Stage2specific Expression Analysis of Mouse Testis2specific Genes in Spermatogenic Cells. GUO Rui LI Xi2Xia WANG Hui2Zhen. , ; Prm1 Prm2 Tnp1 Tnp2

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1 Chinese Journal of Zoology 2009,44 (1) :39 46 ( ) : BalbΠc, RT2PCR 12, SYBR Green PCR, ; Prm1 Prm2 Tnp1 Tnp ; Dnajb3, 215 ; Akap4, 515 ; Spata3 Spata4, ; hils1 Tex24, ; Spag4l Papolb, 45 % 34 %,,, PCR : ; PCR ; ; :R32111 :A : (2009) Stage2specific Expression Analysis of Mouse Testis2specific Genes in Spermatogenic Cells GUO Rui LI Xi2Xia WANG Hui2Zhen ( Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan , China) Abstract :Many testis2specific and stage2specific genes are required for mouse spermatogenesis. In this study, semi2 quantitative RT2PCR was used to confirm the tissue distribution of 12 mouse testicular genes, and fluorescence quantitative PCR labeled with SYBR Green was applied to demonstrate the stage2specific expression of candidate genes at different spermatogenic stages. The results showed that all the genes examined were highly expressed in testis. The expression of Prm1, Prm2, Tnp1, and Tnp2 was predominant in elongating spermatids, 1192, 2182, 3122, and 22fold increase as compared with that in pachytene spermatocytes, respectively. Dnajb3 expression was up2 regulated 2152fold from pachytene spermatocytes to elongating spermatids. Akap4 was strongly expressed at elongating spermatid stage, with 5152fold increase than that in pachytene spermatocytes. Both Spata3 and Spata4 were equally expressed in round and elongating spermatids, 32 and 1152fold increase as compared with pachytene spermatocytes, respectively. The expression levels of hils1 and Tex24 were the highest in round spermatids, with 1192 and 1142fold (No ) ;,,,; :; E2mail com. cn : , :

2 40 Chinese Journal of Zoology 44 increase from pachytene spermatocytes, respectively. Spag4l and Papolb were down2regulated from pachytene spermatocytes to elongating spermatids, decreasing 45 % and 34 %, respectively. The results not only demonstrate the stage2specific expression of these genes, but also provide new data for further investigations into the functions of these specific genes during spermatogenesis. Key words :Spermatogenesis ; Fluorescence quantitative PCR ; Mouse ; Spermatogenic cell,,, [1 ] BalbΠc,,,, Northern RT2PCR, PCR, Northern PCR,, [2 ] PCR,12 PCR ( Protamine gene, Prm ) ( Transition protein gene, Tnp),,,, Hsp40 DnaJ B 3 [ DnaJ ( Hsp40 ) homolog, subfamily B, member 3, Dnajb3 ] A 4 [ A kinase ( PRKA) anchor protein 4,Akap4 ] H1 1 (histone H12like protein in spermatids 1, hils1) A [ poly (A) polymerase beta (testis specific), Papolb ] 4 ( sperm associated antigen 42 like, Spag4l) 24 (testis expressed gene 24, Tex24),, ; PCR,, ;, 3 (spermatogenesis associated 3, Spata3) 4 ( spermatogenesis associated 4, Spata4) [3,4 ],, g BalbΠc,

3 1 : 41,, MMLV ( Promega), RNAlater, RNA RT2PCR RNA RNeasy (Qiagen) RNase2free DNase, ( Eppendorf),1 % RNA, 1 g RNA cdna GenBank cdna, Primer Express 310 (ABI ) ( 1),Takara PCR :94 20 s,52 30 s,72 30 s, 32 2actin, 25 PCR 3 %, () Gene name (Accession No. ) 1 RT2PCRPCR Table 1 Primer pairs used for RT2PCR and quantitative PCR (5 3 ) Primer sequence (5 3 ) Relative position Product size (bp) Akap4 Forward CAA CCA GAA GCA AGG AAA C (NM ) Reverse GCA TAA GGG CAG AGA CAA Spag4l Forward TGA CAC CTG GGA ACT GCT (NM ) Reverse TGG CTG AAA CTG GAA TGC Tex24 Forward CCC TGA CCC TAA TCC AAG C (NM ) Reverse CCC AAC AAA TGT CTG CCT G Papolb Forward TCA GTC GTT TCC AGC ACC (NM ) Reverse GCT TCT TCA ACA CTA ACC CG Spata3 Forward ATT TCA CCG CAG ACT AGC TC (NM ) Reverse CAC TGC TGC TTT CCT GCT TG Spata4 Forward TGT CGG AAC TAC TAA GCA ATC (NM ) Reverse CCT CTT GAC TTG TAT CTG CG Tnp1 Forward AGG AGA GGC AAG AAC CGA GC (NM ) Reverse TCA CAA GTG GGA TCG GTA Tnp2 Forward CCT CAA AGT CAC ACC AGT AAC CAG (NM ) Reverse GTC TTC CTG TTC TTG GGG CA Prm1 Forward AGA TAC CGA TGC TGC CGC (NM ) Reverse ACC TTA TGG TGT ATG AGC G Prm2 Forward CAC CAC CAC CAC AGA CA (NM ) Reverse ATC TCC TCC TCC TTC GG Dnajb3 Forward GGC TTC GCT TCG TTA GAT AC (NM ) Reverse TTT CGG TGG AGG TTG ACA hils1 Forward TTG TGT GTC CCT TGC TAC C (NM ) Reverse TGG CTC TTC TGT CTG TCC T actin Forward CTA TTG GCA ACG AGC GGT (NM ) Reverse GGT CTT TAC GGA TGT CAA CG [5 ] d, :,, PBS,,1 cm,,pbs 0125 %,33,,,

4 42 Chinese Journal of Zoology rπmin 5 min,,pbs, 200, 015 % BSA 10 ml PBS,, 4 % 2 % BSA ml,, 15 mlπmin, 2 % 4 % BSA 4 3 h, BSA Akap4 Spata4,, 85 %, 10 ( ), 2actin PCR,,, 20 l, 2 SYBR R Premix Ex Taq TM 10 l,cdna 2 l, 014 l (10 pmolπl) : s,94 15 s,52 25 s, s,40 3,,, Ct, Ct,, 2 2 Ct 1, 3 % RT2PCR RT2PCR Prm1 Prm2 Tnp1 Tnp2 Papolb Tex24 Dnajb3 hils1 Spag4l Spata3, ( 1),37 5 % CO PCR 6 8 h,, 2actin (,, 2), 100 cdna, : 90 % 88 % 95 % 96 % 92 %, RNeasy RNA MMLV cdna 114 PCR PCR Ct SYBR Premix Ex Taq TM ( Takara) StepOnePlus TM Real Time PCR (ABI) cdna,,,, 3 %, ( 3) 1, , Dnajb3 Spata3 Spata4 Tex24 Spag4l, Prm1 Prm2 Tnp1 Tnp2, Prm1 119 ; Prm2 218 ; Tnp1, ; Tnp2 2,, Akap4, 515

5 1 : cdna 2actin Fig. 2 Amplification of 2actin with diluted cdna samples at RT2PCR Fig. 1 Tissue distribution of candidate genes as confirmed by RT2PCR M: 50 bp DNA ; 1 : ; 2 : ; 3 : ; 4 : ; 5 : ; 6 : ; 7 : ; 8 : ; 9 : ; 2actin M: 50 bp ladder DNA marker ; 1 : Brain ; 2 : Heart ; 3 : Lung ; 4 : Liver ; 5 : Spleen ; 6 : Kidney ; 7 : Ovary ; 8 : Uterus ; 9 : Testis ; 2actin is a positive control. 313 ; Dnajb3, 215 ; Spata3, 3, ; Spata4, 50 % ; hils1 Tex24 3 PCR Fig. 3 Stage2specific analysis of candidate genes by fluorescence quantitative PCR 1 : ; 2 : ; 3 : ; 4 : ; 5 : ; 2actin 1 : Spermatogonia ; 2 : Preleptotene spermatocytes ; 3 : Pachytene spermatocytes ; 4 : Round spermatids ; 5 : Elongating spermatides ; 2actin is a positive control.

6 44 Chinese Journal of Zoology 44, hils1 2 ; Tex % 45 % ; Papolb 214 ; Spag4l Papolb, Spag4l 34 % 3 4 PCR Fig. 4 Relative quantitation of candidate genes by fluorescence quantitative PCR,,,,RT2PCR 12,, 2 1 (testis2 specific histone 1, H1T), (transition protein, TP) H1T, (protamine,mp) TP Tnp1 Tnp2 MP Prm1 Prm2 : Prm1 Prm2 Tnp1 Tnp2 mrna HILS1 [6, 7 ] hils1 mrna HILS1 H1T, 2,, mrna Tnp Prm Prm1 Prm2 Tnp1 Tnp2 mrna, hils1 [8 ],mrna poly A, PAPOLb

7 1 : 45 Poly A, 3, Papolb [9, 10 ] Papolb,,, AKAP4,Northern blot Akap4 [11 ] , AKAP4 DNAJb 3 DNAJ, [12 ] Dnajb3, Tex24,RT2PCR Tex24 mrna [13 ], Tex24 Sgap4l 3, [14 ] Sgap4l, 50 % Spata3 Spata4, [3, 4 ], RT2PCR Spata3 Spata4, Spata3,3 ; Spata4, 115, PCR,, Northern PCR, PCR, [ 1 ] Sluka P, O Donnell L, Stanton P G. Stage2specific expression of genes associated with rat spermatogenesis : characterization by laser2capture microdissection and real2time polymerase chain reaction. Biology of Reproduction, 2002, 67 (3) : [ 2 ] Vanguilder H, Vrana K, Freeman W. Twenty2five years of quantitative PCR for gene expression analysis. Biotechniques, 2008, 44(5) : [ 3 ],,. TSARG1 Mtsarg1 Mtsarg1., 2003, 30(1) : [ 4 ] Liu S F, He S, Liu B W, et al. Cloning and characterization of testis2specific spermatogenesis associated gene homologous to human SPATA4 in rat. Biol Pharm Bull, 2004, 27 (11) : [ 5 ] Yu Z, Guo R, Ge Y, et al. Gene expression profiles in different stages of mouse spermatogenic cells during spermatogenesis. Biology of Reproduction, 2003, 69 (1) : [ 6 ] Wei Y, Lang M, Burns K H, et al. HILS1 is a spermatid2 specific linker histone H12like protein implicated in chromatin remodeling during mammalian spermiogenesis. PNAS, 2003, 100 (18) : [ 7 ] Depa2Martyn w M, Kempisty B, Lianeri M, et al. Association between fertilin beta, protamines 1 and 2 and spermatid2specific linker histone H12like protein mrna levels, fertilization ability of human spermatozoa, and quality of preimplantation embryos. Folia Histochem Cytobiol, 2007, 45 (Suppl 1) : S79 S85. [ 8 ] Hecht N B. Molecular mechanisms of male germ cell differentiation. Bioessays, 1998, 20(7) : [ 9 ] Choi H S, Lee S H, Kim H, et al. Germ cell2specific gene 1 targets testis2specific poly (A) polymerase to the endoplasmic reticulum through protein2protein interactions. FEBS Lett, 2008, 582(8) : [10 ] Kashiwabara S, Noguchi J, Zhuang T, et al. Regulation of spermatogenesis by testis2specific, cytoplasmic poly ( A ) polymerase TPAP. Science, 2002, 298 (5 600) : 1 999

8 46 Chinese Journal of Zoology [11 ] Brown P R, Miki K, Harper D B, et al. A2kinase anchoring protein 4 binding proteins in the fibrous sheath of the sperm flagellum. Biology of Reproduction, 2003, 68 (6) : [12 ] Meccariello R, Cobellis G, Berruti G, et al. Mouse sperm cell2specific DnaJ first homologue : an evolutionarily conserved protein for spermiogenesis. Biology of Reproduction, 2002, 66 (5) : [13 ] Fan J, Graham M, Akabane H, et al. Identification of a novel male germ cell2specific gene TESF21 in mice. Biochemical and Biophysical Research Communications, 2006, 340 (1) : [14 ] Xing X W, Li L Y, Liu G, et al. Identification of a novel gene SRG4 expressed at specific stages of mouse spermatogenesis. Acta Biochimica et Biophysica Sinica, 2004, 36 (5) : , (Hong Meigui Cave Exploration Society),, 2 3 ;,, 3 ; 2,( Aselliscus stoliczkanus Dobson, 1871) ( 1,M Ficco ), ( E ,N ; 256 m) (,2003) Smith & Xie (A Guide to the Mammals of China,2008), (,2007),, 1 LYNCH Erin KREJCA K Jean FICCO Mike ( ; Hong Meigui Cave Exploration Society ; Zara Environmental LLC, Buda, Texas, USA)