BIOLOGY OF REPRODUCTION 48, 1219-1227 (1993)
Immunohistochemical and Nucleic Acid Analysis of Somatotropin Receptor Populations
in the Bovine Ovary
MATTHEW C. LUCY, '
2
ROBERT J. COLLIER, 2 MARIANNE L. KITCHELL,4 JULIA J. DIBNER, 4 SCOTT D. HAUSER, 3
and GWEN G. KRIVI 3
Animal Sciences Division2 and Cellular and Molecular Biochemistry,3 Monsanto Company
St. Louis, Missouri 63198
4
Cell Biology Division, Novus International, Inc., St. Louis, Missouri 63198
ABSTRACT
Ovaries were analyzed for somatotropin receptor protein and mRNA through use of immunohistochemistry, solution hybridization/nuclease protection, Northern blotting, and reverse transcriptase polymerase chain reaction (RT-PCR). As indicated by
immunoperoxidase staining, CL expressed immunoreactive somatotropin receptor (positive stain). Ovarian stroma, connective
tissue, endothelium, and erythrocytes did not express somatotropin receptor (negative stain). Within the CL, somatotropin receptor protein was expressed primarily in large luteal cells whereas small luteal cells were negative. Most follicles (1-5 mm,
after fixation) were negative for somatotropin receptor. On the basis of solution hybridization/nuclease protection, the mRNA
for somatotropin receptor was found in greatest abundance in CL and large luteal cells and was nearly undetectable in small
luteal cells or follicles (class 1, 3-5 mm; class 2, 6-9 mm; and class 3, - 10 mm). Northern blotting of mRNA for somatotropin
receptor showed expression of somatotropin receptor mRNA transcripts in whole ovary (4.7 and 4.4 kb), CL (4.7 and 4.4 kb),
and liver (4.4 kb); and RT-PCR amplified a single amino acid coding region for somatotropin receptor in CL and liver. In summary,
somatotropin receptor (both immunoreactive protein and mRNA) is found primarily in the large luteal cell, and lesser amounts
of the expressed receptor or its message are found in the follicle. Alternative sizes of mRNA for somatotropin receptor suggest
novel mRNA processing in the bovine ovary.
INTRODUCTION
Somatotropin has diverse functions in the cow [1-21. These
include the regulation of growth and differentiation of various cell types and the control of metabolic activity of organs and tissues. Many of the actions of somatotropin are
believed to be mediated by the insulin-like growth factors
(IGF-I and IGF-II) that are secreted from various tissues in
response to somatotropin and can have paracrine and autocrine actions [3]. The majority of cell-surface receptors for
somatotropin in the cow are found in the liver [4], where
binding of somatotropin to its receptor induces the synthesis of mRNA for IGF-I that leads to the secretion of IGF-I
protein. Although one of the primary sites for somatotropin
action (and IGF-I release) is the liver, other tissues contain
somatotropin receptors [4-6]. For example, the ovaries of
the cow contain somatotropin receptors [7, 8] and are potential sites of action for endocrine effects of somatotropin
as well as paracrine or autocrine effects of IGF-I. Direct (via
somatotropin) or indirect (via somatomedins or their binding proteins) actions of somatotropin on the ovary are suggested by studies of cattle either immunized against somatotropin releasing factor (SRF) or treated with
recombinant somatotropin (bST) [9-11]. Immunization of
heifers against SRF delays puberty and decreases follicular
growth [9], whereas administering bST increases numbers
Accepted February 15, 1993.
Received January 5, 1993.
'Correspondence: Dr. Matthew C. Lucy, Monsanto Co., AA3C, 700 Chesterfield
Parkway North, St. Louis, MO 63198. FAX: (314) 537-6480.
1219
of ovarian follicles during an estrous cycle in lactating cows
[10] and heifers [11]. Exogenous bST also increases concentrations of progesterone in plasma [12, 13] as well as
increasing the weight of the CL [14]. Responses of the ovary
to bST suggest that endogenous somatotropin may regulate
ovarian function. Furthermore, somatotropin may differentially affect luteal and follicular cells depending on the
relative abundance of receptors within each cell type.
Therefore, the objectives of the present studies were to locate cell types within the bovine ovary that express the somatotropin receptor protein, to correlate the presence of
somatotropin receptor protein with somatotropin receptor
mRNA, and to measure the sizes of somatotropin receptor
mRNA transcripts in the ovary of cattle.
MATERIALS AND METHODS
Immunohistochemistry
Ovaries were collected at slaughter from nonlactating beef
or dairy cows (n = 7), and a cross section of the ovary
containing the CL was fixed in neutral-buffered formalin.
Stages of estrous cycle or pregnancy were midluteal phase
(n = 5), proestrus (n = 1), and late pregnancy (n = 1).
Tissue was embedded in paraffin and sections containing
the CL and surrounding ovary were dried, deparaffinized,
hydrated in H20, washed with PBS, and blocked with PBS
and 0.1% BSA Somatotropin receptor was localized in ovary
sections with avidin-biotin immunohistochemistry using the
Vector Laboratories (Burlingame, CA) glucose oxidase ABC
kit. The primary antibody used for immunohistochemistry
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LUCY ET AL.
(denoted RI-M1-B11) was a rat monoclonal antibody that
binds the extracellular domain of the somatotropin receptor. Analysis of the binding of R1-M1-B11 to the somatotropin receptor has been reported previously [15]. R1-M1-B11
immunoglobulins were purified from mouse ascites fluid
through use of a Pharmacia FPLC protein G column (Pharmacia LKB Biotechnology, Piscataway, NJ) with Immunopure Gentle binding and elution buffers (Pierce, Rockford,
IL). Ovarian sections were incubated with the primary antibody (R1-M1-B11) for 2 h at 37°C at a concentration of
130 pLg/ml immunoglobulin protein. Incubation of the primary antibody was followed by sequential incubation with
biotinylated anti-rat IgG (30 min, 230C), ABC reagent (1 h,
23 0C), biotinylated anti-avidin D (1:50 dilution, 30 min, 23 0C),
ABC reagent (30 min, 23°C), and glucose oxidase substrate.
The negative control for immunohistochemistry was the same
procedure carried out in the absence of primary antibody
or with normal rat serum (NRS) substituted for the primary
antibody. Sections were counterstained with methylene green
and alcian blue.
Messenger RNA Analyses for Somatotropin Receptor
Isolation of RNA. Unless otherwise noted, chemicals
and reagents were purchased from Sigma Chemical Company (St. Louis, MO). Whole ovaries and liver were collected from cattle after slaughter, placed on ice, and transported to the laboratory. The CL, liver, and ovary with the
CL removed were placed in a plastic bag and immersed in
liquid nitrogen until frozen. The total time during which
tissue remained on ice before freezing in liquid nitrogen
was about 1 h. Tissue was collected from 6 and 4 cows in
the midluteal phase of the estrous cycle for analysis of somatotropin receptor mRNA by nuclease protection assay and
Northern blotting, respectively. Follicles (36 class 1 [3-5
mm], 14 class 2 [6-9 mm], and 11 class 3 [ 10 mm]) were
dissected free from surrounding stroma, measured, aspirated to remove follicular fluid, placed into individual polypropylene tubes, and frozen on liquid nitrogen. Large and
small cells of the CL were separated on a Percoll gradient
[16] from a cow in the third month of pregnancy. Morphology showed that the separation resulted in a nearly pure
population of small luteal cells but that the large luteal cell
fraction contained some cross-contamination with small cells.
Each fraction (about 5 x 106 cells) was frozen in liquid
nitrogen. After freezing, all samples were stored at -80°C
until RNA extraction. Total cellular RNA was isolated from
liver, CL, large and small luteal cell fractions, ovary minus
the CL, and follicle walls. Frozen tissue was removed from
-80°C and mortared to a fine powder under liquid nitrogen. The powder was then homogenized in 4 M guanidinium thiocyanate (Fluka Biochemika, Ronkonkoma, NY) and
RNA was isolated according to Chomczynski and Sacchi [17].
Integrity of ribosomal RNA was checked by electrophoresis
of an aliquot of the RNA preparation through 1% agarose
(FisherBiotech, Fairlawn, NJ) in Tris-borate/EDTA (0.09 M
Tris-borate, 0.002 M EDTA) with 1.25 M ethidium bromide
followed by ultraviolet illumination. Purified RNA was dissolved in H20, quantitated on the basis of A260 measurement, and stored at -80°C.
Poly(A) + mRNA was isolated for nuclease protection and
Northern blots using oligo(dT)-cellulose chromatography
[18]. Total cellular RNA was dissolved in binding buffer (0.01
M Tris-HCI, 0.5 M NaCl, 0.5% SDS, 1 mM EDTA; pH 7.5)
and applied to an oligo(dT)-cellulose column (Collaborative Biomedical Research, Bedford, MA). The column was
then washed with 10 bed volumes of binding buffer and
poly(A) + mRNA eluted by application of elution buffer (0.01
M Tris-HCl, 1 mM EDTA; pH 7.5). The poly(A) + mRNA was
precipitated with 0.1 volume 3 M sodium acetate (pH 5.2)
and 2 volumes absolute ethanol, dissolved in H20, quantitated on the basis of A260 measurement, and stored at -80°C.
Ribonuclease protection assay. A 460-nucleotide 32p_
labeled antisense probe to the extracellular domain of the
bovine somatotropin receptor as well as an 80-nucleotide
antisense bovine actin probe was prepared as previously
described [4]. Ribonuclease protection assays [19] were performed using the RPA II kit (Ambion Inc., Austin, Texas).
Essentially, 5 ug poly(A) + mRNA from CL or follicles of different size classes or 10 [Lg total cellular RNA from large
and small luteal cell fractions was allowed to hybridize in
solution for 15 h at 45 0C with antisense bovine somatotropin and bovine actin RNA probes. Following hybridization,
unhybridized RNA and probe were digested with RNAse A
and RNAse T1. The digestion reactions were precipitated,
and protected (hybridized) fragments (corresponding to
bovine somatotropin receptor and bovine actin mRNA) were
identified by their electrophoretic mobility through a 6%
acrylamide, 8 M urea gel. Gels were dried and autoradiography was performed with XOMAT-AR film (Eastman Kodak, Rochester, NY) for 24 h (actin) or 72 h (somatotropin
receptor) at -80 0C with intensifying screens. As a positive
control, RNA extracted from bovine liver (tissue with abundant somatotropin receptor) was assayed. The negative
control was analysis of 10 ,ag of tRNA. Relative amounts of
32
P-labeled probe hybridized to mRNA for somatotropin receptor and actin were quantitated via phosphor imaging
(PhosphorImager, Molecular Dynamics, Sunnyvale, CA) [20].
Northern blot. Analysis of RNA for somatotropin receptor mRNA by Northern blotting was performed as previously described [4]. Ten micrograms poly(A) + mRNA (ovary
and CL) or 10 ,ug total cellular RNA (liver) was electrophoresed after denaturation with 1 M glyoxal (Eastman Kodak)
and 50% dimethyl sulfoxide in a 1.2% agarose gel in 10
mM sodium phosphate (pH 6.5). The RNA was transferred
to nitrocellulose (Schleicher and Schuell, Keene, NH) by
capillary transfer for 15 h in 20-strength SSPE (3 M NaCl,
0.2 M NaH2PO 4, 0.2 M EDTA, pH 7.0), and the nitrocellulose
was prehybridized for 2 h at 42 0C in 50% (v/v) formamide
(FisherBiotech), 5-strength SSPE, 5-strength Denhardt's solution [21], 0.1% (w/v) SDS, and 100 ptg/ml tRNA. After
SOMATOTROPIN RECEPTOR IN BOVINE OVARY
prehybridization, the filter was incubated in the prehybridization solution for 24 h at 42°C with a 3 2 P-labeled cDNA
probe corresponding to a 780-bp region of the bovine somatotropin receptor mRNA coding for the extracellular domain of the receptor [4]. Labeling of the bovine cDNA probe
was by random priming [22] (Multiprime DNA Labelling
Systems, Amersham International, Amersham, UK) using [32
P]-dCTP (3000 Ci/mmol). Blots were washed two times
(15 min each) in single-strength saline sodium citrate (SSC),
0.1% SDS, 50°C followed by one wash (5 min) in 0.1-strength
SSC, 0.1% SDS, 50 0C. Autoradiography was carried out
through use of XOMAT-AR (Eastman Kodak) film for 168 h
at -80 0C with an intensifying screen. Blots were also exposed by phosphor imaging [20].
Reverse transcriptasepolymerasechain reaction. Primers
for polymerase chain reaction (PCR) were selected to specifically amplify 1.8 kb of amino acid coding region for the
somatotropin receptor cDNA [4] via reverse transcriptase
(RT) PCR. On the basis of information from the human somatotropin receptor gene [23], the forward PCR primer was
located within exon 2 of the somatotropin receptor gene
and the reverse primer was located within exon 10. One
microgram of poly(A) + RNA from CL and liver was incubated with 20 units RNAsin (Promega Corporation, Madison, WI), 5 mM dithiothreitol, 500 KM dATP, 500 p.M dCTP,
500 ,uM dGTP, 500 M dTTP, 20 pmol of reverse primer
(5'-CAGGGCAGTCGCATTGA-3'), and 10 units avian myeloblastosis virus (AMV) reverse transcriptase (Promega) in
single-strength buffer for PCR (10 mM Tris-HCI, 50 mM KCI,
1.5 mM MgCl 2, 0.001% gelatin; pH 8.3; total volume = 20
l1)for 1 h at 420C. After the incubation, 5 pIl of the RT-PCR
solution was added to 75 1 H 20, 10 pl 10-strength PCR
buffer (100 mM Tris-HCI, 500 mM KCI, 15 mM MgC12, 0.01%
gelatin; pH 8.3), 5 l1 of the forward PCR primer (5'CTTGGCAGTGGCAGGCT-3'; 10 pmol/pl), 5 l1of reverse
PCR primer (10 pmol/pl), and 0.25 1l of Taq DNA polymerase (Perkin-Elmer Amplitaq DNA Polymerase, 5 U/ul;
Roche Molecular Systems, Branchburg, NJ). The mixture was
vortexed and overlaid with 2 drops of mineral oil prior to
amplification. Amplification was carried out in a Perkin-Elmer DNA thermalcycler. Thirty cycles, 94°C for 1 min, 60°C
for 1 min, and 72°C for 1 min, were performed.
RESULTS
Immunohistochemistry of Ovarian Tissue
Immunohistochemical analysis of the bovine ovary for
the somatotropin receptor is presented in Figures 1 and 2.
Immunohistochemistry in the absence of primary antibody
or after substitution of NRS for the primary antibody did
not result in immunostaining of the ovarian tissue (CL,
ovarian stroma, or follicles; Figs. 1A and 2A). The presence
of somatotropin receptor (positive staining) in adjacent sections that had been incubated with R1-M1-B11 was indicated by dark reaction products (Figs. 1B and 2B). Positive
1221
staining for somatotropin receptor protein was found in the
CL (Figs. B and 2B), whereas surrounding ovarian stroma,
follicles (Fig. 1B), or blood vessels (Fig. 2B) were negative.
Corpora lutea from each of 7 cows analyzed showed positive staining for somatotropin receptor. Most follicles (25
out of 26; granulosa and theca cell layers) were negative
for somatotropin receptor. Identification of large cells within
the CL was based on their unique morphology (size, polyhedral shape, and central nucleus) compared to that of small
cells (spindle shaped). It appeared that somatotropin receptor was localized within the large but not the small luteal cell (Fig. 2B; darkened reaction products in large luteal
cells). Staining for somatotropin receptor was diffusely localized throughout the cytoplasm of the large luteal cells
(Fig. 2B). Most large luteal cells stained positive for somatotropin receptor, but intensity of staining varied from
cell to cell (Fig. 2B).
Analysis of Somatotropin Receptor mRNA
Nuclease protection analysis of the expression of somatotropin receptor mRNA within the bovine ovary is presented in Figure 3. The mRNA for somatotropin receptor
was found in greatest abundance in CL tissue as compared
to follicle wall tissue. For follicles of all size classes, somatotropin receptor mRNA was difficult to detect by nuclease protection assay. When expressed as a ratio to actin
mRNA, an average of 26-fold less mRNA for somatotropin
receptor was present in follicles of different size classes than
in CL. On the basis of morphological examination of luteal
cell fractions, the population of small luteal cells was essentially pure, whereas some small cells contaminated the
large luteal cell fraction. The mRNA for somatotropin receptor was concentrated primarily in the large luteal cell
fraction as compared to the small luteal cell fraction. As a
ratio to actin, somatotropin receptor mRNA was 3.6-fold
greater in the large than in the small luteal cell fraction.
The detection of some message in the small luteal cell population may have been due to low level contamination of
the small luteal cell population with some large luteal cells.
Northern blot analyses of mRNA for somatotropin receptor in prepubertal ovary, CL, and liver are presented in
Figure 4. The somatotropin receptor cDNA probe hybridized to mRNA transcripts in each of the three tissues tested
(Fig. 4A), with greater hybridization occurring in CL (10 pug
poly(A)+ RNA) compared to prepubertal ovary (10 g
poly(A) + RNA) or liver (10 ,ug total cellular RNA). Liver was
included in these analyses because it is a tissue that has
abundant mRNA for somatotropin receptor [4]. Liver tissue
contained a single band of mRNA that was 4.4 kb in length.
Two distinct transcripts were detected for somatotropin receptor mRNA in the CL and ovary (Fig. 4B). Corpus luteum
and ovary contained a 4.4-kb band of mRNA that was equivalent in size to the somatotropin receptor mRNA found in
liver and an additional transcript of greater size (4.7 kb)
that was not detected in liver tissue.
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LUCY ET AL.
FIG. 1. Immunoperoxidase staining of the bovine ovary for somatotropin receptor. Immunoperoxidase staining of adjacent ovarian sections was performed in the absence of primary antibody (negative control; A, x10) or with an antibody to the somatotropin receptor (B, x10). Positive staining (indicated
by dark reaction products) is present in the corpus luteum (CL) but not the surrounding connective tissue or follicle (FOL).
SOMATOTROPIN RECEPTOR IN BOVINE OVARY
1223
FIG 2. Immunoperoxidase staining of the bovine ovary for somatotropin receptor. Immunoperoxidase staining of adjacent ovarian sections was performed in the absence of primary antibody (negative control; A, x50) or with an antibody to the somatotropin receptor (B, x50). Positive staining (indicated
by dark reaction products) was localized in the large luteal cells (arrows). BV = blood vessel.
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LUCY ET AL.
FIG. 3. Nuclease protection assay for somatotropin receptor (bSTRc) and actin mRNA in separated small and
large luteal cell fractions, follicles of different size classes (class 1, 3-5 mm; class 2, 6-9 mm; class 3, - 10 mm),
and CL. Somatotropin receptor mRNA is found in greatest amounts in the large luteal cell fraction and intact CL.
Follicles of different size classes and the small luteal cell fraction had small amounts of somatotropin receptor mRNA.
Ratio of somatotropin receptor to actin mRNA is given. Autoradiography was for 24 h (actin) or 72 h (somatotropin
receptor).
Analysis of mRNA from CL and liver by RT-PCR is presented in Figure 5. Amplification of the amino acid coding
region for the somatotropin receptor from RNA from either
liver or CL resulted in a cDNA fragment of correct size (1.8
kb). Omission of RNA from the RT reaction prevented amplification of somatotropin receptor cDNA. There was no
evidence for an insertion or deletion within the amino acid
coding region of somatotropin receptor mRNA in CL, as major
cDNA products of alternate sizes were not detected. Re-
striction enzyme mapping as well as Southern blotting confirmed that the amplified cDNA fragment was somatotropin
receptor cDNA (data not shown).
DISCUSSION
Immunohistochemical and nucleic acid analyses of the
bovine ovary indicate that the somatotropin receptor is found
primarily within the large luteal cell. Ovarian follicles as
FIG. 4. Northern blot analysis of somatotropin receptor mRNA in prepubertal ovary, CL, and liver of cattle. Ten
micrograms of polyadenylated RNA was analyzed for ovary and CL, and 10 pg of total cellular RNA was analyzed for
liver. The blot was exposed by autoradiography (A) and phosphorimager (B; imaging shows two distinct mRNA bands
in CL). Sizes of somatotropin receptor mRNA were 4.7 and 4.4 kb for CL and ovary, 4.4 kb for liver,
SOMATOTROPIN RECEPTOR IN BOVINE OVARY
1225
FIG. 5. Somatotropin receptor cDNA amplified by RT-PCR and visualized by agarose gel electrophoresis and ethidium
bromide staining. Amplification of the amino acid coding region of the somatotropin receptor gave a single PCR
fragment (1.8 kb) in both CL and liver. Amplification was carried out in the absence of RNA (No RNA) as a negative
control for the RT PCR.
well as small luteal cells express little mRNA for somatotropin receptor and have undetectable somatotropin receptor protein expression. These data expand the findings of
Tanner and Hauser [7], who detected somatotropin receptor mRNA in the ovary. In addition, the immunohistochemical analyses agree with similar studies of rat ovary in which
somatotropin receptor/binding protein was found in greater
abundance in the granulosa-lutein cell of the CL than in
follicles [24]. It is unknown why cells within the follicle do
not express somatotropin receptor. Local concentrations of
estrogen within the follicle may be inhibiting the expression of the somatotropin receptor gene; estrogen has been
shown to block expression of somatotropin receptor in the
liver [25]. Partial luteinization of the follicle wall may, however, result in expression of the somatotropin receptor (observed in one follicle in the present study). Across all reproductive tissues, mRNA for somatotropin receptor was
found in greatest abundance in CL compared to ovary minus the CL, uterus, or oviduct ([14] and unpublished observation, Lucy and Collier). Therefore, within the reproductive tract of the cow, the somatotropin receptor appears
to be most concentrated in the CL.
When administered to cattle, exogenous bST exerts a wide
range of effects on the ovary, not all of which are related
to CL function. For example, exogenous bST stimulates the
growth of follicles on the ovary [10, 11]. The results of the
present study indicate that very few somatotropin receptors
exist within the follicle. Therefore, bST probably stimulates
follicular development through alternate mechanisms including increased IGF-I in blood or follicular fluid and al-
tered binding protein dynamics. IGF-I has numerous effects
in vitro that are stimulatory to the function of the ovarian
cells [26]. For this reason, increased IGF-I in follicular fluid
or blood could conceivably increase numbers of growing
follicles on the ovary. Administration of galactopoietic doses
of bST to lactating cows also increases weight of the CL [14]
and has been shown to increase concentrations of progesterone in plasma [12, 13]. These effects may represent direct actions of bST, because the somatotropin receptor is
found within the CL. Alternatively, these may also represent
indirect actions of bST through IGF-I or its binding proteins acting via autocrine, paracrine, or endocrine mechanisms. The latter possibility is supported by the finding that
IGF-I increases progesterone secretion from bovine corpora lutea in an in vitro microdialysis system [27].
The importance of mRNA transcripts of different sizes
within ovarian and CL tissue is not resolved at this time.
On the basis of the Northern analyses, it cannot be definitively stated that the 4.7-kb message does not exist in liver.
However, it appears that the 4.4-kb form predominates in
liver while 4.7- and 4.4-kb forms are expressed equivalently
in CL and ovary. Three forms of the somatotropin receptor
mRNA are found in the pregnant mouse liver (8.0, 4.2, and
1.4 kb [28]). The 1.4-kb message corresponds to a somatotropin-binding protein that is expressed in numerous tissues in the mouse and rat [29]. In addition, alternate splicing of the somatotropin receptor mRNA exists in tissues of
both humans [30] and pigs [31] and is primarily associated
with deletion of exon 3 of the somatotropin receptor gene.
The 4.7-kb message found in the CL may result from alter-
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LUCY ET AL.
nate splicing of introns and exons, possibly leading to novel
forms of the somatotropin receptor protein within the CL.
Although this possibility is appealing, RT-PCR of the coding
region for the somatotropin receptor using mRNA from liver
or CL yielded a cDNA of a single length (1.8 kb; Fig. 5).
The PCR with the primers specified should have detected
an alternate sized cDNA in CL if an insertion or deletion
occurs in the amino acid coding region of the somatotropin
receptor mRNA. Since this was not found, modifications of
the somatotropin receptor mRNA within the CL probably
occur upstream (5') or downstream (3') from the coding
region for the somatotropin receptor protein. The additional 300 bp may not result in an altered somatotropin
receptor protein but may in some way regulate cellular
processing of the mRNA.
The physiological significance of the presence of the somatotropin receptor on the large luteal cell is under investigation at this time. Somatotropin has been shown to
stimulate protein synthesis in Chinese hamster ovary cells
that have been transfected with somatotropin receptor cDNA
[32]. Progesterone synthesis was not increased, however,
when bovine luteal tissue slices were treated with somatotropin [33], whereas increases in progesterone have been
reported in luteal cells treated with IGF-I [27]. Given these
data, bST may be acting through its receptor on the large
luteal cell to promote cell function while not increasing
steroidogenesis. It is most likely, therefore, that increased
concentrations of progesterone in the blood of bST-treated
cows [12, 13] are associated with either heavier CL [14], increased IGF-I in blood [1, 2, 27], or altered steroid clearance associated with bST treatment.
In summary, immunohistochemistry of ovarian tissue from
cattle using a monoclonal antibody against the somatotropin receptor showed that the primary location of somatotropin receptor in the ovary is the large luteal cell. Ovarian
stroma, follicles, connective tissue, endothelium, and erythrocytes did not express somatotropin receptor. Analysis of
mRNA from follicles and CL by nuclease protection assay
demonstrated that mRNA for the somatotropin receptor exists primarily in the large luteal cell. Two sizes of mRNA
(4.7 and 4.4 kb) for somatotropin receptor were found in
CL and ovary but not in liver (4.4 kb only). Therefore, alternate mRNA processing mechanisms may exist in ovary
that yield novel forms of somatotropin receptor mRNA. These
data suggest that somatotropin may have a physiological
function associated with the development or function of the
CL.
ACKNOWLEDGMENTS
The authors would like to express their sincere appreciation to Dr. William W.
Thatcher and Dr. Michael J. Fields for contributions of CL and ovary samples for
immunohistochemical and nucleic acid analyses.
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