A morphometric study of the pituitary cell types in the freshwater stickleback, Gasterosteus...

Cell Tiss. Res. 152, 69--92 (1974) �9 by Springer-Verlag 1974

A Morphometric Study of the Pituitary Cell Types in the Freshwater Stickleback, Gasterosteus aculeatus,

form leiurus *

Michael Ben jamin

Department of Zoology, University College of Wales, Aberystwyth, Wales

Received March 29, 1974

Summary. A new approach to the ultrastructure of fish pituitary glands is presented. A morphometric analysis of the cell types in the pituitary gland of the adult, winter, freshwater stickleback, Gasterosteus aculeatus form leiurus, reveals differences between both the relative and absolute volumes of the various organelles in different cell types. The morphometric data on the relative volumes of the organelles, together with section profile diameters of the secretory granules and information on the surface area: volume ratio of the nuclei are then used to build "reconstruction drawings" of "average" cells. A distinction is made between the ultrastructural description and identification of cell types.

Key words: Pituitary - - Freshwater stickleback - - Morphometry - -Elect ron microscopy.

Introduction

Heut s (1947), who s tudied the d i s t r ibu t ion of the euryhal ine threespine st ickle- back, Gasterosteusaculeatus L. in Wes te rn Europe , showed this species to be d iv ided into two d is t inc t groups on the basis of the number of l a te ra l bony plates. Group A had a mean la te ra l p la te number of 5, while group B had a mean la te ra l p la te number of 32. These two forms of G. aculeatus have different life cycles. Group A spends i ts ent ire life in freshwater , while group B overwinters in the sea and re turns to f reshwater in the following spring to breed. I n accordance with Miinzing (1963) and Hagen (1967), the mig ra to ry form is referred to as trachurus and the f reshwater fo rm as leiurus.

I n con t ras t to the mig ra to ry form, l i t t le a t t en t ion has been given to the p i t u i t a r y g land of the f reshwater animal , Bock (1928) being the only worker to s t u d y it. Mullem (1959) has given a de ta i led account of the LM s t ruc ture of the p i t u i t a r y g land of the trachurus form, while Foll6nius (1968) has given a brief account of the u l t r a s t rue tu re of the cell t ypes in the adenohypophys i s of an unnamed form of G. aculeatus. Lea the r l and (1970a, b) has descr ibed the seasonal changes in the s t ruc ture and u l t r a s t rue tu re of the cell t ypes in G. aculeatus, form trachurus, and s tudied (Leather land, 1970c) the effect of p i t u i t a r y t r ansp l an t a t i on on the s t ruc ture of the adenohypophys ia l cells. Lea the r l and and L a m (1971) have inves t iga ted the effect of ACTH and cortisol on the adcnohypophys i s and interrenal g land of this animal .

* This work formed part of a thesis submitted for the degree of Doctor of Philosophy in 1973 and for which the author was in receipt of an S.R.C. studentship. I should like to thank Dr. R. J. Wootton and Dr. J. Savidge of the University College of Wales, Aberystwyth for their help with the computer programming and Dr. M. P. Ireland for his support and supervision throughout the project.

70 M. Benjamin

I n recent years methods have become available which permit efficient and reliable measurement of s tructures by simple count ing or measuring procedures applied to electron micrographs of sectioned tissue (Weibel, 1969). Loud et al. (1965) have used quant i ta t ive methods to compare the effect of different f ixat ion and embedding media on the u i t ras t ructure of rat liver cells, while Hol lman (1968) has compared morphometr ic data on m a m m a r y cancers in the mouse with data from normal lactat ing tissue. I n the field of endocrinology, Weatherhead and W h u r (1972) have used quan t i t a t ive methods to analyze the t ime sequence of the s t ructural changes in Xenopus laevis "MSH" cells when the animals were placed on black and white backgrounds. The a t t empts to ident ify and describe the ul tra- s t ructure of the cell types in the adenohypophysis of fish have been unsatisfactory. The difficulties are firstly tha t so few parameters define the cell type and not the physiological condition, and secondly tha t the pseudoquant i ta t ive descriptions still cus tomary among m a n y morphologists have not been replaced in the field of p i tu i t a ry cytology by true morphometr ic data which can be stat ist ically tested. I n an a t t empt to improve mat ters the normal s i tuat ion was reversed and the cell types were defined by their physiological condition, and fur thermore a quan t i t a t ive as well as a qual i ta t ive analyt ical approach was adopted.

Materials and Methods

Collection o/Fish. Sticklebacks ~ 45 mm in length were collected in late November and early December by hand netting in the river Rheidol, Cardiganshire, about 1 mile from its mouth at Aberystwyth harbour. As these fish had a modal number of 4 lateral plates and could be collected from the same habitat at all times of the year, they were considered to be non- migratory and to belong to group A of Heuts (1947).

Light Microscopy. The brains with pituitaries attached were dissected out and fixed in Bouin's fluid. 8 ~m paraffin wax sections were stained with Alcian blue-PAS-orange G. For each cell type, 20 cells from each of 5 animals were photographed at • 400 magnification and the negatives printed at • 1000 final magnification. The outlines of the cells were then accurately cut out and weighed. The average area of each cell type was then calculated from the area of a known weight of the same photographic paper. The areas of the cells were as- sumed to be directly proportional to their relative volumes.

Electron Microscopy. Pituitaries were fixed for 2 h in 3% glutaraldehyde in 0.05 M eaco- dylate buffer at pH 7.4, washed overnight in cacodylate buffer and postfixed in 1.33 % osmium tetroxide in cacodylate buffer for 3 h and stained in 3 % aqueous uranyl acetate for 1 h. The material was then dehydrated in graded alcohols, treated with propylene oxide and embedded in TAAB resin. Ultrathin sections were cut on an LKB ultrotome, double stained with lead citrate (Reynolds, 1963) and uranyl acetate and examined on an AEI EM6B electron microscope.

Cell types were identified with their LM counterparts by several methods. Adjacent thick (1.0 ~tm) and thin (silver interference colour) sections were compared, particular regions only of the pituitary were fixed, e.g., rostral pars distalis, or they were identified by their topographical relationships to one another, e.g., ACT]-I cells formed a dorsal layer of cells above the prolaetin cells in the rostral pars distalis.

For each cell type, pituitary glands from 3-6 animals were sectioned at random, and 30 random electron mierographs were taken at • 8100 magnification. The final magnification of the prints was • 16200. The relative area of the micrographs occupied by the various organelles shown in Figs. 1 and 2 was determined by the point-counting method of Weibel (1969). The surface-area: volume ratios of the nuclei were determined by a combination of point-counting vohimetry and surface estimations by intersection counts (Weibel, 1969). The test system consisted of two super-imposed quadratic lattices of lines, the cross points of

Pituitary Gland of the Freshwater Stickleback 71

which served as markers for point-counting volumetry, and the lines for intersection counting. The relative area occupied by a particular organelle in each of the 30 micrographs of a given cell type was first estimated by means of the thick line test system where the distance between the lines was 15 mm. Organelles which occupied < 1% of the total cell volume were re- estimated using the thin line test system, where the distance between the lines was 7.5 mm.

A centimetre scale photographically reduced to give 0.5 mm divisions was used with a x 5 magnifier to measure the diameters of the secretory granules on printed micrographs at

the final magnification of X 16200. For this purpose the microscope was accurately calibrated using line gratings (2160 lines/ram). Measurements were made along the longest axis of the granule to the outside of the membrane, or to the edge of the granule if no membrane was visible. 10 recognizable granules randomly chosen from each micrograph were measured, giving a total of 300 measurements per cell type.

Statistics. The mean value for the intersection counts represents the percentage of the total cell volume occupied by a given organelle, and is quoted • standard error. Whenever the intersection counts were not normally distributed, an arc-sine angular transformation of these values was necessary before significant differences between cell types could be assessed. A one-way Analysis of Variance and Duncan's (1955) New Multiple Range Test were used to assess the significance of differences between cell types. I t is important to remember that the percentage volume estimates and their standard errors cannot be interpreted in terms of suitable confidence limits, as the latter can only be given for particular types of distribution. A series of algol computer programmes was used for all the above stages.

In order to compare the absolute amounts of organelles in the cell types rather than the percentage volume figures, the latter were multiplied by a series of correction factors which were based on the sizes of the cells as determined at LM level. Since these correction factors also had standard errors, a new standard error for the corrected figure was calculated according to the method of Jarman (1970). A one-way Analysis of Variance was not possible in this case, and thus only large scale differences were considered important.

Reconstruction Drawings. In the morphometric study it was attempted to visualize the quantitative data by a series of reconstruction drawings. These were based on the percentage volume figures, the frequency histograms of the secretory granule sizes and the surface area: volume ratios of the nuclei. Pencil lines were cross-hatched 1 cm apart as temporary guides on large sheets of good quality board. The cell shape was drawn arbitrarily, but the cell outline was drawn so as to include 800 intersections. For every 1% volume density of a given organelle, 8 intersections were allocated. The secretory granules were drawn to scale and the shape of the nucleus was based on its surface area: volume ratio. Differences in the electron density of the secretory granules were not shown, and the number of ribosomes drawn on the outer membrane of the R E R was completely arbitrary.

Results T h e r e is a d i f fe rence b e t w e e n desc r ib ing cell t y p e s and i d e n t i f y i n g t h e m .

P e r h a p s t h e cell t y p e s can best be iden t i f i ed w h e n t h e y are cons ide red jo in t ly .

Description o/ the Cell Types T h e resul t s of t he m o r p h o m e t r i c ana lys i s a re s u m m a r i s e d in Tab les 1-6 a n d

t h e f r e q u e n c y d i s t r i bu t i ons of t he s ec re to ry g ranu le sizes are shown in Figs . 3-10.

Figs . 11-17 are s e m i - d i a g r a m m a t i c r e cons t ruc t i ons of t h e cell t y p e s based on b o t h

of t h e a b o v e sets of i n f o r m a t i o n a n d on t h e sur face area: v o l u m e ra t ios of t he

nuclei . R o s t r a l P a r s dis ta l is

Prolactin Cells. P r o l a c t i n cells c o n t a i n e d n u m e r o u s e l ec t ron dense s ec re to ry g ranu le s t h a t were d i s t r i b u t e d t h r o u g h o u t t he c y t o p l a s m , e x c e p t in t h e Golgi

r eg ion a n d in t he p o r t i o n of t he cell wh ich c o n t a i n e d R E R . T h e nuc leus was s l igh t ly

e l o n g a t e d a n d of i r r egu la r ou t l ine (surface a rea : v o l u m e ra t io ~ 0.50 • 0.12).

cell

typ

e

cyto

tlie

sm

Gol

gi

reg

ion

--

nu

cle

us

(in

r n

ucl

ea

r m

em

bra

ne

) -

hu

e

L nu

cieO

lus

- n

--g

rou

nd

su

bst

an

ce

- gs

__

m

ito

cho

nd

ria

-

mit

--

RE

R

incl

udes

ri

b~.c

.mes

m

em

bra

ne

s &

cis

tern

ae

free

ri

bo

som

es

- r

ela

tu ~re �9

ra

und

secr

etor

y g

ran

ule

s -

sg 1

ma

ture

p

ea

r-sh

ap

ed

or

ob

long

se

cret

ory

gran

ules

sg

2

tra

nsl

uce

nt

vesi

cles

-

tv

sma

ll

Go

lgi

vesi

cle

s -

s gl

aca

.lh

oso

me

s -

a

imm

atu

re

secr

eto

ry

gra

nu

los

- sg

3

larg

e

dil

ate

d

Gol

gi

vacu

ole

s-

I gl

fla

tte

ne

d

Gol

gi

cisl

ern

ae

--

f gl

mu

lllv

esi

ctd

ar

bo

die

s-

mvb

dens

e h

od

ies-

db

)ara

llel

arr

ays

a

rou

nd

th

e

nu

cle

us

- re

r 1

;mal

l is

olat

ed

piec

es

wit

ho

ut

dila

ted

ca

viti

es

- re

r 2

dila

ted

piec

es

wit

ho

ut

de

nse

co

nte

nts

-

rer

3

dila

ted

pie

ces

wit

h

de

nse

co

nte

nts

- re

r 4

pa

rall

el

arr

ays

in

th

e

ma

in

cell

bo

dy-

re

r 5

pa

rall

el

arr

ays

n

ext

to

th

e

cell

m

em

bra

ne

-re

r6

curv

ilin

ea

r w

ho

rls

- re

r 7

sO 1

�9

C)-

-tv

re

r 7

~.

s o

--g

o

o

sg 3

--~

p

f g

l-

-~

(~ --

mvb

~ --

db

Fig

s. 1

and

2.

Cla

sses

of

orga

nell

es s

elec

ted

for

anal

ysis

in

the

pitu

itar

y ce

ll ty

pes

of t

he f

resh

wat

er s

tick

leba

ck

t gs

Table I.

Percentage

of the total cell volume occupied

by various

organelles

in the Golgi region

(means ~

s.e.)

Organelle

Nucleus

Nucleolus

Golgi

(a)

small vesicles

(b)

large,

dilated

vacuoles

(c)

flattened

cisternae

(d)

total

Multivesicular bodies

Dense bodies

Immature

secretory

granules

Acanthosomes

Prolactin

25.7 ~i.15

0.2

/0.i0

5.0 ~0.60

2.3,

2-0.

35

o.o

*

a.o

o

v.3

ma.

8o

0.2

-*o

. os

0.4

/0.1

0

O.l Zo.o2

0.i2/0.i2

ACTH

25.2 -+

1.45

0.2

20.10

1.5

So.

35

1.4

/0.2

5

0.2

/-9

.05

3.1

2-0

.45

0.5

:-o. io

0.4 /0.10

o.o~

/0.o

o. ~

_o_. o_~_._oo_

STH

23.9 ~1.75

_o=8

_~_.

Zo ......

2.6

-+0.

4o

i.i

2..0

.35

O.i

&o.

o5

3.8

/0.6

5

o.i

/0.0

5

0.2

/0.0

5

o.o

4~

0.0

i

_o~o

_/0_

.~o

GTH

22.2 ~2.05

o.i

zo.0

5

2.4

"tO

. 35

0.5

-+0

.15

0.I ~0.05

3.0

zo.4

5

o.1

Zo.o

s

o.4

~.l

O

o.o

Zo

.oo

o.ov

~o.o

3

TSH

26.9 --+Z. 25

o.4

/0.1

5

3.1

/0

.60

z.o

-+

0.45

_o~o_~_.~o .

..

..

.

4.1

-+o.

85

0.2

/0.05

_0.2__/'(3__.__05

0.0/0.00

o.o

Zo

.oo

Cell types where mean values did not differ significantly

(P (0.05%)

are underlined.

are joined by dotted lines.

'fab

le 2.

Percentage

of the total cell volume occ%

PI 1

PI

2

24.2

+1.60

23.1 -+

1.45

0.4/

0.1o

o.

4 .t

4.1o

3.7 /0.50

3.6 ~0.45

0.9 /0.25

0.6 ~0.15

08/0

.25

0.2~.o5

S.l

/0.7

5 4.

3 /0

.55

0.2

/0.i

o 0.

5 /0

.1o

O_.l_~_.lo

o.3

~O

.lO

o.0

/0.0

0 o.

o ~o

.o0

0.o

/0.0

0 o.

o ~0

.0o

Non-adjacent

groups

Isolated pieces without

dilated cavities

~ied

by the various

forms of RER

(means -+ s.e.)

Organelle

Prolachin

I ACTH

PI 1

PI 2

_5~o

_-+o

_.15

4.7/O.4o

o.6

/O.2

o

7.9

zo

.5o

0.5 ~.15

Isolated

pieces with

dilated cavities

7.5

zo.8

o

STH

GTH

TSH

7.7

2..0

.60

1.8

-t

0.55

8

.5 /

-0.8

5

Perinuclear

arrays

6.8 +1.4

4.5

~.

9o

_o_.

o_~_

._oo

4.3 ~.!%

_3._

i_~.

!5 - _3

_. 6_~_

.!o -

_o_.

4_~_.!s

5.4

+i.3

0._0 ~

0.O0_

0.0 -+

0.00

o.o

Zo.o

o o.

o /0

.0o

o.o

~o.o

o

0.o

/0.0

0 o.

o 24

.00

3o.7

-+2

.39

o.o

/0

.00

o

.o

/0.0

0

o.o

to

.oo

18.0

+1

.25

!1

.4_2

-0_.

70

37.2

--+

2.15

1.i

io

.3o

o.8

/O

.35

6.7

/0

.95

o.~

-*

o.2o

8.3 ~I.05

Parallel

arrays in the

main cell body region

Curvilinear

whorls

0.0 /O.00

3.7 ~1.45

0.0 /O.00

2.2 ~0.65

Parallel

arrays next to

0.0~0.00

0.0 ~0.00

5.5 ~1.50

2.4 ~0.60

the cell membrane

Large,

dilated pieces

0.0

~.00

0.0

-+

Q.O

0 0.0 ~0.00

0.0 ~0.00

with dense contents

0.0

Zo

.oo

2.

5 •

0.0

/0

.00

0

.0

-+0.

00

17.7 ~1.30

Total

21.8 +2.15

16.0

/O.

8O

17.6 ~i.o5

q o m-

Cell types where mean values

did not differ significantly

(P < 0

.05%)

are underlined.

Non-adjacent

groups


Table 3.

Percentage

of the total cell volume~

occupied

by the remaining

cell organelles

(means +- s.e.)

Tab

le 4

. R

elat

ive

Organelle

Mature round secret~ry

granules

Mature oblong

secretory

granules

Translucent

vesicles

M[tochondria

Free cibosomes

Cytoplasmic

ground

substance

Prolactin

16

.4 ~

o.9o

o.o ~o.oo

GTH

o.o ~o.oo

ACTH

STH

ii.0 ~0.95

25.5 +-

1.2

i0.i ~1.4

0.0 ~0.00

0.0 -+0.00

0.0 Zo.oo

0.0 ao.oo

5.4

+--0

.45!

2.

9 :-

o.35

....

. t

=--

:=-:

=-

.....

16.9 ~O.S0"

14.6 +--l.l%

25.3 -+

l.1

- 22.9 -+

1.4

TSH

5.0 -+

1.15

o.o ~o.oo

PI 1

16.3 -+

1.50

O. 93~0.31

PI 2

0.3

Zo

.to

o.o ~o.oo

0.0

+-

0.00

0

.0

+-0.

00

0.0

+-

0.00

0

.0

+-0

.00

13.4

~*

1.64

- 4

.2--

+0

.45

2

.3

~0

.40

5

.4

+-0~

3

.7

+-0

.50

s,a

~0.s

s 15

.v z~

.~0

18.5 +-

l.1

,16.

6 +I.i

20.4 --+1.30

21.0 +-

1.35

15.5 +--1.3

~2~7 +-

l.~0

s.8

~o.6

s

25.9

z~.9o

Cell types where mean values did not differ significantly

(P < 0

.05%)

are underlined.

Non-adjacent

groups


volu

mes

of

cell

typ

es o

ccup

ied

by

vari

ous

orga

nelI

es

in t

he G

olgi

reg

ion-

-cor

rect

ed t

o ac

coun

t fo

r di

ffer

ence

s in

cel

l si

ze (

ratio

s no

t ab

solu

te v

alue

s).

Mea

ns 5

= s.

e.

.=.

~a

Org

anel

le

Pro

lact

in

AC

TH

S

TH

G

TH

T

SH

P

I 1

PI

2

Nuc

leus

11

.1

~:1.

17

5.5

5=0.

65

18.1

~:

2.40

17

.3

5=2.

4 14

.9

5=1.

74

14.8

~:

1.26

16

.9

~:1.

78

Nuc

leol

us

0.14

•

0,06

i0

,02

0,

78 •

0,

11 5

=0,0

6 0,

25 •

0,

29 5

=0,1

1 0,

42 •

G

olgi

(~,)s

mM

1 ves

icle

s 2.

8 5=

0.42

0.

42j:

0,10

2.

5 5=

0,48

2.

4 :h

0,42

2.

0 5=

0.41

3.

0 ~:

0.44

3.

6 J:

0.50

(b)

larg

e, d

ilat

ed

vacu

oles

1.

3 5=

0.22

0.

405=

0.08

1.

1 5=

0.34

0.

505=

0.15

0.

655=

0.28

0.

735=

0.19

0.

615=

0.16

(c)

flat

tene

d ci

ster

nae

0.00

~: 0

.00

0.05

~: 0

.02

0.06

•

0.11

~: 0

.03

0.00

t:0

.00

0.63

~: 0

.20

0.15

~0.

04

(d)

tota

l 4,

2 5=

0,54

0,

875=

0,14

3,

7 5=

0,71

3,

0 5=

0,51

2,

6 !0

.57

4.4

5=0.

62

4.3

:[:0

.61

Mul

tive

sicu

lar

bodi

es

0,10

10.0

4 0,

13i0

.01

0,08

5=0

.03

0.14

5=0.

06

0,11

5=t=

0.03

0.

135=

0.06

0,

50:i

0.12

D

ense

bod

ies

0.22

5=0.

04

0.12

• 0.

175=

0.07

0.

385=

0.10

0.

11 i

0,0

5

0,11

5=0.

08

0.32

5=0.

08

Imm

atur

e se

cret

ory

gran

ules

0.

06 5

=0.0

2 0.

00 5

=0.0

0 0.

04 •

0.

00 5

=0.0

0 0.

00 5

=0.0

0 0.

00 5

=0.0

0 0.

00 ~

0.00

A

cant

hoso

mes

0.

00 ~

0.00

0.

00 5

=0.0

0 0.

00 5

=0.0

0 0.

00 5

=0.0

0 0.

00i0

.00

0.00

•

0.00

~0.

00

m

Tab

le 5

. R

elat

ive

volu

me

of c

ell t

ypes

occ

upie

d by

var

ious

form

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76 M. Benjamin

The Golgi apparatus was very prominent and consisted of small vesicles and large, dilated vacuoles, but no flattened, plate-like cisternae. Images of condensing secretory granules were seen in the Golgi apparatus and the large, dilated Golgi vacuoles bore thick-walled vesicles. Acanthosomes and microtubules also featured in the Golgi region.

RER was mainly organized as parallel arrays in the perinuclear region of the cell, otherwise it was present as isolated tubules. The numerous images of formation and release of secretory granules, the total amounts of mitochondria, RER, and Golgi apparatus, all suggested a turnover of secretory products and hence an active prolactin cell.

Occasionally, there were desmosomes between prolactin cells, hut not between prolactin cells and chromophobes. Deeply invaginated cilia were also found in prolactin cells. At the base of these cilia the plasma-membrane was swollen into a sac-like structure. The cilia usually projected into an adjacent chromophobe and had a 9 ~- 0 arrangement of microtubules. Each cilium was associated with a basal body and a centriole.

A C T H Cells. The ACTH cells contained small, electron-dense granules with a space between the central dense core and the limiting membrane of the granule that was slightly wider than in other cell types. In those cells which bordered the neurohypophysis, there was often an accumulation of secretory granules at the neurohypophysial pole of the cell. In other cells the secretory granules were evenly distributed.

The Golgi region was small and consisted of small vesicles, large dilated vacuoles, and flattened cisternae. Immature secretory granules were rare in the Golgi apparatus. Dense bodies - usually round in shape and of various electron densities - and multivesicular bodies, were characteristic features of the Golgi zone of this cell type. The former were surrounded by a single, thick membrane imme- diately below which was a less dense area.

More of the RER of the ACTH cells was arranged as curvilinear whorls than as parallel arrays in the perinuclear region. There were also a considerable number of scattered tubules of RER that had dilated cisternae. Mitochondria were abundant and pleomorphic. They were sometimes bifurcated and occasionally contained 1 or 2 dense intracisternal granules. The nucleus had an irregular outline and a surface area: volume ratio of 0.53 ~ 0.09. At many points the RER and the nuclear membrane were continuous.

At the junction of the neurohypophysis and the RPD, a basement membrane bordered the ACTH cell region. This presumably corresponded to the PAS- positive basement membrane seen in this region in LM preparations. In suitable sections the interface appeared as a double, electron dense, amorphous membrane enclosing an electron translucent space. The total width was 100-150 nm. This basement membrane was in direct continuity with that of the blood vessels at the periphery of the pituitary gland. I t was also continuous with the intercellular spaces of the ACTH ceils.

Proximal Pars distalis S T H Cells. The STH cells contained densely packed secretory granules and

a round nucleus that had a surface area: volume ratio of 0.68 • 0.12. The nucleolus of the STH cell was more prominent than in any other cell type.

Pituitary Gland of the Freshwater Stickleback

ACTH 4o f . PROLACTIN

77

2(3

5O 150 250 35O 3 4

,01 STH 4o GTH

20

50 150 250 350 50 150 250 350 5 6

40 f TSH 401 PI1

20 2e

50 150 250 350 50 150 250 350 7 8

Figs. 3--8. Percentage frequency distribution of the profiles of secretory granule diameters in the ACTH, Prolactin, STH, GTH, TSH and PI 1 cells. Ordinate, percentage frequency;

abscissa, diameter (nm)

78 M. Benjamin

40 PI 2 ( a ) 4(] PI 2

20 20

50 1150 " 250" 35~0 50 150 250 350 9 10

Figs. 9 and 10. Percentage frequency distribution of the profiles of secretory granule (PI 2 (a)) and translucent vesicle (PI 2 (b)) diameter of the second type of pars intermedia cell. Ordinate,

percentage frequency; Abscissa, diameter (nm)

There was little RER and most of it was arranged as small isolated tubules, although there were some parallel arrays in the perinuclear region. The Golgi apparatus was small and showed few images of newly-formed secretory granules. There were only a few mitochondria, and in general the STH cell of the winter stickleback was inactive.

GTH Cells. The secretory granules of the GTH cells were of moderate and variable electron density. There were few deep indentations in the surface of the GTH cell nucleus {surface area: volume ratio = 0.66 ~ 0.12). The Golgi apparatus was poorly developed and mostly consisted of small vesicles, with no signs of newly- formed secretory granules. The most conspicuous feature of the GTH cells was the extensive RER. Most of the RER had large, dilated cavities filled with slightly electron dense, flocculent material (30.7 • 2.39 per cent of the cell volume). Although there was an irregular scattering of ribosomes on the outer membranes of the RER, gaps free of ribosomes were often found. Sometimes the large cavities of the RER were connected together and they were often close to the cell membrane. Fusions with the latter were not observed. The gap between the inner and outer nuclear membranes contained dense material--a reminder of the continuity of the outer nuclear membrane and the RER. There was little perinuclear RER and there were few ribosomes or mitochondria, although there were one or two dense granules in the mitochondrial matrix.

T S H Cells. The TSH cells contained relatively few secretory granules compared with other cell types. As in the ACTH cells, the membrane around the granules was not closely applied to the central core. The nucleus was relatively large and had a surface area: volume ratio of 0.58 • 0.09. There was also a fairly prominent nucleolus. Although there was plenty of RER, most of it was dispersed as small tubules or dilated cisternae. Some of the RER was arranged as curvilinear whorls or as parallel arrays in the main body of the cytoplasm. There was a relatively small amount of perinuclear RER. Mitoehondria were abundant and occasionally contained l or 2 dense granules.

\


Fig. 11, Reconstruction drawing of a prolactin cell from an adult stickleback collected in the winter

Pars intermedia

P I 1 cells. The P I 1 cells contained electron dense secretory granules of slightly variable shape. While the major i ty were round in section profile, a few were oblong or pear-shaped. There was a well developed Golgi apparatus~ a significant propor- t ion of which (~ppr~x. 15% ~ co~isted of f l a t t ~ a d p~a~es. Some%imes the secretory granules fused with each other in the Go]gi region, but immature secretory granules were still present. R E R was conspicuous as perinuclear arrays or as parallel arrays next to the cell membrane. The surface area: volume ratio of the nucleus was 0.56 • 0.06.

80 M. Benjamin

Fig. 12. Reconstruction drawing of an ACTH cell from an adult stickleback collected in the winter

P I 2 Cells. The remarkab le features of the P I 2 cells were the a b u n d a n t , m e m b r a n e - b o u n d vesicles, which occupied 13.4 ! 1.64 per cent of the cell volume. These vesicles were ei ther empty , i .e. , e lectron t rans lucent , or conta ined s l ight ly f locculent mater ia l , the electron dens i ty of which was s imilar to t h a t of the ground substance. The membrane a round the vesicles was often incomplete . The nucleus was re la t ive ly small and of i r regular shape (surface area: volume ra t io = 0.52 ! 0 - 0 9 ) .

R E R was well organized, e i ther as para l le l a r rays in the per inuelear region, as curvihnear whorls, or as para l le l a r rays nex t to the cell membrane . Occasional ly there were in t rac i s te rna l granules in the R E R . More t han 80 % of the Golgi appa- ra tus consisted of small vesicles whereas only abou t 4% consisted of f l a t t ened


�9 .: O � 9

/ . o o . . o O �9 o++. p ~

"+IA" ~ ": :!|

~' *-~.+o"2+�9 - e - . . ' . . . �9 | . . | U . W

�9 .,. �9 D e * : " �9

; . . . m -. . . . . .

;,',,) /

. i g O r % W . U ' : U w w �9 �9 �9 .,+ �9 O ~ O t

~A. "- "" o~ ~ .~ ~. eeOo. o" . |174 . ~ o O o

Fig. 13. Reconstruction drawing of an STH cell from an adult stickleback collected in the winter

cisternae. I t is strange tha t a l though electron dense secretory granules were rare, there were still immature secretory granules in the Golgi region. Unfor tunate ly it is not known how the translucent vesicles are formed. On some of these vesicles there was a bristle coating. Mitochondria were abundant and frequently contained small, dense granules.

Identi/ication o/the Cell Types There are 2 groups of characteristics t ha t should be used to identify cell types. 1. Qualitative differences between the cell types - -e , g., the presence or absence

of a part icular class of organelle.

6 Cell Tiss. Res . 152

82 M. Benjamin

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

Fig. 14. Reconstruction drawing of a GTH cell from an adult stickleback collected in the winter

2. Quaatitative differeacez between the cell types--(a) the relative prop~rti(ms of a particular organelle to the other organelles in the same cell type, i.e., percentage volumes. (b) the absolute amounts of a given organelle per cell.

The relative size of the cells was important for the comparison indicated in 2 (b). Here, each volume density measurement was multiplied by twice the relative ,size of the cell from which thar orga~elle came. The r~tio oI She values is the ratio of their abselute volumes. It is important to account for ce[[ size differences when identifying ACTH and TSH cells. Whereas there was no adequate distinction between the relative volumes of the cells occupied by the Golgi apparatus, there was a difference in the absolute volumes. There were more mitochondria in the


) Fig. 15. Reconstruction drawing of a TSH cell from an adult stickleback collected in the

winter

T S H cells and more R E R t h a t had d i la ted cavi t ies t han in the ACTH cells. There was no d is t inc t ion in the re la t ive amount s of these organelles in the 2 cell types . Similar ly , the d is t inc t ion between the re la t ive volume of the Golgi a p p a r a t u s in the pro lac t in and S T H cells was no t a p p a r e n t in absolute terms.

There is no r~eed for a q u a n t i t a t i v e analys is ~,o see t h a t t~e P I 2 ce~is are the cells t h a t conta ined the grea tes t numbers of mi tochondr ia and the largest amoun t of perh~uclear R E R . However , if the re la t ive and absolu te volumes of these organ- ellcs are compared , i t can be seen t h a t i t was the absolute amoun t s of these organelles which gave this impression. Al though the re la t ive volumes of these

6 *

84 M. Benjamin

Fig. 16. Reconstruction drawing of a pars intermedia type 1 cell from an adult stickleback collected in the winter

organelles in the P I 2 cells were also large, the prolactin cell had a similar relative volume of perinuclear R E R and the TSH and ACTH cells had similar relative volumes of mitochondria. When cell sizes were accounted for, there were differences in the absolute volumes of the cell types occupied by m a n y other organelles, e.g., secretory granules and isolated small tubules of R E R without dilated cavities. The distinctions so created were not part icularly useful for identifying cell types. Perhaps this is because these organelles were not localized in certain parts of the cell, bu t were generally distributed.

I t must be remembered tha t cell types are also characterised by the relative proportions of one organelle to another, e.g., total Golgi : to ta l R E R . Cells may

1)ituitary Gland of the Freshwater Stickleback 85

Fig. 17, ]~econstruction thawing of a pars intermedia type Z cell from an adult *tickleback collected in the winter

also differ in the degree to which their cytoplasm is occupied by orga.elles. Thus the percentage vQiume ~ccu~ie4 by cyt~(~s~c~c grvund ~uh~tunce is ~lso imt~or- tant, a~ it indicates the degree of diilcrentiation of the cell.

Discussion

One of the most important principles in cell bigtogy is that the majority of d i f [ e ren t~ed cel~s r 2.[[ the di.fferent ~yp~ v~ <~rg~e~les. Diflervnces %eLw~en specialized cells result ir~m differences in the balance of the organelles that they contain; in other words from quantitat ive rather than quahtative variations in their composition (Weibel, 1972). I t is thus unfortunate that so few authors have adopted a quantitative approach to electron microscopy, particularly as methods

86 M. Benjamin

have been available for several years (Loud et al., 1965; Weibel, 1969). Hope (1970) considered that a quantitative approach was essential to his study of rat liver cells during pregnancy and lactation, as the ultrastructural changes in such normal physiological conditions would be minimal. Nussdorfer (1970a) has evaluated the ultrastructural changes in the adrenocortical cells of prednisolonc-treated rats by morphometric methods. Previous experiments (Nussdorfer, 1969, 1970b) showed a quantitative approach was necessary because the changes in adrenocortical cells under the experimental conditions of stimulation and inhibition were prevalently quantitative. An important advantage of the quantitative approach is that it reveals the topographical relationship between components of ceils in its integrity.

The quantification of data using standard techniques is essential before it will be possible to establish the exact degree of morphological equivalence at the ultrastructural level between pituitary cell types that are functionally equivalent in different species. Purves (1966) has pointed out that such a morphological equivalence at LM level can only be expected between closely related species. Although the degree of subcellular organisation may not in all cases reflect the exact functional performance, a quantitative analysis undoubtedly provides a better indi- cation of the functional state of an adenohypophysial cell at a particular time of year. Hollman (1968) advocated the use of high resolution autoradiography or cytochemistry to narrow the gap that still exists in our knowledge of structure and function. Indeed Nussdorfer et al. (1971) have combined autoradiography and ultrastructural morphometry to study the effect of ACTH on rat adrenocortical cells. Cook and Overbeeke (1969) have pointed out the need to correlate information from ultrastructural studies with data concerning the actual concentration of hormones in the pituitary gland. The morphometric approach would be ad- mirably suited to this need.

According to Weibel (1969) a rigorous, random sampling procedure is necessary during all stages of a morphometric analysis from the choice of material to the recording and analysis of electron micrographs. In order to select cells for analysis, he used systematic random sampling of cells rather than simple random sampling. Systematic random sampling was not practical in the present investigation because of the heterogeneity of cell types in the adenohypophysis. As no correlation was attempted between biochemical and ultrastructural data, a simple random sampling procedure should not impair the value of the results. Practical considera- tions have also led Mayhew and Williams (1971) to compromise with sampling procedures.

Granule sizes should not be used to identify adenohypophysial cell types to the exclusion of all other morphological parameters. Although there are significant differences in mean granule size in different cell types, the variability within any one cell type invalidates the use of mean granule size as the sole criterion for identification (Pooley, 1971). Nakane (1970) also considered that other features should bu used to distinguish cell types because of the considerable overlap in the sizes of secretory granules, and the variation that can exist in mean granule size within the same cell in different physiological states and according to different methods of fixation, staining, etc. Doerr-Schott (1962) pointed out that the diameter of the secretory granules in the pituitaries of lower vertebrates varies during the annual sex cycle.


In most works involving the description of cell types, organelles are often described as "extensively developed" or "numerous". Although no statements could be found to this effect in the literature, the above phrases can only be interpreted as referring to the relative volume or numbers of a particular organelle. An impor- tan t facet, of the identification problem is therefore overlooked, as no account is taken of the effect of cell size on the appearances of cells as seen in section. I t is quite possible for two cells to have the same absolute volume of a given organelle, but different relative volumes (i. e., if their cell volumes differ). Obviously both absolute and relative parameters are important, especially when that organelle is packaged within one particular region of the ceil, e.g., perinuclear RER, curvilinear whorls of RER, Golgi apparatus, etc.

The lack of similar quanti tat ive studies on other fish pituitary glands, makes it difficult to compare in detail the ultrastructure of corresponding cell types in the freshwater stickleback and other teleosts. In addition there is some variation in the number of cell types in the adenohypophysis of fish. Only seven ceil types were recognizable at EM level in the freshwater stickleback, unlike Zoarces viviparus (0ztan, 1966) and Carassius auratus (Leatherland, 1972) where there are eight cell types. Two types of GTH cells in these species account for the additional cell type. In Anguilla anguilla and Conger conger, Knowles and Vollrath (1966) have also described two types of GTH cells, and in the latter fish it is also possible there are three cell types in the PI.

The prolactin cells of the freshwater stickleback closely resemble those of other teleosts, particularly in the size of the secretory granules and the form of the RER. In Lebistes reticulatus, Xiphophorus helleri, and Mollienisia sphenops (FollSuius and Porte, 1960), Perca /luviatilis (Foll~nius and Porte, 1961), Tilapia mossambica (Dharmamba and Nishioka, 1968), Oncorhynchus nerka (Cook and Overbeeke, 1969) and Mugil cephalus (Abraham, 1971), perinuclear R E R is prominent as in the stickleback. In Oncorhynchus nerka, nebenkern whorls were also present. However the prolactin cells of Zoarces viviparus contained R E R in a diffuse form as irregular or rounded sacs and lamellae ((}ztan, 1966).

A characteristic feature of the prolactin cells of the freshwater stickleback was the presence of desmosomes between adjacent cells. Cook and Oberbeeke (1969) also described desmosomes between prolactin cells in the sockeye salmon, while Nagahama and Yamamoto (1969) noticed them in the kokanee. In these teleosts the prolactin cells are arranged in follicles and the desmosomes are particularly common near the follicle lumen.

According to Lain and Hoar (1967) and Lam and Leatherland (1969), the pituitary gland of the migratory stickleback, Gasterosteus aculeatus form trachurus, is "physiologically hypophysectomized" in the winter. This is particularly so with regard to prolactin secretion, and Leatherland (1970a) found that the prolactin cells were neither forming nor releasing secretory granules in the winter. In the freshwater stickleback it is evident that the prolactin cells actively synthesize and release secretory granules in the winter and thus the pituitary gland cannot be considered as "physiologically hypophysectomized". A full discussion of this finding will not be presented here, as it is intended as the subject of the following paper.

Unlike the ACTH cells of Perca ]luviatilis (Foll~nius and Porte, 1961) and Anguilla anguilla and Conger conger (Knowles and Vollrath, 1966), the correspond-

88 M. Benjamin

ing cell type in the freshwater stickleback was not characterized by vesicles, tubules or fibrils. As in Perca /luviatilis many of the cavities of the RER were dilated. The loose fitting membrane around the dense core of the secretory granules in the ACTH cell of the stickleback is also characteristic of many other teleosts, e.g., Tilapia mossambica (Dharmamba and Nishioka, 1968), Oncorhynchus nerka (Nagahama and Yamamoto, 1969; Cook and Overbeeke, 1972), and Mugil cephalus (Abraham, 1971).

The STH cells of the freshwater stickleback resembled those of Anguilla anguilla (Knowles and Vollrath, 1966) as the cells of both fish were packed with spherical, secretory granules and contained little RER or Golgi apparatus, and few mitochondria. They were not preferentially arranged around portions of neurohypophysial tissue as in the perch (Foll6nius and Porte, 1961) and consequently did not show the distinct polarization of secretory granules typical of this species. In his study of an unnamed form of G. aculeatus, Foil~nius (1968) considered the distinction between the prolactin cell and the STH cell to be slight. He could go no further than to distinguish these cells ultrastructurally on the basis of a slight difference in the size distribution profiles of the secretory granules. In the present work however, involving a quantitative approach to EM, numerous differences were apparent, e.g., the more rounded nucleus and conspicuous nucleolus of the STH cell, the relatively greater volume of the cell occupied by secretory granules in the STH cell, and the relatively greater volume of the Golgi apparatus in the prolactin cell. Thus it is easier to distinguish between these cell types than Foll~nius (1968) supposed.

The accumulation of material in the cavities of the RER seems characteristic of the GTH cells of several species of teleosts, including Perca/luviatilis (Fol]~nius and Porte, 1961), Carassius auratus (Leatherland, 1972), and Oncorhynchus nerlca (Cook and Overbeeke, 1972). In contrast to this extensive development of RER the Golgi apparatus was poorly developed. Farquhar (1971) also found a poorly developed Golgi apparatus in mammalian TSH cells where there was abundant RER. Echave Llanos and G5mez Durum (1971) reported that the STH cells of hepatectomized mice contained dilated RER filled with electron dense material. In these ceils the dilated cisternae were frequently in contact with the plasma- lemma, suggesting a direct release of the hormone by the RER, bypassing the Golgi complex. I t is possible that the Golgi apparatus in the GTH cells of the stickleback is not so important in the secretory processes as in other cell types, e.g., prolactin cells. Lavallard and Campiglia (1971) pointed out that the cells in the slime glands of Peripatus acacioi accumulate protein in their apical regions without the participation of the Golgi apparatus. On the basis of an autoradiographic study of fibroblasts, Ross and Benditt (1965) considered that glycoproteins arc discharged directly from the RER. Non-participation of the Golgi apparatus in the secretory process could facilitate the rapid and continuous synthesis of proteins. However, Doerr-Schott (1963) has found a conspicuous dilation of RER in the frog GTH cell after castration, where the Golgi apparatus was well developed.

Whatever the significance of the process, the accumulation of material in the cavities of the RER must result from an inability of the GTt t cell to transport protein from the RER quickly enough. I t would seem reasonable to suppose that a ready route does exist for the product to be transported away, especially as the


once favoured idea of a continuity between the cell membrane and the R E R has now been generally abandoned by electron microscopists (Birbeck and Mercer, 1961). Either a local feedback mechanism does not exist to prevent over-produc- tion, or it is prevented from functioning properly. Jamieson and Pa]ade (1968) have shown tha t the removal of the product from the cavity of the R E R is a step requiring energy. The morphometric analysis of pituitaries from adult, freshwater sticklebacks collected in the winter showed that there were fewer mitochondria in the GTH cells than in any other cell type. I t is possible that so much of the energy produced by the mitochondria is used to synthesize secretory proteins that little remains to remove the formed secretory product.

Two cell types in the pituitary of the stickleback had enough acanthosomes in their Golgi regions to be detectable by quantitative means. I t is interesting to note that Farquhar (1971) also found these organe]les were most characteristic of the mammotropes and FSH cells of mammalian pituitaries. Hopkins (1969) reported acanthosomes in the prolactin cells of Poecilia reticulata adapted to freshwater. Acanthosomes have previously been described as fuzzy vesicles, coated vesicles, or alveolate vesicles, by numerous workers, and they are widely distributed throughout the animal kingdom. Dumont (1969) and Nevalainen (1969) noticed acanthosomes in the Golgi regions of hamster peritoneal macrophages, and hen parathyroid glands respectively, and Teitelbaum et al. (1970) have described them at the basal and luminal surface of the parafollicular cells in the dog. Similarly in the present investigation the acanthosomcs were either found in the Golgi region or near the cell membrane at the sites of granule release (in prolactin ceils). Roth and Porter (1964) have reported coated vesicles in cells known to accumulate protein, and have inferred that they are specifically engaged in protein uptake. Obviously the acanthosomes in pituitary cells deserve greater attention than they have yet received, as these cells frequently synthesize hormones that consist part ly or wholly of protein.

One of the features that has emerged from quantifying the ultrastructural data, has been the relatively large amount of the cell volume occupied by mitochondria in the TSH cells of the freshwater stickleback. This is in direct contrast to the findings of Foll@nius (1968) who cited the mitochondrion as an inconspicuous organelle in the TSH cells of an unnamed form of G. aculeatus. In many respects the TSH cells of the stickleback resembled those of Anguilla anguilla and Conger conger (Knowles and Vollrath, 1966). The secretory granules were roughly the same size (100-200 nm) and both contained diffuse l~ER with wide cisternae. I t was possible to confuse the ACTH and TSH cells in the stickleback at the ultrastructural level. Foster (1971) has also recorded a similarity in the ultrastructure of these cell types in the rabbit adenohypophysis, and according to Kurosumi and Kobayashi (1966) the granule size in the ACTH and TSH cells of the rat is similar.

The PI of the migratory (Leatherland, 1970b) and the freshwater stickleback both contain two cell types which although vastly different from one another, are very similar in these two fish. However, the extensive nebenkern whorls of R E R characteristic of the PI 2 cells of the freshwater stickleback were not described by Leatherland (1970b) in the corresponding cell type of the migratory form. Neben- kern whorls have been described by Herman and Fitzgerald (1962) in the pancreatic acinar cells of the rat after ethionine treatment, by Morimoto et al. (1968) in the

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poster ior silk glands of Bombyx mori, and b y Nickerson and Curtis (1969) and Nickerson (1970, 1972) in the adrenocor t ica l cells of Meriones unguiculatus Staubl i et al. (1966), who observed similar concentr ic a r rays of R E R in in tes t ina l epi thel ia l cells of Aedes aegypti after the mosqui toes were fed a blood meal, suggested these a r rays served as a reserve for R E R t h a t synthes ized digest ive enzymes when s t imu- l a t ed b y feeding. The d i sappearance and subsequent r e -appearance of this form of P~ER af ter ACTH t r e a t m e n t led Nickerson (1970) to suggest t h a t the nebenkern whorls m a y be a readi ly avai lable source of bo th S E R and R E R . H e r m a n and F i t zge ra ld (1962) considered them as prol i fera t ion centres for R E R .

In t r a -mi tochondr i a l granules were found in the ACTH, T S H and P I 2 cells, a l though t hey were most a b u n d a n t in the P I 2 cells. I t has been suggested t h a t t hey accumula te d iva len t cat ions (Peachey, 1965; Schracr et al., 1973). I t is perhaps no tewor thy t h a t the ACTH, T S H and P I 2 cells are the cells t h a t conta ined the greates t re la t ive volume of mi tochondr ia . Observat ions on the numbers of in t r a -mi tochondr ia l granules in different s ta tes of cell a c t i v i t y m a y provide morphological clues t h a t could u l t ima t e ly lead to a be t t e r unde r s t and ing of thei r b iochemical significance in the secre tory process.

References

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Dr. Michael Benjamin Department of Cellular Biology and Histology St. Mary's Hospital Medical School Paddington, London W2 England

A morphometric study of the pituitary cell types in the freshwater stickleback, Gasterosteus...

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