The Study Of Testicle Sertoli Cell And Spermatogonia Co-culture System In Rabbit

The　research　on　Spermatogonial　Stem　Cells　(SSC)　is　evoking　more　and　more　interests　of　people　because　of　the　development　of　other　stem　cells　and　the　appearance　of　many　new　research　tools　and　experimental　techniques　in　recent　years.　The　spermatogonia　of　some　kinds　of　animals　such　as　rat,　mouse,　pig　and　cattle　have　been　isolated,　cultured　and　transplanted.　Some　kinds　of　different　culture　systems　for　the　culture　of　SSC　in　vitro　have　been　built　such　as　the　co-culture　system　with　Sertoli　cells,　the　germ　cell　culture　system　with　STO　(mouse　embryonic　fibroblast　lines)　as　feeder　layer　and　so　on.　The　biochemical　and　molecular　characteristics　and　detailed　biological　mechanism　of　differentiation　and　proliferation　of　SSC,　which　is　the　foundation　and　the　premise　of　the　complicated　spermatogenesis,　have　not　been　deep　studied.　According　to　the　present　situation　for　the　research　of　SSC,　this　study　planed　to　gain　the　higher　purification　of　SSC　from　rabbit　tissue　by　several　steps　of　isolation,　and　then　to　culture　the　SSC　for　a　long　time　using　the　different　culture　media(DMEM,DMEM/F,serum-free　medium　and　KSOM)　in　different　culture　systems　for　seeking　the　long-time　optimal　culture　system　for　human　SSC　in　vitro.　This　study　established　a　foundation　for　the　further　research　on　spermatogonial　transplantation　and　functional　assessment　of　rabbit　SSC.　　The　SSC　was　taken　from　rabbit　testicular　tissue.　Sequential　two-step　enzymes　digestion,　Percoll　uncontinuous　density　gradient　centrifugation　and　isolation　according　to　the　different　adhesiveness　were　used　to　purify　the　SSC,　then　the　purified　SSC　was　planted　into　three　kinds　of　culture　media　of　DMEM,　DMEM/F12　or　DMEM-SF　in　which　the　SC　the　single　cell　suspension　originate　from　the　two-step　enzymes　digestion　was　used　as　the　Sertoli　cell-germ　cell　co-culture　in　three　kinds　of　culture　media.　The　spermatogonia　were　identified　by　the　characterized　shape　and　size　with　microscope,　and　the　proportion　of　the　survival　spermatogonia　in　the　seeded　cells　in　all　groups　were　observed　and　assessed　within　a　short-term　culture.　The　result　shows.　1.　After　low　osmosis　preparation,　we　can　obtain　a　high　purity　SC.　Especially　when　the　ratio　between　Hanks　and　super-purity　water　is　1/3,　the　purity　and　viability　were　the　highest.　They　can　reach　72.04%　and　84.5%　separately(P<0.05).　2.　The　methods　such　as　Percoll　uncontinuous　density　gradient　centrifugation,　isolation　according　to　the　different　adhesiveness　can　be　used　to　isolate　and　purify　rabbit　SSC　effectively.　Spermatogonia　mainly　distribute　between　the　gradient　27%　to　35%　(density　between　1.0410g/ml　to　1.0508g/ml).　The　average　purity　of　spermatogonia　is　60.42%　in　this　study(P<0.01).　3.　The　proportion　of　spermatogonia　was　significantly　decreased　in　the　co-culture　system　using　DMEM-SF　medium　after　3　days　culture.　When　cultured　in　DMEM/F12　medium,　the　proportion　of　spermatogonia　in　co-culture　system　with　Sertoli　cell　is　higher.　4.　The　spermatogonia　can　survive　for　a　long　period　of　time　in　the　co-culture　system　with　Sertoli　cell.　The　morphology　characteristics　of　the　spermatogonia　are　showed　round,　ellipsoidal　shape　with　18-24um　in　diameter,　or　becoming　thin　and　flat　with　obvious　processes,　attaching　on　or　between　Sertoli　cells.　Though　the　relative　numbers　of　the　spermatogonia　decrease　as　the　time　goes　on,　the　spermatogonia　can　survive　and　proliferate　over　a　long　period　of　time　in　vitro.