Download Effects of Growth Hormone and Pregnancy on Expression of Growth

BIOLOGY OF REPRODUCTION 55, 996-1002 (1996)
Effects of Growth Hormone and Pregnancy on Expression of Growth Hormone
Receptor, Insulin-Like Growth Factor-I, and Insulin-Like Growth Factor Binding
Protein-2 and -3 Genes in Bovine Uterus, Ovary, and Oviduct'
Crystal J. Kirby,3 William W. Thatcher, 4 Robert . Collier,5 Frank A. Simmen,
4
and Matthew C. Lucy2 ,3
Department of Animal Sciences, 3 University of Missouri, Columbia, Missouri 65211
Department of Dairy and Poultry Sciences, 4 University of Florida, Gainesville, Florida 3261 1
Monsanto Company,s St. Louis, Missouri 63198
ABSTRACT
The effects of growth hormone (GH) and pregnancy on insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-2,
and IGFBP-3 mRNA in reproductive tissues were studied in cattle. Lactating dairy cows were inseminated at estrus and treated
with 25 mg/day GH (n = 8) or saline (n = 8) for 16 days. Corpus
luteum (CL), ovary (CL removed), oviduct, endometrium, and
myometrium were collected at the end of treatment. Messenger
RNA for GH receptor, IGF-I, IGFBP-2, IGFBP-3, and actin were
measured by nuclease protection assays. The CL contained more
GH receptor mRNA than the other reproductive tissues examined. Expression of IGF-I mRNA was highest in myometrium,
with lower amounts found in endometrium; the CL expressed
the least amount of IGF-I mRNA. The IGFBP-2 mRNA was most
abundant in endometrium and least abundant in CL. Expression
of IGFBP-3 mRNA was detected in all reproductive tissues examined. However, endometrium, a tissue that expressed the
most IGFBP-2 mRNA, had the lowest amount of IGFBP-3 mRNA.
The GH receptor mRNA was decreased in cows treated with
GH whereas the mRNA for IGF-I, IGFBP-2, or IGFBP-3 was not
changed. In the reproductive tissues evaluated, cows that contained a conceptus at tissue collection (pregnant) had higher
amounts of IGF-I mRNA than did nonpregnant cows. In summary, the level of mRNA encoding GH receptor, IGF-I, IGFBP-2,
and IGFBP-3 varied within the tissues examined, suggesting that
these genes may play a variety of roles in the bovine female
reproductive tract. Supplemental GH failed to change the expression of IGF-I, IGFBP-2, and IGFBP-3 mRNA, possibly because of low GH receptor mRNA levels in tissues other than CL.
A direct action of GH on IGF-I, IGFBP-2, or IGFBP-3 gene expression within cow reproductive tissues was not supported because the amount of IGF-I, IGFBP-2, or IGFBP-3 mRNA was not
altered by GH.
INTRODUCTION
Components of the insulin-like growth factor (IGF) axis
play an important role in follicular growth and embryonic
development [1-5]. Most reproductive tissues are capable
of IGF-I synthesis, and receptors for IGF-I are expressed
in the ovary, uterus, and embryo [1-11]. Therefore, ovarian
and uterine function, and embryonic growth and differentiation, may be dependent partially on local (autocrine) and
systemic (endocrine) IGF-I. As an example, ovarian folli-
cles synthesize IGF-I and IGF-II [1, 2, 5, 11], and the synergistic actions of IGF, LH, and FSH are involved in follicular growth [1]. In the uterus, elongating pig blastocysts
secrete estradiol that increases IGF-I secretion into the uterine lumen [7, 12]. Unlike the pig, in the cow, endometrial
IGF-I mRNA abundance is not increased during early pregnancy [8]. Additional control of reproductive function may
occur through the actions of IGF binding proteins (IGFBP)
[13]. These IGFBP are found within the ovary and uterus
and can modulate IGF action [1, 2, 5, 9, 13].
Little is known about the endocrine mechanisms that
control the local production of IGF-I, IGFBP-2, or IGFBP-3
within the female reproductive system. One possibility is
that the local production of IGF-I, IGFBP-2, and IGFBP-3
in the ovary and uterus is controlled by growth hormone
(GH). GH receptors are found in the bovine corpus luteum
(CL) [14], and an alternate GH receptor (exon I variant) is
found in CL and endometrium [15, 16]. In the rat, GH receptors occur within all parts of the reproductive tract, but
receptor levels vary across different tissues [17]. When GH
was administered to laboratory animals, IGF-I mRNA was
increased in hepatic tissue (major site of GH receptor
mRNA) [18, 19], and it may be increased similarly in reproductive tissues. Furthermore, production of IGFBP-2 or
IGFBP-3 in reproductive tissues may be altered in response
to GH or IGF-I. In the present study, tissues were identified
that express GH receptor, IGF-I, IGFBP-2, and IGFBP-3
mRNA in the cow reproductive tract on Day 17 of the
estrous cycle. At the same time, the expression of IGF-I,
IGFBP-2, and IGFBP-3 in the ovary and uterus after the
administration of either GH or saline (control) was evaluated. This was done to determine whether GH controlled
transcription of IGF-I, IGFBP-2, or IGFBP-3 mRNA within
several cow reproductive tissues. The GH receptor, IGF-I,
IGFBP-2, and IGFBP-3 mRNA also was evaluated in pregnant compared to nonpregnant cattle.
MATERIALS AND METHODS
Animals and Treatments
Sixteen lactating Holstein dairy cows (> 250 days of
lactation) from the University of Florida Dairy Research
Unit at Hague, Florida, were used. Cows were housed outdoors in concrete lots with access to dirt exercise yards and
were fed diets balanced nutritionally according to National
Research Council [20] guidelines. Estrous cycles were synchronized by using a combination of norgestomet ear implant (Synchromate-B implant; Sanofi Animal Health Inc.,
Overland Park, KS; inserted for 6 days) and prostaglandin
F 2. injection (25 mg/cow; Lutalyse, Pharmacia and Upjohn
Inc., Kalamazoo, MI; administered 5 days after norgestomet implant). Cows were inseminated artificially at 12 and
Accepted June 20, 1996.
Received November 16, 1995.
'Contribution from the Missouri Agricultural Experiment Station, Journal Series Number 12422. This project was partially supported by the
USDA under the National Research Initiative Competition Grants Program. Grant number 92-37203-8341 awarded to M.C.L.
2Correspondence: Matthew C.Lucy, Department of Animal Sciences,
164 Animal Science Research Center, University of Missouri, Columbia,
MO 65211. FAX: (573) 882-6827; e-mail:
[email protected]
996
GROWTH HORMONE, IGF, AND REPRODUCTION
997
24 h after the first behavioral sign of estrus (cow remained
motionless during a mount by a herd-mate) with semen
from the same bull. Beginning on the day of behavioral
estrus, cows were treated daily with either 25 mg methionyl
bovine GH (sometribove; Monsanto Company, St. Louis,
MO; n = 8 cows) or saline (SAL; n = 8 cows). Daily
injections of either GH or SAL continued for the next 16
days.
TABLE 1. Bovine cDNA and subcloning vectors used for the production
of antisense ribonucleotide probes for analyses of bovine mRNA.*
Ovary and Embryo Removal
* Size of the protected fragment was shorter than the ribonucleotide probe
because of RNA synthesis from the polymerase start site and additional
polylinker sequences.
On Day 17 after estrus and insemination, the ovaries and
uterus were collected from each cow after cows were killed
by captive bolt stunning and exsanguination. Time of
slaughter was approximately 24 h after the last injection of
GH or SAL. Tissues were immersed in ice and brought to
a laboratory next to the slaughter facility. Ovaries were kept
on ice, and the CL was dissected free from surrounding
ovarian stroma. None of the CL had undergone luteolysis
before slaughter. This was confirmed by plasma progesterone analyses that showed progesterone concentration to be
at least 6.6 ng/ml [21]. Embryos were flushed from the tip
of the uterine horn ipsilateral to the ovary bearing the CL
by inserting 40 ml of a mixture (1:1) of F-12 Ham and
Dulbecco's Modified Eagle's Medium (Sigma Chemical
Company, St. Louis, MO) into the contralateral uterine horn
at the uterotubal junction. Flushing medium was massaged
gently through the uterine horn and the uterine body to the
ipsilateral uterine horn to recover the embryo. A second
flush (40 ml) was done if no embryo was recovered in the
first flush. The cow was classified as nonpregnant if no
embryo was found in the second flush. On Day 17 of pregnancy, six embryos were recovered from cows treated with
GH (75% conception rate), and five embryos were collected
from cows treated with SAL (63% conception rate).
Processing of Reproductive Tissues
After the embryos were collected, reproductive tissues
(CL, ovary ipsilateral to the CL [luteal tissue removed],
ovary contralateral to the CL, ipsilateral oviduct, contralateral oviduct, ipsilateral endometrium, contralateral endometrium, ipsilateral myometrium, and contralateral myometrium) were placed in a plastic bag, immersed in liquid
nitrogen until frozen (approximately 5 min), and stored at
-80°C. The total time from animal slaughter to liquid nitrogen freezing was 1 h. Total cellular RNA was extracted
from all tissues. Ipsilateral and contralateral tissues were
combined after preliminary analyses indicated no effect of
location (ipsilateral versus contralateral). Frozen tissue was
removed from -80°C storage, placed in a mortar, covered
with liquid nitrogen, and crushed to a fine powder with a
pestle. The powder was then transferred to a hand-held homogenizer and dissociated in 4 M guanidinium thiocyanate
(Sigma). Total cellular RNA was isolated according to
Chomczynski and Sacchi [22]. Cellular RNA that had 28S
and 18S ribosomal bands readily visible under ultraviolet
illumination after electrophoresis and ethidium bromide
staining was classified as intact and used in subsequent
analyses. Purified RNA was dissolved in water, and concentration was determined from an A260 measurement. Storage was at -80°C.
Analysis of RNA by Nuclease Protection Assay
Subcloning of bovine cDNA fragments into plasmid vectors for production of complementary ribonucleotide probes
Bovine gene
Actin
GH Receptor
IGF-I
IGFBP-2
IGFBP-3
Vector
pGEM
pGEM
pGEM
pGEM
pGEM
3Z
Blue
3Z
3Z
3Z
Riboprobe
length
137
498
86
126
177
bp
bp
bp
bp
bp
Protected
length
80
460
63
108
136
bp
bp
bp
bp
bp
Reference
[23]
[231
[231
[24]
[25]
has been described previously for GH receptor, IGF-I, and
actin [23]. For the GH receptor, the ribonucleotide probe
was specific for the extracellular domain. Therefore, the
probe was specific for both the GH receptor and the GH
binding protein mRNA. These are not processed as separate
mRNA in the cow [23]. For IGF-I, the probe was specific
for the "A" domain of IGF-I protein and should detect all
of the IGF-I transcripts [19]. Additional plasmids were constructed from bovine cDNA for IGFBP-2 and IGFBP-3.
One hundred-eight base pairs (bp) of the IGFBP-2 and 136
bp of the IGFBP-3 cDNA were amplified by polymerase
chain reaction (PCR). Primers for the PCR were designed
to amplify cDNA nucleotides 1084-1192 of IGFBP-2 [24]
and 845-981 of IGFBP-3 [25]. Amplified cDNA was subcloned (Table 1), and orientation and integrity were verified
by DNA sequencing. Before their use for ribonucleotide
probe synthesis, plasmids were linearized by digestion with
appropriate restriction enzymes, extracted with a 1:1 mixture of phenol and chloroform, precipitated with 0.1 volume
3 M sodium acetate (pH 5.2) and two volumes absolute
ethanol, dissolved in water, and stored at -20°C.
Antisense ribonucleotide probes were generated by using
a Riboprobe Gemini II Core system (Promega Corporation,
Madison, WI). Two hundred nanograms of linearized plasmid were incubated with either SP6 or T7 polymerase (Promega), [ 32 P]-rCTP (New England Nuclear, Boston, MA),
and appropriate buffers to yield a ribonucleotide probe that
was antisense to the specific mRNA. Before use in nuclease
protection assays, ribonucleotide probes were extracted
with phenol:chloroform (1:1) and then chloroform. Unincorporated [3 2 P]-rCTP was removed by centrifugation
through a G50 Sephadex spin column (Boehringer Mannheim, Indianapolis, IN). Ribonuclease protection assays
[26] were done on 20 ug of total cellular RNA by using
the RPA II kit (Ambion Inc., Austin, TX). Protected mRNA
fragments were identified by their electrophoretic mobility
through 8% acrylamide, 8 M urea gels (Acryl-A-Mix 8,
Promega). Gels were dried and autoradiography was done
by using X-OMAT-AR film (Eastman Kodak, Rochester,
NY) for 48 h (GH receptor, actin, IGFBP-2, and IGFBP-3)
or six days (IGF-I) at - 80C with intensifying screens. Size
of the protected fragment was shorter than the ribonucleotide probe because of RNA synthesis from the polymerase
start site and additional polylinker sequences (Table 1). The
difference in probe length and protected fragment length
was verified by counting single bases on a nucleotide ladder
from the full-length probe to the protected fragment. Relative amounts of [ 32 P]-labeled probe hybridized to mRNA
were measured by using GP Tools, version 3.0 (BioPhotonics Corporation, Ann Arbor, MI).
998
KIRBY ET AL.
FIG. 1. Expression of GH receptor mRNA in reproductive tissues (CL, ovary [Ov; minus luteal tissue], oviduct [Ovid]; endometrium [Endol, and
myometrium [Myo]). Samples were collected on Day 17 after estrus from cows treated with either GH (n = 8 samples/tissue) or SAL (n = 8 samples/
tissue) from Days 0 to 16 after estrus. A) Means for cows treated with GH or SAL. Superscripts on x-axis label denote differences in mRNA expression
among tissues (tested by Duncan's Multiple Range test; tissues with different letters are not the same with p < 0.05). B)Representative autoradiograph
from nuclease protection assay of 2 of 16 cows analyzed. Probe, undigested probe; tRNA, negative control.
Statistical Analysis
Eighty samples of RNA (five tissues from each of sixteen cows) were assayed by nuclease protection. The tissue
means were tested for homogeneity of variance by using a
test initially described by Bartlett [27]. Variances of tissue
means for actin mRNA were homogeneous. However, variances of tissue means for GH receptor, IGF-I, IGFBP-2,
and IGFBP-3 were heterogeneous. Therefore, these data
were logo1 -transformed. After transformation, the variances
of tissue means were homogeneous for GH receptor, IGF-I,
and IGFBP-2. However, variance for IGFBP-3 remained
heterogeneous because of smaller myometrium tissue variance. Alternative transformations (natural log and square
root) failed to reduce the heterogeneity of variance. Therefore, the IGFBP-3 data were analyzed without myometrium. Data were analyzed by least squares analysis of variance by using the Statistical Analysis System (SAS; [28]).
The statistical model for mRNA (actin) or loglo mRNA
(GH receptor, IGF-I, IGFBP-2, and IGFBP-3) analyses was
Yijkl = I. + Ai + Bj + ABp + C(AB)ijk + Dl + ADil +
BDjI + ABDijl + eijkl, where Yijkl = dependent observation,
I. = overall mean, A i = effect of treatment i (GH or SAL),
Bj = effect of pregnancy status j (pregnant or nonpregnant),
ABij = treatment-by-pregnancy interaction, C(AB)ijk = effect of animal k nested within treatment i and pregnancy j,
D = effect of tissue 1 (CL, ovary, oviduct, endometrium,
myometrium), ADil = treatment-by-tissue interaction, BDjl
= pregnancy-by-tissue interaction, ABDij = treatment-bypregnancy-tissue interaction, and eijkl = residual. A i, Bj, and
ABij were tested by using C(AB)ijk. Remaining terms were
tested with the residual. Tissue means were separated by
Duncan's multiple comparison procedures of SAS. For clarity, actual means of the untransformed data are presented
in figures. Standard errors are not presented for GH receptor, IGF-I, IGFBP-2, and IGFBP-3 because tests of significance were based on log 10-transformed data. Means for
log 1o-transformed data for IGFBP-3 within myometrium
were not heterogeneous. Therefore, a single analysis of
loglo myometrial mRNA for IGFBP-3 was done by using
a statistical model without tissue or tissue interactions.
RESULTS
Statistical Interactions of Treatment, Pregnancy, and
Tissue Source
The solutions to the statistical model for GH receptor,
IGF-I, IGFBP-2, and IGFBP-3 each showed a main effect
of tissue. For some genes, an effect of pregnancy or treatment was detected. The effects of treatment by pregnancy,
treatment by tissue, pregnancy by tissue, and treatment by
pregnancy by tissue were not significant (p > 0.10) for
actin, GH receptor, IGF-I, IGFBP-2, and IGFBP-3. Therefore, only the main effects of treatment, pregnancy, and
tissue are presented in the subsequent sections.
Actin
Actin was measured in all RNA samples. Tissue means
for actin mRNA tended to be different (p < 0.07). Mean
actin was (arbitrary units) 17.5 + 1.8, 18.6 ± 1.7, 15.3 +
1.6, 17.4 + 1.5, and 16.4 + 1.3 for CL, ovary, oviduct,
endometrium, and myometrium, respectively. Treatment
and pregnancy were not significant (p > 0.10).
GH Receptor
There was an effect of tissue on GH receptor mRNA (p
< 0.001). The mRNA level for GH receptor was higher in
the CL compared to the ovary (CL removed), oviduct, endometrium, or myometrium (Fig. 1). Expression of the GH
receptor mRNA within endometrium and myometrium was
similar, and these tissues contained more mRNA for the
GH receptor than ovary or oviduct. Treatment with GH
GROWTH HORMONE, IGF, AND REPRODUCTION
999
tended (p < 0.07; Fig. 1A) to decrease GH receptor mRNA
in all tissues. Pregnancy had no effect on GH receptor
mRNA expression (p > 0.10).
IGF-I
There was an effect of tissue on IGF-I mRNA (p <
0.001). Highest expression of IGF-I mRNA was found
within the myometrium (Fig. 2). Endometrium contained
less IGF-I mRNA than did myometrium but more IGF-I
mRNA than did the oviduct, ovary, or CL. The lowest
amounts of IGF-I mRNA were in the CL. There was no
effect of treatment on IGF-I mRNA within reproductive
tissues (p > 0.10; Fig. 2A). Tissues from pregnant animals
had higher amounts of IGF-I mRNA (p < 0.003; Fig. 2B)
compared to nonpregnant animals on estrous cycle Day 17.
IGFBP-2
There was an effect of tissue for IGFBP-2 mRNA (p <
0.001). Messenger RNA for IGFBP-2 was detected in highest amounts in endometrium (Fig. 3), with the least amounts
in CL. The ovary, oviduct, and myometrium had intermediate amounts of mRNA for IGFBP-2. There was no effect
of treatment (p > 0.10; Fig. 3A) or pregnancy (p > 0.10)
on the amount of IGFBP-2 mRNA.
IGFBP-3
The tissue means for IGFBP-3 mRNA were heterogeneous for variance. This was caused by the smaller variance
in myometrium. Removal of myometrium from the analyses led to homogeneous variance. Therefore, myometrium
was removed from statistical analyses of other tissues.
There was an effect of tissue (p < 0.001) for expression of
IGFBP-3 mRNA (Fig. 4). Ovary contained the highest
amount of IGFBP-3 mRNA. Endometrium contained the
least amount of IGFBP-3 mRNA. There was no effect of
treatment (p > 0.10; Fig. 4A) or pregnancy (p > 0.10) on
the amount of IGFBP-3 mRNA. The IGFBP-3 mRNA in
myometrium was analyzed separately from that in other
tissues. There was an effect of pregnancy (p < 0.035) on
myometrial IGFBP-3 mRNA: The level of IGFBP-3 mRNA
was higher in pregnant (18.9 ± 1.4) compared to nonpregnant (13.2 ± 2.2) cows.
DISCUSSION
The cow reproductive tract expressed GH receptor,
IGF-I, IGFBP-2, and IGFBP-3 mRNA in a tissue-specific
manner (effect of tissue, p < 0.001, for each mRNA). Although the expression of IGF-I and IGFBP-2 has been compared within the ruminant ovary and within the uterus [8,
10, 11], no studies have compared ovarian, oviductal, and
uterine tissue expression of mRNA. The results of the present study demonstrated diverse expression of GH receptor,
IGF-I, IGFBP-2, and IGFBP-3 mRNA in the tissues that
we tested. Actin mRNA was measured as a marker for
mRNA extraction, measurement, and loading. There was a
tendency (p < 0.07) for actin mRNA amount to differ
among tissues. However, the difference in actin mRNA between tissues (1.2-fold difference) was minor compared to
the differences in GH receptor, IGF-I, IGFBP-2, or
IGFBP-3 mRNA among tissues (5- to 10-fold difference).
The differences in mRNA expression for GH receptor,
IGF-I, IGFBP-2, and IGFBP-3 mRNA among individual
tissues suggest roles of variable importance for GH, IGF-I,
IGFBP-2, and IGFBP-3 in the ovary, oviduct, and uterus
FIG. 2. Expression of IGF-I mRNA in reproductive tissues (CL, ovary
[Ov; minus luteal tissue], oviduct [Ovid]; endometrium [Endo], and myometrium [Myol). Samples were collected on Day 17 after estrus from
cows treated with either GH (n = 8 samples/tissue) or SAL (n = 8 samples/tissue) from Days 0 to 16 after estrus. Bars represent means for (A)
cows treated with GH or SAL or (B)cows that were either pregnant (n =
11 samples/tissue) or nonpregnant (n = 5 samples/tissue) at the time of
tissue collection. There was a significant effect of pregnancy (p < 0.003)
and tissue (p < 0.001). Superscripts on x-axis label denote differences in
mRNA expression among tissues (tested by Duncan's Multiple Range test;
tissues with different letters are not the same with p < 0.05). C) Representative autoradiograph from nuclease protection assay of 2 pregnant
cows of 16 cows analyzed. Probe, undigested probe; tRNA, negative control.
1000
KIRBY ET AL.
FIG. 4. Expression of IGFBP-3 in reproductive tissues (CL, ovary [Ov;
FIG. 3. Expression of IGFBP-2 in reproductive tissues (CL, ovary [Ov;
minus luteal tissue], oviduct [Ovid]; endometrium [Endo], and myometrium [Myol). Samples were collected on Day 17 after estrus from cows
treated with either GH (n = 8 samples/tissue) or SAL (n = 8 samples/
tissue) from Days 0 to 16 after estrus. A)Means for cows treated with GH
or SAL. Superscripts on x-axis label denote differences in mRNA expression among tissues (tested by Duncan's Multiple Range test; tissues with
different letters are not the same, with p < 0.05). B) Representative autoradiograph from nuclease protection assay of 2 of 16 cows analyzed.
Probe, undigested probe; tRNA, negative control.
of cattle. These data represent gene expression on a single
day of the estrous cycle (Day 17; late luteal phase, before
CL regression). In previous studies, the mRNAs for IGF-I
and IGFBP-2 in reproductive tissues were similar during
the luteal phase [8, 10, 11], but IGF-I mRNA changed at
estrus [10, 29]. Therefore, extrapolation of these data to
nonluteal phases of the estrous cycle may not be appropriate.
An increase in IGF-I gene expression in response to GH
supplementation was not observed in the present study. The
change failed to occur despite a more than 2-fold increase
in IGF-I protein in follicular fluid of these cows [21]. In a
separate study of mice, hepatic IGF-I mRNA was increased
in response to GH whereas IGF-I mRNA in nonhepatic
tissues was unaffected [18]; GH also failed to increase
minus luteal tissue], oviduct [Ovid]; endometrium [Endo], and myometrium [Myol). Samples were collected on Day 17 after estrus from cows
treated with either GH (n = 8 samples/tissue) or SAL (n = 8 samples/
tissue) from Days 0 to 16 after estrus. A)Means for cows treated with GH
or SAL. Superscripts on x-axis label denote differences in mRNA expression among tissues (tested by Duncan's Multiple Range test; tissues with
different letter are not the same, with p < 0.05). B)Representative autoradiograph from nuclease protection assay for both IGFBP-3 and actin
from 2 of 16 cows analyzed. Probe, undigested probe; tRNA, negative
control.
IGF-I in the ovine ovary [30]. Perhaps the low level of GH
receptor mRNA expression (2-22% of liver; Lucy, unpublished data) in ovary, oviduct, and uterus is insufficient to
yield measurable increases in IGF-I mRNA in response to
GH. Alternatively, the GH signal transduction system,
which stimulates IGF-I release in liver [18, 19], may not be
present in other tissues. The low expression of IGF-I
mRNA in CL compared to endometrium or myometrium
shows that the CL is not a major source of IGF-I in the
cow. A similar observation was made in sheep, in which
luteal expression of IGF-I mRNA was low and did not
change during the estrous cycle [11].
The GH receptor mRNA was found in all reproductive
tissues that we tested; CL expressed highest levels. We previously observed that the CL expressed the highest level of
GH receptor protein [14]. This supports the hypothesis that
GH (or possibly a related molecule, placental lactogen [31 ])
is involved in CL development or function. Endometrium
and myometrium had equivalent GH receptor expression,
GROWTH HORMONE, IGF, AND REPRODUCTION
whereas oviduct and ovary had lower levels of this mRNA.
Treatment of cattle with GH tended to decrease the amounts
of GH receptor mRNA. The decrease suggests that high
concentrations of GH, found in GH-treated cattle, may
cause a decrease in GH receptor mRNA expression. Similar
down-regulation of receptor mRNA was observed in other
systems in which hormones exceeded normal physiological
concentrations [32-34].
Although we had a limited number of samples, we found
that pregnant cows had higher IGF-I mRNA levels in reproductive tissues. Geisert et al. [8] examined endometrial
IGF-I mRNA expression on different days of pregnancy in
cattle and concluded that IGF-I mRNA expression was not
increased by the conceptus. An alternative hypothesis is
that cows with more IGF-I in either reproductive tissue or
blood may be most likely to support early embryonic
growth and establish pregnancy. Although direct evidence
for this idea is not available, it is supported conceptually
by the general link between higher concentrations of blood
IGF-I and increased fertility in cows and humans [2, 4, 5,
35].
Neither IGFBP-2 nor IGFBP-3 mRNA was affected by
either treatment or pregnancy. However, mRNA expression
in the various tissues was different for IGFBP-2 and
IGFBP-3. For example, CL, a tissue that expressed the least
IGFBP-2 mRNA, was intermediate among tissues for
IGFBP-3 mRNA, whereas IGFBP-2 mRNA predominated
in endometrium and IGFBP-3 mRNA predominated in myometrium. This is consistent with pig data, in which the
IGFBP-2 mRNA level was higher in endometrium than in
myometrium [9]. These tissue differences may reflect different roles played by IGFBP-2 and IGFBP-3 in reproductive function.
There were clear differences among tissues for the genes
analyzed. However, the technique used (whole tissue homogenization and nuclease protection analyses) did not address potential cellular changes that might have occurred
for these genes. This is particularly true for ovary because
differences among cows for numbers and sizes of follicles
may have increased the variability in mRNA expression.
Therefore, the sensitivity of our analyses may have been
increased if in situ hybridizations had been used instead of
nuclease protection analyses. A second concern is the accuracy of mRNA analyses for the prediction of protein expression within these tissues. Some variation probably exists in the actual relationship between mRNA levels and
protein synthesis within specific tissues. One additional approach would be to examine the protein expression for
these genes by immunocytochemistry. This would localize
the protein to specific cells. It may not be appropriate, however, to examine protein in either follicular or uterine fluids
since proteins may arise at least in part from hepatic synthesis. Therefore, measurement of mRNA may be the most
reliable method for detecting potential differences in local
tissue protein production. Finally, it is possible that collection of mRNA on Day 17 may have compromised the treatment effects because of GH receptor down-regulation. Perhaps collection of tissues at shorter intervals after GH treatment would have demonstrated a biphasic response in
which genes were initially induced by GH. This should be
investigated in subsequent studies.
In summary, mRNA amount for GH receptor, IGF-I,
IGFBP-2, and IGFBP-3 was affected in a tissue-specific
fashion within the cow reproductive tract. Treatment with
GH tended to decrease GH receptor mRNA in reproductive
tissues but failed to up-regulate IGF-I, IGFBP-2, or
1001
IGFBP-3 mRNA. Pregnant cows appear to express elevated
levels of IGF-I mRNA within reproductive tissues. These
data suggest that GH does not directly stimulate the IGF
system in the reproductive tissues examined.
ACKNOWLEDGMENTS
The authors would like to express their appreciation to J. Savio, G.
Danet-Desnoyers, M.T. Moser, M.D. Meyer, and R.L. De La Sota of the
Dairy and Poultry Sciences Department, University of Florida, Gainesville, for assisting during embryo collection and processing of tissues.
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