| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pituitary |
Division of Endocrinology & Metabolism, Department of Medicine,3 University of Louisville, Louisville, Kentucky 40202
Department of Internal Medicine,4 University of Virginia, Charlottesville, Virginia 22908
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
follicle-stimulating hormone, follistatin, neuroendocrinology, neuropeptides, pituitary
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Substantial evidence indicates that PACAP regulates the expression and secretion of the gonadotropins, LH and FSH, and may play a key role in the regulation of reproductive function [9]. In cultures of anterior pituitary cells, PACAP induces an immediate accumulation of LH and free 
subunit in the extracellular medium [10]. In addition to this direct effect, PACAP augments the gonadotroph response to GnRH. Continuous treatment of cultured pituitary cells with PACAP produces a dose-dependent increase in the sensitivity of gonadotrophs to pulsatile GnRH signaling [10]. Continuous exposure to PACAP also increases subunit mRNA levels, lengthens LHß mRNA transcripts, but reduces FSHß mRNA levels [10], thereby differentially regulating the expression of the gonadotropins.
In spite of all that is known about PACAP signaling in gonadotrophs, very little is known about the role of PACAP in the in vivo regulation of gonadotrophs and reproductive function. Because PACAP differentially regulates expression of subunit, LHß, and FSHß mRNAs, it may affect the differential secretion of LH and FSH. One prominent example of differential changes in secretion of the gonadotropins is during the process of sexual maturation in the male rat. Plasma FSH levels rise rapidly around Day 20 to reach peak values around Day 35, followed by a decline to adult levels by Day 50. In contrast, circulating levels of LH rise gradually to a peak and plateau level around Day 50 [11–16]. To our knowledge, only one study appears to have examined the expression of the gonadotropin subunit genes during maturation [17]. In addition, no studies, to our knowledge, have examined the maturational pattern of GnRH receptor (GnRH-R) mRNA levels, which also may be regulated by PACAP [18–21].
The experiments of the present study were performed to characterize the gene expression patterns of the gonadotropins and to pursue the hypothesis that PACAP expression in the anterior hypothalamus is related to the developmental pattern of FSHß and GnRH-R expression during sexual maturation in the male rat. Because these changes may be caused by the effects of PACAP on follistatin production [22, 23], follistatin mRNA levels were also monitored to determine whether a relationship between PACAP and follistatin mRNA expression exists during development.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Male Sprague-Dawley rats (23, 33, 43, and 53 days old) were obtained from Charles River Laboratories (Wilmington, MA) and housed for 1 wk before tissue collection. Animals in a 20-day-old group were shipped to our animal facility at 13 days of age and were kept with a lactating female until they were killed on Day 20. The supplier weaned all other animals on Day 20. Animals were housed in a 12L:12D photoperiod and were provided laboratory chow and water ad libitum. Six animals per group, on Days 20, 30, 40, 50, and 60, were decapitated after lethal exposure to carbon dioxide according to a protocol approved by the Animal Care and Use Committee of the University of Louisville. The trunk blood was collected, the pituitaries were harvested and rapidly frozen in RNA extraction buffer, and the brains were removed and rapidly frozen in mounting medium on an ethanol/dry-ice slurry and then stored at -80°C.
Immunoassays
Enzyme immunoassays developed by Amersham Pharmacia Biotech (Peapack, NJ) were used to measure FSH and LH protein in serum. The within-assay coefficients of variation in the standard curves were 11.4% for FSH and 7.6% for LH. The ranges of the standards for the assays were 6.25–400 ng/ml for FSH and 0.41–100 ng/ml for LH.
RNA Extraction and Northern Hybridization
The RNA was extracted using the Perfect RNA total RNA Isolation kit (5 Prime-3 Prime, Boulder, CO). The concentration of total RNA was determined by reading the optic density at 260 nm. Sample purity was determined by calculating the ratio of sample absorbance at 260:280 nm, and samples were rejected if the ratio was less than 1.8. For Northern analysis, aliquots of pituitary RNA samples were subjected to electrophoresis on 1.2% agarose-formaldehyde gels. The RNAs were transferred to Nytran membranes (Schleicher & Schuell, Keene, NH) and cross-linked to the membranes by baking for 2 h at 80–90°C, followed by irradiation for 2 min with ultraviolet light. Purified cDNAs for rat FSHß (Dr. Richard Maurer, Oregon Health Sciences University, Portland, OR), LHß (Dr. James Roberts, Mount Sinai School of Medicine, New York, NY), and rat -subunit (Dr. William Chin, Harvard Medical School, Boston, MA) were labeled by the random primer method with [32P]dCTP (3000 Ci/mmol; New England Nuclear Research Products, Boston, MA) to a specific activity of 6–8 x 108 dpm/µg, as described previously [10]. The GnRH-R mRNA was measured by Northern analysis using an antisense riboprobe synthesized via the T7 promoter from pCDM8-mouse GnRH-R (Dr. Kevin Catt, NICHD, NIH, Bethesda, MD). Labeled probes were added to the hybridization solutions at a concentration of approximately 5 ng/ml for 48–72 h. Membranes were washed, autoradiographed, and analyzed using a BioRad GS-700 Imaging Densitometer (Hercules, CA). Membranes were rehybridized without stripping for normalization to cyclophilin cDNA.
In Situ Hybridization
Preparation of tissues Cryostat-cut, fresh-frozen coronal sections (thickness, 14 µm) were saved at levels from the optic chiasm to the mamillary bodies. Sections were thaw-mounted onto chrom-alum/gelatin subbed slides, dried, and stored at -80°C before processing. Tissue sections were fixed by immersion in 4% paraformaldehyde in 0.1 M PBS (pH 7.2) for 5 min and then rinsed with 0.1 M PBS for 3 min. Slides were then immersed for 10 min in a solution of 0.25% acetic anhydride containing 0.1 M triethanolamine (pH 8.0). This step decreases nonspecific binding of the probes by neutralizing positive charges on the tissues and slides. Sections were rinsed with 0.2x SSC (30 mM sodium chloride and 3 mM sodium citrate) for 10 min, dehydrated through a graded series of ethanols, and dried in a dessicator.
Preparation of radiolabeled probes
The PACAP mRNA in the PVN was detected using a 33P-labeled riboprobe [24]. A 670-base pair (bp) PACAP cDNA subcloned into pGEM-3Zf(-) was received from Dr. A. Arimura (Tulane University, Belle Chasse, LA). The plasmid was linearized with BamH1 and transcribed with T7 RNA polymerase using the MAXIscript kit (Ambion, Austin, TX) to produce a 670-bp, 33P-labeled antisense riboprobe. Labeling was accomplished by adding [-33P]uridine 5'-triphosphate to produce probes with a specific activity of approximately 2 x 107 cpm/pmol. A sense probe was used to document background hybridization.
Hybridization Groups of slides for hybridization consisted of six animals per group (Days 20–60), with three slides from each animal containing three 14-µm sections. The specific slides for each animal correspond to 1) 42–84 µm caudal to the optic chiasm, 2) 28–56 µm rostral to the start of the arcuate nucleus, and 3) the slide containing sections midway between the first two. Matched sections through the hypothalamus were hybridized with 100 µl of heat-denatured hybridization buffer containing 0.2 pmol of sense or antisense probe pipetted onto each section at 55°C for 16 h. Following hybridization, sections were rinsed with 2x SSC for 30 min and then incubated with 5 µg/ml of ribonuclease A (RNase A; Sigma type X-A; St. Louis, MO) in RNase buffer (10 mM Tris, 0.5 M NaCl, and 1 mM EDTA, pH 8.0) for 30 min at 37°C followed by a 30-min rinse with RNase buffer. Sections were then rinsed with 0.1x SSC at 60°C for 1 h, dehydrated through ethanol, and dried in a dessicator.
Slides were opposed to Kodak X-OMAT film (Eastman Kodak, Rochester, NY) for 7 days; the film was developed and later analyzed. Slides were then dipped into Kodak NTB-3 emulsion at 44°C and stored in light-tight boxes at 4°C. Slides were developed after 21–28 days with Kodak D-19 developer for 4 min at 15°C, fixed with Kodak fixer for 6 min, stained with hemotoxylin, coverslipped with DePeX (Gurr-BDH Chemicals Ltd., Poole, U.K.), and examined with a Nikon photomicroscope (Nikon Corporation, Tokyo, Japan).
Analysis The PACAP labeling within the PVN was quantified at the population and single-cell levels. Digital images of the PVN on each section were produced utilizing a Nikon microscope and image-analysis software. The PACAP labeling in the PVN was evaluated by analyzing the optical densities of the areas corresponding to the PVN in each section utilizing Scion Image analysis software (Scion Corp., Frederick, MD). Numbers of PACAP mRNA-containing cells were determined by counting all labeled cells in each of three sections/animal. Individually labeled cells were identified under high power using bright-field microscopy. Only labeled cells with an identifiable nucleus were included in the analysis. The number of pixels covered by reduced silver grains overlying each labeled cell was counted for all labeled cells in each of three regions per PVN per animal using a Scion Image analysis system. Extreme care was taken to ensure that lighting was constant for all sections analyzed within a given series. Background labeling per unit area was estimated for each section by averaging the number of pixels covered by silver grains in each of 10 fields without labeled cells across the cortex. Estimated background was calculated and subtracted from each cell. Only those cells for which the number of overlying pixels exceeded the background by 4 SD were considered to be labeled and were included in the analysis. In this way, the sections served as their own internal standard.
Measurement of Follistatin mRNA
Quantitative reverse transcriptase-polymerase chain reaction A previously described competitive template quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to measure total follistatin mRNA levels [25, 26]. Follistatin cDNA (Dr. Kelly Mayo, Northwestern University, Evanston, IL) was size-altered by substituting a 163-bp fragment of unrelated DNA for a 72-bp SplI/AccIII segment to create a competitive template that was used as an internal standard. The method therefore allows the same oligonucleotide primers to be used to amplify the native and competitive template cDNAs. Samples from each experimental group were analyzed simultaneously.
Semiquantitative RT-PCR First-strand cDNA was synthesized with a reverse transcription kit (Life Technologies, Inc., Rockville, MD) using 0.5 µg of total RNA, 1 µl of oligo(dT)12–18 (0.5 µg/µl), and 1 µl (50 U) of SuperScript II in a total volume of 20 µl following the manufacturer's suggested procedure. The PCR primers specific for follistatin (forward, 5'-GGGCAGGATCCATTGGATCCATTGGATTAGCCTAT-3'; reverse, 5'-ACACTGCTGGACAGTTTACCACTCT-3') were designed based on the published sequence of rat follistatin cDNA [27]. The cDNA was amplified in a 25-µl PCR reaction containing 1 U of Taq DNA polymerase (Promega, Madison, WI) and its buffer, 1.5 mmol/L of MgCl2, 0.2 mmol/L of each deoxy-NTP, and 0.2 µmol/L of the respective specific primers. Thirty-two cycles of amplification were carried out by denaturing for 30 sec at 94°C, annealing for 30 sec at 58°C, extension for 45 sec at 72°C, and a final extension for 7 min at 72°C. The PCR for glyceraldehyde phosphate dehydrogenase (GAPDH; 5'-GGCATTGCTCTCAATGACAA-3'; reverse, 5'-TGTGAGGGAGATGCTCAGTG-3') was run in parallel to rule out the possibility of RNA degradation. All primer pairs used spanned at least one intron, so the sizes of the PCR products rule out the presence of contaminating genomic DNA in the RNA samples. The RNA not subjected to RT was used as a specific control. Each PCR product was separated on a 1.8% agarose gel in Tris-borate-EDTA buffer and visualized by ethidium-bromide staining. Digital images of the gels were produced utilizing a gel documentation system (Genomic Solutions, Ann Arbor, MI), and the images were analyzed with Scion Image analysis software.
Sequencing the Rat Follistatin Transcript Variant, FST317, cDNA
To obtain a significant quantity of the putative follistatin (FS)-288 PCR product for sequencing, the products of several reactions identical to the one described for semiquantitative RT-PCR were combined and subjected to gel electrophoresis. The two PCR products (FS-288 and FS-315) were cut from the agarose gel and purified utilizing the QIAquick PCR purification kit (Qiagen, Valencia, CA) following the instructions of the manufacturer. The purified FS-315 PCR product was cloned and sequenced (see below), whereas the purified putative FS-288 PCR product was subjected to subsequent nested PCR amplification because of low abundance. The forward primer (5'-AGGTCGCTGCTCTCTCTGC-3') was complementary to the midregion of exon 4 of the published sequence of rat follistatin cDNA [27]. The reverse primer (5'-ATGGCTAGATGGGGGAATACAGG-3') included the first four nucleotides encoding exon 5 and the last 129 nucleotides encoding the splice variable region in intron 5 of the published sequence of human follistatin transcript variant, FST317, mRNA [28] (accession no. NM_006350). The single PCR product amplified by nested PCR of the suspected FS-288 purified PCR product was then gel excised and purified. The purified nested PCR product was then cloned utilizing the Acceptor Vector Kit (Novagen, Inc., Madison, WI), which included pSTBlue-1 vector and NovaBlue Singles competent cells. After ligation and transformation of the nested PCR insert, the cells were processed with the QIAprep Spin Miniprep Kit (Qiagen) for purification of plasmid DNA. Sequencing was performed in opposite directions by the Sequencing Core Facility (University of Louisville) utilizing primers designed to the SP6 and T7 sites of the pSTBlue-1 vector.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Analysis of serum LH and FSH values during sexual maturation in the male rat revealed a significant effect of age (Table 1). Whereas LH levels underwent a gradual developmental increase and peak at Day 50, FSH levels increased between Days 20 and 30 to reach peak concentrations, followed by a gradual yet significant decline to adult values. These results confirm previous reports by other investigators [11–16].
|
To correlate LHß and FSHß expression patterns with serum levels in the maturing male rat, Northern hybridization analysis was performed with total RNA isolated from the anterior pituitaries (Fig. 1). To correct for differences in the size of the anterior pituitaries of maturing rats, mRNA values were normalized by rehybridization to a cyclophilin cDNA. Northern analysis of pituitary mRNA levels revealed no significant effect of age on LHß mRNA expression. A small but nonsignificant rise was observed in LHß mRNA from 20 to 40 days, followed by a return to 20-day values by 60 days.
|
The FSHß mRNA levels in the anterior pituitary during sexual maturation in the male rat, by contrast, were significantly affected by age. The FSHß mRNA levels were lower (P < 0.0001) on Day 20 compared to all other ages examined and rose dramatically and significantly (7.5-fold) from Day 20 to Day 30. Thereafter, a gradual and significant decline in FSHß mRNA levels occurred between Days 30 and 50, at which time adult values (5.2-fold higher than on Day 20) were attained. Although the absolute changes in FSHß mRNA levels exceeded those for serum FSH concentrations, the developmental patterns were identical.
The subunit mRNA levels in the anterior pituitary during sexual maturation also changed significantly. The subunit mRNA levels increased sharply and significantly (5.7-fold) from Day 20 to Day 30. Afterward, a significant decline in subunit mRNA levels was observed between Days 30 and 50, when adult values (77% of the level on Day 20) were attained.
From the serum gonadotropin concentrations and their pituitary mRNA expression levels, it is apparent that the two gonadotropins are differentially regulated during sexual maturation in the male rat. In addition, our results reveal that serum FSH concentrations during development closely reflect pituitary mRNA expression, whereas serum LH concentrations and LHß mRNA levels are not similarly related. These findings suggest that more than one factor is contributing to the differential regulation of the gonadotropins during sexual maturation in male rats.
Expression of PACAP mRNA in the PVN During Sexual Maturation of the Male Rat
To examine a possible role for PACAP in gonadotropin regulation during sexual maturation, in situ hybridization for PACAP mRNA was performed on serial coronal brain sections through the regions of the PVN. Film autoradiography demonstrated intense PACAP labeling in the mediodorsal thalamus; in CA1 and CA4 regions of the hippocampus; in cortical layers 1, 3, and 5; at the amygdalostriatal transition area; and in the anterior and retrochiasmatic areas of the hypothalamus (Fig. 2).
|
To obtain higher regional resolution, PACAP mRNA-labeled brain sections were subjected to emulsion autoradiography and microscopic analyses. To examine age-related differences in PACAP mRNA expression in the PVN, detailed silver grain analysis was performed. At each age examined, PACAP mRNA was found in individual cells in the lateral and posterior portions of the PVN. The PACAP mRNA-positive cells were infrequent within the medial part of the PVN. Quantification of PACAP-positive cells revealed no differences between age groups in the average number (10.000 ± 0.592 SEM) of PACAP-labeled cells per PVN per tissue section (thickness, 14 µm). However, optical density analysis of silver grain accumulation within individual PVN revealed a significant decrease in PACAP mRNA between 20 and 30 days, followed by slowly rising levels that approached Day 20 values by Day 50 (Fig. 3).
|
To evaluate age-related changes in PACAP expression at the single-cell level, silver grain accumulation over individual PACAP mRNA-labeled cells in the PVN was examined (Fig. 4). Analysis of individual cells revealed that the number of grains per PACAP mRNA-positive cell was significantly greater at 20 days (42.7 ± 2.1 SEM grains/cell) compared to all other ages examined. A significant decline was observed in the number of grains from Day 20 to Day 30 (20.20 ± 1.44 SEM grains/cell), followed by a significant rise in PACAP labeling between 30 and 40 days (28.3 ± 1.5 SEM grains/cell). The number of grains per cell then gradually declined to Day 30 values by 60 days of age. This analysis of PACAP mRNA expression in the PVN reveals a reciprocal relationship with FSHß expression in the anterior pituitary (Fig. 1).
|
Follistatin mRNA Expression in the Pituitary During Sexual Maturation in the Male Rat
Previous studies support the hypothesis that PACAP selectively suppresses FSHß by up-regulating follistatin gene expression [22, 23]. Accordingly, anterior pituitary follistatin mRNA levels were examined during sexual maturation in the male rat. Total follistatin mRNA was first evaluated utilizing a quantitative, competitive PCR assay. This analysis revealed no significant change in expression during sexual maturation (Table 2). The mean concentration of anterior pituitary follistatin mRNA during sexual maturation was 3.81 ± 0.32 SEM fmol per 100 µg of RNA.
|
Follistatin mRNA was subsequently evaluated utilizing semiquantitative RT-PCR with primers designed to detect possible splice variants of follistatin mRNA similar to those demonstrated in RNA preparations in other species. This technique produced two different PCR products (Fig. 5). The most abundant band (370 bp) was sequenced and corresponded to the expected size of the mRNA species that encodes for the FS-315 protein. The second band (
720 bp), accounting for approximately 10% of follistatin mRNA, was slightly larger than the expected size (640 bp) for the human mRNA species that encodes for the FS-288 protein [28]. Both PCR products were analyzed for developmental changes. Differences in PCR starting material were corrected for by normalization with a GAPDH PCR product.
|
Analysis of the FS-315 product revealed no significant change during sexual maturation and confirmed the results of the competitive PCR analysis. On the other hand, analysis of the putative FS-288 bands revealed higher levels at Day 20 compared to all other days and significantly higher values at Day 20 than at Days 30 and 60. Comparison of the expression of pituitary follistatin with PVN PACAP revealed similar developmental expression patterns of FS-288 and PACAP mRNA in the PVN.
Sequencing Rat Follistatin Transcript Variant
Nested PCR amplification of the suspected FS-288 PCR product produced a single band of the appropriate size. Sequence analysis (Fig. 6) of this product revealed a region consisting of 354 nucleotides between the cDNA encoding rat follistatin exons 4 and 5 [27]. Consistent with the human follistatin mRNA transcript variant, FST317, this inserted stretch of cDNA began with a stop codon immediately after the codon encoding amino acid 288 of the rat follistatin peptide. Whereas the first 70 and last 164 nucleotides of this inserted region in the rat follistatin cDNA are 90% homologous to the human follistatin mRNA transcript variant, FST317, the remaining nucleotides in the center of this region are not homologous but are similar in that this region is A/T rich in both species. The rat follistatin mRNA transcript variant is nearly 90 bases longer than the human form, as predicted from the size difference in the PCR products amplified during semiquantitative RT-PCR. This alternatively spliced region of rat follistatin cDNA accounts for 60% of the unpublished 0.6-kilobase sequence of intron 5 of the FST344 variant of rat follistatin.
|
GnRH-R Expression in the Pituitary During Sexual Maturation in the Male Rat
Expression of PACAP may influence gonadotroph sensitivity to GnRH signaling through changes in pituitary GnRH-R either by directly activating the GnRH-R promoter [18–20] or through modulation of the follistatin/activin system [21]. Therefore, GnRH-R mRNA expression was also examined during development. As shown in Figure 1, GnRH-R levels at Day 20 were significantly lower than at any subsequent age. Expression of GnRH-R increased significantly (5.8-fold) between Days 20 and 30, followed by a significant decrease between Days 30 and 40 and a gradual decline from Day 40 to Day 60. The developmental pattern of pituitary GnRH-R expression is strikingly similar to that of FSHß expression and is reciprocal to the expression pattern of PACAP mRNA within the PVN.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Our analyses of serum gonadotropin concentrations throughout sexual maturation confirm the previous findings of discordant secretion of LH and FSH [11–16]. We observed that LHß and FSHß mRNA levels are likewise divergent during maturation and that expressions of the subunit and GnRH-R mRNAs more closely approximate that of FSHß mRNA. Furthermore, changes in pituitary FSHß mRNA levels are predictive of serum FSH concentrations, whereas serum LH varies significantly throughout development despite the lack of significant changes in pituitary LHß levels. In fact, the developmental increase in serum LH is parallel to the previously reported increases in hypothalamic GnRH content [30]. These results suggest that posttranslational processes modulate LH secretion whereas pretranslational mechanisms are the primary determinant of FSH secretion in male rats. This fundamental difference may be central to the differential secretion of FSH and LH during maturation.
Previous in vitro investigations have demonstrated that PACAP differentially regulates gonadotropin subunit gene expression in primary rat pituitary cells, effectively causing increased subunit mRNA levels and lengthened LHß mRNA transcripts but reduced FSHß mRNA levels [10]. In vivo, PACAP-immunoreactive cells have previously been demonstrated within the magnocellular or lateral part of the PVN as well as within nerve terminals within the median eminence [31]. Furthermore, in adult rats, the concentration of PACAP within the portal circulation is 2- to 4-fold the levels observed in the peripheral circulation, suggesting that PACAP has a hypophysiotropic function [4]. Therefore, changes in PACAP expression within the PVN could effectively translate into changes in the concentrations of PACAP within the pituitary milieu and contribute to the differential regulation of LH and FSH. Our analysis of the developmental expression pattern of PACAP mRNA within the PVN revealed a reciprocal relationship with pituitary expression of FSHß. Levels of PACAP mRNA were highest when levels of FSHß were lowest, and vice versa. These data are in agreement with the effects of PACAP on FSHß expression in rat pituitary cell cultures [10]. These correlations imply that PACAP may also play a role in regulating FSHß expression in vivo, although we acknowledge that the neurons we studied have not been proven to be hypophysiotropic.
Rather than a direct effect on FSHß expression, data from our laboratory suggest that PACAP decreases FSHß through increased production of the inhibitory paracrine factor follistatin [22, 23]. Follistatin inhibits FSHß expression by binding to and neutralizing activin [32]. Alternative splicing of the follistatin gene produces two mRNA species that encode for FS-315 and FS-288 [28]. Moreover, recombinant human FS-288 is 10-fold more potent than FS-315 in suppressing FSH release from rat pituitary cells in vitro [33]. The possibility of two mRNA species for follistatin in the rat was previously observed through PCR analysis of liver after treatment with carbon tetrachloride [34]. Although many species have been found to express two alternative splice forms of follistatin, to our knowledge the presence of two follistatin mRNA species in the rat pituitary has not previously been reported.
In the present study, we report the expression of the follistatin mRNA species that encodes for the FS-288 peptide within the rat pituitary gland. Moreover, our results demonstrate that the FS-288 mRNA form is developmentally regulated. On the other hand, no change in the level of expression of the predominant mRNA species encoding FS-315 peptide was detected by two different sensitive PCR assays. To our knowledge, differential regulation of the two species of follistatin mRNA has not been previously reported. From the data presented herein, we hypothesize that the relatively high expression of PACAP mRNA at Day 20 stimulates increased expression of FS-288 and that this mechanism contributes to the relative suppression of FSHß at this time point in development. The signal for decreased PACAP and follistatin expression from Days 20 to 30 is unknown but may be related to weaning; however, further investigations of this complicated transitional period are required to evaluate the specific effects of weaning on gonadotropin expression.
In summary, the differential secretion of FSH during sexual maturation in the male rat, as compared to LH, appears to be a consequence of differential expression of the FSHß and LHß genes. Our analysis of expression of the hypophysiotrophic peptide PACAP in the PVN during sexual maturation in the male rat revealed a reciprocal relationship between PACAP and FSHß expression. These data suggest that PACAP may have a role in the regulation of FSH during sexual maturation. The exact mechanism by which PACAP differentially regulates the gonadotropins is unclear but may involve modulation of activin signaling in the pituitary by selective regulation of different splice variants of follistatin.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Joseph P. Moore, Jr., University of Louisville, School of Medicine, Division of Endocrinology and Metabolism, ACB, Third Floor, Room A3G11, 530 South Jackson Street, Louisville, KY 40292. FAX: 502 852 2492; jpmoor03{at}louisville.edu
Received: 29 October 2002.
First decision: 28 November 2002.
Accepted: 26 February 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-DHT from 21 to 1000 days of age in ad libitum fed and dietary restricted rats. Exp Gerontol 1981 16:431-444[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  J. P. Moore ,Jr. and S. J. Winters Weaning and the Developmental Changes in Follicle-Stimulating Hormone, Pituitary Adenylate Cyclase-Activating Polypeptide, and Inhibin B in the Male Rat Biol Reprod, April 1, 2008; 78(4): 752 - 760. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Fernandez-Fernandez, M. Tena-Sempere, J. Roa, J. M. Castellano, V. M Navarro, E. Aguilar, and L. Pinilla Direct stimulatory effect of ghrelin on pituitary release of LH through a nitric oxide-dependent mechanism that is modulated by estrogen Reproduction, June 1, 2007; 133(6): 1223 - 1232. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Moore Jr, L. L. Burger, A. C. Dalkin, and S. J. Winters Pituitary Adenylate Cyclase Activating Polypeptide Messenger RNA in the Paraventricular Nucleus and Anterior Pituitary During the Rat Estrous Cycle Biol Reprod, September 1, 2005; 73(3): 491 - 499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Navarro, J. M. Castellano, R. Fernandez-Fernandez, S. Tovar, J. Roa, A. Mayen, M. L. Barreiro, F. F. Casanueva, E. Aguilar, C. Dieguez, et al. Effects of KiSS-1 Peptide, the Natural Ligand of GPR54, on Follicle-Stimulating Hormone Secretion in the Rat Endocrinology, April 1, 2005; 146(4): 1689 - 1697. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J Winters and J. P Moore Intra-pituitary regulation of gonadotrophs in male rodents and primates Reproduction, July 1, 2004; 128(1): 13 - 23. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) after amino acid 288. Underlined sequences share 90% homology to human follistatin transcript variant, FST317, mRNA (accession number NM_006350). The boxed sequences denote the primers utilized for nested PCR