Yoshikazu Nagao

EFFECTS OF CUMULUS CELL DENSITY DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

To determine the role of cumulus cells in oocyte maturation, we carried out an investigation on the effects of addition of cumulus cells to the maturation medium on the developmental competence of corona-enclosed oocytes and oocytes denuded from their somatic cells. The addition of cumulus cell (1.6 x 10(6) cells/mL) improved the development of bovine corona-enclosed oocytes, however, addition of a similar number of cumulus cells as cumulus-oocyte-complexes (COCs, cumulus cell density: 4.2 x 10(6) cells/mL) had no effect on the development of oocytes denuded from their somatic cells. To determine if corona-enclosed oocytes can obtain developmental competence without the addition of extra cumulus cells, the effects of cell density during in vitro maturation on the developmental competence were studied. A density of 1.6 to 3.2 x 10(6) cumulus cells/mL was the most effective for in vitro maturation of oocytes with intact gap junctions. The effects of the medium conditioned by COCs on the developmental competence of oocytes was also examined. It was demonstrated that COC-conditioned medium improved the development of bovine oocytes to the blastocyst stage. These data suggest that the developmental competence of bovine oocytes surrounded with corona cells is supported in a cell density-dependent manner in the maturation medium. In addition, the data indicate that cumulus cells benefit bovine oocyte development either by secreting soluble factors which induce developmental competence or by removing an embryo development-suppressive component from the medium.

Effects of oxygen concentration and oviductal epithelial tissue on the development of in vitro matured and fertilized bovine oocytes cultured in protein-free medium

Examination was made of the effects of oxygen concentration and supplementation of bovine oviductal epithelial tissue (BOET) on the development of bovine in vitro matured and fertilized (IVM/IVF) oocytes in a protein-free medium. The IVM/IVF embryos were cultured in protein-free tissue culture medium 199 supplemented with or without BOET under 5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2). We found that blastocyst development without BOET at 5% O2 was the same as that with BOET at 20% O2 (30 vs 33%); BOET suppressed blastocyst development at 5% O2 (4%). Blastocysts cultured without BOET at 5% O2 developed into normal fetuses after transfer to recipient heifers. Examination was also made of oxygen pressure in the medium cultured with or without BOET at 20% O2 or 5% O2 by a blood gas analyzer. Oxygen pressure in the medium cultured with BOET at 20% O2 was lower than that without BOET (111.0+/-13.3 vs 149.2+/-1.3 mmHg). These results indicate that bovine IVM/IVF embryos can develop to the blastocyst stage in a protein-free medium without somatic cell support at low oxygen concentration (5%) and that the beneficial role of BOET for embryonic development may be to reduce oxygen concentration in the medium.

Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide

The influence of equilibration time before vitrification on the viability of vitrified morula- to blastocyst-stage bovine embryos and in vivo viability of vitrified embryos following transfer to recipients were investigated. In experiment 1, the embryos were exposed to an equilibration solution (50% VSED) containing 12.5% v/v ethylene glycol and 12.5% v/v dimethyl sulfoxide in modified Dulbeccos phosphate buffered saline with 4 mg/ml BSA (m-PBS) for 1, 2 and 5 minutes at room temperature (22 to 24 degrees C). The embryos were then placed in 15mul vitrification solution (VSED) consisting of 25% v/v ethylene glycol and 25% v/v dimethyl sulfoxide in m-PBS and were loaded into 0.25 ml plastic straws at room temperature. After 30 seconds, the straws were placed in liquid nitrogen (LN(2)) vapor for 2 minutes, plunged and stored in LN(2). To thaw, the straws were warmed in water at 20 degrees C for 15 seconds and the contents of the straws were expelled into a plastic dish. The embryos were diluted in 0.5 M sucrose + m-PBS for 5 minutes and were cultured in TCM-199 supplemented with bovine oviductal epithelial tissue. Viability of the embryos was assessed by the forming or reforming of the blastocoele after 24 hours of culture. High in vitro survival rates (73 approximately 90%) of vitrified embryos were obtained after 1 and 2 minute equilibrations, but was reduced (P<0.05) after 5 minute equilibration. In Experiment 2, morula- to blastocyst-stage embryos were vitrified after 1 minute equilibration in 50% VSED and 30 seconds of exposure to VSED. The vitrified-warmed embryos were transferred to recipient heifers at 7 days after estrus (1 embryo per recipient). Five (38%) of 13 (40%) of 10 recipients that had received blastocysts were diagnosed as pregnant using ultrasonography 60 days following transfer.

DEVELOPMENTAL CAPACITY OF BOVINE OOCYTES FOLLOWING INHIBITION OF MEIOTIC RESUMPTION BY CYCLOHEXIMIDE OR 6-DIMETHYLAMINOPURINE

This study was conducted to assess the fertilizability and developmental capacity of bovine oocytes which had been maintained in meiotic arrest by either a protein synthesis inhibitor, cycloheximide (CHX), or an inhibitor of serine/threonine protein kinases, 6-dimethylaminopurine (6-DMAP). Both CHX and 6-DMAP reversibly prevented nuclear maturation of nearly all oocytes for 24 h. After the reversal of arrest, CHX-treated oocytes could be successfully matured and fertilized. They developed to the blastocyst stage at slightly lower rates than oocytes cultured without inhibition for 22 h prior to sperm addition but at higher rates than those incubated in a medium containing no inhibitors for 46 h prior to fertilization. Oocytes inhibited by CHX for 48 h matured and fertilized normally but failed to develop into blastocysts. Even though 6-DMAP-treated oocytes completed meiosis I after removal from the drug, the rates of fertilization and blastocyst formation were lower than for untreated oocytes or CHX-treated oocytes. Effects of adding FSH and/or estradiol-17 beta (E(2)) during CHX-inhibition for 24 h were also examined. Embryos from oocytes treated with CHX and E(2) or with CHX and FSH + E(2) developed into blastocysts at similar rates as the controls. Further development of inhibited oocytes was examined by transferring blastocysts derived from oocytes inhibited by CHX with FSH and E(2) for 24 h to recipient heifers. Two calves were obtained following transfer. These results indicate that CHX-inhibited oocytes retain developmental competence, while 6-DMAP-inhibited oocytes after the reversal of arrest have reduced capacities for fertilization and further development. The addition of FSH and E(2) during CHX-inhibition improves development to the blastocyst stage of the oocytes that are capable of initiating and maintaining pregnancy after embryo transfer to recipient animals.

Interaction between embryos and culture conditions during in vitro development of bovine early embryos

Various factors such as embryo density and substances in the medium can influence embryo development in vitro. These factors and the embryos probably interact with each other, however the interactions are not fully understood. To investigate the interactions, we examined the effects of the number of embryos, drop size, oxygen concentration and glucose and inorganic phosphate in the medium during protein-free culture of bovine IVM/IVF embryos. In Experiment 1, different numbers of embryos were cultured in a 50 microl drop of medium. The frequencies of blastocyst development in the groups with 25, 50 and 100 embryos per drop were higher than in the other groups. One, five and 25 embryos were cultured in different drop sizes (Experiment 2), a 50 microl drop of medium at different O2 concentrations (Experiment 3) and a 50 microl drop of medium excluding glucose and/or inorganic phosphate (Experiment 4). In Experiment 2, the size of the medium drops did not improve blastocyst development. In Experiment 3, the highest frequency of blastocyst development for one, five and 25 embryos per drop was obtained at 1, 2.5 and 5% O2, respectively. In Experiment 4, blastocyst development for one and five embryos per drop were improved in the medium excluded inorganic phosphate. These results indicate that there is a cooperative interaction among embryos during culture and that this interaction may be mediated by reduction of toxic factors in the medium. At low embryo density, reduced oxygen concentration or the exclusion of inorganic phosphate enhanced blastocyst development.

Effects of water quality on in vitro fertilization and development of bovine oocytes in protein-free medium

Examination was made of the effects of water quality in medium preparation on fertilization and early development of bovine in vitro matured (IVM) oocytes in a protein-free medium. The IVM oocytes were inseminated and cultured for 7 d in protein-free media prepared with 4 different types of water preparations: tap, deionized, twice-distilled, and purified water using the Milli-Q system (Milli-Q water). High frequencies (70 to 83%) of normal fertilization were obtained in media prepared with all types of water. However, the frequency of development to the blastocyst stage in media prepared with Milli-Q water (31 +/- 3%) was significantly higher than with the 3 other types of water (11 to 13%). Moreover, the effects of storage period of Milli-Q water on early development of bovine embryos was also examined. The frequency of development to the blastocyst stage in media prepared with Milli-Q water immediately after preparation (fresh Milli-Q water; 35 +/- 4%) was significantly higher than for Milli-Q water stored for 1 wk (18 +/- 4%) or 2 wk (18 +/- 3%). Effects of commercially available purified water on early development of bovine embryos were also examined. The frequency of development to the blastocyst stage in media prepared with Milli-Q water (33 +/- 5%) was significantly higher than for purified water purchased from 3 different suppliers (Brand A; 21 +/- 6%, Brand B; 21 +/- 2%, Brand C; 21 +/- 4%). Each water sample was analyzed by the measurement of electrical conductivity, organic compounds and/or inorganic ion and endotoxin concentrations to evaluate purity. Fresh Milli-Q water showed the lowest level of electrical conductivity and contained the lowest concentration of organic compounds. These results indicate that in vitro fertilization of bovine oocytes is not affected by the water quality in the preparation of medium; however, early development of bovine embryos is seriously affected by the purification method and the storage period of water used for medium preparation.

Effects of heparin, sperm concentration and bull variation on in vitro fertilization of bovine oocytes in a protein-free medium

The present study was conducted to examine the effects of heparin, sperm concentration and bull variation on the fertilization of bovine oocytes in a protein-free medium supplemented with polyvinyl alcohol and subsequent in vitro development of fertilized embryos. The effects in protein-free medium were compared with those in medium supplemented with bovine serum albumin (BSA). In the presence of heparin (1, 10 and 100 microg/ml), nearly all the oocytes were fertilized with and without BSA. In the absence of BSA, polyspermy was lower (4 to 15%) than in its presence (15 to 48%; P < 0.05). An increase in sperm concentration from 1 x 10(4) cells/ml during insemination enhanced fertilization rate up to 1 x 10(6) cells/ml with and without BSA (14 to 90% and 3 to 77%, respectively). In the absence of BSA, the highest concentration of spermatozoa (1 x 10(7) cells/ml) gave a lower fertilization rate (55%) than that at 1 x 10(6) cells/ml (77%; P < 0.05). Polyspermy neither increased nor decreased sperm concentration without BSA (0 to 8%; P > 0.05). The effects of spermatozoa from 5 different bulls chosen randomly on in vitro fertilization in medium without BSA were examined. Individual bull variation in fertilization rate (36 to 95%) was noted at 3 different heparin concentrations (1, 10 and 100 microg/ml). Polyspermic fertilization was low (0 to 14%) and was the same for all bulls at all heparin concentrations. Embryos fertilized without BSA developed to the blastocyst stage at the same rate (27%) as those with BSA (33%; P > 0.05).

Production of bovine identical twins via transfer of demi-embryos without zonae pellucidae

Embryos were collected nonsurgically from nåturally-cycling or superovulated donors 7 d after estrus. Forty-four morulae, early blastocysts and blastocysts classified as good to excellent were bisected using a fine glass needle to produce forty-four identical demi-embryos. The bisected demi-embryos, without zonae pellucidae, were nonsurgically transferred, either by twin or single transfer. An additional forty-eight embryos were collected from the same donors and transferred as a control. Among the twin transfers, 8 of the 13 recipients became pregnant (61.5%). Seven of them conceived twin fetuses (87%) and one a single fetus. However, only two sets of normal identical twin calves were obtained. Among the single transfers, 72.6% (45/62) of bisected embryos without zonae pellucidae resulted in pregnancy, of which 48.4% (30/62) were identical twins, and 24.2% (15/62) were singletons. Another 27.4% (17/62) of the recipients did not became pregnant. The pregnancy rate for whole embryos with zonae pellucidae was 72.9% (35/48). These data show that there was no significant difference between the pregnancy rates of bisected embryos without zonae pellucidae and whole embryos with zonae pellucidae transferred 7 d after estrus. Bisection of bovine embryos was simplified and even morula stage embryos were transferred without zonae pellucidae.

Effect of PVP on sperm capacitation status and embryonic development in cattle

The objective of this study was to investigate the effects of polyvinylpyrrolidone (PVP) on sperm capacitation status and embryonic development in cattle (Bos taurus). Acrosome-reacted sperm (chlortetracycline stain) and the fertilization rate after intracytoplasmic sperm injection (ICSI) were enhanced (P<0.05) when sperm were cultured in a medium containing 10% PVP. However, injection of bovine in vitro-produced (IVP) embryos with medium containing 10% PVP suppressed development of these embryos to the 2-cell, morula, and blastocyst stages and cell number at the blastocyst stage (P<0.01) but did not affect chromosomal integrity (P>0.05). Embryo developmental capacity differed (P<0.01) among PVP from three suppliers, but there were no significant differences among three solvent media. The PVP remained localized in 40.9% of PVP-injected IVP embryos. In conclusion, PVP affected the acrosome reaction and enhanced the fertilization rate after ICSI. However, PVP remained detectable in IVP embryos and suppressed their developmental capacity.

Ex vivo expansion of human HSCs with Sendai virus vector expressing HoxB4 assessed by sheep in utero transplantation

OBJECTIVE The homeobox B4 (HoxB4) gene promotes expansion of hematopoietic stem cells (HSCs). However, frequent development of leukemia in large animals due to retrovirally transduced HoxB4 gene has been reported. To prevent tumorigenesis, we developed a nonintegrating and nonreplicating Sendai virus vector that did not contain the phosphoprotein gene (SeV/ΔP), which enabled clearance of the vector and transgene shortly after transduction. We tested the SeV/ΔP vector expressing the HoxB4 gene (SeV/ΔP/HoxB4) for the ex vivo expansion of human cord blood CD34(+) cells (HSCs) using a sheep in utero transplantation assay. MATERIALS AND METHODS Human HSCs were ex vivo-expanded by transduction with SeV/ΔP/HoxB4 vector and transplanted into the abdominal cavity of fetal sheep. The engraftment of human HSCs in the lambs was quantitatively evaluated by hematopoietic colony-forming unit assays. RESULTS After transplantation, the HoxB4-transduced HSCs contributed to longer-period (up to 20 months) repopulation in sheep, and human hematopoietic progenitors were detected more frequently in the bone marrow of the HoxB4 group as compared with the control untreated group (p < 0.05). The expansion of human HSCs with the SeV/ΔP/HoxB4 vector was comparable with previously reported retroviral vectors expressing HoxB4. The SeV/ΔP/HoxB4 vector and the transgene were cleared from the recipient sheep and leukemia was not detected at 20 months post-transplantation. CONCLUSIONS The SeV/ΔP vector would be suitable for transient expression of HoxB4 in human CD34(+) cells. In addition, the SeV/ΔP vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors.