Tadashi Mori

Roles of Gap Junctional Communication of Cumulus Cells in Cytoplasmic Maturation of Porcine Oocytes Cultured In Vitro

Abstract Cumulus cells of the oocyte play important roles in in vitro maturation and subsequent development. One of the routes by which the factors are transmitted from cumulus cells to the oocyte is gap junctional communication (GJC). The function of cumulus cells in in vitro maturation of porcine oocytes was investigated by using a gap junction inhibitor, heptanol. Cumulus-oocyte complexes (COCs) were collected from the ovaries of slaughtered gilts by aspiration. After selection of COCs with intact cumulus cell layers and uniform cytoplasm, they were cultured in a medium with 0, 1, 5, or 10 mM of heptanol for 48 h. After culture in vitro, one group of oocytes was assessed for nuclear maturation and glutathione (GSH) content, and another group was assigned to in vitro fertilization and assessed for the penetrability of oocytes and the degree of progression to male pronuclei (MPN) of penetrated spermatozoa. At the end of in vitro maturation, the oocytes reached metaphase II at a high rate (about 80%) regardless of the presence of heptanol at various concentrations. Cumulus cell expansion and the morphology of oocytes cultured in the medium with heptanol were similar to those of control COCs matured without heptanol. The amount of GSH in cultured oocytes tended to decrease as the concentration of heptanol in the medium was increased. Although there was no difference in the rates of penetrated oocytes cultured in media with different concentrations of heptanol, the proportion of oocytes forming MPN after insemination decreased significantly (P < 0.01) at all concentrations tested. A higher rate of sperm (P < 0.01) failed to degrade their nuclear envelopes after penetration into the oocytes that were treated with heptanol. GJC between the oocyte and cumulus cells might play an important role in regulating the cytoplasmic factor(s) responsible for the removal of sperm nuclear envelopes as well as GSH inflow from cumulus cells.

In vitro fertilization of porcine oocytes in chemically defined medium

Abstract Porcine follicular oocytes matured in vitro were inseminated with ejaculated boar spermatozoa. We examined whether or not protein is essential for spermatozoa to penetrate oocytes when added during preincubation of spermatozoa, during sperm-oocyte coincubation, or during both periods. The absence of additional protein (bovine serum albumin; BSA) in preincubation media had no effect on the sperm penetration of oocytes, although fetilization media with protein showed higher rates of penetrated oocytes than media without additional protein (77% with BSA and 57% with fetal bovine serum; FBS, vs 14% with no macromolecule, 19% with polyvinylalcohol; PVA, and 27% with polyvinylpyrrolidone; PVP). A high concentration of spermatozoa at insemination increased the rate of sperm penetration even in medium without additional protein. Oocytes with or without cumulus cells were inseminated with preincubated spermatozoa in the fertilization medium without additional protein. The results showed no difference in the penetration rate between oocytes with and those without cumulus cells. In conclusion, although BSa in the fertilization medium was found to accelerate the ability of porcine spermatozoa to penetrate porcine oocytes matured in vitro, protein and cumulus cells were not essential for in vitro penetration of oocytes.

The suppression of fragmentation by stabilization of actin filament in porcine enucleated oocytes

A thorough understanding of the mechanism underlying fragmentation would contribute to the improvement of the developmental ability of reconstructed embryos after nuclear transfer. We conducted the present study to elucidate the influence of the nuclear transfer method on fragmentation of enucleated oocytes and the relationship between change in actin filament distribution and fragmentation. In Experiment 1, we examined activation rates of in vitro matured oocytes. These were 12.9% in maturation alone, 75.7% in electrical stimulation, and 57.9% in ethanol/cycloheximide treatment. In Experiment 2, we observed a higher rate of fragmentation (P < 0.05) in cultured oocytes that had been enucleated and electrically stimulated than in oocytes subjected to the other treatments (maturation alone, enucleation alone and enucleation plus ethanol/cycloheximide activation). In Experiment 3, we stained enucleated and electrically stimulated oocytes with rhodamine/phalloidin dye to show discontinuous distributions in the ooplasm of treated oocytes; oocytes in the other treatment groups showed homogenous distributions of actin filaments (AFs). In Experiment 4, we added cytochalasin B, an inhibitor of AF polymerization, to the culture medium, which prevented fragmentation of enucleated plus electrically stimulated oocytes (cytochalasin B, [+] 0.0%, [-] 60.7% at 24 h after treatment, P < 0.05). In Experiment 5, we investigated the relationship between fragmentation and alteration in AF distribution in enucleated plus electrically stimulated oocytes. At 0 h of culture, enucleated plus electrically stimulated oocytes showed discontinuous distributions of AFs, while nontreated oocytes showed homogenous AF distributions. At 24 and 48 h of culture, fragmentation proceeded in enucleated plus electrically stimulated oocytes and the discontinuous AF distribution diminished with time. In Experiment 6, we added hyaluronic acid (HA) to the culture medium, which suppressed fragmentation of enucleated plus electrically stimulated oocytes (HA, [+] 28.5%, [-] 66.4% at 24 h after treatment, P < 0.05). The results suggest that electrical stimulation induces a change in the AF distribution of oocytes, resulting in fragmentation, and that the addition of HA to the culture media is effective for the suppression of fragmentation.

Relationship of gap junction formation to phosphorylation of connexin43 in mouse preimplantation embryos

To clarify the relationship of gap junction formation to phosphorylation of connexin43 (Cx43) in mouse preimplantation embryos, immunofluorescence and Western blot analysis were conducted. Immunofluorescence showed Cx43 positive spots first at the mid‐eight‐cell stage (6 hr postdivision to the eight‐cell stage). The number of spots increased from 6 to 15 hr postdivision to the eight‐cell stage. Western blot analysis suggested Cx43 to possibly be present in the nonphosphorylated form at the mid‐four‐cell stage (6 hr postdivision to the four‐cell stage), and phosphorylated Cx43 to increase from the mid‐eight‐cell stage (6 hr post‐division to the eight‐cell stage) onward. Dibutyryl cAMP (dbcAMP), a protein kinase A (PKA) activator, added to the culture medium increased the phosphorylation of Cx43 and Cx43 positive spots. The tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a protein kinase C (PKC) activator, increased the phosphorylation of Cx43, but decreased Cx43 positive spots. These results suggest that the phosphorylation of Cx43, induced by different protein kinase, leads to a different effect on gap junction formation in mouse preimplantation embryos. Mol. Reprod. Dev. 55:393–398, 2000.

Activation and development of porcine oocytes matured in vitro following injection of inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (IP3) is considered to be important for activation of mammalian oocytes at the time of fertilization, and activation induces a rise in intracellular Ca2+ concentration ([Ca2+]i) by release from the Ca2+ stores in the oocytes. Therefore, IP3 could act as an artificial activator of porcine oocytes. Activation and development, and rise in [Ca2+]i in matured oocytes injected with various concentrations of IP3 were investigated in this study. Porcine oocytes were recovered from the ovaries of prepubertal gilts, matured for 46-48 h and cultured in vitro for 7 days in following treatments as non-injected oocytes (NI), injected with carrier buffer, 2.5, 5 and 500 microM of IP3. The result showed that IP3 activated porcine oocytes matured in vitro (NI 3.8%, buffer 7.1%, 2.5 microM IP3 73.5%, 5 microM IP3 76.2%, 500 microM IP3 85.2%). There was a slight but not significant increase in the proportion of oocytes activated as the level of IP3 increased. The rate of development to the cleavage stage increased remarkably when the concentration of IP3 increased (NI 4.9%, buffer 5.7%, 2.5 microM IP3 30.3%, 5 microM IP3 47.1%, 500 microM IP3 78.1%). Blastocyst development was only observed in oocytes that had been injected with a higher concentration of IP3 (5 microM IP3 6.1% and 500 microM IP3 5.3%). Both the peak value and duration of [Ca2+]i rise also increased as the concentration of IP3 increased. Baseline values (ratio value, R) for [Ca2+]i ranged from 1.51 to 1.57 and was not affected by the buffer treatment. The peak value of [Ca2+]i rose significantly with increasing level of IP3 treatment (2.5 microM IP3, 3.54 +/- 0.32; 5 microM IP3, 7.50 +/- 0.37; 500 microM IP3, 8.54 +/- 0.33). Similarly, the duration of the [Ca2+]i rise increased as the level of IP3 increased (2.5 microM IP3, 43.7+/- 7.00 s; 5 microM IP3, 93.5 +/- 9.17 s; 500 microM IP3, 160.6 +/- 18.9 s). It was concluded that injected IP3 promotes the development of porcine matured oocytes and that their developmental ability is positively correlated with the rise in [Ca2+]i induced by IP3.

Effects of culture time, ovarian activity, cumulus cells and sera on the nuclear and cytoplasmic maturation of pig oocytes in vitro

Pig cumulus-oocyte complexes (COC) were used in this study to assess the effect of length of culture, ovarian activity, cumulus cells and sera on maturation and fertilization in vitro. In Experiment 1, the incidence of nuclear maturation increased (P<0.01) from 24 to 48 h of culture and the ability for male pronucleus (MPN) formation peak at 42 h (86.6%). The penetrability of immature COC and those at the metaphase 2 (M2) stage appeared to be not influenced by the stage of meiosis (75.3–89.3%). In Experiment 2, no significant difference in the nuclear maturation (84.8–88.0%) and penetration (82.1–92.0%) rates of COC collected from ovaries with or without corpora lutea (CL) or corpora albicans (CA) was observed, though MPN formation differed (P<0.05) between O1 (82.6%) and O3 (61.5%) oocytes. In Experiment 3, significant differences (P<0.01) on the nuclear maturation and MPN formation of oocytes cultured with cumulus cells versus those without or lacking cumulus cells was observed. It shows that the presence of cumulus cells is important in supporting maturation in vitro. In Experiment 4, serum supplementation of the medium improved the rate at which COC reached the M2 stage (P<0.05) and formed MPN except at the 5% level when using fetal bovine serum (FBS) and calf serum (CS). The use of porcine serum (PS) consistently supported the maturation of COC. Serum supplementation is thus necessary for maturation in vitro and the concentration and type of serum to be used is important.

Timing of sequential changes in chromosome configurations during the second meiotic division and cytoplasmic events of pig oocytes matured and fertilized in vitro

Abstract The present study determined the specifics of early events of fertilization of pig oocytes matured in vitro (IVM) and subsequently fertilized in vitro (IVF). The IVF system used for sperm capacitation, time of fertilization and fixation of eggs at 1–3 h intervals during the 30 h period after insemination precisely measured the timing of each critical stage of fertilization with precision. The first evidence of sperm penetration was observed 3 h after insemination. Attachment of the spermatozoa in the ooplasm caused resumption of meiosis (A2/T2 stage of second meiotic division), and was commonly observed from 4 to 5 h after insemination. The sperm heads of penetrating spermatozoa started to decondense, tails became detached and completion of the second meiosis (second polar body extrusion) occurred from 6 to 9 h. Further decondensation of the sperm head and female chromosomes took place from 9 to 12 h. Opposing and apposing pronuclei were frequently observed from 12 to 18 h and 18 to 24 h after insemination, respectively. Syngamy and prophase stage of first mitotic division, including first cleavage were observed from 24 to 30 h.

A Functional Truncated Form of c-kit Tyrosine Kinase Is Produced Specifically in the Testis of the Mouse But Not the Rat, Pig, or Human

The complete nucleotide sequence of mouse-truncated mRNA of c-kit, tr-kit, has been determined using the CD1 strain. In this study, the nucleotide sequences of tr-kit from AKR/N, C57BL/6, and ICR strains of mice were determined and found to be identical, although many silent variations were found compared with the sequence in a database for CD1. Tr-kit protein consists of 12 amino acids encoded by the 16th intron and the following 190 amino acids of c-kit. In the sequences of tr-kit encoding 12 specific amino acids, no substitution was detected among the three strains and CD1. Furthermore, RT-PCR analysis clearly showed that tr-kit mRNA expression was present only in testis. No nucleotide mutation in two important regions of the presumptive promoter for tr-kit mRNA was detected within the 16th intron of the mouse strains examined. However, no functional form of tr-kit was found in the rat, pig, or human by sequence analysis and homology testing.

Blastocyst formation of pig embryos derived from in vitro fertilization of in vitro matured pig oocytes in the amniotic fluid of a developing chick embryo

In this study, the acquisition for developmental competence of early-stage pig embryos derived from in vitro fertilization of in vitro matured pig oocytes was determined by culturing in a modified Whittens medium (mWM) or amniotic fluid of a developing chick embryo (CEAm fluid). Of 1080 oocytes inseminated, 420 were fixed after 15 h revealing a nuclear maturation rate of 95.2% (400420) with a fertilization rate of 84.3% (354420). The monospermic fertilization rate was 36.4% (129354). Of 660 oocytes that were allowed to continue development, 74.2% (490660) cleaved. Of these, 220 and 157 embryos were cultured in mWM and CEAm fluid with 19.5% and 25.7% progressing to blastocyst stage, respectively. The mean (±standard error of the mean) cell number of blastocyst stage embryos derived from CEAm fluid (52.0 ± 3.0) was significantly higher (P < 0.001) than those obtained from mWM (39.0 ± 1.7). The results suggest that CEAm fluid is a better in vitro culture system for pig embryo development than mWM.