The linker histones constitute the major proteins bound to linker DNA, the DNA bridging nucleosome core particles. The composition of the linker histone fraction is tissue- and species-specific, as well as developmentally-regulated. As such, linker histones play a critical role in the higher order packaging of chromatin and thus, inevitably, in an impressive array of regulatory functions. Recently, we uncovered a mammalian oocyte-specific linker histone H1oo homologous to the cs-H1 of sea urchin and to the B4 of the frog in the course of a differential screening project. H1oo protein localized to the intact germinal vesicle (GV) of preovulatory oocytes, to the condensed chromosomes of ovulated oocytes arrested at metaphase (M) II, and to the first polar body. The pronuclei were both positive at the 1-cell stage. Nuclear staining, however, was reduced in 2-cell embryos and was no longer detectable at the 4-cell stage of embryonic development. These findings indicate oocyte-specific linker histones may play some roles in genome-wide reprogramming in vertebrate eggs.

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The number of cells and the rate of cell division were observed in diploid parthenogenetic mouse embryos during the course of blastocyst formation, and were compared with those in embryos developed from fertilized ova (fertilized embryos), in order to identify the stage at which the number of cells begins to differ and the cause of difference in cell number between parthenogenetic and fertilized embryos. The number of cells and the rate of cell division did not differ between parthenogenetic and fertilized embryos at the 8-cell and compacted morula stages. The numbers of inner-cell-mass and trophoblast cells did not differ between parthenogenetic and fertilized embryos at the early blastocyst stage, but were significantly fewer in parthenogenetic embryos (11.1 and 32.7) than in fertilized embryos (14.9 and 52.0) at the expanded blastocyst stage. The numbers of dead inner-cell-mass and trophoblast cells were significantly more in parthenogenetic embryos than in fertilized ones at the early and expanded blastocyst stages. At the early blastocyst stage, although no difference was observed between parthenogenetic and fertilized embryos in the rate of division of trophoblast cells, the rate of division of inner-cell-mass cells was significantly lower in parthenogenetic embryos (4.4%) than in fertilized ones (9.7%). There was no difference in the rate of cell division between parthenogenetic and fertilized embryos at the expanded blastocyst stage. From these results, it was inferred that the number of cells in diploid parthenogenetic embryos does not differ from fertilized embryos until blastocyst formation, but becomes fewer than in fertilized embryos because more cells are dying in parthenogenetic embryos during the expansion of blastocysts. A low rate of cell division in the early blastocyst stage is thought to be one reason for the fewer number of inner-cell-mass cells in parthenogenetic embryos at the expanded stage, in addition to the presence of a large number of dead cells.

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This study was conducted to examine if oocytes obtained from one day preserved equine ovaries could be used for IVM and IVF research. Ovaries were transported at 20°C over a long distance from a slaughterhouse to a laboratory taking about 24 h. Oocytes with compact cumulus cell layers were collected from ovaries and cultured for 42 h in IVMD101 medium at 38.5°C in 5% CO₂ in air. The proportions of metaphase-II stage oocytes increased significantly between 24 and 30 h of culture and reached a plateau at 30 h. After 30 h of culture, the denuded oocytes with a first polar body were subjected to intracytoplasmic sperm injection (ICSI) using frozen-thawed stallion spermatozoa. The ICSI oocytes were activated by 10 min treatment with ionophore A23187 and 6 h treatment with cycloheximide, and cultured for up to 10 days in CR1aa medium. The proportion of ICSI oocytes fertilized normally (defined as those with 2 pronuclei and 2 polar bodies) was 44%. In vitro culture of ICSI oocytes resulted in a cleavage rate of 52% and a blastocyst development rate of 9%. These results indicate that equine oocytes derived from one day preserved ovaries can reach metaphase-II stage during 30 h of IVM, and that IVM oocytes can develop into blastocysts after ICSI.

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The aim of this study is to induce effective superovulation in Japanese Black cows with hMG and to produce high-quality embryos in a short interval of superovulation treatment. In the first superovulation treatment, a total of 750 IU hMG was used and after collection, 50 ml of iodine was infused into the uterus and a 25 ml PGF_2α intramuscular injection was also given. After confirming the return of oestrous, a second treatment was conducted with a total of 600 IU hMG. Although the total number of collected ova/embryos and transferable embryos was less in the second treatment than in the first one, the ratio of transferable and excellent embryos was considered to be high. In conclusion, it was indicated that 600 IU hMG is effective for superovulation and that iodine preparation and PGF_{2 α} produce high-quality embryos in a short interval of superovulation treatment.

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We investigated the role of cAMP-Protein kinase A pathway and Ca²⁺-Protein kinase C pathway which were activated by FSH and/or LH in the progesterone production by cumulus cells during in vitro maturation of porcine oocytes. The level of progesterone in the medium in which COCs had been cultured for 24 hr without FSH was 7.8 ± 1.5 ng/ml. The addition of FSH significantly increased the progesterone level in a dose-dependent fashion during 24-hr cultivation of COCs; a plateau was detected in 0.02 μg/ml, but no significant increase in the level of progesterone was observed in the medium in which COCs had been cultured for 24 hr with 1 or 10 μg/ml LH. When COCs were cultured with both 0.02 μg/ml FSH and 1.0 μg/ml LH for 24 hr, the maximal level of progesterone was detected. The effects of the addition of LH to FSH-supplemented medium on the response of cumulus cells were affected by the suppression of the Ca²⁺-Protein kinase C pathway. Forskolin-induced progesterone production was not affected by suppression of the Ca²⁺-Protein kinase C pathway but was reduced by Protein kinase A inhibitor. These results showed that Ca²⁺-Protein kinase C pathway in cumulus cells stimulated by FSH, enhanced LH-induced progesterone production via a cAMP-Protein kinase A dependent pathway.

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We identified a novel gene, termed GSE (gonad-specific expression gene). Nucleotide sequence analysis of GSE cDNA revealed that the open reading frame of 745-bp encodes a protein of 247 amino acids with a predicted molecular mass of 27.6 kDa. The deduced amino acid sequence indicated that GSE protein might be a soluble protein in the cytoplasm without a signal peptide. Northern blot analysis showed that this gene was abundantly expressed in mouse testis and slightly expressed in the mouse ovary. RT-PCR analyses indicated that the GSE mRNA in the testis was first detected at Day 14 postpartum, when spermatocytes at mid-pachytene are likely to appear. In situ hybridization confirmed its expression at this stage of spermatogenesis. On the other hand, the GSE mRNA in the ovary was already present at birth, when germ cells are in meiosis. These observations suggest that GSE may be associated with meiosis during gametogenesis.

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A variety of physical and chemical stimuli initiate egg activation events. This study was designed to investigate the rates of pronuclear formation and the cytoskeletal organization of porcine oocytes activated by various stimuli. Oocytes matured for 44-60 hrs were activated by one of the following treatments: double electric pulses (EP, 150 V/mm for 60 μsec, 1 sec apart), ethanol (7% for 5 min), calcium ionophore A23187 (CaA, 50 μM for 2 min), cycloheximide (CHX, 5 μg/ml for 6 hrs), or 6-dimethylaminopurine (DMAP, 2.5 mM for 4 hrs). EP yielded the highest activation rate (97%). CaA and DMAP activated the oocytes in relatively higher proportions than did ethanol and CHX (74 and 83% vs. 55 and 41%, respectively). After CHX and DMAP treatments, egg fragmentation was significantly increased (65 and 82%, respectively) and the polar body emission was restrained. After activation with EP, ethanol or CaA, the pronucleated oocytes possessed a microtubule-rich domain around the pronucleus, which was concomitantly surrounded by microfilaments. In oocytes activated with CHX or DMAP, however, microtubules and microfilaments did not synchronously form a concentrated domain. We suggest that activation with a protein synthesis/phosphorylation inhibitor may have affects on distribution and function of the cytoskeleton of the oocyte different from those elicited by treatments with EP, ethanol or CaA, which result in higher proportions of eggs with the pronucleus restrained in the peripheral ooplasm and greater egg fragmentation.

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There are cases in which the 1-day-old unfertilized oocytes after IVF, or in vitro matured oocytes obtained from denuded immature oocytes after ICSI, are used clinically. We investigated the morphological change of metaphase II spindle and chromosomal alignment in those oocytes. The spindles and chromosome were stained using an anti-α-tubulin antibody and Hoechst 33258 respectively. One-day-old oocytes displayed significant increases in abnormalities of spindle structure (40.4% vs. 17.6%) and chromosomal alignment (40.4% vs. 29.4%). Oocytes matured in vitro from MI and PI oocytes displayed spindle morphological abnormalities at the rates of 41.7% and 36.0% respectively, and chromosomal alignment abnormality at the rates of 45.8% and 44.0% respectively. These results suggest that 1-day-old oocytes and in vitro matured oocytes obtained from denuded immature oocytes after ICSI might have lowered developmental potential. More than 50% of those oocytes were determined to be unsuitable for use as gametes.

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The aim of this study was to recover spermatozoa easily from fresh or frozen-thawed testicular sperm extraction (TESE) samples. We simply minced and washed the TESE samples in medium. Spermatozoa were recovered and washed in the bottom of a PVP droplet with an injection pipette and injected into oocytes. In all cycles 100% of spermatozoa (motile and/or immotile) could be recovered. Injected oocytes were fertilized (100% per cycle and in 41.9% of oocytes) and fertilized oocytes were cleaved (100% per cycle; 38.5% of injected oocytes; 91.8% of fertilized oocytes) after ICSI with fresh or frozen-thawed TESE spermatozoa. The rates of progression to the embryo stage (2-8 cell) and blastocyst formation and their quality were similar in both the fresh sample group and the frozen-thawed sample group. Both of embryo transfer (73.5%) and embryo-blastocyst transfer (26.5%) were performed successfully. Thirty-four cycles of ICSI with testicular spermatozoa resulted in 23.5% pregnancy rates (fresh sample group, 22.2%; frozen-thawed sample group, 24%) with both embryo transfer (8%) and embryo-blastocyst transfer (66.7%). This new sperm preparation method is very simple, easy, effective and rapid for recovering spermatozoa from TESE samples for ICSI.

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In this study we sequentially investigated the expressions of CD44, the principal hyaluronan (HA) receptor, in porcine cumulus-oocyte complexes (COCs) during in vitro maturation. The mRNA expressions of CD44 were analyzed in porcine cumulus cells and oocytes. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, the expression of CD44 was identified only in cumulus cells, indicating that cumulus cells express CD44 mRNAs. The transcript of CD44 was weakly detectable in fresh (0 h) cumulus cells, but was clearly detected after 12 h of culture. In immunostaining, CD44 was distributed on the cytoplasm along the perimembrane of cumulus cells and at the junctions between cumulus cells and oocytes. The level of CD44 expression reached a peak at 24 h of culture although its expression was very weak at 0 h. These findings implied that the level of CD44 expression depends on the extent of cumulus expansion. These results suggest that CD44 may be involved in HA retention in the extracellular matrix of COCs during oocyte maturation.

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We noted varying success rates with IVF on a month-by-month basis from 1998 through 1999. During that interval, we had no control over the room temperature and results appeared to be affected by seasonal temperature fluctuations. Since 2000, we have maintained a constant temperature of 25°C in our operating and culture rooms throughout the year. During that time, our pregnancy rate has been significantly higher than in previous years. Specifically, we noted an improved pregnancy rate during the summer season (June and July). A constant room temperature of 25°C significantly enhances the efficacy of in-vitro fertilization.

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