Talk:Paper - The early appearance of the anlagen of the pars tuberalis in the hypophysis of the chick (1918)

anlagen of the pars tuberalis in hypophysis of chick

UAYNK J. AfWKLF- AND IDA SITLKU

Dr.pnrliiiriil •>) Aiiiilninii, Meilinil Sihiiiil nf the I'niverxili/ nf Miih!ijiin

FIVe PICIHKS

[t is now well n'('()>riuz((l lli:it tlic cpithcliiil portion of tlip liyiJophysis consists of three ilistinct parts. The pars anterior propria is the principal cpitheHal lobe and constitutes the main l)iilk of the ulaml, the i);'.rs int(>nii( dia is a thin lavt'r. epithehal in iiatun". which becomes iiitiniately associated with the neural lobe. 'rh(» nicst recenth" rec(jgnizeil epithelial lobe is the pars tul)eralis — so named by Tilney ('13) on account of its close relation to the tuber cinereum. It extends forwaril from the junction of the pars intermedia anil the pars anterior propria, surrounding the infundil)ular stalk and spreading out for .some di.staiice under the brain floor.

The jnirs tubcralis has been .sometimes confu.sed with the pars intermedia- Lothringer ('86) and Herring ('08) — but recent studies have .shown conclusively that these two parts are different both in adult structure and in developmental historj-. Tilney, in summarizing the develo])ment of tlie pars tuberalis in the chick and the cat, states:

In atUlition to (In- histological ditTorcnccs between these two parts, llic ontogenesis of tlie organ as observed in the cat and the fowl still furtluT einpiiasizes the fact .that the pars tubcralis and the pars infmnlibularis (pars inlernuMlia) are morphologically distinct elements. Till- |)ars infiiiidibularis makes its appearance inniiediately after the aiiianc of the buccal portion of the liypopliysis is fornietl. The pars tubcralis ai'ises as a relatively late stniciiirc. It has its origin in two sccond.aiy ilivcrliciila or sprouts from the ImkIv of the pituitary sac. These sprouts, the tubcral processes, ultimately fuse with each other across the median line, displace the body of the pituitary sac vcntrad and thus secondarily a.ssume their juxta-iiein-al position.

Out" (if lis Al\V(>ll CIS) — ill ;i recent study of tlie tlevelopiiii'iit of tlie liypopliysis in the nil)l)if has obtainod somewhat (lifTereiit results. Wliile afireeiiifi with Tilney as to the distinctness of the pars tul)erahs and the pars intermedia, and also conHrminK the statement tliat the pars tulieralis is late in acquiring its adult relationship with the tuber cinereum, it has been found thai in the rabbit the anlagen of the pars tuberalis may 1)1' discerned \erv early. They were found to precede* the definite pars intermedia by a considerable period of time.

It was with the lio|)e of throwiiifi some light upon this point that the j)resent study was imdertaken. Accordingly we have been led to construct a luimber of wax-plate models of the epillielial hypophysis from chick emiiryos, l)eginning with .stages in whicii the tuberal processes might be recognized easily and then j)roceeding to succes.sively younger (>mbryos in an effort to determine the earliest appearance of th<' anlagcu.

Tlie literature relating to the lateral lobes and the pai's tuberalis in the hypophysis of the chick is not extensive.

Rossi ('{)()) speaks of a median and two lateral parts in the early hypophj'sis of the chick embryo, .\ccording to Ro.ssi the lateral lobes are secondary structures.

iM'onomo ('!)!)) observed a pair of "Seitenspro.ssen' in the hypophysis of the dove and of the domestic fowl. In dove embryos the sprouts appear b(>tween the fourth and seventh days of incubation. \o definite statement is made concerning the first ai)pearance of the sprouts in the chick.

Tilney ('13) first ob.served the 'tuberal processes' in a chick embryo of o days and 20 hours of incubation. From this stage the jiroces.ses were traced to the formation of the pars tuberalis of the adult fowl, .\lthough a reconstruction was prepared from an embryo of fourda3's of incubation the tul)eral proces-ses were not seen in this stage. Embryos younger than four days were not studied.

Woerdeman ('14) notes that the lateral lobes are forming in a chick embryo of about 72 hours of de\-elopment. The thickened epithelium which lies in front of Hathke's pocket is constricted off from the mouth cavity by two lateral folds. Woerdeman considers that the lateral lobes so formed arise independently of Rathko's pocket.

Bruni ('15) observes the presence of two 'lobi laterali' in the chick at 82 hours of incubation. He also figures and describes the latrral lobes in older embryos but does not trace them into the formation of the pars luberalis.

OBSERVATIONS

We have prcpan-d wax-plate reconstructions of the epithelial portion of the hypophj'sis from cliick embryos of 48, 59, 67, 72, 96, 120 and 144 hours of incubation. A relatively high magnification was chosen for the construction of the models in order that all details of structure might be shown as accurately as possible. For all younger stages, including the 72 hour embryo, the magnification was 300 diameters. For the older embryos the magnification was reduced to 200 diameters.

Chick embryo, 4^ hours of incubation {21-2 pairs of primitive segments). Fig. 1. The hj^jophyseal pouch is well formed but opens widely into the mouth invagination. There is no indication of the lateral lobes. The anterior end of the fore-gut, which will later form Seessel's pouch, extends farther cranially than does the hj-pophyseal pouch. At this time the oral membrane is intact.

Chick embryo, 59 hours of incubation (30 pairs of segments). The hypophyseal pouch (Rathke's pocket) has deepened and now exhibits two lateral enlargements near its attachment to the oral epithelium. As later models show, these are theanlagen of the lat«'ral lobes from which the tuberal processes develop. .As may be seen from figure 2, Rathke's pocket is shghtly constricted just above the lateral lobes. The lateral lobes have the fomi of blunt ridges which protrude laterally and also somewhat nas^iUy. Their long axes lie parallel with the long axis of the entire h.vpophyseal pouch. This embryo shows one small perforation in the oral membrane.

Chick embryo, 67 hours of incubation. The lateral lobes are more prominent at this stage due to the fact that the hypophyseal pouch is beginning to be constricted somewhat from the oral ojivity. The constriction of Rathkc's pocket dorsal to the lateral lobes is also more distinct than previously. Each lateral lobe contains a lumen communicating with the cavity of the main h}7iophyseal sac. Seessel's pouch is in contact with the dorsal wall of Hathke's pocket for a considerable area. This is the eeto-entodennal fusion which has been recorded by numerous observers.

Chick embryo, 72 hours of incubnlion, figure 3. The hypophysis anlage is closely appUed to the brain wall, causing the nasal surface of the pouch to be sharply concave. The lateral lobes are more prominent than in the preceding .stage. The lumen of the pouch extends well into each lateral lobe. One striking feature is the extensive degree of communication between the cavity of Seessel's pouch and the hypophyseal sac. The two open into each other almost to the summit of the ecto-entodemial fusion. This cau-ses the opening of the hypophyseal sac into the oro-phuryn.\ to be relatively larger than in pre\-ious stages. From an examination of embryos of this age alone the impression might be gained that the lateral lobes are being added to Rathke's pocket. A critical comparison of this and younger stages, however, indiates strongly that the lateral lobes of the chick do not arise independently of Rathke's pouch, but that they are formed from it. In this we support the observations of Rossi.

Chick cinhryo, 96 hours of incubaUon. The principal feature of interest in this stage is the beginning recession of Seessel's pouch and its separation from the hj'pophyseal .sac. The lateral lobes are more sharply marked off from the superior part of the hypophysis, but otherwise this stage does not exhibit any striking differences from the preceding. .

.\11 figures represent wux-pliite recon-structions of the epithelial hypophysis as viewed from in front and from the left side. S, Seessel's pouch, R, Rathke's pouch, /./., lateral lobes, t.p., tuberal processes, st., hypophyseal stalk.

Fig. 1. Hypophysis region from chick embryo of 21-2 pairs of primitive segments (end of second day of incubation). X 100.

Fig. 2. Hypophysis from chick embryo of 30 pairs of primitive segments (59 hours of incubation). X 100.

Fig. 3. Hypophysis from chick embryo of 72 hours of incubation. X 100.

Fig. 4. Hypophysis from chick embryo, o days (120 hours) of incubation. X 75.

Fig. 5. Hypophysis from chick embryo, 6 days (144 hours) of incubation. X 75.

Chick embryo. 5 days (ISD hoiirn) of incubation. By tliis lime a definite h yp< )pliysoal stalk has been fonned. It is hollow and affords a coniniunioation between the lumen of the hypophysis and the oral cavity. The lateral lobes have increased in size so that the transverse diameter of the gland, measured between the lateral extremities of the two lobes, is almost twice the transverse diameter of the superior part of Rathke's pocket. The lateral lobes ar<^ united by a prominent ridge around the inferior and nasal enil of the hj-jiophysis. This solid median protuberance doubtless corresponds to a vestigial '\'orraum' or 'corpus I«)buli bifiu-cati' of other vertebrates as described by Woerdeman. The lateral lobes are beginning to be solid, also. At this stage they sometimes contain lumina, which, however, no longer conmiunicate clearlj- with the main h3^pophyseal cavity.

Seessel's pouch is represented by a solid bud of epithelial cells just dorsal to the hypophyseal stalk (S, fig. 4). Curiously enough Economo labels this bud the remains of Rathke's pocket.

Chid: embryo. 6 days (144 hours) of incubation. The hypophyseal stalk is much elongated and has become solid. Near its connection with the oral epithelium may be seen the bud-like remains of Seessel's pouch. The superior, or distal, half of the hjT)ophysisis is bent dorsalh" and forms an angle of about ninety degrees with the inferior or proximal half of the gland. The constriction near the middle of the gland is pronounced. Distinct 'tuberal processes' have formed from the lateral lobes. Instead of projecting so much laterally-, thej' are now directed toward the brain wall. The tub(>ral proc(\sses are not located at the extreme nasal end of the gland but are seen to protrude from about the middle of the inferior half (fig. 5).

SUMMARY

The lateral lobes, from which the tuberal processes arise, may be distinguished in a chick embryo having 30 pairs of primitive segjiients. From a careful study of stages preceding and following the rupture of the oral membrane it is evident that the lateral lobes are not fonned independently of Rathke's pocket and later added to it, but are rather fonried secondarily from the nasjil wall of the early hypophyseal anlaK*'.

The lateral lobes, in all foniis studied, appear earlj- in devel«»pment. This would indicate that they and their derivative in higher vertebrates, the pars tuberahs, are of fundamental phylf>genetie importance. Thus gwat interest is attached to the broad homologies drawn by Woerdeman ('14).

LITERATURE

Atwell, Wayne J. 1918 The development of the hypophysis cerebri of the rabbit (Lepus cuniculus L.). Amer. Jour. Anat., vol. 24, p. 271.

Brum, A. C. 1915 Sullo sviluppo del lobo ghiandolare dell' ipofisi negli Amnioti. Internat. Monutschr. f. Anat. u. Physiol., Bd. 31, S. 129.

EcoNOMO, C. J. 1899 Zur Entwicklung der Vogelhj-pophyse. Sitzber. d. kais. Akad. d. Wiss., math.-naturw. Classe, Bd. 108, Abth. 3, S. 381.

Herring, P. T. 1908 The histological appearances of the mammalian pituitary body. Quar. Jour. Ex. Physiol., vol. 1, p. 121.

LoTBRiNGER, S. 1886 Untersuchungcn an der hypophyse einiger Saugetbiere und des Menschen. Arch. f. mikr. Anat., Bd. 28, S. 257.

Rossi, U. 1896 Sui lobi latcrale della Ipsofisi. Monit. Zool. itul., 7, p. 240.

TiiJJET, Frederick 1913 An analysis of the ju.xtra-neural epithelial portion of the hypophysis cerebri, with an embryological and histological account of an hitherto undescribed part of the organ. Internat. Monatschr. f. Anat. u. Physiol.. Bd 30. S. 258.

Woerdeman, Martin W. 1914 Vergleichende Ontogenie der Hypophysis. Arch. f. mikr. Anat., Bd. 86, ,S. 108.

THE IDENTIFICATION OF ENDOTHELIAL LEUCOCYTES IN HUMAN TISSUE

THIRD REPORT OF STUDIES ON THE MONONTJCLEAR CELLS OF THE

BLOOD

F. A. McJUNKIN Deparlmenl of Pathology, Marquette Univerrily School of Medicine

TWO FI0UTIE8

In an earlier report by the writer ('18) it was shown that the phagocytic mononuclear cells present in the peripheral blood arise by mitosis from the endothelium of the blood vessels. The method that was devised for this purpose consists of the intravenous injection of lampblack suspensions and is not, therefore, applicable to human tissues. The tis.sues of animals injected with carbon suspensions in which the endothelial leucocj-tes and cells are characterized bj' carbon particles ingested by phagocj-tosis are, however, well adapted for testing the action of various stains on these cells. It has been found that the staining method u.sed by Craham ('16) colors these leucocytes in a characteristic way in both animal and human tissue. Since paraffin or celloidin sections caiuiot be used, Graham emploj-ed frozen sections for his stain but owing to their thickness, they are not suitable for accurate cell identification. The purpose of this paper is to record a new method of tissue imbedding for obtaining thin sections to which the stain is applicable.

The .staining method of (iraliam depenils on the action of solutions of alphanaphthol on parts of the cytoplasm of cells. It was shown by O. Witt ('82) that a blue dye (indophenol blue) is formed by the oxidation in dilute alkaUne or acid solution of alphanaphthol and dimethyl-para-phenylenediamine. Winkler ('07) and others found that myeloblastic cells react in a characteristic way in tissues treated with these two substances and the

189

190 F. A. MrJUNKIN

grunulcs of the reacting rolls wore said to contain an oxidizing frrniont (oxydase or peroxidase) that oxidized (he two conii)ounds and caused them to unite with the production of a blue color in the neutrophilic and eosinophil granules. Later Loele ('14) found that the treatment of mjeloblastic cells with alphanaphthol solutions alone produced the same blue color in the granules. The part played bj- the cell granules has usually been regarded as an oxidizing one ('oxydase or peroxydase reaction). As applied by Loele the phenomenon consists of a pur])lish or bluish color in the cytoplasmic granules of the cell produced by treatment with old alphanaphthol solutions. If a dj'ostuff is produced by alphanaphthol alone, and this seems likely, an aromatic compound or compounds must be supplied by the leucocytic granules. If such is the case "indophenol reaction" is a better term than "oxydase reaction."

Leucocytic granule stain (Ciraham). Since hydrogen peroxide is added to the alphanaphthol solution to make it immediately activ(> and the swollen granules are heavily and permanently stained by treating the preparations with an aniline dye, the method of Graham is better than the other indophenol staining methods devised. To applj' this method, remove thin sections of formalin-fixed tissue attached to slides from the distilled water and stain in dilute Q-5) hematoxylin (Dclafield) for five minutes; wash them in distilled water, place in a saturated solution of lithium carbonate for five minutes, wash in distilled water for two minutes, and stain for ten minutes in 10 cc. alphanaphthol .solution to which 10 drops of 1 per cent pyronin (Griibler) have been added immediately before placing the preparations in it. Prevent evaporation by covering the dish. Wash the sections in distilled water, place in saturated aqueous solution of lithium carbonate for from five to ten minutes, wash in water and differentiate and dehydrate in 95 per cent alcohol for one-half minute. Complete the dehydration by inniiersing the slides in xylol and raising them above the surface of the liquid two or three times and blotting with smooth, soft filter paper. A flat oblong .staining di.sh, the .size and width of a slide, is used for the staining and differentiation of the slides. Mount in

ENDOTHEUAL LEUCOCYTES IN HUMAN TISSUE 191

coloplioniuni-xylol or acid-fr(M> balsam. Tho tinu' that tlio prpparations remain in tin* saturatod lithium carbonate aftor troatmont with alphanaphthol is important because this removes the excess of pyronin. With some tissue better results are obtained by staininp only five minutes in the alphanaphthol-pjTonin solution and differentiating for a shorter time in the lithium carbonate after this stain. If the one per cent pyronin solution is to be kept for some time, sufficient formalin should be added to make it 10 per cent formalin.

The alphanaphthol solution is made by dissolving 1 gm. alphanaphthol (Merck Reagent) in 100 cc. 40 per cent ethyl alcohol (made from absolute alcohol) at a temperature of 50°C. and adding 0.2 cc. hydrogen peroxide. Commercial hydrogen peroxide containing approximately 3 per cent hydrogen peroxide, as detennined by titration with decinomial potassium permanganate, should be used.

Soap method of imbedding. 200 grams transparent glycerine toilet soap are placed in a 5(X)-cc. Erlenmeyer or Florence flask, that contains 200 cc. distilled water. The soap must be so hard that it is brittle and cracks apart when cut with a knife, otherwise the soap solution will not be of the proper consistency. The flask is placed in the paraffin oven at 52°C. overnight in order to dis,solve the soap. Remove it from the oven and place for three hours in an incubator at 37.o°C. The contents should be a syrupy liquid and should solidify when a small amount is poured into a paper boat and allowed to stand at room temperature for one-half hour. If solidification does not take place, 20 grams of soap should be added to the flask, and the contents again melted in the paraffin oven. After the correct consistency has been obtained the soap solution is placed in 100-cc. wide-mouth bottles with cork or glass stoppers, with about 50 cc. to a bottle.

To imbed the tissue small pieces are taken from 10 per cent fonnalin and dropped into the melted soap contained in one of the bottles which is placed at 37.5°r. two to three hours and occasionally shaken. The liquid soap in the bottles usually become solid jifter remaining at 37.5°C. for a day or more. To melt the solidified soap, the bottles are placed in the paraffin oven for

192 F. A. McJUNKIN

an hour, the s(jap coolctl to A't'^C, the tissue added, and the Inittles rephic«'d in the incubator at 37.5°C. The soap solution with the tissue in it is eniplitd into a box of suitable size made from paper as in paraffin imIjediLing and the tissue arranged on the bott«.>m of the box with forceps. The box should be made from paraffined paper or the paper may be coated by pouring melted paraffin into it. At the end of about one hour the paper is removed, the soUd soap trinuned with a knife to thi; desired size about the tissue and the blocks attached to a heated metal disk just as paraffin blocks are attached. The blocks after about one hour are dropped into a siiturated solution of sodium chioriile in a pint Mason jar, and the jar placed hi the incubator at 37.5°C. overnight.

With forceps remove the block from the saturated salt solution, attach to rotarj- microtome and cut away the block until the tissue is reached. Carefullj' trim the block and allow to dry for from three to six hours until a ribbon 6 to 8 microns thick cuts perfectly. The di.sks maybe detached from the microtome and after the proper drj-ing again attached, so that the ribbon comes from the very surface of the block. The ribbon is placed in distilled water in a fiat dish more than 6 inches in diameter, and sections floated on shdes on which there is a thin coating of fixative made by adding 4 cc. of a very thick .syrupy celloidin to 16 cc. oil of cloves. The preparations after being pressed out and carefully blotted with filter-paper are placed in the paraffin oven for fifteen minutes, when they are removed, washed in 95 per cent alcohol for thirty seconds, and placed in distilled water where they remain less than five minutes. It)nization in the large volume of water in which the soap sections Jire first placed develops only a shght alkalinity, and in the thick soap solution ionization is practically absent. The sjiturated salt solution hardens the blocks since it prevents hydrolysis by mass action. If the tissue is quite fragile the ribbon may be placed in sjiturated salt .solution instead of distilled water.

Reaction of sectio7i.s obtained by the soap method of imbeddimj to the stain. The nuclei are blue and the granules of myeloblastic cells, eudothehal cells and endotlielial leucocytes arc red. (iraham

ENDOTHELIAL LEUCOCYTES IN HUMAN TISSUE 193

notrd fh<' red granuli-s in both ciidothclijil cells and ondothcliiil leucocytes and ixpluincd their presence there as the result of ingestion by phagocytosis of niyeloblastic ceils or the cytoplasmic granules of these cells. He does not make it clear whether the grannies were found in all endothelial cells and leucocytes.

In the thin .soap sections it is evident that many cells of endothelial origin contain the granules and that they are not present here as the result of an accidental phagocytic phenomenon is shown by the small size of the granules and their even distribution in the cell cjioplasm. The granules difTer from the myelobla.stic granules .seen in neutrophiles and eosinophiles in being f(>wer in number, snudler in size and more di.scretely distributed. In ti-ssues in which only mature ipol\Tnorphonuclear) niyeloblastic cells are present a casual glance is sufficient to distinguish the endothelial leucocytes containing the red granules since they are mononuclear. In ti.ssue containing myeloc.vtes, the heavier staining and greater number of granules of the neutrophilic and eosinophihc myelocytes separate them from the endothelial leucoc\-tes. The differentiation between myelocytes and endothelial leucocytes is well shown in sections of bone-maiTow

(fig. 2).

The characteristic action of the stain is best .seen in sections of the liver or other organ of an animal that has received intravenous injwtions of a lampblack suspen.sion according to a method devised by the wTiter ('18). Endothelial cells containing carbon and definiieiy lining the sinusoids have nnl granules scattered in their cytoplasm (fig. 1). Likewise carbon-containing endotheUal leucocytes in the vessels show the discrete red granules. The neutrophilic and eosinophilic leucocytes are more conspicuous than the cells of endoihdial origin owing to the greater number and larger size of their granules. There are a certain number of neutrophiles, eosinphiles, and endothelial leucocytes that do not take the stain; anil it is only in the endotheUal cells with a distinct amount of visible cytoplasm that the granules may be distinguished. The failure of some C(>lls to stain appears to be due to an error in technic but all attempts to correct this have failed.

194 F. A. McJUNKIN

BIBLIOGRAPHY

Witt, O. 1882 J. Soc. Chem. Ind., p. 255.