Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (12): 2389-2396.doi: 10.3864/j.issn.0578-1752.2016.12.014

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Research on the Expression and Localization of CART in Bovine Granulosa Cells at Different Developmental Stages

LI Peng-fei1, BI Xi-lin2, WANG Kai2, JING Jiong-jie2, LÜ Li-hua2   

  1. 1College of Life Science, Shanxi Agricultural University, Taigu 030801, Shanxi
    2College of Animal Science and Technology, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2015-12-01 Online:2016-06-16 Published:2016-06-16

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Abstract: 【Objective】The objective of this paper is to investigate the effect of CART (cocaine-and amphetamine- regulated transcript) expression in granulosa cells on bovine follicular development at different stages.【Method】 Two RNA libraries from ODF1 (the largest onset of deviation follicle) and ODF2 (the second largest onset of deviation follicle) granulosa cells were prepared and sequenced using Illumina HiSeq 2000 platform. Cows were slaughtered, and both ovaries were collected, then the largest and the second largest follicles were screened through the morphology of corpus luteum in the first follicular wave. Follicular fluid was extracted for the concentration of E2 and P measurement using competitive ELISA method to determine DF (dominant follicles) and SF (subordinate follicles). Granulesa cells were isolated from DF and SF, and total RNA were extracted, respectively. Using RPLP0 as a reference gene, primers were designed. qRT-PCR was performed to detect the expression of CART in DF and SF, GraphPad Prism 5.0 was used for construction and significant analysis. Rabbit anti-CART antibody was used to detect the CART expression in cow follicles.【Result】Deep sequencing results showed that CART amounts in ODF1 were 38.2 times more than that in ODF2. Ovaries of 3 cows were screened out which were in the first follicular wave according to the observation of the morphology of corpus luteum, and the DF and SF were obtained. Real-time PCR results showed that CART expressed in DF was extremely significantly higher than that in SF, up to 2310-fold, which is consistent with the deep sequencing results. Immunohistochemistry results showed that CART expressed both in DF and SF, and the amounts in DF was significantly greater than that in SF, at the same time, CART was mainly expressed in granulosa cell layer.【Conclusion】During the process of follicular development in cattle, other inhibitors have suppressed most of the follicles, and prevented them to develop into DF; when the deviation appears, CART inhibited DF to further develop into ovulatory follicle, so it is considered that CART possibly by promoting granulosa cells apoptosis, and suppresses the E2 secretion in granulosa cell layer, eventually leads to DF atresia.

Key words: bovine, follicle, development, CART, expression

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