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Journal of Molecular Endocrinology (2010) 44 45-53    DOI: 10.1677/JME-08-0179
© 2010 Society for Endocrinology
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Identification of protein kinases that control ovarian hormone release by selective siRNAs

 Alexander V Sirotkin1,2, Dmitriy Ovcharenko3 and Milos Mlyncek4

1 Institute of Genetics and Reproduction, Animal Production Research Centre, Hlohovecká 2, 951 41 Luzianky near Nitra, Slovakia
2 Constantine the Philosopher University, 949 74 Nitra, Slovakia
3 Altogen Biosystems, Austin, Texas, USA
4 Polyclinics and Hospital of Nitra, 94901 Nitra, Slovakia

(Correspondence should be addressed to A V Sirotkin at Research Institute of Animal Production Nitra, Animal Production Research Centre; Email: sirotkin{at}scpv.sk)

The goal of this study was to identify protein kinases (PKs) that control the secretory activity of human ovarian cells. Cultured ovarian granulosa cells were transfected with 264 siRNA constructs that selectively block the expression of 88 known PKs. The efficiency of transfection and of silencing marker molecules (glyceraldehyde 3-phosphate dehydrogenase, GAPDH and CDC2/p34 PK) was validated by fluorescence microscopy, real-time reverse transcription-PCR, and immunocytochemistry. Release of steroid hormones (progesterone, P4) and IGF1 was determined by RIA. siRNA suppressed the expression of marker molecules by up to 84%. P4 release was suppressed after inhibiting 34 individual PKs and was stimulated after inhibiting 12 PKs. Blocking nine individual PKs inhibited IGF1 release, while the inactivation of 17 others stimulated IGF1 release. Together, these results demonstrate that the release of both steroid and peptide hormones by human ovarian cells is controlled by a large number of PKs, and that siRNA constructs may be useful tools for further defining the role of PKs in controlling ovarian secretory function.






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