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Department of Anatomy and Neurosciences, University of Texas Medical Branch (G.V.C., B.T.M.), Galveston, Texas 77555-1043; and the Department of Biochemistry, North Carolina State University (W.L.M.), Raleigh, North Carolina 27695
Address all correspondence and requests for reprints to: Gwen V. Childs, Ph.D., Department of Anatomy and Neurosciences, MRB 10–104, 303 University Boulevard, University of Texas Medical Branch, Galveston, Texas 77555-1043. E-mail: childs{at}mbian.utmb.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
one of two ß-subunits (ßA and ßB) (1, 2, 3, 4, 5, 6). Inhibin belongs to the
transforming growth factor-ß superfamily, which plays a role in
differentiation and development in a number of tissues. A primary
source of inhibin production is in the testes or ovary, but both -
and ß-subunits may also be produced in the pituitary (6).
Inhibin inhibits basal and GnRH-mediated FSH synthesis and release
(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Although it does not block LH synthesis, it also inhibits LH
secretion (14, 15, 16, 17, 18, 19). It causes decreases in FSHß (12, 20),
-subunit, and FSHß messenger RNA (mRNA) (20, 21, 22, 23). Its effects on
LH release suggests that its target cells may include gonadotropes that
carry both hormones (bihormonal cells) as well as gonadotropes that
store only FSH. However, no one has identified specific inhibin target
cells in the pituitary.
Inhibin may act on GnRH-mediated gonadotropin secretion partly by a reduction in pituitary receptivity to GnRH (24, 25, 26, 27, 28). Wang et al. reported that exposure to maximal doses of inhibin reduced binding to a GnRH analog to 42% of control values after 72 h (24, 25) and blocked up-regulation of GnRH-binding sites by the calcium ionophore A23187 (24, 25). Inhibin did not compete for GnRH-binding sites, nor did it affect binding affinity, cell number, or cell viability (24, 25). Braden et al. reported that inhibin did not block the rate of GnRH receptor synthesis (26). However, inhibin did block the stimulation of homologous receptor synthesis by GnRH (26, 27, 28). Thus, inhibin’s effects are seen at the level of transcription of FSH mRNA and down-regulation of GnRH receptors.
During the past decade, we have been interested in defining factors that mediate structural and functional changes in pituitary gonadotropes as they approach the proestrous and estrous surges. One of these changes is an increase in the expression of GnRH receptors during diestrus (reviewed in Ref.29). Our cytochemical studies showed that this was due to a 4-fold increase in the percentage of cells that bound a biotinylated analog of [D-Lys6]GnRH (Bio-GnRH) from metestrus (diestrous day 1) to the morning of proestrus (29).
In subsequent studies, the cells were further identified with dual labeling for gonadotropins (30). During peak expression of GnRH receptivity, there was a parallel increase in Bio-GnRH-bound cells containing LHß or FSHß antigens. By the time of the peak period, over 90% of the new Bio-GnRH target cells were gonadotropes. Earlier studies had shown that cells with GH antigens expressed LH or FSH mRNA during this same period (31), and they express Bio-GnRH receptors as well.
Recent ongoing cytochemical studies have focused on studies of cellular mechanisms behind the inhibin-mediated decline in GnRH receptivity (24, 25, 26, 27, 28). Inhibin could cause a decrease in the percentage of Bio-GnRH-receptive cells by reducing receptivity in selected target cells, or it may cause a decline in the receptivity in each target cell. Because of its more pronounced effects on regulation of FSH cells, the major cell type affected by inhibin might be a monohormonal FSH cell. This hypothesis would be proved by a reduction in Bio-GnRH binding to cells with FSH antigens along with little or no changes on cells with LH or GH antigens. However, its effects on LH secretion (14, 15, 16, 17, 18, 19) suggest that these cells may be targets as well.
The studies presented in this report identify inhibin’s effects on Bio-GnRH target cells during the period when GnRH receptivity is increasing to a peak (from diestrous morning to proestrous morning) (29, 30). We also tested inhibin’s effects on storage of FSH and LH in individual cells to learn more about the mechanisms behind its inhibitory effects.
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Materials and Methods |
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Synthesis and characterization of Bio-GnRH
The Bio-GnRH analogs were produced and characterized as
described in previous studies (32). In this
D-Lys6 analog of GnRH, the valeric acid
side-chain of biotin was directly attached to the 
amino group of
D-Lys6. We also employed a new Bio-GnRH
derivative with a spacer arm of 6-aminohexanoic acid between the biotin
moiety and the D-Lys6 residue (32). This long
chain biotinylated [D-Lys6]GnRH analog
(LCBio-GnRH) was synthesized and purified by reverse phase HPLC as
previously described (30). As in our recent report, amino acid
compositional analysis was used to verify monobiotinylation and to
quantify the purified peptide derivatives (32).
The previous report showed that the LCBio-GnRH was as active as our previous probe, which lacked a spacer arm (0.9–9 nM stimulated 2- to 4.5-fold increases in LH release from cultured pituitary cells). It was as sensitive as our original biotinylated probe in cytochemical tests (30). Analysis of different concentrations of the Bio-GnRH analog showed that maximal numbers of labeled cells (16–18% of a proestrous pituitary cell population) were detected after exposure to 0.9 nM LCBio-GnRH for 10 min. For the purpose of simplicity, we refer to both analogs as Bio-GnRH.
Treatment of the pituitary cells with inhibin
The pituitary cells were dispersed and plated on glass
coverslips in 24-well trays as described previously (30, 31). They were
divided into 3 experimental groups. Group 1 received vehicle, and
groups 2 and 3 received either porcine follicular fluid (100 ng/ml) or
recombinant inhibin (from Genentech, South San Francisco, CA; 10
ng/ml). The 32-kDa human recombinant inhibin or porcine follicular
fluid was diluted in DMEM containing insulin, transferrin, sodium
selenite, 0.25% BSA, and aprotinin (100 kallikrein inhibitor units).
The incubation period for these peptides was 24 h. At the end of
the pretreatment period, media were collected and frozen at -20 C for
LH and FSH RIAs to learn whether inhibin affected basal secretion. The
RIAs were performed with kits from the Hormone Distribution Office,
NIH, as previously described (33, 34).
To learn whether the population retained the memory of their level of GnRH receptor expression, we labeled parallel groups of cells either 1 or 24 h after plating. There were no changes in the percentages of Bio-GnRH-bound cells during the 24-h plating period. This agreed with previous studies which showed that the proportion of Bio-GnRH target cells expressed at each stage of the cycle was retained during the 24-h incubation needed for the tests of effects of inhibin (29, 30).
Cytochemical detection of Bio-GnRH and antigens
The protocol was similar to that first published in 1983 (35, 36). On the day of the experiment, the cells were washed gently in two
changes of DMEM. Then they were stimulated with 1 nM
Bio-GnRH for 10 min. This time has been shown to result in maximal
labeling of target cells (35, 36). The cells were then fixed
immediately in 2% glutaraldehyde. Bio-GnRH was detected with
avidin-biotin peroxidase complex, as described previously (29, 30, 31, 32, 33, 34, 35, 36). The
controls for the detection protocol for Bio-GnRH binding involved
omission of Bio-GnRH from three wells per tray. Previous studies had
shown that excess nonbiotinylated GnRH competed for binding sites and
prevented labeling (30, 35, 36).
After the detection of Bio-GnRH, the cells were immunolabeled for LHß, FSHß, or GH as described previously (30, 35, 36). The antiserum sources and dilutions were described recently (29, 30). Immunolabeling controls in a dual labeling protocol included the use of primary antisera absorbed with 100-1000 ng/ml specific antigens. This absorption abolished labeling for each antigen. This confirmed the specificity of the immunolabeling protocols and showed that the second label was the result of binding by the primary antibody and not of the residual activity from the first detection system.
Density measurements
Analysis of the density of antigen storage or Bio-GnRH binding
was performed with the BioQuant MEG IV system, which includes a 486–66
PC and a Sony color videocamera. The system has an automatic background
correction that prevents differential readings due to different
lighting conditions. The corrective common background was read on an
empty brightfield view of a sample slide and then used to correct the
background in each analyzed image. This did not affect the density of
the label over the cells. No further image processing was performed.
Each measurement session collected data from all experimental groups,
sampling at least 20 cells/group during each session.
To detect changes in label density, the thresholding functions were activated. The computer then read the entire range of densities of the label. The pixels over the label were highlighted automatically after the cell was drawn. The read-out was the average density of pixels over the label. At the same time, the area of the labeled cell was obtained by drawing around it. In the analysis of the Bio-GnRH label, the area of the label was calculated after its detection. Essentially it was the area of the highlighted pixels. The analysis surveyed 100 cells/experimental group from 3 separate experiments.
Statistical analysis of data
In the group of proestrous rats, there were a total of 5
separate experiments that included inhibin treatments. The experiments
with diestrous rats were repeated 4 times. Cells from at least 8
rats/stage of the cycle were tested. We counted at least 200 cells on
each coverslip for 3 coverslips/experiment. A single experiment yielded
an average of these 3 coverslips, which was later averaged with
averages from 4–5 replications to produce the final data point.
One-way ANOVA detected significant changes with the stage of the cycle.
Duncan’s multiple range test was used (at the 5% level) to learn
which data points were different.
After the counts, we inserted the raw data in an Excel spreadsheet, which included formulas designed to calculate the percentage of each subtype of labeled cell. This allowed a comparison of counts from single and dual labeling protocols to learn whether the dual labeling protocol had interfered with either the detection of Bio-GnRH or the antigens.
In addition, separate single labeling protocols were run to detect the antigens only. These data were correlated with those from the dual labeling protocols to learn whether the antigens had been washed out or masked by the double labels. This also provided a set of cells that could be analyzed by densitometry for changes in cell area and density of hormone stores.
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Results |
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Quantitative analysis of Bio-GnRH binding to cells from diestrous
or proestrous female rats
Inhibin caused a reduction in the overall percentages of Bio-GnRH
target cells in the pituitary cell population. In diestrous rats, the
average percentages of cells with Bio-GnRH-binding sites was 10.84
± 1% (±SD). After 24 h in inhibin, a slight
reduction in binding to 7.6 ± 0.9% was seen (P
< 0.02; Fig. 1
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was 16.42 ± 1%
Bio-GnRH-bound cells. Inhibin caused a pronounced decrease in the
percentage of Bio-GnRH-target cells in the proestrous group. The
percentage was reduced to 7.5 ± 0.25% (P <
0.0001).
Image analyses focused on the density and area of label for Bio-GnRH on
target cells. The density of labeling for Bio-GnRH was decreased in
inhibin-treated cells from proestrous rats. On a scale of 0–255, the
mean density reading was 88 ± 2 in the control animals. After
inhibin treatment, the density was reduced to a reading of 115 ±
4 (P < 0.05). (Please note that a higher optical
density number means that more light was transmitted, which translates
to a lower density.) This change is best seen as a histogram showing
the range in densities of the label for Bio-GnRH on individual cells.
This figure illustrates the inhibin-mediated shift to target cells
labeled at lower densities (Fig. 2).
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Figures 3 and 4 illustrate binding by
Bio-GnRH with the long chain analog to a cell with FSH antigens. Label
for Bio-GnRH is seen as a patch of purple (black in this micrograph) on
the cell surface. Each figure shows two attached FSH cells. However,
Bio-GnRH binding is found on only one of the cells in each field. The
labeling characteristics are identical to those previously described
(29, 30, 35, 36). The gray label in the cell is the immunolabel for
FSHß. These cells were from proestrous female rats.
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is from a field treated for 24 h with
inhibin, also taken from proestrous rats. The label for Bio-GnRH is
faint on the cell with FSH antigens. It appears as a faint purple
(light gray) line that is difficult to distinguish in light
micrographs. Labeling for FSH is either very light or more dense, as
shown by the arrow.
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shows a cell with LH antigens that is
also densely labeled for Bio-GnRH. It resembles cells from the control
field. Some LH cells, however, have no labeling for Bio-GnRH. The cells
labeled for GH antigens also showed a mixed pattern, similar to that
seen in LH cells (data not shown).
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). Thus, part of the change in FSH hormone content
measured by other workers (12) reflects an actual decline in the number
of FSH-expressing cells during that 24-h treatment period.
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that depicts the shift in the OD of FSH stores from control and
inhibin-treated samples. Note that the cells that label most intensely
for FSH are no longer seen in the inhibin-treated population. Also,
whereas cells with stores in the middensity range remain, most of the
remaining cells have lower FSH storage levels than those found in the
control fields.
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). However, there was a slight reduction in the average area of
LH antigen-bearing cells from 86 ± 3 to 72 ± 2
µm2 (P < 0.05). There was also a slight
reduction in the density of label for LH stores from an OD of 95
± 1 to 100 ± 1 (P < 0.05). This shift in
labeling density is illustrated by the histogram in Fig. 9. Whereas the shift is not as pronounced as that for
FSH, one can see more cells with low stores of LH after inhibin
treatment. Finally, there were no changes in the percentages of GH
cells or the density of GH stores after inhibin treatment.
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shows that 42 ± 5% of FSH
cells and 52 ± 8% of LH cells bound Bio-GnRH in populations from
control diestrous rats. Inhibin caused a slight reduction in Bio-GnRH
binding by both cell types. The fields dual labeled for Bio-GnRH and GH
showed no effect of inhibin on Bio-GnRH binding by GH-bearing
cells.
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shows that in control
fields, over 80% (83–85%) of LHß or FSHß antigen-bearing cells
bound Bio-GnRH, as previously described (30). However, inhibin reduced
binding to only 32 ± 11% of that to FSH cells (P
< 0.001) and 40 ± 11% of that to LH cells (P <
0.001). Figure 11 also shows that inhibin caused a decrease in Bio-GnRH
binding to somatotropes from 38% ± 4% to 21 ± 3% of that to
GH cells (P < 0.002).
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Discussion |
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This study has identified some of the specific cellular changes behind these reductions. These could include reduced numbers of hormone-storing cells, levels of storage in each cell, and/or receptivity to GnRH. A reduction in GnRH receptivity could reflect a loss in Bio-GnRH target cells, or it could signify lowered receptivity by preexisting cells. Each of these results would result in a different change in the cytochemical labeling patterns.
In this study, inhibin not only decreased the percentage of Bio-GnRH receptive cells, but it also decreased the area and density of the label for Bio-GnRH on the remaining cells. Thus, the inhibition is the combined effect of losses in target cells and losses in receptivity on individual LH or FSH cells.
Attardi et al. have shown that inhibin decreased the production of FSHß mRNA by 62% as early as 2 h after exposure (22). It was undetectable after 6 h. This correlates well with the multiple actions on FSH cells assayed in the present study. These include a reduction in FSH ß-subunit stores assayed by densitometry, the area of FSH cells, and the percentages of gonadotropes that express FSH antigens, especially in populations from proestrous rats. Finally, inhibin had a severe effect on Bio-GnRH binding by FSH cells. The decrease in binding to 32% of FSH cells correlates well with its known inhibitory effects on GnRH-mediated FSH secretion. Thus, FSH cells contributed significantly to the overall loss in Bio-GnRH-receptive cells seen in the counts.
In contrast, inhibin did not have as broad an effect on LH cells. Whereas it did reduce LH cell area and LH ß-subunit stores slightly, it had no effect on the percentages of LH cells. Thus, the mechanisms behind inhibin’s inhibitory actions on LH cells may focus on a decrease in Bio-GnRH binding, because inhibin causes a 50% reduction in binding by cells with LH antigens. These data correlate well with those of earlier studies that demonstrated inhibin’s suppressive effects on GnRH-mediated LH secretion (19). Thus, the reduction in LH secretion may be the combined effects of a reduction in Bio-GnRH receptivity and LH stores.
Inhibin’s more pronounced effect on FSH cells compares well with those reported by Kotsuji et al. (19), who found that inhibin suppressed LH release to 80% of control values and suppressed FSH release to 68% of control values. Their studies were performed on male rat pituitary cells. Nevertheless, the differential responses by LH and FSH cells compare favorably with the changes reported for the female. It is interesting to note that the suppressive effects reported by Kotsuji et al. (19) were only seen in the cultures exposed to inhibin for 3 h. In the 24-h exposure, only GnRH-mediated FSH secretion was suppressed. The difference could reflect sex differences in responses to inhibin or the fact that we used cells from proestrous rats, which were at their peak levels of GnRH receptivity. Perhaps they were more responsive to the prolonged inhibin pretreatment.
It is also interesting to compare the responses to inhibin of rats and sheep (37). Whereas inhibin continues to suppress FSH expression by 60–80% in estrous sheep pituitaries, it actually stimulates GnRH binding 3- to 6-fold (38, 39, 40, 41, 42), increases GnRH-stimulated calcium signaling, and enhances LH expression by at least 60%. Thus, it may play a dynamic role in the facilitation of LH production during sheep estrous cycles (37, 38, 39, 40, 41, 42).
Our dual labeling evidence allows us to predict that most inhibin target cells are bihormonal gonadotropes. We already know that over 80% of Bio-GnRH-receptive cells from proestrous rats contain LH or FSH antigens (in a given dual labeling protocol) (30) and that 75% of antigen-bearing gonadotropes are bihormonal (43). Thus, a 60% reduction in the percentage of target cells during proestrous probably reflects this abundant subset. The more pronounced suppressive effects on FSH cell numbers and Bio-GnRH binding would then reflect a selective inhibition of FSH activity in these same bihormonal cells. However, it may also reflect inhibition of activity in monohormonal FSH cells. Monohormonal FSH cells represent about 15–25% of the entire gonadotrope population depending on the stage of the cycle (43). Future cytochemical studies would be needed to test specific effects of inhibin on this subset.
Finally, the effects on GH cells from proestrous rats correlate well with our recent studies of their expression of gonadotropin mRNAs (31) and Bio-GnRH receptors (30) during late diestrus and early proestrus. GH cells with gonadotropin mRNAs represent a subset of somatotropes (40–60% of GH cells) or mRNA-bearing gonadotropes (31). This transient expression augments the gonadotropes during this period of the cycle. Inhibin’s effects on Bio-GnRH binding by GH cells probably reflect its actions on the bihormonal gonadotropes that contain GH antigens (30).
To summarize, our working hypothesis for this study was that inhibin might preferentially act on cells with FSH antigens. This was tempered by the fact that early studies had shown effects on LH secretion (14, 15, 16, 17, 18, 19). Thus, an alternative hypothesis was that inhibin might affect bihormonal cells with LH and FSH stores. Because the data show multiple effects of inhibin on cells with both LH and FSH stores, they support the latter hypothesis. Collectively, the effects of inhibin suggest actions on the multihormonal gonadotrope that predominates late in proestrus.
We also hypothesized that inhibin might down-regulate GnRH receptors by either reducing the number of target cells or reducing the Bio-GnRH binding by individual cells. The data support both hypotheses. Finally, in all parameters measured, the most profound effects are seen in the population from proestrous rats. Whereas there is no evidence for a change in activin receptors (which might bind inhibin) during the cycle (44), these data suggest a differential responsiveness to inhibin that might allow it to act to control levels of gonadotropins after the high proestrous secretory activity.
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Acknowledgments |
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Footnotes |
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Received November 11, 1996.
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and ß subunits in various
tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247–251This article has been cited by other articles:
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