Kazuhiro Kikuchi

Effect of trehalose on DNA integrity of freeze-dried boar sperm, fertilization, and embryo development after intracytoplasmic sperm injection.

Freeze-drying (FD) medium containing ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) is reported to be beneficial for maintenance of sperm DNA integrity after FD. Recently, trehalose has also been reported to have notable ability to stabilize the protein structure and biomembranes of sperm in a dry state. In this study, we examined the effect of a combination of EGTA and different concentrations of trehalose in FD medium on sperm DNA integrity and the inxa0vitro development of IVM porcine oocytes after intracytoplasmic sperm injection (ICSI) using freeze-dried boar sperm. Ejaculated sperm from a boar were suspended in basic FD medium supplemented with 0, 3.75, 7.5, 15, 30, 60, or 90 mM trehalose and freeze-dried. After rehydration, the sperm in all groups were subjected to DNA damage detection using a Halomax kit. It was found that the level of DNA damage in 15-mM group was significantly lower than that in 0-mM group, and no difference was observed between the 15-, 7.5-, and 3.75-mM groups. Moreover, there were no significant differences in the DNA damage level among 0, 3.75 mM, and other groups treated with trehalose. When freeze-dried sperm were used for ICSI, the fertilization rates and blastocyst formation rates (observed at 10 hours and 6 days of IVC after ICSI, respectively) in the 7.5- and 15-mM groups were not different from those in 0-mM group. These results suggest that FD medium supplemented with trehalose at appropriate concentrations improves sperm DNA integrity, but does not improve fertilization and preimplantation embryo development of IVM oocytes following ICSI.

Generation of live piglets from cryopreserved oocytes for the first time using a defined system for in vitro embryo production.

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42°C during warming prevented temperature drops in a medium below 34.0°C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38°C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0u200a=u200aIVF) of the surviving oocytes after warming at 38°C and 42°C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.

Generation of live piglets for the first time using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice.

Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230−350 (day 0u200a=u200a grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230−350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.

Comparison of cytoskeletal integrity, fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.

Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

Abstract In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.

Xenografting of gonadal tissues into mice as a possible method for conservation and utilization of porcine genetic resources

In vitro production of embryos, including in vitro maturation, fertilization of oocytes and their subsequent culture to the embryo stage, has become the most popular method of studying gametogenesis and embryogenesis in pigs. As well as their utility for basic studies, these procedures now enable us to generate viable embryos and offspring as a means of conserving genetic resources and rare animal breeds. Recently, more advanced technologies such as xenografting of gonadal (testicular and ovarian) tissues into immunodeficient experimental animals have been developed. In combination with in vitro embryo production techniques, this approach may provide many benefits. We have been carrying out studies to acquire basic information about the application of this method to porcine species, and to improve the existing techniques. Recently, we obtained oocytes from ovarian tissue xenografted and grown in nude mice that had the capacity to be fertilized and the ability to develop into early-stage embryos. We also obtained spermatozoa from the xenografted testicular tissues and injected them intracytoplasmically into in vitro-matured oocytes to produce piglets. Here we discuss the further possibilities of conservation and utilization of porcine gonadal tissue by xenografting into immunodeficient mice.

Normal reproductive development of pigs produced using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice

Xenografting of immature testicular tissue combined with cryopreservation can preserve and use genetic information of prepubertal animals. For establishment of this new approach, it is essential to clarify whether offspring derived from sperm grown in host mice harboring cryopreserved xenografts show normal reproductive development. This study examined serum profiles of gonadal hormones during sexual maturation in pigs generated by intracytoplasmic sperm injection using sperm derived from cryopreserved xenografts (CryoXeno pigs; three males and three females). We also assessed the reproductive abilities of the male CryoXeno pigs by mating them with conventionally produced (conventional) pigs, and by examining the in vitro fertilizing ability of their sperm. For female CryoXeno pigs, reproductive ability was evaluated by artificial insemination with semen from a conventional boar. During the growth of male CryoXeno pigs, the serum concentrations of inhibin and testosterone showed similar changes (P > 0.17) to those in conventional pigs (n = 4). Histologic analyses of the testes revealed no differences (P > 0.2) in the growth and differentiation of seminiferous tubules between CryoXeno and conventional pigs. Three conventional sows delivered 13.0 ± 1.0 (mean ± standard error of the mean) live piglets after being mated with the three CryoXeno males. Sperm obtained from all CryoXeno pigs had the ability to penetrate oocytes, and these fertilized oocytes reached the blastocyst stage in vitro. During the growth of female CryoXeno pigs, the serum inhibin profile was similar (P > 0.17) to that observed in conventional pigs (n = 5). The first rise in serum progesterone concentration to more than 2 ng/mL was noted at 32.0 ± 2.3 weeks of age in the CryoXeno pigs and at 32.0 ± 3.3 weeks in the conventional pigs, suggesting that both pigs reached puberty at a similar age. After puberty, female CryoXeno pigs farrowed 8.3 ± 1.7 (mean ± standard error of the mean; n = 3) live piglets after artificial insemination with semen from a conventional boar. In conclusion, these findings demonstrate that both male and female CryoXeno pigs have normal reproductive abilities.

Contribution of in vitro systems to preservation and utilization of porcine genetic resources

Historically, the conservation or preservation of mammalian genetic resources, especially farm animals, has been conducted under in situ conditions by maintaining living individuals as livestock. However, systems for laboratory inxa0vitro embryo production using gametes such as spermatozoa and oocytes are now available, in addition to ex situ preservation methods for mammalian genetic resources. One of these methods is the cryopreservation of gametes, embryos, and gonadal tissues. In pigs, freezing of sperm is the most reliable and well-established method for this purpose. On the other hand, cryopreservation of female gametes (oocytes) and gonadal tissues-usually by vitrification-has been associated with very low efficacies. Recently, in our laboratory, some research themes related to this issue have been pursued. We have been focusing on advances in porcine inxa0vitro embryo production systems, and here, we introduce recent data on the vitrification of porcine immature oocytes and gonadal tissues followed by their xenografting into host mice to produce gametes.

Expression of DNA repair genes in porcine oocytes before and after fertilization by ICSI using freeze-dried sperm.

Boar sperm freeze-dried with trehalose showed a protective effect against sperm DNA fragmentation. However, normal fertilization and embryonic development were not improved. Damaged sperm may activate maternal DNA repair genes when injected into oocytes. Therefore, we investigated the expression profile of some DNA repair genes in porcine oocytes after intra-cytoplasmic sperm injection. First, the expression levels of MGMT, UDG, XPC, MSH2, XRCC6 and RAD51 genes that are concerned with different types of DNA repair were examined in in vitro mature (IVM) oocytes injected with ejaculated sperm, or freeze-dried sperm with or without trehalose. Quantitative reverse transcription polymerase chain reaction revealed that expression of six DNA repair genes in the oocytes at 4u2009h after injection did not differ among the four groups. Next, we investigated the gene expression levels of these genes at different stages of maturation. The relative expression levels of UDG and XPC were significantly up-regulated in mature oocytes compared with earlier stages. Furthermore, there was an increased tendency in relative expression of MSH2 and RAD51. These results suggested two possible mechanisms that messenger RNA of DNA repair genes are either accumulated during IVM to be ready for fertilization or increased expression levels of DNA repair genes in oocytes caused by suboptimal IVM conditions.

Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations

Abstract Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.