Potential targets of transforming growth factor-beta1 during inhibition of oocyte maturation in zebrafish
© Kohli et al; licensee BioMed Central Ltd. 2005
Received: 11 August 2005
Accepted: 30 September 2005
Published: 30 September 2005
TGF-beta is a multifunctional growth factor involved in regulating a variety of cellular activities. Unlike mammals, the function of TGF-beta in the reproduction of lower vertebrates, such as fish, is not clear. Recently, we showed that TGF-beta1 inhibits gonadotropin- and 17alpha, 20beta-dihydroxyprogesterone (DHP)-induced maturation in zebrafish. The aim of the present study was to investigate the mechanisms underlying this action.
To determine if the effect of TGF-beta1 on oocyte maturation involves transcription and/or translation, ovarian follicles were pre-treated with actinomycin D, a blocker of transcription, and cyclohexamide, an inhibitor of translation, and incubated with hCG or DHP, either alone or in combination with TGF-beta1 and oocyte maturation scored. To determine the effect of TGF-beta1 on mRNA levels of several key effectors of oocyte maturation, three sets of experiments were performed. First, follicles were treated with control medium or TGF-beta1 for 2, 6, 12, and 24 h. Second, follicles were treated with different concentrations of TGF-beta1 (0 to 10 ng/ml) for 18 h. Third, follicles were incubated with hCG in the absence or presence of TGF-beta1 for 18 h. At the end of each experiment, total RNA was extracted and reverse transcribed. PCR using primers specific for 20beta-hydroxysteroid dehydrogenase (20beta-HSD) which is involved in DHP production, follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), the two forms of membrane progestin receptor: mPR-alpha and mPR-beta, as well as GAPDH (control), were performed.
Treatment with actinomycin D, a blocker of transcription, reduced the inhibitory effect of TGF-beta1 on DHP-induced oocyte maturation, indicating that the inhibitory action of TGF-beta1 is in part due to regulation of gene transcription. Treatment with TGF-beta1 caused a dose and time-dependent decrease in mRNA levels of 20beta-HSD, LHR and mPR-beta in follicles. On the other hand, TGF-beta1 had no effect on mPR-alpha mRNA expression and increased FSHR mRNA levels. Furthermore, hCG upregulated 20beta-HSD, LHR and mPR-beta mRNA levels, but this stimulatory effect was blocked by TGF-beta1.
These findings suggest that TGF-beta1 acts at multiple sites, including LHR, 20beta-HSD and mPR-beta, to inhibit zebrafish oocyte maturation.
Transforming Growth Factor-β1 (TGF-β1) is the prototypical member of the TGF-β family [1, 2]. Members of this family are implicated in diverse physiological processes, including reproduction. Three isoforms of TGF-β (TGF-β1, -β2, and -β3) are expressed in the mammalian ovary [2–4]. They have been shown to regulate follicle development, steroidogenesis, oocyte maturation, ovulation and follicular atresia [2–4]. There is molecular evidence for the presence of TGF-β1–3 in fish [5–7]. However, the role of TGF-β in fish reproduction is not well understood. Studies in zebrafish have suggested that TGF-β inhibits oocyte maturation . In the goldfish, TGF-β has been reported to inhibit ovarian steroid production .
Ovarian development in fish is broadly divided into two major phases: growth and maturation. During oocyte growth, follicle stimulating hormone (FSH) stimulates production of estradiol-17β from the ovary. Estradiol-17β stimulates the production of vitellogenin by the liver. Vitellogenin is taken up by the developing oocyte and cleaved to yolk protein, which serves as a nutritional reserve for the developing embryo [8, 10, 11]. Oocyte maturation in teleosts is triggered by the release of leutinizing hormone (LH) from the pituitary. LH stimulates a number of signaling cascades culminating in the production of 17α-hydroxyprogesterone (17α-HP). In the granulosa cells, under the action of 20β-hydroxysteroid dehydrogenase (20β-HSD), 17α-HP is converted to 17α, 20β-dihydroxyprogesterone (DHP), the maturation inducing hormone (MIH) in cyprinids, such as zebrafish and goldfish. MIH activates the cytoplasmic maturation promoting factor (MPF), which is made up of two subunits: cyclin B (a regulatory subunit) and cdc2 (a catalytic subunit). MIH stimulates the de novo synthesis of cyclin B. Cyclin B protein binds to cdc2 to form MPF. The newly formed MPF is activated by phosphorylation of cdc2 on threonine 161. The active MPF, then, stimulates all the changes associated with oocyte maturation, such as germinal vesicle break down (GVBD), spindle formation, chromosome condensation and allows the transition from G2/M phase of meiosis [12–15].
Two isoforms of the MIH receptor, designated as membrane progestin receptor-α (mPR-α) and mPR-β, have recently been cloned in zebrafish . Microinjection of zebrafish oocytes with antisense oligonucleotides to either mPR-α or mPR-β or both receptors has been shown to block MIH-induced maturation, indicating that both play a role in zebrafish oocyte maturation . Originally discovered in sea-trout oocytes, several isoforms of mPR have also been discovered in humans and other vertebrates [16–20].
The zebrafish model has been used extensively for studies on early embryonic development. This model is also very useful for the investigation of ovarian follicle development and maturation because the zebrafish ovary contains ovarian follicles at different stages of development. We and others have been using zebrafish to examine the role of TGF-β superfamily in oocyte maturation [8, 21–23]. Our previous work has shown that TGF-β1 inhibits human chorionic gonadotropin (hCG) and MIH-induced maturation in zebrafish . We have also observed a decrease in TGF-β1 mRNA expression in maturing follicles. These findings suggest that TGF-β1 may play a role in preventing premature oocyte maturation in zebrafish. The present study is an attempt to elucidate the mechanisms underlying the inhibitory effect of TGF-β1 by identifying the potential target genes of TGF-β1. We report here our recent findings on the effect of TGF-β1 on the important effectors of oocyte maturation in zebrafish, including: 20β-HSD, LHR, mPR-α, and mPR-β.
Materials and methods
Adult zebrafish, Danio rerio, were purchased from a local pet supplier (Fish & Bird Emporium, Brampton, ON) and maintained in 10 L tanks of an AHAB System(Aquatic Habitats, FL) at 26°C, under a 14-h light, 10-h dark photoperiod. The fish were fed twice daily with tropical fish food and supplemented with freshly hatched brine shrimp two or three times a week. Experiments were performed according to the Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care.
In vitro culture of zebrafish follicles
Female zebrafish that have a full-grown ovary were anesthetized using 3-aminobenzoicacid ethyl ester (Sigma-Aldrich Canada Inc., Oakville, ON) and decapitated. The ovaries were removed and follicles greater than 0.52 mm in diameter were collected, since previous studies in the zebrafish have shown that only follicles of this size can undergo maturation in vitro in response to hormones . Approximately 20 follicles were placed into each well of a 24-well culture plate and pre-treated at 26°C in either 1 ml of modified Cortland's medium (MCM) or MCM + chemicals such as Actinomycin D or Cyclohexamide (Sigma-Aldrich, Mississauga, ON) for 2 hours. Pre-treated follicles were then incubated with the control medium, human recombinant TGF-β1 (R&D Systems, Minneapolis, MN), hCG (kindly provided by Dr. A. F. Parlow, National Hormone and Peptide Program, Torrance, CA), MIH (17α, 20β-DHP;Sigma-Aldrich Canada), either alone or in combination, as previously described . Maturation was scored after 18 hours of incubation. Follicles that underwent germinal vesicle breakdown (GVBD) could be identified by their acquired translucency.
Total RNA extraction
Approximately 80 follicles per treatment group were used for RNA extraction. Total RNA was extracted using an RNeasy Mini kit (Qiagen Inc., Mississauga, ON) according to the manufacturer's suggested protocol. The RNase-free deoxyribonuclease Set (Qiagen) was also used during RNA isolation to remove any potential genomic DNA contamination.
Reverse Transcription (RT) and polymerase chain reaction (PCR)
List of primers and their sequences
Forward: TGC ACG AGT GGT CAA TGT GTC
Reverse: ACT AGC TGT CCA TGC GGC TCT
Forward: GGA TTC TTC ACC GTC TTC TCC
Reverse: TGT AGC TGC TCA ACT CAA ACA
Forward: GGC GAA GGC TAG ATG GCA CAT
Reverse: TCG CCA TCT GGT TCA TCA ATA
Forward: CAG CGC CTA CTT CTT CTC GT
Reverse: CAC TGC ATC ATG AGC CAA AT
Forward: ACA ACG AGC TGC TGA ATG TG
Reverse: ATG GGC CAG TTC AGA GTG AG
All values are expressed as mean ± SEM of 3–4 replicates in one representative experiment. All experiments were performed three times to confirm the results using different batches of animals. To determine the statistical difference among different groups at the same time point, multiple group comparisons were performed by one-way ANOVA, followed by a Student-Newman-Keuls multiple group comparisons test, using the GraphPad InStat Software (GraphPad Inc., San Diego, CA). P < 0.05 was considered significant.
Effects of transcriptional and translational inhibitors on final oocyte maturation
Semi-quantitative RT-PCR Validation
Effect of TGF-β1 on the mRNA expression of 20β-Hydroxysteroid dehydrogenase
Effect of TGF-β1 on the mRNA expression of follicle stimulating hormone receptor
Effect of TGF-β1 on the mRNA expression of the luteinizing hormone receptor
Effect of TGF-β1 on the mRNA expression of membrane progestin receptors
Currently, little is known about the role of TGF-β in regulating ovarian functions in lower vertebrates, such as fish. Recent studies have suggested that TGF-β inhibits steroid production in the goldfish ovary  and oocyte maturation in zebrafish . In the present study, we further examined the cellular mechanisms underlying the inhibitory effect of TGF-β in oocyte maturation. We have shown that TGF-β1 inhibits mRNA expression of 20β-HSD, the key enzyme involved in MIH production, as well as LHR and mPR-β. These novel findings suggest that TGF-β1 inhibits multiple targets in the oocyte maturation pathway, both upstream and downstream of MIH.
One of the potential targets of TGF-β1 identified in this study is 20β-HSD. We found that TGF-β1 inhibited basal and hCG-induced 20β-HSD mRNA levels. TGF-β1 alone induced a dose- and time-dependent inhibitory effect on 20β-HSD mRNA expression. Treatment with hCG increased 20β-HSD mRNA levels; however, in the presence of TGF-β1, the effect of hCG was blocked. 20β-HSD activity in the ovary and its stimulation by gonadotropins and cAMP-enhancing drugs has been demonstrated in many species [10, 11, 24–28]. Our finding that hCG stimulates 20β-HSD is consistent with studies in mammals [24, 28] and a recent study in Nile Tilapia, which reported an increase in the mRNA expression of 20β-HSD in response to hCG treatment . The observation of decreased 20β-HSD mRNA levels after TGF-β1 treatment suggests that TGF-β1 may inhibit 20β-HSD activity, leading to a decrease in MIH production. This notion is supported by a recent report that TGF-β1 inhibits the conversion of 17α-HP to DHP in the goldfish . It is possible that one of the actions of TGF-β1 is to decreases MIH production, and thus inhibits oocyte maturation.
In this study, we observed that hCG increased LHR but had no effect on FSHR mRNA levels. Several studies conducted in fish on the effect of gonadotropin on LHR and FSHR expression have yielded inconsistent results. In African and Channel catfish, it has been reported that hCG treatment caused an activation of the LHR and cAMP mediated pathways, as well as a slight increase in FSHR mRNA levels [29–31]. However, a two-fold increase in FSHR expression, but no change in LHR expression in pre-maturational follicles of rainbow trout in response to treatment with partially purified gonadotropins has also been reported . The reason for such discrepancy is unclear, however, it could be due to species specificity or variation in treatment conditions.
An interesting finding from this study is that TGF-β1 differentially regulates FSHR and LHR mRNA levels. We observed that basal FSH receptor mRNA levels were increased, while basal- and hCG-induced LH receptor mRNA levels were decreased, upon incubation of follicles with TGF-β1. TGF-β1 has been reported to regulate LH and FSH binding sites in mammals, however, whether TGF-β1 has a stimulatory or inhibitory effect appears to be species-dependant. TGF-β1 decreased basal and FSH-induced LH binding sites in porcine granulosa cells but enhanced FSH-induced LH binding in rat granulosa cells . FSH induced FSH binding in porcine granulosa cells and this effect was attenuated by TGF-β1. On the other hand, FSH decreased its own binding levels in rat granulosa cells and this effect was also blocked by TGF-β1 . Our finding that TGF-β1 increased FSHR mRNA levels is consistent with previous studies in rat granulosa cells [34, 35]. However, our observation that TGF-β1 decreased LHR mRNA levels is opposite to studies in rat  and chicken  granulosa cells. In these studies, it was reported that TGF-β1 induced LHR mRNA expression. Whether this is due to species variation as in the case of rat and pig or to the difference in follicle development between fish and higher vertebrates awaits more studies in fish species. The finding that TGF-β1 inhibited LHR mRNA expression suggests that the inhibitory effect of TGF-β1 on hCG-induced oocyte maturation may be due, in part, to the downregulation of LH receptor by TGF-β1. Since FSH is known to be a major regulator of oocyte growth [10, 11], by stimulating FSH receptor expression TGF-β1 may play a role in promoting follicle growth. These findings, together with our previous observation that TGF-β1 mRNA levels are higher in growing follicles than in maturing follicles, suggest that TGF-β1 may stimulate follicle development and inhibit precocious oocyte maturation in the zebrafish ovary. This possibility will be investigated further in the future.
A recent study has shown that microinjection of zebrafish oocytes with antisense oligonucleotides to either mPR-α or mPR-β or both causes similar marked decreases in the rates of oocyte maturation, suggesting that both subtypes are obligatory for oocyte maturation in zebrafish . In this study, we observed a strong induction of mPR-β mRNA levels by hCG and an inhibitory effect of TGF-β1 on both basal and hCG-induced mPR-β mRNA expression. These findings support the role of mPR-β in oocyte maturation and suggest that one of the mechanisms by which TGF-β1 inhibits hCG- and MIH-induced oocyte maturation is by the downregulation of MIH receptors, specifically mPR-β. However, we found that neither hCG nor TGF-β1 had an effect of mPR-α mRNA levels, suggesting that the two membrane progestin receptors are under differential regulation. It has been reported that hCG caused an increase in the mPR protein expression in sea-trout oocytes . The sea-trout mPR has a higher homology to mPR-α (80%) than to mPR-β (46%). It remains to be determined if hCG regulates mPR-α and mPR-β protein levels in the zebrafish. Recently, Kazeta et al. (2005) reported that hCG did not change mRNA levels of mPR-α and mPR-β at 5 and 10 h after hCG treatment . The difference between this and our studies may be due to the duration of hCG treatment as 18 h was used in our study.
Based on our findings that TGF-β1 downregulates basal and hCG-induced LHR, 20β-HSD, and mPR-β mRNA levels, we propose that TGF-β1 acts upon multiple targets to exert its inhibitory effect on oocyte maturation. TGF-β1 may downregulate LHR, leading to decreased signal transduction and decreased production of 17α-hydroxyprogesterone. TGF-β1 may inhibit basal and gonadotropin-induced MIH production by inhibiting 20β-HSD. Finally, TGF-β1 may inhibit the expression of the MIH receptor, such as mPR-β, on the oocyte surfaces, leading to a reduction of MPF activation and subsequent oocyte maturation. This model can be tested in the future by examining the protein levels on these molecules once antibodies become available. Similarly, the physiological significance of TGF-β on oocyte maturation will be confirmed using loss-of-function approaches.
This study was supported by a grant from Natural Science and Engineering Research Council (NSERC) of Canada and an Ontario Premier's Research Excellence Award to CP. CP is a recipient of a Mid-Career Award from Ontario Women's Health Council and CIHR. We thank Dr. A.F. Parlow and NHPP for providing human chorionic gonadotropin.
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