Classification of Germ Cells Reproductive Cycle Chaitip WanichanonVi - PDF document

Classification of Germ Cells, Reproductive Cycle, Chaitip WanichanonVichai Linthong, Amporn Thongkukiatkul, Padermsak Jarayabhand and Tanate Poomthong of knowledge that could aid the large scale productionof larvae for aquaculture are still lacking. These are:1) the probable spawning periods and the frequenciesof spawning of land-culture broodstocks during theyear; 2) the age when the abalone reaches full sexualmaturity and could be used as broodstocks; and 3)the possibility of using artificial means to inducespawning when the gonads are fully developed inorder that mature gamete cells from both sexes couldbe obtained simultaneously.preliminary study of around Bon Island,Phuket, revealed that spawning occurred at severalintervals throughout the year during January-February, April-May, June-July and November-December. Gametogenic cycle was also studiedChonburi province, in which the spawningoccurred between June and November. So far thereas well as the development of reproductive organsTherefore, the aims of the present studyare to investigate the reproduction of thathave been reared in land-based culture system withrespect to 1) the gonadal histology and the gameto-genic processes, especially the classification ofvarious stages of germ cells in the testis and ovarybased on light and electron microscopic observa-histology during different months of the year; andbe applied in determining the appropriate time forinduction of spawning, and to increase gameteproduction leading to the improvement ofaquaculture system of this abalone species.ATERIALSAbalone from land-based culture system areprovided by the Coastal Aquaculture DevelopmentCenter, Prachaubkirikhun province, and MarineBiological Station, Chulalongkorn University,Angsila, Chonburi province. They are kept inconcrete tanks housed in the shade and well flushedwith mechanically circulated filtered sea water as wellas air delivery system to maintain the controlledenvironment. The optimum level of salinity is about22.5-32.5 ppt and the temperature is about 22-26¡C.They are fed with macroalgae (usually spp. spp.), supplemented with artificialFor the study of the gonadal histology, ultra-structure and the cyclical changes during the year,adult abalone, aged at least 24 months, were collectedmonthly for a period of one year. The fixed gonadswere prepared for light and electron microscopico

bservations by the paraffin, semithin, andjuvenile abalone reared in the closed-culture systemas mentioned above were collected monthly fromthe age of 1 to 12 months, and the gonads wereprocessed for light microscopic observations.Light MicroscopyAbalone were anesthetized in 5% magnesium) for one hour, for paraffin sectionsthe gonads were cut and fixed in either BouinÕscacodylate buffer pH 7.4, at 4¡C, for overnight. Thetissue blocks were then washed in 70% ethyl alcoholfor removal of the BouinÕs fixative, andglutaraldehyde fixative was removed by washingwith phosphate buffer three times. Then, thespecimens were dehydrated in graded series of ethylalcohol (70-100%) for 30 minutes each, cleared withdioxane, infiltrated and embedded in paraffin wax.Blocks of specimens were sectioned at 5-micronor PAS-heamatoxylin, and observed in an OlympusVanox light microscope.Transmission Electron Microscopywere cut into very small pieces and fixed in a solutionbuffer pH 7.4, at 4¡C, for overnight. The specimenswere post-fixed in 1% osmium tetroxide in 0.1Msodium cacodylate buffer, at 4¡C, for 2 hours. Then,they were dehydrated in graded series of ethanol (50-100%) for 30 minutes each, cleared in two changesof propylene oxide, infiltrated in a mixture ofpropylene oxide and Araldite 502 resin at the ratiosof 3:1 for 1 hour, 2:1 for 2 hours and 1:2 forovernight, then embedded in pure Araldite 502 resin45¡C and 60¡C for 24, 48 and 48 hours, respectively.Blocks of specimens were sectioned at 1-micronthickness by ultramicrotome and stained withMethylene blue for light microscopic observations,and ultrathin sections were cut and stained with leadTEM H-300 at 75 kV. ESULTS1.Gonadal HistologyThe conical organ consists of the hepatopancreassurrounded by the testis or ovary (Fig 1C,D). At thebase of the organ, the hepatopancreas appears largeand occupies most of the cross-sectional profile (Fig1C); while it becomes smaller towards the taperedend of the organ where most of the tissue belongs tothe gonads (Fig 1D). Both testis and ovary aresurrounded by a capsule which is composed of thevaries according to the gonadal cycle during the year.The connective tissue from the capsule extendssepta or trabeculae that are branched, and connectedhepatopancreas. As a result, the gonads arepartitioned into small compartments, each1E, 1J). Within the connective tissue of e

achtrabecula, there are small vessels running throughits whole course (Fig 1F, 1L,M), which may becapillaries that are branched out from the largersubcapsular vessels. Around the capillaries andparallel to the long axis of the trabeculae, there areare intermingled with small cells exhibiting densebe fibroblasts, while others may be follicular orsupporting cells that surround oogonia andEach trabecula acts as the axis on which growinggerm cells are attached (Fig 1E,F, 1J,M). Early stagecells, such as spermatogonia, initial stages of primaryspermatocytes and oogonia, are closely adhered tosecondary spermatocytes and developing oocytes, aremore detached and appear further away from thespermatozoa and mature oocytes, are completelydetached and move to the outermost region fromthe axis. Such an appearance gives rise to a discretegroup of germ cells surrounding each trabecula,2.Classification of Germ Cellsclassified, according to their structural features asobserved under the light and transmission electronmicroscopes, as follows:2.1Spermatogenic cells Based on the nuclearSpermatogonium (Sg). (Fig 1G) Sg is a8-10 µm. Its nucleus is round or slightly indentedmostly euchromatin with only small chromatinblocks attached to the inner surface of nuclearenvelope. The nucleolus is prominent and standsout from the rather transparent nucleoplasm. Sg arePrimary spermatocytes (PrSc). cells (from LSc to PSc) are round and becomeincreasingly larger, then they (from DSc to MSc) aregradually decreased in size. The distinctivedifferences among various stages of PrSc are thepattern of chromatin condensation and the relativeamount of euchromatin versus heterochromatin.Leptotene spermatocyte (LSc). (Fig 1G, H,4A) These round-shaped cells are larger than Sg withdiameter about 10-12 µm and also contain largeround nuclei, each with diameter about 8 µm. Thereis a thin rim of heterochromatin along the nuclearenvelope and small blocks of heterochromatinscattered evenly throughout the nucleus. Thenucleolus is still present but not as prominent as inZygotene spermatocyte (ZSc). (Fig 1G, H,4A) ZSc has approximately the same size as LSc. Thedistinguishing features of ZSc is the heterochromatinblocks which are increasing in size and density, andthey are coupled at many points by synaptonemalcomplexes. The nucleolus disappears completely.Pachytene spermatocyte (PSc). PSc st

illshows round shape with slightly smaller size thanby the heterochromatin which appears as longthreads or thick fibers that are entwined into ÒbouquetpatternÓ, and becoming visible throughout thenucleus. Under TEM (Fig 4A-C) these chromatinÒthreadsÓ are actually thick blocks consisting oftightly packed 30 nm fundamental chromatin fibers.Diplotene spermatocyte (DSc). (Fig 1G, H,4A-C) This cell resembles PSc, except the nucleusbecomes smaller (about 4 µm), and the chromatinblocks become increasingly thicker and packed Diakinetic and Metaphase spermatocytes (Fig 1H, 4B,C) These stages exhibit thickchromosomes that move to the equatorial region,Secondary spermatocyte (SSc). (Fig 4B,C)SSc is a small round cell about 7 µm in diameterchromatin blocks that are crisscrossing one another,thus appearing as checker-board or XY figures. Theindividual chromatin fibers in the block are loosenedSpermatids (St). (Fig 1F-H, 4B,C) There aredepending on the size, chromatin granulation andcondensation. All stages are round or oval, andranging in size from 6 µm in StSpermatid I (St (Fig 1G) St can bedistinguished by their chromatin which appears asfine granules under LM, that are uniformly spreadthroughout the nucleus. As a result, the whole nucleusappears moderately dense without any interveningtransparent areas of nucleoplasm. Under TEM the30 nm chromatin fibers becomes loosely packed anduniformly distributed throughout the nucleus.Spermatid II (St (Fig 1G,H) The generalfeatures of St are similar to those of St but thenucleus, which remains round, decreases in size andis located eccentrically within the cell. As a result,the chromatin fibers become more closely packed,Spermatid III (St (Fig 1G,H, 4B,C) The cellbecomes smaller and assumes more oval shape withchromatin begins to condense into dark blocks withintervening light area of nucleoplasm, individualchromatin fiber is enlarged to 40 nm.Spermatid IV (St (Fig 1H) The cellbecomes smallest but still appears oval. Its chromatinappears rather opaque; however, the outlines ofindividual chromatin fibers could still be observed,and each is enlarged to 60 nm.Spermatozoa (Fig 1F-I,4D) There are 2stages of spermatozoa: Sz is the immature sperma-with completely dense chromatin, thus the outlinesof chromatin granules are barely discernible. Thereis a cap-like structure apposing on one side of theellipsoid nucl

eus, which is the maturing acrosome.The tail is short with a pair of centrioles moving tothe neck region, from which the axonemalmicrotubules start to form.In mature spermatozoa (Sznucleus is fully elongated and slightly tapered at thechromatin is completely dense and the anteriorportion of the head is covered by acrosome withcentral core element (Fig 4D). Five globularmitochondria surround the centrioles in the neckregion. Zig-zag microtubules link mitochondria toaxonemal microtubule doublets surrounded byplasma membrane. Both immature and mature spermare completely detached from the germinal2.2Oogenetic cells. There are 6 stages of femalestages of growing oocytes. (Fig 1K,L) Og is a round ornucleus is round and about 7 µm in diameter. Itcontains small blocks of heterochromatin attachedto the inner surface of nuclear envelope, with theremaining majority appearing as euchromatin. Thenucleolus is present but may not be as prominent asimplies its basophilic property due to the presenceof moderate amount of ribosomes. Og are attachedto the capsular side of trabeculae and usually areconcentrated in groups (Fig 1K,L). Each Og issurrounded by flat, squamous-shaped follicular cells. (Fig 1K,L,5A-C) Oca round or scallop-shaped cell that is closely adheredround nucleus about 12 µm in diameter. The nucleusexhibits densely packed chromatin in the form ofnumerous lampbrush chromosomes. The nucleolusis present but tends to be obscured by the ratherdense chromatin and nucleoplasm. The cytoplasmbasophilic property, reflecting the presence ofnumerous polysomes, newly developed roughendoplasmic reticulum (RER) and Golgi complexes(Gc) as observed in TEM (Fig 5C). Newly releasedribosomes are packed into large mass around nuclearenvelope (Fig 5B). There is very few secretorygranules. Due to its enlarged size each Oc issurrounded by few follicular cells. (Fig 1K,L, 5D, 6A) Ocbecomes larger and transforms into columnar shape,with the cell size around 30x55 µm, and nuclear sizetissue of trabecula by the narrow part, and each Ocis surrounded by several follicular cells. The nucleusexhibits increasingly decondensed chromatin andmembrane are clearly distinct due to the moretransparent nucleoplasm and the presence of mostlyeuchromatin. The cytoplasm is stained light bluedroplets (Fig 5D). At TEM level it was observed tocontain numerous well-developed Gc, RER and

stillabundant ribosomes. There are 2 types of secretory and SG (~330 and 450 nm indiameter) with electron lucent and electron densematrix, respectively (Fig 6A,B). (Fig 1M, 6B) This cellbecomes increasingly larger and assumes flask orpear shape, with the narrow side or base still attachedThe nucleus contains mostly euchromatin, as mostof the lampbrush chromosomes become almosttransparent. The nucleolus is distinct and becomesenlarged due to the uncoiling of nucleolar chromatin.In addition to increasing number of clear lipiddroplets, the cytoplasm begins to show reddish yolkplatelets (Fig.1M) which are electron dense underTEM. Fine blue granules representing SG and SGare evenly distributed between lipid droplets andconcentrated around Gc (Fig 6B). Follicular cellssurround both the cell body and its base near (Fig 2A,C, 6C) Thiscell is large and assumes a pear or polygonal shape,process. The cell size is about 60-80 µm, with nucleareuchromatin and completely transparent nucleoplasmand it also becomes enlarged due to the completeuncoiling of its chromatin. The cytoplasm is filledwith reddish and electron dense yolk platelets (eachnumerous lipid droplets (each about 1500-3000 nmwhich represent SG and SG are decreased in centralarea of the cytoplasm, since most are probablytranslocated to the area underneath the plasmaon the outer surface of the cell membrane (Fig 2C).This coat is PAS positive and may be formed by thereleased content of SG, which were seen exocytosedat the oocyteÕs plasma membrane (Fig 6D). The coatis in turn surrounded by follicular cells. (Fig 2B-D) This is thefully mature oocyte before being released from the is the largest cells with polygonalor round shape, with the cell size about 80-140 µmwith completely enlarged and clear nucleolus. Occould be divided into 2 subgroups based on thecharacteristics of yolk platelets observed under LM(Fig 2D). The first subgroup contains small andsimilar size yolk platelets that are scattered evenlythroughout the cytoplasm. In the second subgroup,the yolk platelets are variable in size, and most arelarge bodies that could be formed by the coalescencegranules are also located underneath the cell (Fig 2C,D). The thick PASis uniform around the outer surface of the cellmembrane, but without the surrounding layer offollicular cells. Under TEM jelly coat appears fibrous are completely detache

d from the3.Reproductive CycleThe reproductive cycle of was assessedby observing the changes in the gonad histology,maturation during one reproductive cycle of theabalone cultured in a closed land-based system could (Fig 2E-I) This is a periodin which gamete cells begin to regenerate tocommence a new reproductive cycle. At the initiationcells, and all of them are closely attached to thetrabeculae. The ovary (Fig 2E,F) contains primarily and Oc which are rapidly increasedin number. In the testis (Fig 2G-I) there are mostlySg and PrSc, but neither St nor Sz are present. Theclusters of these early stage cells are located aroundthe short and dilated trabeculae. The hepatopancreasis quite large in size and occupies most of the crosssectional profile of the conical organ when compared Dorsal views of shell-freed male abalone (in showing testis (te), ovary (ov), hepatopancreas (HP), A cross-section of the testis showinghepatopancreas (HP) surrounded by testicular tissue which is, in turn, surrounded by a thin connective tissue capsule (cp); a cross-section of the ovary showing hepatopancreas (HP) surrounded by ovarian tissue and fibrous capsule; a spermatogenicunit consists of a central trabeculae (tr) arising from capsule (cp), surrounded by various stages of germ cells; in a capillary(ca) is present inside each trabeculae, and successive maturing stages of germ cells lie at different distance from the connect sections showing various stages of male germ cellssurrounding each trabecula; they are spermatogonia (Sg), primary spermatocytes (LSc-leptotene; ZSc-zygotene; PSc-pachytene;DSc-diplotene; MSc-metaphase stage), spermatid (St), and spermatozoa (Sz there are rows of fully mature spermatozoa), which are the most typical characteristic in mature phase of male abalone; ). The fully mature oocytes (Oc) are released into the central areaof the compartment partitioned off by adjacent trabeculae; sections showing stage I, II and III oocytes (Oc there are stage III oocytes (Oc) showing the presence of eosinophilic yolkgranules (arrows) in the cytoplasm when compared with the former stage oocytes. Fig 2.A-D) Sections showing stage IV and V (Oc): notice the first appearance of a thin jelly coat (jc), which is PAS positive, andincreasing number of eosinophilic yolk granules (yg) in Oc. The increasing amount of euchromatin, which is pale stained,and

the enlargement and vesiculation of nucleolus are also noticeable. Blue stripe underneath the oocyteÕs plasma membrane(arrow) is present in Oc there are two subtypes of stage V cells: the upper cell (1) shows small and evenly distributed Sections of Òproliferative phaseÓ, showingthe regeneration of gamete cells after spawning and spent phases. The ovary (, which are rapidlyincreased in numbers. The testis () contains mostly Sg and LSc. Trabeculae, which are depleted of cells and breaking downin spent phase, start to regenerate and appear short and dilated. Sections of Òpremature phaseÓ, showing rapid increase innumbers and sizes of various cells. The ovary (start to appear and gradually increase in numbers. The testis () contains various stage of primary spermatocytes (PrSc),spermatid (St) together and a few spermatozoa (Sz); all of which are located close to the trabeculae. Fig 3.A-E) Sections of Òmature phaseÓ, showing rapid growth of the gonads. The ovary () contains primarily fully mature Oc) contains mostly late spermatids (St) and spermatozoa(Sz), which lie in rows and at low power appear streaky (). Finally they become dispersed and released into luminal area of Sections of Òspawning phaseÓ, showing the period when abalone release the viable sperm or eggs from thegonads. The ovary (F,G) contains only the earlier stage oocytes which are still attached to the dilated trabeculae. Some yellowishgranular substances (arrow) is present in the ovarian lumen. The testis () contains only early stage of male germ cells with Sections of Òspent phaseÓ, showing the complete discharge of gamete cells, and the breakingdown of trabeculae and associated connective tissues in both sexes. Notice the hepatopancreas which becomes larger in relative Fig 4.A-C) Electron micrographs showing various stages of male germ cells, including leptotene (LSc), zygotene (ZSc), pachytene(PSc), diplotene (DSc), secondary spermatocyte (SSc), spermarids (St). Spermatozoa exhibiting dense nucleus (Nu), acrosome Fig 5.A,B) Early stage I oocyte (Oc), exhibiting nucleus with lampbrush chromosomes (Ch), dense nucleolus (no), and cytoplasm(Cy) with abundant ribosomes, some of which are aggregated in crystal-like bodies (arrows). Late Oc Stage II oocyte (Oc), exhibiting lipid droplets Fig 6.A,B) The cytoplasm of Oc (A) and Oc (B) exhibiting high concentration of d

ense jelly coat granules (SG) and lighter cortical) around Golgi complexes (Gc). Fourth stage oocyte (Oc) exhibiting very light nucleus (nu) due to completelyuncoiled chromatin. The cytoplasm contains numerous large yolk granules (yg), small SG and SG granules. The homogeneous) and fibrous jelly coat of Oc (arrow). the separation of gonadal capsule (cp) from hepatopancreas (HP) at 2 months; the presence on the gonadal capsule bordering the hepatopancreas (HP); the ovary at 6 monthsshowing the presence of early oocytes, mostly Oc the testis at 4 months (in and 6 months (in showing full rangeof spermatocytes (Sc), spermatids (St) and some spermatozoa (Sz); ovary at 7 months showing the formation of trabecula(tr) and the presence of early oocytes, mostly Oc and Oc ovary at 11 months in mature phase, containing mostly Oc and to the total gonad area. This phase usually occursaround April to May and October to November.Premature phase. (Fig 2J-M) This phase is theperiod when gametogenesis proceeds at full speedwith rapid increase in numbers and sizes of variouscells, while hepatopancreas is slowly reduced in itsrelative size; the gonads become enlarged in volumeand trabeculae become thinner. At the beginning, andpredominantly Oc, most of which are still attached and Oc cells occur.aggregate around the trabeculae. This phase lastsusually around May to June and January to Februaryin female; and it takes place around April to Mayand December to January in male.Mature phase. (Fig 3A-E) This phase is aperiod of rapid growth of gonads which are reflectedby striking differences in color between the twosexes. The rates of cells proliferation start toare still present and restricted to area immediatelyaround trabeculae. Hepatopancreas is furtherdecreased in size, and trabeculae become slimmer.In the ovary (Fig 3A,B) there are abundant Oconly few remaining and widely scattered Oc appear fully mature and are liberated into thelumen of oogenetic compartment. In the testis (Fig3C-E) there are mostly late stage male germ cells, whichlie in rows that in turn surround the earlier cell stageswhich are still closely attached to the trabeculae (Fig3D). As a result the testis appears to have maximum are dispersed into gonadal lumen and inter-bands of Sg and PrSc surrounding the trabeculae arefrom June to July and February to March in both (Fig 3F-J) This i

s the periodwhen abalone are ready for breeding, during whichthe completely mature and viable eggs or sperm arereleased from the gonads. The gonads are significantlydecreased in size, and the gonadal wall becomeswrinkle when compared with the former phase (Fig3H). Mostly ripen sperm or eggs are discharged whilethe earlier stages of gamete cells are still attached togranular substances (Fig 3G) remain in the lumenof gonadal compartments in both sexes. Spawningof observation, usually from August to October andMarch to April in female, and around August toNovember and February to April in male. Inaddition, partial spawning could be observedthroughout the year in some males. (Fig 3K-N) This is the periodafter spawning when fully mature gamete cells arecompletely discharged. The gonads exhibit thebreaking down of connective tissue stroma, andgametogenic activity momentarily cease. However,there are still clusters of gonial cells remain attachedto parts of the gonadsÕ capsule. As a result the gonadsare greatly decreased in size and become creamy insmall cross-sectional profiles in contrast to those ofthe hepatopancreas, which becomes very large inrelative size (Fig 3M). This phase occurs afterspawning around September to November andFebruary to April in both sexes.4.Maturation of Gonadsof hepatopancreatic capsule into 2 separate layersbe distinguished from the testis by the presence offew Og in contrast to fairly numerous primarymature spermatozoa are increasing in number during6 to 7 months. While testis are rapidly enlarging andsurrounding almost half of the circumference of theconical organ, ovary is much less developed and8 to 9 month the testis becomes enlarged to almostcompletely surround the hepatopancreas, and italready contains fully mature spermatozoa; while theovary tends to be delayed in development andovary starts to enlarge substantially and mature) begin to appear. Thus the maleanimals tend to reach full sexual maturity and startnormal reproductive cycle as early as 7 to 8 months,while female animals reach sexual maturity and startreproductive cycle around 11 to 12 months (Table 25 (1999) Gonadal Structure and Classification of Cells inThe first accounts of reproductive biology on anH tuberculatasince 1924, and Croft in 1929, whocomposed of fibrous capsular and trabecularsupports, from which germ cells appear to generate.Similar

histological studies in other species were laterperformed by many investigators. More recently,a fine structural study of the ovarian cells in the redH rufescens, was also undertaken by Martin All of these studies confirmed similar patternof structural organization of the gonads; however,there are some disagreements on the classificationspermatogenic processes. Utilizing a highresolution TEM to study the relative abundance ofvarious organelles, particularly ribosomes and thedevelopment of rough endoplasmic reticulum andGolgi complexes in the cells, Martin suggestedthat there were 5 stages of female germ cells in rufescens, which they termed oogonium, presyntheticand fully developed postsynthetic oocyte. We feeldividing cells in a single line of differentiation intovarious stages, because in reality these cells areundergoing continuous development. A bettercriterion would be to divide the cells according tofeatures which reflect the beginning of different, light and electronmicroscopic characteristics have been used forregard to the uncoiling of chromatin, as reflected bythe clarity of the two structures; 2) the clarity ofnuclear membrane which is the result of the densitydifference between the condensed chromatin in thenucleus and the surrounding cytoplasm; 3) thebasophilia or the bluishness imparted to thereflect the abundance of ribosomes in the cytoplasm;4) the presence of lipid droplets; 5) the developmentof secretory organelles particularly rough endoplasmicreticulum and Golgi complexes; 6) the occurrenceof basophilic secretory granules including corticalrelative abundance; and 7) the presence of jelly coatsurrounding the egg cells. By using these rather5 stages of egg cells, starting from oogonia (Og)which are the smallest cells closely attached to themaintain a constant pool of early stem cells,Table 1.Summary of the key features during the course of development of gonads in General StructureGametogenic UnitCell TypesPhases of Cycle 2-Separation of gonadal capsule fromonly few undifferentiated hepatopancreatic (HP) capsule.gonial cells attached to capsules-Development of few muscle cells none none: testicular tissue covering a quarterincompleteSg, PrSc, SSc, Stproliferative: ovary shows no further development.noneOgnone: testis covering half of HP capsule.completeSg, PrSc, SSc, Stpremature: ovary still small and not wellnoneOg,

Oc: testis covering half of HP capsule.completeSg, PrSc, SSc, Stmature: ovary still small.begin to develop fromOg, Ocvery early proliferativesprouting trabeculae: testis covering slightly over halfcompleteSg, PrSc, SSc, Stmature: ovary covering about a quarter incompleteOg, Oc early proliferative: testis covering all HP capsule.completeSg, PrSc, SSc, Stmature: ovary covering half of HP capsule.incompleteOg, Ocproliferative /premature11-: testis covering all HP capsulecomplete and numerousSg, PrSc, SSc, Stmature and much thickened.complete and: ovary covering slightly over halfincreasing in numbermature of HP capsule. particularly those that are clustered towards thewhen most mature oocytes are released from theovary and the connective tissues of trabeculae arebreaking down, these cells are the only remaininggroup of germ cells. The restoration of gonadalstructure during proliferative phase is carried outdifferent sizes ranging from 20-24 µm. The mostpronounced characteristics that they exhibit is thecytoplasm. And because of the similar degree ofthe partially condensed chromatin and densenucleoplasm on the other, the outline of nuclearThe nucleolus, while present, is not outstanding. All are surrounded by a single layer of flat follicularcells. Under TEM we found that there is increasingamount of ribosomes which reflects the intensecytoplasmic basophilia. While ribosomes are rapidlysurge in the number and degree of development ofGolgi complexes and RER are observed only in late. These two subgroups of Oc do not yet exhibitany secretory granules. Thus they may correspondto the presynthetic oocytes as described by Martin when cells are preparing themselves for the is the stage that first shows the presence oflipid droplets in the less intense basophilicchromatin, and the increased translucence of thenucleoplasm, the nuclear boundary could be clearlyobserved under LM. For similar reasons thenucleolus also becomes more distinct; and becauseof its enlargement the nucleolar activities for and SGgranules start to appear in this stage, by clusteringaround Golgi complexes. Thus Oc could represent) are first is the stage which eosinophilic yolk granulesfirst appear, and later is increasing in number; hencerendering the cytoplasm of Oc more reddish inSG granules are seen scattered evenly between yolkgranules and lipid droplets. We believed, there

fore,that this is the stage where there is intense syntheticactivities, since under TEM numerous SGas well as yolk granules appear in large numbers; and SG were seen concentratingaround Golgi complexes. Oc is still surrounded byconsists of several cells because of the increase in is further detachedfrom the connectives of trabeculae and assumes apear or even tear-drop shape. The chromatinbecomes completely euchromatic and the nucleolusis enlarged further as its chromatin are almost is the stage where a thin jelly coat is firstcell membrane and the surrounding layer of follicularcells. Under LM the cytoplasm of Oc becomesincreasingly eosinophilic and appears more reddishdue to the staining of numerous yolk granules byeosin. While the jelly coat is intensely PAS positive,the yolk granules are completely PAS negative. Thecontrasting feature implies that there may be verygranules, while these are the major constituent of is and yolk granules, which reflectchromatin of Oceuchromatic state and the nucleolus is fully enlargeddue to the complete uncoiling of its chromatin, andtranscriptional activities. Another remarkable feature under LM is the appearance of a narrow bluishmembrane, while the bluishness of the remainingmass of cytoplasm is much decreased in comparison and Oc and SG granuleswhich are translocated to this area as observed underTEM. Some of the more electron SG granules arealso seen exocytosed to the cellÕs periphery, and thus contains moreelectron lucent material than SGactual cortical granules that are concentrated in thenarrow cytoplasmic zone underneath the plasmamembrane, and thus are kept in reserve for corticalreaction upon fertilization of the egg by the sperm. is the stage where the jelly coat becomesuniformly thick and deprived of surrounding layertransformed from homogeneous in Oc to fibrous structure in Oc. There is no division of this cell coatinto jelly and vitelline layers as reported in other appears completely mature andis fully detached from the trabeculae. The absenceinto space between trabeculae and ready them forrelease from the ovary. From this appearance it couldbe speculated that the major roles of follicular cellsare protective and helping to maintain the adherencewhile the former are undergoing maturation. Innutritive function for oocytes, and their roles in is laden withreddish yolk granules. Based on the

size of these yolkgranules there could be 2 subgroups of Ocother contains very large granules, both of whichappear very electron opaque under TEM. It is stillnot possible to confirm whether these are two separate, or that the latter merely represent thefinal stage in which small yolk granules are coalescedto form larger ones. In any cases these two subgroups should represent fully mature cells. In Oc couldrepresented the early postsynthetic cells and Oc latepostsynthetic cells; even though, judging fromultrastructural features certain degree of syntheticUp to now most studies have not rigorouslyapart from suggesting broadly that there are 4 stages,spermatozoa. In the present study, the male could be classified into 13specific stages according to the size, shape,appearance of chromatin and the presence or absencewhose nucleus contains almost all euchromatinwhich results in the nucleus being very clear andnucleolus is prominent. Spermatogonia dividedmitotically to give rise to primary spermatocytes,which pass through 5 stages as in the first meioticdivision of vertebratesÕ germ cells. These prophasecells exhibit different forms of chromatincondensation, beginning with small to larger blocksof heterochromatin that are evenly scatteredthroughout the nucleus in LSc and ZSc. Hetero-chromatin blocks transform to thread-like patternthat are increasing in thickness and length, andbecome more entwined in PSc and DSc. Finally indiakinetic and MSc stages chromatin appears as pairsof chromatids that are translocated to the equatorialregion. Secondary spermatocytes are quite numerousin comparison to those in vertebrates and they haveheterochromatin that exhibit checker-board or XY-figure pattern. based on the nuclear size, shape andchromatin condensation. Under LM the first twostages exhibit finely granulated chromatin thatthroughout the nuclei. Thus St and St could bedistinguished by the difference in size (St about 6 about 4 µm), and by the denser nuclear. The latter is due to the reduction ofnuclear volume which results in the closer packingof chromatin fibers, even though each fibers stillmaintain their width of 30 nm. In the third stagetogether into large dense blocks, particularly the nuclear envelope, leaving clear areas between theblocks. At this stage individual fiber increases in sizeto 40 nm. Eventually, the decrease in volume ofnucleus an

d its more ellipsoid shape results in thetotal condensation of chromatin mass in Stindividual chromatin fiber is enlarged to 60 nm.The two stages of spermatozoa are distinguished also shows the initialformation of acrosome as a clear cap-like structureshort tail. Under TEM, there is the formation ofaxonemal complexes from centriolar pair that moveto the neck area just distal to the nucleus. Later, threearound the centrioles. In Sz the nucleus is elongatedfurther and chromatin appears completely densewith the outline of 60 nm fibers (or granules) barely exhibits a completely formed tail thatis long and point outwards from each trabecula.Reproductive CycleThere have been a number of studies on thecourse of reproductive cycle in various abaloneare frequently used for determining a reproductivecycle of a population are: 1) the measuring of therelative size of gonads with respect to the size ofconical organ which is termed gonad indices (GI); GI is not always a valid index fordevelopment of the gonads because GI only relatesgonad area to constant parameters ( the size ofconical organ) of the animal, and it does not takevariation in hepatopancreas size into account. The more precise index that can define of reproductiveof cellular association and the time interval betweenTomita, Lee, Giorgi & DeMartini, Ault,classified the reproductive stages in various temperateare more clearly defined in females. In the presentstudy, these various phases were also observed in samplings of the brooding stocks cultured in theland-based culture system reveal 5 distinctivegonadal patterns during the year, , proliferative,premature, mature, spawning and spent phase.Proliferative phase is characterized by theregeneration of gamete cells for the new cycle. The in the ovary, and mainlySg, PrSc without St and Sz in the testis. Giorgi &DeMartini and Ault, on studying found that the ovary contained primarily smallTomita, on studying , reported thatthere were mainly oogonia, yolkless and oil dropoocytes in this stage. Another remarkable featureduring this phase is the reciprocal relationshippancreas, which is similar to that found in other That is the hepatopancreas is relativelylarge when compared to the total area of conicalorgan. Boolootian also reported that, in cracherodii and H rufescens, the size of hepatopancreasexhibits an inverse relationship to gonadal activit

y.During this phase, the hepatopancreas attainsquiescent. The precipitous drop in the size ofhepatopancreas will occur during the subsequentphase when there is a rapid growth of the gonads.This implies that hepatopancreas may act as anutrient storage that is necessary for gamete cellsthe proliferation of gonad cells start to surge. Anotherremarkable histological feature observed during thiscontain large amount of granular materials. This mayrepresent the turgid state of the vessels that arePremature phase is the period of rapid increasein numbers and sizes of gamete cells. The ovary and few which is similar to those reported in the pre-mature stage of while Sg, Sc andonly few of St and Sz are evident in the testis during in studying reported that there were numerous developing earlydevelopment in this phase involve the rapid growth ofthe gonads due to fast proliferation of early germ cells.enlargement of the gonads which exhibit strikingdifferences of color between both sexes: greenish infemale and yellowish in male. The ovary contains with widelyscattered Ocand Sz. Before spawning occurs, Oc are detachedfrom trabeculae and released into the gonadal lumen.trabecula is surrounded successively by a few rowsof Sg, PrSc which are closely bound to trabecularconnectives, and middle Sc, St appear further away,and Sz are completely detached from trabeculae. Incomparison, during the differentiation of Oc to Ocfrom Og, the cells move along the trabeculae fromcapsular side towards the hepatopancreas side, until become detached from trabeculae.start to release their ripened gametes. The period ofspawning is the most important criterion for successof reproduction of various abalone species reared inclose aquaculture system. From manyprevious studies, spawning periods have been foundabalone, and from year to year according to geogra-phical locations, and local environment, such as foodsupply, temperature and the day length. Thus, have classified various spp. into 3 groups according to their spawningseason: those spawn during summer, those spawnduring seasons other than summer, and those thatexhibit year-round spawning. Earlier, Singhagraiwan reported the spawning period of some wildbroodstocks of to peak around October,while the pond-reared broodstocks could spawningthroughout the year with several minor peaks duringMarch through September. In contras

t, the spawning kept in land-based closed culturesystem in the present study occurs twice a year:around August to October and March to April infemale, and around August to November andFebruary to April in male. While this is the generalpopulation, some individual may show irregularperiodic spawning throughout the year, especially gamete cells and the breakdown of connective tissuein the gonads, which is similar to that previouslyH rufescens. According to Shepherd & spent phase is expressed when there is acomplete discharge of gamete cells following defined spent phase in marineinvertebrates as a postspawning quiescent stageIn present study, it was observed that during the are greatlyreduced in size and become creamy in color, andcontrast, hepatopancreas is relatively increased insize which may be filled up with food reserve.From the data collected during one year period,reared in the closed culture system can occur at leasttwice yearly providing that the culturing conditionand food supply are optimal. And that each repro-In previous studies of the gonadal development, fecundity was observed in females withbroodstock, and 44 mm of the hatchery-rearedbroodstock, which was about nine months old.On the other hand, the mature gonad of males The data collected in the presentstudy indicate the same trend. Furthermore, detailedbecome clearly separated from the hepatopancreasat 2 month. Testis and ovary could be distinguishedby the presence of their initial stages of germ cells asearly as 4 month. Testis tends to reach maturitywhich St and Sz are found to be abundant. Ovarytends to mature at 10 to 11 months when it starts tocontain mature oocytes (Oc and Octend to reach maturity and assume reproductive cycleThis investigation was supported by the ThailandResearch Fund (Contract BRG4080004 and SeniorResearch Scholar Fellowship to Prasert Sobhon).1.Nateewathana A and Hylleberge J (1986) A survey on Thaiabalone around Phuket Island and feasibility study of abaloneculture in Thailand. 2.Tookvinart S, Leknim W, Donyadol Y, Preeda-Lampabutra Yand Perng mak P (1986) Survey on species and distribution spp.) in Surajthani, Nakornsrithammarajand Songkhla provinces. Technical Paper No.1/1986, NationalInstitute of Coastal Aquaculture, Department of Fisheries,Ministry of Agriculture and Cooperatives, Thailand. 16p.3.Nateewathana A and Bussarawit S (1988) Abunda

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