LH signaling induced ptgs2a expression is required for ovulation in zebrafish

https://doi.org/10.1016/j.mce.2017.02.042Get rights and content

Highlights

  • Of the three zebrafish cox genes, the ptgs2a is most dominantly expressed in the ovary with a maximal level at M stage.

  • The ptgs2a expression is significantly higher in the follicular cells layers than the oocyte.

  • The ptgs2a expression is regulated by LH signaling through the cAMP-PKA pathway.

  • Co-injection of the Ptgs2 inhibitor with hCG could block the hCG-induced ovulation.

Abstract

It is well known that ovulation is induced by luteinizing hormone (LH) surge. However, the down-stream factors that mediating LH surge induced ovulation are less clear. The cyclooxygenases (also known as PTGS) as key enzymes for prostaglandins synthesis appear to be important for ovulation in mammals, but their functional roles and molecular mechanism in regulation of fish ovulation are largely unexplored. In this study, we have systematically investigated the expression, regulation and functional roles of cox genes during zebrafish ovulation. Three types of cox genes including ptgs1, ptgs2a and ptgs2b have been identified in zebrafish. The ptgs2a was dominantly expressed in the ovary with a maximal level at the maturation stage of the follicles. In addition, the ptgs2a expression is up-regulated by LH signaling in vitro and in vivo. Moreover, co-injection of a selective Ptgs2 inhibitor and non-selective Ptgs inhibitor with hCG could significantly block the stimulatory effect of hCG induced ovulation in vivo. Collectively, our findings indicate that 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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