Effect of nucleocytoplasmic ratio on the in vitro porcine embryo development after in vitro fertilization or parthenogenetic activation.

This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

Combined refinements to somatic cell nuclear transfer methods improve porcine embryo development.

The discovery of how to utilize CRISPR (clustered, regularly interspaced, short, palindromic repeats)-Cas (CRISPR-associated) systems for genome modification has accelerated development of the field of genome editing, especially in large animals such as pigs. The low efficiency of somatic cell nuclear transfer (SCNT) is now becoming a major obstacle in the production of genome-edited animals via cell-mediated approaches and improving efficacy of this technique is crucial. In this study, we propose a few simple modifications to a zona-free SCNT protocol that are effective to produce numerous high-quality blastocysts. To refine the SCNT protocol we modified the following steps/factors: 1) culture medium for SCNT embryos, 2) chemical treatment to prevent precocious activation of the manipulated/reconstructed oocytes and 3) donor cell serum starvation treatment. Although changes in each of these steps only resulted in small improvements, the combination of all modifications altogether significantly enhanced developmental competence of SCNT embryos. Our modified method yielded approximately three times greater blastocyst formation rates. Moreover, resulting blastocysts had roughly twice as many cells as compared to blastocysts produced by the conventional SCNT method. With these significant in vitro improvements, our refined SCNT method is potentially suited for use in the production of genome edited pigs.

Improvement of the developmental competence of porcine oocytes collected from early antral follicles by cytoplast fusion.

In the present study, we propose an alternative technique called cytoplast fusion to improve the maturation rate and developmental competence of growing oocytes collected from early antral follicles in pigs. We examined whether the fusion of a growing oocyte with the cytoplast from a fully-grown oocyte (CFR group) could better promote maturation and developmental competence of the growing oocyte compared to germinal vesicle (GV) transfer (GVTR group). After 44 h of in vitro maturation (IVM), most growing oocytes (GR group) were still arrested at the GV stage (64.0 ± 5.1%); this number was significantly higher (P < 0.01) than that of the other groups. No matured oocyte was observed in the GR group. The maturation rate of GVTR oocytes was significantly improved (18.8 ± 3.5%) compared with that of growing oocytes. The proportion of oocytes that reached the metaphase-II (M-II) stage in the CFR group (37.8 ± 2.0%) was significantly higher (P < 0.05) than that in the GVTR group, although still lower than that in the control group (75.2 ± 4.4%). No blastocyst was derived from growing oocytes. Among in vitro fertilized GVTR oocytes, 3.0 ± 1.9% developed into blastocysts; however, this percentage showed an insignificant increase compared with the GR group. On the other hand, the percentage of CFR embryos that developed into blastocysts (12.0 ± 4.3%) was significantly higher than that of GR embryos (0.0%), although still lower than that of control embryos (27.0 ± 5.5%). Total cell number in blastocysts in the GVTR group (23.3 ± 6.9) was significantly lower (P < 0.05) than that in the control group (50.4 ± 5.0). Meanwhile, the total cell number in blastocysts derived from CFR oocytes (36.3 ± 4.8) was comparable to that of the control group. In summary, cytoplast fusion significantly improves maturation rate and developmental competence of growing oocytes compared with GV transfer.

Optimization of cryoprotectant treatment for the vitrification of immature cumulus-enclosed porcine oocytes: comparison of sugars, combinations of permeating cryoprotectants and equilibration regimens.

Our aim was to optimize the cryoprotectant treatment for the preservation of immature porcine cumulus-oocyte complexes (COCs) by solid surface vitrification. In each experiment, the vitrification solution consisted of 50 mg/ml polyvinyl pyrrolidone, 0.3 M of the actual sugar and in total 35% (v/v) of the actual permeating cryoprotectant (pCPA) combination. After warming, the COCs were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, trehalose and sucrose were equally effective during vitrification and warming in terms of facilitating oocyte survival and subsequent embryo development. In Experiment 2, when equilibration was performed at 38.5 C in a total of 4% (v/v) pCPA for 15 min, the combination of ethylene glycol and propylene glycol (EG + PG = 1:1) was superior to EG and dimethyl sulfoxide (EG + DMSO = 1:1) in terms of oocyte survival after vitrification and the quality of resultant blastocysts. In Experiment 3, equilibration in 4% (v/v) pCPA for 15 min before vitrification was superior to that in 15% (v/v) CPA for 5 min for achievement of high survival rates irrespective of the pCPA combination used. In Experiment 4, when equilibration was performed in 4% EG + PG for 5 min, 15 min or 25 min, there was no difference in oocyte survival and subsequent embryo development after vitrification and warming; however, the developmental competence of cleaved embryos was tendentiously reduced when equilibration was performed for 25 min. In conclusion, trehalose and sucrose were equally effective in facilitating vitrification, and the optimum pCPA treatment was 5-15 min equilibration in 4% (v/v) of EG + PG followed by vitrification in 35% (v/v) EG + PG.

Fertilization ability of porcine oocytes reconstructed from ooplasmic fragments produced and characterized after serial centrifugations.

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.

Excess polyspermy reduces the ability of porcine oocytes to promote male pronuclear formation after in vitro fertilization.

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = -0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = -0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.

Embryo production by intracytoplasmic injection of sperm retrieved from neonatal testicular tissue of Agu pigs after cryopreservation and grafting into nude mice.

The Agu is the only indigenous pig breed in Japan but its population is very small. In order to estimate the efficacy of testicular xenografting for the conservation of Agu pigs, we investigated whether neonatal testicular fragments would acquire the capacity to produce sperm after they had been cryopreserved and grafted into nude mice. Although on day 180 (day 0 = xenografting), grafts showed a low proportion of seminiferous tubule cross-sections containing sperm (0.1 ± 0.1%, mean ± SEM for four mice), the proportion reached 36.9 ± 16.7% (n = 4 mice) by day 240. When single sperm obtained on day 240 was injected into individual porcine oocytes, 28.2% of the oocytes were found to contain one male and one female pronuclei with the second polar body. Moreover, the blastocyst formation rate after injection of the xenogeneic sperm was 28.4%, whereas that in the absence of sperm injection (attributable to parthenogenesis) was 13.3%. These findings suggest that more than half of the blastocysts resulted from fertilization. Thus, testicular xenografting could assist the conservation of Agu pigs by salvaging germ cells present in neonatal testes even after cryopreservation.

Pluripotency-associated genes reposition during early embryonic developmental stages in pigs.

We examined the allelic expression and positioning of two pluripotency-associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4- and 8-cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi-allelically increased from 45% at the 4-cell stage to 60% at the 8-cell stage. Moreover, in 8-cell embryos, SOX2 was expressed bi-allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4- to 8-cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4- or 8-cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency-associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.

Selection based on morphological features of porcine embryos produced by in vitro fertilization: Timing of early cleavages and the effect of polyspermy.

The aim of this study was to examine whether a morphological approach is efficient for selecting high-quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen-thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105  sperms/ml). Under conditions of moderate polyspermy, 4-cell embryos selected at 48 hr after IVF (single selection) and 8-cell embryos selected at 79 hr after IVF from the collected 4-cell embryos (double selection) showed high developmental competence. Likewise, 4- and 8-cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4- and 8-cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4- and 8-cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.

Developmental ability of oocytes retrieved from Meishan neonatal ovarian tissue grafted into nude mice.

Ovarian xenografting makes it possible to obtain oocytes with fertilization ability from immature pigs of Western breeds. In this study, we applied these methods to the Meishan, an indigenous Chinese pig breed, and investigated the developmental competence of oocytes grown in their neonatal tissue after grafting into nude mice. First, mice harboring neonatal ovarian tissue were infused with follicle stimulating hormone (FSH) (62.5 U/ml) for 13 days starting at 10, 30, and 60 days after vaginal opening (D10-, D30-, and D60-FSH groups, respectively). Development of antral follicles and their oocytes was most enhanced in the D60-FSH group. For the next step, we examined the in vitro maturation ability of the oocytes recovered from host mice after infusion with FSH at a dose of 62.5 U/ml or 125 U/ml (FSH-62.5 or -125 group) for 13 days starting at 60 days after vaginal opening. Many more oocytes with maturation ability were obtained from the FSH-125 group. The FSH-125 mature oocytes were fertilized in vitro, as shown by formation of male and female pronuclei, but did not reach the blastocyst stage. These results indicate that Meishan neonatal ovaries are able to produce oocytes with fertilization ability after being grafted into nude mice.

Embryo production by intracytoplasmic injection of sperm retrieved from Meishan neonatal testicular tissue cryopreserved and grafted into nude mice.

Testicular xenografting, combined with cryopreservation can assist conservation of the genetic diversity of indigenous pigs by salvaging germ cells from their neonatal testes. Using Meishan male piglets as an example, we examined whether testicular tissue would acquire the ability to produce sperm after cryopreservation and grafting into nude mice (MS group). For comparison, testicular tissue from neonatal Western crossbreed male piglets was used (WC group). Sixty days after xenografting (day 0 = grafting), MS grafts had already developed seminiferous tubules containing sperm, whereas in the WC grafts, sperm first appeared on day 120. The proportion of tubules containing spermatids and sperm was higher in the MS group than in the WC group between days 90 and 120. Moreover, in vitro-matured porcine oocytes injected with a single sperm obtained from the MS group on day 180 developed to the blastocyst stage. The blastocyst formation rate after injection of the xenogeneic sperm was 14.6%, whereas the ratio in the absence of such injection (attributable to parthenogenesis) was 6.7%. Thus, cryopreserved Meishan testicular tissue acquired spermatogenic activity in host mice 60 days earlier than Western crossbreed tissue. Such xenogeneic sperm are likely capable of generating blastocysts in vitro.

Sucrose assists selection of high-quality oocytes in pigs.

We investigated whether high-quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis-shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis-shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis-shapened aged oocytes. In an attempt to find out why high-quality oocytes maintain a round shape whereas poorer oocytes become mis-shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis-shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high-quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis-shapened oocytes.

Production of sperm from porcine fetal testicular tissue after cryopreservation and grafting into nude mice.

A major goal of testicular xenografting is to salvage germ cells from immature animals that cannot be used for reproduction and generate their offspring. In this study, we investigated whether porcine fetal testicular tissue would acquire the ability to produce sperm with full developmental competence after they had been cryopreserved and grafted into nude mice. Testicular fragments from fetuses at 35, 55 and 90 days postartificial insemination (dpi) were vitrified and stored in liquid nitrogen. Immediately after warming, testicular fragments at each fetal stage were transplanted under the back skin of castrated nude mice (Crlj:CD1-Foxn1nu) (35-, 55- and 90-dpi groups, respectively) (day 0 = grafting). Before grafting, the testicular fragments contained seminiferous cords consisting of only gonocytes and Sertoli cells. Histological analyses of the testicular grafts revealed that the differentiation of seminiferous tubules was largely dependent on the time after grafting, and not on donor age. On day 180 in each group, 10-20% of the total number of tubule/cord cross-sections examined had germ cells that had progressed beyond the spermatogonial stage. Fewer than 5% of tubule cross-sections contained elongated spermatids or sperm. Between days 360 and 420, tubule differentiation advanced further, until more than 45% of the tubule cross-sections contained elongated spermatids or sperm. Sperm were recovered for the first time from a single mouse in the 55-dpi group on day 180, although on days 360-420 sperm were recovered from most mice in all of the groups. Serum concentrations of inhibin and testosterone in host mice in all of the groups were higher than those in castrated mice that had received no testicular grafts. Single sperm collected from mice in each group on day 300 or later were injected into individual in vitro-matured oocytes, and these sperm-injected oocytes were transferred to the oviducts of 2 or 3 estrus-synchronized recipient gilts. None of the recipients in any of the groups produced piglets. The present results clearly indicate that porcine fetal testes during the gestational period acquire endocrine and exocrine functions after being cryopreserved and grafted into nude mice. However, the ability of xenogeneic sperm derived from fetal testis to generate piglets was not confirmed in the present study.

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 4 h 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.

Production of Ban miniature pig embryos by in vitro fertilization: a comparative study with Landrace.

Ban is an endangered miniature pig breed in Vietnam. This study aimed to set up an in vitro embryo production (IVP) system for this breed. Ban's epididymal sperm concentration (1240 ± 35 × 10(6) /mL) was lower (P < 0.01) compared with Landrace (4160 ± 42 × 10(6) /mL). However, sperm characteristics before and after freezing in Ban and Landrace were similar. The numbers of follicles with diameter larger than 2 mm per ovary in Ban females treated with equine chorionic gonadotropin and human chorionic gonadotropin (27.1 ± 1.3) were higher (P < 0.05) than those in Landrace (12.9 ± 2.0) and in non-hormone stimulated Ban (no > 2 mm follicles). After in vitro maturation, the percentages of oocytes with expanded cumulus cells and the first polar body (matured oocytes) were not different among Ban, hormone-stimulated Ban and Landrace. The percentages of two-cell embryos and morulae derived from oocytes collected from three sources did not differ. However, the rate of blastocysts derived from oocytes in non-stimulated Ban (4.0 ± 3.8%) was lower (P < 0.05) than that in Landrace (15.3 ± 1.8%). In conclusion, an effective IVP system for good quality embryos in Ban, that is essential for genetic conservation of this breed, was established.

Roles of the zona pellucida and functional exposure of the sperm-egg fusion factor 'IZUMO' during in vitro fertilization in pigs.

The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm-egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP-intact and ZP-free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44-0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti-IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti-IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP-free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.

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 in vitro 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.