IL 
 
 J C49UJ 
 ser.2 
 no, 21 
 
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 University of Cincinnati 
 
 -BulletiruNo. 21. 
 
 Nov. J 902. 
 
 Publications of the University of Cincinnati, 
 
 Series II. Vol. II. 
 
 Hybridism and the Germ-Cell. 
 
 M. R GUYER, Ph. D. 
 
 The University Bulletins are Issued Monthly 
 
 Entered ?,t the Post Office at Cincinnati, Ohio, as second-class matter 
 
University of Cincinnati 
 
 Bulletin No, 21. Nov. 1902. 
 
 Publications of the University of Cincinnati, 
 Series II. Vol. II. 
 
 Hybridism and the Germ-Cell. 
 
 M. F. GUYER, Ph. D, 
 
 The University Bulletins are Issued Monthly 
 
 Entered at the Post Office at Cincinnati, Ohio, as second-class matter 
 
$c 
 
 c < 
 
 Hybridism and the Germ-CelL 
 
 MICHAEL F. GUYER. 
 
 The writer prepared a paper on "The Spermatogenesis of 
 Hybrid and of Normal Pigeons" in May, 1900, and placed it 
 with The Journal of Morphology. On account of the temporary 
 suspension of that periodical it is deemed desirable to publish 
 an abridged account of the results of the investigation,* leaving 
 details to appear in the original paper whenever The Journal 
 shall resume publication. The chief interest of the investiga- 
 tion centers in the peculiarities met with in the transformation 
 of the germ-cells of hybrid pigeons, hence the present paper will 
 emphasize this phase of the subject. 
 
 The germinal cells of the male pigeon are laid down in a 
 great number of delicate convoluted tubules, which wind back 
 and forth throughout the interior of each testis and make up its 
 main bulk. Near the periphery of each tubule are the sperma- 
 togonia, or parent cells (Fig. 1, sg.), which through growth and 
 division give rise to the various generations of germ-cells lying 
 inward toward the lumen of the tubule. The adult spermatozoa 
 are formed through the final transformation of the spermatids, 
 or cells which lie nearest the center of the tubule, the product 
 of the last cell-division. As in many forms, the spermatozoa 
 attach themselves to a supporting cell (Fig. 1, s.) for a period 
 before their complete maturation and ejection from the testis. 
 
 The usual four phases or types of the germinal cells are 
 recognizable, viz.: (1) spermatogonia (Fig. 1, sg.), a more or 
 less regular layer of cells lying next to the wall of the tubule, 
 each cell of which through division gives rise to two new cells. 
 
 * The original paper is the thesis submitted by the writer as a 
 candidate for the degree of Ph.D., at the University of Chicago. It 
 was accepted by the faculty in March, 1900. A few copies of a 
 limited edition of the thesis, published by the author, are available 
 for investigators who are specially interested in the particular 
 prcblems under discussion. Address the writer at the University 
 of Cincinnati. 
 
 3 
 
One or both of these may increase in size and become (2) pri- 
 mary spermatocytes (Fig. 1, scy. 1), or remain in the layer and 
 continue as spermatogonia. The primary spermatocytes, after 
 some interesting changes, divide to form (3) the secondary sper- 
 matocytes (Fig. 1, scy. 2), which divide again shortly and give 
 rise to (4) the spermatids (Fig. 1, st.), through the transforma- 
 tion of which the spermatozoa are developed. The number of 
 chromosomes in each type as seen at the equator of the spindle 
 before division is, in the spermatogonia sixteen loops, in primary 
 spermatocytes eight rings, and in secondary spermatocytes four 
 rings. The nurse cells, or Sertoli cells, to which the sperma- 
 tozoa become attached at one period of their transformation 
 (Fig. 1, s.), are irregularly disposed among the other cells. 
 
 THE SPERMATOGONIA. 
 
 The spermatogonia lie in a more or less regular layer along 
 the wall of the tubule. In early stages they are far apart and 
 possess small nuclei, which are oblong with the long axis par- 
 allel to the tubule wall. The cell boundaries are at first very 
 indistinct or seemingly absent, and gaps frequently intervene 
 between the individual spermatogonia, so that the latter seem 
 to have been left behind from a preceding set, or to have 
 recently settled in their present position. In later stages the 
 cells are crowded together till they become columnar in shape, 
 while the nuclei increase in size and become very distinct. A 
 condensation of the cytoplasm., or mass of sphere substance 
 (idiozome of Meves), makes its appearance, and gradually in- 
 creases in size till it becomes a well defined area (Fig. 2, i). 
 Near the center of this mass is generally a clear area, in which 
 a minute centrosome (c) is discernible. A nucleolar-like mass 
 is usually visible within the nucleus, but judging from its reac- 
 tions to various stains, it is nothing more than a clump of the 
 same material that composes the linin. This mass usually takes 
 part, together with the linin network, in forming the achromatic 
 sheath within which each individual chromosome is incased 
 when ready for division. Before division the nucleus passes 
 through an incomplete spirem stage. The spirem breaks up 
 into individual chromosomes, which are visible as irregular 
 threads and loops scattered throughout the nucleus. A filament 
 two or three times as long as the other chromatic bodies is to 
 
be seen at times. Before the formation of the spindle for the 
 ensuing cell division, this body is ejected into the sphere sub- 
 stance, where it seems to ultimately break up and become scat- 
 tered throughout the cytoplasm. The significance of this phe- 
 nomenon could not be determined. It seems improbable at 
 present that the extruded body can be homologized with the 
 ''accessory chromosome" of McClung 1 , a curious nuclear ele- 
 ment, which he describes as occurring in Xiphidium fasciatum, 
 one of the Locustidae. The " accessory chromosome," accord- 
 ing to his account, does not disintegrate and disappear, but 
 retains its individuality, and persists throughout the entire 
 period of spermatogenesis, to take part finally in the formation 
 of the spermatozoon. 
 
 After the formation of the chromosomes, the centrosome, 
 which lies in a clear area of the sphere, divides into two, one of 
 which moves along the outer periphery of the nucleus to the 
 opposite pole. The first appearance of the spindle fibers is as 
 radiations which spread around the nucleus from the centro- 
 somes. When the mitotic figure is fully formed, the spindle is 
 short and broad and the chromosomes lie in a confused band at 
 the equator (Fig. 3). The individual chromosomes are loop- 
 shaped, with the closed end of the loop toward the center of 
 the spindle. While moving toward the poles after division, not 
 infrequently the free ends of a chromosome fuse to form a small 
 ring. After repeated attempts at counting, it was determined 
 that sixteen chromosomes are present at the equator of the 
 spindle before division. 
 
 PRIMARY SPERMATOCYTES. 
 
 The primary spermatocytes originate from the cells of the 
 last spermatogonial division through a process of growth. The 
 chromatin passes into the resting condition, and an increase in 
 bulk of both the nucleus and the cytoplasm begins. The sphere 
 first appears as an indistinct granular crescentic area closely 
 applied to the nucleus, with the horns of the crescent so ex- 
 tended as to inclose more than half of the nuclear surface. As 
 the young spermatocyte grows, the sphere also increases in 
 
 1 McClurg, E. C. A Peculiar Nuclear Element in the Male 
 Reproductive Cells of Insects. Zool. Bui. II. 4, 1899. 
 
size, becoming more and more rounded. From an early stage 
 a minute centrosome is visible in the midst of the sphere sub- 
 stance. It is surrounded by a clear area, which becomes more 
 pronounced as the sphere grows older. Thus the developing 
 cell gradually acquires characteristics of size, shape and general 
 appearance, that differ markedly from those of the previous 
 generation. 
 
 Synapsis. — Synapsis occurs in the primary spermatocytes, 
 during which there is a marked drifting of the chromatin to the 
 side of the nucleus in contact with the sphere (Fig. 4). Some 
 substance from the nucleus apparently passes out into the 
 sphere: it may possibly be concerned in the formation of the 
 extremely coarse-fibered spindle, for almost immediately the 
 centrosome divides and the spindle appears. In the ensuing 
 division of the spermatocyte only eight chromosomes are pres- 
 ent, but they are in the form of heavy rings, and are evidently 
 bivalent (Fig. 7). 
 
 During division the eight-ring chromosomes, which are 
 incased in capsules of linin, break transversely, and as they 
 move apart remain connected by threads of the linin casing. 
 These threads constitute the interzonal fibers (Fig. 8, if). An 
 intermediate body is present at the equator of the interzonal 
 fibers and marks out the path of the new cell walls (Fig. 8, ib). 
 The ring type of chromosome seen at this division is formed 
 through the breaking up or rearrangement of the prominent 
 spirem (Fig. 5), which forms immediately after synapsis. The 
 spirem-like appearance inside the nucleus disappears gradually, 
 until by the time the centrosomes reach their positions at oppo- 
 site poles of the nucleus, the components of the spirem are seen 
 as eight elongated, irregular rings (Fig. 6), which consist of a 
 linin groundwork, in which are imbedded numerous granules 
 and lumps of chromatin. The rings gradually condense into 
 a shorter, heavier type, and the chromatin fuses in such a way 
 that distinct granules are no longer visible. In a few instances 
 rings were found to consist of four more or less spherical, 
 densely staining areas, connected by lighter bands of linin. It 
 is possible that this is comparable to the tetrad formation so 
 frequently observed in maturation phenomena (Fig. 6, tr). 
 
SECONDARY SPERMATOCYTES. 
 
 The product of the division just discussed consists of two 
 cells, each of which is considerably smaller than the primary 
 spermatocyte, and which never attains to its volume. These 
 cells are the secondary spermatocytes. They go into a resting 
 stage, which is of very short duration. When the secondary 
 spermatocyte is ready for division, curiously enough only four 
 chromosomes appear (Fig. 9). They are of the same shape 
 and size as those in the division of the primary spermatocyte. 
 In dividing, the chromosomes each break in such a way that a 
 stringing out of the sheaths of the chromosomes gives rise, as 
 in the primary spermatocytes, to a system of interzonal fibers, 
 which, as division proceeds, constrict at the equator to form 
 a large intermediate body. For some time after division the 
 divided chromosomes are seen as four hollow vesicles within 
 the daughter nuclei. They fuse later, ordinarily, into one large, 
 hollow sphere of chromatin near the center of the nucleus 
 (Fig. 10). Numerous fine fragments of chromatin migrate to 
 the nuclear membrane, which has appeared in the meantime, 
 and form a thin shell of chromatin along its inner surface. The 
 centrosome (Fig. 10, c) persists, and together with the tip of 
 the spindle moves out into the cytoplasm. The tip of the 
 spindle seemingly becomes re-converted into sphere substance. 
 
 THE SPERMATID AND ITS TRANSFORMATION. 
 
 The new cell formed from the division of the secondary 
 spermatocyte is the spermatid, and is the cell which will ulti- 
 mately be transformed into the spermatozoon. An adult sperma- 
 tozoon as it exists in the vas deferens of the pigeon is shown in 
 Fig. 14. The head is long and narrow, and is intensely stained 
 by nuclear dyes. Favorable preparations show the chromatin 
 arranged in a series of vesicles within the head. Each vesicle 
 of this chain-like series incloses a clear area, which in some 
 preparations appears highly refractive. A remarkable fact is 
 that the number of vesicles is apparently the same as the re- 
 duced number of univalent chromosomes should be, namely, 
 eight. In some instances, where only six or seven vesicles were 
 present, it was observed that one or two were unusually large, 
 and hence probably equivalent to two. It will be recalled that 
 
in the secondary spermatocyte there were only four chromo- 
 somes, but that they were of the bivalent type, or really com- 
 parable to eight ordinary chromosomes. There is no positive 
 evidence that the vesicles in the head of the spermatozoon cor- 
 respond to individual chromosomes, but the striking coincidence 
 in number is at least very suggestive, and it would not be sur- 
 prising if the fact develops later that after the entrance of the 
 spermatozoon into the egg the vesicles resolved themselves into 
 eight distinct chromosomes. 
 
 At the anterior end of the head is a slender, fine-pointed 
 head-spine. The head posteriorly connects directly with the 
 long cytoplasmic tail. No middle piece is visible. The tail and 
 the head-spine are very difficult to observe accurately, and but 
 little of the details of their structure could be worked out. The 
 only way to gain a satisfactory knowledge of the spermatozoon 
 at all is through a study of its development. 
 
 In the transformation of the spermatid to form the sper- 
 matozoon the first change to be observed is in the centrosome. 
 It divides, and one of the resulting centrosomes enlarges and 
 becomes ring-shaped (Fig. n, c). The axial filament of the 
 tail first appears as a thread connecting the two centrosomes, 
 and later continues backward through the ring-like centrosome 
 and out of the cell (Fig. n, ax). The smaller centrosome, 
 together with material of cytoplasmic origin, finally comes to He 
 within the nuclear membrane. It may be regarded perhaps as 
 a middle piece, which becomes obscured by a covering of chro- 
 matin, and consequently appears to be absent in the adult 
 spermatozoon. 
 
 The long head of the spermatozoon is the transformed 
 nucleus. In the process of elongation only what may be termed 
 the anterior and the posterior ends of the nucleus extend at 
 first, but in a short time the entire nucleus begins to narrow. 
 At the same time the mass of chromatin at the center sprouts 
 out both anteriorly and posteriorly to form a central, thread- 
 like core (Fig. 12). As the process of elongation continues, a 
 narrowing of the sides of the nucleus takes place to some extent, 
 but when one takes into account the enormous elongation that 
 occurs, together with the relatively slight diminution of the 
 transverse diameter, it becomes evident that there must be con- 
 siderable increase in the volume of the nucleus. The heavy cen- 
 tral chromatic filament after a time becomes arranged in a wavy 
 
or spiral manner. As the transformation progresses the spiral 
 design, although often very irregular, becomes more perceptible. 
 A splitting of this spiral core finally occurs, and thereafter the 
 chromatin exists as two threads laid down in an irregular double 
 spiral (Fig. 13). 
 
 The elongation of the nucleus ceases at about the time the 
 bisection of the central filament has been accomplished, and 
 the nucleus displays itself as an enormously long, sinuous head, 
 which may measure twice the length of the head of the adult 
 spermatozoon. A dense protoplasmic mass encases it and 
 extends backward along the axial filament. A shrinkage of 
 the nucleus follows, in the course of which the double spiral of 
 chromatin shortens and widens until the exact relationship of 
 the chromatin of the two filaments can no longer be deter- 
 mined. The final appearance is that of a chain-like series of 
 vesicles, as described for the mature spermatozoon (Fig. 14), 
 the clear area in the center of each vesicle corresponding to 
 the openings between the respective points of intersection of 
 the two spirals of chromatin. 
 
 The head-spine originates from a bubble-like mass of mate- 
 rial (Fig. ii', v) which arises in the sphere. This bubble or 
 vacuole moves slowly around the periphery of the nucleus until 
 it lies at the pole opposite the point at which the centrosome, 
 which marks the anterior end of the axial filament, will enter 
 the nucleus. 
 
 the; gekm-ce)ll,s op hybrid pigeons. 
 
 In the pigeon some crosses are fertile, others are not. The 
 sterile hybrids show a greater or less degeneration of the germi- 
 nal cells. The general rule seems to be that the more divergent 
 the parent forms, the more marked is the degeneration of the 
 germinal cells. From parents which differ widely in structure 
 or habits there seems to be much greater difficulty in securing 
 female than male offspring. I have been able to obtain but one 
 female offspring of very distinct species for microscopical exam- 
 ination, while, on the other hand, I have had six males. For 
 all of this material I am indebted to Professor Whitman. From 
 the testis of the offspring of the common ring dove, Turtitr 
 risorins, and the white ring dove, Columba alba, a large number 
 of sections were made for microscopical study. These two 
 
forms are perfectly fertile when crossed, and the fertility of 
 their offspring seems in no wise diminished. The germ-cells 
 show some of the same phenomena as those of the sterile birds, 
 though in a less marked degree. 
 
 The common brown ring dove when crossed with the white 
 ring dove produces hrowm offspring. One member of the re- 
 sulting pair is usually a few shades lighter in color than the 
 other. In the next or third generation there is generally a 
 return to the original colors of the grandparents ; one of the 
 young is white, the other brown. There is a marked tendency 
 for the white ones to be female and the brown ones male, this 
 being true, at least of the nine pairs killed by the writer. Occa- 
 sionally in the third generation both of the young are white or 
 both brown. 
 
 Of the sterile hybrids, whether male or female, the sexual 
 products were abnormal. The abnormalities of male hybrids 
 may be classified conveniently under three heads': (i) Abnor- 
 malities in mitosis ; (2) abnormalities in the structure of the 
 spermatozoon ; (3) degeneration of the germinal cells. Not all 
 hybrids show these various irregularities in the same degree. 
 All three kinds of the phenomena just mentioned are observ- 
 able in the sterile forms, but the fertile birds differ, for the 
 most part, from normal pigeons only in the slightly irregular 
 character of the mitosis. 
 
 The abnormalities in -mitosis arc in the nature of multipolar 
 spindles and asymmetrical division and distribution of the chro- 
 mosomes (Figs. 15-19). They are more pronounced in sterile 
 birds, but may be met with in fertile hybrids also. It is a 
 curious fact that the multipolar spindles are confined largely to 
 the primary spermatocytes, and one is inclined immediately to 
 associate the fact with the pseudo-reduction or formation of 
 bivalent chromosomes, which occurs normally at this stage of 
 spermatogenesis. Figs. 15-18 show some of the different forms 
 of multipolar spindles. The tripolar types are by far the most 
 common. Fig. 15 represents perhaps the most prevalent struc- 
 ture. It was not unusual to observe two spindles in one cell, 
 as shown in Fig. 16. When two such spindles exist independ- 
 ently in one cell, they may each have a small number of the 
 large bivalent ring-form chromosomes, or a greater number of 
 small chromosomes, which are apparently univalent. More 
 rarely both large and small chromosomes occupy one or both 
 
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of the spindles. The facts indicate that a pseudo-reduction has 
 not occurred, or that it is incomplete or abnormal. What has 
 just been said regarding the chromatin arrangement, where two 
 separate spindles occur, is equally applicable to the multipolar 
 forms, only there is generally more variation in the size of the 
 -chromosomes. In a tripolar type like Fig. 15 the chromosomes 
 are almost invariably numerous and of small size. 
 
 In a few instances two nuclei were present in the primary 
 spermatocyte, and it seems probable that such cells give rise to 
 the multipolar spindles, bearing an excessive amount of chro- 
 matin, which are sometimes seen. Fig. 18 shows a tripolar 
 spindle in a primary spermatocyte, where there is much variation 
 in the size of the chromosomes. 
 
 An asymmetrical distribution of chromatin results, of 
 course, in many cases where the division is by means of multi- 
 polar spindles, but in addition to this there is very frequently 
 an unequal division of the chromosomes themselves. This 
 occurs as often where the spindle is single as in any other case. 
 In dividing, perhaps only one-quarter of a chromosome will go 
 to one pole and the other three-quarters to the opposite pole, 
 or the division may be such that a portion of the chromatin is 
 cut out entirely and left behind in the cytoplasm. Fig. 19 rep- 
 resents a secondary spermatocyte, of which one chromosome 
 is very minute, as if part of its material had been lost in the 
 preceding division. 
 
 In two or three instances one of the large chromosomes of 
 the primary spermatocyte was observed to be made up of four 
 small rings. Whether this indicates an exaggerated demarca- 
 tion into tetrads it is impossible to affirm, though the fact is a 
 significant one. 
 
 Abnormalities in the structure of the spermatozoa are present 
 in sterile hybrids. There is a curious varicosity about the 
 middle of the spermatozoon head in such forms that attracts 
 the attention immediately, when the objects are examined under 
 the microscope (Fig. 20). This enlargement seems to be almost 
 universal amongst the spermatozoa, and is sufficient of itself to 
 produce sterility, for such a malformation would prevent its 
 possessor from entering the egg. In a very few instances what 
 appears to be a normal spermatozoon may be observed among 
 the misshapen ones, and it is possible that if these reached a 
 
 11 
 
suitable egg, fertility might result. Their chance of meeting:; 
 with the egg, however, is very slight. 
 
 A study of the development of these deformed spermatozoa- 
 reveals the fact that the bead-like enlargement results from the 
 incomplete development of the nucleus in the formation of the 
 head. The two ends of the nucleus sprout out, as it were, and 
 grow for a short distance, but the remainder of the nuclear-wall 
 retains its original form and position. The arrangement of the 
 chromatin is very irregular. A deeply staining mass is visible 
 in the bulb-like swelling, from which thick filaments spring out 
 forward and to the rear. 
 
 Degeneration of the germinal cells was in progress in the testes 
 of all sterile forms, but was most pronounced in hybrids from 
 birds which were of very divergent species, or hybrids which, 
 were themselves descendants of fertile hybrids. There were 
 some cases of such extreme degeneration that only the layer of 
 cells lying along the wall remained in the tubule. Where such 
 a degree of degeneration exists there is of course no approach 
 to the formation of spermatozoa. There is often a strong inva- 
 sion of wandering cells into the tubules, especially where the 
 degenerative activities have become extensive. The inter- 
 spaces between such tubules are also usually packed with cells 
 which resemble white blood corpuscles. In some preparations 
 it looks really as if the germinal cells themselves lose their walls 
 and characteristic appearance and become leucocytes, though 
 no definite conclusions could be reached regarding this point. 
 Some tubules are occupied almost entirely by cells which have 
 the exact appearance of the large stroma cells present outside 
 the tubules. 
 
 The primary spermatocytes seem to be the cells most sus- 
 ceptible to decay. Frequently the nuclear contents have a 
 watery and disintegrated appearance and the sphere substance 
 is marked by the presence of a large vacuole in its center. In 
 testes where degenerative processes are pronounced a number 
 of cells may run together to form a giant cell, in the center 
 of which is an enormous vacuole surrounded by the nuclei 
 of the original cells. The mass thus formed is very similar in 
 appearance to the giant cells found in many pathological tissues. 
 
 A detailed description of the individual hybrids will not be 
 entered into at present, but will be left to the original paper. 
 The crosses were : (i) Male black tumbler, female brown ring 
 
 12 
 
dove ; (2) male black tumbler, female brown ring dove ; (3) 
 male hybrid from white and brown ring dove, female homer; 
 (4) male wild passenger pigeon, female brown ring dove; (5) 
 male wild passenger pigeon, female brown ring dove ; (6) male 
 turtle doYQ, female Japanese turtle dove. The hybrid from the 
 last named was a female. The ovary and oviduct were rudi- 
 mentary. Occasional small ova with an incomplete follicle were 
 present. The largest egg had attained to a diameter of only 
 seventy-five micra ; (7) numerous crosses of white and brown 
 ring doves. 
 
 CONCLUSIONS FROM TH£ STUDY OF HYBRIDS. 
 
 It appears that in crosses of very divergent species the 
 degenerative processes in the germ-cells are at a maximum. 
 In closely related forms like the brown and the white ring 
 doves fertility is not diminished, and the testes seem to be 
 normal, except for occasional irregularities in mitoses. 
 
 The formation of multipolar spindles in division and the 
 unequal distribution of chromosomes seen in many instances 
 are among the most interesting phenomena presented. Such 
 irregularities, however, occur to a slight extent in normal birds. 
 A very careful study reveals the fact that they are more preva- 
 lent in the ordinary dove-cot pigeons than in pure breeds of 
 doves, which have bred true for many generations. 
 
 That the irregularities of division in hybrids are due to 
 degenerative processes going on in the tubules, which give rise 
 to deleterious chemical substances, is the first thought that pre- 
 sents itself. This seems a very acceptable idea from what we 
 know oi the effects of drugs upon cell division, but it does not 
 account for the fact that the primary spermatocytes are the 
 cells attacked in the great majority of cases. 
 
 A hybrid offspring is really a compound of two very dif- 
 ferent individual plasmas, hence conflicting tendencies must nec- 
 essarily have been induced within its body. The abnormality 
 in division may be but an attempt of each plasma to assert its 
 individual activity. But why does this effort become apparent 
 only in the primary spermatocytes? The answer appears to be 
 simply that there is no necessity for fusion of the components 
 of the chromatin of cells except in the germ-cells at one stage 
 of their maturation. Investigators have found that in matura- 
 
 13 
 
tion of germ-cells a reduction of the ordinary number of chro- 
 mosomes to one-half occurs. Before this actual reduction there 
 is ordinarily a so-called pseudo-reduction, in which the chromo- 
 somes fuse in pairs, so that when the cell is ready for division, 
 although only half of the regular number of chromosomes 
 appear, each is really double (bivalent), and equivalent to two 
 of the simple (univalent'! type. In spermatogenesis this change 
 generally comes about in the primary spermatocyte. Now, in 
 hybrids it may be supposed that in the ordinary cells of the body 
 the chromosomes from the paternal and the maternal species 
 lie side bv side and carry on the customary functions of the 
 cells, but when it comes to an actual fusion of chromosomes 
 to form the bivalent type necessary for reduction, the incom- 
 patibility of the two different plasmas renders the union incom- 
 plete or prevents it entirely. That the ordinary somatic cells of 
 hybrids, either plants or animals, are under the influence of two 
 distinct tendencies is well shown in the decided mosaic-like 
 structures which frequently occur, the classic figure being to 
 liken hvbrids to warp and woof. To cite but one example, 
 Macfarlane found that a hybrid of the gooseberry and black 
 currant, instead of being a strictly intermediate type, really pos- 
 sessed, side by side/organs characteristic of each parent; the 
 leaves bore both the shield-shaped, oil-secreting hairs of the 
 currant and the simple hairs of the gooseberry, though each 
 hair was but half the size of the parent type. We may infer, then, 
 that in certain hybrid pigeons the univalent chromosomes from 
 each of the parents may lie side by side in the ordinary cells of 
 the body and divide normally, but when it comes to the period 
 of fusion in the germ-cell, they will not unite to form the biva- 
 lent type, or else they unite incompletely. The result is that in 
 the primary spermatocyte, instead of one spindle bearing eight 
 bivalent chromosomes, a multipolar spindle, or not infrequently 
 two separate spindles, bearing two groups of univalent chro- 
 mosomes, may appear. In cases where both large and small 
 chromosomes are seen, it is necessary to suppose that a loose 
 union has occurred in some chromosomes. The unequal divis- 
 ions of the bivalent chromosomes of many hybrids indicate that 
 such chromosomes have in some way been rendered very 
 unstable. 
 
 If we accept the view that chromatin is a substance capable 
 of varying in qualities in the different regions of the chromo- 
 
 14 
 
some, then in fertile hybrids, where irregular mitoses occur, the 
 different germ-cells will certainly not be qualitatively similar 
 after division, and one would expect, the offspring produced 
 from such cells to be variable. That the chromatin of each 
 parent species often retains its individuality is indicated by the 
 fact observed in many primary spermatocytes where two sep- 
 arate groups of the small or single type of chromosome exists. 
 The division of such a cell into three or four, as the case may 
 be, results in the formation of new cells, some of which will 
 manifestly contain chromatin from only one of the original 
 parent species, and some only from the other. Some of the 
 spermatozoa, then, will bear chromatin from only one of these 
 species. In the offspring from such a cell one would expect a 
 much closer return to whichever one of the parent forms it 
 represented than in the offspring of a "mixed" spermatozoon. 
 In discussing irregular divisions, however, it must not be 
 forgotten that many apparently normal divisions of the primary 
 spermatocytes also occur in all hybrids, and constitute by far 
 the predominant kind of division in hybrids from closely related 
 forms. Unequal distributions of chromatin cannot there- 
 fore play the most important part in variation or rever- 
 sion. There seems to be no other interpretation, indeed, than 
 that in the many normal mitoses of the bivalent chromosomes 
 which occur, the chromatin of the father and of the mother is 
 set apart so that the ultimate germ-cells are what might be 
 termed "pure" cells; that is, a given egg or sperm-cell con- 
 tains exclusively or at least predominantly qualities from one 
 parent. The offspring from fertile hybrids of the same parent- 
 age might then be similar to the mixed type of the original 
 hybrid, or revert to one of the grandparent types, dependent 
 upon the chances of the various cells for union at fertilization. 
 Tf a spermatozoon and an egg containing characteristics of the 
 same species unite, then the reversion will be to that species ; 
 if a sperm-cell containing the characteristics of one species hap- 
 pens to unite with an ovum containing characteristics of the 
 other species, then the offspring will be of the mixed type again. 
 By the law of probability the latter will be the more prevalent 
 occurrence, because there are four combinations possible, and 
 two of the four would result in the production of mixed off- 
 spring, while only one combination could result in a return 
 to one of the ancestral species. 
 
 15 
 
From the fact that in cases of apparently complete return to 
 one parent type, characteristics of the other parent may never- 
 theless crop out from time to time in succeeding generations, 
 it is evident that all ot the germ- cells are not absolutely "pure." 
 The occasional inequalities in the division of individual chromo- 
 somes, as already mentioned, may account for this fact. It is 
 probable that the irregular distributions of chromatin, where 
 such occur, have more to do with such succeeding offspring as 
 show variation, and less with those which return to the specific 
 types. In the latter case the chromatin of each species has 
 remained entirely distinct (in marked hybrids), or has normally 
 separated again at the sundering of the bivalent chromosomes 
 (in mild crosses) into the two original plasmas. It is very 
 obvious, of course, that most of the variation seen in the off-, 
 spring of fertile hybrids is due to the union again of two "pure" 
 germ-cells, each of which represents a different one of the 
 original parent species. 
 
 That irregular divisions can not account entirely for rever- 
 sions to grandparent types is very evident in the crosses of 
 brown and white doves, where the irregularities are by far too 
 few to equal the percentage of reversions. There is but one 
 ultimate conclusion, then, namely, that the irregularity in divis- 
 ion of the primary spermatocytes, which appears in hybrids 
 between very different species, is but an index to what occurs 
 in ordinary crosses. In the latter, instead of separate spindles 
 and non-fusion of chromosomes, a true union occurs, but the 
 bivalent chromosomes ultimately divide in such a way that the 
 respective plasmas occupy different cells. There is a separation 
 of the paternal and the maternal chromosomes which had fused 
 during synapsis. 
 
 With regard to the question of the persistence of chromo- 
 somes, the evidence is becoming stronger every day that these 
 elements do retain their individuality. Riickert, 2 for instance, 
 in his study upon the fertilization of cyclops, was able to follow 
 the maternal and paternal chromosomes very distinctly in cleav- 
 age. Again, to cite only one or more of the rapidly multiplying 
 examples, Herla 3 and Zoja 4 have shown that in the hybrid fer- 
 
 2 Riickert, J. Zur Eireifung bei Copepoden: Qu. Hefte, 1S94. 
 
 3 Herla, V. Etude des variations de la mitose chez l'ascaride 
 megalocephala: Arch. Biol., XIII. , 1893. 
 
 4 Zoja. R. Sullo independen>.a della cromatina paterna e materna 
 nel nucleo delle cellule embrionali: Anat. An. XL, 1895. 
 
 16 
 
tilization of Ascaris, if the eggs of variety bivalens is fertilized 
 with the spermatozoon of variety univalens, the three chromo- 
 somes trms brought together retain their individuality and re- 
 appear at each cleavage, at least to the twelve-cell stage. Zoja 
 affirms that the paternal chromosome is of smaller size and is 
 thus distinguishable from the two maternal chromosomes. 
 
 The above interpretations are offered with the hope that 
 they may perhaps lead to some clew concerning the real nature 
 of the material basis of heredity. If the conception proves to 
 be a true one, then it doubtless affords a key, among other prob- 
 lems, to the long-standing one as to why many plants will come 
 true from slips or grafts, but not from seed. The reason may 
 be sought in the pseudo-reduction period of the germ-cell. 
 Plants such as the apple, for example, which do not come true 
 from seed, are practically multi-hybrid. In the germ-cells there 
 will be numerous incompatibilities due to the fact that the plant 
 has been miscellaneously fertilized for a number of generations. 
 In propagation by means of slips, the chromosomes lie side by 
 side and divide in the ordinary way to construct and maintain 
 the new body, so that it is practically a continuation of the old 
 one ; but when the time comes for maturation of the germ-cells, 
 the lack of harmony between the various plasmas represented 
 asserts itself, with the result that bivalent chromosomes are 
 formed, which divide in such a manner as to segregate different 
 sets of ancestral qualities. The resulting combinations in fer- 
 tilization will give rise to seed many of which may possess 
 dissimilar sets of qualities. 
 
 As to the other abnormalities met with in the spermato- 
 genesis of hybrids, about all that can be said is that the whole 
 phenomena show lack of vigor in the development of the germ- 
 cells, whatever this may mean. The deformed spermatozoa 
 indicate want of sufficient vitality to push the development 
 through to completion. The germ-cells start out apparently 
 to perform their functions normally, but later succumb to the 
 conflicting forces at work within their boundaries. 
 
 As to why the reoroductive organs should be more sus- 
 ceptible to abnormal changes than other regions of the body, 
 we have no clew. Darwin has pointed out repeatedly the 
 curious parallel between crossing and the change produced by 
 physical conditions. Animals and plants removed from their 
 natural environment are extremely liable to have their repro- 
 
 17 
 
ductive systems affected . Still he recognizes that sterility is 
 incidental and not a necessary concomitant of hybridism. 
 Hybridization in some forms, indeed, increases fertility. 
 
 SUMMARY. 
 
 i. The usual four types of germinal cells are recognizable, 
 viz: (i) spermatogonia, (2) primary spermatocytes, (3) sec- 
 ondary spermatocytes, and (4) spermatids. Sertoli or nurse 
 cells are likewise present. 
 
 2. The number of chromosomes in the spermatogonia is 
 sixteen, in primary spermatocytes eight, and in secondary 
 spermatocytes four. 
 
 3. The spermatogonia vary considerably in appearance at 
 different phases of their activity. A sphere (idizome), within 
 which the centrosome lies, is visible before division. 
 
 4. A filament two or three times as long as the other 
 chromatic bodies in the nucleus is cast out into the cyto- 
 plasm before the formation of the spindle for division of the 
 spermatogonium. 
 
 5. Synapsis occurs in the primary spermatocytes, during 
 which there is a marked drifting of the chromatin to the side 
 of the nucleus in contact with the sphere. 
 
 6. At the division of the primary spermatocyte, only eight 
 chromosomes are present, but they are in the form of heavy 
 rings, and are evidently bivalent. 
 
 7. In division the chromosomes break transversely, and as 
 they move apart, remain connected by threads of the linin casing 
 which encapsuled the chromosomes. These threads form the 
 interzonal fibers. 
 
 8. Intermediate bodies are present at the equator of the 
 interzonal fibers, and mark out the path of the ensuing division 
 of the cvtoplasm. 
 
 9. At the division of the secondary spermatocytes, the four 
 chromosomes which appear are of the same size and shape as 
 those of the preceding division. 
 
 10. In the transformation of the spermatid the first per- 
 ceptible change is in the centrosome. It divides, and one of 
 the resulting centrosomes enlarges and becomes ring-shaped. 
 The axial filament of the tail first appears as a thread connect- 
 ing the two centrcsomes, but later continues backward through 
 the ring-like centrosome and out of the cell. 
 
 18 
 
11. The smaller centrosome, together with material of 
 cytoplasmic origin, finally comes to lie inside of the nuclear 
 membrane. Although a middle piece appears to be absent in 
 the adult spermatozoon, it seems probable that this centrosome 
 within the nucleus may function as a middle piece which has 
 become obscured by a covering of chromatin. 
 
 12. The nucleus elongates to form the long head. It con- 
 tains a central core of chromatin in the form of a spiral filament, 
 which splits later to form a double spiral. 
 
 13. The head, during the later stages of development, 
 undergoes a very great contraction, but the spiral arrangement 
 of the 1 chromatin still persists in a modified form. 
 
 14. The chromatin appears finally to be arranged within 
 the head in a series of vesicles. A remarkable fact is that the 
 number of vesicles is the same as the reduced number of 
 univalent chromosomes should be, namely, eight. 
 
 15. The head-spine originates from a bubble-like mass of 
 material which arises in the sphere of the spermatid. 
 
 16. The general plan of spermatogenesis in hybrid pigeons 
 is not essentially different from that of normal pigeons. ' 
 
 17. All hybrid pigeons exhibit multipolar spindles and 
 asymmetrical distributions of the chromatin in cell division. 
 These irregularities are much more infrequent in fertile hybrids. 
 
 18. Infertile hybrids show in addition a deformed sperma- 
 tozoon, and often a marked degeneration of the germinal cells. 
 
 19. The irregularities of division are confined for the most 
 part to the primary spermatocytes. Likewise it is in these cells 
 that the formation of bivalent chromosomes occurs normally. 
 In hybrids, it would seem that the conflicting tendencies of the 
 two parental plasmas frequently render the union of the single 
 chromosomes to form the double (bivalent) types impossible or 
 abnormal. There seems to be an attempt on the part of each 
 plasma to assert its individuality. This visible incompatibility 
 of the chromosomes from widely different species serves as an 
 index to a kindred lack of harmony between the plasmas of more 
 nearly related forms, so that even though pseudo-reduction does 
 occur and normal division of the bivalent chromosomes follows, 
 the identity of the individual species is still retained through the 
 segregation of the maternal and paternal chromosomes into sep- 
 arate cells, which may be considered "pure" germ-cells (con- 
 taining qualities of only one species). 
 
 19 
 
20. Union of two cells containing characteristics of the 
 same species would occasion a reversion to that species. Union 
 of two cells representing each of the two original species would 
 yield an offspring of the mixed type. The latter would pre- 
 dominate because of the greater probability of such union. 
 Besides, through the mixing just indicated, variability may be 
 due also in some cases to the not infrequent inequalities in the 
 division of individual chromosomes, through which varying pro- 
 portions of the chromatin of each species may appear in certain 
 of the mature germ-cells. 
 
 21. Irregular divisions can not of themselves account en- 
 tirelv for reversion and variations, because double spindles and 
 irregularities in the formation of bivalent chromosomes are by 
 far too few to equal the percentage of reversions seen in such 
 mild crosses as the brown and the white ring dove. One is 
 forced to the conclusion expressed above, that the double- 
 spindled and multipolar types of cells which occur in hybrids 
 between very divergent forms are but exaggerated images of 
 a tendency which exists in the primary spermatocytes of normal 
 appearance, which are to be found in all hybrids. 
 
 22. The above conception may likewise afford a clew to 
 the problem of why certain plants will come true from slips or 
 grafts, but not from seed. The explanation may be sought in 
 the pseudo-reduction period of the germ-cell. 
 
 Department of Biology. 
 University oe Cincinnati. 
 October. IQ02. 
 
 Note. — Inasmuch as the present paper is a resume of the writer's 
 thesis of 1900, it is not deemed advisable to enter into a discussion 
 of any of the more recent papers which have a bearing on the 
 results obtained. Juel's paper on hybrids of Syringa, which did not 
 appear until later, is perhaps the most significant. His hybrids 
 exhibited abnormal mitoses similar to those that are found abund- 
 antly in pronounced pigeon-hybrids. From his facts I would sug- 
 gest that the same interpretation as set forth in my conclusions 
 holds true, although it can not be extended in the same detail to 
 fertile forms, because his hybrids were sterile. Two very brief ab- 
 stracts of one or two of the more unusual points presented in the 
 writer's thesis have appeared in Science, but thus detached from the 
 body of the work, it would seem from the tenor of letters which 
 have been received that in some cases a misunderstanding of 
 the writer's exact position has arisen. It is hoped that the present 
 paper will set forth the main facts in their true perspective. 
 
 20