2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD or dioxin) is regarded as an endocrine-disrupting chemical with the ability to disrupt reproductive systems [1,2]. A single high dose of TCDD induces abortion, alters sexual behavior, decreases spermatogenesis and diminishes fertility [3][4][5][6]. In the rat, TCDD compromises ovarian follicular development and function including a premature transition to reproductive senescence [5] and a disruption of estrous cyclicity with prolonged periods of diestrus [7]. An acute prepubertal exposure to TCDD in intact and hypophysectomized rats reduces ovulation directly by blocking the follicular rupture and indirectly by blocking the luteinizing hormone (LH) surge [8][9][10]. reduces ovulation rate in rats. The present study was to investigate whether TCDD alters the progression of cell cycle, and thus resulting in the blockade of ovulation in gonadotropin-primed, immature rats. The ovulation rate and ovarian weight were reduced in intact rats given TCDD (32 µg/kg BW in corn oil) by gavage one day before pregnant mare's serum gonadotropin (PMSG; 5 IU/rat) injection. Flow cytometry demonstrated that the percentage of granulosa cells in S-phase was increased at 24 h following PMSG treatment, but declined at 8 h following hCG treatment in corn oil-treated rats. Interestingly, the number of S-phase cells in TCDD-treated rats was reduced 24 and 48 h following PMSG treatment. TCDD, however, increased the percentage of cells in G2/M-phase at 24 h following PMSG treatment. TCDD inhibited the mRNA levels of Cdk2 at 0 h and 24 h, and cyclin D2 at 24 h and 48 h following PMSG treatment. Protein levels of aryl hydrocarbon receptor in granulosa cells were elevated in TCDD-treated rats at 12 h and 24 h following PMSG treatment. The present study indicates that TCDD reduces S-phase cells and inhibits levels of Cdk2 and cyclin D2 at 24 h following PMSG treatment, implying the ovulation-inhibiting action of TCDD may be exerted through the attenuation of cell cycle progression via AhR-mediated cascade.Key words: Dioxin, AhR, Ovary, Cell cycleAlthough it is apparent that TCDD affects follicular maturation and ovulation, the mechanisms that underlie these reproductive toxicities are poorly understood. It is generally accepted that TCDD action is mediated by the aryl hydrocarbon receptor (AhR)-signaling cascade, a transcription factor whose natural ligand remains unknown [11,12]. Since the presence of AhR has been reported in the ovary [13][14][15], TCDD could directly act at the ovary to disrupt critical cellular signals via AhR-mediated alterations in gene transcription, thereby contributing to the observed impairment of follicular maturation and ovulation. TCDD administration to immature rats primed with gonadotropins inhibits ovarian Ptgs2 expression [16], a requisite gene for ovulation [17]. TCDD stimulates Cyp1a1 and Cyp1b1 expression in rat granulosa cells, thereby reducing estrogen secretion by catalyzing estrogen metabolism [18,19]. Moreover, TCDD decreases the expression of Cyp17 in human lu...

The present study was designed to test whether GnRH regulates pituitary adenylate cyclase-activating polypeptide mRNA levels in a stage-dependent manner during follicle development in the rat ovary. The granulosa cells of preovulatory and immature follicles obtained from PMSG- and estrogen-treated rats, respectively, were cultured in serum-free conditions in the presence of various hormones. GnRH receptor mRNA expression was detected in both preovulatory and immature granulosa cells and was down-regulated by gonadotropins. Treatment of preovulatory granulosa cells with GnRH agonist stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner. In situ hybridization analysis of cultured preovulatory follicles revealed that GnRH-induced pituitary adenylate cyclase- activating polypeptide signals were detected in granulosa cells, but not thecal cells. In immature granulosa cells, cotreatment with GnRH agonist suppressed FSH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner, whereas treatment with GnRH alone had no effect. Furthermore, treatment with GnRH antagonist inhibited LH-induced pituitary adenylate cyclase-activating polypeptide gene expression in preovulatory granulosa cells, whereas it stimulated FSH-induced pituitary adenylate cyclase-activating polypeptide gene expression in immature granulosa cells. Interestingly, GnRH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in preovulatory granulosa cells was inhibited by arachidonyltri fluoromethyl ketone, an inhibitor of phospholipase A(2), but not by an inhibitor of protein kinase A or C. Lastly, treatment of preovulatory follicles with pituitary adenylate cyclase-activating polypeptide antagonist suppressed GnRH-stimulated progesterone production during 6--9 h of culture. Taken together, these results demonstrate the stage-dependent regulation of pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH, the stimulatory and inhibitory effect in granulosa cells of preovulatory and immature follicles, respectively.