Expression and cellular pattern of relaxin mRNA in porcine corpora lutea during pregnancy
Expression and cellular pattern of relaxin mRNA in porcine corpora lutea during pregnancy
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DOI：
10.1007/s00441-007-0492-5
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发表时间：
2007-11-01
影响因子：
3.6
通讯作者：
Hashizume, Kazuyoshi
中科院分区：
文献类型：
--
作者：
Kohsaka, Tetsuya;Singh, Udai Pratap;Hashizume, Kazuyoshi
采用非放射性探针和微波固定技术，建立了猪黄体松弛素mRNA的原位杂交检测方法。随后，我们研究了松弛素mRNA的表达和细胞模式在CL在怀孕期间，然后评估松弛素mRNA是否是一个限制激素生产的CL的因素。地高辛（DIG）标记的RNA探针互补的猪松弛素mRNA的体外转录。通过北方分析证实，反义探针与CL中的1.0-kb松弛素转录物杂交，从而验证了特异性。微波固定（在常规微波炉中照射2分钟）与地高辛标记的cRNA探针相结合，可以精确可靠地分析松弛素mRNA，具有上级mRNA保留和更高的分辨率。应用这种方法在猪CL在怀孕期间表明，松弛素mRNA水平每细胞和mRNA表达细胞的百分比随着妊娠的进展而增加，与近期显着下降。北方分析揭示了松弛素mRNA定位的细胞模式，表明松弛素mRNA随着妊娠的进展而增加，这归因于细胞mRNA水平和mRNA表达细胞百分比的增加。目前的研究结果，连同已知的松弛素水平的CL，揭示松弛素mRNA的变化与变化的激素在CL在怀孕期间，这表明松弛素水平是由mRNA的量可用于翻译。
We developed an in situ hybridization method for detecting relaxin mRNA in the porcine corpus luteum (CL) by employing a non-radioactive probe and microwave fixation. We subsequently examined the expression and cellular patterns of relaxin mRNA in the CL during pregnancy and then evaluated whether relaxin mRNA was a factor limiting hormone production by the CL. Digoxigenin (DIG)-labeled RNA probes complementary to porcine relaxin mRNA were produced by in vitro transcription. The specificity was validated by showing, by Northern analysis, that the anti-sense probe hybridized to a 1.0-kb relaxin transcript in the CL. Microwave fixation (2-min irradiation in a conventional microwave oven) combined with DIG-labeled cRNA probes allowed precise and reliable analysis of relaxin mRNA, with superior retention of the mRNA and a higher resolving power. Application of this method to the porcine CL during pregnancy demonstrated that the relaxin mRNA level per cell and the percentage of mRNA-expressing cells increased as gestation progressed, with a marked decline near term. Northern analysis revealed the cellular pattern of relaxin mRNA localization, showing that the increase of relaxin mRNA with advancing pregnancy was attributable to an increase of both the cellular mRNA level and the percentage of mRNA-expressing cells. The present findings, taken together with known relaxin levels in the CL, reveal that changes of relaxin mRNA are correlated with changes of the hormone in the CL during pregnancy, suggesting that the relaxin level is determined by the amount of mRNA available for translation.