Immunocytochemical Evidence for Production

BIOLOGY
OF
45, 788-796
REPRODUCTION
(1991)
Immunocytochemical
and
ERIC
Service
Evidence
Follicle-Stimulating
BASTINGS,2
for Production
Hormone
ALBERT
BECKERS,1’2
of Luteinizing
in Separate
MICHEL
Cells
REZNIK,3
and
Hormone
in the
Bovine
JEAN-FRANCOIS
BECKERS4
and Service
de Neuropathologie,3
Facult#{233} de M#{233}decine, and
Facult#{233} de M#{233}decine V#{233}t#{233}rinaire,
Universit#{233} de Liege, Liege Belgium
d’Endocrinologie2
Service
d’obst#{233}trique4
ABSTRACT
In all mammalian
LH.
females,
glycoprotein
These
receptors,
quite
are
of gonadotrophs
copy
different.
(immunogold
single
and
antisera.
double
pituitary
otropins
in the
cells
was
their
to perform
cellular
localization
imals
[1-13].
munocvtochemical
were
positive
recent
studies
of gonadotropins
in the
matter
the last
for
that
view was partially
challenged
investigations
in various
authors,
however,
LH (bLH)
and
described
bovine
hybridization
in pig pituitary,
mone
(LH or FSH) and double
expressing
cells.
Few
Mikami
studies
have
[7], studying
and electron
changes
after
three
imsug-
cell
for
mone
concept,”
nadotroph
he
cell
FSH
recently
hormone
type
gonadotropin-
(bFSH)
and
described
(LH and
levels,
described
In line with the “one
hypothesized
types
and
The
July
23,
1991.
July
25,
1990.
Dr.
Sart-Tilman,
A. Beckers.
B4000
Service
Liege,
FSH
to specific
and
LII
and
and
membrane
in secretory
(PAP)
of cells
were
UI-containing
finding
role
the
presented
existence
interpretation
granules
electron
positive
cells
that
FSH
and
regulatory
micros-
for
LII
FSH.
With
FSH-containing
and
and
are
produced
process
in
for gonad-
d’Endocrinologie.
Centre
Hospitahier
difficult
study.
et al.
techniques
because
cross-reaction,
are
highly
not
acid
mon
sequence
a subunit.
suitable
and carboxylate
Specific
antisera
for the
content,
against
localization
of the
specific
readily
The primary
structure
of FSH, thyroid-stimulating
(TSH),
and LH exhibits
many
similarities
taming
antisera
cells
and
tained
avail-
horin amino
including
a comthe 13 subunit
were
native
hormone,
in the bovine
pituitary
using
highly
to establish
whether
gonadotropins
in a single
or
in two
distinct
populations
Our immunocytochemical
observations
the first time that LH and FSH are produced
in the bovine
species.
The FSH-containing
fied
and
is described
cytological
cell-one
hor-
Universitaire
no
immunocytochemical
be
able.
mone
not
in situ
a morphological
findings
of the current
techniques,
Dacheux
par-
ticularly
for FSH. This led many authors
to avoid giving
definitive
value
to their
observations.
Because
of these
ambiguities,
it was decided
to revise
this subject.
Our project
was to recognize
and localize
accurately
LH- and FSH-con-
single
horFSH) gene-
of two
the
of
may
yielding
in this
MATERIALS
specific
are conof cells.
demonstrate
for
by distinct
cells
cell was identi-
article.
AND
METHODS
gode-
MateriaLs
Pituitaries
Received
54%
distinct
gonadotropins
2 cows
Accepted
of FSH
consistent
with
immunohistochemical
antisera,
been
devoted
to bovine
gonadotrophs:
bovine
pituitary
at light
microscope
microscope
castration.
gonadotropins,
binding
microscopy
a specific
tuitary.
The
containing
cells possess
the capacity
to produce
and store
both hormones
but perform
these functions
in separate
areas
of the same
cell. Liu et al. [28], using
specific
cDNA probes
bovine
pituitary
[321-by
electron
microscopy-and
Girod
et al. [33] by immunofluorescence-recognized
LH-containing
cells but were
not able to identify
FSH-containing
cells in the bovine
pi-
nuIn
containing
cell types:
specific
LH-containing
cells,
specific
FSH-containing
cells, and a third
bihormonal
cell type [2430]. Childs
et al. [31] considered
that the percentage
of bihormonal
gonadotrophs
in the rat varies
according
to the
physiological
state, and suggested
that most gonadotropin-
for
light
only
suggested
scription
Using
was
an-
when
species
gested
the presence
of a single
gonadotroph
containing
both LH and FSH [14-23].
Some
the
affinity
localization
included
LII, whereas
have
pitui-
despite
the
two decades.
concept
of ‘one cell-one
hormone”
in human
as well as nonhuman
This
a sensitive
high
procedures.
of cells
with
on
on
species.
tary gland
remains
a controversial
merous
studies
performed
during
the early 1970s, the
widely
acknowledged
based
study
morphological
INTRODUCTION
The
dependent
actions,
allowed
identification
of morphologically
that no cells contained
both
hormones.
is in agreement
bovine
essentially
are
but
study
This
labeling)
11.5%
maturation
similarities,
of this
purpose
microscope,
single
labeling
immunostaining
confirmed
separate
and
many
specific
approximatively
the electron
cells.
Double
growth
have
The
highly
using
Histologically,
follicular
hormones
and
were
2 bulls.
removed
Both
immediately
males
case 2). One female
was 4 yr old
was 2.3 yr old and was slaughtered
du
(case
Belgium.
788
4).
were
after
slaughter
2 yr old
(case
3); the
2 wk after
(case
from
1 and
other
female
first delivery
BOVINE
GONADOTROPHS
Methods
antiserum.
Preparation
bit,
of antisera:
Bovine
achieved
munized
2-wk
789
LH
was
as previously
by mntradermal
intervals
Anti-LH
antisera
purified
until
described
injection
[341.
according
raised
in rab-
homogeneity
was
Ten rabbits
were
imof 200 pg of pure
LH at
to Vaitukaitis
[351.
et a!.
1: 100 for
were
then
rabbit
lution
were
extensively
characterized
for titer,
sensitivity,
and
specificity
by R]A as described
previously
[36]. Using
the
cross-reaction
between
LH of rat, sheep,
pig, and cow, we
serum
measured
serum.
ualized
the
This
bLH with labeled
rat LH and ovine
preceding
radioimmunoassay
appeared
specific.
No reaction
was observed
with FSH
Anti-FSH
antisera
raised
in guinea
pig.
was purified
until homogeneity
was achieved
method
developed
in our
laboratory
[381.
LH antito be
and emaciation.
Antisera
were studied
antisera
presented
lower
titer than
lacked
(10%)
specificity:
inhibition
of the
native
FSH. The
chromatography.
covalently
sepharose
pituitary
anti-FSH
antiserum
Two milligrams
bound
according
crude
antiserum
100 il/h.
The
purified
preparations
B4), growth
hormone
detectable
tested
Light
inhibition
up
hibited
was
LH, TSH,
NIH-B18)
observed
a subunit
from low
any
Microscope
of pituitary
were
in Bouin’s
solution.
used
for application
complex.
rehydrated.
by
Immunocytochemical
the
6B
of
rate of
highly
(PRL; NIHsubunit.
No
preparation
Sections
were
Endogenous
incubation
in
Procedure
cut into 5-mm-thick
Fourto six-.tm-thick
of peroxidase-antiperoxi-
deparaffinized
peroxidase
a methanol-H202
specific
binding
sites for immunoglobulmns
by incubation
in swine
normal
serum
for
sera
with
a flow
with
prolactin
and a
with
dilutions,
we
used
0.05
M Tris
at pH
in rabbit
Peroxidase
sections
with
peroxi-
(Sigma,
activity
0.01%
St. Louis,
was vis-
H202
and
0.5%
Microscope
to 1 pg/tube.
Specimens
and placed
tions
were
dase
then
of bovine
(GH;
developed
30 mm).
anti-
with
Electron
for titer by RIA;
did guinea
pig
with
tested
by anti-FSH
of specific
together
was
treated
temperature
in place
was purified
by affinity
of pure
a subunit
were
chromatographed
antiserum
Sections
at room
and
with
cyanogen
bromide-activated
to Axen et al. [39]. Two microliters
were
purified
by incubating
and
antiserum
swine
anti-
rabbits
Ten
LH and
of binding
glycoprotemns
for 30 mm).
antibody
1:20 for
anti-LH
(Dako,
G. Postrup,
Denmark;
diand thereafter
with
PAP (Dako,
mm),
incubated
conjugate
dilution
for
anti-LH
with
FSH
the
doses
(200
pg).
This
low inhibition
of binding
was not
modified
if the dose of antigen
was increased
(up to I g).
This observation
was compatible
with
the existence
of a
restricted
population
of immunoglobulins
recognizing
the
a subunit
dase
MO;
1: 100
[37].
Bovine
by using
antisera.
Therefore,
guinea
pig antisera
were
characterized
for sensitivity
and specificity.
The most
sensitive
(80 pg/
tube)
antiserum
was used
at a final dilution
of 1:400000.
The system,
however,
presented
a restricted
1:20
were
were
treated
by
temperature
diaminobenzidmne
tetrachlorhydrate
in 0.05 M Tris at pH
7.2 for 10 mm. Sections
were
then counterstamned
with hematoxylin.
Controls
were performed
by incubating
sections
with normal
rabbit
serum
or with normal
guinea
pig serum
4 guinea
pigs were
immunized
according
to the mntraderma! injection
method,
with the difference
that guinea
pigs
received
incomplete
Freund’s
adjuvant
to avoid necrotic
lesions
rabbit
dilutions
Sections
at room
immunoglobulmns
1:20
for 30
dilution
Antisera
Working
anti-FSH.
incubated
pieces
sec-
with xylene
and
activity
was inmixture.
were
30 mm.
Specimens
(in
7.2.
The usual
peroxidase-antiperoxidase
(PAP) method
[40]
was used
as previously
described
[411. Sections
were
first
incubated
for 17 h at 4#{176}C
with the anti-LH
or the anti-FSH
Immunocytochemical
of pituitary
were
Procedure
fixed
in 4%
at 4#{176}C
for 2 h. Post-fixation
ted. Tissues
were
graded
isopropylic
embedded
rinsed
with
alcohol
in Epon.
with
distilled
water,
and
propylene
Ultrathin
sections
grids.
Single
immunostaining.
were
subjected
to similar
glutaraldehyde
osmium
was
omit-
dehydrated
dioxide,
were
picked
in
and
up
on
gold
itary
adenoma
mal
goat
[42].
serum
They
luted
were
using
1:500
for
The
procedures
Sections
(NGS)
were
grid-mounted
as for
first
in PBS for
anti-FSH)
for
4 days
human
treated
with
1 h at room
then
incubated
with the
2% NGS in PBS (dilution
moist
chamber.
The grids
and placed
into droplets
primary
1: 400
at room
sections
pitu2% nor-
temperature.
antiserum
for anti-LH
diand
temperature
in a
were afterwards
washed
with
of PBS for 15 mm (twice),
PBS
and
then incubated
with the second
antibody
(gold-labeled
goat
anti-rabbit
or goat anti-guinea
pig) for 1 h (dilution
1:20).
They were
then washed
with PBS and rinsed
into droplets
of PBS for 5 mm (twice),
and finally
water.
Staining
with
uranyl
acetate
washed
with
was performed
wards.
Double
Both
immunostaining
(rabbit
anti-LH
gether
(dilution
and
[42
guinea
1:400
for
pig
anti-FSH)
anti-LH
and
as well
as both
immunogolds
munocytochemical
procedure,
uranyl
acetate
and observed
croscope.
1200X
Sections
to70000X.
were
distilled
after-
primary
were
1:500
antisera
mixed
for
to-
anti-FSH)
(dilution
1:40).
After
imthe grids were
stained
with
with a Philips
300 electron
mi-
observed
at magnifications
from
Immunogold
Nonblocked
For anti-
PBS)
antibody.
Immunogold
Janssen
goat
tion
and
diameter
cross-reaction
was
raised
in goats
Pharmaceuticals,
anti-rabbit
particles
nm
No
reagents
Research
antibodies
goat
anti-guinea
gold
particles,
between
observed.
Beerse,
linked
pig
to
were
15-nm
antibodies
as described
immunogolds
provided
Belgium.
by
We
used
diameter
linked
previously
at working
gold
to
10[43].
dilu-
790
BASTINGS
TABLE
1.
Counts
of LH- and
FSH-containing
cells
ET AL.
(mean
%).
LH
Subject
Field
Male
Male
1
2
Female
1
Female
2
*Means
are
1
Field
FSH
Field
2
Mean
3
13.15
7.65
11.21
15.25
10.53
13.92
11.63*
11.17
10.38
12.75
11.97
12.76
11.79
significantly
The
specificity
of the
normal
antisera;
antigen;
2) absorbing
3) omitting
cross-antigen
for
with
Quan4fication
Each
slice
ied
performed
fields.
under
cells
for
a light
a calibrated
positive
cells)
ratio
mined
by dividing
with the nucleus
Stastistical
using
Wilcoxon
We compared
for
FSH
of
also
immunoreactive
the nucleus
count
count
of all cells.
at a
reticle.
or
for
The
LH was
cells in
of all cells
realized
in the
cells
was
studdeter-
of immunostained
analysis
of these
counts
was
test after arcsin
transformation
LH with
FSH for each
sex,
each
4)
FSH
microscope
ocular
for
was
cells
also
performed
of the data.
and male
with
Microscopy
The
pituitary
anterior
(PA), the pars
vosa (PN). The residual
between
P1 and
Approximatively
Mean
12.34
2.74
5.8P
4.09
12.22
11.38
5.55
2.57
5.66
3.37
5.31
5.58
5.51*
3.82
Microscopy
Both
antisera
background.
1w ‘estigations
produced
LH cells
in shape,
and
cleus (N) with
small
granules
confident
were
labeling
of medium
a and
b).
That
exclusively
was found
into
three
intermedia
(P1),
lumen
of Rathke’s
PA [5]. All our samples
11.5% of cells were
parts:
and the
pouch
were
positive
the
pars
(N)
(Fig.
type
of very
2a).
The
granules
of immunostaining
with anti-FSH
of another
often
triangular
(g) were electron-dense
2c). Some
granules,
granule
cells. Very
mitochondria
secretory
completely
devoid
Immunostaining
identification
irregular,
irregular
cell
(Fig.
antiserum
and
however,
no
was
also
and
(m)
in distinct
cells
both
hormones.
(Fig.
pars neris located
DISCUSSION
studies
Clinical
for
LH-contamning
cells
the global
population.
to be scattered
and
the LH-contamning
throughout
onal (Fig.
the
lb).
between
percentage
(p
<
sexes
(Table
to be different
0.06),
for
each
FSH
with-
1). There
from that
sex,
and
They
were
oval,
triangular,
Despite
gested
numerous
few
specific
roles
studies
have
studies
for
FSH
in various
species,
been
devoted
in bovine
species
and
LH
the
exis-
of gonadotromatter.
At pres-
in follicular
to
bovine
have suggrowth
for
FSH-contamning
cells often
appeared
rarely were grouped
in small islets. Like
cells,
they were
uniformly
distributed
gland.
anFSH-
ent,
very
gonadotrophs.
a difference
for this
in each
and
usually
Few
showing
a tendency
in the
rod- or heartwere
usually
LH-positive
or heterogenous
population
cells remains
a controversial
out
was
were
cells were
2c). Granules
performed
slices,
were
clearly
located
cells that contained
from the PA.
for LH with
la).
for
cells
rather
small (50-200
nm) (Fig.
appeared
to be a little larger
In all examined
positive
granules
3). There
were
observed
2a).
resulted
type. FSH-containing
or polygonal
(Fig.
immunostamning
pituitary.
was
of adjacent
tence
of unique
pin-containing
or round
(Fig.
to be positive
low
or oval
little or no staining
(m), or the nucleus
no statistical
difference
between
male and female
(Table
1).
LH-contamning
cells were
scattered
or grouped
in very small
islets
uniformly
distributed
throughout
the gland.
They
appeared
to be ova!
cells (5.4%)
appeared
very
round
contained
an eccentrically
located
large
nua prominent
nucleolus
(Fig. 2a). Most of their
(sg),
150-400
nm in diameter,
were
elec-
in LH-containing
in cytoplasm,
Double
is divided
with
size,
tron-dense
and regularly
spherical
(Fig. 2, a and b). Accumulation
of granules
in the periphery
of the cell was not
uncommon
(Fig. 2a). Much
larger
granules
(lg), irregular
in shape
and less dense
than the smallest
granules
(sg) but
more
strongly
stained,
were
observed
in both sexes
(Fig. 2
imal
Investigations
bovine
3
and irregular
in form-such
as elongated
shaped
(Fig. 2, c and d). Mitochondria
rod-shaped
or elongated
(Fig. 2c).
hormone.
RESULTS
Light
Field
2
3.63
3.82
1)
for
LH with
nucleated
immunostamned
slice. A nucleus
count
unstained
The
by
serum
‘e Cells
examined
by counting
of the same
and
female
was
of 400 x using
of pituitary
(stained
pig
LH.
was
determined
three
fields
tested
guinea
each
antiserum
with
its respective
component
of the reaction;
and
one
of Immunoreactit
magnification
was
or normal
absorption
FSH
number
immunostamning
rabbit
Field
8.13
5.63
Electron
substituting
1
(p < 0.06).
different
Controls
and
Field
or polyg-
FIG.
1.
lmmunohistochemical
localization
of LH (Panel A) and FSH (Panel
of bovine
pituitary
demonstrating
the uneven
distribution
of
both cell types. This figure illustrates
the differences
in shape of the labeled
cells (Panel A: LH cells, round
or oval; Panel B: FSH cells, more irregular,
often polygonal).
Peroxidase
anti-peroxidase
complex
technique.
x250.
B) in sections
BOVINE
GONADOTROPHS
791
BASTINGS
792
El
AL.
4
4
.q).
r
-
r
‘
-:
#{149}-4-:
.
BOVINE
793
GONADOTROPHS
-c
a,
C
a,
‘
a,
a,
a,
I
a,
CCL.
a,
--
a,
-
0
N
x
-
a,_
-<
!
-
-
a,a,
E
-g
#{176}
a, .8.
.0
a,
c
-
.r
q.
I
.8
-
#{176}
:
0,-
E
0
.
.
a,
0.a,
o
.
a
0)
.
a-
‘
.c
a,
I‘i
E
o
a,
u
0a,
a,
U
a,
,
a,
a,
0.6
2
a-
.0
c
-
>
.0
‘-)
C
“
--
0
U..
C
a
a,
0
I
00
-j
‘S
a,,
.8
-
U)
ca
o
C
U
.8
0
.
-
Ooa,
0
a,0
0
ac
#{176} .
-
0-....
0)0
a,
E
C0
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0-c
c
U
_;
.8
.
0)a,
-
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o)
0-c
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C
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c
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o’
a,0
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U..
0
Eat
a,
794
BASTINGS
[44, 45]. FSH
for increasing
is responsible
the number
for early
of healthy
duces
atresia
during
In view of these
this period
physiologic
to
of LH and
reinitiate
a study
pituitary
tissue
The specificity
using a sensitive
of our antibodies
both
selection
and
while
LH in-
[45].
observations,
FSH
we
localization
electron
was
specific
labeling
allowed
identification
ulations
of gonadotrophs.
Moreover,
recognized
by their
morphological
contained
follicular
follicles,
decided
in bovine
microscopy
method.
assessed
by RIA. The
of two distinct
popthese
cells could
be
appearance.
No cells
hormones.
El
AL.
Our
data
calized
identify
not
confirm
the
LH-contamning
FSH-contamning
differentiate
trophs.
Mikami
scope
and
cal changes
results
cells,
cells
between
[7], studying
one
electron
microscope
after castration
or
types.
He
cells, round
or
bovine
the identification
of six different
interestingly
hypothesized
the
troph
cell
dium-sized
of Dacheux
although
in bovine
described
or oval
two
types
pituitary
10-nm
particles;
x36 450.
recognized
as
an
FSH
cell.
Mitochondria
(m)
and
nucleus
(N)
are
completely
of
at light
types of secretory
existence
of two
lo-
gonadomicro-
cells. He
gonado-
LH gonadotrophs
as mein shape,
with electron-dense
FIG. 3. Double
immunogold
staining
for LH (15 nm) and FSH (10 nm) in bovine
pituitary
cells. Upper part:
A
nucleated
(N) cell containing
both small, dense granules
(sg) and large, light granules
(19) labeled with 15-nm particles;
recognized
as an LH cell. Lower part:
A cytoplasmic
portion
of another
cell containing
only
dense
granules
(g)labeled
nostaining.
who
not able to
and could
levels,
described
cytologithyroidectomy
as a clue for
,::
with
[32]
she was
pituitary
devoid
of
immu-
BOVINE
and
300
regularly
spherical
granules,
p..m in diameter.
These
cells
ranging
contained
in size,
erately
oval
dense
or
polygonal
and rather
11
in size from 250dilated
vacuoles
randomly
scattered
among
the secretory
scription
is consistent
with
our findings
cells. FSH gonadotrophs
were
described
containing
(220-250
H. The
du
13.
concept
contradict
by immunocytochemical
techniques
years
in various
nonhuman
animal
as in humans.
Indeed
most
of cells containing
only
preponderant
gonadotroph
types.
It should,
reports
one
however,
during
species
mention
14.
the
be
remembered
that
sepob-
17.
18.
technical
guity of reported
In conclusion,
problem
could
results.
our results
explain
a
cytochemical
evidence
that
FSH are produced
by two
distinct
gonadotrophs,
with
the
first
cell
20.
the
21.
secific
are dif-
PM, Begeot
in the
1978;
DC.
22.
pituitat’
Hotvath
E, Kovacs
1988;
lnoue
Mozin
for
23.
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