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Interferon tau (IFNT), a type I interferon, is known as a key signal molecule during pregnancy in ruminants because of the necessity in maternal recognition of pregnancy. It is produced in trophectoderm cells of the elongation bovine conceptus at Day 13-21, and peak production is at Day 15 to 17 of pregnancy. In addition, other studies show that it can affect embryonic development and quality. In this study, the effect of IFNT knockout in donor cells to bovine cloned embryonic development by somatic cell nuclear transfer (SCNT) was investigated. To proceed with this experiment, immature oocytes from ovaries at a local slaughterhouse were matured in vitro for 22h. To prepare the donor cell with IFNT knockout, somatic cells were transfected with Cas9 and single guide RNA targeting IFNT, and several single derived colonies with high proliferation were isolated for mutation assay. Finally, one colony that had mono-allelic mutation (4bp deletion) was selected and used as the donor cell for SCNT. A donor cell was injected into an enucleated oocyte. Reconstructed oocytes with the donor cell were fused by electrical shock, activated by chemical stimulation, and cultured for 7 days in chemically defined medium. For this study, control (n=94) and IFNT knockout groups (n=140) were compared with 4 replications. The results showed no significant difference between control and IFNT knockout groups not only in cleavage rate, but also in blastocyst formation rate (control: 15.7±8.3%, IFNT knockout group: 26.3±13.1%). In addition, the number of blastocyst cell was not different between control (88.2±27.0) and IFNT knockout group (88.0±21.1). Some IFNT mutated blastocysts from SCNT were randomly selected for confirmation of the deletion of IFNT, and all samples were positive for mutation. In conclusion, these data demonstrated that the disruption of IFNT did not affect embryonic development. In future study, we would transfer these embryos and check the effect of IFNT during pregnancy status. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.

The p53 gene is a tumour suppressor gene with many roles in cellular reprogramming, senescence, or cancers. Knockdown of p53 in somatic cells improves generation of induced pluripotent stem cells and developmental competence of cloned embryos. In this study, we hypothesised that disruption of p53 in bovine somatic cells using CRISPR-Cas9 may improve reprogramming and result in increased blastocyst formation. For the study, ovary-derived immature bovine cumulus-oocyte complexes were matured in vitro. To prepare the donor cells with disruption of p53, primary cells from the skin tissue were transfected with spCas9 and single guide RNA for bovine p53. Two days later, genomic DNA was extracted and the mutation was evaluated on the target region using the T7E1 enzyme. Several single cell colonies were isolated to investigate the mutation and sequencing for mutation positive colonies was performed. One colony with high proliferative activity and heterozygotic mutation (−7bp deletion) was selected and used as the donor cell for somatic cell nuclear transfer. The donor cell was microinjected into an enucleated oocyte. Reconstructed oocytes were fused by electrical pulse, activated by chemical stimulation, and cultured in chemically defined media for 7 days at 5% CO2 and 5% O2. Data were subjected to ANOVA and least significant different test to determine differences between experimental groups by using SAS software (SAS Institute Inc., Cary, NC, USA). Statistical significance was determined when the P-value was less than 0.05. Blastocyst formation rate (13.5±7.6%) from the mutated cell line was significantly lower than wild type cells (43.9±12.1%). Moreover, total cell number (115.4±13.3) of the blastocysts from knockout cells was significantly decreased compared with the control group (143.6±22.5). Some randomly selected blastocysts derived from mutated cells were subjected to mutation assay to confirm deletion of p53. As expected, all the samples were positive to mutation. These data did not support our hypothesis that p53 disruption could improve blastocyst formation competence of bovine cloned embryos. In the future, the reason for impaired embryonic development of clones of p53 deleted somatic cells will be further investigated. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.

A large number of oocytes and adequate recipient conditions are required to produce genetically modified rats by embryo transfer. In this study, we investigated the effect of a synthetic gonadotropin-releasing hormone (GnRH) agonist, buserelin, on (1) superovulatory response in donor rats, and (2) oestrous cycle synchrony in recipient female Sprague Dawley rats at 8 weeks of age. First, for superovulation, donors were divided into 2 groups: control and buserelin injection group (42 μg mL−1). Superovulation was performed in both groups by injection of pregnant mare’s serum gonadotropin (PMSG, 150 IU) at Day −3 and human chorionic gonadotropin (hCG, 150 IU) at Day −1 (Day 0 = oocyte collection). In addition, the buserelin group was given 42 μg mL−1 buserelin on Day −4. Oviduct and ampulla were removed surgically to collect the oocytes. As results, more ovulations were detected in the 42 μg mL−1 buserelin group (6.18 ± 1.10 oocyte/rat, n = 240) than in the control group (5.06 ± 0.82 oocyte/rat; n = 124; t-test P = 0.038). Second, 2 groups were set up to investigate the effect of administration of buserelin on synchronizing the oestrus cycle of recipients. Because the oestrous cycle of the rat was 4 to 5 days, we examined the difference of vaginal impedance between the 2 groups on Day 4 after injection and checked whether there was a significant difference in the synchronization of the oestrous cycle. An impedance value >3.0 kΩ was considered as an indication for proestrus stage; therefore, we analysed the data >3.0 kΩ in both groups. In results, the incidence of proestrus was significantly increased in buserelin injection group (n = 128) than in the control group (n = 70). In addition, there was a significant difference in mean impedance in the 2 groups (buserelin injection group: 4.25 ± 3.79 kΩ; control group: 1.43 ± 1.20 kΩ; P

By subtraction screening methods, we identified a novel mouse and its counterpart, human MOPT gene. The mouse and human MOT gene is localized on mouse chromosome 17E3 and human chromosome 2p22, and spans approximately 7 kb. Analysis of the mouse Mopt sequence revealed the existence of an ORF of 240 bp encoding a putative protein of 79aa amino acids. The predicted protein has a theoretical molecular mass of 8.89 kDa and a calculated isoelectric point of 5.82. The protein was unique; it did not show any similarities with other known protein except for Morn motif domain. Real-time reverse transcriptase polymerase chain reaction and Northern blot analysis revealed that mouse Mopt transcripts are highly and specifically expressed in adult testis and skeletal muscle. In situ hybridization and immunohistochemistry studies showed that mouse Mopt transcript and protein was confined mainly to round and elongated spermatids, except for a few individual dispersed spermatocytes, and increases in abundance in subsequent stages. To characterize Mopt functions in spermiogenesis, we examined Mopt protein distribution in late spermiogenesis by using immunogold electron microscopy: Mopt protein first appeared in the proacrosomic vesicles of the early Golgi phase spermatids. In the final step of spermiogenesis, Mopt expression was translocated from the head cap of an elongated spermatid to the nucleus of mature spermatozoa. However, no other testicular cell types, including somatic cells, spermatogenic cells, and residual cytoplasm that ultimately is engulfed as residual bodies into Sertoli cells, were found to bear Mopt-positive staining. This observation suggested that Mopt may play an important role in dynamic regulation of acrosome biogenesis during late spermiogenesis and an as yet uncharacterized role in oocyte activation during capacitation, after fertilization, or both.