Analyses of physiological factors regulating oocyte maturation and early development of a hermaphrodite pond snail Lymnaea stagnalis

(1)钉螺从被污染的死水中转移到清洁的充气水中，可在3h内刺激其产卵。MALDI-MS分析显示，脑连接（COM）后30 min内，尾背细胞激素（CDCH）从脑连接（COM）释放到血淋巴（HL）。背体COM粗提物可诱导卵细胞-消化腺复合物体外释放配子（卵母细胞和精子）。(2)卵细胞在精管远端很快出现、移动并受精。DSO卵母细胞浸泡在蜗牛盐水、HL和蒸馏水中均可诱导减数分裂和卵裂，但在体内直到产卵前均受到严格抑制。刚有卵囊（卵膜+卵周液）的离体卵母细胞在低渗甘露醇溶液中能出现极体形成和卵裂，而在高渗甘露醇溶液中则没有。浸渍试验结果表明，低分子量的渗透物大多能立即渗透到囊内，通过内部胶体渗透压使卵泡周液始终保持比HL高渗。这似乎是卵母细胞在产卵前不能进行减数分裂的主要原因。(3)卵螺浸泡在10^<-3> ~ 10^<-1>M β-蜕皮激素溶液中，卵囊整体体积略有增加。卵泡周围液体的形成可能被这种类固醇刺激。(4)从成熟蜗牛生殖道提取液中，RT-PCR未获得蜕皮激素受体的互补DNA。(5) HPLC-EIA显示，HL中β-蜕皮激素水平在卵母细胞包装早期（蛋白腺分泌时）达到峰值，随后迅速下降。α -蜕皮激素水平逐渐升高，在产卵前后达到峰值。β-蜕皮激素可能通过卵泡周液的形成在阻止减数分裂中起重要作用。

(1) Snails could be stimulated to oviposit egg-mass within 3h by their transfer from polluted stagnant water into clean aerated one. MALDI-MS analysis revealed that caudo-dorsal cell hormone (CDCH) were released from cerebral commissure(COM) into haemolymph (HL) within 30 min after transfer. The crude extracts of COM with dorsal bodies (DB) could induce in vitro gamete-release(oocytes and sperm) from the dissected ovotesis-digestive gland complex.(2) Ovulated oocytes soon appeared, moved and fertilized in the distal part of spermoviduct (DSO). Meiosis and cleavage could be induced by immersing DSO oocytes in snail saline, HL and distilled water, while they were strictly inhibited in vivo till spawning. Isolated oocytes just spawned with capsule (egg membrane + perivitelline fluid) could show polar body formation and cleavage in hypotonic solution of mannitol but in hypertonic ones they could not. Immersion test showed that most osmolytes with low molecular weight immediately penetrate the capsule, which always keep perivitelline fluid hypertonic than HL by inner colloid osmotic pressure. This seems to be the major reason why oocyte meiosis can never be allowed before spawning.(3) Immersion of spawning snails in 10^<-3>〜10^<-1>M solution of β-ecdysone slightly increased the whole volume of egg capsule. Formation of perivitelline fluid may be stimulated by this steroid.(4) Complementary DNA of ecdysone receptor was not obtained by RT-PCR from the extract of the reproductive tract of mature snails.(5) HPLC-EIA demonstrated that β-ecdysone level in HL reached the peak at the early phase of oocyte-packaging (at the secretion of albumen gland) and rapidly decreased. Alpha-ecdysone level gradually increased and showed the peak around spawning time. Then β-ecdysone may play an important role in preventing meiosis through the formation of perivitelline fluid.