非洲鲶鱼(Clarias gariepinus, Burchell 1822)垂体细胞原代培养中产生的激素诱导排卵

O. O. Oyeleye, S. I. Ola, O. Omitogun
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引用次数: 6

摘要

本研究考察了非洲鲶鱼垂体细胞在培养基中增殖的潜力,以及在其原代培养中产生的激素诱导产卵的潜力。将胰蛋白酶化的垂体细胞分别培养于RPMI 1640、McCoy 5a和M2培养基中,并添加10%胎牛血清。雌性鲶鱼(800±200 g)采用群体倍增时间最短、细胞数最高(RPMI 1640)的培养基进行诱导。卵子在体外与精子细胞受精。测定受精率和孵化率。分别于注射前0、注射后3、6、9、12 h采集诱导产卵卵的血样。采集血浆样品,采用酶联免疫吸附法定量测定促卵泡激素(FSH)和促黄体生成素(LH)。在三种培养基中,每一种培养基在培养12 h后,细胞计数在初始播种密度4.1 × 106/ml的基础上显著增加。作为促性腺激素特异性功能生化指标的诱导产卵生物试验表明,培养垂体细胞(CPC)的成熟卵数为97.5 g/kg,新鲜垂体(FPG)诱导的成熟卵数为127.5 g/kg,促性腺激素释放激素类似物(Ovaprim)诱导的鲶鱼(800±200 g)的成熟卵数为157 g/kg,培养垂体细胞(CPC)的受精率差异显著(P < 0.05)。血浆促性腺激素的结果显示,在所有使用的诱导剂中,血浆LH和FSH在排卵的第9和12小时达到最高水平。两种垂体激素(FSH和LH)在鲶鱼卵的排卵和产卵过程中起互补作用。本研究提供了利用垂体细胞原代培养的激素诱导非洲鲶鱼产卵的可能性。关键词:垂体细胞,原代培养,诱导产卵,排卵。
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Ovulation induced in African catfish (Clarias gariepinus, Burchell 1822) by hormones produced in the primary culture of pituitary cells
This study examined the potential of catfish pituitary cells to proliferate in culture media and that of hormones produced in their primary culture to induce spawning in African catfish. The trypsinized pituitary cells were cultured in three culture media RPMI 1640, McCoy 5a and M2 media with addition of 10% fetal bovine serum. Female catfish (800±200 g) were induced using the culture medium that gave the shortest population doubling time and highest cell count (RPMI 1640). The eggs were fertilized with sperm cells in vitro. The fertilization and hatchability rates were determined. The blood samples of the induced spawners were collected at O (before injection), 3, 6, 9 and 12 h, respectively after injection. The collected samples of blood plasma were analyzed using enzyme-linked immunosorbent assay for quantitative determinations of Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH). A significant increase in the cell counts over the initial seeding density of 4.1 × 106/ml after 12 h of culture was established in each of the three culture media used. The induced spawning bio-assay which served as a biochemical marker for gonadotrophin specific function showed that  97.5 g/kg of mature oocytes were spawned from cultured pituitary cells (CPC), 127.5 g/kg from induction with fresh pituitary gland (FPG) and 157 g/Kg from gonadotrophin releasing hormone analogue (Ovaprim) induced catfishes (800±200 g). The fertilization rate of cultured pituitary cells (CPC) differed significantly (P 0.05). The results of the plasma gonadotrophin revealed that the highest levels of plasma LH and FSH were reached between 9 and 12 h of ovulation in all the inducers used. The two pituitary based hormones (FSH and LH) played complementary roles during ovulation and spawning of catfish eggs. This study provides an insight to the possibility of using hormones from the primary culture of pituitary cells to induce spawning in African catfish. Key words: Pituitary cells, primary culture, induced spawning, ovulation.
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