of others that may also be suit- able. 2Correspondence: James. E. Kinder,. A2241 Animal. Science,. Univ. of .... by Dr. Richard. Maurer,. University of Iowa, Iowa.
BIOLOGY
OF
Bovine
REPRODUCTION
Luteinizing for Alpha-
T.T.
776-781
47,
(1992)
Hormone (LH) Isoforms and Amounts of Messenger and LH Beta-Subunits in Pituitaries of Cows Immunized LH-Releasing Hormone1 M.W.
STUMPF,#{176}’7
WOLFE,4’7
M.S.
ROBERSON,5’7
H.E. Department USD8
of Animal Agricultural
Science,7 Research
CADDY,7
G.
GROTJAN,7
and
KITrOK,7
RJ. KINDER2’7
J.E.
Universiiy of Nebraska-Lincoln, Service, Roman L. Hruska US. Clay Center, Nebraska 68933-0166
B.D.
Ribonucleic against
Acid
SCHANBACHER,6’8
Lincoln, Nebraska 68583-0908 Meat Animal Research Center
ABSTRACT Ovariectomized yr)
beef
would
tropes
ovariectomized
serum
globulin
LHRH
(1.0
subunit
(n
UI
were
and
randomly
and
were
assigned
concentrations
relative altered
amount
without
into pH
of isoform
1) circulating
isoforms
of Lii
resolved
the
by
controls
2) amounts
added
to subunit
process
peptides
of subunit
plasmic
reticulum
gosaccharides apparatus
[8].
geneous
and
observed
charge
The are
April
the
reticulum
a major
are component
[9, 10]. Whether
LHRH
in U-I biosynthesis
address:
remains
A2241 Animal
Science,
Univ.
endo-
Route
I, Box
of Animal
Science,
of Pharmacology, of Physiology 4A,
University Case Western
and Biophysics,
Clay Center,
of Missouri,
Golgi
from
with
the
the
immunized
of UI,
3) distribution
13-
Lii weight
two
groups.
were
determined
most
basic
against
immunization
and
and
However,
fractions
In summary,
but
has
it
secretion with
been from
the
the
form).
LHR1-I
of cows
had
against
of intrapituitary
clearly the
secretory
of the
pattern and
established
anterior pattern
of UI
that
pituitary
the
pat-
is closely
of LHRH
isoforms
cryptorchidism
secretion of basic
of the vation
in the
are
[4, 5].
to be
pituitary.
associated
of LHRH and UI as well isoforms of UI [14, i7-i9].
present study of LHRJ-I would
ovariectomized cows to reduce stimulation
were actively immunized of the pituitary by
Go-
with
in-
as higher perThe objective
was to determine what have on the distribution
for UI and steady-state amounts of alphaunit mRNAs in the pituitary. To accomplish
alters
and
effect depriof isoforms UI beta-subthis objective,
against LHRH LHRH. Our hy-
pothesis was that the decrease in LHRH stimulation of the pituitary would alter distribution of isoforms for UI and content
of UI
subunit
mRNAs.
MATERIALS
AND
METHODS
Lin-
Colum-
Eight were
Reserve University, University
Cows
cows.
cows
Z beginning
0.05).
subunits
of UI
creased centages
hetero-
of Nebraska,
among
against
= 0.13)
pituitary.
Design
Experimental Department
bia, MO 65211. Current address: Department Cleveland, OH 44106. SCurrent address: Department Iowa City, IA 52242. address:
for the
nadectomy
no. 9841,Joumal Ser. Nebraska Agr. Res. Div. Research supUSDA CRGO 86-01108, 88-37240, and NIH HD18879. Mention of names to report factually on available data; however, the USDA neither guarwarrants the standard of the product, and the use of the name by USDA approval of the product to the exclusion of others that may also be suitE. Kinder,
H and =
1.4%).
±
modulate
the
of ohand
0.90)
immunized
(p
(3
to human
Luteinizing hormone exists as a series of charged isomers or “isohormones” [11-14]. Stage of the estrous cycle [is], gonadectomy [12, 14, i6, i7], and cryptorchidism [18, 19]
6, 1992.
James
(37.2
of a-
of LH in eluent
(p
correlated
as paper
ported by is necessary antees nor Implies no able. 2Correspondence: coln, NE 68583-0908.
6Current
to be
events
June 30, 1992.
in
oligosaccharides
thought
Received:
and
processing
in the endoplasmic
heterogeneity
Accepted:
3Current
is initiated
resulting
posttranscriptional
‘Published
translation
[6, 7]. Posttranslational
occurs
=
determined,
during
combination
(p
long-term
conjugated
LH in cows to control
of gonado-
Eight
LHRII
Amounts
compared
A through
tern
Both the synthesis of gonadotropin subunit mRNA in the pituitary and the secretion of U-I are dependent upon stimulation of gonadotropes by LHRH [1-5]. The mRNAs for the subunits are transcribed and then translated by ribosomes bound to the endoplasmic reticulum [3]. Oligosaccharides are
differ
anterior
INTRODUCTiON
LHRH
treatment
of mRNA
in the
ng/ml).
stimulation
pituitary.
against
of serum
0.83
concentrations
(designated
than
of UI
not
and
influenced
±
decreased
of LH in the
immunization
against did
that
hypothesis
subunits
concentration
(5.0
gradients,
1.4%)
concentrations
Mean
cows
isoforms
of UI,
4).
gland
the
for
treatments:
immunized
in this
was
±
concentrations
changing
nine
F (42.1
of two
n =
10.5-7.0
= 8.8)
of mRNA
in control
in cows
on pH
elution,
to one
than
to test
LHRH
amounts
(control,
chromatofocused
F (mid-alkaline
against
and
nonimmunization
of all pituitaries
isoform
LHRII
4)
pituitary
extracts
a greater
=
immunized isoforms
ng/ml) was less (p = 0.01) = 0.10) mRNA tended to be reduced
by RJA. Extracts Only
actively of Lii
.83
anterior
Pituitary
were
cows
beef
±
(p
of the
cows
the distribution
alter
LHRH
of Iowa,
human reaction
NE 68933.
776
beef
assigned (n = serum [20],
cows to one 4)
that
had
of two
been treatments:
ovariectomized immunization
for
3 yr
against
or control (n = 4). LHRH, conjugated to globulin (LHRH-hSG) by the carbodiimide was emulsified by sonication in equal vol-
ISOFORMS
umes of sterile saline and complete s.c. injections were given between rear flank. Cows were hSG) using incomplete
LHRH
antibody
titers
suppressed. Six months munized
after
against
lected
from
animals collected allowed
was
g for
last
the
intervals
it
was
was
were
and LHRH
Antibody
Titers
frozen
titers
and
glands were snap-frozen,
coland
to LURII
Antibody titers to LHRH in cows immunized against LI-IRH were determined in a single assay. Serum from all cows was diluted 1:1000 with PBS containing 0,2% gelatin. The diluted
serum
(700
1)
was
placed
in culture
20000 cpm of ‘251-LHRI-I (100 pA) and at 4#{176}C. Labeled LHRH used for binding glucose
oxidase/lactoperoxidase
iodinated products chromatography arated (15-mm
by
[22].
Free
the addition incubation).
pA of each radioactivity
the
percentage
incubated for was prepared
from (Pharmacia,
and
bound
the
[21].
with 18 h by
was
Mono-
other reaction Piscataway, NJ)
‘251-LHRH
was
transferred
determined.
to
Titers
of radiolabeled
higand
were
a culture
were
tube
expressed
bound
as
at a 1:1000
Collection
Anterior arated
and
and
posterior
anterior
lobes lobes
of the
were
70#{176}C. One hemisection from assigned to either chromatofocusing ysis. -
pituitary
were
hemisected
each
Size-separated
tcRNA filters
University
of Iowa,
was
subse-
by capillary
action
provided
Iowa
City,
linearized
(EcoRI
by Dr.
Richard
IA) to bovine
with the for a-subunit;
a- [25] appropriXba I for
LU 13-subunit). Riboprobes were labeled to high specific activity with 32P-labeled a-CTP (NEN DuPont, Boston, MA) using a Riboprobe II transcription kit and SP6 polymerase
and
pituitary was or Northern
a-AlP. Radiolabeled R1”IA and ohigonucleotide isolated with a NENSORB 20 nucleic acid tridge
(NEN
beled cpm/ml
a-
sepstored
randomly blot anal-
DuPont).
The
hybridization
quantified
directly
bis Radioanalytic radiolabeled cellulose
probe was
the
from
the
Imaging
nitrocellulose
System.
The
expressed area
were car-
of the
la-
probes (106 total probe) to nitrocel-
to continue reaction, radiolabeled
hybridizing
discrete
probes purification reaction
LU and 13-subunit and a-tubulin hybridization buffer for each
lulose-bound tcRNA was allowed 42#{176}C. Following the hybridization filters were washed [24] and specific
for 24 h at nitrocellulose bands were utilizing
an Am-
quantification
to mRNA
as net
counts
of the
radiolabeled
per
of the
bound
to nitro-
minute
detected
band.
Chromatofocusing
sep-
dilution. Tissue
kb.
to nitrocellulose
baked at 80#{176}C for 2 h. The cDNA probes (generously
within
of 200 pA dextran-coated charcoal Samples were then centrifuged; 600
supernatant
and
tubes
radioiodination
UIRH was separated by QAE Sephadex
5.0
(Promega, Madison, WI). Heterologous oligonucleotide were to rat a-tubulin (NEN DuPont; nucleotides + 958 to at 4#{176}C. probes +987 of rat cDNA) were 3’ end-labelled (3’ end-labelling at 1520 X kit; NEN DuPont) to high specific activity with 35S-labeled at -20#{176}C
antibody
concentrations of LU. Anterior pituitary lected within 15 mm of exsanguination, stored in liquid nitrogen.
and
from hemiof approxi-
stored
36 h of collection for
transferred
im-
samples
decanted
2.0
quently
col-
for 5 h. All blood
assayed
mately
control. All tcRNA extracted obvious ribosomal bands
cows
while
within
used as a negative pituitaries contained
were
cannula
then
777
IMMUNIZATION
and UI 13-subunits [26] were ate restriction endonuclease
samples
and
LHRH
Maurer,
Blood
temperature
serum
time
via an indwelling
in stanchions.
centrifuged
to
AFFER
UI
was
of UI
samples
OF
and
injections until actual
injection blood
Four and
p..g LHRHfollowing
secretion
booster
vein
at room
15 mm;
until
booster for 9 mo
and
sequential
jugular
to clot
Blood
the
restrained
at 12-mm
Thereafter, intervals
plateaued
LHRI-I,
the
were
Freund’s adjuvant. the mammary gland
given booster injections (250 Freund’s adjuvant 30 days
the primary immunization. were administered at 3-mo
mRNAs
SUBUNIT
AND
was
Pituitary extract subjected to
dients
(100 mg tissue chromatofocusing
as described
by Zalesky
focusing fractions (75 fractions lected until a stable, lower-limiting bound to the column with 1.0 M NaCI and
at this collected
tions
Samples
(1.5
ml each).
equivalent) on pH and
Grotjan
from all cows 10.5-7.0 gra[27].
of 1.5 ml each) pH was obtained.
Chromatowere colProteins
lower-limiting pH were eluted in an additional twenty fracwere
allowed
temperature before the pH of each fraction mined. Samples were then buffered by adding
to reach was 0.15
room determl 1.1
M Iris (pH 7.0) to each fraction. All buffers contained 1% glycerol and were completely degassed before use. Recovery of immunoreactive UI from the columns averaged 62%.
Radioimmunoassays Northern Total
Blot
Analysis
cellular
hemisection
RNA
of each
[23] and prepared scribed by Roberson
was
(tcRNA)
cow
by the
isolated
procedures
for electrophoretic et al. [24]. Ten
from
a single
of Nilson
separation micrograms
et al.
as deof tcRNA
from individual hemipituitaries phoresis in 1% agarose gels
was containing
separated by electroformaldehyde. Total
cellular
a bovine
cerebellum
RNA
isolated from
pool was
Concentrations cusing fractions, ifiA [28,29]. This
of UI in pituitary and serum samples assay utilized rabbit
extracts, chromatofowere determined by antiserum against ovine
UI (TEA-RAoLH #35), highly purified ovine UI (LER-1056C2) as radiolabeled tracer, and NIH-LH-B7 as standard. Intra- and interassay coefficients of variation were 2.6 and 7.5%, respectively. Secretion of U-I was characterized by determination of mean concentrations (ng/ml), pulse frequency
778
STUMPF
TABLE
1.
Anterior
serum, and ovariectomized
pituitary
weight,
concentrations
of LH in pituitary
and
relative amounts of LH subunit mRNA in pituitaries of cows and ovariectomized cows immunized against LHRH.
Immunized
against LHRH
Control weight
Pituitary
LH tissue
pg/mg
1mg)
p.9/pituitary Serum LH mean concentration (ng/ml)* pulse frequency (pulses/5 h) pulse amplitude (ng/ml) mRNA alpha-subunit (CPM)** LH beta-subunit (CPM)*** alpha-tubulin (CPM) Mean pulse culated only 0.04);
=
(pulses ods
in 5 h),
and
of Goodman
Statistical
pulse
and
Effects
of treatment
0.6 1069
0.6
0.2
1433
480
5.0 6.5 5.3
1.0 2.5 0.5
0.8 1.1 2.0
1100 1598 96
638
192
539 94
402
LHRH
forms eluted in the (isoforms A through
by the
was
cal-
subjected
to analysis
of variance
subjected
to arc sine
transformations
of the
percentage)
of pituitary
[3i].
subunits
meth-
LU, se-
values
sine
of the
the
time
bound (approx. centage was itary
were
killed.
UIRH
LHRH, bound
60.2% (i:i000 diluin blood collected at
Percentage
2.0% at all times differences were
concentration
bound
of
in blood
monitored. observed
of UI,
or
labeled
cows’ was from
in pituitary
total
pituitary
UIRH
last booster 71.8%. Percontrol
cows
weight, content
tion
=
0.Oi) than in the control the control group displayed
of LU, whereas
had a pulsatile UI in immunized fold
lower
than
only
pattern cows that
two
cows
immunized
of LH (Table with pulsatile of cows
in the
group (Table 1). pulsatile secreagainst
LURE
i). Pulse amplitude UI secretion was control
of 10-
group
(Table
a- or LU 13-subunit
probes
1). Hybridization resulted subunit tuitary
of tcRNA
to either
for
the column was bound
G (basic elution in each chro-
approximately
60%
of
percentage in cows
of UI
as isoform
immunized
against
F (p LHRH
to cows in the control group (42.1% and tively). Treatment did not affect the relative any LU among other isoforms.
0.05)
=
compared
37.2%, respecdistribution of
DISCUSSION Hypothalamic
UIRH
ated with eminence section disrupts via the
released
regulates pulsatile
the pulsatile [4, 5] and
into
the
secretion release
release is ablated
hypophyseal
por-
of UI from the pituof UI is closely associ-
of LHRH from the stalk median after hypophyseal stalk tran-
[1, 341. However, hypophyseal stalk transection also the blood flow to the pituitary normally occurring long portal vessels [35]. Hypothalamic-pituitary dis-
connection
has
centrations
of gonadotropin
been
found
to markedly subunit
decrease mRNAs
the (>
con-
90%
re-
pituof LH
among cows in the two groups (Table 1). The pattern of LH secretion was altered by treatment (Table 1). Mean concentrations of UI in cows immunized against UIRI-I were 5-fold less (p All cows from
in the
observed
F and isoforms
accounting
UI
UI eluted from the column. Isoform pH range) accounted for approximately LH in each chromatofocusing profile. An
tal blood vessels itary [32, 33]. The
collected 3 wk after these prior to tissue collection)
of labeled
