Activin Subunit, Follistatin, and Activin Receptor Gene Expression in the Prepubertal Female Rat Pituitary¹

^a Program in Molecular Biology and Department of Cell Biology, Neurobiology, and Anatomy, Loyola University, Stritch School of Medicine, Maywood, Illinois 60153

	ABSTRACT

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In the prepubertal female rat, a transient and selective increase in FSH secretion and mRNA expression by the pituitary gland occurs toward the end of the second postnatal week of life. To begin to investigate the possibility that activin may play a role in up-regulating FSH during this time, we have studied the ontogeny of the expression of the activin ß subunits, follistatin, and activin receptor subtypes in the prepubertal female rat pituitary. The levels of expression of ß_A, ß_B, and follistatin mRNAs were determined in the pituitary gland on postnatal days (PND) 8, 10, 12, 15, and 21 by semiquantitative reverse transcription-polymerase chain reaction. All values were compared to those of adult females killed on diestrus. mRNA levels of subunit ß_A were significantly (p < 0.05) elevated on all postnatal days examined; ß_B mRNA levels were elevated above adult levels only on PND 10 (p < 0.05). Follistatin mRNA was high on PND 8 (p < 0.05) and then decreased to adult levels. The level and distribution of activin receptor type II subtype mRNAs were determined by in situ hybridization. Activin receptor type II (Act RII) mRNA expression was diffusely expressed throughout all areas of the pituitary. Activin receptor type IIB (Act RIIB), on the other hand, was highly expressed by a subset of anterior pituitary cells. In situ hybridization for activin receptor subtype mRNAs was combined with immunocytochemistry to detect FSH-containing cells. We determined that in the infantile female pituitary, Act RII mRNA was generally not expressed in FSH-immunoreactive cells, while Act RIIB mRNA was expressed in FSH-immunoreactive cells. Act RII mRNA was lower on PND 10 and 15 when compared to PND 21 (p < 0.05), whereas Act RIIB mRNA expression did not change with age. These data suggest that the essential components of the activin regulatory system are present in the infantile female pituitary gland and thus may be involved in the differential regulation of FSH at this time.

	INTRODUCTION

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In the infantile female rat (postnatal days [PND] 8–21), the gonadotropins LH and FSH are differentially regulated. There is a dramatic increase in plasma FSH levels as well in FSH-ß mRNA levels in the pituitary [1, 2]. Both peak on PND 12 and rapidly decline by PND 15. During this same time, LH levels, although elevated compared to adult levels, do not exhibit such a peak. Similarly, LH-ß mRNA levels do not change across the infantile period. The mechanisms that allow for this selective regulation of FSH are not known.

In the adult, selective regulation of FSH without changes in LH are due, in part, to an activin regulatory system consisting of activin and its receptor as well as follistatin. Activin is a gonadal peptide hormone and a member of the transforming growth factor family. The activin hormone is composed of two inhibin ß subunits termed ß_A and ß_B. The subunits heterodimerize or homodimerize to form activin A (ß_A-ß_A), activin AB (ß_B-ß_A), or activin B (ß_B-ß_B) [3]. Follistatin, which was first isolated from ovarian follicular fluid, also inhibits FSH secretion [3]. Both activin and follistatin are locally produced in the pituitary, as well as in the ovary, of the female rat, making them potential paracrine effectors of FSH synthesis and secretion [4, 5].

At the surface of target cells, activin binds to a dimeric receptor complex similar to the transforming growth factor family of receptors (for review see [6]). The receptor complex consists of a type I and a type II serine/threonine kinase receptor. Both forms are necessary for proper signaling following activin binding. In the rat, there are multiple forms of both type I and type II activin receptors. There are two subtypes of the type II receptors, Act RII and Act RIIB. The primary type II receptor is thought to be Act RII, although it has not been definitively shown in the developing pituitary.

Both activin and follistatin have been shown to be produced in gonadotrophs of adult female rat pituitary as well as in other pituitary cell types [7, 8]. Activin receptor mRNA expression has also been demonstrated in the pituitary by in situ hybridization [9]. To date, the distribution of the individual receptors in specific cell types within the anterior pituitary has not been determined. However, since both the ligand and the receptors are expressed in the pituitary, the possibility that they act in a paracrine or autocrine fashion to locally regulate FSH exists [10, 11].

At present, no studies have described the expression of activin, follistatin, or activin receptor mRNAs in the pituitary gland of the prepubertal rat. In the mouse ovary, however, activin subunits are expressed early in postnatal life [12]. Whether or not this ovarian expression results in physiologically relevant changes in circulating levels of activin remains to be determined. Ovariectomy of rats during early infantile life does not consistently result in changes in levels of circulating FSH or FSH-ß mRNA [13, 14], suggesting that ovarian-derived activin may not play a major role in the regulation of FSH during this time.

We have hypothesized that locally produced activin and/or follistatin may play a role in the selective increase in FSH-ß mRNA levels at this time. To begin to investigate this possibility, here we describe the ontogeny of activin subunits, follistatin, and activin receptor type II mRNAs, as well as the distribution of activin type II receptor subunit mRNAs, within gonadotrophs of the infantile female rat pituitary. Because activin subunits have previously been shown to be expressed in gonadotrophs, we measured their expression in the anterior pituitary by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), whereas we determined the cellular distribution and level of expression of the activin receptor subtypes by in situ hybridization.

	MATERIALS AND METHODS

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Animals

Pregnant dams were purchased from Charles River Laboratories (Portage, MI) and delivered to the animal facilities at Loyola University when 1 wk pregnant. The day of birth was designated PND 0. On PND 1, litters were culled to 10 (mixed-sex). Pups were killed on PND 8, 10, 12, 15, and 21, and on each of these days, pups were selected from different litters so that multiple litters would be represented for each time point. Pituitaries were removed and frozen immediately on dry ice and stored at -70°C. For some studies, animals were anesthetized with ether and killed by perfusion with saline followed by 4% buffered paraformaldehyde (pH 7.0). This tissue was stored in a 30% sucrose solution at 4°C. Adult pituitaries were collected in a similar manner. Since activin subunit mRNA expression does not vary greatly in the pituitaries of adult female rats across the estrous cycle, and follistatin mRNA is at basal levels at diestrus [15], diestrous adult females were selected for comparison of their levels to infantile levels of ß_A, ß_B, and follistatin mRNA.

RT-PCR

Total RNA was isolated from frozen pituitaries using guanidium isothiocyanate by a method previously described [16]. One microgram of total RNA was reverse transcribed using an oligo dT(12–15) primer and Superscript RT (Life Technologies, Gaithersburg, MD) in a final reaction volume of 20 µl. Two microliters of the cDNA was then amplified by PCR in the presence of 2 µCi [³²P]dCTP as previously described [16] using primers specific for the ß_A, ß_B, or follistatin genes [17]. Reaction products were analyzed by nondenaturing PAGE and dried, and the amount of radioactivity in the reaction products was counted using a betascope (Betagen, Waltham, MA). To control for loading differences, samples were normalized to the control histone 3.3 gene from the same sample as previously described [16].

In Situ Hybridization

In situ hybridization for activin receptor mRNAs was performed by methods previously described [18]. For the activin receptor subtypes, an antisense cRNA probe was generated by in vitro transcription of a specific PCR fragment that contained an SP6 promoter on the 3' end. The 5' end contained a T7 promoter so that sense strand probes could also be generated. These promoters were added by PCR with receptor-specific primers that contained an eight-base pair overhang complementary to the primers containing the promoters [2]. The primers used to generate the cDNAs were previously published: Act RII [19] and Act RIIB [20]. The Act RII probe recognizes the intracellular domain. The Act RIIB probe hybridizes to the kinase domain common to all four potential RIIB isoforms. Pituitaries were sectioned at 20 µm on a Leitz (Wetzlar, Germany) cryostat and mounted onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). Tissue was acetylated with 0.25% acetic anhydride. Tissue was then dehydrated in graded alcohols, air dried, and hybridized in hybridization solution (50% formamide, 0.60 M NaCl, 0.02 M Tris, 0.01 M EDTA, 10% dextran sulfate, double-strength Denhardt's, 50 mM dithiothreitol, 0.2% SDS, 100 mg/ml salmon sperm DNA, 500 mg/ml total yeast RNA, and 50 mg/ml yeast tRNA) containing the radiolabeled probe at a concentration of 2 x 10⁷ cpm/ml. After hybridization, the slides were rinsed in double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), and nonhybridized RNA was digested with 30 mg/ml RNase A for 30 min at 37°C. Final wash stringency was 0.1-strength SSC (60°C). Slides were rinsed and dehydrated in ascending alcohols. For autoradiographic detection of hybridization, slides were dipped in nuclear tract emulsion (Kodak NTB-3; Eastman Kodak, Rochester, NY), air dried, and allowed to expose for 1–4 wk at 4°C.

Immunocytochemistry

Slides for FSH immunocytochemistry were then incubated overnight with the NIDDK (Rockville, MD) rabbit anti-rat FSH primary antibody (1:500) at room temperature after in situ hybridization, but prior to autoradiography. An anti-rabbit secondary antibody coupled to biotin was visualized by diaminobenzidine using the Vectastain ABC kit (Vector Labs., Burlingame, CA). After detection of FSH-immunoreactive cells, slides were dipped in emulsion. The radiolabeled cells were detected by autoradiography and the FSH-containing cells by light microscopy. A brown oxidation product overlaid by silver grains was indicative of a double-labeled cell.

Image Analysis

The number of grains per fixed area was counted using a video camera (Sony XC-77; Tokyo, Japan) connected to a Zeiss (Carl Zeiss, Thornwood, NY) microscope and an Apple Power Macintosh 7100 (Cupertino, CA) computer and utilizing National Institutes of Health Image (version 1.57; Bethesda, MD) software. A grain-counting macro originally written by Dr. Karl Beykirch (UCLA School of Medicine, Los Angeles, CA) and adapted for our use by Dr. Alan Nagahara (Loyola University, Chicago, IL) was used. The macro counted the density of silver grains, identified by darkfield microscopy, by calculating the number of pixels of label above the user-defined threshold. A region corresponding to 40 µm² was analyzed. Measurements were taken from 25 regions from five different sections for each animal, and the means of the five sections were averaged together to give the mean for that animal. Since the distribution of grains for Act RIIB was more restricted, counts were made from labeled areas only. Since cells were densely packed, an area roughly the size of a single cell was selected based on the discretion of the experimenter. The data were expressed as the percentage of area covered by grains (mean grain area).

Statistical Analysis

Data were analyzed across age by one-way ANOVA. Post hoc comparisons were made using the Student-Newman-Keuls test.

	RESULTS

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Ontogeny of Activin Subunits ß_A and ß_B, and Follistatin mRNA

Figure 1 shows pituitary mRNA levels of ß_A and ß_B on PND 8, 10, 12, 15, and 21. The mRNA levels are expressed as a percentage of those in the diestrous adult female (n = 4). The data represent the mean of triplicate determinations from four separate animals. ß_A levels were significantly elevated on PND 8, 10, 12, and 15, but not PND 21, as compared to adult levels (p < 0.05). In general, ß_A levels are 1–1/2 to 2 times higher in the infantile pituitary than in the adult. For ß_B subunit, there was a significant effect of age across the infantile period [F(4,9) = 4.434; p < 0.03]. mRNA levels of ß_B were elevated on PND 10 (p < 0.05) as compared to PND 8 and PND 15. At all infantile ages, ß_B levels were not different from adult levels. Based on the length of exposure times, the level of ß_B mRNA expression was considerably greater than that of ß_A mRNA.

View larger version (19K): [in this window] [in a new window]   FIG. 1. ßA (A) and ßB (B) subunit mRNA expression in the infantile period determined by semiquantitative RT-PCR. ßA levels were significantly elevated compared to adult levels (p < 0.05). One-way ANOVA revealed a significant age effect for ßB mRNA levels (p < 0.03). Values represent the mean of four animals performed in triplicate ± SEM. C) Representative lanes from autoradiogram of PCR products. The same animal for each time point is shown in the example.

The ontogeny of pituitary follistatin mRNA levels is shown in Figure 2. One-way ANOVA revealed a significant effect of age [F(4,10) = 5.035; p < 0.02]. Follistatin mRNA levels were initially elevated on PND 8 (p < 0.05) and then dropped to adult levels by PND 10.

View larger version (19K): [in this window] [in a new window]   FIG. 2. Follistatin mRNA expression as determined by semiquantitative RT-PCR. Values represent the mean of triplicate PCR replications (for each replicate, n = 4). *Significantly different (p < 0.02). Inset: A representative autoradiogram.

Activin Receptor Gene Expression

The cellular distribution of activin receptor subtypes RII and RIIB mRNAs was examined. In the infantile pituitary, Act RII mRNA was diffusely expressed in the posterior and anterior lobes (Fig. 3a). Act RIIB expression was limited to the anterior pituitary and appeared to be expressed in select cells (Fig. 3b). In addition, Act RIIB expression was much higher than that of Act RII, requiring one third the time for visualization using emulsion autoradiography. Sense strand probes showed no specific hybridization and served as negative controls (Fig. 3, c and d).

View larger version (93K): [in this window] [in a new window]   FIG. 3. Photomicrographs of PND 12 female rat pituitary sections hybridized with riboprobes to Act RII (a) and Act RIIB (b). Anterior lobe (a.l.), posterior lobe (p.l.), intermediate lobe (i.l.). Hybridization with sense strand negative control riboprobes to Act RII (c) and Act RIIB (d). x200 (reproduced at 83%).

To determine whether or not the activin receptors are expressed in FSH-positive gonadotrophs, some sections were subjected to immunocytochemistry for FSH following in situ hybridization prior to autoradiography. FSH-immunoreactive cells coexpressed Act RII mRNA subtypes only 24.4 ± 2.8% of the time. Act RIIB mRNA was expressed in 85.7 ± 1.3% of FSH-positive cells (Fig. 4). A total of 94.3 ± 1.4% of Act RIIB mRNA-positive cells were also FSH positive, suggesting that Act RIIB mRNA expression appeared to be limited to FSH-expressing gonadotrophs.

View larger version (126K): [in this window] [in a new window]   FIG. 4. Photomicrographs of PND 12 pituitary sections labeled immunocytochemically for FSH protein and by in situ hybridization with riboprobe for Act RIIB. Black silver grains are indicative of activin receptor mRNA hybridization. Gray cytoplasmic precipitate is indicative of FSH immunoreactivity. Black arrows indicate representative double-labeled cells. White arrows point to cells positive for FSH immunoreactivity but negative for activin receptor expression. x400 (reproduced at 72%).

The levels of expression of the type II subtypes were compared across the days of the infantile period by quantification of in situ hybridization density in sections not subjected to immunocytochemistry (Fig. 5). There were no significant effects of age for Act RIIB. Act RII mRNA expression, however, did differ significantly across time as determined by one-way ANOVA [F(4,10) = 7.713, p < 0.005], with levels on both PND 10 and 15 being significantly lower than those on PND 21 (p < 0.05).

View larger version (18K): [in this window] [in a new window]   FIG. 5. Quantification of activin receptor RII (a) and RIIB (b) in situ hybridization in infantile pituitary sections. Values represent the mean ± SEM, n = 3. *Significantly different from PND 21 (p < 0.05).

	DISCUSSION

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In these studies we describe for the first time the expression of activin subunit, follistatin, and activin receptor mRNAs in the prepubertal female rat pituitary. Expression for each is developmentally regulated. In addition, FSH-producing gonadotrophs express the mRNA for Act RIIB receptors. These data suggest that the activin regulatory system is operable in the pituitary gland early in postnatal life.

In the adult female rat, ß_B homodimers have been identified from cultures of pituitary cells [21], and ß_B mRNA has been detected in vivo [4]. Thus, activin B is thought to be the form that acts locally at the level of the pituitary. While the activin A protein subunit (ß_A) is found in pituitary cultures [21], only very sensitive techniques, such as RT-PCR, have detected its mRNA [15]. Activin A, therefore, is thought to be primarily of ovarian origin and may not influence pituitary gene expression. In contrast to observations in the adult, both ß_A and ß_B subunit mRNAs are expressed in the infantile rat pituitary. ß_B is expressed at approximately adult levels. However, when compared to adult levels, ß_A expression is about twofold greater. This raises the possibility that in the infantile rat, both activin A and activin B may function as paracrine regulators of FSH expression. These data also demonstrate the differences between the pre- and postpubertal female pituitary. The possibility exists that the mechanisms of FSH-ß regulation are different under these conditions.

Additionally, both ß_A and ß_B are developmentally regulated in the pituitary. Their expression is highest on PND 10 just prior to the FSH-ß mRNA peak on PND 12 [2]. This suggests the possibility that activin A or B may play a role in the selective up-regulation of FSH-ß mRNA. Activin A has been shown to increase FSH-ß mRNA as well as secretion in vivo [10, 22] and in vitro [3]. Activin B is also involved in stimulating a selective increase in FSH secretion in vitro [10]. In these experiments, the effects reported were rapid, within 2–12 h. In contrast, in the infantile rat, the peak of FSH is seen 2 days after the highest levels of activin mRNA are achieved. Since very little is known about the relationship of mRNA increases to activin secretion or clearance, the time frame for its effects remain unclear. It seems likely, however, that the time frame reported here could complement the FSH-ß mRNA increases.

During infantile development, follistatin mRNA levels are similar to those of the adult, except on PND 8, when levels are 2 times higher. The elevation of follistatin precedes that of activins by 2 days, and this could potentially alter activin action. The high levels of follistatin mRNA decrease at the time activin peaks. If this follistatin indeed binds to activin to prevent its action, this rapid decrease could make more free activin available to bind to its receptor.

In the present studies we describe the ontogeny of the mRNA levels of ß_A, ß_B, and follistatin. Whether or not the changes in mRNA levels reflect levels of secreted protein remains to be determined. Translation efficiency, posttranslational modifications, and secretion could also play a role in the regulation of the secreted protein product. In this study, we did not investigate the expression of the inhibin subunit, which combines with the ß subunits to form inhibin. Previous studies have suggested that inhibin is not physiologically important in FSH regulation before PND 20 [23, 24]. Since the ß subunit forms a dimer with the subunit to form the inhibin molecules, it remains possible that the level of subunit expression relative to ß subunits could modulate the amount of the ß dimers present, and thus the amount of biologically active activin.

Our data demonstrate that Act RII and Act RIIB mRNAs are present in the infantile female rat. They have previously been identified in the adult rat pituitary [9, 20]. Our data suggest that of the two type II receptors, Act RIIB is more likely associated with activin signaling for regulating FSH expression. This is further emphasized by the fact that virtually of all RIIB mRNA-positive cells are also FSH positive.

Our studies show that no significant developmental changes occur in Act RIIB mRNA expression. Act RII mRNA levels change with age, but because Act RII is rarely expressed in gonadotrophs, this developmental change does not appear to be relevant for selective FSH regulation. Thus, if the activin system is involved in the developmental regulation of FSH, this probably occurs through changing levels of ligand concentration. Physiologically important interactions between activin and follistatin proteins may occur at this time to enhance activin efficacy, but this possibility remains to be determined.

In the infantile pituitary, activin subunit ß_A mRNA is expressed at a higher level than in the adult pituitary. Differences between infantile animals and adults are also seen with the activin receptor subtypes. It has previously been reported that Act RII is expressed at a much higher level than Act RIIB [20]. In the infantile female pituitary, Act RIIB is expressed at a much higher level than Act RII. This is consistent with the observation that during fetal development, only Act RIIB mRNA can be detected in the pituitary [25]. These observations suggest that at some critical period in prepubertal development, changes occur in the pituitary that result in different adult regulation of the activin system, in particular ß_A and Act RIIB mRNA expression.

A previous study reported the distribution of Act RII and Act RIIB mRNA by in situ hybridization in the adult pituitary [9]. In that study, Act RII mRNA expression was similar to what we report: a diffuse expression throughout the pituitary. Act RIIB distribution was different in that our data show there to be punctate expression in the anterior lobe only. Previously, it was reported that RIIB was diffusely expressed throughout the pituitary, with high levels in the intermediate lobe [9]. In our study we examined RIIB expression in infantile females, whereas the previous study used adult females. It remains possible that Act RIIB distribution changes during pubertal development.

These data demonstrate that the activin subunits and follistatin are developmentally regulated at the mRNA level in the prepubertal pituitary. One possible source of this regulation is estrogen. Estrogen has been shown to stimulate activin A in ovarian granulosa cells [26] and may regulate pituitary follistatin mRNA [15]. We have evidence showing that estrogen regulates FSH during this time [27], but it remains to be seen whether its effect is mediated directly or through the activin system.

The present studies provide important insight into the development of the activin system in the prepubertal pituitary gland. The infantile female rat presents another situation in which gonadotropins are differentially expressed. An understanding of the development of the activin system in this model will be important in understanding the role activin may play in such regulation.

	FOOTNOTES

 
¹ This material is based on work supported by the USPHS grant AA08696 (R.J.H.) and NRSA predoctoral fellowship F31-AA05395 (M.E.W.).

² Correspondence: Robert J. Handa, Department of Cell Biology, Neurobiology, and Anatomy, Loyola University Chicago, Stritch School of Medicine, 2160 South First Ave., Maywood, IL 60153. FAX: (708) 216–3913; rhanda{at}wpo.it.luc.edu

³ Current address: Department of Physiology, University of Kentucky, Lexington, KY 40536.

Accepted: March 16, 1998.

Received: November 21, 1997.

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