Bulletin of the Museum of Comparative Zoology AT HARVARD COLLEGE Vol XLVI1I No THE SPERMATOGENESIS OF SCOLOPENDRA HEROS By Madlsbt W Blackman With Nine Plates CAMBRIDGE, MASS., U.S.A.: PRINTED FOR THE MUSEUM October, 1905 MiCROHLMEP AT HARVARD No — CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE, UNDER THE DIRECTION OF E L MARK, No 170 TJie III Spermatogenesis of the Myriapods TJie Spermatogenesis of Scolopendra heros By Maulsby W Blackman TABLE OF CONTENTS I bulletin: museum of comparative zoology many of the figures were later redrawn and a number of additional obserPractically all of the work upon the metamorphosis of vations made the spermatid was done in the Harvard laboratory It is with pleasiwe that I take this opportunity of expressing my gratitude to Dr E L Mark for much valuable advice and counsel in the work upon Scolopendra, and for his careful reading and criticism of my paper My thanks are also due to Dr C E McClung for his advice and encouragement during the early part of the work Comparatively little cytological work has ever been done upon the to the difficulty of obtaining material, in the myriapods, probably owing required stages, of the more But such common members (Geophilus, Julus) of difficulties are not experienced in working upon This apthe various species of Lithobius, Scutigera, and Scolopendra is certainly not due to any lack of excellence in the neglect parent this group material, for while the cells some of chromosomes are not insects (Acrididae), they are as large as in the still sperm of such size that their may be followed in the minutest detail, and they are in some more favorable than those of other arthropods Then, too, in the study of other problems than those depending on the size and such as the behavior of the cytoplasmic number of the chromosomes, of the and the evolution structures spermatid into the spermatozoon, behavior respects — — - the cells of Scolopendra are decidedly superior to those of any other arthropod I have examined The published works upon the spermatogenesis of myriapods may be, divided into two groups the first comprising the early works by Gilson the second including the later ('84), Carnoy ('85), and Prenant ('87) : ; works by P Bouin (:00, :01, :03), P et M Bouin ('99, Bouin et Collin (:Ol), Collin (:Ol), Meves und von Korff :02, :03), (:Ol), and Blackmail (:01, :03) The work of Carnoy upon the spermatocytes of Lithobius and Scolopendra dalmatica, while in many respects valuable, contains numerous inaccuracies, as These mistakes, also the observations of Gilson ('84) and Prenant ('87) early ('85) forficatus, Geophilus, Scutigera arachnoides, which in many cases are doubtless due to the imperfect technique of the time, have at several points been the cause of errors of interpretation The results of these authors will be discussed later in connection with the observations upon which they have a bearing In 1901 appeared a short paper by Meves und von Korff upon mitosis Their observations are concerned principally with the in Lithobius blackman: the spermatogenesis of scolopendra formation of the spindle and the its peculiar modification in the division of the early prophase the centrosomes During spermatocyte never rest directly upon the nuclear membrane, but remain at a considerable distance from it, and in the later prophase they migrate in first to lie very close to the cell memopposite directions, until they come From these proceed the astral rays, none of which seem to brane connect in later stages with the spindle fibres The linin of the nucleus gives rise to the fibres of the spindle, which, though the poles are truncate, are directed respectively toward the two (now double) centrosomes, The astral fibres radiating often seen in plant cells from those of the spindle remain distinct from the centrosomes entirely a condition that is Concerning the origin of the chromatin the authors have little to say, the earliest stages studied by them the chromosomes were At this stage the " nucleolus " in the form of distinct elements already has already broken up into several fragments, which are colored red by since in the Ehrlich-Biondi stain I should like to mention in this connection that in preparations of three species of Lithobius collected at three widely separated localities, Kansas, Massachusetts, and Bermuda, I have observed that the chromosomes arise from the nucleolus-like body or This is shown in material fixed by various reagents and karyosphere stained by several methods, including Heidenhain's haematoxylin and In a later paper I hope to discuss at the Ehrlich-Biondi triple stain length the origin of the chromosomes In a series of short papers from the University of Nancy, P Bouin, M Bouin, and R Collin have dealt with various problems connected with The first the spermatogenesis of Lithobius, Geophilus, and Scolopendra of these (P et M Bouin, '99) is concerned with the presence and evoThese the lution of certain irregularly formed bodies in the cytoplasm brothers Bouin believe to arise by the breaking later they undergo a down of the astral fibres ; metamorphosis, and in the early Meves und von Korff (:01, p 482), however, find sort of gelatinous prophase disappear these bodies throughout the whole period of mitosis of the first spermatocyte, although during the later stages they break up into smaller granules In several later papers Bouin and his colleagues have described modifiLithobius forfications of the spindle in several species of myriapods to those and similar morsitans catus, Geophilus linearis, Scolopendra — — described by Meves und von Korff Other results of these authors will be mentioned later at various places in this paper bulletin: museum of comparative zoology II Material and Technique The material upon which these observations were made was obtained from Scolopendra heros, the large centipede most common in the southwestern part of the United States The greater part of the work was done upon material obtained from Russel County, Kansas, in June, 1900, through the kindness of Mr W S Sutton Later a number of speci- mens of the through Mr The same variety of S heros were received from Beulah, Colorado, B E Scammon testis of S heros consists of a variable number of follicles lying near the dorsal wall of the body-cavity and communicating with the exterior at the hind end of the body by means of the vasa deferentia Usually the follicles are united in pairs to constitute a lobe, but occasion- Each follicle is in effect a ally there is only a single follicle to a lobe blind tube, its only connection being with the vas deferens The arrangement of the various cell generations within the follicle is from that existing in insects This would naturally be view of the fact that the centipede is a perennial animal and quite different expected in that the testicular elements must therefore be so arranged as to permit the annual regeneration of the organ The extreme periphery of the follicle is occupied by spermatogonia in various stages of so-called rest and In the mature testis these never (Plate 1, Fig 1) of cell division form a continuous layer From the periphery to the centre of the follicle, there are found in the order given (1) young spermatocytes, of : many sizes and stages of growth, " vesicle " (2) spermatocytes in the stage and in various phases of the maturation divisions, (3) spermatids in different stages of metamorphosis, and (4), in the centre of the follicle, mature spermatozoa In a series of preparations made in June, about one half of the volume the follicle is occupied by spermatozoa In preparations of main and the follicles are smaller and terial, captured August September, of the space occupied by spermatozoa is relatively somewhat less It is of note that the of the testis have not all follicles worthy developed at the same rate, so that in the ripe testis all represented In the preparation of the material two stages are abundantly fixing fluids were used : Flem- ming's chromic-osmic-acetic mixture and Gilson's nitric-acetic-sublimate mixture The results with Flemming's fluid, while fairly # good, were so inferior to those obtained rations with Gilson's mixture, that in the later prepathe latter reagent was used exclusively "With Gilson's fluid BLACKMAN : THE SPERMATOGENESIS OF SCOLOPENDRA the fixation in favorable material perceptible shrinkage, and identical with is apparently perfect There is at least the grosser structure of the cells that observed in cells living no is derived from the same source The material was left in this killing fluid for lengths of time varying from twenty-four to sixty hours Probably the best results were obtained with material fixed forty-eight hours, although there is little It is apparent difference due to variations in the length of exposure always well to use a large amount of fluid, thirty to fifty times the volume of the object, and if left for more than twenty-four hours in the fixing reagent, this should be renewed washed for several hours After fixation the objects were running water and then gradually dehy- in drated in alcohol of the grades of thirty per cent, fifty per cent, and To remove the sublimate and prevent the formation of seventy per cent few drops of an alcoholic solution of iodine were added to the seventy per cent alcohol In order to obtain the best results with material fixed in Gilson's mixcrystals, a ture, I have found that it is necessary to employ for infiltrating and embedding, the combined celloidin and paraffin method described in a While this fixation if properly former paper (Blackman, :01, p 62) is nearly perfect, it leaves the tissues very soft, so that, if the ordinary paraffin method is employed, some shrinkage must ineviWith the celloidin-paraffin method this disadvantage is tably occur carried out obviated The sections were cut with the Minot precision microtome, the thick- ness varying from to ruicra, and were affixed to the slide in the ordinary manner by using very dilute albumen water (two drops of Then the paraffin was removed by means of xylol and the sections stained as noted later In the finer work it was found desirable to remove the celloidin also Mayer's albumen to one ounce of distilled water) This for a may be done, either before or after staining, by placing the slide few minutes in a mixture of absolute alcohol and ether In staining the sections a ings number of methods were used and photomicrographs accompanying this paper The draw- were made exclu- sively from sections stained in Heidenhaiu's haematoxylin, either used alone or in conjunction with Congo red For microchemical tests, Bis- mark brown, cyanin, methyl green, Auerbach's raethyl-green-acid-fuchsin, three-color For stain, and numerous other dyes were used Flemming's the study of the chromatin structures the preparations stained with Heidenhain's haematoxylin were the most favorable, although in some bulletin: museum of comparative zoology cases Congo red was of counterstaining with chief value of the latter stain was found in the value However, the work upon the meta- morphosis of the spermatid, where it was nearly indispensable in studyFor in ing the early stages in the formation of the axial filament sections stained with haematoxylin alone the very young axial filament could hardly be distinguished from the cytoplasm, bui, when Congo red stain, the cytoplasm in well-decolorized sections was used as a counter was stained orange-red and thus served as an excellent background I believe that the value of Congo red as a coun- for the black filament ter stain for haematoxylin, when used after Gilson's fluid or other sub- limate-acetic mixture in the study of the spermatid changes, can hardly be overrated III Observations Introductory In studying the spermatogenesis of Scolopendra one is surprised at the striking similarity of the cells in their general appearance and in their behavior to those of the female germ elements of various animals Indeed, it is often the case that, were the cells isolated and mounted by themselves, they would be immediately and invariably mistaken for This resemblance is most striking in the stages stages in oogenesis beginning immediately before the prophase of the first spermatocyte and extending to the completion of the second spermatocyte, but it is also very pronounced even in the earlier phases After the completion of the "division period" the cells, which are at that time very small, immediately begin to increase rapidly in size They continue to grow until finally, when they are ready for the maturation divisions, their bulk is many times that of the spermatogonium This enormous growth suggests of itself a comparison of the spermatoBut there is also a surprising cytes of Scolopendra with egg cells similarity in the structure of the cells themselves as well as in their general appearance By the time the two cells arising from the last spermatogonial division are really separated by a membrane and the nucleus has been reconstructed, the cytosome has already increased conThe chromatin at this stage, as in eggs, is in the form siderably in size number of granular segments distributed irregularly throughout the nuclear space, and upon one side of the nucleus, in close contact with the membrane, there is a chromatin body which in general shape reof a sembles the karyosomes often seen in growing eggs In later stages the BLA.CKMAN: THE SPERMATOGENESIS OF SCOLOPEND1IA spermatocyte resembles the egg cell still more It has become much enlarged, and by its growth the cytosome has increased greatly, and out of proportion to the nucleus Thus, even in size, the spermatocyte at the completion of the growth period resembles the egg The elements within the nucleus have become so arranged that this space is now occupied by a very fine reticulum of granular liuin, which is no more dense than the cytoplasm outside the nucleus and stains in a similar manner All the chromatin has apparently been withdrawn from this network and is now contained in a large, peripherally placed, nucleolus-like body, which stains in such a manner as to leave no doubt wdiatever as to its richness in From chromatin the striking resemblance of the cells at this time to egg cells during the stage of the germinative vesicle I have called this the pseudo-germinal vesicle stage of the spermatocyte, but shall in future " vesicle " call it simply the stage During all in these cells the growth period the centrosomes can generally be found and can, indeed, be identified at all stages from the pro- last spermatogonium up to the formation of the spermatozoon the During growth period they are contained in a centrosphere of slightly modified archoplasm, which is often readily distinguishable from the phase of the remaining archoplasm of the cell Throughout the following division the chromatic and archoplasmic structures behave in a manner similar to that often recorded of egg cells daring the maturation and cleavage Indeed, the division figures resemble those characteristic of cleavage stages much more closely than they the maturation mitoses of eggs This is of course to be expected, since here there is to be an stages equal division of the cytoplasm During the changes following the beginning of the growth period there arise what are apparently two types of spermatocytes These are probdifferences in the environmental surroundconditions ably produced by ing the different cells That they result in the formation of fundamentally different kinds of spermatozoa, is very improbable when we consider their later behavior They, however, show notable differences at various and it is therefore convenient to The recognize the two kinds the larger type resemble egg cells much more closely than those of the small type This is true both as regards general appearance and specific behavior stages, cells of Xot only are the division figures of these spermatocytes very similar germ cell, but the cells arising by these divisions to those of the female are also at first very similar The nuclei of the second spermatocyte like the female pronucleus and the and the spermatids are peculiarly BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY later cleavage nucleus of the egg They are comparatively small vesicles which the chromosomes are closely aggregated and in which the amount of linin is much smaller than in the "vesicle" stage Thus in we see that during all the spermatocyte nomena similar to those characteristic of stages the cells exhibit phethe female germ cell under- going maturation and cleavage What is the explanation of this resemblance In my description of ? the structure of the testis I have given in a general way the position of the parts and the conditions surrounding the cells at various stages I will repeat this description here from The it and see testis is divided into a what conclusions can be drawn number of follicles, each one of connected to the vas deferens by a tube which is continuous with the wall of the follicle at one end (Fig 1) The lumen of this duct which is is continuous with that of the length of this structure and treme periphery of each follicle, is filled which extends nearly the entire with spermatozoa Upon the ex- are arranged the spermatogonia, either in the resting stage or in various phases of mitosis Next to these are from to centre in sequence disposed periphery (1) spermatocytes in various stages of growth ; (2) first spermatocyte in the vesicle stage and follicle : in various phases of the first maturation division ; (3) second spermato- (4) spermatids; (5) young spermatozoa; and lumen, mature spermatozoa cytes in various stages; (6), in the These different cells in various stages are distributed in more or less There are, to be sure, many irregularities in this distriregular layers bution, the most pronounced appearing in follicles which are far advanced Here the closely packed spermatozoa fill the lumen development and show a tendency near the middle of the follicle to encroach upon the younger cells, so that these are frequently forced aside, and the mass of in spermatozoa thus comes more or less close to the follicular sheath However, the most typical arrangement is that which I have (Fit, 1) r mentioned This is especially cytes, which, as I shall manner show marked for the larger type of spermato- later, are always arranged in a definite in the follicle In the interstices between the cells, there is present at all stages a substance of a more or less viscid consistency, which very probably serves In testes which have been fixed this material consists as nourishment of a basis which in general structure very much resembles cytoplasm is not, however, of the same nature as cytoplasm, This reticular substance its reaction to various stains being different affinity for It does not show a strong iron-haematoxylin nor for other similar stains, but is colored Bigelow — Nuclear Cycle of Gonionemus murbacbii PLATE All figures are magnified 2600 diameters Figures 92-108 and 120-123 from isolated cells figures 109-119 from sections ; Fig 92 Spermatid Fig 93 Spermatid The The interzonal remnants have dwindled to a small sphere axial filament extends inward from the centrosome divided, and the halves lie The centrosome has the cell Thenucleus in main the greatest size The interzonal remnants have disappeared, hut there are two arclioplasmic masses lying one on either side of the axial filament axis of has attained its Fig 95 Spermatid The inner centrosome has nearly reached the nucleus it is connected with the outer centrosome hy the axial filament In this cell the interzonal remnants still persist Spermatid The inner centrosome has reached the nucleus and become Fig 96 Metamorphosis Fig 94 ; flattened against it The tail has now grown to a conof the spermatid siderable length the acrosome has appeared at the anterior pole of the nucleus The chromatin is collected in several masses ; The acrosome is a sphere of archoplasm The chromatin has become diffused so that the nucleus is uniformly deeply stained The two arclioplasmic masses have grown and occupy most Fig 97 Slightly later stage Fig 98 The same Fig 99 two arclioplasmic masses nearly cover each other, one lying at high, the other at low focus, and the axial filament running between them Later stage The nucleus has assumed a conical form, and shows a of the space between the nucleus and the posterior margin of the cell stage, seen in a plane perpendicular to that of Figure 97 The Fig 100 deeply stained basal plate Polar view of this same stage Fig 101 Still later stage Fig 102 Adult spermatozoon Fig 103 arclioplasmic masses Giant spermatid with two centrosomes and two tail filaments Fig 104 The cytoplasm has become vacuolated The middle piece consists of three separate Giant spermatid with three centrosomes and three tail filaments Later stages in the metamorphosis of giant spermatids, showing evidences of degeneration Figs 105, 106 Fig 107 Fig 108 Multiple spermatozoon with two nuclei, two centrosomes, and two tails Multiple spermatozoon with two nuclei, three centrosomes, and three tails " Fig 112 Resting" stage of oogonium The nucleolus is compound Early prophase of oogonium The karyoplasm has disappeared, and the karyosomes increased in size Prophase of oogonium, showing contraction Prophase The karyosomes have partly condensed to form chromatin Fig 113 Prophase Fig 114 attached to the margin of the persistent plasmosome Later prophase The nucleolus has disappeared The chromatin Fig 109 Fig 110 Fig 111 segments are The nucleolar shell has broken down, but chromatin masses still condensed into segments of the reticulum is Bioelow Fig 115 — Nuclear Cycle of Gonionemus murbachii Still later prophase The nuclear membrane has broken down eral dumb-bell-shaped Fig 116 chromosomes have been formed by the Sevcon- densation of the chromatin segments The chromosomes are dumb-bell-shaped; polar view Metaphase ; Fig 119 them have divided Most of the chromosomes are in process of splitting The chromosomes appear as spheroidal Metaphase in side view masses The centrosomes are now visible Most of them Anaphase The chromosomes form daughter plates Fig 120 preserve their orientation parallel to the plane of future cell division Stout interzonal filaments connect the two daughter groups " " Resting stage of oocyte The nucleolus is compound several of Fig 117 Fig 118 Fig 121 Fig 122 Fig 123 Late metaphase Early pseudoprophase of oocyte Pseudoprophase Several chromatin segments have been formed by the coalescence of the karyosomes Later stage of the same The nucleolar shell has partly broken down BlGELOW - Nuclear Cycle TE Gomonemus 105 •»•• tt*&*t ***** 117 116 115 121 120 H.B.B del 118 119 123 7i 7S- oc / Btgelow — Nuclear Cycle of G-onionemus murbachii PLATE Figures 124-130 from isolated cells; figures 131-139 from sections Figs 124-126 Late pseudoprophase The nucleolar shell has broken down, The chromatin is entirely leaving a persistent plasmosome Fig 127 " " condensed into beaded segments X 2600 The chromatin segments are irregular of Regression pseudoprophase and have commenced to break down X 2600 Fig 128 Later stage in regression The chromatin segments have largely broken down The plasmosome has grown much larger X 2G00 Fig 129 " " Resting olus Figs 130-138 Fig 130 The is stage of oocyte, following the pseudoprophase 2600 a homogeneous structure Growth stage nucle- of the oocyte become vacuolate, and an accessory nucleolus The chromatin is finely diffused and has largely chief nucleolus has has been formed lost its staining capacity Fig 131 The X Later stage X 900 The chromatin, which has regained a large number of angular masses of its affinity for stains, granules Both chief and accessory nucleoli, as well as the nucleus as a whole, have increased greatly in size X 900 consists of Fig 132 The chromatin masses have become arranged in which are in the form of Y's and Vs X 900 Section of late stage in the growth period, showing segments of numerous chromatin strands The chief nucleolus contains five vacuoles Still later strands, Fig 133 X Figs 134, 135 stage many of 900 Chief nucleolus of oocyte Fig 136 2600 Chief nucleolus, containing many X Fig 137 Fig 138 The ground X stained strands substance contains deeply deeply stained strands and granules 2600 Chief nucleolus, ordinary type, containing vacuoles X 2600 The walls of the vacuoles are deeply stained Chief nucleolus X 2600 BlGELOW.— Nuclear Cycle Gomonemus 124 125 127 130 12 •/ 134 vVtS % - • • * 137 /3J • *» • V * * •• : a ;:? - » * /T'£: Jet ** w'vt 132 129 /j/ E del Bioelow — Nuclear Cycle of Gonionemus murbachii PLATE All figures are from entire eggs stained in borax carmine The egg nucleus is in the metaphase of the second The sperm structure consists of head and maturation division Fig 139 Fertilization Fio 140 Fertilization Fio 141 The sperm middle piece, and surrounded by astral radiations is The egg nucleus division The middle X 500 anaphase of the second maturation piece of the sperm structure has disappeared, and the astral radiations focus at some distance from the sperm nucleus aster X is in the 500 has divided, and the two resultant asters lie at A supernumerary spermaopposite sides of the sperm nucleus tozoon is attached to the vitelline membrane The egg nucleus is anaphase of the second maturation division X 500 polyspermy Each supernumerary sperm nucleus is accompanied by an aster One sperm nucleus has joined the egg in the Fig 142 Fig 143 Fig 144 Fig 145 Fig 140 An egg showing X 500 nucleus, and its two asters lie on either side of the latter Union of the germ nuclei The sperm asters lie in the plane of union " The egg nucleus is in the reticulate " resting condition X 1780 Union of the germ nuclei The sperm nucleus, which lies in a clear The sperm area, has increased in size, and become more spongy asters lie on opposite sides of the egg nucleus X 1780 Later stage of the same The sperm nucleus forms a cap over part of the egg nucleus, and has become less dense X 1780 Fusion of the germ nuclei The sperm nucleus has broken up into a large number Fig 147 The egg chromatin forms and both maternal and paternal chromatin of small chromatin masses an irregular threadwork ; are enclosed within a single membrane X 1780 Later stage in fusion The maternal and paternal chromatin form threadworks which are no longer distinguishable, though the fusion nucleus still has a constricted form X 1780 BlGELOW.— Nucle ar Cycle Gonionemus Plate *« 139 141 143 He 147 145 YV 144 142 B-B del 140 Bioelow — Nuclear Cycle of Gonionemus murbachii PLATE All figures are from entire eggs stained in borax carmine Fig 148 Union of the germ nuclei by apposition In the egg nucleus the chromatin forms a threadwork in the sperm nucleus it consists of many ; Fig 149 small masses connected with one another by linin strands X 1780 Union of the germ nuclei by apposition The two nuclei are of nearly X 1780 equal sizes and are both in the reticulate resting condition Fig 150 First cleavage nucleus Fig 151 Fig 152 formed by the fusion of the germ nuclei The chromatin forms an irregular threadwork, in which the maternal and X 2600 paternal components are not distinguishable Prophase of first cleavage The nuclear membrane has broken down, and the chromatin forms separate segments X 2600 Late prophase of first cleavage There are twenty-four chromosomes formed by the condensation of the chromatin segments Maternal and paternal groups are not distinguishable X 2600 Fig 153 Metaphase of first cleavage spindle The chromosomes are somatic number The asters are small X 1780 Fig 154 Oblique polar view of daughter plate of first cleavage, showing the somatic number of chromosomes X 2600 Fig 155 Metaphase of second cleavage The chromosomes are number The asters are small X 1780 Fig 156 Anaphase daughter X of second cleavage The asters are much in in the full a reduced larger, chromosome groups form minor centres of full and the radiation 1780 Fig 157 Polar view of daughter chromosome group of second cleavage There are twelve bivalent chromosomes, several of which show their double Fig 158 Polar view of daughter chromosome group of second cleavage nature by their constricted outlines X 2600 are nine chromosomes which are clearly bivalent are probably univalent 2600 ; There the remaining five X Fig 159 Side view of fourth cleavage spindle in the metaphase somes are present in the full somatic number X 1780 Fig 160 Polar view of daughter chromosome There are twenty-four chromosomes group X 2600 of The chromo- fourth cleavage BlGELOW - Plate Nuclear Cycle Gomonemus m 148 149 151 152 154 • •• • 157 158 ISO « 155 153 f i * ;^