Humdai Park

Aberrant Allocations of Inner Cell Mass and Trophectoderm Cells in Bovine Nuclear Transfer Blastocysts

Abstract Abortions of nuclear transfer (NT) embryos are mainly due to insufficient placentation. We hypothesized that the primary cause might be the aberrant allocations of two different cell lineages of the blastocyst stage embryos, the inner cell mass (ICM) and the trophectoderm (TE) cells. The potential for development of NT embryos to blastocysts was similar to that for in vitro fertilized (IVF) embryos. No difference in the total cell number was detected between NT and IVF blastocysts, but both types of embryos had fewer total cells than did in vivo-derived embryos (P < 0.05). The NT blastocysts showed a higher ratio of ICM:total cells than did IVF or in vivo-derived embryos (P < 0.05). Individual blastocysts were assigned to four subgroups (I: <20%, II: 20–40%, III: 40–60%, IV: >60%) according to the ratio of ICM:total cells. Most NT blastocysts were placed in groups III and IV, whereas most IVF and in vivo-derived blastocysts were distributed in group II. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocations of NT embryos to the ICM and TE cells during early development.

Iloprost, a prostacyclin analogue, stimulates meiotic maturation and early embryonic development in pigs

Oviduct fluid contains various cytokines and growth factors that enhance the embryo development during the preimplantation period. In hatched embryos, prostacyclin (PGI(2)) improves implantation, but its role during oocyte maturation and early embryo development remains contentious. Therefore, in the present study, we examined the effects of a PGI(2) analogue (iloprost) on meiotic maturation and early embryonic development in pigs, as well on the structural integrity, mitochondrial membrane potential and apoptosis in blastocysts. First, meiotic maturation in pig oocytes was examined in the presence of increasing concentrations of iloprost (1, 5 and 10 muM). After IVM, a higher proportion of iloprost-treated compared with untreated oocytes was in MII (90.0% v. 65.7%, respectively; P < 0.05). In addition, protein kinase A activity increased in iloprost-treated oocytes, indicating increased intracellular cAMP concentrations. After 22 h iloprost treatment (44 h total incubation time), western blotting demonstrated increased expression of extracellular signal-regulated kinase (ERK) 1/2, phosphorylated (p-) ERK1/2, cAMP response element-binding protein (CREB), p-CREB and cyclo-oxygenase-2, indicating activation of the mitogen-activated protein kinase and PGI(2) pathways. In addition, the frequency of polyspermy decreased in iloprost-treated oocytes (19.9%) compared with control (35.8%), whereas the rate of blastocyst formation increased (P < 0.05). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) showed that the number of nuclei containing fragmented DNA at the blastocyst stage decreased in the iloprost-treated group compared with control (1.2% v. 3.6%, respectively). In conclusion, iloprost appears to play a direct role in porcine oocyte maturation by enhancing blastocyst structure and survival.

Mdivi-1, mitochondrial fission inhibitor, impairs developmental competence and mitochondrial function of embryos and cells in pigs

Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 μM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 μM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 μM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 μM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 μM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells.

Forced Collapse of the Blastocoel Cavity Improves Developmental Potential in Cryopreserved Bovine Blastocysts by Slow‐Rate Freezing and Vitrification

This study was conducted to evaluate the effectiveness of forced collapse of the blastocoel before slow-rate freezing and vitrification of bovine blastocysts. Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production using the embryo transfer technique. However, the low efficiency of frozen-thawed embryos survival and further development is a crucial problem. In this study, bovine in vitro and in vivo blastocysts were slow-rate frozen and vitrified after forced blastocoele collapse (FBC) of the blastocyst cavity by puncturing the blastocoele with a pulled Pasteur pipet. Differences in the developmental potential of frozen-thawed blastocysts derived from FBC and non-FBC groups were found in both slow-rate freezing and vitrification. Furthermore, we found that the total cell number of blastocysts in FBC groups was increased and the index of apoptosis in FBC groups was decreased. Consistent with these results, real-time RT-PCR analysis data showed that expression of the anti-apoptotic Bcl-XL gene was significantly increased by FBC groups, whereas expression of the pro-apoptotic Bax gene was significantly decreased by FBC groups. Our results also showed that pregnancy outcomes in both slow-rate frozen and vitrified bovine in vivo blastocysts could be improved by reducing the fluid content after FBC of the blastocyst cavity. Therefore, we suggest that FBC of the blastocyst cavity with a pulled Pasteur pipet is an effective pre-treatment technique for both slow-rate freezing and vitrification of bovine blastocysts.

Identification and characterization of a novel mouse and human MOPT gene containing MORN-motif protein in testis.

A novel testis-derived membrane occupation and recognition nexus (MORN)-motif protein was identified in mouse testis (MOPT) by subtraction screening methods and found to be localized on chromosome 17E3, spanning approximately 7kb. Sequence analysis showed that MOPT contains 669 base pair nucleotides of open reading frame and the corresponding 79 amino acids. The protein is predicted to have theoretical molecular mass of 9000 Da and an expected isoelectric point of 5.8 and seems to have unique sequences except for MORN-motif domain. The transcript of MOPT is highly and specifically expressed in adult testis as well as skeletal muscle. Moreover, MOPT transcript and protein are confined mainly to round and elongated spermatids, except for a few individual dispersed spermatocytes, and increase in abundance at subsequent stages. MOPT first appeared in the proacrosomic vesicles of the early Golgi phase spermatids and was translocated from the head cap of elongated spermatid to the nucleus of mature spermatozoa at the final stage of spermiogenesis. Our study suggests that MOPT may play an important role in dynamic regulation of acrosome biogenesis during late spermiogenesis.

ICSI significantly improved the pregnancy rate of patients with a high sperm DNA fragmentation index

Objective Correlations between semen parameters and sperm DNA fragmentation index (DFI) were investigated to identify characteristics of sperm without DNA damage that could be used in selecting sperm for intracytoplasmic sperm injection (ICSI). Pregnancy outcomes were compared to determine whether in vitro fertilization (IVF) or ICSI is a better choice for patients who have sperm with a high-DFI. Methods Semen analysis was carried out in 388 patients who visited our IVF center for the first time to investigate correlations between sperm DFI and semen parameters. In addition, 1,102 IVF cycles in 867 patients were carried out in the present study; 921 cycles in the low-DFI group (DFI <30%) and 181 cycles in the high-DFI group (DFI ≥30%). Both the low- and high-DFI groups were subdivided into IVF and ICSI cycle groups. Results Sperm DFI showed significant inverse correlations with sperm motility (r=−0.435, p<0.001) and morphology (r=−0.153, p<0.05). Sperm DFI also showed significant correlations with rapid motility (r=−0.436, p<0.001), and the kinetic parameters of average-path velocity (r=−0.403) and linearity (r=−0.412). Although there was no significant difference in the pregnancy rates between IVF (48.6%) and ICSI (44.8%) in the low-DFI group, the pregnancy rate of ICSI cycles (44.8%, p<0.05) was significantly higher than IVF cycles (25.0%) in the high-DFI group. No significant difference was observed in the abortion rates between the low-DFI (52 of 921, 5.6%) and high-DFI groups (7 of 181, 3.8%). Conclusion ICSI is a better choice than IVF for improving the pregnancy outcomes of patients who have sperm with a high DFI.

Early fragment removal on in vitro fertilization day 2 significantly improves the subsequent development and clinical outcomes of fragmented human embryos

Objective To determine whether fragment removal on in vitro fertilization (IVF) day 2 improved the subsequent development and pregnancy outcomes of fragmented embryos compared to similar-grade embryos without fragment removal. Methods This study was a retrospective analysis involving 191 IVF cycles in which all embryos had over 10% fragmentation (grade 3 or 4) on day 2 of the IVF-embryo transfer cycle from March 2015 to December 2017. IVF cycles were divided into the fragment removal group (n=87) and the no fragment removal group (n=104) as a control cohort. Before fragment removal, embryos with fragmentation on day 2 were incubated in Ca2+- and Mg2+-free biopsy medium under paraffin oil for 30 minutes. Microsurgical fragment removal was performed with later-assisted hatching and a handmade suction micropipette that had an outer diameter of 30 µm. Results There were no significant differences in the characteristics of the patients between the control and the fragment removal groups. After fragment removal and subsequent in vitro culture for 24 hours, the number of blastomeres (7.1±1.7 vs. 6.9±1.6) was comparable between the transferred embryos in the two groups, but the morphological grade of the embryos in the fragment removal group (1.9±0.7) was significantly higher than that of the control group (3.1±0.5, p<0.01). The clinical pregnancy (43.7%) and implantation rates (25.8%) in the fragment removal group were significantly higher than those in the control group (28.8% and 14.0%, respectively; p<0.05). Conclusion Early fragment removal on day 2 significantly improved the subsequent development and pregnancy outcomes of fragmented embryos.