Cytochemical Studies of the Effects of Activin on Gonadotropin-releasing Hormone (GnRH) Binding by Pituitary Gonadotropes and Growth Hormone Cells

Abstract

Activin stimulates the synthesis and secretion of follicle-stimulating hormone (FSH). It inhibits the synthesis and release of growth hormone (GH). It acts on gonadotropes by stimulating the synthesis of gonadotropin-releasing hormone (GnRH) receptors. To test activin's effects on GnRH target cells, pituitary cells from diestrous or proestrous rats were exposed to media with and without 60 ng/ml activin for 24 hr and stimulated with biotinylated GnRH (Bio-GnRH). The populations were double-labeled for Bio-GnRH and/or luteinizing hormone-β (LH-β), FSH-β, or GH antigens. In both diestrous and proestrous rats, activin stimulated more LH and FSH cells and increased the percentages of GnRH target cells. In diestrous rats, activin stimulated increases in the average area and density of Bio-GnRH label on target cells. In addition, more FSH, LH, and GH cells bound Bio-GnRH. The increment in binding by gonadotropes was not as great as that normally seen from diestrus to proestrus, suggesting that additional factors (such as estradiol) may be needed. These data suggest that activin plays an important role in the augmentation of Bio-GnRH target cells normally seen before ovulation. Its actions on GH cells may reflect a role in the transitory change from a somatotrope to a somatogonadotrope that is seen from diestrus to proestrus.

Keywords

anterior pituitary activin gonadotrope growth hormone follicle-stimulating hormone luteinizing hormone gonadotropin-releasing hormone receptors rat

Activin is a regulatory peptide belonging to the transforming growth factor-β (TGFβ) superfamily that plays a role in differentiation and development in a number of tissues (Kingsley 1994). It was discovered during purification of inhibin from ovarian follicular fluid and is a dimer of the two inhibin β-subunits (Ling et al. 1986; Ying 1988; De Kretser and Robertson 1989; Vale et al. 1986, 1990). Activin stimulates follicle-stimulating hormone (FSH) synthesis and release (Ling et al. 1986; Vale et al. 1986; Carrol et al. 1988; Kitaoka et al. 1988). Less striking effects are also seen on luteinizing hormone (LH) secretion (Kitaoka et al. 1988; Vale et al. 1988; Stouffer et al. 1993; Knight 1996).

Activin also stimulates gonadotropes by increasing the synthesis of gonadotropin-releasing hormone (GnRH) receptor mRNA and proteins (Braden and Conn 1990, 1991; Conn et al. 1995; Fernandez-Vazquez et al. 1995). Experimental evidence has suggested additional roles for activin, however (Kitaoka et al. 1988; Billestrup et al. 1990; Struthers et al. 1992; Bilezikjian et al. 1990, 1991, 1993). Among these are inhibition of growth hormone (GH) synthesis and release (Kitaoka et al. 1988; Struthers et al. 1992; Billestrup et al. 1990; Bilezikjian et al. 1990, 1993) and proliferation of somatotropes (Billestrup et al. 1990).

In cycling female rats, the gonadotrope population increases in number, storage, and GnRH receptivity during diestrus in preparation for the peak secretory activity before ovulation (proestrus) (Lloyd and Childs 1988; Childs et al. 1987, 1992a,b, 1994a,b). Some of this increase is due to the onset of expression of LH-β and FSH-β mRNAs by somatotropes (Childs et al. 1994a). These somatotropes also increase their expression of GnRH receptors (Childs et al. 1994b) during diestrus and proestrus, which suggests they may function as transitory somatogonadotropes. We hypothesized that, because of its stimulatory effects on the production of GnRH receptor mRNAs and proteins (Braden and Conn 1990, 1991; Conn et al. 1995; Fernandez-Vazquez et al. 1995), activin may mediate some of these changes in the somatotrope population. This report presents the results of studies that tested this hypothesis. As in our recent studies (Childs et al. 1994b, 1997), GnRH-receptive cells were identified by affinity cytochemistry during the period of upregulation of GnRH receptors (diestrus to proestrus) (Park et al. 1976; Clayton et al. 1980; Savoy-Moore et al. 1980; Lloyd and Childs 1988; Funabashi et al. 1994). They were further identified by their content of gonadotropins or growth hormone antigens with double-labeling techniques (Childs et al. 1983a,b, 1994a,b, 1997). The purpose of this study was to learn if activin changes the expression of GnRH receptors by any of the GnRH target cells.

Materials and Methods

Animal Care and Handling

Female rats (Harlan Sprague-Dawley; Houston, TX) were acclimated in a 14-hr on (0600 hr), 10 hr off (2000 hr) light-dark cycle for 7–10 days with food and water ad libitum as previously described (Childs 1992a,b, 1994a,b). The stage of the cycle was determined via daily vaginal smears. The rats were used 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.

The rats were sacrificed during the morning of diestrus II or proestrus (1000 hr). This was to test affects of activin during the period of upregulation of GnRH receptors. GnRH receptor assays show that the receptors are low during the morning of diestrus II. Receptors then rise to reach a peak by the morning of proestrus. This is followed by a decrease by the afternoon of proestrus, which reaches a nadir during estrus (Park et al. 1976; Clayton et al. 1980; Savoy-Moore et al. 1980).

Synthesis and Characterization of Biotinylated GnRH (Bio-GnRH)

The Bio-GnRH analogues were produced by Dr. Brian Miller as described in previous studies (Childs et al. 1994b, 1997). In the [d-Lys⁶] analogue of GnRH, the valeric acid side-chain of biotin was directly attached to the ε-amino group of d-Lys⁶. After synthesis, the analogue was purified by reverse-phase HPLC. Monobiotinylation was verified by amino acid composition analysis. Tests and controls were described in our recent reports (Childs et al. 1994b, 1997).

Treatment of the Pituitary Cells with Activin

The pituitary cells were dispersed and plated on glass cover-slips in 24-well trays as described previously (Childs et al. 1992a,b, 1997). Plating density was 40,000–50,000 cells/well. They were divided into two experimental groups. Group 1 received vehicle and Group 2 received 60 ng/ml human recombinant activin (from Genentech; San Francisco, CA). The activin was diluted in Dulbecco's minimal essential medium (DMEM) containing insulin, transferrin, sodium selenite, 0.25% bovine serum albumin, and aprotinin (100 KIU), as described previously (Childs et al. 1992a,b, 1994a,b, 1997). The incubation in these peptides was 24 hr. We found no changes in the percentages of Bio-GnRH-bound cells during the 24-hr plating period. This agreed with previous studies showing that the percentages of Bio-GnRH target cells expressed at each stage of the cycle were retained during the 24-hr incubation period needed for the tests of effects of activin (Lloyd and Childs 1988).

Cytochemical Detection of Bio-GnRH and Antigens

The protocol was similar to that in our first reports published in 1983 (Childs et al. 1983a,b). After a wash in two changes of DMEM, the cells were stimulated with 1 nM Bio-GnRH for 10 min, which results in maximal labeling of target cells (Childs et al. 1994b). The cells were then fixed immediately in 2% glutaraldehyde and the Bio-GnRH was detected with the avidin-biotin–peroxidase complex, as described in previous studies (Lloyd and Childs 1988; Childs et al. 1983a,b, 1994b, 1997). After a 1-hr incubation in ABC solution, the cells were washed and the peroxidase detected by nickel-intensified diaminobenzidine, a substrate that yields a blue-black reaction product.

The controls for the detection protocol for Bio-GnRH binding involved omission of Bio-GnRH from 3 wells/tray. Previous controls had shown that 100–1000-fold excess nonbiotinylated GnRH (100–1000 nM) competed for binding sites and prevented labeling (Childs et al. 1983a,b, 1994b). The cells were then immunolabeled for LH-β, FSH-β, or GH as described in previous studies (Childs et al. 1983b, 1994a,b). The reaction involved a 2-hr incubation in the primary antiserum, followed by a 10-min incubation in the components of the DAKO (Carpinteria, CA) Rapid Immunoperoxidase kit. The peroxidase was detected with diaminobenzidine, which produced an orange-amber reaction product. This dual labeling approach was validated 14 years ago (Childs et al. 1983b)

The antiserum against LH-β was a gift from Dr. J.G. Pierce and was diluted 1:30,000–1:40,000. Antisera against rat FSH-β were a gift from Dr. Parlowe and the Hormone Distribution Program, NIH. They were diluted 1:20,000. Antisera raised against GH were purchased from Chemicon (Temecula, CA) and diluted 1:35,000. Immunolabeling controls in a double labeling protocol tested primary antisera absorbed with 100–1000 ng/ml specific antigens (Childs et al. 1994a,b). The absorption abolished labeling for each antigen. This confirmed the specificity of the immunolabeling protocols. In addition, 1-μl drops of GH, LH, or FSH (1–100 ng/ml) were added to Immobilon nylon filter paper. After drying, these drops were immunocytochemically labeled with antiserum to LH-β, FSH-β or GH. Each antiserum labeled only the dots containing their specific antigen. Furthermore, there were no changes in labeling intensity on either the cells or the dots when the antiserum to GH was absorbed with LH or FSH, or when the antiserum to the gonadotropins was absorbed with GH.

Density Measurements

The labeling for Bio-GnRH binding was analyzed with the BioQuant MEG IV system, which includes a 486–66 PC and a Sony Video color camera as described previously (Childs et al. 1987, 1997). The system captures each image and automatically corrects for background. This prevents differential readings due to different lighting conditions. No further image processing was done. Analysis of the average density was then done after the thresholding function was activated. This function detected all label in a given cell and automatically subtracted any background densities. The template for the detection system was set to read both the average density of all pixels covering the label and the area of the label detected in a given cell. During the same measurement period, the cursor was used to draw around each labeled cell and the cell area was read automatically by the computer. Each measurement session collected data from all experimental groups, sampling at least 20 cells/group during each session. At least 100 cells/group from three separate experiments were evaluated.

Statistical Analysis of Data

The experiments were repeated four or five times with one or two rats per stage of the cycle. Cells from at least 8 rats/stage of the cycle were tested. Cells were counted under a x100 oil immersion objective with Nomarski optics. The fields were scanned across the slide to avoid overlap and at least the first 200 cells encountered were counted and analyzed as to the presence and type of label. Cell counts sampled at least 3 coverslips/experiment. During the analyses, the raw data from each coverslip were inserted into an Excel spreadsheet that included formulas designed to calculate the percentages of each subtype of labeled cell. This enabled us to compare counts from single and double labeling protocols to learn if the double labeling protocol had interfered with detection of either the Bio-GnRH or the antigens. Separate single labeling protocols had been run to detect the antigens only. These data were correlated with those from the dual labeling protocols to learn if the antigens had been washed out or masked by the double labels.

A single experiment yielded an average from these three coverslips. This value was then averaged with values from the four or five replications to produce the final data point. The averages were compared with ANOVA, followed by the Fischer's least significant difference (LSD) post hoc test to detect differences between groups. Values were considered significant at p >0.05.

Results

Effects of Activin on Expression of Bio-GnRH Binding

There was a significant 40–50% increase in the percentages of cells that bound Bio-GnRH (p >0.001; Figure 1) from diestrus to proestrus. Activin stimulation of cells from diestrous rats mimics this increase (p >0.001). In the proestrous group, activin stimulated more Bio-GnRH target cells than are normally found (Childs et al. 1994b), resulting in peak percentages of 21% (p >0.001).

Figure 1

Single labeling and double labeling for biotinylated GnRH (Bio-GnRH) provided fields for analysis of the changes in the percentages of Bio-GnRH expressing cells in control and activin-treated populations. There is an increase when diestrous and proestrous groups are compared (b). In addition, activin stimulates an increase in percentages of GnRH target cells in both groups (a). (The letters “a” and “b” indicate significant differences.)

Analysis of the average density of label (optical density, OD) for Bio-GnRH on individual target cells showed that activin increased the average density of label from an OD value of 98 ± 1 to 90 ± 2 (p>0.001; diestrous rats) or 99 ± 2 to 93 ± 2 (p>0.047; proestrous rats). (Note: A lower number means less light transmitted and this translates to a higher density value.) The density increase in the diestrous population is shown by a histogram that illustrates the activin-mediated shift in the GnRH target cell population to one with more densely labeled cells (Figure. 2).

When proestrous cells were compared to diestrous cells, the average area of label for Bio-GnRH in individual target cells was significantly increased from 8 ± 0.2 nm² to 16 ± 2 nm² (p>0.001). Activin also increased the average area of label to 16 ± 1 nm² when applied to cells from diestrous rats (p>0.003). Activin decreased the area of label to 12 ± 2 nm² in populations from proestrous rats (p>0.047 compared with controls). When the average area of GnRH target cells was measured, activin stimulated an increase in average area of GnRH target cells in the diestrous group from 105 ± 4 μm² to 118 ± 5 μm² (p>0.05). Activin did not affect areas of GnRH target cells from proestrous rats.

Effects of Activin on Percentages and Areas of Cells with LH, FSH, or GH Antigens

Activin exposure resulted in higher percentages of FSH-β and LH-β antigen-bearing cells (detected by immunolabeling) in both the diestrous (FSH, p>0.001; LH, p = 0.003; Figure 3) and the proestrous (FSH, p>0.001; LH, p = 0.022; Figure 4) populations. Percentages of growth hormone (GH) cells were not changed by activin treatment of either group (p = 0.797) (Figures 3 and Figures 4).

Figure 2

Histogram of the population of cells analyzed for the density of label for Bio-GnRH. The x axis shows the different bins, each including a range of densities. The Y axis is the percentages of cells that expressed label within a particular density range. The control values (dotted lines) show that most cells had densities in the range of 104–120. However, after activin treatment (solid lines), this had shifted so that the peak densities fell into the 96–103 range. (As stated above, a lower optical density value represents higher density label, or less light transmitted.).

Activin had no effects on the average area of FSH cells or LH cells from diestrous rats (p = 0.319). However, activin decreased the average area of FSH cells from proestrous rats from 97 ± 4 nm² to 79 ± 3 nm² (p>0.001). In contrast, it increased the average area of LH cells from proestrous rats (from 86 ± 3 nm² to 93 ± 3 nm²) (p>0.047). Activin did not affect the average area of GH cells in either of the experimental groups (p = 0.6).

Effects of Activin on the Proportion of Gonadotropes or GH Cells that Bound Bio-GnRH

Figures 5 and Figures 6 illustrate fields that are double labeled for Bio-GnRH and LH-β, FSH-β, or GH. The double labeling for Bio-GnRH and gonadotropins or GH was run to learn if activin stimulated binding by specific types of target cells identified by hormone content. The dense lines or patches of labeling represent the dark-blue label for Bio-GnRH. The gray label represents the orange-amber label for the antigens. The increased area of label for Bio-GnRH discussed in the first section of these results can be seen in the activin treated fields (Figures 5A and 5D).

Figure 3-4

Cells bearing LH-β, FSH-β, or GH antigen were counted in control and activin-treated populations. Significant increases in both LH and FSH cells were seen in the activin treated diestrous ( Figure 3 ) and proestrous ( Figure 4 ) populations. (The letter “a” indicates a significant difference.)

When double labeled fields from diestrous rats were analyzed, 40–52% of gonadotropes and 18% of GH cells bound Bio-GnRH in the control populations (Figure 7). Activin treatment increased GnRH binding to 72 ± 5% of FSH cells (p>0.01), 73 ± 12% of LH cells (p>0.05), and 46 ± 7% of somatotropes (p>0.018). In contrast, activin did not stimulate more gonadotropes from proestrous rats to bind GnRH (p = 0.5, FSH; p = 0.7, LH) (Figure 8). However, activin increased binding by GH cells from proestrous rats from 34 ± 3% to 68 ± 5% of GH cells (p>0.001).

Discussion

Activin is actually produced by gonadotropes and therefore functions in an autocrine regulatory loop to control the synthesis, storage, and release of FSH (Bilezikjian et al. 1993; Halvorson et al. 1994; Besecke et al. 1996; Knight 1996). However, activin's effects may be broader and may include inhibitory effects on somatotropes (Bilezikjian et al. 1993). In this study, we focused on activin target cells among somatotropes and gonadotropes and tested activin's effects on the capacity of the cells to bind Bio-GnRH.

Figure 5

Cells in A-C show control fields from diestrous populations with dense black immunoperoxidase label for Bio-GnRH in a linear pattern on or near the cell periphery (arrows) and gray (amber) label for LH-β (L) or FSH-β (F) antigen. A and B detect Bio-GnRH label with FSH-β antigen and C detects Bio-GnRH with LH-β antigen. D shows a cluster of FSH cells from an activin-treated diestrous population. Note the spread of the label for Bio-GnRH over more of the cell surface. Bar = 5 μm.

Figure 6

Activin-treated fields from proestrous rats. (A, B) A cluster of three gonadotropes is labeled for Bio-GnRH (arrows) and FSH-β (F) (A) or LH-β (L) (B) antigens (gray label). The increased area of label for Bio-GnRH is prominent on both sets of cells. (C) A double labeled GH cell with prominent labeling for Bio-GnRH. U, unlabeled cell. Bar = 5 μm.

Figure 7

Counts of cells double labeled for Bio-GnRH and LH-β, FSH-β, or GH antigen in populations from diestrous rats. Activin stimulated increased binding of GnRH to all three cell types. The increment in binding by LH and FSH cells was not as great as that normally seen from diestrus to proestrus (see Figure 8). (The letter “a” indicates a significant difference.)

Braden and Conn had shown previously that activin stimulates the synthesis of GnRH receptor mRNA and proteins (Braden and Conn 1990, 1991; Conn et al. 1995; Fernandez-Vazquez et al. 1995). The present studies were designed to learn if this stimulation reflected changes in the overall numbers of GnRH-bound target cells and/or the numbers of binding sites per cell, or both. Such answers can be obtained only by analysis of cytochemically labeled fields. For example, if the latter case were true, no additional GnRH target cells would be counted after activin treatment. However, the densitometric studies might show increases in the average area or density of label for the Bio-GnRH on individual target cells. This increase may or may not be combined with an increase in area of the GnRH target cell. Collectively, our data show that activin both recruited additional Bio-GnRH-target cells and increased the area and density of label on individual cells.

Cells from diestrous rats appeared to be very sensitive to the stimulatory effects of activin. Activin increased the percentages of Bio-GnRH target cells to proestrous values. This supports the hypothesis that activin may help mediate the increases in expression of GnRH receptors.

Figure 8

Counts of cells double labeled for Bio-GnRH and LH-β, FSH-β, and GH in populations from proestrous rats. Activin did not cause a further increase in the percentages of LH or FSH cells that bind GnRH. However, it did increase the percentages of GH cells that bound the hormone beyond levels normally seen in proestrus (Childs et al. 1994b). (The letter “a” designates a significant difference.)

Activin's Specific Effects on LH or FSH Antigen-bearing Cells

Activin also stimulated the appearance of more LH and FSH cells in diestrous rats, resulting in percentages of LH cells similar to those normally seen during proestrus. However, the resulting percentages of FSH cells exceeded those of proestrous rats. This may reflect activin's possible differentiating or mitogenic effects described previously for FSH cells in culture (Katayama et al. 1990).

However, the double labeling analyses showed that activin's effects on GnRH binding by LH or FSH cells fall short of the changes normally seen from diestrus to proestrus (Childs et al. 1994b). Perhaps estradiol is needed to further enhance GnRH binding by gonadotropes. Our previous studies showed that it also increases the number of GnRH-receptive cells. The resulting numbers are not as high as those normally seen, suggesting that additional factors are involved (Lloyd and Childs 1988). Perhaps the effects of activin and estradiol on GnRH receptors are additive. Ongoing studies address this possibility.

Specific Effects of Activin on Monohormonal FSH Cells

Activin regulates FSH synthesis and secretion. Therefore, it is not surprising that it had potent effects on FSH cells in almost all tests in this study. It increased percentages of FSH cells beyond values normally seen during proestrus. The fact that activin did not cause the same increase in percentages of LH cells suggests that some of the activin target cells may be monohormonal FSH cells.

The activin-mediated decrease in area of these new FSH cells from proestrous rats also supports possible mitogenic or differentiating effects (Katayama et al. 1990). Newly divided FSH cells may be smaller. We have previously shown that small size is also a feature of developing monohormonal FSH cells (Childs et al. 1992b). A reduction in average area of cells with FSH-β mRNA was also seen in earlier studies during an intermediate period after castration, as new mRNA bearing cells were being added to the population (Childs et al. 1990). Finally, the slight decrease in the average area of label for Bio-GnRH in the proestrous group also correlates with activin effects on FSH cells from this group. The small immature FSH cells may express smaller areas of binding sites for GnRH.

Activin did not cause a decrease in average area of LH cells, which again points to its selective action on monohormonal FSH cells. Collectively, it appears that activin may enhance FSH secretion by stimulating the development of undifferentiated gonadotropes and/or the division of pre-existing FSH cells.

Specific Effects of Activin on LH Cells

As stated above, activin caused significant increases in percentages of LH cells in populations from both diestrous and proestrous groups, although the increment in percentages of LH cells was not as high as that for FSH cells. Some of these cells are undoubtedly the bihormonal gonadotropes that predominate in the proestrous gonadotrope population (Childs et al. 1994a). In addition, activin had unique effects on LH cells from proestrous rats. It increased the average area of LH cells. This increase coincided with a decrease in the average area of FSH cells. The lack of parallelism again suggests that activin may have an effect on a subset of monohormonal LH gonadotropes.

Activin May Stimulate Other GnRH Target Cells

One finding that was difficult to explain was the stimulatory effect of activin on the average area of GnRH target cells from diestrous rats. This change was normally not seen when diestrous and proestrous populations were compared. Activin did not increase average areas of the target cells tested (FSH, LH, or GH cells) from these same diestrous groups. Our cell counts showed that there were no losses in FSH, LH, or GH antigen-bearing cells. Therefore, the increased areas are not due to larger gonadotropes or GH cells that can not be identified. It is possible that the increase in average area comes from another type of activin target cell that is also GnRH receptive, such as thyroid-stimulating hormone (TSH) cells (Childs et al. 1994b), which are among the largest in the pituitary (Childs et al. 1983c).

Activin May Mediate the Conversion from Somatotropes to Somatogonadotropes

Activin inhibits GH synthesis, secretion, and mitoses (Kitaoka et al. 1988; Bilezikjian et al. 1990; Billestrup 1990). The present studies now show that activin increases Bio-GnRH binding by somatotropes from both diestrous and proestrous rats. The increase may account for the overall increase in GnRH receptive cells seen in the proestrous rat pituitary.

These data correlate with recent studies in which somatogonadotropes were discovered in diestrous and proestrous rat cell populations by double labeling (Childs et al. 1994a,b). More recent findings also show that inhibin reduces GnRH binding by GH cells from proestrous rat populations (Childs et al. 1997). Collectively, these findings suggest that one of activin's target cells is the transitional somatogonadotrope. Perhaps activin helps mediate the transition from a somatotrope to a somatogonadotrope.

Further support for this hypothesis is found in recent studies reported by Tilemans et al. (1992) and Van Bael et al. (1995), who showed inhibitory effects of GnRH itself on GH mRNA, proteins, and GH cell mitoses. Activin could begin the transition process during diestrus by stimulating the production of GnRH receptors and FSH-β mRNA in somatotropes. At the same time, activin could inhibit the synthesis of GH (Bilezikjian et al. 1990). As the levels of GnRH receptors rise in GH cells, GnRH itself could stimulate LH and FSH synthesis while inhibiting that of GH. Therefore, GnRH might complete the transition from a somatotrope to a somatogonadotrope, allowing the cells to support the gonadotrope population during late proestrus. After the gonadotropin surge, the loss of GnRH receptors late in proestrus (Clayton et al. 1980; Savoy-Moore et al. 1980; Lloyd and Childs 1988), coupled with the production of follistatin, which binds activin (Halvorson et al. 1994; Besecke et al. 1996), may reverse this process, enabling the somatogonadotropes to revert to their growth hormone function.

Footnotes

Acknowledgements

Supported by NIH R01 HID 15724, by a developmental grant from the Sealy Smith foundation, and by NIH R01 33915.

We acknowledge the technical help of Diana Rougeau. We also thank Dr Parlowe and the NIH Hormone Distribution program for antisera and for rat GH, LH-β, and FSH-β antigens. We thank Dr J.G. Pierce for the antisera to bovine LH-β and appreciate the gift of activin from Genentech.

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