Sueo Niimura

Sericin Accelerates the Production of Hyaluronan and Decreases the Incidence of Polyspermy Fertilization in Bovine Oocytes During In Vitro Maturation

Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.

Size of Perivitelline Space and Incidence of Polyspermy in Mouse Oocytes Matured In Vivo and In Vitro

ABSTRACT In mouse oocytes soon after collection, perivitelline space could be seen in about half of them and its mean size was 0.28 μm. After 6 hrs of in vivo and in vitro maturation, perivitelline space appeared in all oocytes. In oocytes in the process of maturation in vivo the mean size of perivitelline space significantly increased 6 hrs after hCG injection and reached 7.00 μm at 14 hrs after the hCG injection. On the other hand, perivitelline space in cultured oocytes enlarged significantly at 2 hrs culture. Thereafter, perivitelline space in oocytes continued to enlarge with culture time and reached 5.40 μm at 14 hrs of maturation culture. The size of perivitelline space in oocytes 14 hrs after the hCG injection was significantly larger than that in oocytes cultured for 14 hrs. The fertilization rates of oocytes matured in vivo (85%) did not differ from that of oocytes matured in vitro (80%). However, the incidence of polyspermy in oocytes matured in vivo was 4%, which was significantly lower than the 16% of oocytes matured in vitro. These findings suggest there could be a relationship between the size of the perivitelline space and the incidence of polyspermy in mouse oocytes.

Changes in the amount of cytoplasmic inclusions in mouse oocytes during meiotic maturation in vivo and in vitro

Background and aimsThe changes in cytoplasmic inclusions during meiotic maturation have only been examined in porcine oocytes. In the present study, the amount and the number of cytoplasmic inclusions (glycogen granules, lipid droplets and fibrous structures) were examined in mouse oocytes in the process of in vivo and in vitro maturation. For those inclusions that changed in amount during maturation, we also examined their content in oocytes treated with olomoucine, an inhibitor of cyclin-dependent kinase, in order to clarify the relationship between nuclear maturation and changes in the inclusions.MethodsNuclear maturation in the oocytes cultured for various periods and those collected from antral follicles and oviducts was examined after staining with aceto-orcein. For the demonstration of glycogen granules and lipid droplets, oocytes were stained with periodic add-Schiff or Sudan IV. Fibrous structures in the oocytes were observed under an electron microscope.ResultsThe amount of glycogen granules, Sudanophilic lipid droplets and fibrous structures did not change in the oocytes matured in vivo and in vitro, whereas the number of the lipid droplets increased during maturation. In the oocytes treated with olomoucine, the resumption of nuclear maturation was inhibited, whereas the increase in the number of Sudanophilic lipid droplets was not inhibited.ConclusionPresent findings suggest that the increase in the number of Sudanophilic lipid droplets occurs in the cytoplasm of mouse oocytes during maturation, regardless of in vivo or in vitro maturation, and that such the change in the inclusion is not related to nuclear maturation.

Effect of supplemented sericin on the development, cell number, cryosurvival and number of lipid droplets in cultured bovine embryos

Sericin was investigated as an alternative to fetal bovine serum (FBS) for bovine embryo culture. In vitro matured oocytes were developed using 0.05%, 0.1% or 0.15% sericin. The developmental rate, cryosurvival rate and blastulation time of these embryos were compared with those of embryos developed using 5% FBS. The number of lipid droplets was compared among the blastocysts developed using 5% FBS, using 0.05% sericin and in vivo. The rate of cleavage and blastocyst formation was similar among all groups. Blastulation occurred significantly earlier in the embryos developed using 5% FBS than in those developed using sericin at any concentration (P < 0.05). At 72 h after thawing, the cryosurvival rate of the blastocysts developed using 5% FBS and 0.05% sericin were significantly higher compared with those developed using 0.1% and 0.15% sericin (P < 0.05). The blastocysts developed using 0.05% sericin and in vivo produced a significantly fewer number of medium and large lipid droplets than those developed using 5% FBS. These results suggest that the blastocysts developed using 0.05% sericin show characteristics similar to those of the blastocysts developed in vivo and that the use of sericin as an alternative to FBS is feasible.

Relationship between BCB Stainability and Developmental Competence to the Blastocyst Stage in Mouse Early Embryos

Abstract: The stainability of mouse embryos with brilliant cresyl blue (BCB) and the development to blastocysts of BCB-positive and BCB-negative embryos were examined. From the 2-cell to the morula stages, the incidences of BCB-positive embryos collected from mature mice were 94.2 to 96.4%. These rates were significantly higher at all stages than those of BCB-negative embryos. The rates of development to blastocysts of BCB-positive embryos (85.7 to 96.7%) were significantly higher than those of BCB-negative embryos (0 to 50.0%) at all stages. In 2-cell embryos and morulae collected from aged mice, the incidences of BCB-positive embryos (79.5 and 58.6%) were significantly higher than those of BCB-negative embryos (20.5 and 41.4%, respectively). The rates of development to blastocysts of BCB-positive 2-cell embryos and morulae collected from aged mice were 77.1 and 79.4%, which were significantly higher than those of BCB-negative embryos, 0 and 45.8%, respectively. Although the incidences of BCB-positive embryos collected from aged mice were significantly lower than those collected from mature mice, the rates of development to blastocysts in BCB-positive embryos did not differ between the embryos collected from aged and mature mice. In these findings, we demonstrate that BCB-positive embryos have high developmental ability to the blastocyst stage. A correlation was also found between BCB stainability and G-6-PDH activity in mouse eggs and embryos.

Correlation between the Distribution Pattern of Cortical Granules and the Incidence of Polyspermy in Ovulated Hamster Eggs

ハムスターの過排卵卵子と自然排卵卵子について,Lens culinaris agglutinin (LCA)を用いて表層粒をレクチン組織化学的に検出するとともに多精子受精を観察し,表層粒の状態(分布と量)と多精子受精の頻度との相関の有無を調べた.未受精卵子において,LCAに陽性の表層粒は主として細胞質表層に存在していたが,量が多く単独で一様に分布している卵子(I型),量は多いが,分布が一様でない卵子(II型),および量がやや少なく単独で一様に分布している卵子(III型)の3型が区分できた.表層粒の分布が一様でない卵子(II型)の割合は,自然排卵の卵子群では0.6%であったが,過排卵のものでは高く,5.0%であった.また,表層粒がやや少ない卵子(III型)の割合は,自然排卵の卵子群では1.6%であったが,過排卵のものでは有意に高く,4.4%であった.一方前核期卵子において,多精子受精は自然排卵の卵子群では0.3%みられたが,過排卵のものでは有意に高く,2.6%みられた.このように,ハムスターでは,表層粒が少ない卵子や分布が一様でない卵子が多いと,多精子受精の頻度が高くなり,両者の間には相関のあることが示唆された.

The Immunohistochemical Detection of Prostaglandins in the Granulosa Cells of Hamsters

原始卵胞から胞状卵胞までのハムスターの顆粒層細胞と排卵後の卵丘細胞について,プロスタグランジン(PG) E2とPGF2aの検出を間接蛍光抗体法を用いて免疫組織化学的に行ない,顆粒層細胞におけるPGの出現時期と量的変動を調べた.PGE2およびPGF2aの局在を示す蛍光は原始細胞の顆粒層細胞には観察されなかったが,いずれの種類のPGも小型二次卵胞の顆粒層細胞に弱く出現した.胞状卵胞において,PGE2とPGF2aの局在を示す蛍光はいずれも排卵前9時間までの顆粒層細胞では強かったが,排卵前6時間と3時間では著しく弱まり,蛍光を持つ細胞を含まない卵胞が出現した.また,排卵後12時間の卵丘細胞には蛍光は全くみられなかった.これらの結果から,ハムスターの顆粒層細胞は小型二次卵胞の時期からPGE2とPGF2aを持ち始めること,LHサージ後の胞状卵胞の顆粒層細胞ではPGの含量が著しく減少し,PGを含まない細胞が現れることが明らかとなった.

The Immunohistochemical Detection of Prostaglandin E2 in Hamster Oocytes

原始卵胞から胞状卵胞までのハムスターの卵母細胞について,プロスタグランジン(PG) E2の検出を間接蛍光抗体法を用いて免疫組織化学的に行ない,卵母細胞におけるPGE2の出現時期を調べるとともに,PGE2を含有する卵母細胞の割合を排卵まで経時的に調べた.原始卵胞および小型二次卵胞の卵母細胞にはPGE2の局在を示す蛍光は観察されなかったが,大型二次卵胞では28.6%の卵母細胞に蛍光が出現した.排卵前87時間,63時間,39時間および15時間の胞状卵胞では,蛍光を示す卵母細胞の割合はそれぞれ25.0%,36.4%,34.8%および33.3%で大きく変動しなかった.しかし,排卵前9時間の胞状卵脆から,蛍光をもつ卵母細胞の割合は徐々に増加し,排卵前6時間では60.0%,排卵前3時間では72.7彰の卵母細胞に蛍光が観察された.これらの結果から,ハムスターの卵母細胞は大型二次卵胞の時期からPGE2を持ち始めることが明らかとなり,卵母細胞に含まれるPGE2は,LHサージ後の卵母細胞の成熟に関与していることが推察された.

Histochemical Studies of Hydroxysteroid Dehydrogenases in Delayed Implanting Mouse Blastocysts

マウスの無処置胚盤胞(Day 4),着床遅延胚盤胞(Day 10)およびestradiol benzoate投与後18時間の着床遅延胚盤胞(Day 10)について,Δ5-3β-hydroxysteroid dehydrogenase (Δ5-3β-HSD),17β-hydroxysteroid dehydrogenase (17β-HSD),20α-hydroxysteroid dehydrogenase (20α-HSD)および20β-hydroxysteroid dehydrogenase (20β-HSD)の活性を組織化学的に検出した.無処置胚盤胞において,dehydroepiandrosterone (DHA)とpregnenoloneを基質としたΔ5-3β-HSD,testosteroneとestradiol-17βを基質とした正7β-HSD, 20α-HSDおよび20β-HSDの活性は強かったが,17α-hydroxypregnenoloneを基質としたΔ5-3β-HSDの活性は認め間れなかった.着床遅延胚盤胞において,DHAを基質としたΔ5-3β-HSDと17β-HSDの活性は弱く,20α-HSDと20β-HSDの活性は弱いもののほかに陰性のものが少数出現した.pregnenoloneと17α-hydroxypregnenoloneを基質としたΔ5-3β-HSDの活性はみられなかった.estradiol benzoate投与後18時間の着床遅延胚盤胞において,すべてのHSD活性は着床遅延胚盤胞のものに類似であった.以上のことから,着床遅延胚盤胞のステロイド代謝能はDay 4のものに比べて低下しており,エストロジェンを投与して着床を開始させても回復しないことが推察された.

Appearance of the Tubular-Typed Smooth Endoplasmic Reticulum and Annulate Lamella in the Hamster Oocytes 10 Hours before Ovulation

排卵前36時間から1時間までのハムスターの胞状卵胞卵子には,内腔の電子密度の低い小胞状の滑面小胞体が細胞表層部に常に散見されたが,排卵前10時間の卵子に限って,このほかに,内腔の電子密度の高い管状の滑面小胞体が細胞表層部と核周辺部に多数出現し,さらに,有窓層板が細胞表層部に認められた.内腔の電子密度の高い管状の滑面小胞体は有窓層板と連絡しており,有窓層板から形成されることが推察された.これらの小器官の出現はLHサージの時期に一致しており,卵子に取り込まれたLHの処理に関与していることが考えられた.