Alterations in the expression of genes involved in lipid metabolism during bovine oocyte maturation and at the blastocyst stage in vivo vs.

Transcription

1 Chpter 5 Altertions in the expression of genes involved in lipid metolism during ovine oocyte mturtion nd t the lstocyst stge in vivo vs. in vitro O.A. Algriny 1, P.L.A.M. Vos 1, M.A. Sirrd 2, S.J. Dielemn 1 1 Deprtment of Frm Animl Helth, Section Reproduction, Fculty of Veterinry Medicine, Utrecht University, The Netherlnds 2 Centre de Recherche en Biologie de l Reproduction, Déprtement des Sciences Animles, Universitéé Lvl, Sinte Foy, Quéec, Cnd (Sumitted for puliction)

2 Astrct Lipids, minly long chin ftty cids, re in ddition to their role s energy source implicted in mny physiologicl processes such s precursors for the iosynthesis of memrne lipids, signling trnsduction molecules nd lignds for trnscription fctors. Ftty cids (FAs) nd lipid trnsport, ctolism nd synthesis require the correct temporl ctivity of severl gene products. While the control of FAs trnsport nd metolism hs een exmined in different mmmlin cells, regultion nd coordintion of lipid metolism throughout mturtion of mmmlin oocytes nd culture of erly emryonic development is widely unknown. Therefore, trnscript undnce of mrnas involved in, 1) FAs trnsport, including FA trnslocse (FAT/CD36), FA trnsport proteins 1 (FATP1), 2) FAs β-oxidtion like AMP-ctivted protein kinse (AMPK), crnitine plmitoyltrnsferse 1 (CPT-1), peroxisome prolifertor-ctivted receptors αα (PPARα) nd cetyl CoA croxylse β (ACCβ), nd 3) de novo FA synthesis including cetyl CoA croxylse α (ACCα) nd ftty cid synthse (FAS), were nlyzed throughout oocyte mturtion (2 h pre LH, 6 nd 22 h fter LH) nd t the lstocyst stge (dy 7) in vivo, using quntittive rel time PCR. Presumptive competent oocytes were selected fter ofsh tretment on the sis of the steroid profile in the enclosing follicle. Moreover, to determine the role of these trnscripts in developmentl competence, mrna expression ws compred with tht of non-competent oocytes from preovultory follicles of the sme group of cows, nd to tht of cultured oocytes from slughterhouse ovries t corresponding times of mturtion nd in vitro derived lstocysts. Tken together, our results showed n increse in mrna implicted in FAs trnsport (CD36 nd FATP1) nd ctolism (AMPK, CPT-1) during oocyte mturtion compred with the lstocysts, where there is mrked decrese or lck of these trnscripts. In contrst, shrp nd significnt increse ws oserved in mrnas involved in de novo FA synthesis (FAS nd ACCα) t the lstocyst stge compred to the oocytes. Moreover, our results showed tht different oocyte microenvironments cn hve profound effect on mrna encoding components of FA utiliztion, ctolism nd synthesis. In vitro produced lstocysts showed significnt decrese in ACCα compred to the in vivo derived ones. Our results suggest tht n errnt expression of these trnscripts might contriute to oocytes nd lstocyst developmentl competence. Key words: gene expression; lipid metolism; CD36; FATP1; CPT-1; ACCα 116

3 Chpter 5 Introduction Lipids, s well s crohydrtes nd mino cids, re the three sic mterils providing n importnt source of energy for ll mmmlin cells. Beside their crucil role s energy source, lipids, minly long chin ftty cids (LCFAs) re precursors for the iosynthesis of memrne lipids, lipid signling molecules nd lignds for trnscription fctors tht control metolic ctivity. Depending on the cell type, nd the energy needs, LCFAs cn e imported into the cell from exogenous source, or generted endogenously from de novo synthesis, hydrolysis of triglycerides. Although, energy contined in lipids exceeds tht in crohydrtes nd proteins, much reserch relevnt to energy needs during mmmlin oocyte mturtion nd erly emryonic development in vitro hs een directed towrds crohydrtes nd mino cids nd we know much less out the possile regultory roles of lipids during mmmlin oocyte mturtion nd erly emryonic development. Structurlly, lipid content in ovine oocytes tends to increse progressively with oocyte growth [1], In ddition, rnking the oocytes sed on the commonly used indices for COCs qulity [2] demonstrted difference in ftty cids (FAs) composition nd indictes tht the ppernce of the cytoplsm my reflect its lipid, FAs content nd developmentl competence [3, 4]. Biochemiclly, higher lipse ctivity [5] nd decrese in triglyceride content recorded during in vitro mturtion of ovine oocytes [4] indicte tht lipid my ct s energy source during ovine oocyte mturtion. Oocyte nd emryo FAs profiles nd their rte of uptke hve een proposed s tool to predict oocytes nd emryo qulity. Hggrty et l [6] showed reduction in the concentrtions of linoleic nd oleic cid in humn emryos tht filed to develop eyond 4- cell stge compred to tht developed normlly. Similrly, Zeron et l [7] found evidence tht the sesonl chnges in the ovine oocyte developmentl competence might rise primrily due to chnges in FAs profiles of ovrin folliculr fluid nd oocytes. Moreover, mrked chnges in linoleic cid levels of NEFA nd phospholipids concentrtions hve lso een reported etween estrdiol ctive nd inctive ovine preovultory follicles [8]. On the other hnd, lipid ccumultion during in vitro culture of erly ovine emryos in medium contining FCS compred to serum free medium remins source of dete for numer of studies. Erlier studies hd indicted tht culture in the presence of serum results in intrcellulr lipid ccumultion nd greter sensitivity to freezing [9-11] ut more recent work suggests no difference in the post-cryopreservtion survivl [12]. Regrdless of the presence or sence of serum, it is cler tht culture of ovine emryos in vitro is 117

4 ssocited with greter sensitivity to freezing during cryopreservtion compring to those mtured in vivo [13]. Despite this evidence, the necessity of FAs nd the pthwys involved in regultion nd coordintion of lipid metolism t the moleculr level throughout mturtion of oocytes nd culture of erly emryonic development remins lrgely unknown. Exmining some of the key genes controlling the mjor pthwys of 1) FAs trnsport like FA trnslocse (FAT/CD36), FA trnsport proteins 1 (FATP1), nd 2) FAs β-oxidtion like AMP-ctivted protein kinse (AMPK), crnitine plmitoyltrnsferse 1 (CPT-1), peroxisome prolifertor-ctivted receptors α (PPARα) nd cetyl CoA croxylse β (ACCβ), nd 3) de novo FA synthesis including cetyl CoA croxylse α (ACCα) nd ftty cid synthse (FAS), my elucidte the possile role of FAs during oocyte mturtion nd erly emryonic development, nd the tsks plyed y these genes with regrd to the control of lipid metolism. Although the Free FAs cn e tken up y diffusion through the plsm memrne [14], different proteins integrlly ssocited with the plsm memrne tht hve een identified [15] cn fcilitte uptke nd coordinte the import of Free FA in hrmony with metolic demnds. The est chrcterized, including FAT/CD36 [16] nd FATPs [17], chnges in LCFA uptke hve een oserved to occur in prllel with chnges in FAT/CD36 expression in rt hert nd skeletl muscle [18], nd null muttion in FAT/CD36 reduced the uptke of FAs [19]. FATP1 is well chrcterized memer of lrge fmily of plsm memrne proteins tht increse FA import when expressed in cultured cells [17], disruption of the yest Scchromyces cerevisie FATP1 hs een shown to impir LCFA uptke growth [20]. Within most mmmlin cells, controlling energy demnds in the form of ATP re done through β oxidtion of FAs in the mitochondri. Different genes hve een involved in the trnscriptionl control of β oxidtion. AMPK is n ttrctive potentil cndidte, nd its role in this process hs een suggested [21, 22]. AMPK is ctivted y low ATP/AMP rtios to serve s fuel guge for mmmlin cells to protect ginst energy deprivtion [23], its ctivtion is ssocited with incresed mitochondril iogenesis [24] nd enzyme content [25] in rt skeletl muscle. Activtion of this enzyme in muscle increses FAs oxidtion [26-28] nd inhiits cetyl CoA croxylse [25, 29]. Within the cytoplsm, FAs re ctivted to long-chin cyl-coa y n cyl-coa synthetse. The cyl prt is then trnsferred into the mitochondri for oxidtion y complex of enzymes involving CPT-1 on the outer spect of the inner mitochondril memrne. Inside the mitochondril mtrix, long-chin cyl-coa psses through the β-oxidtion enzyme system to produce cetyl- CoA. It is generlly ccepted tht in different mmmlin cells β oxidtion is regulted 118

5 Chpter 5 through CPT 1 through different mechnisms, including chnges in the ctivity nd trnscription rte of CPT 1, or its inhiitor mlonyl-coa the end product of ACCβ ssocited with mitochondril memrne [30]. The trnscription fctor PPARα is memer of PPAR fmily tht functions s lignd-dependent trnscription fctor [31], its expression is more undntly in tissues tht re chrcterized y high rtes of FAs oxidtion (FAO), nd is considered s the min sutype tht medites lipid-induced ctivtion of FAO genes [32]. FAs re synthesized y common iochemicl pthwy in ll mmmlin cells involving two key enzymtic rections, the first is the croxyltion of cetyl-coa in the cytosol to form mlonyl CoA y ACCα [30], then FAS ctlyzes the synthesis of LCFAs, y using cetyl-coa s primer, mlonyl-coa s two-cron donor for chin elongtion, nd NADPH for the reduction rections [33]. While the control of ftty cid trnsport nd metolism hs een exmined in different mmmlin cells, little is known out the oocyte control of ftty cid entry during the finl stges of mturtion. It is lso well documented tht oocyte qulity otined from gondotropin-stimulted cows or mtured in vitro cn e vrile due to mny fctors, mong these fctors which hve een clerly demonstrted re the ATP content [34, 35] nd lipid composition of the oocyte [7]. To djust to chnges in the demnd of lipids during mturtion nd erly emryonic development, oocytes nd erly emryos must precisely regulte nd coordinte the expression of severl genes. Therefore, this study investigtes the expression of key mrnas involved in lipid metolism throughout ovine oocyte mturtion nd lstocysts stge in vivo. Additionlly, we lso nlyzed the expression of these genes under in vivo nd in vitro culture conditions to unrvel prt of the signls nd mechnisms tht prticipte in developmentl competence. Mterils nd Methods Experimentl design At ech time point of mturtion, oocyte mrna expression ws compred with tht of non-competent oocytes from preovultory follicles of the sme group of cows nd with tht of cultured oocytes from slughterhouse ovries t corresponding times of in vitro mturtion. Secondly, mrna expression of the competent oocytes ws compred with tht 119

6 of in-vivo derived lstocysts. Finlly, mrna expression of dy 7 in-vitro produced lstocysts ws compred to in-vivo derived counterprts. Follicle development ws stimulted in Holstein-Friesin cows using our stndrd protocol [36] with ofsh nd Crestr/GnRH-controlled LH surge. Cows were llocted t rndom to three experimentl groups for ovriectomy (OVX): 1) t onset (2 h efore LH), 2) fter initition (6 h fter LH), nd 3) t completion (22 h fter LH) of finl mturtion to determine chnges in mrna expression throughout mturtion in vivo. Oocytes were selected on the sis of follicle size, the steroid profile in the enclosing follicle s shown in tle 2 nd, were ssigned to replictes for QPCR nlysis in such wy tht within group replictes were equivlent with regrds to steroid profile nd represented mximum numers of cows. In vivo lstocysts were collected from superovulted cows y flushing the uterus t dy 7 fter insemintion. In-vitro oocytes were produced fter mturtion in vitro (IVM) using oocytes otined from n ttoir, while in-vitro produced lstocysts were collected fter fertiliztion nd culture in-vitro of the in-vitro mtured oocytes till the lstocysts stge. The experiment ws crried out s pproved y the Ethicl Committee of the Veterinry Fculty of Utrecht University. Animls nd tretment Normlly cyclic Holstein-Friesin cows (n=36) were selected nd treted for superovultion using the protocol s descried efore [36] with ofsh (Ovgen ICP, Aucklnd, New Zelnd), prostglndin (PG; Prosolvin; Intervet Interntionl B.V., The Netherlnds) nd timed LH surge controlled y norgestomet/gnrh (Crestr er implnt/receptl; Intervet Interntionl B.V.). Cyclicity ws verified y mesuring the concentrtion of progesterone in peripherl plsm during t lest 6 weeks efore FSHstimultion [37] nd the LH surge ws monitored in plsm from 38 h fter PG until OVX using vlidted RIA with ovine LH (LH-7981) for iodintion nd stndrds, nd rit nti-lh (8101) s ntiserum [38]. Oocytes nd folliculr fluids were collected following OVX t 50, 58 nd 74 h fter PG corresponding with 2 h efore, 6 nd 22 h fter the mximum of the LH surge, respectively. Collection nd selection of in-vivo preovultory oocytes For every tretment run with group of 4 cows OVX ws performed t 1 h intervls, the time needed to collect ll oocytes nd follicles from one cow y lprotomy through flnk incision under locl infiltrtion nesthesi [39]. Ovries were collected in 0.9% (w/v) NCl t 37 o C nd immeditely trnsported to the lortory. The contents of ech follicle > 120

7 Chpter 5 9 mm were spirted using n 18-g winged infusion set needle ttched to 15 ml polystyrene conicl tue under low pressure y mens of suction pump, nd were then immeditely stored on ice t 4 o C. The size of the follicles ws clculted from the volume of folliculr fluid fter collection. After retrievl of the cumulus oocyte complexes (COC) under stereo microscope, the folliculr fluids were centrifuged 3,000 g for 10 min t 4 o C nd stored t -25 o C until nlysis for steroids. Collected COCs were rinsed twice in 700 µl PBS (Sigm-Aldrich, St. Louis, MO, U.S.A.) supplemented with 5% (w/v) polyvinyl lcohol (PVA; Sigm). Cumulus cells were removed y continuous pipetting fter incution for one minute with 100 µl 0.1% (w/v) hyluronidse (Sigm-Aldrich, St. Louis, MO, U.S.A.) nd denuded oocytes were checked for remining cumulus cells nd wshed three times with PBS-PVA nd stored individully in t -80 C until RNA extrction. For ech collection group reltive to the LH surge, oocytes were su-divided on the sis of the concentrtion of steroids in the fluid of the enclosing follicle. Briefly, oocytes from follicles with estrdiol 17β > 0.9 µmol/l efore LH, > 0.5 µmol/l 6 h fter LH, nd progesterone > 0.5 µmol/l 22 h fter LH were considered to e competent (Tle 2). Oocytes from follicles with unmistkly deviting steroid concentrtions were ssigned to the respective non-competent su-groups, tht is with estrdiol < 0.37 µmol/l efore nd 6 h fter LH, nd with progesterone < 0.38 µmol/l 22 h fter LH. The few oocytes tht hd hevy tretic fetures (expnded cumulus scttered in drk clumps in jelly-like mtrix) were excluded. Blood smpling Heprinized lood smples were collected from the jugulr vein every dy during the experimentl cycle, every 3 h strting 12 h efore removl of the second implnt nd every hour therefter for 6 h. After immedite centrifugtion t 4 ºC, plsm ws stored t -25 ºC. RIA of steroids in folliculr fluid Concentrtions of the steroid hormones estrdiol 17β nd progesterone in folliculr fluid were determined in liquots of 1 to 25 µl fluid dependent of the hormone nd the size of the follicle y solid-phse 125 I RIA methods (Cot-A-Count, Dignostic Products Corportion, Los Angeles, CA, U.S.A; estrdiol 17β: TKE2; progesterone: TKPG) s vlidted for lood plsm of cows [37] with slight modifictions such s extrction with diethyl ether (BDH Lortory Supplies, Poole, Englnd). 121

8 In vitro mturtion (IVM) of immture oocytes Bovine ovries were collected from ttoirs, nd trnsported in thermos flsk within 3 hours of collection. After wshing with wter nd sline solution, cumulus oocyte complexes (COCs) were spirted from follicles 3-6 mm in dimeter, wshed in HEPESuffered TCM-199 (Gico BRL, Pisley, UK), nd selected on the sis of their morphology for in vitro mturtion ccording to the density of their cumulus cell lyers, nd rndomly llocted in groups of 50 COCs per well to 4-well culture plte (Nunc A/S, Roskilde, Denmrk). In vitro mturtion of the COCs ws performed in 500 µl TCM-199 per well, supplemented with 10% (v/v) fetl clf serum, 4 µg FSH/ml, nd 6 µg LH/ml (Sioux Biochemicl Inc., Iow, U.S.A.), nd 0.1 mm cystemine (Sigm Chemicl Co., St Louis, MO, U.S.A.) for 22 h t 38.5ºC under n tmosphere of 5% CO2 in ir with mximum humidity. At the eginning (0 h), 6 h nd 22 h of the mturtion, cumulus cells of 13 oocytes from three different tches were denuded y vortexing nd stored t -80 ºC. In vitro emryo production (IVP) Procedures for in vitro mturtion were performed s descried previously, fter mturtion, oocytes in COCs were fertilized in vitro ccording to the procedure descried y Prrish et l. [40] with minor modifictions [41] using frozen-thwed semen from ull of proven fertility. The presumptive zygotes were freed from cumulus cells 20 h fter IVF y vortexing, nd mximum of ten zygotes ws plced in 20 µl droplet of synthetic oviductl fluid (SOF) medium [42] supplemented with essentil nd non-essentil mino cids (Sigm-Aldrich, St. Louis, MO, U.S.A.) nd 0.1% (w/v) BSA (Sigm-Aldrich), under oil (Reproline medicl GmBH, Rheinch, Germny) nd cultured t 39 C, in humidified ir contining 5% CO 2 nd 7% O 2. On dy 4 fter IVF the numer of cells per cleved emryo ws scored nd ll cleved emryos were trnsferred to fresh SOF droplets. The developmentl stge of the emryos ws ssessed t dy 7 fter IVF. Four groups of 5 expnded lstocysts were rinsed in PBS nd stored t -80 C until RNA extrction. Collection of in-vivo derived lstocysts Superovultion ws induced using n ecg/monoclonl nti-ecg/pg tretment scheme [37]. Cows were inseminted with one strw into ech uterine horn, 10 h fter the LH pek. Seven dys lter the emryos were non-surgiclly recovered, emryonic developmentl stge nd generl morphologicl ppernce were ssessed y stereo microscopy, once 122

9 Chpter 5 qulified to those in morphologicl grdes I nd II [43], frozen for storge in liquid nitrogen until use. RNA isoltion, precipittion Totl RNA ws prepred from t lest three replictes t ech mturtion stge, except 22 h norml, 6 nd 22 h devint oocytes, where 20, 18 nd 11 oocytes were collected, respectively. Ech replicte contining from 9 to 13 pooled oocytes. Four replictes of dy 7 lstocysts were used ech contining 5 lstocysts, the RNA ws then isolted using microspin column nd DNA ws digested with Dnse1 to eliminte possile genomic DNA contmintion ccording to mnufcturer s instruction (Asolutely RNA Microprep Kit, Strtgene, Sn Diego, CA, U.S.A.). The RNA ws recovered y two susequent 50-µl elutions with wrmed (60 C) elution uffer provided in the kit. RNA ws then precipitted with 250 µl of 100% ethnol (EtOH) nd 10 µl of 3 M sodium cette ph 5.2, using 1 µl of 1 mg/ml liner crylmide (Amion, Austin, TX, U.S.A.) s co-precipitnt. The mixture ws chilled t -80 o C for 30 min, centrifuged for 20 min t 4 o C t g. The pellet ws then wshed with 75% EtOH nd resuspended in 15 µl of wter for rel-time PCR nlysis. Rel-time Polymerse Chin Rection Reverse Trnscription nd primers design Totl RNA ws reverse-trnscried in totl volume of 20 µl using the iscript cdna Synthesis Kit (Bio-Rd Lortories, Hercules, CA, U.S.A.) contining oligo-dt nd rndom hexmer primers. Rections were incuted for 5 min t 25 C, 30 min t 42 C, nd 5 min t 85 C. Primer sets were designed y using Becon Designer 4 softwre (PREMIER Biosoft Interntionl, Plo Alto, CA, U.S.A.), from ovine sequences from NCBI, the primers used in the study re shown in tle 1. The specificity of the primers ws confirmed y sequencing nd confirmtion of the PCR product size on stndrd 2% grose gel with ethidiumromide (EtBr). The PCR products were sequenced fter purifiction with the QIquick PCR Purifiction Kit (Qigen, Vlenci, CA, U.S.A.) nd quntifiction with spectrophotometer (Nnodrop ND 1000, Isogen, IJsselstein, the Netherlnds). PCR products were then diluted from 100 to 0.01 fg s stndrds to construct the stndrd curve. 123

10 Rel-time Polymerse Chin Rection Rel-time PCR ws performed on Bio-Rd MyiQ system using the 2X iq SYBR Green Supermix regents, following the mnufcturer s protocols. Rections were performed using 25 µl duplicte rections with the quntity of diluted cdna corresponding to 0.1 to 0.3 of n oocyte, depending on the mrna undnce s determined using oocytes from slughter house ovries mtured in vitro, nd 0.5 µm of ech primer. Ech trnscript ws mplified from t lest three different groups of pooled oocytes, except 22 h norml, 6 nd 22 h devint oocytes, where 2, 2 nd 1 group were used, respectively (see tle 2). Tle 1. Informtion on the primers used for rel-time PCR Genes GeneBnk ccession numer Oligos sequences CD36 NM_ F 5 -tcttgctggtgctgtcttgg-3 R 5 -ctgtccttctctggttctgc-3 FATP1 NM_ F 5 -cggcctggtcgttc-3 R 5 -cgtgtgtgtgggtcg-3 AMPAKγ1 BT F 5 -gtgcttggggggtg-3 R 5 -cttgggcttgggtgc-3 CPT-I NM_ F 5 -ggtccgcctctcg-3 R 5 -tgctcctctcctctgg-3 PPARα NM_ F 5 -tcgtttcctcctttccttc-3 R 5 -tctgtgtcccctctcc-3 ACCβ AJ F 5 -tcctgcgggcgtctgg-3 R 5 -gctgttctggtgtgtcttgg-3 ACCα NM_ F 5 -gctggtgccttcttc-3 R 5 -gtgcccgtcgggc-3 FAS NM_ F 5 -ctctccctcgccgttcg-3 R 5 -gcctgtctctctgtccc-3 GAPDH BTU85042 F 5 -cccgggttcccc-3 R 5 -gccgtggcgggtg-3 Product size (p) Anneling temperture ( o C) Bos turus CD36, Ftty cid trnslocse; FATP1, Bos turus ftty cid trnsport protein 1; AMPAKγ1, Bos turus AMP-ctivted protein kinse gmm 1; CPT-1, Bos turus crnitine plmitoyltrnsferse 1; PPARαα, Bos turus peroxisome prolifertor-ctivted receptors α; ACCβ, Bos turus cetyl-coa croxylse β; ACCα, Bos turus cetyl-coa croxylse α; FAS, Bos turus ftty cid synthse; GAPDH, Bos turus glycerldehyde-3-phosphte dehydrogense 124

11 Chpter 5 Smples nd stndrd curves were mplified on the sme run with the sme PCR mster mix. The therml cycling progrm strts with n initil denturtion step t 95 C for 3 min, nd is followed y 45 PCR cycles (dissocition for 5 sec t 95 o C, nneling for 5 sec t temperture showed in tle 1, nd elongtion for 20 sec t 72 o C), one melting cycle consisting of 5 sec t 95 o C, 30 sec t 72 o C, nd step cycle up to 95 o C (0.3 o C/sec trnsition rte), nd finlly cooling down cycle t 40 o C. Amplifiction of the GAPDH mrna [44, 45] ws performed for ech reverse trnscried smple s n endogenous quntifiction stndrd. These rw CT vlues were then nlyzed with modified delt-ct method using PCR dt nlysis progrm, qbse (version 1.3.2) ( to otin reltive quntifiction vlues. PCR product ws nlyzed on 1.5% grose gel with EtBr to confirm mplifiction. Product sizes nd nneling tempertures for ech gene re presented in tle 1. Sttisticl nlysis Dt re presented s the men ± SEM. Initilly, expression nlysis dt of the oocytes from follicles with norml profile were sujected to one-wy ANOVA nd Bonferroni test ws used s post hoc for comprison of individul mens to ssess the effect of mturtion stges (-2, 6, 22). Secondly, dt from oocytes clssified norml in the premturtion stges (-2) were compred using un-pired Student s t-test to the in vivo derived lstocysts to study the difference in gene expression etween oocytes nd lstocysts. Thirdly, differences within ech oocyte time group (follicles with norml profile, devint profile nd oocytes mtured in vitro) were nlyzed using one-wy ANOVA. Difference etween in vivo nd in vitro derived lstocysts ws nlyzed using Student s t-test. Differences were considered sttisticlly significnt t the 95% confidence level (P < 0.05). Results In totl 171/241 efore LH, 135/183 t 6 h fter LH nd 60 oocytes/ 93 follicles were retrieved resulting in 71, 74 nd 65% recovery, respectively. The numer of oocytes clssified s competent nd non-competent sed on steroid nd follicle size re presented in tle

12 Tle 2.: Steroid concentrtions in follicles with competent nd non-competent oocytes recovered during mturtion in FSH-stimulted cows with controlled LH surge notes 2 h efore LH (Pre) 6 h fter LH (Post) 22 h fter LH (Post) Norml follicles - numer - estrdiol 17β - progesterone - follicle dimeter - numer of cows [1] [2] [3] [4] ± ± ± ± ± ± ± ± ± Devint follicles - numer - estrdiol 17β - progesterone - follicle dimeter - numer of cows ± ± ± ± ± ± ± ± ± [1] competent: oocytes from functionl follicles on the sis of steroid profile [2] steroid concentrtions ± SEM in µmol/l folliculr fluid [3] men dimeter in mm s clculted from volume of fluid of follicles from which oocytes were used [4] numer of cows from which oocytes were retrieved Results of gene expression were used to orgnize group of genes ccording to their function. Group 1. Ftty cid trnsport: within the oocytes selected from norml follicles, levels of CD36 (fig. 1, A) nd FATP1 (fig. 1, B) mrna were reltively high efore, 6 nd 22 h fter mturtion, ut no significnt (P < 0.05) differences were oserved etween the different time groups (from norml follicles). The expression pttern of this group t the lstocysts stge chrcterized y the sence (CD36) or low levels of mrna (FATP1). Within ech time group, there were mrked chnges in the expression etween the groups ut it ws significnt only etween oocytes collected from FSH stimulted cows clssified s norml nd devint in the pre-mturtion groups for oth mrna. Group 2. Ftty cid β-oxidtion: mrna levels vried cross this group, with the level of AMPK (Fig.1, C) nd CPT-I (fig. 1, D) high during the pre-mturtion nd 6, 22 h fter mturtion ut there were no sttisticl significnt differences etween the time groups (P < 0.05). A mrked reduction in the mrna expression for oth mrna t the lstocyst stge, 126

13 Chpter 5 CPT-1 mrna ws not detected in in vivo derived lstocysts. The gene expression of AMPK nd CPT-1 ws significntly (P < 0.05) reduced in the oocyte scored s d during pre-mturtion compred to the good one, CPT-1 expression in the oocyte mtured in vitro ws significntly ltered compred to the oocytes clssified s norml, wheres oth trnscripts remined unchnged during 6 h of mturtion. A significnt difference within the 22 h group in the expression of AMPK, etween in vivo norml, devint, nd those mtured in vitro ws oserved. The levels of PPARα (Fig. 1, E) mrna remined unchnged etween the different time groups nd lso etween oocytes nd lstocysts. A significnt difference etween the oocytes collected from follicles with norml profiles ws seen compred to those scored s devint nd mtured in vitro (P < 0.05). Wheres no significnt ltertions in PPARα mrna expression etween in vivo nd in vitro lstocysts were detected. Similrly, ACCβ (Fig. 1, F) mrna levels were not significntly different (P < 0.05) within the oocyte time groups (-2, 6, 22) nd lso etween the oocytes nd the lstocysts. Group 3. Ftty cid synthesis: A similr pttern of chnges chrcterize oth memer of this group, ACCα (Fig. 1, G) nd FAS (Fig. 1, H). No differences etween different time groups during oocyte mturtion nd shrp nd significnt increse in oth mrna levels during the lstocysts stges compred to oocyte mturtion. A ststiclly significnt difference (P < 0.05) ws oserved in the expression of ACCα etween in vivo derived lstoysts nd those cultured in vitro. 127

14 Figure 1. Quntifiction of mrna levels y rel-time PCR of genes involved in lipid metolism (A to H) in ovine oocytes from cows undergoing superovultion with FSH scored s norml or devint sed on steroid profile nd follicle sizes nd oocytes collected from slughterhouse ovries nd mtured in vitro.,, c: significntly different within the time groups ( in vivo norml, in vivo devint nd in vitro oocytes) (P < 0.05). * Significntly different from the oocytes CD36 mrna reltive expression invivo norml invivo devint invitro Pre mturtion 6 h mturtion 22 h mturtion Dy 7 lstocyst * A. Ftty cid trnslocse/cd36 mrna FATP1 mrna reltive expression invivo norml invivo devint invitro Pre mturtion 6 h mturtion 22 h mturtion Dy 7 lstocyst * B. Ftty cid trnsport protein1 128

15 Chpter 5 AMPAK mrna expression invivo norml invivo devint invitro Pre mturtion 6 h mturtion 22 h mturtion Dy 7 lstocyst c * C. AMP-ctivted protein kinse γ1 CPT-I mrna reltiveexpression 0.9 invivo norml 0.8 invivo devint 0.7 invitro * 0.0 Pre mturtion 6 h mturtion 22 h mturtion Dy 7 lstocyst D. Crnitine plmitoyltrnsferse I PPARα reltive mrna expression , invivo norml invivo devint invitro Pre mturtion 6 h mturtion 22 h mturtion Dy 7 lstocyst E. Peroxisome prolifertive ctivted receptor lph 129

16 invivo norml invivo devint invitro ACAC et mrna expression , Pre mturtion 6 h mturtion 22 h mturtion Dy 7 lstocyst F. Acetyl CoA croxylse et invivo norml invivo devint invitro ACACα mrna reltive expression , Pre mturtion 6 h mturtion 22 h mturtion Dy 7 lstocyst * G. Acetyl CoA croxylse Alph FAS mrna reltive expression , invivo norml invivo devint invitro Pre mturtion 6 h mturtion 22 h mturtion Dy 7 lstocyst * H. Ftty cid synthse 130

17 Chpter 5 Discussion Our dt suggest tht, under physiologicl conditions, 1) during oocyte mturtion, exogenous FAs supply is proly the preferentil source of LCFA nd energy source, sed on the higher expression of trnscripts involved in FAs trnsport nd key regultory enzymes involved in β-oxidtion of FAs. 2) At the lstocysts stge, the prllel increse in the mrna undnce of FAS nd ACCα suggests n incresed rte of lipogenesis which my e needed to support erlier emryogenesis. 3) A lower relince on FAs s source of energy t the lstocysts stge s shown y lck or shrp decrese of mrnas implicted in FAs ctolism. 4) Exogenous FSH stimultion nd in-vitro culture conditions cn results in extreme devitions in severl trnscripts involved in lipid metolism during oocyte mturtion nd lstocysts formtion. CD36 expression fvors tissues with high metolic cpcity for LCFA, such s dipose, hert nd muscle, wheres it is sent from tissues like rin tht do not utilize LCFA [16], its mrna expression is induced y LCFA nd not y short chin FAs [46]. The CD36 mrna hs een reported previously in mouse oocytes using the microrry technique [47]. The mechnisms y which oocyte competence is ffected y FAs re not completely understood. However, the higher undnce of plsm memrne ftty cid trnsport mrnas (CD36 nd FATP1) suggests tht the effect of LCFAs on oocyte mturtion is direct nd not medited y cumulus cells. It lso my indicte tht the oocyte cn not synthesize certin LCFA nd tht exogenous FAs re needed to support certin oocyte requirements. It is well known tht nimls cn synthesize most ftty cids, with the notle exceptions of polyunsturted ftty cids (PUFA) like linoleic nd linolenic cids, since nimls must hve dietry source of these FAs nd therefore they re considered s essentil FAs. The essentil FAs hve different importnt functions, the most importnt is structurl contriution to the mintennce nd function of the cellulr memrnes [48]. Altertions in FAs composition nd lipid contents during oocyte growth re well documented phenomenon. Zeron et l [7] reported tht the decline in the developmentl competence of oocytes Holstein cows ws minly ssocited with significntly lower linoleic cid content in the oocyte memrne. The sme uthor reported chnges in the ewes oocyte s FAs profile y PUFA diet supplementtion which ssocited with etter oocyte qulity nd chilling resistnce [49]. As result, it ppers likely tht the reduction in the synthesis of mrna for the genes encoding CD36 nd FATP1 oserved in oocytes clssified s devint nd oocytes mtured in vitro my reflect lter reduced LCFAs import into the oocyte tht my ffect fundmentl cellulr processes such s FA incorportion into triglycerides, memrne components, signl trnsduction pthwys, gene expression 131

18 nd FAs generted energy. Moreover, the co-expression of oth CD36 nd FATP1 in the oocyte my indicte tht they hve different function since numer of studies using 293 cells [50] nd FATP1 knock-out mice hve reported tht LCFAs tken y FATP1 were chnneled into triglycerides synthesis. This synthesis exceeded oxidtion in the mitochondri [51], wheres FA/CD36 ws identified recently on mitochondri isolted from rt nd humn skeletl muscle nd found to e involved in LCFA oxidtion [52, 53]. Little is known out the nutritionl requirement of the ovine oocyte. Using FCS s IVM medium supplement cn promote oocyte developmentl competence. Its efficcy hs een scried to its superior protein qulity, mino cids, vitmins, hevy-metl cheltors nd unknown growth fctors, mking it the most effective IVM component. Oocyte triglyceride nd the totl lipid content hve een shown to increse in oocytes mtured in vitro in medium contining FCS compred to those mtured in serum free medium [4]. This my strongly indicte indirectly tht the eneficil effect of FCS s mjor component of ovine oocyte IVM medium my e lso due to FAs contents. It hs een suggested tht decrese or increse in the expression of different lipid metolic genes re modulted y direct contct with FA, tht lter chromtin structure nd trnscription through direct effect on histone decetylse ctivity, for instnce, FATPs, CPT-1, PPAR hs een shown to e upregulted y FAs wheres FAS, ACC re down-regulted (for review see [54]. Bsed on the ove, decresed FA oxidtion my reflect defect in ftty cid oxidtion genes ctivtion due to ltertions in CD36 nd FATP1 undnce s result of poor culture conditions. AMPK ctivity depends on the AMP/ATP rtio tht is chnged during oocyte mturtion. Cyclic AMP degrdtion results in the ccumultion of AMP which inds nd ctivte AMPK directly y inding to its γ suunit, mechnisms other thn chnges in the cellulr AMP-to-ATP rtio hve lso een reported to ctivte AMPK [55]. However, our results showed higher expression of AMPK mrna even in the oocytes collected efore LH surge nd mturtion in vitro, which is consistent with the recent finding using mouse oocytes [56]. AMPK is stress-ctivted enzyme, nd even gentle physicl mnipultion cn stimulte the kinse [57, 58]. In mouse oocytes, AMPK ctivtion hs een shown to precede GVBD nd to ply n importnt role in meiotic resumption [56]. The simultneous increse of AMPK, CD36, FATP1, nd CPT-1 suggests tht AMPK is ssocited with FAs ctolism s shown previously in most mmmlin cells. Cyclic AMP-dependent protein kinse (PKA) nd AMPK phosphoryltes nd inctivtes ACC, decreses mlonyl-coa levels nd increses ftty cid oxidtion oth in vivo nd in vitro [59, 60]. This phosphoryltion regultion my e lso n importnt regultory spect 132

19 Chpter 5 of ftty cid metolism during oocyte growth nd mturtion. Higher camp within the oocytes ctivtes PKA efore mturtion nd mintin meiotic rrest while decrese in intr-oocyte camp initite oocyte mturtion [61]. The expression of oth ACC nd AMPK my suggest tht the oocyte uses the sme mechnism to phosphorylte nd inctivte ACC, which stimultes ftty cid oxidtion y different pthwys s for other somtic cells through stimultion of PKA following elevtion of camp levels. Thus, camp signling is required not only for meiotic rrest nd resumption ut lso for their intimte coordintion of the severl oocyte metolic processes. CPT-1 resides on the outer mitochondril memrne nd ctlyses the conversion of plmitoyl-coa to plmitoylcrnitine, which is the rte limiting step in the trnsfer of longchin ftty-cylcoas from the cytosol to the mitochondri for oxidtion [30, 62]. Its ctivity is inhiited y mlonyl-coa, therefore, decrese in the mlonyl-coa removes inhiition of CPT-1 nd llows ftty cid oxidtion to increse to meet the incresed energy requirement. In mmmls, two different CPT-1 enzymes re encoded y two different genes, liver CPT-1 nd muscle CPT-1 [63]. Muscle CPT1 hs crucil role in controlling the rte of β-oxidtion in hert nd skeletl muscle nd is more sensitive to mlonyl-coa inhiition [64]. Therefore, we decided to specificlly mplify the muscle isoform. In the present study, we found tht generl increse of CPT-1 mrna levels tkes plce during erly oocyte mturtion or results from the polydenyltion of stored pools nd lck of expression t the lstocysts stge. These findings together with the results pulished recently, using methyl plmoxirte (MP), the inhiitor of CPT-1 enzyme ctivity [65], indicte n increse in the rte of β-oxidtion nd importnt role of ftty cids s source of energy during oocyte mturtion. The lower expression of CPT-1 during premturtion in the oocytes mtured in vitro nd oocytes from devint follicles my indicte dysregultion of ftty cid oxidtion ctivity cusing shortge of ATP nd hence lower oocyte developmentl competence [34]. These results re not surprising, steroid hormones hve significnt control on cellulr lipid in metoliclly ctive tissues [66]. In skeletl muscle, ovriectomy suppresses the mximl ctivity of CPT-1 nd the ctivity of this enzyme is up-regulted following tretment with E2. Moreover, In romtse deficient mice (ArKO), exogenous E2 is necessry to mintin the gene expression nd enzyme ctivity of the heptic β-oxidtion pthwy [67] suggesting cpcity of E2 to regulte the expression of genes implicted in lipid metolism. Low mitochondril β-oxidtion could lso chnge the ftty cid composition within the oocyte since mrked chnge hs een reported in the linoleic cid levels occuring etween E2 ctive nd inctive ovine preovultory follicles [8]. In ddition to its role in β-oxidtion, lower CPT-1 ctivity hs lso een reported to enhnce plmitic cid (PA) induced cell deth nd PA nti- 133

20 prolifertive effect [68]. Possily, this is lso the cse in the oocytes from devint follicles nd in those mtured in vitro, the decrese in CD36 nd FATP1 my result in the preferent use of FAs other thn LCFA through pssive diffusion, nd lower CPT-1 mrna expression my decrese the utiliztion of LCFA. This ltered mrna expression my ffect the lnce in the lipid FAs composition rtios t the physiologicl level. Thus, understnding of the import of FAs, the specific metolic ftes of different FAs nd the mnner in which these re regulted my contriute to the etter design of oocyte IVM medium. At the lstocysts stge, we found smll increse of CPT-1 mrna in the ones cultured in vitro compred to the in vivo counterprts. This my contriute to incomplete lockge of β-oxidtion pthwy in lstocyst cultured in vitro. It hs een hypothesized tht metoliclly ctive lstocysts hve less chnce to develop to term fter trnsfer nd the metoliclly inctive re more likely to develop fter trnsfer [69, 70]. This slight increse in CPT-1 mrna in the lstocysts cultured in vitro my e lso relted to the lower levels of ACCβ mrna expression in in vitro lstocysts tht proly ws not enough to lock CPT-1 ctivity. The lock of CPT-1 ctivity proly induces more cetyl- CoA to go to lipid synthesis insted of β-oxidtion. Our finding tht the oocytes express CPT-1 mrna efore nd during mturtion nd the lck of the expression t the lstocysts stge, support nd explin recent findings tht locking of β-oxidtion using CPT-1 inhiitor methyl plmoxirte efore or during mturtion of ovine oocytes significntly decreses oxygen consumption, while no difference ws reported in the oxygen consumption of dy 7 lstocysts [65]. Furthermore, sed on the lck of CPT-1 mrna expression in lstocysts, it ppers tht the lipids synthesized during erly emryonic development were not used s source of energy, nd my e directed for memrne structure nd orgnogenesis. Therefore, energy requirements t this stge re preferentilly supplied y crohydrtes nd mino cids s shown previously [71, 72] The potentil role of PPARα in regulting ftty cid oxidtion hs een well demonstrted. Activtion of PPARα results in increse in the gene expression of mny FAO enzymes [73, 74]. In the present study, the synergisticlly up-regultion of ftty cid trnsport nd oxidtion genes nd PPARα mrna my indicte tht these genes re trgets for the ctivtion y PPARα. Ftty cids especilly highly unsturted ftty cids hve profound effects on gene expression, cting on the genome through PPAR leding to chnges in metolism, growth nd cell differentition [75, 76]. Fts rich in PUFA like fish oil increse ctivity nd mrna levels of heptic FAO enzymes [77]. Polyunsturted ftty cids hve lso een considered s nturl lignds or ctivtors of PPAR [78]. A study 134

21 Chpter 5 using PPARα-deficient mice suggested tht PUFA (fish oil) up-regultes FAO enzyme gene expression through PPAR dependent mechnism [79]. In the sme experiment, it hs een shown tht PUFA-medited suppression of lipogenic gene expression does not require PPARα. Therefore, the lck of certin FAs my explin the difference in the undnce etween oocytes from norml or devint nd those mtured in vitro. In the present study, the expression of oth isoforms of ACC ws identified in the oocyte nd the lstocyst stge. The mlonyl-coa, the product of ACCα nd ACCβ, exists in two seprte comprtments in the cell, cytosol nd mitochondri, respectively [80] nd do not mix. The cytosolic mlonyl-coa is used in FAs synthesis, while the mitochondril prt regultes CPT-1 nd hence, FAs oxidtion. ACCα is the dominnt isoform nd contriutes to most of the mlonyl-coa. A study using ACCα knockout mice reported n essentil role in emryo development [80] wheres ACCβ works s regultor of FAs oxidtion [81]. Oocytes from devint follicles nd those mtured in vitro, showed lower mrna level of ACCα t the GV nd MII thn those clssified s norml. Since ACCα is involved in ftty cid elongtion, this indicted tht the ctivity of ACCα is not completely locked nd certin enzyme ctivity is needed. A Scchromyces cerevisie mutnt of ACCα grown t restrictive temperture developed n ltered nucler envelope nd showed severe normlities in spindle formtion to ecome rrested in the G2-M phse of the cell cycle loosing viility [82]. In ddition, our study showed sttisticl significnt decrese in ACCα mrna undnce in the lstocysts otined using IVP system. This my indicte chnge in the quntity of synthesized lipid, resulting in lck of certin LCFAs needed for memrne integrity nd structure. In yest, inctivtion of ACCα completely inhiits growth nd cuse cell deth even fter supplementtion of FAs [83]. Moreover, inhiition of ACCα in prostte tumor cell line hs een shown to induce growth rrest nd poptosis [84]. LCFAs nd the degree of unsturtion of memrne lipids re the mjor fctors determining the structure nd fluidity of lipid memrnes nd hence chilling sensitivity [85, 86]. Thus, memrnes with unsturted cyl chins in phospholipids remin fluid t lower tempertures thn do memrnes with sturted lipids [87, 88]. In view of the high degree of conservtion of ACCα in most living orgnisms, similr function of this gene is possile in the oocytes nd erly emryos. Collectively, these findings my explin prtly the lower developmentl competence of the in-vitro mtured oocytes nd those from devint follicles with FSH stimulted cows. They proly explin the sensitivity of IVP lstocysts to chilling compred to the in-vivo derived ones, nd represent strong cndidte mrkers for developmentl competence of oocytes nd lstocysts. 135

22 FAS, ctlyses the de novo synthesis of sturted FAs such s, myristte, plmitte nd sterte using cetyl- nd mlonyl-coa. Trgeted deletion of the FAS gene results in mice tht die in utero [89], nd more thn 50% of heterozygous FAS knockout mice fil to survive emryonic development [89]. Expression of FAS is controlled primrily t the level of trnscription nd in response to oth hormonl nd nutritionl signls [90, 91]. In the present study, contrry to our nticiptions, there ws mrked increse of FAS mrna undnce t the lstocysts stge compred to tht in oocytes suggesting n importnt role in the FAs or triglycerides synthesis. However, the sence of difference in the undnce of FAS mrna etween the in vivo derived lstocysts nd those otined from in vitro, my suggest less criticl role in the developmentl competence of erly emryos. Chnges in trnscription cnnot e equted to chnges in functionl pthwys ecuse trnscripts hve to e trnslted, nd proteins my require post-trnsltion modifiction in order to ecome functionl, [92]. Miniml polydenyltion is required for reverse trnscription ut it does not ensure trnsltion. To vlidte trnsltion, protein levels hve to e mesured nd often they increse s the RNA level drops efore the mternl-zygote trnsition (MZT) in ovine while it is the opposite fter the MZT [93]. Therefore, chnges in mrna levels needed the support of functionl iochemicl ssys. Unfortuntely, current protein nd enzymtic ctivity evlution techniques will require lrge quntity of oocytes, which re very difficult to otin nd limit such ssys in the cse of oocytes from in vivo mteril. Identifiction of mrna expression will provide the sis to define the complex regultion of lipid metolism. In conclusion, the increse in mrna levels implicted in FAs trnsport nd ctolism during oocyte mturtion compred to tht during erly emryonic development is proly due to n incresed metolic ctivity resulting from higher energy requirements. Moreover, we hve shown tht different oocyte nd lstocyst microenvironment nd culture conditions cn hve profound effect on mrna encoding components of FA utiliztion nd synthesis. We propose tht the resultnt chnges in the oocyte nd lstocyst gene expression in vivo vs. in vitro contriute to the oserved decrese in the developmentl competence of emryos y ltertion in lipid metolism. Acknowledgements Mrs. D.M. Blnkenstein nd Mrs. C.H.Y. Oei for their excellent technicl ssistnce with ll the endocrine monitoring of cows nd follicles; post-grdute students H. 136

23 Chpter 5 Groenendl, A.C.T.M. vn Gstel nd Mrs. E. vn Reenen for round-the-clock ssistnce with mnging the experiments; J.H.M. Lutz nd niml cretkers for their skilful treting of the nimls; F.J. vn Kooi nd ssistnts for help with surgery; Dr B. Aguilr et l. (Theriogenology, ;111-20) for kindly donting in vivo derived lstocysts, nd S. Merton from Hollnd Genetics Arnhem, The Netherlnds, for the generous supply of in vitro produced lstocysts; Dr B.A.J. Roelen for providing fcilities for IVM. We lso thnk Dr. Iselle Dufort for criticl review of the mnuscript. References 1. Fir T, Hulshof SC, Hyttel P, Greve T, Bolnd M. Oocyte ultrstructure in ovine primordil to erly tertiry follicles. Ant Emryol (Berl) 1997; 195: de Loos F, vn Murik P, vn Beneden T, Kruip TA. Structurl spects of ovine oocyte mturtion in vitro. Mol Reprod Dev 1992; 31: Ngno M, Ktgiri S, Tkhshi Y. Reltionship etween ovine oocyte morphology nd in vitro developmentl potentil. Zygote 2006; 14: Kim JY, Kinoshit M, Ohnishi M, Fukui Y. Lipid nd ftty cid nlysis of fresh nd frozen-thwed immture nd in vitro mtured ovine oocytes. Reproduction 2001; 122: Cetic P, Pintos L, Dlvit G, Beconi M. Activity of key enzymes involved in glucose nd triglyceride ctolism during ovine oocyte mturtion in vitro. Reproduction 2002; 124: Hggrty P, Wood M, Ferguson E, Hod G, Sriknthrjh A, Milne E, Hmilton M, Bhttchry S. Ftty cid metolism in humn preimplnttion emryos. Hum Reprod 2006; 21: Zeron Y, Ocheretny A, Kedr O, Borochov A, Skln D, Arv A. Sesonl chnges in ovine fertility: reltion to developmentl competence of oocytes, memrne properties nd ftty cid composition of follicles. Reproduction 2001; 121: Mollem U, Folmn Y, Bor A, Arv A, Skln D. Effect of clcium sops of ftty cids nd dministrtion of somtotropin on milk production, preovultory folliculr development, nd plsm nd folliculr fluid lipid composition in high yielding diry cows. J Diry Sci 1999; 82: Ae H, Ymshit S, Stoh T, Hoshi H. Accumultion of cytoplsmic lipid droplets in ovine emryos nd cryotolernce of emryos developed in different culture systems using serum-free or serumcontining medi. Mol Reprod Dev 2002; 61: Fir T, Lonergn P, Dinnyes A, Cottell DC, Hyttel P, Wrd FA, Bolnd MP. Ultrstructure of ovine lstocysts following cryopreservtion: effect of method of lstocyst production. Mol Reprod Dev 2001; 58: Reis A, Rooke JA, McCllum GJ, Stines ME, Ewen M, Lomx MA, McEvoy TG. Consequences of exposure to serum, with or without vitmin E supplementtion, in terms of the ftty cid content nd viility of ovine lstocysts produced in vitro. Reprod Fertil Dev 2003; 15: Mucci N, Aller J, Kiser GG, Hozor F, Codevil J, Alerio RH. Effect of estous cow serum during ovine emryo culture on lstocyst development nd cryotolernce fter slow freezing or vitrifiction. Theriogenology 2006; 65:

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