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1 J. of Aquaculture Vol. 19(4) : , 2006 µ Journal of Aquaculture Korean Aquaculture Society ã(misgurnus mizolepis) : II. sƒ û«*, 1, y 2,, ½ w w/wxy, 1ww wœw, 2 w w Intraspecific Androgenesis in Mud Loach (Misgurnus mizolepis): II. Diploid Restoration and Viability Assessment Yoon Kwon Nam*, In-Chul Bang 1, Choong Hwan Noh 2, Young Sun Cho and Dong Soo Kim Department of Aquaculture & Institute of Marine Living Modified Organisms (imlmo), Pukyong National University, Busan , Korea 1 Department of Marine Biotechnology, Soonchunhyang University, Asan, , Korea 2 Marine Resources and Research Department, Korea Ocean Research and Development Institute, Ansan , Korea Intraspecific diploid androgenesis was achieved in mud loach (Misgurnus mizolepis) by the inhibition of the first mitotic division using combined thermal treatment. A combined thermal treatment (heat shock at 40.5 o C for 120 sec followed by cold treatment at 1 o C for 45 min) applied to the 1st metaphase of cell division (28 min post insemination at 25 o C) successfully recovered viable androgenetic diploidy. Mean hatching success of the androgenetic diploid group was 29.6%, and the average yield out of total eggs taken was about 7% assessed at 1 week of age. However, relatively large variations in the yield of diploid androgenesis were observed among different egg batches used as cytoplasmic donors. Successful diploidization was confirmed by flow cytometric analysis, and parthenogenic reproduction in a paternal exclusive manner was verified with transgene dosage. Significant mortality was found in most androgenetic groups especially from hatch to 1 month of age, although such mortality was stabilized later. Keywords: Diploid androgenesis, Mud loach, Parthenogenic reproduction, Viability 1ùw»(blocking of the first mitotic division) œ (induced artificial parthenogenesis) 4 (genome duplication; tetraploidy) w» ù w 1s» 2s» (embryonic development) jš, (chromosome set)ƒ ƒ(doubling) w»(thorgaard, 1986; Arai, 2001). p (induced androgenesis) k (loci), xw(homozygous)yw š mw p x šw (Bercsenyi et al., 1998; Kirankumar and Pandian, 2003). *Corresponding author: yoonknam@pknu.ac.kr 281» š m wš w, p (genome-restoration) xw w r ƒ yš (Zhang et al., 2000; Babiak et al., 2002a; Kirankumar and Pandian, 2004; David and Pandian, 2006). ù z û 1) yù ù w w, 2) ù yy ùy RNA q w, 3) w s w (nuclearcytoplasmic instability, NCI) š 4)»x û»»w(bercsenyi et al., 1998; Babiak et al., 2002b). s mw

2 282 û«,, y,, ½ w { û šš (Bongers et al., 1994; Babiak et al., 2002b). z l w» w, ù w (intraspecies androgenesis) œ w tƒ, ù w NCI z yw x w w k., yy ù 1ùw wš, 1ùw w š w zƒ y z» z yw sƒ w w w ƒ y w. ù š ã (Misgurnus mizolpis) ã (interspecies androgenesis) û z( 4%; Nam et al., 2002) w» w y ü ã» wš w. w ã xy t, intraspecies haploid androgenesis yw (Nam et al., 2006), 1ùw mw ã wš» z» sƒwš w. x I: ù yy mw x ã w w w 5 e s³ (body weight BW) 45Û 13 g f 15Û5 g f e w. el Kim et al. (1994) k y(hcg) w ww. w ù yw. 8 fl ù j»» ù 4 f w, 5 f yww x w. ù yy j» w Nam et al. (2006) w (UV) ww. UV 10,800 ergs/mm 2 w yy ù 3, µl (1:30 in saline), mw œ w. 25 o C w ww 1ùw» 28 z Nam et al. (2004) w š (40.5 o C 2) (1 o C 45) ww ww. 1ùw ƒ óù 25 o C w y¾( z 24~27 ) w. z sƒw» w ƒ 3 w, y ù y»(y z 36 )¾» w. x II: f e z w ã f el ù ywš ù e w w. wš m w yw 16 f( 36~61 g)l œ» mw ƒƒ ù ywš» ù yy w. œ w w 12 fl yw w. ƒ w ƒ 3,000Û120 ù swm w. œ,, y, y»» x I w wwš, ƒ x 3 y w z w f e z w. x III: sƒw» w ùy zl» z» w. x I II k 3 f 3 fl w wš ùy ƒ 1, 2, 3 4» w. z 1 y 4 ¾ z» 3z w. ùy 36 ƒ ƒ 50 L 3 w w 25Û1 o C wš k x Kim et al. (1994) w. x IV: flow cytometry z yw» w flow cytometry w ã s DNA w dw. e homogenization (10 mm Tris ph 7.8, 5 mm EDTA ph 8.0, 50 mm NaCl) w z 20 µm mesh w s zw. z s 50 µg/ml propidium iodideƒ sw (1x PBS ph 7.6, 0.01% NP 40) 1 w z WinBryte HS flowcytometer (BioRad, USA) w s s³ DNA w w., ã (1.4 pg/cell), š x

3 s(2.8 pg/cell) w. y 2 w l x s w homogenization» w flow cytometry w. x V: t w œ xy t w, duplication yw. x w xy chloramphenicol acetyltransferase (CAT) reporter construct xw(homozygous) xy m w w p Nam et al. (1999, 2000)». x I k xy f l 3 fl yy ù» w y 23 flow cytometry mw yw y ƒ 12 transgene dosage w. ƒ (fin)l Nam et al. (1999) w genomic DNA w genomic DNA 2 µg dot blot hybridization w. genomic DNA w positively charged nylon membrane (Roche, Germany) dot blot apparatus (Bio- Rad) w 3 spotting w CAT-transgene probe (Nam et al., 2000) w hybridization w. Hybridization sww stringent washing signal detection (DIG Non-radioactive DNA Labeling and Detection kit, Roche) instruction manual w ww. Internal control ã growth hormone gene probe (Nam et al., 2002) w CAT transgene signal normalizationw. yw» w (doubled transgene dosage; homozygous at transgene loci) bi-parental (heterozygous transgene dosage) signal image analysis software Quantityone (Bio-Rad) w. m x I z sƒ w ã 283 w y» x (ANOVA) Duncan s multiple range test w P<0.05 sƒ w. x III student s t-test mw wš(p<0.05), x V transgene dosage Student s t-test w bi-parental transgene signal (P<0.05) w. š w x I w ù z s³ 85% ùkù(figures not shown) w Nam et al. (2006) w z UVƒ. 1ùw» mw w, 90% y 10,800 ergs/mm 2 UV 51.4% s³ y, š UV ƒ w 29.6% s³ y ù kü(table 1). yz» s. y s³ 87.5%ƒ ùy»¾ wù 37.5% š ù ùy w w sw. k 1ùw w w w ƒ qù(pandian and Koteeswaran, 1998) ù potential aneuploidy hypodiploidy x, w r w s w w (Corley-Smith et al., 1996; Maremgpmo and Onoue, 1998; Tanck et al., 2001). xwyw» x(recessive traits)» w w q(lin and Dabrowski, 1998; Paschos et al., 2001). y 1 Table 1. Percent hatching, early survival, incidence of 2N and yield of intraspecific androgenesis in mud loach 1 Genotype Hatching success (%) 2 Early survival rate (%) 2 Incidence of diploidy (%) 3 Normal diploid control 91.2±5.8 a 87.5±6.8 a 100±0.0 a Androgenetic haploid 51.4±9.2 b - 7.1±2.7 b Androgenetic diploid 29.6±8.1 c 37.2±5.4 b 98.1±2.1 a 1 Mean±SDs are based on at least three replicate examinations. Means with same letters within a column did not differ significantly at P=0.05 based on ANOVA. 2 Hatching success and early survival up to yolk sac absorption were estimated as percentages of eggs inseminated and hatched larvae, respectively. 3 Ploidy estimation was carried out using flow cytometry with 1-week-old fish for diploid groups, while the score for androgenetic haploid group was based on the examination of external morphology at 1 day post hatching.

4 284 û«,, y,, ½ l 3z w 54 flow cytometry ww aneuploidy peak 1 wš 53 (98.1%) yw DNA w y z ùkû (Table 1; peaks not shown). ù 1ùw w ƒ 7.1% w ƒ w wš. û xw w UVƒ 100% ù yy j w» w k (Maremgpmo and Onoue, 1998; Tanck et al., 2001; Nam et al., 2006). Cytoplasmic donor z» x k w f el ù(cytoplasmic donor) w z w. (yield) w sƒw w w š w ù ùkü. (yield) = [y(%) ùy» ¾ (y w ) ùy»l y 1¾ (%) x (y 1 flow cytometry ; group 1220 )] x w f e y 5894% w ùkû(histograms in Fig. 1a) ƒƒ w ù (egg batch) w ww, ù w 0.05% (f no. 5) 10.3% (f no. 3)¾ w egg batch j ùkü(line drawing in Fig. 1a). x y, y û z w w ƒ. Fig. 1b mw» y (X ) w ƒ e(y ) txw, Fig. 1b y 70% w œ ƒ w ùkû. w y 70% y y w ùkü, 7% y» w y 90% w ƒw (Fig. 1b). w e Fig. 1. Maternal influences on the yield (%) of diploid intraspecific androgenesis in mud loach. UV-inactivated egg batches from 16 independent females were separately inseminated with the pooled sperm from 9 males, and subjected to thermal treatment for blocking the 1st cleavage. a) Yields (mean±sds based on triplicate examinations per cross) of diploid androgenesis at 1 week of age (line drawing) addressed for each female with the comparison of percent hatching for the non-treated control group (histograms) from the same female. b) Percent yields represented as function of the hatching success in diploid control groups without data separation into each female group. Details for calculating the yield of androgenesis can be referred to the equation in Materials and Methods. m ù j w x wš (Bongers et al., 1995), p y w w ù w w wš. ã ùy z w, w û (Table 2). ùy z s ùk ü p sƒ»» w ùkü. ùy z 1, s³ 97.2%

ã 285 Table 2. Percent survival of androgenetic diploid mud loaches along with normal diploid controls as function of age Age Normal diploid control Androgenetic diploid 1 week 98.1±1.6 a 69.4±7.

5 ã 285 Table 2. Percent survival of androgenetic diploid mud loaches along with normal diploid controls as function of age Age Normal diploid control Androgenetic diploid 1 week 98.1±1.6 a 69.4±7.3 a 2 week 95.4±1.6 a 54.6±5.8 a 3 week 91.7±2.8 a 48.1±4.2 a 4 week 88.9±2.8 a 38.9±7.3 a 2 month 85.2±1.6 a 37.0±4.2 a 3 month 84.3±3.2 a 34.3±4.2 a 4 month 83.3±2.8 a 34.3±4.2 a Fish were allocated into replicate tanks after yolk sac absorption (see Materials and methods). Means with same letters within a row were not significantly different at P=0.05 based on student s t-test. û 69.4% ùkü(p<0.05). z 2, 3 4¾ ƒ 6.5~14.5% sƒ ƒ wš, y 1 s 12% 61% w e ùkü. wš y 1 zl y w ùkü y 4 ¾ s(0~2.8%) š s(0~3.7%) m ùkü (P>0.05). ù ùy l y 4 ¾ (34.3%) (83.3%) 41% ww ùkû(table 2). e x ù» w šwš w w ù kü(bercsenyi et al., 1998; Babiak et al., 2002b). w m w xw z xw k tx (recessive genes) x wy» x q p», e k s 1ùw w z(adverse effect)ƒ y z» ùküš. ƒw, š ƒ y z» w w öe ã k š (Thorgaard, 1986; Kim et al., 1994).» w w š» z» x ùkù š m ƒ vw š. Transgene dosage w xy t w m w y( duplication) w. xw CAT-transgenic Fig. 2. Transgene dosage assessment in androgenetically derived homozygous (Andro-2N) and bi-parental hemizygous (Bi-parental 2N) mud loaches. a) Representative dot blots showing the difference in hybridization signal between homozygous and hemizygous fish. b) Densitometric analysis of the hybridized signals using the GH gene probe as a normalization control. Statistical difference was found between androgenetic diploid and bi-parental diploid groups based on student s t-test (P<0.05). fl f(non-transgenic)l yy ù w y z 2~3 z CAT xy w x y s copy (transgene dosage) ƒ(homozygous y) w(fig. 2). e w w (bi-parental 2N)l ƒ 12 w dot blot hybridization xy x w xy f xw k wš (photographs not shown). ù xy ùkû (chromosome-set) w z (Fig. 2a). y mw x y xy xw xk ùkü xy f f w transgenic hemizygous k ùkü (see Nam et al., 2002). GH gene ü w CAT-xy normalizationw

6 286 û«,, y,, ½ w 2 w e ùkü ew (Fig. 2b). ù Fig. 2b internal control normalization ww wš r ƒ j ùkù xy copy w d ƒw. blot hybridization ƒ w»w q(see Cho et al., 2005) wz real-time PCR w w w vƒ. mw ü ã ƒ ƒw w s œ w w ù w ùkù û w ƒ ƒw q. ù ù w yy w» bi-parental x, š û» wz w m mw w w w û. w k x w sƒ( ) mg DNA sƒw. ù ã(misgurnus mizolepis) w» w y ü wš sƒw. (UV; 10,800 ergs/mm 2 ) w ù yyjš œ 28 z(1ùw») (š 40.5 o C 120 z 1 o C 45) mw 1ùw w. mw š(s³ y, 26.9%), y z 1» s³ ù 7%ü ùkû. ù z cytoplasmic donor j w w f e j rƒ. Flow cytometry w y xy t w xw ƒ y. w û p y z 1 ¾ û ùkü. ù z» w w w w w w (KRF F00041). šx Arai, K., Genetic improvement of aquaculture finfish species by chromosome manipulation techniques in Japan. Aquaculture, 197, Babiak, I., S. Dobosz, H. Kuzminski, K. Goryczko, S. Ciesielski, P. Brzuzan, B. Urbanyi, A. Horvath, F. Lahnsteiner and J. Piironen, 2002b. Failure of interspecies androgenesis in salmonids. J. Fish. Biol., 61, Babiak, I., S. Dobosz, K. Goryczko, H. Kuzminski, P. Brzuzan and S. Ciesielski, 2002a. Androgenesis in rainbow trout using cryopreserved spermatozoa: the effect of processing and biological factors. Theriogenology, 57, Bercsenyi, M., I. Magyary, B. Urbanyi, L. Orban and L. Horvath, Hatching out goldfish from common carp eggs: interspecific androgenesis between two cyprinid species. Genome, 41, Bongers, A. B. J., E. P. C. in t Veld, K. Avo-Hashema, I. M. Bremmer, E. H. Eding, J. Komen and C. J. J. Richter, Androgenesis in common carp (Cyprinus carpio L.) using UV irradiation in a synthetic ovarian fluid and heat shocks. Aquaculture, 122, Bongers, A. B. J., J. B. Abraca, Z. Doulabi, E. H. Eding, J. Komen and C. J. J. Richter, Maternal influence on development of androgenetic clones of common carp, Cyprinus carpio L. Aquaculture, 137, Cho, Y. S., B. N. Choi, E.-M. Ha, K. H. Kim, S. K. Kim, D. S. Kim and Y. K. Nam, Shark, Scyliorhinus torazame metallothionein: cdna cloning, genomic sequence and expression analysis. Mar. Biotechnol., 7, Corley-Smith, G. E., C. J. Lim and B. P. Brandhorst, Production of androgenetic zebrafish (Danio rerio). Genetics, 142, David, C. J. and T. J. Pandian, Cadaveric sperm induces intergeneric androgenesis in the fish, Hemigrammus caudovittatus. Theriogenology, 65, Kim, D. S., J.-Y. Jo, and T.-Y. Lee, Induction of triploidy in mud loach (Misgurnus mizolepis) and its effect on gonad development and growth. Aquaculture, 120, Kirankumar S. and T. J. Pandian, Interspecific androgenetic restoration of rosy barb using cadaveric sperm. Genome, 47, Kirankumar, S. and T. J. Pandian, Production of androgenetic tiger barb Puntius tetrazona. Aquaculture, 228, Lin, F. and K. Dabrowski, Androgenesis and homozygous gynogenesis in muskellunge (Esos masquinongy): evaluation using flow cytometry. Mol. Reprod. Dev., 49, Maremgpmo, M. G. and Y. Onoue, Ultraviolet-induced androgenesis in nile tilapia, Oreochromis niloticus (L.), and hybrid nile x blue tilapia, O. aureus (Steindachner). Aquaculture Res., 29, Nam, Y. K., C. H. Noh and D. S. Kim, Transmission and expression of an integrated reporter construct in three generations of transgenic mud loach (Misgurnus mizolepis). Aquaculture, 172,

7 ã 287 Nam, Y. K., C. H. Noh and D. S. Kim, Intraspecies androgenesis in mud loach (Misgurnus mizolepis): I. Optimization of the egg inactivation and haploid androgenesis using transgene marker. J. Aquacult., 19 (in press) Nam, Y. K., G. C. Choi and D. S. Kim, An efficient method for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach (Misgurnus mizolepis). Theriogenology, 61, Nam, Y. K., Y. S. Cho, H. J. Cho and D. S. Kim, Accelerated growth performance and stable germ-line transmission in androgenetically derived homozygous transgenic mud loach, Misgurnus mizolepis. Aquaculture, 209, Nam, Y. K., Y. S. Cho, Y. J. Chang, J.-Y. Jo and D. S. Kim, Generation of transgenic homozygous line carrying the CAT gene in mud loach Misgurnus mizolepis. Fish. Sci., 66, Pandian, T.J. and R. Koteeswaran, Ploidy induction and sex control in fish. Hydrobiologia, 384, Paschos, I., L. Natsis, C. Nathanailides, I. Kagalou and E. Kolettas, Induction of gynogenesis and androgenesis in goldfish Carassius auratus (var. oranda). Reprod. Dom. Anim., 36, Tanck, M. W. A. P. Palstra, M. van der Weerd, C. P. Leffering, J. J. van der Poel, H. Bovenhuis and J. Komen, Segregation of microsatellite alleles and residual heterozygosity at single loci in homozygous androgenetic common carp (Cyprinus carpio L.). Genome, 44, Thorgaard, G. H., Ploidy manipulation and performance. Aquaculture, 57, Zhang, H., B., Robison, G. H. Thorgaard and S. S. Ristow, Cloning, mapping and genomic organization of a fish C-type lectin gene from homozygous clones of rainbow trout (Oncorhynchus mykiss). Biochim. Biophys. Acta, 1494, š : :

14.531~539(08-037).fm

14.531~539(08-037).fm G Journal of the Korea Concrete Institute Vol. 20, No. 4, pp. 531~539, August, 2008 š x y w m š gj p { sƒ z 1) * 1) w w Evaluation of Flexural Strength for Normal and High Strength Concrete with Hooked