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Expression and cellular pattern of relaxin mRNA in porcine corpora lutea during pregnancy

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Abstract

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.

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Authors and Affiliations

  1. Laboratory of Animal Reproduction, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-Ku, Shizuoka, 422-8529, Japan

    Tetsuya Kohsaka & Keiichiro Yogo

  2. Centre of Advanced Study in Zoology, Banaras Hindu University, Varanashi, 221-005, India

    Udai Pratap Singh

  3. Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai, 981-8555, Japan

    Hiroshi Sasada

  4. Laboratory of Veterinary Physiology, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan

    Kazuyoshi Taya

  5. Laboratory of Veterinary Physiology, Faculty of Agriculture, Iwate University, Iwate, 020-8550, Japan

    Kazuyoshi Hashizume

Authors
  1. Tetsuya Kohsaka
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  2. Udai Pratap Singh
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  3. Keiichiro Yogo
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  4. Hiroshi Sasada
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  5. Kazuyoshi Taya
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  6. Kazuyoshi Hashizume
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Correspondence to Tetsuya Kohsaka.

Additional information

This work was supported in part by a Grant-in aid for Scientific Research from the Japan Society for the Promotion of Science (to T.K.) and by a postdoctoral fellowship from the Japan Society for the Promotion of Science (to U.P.S.).

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Kohsaka, T., Singh, U.P., Yogo, K. et al. Expression and cellular pattern of relaxin mRNA in porcine corpora lutea during pregnancy. Cell Tissue Res 330, 303–312 (2007). https://doi.org/10.1007/s00441-007-0492-5

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  • DOI: https://doi.org/10.1007/s00441-007-0492-5

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