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Objective To demonstrate that functional spermatids can be derived in vitro from nonhuman primate pluripotent stem cells. Design Green fluorescent protein-labeled, rhesus macaque nonhuman primate embryonic stem cells (nhpESCs) were differentiated into advanced male germ cell lineages using a modified serum-free spermatogonial stem cell culture medium. In vitro-derived round spermatid-like cells (rSLCs) from differentiated nhpESCs were assessed for their ability to fertilize rhesus oocytes by intracytoplasmic sperm(atid) injection. Setting Multiple academic laboratory settings. Patient(s) Not applicable. Intervention(s) Intracytoplasmic sperm(atid) injection of in vitro-derived spermatids from nhpESCs into rhesus macaque oocytes. Main Outcome Measure(s) Differentiation into spermatogenic cell lineages was measured through multiple assessments including ribonucleic acid sequencing and immunocytochemistry for various spermatogenic markers. In vitro spermatids were assessed for their ability to fertilize oocytes by intracytoplasmic sperm(atid) injection by assessing early fertilization events such as spermatid deoxyribonucleic acid decondensation and pronucleus formation/apposition. Preimplantation embryo development from the one-cell zygote stage to the blastocyst stage was also assessed. Result(s) Nonhuman primate embryonic stem cells can be differentiated into advanced germ cell lineages, including haploid rSLCs. These rSLCs undergo deoxyribonucleic acid decondensation and pronucleus formation/apposition when microinjected into rhesus macaque mature oocytes, which, after artificial activation and coinjection of ten-eleven translocation 3 protein, undergo embryonic divisions with approximately 12% developing successfully into expanded blastocysts. Conclusion(s) This work demonstrates that rSLCs, generated in vitro from primate pluripotent stem cells, mimic many of the capabilities of in vivo round spermatids and perform events essential for preimplantation development. To our knowledge, this work represents, for the first time, that functional spermatid-like cells can be derived in vitro from primate pluripotent stem cells.

The aim of this study was to evaluate the effect of L-carnitine (LC) supplemented in vitro maturation (IVM) medium on the cryotolerance and development of buffalo oocytes. Mitochondrial activity, reactive oxygen species (ROS) content, and lipid droplet distribution of vitrified-warmed buffalo oocytes were also observed. Oocytes were matured in IVM medium supplemented with 0, 0.3, 0.6, and 1.2 mg/mL LC and some of them were vitrified by the Cryotop® method. The highest survival, morula, and blastocyst rates were obtained with 0.6 mg/mL LC, irrespective of whether oocytes were fresh or vitrified-warmed. The density of active mitochondria in vitrified oocytes was significantly higher with 0.6 mg/mL LC than without LC treatment. In contrast, H2O2 levels and lipid droplet content were significantly lower following LC treatment than without it. In conclusion, supplementation with 0.6 mg/mL LC during IVM improves the buffalo oocytes’ survival rate and subsequent embryo development after in vitro fertilization. This may be linked to improved mitochondrial activity, enhanced β-oxidation, and reduced ROS levels.

The stabilization of spindle fibersis important for successful vitrification of bovine oocytes because microtubules and other cytoskeleton fibers (CSF) can be damaged during vitrification, resulting in failure of fertilization after thawing. Docetaxel, a stabilizing agent, could potentially reduce CSF damage of bovine oocytes induced during vitrification. However, there have been no reports on the effects of docetaxel on their vitrification. Experiment 1 was conducted to investigate the effects of various doses of docetaxel (0.0, 0.05, 0.5, 5.0 and 50 μM) in preincubation medium of in vitro matured (IVM) bovine oocytes on their developmental ability after in vitro fertilization (IVF). The results show that 0.05 μM docetaxel had no adverse effect on embryo development, while docetaxel at a concentration of ⩾0.5 μM inhibited development. Experiments 2 and 3 were conducted to investigate the effects of preincubation of IVM bovine oocytes with 0.05 μM docetaxel for 30 min prior to vitrification-warming on CSF integrity (Experiment 2), and on oocyte survival and viability after IVF (Experiment 3). When preincubated with 0.05 μM docetaxel for 30 min before vitrification, post-thawed oocytes had less CSF damage and higher survival rates compared with those untreated with docetaxel before vitrification. Surviving oocytes also had higher rates of cleavage and development to the blastocyst stage after IVF. In conclusion, preincubation of IVM bovine oocytes with 0.05 μM docetaxel for 30 min before vitrification was effective at preventing CSF damage during vitrification, and improving oocyte viability after warming and subsequent cleavage and blastocyst formation after IVF.

In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing.

a b s t r a c t Oocyte cryopreservation is an alternative tool in assisted reproductive technology. The objective of this study was to determine the developmental potential of vitrified goat oocytes after somatic cell nuclear transfer (SCNT). In vitro matured goat oocytes were vitrified by Cryotop method. The survival rate of vitrified oocytes at 1 h after thawing determined by fluorescein diacetate staining was 92.1% which was significantly lower than that of fresh oocytes (99.3%). Live oocytes from both vitrified and fresh groups were used as recipient cytoplasts for SCNT. No significant difference in fusion rate was found between vitrified (97.6%) and fresh (93.2%) oocytes. The cleavage rates of vitrified oocytes in the SCNT and parthenogenetic activation (PA) embryos (29.6% and 27.9%) were significantly lower than those from fresh SCNT and PA oocytes (80.9% and 91.1%). The developmental rates to 8cell stage of SCNT and PA embryos from vitrified oocytes were also lower than that of fresh oocytes. There was no significant difference among the groups in the development to morula stage (11–27%). However, the blastocyst rate of PA embryos derived from fresh oocytes (12.5%) was significantly higher than the other groups (1.2–3.3%). Although high survival rate of vitrified oocytes was obtained, the cleavage, 8-cell, and blastocyst formation rates of SCNT and PA embryos from vitrified oocytes were still lower than those of fresh

In vitro matured (IVM) buffalo oocytes at the metaphase of the second meiotic division (MII) were vitrified in 20% Me(2)SO: 20% EG (v/v) and 0.5M sucrose (VA), or 35% EG (v/v), 50mg/mL polyvinylpyrrolidone (PVP), and 0.4M trehalose (VB), either on cryotops or as 2μL microdrops. The viability was assessed after warming by fluorescein diacetate (FDA) staining and all surviving oocytes were subjected to ICSI and ethanol activation. All vitrified groups had similar recovery rates but both VA groups had significantly higher survival and pronuclear formation rates than either of the VB groups. Non treated control oocytes and non cryopreserved oocytes exposed to FDA had significantly higher survival, 2nd polar body extrusion, PN and blastocyst formation rates than any of the four vitrified groups (P

Embryonic Stem-derived Neural Cells (ESNCs) hold potential as a source of neurons for a cell-based therapy for the treatment of brain tumors, and other neurological diseases and disorders in the future. The sorting of neural cell types is envisaged to be one of the most important processed for clinical application of these cells in cell-based therapies of the central nervous system (CNS). In this study, laboratory-based FTIR and Synchrotron-FTIR (SR-FTIR) microspectroscopy were used to identify FTIR marker for distinguishing different neural cell types derived from the differentiation of mouse embryonic stem cells (mESCs). Principal Component Analysis (PCA) and Unsupervised Hierarchical Cluster Analysis (UHCA) were shown to be able to distinguish the developmental stage of mESCs into three cell types: embryoid bodies (EBs), neural progenitor cells (NPCs), and ESNCs. Moreover, PCA provided the mean for identifying potential FTIR “marker bands” that underwent dramatic changes during stem cell differentiation along neural lineages. These appeared to be associated with changes in lipids (bands from CH 2 and CH 3 stretching vibrations at ∼2959, 2923 and 2852 cm −1 ) and proteins (changes in the amide I band at ∼1659 and 1637 cm −1 ). The results suggested that lipid content of cells increased significantly over the time of differentiation, suggesting increased expression of glycerophospholipids. Changes in the amide I profile, suggested concomitant increases in α-helix rich proteins as mESCs differentiated towards ESNCs, with a corresponding decrease in β-sheet rich proteins, corresponding with changes in cytoskeleton protein which may have been taking place involved with the establishment of neural structure and function.

Cloned animals often suffer from loss of development to term and abnormalities, typically classified under the umbrella term of Large Offspring Syndrome (LOS). Cattle are an interesting species to study because of the relatively greater success rate of nuclear transfer in this species compared with all species cloned to date. The imprinted insulin-like growth factor receptor (IGF2R; mannose-6-phosphate) gene was chosen to investigate aspects of fetal growth and development in cloned cattle in the present study. IGF2R gene expression patterns in identical genetic clones of several age groups were assessed in day 25, day 45, and day 75 fetuses as well as spontaneously aborted fetuses, calves that died shortly after birth and healthy cloned calves using single stranded conformational polymorphism gel electrophoresis. A variable pattern of IGF2R allelic expression in major organs such as the brain, cotyledon, heart, liver, lung, spleen, kidney and intercotyledon was observed using a G/A transition in the 3'UTR of IGF2R. IGF2R gene expression was also assessed by real time RT-PCR and found to be highly variable among the clone groups. Proper IGF2R gene expression is necessary for survival to term, but is most likely not a cause of early fetal lethality or an indicator of postnatal fitness. Contrary to previous reports of the transmission of imprinting patterns from somatic donor cells to cloned animals within organs in the same cloned animal the paternal allele of IGF2R can be imprinted in one tissue while the maternal allele is imprinted in another tissue. This observation has never been reported in any species in which imprinting has been studied.

Synchrotron-Infrared (SR-IR) mapping and Focal plane array (FPA) imaging have been applied for discrimination of the three biochemical components of the mouse blastocyst. The mouse blastocyst consists of two clusters of cells known as the inner cell mass (ICM) formed within the blastocoel cavity and the thin layer of outer cells called the trophectoderm. Using Hierachical Cluster Analysis (HCA) and Principle Component Analysis (PCA), it can be shown that the composition and distribution of biochemical components within the blastocyst show differences in the protein secondary structure and the lipid content. It is worth noting that the secondary structure of the outer layer cells indicates more distinctive β-type secondary structure. The blastocoel cavity was observed to be predominantly α-helix. Significantly, the ICM region showed the predominant high absorption intensities of lipid content (CH2, CH3 symmetric and asymmetric stretching around 3000–2800 cm−1). The results show agreement between both SR-IR mapping and FPA-IR imaging. We propose that the biochemical difference within the blastocyst, especially the high lipid content in the ICM region, could be involved in the process of lipid signaling during pre-implantation. The use of both techniques is shown to be significant approach for revealing the biochemical components within the blastocyst.

We investigated the potential of vitrified-warmed buffalo oocytes to develop to blastocysts after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). In vitro-matured oocytes before and after enucleation (M-II oocytes and enucleated oocytes, respectively) were put in 7.5% DMSO and 7.5% ethylene glycol (EG) for 4, 7 and 10 min, and then vitrified (Cryotop device) after 1-min equilibration in 15% DMSO, 15% EG and 0.5M sucrose. Following 4-, 7- and 10-min exposure, proportions of the post-warm oocytes with a normal vitelline membrane were similar (66-71% in M-II oocytes and 69-71% in enucleated oocytes). However, 18-20% of the normal M-II oocytes had no detectable first polar body in their perivitelline space (no potential for subsequent enucleation). When the post-warm M-II oocytes were treated for PA by 7% ethanol, 10 microg/mL cycloheximide and 1.25 microg/mL cytochalasin-D, parthenogenetic development into Day-7 blastocysts occurred in 10-13% of cultured oocytes, lower (P0.05) than fresh (control) oocytes (24%). In the absence of the cooling and warming, blastocyst rates in the 4-min exposure group (22%), but not in the 7-min and 10-min exposure groups (14-15%), were similar to that in the fresh group (23%). The total cell number (group average 117-132 cells) and the ICM ratio (22-24%) of the PA blastocysts derived from vitrified M-II oocytes were comparable with fresh oocytes (127 cells and 25%). After SCNT (with fibroblast cells and vitrified-warmed oocytes), blastocyst rates were similar for the three exposure periods for M-II oocytes (8-10%) and enucleated oocytes (7-9%), but were lower (P0.05) than in the fresh group (15%). The total cell number of the SCNT blastocysts derived from vitrified M-II and enucleated oocytes (80-90 and 82-101 cells) was smaller (P0.05) than from fresh oocytes (135 cells); the ICM ratio of blastocysts derived from the M-II and enucleated oocytes after vitrification in 7- or 10-min exposure groups (20-22%) was not different (P0.05) from fresh control oocytes (24%) or those in 4-min exposure group (M-II 23%, enucleated 24%). Thus, SCNT of swamp buffalo oocytes following vitrification before or after enucleation resulted in blastocysts with a slightly decreased cell number.

The aim of the present study was to optimize the temperature for the temporal storage of matured bovine oocytes. In vitro-matured bovine oocytes were preserved in HEPES-buffered TCM199 medium supplemented with 10% newborn calf serum at different temperatures (4 °C, 15 °C, 25 °C, and 38.5 °C) for 20 hours. Embryo development and blastocyst quality after in vitro fertilization, cytoplasmic ATP and glutathione levels in oocytes, and the frequency of apoptotic oocytes were compared among storage groups and a control group without storage. Among the storage groups, those at 25 °C and 38.5 °C showed the highest rates of blastocyst development (19.3% and 24.5%, respectively) compared with those stored at 4 °C and 15 °C (8.5% and 14.9%, respectively); however, blastocyst formation rates in all storage groups were lower than that in the control group (39.8%; P0.05). Storage at 38.5 °C and 15 °C was associated with reduced cell numbers in resultant blastocysts compared with the control and the 25 °C storage groups. Storage at 4 °C reduced metabolic activity of oocytes characterized by their lower ATP levels compared with the other groups. Storage for 20 hours significantly reduced the glutathione content in oocytes in all groups in a similar manner, irrespective of the temperature. Storage at 4 °C or 15 °C but not at 25 °C and 38.5 °C significantly increased the percentage of apoptotic oocytes compared with the control group. In conclusion, 25 °C was found to be the most suitable temperature for the temporal storage of matured bovine oocytes regarding both the developmental competence of oocytes and the quality of resultant blastocysts.