Reprod. Boar ejaculated spermatozoa were incubated with CAMP analogs and then used for the immunodetection of serine/threonine-phosphorylated proteins and assessment of acrosome morphology. Moreover, tyrosine phosphorylation of the proteins (eg, >220 kc, 190 kc, 93 kd, 59 kd, 54 kc, and 32 kd) was induced intensely in the connecting and principal pieces and moderately in the middle piece of almost one half of the agglutinated spermatozoa after incubation with cBiMPS for more than 30 minutes, but rarely in those of the free spermatozoa. Boar ejaculated spermatozoa were pre-incubated with a cell-permeable cyclic adenosine monophosphate (cAMP) analog cBiMPS' and without CaCl2 to induce the cAMP-triggered events including capacitation-associated changes. The quantitative analyses with real-time polymerase chain reaction using the testicular RNA from the same bulls revealed that the relative expression levels of activator CREM variants in testes varied significantly among these bulls in the range from 0.56 to 1.64 (P < 0.05). In the samples after the treatments to induce the acrosome reaction, the percentages of spermatozoa without acrosomes and with IZUMOI in whole equatorial segment (pattern P2 of IZUMOI) significantly increased. The aim of the present study was to investigate the effects of cyclic adenosine 3',5'-monophosphate (cAMP) and serum albumin on sperm agglutination and to discuss a possible mechanism for sperm agglutination.
In addition, a protein 'anti-agglutinin' inhibiting sperm agglutination, was extracted from spermatozoa incubated with serum albumin or MBC and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Indirect immunofluorescence with specific polyclonal antibodies suggested possible co-localization of Epac1 and Rap1 proteins in the heads of epididymal spermatozoa. It also appears that this event is mediated by increasing the concentration of intracellular cyclic adenosine 3',5'-monophosphate. Western blotting showed that the antiserum selectively recognized a band of the anti-agglutinin in the corpus and cauda epididymal plasma, although no band was detected in the caput epididymal plasma. 実験2.電位依存性カルシウムチャンネル(VDCCs)の役割:昨年度の研究において10〜100μMの濃度では精子の頭部間凝集に有意な抑制作用を示さなかったL型VDCCs阻害剤のNifedipineとNitrendipineをより高い濃度(1mM)で添加したところ,頭部間凝集率は有意に低下した.他方,T型VDCCsの役割については,予備実験で精子に対する毒性が比較的少ないことが判明した阻害剤のPimozideとAmiloride(いずれも0.1〜10μM)を用いて検討したが,いずれも凝集精子率に有意な変動は認められなかった.以上の結果から,精子の頭部間凝集の発生においてVDCCsが機能している可能性が示唆されたが,L型阻害剤で抑制効果が認められた濃度は1mMと高く,またT型阻害剤(0.1〜10μM)には効果が認められなかったことから,VDCCsの種類を特定するには至っていない.三宅 正史, 紅林 賢臣, 原山 洋電気刺激により高率に活性化したブタ単為発生2倍体を用いて,1.ブタ初期胚の培養系を確立するために,浸透圧,アミノ酸の胚盤胞への発生に及ぼす影響,2.単為発生2倍体の体内発生限界と単為発生胎子の生産効率,3.ブタにおけるインプリント侯補遺伝子の単為発生胎子における発現性の3点について検討し,以下の点について明らかにした。 [査読有り]研究論文（学術雑誌）Hiroshi HARAYAMA, Masashi MIYAKE, Seishiro KATO[査読有り]研究論文（国際会議プロシーディングス）Seishiro KATO, Koichiro KANO, Hiroshi HARAYAMA, Hiroshi KUSUNOKI, Iwao NANJO[査読有り]研究論文（国際会議プロシーディングス）Hiroshi HARAYAMA, Seishiro KATO[査読有り]研究論文（学術雑誌）原山 洋, 筒井 昭夫, 加藤 征史郎[査読有り]研究論文（学術雑誌）Hiroshi HARAYAMA, Iwao NANJO, Sunao KANDA, Seishiro KATO[査読有り]研究論文（学術雑誌）H HARAYAMA, NANJO, I, S KANDA, S KATOThe developmental process of the testis and age-related changes in the morphology of rete testicular spermatozoa were investigated in Meishan boars at 1 to 364 days of age. The obtained Western blots revealed that sperm-bound anti-agglutinin was detected less in the samples incubated with either BSA (4 mg mL(-1)) or MBC (5-10 mg mL(-1)), compared with control samples. Dev. 受精能獲得誘起処理に伴う精子結合AAの変化を観察した。ウエスタンブロッティングにより検出されたAAをデンシトメーターで解析したところ,精子結合AAは処理開始当初の45分間で処理前の試料の約半分まで減少したが,その後の減少は緩慢であった。間接蛍光抗体法による観察結果によると,処理に伴う精子結合AAの減少は精子頭部の先体において認められた。以上の結果から,精子結合AAの多くは受精能獲得過程の初期段階において精子先体から解離することが明らかになった。 The SPACA1 indexes were significantly correlated with developmental rates of embryos to blastocysts (r = 0.829, P = 0.00162), although they were barely associated with fertilization rates at 19 h after insemination (r = 0.289, P = 0.389). This shows that expression of Epac mRNAs is present in mouse testicular germ cells. H18年度検討内容(2)と(3):上述の好適条件で処理した精子では少なくとも9種類のタンパク質でcAMP依存的なチロシンリン酸化(pY)反応が認められたが,pYの程度はブタ精子よりかなり低レベルであった。また黒毛和種精子ではcAMP-PKA-SYKシグナリングは検出されなかったが,これとは別にcAMP-FAK様pYタンパク質シグナリングが存在することを指摘した。以上の結果は,黒毛和種精子でのHAがブタ精子とは異なる細胞内シグナリングにより制御されることを示唆している。 2. Ejaculated spermatozoa were collected from six mature boars, washed, and incubated to promote capacitation. These results indicate that increasing numbers of goat spermatozoa improve in the functions related to the acrosome reaction and subsequent fusion with the egg plasma membrane during their transit through the caput epididymidis. 2.SYKの活性化と精子受精能力発現状態との相関性の観察:活性化型SYKが出現するcAMPアナログ処理2〜3時間後の精子では超活性化運動の発現が認められた。超活性化運動は精子の卵母細胞への侵入に必要な推進力を生み出すという事実を考え合わせると,SYKは精子の受精能力発現制御因子のひとつといえる。 Based on these results, the following matters concerning the anti-agglutinin are discussed: (1) the importance of its association with the acrosome of spermatozoa in inhibiting sperm head-to-head agglutination; (2) its origin in the epididymis; and (3) its tissue specificity. Moreover, the addition of W-7 (a calmodulin antagonist, 24 mu M) enhanced the percentages of hyperactivated spermatozoa after incubation with cBiMPS and CaCl2, independently of protein tyrosine phosphorylation.