Michiko Nakai

Production of good-quality porcine blastocysts by in vitro fertilization of follicular oocytes vitrified at the germinal vesicle stage

We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro-matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro-matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.

Pre-treatment of sperm reduces success of ICSI in the pig

In pigs, although ICSI is a feasible fertilization technique, its efficiency is low. In general, injected pig sperm are insufficient to induce oocyte activation and embryonic development. Pretreatments for disrupting sperm membranes have been applied to improve the fertility of ICSI oocytes; however, we hypothesize that such pretreatment(s) may reduce the ability of the sperm to induce oocyte activation. We first evaluated the effects of sperm pretreatments (sonication (SO) to isolate the sperm heads from the tails, Triton X-100 (TX), and three cycles of repeated freezing/thawing (3×-FT) for disrupting sperm membranes) on the rate of pronucleus (PN) formation after ICSI. We found that oocytes injected with control (whole) sperm had higher rates of PN formation than those obtained after subjecting the sperm to SO, TX, and 3×-FT. The amounts of phospholipase Cζ (PLCζ), which is thought to be the oocyte-activating factor in mammalian sperm, in sperm treated by each method was significantly lower than that in whole untreated sperm. Furthermore, using immunofluorescence, it was found that in pig sperm, PLCζ was localized to both the post-acrosomal region and the tail area. Thus we demonstrated for the first time that sperm pretreatment leads to a reduction of oocyte-activating capacity. Our data also show that in addition to its expected localization to the sperm head, PLCζ is also localized in the tail of pig sperm, thus raising the possibility that injection of whole sperm may be required to attain successful activation in pigs.

Selected aspects of advanced porcine reproductive technology.

In vitro fertilization (IVF) of in vitro matured (IVM) oocytes in pigs has become the most popular method of studying gametogenesis and embryogenesis in this species. Furthermore, because of recent advances in in vitro culture (IVC) of IVM-IVF embryos, in vitro production (IVP) of embryos now enables us to generate viable embryos as successfully as for in vivo-derived embryos and with less cost and in less time. These technologies contribute not only to developments in reproductive physiology and agriculture but also to the conservation of porcine genetic resources and the production of cloned or genetically modified pigs. However, in IVP, there still remains the problem of abnormal ploidy, which is caused by performing procedures under non-physiological conditions. In recent years, unique technologies such as intracytoplasmic sperm injection (ICSI) or xenografting of gonadal tissue into immunodeficient experimental animals have been developed to help conserve gamete resources. These technologies combined with IVP are expected to be useful for the conservation of gametes from important genetic resources. Here, we discuss the developmental ability and normality of porcine IVP embryos and also the utilization of ICSI and xenografting in advancing biotechnology in pigs.

Generation of porcine diploid blastocysts after injection of spermatozoa grown in nude mice

It is anticipated that the utilization of spermatogonia through testicular xenografting will open new avenues for the conservation of male gametes. With the aim of establishing this new technique for genetic preservation of pigs, we used it in combination with intracytoplasmic sperm injection (ICSI). Testicular tissues derived from neonatal piglets, which contained seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 125 and 192 d after xenografting, sperm (morphologically similar to epididymal sperm) were recovered from 41 of the 65 host mice (63.1%). Testicular spermatozoa from adult boars were used as a positive control. A single spermatozoon was injected into an in vitro matured porcine oocyte, and the oocytes were electro-stimulated and cultured (graft-ICSI and testis-ICSI, respectively). Blastocyst rates in both ICSI groups (24.9% and 37.4%, respectively) were higher (P<0.05) than those without the injection procedure (parthenogenetic; 12.7%) and after injection of a small amount of injection buffer (sham; 13.0%). Rates of diploid blastocysts in both graft-ICSI and testis-ICSI groups (48.9% and 60.6%) were higher (P<0.05) than those in the parthenogenetic and sham groups (13.5% and 28.0%). Therefore, we demonstrated that porcine oocytes injected with xenogeneic sperm have in vitro developmental ability to the blastocyst stage.

Nuclear Replacement of In Vitro-Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method

The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2-32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0-15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.

Treatment with protein kinase C activator is effective for improvement of male pronucleus formation and further embryonic development of sperm-injected oocytes in pigs

To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm-injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 μM) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-μM PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 μM, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a combination of electrical stimulation and PMA is a fairly effective artificial protocol for promoting 2PB+2PN and embryonic development in sperm-injected porcine oocytes.

Lack of calcium oscillation causes failure of oocyte activation after intracytoplasmic sperm injection in pigs

In pigs, the efficiency of embryo production after intracytoplasmic sperm injection (ICSI) is still low because of frequent failure of normal fertilization, which involves formation of two polar bodies and two pronuclei. To clarify the reasons for this, we hypothesized that ICSI does not properly trigger sperm-induced fertilization events, especially intracellular Ca2+ signaling, also known as Ca2+ oscillation. We also suspected that the use of in vitro-matured oocytes might negatively affect fertilization events and embryonic development of sperm-injected oocytes. Therefore, we compared the patterns of Ca2+ oscillation, the efficiency of oocyte activation and normal fertilization, and embryo development to the blastocyst stage among in vivo- or in vitro-matured oocytes after ICSI or in vitro fertilization (IVF). Unexpectedly, we found that the pattern of Ca2+ oscillation, such as the frequency and amplitude of Ca2+ rises, in oocytes after ICSI was similar to that in oocytes after IVF, irrespective of the oocyte source. However, half of the oocytes failed to become activated after ICSI and showed no Ca2+ oscillation. Moreover, the embryonic development of normal fertilized oocytes was reduced when in vitro-matured oocytes were used, irrespective of the fertilization method employed. These findings suggest that low embryo production efficiency after ICSI is attributable mainly to poor developmental ability of in vitro-matured oocytes and a lack of Ca2+ oscillation, rather than the pattern of oscillation.

Efficient pig ICSI using Percoll-selected spermatozoa; evidence for the essential role of phospholipase C-ζ in ICSI success

In pigs, the damaged sperm membrane leads to leakage of phospholipase C-ζ (PLCζ), which has been identified as a sperm factor, and a reduction of oocyte-activating ability. In this study, we investigated whether sperm selected by Percoll gradient centrifugation (Percoll) have sufficient PLCζ, and whether the efficiency of fertilization and blastocyst formation after intracytoplasmic sperm injection (ICSI) using Percoll-selected sperm can be improved. Percoll-selected sperm (Percoll group) or sperm without Percoll selection (Control group) were used. A proportion of the oocytes injected with control sperm were subjected to electrical stimulation at 1 h after ICSI (Cont + ES group). It was found that the Percoll group showed a large amount of PLCζ in comparison with the Control group. Furthermore, application of Percoll-selected sperm for ICSI increased the efficiency of fertilization and embryo development. Thus, these results indicate the Percoll-selected sperm have sufficient PLCζ and high oocyte-activating ability after ICSI in pigs.

Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation

TP53 (which encodes p53) is one of the most frequently mutated genes in cancers. In this study, we generated TP53-mutant pigs by gene editing via electroporation of the Cas9 protein (GEEP), a process that involves introducing the Cas9 protein and single-guide RNA (sgRNA) targeting exon 3 and intron 4 of TP53 into in vitro-fertilized zygotes. Zygotes modified by the sgRNAs were transferred to recipients, two of which gave birth to a total of 11 piglets. Of those 11 piglets, 9 survived. Molecular genetic analysis confirmed that 6 of 9 live piglets carried mutations in TP53, including 2 piglets with no wild-type (WT) sequences and 4 genetically mosaic piglets with WT sequences. One mosaic piglet had 142 and 151 bp deletions caused by a combination of the two sgRNAs. These piglets were continually monitored for 16 months and three of the genome-edited pigs (50%) exhibited various tumor phenotypes that we presumed were caused by TP53 mutations. Two mutant pigs with no WT sequences developed mandibular osteosarcoma and nephroblastoma. The mosaic pig with a deletion between targeting sites of two sgRNAs exhibited malignant fibrous histiocytoma. Tumor phenotypes of TP53 mosaic mutant pigs have not been previously reported. Our results indicated that the mutations caused by gene editing successfully induced tumor phenotypes in both TP53 mosaic- and bi-allelic mutant pigs.

Establishment of a strain of haemophilia-A pigs by xenografting of foetal testicular tissue from neonatally moribund cloned pigs

Grafting of testicular tissue into immunodeficient mice makes it possible to obtain functional sperm from immature donor animals that cannot be used for reproduction. We have developed a porcine model of human haemophilia A (haemophilia-A pigs) by nuclear transfer cloning from foetal fibroblasts after disruption of the X-linked coagulation factor VIII (F8) gene. Despite having a recessive condition, female F8+/− cloned pigs died of severe bleeding at an early age, as was the case for male F8−/Y cloned pigs, thus making it impossible to obtain progeny. In this study, therefore, we produced sperm from F8−/Y cloned pigs by grafting their foetal testicular tissue into nude mice. Two F8+/− female pigs were generated from oocytes injected with xenogeneic sperm. Unlike the F8+/− cloned pigs, they remained asymptomatic, and delivered five F8−/Y and four F8+/− pigs after being crossed with wild-type boars. The descendant F8−/Y pigs conserved the haemophilia phenotype. Thus, the present F8+/− pigs show resolution of the phenotypic abnormality, and will facilitate production of F8−/Y pigs as founders of a strain of haemophilia-A pigs for the development of new therapeutics for haemophilia A. This strategy will be applicable to other genetically modified pigs.