Effect of CDK9 inhibition on transcriptional activity of GV oocytes.

<p><b>A)</b> COCs were collected from different size of follicles and immediately cultured in a complete maturation medium in presence of 5 mM FU for 1 hour. Then, COCs were denuded and oocytes were fixed and subjected to immunostaining. In NSN oocytes in which CDK9 is distributed throughout the GV, transcriptional activity is at the highest level. In SN oocytes, CDK9 accumulates around the NLB and nascent RNAs become undetectable. <b>B)</b> COCs were subjected or not to α-amanitin, actinomycin D (ActD), or Flavopiridol for 1 hour. Treatment with α-amanitin started 1 hour ahead of the other compounds. At the same time, nascent transcripts were labeled with FU. Then, COCs were denuded and oocytes were fixed and subjected to immunostaining. In α-amanitin-treated oocytes, nucleoplasmic transcription decreased dramatically, but a rim of transcripts remained around the nucleolus. In oocytes treated with ActD, no transcriptional activity was detected. In Flavopiridol-treated oocytes, similarly, almost all the RNA transcription was abolished. Similar result was obtained when oocytes treated with CDK9 Inhibitor II (CAN508).</p

Effect of CDK9 inhibition on transcriptional activity of preimplantation embryos.

<p><b>A)</b> The susceptibility of FU labeling was determined in preimplantation embryos. Embryos were cultured in PZM-3 medium. For nascent RNA labeling, embryos were transferred to the same medium plus 5mM FU and cultured for additional 1 hour. Then embryos were fixed and subjected to immunostaining. While 2-cell embryos showed very faint, if any, nascent RNA level, 4-cell stage embryos and blastocysts exhibited strong nuclear signals from FU incorporation into nascent RNAs. <b>B)</b> IVF embryos were cultured in PZM-3 medium in presence or absence of 100 nM Flavopiridol. One hour before fixation, embryos were transferred to the same media supplemented with FU. CDK9 inhibition dramatically decreased the level of nascent RNAs in late 4-cell stage embryos. The level of nuclear CDK9 also decreased slightly in these embryos. <b>C)</b> Treatment of late 4-cell embryos for 1 hour with Flavopiridol abolished the pre-rRNA transcription. Although the signal corresponding to both 5’ETS and 28S rRNA was evident in the center of NPBs in most blastomeres’nuclei of untreated control embryos, such signals were not observed in their treated counterparts. <b>D)</b> Blastocysts were treated or not with Flavopiridol for 1 hour and simultaneously were labeled with FU. Then embryos were fixed and co-immunostained against FU and CDK9. In control group, the transcriptional activity was at higher level in the areas with less DAPI staining and corresponding to nucleoli. CDK9 also predominated in these areas. In treated embryos, both nucleoplasmic and nucleolar nascent RNAs were declined. Also nucleolar localization of CDK9 was unraveled. Only individual cells from the whole blastocysts are shown.</p

P-TEFb association with nucleolar proteins in the germinal vesicle oocytes at different stages of growth.

<p><b>A)</b> CDK9 is colocalized with the DFC component, fibrillarin, only at the periphery of the NLB in NSN oocytes. In pNSN oocytes, nucleolar structure is changed and fibrillarin and other nucleolar components move to the periphery of the NLB. In this stage, CDK9 shows partial colocalization with fibrillarin (inset). In SN oocytes, fibrillarin is mostly gone, but CDK9 remains bound to the nucleolar structure. At the periphery of the NLB, CDK9 shows more colocalization with the FC component, UBF, in pNSN (inset). <b>B)</b> The same pNSN oocytes as in A was analyzed further. Eight optical sections, Z-stacks with 0.5 μm, were taken by confocal microscope confirming the colocalization of CDK9 and fibrillarin at the periphery of NLB. <b>C)</b> X and Y axes of a single focal plane of the same image as B focusing on an area corresponding to the NLB periphery, indicating the spatially colocalization of CDK9 and fibrillarin in that area. <b>D)</b> Cyclin T1 also shows colocalization with nucleolar factor, UBF, in pNSN and pSN oocytes. DNA counterstained with DAPI. <b>E)</b> COCs were collected separately from small or large follicles and then were denuded and subjected to Western blotting. The same anti-CDK9 antibody as used for immunostaining was used for Western blot. The bands correspond to two CDK9 isoforms, 42 kD and 55 kD. Fifty oocytes were collected for each group.</p

Subcellular localization of P-TEFb components in GV of fully grown pig oocytes.

<p><b>A)</b> CDK9, Cyclin T1 and Cyclin T2 are present and located predominantly in the nuclei of pig GV oocytes. CDK9 and Cyclin T1 also show speckles in GV oocytes. Rabbit polyclonal antibodies were used for immunostaining. <b>B)</b> CDK9 and Cyclin T1 speckle-like structures are colocalized with Pol II CTD Ser2p in GV of NSN and pNSN oocytes. In SN oocytes, CDK9 tends to accumulate at the periphery of the NLB and also dissociate from Pol II CTD Ser2p speckles. <b>C)</b> CDK9 also colocalizes with the splicing speckle marker SC-35 protein (red) in GV of pNSN oocytes. In transcriptionally inactive SN oocytes, SC-35 is dispersed throughout the nucleoplasm but CDK9 aggregates around the NLB. SC-35 is immunostained with a mouse monoclonal antibody. DNA is counterstained with DAPI.</p

The presence and the necessity of CDK9 in preimplantation embryos.

<p>Confocal immunofluorescence analysis of CDK9 in whole-mount IVF and parthenogenetic embryos. <b>A)</b> CDK9 nuclear localization in IVF embryos. Pol II (red) was used as the nuclear marker. No intense signal at the periphery of the nucleolar precursor bodies was observed for CDK9. <b>B)</b> CDK9 nuclear localization in parthenogenetic embryos. Nucleolin (red) was used as the nucleolar marker. In 1-cell embryos, CDK9 was evenly distributed throughout pronuclei. At the 2-cell stage, CDK9 showed some localization at the periphery of NPBs in some cases. In late 4-cell embryos, CDK9 exhibited more profound nucleolar association and colocalized with nucleolin. As nucleolar precursors transformed to matured nucleoli in morula and blastocysts, CDK9 localized within the nucleoli with and surrounded by nucleolin. DNA was counterstained with DAPI. Enlarged views of the small insets are shown in the right panels. <b>C)</b> Embryos were produced via either IVF or parthenogenesis and cultured absence or presence of Flavopiridol (100nM) or CAN508 (10 μM) for 7 days. Then, the embryos were counterstained with DAPI and were analyzed under a fluorescent microscope. Embryos with zero nuclei were excluded. The first cleavage rate was not significantly changed in treated embryos. However, the majority of treated embryos tended to remain at 4-cell stage. Also, progress to next cell cycles was very poor in treated embryos compared with their control groups. No blastocyst was observed in groups treated with either CDK9 inhibitor.</p

Effect of CDK9 inhibition on oocyte maturation and the MPF kinase activity.

<p><b>A)</b> COCs were obtained from small and medium size antral follicles and matured in absence or presence of increasing concentration of Flavopiridol or CAN508. Then COCs were denuded and fixed and subjected to immunostaining against nuclear lamin A/C. DNA counterstained with DAPI. Oocytes with two distinct metaphase chromosomes and polar body were considered as MII. Oocytes with only one metaphase chromosome and no polar body were considered as MI. Oocytes with intact germinal vesicle and positive lamin A/C staining were considered as GV. The figure shows that inhibition of CDK9 strictly inhibits GVBD in pig oocytes. <b>B)</b> COCs were obtained from large antral follicles and treated or not with CDK inhibitors, Flavopiridol, Dinaciclib and JNJ-7706621 for 6 hours in complete maturation medium. Then, COCs were denuded and subjected to immunostaining. MPF/CDK1 kinase activity was measured using a monoclonal antibody recognizing the proteins that share two peptide motives LTPLK and FTPLQ. CDK1 phosphorylates the Thr residue of these motives and promotes GVBD. MPM-2 (green) recognizes these motives only when they are phosphorylated. Although treatment with Flavopiridol blocked GVBD, but the level of MPM-2 signal did not changed significantly compared with untreated oocytes. On the other hand, both Dinaciclib and JNJ-7706621 (JNJ) decline the level of CDK1 kinase activity. <b>C)</b> CDK1 activity is inhibited by phosphorylation of Thr14/Tyr15 residues. Dephosphorylation of these residues activates CDK1 and promotes GVBD. In Flavopiridol-treated oocytes, the level of pCDK1 did not change compared with the control group, but Dinaciclib, and less potently JNJ, elevated the level of inhibitory phosphorylation of CDK1. In all groups, the level of pCDK1 was normalized with pan CDK1. <b>D)</b> COCs were obtained from small or medium size antral follicles. Inhibition of CDK9 by Flavopiridol or Dinaciclib inhibited transcriptional activity of GV oocytes, but inhibition of CDK1 by JNJ did not changed the level of nascent RNAs compared with untreated oocytes. These experiments totally showed that GVBD block by Flavopiridol was due to inhibition of transcription but not inhibition of CDK1 activity.</p

Method To Purify and Analyze Heterogeneous Senescent Cell Populations Using a Microfluidic Filter with Uniform Fluidic Profile

To precisely purify and study aged (senescent) cells, we have designed, fabricated, and demonstrated a novel diamond-structure (DS) microfluidic filter. Nonuniform flow velocities within the microfilter channel can compromise microfluidic filter performance, but with this new diamond structure, further optimized via simulation, we achieve a uniform microfilter flow field, improving the throughput of size-based separation of senescent cells, as obtained by 39-passaged human dermal fibroblasts. After separating these aged cells into two groups, consisting of large- and small-sized cells, we assessed senescence by measuring lipofuscin accumulation and β-galactosidase activity. Our results reveal that even though these senescent cells had been equivalently passaged in culture, a high degree of size distribution and senescent phenotype heterogeneity was observed. In particular, the smaller-sized cells tended to express a younger phenotype while the larger aged cells demonstrated an older phenotype. We suggest that size-based separation of senescent cells, subtyped into small- and large-sized cohorts, offers an alternative method to purify such aged cells, thereby enabling more precise study of the mechanisms of aging, autophagy impairment, and rejuvenation