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Ukuchuma kweentaka kuxhomekeke ekukwazini kwazo ukugcina isidoda esaneleyo sexesha elide kwii-tubules zokugcina amadlozi (SST).Indlela echanekileyo apho i-spermatozoa ingena ngayo, ihlale, kwaye ishiye i-SST ihlala iphikisana.Amadlozi eenkukhu ze-sharkasi abonise ukuthambekela okuphezulu kwi-agglutination, ukwenza i-filamentous bundles ehambayo equlethe iiseli ezininzi.Ngenxa yobunzima bokuqwalasela i-motility kunye nokuziphatha kwe-spermatozoa kwi-opaque fallopian tube, sasebenzisa isixhobo se-microfluidic kunye ne-microchannel cross-section efana ne-spermatozoa ukufunda i-spermatozoa agglutination kunye ne-motility.Olu phononongo luxoxa ngendlela iinkunzi zesidoda ezenza ngayo, indlela ezihamba ngayo, kunye nendima yazo enokubakho ekwandiseni ukuhlala kwesidoda kwi-SST.Siphande isantya samadlozi kunye nokuziphatha kwerheological xa ukuqukuqela kolwelo kwenziwa ngaphakathi kwitshaneli yemicrofluidic ngoxinzelelo lwe-hydrostatic (isantya sokuhamba = 33 µm/s).I-spermatozoa ivame ukudada ngokubhekiselele kwi-current (i-rheology ephilileyo) kunye nesantya se-spermatozoon bundle siyancipha kakhulu xa kuthelekiswa ne-spermatozoa enye.Iinyanda zesidoda ziye zabonwa ukuba zihamba kwi-spiral kwaye zonyuka ngobude kunye nobukhulu njengoko kufunwa isidoda esinye. Iinyanda zamadlozi ziye zajongwa zisiza kwaye zibambelela emacaleni emisele yemicrofluidic ukunqanda ukutshayelwa ngesantya sokuqukuqela kolwelo> 33 µm/s. Iinyanda zamadlozi ziye zajongwa zisiza kwaye zibambelela emacaleni emisele yemicrofluidic ukunqanda ukutshayelwa ngesantya sokuqukuqela kolwelo> 33 µm/s. Было замечено, что пучки сперматозоидов приближаются и прилипают к боковым стенкам микрофлюидных каналов, чтобы избениять 3 смета мкм / p. Iinyanda zesidoda ziye zaqatshelwa ukuba zisondele kwaye zibambelele kwiindonga ezisecaleni zemijelo ye-microfluidic ukuphepha ukutshayelwa kumazinga okuhamba kolwelo>33 µm/s.观察到精子束接近并粘附在微流体通道的侧壁上，以避免被流体流速> 33 µm/s 扫过.33 µm/s 扫过. Было замечено, что пучки сперматозоидов приближаются и прилипают к боковым стенкам микрожидкостного канала, чтобы избежать 3 сместо 3 км/с. Iinyanda zesperm ziye zaqatshelwa ukuba zisondele kwaye zibambelele kwiindonga ezisecaleni ze-microfluidic channel ukunqanda ukutshayelwa kukuhamba kolwelo kwi>33 µm/s.Ukuskena kunye nokusasaza i-electron microscopy ibonise ukuba iinyanda zamadlozi zixhaswa zizinto ezininzi ezixineneyo.Idatha efunyenweyo ibonisa ukuhamba okuyingqayizivele kwe-Sharkazi chicken spermatozoa, kunye nokukwazi kwe-spermatozoa ukuba i-agglutinate kwaye yenze i-bundles mobile, enegalelo ekuqondeni kakuhle ukugcinwa kwexesha elide le-spermatozoa kwi-SMT.
Ukuze kuqhamiseke ebantwini nakwizilwanyana ezininzi, amadlozi namaqanda afanele afike ngexesha elifanelekileyo kwindawo yokuqhama.Ngoko ke, ukukhwelana kufuneka kwenzeke ngaphambi okanye ngexesha le-ovulation.Kwelinye icala, ezinye izilwanyana ezanyisayo, ezinjengezinja, kwakunye nezilwanyana ezingezozanyisayo, ezinjengezinambuzane, intlanzi, izilwanyana ezirhubuluzayo neentaka, zigcina amadlozi kumalungu azo okuzala kangangexesha elide de amaqanda azo alungele ukuchumisa ( asynchronous fertilization 1 ).Iintaka ziyakwazi ukugcina ukusebenza kwe-spermatozoa ekwazi ukuchumisa amaqanda kwiiveki ezi-2-102.
Olu luphawu olukhethekileyo olwahlula iintaka kwezinye izilwanyana, njengoko libonelela ngamathuba aphezulu okuchumisa emva kokufakwa kwelinye iiveki ezimbalwa ngaphandle kokudibana kunye kunye ne-ovulation.Elona lungu logcino lwesidoda, elibizwa ngokuba yi-sperm storage tubule (SST), likwimigoqo yangaphakathi ye-mucosal kwindawo ekudityaniswa kuyo isibeleko.Ukuza kuthi ga ngoku, iindlela zokungena kwesidoda, ukuhlala, kunye nokuphuma kwibhanki yesidoda aziqondi ngokupheleleyo.Ngokusekelwe kwizifundo zangaphambili, ezininzi iingcamango zibekwe phambili, kodwa akukho nanye kuzo eqinisekisiweyo.
I-Forman4 icinge ukuba i-spermatozoa igcina indawo yayo yokuhlala kwi-SST cavity ngokusebenzisa intshukumo eqhubekayo ye-oscillatory ngokuchasene nesalathiso sokuhamba kwamanzi kwiitshaneli zeprotheni ezibekwe kwiiseli ze-SST epithelial (rheology).I-ATP iphelile ngenxa yomsebenzi oqhubekayo we-flagellar ofunekayo ukugcina i-sperm kwi-lumen ye-SST kunye ne-motility ekugqibeleni iyancipha de i-sperm iqhutywe ngaphandle kwe-sperm bank ngokuhamba kolwelo kwaye iqalise uhambo olutsha ukuhla kwi-fallopian tube enyukayo ukuze ichumise isidoda.Iqanda (Forman4).Lo mzekelo wokugcinwa kwesidoda uxhaswa ngokufunyanwa yi-immunocytochemistry ye-aquaporins 2, 3 kunye ne-9 ekhoyo kwiiseli ze-epithelial ze-SST.Ukuza kuthi ga ngoku, izifundo malunga ne-rheology yesidoda senkukhu kunye nendima yayo kugcino lwe-SST, ukhetho lwedlozi lobufazi, kunye nokhuphiswano lwesidoda alukho.Kwiinkukhu, isidoda singena kwilungu lobufazi emva kokukhwelana kwendalo, kodwa ngaphezu kwe-80% ye-spermatozoa ikhutshwa kwilungu lobufazi kungekudala emva kokudibana.Oku kubonisa ukuba ikuku yeyona ndawo iphambili yokukhetha amadlozi kwiintaka.Ukongezelela, kuye kwaxelwa ukuba ngaphantsi kwe-1% ye-spermatozoa ekhuliswe kwi-vagin iphelela kwi-SSTs2.Ekufakeni amantshontsho okwenziwa kwilungu lobufazi, inani le-spermatozoa elifikelela kwi-SST lidla ngokunyuka kwiiyure ezingama-24 emva kokufakwa.Ukuza kuthi ga ngoku, indlela yokukhethwa kwesidoda ngexesha lale nkqubo ayicacanga, kwaye i-sperm motility inokudlala indima ebalulekileyo ekufumaneni isidoda se-SST.Ngenxa yeendonga ezishinyeneyo nezingacacanga zemibhobho yefallopian, kunzima ukujonga ngokuthe ngqo ukuhamba kwedlozi kwityhubhu yeentaka.Ngoko ke, asinalo ulwazi olusisiseko malunga nendlela i-spermatozoa eguqulela ngayo kwi-SST emva kokuchumisa.
I-Rheology isandula ukuqatshelwa njengeyona nto ibalulekileyo elawula ukuthuthwa kwesidoda kwi-mammalian genitalia.Ngokusekwe kwisakhono se-motile spermatozoa ukufuduka ngokuchaseneyo, u-Zaferani et al8 wasebenzisa inkqubo ye-corra microfluidic ukwahlula ngokuzimeleyo i-motile spermatozoa kwiisampulu zesidoda esibhaliweyo.Olu hlobo lokuhlela amadlozi lubalulekile kunyango lobudlolo lwezonyango kunye nophando lwezonyango, kwaye lukhethwa kuneendlela zesintu ezithatha ixesha kunye nomsebenzi onzima kwaye zinokubeka esichengeni ukumila kwedlozi kunye nokuthembeka kwesakhiwo.Nangona kunjalo, ukuza kuthi ga ngoku, akukho zifundo ezenziweyo malunga nefuthe leemfihlo ezivela kwizitho zangasese zeenkukhu kwi-sperm motility.
Nokuba yeyiphi na indlela egcina amadlozi agcinwe kwi-SST, abaphandi abaninzi baye baqaphela ukuba i-spermatozoa ehlala i-agglutinate intloko-to-intloko kwi-SST yeenkukhu 9, 10, izagwityi 2, kunye ne-turkeys 11 ukwenza i-agglutinated sperm bundles.Ababhali bacetyisa ukuba kukho unxibelelwano phakathi kwesi sigqubuthelo kunye nokugcinwa kwexesha elide le-spermatozoa kwi-SST.
ITingari kunye neLake12 zanika ingxelo yonxulumano olomeleleyo phakathi kwespermatozoa kwidlala lenkukhu elamkela isidoda kwaye yathandabuza ukuba ingaba i-avian spermatozoa agglutinate ngendlela efanayo neye mammalian spermatozoa.Bakholelwa ukuba unxibelelwano olunzulu phakathi kwesidoda kwi-vas deferens lusenokuba ngenxa yoxinzelelo olubangelwa kubukho benani elikhulu lamadlozi kwindawo encinci.
Xa uvavanya isimilo se-spermatozoa kwizilayidi zeglasi ezijingayo, iimpawu ezidlulayo ze-agglutination zinokubonwa, ngakumbi emaphethelweni amathontsi amadlozi.Nangona kunjalo, i-agglutination yayihlala iphazamiseka yisenzo sokujikeleza esihambelana nokunyakaza okuqhubekayo, okuchaza ubume bexeshana le nto.Abaphandi baye baqaphela ukuba xa i-diluent yongezwa kwi-semen, i-cell aggregates emide "efana nomsonto" ibonakala.
Iinzame zakwangoko zokulinganisa i-spermatozoon zenziwe ngokususa ucingo olucekethekileyo kwithontsi elijingayo, nto leyo ekhokelele kwi-vesicle emide efana nedlozi ephuma kwithontsi lamadlozi.I-spermatozoa ngokukhawuleza ifakwe ngendlela ehambelanayo ngaphakathi kwe-vesicle, kodwa yonke iyunithi yanyamalala ngokukhawuleza ngenxa yokukhawulelana kwe-3D.Ngoko ke, ukufunda i-spermatozoa agglutination, kuyimfuneko ukujonga ukuhamba kunye nokuziphatha kwe-spermatozoa ngokuthe ngqo kwii-tubules zokugcina zesidoda, okunzima ukufezekisa.Ngoko ke, kuyimfuneko ukuphuhlisa isixhobo esilinganisa i-spermatozoa ukuxhasa izifundo ze-sperm motility kunye nokuziphatha kwe-agglutination.UBrillard et al13 unike ingxelo yokuba umndilili wobude beetyhubhu zokugcina amadlozi kumantshontsho amadala yi-400–600 µm, kodwa ezinye ii-SSTs zinokude zibe yi-2000 µm.I-Mero kunye ne-Ogasawara14 yahlula i-seminiferous glands kwi-tubules yokugcina i-sperm eyandisiweyo kunye ne-non-eyandisiweyo, zombini zifana ngobude (~ 500 µm) kunye nobubanzi bentamo (~ 38 µm), kodwa i-lumen ye-lumen ye-tubules yi-56.6 kunye ne-56.6 µm.., ngokulandelelanayo 11.2 μm, ngokulandelanayo.Kuphononongo lwangoku, sisebenzise isixhobo se-microfluidic esinobungakanani betshaneli ye-200 µm × 20 µm (W × H), icandelo layo elinqamlezayo lisondele kakhulu kwi-SST ekhulisiwe.Ukongeza, siye savavanya i-sperm motility kunye nokuziphatha kwe-agglutination kulwelo oluqukuqelayo, oluhambelanayo nengcamango kaForeman yokuba ulwelo oluveliswa ziiseli ze-SST epithelial zigcina isidoda kwilumen kwicala eliphikisanayo (rheological).
Injongo yolu phononongo yayikukunqoba iingxaki zokujonga ukuhamba kwe-spermatozoa kwityhubhu ye-fallopian kunye nokuphepha ubunzima bokufunda i-rheology kunye nokuziphatha kwe-spermatozoa kwindawo eguquguqukayo.Kwasetyenziswa isixhobo esine-microfluidic esenza uxinzelelo lwe-hydrostatic ukulinganisa ukuhamba kwedlozi kumalungu obuni enkukhu.
Xa ithontsi lesampulu yamadlozi ehlanjululweyo (1:40) yalayishwa kwisixhobo se-microchannel, iindidi ezimbini ze-sperm motility zazinokuchongwa (i-sperm eyedwa kunye nesidoda esibophelelekileyo).Ukongeza, i-spermatozoa ithande ukuqubha ngokuchasene nangoku (i-rheology enhle; ividiyo ye-1, i-2). Nangona i-sperm bundles yayinesantya esisezantsi kuneso se-sperm eyedwa (p <0.001), yandisa ipesenti yesidoda ebonisa i-rheotaxis enhle (p <0.001; Itheyibhile 2). Nangona i-sperm bundles yayinesantya esisezantsi kuneso se-sperm eyedwa (p <0.001), yandisa ipesenti yesidoda ebonisa i-rheotaxis enhle (p <0.001; Itheyibhile 2). Хотя пучки сперматозоидов имели более низкую скорость, чем у одиночных сперматозоидов (p < 0,001), они увеличивали процонтов, проценты, увеличивали процеман ложительный реотаксис (p < 0,001; таблица 2). Nangona i-spermatozoa bundles inesantya esisezantsi kuneso se-spermatozoa enye (p <0.001), yandisa ipesenti ye-spermatozoa ebonisa i-rheotaxis enhle (p <0.001; Itheyibhile 2).尽管精子束的速度低于孤独精子的速度（p <0.001），但它們增加了显示阳性流变性生的。表2）.尽管 精子束的 速度 低于 孤独的 速度 （p <0.001） , 但 增加 显示 阳性 流口.001 ; 2....."..((())) Хотя скорость пучков сперматозоидов была ниже, чем у одиночных сперматозоидов (p <0,001), они увеличивали процентоложенный (p <0,001) p <0,001; таблица 2). Nangona isantya se-sperm bundles sasiphantsi kuneso se-spermatozoa enye (p <0.001), yandisa ipesenti ye-spermatozoa nge-rheology enhle (p <0.001; Itheyibhile 2).I-rheology enhle ye-spermatozoa enye kunye ne-tufts iqikelelwa malunga ne-53% kunye ne-85%, ngokulandelanayo.
Kuye kwaphawulwa ukuba i-spermatozoa yeenkukhu ze-sharkasi ngokukhawuleza emva kokuba i-ejaculation yenza i-bundles ehambelanayo, equka abantu abaninzi.Ezi tufts zanda ngobude kunye nobukhulu ngokuhamba kwexesha kwaye zinokuhlala kwi-vitro iiyure ezininzi ngaphambi kokuchitha (ividiyo 3).Ezi bundles ezinefilamentous zifana ne-echidna spermatozoa eyenza ekupheleni kwe-epididymis.Imbewu ye-Sharkashi hen ifunyenwe inomdla ophezulu wokudibanisa kunye nokwenza i-bundle ye-reticulate ngaphantsi komzuzu omnye emva kokuqokelela.Le miqadi inamandla kwaye iyakwazi ukunamathela kuzo naziphi na iindonga ezikufutshane okanye izinto ezimileyo.Nangona iinyanda zamadlozi zinciphisa isantya seeseli zesidoda, kuyacaca ukuba ngokwemacroscopically zonyusa umgca wazo.Ubude bama-bundle buyahluka ngokuxhomekeke kwinani lamadlozi aqokelelwe kwimiqulu.Iinxalenye ezimbini ze-bundle zazibekwe zodwa: inxalenye yokuqala, kuquka nentloko ekhululekileyo ye-sperm edibeneyo, kunye ne-terminal part, kubandakanywa nomsila kunye nesiphelo esipheleleyo se-sperm.Ukusebenzisa ikhamera yesantya esiphezulu (i-950 fps), iintloko zamahhala ze-spermatozoa ezixubileyo zabonwa kwindawo yokuqala ye-bundle, ejongene nokuhamba kwe-bundle ngenxa yentshukumo yabo ye-oscillatory, idonsa eziseleyo kwi-bundle nge-helical motion (Ividiyo 4).Noko ke, kwimibhobho emide, kuye kwafunyaniswa ukuba ezinye iintloko zamadlozi ezikhululekileyo zibambelela emzimbeni yaye indawo ekuphela kwayo isebenza njengee-vane ukunceda ukuqhubeleka.
Ngelixa ukuhamba okucothayo kolwelo, iinyanda zesidoda zihamba zihambelana enye kwenye, nangona kunjalo, ziqala ukugqithisa kwaye zinamathele kuyo yonke into ekhoyo, ukuze ingahlanjwa ngokuhamba kwangoku njengoko isantya sokuhamba sikhula.Iinyanda zenza xa iiseli zamadlozi ezimbalwa zisondela enye kwenye, ziqala ukushukuma kwi-synchrony kwaye zijijelane, zize zibambelele kwinto encangathi.Amanani 1 kunye ne-2 abonisa indlela i-sperm esondelana ngayo, yenza i-junction njengoko imisila ijikelezana.
Abaphandi basebenzise uxinzelelo lwe-hydrostatic ukwenza ukuhamba kwamanzi kwi-microchannel ukufunda i-rheology yesidoda.I-microchannel enobungakanani be-200 µm × 20 µm (W × H) kunye nobude be-3.6 µm yasetyenziswa.Sebenzisa i-microchannels phakathi kwezikhongozeli ezinesirinji ezifakwe eziphelweni.Umbala wokutya wawusetyenziselwa ukwenza imijelo ibonakale ngakumbi.
Bopha iintambo zoqhagamshelwano kunye nezixhobo edongeni.Ividiyo ithathwe ngemakroskopu yesigaba sokuchasana.Ngomfanekiso ngamnye, i-microscopy yesigaba sokuchasana kunye nemifanekiso yemephu iyaboniswa.(A) Uqhagamshelwano phakathi kwemijelo emibini luxhathisa ukuhamba ngenxa yentshukumo ye-helical (utolo olubomvu).(B) Uxhulumaniso phakathi kwe-tube bundle kunye nodonga lwesiteshi (iintolo ezibomvu), ngexesha elifanayo lidityaniswe kwezinye iinyanda ezimbini (iintolo ezimthubi).C(D) Ukwenziwa kothungelwano lwamadlozi.
Xa ithontsi ledlozi elihlanjululweyo lalayishwa kwisixhobo se-microfluidic kwaye kwadalwa ukuhamba, umqadi wesidoda wawujongwa ukuba uhambe ngokuchasene necala lokuhamba.Iingxowa zihambelana ngokufanelekileyo kwiindonga ze-microchannels, kunye neentloko zamahhala kwinxalenye yokuqala yeenyanda zihambelana nazo (ividiyo 5).Bakwancamathela kuwo nawaphi na amasuntswana amileyo endleleni yabo, njengobutyobo, ukuxhathisa ukukhukuliswa ngumsinga.Ngokuhamba kwexesha, ezi zixhobo ziba yimicu emide ebambe enye i-spermatozoa enye kunye neengqungquthela ezimfutshane (Ividiyo 6).Njengoko ukuhamba kuqala ukucotha, imigca emide yesidoda iqala ukwenza inethiwekhi yemigca yesidoda (Ividiyo 7; Umfanekiso 2).
Kwisantya sokuqukuqela okuphezulu (V > 33 µm/s), iintshukumo ezijijekileyo zemisonto ziye zandiswa njengelinge lokubamba amadlozi amaninzi enza amanqwanqwa ngcono ukuxhathisa amandla akhukulisekayo okuqukuqela. Kwisantya sokuqukuqela okuphezulu (V > 33 µm/s), iintshukumo ezijijekileyo zemisonto ziye zandiswa njengelinge lokubamba amadlozi amaninzi enza amanqwanqwa ngcono ukuxhathisa amandla akhukulisekayo okuqukuqela. При высокой скорости потока (V > 33 мкм/с) спиралевидные движения нитей усиливаются, поскольку они пытаются поймать множествоы оздельков, другий х пучки, которые лучше противостоят дрейфующей силе потока. Kumazinga aphezulu okuhamba (V > 33 µm/s), iintshukumo zehelical zemisonto ziyanda njengoko zizama ukubamba i-spermatozoa zenza iinyanda ezikwaziyo ukumelana nokukhukuliseka kokuhamba.7漂移力。在 高 流速 (v> 33 µm/s) 时 ， 的 螺旋 运动 增加 ， 以漂移力。。。。。. При высоких скоростях потока (V > 33 мкм/с) спиральное движение нитей увеличивается в попытке захватить множество отделььное движение нитей увеличивается чтобы лучше сопротивляться силам дрейфа потока. Kumazinga okuqukuqela okuphezulu (V > 33 µm/s), intshukumo yehelical yeentsinga iyenyuka kwilinge lokubamba uninzi lwe-spermatozoa lwenza iinyanda ukuxhathisa ngcono amandla okukhukuliseka okuhamba.Baphinde bazama ukuncamathisela i-microchannels emacaleni.
Iinyanda zesidoda zachongwa njengeqela leentloko zesidoda kunye nemisila egobileyo kusetyenziswa i-microscopy ekhanyayo (LM).Iinyanda zesidoda ezinee-aggregates ezahlukeneyo ziye zachongwa njengeentloko ezijijekileyo kunye ne-flagellar aggregates, imisila yesidoda edibeneyo emininzi, iintloko zesidoda ezincanyathiselwe emsileni, kunye neentloko zesidoda ezinenuclei egobileyo njengeenuclei ezininzi ezidityanisiweyo.usasazo lwe-electron microscopy (TEM).Ukuskena i-electron microscopy (SEM) yabonisa ukuba amadlozi adityaniswe ziintloko zamadlozi kwaye ii-aggregates zamadlozi zibonisa uthungelwano oluncamatheleyo lwemisila esongelwe.
I-morphology kunye ne-ultrastructure ye-spermatozoa, ukubunjwa kweenyanda ze-spermatozoa zaphononongwa kusetyenziswa i-microscopy yokukhanya (isiqingatha secandelo), i-electron microscopy (SEM) yokuskena kunye ne-electron microscopy (TEM), i-sperm smears yangcoliswa nge-acridine orange kwaye yavavanywa ngokusebenzisa i-epifluorescence microscopy.
I-sperm smear staining kunye ne-acridine orange (Fig. 3B) yabonisa ukuba iintloko ze-sperm zinamathele kunye kwaye zigqunywe ngezinto ezifihlakeleyo, ezikhokelela ekwakhiweni kwee-tufts ezinkulu (Fig. 3D).I-sperm bundles yayiquka i-sperm aggregates kunye nenethiwekhi yemisila eqhotyoshelweyo (Fig. 4A-C).Izigaqa zesidoda zenziwe ngemisila ye-spermatozoa ezininzi ezinamathele kunye (Umfanekiso we-4D).Iimfihlo (Fig. 4E,F) zigqume iintloko ze-spermatozoa bundles.
Ukuqulunqwa kwenqwaba ye-spermatozoa Ukusebenzisa isigaba sokuchasa i-microscopy kunye ne-sperm smears efakwe i-acridine orange, ibonise ukuba iintloko ze-spermatozoa zinamathelana.(A) Ukwakheka kwedlozi kwakwangoko kuqala ngedlozi (isazinge esimhlophe) kunye namadlozi amathathu (isangqa esimthubi), yaye ujiko luqala emsileni luphele entloko.(B) Ifotomicrograph ye-sperm smear ene-acridine orange ebonisa iintloko zesidoda ezibambelelayo (iintolo).Ukukhutshwa kugqume intloko (iintloko).Ukwandiswa × 1000. (C) Ukuphuhliswa komqadi omkhulu othuthwa ngokuhamba kumzila we-microfluidic (usebenzisa ikhamera yesantya esiphezulu kwi-950 fps).(D) I-Micrograph ye-sperm smear ene-acridine e-orenji ebonisa ii-tufts ezinkulu (iintolo).Ukwandiswa: × 200.
Ukuskena i-electron micrograph ye-sperm beam kunye ne-sperm smear ene-acridine orenji.(A, B, D, E) zii-digital color scanning electron micrographs of spermatozoa, kunye no-C no-F ziimicrographs ze-acridine orange stained sperm smears ezibonisa ukuncamatheliswa kwe-spermatozoa ezininzi ezisonga i-caudal web.(AC) Udityaniso lwamadlozi luboniswa njengothungelwano lwemisila encanyathiselweyo (iintolo).(D) Ukuncamathela kwespermatozoa ezininzi (ezinento encamathelayo, ulwandlalo olupinki, utolo) ezisonga umsila.(E kunye no-F) Iintloko zesidoda (izikhombisi) ezigqunywe ngezinto ezincamathelayo (izikhombisi).I-spermatozoa yenze izibophelelo ezinezakhiwo ezininzi ezifana ne-vortex (F).(C) × 400 kunye (F) × 200 ulwandiso.
Ukusebenzisa i-electron microscopy yokudluliselwa, safumanisa ukuba i-sperm bundles yayinemisila eqhotyoshelweyo (Fig. 6A, C), iintloko ezifakwe kwimisila (Fig. 6B), okanye iintloko ezifakwe kwimisila (Fig. 6D).Iintloko ze-spermatozoa kwi-bundle zigobile, zibonisa kwicandelo leemimandla zenyukliya ezimbini (Fig. 6D).Kwi-incision bundle, i-spermatozoa yayinentloko ephothiweyo kunye nemimandla emibini yenyukliya kunye nemimandla emininzi ye-flagellar (Fig. 5A).
I-digital color electron micrograph ebonisa imisila edibanisayo kwi-sperm bundle kunye ne-agglutinating material edibanisa iintloko zesidoda.(A) Umsila oqhotyoshelweyo wenani elikhulu le-spermatozoa.Qaphela ukuba umsila ujongeka njani kwi-portrait (utolo) kunye nembonakalo yomhlaba (utolo).(B) Intloko (utolo) lwedlozi ludityaniswe nomsila (utolo).(C) Iintlobo ezininzi zemisila yamadlozi (iintolo) zincanyathiselwe.(D) Izinto ze-agglutination (AS, blue) zidibanisa iintloko ezine zamadlozi (ezimfusa).
Ukuskena i-electron microscopy yayisetyenziselwa ukukhangela iintloko zesidoda kwimiqulu yesidoda egqunywe ngeemfihlo okanye i-membrane (Figure 6B), ebonisa ukuba i-sperm bundles yayigxininiswe yi-extracellular material.I-agglutinated material yayigxininiswe kwintloko yesidoda (indibano efana nentloko yejellyfish; Fig. 5B) kwaye yandiswa kude, inika inkangeleko eqaqambileyo emthubi phantsi kwe-fluorescence microscopy xa ifakwe i-acridine orange (Fig. 6C).Le nto ibonakala ngokucacileyo phantsi kwe-microscope yokuskena kwaye ithathwa njenge-binder.Amacandelo amancinci (Umfanekiso 5C) kunye ne-sperm smears engcoliswe nge-acridine orange ibonise ama-sperm bundles aneentloko ezixineneyo kunye nemisila egobileyo (Fig. 5D).
Iifotomicrographs ezahlukeneyo ezibonisa ukudityaniswa kweentloko zesidoda kunye nemisila esongekileyo kusetyenziswa iindlela ezahlukeneyo.(A) Ukuhanjiswa kombala wedijithali we-electron micrograph yamacandelo anqamlezileyo wenqwaba yamadlozi ebonisa intloko yedlozi ebhijeneyo enenucleus enamacala amabini (bhlowu) kunye neenxalenye ezininzi zeflegi (eziluhlaza).(B) I-digital color scanning electron micrograph ebonisa iqela leentloko zamadlozi ezifana nejellyfish (iintolo) ezibonakala zigqunyiwe.C(D) I-Micrograph ye-sperm smear ene-acridine orange ebonisa i-aggregates yeentloko zamadlozi (iintolo) kunye nemisila ebambeneyo egobileyo (iintolo).Qaphela ukuba into encangathi (S) igquma intloko yedlozi.(D) × 1000 ukukhulisa.
Ukusebenzisa i-electron microscopy yokudluliselwa (Fig. 7A), kwaphawulwa ukuba iintloko zamadlozi zijijekile kwaye i-nuclei yayinemilo ejikelezayo, njengoko ingqinwe yi-sperm smears efakwe i-acridine orange kwaye ihlolwe kusetyenziswa i-fluorescence microscopy (Fig. 7B).
(A) I-Digital color transmission electron micrograph kunye (B) ne-Acridine orange stained sperm smear ebonisa iintloko ezibhijeneyo kunye nokuncamatheliswa kweentloko zedlozi kunye nemisila (iintolo).(B) × i-1000 yokwandisa.
Uphando olunika umdla kukuba idlozi likaSharkazi liyadityaniswa ukuze zenze imiqulu eshukumayo enefilamentous.Iimpawu zale miqulu zisivumela ukuba siqonde indima yazo enokwenzeka ekufunxeni nasekugcinweni kwe-spermatozoa kwi-SST.
Emva kokukhwelana, isidoda singena kwilungu lobufazi kwaye singene kwinkqubo yokukhetha kakhulu, nto leyo ekhokelela kwinani eliqingqiweyo ledlozi elingena kwi-SST15,16.Ukuza kuthi ga ngoku, iindlela zokungena kunye nokuphuma kwesidoda kwi-SST azikacaci.Kwiinkukhu, i-spermatozoa igcinwa kwi-SST ixesha elide lee-2 ukuya kwiiveki ze-10, kuxhomekeke kwiintlobo6.Ingxabano ihlala malunga nemeko yesidoda ngexesha lokugcinwa kwi-SST.Ngaba bakwintshukumo okanye baphumle?Ngamanye amazwi, iiseli zesidoda zigcina njani indawo yazo kwi-SST ixesha elide kangaka?
UForman4 ucebise ukuba indawo yokuhlala ye-SST kunye nokukhutshwa kungachazwa ngokwemigaqo ye-sperm motility.Ababhali baqikelela ukuba isidoda sigcina isikhundla sazo ngokuqubha ngokuchasene nokuhamba kolwelo oludalwe yi-SST epithelium kwaye eso sidoda siyakhutshwa kwi-SST xa isantya sazo siwela ngaphantsi kwendawo apho ziqala khona ukubuyela ngasemva ngenxa yokungabikho kwamandla.I-Zaniboni5 iqinisekisile ubukho be-aquaporins 2, 3, kunye ne-9 kwindawo ye-apical ye-SST epithelial cells, enokuthi ixhase ngokungathanga ngqo imodeli yokugcina isidoda se-Foreman.Kuphononongo lwangoku, siye safumanisa ukuba phantse isiqingatha spermatozoa Sharkashi kaThixo ukubonisa rheology elungileyo kulwelo oluqukuqelayo, kwaye amadlozi agglutinated kwandisa inani spermatozoa ebonisa rheology positive, nangona agglutination icothisa kubo.Indlela iiseli zamadlozi ezihamba ngayo zinyuka kumbhobho wentaka ukuya kwindawo eziqhame ngayo ayiqondwa ngokupheleleyo.Kwizilwanyana ezanyisayo, i-follicular fluid chemoattracts spermatozoa.Nangona kunjalo, ii-chemoattractants zikholelwa ukuba zikhokelela kwi-spermatozoa ukuba isondele kumgama omde7.Ngoko ke, ezinye iindlela zinoxanduva lokuthutha isidoda.Ukukwazi kwesidoda ukuqhelanisa kunye nokuhamba ngokuchasene ne-fallopian tube fluid ekhutshwe emva kokukhwelana kuye kwabikwa ukuba yeyona nto iphambili ekujoliseni isidoda kwiimpuku.UParker 17 ucebise ukuba i-spermatozoa iwele i-oviducts ngokuqubha ngokuchasene nomsinga weciliary kwiintaka kunye nezirhubuluzi.Nangona ingakhange ibonakaliswe ngokomfuniselo kwiintaka, i-Adolphi18 yaba yeyokuqala ukufumanisa ukuba isidoda se-avian sinika iziphumo ezilungileyo xa umaleko obhityileyo wolwelo phakathi kwe-coverlip kunye nesilayidi senziwa ngomcu wephepha lokucoca.I-Rheology.I-Hino kunye ne-Yanagimachi [19] babeke i-ovary-tubal-uterine ye-mouse kwi-ring ye-perfusion kunye nokutofa i-1 µl ye-inki kwi-isthmus ukujonga ukuhamba kwamanzi kwi-fallopian tubes.Baqaphela intshukumo esebenzayo kakhulu yokunciphisa kunye nokuphumla kwi-fallopian tube, apho zonke iibhola ze-inki zazihamba ngokuthe ngcembe ukuya kwi-ampulla ye-fallopian tube.Ababhali bagxininisa ukubaluleka kokuhamba kwe-tubal fluid ukusuka ezantsi ukuya kwi-fallopian tubes ephezulu ukwenzela ukunyusa isidoda kunye nokuchumisa.I-Brillard20 ibike ukuba kwiinkukhu kunye ne-turkeys, i-spermatozoa ifuduka ngokushukuma okusebenzayo ukusuka kumnyango wangasese, apho igcinwe khona, ukuya kwi-junction ye-utero-vaginal, apho igcinwe khona.Nangona kunjalo, le ntshukumo ayifuni phakathi kwe-junction ye-uterovaginal kunye ne-infundibulum kuba i-spermatozoa ihanjiswa nge-passive displacement.Ukwazi ezi ngcebiso zangaphambili kunye neziphumo ezifunyenweyo kwisifundo samanje, kunokucingelwa ukuba amandla e-spermatozoa ukunyuka phezulu (rheology) yenye yeempawu apho inkqubo yokukhetha isekelwe khona.Oku kugqiba ukuhamba kwe-spermatozoa ngesondo kunye nokungena kwabo kwiCCT yokugcina.Njengoko Forman4 icetyisiwe, oku kusenokwenza kube lula inkqubo yokungena kwedlozi kwi-SST nendawo yayo kangangexesha elithile kwaye iphume xa isantya sazo siqala ukucotha.
Ngakolunye uhlangothi, uMatsuzaki kunye noSasanami 21 bacebise ukuba i-spermatozoa ye-avian itshintshe utshintsho ukusuka kwi-dormancy ukuya kwi-motility kwiindlela zokuzala zamadoda nabasetyhini.Ukuvinjelwa kwe-sperm motility yokuhlala kwi-SST kuye kwacetywa ukuba kuchazwe ixesha elide lokugcina isidoda kunye nokuvuselela emva kokushiya i-SST.Ngaphantsi kweemeko ze-hypoxic, uMatsuzaki et al.1 ichaze imveliso ephezulu kunye nokukhutshwa kwelactate kwi-SST, nto leyo enokukhokelela ekuthinteleni ukuhamba kwedlozi labahlali.Kule meko, ukubaluleka kwe-rheology yesidoda kubonakaliswa ekukhethweni nasekufakweni kwe-spermatozoa, kwaye kungekhona ekugcinweni kwabo.
Ipateni ye-sperm agglutination ithathwa njengenkcazo ecacileyo yexesha elide lokugcina isidoda kwi-SST, njengoko le ndlela iqhelekileyo yokugcinwa kwesidoda kwinkukhu2,22,23.Bakst et al.I-2 yaqaphela ukuba uninzi lwe-spermatozoa lunamathele omnye komnye, lwenza i-fascicular aggregates, kunye ne-spermatozoa enye yayingafane ifumaneke kwi-CCM yezagwityi.Kwelinye icala, uWen et al.I-24 yaqaphela ngakumbi i-spermatozoa esasazekileyo kunye ne-spermatozoa embalwa kwi-lumen ye-SST kwiinkukhu.Ngokusekelwe kolu qwalaselo, kunokucingelwa ukuba ukuthambekela kwe-sperm agglutination kuyahluka phakathi kweentaka kunye naphakathi kwe-spermatozoa kwi-ejaculate efanayo.Ukongeza, uVan Krey et al.I-9 iphakamise ukuba ukuchithwa okungahleliwe kwe-spermatozoa edibeneyo kuxanduva lokungena ngokuthe ngcembe kwe-spermatozoa kwi-lumen ye-fallopian tube.Ngokwale ngcamango, i-spermatozoa enomthamo ophantsi we-agglutination kufuneka ikhutshwe kwi-SST kuqala.Kulo mongo, ukukwazi kwe-spermatozoa ukuba i-agglutinate inokuba yinto echaphazela umphumo wokhuphiswano lwesidoda kwiintaka ezingcolileyo.Ukongezelela, ixesha elide i-sperm ehlanganisiweyo ihlukana, ukuchuma kwexesha elide kugcinwa.
Nangona i-spermatozoa aggregation kunye nokuhlanganiswa kwiingxowa ziye zabonwa kwizifundo ezininzi ze-2,22,24, azizange zichazwe ngokweenkcukacha ngenxa yobunzima bokubonwa kwe-kinematic ngaphakathi kwe-SST.Iinzame ezininzi zenziwe ukufunda i-sperm agglutination in vitro.Ukudityaniswa okubanzi kodwa okwethutyana kuye kwabonwa xa ucingo olucekethekileyo lususiwe kwimbewu ejingayo.Oku kukhokelela ekubeni iqamza elide liphuma kwithontsi, lilinganisa idlala lobudoda.Ngenxa yemida ye-3D kunye namaxesha amafutshane okumisa i-drip, yonke ibhlokhi yakhawuleza yawela ekudakaleni9.Kuphononongo lwangoku, sisebenzisa iinkukhu ze-Sharkashi kunye ne-microfluidic chips, sakwazi ukuchaza indlela ezi zixhobo zenza ngayo kunye nendlela ezihamba ngayo.Izigaqa zesidoda zenziwe ngokukhawuleza emva kokuqokelela amadlozi kwaye zifunyenwe zihamba kwi-spiral, zibonisa i-rheology efanelekileyo xa ikhona ekuhambeni.Ngaphaya koko, xa kujongwe ngokugqithisileyo, iinyanda zesidoda ziye zajongwa ukuba zandise umgca wokuhamba xa kuthelekiswa ne-spermatozoa ekwanti.Oku kuphakamisa ukuba i-sperm agglutination ingenzeka ngaphambi kokungena kwe-SST kwaye ukuveliswa kwesidoda akukhawulelwanga kwindawo encinci ngenxa yoxinzelelo njengoko bekucetyisiwe ngaphambili (iTingari kunye neLake12).Ngexesha lokwakhiwa kwe-tuft, i-spermatozoa idada kwi-synchrony de ibe yinto edibeneyo, emva koko imisila ijikeleze enye kwenye kwaye intloko ye-spermatozoon ihlala ikhululekile, kodwa umsila kunye nenxalenye ekude ye-spermatozoon inamathela kunye nento encangathi.Ngoko ke, intloko ekhululekile ye-ligament inoxanduva lokunyakaza, ukudonsa yonke i-ligament.Ukuskena i-electron microscopy ye-sperm bundles ibonise iintloko zamadlozi ezincanyathiselweyo zigqunywe ngezinto ezininzi ezincangathi, ebonisa ukuba iintloko zesidoda zincanyathiselwe kwimiqulu yokuphumla, enokuthi yenzeke emva kokufikelela kwindawo yokugcina (SST).
Xa i-sperm smear ingcoliswe nge-acridine e-orenji, izinto zokuncamathelisa ezingaphandle kweeseli zesidoda zinokubonwa phantsi kwemakroskopu enefluorescent.Le nto ivumela i-sperm bundles ukuba ibambelele kwaye ibambelele kuyo nayiphi na indawo ejikelezileyo okanye amasuntswana ukuze ingakhukuli kunye nokuhamba okujikelezayo.Ngaloo ndlela, ukujonga kwethu kubonisa indima ye-spermatozoa adhesion ngendlela ye-bundles mobile.Ukukwazi kwabo ukuqubha ngokuchasene nomsinga kunye nokunamathela kwiindawo ezikufutshane kuvumela isidoda ukuba sihlale ixesha elide kwi-SST.
I-Rothschild25 isebenzise ikhamera ye-hemocytometry ukufunda ukuhanjiswa okudadayo kwe-semen yenkomo kwithontsi lokunqunyanyiswa, ithatha iifotomicrographs ngekhamera ene-axis ebonakalayo ethe nkqo kunye nethe tyaba ye-microscope.Iziphumo zabonisa ukuba i-spermatozoa yakhangwa kumphezulu wegumbi.Ababhali bacebisa ukuba kunokubakho ukusebenzisana kwe-hydrodynamic phakathi kwesidoda kunye nomphezulu.Ukuthathela ingqalelo oku, kunye nokukwazi kwembewu yentshontsho yaseSharkashi ukwenza iziqwenga ezincangathi, kunokonyusa amathuba okuba amadlozi abambelele kudonga lwe-SST kwaye agcinwe ixesha elide.
I-Bccetti kunye ne-Afzeliu26 ibike ukuba i-sperm glycocalyx iyadingeka ukuze kuqatshelwe i-gamete kunye ne-agglutination.I-Forman10 yaqaphela ukuba i-hydrolysis ye-α-glycosidic bonds kwi-glycoprotein-glycolipid coatings ngokunyanga incindi ye-avian nge-neuraminidase ibangele ukuzala okuncitshisiweyo ngaphandle kokuchaphazela ukuhamba kwedlozi.Ababhali bacebisa ukuba isiphumo se-neuraminidase kwi-glycocalyx yonakalisa ukuhluthwa kwesidoda kwindawo ye-utero-vaginal junction, ngaloo ndlela kunciphisa ukuchuma.Uqwalaselo lwabo alunakuyihoya into yokuba unyango lwe-neuraminidase lunokunciphisa ukuqondwa kwedlozi kunye ne-oocyte.I-Forman kunye ne-Engel10 yafumanisa ukuba ukuchuma kuyehla xa iinkukhu zifakwe kwi-intravaginal nge-semen enyangwa nge-neuraminidase.Nangona kunjalo, i-IVF ene-neuraminidase idlozi elinyangwayo ayizange ichaphazele ukuchuma xa kuthelekiswa nokulawula iinkukhu.Ababhali bagqibe kwelokuba utshintsho kwi-glycoprotein-glycolipid coating ejikeleze inwebu yesidoda yanciphisa amandla e-sperm ukuchumisa ngokuphazamiseka kokuhluthwa kwesidoda kwindawo ye-utero-vaginal junction, nto leyo eyandisa ilahleko yesidoda ngenxa yesantya se-uterus-vaginal junction, kodwa ayichaphazeli isidoda kunye nokubonwa kweqanda.
Kwii-turkeys u-Bakst kunye ne-Bauchan 11 bafumana ama-vesicles amancinci kunye ne-membrane fragments kwi-lumen ye-SST kwaye baqaphela ukuba ezinye zezi granules ziye zadibana ne-sperm membrane.Ababhali bacebisa ukuba obu budlelwane bunokuba negalelo ekugcinweni kwexesha elide le-spermatozoa kwi-SST.Nangona kunjalo, abaphandi abazange bacacise umthombo wale micu, nokuba ifihliwe ngamaseli e-CCT epithelial, eveliswa kwaye afihliweyo yinkqubo yokuzala yamadoda, okanye aveliswe yi-sperm ngokwayo.Kwakhona, la maqhekeza anoxanduva lwe-agglutination.I-Grützner et al27 ibike ukuba iiseli ze-epididymal epithelial zivelisa kwaye zikhuphe iprotheni ethile efunekayo ukuze kuqulunqwe amaphecana e-single-pore seminal.Ababhali baphinda babike ukuba ukusasazwa kwezi zibopho kuxhomekeke ekusebenzisaneni kweeprotheni ze-epididymal.I-Nixon et al28 yafumanisa ukuba i-adnexa ikhupha iprotheni, i-osteonectin ene-acidic cysteine-rich;I-SPARC ibandakanyeka ekwenziweni kwe-sperm tufts kwii-echidnas ezinomlomo omfutshane kunye ne-platypus.Ukusasazeka kwale miqadi kunxulunyaniswa nokulahleka kwale proteni.
Kuphononongo lwangoku, uhlalutyo lwe-ultrastructural usebenzisa i-electron microscopy lubonise ukuba i-spermatozoa ibambelele kwisixa esikhulu sezinto ezixineneyo.Ezi zinto zicingelwa ukuba zinoxanduva lokudibanisa okuhlangana phakathi kunye nokujikeleza iintloko ezibambelelayo, kodwa kwiindawo ezisezantsi kwingingqi yomsila.Sicinga ukuba le nto i-agglutinating ikhutshwe kwinkqubo yokuzala yamadoda (epididymis okanye vas deferens) kunye ne-semen, kuba sihlala sibona amadlozi ahlukana ne-lymph kunye ne-seminal plasma ngexesha lokuphuma.Kuye kwaxelwa ukuba njengoko i-avian spermatozoa idlula kwi-epididymis kunye ne-vas deferens, bafumana utshintsho olunxulumene nokuvuthwa okuxhasa amandla abo okubopha iiprotheni kunye nokufumana i-plasma lemma-associated glycoprotein.Ukuphikelela kwezi proteni kwiimbumba zesidoda ezihlala kwi-SST zibonisa ukuba ezi proteni zinokuchaphazela ukufunyanwa kwe-sperm membrane ukuzinza kwe-30 kwaye zinqume ukuzala kwazo i-31.U-Ahammad et al32 uchaze ukuba i-spermatozoa efunyenwe kwiindawo ezahlukeneyo zenkqubo yokuzala yamadoda (ukusuka kumatyhalarha ukuya kwi-distal vas deferens) ibonise ukunyuka okuqhubekayo kwimeko yokugcinwa kolwelo, kungakhathaliseki ukuba iqondo lokushisa lokugcina, kunye nokusebenza kweenkukhu kukwanyuka kwimibhobho ye-fallopian emva kokufakwa kwe-artificial insemination.
Amadlozi enkukhu e-Sharkashi aneempawu ezahlukeneyo kunye nemisebenzi kunezinye iintlobo ezifana ne-echidnas, i-platypus, iigundane zokhuni, iimpuku zenyamazane kunye neehagu ze-guinea.Kwiinkukhu ze-sharkasi, ukubunjwa kwe-spermatozoa bundles kwanciphisa isantya sabo sokubhukuda xa kuthelekiswa ne-spermatozoa enye.Nangona kunjalo, le miqulu yandisa ipesenti ye-rheologically positive spermatozoa kwaye yandisa amandla e-spermatozoa ukuzinzisa kwimeko eguquguqukayo.Ngaloo ndlela, iziphumo zethu ziqinisekisa isiphakamiso sangaphambili sokuba i-sperm agglutination kwi-SST ihambelana nokugcinwa kwesidoda sexesha elide.Siphinde sicinge ukuba ukuthambekela kwesidoda ukwenza ii-tufts kunokulawula isantya sokulahleka kwesidoda kwi-SST, enokutshintsha isiphumo sokhuphiswano lwesidoda.Ngokwale ngcinga, i-spermatozoa ene-low agglutination capacity ikhupha i-SST kuqala, ngelixa i-spermatozoa ene-agglutination umthamo ophezulu ivelisa uninzi lwenzala.Ukubunjwa kweenyanda zesidoda esine-pore enye kunenzuzo kwaye kuchaphazela umlinganiselo womzali nomntwana, kodwa kusetyenziswa indlela eyahlukileyo.Kwi-echidnas kunye ne-platypus, i-spermatozoa ihlelwe ngokuhambelanayo ukuze inyuse isantya sangaphambili se-beam.Iinyanda ze-echidnas zihamba ngokuphindwe kathathu ngokukhawuleza kune-spermatozoa enye.Kukholelwa ukuba ukuvela kwezi qhuma zamadlozi kwi-echidnas kukuziqhelanisa nokuziqhelanisa nokugcina ubukhosi, ekubeni amabhinqa aziphethe kakubi yaye edla ngokukhwelana namadoda aliqela.Ngoko ke, i-spermatozoa evela kwi-ejaculates eyahlukeneyo ikhuphisana ngokukrakra ngokuchumisa kweqanda.
I-Agglutinated spermatozoa yeenkukhu ze-sharkasi kulula ukuzibona usebenzisa i-phase difference microscopy, ebonwa njengenzuzo kuba ivumela ukufunda ngokulula ukuziphatha kwe-spermatozoa in vitro.Indlela ekwenziwa ngayo i-sperm tuft ikhuthaza ukuzala kweenkukhu ze-sharkasi ikwahlukile kuleyo ibonwa kwezinye izilwanyana ezincancisayo ezimele intsebenziswano yamadlozi afana neempuku zomthi, apho i-spermatozoa ethile ifikelela emaqandeni, inceda abanye abantu abazalanayo bafikelele kwaye bonakalise amaqanda abo.ukuze uzibonakalise.ukuziphatha ngokungenabubele.I-self-fertilization 34. Omnye umzekelo wokuziphatha ngokubambisana kwi-spermatozoa ifunyenwe kwiigundane zenyamazane, apho i-spermatozoa yayikwazi ukuchonga kunye nokudibanisa kunye ne-spermatozoa ehambelana kakhulu ne-genetically kunye nokwenza amaqela okubambisana ukuze kwandiswe isantya sabo xa kuthelekiswa ne-spermatozoa35 engahambelaniyo.
Iziphumo ezifunyenwe kolu cwaningo aziphikisani nethiyori kaFoman yokugcina ixesha elide le-spermatozoa kwi-SWS.Abaphandi baxela ukuba iiseli zesidoda ziyaqhubeka nokuhamba ngokuhamba kweeseli ze-epithelial ezibeka i-SST ixesha elide, kwaye emva kwexesha elithile, izitora zamandla zeeseli zesidoda ziphelile, okukhokelela ekunciphiseni kwesantya, esivumela ukugxothwa kwezinto ezincinci ze-molecular weight.amandla e-spermatozoa kunye nokuhamba kolwelo olusuka kwilume ye-SST Umgodi wetyhubhu yesibeleko.Kuphononongo lwangoku, siye sabona ukuba isiqingatha sesidoda esinye sibonise ukukwazi ukuqubha ngokuchasene nolwelo oluqukuqelayo, kwaye ukuncamathela kwabo kwinqwaba kwandisa amandla abo okubonisa irheology elungileyo.Ngapha koko, idatha yethu iyahambelana nezo zikaMatuzaki et al.1 onike ingxelo yokuba ukwanda kwencindi ye-lactate kwi-SST kunokuthintela ukuhamba kwedlozi labahlali.Nangona kunjalo, iziphumo zethu zichaza ukubunjwa kwe-sperm motile ligaments kunye nokuziphatha kwabo kwe-rheological phambi kommandla oguqukayo ngaphakathi kwe-microchannel kumzamo wokucacisa ukuziphatha kwabo kwi-SST.Uphando lwexesha elizayo lunokugxila ekumiseleni ukubunjwa kweekhemikhali kunye nemvelaphi ye-agglutinating agent, eya kunceda abaphandi ukuba baphuhlise iindlela ezintsha zokugcina isidoda solwelo kunye nokwandisa ixesha lokuzala.
Ishumi elinesihlanu leeveki ezingama-30 ubudala i-sharkasi eyindoda (i-homozygous elawulayo; i-Na Na) yakhethwa njengabanikezeli besidoda kwisifundo.Iintaka zazikhuliswe kwiFama yeeNkukhu zoPhando kwiFakhalthi yezoLimo, kwiYunivesithi yaseAshit, kwiRhuluneli yaseAshit, eYiputa.Iinyoni zazigcinwe kwiikheji ezizimeleyo (30 x 40 x 40 cm), ziphantsi kweprogram yokukhanya (iiyure ze-16 zokukhanya kunye neeyure ze-8 zobumnyama) kwaye zondliwe ukutya okuqukethe i-160 g yeprotheni ekrwada, i-2800 kcal yamandla e-metabolizable, i-35 g ye-calcium nganye.I-5 grams ye-phosphorus ekhoyo ngekhilogram yokutya.
Ngokwedatha ye-36, 37, isidoda saqokelelwa kumadoda nge-massage yesisu.Kuqokelelwe iisampulu ezingama-45 kumadoda ali-15 kwiintsuku ezi-3.I-Semen (n = 15 / imini) yaxutywa ngokukhawuleza i-1: 1 (v: v) kunye ne-Belsville Poultry Semen Diluent, equkethe i-potassium diphosphate (1.27 g), i-monosodium glutamate monohydrate (0.867 g), i-fructose (0.5 d) i-sodium anhydrous.i-acetate (0.43 g), i-tris (i-hydroxymethyl) i-aminomethane (0.195 g), i-potassium citrate monohydrate (0.064 g), i-potassium monophosphate (0.065 g), i-magnesium chloride (0.034 g) kunye ne-H2O (100 ml), pH = 7, 5, osmolarity 3/33 m33 m33 m8 osmolarity.Iisampulu zamadlozi ezihlanjululweyo zaqale zavavanywa phantsi kwe-microscope ekhanyayo ukuqinisekisa umgangatho wobudoda obulungileyo (ukufuma) kwaye emva koko zigcinwe kwindawo yokuhlambela yamanzi ngama-37°C de zisetyenziswe kwisiqingatha seyure emva kokuqokelela.
I-kinematics kunye ne-rheology ye-spermatozoa ichazwa ngokusebenzisa inkqubo yezixhobo ze-microfluidic.Iisampulu zamadlozi ziye zaphinda zahlanjululwa zaya kutsho kwi-1:40 eBeltsville Avian Semen Diluent, elayishwe kwisixhobo se-microfluidic (jonga ngezantsi), kwaye iiparamitha zekinetic ziye zamiselwa kusetyenziswa inkqubo yeComputerized Semen Analysis (CASA) eyayiphuhliswe ngaphambili ukubonakaliswa kwemicrofluidics.ekuhambeni kwe-spermatozoa kumajelo eendaba (iSebe lezobuNjineli boMechanical, i-Faculty of Engineering, iYunivesithi yase-Assiut, eYiputa).I-plugin inokukhutshelwa apha: http://www.assiutmicrofluidics.com/research/casa39.Isantya seCurve (VCL, μm / s), isantya somgca (VSL, μm / s) kunye nesantya somzila wendlela (VAP, μm / s) silinganiswe.Iividiyo ze-spermatozoa zithathwe kusetyenziswa i-Optika XDS-3 isigaba sokungafani semicroscope (enenjongo ye-40x) eqhagamshelwe kwikhamera ye-Tucson ISH1000 kwi-30 fps kwi-3 s.Sebenzisa i-software ye-CASA ukufunda ubuncinane iindawo ezintathu kunye ne-500 ye-sperm trajectories kwisampuli nganye.Ividiyo erekhodiweyo yaqhutywa kusetyenziswa iCASA yasekhaya.Inkcazo ye-motility kwi-plug-in ye-CASA isekelwe kwisantya sokubhukuda kwesidoda xa kuthelekiswa nesantya sokuhamba, kwaye ayibandakanyi ezinye iiparitha ezifana nokunyakaza kwecala ukuya kwicala, njengoko oku kufunyenwe kunokwethenjelwa ngakumbi ekuhambeni kwamanzi.Intshukumo ye-Rheological ichazwa njengentshukumo yeeseli zamadlozi ngokuchasene necala lokuhamba kolwelo.I-Spermatozoa eneempawu ze-rheological yahlulwe ngenani le-spermatozoa ehamba phambili;isidoda ebesiphumle kwaye sishukuma isidoda ebesingabandakanywa ebaleni.
Zonke iikhemikhali ezisetyenzisiweyo zifunyenwe kwi-Elgomhoria Pharmaceuticals (Cairo, Egypt) ngaphandle kokuba kuchazwe ngenye indlela.Isixhobo senziwa njengoko kuchazwe ngu-El-sherry et al.40 kunye nohlengahlengiso oluthile.Izinto ezisetyenziselwa ukwenza i-microchannels zibandakanya iipleyiti zeglasi (i-Howard Glass, i-Worcester, MA), i-SU-8-25 yokumelana ne-negative (MicroChem, Newton, CA), i-diacetone alcohol (Sigma Aldrich, Steinheim, Germany), kunye ne-polyacetone.-184, eDow Corning, eMidland, eMichigan).Ii-Microchannels zenziwe kusetyenziswa i-lithography ethambileyo.Okokuqala, imaski yobuso obukhuselayo obucacileyo kunye noyilo olufunwayo lwe-microchannel yaprintwa kwiprinta yesisombululo esiphezulu (Prismatic, Cairo, Egypt kunye nePacific Arts and Design, Markham, ON).Iinkosi zenziwa ngokusebenzisa iipleyiti zeglasi njengama-substrates.Iipleyiti zahlanjululwa kwi-acetone, i-isopropanol kunye namanzi e-deionized kwaye emva koko zigqunywe nge-20 µm umaleko we-SU8-25 nge-spin coating (3000 rpm, 1 min).I-SU-8 layers ke yomiswa ngobumnene (65 ° C, 2 min kunye ne-95 ° C, i-10 min) kwaye ibonakaliswe kwimitha ye-UV ye-50 s.I-post-exposure exposure bhaka kwi-65 ° C kunye ne-95 ° C ye-1 min kunye ne-4 min ukuya kwi-crosslink evezwe i-SU-8 layers, ilandelwa kuphuhliso kwi-diacetone alcohol ye-6.5 min.Ukubhaka kanzima i-waffles (200 ° C kwi-15 min) ukuqhubela phambili ukuqinisa i-SU-8 layer.
I-PDMS yalungiswa ngokuxuba i-monomer kunye ne-hardener kumlinganiselo wobunzima be-10: 1, emva koko i-degassed kwi-vacuum desiccator kwaye igalele kwi-SU-8 isakhelo esikhulu.I-PDMS iphiliswe kwi-oven (i-120 ° C, i-30 min), emva koko iziteshi zinqunywe, zihlulwe kwi-master, kunye ne-perforated ukuvumela iibhubhu ukuba zifakwe kwi-inlet kunye nokuphuma kwe-microchannel.Ekugqibeleni, i-microchannels ye-PDMS ifakwe ngokusisigxina kwiislayidi ze-microscope zisebenzisa iprosesa ye-corona ephathekayo (Iimveliso ze-Electro-Technic, Chicago, IL) njengoko kuchazwe kwenye indawo.I-microchannel esetyenziswe kolu phononongo ilinganisa i-200 µm × 20 µm (W × H) kwaye yi-3.6 cm ubude.
Ukuhamba kwamanzi okubangelwa uxinzelelo lwe-hydrostatic ngaphakathi kwe-microchannel kufezekiswa ngokugcina inqanaba le-fluid kwi-inlet ye-inlet ngaphezu kokuhluka kobude Δh39 kwi-reservoir yokuphuma (umzobo 1).
apho i-f i-coefficient of friction, ichazwa njenge-f = C / Re ye-laminar flow in the channel ye-rectangular, apho i-C isigxina ngokuxhomekeke kwi-aspect ratio ye-channel, i-L ubude be-microchannel, i-Vav yi-avareji yesantya ngaphakathi kwe-microchannel, i-Dh yi-diameter ye-hydraulic ye-channel, i-g - i-acceleration of gravity.Ukusebenzisa le nxaki, umndilili wesantya setshaneli unokubalwa kusetyenziswa le nxaki ilandelayo: