Highlights d ATAC-seq accessibility at sperm and oocyte promoters is maintained in the embryo d Sperm enhancers containing transcription factors are conserved in mammals d Accessible sperm enhancers are also open in oocytes and preimplantation embryos d Interactions mediated by FoxA1 and CTCF and cohesin persist from gametes to embryos

This study was designed to compare the efficiency of the Cryotop method and that of two methods that employ a micro volume air cooling (MVAC) device by analyzing the survival and development of bovine oocytes and blastocysts vitrified using each method. In experiment I, in vitro-matured (IVM) oocytes were vitrified using an MVAC device without direct contact with liquid nitrogen (LN2; MVAC group) or directly plunged into LN2 (MVAC in LN2 group). A third group of IVM oocytes was vitrified using a Cryotop device (Cryotop group). After warming, vitrified oocytes were fertilized in vitro. There were no significant differences in cleavage and blastocyst formation rates among the three vitrified groups, with the rates ranging from 53.1% to 56.6% and 20.0% to 25.5%, respectively; however, the rates were significantly lower (P < 0.05) than those of the fresh control group (89.3% and 43.3%, respectively) and the solution control group (87.3% and 42.0%, respectively). In experiment II, in vitro-produced (IVP) expanded blastocysts were vitrified using the MVAC, MVAC in LN2 and Cryotop methods, warmed and cultured for survival analysis and then compared with the solution control group. The rate of development of vitrified-warmed expanded blastocysts to the hatched blastocyst stage after 24 h of culture was lower in the MVAC in LN2 group than in the solution control group; however, after 48–72 h of culture, the rates did not significantly differ between the groups. These results indicate that the MVAC method without direct LN2 contact is as effective as the standard Cryotop method for vitrification of bovine IVM oocytes and IVP expanded blastocysts.

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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.