ARTICLE |
Correspondence to: Gwen V. Childs, Dept. of Anatomy and Neurosciences, U. of Texas Medical Branch, Galveston, TX 77555-1043.
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Summary |
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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-ß (LH-ß), FSH-ß, 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. (J Histochem Cytochem 45:1603-1610, 1997)
Key Words: anterior pituitary, activin, gonadotrope, growth hormone, follicle-stimulating hormone, luteinizing hormone, gonadotropin-releasing, hormone receptors, rat
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Introduction |
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Activin is a regulatory peptide belonging to the transforming growth factor-ß (TGFß) superfamily that plays a role in differentiation and development in a number of tissues (
Activin also stimulates gonadotropes by increasing the synthesis of gonadotropin-releasing hormone (GnRH) receptor mRNA and proteins (
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) (
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Materials and Methods |
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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 (
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 (
Synthesis and Characterization of Biotinylated GnRH (Bio-GnRH)
The Bio-GnRH analogues were produced by Dr. Brian Miller as described in previous studies (-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 (
Treatment of the Pituitary Cells with Activin
The pituitary cells were dispersed and plated on glass coverslips in 24-well trays as described previously (
Cytochemical Detection of Bio-GnRH and Antigens
The protocol was similar to that in our first reports published in 1983 (
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 (
The antiserum against LH-ß was a gift from Dr. J.G. Pierce and was diluted 1:30,000-1:40,000. Antisera against rat FSH-ß 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 (
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 (
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.
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Results |
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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 (
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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).
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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-ß and LH-ß 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) (Figure 3 and Figure 4).
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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
Figure 5 and Figure 6 illustrate fields that are double labeled for Bio-GnRH and LH-ß, FSH-ß, 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 (Figure 5A and Figure 5D).
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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).
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Discussion |
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Activin is actually produced by gonadotropes and therefore functions in an autocrine regulatory loop to control the synthesis, storage, and release of FSH (
Braden and Conn had shown previously that activin stimulates the synthesis of GnRH receptor mRNA and proteins (
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.
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 (
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 (
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 (
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 (
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 (
Activin May Mediate the Conversion from Somatotropes to Somatogonadotropes
Activin inhibits GH synthesis, secretion, and mitoses (
These data correlate with recent studies in which somatogonadotropes were discovered in diestrous and proestrous rat cell populations by double labeling (
Further support for this hypothesis is found in recent studies reported by
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Acknowledgments |
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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-ß, and FSH-ß antigens. We thank Dr J.G. Pierce for the antisera to bovine LH-ß and appreciate the gift of activin from Genentech.
Received for publication April 11, 1997; accepted June 25, 1997.
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