Contents lists available at ScienceDirect Reproductive Biology journal homepage: www.elsevier.com/locate/repbio Original article Effects of CDK inhibitors on the maturation, transcription, and MPF activity of porcine oocytes Reza K. Oqani, Tao Lin, Jae Eun Lee, So Yeon Kim, Jung Won Kang, Dong Il Jin ⁎ Department of Animal Science and Biotechnology, Research Center for Transgenic Cloned Pigs, Chungnam National University, Yuseong-gu, Daehak-ro 99, Daejeon, 34134, Republic of Korea ARTICLE INFO Keywords: CDK Cumulus cell Germinal vesicle Transcription Porcine oocyte ABSTRACT In mammals, cyclin-dependent kinases (CDKs) are involved in regulating both the cell cycle and transcription. Although CDK1 is known to act as the kinase subunit of maturation-promoting factor (MPF), the roles of the other CDKs in mammalian oocyte maturation are not yet understood. Here, we show that inhibition of various CDKs by small molecule inhibitors has different effects on the maturation and transcriptional activity of pig oocytes in vitro. Inhibition of CDK1 did not significantly affect cumulus cell expansion, but its kinase activity was necessary for germinal vesicle breakdown (GVBD). The inhibitions of CDK2, CDK4, or CDK6 had no effect on cumulus expansion or GVBD. The catalytic activity of CDK7 was crucial for GVBD but less important for cumulus expansion, whereas inhibition of CDK9 severely blocked both cumulus cell expansion and GVBD. CDK1, -2, -4, and -6 appeared to be dispensable for nuclear transcription, as their inhibitions did not affect nascent RNA production in oocytes. However, inhibition of CDK7 or CDK9 dramatically decreased the transcriptional activity in oocytes. Finally, we found that the GVBD arrest triggered by CDK9 inhibition was not due to altered MPF activity, but rather the inhibition of transcription. Overall, our results show that CDK7 and CDK9 are important for the nuclear maturation and transcriptional activity of pig oocytes. 1. Introduction Mammalian oocytes are transcriptionally active, producing and storing maternal factors (mainly mRNAs and proteins) that support the early stages of embryonic development. Studies have shown that treating mammalian oocytes with transcription inhibitors triggers de- fects in their subsequent growth and maturation. For example, treat- ment of mouse oocytes with the positive transcription elongation factor b (P-TEFb) inhibitor, DRB (5,6-dichloro-1-beta-D-ribofuranosy-benzi- midazol), leads to failure of germinal vesicle breakdown (GVBD) [1]. The oocytes of large animals have also been found to arrest at the GV stage when treated with transcription inhibitors. Bovine oocytes, for instance, arrest at the GV stage in the presence of α-amanitin [2] or DRB [3], while porcine oocytes show GV arrest when they are exposed to α-amanitin in vitro [4]. Moreover, protein synthesis inhibitors (e.g., cycloheximide) cause GV arrest in both bovine and porcine oocytes [2,5,6]. Therefore, it seems that both mRNA transcription and protein synthesis are needed for oocyte maturation in mammals. The numerous maternal proteins that are produced and stored during oocyte growth include various protein kinases. Among them are the cyclin-dependent kinases (CDKs), which are predominantly involved in cell cycle regulation [7]. The meiotic maturation of an oocyte involves dynamic waves of protein phosphorylation that are partially regulated by the activation of CDKs. The first and best-char- acterized oocyte-maturation-associated CDK is CDK1. This catalytic subunit of maturation-promoting factor (MPF) was initially obtained from frog egg extracts [8,9] and is activated by its binding to a cyclin subunit; this activation triggers GVBD and the transition from GV stage to metaphase I. CDK1 is activated by the phosphorylation of its Thr161 residue by CDK-activating kinase (CAK) [10,11]. For meiotic prophase I arrest to occur, CDK1 must be inactivated; this occurs mainly via the phosphorylations of the Thr14/Tyr15 residues of CDK1 by the mem- brane associated tyrosine/threonine 1 (MYT1) and Wee1B kinase, re- spectively [12,13]. Among the other CDK family members, CDK2 is known to form a complex with its regulatory partners, cyclin E or cyclin A, to regulate several cell cycle events [14], such as the phosphorylation of retino- blastoma protein (Rbp), which supports the transition from G1 to S phase. Cdk2-knockout mice are viable, however, and it seems that CDK1 compensates for their lack of CDK2 activity [15,16]. Both CDK4 and CDK6 bind to cyclin Ds and act in the G1 phase of the cell cycle. These two CDKs also phosphorylate Rbp and induce DNA synthesis and http://dx.doi.org/10.1016/j.repbio.2017.09.003 Received 19 May 2017; Received in revised form 1 September 2017; Accepted 2 September 2017 ⁎ Corresponding author. E-mail address: dijin@cnu.ac.kr (D.I. Jin). Reproductive Biology xxx (xxxx) xxx–xxx 1642-431X/ © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved. Please cite this article as: Oqani, R.K., Reproductive Biology (2017), http://dx.doi.org/10.1016/j.repbio.2017.09.003