Anatomical Record 13 (1917)

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THE ANATOMICAL RECORD

Editorial Hoard

Irvine Hardesty Tulano University

Warren H. Lewis Johns Hopkins University

Clarence M. Jackson University of Minnesota

Charles F. W. McClure Princeton University

Thomas G. Lee University of Minnesota

William S. Miller University of Wisconsin

Frederic T. Lewis Harvard University

Florence R. Sabin Johns Hopkins University

George L. Streeter

University of Michigan

G. Carl Huber, Managing Editor

1330 Hill Street. Ann Arbor. Michigan

Volume 13 JUNE-DECEMBER, 1917

PHILADELPHIA THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY

Contents

No. 1 JUNE

II. 10. JoKDAN. Studies on striped niusolo .structure. III. 'I'lie comparative histology' of ciirdiiic and skeletal muscle of scorpion. Twenty-one figures 1

Margaret Hekii Lkwis. fhe effect of certain vital stains upon the development of the eggs of cerebratulus lacteus, Kchinorachnius ])armii and Lophius piscatorius. . . 21

Ai>nERT M. Reese. The blood of alligator Mississippiensis. Kight figures 37

S. B. Grant. A persistent superior vena cava sinistra in the cat transmitting coronary blood. One figure 45

Hollo E. McCottkk. The vomero-nasal apparatus in Chrysemys punctata and Kana catesbiana. Seventeen figures 51

No. 2 JULY

IIouEK Ci. KisHEU. Histological structure of the retractor penis muscle of the dog. Four figures (two plates) 69

\Vak(j Nakahara. Preliminary note on the nuclear division in the adipose cells of insects. Eleven figures 81

J. T. Patterson and C. G. Hartman. A polyembryonic blastocyst in the opossum. (Jne text figure and two plates 87

M. Stkonc;. Some observations on the origin of melanin pigment in feather germs from the Plymouth Hock and Brown Leghorn fowls. Six figures 97

Corner GW. Maturation of the ovum in swine . (1917) Anat. Rec. 13(2): 109-112.

Lloyd K. Reynolds. Hyperphalangism accompanied by supernumerary epiphyses and muscular deficiencies. Twelve figures 113

Ivan E, VVallin. An inexpensive projection apparatus. Three figures 127

No. 3 AUGUST

Ralph D. Lillie. Variations of the canalis hypoglossi 131

Mary T. Harman. A case of superfetation in the cat. Three figures (two plates) . 145

Laboratory Apparatus.

I. A simple electric thermo-regulator. H. S. Bxtrt. One figure.

II. An automatic air pressure valve.

III. A laboratory timing clock. One figure. .VoonsTUs'F. Scharfk 159

Marsh Pitzman. Some suggested changes in nomenclature 165

Charles Eiioene Johnson. On the development of the liver in the genus Citellus. Four figures 166

Newton Miller and James S. Godfrey. A note on the anastomosis of arteries and veins in a cat. One figure 177

No. 4 SEPTEMBER

.\i.iiert M.' Reese. The anatomy of a two-headed lamb. Eight figures 179

Studies On The Mammary Gland III. A Comparison Of The Developing Mammary Glands In Male And Female Albino Rats From The Late Fetal Stages To Ten Weeks Of Age.

«. -. vrii ligtin'M 205

II I ltM.<-.M vMi.l I. Kw/. Vn iiHjimliilile slide liiisket. Five, figures . 227

No. 5 OCTOBER

J A I iiiifiiiul study of the effect of stress and strain on bone de VI irns 233

... AM) JtAYMiiM) Pkaki.. Sex studies. IX. Interstitial cells in the organs of the rhickcn. Six figures 253

i.i.-ii-ii. li. .\\it.\ . rhiigocytoHi.s liv ostoorlasts. Four figures 269

S K Wiluamh AMI |{ \\ Haiiii. The anatomy of a double pig (Synrephalus thoracopagus I . Seven figiircn 273

KiMiKiui K. Oi.ivKii Mi-iiil>r:ini-.s nf the right iliac region.. . . . 2S1

('«KHo\ (iiixArtni: »M) lloHMiii llii,i. IlKfsToN. Detailed study of a monster with

Taniorarhischisi.i ami nlhrr anomalies. Six figures 289

Iv*\ K. Wai.i.in. .V teaching model of a 10 mm. pig embryo. Three figures. 295

KdwamI) t'AKKiiLt, Dav. Two conveniences for the laboratory. Three figures. 299

MAmin W. Lton, Jk. An hereditary case of congenital absence of one kidney. One figure " 303

No. 6 NOVEMBER

G. ("aki. HuBKR. On the morphology of the renal tubules of vertebrates. Twenty-two figures 305

C Caki. llrnr.K and Arnold H. Egoerth. On the morphogenesis of the papilla foliata of the rabbit. Six figures 341

\V«hh»:n H. Lcwi.s and Maroaret R. Lewis. The duration of the various phases of mitosis in the mesenchymp cells of tissue cultures 3.i0

I'aii. B. Katon. The cfM-liac axis. One figure 369

F. IV>TAi.r-. IVsarrollodo los mil.sculos oculares recto cxterno y recto interne, on el

rmbritin humnno. Diei figuras ". 375

John a. KiTTKi-xoN. The poatnatjil growth of the kidney of the albino rat, with observations on an adult human kidney 385

II. S. f;t"r>Ei.i.. An anomalous case of blood formation in the connective tissue of the

sciatic nerve in man. Six figures 409

I*. K. Unkback. A scheme for drawings in a course in embryology. Three figures. . . . 419

No. 7 DECEMBER

.\|»m T. IIahwan. .Vnother case of gynandromorphism. One figure 12.')

J .M. .VtNimt (iAi.vAo. The finer structure of the I'iliary ganglion of Ophidians 437

A. or. I.KMiw ToHKi'-H. On the ciirlrlaginoiis tissue of the heart of Ophidia 443

J MoHKiHA MA ItcH'iiA. Slaiiiingof adult cartilage by Lundwall's methods 447

J«ur... |{ ('aim l)n the ilevflopmenl of the lymphatics in the heart of the embryo pig 451 KiTii It^Nh I III the relation of the licad chorda to the pharyngeal epithelium in the IMKpnilirvo: » contrilmtioii to the development of the bursa pliaryngca .iiid the toii.silla ' * .'••1. Ti-n figures 465

• iiAi.i. I)»:TWii.rR. «ln the use of Nile blue sulphate in embrvonic tissue >. iM..|ii iiitniiofi . 493

sTrDn:s ox sthiim:!) muscle sTHrcTURK

III. THE COMPARATIVE Histology OF CARDIAC and SKELETAL MUSCLE OF scorpion

II. K. JORDAN

Department of Anatomy, I'nivcrsity of Virginia

TWENTY-O.NE FIGURES

I. ixTHonrrTiox

Tho present investiiiatioii tleiils witli the striped muscle of the Floruhi scorpion.- iuchicling tlie po.st-abiloniinal ('tail'), prealuloininal, ceplialo-thoracie. leg. and heart muscle of both the adult and the new-born. In the latter material occur unportant developniciital stages, which tlir()\v valual)le light uptm the more involved adult structure.

As was to be expected the strijied nuiscle of scorpion is ver\' similar to that of limulus, and accordingly confonns more closely to the vertebrate tyjje of strijx^d muscle than to the arthro])(Kl type as excmplilied, for example, by the sea-spider, .\iiopoldact j'lus lentus, already describetl, and by certain insects.

Aj)art from being, as far as I am aware, the first detailed microscopic stmly of this muscle, the chief \alue of this investigation inhei"es in the information it yields, by reason of the greater

' The two earlier studies of this series eoiieeriied the musc-le of the king crab (I'roc. .\iii. .\ssoc. .\iiat.. .Viiat. Hec, Jan., I'.llll; eoniph'te pafH-r in a forlheoiuiiiK piililieation l)y the Carnegie Institution of Washingtonl. anil that of the seaspider (.\nat. Hec, vol. 10, pp. 493-508, 1916). The pertinent literature is fully reviewed in these papers, and a l>ililiographic list ineludeil, and will not here l>c repeated in detail.

' This tnaterial was very kindly furnished alive liy Dr. William I'atten, of Dartmouth College. It was variously fixed in the strong rhrom-aeeto-osmic solution of I'lcinniiiig, and in the alcolinl-nitric-arid solution of /immerinann, and stained liy the lleideidiain iron-hemaloxylin method, in eertain eases followed hy Van (lieson's stain.

1

TIO: AKATOUICAL HETOKO. VOL. 13, No. I JISK, 1017

'2 II. K. joiin.vx

il<;irn<>>s in irs|H'ct of soinc importunt details in this tissue, n-nanliiiK the minute stmetiue of slripe«l muscle in general. TIM'S*" »iet nils conceni es|H'cially : (a) the nat ui-e of the sarcolemma ; (h) the n'lation of tin- telophragma to the sarcolemma and to the nuclear wall: (e) the mode of muscle giowth and development, hoth with n«s|x>ct of the multii)lication of the nuclei, and the iiicrejiso in the numher of the myofibrils; (d) the decisive evidenc*' in contravention of the interpretation of striped muscle structuH' in ti-rms of 'muscle cells' (Ajiatliy; Baldwin) andextrar4>lluhir myolihrillae. in analogy with connective tissue; and (e) the relation l)<'tw«><>n the nuiscle fibrils and the tendon fibrils. The cardiac muscle of scorpion and of Limulus conforms with vertebrate heart muscle in its sjTicytial structure, in the more or less n'Rular railial arrangement of the myofibril bundles (lamellae) and in the definite character of the relatively less complex cross striping. I'he skeletal nuiscle also of these fonns agrees with vortobnite skeletal muscle with regard to the major stripes, and in the ab.^'iice (or exti-eme temiity) of the mesophragma and the ucces.sory disk (N-disk) of l^ngelmann and Rollet, conspicuous in certain insect muscles. However, the cardiac and skeletal muscles of these forms are, in respect of minute structure, somewhat mor«' clos<'ly similar than is the case in vertebrates generally, possil)ly the ivsult of relatively clo.ser functional similarity. Whether the above-named similarities between arachnitl and vertebrates' muscles have genetic significance, as would s<>em probable on the basis of many other observations on other structures eiuunerateil by Patten,' or whether they indicate only functional eciuivalence remains an o]kmi (luestion.

II DK.SCUII'TIVK

The .skeletal muscle fibers are long multinucleated elements tap«>rinK towards their extremities. They vary greatly in diameter in difTen*nt r<'gions of tin- body. Those of the post • Pntti-n. W. Thr Kvoluticiii nf ilif \'crtcl>ratca and tlirir Kin. Ulukiston, I'hilailrtiihin. IUt2.

STRIPED MUSCLE OF SCORPION 3

ahdonion arc very stout and of irregular form in eross-soction (fig. 1 a and !>); elsewliciv in tlic Ixxiy tlic HIxts aro of much smaller girth, more unifonn in size and of an a])i)roxiinateIy circular outline in cross-section. In resjjcct of minute structure the skeletal stri])ed muscle Hljers of the .sevend regions of the body are essentially identical. Hence the description may confine itself largely to the coarser fibers of the post-abdomen.

In ligure 1 a and b are illustrated two larger and more irregular fibers, in transvei-se section. 'Hw vesicular nuclei are located centrally within an iiregular core of finely granular, verj-^ delicately reticular, saicoplasm. Peripheral to this is a wide border in which the myofil)rils are arranged in the fonn of lamellae radially dispo.setl. Many of these lamellae show a peripheral split of gi'eater or less depth, giving to these lamellae a Y oi' \' shape. Certain lamellae show a double ])erii)heral sjjlitting. This condition most probably is a gi'owth phenomenon, increa.se in the number of the lamellae, ami the size of the fiber, being attained by a radial longitudinal fission of parent lamellae, as is the case in growing skeletal fibers of certain teleo.-<t fi.'^hes, e.g., the rain-bow trout. Certain iil)ers show a grouping of the lamellae into Imndles, suggestive of K()lliker's columns (Cohnheim's areas, in cross-.'^ection ; fig. 1 b) of mammalian skel<>tal mu.scle. The irregular form of the fiber in cross-section exjilains the variation in wiilth of fibers as seen in longitudinal sections.

The fibers are invested by a distinct sarcolenuna, which varies in difTer(Mit fib«>rs between a relatively coarse memltrane and one relatively tlelicate. Closely atUierent to the sarcolenuna superficially is a more or less delicate and wide-meshed fii>rillar connective tissue, with smaller more elongate and generally more chromatic nuclei. .Vfter Van Cieson's stain the connective tissue (iK>rimysium) is coloretl red, while the sarcolenuna remains unstained. After prolonged staining in a solution concentrated with acitl fuchsin, both the telopliniginata i ground membranes; z-meml)ranes) and the sarcolenuna stain pink or red.

In longitudinal sections the nuclei ai'e seen to be elongate elements with irregular sinuous or serrated contour (figs. 2 and 3). Such sections, jjroperly stained with iron-hematoxylin, show a

4 H. K. JOltU.VN

(li.stiiict cnKss-st nation <»ss«Mitially like lliut of vortolirato voluntan- striiM^d inuscli>. Tlio st nations of the relaxod fiber include the linliter .l-«lisk. tlie darker C^-disk, and the Kranular telophnifnna. 'rh«'re is not the slightest indication of an additional niesophragina ( M-nienil)rane) and tlic accessory disk of ICngelinann and Hollet (^N-lineJ. The granular teloiihragnia stains n-latively intensely. The granules represent swellings at the |M»ints when' the inyotihrils are attached to the telophragma. Centrally the telophragma passes through the sarcoplasmic iion-tilirillar con> and becomes intimately attached to the tips of the nuclear s«>rrations. \\'ithiM the core the telophragma ap|)ears non-gr;iiiular and stains less intensely (fig. 2). Peripherally the telophragma makes a close union with the sarroleninia (Rg. li). This extra-fihrillar portion of the telophragmata is also non-granular and less deeply .staining. The sarcoI«>mnia is usually festooned hetwcn^n successive telophragmata, |)roital)iy a fixation artifact. The serrated character of the nuclear wall may he in jmrt similarly an artifact.

Figure :\. at the left, shows a iM'culiar condition freciuently met with. Here the sjircolenunas of two adjacent fibers have become blended, and the common membrane has been thrown into a zig-ziig fonn, the apices with their attached telophragmata altenuiting in the apposed fibers. Such result is made ])ossible by n'a.^»n of a local ])aucity or practical absence of inter-fil)er connective tis.sue allowing thus of a fusion of the adjacent sarcolenunas. The process of such fusion can be pictured by reference* to illustration figure 4, which represents the tajiering end of a lilwr and part of an adjacent fiber. Here the connective tissue l)etwe<>n the two filn^rs is relatively abundant and closely wiherent to the highly festooned sarcolemmas. If we imagine the conne<-tive ti.s.>iue n>moved, ;ind tiie adjacent san'olemmas thus l>rought together .so that the crest of a festoon on one side fits into the trough W'tween successive festoons in tiie ai)posed win-olemma. then by a blending of the two .sarcolenunas, and under the opposed lateral tractions of the attached telophragniata, the conditijui shown in figure ^ would become realizinl. Certain steps in .such a process, esfMH-ially the one where a doulile

STRIPED MUSCLE OF SCORPION O

.sarcolciiiina witliout iutervoning conuectivo tissue occurs, can actually be traced in the sections. Similar conditions have been described in inamnia'ian heart muscle (Heidenhain, 'Plasma undZclIe'j.

b. Skeletal ruuscle of (he new-hnni

In the no\v-l)orn scorpion not all of the muscles ;iri' <'(|ually well developed. Those (jf the cephalo-thorax, abdcMiicn and legs seem in general functionally mature; though the oval, regularly contoured, and more chromatic character of the nuclei (figs. 5 and (i) indicate a lesser degree of progressive differentiation. (Vrtain fibers in the region of the head are in the early stages of transformation of myoblasts into multinucleated fibers.

Figine ."> illustrates a lightly-stained mature fiber of this stage, with a festooned sarcolemma closely adherent to which is a fibrillar connective tissue. Figure 6 is more deeply stained and thus shows a more conspicuous Q-disk and the granular tt^lojihragmata. Tliis fiber is moreover distorted, apparently l)y a force causing compression along the longitudinal axis. The folding or bending thus produced caused an alternate bulging on ojiposite sides, and the liend and the bulge are always along the levels of the telophragmata. This i)henomenon demonstrates a firm connection between myofibrils and the telol)hragmata. It demonstrates also the absence of a mesophragma of a structure similar to that of the telophragma.

In figure 7 is illusti'ated a mid-phase of contraction. Here a new disk, the H-disk (of Hensen) becomes conspicuous. The ll-disk is an incident of contraction. Figure S illustrates the completely contracted condition, in which ilarker contraction bands alternate with lighter disks of apjiroximatc^ly eiiual or somewhat gieater longitudinal width.

Figinv 9 represents a contracted hl)er ujion which is sui^erposed terminally a traction stress. I'nder .<uch conditions a flexible mesophragma (M-membrane) if present, but suppi)s*xlly ordinarily invisible in vertebrate strij>ed nuiscle because of its alleged tenuity, should be brought into view l)y reason of a tliicUening after releas(> of lat(>ral tension. Such was the case,

ft M. K. JOKDAN

jiH pn'vioiisly (l<'scrili<Ml. in the leu imisclc of the soa-spidor. Hut ill 111*' scorpion striiH'<l inusch- no iiu'soplirugina becomes di.sn'rniliU'.

In linun- 10 is shown the region of transition from a small RhfT of the jaw to its connectinK tendon. The myofibrils and tendon fibrils an- in direct continuity, the latter apparently n'pn*s«>ntinK modification products of the former. The tendon stains m<m> deeply than the muscle in iron-hematoxylin preparations, and faintly red after \'an (liesons stain. The cros.s striiM>s fade out at the level of transition from the muscle to the tendon.

In the head region certain Hbers can be seen representing early stap-s in the metamorjjhosis of a myoblast into a. long multinucleatiHl slriiK'd fiber. Figure 1 1 illustrates a myoblast with four miclei, the next to the lo\v<>nnost one in process of amitotic division. The lower extremity shows also a transition to the developing tendon. The sarcolenmia is relatively delicate. The myofibrils are relatively comjiuct. and give to the cell a rt'latively dwi^^r color. No cross striations have yet appeared. Such ari.se first terminally and apjKar progressively towards the center. The first striations are the telophragmata; the Q-disks apjK'ar only some time later. Tlic nuclei imiltii)!y by direct division. Coincident with the nuclear multiplication and the apjK'arance of striations, the fiber elongates. Diu-ing this process {il)ers occur in which as many as twenty nuclei may l)e count<>d in close series in a relatively short extent of the central sarcoplasmic core.

Figure 12 shows two atljacent young fibers from the anterior n-gion of the cephalo-thorax. The jieripheral lamellae of myofibrils <H'cupy less than a third of the radius. A few lam<'llae may he seen in pn)cess of radial longitudinal splitting, by which event the lamelhu* are increa-sed in number and the fiber grows in diameter.

c. Cardiac muscle of the adult

The heart tul)e consists of a single layer of muscle fibers ftralM'culae), invested ix-ripherally liy a layer of connective

STKIPKD MUSCLE OF SCORPION /

tissue (pericardium), l^oth the scorpion and the Liinulus heart hick iin eiulothcHal lining. The myocardial fillers are for the most part arranged in an ol)li(iuc circiihir direction. The original fil)crs lia\c anastomost'd in the adult heart so a.s to form a continuous, hrandiing, spirally disposed, syncytial muscular membrane, 'llie fibers of this meml)rane vary in the degree of compactness and the diameter of their cross-section. In a medial longitudinal section of the heart tube groups of from three to four or more, compacter cro.ss-sections alternate with a similar numl)(>r of looser cros.s-sections. In the transverse direction al.so the more compact portions of the fiber alternate with the loose portions due to the spiral arrangement of the trabeculae. .\nd in paratangential section, the compacter portions may be seen to pass by gradual stages into the looser portions of the opposite side. It appears probable that the compacter portions represent the extremities of the original myoblasts. According to this interpretation the derivatives of an original myoblast would extend for about two and one-half turns around the adult heart.

Figure 13 illustrates a transverse section through a more compact portion of a myocardial fiber. The midei are located centrally in a coarsely granular sarcoplasni. The delicate sarcolemma bounds a peripheral layer of similar construction. Between the peiii)heral and the central granular sarcoplasmic areas, the plates of myofibrils are radially disposed, and grouped into smaller bundles which anastomose internally, thus giving to the heart nmsculature a double-syncytial character, as in Limulus. A few of the lamellae are split peripherally. At the lower pole of the ilrawing, the point where the liber curves from a lateral to a ventral jmsition, the lamellae are cut very obliquely.

The heart muscle fiber in longitudinal section is practically identical with the skeletal nniscle fiber (figs. 14 anil 1.")). This very close similaritj- between heart and skeletal muscle filler was striking also in the ca.se of Limulus. In figure lli is shown a bundle of fibrils in extreme contraction. The change in .shape of the nucleus, and the finely serrateil character of its wall, as compar(>d with conditions in figure 14, demonstrate a very firm iMiion IxMween the telophragmata and the nuclear membi-ane.

{i H. K. JORDAN

d. Caniiac muscle of the new-born

The hoart tube of tlic new horn scorpion is very like tlial of the lulult, t'xrept that tlic syncytium is less compact, tlic fihcrs an" smaller and more rcgvilar in cross-section, the nuclei are utoutor. have a more regular contour, and are somewhat more chromatic, indicating a lesser degree of differentiation; and the inyoliliril lamellae are relatively few in number and more regularly disposed radially without any segregation into smaller bundles (fig. 17). A few lamellae are undergoing a longitudinal fission.

.\s indicated by the form of the nuclei, different portions of the myocanliuni are at different stages of differentiation. In the younger portions, the nuclei are actually multiplying by direct division (lig. IS); in other portions, practically adult conditions obtain (fig. 19). Certain portions have suffered contraction, imposed extraneously pos.sibly by the coagulation effect of the fixing fluid; in such portions the nuclei are strikingly modifietl (fig. 20), in that they are shortened longitudinally, caused to widen transversely, and the peripheral serrations are greatly accentuated. .Mso the telophragmata are less widely spaced in .such regions. The conditions shown in figure 20 can be readily conceived as derived from figure 11), where also four serrations with attached telophragmata are present, by compression exerted at right angles to the long axis of the fiber. Figure 21 illustrates a similar condition, and shows besides a stage in the direct division of the larger imcleus; and a mass of nucleated connective ti.ssue is shown at the right. The plane of tiudear division pa.vses between successive .serrations, which brings it to pass that the daughter nuclei retain their connection with the telophragmata.

m. Disri'ssioN .\nd conclusions

One f)f the most striking facts regarding the scorpion muscle, as also regarding the Linnihis muscle which it very closely resembles, is the es.sential identity in microscopic structure between the cardiac and the skeletal muscle. The axial location of the nuclei, the radial lamellar arrangement of the myo

STRIPED MUSCLE OF 8COKPION 9

fibrils, and the relative simplicity of the striations all inriicate a relatively low degree of diflerentiatioii. This circumstance is probably a reflection of a relatively low functional ref|uirernent and a coincident approximate similarity of action. Muscular movement is in both instances leisurely, and the meagre reciuirements are presumal)ly satisfactorily met bj- a relatively simple structural condition.

Furthermore, the minute structure of the scorpion muscle resembles closely vertebrate striped muscle (especially cardiac muscle; note figs. 1 to 3). ,\s a subdivision" of the arthrofxxls the muscle of the arachnids might be expected to resemble closely that of insects. But there is not the slightest indication of the mesophragnia and the accessory disk of Engelmann and Rollet (N-disk), (•()nsi)icuous in certain insect muscles. The fact that the mesophragnia is not discernible in a fil)cr like the one shown in figure 9, where the contracted fiber has been stretched terminally, leads me to conclude that a mcsophragma is actually absent in this muscle; and that this membrane is characteristic only of very highly difTerentiated fibers. Moreover if a mesophragma occured, of the same nature as the telophragma as is claimed by Heidenhain, we should expect, under the conditions represented in figure ti, a double series of foldings, that is, at the levels of the alleged mesophagmata as well as at those of the telophragmata. Whether the similarity here also, that is. between the muscle structure of certain arachnids (scorpion; Linmlus) and vertebrates (as is more probably the case as between cardiac and skeletal muscle of these forms) signifies likewise only a functional eciuivalence. or whether it has any phylogcnetic significance, —or po.s.sibly both — is a (juestion whose further consideration lies outside the scope of this investition. All thai need be said by way of interpretation is that the-^ie additional facts harmonize with Patten's theory of the arachnid origin of \'ertel)rates.' Moreover, lioth tlie ■<ct)rpion

Among the large body of evidence presented in support of this theory Patten noted that "The iiriiohnids resemble the vertebrates in more general ways, as in the minute structure of curtilage, muscle, nerves, digestive and sexual organs,' (p. XVIII, Historical Sketch); but he nowhere illustratesor describes in detail the microscopic structure of the arachnid striped muscle.

in M. K. JOKDAN

niul Limuliis mytK-artlia arc .syncytial in stiuc-turt", very like thr iiiyocartliuni of vortphratos; but the scorpion cardiac musculature is only one filxT in thickness, while that of Linuilus is many fihers thick: and hoth imisculatures practically lark lon^iludinally disposed iihers.

'I'hc observations al)ove recorded repardinij the relation of the telophrapmata to the nucl(>ar wall, when taken in connection with sin ilar observations in Linuilus nuuscle and that of cat and mouse,' wouKl seem to definitely dispose of any further profitable attempt to interpret striped muscle in terms of 'muscle cells.' and extra-cellular niyofilirillae. The close union between the telophragmata and the myohbrils, sarcolemma, and serrations of the nuclear membrane, above described, demonstrate that the fiber is the cellular unit. There can remain no longer any (|uestion of a smaller definitive cellular element. -Moreover, the history of the development of the myoblast disproves .such interpretration. The myoblast is a stout fusiform cell, envelope<l by a membrane which becomes the definite sarcolemma and .supplied with a nucleus which divides re]ieatedly by amitosis. The cell becomes measurably elongated, showing first more anil more conspicuously the telophragmata and then the (^-disks appearing in a medial jirogression, the simple myoblasts thus becoming transformed into the definite multinucleated striatetl muscle fiber.

.\nother interesting similarity between this muscle and that (tf vertebrates concerns the manner of growth. This is by radial longitudinal splitting of the myofibril-lamellae, a process .substantially identical with that occurring in embryonic fibers of certain teleost fishes, e.g., the rain-bow trout.

This radial disposition of the myofiliril-lamellae resembles also the condition in the adult heart muscle of vertebrates. liut in the latter the fibrillar constitution of the lamellae is readily di.s<'ernible in cross-sect irms of the fibers. In the case of the scorjnon and Linuilus skeletal fibers the finer fibrillar condition

' Jordnn. H. K. Tlir mirrosropic structure- of niammnliun ciirdiar muscle with aperiBl rcfrrcncp to Ro-callcd muscle cells. Annt. Roc, vol. 8. 1914.

STHIPKU MUSCI.K OF SCORPION I I

of the lamellae is much less conspicuous and occasionally quite impossible to recognize in transverse sections: in the cardiac muscle of these forms the myofil)ri!s are less compactly aRgregated into lamellae, and in consequence are easier to distinguish. The condition in the post-abdominal muscles approaches that described for certain insect wing-muscle, e.g., Libella, in which, according to E. Holmgren*^ the radial lamella cannot be further analyzed into constituent hbrillae. Hut when one observes the .scorpion muscle very carefully, especially in thin longitudinal section under the oil-inunersion lens, one may .satisfy himself that the lamellae here actually do consist of fine myofibrillae, some of which appear to cross between adjacent lamellae. Moreover when the coarser myofibrils are carefully followed for some distance, they may be seen to resohe ir)to still finer hbrillae, to the limit of visibility. The internal fibrillar (lamellar) constitution of this muscle fiber thus appears to be of a syncytial character, and the evidetice with respect to the successively finer resolution of the lamellae into more and more delicate myofil)rillae offers support to Heidenhain's protomere hypothesis of histologic structure as applied to striped muscle, namely, that the ultimate vital units are metamicro.scopic fibrils. '

This material shows also very clearly that the tendon is a derivative of the myofibrils. Myofibrils and tendon fibrils are in direct continuity: the sarcolemma apparently blends with the more peri])lu'ral tendon fibrils.

A feature of Limulus heart muscle which brought still closer the similarity between its structure and vertebrate cardiac muscle was a simple type of intercalated disk. Such di.sks could not be detecteil in the scorpion heart. This does not necessarily mean that they were not actually present somewhere, nor that they could never appear in any heart of any age or any condition of functional strain, ."^uch a statement could only be made with approximate certainty if a complete heart tube 'of an old specimen were carefully searched, which has not been done. Even

« Sec llciclcnliaili. M. •l'l:i.«m.i uiul 7a-\U\' p. .iSI, 1!I|I. • lli'idfiiliiiiii. .M. 'Plasma and Zcllc," p. 5S2.

12 II. K. JORDAN

III mliilt 1-iiiuilus such disks an- coinparatively rare. If my hy|HillM>sis. which is supported by a number of suggestive obserVHtidiis, and is in harmony with the actual histologic facts, is rorn-ct. namely, that tiicsc disks represent irreversible contracliitn bands, possibly the result of a local impairment of function, then one would perhaps hardly expect such disks ordinarily ill Ihe scorjiion heart: but they might possibly occur sparsely in aged specimens.

The blending of the sarcolemmas of two adjacent fibers, and the ensuing alternation of the tips of the resulting zig-zag membrane, and of the attached telophragmata calls for discu.ssion. It is interesting to note that essentially identical structures occur in vertebrate and human heart muscle." The interpretation which at once suggests itself regarding this alternation of structure between the apposed .surfaces, is that the adjacent fibers have been drawn slightly in opposite directions by the tensions exerted during de\elopment or function. Rut Ileidenhain calls attention to the fact regarding vertebrate cardiac muscle, in o|)position to this interpretation, that the numerical relationship of the serrations and the attached telophragmata on the two surfaces of the common sarcolemma is always as n to n + I; and he is led to the explanation of this j)heiiomcnon as the result of a spiral distortion of a fiber about a longitudinal axis.

The sim|)ler conditions in the scorpion skeletal muscle lend support to Hcidenhain's hypothesis. In the case illu.strated in part in figure 3 the condition.s are as follows: the two fibers are from the peripheral region of a post-abdominal segment : the fiber at the left comes to a point above, the fiber at the right terminates similarly below; above and below near the termini of the pointed ends the telophragmata of the two fibers are at the same level; between the.se j)oints, the telophragmata of the adjacent fibers alternate in such a manner as to leave fifteen sarcomeres on the left and only fourteen on the right. The pointed termini of the two fibers at the oppo.site end of this field may be accounted for by the a.ssumjition of a spiral twisting about a

• Hridpiihitin. .M. 'Plaatna iind Zcllc,' pp.641 to 544; 615 to 617.

STRIPED MUSCLE OF SCORPION I'l

common axis, producing thus in a longitudinal section the asyninu'trical alignment of the opposite sarcomeres, according to the conception of Heideiihain. It should again he noted that there is with respect of this phenomenon also a very close similarity between scorpion skeletal muscle and niamn)alian heart muscle.

One of the most puzzling and interesting features of the developing scorpion nniscle. both skeletal and cardiac, concerns the multiplication of the nuclei hy the direct method of division. Not a single mitotic figure api)ears in the muscle tissue of this form; but abundant instances of amitotic nuclear division may be seen. The plane of division is alwaj's between successive points of attachment of the telophragmata, so that the daughter nuclei remain susj)ended by these membranes. But in this connection it should I)e noted that in the earlier stages of metamorphosis of the myoblast into the muscle fibers, when nuclear multiplication is most active, telophragmata are not present, at least not in such complete form as to be recognizable under the ordinary magnification. Such dexice would .^^eem to aid, by rea.M)n of the absence of possible restraining membranes, the intensive and raf)id multiplication of the nuclei, necessitated by the metabolic requirements of the 3'oung, rapidlj* differentiating muscle fiber.

This material would seem to offer a favorable opportunity for the study of the underlying causes of amitotic nuclear division as opposed to karyokinetic <livision. This may constitute the basis of a future investigation, .~^in(•e the technic here employed failed to disclose a centrosome or archoplasmic .substance, it must remain uncertain whether such is actually lacking or whether it simply was not visibly preserved. In consetiuence no final argument can be made on the basis of this material for or against the hypothesis that amifosis is the consequence of some factor which puts a restraint upon the activity of the centrosome substance in a growing tissue. However, in the certain absence of jKithologic factors here, it n\ight with some reason be a.s.sumed that the possible restraining agent might be lack of relatively adeciuate luitriment, caused by the rapidity of growth

14 II. K. JOKDAN

mill (litTtTiTitiatiDii of tin- myoblast, ami tlius intorforing with rfi\tr«»«iinal activity. However tliis may l>e, it .seenis oloar, in view of the doiihlo function of the nudcu.s, namely, as an organ in control of constru<-tive tnptal)oli.<m and of specific heredity, that the nniuiremenls of growth and differentiation are am|)ly met by the increa.><e and distribution of nuclear materials by din«ct division. The cause of amitosis in this muscle may inhen' in an economic adaptation to essential needs, rather than in an indirect restraining influence upon the activities of the karyokinet ic ntechanism.

The regular reticular system of telophragniata and sarcolenuna is so conspicuous and robust that one feels inclined at first sight to interpret the whole mechanism as a connective tis.sue derivative. And the absence — whether apparent or real is uncertain of the telophragmata in the myobla.st and in early stages of transformation into the muscle fiber, gives further stn-ngth to this inclination; as also the fact of the very close union between connective tissue and sarcolemma. Hut two chief facts negative any .such interpretation: (1) the intimate union between telophragniata and the nuclear membrane; (2) the difference in staining reaction to \'an Gieson's solution of the connective tissues and the sarcolemma: and the similar lack of affinity of the sarcolemma and the telophragniata for the acitl fuchsin. The first point is in harmony with the derivation of the telophragmata from the cytoreticuluni of the myoblast, by |)roce,s.s of condcn.sation and growth and a rearrangement into n'ctiiinear me.shes. somewhat according to the suggestion first nuule by MacCallum." Similarly, the sarcolemma is the adult repn-sentative of the original cell meiiibraiic.

The decided festooning of the sarcolemma in this material niises the riue.stion of the cau.sal factor in the production of this condition. Huber'" suggests on the basis of his studies of rabliit voluntary striped mu.scles in teased prejiaration th:it the

• MBrCiillum, J, B. On the histoloiO' und hisluKcncsis of the heart muscle cell .Xnnl. Ani.. IW III IS'tT.

'* Hutior. f!. Carl. On the form nn<l iirninKcmpnt in fasciculi ofsfriatcd volun»ry muiirlr IiIhtb. Aniit. Iter., vol. II, p. IOC.

,\

STKII'ED MUSCLE OF SCORPION 15

festooiiiiijr (il the sarcolemnia, desfribod l)y certain authors, may perhaps be brought in relation with the ends of myofibrils which do not extend the entire length or tlie muscle fiber.' It is impossibh- for me to speak witli certainty renardiiiK the h-ngth of the separate myoftbrils in the scorpion or the I.inmhis nuisde. One gels the very clear impression from a study of thin iotiKitudinal sections that the hirfje myofibrils are divisible into fine fibrillar components, and that the entire fibrillar constituent forms a loose meshwork or syncytium. On this basis one miRht perhai)s lefjitimately conclude that many of the myofibrils at least do not extend the entire leiiffth of a fiber. Hut the rather intimate interconnection of the entire fibrillar component of the fiber, coupled with the fact of the continuity of the terminal myofibrils and the sarcolemnia with the tendon fibrils, makes difficult the application of Huber"s sugp;estion in regard to the formation of the festoons of the sarcolennna. The explanation that most strongly suggests itself to me, as the result of my observations in the scorpion muscle, is that, due to the intimate imion between myofibrils and the telo])hragmata and between the l.'ilter and the sarcolemnia; and the difTerence between the contraction etTect of the fixing fluid u])on th<' myofibrils and the sarcolemnia, the latter is forced to bulge in order to adapt itself to the greater shortening of the successive sarcomeric portions of the involved myofibrils. The same jihenomenon would result in the case of functional contraction.

KXri.ANATKlX OF Iir.fUK.S

Tlir illu.slrnlioiis wiTc inaili- with tliu aid <if llic H, and I.. drawiriK i-ainorii. tlic del ails l)ciii(5 added frcr li:irid. Tlic tii:iKi>>'<<">t>i»> <>> "H ca.Mc.s is I.VX) diamplcrs, rcduci'd oni'-lliird in rcprddiirtiiin in liKurrs 1 to 1'.'. oiit'-fourlli in timircs 13 to '21. fnlcss ollicrwisc spiM-iKrd the Iccliiiic cniployrd was tixation l>y Zimnirnnanii's alfoliol-nitric-acid soliilion fullowcd liy tlir inin-lipinatoxylin and \*:in (lirsitn's stains.

If) H. E. JORDAN

Kig. 1 Triinsvcrse sections of two liirRc fihors from the poHt-ubdomon. The filx-rs viiry uri'ntly in sliiipo iinil dianietiT in cross-scrtion. The nurici iirc rontr:illy pliiccd in n very (Icliciilcly reticular siircopliisni free of myofibrils; they are rireulnror oval in eross-seetion. The fibers are elosely invested by a sareolemnia of varying delieaey. which does not readily take the Van (iieson's stain. Intimately adherent to llie sarcolemnia is a more superficial fibrillar connective tissue ('perimysium') which stains a more or less deep red in Van (iieson's mixture. The myofibrils are arranged in the form of radially disposed |)lntes, many of which lamellae are in process of longitudinal fission, probably a growth phenomenon. The cleavage is initiated peripherally so that many of the plates have the form of a V or V in cross-section. In b the myofibril-plates are aggregated into columns (the probable analogues of Kiilliker'seolumnsof mntnmalian striped muscle) siiggi'sting in cross-section Cohnheim's areas. The smaller cephalo-lhoracic and leg muscles are nearer the size of the.se myofibril columns. The shape of their cross-section c.\plains the variations in width seen in longitudinal sections of the post-abdominal muscles.

Fig. 2 Portion of a longitudinal section of a post-abdominal muscle in the relaxed condition, including three nuclei. The sarcolemraa is not indicated. The fiber contains centrally a continuous non-fibrillar sarcoplasm; this sarcoplasmic core contains an extremely delicate cytoreticulum and occasional small masses of granules (myochondria). The nuclei have an irregular or spinous contour in longitudinal section, in pari possibly the result of fixation distortion. To the apices of the peripheral projections or ridges are attached the tclophragmata or ground (Zi mendjranes. Where the latter |)ass among the peripheral myofdirils they have a granular appearance. The gramiles stain more deeply and mark the point of attachment of the niyofibril to the telophragmaia (fig. 6). Uesides the telophragmata there can be distinguished in the relaxed myofibrils also the J-and Q-disks. Not the slightest indication of an H-disk or a mesophragma (.M-membrane) appears.

Fig. '.i Portion of a longitudinal section of a fiber including both surfaces, and the i)eriphery of an adjacent fiber, showing besides the above-enumerated features also the sarcolemnui. The latter is regularly festooned between successive telophragmata, to which it is intimately attached. Peripherally, beyond the myofibrils, the telophragma is non-granular. At the left where the two fibers are apposed, the intervening common sarcolenuna a.ssumes a zig-zag character, the telophragmata of the adjacent fibers alternating, and so drawing the crests of the 'festoons' into sharp points. It seems probable that in such cases the adjacent sarcolemmas become intimately blended through the medium of a sparse amount of intervening connective tissue (fig. 4), the close ap|)roximation (and fusion) and the alternation of the opposite telophragmata being the result of a spiral torsion about a common axis of the two adjacent fibers.

Fig. 4 Portion of a paratangential section through the tapering end of a filH>r, and the periphery of .an adjacent fiber. In this case the inter-fiber connective tissue is more abundant, and so ])revents a blending of the sarcolemmas. This illustration shows well also the non-granular character of the extra-librillar portion of the telophragmata. The condition shown in figure '.i coidd be derived from the one here illustrated by an interlocking of the festoons of the adjacent

%$55^ [*|M»(;iIi|'(i"( '//i»//;i;iflll % "' I li!i:-:i i»H u»»»\ ^ f '"I « I q > ,/^' A* ■tJ.^^ v:V KH" ^ 8 10 •CXI^o ...^.H 7 la IS II. K. JOIID.W tiU-r-H. Ml tliHl llif crcHls of mir would lit into (lie troiiKlis lit'lvvccii successive >|i|Miiii(<- fcMtoniiH, ami a 8uliiir(|uriil lilriidiiiK of the siin-olcliinias. FilC. ■*> I'ortion of ii loiiKiluiliiial scclioti of a filicr from the ccplialo-tliorax of Ihc iH"W-lHirii. This lilicr is ill tlu- rcla.vii coiulition. Note the oval, refjularly I'ontiiiireil, a ml more cliroiiiatic character of the nucleus in these youiiuer fillers. At the left is shown the connective tissue, with one nucleus, closely ailherent to the fistooncil luircolemimi. The connective tissue stains red in \un Ciieson's solution: the sarcolenuna remains unstained. When the staining; ))rocess is greatly proIonKi'd the telopliraKXiata as well as the sarcolemiiia arc colored red. I'iK. ti i'ortion of a loiiKitudinal section of a relaxed fiber from the same region, <lis(or(ed liy a coiiii)ression-8( ress aetini; at risht angles to the long axis of the filMT. The line of transverse hending is always at the level of the telophragma, thus demonstrating the close attachment of the myotihrils to the ground-mendirane, and thi' aiisence of an additional niemlirane Iniesophragma) of the nature of the telophragma. Kig. 7 I'ortion of a longitudinal section of a fiher from the .same region at a mid-phase of contraction. .\n additional II-<lisk has now made its appearance. Fig. N I'ortion <if a longitudinal .section of a contracted fiher from the same region, showing the contraction hands alternating with lighter disks of approximately twice the longitudinal width. The positions of the telophragmata are indicated l>y the contraclion-liaiids. At the right is shown the relatively coarser sarcolemma. Kig. !> I'ortion of a longitudinal section of a contracted fihcr from the jawregion. I'pon the contracted condition is superposed a longitudinal traction. This circumstance should l>ring into view the mesophragmata if actually present (as is the cas<> in certain in.sect muscles); but not the slightest indication of such a membrane api>ears. Fig. 10 Longitudinal section of a small fiber from the head region at the exlri'tiiity where it is attached to the chitinous cxoskeleton by a short tendon. The fiU-rs of the latter are directly continuous witli the myofibrils; they stain ~<>mewhal more tleeply in ordinary preparations, and pink after Van Gieson's ?<tain. Fig. II Longitudinal section of a large multinucleated myoblast from the head region. The myofibrils are more compactly arranged and hence caii.se the (iU-r to stain relatively more deeply. \o cross-striations have yet made their ippearaiice. Such appear first towards the extremities in slightly older myoi'hists. and progress centrally. M the lower extremity the tendinous connection is diffcretitiating from the myoblast fibrils. The definitive striped muscle fiber uriws by process of elongat ion of a single myoblast. Such fibers become multinurlented by a process of nuclear amitosis. In slightly older stages as many as 20 nuclei have lieen counted in .scries in the same, relatively short, sarcoplasmcore. Fig. 1'2 Transvers<- .sections of two adjacent young muscle fibers from the hcnd region of this same s|H'cimen. The myolibril-platt's are radially disposed, of relatively short width, and only a few are in process of longitudinal fi.ssi(m. Fig. 13 Transverse section of a smaller traU'cula ( fiber i of the cardiac syncytium from a paramedial longitudinal s<'ction of the heart tube. (Flemming's fluid ;iron-hcnintoxylin stain I. The heart -tubi' wall is one trabccuhi in thickness. STKII'EU Mrs<I,i; OF SCORPION l'.» ^v //, %rj 13 15 &^ /<<lt^r> 14 16 ,' 17 18 19 20 The miclci arc ccnl rally IdcalctI in a granular, very dolicati-ly reticular, sjircopiasin, froc of myotihrlls. The myofibrils are pcripluTally arrangfil in radial InmoUno, which increase in nunil>pr liy InnRitudinal fission. The lamellae are Kalhereil into smaller l)iin«lles, or siil)-tralieciilae. which nnastomos*- freely and thus form with the main traheeulao or fihers a <loul)le syncytium, as in the Limulus heart. The 'fibers' are invested by a delicate sarcolemina, In-tween which and the pericentral myofihril-lamellae is a frequently relatively extensive, coarsely Kranular. non-filirillar sareoplasm. .\t the lower Imrder of this section, at which point the 'fiher' turns from the lateral to the ventral wall of the heart IuIh«, the 20 II. K. J OKI) AN liiiiiflliif iirr cut very iil>li(|Ui'ly. Thin (ilnT is cut »l n piiiiil wliorc the iiitcriiiil synrytiiiiii in iiiiiri* coinimrt, pnilmlily nciir llu'fxtrrinity of the i>ri|;iiiiil myiiMiist. Kig. 14 Portion of n loiiKitniliiinl Brrlioii of a ciiniiiK- tnilx-riila (filirrl from tlip niinu- lioarf. nntcriorly. Tin- intoriiiil sym-ytiiil structure i» horo Honiowlint loowr. (Itlu'r portioiiM iirr still more loosely nrr»nn<'<l. This filipr is in the n-laxfd i-oiulilion. Note tli<- close similarity, an essential iilentity, as also in the ease of I.imuliis, lietwi^en cardiac alitl skeletal muscle I fi(js. 2 and ^), especially from the standpoint of sliajM' and character of these nuclei, and the relation of the telnphraKinata to the luicle.'ir wall and the sarcolemma. I'ii;. I.i Portion of a lon)(itndinal .section of n rnrdiac lrali<'cul,'i in the relaxed rondition, from the same heart. Compare with skeletal fillers, (ignres 3 and 6. Fi(t. 16 Portion of a longitudinal section of a contracted .smaller cardiac tralH'cula. from the same liearl. Note the close series of contraction hands, and compare with skeletal muscle, figures. The shape of the nucleus and the serrated character of its wall, can lie ri'adily intiTpreted in terms of the condition illustrated for the relaxed fiber (liu- ' ' ' ^ which condition can lie conceived to pass into that of this illustration liy .a process of a coincident longitudinal short eiiinf; and a transv<-rse widening of the .sarcomeres (inokommata) and the nucleus. Kin- 1" Transverse section of five adjacent earrliae trabeculae ('fihers'l from a loiiKitudinal .section of the heart tulie of the new-horn. To the left is shown a portion of the peripheral connective tissue ('pericardium'). The traheculae are oval in cross-section with a large central non-fihrillar, hut granular nnd very delicately reticular, sarcoplasmic area, in which the oval nucleus is generally locale<l. The nuclei show the regular contour and the relatively more chromatic condition, characteristic of the earlier stapes of development. The myofiliril-l.'imellae are peripherally arranged in a radial manner. Only a few of the lamellae are untU'rgoiiig a loiigit\idinal radial splitting at this stage. The filler is invested liy a delicate s.arcolemma. immediately internal to which is a narrow granular zone of non-fihrillar sarcoplasm. Fig. IS Portion of a longitudinal .section of a cardiac fiber from the same heart tube, showing the amitotic multiplication of the nuclei, and the intimate continuity between the telo|)hragmata and the sarcolemma peripherally, and the nuclear wall centrally. Pig. ID Portion of a longitudinal .section of a more differentiated cardiac fiber of the same heart, in the relaxed condition. The mn-lear wall is thrown into spinous processes or ridges ti. which are attached the telophragmata. Kig. 'in Tiber from the same heart ami in the same ri'laxed condition, but niechani<'ally shortened iprobably thnnigh the contracting influence of the fixing fluidi, causing thus a longitudinal shortening and transverse widening of the nin-leiiH, and a coincident accentuation of the peri])heral serrations, but without a detachment of the supporting telophragmata. Fig. 21 Similar fiber, fnmi same heart, showing in addition a stage in the process of nuclear amitosis; and a peripheral mass of nucleated connective tissue lat right) closely adherent to the festooned .sarcolemma. IHK EFFECT OF CERTAIN VITAL STAINS UPON THE DKVKLOPMKNT OF THE ECf;s OF CKHKHH ATIM'S LACTEIS. JXHINOKACHNIUS I'AH.MA AND I.OPHUS PISCATOIUUS MARGARET RKED LEWIS From the Harpswell Laboratory, South Har/iiiretl, Me. The stains which were used in the following experiments were those which have been frequently used in observations upon tissue cultures of chick embryos, namely Janus pjeen, neutral red, brilliant cresyli)lue 21), gentian violet, pjrol blue and others. The object was to determine whether any new light as to the action of these stains could be obtained by a study of the effect of these stains ujion the fertilization of eggs and also upon the later development of embryos, especially as to whether the effects of these stains is in any manner harmful or whether these stains are in reality vital stains, that is whether it is possible to stain the granules of a cell without injury to either the cell or the developing embryo. CEREBRATl'LUS LAC'TEfS The cerebratulus eggs proved to be the most plentiful as well as the most favorable material for ob.^ervations upon the effect of the stains upon fertilization and development. The eggs lived and (l(n'(>lope(l normally in the laboratory and were fre(|uently kept in a healthy state until in the late pihdia stage or for time or four weeks. These eggs have a so-called gelatinous membrane surrounding the characteristic egg membranes, which enclo.-^e the egg. The egg membranes have a tube-like protuberance at the end of which is an opening. The gelatinous membrane has been suppo.<ed to become dissohed off after the eggs have lieen in sea water for some (ime (Wilson), but this is not the case as can easily be demon 21 Till: ANAT(IMI|-AI. RECORD, VOL. 13. NO. I 22 MARGARET RKKD LEWIS stnitod by flio uso of stains. The gelatinous inetnbranc Ix'conios inucli loss visible after the e^gs are placed in sea water, but when a few tlrops of .lanus preen are acliled to the sea water this membrane takes up the stilin and can be distinguished as a pinkish blue layer, which has become separated quite some distance from the enclosed egg. The membrane takes up the Janus green stain, but does not readily permit it to pass through, and thus protects the egg from the toxic effect of the Janus green so that unstained eggs may develoj) up to 4 or 8 cell stage within the deeply stained gelatinous membrane before they are killed b}- the stain. Dr. Robert Chambers found that the gelatinous memlirane can be removed without causing any injury to the eggs by l)assing the eggs in sea water througli a ilouble laj'or of ordinary laboratory gauze. The egg membranes can be removed by using a tine cambric handkerchief in place of the gauze. When the handkerchief is used care must be taken not to break the eggs, as fre(|uently happens when the eggs are handled roughly. Bj* thus removing the membranes naked eggs are obtained upon which the stains act more rapidly than ui)on tho.so protected by the egg membranes. Also these eggs which are thus unprotected by the membranes can be stained by Janus green, in which case the toxic effect of the Janus green kills the egg in the same manner as it kills the tissue culture cells. The develoj)ment of the naked eggs obtained in this manner was normal; the j)olar boilies were given off; and the segmentation planes came in as usual, except that the ciliated blastula. instead of being confined within the membranes until the ])iliiiia stage, became at once free swimming and developed into normal pilidia. The eggs of cerebratulus are relatively large spheres full of various granules and globules. The unfertilized egg has a clearer space at one side where there were fewer granules. The unfertilized eggs were subjected to the action of these various stains. In no case throughout the.se observations was the cytoplasm or nucleus stained. Occasionally the nucleolus was faintly stained. When the eggs were placed in a weak solution of Janus green numerous minute granules in the cytoplasm became blue; when VITAL STAIXS TPOX DKVELOPMENT OF EGOS 2!? ))lacp(l in JU'utral rod solution thoro appeared an eciuallj* Rrcal nuniljor of larger rod glohulos; when placed in a double stain of Janus green and neutral red in addition to the blue granules and red globules many unstained globules scarc-ely evident before l)ooamo quite prominent. These are proi)al>ly yolk gloljulos. Kggs stained from ten minutes to one hour indicated no localization of the cj'toplasm. Detailed observations were not made upon the unfertilized eggs. The spermatozoon is larger than that of the sanddollar and the head is sickle shaped. The middle piece is in the form of a niuiid disc, as is so characteristic of this body for most Echinodorms as has been shown by Retzius, and to it is attached a rai)idly moving flagellum or tail. With Janus green, the whole mifldle piece immediately stains blue. In a few moments the blue staining material clumps into one or more ma.sses of granules, which later become more or less extruded from the spermatozo(')n due to the toxic effect of the Janus green. Retzius figures the middle piece of many invertebrate spermatozoa in the form of usually 4, sometimes 5 or granules. In some ca.ses as for instance his drawing of Lucina, shows those granules more or less extruded to one side of the head of the spermatozoiin. This is probably due to a less rapid fixation of these particular spermatozoa. Whoi\ i)lacod in neutral red a small rounil graiuile just above the collar and on one side of the sickle shaped head becomes bright red. There was usually only one such granule, but in a few cases it appeared to lie double. Brilliant cresylbluo 2b stains a granule purple in the same position as that which is stained red with neutral rod and in all probability the same granule stains red with neutral red and purple with brilliant cresylblue 21). With gentian violet the middle piece tirst stains violet, but very shortly the whole spermatozoon becomes a stained diffuse violet, especially the head, the middle piece, and even the small granule above the latter. 24 MAHUAKET KKEU LKWIS KruixouAniNirs tahma (SANnnoi.i.AR) riir ('UK is quite (liffcroiif in sliapc and has differont cf^n imMiibrancs fronj that of the eerel)ratuhis and yet the results in regard to the various stains are practically the same. The sperniutoz()<)n is smaller than that of the cerebr^itulus and the head inst<'ad of heinp sickle sluiped is sjiear shaped, hut the arrangement of the stainahle material is the same, i.e., there is similar shaped middle piece whicli stains with Janus green and above the middle piece on one side of the spear shaped head is a small granule which stains red with neutral red and purple with brilliant cresylblue 2b. Meves ('12) pictures the spermatozoon of Parechiiuis miliaris nuich the same as that of Echinorachnius. Altliough Meves finds that the mitochondria occujiy the middle piece, he does not ilescribe any graiuile on tlie side of the head where the neutral red granule is located in the spermatozoon of Echinorachnius and also in that of Orel)ratuIus. Meves describes only two types of granules in the egg of Purechinus, one the mitochondria, which correspond with the Janus green granules of the egg of Echinorachnius, and the other the yolk granule. The reason that Meves does not describe the neutral red granule either in the spermatozoitn or in the egg may be due to the fact that it is difficult to fix the neutral red granule. The eggs of the cerebratulus and also tho.se of the sanddollar were studied after fertilization in various stages of development, such a,s S cell. Hi cell, etc. The granules which stained with the difTerent vital stains could be differentiated from one another in (lie diffen'iit typesof cells of the embry.os. The stained granules differ in shades of color and size. .VU granules are more deeply stained on the surface of the egg than in the center. The Janus green granules are very small and often are around the edge of the neutral red graimles and in such cases the neutral red granules are .sometimes slightly bluish. I'nder the conditions in which the.se observations were carried out, it was imp()ssil)le to weigh out the stains in order to determine the exact solution of tin- various stains used. .\ standard solution of each stain was made up in sea water, or in a few ca.ses in distilled water, and from this standard solution three VITAL STAINS UPON DEVELOPMENT OF EGOS 25 solutions of oaoh stain wore made by adiliiiK duv or more drops of the standard solution to a given amount of fresh sea water. The three solutions were as follows: a weak solution or one whieh contained so little of the stain that the color could scarcely l>e detected when held over a white surface; a medium solution or one which had a slight color; and a strong solution or one which contained enough of the stain to give it a distinct color. Thi.s strong solution corresponds in color with a 1 lOO.OOO .solution of Janus green, a 1 100, (MM) solution of brilliant cresyiblue 2b, a l-oO,0(M) solution (»f neutral red. and a 1 .lO.OOO solution of gentian violet. By this method the amount of a given stain in the solutions of the same strength in the different experiments remained the same throughout the experiments, although the exact amount of stain in gram weight present in the solution was not determined. Pyrol blue was not soluble in sea water and even when a few drops of a solution of the dye in distilled water was added to the sea water the stain was at once precipitated. This precipitate was not toxic as the eggs continued to develop normally, although th{\v remained unstained. Numerous experiments to determine the effect of the stain upon the fertilization and later develo])ment were carried out with both the cerebratulus and the sanddoUar eggs and spermatozoa. These experiments were repeated many times and were as follows: 1. Spermatozoa were i)laced in strong, medium and weak solutions of the various stains and then after different periods of time a dniji of these stained spermatozoa was placed in fresh sea water together with fresh unfertilized eggs, either with or without the egg meml)ranes. 2. Fresh unfertilized eggs were placed in strong, medium and weak solutions of the stains and after different periods of time were taken out and placed in fresh sea water to which a drop of sjiermatozoa had been adiled. '^. I'nfertilized eggs which had been tn-ated with the stain were placed in fresh ,sea water to which a drop of stained spermatozoa was added. 20 MAHCAKKT HKKD LEWIS 4. I'nfoitilizoil eggs ami sporinatozoa were placed in strong, medium and weak solutions of the stain and left in these solutions. 0. CentrifuRod eggs w(>r(' treated with the stain and later fertilized. (). Fertilized eggs in \arious stages of development, such as the jiolar liodies forming. 1st division plan(\ '2-cel!, 4-coIl, S-cell, Ui-coll, 3'J-eell. t)4-eell, hlastula, gastrula. pilidia antl later free swimming pilidia, were placed in strong, medium and weak solutions of the .stain and left in them. Controls were always made with either normal eggs or normal spermatozoa in place of the staineil ones and only the results of those experiments who.>;e controls developed normally were consiilered. The results of the ten al)ove experiments were as follows: 1. In only a few cases did spermatozoa which had been placed in a solution of Janus green fertilize the eggs, while those which had been placed in weak solutions of neutral red or lirilliant cresylblue 21) for perioils of time varying from two minutes to fifteen minutes did fertilize the eggs even though the granule on one side of the heail of the spermatozocui was colored in the typical manner. SiX'rmatozoa placeil in strong solutions of the stains for long periods of time did not fertilize the eggs. The presence or absence of the egg membranes did not appear to effect the ability of the spermatozoa to enter the eggs. 2. ^Vhen the unfertilized eggs were placed in the .Janus green solution the egg membranes protected the egg from the stain and, even though the outside gelatinous membrane itself became deeply stained, the egg remaineil unstained for some time. When the egg which had Ix'en placed in weak solutions of .lanus green for short periods of time (5 to 15 minutes) were taken out and i)laced in fresh sea water together with spermatozoa, they became fertilized and developed as far as 4 or S cell stage and in one or two cases as far as the pilidia. When the egg membranes had been removed before the eggs were placeil in the .Janus green solution, the eggs were not fertilized and so did not develop. ^^'hen the eggs and the spermatozoa were placed in the VITAL STAINS UPON DEVELOrMKNT OK F.dC.S 27 solution of Janus groon at the same time, the eggs oecasionally became fertilized and developed jiolar l)odies and sometimes (li\ ided once or twice before the Janus green killed them. The lirilliant cresyll)lue 2b and neutral red on the other han<l did not kill the eggs, although the egg membranes did not protect the eggs from the stain. When the eggs were placed in weak solutions of these stains for jieriods of time extending from five to fifteen minutes the eggs became faintly stained, but could be fertilized and frequently developed into normal free swimming l)ilidia. A\'hen the spermatozoa and eggs were placed in the weak solution of the stain at the same time the eggs became fertilized and later develdju'd into free swimming pilidia, although the pilidia were red when in neutral red and blue when in brilliant cresylblue 2b. When a strong solution of neutral red or brilliant cresylblue 2b was u.seil the eggs usually died. 3. \Mien the .spermatozoa and the eggs had each been treated with the stain before they were placed together the results were not so good, although occasionally the eggs stained with either neutral red or brilliant cresylblue 2b were fertilized by si)erniatozoa correspondingly stained and developed into pilidia. When Janus green was u.^^ed fertilization did not take place. 4. The results in this case were much the same as those of 2 and '.i except that only eggs left in the weak solutioas of the stain developed. r>. The centrifuged eggs d('$$'loped ]iracti<'ally the same as the normal eggs except that there was a much larger percentage which died. The centrifuged eggs did not show any localization of the stain. (). The fertilized eggs became increasingly resistant to the toxic efTect of all the stains except .lauus green as development proceeded. AA hile the early stages of development up to the blast ula were easily killed by even the medium solutions of the stains, the later stages of development lived and contimied to develop in the medium solutions and frequently pilidia liv»>d for .some days in the strong solutions of the stains except Janus green and gentian violet. .lanus green, on the other hand, was more toxic for the pilidia than for the unfertilized eggs. All 28 MAIUJAUKT UKKl) LEWIS staK«'!< of tlrvclopiiu'iit wliicli wcri' |)n)t('i't('il by tlio ckk incml)rancs lived for a short wliilc in the modiuni or in the weak solutions of the Janus preen, but the hiter stages of developni('i\t which were free swinuiiing, were rai)idly killed, even by the weakest solution of the stain. The free ends of the eilia ininiediately nmtted together into deeply stained blue clumps which shortly became torn away from the emliryo. A few experiments serveil to demonstrate the fact that .Janus green cannot be considered a vital stain in the above sense, for not only did it usually prevent fertilization of the- eggs but also in every rase where the solution of the stain was of sufficient strength to color any of the granules of a cell of the embryo, not only was the particular cell killed, but also the embryo itself died. The same was true in most instances where this particular gentian violet was used. The gentian violet was not the same as that used in tissue cultures, but was a stain acquired through the kindness of Dr. Neil at the Harpswell laboratory and was in all ])robability a more toxic stain than the one previously used in tissue cultures in wliich growth of cells occurred when the medium contained a very small amount of gentian violet. On the other hand brilliant cresylblue 2b and neutral red each proved to be true vital stains, for the embryo continued to develop and the cells to divide, although certain granules of the ceils were colored and the embryos were kept in a weak solution of the dye. In many exi)eriments the granules of the cells were .so deeply colored that the red (neutral red) or blue (brilliant cresylblue 2b) embryo which resulted could be seen with the naked eye. While the toxic effect of the \arious stains is in a way in proportion to the concentration of the stain in the .solution, the depth of color with which the stain appears in the granule is only to a small extent dcjjendeiit upon the concentration of the stain in the .solution. When the piUdia are left for twenty-four hours or longer in a solution of either neutral red or brillant cresylblue 2b, which contains so little of the stain that the color can hardly be detected, the granules of the embryo take up the stain until it api)ears in the granules in so nmch greater concen VITAL STAINS UPON DEVEL0PMP;NT OK EGOS 29 tratioii thiit the embryos can l)e seen as miiiuto rod or blue bodies in the solution. One of the intcrestitiK results of these observations is the fart tliat the spermatozoon and the unfertilized egK proved to be the least resistant to the toxic effect of all the stains, while the older the embr\o develoj)ed the more resistant it became. However a sufficiently stronp; solution of any of the above stains, except pyrol blue, was toxic and caused the death, not only of the egg or young embryo, i)ut also of the later embryo. Each stain seemed to have an affinity for a certain type of granule. In the egg and later stage of development .hmus green stained the small round granules, which are scattered abundantly throughout the cytoplasm. In the spermatozoon the Janus green in every case stained only the granules, which lie within the middle piece. In the fertilized egg and later stage of development botli the neutral nnl and the brilliant cresylblue stained definite large and medium sized granules and in the spernuitozoon only a very small granule .situated at one side of the head and just above the middle piece. .V double stain could be obtained by means of .lanus green and neutral retl or brilliant cresylblue 2b. Neutral red stained the large granules dilTerent .shades of red. pink or yellow, while brilliant cresylblue 2b stained the same type of granules various shades of lavender or purple and with this stain the large granules frequently became difT(>rentiated into a pink globule or vacuole which contained one or more puri)le granules. If an embryo was stained with neutral red and then later by brilliant cresylblue, the blue stain replaced the red in the granviles and so far as could be observed diil not stain any difYerent types of granules from those previously stained by the neutral red. Cientian violet stained diffu.sely both the granules which can be stained by .lanus gnvn and also those which can be stained by neutral H'd or brilliant cresylblue "Jl). In the sjiermatozoon only two kinds of granules wea-e ob.served, thos«> of the mi<ldle piece which can be stained by means of .lanus gi-(H>n and the one above the middlepiece which can Im> stained bv means of neutral red or brilliant cresylblue 2b. In 30 MAIUJAUKT KKKD LEWIS the t'Rg thoro were observeil tlire*^ kinds of granules, those which cjin l)e stained by meatus of Janus preen, those which can lie stained hy means of neutral red or brilliant cresylblue 21) aJid also a large granule which reniainiHl unstaiiu^il and which may be a yolk granule. The behavior of the various sizeti gi-anules or globules which stain with the neutral red or the brilliant cresylblue 2b corresponds with that of the so-called vacuole described by lx>wis and Lewis in th(> cells of tissui^ cultures. I.ul'llIlS nSCATOIiUS lANCLKK I'l.sil OK (lOOSl:! KISII) A few obser\ations were made upon the eggs of Lopliius piscatorius, which were brought ijito the laboratory in the gastrula stage. Unfortutiately these eggs were not obtaiiied again during the sinnmer. so that observations could Jiot b<^ made upon yoiuiger stages, and these obser\ations upoji the older embryos covdd not be repeated. However, they are given below l>ecau.><e they suggest that certain extremeh' interostuig results may be obtained by the use of neutral red upon the developing eggs of this form and possibly other fish eggs. A mass of the eggs of Lophius piscatorius was put into a weak solution of neutral red in sea water and the remainder of th«> eggs was kept iji normal .sea water. The water was chaitged once or twice a day and the eggs in the normal se.a water develo|)ed into normal fre«> swinunijig embryos, while tho.se in the neutral i-ed develojx'd marked abjiormalities. The mo.st evident of these was that the chromatophores did not develop in the normal number aixd what chromatophores did develop remained small more or less round cells with black granules. The chromatojjhores of the normal embryo develop into large cells with many ramifying processes full of black gi-anules which fonn a gray network over the great(M' part of the embryo. The neutral red embryo remained more or less transparent, while the nonnal (^mbryo b(>came dark gray and more or less opaiiue. The yolk sac of Lojiliius is filled with a colorless transjmrent fluid in which is sus]K>nded a consjiicuous yell()W oil droplet and in the normal embryo it is not possible to di.stinguish the boundaries of the yolk sac. When the young embryos were VITA I, STAINS UPOV DKVKLOI'MKNT OK KtJGS 'M placed iji tlic luutral rod solution, certain numerous granules within the cpitlicliul cells became stained red, although the cytoplasm of the ceil remained unstained and very shortly after this the red color appean-d in the yolk and contijiued to U- taken up by the yolk until the yolk became many times pinker than the solution of neutral red in which the embryos were placed. This sphere of reil fluid is cojisiderably smaller than the membranes around it and the oil droplet did not take up the stain Imt remained a clear yellow. When such a pink stained yolk sac was punctured the epp; membranes collapsed and the deeply stained, transparent fluid nii)idly flowed out and quickly became .so diluted with the surrounding medium that it could no longer be distinguished from it. The clear, yellow oil droplet usually remain(>tl within the collapsed egg membranes. The action by means of wliich the neutral red was deposited in the fluid within the yolk did not correspond with that of a simple (lifTusion, Init it apiiear(>d to be ii-ssociated entirely with th<> large neutral red grauuU's which seemed to take up the stain from the solution of the stain in the sea water which surroimded the egg and to give it up again to the fluid within the yolk sac. In the embryos wliich had develoixnl in the neutral red solution the cells of the central nervous system contained so many red granules that the neural tube appeared pijik. This pink color of the central nervous system persisted throughout the various stages of de\elopment. .•Vnother esjiecially interesting abnormality in the development of these eggs of Lophius in a solution of neutral red was that, although the heart and the chief blood ve.>*sels ileveloped, there was jio circulation of blooil cells withux the blood vessels and heart. The heart did not begin to beat quite as soon as did the heart of th(> normal eml)ryo, but eventually the heart contracted normally. No blootl cells were ever ob.-^erved to pjiss through the heart or the blood ve.ssels, although the embryos romained alive in the neutral red solution for several days after the circulation of blood cells had Ihmmi establi.><hed in the normal embrvos. 32 MARGARET UKED LEWIS Tlio lM>lmvior of tlic (•hr<>iiuitoi)h(»res and thv lack of a cirrulatioii of blood cells sopin to indicate that the neutral red may have sonunvhat the same ('ffect upon the developinn fish einliryo as that of alcohol upon I'undulus e^K^ observed bj- Stockard, in which case the chroniato|)hores remained small round cells and no lilood cells were observed in circulation, although the heart contracted rhythmically. l-'ischel ("Oil carried out lunnerous experiments with various stains upon the embryos of the frog and also those of the salamanth'r. Fischel found that Ris!narckl)r;uiii. neutralrot, neutralviolett, nilblauchlorhydrat and nilbluusulfat are all true vitals stains, i.e., that the embryo continued to live and develop although stained. Fischel describes the same tj'pe of neutral retl granule as those described above in the embryo of Lophius. From his observations he concludes that the neutral red granule is a living plasmatic element. Fischel does not describe any changes in the chromat<)i>hores or in the circulation of the blood in the amphibian embryos which were stained with neutral red. This may be due to the fact that the embryos were first stained and then transferred to clean water, while in these observations the embryos were permitted to develop in a weak solution of neutral red. It was impossible to obtain these eggs again, but it is hoped that some future summer it may be jiossible to acquire enough of the.se fish eggs to carry out a more elaborate series of exix?riments. The study of the spermatozoa, the unfertilized eggs and the later stages of development led to the conclusion that not only the embryo, but also the unfertilized egg and the sjx'rmatozoon react in the same maiuier as the cells of tissue cultures of the chick embryo do to the stains, that is they contain granules which are stained blue with Janus green, probably the mitochondria, and others stained red by neutral red or purple with brilliant cre.sylblue 2b. The Janus green staiii is toxic and resulted in the death of the embryo just as it caused the death of the ti.ssue cultures, while the neutral red and brilliant cresylblue 2li are less toxic and the embryo may continue to live and develop, although the granules in' the cells are stained. These two large groups of VITAL STAINS UPON DEVELOPMENT OF EGOS 33 granvilos, tho Jiinus proon pranulo und tho nrntral rod (franiilft, may \h' furtlicr ditTcn-iitiatcd liv tlic uso of other staiit- Midi as p>Tol blue, l)rilliant cresylblue 2b or iiile blue. In addition to these two types of K'a""'*'^- the e^R and certain cells of the enibrj'o contain yolk rIoIjuIcs or fat glol)ules. Dr. Paul CI. Shipley, who has made a careful study of the neutral roil Rranule, has suggested the te^rm vacuoles of segregation for this body and has studied its behavior in detail. The results of Dr. Shipley's obsorvatiojis will l)e i)ut)lisli('([ iji the near future. In the discussion of the plastosomes of the living cell (p. tiOS) Prof. .1. Duesberg states that a number of authors have claimed to have stained the jilastosomes in the living cell. The mo.st used dyes according to Professor Duesberg are neutral red and methyleneblue, whil(> dahiiaviolett appears to be the most satisfactory stain. Daliliavioiett wixs first u.><ed by von la Valet ta St. George and after him by Hemieguj' i'Otij ajid Fauro Fremiet ('10). Michaelis ('00) and Laguesse ('05) found Janus green very satisfactorj', wliile Renaut ('11 ) used methylviolett and C'iaccio Cll) used brilliant cresylblue. The graiuiles stained l)y these different ob.servers cjuoted by Professor Duesberg camiot be the same granules and therefore not in every ca.se the mitochondria for n<Hitral red d<K\s not stain the same granules as those staineil by Janus greeii except in the ca.se of the granules within certain, not all, of the vacuoles described by I^wis and I.ewis ('15) (p. 3S2). The rea.son for the discrepancy in the results of the abo\e writers is without doubt due to the condition of the tissue sujjjjosed to be livijig but which iji many ca.ses wjls probably dying, as can be seen from the following facts. The action of most of the socalled vital stains cau.^es tho death of the cell more or le.ss rapidly and a concentrated .solution of almost any of these stains will kill the cell. In ca.ses where animals are given increasing doses of stain until the body tissues are stained, the cells when i)la((>d und< r the microscojH' show first one |)i<-ture ajid later as the cells begin to die the siime cells show (piite a different picture. This can easily be tlemonst rated by a study of a living embryo such as the ceivbratulus pilidia in which the death of tlie i)iiidia cause s brilliant cresylblue 2b to fade out }4 MAK(;.\ltKT iu;ki) i.kwis of tlic Iiirne uninuU's aiid at the same time to bo taken uj) l)y the niitochoiidria. This sjiine phoiinmenon has boon dosfribcd by I^'wis and I^wis for the action of iiile l)hu' upon tii<> grajuiles of the tissue culture cf'lls, in wliich ease the vacuoles of the living cell were stained i)ink l)y means of nile blue but when formaldehyde vapor was ])assed ujuler tlu^ cover slip the color faded out of the vacuoles and the mitochondria then became stained a blue color. The results obtained with pyrol blue and possibly with other staiiLs may also lead to confusion because of the change iji color shown by certain of the granules (I^vi, '16). I have observed in tissue cultures that when th(> cells first grow out (12 to 24 hours) into a medium which contaijis jiyrol blue, lioth the many mitochonilria and the few neutral red graimles are stained blue. As the growth increases in age the neutral red granules become more and more dwply stained and also increase in uuiHlx^r, while the color fades out of the mitochondria, so that the cells of a seventy-two-hour growth contaui numerous very dark blue granules and many vnistained mitochondria. rischel states that the nucleus becomes diiTusely staiixed when dying and in previous ob.servations upon the cells of tissue cultures, the germ cells of the grasshopper as well as in the observations above, it has been fovmd that the slightest trace of color in tlie nucleus is an indicatioji that the cell is ijijured. This does not hold however for the nucleolus, for the nucleolus maj- remain faintly stained with pyrol blue through several gcncratioas of cells and also the chromosomes may be faintly stained with certain stains during division. Observations upon the living embryo and also upon the cells of tissue cultiu-es show, when the cell has not been injured by the staiji and the nucleus is not stauied, that the cytoplasm of the cell consists of a homogeneous ground substance which contains certain gianul«>s which may l)e stained by means of neutral red and certain others which may lie stainetl by meaas of Janus green, and in addition to these two tyjx-s of granules there may Im' pre-sent fat globules which do not stain with either neutral red or .lanus green. VITAI, STAINS ll'O.V DKVKLOPMKNT <IF y.C.iiH 35 ()l)s<'r\iitit)iis li;i\(' shown tlic proscnf*^ of tlic .hirius Kn***!! KruiuiK' ami also tlu^ neutral rcil granule in the nerves, the red blood eells, the endothelial cells, the ectodenn, the endodenn, the suKtoth muscle eells, the striated nuisde eells and the connective tissue of tlie chick embryo and also in various embryonic tissues of tiie i)ifj;, th(^ (ish, the firasshoi)|)er. the hermit crab, the cerel)ratutus, the sanddollar and the sea urchin. Thc-e granules an* i)r('sent in tlie germ cells of the grasshopiMT, the cerebratulus and the sandilollar and in all probability they are jiresent in all embryonic animal cells. 1,1 IKUATIKK CITKl) fl) DcKsiiKKii. J. I'M- I'lastosonicri ".\pparato Kotirolare Iiitorrio unci Chromidialapparat." Er|;cb. dcr Anat. und KntwirkliinRResch.. lid. 20. l2) FisciiKl.. .\. l'.M)l rntcrxuchiiiigi-n iibor vitaU- riirlmiig. .Viiat. Ileftp, Bd. 16. Heft. I. (3) IiK.vi, Ci. 10l(> Dciiioii.strazionp dclla lint lira rondrinscimiru drgli oricanuli (■('llulari I'liIoraMli col Moil pirrolo in cellule coltivati 'in vitni.' Ueale .\ccadeinia doi Liiicei, vol. 25. (4) I.KWis, .\t. It. .\\t) LKWl.-i, W. H. 191.1 Mitochondria and other ryto plasmic structures in tissue cultures. .Vni. Jour. .\naf., vol. 17. (5) Lkwis, .M. K. and Robertson, W. R. 1916 The mitochondria and other structures ob.served by the tissue culture method on the male K(^rni cells of t'hortliippus curtipennis Scucld. Biol. Bull., vol. 2.'), no. 2. (6) Mevbs, Kn. 1912 \'erfolKung des sogenannten .Mittelstiickes ties Kchini dens periniuins in befruchteten Ki bis zum Knile der Furchengsteilung. Arch fiir .\Iikr. Anat.. Bd. SO. (7) Hetzhs, (i. 190I Zur Kenntni.'j der S|K'rmicn der Kvertebrateii. Biol. I'nters., Xeuc Kolge Bd. II. (8) Retzics. (!. 1910 t'ber den Bau des Kies der Echinodcrmen in unlw fruchteten und Ijefruchtclen Zustand. Biol. Unters., Neue Folge Bd. 1.-.. (9) StockaUD, t". R. 191.") .\n experimental analysis of the origin and rela tionship of blood corpuscles and the lining cells of these vessels. Proceedings of the National .Vcadeiiiy of Sciences, vol. 1. p. 556. lit)) WiusD.N, I'. B. The habits and early development of Cerebratulus lacteiis iVerrill). The (^uarl. .lourii. of Mirr. Science, vol. I;{. p:irl I. new series. TUV: l^LOOI) OF ALI.IC.ATOll MIS.SISSIPPIKNSIS AMIKKT iM. KKP:SE DeparlmenI of Zoology, West Virginia Univeritily F.IOHT FIGURES METHODS In this study of the blood of the alligator both fresh and stained prejiarations were useti. The blood was obtained from an animal kept in the aboratory, by making a small slit between the ventral abdominal scales; the wound ([uickly healed. This operation was reiM»atetl whenever a new preparation was needed. By sealing the cover with a ring of oil, to prevent access of air, the fresh blood could be kejit in normal condition for many days, during which ix^riod liie amoelioid activity of the leucocytes could l)e studied. In making stained preparations various fixing fluids were used, but the ordinary dried smears gave the liest results. Of the stains used the best results were obtained with hematoxylin and eosin and with Wright's stain. THK l•;K^■ll^HOc^TKs The red cells of the alligator's blood are, as is well known, of the usual elli])tical form seen in the lower vertebrates. According to (iulliver (2) there is some variation in the cells of closely related species. He found (3), as will also be noted below, that the corinisdes of dried blood are appreciably smaller than those of fresh blood. He says that in Crocodilus acutus and in an unknown s|x»cies from V< ra Cruz the length of tlic corpusclf is somewhat less than twice the breadth. TIIC ANaTOSIIC-AL MECORD, vol.. 13, N(l. 1 JS ALBKUT M. RKESE Oil the other h:ii\(l, Maiull (4) wlio studied C'. lucius says the lonntli is two or three times the width. In only three cases of several dozen nieasurenu'iits made of the corpuscles of A. mississippiensis did the writer find the length as much as twice the width, and in no case did it aiijiroarh three times the width. In ( ". acutus tlulliver gives tiie avera^jje leii{i;th of the corpuscles as 20.31+ micra; the average width as 10.93+ micra. In C. fissi|x^s the average length was 1".).S5+ micra; the average width was lO.T'.t + micra. .Vs will be seen below, these variations are not nearly so great as those seen among corpuscles from the same individual animal in the Florida .Vlligator, A. mississippiensis. Milne-Kdwards (5) gives the following measinrments, presumal)ly averages; A. sderops, length 123. <S(). micra; width 13.33 micra; \. lucius length 20.83 micra; width 11.11 micra. In A. missis.sippionsis a considerable number of measurements was made, both of fresh and of stained corpuscles. The thickness of the corpuscles was also measured in a number of cases; this can be ilone only with the fresh blood where corpuscles can occasionally l)e seen in jirofile. The average length of the fresh corpuscles was found to be JO. 77 micra the average width 12.78 micra; and the average thickness 4.17 micra. .\s noted above, and as will be seen later, these measurements are apparently greater than those of .stained corjiusdes. The longest corpuscle found in the fresh blood was 24 micra; the widest was 14.00 micra: the thickest was 5 micra; the shortest was 18.50 micra; the narrowest was 11 micra; and the thinnest was 3.60 micra. .\mong the stained corpuscles the average length was 18.69 + micra; the average width was 10.8.5 micra, both measurements being noticeably less than in the fresh blood. The longest stained corpuscle measured was 22.10 micra; the shortest stained corpuscle was 14. SO micra in length, only 0.8 micra more than the width of the widest fresh corpuscle. The widest stained corpuscle was 12.80 micra; the narrowest was 8.10 micra. BLOOD OK ALLIGATOIt MISSISSIFPIENSIS 39 Among all the forpusclos. hoth frosh and stainod, tlu> one that showed the greatest dilTerenee Ix'tweeii the length and the width was one that wan 22.10 niicra long and 10.40 niiera wide. On the stained slides, for obvious reasons, it was not possible to measure the thickness of the corpu.scles, but the length and width of the nucleus were measured in each case. The greatest variation in the length of the nuclei was from 3.S0 micra to ti miera; in width from 2. SO miera to 4.30 micra: the average length was 4.S.") micra, the average width was H.titi micra. The appearance of the erythrocytes, as seen in the flat. Is shown in figures 1, la, \h, and Id-h, drawn, as were all of the figures, with a camera lucida under an oil immersion objective from a stained slide. The proHle view, figure 1 c, wis, of course, drawn from a slide of fresh blood. .\s would l>e expected from the measurements given aliove. the ellipse varies considerably in ditTerent corpuscles. In the proHle the central thickening is jilain but the nucleus could not be determined. The cytoplasm, so far as could l>e determined, was homogeneous, though it was examined under a magnification of 23(M) diameters. The cytoplasm was so transparent that, on the ordinary stained slide, when one corpuscle lay over another the nucleus of the imder corpuscle showed witli the same apparent distinctness as that of the upper cell. This .structureless condition is in contrast to that descriljod by Bryce (1) in I>epidosiren, where he figures a clear band lx»neath the membrane, a fine network throughout the cytopla.sm, an<l one or more clear areas and vacuoles in the cytoplasm. Whether examined under a magnification of KMM) or of 2300 diameters, the cytoplasm in .\. mississippiensis apixvin-d the same. Possibly more R'fined methods of technic might have brought out some details of structiu-e in the cytoplasm. The nuclei, as indicated by the measurements and as seen in figures 1, \(i. Ih, etc., vary both in size anil shajx*. though they are usually ellii)soidal. They stain easily and darkly but not homogeneously; sometimes small unstained or more lightly stained areas are scattereil fairly evenly throughout the lujcleus, as in figure I; sometim(>s larger and more irn^gular artvis are 40 ALKKKT M. UKKSE so<Mi ill Viirious parts <if tlic iiudcus as in linurc \ii. Occasionally a iiucU'Us is located at one cml of tlu' cell instead of its usual central position, and soniotiines a nucleus is seen at each end of the coll, figure 1 g. No instance of mitosis was seen on any slide exaniin(>d, l)Ut erythrocytes are occasionally found with two closely adjacent nuclei, figinc 1 <■. which would s('<>in to have just resultetl from an amitotic dixision. I'Ijiiut If seems an evid<>nt case of amitotic tlivision of the nucleus just before the completion of the process, ^^^lile the cells in which two nuclei are found are usually of large size and elongated form, but one case could be found in which there was indication of a division of the cell body. This cell is .shown in figure 1 /i.it is j)ossibly an artifact, but it is difhciilt to .see how the nucleus could have been pulled apart by artificial means as is shown in tlie figure. Quite infrequently erythrocytes of the form shown in figure 1 (/ ai"e seen. It would, at first, seem possible that these were halves of just-di\-ided cells, but if this were the case they should frequently be founds in pairs, while, as a matter of fact, it is but seldom that two of them are found in the same micro.scopic field. It is possible that they may be comparable to the spindle cells found in frog's blood, but it seems more likely that thej' are merely artifacts. 'lUK I.KfCOCVTK.S On a slide of fresh blf)od, mounted with a ring of oil to j^revent access of air, as noted al)o\e, the amoeboid activities of the same white corpuscle may be studied for several days, though after a few days the motion is so slow that it can only be determined by making a .series of drawings at intervals of several mimites, as must sometimes be done to demonstrate the changes in shajM^ in amoeba. In the fresh blood it is diflicult to identify the various types of leucocytes that may be seen in the stained blood, not only because of the lack of stain but also because the pseudopodia are usually more or less withdrawn in the stained l)lood. I'igure 2 shows one type of leucocyte as .seen in fresh blood. The general outline of the cell is circular and a number of small, RLOOl) OK ALLKJATOl: MISSISSIPPIKNSIS 41 sliiirply-poiiitcd i)scu(l()j)n(liii project frimi its ix-riplicry. lifinn uiistuiiicd, tlic nucleus is indistinct or invisihle, hut one or more small vacuoles may be soon. The cell is filled with fine, unevenly distributed graiuiles which chjuiKe their appearance as the cell rh.iiiKes its sh:i])e. Sucli Ji c<'ll, while it chanfjes its shape but little, changes (juite rapidly — about as fast as the changes se<»n in an active leucocyte in frog's blood. Another type of leucocj'te, seen in fresh blood, is shown in figure :{; it is coarsely granular and changes its shape quite rapidly and markedly. In stained preparations of alligator's blood .several types of l<>ucocytes may he distinguished. Of these the most numerous is shown in figure 4; since it stains with hemato.xyliii rather than with eosin it miglit he thought to be an extruded nucleus from an erythrocj'te exce])t that it is several times the bulk of su<'h a nucleus. It is of fairly large size and is usually circular in outline though the sha]je is variable. It is possible that it is a cori)Uscle in which the nucleus is verj' large and tlH> cytoplasm is so reduced as to he invisible. This would seem possible from the fact that occasional cells are found with a very large nucleus and a very thin peripheral zone of protoplasm. Of almost, if not (juite, as frecjuent occurrence as the cell just described is a smaller tyjw shown in figures rt. n n, 5 b, and 5 r. This cell may, ijerhaps, correspond to the lymphocyte in the human blood. It varies considerably in size and shape but contains an oval or circular nucleus and a small amount of cytoplasm which generally gives the cell a pointed or spindle form, as seen in figures 5 b and 5 c. Another type of leucocyte that is fairly coimiion is shown in iigures (> and ti a. These forms might, perhaps, be called mononuclear leucocytes: they are large, some of them being larger than any of the other types. The cytoplasm is clear or very finely graiuilar, and stains, with eo.sin, a pale pink color. The nucleus is very large and of an oval or circular outline: it does not stain so darkly as the nucleus of the erythrocyte. The outline of the cell is usually circular or polygonal. The most striking in appearance of all the leucocytes. an<l, possibly witii one excei)ti(>n, the least numerous, is the ty|)«« 42 ALUEKT M. K£ESE that may ho coniparod to tlio rosiiiophilc roll of niainnials. It is a largo, usually circular coll tiiat may at once bo rocogiiizod by its coarsoly-gratmlar cytoplasm that tako a strong pink color with oosin. The nucleus is usually round or oval, and generally lies cl()S(» to one side nf tlic coll. as shown in figure 7. Occasionally two nuclei in a single cell may 1)0 soon, figure 7 h, as tiiough li\ di\ision of the larger nucleus; and an occasional elongated nucleus, as seen in figure 7 a, would seem to indicate an impending amitotic division. No ca.se was .soon in which there was any indication of the division of the coll as a whole. In many of these eosinophile cells, especially in those in which the cytoplasm did not take the stain, there was seen a heavy outline, like a thick coll wall, possibly caused l)y a iiorijjhoral zone of denser protoplasm; this appearance was usually most marked on the side of the cell farthest from the nucleus. This type is the most imiform in size and shape of any of the leucocytes. The least immerous typo of leucocyte, if indeed it be a distinct tj'pe, is shown in figures 8, 8 a. and 8 b. Among the tens of thousands of erythrocytes seen, on several difTorent preparations, but throe of this possible type of leucocyte were seen; it is this extreme rarity that raises the doubt as to their being a normal type of corpuscle. They are all of rather small size and not very irregular outline. The cytoplasm is clear or verj' finely granular. The nucleus, or nuclei — figure 8 h shows no less than eightare so dark as to be almost a solid black. Whether these are really a nomial element of the blood or are some artifact or other abnormality it is difKcult to determine. LITER.VnUK CITKl) (1) Bryce, T. II. 1904-5 Tho histology of the Mood of the larva of I^-pido siren parudoxu. Trans. Hoy. Soc. Kdiiil)., vol. 41, no. 9, pp. 201-311 and 435-69. (2) GcLI.IVER, G. 1840 On tho l>lood corpuscles of the Crocodilia. Pro. Zool. Soc. London, vol. 8, p. 131. (3) IS42 On the blood corpuscles of the British Ophidians, Ucptilcs and other oviparous vertebrates, ibid, vol. 10, pp. 108-11. (4) Mandl, 1S.39 Note sur les Rlobules sanguine du Protde et des Croco diliens. Ann. dcs He. Nat., 2 .^eric, T. 12. p. 289. (5) Milne-Rdward.h. A. 1856 Note sur les dimensions de globules du sang chcz quclquc vertcbres. Ann. des Sc. Nat, T. 5, pp. 165-7. BLOOD OF MAAC.ATOH MISSISHIPPFENSIS U KXPLA NATION OF IICilTtES All i)f the figures \viT(> drtiwii with a canicrii liicida under the same mugoification, I'j oil immersion olijoelive uml no. S e(>iii|M>nsating; orular. FIrh. I, 1 u, 1 6 Three views of erythrocytes seen in the flat ; figure 1 c is a normal erythroeyte seen in profile, drawn from a slide of unstained and living Motni Figure 1 (/ is one of the rather unronimon pointed erylhrocytca, which may be 44 ALUEllT M. KEESK Miiiiply an iirtifart. I'iKuri- I <' is ii ri'il im-II willi, apparently, a jiisl -divided nu<*lcii8. I'imirc I /i.x a cell in wliieh the niiflruH is in process of division. Kin'irc 1 (7 in n rod cell with a nuclens at each end. KiRiire 1 h represents the single ease that was found that seemed to show an erythrocyte in which the entire cell was in process of division. Figs. - and '.i Uepresenl two types of leucocytes drawn from living blood while CNhiliiling amoel>oid niolinns; figure .'? represents a more active cell than figure 2. and one in which the granules are coarser. Fig. 4 Heprescnia a type of doiihtful character, which may he simply a cell with an enormous nucleus and almost no cytoplasm; it is the most common of the leucocytes. F'igs. 5, .") n, ,")/>, and ,5 c Represent a type of small leucocytes very alnindantly represented. Figs, (i and 6 a Represent a type of large mononuclear cells that may possibly lie the same as the one shown in figure 4. Figs. 7, 7 a. and 7 l> .Show three very charaelcristic cells that seem to correspond to the eosinophile cells of mammalian blood; they are very coarsely granular and generally slain strongly with Eosin. Figs. .S. S n, and S b Represent a type of small and unusual cells that arc so s<>ldom seen as to make it seem doubtful that they are a normal constituent of blood. A PERSISTENT SUPERIOR VKNA C.W A SINISTRA IN THE CAT TRANSMITTINCi CORONARY HLOOI) S. B. GUANT Washington University ONK FIIJI'KK The anomaly of the vascular system stated in the title was foimd (hiriiiR a oourse in comparative anatomy in the Zoolop^Department of Washington University. At the suggestion of Dr. E. A. Baumgartner, a study of the literature of this form of variation was untlertaken, the results f)f which are Iiere given. This anomaly was found in a young and a})parenlly nonnal eat. Upon ojiening the thoracic cavity, a long slender vein, of uniform diameter, was exposed, which reached from the left innominate vein to the coronar>' sinus, about 4 mm. distant from its atrial end. This was recognized as a left suix-rior \ena c&va. The coronar>' veins were all of normal size ami (hstrihution, antl their oix>nings into the coronaiy sinus were nonnal. But, the coronary sinus ended blindly about o nun. short of the jwint at which it should have opened into the right atrium. At this end of the sinus it received the vena cordis media. No rr>mnant of a connection could ho found upon the l)lind sinus end or the riglit atrial wall. Upon tracing the anomalous vena cava upward it was found to ojien into the left innominate vein at the point where this vein joined with the right to form the nonnal superior vena cava (fig. 1). In the upiM>r third of its extent the left superior vena cava received the sujx^rior intercostal vein, which was coTuposed of two intercostal branches. Ui)on examining the ulterior of the right atrium, no trace of an o]x>ning of the coronarj' sinus was seen. However, the wall 4(> S. n. (iRANT hrn* was thin. No Tlu'lK'sinii valvo was prrsent. The valve of the inferior vena cava, howcMT, was well (level<»|)e<l. A large ilistinct foramen ovale was present. I have found two references besides those p^iven by McCotter CIO)— l>eCat, Beyerlein and Hutton — of anomalies similar to the one under consiiieration. According to Marshall ('50), W. ^fien. D, y'^non S Fig. 1 The heart and vessels viewed from the left and dorsaliy. It shows the course of the coronary sinus and left superior vena cava. Enlarged one-half. I^eCat ol)st>rved (17."^S) a coronary vein which emptied into the left subclavian vein in an eight days old child. Hutton ('15) found a case in which the coronarj' sinus ended in a sunken pitted area against the wall of the right atrium. Within th(> riglit atrium there was a shallow circular deiiression c(jrrespt)nding to the coronary oix-ning. There was no Thebesian valve, but a considerable remnant of the left venous valve. Hutton suggested two explanations of the closure of the coronary' sinus. The partition between the sinus and atrium was either; first, a composite structure, the result of fusion between VENA CAVA DRAINING CORON'AKY SINUS 47 tli<' coronury scfniK'nts of the ri^lit hikI left vonous valves; or, socomi, tho rosiilt of an unusually voluminous 'riwlx'sian valvp, which ha<l cvcnlually fusod with tho marRins of the ostium of the sinus. l^oyorlein ('14) described a case in a fift<'<'n months old child in which the coronary sinus extended to the wall of the ri^ht atrium. I'pon ohsorvinp; the interior of the right atrium, all tlH> openings apix-arod normal, but, upon probing the crironary sinus it was found to b(^ closeii. The Theijcsian valve. nf)t iK'ing mentioned, was evidently normal. Beyerlein offered two possible ('xplanatif)ns of his case: viz.: inflammation of the endocardium and atresia, although he siiw no evidence of disea.se; or, a mechanical influence due to suction after the formation of the left innominate, plus pressure in the atrium from the right superior and inferior venae cavae. (iruber CSo), descrilx'd, in a fifty years old man, a coronary sinus without any atrial opening. In the right atrium in the plac(> of the usual coronary afXTture, there was a small groove leading into a lilind i)ouch (i nun. deep, (iruber tentatively called the free membranous margin of the groove the Thebesian valve, .\nton Siding ('9lj) also observed a case in an adult male in which the coronary sinus ended blintlly lo mm. short of the site of its o])ening into the right atrium. Within the right atrium there was a narrow aperture, guardetl b^- a feebly developed Thebesian valve, opening into a blind sac 10 mm. long In all of these cases the coronary simis drained through a {M>rsisting left superior vena cava. The fact that the left .superior vena cava, of the present case, opened into the left innominate so close to the right superior vena cava, instead of some distance from it, is oiusily explained when one reflects that in the cat, with its narrow thorax, the transverse branch coimecting the two superior venae cavae becomes shorter and larger as the embryo increa.ses in size until the left supi-rior v<>na cava may ai)]H>ar to ojx^n into the right at an acute angle. The closure of the normal ostium of the coronary sinus probably took place after the transverse branch betwe<Mi the two 48 S. B. CiHANT siijH'rior vonao cavac had Ikmmi foniuHl. As Ihitton Clo) su^H«'sts. (his closun' may liavi- lu't-ii duo to an visually lar{;(' Theliosiau valve, which may have closed it too effectively when j)rossun> was exerted upon it from the inside hy the flow of blood into the atrium. The left superior vena cava being still open, the coronary blood would tind an easy path to the right atrium through it, the left iimominate, and the right superior vena cava. If this suiiposedly large 'I'hebesian valve fused with the wall and pennanently closed the normal opening of the coronary sinus, the portion of the latter, or left duct of Cuvder, between the wall of the right atrium and the proximal end of the coronary sinus may have ilegenerated. It has been suggested by Beyerlein ('14), that the suction in the left .superior v(>na cava, caused by the ru.sh of blood in the left innominate vein would aid in closing the normal sinus oix^ning. That the Thebesian valve, which is foniied froni the caudal portion of the right valve of the sinus venosus, may have been abnormal and possibly concerned with the closure of the coronary opening, is indicated by its absence and also by the presence of a patent foramen ovale. If th«' left venous valve were abnonnally small, it may have resulted in the failure of the foramen ovale to close, and in such a case, the right venous valve would be likely to be unusually large, anil, consequently, the Thebesian valve as well. Gruber's and Siding's cases are similar to the present, in that the coronary sinus ended before reaching the wall of the right atrium; but differ in that in my case there is no cul-de-sac or ojHMiing in the atrium at the site of the normal ostium of the sinus. Ilutton's and Beyerlein's cases differ from the writer's and the other two, in that the coronary sinus in these cases extends to the wall of the right atrium, where it is closed. Hutton's second suggestion as to causes of the anomaly seems to suit the case here described, but the fusion of tlu^ Thebesian valve to the atrial wall in my case must have taken place so early in development that no traces of it are left. Coupled with this may have been the mechanical causes suggested by Beyerlein. VENA CAV.V OKAININd fORONARY SIVUS 4'J MTKKATl lii; ( ITKIJ Hkykrlkin, K. I'.il I Die iM-rsistirreiule Venn oiiv.i mipcriorc Bin iatrn nia AbfltiHsrolir fiir(ln.sCoronarvcncnl)liit. Fninkfurtfr Zeitxeh. f. Path., Hd. 15. CIrvukk, \V. IS85 Anittoiiiischc Notizcn. An-li. f. path. Aniil., Hd. 19. HuTTO.N, \V. K. Ull.'i .\ii iin<>ii>!ilo\i8 roroimry sinus. Jour. .Vniit. I'liyn., vol. 4'J. Keibel and M.\ll 1912 Muniiid of Humiin KnihrynloKv. I.ippincott. Philadelphia. LeCat, 17.'{8 Hi.stoirc dc raeudemic dcs Scicndos. Pari.s, IS40. (Quoted from Marshall.) Makshall, J. 1S50 On the development of the Rrcal anterior veins, etc. Phil. Trans. Royal Soc, London, vol. HI. McCoTTER, U. K. 1916 Three ea.ses of the persistence of the left superior vena cava. Anat. Kec, vol. 10. Siding, A. 1890 Ueher den .Vhfluss dcs Sinus coronarius cordis gegen den rcehten Vorhof. .\nat. .\n7,.. Bd. 12. THE VOMF.HO-NASAL APPARATl'S IX r-HnVSKMVS PUNCTATA AND RANA CATE.SiilANA ROLLO E. M COTTER Department of Anatomy, University of Michigan, Ann Arbor SEVENTEEN- FIGURES In a previous communication the writer ('12) described the vomoro-nasal apparatus in the opossum and other mammals. It was sliown that the Noniero-nasal organ, the voiner()-nasal nerves and the accessory olfactory bulb are parts of a special olfactory jiicchanisin the sjx'cific function of which still nMuains doubtful. It is with the ide^i that a careful comparative study of this apparatus in the different animals may lead to a more definite understanding of its function that this study was undertaken. The obser\'ations about to be reported were based in part on serial sections of the heads of turtles and frogs and in part on ilissections of jirepared s|x>cimens of the .siime six>cies. Wax ]ilate reconstructions of the olfactory apparatus were made to show, so far as possible, the form and comjiarative size of its compojient parts. Figures 7, S. 1.") and Ki represent drawings of these reconstructions. CHELONI.V Two views have been advanced as to the structure that should be designated the vomero-nasal organ in these forms. One giouji of observers believe that the vomero-na.sal organ exists iji a very simple cojulition, and that in some sixM-ies it forms a shallow fossa covered by neuro-epithelium situated on the metlial wall of the nasal cavity, while in others the neun>-epithelial 52 . Ill >I. 1,1 1 K. M( CO'ITKIt an>a Ims <>xl<'iul('<l on to tlic anterior and latofal walls of the na>;il fossji. Another kiou]) of workers cluiin that the voineronnsnl organ is a rudimentary structure and consists of a small duct that extends from tlie surface of the septal mucosa caudalwanl in the sMhnnicosji and ends hlimlly. AccordijiK to Seyilel's {'{Hi) conununication the nasal cavity m Chclonia may be suhdi\ idotl into a cranially situated pars olfactoria and a mow caudally situated pars respiratoria. The vomeronasal *)rgiui belongs to the last named subdivision. In testudo graeca one can obser\e the vomero-nasal organ occupying a sliallow fossa on the medial wall. The separation of its epithelium from the neuro-ei)it helium of the olfactory region is completed through a narrow intervening zojie of indifferent epitluv lium. The \'entral and lateral portions of the pars respiratoria exhibits jio iunu-o-e]iit helium. II<> states that in emys europaea the pars respiratoria is more comjilicated than iji te.studo. Here the neuro-ejiitheUiun that comprises the vomero-nasal organ is foimd in four fo.s.sae which occupy the medial, the two sitle walls ajid \\\v floor of the ]>ars respiratoria. It is separated from the neuro-i'pit helium of the pars olfactoria by a low ridge that is covere<l by inditTerent epithelium. iSeydel accepts the view that the neuro-(>pit helium iji emys has extended from the medial walls onto tlie floor and side walls of the pars respiratoria. For an explanation of this ^'ie\v he refers to the course of the ner\'e fibers of the pars respiratoria. The olfactory fibers extend from the medial wall downward ajid cm-xe lateralward IxMieath the floor and upward on the lati^ral wall where they subdivide mto branches. These relations have resulted from a condition where the neuro-epithelium oc(U|)ied a small an^a on the medial wall juul has extended to the anterior and lateral walls. In one embryo of C'hrysemys punctata Seydel observed that the neuro-epithelium of the vomero-nasal organ occupied a small area on the medial wall while on the floor indifTcrent epithelium was foiuid. In another the neuro-epithelium had ext<'nded from the medial wall onto the floor of the pars respirat oria. VOMERO-NASAU AI'FAKATUS 5^} Miluilkovics researches on emys europaea has lead hini to differ us to the position, fonn and structure of the vomero-nasal ornan. He lieliexcs that the \()nier(>-n;is!il ornan is a rudiinentury structure in tliese forms. He descrihcs this organ a« a small, blind, tubular structure extending from the surface of the septal mucosa caudahvard in tiie submucosa. It re<'eivcs at it« distal extromity the ducts of the medial nasal glands. Zuckcrkaiidl (10) observed a sjx cimcn of em\'s europaea and corroborates Seydel's important anatomical observations. In regard to the structure that Mihalkovics has dcsigruited the vomero-nasul organ lie is of the opijiion that it is the duct of the medial nasal glands. Zuckerkandl further observed that the olfactory nerv'es arise from two areas of nasal nmcosa. A dorsal branch aris?s from the mucosa of the j)ars olfactoria and a ventral branch receives tilamcjits from the vomero-nasal area. The dorsal and ventral branches unite to form a common olfactory nerve that jmsses through a large opening in the cranium togetlier with the nerve of the opposite side. .As these nerves ajjproach the olfactory bulb in their course caudahvard the nerve bundles become separated, the dorsal branch forms a large lateral bundle that distributes filaments to the ai)ex and tlie ventral surface of the olfactory bulb and extends tlorsiilwards over the medial and lateral surfaces. The \entral branch becomes the more slender medial ramus that sends filaments to the upper half of the inedial surface and to the dorsal surface of the olfactory bulb. cnUYSKMVS pinc:tata The naj^al fossa in chrysemys punctata consists of a principal nasjil chamber that conununicates anteriorly with a circular naris by means of a small cylindrical nasal pas.sjige and posteriorly with the choana through a larger posterior na-^il canal. Hy referring to figure '■] it will be seen that the princijuil nasal chamber is oval iji transvers<> .section with the greatest diameter in the ]M'rpt>ndicular direction and its .shortest diameter in a horizontal plane. The anterior i'a.«al pas.sjige conununi TUK ANATOMICAI. HKCORD. VUL 13, No. 1 54 KOLI.O K. M.COTTKR call's \vitl\ it oil the iintrrior wall al)<)Ut half tlic distance Ix'twocii the roof and tlie floor. 'I'hc posterior nasal eanal extends horizontally caudalward on a level with the floor. The otherwise smooth interior is interrui)ted hy many low ridges which course generally in an antero-posterior direction and suljdivide the cavity ijito numerous fossjie of varying sizes and depths. H«'giiuiing just above the communication between the prineijMil i\iv.sjil chamber and the anterior nasjil jiassage are two ridges, one of which (>xtends caudalward over the medial wall, the other in the s;inie direction over the lateral wall ajid become less proi\otmced as they ajiproach the i)osterior wall. The.se ridges which are covered l)y respiratory epithelium separate completely a large fossa in the roof of the principal nasal chamber. This fossa is covered by olfactory neino-epithelium and gives origin to the olfactory nerves, lielow the two ridges mentioned aljove is an extensive fossa that occupies the lower half of the anterior wall, the anterior jiortioji of the floor and adjacent portioii.s of the medial and lateral walls. This area which has a very irregular outlijie is covered by the ^omcro-nasal neuroepithelium and gives origin to the vomero-nasal nerves. By ref<^rring to figure 2, which is a transverse section through the anterior jiortion of the jirincipal nasal chamber, it will be seen that the vomero-nasal oigan occupies a suigle and extensive fossa situated on the lateral, medial and anterior walls of the nasjil chamber. By following this fossa caudalward in serial section it will be found that it becomes subdivided into four areas (fig. 3) by the apparent invasion from the cautlal direction of three low ridges capped by respiratory epithelium and thereby giving to the vomero-nasal organ the apix^arauce of occujiying four s<^j)arate foss;ie. The olfactory neuro-epithelium (figs. 7 and 8) occupies the roof and adjacent j)ortions of the meilial, anterior and lateral walls of the nasal cliamber. It extends lowest on the anterior wall where it covers one-third the ilistance from roof to floor. From this jioijit the border grailually recedes donsjillj- until the caudal wall is reached. The vomero-nasal neuro-ei)ithelium VOMERONASAL APPARATUS :Vi occupirs the lowor p<irtioji of tlio antrrior, modial and Iat4>ral walls ami (loor of tlic nasal cliainlKT. It ocfui)i(>s a siiigU- irn-RUiar fossa and is separated from tlic olfactory neuro-«pitholium by low rid)j;f's covorod 1)>' respiratory epitlicliuni. The reinairiinj? portion of the wall of th<^ nasal chaiuijer is covered tjy respiratory epithelium. I'ii;. 1 A Iransvfisi' si-ctioii of tlio hoad of a turtle at aliout the middle of the anterior nasal canal. It shows the form of this portion of tiie nasal fos.ia. X 10. Fig. 2 .V transverse section of the head of a turtle pa.ssiiig througli the anterior portion of the principal nasal chamber to show the form of the na.sal fossa and the [losilion and distribution of the voinero-na.sal mucosa. X 10. The anterior na.sal pa.ssage extends nearly horizontally eaiidalwaril from the naris ;md communicates with the princijial nasal chamher about midway betwivn the roof and tloor. It is nearly cylindrical in outline and presents a low ridge that courses oblicjuely in a caudo-lateral direction from a metlit>cephalic ori{!;iir. 50 Udl.I.O E. McCOTTEK The i><)storior nasjil cjuuil ('Xt«')i(ls nearly horizdntnlly caudalward from the ])riiu'i|)al luisal (•lian\l)C'r. It is seinicircular in cross section. Attachcil to the anterior half of the lateral wall and to the cephalic portion of the roof is a crescentic valve-like fold that separates a dorsjvlly pla('<'d blind pouch that opens caiidahvard. Fir. .1 A transvonto sprtion of the head of a turtle passing through the middle iif the prinripal tmsal chainlirr showing its form and size, the distriliution of the vomero-nasal and olfactory ncuro-fpit helium and peripheral course of the vomero-nasal ner\'e8. X 10. The nerse fibers from that portion of the ncuro-epit helium of the vomero-na.sal orsan situated on the lateral wall collect into two limbs :ui anteri()r and a posterior. The former is the smaller and courses downward in the lateral wall of the nasal fossa. The latter is a broad flat band that passes medially lieneath the floor and is joined by the anterior limb. VOMERO-NASAL APPARATUS 57 Thoso c<)inl)iiicd Hlamunts form u broad shwt of imrvo fil>ers coursing mediallj' Ijeneath the floor to the iiiediul wall aiid at the same time receiving iidditioiuil fibers from the neuro-epitheiiun). In the medial wall the broad flat band of nerve filx^rs b«'come» sonuiwhat narrowetl and thickened and courses dorsally and somewhat caudally to the roof of the nasal fossa. Here it is joined by the bundle of olfactory nerves of the same side and the Kij!. 1 A transverse seotion of the head of a turtle passing through the middle of the posterior nasal oanal. It shows the form of this passage and the relation of vomcro-nasal and olf.ntDrv iii-rves in tlic^ir cuiirsi' tlinmuh tin- ininiiim. X 10. combijied filaments of vomcro-nasal and olfactory fibers of the opposite side. The combiiie<l filaments form a large round nerve bundle that courses caudahvard through a large o|x>ning in the cranium to the cranial cavity, .\lthough the right ami the left vomero-nasal and olfactory nerves course tlirougli the cranium together the nerve fil)ers of the lUtTerent bundles do not intcrmijigl(\ They lie contiguous to one another and may lx» separated from each other with little effort ivs will be s<H'n by ix'fcrriiig to figure 4. 58 UOI.LO K. McCOTTER As th»'y «!nter tho cniiiiHl rnvity tho vnmoro-nasal uml olfat'tory nones soimrato: the fornuT jmss dorsiil to tlic ulfactory nerves and to the ilorsnl surfuco of the olfactory l)ull) where the filaments spread out o\'er tlie \'oiner<>-iuisaI area. Thi> ner\e libers of tlie olfactory neuro-epithelial area collect into numerous filaments that course tlorsahvard and converge to form a large oval bundle above the roof of the nasal fossa. The bujulle of olfactory nerves lie lateral to the vomero-nasal fibers in its course through the large opejiLng in tlie crauiuni. P'ig. 5. A transverse sertion through tho anterior portion of the olfactory bull>s of the turtle to show the relation of olfactory and vomero-nasal nerves. X 20. I poll reiidaiig the cranial cavity olfactory^ fibers separate from the vomero-nasal fibers and pass ventralward to be distributed to the olfactory area on the apex and ventral surface of the olfactory bulb. The olfactory l)ulii is an ovoid mass extending horizontally forward from the forebrain. It is separated from the latter by a well definetl f)bli(iu<> groo\e that defines a verj* short olfactory peduncle. By referruig to figures .">, (>, 7, and 8 it will be seen that the surface of the bulb is sub-divided into two definite and separate arenas. An oval area occujiyijig the entire dorsal surface and upper half of the medial surface of the bulb to which the vomero-na.sal filaments are distributed is the vom«*ro-nasal V< >.MKIU)-NASAL Al'i'.VUATL'.S 59 ana of the olfactory 1)1111). This arra is homologous to the ufcessory olfactory hull) of inainnials. Thcr olfactory an-u <)f the bulb is somcwluit inoro extensive. It occupies the apex, aii<l ventral surface ami extends 8onie distance upwaril on the fi' .■»«• Set molnuiM* Ki^. (■) .\ trnii.sversc section fhrough the niiddle portion of the olfactory bulb to shiiw the relation nf the vnnioro-nasnl ami olfactory areas. X 20. lateral and medial surface. The filaments of the olfactory nerves are dislrit)Uted to this area. \n extension forward of the fore hrahx cortex separates the vomero-nasal and olfactory areas of the olfactorv bulb. Viii- ~ -V nieilial view of a wax plate reconstruction of the olfactory apparatus of the turtle to show the origin cour.xc and distribution of the vomero-nasal and olfactory nerves. X 3. Fin. .S A lateral view of a wax plate reconstruction of the olfactory appuratu."! of the turtle to show the oriisin, course and distribution of the voniero-n;is;i! and olfactory nerves. X 3. 60 ROI.LO K. McCOTTKR 111 transverse section (fig. (>) the olfactory bulb oxhil>its a large ventricle oval in form. One can (listinpuish tlie (liff«'rent concentric lay«>i-s that have Ixm-ii ilescrilxHl for tiiis portion of the brain in other forms. The ntMve fiber layer is incomplete and presents a dorsjvl .•ieRnient of vomero-nusal filxTs and a ventral st^gnient of fila olfactoria. The latter complet(^ly encircles the olfactory bulb in the lower mammals. The glomerular layer presents a dorsal and a \entral segment. The former is much thicker than the latter. Then follow the molecular, nerve cell, tile granular anil <']M'n(lyiiial layers in order. U.VNA The excellent ilescription of the nasal fossa in Rana by Kcker and ( laupp has been frequently consulted and, as far as possible, the same terminology has been used in this conununication. While he recognized tho origin of the olfactory nerves from the olfactory mucosa by two l)ranch<'s -a large dorsal and a small ventral —and that the vomero-na.sal nerves join the dorsal ramus, he faileil to determine the further course and termination of the vomero-na.sal nerves as a separate bundle. He states that the olfactory nerves on enterijig the cranium separate into two roots, an anterior distributed to the antero-ventral surface of the olfactory bulb, and a posterior root that is distribut(>d to the acces.><ory olfactory bulb. Zuckerkandl ('10) was the first to recognize a separate vomeronasjil ajjparatus for the amjihibians. He describes the formation of the common olfactory nerves as of fibers formed by the union of the olfactory and vomero-nasal nerves but fails to state the relations of vomero-nasal and olfactory nerves in their pas.sage from the nasal cavity to the brain. RAN.V C.\TKSBIANA .Although the na.sal cavity of the frog has been very carefully descrilied by Kcker and (;au!)p and in text books on comparative anatomy it apjx-ars to me advisable, owing to the complexity of the arrangement of its subilivisions, to summarize briefly its more important features. VOMERO-VASAr- AI'PAUATI'S 61 The nasul cavity of tho frog is situated in tin; ant<Tior part of the crauiuiii. It is very much flattmed dorso- vent rally and expands anteriorly anil laterally so that m a dorsal view it presents a semicircular outline. The anteriolateral curved Fig. A triinsver8e section throURli tho head of the frog in the region of the external nasal opening. It shows the furni of the principal and middle nasal chambers and the distribution of th(j olfactory mucosa. X 2.5. Fig 10 A transverse section through the head of a frog posterior to nuris. It shows the relation between the superior, middle and inferior nasal cavities and the distribution of the olfactory mucosa. X 2..5. Fig. 11 .\ transverse section through the vomero-nasal organ of the frog showing the relation of the superior, middle and inferior na.sal cavities and the distribution of the vomero-nasul and olfactory mucosa. X '2.'i. Fig. 12 .\ tnmsverse section tlirough the middle of the principal nasul cavity of the frog showing distribution of olfactory mucosa and thejrelation of vomeronasal and olfnctorv nerves. X 2. .5. margins follow closely the curvature of the maxillae. It consists of two parts, the ri^iiht and the left nasal fossae. l'>ach fossa comnumicates with the exterior by an external luksal aiH>rtu«\ the naris, and with the oral cavity by an oval internal na.sal ajierture the choana. (•.2 Kiil.l.it K. MiCOlTKK IIk' nnsjil fossa is subdivided into several irn'Kiilar foninuinicatiiip cliainlicrs whicli lia\e lu'come separated to a greater or less extent dvirinn the pn)eess of development, by tlie ingrowth of septa anil ridges. It, therefore', presents for description a Fi(j. 13 A transverso soction tlir«uf;li llic clioana of thr hoad of a froj;. It shnnx the ilistrilmtion of tlio olfactory murosa anil the relation of the vomeronasal and olfactory nerv-es. X -.5. suix'rior or principal, a middle and an mferior nasal chamber, a lateral recess, the \omero-nasul organ, and two well defined narrow connecting channels, the infunilibulum and the isthmus. The principal na.sal cluimber occupies a dorso-medial jiosition and comi)rises al)out time fourths of the fossa (figs. 15 and 10). By referring to figure it, it will be seen that a transverse section FiK. 14 \ transverse section of the head of a frog passing through the posterior part of the !<up<'rior nasal cavity. It shows the distribution of the olfactory mucosa and the relation of the vomero-nasal and olfactory nerves. X 2..5. of the cophalic portion of the superior cliambor has a circular outline and coninumicates with the exterior by means of the naris. .\f the lower part of the lateral wall can be seen the V< t.M KKO-NASA U A I'PARATIS 63 plifu (crniijuilis whicli iiuirks off a dcn^p Kroo\(> which is the ix'Hijiniiig of th«' iiifuiidihuUini. In this rcj^ion the principal nasal cavity is covered for abtmt tliree-fourths of its circumference l>y olfactory neuro-ei)itheliuni. Ccv.Np pa*. IB HjmwMo-fimtmi. Fig. 15 A modial view of a wax plate reconstruction of the nasal fossa of the frog. It shows the origin and peripheral course of the vomero-nasal and olfactory nerves. .\l)Oiit three limes natural size. V'tR. Ill .V dorsal view of a wa.\ plate reconstruction of the nasal fossa of a frog showing the origin and peri|)lieral course of ihc vomero-nasal and olfactory nerves. About three times natural sine. Fig. 17 Kepresentsa dissection of theolfaclory apparatus of the frog showing the origin course and distribution of the vomero-nasal and olfactory nerves. Two and one-half times natural size. Figure 10 r(>pix'sents a cross-section of the principal luisal chaiiilHTs ixisterior to the naris. It pre.>ients the liepinning of the ijifunihbuluni separated from the principal luusjd chamlMT by 64 KOLLO K. McCOrrEH the broadenctl plica tennlrmlis. It may be seen tliat the olffartory iicunM'pithi'liiiin covers tlie floor, the medial wall ami tlu' unat^T portion of the roof. The remaining one-thinl of the cireumfenMice is clothed hy respiratory mueo.sa. The principal na.sjil chamber in liKure II present.s a n«'arly circular outline and communicates at its ventro-latcral margin with the middle nasal chamber by means of the infundibulum. The olfactory neurorpit helium covers about three-fourths of its circumference. The lateral wall is covered by simjile mucosa. In figure 12 the principal I'.a.-^jd chamlxr presents the form of an inverted hor.se shoe. The lateral limb of which comnumicates through the isthmas with th«> lateral recess. The olfactory eminence which extends dorsjilly from the floor aids materially iji givijig the peculiar form to this jwrtion of the cavity. It will be seen tliat the olfactor>- neuro-epithelium has become separated into two areas by a narrow intervening zone of ijidifferent mucosa. One area covering the olfactory eminence and a more extensive layer lining nearly all of the medial and lateral walls and the roof. Figure \'A repn^s(>nts a section passing tlu-ough the choana. The principal nasitl chamber has an outline similar to that of figure 12. It conununicates directly with the choana. The olfactory eminence has increased ui height and breadth. The olfactory neunM'pit helium occupies two areas on the circumference of the chami)er. One caps the olfactory eminence, the other covers the upjjer part of the medial wall and about the medial two-thirds of the roof. The former is more extensive and the latter less extensive, than showji in figure 12. These two areas are separated by a broad intervening zone of respiratory epithelium. Figure 14 re])r( sents a section posterior to the choana. The princijjal na.>*al chamber sIkjws marked reduction ui size and has a semihuuir outline. The olfact<jry eminence is very much flattened. The olfactory neuro-epithelium caps the olfactory (•minence and covers a |)ortion of tlu> medial wall and roof of the na.sal chamber; the n-maining circumference in this region is covered by respiratory epithelium. VOMERONASAL APPARATUS 05 The small iniddU' nasal rhambor is sitnatfii vfntro-lateral to the cephalic cxlrciiiity of the princiiKil chainhcr. It is much flatt<»ne(l ilorso-ven trail y and broad in a trans^'erse direction (figs. 15 and Ki). The naso-iachrymal duct communicates with its postero-lateral angle (fig. 10). Posteriorly it conununicatcs with the principal nasal chamber through the anterior part of the infundibulum (fig. 11), and more caudally with the inferior nas;il chainl)cr. In fact, the middle chamber appears to be merely an anterior sacculated expansion of the tear duct. It is lined by simple mucosa. The inferior nasal chamber is an elongated, tran.sversel\' placed cavity lying ventral to the cephalic extremity of the j)rincipal nasid chamber. It is directly contijmous laterally with the lateral recess and medially with the vomero-nasal organ. The inferior nasal chamber as shown in figure 10 is an obliquely placod cavity, oval iji outline, and clothed by ordinary mucosii. In figure 1 1 this cavity is shown in direct conmiunication laterally with the lateral recess and metlially with the medial recess, the vomero-nasal organ. It is everywhere covered by simple mucosa. The lateral recess is the direct latero-caudal extension and expansion of the inferior nasal chamber. It is oval in outline and follows the curvature of the maxilla. It communicates anteriorly with the inferior nasal chamber (fig. 11). medially with the iirincipal chamber through the i-sthmus (fig. 12), and more caudally with choana and oral cavity (figs. 13 and 14). It is lined by simple mucosa. The vomero-nasal organ is a cup shaped structure that lies at the medial extremity of the inferior nasal chamber and communicates directly with it laterally. It is clothed by neuroepithelium (fig. 11). The infundibulum is a broad flattened chaimel which permits communication between the anterior portion of the principal nasal chamber and the middle and the inferior nasal chambers. The isthmus is an obiitiuely placed .slit (fig. 12), broad in a sagittal plane that serves as a meiuis of communication between the i)rincipal na.sal chamber anil the lateral recess. 6(5 nOLLO E. McCOTTKR It will 1)0 soiMi from the fon-noijiR Jiiul by rcforriiiK to fipiires 9 to l(i iiiclusivo timt the i»<Miro-(>i)it helium is found in two sopanito and distinct ronions of thr nasal fossa. Ihv olfactory niucosii consists of a very oxtonsivc and irregular area oi\ the wall of the principal nasal chamber. From an extensive area covering tiie anterior wall and tlie adjacent portions of the floor, medial wall and roof as shown in figures 9, 10, and 11, the olfactory mucosa extends caudahvard in two strips or zones separated by intervening zones of respiratory epithelium, figures 12, 13. and 14. The vejitral, caudal prolojigation cov<>rs the olfactory eminence and gives origin to the small ventral branch of the olfactory ner\'es. The dorsal, caudal prolongation covers a variable jxirtion of the medial juid lat«'ral walls and the roof of tlie principal nasal chamber. This portion of the olfactory mucosa together with the extension forward on to the anterior wall gives rise to the large dorsal brancli of the olfactory nerves. The vomero-nasal nuicosa lijies the wall of the cui>shaped vomero-nasal organ and gives rise to the vomero-nasal nerves. .Ml the remaining portion of the nasal cavity is lined by respiratory ej)ithelium. It will be seen from the foregouig description that the vomerona.sal apjmratus in the turtle eriuals in size and importance that of the ordinary olfactory mechanism. That th*^ olfactory bulb exhibits an olfactory and a Aoniero-nasal area which share about equally in its formation. In the frog, however, the vomeronasal ap|)aratus apparently performs a secondary roll in olfaction. It is very small comjwired to th(> olfactory mechanism. The accessor>' olfactory bull) is situated on the lateral surface of the hemispiiere caudal to the olfactory bulb and is only about one twenty-fifth the size of the olfactory Indb. Hy referrijig to figures IJ, 13, and 14 it will be seen that the olfactory ner\'es collect into two sejiarate groups. Filaments collecting into nerve bundles on the dorso-medial wall of the suiM-rior chamber fonn the large ilor.sal ranuis of olfactorj' nerves and filaments collecting ijito ner\e bundles from the mucosa of the olfactory eminence form the small ventral branch VOMEUO-NASAI- AIMWRATUS 07 of tho olfactory iutvcs. Tlwsc Ijiiiiiclics coursf cuiul.ilwanl and join to form a single bundle at the caudal cxtroinity of the principal nasal chamber. From this point it courses to tho olfactory bulb where it becomes distributed over the antero- ventral surface. The vomero-nasal nerves formed by filaments from the dorsal, ventral and medial walls of the vomero-na.sal organ form a single roujuled bundle that courses dorso-caudally in the medial wall of the principal nasal chaml)er. At the caudal extremity of this chamber it joins the bundle of olfactory nerves. Although there is a slight intermingling of the vomero-nasal ajid olfactory bundles the mriority of the fibers of the former can l)e followed in their spiral course caudalward where they wind Ix'neath the olfactory nerves to gaui the lateral side of this bundle and course over the lateral surface^ of the olfactory bulb to reach the acce.s.sory olfactory bull), which lies more caudally (fig. 17). liti:r.\turk citkd Ecker-Gaupp. Anatomic des Frosches. MrCoTTEK, R. E. 1912 The connection of the vomcro-nasul ni-rvcs with the accessory olfactory hull) in the opossum anil other mammals. .\nat. Rec, vol. 6, p. 2(t!Ml.S. MlHALKovK's, v. 1S9S Na.senhiihle uiid Jacobsonsche Organ. .\nat. Hefte, B(i. 2. Aht. 1. s. .-J-IOT. Seydki,, O. I.S9.5 (Ibor die Nasenhohle und das .Jucohaon'sche Orpin der Amphihien. .\Iorph. .Jahrh., Bd. 2.3, S. 4o.'}-")4.i. l.S9() Cher die Nu.senhohle und Jacobsonsche Organ dor Land und Sumpf-schildkroten. Festschrift zum 70 Geburtstage von C. Clegenbaur, B. 2., 1896. ZucKEKKANDL, E. 1910 Cber die Wechselbeziehung in der .Vusbildung dcs .lucobsonschen Organs vmd des Riechlappens. .\nat. llefte. Hd. 41, Abt. 1, S. 3-73. u HISTOLOC;UAL STUICTIKK UF TIIK UKl llACTOIl PENIS MUSCLE OF THE 1)0(1 HOMKR G. FISHER From the Anatomical Laboratory of the Johns l/upkins University FOUR FIOUnES (two PLATES) During the past few yearti, physiologists and chemists have been working with the retractor penis muscle of various animals, chiefly of the clog. The muscle has in every case been rcgarcieil by these workers as composed of non-striated fibers. The result* ()l)tained in these investigations have been discordant, (liiTcriii^ in some regards from the customary reactions, both physiological and chemical, of other smooth muscles. At the suggestion of Dr. Charles D. Snyder, this study of the histolog>' of the retractor penis muscle was undertaken, for it was felt that any theories of smooth muscle contraction, arising from a study of this retractor nuiscle, must be based upon an established histology. The retractor penis nuisde in the dog is a cord-like structure, pale and translucent in its anterior portion but somewhat darker and more fleshy in its posterior fraction. It has it* origin by two separate bundles of fibers, one from each side of the sphincter ani nuiscle. These bundles pass ventrally and at a distance of about 1 cm. from the sphincter join in the median plane and run forward o\('r the ventral surface of the corpus spongiosum to the base of the glans where the fusetl bundles are insert eil into the corjnis spongiosum. In the medium-sized dog of 6 to 10 kilogiams, the nmscle has a length of about 50 nmi. while its diameter is about 3 nun. throughout its whole extent. The nuiscle is .^jurrounded by a dense fibrous sheath, continuous with the fascia covering the sphincter. On both sides of this are the bulbocavernosus muscles; these are attached partly into th(> sheath of the retractor penis muscle and partly into the (ill Tin: ANATllUICAL RBCOnD, VOL. 13, NO. 2 jvLT. igi7 70 HOMKR (i. FISHER fihnnis tissue of a incdian rai)li(> mi the ventral side of that muscle. In tliis study the retractnr iicnis muscles of fi\'e adult dogs and of one puppy were examined. The nmscles were removed, fi.\pd in Houin's picro-formalin-acetic fluid and embedded in paraffin. The sections were stained in hematoxylin and eosin, iron lumatoxylin and Mallory's connective tissue stain. One m\iscle was cut serially and another was removed with the adjacent part of the bulbocavemosus intact; this block was sectioneil for the jnirpose of determining the r(>lations of the two nmscles. Representati\e sections were examined from different parts of the other retractor muscles. A study of the sections showed in every instance that the retractor nuiscle is mixed, i.e., composed of both smooth and striated fibers. The anterior three-fifths of the muscle is Composed entirely of smooth fibers (fig. 1) while the posterior twofifths is made uji of fibers of both types (fig. 3). The mimber of striateil fibers in the jjosterior part is variable but their presence is constant. The proportion of striped muscle varied in the specimens studietl from one-third to one-half. The proportion of striated fibers was least in the muscle from the ]iuppy. .\ characteristic field in the anterior j)art of the nuisde (figs. 1 and 2) shows large spindle-shaped smooth muscle cells varying in none of the essential features fnmi the usual text-book descriptions. The cells for the most part have their long axes parallel to the long axis of the muscle but there is some tendency to interlacing of the fibers and bundles of fibers (figs. 1). With Mallory's connective tis.sue stain the muscle plasma takes the characteristic red color. Sections treated witii this stain show the amount of connective tissue to be abundant. This tissue is partly of the white fibrous variety, but there is distributed throughout a relatively great number of elastic fibers. In certain of the sections (fig. 2) the.se fibers have an undulating, wavy appearance. The nuclei of many of the smooth muscle cells in such a region tend to as,»!ume spiral fonris. Mcdill ('09) has described this phenomenon in smooth muscle from the walls of arteries and has reviewed the literature on the subject KETRACTOR PENIS MUSCLE OF DOG 71 particularly in regard to its rauso and signifirance. The spiral form may$$v duf, as is suggested, to tlie 'active or pa.s.<ive" contraction of the muscle cells, or it may be related to the shortening of the elastic tissue. While the cause of these spiral nuclei cannot be determined from the evidence at hand, it may be noted that in the tissues in which McCiill described the phenomenon, there is also associated with the muscle element a relatively large amount of elastic tissue. In the sections from the retractor muscle studied, the spiral form of the nuclei is most abundant where the elastic tissue is most undulating and presumably in the most shortened state. The microscopic structure, then, of the anterior three-fifths of this nuiscle is (juitc characteristically that of any smooth nmsde, with, however, a considerable interlacing with yellow elastic tissue. In the posterior portion of the muscle, the histology is (luite different (figs. 1 and 3). Interspersed with the typical Inindlcs of smooth muscle cells are frequent small fasciculi of striated nmscle fibers. These fasciculi are often broken up with the individual fibers diverging somewhat from the original j)lane. In other i)laces, a few single anil isolated cro.ss-striated fibers are found in the midst of dense smooth muscle fascicuU. In gen(>ral, however, the grouping of the striated fibers is in small fasciculi, alternating with eciually small bundles of the non-striated variety. This rather general arrangement can be made out in a low-power photomicrograph of a characteristic field in the posterior two-fifths of the muscle (fig. 3). In this, it is seen that the general direction of tlie fasciculi is parallel to the long axis of the muscle. Under higher magnification, the cross-striated fibers are found to possess the typical structure of such voluntary muscles from other parts of the body. The .striation (tig. 4) is very distinct and outspoken, very regular and at right angles to the long axis of the fiber. The alternation of light and dark bands is entirely similar to that of typical cross-striated muscle. The nuclei, oval and with only a small amount of chromatin, are found solely in the peripheral portions of the fibers. Between these fii)ers, a niininnnn amount of white fibrous connective tissue 72 IIOMKK (i. 1 ISIIKK will) hut few yellow elawtio filirils is foiiml. ^^'hen stained by Mallory's niothod, the cross-striations of these libers are beautifully brouuhl oul, if Kiunery's ('l(i) use of this -itain after Bouin'a fixation be followed. Inder this higher niaRnifieation, also, the smooth musele fibers are foiiiid to he ([uite similar to those of other organs (fig. 4). In the posterior two-lifths of the retractor muscle, the projjortion of stripetl fibers to the unstriped is about equal, or the unstriped lihers may he somewhat in excess. This ]iroportioTi has been ealculateil from a study of the serial sections through the whole retractor muscle of an adult dog: it represents not an exact detennination of the amount of either tyjjc of muscle but rather a rough judgment of the jiroportion. In many single sections, as illustratetl by hgure 3, the relative amounts of striated and smooth muscle may be easily estimated. Apjiarently, the amoimt of striped nmscle in the one puppy stuilied was somewhat less than in the adult animals. In the gross, it seemed possible that the striated element of the retractor muscle might be deriveil from direct extensions of the bulbocavemosus fibers which at their insertions attach partly to the sheath of this nmscle in its posterior portion. Serial sections, however, of a block made up of the two muscles removed intact failed to demonstrate any such origin; there was always a definite fibrous sheath interposed between the fibers of the retractor and those of the bulbocavemosus. While no definite continuous prolongation of the striated fibers from the adjacent structures was demonstrated, it is possible that they are derived from the sjiine anlage as the sjjhincter ani nmscle with which the retractor is so closely associated. The exact origin of the retractor jxTiis nmscle is rather indefinite, for the fillers arise gradually out of the sphincter. The nerve supply to the retractor penis muscle was not (i( inonstratcd. It was suggested that if the nmscle was supplied by the sacral autonomics (as seems most likely), one might possibly he able to (ind nerve ganglia either in the muscle or in its sheath. Systematic search was made in serial sections for nervecells hut with negative resvilts as regards the demonstration of such collections of nerve-cells. RETRACTOK PENIS MUSCLE OF I)()C; 73 Thus it sof'iiis (juito iifrcssary to roiisidi r this n-tractor penis muscle us heiii(j; mixed, eomposcd of both smootli and striated fil)ers. Until 1915, however, this muscle, so far as it is possible to determine from the literature, was considered as a typical smooth nuiscle. This conception of its histology has led to its use by chemists as a i-elati\ely large mass of smooth nuiscle tissue, and determinations of the creatin, camosine and other" nitrogenous extracti\'cs in it have been made. The finding of a disprop-ortionatc amount of these chemical bodies in this nuiscle difTerentiated it chemically from other smooth muscles. The chemists. liii\\c\-ei-, haxc coiisideiid tliat tl:c findings harmonize with Botazzi's observation that tlie muscle is tlitTerent physiologically from intestinal or other smooth muscle, for it has a shorter latent ])eriod and sometimes presents two types of contraction, viz., the clonic and tonic. But it seems proper to suggest here that the hnding of a somewhat larger amount of creathi and other nitrogenous bodies in the retractor muscle was due to the presence of cross-striated fibers. Retterer and his co-workers ('00-'1.5), in a series of articles dealing with the structure of muscle, has described the retractor muscle anatomically, both from a gross and microscopical standpoint, and has reviewed the literature on the subject. Chauveau was apparently the fii'st to examine the muscle histologically, and in 1S,")7 ileseribed it in the horse as composed of smooth muscle fibers (fibres musculaires ile la vie organi(iue). Comparative anatomists in subsernient jiublicatioiis either ditl not comment on the microscopic structure of the nuiscle or merely stated that it was involuntary. Retterer Clo) concluded that the nuiscle was striated but attributed the striations to the branching of the elastic tissue fibers ami not to the characteristic cro,<s-striations of voluntary muscle. These branches were described as leaving the longitudinal elastic fibers at right angles and encircling the nuiscle fibers or even passing into their substance, so as to give the appearance of heart muscle. Such ajijiearances have also been observed in liic present study, but the plienomenon of the cross branching ot eia>lic fibers was found to occur only in the smooth portion 74 HOMKU <:. KISHKK iif tlic muscl(>. Il must lie cniphasizcd, liowcxcr, tli;il these lateral liiaiichin(;s of tlic elastic elements (»f tiie muscle are entirely (litTereiit from the striations of the striated fibers in the |>osterior ])art of the muscle where the cross-st nations oi)served are evidently due to the alternation of lipht and dark hands in the mynlihrils, as in typical cross-striated muscle tihers. l^ota/./i ri.")). who made an extensive study of the retractor muscle from a ])hysioloKi( al sland])oint, divided the muscle into three j)arts an anterior or pn pucial end, a middle ])ortion, and a jiosterior or i)erineal end. He stated that the posterior fraction is darker than the other portions and suj^gested that it resembles striated muscle in its gross api)earance. On direct stinuilation he obtained from some s])ecimens two ty])es of contraction a clonic or twitch contiaction, and a tonic contraction (slow and gradual) while from other preparations he obtained only the tonic contraction. He writes (page 11) that "if the animal is very large the muscle is excessively long and tin II I take only the i)repucial and middle parts. If instead the animal is small, I isolate and take out also the extreme perineal end." Wliile he docs not state with which muscle ])re])arations he obtained the two types of contracti(jn, it is jiossible that thej' were obtained only from those preparations comprising the entire muscle. This would include the portion of the muscle containing the striated fibers which on direct stimulation should give a clonic type of contraction. On the other hand, the smooth muscle fibers might well lie expected to yield only the tonic tyjM' of contraction. It seems, then, justifiable to conclude that the retractor i)eni.s muscle of the dog is mixed. In the anterior three-fifths the fibers are wholly smooth while in the posterior two-fifths the fibers are both smooth anil cross-striated. The divergent chemical and physiological ol)ser\ations b;i,sed on the assumption that this muscle was wholly smooth, have a possible explanation in the mixture of the two kinds of muscle fibers here described. RETKACTOK PKMS MIS<LK OK DOG 75 BIllMOdl! Al'll'l BoTTAZZi, I'li.iriMi I'.il.") Kiccrclic sill M. Ucir.'iclor IVnia v hu iiliri prcpurati Musroluri Lisci. TipoKrufiu dcllu U. Aceudcmia dpi Lineci, v. 2, p. 43. Hicii.iA. (!. UND Constantino, A. liU'J HeitniRp zur Muski-lchemic. Zcit schrift fiir PhyaioloKisclu- Clipniic. Bd. 81, p. 120. KiN'UEKY, H. M. 1910 Some uses of .Mallory's connective tissue stain. Anat. Roc, vol. 12, p. 2<.»1. McGiLL, Caiiolink UI09 The structure of smooth muscle in the restinf; and in the contracted condition. .\m. Jour. .Vnat., vol. 0, p. 493. Ketterkh, K. et Lklikvre, a. 1909 Structure du tissu musculaire lisse. Comptes Rend. Soc. do Biol., T. 61, p. 244. 191.5 Ivcs fibres musculaires des cordons retractor penis sont des fibre cellules strics en travers. Comptes Rend. Soc. de Biol., T. 78, p. 136. ET Neiville. H. 191.1 Des connexion et de la structure des cordons musculoK'lastiques ou retracteurs du penis. Comptes Rend. Soc. de Hii.l.. T. 7S, p. (iO. iM.A'ii': 1 KXri.ANATION- OK FIfiUREa 1 HctoudiPil pilot omicrograpli of typical field in anterior two-fifths of the retractor penis muscle of the dog. The smooth muscle fibers arc shown collected in bands and converging somewhat at the anterior termination of the muscle. KnlargcMienl . 240 diameters. 2 Hetouclicd photomicrograph, under higher iiiagnilication. of the rectangular area shown in figure 1. The fasciculi of smooth muscle fillers are shown separate<l by somewhat abundant connective tissue. The miclci of the nonstriated fibers exhibit a spiral form. Eidargement, 750 diameters. 76 HICTKAf rule I'KNIS MISCI-E OF rx)c; PLATE 1 -> 1 V 1*"^"^ % > .^-, ^^ ^'^1 '*^.l 12, \ PLATIC 2 KXPLA.NATIO.N l)K KIorRES j Retouched photomieroRraph of a typical field in the posterior two-fifths of the retractor i)onis nmsde of the dog. The alternation in the section of striated and non-striated fillers is well liroURht out and the e(iual proportion of the two types of fibers is indicated. Enlargement, ISO diameters. 4 Retouched photomicrograph of the squared area in figure 3. The typical cross striations of the cross-striated muscle fibers and the peripheral distribution of their nuclei is well lirought out. On both sides of these striated fibers are dense bundles of smooth muscle. Enlargement, 7.50 diameters. 78 RETRACTOR PKMS MrST'I.K OF DDG nOMKII <l » IHIIKII I > '1.4 TE J '<i'w:'r'fir'} PRELIMINARY NOTE ON THE NIC'LEAH DIMSlON IN THE ADll'OSE CELLS OF INSECTS WAUO NAKAllAKA From the Deparlmenl of Enlomology, Cornell Univerttity ELEVEN FIGURES As to the liiological significance of aniitosis, Fleniming's ('91) theory that "it represents either degeneration or an aberration, or perhaps in many cases is tributary to metabolism through the increase of nuclear surface" (Wilson, '00, p. 117) is generally regarded as rcjirespnting the truth. Cells which have divided amitotically and arc active in their metabolic processes may eventually degenerate and perish. This, however, is no direct proof of the first i)art (jf Flcnuning's statements here cited. My study on the relation of nuclear divisions and metabolic activity in the adipose cells of various insects furnishes good evidence to show that amitosis docs not mean the approach of degeneration, or aix'rration at all. but this kind of nuclear division may be chiefly, if not entirely, to .secure the increase of the nuclear surface to meet the jihysiological necessity which is ilue to active metai)olic interchanges between nucleus and cytoplasm. This is the theory first suggested by Chun more than twentyfive years ago {'^)0), when he studied amitotic nuclear division in a giant cntodermic cell of the radial canal of Siphonophores (Flenuiiing. '91, 'i)'J; Wilson, '00), but was somewhat neglected l)y subse(juent writers. According to this theory, amitosis is primarily concerned with the vegetative function of individual cells, and so amitosis can no longer be regarded as one of the two es.sential methods of cell-mult ijjHcation. It may perhaps be consid(>red in association with such phenomenon as the ramification of the nucleus with its increasing function.nl activity, as in the case of silk-gland cells of many insects, ami the division of SI S2 WAIU) \.\K.\H.\K.\ the cell-hody following that nf nucleus is a relatively subordinate phononionon. In this i)ai)er I wish to ])()int out thiit in the case of the adipose cells of insects, amitotic nuclear di\'ision occurs preparatory to as well as sinuiltaneously with a certain metabolic activity of the cell, in which niudei take the r61e of essential importance. Fuller accounts of the relation of nuclear diNision and the metabolism of the cell in question, together with more extensive discussion on the general subject of aniitosis. \\nll be given in my further jjajjer. The following observation done primarily on lar\ae of Pieris rapae, brings out the general feature of the changes observable in larval adipose cells during their activity. Changes similar to those described below, have been observed by many previous writers and also by myself in the case of various other insects. In an adipose cell from a larva of the first stage the nucleus is round and shows no sign of division; the cytoplasmic area is small in most cells and contains only a few vacuoles. These vacuoles we interpret as indicating the places occupied l)y fatdroplets. This rather unspecialized condition of the cell changes in the following stage by areal expansion and more vacuolate appearance of the cell-body and by frequent occurrence of peculiarly sha])ed nuclei. Sonic few of these nuclei show nothing but their irregularity in shape, while most of the others apparently represent different stages in the process of amitosis. I have shown in figures 1 to 7 sketches of nuclei, all representing possible stages of amitosis, and many of which one can find in every section. I have not been able to find any particular way in which these nuclei diAide, except that the division itself is effected by the constriction of the nucleus across its longitudinal axis, thus making the nucleus show a bilobed condition. More rarely, in the case of long, slender nucleus, constriction may take place at more than two different places in the nucleus, and in that case the latter shows multilobed appearance. Nucleoli and chromatin grainiles are apparently evenly distributed throughout the nucleus, and neither of them seems to behave unusually during the process. NUCLEAK DIVISION I.V ADIPOSE CELLS 83 In tlic tliird stage larva, one may notice the fact that some of the cells begin to show peculiar spherical granules in the cell-body. As pointed out by Herlese ('99), P6rez ('02, '10), Henneguy ('04), and others, these granules are of albuminous nature, and occur more abundantly in close proximity t(j nucleus, than in the periphery of the cell. They become very abundant at the fourth stage and at the last larval stage almost all of the adipose cells are seen to be hlltd with the granules. The cell Fins. 1 to 7 Nuclei of ailipo.-ii- (.'ells, representing possible stages of amitosis. X 7.tO. Kigiire G represents predoniiii.'itinK type. body becomes larger and larger with the advancing stages of the insect; ajiparently correlating \\'ith this, the occurrence of nuclear division is seen more and more freciuently, and finally, in old larvae, we find the condition as shown in figure S to be met with very commonly. Sunmiarizing the facts, we may say that, in the adijiose cell, the luicleus continues to divide amitotically from early in the .second .stage, and the cell stores up albuminous gramiles in its cell-body, commencing the process late in the thinl stage. This 84 WAIUJ .NAK.VIIAKA shows tliat the foll-miclci which have undorgoiip amitotic division, without lOKaril to wlicthcr tho cells rcinaiii nmhimiclcato or not, do not degenerate, and theceils proceed witii their active functional processes. This can he more strongly emphasized because we now see the fact that nuclei themselves take a direct part in the formation of alhumiiious granules, giving most conclusive evidence in support of the theory. Fig. 8 A multinurleatc adipose cell from iin old liirva, contnining iillitiiiiinous granules in its cell-body. X 250. It has been supposed bj^ some writers that albuminous granules are derived from the blood. HoUande's ('14) recent work. from the cliemical viewpoint, casts grave doubt on this assumption, and although lie has given no evidence, he has suggested that the granules may be of nuclear origin. It seems quite probable, from a cytological point of view, that such might really be the case, and especially so when we recall the fact that, in the ca.se of .silk gland cells of Pieris and Xeuronia at lea-st, the nucleoli, after eliminating their phosphorus, change into albuminous graiuiles and then extrude bodily out of the nucleus (.Nakahara, 17;. NtrCLEAR DIVISION IN ADIPOSE CELLS 85 Examining the nuclei of such cells as contain the granules, one can distinguish three different kinds of granules within them. One of the three kinds undouhtedly represents chromatin and another nucleoli, as can he judged from their appearances and staining reactions, characteristic of such elements. The third kind of granule is of nearly the same size as nucleoh but differs from tlio latter in that it shows stronger affinity for certain acid • ' V \ - ' • • • • V Figs. 9 to 11 Nuclei of adipose cells, showing the extrusion of acidophile granules. X 7.50. and weaker for basis stains. This is exactly the sort of reaction shown by the cjioplasmic albuminous granules, and one may here a.'^suinc that these acidophile granules in the nucleus may be extruded into the cell-body, constituting the albuminous granules in question. As e\-idence in support of this theory, I have .shown in figures 9 to 11 some unmistakable cases of extrusion of the acidophile granules into the cell-body through the nuclear mcmbram-. These are not cases rarely obser\able but are those of very frequent occurrence. Tlir. WATOUICAL RECORD, VOL. 13, NO. 2 86 WARO NAKAHAIIA LITKUATl-UK C'lTIOD Berlese, a. 1S90-00 Obsorvazioni Mil fononiciii ('he avvoDKniio (liiraiilc la ninfosi degli inaetti mctaholici. Utvista di Patologia VcKetalc, T. 8, pp. 1-444. Flemmino, \V. 1891 I'cbcr TheiluiiK und Kornformen Im Leukocytoii und dorcn Attractionssphiirpn. .\roh. f. inikro.skop. Annt., Bd. 37. pp. 249-298. 1.S92 Zellc. EiitwicklunK und Stand dor Kcnntniaso iilxT Aniitose. Morkol 11. Bonnet's Kruchnisse der Aiiat. u. Entwick., Bd. 2, pp. 3782. Henneoi'y, L. F. 1904 Lps inscctes. Paris. HoLL.\NDB, A. Ch. 1914 Formations endoni'iics dcs cristBlloidcs allmniinoides et des urates des cellules adipcuses dcs chenilles dc Vanessa io et Vanessa urticae. Arch. Zool. Exp. Gener., T. 53, pp. 559-578. >;.\K.Mi.\nA, W. 1917 On the physiology of the nucleoli as seen in the silkgland cells of certain insects. Journ. Morph. (in press). P£iiEZ, Ch. 1902 Contribution d I'^tude dcs metamorphoses. Bull. Sci. de la France et de la Belgique. T. 37, pp. 195-427. 1910 Recherches histologiques sur la m^^tamorphose des niuscides, Calliphora erythri>cei)hala .Mg. .Vrch. de Zool. Exp. et Gener. T. 4, pp. 1-274. Wilson, E. B. HKX) The cell in development and inheritance. 2nd ed. New York. A POLYEMBRYONIC BLASTOCYST IN THE OPOSSUM J. T. pattp:r.son and c. g. hartman Contribution from the Zoological Laboratory of the University of Texas, So. ISi ONE TEXT FIGURE AND TWO PLATES INTRODrCTION WTiile engaged in collecting material to complete the series of stages on the development of the opossum, Didelphys \-irginiana, we have recently come upon a polyembrA'onic blastocyst containing four enibr>'os. The history of the female from which this specimen was taken is as follows: The female (no. 300) came from the wild, and was received at the laboratory on January 20. It was anaesthetized and the left utenis removed at y.2() p.m. that evening. This uterus had six normal blastocysts, each measuring between 10 and 11 mm. in diameter and containing an embrj-o in the early somite stage. The incision was closed and the animal allowed to live vuitil the next day, when she was killed at 12.15 p.m. In addition to the polyembryonic blastocyst, the right uterus was found to contain one dead vesicle al)ove 7 mm. in diameter, and six normal blastocysts, each with an embryo. The average normal blastocyst from the right uterus measured about 14.9 mm. in diameter (fig. 7), while the polyembryonic specimen is not quite as large, measuring only about 12.88 mm. in diameter (fig. 4). For several reasons this case is of especial interest. In the first place, it represents one of those rare cases in which a polyembryonic blastocyst has been discovered in a multiparous mammal. In the second place, it may have a bearing on the possibility of normal polyembrj'ony occurring in Marsupials, as reported by Bluntschli ('13) for the South American opos.sum, Didelphys marsupiales. Finally, as will be .shown later, the 88 J. T. PATTERSON AND C. O. HAHTMAX arrannenicnt of the embryos suggests a close similarity to the paired coiulitioii tlial is normally found in tlie four embryos of the Armadillo, Tatusia novemcinrta. DESCUIITION OK THK POLYKMHRYONIC BLASTOCYST In the normal blastocyst of this litter the eml)ryo has already sunk beneath the surface of the blastoilerm, and owin^ to the well-developed cervical flexure, the anterior portion of the embryo with its membranes ])rojects into the cavity of the blastocyst (fig. 8). In the surface view the posterior end of the embryo is dimly seen tlu-ough the membranes (fig. 7). The area vasculosa covers almost the entire upper half of the blastocyst. It is limited by a sinus terminalis, which forms a distinct marginal notch at tlie posterior margin where the vessels lead to the embryo. In the polyembryonic blastocyst there is also a well-formed area vasculosa, with a less distinct notch on the lower side (figs. 4, 5). There is a bajing in of the sinus terminalis on the right side, suggesting a second notch, but no vessels pass from this to the embryos (fig. 6). With the exception of the united posterior enils of the two embryos lying al)ove in the figin-e, the embryos have only begim to sink down into the bla.stoderm (fig. o). The four embryos are arranged ajiiiroximately in the form of a square, -with each embryo constituting one side (fig. 6). The area included within the square is somewhat higher than the general level of the embryos, giving to this central area the appearance of a vesicle. This is produced l)y the sinking of the embryos into the blastoderm. In sections the central area is seen to be structurally of the same character as the vascular area hnng just outside the square. The exact nature of the paired arrangement of the embryos can best be descril)ed by reference to the sketch shown in text figure 1. For the sake of convenience in description, the embrj'os are designated by the Roman numerals 1-I\'. Embryos I and II are united at their posterior ends, and constitute one pair. Embr\'os 1 1 1 and IV are similarly united, and POLYEMBKYUNIC BLASTOCYST IN THK OPOSSl'M 89 (•((iistitutc the <illu'r pair. All of ilie (Miibryos have the foreliiiil) hmls well started. The components of each pair are not equal in size, enihryn I being somewhat smaller than its twin, and ('ml)ryo III (Iccidedly smaller than IV. DKSCHIl'TION OF EACH KMBKVO In order to be able to give a brief description of each inthvidual embryo, it is necessary to have recourse to sections. The embryos were cut into a series of sections (12 n). Tin- plane of -96 .331 I'itJ. 1 Outline sketch of the four embryos. Note tliat tliey are in two pairs, eniliryos I and II l)einn united at posterior ends, and likewise III and IV. The broken lines represent the planes of the three sections illustrated in figures 1 to 3. The numbers at the ends of these lines correspond to the sections in the series. X S. section is .ipiiroximately transverse to the long axis of embryo II (fig. 1). There are .^Ot) sections in the .series, three of which (5)(), 242, '.V.W) are drawn in figures 1 to 8. Section 9() jia.sses through heart of embryo II, obliciue through embryo III, and transverse through the posterior enil of embryo 1\'. Section 242 passes through the 12fh pair of somites of embryo II. and the fore-limb buds of embryo 1\'. Section XM cuts the posterior end of embryo II, anil passes obliquely through embryo I. Embryo I. This embryo is 4 nun. long, and has 13 to 14 pairs of somites. The neural tube is closed in the brain region, but is represented In- a Hat plate posteriorly. The heart is 90 .1. T. I'.VTTEIISD.N AM) C. C. IIAUTM.W jjrcscnt Init hinlily :il)ii()iin;il. Tlu' anterior half of the iiotochortl is formed, and the WOlfiian ihicts are well ileveloped. Embryo II. This einhryo is iiraotically normal, and bilaterally synnnetrical. It is 5 mm. long, and has 20 pairs of somites. The neural folds, anterior to the level of the first jjair of somites, is open, hut a tyi)ical neural tube extenils from this point hack to the 13th jiair of snuiito. Posterior to this a eharacteristic open myelon extends to the end of the embryo. It has a welldevelopeil lu-art. notoelujrd, auditory vesicles, and NN'olttian ducts. Embryo III. This embryo is 3.25 mm. long and has (i pairs of somites. The embryo is so hiffhly abnormal that it is difficult to make out all the details of structure in obliciue sections. However, the central nervous system is represented by a flat plate throughout the entire length of the embryo, except at extreme anterior end, where the folds are slightly elevateil. The heart is very rudimentary, but the Wolffian ducts are present. No notochord nor auditory vesicles could be distinguished in the sections. Einhryo I\'. This embryo is 4..") nun. long, and has 15 pairs of poorly developed somites. The neural folds are open at the anterior end, but doseil at the level of the midbrain region. Posterior to this there is a flat neural plate, with a .slight indication of folding in the region of the limb buds (fig. 2, IV). A notochord is present for a short distance in the region of the heart. The WolfTian ilucts and auditory vesicles are both present. DISCfSSION At least three of the embryos are abnormal and would not have formed normal individuals. So far as one can tell from a study of the fourth embryo (II), it is normal, and prol)ably would have completed its develojiment. Regardless of whether or not any of the embryos would have reached full term, the ca.se is none the less inten-sting, because it shows that the blastocyst of the opos.sum can j)roduce more than a single embryo. There is no good reason to doubt that the blastocyst of this species may sometimes produce a normal jiolyt nibryonic group of indi POLYKMHKYONIC «L.\STOCYST IN TUK (JPOSSfXl 91 viduals. lint should swell ii fjroup coinc to f\ill Iciin, the iiidividuals could not he distiiinuislii'tl from tliosi' in the litter arising singly from tlic other blastocysts. This fact probably accounts for the extreme rareness of observed cases of polyembryony amonp; multiparous mammals, in contrast to the numerous cases on record for imiparous mammals. In reference to the bearing of this case on the ])robability of polyembryony occurring in the South American species, we may call attention to the brief report of Bluntschli's Cl'.i) denumstration at the 27th meeting of the Anatoniische Gesellschaft, held at CJreifswald. This investigator demonstrated several embryos and young from the pouch, as well as one opened uterus. It is stated that the latter contained four embryos, each in its own amnion, i)ut all lying within a single chorionic cavity. The condition in this uterus was corroborated by analogous preparations of yovmger stages. Bluntschli con.siders the occurrence of jiolyembryony in Dideljihys marsupiales as probable, not only because of the conditions of the young in the uterus, but also because he had always encountered the young from the pouch of the same se.\. In the light of these facts it may be that the rare occurrence of multiple-embryo formation in D. viginiana has become a permanent phenomenon in the development of D. marsupiales. As to the manner in which multiple embryos are formed in the blastocyst of the opossum, we cannot, from the study of a single, comjiaratively advanced stage, offer any definite explanation. It is, however, a significant fact that there are four embryos, and that the arrangement of these on the blastoderm suggests a certain similarity to the condition in the armadillo. .\s in the armadillo, the four embryos of the opossum are clearly in two pairs. Furthermore, one of the two embryos in each pair is larger than its twin, exactly as has been descrilied for an early stage of the armadillo (Patterson. '13). i.rn.uAriKK cithd Hi.rNTsnii.i. II. liii;< DciiKiiiMtratidii of cmhryoH of DulclplivH iiiursiipinles. I'roc. of the Aimtomischcn (iosfllaolmft, ?7tli moftinR, (ircif.swiild, May 10-13. 1913. Sii|>i>1<miiiiiI. Aunt. Anz.. vol. 44. p. •-•00. II.\RTMAN. C. Ci. 101(> Sludirs oil I lie (li'Vi'lopiiu'iil of tlio f)i)ossiiin Didi-lphys virRiiii.'inii. Jour. Morpli.. vol. '11. p. 1 .s;j. Patterson, J. '1'. liU3 rolyciiitiryoiiic (li'vplopiiu-iit in Tiitu.siii iiuvomcincta. Jour. Morpli.. vol. 24. ]>. .55i)-(>.S.3. PLATE 1 EXPLANATION OF FIGURES lto3 Sections %. 242. 331, respectively, of the series. The Roman numerals indicate sections through the four einliryos. X 37. 92 A |-ol,Yi:.MBI<VONIC BI.AsnXVST IN THK OPOSSIM J. T. I'ATTKIWON ANUt. <J. IIAItrMAN IMJ^TK I !»:t PLATE 2 KXI'LANATION OF FIcrRES 4 Dorsal view of tlic entire polypmbryonic blastocyst, showinp the four emliryos arranged in the form iif a .s(|\iare. X 2.95. 5 The area vaseiihisa with eniliryo.s. X 4. 6 The same, after staining aii<l cleariiiK. photographed by transmitted light. X 6.5. 7 Dorsal view of a monembryonie blastocyst from the same litter. The back of the embryo, lying in the center of the area va.sciilosa, is dimly seen through the membranes. X 2.95. 8 View of the inside of the same blastocyst, from which the lower wall has been cut away. It clearly shows the embryo projecting into the cavity of the blastocyst. X 2.95. 4, 5. 7 and 8 are each one-half of stereoscopic photographs. For all of these photographs we are indebted to our frii'iid Dr. Chester Ileuser of the Wistar Institute. (M J. T. I'ATTKIIUMN- \Hlt C. <i. IIAHTM \N SOME OBSERVATIONS OX THE ORKIIX OF MELANIN PIGMENT IN FEATHER GERMS FROM THE PLYMOUTH ROCK AND BROWN LEGHORN FOWLS R. M. STRONG From the Anatiimical Laboratories of Vanderbitt University Medical .School SIX FiatTRES I INTRODUCTION This paper is a consequence of work done under my direction by Katherine Knowlton. a candidate for a Master's decree at the University of Chicago during the year 1912-11)13. She was assigned comparative research on the pigmentation phenomena of feather germs from the Plymouth Rock and Brown Leghorn fowls. In the course of this work some new facts concerning the origin of pigment in feather germs were discovered. A preliminary statement concerning these results has been made (Strong, '15). I ha\e recently restudied Mrs. Knowlton's material, anil I have prepared the illustrations which accompany this paper, as well as the manuscript. The development of melanin pigment in feathers has been described in detail in a jM-eceding paper (Strong, '02). Others who have wTitten on the subject are mentioned in that publication. Since then, an important ))aper by Lloyd-Jones ('15) has appeared. This discusses inciilentall}' the origin of melanin l)igment in various varieties of domestic pigeons. .Vs stated in pre\'ious publications, the feather is purely epidermal in origin. It is ditTerentiated from tissues which make up a cylinder surrouniling a central core of dense and \ery vascular mesenchyma, the dermal pulp (figs. 1 to (i). The meseiichyma is se])arated from the epidermal cylinder by a ba.<ement menii)rane (figs. 1, 2, 5 and li) which is well delined except !»7 98 K. M. STRONt; at the proximal end of the feather germ. The epidermal tissue includes an external sheath and a layer of stratified s(|uanious ei)itheliuni under the sheath, tiie inner sheath eells, neitlier of which become parts of the feather structure. Between the dermal pulp and the iiuier sheath c(>lls lies a broad zone of cells the intermediate cell region, (Hg. 1 ). This zone isdivided by radial extensions of the basement membrane (figs. 2 and 6) into sections called barb-vane ridges by Jones ('07), except at the proximal end of the feather germ. Each barb-vane ridge consists of so-called cylinder and intermediate cells. The cylinder Basemeinl mernbrane; Dermal Tnelanopnorea ;' tpid eriTial melanophorea / /' ' iTiferior umDilicxi* / 'IriteTtnediate-cell region 'jinrier »lieatn-ce1l« Kijt. 1 Tartly diagrammatir longitudinal section of proximal end of foiitlicr germ. X 30. Melanophoros arc indicated l>y dots. Drawn with aid of camera liicida. cells make up a layer, one cell deep (figs. 2. 4, and (i) which lies next to the basement membrane and its radial extensions. This layer has the form of a deep and narrow gutter or a letter U in cross section. The base of the U or gutter lies next to the dermal pulp except for the intervening basement membrane, and the other end is next to the inner sheath cells. The space inside the limbs of the U is filled by intermecUate cells (figs. 2, 4, and 6). .\11 of the feather structure except the loose pith in the quill is developed from these intermediate cells. Some of the intermediate cells are differentiated into melanophores which supply melanin granules to the differentiating feather cells. MELANIN PIGMENT IN FEATHER GERMS 99 For details conccriiing these remarkable pigmentation processes, the reader is referred to a previous i)ai)er (Strong, '02). There the histogenesis of the feather also is described in detail except in the case of the shaft and (luill whose development was worked out by Davies ('S*)j. Sheatli Inner <keath-celU Dasemenl membrine extension lnterine(Jiste ceU nuclei B^ooJ c«pill«rv- Fi(?. 2 Siniill portion of truiisvi>rse section of feather germ showing an oblique section of a barli-vanc ridge and cylinder cell nuclei of adjacent l)arb vane ridges. X o(X). Back covert of Plymouth Rock fowl. I.«vel of section indicated by the line a in figure 1. This view was selected from a non-pigmented region. Drawn with aid of camera lucida. H. MATERI.VL AND METHODS The material used consisted of feather germs pulled from their follicles and placed in the fixing fluid inuuediately. The regions of the integument which sujiplied the feather germs were the back, breast, neck and wings. The scajnilars also were used. Two Plymouth-Rock males, two Tlymouth-Rock females, one Brown-Legliorn male, and one Brown-Leghorn female furnished the material. .\ majority of the feather germs came from the females as the males did not molt so extensively tluring the perioils when the material wa.s obtained. 100 II. M. STKONG The feather germs were mostly 1 to 2 cm. in length. In most cases they had just begun to expose the barbs at the distal end of the slu'ath. A few feathers in more advanced stages were also used. (Jilson's fixing fluid was enijiloyed largely, the material remaining in it for three hours. Otherwise the technique was that described by Strong ('02). The sections were cut in paraffin G to 10 microns thick. About forty of the sections at the proximal end of the feather germs remained regularly in the i)araflin ril)l)oii when 10 microns thick. Succeeding sections were more and more apt to drop out of the ribbon as more and more cornified material was reached. III. Ki:srLT.s A. Origin of epidermal pigment 1. In intermediate cells. All of the germs studied were of feathers which contain large amounts of melanin jiiginent. Consequently, melanophores crowd the intermediate cell region in the preparations from the two fowls. No e\'idence was obtained that these melanophores were not of epidermal origin, and I believe that they are specialized intermediate cells as I have stated before (Strong, '02). Above the region where the melanophores ileveloj), the intermediate cells which enter into the feather structure have their cytoplasm characteristically jiacked with melanin granules. The pigmentation phenomena here are essentially like those described in previous publications. According to Lloyil-Jones ('15) many ordinary intermediate cells may develop melanin granules autoctlionously. It is of course imjiossible to prove that this is not a frcciuent occurrence and I have indicated its po.ssibility (Strong, p. 170, '02). However, the mere fact that a cell containing melanin iiigment is not sometimes in contact with a chromatophore process in a microscopic preparation, does not prove that this pigment may not have come from a melanophore before the material was fixed MELANIN IMCMKNT IN KEATHKK GKKMS 101 unless it ran ho shown that no inclanophorcs arc nrar onou^'h to have siii)pncil the pij^nicnt. 'Ihc niclanojihoro relationship is obvious at many points in a niicrosco])ic field. The evidence in my judgment points towards the orij^in of the melanin ])i{iment in intermediate cells from epidermal melanophores as the rule. This position is not inconsistent with the ol)servations made by Lloyd-Jones. I'i)!. ',i riiiitiiinii'niKrapli .slion iiig a portion l4 nun. longi of u lon(;itU(lina| !<i-clion of feather (jiTni from near its proximal end. X I5(). Baekeoverl. I'lymouth Ilork fowl. The rejtion photographed was seleeted l>eeailse it had fewer melanophores than ii.siial. with eonse(iiient better outlines of individ\ial melanophores. Numerous melanophores whose hranehin^; processes connect with difTerentiating feather barhule cells may he seen. The lower end of the view corresponds with the line o in figure 1. and the area extends somewhat above the region indicated in figure I. Ocular l();olij. I mm. Hausch and I.omli: picture reduced in publication. 2. In cylinder cells. A small luimber of cylinder cells were found ctmtaining clearly dcKnod melanin granules in their cytol)lasm. These cells occurred at the level in the feather germ where the meliinojihorrs were begimiinn to send out processes. .\ few of these cells were at the de())er end of the barb-vane ridges ne.xt to the pulj) except for the intervening basement membrane. Other cylinder cells containing melanin gi-anules were at the sides of the I ail -vai:e ridge, some distance from the TUi: ASATOUICAL H>l<t>lu>, vol 13 Ni>. lOJ U. M. SIKONt; pul|). Till' iiu'laiiiii K'w'i""'^ wen* always found on tlio outer sill*' of the luiflcus. i.e.. tlio side away from the dornial pulp. The Kraiuiles occurred in small tlense groups. It was not possible to determine their origin but we believe that they were formed in situ and did nf)t come from mclanophorcs. .i". In itinrr slniith nils MclaniM firanuics occurred in some of the inner sheath cells of seven out of sixteen series of feather SUatK Inner sneiln-celU riOmenlec) oarDule oelU.; Intermeaiate-cell nuclei Lpioermal mfclanopnoTes. DerTTiii puip -fiipC?' Fi({. 4 I'li<itoiiii<TiiKr:ipli of .small ixirtiini of transverse section of feather Kcrm. Neck covert. Brown IcKhorn fowl. X 'M). Level of line a in figure 1. Kpidermal melannphores may lie seen in their usual abundance, more or leas monopolizing the intermediate cell region. Intermediate cells differentiating into feather l>arl)ules are shown with their cytoplasm packed with melanin granules received from mclanophorcs. .\l the left, histological details have been ndih'il with a pen. a camera lucitla being employed. (>c. 10; obj. 1.9 mm. oil inuner.sion i Hausch A- Lombi. The picture was much reduced in publication. One barb-vane ridgi' appears cotnplctcly, another at the left is almost all in the picture, and a large portion of a third at the right is included. germ sections, according to Mrs. Knowlton. In all cases the pigmented inner .sheath cells were found within the first millimeter from the inferior uml)ilicus, i.e., at the proximal end of the feather germ, but not in every section of this region. This pigmentation was irregular and sjjarse. The granules were of the same size, form, and color as those of the intermediate and cylinder cells. The melanin granules were found usually on the outer or peripheral side of the nucleus, as in the case of pigmented cylinder MELANIN I'KiMKNT I.\ KKATHKK (iEUMS 103 cells. As there wore no mclaiioplioros near crKHinh t<> have distributed niclaiiiii niaimli's to these cells, we eoiiehjded that they also must have a capacity for forming melanin granules. Such cells never accumulate many Kranules. ami they always retain tlieir normnl character as stratified s(|uaiiHiiis e])ithelium. B. Origin of dermal pulp pigment Melaiiopliores were found at tlie i)roximal end ot the dermal pulp near the inferior umbilicus of feather germs from both the Plymouth Rock and Brown Lcfrhorn fowls (figs. 1, 3, and 4). Dermal pulji pigment was not found in the feather germs of over ten species of birds previously studied by me, and 1 know of no other ol)sci\ations of it, Ral)l ('94) found no melanin pigment in the dermal pulp of nestling-down feather germs from the chick. The dcinial iii(lan()])hores we observed occurred in the mesenchyma not far from the ba.sement membrane, as may be seen in figiUTs 1, ") and (>. Usually a good many were grouped near the shaft-forming epidei-niis. 'I'hat these bodies are inelano])liores is indicated by the following observations. 1) They are similar in shape and size to some ejiidcrmal melanophores. 2) The nucleus is often clearly seen, proving that they are cells, which would not be the case if they were artifacts. 3) The.se bodies are made up of granules of the same size, shape and color as the epidermal melanin granules. 4) The arrangement of the granules is characteristic of a melanophore. The granules surround the nucleus, where they are packed densely. Naturally, some artifacts and foreign l)odies resembling melanophores superficially, have been found in the sections, l)ut they have always Iteen distinguished without serious difficulty. .V ([Uestion naturally arises as to whether these jiulp melanophores may not have been dragged in from the epidermal cylinder in cutting sections, .\gainst such an interpretation we have several arguments. 1) There are too many of the melanojihores to warrant concluding that their positions are accidental. 2) There is no evid(>nce of distortion or tearing of the surrounding tissue such as 1U4 U. M. .STK()N(i would lio oxportod if tlicy had lu'cu moved thoro l)y the miorotouif knifo or l)y any other artificial eause. 3) These inelanophores lit in too naturally in the svuTounding tissue. 4) They are arranged with considerate uniformity around the periphery of the pulj) near the basement membrane. If their position had ^jU, m. '■"'It ermal Tne^anophore Deepest lay^r ofepidermis Kift. .5 Small portion of Iraiisvorsc section of feather Keriii near il.s proximal pikI. Breast covert of I'lymontli Kock fowl. X 700. Level iiulicated liy line 6 in figure 1. The dee|H'sl portion of the epidermal cylinder next to the hasenient menilirane is shown at the right. The elongated nuclei are separated by radiations which simulate cell boundaries. The basement membrane is poorly defined at this level. .\t the left the mesenchymal pulp is drawn partly diagranunatically. and its den.se character is not indicated. Kour dermal nielanophorcs app<'ar. been the eonsequenee of eutting, it would be expected that all of them would l)c on one side of the pulp. We ol)taincd no e\i(leiicc as to what particular cells form these dermal pulp melai:oi)hores. The de\elo])nicntal stages resemble those of the epidermal melanophores, but the pulji melanophores do not become so large as the latter, and they do not form processes so profusely. In fact, the majority of the pulp n.elanophores apparently do not form processes at all. Son.e have small inconspicuous processes, and a few have larg( r i)rocesses. The largest melanophores observed had no processes. MELANIN PKJMENT IN KEATHKH C;KKMS 105 It is charact eristic of tiic inilj) in('laiioj)horcs observed that such processes as occur are directed towards the ejHdermis either radially <ir in the line of a secant. These ])rocesses, as pre\iously slalcil, aic usually short, thick, and closely packed with melanin granules that never .■^eeiii to he distrihiited to other cells. The following talilc gives observations made by Mrs. Knowlton. SECT IONS Fulp motano phorra with no procpwn With nvlially dircctMl proceimem With proce— ea at hcnt ancln to raditu 15() transverse. . . 00 LonKitudinul pw cent 65 67 prr ctnt 16 13 per cent 19 20 The melanophores with longer processes lie near the basement membrane, and they are not frequent (figs. 2 and 3). Mrs. Knowlton found them in tliree feather germs Lying so near to the epidermis, it is important to know whether they penetrate the basement membrane or not. We have been unable to find any such penetration, even when the melanophore or its process was against the basement membrane or apparently in its barbvane extensions. These melanophores are located either near the apex of a barb-vane ridge or between two barb-vane ridges as just noted. In the former case the process follows the ba.sement membrane until it reaches a barb-vane extension of the latter, where it runs up between the two adjacent barb-vane ridges apparently splitting the basement membrane partition. In tlie other type (fig. (l). the body of the melanophore lies at the deeper end of the ba.sement memlirane i)artition, and its process runs straight out, i.e., radially, between the adjacent barb-vane ritlges. No such process was found extending more than threefourths the radial extent of the ailjacent ridges. ^^'e are unable to explain the location and behavior of these melanophores. They are so rare that we have considered them more or less abnormal. In the region where they occur, various features indicate activitv in a radial direction Here and more Ktti K. M. STKONU imiximally, tlu> cells of the syncytial cijitlK*!!!!!!! arc undorKoinR coiisuh'ral)lc chaiiRes in position. Proxinuiliy, pale refraction lines simulating inconiplcto cell boundaries lie radially between the nuclei, which are narrow and arranged radially. In figure 5 such lines have l)een drawn for the deepest nuclei at the right. They have not i)een ol)served to form comi)lete cell boundaries , Inter rnedi ate ce\n Inter mediate cell layer \ "Cvlmoei^ceu layer Basemenl membrane extension melanopnore _ aaemer.t ■membrane Fig. 6 Sninll portion of transverse section from feather germ of Plymouth Rook fowl. Near level n in (igiirc 1. X 700. Drawn with aid of camera liicida and reduced in pviVilieation. Oc. 10; oI>j. 1.0 mm. oil immersion, (Raiisch and Ix)ml>i. .\ Imwment membrane extension separating two barb-vane ridges is shown with a dermal melanophore process lying in its inner two thirds. The cell body of the melanophore is only partly in this section. Adjacent layer.s of cylinder cells and iiiiiriiHili:iti> cells ,iri- imlic-itnl by their nuclei. often if ever, and they disappear at the level where the radially directed basement membrane extensions develop. The frccjuent occurrence of melanin pigment only on the outer side of nuclei, mentioned in this paper, is very likely another illustration of radially acting forces. It is conceivable that melanophore proce.«<ses tire carried radially by such forces or by the developing barb-vane extensions of the basement membrane. MELANIN PIGMENT IN f-EATHER GEKMS 107 In longitudinal srctions some liKht appears to he thrown on the signilicance of the dernial pulp nielanophores in general. In a few sections, I have found an essentially continuous series of these bodies extending to the inferior uinhilicus as seen in figure 1. It is my opinion that these nielanophores are homologous with those of the skin dermis. The dermal pulp being continuous with the skin dermis, such a continuance into the proximal end of the feather germ is not surprising. C. Observations on the melanin granules So far as I could determine, all of the granules were rod shaped as is usual in birds. Even when e\ndently fully developed, there appears to be considerable variation in size. The longest are slightly over 1 micron in length. Others may not be more than 0.5 micron long. They are about two to four times^their thickness in length. It is not easy to make exact measurements of such bodies because of their minute size and their arrangement at all possible angles with reference to the plane of the section. In the Plymouth Rock and in some Brown Leghorn feather germs, the granules were of the usual brown, almost black color. SUMMARY 1. ^lelanin pigment granules occur occasionally in the socalled cylinder and inner-sheath cells of feather germs from the common fowl. 2. Further evidence was obtained that the melanin pigment of feathers is epidermal in origin. 3. Melanoj)hores were found in the dermal pulp at the proximal end of feather germs from the common fowl. They are presumably homologous with the dermal mfelanophores of the skin. Some of these pulp nielanophores have processes which are usually relatively short, but they do not appear to distriliute pigment to other cells, and they have no part in the histogenesis of the feather or its pigment. A few of these dermal nielanophores were found in contact with the basement membrane but none had penetrated it. KtS R. M. STRONG MTKUATrUK flTKO Daviks. II. U. l>vS<.) Dii- KiitwirkliitiK <I<t Kcdor iind ihro HczioliiinKPn fu aiulrrn InlcKUiii<-iitK<'l>>l<l>'»' M<>rp. Juhrli., Bd. 15, pp. rM) lAh. JuNEit. I., 'rill- ili'Vrli)pini-nt of iiratlinK feiithrrs. Liib<>rati>ry Hull., no. 13, OImtIih CollfKc IS pp. I.i.iivD-.rnNKs. (). l!)l,5 Stiidirs nn iiiluTitaiirr in piRooiw. II. A microscopical and chemical study of llic fcutlior pinmonts. Jour. ICxp. Zoo!., vol. IS, no. 3. April, pp. 4.>{-»05. R.Kiii.. II. ISm I'dM-r die KntuicklunR dcs Pigmentos in dcr Diincnfeder des llulinclicns. Ontral)!., f. Phy«'<>l • Bd. S, p. 250. Strosc. U. M. I902 The development of color in the definitive feather. Bidl. Mils. Comp. Zool.. Harvard, vol. 40. no. 3. October, pp. 145-185. 1915 Further oliservations of the oriRiii of melanin pigments. Anat. Ree., vol. 0, no. I, January, .Mistract o'J. Maturation of the ovum in swine GEORGE W. CORNER Frniii lite Aniilnmical Lnl)oratory, Universilij of California Since the first description of maturation of the ovum of the rabl)it l>y \'an Beneden in 1875, the study of this remarkable phenonieiioii lias boon extended to certain other niannnals. with the result that practically all observers now agi'ee that two polar bodies are formed by every o\'um, the first of which is extruded from the egg while still in the ovary, but immeiliately before rupture of the follicle. Formation of the second jwlar body then proceeds to the formation of a division-spindle, but goes no farther until the egg is discharged and fertilized. .Vfter the entrance of the spermatozoon the .seconil polar body is completed and extrudcil. In the event that fertilization does not occur, the ovum degenerates in the state in which it was freed from the ovary. Full and comprehensi\-c observations of the proc(>ss of maturation have been made in the rat (Sobotta and Hunkhard '10), guinea-pig (Rubaschkin '()o), opos,sum (Hartman 'IG), a bat, Vesperugo noctula (\'an der Stricht '08), the domestic cat (Longley '11), and the mouse. The studies of Sobotta ('95) and others, lead them to believe that in the last named species many eggs form but one polar body. The interest excited by this statement has caused the mouse to be more fully investigated than any other, with tlie result that the views ciuoted have been proven ineoiTeet. The reader is referred to the work of I>ong and Mark Cll) for details of the question together with the most comprehensive account of maturation yet presenteti in any nuimmal. The final disproof of this supposed exceptional case raises the (|uestion as to whether the process may not be identical in all manunals. To answer this a wider .search will be necessary, for the species named above comprise but three rodents, one marsupial, one carnivore, and one chiropter. There are also old or undetailed accounts or ])reliniinary notes ujion the ova of the dog (O. \ an der Stricht 'OS), tlie rabbit (Van Beneden 75, Heape '05), the niarsuiual Dasyurus viverrinus or Australian "native eat" (Hill '10). an insectivore. Tupaia javanica (Hubrecht '95), and of a lemuroid ape. Tarsius spectrum (Hubrecht '02), and thus of the twelve orders of mammals there are four in which the process of maturation is well known in one species or more, two in which it is very obscure, anil six in which ripening ova have never been seen. Amonp the la.«t are the ungulates, in which the species are either rare and inacces.*<ible, or if conunon, are so large that the search for the ova is very difficult. For these reasons it will be of interest to describe a small series consisting of 15 ova from 7 sows (sus scropha domesticus). I am indebted to Mr. Ralston B. Brown, Sui)eriiiten(lent of the Oakland Meat and Packing Company of Oakland. California, for the opportunity to collect the material, and to Mr. A. E. Amsbaugh and Mr. Felix H. Hurni for a.ssistance in its preparation. The sows were selected and the tubal ova found by the method given in a previous paper (Corner and Amsbaugh '17). Ovarian ova wer6 studied in serial sections of the follicles (celloidin) or by cutting hardened follicles into sliees, locating the discus proligerus and sectioning it in paraffin. 1. Killed during oestrus, probably on the first day. Unruptured follicles. Three ovarian ova sectioned, two show germinative ve.sicles, the other shows the first polar body and the second polar spindle. 2. First or second day of oestrus. One follicle ruptured, its ovum not found in the tubes. An ovarian ovum shows the first polar body and the second spindle. 3. Second or third day of oestrus. All follicles ruptured. Four imfertilized tubal ova sectioned: all show the first polar body and the second .sj)indle. 4. History unknown. Five unfertilized tubal ova found, of which four (studied fresh) show one polar body extruded. 5. Probably socoml day of oostrus. Copulation obsen'ed 24 hours before killing. One tubal ovum found, covered with spermatozoa. In sections, the male and female pronuclei are in contact. Two polar bodies. 0. Second or third day of oostrus. Copulation observed 10 hours before killing. Two ova found in the tubes; both were covered by spermatozoa. One of them, studied fresh, showed two polar bodies. The other was sectioned, and showed the pronuclei in contact, but unfortunately the polar bodies were obscured by damage to the sections. 7. Tubes contained segmenting ova of 2, 4. and G blastomeres. One of the two-celled embryos (studied fresh) showed two polar bodies; a favorable view of the others could not be obtained. COXCLUSIOXS The first ova of an ungulate mammal to be studied indicate that the sequence of maturation is the same in swine as in previously stuilictl forms of other orders, the first polar body being extrudetl and the second polar division proceeding as far as spindle formation before fertilization, the second polar body being cut off only after the entrance of the spermatozoon. LITERATURE CITED « VAN Benkden, E. 1S7.3 La maturation de I'oeuf. la fecondation, Cetc.) chez le Lapin. l$iill. do I'.Vcad. Royale de Bclpiqup. T. 40, no. I'J, p. fiSfi. CoUNER, G. W. AM) .Vmsuaui;!!, a. E. 1917 Oestrus and ovulation in swine.

Anat. Rec, vol. 12, p. 287. Hakt.max, v. O. 191C Studies in the development of the opossum, Didelphys

virjiiniana L. Jour. Morph., vol. 27, p. 1. Heape, W. 1905 Ovulation and deffcneration of ova in the rabbit. Proc. Royal

Soeiety, London, vol. 71), B, p. 2()0. Hli.l,, J. P. 1910 The early development of the marsupialia with especial reference to the native cat I Dasyurus viverrinusi. Quart. Jour. Micros.

Sci., vol. 5f), p. 1. HuBRECHT. .\. A. W. 1,S95 Die Phylogenose des .^mniros und die Bedeutung des Trophoblasts. Verhandelingen der Kon. .\kad. van Wctenschap pcn. te .\ni8terdam. Tweedo .'^ectie, Dec!. 4, No. 5.

1902 Furrhung und Keimblattbildung bei Tarsius spectrum. Ibid., Dccl 8, No. 6.

Long, J. A. and Mahk, E. L. ISMl Tin- imitiirntion of tlif fn\i of llie nio\i8C.

Puliliriilion N<i. 142 of the CariicKii' Iiuslitiition of WaKhiiiKton. LoNfiLEY, W. H. mil Till- iiiiktiiratioii of tlii> ccfc nnd oviilution in the (lumestir

cut. Am. Jour. Aniil.. vol. V2. p. 13'J. RrBASciiKiN. \V. 10(15 I'i'Imt <lir Ki'ifuiiR.s — iiiul I)<>fru(-htiiii):i<|)rozes8c dcs

Mccrsrhweinclu'iK'icK. Anat. Ilcflc, Bd. 29, S. 507. SoDOTTA. J. 1895 Die HefriirhtunK und Furchung dcs Eic8 dcr Mans. Arcli. f.

Mikr. Anat., Hd. J.i. S. 15. SoBOTTA, J. AND BrHrHKAiiD. (i. L. 1910 Kcifung und Bofruchtiing dcs Eies

dcr Wcissi-ii Ilattr. Aiml. Hcftc. Bd. 42, S. 43.3. VA.N DEK i^THKiiT. O. 1!K)S La Structure <ic I'ocuf chcz la chioniic. C. R.

dc I'Assoc. dcs .Vnatoiiiists. Dixicmc Hounion. Marseille.

1910 La structure <le I'oeuf des inaniiniferes, (C'hauve-souris, Vcspc rugo noctulai. Troisieme partie. Memoire presentc a la classc des

isciences, Academic Hoyalc de Bel|{i(|ue, 2 ""^ Serie, Tome 2.

Hyperphalangism accompanied by supernumerary epiphyses and muscular deficiencies

Lloyd K. Reynolds. From the Division of Anatomy of the Stnnjitni Medical School

TWELVE FIGURES

This interesting (IcvcloiJUK'ntal anomaly, was observed in the practise of Dr. Robert (1. Reynolds of Palo Alto in March of H)l(). The x-ray photographs acconipan>-ing this article were kindly taken by Dr. l^cyiioids, through whose courtesy I am enabled to report the case. .\s it usually happens in these instances the family history reveals related abnormalities. On the father's side of the family there have been se\cral cases of abnormal development of the skeleton of the upper extremities. The paternal grandfather is said to have had syndactyly of the index finger anil the thumb of the right hand, up to the interphalangeal joint of the thuml). The terminal phalanx of the thumb is said to have been flexed at right angles on the proximal phalanx so as to lie in the palm. The left hand and both feet were said to have been normal.

The father's oldest sister who died at the age of six is said to have had a deformity of the right hand similar to that of her father. Otherwis(> she was normal.

The father's second .Mster who died soon after l)irth is said to have had congenital absence of radius and ulna on the right arm and the father's older brother is said to have had similar deformities as the jiresent case.

The father's hands both show muscular defects. On the left hand the thenar eminence is absent, and the opponens and abductor jiollicis brevis apparently are lacking. The flexor brevis may be present. '1 here are two gi-oups of intcrphalangeal

aiul one of iiu'ta(';irpoi)lial;iiint';il -ulii. in tlic tliuiuli. ^ Ct Iho x-r:iy pliotoKraj)!! itifi. 1 i sliows that there arc only two plialanjrcs. The h'ft thuinl) is strai^lit and tapering; only al)out two-tliirds normal sizo and tho skin is smooth, shiny and atrophic. It is (1.4 cm. lonji and is rotated out a little. The mobility in the second terminal phalan.x of the thnml) is somewhat limited. The width of the left hand in the line of the metacarpophalangeal articulations is S.li cin. There is no wehhiiig of the fingers: the rest of the lingers of the left hand lieing apparently normal. The thumi) of the right hand is somewhat longer than that of the left. l>ut the thenar eminence is also absent; the regionbeing marked liy a (Icciilcd llatlcniiin. Tlic rinlit thumb is 7.4 cm. long and is rotated in rather than out, as is the ca.se with the h'ft. It is tapcriiiK and atropine as in the other htind, pf)s.se.s.siuK a small l)Ut ap])anMitly normal nail. The i)ulp of the thumb is (-(impressed from side to side and looks atrophied. 'I'liere are two prominent sulci opposite the interphalanj^eal joint of the thmnb and the width of the hand at the meta('ari)al|)lialanneal articulations i.s decidctlly greater than in the left hand. The other fingers are normal. The width of the right hand in the line of the metacari)alphalangeal articulations is 9.2 cm. or 0.0 em. more than the left. The medial surfaces of both thumbs, a little distal to the metacarpn|)lialanf?eal articulations are marked by an oval callosity about '2 cm. in diameter and O.o cn\. thick which forms a prominent pad. .Vs shown in itig. 2) the medial surface of the thumb is concave, the distal portion being bent medially, that is toward the ulna, the distal phalan.x being (Hrected medially at ai\ angle of about 10 degrees.

In the x-ray jihotographs itigs. 1 and 2) there is a pronounced mortising of the basal ends of some of the metacarpals of the thumb with the basal ends of the metacarpals of the index fingers. This condition seems to be the result of a general naiTowing of the wrist bones. In each hand the greater nmltangular is placed medially until its medial border is almost even with the medial border of the lesser multangular. Hence the position of these bones and the con.scMiuent jKisition of the respective metacarpals may jio.ssibly account to some extent for the lack of mobility of the metacarpals of the thumbs. The proximal phalanx in each thumb is markedly concave on its palmar surface and has a slightly bent shaft. This is more noticeable in figiu-e 2. These peculiarities in the thmnb may be ex|)lained in part by the fact that the lack of mobility of the metacarpals has made it nec(>ssary to rotate the j^roximal phalanx medially in order to use the thumb in conjimction with the other lingers. As a result of this rotation we see the phalanges from a lateral view rather than a dorsal and therefore the concave surface is the ventral and not the medial .surface of the phalanx. There doesn't .s(>cm to be anything to account for the three distinct

liciiiy tuborcules shown in outline in the x-ray plate on the ventral surface of the distal phalanx nf eaeh tlninil) (fips. 1 and 2). The distal ends of tlie i)lialanges seem large and irref^uiar. This is more noticeable in the rigiit (fig. 2) than in llie left

Fin- 2 Hinlit h.ilMl of f:illirr

(fig. 1) hand. l>ul this may he due to the fact that in figure 2 the hand was not held as flatly on the plate as in figure 1.

The son, the subject of this rej)ort is 13 years of age, ") feet, 8^ inches in height and weiglis \'M) jjounds. The ])hotograi)hs (figs. 3 and 4) show the marked deformity in the general appearance of the left hand and I figs. 5 and (i) the lesser deformity in the right. The I! \' fingers are developmenlally (|uite

normal for the iiiidtUc iingcr of the li-ft liaiui was injured in a recent accident. There is no webhing of the fingers and with the excei)tion of the second and fourth fingers the sulci are normal. On the palmar surface of the fourtli finger there are two sulci about 1 cm. apart opjxtsite the first intcrphalaiigeal joint. The index finger has a similar peculiarity. The thumb has the appearance of the little finger of either liand. It would indeed be im

Figs. 3 and t Left hand of son

possible to identify it by means of a photograph of these digits alone, in the ca.se of the left hand. The terminal phalanx and nail of the thumb are shaped exactly like the terminal phalanx and the nail of the little linger.

On the right hand the nail ui the thumb, although smaller, is shaped more nearly like the normal thumi) nail, and the terminal phalanx is also shorter and heavier than the terminal phalanx of the little finger. In each instance the thumb is 0.7

("III. loiipcr than tin- little (inner and markedly coiicavo medially. The space l)i't\veen llie index and middle linnt'is of the left hand is (greater than normal and the index tinp;<'i' is ooncavo medially. This is jirohahly due mostly to the fact that the boy usually prasps objects between the iiulex and middle fingprs, rather than between the thumb ai\d ind(>x (infers, because of the abnormal tlovolopn)rnt and relation of the thumb. I Ix- thumb

l"i(»s. ."i :iml tl liiRht liiiiul of son

of tlie rifiht haml is also concaxc medially and small objects, such as a coin, when nrasj)ed by the ri>;lit hand ai'e ])lace(i between the dorsal surface of the ternunal phalanx of the thumb and the tips of the fingers

The thenar eminencfs are absent with ai)i)arently an absence of all the thenar nmscles (figs. 4 and (i). Even the adductors appear to be ab.sent and the.se facts no doubt account for the jMculiarily in mobility. There is a lack of mobility of the metacarpals of the Ihumlis which is very noticeable because it limits the motion f)f the tlninilis to tlio niovonionts possihlo in the othor {iiifijcrs.

The position of the thunil) in relation in the other fingers gives the forearm tlic apix'aiance of Iw'ing much longer than normal even though measurements show it not to l)e exceptionally long for a hoy of his height. The hand is markedly widened at the metacarpo])halangcal articulations. The left hand is 9 cm. wide. The width of the four fingers lieing S cm. The right lianil is [).'.i cm. wide and the width of the four hngers S.4 cm. A similar tlifferenee was present in the father's hands as stated above.

The jihotogiaph in figure 4 shows the smooth, glistening appearance of the skin in the ]ialm of the hand. This is characteristic of both hands although a slight movement during the taking of the picture prevented its showing in figure 0. The skin seems to be drawn tightly so that very few sulci apjiear.

In the x-ray photographs (figs. 7 and 10) it is shown that the thumb of the left hand has three, well-formed, phalanges which in large part accounts for its length. In this case the age is such that the epii)hyses show exceptionally well. There is nothing to indicate that the middle phalanx of the left thumb is rudimentary and will later fuse with the terminal jihalanx. Indeed the middle is longer than the terminal phalanx anil has a we'll-formed, normally-located epiphysis (fig. 7). Each of the other phalanges has al.so a well-formed epiphysis and seems entirely separate from the middle phalanx. The metacarpal of the thumb on this, the left hand, also is of interest for its form and length seem inoic nearly like a true metacarpal than the corresi)on(ling bone in a normal hand. Besiiles, the epiphysis is at the distal instead of the proximal end of the bone, the same as the ejiiphy.ses on the metacarpals of the other tiiigers (figs. 7 and 10). The metacari)al of the thumb is relatively long. The x-ray i)hotographs were taken with the hands lying directly on the plate and with the machine o\-er the .<ame part of each hand. Measuring the negative we find that the nietaeari)al of the thumb is 0.7 cm. loiifr while the metacarpal of the little finger is only (1 cm. long. The circumference of the first meta

(•ari)al al.>*o seems less than the circunifoionce of the correspondiiiH liDiie of the little fiiiiicr. Xoiinally the inctacariJal of the thunil) is sliortcr and tliickcr than any of llic other in('tacari)als. The x-ray ])hotogi'ai)h of tlic i-i^ht liaiid is in sonic i-cspccts more interestiiip; than lliat of the left. The extra phalanx of this tliuinb is not so well formed as that on the other thiimh or

Figs. 7 iiiul .s Left and ii^;lii li.-mds of .son

those of the other corresponding phalanfjes liut nevertheless as .shown in hunre '.• it is a separate bone with a distinct diajihysis and epiphysis, articular surfaces and base and head. Besides showing the .same peculiarities in regard to the extra i)halanx, the metacarpal of this thumb shows a double epiphysis (figs. S and 11).

In all of the cases of three-jointed thumbs reported, heretofore, the middle i)halanx is con>idered the extra phalanx.

Pfitziior, '!)() who has done more work than anyone else on the (Icvclopiiicnt of tho oxtreinitios. has roportod several rases of tlircc-joiiitcd tiiiinilis, hut in all of these cases the three joints were only temporary for the extra, middle phalanx later fused with the end phalanx to form one bone. In no ease did Pfitzner find an extra jjhalanx that hail a distinct epiphysis, articular surface, head, and which in general had the shajje of a normally developed phalanx as in the present ease.

Kig- !' Thumb of right h:iti(l taken latorally

Rieder '00. rejiorted a family, most of whom had three-jointed thumbs. The father ami four of tlie children had an extra phalanx in the thumb. Kieder claims that his cases ditTcr from those reported by Ptitzner in that each end i)halatix did have a distinct epiphysis entirely separate from the middle lihalanx.

Dwijrht '07 in his report on the variations of the bones of the hanil and foot considers the jiresence of an extra i^halanx in the tliuiiil). lie says that in no cas(> reixirtcd diil the extra jihalaiix have an ei)iphysis in those of an a^je at which it could be expectctl to l)e present. He supports Phtzner's theory tliat the extra phalanx is later fuseil with the terminal ])halanx.

Salzar 'OS, also r('pt)rtr(l a case of a tlirco-joiiitcd thuiiil) on oach hand. In each instance the second phalanx was short and ajjpaniitly ludinifntary. The so-callid ps(udo-e])i])hysis shown in hjtures S and 11 is distinct and has the cliaractcristics of a normal ejMphysis. It woulii be hard to consider one a secondary e|)ii)hysis as has been (h)ne in most of the cases heretofore reported and in this case it seems (juite evident that the first metacarpal had two true epiphyses. The presence of an extra ei>iphysis. however, has not caused any increased length as might be sui)posed. f<ir although this metacarpal is longer than the metacarpal of tiie fifth linger of the same hand, it is not as long as the metacarpal of the thumb on the left hand, which has only one epiphysis. I am leail to believe that both epiphysis are true epiphyses, by comparing the x-ray photographs taken nine months apart. In figure 11, taken in November of liUti, both epiphyses h;ive the appearance of true epiphyses as much as they did in the photogiaph taken in March of 191(i (fig, 8) or almost a year before. Hence although the supernumerary or cephalic epiphysis may fuse earlier than the basal or normal one it is clear that the former does not fall under what Thomson '06 .speaks of as "scale-like epiphyses on the head of the first metacarjjal which makes its apjiearance about eight or ten, and rapidly unites with the head. Since the first metacarpal in the left hand of this case, has a distally located epiphysis an interpretation of the latter in Thomson's sense would leave this metacarpal without an epiphysis.

Freund '05 also reporteil a case in which the midiUe phalanx had an epiphysis at the distal as well as at the proximal end. He compares his case to those reported by P(it/ner (HI, and arrived at the same conclusion regarding the spurious character of the.«^ epijjhyses Pfitzner ba.ses his conclu.sion ujion the variation in ossification foimd in many ca.ses which came under his observation. He thinks that in the.se ca.ses of apparent supernumerary epiphyses the oestodasts, instead of breaking down the epijihysral cartilage in a straight-line front, extend into it in the fmin of luoccssc s or projections and thus ])roduce an appearance which roughly simulatrs an epiphyseal line.

But as the photographs (figs. 8 and 1 1 ) show the breaking down of the cartilage in tliis case is being earricd on in a straight line front. The portion of cartilage between the diaphysis and the extra epiphysis is as even and regular as in any normally developing bone. Hence Pfitzner's explanation does not apply here and this epiphysis seems to be a true ami not a pseudoei)iphysis.

l-iKsi. Ill :iiul II Saliio Imiul^i nboiit one vcar hiUT

'l"lu' ini)rtisiii>r of llic l)j\sos of sonic of tlw metacarpals is fiuite proiioimccd. On I lie iiicdial side of I tic liasal ciiii of the thinl left nietacar))al. for example, tlierc is a dee]) notch for tlie articulation of the fourtli metacari)al (fig. lOj. There doesn't seem to be any explanation for the jMesencp of this unusually deep notch.

Scrutiny of figures 10 and 11 will show that the carjial hones in tlie right and left hand vary both as to shape and relative position. In (fig. 10) of the left liand the greater multangular is considerably larger and comiiletcly o\ershadows the le.sser multangular bone. In the riglit hand (fig. 11) the lesser multangular is larger than the greater nuiltangular but the greater multangular is more laterally placet!. Figure 10 also shows the tri(|uetral bone to have a rectangular shape. Indeed.it looks not luilike a short phalanx. The triiiuetral bone shown in figure 11 is triangular and considerably larger than that in figure 10.

The thumbs in this case es|)ecially the left seem to be like a little finger in most respects except their position on the hand. Their movements are limited to the normal movements of a finger; they have the same number of phalanges as the other fingers: the nails resemble the nails of other fingers rather than thumb nails and their metacarpals have flic characteristics of the metacarpals of fingers rather than those of normal thumbs. I df) not think that the extra phalanx in either case can be cont<idered as rudimentary becau.se of the perfect development of each individual phalanx, the presence of a di.stinct normallylocated epiphysis and the absence of any indication of fusion.

Professor Meyer to whom 1 am indebted for suggestions and assistance has offered a i)o>sible explanation for the occurrence of sueh a finger or thimib. If 1 understood him correctly he thinks that the ai)pareiit absence of the thenar muscles may indicate that the normal impulse to develojiment of the thumb was absent and that hence the phalanges and also the metacarpals of the thumbs, assumed the form and relations characteristic f)f the fingers rather than of the thumb thus accounting for the extra phalanges in the thumb, the epi|)hyses in the supernumerarj' phalanges and also for the superinunerary epiphy.ses

Fig. 12 Normal left liiiiid of u boy of approximately the samp age, height and

weight for comparison

ill the nu'tacarpal of the rifiht thuinh as well as the distallyplaced, single e])iphysis in the metacarpal of the left thumb.

Although Professor Meyer's suggestions seem to account for the results found in this case, he feels that we can not he certain that the thenar nutsdcs are entirely absent. However, sis far as can be determined from external apjicarances and from the limited mobility of the thumb thiy must lie exceedingly rudimary indeed if present.

IJli I.I.OVl) K. ItKVNOI-ns

lUHMiiCK M'llV

Annadalk, Til. 1S<)5 The iniilformiitions. dispiisofi and injiiries of the finf^ers

nnil locH and their siirKieiil treatment. KdiiiliurRh. Hi.iFF. M. .1. l.SLt) riM-r das xoiteiianiite ()s nietanirpi piillicis mit 3 .\l)l)il diincen. .\rchiv fiir An.ntoniie iinil Pliy.-iUilouie. nwHiiiT, Till >M AS liHlT Clinical atlas of the hones of the hand and fool. Philadelphia and London. P'HEfND, L. MKI.") l"l)er IVeiKhiepiphy.sen. Zeitsehrift fiir .Morpholojiie iind

AnthrnpoloKie. vol. S. Kaltin. U. I'.HM Kin Fall von .Misl>ilduiiKen der olieren lOxtremitiit d\irc'h

I'lxjrznhl. .\rchiv fiir .Vnatoniie iind Physiologic Jo.M'illMSTHAL. G. I'ljcr Brachydaktylie iind Hyperphalan(ric. Virohow's

Arehiv. Bd. 151 Heft 3. Ki'MMKl.. W. ISSI.i Die Mi.ssliildiinKcii der Kxtreniitiiten diireh Defeckte,

Vervvai-hsunK und (Mierz;ihl. Hililioth. Med. K., Heft. 3. Leboitq, H. I.SOli De la hraehydaetylie et de I'liyperphalaiipie ehez I'lioinine.

Hidl. lie I'aeadeniie royale de medicine de Helfsicine .S-anee du. 30 Mai. Pfitzxf:r. \V. IS'X) Kpiphyseiil>ildiinpen. Mannigfaltigkeit dersclhon. Arehiv

fiir .Vnatomie iind Phy.siolopie. .Vnat. .Vlith.

1892 Beitriine zur Kenntniss des nienschliehen E.xtremitatenskelettes.

Schwalbe's MorpholoRische .\rl)eiten, Bd. 1.

Normalmasse der Phalangen o. Beitrag zur Kenntniss dcs mensch lichen Extremitatenskelettes. Sch\vallie"s Morphologische Arbeiten,

Bd. 2.

1S94 I'ber das Skelett der nienschliehen Hand. .*<ch\valbe's Mor pholoijische .\rbeiten. Bd. 4.

1S9.5 Kin Fall von beider.seitner Doppelbildungen tier 5 Zelie.

Schwalbe's .MorpholoRi.sche .Vrbi'iten, Bd. 5.

1897 Kin Fall von VerdopiM'hinn des ZeiRefingers. Schwalbe's Morphologische .\rbeiten, Bd. 7, Heft 2. HiEDKR, H. UKX) Eine Familie mit dreigliedrigen Daiiiuen. Zeil.schrift fiir

.Morphologic und .Vnthropologie. Salzkii, H. Zwei Fiille von dreigliedrigen Daiinicn. .Vnatomischer .\nzciger,

Bd. 14. .'^tliAKFK. .\. 1912 Zwei Falle von symmetrisclien Mi.ssbildiingen der Finger.

Ztschr. f. orth. Chir.. Bd. 20. TiioMaox. .\nTnfK liKXi Chapter on osteology in Textbook of .\naloniy,

Cunningham, London. Second revised e<lition. l'KiKi..\iA.\x, J. isi;2 Der .Mittelhandknoehen des Daumens, seine ICntwickc lungsgeschichte unci Bedeutung. (iottingen. WiMJLE, B. 1891 Thi- occurrence of an additional phalanx in the human

pollcx. Jour, of .\nat. and PhjsioL, vol. 20. \Vi:u:KEN, il. 1884 Die morphologi.sche Bedeutung des 1. Daiimengliedcs.

Prcisvertheilungs progr:inun der l'nivcr.-<ilat Halle.

AX INKXPKXSIVK PRO.TIX 'TIOX AI'PAH ATI'S

IVAN K. WALLIN Departmetit <ij Aiiiitoiinj and liiologij, Marijuittc Cnivcrttity School of Medicine

THKKE riGrRES

Tlu ohJMt of this iijiparatus is the siil)stituti<iii of an Edison Mazda liimi) for the arc lain]! and the r( diiction in (Mist of a scrvircalilc apijaratns for drawinj;. The arc Ian )) as an illun inatinn unit in a i)rojcctior ap]>aratus has certain ohji ctionaMc features; the light is not always steady and it re(|uires aln'ost constant attention when in use. The perftction of the nitroRcn-fiiled electric Inill) has supjilied a lijihtinn unit which is only slightly less intense than the arc light.

A siir])lc and inexpensive jirojection apparatus would he valuable for students' use in the lahoratcry. T.he ajiparatus di scribed below, it seems to inc. will supi)ly a serviceable ai)|)aratus for class use at a very moderate cost.

This a])paratus is construct((l on the plan of the Edinger apjiaratus (fig. 1). It consists of an ui)right oak lieani (u) attached to an ordinary table, a microscope .support (;/;.. v.) which also supports a rod (.•-■./•.) carr>-ing n flcctor, lamp, condensors and diaiihragni.

The upright (u) is an oak bean', 2 by 4 by 42 inchis, containing a slot for the reeei)tion of the U'icroscoiie support. It is bolted to the sides of the tai)le and braced underneath to the top of the table.

The niicroscoiie sui)port (in.s.) is a steil i)ean'. The l>ack has a flange which fits into the slot of the U])right and serves to hohl it firm. The free end is constructed in the form of a clamp to fit around the limb of the n'icroseo]ie and is n'ade firm by means of a bolt (/>). The body of the supjiort has a slot (.•<) to receive the upright siipjxirt rod (s.r.). The n;icre..scope siipport beam is movable up and down on the wooden ujiright and can be trade secure at any iitint by ireans of the han<l nut (li.n.).

A different ty]>e of microscoix- supjiort is illustrated in figure 3. This t^^^e of su])port has the advantage that the iricroscojie may be attacheil or removed in an instant. The stage of the M'icrosco))e slides iiite) the sle'cve e)f the' I'-shapeel suppeirt. The- feieit allel pillar of the- micreisce)pe" are' turneel bae'k at right angles te) the l><)ely tube'. A sle)t in the' dia])hragm weiulil ace'eimnieulate' the' feieit. (It is an aelvantage te) have- a large diaphragm as it increa.ses the> we)rking sjiace.)

The sui)peirt roel (.s.r.) and the .sujiport rings which heilel the le'nses, diaphragm, lamj) heuise and re'tlee'tor wire' taken freun an e)relinar>

IJS

lUAN K. WAI.I.IN

AN INEXPENSni; I'KOJECTIOX AI'l'AKATrs IJ'.t

hiliorsitdiy support or rinn stand. Tin- suijport riiijis in this type :ill have a coiiiniDn center. The siii)|)()rt rod is movable on the tnicro.icopc support heain so that lanip and eondensors may he properly centeri-d \\ it li the microscope.

The dia))luanm is a circular |)iece of wood which is held in jjhice by a su])port riiin. Curtains are attached to the diai)hrat;m and hanj: <lown over the edne of the tal)le. The curtains may be attached by means of hooks and eyes or nl<'v<' fasteners.

The reflector (r) and its housing are a jiart of an old style gas automobile lamp.

The linhtinji luiit is a 500 watt Edison Mazda steroopticon lan)j). The fdaments in this lamp are concentrated and form a compact unit. The lamj) house is made of sheet iron and is held in jilace by a ring support. It al.so serves as a sui)i)ort for the lamp.

The condensor (r) consists of two 4-inch stereoi)t icon lenses. These can be obtained from stereo])ticon supi)ly houses. Thi' adjustieent of the lenses is illustrated in fiinu'c 2. The distance between the two lenses H and (' is arbitrary. The filament of the lamp (.1) should be at the point of focus of lens (fi). The focus of lens (D shoulil fall within the objective of the microscope (D). The focal length may be determined by holdinp, the lens to the sunlight in a smoky or tlusty atmos])here and mea.suring the distance from the point where the rays cro.ss to the center of the lens.

The microscojie is an old tyiie from which the foot and iiiiiar have been removed. The stage has been reversed so that the sliile rests tm top of the stage. The fine adjustuK nt extension (cm.) c-onsists of a rod, a pi])e, a steel sprinp and a ca]). The ca)) is secured to the fine adjustment by means of a .set screw. The rod fits into the jiipe and is made secure for any length by a set screw.

The fine adjustment extension was made in the laboratory. The microscojie suiijxjrt was made by a blacksmith. If re(|uired in (|uantity the microscoiie supports could be cast in a foundry and obtaineil at a smaller cost.

The cost of the jiarts of this ajiparatus is as follows:

tprinlil iliard oak) $1 2."i Micniscopc support, holts nnd nuts 3 50 Lenses for condensor 2 7.i Keneclor .. 100 Ring supports and support rod \ ~r> Lump ;{ IK) Fine adjustment extension. ■")()$14 35

Baiiseh and I.nml> Optical ('om])any liave recentl\' p(rf(i-ted a lamji house witli relleiti r and eondensors which might i>e adapteil to this a])|)aratus.

I am greatly indebted to Dr. 11. ('. Tracy for a.ssistanci' in the optical adjustnu'Ht of the ai)paratus.

AnTnoR*"* AiiHTKACT or mm papkh i^bdbd

BT TIU: hIKl rf)(iftAPniC BtHVK E AL'Ol^T 11.

VARIATIONS OF THE CANALIS HYPOGLOSSI

HALPH D. LILLIE From the Division of Anatomy of the Stanford Medical School

My attention was attracted to the sul)ject of tho variations of tho hypoglossal canal by the discover)^ of a double hypoglossal canal in the dissecting room. Subsequent examination of about thirty skulls, about twenty of them Eiu-opean, revealed ten cases of complete division of the canal. Consultation of the ordinary reference books failed to give definite figm-es. So Dr. Meyer placed the collection of skiiUs in the anatomical museum and Indian skulls in the general nuiseum at my disposal.

Battels ('04) found the hjTioglossal canal doubled in 117 cases in 05 canal was double it was frequently dividetl into unequal parts so that the posterior canal was about twice the size of the anterior, or more rarely in the reverse ratio. Weigner believes that the tlivi.sion of the canalis hypoglossi is one ot the manifestations of an occi]iital vertebra. Graf v. Spec {'96) notes the division of the canal anil says that indications of division are almost constant in the adult but makes no mention of tho character of th(> indications. .Taboiilay and Lucy ('11) attribute the division of the canal to that of the nerve and Froriep ('11) showed

i:ii

THr ANATOMICAL RICORD, VOL. 13. No. 3 AUatTBT, 1017

132 KM. I'll D. I. II. I. IK

that in ihr hoviiK' oinl)ry<) tho hy]>oglossal n('r\c corresponds to three segmental iier\-es. Schiifer aiul Syniiiintoii in Quain's Anatomy III, part '2. lOO'J. also regard the hjixjglossal nerve as representing tliree segmental nerves. Prentiss (10) working on pig emhrj'os, found eight ganglionic masses correspontling to four ganglia associated with the In'iioglossal nen'es as their dorsal root ganglia. These ga'nglia were in series between those of the \agus and of the first eerv-ical ner\'es. They are interpreted as representing four ln^^oglossal ner\-es. Five or six ventral roots of the In^ioglossal e.>dst, but according to Bremer ('08) the more anterior of these represent ventral roots of the vagus and glossopharjTigeal nerves. Martin ('91) working on cats, found live ganglia and five ventral roots and concluded all were hj^ioglossal. So it woukl seem that either tliree or four nerves exist developmentally and this could account for either three or four somites and their corresponding neural foramina and their corresponding vertebrae. Hence between the jugular foramen and the synchondroses petro-occipitalis and occipitosphenoidalis on one hand, and the atlas on the other, there must have existefl four or five occipital vertebrae with three or four neural foramina or hj'poglossal canals between them. Consequently we should expect to find traces of di\-ision of the hypoglossal canal into either three or four parts.

In fact we do find such divisions. As stated above Weigner noteil indications of cUvisions into three parts, also into two parts, the posterior of which was twice the size of the anterior. I also have seen a number fif such cases. Such occurrences could be interjjreted as jnutial or complete separation of the first hM'oglossal nerv^e while the two following remain together. I have seen indications of dixisiou into three equal parts two of which may be comjiletely separated. However, complete division into three i)arts has not b en obsen'ed.

McMurrich ('05) says "dujnng the cartilaginous stage of the skull the antcT-ior condyloid foramina are divided into three portions by two cartilaginous p.utitions which separate the three roots of the hj'poglossal nerve," and considers this as evidence of the existence of four fused vertebrae in the occipital bone.

VARIATIONS OF THK CANALIS HYl-OGLOSSl 133

These facts point toward tlirce original hyjioglossal canals. But a mniilx-r of oases wore ohscrvcd in which tlic canal was conij)lct('ly or inconii)lctoly divided into two ecjual parts and in which the anterior canal showed indications of further division into two equal parts. In the light of the work of Prentiss and Uremer I nnist look ujiou such instances as an indication of division of the canal into four equal parts. Of course, it may be in this case that it is really di\'ided into three and that the third h^-poglossal nerve is larger than the others. Poirier et C"liari)y report cases in which the canal was divided into four parts. I'rofessor Meyer suggested to me that some of the osseous processes observed may be secondary' ossification in the dural septa extending between the fasciculi of the roots instead of remnants of arches of occipital vertebrae. Such a ca.'^e was noted in No. 42, a dissecting-room specimen. In this skull the dried remains of a dural or connective tissue septum extended between two spurs on opposite sides of the foramen. Whether these spurs are to be looked upon as secondary ossifications of a dural septum or whether such septa are to be considered as unossified remnants of vertebral arches can not be decided in dried skulls.

It is also probable, indeed almost certain, that in some eases these di\'isions are significant not of diAision of the nen'e, but of vascular variation. Some of the variations recorded here are unquestionably due to aberrant vessels and not to diAision of the nerve. I refer here to those cases in which tortuous canals leave the main canal more or less obliquely to reenter in another place in similar fashion. On the other hand a large number of these variations are in all probabilitj' due to di\-ision of the nerve, for the nerve has been observed to pass through as two separate nerves, each in a canal of its own. In the more caudal parts of the body the metamerism of nerves and vertebrae iiolds strictly. Metamerism of the nerves exists in the head, showing best in the embrA'o. Indications of bony segmentation of the canal corresponding to the nervous segmentation are al.>^o found. Moreover an occipital vertebra may be partially separateVl. The.se facts all point in the same direction.

\M

HALPH D. LILLIE

OB.SKUVATIONS

SS Indian akitlh from Jersey County, Illinois

Character of hypoRloBHnI cunals

LKfT

RIOUT

7616

A sliRlit suiK-rior spur not

\ septum a few millimeters thick at inter

at cither opciitii):

nal opening. Tlir for.imina equal

7613

Simple

Simple

7612

Simple, smaller

Simple, larger

7630

A slight superior, promincneo near tlie margin

A slight superior tubercle at the inner margin

7609

Simple, n>iinJ

An inferior spine, a flattened internal opening and a slight indication above at the internal margin. Hound externally

7607

A small a'ntero-siiperior spur

A small antero-superior spur

7605

•Simple

.\ rough superior internal margin

7608

A heart shaped internal

An anterior foramen right through to a

opening with a promi

common external opening. A larger pos

nence sui>crior

terior foramen giving rise to a small anterior canal through to the common external opening and a large posterior blind pit. In the external opening are three small pits above and posterior to the two canals

f606

A small antero-suix^rior notch

A small antero-superior notch

7604

The internal margin is

A marked internal superior spine with an

rough superiorly

indication opposite on the lower margin. The anterior division is half the size of the posterior

70IC

A septum 3 mn>. wide by 1 mm. thick internally. The anterior canal is smaller than the posterior

A projection on the superior margin

7618

A slight su|)erior spur

Two equal canals

7610

Opposed spurs almve and below

Compre.s.scd dorso-vent rally

7620

.\ broad septum within the canal. The

canal. Anterior canal

anterior canal half the size of flic posterior

half the size of the

posterior

7627

Simple

Simple

7620

A slight superior tul>crcle

\ slight superior spur

762,1

Simple

Simple

VARIATIONS OF THE CANALIS HYP0GL08SI

135

OBSERVATIONS-Condnut./

WDMBCR

Lcrr

RIOHT

7624

■ Simple

A deep septum half the length of the canal. I'lqual division.

7623

Simple

A slight double spur on the internal HUficrior margin

7622

Simple

.\ slight spur from the roof near the internal orifice

7639

A superficial internal sep

A rough su|K."rior margin internally. Tlie

tum. Small anterior,

medial side of the posterior condyloid

large posterior canal

canal opens into the skull posterior to canalis hypoglossi

7638

.\ slight superior spur. The anterior divi

nal septum. Anterior

sion half the posterior

canal smaller

7637

Slight antero-supcrior spur on roof

Slight antero-suix-rior notch

7635

Simple

Simple

7KU

Sim|)le, larger

Simple, smaller

7(«3

Anterior superior spur. One to two division

Slight double superior spur

7r)31

Antero-superior notch

Two slight superior elevations

7628

Simple

Simple

7632

Simple

Simple

7610

Simple

Simple

7624

A ridge below, a spur above

Simple

7641

Simple

Simple

7640

Simple

Simple

7643

A marked superior spur

Simple

7642

Simple

Simple

7'iro Indian skulls from La Conner, Washington

7645 7644

An internal septum. Anterior canal half the posterior

Simple

Simple Simple

Three Indian skulls from the Longueville graves, Plumas County, California

7647

A superior spur

A narrow, sept urn

median,

superficial

internal

7648

Simple

7649

Simple

Simple

VMf

UAI.l'lI 1). I.II.ME

(iBSKUVATIONS-ronlinurrf 16 Iniiian skulls from I'onct mound near MayficUl, California

KOM

Lsrt

■laRT

K.

Simple

Simple

7650

A thin, narrow srptuni

Superior tongue-like process and

1 sIIkIk

in (he niid-pnrtion of

inferior (ulierclc

the oiiniil

7651

Clear

Complete septum, .\nterior canal size of the posterior

half the

7652

Spur on the superior internal niarfdn

Opposed spurs on internal .margin

7G53

Simple

Simple

7654

Simple

A superior anterior roughening

7655

Simple (young, first permanent molar)

Simple

7657

Simple

Simple

7056

Simple

Simple

7660

Simple, flattened

Simple, flattened

7658

Simple

Simple

7662

A deep internal septum in inner third of canal

Simple

7661

Simple

Simple

Ml

A thin, superficial internal septum. Anterior foramen half the size of the posterior

Simple

M2

Both foramina round. Septum deep' in middle third of canal. Anterior canul larger

Flattened from top down.

Fire Indian skulls .

'rom near San Jose, California

M3

Median .septum lu-ar in

.Median septum near internal foramen.

ternal foramen

Opposed spurs divide anterior canal into two

M4

Antero-£U|K'rior spur and notch

Simple

M5

Simple, round

Opposed spines. Anterior division half posterioV

M6

Broad flat sepliun. Two internal foramina arc equal. External foramen single

Basioccipital of this side is destroyed

M7

Simple

Strong sup«'ri<>r and .slight inferii>r spines on internal margin. Anterior division half poi<terii>r

VARIATIONS OF THE CAN ALLS HYI'OGLOSSI

137

Twelve whole skitlU or entire occipital bonea

KUMBKR

9 10

11 12

ronipletely dividoil iiorve ill liDth cunnls

RoukIi superior internal margin

Slight median superior spur

(102.) Clear

Antero-superior notch

Medium superior spur. .\Iso superior spur in middle of anterior division

Clear

(13.) Strong septum anterior canal 2-3 times as large as posterior

An anterior-superior notch

Clear. (Occipital only)

Clear

.Slight median sui)erior elevation. Two small orifices just above internal opening

Clear. Only one hole in dura

Hough su|>erior internal margin

Clear. (Anterior part atlas assimilated)

Clear Clear Opposed median spurs

Clear

Septum in middle third of canal. Anterior canal is twice the size of the posterior

Anterio-siiperior notch. UJccipital only)

Clear internally. At external opening a small superior canal cut off

Clear

Clear

Twenty-two European skulls

1

IV. Clear

Antero-superior cleft and spur

2

V. Thin median septum

Thicker median septum in middle third.

in inner third

Spur in middle of superior margin. Small inferior spur and opposed superior ridge in middle of anterior canal

3

XIII. Large and clear, posterior condyloid canal opens laterallv

Large and clear

4

II. Clear

Median su[)erior tuljcrcle

5

XV. .\ntero-superior ir

Antero-superior and antero-medial spurs.

regular process 1 to 2

Small canal antero-superior to two spurs

nun. long

6

Thin septum in middle

Opposed elevations. Anterior part con

third of canal, .\nte

stricted and half the size of the posterior

rior two-thirds size of

posterior. ((> on medial

side of left mastoid;

l.-iS

ItAI.I'Il I). I.II.I.IK

OBSEIIVATIONS— Coniinu*^

HenBill

Lurr

MOHT

7

XII. Oppo.m-d iiiitoro-«iiIH-rior itiul -iiiotiiiil spiirs which enclose n Hinitll caniil

Same as left but enclosed canal smaller

8

XIV. Clear

Clear

VII. .\htero-8iipcrinr nmri;inal elevalicin

Clear

10

III. Slight nntcro-su|wrior tuljcrcle

Clear

11

XVII. TriaiiKular. A fossa runs liack from posterior suix-rior corner into a small pit

Antcro-superior notch clear through

12

IX. Clear openin):. A small venous canal runs antero-medially from roof near posterior orifice to near the external orifice of canal

Clear

13

XVI. Clear

Clear

14

VIII. Clear

Clear

15

Heavy septum. Anterior canal small, almost slitlike. The slit points toward the condyle

Clear

16

V. Clear

Clear

17

III. Clear

A long superior spine which almost divide«  the main canal into a smaller anterior canal and a larger posterior. .\ wide opening in the postcro-latcral wall <)i>cning into the posterior condyloid canal.

18

II. Strong septum in inner half. Anterior canal slightly smaller

Clear

19

(04.) (Wcfxarate occipital) Clear

Median superior spur

20

BU. Opposed nieilian spurs, the su|M-ri(ir being stronger

Clear

21

Cll. Clear

Clear

22

Clear

Clear

VAUI.XTIONS OK TIIK CANALIS HYPOGLOSSI

139

Summary

IXK Al.lTf

■IDK

X

M

a.

a

H

n n

a

n

Si 8^

H

X a!

X

Illinois Indians <

California Indians \

{

Specimens from dissecting room I

European skulls

Total 1

Left

Right

Both

Left

Right

Both

Left

Right

Both

Left

Right

Both

Left

Right

Both

17 15 32

14 16 30

31 31 62

13 13 26

75

75

150

48.6 42.9 45.7

56.0 66.7 61.2

43.7 43.7 43 7

59 1 59 1 59 1

49.02 49.34 49 18

14 15 29

3 5 8

30 31 61

5

8

13

52

59

111

40

42 9

41 4

12.0 20.8 16.3

42.2

43 7

42 9

22.7 36 4 29.5

33.99 38.82 36.39

4

5 9

8

3

11

10

<)

19

4 1. 5

26 18 44

11.4 14 3 12.9

32.0 12.5 22,4

14 1

12.7

13 4

18.2

4.5

11.4

16 99 11 84

14 43

35 35 70

25 24 49

71

71

142

22 22 44

153 152 305

Unpaired series. Fifty-nine left sides of skulls or of occipital bones from cadavers dissected in the dissecting room

1. .Viitorior superior spine and notch.

2. Anterior superior spine and notch.

3. Clear. .\ small canal from the lateral xvall of the canal opens behind the occipital condyl. Anterior to this are two other apparently blind small canals.

4. A superior tubercle in the middle of the internal margin and another midway between the first and the antero-medial internal margin.

5. Clear.

6. \ strong septum with a suiH'rior spur projecting inward. The anterior canal is smaller.

7. The superior wall of the canal is destroyed, the remainder appears clear.

8. A strong superior spur internally. The anterior division is half the posterior.

9. Clear.

10. .\ small au|M'rior elevation between anterior and middle thirds of the internal nuirgin.

11. Clear.

12. Large. Rough superior and aiiteru-mcdial margin.

13. (13L.) .\ strong complete septum clear through to the external opening. The anterior canal is half the posterior. .\ canal from the postero-lateral

141) UALl'Il I). l.n.I-IE

wall of thi- piiatorior riitial oimtis into the mcditil siilo of tho posterior roiulyloid canal. Anterior ciiniil has niarked anterior eonsi ruction. From anterior opening the anterior canal lias marked superior diverticulum which communicates with top of posterior canal. Latter has smaller anterior opening.

14. Clear.

15. I^arge, clear.

16. Posterior condyloid canal opens just posterior to posterior internal margin. The internal orifice had sliRht antero-supcrior notch. Opposed superior and inferior ridges near the external orifice divide the canal evenly.

1/. Antero-superior cleft.

18. Clear. One pit in sujiefior, another in postero-lateral wall of the canal.

10. Hough suj)crior margin. .\ small vascular canal goes laterally then for\vard from the posterior internal margin and reenters the canal at the external orifice.

20. Antero-superior cleft internally.

21. The superior wall is destroyed. Uest clear.

22. Strong median superior spine internally.

23. Small antero-superior cleft.

24. Clear.

25. Clear.

26. Clear.

27. (27L. ) A strong internal septum divides the canal equally. The internal orifice of the posterior canal is much larger. A wide postero-lateral opening into the posterior condyloid canal and a large superior diverticulum opening posteriorly and internally. The internal opening of the anterior canal has a sharp spine from the antem-niedial wall with a deep cleft below. A superior diverticuliun from the common external opening.

28. Clear.

29. Clear Slight antero-medial constriction.

jO. .\ septum in the outer third or half of the canal. The posterior canal is half the anterior.

31. Antero-medial or -superior notch.

32. Large. .Vntero-medial notch. Posterior condyloid canal has ot>ening into postero-lateral wall.

33. Opposed spurs. Anterior division is half or third the size of the posterior.

34. Antero-suijerior spur.

35. (35L.) Internally an antero-superior spur has bridged the canal completely, cutting off a small antero-sujicrior canal which has a median superior spur that makes it I'-shaix^d.

3)>. Hough sujierior margin. Anterior constriction. Two small canals from postero-supcrior wall probably to the posterior condyloid canal.

37. Clear.

38. Clear.

39. Antero-suiK-rior notch. Canal from postero-supcrior internal margin probably to posterior condyloiil canal.

40. Clear.

41. Median and anterior superior notches.

42. Clear.

VAKIATIONS OK THK CANALIS HYPOGLOSSI 141

43. Clciir. •M. Clear.

45. Internal .siiprrior iiinreinal spur, l)ctwccii iniddle and postprior thirds.

46. Clear.

47. .Vnlero-sniM'rior spur.

48. .Aiitcro-iiu'dial tiit)ercle.

40. \ superior spine and an oppiwed inferior elevation. .Vlniost complete division, so that the posterior division is one and half times the size of (he anterior division.

o(l. .\iitero-superior internal noteh includes a small spine and opposed spine.s in the middle of the canal dividing it equally into postcro-superior and anteroinferior parts.

51. .\ntero-8uperior notch.

52. Opposed spurs. .Vnterior canal smaller.

53. Clear.

54 Median superior notch.

55. Strong median septimi.

56. Opposed spines — sujierior longer. Anterior canal smaller.

57. Clear.

58. Clear. Only one hole in dura mater.

59. Large. Strong slightly anterior septum in middle third of canal.

Fifty-nine right halves

1. .\nlero-sui>erior cleft.

2. Thin septum which is incomplete at the inner opening. .Vnterior canal «maller with further anterior notch.

3. Flattened from ahove down. .\n indicated spur in the middle of the su|)crior internal margin. .Vnterior half of orifice especially flattened. P^xternal orifice rounded.

4. Superior median elevation and an anterior superior spur with an opposed inferior elevation.

5. Narrow septum. .Vnterior canal smaller.

6. Clear.

7. Clear.

8. Hough superior margin. Klevation between anterior and middle thirds of superior internal margin.

9. .Vntero-superior notch. 10. Constricted anteriorly.

U. Strong septum with superior internal spur. Kqual canals.

12. Clear.

13. Snmll antero-superior cleft.

14. Opposed spurs which seiKirate the anterior third of the canal. In addition there is an antero-medial spur.

15. Clear.

16. Greatly flattened. Median spur within the canal on its roof.

17. Clear. Klatteneil.

14'J UAI.ni 1). i.llAAK

IS. (ISH.i I^irKt' nv!il iiiiuT iirilico. Al llir oiitor orifico ii thin strand of bono divide!* off a Hnmllcr posterior diviHiun. Postcro-liitornl to this is a pocket opening liack and inward with a round liolo 1 inin. in diameter in the middle which ofH'ns through into the externul orilice and a pit 'J mm. in diameter ahove, which opens anteriorly into a large superior diverticulum from the anterior canal.

10. Median suiierinr spine.

20. Clear. Large canal from posterior wall to posterior condyloid canal.

21. Clear.

22. Clear.

2;l. .\iitero-superior spur and notch.

2-1. Strong st'ptum. .Vnterior canal smaller. Common external opening has 8up«'rior diverticulum.

25. Slight spvir internally on upper third of antero-mediul margin.

26. Clear.

27. Superior spur on inner margin posterior division half the size of the anterior.

28. Clear.

29. .\ntero-8ui)crior spur and notch.

30. Clear. Foramen magnimi and condyle malformed.

31. Clear.

.32. Large internal opening clear. .\ canal starts off from the middle of the postero-sujx'rior wall o|x"ning separately at outer opening. .\n antcro-medial notch. Antero-supcrior diverticulum in middle of canal.

33. Clear.

34. Slight opposed elevations anteriorly on inner margin. Inferior elevation stronger.

.'tS. I.4irge. Clear.

35. Large. Opposed spurs between anterior and middle thirds.

37. Clear.

38. Large. Antero-superior spur.

39. Slight anterior superior eminence between anterior and middle thirds. 4fl. Clear.

41. Clear.

42. Opposed elevations and dried remains of a connective tissue septum. Anterior canal half posterior.

43. Slight superior spur lietween anterior and middle thirds of internal m.'irgin.

44. Merlinn and anterior superior spurs with slight opposed inferior ridges. A snial! canal from anterior superior corner returns by a curved course to superior wall near external orifice.

45. (IV.) .Vntero-superior notch.

46. Two su|H>rior internal spurs equally spaced with an anterior medial spur opposed to the anterior superior spur. .\ canal from posterior wall probably to posterior condyloid i-anal.

47. .Vn anterir)r constriction.

48. Snudl 1 to 1.5 mm. roiiiiil nolch on antero-medial internal margin which is set off by two spurs.

49. /Vntero-suiierior nolch.

\

VAHIATIOXS OK THK < AXAMS HYI'OOLOSSI 143

50. .\Icili;in 8ii|MTi(ir lulnTcle.

51. (51R.I Strorin liDrizontal septum. SinallLT infi-rior, liirRt'r >iii|M.Tior ciinnl.

52. Larue, f'lcar.

53. Septuin in internal part of canal not quite to inner orifice. .Vntcrior cunul half the size of the posterior.

54. A complete thin, 2 mm. wide, superficial infernal septum. .Vntcrior canal slif^htly smaller.

55. Two equally spaced superior internal tulxTcles. .Vntero-infcrior spur opposes anterior one.

an. .\nteriorly constricted internal opening.

57. Clear.

58. Clear.

59. Clear

Althougli the uuniber of skulls from different gioujis or types was too small for a comprehensive Aaew, the above table indicates a variation in the frequency of division among the different racial tyi)es. This was also found to be true iiy Cartels ('04). Complete di\'ision shows itself in the Illinois Indians in only 12.9 per cent of the cases, while in the California Indians it was seen in 22.4 per cent. In the dissectmg room si)eeimens and in the Eurojjean skulls the incidence was 13.4 per cent and 11.4 per cent respectively.

Distinct differences between the right and left sides e.xist. Complete di^■ision is more frequent on the left side — 10.99 per cent as against 11.84 per cent — while indicated di^^sion is more common on the right — 38.82 per cent as compared with 33.99 per cent. The canal is clear in an equal numlier of cases on each side. Racial differences seem to l)e indicated here, too. In the Illinois IntUan skulls and in those from our dissecting room there is ])ractically no difference between the two sides, but in the Cahforuia Indians anil in the European skulls decided differences were found.

The canal is clear in an equal unmber of cases on both sides This agrees very well with Weigner's CM) who found the canal clear in 02.1 per cent of the skulls on the left and in .")9.2 per cent on the right. I find 49.02 per cent on the left and 49.34 per cent on the right. Weigner found indicated division in 19.4 per cent on the left ami in 18..") per cent on the right — praeti

144 KAM'M D. l.II.I.tK

(•ally in qcihuI nuinbors in 103 skulls. I (ind 3.^.99 per cent and 3S.S2 per cent for the left and right sides respectively. Complote division of the canal was present on the left iu 1.S.5 per rent and on the right iu 2'2.'.i p<>r cent of Weigner's specimens, hut in only 17.0 per cent oii tiic left and in 1 l.S per cent on the right side in my series.

Freciuently in ca.ses of complete or indicated di\asions the two canals are unequal, one being about twice the size of the other. The anterior usually is the smaller. Indications of further division of the larger of the two canals occurs in a few cases. The two canals are often equal in size. In the latter case some few examples of further indicated di\'ision of the anterior canal were seen.

From the variations in form of the hyjjoglossal canal in adult skulls I am unable to decide whether there were three or four h>7)()glossal canals developmentally.

I take pleasure in thanking Professor Meyer for his assistance and suggestions.

LiTKK.vniU': citi:d

Bahtkus, I'. 1904 Ul>i'r U!is.sonuntpr.s<'hcidc am Srhiidcl I. Iiiterti .MoiLsi-lir. f. .Vnat. u. Physiol. Bd. 21.

Bremer, J. L. 190S .\herraiit roots and braiiclu'S of the abdiircnt and hypoglossal nerves. Jour. Conip. Xeur. Psychol., vol. 18.

Frokiep 1911 Poirier et C'harpy. Tr. d'anat. hum. Tome 1. Paris.

Jaboclay et LrcY 1911 Poirier ct Charpy. Tr. tl'anat. hum. Tome 1, Paris.

Martin 1891 Die EntwickehinR der neunten bis zwoiften Kopfnerven l>ei der Katze. .\nat. .\nz. Ud. (>.

McMl'RRlCH, J. P. 1905 The development of the human body. Phila.

Poirier et Charpv 1911 Trait*- d'.\natomie humaine. Tome 1, I'aris.

Pke.nti.ss, C. W. 1910 The development of the h.vpoglossal K'tn^li" of P'K embryos. Jour. Com]). Xeur. Psychol., vol. 20.

ScHAFER ANi> Symington 190!t (^uain's .Vnat.. vol. 3, pi. 2. lllhed. London.

Graf vox Spek 1.S96 Bardeleben Ilandb. d. Anal. d. Menschen. Bd. 1, Abfh. 2.

WeiCiNER, K. 1911 Clier die .Vs.similalion des .Vila.-) und iiber die Variatiunen am Os occipitale beim .\Ionsehen. .\iiat. IJefte Bd. A'y.

At'THOIlH AIIHTKACT OF THIH PAPKR DWUKO BT TflK BrilLIOOHAPHIC HEHVICE AI.'aUAT 6.

A CASE OF SUPERFETATION IN THE CAT

MAHV T. IIAUMAN Kansas State Agricultural College'

THRKK FIGURES (TWO PLATEs)

DKFIXniON OK .srF'EHFETATIOX

There soems to be a lack of unity among authors as to what the phenonuMion of superfetation really is. Marshall ClO) defines superfetation as a condition in which "foetuses of different ages may be present in the same uterus."' He does not say that a second coition is necessarj' to produce this condition, but from his statement he imphes that it is. He says: "If oA-uIation takes place during pregnane}-, and if, owing to the occurrence of coition the o\a become fertilized, the phenomenon of superfetation maj' take place." At least, he makes it perfectly plain that the eggs belong to different periods of o\'ulation.

McMumch ('13), suggests that many cases which have been reported as superfetation are due to differences of nutrition, and are the result of the simultaneous fertilization of different ova. He says that it is not impossible for a second o^'^ml to be fertiUzed as the result of a coition at an appreciable inter\al of time after the first ovum has started upon its development. Howe\er, the difference between the normal emlirj'o and that due to superfetation is comparatively small, pro\'ided that the nutrition to both embryos are the same. He further states that for physiological reasons, the jiassage of t\w sjierniatozoiui to the egg is very soon impossible.

Longley ('10 and 'ID finds that coition is neces.>;ar\- to o\-ulatioii in the cat, and jiarticularly to maturation; therefore, in

' ('t)iitributioii from llic Zoulugiuul Laboriitorv, Kaiisiis ."^talc AKricultiiral C'dllcRC, No. 15.

145

14t> M AlC^ 1. 11 ARM A.N

or(l(>r to have suporfctation, it is necpssarv to have a second coition.

AiTowsniith {'34) gives an account of a number of cases of su])erfetation and suggests that it is impossible that they could be the result of the simultaneous fertilization of (HfTerent ova.

lionner (•')) classifies the reported cases of superfetation in man in tin-ee groujis: 1; His first grouj) is composed of those cases in which two mature children are Ijorn at the same time but bear marks of different jiarents. This grou]) of cases, of course, is due to ditTerent coitions, but not necessarily due to dilTerent jierioils of cn-iilation. In fact, they could not be due to very different periods of ovulation and be born at the same time in the same degree of development. 2) His second gi-oup includes twins liorn at difTerent times. He would saj' tliat these are the result of difTerences in development. 3) His third group, and the group which he would consider true cases of superfetation are those in which embryos are born at intervals too .small for a second conception. He cites a number of cases in the human, ranging in inters'als from two and one-half months to five and one-half months.

King ('13) distinguishes between what she considers superfetation and superfccundation. She defines superfecundation as the fertilization by successive matings of ova belonging to the same period of ovulation; and superfetation as the fertilization of ova of different periods of ovulations, followed by copulation occurring during pregnancy wliich leads to the simultaneous develojiment of two sets of ova. From King's definition, it is seen, that she would confine the term superfetation to those cases in which there has not only been a second coition but also a second period of ovulation.

Sumner ('!(») believes that superfetation may be the result of a .second coition, but not necessarily so. He thinks that the spermatozoa (in case of mice) may be retained in the uterine pa.ssage of the female for a length of time and still be functional, and that true casc-^ nf superfetation may be the result of a single coition.

A fASE OF sri'EUKETATIOX I\ THE CAT 147

Schultzf ('()(■>) (1()( s not say that suporfetation is iinpossihle; hut he explains all of tho cases reported in man up to that time on tiic l)asis of either the death or the retardation of the less advanc(tl fetus. Tiiis also seems to he the jxisition taken by Kuntz ('10). He states that the "two cases of apparent superfetation in cats which were examined hy the writer, afforded no evidence in favor of the occim-enee of superfetation."

Clodlewski ('14) believes that the process of suixrfetation is a physioiofricai impossibility.

It will be seen from the above that there is a pi-eat. difference of opinion as to what superfetation is; but all agree, at least, in one particular, that is, that embryos of different degrees of development are in the uterus at the same time. The case of the cat, which the writer is al)out to report, has also this characteristic in connnon with the other cases. There is a decided difference in tiic degi'ee of development in the embrj'os, and neither set shows lack of nourishment, if the blood supply may be taken as a criterion, or any dc gi'ce of decomposition.

The writer will use the word 'superfetation' to mean that condition in which the uterus contains embryos of different degrees of de^•e]opment. This condition may be the result of a second coition or a second concejjtion may have taken ])lac(' without a second coition.

SIPERFETATIOX OF THK C'.\T

During the dissection of the cats in the class work of general zoology, a marked difference in the size of the enlargeil ])laces of the uterus was noticed in one of them. This leil to an examination of the different embryos which revealed that three embryos, were developed near to term, and one apparently normal, was much smallc r and showed a much less degree of development. Nothing is known concerning the periods of heat or time of coition.

Figure 1 shows the gravid uterus about one-half natural size. The enlargements. A, R, C, and T). contain embryos but no trace of an ernbryo could b(> fouiul in enlarg(Mnent K. The blood ves.sels had been injected and the blood sujijily to tho uterus

Tlir. ANATOMII'AI. HKCOHh, VOL. 13, NO 3

148 MAUY T. llAltMW

is plainly shown. The supply to the smaller oiilarRoiiiPiits seems to he eciually as good as to the larper ones, if the size of the blood ves.sels may he taken as an iiulic-ation.

The right horn of the uterus contained one of the larger emhryos and the smaller one. The larger emhryo was next to the P'alloiMan tube wliile the smaller one was near the vagina. The left horn of the uterus had three enlargements, two of which contained embrj'os of the same degree of development as the larger one in the right horn. No trace of an embryo was found in the enlargement next to the vagina, although the fetal membranes extended into it and there also seemed to be an abundant blood supply.

The large embryos (fig. 2) are about DU nun. long, not including the tail, and from the external features appear to be near to term. The limbs are well formed and normal, ha\-ing joints, and on the ends of the digits are claws. The whole surface of the skin is covered with pits, althougli there is very little hair. Th(> nose and mouth resemble the nose and mouth of a newly born kitten and the external ears are very prominent. The tail is more than one-third the length of the remainder of the body. The body is closed ventrally. The one represented in the drawing differs from the other large embryos in that a short distance from the body the umbilical cord divides, and a part passes around either side of the body to connect with the placenta. The fetal membranes fill the entire enlargement of the uterus and fit very closely to its walls.

The snuill embryo which to all appearances shows no decompo.sition, is only 10 mm. in length. The umbilical cord occupies about one-sixth of the ventral surface. The limbs, fore and hind, are merely buds. The tail is about one-fifth the length of the remainder of the body. There are no indications of hair follicles. The mouth is in the process of formation. The mandibular ])roces.ses have met in the midventral line, hut they extend only about one-third as far as the maxillary processes. The lip gi-oove is shallow, in fact, there is merely the beginning of the separation of the lips and cheeks from the jaw proper. The oral pit is yet rectangular in shape. There is no indication

A CASE OF SUPERFETATION IN THE CAT 149

of eyelids; hut tho eyes are plainly visiMe from the outside as small dark splieres.

It seems that the smaller embryo is at a stage of not more than throe weeks' development, while the larger embryos represent seven or possibh' eight weeks' development.

Owing to the fact that the material was presented merely for the examination of thegross anatomy of the mother, the tissue is not favorable for microscopie study, but the conditions for the less athanceil fetus are the same as those for the larger ones. The fetal membranes of the one is in as good a state of preservation as those of tlie other. Both the anmion and the chorion of the smaller embryo has the appearance of being younger than they do in the case of tlie larger embryos, but they have no greater e\'idence of necrosis. The injection of the blood system of the mother l>efore jireservation has made jiossible a better study of tlie blood supjily to the embryos than could have been made otherwise. If the smaller embrjo should have been the result of an early death or retardation of development, this is not evidenced by any lack of blood supply.

Altliough, as was stated before, nothing is known concerning the periods of heat and time or times of coition, yet it seems that the condition of the fetal membranes of both the smaller and tlie larger embryos, the abuiulaiit blood supply to the smaller embryo as well as the lack of decomposition are e^^dences that it is not a result of early embryonic death, but that it is a result of a later conception.

DI.SCUSSION

The explanations of reported cases of superfetation are almost as numerous as the authors who have rei)orted them. There are those who do not believe that the supernumerary brood or accessory birth, if the writer may use that term, is (Schultze, '60) or can be (Clodlewski, '14) a result of concejition during pregnancy. As has been prexiously stated, Schultze (in the human) considers these cases as twins in which the one has been retained in the uterus for a longer jieriod than the other. He exjiluins the dilTerenee in the degiee of deveIoi)ment on tly? basis of lack

l-')<t MARY T. HAUMAN

of f<Mi(l supply, tlunforc, (l('V('lnj)mcul is retarded. If this ho true of the cases reported in the human the gestation )>( riod lias been prolonged as niueh as five and one-half months. So far as the author is aware, this is a much longer period than i.s recorded for prolonged pregnancies. Furthermore, if this explanation should be applied to the cases of the mouse as rrporl(d l)y Sunnier ("Ki) the period of gestation would l)e almost three tinus the normal iKriod of gestation. .\t liast, tlie ])( riod from the birth of the last normal litter until the birth of the suptrninnc rary litter in one ease was as nmch as thirty-.st\en days, which, if adtled to the period of gestation for the normal litter which is twenty-two days, would be fifty-nine days. This lacks only one week of being three times the normal period of gestation. It might lie added that the male was removed from the nest !)efor(' the birtli of the lirst litter. The writer, in giving extremes, would not i)resume to say that such a phenomenon is impossible; but it seems that the e\ndence is scarcely enough to justify the belii f in such an extended prolongation of the time of gestation.

Another explanation of the cause of superfetation which has been freeiuently given is that the organs of reiiroductinn are abnormal, such as a Intid uterus, etc. AiTowsmitli and others have shown that in a number of cases, at least, this could not aceoimt for the phenomenon. In the case of the sheep as reported by the above mentioned author, an examination of the slaughtered animal revealed no abnormality. While this explanation may soem perfectly plausible, it does not always agree with fact, and coidd not be accepted as a universal cause.

Whether or not ovulation takes place during pngnancy, has been a subject for much discussion and investigation. It is the general belief, one is safe in saying, that ordinarily during pregnancy, in man, oAiilation cea.ses but if the condition of corpora lutea may be a safe criterion (McMunich '\'-h this is not always true. Chinstophcr ('8(5) reports having found a cat far advanced in imgnancy with four mature flraafian follichs each containing a mature ovule. There is also some evidence that ovulation may occur during pregnancy in the horse.

A CASE OF sri'EHFETATIOX IX THE CAT l')l

According to Marshall CIO), McMurrich ('13), Bonnar ('65), Arrowsinith (':U), King ('13), Hcrzog ('98), and Sinnncr f'16), supcrfctation may he caused by the fertilization of an egg ovulated during pregnancy. Marshall, McMurrich, and King further state tliat the plienonienon is also ilue to a second coition. Sumner suggests that perhaps there is a periodicity of ovulation which may not be interrupted by gestation, and that the spermatozoa may be retained in the reproductive organs of the female for a consideral)le length of time, and yet remain functional. His data show that either this must be true, or that the perif)d of gestation is greatly prolonged, even more than doubled. In one ca.se the last ojiportunity for copulation was nineteen days before the birth of the first litter, and forty-seven days before the birth of the second litter. Since spermatozoa have been found alive and acti\-e in tlie female reproductive organs some time after coition, it seems as reasonal)le to i)eliev(> that these spermatozoa would function as that development would be prolonged to such an extent.

However, in the case of the cat described by the wTiter, another difficulty arises. The le.ss mature embryo is next to the vagina, and in the same horn of the uterus between it and the Fallopian tube, is an embryo almost at maturity. While one would scarcely be justified in sajnug that the passage of an egg from the ovary to this po.sition in the uterus under these conditions, is imjiossible, yet it hardly seems probal)le, for the fetal membranes are in very close contact with the uterine walls.

King suggests, in case of her rats, that perha])s the ovaries I'xuicliimed iuilependently, and that the first litter was the result of the ()\ul:ition of one ovarj' and the second litter the result of the ovulation of the other ovarj'. So far as the data given in her paper shows, this is merely a suggested jiossibility. No structures were examined to sui)i)ort the theory.

In the caseof the cat, this would be equallyasimprobableasthat the egg was fertilized by sjKrmatozoa which have lieen retained in the uterus of the mother for, as has been stated before, in the same horn of the uterus between the vagina and the small emhrvo was an embrvo which was near to term.

15'J MAllY T. JIAUMAN

'I'lic writer would raise tlie (|ue.sti<>ii whctlur it would not lie as rrasoimhlp to beliovo that the egji ininht be retained in the uterus some time before it was fertiHzed and yet bo able to develop into an embryo, as that the si)erniatozoa eould function some time after being diseharged. The evidence seems to point to a good blood supjily to the smaller embryo, and the implantation seems as good as the more ailvanced eml)rj'os. If it is small on account of retardation of develojiment, some other cause for this arrested (levelo])ment must be sought. If the egg whicli gave rise to this embryo is of a much later ])('riod of oNiilation than the eggs which gave rise to the other embryos, there is the mechanical difficulty of the passage of the egg from the ovary to the position which the embn'o occupies in the uterus. This is tnic whether or not the egg was fertilized by a spermatozoon from a second coition, or from a spermatozoon which has been retained in the uterus for some time.

It seems reasonable to the writer that the different cases of superfetation may l)e the result of dift'erent causes and that it is hardly necessary to ascribe all cases to the same cause. C'onsitlering all the conditions the writer would suggest that the less developed fetus could be accounted for as a result of delayed fertilization.

Sr.M.M.VK'i .VND C-ONCIA'.SIONS

1. The word 'superfetation' has been used to mean that condition in which the uterus contains embryos of different degrees of development. This condition may be the results of a second coition or a second conception may have taken ])lace without a second coition.

2. .Ml hough superfetation is rare and abnormal, many cases have been reported in man as well as in a number of other mammals, which do not seem to be satisfactorily explained in any other way than that a second conception has taken place.

3. Perhaps not all cases of superfetation may be attributed to the same cause.

A CASE OF SUPERFETATIOX I\ THK CAT I .").'i

4. In the caso of tho rat doscribod in this paper,' it seems as reasonalile to tliiiik of tlie less advaiieed eiiihryo as the result of delayed fertilization as to account for it on the ground of delayed development oi' a scconil coition.

LITERATURE CITED

Arrowsmith, R. 1S;W .S\iperfetafion (?) in the sheep. London Med. Gazette,

vol. 14. Bonnah. Okohgk Lindsay 1S0.5 .\ critical in(|uiry of superfctiition, with cases.

KdinbtirRh -Mi-d. .J<mr., vol. 10, p;irl 2. CliRlsToPHKK, W. .S. lS,S(i Uvuhilion during proRnancy. .\mer. Jour. Oli stetrics, vol. 19. Herzoo, Maximillia.n 1898 Supcrfetation In the liuniun race. Chicago .Med.

Recorder, vol. 15. Kino, Helk.n Df.ax 1913 Some anomalies in the gestation of the all>ino rat

(Mus NorwpRiciis albinus). Biol. Bull., vol. 24. KiuKiiAM, \V. B. 1907 The maturation of the e(jf; of the white mouse and rat.

Biol. Bull., vol. 1.1

1910 Ovulations in nianiinals with special reference to the mouse and rat. Biol. Bull., vol. IS.

KiNTz, .Vlhkht 19l(i A note on supcrfetation. Interstate Med. Journ., vol.

23, no. 3. Lo.N'GLEY, VV. H. 1910 Factors which influence the maturation of the egg and

ovulation in the domestic cat. Science, X. S., vol. 31, no. 79.5.

1911 The maturation of the egg and ovulation in the domestic cat. Am. Jour. Anat., vol. 12, no. 2.

Marshall, Fra.ncis H. A. 1910 The physiology of reproduction. Longmans.

Cireon and Co., New York. McMurrich, J. Plavkair 1913 The development of the human body. P.

Blakiston's .Son and Co., Philadplphia. .SiMNKH, V. B. 1910 Notes on the supcrfetation and deferred fertilization

among mice. Biol. Bull., vol. 30. ScHi'LTZE, B. S. 18(56 I'cber Superfoccundation und Supcrfoetation. Jen aischc Zeitschrift fur Medicine und Xatur wissenschaft, Bd. 2.

'The author wishes to express her indebtedness to Mr. Wallace Park, .\ssislant in Zoology, for calling her attention to this case.

I'L.VTK 1

EXPLANATION OK FIGl'RES

The prognaiit uterus of tlic cat, one-half natural size. EnlarRements A, C, and I) rontain embryos IH) mm. in length. Enlargement D contains the embryo shown in figure 3. Enlargement E does not contain an embryo.

l.>i

A CASE OK SUI'KUKF.TATIIIN IN TllK CAT

MAItr T. llAltMAN

PLATE 1

15")

PLATE 2

EXPLANATION OF FIQUKES

The embryo of enlarpeiiicnt D, fiKiire 1, two-thirds nntural size. The embryo is surrounded by the fetal membranes and a portion of the uterus. A part of the umbilical cord extends around one side of the body and a part of it extends around the other.

The eml)ryo of enlargement B. figure 1. enlarged 5 diameters. The embryo is surrounded by the fetal membranes and a portion of the uterus.

186

A CASE OF SLPERFETATION IN THE CAT

MAHT T IIAHUW

PLATE 2

157

AL'TIKMt- *ir*TH*cr fiV TIIW PAPER OMUKD BY Till; hiiii.UMiKM'llir skhvkK AUGUST It.

LABORATORY APPARATUS I. A SIMPLK ELECTRIC THERMO-UEGULATOK

H. S. BURR From the Anatomical Laboratory of llie Yale School of Medicine

ONE FIGURE

The electric f liermo-refjiilalor liere (le.-<cril)efl is the result of several years (if sixiradic expeiiiiients. The object was to design an insiruiiieiit tiiat could I)e readily huili by any laboratory technician; that coulil be run on an ordinary liKhiiuK circuit without the use of a relay, .condensor or other apparatus of that sort; that would bo more positive and less subject tfi tlucluations due to jar than the tnorcury cohniin; and finally that would be more accurate than a bi-metallic renulator. It is Ix'lieved that this instrument fulfills many of these re(iuirenir>nts.

The principle is simple. It consists of a walking beam actuated by an (>ther taml)our. The expansion of the taml)our forces down the reach of the walkinj; beam so that the consec|uent raising of the other reach breaks the electric circuit. Finure 1 gives the details of its construction. The whole apparatus should be built of brass with the exception of the .stool pin which acts as the Iwaring of the walking beam, anil the platinmn-iridium contact points which are forced under pressure into the i-nds of the walking beam and adjiustinont screw. The dimensions given may be varieil to suit the convenience of the material at liand although the measurements given have lieen found to be the most satisfactory.

The most difficult pail of the construction is the ether tambour. While it is possible to make ii in a well equipped shop it will be found more satisfactory to purchase a thermostat manufactured by the Banner Incubator Company, Baraboo, Wisconsin or a Newiown thermostat manufacttired by the (!iant Incul)ator Company. .\ good mechanic, however, can spin a tambour i)rovided he has a sjieed lathe available. The method is as follows: In the end grain of a hard wood block a series of concentric giooves and ridges of semi-circular cros,s section are worked. Then by fastening a slit>ot of thin soft bras,s against the wood block, the whole being fastened to the face plate of the lathe, the soft bra.ss can be worked into the grooves. Two plates are crimped in this manner, cut into circular disks slightly larger than the diameter of the tambour and soldered gas tight over a spreader pl.'ite.

159

1()()

H. s. mint

Tlic :is,s('iiil>l('ii inslniiucnt slmiild lie siipijoilcd iipsidc down in the fonslanl t(Mii|M'i;ituif Imx or inoni, thai is to say, it siiould lie fastened to tlio sidfwall with llio wooden liase up and the instrunienl lian^inn down so tlnit K'iivity will ke<'p tlie loan reach of the walivinfi Ix-ani pressed against the contact ailjustinent screw. The expansion of the ether tlien depr(>sses the shoU arm, raises the lonK arm and hreaks the circtiit. Two adjustments are provid(>d so tiiat any tcniiierature witiiin the ranp»' of the lieatinjr element may l>e maintained.

Almost an>- electric heating ai)paratus from simple batteries of carl)on lights to an electric ra(li;itor may he used with complete satisfaction. Where large sjiaces are to ho heated it will he found l)e.'<t to have sufficient heat on all the time to keep the temperature up to

\OooJg 7 Boae

fe"

\od,oror '^a\^^_^^^'^\{

witliin 5° of the rtiiuired temperature, the regulator being then cut in on a circuit with a sufficient heating element to readily raise the temperature the remaining .5°.

In installing this instrument the most satisfactory result will be obtained if the box or room is thoroughly insulated from the surrounding temiK-ratuif. For ordinary paratliiic work in the class room or research laboratory, a light, thick-walleil wooden box serves admirably. Such an oven with inside dimensions of 12 by 12 by 18 inches is especially adapted for parafline work since a temiK>ralure of .54° on a shelf placeil just above the heating element gives a temp<'rature on a shelf () inches higher that will flatten our paraffine .sections and dry them. A small room may be kept at a constant temperature provide11 iii.siil:tt('(| Willis :iiiy jrivcn space cun Ik- initintuintKl at a constant t^-nipoiatuii' with lcs,s than 0.5° of variation. Little work is rec|uir('<l to kt'c[) the ajjparatus in ^ood working order: it is only necessary at intervals to dean the platinum contacts.

The author is indeliled t.o Mr. \. K. ScharfT, Laboratory Technician of the .Vnatoinical Laboratory of tlie Vale Scliool of Medicine for help and many supne.stions in making tliis instrument.

II. .\N AITO.MATIC AIR PRESSURE VALVE

AUGUSTUS E. SCHARFF ONE FIGURE

Those who have had to dciJond upon the ordinary means for applj-ing air pressure for injectinK such iis n hand or water pump, recoKniz(^ the difficulty of accurately regulating the pressure or maintaining known constant i)re.ssure over any length of time. The e.^sential feature of an apparatus used in this lal)oratory for several years which accomplishes the above object is the automatic air pre.-ssure valve described below (fig. 2).

The valve consists of a piston working against a spring within a cylinder and connected with the lever of an air cock. The air from the reservoir, under high pressure, is led through the air cock to the rear of the cylinder ;ind forces the piston outward, compre.s.sing the spring, whicii ])ai-lially shuts off the supply of air through its connection with the lever of the cock.

The iiressure valve was made from a steam radiator air valve. The base is unscrewed from the bowl, the parts within removwi and a spring and piston in.serted into the bowl. Tlic spring is of Xo. 14 bra.ss wire, coiled J inch in diameter and Ij inches in length. The piston is of leather, fa-stentnl to the pi.ston rod l)y a nut al)ove and below it. The piston rod is of i inch round brass, '.i\ inches in length, threaded for § inch at the end to which the pi.ston is attached and for 1] indues at the other end, which is fa.<tened to the connecting nxl. The vent of the air valve is enlargeil to allow the piston roil fri-e i)lay. A connecting rod of Yi ifl' round brass. 2] inches in lengtli, with a shar]> right angle bend 1 inch long at (>ach end, is fa.stened to the pislon rod by a link at one end and to the lever of the cock at the other. Both ends are burred to hold them in position. The link is made of bra.ss. ^ by f by 1 inch in size, b(>nt in the mifldle at an angle of 00°. A hole in one end admits the <-onnecting rod, allowing (rev sideways motion. Through a holi' in the other end, the |)iston roil is ]>as.s('d and clamped by a thumb nut on each side. Hy shifting these thumb sitcws along the piston rod, the distance between the piston and the lever of the air cock may be lengthened or shorteniHl, thereby changing the tension on the spring within the valv<> which thus decreases or increases the pressure.

1(>- ArC.USTl'S E. SCHAUKK

The pi|H' convfviitK tin- :iir from tlic rosciNoir lo the r yliiitlcr is lc<l to 11 Y lulii'. Olio hniiicli of wliich loads through a low pressure reservoir (any larpe hottle, wliich acts as an air cushion on the mercury) to a l'-nianon»eter graduated to inillimetei-s and indicating the pre.ssurc beyond the valve. The other hraiich of the Y tuiie leads to the receptacle contaiiiinn the injectiiiu fluid. .\s the jiressure in the cylinder is rcduct'd liy the decrease in the amount of injecting fluid, the spring is released pulling on the lever of the air cock, opening it and a<lniitting a fresh supply of air. This maintains a balance between the air pressure in the eyjinder and a tciisinn on llie spring.

Ill .'. I.AIioKAlDlJV ll.\ll\<, CLOCK

.\U(ir.STi;s F SCHAUFF

The device ilescribed below is of gi-eat value in niea.suriiig the length of time that pieces of tis.sue or slides are to he left in the dilTerent reagent»s. It is the experience of every laboratory worker that either his entire time must be devoted to the staining of slides or great variations in the length of time in which they remain in the different reagents are almost sure to follow divided attention. The clock a?i described below has Invn used in this laboratory for over a year and it has been found that much time may be saved in .staining sections and great^er accuracy in their treatment obtained from its use.

The works of an ordinary clock are reniove<l from the case. With a number 28 drill make a hole through the face, just above the twelve o'clock mark and about J inch from the end of the minute hand. Through this hole a piece of insulated annunciator wire ti inches in length is passed. Strip the insulation for about | inch from the end of the wire and bend the bared poi-tion in the form of a flat looj) so that (he minute hand in pa.-ising will rub over it. The insulation mu.st not be removefl from that jiortion of the wire which passes through the liole in the clock face. \Vr;i|) the end of thi-; wire behind the clock face, around one of the cross stays of the works and leave the end free.

Drill a hole through the sliell of the clock through which the loose end of the anmmciator wire may be passed. If the clock used is an alarm clock, the clap|>er may be removed and the wire passed out through the clapjier slit. Place the works within the shell.

Fasten one wire of a 2 or 3 foot piece of double, flexible, electric hght cord to the end of the annunciator wire from which the insulation has been stripfwd f<ir the purpos<>. Wrap the union with tape. Fa.sten the stripiK'd end of the other wire to any convenient |)ortion of the clock. Fasten one wire from the other end of the flexible cord to a buzzer or bell, the other two to dry cells. Complete the circuit, interposing a simple switch so that the circuit may be broken at will.

For convenience in turning the minute hand a piece of bnuss tubing may Iw fa.stened to the thumb screw controlling it.

LABORATOItY APPARATUS HV.i

To uso tlio clock: wind it, turn tho niinutc hand to the (icsircd number of minutes hoforo the oven hour that it is dosirod to Icavo thr slides in the stain. Place slides in stain, ("lo.sc the switch. When the time hits elapsed, liie minute hand connectinn the contact with the wire looj) will sound the i>uzz('r, which may he stoppe<l by openinft the switch. IJemove th(> slides from the stain and reset the clock for the next slatje. In the intervals Ix-twe^Mi the ac-tual proces.sos of setting the clock and chaniring the slides from one reagent to another, the mind is left free for other things.

By fa-stening a piece of sheet metal to the bottom of the shell of the clock so that it will project backward just behind the clock anrl fastening a strip of metal to the table so that the projection on the bottom of the clock may be slipped under it, the clock is rendered more stable.

TUC ANATtllllCAL BCCOltD, VOL. 13. NO. 3

AUTHOR M AIIHTHAfT or TIIIH PAI'EK lAArKD Ur TIIK lllULIOOKAi'lllC HEHVICE AUntKT 11.