Potential targets of transforming growth factor-beta1 during inhibition of oocyte maturation in zebrafish.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
These findings suggest that TGF-beta1 acts at multiple sites, including LHR, 20beta-HSD and mPR-beta, to inhibit zebrafish oocyte maturation.
Kohli
Gurneet
G
Department of Biology, York University, Toronto, Ontario, M3J 1P3, Canada. gkohli@yorku.ca
Clelland
Eric
E
Peng
Chun
C
D000818:Animals; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D003349:Cortisone Reductase / Q000096:biosynthesis; D003350:Cortodoxone / Q000031:analogs & derivatives; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D015536:Down-Regulation; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006908:Hydroxyprogesterones / Q000037:antagonists & inhibitors; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D006080:Ovarian Follicle / Q000187:drug effects / Q000502:physiology; D011962:Receptors, FSH / Q000096:biosynthesis; D011974:Receptors, LH / Q000096:biosynthesis / Q000378:metabolism; D011980:Receptors, Progesterone / Q000096:biosynthesis; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000096:biosynthesis
D006063:Chorionic Gonadotropin; D006908:Hydroxyprogesterones; C511728:Paqr8 protein, zebrafish; D011962:Receptors, FSH; D011974:Receptors, LH; D011980:Receptors, Progesterone; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D029961:Zebrafish Proteins; C511727:paqr7b protein, zebrafish; C007433:17,20-dihydroxy-4-pregnen-3-one; D003609:Dactinomycin; C051954:17,20,21-trihydroxy-4-pregnen-3-one; D003513:Cycloheximide; D003349:Cortisone Reductase; D003350:Cortodoxone