Multifactorial control of reproductive and growth axis in male goldfish: Influences of GnRH, GnIH and thyroid hormone

Highlights

• GnRH elevates both LH and GH serum levels in a seasonally dependent manner.

• GnIH both stimulates and inhibits GnRH-induced response depending on the season.

• T3 exerts seasonal effects on basal, and LH and GH responses to GnRH and GnIH.

• Liver and testis transcripts vary in a seasonal-, sex- and tissue-specific manner.

• GnRH and GnIH actions on testis and liver may be direct and/or indirect.

Abstract

Reproduction and growth are under multifactorial control of neurohormones and peripheral hormones. This study investigated seasonally related effects of GnIH, GnRH, and T3 on the reproductive and growth axis in male goldfish at three stages of gonadal recrudescence. The effects of injection treatments with GnRH, GnIH and/or T3 were examined by measuring serum LH and GH levels, as well as peripheral transcript levels, using a factorial design. As expected, GnRH elevated serum LH and GH levels in a seasonally dependant manner, with maximal elevations of LH in late stages of gonadal recrudescence (Spring) and maximal increases in GH in the regressed gonadal stage (Summer). GnIH injection increased serum LH and GH levels only in fish at the regressed stage but exerted both stimulatory and inhibitory effects on GnRH-induced LH responses depending on season. T3 treatment mainly had stimulatory effects on circulating LH levels and inhibitory effects on serum GH concentrations. In the liver and testes, we observed seasonal differences in thyroid receptors, estrogen receptors, vitellogenin, follicle-stimulating hormone receptor, aromatase and IGF-I transcript levels that were tissue- and sex-specific. Generally, there were no clear correlation between circulating LH and GH levels and peripheral transcript levels, presumably due to time-related response and possible direct interaction of GnRH and GnIH at the level of liver and testis. The results support the hypothesis that GnRH and GnIH are important components of multifactorial mechanisms that work in concert with T3 to regulate reciprocal control of reproduction and growth in goldfish.

Introduction

Control of reproduction and growth are multifactorial and involve hormones of the brain-pituitary-peripheral target axis (Blazquez et al., 1998; Klausen et al., 2003; Chang et al., 2000; Trudeau, 1997). In fish and in a number of other vertebrates, reproduction and growth follows a clear seasonal cycle involving changes in gonadal development, circulating hormones and metabolism. A key regulator of reproduction is gonadotropin-releasing hormone (GnRH), which stimulates production and secretion of the gonadotropins (follicle-stimulating hormone, FSH and luteinizing hormone, LH). LH and FSH, in turn, stimulate gametogenesis and hormone production in the ovary and testis. These components form the hypothalamo-pituitary-gonad (HPG) axis important for the control of reproduction (for review see: Zohar et al., 2010; Trudeau, 1997). Growth is regulated by growth hormone (GH); its synthesis and secretion are also controlled by various neurohomones (for review see: Klausen et al., 2003; Chang et al., 2012; Canosa et al., 2007). During the reproductive season in oviparous species, significant growth of gonads occurs which requires investment of metabolic energy to sustain development of eggs and sperm. Therefore, maximal growth and reproduction do not occur simultaneously in seasonally breeding animals and co-ordinated endocrine changes would be needed to achieve these seasonal reproductive changes (Sohn et al., 1999; Pasmanik and Callard, 1988).

GnRH homologs in vertebrate species are categorized into three main types, GnRH1, GnRH2, and GnRH3, where one species can express multiple forms (for review see: Klausen et al., 2003; Chang and Pemberton, 2018; Zohar et al., 2010; Okubo and Nagahama, 2008). GnRH1 is the main pituitary regulator for mammals and includes the isoform found in humans (Okubo and Nagahama, 2008). GnRH2 is found in all vertebrate classes and is thought to be involved in changing behaviour, feeding activities, and energy balance due to expression found in a variety of regions in the mid brain (Yu et al., 1987; Schneider and Rissman, 2008; Xia et al., 2014). GnRH3 is found in teleost species and in the absence of GnRH1 expression, GnRH3 compensates and regulates pituitary action (Okubo and Nagahama, 2008). In goldfish specifically, chicken GnRHII (cGnRHII, GnRH2) and salmon GnRH (sGnRH, GnRH3) are the two native isoforms expressed in the brain and both GnRH forms reach the pituitary (Peter et al., 1985; Kim et al., 1995).

The multifactorial control of reproduction also involves other neurohormones such as the RF-amide gonadotropin-inhibitory hormone (GnIH), which was first discovered in Japanese quail and was reported to inhibit gonadotropin synthesis and release from the pituitary (Tsutsui et al., 2000). Many species have genes for multiple forms of GnIH (for review see: Ullah et al., 2016). Specifically in goldfish, there are three GnIH genes but only GnIH-3 (GnIH: SGTGLSATLPQRF-NH2) expression has been detected in the hypothalamus (Sawada et al., 2002). Although most mammalian and bird studies of GnIH have yielded inhibitory effects on gonadotrope function, studies in other vertebrates like fish have found both stimulatory and inhibitory effects of GnIH (for review see: Muñoz-Cueto et al., 2017; Tsutsui and Ubuka, 2018; Ubuka and Parhar, 2018). In goldfish, GnIH was shown to inhibit both synthesis and release of gonadotropins in early stages of gonadal recrudescence, but not in pre-spawning fish (Moussavi et al., 2012). In the cinnamon clownfish (Amphiprion melanopus) intraperitoneal (ip) injection of GnIH was shown to inhibit expression of gonadotropin α and β subunits (Choi et al., 2016). In cichlid fish, cichlid GnIH1 (cdGnIH1) inhibited the expression of both gonadotropins, but cdGnIH2 was stimulatory for FSHβ subunit expression (Di Yorio et al., 2016). In grass puffer (Takifugu niphobles), increased FSHβ and LHβ mRNA expression during spawning season coincide with increased GnIH and GnIH receptor (GnIHR) mRNA expression (Ando et al., 2018). In male A. altiparanae, zebrafish (z)GnIH-3 had no effects on basal gonadotropin expression but inhibited cGnRHII-induced gonadotropin subunits transcript expression, and increased cGnRHII transcript expression (Branco et al., 2019). Despite the fact that a number of investigators have demonstrated that GnIH exerts both stimulatory and inhibitory actions, depending season and species, both GnRH and GnIH are accepted as important components of multifactorial control of reproduction.

There is also evidence that GnRH and GnIH may participate in the multifactorial control of somatotrope functions in some fish species. Studies in goldfish have shown that GnRH exerts stimulatory actions on GH synthesis and release (Marchant et al., 1989; Moussavi et al., 2014; Klausen et al., 2003). Binding sites for GnRH isoforms have been identified in somatotropes in goldfish (Cook et al., 1991), cichlid fish (Parhar et al., 2002), and pejerrey (Stefano et al., 1999). Direct actions of GnRH isoforms on GH release and synthesis have been demonstrated and particularly well characterized in goldfish (Habibi et al., 1992; Marchant et al., 1989; Klausen et al., 2001, 2005; Chang et al., 1996; Chang and Pemberton, 2018). GnRH has also been shown to stimulate GH production in other fish species, including tilapia (Melamed et al., 1995), common carp (Li et al., 2002), pejerrey (Montaner et al., 2001), and masu salmon (Bhandari et al., 2003). In contrast, other studies observed no increase in serum GH levels after GnRH treatment in the turbot (Rousseau et al., 2001), eel (Rousseau et al., 1999), or catfish (Lescroart et al., 1996); these results may indicate the presence of species and/or experimental condition differences.

Likewise, although GnIH has been shown to be stimulatory to GH release in mammals (Johnson et al., 2007; Johnson and Fraley, 2008), the situation in fish is not as straight forward. Briefly, although GnIH receptors were reported to be absent in tilapia somatotropes (Biran et al., 2014), GnIH neurons have been shown to project to GH cells in the European sea bass (Paullada-Salmerón et al., 2016a) while brain injections of GnIH stimulated pituitary GH mRNA expression (Paullada-Salmerón et al., 2016b). GnIH also elevated GH mRNA expression in primary pituitary cell cultures prepared from grass puffer (Shahjahan et al., 2016), as well as GH release from pituitary cultures of cichlid C. dimerus (Di Yorio et al., 2016). In vitro GnIH applications generally stimulated GH accumulation in media in 24-hr static incubation experiments with primary cultures of goldfish pituitary cells; however, these responses were accompanied by elevations in GH mRNA expression in cells prepared from goldfish at late recrudescence but reduced GH mRNA expression in cells prepared from fish at mid recrudescence and regressed states (Moussavi et al., 2014). In contrast, GnIH reduced basal GH release from goldfish pituitary cells prepared from fish at mid recrudescence when applied in rapid cell column perfusion studies (Moussavi et al., 2014). Thus, GnIH influences on fish GH release and synthesis are species-specific, seasonally dependent, may consist of a combination of direct pituitary and non-pituitary effects, as well as exhibit treatment protocol and time-course differences. Adding to the possible complexity of the results on GnRH and GnIH effects, many of these studies have used mixed sex groups. The full picture of somatotrope regulation (and to certain extends, that of gonadotropes) by these factors within males and seasonally reproducing animals remains unclear.

Thyroid hormones (T3 and T4) are also known to affect reproduction and growth in different species, including fish (for review Cyr and Eales, 1996; Cyr and Eales, 1988; Nelson et al., 2010; Habibi et al., 2012; Tovo-Neto et al., 2018). In goldfish, blood thyroid hormone levels are highest during regressed gonadal stage and decrease to minimum levels during spawning (Sohn et al., 1999). Treatment with T3 was shown to decrease gonadal expression of estrogen receptors and aromatase in male goldfish (Nelson et al., 2010). Furthermore, T3 injection was shown to significantly reduce sex hormone production in male goldfish at early and mid-stages of recrudescence (Allan and Habibi, 2012). In zebrafish, T3 was shown to stimulate the proliferation of Sertoli cells and spermatogonia type-A in testis (Morais et al., 2013). While T3 reduced pituitary LH and gonadal estrogen receptor expression, it enhanced vitellogenesis by increasing liver estrogen receptors, which primes the liver for reproduction at early stages of gonadal recrudescence (Nelson and Habibi, 2016). Thyroid hormones are also known to work synergistically with GH to increase overall growth (Louis et al., 2010; Lostroh and Li, 1958; for review see: Gouveia et al., 2018; Cabello and Wrutniak, 1989), and are crucial for metamorphosis of in amphibians and certain fish species (Power et al., 2001; Einarsdóttir et al., 2006; Manzon and Manzon, 2017). In addition, treatment with T3 increased GH mRNA expression in the rainbow trout (Moav and McKeown, 1992), although no such effects were seen in goldfish (Allan and Habibi, 2012). In contrast, thyroid hormones inhibited GH release and synthesis by direct actions at the pituitary level in eel (Rousseau et al., 2002). Thus, despite possible species differences, thyroid hormones are also factors in the control of reproduction and growth.

In the present study, we have used male goldfish as a model organism that undergoes seasonal reproductive cycling to test the hypothesis that GnRH, GnIH and thyroid hormones are important components of the multifactorial mechanisms underlying reciprocal control of reproduction and growth. The effects of GnRH and GnIH, applied alone or in combination in vivo, on several indices including, serum LH and GH levels; mRNA expression of FSH receptors (FSHR), estrogen receptors (ERα, ERβ1), and aromatase (Cyp19a) in testes; and mRNA expression of thyroid hormone receptors (TRα1, TRβ), vitellogenin (Vtg), insulin-like growth factor-I (IGF-I), ERα, and ERβ1 in liver were monitored at three distinct stages of gonadal recrudescence. In addition to examining the changes in liver thyroid receptors, the effects of T3 injection on GnRH and/or GnIH treatment-induced serum LH and GH responses were also investigated as an attempt to more directly examine thyroid hormone influences.