Differential Effects of Inhibin on Gonadotropin Stores and ...

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0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 4 Printed in U.S.A.

Differential Effects of Inhibin on Gonadotropin Stores and Gonadotropin-Releasing Hormone Binding to Pituitary Cells from Cycling Female Rats* GWEN V. CHILDS, BRIAN T. MILLER,

AND

WILLIAM L. MILLER

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 ABSTRACT Numerous studies of rat pituitaries have reported that inhibin suppresses the synthesis and release of FSH and decreases the release of LH. The latter effect seems to be related to the down-regulation of receptors for GnRH. The studies reported here identified cellular changes behind the inhibitory effects of inhibin on gonadotropes to learn whether its effects are mediated by changes in subtypes of gonadotropes. Cell populations from diestrous day 2 and proestrous (morning) rats were collected, dispersed to single cell populations, and plated in medium containing either recombinant 32kDa inhibin or porcine follicular fluid for 24 h. GnRH binding was detected by exposing the cells to a biotinylated analog (Bio-GnRH) for 10 min before fixation, followed by avidin-peroxidase labeling protocols to detect the biotin on the analog. In parallel fields, the cells were further identified by immunolabeling for LH or FSH b-subunits or for GH with a different colored reaction product. The most striking changes were seen in cells from proestrous rats. Inhibin reduced the percentages of Bio-GnRH target cells in the population by 60% and the area and density of Bio-GnRH label on the remaining cells. Inhibin reduced the percentages of FSH cells by 30% and caused nearly a 60% reduction in the binding of Bio-GnRH by this cell type (from 83% of

FSH cells to 32% of FSH cells). Inhibin also reduced the area of FSH cells and the density of FSH stores. Inhibin’s effects on LH cells were limited to a reduction in the area of the cells and the density of LH stores, but not the number of LH cells. In addition, it reduced the percentages of LH cells with Bio-GnRH receptors from 84% to 40%. When cells with GH were analyzed, inhibin had no effect on their percentages, areas, or GH stores. In populations from proestrous rats, inhibin reduced the percentages of GH cells with Bio-GnRH binding from 38% to 21%. These data suggest that inhibin’s target cell is the abundant multihormonal gonadotrope that contains LH, FSH, and GH and predominates during proestrus. Inhibin’s effects are most severe on FSH cells, which suggests that it may either selectively affect FSH synthesis and stores in bihormonal gonadotropes and/or affect monohormonal FSH cells. Thus, mechanisms behind its inhibitory effects include 1) a reduction in the percentage of Bio-GnRH target cells, 2) a reduction in the area of Bio-GnRH-binding sites on individual cells, and 3) a reduction in the stores of FSH and the percentages of FSH cells. These last effects are consistent with known reductions in FSH synthesis. The effects of inhibin on LH secretion may be secondary to the effects on Bio-GnRH receptors in bihormonal gonadotropes. (Endocrinology 138: 1577–1584, 1997)

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mones (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 –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 –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

NHIBIN IS a regulatory peptide that inhibits FSH synthesis and release (1–3). It was discovered in 1932 as a factor that suppressed pituitary gonadotrope hypertrophy after castration and was eventually identified as a heterodimer of an a and one of two b-subunits (bA and bB) (1– 6). Inhibin belongs to the transforming growth factor-b 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 a- and b-subunits may also be produced in the pituitary (6). Inhibin inhibits basal and GnRH-mediated FSH synthesis and release (7–20). Although it does not block LH synthesis, it also inhibits LH secretion (14 –19). It causes decreases in FSHb (12, 20), a-subunit, and FSHb messenger RNA (mRNA) (20 –23). Its effects on LH release suggests that its target cells may include gonadotropes that carry both horReceived November 11, 1996. 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: [email protected]. * This work was supported by NIH Grants R01-HD-15472 and R01HD-33919 (to G.V.C.) and a developmental grant from the Sealy Smith Foundation. This study was presented in poster format at the 1996 meeting of The Endocrine Society.

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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 LHb or FSHb antigens. By the time of the peak period, over 90% of the new BioGnRH 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 BioGnRH receptors as well. Recent ongoing cytochemical studies have focused on studies of cellular mechanisms behind the inhibin-mediated decline in GnRH receptivity (24 –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 –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. Materials and Methods Animal care and handling Female Sprague-Dawley rats were purchased from Harlan SpragueDawley (Houston, TX). They were acclimated to a 12 h on, 12 h off light-dark cycle for 7–10 days with food and water ad libitum, as previously described (29 –31). Vaginal smears were performed daily, and we used the rats only after they had completed two sequential normal 4-day cycles. The institutional review committee reviewed and approved the animal care and use protocol annually. In this study, we used rats taken during the morning of either diestrous day 2 or proestrus (1000 h).

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 e 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 2to 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.

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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 220 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 –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 LHb, FSHb, 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

EFFECTS OF INHIBIN ON GONADOTROPIN STORES AND GnRH BINDING 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.

Results Effects of inhibin pretreatment on gonadotropin secretion and Bio-GnRH binding

In agreement with earlier studies (14 –19), inhibin caused a significant 30 – 60% decrease in basal FSH secretion in cell populations from both diestrous and proestrous rats (by Duncan’s multiple range test, P , 0.05). However, it did not affect basal LH secretion. Inhibin also decreased LH or FSH secretion mediated by Bio-GnRH (during the 10-min pulse). This correlated with its effects on Bio-GnRH binding. 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 BioGnRH-binding sites was 10.84 6 1% (6sd). After 24 h in inhibin, a slight reduction in binding to 7.6 6 0.9% was seen (P , 0.02; Fig. 1). As previously reported (29, 30), peak expression of BioGnRH binding is normally seen on the morning of proestrus (16 –18% Bio-GnRH-bound cells). The average shown in Fig. 1 was 16.42 6 1% Bio-GnRH-bound cells. Inhibin caused a pronounced decrease in the percentage of Bio-GnRH-target

FIG. 1. Analysis of the percentages of Bio-GnRH-bound cells in cell populations from diestrous day 2 (mornining) or proestrous (morning) rats after inhibin treatment. *, Significantly different from untreated control values (P , 0.02).

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cells in the proestrous group. The percentage was reduced to 7.5 6 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 BioGnRH was decreased in inhibin-treated cells from proestrous rats. On a scale of 0 –255, the mean density reading was 88 6 2 in the control animals. After inhibin treatment, the density was reduced to a reading of 115 6 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). The average area of label on Bio-GnRH target cells from proestrous rats was reduced by 50% from 16 6 1 to 8 6 1.3 mm2 after inhibin treatment (P , 0.005). However, when we measured the area of each Bio-GnRH target cell, there were no changes after inhibin treatment. Among diestrous female rats, there were no changes in the density or area of label for Bio-GnRH, nor were there changes in the average area of the Bio-GnRH target cells. 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 FSHb. These cells were from proestrous female rats. Figure 5 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. A similar pattern is seen in inhibin-treated cells labeled for LHb antigens. Figure 6 shows a cell with LH antigens that is

FIG. 2. The density of label for Bio-GnRH was assayed on individual target cells after the cytochemical detection protocol was run. This histogram shows the shift in the density to lower values after inhibin treatment. A lower density allows more light to be transmitted. Therefore, the optical density reading is actually higher and shifted to the right.

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FIG. 3. Shown are two attached FSH cells. Labeling for Bio-GnRH (arrows) is dense on one of the FSH gonadotropes in a patch. The FSH label (F) is gray and distributed throughout the cells. U, Unlabeled cell; n, nucleus. Magnification, 32700; bar 5 5m.

FIG. 4. Shown are two attached FSH cells. Labeling for Bio-GnRH (arrows) is distributed over a broader area than that shown in Fig. 3. See Fig. 3 for details.

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). Effects of inhibin on FSH storage

The counts of FSH antigen-bearing cells showed that inhibin did not reduce the percentage of FSH cells in popula-

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FIG. 5. After inhibin treatment, the labeling for Bio-GnRH appears to be a faint line on some of the cells. On others, it is missing altogether. Shown is a cluster of four cells. One FSH cells contains faint label for Bio-GnRH (arrow) and a denser label for FSH. The other cell contains very pale label for FSH and no detectable Bio-GnRH binding. n, Nucleus. Magnification, 32700. Bar 5 5 mm.

FIG. 6. After inhibin treatment, the labeling for Bio-GnRH appears to be a faint line on some of the cells. On others, it is missing altogether. Shown is a LH cell with patches of label for Bio-GnRH (arrow). It is attached to an unlabeled cell (U). See Fig. 5 for details.

tions from diestrous rats (P 5 0.233; data not shown). However, inhibin did cause a significant reduction in the percentage of cells expressing FSHb from proestrous rats (P , 0.01; Fig. 7). 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.

EFFECTS OF INHIBIN ON GONADOTROPIN STORES AND GnRH BINDING

Because inhibin’s effects were most pronounced on cells from proestrous rats, the density measurements of FSH and LH b-subunit stores focused only on this group. When the average area of FSH cells was measured after inhibin treatment, there was a slight decrease from an average of 97 6 4 to 87 6 3 mm2 (P , 0.05). There was also a decline in the density of the label for FSH from an optical density (OD) of 96 6 1 to 107 6 1 (P , 0.05; note, as stated above, a lower density is read as a higher OD value). This overall shift in the FSH cell population is shown by the histogram in Fig. 8 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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Effects of inhibin on LH and GH stores

The cell counts showed no changes in the percentages of LH cells from either diestrous or proestrous populations pretreated with inhibin (Fig. 7). However, there was a slight reduction in the average area of LH antigen-bearing cells from 86 6 3 to 72 6 2 mm2 (P , 0.05). There was also a slight reduction in the density of label for LH stores from an OD of 95 6 1 to 100 6 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. Effects of inhibin on Bio-GnRH target cells identified by their content of hormone antigens

An analysis of cells dual labeled for Bio-GnRH and LH, FSH, or GH antigens was performed to identify the target cells most affected by inhibin. Figure 10 shows that 42 6 5% of FSH cells and 52 6 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. The most profound changes were seen after inhibin treatment of cells from proestrous rats. Figure 11 shows that in control fields, over 80% (83– 85%) of LHb or FSHb antigenbearing cells bound Bio-GnRH, as previously described (30). However, inhibin reduced binding to only 32 6 11% of that to FSH cells (P , 0.001) and 40 6 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% 6 4% to 21 6 3% of that to GH cells (P , 0.002). Discussion FIG. 7. In cell populations from proestrous rats, inhibin has no effect on LH or GH cells. However, there is a decline in the percentage of cells with FSH stores. *, Significantly different from control values (untreated), P , 0.01.

FIG. 8. Image analysis was used to measure the density of FSH stores in individual cells. After inhibin treatment, the histogram shows a shift to a lower density (seen as a higher optical density reading). The cells with high density stores seen in the control population are missing from the inhibintreated population.

Inhibin affects FSH cells by reducing basal and GnRHmediated FSH secretion (1–15). It also reduces FSH stores and mRNA (20 –23). It has a less pronounced and variable effect

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FIG. 9. When image analysis of LH stores in individual cells was assayed, there was no shift to the right as is seen in the histogram illustrating the changes in the density of FSH stores. However, there were fewer cells with high density stores and more with stores of the lowest density in the inhibintreated fields.

FIG. 10. Dual labeling was used to further identify Bio-GnRH target cells. Among diestrous rats, there was a significant reduction in the percentages of cells with FSH or LH antigens that bound Bio-GnRH. Thus, whereas inhibin does not cause a reduction in the percentages of LH or FSH cells in this population, it does reduce their expression of Bio-GnRH binding. *, Significantly different from untreated cells.

on LH secretion (14 –19). In addition, inhibin decreases GnRH receptivity in the rat pituitary population by downregulating GnRH receptors (24 –28). 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.

FIG. 11. Analysis of dual labeling in the proestrous rat populations showed a striking decrease in the percentage of LH or FSH cells bound by Bio-GnRH. In addition, there was a significant decline in the percentage of cells with GH antigens that bound GnRH. This may reflect changes in the multihormonal gonadotropes that contain GH antigens. *, Significantly different from untreated cells.

Attardi et al. have shown that inhibin decreased the production of FSHb 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 b-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 b-subunit

EFFECTS OF INHIBIN ON GONADOTROPIN STORES AND GnRH BINDING

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 – 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– 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 an-

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tigens. This was tempered by the fact that early studies had shown effects on LH secretion (14 –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. Acknowledgments The authors acknowledge the excellent technical assistance of Geda Unabia and Diana Rougeau throughout this study. We also thank Genentech for the recombinant inhibin and the Hormone Distribution Program (Dr. A. F. Parlow) for the antisera to rat and human FSH or rat GH. We thank Dr. J. G. Pierce for the antisera to bovine b-subunit of LH.

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