Research Article A Tissue Retrieval and Postharvest

Research ArticleA Tissue Retrieval and Postharvest ProcessingRegimen for Rodent Reproductive Tissues Compatible withLong-Term Storage on the International Space Station andPostflight Biospecimen Sharing Program

Vijayalaxmi Gupta1 Lesya Holets-Bondar1 Katherine F Roby23

George Enders2 and Joseph S Tash1

1Department of Molecular amp Integrative Physiology University of Kansas Medical Center Mail Stop 3050 3901 Rainbow BoulevardHLSIC 3098 Kansas City KS 66160 USA2Department of Anatomy and Cell Biology University of Kansas Medical Center Kansas City KS 66160 USA3Institute for Reproductive Health and Regenerative Medicine University of Kansas Medical Center Kansas City KS 66160 USA

Correspondence should be addressed to Joseph S Tash jtashkumcedu

Received 6 June 2014 Revised 18 September 2014 Accepted 20 October 2014

Academic Editor Jack J W A Van Loon

Copyright copy 2015 Vijayalaxmi Gupta et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Collection and processing of tissues to preserve space flight effects from animals after return to Earth is challenging Specimensmust be harvestedwithminimal time after landing tominimize postflight readaptation alterations in protein expressiontranslationposttranslational modifications and expression as well as changes in gene expression and tissue histological degradation aftereuthanasia We report the development of a widely applicable strategy for determining the window of optimal species-specific andtissue-specific posteuthanasia harvest that can be utilized to integrate into multi-investigator Biospecimen Sharing Programs Wealso determined methods for ISS-compatible long-term tissue storage (10 months at minus80∘C) that yield recovery of high qualitymRNA and protein for western analysis after sample return Our focus was reproductive tissues The time following euthanasiawhere tissues could be collected and histological integrity was maintained varied with tissue and species ranging between 1 and3 hours RNA quality was preserved in key reproductive tissues fixed in RNAlater up to 40min after euthanasia Postfixationprocessing was also standardized for safe shipment back to our laboratory Our strategy can be adapted for other tissues underNASArsquos Biospecimen Sharing Program or similar multi-investigator tissue sharing opportunities

1 Introduction

With the current paucity of opportunities for studying wholeanimal mammalian physiology in space flight the Biospec-imen Sharing Program (BSP) for postflight tissue collectionoffers the opportunity to broaden access to biological samplesshortly after return and maximize the data generated fromflight animal payloads The logistics of space flight experi-ments involving live animals often requires harvesting tissuesat a remote site followed by shipping the specimens to thePrinciple Investigatorsrsquo laboratories for detailed analysis Fur-thermore as the capabilities to house rodent andother animalson the International Space station (ISS) and to conduct long-term space flight experiments using animals are enabled the

need to harvest and fix tissues for long-term storage on ISSthat will retain high quality RNA and protein for subsequentanalysis in laboratories on Earth is also required Theseapproaches to live animal experimentation in space flightthat include tissue harvest for multiple investigators requiredetermination of (1) tissue-specific windows of time betweeneuthanasia and tissue fixation that retain quality of histologyand (2) tissue-specific windows of time for tissue fixation thatretain high quality protein and RNA for subsequent analysisDetermination of these windows provides a quantitativelogical approach to generate appropriately prioritized andoptimized tissue harvest and fixation logistics in a multi-investigator Biospecimen Sharing Program scenario be it onEarth or during tissue harvest on the ISS In addition tissue

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 475935 12 pageshttpdxdoiorg1011552015475935

2 BioMed Research International

storage methods should retain high sample quality underlong-term storage as samplesmay be harvested and stored onthe ISS but may not be returned to Earth for many monthsdepending on ISS to Earth flight frequency and payloadcapacities This issue has been addressed for preserving plantmaterial for gene expression analysis [1] but there is nodata available for animal tissues Our participation in theBSP program involved tissue harvest from female mice atKennedy SpaceCenter Florida USA (KSC) for animals flownfor 12ndash15 days in orbit on three space shuttle flights STS-131STS-133 and STS-135 In addition we harvested tissues frommale mice at the Institute for Biomedical Problems (IMBP)laboratory in Moscow RU for animals flown for 30 daysin orbit on the BION M1 satellite Both the STS and BIONseries of flight experiments involved age- and time-matchedground control groups of animals During the early flightplanning phase of BION there were possibilities that maleor female mice would be flown and that male gerbil tissuesmay also be provided to the BSPThus to be prepared for anyof these possibilities we undertook to determine the optimaltissue harvest windows for all of the species and reproductivetissues that we might be able to obtain Our participationin these multi-investigator specimen sharing efforts coveringfour different primary flight PI experimental designs andharvest logistics necessitated a determination of the windowof time between euthanasia and harvest and preservation ofour tissues of interest that would allow us sufficient flexibilityto obtain the highest possible quality of tissue for histopathol-ogy RNA for gene-transcription analysis and protein forexpression and posttranslational modification analysis Adetermination of these time windows is essential since thetissues that are made available to the BSP investigators areprovided after the primary flight PIrsquos have obtained their tis-sues Knowledge of the optimal windows for all of the tissuesof interest aids in the preparation of targeted tissue harvestflow logistics that can provide each of the BSP teammembersthe highest possible quality tissue respectively Therefore aswe report here we developed a strategy to determine thewindowof time between euthanasia and fixation for retentionof high quality histology for male and female reproductiveorgans We also determined methods for long-term tissuestorage for 10 months that provide for recovery of both highquality protein and RNA These strategies are adaptable andcan be applied to harvest and storage of other time-sensitivelabile tissues from animals and plants Furthermore thesemethods can be used to optimize logistics and data collectionundermulti-investigator tissue harvest and sharing programsoperated by any space agency commercial entity or flightplatform

2 Materials and Methods

21 Animals Approximately 8 wk old male and femaleC57Bl6J (Jackson Lab Bar Harbor ME) and sim10-month oldmale and female Mongolian gerbils (Charles River Wilming-ton MA) were used throughout this study All animal useprotocols were approved by the University of Kansas Insti-tutional Animal Care andUse Committee (IACUC) Animals

were maintained in standard cages with 12 12 h dark lightcycle and standard food and water were provided ad libitumAll animals were euthanized using CO

2asphyxiation fol-

lowed by cervical dislocation prior to tissue harvest

22 Determination of the Limit of Time andTemperature between Euthanasia and Tissue Fixation

221 Male Mice Our protocol consisted of three groups ofsix mice each with one mouse for each time point In groupA all mice were euthanized at one time and the testes andepididymides were harvested and separated and then imme-diately placed in Hamrsquos F-10 medium (Sigma Aldrich StLouis MO) on ice At time intervals of 0 05 10 15 20 and25 hr each testis (one mouse per time point) and epididymis(2 mice per time point) were separated and then transferredfrom Hamrsquos F-10 medium to Bouinrsquos fixative (Sigma AldrichSt Louis MO) at room temperature (RT 21∘C) In group Bthe same procedure was followed except that the testes andepididymides separated were placed in Hamrsquos F-10 mediumat RT until transfer to Bouinrsquos at the same time interval asabove In group C all mice were euthanized at one time thecarcasses were maintained at (RT) and then at time 0 05 1015 20 and 25 hr the testis and epididymis were harvestedfrom the carcasses respectively At each of the time pointsabove groupC testes were placed in Bouinrsquos solution and pro-cessed as per standard histology protocols as detailed belowFor all groups the tissues fixed in Bouinrsquos were processed asdetailed in Section 23 below At the same time point groupC epididymides were processed to obtain cauda sperm toassess sperm motility by computer assisted sperm analysis(CASA) [2] For animals used for collection of cauda epi-didymal sperm formotility analysis two animals were used ateach time point

222 Female Mice Female mice were euthanized (carcassesmaintained at RT as per groupC above) and their ovaries anduteri were harvested at 0 05 10 15 20 25 and 30 hr aftereuthanasia (one animal per time point) At the times indi-cated the tissues were fixed at RT in Bouinrsquos overnight andthen processed for histology as detailed below (Section 23)

223 Mongolian Gerbils The six male gerbils were euth-anized at one time and the carcasses were kept at roomtemperature At 0 05 10 15 20 and 30 hr after euthanasiatestis and epididymis were harvested from the carcasses andseparated respectively (one animal per time point) Similarlyall six female gerbils were euthanized and the carcasses werekept at RT Ovaries and uterine horns were harvested fromeach carcass at the same time interval as the males At thetimes indicated above the tissues were placed in Bouinrsquos fixa-tive at RT and then processed for histology as detailed below(Section 23) Sperm were immediately harvested from caudaepididymis as mentioned above and sperm motility analysiswas carried out using CASA as described above [2]

23 Postharvest Processing of Mouse Testicular Tissues Testesand epididymides from the mature mice were harvested as

BioMed Research International 3

detailed above Unless indicated otherwise all procedureswere done at RT Tissues were fixed in Bouinrsquos solution for48 h washed in 70 ethanol (ETOH) until the yellow colorof Bouinrsquos disappeared (sim48 hr with frequent changes of 70ETOH and gentle agitation) and divided into four groupsControl group tissue was stored in 70 ETOH until paraffinembedding Rapid transition 70 ETOH was immediatelyreplaced with PBS (Sigma Aldrich St Louis MO) pH 74for one wk and then rapidly replaced with 70 ETOHSlow Re-ETOH only 70 ETOH was immediately replacedwith PBS for one wk and then sequentially replaced at 2 hrintervals with each of 10 30 50 and 70 ETOH Slowrehydration-dehydration 70 ETOH was sequentially (7050 30 10 then PBS at 2 hr each) substituted with PBSfor one wk and then replaced at 2 hr intervals with each of10 30 50 and 70 ETOH To prevent contaminationwe kept tissue in PBS at 4∘C and ETOH replacement wascompleted at RT with 2 hr intervals between changes asdetailed above After each of the respective final dehydrationsteps above the tissues were paraffin-embedded and pro-cessed for histology and hematoxylin and eosin staining (HE)using standard methods as performed previously [3]

24 Total RNA Extraction and Preservation Freshly harves-ted mouse testes were immediately stabilized in 10 volumesof TRIzol reagent (Invitrogen Carlsbad CA) or RNAlatersolution (Ambion Austin TX) The samples placed inRNAlater were stored for (1) two wks at 4∘C (2) one wk at RTand one wk at 4∘C or (3) two wks at RT Before RNAextraction tissues were retrieved from RNAlater solutionwith sterile forceps and then submerged in TRIzol reagentRNAwas isolatedwithTRIzol reagent according to themanu-facturerrsquos instructions Time of placement into RNAlater wasnoted to determine if RNA quality was related to the durationof window from euthanasia to placement in RNAlater RNAintegrity and quantity were determined using Agilent RNAkit andAgilent Bioanalyzer 2100 (Santa Clara CA) One 120583g ofRNA was subjected to RT PCR with the primers specific formouse GAPDH (Forward 51015840CCTTCATTGACCTCAAC-TAC Reverse 51015840ATGACAAGCTTCCCATTCTC) and inte-rleukin-1alpha (IL-1120572) (Forward 51015840ACTTGTTTGAAGAC-CTAAAG Reverse 51015840GTTTCAGAGGTTCTCAGAG) Pri-mers were designed using online Primer Design Tool Primer3 Uteri and ovaries from STS-135 were harvested in RNAlatersolution and stored at minus80∘C for 10 months Total RNA wasisolated from ovaries and uterine horns with Gene EluteMammalian RNA kit (Sigma Aldrich St Louis MO) PCRproducts were verified on 30 agarose gels using standardprocedures

25 Protein Extraction from Ovaries and Uterus Tissue Pre-served in RNAlater Ovaries stabilized in RNAlater for onewk at RT and two wks at 4∘C were used for protein extrac-tion The tissue was removed from RNAlater briefly rinsedwith ice-cold PBS pH 74 and then homogenized in RIPAbuffer containing protease inhibitor cocktail (all from SigmaAldrich St Louis MO) or ProteoJet Mammalian Cell LysisReagent (Fermentas Pittsburgh PA) Uteri from STS-135

mission mice were preserved in RNAlater for 10 months atminus80∘C prior to processing in RIPA as described above Pro-tein concentration was determined using the DC Assay (Bio-Rad Hercules CA) and 15 120583g protein was electrophoresedunder denaturing conditions on 4ndash15 polyacrylamide geland transferred to nitrocellulose membrane (Bio-Rad Her-cules CA) The membranes were blocked for 1 hr with 5nonfat milk in TBS-T (Tris buffered saline with Tween-20SigmaAldrich St LouisMO) and probedwith 1120583gmL rabbitanti-mouse estrogen receptor alpha (ER120572) antibody (SantaCruz Biotechnology CA) To verify equal loading of proteinsmembranes were stripped and reprobed with a goat anti-120573-actin antibody (Santa Cruz Biotechnology CA) Incubationswith primary antibodies were carried out overnight at 4∘CAfter washing in TBS-T and probing with the correspondinghorseradish peroxidase-labeled secondary antibody (PierceBiotechnology Rockford IL) bound antibodies were iden-tified using AmershamACL PlusWestern Blotting DetectionReagents (GEHealthcare Pittsburgh PA) and luminographyWestern blots were quantitated by densitometric analysis

3 Results

31 Effect of Delayed Processing on Quality of Mouse andGerbil Reproductive Tissue Histology

311 Male Mouse Testes harvested any time between time0 after euthanasia up to 25 hr after euthanasia showedhistological properties comparable to the time 0-harvestedmice For comparative purposes the 0min harvested testesrepresent the control for subsequent time points for eachstorage treatment respectively Histological quality of thetestes was excellent in all postharvest treatment proceduresnamely tissues were kept in the carcass until fixation or fixedin Bouinrsquos immediately or stored in Hams F10 on ice or RTbefore fixation Testis tubules appeared normal in all treat-ment groups compared to the 0min controls with noshrinkage of tissue and complete retention of histologic archi-tectural details (Figures 1(a) 1(b) and 1(c))

Total motility and progressive motility of cauda epididymalmouse sperm at times 0 05 10 15 20 and 25 hr were notsignificantly different between any of the tissue harvest timepoints and also not significantly different between the threetissue harvest scenarios Table 1 shows the percent progressivemotility for each time point under all three tissue storageconditions Table 2 shows the percent total motility for eachtime point under all three tissue storage conditions Based oncomparable results between the three tissue harvest regimenswe focused on the ldquotissue in carcassrdquo at RT regimen forsubsequent experimentswith femalemice aswell asmale andfemale gerbils

312 Male Gerbil Testis showed normal distinct histologicaldetails with all spermatogenic cells arranged in a normal pat-tern in the tubule when collected up to 25 hr after euthanasia(Figure 2) Total motility of the gerbil cauda epididymalsperm harvested at each at time point was analyzed and ispresented in Table 3 Since the data represent a single animal


(a) (b)

(c)

Figure 1 Effect of delayed processing on C57Bl6J testicular morphology Each panel represents light microscopy (40x objective) of sectionsof adult mouse testis stained with hematoxylin and eosin (HE) (magnification bar is 100 120583m) (a) Tissues kept on ice for 0 to 25 hr afterharvesting (b) tissues allowed to remain in the carcass for 0 to 25 hr (c) tissues removed and kept at RT for 0 to 25 hr in Hamrsquos F10 afterharvesting


Figure 2Gerbil testicularmorphology (40x objective) at various time points after tissue harvest from the carcass (magnification bar is 50 120583m)All gerbils were euthanized at once and tissues were harvested from the carcass at 05 hr interval from 0 to 25 hr HE staining demonstratedthe retention of histological features at every time point

Table 1 Percent progressivemotility (plusmnSD) ofmouse cauda epididy-mal sperm after exposure of epididymis to various conditions

Time aftereuthanasia In carcass On icelowast

lowastAt roomtemperature

0 h 23 plusmn 18 25 plusmn 26 26 plusmn 1905 h 28 plusmn 13 22 plusmn 13 27 plusmn 1210 h 31 plusmn 3 32 plusmn 19 31 plusmn 915 h 27 plusmn 6 47 plusmn 9 30 plusmn 1120 h 26 plusmn 7 18 plusmn 9 28 plusmn 1425 h 26 plusmn 9 23 plusmn 12 25 plusmn 16lowastTissue was submerged in Hamrsquos F-10 medium in a 15mL tissue culture tubewhich was placed on ice or at room temperature Values are mean plusmn standarddeviation (119899 = 2mice at each time point)There was no significant differencein motility between the three testing conditions at each time point

statistical analysis cannot be done Given the variation inmotilitywith time the data suggest thatmotilitywas relativelystable at all time points except with perhaps a drop at 25 hr

313 Female Mouse The ovaries (Figure 3(a)) and uteri(Figure 3(b)) harvested from female mice up to 3 hr aftereuthanasia showed excellent histological properties devoid ofapparent tissue degradation

314 FemaleGerbil Gerbil ovaries (Figure 3(c)) harvested upto 1 hr after euthanasia showed normal healthy follicles and

Table 2 Percent total motility (plusmnSD) of mouse cauda epididymalsperm after exposure of epididymis to various posteuthanasiaconditions



0 h 48 plusmn 18 49 plusmn 29 45 plusmn 2105 h 51 plusmn 12 47 plusmn 11 50 plusmn 1510 h 53 plusmn 6 60 plusmn 21 40 plusmn 915 h 57 plusmn 12 67 plusmn 16 54 plusmn 1120 h 51 plusmn 14 46 plusmn 14 43 plusmn 1425 h 50 plusmn 17 49 plusmn 18 52 plusmn 12lowastTissue was submerged in Hamrsquos F-10 medium in a 15mL culture tube whichwas placed on ice or at room temperature as indicated Values are mean plusmnstandard deviation (119899 = 2mice at each time point) There was no significantdifference inmotility between the three testing conditions at each time point

Table 3 Percent total motility of gerbil cauda epididymal spermrecovered from the epididymis after storage in the carcass at RT forthe times indicated (119899 = 1 at each time point)

Time aftereuthanasia

Total motility()

Progressivemotility ()

0 h 983 85005 h 824 58010 h 826 61015 h 952 86220 h 879 73225 h 756 475Since we used one animal per time point standard deviation could not bedetermined


(a)

(b)

(c)

(d)

(e)Figure 3 Mouse and gerbil ovarian and uterine horn histology HE staining was used to evaluate quality of oocyte and follicles Rowsrepresent HE staining of (a) mouse ovary up to 3 hr after euthanasia (4x objective magnification bar is 500120583m) (b) mouse uteri up to3 hr after euthanasia (10x objective magnification bar is 500120583m) (c) gerbil ovary up to 15 hr after euthanasia (4x objective magnificationbar is 500 120583m) (d) significantly high number of vacuoles are indicated in the yellow circles in gerbil ovaries from 15 h after euthanasia (40xobjective magnification bar is 100 120583m) (e) gerbil uteri up to 25 hr after euthanasia (10x objective magnification bar is 500 120583m)

healthy oocytes devoid of signs of tissue degradation how-ever at 15 hr after euthanasia high numbers of unhealthy fol-licles and shrunken oocytes were seen suggesting tissue dete-rioration due to the delay in fixation process after euthanasiaSignificantly high numbers of ldquovacuoles-likerdquo structures werealso seen at 15 hr after euthanasia (Figure 3(d)) which isindicative of tissue degradation (indicated by the yellow cir-cles) Control (0min) gerbil ovaries had negligible ldquovacuolesrdquo(indicated by two black arrows) The ovary harvested after

15 hr did not sustain the histological processing as they weretoo fragile and degraded implying that gerbil ovaries weremore sensitive and should be harvested within 1 hr of euth-anasia Uterine histology indicated tissues were intact andcomparable to control at all tested time points (Figure 3(e))

32 Effect of Different Postfixation Procedures on Quality ofTesticular Histology Testicular and epididymis morphology


60x

(a)

60x

(b) (c)

(d)

Figure 4 Morphological analysis of mouse testis after different postfixative manipulations Sections of adult mouse testis were stained withHE (all at 60x magnification bar is 40 120583m) (a) Control (b) slow rehydration-dehydration stepwise replacement of ETOH-PBS-ETOH (c)slow Re-ETOH only stepwise replacement (d) rapid ETOH-PBS-ETOH transition Sertoli cell (Se) spermatogonia (Sp) spermatocytes (Sc)spermatids (Sd) and Leydig cell (L) Black arrowsmdashabnormal open spaces in seminiferous epithelium yellow arrowsmdashabnormal wavy andthinner basement membrane

was evaluated for histological changes after different post-fixation processing (Figures 4 and 5) In control testiculartissue (Figure 4(a)) all types of spermatogenic cells sper-matogonia (Sp) Sertoli cells (Se) spermatocytes (Sc) andspermatids (Sd) were evident Lymphatic spaces betweenseminiferous tubules and adjacent to Leydig cells (L) clustersare clearly defined After the slow rehydration-dehydrationstepwise replacement with ETOH testis tubules appearednormal with histological architecture similar to the controlgroup (Figure 4(b)) Although all types of spermatogenic cellwere identified after slow rehydration-dehydration stepwisereplacement spermatogonia and Sertoli cell nuclei weremoredifficult to distinguish compared to control The quality oftesticular histology observed after slow Re-ETOH only (Fig-ure 4(c)) was similar to that observed in the slow rehydration-dehydration stepwise replacement group However openspaces (black arrows) were observed within portions ofseminiferous tubules in the slow Re-ETOH only group andthe seminiferous tubule basement membrane in this groupoften appeared wavy and thinner (yellow arrows) comparedto the control (Figure 4(a))The histologic quality was poor inthe group treated by single step (rapid transition) change ofsolution (Figure 4(d)) as evidenced by a diffuse appearance ofthe tissue (not due to focus) and limited clarity of nucleardetails in spermatogonia and Sertoli cells In addition theseminiferous tubule basement membrane was occasionallyindistinct and some spermatid artifactual loss of residualbodies is also observed

We found epididymal tissue to be sensitive to dehy-drationrehydration shock (Figure 5) Slow rehydration-dehydration ETOHreplacement had no visible negative effect

on the quality of epididymal morphology (Figure 5(b)) how-ever histological examination revealed differences in epi-didymal morphology after slow Re-ETOH only (Figure 5(c))and rapid transition procedures (Figure 5(d)) compared tocontrol (Figure 5(a)) The differences included alterations inthe thickness of columnar epithelium and basal and principalcells are not very sharp The slow rehydration-dehydrationcaused less destruction of testicular and epididymal tissuethan the rapid single-step changes of solution and has anoverall better morphological detail preservation compared tothe rapid ETOH-PBS solution change

33 Total RNA and Protein Evaluation after TissuePreservation in RNAlater under Long-Term Storage

331 RNA Stability We compared RNA integrity afterpreservation testicular tissue in TRIzol reagent and RNAlater(Figure 6(a)) Total RNA integrity analysis demonstrated highquality RNA after storing tissue in RNAlater for 1-2 wk at RTor 4∘C compared to TRIzol preservation Distinct 28S and 18Sribosomal RNA and absence of degraded RNAwere observedon the gel RT-PCR analysis of RNAwith primers forGAPDHand IL-1120572 indicated high quality of expected PCR products inall analyzed samples (Figure 6(b)) Integrity analysis of totalRNA isolated from STS-131 ground controls (G10 G11 andG12) and flight (F10 F11 and F12) mouse uteri after 30 to40min after euthanasia demonstrate high RNA quality inthese samples (Figure 6(c)) We found no differences in RNAquality between all analyzed uteri and ovaries samples placedinto RNAlater from 15min to 40min after euthanasia


40x

(a)

40x

(b) (c)

(d)

Figure 5 Morphological analysis of mouse epididymis after different postfixative modifications Sections of adult mouse epididymis werestained with HE (all at 40x magnification bar is 50 120583m) (a) Control (b) slow rehydration-dehydration stepwise replacement of ETOH-PBS-ETOH (c) slow Re-ETOH only replacement (d) rapid ETOH-PBS-ETOH transition Epithelium (E) sperm (S) basal cell (B) and principalcell (P)

332 Long-Term Storage for RNA Analysis Using ovary anduteri harvested from STS 135 mice we also determined theeffect of long-term (10 months) preservation in RNAlater onRNA and protein quality (Figure 7) This time frame waschosen to mimic a possible storage scenario that could occuron the ISS Ovaries and uterus from STS-135 ground controlsstabilized in RNAlater for 10 months showed excellent RNAquality (Figure 7(a)) and yield in range 6ndash8 120583g

333 Protein Stability Although not commonly done tissuestabilized in RNAlater can be used for subsequent proteinextraction Protein obtained from samples stored inRNAlateris suitable for western blotting or 2D gel electrophoresis butnot for applications that require native protein (AmbionGuideline for RNAlater) We optimized the protocol for pro-tein extraction from ovaries preserved in RNAlater for onewk at RT or two wks at 4∘C by using RIPA buffer or ProteoJetLysis reagent (LR) Western analysis of 120573-actin integritydemonstrated that one wk ambient storage of testicular tissuein RNAlater did not affect actin integrity as evidenced by theabsence of proteolytic fragments and consistent signal inten-sity in replicate samples (Figure 6(d))

334 Long-Term Storage for Protein Analysis Protein fromuteri stabilized in RNAlater for 10 months after STS-135mission was extracted with RIPA buffer Immunoblot forER120572 and 120573-actin verified excellent expression levels of bothproteins with no evidence of proteolytic degradation of bothproteins (Figure 7(b)) Quantitative densitometry analysis ofwestern blots indicated reduced levels ER120572 in mostly flight

animals These results demonstrate that RNAlater is aneffective sample collection and stabilization reagent for pro-tecting both RNA and protein under long-term conditionscompatible for the ISS

4 Discussion

We report here a logical method to determine the optimaltime window of tissue harvest and fixation after euthanasiafor use in multi-investigator tissue harvest programs that iscompatible with processing of tissue sample obtained fromspace flight animals We also demonstrate here a long-termstorage regimen for animal tissues compatible with recoveryof high quality RNA and protein under conditions similar tothat on the ISS when there may be many months betweensample collection and return to Earth The procedures setforth also include methods for tissue harvest at the site ofreturn and for safe shipment to external laboratories forfurther processing for histopathology and recovery of proteinand RNA Several fixationpreservation studies have beencarried out for plant samples [1 4 5] One European SpaceAgency report included a brief discussion on fixation ofmammalian cells in tissue culture for microscopy [6] Freidinet al [7] have demonstrated significant alterations on geneexpression in lung carcinoma tissues collected about 30minutes after harvest Durrenberger et al [8] reported thatin human brain samples collected from several brain banksantemortem events appeared to negatively affect the RNAquality but postmortem delays caused no significant dete-rioration This observation supported earlier report that


Ladd

er

1 2 3

(s)

70

65

60

55

50

45

40

35

30

25

20

4

120573-Actin

RIPA-R RIPA-4∘C LR-RT LR-4∘C

IL-1120572 GAPDH1 2 3 4 1 2 3 4

Ladd

er

G10

ut

G11

ut

G12

ut

F10

ut

F11

ut

F12

ut

4000

2000

1000

500

200

25

(nt)

(a)

(b) (d)

(c)

Figure 6 Effect of different extraction and storage methods on RNA and protein quality in ovaries and testes extracted andor stabilized inTRIzol or RNAlater (a) Total RNA integrity analysis Total RNA was isolated from mouse testis and stabilized in TRIzol reagent (RNA yieldand quality control (lane 1) RNAlater for 2wks at 4∘C (lane 2) RNAlater for 1 wk at room temperature and 1 wk at 4∘C (lane 3) RNAlater for2 wks at RT (lane 4) (b) Agarose gel electrophoresis of PCR products with primers for IL-1120572 and GAPDH For RT PCR total RNA was usedafter stabilization in TRIzol reagent (lane 1) RNAlater for 2wks at 4∘C (lane 2) RNAlater for 1 wk at RT and 1wk at 4∘C (lane 3) RNAlater for2 wks at RT (lane 4) (c) Total RNA integrity analysis STS-131 ground (G10 G11 and G12) and flight (F10 F11 and F12) uteri fixed in RNAlaterafter 30ndash40min after euthanasia (d) Comparison of buffers to remove RNAlater for subsequent western analysis of 120573-actin integrity inovaries Mouse ovaries were stored at RNAlater for 1 wk at RT or 2wk at 4∘C tissues were homogenized in RIPA lysis buffer (lanes 1 2) orProteoJET Lysis reagent (LR) (lanes 3 4) Total cell lysates were prepared and subjected to SDS-PAGE Western for 120573-actin is presented

postmortem delay had negligible effect on RNA quality [9]Human stomach has been described as the tissue showingthe earliest sign of postmortem [10] Presnell and Cinadescribed stomach and pancreas as the earliest human tissuesto deteriorate following death [11] The significance of quickprocessing of histopathological specimen in a clinical settinghas been identified by Rohr et al [12] However we havenot come across similar studies for animal tissues used inbiomedical research Prior to our study reported here therewas a significant knowledge gap in the literature for methodsto process animal tissues compatible for multi-investigatorBiospecimen Sharing Programs for space flight logistical sce-narios For our flight studies using male and female mice onthree different space shuttle flights and the BIONM1 flight itwas critical to determine the optimum conditions of tissueharvest and processing for tissues of our interest namelytestis epididymis ovary and uteri

Space flight studies usually comprise remotely dispersedmulti-investigator collaborationsThus there is a need to ship

tissue samples from the site of collection at the return-to-Earth laboratory facility for initial tissue harvest to the site offinal processing and detailed data collection With respect tocollection of RNA samples TRIzol reagent gives excellentRNA integrity however it is a phenol-based solution andtissue preserved in TRIzol cannot be shipped internationallydue to airline safety restrictions Lyophilization of fresh tissuespecimen has been shown to preserve RNA and protein qual-ity and levels by Wu et al [13] Though lyophilization makesshipping easier especially across international borders itis not a viable option for preservation of highest qualityhistological analysis To obtain optimum RNA and proteinquality our results demonstrate that mouse testicular tissuecan be submerged in RNAlater and stored successfully foranalysis at a later time point at least 10 months at minus80∘C Itshould be noted that 10 months represents a minimum limitand longer storage intervals would still need to be directlyassessed Given the current estimate for ISS SpaceX Dragonflights at approximately 3-month intervals this would span


(nt)La

dder

G16

OV

G18

OV

G20

OV

G22

OV

G24

OV

G26

OV

G16

utG18

ut

G20

ut

G22

ut

G24

ut

G26

ut

L 1 2 3 4 5 6 7 8 9 10 11 12

4000

20001000

500200

25

(a)

16

14

12

10

08

06

04

02

00

G16

G18

G20

G22

G24

G26

G28

G32

G34

G40

G42

G44

F46

F50

F52

F54

F56

F58

F60

F62

F64

F66

F68

F70

Ground control group Flight group

Rela

tive E

R120572120573

-act

in p

rote

in ex

pres

sion

G1618 20 22 24 26 G28 32 38 40 42 44 F46 F6050 52 54 56 58 62 64 66 68 70

ER120572

120573-Actin

(b)

Figure 7 RNA and protein quality of STS-135 uteri and ovaries stabilized in RNAlater for 10 months at minus80∘C (a) STS-135 ground control(G16ndashG26) ovarian and uterine RNA integrity analysis Total RNA was extracted and examined for RNA quality (b) Western blot analysis ofER120572 and actin in STS-135 mouse uterus Total cell lysates were prepared and subjected to SDS-PAGE (15120583glane) Western blot analysis wasperformed using the corresponding antibodies to check expression levels of the proteins Representative Immunoblot (top) and its graphicalpresentation (bottom) Densitometric intensities of specific protein bands were digitally obtained and normalized to 120573-actin

three opportunities for sample return after an experimentis terminated and ensure maintenance of RNA and proteinsample quality It is known that in order to isolate high qualityRNA and protein from mammalian tissue the tissue mustbe processed directly after harvest We determined for ourtissues of interest that excellent RNA stability was achieved ifthe tissue samples were placed into RNAlater up to 40minafter euthanasia RNAlater is a popular reagent that inac-tivates all cellular enzymes including RNAses thus RNAexpression profiles can be preserved in situations whenimmediate RNA isolation is not feasible The tissue can bestored in RNAlater for a long time without nucleic acid deg-radation RNAlater has been used by investigators for col-lection of human tissue [14] and used in RNA expressionmicroarrays [15] Our results indicated that RNAlater enableslong-term tissue preservation for RNA and protein extractioncompatible with delayed sample return from flight Thisprocess should be evaluated for use on other tissues to max-imize optimal histology and gene transcription data collec-tion in the primary flight experiments as well as the Biospec-imen Sharing Programs (BSP) investigators Freidin et al [7]have confirmed the significance of RNAlater as a medium topreserve gene expression of lung tissues Tissue processingmethods should be standardized for best storage and analysisof harvested tissues Our methods for tissue fixation long-term storage and recovery of protein and RNA are compati-ble for planned in-flight tissue harvest on ISS

With regard to obtaining the best possible tissue fixationfor histology collected under a multi-investigator Biospec-imen Sharing Program obtaining many tissues of interest

to the participating investigators has to be considered in anintegrated way to accommodate the scientific requirementsof the overarching flight project as well as the natural degra-dation process that tissues undergo as soon as euthanasia hasoccurred Depending on the tissue and species differentwindows of time from euthanasia to fixationmay exist withinwhich histological architecture (as well as RNA and proteinintegrity) is stable In the study design reported here we havedetermined the window of time and temperature for optimalpostharvest maintenance of male and female tissue quality(testis epididymis ovary and uterine horn) and sperm via-bility in mice and gerbils These harvest protocols provide alogical method for integrating the tissue flow logistics forpostflight animals for any project involving multiple inves-tigators Protocols compatible with investigators who requiremore rapid tissue retrieval can be identified and prioritized toensure data preservation We determined that mouse testeswere able to retain excellent histological details when pro-cessed up to 3 hr after euthanasia Sperm motility showedgradual decline with time The authors would reiterate asmentioned in the Results section that sperm motility is ahighly sensitive and variable parameter It is normal to seemajor differences inmotility of sperm obtained fromnot onlyone mouse to another but also within samples obtained fromthe same mouse or gerbil Nonetheless even within the spanof variability seen our results indicate that in case of mousethere was no major difference in sperm motility between anyof the time points whereas in case of gerbil we saw sug-gestions of a slight drop at 25 hr after harvest Future studiesfor flight will require analysis of larger 119899rsquos during definition


phase if gerbils will be used Of the tissues studied hereMongolian gerbil ovaries appear most sensitive to delay inprocessing and require more rapid posteuthanasia process-ing than mouse ovaries Determining optimum conditionsfor tissue handling after harvest is very crucial and can help inmaximizing tissue retrieval form animal models therebymaximizing data output Based on the time-sensitivity inves-tigators may be able to plan the sequence in which the tissuesare harvested starting with the most sensitive tissue to leastsensitive

Finally with regard to the requirement to ship tis-sues fixed for histopathology airline and ground transportproviders (especially international carriers) have specificsafety regulations that prohibit shipment of samples contain-ing many widely used fixatives and preservatives of tissuehistologic integrity Science requirements may present chal-lenges in using alternative fixatives In this regard Bouinrsquosfixative (which contains picric acid) is the best fixative for thetestis if the tissue is to be embedded in paraffin [16] Howeverin space flight experiments safety regulations prevent its useon flight platforms and its presence in tissue samples beingshipped Thus we optimized protocols for fixative removaland storage of tissues in PBS for safe shipping as well asreconstitution protocols for storing tissues in 70 ethanol toretain excellent histology

5 Conclusion

Optimal time frames for harvesting testis epididymis ovaryand uteri without compromising the histological qualitysperm motility and RNA quality have been determinedDifferences in tissue-specific optimal fixation time windowswere noted between mice and gerbils We provide here newmethods for (1) fixative removal and transfer of tissues intoaqueousmedia for safe shipping and (2) reconstitution proto-cols into 70 ethanol that retains excellent histologyWe con-clude that stepwise replacement of ETOH-PBS-ETOHcausedless degradation of histological quality of tissue than a single-step change of solution Our results demonstrate that maleand female mouse reproductive tissues stored in RNAlatersolution were stable and gave high quality RNA and proteinafter 10months of storage atminus80∘CThuswe have determinedmethods for postharvest tissue processing to replace Bouinrsquoswith 70 ethanol for safe shipping across USA and alsoreplace 70 ethanol with PBS to enable shipping of tissuesacross international borders These protocols will facilitateintegration of tissue harvest logistics in multi-investigatorBiospecimen Sharing Programs for optimal tissue histologyand retention of high quality RNA and protein recovery fromanimal tissues on long-term space flight experiments on ISSas well as other flight platforms

Abbreviations

TBS-T Tris buffered saline with 01 Tween 20BSP Biospecimen Sharing ProgramETOH Ethyl alcoholSTS Space Transport System

PBS Phosphate buffered salineISS International Space StationCASA Computer assisted sperm analysisWk WeekRT Room temperatureIACUC Institutional Animal Care and Use CommitteeKSC Kennedy Space CenterRNA Ribonucleic acidCO2 Carbon dioxide

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study is supported by NASA Grant NNX09AP04G toJoseph S TashThe authors wish to acknowledge the excellentsupport for the project from Richard Boyle Paula DumarsVera Vizir Gwo-Shing and Kenny Vassigh from AmesResearch Center NASA and from Stacy Engel AshleighRuggles and Ramona Bobber at Kennedy Space CenterFlorida The authors thank Stanton Fernald of the KUMCImaging Core for assistance in preparation of final figures

References

[1] A-L Paul H G Levine W McLamb et al ldquoPlant molecularbiology in the space station era utilization of KSC fixation tubeswith RNAlaterrdquo Acta Astronautica vol 56 no 6 pp 623ndash6282005

[2] J Luo V Gupta B Kern et al ldquoRole of FYN kinase inspermatogenesis defects characteristic of FYN-null sperm inmicerdquo Biology of Reproduction vol 86 no 1 article 22 2012

[3] J S Tash B Attardi S A Hild R Chakrasali S R Jakkaraj andG I Georg ldquoA novel potent indazole carboxylic acid derivativeblocks spermatogenesis and is contraceptive in rats after a singleoral doserdquo Biology of Reproduction vol 78 no 6 pp 1127ndash11382008

[4] M Braun B Buchen and A Sievers ldquoFixation procedure fortransmission electronmicroscopy ofChara rhizoids under mic-rogravity in a Spacelab (IML-2)rdquo Journal of Biotechnology vol47 no 2-3 pp 245ndash251 1996

[5] V D Kern F D Sack N J White K Anderson W Wells andCMartin ldquoSpaceflight hardware allowing unilateral irradiationand chemical fixation in petri dishesrdquo Advances in SpaceResearch vol 24 no 6 pp 775ndash778 1999

[6] F J Medina A Cogoli C Dournon et al ldquoPreservation ofsamples during space experimentsrdquo in Topical Teams in theLife amp Physical Sciences Towards New Research Applications inSpace pp 200ndash208 European Space Agency 2005

[7] M B Freidin N Bhudia E Lim A G Nicholson W OCookson andM FMoffatt ldquoImpact of collection and storage oflung tumor tissue on whole genome expression profilingrdquoJournal ofMolecular Diagnostics vol 14 no 2 pp 140ndash148 2012

[8] P F Durrenberger S Fernando S N Kashefi et al ldquoEffects ofantemortem and postmortem variables on human brainmRNAquality a brainNet Europe studyrdquo Journal of Neuropathology ampExperimental Neurology vol 69 no 1 pp 70ndash81 2010


[9] J F Ervin E L Heinzen K D Cronin et al ldquoPostmortemdelay has minimal effect on brain RNA integrityrdquo Journal ofNeuropathology and Experimental Neurology vol 66 no 12 pp1093ndash1099 2007

[10] A R Thomas A Practical Guide for Making Post-MortemExaminations BiblioBazaar 2009

[11] S E Presnell and S J Cina ldquoPostmortem changesrdquo MedscapeDrugs amp Diseases 2013 httpemedicinemedscapecomarticle1680032-overview

[12] L R Rohr L J Layfield DWallin andDHardy ldquoA comparisonof routine and rapid microwave tissue processing in a surgicalpathology laboratory quality of histologic sections and advan-tages of microwave processingrdquoThe American Journal of Clini-cal Pathology vol 115 no 5 pp 703ndash708 2001

[13] YWuMWuY Zhang et al ldquoLyophilization is suitable for stor-age and shipment of fresh tissue samples without altering RNAand protein levels stored at room temperaturerdquo Amino Acidsvol 43 no 3 pp 1383ndash1388 2012

[14] S R Florell C M Coffin J A Holden et al ldquoPreservation ofRNA for functional genomic studies a multidisciplinary tumorbank protocolrdquo Modern Pathology vol 14 no 2 pp 116ndash1282001

[15] G L Mutter D Zahrieh C Liu et al ldquoComparison of frozenand RNALater solid tissue storage methods for use in RNAexpressionmicroarraysrdquoBMCGenomics vol 5 article 88 2004

[16] L D Russell R A Ettlin H A Sinha and E D Clegg EdsHistological andHistopathological Evaluation of the Testis CacheRiver Press Clearwater Fla USA 1990

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


storage methods should retain high sample quality underlong-term storage as samplesmay be harvested and stored onthe ISS but may not be returned to Earth for many monthsdepending on ISS to Earth flight frequency and payloadcapacities This issue has been addressed for preserving plantmaterial for gene expression analysis [1] but there is nodata available for animal tissues Our participation in theBSP program involved tissue harvest from female mice atKennedy SpaceCenter Florida USA (KSC) for animals flownfor 12ndash15 days in orbit on three space shuttle flights STS-131STS-133 and STS-135 In addition we harvested tissues frommale mice at the Institute for Biomedical Problems (IMBP)laboratory in Moscow RU for animals flown for 30 daysin orbit on the BION M1 satellite Both the STS and BIONseries of flight experiments involved age- and time-matchedground control groups of animals During the early flightplanning phase of BION there were possibilities that maleor female mice would be flown and that male gerbil tissuesmay also be provided to the BSPThus to be prepared for anyof these possibilities we undertook to determine the optimaltissue harvest windows for all of the species and reproductivetissues that we might be able to obtain Our participationin these multi-investigator specimen sharing efforts coveringfour different primary flight PI experimental designs andharvest logistics necessitated a determination of the windowof time between euthanasia and harvest and preservation ofour tissues of interest that would allow us sufficient flexibilityto obtain the highest possible quality of tissue for histopathol-ogy RNA for gene-transcription analysis and protein forexpression and posttranslational modification analysis Adetermination of these time windows is essential since thetissues that are made available to the BSP investigators areprovided after the primary flight PIrsquos have obtained their tis-sues Knowledge of the optimal windows for all of the tissuesof interest aids in the preparation of targeted tissue harvestflow logistics that can provide each of the BSP teammembersthe highest possible quality tissue respectively Therefore aswe report here we developed a strategy to determine thewindowof time between euthanasia and fixation for retentionof high quality histology for male and female reproductiveorgans We also determined methods for long-term tissuestorage for 10 months that provide for recovery of both highquality protein and RNA These strategies are adaptable andcan be applied to harvest and storage of other time-sensitivelabile tissues from animals and plants Furthermore thesemethods can be used to optimize logistics and data collectionundermulti-investigator tissue harvest and sharing programsoperated by any space agency commercial entity or flightplatform

2 Materials and Methods

21 Animals Approximately 8 wk old male and femaleC57Bl6J (Jackson Lab Bar Harbor ME) and sim10-month oldmale and female Mongolian gerbils (Charles River Wilming-ton MA) were used throughout this study All animal useprotocols were approved by the University of Kansas Insti-tutional Animal Care andUse Committee (IACUC) Animals

were maintained in standard cages with 12 12 h dark lightcycle and standard food and water were provided ad libitumAll animals were euthanized using CO

2asphyxiation fol-

lowed by cervical dislocation prior to tissue harvest

22 Determination of the Limit of Time andTemperature between Euthanasia and Tissue Fixation

221 Male Mice Our protocol consisted of three groups ofsix mice each with one mouse for each time point In groupA all mice were euthanized at one time and the testes andepididymides were harvested and separated and then imme-diately placed in Hamrsquos F-10 medium (Sigma Aldrich StLouis MO) on ice At time intervals of 0 05 10 15 20 and25 hr each testis (one mouse per time point) and epididymis(2 mice per time point) were separated and then transferredfrom Hamrsquos F-10 medium to Bouinrsquos fixative (Sigma AldrichSt Louis MO) at room temperature (RT 21∘C) In group Bthe same procedure was followed except that the testes andepididymides separated were placed in Hamrsquos F-10 mediumat RT until transfer to Bouinrsquos at the same time interval asabove In group C all mice were euthanized at one time thecarcasses were maintained at (RT) and then at time 0 05 1015 20 and 25 hr the testis and epididymis were harvestedfrom the carcasses respectively At each of the time pointsabove groupC testes were placed in Bouinrsquos solution and pro-cessed as per standard histology protocols as detailed belowFor all groups the tissues fixed in Bouinrsquos were processed asdetailed in Section 23 below At the same time point groupC epididymides were processed to obtain cauda sperm toassess sperm motility by computer assisted sperm analysis(CASA) [2] For animals used for collection of cauda epi-didymal sperm formotility analysis two animals were used ateach time point

222 Female Mice Female mice were euthanized (carcassesmaintained at RT as per groupC above) and their ovaries anduteri were harvested at 0 05 10 15 20 25 and 30 hr aftereuthanasia (one animal per time point) At the times indi-cated the tissues were fixed at RT in Bouinrsquos overnight andthen processed for histology as detailed below (Section 23)

223 Mongolian Gerbils The six male gerbils were euth-anized at one time and the carcasses were kept at roomtemperature At 0 05 10 15 20 and 30 hr after euthanasiatestis and epididymis were harvested from the carcasses andseparated respectively (one animal per time point) Similarlyall six female gerbils were euthanized and the carcasses werekept at RT Ovaries and uterine horns were harvested fromeach carcass at the same time interval as the males At thetimes indicated above the tissues were placed in Bouinrsquos fixa-tive at RT and then processed for histology as detailed below(Section 23) Sperm were immediately harvested from caudaepididymis as mentioned above and sperm motility analysiswas carried out using CASA as described above [2]

23 Postharvest Processing of Mouse Testicular Tissues Testesand epididymides from the mature mice were harvested as






3 Results






(a) (b)

(c)

















Total motility()




(a)

(b)

(c)

(d)






60x

(a)

60x

(b) (c)

(d)








40x

(a)

40x

(b) (c)

(d)






4 Discussion



Ladd

er

1 2 3

(s)

70

65

60

55

50

45

40

35

30

25

20

4

120573-Actin


IL-1120572 GAPDH1 2 3 4 1 2 3 4

Ladd

er

G10

ut

G11

ut

G12

ut

F10

ut

F11

ut

F12

ut

4000

2000

1000

500

200

25

(nt)

(a)

(b) (d)

(c)






(nt)La

dder

G16

OV

G18

OV

G20

OV

G22

OV

G24

OV

G26

OV

G16

utG18

ut

G20

ut

G22

ut

G24

ut

G26

ut

L 1 2 3 4 5 6 7 8 9 10 11 12

4000

20001000

500200

25

(a)

16

14

12

10

08

06

04

02

00

G16

G18

G20

G22

G24

G26

G28

G32

G34

G40

G42

G44

F46

F50

F52

F54

F56

F58

F60

F62

F64

F66

F68

F70


Rela

tive E

R120572120573

-act

in p

rote

in ex

pres

sion

G1618 20 22 24 26 G28 32 38 40 42 44 F46 F6050 52 54 56 58 62 64 66 68 70

ER120572

120573-Actin

(b)








5 Conclusion


Abbreviations





Acknowledgments


References

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






3 Results






(a) (b)

(c)

















Total motility()




(a)

(b)

(c)

(d)






60x

(a)

60x

(b) (c)

(d)








40x

(a)

40x

(b) (c)

(d)






4 Discussion



Ladd

er

1 2 3

(s)

70

65

60

55

50

45

40

35

30

25

20

4

120573-Actin


IL-1120572 GAPDH1 2 3 4 1 2 3 4

Ladd

er

G10

ut

G11

ut

G12

ut

F10

ut

F11

ut

F12

ut

4000

2000

1000

500

200

25

(nt)

(a)

(b) (d)

(c)






(nt)La

dder

G16

OV

G18

OV

G20

OV

G22

OV

G24

OV

G26

OV

G16

utG18

ut

G20

ut

G22

ut

G24

ut

G26

ut

L 1 2 3 4 5 6 7 8 9 10 11 12

4000

20001000

500200

25

(a)

16

14

12

10

08

06

04

02

00

G16

G18

G20

G22

G24

G26

G28

G32

G34

G40

G42

G44

F46

F50

F52

F54

F56

F58

F60

F62

F64

F66

F68

F70


Rela

tive E

R120572120573

-act

in p

rote

in ex

pres

sion

G1618 20 22 24 26 G28 32 38 40 42 44 F46 F6050 52 54 56 58 62 64 66 68 70

ER120572

120573-Actin

(b)








5 Conclusion


Abbreviations





Acknowledgments


References

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c)

















Total motility()




(a)

(b)

(c)

(d)






60x

(a)

60x

(b) (c)

(d)








40x

(a)

40x

(b) (c)

(d)






4 Discussion



Ladd

er

1 2 3

(s)

70

65

60

55

50

45

40

35

30

25

20

4

120573-Actin


IL-1120572 GAPDH1 2 3 4 1 2 3 4

Ladd

er

G10

ut

G11

ut

G12

ut

F10

ut

F11

ut

F12

ut

4000

2000

1000

500

200

25

(nt)

(a)

(b) (d)

(c)






(nt)La

dder

G16

OV

G18

OV

G20

OV

G22

OV

G24

OV

G26

OV

G16

utG18

ut

G20

ut

G22

ut

G24

ut

G26

ut

L 1 2 3 4 5 6 7 8 9 10 11 12

4000

20001000

500200

25

(a)

16

14

12

10

08

06

04

02

00

G16

G18

G20

G22

G24

G26

G28

G32

G34

G40

G42

G44

F46

F50

F52

F54

F56

F58

F60

F62

F64

F66

F68

F70


Rela

tive E

R120572120573

-act

in p

rote

in ex

pres

sion

G1618 20 22 24 26 G28 32 38 40 42 44 F46 F6050 52 54 56 58 62 64 66 68 70

ER120572

120573-Actin

(b)








5 Conclusion


Abbreviations





Acknowledgments


References

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Total motility()




(a)

(b)

(c)

(d)






60x

(a)

60x

(b) (c)

(d)








40x

(a)

40x

(b) (c)

(d)






4 Discussion



Ladd

er

1 2 3

(s)

70

65

60

55

50

45

40

35

30

25

20

4

120573-Actin


IL-1120572 GAPDH1 2 3 4 1 2 3 4

Ladd

er

G10

ut

G11

ut

G12

ut

F10

ut

F11

ut

F12

ut

4000

2000

1000

500

200

25

(nt)

(a)

(b) (d)

(c)






(nt)La

dder

G16

OV

G18

OV

G20

OV

G22

OV

G24

OV

G26

OV

G16

utG18

ut

G20

ut

G22

ut

G24

ut

G26

ut

L 1 2 3 4 5 6 7 8 9 10 11 12

4000

20001000

500200

25

(a)

16

14

12

10

08

06

04

02

00

G16

G18

G20

G22

G24

G26

G28

G32

G34

G40

G42

G44

F46

F50

F52

F54

F56

F58

F60

F62

F64

F66

F68

F70


Rela

tive E

R120572120573

-act

in p

rote

in ex

pres

sion

G1618 20 22 24 26 G28 32 38 40 42 44 F46 F6050 52 54 56 58 62 64 66 68 70

ER120572

120573-Actin

(b)








5 Conclusion


Abbreviations





Acknowledgments


References

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

(b)

(c)

(d)






60x

(a)

60x

(b) (c)

(d)








40x

(a)

40x

(b) (c)

(d)






4 Discussion



Ladd

er

1 2 3

(s)

70

65

60

55

50

45

40

35

30

25

20

4

120573-Actin


IL-1120572 GAPDH1 2 3 4 1 2 3 4

Ladd

er

G10

ut

G11

ut

G12

ut

F10

ut

F11

ut

F12

ut

4000

2000

1000

500

200

25

(nt)

(a)

(b) (d)

(c)






(nt)La

dder

G16

OV

G18

OV

G20

OV

G22

OV

G24

OV

G26

OV

G16

utG18

ut

G20

ut

G22

ut

G24

ut

G26

ut

L 1 2 3 4 5 6 7 8 9 10 11 12

4000

20001000

500200

25

(a)

16

14

12

10

08

06

04

02

00

G16

G18

G20

G22

G24

G26

G28

G32

G34

G40

G42

G44

F46

F50

F52

F54

F56

F58

F60

F62

F64

F66

F68

F70


Rela

tive E

R120572120573

-act

in p

rote

in ex

pres

sion

G1618 20 22 24 26 G28 32 38 40 42 44 F46 F6050 52 54 56 58 62 64 66 68 70

ER120572

120573-Actin

(b)








5 Conclusion


Abbreviations





Acknowledgments


References

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


60x

(a)

60x

(b) (c)

(d)








40x

(a)

40x

(b) (c)

(d)






4 Discussion



Ladd

er

1 2 3

(s)

70

65

60

55

50

45

40

35

30

25

20

4

120573-Actin


IL-1120572 GAPDH1 2 3 4 1 2 3 4

Ladd

er

G10

ut

G11

ut

G12

ut

F10

ut

F11

ut

F12

ut

4000

2000

1000

500

200

25

(nt)

(a)

(b) (d)

(c)






(nt)La

dder

G16

OV

G18

OV

G20

OV

G22

OV

G24

OV

G26

OV

G16

utG18

ut

G20

ut

G22

ut

G24

ut

G26

ut

L 1 2 3 4 5 6 7 8 9 10 11 12

4000

20001000

500200

25

(a)

16

14

12

10

08

06

04

02

00

G16

G18

G20

G22

G24

G26

G28

G32

G34

G40

G42

G44

F46

F50

F52

F54

F56

F58

F60

F62

F64

F66

F68

F70


Rela

tive E

R120572120573

-act

in p

rote

in ex

pres

sion

G1618 20 22 24 26 G28 32 38 40 42 44 F46 F6050 52 54 56 58 62 64 66 68 70

ER120572

120573-Actin

(b)








5 Conclusion


Abbreviations





Acknowledgments


References

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


40x

(a)

40x

(b) (c)

(d)






4 Discussion



Ladd

er

1 2 3

(s)

70

65

60

55

50

45

40

35

30

25

20

4

120573-Actin


IL-1120572 GAPDH1 2 3 4 1 2 3 4

Ladd

er

G10

ut

G11

ut

G12

ut

F10

ut

F11

ut

F12

ut

4000

2000

1000

500

200

25

(nt)

(a)

(b) (d)

(c)






(nt)La

dder

G16

OV

G18

OV

G20

OV

G22

OV

G24

OV

G26

OV

G16

utG18

ut

G20

ut

G22

ut

G24

ut

G26

ut

L 1 2 3 4 5 6 7 8 9 10 11 12

4000

20001000

500200

25

(a)

16

14

12

10

08

06

04

02

00

G16

G18

G20

G22

G24

G26

G28

G32

G34

G40

G42

G44

F46

F50

F52

F54

F56

F58

F60

F62

F64

F66

F68

F70


Rela

tive E

R120572120573

-act

in p

rote

in ex

pres

sion

G1618 20 22 24 26 G28 32 38 40 42 44 F46 F6050 52 54 56 58 62 64 66 68 70

ER120572

120573-Actin

(b)








5 Conclusion


Abbreviations





Acknowledgments


References

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Ladd

er

1 2 3

(s)

70

65

60

55

50

45

40

35

30

25

20

4

120573-Actin


IL-1120572 GAPDH1 2 3 4 1 2 3 4

Ladd

er

G10

ut

G11

ut

G12

ut

F10

ut

F11

ut

F12

ut

4000

2000

1000

500

200

25

(nt)

(a)

(b) (d)

(c)






(nt)La

dder

G16

OV

G18

OV

G20

OV

G22

OV

G24

OV

G26

OV

G16

utG18

ut

G20

ut

G22

ut

G24

ut

G26

ut

L 1 2 3 4 5 6 7 8 9 10 11 12

4000

20001000

500200

25

(a)

16

14

12

10

08

06

04

02

00

G16

G18

G20

G22

G24

G26

G28

G32

G34

G40

G42

G44

F46

F50

F52

F54

F56

F58

F60

F62

F64

F66

F68

F70


Rela

tive E

R120572120573

-act

in p

rote

in ex

pres

sion

G1618 20 22 24 26 G28 32 38 40 42 44 F46 F6050 52 54 56 58 62 64 66 68 70

ER120572

120573-Actin

(b)








5 Conclusion


Abbreviations





Acknowledgments


References

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(nt)La

dder

G16

OV

G18

OV

G20

OV

G22

OV

G24

OV

G26

OV

G16

utG18

ut

G20

ut

G22

ut

G24

ut

G26

ut

L 1 2 3 4 5 6 7 8 9 10 11 12

4000

20001000

500200

25

(a)

16

14

12

10

08

06

04

02

00

G16

G18

G20

G22

G24

G26

G28

G32

G34

G40

G42

G44

F46

F50

F52

F54

F56

F58

F60

F62

F64

F66

F68

F70


Rela

tive E

R120572120573

-act

in p

rote

in ex

pres

sion

G1618 20 22 24 26 G28 32 38 40 42 44 F46 F6050 52 54 56 58 62 64 66 68 70

ER120572

120573-Actin

(b)








5 Conclusion


Abbreviations





Acknowledgments


References

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




5 Conclusion


Abbreviations





Acknowledgments


References

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article A Tissue Retrieval and Postharvest