LH signaling induced ptgs2a expression is required for ovulation in zebrafish
Introduction
Ovulation is a tightly regulated process during which one or more mature fertilizable ova are liberated from the ovarian follicle. In mammals, the luteinizing hormone (LH) surge induces the coordinate expression of a plethora of genes, initiates a cascade of events, and finally results in follicle rupture and release of the mature oocyte from the ovary (Richards et al., 2002). LH is also considered to be essential for ovulation in teleost fish (Ogiwara et al., 2013, Crespo et al., 2013). More recent studies showed that ovulation is disrupted in lhβ knockout zebrafish and medaka (Takahashi et al., 2016, Zhang et al., 2015, Chu et al., 2014). These findings suggest LH surge plays a conserved role in regulation of ovulation across species.
The functional roles of prostaglandins in regulation of ovulation have been investigated intensively in mammalian species (Tsafriri et al., 1972, Orczyk and Behrman, 1972, Armstrong, 1981, Murdoch et al., 1993). The cyclooxygenase (COX, also known as prostaglandin endoperoxide synthase, PTGS) enzyme catalyzes the limiting step of prostaglandin synthesis. Two COX genes (COX-1 and COX-2) have been identified in mammals (DeWitt et al., 1989, Funk et al., 1991, Smith et al., 2000). The COX-1 null mutants are fertile whereas COX-2 null mutants are infertile due to impaired ovulation (Lim et al., 1997). Treatment with PGE2 rescues the ovulation failure in COX-2 null mice (Davis et al., 1999). Moreover, gene knockout of PGE2 receptor lead to ovulation failure in mice (Hizaki et al., 1999, Tilley et al., 1999), indicating that PGE2 is the key ovulatory prostaglandin. The follicular PGs levels were stimulated by LH surge, which controls the timing of key ovulatory events (Richards, 1997). Similar to mammals, ovarian PGs synthesis is stimulated during ovulation process in non-mammalian vertebrates (Fujimori et al., 2011, Crespo et al., 2015), suggesting that prostaglandins might play an important role in regulation of ovulation in different species.
The cox genes have been also identified in teleosts. Interestingly, teleosts possess additional copies of cox gene that may result from the teleost-specific genome duplication event (Jarving et al., 2004, Ishikawa and Herschman, 2007, Ishikawa et al., 2007, Havird et al., 2008). Two cox-1 genes (ptgs1a and ptgs1b) and one cox-2 gene (ptgs2) have been identified in Japanese medaka (Fujimori et al., 2011, Takahashi et al., 2013), while one cox-1 gene (ptgs1) and two functional cox-2 genes (ptgs2a and ptgs2b) have been identified in the zebrafish genome (Ishikawa et al., 2007, Grosser et al., 2002, Pini et al., 2005). Previous studies showed that the synthesis of ovarian prostaglandin is involved in the regulation of oocyte maturation, ovulation and spawning in zebrafish (Lister and Van Der Kraak, 2008, Lister and Van Der Kraak, 2009, Knight and Van Der Kraak, 2015), but the precise role of the ptgs genes in mediating the pro-ovulatory action of LH has not been explored in zebrafish. In this study, we have systematically investigated the expression, regulation of ptgs in the ovary and their potential roles in mediating LH action on ovulation in zebrafish. We provided in vitro and in vivo evidence that the LH signaling induced ptgs2a expression is required for ovulation in zebrafish.
Section snippets
Chemicals and hormones
Human chorionic gonadotropin (hCG), Rp-cAMP, Forskolin, IBMX and Indomethacin were purchased from Sigma-Aldrich, SC-560 and NS-398 were obtained from Cayman Chemical. hCG and Rp-cAMP were first dissolved in water, whereas Forskolin, IBMX, SC-560, NS-398 and Indomethacin were first dissolved in dimethylsulfoxide (DMSO). They were diluted to the desired concentrations with the water or medium before use.
Zebrafish husbandry
AB strain zebrafish were reared in the laboratory of the Sun Yat-Sen University, following the
Gene expression of ptgs in zebrafish ovarian follicles
The zebrafish genome contains one ptgs1 gene and two ptgs2 genes (ptgs2a and ptgs2b). Firstly, we examined the relative levels of these genes in the ovarian follicles of different developmental stages using real-time PCR. The ptgs1 and ptgs2b expression were not significantly changed whereas the ptgs2a expression was significantly increased during folliculogenesis (Fig. 1b).
The ptgs1, ptgs2a and ptgs2b expression levels were further investigated from the FG stage to the maturation stage (prior
Discussion
It is well established that the LH surge is essential for ovulation in teleosts (Takahashi et al., 2016, Zhang et al., 2015, Chu et al., 2014, Tang et al., 2016). The cyclooxygenases and prostaglandins act as important down-stream mediators of LH signaling, their functional roles and molecular mechanism in mediating pro-ovulatory actions of LH on ovulation is not well established in non-mammalian vertebrates. In this study, we have demonstrated that the ptgs2a is stimulated by LH signaling and
Acknowledgements
We thank Mi Yao and Xi Wu for expert technical assistance. The research was supported by National Natural Science Foundation of China (NSFC Grant No. 31372512).
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