Loyola University ChicagoLoyola eCommons

Master's Theses Theses and Dissertations

1971

The In Vitro Synthesis of Melanin in the AvianPineal When Treated with D, L-DopaBernadette Ann NowickiLoyola University Chicago

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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.Copyright © 1971 Bernadette Ann Nowicki

Recommended CitationNowicki, Bernadette Ann, "The In Vitro Synthesis of Melanin in the Avian Pineal When Treated with D, L-Dopa" (1971). Master'sTheses. Paper 2588.http://ecommons.luc.edu/luc_theses/2588

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THE IN VITRO SYNTHESIS OF' MELANIN IN THE AVIAN PINEAL

WHEN TREATED WITH D,L-DOPA

BERNADETTE ANN NOWICKI

A THESIS SUBMITTED T'.0 THE FACULTY OF THE GRADUATE

SCHOOL O;r- LOY·OLA UNIVERSITY IN PAR~IAL FULFILLMENT'

OF THE REQUIREMENTS F:OR THE 1 DEGREE OF.;·

MASTER OF SCIBNOE

1971

ACKNOWLEDGEMENT

The author wishes to express her gratitude to

Doctor Boris Spiroff for his valuable guidance throughout

the course of this investigation and for his assistance,

in the preparation of this manuscript. Sincere appreciation

is extended to.Miss C~rol Waldock for her help in the

preparation of the slides.

For their help in the completion of this thesis, I

wish to thank Mrs. Andrea Schneider and Mr. Anthony

Marchese.

My special thanks are to my mother for her patience

and encouragement.

ii

TABLE OF CONTSNTS

LIST OF PLATES • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

. . . . . . . . . . . . . . . . . . . . . . . . . . INTRODUCTION ••.••••••• REVIEW OF LITERATURE • • • • • • • • • • • • • • • • • • • • • • • • • • • •

MATERIALS AND METHODS • • • • • • • • • • • • • • • • • • • • • • • • 4 • •

RESULTS •••••••••••••••••••••••••••••••••••••••••

Morphology of the Pineal • • • • • • • • • • • • • • • • • • •

The DOPA Reaction • • • • • • • • • • • • • • • • • • • • • • • • • •

Page

iv

1

3

14

17

17

18

The Control Group ••••••••••••••••••••.••••• 21

The Osmic Acid Reaction • • • • • • • • • • • • • • • • • • • •

• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • DISCUSSION

SUNMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LITERATURE CITED • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iii

21

23

31

33

38

PLATE I

Fieure 1

Fisure 2

Figure 3

Figure 4

PLATE II

Figure 5

Figure 6

Fie;ure 7

Fic;ure 8

PLATES

DGPA Treated Pineal-72 hours of incubation (800x)

DOPA Treated Pineal-84 hours of incubation (800x)

DOPA Treated Pineal-96 hours of incubation (800x)

DOPA Treated Pineal-108 hours of incubation ( aoox) • . • • • • • • • • • • • • • • • • • • • 39

DOPA Treated Pineal-120 hours of incubation (800x)

DOPA Treated Pineal-7 days of incubation (800x)

D0PA Treated Retina-108 hours of incubation (8oox)

Control Pineo.1-96 hours of incubRtion (800x) ••..••••.•..••.•••••• 41

iv

PLATE III

Figure 9

Figure 10

Figure 11

Figure 12

PLATE IV

Figure 13

Figure 14

Fie;ure 15

Control Pineal-108 -hours of incubation (~OOx)

Control Pineal-120 hours of incubation (800x)

Control Pineal-7 days of incubation (800x)

Osmic Acid Treated Pineal-84 hours of incubation

Page

(800x) ••••••••••••••••••••• 43

Osmic Acid Treated Pineal-96 hours of incubation (800x)

Osmic Acid Treated Pineal-108 hours of incubation (800x)

Osmic Acid Treated Pineal-120 hours of incubation (800x) ••••••••••••••·•••••• 45 . .

v

INTRODUCTION

The pineal organ has held the fascination of scientists

and non-scientists alike since the 2nd century A.D. This

structure has been considered a "third" eye (Eakin, 1970),

a secretory organ (De Robertis and Pelligrino De Iraldi, 1961;

Beattie and Glenny, 1966; Quay, 1968), a photoreceptive

device (Kelly, 1962; Quay and Renzoni, 1963; Quay, 1965;

Bischoff, 1969), an evolutionary vestige (Krabbe, 1955), and

even a "biological clock" (Wurtman and .Axelrod, 1965;

Gaston and Menaker, 1968). Some of the above differences

may lie in the fact that this structure, together with the

parietal body, have assumed diverse cytological appearances

in the vertebrates.

It has been said by some authors that the avian pineal

is an evolutionary link between the·photoreceptive, saccular

pineal organs of the lower vertebrates and the secretory,

parenchymal pineal organs of the mammals (Wurtman, Axelrod

and Kelly, 1968; Bischoff, 1969). Assuming the above, one

should be able to find evidence of both photoreceptive and

secretory structures within the avian pineal. Several

investigators have reported the presence of photoreceptive

cells (Quay and Renzoni, 1963; Collin, 1967, 1968; Bischoff,

1969), while others have reported the presence of secretory

cells (Quay, 1965; Wurtman, Axelrod and Kelly, 1968;

............ -----------------------------------------------------

B~schoff, 1969). It can thus be concluded that the avian

pineal contains both photoreceptive and secretory cells.

In various photoreceptors, for example the Vertebrate

eye, pigmentation is either found in situ (Hamilton, 1952) . . .

or else is brought in by neural crest cells, as in the

Amphibian eye (Horstadius, 1950). The synthesis of melanin

might clarify this role for the avian pineal. The purpose

of this investigation would involve not only the in vitro

technique of melanin synthesis by D,L-DOPA but also the

probable role of the Golgi Apparatus in its formation.

REVIEW OF LITERATURE

The early history of the pineal dates back to Galen,

who in the second century A·.D., noted that the pineal was

a single struc~ure. Ke concluded that it functioned as a

valve to regulate the flow of thoughts out of its "storage

bin," located atop the- aqueduct of the cerebrum.

Rene Descartes attached a metaphysical significance

to the pineal by calling it the "seat of the rational soul."

However, in the past decade interest in the pineal has,

so to speak, been revived, when in 1958, Lerner and his

o o-workers discovered melatonin in bovine pineal organs.

The pineal was thus elevated from its status as a mere

vestige to an organ of significant physiological activity,

as well as a source of considerable interest.

EMBRYOLOGY

The embryological development of the avian pineal has

been studied in numerous species some of which are: Gallus

domesticus (Spiroff, 1958; Campbell and Gibson, 1970),

Larus canus (Wetzig, 1961) and Phalacrocorax carbo (Charvat,

1954, as cited by Ralph, 1970).

However, generalizations cannot be made concerning all

other avian species from a study of a few species.

The avian pineal originates as a single, small

3

evagination from the roof of the diencephalon between the

posterior and habenular commissures.

The pineal of the chick first appears at approximately

48 hours of incubation, showing little change through 78 hours

of incubation, although doubling in size (Spiroff, 1958).

This prominence becomes a hollow sac which communicates with

the third ventricle as a broad pineal recess (Krabbe, 1955).

This connection exists for the first two or three weeks in

Gallus domesticus (Romieu and Jullien, 1942). In some species

this connection was seen to persist for three months- post-

hatching (Spiroff, 1958).

Eventually the pineal is seen to germinate a large

number of primary vesicles at its distal end. According to

Tililey and Warren (1919) and Gladstone and Wakeley (1940)

these vesicles are lined by ependym~l cells and separated

from each other by a loose mesenchyme. The cells forming the

vesicles are seen to assume a radial arrangement around a

central lumen. There is no evidence of continuity between

the lumina of the vesicles and the pineal recess (Spiroff,

1958). The formation of the primary vesicles continues

through 120 hours of incubation.

Secondary vesicles are seen to arise as outgroWths of

the ventral pineal surface. At about ten days of incubation,

tertiary vesicles form at the periphery of the secondary

vesicles. The lumina of the tertiary vesicles are not

c onnec te d with the lumina of the secondary vesicles. By the

sixteenth day of incubation, the pineal has become a compact

or0an (Spiroff, 1958). Only sli3ht morphological changes

occur in the pineal development hereafter.

AUATO}fi

Studn1cka (1905, as cited by Ralph, 1970) has classified

avian pineal into three structural types: (1) saccular

•;1th thick walls; (2) follicular and tubular; and (3) solid.

There are also intermediate, as well as mixed structural

types. Romieu and Jullien (1942) and Spiroff (1958) noted a

change from the vesicular to the compact type of pineal in

the domestic fowl. In Passer domesticus the pineal of

younger animals is markedly follicular and larger than the

more solid orean of older birds (Ralph and Lane, 1969).

Shellabarger (1953) has also noted that in male W'hite

Legh~rns, the pineal looses its follicular arrangement by

the nineteenth day posthatching.

Aside :frcm a difference in basic structure, the pineals

of avian species differ in size and shape (Quay and Renzoni,

1963). The larcest pineal; in terms of actual size (lOOm~)

has been found in the emu· (Dro:naeus novaehollandiae) (Cobb

and Edinger, 1962). However, in terms of body size the

parakeet pineal is seen to be the larcest.

5

Ill""'"-------------------------------------------------------------,

In certain avian s:pecies, particulo.rly the Stric:iformes

c~vls) the pine~l is rudimentary or sometimes even absent

(Krabbe, 1955).

The pineal of certain species is seen to bear a

resemblance to the third eye of lower vertebrates. Krabbe

(1955) describes the development of an enlarGement from the

distal end of the pineal in the swan (C'yp;nus olor) which

detaches and forms a "parietal corpuscle" resembling the

parietal eye of saurians. In the parakeet embryo

(!-:elousittacus -:.indulatus), Krabbe (1955) describes the

existence of an eye-shaped enlargemei;t that remains gttached

to the pineal.

INNERVATION

It has now been commonly agreed that the avian pineal

is an innervated organ (Quay and Renzoni, 1963; Kappers, 1965).

~uay and Renzoni (1963) have described non-myelinated fibers

surrounding and penetrating the pineal follicles in the Order

Passeriformes. They also stated that "the parenchyma of

the distal part of the pineal in some species receives an

innervation anteriorly from a (sympathetic?) trunk accom:pan:,rins

a ven:::us sinus."

~rerve cells have been observad to f0rm a system of

S'.".'!aller and l".'.r:er cells havinc: sym-pa.thetic relations w;I.th

~r~bn~le photoreceptive cells. Neurons send axons down the

6

pineal stalk to the habenular and post~rior cu.wr1111::1isurea

(Quay and Renzoni, 1963).

It has also been observed that bilateral cervical

Ganglionectomy resulted in complete sympathetic denervati on

of the pineal (Hedlund, Nalbandov, 1969).

CYTCLCGY

The three basic cell types found in the avian pineal

have been described as ependymocytes, hypendymocytes, and

pin~alocytes. Initially the ependymocytes are radially

arransed around the lumina of the primary vesicles

( Studnicka, 1905, as cited by Ralph,· 1970). Hcrwever, at

ten days of incubation, two distinct cell types become

visible within the vesicular wall: (1) ependjrmocytes, cells

which border the lumina and have elongated nuclei, ?...nd (2)

hypendymocytes, cells which are peripheral to the ependymocytes

and are not radially arraneed (Spiroff, 1958). Romieu and

Jullien (1942) also consider the hypendymocytes ~o o~

peririhe~al, as well as much smaller in size.

The pinealocyte, acccrding to Romieu and Jullien (1942),

is the definitive cell type of the adult avian pineal.

Fujle (1958) c 1Jnsiders the pinealocyte to be the most

nu.'ner()US ·cell t:rpe and to have a cilium r>ro~ecting into

the lumen.

7

,...---------------------------------------------------------.

SevGral other cytol0~ical c1Rss1f1c~t1nns have been

presented. Fujie (1968) describes three types of cells in

the pineal of the chicken: (1) pinealocytes- parenchymal

cells that are very numerous and possess a cilium that

projects into the lumen, (2) supportive cells- these cells .. . are found between the pinealocytes, and (3) glial cells-

very uncommon and possessing filaments.

Other specialized types of cells have been reported

by Quay (1968) in the parakeet pineal: Type I- ciliated

cells, having whorls protruding into the lumen and Type II-

cells containing microvilli projecting into the lumen and

exhibiting abundant microfibrils indicating a secretory

fwiction.

In a recent ultrastructure study, Bischoff (1969)

.designated three types of cells in the Japanese ~uail ~.nd

the 1i!11te Lechorn Chicken: (1) ependymal- th~se cells

!)assessing cilia, fe":·:r mictov1111 and projecting into the

lumen, (2) secretory- those cells containing .a.ui.at:rous

membr~ne. bound granules, and (3) photoreceptive- those cells

with a modified cilium at the apical end proje~ting into

the lumen.

Photoreceptive cells have been reported by several

investi3ators. ~uay and Renzoni (1963) found cells which

a:;:parently are photorace,tive on the basis of t:1eir

8

·-·----------------------------------------~------. similarity to amphibian and reptilian photoreceptors and

their nervous connections. Such cells are concentric and

possess irregular lamellar whorls. /

Oollin (1966) stated: "L'ultrastructure comparee / . / ./ / /

photorecepteurs des organes parietaux des Vertebres inferieurs /

et des pbotorecepteurs rudimentaires chez l'embryon et le

jeune Pie."

In-1969, Bischoff noted photoreceptive cells that

resembled retinal cone cells, although they were not as

organized as those of the lower vertebrates. These cells

were located singly within the follicles and are the

least numerous of the cell types observed previously.

Bischoff also indicated that the photoreceptive cells are

degenerate or rudimentaJ.7, but st111 functiona~.

Other investigators have reported that the avian pineal

can respond to light even when the eyes have been removed,

thus suggesting that the skull allows the penetration of

light (Lauber, Boyd ahd Axelrod, 1968). Benoit (1964) has

postulated the presence of photoreceptive sites within the

diencephalon of duck~. The pineal or some other ·structure

that can influence it, apparently can act as a photoreceptor.

Therefore, it would n_ot be surprising to find that the

av1z..n pin.e3.l can respond to light (Lauber, Boyd and

9

.,.._. ~~ ...... i-!lilll---~--~-~-~--~·····.,.."""'"~---.................... ~~ ...... ...-............. """" ... """' ... ,...., .... . Fi- tic" :; ... ~

Ax.:;lro-d, 1968)..

Accwrding to Ralph (1970): "If the avie.n·pim.:al is

a photosensory orgnn, its receptor uni ts rtre ·not a

c onv:;ntional kind."

J.!any investigators have concu·rred w·ith the theory that

the avian· pineal is a secretory organ. As noted previously,

several 1nvest1.gators have· reported the presence of

secretory cells (Quay and Renzoni, 1963; ::uay, l968T ·

3isc'loff, 1969). ~uay (1956) conducted cytolo51cal stud1e-;e

on .various •Vian species. These· studies revealed structural,.

details t.hat- indicate _a secretory activity by the pineal

cells. , Ilo~-;ever~ these ob.serv-ations are· not a dB:f'1n1t1ve

indioation··ar the g:laildular nature_- of the i)1neai~ Rom1eu~"and cT.ullien (1942) and 'Wetzig· (1961) ncted a

f1br.ous, globular accumulation of ma:tarial in the J:umina

of both -e'he ~:'&mbry onic'~:ana ad:Ul t avitill :pine :=i.ls and

· sucsested that the material wa.e a secretion of the·

e pendym.oeytes.

S1m116.rly, :Be.attie and Glenny (1966) noted a large .

amoant ot !JN..t,. and muoin-like substance in the lumen and

believed' it to be a 'secretory produc.t. Often secretory

e-ranule s ·are scten w1 thin sm,ill ve siole·s 1n close !)rOx1m1 ty

to the Golg1 Apparatus (Bischoi'f ,· 19G9). ·

r=..--------~---------------.......... ..,,... ............... -~~!1111!1-- -~-., ..... r:-.1 ~·" . . "

Bowen (1929, as cited by W'olfe, 1965) considered

secretion to be a phenomenon of granule or droplet

f orm.ation. The secretion could be observed in a gland

cell as an accumulation of granules produced by the cell.

Therefore, the material described by Romieu and

Jullien (1942), Wetzig (1961) and Beattie and Glenny (1966)

could be the 'granule' or 'droplet' formation described by

Bowen.

Thus the evolutionary significance of the avian pineal

stems from its close association with the third eye of •

reptiles and the frontal organ (Stirndriise) of the

amphibians (Lauber, Boyd and Axelrod, 1968). The av1an

pineal, having both secretory and photoreceptor cells is

considered to be intermediate in the development of cold-

blooded vertebrates and mammals.

· MEL.A.T ONIN

One of the major advances in pineal biology has been

the discovery of melatonin (Lerner~ 1958). This indole,

which was initially dlscovered in bovine pineals, is

characterized as 5-methoxy-N-acetyltryptamine.

Experimentation on mammalian melatonin has indicated

that it -is the most potent ac;ent causin.t; contraction of

frog melanophores.

11 .._ _______ '~-,.--------... ----~-.. _~ __ """!'.--""'-_..,...,_....,~!""!'!"!'!-...~ ..... -----111111,....,.,., .... ~ . .,--.,,

The presence of melatonin in the domestic !owl was

demonstrated-,by Beattie and Glenny (1966). In 1967,

Ralph measured the melatonin content of four species of birds.

According to Axelrod (1970) the actual biosynthesis

of melatonininvolves·the following reaction:

N-acetylserotonin + S-adenosylmethionine ~Melatonin. Axelrod and· Lauber (1968) have observed that serotonin

is also a suitable substrate in several avian species.

HIOMT, hydroxyindole-0-methyltrans!erase, the enzyme

· responsible !or the formation of melatonin was found to

have a characteristic location in mammalian and avian

(.Axelrod and Lauber, 1968) pi.neals. Quay (1965) reported

that only one other tissue, the retina, contains HIOMT,

however, HIOMT has never been detected in avian retinas.

The activity of HIOMT has been studied by several

investigators (Axelrod, 1964; Quay, 1965; Lauber, 1968)

who have concluded that it is very active in avian pineals.

This high activity indicates that avian pineals have a nigh

capacity to ~Jnthesize melatonin (Lauber and Axelrod, 1968).

The large amount· of melatonin synthesized suggests

that it may play a ~hysio~osical role in the avian pineal.

1-:e,latonin is seen to cause a decrease in the weieht of

avian'ovaries, oviducts, and testis. Under certain lie;htine

conditions small doses of melatonin 1·:-ere seen to ·inhibit

growth in the Japanese Quail (H?mma, McFarland and Wi_lson,

1967). Since env1ronmental lighting influences gonads as

well as melatonin synthesis,- it is probable that certain

lighting effeets on a11ian gonads could be controlled by··-

changes in ~he rate of synthesis of avian melatonin

(Axel3:od and ;.rurtman,. 1964). Thus the chemistry of the

pineal gland seems to be affected by environmental lighting.

BIOLOGICAL CLOCK

Closely associated with the effects of environmental

liehting on the pineal is the theory that suggests that

the pineal is a biological clock. Wurtman·and Axelrod (1965),

indicated that the pineal is an intrinsic and sansitive

"Biological Clock" which secretes hormones in z:-esponse to

cyclic nervous activity and environmental lighting.·

Gaston and Menaker (1968) have reported that the pineal

of Passer domesticus is necessary for the operations of the

circadian locomotor rhythms in constant conditions. They

concluded that the nineal was a crucial comuonent of the . . . time measuring system of the sparrow •

. 1

,..-' --------------------------------------------------------------....

MATERIALS AND METHODS

Since the purpose of this investigation is to clarify

the function of the avian p1nea1, a study of the in vitro

synthesis of melanin, using the DOPA Reaction (Bloch, 1917,

as cited by Laidlaw, 1932) was used. -

White Leghorn embryos were randomly divided into three

groups: (1) those incubated with D,L-DOPA (3,4-dibydroxy-

phenylalanine); (2) a control group; and (3) those

impregnated with osmic acid to demonstrate the Golg1 Zone.

The procedu!e for Group 1 consisted of a modified

adaptation of Bloch's technique (Laidlaw, 1932; Guttes and

Brandt, 1961) in which the underlying principle supposes

the presence of an intracellular enzyme within the cells

·which oxidizes DOPA to DOPA Melanin. A brown or black

stain appears in the area where the copper.containing

enzyme, tyrosinase and the substrate DOPA react in the

presence of molecular oxygen. This darkening of the cell _

is the DCPA Reaction. Further investigation validated t.he

reliability of the DO.PA reaction as an indicator of the

presence of the specific enzyme,· tyrosinase within (1) a

specialized cell, the melanocyte; (2) at a site where melanin

formation is occuring; or (3) at a site where it can be

produced by specific stimulation (Laidlaw, 1932). The

14

II"'"':-------------------------------------------------------.

reaetion is also an indicator of the capacity of a tissue

to produce pigment.

The reaction proceeds as follows:

Tyroslne . + (Tyrosinase)

DoPA t

Intermediates +

Melanin

(Lerner and Fitzpatrick, 1950).

The DOPA reaction itself consists of incuba~ing chick

embryos which have been previously fixed for one hour :tn

formalin (diluted 1:3 in distilled water), in an aqueous Cl

solution of 0.2% DOPA at 38 c. The solution was adjusted te pit 7. 2 (S·orensen' B" Buffer).

After approximately two hours of incubation, the

solution turns a reddish orange, and after three to four

hours of incubation, a sepia color, indicating the reaction

of the copper containing enzyme, tyrosinase, with the

substrate, DOPA, in the presence of oxygen.

The appearance of the sepia color marks the end of

the reaction (Mccurdy, 1969). The embryos were. then fixed

in formalin, dehydrated, cleared and embedded in paraffin.

The embryos were sectioned at sev2n microns and stained

with Fast Green.

lS

~-·--------------------------------------------------.

Embryos in the control croup were treated in a similar

manner, thou:3h not 1ncubn.ted >rith the DOPA soluticn.

As a further control measure, pieces of filter paper

·were incubated -:·;1th the DC.PA. treated embry0s. The filter

paper remained white, thereby indicating that the DOPA

solu~1on was not autooxidized.

~he third group of embryos were fixed in Ohampy 1 s

solution tor 24 hours. After being washed in running water

for 24 hours, there were impregnated with 1% osmic acid 0

at 38 o··. for six days 1n orde;r to demonstrate the site of the Golgi Apparatus as the suggested location of melanin

synthesis.

The stages used in this experiment were :

72 hours of incubation (5 embryos), 78 hours (7), 84 hours

(10), 90 hours (10), 96 hours (9), 102 hours (9), 108 hours

(9), 114 hotirs (14), 120 hours (15), 6 days (5), 7 days (7),

and 8 days (4).

16

~~, -----------------~ ~,, £:"• ,, . ~-",::,;,,

•

~ULTS

The a~an pineal between 72 and 102 hours of incubation

occurs as· an evac.1nat.1on from the roof of the diencephalon

(Pi~Tes 1,. ·3·' ·and l~). !eC"ortdary vesi-c-les begm to .form

at· the. distal end of _the main pineal ou:tgrowth at

approximately 84 hours of'· incubation .. (Figures 2 and 3) • .

Cells are assuming a radial arrangement around isolate.a

spaces. At each progressive stae;e, the pineal is increilelne

in length as well. as width.

Between 108 and 120 hours of incubation,. the seco~dary

vesiclee bee ome more distinct ent·1 ties, although they are

st·ill atti.~hed to the distal end of the main pineal

·outgrowth· (jt1gures 4 • 10 1 14 and 15) • · When cut 1n cross

seotion, .the pineal assumes' a rosettt·l'!"11ke appearfi,nce 'Wi'th . /

tha. pineal :rece.ss (the lumen of the main pineal outgro-vrth)

in the c.enter and. the- secondary vesicles surroundine 1 t

(P1c;ures 4, 14 and 15). ·;.;1th the increase in ace, the

pineal~.oont!nues to ~row, th1'ttgh still reta.1n1ne; a club•

111te appearance.

Throu.gh six, seven,and eight dafs of incttbation, the

. :;in~al is n,c;·i d11·t:cted an:tericrly with distinct, uum.G rc·us

·.-·~ '·

. .

f{.·:::· ! ·.~ ' ::.;··· ~;:,.·. - , .. ! ;:.:·. secondaey vesicles at 1ts distal end. The lumina of the

sec ondaey vesicles are now clearly vis1bl~ (Figures 6 and 11). - According to Bischoff (1969), the cells of the pineal

can be divided into three regions. The basal segment

contains the nucleus and _rests upon the folliouLar

b-oonda:r,r. ~he eollicular or neck region is laterall7

constricted and connects the basal to the apical segments.

The apical or polar terminal is an expanded area that

extends into the follicular lumen. On this basis, the

pineal ·-tissue itself' can be subdivided into the same three

. regions: the apical; the collicular; and the basal.

B. THE DOPA REACTION:

~he DOPA reaction is a constant ~ reliable indicator

of the presence of;,naturalmelanin pigment (Laidlaw,- 1932).

Each blac~ene;cl cell contains the ettZ:yme* tyro-sina·se and is

therefore, an active -site ot melanin.production.

l. PIGMENT GRANULES AND LA.aGE PARTI0!.3S:

Embryos of 84 ·throuf!h 102 hours of incubation

_ pra sented a dense- granular a.ccumulat1on of material :forming

a circular mass in'the collicularreg1on of the main pineal

out3rcwtb'~(Figures a and 3)~ This dense accumule.t1on term111ated'. at the junc.ticn of the pineal and the d1ence11halo.u ·

l,

r;_ r:·:r ~: .

~··· '

(Fi.zure 3). Rows o! pigment granules, visible in the

oollioular r~s1on., were seen t.o radiate from the dense

accumulation. At approximately the basal reeion, the rows .,.

abruptly termllla.ted (Fl3ures;·2 and 3). A few, large blaclt

spherical particles were superimposed on the eranules

com.prising the radiating rows (Figure 3).

The DOPA reaction was also observed as a black,

eranular·area surrounding the lum:Lna of the secondary,

vesicles (Figures2 and 3).

The DOPA reactidn was negative .1n the f3mbryos of 72

alid 18 hours of iihcubation (Fi~re 1).

2 ~ THE ·pRESENOE'. o:r i YELLcir. ooLOR: Laidlaw (1932) stated that the appearanc~ of a

yellow-brolfli ooloratlon beneath .the DOPA positive cells

indicates a correct'DOP! reaction.· Re further stated that

the y:e11:6w.;.br~'im·coio'r .. deriot_es the pre-sence ;of. melaniii .. that

has ·retained: 1.ts natural color ou·tside of the DOPA positive cells.

. .

:Bet1·ieen 90 and i~o hours of incubation, such a coloration was visible beneath the DOPA positive cells

in the main :pinea.l outgr.owth (Ficure 4). Beeinnine: at 96 ..

hours o:e · ~ubation~ the color:ition ·was detected around the

lu:nina of the·:' sec'.'lndttfy pine"Etl vesicles (Fi,:ures 3 and 4).

19

,.,:.---------------------------------------------. Although a positive DOPA reaction occurred 1n the

embryos of 6, 7 and 8 days of incubation, no yellow-brown

underlying color was observed (Figure 11).

3. LARGE PARTICLES:

The DOPA posi t1v-e· area in embryos of '108 to 120 hours

of incubation consisted of large black particles extending

from the apicai region, immediately surrounding the pineal

recess. The particles formed radiating rows, one or two

:particles thick, and extended to the basal region of the

pineal tissue (Figures 4 and 5). Dense black bands of

material located in the apical region were also visible

within some of the radiating rows (Figure 5).

The same type of particle arrangement surrounded the

lumina of the secondary pineal vesicles (Figures 4 and 5).

At 6, 7 and 8 days of incubation, the pineal recess

was still Surrounded by DOPA positive cells. The material

was composed of large black particles accumulated in the

apical region surrounding the pineal recess (Fieure 6).

The particles appear to be on the pineal tissue, rather

than within it. Laidlaw (1932) noted that newly formed DOPA

melanin can remain in or on the cell that has produced it.

The particles surrounding t}le lumina of the secondary

vesicles still ap;:>ear in radiatine: rows. The superposition

of the particles is also visible in the secondary vesicles

20

Fi . . ~ .. · .. ·'"

'-'.· ' · .. '~ ... : . ,,, f;.':.:-" ....... ('··· .··

{Fie:ure ·· 6).

C~ THE CCl'iTRCL GRGUP:

The. pin.ea.l .bodies of chick embryos of 72 through 114

hours of incubation Md 1 not possess allY'"Pigmen:t materiaJ.

(Figures 6 and 9) •.... -~

H'OWever, at 120 _hours of incubation a sJ1).all number of

black particles were observed as superimposed on the pineal

tissue. These particles were located 1n the apical reg19ll;

of both the,p1n pineal outgrowth and the secondary vesicles

(Figure 10). Wetzie; (1961) noted the ·preaence ·Of pigment ·

in and around the develoJ>ing avian pine_al.

At 6, 7 and 8 days of incubation, the laree pigment ' particles had increased in number• The superpos! ti on of

th~ particles was still. evident (Fie;ure 11).

D. ·CS?·:IC AO!j) REA.CTI :::1I: · ·

O~ic ao~d impregnation localizes the Golgi Zone

within a specific tissue by staining it an intense black

while the rema1nill:g"41:ssue ·retains a 7ellow color (Gut'tes

and Brandt .. 1961; Huma1son, 1967).

Ch~ckeinbryos.fz:om 84 through 120 hours of incubation

were ir.1pregnatect with. osmtc acid and t;ave the following

rasults.

21

~....-------~--.. ~r~· ~'':·· ?i Z1;F ::;k A very dense black accumulatio~ of material was

visible within the apical region of the main pineal out-

~routh. EmeJ;"c1ng frym this. accu:nulation were radiating

rows of dense black material located in the coll1cular zone

•• of the pineal tissue. The radiating rows extended to the

basal area .(F1eures 12, 13, 14 and 15).

The lu:nina of the seo.cndary vesicles were also

surrounded by the same material. Radiatine; rows ·emerged as

in the primaryv out gr ovrth (F.igure s 4.4 and 15) • The

background tissues were stainedca yellow~gray color.

DISCUSSION

The pineal organs of the higher vertebrates (birds

· and mammals) have been considered parenchymal as opposed

to the saccular pineal glands of the lower vertebrates

' (Kelly, 1962). The parenchymal systems function mainly as

secretory organs. while the saccular pineals function as

photoreceptors.

However, recent embryol.ogical and f1ne structure

details have indicated that cellular homologies may exist

between the two pineal systems (Wurtman, Axelrod and Kelly,

1965). Although the vast majority of pineal organs are

distinctly parenchymal or distinctly saccular, there is

evidence for the existence of intermediate forms.

Avian pineal organs, for example, may exhibit the

·full spectrum of pineal systems from saccular through

parenchymal. Krabbe (1955) noted a distal swelling on the

parakeet pineal reminiscent of the third eye. A distal

enlargement was also noted on the swan pineal, which

detached and formed a "parietal corpuscle" resembling the

parietal eye of the saurians (Krabbe, 1955). While at the

oth~r end of the spectrum, several species of owl revealed

a compact, tiny organ. Ralph and Lane (1969) observed that

in Passer domesticus, the older birds retn.in a solid pineal.

23

Quay and Renzon1 (1963) indicated the presence of both

sensory and secretory cells within the.pineals of several

passeriform birds. Recent ul trastructural ·evidence has

shown the existence of photoreceptive and secretory cells

in the ~lhi te Lee;ho:tn Chicken and the Japanese Quail

(J3lschoff, ·1969). Hence, the possibility exists that

certain species. of bi:rds may possess characteristics of

both types of pineal systems.

rn this investigati~, the darkening of certain cells,, indicative of a positive DOPA· reaction, was interpreted a.s

demonstrating the presence of _tyrosinase in an area "- ' capable of producing melanin pigment.in the avian pineal.

Ih order for the pigment to form, three basic

re1uirements must be fulfilled: (1) the presence of the

initial substance, tyrosine; (2) a particular quantity of

the enzyme, tyrosinase, capable of catalyzing the reaction;

and ( 3) a sufficient amount· of the substrate, DOPA. 1'1'hen

the substrate, DOPA was provided in a sufficient quantity

the avian pineal was able to synthesize melanin piement,

thereby con!irminz the fact. that the avian r-ineal cf the

embryonic chick has the capacity to form melanin pigment.

The radial arrangement of the D8PA positive cells

can be attrtbuted to the presence of sensory cells within

t.he avi~n pineal •. Oyt0plasmic processes radiatins from

24

r..---------------------the piemented epithelium in the r~t1na are known to

extend bet·~~een the rods and the cones (Leeson and Leeson,

1966). It is possible that the radiating rows of pigment

observed in the av1a·n pineal are also extendine between

sensory cells.· The r"bws of pigment are not densely packed.-

together in the pineal, thereby indicating that the

sensory cel!s are interspersed in the pineal tissue. Ina

recent ultrastructure study on the pineal of the White

Leghorn Ch~cken and the J~panese Quail, Bischoff (1969)

noted that the photoreceptor cells occurred sine;ly within

the follicles.

Naturally produced pie;ment was also observed within

the pineal of the control embryos beginning at five days

of incubation. However, at this time, the amount of pigment

was extremely small compared to that produced in.the DOPA

reattion. With the increase in age, the amount of naturally

produced pigment in the control embryos was also seen to

increase, but never to the extent of that found in the

Dopa treated embryos. The presence of pic;ment in and around

the develo:pinc pineal of Sterna,_ Larus and Anser was also

noted by Wetzig (1961).

The re"3ults of this present.investi~ation indicate

that the pineal body of t:-ie embryonic chick may be an

·. i!l.temediate form, basically parench.ymal but posse ssinz

2

~----------------------------------------~ r-~ functioning photorecept1ve and secretory elements.

Quay and Renzoni;~,(1963) have indicated the presence of

sensory cells within the pineal of several passeriform birds.

They described the sensory cells as lining the lumina and

c onta1n1ng modified cells with basal proce-sses. -ending in a ~----"

probable synaptic relation with other possible secretory

cells. Quay· and Renzoµi (1963) believed these cells to be

sensory on the basis of their apical processes and their

basal. synaptic mGdifications. The apical processes of

the avian sensory cells are similar by light microscopy to

the apical processes of reptilian and amphibian

photoreceptive cells.

Recent ultrastructure evidence has indicated that the

sensory ce 11 s of the J's.pane se ~uail and the ·white Leghorn

Chicken resemble retinal cone cells (Bi sch off, 1969).

o·orrelat1ng the data presep.ted by ~uay and Renzoni

(1963) and Risch.Off (1969) with the presence of pigment in

the avian pineal suggests a functi·o?al photoreceptlve

ability in the avian ~ineal.

An analogy with the reti:".la of the eye may further

illustrate the above observations. The eye is a hollow

sphere consisting of a three layered wall: the outer

fibr:u s tunic; the middle uvea; and the innermost retina,

which borders the vitrei:us cha::iber. The retina is the

26

~·..------------------------------------------. photorecept1ve device. The two strata which comprise the

retina are an outer piemented layer and an inner sensory

(nervous) layer containing the light receptive units, the

rods and the conas, and neurons in a synaptic series

(Figure 7).

The free processes of the light sensitive cells are

directed into the pigment layer. Oentrally, the rods

and the cones send processes into the outer synaptic layer

where they form ,,the synapses with the bipolar neurons. In

the inner synaptic layer,· the bipolar neurons form synapses·

w1,th the ganglion cells. The· axons of the e;an~lion_ cells

c c1nverge at one point to form the optic nerve (Leeson and

Lee son, 1966; Torrey, 1971).

Within the avian pineal, a similar arrangement can be

.observed •. Beginning in the apical region of the pineal,

near the pineal recess, a layer of pigment is seen, analogous

to the outer pigment layer in the retina of the eye

(Figures 3 and 4). ~he apical portions of the photoreceptive

cells line the llimina while the basal segments are presumed

to send axons into a nerve fiber layer, forming the

traotus pinealis (Quay and Renzoni, 1963).

At first glance, the arrangement of the retinal layers

in the eye seem to be- opposite to the layering in the pineal<

body. However, upon further examination, it has been noted

that· the light- entering t·hey. eye must pass through the layer

of gg,ne;lion and bipolar neurons in order to reach the rods

and the cones and fi:rtally the pigment (Rol!ler, 1962; Torrey,

1971). After the lieht has stimulated the rods and the

cones, the impulse m~st pass .b.ac.k through .these layers t-a-

reach the opt.le nerre (Figure 7).

A similar situation is observed in the pineal. It

has been postulated that the skull of birds is thin enough

to permit light to enter (Lauber, Boyd and Axelrod, 1968).

Tracing the path of the light as it enters the skull above

the pineal, one. can postulate that the· light first. passes

the neurons and the nerve fibers surroundine the basal .

segments 0-f 'the sell-s·oey cells. The light the:ri passes over

the sensory cells and the piement layer. The impulse must

then travel back over these areas. This sequence.is very

similar to .that observed in the eye. However, in the case

of the pineal,_the impulse cQ\ild continue into the diencephalon

since Benoit (1964) has postulated the presence of

photoreceptive cells within the diencephalon of ducks.

Quay a~_cl Renzoni (19q3) have suggested that the nerve

fibers of the pineal stalk can be traced into the posterior

and habenular ccmmissliras.

Ralph (1970) has suec;ested that the pineal might be in

an ap!'ropria:te posi tir.Jn to serve as a "light funnel"

conducting light via 1ts distal end through the pineal stalk

to the diencephalon. The experiments performed by Lauber,

Boyd and Axelrod, 1968), also lend credence to the idea of

pineal photoreception. Blinding chicks or severing their

· - -sympathetic ganglia had ·no effect on the synthesis of'"-- - --

melatonin Which was still affected by changes in light and

dark. They concluded that the chicks perce~ved light

through non-retinal.photoreceptors, possibly the pineal.

According to Quay and Renzoni (1963) the anatomical position

ot the pineal, the demonstrated photosensory nature of the

pineal in lower vertebrates, and especially the suggestive

structure of the presumed pineal sensory cells plus their

nervous connections suggest for the avian pineal a photo-

receptor functional component.

The present study also localizes the Golgi Zone within

the avian pineal as a black accumulation of radiating rows

extending from the pineal recess to the basal region of the

pineal tissue. Following impregnation, the Golgi Zone was

thus visible surrounding the pineal recess and the lumina

of the secondary_ vesicles. Tbe DOPA positive cells and the

Golgi Zone were thus located in the s~me area within the

avian pineal.

A similar observation was made in the retina of the

chick by Guttes and Brandt. (1961). They noted that the

. 29. -· ·' • ...

Do.PA positive cells and the Golg1 material were localized

in the same position in the avian retina. From their

observations they concluded that the DOPA positive grains

containing pigment are bound to the Golgi Zone at the

beginning o:r· pigment formation and the "'mature" Golgi bodies

are leaving. the Golgi Zone and becoming melanize·d when

pigmentation in the retina is in full progress. Therefore,

the Golg1 bodies are considered to be the granular

precursors of the pigment (Guttes, 1953; Barnicot and . .

· Birbeck, 1958; Guttes and Brandt, 1961; Hu and Cardell, 1967).

Further investigations have localized the Golgi Zone

as a series ·of parallel double membranes associated with

vacuoles. Places can be found where vacuoles.containing

rudimentary pigment granules appear to be contained within

the Golgi vacuoles (Barnicot and B1rbeck, 1958).

Due to the similarity between melanin granule formation

and the formation of other secretory products, Barnicot and

Birbeck (1958) have suggested that pigment production is a

specialized form of secret.ion.

Since the avian pineal contains melanin pigment which

seems to be localized within the Golgi Zone, it can be

suggested that the pigment is of Golgi origin and the pineal

has the capacity to form a secretory product--pigment.

SUMMARY

1. The DOPA reaction with adequate controls is a

positive indicator of the presence of the enzyme, tyrosinase,

within the avian pineal • . 2. The pineal body of the chick has the capacity to

synthesize melanin pigment in vitro.

3. The=presence of naturally formed melanin was

observed in the pineal organs of the control embryos

beginning. at five days of incubation.

4. The amount of naturally formed melanin was less

than that £ormed in vitro.

5. Osmic acid impregnation localized the Golgi Zone

in the avian pineal centrally around the pineal recess and

the lumina of the secondary :vesicles.

6. The location of the DOPA positive cells and the

Golgi Zane were observed to be in the same area within the

avian pineal.

7. The above observations suggest that the avian pineal

has the capacity to produce melanin. Due to the presence

of melanin and sensory cells (Quay and Renzoni, 1963)

within the avian pineal, it has been suggested that the

avian pineal is.a photoreceptive organ, similar in structure

to the .retinal layer of the eye.

8. Since the Golgi Zone was localized in the same

area as the DOPA positive cells, it has been suggested that

the Golgi Zone is the site of pigment granule formation.

The secretory nature of the pineal can also be identified,

_since :Barnicot- and :Birbeck (1958) have cons1derecr,p1gment

production as a specialized type of secretion.

9. Therefore, the pineal of the domestic fowl- can be

considered intermediate in form, neither distinctly or

solely parenchymal (secretory) nor distinctly or solely

saccular'(photoreceptive).

10. Further studies in this area would include(l)

a continuation of the DOPA reaction using later embryonic

stages as well as post~hatch1ng _and adult birds; and (2) an

electron microscopical study of the pigment granules and

their suggested association with the Golgi Zone within the

avian pineal.

32

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35

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:rolfe, D. 1965. The . .E~i:pcysaa.1 0~11: an El~ctro:..unict":o~·opJ;ci,~:i;~~ Study of .. its InterceJJ.ular Relationshlps and Intra• . cellul~~r ?'.orphology in the· :Pineal Body ... of the .b.lbino Ra1;. Pro.:;rass in Bt"aiil. Resea;roh 1¢''~ 3?2-:5'7'.6.

. ' ~ .

~-Turtman, Richard, and J'Ulius Axelrod. 1965... T~ Pineal Gland. Sc. Am. 50-60. , · ·

... ...

Fic:;ure 1 . . ..

Fis·ure 2 '-'

Fie;ure 3

PLAT E I

EXFLANAT i r; N JF FI"U~2S

Sagittal secti on of DO?A treat e d Plne al, 7~ h our s of incuba ti on, sta i ne d with Fast Green. 800x

Cross section of DOPA treated Pineal, 84 hours of incubation, stained with Fast Green, demonstrating a circular accumula tion of pigment in the collicular zone (C), secondary vesicles (V), and large pigment particles (P). 800x

Sar, ittal section of DOPA treated Pineal, 96 hours of incubation, stained with Fast Green, demonstra ting the circular accumulation of pivnent in the c oll icu lar zone (C), sec onda ry vesicles (V), lar~e pi gment particles (P) and the dience phal on (DJ. 800x

Cross secti on of D0 PA treated Pineal , 108 h ours of incub ;:i. ti Gn, st ;:d !'le d ~·; ith F3. st Gr een, de m 0nstr~tinc 18 r e e pi 3ment na rticles (P), the yell ow unde r lyins c ol or (Y) and the s ec onda ry ves icle s (V). 200x

"38

p

"!:Ii :i.-~- 1 ._ - ~i u ... - 2

p

F ....,ur

FiGure 6

Fisure 7 ·

· Fic.;ur e 8

press secti on of n: PA treate d Pineal , 1 20 h ours of incubati 0n, sta ined with Fast Green, de monstrat ine: se c ond ~1 ry vesic les (V), and D2rk bands of pi cment (B). Soox

Sacitta l secti on of DCPA treated Pineal, 7 days of i ncubati on, sta i ne d uith Fa st Green , demon str~tin~ la~ [e pl smen t particles (P) surroundins the pins~l r ecess (R) and the lura ina of the sec ondar y vesicles (L). 800 :.-:

Cross secti on of ~CPA tre ated retina , 103 h ours of incub ati on, stainsd with Fast Gre en de monstr&tins the pr e sence of pi ,snent (P). 800x

Sa;:si t t a l s ecti on of Control Pine2.l, 96 h ours of incub :t ti on, st a i::1e d ·-1th F8. st Green. 300x

40

!.\ '.!:

v

r~ 5

4

p

F-

p

:.U:.'.''

. .

... Fis ure 9 . ·

.,

Fi;:;ure 10

Fi :_:ure 11

Fic;ure 12

PL\.T3 III

Cr oss secti on of Cont r ol Pineal, 163 h ours of incub~ti on, st a i ned ~ith Fa st Gre en, demonstratinc the Pinaul Recess (R). 800x

Cross secti on of Control Pineal, 120 h ours of incubs.ti on, stained with Faqt Gre en, de!n(m stratin0 t h e sec o::-idary ve siclG s (V) and the l a rLe p i cfilent particle s (P). 800x

Sugittal secti on of Cont r ol Pine a l, 7 days of i ncub ation , st a i ned ~ith Fast Gr.~ en, de ill. on stra tinc the pi c;ment 92.rticles surr :;undinc; t he s ec onda l'ij v e sicle s (V) and the lumina of the se c ~n dary vesicles (L). 800x

Cross ss cti on cf Gsmic Acid treat e d Pinsal, 84 h ours of incubatl r::n. 800x

42

.L .L J..

F_ J.rr:i

-

F re 11 3 '"Ur,.,

Fie;ure 13 · ..

Flc;ure 14

Fic;ure 1 5

PLAT"E IV

EXPLANATI ON OF FIGU ::P~S

' Oro~s section of Osm1c Acid tre: ted Pine al, 96 h ours of incubati on, dem onstrating the darkened centra l a re a conta ining the Gol ei Zone (G}. Boox ·

Cross secti on of Osmic Acid Treate d Pineal, 108 h ours of incubqti on, deI'.lonstro. ting the Gol gi area (G) and the sec ondary ve sicles (V). 8oox

Sagitta l secti on of Osmic Acid tre a ted Pineal, 1 20 h ours of i n cub ati on. 800x

•

44

Fie;ure 3

r

(

APPROVAL SHEET

The thesis submitted by Bernadette Ann Nowicki

has been read and approved by members of the Depai'~ent

of Biology.

The final copies have l:lieen examined by the

director of the thesis and the signature which .. ,..,.ara,

below verifie$ the fact that any necessary changes have' '

been incorpor'9-ted and that the thesi.s is now given fi~l

l

approval with reference to content and form. I I

The the1

sis is therefore accepted in p~rtial 1 I' '

fulfillment o,; the requirements fr the deq,ree• of !i

t{aster of science. il '' '

':'I

~2,'f.Ji'l'.11··· . ' . , ·· .. · · . Date ·1 ' " : I

" i j1 i '

I" I

"

Signatpre.of : I

1:

.~

Loyola University ChicagoLoyola eCommons1971

The In Vitro Synthesis of Melanin in the Avian Pineal When Treated with D, L-DopaBernadette Ann NowickiRecommended Citation

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