Endocrinology Vol. 141, No. 4 1560-1570
Copyright © 2000 by The Endocrine Society
Differential Expression of Growth Hormone Messenger Ribonucleic Acid by Somatotropes and Gonadotropes in Male and Cycling Female Rats1
Gwen V. Childs,
Geda Unabia and
Ping Wu
Department of Anatomy and Neuroscience (G.V.C., G.U., P.W.),
University of Texas Medical Branch, Galveston, Texas 77555; and
Department of Anatomy (G.V.C.), University of Arkansas School for
Medical Sciences, Little Rock, Arkansas 72205
Address all correspondence and requests for reprints to: Gwen V. Childs, Ph.D., Professor and Chair, Department of Anatomy, University of Arkansas School for Medical Sciences, 4301 W Markham, Slot 510, Little Rock, Arkansas 72205. E-mail: childsgv{at}exchange.uams.edu
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Abstract
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Past studies have reported the appearance of cells sharing phenotypic
characteristics of gonadotropes and GH cells. During diestrus and early
proestrus, a subset of somatotropes (4060%) expressed both GH
antigens and gonadotropin (LH-ß, LHß, or FSH-ß) messenger RNAs
(mRNAs) or GnRH receptors. More recently, we reported that subsets of
gonadotropes identified by LHß or FSHß antigens expressed GH-
releasing hormone (GHRH) binding sites. The present studies were
designed to learn if these putative multipotential cells also expressed
GH mRNA. Biotinylated sense and antisense oligonucleotide probes were
developed and cytochemical in situ hybridization tests
were optimized for the detection of GH mRNA with GH, LHß, and FSHß
antigens. RNase protection assays were developed with a complementary
RNA probe that detected a 380-bp region at the 5' end of the GH mRNA.
Both the in situ hybridization and RNase protection
assays detected changes in expression of GH mRNA during the estrous
cycle with the lowest expression occurring during metestrus and peak
expression occurring on the morning of proestrus. Cell counts confirmed
the results of the RNase protection assays showing that increases in
mRNA levels seen from metestrus to proestrus reflected increased
percentages of GH mRNA-bearing cells. In addition, densitometric
analyses demonstrated that the higher GH mRNA levels assayed from
diestrus to proestrus reflected increased area and density of label per
cell. Both types of assays showed sex differences in expression of GH
mRNA; male rat cell populations had higher values than female rats in
metestrus, diestrus, or estrus. However, percentages of GH cells in
male rats were equal to those from proestrous female rats and levels of
GH mRNA were lower in male rats than proestrous females. Dual labeling
experiments showed that, in male rats and diestrous, proestrous, or
estrous females, GH mRNA was expressed in over 70% of GH cells.
Expression of GH mRNA was also found in 5057% of cells with LHß or
FSHß antigens in the same groups. The lowest expression was seen in
the metestrous groups (3040% of GH cells or gonadotropes expressed
GH mRNA). Expression of GH mRNA was first increased from metestrus to
diestrous largely in GH cells, and slightly in cells with LHß
antigens. Further increases were seen in GH and LH cells by the morning
of proestrus. In contrast, FSH gonadotropes did not show an increased
expression of GH mRNA until the morning of proestrus (reaching the same
peak reached by LH cells). These data confirm the working hypothesis
that a multihormonal cell type develops during diestrus to support both
the somatotrope and gonadotrope populations. Collectively, our studies
suggest that this multihormonal cell may function to help support the
regulatory functions of the gonadotrope during the periovulatory
period. In addition, the appearance of significant levels of expression
of GH mRNA by male rat gonadotropes suggests that this multihormonal
cell may play a role in regulation of the male reproductive system as
well.
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Introduction
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STUDIES DURING THE past decade have
reported that the expression of GH messenger RNA (mRNA) varies with
age, gender and exposure to sex steroids (1, 2, 3, 4, 5, 6, 7, 8, 9). In rats, GH secretory
patterns become sexually dimorphic after the onset of puberty (1, 2, 3, 4),
and studies during the past 30 yr show that this is mediated by the sex
steroid environment (3, 4, 5). Ibrahim et al. (6), from our
laboratory, reported that male rat pituitaries have 3036% GH cells,
whereas female rat pituitaries may have 2628% GH cells (identified
by immunolabeling). Removal of steroids by ovariectomy stimulated an
increase in the percentages of GH cells after one month to levels like
those of the intact male rats. Castration caused a decrease in
percentages of GH cells after 1 month to 2628% (6).
Recently, Gonzalez-Parra et al. (7) showed that castration
of adult or neonatal rats decreased percentages of GH cells and levels
of mRNA for GH and Pit 1, which encodes a transactivating factor for
the GH gene. Normal percentages of GH cells and levels of mRNA could be
restored if neonatal castrates received testosterone replacement.
The mechanisms behind the sexual dimorphism appear to be complex.
Gatford et al. (9) tested sex differences in circulating
patterns of somatostatin and GH-releasing hormone (GHRH) in growing
lambs. In experiments that controlled for sex differences in food
intake, they found that rams had higher levels of the following: plasma
and pituitary GH, median eminence content of GHRH and somatostatin, GH
pulse amplitudes, and integrated plasma GH. However, the steady-state
levels of GH mRNA or the portal blood levels of somatostatin did not
differ between the sexes. Thus, they concluded that the sex differences
may also be driven by GHRH pulses or changes in pituitary sensitivity
to the pulses.
Recent studies in our laboratory have provided additional clues to
possible cellular mechanisms behind the sexual dimorphism. In the early
1990s, we discovered that, in diestrous and proestrous female rats,
4060% of cells with GH antigens expressed gonadotropin ß subunit
mRNA. These cells contributed significantly to the overall increase in
the percentages of gonadotropes seen late in diestrus and early in
proestrus (10). Further tests of females during the same period of the
cycle showed that about 3038% of cells with GH antigens expressed
receptors for GnRH (11), suggesting that they could function as
multipotential gonadotropes. In subsequent studies, we reported that
the expression of GnRH receptors by GH cells could be modulated by
inhibin (12) or activin (13). We hypothesized that a subset of
somatotropes might be stimulated to support the gonadotrope population
(by the production of LH and FSH) and produce a cocktail of hormones
that could be used by the ovary or testis. This multihormonal cell
might also be a logical cellular site for steroid induced sex
differences in the expression of GH.
We recognized, however that the expression of GH antigens cannot be
used to prove that the cell produces GH. GH could be present in
gonadotropes as a regulatory hormone bound to receptors or GH binding
proteins. Therefore, our studies have sought to determine if the
multihormonal cells retain their GH phenotype. In recent studies, we
reported that cells with gonadotropin antigens expressed GHRH receptors
during proestrus, which is an important characteristic of somatotropes
(14). The present report presents parallel studies to learn if these
multihormonal gonadotropes express GH mRNA. Biotinylated oligoprobes
complementary to GH mRNA were produced and applied to cell populations
from male rats and cycling female rats. The cytochemical data were then
correlated with RNase protection assays for GH mRNA to learn if the
changes in the cell populations corresponded to overall changes in GH
mRNA levels in the pituitary. Our studies provide new evidence for the
differential expression of GH mRNA during the estrous cycle as well as
significant expression of GH mRNA by gonadotropes from both male and
proestrous female rats. Furthermore, this report will confirm previous
studies that reported sex differences in expression of GH mRNA.
However, these differences depend on the stage of the estrous cycle
being studied. Male rats express more GH mRNA only if they are compared
with females in estrus, metestrus, and diestrus.
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Materials and Methods
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Pituitary cells from male and cycling female Sprague Dawley rats
were used for these studies. The rats were acclimated for 10 days in a
constant light controlled environment (10 h on, 14 h off). Cycling
female rats were tested daily for the stage of the estrous cycle, by
vaginal smears. Male or female rats were killed between 09001000 h.
Female rats were candidates after each had completed at least two
normal 4-day cycles. The Animal Use and Care (ACUC) Protocol has been
approved annually by the University ACUC committee. All experiments
sampled 57 rats/experimental group.
Rats were killed by decapitation within seconds of removal from their
cages. The pituitaries were then removed and placed in defined medium
as previously described (10). They were dissociated and the cells were
plated overnight in defined media (10).
For the RNase protection assays, normally cycling female or male rats
were killed as described above. Their pituitaries were collected and
stored in RNAlater (Ambion, Inc.), a solution that
protects cellular RNA from degradation. At the time of extraction, RNAs
from each pituitary were extracted with RNAwiz (Ambion, Inc.) according to manufacturers instructions. Typical
yields were 3050 micrograms RNA per pituitary.
In situ hybridization
The protocol for in situ hybridization is described
in two recent techniques papers (15, 16). The probe for GH was an
oligonucleotide synthesized with biotin attached to the 5' end (DNA
International, Inc.). It was complementary to mRNA encoding amino acids
40 to 54 on the GH molecule. This is a region that carries no homology
with PRL. The biotinylated sense oligonucleotide sequence was also
produced and used as a control probe.
The pituitary cells were fixed in 2% glutaraldehyde as described
previously (10). They were then stored for use in the in
situ hybridization protocol for no longer than a week.
Prehybridization steps were as described in techniques papers 15 and 16
and the cells were incubated with the probes overnight at 37 C. The
biotinylated sense and antisense probes were then detected by antibody
to biotin in a sandwich technique that ultimately used streptavidin
peroxidase as the reporter molecule (15, 16). This produced a sensitive
blue-black reaction in patches or linear patterns in the cell.
In parallel experiments, cells were labeled for the GH mRNA, after
which the population was prepared for dual labeling for LHß, FSHß,
or GH antigens. The immunocytochemical technique was identical to that
described previously (15, 16, 17, 18). It produced an orange-amber reaction
product that usually filled the entire cytoplasm.
Counts of at least 150 cells/slide provided the quantitative data for
the in situ hybridization studies. Each set of counts
represented cell populations from three different slides. Each
experimental group was repeated at least three times with two
rats/group. We calculated percentages of cells that contained label for
GH mRNA with or without label for one of the antigens as described in
previous studies (10, 11). ANOVA (1x) was run to compare averages from
each set of rats (n = at least three groups) and, if the F value
was significant (P <0.05), the Fishers least significant
difference post hoc test was run to identify the values that were
different.
To detect differences in density or area of label, the threshold
density of the GH mRNA label was first established with a Bioquant
Windows 95 computer-based densitometer (R & M Biometrics, Nashville,
TN). This provided automatic measurements of the area of the label in
each cell as well as the average density of the pixels on a scale of
0255, where 0 is maximal density (no light transmitted) and 255 is
the lowest density (maximal light transmitted). The light meter was set
to the same reading each day, and the first 25 labeled cells/coverslip
were analyzed. Periodic checks of the threshold were done to highlight
the pixels over the label. This helped ensure that all label was
detected. During each measurement, the area of the cell was calculated
after drawing around its perimeter. Then, the computer automatically
calculated the density of label inside the cell. Area of label was
calculated by the "video count" reading that provided the number of
pixels that covered label for GH mRNA. The area of these counts was
calculated automatically. These measurements were done only on cell
populations that were exposed to single labeling protocols. Averages
were obtained for each experimental group (sampling 4 rats/group), and
the averages for the diestrous and proestrous populations were compared
by Students t test. A P < 0.05 was
considered significant.
Assays for pituitary GH mRNA
Production of the cRNA probes. To make the GH cRNA probe, we
chose the 5' end of rat GH cDNA as a template. This 380-bp fragment was
first removed from pRGH-1 (ATCC) by PstI and
KpnI digestion, and then inserted into pBluescript II SK (+)
(Stratagene, pBSGH). The design ensured that the GH
fragment was under the control of the T7 promoter in an antisense
orientation. The resulting pBSGH plasmid was amplified in
Escherichia coli DH 5
, and purified with
QIAGEN Plasmid Midi kit (QIAGEN). An aliquot
of pBSGH was linearized with BamHI, and then purified using
QIAquick PCR Kit (QIAGEN) to remove the salt and
enzymes.
32P-labeled antisense RNA probes and RNA markers
were produced using a MAXIscript T7 in vitro transcription
kit (Ambion, Inc.) according to manufacturers
instruction. The GH cRNA probe was made with the use of the linearized
pBSGH as a template. In addition, pTRI-RNA-28S purchased from
Ambion, Inc. was applied to make a 28S probe, which
targets cellular 28S rRNA. We chose 28S as an internal control for
quantitative studies since it is rarely influenced by changes in cell
stages or different tissues. RNA Century Marker Templates
(Ambion, Inc.) were used for the synthesis of labeled RNA
that provided molecular size standards.
Initially, the conditions for the production of the labeled probe were
optimized by varying the concentrations of labeled vs.
unlabeled UTP in the reactions. Concentrations of 3.1
µM of
-32P-UTP and 50
µM of unlabeled UTP were found to be optimal
for the synthesis of the GH rRNA probe. Similarly, 0.625
µM of labeled and 500
µM of unlabeled UTP were optimal for the 28S
probe. Both GH and 28S probes were gel-purified to ensure the recovery
of only full length probes.
The Ribonuclease protection assay (RPA). The RPA was
performed using the RPA III kit (Ambion, Inc.). In the
initial phases of the study, pilot experiments were set up to determine
the optimal ratios between the amounts of sample RNA and radiolabeled
probes. This was done by applying pituitary RNA ranging from 0.25 µg
to 10 µg in these pilot RPAs with a constant amount of radiolabeled
GH or 28S probe. Our preliminary study indicated that 1 µg of
pituitary total RNA is sufficient for the detection of GH mRNA by RPA.
The specificity of the GH rRNA probe was verified by size fragmentation
on a polyacrylamide gel, as well as by RPA run on samples of yeast RNA
or on samples that omitted the target RNA.
After the pilot studies optimized the reaction conditions and also
showed the specificity of the reaction, GH mRNA was assayed following
detailed procedures described in the kit instruction. Briefly, 1 µg
of sample RNA plus 10 µg of yeast RNA (for expansion) were mixed with
4 x 104 cpm of GH probe and 2 x
104 cpm of 28S probe. After ethanol
precipitation, they were resuspended in 10 µl of the kit
hybridization buffer and incubated at 56 C for about 19 h.
Unhybridized RNAs and probes were then digested with RNase A/RNase T1
at a dilution of 1:100 at 37 C for 30 min. Following RNase inactivation
and precipitation, sample pellets were resuspended in 10 µl of Gel
Loading Buffer II, and then run on a 5% acrylamide/8 M
urea gel at 200 V for 1.5 h. Autoradiography was carried out by
exposing the gel to an x-ray film with an intensifying screen at -70 C
for 11.5 h. The radioactive signals on the gel were also quantified
by an automated system, InstantImager (Packard Instrument Co., Meriden,
CT). The intensity of signals for GH mRNA was depicted as cpm and
normalized against signals for 28s rRNA.
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Results
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Analysis of in situ hybridization labeling for GH mRNA
Counts of single labeled fields showed that male rat pituitaries
contained 30.9 ± 3% cells with GH mRNA. (Fig. 1
). This value is significantly higher
than those from metestrous (14.9 ± 0.7%) or estrous (23 ±
3%) females. Diestrous or proestrous female rats had comparable
percentages of GH mRNA-bearing cells (29 ± 3%). When the
biotinylated sense sequence was substituted for the antisense sequence,
the percentages of labeled cells averaged 3 ± 1.5% (three
separate experiments, cells from male rats). Cells exposed to
hybridization buffer alone during the hybridization had minimal
labeling (1.52.4%; examples from all sexes and experimental
groups).

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Figure 1. Single labeling for GH mRNA or GH antigens (Ag) in
populations of pituitary cells from male or cycling female rats. The
lowest percentages of cells with GH mRNA is seen in metestrous rats.
Values are lower than all other groups (a). Percentages of GH
mRNA-bearing cells from rats in estrus are lower than those from male
or female rats (diestrus and proestrus) (b). Students
t test showed that populations from metestrus rats had
lower percentages than those from proestrous female or male rats (c).
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Figure 1
also shows the counts of GH antigen-bearing cells from these
same groups (taken from the dual-labeling experiments). Male rats had
35.7 ± 1.4% GH cells. Female rats had percentages of GH cells
that ranged from 26 ± 1.4% (metestrous rats) to 34.7%
(proestrous rats). The application of the Students t test
showed that metestrous female rats had significantly lower percentages
of GH cells when compared with male rats (P < 0.01) or
proestrous female rats (P <0.05).
Figure 2
is a plate that illustrates the
GH mRNA labeling data. As in our previous studies (10), the label is in
dark spots or lines in the cells and it varies in density from cell to
cell. Figure 2
, ac, compares the labeling in the male rat cells with
the use of antisense (Fig. 2a
) and sense probes (Fig. 2
, b and c).
Figure 2d
shows that the expression in proestrous females compares
favorably with that in the male rats (Fig. 2a
). Figure 2e
shows that
the expression in some regions from metestrous rat populations is
virtually absent. In other fields, there are only one to two cells.

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Figure 2. Illustrations of populations labeled for GH mRNA
in single labeling protocols. Labeled cells are noted by
arrows. ac, Fields from male rats. Figure 2
illustrates two GH mRNA-bearing cells (also shown in
inset) after exposure to 10 nM biotinylated
oligonucleotide complementary to GH mRNA. b and c, Fields from male rat
cells following exposure to the same amount of biotinylated sense
sequence. The field in 2b is a higher magnification of an area from
Fig. 2c . Figure 2d shows a field from proestrous rat pituitaries
labeled with the biotinylated antisense probe for GH mRNA. GH mRNA
bearing cells are noted by arrows (and the
inset). The counts of GH mRNA-bearing cells are
comparable to those in the male rat (Fig. 2a ). A field from metestrous
rat pituitaries is shown in Fig. 2e . The inset shows one
of the cells (arrowhead) that has a gray
label. In another cluster of cells (arrowhead) there are
small patches of label in the cells. However, overall labeling is
significantly reduced in the metestrous group. a, c, and d,
Magnification, 451x, bar = 10 µm;
inset magnification, 902x; b, magnification 825x; e,
magnification 1155x; inset magnification 1546x.
Bar, 10 µm.
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Correlative RNase protection assays
RNase protection assays were applied to total RNA purified from
additional pituitaries from the same experimental groups to learn if
the changes in percentages of GH mRNA bearing cells reflected changes
in overall levels of GH mRNA. Figure 3
shows an autoradiogram that includes some of the tests of specificity
of the antisense RNA probes following the manufacturers instructions.
Lane B shows a strong signal on the gel when 10 µg yeast RNA was
hybridized with 4 x 104 cpm of the GH probe
and no RNase treatment was used (Lane B). In contrast, Lane A shows
that, when RNase A/RNase T1 at 1:100 was added to digest all
nonhybridized RNAs, it completely abolished the signal, indicating that
no hybrids had formed from the yeast RNA and the GH probe. The 400-bp
band seen in Lane B includes both the 380-bp GH probe fragment and
20-bp of flanking sequences from the pBluescript II SK (+) plasmid.
Lane C contains a distinctive band (380-bp) from a sample containing
total RNA (from male rat pituitary) and the GH probe. The sample was
treated with RNase, and the radioactive band indicates that hybrids had
formed. The extra bands are believed to be a nonspecific background.
This is most likely due to the excess amount of probes used to ensure a
quantitative analysis. Lane D shows the effect of adding a sample
containing male rat pituitary RNA without the GH probe. No signal was
detected when 1 µg of total RNA from a male rat pituitary was
digested with the same amount of RNase in the absence of the GH
probe.

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Figure 3. RNase protection assay on yeast RNA and a total
RNA sample from male rat pituitary. Lane A, 10 µg yeast
RNA + 4 x 104 cpm GH probe + 1:100 RNase A/T1. Lane
B, 10 µg yeast RNA + 4 x 104 cpm GH probe. Lane C, 1
µg pituitary total RNA + 4 x 104 cpm GH probe +
1:100 RNase A/T1. Lane D, 1 µg pituitary total RNA + 1:100 RNase
A/T1.
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Figure 4a
illustrates a representative
autoradiogram showing samples containing total rat pituitary RNA plus
GH and 28S probes (and RNase A/RNase T1 treatment) from each
experimental group. The quantification of RNA from at least six
rats/experimental group is shown in Fig. 4b
. The statistical analysis
shows that levels of GH mRNA in male rats are greater than those in
diestrous, metestrous, and estrous female rats. However, proestrous
female rats have higher levels of GH mRNA than all other groups,
including male rats. Metestrous rats contain levels of GH mRNA that are
lower than all but the estrous group.

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Figure 4. Autoradiogram showing detection of GH mRNA (GH)
and 28S rRNA (28S, as an internal control) by an RNase protection assay
is shown in a. Experimental groups include male, and Metestrous (M),
Diestrous (D), Proestrous (P), and Estrous (E) females. The highest
values are seen in male and proestrous female rats. 4b, Quantification
of RNase protection assays on pituitaries from 56 rats/group show
that, as with the in situ hybridization studies, the
highest levels of GH mRNA are found in proestrous female rats. Male
rats express more GH mRNA than females in all stages but proestrus.
Values on y-axis are GH mRNA signals (cpm) normalized
against 28S rRNA (cpm). a = the highest values; b = the next
highest values; c= lower than values from males or proestrous females;
d = the lowest values.
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Densitometric analysis of GH mRNA in individual cells
The RPA data suggested that the rise in levels of GH mRNA peaks at
proestrus; however, the cell counts in Fig. 1
indicate that similar
percentages of GH mRNA-bearing cells are found in diestrous and
proestrous groups. Densitometric analyses were conducted to learn if
the difference in levels assayed by the RPA reflected lower levels of
GH mRNA/cell. Table 1
shows that values
were higher in proestrous rats for all types of measurements, including
average areas of GH mRNA bearing cells, average areas of label for GH
mRNA/cell, and the average density of the label. When evaluating
density measurements, it is important to note that a lower number
reflects less light transmitted. Therefore, labeling density that is
more intense (darker blue or black) would let less light through and
give a reading closer to 0. Weaker labeling would let more light
through and give a higher value in the reading.
Identity of cell types expressing GH mRNA: analysis of dual
labeling
Dual labeling showed that changes in expression of GH mRNA
occurred in cells with GH or gonadotropin antigens. Figure 5
shows that over 7080% of cells with
GH antigens expressed GH mRNA in all groups, except metestrous females.
Fewer than 30% of GH antigen-bearing cells expressed GH mRNA in the
metestrous rats, which is significantly lower than values from all
other groups.

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Figure 5. Counts of GH antigen-bearing cells dual labeling
for GH mRNA. This graph illustrates the percentages of GH
antigen-bearing cells that also express GH mRNA. The lowest level of
expression is seen in metestrous rats (a).
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To see the impact of the changes on the entire pituitary cell
population, the data are also calculated as the percentage of dual
labeled cells/total pituitary cells. Figure 6
shows that, in metestrous rats, cells
with GH antigens and GH mRNA are only 7% of the pituitary cells. In
contrast, these dual-labeled cells represent 2229% of pituitary
cells in all other groups.

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Figure 6. Counts of cells dual-labeling for GH mRNA and GH
antigens. This graph illustrates the percentages expressed as the
proportion of total pituitary cells. The lowest levels of expression
are seen in metestrous rats (a). Estrous rats have 22% cells with GH
mRNA and GH antigens, which is lower than cells from either proestrous
female or male rats (b).
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Among gonadotropes in male rats, over half of the cells with LHß
antigens (55%) and 50% of those with FSHß antigens expressed GH
mRNA (Fig. 7
). In metestrous females, only 3035% of cells with
FSHß or LHß antigens contained GH mRNA. Expression of GH mRNA by LH
cells rose significantly during diestrus to reach a peak of 57% of
cells with LHß antigens by the morning of proestrus, and it remained
high during estrus. Cells with FSH antigens exhibited a slightly
different pattern. The rise to 57% of FSH cells did not occur until
the morning of proestrus, and this peak level persisted until the
morning of estrus (which is not different from the values in proestrous
rats).

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Figure 7. Counts of antigen-bearing gonadotropes that also
express GH mRNA. The percentages of each type of gonadotrope
(immunolabeled for LHß or FSHß) that express GH mRNA are shown. The
lowest expression of GH mRNA among FSH cells is seen in metestrus and
diestrus (a). There is a significant increase on the morning of
proestrus to values similar to those seen in the male rat. Among LH
cells, the lowest expression is seen in metestrous groups (a). The
first increase in expression of GH mRNA by LH gonadotropes is seen on
the morning of diestrus (b). These levels are higher than those seen in
metestrous groups. Peak expression of GH mRNA by LH gonadotropes is
seen in proestrous and estrous groups. Values are not different from
those in male rats, however.
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Because they have actively secreted their stores, percentages of
immunoreactive gonadotropes are at their lowest during metestrus (7%,
10, 17, 18). Therefore, the dual labeling data were also calculated as
percentages of dual labeled cells/total pituitary cells to show the
impact of the changes in gonadotropes on the entire pituitary
population (Fig. 8
).

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Figure 8. This graph shows the changes in percentages of LH
or FSH gonadotropes with GH mRNA. Values are expressed as a percentage
of the total pituitary cell population. The data reflect the overall
changes in gonadotropin-antigen bearing cells described previously (10 17 18 ). Male rat pituitary populations contain 67% cells with LHß
or FSHß antigens and GH mRNA. Among metestrous female groups,
22.75% of pituitary cells show gonadotropin antigens and GH mRNA
(a). Diestrous and estrous female rats have 46% cells with
gonadotropin antigens and GH mRNA (b). The highest levels of cells with
gonadotropin antigens and GH mRNA is seen on the morning of proestrous
(c). The percentages of these multihormonal cells are similar to those
from previous studies (10 ) which defined somatogonadotropes by their
expression of GH antigens and LHß or FSHß mRNA. a =
percentages lower than male and proestrous female rats. b =
percentages lower than proestrous female rats.
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In male rat populations, 67% of the pituitary cells contain LHß or
FSHß antigens and GH mRNA. Metestrous populations have only
2.52.75% cells dual labeled for gonadotropin antigens and GH mRNA.
The percentages of cells with GH mRNA and LHß antigens rise rapidly
during diestrus to reach a peak of nearly 11% of the pituitary
population by the morning of proestrus. A slower rise is seen in
percentages of cells with GH mRNA and FSHß antigens, although the
peak values reached by the morning of proestrus are similar. This is
followed by a decline in percentages during estrus to 4.5% cells with
FSHß antigens and GH mRNA and 6% cells with LHß antigens and GH
mRNA.
Finally, the data also calculated changes in percentages of GH
mRNA-bearing cells that contained each of the antigens tested. Figure 9
shows the data. In all stages of the
cycle but estrous, over 90% of GH mRNA bearing cells had translated
the mRNA and stored GH antigens. During metestrus, this value declined
significantly to 60 ± 15%. This suggests that 40% of GH
mRNA-bearing cells have stores of GH below threshold levels needed for
detection by immunolabeling. This could be the result of secretion, or
it could be that the cells have simply not translated the mRNA. When
gonadotropins were analyzed, 1826% of GH mRNA bearing cells stored
LH or FSH during all phases of the cycle except proestrus when the
values increased to 41 ± 2%. Recall that percentages of cells
with gonadotropin antigens are lowest during estrus and metestrus
because of loss of gonadotropin stores. Thus, as the percentage of GH
mRNA bearing cells reaches its nadir, so do percentages of LH or FSH
cells. This parallel reduction maintains the low proportion of GH mRNA
bearing cells that store gonadotropins.

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Figure 9. Graph illustrates data showing the changes in the
percentages of GH mRNA bearing cells that contain GH, LHß, or FSHß
antigens. The significant loss in GH mRNA bearing cells during
metestrus is accompanied by a loss in cells that also bear GH antigens.
At least 40% of GH mRNA cells appear to not have translated the GH (or
have secreted it). The significant increase in GH mRNA-bearing cells
with LH and FSH antigens during proestrus agrees well with previous
studies showing the same increase in cells with GH antigens and
gonadotropin mRNAs (10 ). Star, Significantly different from all other
values.
|
|
Figure 10
is a plate illustrating
examples of dual-labeled cells. Figure 10
, a and b, compares fields
labeled for GH mRNA and GH antigens in metestrous (Fig. 10a
) and
proestrous (Fig. 10b
) rats. Fewer dual labeled GH cells are seen in the
metestrous field as would be expected from the values in Fig. 9
. In
contrast, numerous cells with GH mRNA and/or GH antigens are evident in
the field from proestrous rats (10b).

View larger version (84K):
[in this window]
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|
Figure 10. Illustrations of dual labeling for GH mRNA
and GH, LHß, or FSHß antigens. The different colored backgrounds
reflect differences in the filters used during photography that did not
affect the visualization of the label. Labeled cells are noted by
arrows. a and b compare fields from metestrous (a) and
proestrous (b) rats labeled for GH mRNA and GH antigens. Five GH cells
(G) are seen in a; however, the label is so faint for GH in three of
the cells that it cannot be detected well in the low magnification view
(also shown in inset). One of the cells also has GH mRNA (cell a) and
one of the cells shows labeling for GH only (cell b). Cell a is also
shown in inset. b, Stronger label for GH mRNA in proestrous rats. Most
of the labeled cells have a blend of label for GH mRNA and antigens
which gives them a muddy appearance in the low magnification views. The
inset shows that two of the cells in the cluster have
black linear patterns for the GH mRNA label. c and d, Fields labeled
for GH mRNA and LHß antigens from metestrous (c) and proestrous (d)
populations. Only one labeled cell is seen in the field in c, and it
shows a linear and patchy label for GH mRNA (black) in a
cell with orange label for LHß. The cell has extended
a process, a feature that is typical of gonadotropes (also shown in the
inset). In d, three labeled cells are evident. The cell
that contains both GH mRNA (black patch or
line) and LHß antigens is also shown in the
inset. This cell also has extended a process. Cells
labeled gray for GH mRNA are also seen in the field
(arrows). eh, Added to illustrate the differential
labeling pattern from these groups. Label for GH mRNA and LHß
antigens in male rat cells is seen in e and f. e, Shows one cell
densely labeled for GH mRNA (gray) with some faint
orange label seen (out of the focal plane). It lies
adjacent to a cell with dense orange label for LHß
that also includes patches of black label for GH mRNA.
Another cell contains dense gray label and faint
orange label for the same combination of hormones. g,
Shows that the label for GH mRNA appears in vacuoles around the central
nucleus (n). This cell (from a male rat) is also labeled
orange for GH. h, Dual labeling for GH mRNA
(black) and FSH in one of the three cells taken from a
proestrous rat. All three cells contain orange label for
FSH, but only one shows dense black label for GH mRNA. a
and b, Magnification 451x, bar = 10 µm;
inset magnification 902x; c and d, magnification 825x,
bar = 10 µm; inset magnification
1,155x; eh, magnification 1,546x, bar = 5 µm.
|
|
Figure 10c
shows a cell with GH mRNA and LHß antigens in the
metestrous field. It is extending a process and strands of linear
arrayed label are evident in the cytoplasm. The field in 10d
illustrates a cell with GH mRNA and LHß antigens (also shown in the
inset). Also illustrated are two cells with label for GH
mRNA only (gray label).
Figure 10
, eg, are views from male rats taken with oil immersion
objectives to show examples of the differential labeling patterns.
Figure 10e
shows two labeled cells. One cell is labeled intensely
gray for GH mRNA next to a cell labeled orange
for LHß and black for GH mRNA. Faint orange label is seen
in the adjacent cell when one focuses through the gray label. Figure 10f
is also from male rats and shows a cell labeled gray for GH mRNA
(with a faint orange for LH antigens) and another cell labeled dense
orange for LHß and black for GH mRNA (in a linear pattern). Figure 10
g shows a view of the circular pattern of label for GH mRNA
(vacuoles) in a cell that contains GH antigens. Figure 10h
shows three
labeled cells from proestrous rats. Two of the cells are labeled
orange for FSHß only. The cell in the center contains
FSHß antigens and a dense patch of label for GH mRNA.
 |
Discussion
|
|---|
The present study adds further information about common phenotypic
characteristics shared by members of the somatotrope and gonadotrope
populations. This phenomenon was first described in 1994 when we
demonstrated the transient expression of LHß and FSHß mRNA (10) and
GnRH receptors (11) by cells with GH antigens during diestrus that
reached a peak during proestrus. Later studies showed that the rise in
expression of GnRH receptivity by cells with GH antigens could be
mimicked by treating diestrous rat pituitary populations with activin
(13).
In our earlier studies (10, 11), we recognized that the presence of GH
antigens in the putative multihormonal cell did not necessarily prove
that the cells were somatotropes. We reasoned that GH could be present
in gonadotropes as a regulatory hormone bound to GH binding proteins
(reviewed in Ref. 11). Therefore, during the past 3 years, we have
continued to look at the extent to which gonadotropes might express
characteristics unique to GH cells, to learn more about the
significance of this coexpression of GH and gonadotropin hormones and
receptors.
The first studies of GH cell phenotypes showed that GHRH binding sites
could be found on GH cells and gonadotropes (14). We also reported that
the highest expression of GHRH receptors by cells with gonadotropin
antigens occurs in early proestrus, when the putative multihormonal
cells emerge. Significant numbers of gonadotropes in male rats
(2030% of gonadotropes) also expressed GHRH. The presence of GHRH
receptors added support for the hypothesis that the cells were
somatotropes and not gonadotropes with endocytosed GH antigens. It also
added information suggesting that these cells could respond to multiple
secretagogues.
The findings from the present study provide the strongest support for
the hypothesis that the multihormonal cells represent a unique subset
that functions to support both the GH and gonadotrope population. In
all but the metestrous groups, over 91% of the mRNA-bearing cells
contain GH antigens (Fig. 9
). At the same time, 41% or 32% of GH
mRNA-bearing cells contain LHß or FSHß antigens, respectively.
Adding these percentages accounts for over 100% of the GH mRNA cell
population. Therefore, it is likely that 3040% of these GH
mRNA-bearing cells are multipotential and can store all three hormones.
These data correlate well with percentages published in 1994 (10),
which showed that 40% of cells with GH antigens contain LHß mRNA and
34% of cells with GH antigens express GnRH receptors (11).
At this point, there are four combinations of phenotypes found in these
pituitary cells that point to a multihormonal function for this subset.
These include expression of: GH antigens with LHß or FSHß mRNAs
(10); GH antigens and GnRH receptors (11, 12, 13); GHRH receptors and LHß
or FSHß antigens (14); and GH mRNA and LHß or FSHß antigens (the
present report). A fifth phenotypic characteristic common to this
subset is the response to inhibin and activin (12, 13), which decreases
or increases expression of GnRH receptors in GH cells,
respectively.
Sexual dimorphism in expression of GH mRNA
Sexual dimorphism appears during pubertal development (1, 2, 3, 4) and,
as discussed in the introduction, removal of steroid feedback produces
expression of GH mRNA or antigens more like that in the opposite sex
(6, 7). Similarly, restoration of steroid feedback allows recovery to
the sex-specific level of expression (7). The results from this study
also partially confirm studies that show sexual dimorphism in the
expression of GH mRNA by adult rat pituitaries (7, 8, 19). However, in
our experiments, the female rats were separated into groups based on
the stage of the estrous cycle. New findings in the present study show
that male rat pituitaries have more GH mRNA only when compared with
female rats in metestrus, diestrus, or estrus. Because none of the
previous studies of adult rats identified the stage of the cycle (7, 8, 19), it is likely that they assayed GH mRNA in a mixed group of females
that might contain proestrous rats in a minority of the population.
Differential expression of GH mRNA during the estrous cycle
Both the RNase protection assays and counts from the in
situ hybridization studies agree that there is an increase in GH
mRNA transcripts (or their stability) when metestrous and proestrous
groups are compared. However, the RNase protection assays showed
intermediate values in the diestrous group, whereas the counts from
in situ experiments showed maximal percentages of cell
labeled for GH mRNA. Densitometric studies were then done to learn if
the differences between the assays were due to changes in the amount of
GH mRNA/cell (as detected by area and density). The stereometric and
densitometric data showed that GH mRNA bearing cells from proestrous
rats were larger and contained more GH mRNA (detected by area and
density) than their counterparts from diestrous rats.
The current study is the first to show differential expression of GH
mRNA during the estrous cycle. Past studies of the expression of GH
secretory activity using reverse hemolytic plaque assays (20) showed no
changes in the number of GH-secreting plaques with the bovine estrous
cycle. Indeed, most studies that have counted percentages of GH cells
in the cycling female rat have reported no significant differences (10, 11). The present studies reported differences in GH antigen-bearing
cells only when values from metestrous rats were compared with those
from males and proestrous female rats.
This study also is the first to show that differences in GH mRNA are
expressed by cells bearing GH or gonadotropin antigens. The lowest
expression of GH mRNA was in both GH cells and gonadotropes during
metestrus. The first increase in GH mRNA-bearing cells during diestrus
came from cells that stored GH antigens as well as those storing LHß
antigens. The timing of the increase in expression varied with the cell
type, however. This suggests a sequence of events that may underlie the
differentiation of this multihormonal cell.
Proposed steps toward differentiation of the multihormonal
subset
Significant changes in GH mRNA appeared first in the GH cell
population early in diestrus. This is seen as an increase in expression
of GH mRNA from 30% of GH cells during metestrus to 70% of GH cells
by the morning of diestrus. GHRH is known to be an important regulator
of the synthesis of GH mRNA (21, 22, 23, 24, 25, 26, 27, 28, 29, 30). These data suggest that the
GHRH-GH cell axis may be triggered early in the cycle to stimulate an
increase in GH mRNA synthesis.
Comparing these results with those in the previous study (10) also
showed that this increased transcriptional activity from metestrus to
diestrus occurs before the appearance of significant numbers of GH
cells with LH or FSH mRNAs. Hence, the data suggest that GH mRNA is the
first product from this multihormonal subset. At the same time,
however, there is a significant increase in the percentages of LH cells
with GH mRNA. Thus, collectively this suggests that the diestrous
multihormonal cells are producing three products: GH and LH antigens
and GH mRNA.
In our 1994 studies (10), we showed that 30% GH cells (defined by
antigen content) begin to express GnRH receptors (11) late in diestrus.
These data are consistent with a multihormonal cell type that contains
LH antigens. Thus, by late diestrous, we hypothesize that a fourth
product (GnRH receptors) is made that allows the population to respond
to GnRH pulses. On the morning of proestrus, there is an expansion in
the population of cells with GH antigens and LHß and FSHß mRNA. The
timing of appearance suggests that the GnRH receptors produced during
the previous 12 h have allowed the expression of gonadotropins
that eventually reach a peak of 12.5%15% of pituitary cells by the
afternoon of proestrus (10).
Finally, the present studies show some nonparallelism when LH and FSH
cells are compared. Whereas LH cells show an increase in GH mRNA during
diestrus, the same change is not seen in FSH cells until proestrus.
Furthermore, during peak expression periods late in proestrus, FSH is
found in a higher percentage of GH cells (60%) than LH (40%, 10).
Collectively, these findings suggest the later appearance of a subset
of GH-FSH bearing cells. Perhaps these cells support unique FSH
functions during early estrous.
Significance of the appearance of the multihormonal cells
The significance of the coexpression of GH and gonadotropins may
be related to their ability to secrete a "cocktail" of hormones
needed by the gonads, including GH. In 1993, Blumenfeld presented
evidence that GH plays a role in ovulation induction. He suggested that
GH may be a "co-gonadotropin" because it adds to or synergizes with
gonadotropins (31). There is evidence that GH is involved in a variety
of ovarian functions (31, 32, 33, 34, 35, 36). In a recent review Adashi (37) cited
several studies showing that GH is therapeutic in humans, particularly
in hypogonadotropic conditions. For example, GH may play a permissive
role in the onset of puberty. Also, patients resistant to ovulation
induction therapy could ovulate if given GH.
In rodents, human GH (hGH) prolongs the cycles (from 5 to 10 days) and
the period of fertility (37). In addition, partial suppression of
endogenous GH secretion resulted in delayed puberty in female rats. Rat
GH replacement therapy increased ovarian progesterone production (in
response to gonadotropins) and allowed normal puberty to occur.
Finally, GH replacement therapy restored follicular development in some
GH deficient mice (reviewed in Ref. 37). Recent studies of the Wistar
derived dwarf rat reported that rhGH therapy restored the reduced
ovarian weights and promoted development of intermediate follicles
(38). The phenotypic defects in the reproductive system of these
GH-deficient animals (39, 40) and the fact that some defects can be
corrected with GH therapy (37, 38, 41, 42) points to the importance of
GH in normal development of sperm and ovarian follicles.
To summarize, this study has added more information about the GH
phenotypic characteristics shared by subsets of GH and gonadotropin
cells in adult pituitaries. The study shows that gonadotropes may
express significant levels of GH mRNA in both male and female rats,
especially females in early proestrus. This supports the working
hypothesis that the multihormonal cell is derived from a subset of
cells that can express normal somatotropic and gonadotropic phenotypes.
It adds information about the significant expression of GH mRNA in male
rat gonadotropes that suggests that GH may be important in the
regulation of the male reproductive system as well. The appearance of
this multihormonal cell may not be solely the result of GH binding and
endocytosis by gonadotropes as was suggested after our 1994 studies
(10, 11), although autocrine or paracrine regulation by GH cannot be
ruled out (43, 44).
Finally, the study confirms sex differences in the expression of GH
mRNA reported previously, but only if male rats are compared with
females rats in metestrus, diestrus, or estrus. This study is also the
first to show differential expression of GH mRNA during the estrous
cycle that suggests that expression might be regulated by hormones in
the reproductive axis. Because the sex differences in GH expression are
clearly affected by changing the steroid feedback (gonadectomy), this
presents candidate hormones for further tests of this hypothesis.
 |
Acknowledgments
|
|---|
The authors would like to thank Diana Rougeau for technical
assistance and would like to acknowledge the gift of antisera to bovine
LHß from Dr. J. G. Pierce and that to human FSHß from Dr.
A. F. Parlow.
 |
Footnotes
|
|---|
1 This study was supported by NIH Grant HD-33915 along with salary
support from NSF-IBN-9724066. 
Received October 27, 1999.
 |
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