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Ukuchuma kweentaka kuxhomekeke ekubeni zikwazi ukugcina isidoda esaneleyo ixesha elide kwi-tubules yokugcina isidoda (SST). Indlela echanekileyo i-spermatozoa engena ngayo, ihlale, kwaye iphume kwi-SST isaphikisana. Isidoda seenkukhu ze-sharkasi sibonakalise ukuthambekela okuphezulu kokuhlanganiswa, senza ii-bundles ezihambayo ezine-filamentous bundles eziqulethe iiseli ezininzi. Ngenxa yobunzima bokujonga ukuhamba kunye nokuziphatha kwe-spermatozoa kwi-opaque fallopian tube, sisebenzise isixhobo se-microfluidic esine-microchannel cross-section efana neye-spermatozoa ukuze sifunde i-spermatozoa agglutination kunye nokuhamba. Olu phononongo luxoxa ngendlela ii-sperm bundles ezakheka ngayo, indlela ezihamba ngayo, kunye nendima yazo enokwenzeka ekwandiseni ukuhlala kwesidoda kwi-SST. Siphande isantya sesidoda kunye nokuziphatha kwe-rheological xa ukuhamba kolwelo kwenziwa ngaphakathi kwe-microfluidic channel ngoxinzelelo lwe-hydrostatic (isantya sokuhamba = 33 µm/s). I-spermatozoa ithambekele ekudada ngokuchasene nomsinga (i-rheology elungileyo) kwaye isantya se-spermatozoon bundle sinciphile kakhulu xa kuthelekiswa ne-spermatozoa enye. Kuye kwabonwa ukuba ii-sperm bundles zihamba ngendlela ejikelezayo kwaye zikhula ngobude nangobukhulu njengoko kuqokelelwa isidoda esinye. Kubonwe iimbumba zesidoda zisondela kwaye zinamathele emacaleni emijelo ye-microfluidic ukuze zingakhukuliswa ngesantya sokuhamba kolwelo > 33 µm/s. Kubonwe iimbumba zesidoda zisondela kwaye zinamathele emacaleni emijelo ye-microfluidic ukuze zingakhukuliswa ngesantya sokuhamba kolwelo > 33 µm/s. Было замечено, что пучки сперматозоидов приближаются и прилипают к боковым стенкам микрофлюидных каналов, чтобы избениять смета жидкости> 33 мкм / с. Kuye kwabonwa ukuba ii-sperm bundles zisondela kwaye zinamathela eludongeni olusecaleni lwemijelo ye-microfluidic ukuze zingakhukuliswa xa ulwelo luhamba ngesantya esingaphezulu kwe-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 компания сметания 33 мкм/с. Kuye kwabonwa ukuba ii-sperm bundles zisondela kwaye zinamathela eludongeni olusecaleni lomjelo we-microfluidic ukuze zingakhukuliswa kukuhamba kolwelo kwi->33 µm/s.Ukuskena kunye ne-transmission electron microscopy kubonise ukuba ama-sperm bundles axhaswe zizinto ezininzi ezixineneyo. Idatha efunyenweyo ibonisa ukuhambahamba okukhethekileyo kwe-Sharkazi chicken spermatozoa, kunye nokukwazi kwe-spermatozoa ukuhlanganisa nokwenza ama-mobile bundles, nto leyo enegalelo ekuqondeni ngcono ukugcinwa kwe-spermatozoa ixesha elide kwi-SMT.
Ukuze kufunyanwe ukuchumisa ebantwini nakwizilwanyana ezininzi, isidoda kunye namaqanda kufuneka afike kwindawo yokuchumisa ngexesha elifanelekileyo. Ke ngoko, ukudibana kufuneka kwenzeke ngaphambi okanye ngexesha lokuvundiswa kweqanda. Kwelinye icala, ezinye izilwanyana ezanyisayo, njengezinja, kunye neentlobo ezingezizo izilwanyana ezanyisayo, ezifana nezinambuzane, iintlanzi, izilwanyana ezirhubuluzayo, kunye neentaka, zigcina isidoda kwizitho zazo zokuzala ixesha elide de amaqanda azo alungele ukuchumisa (ukuchumisa okungahambelaniyo 1). Iintaka ziyakwazi ukugcina amandla e-spermatozoa ekwaziyo ukuchumisa amaqanda iiveki ezi-2 ukuya kwezi-102.
Olu luphawu olulodwa olwahlula iintaka kwezinye izilwanyana, njengoko lubonelela ngamathuba aphezulu okuchumisa emva kokufakelwa isidoda kube kanye kangangeeveki ezininzi ngaphandle kokudibana ngaxeshanye kunye nokukhutshwa kwesidoda ngaxeshanye. Ilungu eliphambili lokugcina isidoda, elibizwa ngokuba yi-sperm storage tubule (SST), likwiindawo zangaphakathi ze-mucosal kwindawo yokudibana kwe-uterovaginal. Ukuza kuthi ga ngoku, iindlela isidoda esingena ngazo, esihlala ngazo, nesiphuma ngazo kwi-sperm bank azikaqondwa ngokupheleleyo. Ngokusekelwe kwizifundo zangaphambili, kuye kwavezwa iingcamango ezininzi, kodwa akukho nanye kuzo eqinisekisiweyo.
UForman4 ucinge ukuba ispermatozoa igcina indawo yayo kwi-SST cavity ngokusebenzisa intshukumo eqhubekayo ye-oscillatory ngokuchasene necala lokuhamba kolwelo ngemijelo yeproteni ekwiiseli ze-SST epithelial (rheology). I-ATP iyaphela ngenxa yomsebenzi we-flagellar oqhubekayo ofunekayo ukugcina isperm kwi-SST lumen kwaye ukuhambahamba ekugqibeleni kuncipha de isperm iphume kwibhanki yesperm ngokuhamba kolwelo kwaye iqale uhambo olutsha phantsi kwityhubhu ye-fallopian enyukayo ukuze ichumise isperm. Iqanda (Forman4). Le modeli yokugcina isperm ixhaswa kukufunyanwa yi-immunocytochemistry ye-aquaporins 2, 3 kunye ne-9 ekhoyo kwiiseli ze-SST epithelial. Ukuza kuthi ga ngoku, izifundo kwi-rheology yesperm yenkukhu kunye nendima yayo ekugcinweni kwe-SST, ekukhethweni kwesperm yesini, kunye nokhuphiswano lwesperm azikho. Kwiinkukhu, isperm ingena kwi-vagina emva kokudibana kwendalo, kodwa ngaphezulu kwe-80% yespermatozoa ikhutshwa kwi-vagina kungekudala emva kokudibana. Oku kuthetha ukuba i-vagina yindawo ephambili yokukhethwa kwesperm kwiintaka. Ukongeza, kuye kwaxelwa ukuba ngaphantsi kwe-1% yesidoda esichumisiweyo kwisini siphelela kwi-SSTs2. Ekufakeni amantshontsho ngokwenziwa kwisini, inani lesidoda esifikelela kwi-SST lidla ngokwanda kwiiyure ezingama-24 emva kokufakelwa. Okwangoku, indlela yokukhetha isidoda ngexesha lale nkqubo ayicacanga, kwaye ukuhamba kwesidoda kunokudlala indima ebalulekileyo ekuthathweni kwesidoda kwi-SST. Ngenxa yodonga oluqinileyo nolungacacanga lweetyhubhu ze-fallopian, kunzima ukujonga ngokuthe ngqo ukuhamba kwesidoda kwiityhubhu ze-fallopian zeentaka. Ke ngoko, asinalo ulwazi olusisiseko malunga nendlela i-spermatozoa etshintshela ngayo kwi-SST emva kokufakelwa.
I-Rheology isandula ukuqatshelwa njengento ebalulekileyo elawula ukuthuthwa kwesidoda kwi-genitalia yezilwanyana ezincancisayo. Ngokusekelwe kubuchule be-spermatozoa ehambahambayo bokuhamba ngokuchaseneyo, uZaferani et al8 basebenzise inkqubo ye-corra microfluidic ukuze bahlukanise i-spermatozoa ehambahambayo kwiisampuli zesidoda ezibhaliweyo. Olu hlobo lokukhetha isidoda lubalulekile kunyango lokungazali kwezonyango kunye nophando lweklinikhi, kwaye lukhethwa kuneendlela zendabuko ezithatha ixesha kwaye zifuna umsebenzi omninzi kwaye zinokubeka emngciphekweni imo yesidoda kunye nokuqina kwesakhiwo. Nangona kunjalo, ukuza kuthi ga ngoku, akukho zifundo zenziweyo malunga nempembelelo yokukhutshwa kwesidoda kwi-genitalia yenkukhu ekuhambekeni kwesidoda.
Nokuba yeyiphi indlela egcina isidoda sigcinwe kwi-SST, abaphandi abaninzi baye baphawula ukuba i-resident spermatozoa ihlanganisa intloko nentloko kwi-SST yeenkukhu 9, 10, quails 2, kunye neeturkeys 11 ukuze zenze ii-agglutinated sperm bundles. Ababhali bacebisa ukuba kukho unxibelelwano phakathi kolu agglutination kunye nokugcinwa kwexesha elide kwe-spermatozoa kwi-SST.
UTungari kunye neLake12 babike unxibelelwano oluqinileyo phakathi kwe-spermatozoa kwi-gland yenkukhu efumana isidoda kwaye babuza ukuba ingaba i-avian spermatozoa ihlangana ngendlela efanayo ne-mammalian spermatozoa. Bakholelwa ukuba unxibelelwano olunzulu phakathi kwesidoda kwi-vas deferens lunokubangelwa kuxinzelelo olubangelwa bubukho benani elikhulu lesidoda kwindawo encinci.
Xa kuvavanywa indlela i-spermatozoa eziphatha ngayo kwiislayidi zeglasi ezixhonyiweyo ezintsha, iimpawu ze-agglutination ezidlulayo zinokubonakala, ingakumbi emaphethelweni amathontsi esidoda. Nangona kunjalo, i-agglutination yayisoloko iphazanyiswa yintshukumo yokujikeleza enxulumene nokuhamba okuqhubekayo, nto leyo echaza uhlobo lwe-agglutination yale nto. Abaphandi bakwaphawula ukuba xa i-diluent yongezwa kwi-semen, kwavela ii-aggregates zeeseli ezinde "ezifana nomsonto".
Imizamo yokuqala yokulinganisa i-spermatozoon yenziwa ngokususa ucingo olubhityileyo kwithontsi elixhonyiweyo, nto leyo eyabangela ukuba i-vesicle ende efana ne-sperm iphume kwithontsi le-sperm. I-spermatozoa yakhawuleza yahlangana ngendlela efanayo ngaphakathi kwi-vesicle, kodwa yonke iyunithi yanyamalala ngokukhawuleza ngenxa yokunciphisa i-3D. Ke ngoko, ukuze kufundwe i-spermatozoa agglutination, kuyimfuneko ukujonga ukuhamba kunye nokuziphatha kwe-spermatozoa ngokuthe ngqo kwii-tubules zokugcina isidoda ezizimeleyo, nto leyo enzima ukuyenza. Ke ngoko, kuyimfuneko ukuphuhlisa isixhobo esilinganisa i-spermatozoa ukuxhasa izifundo zokuhamba kwesidoda kunye nokuziphatha kwe-agglutination. UBrillard et al13 baxele ukuba ubude obuqhelekileyo bee-tubules zokugcina isidoda kumantshontsho amadala yi-400–600 µm, kodwa ezinye ii-SST zingaba zide njenge-2000 µm. I-Mero kunye ne-Ogasawara14 zahlulahlula ii-seminiferous glands zaba zii-tubules zokugcina isidoda ezinkulu nezingengomkhulu, zombini ezazilingana ngobude (~500 µm) kunye nobubanzi bentamo (~38 µm), kodwa ububanzi be-lumen ephakathi yee-tubules yayiyi-56.6 kunye ne-56.6 µm. . , ngokulandelelana yi-11.2 μm, ngokulandelelana. Kolu phononongo lwangoku, sisebenzise isixhobo se-microfluidic esinobukhulu betshaneli ye-200 µm × 20 µm (W × H), esine-cross section esondele kancinci kwe-SST ekhulisiweyo. Ukongeza, sihlolisise ukuhamba kwesidoda kunye nokuziphatha kwe-agglutination kulwelo oluhambayo, okuhambelana nengcamango kaForeman yokuba ulwelo oluveliswa zii-SST epithelial cells lugcina isidoda kwi-lumen kwicala elichaseneyo (rheological).
Injongo yolu phononongo yayikukoyisa iingxaki zokujonga ukuhamba kwe-spermatozoa kwi-fallopian tube kunye nokuphepha ubunzima bokufunda i-rheology kunye nokuziphatha kwe-spermatozoa kwindawo eguquguqukayo. Kusetyenziswe isixhobo se-microfluidic esenza uxinzelelo lwe-hydrostatic ukuze kulinganiswe ukuhamba kwe-sperm kwizitho zangasese zenkukhu.
Xa ithontsi lesampulu yesidoda esixutyiweyo (1:40) lifakwe kwisixhobo se-microchannel, iintlobo ezimbini zokuhamba kwesidoda zinokuchongwa (isidoda esihlukanisiweyo nesidoda esibotshelelweyo). Ukongeza, i-spermatozoa yayidla ngokudada ngokuchasene nomsinga (i-rheology elungileyo; ividiyo 1, 2). Nangona ii-sperm bundles zazinesantya esiphantsi kunese-lonesome sperm (p < 0.001), zonyuse ipesenti ye-sperm ebonisa i-rheotaxis entle (p < 0.001; Itheyibhile 2). Nangona ii-sperm bundles zazinesantya esiphantsi kunese-lonesome sperm (p < 0.001), zonyuse ipesenti ye-sperm ebonisa i-rheotaxis entle (p < 0.001; Itheyibhile 2). Хотя пучки сперматозоидов имели более низкую скорость, чем у одиночных сперматозоидов (p < 0,001), они увеличивали процемант, процеман демонстрирующих положительный реотаксис (p < 0,001; таблица 2). Nangona ii-spermatozoa bundles zazinesantya esiphantsi kunese-single spermatozoa (p < 0.001), zonyuse ipesenti ye-spermatozoa ebonisa i-rheotaxis entle (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的美生0.001;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 流古(p <0.001 ; 2...."....) Хотя скорость пучков сперматозоидов была ниже, чем у одиночных сперматозоидов (p <0,001), они увеличивали процент спедрмато реологией (p < 0,001; таблица 2). Nangona isantya see-sperm bundles sasiphantsi kuneso se-single spermatozoa (p < 0.001), bonyuse ipesenti ye-spermatozoa ene-rheology eyakhayo (p < 0.001; Itheyibhile 2).I-rheology elungileyo ye-spermatozoa enye kunye nee-tufts iqikelelwa malunga ne-53% kunye ne-85%, ngokulandelelana.
Kuye kwabonwa ukuba i-spermatozoa yeenkukhu ze-sharkasi emva nje kokuphuma kweqanda yenza ii-bundles ezithe tyaba, eziquka izilwanyana ezininzi. Ezi ziqu zikhula ngobude nangobukhulu ngokuhamba kwexesha kwaye zinokuhlala kwi-vitro iiyure ezininzi ngaphambi kokuba ziphele (ividiyo 3). Ezi ziqu zi-filamentous bundles zimile njenge-echidna spermatozoa eyenzeka ekupheleni kwe-epididymis. I-Sharkashi hen semen ifunyenwe inomdla ophezulu wokudibanisa kwaye yenze i-reticulate bundle ngaphantsi komzuzu omnye emva kokuqokelelwa. Ezi beams zinamandla kwaye ziyakwazi ukunamathela nakweyiphi na indonga ezikufutshane okanye izinto ezingashukumiyo. Nangona ii-sperm bundles zinciphisa isantya seeseli ze-sperm, kuyacaca ukuba ngokwe-macroscopic ziyandisa umgca wazo. Ubude bee-bundles buyahluka ngokuxhomekeke kwinani le-sperm eqokelelwe kwii-bundles. Iindawo ezimbini ze-bundle zahlulwe: inxalenye yokuqala, kubandakanya intloko ekhululekileyo ye-sperm eqokelelwe kwi-bundles, kunye nenxalenye yokugqibela, kubandakanya umsila kunye nesiphelo sonke se-distal se-sperm. Kusetyenziswa ikhamera ekhawulezayo (950 fps), iintloko ezikhululekileyo ze-spermatozoa ezihlanganisiweyo zabonwa kwinxalenye yokuqala ye-bundle, ezinoxanduva lokushukuma kwe-bundle ngenxa yokuhamba kwazo okujikelezayo, zirhuqa eziseleyo kwi-bundle ngentshukumo ejikelezayo (Ividiyo 4). Nangona kunjalo, kwii-tufts ezinde, kuye kwabonwa ukuba ezinye iintloko ze-sperm ezikhululekileyo zinamathele emzimbeni kwaye inxalenye yokugqibela ye-tuft isebenza njenge-vanes ukunceda ukuhambisa i-tuft.
Nangona kukuhamba kancinci kolwelo, amaqhuqhuva esidoda ahamba ngaxeshanye, nangona kunjalo, aqala ukunamathelana aze anamathele kuyo yonke into emileyo, ukuze angakhukuliswa kukuhamba kwamanzi njengoko isantya sokuhamba sisanda. Amaqhuqhuva akheka xa iiseli zesidoda ezimbalwa zisondelana, ziqala ukuhamba ngendlela efanayo kwaye zijikelezene, zize zinamathele kwinto enamathelayo. Imifanekiso 1 kunye no-2 ibonisa indlela isidoda esisondelana ngayo, senza ukudibana njengoko imisila ijikelezana.
Abaphandi basebenzise uxinzelelo lwe-hydrostatic ukudala ukuhamba kolwelo kwi-microchannel ukuze bafunde i-sperm rheology. Kusetyenziswe i-microchannel enobukhulu be-200 µm × 20 µm (W × H) kunye nobude be-3.6 µm. Sebenzisa ii-microchannels phakathi kwezikhongozeli ezineesirinji ezifakwe eziphelweni. Umbala wokutya usetyenzisiwe ukwenza ii-channels zibonakale ngakumbi.
Bopha iintambo zokudibanisa kunye nezixhobo eludongeni. Ividiyo ithathwe nge-phase contrast microscope. Ngomfanekiso ngamnye, kuboniswa i-phase contrast microscopy kunye nemifanekiso yemephu. (A) Unxibelelwano phakathi kwemijelo emibini lumelana nokuhamba ngenxa yentshukumo ye-helical (utolo obomvu). (B) Unxibelelwano phakathi kwe-tube bundle kunye nodonga lwe-channel (iintolo ezibomvu), kwangaxeshanye ziqhagamshelwe kwezinye ii-bundles ezimbini (iintolo ezityheli). (C) Ii-sperm bundles kwi-microfluidic channel ziqala ukunxibelelana (iintolo ezibomvu), zenze i-mesh yee-sperm bundles. (D) Ukwenziwa kwenethiwekhi yee-sperm bundles.
Xa ithontsi lembewu exutyiweyo lifakwa kwisixhobo se-microfluidic kwaye kwadalwa umjelo, umqadi wembewu wabonwa ukuba uhamba ngokuchasene necala lokuhamba. Iinqwaba zilingana kakuhle neendonga zeendlela ezincinci, kwaye iintloko ezikhululekileyo kwinxalenye yokuqala yeenqwaba zilingana kakuhle nazo (ividiyo 5). Zinamathela nakwiziphi na iinxalenye ezimileyo endleleni yazo, njengenkunkuma, ukuze zimelane nokukhukuliswa ngumsinga. Ekuhambeni kwexesha, ezi ziqu ziba yi-filaments ende ebamba ezinye ii-spermatozoa kunye nee-tufts ezimfutshane (iVidiyo 6). Njengoko ukuhamba kuqala ukucotha, imigca emide yembewu iqala ukwenza uthungelwano lwemigca yembewu (iVidiyo 7; Umfanekiso 2).
Kwisantya esiphezulu sokuhamba kwamanzi (V > 33 µm/s), iintshukumo ezijikelezayo zemisonto ziyanda njengoko kuzanywa ukubamba isidoda ngasinye esenza amaqhuqhuva ukuze simelane ngcono namandla okuhamba kwamanzi. Kwisantya esiphezulu sokuhamba kwamanzi (V > 33 µm/s), iintshukumo ezijikelezayo zemisonto ziyanda njengoko kuzanywa ukubamba isidoda ngasinye esenza amaqhuqhuva ukuze simelane ngcono namandla okuhamba kwamanzi. При высокой скорости потока (V > 33 мкм/с) спиралевидные движения нитей усиливаются, поскольку они пытаются поймать множествоы оздень образующих пучки, которые лучше противостоят дрейфующей силе потока. Xa isantya sokuhamba kwamanzi siphezulu (V > 33 µm/s), iintshukumo ezijikelezayo zemicu ziyanda njengoko zizama ukubamba ii-spermatozoa ezininzi ezibumba amaqoqo anokukwazi ukumelana namandla okuhamba kwamanzi.在高流速(V > 33 µm/s)时，螺纹的螺旋运动增加，以试图捕捉许多形成束的单个精子，从而更好地抵抗流动的。在 高 流速 (v> 33 µm/s) 时 ， 的 螺旋 运动 增加 , 以抵抗 的 漂移力。。。。。. При высоких скоростях потока (V > 33 мкм/с) спиральное движение нитей увеличивается в попытке захватить множество отделььное движение нитей увеличивается пучки, чтобы лучше сопротивляться силам дрейфа потока. Xa isantya sokuhamba kwamanzi siphezulu (V > 33 µm/s), intshukumo ye-helical yee-filaments iyanda xa kuzama ukubamba ii-spermatozoa ezininzi ezibumba amaqoqo ukuze zimelane ngcono namandla okuhamba kwamanzi.Bazama nokudibanisa ii-microchannels kwiindonga ezisecaleni.
Ii-sperm bundles zichongiwe njengee-clusters zeentloko zesidoda kunye nemisila egobileyo kusetyenziswa i-light microscopy (LM). Ii-sperm bundles ezinee-aggregates ezahlukeneyo zichongiwe njengee-twisted heads kunye ne-flagellar aggregates, i-multiple fused sperm tails, ii-sperm heads zinamathele kumsila, kunye nee-sperm heads ezine-bent nuclei njenge-multiple fused nuclei. transmission electron microscopy (TEM). I-scanning electron microscopy (SEM) ibonise ukuba ii-sperm bundles zazizi-sheathed aggregates zeentloko zesidoda kwaye ii-sperm aggregates zibonise inethiwekhi edibeneyo yemisila egobileyo.
Imo kunye nolwakhiwo oluphezulu lwe-spermatozoa, ukwakheka kwee-bundles ze-spermatozoa kufundwe kusetyenziswa i-light microscopy (isiqingatha secandelo), i-scanning electron microscopy (SEM) kunye ne-transmission electron microscopy (TEM), ii-sperm smears zadaywa nge-acridine orange zaza zahlolwa kusetyenziswa i-epifluorescence microscopy.
Ukufakwa kwe-sperm smear nge-acridine orange (Umzobo 3B) kubonise ukuba iintloko ze-sperm zazinamathelene kwaye zigqunywe ngezinto ezikhupha ulwelo, nto leyo eyakhokelela ekwakhiweni kwee-tufts ezinkulu (Umzobo 3D). Ii-sperm bundles zaziquka ii-sperm aggregates ezinenethiwekhi yemisila enamatheleyo (Umzobo 4A-C). Ii-sperm bundles zenziwe yimisila ye-spermatozoa ezininzi ezinamatheleneyo (Umzobo 4D). Iimfihlo (Umzobo 4E,F) zazigubungela iintloko ze-spermatozoa bundles.
Ukwakheka kwe-spermatozoa bundle Ukusebenzisa i-phase contrast microscopy kunye ne-sperm smears ezidaywe nge-acridine orange, kubonise ukuba iintloko ze-spermatozoa ziyanamathelana. (A) Ukwakheka kwe-sperm tuft kwasekuqaleni kuqala nge-sperm (isangqa esimhlophe) kunye ne-sperm ezintathu (isangqa esityheli), apho i-spiral iqala emsila ize iphele entloko. (B) I-Photomicrograph ye-sperm smear edaywe nge-acridine orange ebonisa iintloko ze-sperm ezinamatheleyo (iintolo). Ukukhupha kugubungela intloko (iintloko). Ukukhulisa × 1000. (C) Uphuhliso lomqadi omkhulu othuthwa ngokuhamba kwi-microfluidic channel (kusetyenziswa ikhamera yesantya esiphezulu kwi-950 fps). (D) I-Micrograph ye-sperm smear edaywe nge-acridine orange ebonisa ii-tufts ezinkulu (iintolo). Ukukhulisa: ×200.
Ukuskena i-electron micrograph ye-sperm beam kunye ne-sperm smear enemibala ye-acridine orange. (A, B, D, E) zii-electron micrographs ze-spermatozoa ezinombala wedijithali, kwaye u-C no-F zii-micrographs ze-acridine orange stained sperm smears ezibonisa ukuncamatheliswa kwe-spermatozoa ezininzi ezigquma i-caudal web. (AC) Ii-sperm aggregates ziboniswa njengenethiwekhi yemisila eqhotyoshelweyo (iintolo). (D) Ukuncamatheliswa kwe-spermatozoa eliqela (enento enamathelayo, umgca opinki, utolo) ejikeleza umsila. (E no-F) Ii-sperm head aggregates (iinkhomba) ezigqunywe ngezinto ezinamathelayo (iinkhomba). I-spermatozoa yenze ii-bundles ezinezakhiwo ezininzi ezifana ne-vortex (F). (C) ×400 kunye (F) ×200 magnifications.
Sisebenzisa i-transmission electron microscopy, sifumanise ukuba ii-sperm bundles zinemisila enamatheleyo (Umzobo 6A, C), iintloko ezinamatheleyo kwimisila (Umzobo 6B), okanye iintloko ezinamatheleyo kwimisila (Umzobo 6D). Iintloko ze-spermatozoa kwi-bundle zigobile, zibonakala kwicandelo lesibini lemimandla yenyukliya (Umzobo 6D). Kwi-incision bundle, i-spermatozoa yayinentloko ejijekileyo enemimandla emibini yenyukliya kunye nemimandla emininzi ye-flagellar (Umzobo 5A).
I-electron micrograph enemibala yedijithali ebonisa imisila edibanisayo kwi-sperm bundle kunye nezinto ezidibanisayo ezidibanisa iintloko zesidoda. (A) Umsila oncamathelisiweyo wenani elikhulu lesidoda. Qaphela indlela umsila obonakala ngayo kwi-portrait (utolo) kunye ne-landscape (utolo) projections. (B) Intloko (utolo) yesidoda iqhagamshelwe kumsila (utolo). (C) Imisila emininzi yesidoda (iintolo) iqhagamshelwe. (D) Izinto ezidibanisayo (AS, luhlaza okwesibhakabhaka) ziqhagamshelwe iintloko zesidoda ezine (ezimfusa).
I-scanning electron microscopy yasetyenziswa ukufumanisa iintloko zesidoda kwii-sperm bundles ezigqunywe yi-secretions okanye ii-membranes (Umfanekiso 6B), nto leyo ebonisa ukuba ii-sperm bundles zazinamathele kwizinto ezingaphandle kwe-extracellular. Izinto ezihlanganisiweyo zazigxile kwi-sperm head (i-jellyfish head-like assembly; Umzobo 5B) kwaye zandiswa kude, zinika imbonakalo etyheli eqaqambileyo phantsi kwe-fluorescence microscopy xa zidaywe nge-acridine orange (Umzobo 6C). Le nto ibonakala ngokucacileyo phantsi kwe-scanning microscope kwaye ithathwa njenge-binder. Iindawo ezincinci (Umzobo 5C) kunye ne-sperm smears ezidaywe nge-acridine orange zibonise ii-sperm bundles eziqulethe iintloko ezixineneyo kunye nemisila egobileyo (Umzobo 5D).
Iifotomicrograph ezahlukeneyo ezibonisa ukuhlanganiswa kweentloko zesidoda kunye nemisila egobileyo kusetyenziswa iindlela ezahlukeneyo. (A) I-electron micrograph yedijithali yokudlulisa umbala wedijithali enqamlezileyo ye-sperm bundle ebonisa intloko yesidoda egobileyo ene-nucleus enamacandelo amabini (eluhlaza okwesibhakabhaka) kunye neendawo ezininzi ze-flagellar (eziluhlaza). (B) I-electron micrograph eskena ngombala wedijithali ebonisa iqela leentloko zesidoda ezifana ne-jellyfish (iintolo) ezibonakala ngathi zigqunyiwe. (C) Icandelo elincinci kakhulu elibonisa iintloko zesidoda eziqokelelweyo (iintolo) kunye nemisila egokelelweyo (iintolo). (D) I-Micrograph ye-sperm smear enemibala ye-acridine orange ebonisa ukuhlanganiswa kweentloko zesidoda (iintolo) kunye nemisila egokelelweyo (iintolo). Qaphela ukuba into enamathelayo (S) igubungela intloko yesidoda. (D) × 1000 magnification.
Ngokusebenzisa i-transmission electron microscopy (Umzobo 7A), kwaphawulwa nokuba iintloko zesidoda zazijijekile kwaye iinuclei zazinesimo esijikelezayo, njengoko kuqinisekiswa yi-sperm smears edaywe nge-acridine orange kwaye yahlolwa kusetyenziswa i-fluorescence microscopy (Umzobo 7B).
(A) I-digital color transmission electron micrograph kunye (B) I-Acridine orange stained sperm smear ebonisa iintloko ezijijekileyo kunye nokuncamatheliswa kweentloko zesidoda kunye nemisila (iintolo). (B) × 1000 magnification.
Into enomdla efunyenweyo kukuba isidoda sikaSharkazi siyahlangana ukuze senze iibhanti ezithambileyo ezihambayo. Iimpawu zezi bhanti zisenza siqonde indima yazo enokubakho ekufunxeni nasekugcinweni kwe-spermatozoa kwi-SST.
Emva kokudibana, isidoda singena kwilungu lobufazi size singene kwinkqubo yokukhetha enzulu, nto leyo ebangela ukuba kubekho inani elilinganiselweyo lesidoda esingena kwi-SST15,16. Ukuza kuthi ga ngoku, iindlela isidoda esingena nesiphuma ngazo kwi-SST azicaci. Kwiinkuku, isidoda sigcinwa kwi-SST ixesha elide leeveki ezi-2 ukuya kwezili-10, kuxhomekeke kuhlobo6. Impikiswano isasele malunga nemeko yesidoda ngexesha lokugcinwa kwi-SST. Ngaba ziyashukuma okanye ziphumle? Ngamanye amazwi, iiseli zesidoda zigcina njani indawo yazo kwi-SST ixesha elide kangaka?
UForman4 ucebise ukuba ukuhlala kunye nokukhutshwa kwe-SST kunokuchazwa ngokwendlela yokunyakaza kwe-sperm. Ababhali bacinga ukuba i-sperm igcina indawo yayo ngokudada ngokuchasene nokuhamba kolwelo oludalwa yi-SST epithelium kwaye i-sperm ikhutshwa kwi-SST xa isantya sayo siwela ngaphantsi kwendawo apho iqala ukubuyela umva ngenxa yokunqongophala kwamandla. UZaniboni5 uqinisekisile ukubakho kwe-aquaporins 2, 3, kunye ne-9 kwinxalenye ye-apical yee-SST epithelial cells, ezinokuxhasa ngokungathanga ngqo imodeli yokugcina i-sperm kaForeman. Kolu phononongo lwangoku, sifumanise ukuba phantse isiqingatha se-spermatozoa kaSharkashi sibonisa i-rheology entle kulwelo oluhambayo, kwaye ii-sperm bundles ezihlanganisiweyo zonyusa inani le-spermatozoa ezibonisa i-rheology entle, nangona i-agglutination izicothisa. Indlela ii-sperm cells ezihamba ngayo kwi-fallopian tube yentaka ukuya kwindawo yokuchumisa ayiqondakali ngokupheleleyo. Kwizilwanyana ezanyisayo, i-follicular fluid chemoattracks spermatozoa. Nangona kunjalo, ii-chemoattractants zikholelwa ukuba zikhokelela i-spermatozoa ukuba isondele kumgama omde7. Ngoko ke, ezinye iindlela zinoxanduva lokuthuthwa kwesidoda. Amandla esidoda okujika nokuhamba ngokuchasene nolwelo lwetyhubhu ye-fallopian olukhutshwa emva kokudibana kuye kwaxelwa ukuba yeyona nto iphambili ekujoliseni isidoda kwiimpuku. UParker 17 ucebise ukuba isidoda siwele ii-oviducts ngokudada ngokuchasene nomsinga we-ciliary kwiintaka nakwizilwanyana ezirhubuluzayo. Nangona kungakhange kuboniswe ngovavanyo kwiintaka, u-Adolphi18 wayengowokuqala ukufumanisa ukuba isidoda seentaka sinika iziphumo ezilungileyo xa umaleko omncinci wolwelo phakathi kwe-coverlip kunye ne-slide wenziwa ngephepha lesihluzo. I-Rheology. UHino noYanagimachi [19] babeke i-ovary-tubal-uterine complex yegundane kwiringi yokuphalaza amanzi kwaye bafaka i-1 µl ye-inki kwi-isthmus ukuze babone ukuhamba kolwelo kwityhubhu ye-fallopian. Baqaphele intshukumo esebenzayo kakhulu yokuxinana nokuphumla kwityhubhu ye-fallopian, apho zonke iibhola ze-inki zazisiya ngokuthe chu kwi-ampulla yetyhubhu ye-fallopian. Ababhali bagxininisa ukubaluleka kokuhamba kolwelo lwe-tubal ukusuka kwityhubhu esezantsi ukuya kwityhubhu ephezulu ye-fallopian ukuze kunyuswe isidoda kwaye kuchumiswe. UBrillard20 uxele ukuba kwiinkukhu nakwii-turkeys, i-spermatozoa ifuduka ngokushukuma okusebenzayo ukusuka emnyango we-vaginal, apho igcinwa khona, ukuya kwindawo yokuhlangana kwe-utero-vaginal, apho igcinwa khona. Nangona kunjalo, le ntshukumo ayifuneki phakathi kwe-uterovaginal junction kunye ne-infundibulum kuba i-spermatozoa ithuthwa ngokufuduka okungenamsebenzi. Ukwazi ezi ngcebiso zangaphambili kunye neziphumo ezifunyenweyo kuphononongo lwangoku, kunokucingelwa ukuba amandla e-spermatozoa okuhamba phezulu (rheology) yenye yeempawu apho inkqubo yokukhetha isekelwe khona. Oku kumisela ukudlula kwe-spermatozoa kwi-vaginal kunye nokungena kwayo kwi-CCT ukuze igcinwe. Njengoko uForman4 ecebisile, oku kunokwenza lula inkqubo ye-sperm ingena kwi-SST kunye nendawo yayo yokuhlala kangangexesha elithile ize iphume xa isantya sayo siqala ukuhla.
Kwelinye icala, uMatsuzaki noSasanami 21 bacebisa ukuba i-spermatozoa yeentaka itshintshe indlela eshukuma ngayo ukusuka ekubeni buthathaka ukuya ekubeni buthathaka kwindlela yokuzala yamadoda nabafazi. Ukuthintelwa kwendlela eshukuma ngayo i-sperm kwi-SST kuye kwacetyiswa ukuchaza ixesha elide lokugcina i-sperm kunye nokuvuselelwa kwayo emva kokuphuma kwi-SST. Phantsi kweemeko ezinobungozi, uMatsuzaki et al. 1 baxele ukuba i-lactate ifumaneka kakhulu kwaye ikhutshwa kwi-SST, nto leyo enokubangela ukuba i-sperm ishukume. Kule meko, ukubaluleka kwe-sperm rheology kubonakala ekukhetheni nasekufunxeni i-spermatozoa, kungekhona ekugcinweni kwayo.
Ipateni yokuhlanganiswa kwesidoda ithathwa njengengcaciso enokwenzeka yexesha elide lokugcina isidoda kwi-SST, njengoko le yindlela eqhelekileyo yokugcina isidoda kwiinkuku2,22,23. UBakst et al. 2 baqaphele ukuba uninzi lwe-spermatozoa lunamathelana, lwenza ii-aggregates ze-fascicular, kwaye i-spermatozoa enye yayingafumaneki rhoqo kwi-CCM ye-quail. Kwelinye icala, uWen et al. 24 baqaphele i-spermatozoa esasazekileyo kunye nee-tufts ezimbalwa ze-spermatozoa kwi-lumen ye-SST kwiinkukhu. Ngokusekelwe kwezi ngqwalasela, kunokucingelwa ukuba ukuthambekela kokuhlanganiswa kwesidoda kwahlukile phakathi kweentaka kunye naphakathi kwe-spermatozoa kwi-ejaculate efanayo. Ukongeza, uVan Krey et al. 9 bacebise ukuba ukuhlukana okungacwangciswanga kwe-spermatozoa edibeneyo kunoxanduva lokungena kancinci kwe-spermatozoa kwi-lumen ye-fallopian tube. Ngokwale ngcamango, i-spermatozoa enekhono eliphantsi lokuhlanganiswa kwesidoda kufuneka ikhutshwe kwi-SST kuqala. Kule meko, amandla e-spermatozoa okudibanisa anokuba yinto echaphazela iziphumo zokhuphiswano lwe-sperm kwiintaka ezingcolileyo. Ukongeza, okukhona i-sperm edibeneyo ihlukana ixesha elide, kokukhona inzala ihlala ixesha elide.
Nangona ukuhlanganiswa kwe-spermatozoa kunye nokuhlanganiswa kwayo kube zii-bundles kuye kwabonwa kwizifundo ezininzi2,22,24, azichazwanga ngokweenkcukacha ngenxa yobunzima bokuqwalaselwa kwayo kwi-kinematic ngaphakathi kwe-SST. Kuye kwenziwa imizamo emininzi yokufunda ukuhlanganiswa kwe-sperm in vitro. Ukuhlanganiswa okukhulu kodwa okwethutyana kwabonwa xa ucingo oluncinci lususwa kwi-drop yembewu exhonyiweyo. Oku kukhokelela kwinto yokuba i-bubble ende iphuma kwi-drop, ixelisa i-seminal gland. Ngenxa yokunqongophala kwe-3D kunye namaxesha amafutshane okomisa nge-drip, yonke ibhloko yawa ngokukhawuleza9. Kolu phononongo lwangoku, sisebenzisa iinkukhu zeSharkashi kunye nee-microfluidic chips, sikwazile ukuchaza indlela ezi tyhufts ezakha ngayo kunye nendlela ezihamba ngayo. Ii-sperm bundles zakhiwa kwangoko emva kokuqokelelwa kwe-sperm kwaye zafunyaniswa zihamba kwi-spiral, zibonisa i-rheology entle xa ikhona kwi-flow. Ngaphezu koko, xa zijongwa nge-macroscopically, ii-sperm bundles ziye zabonwa ukuba zonyusa ulungelelwaniso lwe-motility xa kuthelekiswa ne-spermatozoa ehlukanisiweyo. Oku kuthetha ukuba ukuhlanganiswa kwesidoda kuyenzeka ngaphambi kokuba kungene i-SST kwaye ukuveliswa kwesidoda akupheleli kwindawo encinci ngenxa yoxinzelelo njengoko bekucetyisiwe ngaphambili (iTingari kunye neLake12). Ngexesha lokwakheka kwe-tuft, i-spermatozoa idada kwi-synchrony ide yenze i-junction, emva koko imisila yayo iyajikelezana kwaye intloko yesidoda ihlala ikhululekile, kodwa umsila kunye nenxalenye ekude yesidoda zinamathelana kunye nento enamathelayo. Ke ngoko, intloko ekhululekileyo yesidoda inoxanduva lokuhamba, itsala yonke i-ligament. Ukuskena i-electron microscopy yee-bundles zesidoda kubonise iintloko zesidoda ezinamatheleyo zigqunywe ngezinto ezininzi ezinamathelayo, nto leyo ebonisa ukuba iintloko zesidoda zazinamathele kwii-bundles eziphumlayo, okunokwenzeka ukuba kwenzeke emva kokufika kwindawo yokugcina (i-SST).
Xa i-sperm smear ipeyintwe nge-acridine orange, izinto ezinamathelayo ezingaphandle kweseli zesidoda zinokubonwa phantsi kwe-microscope ye-fluorescent. Le nto ivumela ii-sperm bundles ukuba zinamathele kwaye zinamathele kuyo nayiphi na indawo okanye amasuntswana ajikelezileyo ukuze angashukumi kunye nokuhamba okujikelezileyo. Ngoko ke, izinto esizibonileyo zibonisa indima yokunamathela kwe-spermatozoa ngendlela yee-mobile bundles. Ukukwazi kwazo ukudada ngokuchasene nomsinga kunye nokunamathela kwiindawo ezikufutshane kuvumela isidoda ukuba sihlale ixesha elide kwi-SST.
URothschild25 usebenzise ikhamera ye-hemocytometry ukuze afunde ukusasazwa okudadayo kwesidoda senkomo kwithontsi lokumiswa, ethatha iifotomicrographs ngekhamera ene-axis ethe nkqo nethe tye ye-microscope. Iziphumo zibonise ukuba isidoda sitsaleleke kumphezulu wegumbi. Ababhali bacebisa ukuba kunokubakho ukusebenzisana kwe-hydrodynamic phakathi kwesidoda nomphezulu. Ukuqwalasela oku, kunye nokukwazi kwesidoda senkukhu sikaSharkashi ukwenza ii-tufts ezinamathelayo, kunokunyusa amathuba okuba isidoda sinamathele eludongeni lwe-SST kwaye sigcinwe ixesha elide.
UBccetti kunye no-Afzeliu26 baxele ukuba i-glycocalyx yesidoda iyimfuneko ukuze kuqatshelwe i-gamete kunye ne-agglutination. UForman10 waphawula ukuba i-hydrolysis ye-α-glycosidic bonds kwi-glycoprotein-glycolipid coatings ngokunyanga isidoda seentaka nge-neuraminidase ibangele ukuncipha kokuzala ngaphandle kokuchaphazela ukuhamba kwesidoda. Ababhali bacebisa ukuba isiphumo se-neuraminidase kwi-glycocalyx siphazamisa ukuhlukaniswa kwesidoda kwindawo yokudibana kwesidoda nesidoda, ngaloo ndlela kunciphisa ukuzala. Izinto abazibonileyo azinakuyityeshela into yokuba unyango lwe-neuraminidase lunokunciphisa ukwamkelwa kwesidoda kunye ne-oocyte. UForman no-Engel10 bafumanise ukuba ukuzala kunciphile xa iinkukhu zifakwa isidoda ngaphakathi kwesidoda nge-semen ephathwa nge-neuraminidase. Nangona kunjalo, i-IVF ngesidoda ephathwe nge-neuraminidase ayizange ichaphazele ukuzala xa kuthelekiswa neenkukhu ezilawulayo. Ababhali bagqibe kwelokuba utshintsho kwi-glycoprotein-glycolipid coating ejikeleze i-sperm membrane lunciphise amandla esidoda okuchumisa ngokuphazamisa ukuthuthwa kwesidoda kwindawo yokudibana kwesidoda nesidoda, nto leyo eyandisa ukulahleka kwesidoda ngenxa yesantya sokudibana kwesidoda nesidoda, kodwa ayichaphazeli ukuqondwa kwesidoda neqanda.
Kwiiturkeys uBakst noBauchan 11 bafumene amaqhekeza amancinci e-vesicles kunye ne-membrane kwi-lumen ye-SST baza baphawula ukuba ezinye zezi granules zidibene ne-sperm membrane. Ababhali bacebisa ukuba olu lwalamano lunokuba negalelo ekugcinweni kwexesha elide kwe-spermatozoa kwi-SST. Nangona kunjalo, abaphandi abazange bachaze umthombo wezi nxalenye, nokuba zikhutshwe zii-CCT epithelial cells, eziveliswa kwaye zikhutshwe yinkqubo yokuzala yamadoda, okanye eziveliswa yi-sperm ngokwayo. Kwakhona, ezi nxalenye zinoxanduva lwe-agglutination. UGrützner et al27 baxele ukuba ii-epididymal epithelial cells zivelisa kwaye zikhuphe iproteni ethile efunekayo ekwakhiweni kwe-single-pore seminal tracts. Ababhali baxela nokuba ukusasazeka kwezi nqwaba kuxhomekeke ekusebenzisaneni kweeproteni ze-epididymal. UNixon et al28 bafumanise ukuba i-adnexa ikhupha iproteni, i-acidic cysteine-rich osteonectin; i-SPARC ibandakanyeka ekwakhiweni kwee-sperm tufts kwi-echidnas ezimfutshane kunye ne-platypuses. Ukusasazeka kwezi beams kunxulunyaniswa nokulahleka kwale protein.
Kolu phononongo lwangoku, uhlalutyo lwe-ultrastructural olusebenzisa i-electron microscopy lubonise ukuba i-spermatozoa inamathele kwinani elikhulu lezinto ezixineneyo. Ezi zinto zicingelwa ukuba zinoxanduva lwe-agglutination ehlangana phakathi nangaphakathi kweentloko ezinamathelayo, kodwa xa ziphantsi kakhulu kwindawo yomsila. Sicinga ukuba le nto idibanisayo ikhutshwa kwinkqubo yokuzala yamadoda (i-epididymis okanye i-vas deferens) kunye ne-semen, kuba sihlala sibona i-semen yahlukana ne-lymph kunye ne-seminal plasma ngexesha lokuphuma kwe-ejaculation. Kuye kwaxelwa ukuba njengoko i-avian spermatozoa idlula kwi-epididymis kunye ne-vas deferens, zenzeka utshintsho olunxulumene nokuvuthwa oluxhasa amandla azo okubopha iiproteni kunye nokufumana i-plasma lemma-associated glycoproteins. Ukuhlala kwezi proteni kwi-resident sperm membranes kwi-SST kubonisa ukuba ezi proteni zinokuchaphazela ukufunyanwa kozinzo lwe-sperm membrane 30 kwaye zichonge ukuzala kwazo 31. U-Ahammad et al32 baxele ukuba i-spermatozoa efunyenwe kwiindawo ezahlukeneyo zenkqubo yokuzala yamadoda (ukusuka kuma-testes ukuya kwi-distal vas deferens) ibonise ukunyuka okuqhubekayo kokuphila phantsi kweemeko zokugcina ulwelo, nokuba ubushushu bokugcina bunjani, kwaye ukuphila kwiinkukhu kuyanda nakwiityhubhu ze-fallopian emva kokufakelwa i-artificial insemination.
Ii-tufts ze-chicken sperm tufts zeSharkashi zineempawu kunye nemisebenzi eyahlukileyo kunezinye iintlobo ezifana ne-echidnas, i-platypuses, iimpuku zomthi, iimpuku ze-deer, kunye nee-guinea pigs. Kwiinkukhu ze-sharkasi, ukwenziwa kwee-bundles ze-spermatozoa kunciphisa isantya sazo sokudada xa kuthelekiswa ne-single spermatozoa. Nangona kunjalo, ezi bundles zonyuse ipesenti ye-rheologically positive spermatozoa kwaye zonyusa amandla e-spermatozoa okuzinza kwindawo eguquguqukayo. Ke ngoko, iziphumo zethu ziqinisekisa isiphakamiso sangaphambili sokuba i-sperm agglutination kwi-SST inxulunyaniswa nokugcinwa kwesidoda ixesha elide. Sikwacinga ukuba ukuthambekela kwesidoda ekwenzeni ii-tufts kunokulawula izinga lokulahleka kwesidoda kwi-SST, okunokutshintsha iziphumo zokhuphiswano lwesidoda. Ngokwale ngcinga, i-spermatozoa enekhono eliphantsi le-agglutination ikhupha i-SST kuqala, ngelixa i-spermatozoa enekhono eliphezulu le-agglutination ivelisa uninzi lwenzala. Ukwenziwa kwee-bundles ze-sperm ezine-pore enye kuluncedo kwaye kuchaphazela umlinganiselo womzali nomntwana, kodwa kusebenzisa indlela eyahlukileyo. Kwi-echidnas kunye ne-platypuses, i-spermatozoa icwangciswe ngaxeshanye ukuze kwandiswe isantya sokuya phambili kwe-beam. Iibhanti ze-echidnas zihamba ngokukhawuleza ngokuphindwe kathathu kune-spermatozoa enye. Kukholelwa ukuba ukwakheka kwezi sperm tufts kwi-echidnas yindlela yokuziqhelanisa ukuze kugcinwe ulawulo, kuba amabhinqa aziphatha kakubi kwaye adla ngokudibana namadoda amaninzi. Ke ngoko, i-spermatozoa evela kwii-ejaculates ezahlukeneyo ikhuphisana ngamandla ngokuchumisa iqanda.
I-spermatozoa ehlanganisiweyo yeenkukhu ze-sharkasi kulula ukuyicinga kusetyenziswa i-phase contrast microscopy, ethathwa njengenzuzo kuba ivumela ukufunda lula ukuziphatha kwe-spermatozoa kwi-vitro. Indlela yokwenziwa kwe-sperm tuft ekhuthaza ngayo ukuzala kwiinkukhu ze-sharkasi nayo yahlukile kuleyo ibonwa kwezinye izilwanyana ezincancisayo ze-placenta ezimele ukuziphatha kwe-sperm okusebenzisanayo njengeempuku zomthi, apho ezinye ii-spermatozoa zifikelela emaqandeni, zinceda abanye abantu abanxulumene nabo ukufikelela nokonakalisa amaqanda abo. ukuze uzibonakalise. ukuziphatha okunobubele. Ukuzichumisa 34. Omnye umzekelo wokuziphatha kwentsebenziswano kwi-spermatozoa wafunyanwa kwiimpuku zenyamakazi, apho i-spermatozoa yakwazi ukuchonga nokudibanisa ne-spermatozoa enxulumene kakhulu nofuzo kwaye yenze amaqela entsebenziswano ukunyusa isantya sazo xa kuthelekiswa ne-spermatozoa enganxulumananga35.
Iziphumo ezifunyenwe kolu phononongo aziphikisani nengcamango kaFoman yokugcina i-spermatozoa ixesha elide kwi-SWS. Abaphandi baxela ukuba ii-sperm cells ziyaqhubeka nokuhambahamba kwii-epithelial cells ezigqume i-SST ixesha elide, kwaye emva kwexesha elithile, iindawo zokugcina amandla ee-sperm cells ziyaphela, nto leyo ebangela ukwehla kwesantya, okuvumela ukukhutshwa kwezinto ezincinci ezinobunzima be-molecular. amandla e-spermatozoa ngokuhamba kolwelo oluvela kwi-lumen ye-SST. Kwisitho setyhubhu ye-fallopian. Kolu phononongo lwangoku, siqaphele ukuba isiqingatha se-sperm enye sibonisa amandla okudada ngokuchasene nolwelo oluhambayo, kwaye ukunamathela kwazo kwi-bundle kwandisa amandla azo okubonisa i-rheology elungileyo. Ngaphezu koko, idatha yethu iyahambelana naleyo kaMatsuzaki et al. 1 ababike ukuba ukwanda kwe-lactate secretion kwi-SST kunokuthintela ukuhambahamba kwe-sperm ehlala apho. Nangona kunjalo, iziphumo zethu zichaza ukwakheka kwe-sperm motile ligaments kunye nokuziphatha kwazo kwe-rheological xa kukho indawo eguquguqukayo ngaphakathi kwe-microchannel ukuzama ukucacisa ukuziphatha kwazo kwi-SST. Uphando lwexesha elizayo lunokugxila ekumiseleni ukwakheka kweekhemikhali kunye nemvelaphi yearhente edibanisayo, nto leyo ngokungathandabuzekiyo eya kunceda abaphandi ukuba baphuhlise iindlela ezintsha zokugcina isidoda esimanzi kunye nokwandisa ixesha lokuzala.
I-sharkasi yendoda ene-braai ene-homozygous dominant (i-Na Na) eneeveki ezingama-30 ubudala ikhethwe njengabanikeli besidoda kolu phando. Iintaka zikhuliswe kwiFama yeeNkukhu zoPhando lweSebe lezoLimo, kwiYunivesithi yaseAshit, kwiPhondo laseAshit, eJiphutha. Iintaka zagcinwa kwiikheyiji ezahlukeneyo (30 x 40 x 40 cm), zafakwa kwinkqubo yokukhanya (iiyure ezili-16 zokukhanya kunye neeyure ezisi-8 zobumnyama) kwaye zondliwa ukutya okuqulethe i-160 g yeproteni eluhlaza, i-2800 kcal yamandla anyibilikiswayo, i-35 g ye-calcium nganye. Iigram ezi-5 ze-phosphorus ekhoyo ngekhilogram yokutya.
Ngokwedatha 36, ​​​​37, i-semen yaqokelelwa kumadoda ngokuhlikihla isisu. Iisampulu ze-semen ezingama-45 zaqokelelwa kumadoda ali-15 kwiintsuku ezi-3. I-semen (n = 15/ngosuku) yaxutywa ngoko nangoko nge-1:1 (v:v) nge-Belsville Poultry Semen Diluent, equlethe i-potassium diphosphate (1.27 g), i-monosodium glutamate monohydrate (0.867 g), i-fructose (0.5 d) i-anhydrous sodium. acetate (0.43 g), i-tris(hydroxymethyl)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), i-pH = 7, 5, i-osmolarity 333 mOsm/kg38. Iisampulu zesidoda ezixutyiweyo zaqala zahlolwa phantsi kwe-microscope ekhanyayo ukuqinisekisa umgangatho olungileyo wesidoda (ukufuma) zaza zagcinwa kwindawo yokuhlambela ngamanzi kwi-37°C de zisetyenziswe kwisithuba sesiqingatha seyure emva kokuqokelelwa.
I-kinematics kunye ne-rheology ye-spermatozoa zichazwe kusetyenziswa inkqubo yezixhobo ze-microfluidic. Iisampuli ze-spermatozoa zaphinda zaxutywa ukuya kwi-1:40 kwi-Beltsville Avian Semen Diluent, zafakwa kwisixhobo se-microfluidic (jonga ngezantsi), kwaye iiparameter ze-kinetic zachongwa kusetyenziswa inkqubo ye-Computerized Semen Analysis (CASA) eyayiphuhliswe ngaphambili ukuze ichaze i-microfluidics. kwi-mobility ye-spermatozoa kwi-liquid media (iSebe lezobuNjineli boMatshini, iFakhalthi yoBuNjineli, iYunivesithi yase-Assiut, e-Egypt). I-plugin ingakhutshelwa apha: http://www.assiutmicrofluidics.com/research/casa39. I-curve velocity (VCL, μm/s), i-linear velocity (VSL, μm/s) kunye ne-avareji trajectory velocity (VAP, μm/s) zalinganiswa. Iividiyo ze-spermatozoa zathathwa kusetyenziswa i-inverted Optika XDS-3 phase contrast microscope (ene-40x objective) eqhagamshelwe kwikhamera ye-Tucson ISH1000 kwi-30 fps imizuzwana emi-3. Sebenzisa isoftware yeCASA ukufunda ubuncinane iindawo ezintathu kunye neendlela zesidoda ezingama-500 kwisampulu nganye. Ividiyo erekhodiweyo icutshungulwe kusetyenziswa iCASA eyenziwe ekhaya. Inkcazo yokuhambahamba kwi-CASA plug-in isekelwe kwisantya sokudada kwesidoda xa kuthelekiswa nesantya sokuhamba, kwaye ayibandakanyi ezinye iiparameter ezifana nokuhamba ecaleni, njengoko oku kufunyenwe kuthembekile ngakumbi ekuhambeni kolwelo. Ukuhamba kwe-Rheological kuchazwa njengokuhamba kweeseli zesidoda ngokuchasene necala lokuhamba kolwelo. I-Spermatozoa eneempawu ze-rheological yahlulwe ngenani le-spermatozoa ehambayo; i-spermatozoa eyayiphumle kwaye i-spermatozoa ehamba ngokuguquguqukayo ayifakwanga kwinani.
Zonke iikhemikhali ezisetyenzisiweyo zifunyenwe kwi-Elgomhoria Pharmaceuticals (eCairo, e-Egypt) ngaphandle kokuba kuchazwe ngenye indlela. Esi sixhobo senziwe njengoko kuchaziwe ngu-El-sherry et al. 40 kunye notshintsho oluthile. Izinto ezisetyenzisiweyo ukwenza ii-microchannels ziquka ii-glass plates (Howard Glass, Worcester, MA), i-SU-8-25 negative resist (MicroChem, Newton, CA), i-diacetone alcohol (eSigma Aldrich, eSteinheim, eJamani), kunye ne-polyacetone. -184, eDow Corning, eMidland, eMichigan). Ii-Microchannels zenziwe kusetyenziswa i-soft lithography. Okokuqala, i-face mask ecacileyo ekhuselayo enoyilo lwe-microchannel olufunwayo yaprintwa kwiprinta enesisombululo esiphezulu (ePrismatic, eCairo, e-Egypt nasePacific Arts and Design, eMarkham, ON). Ii-masters zenziwe kusetyenziswa ii-glass plates njenge-substrates. Ii-plates zacocwa kwi-acetone, i-isopropanol kunye namanzi acocekileyo zaza zagqunywa ngomaleko we-20 µm we-SU8-25 nge-spin coating (3000 rpm, 1 min). Emva koko iileya ze-SU-8 zomiswa kancinci (65°C, 2 min kunye 95°C, 10 min) zaza zavezwa kwimitha ye-UV imizuzwana engama-50. Bhaka emva kokuvezwa kwi-65°C kunye ne-95°C imizuzwana e-1 kunye nemizuzu emi-4 ukuya kwiileya ze-SU-8 ezivezwe yi-crosslink, kulandele uphuhliso kwi-diacetone alcohol imizuzu eyi-6.5. Bhaka ii-waffles ngamandla (200°C imizuzwana eli-15) ukuze uqinise ngakumbi umaleko we-SU-8.
I-PDMS yalungiswa ngokuxuba i-monomer kunye ne-hardener ngomlinganiselo wobunzima we-10:1, yaze yakhutshwa igesi kwi-vacuum cleaner yaza yagalelwa kwisakhelo esiphambili se-SU-8. I-PDMS yanyangwa kwi-oven (120°C, imizuzu engama-30), emva koko imijelo yasikwa, yahlulwa kwi-master, yaza yagqobhozwa ukuze kuvunyelwe ukuba iityhubhu zincamathiselwe kwindawo yokungena kunye neyokuphuma ye-microchannel. Ekugqibeleni, imijelo emincinci ye-PDMS yaqhotyoshelwa ngokusisigxina kwi-microscope slides kusetyenziswa iprosesa ye-corona ephathekayo (i-Electro-Technic Products, eChicago, IL) njengoko kuchaziwe kwenye indawo. I-microchannel esetyenziswe kolu phononongo ilinganisa i-200 µm × 20 µm (W × H) kwaye ubude bayo buyi-3.6 cm.
Ukuhamba kolwelo okubangelwa luxinzelelo lwe-hydrostatic ngaphakathi kwe-microchannel kufezekiswa ngokugcina inqanaba lolwelo kwi-inlet reservoir ngaphezulu komahluko wokuphakama Δh39 kwi-outlet reservoir (Umzobo 1).
apho u-f yi-coefficient of friction, echazwa njenge-f = C/Re yokuhamba kwe-laminar kwitshaneli engunxande, apho u-C eyinto engaguqukiyo ngokuxhomekeke kumlinganiselo we-aspect yetshaneli, u-L bubude betshaneli encinci, u-Vav yi-avareji yesantya ngaphakathi kwetshaneli encinci, u-Dh yi-hydraulic diameter yetshaneli, u-g - ukukhawulezisa umxhuzulane. Ukusebenzisa le nxaki, i-avareji yesantya setshaneli ingabalwa kusetyenziswa le nxaki ilandelayo: