocytes without any damage. We succeeded in visualizing and removing the MII chromosome in matured bovine oocytes. This experimental system consists of injecting fluorescence-labeled antibody conjugates that bind to chromosomes and fluorescent observation using a conventional halogen-lamp microscope. The cleavage rates and blastocyst rates of bovine embryos following in vitro fertilization (IVF) decreased as the concentration of the antibody increased (p<0.05). The enucleation rate of the conventional method (blind enucleation) was 86%, whereas all oocytes injected with the antibody conjugates were enucleated successfully. Fusion rates and developmental rates of SCNT embryos produced with the enucleated oocytes were the same as those of the blind enucleation group (p>0.05). For the production of SCNT embryos, the new system can be used as a reliable predictor of the location of metaphase plates in opaque oocytes, such as those in ruminant animals.

Production of somatic cell nuclear transfer embryos using in vitro-grown and in vitro-matured oocytes in rabbits.

We examined growing oocytes collected from follicles remaining in superovulated rabbit ovaries, that were grown (in vitro growth, IVG) and matured (in vitro maturation, IVM) in vitro. We produced somatic cell nuclear transfer (SCNT) embryos using the mature oocytes and examined whether these embryos have the ability to develop to the blastocyst stage. In addition, we examined the effects of trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), on the developmental competence of SCNT embryos derived from IVG-IVM oocytes. After growth for 7 days and maturation for 14-16 h in vitro, the growing oocytes reached the metaphase II stage (51.4%). After SCNT, these reconstructed embryos reached the blastocyst stage (20%). Furthermore, the rate of development to the blastocyst stage and the number of cells in the blastocysts in SCNT embryos derived from IVG-IVM oocytes were significantly higher for TSA-treated embryos compared with TSA-untreated embryos (40.6 versus 21.4% and 353.1 ± 59.1 versus 202.5 ± 54.6, P < 0.05). These results indicate that rabbit SCNT embryos using IVG-IVM oocytes have the developmental competence to reach the blastocyst stage.

Mouse zygote-specific proteasome assembly chaperone important for maternal-to-zygotic transition.

During the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin-proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT.

Development of interspecies cloned embryos reconstructed with rabbit (Oryctolagus cuniculus) oocytes and cynomolgus monkey (Macaca fascicularis) fibroblast cell nuclei.

Interspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus-cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey-rabbit embryo). The monkey-rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey-rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey-rabbit ISCNT and rabbit-rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey-rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit-rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey-rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey-rabbit ISCNT embryos is similar to that of rabbit-rabbit SCNT embryos; and (4) the mitochondrial fate of monkey-rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.

Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

Selection of high-potential embryos by culture in poly(dimethylsiloxane) microwells and time-lapse imaging.

OBJECTIVE: To assess the developmental kinetics of human embryos and their ability to develop to morphologically normal blastocysts. DESIGN: Experimental study on human embryos donated for research using a time-lapse imaging system based on individual embryo culture in poly(dimethylsiloxane) microwells and monitored using a microscope inside the incubator. SETTING: Private fertility clinic. PATIENT(S): Surplus embryos donated by couples after undergoing fertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Blastocyst score and times required from beginning to completion of the second and third mitotic divisions. RESULT(S): The time required for completion of the second division (the three- to four-cell stage) was shorter in embryos that developed to high-scoring blastocysts (0.7 hours, n = 17) than in those forming low-scoring blastocysts (3.7 hours, n = 24). Similarly, the mean time required to completion of the third division (five- to eight-cell stage) was also significantly shorter in embryos forming high-scoring blastocysts (5.7 hours) than among those forming low-scoring blastocysts (16.9 hours). CONCLUSION(S): Individual embryos with the potential to develop to high-scoring blastocysts could be selected at 2-3 days of culture using this system by examining the times required to complete the second and third mitotic divisions.

Donor cells at the G1 phase enhance homogeneous gene expression among blastomeres in bovine somatic cell nuclear transfer embryos.

The success rate of bovine somatic cell nuclear transfer (SCNT) embryos to full term has been reported to be higher with G1 cells than with G0 cells. To better understand the reason for this, we analyzed the kinetics of luminescence activity in bovine SCNT embryos from G0 and G1 cells carrying a luciferase gene under the control of the ß-actin promoter during early embryonic development. At 60-h postfusion, when bovine embryonic gene activation (EGA) begins, the luminescence activity was higher in G1-SCNT embryos than G0-SCNT embryos. Moreover, half of the G1-SCNT embryos exhibited homogeneous luminescence among the blastomeres, whereas more than half of the G0-SCNT embryos exhibited mosaic luminescence. To characterize the differential luminescence pattern in SCNT embryos, the expressions of several endogenous genes and the level of DNA methylation were determined in all blastomeres of SCNT embryos with or without luminescence. The expressions of several development-related genes (H2AFZ, GJA1, and BAX) and level of DNA methylation of the SCNT embryos with luminescence were the same as those of normal embryos produced by in vitro fertilization. A higher success rate in G1-SCNT embryos is thought to contribute to homogeneous expression among all blastomeres at EGA.

Inhibition of the ubiquitin-proteasome system leads to delay of the onset of ZGA gene expression.

In mammalian oocytes, the ubiquitin-proteasome system (UPS) is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation. However, little is known about the direct relationship between zygotic gene activation (ZGA) and degradation of maternal proteins. Here, we investigated the role of the UPS in the onset of ZGA in early mouse embryos. First, we found degradation of cyclin B1 protein in fertilized oocytes at 1 hpi by western blot analysis and used these oocytes throughout this study. Subsequently, we determined optimal experimental conditions for transient inhibition of proteasomal activity by specific and reversible proteasomal inhibitor MG132 in the G1 phase of the first cell cycle. Under the selected optimal conditions, we subjected transient MG132-treated embryos to reverse transcription (RT)-PCR analysis of expression of four ZGA genes, i.e., the hsp70.1, MuERV-L, eif-1a and zscan4d genes. As a result, we found that onset of expression of the four examined ZGA genes was delayed in both normally developed 2-cell embryos and arrested 1-cell embryos. Our results indicate that proteasomal degradation of proteins by the UPS plays a pivotal role in the molecular mechanisms of ZGA in early mouse embryos.

Deposition of acetylated histones by RNAP II promoter clearance may occur at onset of zygotic gene activation in preimplantation mouse embryos.

We investigated the contribution of phosphorylated RNA polymerase II (RNAP II) and dynamic epigenetic changes to the onset of minor zygotic gene activation (ZGA). Using immunofluorescence staining, we observed that the nuclear localization of RNAP II was initiated by 6 hours post insemination (hpi), whereas RNAP II phosphorylated at serine residue 5 of the carboxyl-terminal domain (CTD) was localized by 9 hpi, and then RNAP II phosphorylated at serine residue 2 of the CTD was localized in the nucleus of embryos by 12 hpi. In a transient gene expression assay using a plasmid reporter gene (pß-actin/luciferase+/SV40) injected during 6-9 hpi into the male pronucleus, the luciferase+ gene was actively transcribed and translated by 13 and 15 hpi, respectively, indicating that a transcriptionally silent state persisted for at least 4 hours after injection. We found that the methylation status in the chicken ß-actin promoter region of the plasmid reporter gene may not be associated with the transcriptionally silent state before minor ZGA. Exposure to trichostatin A did not induce premature expression of the silent reporter gene injected into 1-cell embryos containing histone deacetylase activity and did not affect the amount of luciferase produced per embryo. Acetylated histone H3 lysine 9/14 and acetylated histone H4 lysine 12 and 16 were enriched preferentially in the injected reporter gene at least until 13 hpi, which coincided with the transcriptionally active state. Taken together, these results suggest that deposition of selectively acetylated histones onto the chromatin of 1-cell embryos functions together with transcriptional elongation by RNAP II and that this sequential chromatin remodeling is involved in the molecular mechanism associated with the onset of minor ZGA in the preimplantation mouse embryo.

Expression analysis of circadian genes in oocytes and preimplantation embryos of cattle and rabbits.

We previously showed that circadian genes clock, bmal1, cry1, cry2, per1, and per2 are expressed and function as maternal mRNA regulating events in the oocytes and preimplantation embryos of mice. Recent evidence indicates however that either or both expression profiles of circadian genes in some tissues, and transcript sequences of circadian genes, differ to generate the physiological differences between diurnal and nocturnal species. We therefore investigated the expression profiles of circadian genes in oocytes and preimplantation embryos of species other than mice, namely cattle and rabbits, representing diurnal and nocturnal species, respectively, and determined the protein sequences of circadian genes in these species. Quantitative real-time PCR revealed that all circadian genes considered in this study were present in the oocytes and preimplantation embryos of both species, and the transcript amounts of clock, cry1 and per1 contained in oocytes were significantly higher than in preimplantation embryos of both species. The transcripts of clock, cry1, and per1 of cattle and rabbits were determined by primer walking, and functional domains in the estimated amino acid sequences were compared between cattle and rabbits and with those of humans and mice. The sequences of clock, cry1, and per1 in cattle and rabbits closely resembled those in mice (85-100% homologies), and no difference based on diurnality or nocturnality was observed. These findings suggest that circadian genes in the oocytes and preimplantation embryos of mammals fulfill the same functions across species as maternal mRNA.

Comparison of early development in utero of cloned fetuses derived from bovine fetal fibroblasts at the G1 and G0/G1 phases.

In bovine somatic cell nuclear transfer (NT), embryos are more likely to develop to full term when they are derived from fibroblasts at the G1 phase instead of cells at the G0/G1 phase. To better understand the reason for this difference, we examined morphological development in the early pregnancy of NT embryos using G1 phase cells (G1-NT embryos) and G0/G1 phase cells (G0/G1-NT embryos). Blastocysts derived from G1 and G0/G1-NT embryos were transferred to recipient heifers, and the conceptuses at day 50 of gestation were retrieved non-surgically using prostaglandin F(2alpha) and oxytocin. In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. The percentages of embryos that developed to the blastocyst stage did not differ between G1 and G0/G1-NT embryos. Pregnancy rates at day 30 of recipient heifers carrying G1-NT, G0/G1-NT, IVF, parthenogenetic and AI embryos were similar (57-100%). Two recipient heifers carrying parthenogenetic embryos returned to estrus between days 30 and 50 of gestation, whereas all other pregnancies remained viable. Most fetuses at day 50 of gestation of all experimental groups (83%) were recovered non-surgically by several PGF(2alpha) and oxytocin treatments. Recovery rates of normal fetuses derived from G1-NT embryos (83%), IVF embryos (80%) and AI embryos (88%) were greater than those of G0/G1-NT embryos (33%) and parthenogenetic embryos (0%). Our results suggest that NT embryos reconstructed with cells at the G1 phase have a high developmental competence from the time of embryo transfer to day 50 of gestation.

Potential existence of stem cells with multiple differentiation abilities to three different germ lineages in mouse neurospheres.

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential in brain, and are committed cells of the central nervous system. Recently, some reports have suggested the possibility of the NSCs to differentiate into non-CNS mesodermal derivatives, such as blood cells and skeletal muscle cells. Here we isolated NSCs as neurospheres from a neonatal mouse brain using serum replacement medium, and demonstrated that the stem cell population expressing pluripotent-related genes such as Oct-4, Sox-2, and Nanog possess multiple differentiation potentials to ectodermal, mesodermal, and endodermal lineages, that is, some neural cells, beating cardiomyocytes, adipocytes, and insulin-producing cells. The results of the present study partly provide further evidence for multiple differentiation properties of NSCs and suggest common characteristics between NSCs and other pluripotent stem cells.

Functional expression of a humanized gene for an omega-3 fatty acid desaturase from scarlet flax in transfected bovine adipocytes and bovine embryos cloned from the cells.

Long-chain n-3 fatty acids can lower the risk of lifestyle-related diseases, therefore, we introduced a plant fatty acid desaturation3 (FAD3) gene into mammalian cells. The FAD3 cDNA was isolated from the immature seeds of scarlet flax and optimized to human high-frequency codon usage for enhancement of its expression levels in mammalian cells (hFAD3). We introduced the gene into bovine muscle satellite cells, which can be differentiated into multilocular adipocytes in vitro. After hFAD3 transfection, the cells were differentiated into adipocytes and their fatty acid composition was analyzed by gas chromatography. The level of alpha-linolenic acid (18:3n-3) in transfected adipocytes increased about ten-fold compared with non-transfected adipocytes. In addition, the levels of docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in transfected adipocytes were significantly higher than those in non-transfected adipocytes. Moreover, we produced bovine cloned embryos from the hFAD3 cells by somatic cell nuclear transfer. Blastocyst rates of hFAD3 clones were the same as the control clones using the non-transfected cells (21% vs 27%, P > 0.05). hFAD3 transcripts were detected in all of the blastocysts. These results demonstrate the functional expression of a plant hFAD3 in mammalian adipocytes, and normal development of cloned embryos carrying the hFAD3 gene.

Enhancement of histone acetylation by trichostatin A during in vitro fertilization of bovine oocytes affects cell number of the inner cell mass of the resulting blastocysts.

Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genomes after fertilization to establish a totipotent state for normal development. In the present study, the effects of trichostatin A (TSA), an inhibitor of histone deacetylase, during in vitro fertilization (IVF) of bovine oocytes on subsequent embryonic development were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were matured in vitro and subjected to IVF in a defined medium supplemented with 0 (control), 5, 50, and 500 nM TSA for 18 h. After IVF, presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF) medium until 168 h postinsemination (hpi). Some oocytes were immunostained using antibody specific for histone H4-acetylated lysine 5 at 10 hpi. Cleavage, blastocyst development and cell number of inner cell mass (ICM) and trophectoderm (TE) of blastocysts were assessed. TSA treatment enhanced histone acetylation that was prominent in decondensed sperm nuclei. TSA did not affect the postfertilization cleavage, blastocyst rates, and TE cell number. However, it significantly enhanced ICM cell number (p < 0.05). These results indicate that TSA treatment during IVF of bovine oocytes does not affect blastocyst development but alters the cell number of ICM, suggesting that overriding epigenetic modification of the genome during fertilization has a carryover effect on cell proliferation and differentiation in preimplantation embryos. Thus, further environmental quality controls in assisted reproductive technologies are needed in terms of factors which affect chromatin remodelling.

Proteomic analysis of the mouse ovary in response to two gonadotropins, follicle-stimulating hormone and luteinizing hormone.

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.

Mouse androgenetic embryonic stem cells differentiated to multiple cell lineages in three embryonic germ layers in vitro.

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.

Resurrection of a bull by cloning from organs frozen without cryoprotectant in a -80 degrees c freezer for a decade.

Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a -80 degrees C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.

Expression and functional analyses of circadian genes in mouse oocytes and preimplantation embryos: Cry1 is involved in the meiotic process independently of circadian clock regulation.

In mammals, circadian genes, Clock, Arntl (also known as Bmal1), Cry1, Cry2, Per1, Per2, and Per3, are rhythmically transcribed every 24 h in almost all organs and tissues to tick the circadian clock. However, their expression and function in oocytes and preimplantation embryos have not been investigated. In this study we found that the circadian clock may stop in mouse oocytes and preimplantation embryos. Real-time PCR analysis revealed the presence of transcripts of these genes in both oocytes and preimplantation embryos; however, their amounts did not oscillate every 24 h in one- to four-cell and blastocyst-stage embryos. Moreover, immunofluorescence analyses revealed that CLOCK, ARNTL, and CRY1 were localized similarly in the nuclei of germinal vesicle (GV) oocytes and one-cell- to four-cell-stage embryos. Because CRY1 is known to interact with the CLOCK-ARNTL complex to suppress transcription-promoting activity of the complex for genes such as Wee1, Cry2, Per1, Per2, and Per3 in cells having the ticking circadian clock, we hypothesized that if the circadian clock functions in GV oocytes and one-cell- to four-cell-stage embryos, CLOCK, ARNTL, and CRY1 might suppress the transcription of these genes in GV oocytes and one-cell- to 4-cell-stage embryos as well. As a result, knockdown of CRY1 in GV oocytes by RNA interference did not affect the transcription levels of Wee1, Cry2, Per1, Per2, and Per3, but it reduced maturation ability. Thus, it seems that circadian genes are not involved in circadian clock regulation in mouse oocytes and preimplantation embryos but are involved in physiologies, such as meiosis.