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Ukuzala kwezinyoni kuncike emandleni azo okugcina isidoda esanele esiphilayo isikhathi eside kuma-tubules okugcina isidoda (SST). Indlela eqondile i-spermatozoa engena ngayo, ihlala kuyo, futhi iphume ngayo i-SST isalokhu iphikisana. Isidoda sezinkukhu ze-sharkasi sibonise ukuthambekela okuphezulu kokuhlanganiswa, sakha ama-bundles ahambayo ane-filamentous aqukethe amaseli amaningi. Ngenxa yobunzima bokubuka ukunyakaza kanye nokuziphatha kwe-spermatozoa epayipini le-fallopian elingacacile, sisebenzise idivayisi ye-microfluidic ene-microchannel cross-section efana neye-spermatozoa ukuze sifunde ukuhlanganiswa kwe-spermatozoa kanye nokuhamba kwayo. Lolu cwaningo luxoxa ngendlela ama-bundles esidoda akha ngayo, indlela ahamba ngayo, kanye nendima yawo engaba khona ekwandiseni ukuhlala kwesidoda ku-SST. Sihlole ijubane lesidoda kanye nokuziphatha kwe-rheological lapho ukugeleza koketshezi kukhiqizwa ngaphakathi kwesiteshi se-microfluidic ngengcindezi ye-hydrostatic (isilinganiso sokugeleza = 33 µm/s). I-spermatozoa ivame ukubhukuda ngokumelene nogesi (i-rheology enhle) futhi ijubane le-bundle ye-spermatozoon lincishiswe kakhulu uma kuqhathaniswa ne-spermatozoa eyodwa. Kuye kwabonakala ukuthi izinqwaba zesidoda zihamba ngendlela ejikelezayo futhi zanda ngobude nangobukhulu njengoba kutholwa isidoda esisodwa esiningi. Kubonwe izinqwaba zesidoda zisondela futhi zinamathela ezindongeni zemisele ye-microfluidic ukuze kugwenywe ukukhukhulwa ngesivinini sokugeleza koketshezi > 33 µm/s. Kubonwe izinqwaba zesidoda zisondela futhi zinamathela ezindongeni zemisele ye-microfluidic ukuze kugwenywe ukukhukhulwa ngesivinini sokugeleza koketshezi > 33 µm/s. Было замечено, что пучки сперматозоидов приближаются и прилипают к боковым стенкам микрофлюидных каналов, чтобы избениять смета жидкости> 33 мкм / с. Kuye kwabonakala ukuthi izinqwaba zesidoda zisondela futhi zinamathela ezindongeni eziseceleni zeziteshi ze-microfluidic ukuze kugwenywe ukukhukhulwa ngamazinga okugeleza koketshezi >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 сместо смести 33 mkm/s. Kuye kwabonakala ukuthi izinqwaba zesidoda zisondela futhi zinamathela ezindongeni eziseceleni zomsele we-microfluidic ukuze kugwenywe ukukhukhulwa ukugeleza koketshezi ku->33 µm/s.Ukuskena kanye ne-transmission electron microscopy kwembule ukuthi izinqwaba zesidoda zazisekelwa izinto eziningi ezixinene. Imininingwane etholakele ikhombisa ukuhamba okuyingqayizivele kwesidoda senkukhu iSharkazi, kanye nekhono lesidoda lokuhlanganisa nokwakha izinqwaba ezihambayo, okusiza ekuqondeni kangcono ukugcinwa kwesikhathi eside kwesidoda ku-SMT.
Ukuze kufezwe ukukhulelwa kubantu nasezilwaneni eziningi, isidoda namaqanda kumele kufike endaweni yokukhulelwa ngesikhathi esifanele. Ngakho-ke, ukukhulelwa kumele kwenzeke ngaphambi noma ngesikhathi sokuvuthwa. Ngakolunye uhlangothi, ezinye izilwane ezincelisayo, njengezinja, kanye nezinhlobo ezingezona izilwane ezincelisayo, njengezinambuzane, izinhlanzi, izilwane ezihuquzelayo, nezinyoni, zigcina isidoda ezithweni zazo zokuzala isikhathi eside kuze kube yilapho amaqanda azo eselungele ukukhulelwa (ukukhulelwa okungahambisani neminyaka 1). Izinyoni ziyakwazi ukugcina amandla e-spermatozoa ekwazi ukuvundisa amaqanda amasonto ama-2-102.
Lesi yisici esiyingqayizivele esihlukanisa izinyoni kwezinye izilwane, njengoba sinikeza amathuba aphezulu okukhulelwa ngemva kokutshalwa kanye amasonto amaningana ngaphandle kokuhlangana kanye nokuvuthwa ngesikhathi esisodwa. Isitho esiyinhloko sokugcina isidoda, esibizwa ngokuthi i-sperm storage tubule (SST), sitholakala emafolweni angaphakathi e-mucosal endaweni yokuhlangana kwe-uterovaginal. Kuze kube manje, izindlela isidoda esingena ngazo, esihlala futhi siphume ngazo ebhange lesidoda azikaqondakali ngokugcwele. Ngokusekelwe ezifundweni zangaphambilini, kuye kwavezwa imibono eminingi, kodwa ayikho neyodwa yazo eqinisekisiwe.
UForman4 ucabange ukuthi i-spermatozoa igcina indawo yayo emgodini we-SST ngokunyakaza okuqhubekayo kwe-oscillatory ngokumelene nokuqondiswa kokugeleza koketshezi ngeziteshi zamaprotheni ezitholakala kumaseli e-epithelial e-SST (rheology). I-ATP iyaphela ngenxa yomsebenzi oqhubekayo we-flagellar odingekayo ukugcina isidoda ku-lumen ye-SST futhi ukuhamba kwayo kugcina kwehla kuze kube yilapho isidoda senziwa ngaphandle kwesidoda ngokugeleza koketshezi futhi siqale uhambo olusha phansi kwepayipi le-fallopian elikhuphukayo ukuze livundise isidoda. Iqanda (Forman4). Le modeli yokugcina isidoda isekelwa ukutholakala kwe-immunocytochemistry yama-aquaporins 2, 3 kanye no-9 akhona kumaseli e-epithelial e-SST. Kuze kube manje, izifundo nge-rheology yesidoda senkukhu kanye nendima yayo ekugcinweni kwe-SST, ekukhethweni kwesidoda sesitho sangasese sowesifazane, kanye nokuncintisana kwesidoda azikho. Ezinkukhu, isidoda singena esitho sangasese sowesifazane ngemuva kokuhlangana ngokwemvelo, kodwa ngaphezu kuka-80% we-spermatozoa ikhishwa esitho sangasese sowesifazane ngemuva nje kokuhlangana. Lokhu kusikisela ukuthi isitho sangasese sowesifazane siyindawo eyinhloko yokukhethwa kwesidoda ezinyonini. Ngaphezu kwalokho, kubikwe ukuthi ngaphansi kuka-1% wesidoda esivundisiwe esithweni sangasese sowesifazane sigcina sise-SST2. Ekutshalweni kwezinkukhu ngesitho sangasese sowesifazane, inani lesidoda esifinyelela ku-SST livame ukwanda emahoreni angama-24 ngemva kokufakelwa isidoda. Kuze kube manje, indlela yokukhetha isidoda phakathi nale nqubo ayicaci, futhi ukuhamba kwesidoda kungadlala indima ebalulekile ekuthathweni kwesidoda se-SST. Ngenxa yodonga olujiyile nolungacacile lwamapayipi esitho sangasese sowesifazane, kunzima ukuqapha ngqo ukuhamba kwesidoda emapayipini esitho sangasese sowesifazane ezinyonini. Ngakho-ke, asinalo ulwazi oluyisisekelo lokuthi isidoda sishintshela kanjani ku-SST ngemva kokufakelwa isidoda.
I-Rheology isanda kuqashelwa njengesici esibalulekile esilawula ukuthuthwa kwesidoda ezithweni zangasese zezilwane ezincelisayo. Ngokusekelwe ekhonweni le-spermatozoa ehambayo lokufuduka ngokumelene nalokhu, uZaferani nabanye basebenzise uhlelo lwe-corra microfluidic ukuze bahlukanise i-spermatozoa ehambayo emasampeni esidoda abhalwe phansi. Lolu hlobo lokuhlunga isidoda lubalulekile ekwelashweni kokungabi nabantwana kwezokwelapha kanye nocwaningo lwezokwelapha, futhi luthandwa kunezindlela zendabuko ezithatha isikhathi futhi ezidinga umsebenzi omningi futhi ezingalimaza isimo sesidoda kanye nobuqotho besakhiwo. Kodwa-ke, kuze kube manje, azikho izifundo ezenziwe ngomphumela wokukhishwa kwezitho zangasese zezinkukhu ekunyakazeni kwesidoda.
Kungakhathaliseki ukuthi iyiphi indlela egcina isidoda sigcinwe ku-SST, abacwaningi abaningi baye baphawula ukuthi i-resident spermatozoa ihlangana ikhanda nekhanda ku-SST yezinkukhu ezingu-9, 10, quails 2, kanye nama-turkeys 11 ukuze kwakheke ama-agglutinated sperm bundles. Abalobi basikisela ukuthi kukhona ukuxhumana phakathi kwalokhu kuhlangana kanye nokugcinwa kwesikhathi eside kwe-spermatozoa ku-SST.
I-Tingari ne-Lake12 babike ubudlelwano obuqinile phakathi kwe-spermatozoa endlaleni ethola isidoda senkukhu futhi babuza ukuthi i-avian spermatozoa ihlangana ngendlela efanayo ne-mammalian spermatozoa. Bakholelwa ukuthi ukuxhumana okujulile phakathi kwesidoda kuma-vas deferens kungenzeka kungenxa yokucindezeleka okubangelwa ukuba khona kwenani elikhulu lesidoda endaweni encane.
Uma kuhlolwa ukuziphatha kwe-spermatozoa kuma-slide engilazi alengayo amasha, izimpawu zesikhashana ze-agglutination zingabonakala, ikakhulukazi emaphethelweni amaconsi esidoda. Kodwa-ke, i-agglutination yayivame ukuphazamiseka isenzo sokujikeleza esihlotshaniswa nokunyakaza okuqhubekayo, okuchaza uhlobo lwesikhashana lwalesi simo. Abacwaningi baphinde baphawula ukuthi lapho i-diluent ingezwa esidoda, kwavela ama-aggregate amaseli amade “afana nentambo”.
Imizamo yokuqala yokulingisa isidoda yenziwa ngokususa ucingo oluncane ethonsini elilengayo, okwaholela ekutheni i-vesicle ende efana nesidoda iphume ethonsini lesidoda. I-spermatozoa yavele yaqondana ngendlela efanayo ngaphakathi kwe-vesicle, kodwa yonke iyunithi yanyamalala ngokushesha ngenxa yomkhawulo we-3D. Ngakho-ke, ukuze kufundwe i-spermatozoa agglutination, kubalulekile ukubona ukunyakaza kanye nokuziphatha kwe-spermatozoa ngqo kuma-tubules okugcina isidoda ahlukanisiwe, okunzima ukukufeza. Ngakho-ke, kuyadingeka ukuthuthukisa ithuluzi elilingisa i-spermatozoa ukusekela izifundo zokunyakaza kwesidoda kanye nokuziphatha kwe-agglutination. UBrillard et al13 babike ukuthi ubude obujwayelekile bama-tubules okugcina isidoda kumachwane amadala bungu-400–600 µm, kodwa amanye ama-SST angaba amade angu-2000 µm. I-Mero ne-Ogasawara14 bahlukanise izindlala ezi-seminiferous zaba ama-tubules okugcina isidoda akhulisiwe nalawo angakhulisiwe, womabili ayefana ngobude (~500 µm) kanye nobubanzi bentamo (~38 µm), kodwa ububanzi be-lumen obumaphakathi bama-tubules babungu-56.6 no-56.6 µm. . , ngokulandelana okungu-11.2 μm, ngokulandelana. Esifundweni samanje, sisebenzise idivayisi ye-microfluidic enosayizi wesiteshi ongu-200 µm × 20 µm (W × H), ingxenye yayo eseduze kancane neye-SST ekhulisiwe. Ngaphezu kwalokho, sihlole ukuhamba kwesidoda kanye nokuziphatha kwe-agglutination ku-fluid egelezayo, okuhambisana nombono kaForeman wokuthi uketshezi olukhiqizwa amaseli e-epithelial e-SST lugcina isidoda ku-lumen ngendlela ephikisanayo (ye-rheological).
Inhloso yalolu cwaningo kwakuwukunqoba izinkinga zokubuka ukunyakaza kwesidoda emhubheni we-fallopian nokugwema ubunzima bokufunda i-rheology kanye nokuziphatha kwesidoda endaweni eguquguqukayo. Kwasetshenziswa idivayisi encane ekhiqiza ingcindezi ye-hydrostatic ukuze ilingise ukunyakaza kwesidoda ezithweni zangasese zenkukhu.
Lapho ithonsi lesampula yesidoda esixutshwe (1:40) lifakwa kudivayisi ye-microchannel, izinhlobo ezimbili zokuhamba kwesidoda zingatholakala (isidoda esihlukanisiwe nesidoda esiboshiwe). Ngaphezu kwalokho, isidoda sasivame ukubhukuda ngokumelene nomsinga (i-rheology enhle; ividiyo 1, 2). Nakuba izinqwaba zesidoda zazinejubane eliphansi kunelesidoda esinomuntu oyedwa (p < 0.001), zandise iphesenti lesidoda esibonisa i-rheotaxis enhle (p < 0.001; Ithebula 2). Nakuba izinqwaba zesidoda zazinejubane eliphansi kunelesidoda esinomuntu oyedwa (p < 0.001), zandise iphesenti lesidoda esibonisa i-rheotaxis enhle (p < 0.001; Ithebula 2). Хотя пучки сперматозоидов имели более низкую скорость, чем у одиночных сперматозоидов (p <0,001), они увеличивали процент, сперматозоидов демонстрирующих положительный реотаксис (p < 0,001; таблица 2). Nakuba ama-spermatozoa bundles ayenejubane eliphansi kunele-single spermatozoa (p < 0.001), andise iphesenti le-spermatozoa elibonisa i-rheotaxis enhle (p < 0.001; Ithebula 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羾） 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 流叾(p <0.001 ; 2.......)）) Хотя скорость пучков сперматозоидов была ниже, чем у одиночных сперматозоидов (p <0,001), они увеличивали процент спедрман реологией (p < 0,001; таблица 2). Nakuba ijubane lama-sperm bundles laliphansi kunele-single spermatozoa (p < 0.001), bandise iphesenti le-spermatozoa nge-rheology enhle (p < 0.001; Ithebula 2).I-rheology enhle yesidoda esisodwa kanye nama-tufts ilinganiselwa cishe ku-53% kanye no-85%, ngokulandelana.
Kuye kwaphawulwa ukuthi i-spermatozoa yezinkukhu ze-sharkasi ngokushesha ngemva kokukhipha iqanda yakha izinqwaba eziqondile, eziqukethe inqwaba yabantu. Lezi zinqwaba zanda ngobude nobukhulu ngokuhamba kwesikhathi futhi zingase zihlale zise-vitro amahora amaningana ngaphambi kokunyamalala (ividiyo 3). Lezi zinqwaba ezinemicu zibunjwe njenge-spermatozoa ye-echidna eyakheka ekugcineni kwe-epididymis. I-Sharkashi hen seed itholakale inesimo esiphakeme sokuhlangana futhi yakhe inqwaba eqondile ngaphansi komzuzu owodwa ngemva kokuqoqwa. Lezi zinhlayiya zinamandla futhi ziyakwazi ukunamathela kunoma yiziphi izindonga eziseduze noma izinto ezimile. Nakuba izinqwaba zesidoda zinciphisa ijubane lamaseli esidoda, kusobala ukuthi ngokwe-macroscopic zikhulisa ukulingana kwazo. Ubude bezinqwaba buyahlukahluka kuye ngenani lesidoda esiqoqwe ngamaqoqo. Izingxenye ezimbili zesixha zahlukaniswa: ingxenye yokuqala, kufaka phakathi ikhanda elikhululekile lesidoda esihlanganisiwe, kanye nengxenye yokugcina, kufaka phakathi umsila kanye nokuphela konke okukude kwesidoda. Kusetshenziswa ikhamera yesivinini esikhulu (950 fps), amakhanda akhululekile e-spermatozoa ahlanganisiwe abonwa engxenyeni yokuqala ye-bundle, ephethe ukunyakaza kwe-bundle ngenxa yokunyakaza kwawo okuguquguqukayo, ehudula asele e-bundle ngokunyakaza okujikelezayo (Ividiyo 4). Kodwa-ke, kuma-tuft amade, kuye kwabonwa ukuthi amanye amakhanda e-sperm akhululekile anamathele emzimbeni kanti ingxenye yokugcina ye-tuft isebenza njengama-vanes ukusiza ukuhambisa i-tuft.
Ngesikhathi kugeleza kancane koketshezi, izinqwaba zesidoda zihamba zihambisana, nokho, ziqala ukugoqana futhi zinamathele kukho konke okusamile, ukuze zingagezwa ukugeleza kwamanje njengoba ijubane lokugeleza landa. Izinqwaba zakheka lapho inqwaba yamaseli esidoda isondelana, ziqala ukuhamba ngokuvumelana futhi zigoqene, bese zinamathela entweni enamathelayo. Izibalo 1 no-2 zibonisa indlela isidoda esisondelana ngayo, sakha ukuhlangana njengoba imisila igoqana.
Abacwaningi basebenzise ingcindezi ye-hydrostatic ukudala ukugeleza koketshezi ku-microchannel ukuze bafunde i-sperm rheology. Kusetshenziswe i-microchannel enobukhulu obungu-200 µm × 20 µm (W × H) kanye nobude obungu-3.6 µm. Sebenzisa i-microchannels phakathi kwezitsha ezine-syringe ezifakwe emaphethelweni. Umbala wokudla wasetshenziswa ukwenza iziteshi zibonakale kakhudlwana.
Bopha izintambo zokuxhumanisa kanye nezesekeli odongeni. Ividiyo ithathwe nge-phase contrast microscope. Ngesithombe ngasinye, kwethulwa i-phase contrast microscopy kanye nezithombe zemephu. (A) Ukuxhumana phakathi kwemifudlana emibili kumelana nokugeleza ngenxa yokunyakaza kwe-helical (umcibisholo obomvu). (B) Ukuxhumana phakathi kwe-tube bundle kanye nodonga lwesiteshi (imicibisholo ebomvu), ngesikhathi esifanayo zixhunywe kwezinye izinqwaba ezimbili (imicibisholo ephuzi). (C) Izinqwaba zesidoda emgudwini we-microfluidic ziqala ukuxhumana (imicibisholo ebomvu), zakha i-mesh yezinqwaba zesidoda. (D) Ukwakheka kwenethiwekhi yezinqwaba zesidoda.
Lapho ithonsi lesidoda esincibilikisiwe lifakwa kudivayisi ye-microfluidic futhi kudalwa ukugeleza, kwabonakala ukuthi umsebe wesidoda uhamba ngokumelene nokuqondisa kokugeleza. Amaqoqo alingana kahle nezindonga zama-microchannel, kanti amakhanda akhululekile engxenyeni yokuqala yamaqoqo alingana kahle nawo (ividiyo 5). Anamathela futhi kunoma yiziphi izinhlayiya ezimile endleleni yawo, njengemfucumfucu, ukuze amelane nokukhukhulwa umsinga. Ngokuhamba kwesikhathi, la ma-tuft aba yizicucu ezinde ezibamba ezinye izitho zobulili ezilodwa kanye nama-tuft amafushane (Ividiyo 6). Njengoba ukugeleza kuqala ukwehla, imigqa emide yesidoda iqala ukwakha inethiwekhi yemigqa yesidoda (Ividiyo 7; Umfanekiso 2).
Uma ijubane lokugeleza liphezulu (V > 33 µm/s), ukunyakaza okujikelezayo kwezintambo kuyandiswa njengoba kuzanywa ukubamba izinqwaba eziningi zesidoda ngasinye ukuze kulwiswane kangcono namandla okukhukhuleka okugeleza. Uma ijubane lokugeleza liphezulu (V > 33 µm/s), ukunyakaza okujikelezayo kwezintambo kuyandiswa njengoba kuzanywa ukubamba izinqwaba eziningi zesidoda ngasinye ukuze kulwiswane kangcono namandla okukhukhuleka okugeleza. При высокой скорости потока (V > 33 мкм/с) спиралевидные движения нитей усиливаются, поскольку они пытаются поймать множествоы озден образующих пучки, которые лучше противостоят дрейфующей силе потока. Uma kugeleza kakhulu (V > 33 µm/s), ukunyakaza kwe-helical kwezintambo kuyanda njengoba zizama ukubamba izitho eziningi ezikhiqiza isidoda ezikwazi kangcono ukumelana namandla okukhukhuleka kokugeleza.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 в попытке захватить множество отделььное движение нитей увеличивается пучки, чтобы лучше сопротивляться силам дрейфа потока. Uma kugeleza kakhulu (V > 33 µm/s), ukunyakaza kwe-helical kwe-filaments kuyanda emzamweni wokubamba ama-spermatozoa amaningi akha ama-bundle ukuze amelane kangcono namandla okugeleza kokugeleza.Bazama nokuxhuma ama-microchannel ezindongeni eziseceleni.
Amaqoqo esidoda ahlonzwe njengamaqoqo amakhanda esidoda kanye nemisila egoqekayo kusetshenziswa i-light microscopy (LM). Amaqoqo esidoda anezinhlanganisela ezahlukahlukene nawo ahlonzwe njengamakhanda asontekile kanye nama-flagellar aggregates, imisila yesidoda ehlanganisiwe eminingi, amakhanda esidoda anamathele emsila, kanye namakhanda esidoda ane-nuclei egobile njenge-nuclei ehlanganisiwe eminingi. i-transmission electron microscopy (TEM). I-scanning electron microscopy (SEM) ibonise ukuthi amaqoqo esidoda ayengamaqoqo ahlanganisiwe amakhanda esidoda kanti amaqoqo esidoda abonise inethiwekhi enamathiselwe yemisila egoqekile.
Ukwakheka kanye nesakhiwo se-ultrastructure se-spermatozoa, ukwakheka kwama-spermatozoa bundles kwafundwa kusetshenziswa i-light microscopy (ingxenye yesigamu), i-scanning electron microscopy (i-SEM) kanye ne-transmission electron microscopy (i-TEM), ama-sperm smears afakwe i-acridine orange futhi ahlolwa kusetshenziswa i-epifluorescence microscopy.
Ukufakwa kwe-sperm smear nge-acridine orange (Isithombe 3B) kubonise ukuthi amakhanda esidoda ayenamathele ndawonye futhi embozwe ngezinto ezikhipha uketshezi, okwaholela ekwakhekeni kwama-tuft amakhulu (Isithombe 3D). Ama-sperm bundles ayehlanganisa ama-sperm aggregates anenethiwekhi yemisila enamathiselwe (Isithombe 4A-C). Ama-sperm bundles akhiwa yimisila yama-spermatozoa amaningi anamathele ndawonye (Isithombe 4D). Izimfihlo (Isithombe 4E,F) zazimboze amakhanda ama-spermatozoa bundles.
Ukwakheka kwenqwaba yesidoda Ukusebenzisa i-phase contrast microscopy kanye nama-sperm smears anombala we-acridine orange, kubonise ukuthi amakhanda esidoda ayanamathelana. (A) Ukwakheka kwesidoda sokuqala kuqala ngesidoda (indilinga emhlophe) kanye nesidoda ezintathu (indilinga ephuzi), lapho i-spiral iqala emsileni iphele ekhanda. (B) I-Photomicrograph yesidoda esinombala we-acridine orange ekhombisa amakhanda esidoda anamathelayo (imicibisholo). Ukuphuma kwamanzi kuhlanganisa ikhanda/amakhanda. Ukukhulisa × 1000. (C) Ukuthuthukiswa kogongolo olukhulu oluthuthwa ukugeleza kumzila we-microfluidic (kusetshenziswa ikhamera yesivinini esiphezulu ku-950 fps). (D) I-Micrograph yesidoda esinombala we-acridine orange ekhombisa ama-tuft amakhulu (imicibisholo). Ukukhulisa: ×200.
Ukuskena i-electron micrograph ye-sperm beam kanye ne-sperm smear efakwe i-acridine orange. (A, B, D, E) yi-digital color scanning electron micrographs ye-spermatozoa, kanti u-C no-F yi-micrographs ye-acridine orange stained sperm smears ekhombisa ukunamatheliswa kwe-spermatozoa eminingi egoqa i-caudal web. (AC) Ama-sperm aggregates aboniswa njengenethiwekhi yemisila enamathiselwe (imicibisholo). (D) Ukunamathela kwe-spermatozoa eminingana (enento enamathelayo, uhlaka olupinki, umcibisholo) egoqa umsila. (E no-F) Ama-sperm head aggregates (izikhombisi) ambozwe ngezinto ezinamathelayo (izikhombisi). I-spermatozoa yakha amaqoqo anezakhiwo eziningana ezifana ne-vortex (F). (C) ×400 kanye (F) ×200 magnifications.
Sisebenzisa i-transmission electron microscopy, sithole ukuthi izinqwaba zesidoda zinemisila enamathiselwe (Isithombe 6A, C), amakhanda anamathiselwe emisileni (Isithombe 6B), noma amakhanda anamathiselwe emisileni (Isithombe 6D). Amakhanda esidoda esisesixhantini agobile, avela esigabeni sesibili sezifunda zenuzi (Isithombe 6D). Esixhantini sokusika, isidoda sasinekhanda elisontekile elinezifunda ezimbili zenuzi kanye nezifunda eziningi ze-flagellar (Isithombe 5A).
I-micrograph ye-electron yombala wedijithali ekhombisa imisila exhumanisayo esiqwini sesidoda kanye nezinto ezihlanganisayo ezixhumanisa amakhanda esidoda. (A) Umsila onamathiselwe wenani elikhulu lesidoda. Qaphela ukuthi umsila ubukeka kanjani kokubili ezithombeni (umcibisholo) kanye nasezindaweni (umcibisholo). (B) Ikhanda (umcibisholo) lesidoda lixhunywe emsila (umcibisholo). (C) Kunemisila eminingana yesidoda (imicibisholo). (D) Izinto ezihlanganisayo (AS, okwesibhakabhaka) ezixhumanisa amakhanda amane esidoda (onsomi).
I-scanning electron microscopy yasetshenziswa ukuthola amakhanda esidoda ezinqwabeni zesidoda ezimbozwe uketshezi noma ulwelwesi (Isithombe 6B), okubonisa ukuthi izinqwabeni zesidoda zazinamathele ngezinto ezingaphandle. Izinto ezihlanganisiwe zazigxile ekhanda lesidoda (i-jellyfish head-like assembly; Isithombe 5B) futhi zandiswa kude, zinikeza ukubukeka okuphuzi okukhazimulayo ngaphansi kwe-fluorescence microscopy uma zifakwe i-acridine orange (Isithombe 6C). Lesi sici sibonakala ngokucacile ngaphansi kwe-scanning microscope futhi sibhekwa njengesibopho. Izingxenye ezincane kakhulu (Isithombe 5C) kanye ne-sperm smears ezifakwe i-acridine orange zibonise izinqwabeni zesidoda eziqukethe amakhanda agcwele kakhulu kanye nemisila egobile (Isithombe 5D).
Ama-photomicrograph ahlukahlukene abonisa ukuhlanganiswa kwamakhanda esidoda kanye nemisila egoqiwe kusetshenziswa izindlela ezahlukene. (A) I-digital color transmission electron micrograph ye-cross-sectional ye-sperm bundle ekhombisa ikhanda lesidoda eligoqiwe eline-nucleus enezingxenye ezimbili (eluhlaza okwesibhakabhaka) kanye nezingxenye eziningana ze-flagellar (eluhlaza okotshani). (B) I-digital color scanning electron micrograph ekhombisa iqoqo lamakhanda esidoda afana ne-jellyfish (imicibisholo) abonakala embozwe. (C) Ingxenye encane kakhulu ekhombisa amakhanda esidoda ahlanganisiwe (imicibisholo) kanye nemisila egoqiwe (imicibisholo). (D) I-Micrograph ye-sperm smear efakwe i-acridine orange ekhombisa ukuhlanganiswa kwamakhanda esidoda (imicibisholo) kanye nemisila enamathelayo egoqiwe (imicibisholo). Qaphela ukuthi into enamathelayo (S) imboza ikhanda lesidoda. (D) × 1000 magnification.
Kusetshenziswa i-transmission electron microscopy (Isithombe 7A), kwaphawulwa nokuthi amakhanda esidoda ayesontekile kanti ama-nuclei ayenesimo esijikelezayo, njengoba kuqinisekiswa yi-sperm smears efakwe i-acridine orange futhi yahlolwa kusetshenziswa i-fluorescence microscopy (Isithombe 7B).
(A) I-micrograph ye-electron yokudlulisa umbala wedijithali kanye (B) I-Acridine orange stained sperm smear ekhombisa amakhanda ahlanganisiwe kanye nokunamathela kwamakhanda nemisila yesidoda (imicibisholo). (B) × 1000 magnification.
Okutholwe okuthakazelisayo ukuthi isidoda sikaSharkazi siyahlangana ukuze sakhe ama-filamentous bundles ahambayo. Izakhiwo zala ma-bundles zisenza siqonde indima yawo engaba khona ekumuncweni nasekugcinweni kwe-spermatozoa ku-SST.
Ngemva kokuhlangana, isidoda singena esithweni sangasese sowesifazane bese sidlula enkambisweni yokukhetha ejulile, okuholela enanini elilinganiselwe lesidoda esingena kwi-SST15,16. Kuze kube manje, izindlela isidoda esingena futhi siphume ngazo kwi-SST azicaci. Ezinkukhu, isidoda sigcinwa kwi-SST isikhathi eside esingamaviki amabili kuya kwayi-10, kuye ngohlobo lwaso6. Impikiswano isasele ngesimo sesidoda ngesikhathi sigcinwa kwi-SST. Ingabe siyanyakaza noma siphumule? Ngamanye amazwi, amaseli esidoda asigcina kanjani isikhundla sawo kwi-SST isikhathi eside kangaka?
UForman4 uphakamise ukuthi ukuhlala kanye nokukhishwa kwe-SST kungachazwa ngokwendlela yokunyakaza kwesidoda. Abalobi bacabanga ukuthi isidoda sigcina indawo yaso ngokubhukuda ngokumelene nokugeleza koketshezi okudalwe yi-SST epithelium nokuthi isidoda sikhishwa ku-SST lapho ijubane laso liwela ngaphansi kwendawo lapho siqala khona ukubuyela emuva ngenxa yokuntuleka kwamandla. UZaniboni5 uqinisekise ukuba khona kwama-aquaporins 2, 3, kanye no-9 engxenyeni ye-apical yamaseli e-SST epithelial, okungase kusekele ngokungaqondile imodeli yokugcina isidoda sikaForeman. Esifundweni samanje, sithole ukuthi cishe ingxenye yesidoda sikaSharkashi ikhombisa i-rheology enhle kuketshezi olugelezayo, nokuthi izinqwaba zesidoda ezihlanganisiwe zandisa inani lesidoda ezibonisa i-rheology enhle, yize i-agglutination izinciphisa. Indlela amaseli esidoda ahamba ngayo epayipini lenyoni le-fallopian aye endaweni yokukhulelwa ayiqondakali ngokugcwele. Ezincelisayo, i-follicular fluid chemoattracts spermatozoa. Kodwa-ke, kukholelwa ukuthi i-chemoattractants iqondisa isidoda ukuba sisondele amabanga amade7. Ngakho-ke, ezinye izindlela zibangela ukuthuthwa kwesidoda. Amandla esidoda okuhamba nokugeleza ngokumelene noketshezi lwe-fallopian tube olukhishwa ngemva kokuhlangana kuye kwabikwa ukuthi luyisici esiyinhloko ekuqondiseni isidoda emagundwini. UParker 17 uphakamise ukuthi i-spermatozoa iwele ama-oviducts ngokubhukuda ngokumelene nomsinga we-ciliary ezinyonini nasezihuquzelayo. Nakuba kungakaze kuboniswe ngokuhlola ezinyonini, u-Adolphi18 waba ngowokuqala ukuthola ukuthi isidoda sezinyoni sinikeza imiphumela emihle lapho ungqimba oluncane loketshezi phakathi kwe-coverlip kanye ne-slide ludalwa ngomugqa wephepha lokuhlunga. I-Rheology. UHino noYanagimachi [19] babeke i-ovary-tubal-uterine complex yegundane endandatho yokugeleza kwamanzi futhi bafaka i-1 µl yeyinki esiqwini ukuze babone ukugeleza koketshezi emathunjini e-fallopian. Baqaphele ukunyakaza okusebenzayo kakhulu kokufinyela nokuphumula ethunjini le-fallopian, lapho wonke amabhola eyinki ayehamba kancane kancane eya e-ampulla yethubhu le-fallopian. Abalobi bagcizelela ukubaluleka kokugeleza koketshezi lwe-tubal kusuka ephansi kuya emathunjini e-fallopian aphezulu ukuze kukhuliswe isidoda futhi kukhule. UBrillard20 ubike ukuthi ezinkukhu nasezinkanini, i-spermatozoa iyafuduka ngokunyakaza okusebenzayo isuka emnyango wesitho sangasese sowesifazane, lapho igcinwa khona, iye endaweni yokuhlangana kwesitho sangasese sowesifazane, lapho igcinwa khona. Kodwa-ke, lokhu kunyakaza akudingeki phakathi kwendawo yokuhlangana kwesitho sangasese sowesifazane kanye ne-infundibulum ngoba i-spermatozoa ithuthwa ngokufuduka okungenamsebenzi. Ukwazi lezi zincomo zangaphambilini kanye nemiphumela etholwe ocwaningweni lwamanje, kungacatshangwa ukuthi ikhono le-spermatozoa lokuhamba phezulu (rheology) lingenye yezimpawu inqubo yokukhetha esekwe kuzo. Lokhu kunquma ukudlula kwe-spermatozoa ngesitho sangasese sowesifazane kanye nokungena kwayo kwi-CCT ukuze igcinwe. Njengoba uForman4 ephakamise, lokhu kungasiza futhi inqubo yesitho sangasese sowesifazane singene kwi-SST nendawo yaso yokuhlala isikhathi esithile bese siphuma lapho ijubane laso liqala ukwehla.
Ngakolunye uhlangothi, uMatsuzaki noSasanami 21 basikisela ukuthi i-spermatozoa yezinyoni ishintshe ukuhamba kwayo kusukela ekungalali kuya ekunyakazeni ezindaweni zokuzala zabesilisa nabesifazane. Ukuvinjelwa kokuhamba kwesidoda esihlala ku-SST kuye kwaphakanyiswa ukuchaza isikhathi eside sokugcina isidoda bese sivuselelwa ngemva kokuphuma ku-SST. Ngaphansi kwezimo ezingenayo i-hypoxia, uMatsuzaki nabanye babike ukukhiqizwa okuphezulu nokukhululwa kwe-lactate ku-SST, okungaholela ekuvinjelweni kokuhamba kwesidoda esihlala ku-SST. Kulokhu, ukubaluleka kwe-rheology yesidoda kubonakala ekukhetheni nasekumuncweni kwe-spermatozoa, hhayi ekugcinweni kwayo.
Iphethini yokuhlanganiswa kwesidoda ibhekwa njengencazelo enengqondo yesikhathi eside sokugcina isidoda ku-SST, njengoba lokhu kuyiphethini evamile yokugcina isidoda ezinkukhu2,22,23. UBakst nabanye 2 baphawula ukuthi iningi le-spermatozoa linamathelana, lakha ama-fascicular aggregates, kanti i-spermatozoa eyodwa yayingavamile ukutholakala ku-CCM yezigwaca. Ngakolunye uhlangothi, uWen nabanye 24 babonile ukuthi i-spermatozoa esakazeke kakhulu kanye nama-tufts ambalwa e-spermatozoa ku-lumen ye-SST ezinkukhu. Ngokusekelwe kulokhu okubonwe, kungacatshangwa ukuthi ukuthambekela kokuhlanganiswa kwesidoda kuhlukile phakathi kwezinyoni naphakathi kwe-spermatozoa e-ejaculate efanayo. Ngaphezu kwalokho, uVan Krey nabanye 9 baphakamise ukuthi ukuhlukaniswa okungahleliwe kwe-spermatozoa ehlanganisiwe kunesibopho sokungena kancane kancane kwe-spermatozoa ku-lumen ye-tube fallopian. Ngokusho kwalo mbono, i-spermatozoa enekhono eliphansi lokuhlanganiswa kufanele ikhishwe ku-SST kuqala. Kulesi simo, ikhono le-spermatozoa lokuhlangana lingaba yisici esithonya umphumela wokuncintisana kwesidoda ezinyonini ezingcolile. Ngaphezu kwalokho, uma isidoda esihlangene sihlukana isikhathi eside, kulapho ukuzala kugcinwa khona isikhathi eside.
Nakuba ukuhlangana kwe-spermatozoa kanye nokuhlangana kube yizinqwaba kuye kwabonwa ezifundweni eziningana2,22,24, azichazwanga ngokuningiliziwe ngenxa yobunzima bokubuka kwazo kwe-kinematic ngaphakathi kwe-SST. Kuye kwenziwa imizamo eminingana yokufunda ukuhlangana kwesidoda ngaphakathi kwe-vitro. Ukuhlangana okukhulu kodwa kwesikhashana kwabonwa lapho ucingo oluncane lususwa ethonsini lembewu elilengayo. Lokhu kuholela eqinisweni lokuthi i-bubble ende iphuma ethonsini, ilingisa i-seminal gland. Ngenxa yemikhawulo ye-3D kanye nezikhathi ezimfushane zokomisa amaconsi, lonke ibhloko lawa ngokushesha9. Esifundweni samanje, sisebenzisa izinkukhu zeSharkashi kanye nama-microfluidic chips, sikwazile ukuchaza ukuthi lawa ma-tufts akha kanjani nokuthi ahamba kanjani. Izinqwaba zesidoda zakhiwa ngokushesha ngemva kokuqoqwa kwesidoda futhi zatholakala zihamba ngendlela ejikelezayo, zibonisa i-rheology enhle uma zikhona ekugelezeni. Ngaphezu kwalokho, lapho zibukwa nge-macroscopically, izinqwaba zesidoda ziye zabonwa ukwandisa ukulingana kokuhamba uma kuqhathaniswa ne-spermatozoa ehlukanisiwe. Lokhu kusikisela ukuthi ukuhlangana kwesidoda kungenzeka ngaphambi kokungena kwe-SST nokuthi ukukhiqizwa kwesidoda akugcini endaweni encane ngenxa yokucindezeleka njengoba kwasikiselwa ngaphambilini (iTingari neLake12). Ngesikhathi sokwakheka kwe-tuft, i-spermatozoa ibhukuda nge-synchrony ize yakhe i-junction, bese imisila yayo igoqana futhi ikhanda lesidoda lihlala likhululekile, kodwa umsila kanye nengxenye ekude yesidoda kunamathelana nento enamathelayo. Ngakho-ke, ikhanda elikhululekile lesidoda linesibopho sokunyakaza, lihudula lonke i-ligament. Ukuskena i-electron microscopy yezinqwaba zesidoda kubonise amakhanda esidoda anamathele ambozwe ngezinto eziningi ezinamathelayo, okuphakamisa ukuthi amakhanda esidoda anamathele ezinqwabeni eziphumule, okungenzeka ukuthi kwenzeke ngemuva kokufika endaweni yokugcina (i-SST).
Uma i-sperm smear inombala we-acridine orange, izinto ezinamathelayo ezingaphandle kweseli lesidoda zingabonakala ngaphansi kwe-microscope ye-fluorescent. Lesi sici sivumela ama-sperm bundles ukuthi anamathele futhi anamathele kunoma yiziphi izindawo ezizungezile noma izinhlayiya ukuze angashukumi nokugeleza okuzungezile. Ngakho-ke, okubonile kukhombisa indima yokunamathela kwe-spermatozoa ngesimo sama-mobile bundles. Amandla abo okubhukuda ngokumelene nomsinga nokunamathela ezindaweni eziseduze avumela isidoda ukuthi sihlale isikhathi eside ku-SST.
URothschild25 usebenzise ikhamera ye-hemocytometry ukuze atadishe ukusatshalaliswa okuntantayo kwesidoda sezinkomo ethonsini lokumiswa, ethatha ama-photomicrograph ngekhamera ene-axis eqondile neqondile ye-microscope. Imiphumela ibonise ukuthi isidoda sadonseleka ebusweni begumbi. Abalobi basikisela ukuthi kungase kube nokusebenzisana kwe-hydrodynamic phakathi kwesidoda kanye nobuso. Uma sicabangela lokhu, kanye nekhono lesidoda sezinkukhu sikaSharkashi lokwakha ama-tuft anamathelayo, kungandisa amathuba okuthi isidoda sizonamathela odongeni lwe-SST futhi sigcinwe isikhathi eside.
UBccetti no-Afzeliu26 babike ukuthi i-glycocalyx yesidoda iyadingeka ukuze kuqashelwe i-gamete kanye ne-agglutination. UForman10 waphawula ukuthi i-hydrolysis yezibopho ze-α-glycosidic ezimbozweni ze-glycoprotein-glycolipid ngokwelapha isidoda sezinyoni nge-neuraminidase yabangela ukwehla kokuzala ngaphandle kokuthinta ukunyakaza kwesidoda. Abalobi basikisela ukuthi umphumela we-neuraminidase ku-glycocalyx uphazamisa ukuhlukaniswa kwesidoda esixhumeni sesibeletho nesitho sangasese sowesifazane, ngaleyo ndlela kunciphisa ukuzala. Ukubuka kwabo akunakuziba ukuthi kungenzeka ukuthi ukwelashwa nge-neuraminidase kunganciphisa ukuqashelwa kwesidoda ne-oocyte. UForman no-Engel10 bathole ukuthi ukuzala kwehla lapho izinkukhu zifakelwa isidoda ngaphakathi kwesitho sangasese sowesifazane ngesidoda esiphathwe nge-neuraminidase. Kodwa-ke, i-IVF ngesidoda esiphathwe nge-neuraminidase ayizange ithinte ukuzala uma kuqhathaniswa nezinkukhu ezilawulwayo. Abalobi baphetha ngokuthi izinguquko embozweni we-glycoprotein-glycolipid ozungeze ulwelwesi lwesidoda zinciphise ikhono lesidoda lokuvundisa ngokuphazamisa ukuqoqwa kwesidoda endaweni yokuhlangana kwesidoda nesitho sangasese sowesifazane, okwakhulisa ukulahlekelwa kwesidoda ngenxa yesivinini sendawo yokuhlangana kwesidoda nesitho sangasese sowesifazane, kodwa akuthinti ukuqashelwa kwesidoda neqanda.
Kuma-turkey uBakst noBauchan 11 bathole ama-vesicle amancane nezingcezu ze-membrane ku-lumen ye-SST futhi baphawula ukuthi amanye ala ma-granule ayehlangene ne-sperm membrane. Abalobi basikisela ukuthi lobu budlelwano bungaba negalelo ekugcinweni kwesikhathi eside kwe-spermatozoa ku-SST. Kodwa-ke, abacwaningi abazange bachaze umthombo walezi zinhlayiya, kungakhathaliseki ukuthi zikhishwa ngamaseli e-epithelial e-CCT, akhiqizwa futhi akhishwa uhlelo lokuzala lwabesilisa, noma akhiqizwa yi-sperm uqobo. Futhi, lezi zinhlayiya zinesibopho sokuhlanganiswa. UGrützner et al27 babike ukuthi amaseli e-epithelial e-epididymal akhiqiza futhi akhipha iphrotheni ethile edingekayo ekwakhiweni kwama-seminal tracts angama-single-pore. Ababhali babika nokuthi ukusabalala kwalezi zinqwaba kuncike ekusebenzisaneni kwamaprotheni e-epididymal. UNixon et al28 bathole ukuthi i-adnexa ikhipha iphrotheni, i-osteonectin ecebile nge-cysteine; i-SPARC ihilelekile ekwakhiweni kwama-tufts esidoda kuma-echidnas nama-platypuses amafushane. Ukusabalala kwale misebe kuhlotshaniswa nokulahleka kwale phrotheni.
Esifundweni samanje, ukuhlaziywa kwesakhiwo kusetshenziswa i-electron microscopy kubonise ukuthi i-spermatozoa inamathele entweni eningi exinene. Lezi zinto kucatshangwa ukuthi zibangela i-agglutination ehlangana phakathi naseduze kwamakhanda anamathelayo, kodwa ngamanani aphansi esifundeni somsila. Sicabanga ukuthi le nto ehlanganisayo ikhishwa ohlelweni lokuzala lwabesilisa (i-epididymis noma i-vas deferens) kanye nesidoda, njengoba sivame ukubona isidoda sihlukana ne-lymph kanye ne-seminal plasma ngesikhathi sokuphuma kwesisu. Kubikwe ukuthi njengoba i-avian spermatozoa idlula ku-epididymis kanye ne-vas deferens, idlula ezinguqukweni ezihlobene nokuvuthwa ezisekela ikhono layo lokubopha amaprotheni futhi ithole ama-glycoprotein ahlobene ne-plasma lemma. Ukuphikelela kwalawa maprotheni kuma-resident sperm membrane ku-SST kusikisela ukuthi lawa maprotheni angathonya ukutholwa kokuqina kwe-sperm membrane 30 futhi anqume ukuzala kwawo 31. U-Ahammad nabanye 32 babike ukuthi i-spermatozoa etholakala ezingxenyeni ezahlukene zesimiso sokuzala sabesilisa (kusukela kuma-testes kuya kuma-distal vas deferens) ibonise ukwanda okuqhubekayo kokuphila ngaphansi kwezimo zokugcina uketshezi, kungakhathaliseki ukuthi izinga lokushisa lokugcina lingakanani, futhi ukuphila ezinkukhu nakho kuyanda emapayipini e-fallopian ngemva kokufakelwa isidoda okwenziwe ngokwenziwa.
Ama-tuft esidoda senkukhu iSharkashi anezimpawu nemisebenzi ehlukile kunezinye izinhlobo ezifana nama-echidna, ama-platypuse, amagundane ezinkuni, amagundane ezinyamazane, kanye nezingulube ze-guinea. Ezinkukhu ze-sharkasi, ukwakheka kwama-bundle e-spermatozoa kunciphisa isivinini sawo sokubhukuda uma kuqhathaniswa ne-single spermatozoa. Kodwa-ke, la ma-bundle andise iphesenti le-spermatozoa ene-rheologically positive futhi andise ikhono le-spermatozoa lokuzinza endaweni eguquguqukayo. Ngakho-ke, imiphumela yethu iqinisekisa isiphakamiso sangaphambilini sokuthi i-sperm agglutination ku-SST ihlotshaniswa nokugcinwa kwesidoda isikhathi eside. Siphinde sicabange ukuthi ukuthambekela kwesidoda ekwakheni ama-tuft kungalawula izinga lokulahlekelwa isidoda ku-SST, okungashintsha umphumela wokuncintisana kwesidoda. Ngokusho kwalokhu kucatshangwa, i-spermatozoa enekhono eliphansi le-agglutination ikhipha i-SST kuqala, kuyilapho i-spermatozoa enekhono eliphezulu le-agglutination ikhiqiza iningi lenzalo. Ukwakheka kwama-bundle esidoda ane-pore eyodwa kuyazuzisa futhi kuthinta isilinganiso somzali nengane, kodwa kusebenzisa indlela ehlukile. Kuma-echidna nama-platypuses, i-spermatozoa ihlelwe ngendlela efanayo ukuze kwandiswe isivinini sokuya phambili kwe-beam. Amaqoqo e-echidnas ahamba ngokushesha okuphindwe kathathu kune-spermatozoa eyodwa. Kukholelwa ukuthi ukwakheka kwama-tufts esidoda anjalo kuma-echidnas kuyindlela yokuziphendukela kwemvelo ukuze kugcinwe ukubusa, njengoba abesifazane beziphatha kabi futhi ngokuvamile behlangana nabesilisa abaningana. Ngakho-ke, i-spermatozoa evela kuma-ejaculate ahlukene incintisana kakhulu ngokukhulelwa kweqanda.
I-spermatozoa ehlanganisiwe yezinkukhu ze-sharkasi kulula ukuyibona ngeso lengqondo kusetshenziswa i-phase contrast microscopy, okubhekwa njengenzuzo ngoba ivumela ukutadisha okulula kokuziphatha kwe-spermatozoa in vitro. Indlela ukwakheka kwe-sperm tuft okukhuthaza ngayo ukuzala ezinkukhu ze-sharkasi ihlukile naleyo ebonwa kwezinye izilwane ezincelisayo ze-placenta ezimele ukuziphatha kwesidoda ngokubambisana njengegundane lezinkuni, lapho ezinye izidoda zifinyelela khona amaqanda, zisiza abanye abantu abahlobene ukufinyelela nokulimaza amaqanda abo. ukuze uzibonakalise. ukuziphatha kokuzinikela. Ukuzizalisa 34. Esinye isibonelo sokuziphatha ngokubambisana ku-spermatozoa sitholakale ezimpukuneni zezinyamazane, lapho izidoda zazikwazi khona ukuhlonza nokuhlangana ne-spermatozoa ehlobene kakhulu nofuzo futhi zakhe amaqembu okubambisana ukuze kwandiswe isivinini sazo uma kuqhathaniswa ne-spermatozoa engahlobene35.
Imiphumela etholwe kulolu cwaningo ayiphikisani nombono kaFoman wokugcina isikhathi eside kwe-spermatozoa ku-SWS. Abacwaningi babika ukuthi amangqamuzana esidoda ayaqhubeka nokuhamba ekugelezeni kwamaseli e-epithelial amboze i-SST isikhathi eside, futhi ngemva kwesikhathi esithile, izitolo zamandla zamaseli esidoda ziyaphela, okuholela ekunciphiseni isivinini, okuvumela ukuxoshwa kwezinto ezincane zesisindo sama-molecule. amandla e-spermatozoa ngokugeleza koketshezi oluvela ku-lumen ye-SST. Esifundweni samanje, sibonile ukuthi ingxenye yesidoda esisodwa ibonise ikhono lokubhukuda ngokumelene noketshezi olugelezayo, futhi ukunamathela kwazo esigxotsheni kwandisa ikhono lazo lokubonisa i-rheology enhle. Ngaphezu kwalokho, idatha yethu iyahambisana naleyo kaMatsuzaki et al. 1 ababike ukuthi ukwanda kokukhiqizwa kwe-lactate ku-SST kungavimbela ukuhamba kwesidoda okuhlala khona. Kodwa-ke, imiphumela yethu ichaza ukwakheka kwemisipha ye-sperm motile kanye nokuziphatha kwayo kwe-rheological lapho kukhona indawo enamandla ngaphakathi kwe-microchannel ngomzamo wokuqonda ukuziphatha kwazo ku-SST. Ucwaningo lwesikhathi esizayo lungagxila ekunqumeni ukwakheka kwamakhemikhali kanye nomsuka we-agglutinating agent, okungangabazeki ukuthi kuzosiza abacwaningi ukuthi bathuthukise izindlela ezintsha zokugcina isidoda esiwuketshezi futhi bandise isikhathi sokuzala.
I-sharkasi yesilisa engamaviki angu-30 ubudala engenalutho (i-homozygous dominant; i-Na Na) yakhethwa njengabanikeli besidoda ocwaningweni. Izinyoni zakhuliswa ePulazini Lenkukhu Locwaningo le-Faculty of Agriculture, e-Ashit University, e-Ashit Governorate, eGibhithe. Izinyoni zagcinwa ezindlini ngazinye (30 x 40 x 40 cm), zafakwa ohlelweni lokukhanya (amahora angu-16 okukhanya namahora angu-8 obumnyama) futhi zondliwa ukudla okuqukethe amagremu angu-160 eprotheyini eluhlaza, ama-kcal angu-2800 wamandla aguqulwayo, amagremu angu-35 e-calcium ngalinye. Amagremu ama-5 e-phosphorus atholakalayo ngekhilogramu yokudla.
Ngokusho kwedatha 36, ​​​​37, isidoda saqoqwa kwabesilisa ngokuhlikihla isisu. Kwaqoqwa amasampula esidoda angu-45 emadodeni angu-15 ezinsukwini ezi-3. Isidoda (n = 15/ngosuku) sancibilikiswa ngokushesha nge-1:1 (v:v) nge-Belsville Poultry Semen Diluent, equkethe 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) kanye ne-H2O (100 ml), i-pH = 7, 5, i-osmolarity 333 mOsm/kg38. Amasampula esidoda axutshwe aqale ahlolwa ngaphansi kwe-microscope ekhanyayo ukuqinisekisa ikhwalithi enhle yesidoda (umswakama) bese egcinwa emanzini okugeza ku-37°C kuze kube yilapho esetshenziswa zingakapheli isigamu sehora ngemva kokuqoqwa.
I-kinematics kanye ne-rheology ye-spermatozoa kuchazwa kusetshenziswa uhlelo lwamadivayisi e-microfluidic. Amasampula esidoda aphinde ancibilikiswa abe yi-1:40 eBeltsville Avian Semen Diluent, afakwa kudivayisi ye-microfluidic (bheka ngezansi), futhi amapharamitha e-kinetic anqunywa kusetshenziswa uhlelo lwe-Computerized Semen Analysis (CASA) olwalwenzelwe ngaphambili ukuchaza i-microfluidics. ekuhambeni kwe-spermatozoa ku-liquid media (uMnyango Wezobunjiniyela Bemishini, i-Faculty of Engineering, i-Assiut University, eGibhithe). I-plugin ingalandwa ku: http://www.assiutmicrofluidics.com/research/casa39. Ijubane lejika (VCL, μm/s), ijubane eliqondile (VSL, μm/s) kanye nejubane elijwayelekile le-trajectory (VAP, μm/s) kwalinganiswa. Amavidiyo esidoda athathwe kusetshenziswa i-inverted Optika XDS-3 phase contrast microscope (ene-40x objective) exhunywe kwikhamera ye-Tucson ISH1000 ku-30 ​​fps imizuzwana emi-3. Sebenzisa isofthiwe ye-CASA ukuze utadishe okungenani izindawo ezintathu kanye nezindlela zesidoda ezingama-500 ngesampula ngayinye. Ividiyo eqoshiwe icutshungulwe kusetshenziswa i-CASA eyenziwe ekhaya. Incazelo yokuhamba ku-plug-in ye-CASA isekelwe esivinini sokubhukuda kwesidoda uma kuqhathaniswa nesilinganiso sokugeleza, futhi ayifaki ezinye izilinganiso ezifana nokunyakaza kohlangothi, njengoba lokhu kutholakale kuthembekile kakhulu ekugelezeni koketshezi. Ukunyakaza kwe-Rheological kuchazwa njengokunyakaza kwamaseli esidoda ngokumelene nesiqondiso sokugeleza koketshezi. I-Spermatozoa enezakhiwo ze-rheological yahlukaniswa ngenani le-spermatozoa ehambayo; i-spermatozoa eyayiphumule futhi i-spermatozoa ehamba kancane ayifakwanga ekubalweni.
Wonke amakhemikhali asetshenzisiwe atholakale kwa-Elgomhoria Pharmaceuticals (eCairo, eGibhithe) ngaphandle kwalapho kuphawulwe ngenye indlela. Idivayisi yakhiqizwa njengoba kuchaziwe ngu-El-sherry et al. 40 kanye nokuguqulwa okuthile. Izinto ezisetshenziswe ukwakha ama-microchannel zazihlanganisa amapuleti engilazi (eHoward Glass, eWorcester, MA), i-SU-8-25 negative resist (eMicroChem, eNewton, CA), i-diacetone alcohol (eSigma Aldrich, eSteinheim, eJalimane), kanye ne-polyacetone. -184, eDow Corning, eMidland, eMichigan). Ama-Microchannel enziwa kusetshenziswa i-soft lithography. Okokuqala, imaski yobuso evikelayo ecacile enomklamo we-microchannel ofunwayo yanyatheliswa kuphrinta enesinqumo esiphezulu (ePrismatic, eCairo, eGibhithe nasePacific Arts and Design, eMarkham, ON). Ama-master enziwa kusetshenziswa amapuleti engilazi njengezinto ezisetshenziswayo. Amapuleti ahlanzwa nge-acetone, i-isopropanol kanye namanzi akhishwe i-ionized abese embozwa ngesendlalelo esingu-20 µm se-SU8-25 nge-spin coating (3000 rpm, 1 min). Izingqimba ze-SU-8 zomiswa ngobumnene (65°C, 2 min kanye no-95°C, 10 min) futhi zavezwa emisebeni ye-UV imizuzwana engu-50. Bhaka ngemva kokuvezwa ku-65°C kanye no-95°C umzuzu o-1 kanye nemizuzu engu-4 ukuze kuhlanganiswe izingqimba ze-SU-8 ezivezwe, kulandelwe ukuthuthukiswa kotshwala be-diacetone imizuzu engu-6.5. Bhaka ama-waffle kanzima (200°C imizuzu engu-15) ukuze uqinise ungqimba lwe-SU-8.
I-PDMS yalungiswa ngokuxuba i-monomer kanye ne-hardener ngesilinganiso sesisindo esingu-10:1, yabe isikhishwa igesi ku-vacuum cleaner yabe isithelwa ohlakeni oluyinhloko lwe-SU-8. I-PDMS yapholiswa kuhhavini (120°C, imizuzu engama-30), yabe isisikwa iziteshi, yahlukaniswa ne-master, yabe isibhobozwa ukuze kuvunyelwe amashubhu ukuthi anamathiselwe ekungeneni nasekuphumeni kwesiteshi esincane. Ekugcineni, iziteshi ezincane ze-PDMS zanamathiselwa unomphela kumaslayidi e-microscope kusetshenziswa iprosesa ye-corona ephathekayo (i-Electro-Technic Products, eChicago, IL) njengoba kuchaziwe kwenye indawo. Isiteshi esincane esisetshenziswe kulolu cwaningo silinganisa u-200 µm × 20 µm (W × H) futhi sinobude obungu-3.6 cm.
Ukugeleza koketshezi okubangelwa ingcindezi ye-hydrostatic ngaphakathi kwesiteshi esincane kufezwa ngokugcina izinga loketshezi echibini lokungenisa lingaphezu komehluko wokuphakama Δh39 echibini lokukhipha (Isithombe 1).
lapho u-f kuyi-coefficient of friction, echazwa ngokuthi i-f = C/Re yokugeleza kwe-laminar kumzila oyisikwele, lapho u-C ehlala njalo kuye ngokuthi isilinganiso se-aspect sesiteshi sinjani, u-L ubude besiteshi esincane, u-Vav yijubane elimaphakathi ngaphakathi kwesiteshi esincane, u-Dh ububanzi be-hydraulic besiteshi, u-g - ukusheshisa amandla adonsela phansi. Ukusebenzisa lesi sibalo, ijubane elimaphakathi lesiteshi lingabalwa kusetshenziswa i-equation elandelayo: