Archiv Tierzucht 55 (2012) 1, 84-96, ISSN 0003-9438 © Leibniz Institute for Farm Animal Biology, Dummerstorf, Germany

Modifications of lampbrush structure of the European domestic goose Anser anser

Katarzyna Andraszek and Elżbieta Smalec

Department of Animal and Horse Breeding, Institute of Bioengineering and Animal Breeding, University of Life Sciences and Humanities, Siedlce, Poland

Abstract Lampbrush (LBCs) represent a new model in avian cytogenetics and are increasingly more often used in poultry chromosome analyses. Additionally, lampbrush chromosomes are considered as model structures in the study of transcription regulation. Changes in transcription activity are reflected as modifications of LBC morphological structure and associated with physiological processes in the organism. The aim of the present study was to compare transcriptional activity of the first five lampbrush macrochromosomes and ZW sex lampbrush bivalents sampled from the oocytes of geese prior to and after the reproductive period. The respective bivalents sampled before and after reproduction have similar sizes but differ in morphological structure. Side loops of lampbrush chromosomes are sites of transcription activity. The activity varies according to the loop size. As the loops become more prominent, the activity grows and vice versa. Lampbrush chromosomes sampled after reproduction have smaller side loops. On the other hand, inactive chromomeres become prominent in the chromosomes. Marker loops are the last structures to be degraded after the end of reproduction. Consequently, they are used for identifying particular bivalents at different stages of cellular transcriptional activity.

Keywords: Anser anser goose, reproduction, meiotic chromosome, lampbrush chromosome, transcription

Introduction High transcriptional activity observed in avian oocytes during previtellogenesis causes decondensation. Elongated and decondensed chromosomal forms appear. These are lampbrush chromosomes (LBCs) with thick mantles of side loops that constitute the transcription units. Each LBC consists of alternately positioned chromomeres (condensed chromatin) with one pair or numerous pairs of loops (transcriptionally active chromatin) (Gaginskaya et al. 1984, Callan 1986, MacGregor 1986, Morgan 2002, 2007, Austin et al. 2009). Lampbrush chromosomes were introduced as an object of cytogenetic study of poultry by Kroptova & Gaginskaya (1984) and Hutchinson (1987). Since 2005 they have become established as a new model in avian cytogenetics (Schmid et al. 2005). Additionally, LBCs are used as model structures in studies of transcriptional regulation, as their transcription activity is visible even under a light microscope. Transcriptional activity analyses are performed based on the assumption that side loops of LBCs are the active sites. Arch Tierz 55 (2012) 1, 84-96 85

Reduction of transcriptional activity corresponds with smaller sizes of side loops (Varley et al. 1980, Jamrich et al. 1983, Scheer et al. 1984, Baldwin & MacGregor 1985, Callan et al. 1987, Scheer 1987, Gaginskaya & Tsvetkov 1988, Gall & Murphy 1998, Morgan 2002, 2007, Gaginskaya et al. 2009). In transcriptionally active regions of LBCs, proteins give place to non-histone proteins, inducing the loss of the nucleosome structure of coding chromosome segments which assume the shape of a loop. However, the exposure of nucleosomes, which enables DNA transcription, does not cause histone dissociation but leads to a kind of spatial rearrangement of the transcribed regions, allowing the entry of RNA polymerase into the promoter sequence. This occurs through the fixation of regulatory proteins at the site of a remote activating sequence. A specific characteristic of lampbrush chromosomes is that they become incorporated in the structure of the chromatin of non-histone proteins (HMG - High Mobility Group). HMGs are structural proteins of chromatin. They reduce its condensation (Hill et al. 1974, Di Mario et al. 1989, Korner et al. 2003). In transcriptional units with insignificant or declining transcription, nucleosomes are present in the axial part of the chromosome between sets of polymerase units. The gaps between the polymerase blocks are not even. After polymerase has passed along the DNA matrix and the regulation proteins have become dissociated, nucleosome structure formation follows (Spring & Franke 1981, Gaginskaya et al. 2009). Despite the fact that increasingly more studies are devoted to LBCs, these structures remain largely unexplored. Neither the exact mechanism of LBC formation out of little mitotic chromosomes, nor the way lampbrush chromosomes function in the oocyte are known. Nevertheless, the scale and form of transcription of these chromosomes suggest that this may be the earliest form of »looking after« a potential embryo (Rodinov 1996). The aim of the present paper was to compare the structure of lampbrush chromosomes isolated prior to and after the reproductive period of the European domestic goose (Anser anser).

Material and methods Lampbrush chromosomes were prepared by manual isolation from oocytes with diameters of 1.5-3.5 mm obtained from sexually mature geese using the standard procedure (MacGregor & Varley 1988) modified for bird oocytes (Solovei et al. 1992, Rodinov & Chechik 2002). The preparations were stained with Coomassie Blue R250. The first five macrobivalents and lampbrush sex bivalents (sex LBCs) were identified on the basis of their length and marker structure determined for avian lampbrush chromosomes (Solovei et al. 1993, Mizuno & MacGregor 1998). The cytological map of Gallus domesticus LBCs described by Chelysheva et al. (1990), Rodinov et al. (2002) and Schmid et al. (2005) was particularly useful. The preparations were analysed under an Olympus BX 50 light microscope (Olympus, Tokyo, Japan). A 100× zoom was used to identify specific marker structures, while the whole bivalent was examined with a 20× zoom. A detailed imaging analysis was carried out using the multiscan image analysis system, the software and graphic software compatible with the system. The analysis of transcriptional activity concerned the position and structure of the side loops, as well as the location of chiasmata and protein bodies in respective bivalent pairs. The pre-reproduction bivalents were marked with lowercase »a« and the post-reproduction ones with lowercase »b«. The marker structures of the bivalents were successively numbered. 86 Andraszek and Smalec: Modifications of LBC structure of the European domestic goose Anser anser 97 reproduction ones with lowercase “b”. The marker structures of the bivalents were 98 successivelyResults numbered. 99 100 ResultsThe LBCs sampled before the reproduction featured regions with well-preserved side loops 101 The(active LBCs transcription sampled areas) before alternating the reproduction with regions featured with reduced regions side with loops well-preserved (transcriptionally 102 sideinactive loops areas). (active Specific transcription marker structures areas) (distinctivealternating side with loops) regions were with identified reduced on theside LBCs. 103 loopsAfter the(transcriptionally egg production inactive season, theareas). LBCs Specific had the characteristicmarker structures atrophic (distinctive side loops. side What 104 loops) were identified on the LBCs. After the egg production season, the LBCs had could be clearly seen were the chromomeres, visible as dark lumps distributed over the whole 105 the characteristic atrophic side loops. What could be clearly seen were the 106 chromomeres,length of the chromosome. visible as dark The lumps following distributed marker over structures the whole were length identified of the in the LBCs 107 chromosome.under analysis: Thegiant following lumpy loops marker (GLLs), structures marker loops were (MLs), identified distal inboundary the LBCs loops under (DBLs), 108 analysis:proximal boundarygiant lumpy loops loops (PBLs), (GLLs), telomeric marker loops loops (TLs), (MLs), telomeric distal boundary bow-like loops loops (TBLs), 109 (DBLs),telomeric proximal lumpy loops boundary (TLLs), doubleloops (PBLs), bridges (DBs),telomeric chiasmata loops (Chs) (TLs), and telomeric protein bodies bow-like (PBs). 110 loops (TBLs), telomeric lumpy loops (TLLs), double bridges (DBs), chiasmata (Chs) 111 andComparison protein ofbodies the structure (PBs). of LBCs 1 prior to and after the reproductive period 112 113 ComparisonSeven groups of of the marker structure loops of(MLa1-MLa7), LBCs 1 prior Ta totelomeric and after loops the inreproductive the distal segment period and 114 TBLa Seventelomeric groups loops of were marker identified loops in(MLa the1 bivalent-MLa7), sampled Ta telomeric before loops the egg in the production distal 115 segmentperiod. Chromatin and TBLa without telomeric the loops,loops limitedwere identified by the DBLa in theand bivalent PBLa markers sampled delineates before the 116 thebivalent egg production centromere. period. Additionally, Chromatin seven without chiasmata the (Cha1-Cha7)loops, limited and by two the proteinDBLa and bodies 117 PBLa(PBa1 andmarkers PBa2) delineates connected withthe bivalent each of thecentromere. bivalent homologues Additionally, (Figure seven 1a) chiasmata were spotted 118 (Cha1-Cha7) and two protein bodies (PBa1 and PBa2) connected with each of the on the bivalent. Remnants of six groups of marker loops (MLb1-MLb6), telomeric loops (Tb) 119 bivalent homologues (Figure 1a) were spotted on the bivalent. Remnants of six 120 groupsin the distal of marker segment loops and (MLb already1-MLb6), fragmented telomeric TBLb loopsloops (Tb)were inidentified the distal on segment the bivalent 121 andsampled already after fragmented the egg production TBLb loops period. were The areaidentified adjacent on tothe the bivalent DBLb1 and sampled PBLb1 markersafter 122 thewas egg identified production as a centromere period. The of the area bivalent. adjacent Additionally, to the DBLb four1 chiasmata and PBLb (Chb1-Chb4)1 markers and 123 wasprotein identified bodies (PBb1as a centromereand PBb2) connected of the bivalent. with each Additionally, of the bivalent four homologues chiasmata (Figure (Chb1 1b)- 124 Chb4)were found and proteinon the bivalent. bodies (PBb1 and PBb2) connected with each of the bivalent 125 homologues (Figure 1b) were found on the bivalent. 126

127 128 Figure 1 Figure 1 Comparison of the structure of LBC 1 prior to (a) and after (b) the reproductive period. Arrows indicate marker 3 structures of bivalents. Arch Tierz 55 (2012) 1, 84-96 87 129 Comparison of the structure of LBC 1 prior to (a) and after (b) the reproductive period. Arrows 130 indicateComparison marker of structuresthe structure of bivalents.of LBCs 2 prior to and after the reproductive period 131 132 ComparisonSix groups of markerof the structure loops (MLa1-MLa6), of LBCs 2telomeric prior to loopsand after (Ta1 theand reproductiveTa2), as well as period TLLa1 and 133 TLLa2Six loops groups located of marker in the terminalloops (MLa regions1-MLa6), were identifiedtelomeric in loops the bivalent. (Ta1 and The Ta2), segment as 134 wellwithout as TLLa the loops,1 and encompassed TLLa2 loops by located the DBLa in the and terminal PBLa markers regions is awere bivalent identified centromere. in the 135 bivalent.Moreover, The protein segment structures without (PBa1 the and loops, PBa2) encompassed connected with by each the of DBLa the homologues and PBLa and 136 markersfive chiasmata is a bivalent (Cha1-Cha5) centromere. were detected Moreover, on the proteinbivalent. structures Figure 2b depicts (PBa1 the and second PBa2) LBC 137 connectedobtained after with the each reproductive of the homologues period. Residual and marker five chiasmata loops (MLb1-MLb5), (Cha1-Cha5) telomeric were loops 138 detected on the bivalent. Figure 2b depicts the second LBC obtained after the (Tb1 and Tb2) and well-preserved TLLb1 and TLLb2 loops were detected on the bivalent. 139 reproductive period. Residual marker loops (MLb1-MLb5), telomeric loops (Tb1 and 140 Tb2)The bivalent and well-preserved centromere was TLLb spotted1 and in TLLb2 the loopless loops segment were detected between on the the DBLb bivalent. and PBLb 141 Themarkers. bivalent In addition, centromere small protein was spotted bodies connectedin the loopless with each segment of the betweenhomologues the (PBb1 DBLb and 142 andPBb2) PBLb were markers. identified. In Five addition, spots likely small to containprotein chiasmatabodies connected (Chb1-Chb5) with were each also of found the on 143 homologuesthe bivalent. (PBb1 and PBb2) were identified. Five spots likely to contain 144 chiasmata (Chb1-Chb5) were also found on the bivalent.

145 146 Figure 2 147 ComparisonFigure 2 of the structure of LBC 2 prior to (a) and after (b) the reproductive period. Arrows 148 indicateComparison marker of the structures structure ofof LBC bivalents. 2 prior to (a) and after (b) the reproductive period. Arrows indicate marker 149 structures of bivalents. 150 Comparison of the structure of LBCs 3 prior to and after the reproductive period 151 ComparisonVery prominent of the structure clusters of LBCs of 3marker prior to loopsand after (MLa the1 reproductive and MLa2), period as well as large 152 GMLa loops were identified on the bivalent. In the distal part of the bivalent there 153 areVery Ta prominent loops that, clusters along of withmarker TLLa loops loops (MLa1 form and the MLa2), distinct as well “prongs”. as large TwoGMLa chiasmata loops were 154 (Chaidentified1 and on Cha2) the bivalent. spots wereIn the founddistal part on theof the bivalent. bivalent In there the areproximity Ta loops of that, Cha2 along there with 155 areTLLa small loops TBLa form theloops. distinct Numerous »prongs«. protein Two chiasmata bodies (PBs) (Cha1 wereand Cha2) identified spots wereclose found to the on 156 bivalent.the bivalent. Figure In the 3b proximity depicts ofthe Cha2 third there LBC are sampled small TBLa after loops. the reproductionNumerous protein period. bodies 157 The(PBs) bivalent were identified is almost close completely to the bivalent. devoid Figure of side 3b loops. depicts Marker the third structures LBC sampled MLb1 after and 158 MLb2the reproduction are present period. as large The chromomeres, bivalent is almost while completely the GMLb devoid structures of side have loops. retained Marker 159 thestructures shape MLb1of large and and MLb2 conspicuous are present loops.as large Moreover, chromomeres, well-preserved while the GMLb TLLb structures loops 160 werehave retainedalso observed the shape on of the large bivalent. and conspicuous The Tb loops loops. alsoMoreover, had the well-preserved shape of distinctive TLLb loops 161 “prongs”, whereas the TBLb loops had the open form that is so uncommon in were also observed on the bivalent. The Tb loops also had the shape of distinctive »prongs«, 162 lampbrush autosomes. Furthermore, well-preserved chiasmata locations (Chb1 and whereas the TBLb loops had the open form that is so uncommon in lampbrush autosomes. Furthermore, well-preserved chiasmata locations (Chb1 and Chb2) were observed on the bivalent. Protein bodies (PBs) were identified close to the bivalent. 4 163 Chb2) were observed on the bivalent. Protein bodies (PBs) were identified close to 164 the bivalent. 163 88Chb2) were observedAndraszek and on Smalec: the bivalent. Modifications Protein of LBC structure bodies of the(PBs) European were domestic identified goose Anserclose anser to 164 the bivalent.

165 166 Figure 3 165167 Figure 3 168166 Comparison of of the the structure structure of LBC of LBC3 prior 3 toprior (a) and to (a) after and (b) afterthe reproductive (b) the reproductive period. Arrows period. indicate Arrows marker 169167 structuresindicateFigure 3 marker of bivalents. structures of bivalents. 170168 Comparison of the structure of LBC 3 prior to (a) and after (b) the reproductive period. Arrows 171169 Comparisonindicate marker of ofthe structures the structure structure of of bivalents. LBCs of 4LBCs prior to4 andprior after to and the reproductive after the reproductive period period 170 172 ThreeThree groups groups of marker of markerloops (MLa1-MLa3) loops (MLa and1-MLa3) the distinct and the TBLa distinct loops wereTBLa identified loops were on 173171 identifiedComparison on ofthe the bivalent. structure In theof LBCs proximity 4 prior of MLa3to and there after isthe a reproductiveunique structure period that the bivalent. In the proximity of MLa3 there is a unique structure that was not observed in 174172 was notThree observed groups in of the marker other loops bivalents. (MLa 1This-MLa3) DB structureand the distinct is a large TBLa “loopless” loops were 175173 thearea,identified other parallel bivalents. on the in bothbivalent. This chromatids DB structureIn the proximity of is the a large bivalent. of »loopless« MLa3 Additionally, there area, is parallel a uniquetwo inchiasmata both structure chromatids (Cha that 1 176174 ofandwas the Cha2),not bivalent. observed as Additionally, well in as the numerous other two chiasmatabivalents. bivalent-specific (Cha1 This andDB structure Cha2),protein as bodieswell is a as large numerous(PBa “loopless”1-PBa5) bivalent- were 177175 specificfoundarea, parallelon protein the bivalent. inbodies both (PBa1-PBa5)chromatids Figure 4b were depictsof the found bivalent. the on fourth the Additionally, bivalent. LBC sampled Figure two 4b afterchiasmata depicts the the egg (Chafourth 1 178176 LBCproductionand sampled Cha2), period afteras well the had as egg numerousended. production Remnants bivalent-specific period hadof two ended. groups protein Remnants of bodiesmarker of two (PBa loops groups1 -PBa5)(MLb of marker1 andwere 179177 loopsMLb2),found (MLb1 on very the and prominent bivalent. MLb2), veryFigure TBLb prominent loops4b depicts and TBLb telomericthe loops fourth and Tb LBCtelomeric loops sampled were Tb loops identifiedafter were the identified eggon the 178 production period had ended. Remnants of two groups of marker loops (MLb1 and 180 onbivalent. the bivalent. In the Inproximity the proximity of one of of one the of marker the marker loops loops there there is a is DB. a DB. Two Two chiasmata chiasmata 181179 (ChbMLb2),1 and very Chb2), prominent as well TBLb as twoloops bivalent-specific and telomeric Tbprotein loops bodies: were identified a large (PBb on the1) 180 (Chb1bivalent. and InChb2), the proximityas well as oftwo one bivalent-specific of the marker protein loops bodies:there is a alarge DB. (PBb1) Two chiasmataand a small 182 oneand (PBb2) a small were one identified (PBb2) onwere the identified bivalent. on the bivalent. 183181 (Chb1 and Chb2), as well as two bivalent-specific protein bodies: a large (PBb1) 182 and a small one (PBb2) were identified on the bivalent. 183

184 5 185 Figure 4 Figure 4 186 Comparison of the structure of LBC 4 prior to (a) and after (b) the reproductive period. Arrows 5 187 indicateComparison marker of the structures structure of of LBC bivalents. 4 prior to (a) and after (b) the reproductive period. Arrows indicate marker 188 structures of bivalents. 189 Comparison of the structure of LBCs 5 prior to and after the reproductive period 190 Two clusters of marker loops (MLa1 and MLa2), TBLa loops and two 191 chiasmata (Cha1 and Cha2) were identified on the bivalent. Figure 5b depicts the 192 fifth LBC isolated from the oocytes after the reproductive period. Remnants of two 193 clusters of marker loops (MLb1 and MLb2) and two chiasmata (Chb1 and Chb2) 194 are visible in the bivalent. Telomeric TBLb loops were identified in the distal part of 195 the bivalent.

196 197 Figure 5 198 Comparison of the structure of LBC 5 prior to (a) and after (b) the reproductive period. Arrows 199 indicate marker structures of bivalents. 200 201 Comparison of the structure of the sex LBCs prior to and after the reproductive 202 period 203 TBLa and TLa structures connected with the free telomere of the Z 204 chromosome were identified in the LBC sex ZWa. A GLLa loop was observed near 205 the chiasm linking chromosomes Z and W. Such loops are very distinctive and easy

6 184 185 Figure 4 186 ComparisonArch Tierz 55 (2012) of the 1, 84-96structure of LBC 4 prior to (a) and after (b) the reproductive period. Arrows 89 187 indicate marker structures of bivalents. 188 Comparison of the structure of LBCs 5 prior to and after the reproductive period 189 Comparison of the structure of LBCs 5 prior to and after the reproductive period 190 Two clustersTwo clusters of marker of marker loops (MLa1 loops and (MLa MLa2),1 and TBLa MLa2), loops TBLa and twoloops chiasmata and two (Cha1 and 191 chiasmataCha2) were (Cha identified1 and on Cha2) the bivalent.were identified Figure 5bon depicts the bivalent. the fifth Figure LBC isolated5b depicts from the the 192 fifthoocytes LBC after isolated the reproductive from the oocytes period. afterRemnants the reproductive of two clusters period. of marker Remnants loops (MLb1 of two and 193 clustersMLb2) and of twomarker chiasmata loops (Chb1(MLb 1and and Chb2) MLb2) are andvisible two in thechiasmata bivalent. (Chb Telomeric1 and TBLb Chb2) loops 194 arewere visible identified in the in thebivalent. distal partTelomeric of the bivalent. TBLb loops were identified in the distal part of 195 the bivalent.

196 197 Figure 5 198 Comparison of the structure of LBC 5 prior to (a) and after (b) the reproductive period. Arrows 199 indicateFigure 5 marker structures of bivalents. 200 Comparison of the structure of LBC 5 prior to (a) and after (b) the reproductive period. Arrows indicate marker 201 Comparisonstructures of bivalents. of the structure of the sex LBCs prior to and after the reproductive 202 period 203 TBLa and TLa structures connected with the free telomere of the Z 204 chromosomeComparison of thewere structure identified of the in sex the LBCs LBC prior sex to ZWa.and after A GLLa the reproductive loop was period observed near 205 the chiasm linking chromosomes Z and W. Such loops are very distinctive and easy TBLa and TLa structures connected with the free telomere of the Z chromosome were identified in the LBC sex ZWa. A GLLa loop was observed near the chiasm linking 6 chromosomes Z and W. Such loops are very distinctive and easy to spot even on bivalents with a dense mantle of loops. Over the entire length of the Z chromosome, in specific places there are clusters of marker loops (MLa1-MLa3). In the vicinity of loop MLa3 small protein bodies were identified (PBa1). Structure of chromosome W in which all the marker structures are in the form of large side loops; chromosome W is present as a heterochromatic lump (Figure 6a). TBLb loops connected with the free telomere of chromosome Z were also identified on the post-reproduction sex LBCs. In the proximity of the TBLb loop a PBb1 was observed. Three clusters of marker loops (MLb1-MLb3) were observed in the Z chromosome. The same as in the pre-reproduction bivalents, the GLL structure is located close to the chiasm linking chromosomes Z and W. After the egg production period, the GLL structure assumes the shape of very large loops with an open structure. As in the pre- reproduction bivalents, the structure of chromosome W depends on the structure of the entire sex bivalent. In bivalent b, the loops of chromosome W are withdrawn towards the axis of the chromosome (Figure 6b). 206 to spot even on bivalents with a dense mantle of loops. Over the entire length of the 207 Z chromosome, in specific places there are clusters of marker loops (MLa1-MLa3). 208 In the vicinity of loop MLa3 small protein bodies were identified (PBa1). Structure of 209 chromosome W in which all the marker structures are in the form of large side 210 loops; chromosome W is present as a heterochromatic lump (Figure 6a). TBLb 211 loops connected with the free telomere of chromosome Z were also identified on 212 the post-reproduction sex LBCs. In the proximity of the TBLb loop a PBb1 was 213 observed. Three clusters of marker loops (MLb1-MLb3) were observed in the Z 214 chromosome. The same as in the pre-reproduction bivalents, the GLL structure is 215 located close to the chiasm linking chromosomes Z and W. After the egg production 216 period, the GLL structure assumes the shape of very large loops with an open 217 structure. As in the pre-reproduction bivalents, the structure of chromosome W 21890 depends onAndraszek the structure and Smalec: of the Modifications entire sex of bivalent.LBC structure In bivalentof the European b, the domestic loops of goose Anser anser 219 chromosome W are withdrawn towards the axis of the chromosome (Figure 6b).

227 The analysis of the structure of microbivalents is possible only at a 100-fold 228 zoom. For a better comparison of the sizes of macro- and microbivalents, the 229 microbivalent images at the 20× zoom were inserted as frames within Figure 7a 230 and 7b. This is the zoom at which the first five macrobivalents and the sex bivalent 231 were analysed. 232 Figure 7a depicts a sample microbivalent isolated from goose oocytes before 233 220the reproductive period. Along the whole axis of the microbivalent there is a mantle 221 Figure 6 234 222of sideComparison loops, large of the marker structure loops of the sex(MLa LBC1 andprior toMLa2) (a) and and after characteristic (b) the reproductive telomeric period. 235 223Figureloops Arrows 6GLLa indicate1 and GLLa2.marker structures Figure of7b bivalents. depicts a sample goose microbivalent obtained 236 224Comparisonafter the egg of the production structure ofperiod. the sex Over LBC prior the towhole (a) and length after (b) of the the reproductive bivalent, the period. side Arrows indicate 237 225markerloops Comparison arestructures withdrawn of bivalents.of the towards structure the of axis the andmicro are LBCs present prior into theand form after ofthe chromomeres. reproductive 238 226Only periodthe marker loops (MLb1 and MLb2) are well-preserved. In the distal parts of 239 the microbivalent there are the gigantic GLLb1 and GLLb2 loops. 7

240 241 Figure 7 242 FigureComparison 7 of the structure of the micro-LBC prior to (a) and after (b) the reproductive period. 243 ComparisonArrows indicate of the marker structure structures of the micro-LBCof bivalents. prior to (a) and after (b) the reproductive period. Arrows indicate 244 marker structures of bivalents. 245 Discussion 246 The available bibliography did not contain any publications analysing the structure 247 and transcriptional activity of goose LBCs or those of any other water poultry 248 species. Moreover, this is the first study comparing the structure of pre- and post- 249 reproduction LBCs. Therefore, the results of the study were compared with the 250 results of experiments analysing the structure of LBCs in hens, quails, turkeys, 251 doves, chaffinches, sparrows and amphibians. 252 The first goose LBC is characterised by a relatively high number of chiasmata 253 and marker loops. These structures remain well preserved on the bivalent after the 254 egg production period. The difference concerns the whole mantle of LBC side loops 255 that recede towards the axis of the chromosome after the egg production. There 256 are also telomeric loops (TBL) on the LBCs both prior to and after reproduction. 257 Such loops had also been observed on the first LBC in hens (Hutchison 1987, 258 Chelysheva et al. 1990, Solovei et al. 1992, 1993, 1995, Rodinov et al. 2002,

8 Arch Tierz 55 (2012) 1, 84-96 91

Comparison of the structure of the micro LBCs prior to and after the reproductive period The analysis of the structure of microbivalents is possible only at a 100-fold zoom. For a better comparison of the sizes of macro- and microbivalents, the microbivalent images at the 20× zoom were inserted as frames within Figure 7a and 7b. This is the zoom at which the first five macrobivalents and the sex bivalent were analysed. Figure 7a depicts a sample microbivalent isolated from goose oocytes before the reproductive period. Along the whole axis of the microbivalent there is a mantle of side loops, large marker loops (MLa1 and MLa2) and characteristic telomeric loops GLLa1 and GLLa2. Figure 7b depicts a sample goose microbivalent obtained after the egg production period. Over the whole length of the bivalent, the side loops are withdrawn towards the axis and are present in the form of chromomeres. Only the marker loops (MLb1 and MLb2) are well-preserved. In the distal parts of the microbivalent there are the gigantic GLLb1 and GLLb2 loops.

Discussion The available bibliography did not contain any publications analysing the structure and transcriptional activity of goose LBCs or those of any other water poultry species. Moreover, this is the first study comparing the structure of pre- and post-reproduction LBCs. Therefore, the results of the study were compared with the results of experiments analysing the structure of LBCs in hens, quails, turkeys, doves, chaffinches, sparrows and amphibians. The first goose LBC is characterised by a relatively high number of chiasmata and marker loops. These structures remain well preserved on the bivalent after the egg production period. The difference concerns the whole mantle of LBC side loops that recede towards the axis of the chromosome after the egg production. There are also telomeric loops (TBL) on the LBCs both prior to and after reproduction. Such loops had also been observed on the first LBC in hens (Hutchison 1987, Chelysheva et al. 1990, Solovei et al. 1992, 1993, 1995, Rodinov et al. 2002, Schmid et al. 2005) and chaffinches (Saifitdinova et al. 2003). On the other hand, they had not been identified on the first LBC of the quail (Rodinov & Chechik 2002, Schmid et al. 2005). What is interesting is the significance and location of PBs. These structures are present in cells only in association with LBCs and their role has not been entirely explained (Morgan 2002, 2007). In the first goose bivalent, two protein bodies (PBs) connected with each homologue of the bivalent were identified. PBs were observed on the chromosomes both prior to and after the egg production period. PBs are not typical of the LBCs of all avian species. Apart from geese, they have only been identified in chaffinches, doves and sparrows (Solovei et al. 1993, Saifitdinova et al. 2003, Krasikova et al. 2004). The second lampbrush chromosome was characterised by a high number of chiasmata and ML loops. The configuration of ML loops was preserved on the bivalent sampled from the geese after reproduction but the number of chiasmata was reduced. The reduction in the number of chiasmata is probably due to their terminalisation. In the region limited by the DBL and PBL markers there are two protein bodies associated with each homologue of the bivalent. The spot is identified as a centromere of the bivalent. The structure of this segment is preserved in the LBC after the egg production period, as well. The same configuration of PBs was observed in chaffinches (Saifitdinova et al. 2003). On the other hand, PBs were 92 Andraszek and Smalec: Modifications of LBC structure of the European domestic goose Anser anser not identified in LBCs of hens, quails and turkeys (Chelysheva et al. 1990, Solovei et al. 1992, Myakoshina & Rodinov 1994, Rodinov & Chechik 2002, Schmid et al. 2005). In the second goose bivalent no telomeric TBL loops were found. These are typical of the second LBC in hens (Chelysheva et al. 1990, Solovei et al. 1992, Schmid et al. 2005). On the third goose LBC, two chiasmata and three marker loop (ML) clusters were identified. Some of the loops were classified as GLL – large marker loops. They were found near the first chiasm, in the analogical bivalent spots. Large GLL loops are also typical of the third bivalent of other bird species mentioned above but in no other species are they as large as in geese. The GLL loops are particularly well visible in the pre-reproduction goose bivalent on which the mantle of side loops and other marker loops are withdrawn towards the chromosome axis. Characteristic telomeric T loops, located in the telomere adjacent to the first chiasm, were observed in the post-reproduction LBC. The loops were folded into the centre of the bivalent and formed the distinctive »prongs«. Such loops were not observed in LBCs of other bird species. Probably, this distinctive configuration is likely to be observed in LBCs after the egg production period, when the other marker and telomeric loops are inactive. In the opposite telomere, open TBL loops were identified. TBL loops had been observed only on the third bivalent of chaffinches (Saifitdinova et al. 2003). Apart form the ML marker loops and numerous protein bodies, the fourth bivalent isolated from the pre-reproduction geese contained the DB structure that was not observed in other goose LBCs. The DB structure remains well preserved on the bivalent after the egg production period. Sizeable DBs had only been identified in the fourth chromosome of hens (Schmid et al. 2005). Small DBs had been observed on the LBCs of chaffinches (Saifitdinova et al. 2003). The same as in hens (Schmid et al. 2005), the fifth goose bivalent had the distinctive TBL loops. The change of bivalent structure after the egg production period as compared to its pre-reproduction state corresponded with the commonly accepted assumption that post- reproduction bivalents do not have the mantle of side loops but only marker structures that are the last to become degraded. The sex bivalent of birds has the shape of an asymmetric univalent. One of the telomeres, in the form of a compact knob, is considered as a condensed microchromosome W (Hutchinson 1987, Mizuno & MacGregor 1998). As for the sex bivalent of geese, its various forms were observed both prior to and after the reproductive period. Diverse shapes and lengths of the ZW bivalent had also been described in other domestic birds: hens, quails, turkeys and doves (Solovei et al. 1993, Mizuno & MacGregor 1998, Schmid et al. 2005) and wild ones: sparrows and chaffinches (Solovei et al. 1993, Saifitdinova et al. 2003). The distinctive TBL structures identified on the sex LBCs of geese prior to the egg production period and present in the free telomere of chromosome Z had otherwise been observed only in hens (Solovei et al. 1993; Mizuno & MacGregor 1998). These loops are not present on the ZW bivalent of the quail, turkey, dove and chaffinch (Solovei et al. 1993, Mizuno & MacGregor 1998, Saifitdinova et al. 2003). Another distinctive structure in lampbrush sex bivalents are the large GLL loops situated in the region of the chiasm linking chromosomes Z and W. In goose LBCs, these loops are large and easy to identify but not as prominent as the giant GLLs in turkeys, sparrows and chaffinches. In quail chromosomes, the structure was not observed. In turn, on the sex Arch Tierz 55 (2012) 1, 84-96 93 bivalents of hens the GLL structure can assume forms ranging from intensively condensed small loops to enormous loops as large as 1/3 of the total length of the bivalent. These loops can have a closed (the distinctive loop) or open structure (Solovei et al. 1993, Mizuno & MacGregor 1998, Saifitdinova et al. 2003). On the sex LBCs, one PB associated with chromosome W was always observed; the other PBs were present in various locations in the bivalent and in different numbers. Protein bodies had not been observed in sex LBCs of hens, quails and turkeys. On the other hand, PBs had been detected in regular locations on sex bivalents of the dove, sparrow and chaffinch. In the species mentioned above, one PB is always associated with chromosome W. Moreover, in doves, sparrows and chaffinches, PBs are present in the centromere segment of the sex bivalent (Solovei et al. 1993, Saifitdinova et al. 2003, Krasikova et al. 2004). A similar configuration of PBs was observed in the sex LBCs of geese. ML loops in the goose sex LBCs were found in different numbers and sizes. No marker loops were identified within the W chromosome in the geese. Such loops are typical of the bivalents of the quail and dove (Solovei et al. 1993, Saifitdinova et al. 2003). Particularly interesting are the sex LBCs after the reproductive period. Very well preserved TBL and ML loops remain on the bivalents. The same as in the case of the pre-reproduction bivalents, two protein bodies (PBs) are associated with the two ML markers. In turn, on the post-reproduction bivalents close to the chiasm linking the Z and W chromosomes there are gigantic GLL loops. GLL loops were also observed in the geese prior to reproduction but they were not as well-developed. In hens, diversified forms of loops (GLL) had also been observed but they were sporadic on pre-reproduction chromosomes (Solovei et al. 1993). A distinctive feature of sex LBCs of the dove, chaffinch and sparrow are the enormous GGL loops present in the proximity of the chiasm linking the Z and W chromosomes (Solovei et al. 1993, Mizuno & MacGregor 1998, Saifitdinova et al. 2003). The different patterns of chromosomes sampled from pre- and post-reproduction geese are due to a modified transcriptional activity that is related to physiological processes in the organism and reflected in the morphological structure of the chromosome. The LBCs of pre- reproduction geese had the distinctive large side loops corresponding with transcriptional activity. The chromosomes sampled from the geese towards the end of the reproductive period had the characteristic small side loops and visible chromomeres resulting from the reduced transcriptional activity of the cell. Regulation of lampbrush chromosome transcription is performed by means of modifications of chromosome structure and the activity of a number of post-transcription factors. The preservation of lampbrush chromosome structure during transcriptional activity is connected with the presence of the so-called »constitutive« nucleosomes (Spring & Franke 1981, Gaginskaya et al. 1984, Scheer et al. 1984, Scheer 1987, Gaginskaya & Tsvetkov 1988, Olins & Olins 2003). The transcription of the oocyte-specific topoisomerase I (topo-I) variant is activated during the formation of lampbrush chromosome structures. This topoisomerase is present in lampbrush chromosome loops and participates in the spatial conformation of these structures. The inhibition of topo-I activity causes the lampbrush loops to recede and stimulates the condensation of nuclear chromatin (Gebauer et al. 1996, Morgan 2002, 2007). The changes in gene expression commence at the moment of fertilisation (Davidson 1986). At this time, chromosome transcription activity almost completely disappears in 94 Andraszek and Smalec: Modifications of LBC structure of the European domestic goose Anser anser most animal species until the first embryo divisions. In this period, chromosomes lose their distinctive lampbrush structure. Nuclear DNA transcription is resumed only after the embryo differentiation process has begun. The moment of DNA transcription reactivation is indeed a critical point in the development of the embryo, with the highest mortality rate. During this critical period for the developing embryo, protein synthesis goes on owing to the translation of mRNA accumulated in the oocyte (Landsberger & Wolffe 1997). This means that an adequate number of ribosomes are necessary for the translation of the accumulated information and these are not produced by the embryo at the initial stage of its development. The effect of the amplification of rDNA genes is that the 18S and 28S ribosomal components become the most numerous population of the oocyte RNA (MacGregor & Kezer 1970, Gaginskaya & Gruzova 1975, Gaginskaya et al. 1984, Gaginskaya et al. 1988). This is particularly evident in birds, reptiles and amphibians whose oocytes accumulate an even 200 thousand times higher number of ribosomes than that of ribosomes in somatic cells. This may be associated with oviparity and the related worse conditions for embryo development than in the case of viviparous organisms. Though lacking the activity of their own nucleolar organizer regions, the zygotes and subsequently the embryos of these animals have to keep developing, which is made possible by the accumulated maternal ribosomes. Lampbrush ribosomes also undergo seasonal changes that modify their morphology and transcriptional activity, which is particularly evident in hibernating amphibians. In the summer, when the animals are the most active, the activity of lampbrush chromosomes is the highest, as well. On the other hand, in the autumn, lampbrush chromosome activity abates. Nevertheless, this is not associated with morphological changes. At that time of the year, each transcription unit contains approximately 10 RNP filaments, while in the summer this number is twice as high. In the winter, transcription substantially declines. Both in chromosomes and in nucleoli numerous and very characteristic morphological changes take place (Tsvetkov & Parfenov 1994) No more than 70 % of the nuclear DNA is subject to transcription at the time. If in the case of physical factors, such as radiation, or numerous chemical factors a similar effect on the structure and activity of lampbrush chromosomes in various groups of animals can be expected, seasonal changes predominantly affect poikilotherms (Morgan 2002). Lampbrush chromosomes are very dynamic structures in which the loops are formed and disappear in response to a number of nucleus-specific and environmental factors (Gall et al. 1999, Gall 2000, Doyle et al. 2002, Saifitdinova et al. 2003, Austin et al. 2009, Gaginskaya et al. 2009). The interrelation of active and inactive genes is controlled by epigenetic mechanisms operative at the level of DNA methylation and histone modification that change chromatin structure. The open conformation of chromatin is transcriptionally active, whereas the »closed« conformation is associated with the so-called transcription decline (Grummt & Pikaard 2003). The study of the influence of particular factors that improve or deteriorate hatching parameters is particularly interesting in the context of the poultry industry. This is due to the fact that the quality of the hatching material constitutes one of the most important aspects of poultry production economics. The influence of lampbrush chromosomes on hatching parameters seems unquestionable. The amount of mRNA that an oocyte produces on the maternal DNA matrix seems to be of critical importance for the development of the embryo whose autotranscription is initiated at a later stage. Arch Tierz 55 (2012) 1, 84-96 95

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Received 28 April 2011, accepted 31 August 2011.

Correspondending author: Katarzyna Andraszek email: [email protected] Department of Animal Genetics and Horse Breeding, Institute of Bioengineering and Animal Breeding, University of Life Sciences and Humanities, Prusa 14, 08-110 Siedlce, Poland