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Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
(Correspondence should be addressed to W Ge; Email: weige{at}cuhk.edu.hk)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activin (ßAßA, ßAßB, and ßBßB) was first identified as an ovarian factor in mammals that specifically stimulated pituitary FSH secretion without any effect on LH (Ling et al. 1986, Vale et al. 1986). Its subunits ßA and/or ßB have later been demonstrated to be expressed in a variety of extra-gonadal tissues including the pituitary in both mammals and fishes (Meunier et al. 1988, Roberts et al. 1989, Lau & Ge 2005), suggesting a wide range of biological activities at various sites. The functionality of the pituitary-derived activin in regulating mammalian FSH secretion has been demonstrated by immunoneutralization of activin both in vitro (Corrigan et al. 1991) and in vivo (DePaolo et al. 1992). The activities of activin are modulated or fine-tuned by its potent binding protein follistatin, which serves as a local modulator of activin activities in various tissues including the pituitary (Welt et al. 2002). In the goldfish, the treatment of cultured pituitary cells with follistatin caused a decrease in FSHß but an increase in LHß expression (Yuen & Ge 2004), which were opposite to the effects of activin (Yam et al. 1999a, Yuen & Ge 2004), suggesting an intra-pituitary activin-mediated autocrine/paracrine system in this species (Yuen & Ge 2004).
The potent modulatory effect of follistatin on activin and their co-expression in the pituitary suggest that the pituitary-derived follistatin may represent a regulatory point subject to the influence by both neuroendocrine and endocrine inputs, which in turn influences gonadotropin biosynthesis by suppressing activin activities. This hypothesis is supported by several studies in mammalian models. In the rat, a pulsatile application of GnRH at high frequency inhibited FSHß by stimulating pituitary follistatin, leading to a decreased activin activity (Kirk et al. 1994, Besecke et al. 1996). Follistatin expression was noted to precede that of FSHß in the rat pituitary during its estrous cycle but dropped drastically when FSH expression reached the maximal level (Halvorson et al. 1994), suggesting that follistatin expression may also be influenced by gonadal sex steroids. The effects of sex steroids on follistatin have been evidenced by the observation that gonadectomy stimulated pituitary follistatin in both male and female adult rats (Kaiser & Chin 1993). In support of this, treatment with testosterone suppressed follistatin expression in the rat pituitary in vitro (Bilezikjian et al. 1996). In contrast, the expression of follistatin in the pituitary of rhesus monkey was stimulated by in vitro testosterone treatment (Kawakami et al. 2002). This discrepancy may account for the difference in the post-gonadectomy increase of FSHß expression and FSH secretion between the rats (2- to 3-fold; Gharib et al. 1987) and primates (40-fold; Attardi et al. 1992). Interestingly, activin has been reported to stimulate follistatin mRNA expression (Bilezikjian et al. 1996), synthesis (Farnworth et al. 1995), and release (Bilezikjian et al. 1993) from cultured pituitary cells, and the effects could be abolished by follistatin itself. These results point to the presence of a local regulatory feedback loop involving activin and follistatin in the pituitary (DePaolo et al. 1991), and this regulatory loop is subject to the regulation by external hormonal factors.
In the goldfish, both activin ßA and ßB have been cloned by our laboratory (Ge et al. 1997, Yam et al. 1999a), and our previous work has demonstrated that different from the situation in mammals, activin differentially stimulates goldfish FSHß but suppresses LHß expression, and its effects can be reversed by follistatin (Yam et al. 1999a, Yuen & Ge 2004). The inverse effects of activin on the expression of the two gonadotropins make goldfish a unique model for studying the role of activin in the differential regulation of FSH and LH. Similar to that in mammals, we have also shown that the transcript of activin ßB subunit, but less likely ßA, is expressed in the goldfish pituitary (Ge et al. 1997, Yam et al. 1999b, Lau & Ge 2005). This, together with the reports in mammals that follistatin is also expressed in the pituitary (Besecke et al. 1996, Bilezikjian et al. 1996), has led us to hypothesize that there may exist a local paracrine regulatory system involving activin and follistatin in the goldfish pituitary, and this system may serve as a regulatory point that relays various neuroendocrine and endocrine inputs to the pituitary to control pituitary hormone biosynthesis, especially that of gonadotropins. In support of this hypothesis would be the evidence for activin and follistatin response to potential factors that control pituitary function. The present study was therefore undertaken to characterize the activin–follistatin system in the goldfish pituitary, with particular emphasis on its regulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Goldfish (Carassius auratus) of about 4–5 inches in body length were purchased from the local pet market and reared in 1000 l flow-through tanks with ample aeration for at least 1 week before use. The conditions for maintaining the fish were at 22 °C water temperature and 14 h light:10 h darkness photoperiod. All experiments were performed under the license from the Government of the Hong Kong Special Administrative Region and endorsed by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong. All fishes were anesthetized with tricaine methanesulfonate (MS222) before killing.
Reagents
Chemicals and enzymes were purchased from Sigma and Promega respectively unless otherwise specified. The medium M199 used in cell culture was obtained from Gibco Invitrogen. The Flp-In CHO cells for recombinant goldfish follistatin production were purchased from Invitrogen. Recombinant human follistatin was kindly provided by Dr A F Parlow (National Hormone and Peptide Program, National Institute of Diabetes & Digestive & Kidney Disease, CA, USA). Recombinant goldfish activin B was produced in our laboratory from an established CHO cell line, and one unit (U) is defined as the amount per ml that induces half maximal erythroid differentiation of F5-5 cells (ED50). Estradiol (E2) and testosterone (T) were purchased from Sigma. All drugs were first prepared as stocks and then diluted to specific working concentrations in culture medium before use.
Extraction of total RNA and reverse transcription
Goldfish tissues were collected and the total RNA from each tissue (10–30 mg) extracted in a 1.5 ml microtube containing 200 µl TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's protocol with some modifications. Following homogenization of the tissue, chloroform (50 µl) was added to each microtube, vortexed, and left at room temperature for 15 min. After centrifugation at 4 °C for 20 min, the aqueous supernatant was transferred to another tube containing 100 µl isopropanol for precipitation (–20 °C, 10 min). The pellet was washed with 75% ethanol prepared with diethylpyrocarbonate (DEPC)-treated water and dissolved in 4 µl RNase-free water. For RNA extraction from single pituitary, the same procedure was followed after the pituitary was homogenized in 200 µl TRI Reagent. The RNA (1 µg) was then subjected to reverse transcription in a total volume of 10 µl containing 1x Moloney murine leukemia virus (MMLV) first-strand buffer, 0.5 mM each dNTP, 0.5 µg oligo-dT, and 100 U MMLV reverse transcriptase (Promega) at 42 °C for 2 h.
Cloning and sequencing of full-length cDNA encoding goldfish follistatin
A pair of degenerate primers (Primer 1 and Primer 2; Table 1) was designed based on the conserved amino acid sequences of follistatin from other species. A goldfish ovarian cDNA library was used as template for PCR amplification, which was performed in 1x PCR buffer, 0.2 mM each dNTP, 2.5 mM MgCl2, 0.6 U Taq polymerase, and 0.2 µM each primer in a total reaction volume of 25 µl. The reaction was run for 31 cycles with the profile of 30 s at 94 °C, 30 s at 56 °C, and 1 min at 72 °C.
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The sequences of both strands of the cloned cDNA were obtained by exonuclease III and mung bean nuclease deletion method. All the sequencing reactions were performed on Peltier Thermal Cycler 100 (MJ Research, San Francisco, CA, USA) using dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin–Elmer, Foster City, CA, USA). The sequence was analyzed by ABI PRISM 310 Genetic Analyzer (Perkin–Elmer) followed by GenBank database search.
Production of recombinant goldfish follistatin
The Flp-In CHO Expression System (Invitrogen) was used to produce recombinant goldfish follistatin. Briefly, a pair of primers (Primer 5 and Primer 6; Table 1) was designed based on the sequence of the cloned cDNA, which flank the open reading frame (ORF) of goldfish follistatin. Two restriction sites, HindIII and BamHI, were added to the two primers respectively for cloning into the expression vector, pcDNA/FRT. A Kozak sequence (CCACC) was included between HindIII and the initiation codon ATG to improve translation efficiency (Kozak 1984). The protocol used for amplifying ORF was the same as described above except that Pfu polymerase (1.5 U) instead of Taq was used to increase fidelity of the reaction. The purified amplicon was digested with HindIII and BamHI and subcloned into the double-digested pcDNA/FRT to obtain the expression construct pcDNA/FRT/goldfish follistatin (gfFS). To facilitate future purification of recombinant follistatin, we also constructed an expression plasmid that contained an insert with six His residues added at the carboxyl terminal (pcDNA/FRT/gfFS-His). The construction of pcDNA/FRT/gfFS-His was the same as that of pcDNA/FRT/gfFS except that the antisense primer had the codons for His residues incorporated.
The cultured Flp-In CHO cells at 
40% confluence were co-transfected with pcDNA/FRT/gfFS or pcDNA/FRT/gfFS-His and pOG44 at the ratio of 1:9 by Lipofectamine (Invitrogen). The plasmid pOG44 provides the recombinase essential for genomic integration. The transfected cells were cultured to confluence in Ham F12 medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (HyClone Laboratories, Logan, UT, USA) at 37 °C with 5% CO2. The cells were then trypsinized, washed, and plated in 10 cm culture dishes at the density of 1x104/ml for selection with 450 ng/ml hygromycin B (Invitrogen). Individual clones were isolated by limited dilution in 96-well plates. The positive clones were identified by PCR on the genomic DNA and RT-PCR on the total RNA isolated from each clone.
Functional assay for recombinant goldfish follistatin
The biological activity of recombinant goldfish follistatin was assessed based on its inhibition of activin-induced differentiation of F5-5 cells, a mouse erythroleukemia cell line that responds to activin by differentiating into hemoglobin-producing cells (Eto et al. 1987, Schwall & Lai 1991). The assay protocol was according to Machida et al. (2000) with some modifications. Briefly, the F5-5 cells were cultured at 37 °C with 5% CO2 in Ham F12 medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (HyClone Laboratories). The cells were plated at a density of 2000 cells/400 µl/well in a 48-well plate. Hemoglobin production reached maximal level after incubation for 6 days with recombinant goldfish activin B. To assay the activity of follistatin produced, the conditioned media collected from untransfected and transfected CHO cells as well as individual clones at 80% confluence were added to the culture wells in the presence or absence of activin B (3 U/ml). Recombinant human follistatin was used as the positive control. After incubation for 6 days, the cells from each well were suspended, transferred to a 1.5 ml microtube, and stained by O-dianisidine/H2O2 solution for 15 min. After centrifugation to remove the supernatant, the pelleted cells were lysed with 100 µl H2O followed by freezing at –80 °C for 1 h and further solubilized with 10% SDS at 37 °C for 30 min. The lysates were transferred to a 96-well plate and the absorbance at the wavelength of 405 nM was measured on SpectraMax 250 microplate spectrophotometer (GMI Inc., Albertville, MI, USA).
Primary culture of dispersed pituitary cells
The primary culture of goldfish pituitary cells was performed as described previously (Yam et al. 1999a). Briefly, the pituitaries were collected from both males and females, washed, minced into small pieces, and trypsinized to disperse the cells. The dispersed cells were seeded in 48-well plates at a density of 0.4–0.6x106 cells/0.5 ml/well in 70% M199 supplemented with Earle's salt and 10% fetal calf serum (Hyclone Laboratories) and incubated at 28 °C with 5% CO2. The plates were pre-coated with 0.01% poly-D-lysine to enhance cell attachment. After pre-incubation for 24 h, the medium was changed before drug treatment.
Real-time RT-PCR assays
Total RNA from the cultured pituitary cells or individual pituitary gland was extracted with Tri Reagent and reverse transcribed as described above. The levels of gene expression were assayed with real-time RT-PCR. All primers used in the assays are listed in Table 2. To prepare standard for real-time PCR assay, the cDNA fragment of each target gene was amplified and purified by phenol/chloroform extraction and ethanol precipitation. The amplicon was then quantified by electrophoresis on a 1.8% agarose gel together with serially diluted MassRuler DNA Marker (MBI Fermentas, Hanover, MD, USA). The analysis and quantification were carried out on the Gel-Doc 1000 system with Molecular Analyst software (Bio-Rad).
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Data analysis
Each experiment was repeated at least twice with each treatment performed in triplicate. Data of follistatin bioassays were expressed as absorbance at OD405 and analyzed with one-way ANOVA followed by Dunnett's test. For the experiments on gene expression, the expression levels of goldfish follistatin, activin ßB, FSHß, LHß, and growth hormone (GH) were normalized to ß-actin, the internal control for RNA loading. Statistical analysis was performed with one-way ANOVA followed by Dunnett's test or Student's t-test using Prism 4.0 on Macintosh (GraphPad, San Diego, CA, USA). The differences were considered significant at P<0.05 and very significant at P<0.001.
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Results |
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A full-length cDNA (1228 bp) encoding goldfish follistatin was cloned by RACE. ORF analysis reveals that the cloned cDNA encodes a precursor protein with 323 amino acid residues (Fig. 1). GenBank database search showed that the deduced amino acid sequence had the highest homology with zebrafish follistatin (accession number: NM_131037 [GenBank] ; Fig. 1). The cloned goldfish follistatin lacks 27 conserved amino acids at the carboxyl terminal, similar to that of Xenopus and zebrafish, which is similar to the form of FS288 but different from FS315 reported in birds and mammals. The secretory nature of goldfish follistatin was suggested by the presence of a potential signal peptide at the amino terminal of the protein, which contains a leucine-rich hydrophobic region (ILLLLWLCYL) and a hydrophilic region (EDQKVQ) located just prior to the putative cleavage site (A/G). The possible cleavage site was identified between residues 33 and 34, rendering the secreted mature peptide 290 amino acids.
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Tissue distribution of follistatin expression in the goldfish
To examine the expression of follistatin in the goldfish, primers specific for follistatin were designed based on the sequence of the cloned cDNA. As shown in Fig. 2, a specific band of expected size (432 bp) was detected in all the tissues of the brain–pituitary–gonadal axis (brain, pituitary, ovary, and testis) although the expression level was relatively low in the pituitary. Expression of follistatin was also noted in the kidney and gill, but not in the liver. No product was observed in RT negative controls, which were the total RNA samples without being reverse transcribed. The expression of ß-actin as the internal control could be detected in all the tissues tested.
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After pcDNA/FRT/gfFS or pcDNA/FRT/gfFS-His was transfected into the Flp-In CHO cells, positive cells were selected and cloned in the presence of hygromycin B. The production and functional identity of goldfish follistatin were examined by the inhibition of activin activity in the erythroid differentiation factor (EDF) bioassay using F5-5 cells. One of the clones (gfFS-C3) that exhibited the best performance in suppressing activin activity was identified and expanded. The conditioned medium from gfFS-C3 significantly suppressed the activin effect on F5-5 cells with an ED50 of about 1.8 µl/ml (Fig. 3). As the positive control, recombinant human follistatin (FS288) also suppressed activin activity with an ED50 of about 3.1 ng/ml (Fig. 3). The amount of recombinant goldfish follistatin in the medium collected after 2-day incubation was therefore equivalent to 1.7 ng/µl human follistatin. Interestingly, addition of the His-tag at the carboxyl terminal appeared to abolish the activity of the recombinant protein because the selected pcDNA/FRT/gfFS-His-transfected cells produced no bioactivity of suppressing activin although the mRNA was abundantly transcribed from the insert (data not shown).
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Follistatin is a potent activin-binding protein and its expression has been demonstrated to be regulated by activin in a few systems (Farnworth et al. 1995, Dalkin et al. 1996, Besecke et al. 1996, Bilezikjian et al. 1996). To examine whether activin has any influence on the expression of follistatin in the goldfish pituitary, the cultured pituitary cells were exposed to different concentrations of goldfish activin B for 48 h after 24-h pre-incubation as described previously (Yuen & Ge 2004). Activin B significantly stimulated the expression of follistatin in a dose-dependent manner, and this effect could be blocked by co-incubation with recombinant human follistatin (Fig. 4A). Furthermore, treatment with human follistatin alone at 100 ng/ml nearly abolished the basal expression of goldfish follistatin in cultured pituitary cells (Fig. 4A).
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The dispersed goldfish pituitary cells were treated with various doses of testosterone or E2 for 48 h after pre-incuba-tion. Treatment with testosterone (1–100 nM) stimulated pituitary follistatin expression in a dose-dependent manner with the maximal response observed at 100 nM. Similarly, E2 also stimulated follistatin expression. Interestingly, none of the steroids had any effect on the expression of activin ßB (Fig. 6A). We also examined the responses of FSHß and LHß expression to the steroids in the same samples. The expression of LHß was significantly up-regulated by both testosterone and E2 with the maximal stimulation being observed at the highest concentration (100 nM). However, the expression of FSHß showed little response to either testosterone or E2 (Fig. 6B).
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The results described above suggested that there existed an activin–follistatin system in the goldfish pituitary and that follistatin could be the point of the system that is subject to regulation by endocrine and paracrine inputs. To provide further evidence for this hypothesis, we examined the annual expression profiles of follistatin, activin ßB, FSHß, and LHß in the individual pituitary glands during a 12-month reproductive cycle. Since GH may have potential roles in vertebrate reproduction (Hull & Harvey 2002), we also included GH in this study. In the spawning period (February and March), both FSHß and LHß significantly increased their expression levels, which remained high in the post-spawning period (April and May). Their expression levels dropped abruptly in June and remained low in the seasons afterward. These profiles were similar to those reported previously in the same species (Sohn et al. 1999). The GH showed relatively high expression levels in both the spring and autumn and low levels in the summer (June to August); however, the differences were not statistically significant. As demonstrated in vitro, the expression level of activin ßB remained rather stable throughout the reproductive cycle with no significant trend of changes. In contrast, follistatin expression in the pituitary showed significant variation in the annual reproductive cycle with the highest levels detected from August to November (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functional identity of the cloned goldfish follistatin homolog was confirmed by establishing a stable CHO cell line (gfFS-C3) that expressed the protein. The conditioned medium from the cloned gfFS-C3 cells significantly suppressed activin-induced F5-5 cell differentiation, while the control CHO cells produced no follistatin activity (data not shown).
Although follistatin was first isolated from the follicular fluid (Esch et al. 1987, Robertson et al. 1987, Ueno et al. 1987), its expression has been shown in other locations of the brain–pituitary–gonadal axis as well as in a variety of nonreproductive tissues (Michel et al. 1990). RT-PCR analysis in the present study showed that goldfish follistatin also had widespread tissue distribution except for the liver, which correlates well with the ubiquitous expression of activin subunits in the goldfish (Lau & Ge 2005). The co-expression of follistatin and activin in various tissues supports the idea that follistatin may be a critical factor in fine-tuning local activities of activin in the target tissues. It is noteworthy that although activin subunits and its receptors were detected in the goldfish liver in our previous studies (Ge et al. 1997, Yam et al. 1999b), the expression of follistatin was undetectable there, which agrees well with a previous study in the rat (Michel et al. 1990).
Similar to reports in mammals (Gospodarowicz & Lau 1989, Kogawa et al. 1991), the present and our previous studies also demonstrated co-expression of activin and follistatin in the goldfish pituitary (Lau & Ge 2005), suggesting an intra-pituitary activin–follistatin regulatory loop across vertebrates. Since activin stimulates goldfish FSHß but inhibits LHß expression in the goldfish (Yam et al. 1999a, Yuen & Ge 2004), the co-expression of activin and follistatin in the pituitary has led us to hypothesize that the relative expression levels of FSH and LH are likely influenced by the balance between activin and follistatin in the pituitary, and any factors that disturb such balance would potentially regulate the expression of the two gonadotropins. An interesting question that follows would be how activin subunits and follistatin in the goldfish pituitary are regulated.
To address this question, we first investigated the effect of activin on follistatin expression in cultured goldfish pituitary cells. In agreement with our previous finding (Yam et al. 1999a), goldfish activin B exhibited differential effects on FSHß and LHß expression. In contrast, both human follistatin and the conditioned medium of gfFS-C3 cells that produced recombinant goldfish follistatin caused opposite responses of FSHß and LHß, and these effects were likely due to the neutralization of endogenous activin in the pituitary. Interestingly, treatment with activin also significantly increased the expression of pituitary follistatin, therefore supporting the existence of a closed activin–follistatin regulatory loop in the pituitary (Fig. 8). The increased follistatin expression in response to activin may help keep the local activities of activin in check. In mammals, it has also been reported that addition of activin to cultured pituitary cells induced parallel increases of follistatin and FSH secretion in the rat (Bilezikjian et al. 1993) and sheep (Farnworth et al. 1995). The stimulatory effect of activin on follistatin has also been demonstrated at the transcriptional level (Besecke et al. 1996, Bilezikjian et al. 1996, Dalkin et al. 1996).
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The increased expression of follistatin but not activin ßB in response to steroid treatments suggests that it is likely follistatin that serves as a regulatory point of the activin–follistatin system in the pituitary. Although activin ßB level remains rather constant, the activities of activin are expected to be fine-tuned by the local level of follistatin in the pituitary. In support of this hypothesis is the in vivo evidence that similar to FSHß and LHß, follistatin in the female goldfish pituitary exhibited significant changes in expression during the annual reproductive cycle whereas activin ßB showed no evident trend of variation. Although it is difficult to assess the importance of follistatin variation in the annual expression profiles of gonadotropins (FSH and LH) at this moment because of the multifactorial nature of FSH and LH regulation, this result at least lends further weight to the hypothesis that follistatin in the pituitary may represent a critical regulatory point.
Both testosterone and E2 stimulated LHß expression but lacked effect on FSHß expression in the present study. In the goldfish, previous studies using incubated pituitary fragments demonstrated stimulatory effects of testosterone and E2 on the expression of both FSHß and LHß; however, FSHß appeared to be less responsive to E2 treatment than LHß (Huggard et al. 1996, Habibi & Huggard 1998, Huggard-Nelson et al. 2002). The in vitro effect of E2 on gonadotropin subunit expression, especially that of FSHß, seemed to be dependent on the stage of sexual maturity. E2 significantly stimulated FSHß expression in cultured pituitary fragments from early but not late recrudescent (vitellogenic) fish (Huggard-Nelson et al. 2002). This stage dependence could also be the reason for the lack of effects of steroids on FSHß in the present study because we performed the experiments in February, when the goldfish in Southern China was near the peak spawning period. Indeed, in a previous unpublished study using slot blot hybridization, we did observe significant stimulatory effects of testosterone and E2 on both FSHß and LHß expression in cultured pituitary cells from sexually regressed goldfish (Yuen & Ge unpublished data). In contrast to the variable response of FSHß to steroids, both testosterone and E2 consistently stimulated goldfish LHß expression in the present as well as our previous unpublished studies. Although these stimulatory effects could be due to direct actions of these steroids on LH cells, the increase might also be linked to the increased expression of follistatin, which could in turn reduce the inhibitory effect of activin on LHß expression. The same speculation does not seem to be applicable to FSHß, whose expression would be expected to decrease in response to an increased follistatin expression. In mammals, the expression level of FSHß has been reported to correlate well with that of pituitary follistatin (Bilezikjian et al. 1996, Kawakami et al. 2002, Leal et al. 2002). One possibility is that goldfish FSHß may be subject to dual controls by both the intra-pituitary activin–follistatin system and gonadal steroids, and the relatively stable FSHß expression in response to steroidal treatments may reflect the net effect of reduced activin stimulation due to the increased follistatin and an increased steroidal stimulation. What is intriguing is that in addition to activin, follistatin has also been reported to bind some members of the bone morphogenetic protein (BMP) family such as BMP-4 and BMP-7 (Iemura et al. 1998), and some BMP members such as BMP-6 and BMP-7 have also been shown to exert a paracrine effect in the pituitary of mammals to regulate gonadotropin expression (Huang et al. 2001). How these BMP ligands fit into the activin–follistatin system in the goldfish pituitary remains to be elucidated.
To summarize, a full-length cDNA for goldfish follistatin was cloned and its expression demonstrated in the pituitary. Further experiments showed that in addition to the differential effects on the expression of goldfish FSHß and LHß, activin also stimulated the expression of its binding protein follistatin within the pituitary, which may represent a local closed-loop negative feedback mechanism to modulate the activities of activin. This mechanism seems to be conserved across vertebrates, suggesting physiological importance of the intra-pituitary activin–follistatin system in controlling pituitary hormone biosynthesis and secretion. Furthermore, the expression of follistatin but not activin itself was regulated by sex steroids, suggesting that it is likely follistatin that serves as the point of the system that is subject to the endocrine or neuroendocrine regulation (Fig. 8). This idea is further supported by the in vivo evidence that the expression of follistatin in the pituitary varied significantly, similar to that of FSHß, LHß, and GH, whereas activin ßB expression remained rather constant during the annual reproductive cycle.
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Acknowledgements |
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Received in final form 7 September 2007
Accepted 24 September 2007
Made available online as an Accepted Preprint 25 September 2007
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