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To date, efforts to establish pluripotent embryonic stem cells from bovine embryos have failed. The lack of reliable pluripotency markers is an important drawback when attempting to derive these cells. This study aimed to identify genes upregulated in the inner cell mass (ICM) of bovine blastocysts, and we selected SOX2 for further characterization. Spatial and temporal localization of the SOX2 protein revealed that its expression starts at the 16-cell stage and then becomes restricted to the ICMs of blastocysts. To study the role of SOX2 during the early development of bovine embryos, we designed siRNA to target SOX2. We began by injecting this siRNA into zygotes; the rate at which blastocysts developed declined compared to noninjected or scramble-injected controls. When only one blastomere of a two-cell embryo was injected with SOX2 siRNA, we observed development rates similar to those of controls. Daughter cells of the injected blastomere were tracked by TRITC fluorescence and found to contribute to the ICM, as select cells also lacked SOX2. Gene expression analysis revealed a decrease in SOX2 and NANOG gene expression in siRNA-injected embryos, but OCT4 expression remained unchanged. We conclude that SOX2 localizes exclusively in the ICM of bovine blastocysts, and its downregulation negatively impacts preimplantation development; however, it is still unclear as to why downregulation of SOX2 in one cell of a two-cell embryo does not affect the composition of the ICM.

To date, efforts to establish pluripotent embryonic stem cells from bovine embryos have failed. The lack of reliable pluripotency markers is an important drawback when attempting to derive these cells. This study aimed to identify genes upregulated in the inner cell mass (ICM) of bovine blastocysts, and we selected SOX2 for further characterization. Spatial and temporal localization of the SOX2 protein revealed that its expression starts at the 16-cell stage and then becomes restricted to the ICMs of blastocysts. To study the role of SOX2 during the early development of bovine embryos, we designed siRNA to target SOX2. We began by injecting this siRNA into zygotes; the rate at which blastocysts developed declined compared to noninjected or scramble-injected controls. When only one blastomere of a two-cell embryo was injected with SOX2 siRNA, we observed development rates similar to those of controls. Daughter cells of the injected blastomere were tracked by TRITC fluorescence and found to contribute to the ICM, as select cells also lacked SOX2. Gene expression analysis revealed a decrease in SOX2 and NANOG gene expression in siRNA-injected embryos, but OCT4 expression remained unchanged. We conclude that SOX2 localizes exclusively in the ICM of bovine blastocysts, and its downregulation negatively impacts preimplantation development; however, it is still unclear as to why downregulation of SOX2 in one cell of a two-cell embryo does not affect the composition of the ICM.

Reproductive health of humans and animals exposed to daily irradiants from solar/cosmic particles remains largely understudied. We evaluated the sensitivities of bovine and mouse oocytes to bombardment by krypton-78 (1 Gy) or ultraviolet B (UV-B; 100 microjoules). Mouse oocytes responded to irradiation by undergoing massive activation of caspases, rapid loss of energy without cytochrome-c release, and subsequent necrotic death. In contrast, bovine oocytes became positive for annexin-V, exhibited cytochrome-c release, and displayed mild activation of caspases and downstream DNAses but with the absence of a complete cell death program; therefore, cytoplasmic fragmentation was never observed. However, massive cytoplasmic fragmentation and increased DNA damage were induced experimentally by both inhibiting RAD51 and increasing caspase 3 activity before irradiation. Microinjection of recombinant human RAD51 prior to irradiation markedly decreased both cytoplasmic fragmentation and DNA damage in both bovine and mouse oocytes. RAD51 response to damaged DNA occurred faster in bovine oocytes than in mouse oocytes. Therefore, we conclude that upon exposure to irradiation, bovine oocytes create a physiologically indeterminate state of partial cell death, attributed to rapid induction of DNA repair and low activation of caspases. The persistence of these damaged cells may represent an adaptive mechanism with potential implications for livestock productivity and long-term health risks associated with human activity in space.

Reproductive health of humans and animals exposed to daily irradiants from solar/cosmic particles remains largely understudied. We evaluated the sensitivities of bovine and mouse oocytes to bombardment by krypton-78 (1 Gy) or ultraviolet B (UV-B; 100 microjoules). Mouse oocytes responded to irradiation by undergoing massive activation of caspases, rapid loss of energy without cytochrome-c release, and subsequent necrotic death. In contrast, bovine oocytes became positive for annexin-V, exhibited cytochrome-c release, and displayed mild activation of caspases and downstream DNAses but with the absence of a complete cell death program; therefore, cytoplasmic fragmentation was never observed. However, massive cytoplasmic fragmentation and increased DNA damage were induced experimentally by both inhibiting RAD51 and increasing caspase 3 activity before irradiation. Microinjection of recombinant human RAD51 prior to irradiation markedly decreased both cytoplasmic fragmentation and DNA damage in both bovine and mouse oocytes. RAD51 response to damaged DNA occurred faster in bovine oocytes than in mouse oocytes. Therefore, we conclude that upon exposure to irradiation, bovine oocytes create a physiologically indeterminate state of partial cell death, attributed to rapid induction of DNA repair and low activation of caspases. The persistence of these damaged cells may represent an adaptive mechanism with potential implications for livestock productivity and long-term health risks associated with human activity in space.