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1 33 1 Vol.33 No Dec. 013 Reproduction & Contraception doi: /j.issn.03-37X randc_journal@163.com Knockout TM SR ; ; ; : FBS Knockout TM SRKSR : FBS KSR HE TUNEL RT-PCR Kit Sycp3 Crisp1 : KSR RT-PCR Kit Sycp3 Crisp1 FBS Sycp3 Crisp1 : KSR : ; ; ; : R697 +.; R : A : 03-37X [1-3] B [4] [6] [7-9] : : ; Tel: ; Fax: ; dw.he@163.com 804 SR KSR [10] Knockout TM FBS

2 1 1.1 SPF 1 Sprague-DawleySD 9 DMEM/F KSR Gibco ; FBSSV HyClone ; A904-10G Sigma ; mdfmodified Davidson s Fluid 30% +1% +% +0% [11] RNA RP-10 Tap PCR Master Mix ; DDR 00374A Takara ; TUNEL Cat. No Roche ; DAB ZLI FBS KSR 10%FBS DMEM/F1 10%KSR DMEM/F1 0. μm 1 g/l 6 10 mm 10 mm mm FBS KSR 1 SD 4~ 1 1A 34 %CO 4 d 1 ; Nikon Elememts-DR 3 mdf 4 h 4 μm HE DAB 1.4 RT-PCR mrna RNA RNA RNA β- β-actin : Kit Sycp3 Crisp1 [1] 1 1. x s SPSS16.0 t P<0.0.1 KSR FBS 1B~E FBS 4 P<0.01 KSR P<0.0 1 RT-PCR Table 1 RT-PCR analysis of gene primer Gene Primer sequence Temperature Length bp β-actin Kit Sycp3 Crisp1 '-GAGATTACTGCCCTGGCTCCTA-3' '-GACTCATCGTACTCCTGCTTGCTTG-3' '-ACAGGACGCCTACTAACAGA-3' '-ATCAAATGTCACGGAAGCAC-3' '-TTTCAAAGCCAGTAACCA-3' '-CTTTCATTCTCTGGCTCT-3' '-AGGTGCATTTACAATCACA-3' '-GATTACAGAAGACCACGA-3'

3 A: testicular tissue is placed in a low melting point agarose gel B: 1 d culture for 1 d; C D: FBS 1 culture for 1 week C and weeks D in FBS group E F: KSR 1 culture for 1 week E and weeks F in KSR group 1 Figure 1 Testicular tissue after cultured 4 x s Table Area and change rate of the testicular tissue before cultured and cultured for weeks 4 weeks and weeks FBS KSR Time μm % μm % Area Rate of reducement Area Rate of increasement * Before cultured weeks $# weeks ## $ weeks $ *: P<0.0 FBS compared with FBS group of testicular tissue for weeks $: P<0.01 KSR compared with KSR group of testicular tissue before cultured #: P<0.0 ##: P<0.01 KSR 4 compared with KSR group of testicular tissue for 4 weeks : P<0.01 KSR compared with KSR group of testicular tissue for weeks. KSR FBS KSR E~H FBS A~D 1 KSR FBS KSR KSR 3A FBS 806

4 A: FBS 1 proliferated spermatogonia B: FBS the testicular tissues were cultured for 1 week in FBS group Sertoli cells closed to the basement membrane after cultured for weeks in FBS group primary spermatocytes were visible in the seminiferous tubule C: FBS 4 after cultured for 4 weeks in FBS group germ cells and Sertoli cells were visible in the seminiferous tubules D: FBS after cultured for weeks in and Leydig cell proliferated in the interstitial tissue FBS group there was no germ cells and Sertoli cells in the seminiferous tubules E: KSR 1 proliferated spermatogonia F: KSR number of primary spermatocytes G: KSR after cultured for 1 week in KSR group Sertoli cells after cultured for weeks in KSR group a large appeared in the seminiferous lumen Sertoli cells 3 and sperm cells H: KSR was near to the basement membrane primary spermatocytes were perpendicular to the basementmem brane after cultured for 3 weeks in KSR group primary spermatocytes appeared in the seminiferous tubules after cultured for weeks in KSR group develop- ment of germ cell was in varying degrees in different seminiferous tubules Figure H showed a large number of primary spermatocytes Figure The testicular histological performance of the cultured tissues A: KSR in KSR group apoptosis was not obvious there were a small number of apoptotic cells in the seminiferous tubule B: FBS in FBS group apoptosis was obvious there were a large number of apoptotic cells in the seminiferous tubules 3 SD Figure 3 The apoptosis results of testicular tissues cultured for weeks in SD rats 807

5 3B 3 KSR FBS P<0.0 FBS 4 KSR Sycp3 Crisp1 SD 3 FBS Kit 3.3 KSR FBS mrna 3 4 KSR Kit 3 x s Table 3 The seminiferous tubule diameter of the testicular tissue before and after cultured Time FBS μm KSR μm Before cultured week * # weeks *# # 3 3 weeks *# # 4 4 weeks *# # weeks *# # *: P<0.0 KSR compared with KSR group at the same time #: P<0.0 compared with before cultured in the same group [1314] Steinberger [1] Trowell - [16] Trowell - DMEM/F1 β-actin: internal reference Kit: meiosis early marker genes Sycp3: marker gene in late phase of meiosis Crisp1: surface marker gene of sperm cell 1 d: 1 d the testicular tissue one day after birth ad: the testicular tissue of adult SD rat K1w Kw K3w K4w Kw: KSR the testicular tissue of KSR group cultured for 1 week weeks 3 weeks 4 weeks weeks respectively K1w Kw K3w K4w Kw: FBS the testicular tissue of FBS group cultured for 1 week weeks 3 weeks 4 weeks weeks respectively 4 Figure 4 The marker genes of the spermatogenesis process in the testis before and after cultured 808

6 [17] 0.4 μm FBS [18] FBS KSR FBS [19] KSR FBS ICM embryonic stem cells ESC [0-] SD KSR FBS KSR 3 4 RT-PCR Kit Sycp3 Crisp1 FBS 4 KSR FBS KSR 8 FBS 7 KSR FBS KSR 8 KSR KSR ICM ESC KSR [1] Steinberger A Steinberger E. Factors affecting spermatogenesis in organ cultures of mammalian testes. J Reprod Fertil 1967 Suppl: [] Steinberger A Steinberger E. In vitro growth and development of mammalian testes. The Testis 1970 : [3] Steinberger A Steinberger E Perloff W. Mammalian testes in organ culture. Exper Cell Res :19-7. [4] :33-7. [] Nagano M Ryu BY Brinster CJ et al. Maintenance of mouse male germ line stem cells in vitro. Biol Reprod : [6] Anderson R Wallace E Groome N et al. Physiological relationships between inhibin B follicle stimulating hormone secretion and spermatogenesis in normal men and response to gonadotrophin suppression by exogenous testosterone. Hum Reprod : [7] Cabrol S Ross JL Fennoy I et al. Assessment of Leydig and Sertoli Cell functions in infants with nonmosaic Klinefelter syndrome: insulin-like peptide 3 levels are normal and positively correlated with LH levels. J Clin Endocrinol Metab :E [8] Hess RA Cooke PS Hofmann MC et al. Mechanistic insights into the regulation of the spermatogonial stem cell niche. Cell Cycle : [9] Rey RA Grinspon RP Gottlieb S et al. Male hypogonadism: an extended classification based on a developmental endocrine physiology-based approach. Andrology :3-16. [10] :61-. [11] Latendresse JR Warbrittion AR Jonassen H et al. Fixation of testes and eyes using a modified davidson s fluid: comparison with Bouin s fluid and conventional davidson s fluid. Toxicol Pathol :4-3. [1] Leeb T Sieme H Töpfer-Petersen E. Genetic markers for stallion fertility lessons from humans and mice. Animal Reprod Sci :1-9. [13] Gonzales GF Del Valle L. Adult rat seminiferous tubules secrete a fraction greater than 30 kda to regulate spermatogenesis. Hum Reprod :

7 [14] Franca LR Ogawa T Avarbock MR et al. Germ cell genotype controls cell cycle during spermatogenesis in the rat. Biol Reprod : [1] Steinberger E Steinberger A Perloff W. Studies on growth in organ culture of testicular tissue from rats of various ages. Anatomical Record :81-9. [16] Trowell O. The culture of mature organs in a synthetic medium. Exper Cell Res : [17]. PGP :-4. [18] Freshney RI. Culture of Animal Cells: A Manual of Basic Technique. New York: Wiley [19] Cheng J Dutra A Takesono A et al. Improved generation of C7BL/6J mouse embryonic stem cells in a defined serumfree media. Genesis : [0] Hong-mei P Gui-an C. Serum-free medium cultivation to improve efficacy in establishment of human embryonic stem cell lines. Hum Reprod :17-. [1] Petkov SG Anderson GB. Culture of porcine embryonic germ cells in serum-supplemented and serum-free conditions: the effects of serum and growth factors on primary and longterm culture. Cloning Stem Cells : [] Fraga AM de Araújo ÉSS Stabellini R et al. Establishment of new lines of human embryonic stem cells: Methods Mol Biol : Influence of Fetal Bovine Serum and Knockout TM SR on Germ Cell Development by Rat Immature Testicular Tissue Culture Chun-hong CAI Da-wei HE Xin WU Yan-xia CHENG Guang-hui WEI Department of Urology Children s Hospital of Chongqing Medical University; Ministry of Education Key Laboratory of Child Development and Disorder; Key Laboratory of Pediatrics in Chongqing Chongqing International Science and Technology Cooperation Center for Child Development and Disorders Chongqing ABSTRACT Objective: To evaluate the effect of fetal bovine serum FBS and serum replacement Knockout TM SR KSR on germ cell development. Methods: The FBS and the KSR were used as supplement of culture medium the testicular tissues were cultured for weeks. Area of the cultured tissues was messured. The testicular histology the development of the germ cell and the diameter of seminiferous tubules were observated by HE staining. The apoptotic cells were observed in the testis cultuted for weeks by apoptosis experiments. The marker genes of spermatogenesis in various stages such as Kit Sycp3 and Crisp1 were identified by RT-PCR. Results: In KSR group the cultured tissues could sustain growth and the seminiferous tubules gradually increased in the period of culture. The spermatogonia primary spermatocytes secondary spermatocytes and round spermatids were observed in the cultured tissues after cultured for weeks and their marker genes Kit Sycp3 and Crisp1 were identified. In FBS group the testicular organization dwindled in the period of culture; the seminiferous tubules appeared obviously necrosis and the expression of the Sycp3 and Crisp1 were instability after cultured for weeks. Conclusion: KSR has more advantages for spermatogonial cells to develop into sperm cells in immature tissue culture and can maintain the germ cell development and the cultured tissues growth development for a long time. Key words: testis; tissue culture; spermatogenesis; rat 810