Failure of interspecies androgenesis in salmonids

Transcription

1 Journl of Fish Biology (2002) 61, doi: /jfbi , vilble online t on Filure of interspecies ndrogenesis in slmonids I. BABIAK*, S. DOBOSZ, H. KUZMINSKI, K. GORYCZKO, S. CIESIELSKI, P. BRZUZAN, B. URBÁNYI, Au. HORVÁTH, F. LAHNSTEINER AND J. PIIRONEN** *Deprtment of Animl Biochemistry, University of Wrmi nd Mzury, Olsztyn, Polnd, Deprtment of Slmonid Reserch, Inlnd Fisheries Institute, Rutki, Zukowo, Polnd, Deprtment of Fish Biology nd Culture, University of Wrmi nd Mzury, Olsztyn, Polnd, Deprtment of Evolutionry Genetics, University of Wrmi nd Mzury, Olsztyn, Polnd, Szent István University, Deprtment of Fish Culture, H-2103 Gödöllõ, Hungry, Institute of Zoology, Slzburg University, A-5020 Slzburg, Austri nd **Finnish Gme nd Fisheries Reserch Institute, Sim Fisheries Reserch nd Aquculture, Enonkoski, Finlnd (Received 31 My 2001, Accepted 21 June 2002) Androgenetic development of slmonid embryos ws induced in recipient oocytes from the sme or other species (intr- or interspecies ndrogenesis). Prmeters for induced ndrogenesis were investigted in brown trout Slmo trutt nd brook trout Slvelinus fontinlis. Reciprocl ndrogenetic nd control crosses were conducted mong fishes from three gener: Oncorhynchus (rinbow trout, O. mykiss), Slmo (brown trout) nd Slvelinus (brook trout), nd within two gener: Slmo (brown trout nd Atlntic slmon, S. slr) nd Slvelinus (brook trout nd Arctic chrr, S. lpinus). Live htched ndrogenetic progenies were obtined in ll intrspecies vrints, where oocytes nd sperm originted from the sme species. Interspecies ndrogenesis resulted in no vible lrve, despite the fct tht most hybrid controls nd intrspecies ndrogenetic individuls were vible. When recipient oocytes originted from other gener (interspecific intergeneric ndrogenesis), embryonic development cesed in erly developmentl stges, except for hploid controls of brook trout produced in eggs of brown trout. Survivl of embryos to the eyed-egg stge ws reltively high in the intrgeneric ndrogenesis experiment. Nevertheless, none of these embryos survived to htching. Some of the presumed Atlntic slmon individuls developing in brown trout eggs contined mternl DNA, questioning the ccurcy of enucletion using irrdition. The inbility to induce interspecific ndrogenesis mong the exmined slmonid species my hve been the result of substntil kriotypicl nd developmentl differences between spermtozol donors nd oocyte recipients, cusing n incomptibility between mternl cytoplsmic regultory fctors nd the pternl nucler genome The Fisheries Society of the British Isles Published by Elsevier Science Ltd. All rights reserved. Key words: ndrogenesis; embryo; fish; hybrid; reproduction; trout. INTRODUCTION Androgenesis, the induced development of individuls inheriting exclusively the pternl nucler genome, hs been ccomplished in only few fish species (Ihssen et l., 1990; Pndin & Koteeswrn, 1998). The difficulties in producing vible ndrogenetic progeny result from the drstic tretments pplied to oocytes before insemintion (mternl nucler DNA eliminted by irrdition) nd to Author to whom correspondence should be ddressed. Tel: ; fx: ; emil: bbik@uwm.edu.pl /02/ $35.00/ The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved.

2 ANDROGENESIS IN SALMONIDS 433 zygotes t the first mitotic division (therml or pressure shock for doubling the hploid chromosome set). Aside from the production of homozygous nd clonl lines nd of monosex popultions using spermtozo from ndrogenetic YY supermles, ndrogenesis is considered relible tool for the recovery of entire fish cryopreserved genome informtion (Thorgrd, 1986; Thorgrd & Cloud, 1993). Most studies on ndrogenesis hve concerned slmonids of the genus Oncorhynchus. Few of the studies hve reported survivl high enough to estblish ndrogenetic stocks (Prsons & Thorgrd, 1985; Scheerer et l., 1986, 1991; Thorgrd et l., 1990; Ngoy et l., 1996; Bbik et l., 2002). Scheerer et l. (1991) first demonstrted tht ndrogenetic rinbow trout Oncorhynchus mykiss (Wlbum) re ble to produce gmetes nd tht ndrogenetic development from cryopreserved spermtozo is possible. Recently, Bbik et l. (2002) estblished stock of ndrogenetic rinbow trout produced using cryopreserved spermtozo. This shows promise for genome bnking progrmmes in rinbow trout, however the technology is not developed in other slmonid species. Aprt from Oncorhynchus, successful ndrogenesis hs lso been induced in brook trout Slvelinus fontinlis (Mitchill) (My et l., 1988). No dt re reported for Slmo. Interspecific ndrogenesis (induced ndrogenetic development in oocytes originting from other species) is considered s mens to restore extinct txons from their cryopreserved spermtozo s well s to investigte nucleocytoplsmic comptibility (Bercsenyi et l., 1998). In slmonids, erly developmentl stges re controlled exclusively by mternl, cytoplsmic fctors; expression of zygotic informtion begins t the blstul stge (Aoygi et l., 1993; Ngler, 2000). Thus, in interspecific ndrogenesis, nucler genomes follow cell cycles regulted in other species mode. There re only few reports on interspecific diploid ndrogenesis. First successful ttempt ws performed on cyprinids (Bercsenyi et l., 1998), where ndrogenetic goldfish Crssius urtus urtus (L.) were produced in crp Cyprinus crpio L. eggs. Recoubrtsky & Grunin (2001) obtined few vible ndrogenetic crp lrve htched out from hybrid goldfish crp eggs. In slmonids, ndrogenetic development of rinbow trout in Yellowstone cutthrot trout Oncorhynchus clrki bouvieri (Richrdson) eggs hs been recently reported (Brown & Thorgrd, 2002). The present study consists of two experiments, ech iming t induction of interspecies (between species) ndrogenesis in slmonid fishes. In Experiment 1, prmeters of gmm irrdition nd pressure shock ppliction were tested in order to induce intrspecies ndrogenetic development in brown trout Slmo trutt L., brook trout nd rinbow trout. Also, ndrogenetic nd control crosses were performed between those species in order to induce interspecific nd intergeneric hybrid development. In Experiment 2, bsed on results from Experiment 1, the optimum time of ppliction of pressure shock for brown nd brook trout intrspecies ndrogenesis ws determined. Androgenetic development of Atlntic slmon Slmo slr L. nd Arctic chrr Slvelinus lpinus (L.) in eggs of brown nd brook trout, respectively, ws lso induced in order to exmine whether interspecies but intrgeneric development in those fishes ws possible.

3 434 I. BABIAK ET AL. TABLE I. Scheme of experimentl design. OM, Oncorhynchus mykiss; ST, Slmo trutt; SF, Slvelinus fontinlis; SS, Slmo slr; SA, Slvelinus lpinus. Gmm irrdition ws pplied to unfertilized oocytes in ndrogenetic hploid nd diploid groups. In Experiment 2, dose of 35 kr ws used in ll vrints. Pressure shock ( kn m 2 lsting 4 min) ws pplied to ll ndrogenetic diploid groups fter insemintion nd incubtion t 10 C. (See Mterils nd Methods) Fctors tested Femles Androgenetic nd control crosses Mles Resulting embryos Experiment 1 1. γ-irrdition dose: () 35 kr or (b) 50 kr* pplied to oocytes; 2. pressure shock pplied to zygotes: () 5 h 50 min (b) 7 h 30 min fter insemintion OM ST SF OM OM OM ST OM SF ST OM ST ST ST SF SF OM SF ST SF SF OM ST SF All possible controls, ndrogenetic hploids nd diploids within nd between species Experiment 2 Pressure shock pplied to zygotes: () 5 h 8 h 30 min (SF) (b) 5 h 10 h (ST) ST SF ST ST ST SS SF SF SF SA ST SS SF SA Control, ndrogenetic hploid nd diploid ST, SF, nd hybrids: ST SS nd SF SA *Irrdition dose 50 kr ws not pplied to OM oocytes. MATERIALS AND METHODS EXPERIMENTAL DESIGN The experiments were conducted in 1998 (Experiment 1) nd 1999 (Experiment 2). Experiment 1 ws performed on brown, brook nd rinbow trout gmetes. It imed t optimiztion of intrspecies ndrogenesis in brown nd brook trout nd induced interspecies intergeneric ndrogenetic development between brown, brook nd rinbow trout (Tble I). Experiment 2 ws performed on brown nd brook trout oocytes nd spermtozo, nd Atlntic slmon nd Arctic chrr spermtozo. Bsed on results of Experiment 1, further optimiztion of time of pressure shock ppliction for intrspecies ndrogenesis in brown nd brook trout ws conducted. Interspecies, intrgeneric (within genus) ndrogenetic development ws lso induced within two gener: Slmo nd Slvelinus (Tble I). Optimiztion of intrspecies ndrogenesis In Experiment 1, the effect of two processing fctors ws tested: gmm irrdition dose pplied to destroy mternl nucler genomes (35 or 50 kr) nd the time of ppliction of hydrosttic pressure shock (5 h 50 min or 7 h 30 min fter insemintion nd incubtion of eggs t 10 C). In vrints where ndrogenesis ws induced in oocytes of brown nd brook trout, fctoril design ws full (two irrdition doses two times of pressure ppliction). Eggs of rinbow trout were irrdited only with dose of 35 kr (Prsons & Thorgrd, 1985) but subjected to the pressure shock in the two previous settings: 5 h 50 min nd 7 h 30 min. The settings of irrdition dose, were chosen following Bbik et l. (1998) who demonstrted tht doses of 35 nd 50 kr were eqully efficient in rinbow trout, wheres doses of 65 kr or higher showed lethl effect despite

4 ANDROGENESIS IN SALMONIDS 435 high percentge of eyed embryos in hploid controls. The settings for time of ppliction of pressure tretment, 5 h 50 min or 7 h 30 min fter insemintion, were chosen becuse they were optiml for rinbow nd brook trout, respectively (Chourrout, 1984; Prsons & Thorgrd, 1985; My et l., 1988), nd no dt were vilble on brown trout. As the pternl effect on the rte of first mitotic division in the interspecific ndrogenetic zygote could not be excluded, those two settings were pplied for ech species, including rinbow trout. In Experiment 2, eggs were subjected to the pressure tretment in 0 5 h intervls within 5 10 h (brown trout) or 5 8 h 30 min (brook trout) fter insemintion t 10 C. Induction of interspecies ndrogenesis In Experiment 1, individuls from the three species were crossed in reciprocl combintions. Also, ndrogenetic development ws induced using spermtozo nd oocytes from ech species in ll possible combintions (three species three species=9 combintions). In Experiment 2, crosses nd induced ndrogenesis were conducted within two gener: Slmo (oocytes of brown trout nd spermtozo from brown trout or Atlntic slmon) nd Slvelinus (oocytes of brook trout nd spermtozo from brook trout or Arctic chrr). BREEDERS AND GAMETE COLLECTION In Experiment 1, gmetes were collected in November 1998 from broodstocks of brown, brook nd rinbow trout kept t the Deprtment of Slmonid Reserch, Inlnd Fisheries Institute, Rutki, Polnd. Oocytes were pooled within ech species from three 3 to 4 yer old femles. Spermtozo were obtined from severl 2 to 3 yer old mles nd single smple from ech species ws chosen for experiment fter exmintion of spermtozol motility. In Experiment 2, gmetes were collected from brown trout nd Atlntic slmon (November 1999), nd brook trout nd Arctic chrr (December 1999). Brown nd brook trout gmetes were obtined from broodstock cultivted in Rutki, Polnd. Atlntic slmon semen ws collected from wild spwners cptured nd kept in the Fishery Sttion t Swibno, Polnd. Arctic chrr semen ws collected from 5 yer old donors cultivted in the Sim Fisheries Reserch nd Aquculture Institute, Enonkoski, Finlnd. Oocytes were pooled within species from four brown trout femles 4 yers old nd 14 brook trout femles 2 yers old. Sperm from brown trout, Atlntic slmon nd brook trout ws collected on the dy of experiment. Arctic chrr spermtozo were obtined 9 dys prior to the experiment nd stored under oxygen t 0 1 C in Ziploc bgs until used. A single smple from ech species ws chosen for experiment fter exmintion of motility. TREATMENT After pooling, oocytes were kept submerged in coelomic fluid on crushed ice (0 2 C) until treted. Prt of the oocytes were left intct to serve s controls, the remining oocytes were subjected to gmm irrdition s described by Bbik et l. (2002). Doses of either 35 kr or, excluding rinbow trout, 50 kr were used in Experiment 1, nd dose of 35 kr ws pplied in Experiment 2. Both control nd irrdited btches contined c. 700 eggs per vrint. They were inseminted with 0 08 ml of semen. Prior to insemintion, 10 ml of fertiliztion diluent (154 mm NCl nd 1 mm C 2+,buffered to ph 9 0 with 20 mm Tris+30 mm glycine; Billrd, 1992) were dded to ech btch. Inseminted eggs were incubted t 10 C. To induce dupliction of chromosome sets during the first mitotic division, btches of irrdited nd inseminted eggs were subjected for 4 min to kn m 2 hydrosttic pressure shock (Chourrout, 1984). This pressure shock ws chosen becuse it proved to be optiml for rinbow trout (Bbik et l., 2002). In Experiment 1, the pressure shock ws used t 5 h 50 min or 7 h 30 min fter insemintion. In Experiment 2, the pressure ws pplied every 30 min within 5 10 h (brown trout eggs) or 5 h 8 h 30 min (brook trout eggs) fter insemintion to

5 436 I. BABIAK ET AL. determine the optiml time of interruption of the first mitotic division. Control hploid btches of irrdited but not pressure-shocked eggs were performed for ll experimentl combintions. Experimentl s well s control vrints were incubted seprtely in three replictes t 7 9 C. Embryos were counted t the mid- to lte eyed-egg stge, when embryo movements inside chorion could be visible. All live embryos, including those showing morphologicl neuploid syndrome, were counted. The progenies were counted gin c. 2 weeks fter htching, t the mid yolk-sc resorption stge (c. 50% of the yolk sc ws resorbed). All living lrve were clssified s htched t this stge. The dte of counting ws corrected for differences in development rtes of studied species. MICROSATELLITE ANALYSIS Microstellite loci STR 73 (Estoup et l., 1993) nd 85 (Estoup et l., 1998) were used for genetic identifiction of prentl, hybrid nd ndrogenetic genotypes in Slmo nd Slvelinus, respectively. These loci showed interspecific polymorphism nd no intrspecific polymorphism. Amplifiction ws bsed on the primers described by Estoup et l. (1993, 1998). Genomic DNA ws extrcted from fin clips (prents) nd from body pieces of eyed embryos (progeny) using WIZARD purifiction Kit (Promeg, Mdison, U.S.A.). Double strnded PCR mplifictions were performed s described by Bbik et l. (2002). The mplified products were seprted on 2% grose gel (Promeg, Mdison, U.S.A.). The gels were stined with ethidium bromide (10 mg ml 1 ) nd photogrphed under UV light. Microstellite nlyses were performed on: seven brown trout femle donors; 14 brook trout femle donors; four Atlntic slmon mles; five Arctic chrr mles; 15 hybrid brown trout Atlntic slmon progeny; 12 presumed interspecies ndrogenetic Atlntic slmon progeny; 15 hybrid brook trout Arctic chrr progeny; nd 16 presumed interspecies ndrogenetic Arctic chrr progeny. STATISTICAL ANALYSIS The dt were orgnized, trnsformed nd nlysed ccording to Bbik (1998). The percentge vlues were trnsformed to follow norml distribution, thus enbling the ppliction of prmetric procedures. The squre root trnsformtion ws used (T= P, where P is proportion of eyed eggs) for htching rtes in ndrogenetic groups, nd rcsine squre root trnsformtion (T=rc sin P) ws used for percentges of eyed embryos (Anderson & McLen, 1974). The trnsformed dt showed norml distribution, nd the vrinces were homogenous (Cochrn & Brtlett s test). Multivrite nlysis of vrince (MANOVA) ws used to determine the effect of tested fctors nd their interctions on the survivl rtes. A post hoc multiple rnge nlysis (Duncn s test) ws used to estimte the significnce of differences mong the groups. Person s product moment correltion coefficient ws used to ssess correltions. RESULTS INTRASPECIES ANDROGENESIS Hploid control individuls did not htch in ny of the species. In Experiment 1, the highest survivl of ndrogenetic eyed embryos ws 42 7, 25 1, nd 20 5% in brown, brook, nd rinbow trout, respectively. Androgenetic brown nd brook trout htched lrve were produced under ech setting of irrdition dose nd ppliction of pressure shock (Fig. 1). Vible ndrogenetic rinbow trout lrve were obtined only when pressure shock ws pplied 5 h 50 min fter insemintion (men S.D., % of lrve t the mid yolk-sc resorption stge).

6 ANDROGENESIS IN SALMONIDS Irrdition dose Time of pressure ppliction Survivl (%) () b 31.7 b kr 50 kr Not pplied d 4.1 f 0.4 d 7.3 f d,e 2.5 c 14.1 e,f kr 50 kr 35 kr 50 kr 5 h 50 min 7 h 30 min Survivl (%) (b) 27.1,b c,d 15.1 d 11.1 b,c e 4.3 e 3.7 e 3.1 e Irrdition dose Time of pressure ppliction 35 kr 50 kr Not pplied 35 kr 50 kr 35 kr 50 kr 5 h 50 min 7 h 30 min FIG. 1. Experiment 1: Survivl of intrspecies ndrogenetic () Slmo trutt nd (b) Slvelinus fontinlis t eyed-egg ( ) nd mid yolk-sc resorption ( ) stges. Gmm irrdition t dose of 35 or 50 kr ws pplied to unfertilized oocytes. Pressure tretment ( kn m 2 for 4 min) ws pplied to ndrogenetic groups 5 h 50 min or 7 h 30 min fter insemintion t 10 C. Not pplied, hploid groups not subjected to the pressure. None of the hploid individuls survived until htching. Brs mrked with the sme letter do not differ significntly from ech other (Duncn s multiple rnge tests, P<0 05). Survivl of respective control groups is shown in Tble II. Anlysis of vrince for brown trout htched lrve reveled tht the ppliction of pressure tretment t 7 h 30 min fter fertiliztion ws significntly superior to 5 h 50 min (F 1,8 =17 6, P=0 003). The effect of irrdition dose ws insignificnt (P=0 15), similrly to the interction between the irrdition dose nd the time of ppliction of pressure shock (P=0 67). The highest ndrogenetic yield in brown trout (men S.D., %) ws obtined

7 438 I. BABIAK ET AL. when oocytes were irrdited with 35 kr nd were then treted with pressure shock 7 h 30 min fter insemintion [Fig. 1()]. Neither irrdition dose nor the time of ppliction of pressure shock significntly ffected ndrogenesis efficiency in brook trout (P=0 31 nd 0 11, respectively). Also, the interction between these two fctors ws insignificnt (P=0 71). The highest survivl rte of brook trout ndrogenetic lrve ( %), not differing significntly from others, ws obtined when 35 kr irrdited eggs were treted with pressure shock 5 h 50 min fter insemintion [Fig. 1(b)]. In Experiment 2, the highest survivl of ndrogenetic eyed embryos of brown nd brook trout ws nd %, respectively (Fig. 2). Androgenetic brown trout showed very poor survivl t htching. Few htched lrve were recorded in vrints where the pressure shock ws pplied t 7 h 30 min or lter fter insemintion [Fig. 2()]. Due to low number of survivors, no sttisticl nlysis ws possible. The highest survivl of ndrogenetic brook trout ( %), not differing significntly from the others, ws obtined in the vrint with the ppliction of pressure shock t 7 h fter insemintion. In this vrint, the eyed-egg stge rte ws lso the highest [Fig. 2(b)]. The survivl of ndrogenetic brook trout ws significntly ffected by the time of ppliction of pressure shock (F 7,16 =12 6, P<0 0001). A post-hoc Duncn multiple rnge test reveled no significnt differences mong the time of ppliction of pressure tretment in the rnge 5 h 7 h 30 min [Fig. 2(b)]. A very high correltion between survivls of brook trout ndrogenetic eyed embryos nd htched lrve (r=0 93, P<0 05) ws found. INTERSPECIES ANDROGENESIS In Experiment 1, where brown, brook nd rinbow trout interspecies ndrogenetic development ws induced in ll reciprocl combintions, four of six hybrid crosses resulted in vible progeny (Tble II). Androgenetic individuls were successfully produced, however, in only intrspecies vrints. No developing embryos t the eyed-egg stge were observed in the interspecies ndrogenetic groups. The only interspecies ndrogenetic hploid controls vible t the eyed-egg stge ( nd %) were obtined fter insemintion of brown trout oocytes (irrdited with 35 nd 50 kr, respectively) with brook trout spermtozo. They did not htch, however. In Experiment 2, contrry to the results of Experiment 1, survivl of interspecific ndrogenetic embryos ws observed t the eyed stge (Fig. 2). The highest percentge of ndrogenetic Atlntic slmon developing in brown trout eggs ws % [Fig. 2()], wheres ndrogenetic Arctic chrr developing in the brook trout eggs yielded % of eyed embryos in the best vrint [Fig. 2(b)]. Despite this, nd the high survivl of control hybrid lrve (Tble II), none of interspecific ndrogenetic individuls survived until htching. The highest survivl t the eyed-egg stge of both interspecies Atlntic slmon nd intrspecies brown trout ndrogenetic embryos occured when pressure shock ws pplied 9 h fter insemintion [Fig. 2()]. These results, however, do not differ sttisticlly from the others. A very high correltion (r=0 89, P<0 05)

8 ANDROGENESIS IN SALMONIDS () b b b b b b b b b b 5 b b Survivl (%) 0 30 b 5,00 (b) 5,30 6,00 6,30 7, , ,00 8,30 9,00 9, ,00 25 b 20 bc 15 cd 10 df de b b b e 5 0 b 1.2 c b 2.0 c ,00 5,30 6,00 6,30 7,00 7,30 8,00 Appliction of pressure shock (h, min fter insemintion) FIG. 2. Experiment 2: The effect of time of pressure tretment ppliction on survivl t the eyed embryos nd htching stge of intr-nd interspecific ndrogenetic () Slmo trutt nd Slmo slr, produced in eggs of S. trutt, nd (b) Slvelinus fontinlis nd Slvelinus lpinus, produced in eggs of S. fontinlis. None of interspecific ndrogenetic individuls survived htching. Within stges (eyed-egg or htching) nd species, vlues mrked with the sme letter do not differ significntly from ech other (Duncn s multiple rnge tests, P<0 05). (), S. trutt htched lrve;, S. trutt eyed embryos;, S. slr eyed embryos. (b), S. fontinlis htched lrve;, S. fontinlis eyed embryos;, S. lpinus eyed embryos. between survivl in these two species t the stge of eyed embryos ws determined. Androgenetic Arctic chrr eyed embryos, similrly to brook trout, showed the highest survivl when the pressure shock ws pplied 7 h fter insemintion [Fig. 2(b)]. A very high correltion between survivl of brook trout nd Arctic chrr ndrogenetic eyed embryos (r=0 93, P<0 05) ws found. 3.3 b 1.3 bc 0.4 c c f c 0.1 8,30

9 440 I. BABIAK ET AL. TABLE II. Men S.D. survivl of intr- nd interspecies control crosses t the eyed-egg nd mid yolk-sc resorption stges. None of interspecies ndrogenetic embryos survived htching. ST, Slmo trutt; SF, Slvelinus fontinlis; OM, Oncorhynchus mykiss; SS, Slmo slr; SA, Slvelinus lpinus Femle Survivl (%) Mle ST SF OM Experiment 1 ST Eyed Htched SF Eyed Htched OM Eyed Htched ST SS SF SA Experiment 2 ST Eyed Htched SF Eyed Htched MOLECULAR EXAMINATION OF ANDROGENETIC INDIVIDUALS Microstellite primers, both STR 73 nd 85, showed species-specific polymorphism nd consistent lck of intrspecies polymorphism. Two lleles t locus STR 73 in smples from the genus Slmo were detected. Homozygous genotypes were reveled for Atlntic slmon mle nd brown trout femles, AA (180/180 bp) nd BB (160/160 bp), respectively [Fig. 3()]. All control hybrid progeny possessed presumed genotype AB (180/160 bp). Interspecific ndrogenetic progeny showed either pternl genotype AA or hybrid genotype AB; the ltter indicted the presence of mternl nucler DNA in the exmined specimens. Three llelic forms were reveled t locus 85 within the Slvelinus genus [Fig. 3(b)]. The Arctic chrr pternl genotype ws heterozygous AB (A=145 bp nd B=130 bp), wheres mternl genotypes were homozygous CC (120 bp). Control hybrid progeny possessed one of two expected genotypes (AC or BC). Interspecific ndrogenetic progeny exhibited only pternl genotypes; individuls were homozygous for either llele A or B (frequencies 0 5:0 5, n=16) DISCUSSION INTRASPECIES ANDROGENESIS This is the first report on induced ndrogenesis in the genus Slmo. The survivl of 42 7% of brown trout eyed embryos in the best vrint of Experiment 1[Fig. 1()] is one of the highest reported on induced ndrogenesis in fishes (Ihssen et l., 1990; Pndin & Koteeswrn, 1998), comprble to the results for

10 ANDROGENESIS IN SALMONIDS 441 () Allele A Allele B (b) Allele A Allele B Allele C FIG. 3. Experiment 2: () interspecific ndrogenetic development of Slmo slr in eggs of Slmo trutt: genotypes for the microstellite locus STR 73 (Estoup et l., 1993). Mternl BB nd pternl AA genotypes re presented in lnes 1 nd 9, respectively, nd their hybrid progeny in lne 2 (AB). Androgenetic progeny exhibited two genotypes: heterozygous AB (lnes 3, 4 nd 5) nd homozygous AA (lnes 6, 7 nd 8). Lne 10: puc 18 DNA/He III DNA ldder; (b) interspecific ndrogenetic development of Slvelinus lpinus in eggs of Slvelinus fontinlis: genotypes for the microstellite locus 85 (Estoup et l., 1998). Mternl CC nd pternl AB genotypes re presented in lnes 1 nd 7, respectively, nd their hybrid progeny in lne 2 (BC). Androgenetic progeny showed either AA (lne 5) or BB (lnes 3, 4 nd 6) genotypes. Lne 8: ΦX 174 DNA/Hinf I DNA ldder. Schemtic representtion of genotypes: mternl, ; pternl, ; hybrid,. rinbow trout (Bbik et l., 2002). Despite this high survivl of brown trout eyed embryos, only 2 5% lrve htched, which is ten times lower thn for rinbow trout (Bbik et l., 2002). As it ws reveled in Experiment 2, optiml time of ppliction would be c. 9 h fter insemintion, thus pressure shock pplied erlier probbly led to n increse of the neuploidy level in treted

11 442 I. BABIAK ET AL. embryos in Experiment 1. Unfortuntely, the poor qulity of oocytes in Experiment 2 did not llow for the improvement of ndrogenesis efficiency. As none of ndrogenetic brown trout survived for more thn 4 months, further optimiztion of the protocol is needed. Htching of ndrogenetic brook trout hs only been reported to dte by My et l. (1988), however, the uthors did not present dt on htching rtes or further survivl. In the present study, htching rtes were reltively low despite high percentges of eyed embryos [Figs 1(b) nd 2(b)]. Nevertheless, few ndrogenetic individuls survived 2 yers. The results of Experiment 2 indicte tht optiml time for ppliction of pressure shock ws 7 h fter insemintion nd incubtion t 10 C[Fig. 2(b)]. My et l. (1988) found 7 h 30 min to be optiml. This 30 min difference might be becuse the rte of the first mitotic division vries mong different strins (Scheerer et l., 1991). The protocol used in the present study proved its suitbility for production of ndrogenetic brook trout. Results obtined with ndrogenetic rinbow trout (4 5% of mid-yolk-sc resorption lrve), lthough comprble with most of others in this species (Pndin & Koteeswrn, 1998), re rther low when compred to Bbik et l. (2002). The reson probbly lies in the genetic origin of oocyte donors, criticl biologicl fctor in induced ndrogenesis (Bbik et l., 2002). Fish used in the present experiment originted from strin not selected for egg qulity, contrry to the strin used by Bbik et l. (2002). INTERSPECIES HYBRIDIZATION AND ANDROGENESIS Htching rtes of control intergeneric hybrids in Experiment 1 exceeded 60% in crosses ST SF, SF ST nd OM SF, while htching of hybrids OM ST ws poor, nd no vible lrve were obtined in crosses ST OM nd SF OM. In Experiment 2, htching rtes of intrgeneric hybrids ST SS nd SF SA were reltively high (Tble II). These dt re in greement with results reported by other uthors (McKy et l., 1992; Gry et l., 1993). High mortlity ws observed in ll htched hybrids nd until they reched levin stge (unpubl. dt). Contrry to the pre-htch mortlity in invible crosses, which is cused by the chromosome elimintion nd resulting lethl neuploidy (Fujiwr et l., 1997), the resons of the post-htch mortlity re uncler. Incomplete kryogmy, cused by different numbers of mternl nd pternl chromosomes, would led to neuploidy (Chevssus, 1983), probbly by chromosome elimintion. If such n event occurs in intergeneric hybrids, which re functionlly sterile (Chevssus, 1983), this does not pply to intrgeneric ST SA nd SF SA hybrids, which cn be fertile (Johnson & Wright, 1986; Johnson et l., 1987) nd possess intermedite number of pternl chromosomes (Disney & Wright, 1987). Interspecific ndrogenesis ws successfully induced in cyprinids (Bercsenyi et l., 1998; Recoubrtsky & Grunin, 2001) nd in Oncorhynchus sp. (Brown & Thorgrd, 2002). The present study demonstrtes tht this does not directly pply to other slmonids. No vible interspecific ndrogenetic individuls were obtined from recipient eggs originting from other gener (Experiment 1). Even using recipient oocytes from other species within the sme genus (Experiment 2) resulted in no vible lrve. Genetic reltionships between the

12 ANDROGENESIS IN SALMONIDS 443 species used were close enough to produce vible hybrids in four of six possible crosses in Experiment 1 nd in two of two crosses in Experiment 2. Moreover, ndrogenetic progeny htched in ll intrspecies vrints, where oocytes nd sperm originted from the sme species. This indictes tht lck of mternl nucler DNA is primry lethl fctor preventing interspecific ndrogenesis in tested species nd not hybrid development (except crosses with rinbow trout mles) or the ndrogenesis process. In Experiment 1 (intergeneric ndrogenesis), the development cesed erlier thn in Experiment 2 (intrgeneric ndrogenesis). The only intergeneric ndrogenetic eyed embryos (ndrogenetic brook trout produced in brown trout eggs) were obtined in hploid controls. A similr low survivl of hploid ndrogenetic brook trout, developing in brown trout eggs, ws observed by My & Grewe (1993). In Experiment 2, reltively high percentge of eyed embryos ws observed in interspecific ndrogenetic groups (Fig. 2). Optiml time of ppliction of pressure tretment ws the sme for ndrogenetic brown trout nd Atlntic slmon [9 h; Fig. 2()], s well s for brook trout nd Arctic chrr (7 h; Fig. 2(b)]. Also, correltions between ndrogenetic eyed embryos rtes in Slmo nd Slvelinus were very high (0 89 nd 0 93, respectively), indicting tht the rte of the first cell division within these two pirs ws pproximtely the sme. Nevertheless, none of interspecific ndrogenetic lrve htched. Non-nucleted slmonid embryos cn develop until the blstul stge (Aoygi et l., 1993) nd the nucler genome is not ctive until blstultion (Ngler, 2000). Mternl cytoplsmic fctors exclusively control erly development in fishes until the midblstul trnsition, MDT (Kne & Kimmel, 1993). It hs been demonstrted tht regultory signls controlling development of zebrfish Brchydnio rerio (Hmilton-Buchnn) embryos re exclusively mternllyderived until MDT (Shimizu et l., 2000; Brt et l., 2000; Gore & Smpth, 2002). Even when zygotic informtion is expressed, number of cytoplsmic signls is involved in modultion of nucler genome in mmmls (Cummins, 2001; Fulk et l., 2001). In the cse of interspecies nucleo-cytoplsmic hybrids, this regultory incomptibility leds to chromosome elimintion during erly development; demonstrted by Fujiwr et l. (1997) on invible msu slmon Oncorhynchus msou (Brevoort) rinbow trout hybrids. These uthors reveled tht the elimintion of chromosomes ws uniprentl in the cse of nucler hybrids, with preference to eliminte pternl chromosomes, yet still occurred in the bsence of the mternl msu slmon genome in hploid ndrogenetic rinbow trout embryos. This effect could result in cesing the development of interspecific ndrogenetic embryos in the present study. Kryotypicl nd developmentl exmintion of embryos t erly stges would help to understnd the resons of invibility of interspecies ndrogenotes. Filure of interspecies ndrogenesis in the present study stnds in contrst to successful ttempts reported in cyprinids nd Oncorhynchus sp. Rinbow trout nd Yellowstone cutthrot trout, used by Brown & Thorgrd (2002), show similr kryotypicl chrcteristics (Hrtley, 1987) nd their hybrids re fully fertile nd developmentlly comptible (Ferguson et l., 1985) s re cyprinids (Kirpichnikov, 1981; Liu et l., 2001). Species used in the present study, even within the sme genus, kryolotypiclly differ much more (Hrtley, 1987) nd their hybrids show poor fertility if ny (Chevssus, 1983; Glbreth &

13 444 I. BABIAK ET AL. Thorgrd, 1995; K. Goryczko, S. Dobosz & H. Kuzminski, unpubl. dt). These dt suggest tht interspecific ndrogenesis is possible only in very closely relted species, thus different gener of cyprinids re in fct geneticlly more closely relted thn slmonid species within the gener Slmo nd Slvelinus. Phylogenic reltionships mong Slmonide re complicted nd, despite extensive studies, no stisfying, unequivocl model hs been defined to dte (Okley & Phillips, 1999; Osinov & Lebedev, 2000). DNA-bsed reserch on phylogeny in Slmonine hs led Okley & Phillips (1999) to the conclusion tht Oncorhynchus nd Slmo re not sister gener, contrry to the commonly ccepted hypothesis. One possible phylogenetic model within Slmonine, proposed by Okley & Phillips (1999), ssumes closer reltionship between Slmo nd Slvelinus thn between Oncorhynchus nd these two gener. The present reproductive dt seem to support this model, s reciprocl hybrids of brown nd brook trout were vible, contrry to hybrids of these two species with rinbow trout mles (Tble II), nd the only interspecific development to the eyed-egg stge ws observed in hploid controls of brook trout developing in brown trout eggs. MOLECULAR EXAMINATION OF THE PROGENY All exmined ndrogenetic Arctic chrr eyed embryos showed homozygous Arctic chrr pternl phenotypes [Fig. 3(b)], wheres the prt of the presumbly ndrogenetic slmon contined mternl brown trout nucler DNA [Fig. 3()]. This indictes n incomplete nucler inctivtion of brown trout oocytes. The need for further development of ndrogenesis protocols for brown trout focusing on irrdition dose seems to be obvious, however, implictions re much broder. The genetic control in single microstellite locus, s in the present study, is sufficient for confirming homozygosity nd pternl origin of resulting embryos, yet probbly insufficient to exclude mternl DNA remins. Using more points of control (more microstellites, RAPD, AFLP) would increse the probbility for detection of mternl contmintion, but still not gurnteeing tht no mternl DNA persists. The irrdition doses used in the present study were equl to or higher thn doses considered s optiml for enucletion of rinbow trout oocytes (Scheerer et l., 1991). Brown trout oocytes re lrger thn those of rinbow trout, but n oocyte size should not be criticl when using permeting irrdition, such s gmm rys. Therefore, the presumed lck of mternl nucler DNA in every ndrogenetic individul produced using oocyte irrdition my be questionble. The intriguing question is wht hppens with mternl DNA frgments fter irrdition. Kryologicl exmintion of dult, 3 yer old ndrogenetic rinbow trout reveled extr chromosome frgment-like structures in some individuls (K. Oclewicz, S. Dobosz, K. Goryczko, H. Kuzminski & I. Bbik, unpubl. dt). This observtion nd the present study do not exclude possibility tht mternl nucler genome frgments cn persist fter numerous cell divisions, therefore, they cn be replicted. Besides n extremely high inbred level (Scheerer et l., 1991), these unintended remins of mternl genome might be n dditionl reson for poor vibility of ndrogenetic lrve, reported by Bbik et l. (2002). Mternl genomic DNA contmintion my result lso in high

14 ANDROGENESIS IN SALMONIDS 445 mortlity of eyed embryos, observed in the present study in both intr- nd interspecies ndrogenetic groups, nd reported by other uthors (Pndin & Koteeswrn, 1998). We re much indebted to T. Hrtmn for criticl reding the mnuscript. The study ws supported by the Polish Committee for Scientific Reserch, Project 5 P06D References Anderson, V. L. & McLen, R. A. (1974). Design of Experiments: A Relistic Approch. New York: Mrcel Dekker, Inc. Aoygi, K., Kojim, Y. & Sneyoshi, M. (1993). Developmentl cpcity of nonnucleted embryos of coho slmon, Oncorhynchus kisutch, induced by γ-irrdition. Cndin Journl of Zoology 71, Bbik, I. (1998). Experimentl design nd sttisticl nlysis in reserch on fish reproduction. Archives of Polish Fisheries 6, Bbik, I., Dobosz, S., Goryczko, K., Kuzminski, H. & Woznicki, P. (1998). Androgenesis in rinbow trout, Oncorhynchus mykiss, using gmm irrdition nd het shock. Specil Publictions, Europen Aquculture Society 26, Bbik, I., Dobosz, S., Goryczko, K., Kuzminski, H., Brzuzn, P. & Ciesielski, S. (2002). Androgenesis in rinbow trout using cryopreserved spermtozo: the effect of processing nd biologicl fctors. Theriogenology 57, Bercsenyi, M., Mgyry, I., Urbnyi, B., Orbn, L. & Horvth, L. (1998). Htching out goldfish from common crp eggs: interspecific ndrogenesis between two cyprinid species. Genome 41, Billrd, R. (1992). Reproduction in rinbow trout: sex differentition, dynmics of gmetogenesis, biology nd preservtion of gmetes. Aquculture 100, Brt, A. K., Vn der Wlter, S., Goos, H., Bogerd, J. & Zivkovic, D. (2000). Vs protein expression nd locliztion in zebrfish. Mechnisms of Development 95, Brown, K. H. & Thorgrd, G. H. (2002). Mitochondril nd nucler inheritnce in n ndrogenetic line of rinbow trout, Oncorhynchus mykiss. Aquculture 204, Chevssus, B. (1983). Hybridiztion in fish. Aquculture 33, Chourrout, D. (1984). Pressure-induced retention of second polr body nd suppression of first clevge in rinbow trout: production of ll-triploids, ll-tetrploids, nd heterozygous nd homozygous diploid gynogenetics. Aquculture 36, Cummins, J. M. (2001). Cytoplsmic inheritnce nd its implictions for niml biotechnology. Theriogenology 55, Disney, J. E. & Wright, J. E. Jr (1987). Cytogenetic nlyses of Slvelinus hybrid revel n evolutionry reltionship between the prentl species. Cytogenetics nd Cell Genetics 45, Estoup, A., Pres, P., Krieg, F., Vimn, D. & Guyomrd, R. (1993). (CT) n nd (GT) n microstellites: new clss of genetic mrkers for Slmo trutt L. Heredity 71, Estoup, A., Rousset, F., Michlkis, Y., Cornuet, J.-M., Adrimng, M. & Guyomrd, R. (1998). Comprtive nlysis of microstellite nd llozyme mrkers: cse study investigting microgrphic differentition in brown trout (Slmo trutt). Moleculr Ecology 7, Ferguson, M. M., Dnzmnn, R. G. & Dorf, F. W. (1985). Absence of developmentl incomptibility in hybrids between rinbow trout nd two subspecies of cutthrot trout. Biochemicl Genetics 23, Fujiwr, A., Abe, S., Ymh, E., Ymzki, F. & Yoshid, M. C. (1997). Uniprentl chromosome elimintion in the erly embryogenesis of the invible slmonid hybrids between msu slmon femle nd rinbow trout mle. Chromosom 106,

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