KIRREL is differentially expressed in adipose tissue from “fertil+” and “fertil-” cows: in vitro role in ovary? Stéphanie Coyral-Castel, Christelle Ramé, Juliette Cognie, Jerôme Lecardonnel, Sylvain Marthey, Diane Esquerré, Christelle Hennequet-Antier, Sébastien Elis, Sébastien Fritz, Mekki Boussaha, et al.
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Stéphanie Coyral-Castel, Christelle Ramé, Juliette Cognie, Jerôme Lecardonnel, Sylvain Marthey, et al.. KIRREL is differentially expressed in adipose tissue from “fertil+” and “fertil-” cows: invitro role in ovary?. Reproduction -Cambridge- Supplement-, Society for Reproduction and Fertility, 2018, 155 (2), pp.181-196. 10.1530/REP-17-0649. hal-02625059
HAL Id: hal-02625059 https://hal.inrae.fr/hal-02625059 Submitted on 26 May 2020
HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright Version postprint Page 1of49 Reproduction AdvancePublicationfirstpostedon23November2017asManuscriptREP-17-0649 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 11 11 18 18 17 16 15 14 13 12 10 10 23 23 25 25 19 19 21 21 22 22 5 5 4 4 3 3 2 1 6 6 9 9 8 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Josas, France France Josas, France France France Dupont Esquerré Coyral Castel S vitro in 7 9 8 7 6 5 4 3 2 1 cows: “fertil-” and “fertil+” from tissue adipose in expressed differentially is KIRREL Short title: KIRREL in bovine adipose tissue and ovary tissueovary and adipose bovineKIRREL in title: Short 24 orsodn ato: r ole uot Uié e hsooi d l Rpouto e des et Reproduction la de Physiologie de Unité Dupont, Joëlle Dr author: Corresponding 20 opreet, nttt ainl e a ehrh Arnmqe 330 ozly France Nouzilly, 37380 Agronomique, Recherche la de National Institut Comportements, (email: (email: INRA, UR83 Recherches Avicoles, F-37380 Nouzilly, France Nouzilly, F-37380 Avicoles, Recherches UR83 INRA, F-78350Jouy-en- Intégrative, et AnimaleBiologie Génétique UMR1313 AgroParisTech, F-78350Jouy-en-Josas, Biologie etIntégrative, Animale Génétique UMR1313, INRA, France12, cedex 75595Paris Bercy,149 de rueGIPSIE, département del’Elevage, Institut France Nouzilly, F-37380 IFCE, Nouzilly,F-37380 desComportements, Reproduction et dela Physiologie UMR85 INRA, Université François Rabelais de Tours, F-37000 Tours, France France F-37000 Tours, deTours, Rabelais François Université FranceNouzilly, F-37380 UMR7247,CNRS, UNCEIA, 149 rue de Bercy, 75595 Paris cedex 12, France12, cedex75595 Paris deBercy, 149rue UNCEIA, ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
role in ovary? inovary? role 1,2,3,4 [email protected] 6,7 C Hennequet Antier C ,
Copyright ©2017 bythe Society for Reproduction and Fertility. 1,2,3,4,5 Comment citer cedocument: C Ramé C , ; Phone: 33 2 47 42 77 89 ; Fax: 33 2 47 42 77 43).4277247 33 Fax: 89; 42 77 2 47 Phone:33 ; 8
1,2,3,4 S Elis S , J Cognié J , 1,2,3,4 S Fritz S , 1,2,3,4 J Lecardonnel J , 9 M Boussaha M , 6,7 6,7 F Jaffrézic F , S Marthey S ,
6,7 n J and 6,7 , D D , 1
Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 35 35 27 27 26 26 28 28 29 29 34 34 30 30 31 31 36 36 32 32 37 37 38 38 33 33 42 42 39 39 43 43 40 40 41 41 44 44 47 47 46 45 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is proliferation ( proliferation greater expressed in “fertil+” than in “fertil ”. “fertil ”. in than “fertil+” in expressed greater uniaie ri lcs fetn fml friiy oae o te oie hoooe three chromosome bovine the on located fertility female affecting locus trait quantitative Abstract (QTL F Fert BTA3) have a significantly lower conception rate and body weight after calving after weight body and rate conception lower significantly a have (QTL F Fert BTA3) than cows carrying the “fertil+” haplotype. “fertil+” the carrying cows than immunoblot we confirmed that adipose tissue tissue adipose that confirmed we immunoblot of genes included in the QTL F Fert BTA3 in “fertil+” and “fertil ” adipose tissue one week week one tissue adipose “fertil ” and “fertil+” in QTL F Fert BTA3 the in included genes of after calving when plasma non esterified fatty acid concentrations were greater in “fertil ” “fertil ” in greater were concentrations acid fatty esterified non plasma when calving after adipose tissue, pituitary, and ovary and detectable in hypothalamus and mammary gland. Its gland. mammary and hypothalamus in detectable and ovary and pituitary, tissue, adipose animals. animals. expression (mRNA and protein) is greater in kidney of “fertil+” than “fertil ” cows ( cows “fertil ” than “fertil+” of kidney in greater is protein) and (mRNA expression KIRREL (mRNA and protein) is also present in the different ovarian cells with a greater greater a with cells ovarian different the in present also is protein) and (mRNA KIRREL xrse i “etl” s oprd o fri cw ( cows “fertil ” to compared as “fertil+” in expressed to a rapidly increase in MAPK1/3 and MAPK14 phosphorylation in granulosa cells and to a a to and cells granulosa in phosphorylation MAPK14 and MAPK1/3 in increase rapidly a to xrsin n rnls cls f fri+ ta “etl” os I clue gauoa cells, granulosa cultured In cows. “fertil ” than “fertil+” of cells granulosa in expression erae n AK/ popoyain n oye Thus, oocyte. in phosphorylation MAPK1/3 in decrease recombinant KIRREL halved steroid secretion in basal state ( state basal in secretion steroid halved KIRREL recombinant metabolic messenger linking body composition and fertility. fertility. and composition linkingbody messenger metabolic ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
We have previously shown that dairy cows carrying the “fertil ” haplotype for one one for haplotype “fertil ” the carrying cows dairy that shown previously have We
We observed that thirty one genes were over expressed whereas twelve were under were twelve whereas over expressed were genes thirty one that observed We P <0.05) and and <0.05) Comment citer cedocument: oocyte maturation ( maturation oocyte vitro in
Here, we compared by tiling array tiling by compared we Here, KIRREL KIRREL P mRNA and protein were significantly were protein and mRNA mRNA is abundant in bovine kidney, kidney, bovine in abundant is mRNA <0.05). These results were associated associated were results These <0.05). P <0.05). By quantitative PCR and and PCR quantitative By <0.05). KIRREL KIRREL P <0.05). It also decreased cell cell decreased also It <0.05). ol b a potential a be could
the expression expression the P <0.05). <0.05). 2 Page 2of49 Version postprint Page 3of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 63 63 64 64 58 58 69 69 70 70 71 71 72 72 59 59 57 57 60 60 65 65 62 62 61 66 66 67 67 68 68 56 56 55 55 54 54 49 49 48 48 50 50 52 52 51 53 53 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is production, locomotion, maintenance or reproduction, are greater than the energy provided by by provided energythe than greater are reproduction, or maintenance locomotion, production, partially due to a lowest quality of the oocytes and consequently of pre implantation embryo embryo pre implantation of consequently and oocytes the of quality lowest a to due partially balance, but its magnitude and duration are quite variable (Butler variable quite are duration and magnitude its but balance, o, t s el nw ta eeg epniue fr hsooia fntos sc a milk as such functions, physiological for expenditures energy that known well is it cow, eeomn (Coyral Castel development ure 90 Shöe & tuebe 20) n cw my oe oy egt n body and weight body lose may cows and 2006) Staufenbiel & Schröder 1980, Currie odto. n al lcain cw my oiie bu 5 k o lpd Bua & Bruce & (Bauman lipid of kg 50 about mobilize may cows lactation, early In condition. Currie 1980) to support lactation. The use of body reserves accounts energetically for about for energetically accounts reserves body of use The lactation. support to 1980) Currie cows for food intake and eating behaviour, milk production, live weight and plasma plasma and weight live production, milk behaviour, eating and intake food for cows metabolites during the first lactation. Interestingly, the body weight of “fertil ” cows in the in cows “fertil ” of weight body the Interestingly, lactation. first the during metabolites feed intake. The maximum dry matter intake is reached about four to ten weeks after peak after weeks ten to four about reached is intake matter dry maximum The intake. feed al. weeks eight first ik Cpok 95. o hg ilig ar cw asm a eid f eaie energy negative of period a assume cows dairy high yielding So, 1985). (Coppock milk h eeg dfct bd rsre ae oiie (y nrae lplss (amn Bruce & (Bauman lipolysis) increased (by mobilized are reserves body deficit, energy the al. waves, length of oestrus cycle...) in “fertil+” and “fertil ” heifers and cows (Coyral Castel cows and heifers “fertil ” and “fertil+” in cycle...) oestrus of length waves, quantitative trait locus affecting female fertility located on the bovine chromosome 3 (QTL F 3 chromosome bovine the on located fertility female affecting trait locus quantitative Introduction Introduction Fert BTA3) had a greater conception rate 35 days after the first artificial insemination than insemination artificial first the after days 35 rate conception greater a had Fert BTA3) mapped (Druet mapped (Coyral Castel haplotype “fertil ” carrying those al. ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 , 2013) suggesting a greater fat mobilization in “fertil ” animals. During early lactation in in lactation early During animals. “fertil ” in mobilization fat greater a suggesting 2013) , 2007) and explained 14% of the total genetic variance (Ben Jemaa(Benvariancetotalgenetic of 14%the 2007)explained and 2011). However, we have demonstrated that the lower fertility of “fertil ” females could be could females “fertil ” of fertility that the lower demonstrated have we However, 2011).
We observed no differences in ovarian activity (number of follicles and follicular follicular and follicles of (number activity ovarian in differences no observed We We have previously shown that primiparous cows carrying “fertil+” haplotype for one one for haplotype “fertil+” carrying cows primiparous that shown previously have We et al. et ot partum post Comment citer cedocument: 2008), was described to affect early reproductive events (Guillaume events reproductive early affect to described was 2008), e al. et was significantly lower than “fertil+” cows (Coyral Castel (Coyral Castel cows “fertil+” than lower significantly was
02. e ae lo hrceie fri+ n “fertil ” and “fertil+” characterized also have We 2012). et al. et 2011). This QTL F Fert BTA3, finely finely QTL F Fert BTA3, This 2011). et al. et et al. et 1981). As a result of result a As 1981). 2008). et et et et 3 Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 81 81 80 80 82 82 83 83 84 84 78 78 77 77 79 79 85 85 86 86 73 73 87 87 74 74 88 88 75 75 76 76 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is and greater fat mobilization of “fertil ” cows, we compared by Tiling array the expression of expression the array Tiling by compared we cows, “fertil ” of mobilization fat greater and genes included in QTL F Fert BTA3 in the adipose tissue of “fertil+” and “fertil ” females females “fertil ” and “fertil+” of tissue adipose the in QTL F Fert BTA3 in included genes one week after calving. We then studied the distribution in bovine tissues of one candidate candidate one of tissues bovine in distribution the studied then We calving. after week one ee Kn f RE ie Doohl)lk ( (Drosophila) like like IRRE of Kin gene, o ert a ubr f omn ie opud ta rglts dpct dvlpet and development adipocyte regulates that compounds hormone like of number a secrete to 2008, Roche 2008, metabolic function (Ouchi function metabolic “fertil+” adipose tissue. Finally, we localized KIRREL by immunohistochemistry in bovine bovine in immunohistochemistry by KIRREL localized we Finally, tissue. adipose “fertil+” 3 o te ik rdcd n h frt ot o lcain Bua & rc Cri 1980). Currie Bruce & (Bauman lactation of month first the in produced milk the of 33% ovarian cells and investigated more precisely its its precisely more investigated and cells ovarian oiiain f a rsls n ees o nnetrfe fty cd (EA i bod which blood, in (NEFA) acids fatty non esterified of release in results fat of Mobilization steroidogenesis and proliferation and oocyte maturation by using recombinant KIRREL. recombinant using maturationby oocyte proliferation and and steroidogenesis were reviewed as indicators of energy status of ruminants (Bowden 1971). It is now well well now is It 1971). (Bowden ruminants of status energy of indicators as reviewed were 89 salse ta ngtv eeg blne mat erdcie ris t aiu lvl o the of levels various at traits reproductive impact balance energy negative that established yohlm iutr oaa ai (em Bte 19, Roche 1999, Butler & (Beam axis hypothalamo pituitary gonadal ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
In order to better understand the molecular mechanisms involved in the lower fertility fertility lower the in involved mechanisms molecular the understand better to order In et al. et 2009). Adipose tissue is not only an energy storage organ but it is also able also is it but organ storage energy onlyan isnot tissue Adipose 2009). Comment citer cedocument: et al.et ) but also fertility (Campos ) fertility but also
KIRREL fet o te rnls cell granulosa the on effects vitro in ), significantly greater expressed in in expressed greater significantly ), etal. 2008, Tersigni2008, t al. et 00 Leroy 2000, et al. et 2011). t al. et 4
Page 4of49 Version postprint Page 5of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 107 107 106 106 105 105 104 104 114 114 103 103 102 102 112 112 101 101 100 111 111 110 110 109 109 108 108 113 113 99 99 98 98 96 95 93 93 92 92 91 90 94 94 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is between the energy intake and the energy requirements for maintenance, milk production, and and production, milk maintenance, for requirements energy the and intake energy the between Animals Animals rgac. codn t h IR sse, h al rqieetfr aneac i 11 * 1.1 is maintenance for requirement daily the system, INRA the to According pregnancy. Ethics Ethics rtcl, hc wr cnitn wt te udlns rvdd y h Fec Cucl for Council French the by provided guidelines the with consistent were which protocols, after calving). Plasmas were stored at 20°C until assay. NEFA plasma concentrations were concentrations plasma NEFA assay. until 20°C at stored were Plasmas calving). after diet distribution, one week after calving and 5 months of pregnancy (about 7 and 8 months 8 and 7 (about pregnancy of months 5 and calving after week one distribution, diet heat detection with the semen of the same bull. Blood samples were taken from the tail before tail the from taken were samples Blood bull. same the of semen the with detection heat was more variable. Animals were artificially inseminated from 55 60 d postpartum 12 h after h 12 postpartum d 55 60 from inseminated artificially were Animals variable. more was morning live body weight was used for weight analyses, because the afternoon body weight weight body afternoon the because analyses, weight for used was weight body live morning each milking, cows were automatically weighted (software RIC version RW1.7). Only the the Only RW1.7). version RIC (software weighted automatically were cows milking, each soybean, 15% concentrate, 10% dehydrated alfalfa, and 0.5% calcium oxide (CaO). After After (CaO). oxide calcium 0.5% and alfalfa, dehydrated 10% concentrate, 15% soybean, fed and yards acltd e w acrig o h IR feig ytm (NA 20) s h difference the as 2007) (INRA, systems feeding INRA the to according wk per calculated were monitored during their second lactation. Dairy cows were managed in straw bedded straw bedded in managed were cows Dairy lactation. second their during monitored were week after calving when the adipose biopsy was performed as described below. It was was It below. described as performed was biopsy adipose the when calving after week Care. Animal 97 corporation, Espoo, Finland). Energy balance (EB, expressed in Mcal/d) was calculated one calculated was Mcal/d) in expressed (EB, balance Energy Finland). Espoo, corporation, determined by enzymatic colorimetry on a multiparameter analyser (KONE instruments instruments (KONE analyser multiparameter a on colorimetry enzymatic by determined (CEEA VdL”)), protocol registered under ref. n° 2012 10 4) approved all experimental experimental all approved 2012 10 4) n° ref. under registered protocol VdL”)), (CEEA methods and Materials ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Thirty six Holstein dairy cows (n=18 fertil+ and =18 fertil animals), born in 2006, 2006, in born animals), fertil =18 and fertil+ (n=18 cows dairy Holstein Thirty six nehc cmite(Cmt dEhqee Eprmnain nml a d Loire de Val Animale Expérimentation en d’Ethique (“Comité committee ethics An d libitum ad Comment citer cedocument: ih ttl ie rto cmoe o 6.% az slg, 10% silage, maize 64.5% of composed ration mixed total a with
5 Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 118 118 116 116 121 121 120 120 131 131 115 115 119 119 130 130 129 129 128 128 133 133 127 127 132 132 138 138 137 137 136 126 126 123 124 124 122 122 135 135 134 134 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Biopsy of subcutaneous adipose tissue tissue subcutaneous adipose of Biopsy Bovine fertility Tiling Array design Array Tilingfertility Bovine 117 Xylazine i.v. (Rompun; Bayer AG, Leverkusen, Germany) and an injection of 200 mg of of 200mg of injection an and Germany) Leverkusen, AG, Bayer (Rompun; i.v. Xylazine fasted for 12 h before surgery. Anesthesia was induced with injections of 12 to 14 mg of of mg 14 to 12 of injections with induced was Anesthesia surgery. before h 12 for fasted having a unique genome sequence match were selected with SSAHA (Ning SSAHA with selected were match sequence genome unique a having expressed in Mcal/d, where kg where Mcal/d, in expressed nml a 1kp oe ek otatm ad mg 5 ots f etto) Cw were Cows gestation). of months (5 5mpg and postpartum) week (one 1wkpp at animals Highly repeated elements in the genome were repeat masked. Concerning uniqueness, probes uniqueness, Concerning repeat masked. were genome the in elements repeated Highly .4 * kg * 0.041 fertility Tiling Array was designed and produced by Roche NimbleGen Inc. (Madison, USA). (Madison, Inc. NimbleGen Roche by produced and designed was Array Tiling fertility a gt rm CC aaae n c. 07 ees bsa4 nldn 3282 t The nt. 3627832 including bosTau4 release 2007 Oct. on database UCSC from got was for probe synthesis. Each probe overlapped its neighbour by about 40 bases. The arrays were arrays The bases. 40 about by neighbour its overlapped probe Each synthesis. probe for QTL F Fert BTA3. The sequence from position 9 887 417 to 13 515 249 on chromosome 3 chromosome on 249 515 13 to 417 887 9 position from sequence The QTL F Fert BTA3. isothermal format (Tm=76°C) and probe length constraint between 50 and 75 bp were used were bp 75 and 50 between constraint length probe and (Tm=76°C) format isothermal maskless process in which the synthesis of each probe is directed by a digital light processor. digitallight by a directed is each probe ofthe synthesis which in process maskless probes oligonucleotide the synthesized NimbleGen 2002). 125 dewlap. the from Lidocaine s.c. (Lurocaïne; Vétoquinol SA, Lure, France). Subcutaneous fat was collected collected was fat Subcutaneous France). Lure, SA, Vétoquinol (Lurocaïne; s.c. Lidocaine yteie o te ras y htltorpy (Singh Gasson photolithography by arrays the on synthesized manufactured by maskless array synthesis technology and the oligonucleotides were were oligonucleotides the and technology synthesis array maskless by manufactured ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
During the second lactation, biopsies of adipose tissue were collected from the same same the from collected were tissue adipose of biopsies lactation, second the During The 385k bovine fertility Tiling Array was designed in both orientations to cover the the cover to orientations both in designed was Array Tiling fertility bovine 385k The 0.75 ad h rqieet o ml pouto i 04 * ik rdcin E is EB production. milk * 0.44 is production milk for requirement the and , Comment citer cedocument: 0.75
indicates metabolic body weight (INRA, 2007). (INRA, weight2007). body metabolic indicates n situ in t al. et using a photo mediated, photo mediated, a using 99 Nuwaysir 1999, et al. et 2001). An 2001). t al. et 6
Page 6of49 Version postprint Page 7of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 153 153 162 162 144 144 150 150 149 152 152 143 143 148 148 161 161 159 159 158 160 160 147 147 154 154 142 142 163 163 141 141 146 146 145 145 157 157 140 140 156 156 139 139 155 155 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is pair. by aligning probe coordinates with annotation data from Ensembl database (release 56). The The 56). (release database Ensembl from data annotation with coordinates probe aligning by ae o Esml oprtv aayi, o ee wt kon tts n nte species another in status known with genes to analysis, comparative Ensembl on based bovine fertility Tiling Array platform has been submitted to the Gene Expression Omnibus Omnibus Expression Gene the to submitted been has platform Array Tiling fertility bovine several probes are available per exon and per gene. The model proposed is a mixed model mixed a is proposed model The gene. per and exon per available are probes several em ewe eo ad odto. Parameter condition. and exon between term transcript with a sequence match in a sequence repository external to Ensembl for the same same the for Ensembl to external repository sequence a in match sequence a with transcript Tiling Array data analysis analysis data Array Tiling 151 loci are classified three types: (1) known protein coding gene, known gene has at least one least at has gene known gene, coding protein known (1) types: three classified are loci genebuild transcript and the Vega manual annotation have the same sequence, for every base base every for sequence, same the have annotation manual Vega the and transcript genebuild omitted here as it is a gene by gene model: gene by gene hereisait as omitted where where (usually human genes). (3) Putative protein coding gene refers to genes where the Ensembl the where genes to refers gene coding protein Putative (3) genes). human (usually including a fixed exon and a random probe effects. In this study, our aim was to detect detect to was aim our study, this In effects. probe random a and exon fixed a including (GEO) repository and the accession number is GPL15186. Annotation of probes was obtained obtained was probes of Annotation GPL15186. is number accession the and repository (GEO) species. (2) Known by projection protein coding gene, refers to genes that are homologous, are that genes to refers gene, coding protein projection by Known (2) species. fet a be cniee fr ah gene each for considered been has effect A hierarchical mixed model with an exon within gene effect and a random probe within exon exon within probe random a and effect gene within exon an with model mixed hierarchical A n vrg eey lvn ae ad 5 6 rnol gnrtd rbs Al nomto of information All probes. generated randomly 961 45 and bases eleven every average on differentially expressed genes between “fertil+” and “fertil ” samples. samples. and “fertil ” genes“fertil+” between expressed differentially The array contained 343 162 50 75 mer oligonucleotides designed on both strands and tiled and strands both on designed oligonucleotides 50 75 mer 162 343 contained array The ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 corresponds to an exon effect effect exon an to corresponds
We developed a new model to perform Tiling array analysis taking advantage that that advantage taking analysis array Tiling perform to model new a developed We corresponds to a condition effect with two levels ( levels two with effect condition a to corresponds Comment citer cedocument:
within gene gene within ( orsod t te rb efc k within k effect probe the to corresponds . For simplicity the index index the simplicity For . c =1.2 for =1.2for " ), and ),
fertil is the interaction the is +" and and " fertil will be be will " ), 7 Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 188 188 187 187 186 186 185 185 184 184 183 183 179 179 182 182 180 178 178 169 169 177 177 166 166 176 176 174 174 175 175 173 173 172 172 171 171 168 168 170 170 167 167 165 165 164 164 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is sample. Tubes were waved for 15 seconds and left at room temperature for 5 minutes before minutes 5 for temperature room at left and seconds 15 for waved were Tubes sample. QIAzol lysis reagent (Qiagen, Courtaboeuf, France). Chloroform (0.2 ml) was added to each each to added was ml) (0.2 Chloroform France). Courtaboeuf, (Qiagen, reagent lysis QIAzol a etatd n c fo 20 g ftsu wt a utaua hmgnzr sn 8 l of ml 8 using homogenizer ultraturax an with tissue of mg 250 from ice on extracted was “fertil+” and 14 “fertil ”), frozen in liquid nitrogen and stored at 80°C until use. Total RNA Total use. until 80°C at stored and nitrogen liquid in frozen “fertil ”), 14 and “fertil+” (18 “fertil+” and 18 “fertil ”) one week after parturition and at 5 months of pregnancy (16 (16 pregnancy of months 5 at and parturition after week one “fertil ”) 18 and “fertil+” (18 Total RNA extraction extraction RNA Total 1.8).release (AmiGO website Ontology Gene 181 Genes were classified according to the Gene Ontology using NCBI, Ensembl, DAVID and the DAVID and Ensembl, NCBI, Ontology using Gene to the according classified were Genes ( Jaffrézic. We focused our study on known and known by projection protein coding genes. genes. coding protein projection by known and known on study our focused We Jaffrézic. variances variances alternative splicing. R functions implementing this model are available upon request from F. from request upon available are model this implementing functions R splicing. alternative Our model could also be used to detect differentially expressed exons and genes with with genes and exons expressed differentially detect to used be also could model Our strand. for the forward strand, and 4 379 probes matching to 62 genes and 352 exons for the reverse reverse the for exons 352 and genes 62 to matching probes 379 4 and strand, forward the for gene and exon information: 5 822 probes matching to 62 genes and 449 exons in the analysis analysis the in exons 449 and genes 62 to matching probes 822 5 information: exon and gene & Hochberg 1995). This model was applied on two datasets containing annoted probes with probes annoted containing datasets two on applied was model This 1995). Hochberg & hypothesis hypothesis adjusted by Benjamini Hochberg’s procedure to control the False Discovery Rate (Benjamini Rate Discovery False the control to procedure Benjamini Hochberg’s by adjusted In this model, testing for differentially expressed genes is equivalent to testing the null null the testing to equivalent is genes expressed differentially for testing model, this In independent and normally distributed such that: that: such distributed normally and independent exon exon that: c ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 =1.2 for =1.2for "
Subcutaneous adipose tissue was sampled at the dewlap of 36 second lactation cows cows lactation second 36 of dewlap the at sampled was tissue adipose Subcutaneous ( fertil . Index . +" , with gene by gene variances variances gene by gene with , and and " Comment citer cedocument: . h poe fet s sue t b a adm fet such effect random a be to assumed is effect probe The ). represents the biological replicates biological the represents fertil , where where , " ). Taking into account multiple testing, testing, multiple account into Taking ).
n and Residuals . . . wt gene by gene with , for two conditions conditions two for are also assumed assumed also are P vle were values 8 Page 8of49 Version postprint Page 9of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 197 197 198 198 196 196 195 195 202 202 201 201 200 200 194 194 206 206 193 193 204 204 205 205 192 192 191 191 190 190 203 203 209 209 208 207 211 211 210 210 189 189 213 213 212 212 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is NanoDrop Spectrophotometer (Nyxor Biotech, Paris, France) and RNA quality with an an with quality RNA and France) Paris, Biotech, (Nyxor Spectrophotometer NanoDrop S1. Table 199 Massy, France). The RNA integrity number (RIN) for each RNA sample is shown in the the in shown is sample RNA each for (RIN) number integrity RNA The France). Massy, Agilent 2100 Bioanalyzer using a RNA 6000 Nano assay protocol (Agilent Technologies, Technologies, (Agilent protocol assay Nano 6000 RNA a using Bioanalyzer 2100 Agilent luhehue a etatd n c wt a utaua hmgnzr n TRIzol in homogenizer ultraturax an with ice on extracted was slaughterhouse ag flils > m) cru ltu, vra cre ad rnls cls from cells) granulosa and cortex ovarian luteum, corpus mm), 7 (> follicles large tissue, kidney, pituitary, lung, skeletal muscle, ovary, hypothalamus, small follicles (< 6 mm), mm), 6 (< follicles small hypothalamus, ovary, muscle, skeletal lung, pituitary, kidney, tissue, was extracted using 1 ml of TRIzol of ml 1 using extracted was System and stored at 80°C until cDNA synthesis. RNA quantity was assessed with a a with assessed was quantity RNA synthesis. cDNA until 80°C at stored and System sur Yvette, France). A treatment with DNaseI using the DNA the using DNaseI with treatment A France). Yvette, sur technologies were evaporated without heating during 1.5 hours in a Thermo Savant SPD1010 SpeedVac SPD1010 Savant Thermo a in hours 1.5 during heating without evaporated were RNase free DNaseI (Qiagen) was performed. After elution with RNase free water, samples water, free RNase with elution After performed. was (Qiagen) DNaseI RNase free according to the manufacturer’s recommendations. During purification, a treatment with a a with treatment a purification, During recommendations. manufacturer’s the to according according to manufacturer’s recommendation (Invitrogen recommendation manufacturer’s to according cDNA synthesis and labeling, array hybridization, washing and scanning scanning and washing hybridization, array and labeling, synthesis cDNA Spectrophotometer. NanoDrop with a nn “etl” n nn “etl” oe ek fe clig ht s sae f nes adipose intense of stage a is that calving after week one “fertil ”) nine and “fertil+” (nine tao 7% vv. hn oa RA a prfe uig RNeasy a using purified was RNA total Then (v:v). 70% ethanol centrifugation (5000 g, 15 minutes, 4°C). Each aqueous phase was mixed to equal volume of volume equal to mixed was phase aqueous Each 4°C). minutes, 15 g, (5000 centrifugation tissue mobilization. Samples were prepared, labelled and hybridized according to the the to according hybridized and labelled prepared, were Samples mobilization. tissue ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
For RT PCR, total RNA from bovine tissues (Liver, mammary gland, heart, adipose adipose heart, gland, mammary (Liver, tissues bovine from RNA total RT PCR, For Array hybridation was performed using cDNA of adipose tissue from eighteen animals eighteen from tissue adipose usingof cDNA performed was Array hybridation ) was performed on the total RNAs. Total RNA from granulosa cells in culture culture in cells granulosa from RNA Total RNAs. total the on performed was ) Comment citer cedocument:
reagent by scratching wells. RNA quantity was assessed was quantity RNA wells. scratching by reagent by Life technologies Life by free Kit Midi Kit (Qiagen) (Qiagen) Kit Midi
(Ambion , Villebon , reagent by Life Life by 9
Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 238 238 235 236 236 234 234 233 233 232 232 231 231 230 230 229 229 228 228 227 227 217 217 222 222 216 216 215 221 221 225 225 220 220 226 226 219 219 218 218 214 214 224 224 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Real-time quantitative PCR (qPCR) (qPCR) PCR quantitative Real-time bromide. bromide. NimbleGen, Inc.). Hybridization solution was prepared from the NimbleGen Hybridization Hybridization NimbleGen the from prepared was solution Hybridization Inc.). NimbleGen, NimbleGen Arrays User’s Guide: Gene Expression Analysis v3.2. cDNAs were synthesized synthesized were cDNAs v3.2. Analysis Expression Gene Guide: User’s Arrays NimbleGen Reverse Transcription and Polymerase Chain Reaction Chain andPolymerase Transcription Reverse KIRREL France). (Charbonnières les Bains, 237 eois Geol, rne. T n PR osmbe wr prhsd rm Promega from purchased were consumables PCR and RT France). (Grenoble, Genomics codn t te auatrrs rcdr. N ws eune b Bcmn Coulter Beckman by sequenced was DNA procedure. manufacturer’s the to according e uig h EN mcolt Gl xrcin i (W, otnysu os France) Fontenay sous Bois, (VWR, kit Extraction Gel microelute EZNA the using gel at 72°C. PCR products were visualized in a 1.5% (w:v) agarose gel stained with ethidium ethidium with stained gel agarose (w:v) 1.5% a in visualized were products PCR 72°C. at minute at 94°C, 1 minute at 58°C, 1 minute at 72°C), with a final extension step of 7 minutes 7 of step extension final a with 72°C), at minute 1 58°C, at minute 1 94°C, at minute described mixture (Coyral Castel mixture described technologies 223 Samples were labelled with Cy3 with a NimbleGen One Color DNA Labeling Kit (Roche (Roche Kit Labeling DNA One Color NimbleGen a with Cy3 with labelled were Samples Technologies 0µ mxue s rvosy ecie (Coyral Castel described previously as mixture µl 20 Inc.). NimbleGen, (Roche software NimbleScan theRoche wereData extracted with nm. i (oh NmlGn n..Ary wr sand ih GnPx40B cne t 532 at Scanner 4000B GenePix a with scanned were Arrays Inc.). NimbleGen, (Roche Kit 42°C for 18 hours. Finally, arrays were washed with solutions of the NimbleGen Wash Buffer Buffer Wash NimbleGen the of solutions with washed were arrays Finally, hours. 18 for 42°C Kit (Roche NimbleGen, Inc.) and Cy3 labeled samples were hybridized on the 385K array at array 385K the on hybridized were samples Cy3 labeled and Inc.) NimbleGen, (Roche Kit (Invitrogen Kit Synthesis cDNA Double Stranded Superscript Invitrogen an using ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Reverse transcription (RT) of total RNA (1 µg) was performed for 1 hour at 37°C in a a in 37°C at hour 1 for performed was µg) (1 RNA total of (RT) transcription Reverse and and ACTR3 Tbe ) oyeaecan ecin PR ws are ot n previously a in out carried was (PCR) reaction chain Polymerase 2). Table , ). They were then purified using a MinElute Reaction Cleanup Kit (Qiagen). (Qiagen). Kit Cleanup Reaction MinElute a using purified then were They ). ACTR3 was used as positive control. Finally, DNA was extracted from the agarose agarose the from extracted was DNA Finally, control. positive as used was Comment citer cedocument: ee mlfe wt seii pies (Invitrogen primers specific with amplified were
et al. et 2010) for 30 ( 30 for 2010) ACTR3 et al. et 00.Snl tadcNs of cDNAs Single strand 2010). ) or 40 ( 40 or ) KIRREL ) PCR cycles (1 cycles PCR ) y Life by by Life by 10 Page 10of49 Version postprint Page 11of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 253 253 252 252 263 263 262 262 261 261 251 251 260 260 250 250 259 259 249 249 255 255 248 248 254 254 247 247 258 258 256 246 246 245 245 244 244 243 243 242 242 239 239 241 241 240 240 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is o h goerc en f PA n EFA (h ms sal cmiain floig the following combination) stable most (the EEF1A1 and PPIA of mean geometric the to changes between « fertil+ » and « fertil » tissues or cells. Therefore, the data were normalized were data the Therefore, cells. or tissues «fertil » »and «fertil+ between changes rne, eiiln 10 Im, A lbrtre, e Mrax Fac) srpoyi (0.1 streptomycin France), Mureaux, Les laboratories, PAA UI/ml, (100 penicillin France), Euromedex, Souffelweyersheim, France), L glutamine (3 mM, Eurobio, Courtaboeuf, Courtaboeuf, Eurobio, mM, (3 L glutamine France), Souffelweyersheim, Euromedex, fiiny n C : expression=E : Cq and efficiency mm) in McCoy’s 5A culture medium enriched with bovine serum albumin (BSA 0.1% (w:v), (w:v), 0.1% (BSA albumin serum bovine with enriched medium culture 5A McCoy’s in mm) E11 wr eaie. o ec gn, xrsin a cluae acrig o primer to according calculated was expression gene, each For examined. were EEF1A1– saline up to the laboratory. Granulosa cells were isolated by puncturing small follicles (< 6 (< follicles small puncturing by isolated were cells Granulosa laboratory. the to up saline the expressions of four housekeeping genes – PPIA (cyclophilin A), RPL19, ACTR3 and and ACTR3 RPL19, A), (cyclophilin PPIA – genes housekeeping four of expressions the normalization factor (Vandesompele (Vandesompele factor normalization serial dilutions of a pool of obtained cDNA and ranged from 1.80 to 2.16. For normalization, normalization, For 2.16. to 1.80 from ranged and cDNA obtained of pool a of dilutions serial report that suggests the geometric mean of multiple housekeeping genes as an accurate accurate an as genes housekeeping multiple of mean geometric the suggests that report following by the acquisition of the melting curve. Primers’ efficiency (E) was performed from performed from was (E) efficiency Primers’ curve. melting the of acquisition bythe following Granulosa cell collection and primary culture culture primary and collection cell Granulosa 257 ujce t 4 cce (0 eod a 9°, 0 eod a 6°, 0 eod a 72°C), at seconds 30 60°C, at seconds 30 95°C, at seconds (30 cycles 40 to subjected incubation for 2 minutes at 50°C and a denaturation step of 10 minutes at 95°C, samples were samples 95°C, at minutes 10 of step denaturation a and 50°C at minutes 2 for incubation with water, instead of cDNA, was performed systematically as a negative control. After After control. negative a as systematically performed was cDNA, of instead water, with were tested in duplicate on the same plate and the CVs was less than 5% . PCR amplification PCR . 5% than less was CVs the and plate same the on duplicate in tested were wkpp and thirty animals (n=16 “fertil+” and =14 “fertil ” animals) at 5 months of pregnancy of months 5 at animals) “fertil ” =14 and “fertil+” (n=16 animals thirty and wkpp (Invitrogen primers specific of nM 250 and France) Coquette, la Marnes (Bio Rad, adipose tissue, samples from thirty six animals (n=18 “fertil+” and =18 “fertil ” animals) at 1 1 at animals) “fertil ” =18 and “fertil+” (n=18 animals six thirty from samples tissue, adipose technologies ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Targeted cDNAs were quantified by real time PCR using SYBR Green Supermix Supermix Green SYBR using PCR real time by quantified were cDNAs Targeted Bovine ovaries were collected at the slaughterhouse and transported in physiological in transported and slaughterhouse the at collected were ovaries Bovine Tbe ) n oa vlm o 2 µ i a yQ yl dvc (i a) For (Bio Rad). device Cycle MyiQ a in µl 20 of volume total in 1) Table , Comment citer cedocument:
Cq et al. et Tee or oskeig ee soe expressional showed genes housekeeping four These . 2002). 2002). by Life by 11 Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 271 271 270 270 269 269 273 273 272 272 278 278 280 280 268 268 277 277 267 267 266 266 287 287 288 288 265 265 286 286 276 276 274 264 264 285 285 281 281 284 284 283 283 282 282 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Systems starved for 18 hours before treatment with a recombinant mouse (rm) KIRREL (R&D (R&D KIRREL (rm) mouse recombinant a with treatment before hours 18 for starved were seeded per well of a 24 well culture plate. After 24 hours of culture, cells were serum serum were cells culture, of hours 24 After plate. culture 24 well a of well per seeded were containing 5%CO containing NDK NH ehsa UA. utrs ee efre a 3° i a uiiid air humidified a in 37°C at performed were Cultures USA). Bethesda, NIH (NIDDK, Thymidine incorporation into granulosa cells into incorporation Thymidine 279 France) and amphotericin B (5 µg/ml, PAA laboratories). Approximately 2 x 10 x 2 Approximately laboratories). PAA µg/ml, (5 B amphotericin and France) cells (blue cells) were counted using a hemocytometer. ahemocytometer. using counted were (blue cells) cells supplemented with 10% (v:v) fetal bovine serum (FBS, PAA laboratories, Les Mureaux, Mureaux, Les laboratories, PAA (FBS, serum bovine fetal (v:v) 10% with supplemented fresh enriched McCoy’s 5A and the pellet was resuspended in enriched McCoy’s 5A 5A McCoy’s enriched in resuspended was pellet the and 5A McCoy’s enriched fresh with each condition in quadruplate. in condition with each Saint Quentin Fallavier, France). Cells were centrifuged at 200 g for 5 minutes, washed with with washed minutes, 5 for g 200 at centrifuged were Cells France). Saint Quentin Fallavier, values, expressed as count per min (CPM), are representative of five independent cultures cultures independent five of representative are (CPM), min per count as expressed values, Cell viabilityCell 275 lrc, an uni alve, rne ad nrseein (. µo/, Sigma Aldrich, µmol/l, (0.1 androstenedione and France) Saint Quentin Fallavier, Aldrich, mg/ml, PAA laboratories), Hepes (20 mM pH = 7.6), bovine apo transferrin (5 µg/ml, Sigma µg/ml, (5 apo transferrin bovine 7.6), = pH mM (20 Hepes laboratories), PAA mg/ml, radioactivity was determined in scintillation fluid by counting in a a in counting by fluid scintillation in determined was radioactivity [ sn cl 5% vv tihooctc cd o 1 mnts n lsd y . N aH The NaOH. N 0.5 by lysed and minutes 10 for acid trichloroacetic (v:v) 50% cold using excess of thymidine was removed by washing cells twice using PBS 1X. Then cells was fixed fixed was cells Then 1X. PBS using twice cells washing by removed was thymidine of excess rm KIRREL (10 ng/ml or 100 ng/ml) in enriched McCoy’s 5A. After 24 hours of culture, culture, of hours 24 After 5A. McCoy’s enriched in ng/ml) 100 or ng/ml (10 KIRREL rm 3 H] thymidine (Perkin Elmer, Courtaboeuf, France) was added in the presence or absence of absence or presence the in added was France) Courtaboeuf, (Perkin Elmer, H] thymidine ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
fe 1 hus f eu savto, utr mdu ws eoe ad µim of µCi/ml 1 and removed was medium culture starvation, serum of hours 18 After Cell viability was determined by Blue Trypan staining. Live (normal cells) and dead and cells) (normal Live staining. Trypan Blue by determined was viability Cell , Lille, France), human recombinant IGF1 (Sigma) and/or ovine recombinant FSH recombinant ovine and/or (Sigma) IGF1 recombinant human France), Lille, , 2 . Comment citer cedocument:
β photomultiplier. The The photomultiplier. 5 live cells cells live 12 Page 12of49 Version postprint Page 13of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 297 297 304 304 303 303 298 298 307 307 302 300 313 313 306 306 299 312 312 305 305 311 311 295 295 310 310 309 309 294 294 308 308 293 293 291 291 290 290 292 292 296 296 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is protein concentration/well. Results are presented as mean mean as presented are Results concentration/well. protein Protein extraction and western-blot western-blot and extraction Protein presence or absence of rm KIRREL (10 ng/ml or 100 ng/ml), IGF1 (10 IGF1 ng/ml), 100 or ng/ml (10 KIRREL rm of absence or presence Progesterone and oestradiol assay oestradiol and Progesterone 2 ad 0, epciey Rsls ee xrse a the as expressed were Results respectively. 10%, and 12% s rvosy ecie (Coyral Castel described previously as ultraturax homogenizer (tissues) or by scratching wells (primary cultured cells) in lysis buffer buffer lysis in cells) (primary cultured wells scratching by or (tissues) homogenizer ultraturax (Coyral Castel 301 onto nitrocellulose membrane and incubated with specific antibodies as previously described described previously as antibodies specific with incubated and membrane nitrocellulose onto in quadruplate. was analyzed condition in each which cultures, Souffelweyersheim, France). Mouse monoclonal antibodies to Vinculin (VCL) and PCNA PCNA and (VCL) Vinculin to antibodies monoclonal Mouse France). Souffelweyersheim, denaturated, submitted to electrophoresis in a 12% (w:v) SDS polyacrylamide gel, transferred gel, SDS polyacrylamide (w:v) 12% a in electrophoresis to submitted denaturated, MAPK14 and KIRREL were obtained from Santa Cruz Biotechnology (Euromedex, (Euromedex, Biotechnology Cruz Santa from obtained were KIRREL and MAPK14 coefficients of variation were less were variation of coefficients Yveline, France). Rabbit polyclonal antibodies to phospho AKT1 (Ser473), MAPK1, MAPK1, (Ser473), phospho AKT1 to antibodies polyclonal Rabbit France). Yveline, (Thr180/Tyr182) were purchased from Cell signalling Technology (Ozyme, Saint Quantin en Quantin Saint (Ozyme, Technology signalling Cell from purchased were (Thr180/Tyr182) 2005). The limit of detection of progesterone was 12 pg/tube and pg/tube 12 was progesterone of detection of limit The 2005). (Thr172), PRKAA, phospho MAPK1/3 (Tyr204/Thr202), phospho MAPK14 phospho MAPK14 (Tyr204/Thr202), phospho MAPK1/3 PRKAA, (Thr172), medium was measured by a radioimmunoassay protocol as previously described (Tosca described previously as protocol radioimmunoassay a by measured was medium 8 289 oestradiol was 25 pg/tube and pg/tube 25 was oestradiol M) in enriched McCoy’s 5A. The concentration of progesterone and oestradiol in the culture the in oestradiol and progesterone of concentration The 5A. McCoy’s enriched in M) ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Lysates of tissues (adipose tissue and kidney) or cells were prepared on ice with an with ice on prepared were cells or kidney) and tissue (adipose tissues of Lysates Granulosa cells were cultured for 48 hours, after 18 hours of serum starvation, in the in starvation, serum of hours 18 after hours, 48 for cultured were cells Granulosa t al. et Comment citer cedocument: 2010). Rabbit polyclonal antibodies to AKT1, phospho PRKAA phospho PRKAA AKT1, to antibodies polyclonal Rabbit 2010).
the intra and inter assay coefficients of variation were less were variation of coefficients inter assay and intra the
than 10% and 11%, respectively. The limit of detection of detection of limit The respectively. 11%, and 10% than t al. et 2010). Proteins extracts (80 µg) were were µg) (80 extracts Proteins 2010). ±
concentration of steroids/cell steroids/cell of concentration .. o fu independent four of S.E.M
the intra and inter assay and intra the 8 M) and/or FSH (10 FSH and/or M) et al. et
than 13
Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 338 338 337 337 336 336 335 335 334 334 333 333 332 332 331 331 330 330 328 327 327 326 326 321 320 320 325 325 319 319 318 318 317 317 324 324 322 316 316 315 315 314 314 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Bovine Oocyte Collection and In Vitro Maturation Maturation InVitro and OocyteCollection Bovine phosphorylation, respectively) and vinculin (for KIRREL) as an internal standard. aninternal (forKIRREL) as vinculin respectively) and phosphorylation, Immunohistochemistry Immunohistochemistry by enhanced chemiluminescence (Western Lightning (Western chemiluminescence byenhanced TCM 199 (Sigma) with 4 mg/ml BSA supplemented or not with different concentrations of concentrations different with not or supplemented BSA mg/ml 4 with (Sigma) 199 TCM 0.4% BSA and gentamycine (2.5ml/L) under mineral oil (Sigma). The COCs were cultured in cultured were COCs The (Sigma). oil mineral under (2.5ml/L) gentamycine and BSA 0.4% eliminated: only intact COCs were washed in TCM Hepes 199 (Sigma) supplemented with supplemented (Sigma) 199 Hepes TCM in washed were COCs intact only eliminated: ee hn eetd ne a iscig irsoe Epne o nnnat Os were COCs nonintact or Expanded microscope. dissecting a under selected then were n t a aum ie 10mg a peiul dsrbd Rvrhn et (Reverchon described previously as (100mmHg) line vacuum a to and from follicles 3–8 mm in diameter using an 18 gauge needle connected to a sterile test tube test sterile a to connected needle 18 gauge an using diameter in mm 3–8 follicles from maintained at 37°C until aspiration. The cumulus oocyte complexes (COCs) were aspirated aspirated were (COCs) complexes cumulus oocyte The aspiration. until 37°C at maintained 329 or rabbit IgG as negative controls. Ovaries from 3 different cows were studied.were cows 3 fromdifferent Ovaries IgG as negative controls. rabbit or were incubated overnight with antibodies against KIRREL (1:100, Santa Cruz biotechnology) biotechnology) Cruz Santa (1:100, KIRREL against antibodies with overnight incubated were Immunohistochemistry was performed as previously described (Tosca described previously as performed was Immunohistochemistry PRKAA and AKT1 MAPK14, MAPK1/3, (for total PRKAA AKT1, MAPK14, nest sga i abtay nt atr omlzto alwd y h peec o MAPK3, of presence the by allowed normalization after units arbitrary in signal intensity uniid ih h GnTos otae rlae .10) Te eut ae xrse a the as expressed are results The 4.01.02). (release software GeneTools the with quantified SynGene (Ozyme) with the GeneSnap software (release 7.09.17). Signals detected were were detected Signals 7.09.17). (release software GeneSnap the with (Ozyme) SynGene 323 and anti mouse IgG were purchased from Eurobio (Les Ulis,France). Proteins were detected were Proteins Ulis,France). (Les Eurobio from purchased were IgG anti mouse and respectively. Antibodies were used at 1:1000. Horseradish peroxidase conjugated anti rabbit anti rabbit peroxidase conjugated Horseradish 1:1000. at used were Antibodies respectively. (proliferating cell nuclear antigen) were purchased from Sigma Aldrich and Ozyme, Ozyme, and Sigma Aldrich from purchased were antigen) nuclear cell (proliferating ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Bovine ovaries were collected from a slaughterhouse in sterile NaCl solution and and solution NaCl sterile in slaughterhouse a from collected were ovaries Bovine Bovine ovaries embedded in paraffin were serially sectioned at a thickness of 7 µm. 7 of thickness a at sectioned serially were paraffin in embedded ovaries Bovine Comment citer cedocument:
Plus ECL, Perkin Elmer) using a G:Box G:Box a using Elmer) Perkin ECL, et al. et al. 2005). Sections Sections 2005). 2014). COCs COCs 2014). 14 Page 14of49 Version postprint Page 15of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 340 340 339 339 346 346 345 345 344 341 342 342 349 349 348 348 347 347 353 353 352 352 351 351 356 356 355 355 354 354 350 350 358 358 357 357 361 361 360 359 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is pipetting with 0.5% hyaluronidase (Sigma), and the DNA was colored with Hoechst before Hoechst with colored was DNA the and (Sigma), hyaluronidase 0.5% with pipetting Statistical analysis Statistical progesterone and oestradiol secretion by bovine granulosa cells in basal state or in response to response in or state basal in cells granulosa bovine by secretion oestradiol and progesterone rgseoe ocnrto i te n ir mtrto mdu ad h lvl f phospho of level the and medium maturation vitro in the in concentration progesterone 2009). The MIXED procedure for linear mixed models was used to determine the changes of : thechanges determine used to was models mixed linear for procedure MIXED The 2009). by denuded were COCs maturation, After oocytes. 50 least at contained group oocyte Each rm KIRREL (10 and 100 ng/ml) for 22 h at 39°C in 5% CO2 in air with saturated humidity. saturated with air in CO2 5% in 39°C at h 22 for ng/ml) 100 and (10 KIRREL rm mounting. 343 mpg), haplotype (fertil +, fertil ) and time after calving×haplotype interaction. interaction. calving×haplotype time after +,and fertil ) (fertil haplotype mpg), adipose tissue KIRREL expression. The initial model included time after calving (1 wkpp, 5 wkpp, (1 calving after time included model initial The expression. KIRREL tissue adipose i) the live body weight; ii) the energy balance ; iii) the plasma NEFA concentrations ; iv) ; concentrations NEFA plasma the iii) ; balance energy the ii) weight; body live the i) various ovarian compartments and in granulosa cells, the effect of rm KIRREL on on KIRREL rm of effect the cells, granulosa in and compartments ovarian various tissues (kidney, hypothalamus, pituitary and mammary), the KIRREL mRNA expression in in expression mRNA KIRREL the mammary), and pituitary hypothalamus, (kidney, tissues granulosa cells and on the amount of PCNA, the amount of oocyte at the GV stage, and the and stage, GV the at oocyte of amount the PCNA, of amount the on and cells granulosa IGF1 and FSH, the effect of rm KIRREL on the amount of 3H thymidine incorporated into into incorporated thymidine 3H of amount the on KIRREL rm of effect the FSH, and IGF1 means±SEM and results were considered statistically significant atsignificant statistically considered were results andmeans±SEM MAPK1/3 in oocyte were assessed using one way ANOVA. Numerical data are expressed as expressed are data Numerical ANOVA. one way using assessed were oocyte in MAPK1/3 ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
l saitcl nlss ee odce uig h SS otae SS nttt INC, Institute (SAS software SAS the using conducted were analyses statistical All The protein amount of KIRREL in adipose tissue, kidney, granulosa cells and various various and cells granulosa kidney, tissue, adipose in KIRREL of amount protein The
Comment citer cedocument:
P <0.05. <0.05. 15 Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 362 362 363 363 376 376 364 364 375 375 374 374 366 366 386 386 385 365 365 371 371 373 373 382 382 370 370 381 381 369 369 380 380 367 367 368 368 379 379 384 384 378 378 383 383 377 377 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is NEFA plasma concentrations, Energy Balance and Live Body Weight of animalsof Body Weight Live and Balance Energy concentrations, plasma NEFA plasma NEFA than “fertil ” cows (n= 18; 860.6 860.6 18; (n= cows “fertil ” than NEFA plasma NEPH1 od n h Tbe ta hd h hget od hne o efr epeso aayi by analysis expression perform to change fold highest the had that 2 Table the in bold Results genes were differentially expressed in adipose tissue of “fertil+” and “fertil ” cows ( cows “fertil ” and “fertil+” of tissue adipose in expressed differentially were genes a 385K array containing the sequence of the QTL F Fert BTA3. We observed that 43 known known 43 that observed We QTL F Fert BTA3. the of sequence the containing array 385K a nine “fertil+” and nine “fertil ” was extracted, reverse transcribed, labelled and hybridized on hybridized and labelled transcribed, reverse extracted, was “fertil ” nine and “fertil+” nine 0.6 Mcal/day, respectively, respectively, Mcal/day, 0.6 respectively, respectively, Tiling array Tiling 372 “fertil+” and “fertil ” animals (n=16 “fertil+” and n=14 “fertil ” ) (Fig. 1). (Fig. ) “fertil ” andn=14 (n=16 “fertil+” animals “fertil ” and “fertil+” olfactory receptors (10 on 12 genes, Table 2). We then selected about 10 genes represented in represented genes 10 about selected then We 2). Table genes, 12 on (10 receptors olfactory reserves, plasma NEFA, energy balance and live body weight were not significant between significant not were weight body live and balance energy NEFA, plasma reserves, 0.7694 to 0.9714 (Table 2). Genes under expressed in “fertil+” adipose tissue were mainly were tissue adipose “fertil+” in under expressed Genes 2). (Table 0.9714 to 0.7694 0.7 kg, respectively, respectively, kg, 0.7 “fertil ” than in “fertil+” cows. At 5 months of gestation (mpg) during reconstitution of body of reconstitution during (mpg) gestation of months 5 At cows. “fertil+” in than “fertil ” Twelve were under expressed in “fertil+” adipose tissue, with fold change varying from from varying change fold with tissue, adipose “fertil+” in under expressed were Twelve Tiling array by qPCR for only one gene, named named gene, one only for qPCR by array Tiling “fertil ” cows, with fold change (“fertil+”/“fertil ”) ranging from 1.0345 to 1.6612 (Table 2). 2). (Table 1.6612 to 1.0345 from ranging (“fertil+”/“fertil ”) change fold with cows, “fertil ” quantitative PCR using specific primers (Table 2). Interestingly, we confirmed the results of results the confirmed we Interestingly, 2). (Table primers specific using PCR quantitative Table 2). Thirty one genes were over expressed in “fertil+” adipose tissue as compared to to compared as tissue adipose “fertil+” in over expressed were genes Thirty one 2). Table ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
One week after calving, “fertil+” cows (n=18) had significant lower concentrations of concentrations lower significant had (n=18) cows “fertil+” calving, after week One To better investigate this difference in mobilization, total adipose tissue RNA from from RNA tissue adipose total mobilization, in difference this investigate better To
. P <0.05, Fig. 1A.) and a greater energy balance ( 10.8 balance energy greater a and 1A.) Fig. <0.05, Comment citer cedocument: P <0.05, Fig.1C), suggesting a greater adipose tissue mobilization in in mobilization tissue adipose greater a suggesting Fig.1C), <0.05, P ± (666.1 weight body live and Fig.1B) <0.05,
KIRREL ± 105.4 µmol/l vs 1247.0 1247.0 vs µmol/l 105.4 (kin of IRRE like) also known as known also like) IRRE of (kin ± 0.7 Mcal/day vs 14.4 14.4 vs Mcal/day 0.7 19.6 kg vs 610.2 vs kg 19.6 ± 27 µmol/l, 72.7 P <0.05, 16 ± ±
Page 16of49 Version postprint Page 17of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 387 387 389 389 388 388 392 392 407 407 391 391 406 406 403 390 390 411 411 402 402 400 400 397 397 396 396 409 409 405 410 410 408 408 404 393 393 394 394 398 398 395 395 399 399 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Expression of KIRREL in subcutaneous adipose tissue of “fertil+” and “fertil-” cows one cows “fertil-” and “fertil+” of tissue adipose subcutaneous in KIRREL of Expression Expression of KIRREL in bovine tissues bovine inKIRREL of Expression balance. Moreover, we noted that in “fertil+” but not in “fertil ” adipose tissue, the mRNA the tissue, adipose “fertil ” in not but “fertil+” in that noted we Moreover, balance.
week after calving and after five months ofpregnancy and fivemonths after calving after week agreement with the Tiling array results, adipose tissue adipose results, array Tiling the with agreement then compared the expression of of expression the compared then genotype used for the tiling array experiment). Indeed, as shown in Fig. 2A and in a good good a in and 2A Fig. in shown as Indeed, experiment). array tiling the for used genotype iny iutr ad vr ad es bnaty n amr gad n yohlms We hypothalamus. and gland mammary in abundantly less and ovary and pituitary kidney, (Donoviel kidney in expressed highly described been has it where confirmed in 18 “fertil ” and 18 “fertil+” animals (including the samples of nine animals per animals nine of samples the (including animals “fertil+” 18 and “fertil ” 18 in confirmed 401 haplotypes at 5 months of pregnancy of months 5 at haplotypes bu tofl getrepesd n fri+ ta i “etl”cw ( cows “fertil ” in than “fertil+” in expressed greater two fold about kidney 3B, Fig. in showed As lactation. fourth or third their 3A, Fig. in shown as RT PCR, hypothalamus, pituitary and mammary gland of “fertil+” and “fertil ” cows slaughtered after after slaughtered cows “fertil ” and “fertil+” of gland mammary and pituitary hypothalamus, By tissues. bovine in investigated been never has KIRREL of distribution protein or mRNA greater expressed in “fertil+” than in “fertil ” in the first week post partum ( partum post week first the in “fertil ” in than “fertil+” in expressed greater difference was also observed at the protein level by immunoblot (Fig. 2B, (Fig. immunoblot by level protein the at observed also was difference xrsin of expression contrary, the mRNA expression of adipose tissue tissue adipose of expression mRNA the contrary, months of pregnancy( of months ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
KIRREL Differential adipose tissue mRNA expression of of expression mRNA tissue adipose Differential KIRREL (also called called (also Comment citer cedocument: P was significantly decreased between one week after calving and 5 5 and calving after week one between decreased significantly was =0.04). NEPH1)
KIRREL expression has been studied in human and mouse tissues mouse and human in studied been has expression
KIRREL (Fig. 2A), when animals were not in negative energy energy negative in not were animals when 2A), (Fig. a srnl dtce i bvn aioe tissue, adipose bovine in detected strongly was mRNA by quantitative PCR in kidney, kidney, in PCR quantitative by mRNA KIRREL KIRREL KIRREL KIRREL a smlr ewe te two the between similar was expression was significantly was expression one week after calving was was calving after week one et al. et mRNA expression was expression mRNA P 00) Hwvr the However, <0.05). 2001). However, the However, 2001). P =0.023). On the On =0.023). P =0.005). This This =0.005). 17 Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 430 430 434 434 436 436 435 433 433 429 429 413 413 427 427 416 416 414 428 428 426 431 431 412 412 417 417 432 432 425 425 424 424 418 418 423 423 422 422 420 420 421 421 419 419 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Expression of KIRREL in bovine ovary bovine inKIRREL of Expression pituitary and mammary gland (Fig. 3B). By immunoblot, we confirmed at the protein level protein the at confirmed we immunoblot, By 3B). (Fig. gland mammary and pituitary precisely its expression in the various compartments of the ovary. By RT PCR, we showed we RT PCR, By ovary. the of compartments various the in expression its precisely collected from random cows and determined whether the two main hormones involved in the the in involved hormones main two the whether determined and cows random from collected shown). affect affect the greater expression of KIRREL in the kidney of “fertil+” cows ( cows ofthe kidney“fertil+” in KIRRELof expression greater the Fig. 4D, 4D, Fig. 415 fold greater expressed in “fertil+” than in “fertil ” cows ( cows “fertil ” in than in“fertil+” expressed greater fold the in shown As lactation. fourth or third their after slaughtered cows “fertil ” and “fertil+” of olclgnss FH n IF, ee be o euae RA xrsin of expression mRNA regulate to able were IGF1, and FSH folliculogenesis, relative expression of this gene was similar between the two haplotypes in hypothalamus, hypothalamus, in haplotypes two the between similar was gene this of expression relative Treatment with FSH (10 FSH with Treatment compared the expression of expression the compared was localized in theca and granulosa cells, oocyte, cumulus cells and follicular fluid. We then We fluid. follicular and cells cumulus oocyte, cells, granulosa and theca in localized was that that KIRREL protein by immunohistochemistry in the ovarian follicle. More precisely, KIRREL KIRREL precisely, More follicle. ovarian the in immunohistochemistry by protein KIRREL compartments or cells ( cells or compartments xrse i gauoa el fo lre olce a cmae t te te ovarian other the to compared as follicles large from cells granulosa in expressed that observed have we qPCR, By 4A). Fig. LF, GC and SF and LF), corpus luteum (CL), cortex (Ctx) and granulosa cells of small and large follicles (GC follicles large and of small cells granulosa and (CL),(Ctx) cortex luteum corpus LF), and ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
KIRREL KIRREL
We next performed primary culture of bovine granulosa cells from small follicles follicles small from cells granulosa bovine of culture primary performed next We As shown in Fig. 3A, Fig. in shown As KIRREL mRNA was present in theca interstitial cells from small and large follicles (SF follicles large and small from cells theca interstitial in present was mRNA expression as determined by qPCR in cultured bovine granulosa cells (data not (data cells granulosa bovine cultured in qPCR by determined as expression mRNA expression in granulosa cells from small follicles was about twelve about was follicles small from cells granulosa in expression mRNA Comment citer cedocument: P 8 <0.02, Fig. 4B). As showed in Fig. 4C, we confirmed the presence of presence the confirmed we 4C, Fig. in showed As 4B). Fig. <0.02, M) and IGF1 (10 IGF1 and M) KIRREL KIRREL
mRNA by qPCR in granulosa cells from small follicles small from cells granulosa in qPCR by mRNA is expressed in bovine ovary. So, we examined more more examined we So, ovary. bovine in expressed is 8 M) alone or combined for 24 or 48 hours did not did hours 48 or 24 for combined or alone M) P <0.002). <0.002). KIRREL P <0.05, Fig. 3C). <0.05, Fig. was significantly greater greater significantly was KIRREL 18 . . Page 18of49 Version postprint Page 19of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 451 451 444 444 443 443 453 453 452 452 439 439 438 438 461 461 445 445 460 460 437 437 442 442 459 459 441 441 440 440 456 456 455 455 450 450 454 454 449 449 448 448 446 446 457 457 458 458 447 447 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is presence or absence of different concentration of rm KIRREL (1, 5, 10 and 100 ng/ml). We ng/ml). 100 and 10 5, (1, KIRREL rm of concentration different of absence or presence Effect of rm KIRREL on primary bovine granulosa cell steroidogenesis and proliferation andproliferation steroidogenesis granulosa bovinecell onprimary KIRRELrm of Effect bovine granulosa cells as determined by trypan blue incorporation (data not shown).not (data trypanblueincorporation by determined as granulosa cells bovine Effect of rm KIRREL on various signalling pathway in primary bovine granulosa cells cells granulosa bovine primaryin pathwaysignallingon KIRRELrm various of Effect h clue eim ( medium culture the in a dose dependent manner (1 to 100 ng/ml) basal progesterone and oestradiol secretion in in secretion oestradiol and progesterone basal ng/ml) 100 to (1 manner dependent dose a in dose dependent manner (Fig. 6A, 6A, (Fig. manner dependent dose observed that rm KIRREL significantly decreased basal proliferation of granulosa cells, in a a in cells, granulosa of proliferation basal decreased significantly KIRREL rm that observed more than 98% identity with bovine KIRREL. Primary bovine granulosa cells were cultured cultured were cells granulosa bovine Primary KIRREL. bovine with identity 98% than more incubated these cells with commercial recombinant mouse KIRREL (rm KIRREL) that shares that KIRREL) (rm KIRREL mouse recombinant commercial with cells these incubated oestradiol secretion was significantly increased by IGF1 and FSH (Fig. 5C and D) compared compared D) and 5C (Fig. FSH and IGF1 by increased significantly was secretion oestradiol inln ptwy (Harita pathways signaling absence of IGF1 (10 IGF1 of absence KIRREL (1, 5, 10 or 100 ng/ml) or with or without rm KIRREL (10 ng/ml) in the presence or presence the in ng/ml) (10 KIRREL rm without or with or ng/ml) 100 or 10 5, (1, KIRREL for 48 hours in serum free medium supplemented with either different concentrations of rm of concentrations different either with supplemented medium serum free in hours 48 for any effects of rm KIRREL (10 and 100 ng/ml for 24h and 48h) on the viability of primary primary of viability the on 48h) and 24h for ng/ml 100 and (10 KIRREL rm of effects any cells. We measured the [ the measured We cells. PCNA level by Western blotting (Fig. 6B). However, all these data were observed without without observed were data these all However, 6B). (Fig. blotting Western by level PCNA investigated whether rm KIRREL affected the basal proliferation of primary bovine granulosa granulosa primarybovine of proliferation basal the affected KIRREL rm whether investigated or oestradiol secretion by primary bovine granulosa cells (Fig.5C and D). We also also We D). and (Fig.5C cells granulosa bovine primary by secretion oestradiol or to the basal state ( state basal the to
ng/ml (data not shown) concentrations was observed on IGF1 or FSH induced progesterone FSH induced or IGF1 on observed was concentrations shown) not (data ng/ml ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
In order to elucidate the effects of KIRREL in bovine granulosa cells, we we cells, granulosa bovine in KIRREL of effects the elucidate to order In n h ltrtr, IRL NP1 hs en ecie t mdlt intracellular modulate to described been has (NEPH1) KIRREL literature, the In P <0.01). However, no significant effect of rm KIRREL at the 10 and 100 and 10 the at KIRREL rm of effect significant no However, <0.01). Comment citer cedocument: 8 M) or FSH (10 FSH or M) P <0.05) as determined by RIA. As expected, the progesterone and and progesterone the expected, As RIA. by determined as <0.05) 3 H] thymidine incorporation into cells after 24 hours of culture in the in culture of hours 24 after cells into incorporation H] thymidine t al. et
2008). Thus, we studied the effects of rm KIRREL on on KIRREL rm of effects the studied we Thus, 2008). P 00) Teerslswr ofre b vlaig the evaluating by confirmed were results These <0.04). 8 M). As shown in Fig. 5A and B, rm KIRREL reduced reduced KIRREL rm B, and 5A Fig. in shown As M). n vitro in 19
Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 478 478 481 481 477 477 485 485 486 486 476 476 484 484 475 475 483 483 474 474 482 482 473 473 480 480 479 479 472 472 471 471 470 470 463 463 462 462 469 469 468 467 467 465 465 466 466 464 464 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is phosphorylation increased in the oocyte from COCs during IVM and the addition of rm rm of addition the and IVM during COCs from oocyte the in increased phosphorylation rgeso o bvn octs n Os uig n ir mtrto (V) Atr 2 of h 22 After (IVM). maturation vitro in during COCs in oocytes bovine of progression pattern of MAPK1/3, AKT1, PRKAA and MAPK14 phosphorylation. As shown in Fig. 7A, Fig. in shown As phosphorylation. MAPK14 and PRKAA AKT1, MAPK1/3, of pattern Effect of rm KIRREL on the nuclear maturation and MAPK1/3 phosphorylation of bovine of phosphorylation MAPK1/3 and maturation nuclear the on KIRREL rm of Effect effects of rm KIRREL on the nuclear maturation of bovine oocytes in COCs by determining determining by COCs in oocytes bovine of maturation nuclear the on KIRREL rm of effects during IVM resulted in meiotic arrest. We studied the molecular mechanisms involved in the the in involved mechanisms molecular the studied We arrest. meiotic in resulted IVM during 8C). 45% of oocytes remained at the GV stage (Fig. 8A). Thus, rm KIRREL treatment of COCs COCs of treatment KIRREL rm Thus, 8A). (Fig. stage GV the at remained oocytes of 45% maturation medium for 22 h significantly decreased progesterone secretion in COCs (Fig. (Fig. COCs in secretion progesterone decreased significantly h 22 for medium maturation matured for 22 h in IVM medium supplemented with 10 or 100 ng/ml of rm KIRREL, 40% to to 40% KIRREL, rm of ng/ml 100 or 10 with supplemented medium IVM in h 22 for matured MAPK3/1 phosphorylation. We also observed that the addition of rmKIRREL to the the to rmKIRREL of addition the that observed also We phosphorylation. MAPK3/1 less than 10% remaining at the germinal vesicle (GV) stage (Fig. 8A). Conversely, if COCs if Conversely, 8A). (Fig. stage (GV) vesicle germinal the at remaining 10% than less KIRREL (10 and 100 ng/ml) to the maturation medium for 22 h significantly decreased decreased significantly h 22 for medium maturation the to ng/ml) 100 and (10 KIRREL culture in IVM medium, about 90% of oocytes had progressed to the metaphase II stage, with with stage, II metaphase the to progressed had oocytes of 90% about medium, IVM in culture in COCs allowed to mature mature to allowed COCs in the MAPK3/1 phosphorylation in the presence or absence of rm KIRREL (10 and 100 ng/ml) ng/ml) 100 and (10 KIRRELrm of absence or presence the in phosphorylation MAPK3/1 the oocytes in COCs and progesterone secretion by bovine COCs during in vitro maturation during in maturation vitro COCs bybovine secretion progesterone andCOCsin oocytes added to the medium culture for different times (0 to 60 minutes) and we analysed the protein the analysed we and minutes) 60 to (0 times different for culture medium the to added various signalling pathways in primary bovine granulosa cells. rm KIRREL (100 ng/ml) was was ng/ml) (100 KIRREL rm cells. granulosa bovine primary in pathways signalling various Conversely, rm KIRREL did not affect AKT1 and PRKAA phosphorylation (data notshown). (data phosphorylation PRKAAand notAKT1affect did KIRREL rm Conversely, fe 5 iue o siuain ( stimulation of minutes 5 after nrae MP1 popoyain rm t 5 iue o tetet ( treatment of minutes 5 to 1 from phosphorylation MAPK14 increased rm KIRREL led to a significant rapid and transient increase of the MAPK1/3 phosphorylation phosphorylation MAPK1/3 the of increase transient and rapid significant a to led KIRREL rm ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
We also studied the effects of different concentrations of rm KIRREL on the meiotic the on KIRREL rm of concentrations different of effects the studied also We
Comment citer cedocument: for 22 h. As shown in Fig.8B, the level of MAPK3/1 MAPK3/1 of level the Fig.8B, in shown As h. 22 for vitro in
P =0.0056). In the same way, rm KIRREL has rapidly rapidly has KIRREL rm way, same the In =0.0056). P 00, i. 7B). Fig. <0.05, 20 Page 20of49 Version postprint Page 21of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 510 510 508 508 507 507 511 511 506 506 505 505 509 509 504 504 503 503 502 502 501 501 500 500 499 499 498 498 497 497 496 496 495 495 491 491 494 494 493 493 492 492 490 490 489 489 488 488 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is rcsl, hryoe ee wr oe xrse wees wle ee ne xrse in under expressed were twelve whereas over expressed were genes thirty one precisely, rtis ifrnily xrse i aioe ise oprd o fri+ aias More animals. “fertil+” to compared tissue adipose in expressed differentially proteins development in “fertil ” compared to “fertil+” heifers (Coyral Castel (Coyral Castel heifers “fertil+” to compared “fertil ” in development (Coyral Castel (Coyral Castel artificial insemination, respectively, compared to the unfavourable (“fertil ”) haplotype haplotype (“fertil ”) unfavourable the to compared respectively, insemination, artificial (QTL F Fert BTA3) had a 31% and 26% greater success rate at 35 and 90 days after the first the after days 90 and 35 at rate success greater 26% and 31% a had (QTL F Fert BTA3) favourable (“fertil+”) haplotype at one QTL of female fertility located on the chromosome 3 3 chromosome the on located fertility female of QTL one at haplotype (“fertil+”) favourable dynamics after after dynamics metabolic messenger linking metabolism, body composition and fertility. fertility. composition and body linkingmetabolism, messenger metabolic el bt also but cells KIRREL was able to decrease decrease to able was KIRREL oprd o fri cw. y sn rcmiat rti, e ae eosrtd that demonstrated have we protein, recombinant using By cows. “fertil ” to compared and its mRNA expression was significantly greater in the granulosa cells of “fertil+” “fertil+” of cells granulosa the in greater significantly was expression mRNA its and KIRREL was expressed in various ovarian cells including oocyte, granulosa and theca cells cells theca and granulosa oocyte, including cells ovarian various in expressed was KIRREL fri+ cmae t “etl” nml. n vr, e ae hw fr h frt ie that time first the for shown have we ovary, In animals. “fertil ” to compared “fertil+” observed that that observed n ptiay n ls audnl i hptaau ad amr gad Itrsigy we Interestingly, gland. mammary and hypothalamus in abundantly less and pituitary and animals. We showed that showed We animals. KIRREL was significantly greater expressed in “fertil+” adipose tissue compared to “fertil ” to compared tissue adipose “fertil+” in expressed greater significantly was KIRREL “fertil+” compared to “fertil ” cows. We confirmed by RT qPCR and immunoblot that that immunoblot and RT qPCR by confirmed We cows. “fertil ” to compared “fertil+” greater plasma NEFA concentrations one week after calving, had 43 genes coding for known known for coding genes 43 had calving, after week one concentrations NEFA plasma greater Discussion Discussion 487 ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
We have previously shown that Holstein cows selected for their homozygous homozygous their for selected cows Holstein that shown previously have We In the present study we reported by using Tiling array that “fertil ” cows, exhibiting exhibiting cows, “fertil ” that array Tiling using by reported we study present the In KIRREL KIRREL oye auain ugsig ht hs rti cud e potential a be could protein this that suggesting maturation oocyte vitro in t al. et n vivo in Comment citer cedocument: 01. utemr, e ae bevd lwr oye maturation oocyte slower observed have we Furthermore, 2011). RA xrsin n iny a sgiiaty rae epesd in expressed greater significantly was kidney in expression mRNA KIRREL maturation and lower blastocyst quality after after quality blastocyst lower and maturation in vitro vitro in
is mainly expressed in bovine kidney, adipose tissue, ovary tissue, adipose kidney, bovine in expressed mainly is progesterone secretion and proliferation in granulosa granulosa in proliferation and secretion progesterone et al. et 2012). In addition addition In 2012). in vitro vitro in embryo embryo 21 Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 536 536 533 533 535 535 532 532 534 534 529 529 528 528 527 527 526 526 531 531 525 525 530 530 524 524 523 523 521 520 520 513 513 519 519 512 512 518 518 517 517 522 522 516 516 515 515 514 514 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is per gene on the array. We performed qPCR on 10 genes that had the highest fold change. change. fold highest the had that genes 10 on qPCR performed We array. the on gene per KIRREL bovine species (Spicer (Spicer species bovine tendency was observed for two other genes (CADM3, (CADM3, genes other two for observed was tendency sensitivity, mainly due to the design of the designthe array.of to duethe mainly sensitivity, adipose tissue of “fertil+” compared to “fertil ” cows one week after calving. Similar Similar calving. after week one cows “fertil ” to compared “fertil+” of tissue adipose aa lal idct ta te w tcnqe (iig ra ad PR hv nt h same the not have qPCR) and array (Tiling techniques two the that indicate clearly data significant. One explanation of this result is that several probes were available per exon and and exon per available were probes several that is result this of explanation One significant. However, the ratios obtained were low (less than 2) even though difference was statistically was difference though even 2) than (less low were obtained ratios the However, were overexpressed whereas 12 were under expressed in “fertil+” compared to “fertil ” cows. “fertil ” to compared “fertil+” in under expressed were 12 whereas overexpressed were were differentially expressed in adipose tissue between the two haplotypes. Indeed, 31 genes 31 Indeed, haplotypes. two the between tissue adipose in expressed differentially were Unfortunately, we significantly confirmed the results of Tiling array for only one gene, named gene, only one array for Tiling of theresults confirmed significantly we Unfortunately, td, ehv son ht 3gnscdn kon rtisot f 2 gns n h array the on genes 124 of out proteins known coding genes 43 that shown have we study, mediates such effects has not been clearly established. Some evidence suggests suggests evidence Some established. clearly been not has effects such mediates reproductive performance (Randel 1990, Dunn & Moss 1992), although the pathway which which pathway the although 1992), Moss & Dunn 1990, (Randel performance reproductive to fertility problems, we have shown that “fertil ” cows had a lower body weight in the first the in weight body lower a had cows “fertil ” that shown have we problems, fertility to week week haplotypes. Various studies indicate that body fat content is associated with changes in in changes with associated is content fat body that indicate studies Various haplotypes. e f ee lctd n h QLFFr T3 y iig ra i te dps tsu o h two the of tissue adipose the in array Tiling by QTL F Fert BTA3 the in located genes of haplotypes could contribute to explain the difference in fertility, we compared the expression expression the compared we fertility, in difference the explain to contribute could haplotypes vitro vitro avn. n re t dtrie f hs ifrne n a mblzto bten h two the between mobilization fat in difference this if determine to order In calving. concentration was significantly greater in “fertil ” compared to “fertil+” cows one week after after week one cows “fertil+” to compared “fertil ” in greater significantly was concentration al. ight weeks after calving than “fertil+” cows and a more negative energy balance in the first the in balance energy negative more a and cows “fertil+” than calving after weeks ight ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 , 2013). In the present study, we confirmed this hypothesis since plasma NEFA NEFA plasma since hypothesis this confirmed we study, present the In 2013). ,
ht dpkns euae h hptaau iutr vr ai i mmas including mammals in axis hypothalamus pituitary ovary the regulate adipokines that post partum post (kin of IRRE like) also known as as known also like) IRRE of (kin , suggesting a greater fat mobilization in this haplotype (Coyral Castel (Coyral Castel haplotype this in mobilization fat greater a suggesting , Comment citer cedocument: et al. al. et 1993, Williams Williams 1993,
NEPH1 et al. et . . 2002, Maillard 2002, KIRREL KIRREL P =0.069 and SLAMF6 SLAMF6 and =0.069 is significantly overexpressed in overexpressed significantly is et al. al. et 2010). In the present the In 2010). P =0.08). These =0.08). in vivo vivo in and and 22 in in et
Page 22of49 Version postprint Page 23of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 555 555 554 554 549 549 547 548 548 561 561 559 540 540 541 541 558 558 560 560 542 542 539 557 557 553 552 552 556 556 546 546 551 551 545 545 550 550 538 538 544 544 543 537 537 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is podocytes, heavy proteinuria, and early postnatal death (Donoviel (Donoviel death postnatal early and proteinuria, heavy podocytes, KIRREL presence of KIRREL in the ovary or pituitary had never been investigated. In bovine tissues, tissues, bovine In investigated. been never had pituitary or ovary the in KIRREL of presence bovine ovary, we detected detected we ovary, bovine xrsin of expression cells. As in adipose tissue one week after calving and in kidney, we have shown a greaterr a shown have we kidney, in and calving after week one tissue adipose in As cells. the kidney (Donoviel (Donoviel kidney the KIRREL plays a pivotal role for the development and maintenance of the filtration barrier in in barrier filtration the of maintenance and development the for role pivotal a plays KIRREL 1 (ZO 1) (Huber (Huber (ZO 1) 1 such as brain, placenta and testis (Donoviel (Donoviel testis and placenta brain, as such ebae ufc o itrellr ih jntos I itrcs ih h gp ucin protein junction gap the with interacts It junctions. tight intercellular of surface membrane occludens zona protein junction Tight and Nephrin with interact to shown been has KIRREL muolblnlk dmis n i srcual rltd o ehi. n ua ad rodents, and human In nephrin. to related structurally is and domains immunoglobulin like that mammalian KIRREL proteins have similar cell cell recognition functions. Furthermore, functions. recognition cell cell similar have proteins KIRREL mammalian that function of KIRREL in extra renal organ systems is almost unknown. Recent studies revealed revealed studies Recent unknown. almost is systems organ renal extra in KIRREL of function In haplotype. inthis filtration renal glomerular better a explain could “fertil+” of kidney in the cows. In mice, the disruption of the the of disruption the mice, In cows. compared to “fertil ” cows (unpublished data) suggesting that the greater KIRREL expression expression KIRREL greater the that suggesting data) (unpublished cows “fertil ” to compared greater mRNA and protein expression of KIRREL in “fertil+” kidney compared to “fertil ” to compared kidney “fertil+” in KIRREL of expression protein and mRNA greater have observed that the plasma concentrations of urea were significantly greater in “fertil+” “fertil+” in greater significantly were urea of concentrations plasma the that observed have and less abundantly in hypothalamus and mammary gland. Interestingly, we have shown a a shown have we Interestingly, gland. mammary and hypothalamus in abundantly less and detected have we (Donoviel screen mutagenesis KIRREL2 and KIRREL3. It is a molecule identified in mice by a retrovirus mediated retrovirus mediated a by mice in identified molecule a is It KIRREL3. and KIRREL2 ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
KIRREL is a member of the nephrin like protein family, which 444 also includes includes also 444 which family, protein nephrin like the of member a is KIRREL is abundantly expressed in the kidney but also found in some reproductive organs reproductive some in found also but kidney the in expressed abundantly is KIRREL KIRREL et al. al. et KIRREL Comment citer cedocument: et al. al. et 03 Liu 2003, n rnls cls rm fri+ cmae t “etl” os The cows. “fertil ” to compared “fertil+” from cells granulosa in KIRREL KIRREL mRNA expression in kidney, adipose tissue, ovary and pituitary pituitary and ovary tissue, adipose kidney, in expression mRNA 2001, Neumann Haefelin Neumann Haefelin 2001,
et al. al. et et al. al. et in different cells including granulosa and corpus luteum luteum corpus and granulosa including cells different in NEPH1 NEPH1 03. O1 s poen oae o a cytoplasmic a on located protein a is ZO 1 2003). 2001). KIRREL contains five extracellular extracellular five contains KIRREL 2001). et al. al. et gene results in effacement of glomerular glomerular of effacement in results gene 2001, Beall Beall 2001, et al. al. et 00. n peiu wr, we work, previous a In 2010). et al. al. et 05. oee, the However, 2005). et al. al. et 01. Thus, 2001). 23 Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 575 575 566 566 574 574 564 564 565 565 573 573 563 563 572 572 562 562 571 571 568 570 570 567 576 576 569 569 577 577 579 579 586 586 578 578 585 585 584 584 583 583 582 582 581 581 580 580 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is efre piay rnls cls n icbtd hm ih ifrn cnetain of concentrations different with them incubated and cells granulosa primary performed family tyrosine kinase, Fyn (Verma (Verma Fyn kinase, tyrosine family contains a large number of tyrosine residues. These residues can be phosphorylated by a Src a by phosphorylated be can residues These residues. tyrosine of number large a contains I odr o eemn te ucin f IRL n h bvn gauoa el, we cells, granulosa bovine the in KIRREL of function the determine to order In cows. IRL a be cniee a a inlig oeue I hs ctpamc oan that domain cytoplasmic a has It molecule. signalling a as considered been has KIRREL contribute to the greater fat mobilization in adipose cells of “fertil ” compared to “fertil+” “fertil+” to compared “fertil ” of cells adipose in mobilization fat greater the to contribute secretion suggesting that rm KIRREL is active in bovine cultured granulosa cells. Recently, Recently, cells. granulosa cultured bovine in active is KIRREL rm that suggesting secretion adhesion and in the signal transduction at cell cell junctions. In this way, KIRREL could could KIRREL way, this In junctions. cell cell at transduction signal the in and adhesion MAPK1/3 and MAPK14 phosphorylation and to decrease progesterone and oestradiol oestradiol and progesterone decrease to and phosphorylation MAPK14 and MAPK1/3 (Schwarz rafts lipid in found are complexes onxn3 Gemn & olna 19) Tu, IRL ol b ivle i te cell the in involved be could KIRREL Thus, 1998). Moolenaar & (Giepmans connexin43 u suy w hv osre ta rcmiat IRL a al t ices rapidly increase to able was KIRREL recombinant that observed have we study, our these and ZO 1 and Nephrin associate to known is KIRREL protein. KIRREL recombinant (Wayne (Wayne assemblies of sphingolipids and cholesterol in the outer leaflet of the plasma membrane. In membrane. plasma the of leaflet outer the in cholesterol and sphingolipids of assemblies intracellular signaling by binding to Grb2 (Harita (Harita Grb2 to binding by signaling intracellular signaling pathways through the tyrosine kinase, Fyn, in bovine granulosa cells. In the present the In cells. granulosa bovine in Fyn, kinase, tyrosine the through pathways signaling novd n h MP13 inln ptwy. hs KRE cud ciae MAPK3/1 activate could KIRREL Thus, pathways. signaling MAPK1/3 the in involved remains to be determined. This pathway has been described to be involved in the differential differential the in involved be to described been has pathway This determined. be to remains mediated by MAPK3/1 in cultured bovine granulosa cells. The involvement of MAPK14 MAPK14 of involvement The cells. granulosa bovine cultured in MAPK3/1 by mediated Thus, it is likely that the inhibitory effect of KIRREL on the progesterone secretion is not not is secretion progesterone the on KIRREL of effect inhibitory the that likely is it Thus, granulosa cells in different species (Gyles species different in cells granulosa Various studies have showed that MAPK3/1 positively regulates progesterone production by production progesterone regulates positively MAPK3/1 that showed have studies Various work, we have observed that recombinant KIRREL protein decreases steroid secretion. secretion. steroid decreases protein KIRREL recombinant that observed have we work, ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
et al. al. et 2007). Once tyrosine are phosphorylated, KIRREL is able to modulate modulate to able is KIRREL phosphorylated, are tyrosine Once 2007). Comment citer cedocument:
et al. al. et 2003) that has been described in rat granulosa cells granulosa rat in described been has that 2003) et al. al. et et al. al. et 2001, Tosca Tosca 2001, et al. al. et 01, mcooan ht osss of consists that microdomain a 2001), 08. r2 s ctslc adaptor cytosolic a is Grb2 2008). et al. al. et 2005, Tosca Tosca 2005, et al. al. et 2007). 24 Page 24of49 Version postprint Page 25of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 593 593 592 592 587 587 591 591 588 594 594 589 590 590 597 597 595 595 602 602 601 601 596 596 600 600 603 603 606 606 608 608 607 605 605 604 604 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is previously shown that progesterone secretion by cultured granulosa cells in basal state or in in or state basal in cells granulosa cultured by secretion progesterone that shown previously by Tiling array. Among these genes differentially expressed by Tiling array, we confirmed the confirmed we byarray, Tiling expressed differentially genes these Among byarray. Tiling regulation of steroidogenesis in rat granulosa cells (Yu (Yu cells granulosa rat in steroidogenesis of regulation compared to “fertil ” animals through its effects on the granulosa cells or oocyte. We have We oocyte. or cells granulosa the on effects its through animals “fertil ” to compared decreases KIRREL rm that shown also have we cells and oocyte maturation. So, KIRREL might not explain the better fertility in “fertil+” as “fertil+” in fertility better the explain not might KIRREL So, maturation. oocyte and cells that observed We . byCOCs secrection progesterone of inhibition throughan response to FSH or IGF1 was similar between “fertil+” and “fertil ” heifers submitted to to submitted heifers “fertil ” and “fertil+” between similar was IGF1 or FSH to response decreases KIRREL recombinant that showed greater is expression 599 598 ovarian stimulation (Coyral Castel (Coyral Castel stimulation ovarian calving and a differential expression of adipose tissue genes located in the QTL as determined determined QTL as inthe located genes tissue adipose of expression differential a and calving KIRREL exhibiting difference in fertility, had also a difference in fat mobilization one week after after week one mobilization fat in difference a also had fertility, in difference exhibiting eut t oh RAad rti mut o oe eenmd IRL Ti gn highly gene This KIRREL. named gene one for amount protein and mRNA both at results
reproduction and could explain some infertilities in dairycows.in infertilities some explain could and reproduction expressed in granulosa cells could be involved in the interactions between metabolism and metabolism between interactions the in involved be could cells granulosa in expressed ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
In conclusion, we have shown that dairy cows selected for one QTL F Fert BTA3, QTL F Fert BTA3, one for selected cows dairy that shown have we conclusion, In in vivo in in ovarian functions in the bovine species. the in functions ovarian in Comment citer cedocument: in granulosa cells of “fertil+” compared to “fertil ” cows but we but cows “fertil ” to compared “fertil+” of cells granulosa in vivo in
et al. et 2012). Thus, it will be interesting to know the role of role the know to interesting be will it Thus, 2012). steroid production in bovine granulosa granulosa bovine in production steroid vitro in in vitro in et et bovine oocyte maturation probably maturation oocyte bovine 494 494 al. 2005). In the present study, present the In 2005). KIRREL KIRREL mRNA mRNA 25 Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 614 614 610 610 613 613 612 611 609 609 623 623 626 626 624 622 622 621 621 620 620 619 619 616 616 618 618 617 615 615 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is
S. Coyral Castel is a PhD student supported by the “Institut de l’Elevage” and the the and l’Elevage” de “Institut the by supported student PhD a is Coyral Castel S. This work was supported by ANR Genanimal and Apis Gene Apis Gene Genanimal andbyANR supported was work This Funding thereported. researchof impartiality the The authors declare that there is no conflict of interest that could be perceived as prejudicing as perceived be could that interest of conflict no is there that declare authors The proposal). (Valoprot 625 funding from ANR (Agence Nationale de la Recherche) Fertilité 1 et 2 and from Apisgene Apisgene from and 2 et 1 Fertilité Recherche) la de Nationale (Agence ANR from funding ntain f h Tln Ary rtcl Te eerh edn t tee eut hs received has results these to leading research The protocol. Array Tiling the of initiation involvement in the experiment. We also acknowledge André Eggen for his implication in the the in implication his for Eggen André acknowledge also We experiment. the in involvement Christophe Mouaze of the Experimental Unit UEPAO for animal management and their their and management animal for UEPAO Unit Experimental the of Mouaze Christophe The authors thank Eric Briant, Mickael Dupont, Mickael Delanoue, Ludovic Métivier and and Métivier Ludovic Delanoue, Mickael Dupont, Mickael Briant, Eric thank authors The Acknowledgements “Association Nationale de la Recherche et deTechnologie”. la et Nationaledela Recherche “Association Declaration ofinterest Declaration ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Comment citer cedocument:
26 Page 26of49 Version postprint Page 27of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 632 632 633 633 649 649 631 631 648 648 650 650 646 646 647 647 645 640 640 637 637 639 639 644 644 636 636 630 630 635 635 641 641 643 643 634 634 642 642 629 629 638 638 628 628 627 627 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Beam SW & Butler WR Butler & SW Beam Campos DB, Palin MF, Bordignon V & Murphy BD Murphy & V Bordignon MF, Palin DB, Campos Butler WR, Everett RW & Coppock CE Coppock & RW Everett WR, Butler ejmn Y Hcbr Y Hochberg & Y Benjamini e Jma , rt S Gilue , re T Dns , ge A Guir M Gautier & A Eggen C, Denis T, Druet F, Guillaume S, Fritz S, Jemaa Ben 2005 Placental and Placental 2005 MG Ross & R Beloosesky SB, Wang DA, Gayle F, Amidi MH, Beall odn DM Bowden amn E Bue ure W Currie Bruce & DE Bauman References ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Journal of the Society for Gynecologic Investigation Gynecologicfor theSociety of Journal powerful approach to multiple testing. testing. multiple to approach powerful Journal of Dairy Science Dairyof Journal Journal of Animal Breeding andGenetics Breeding Animal of Journal reproduction and fertility. fertility. and reproduction fetal membrane nephrin and neph1 gene expression: Response to inflammation. inflammation. to Response expression: gene neph1 and nephrin membrane fetal ik rdcin n Ouain n otatm osen Cows. Holstein Postpartum in Ovulation and Production Milk Science Detection of quantitative trait loci affecting non return rate in French dairy cattle. cattle. dairy French in rate non return affecting loci trait quantitative of Detection REVIEW. REVIEW. 54 B-Methodological BLOOD AS INDICATORS OF NUTRITIONAL STATUS IN RUMINANTS: A A RUMINANTS: IN STATUS NUTRITIONAL OF INDICATORS AS BLOOD ovulation in postpartum dairy cows. cows. dairy postpartum in ovulation atto: Rve o Mcaim Ivlig oesai ad Homeorhesis. and Homeostasis Involving Mechanisms of Review A Lactation: 411 424. 1971 NON ESTERIFIED FATTY ACIDS AND KETONE BODIES IN IN BODIES KETONE AND ACIDS FATTY NON ESTERIFIED 1971
53 742 748. 742 748. Canadian Journal of AnimalScience of Journal Canadian Comment citer cedocument:
1999 Effects of energy balance on follicular development and first and development follicular on balance energy of Effects 1999 57 289 300. 1995 Controlling the false discovery rate A practical and and practical A rate discovery false the Controlling 1995
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International Journal of Obesityof Journal International 1514 1529. 1514 1529. 1980 Partitioning of Nutrients During Pregnancy and and Pregnancy During Nutrients of Partitioning 1980 1981 The Relationships between Energy Balance, Balance, Energy between Relationships The 1981 Journal of Reproduction and Fertility Supplement Fertility and Reproduction of Journal Journal of the Royal Statistical Society Series Society Statistical Royal the of Journal
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51 2008 The 'beneficial' adipokines in in adipokines 'beneficial' The 2008 1 13.
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Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 675 675 670 670 669 669 674 674 653 653 672 672 668 668 652 652 673 673 671 664 664 667 667 654 654 666 666 663 651 651 657 657 665 665 662 656 656 661 661 655 655 660 660 659 659 658 658 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Coyral-Castel S, Rame C, Fatet A & Dupont J Dupont & A Fatet C, Rame S, Coyral-Castel re T Fiz , osaa , e-ea S Gilue , ebl D Zlnk D, Zelenika D, Derbala F, Guillaume S, Ben-Jemaa M, Boussaha S, Fritz T, Druet Donoviel DB, Freed DD, Vogel H, Potter DG, Hawkins E, Barrish JP, Mathur BN, BN, Mathur JP, Barrish E, Hawkins DG, Potter H, Vogel DD, Freed DB, Donoviel Coyral-Castel S, Faverdin P, Ramé C, Fréret S, Guillaume D, Fritz S & Dupont J J Dupont & S Fritz D, Guillaume S, Fréret C, Ramé P, Faverdin S, Coyral-Castel Coyral-Castel S, Ramé C, Monniaux D, Fréret S, Fabre-Nys C, Fritz S, Monget P, P, Monget S, Fritz C, Fabre-Nys S, Fréret D, Monniaux C, Ramé S, Coyral-Castel Coppock CE Coppock orlCse S Biad , oz J, uot , aé , zeoa & uot J Dupont & S Uzbekova C, Ramé M, Dupont JL, Touze D, Brisard S, Coyral-Castel ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Molecular and Cellular andCellular Biology Molecular progesterone secretion and selected protein kinases in goat granulosa cells. cells. granulosa goat in kinases protein selected and secretion progesterone bovine chromosome 3 are not attributable to energy balance, although eating eating although balance, energy to attributable not are 3 chromosome bovine Animal Endocrinology Animal Dairy Science Dairy behaviour isaffected behaviour Lethality in Mice Lacking NEPH1, a Novel Protein with Homology to NEPHRIN. NEPHRIN. to Homology with Protein Novel a NEPH1, Lacking Mice in Lethality using a dense single nucleotide polymorphism map. polymorphism single nucleotidedense ausing ehe D Cao C Bihr D Gt G Egn & ate M Gautier & A Eggen IG, Gut D, Boichard C, Charon D, Lechner aie-oi R Zmrwc B & oel DR Powell & BP Zambrowicz R, Ramirez-Solis mapping of quantitative trait loci affecting female fertility in dairy cattle on BTA03 BTA03 on cattle dairy in fertility female affecting loci trait quantitative of mapping Turner CA, Geske R, Montgomery CA, Starbuck M, Brandt M, Gupta A, A, Gupta M, Brandt M, Starbuck CA, Montgomery R, Geske CA, Turner Significant differences in fertility between dairy cows selected for one QTL located on QTLlocated one for selected dairycows between fertility differences in Significant for one QTL located on located QTLoneBTA3. for Dupont F & Dupont J Dupont & F Dupont located on BTA3 located expression in cumulus cells of dairy cows and heifers selected for one fertility QTL fertility one for selected heifers and cows dairy of cells cumulus in expression 2012 Analysis of in vivo oocyte maturation, in vitro embryo development and gene and development embryo vitro in maturation, oocyte vivo in of Analysis 2012 1985 Energy Nutrition and Metabolism of the Lactating Dairy Cow. Cow. Dairy Lactating the of Metabolism and Nutrition Energy 1985 Comment citer cedocument:
68 3403 3410. Theriogenology Animal
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21
77 4829 4836. 4829 4836. 1822 1833. 1822 1833. 2010 Effects of unsaturated fatty acids on acids fatty unsaturated of Effects 2010
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Page 28of49 Version postprint Page 29of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 693 693 688 688 696 696 695 694 690 689 689 692 692 697 697 691 691 698 698 680 680 679 687 687 686 685 685 684 684 678 678 677 677 676 676 681 681 683 683 682 682 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Leroy J, Vanholder T, Van Knegsel ATM, Garcia-Ispierto I & Bols PEJ Bols & I Garcia-Ispierto ATM, Knegsel Van T, Vanholder J, Leroy Huber TB, Schmidts M, Gerke P, Schermer B, Zahn A, Hartleben Br, Sellin L, Walz G Walz L, Sellin Br, Hartleben A, Zahn B, Schermer P, Gerke M, Schmidts TB, Huber Guillaume F, Gautier M, Ben Jemaa S, Fritz S, Eggen A, Boichard D & Druet T Druet & D Boichard A, Eggen S, Fritz S, Jemaa Ben M, Gautier F, Guillaume aia , uiaa , oao , eua , eie , grsi & atr S Hattori & T Igarashi T, Sekine T, Tezuka H, Kosako H, Kurihara Y, Harita 1998 The gap junction protein connexin43 interacts with interacts connexin43 protein gapThejunction 1998 WH Moolenaar & BNG Giepmans un G Ms GE Moss & TG Dunn Gyles SnL, Burns CJ, Whitehouse BJ, Sugden D, Marsh PJ, Persaud SJ & Jones PM Jones & SJ Persaud PJ, Marsh D, Sugden BJ, Whitehouse CJ, Burns SnL, Gyles ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
eh, Cmoet f h Kde Si Daham I Trsn hshrltd by Tyrosine phosphorylated Is Diaphragm, Slit Kidney the of Component a Neph1, roiiain n ar cw ery otatm Msac bten eaoim and metabolism between Mismatch postpartum: early cows dairy in prioritization PDZ Domain Protein Zonula Occludens 1. Occludens 1. Zonula Protein Domain PDZ 13417 13421. 13417 13421. Grb2. the Src Family Tyrosine Kinase and Modulates Intracellular Signaling by Binding to Binding by Signaling Intracellular Modulates and Kinase Tyrosine Family Src the & Benzing T Benzing & fertility? fertility? 934. 276 the Steroidogenic Acute Regulatory (StAR) Gene. Gene. (StAR) Regulatory Acute Steroidogenic the 2001 ERKs Regulate Cyclic AMP induced Steroid Synthesis through Transcription of Transcription through Synthesis Steroid AMP induced Cyclic Regulate ERKs 2001 the second PDZ domain of the zona occludens 1 protein. protein. occludens 1 zona the of domain PDZ second the efficiency of livestock.of efficiency Refinement of two female fertility QTL using alternative phenotypes in French French in phenotypes alternative using QTL fertility female two of Refinement Holstein dairycattle. Holstein 34888 34895. 34888 34895. Journal of Biological Chemistry of Biological Journal Reproduction in Domestic Animals Domestic in Reproduction Comment citer cedocument: 2003 The Carboxyl Terminus of Neph Family Members Binds to the to Binds Members Family Neph of Terminus Carboxyl The 2003 1992 Effects of nutrient deficiencies and excesses on reproductive reproductive on excesses and deficiencies nutrient of Effects 1992 Animal Genetics Animal Journal of Animal Science ofAnimal Journal
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Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 723 723 703 703 705 705 722 722 704 704 702 702 721 721 701 701 700 700 699 699 706 706 720 720 719 707 707 718 718 710 710 709 709 708 711 711 717 717 715 715 716 716 714 714 713 713 712 712 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Neumann-Haefelin E, Kramer-Zucker A, Slanchev K, Hartleben B, Noutsou F, Martin Martin F, Noutsou B, Hartleben K, Slanchev A, Kramer-Zucker E, Neumann-Haefelin Maillard V, Uzbekova S, Guignot F, Perreau C, Rame C, Coyral-Castel S & Dupont J Dupont & S Coyral-Castel C, Rame C, Perreau F, Guignot S, Uzbekova V, Maillard Reverchon M, Bertoldo MJ, Ramé C, Froment P & Dupont J J Dupont & P Froment C, Ramé MJ, Bertoldo M, Reverchon i ,KwB ufsJ aaaudn ,Kna S&Cuh SS Chugh & YS Kanwar S, Rahamanuddin J, Kurfis B, Kaw G, Liu Randel RD Randel ig , o A & ulkn JC Mullikin & AJ Cox Z, Ning Nuwaysir EF, Huang W, Albert TJ, Singh J, Nuwaysir K, Pitas A, Richmond T, Gorski Gorski T, Richmond A, Pitas K, Nuwaysir J, Singh TJ, Albert W, Huang EF, Nuwaysir 2011 Adipokines in inflammation and metabolic metabolic and inflammation in Adipokines 2011 K Walsh & JJ LugusJL, Parker N, Ouchi ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
permeability. permeability. by Maskless Photolithography. Photolithography. byMaskless maturation and embryo development. development. embryo and maturation K, Wanner N, Ritter A, Gödel M, Pagel P, Fu X, Müller A, Baumeister R, Walz R, Baumeister A, Müller X, Fu P, Pagel M, Gödel A, Ritter N, Wanner K, meiotic progression in bovine species. inbovine progression meiotic 00 fet f dpnci o bvn gauoa el trioeei, oocyte steroidogenesis, cell granulosa bovine on adiponectin of Effect 2010 RRE2 dcess n ir gauoa el trioeei ad lcs oocyte blocks and steroidogenesis cell granulosa vitro in decreases (RARRES2) nephrin interaction in the slit diaphragm is an important determinant of glomerular glomerular of determinant important an is diaphragm slit the in interaction nephrin G & Huber TB Huber & G 853 862. as regulators of neuron and kidney morphogenesis. morphogenesis. kidney and neuron of regulators as Databases. Databases. 2347 2359. 2347 2359. disease. disease. Green RD Green Porter D, Molla M, Hall C, Blattner F, Sussman MR, Wallace RL, Cerrina F & F Cerrina RL, Wallace MR, Sussman F, Blattner C, Hall M, Molla D, Porter T, Berg JP, Ballin J, McCormick M, Norton J, Pollock T, Sumwalt T, Butcher L, Butcher T, Sumwalt T, Pollock J, Norton M, McCormick J, Ballin JP, Berg T, 1990 Nutrition and postpartum rebreeding in cattle. cattle. rebreedingin postpartum and Nutrition 1990 Nature Reviews Immunology Nature 2002 Gene Expression Analysis Using Oligonucleotide Arrays Produced Produced Arrays Oligonucleotide Using Analysis Expression Gene 2002 Genome Research Genome Comment citer cedocument: Ingestigation of Clinical Journal The 2010 A model organism approach: defining the role of Neph proteins Neph of role the defining approach: organism model A 2010
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8 23.
30 68 19
Page 30of49 Version postprint Page 31of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 747 747 744 744 746 746 745 745 736 736 743 743 735 735 734 741 741 742 742 733 733 740 740 732 732 731 731 739 739 738 738 737 737 730 730 729 729 728 728 727 727 726 726 725 725 724 724 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is oc L Carle , zeoa & uot J Dupont & S Uzbekova C, Chabrolle L, Tosca ig-asn , re R, u Y Nlo C Bate F Ssmn R Cria F Cerrina & MR Sussman F, Blattner C, Nelson Y, Yue RD, Green S, Singh-Gasson esgi , i iul F DIplt S ela ,CselciM&D Smn N Simone Di & M Castellucci M, Veglia S, D'Ippolito F, Nicuolo Di C, Tersigni Schwarz K, Simons M, Reiser J, Saleem MA, Faul C, Kriz W, Shaw AS, Holzman LB & LB Holzman AS, Shaw W, Kriz C, Faul MA, Saleem J, Reiser M, Simons K, Schwarz Spicer LJ, Alpizar E & Echternkamp SE Echternkamp & E Alpizar LJ, Spicer crdr J Safnil R Staufenbiel & UJ Schröder oh J, rges C Ky K Fse M, tfod J Bry DP Berry & KJ Stafford MW, Fisher JK, Kay NC, Friggens JR, Roche Roche JF, Mackey D & Diskin MD Diskin & D Mackey JF, Roche ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Animal Reproduction Science Reproduction Animal Gynecological Survey Gynecological rnls Cls trioeei: osbe novmn o Aeoie 5' Adenosine of Involvement Possible Steroidogenesis: Cells Granulosa 1999 Maskless fabrication of light directed oligonucleotide microarrays using a digital digital a using microarrays oligonucleotide light directed of fabrication Maskless 1999 Adipokines: New Emerging Roles in Fertility and Reproduction. Reproduction. and Fertility in Roles Emerging New Adipokines: 108 of Animal Science Animal of diaphragm, interacts with CD2AP and nephrin. and CD2AP with interacts diaphragm, estradiol production, and(or) insulin like growth factor I production in vitro. vitro. in production I factor growth insulin like and(or) production, estradiol udl P Mundel I, and gonadotropins on bovine granulosa cell proliferation, progesterone production, production, progesterone proliferation, cell granulosa bovine on gonadotropins and I, micromirror array. micromirror Backfat Thickness. Thickness. Backfat Reserves in the Dairy Cow with Special Regard to Ultrasonographic Measurement of Measurement Ultrasonographic to Regard Special with Cow Dairy the in Reserves and welfare. welfare. and review: Body condition score and its association with dairy cow productivity, health, productivity, cow dairy with association its and score condition Body review: 1621 1629. 1621 1629. 01 ooi, rf soitd opnn o te lmrlr slit glomerular the of component raft associated a Podocin, 2001 Comment citer cedocument: Journal of Dairy Science of Dairy Journal
71 Nat Biotech Nat Journal of DairyScience of Journal 1232 1241. 1232 1241.
66 47 63 10.1097/OGX.1090b1013e318217b318210a318214. 47 6310.1097/OGX.1090b1013e318217b318210a318214.
06 nie Rve: ehd t Dtrie oy Fat Body Determine to Methods Review: Invited 2006
60-61
2000 Reproductive management of postpartum cows. cows. postpartum of management Reproductive 2000 17 974 978. 703 712. 703 712. 1993 Effects of insulin, insulin like growth factor factor growth insulin like insulin, of Effects 1993
92 5769 5801. 5769 5801.
89 07 fet o Mtomn n Bovine on Metformin of Effects 2007 1 14. The Journal of Clinical Investigation Clinical of Journal The bttia and Obstetrical 09 Invited 2009 Journal Journal 2011 2011 31
Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 760 760 769 769 772 772 771 770 759 759 768 768 757 757 751 751 758 758 752 752 753 753 750 750 762 762 765 765 749 749 748 761 761 754 754 756 756 767 767 755 755 766 766 764 764 763 763 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is
an C, a HY Ceg & ihrs JS Richards & X Cheng H-Y, Fan CM, Wayne Verma R, Wharram B, Kovari I, Kunkel R, Nihalani D, Wary KK, Wiggins RC, Killen Killen RC, Wiggins KK, Wary D, Nihalani R, Kunkel I, Kovari B, Wharram R, Verma oc L Foet , oni P Fre , ofle & uot J Dupont & F Foufelle P, Ferre P, Solnais P, Froment L, Tosca Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A & Speleman Speleman & A Paepe De N, Roy Van B, Poppe F, Pattyn K, Preter De J, Vandesompele Yu F-Q, Han C-S, Yang W, Jin X, Hu Z-Y & Liu Y-X Liu & Z-Y Hu X, Jin W, Yang C-S, Han F-Q, Yu ilas L Asadn , aca R Sak R, iilk S, orsn D & CD Morrison SE, Nizielski RL, Stanko MR, Garcia M, Amstalden GL, Williams ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Domestic Animal Endocrinology Animal Domestic pathway by follicle stimulating hormone regulates steroidogenesis in granulosa cells granulosa in steroidogenesis regulates hormone follicle stimulating by pathway nue Mlil Sgaig acds Eiec ta Atvto o Ru Sarcoma Rous of Activation that Evidence Cascades: Signaling Multiple Induces differentially. differentially. geometric averaging of multiple internal control genes. genes. control internal multiple of averaging geometric granulosa cells. granulosa RESEARCH0034. P & Holzman LB Holzman & P oohsht ciae poen iae euae poetrn scein n rat in secretion progesterone regulates kinase protein monophosphate activated Granulosa Cell Differentiation. Differentiation. Cell Granulosa Monophosphate Activated Protein Kinase (AMPK). (AMPK). Kinase Protein Monophosphate Activated 378. noee RS ad h Eieml rwh atr eetr r Ciia for Critical Are Receptor Factor Growth Epidermal the and RAS, Oncogene, Component Nephrin. Component F Keisler DH Keisler 2002 2002 Accurate normalization of real-time quantitative RT-PCR data by by data RT-PCR quantitative real-time of normalization Accurate 2002 Leptin and its role in the central regulation of reproduction in cattle. cattle. in reproduction of regulation central the in role its and Leptin 2002 Comment citer cedocument: Journal ofEndocrinology Journal Endocrinology
2003 Fyn Binds to and Phosphorylates the Kidney Slit Diaphragm Slit Kidney the Phosphorylates and to Binds Fyn 2003
Journal of BiologicalChemistry of Journal
146 Molecular Endocrinology Molecular
23 4500 4513. 4500 4513. 339 349.
186 85 96. 85 96. 07 olceSiuaig Hormone Follicle Stimulating 2007 2005 Activation of the p38 MAPK p38 the of Activation 2005 Reproduction of Biology
278
21 20716 20723. 1940 1957. 1940 1957. 05 dnsn 5' Adenosine 2005 eoe Biol. Genome 76 368
32 18 Page 32of49 Version postprint Page 33of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 25 25 24 24 19 19 22 22 21 20 20 15 15 16 16 17 17 18 18 13 13 14 14 12 12 11 2 2 1 6 6 3 3 4 4 5 5 7 7 8 8 9 9 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is KIRREL procedure for linear mixed models in the SAS software. Information about effects of the time time the of effects about Information software. SAS the in models mixed linear for procedure pregnancy olce te is we ps pru. C ws sd s laig oto. eut are Results control. loading a as used was VCL partum. post week first the collected Figure 0.05). 23 represented as mean mean as represented iue 1: Figure legends Figure determined on the day of sample collection (1 week postpartum (1 wkpp, n=18 fertil+ n=18 wkpp, (1 postpartum week (1 collection sample of day the on determined Results are presented as means ± SEM and were analyzed using the MIXED procedure for procedure MIXED the using analyzed were and SEM ± means as presented are Results linear mixed models in the SAS software. Information about effects of the time after calving after time the of effects about Information software. SAS the in models mixed linear and n=18 fertil-) and 5 months of gestation of months 5 and fertil-) n=18 and (T, 1 wkpp, 5 mpg), haplotype (H, fertil+, fertil-) and Time x haplotype (TxH) are placed placed are (TxH) haplotype x Time and fertil-) fertil+, (H, haplotype mpg), 5 wkpp, 1 (T, above the graph. B. Protein of KIRREL was studied by western blot in adipose tissue tissue adipose in blot western by studied was KIRREL of Protein B. graph. the above lactation)). Results are presented as means ± SEM and were analyzed using the MIXED MIXED the using analyzed were and SEM ± means as presented are Results lactation)). after calving (T, 1 wkpp, 5 mpg), haplotype (H, fertil+, fertil-) and Time x haplotype (TxH) haplotype x Time and fertil-) fertil+, (H, haplotype mpg), 5 wkpp, 1 (T, calving after TqC i aioe ise smld we ps pru (kp ad t mnh of months 5 at and (wkpp) partum post week 1 sampled tissue, adipose in RT-qPCR interaction on Live Body Weight, EB and plasma NEFA levels are placed above each graph. each placed aboveare plasma levels andNEFA EB LiveWeight, Body on interaction adipose tissue of “fertil+” and “fertil-” dairy cows. dairy “fertil-” and “fertil+” of tissue adipose of expression Relative 2: Figure 10 ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
3: in liver (Li), mammary gland (Ma), adipose tissue (AT), kidney (Kid), pituitary (Pit), (Pit), pituitary (Kid),kidney (AT), tissue adipose (Ma), gland mammary (Li), liver in
Expression of of Expression (mpg). The data were normalized to the geometric mean of PPIA and EEF1A1. EEF1A1. and PPIA of mean geometric the to normalized were data The (mpg).
Plasma NEFA level (A), Energy Balance (EB, B), and Live Body Weight Weight Body Live and B), (EB, Balance Energy (A), level NEFA Plasma Comment citer cedocument: ± SEM. Bars with different superscripts are significantly different ( different significantly are superscripts different with Bars SEM. KIRREL
mRNA in bovine tissues bovine in mRNA KIRREL RA A) n KRE poen B) in (B.) protein KIRREL and (A.) mRNA (mpg, n=16 fertil+ and n=14 fertil-) in second second in fertil-) n=14 and fertil+ n=16 (mpg, A. mRNA of of mRNA A. . A. RT-PCR of the mRNA of mRNA the of RT-PCR A. . KIRREL was analysed by analysed was P < < Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 42 42 35 35 34 34 36 36 43 43 32 32 31 30 30 41 41 44 44 26 26 45 45 46 46 37 37 38 38 39 39 48 48 40 40 49 49 27 27 50 50 28 28 29 29 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is cells; Oo, oocyte; CC, cumulus cells. D. D. cells. cumulus CC, oocyte; Oo, cells; (SF) and large follicle (LF), Corpus luteum (CL), cortex (Ctx), granulosa cells from SF (GC (GC SF from cells granulosa (Ctx), cortex (CL), luteum Corpus (LF), follicle large and (SF) bovine ovary bovine SF), and granulosa cells from LF (GC LF). LF). (GC LF from cells granulosa and SF), granulosa cells from small follicles (< 6 mm) of “fertil+” and “fertil-” dairy cows dairy “fertil-” and “fertil+” of mm) 6 (< follicles small from cells granulosa Figure 4: Figure 0.05). 33 kltl uce S) oay O) n hptaau (yo. CR ws sd s positive as used was ACTR3 (Hypo). hypothalamus and (Ov) ovary (SM), muscle skeletal represented as mean mean as represented a section incubated with rabbit IgG (n=3). FF, follicular fluid; GC, granulosa cells; TC, theca TC, cells; granulosa GC, fluid; follicular FF, (n=3). IgG rabbit with incubated section a data were normalized to the geometric mean of PPIA and EEF1A1. Results are represented as represented are Results EEF1A1. and PPIA of mean geometric the to normalized were data control. B. Relative expression of of expression Relative B. control. mean mean Figure 47 expression of expression h dt wr nraie t te emti ma o PI ad E11 Rsls are Results EEF1A1. and PPIA of mean geometric the to normalized were data The represented as mean as represented secretion of progesterone (A,B) and estradiol (C,D) by bovine granulosa cells granulosa bovine by (C,D) estradiol and (A,B) progesterone of secretion ( cells from small bovine follicles were cultured for 48 h in a medium with serum and then in then and serum with medium a in h 48 for cultured were follicles bovine small from cells and mammary gland of “fertil+” and “fertil-” dairy cows. The data were normalized to the to normalized were data The cows. dairy “fertil-” and “fertil+” of gland mammary and geometric mean of PPIA and EEF1A1. C. Protein of KIRREL was studied by western blot in in blot western by studied was KIRREL of Protein C. EEF1A1. and PPIA of mean geometric kidney of “fertil+” and “fertil-” dairy cows. VCL was used as a loading control. Results are are Results control. loading a as used was VCL cows. dairy “fertil-” and “fertil+” of kidney P ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 < 0.05). C. Localization of KIRREL by immunohistochemistry. Negative controls included included Negativecontrols immunohistochemistry. KIRREL by of Localization C. 0.05). < ±
SEM. SEM. 5:
Expression of of Expression Effect of rm KIRREL treatment on basal and FSH- or IGF1-stimulated IGF1-stimulated or FSH- and basal on treatment KIRREL rm of Effect . A. RT-PCR of RT-PCR A. . KIRREL Comment citer cedocument: ± ± SEM. (n=6). Bars with different superscripts are significantly different different significantly are superscripts different with Bars (n=6). SEM. mRNA in the different compartments or cell types from bovine ovary. bovine from types cell or compartments different the in mRNA SEM. Bars with different superscripts are significantly different ( different significantly are superscripts different with Bars SEM. KIRREL KIRREL
KIRREL mRNA (A. and B.) and localization (C.) of KIRREL in KIRREL of (C.) localization and B.) and (A. mRNA mRNA in theca-interstitial cells from small follicle small from cells theca-interstitial in mRNA Relative expression of of expression Relative mRNA in bovine kidney, hypothalamus, pituitary pituitary hypothalamus, kidney, bovine in mRNA ACTR3 was used as a positive control. B. Relative B. control. positive a as used was KIRREL mRNA in bovine bovine in mRNA . Granulosa Granulosa . . The . P < < Page 34of49 Version postprint Page 35of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 73 73 71 71 52 52 70 70 69 69 60 60 61 61 74 74 62 62 75 75 63 63 64 64 65 65 53 53 66 66 54 54 67 67 55 55 68 68 56 56 59 59 51 51 57 57 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is blot from three independent experiments is shown. Bars with different letters are significantly are letters different with Bars shown. is experiments independent three from blot in primary bovine granulosa cells. granulosa bovine primary in Figure 7: Figure 72 or 10 or different ( different serum-free medium in the presence of different concentrations of rm KIRREL (1, 5, 10 and and 10 5, (1, KIRREL rm of concentrations different of presence the in medium serum-free 0 n/l a dsrbd n aeil ad ehd. eut ae xrse a thymidine as expressed are Results Methods. and Materials in described as ng/ml) 100 uig ifrn tms 0 o 0 iue) n nihd co’ 5 mdu (ihu FBS) (without medium 5A McCoy’s enriched in minutes) 60 to (0 times different during incorporated in cpm (counts per minute). Results are representative of five independent independent five of representative are Results minute). per (counts cpm in incorporated xeiet. h rsls r epesd s en ± E. , fet f m IRL n the on KIRREL rm of Effect B, SEM. ± means as expressed are results The experiments. amount of PCNA protein in bovine granulosa cells. Protein extracts from bovine granulosa granulosa bovine from extracts Protein cells. granulosa bovine in protein PCNA of amount cells cultured for 48 h in the presence or absence of different concentrations of rm KIRREL KIRREL rm of concentrations different of absence or presence the in h 48 for cultured cells then collected and analyzed for progesterone (A and B) and estradiol (C and D) content by content D) and (C estradiol and B) and (A progesterone for analyzed and collected then (1, 5, 10 and 100 ng/ml) were subjected to SDS-PAGE as described in Materials and and Materials in described as SDS-PAGE to subjected were ng/ml) 100 and 10 5, (1, RIA. The results are expressed as the amount of steroid secreted relative to the basal state. state. basal the to relative secreted steroid of amount the as expressed are results The RIA. Methods. The membranes were incubated with antibodies raised against PCNA. Equal protein Equal PCNA. against raised antibodies with incubated were membranes The Methods. The results are means ± SEM of six independent experiments. Bars with different letters are are letters different with Bars experiments. independent six of SEM ± means are results The loading was verified by reprobing membrane with an anti-tubulin-antibody. A representative representative A anti-tubulin-antibody. an with membrane reprobing by verified was loading significantly different ( different significantly serum-free medium in the presence or in the absence of various doses of rm KIRREL (A and and (A KIRREL rm of doses various of absence the in or presence the in medium serum-free Thymidine incorporation was determined in bovine granulosa cells cultured for 24 h in in h 24 for cultured cells granulosa bovine in determined was incorporation Thymidine C) for 48 h, or in presence or absence of 10 ng/ml rm KIRREL, with or without 10 without or with KIRREL, rm ng/ml 10 of absence or presence in or h, 48 for C) Figure 58 ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 -8 M IGF1 (B and D) as described in Materials and Methods. The culture medium was was medium culture The Methods. and Materials in described as D) and (B IGF1 M
6:
P Effect of rm KIRREL on phosphorylation of MAPK1/3 (A.) and MAPK14 (B.) (B.) MAPK14 and (A.) MAPK1/3 of phosphorylation on KIRREL rm of Effect
<0.05). fet f m IRL n h poieain f oie rnls cells. granulosa bovine of proliferation the on KIRREL rm of Effect Comment citer cedocument: P < 0.05). 0.05). <
After 18 hours of serum starvation, cells were stimulated were cells starvation, serum of hours 18 After -8 M FSH, M
A, Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 76 76 78 78 77 77 80 80 81 81 82 82 83 83 84 84 98 98 99 99 85 85 86 86 87 87 88 88 89 89 90 90 91 91 92 92 93 93 94 94 95 95 96 96 97 97 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is bovine oocytes for each set of conditions in each experiment were used. B. Bovine COCs COCs Bovine B. used. were experiment each in conditions of set each for oocytes bovine SEM. The results are representative of 4 independent cultures. Bars with different superscripts different with Bars cultures. 4 independent of representative are results The SEM. iue : fet o r KRE tetet n oie oye ula maturation. nuclear oocyte bovine on treatment KIRREL rm of Effects 8: Figure 79 are significantly different ( different significantly are oie oye wr alwd o aue o 2 h n h peec o asne f various of absence or presence the in h 22 for mature to allowed were oocytes Bovine concentrations of rm KIRREL (1, 10 and 100 ng/ml). The percentage of oocytes at the GV the at oocytes of percentage The ng/ml). 100 and 10 (1, KIRREL rm of concentrations stage in the various conditions is shown. Different letters indicate significant differences with with differences significant indicate letters Different shown. is conditions various the in stage P supplemented with or without rm KIRREL (100 ng/ml). Results are represented as mean ± mean as represented are Results ng/ml). (100 KIRREL rm without or with supplemented
100 were cultured for 22 h in maturation medium in the presence or absence of rm KIRREL (1, 10 10 (1, KIRREL of rm or absence presence the in medium in maturation h 22 for cultured were n 10 gm) CC wr te mcaial sprtd no oye n cmls cells. cumulus and oocyte into separated mechanically then were COCs ng/ml). 100 and Denuded oocytes (50 oocytes per lane) were lysed and subjected to Western blot analysis with analysis Western blot to subjected and lysed were lane) (50 oocytes per oocytes Denuded niois gis popoMP31 n MP3 Rpeettv bos rm three from blots Representative MAPK3. and phospho-MAPK3/1 against antibodies independent experiments are shown. Blots were quantified, and the phosphorylated protein to to protein phosphorylated the and quantified, were Blots shown. are experiments independent total protein ratio is shown. Different letters indicate significant differences with with differences significant indicate letters Different shown. is ratio protein total The results are presented as means ± SEM. C. Bovine COCs were cultured for 22 h in in h 22 for cultured were COCs Bovine C. SEM. ± means as presented are results The maturation medium in the presence or absence of various doses of rm KIRREL (1, 10, and 10, (1, KIRREL rm of doses various of absence or presence the in medium maturation 0 n/l. h clue eim a te cletd ad t poetrn cnet was content progesterone its and collected, then was medium culture The ng/ml). 100 analyzed by RIA as described in Materials and Methods. The results are expressed as ng/ml of ng/ml as expressed are Theresults Methods. and Materials in described by analyzed RIA as 50 COC-equivalent cumulus cells. The results are means ± SEM for three independent independent three for SEM ± means are results The cells. cumulus COC-equivalent 50 experiments. Different letters indicate significant differences with significant indicate differences lettersDifferent experiments. < 0.05. The results are presented as mean ± SEM of three independent experiments. Fifty experiments. independent three of SEM ± mean as presented are results The 0.05. < ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 Comment citer cedocument: P < 0.05). <
P <0.05. P P 0.05. < A A Page 36of49 Version postprint Page 37of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is EF1A1-like EF1A1-like ATP1A2 KCNJ10 EEF1A1 RPL19 PPIA ACTR3 KIRREL Primer name Primer sequence Accession number number Accession sequence Primer name Primer 1 Table Sense 5’- TCG TTG TCA TTG GGC ACG TA -3’ XR_083620 XR_083620 -3’ TA TTGACG GGCTCA TTG TCG 5’- NM_001081524 Sense -3’ TCGGA GCT GAC AGAAGG TTG 5’- -3’ TAT CTGCCCCC GGA CAT CGA 5’- Antisense NM_001034394 Sense -3’CC TAT CAC AGA TGG CCA TGC 5’- -3’ TC CCC TCCGAA GAAGCA GCT 5’- Antisense NM_001081601 Sense CDIE -3’ TCT GGA CAGGC TGT TGA GGT 5’- ATACA-3’ CTC TAG CCG TCG CAG 5’- Antisense NM_001105645 Sense -3’TGGG ATC CAA AGA ACTC GGC 5’- -3’ CT GCAAGA CTT GCTTGG ATT 5’- Antisense NM_174537 Sense COPA -3’ GTGTTGTCGGGTGT TAC TGC 5’- -3’TGC TG CTG TGA GGC ATC CCA 5’- Antisense NM_001040516 Sense CC-3’ATACCTTTA CGCTTT CCC 5’- -3’CTC TGC CAA CGC CAA AAT 5’- Antisense NM_178320 Sense GT-3’ ATG GCA TCA CCA CAG TGT 5’- -3’ CATCT TGGTCC AGG TAC GCA 5’- Antisense Sense NM_174226 ACC-3’ TAT AAC TTC GTC GTC CCA 5’- ATC-3’ ATC TTTCAG CCA GAA ACG 5’- Antisense XM_003585822 Sense AT-3’TTC TGT GAG GTG AAG GGC 5’- AT-3’TTC TGT GAG GTG AAG GGC 5’- Antisense Sense ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 Oligonucleotide primer sequences sequences primer Oligonucleotide Comment citer cedocument: Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is IFI16 IFI16 PEA15 Sense 5’- GGA CAT TAC CGT GCC CAG AT -3’ NM_001206364 NM_001206364 -3’AC GCA TGT GTG TGA GTG CAC 5’- AT-3’ GCCCAG TACCGT CATGGA 5’- Antisense Sense XM_863928 SLAMF6 AC -3’CTGCTC TCA TGG GTC CTC 5’- -3’CGTAA GGA ACCCAA CAC AGC 5’- Antisense NM_001075946 Sense -3’GA CAC GCA AGA CACGTT ATG 5’- -3’CC AAT CTA GGG ATG TCCAGC 5’- Antisense Sense NM_001075456 CADM3 TA-3’ GCT CGATGT AGA CAA TCT AGA 5’- -3’GAG GAA TGA CCCCAG CATGGA 5’- Antisense Sense -3’ACGCCGAT GTTCCC CTT TCT 5’- Antisense ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 Comment citer cedocument:
Page 38of49 Version postprint Page 39of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is PEX19 Protein targeting to peroxisome NM_001034540 1.1232 1.1232 NM_001034540 peroxisome to targeting Protein response Immune PEX19
week after calving, with calving, adjusted week after Table 2 Table Cell development and organization organization and development Cell proteintransport and Ion M ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 etabolism Gene Symbol Gene FCER1A FCER1A KCNJ10 KCNJ10 DARC ATP1A2 ATP1A2 LOC512286 IGSF9 CRP CRP SLAMF1 SLAMF1 TAGLN2 TAGLN2 CD1E CD1E CD1A CASQ1 COPA COPA SPTA1 VANGL2 Genes differentially expressed between “fertil+” and “fertil-” adipose tissue one tissueone and “fertil-” adipose “fertil+” between Genes expressed differentially
Negative regulation of macrophage NM_001144097 1.0970 1.0970 NM_001144097 macrophage of regulation Negative Signal transduction transduction Signal Lymphocyte activation activation Lymphocyte oasu o rnpr NM_001081601 transport ion Potassium Biological process Biological Antigen processing and preresponse immune andprocessing Antigen response Inflammatory organization endoplasmic Reticulum Muscle organe development organe Muscle nie rcsigadpeetto NM_001034394 andpresentation processing Antigen ATP biosynthetic process biosynthetic ATP preresponse immune andprocessing Antigen transport Vesicle-mediated Hemopoiesis development Dendrite 1.0998 NM_001205875 organismaldevelopment Multicellular Comment citer cedocument:
P -value<0.05.
NM_001077877 NM_001102024 NM_001015634 NM_001206588 1.0416 1.0416 NM_001206588 XM_003585820 1.1011 1.1011 XM_003585820 NM_001081524 NM_001100310 1.1468 1.1468 NM_001100310 NM_001105645 NM_001205532 1.0471 1.0471 NM_001205532 NM_001013599 1.0819 1.0819 NM_001013599 NM_174184 1.0469 1.0469 NM_174184 NCBI accession accession NCBI number number
Fold change change Fold “f+”/ “f-” “f+”/ 1.2367 1.2367 0.7694 1.1718 1.2486 1.0774 1.1261 1.1439
Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is
receptors Olfactory
Gene ontology unknown in in unknown ontology Gene process biological Other
ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 PEA15 PEA15 DCAF8 DCAF8 SLAMF6 SLAMF6 EF1A1-like EF1A1-like LO LOC617783 PIGM NCSTN APCS LOC519294 LOC519294 ATP1A4 IFI16 IFI16 LOC530601 LOC514540 LOC514540 LOC522554 KIRREL KIRREL CADM3 CADM3 OR6Y1 OR10K2 OR6P1 OR6P1 OR10R2 OR10T2 C508806
Cell adhesion Cell Anti-apoptosis Anti-apoptosis Gene ontology unknown in ontologyunknown Gene Gene ontology unknownin ontology Gene Translation elongation factor activity XR_083620 1.6612 1.6612 XR_083620 activity factor elongation Translation S transduction Signal transduction Signal transduction Signal G M Response to protein stimulus protein to Response Signal transduction transduction Signal process biosynthetic ATP Signal transduction transduction Signal transduction Signal transduction Signal Gene ontology unknown in unknown ontology Gene transduction Signal Signal transduction transduction Signal transduction Signal Excretion Excretion ignal transduction ignal lycosylphosphatidylinositol biosynthesis lycosylphosphatidylinositol Comment citer cedocument: embrane protein ectodomain proteolysis ectodomain protein embrane
BosTaurus
BosTaurus
Bos Taurus Bos Bos Taurus Bos NM_001206419 1.1878 1.1878 NM_001206419 NM_001206364 XM_863928 1.3068 1.3068 XM_863928
NM_001075456 NM_001015563 NM_001034475 NM_001034466 NM_001075946 XM_002685946 XM_003581920 XM_002685938 XM_002685942 NM_001144103 1.0557 1.0557 NM_001144103 XM_003585822 XM_002685904 1.0901 1.0901 XM_002685904 XM_002685937 0.9449 0.9449 XM_002685937 0.9506 XM_002685943 0.9572 XM_002685925 XM_002685875 0.9442 0.9442 XM_002685875 XM_002685910 0.9377 0.9377 XM_002685910 0.9456 XM_002685948
1.1857 1.1857 1.2057 1.2301 1.2695 0.9617 0.9664 1.0579 1.1 0.9714 0.9455 0.9455 652
Page 40of49 Version postprint Page 41of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is
“f+”/ “f-”, “fertil+”/ “fertil-” “fertil-” “f-”, “fertil+”/ “f+”/ ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 IGSF8 VSIG8 CCDC19 CCDC19 SLAMF8 CD84
Gen in ontologyunknown Gene Gene ontology unknown in ontologyunknown Gene in ontologyunknown Gene in ontologyunknown Gene Comment citer cedocument: e ontology unknown in ontologyunknown e
BosTaurus BosTaurus BosTaurus BosTaurus BosTaurus
NM_001038219 1.0345 1.0345 NM_001038219 1.0932 1.1339 NM_001205794 XM_588136 NM_001082439 NM_001205873
1.1568 1.1660
Version postprint Figure1
NEFA (mM) A. Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet 1000 1200 1400 200 400 600 800 0 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 a 1 1 wkpp T: p<0.0001 T: b Comment citer cedocument: H: p<0.05 H: C.
Live Body weight (kg)
a TxH: p = 0.3 TxH:p = 500 540 580 620 660 700 5 5 mpg “fertil+” “fertil+” “fertil a T: p<0.05 a - ” (n=18 at 1 wkpp and n=14 at 5 mpg)wkpp 5 1 and at at n=14 (n=18 1 1 wkpp (n=18 at 1 wkpp and n=16 at 5mpg ) 5mpg1 wkpp and at at n=16 (n=18 H: p<0.05 H: b TxH : p= 0.6 p=TxH : B. a
5 5 mpg Energy Balance (Mcal/day) - - - - 16 14 12 10 - - - - 0 2 8 6 4 2 a a T: 1 1 wkpp p<0.0001 b H: p<0.05 H: TxH: p = 0.5 TxH:p = a 5 5 mpg Page 42of49 a Version postprint Page 43of49 Figure2 Coyral-Castel, S., Ramé, C., Cognié, J., Lecardonnel, J., Marthey, S., Esquerré, D., Hennequet differentially expressed in adipose tissue from “fertil+” and “fertil-” cows: invitro role in Antier, C.,Elis, S., Fritz,S., Boussaha, M., Jaffrézic, F., Dupont, J.(2018). KIRREL is A. ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649
Relative expression of KIRREL mRNA (x 10-3) T: p<0.05 T: 0.2 0.4 0.6 0.8 1.2 1.4 1.6 1.8 0 1 2 N = N Comment citercedocument : 1 wkpp 5 5 mpg 1 wkpp 81 614 16 18 18 a H: p<0.05 H: b —fertil+“ —fertil+“ —fertil
TxH: p = 0.4 p = TxH: b - “ “ b B. KIRREL VCL Ratio KIRREL/VCL —fertil+“ —fertil+“ 0.02 0.04 0.06 0.08 0.1 0 (n=8) a b —fertil - “ “ (n=8) kDa 110 90 Figure3 Version postprint Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet B. A. differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is KIRREL Relative expression of KIRREL mRNA (x 10-3) ACTR3 ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 0.2 0.4 0.6 0.8 1.2 1.4 1 0 Li Mam Heart AT Kid Pit Lung SM Kid Hypo Lung AT Ov SM Pit Heart Li Mam kidney a b Comment citer cedocument: a,b hypo b “fertil+” “fertil+” “fertil
pituitary b - ” (n=3) (n=4) b b mam b C. KIRREL VCL “fertil+” “fertil+”
Ratio KIRRREL/VCL 188 165 bp 0.05 0.15 0.25 0.35 0.45 0.1 0.2 0.3 0.4 (n=4) 0 a b “fertil - ” (n=3) kDa 110 90 Page 44of49 Figure4 Version postprint Page 45of49 B. A. D. Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet KIRREL ACTR3 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is Relative expression of KIRREL mRNA (x 10-2) 0.5 1.5 2.5 3.5 4.5 ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 Relative expression of
-3 SF KIRREL mRNA (x 10 ) 0 1 2 3 4 0.5 1.5 2.5 3.5 4.5 0 1 2 3 4 SF a a LF LF a a CL CL Comment citer cedocument: a b Ctx Ctx a a “fertil+” “fertil+” “fertil GC GC SF SF a
- ” GC LF (n=3) (n=3) GC LF b 165 188 bp C. FF Control IgG Control Oo CC FF GC GC TC FF TC x20 x20 x10 FF GC KIRREL Oo TC FF GC CC TC FF x10 x20 x20 Figure5 rm KIRREL 10 ng/ml10 KIRREL rm C. Version postprint A. IGF110 FSH FSH 10 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet rm KIRREL ng/mlKIRREL rm differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is - 8 - Progesterone secretion / basal state 8 M M 0 1 2 3 4 5 6 7 8 ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 Progesterone secretion / basal state a 0.2 0.4 0.6 0.8 1.2 0 1 b a Comment citer cedocument: c 1 a c b 5
10 b,c d 100 c d rm KIRREL 10 ng/ml10 KIRREL rm D. B. rm KIRREL ng/mlKIRREL rm IGF110 FSH 10 - 8 -
8 Oestradiol secretion / basal state M M 9 0 1 2 3 4 5 6 7 8 Oestradiol secretion / basal state 0.2 0.4 0.6 0.8 1.0 1.2 a 0 b a a 1 c b 5 c 10 b,c d 100 c d Page 46of49 Figure6 Version postprint Page 47of49 Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet rm KIRREL ng/mlKIRREL rm differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is A. ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 [3H] thymidine incorporation into granulosa cells (CPM x 102) 10 20 30 40 50 60 70 0 a Comment citer cedocument: a,b 1
b 5 10 c 100 c B. rm KIRRELng/mlrm
Ratio PCNA/Vinculin 0.5 1.0 0 Blot: PCNA Blot: Blot: Vinculin Blot: a a 1 b 5 10 c 100 c 30 110 kDa Figure7 Version postprint
Coyral-Castel, S.,Ramé, C.,Cognié, J.,Lecardonnel, J.,Marthey, S.,Esquerré,D., Hennequet Ratio phospho-MAPK1/3 / MAPK1 / basal state A. 0.5 1.5 2.5 differentially expressed in adipose tissue from“fertil+” and “fertil-” cows: invitro rolein Antier, C., Elis,S.,Fritz, S.,Boussaha, M.,Jaffrézic, F.,Dupont,J. (2018).KIRREL is 3 0 1 2 Time of rm KIRREL stimulation (minutes)stimulationKIRREL rm of Time MAPK1 Phospho ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 0 a - a 1 MAPK1/3 b 5 Comment citer cedocument: 10 a,b 30 a
60 a kDa 42 42 44
Ratio phospho-MAPK14 / MAPK14 / basal state B. 0.2 0.4 0.6 0.8 1.2 1.4 1.6 1.8 0 1 2 Time of rm KIRREL stimulation (minutes)stimulationKIRREL rm of Time MAPK14 Phospho a 0 - b 1 MAPK14 b 5 10 a 30 a 60 a kDa 43 43 Page 48of49 Version postprint Page 49of Figure8 Coyral-Castel, S., Ramé, C., Cognié, J., Lecardonnel, J., Marthey, S., Esquerré, D., Hennequet differentially expressed in adipose tissue from “fertil+” and “fertil-” cows: invitro role in Antier, C.,Elis, S., Fritz,S., Boussaha, M., Jaffrézic, F., Dupont, J.(2018). KIRREL is rm KIRREL (ng/ml)KIRREL rm rm KIRREL (ng/ml)KIRREL rm C. A. ovary? . Reproduction, 155 (2), 181-196. , DOI : 10.1530/REP-17-0649 Progesterone
concentration (h) IVM (ng/ml) % oocytes at the GV stage 100 120 20 40 60 80 0 0 2 4 6 a Comment citercedocument : a 22 b 1 a
1 22 b 0100 10 b 0100 10 222 22 c b c rm KIRREL (ng/ml)KIRREL rm B. B. IVM (h) IVM
Ratio phospho-MAPK1/3 / MAPK1 0.5 1 0 lt MAPK1 Blot: lt phospho-MAPK1/3 Blot: a 22 b 22 22 22 22 1 b 0100 10 c c