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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, fresh- water stickleback, Gasterosteus aculeatus form leiurus, reveals differences between both the relative and absolute volumes of the various organelles in different cell types. The morpho- metric 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 be- tween 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 inter- renal 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 micro- scope.
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 topo- graphical 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 inter- section 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 percent- age 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
are joined by dotted lines.
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
are joined by dotted lines.
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
s of
RE
tG--
corr
ecte
d to
acc
ount
for
diff
eren
ces
in c
ell s
ize
(rat
ios
no
t ab
solu
te v
alue
s).
Mea
ns 3
_ s.
e.
Org
anel
le
Pro
laet
in
AC
TI-
[ 8
T[[
G
TH
T
SI-[
P
I 1
PI
2
Isol
ated
pie
ces
wit
hout
di
late
d ca
viti
es
4.5
• 1
.39
3=
0.1
9
7.5
• 1.
8 •
5.3
• 3.
7 •
4.1
•
Isol
ated
pie
ces
wit
h di
late
d ca
viti
es
0.29
• 1.
27:~
0.28
0.
00:E
0.00
4.
3 ={
=2.3
3 4.
3 •
0.64
~:0
.30
0.63
~:0
.23
Per
inuc
lear
arr
ays
4.3
~:0,
30
3.1
:b0,
59
3,5
• 0,
42•
0.70
:[:0
,19
5,3
~:0,
79
8,3
• C
urvi
line
ar w
horl
s 0,
00~
0.00
1.
5:1:
0.40
0,
00 J
:0,0
0 0,
00•
2,3
• 0,
00:]
:0.0
0 2.
2 •
Par
alle
l ar
rays
nex
t to
th
e ce
ll m
embr
ane
0.00
J:0,
00
0.00
:~0.
00
0.00
• 0.
O0•
0.
00~
0.00
4.
5 •
2.5
•
Lar
ge,
dila
ted
piec
es
wit
h de
nse
co~l
tent
s 0.
00•
0.00
J:0.
00
0.00
• 30
.7
~:3.
02
0.00
• 0.
00~
0.00
0.
00:L
0.00
Par
alle
l ar
rays
in
the
mai
n ce
ll bo
dy r
egio
n 0.
00 a
kO.O
0 0.
00 a
kO.O
0 0,
00 •
0.
00 •
1,
6 :k
0.26
0.
00 •
0,
00 :k
O.O
0
q K
Tot
al
9,1
3=0,
76
5.0
:d:0
,53
11,0
•
37,2
•
0.00
d:0.
00
10,5
•
14,1
•
Tab
le 6
. R
elat
ive
volu
me
of c
ell t
ypes
occ
upie
d by
the
rem
aini
ng or
gar~
elle
s--c
orre
cted
to
acc
ount
for
diff
eren
ces
in c
ell s
ize
(rat
ios n
ot a
bsol
ute v
alue
s).
~ ~"
Mea
ns -
4- s
.e.
Org
anel
le
Pro
lact
in
AC
TfI
[ S
T[[
G
T[-
[ T
SI-[
P
I [
PI
2
Mat
ure
roun
d se
cret
ory
gran
ules
9.
4 •
3.1
~0.
37
23.7
~:
2.58
10
.0
• 3.
1 ~:
0.78
13
.1
• 0.
34~
:0.0
8
Mat
ure
oblo
ng s
ecre
tory
gr
anul
es
0.00
~0.
00
0.00
• 0.
00 :L
0.00
0.
00 ~
0.00
0.
00J:
0.00
0.
74:L
0.25
0.
00 ~
0.00
T
rans
luce
nt v
esic
les
0,00
:L0,
00
0,00
&0.
00
0,00
•
0,00
~0,
00
0,00
•
0,00
:t:0
,00
13,4
d:
1.7
8 M
itoc
hond
ria
2,4
:i:0,
29
1.5
~0,
17
2.8
• 2,
3 :t:
0,44
3,
4 3=
0.45
3,
0 :i:
0,41
5,
7 i0
,72
F
ree
ribo
som
es
9,0
• 4.
7 •
14,1
•
8,4
• 9.
8 •
13,2
•
13.5
•
Cyt
opla
smic
gro
und
subs
tanc
e 10
.6 :
t:0.9
7 7.
09~:
0.77
22
.2
:L2.
46
21.0
~:
1.61
14
.3
:t:1.
06
16.3
:E
0.10
25
.9
~1
.64
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 forma- tion 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 mem- brane. 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 com- pared 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 rela- tively small amount of perinuclear RER. Mitoehondria were abundant and occa- sionally contained l or 2 dense granules.
\
Pituitary Gland of the Freshwater Stickleback 79
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
Pituitary Gland of the Freshwater Stickleback 81
�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., percent- age 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
Pituitary Gland of the Freshwater Stickleback 83
) 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 ultra- structural level between pituitary cell types that are functionally equivalent in different species. Purves (1966) has pointed out that such a morphological equi- valence 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 func- tional 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 informa- tion 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 identi- fication (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.
Pituitary Gland of the Freshwater Stickleback 87
In most works involving the description of cell types, organelles are often de- scribed 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, curvi- linear 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 vivi- parus (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 neuro- hypophysial 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 autoradio- graphic 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
Pituitary Gland of the Freshwater Stickleback 89
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 mito- chondria 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 ultra- structural 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
90 M. Benjamin
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
Abraham, M.: The ultrastructure of the cell types and of the neurosecretory innervation in the pituitary of Mugil cephalus L. from fresh water, the sea, and a hypersaline lagoon. Gen. comp. Endocr. 17, 334-350 (1971)
Birbeek, M.S.C., Mercer, E. H.: Cytology of cells which synthesize protein. Nature (Lond.) 189, 558-560 (1961)
Bock, F.: Die Hypophyse des Stichlings (Gasterosteus aculeatus L.) unter besonderer Beriick- sichtigung der jahrescyklischen VerEnderungen. Z. wiss. Zool. 131, 645 710 (1928)
Cook, It., Overbeeke, A. P. van: Ultrastrueturc of the eta cells in the pituitary gland of adult migratory sockeye salmon (Oncorhynchus nerka). Canad. J. Zool. 47, 937-941 (1969)
Cook, H., Overbeeke, A. P. van: Ultrastructure of the pituitary gland (pars distalis) in sockeye salmon (Oncorhynchus nerka) during gonad maturation. Cell Tiss. Res. 130, 338-350 (1972)
Dharmamba, M., Nishioka, R. S.: Response of "prolactin-secreting" cells of Tilapia mossam- bica to environmental salinity. Gen. comp. Endocr. 10, 409420 (1968)
])oerr-Schott, J.: Evolution des cellules gonadotropes fl au cours du cycle annuel chez la grenouille rousse Rana temporaria L. Etude au microscope 61ectronique; observations histochimiques et cytophysiologiques. Gen. comp. Endocr. 2, 541-550 (1962)
Doerr-Schott, J.: Etude au microscope 61ectronique des changements cytologiques des ccllules gonadotropes fl de l'hypophyse aprbs castration, chez Rana temporaria L. m~le. C. R. Soc. Biol. (Paris) 157, 664-666 (1963)
Dumont, A.: Ultrastructural study of the maturation of peritoneal macrophages in the hamster. J. Ultrastruct. Res. 29, 191-209 (1969)
Duncan, D. B.: Multiple range and multiple F tests. Biometrics 11, 142 (1955) Echave Llanos, J. M., GSmez Dumm, C. L.: Release of growth hormone from somatotropin
producing cells of hepatectomized mice without the participation of the Golgi complex. Experientia (Basel) 27, 318-319 (1971)
Farquhar, M. G.: Processing of secretory products by cells of the anterior pituitary gland. In: Subeellular organization and function in endocrine tissues. Mem. Soc. Endocr. 19, 79-122 (1971)
Foll6nius, E.: Analyse de la structure fine des diffbrents types de cellules hypophysaires des poissons t616ost6ens. Path. et Biol. 16, 619-632 (1968)
Foll6nius, E., Porte, A.: Ultrastructure de l'hypophyse des cyprinodontes vivipares. Etude des types cellulaires composant l'ad6nohypophyse. C. R. Soc. Biol. (Paris) 154, 1247-1250 (1960)
Pituitary Gland of the Freshwater Stickleback 91
Foll6nius, E., Porte, A.: Structure fine de l'hypophyse de deux t~l~ost~ens, Lebistes reticulatus et Perca ]luviatilis. Bull. Soe. zool. (France) 86, 295-296 (1961)
Foster, C. L.: Relationship between ultrastructure and function in the adenohypophysis of the rabbit. In: Subcellular organization and function in endocrine tissues. Mem. Soc. Endocr. 19, 125-146 (1971)
Hagen, D. W.: Isolating mechanisms in threespine sticklebacks (Gasterosteus). J. Fish. Res. Bd. Canada 24, 1637-1692 (1967)
Herman, L., Fitzgerald, P. J.: Restitution of pancreatic acinar cells following ethionine. J. Cell Biol. 12, 297-312 (1962)
Heuts, M. J.: Experimental studies on adaptive evolution in Gasterosteus aculeatus L. Evo- lution 1, 89-102 (1947)
Hollman, K. H.: A morphometric study of sub-cellular organization in mouse mammary cancers and normal lactating tissue. Z. Zellforsch. 87, 266-277 (1968)
Hope, J.: Stereological analysis of the ultrastructure of liver parenchymal cells during preg- nancy and lactation. J. Ultrastruct. Res. 33, 292-305 (1970)
Hopkins, C. R.: The fine structural localization of acid phosphatase in the prolactin cell of the teleost pituitary following the stimulation and inhibition of secretory activity. Tissue & Cell 1, 653-671 (1969)
Jamieson, J. D., Palade, G. E.: Intracellular transport of secretory proteins in the pancreatic exocrine cell. J. Cell Biol. 39, 589403 (1968)
Jarman, M.: Examples in quantitative zoology. London: Arnold 1970 Knowles, F., Vollrath, L.: Neurosecretory innervation of the pituitary of the eels Anguilla
and Conger. Phil. Trans. B 250, 329-342 (1966) Kurosumi, K., Kobayashi, Y.: Corticotrophs in the anterior pituitary glands of normal and
adrenalectomized rats as revealed by electron microscopy. Endocrinology 78, 745-758 (1966) Lain, T. J., Hoar, W. S.: Seasonal effects of prolactin on freshwater osmoregulation of the
marine form (trachurus) of the stickleback Gasterosteus aculeatus. Canad. J. Zool. 45, 509-516 (1967)
Lam, T. J., Leatherland, J. F.: Effect of prolactin on freshwater survival of the marine form (trachurus) of the three-spine stickleback, Gasterosteus aculeatus, in the early winter. Gen. comp. Endocr. 12, 385-387 (1969)
Lavallard, R., Campiglia, S.: Donn~es cytochimiques et ultrastructurales sur les tubes s~cr~- teurs des glandes de la glu ehez Peripatus acacioi Marcus et Marcus (Onychophore). Z. Zell- forsch. 118, 12-34 (1971)
Leatherland, J. F.: Seasonal variation in the structure and ultrastructure of the pituitary of the marine form (Trachurus) of the threespine stickleback, Gasterosteus aculeatus L. Z. Zellforsch. 104, 301-317 (1970a)
Leatherland, J. F.: Seasonal variation in the structure and ultrastructure of the pituitary gland in the marine form (Trachurus) of the threespine stickleback, Gasterosteus aculeatus L. Z. Zellforsch. 104, 318-336 (1970b)
Leatherland, J. F.: Histological investigation of pituitary homotransplants in the marine form (Trachurus) of the threespine stickleback, Gasterosteus aculeatus L. Z. Zellforsch. 104, 337-344 (1970c)
Leatherland, J. F.: Histophysiology and innervation of the pituitary gland of the goldfish, Carassius auratus L.: a light and electron microscope investigation. Canad. J. Zool. 50, 835-844 (1972)
Leatherland, J. F., Lam, T. J.: Effects of prolactin, cortieotrophin and cortisol on the adeno- hypophysis and interrenal gland of anadromous threespine sticklebacks, Gasterosteus aculeatus L. form trachurus, in winter and summer. J. Endocr. 51, 425-436 (1971)
Loud, A. V., Barany, W. C., Pack, B. A.: Quantitative evaluation of cytoplasmic structures in electron micrographs. Lab. Invest. 14, 258-270 (1965)
Mayhew, T. M., Williams, M. A.: A morphometric study of the rat peritoneal macrophage following stimulation in vivo. J. Anat. (Lond.) 108, 602 (1971)
Morimoto, T., Matsuura, S., Nagatta, S., Tashiro, Y.: Studies on the posterior silk gland of the silkworm, Bombyx mori. J. Cell Biol. 38, 604-614 (1968)
Miinzing, J.: The evolution of variation and distributional patterns in European populations of the three-spined stickleback, Gasterosteus aculeatus. Evolution 17, 320-332 (1963)
92 M. Benjamin
Mullem, P. J. van: A histo- and cytochemical study on the pituitary of the stickleback, Gasterosteus aculeatus L. forma trachura Cuv. partly based on a new fixation procedure after freeze drying. Arch. n6erl. Zool. 18, 149-195 (1959)
Nagahama, Y., Yamamoto, K.: Fine structure of the glandular cells in the adenohypophysis of the kokanee Oncorhynchus nerka Bull. Fac. Fish. Hokkaido Univ. 20, 159-168 (1969)
Nakane, 1 ). K.: Classifications of anterior pituitary cell types with immunoenzyme histo- chemistry. J. Histochem. Cytochem. 18, 9-20 (1970)
Nevalainen, T.: Fine structure of the parathyroid gland of the laying hen (Gallus domesticus). Gen. comp. Endocr. 12, 561-567 (1969)
Nickerson, P. A.: Effects of ACTH on membranous whorls in the adrenal gland of the mongo- lian gerbil. Anat. Rec. 166, 479490 (1970)
Nickerson, P. A.: Effect of testosterone, dexamethasone and hypophysectomy on membranous whorls in the adrenal gland of the mongolian gerbil. Anat. Rec. 174, 191-204 (1972)
Nickerson, P. A., Curtis, J. C.: Concentric whorls of rough endoplasmic reticulum in adreno- cortical cells of the mongolian gerbil. J. Cell Biol. 40, 859-862 (1969)
Nussdorfer, G. G.: Analisi citometrica della zona reticolare della corticosurrene di ratto normale ed in gravidanza. Arch. ital. Anat. Embriol. 74, 265-279 (1969)
Nussdorfer, G. G.: Effects of corticosteroid-hormones on the smooth endoplasmic reticuhim of rat adrenocortical cells. Z. Zellforsch. 196, 143-154 (1970a)
Nussdorfer, G. G.: The fine structure of the newborn rat adrenal cortex. Z. Zellforsch. 108, 382-397 (1970b)
Nussdorfer, G. G., Mazzochi, G., Rebonato, L.: Long-term trophic effect of ACTH on rat adrenocortical cells. An ultrastructural, morphometric and autoradiographic study. Z. Zellforsch. 115, 30-45 (1971)
Oztan, N.: The fine structure of the adenohypophysis of Zoarces viviparus L. Z. Zellforsch. 69, 699-718 (1966)
Peachey, L. D.: Electron microscopic observations on the accumulation of divalent cations in intramitochondrial granules. J. Cell Biol. 26, 95-109 (1965)
Pooley, A. S.: Ultrastructure and size of rat anterior pituitary secretory granules. Endocrino- logy 88, 400-411 (1971)
Purves, H. D.: Cytology of the adenohypophysis. In: The pituitary gland (Harris, G. W. and Donovan, B. T., eds.), vol. 1, p. 147-232. London: Butterworths 1966
Reynolds, E. S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963)
Ross, R., Benditt, E. P.: Wound healing and collagen formation. J. Cell Biol. 27, 83-106 (1965) Roth, T. F., Porter K., R.: Yolk protein uptake in the oocyte of the mosquito A edes aegypti L.
J. Cell Biol. 26, 313-332 (1964) Schraer, R., Elder, J. A., Schraer, H.: Aspects of mitochondrial function in calcium move-
ment and calcification. Fed. Proc. 82, 1938-1943 (1973) Staubli, W., Freyvogel, T. A., Suter, J.: Structural modification of the endoplasmic reticulum
of midgut epithelial cells of mosquitoes in relation to blood intake. J. Microscopie 5, 189-204 (1966)
Teitelbaum, S. L., Moore, K. E., Stfieber, W.: C cell follicles in the dog thyroid: demonstration by in vivo perfusion. Anat. Rec. 168, 69-78 (1970)
Weatherhead, B., Whur, P.: Quantification of ultrastructural changes in the "melanocyte- stimulating hormone cell" of the pars intermedia of the pituitary of Xenopus laevis, pro- duced by change of background colour. J. Endocr. 58, 303-310 (1972)
Weibel, E. R.: Stereological principles for morphometry in electron microscopic cytology. Int. Rev. Cytol. 26, 235-302 (1969)
Weibel, E. 1~.: The value of stereology in analysing structure and function of cells and organs. J. Microscopy 95, 3-13 (1972)
Dr. Michael Benjamin Department of Cellular Biology and Histology St. Mary's Hospital Medical School Paddington, London W2 England