Characterization of gonadal glucocorticoid-induced leucine zipper (GILZ) protein expression during sex change in the protogynous orange-spotted grouper, Epinephelus coioides

Highlights

• Steroid hormones have multiple mechanisms that influence grouper gonadal sex change.

• Glucocorticoid-induced leucine zipper (GILZ) is expressed during sex change.

• GILZ protein contributes to spermatogenesis.

• GILZ expression involvement in steroidogenesis during sex change.

Abstract

Steroid hormones have been proven as a key drive of sex change in sequentially hermaphroditic organisms. However, the upstream mechanism of sex steroid hormones regulation that affect sex change remain unknown. The main glucocorticoid in teleost fish is cortisol, which both regulates steroidogenesis and has antistress action. Thus, cortisol might be one of the prime factors in sex change. In this study, the glucocorticoid-induced leucine zipper (GILZ) gene, was proven to have a dramatic effect in orange-spotted groupers (Epinephelus coioides) during sex change at the early stage of gonadal transition. The specific action of the GILZ protein is at the pouch-shaped proliferative spermatogonia instead of the degenerative oocyte at the onset of sex change. Immunohistochemical (IHC) evidence revealed that GILZ performs intensively at undifferentiated spermatogonia in the early testis stage. These results imply that cortisol provokes a rise of GILZ through regulation caused by steroid hormones leading to sex change.

Introduction

The sexuality of teleosts appears diversified. Although the majority of vertebrates are gonochoristic, some teleosts display hermaphroditism at sex change. The direction of sex change for these organisms depends on their life expectancy, and such change can be classified into three types: which protogyny; (female to male), protandry (male to female), and serial sex change, (repeated change between male and female) (Devlin and Nagahama, 2002; Kobayashi et al., 2013). No matter the access path, sex steroid hormones play a key role in all possible sex change paths (Frisch, 2004). For example, for protogynous groupers, endogenous estrogens declined during sex change (Bhandari et al., 2003). In contrast to protogynous fish, estrogen increase and androgen decrease, are the causes of protandrous fish sex change (Chang and Lin, 1998). Moreover, the secretion of sex steroid hormones causes the density of follicle-stimulating hormones to increase under the control of gonadotropins, which have been reported to be a trigger for sex change among protogynous grouper fish (Kobayashi et al., 2010). Therefore, steroidogenesis may play a vital role in the onset of sex change during physiological changes in fish (Avise and Mank, 2009). However, little is known about the endocrinological mechanism that controls the steroidogenesis shift at the onset of sex change.

Cortisol is one of the major glucocorticoids, which are extensively involved in the physical regulation, such as osmotic regulation, reproduction, and the immune system. The cortisol hormones of vertebrates are produced by the adrenal cortex. An increase of cortisol is associated with the anti-stress response (Mommsen et al., 1999). Regarding regulation by steroid hormones such as cortisol, long-term exposure to glucocorticoids results in lower amount of gonadotropin-releasing hormone (GnRH) being released in the brain, which reduces overall levels of of gonadotropin and affects the pituitary reaction (Rivier and Rivest, 1991). During the activation of gonadal steroid hormones, cortisol directly affects the circulating levels of luteinizing hormones in the blood (Monder et al., 1994). In cells developed within the pituitary glands of grouper, glucocorticoids have been proven to increase the production of follicle-stimulating hormones. Therefore, the participation of glucocorticoids and GnRH or gonadotropin production controls the activation of steroid hormones.

Studies have indicated that for some teleosts, cortisol is involved in masculinization at sex change, which decreases aromatase activity (Hayashi et al., 2010; Yamaguchi et al., 2010). At the first stage, high-temperature stress increases masculinization of fish. Heat stress increases cortisol levels and results in masculinization due to the direct inhibition of aromatase activity and reduction of estrogen density (Hattori et al., 2007; Kitano et al., 2012). In support of these findings, other researchers have found that the aromatase promoter includes the presence of novel glucocorticoid response elements (GRE) in gobies and pejerreyes and that, cortisol promotes androgen production during the process of masculinization following the induction of thermal stress (Gardner et al., 2005; Hattori et al., 2009). These results indicate that cortisol plays a key role in controlling the production of steroids and that social environmental stimulus affects sex change.

In mammals, the origin of embryo of adrenal cortex is the same as the gonad (Hatano et al., 1996). Meanwhile, adrenal cortex as well as gonad secrets several kinds of steroid hormones, among them, it is well-known that glucocorticoid possesses the effects of anti-inflammation. The performance of glucocorticoid-induced leucine zipper (GILZ) is to imitate Glucocorticoid, which can restrain the activation of inflammation (Ayroldi and Riccardi, 2009). Although most studies related to GILZ focus on anti-inflammatory effects, the function of GILZ at cell regulation stage has been reported about cell differentiation and cell death (Riccardi et al., 2001). Furthermore, the performance of GILZ in mice influences the functions of self-renewal and differentiation of spermatogonial stem cells (SSC) (Fallahi et al., 2010), and it supports germ cell survival (Ayroldi et al., 2007). In the present study, we found that GILZ is expressed in spermatogonia and early meiotic spermatocytes in the grouper testis. To better clarify the role of GILZ involvement in sex change in the protogynous orange-spotted grouper, Epinephelus coioides, we cloned the GILZ gene and examined its expression patterns in the gonad. The majority of groupers are classified as monandric protogynous type, in which adult male fish are converted from mature female fish. By contrast, some groupers are diandric protogynous, where adult male fish can come from juvenile males or develop from female fish. Primary male fish directly become adult male fish after maturation; secondary male fish are converted from female fish and turn into adult male fish after maturation. However, the two types of males cannot be distinguished by appearance or behavior, and thus, they must be identified by observing the microstructural testis. Groupers are an optimal candidate for research into the physiological mechanism relevant to sexual conversion, because they can be examined to reveal the interactions between sex steroid hormones and sex change. Furthermore, the gonadal sex change process is clear in this species. Many studies have shown that both reduction of estrogen and administration of androgen induce sex change from female to male in bony fish. Moreover, Nozu and Nakamura (2015) demonstrated that cortisol administration induced ovary-to-testis sex change in the three-spot wrasse, Halichoeres trimaculatus. Therefore, the present study investigated the expression of a cortisol-induced protein during gonadal sex change in the orange-spotted grouper.

The purpose of this study was to (i) clone grouper GILZ and determine its genomic structure, (ii) use phylogenetic analysis to characterize the divergence of grouper GILZ from ancestral GILZ, (iii) determine the localization of GILZ in different grouper tissues, (iv) clarify the expression of GILZ in the early periods of spermatogenesis, (v) evaluate whether the grouper GILZ is specifically expressed during sex change, and (vi) clarify GILZ expression involvement in steroidogenesis in the ripening female ovary.