Investigating the role of prostaglandin receptor isoform EP4b in zebrafish ovulation

https://doi.org/10.1016/j.ygcen.2019.113228Get rights and content

Highlights

  • Zebrafish ovarian follicles express multiple prostaglandin receptor subtypes.

  • Only the expression of receptor subtype EP4b increased during spontaneous ovulation.

  • Treatment with Ovaprim to induce ovulation increased expression of EP4b.

  • In vitro treatment of full-grown follicles with 17,20βP increased EP4b expression.

  • EP4 receptor antagonists block the effect of Ovaprim but not EP1 or EP2 antagonists.

Abstract

Prostaglandins (PGs) are a class of fatty acid-derived hormones that play an essential role in the regulation of ovulation of teleosts. This study investigated the various isoforms of ovarian PG receptors in the zebrafish ovary and their role in ovulation. Using real time qPCR, six PG receptor isoforms (ptger1a, ptger1b, ptger2a, ptger4a, ptger4b, and ptgfr) were shown to be expressed in the ovary. Only the PG receptor isoform ptger4b was upregulated at the time of ovulation in vivo, or following treatment in vivo with Ovaprim, which contains a gonadotropin releasing hormone analogue and a dopamine receptor antagonist and stimulates ovulation. Treatment of full-grown follicles with the maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) in vitro also induced expression of EP4b mRNA. Females ovulate in vivo after injection with Ovaprim, or injection with Ovaprim and inhibitors of EP1 (ONO-8130) or EP2 (TG4-155) function; they do not ovulate when injected with Ovaprim and an EP4 inhibitor (GW237368x). These findings suggest that the EP4 receptor, in particular the EP4b isoform, is essential for ovulation.

Introduction

Ovulation is the process by which mature oocytes exit their ovarian follicles in preparation for fertilization. In fishes, the process of ovulation is under the control of the ovulatory surge of gonadotropins. Numerous studies, including hormone replacement, gene knockout, and inhibition of downstream signalling have established that ovulation is mediated by the essential actions of luteinizing hormone (LH) (Crespo et al., 2013, Ogiwara et al., 2013, Tang et al., 2016, Zhang et al., 2015). Knockout of both zebrafish gonadotropin releasing hormone isoforms does not inhibit ovulation; however knockout of nuclear progesterone receptor, a mediator of luteinizing hormone activity, leads to anovulation (Marvel et al., 2018, Tang et al., 2016). Additionally, spawning is inhibited in LH knockout zebrafish; however females with knockout of the follicle stimulating hormone (FSH) gene are still fertile and able to reproduce (Zhang et al., 2015).

LH recruits a number of downstream hormones, receptors, and enzymes that mediate the ovulatory process (Goetz and Garczynski, 1997, Patiño et al., 2003, Takahashi et al., 2013, Zhang et al., 2015). The molecular species involved in ovulation in fishes include progestins such as 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) (Knight and Van Der Kraak, 2015, Tokumoto et al., 2011) progestin receptors (Hanna and Zhu, 2011, Tang et al., 2016, Zhu et al., 2015), immune regulators (Berishvili et al., 2006, Crespo et al., 2010), matrix metalloproteases and their inhibitors (Ogiwara et al., 2005, Russell et al., 2015, Shozu et al., 2005), and prostaglandins (Kim et al., 2014, Lister and Van Der Kraak, 2008).

Prostaglandins (PGs) are a class of fatty acid derivatives whose proper function is essential for ovulation (Goetz et al., 1991). PGs are also involved in a variety of additional physiological processes in vertebrates, including inflammation, immune system regulation, and smooth muscle contraction (Simmons et al., 2004). The role of PGs, particularly PGE2, in ovulation have been well characterized (Goetz and Garczynski, 1997, Murakami and Kudo, 2004, Takahashi et al., 2013). Studies in mice have shown that knockout or inhibition of cyclooxygenase (COX) enzymes, which are essential in the production of PGs, leads to infertility in females by blocking ovulation (Lim et al., 1997). Additionally, inhibition of COX enzyme function leads to inhibition of ovulation in zebrafish, an effect which is ameliorated with subsequent addition of PGE2 (Fujimori et al., 2011, Lister and Van Der Kraak, 2008). LH signaling has also been shown to upregulate expression of ptgs2a mRNA, one of the COX isoforms responsible for PG synthesis (Tang et al., 2017). PGs have also been shown to be involved in the ovulation of fishes, as seen in studies in the yellow perch (Goetz et al., 1989), brook trout (Goetz et al., 1982), medaka (Fujimori et al., 2012), and goldfish (Stacey and Pandey, 1975). While it is well established that PGs are essential for ovulation in vertebrates, the nature of the role played by PGs in this process is less well understood, such as which of the various isoforms of PG receptors play a role in mediating ovulation. Teleost fish have several isoforms of prostaglandin receptors – EP1a, EP1b, EP1c, EP2a, EP4a. EP4b and FP all of which are present in the ovary of zebrafish (Kwok et al., 2012, Villablanca et al., 2007). In mammals, there have been several studies investigating the roles of the various PG receptors with EP1, EP2, and EP4 all being implicated as being involved in regulating the process of ovulation (Harris et al., 2011, Kim et al., 2014, Peluffo et al., 2014, Sugimoto et al., 2015); however there has been comparatively less investigation into the roles of the PG receptor isoforms in fish. In medaka, expression of Ptger4 mRNA was upregulated by recombinant luteinizing hormone via a pathway involving the nuclear progesterone receptor (Hagiwara et al., 2014, Tang et al., 2016). Additionally, impairment of the EP4b receptor by a competitive antagonist in vitro led to anovulation in medaka follicles (Fujimori et al., 2012).

This study sought to determine whether any of the EP and FP series receptors were dynamically regulated in zebrafish and changed in expression during the ovulatory period. Additionally, this study examined if any of the EP series receptors are under the hormonal control of gonadotropins. Finally, we investigated whether inhibition of any of the EP1, EP2, or EP4 series receptors would block ovulation in vivo.

Section snippets

Animals

Sexually mature zebrafish were purchased from AQUAlity Tropical Fish Wholesale (Mississauga, ON) and held in an environmentally controlled chamber with a photoperiod of 12 h light:12 h dark in the Hagen Aqualab (University of Guelph, Guelph, ON), with lights turned on at 7:00 am and turned off at 7:00 pm daily. Fish were held in aquatic habitat units (Aquatic Ecosystems, Apopka, FL) with recirculating Guelph well water held at 28 °C. Fish were fed either brine shrimp (AQUAlity) or salmon fry

Temporal

Initial studies examined if prostaglandin receptors were dynamically expressed across the ovulatory period. Whole ovaries of females were isolated at 10:00 pm, 1:00 am, 4:00 am, 7:00 am, and 10:00 am. The gonadosomatic index of the females used in this experiment was 7.28 ± 0.29 (N = 55). No females had ovulated at 1:00 am, 4:00 am, or 10:00 am. All females (N = 11) had ovulated at 7:00 am, while 1 out of 11 females had ovulated at 10:00 pm. Using qPCR, relative expression levels of ptger1a,

Discussion

The purpose of this study was to investigate the role of prostaglandin receptors in the process of ovulation in zebrafish using both in vivo and in vitro methods. Prostaglandins and their receptors have been demonstrated to be essential to properly functioning ovulation in teleosts (Goetz et al., 1989, Goetz and Garczynski, 1997, Lister and Van Der Kraak, 2008, Patiño et al., 2003). In particular, prostaglandin E2 and its EP4b receptor have been highly implicated as playing a crucial role in

Conclusions

In the present study, we have showed that levels of the mRNA transcript for the prostaglandin receptor EP4b increase in the time period leading up to ovulation and spawning in vivo. This increase in ptger4b expression is also seen when female fish are injected with Ovaprim, a potent stimulant of ovulation. Consistent with this, we saw high rates of ovulation in fish injected with Ovaprim. The rate of ovulation was drastically reduced when fish were injected with both Ovaprim and GW627368x, a

Acknowledgements

This study was supported by a Grant to G.V.D.K. from NSERC. We thank Jacquie Matsumoto for contributing her insights and expertise to this project and Irena Papst for assistance with R statistical software.

The authors have no competing interests to declare.

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