Anti-Müllerian hormone (Amh/amh) plays dual roles in maintaining gonadal homeostasis and gametogenesis in zebrafish

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Abstract

Anti-Müllerian hormone (AMH/Amh) plays a role in gonadal differentiation and function across vertebrates. In zebrafish we demonstrated that Amh deficiency caused severe gonadal dysgenesis and dysfunction. The mutant gonads showed extreme hypertrophy with accumulation of early germ cells in both sexes, namely spermatogonia in the testis and primary growth oocytes in the ovary. In amh mutant females, the folliculogenesis was normal in young fish but receded progressively in adults, which was accompanied by progressive decrease in follicle-stimulating hormone (fshb) expression. Interestingly the expression of fshb increased in the pituitary of juvenile amh mutant males but decreased in adults. The upregulation of fshb in mutant male juveniles was likely one of the mechanisms for triggering gonadal hypergrowth, whereas the downregulation of fshb in adults might involve a negative feedback by gonadal inhibin. Further analysis using mutants of fshb and growth differentiation factor 9 (gdf9) provided evidence for a role of FSH in triggering ovarian hypertrophy in young female amh mutant as well. In summary, the present study provided comprehensive genetic evidence for dual roles of Amh in controlling zebrafish gonadal homeostasis and gametogenesis in both sexes. Amh suppresses proliferation or accumulation of early germ cells (spermatogonia in testis and primary growth oocytes in ovary) while promoting their exit to advanced stages, and its action may involve both endocrine and paracrine pathways.

Introduction

Anti-Müllerian hormone (AMH/Amh) belongs to the transforming growth factor-β (TGF-β) superfamily, and it was initially known for its function of inducing Müllerian duct regression in mammals (Behringer et al., 1994; Vigier et al., 1984). Like other TGF-β family members that signal through a receptor complex containing a type I and a type II receptors, AMH signals through a specific type II receptor, AMHRII (Mishina et al., 1996). In addition to inducing Müllerian duct regression during sexual differentiation, AMH is also expressed in adult gonads, specifically the granulosa cells in the ovary and Sertoli cells in the testis (Hirobe et al., 1992; Ueno et al., 1989b). In the ovary, AMH is mainly expressed in preantral and early antral follicles (Gruijters et al., 2003; Ueno et al., 1989a; Weenen et al., 2004), and it plays an important role in primordial follicle recruitment and dominant follicle selection as evidenced in both mice (Durlinger et al., 1999) and humans (Weenen et al., 2004). The recruited follicles continue to express AMH until selection by pituitary follicle-stimulating hormone (FSH) for dominance (Durlinger et al., 2002b; Visser and Themmen, 2005). Studies have shown that AMH exerts multiple effects in gonads, including inhibition of follicle assembly (Nilsson et al., 2011) and initiation of primordial follicle growth (Durlinger et al., 2002a) in females, and promotion of steroidogenesis in the Leydig cells in males (Wu et al., 2005).

Interestingly, teleost fish also have AMH orthologs (Amh/amh) though they have no Müllerian ducts (Pfennig et al., 2015), and its presence has been demonstrated in a variety of fish species including Japanese eel (Miura et al., 2002), medaka (Kluver et al., 2007), Nile tilapia (Poonlaphdecha et al., 2011), European sea bass (Halm et al., 2007), Japanese flounder (Yoshinaga et al., 2004) and zebrafish (Rodriguez-Mari et al., 2005). Amh in fish often shows sexually dimorphic expression patterns with higher expression in testis, suggesting a potential role for Amh in spermatogenesis. This is further supported by the elevated amh expression in testis during sex differentiation in zebrafish (Chen et al., 2017; Wang and Orban, 2007), Japanese flounder (Yamaguchi et al., 2010) and Nile tilapia (Poonlaphdecha et al., 2011). Genetic evidence also supports a role for Amh in fish sex differentiation. An Amh type II receptor (amhr2) mutation in Japanese medaka (hotei) causes a male-to-female sex reversal in XY male individuals (Morinaga et al., 2007). Recently, an extra Y-linked amh gene (amhy) was reported in Patagonian pejerrey (Hattori et al., 2012) and Nile tilapia (Li et al., 2015) as a male-determining gene. Knockout of the amhy in tilapia resulted in male-to-female sex reversal in XY males (Li et al., 2015).

Functional studies in fish have provided substantial evidence for Amh activities in the testis. Amh inhibits spermatogonial proliferation and differentiation in zebrafish testis (Skaar et al., 2011). In agreement with this, excessive proliferation of spermatogonia in the testis and germ cells in the ovary have been observed in both Amh null zebrafish (Lin et al., 2017; Yan et al., 2019) and tilapia (Liu et al., 2019), as well as Amh type II receptor (amhr2)-deficient medaka (Morinaga et al., 2007) and putative type I receptor (bmpr1bb/alk6b)-deficient zebrafish (Neumann et al., 2011). Despite these studies, the mechanisms by which Amh works remain largely unknown in fish species.

Although amh shows lower expression level in the ovary of some fish species, its potential role in ovarian development has been reported. The expression of amh is detectable in primary growth (PG, stage Ib) follicles and increases significantly in pre-vitellogenic (PV, stage II) follicles in the zebrafish (Rodriguez-Mari et al., 2005). These data strongly implicate Amh in regulating early follicle development, especially follicle activation or PG-PV transition and subsequent vitellogenic growth; however, the exact roles of Amh and its action mechanisms in this regard are unclear.

Using CRISPR/Cas9 approach, we undertook this study to disrupt the gene of amh from the zebrafish genome followed by phenotype analysis in both sexes. In addition to describing gonadal phenotypes of amh mutant as reported by others, we focused our attention on how Amh loss disrupted the function of the pituitary-gonad axis and how the gonadal inhibin-mediated negative feedback was involved. Our data strongly suggest dual roles for Amh in regulating gonadal homeostasis and gametogenesis in zebrafish through both endocrine and paracrine pathways.

Section snippets

Fish and maintenance

The AB strain zebrafish was used in the present study to generate amh mutant (+66, −4) (amhumo17). In addition, several other zebrafish mutants were also used in the study, including cyp19a1aumo5 (Lau et al., 2016), fshbumo1 (Zhang et al., 2015b), fshrumo3 (Zhang et al., 2015a), gdf9umo18 and inhaumo19. The mutant lines gdf9umo18 (−5-bp deletion in exon 1) and inhaumo19 (+1-bp insertion and −5-bp deletion in exon 1) were also generated by CRISPR/Cas9 method (details to be published separately).

Roles of Amh in sex determination and differentiation

To disrupt amh gene in the zebrafish, we used CRISPR/Cas9 method to target exons I of the gene. One homozygous mutant line (+66, −4) (amhumo17) was established for functional study (Fig. 1A). Considering potential roles of Amh in gonadal differentiation, we first assessed the impact of Amh deficiency on sex determination and differentiation.

As a male-enriched gene, Amh has been implicated in promoting testis development in fish (Chen et al., 2017; Pfennig et al., 2015; Rodriguez-Mari et al.,

Discussion

In mammals, AMH has diverse functions in reproduction. In addition to its primary role of inducing Müllerian duct regression (Behringer et al., 1994), AMH is also involved in controlling gonadal development and function in both sexes (Durlinger et al., 2002b; Rehman et al., 2017; Visser and Themmen, 2005). Interestingly, Amh/amh also exists in teleosts despite the lack of Müllerian ducts (Pfennig et al., 2015). In this study, we provided genetic evidence for roles of Amh in controlling gonadal

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgment

This study was supported by grants from the University of Macau (MYRG2015-00227-FHS, MYRG2016-00072-FHS, MYRG2017-00157-FHS and CPG2014-00014-FHS) and The Macau Fund for Development of Science and Technology (FDCT/089/2014/A2 and FDCT173/2017/A3) to W.G.

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