Name: inactivation of mtor: a tool to investigate meiotic progression and translational control during bovine oocyte maturation
Uploaded: 2015-09-24T15:03:52
Description: Although routinely used in breeding programs, in vitro maturation (IVM) of bovine oocytes and in vitro production (IVP) of embryos are nevertheless still ...

Manuela Krei&#223;elmeier and Sophia Mayer were scholarship holders of the Dr. Dr. Karl-Eibl-Stiftung. We wish to thank Gesine Kr&#252;ger and Petra Reckling for excellent technical assistance.

All experiments were performed in accordance with the guidelines of the local ethics committee (Landesamt f&#252;r Landwirtschaft, Lebensmittelsicherheit und Fischerei, Mecklenburg-Vorpommern, Germany). The ovaries were obtained from a commercial slaughterhouse. The remaining carcass was used for meat production.
1. Solutions to be prepared.
<ol>
	<li>Prepare PBS for gaining oocytes by weighing Glucose (500 mg), Pyruvate (18 mg), Penicillin (10 mg), Streptomycin (20 mg), Heparin (5.6 mg) and...

<ol>
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M., Motlik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKB/AKT+is+involved+in+resumption+of+meiosis+in+mouse+oocytes.">PKB/AKT is involved in resumption of meiosis in mouse oocytes.</a> Biology of the Cell. 98, 111-123 (2006).</li><li>Mayer, S., Wrenzycki, C., Tomek, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+mTor+arrests+bovine+oocytes+in+the+metaphase-I+stage,+despite+reversible+inhibition+of+4E-BP1+phosphorylation.">Inactivation of mTor arrests bovine oocytes in the metaphase-I stage, despite reversible inhibition of 4E-BP1 phosphorylation.</a> Mol Reprod Dev. 81 (4), 363-375 (2014).</li><li>Dowling, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1-mediated+cell+proliferation,+but+not+cell+growth,+controlled+by+the+4E-BPs.">mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E-BPs.</a> Science. 328, 1172-1176 (2010).</li><li>Susor, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+regulation+of+translation+in+the+mammalian+oocyte+via+the+mTOR-eIF4F+pathway.">Temporal and spatial regulation of translation in the mammalian oocyte via the mTOR-eIF4F pathway.</a> Nat Commun. 28 (6), 6078 (2015).</li><li>Tomek, W., Wollenhaupt, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&amp;#34;closed+loop+model&amp;#34;+in+controlling+mRNA+translation+during+development.">The &amp;#34;closed loop model&amp;#34; in controlling mRNA translation during development.</a> Anim Reprod Sci. 134, 2-8 (2012).</li><li>Ballou, L. 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(44), e2359 (2010).</li><li>Wollenhaupt, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+occurrence+and+physiological+role+of+a+truncated+eIF4E+in+the+porcine+endometrium+during+implantation.">Natural occurrence and physiological role of a truncated eIF4E in the porcine endometrium during implantation.</a> Biochem J. 432, 353-363 (2010).</li><li>Lee, S. E., Sun, S. C., Choi, H. Y., Uhm, S. J., Kim, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR+is+required+for+asymmetric+division+through+small+GTPases+in+mouse+oocytes.">mTOR is required for asymmetric division through small GTPases in mouse oocytes.</a> Mol Reprod Dev. 79, 356-366 (2012).</li><li>Tomek, W., Smiljakovic, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Akt+(protein+kinase+B)+stimulates+metaphase+I+to+metaphase+II+transition+in+bovine+oocytes.">Activation of Akt (protein kinase B) stimulates metaphase I to metaphase II transition in bovine oocytes.</a> Reproduction. 130, 423-430 (2005).</li><li>Tomek, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+translation+during+in+vitro+maturation+of+bovine+oocytes:+the+role+of+MAP+kinase,+eIF4E+(cap+binding+protein)+phosphorylation,+and+eIF4E-BP1.">Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1.</a> Biol Reprod. 66, 1274-1282 (2002).</li><li>Torner, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+subcellular+characterization+of+oocytes+screened+for+their+developmental+competence+based+on+glucose-6-phosphate+dehydrogenase+activity.">Molecular and subcellular characterization of oocytes screened for their developmental competence based on glucose-6-phosphate dehydrogenase activity.</a> Reproduction. , 197-212 (2008).</li><li>Romasko, E. J., Amarnath, D., Midic, U., Latham, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Maternal+mRNA+and+Phosphorylated+EIF4EBP1+Variants+with+the+Spindle+in+Mouse+Oocytes:+Localized+Translational+Control+Supporting+Female+Meiosis+in+Mammals.">Association of Maternal mRNA and Phosphorylated EIF4EBP1 Variants with the Spindle in Mouse Oocytes: Localized Translational Control Supporting Female Meiosis in Mammals.</a> Genetics. 195 (2), 349-358 (2013).</li><li>Morita, M., Alain, T., Topisirovic, I., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+Profiling+Analysis.">Polysome Profiling Analysis.</a> Bio-protocol. 3 (14), e833 (2013).</li><li>Scantland, S., Grenon, J. P., Desrochers, M. H., Sirard, M. A., Khandjian, E. 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In vitro maturation (IVM) of bovine oocytes is an important technique because it is an integral part of the in vitro production of embryos in specific breeding programs. However, a critical limitation is the fact that no method exists to assess the developmental competence of fully grown oocytes directly after follicular release. Furthermore, when the chromatin status of bovine oocytes is analyzed morphologically, more than 90 % have reached the M II stage after 24 h of IVM under standard conditions1. However, transfera...

Fully grown bovine oocytes (arrested at prophase I; germinal vesicle -GV- stage) resume meiosis spontaneously when they are released from their follicles and transferred to a suitable culture medium. Previous investigations using the application of different inhibitors to in vitro culture media1, 2 revealed that activation of protein kinases and de novo protein synthesis trigger the maturation of mammalian oocytes and arrest them in metaphase II (M II), the stage suitable for fertilization. In the present study we describe a method to inhibit the Ser/Thr kinase mTorduring IVM of bovine oocytes. This approach might provide deeper insights into the complex processes involved in the regulation of protein synthesis in the context of meiotic maturation (transition from GV-stage to M II), because mTor links the phosphorylation of specific factors directly to translational control3, 4.
The focus on the investigation of translational control reflects the importance of this process; fully grown oocytes are transcriptionally silent and protein synthesis relies on the activation of stored, dormant mRNAs5. In this context, mTor plays a predominant role. The kinase directly phosphorylates and inactivates repressors of the mRNA cap-binding protein eIF4E, the so-called 4E-binding proteins (4E-BP1-3), and thereby allows the formation of the 5&#180;-mRNA-cap binding complex eIF4F (composed of eIF4E, the scaffold protein eIF4G and the RNA helicase eIF4A). Together with other factors it also stimulates ribosome binding and translation initiation6.
mTor, however, exists as two complexes: mTorC1 and mTorC2. Each complex is composed of different major regulators, differs in sensitivity to Rapamycin and has different cellular targets7. The major regulator of mTorC1, Raptor (regulatory-associated protein of mTOR), phosphorylates components of the translational machinery, namely ribosomal proteins (for instance RPS6 at Ser235/36) and the translational repressor 4E-BP1 (at Thr37/46/65/70). The major regulator of mTorC2, Rictor (Rapamycin-insensitive companion of mTOR), is Rapamycin- resistant and phosphorylates Akt (PKB) which in turn phosphorylates mTorC1. Preliminary investigations in bovine oocytes during IVM revealed different transient activities of mTorC1 and mTorC2 during IVM. In the GV-stage of oocytes the mTorC2 is active3 ; it is inactivated in the course of IVM. In contrast, mTorC1 shows the opposite behavior3. These results correspond with findings showing that 4E-BP1 phosphorylation is lower in the GV-stage, continuously increases during IVM, and is highest in the M II stage8, 9.
However, mTorC1 and C2 both respond to the active site inhibitor Torin210 and might have other (yet unknown) targets. Candidates are meiotic spindle-forming or regulatory proteins, since mTor associates with meiotic spindles during chromatin segregation.
From a practical point of view, it should be noted that in vitro systems yield only 30-40 % transferable embryos in the bovine species 11. The causes for this could be suboptimal in vitro conditions and/or differences in the developmental competence of the starting oocytes which occur despite their selection from follicles of a defined size. However, detailed investigations of meiotic maturation on a molecular level can contribute to the optimization of IVM systems. Furthermore, oocytes might be selected according to their developmental competence, for instance by IVM systems under inhibitory conditions (see discussion). Hence, in the procedure presented here, we used two independent mTor inhibitors, Torin2 and Rapamycin, which resulted in different chromatin statuses and differential phosphorylation of 4E-BP1. Interestingly, approximately 20 % of the oocytes overcame the Torin2 block and might thus be candidates which possess a high developmental competence.

<table border="0" cellpadding="0" cellspacing="0" width="643">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS Dulbecco wCa2+ wMg2+</td>
			<td style="width:141px;">Biochrom AG</td>
			<td style="width:119px;">L1815</td>
			<td style="width:167px;">For gaining of the COC and denudation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-(+)-Glucose</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">G5400-250G</td>
			<td>Supplement to PBS Dulbecco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">P3662-25G</td>
			<td>Supplement to PBS Dulbecco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin G sodium salt</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">P3032-25 MU</td>
			<td>Supplement to PBS Dulbecco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin sulfate salt</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">S1277-50G</td>
			<td>Supplement to PBS Dulbecco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heparin sodium salt</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">H3149-25 KU</td>
			<td>Supplement to PBS Dulbecco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine serum albumin</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">A9647-10G</td>
			<td>Supplement to PBS Dulbecco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">S4019-500G</td>
			<td>Supplement to PBS Dulbecco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gentamicin sulfate salt</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">G3632-250MG</td>
			<td>Component of TCM medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCM-199 Hepes Modification</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">M2520-1L</td>
			<td>Component of TCM medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride (NaCl)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">S5886-500G</td>
			<td>Component of 0.9% NaCl solution</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Rapamycin</td>
			<td style="width:141px;">Cell Signaling Technology (CST)</td>
			<td style="width:119px;">9904 S</td>
			<td>m-Tor-inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:141px;">Serva</td>
			<td style="width:119px;">39757.01</td>
			<td>Used as solubilizer for Rapamycin and Torin 2</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Torin 2</td>
			<td style="width:141px;">R&#38;D Systems</td>
			<td style="width:119px;">4248/10</td>
			<td>m-Tor-inhibitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphat Buffered Saline Tablets (PBS)</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">P-4417</td>
			<td>for PBS-pr&#228;paration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20</td>
			<td style="width:141px;">Serva</td>
			<td style="width:119px;">39796.01</td>
			<td>permeabilization buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Roti-Immuno-Block</td>
			<td style="width:141px;">Roth</td>
			<td style="width:119px;">T144.1</td>
			<td>Blockingsolution+Lsg.f&#252;r AK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X100</td>
			<td style="width:141px;">Serva</td>
			<td align="right" style="width:119px;">37240</td>
			<td>permeabilization buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes</td>
			<td style="width:141px;">Serva</td>
			<td align="right" style="width:119px;">25245</td>
			<td>permeabilization buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Albumin Fraktion V(biotinfrei)</td>
			<td style="width:141px;">Roth</td>
			<td style="width:119px;">0163.3</td>
			<td>permeabilization buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D(+)-Saccharose</td>
			<td style="width:141px;">Roth</td>
			<td style="width:119px;">4621.1</td>
			<td>permeabilization buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nacl</td>
			<td style="width:141px;">Roth</td>
			<td style="width:119px;">3957.1</td>
			<td>permeabilization buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgCl2x6 H2O</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">M2393</td>
			<td>permeabilization buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:141px;">Sigma</td>
			<td style="width:119px;">P-6148</td>
			<td>Fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kaisers Glyceringelatine</td>
			<td style="width:141px;">Merck</td>
			<td align="right" style="width:119px;">109,242</td>
			<td>cover oocytes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alex-Fluor&#160; 546F(ab)2 fragment goat anti rabbit IgG( H+L)</td>
			<td style="width:141px;">MOBITEC</td>
			<td style="width:119px;">A11071</td>
			<td>secondary antibody fluorecent labeled</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SYBR Green nucleic acid gel stain</td>
			<td style="width:141px;">Invitrogen</td>
			<td style="width:119px;">S7563</td>
			<td>DNA staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4E-BP1</td>
			<td style="width:141px;">CST</td>
			<td align="right" style="width:119px;">9452</td>
			<td>primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">p4E-BP1 Thr37/46</td>
			<td style="width:141px;">CST</td>
			<td align="right" style="width:119px;">2855</td>
			<td>primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti rabbit IgG-HRP</td>
			<td style="width:141px;">CST</td>
			<td align="right" style="width:119px;">7074</td>
			<td>secondary antiboday HRP labelled</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ECL prime</td>
			<td style="width:141px;">GE healthcare</td>
			<td style="width:119px;">RPN2232</td>
			<td>WB detection</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Fluid filter Infufil 0,2&#181;m 5,7 cm2</td>
			<td style="width:141px;">Fresenius Kabi</td>
			<td align="right" style="width:119px;">2909702</td>
			<td>Used for sterile filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette pipetman P10 0,1-10&#181;l</td>
			<td style="width:141px;">Gilson</td>
			<td style="width:119px;"></td>
			<td>Used for transferring denudated oocytes and embryos and volumina of 0,1-10&#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette Reference 10-100&#181;l</td>
			<td style="width:141px;">Eppendorf</td>
			<td align="right" style="width:119px;">4920000059</td>
			<td>Used for transferring COC and volumina of 10-100&#181;l and for denudation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette Reference 100-1000&#181;l</td>
			<td style="width:141px;">Eppendorf</td>
			<td align="right" style="width:119px;">4920000083</td>
			<td>Used for transferring volumina of 100-1000&#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro-classic pipette controller</td>
			<td style="width:141px;">Brand</td>
			<td align="right" style="width:119px;">25900</td>
			<td>Used for transferring COC&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micropipettes intraMark 20&#181;l</td>
			<td style="width:141px;">Brand</td>
			<td align="right" style="width:119px;">708718</td>
			<td>Used for transferring COC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Safe-lock tubes 0,5 ml</td>
			<td style="width:141px;">Eppendorf</td>
			<td style="width:119px;">0030 121.570</td>
			<td>Used to store volumina up to 0,5 ml</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Polypropylene centrifuge tube with conical base 50 ml, 30,0/115mm</td>
			<td style="width:141px;">Greiner</td>
			<td align="right" style="width:119px;">210261</td>
			<td>Used for the aspirated fluid to sediment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pasteur pipette 7 ml</td>
			<td style="width:141px;">VWR</td>
			<td style="width:119px;">612-1681</td>
			<td>Used for transferring the sediment to screened Petri dishes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Germ count dish with vents 90/16 mm</td>
			<td style="width:141px;">Greiner</td>
			<td align="right" style="width:119px;">633175</td>
			<td>Used for searching the COC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish with vents 35/10 mm</td>
			<td style="width:141px;">Greiner</td>
			<td align="right" style="width:119px;">627102</td>
			<td>Used for medium preparation, washing the COC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish with vents 60/15 mm</td>
			<td style="width:141px;">Greiner</td>
			<td align="right" style="width:119px;">628102</td>
			<td>Used for medium preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multidish 4 wells</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">176740</td>
			<td>Used for washing the COC, for COC maturation, embryo culture and for denudation of the oocytes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue culture dish 35/10 mm, 4 compartments</td>
			<td style="width:141px;">Greiner</td>
			<td align="right" style="width:119px;">627170</td>
			<td>Used for washing the COC&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope slides 76x26x1mm</td>
			<td style="width:141px;">Thermo Scientific</td>
			<td style="width:119px;">AB00000112E</td>
			<td>For fast morphological inspection by Aceto-Orcein-staining</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope cover glasses, 18x18 mm</td>
			<td style="width:141px;">VWR</td>
			<td style="width:119px;">ECN 631-1567</td>
			<td>For fast morphological inspection by Aceto-Orcein-staining</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Needle 18G x 1 1/2&#34;&#160; 1,2 x 40 mm</td>
			<td style="width:141px;">BD Microlance</td>
			<td style="width:119px;">REF 304622</td>
			<td>For medium preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Incubator inc108med with CO2 control</td>
			<td style="width:141px;">Memmert</td>
			<td align="right" style="width:119px;">84198998</td>
			<td>Used for in vitro maturation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Universal oven model UNB 200</td>
			<td style="width:141px;">Memmert</td>
			<td align="right" style="width:119px;">84193990</td>
			<td>Used for medium preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Control unit HAT 400 W1, 72VA 470 x 263 mm</td>
			<td style="width:141px;">Minitube</td>
			<td style="width:119px;">12055/0400</td>
			<td>Used for keeping the cells warm in the course of treatment</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">confocal laser scanning microscope, phase contrast microscope&#160;</td>
			<td style="width:141px;">Zeiss</td>
			<td style="width:119px;">Model: LSM 5 PASCAL Axiovert 200 M</td>
			<td>Morphological and immuno-histochemical analysis of oocytes</td>
		</tr>
	</tbody>
</table>

inactivation of mtor: a tool to investigate meiotic progression and translational control during bovine oocyte maturation

Although routinely used in breeding programs, in vitro maturation (IVM) of bovine oocytes and in vitro production (IVP) of embryos are nevertheless still the subject of basic research owing to suboptimal IVM conditions and variations in the developmental competence of the starting oocytes. In the present study we provide a method to inhibit the Ser/Thr kinase mTor during IVM using two independent inhibitors, Torin2 and Rapamycin. Both substances have different effects on meiotic progression and translational control and may allow discrimination between the mTorC1 and mTorC2 complex functions. The effects of the inhibitors are monitored by inspection of the chromatin configuration using aceto-orcein-staining as well as Western blotting and immunohistochemical analysis of the phosphorylation state of the translational repressor 4E-BP1, which is a prominent mTor target. Whereas Torin2 arrests bovine oocytes in the M I stage and inhibits 4E-BP1 phosphorylation, Rapamycin inhibits asymmetric division and does not influence 4E-BP1. Investigations utilizing these reactions can provide deeper insights into the regulatory events involved in meiotic maturation. Moreover, special focus can be placed on the temporal and spatial regulation of translational control. Such findings can contribute to the definition of the developmental competence of oocytes and to an improvement of IVM conditions.

Source of oocytes, in vitro maturation and mTor inhibition
Figure 1 illustrates the collection of cumulus oocyte complexes (COCs) and the analysis of the chromatin configuration. Only healthy ovaries (Figure 1A) obtained from a local slaughterhouse were used as oocyte sources. COCswere aspirated from follicles sized 4-8 mm (Figure 1B). Only COCs with compact layers of cumulus cells (Figure 1C) were used fo...

The protocol presented here describes the inhibition of the Ser/Thr kinase mTor during IVM of bovine oocytes. This approach can facilitate the investigation of meiotic progression and translational control. It also contributes to the definition of developmental competence and the improvement of IVM conditions.

Inactivation of mTor: A Tool to Investigate Meiotic Progression and Translational Control During Bovine Oocyte Maturation

Although routinely used in breeding programs, in vitro maturation (IVM) of bovine oocytes and in vitro production (IVP) of embryos are nevertheless still ...

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