Expression Analysis of Growth Differentiation Factor 9 (Gdf9/Gdf9)

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Biology of Reproduction, 2017, 96(2), 401–413 doi:10.1095/biolreprod.116.144964 Research Article Advance Access Publication Date: 3 February 2017

Research Article Expression analysis of growth differentiation factor 9 (Gdf9/gdf9), anti-mullerian¨ hormone (Amh/amh) and aromatase Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021 (Cyp19a1a/cyp19a1a) during gonadal differentiation of the zebraﬁsh, Danio rerio† Weiting Chen1,2,‡,LinLiu2,3,‡ and Wei Ge1,2,∗

1Centre of Reproduction, Development and Aging, Faculty of Health Sciences, University of Macau, Taipa, Macau, China; 2School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China and 3School of Life Science, South China Normal University, Guangzhou, China

∗Correspondence: Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, China. Tel: +853-8822-4996; Fax: +853-8822-2314; Email: [email protected]

†The work was substantially supported by grants from the University of Macau (MYRG2014-00062-FHS, MYRG2015-00227-FHS, and CPG2014-00014-FHS) and The Macau Fund for Development of Science and Technology (FDCT114/2013/A3 and FDCT/089/2014/A2) to W. Ge. The early part of the study was also partially supported by a Focused Investment Scheme C (FIS-C-1903023) grant from The Chinese University of Hong Kong to W. Ge, and Research Grants Council of Hong Kong Areas of Excellence (AoE) Schemes on Marine Environmental Research and Innovative Technology (AoE/P-04/04) and Organelle Biogenesis and Function (AoE/M-05/12). ‡These authors contributed equally to this work.

Received 26 September 2016; Revised 2 December 2016; Accepted 19 December 2016

Abstract In the zebrafish, no sex-determining gene has been identified, while some sex-related genes, such as cyp19a1a and amh, show sexually dimorphic expression. Interestingly, most of these genes are expressed in the somatic cells. With increasing evidence suggesting roles of germ cells in gonadal differentiation, there is an increasing interest in the factors released by the germ cells for the bidirectional communication between the two compartments. We have reported that Gdf9/gdf9 is an oocyte-specific factor in the zebrafish, similar to that of mammals. Whether and how Gdf9 is involved in gonadal differentiation is unknown. In this study, we compared the expression levels of gdf9, cyp19a1a,andamh among several other sex-related genes in the gonads before, during, and after sex differentiation. The expression of gdf9 started in the gonads before sex differentiation, and its level surged in the differentiated ovary. Its expression pattern was similar to that of cyp19a1a, but reciprocal to amh expression. Using recombinant zebrafish Gdf9 (rzfGdf9), we further showed that Gdf9 significantly suppressed the expression of amh while increased that of activin beta subunits (inhbaa and inhbb) in vitro. Although gdf9 and cyp19a1a showed co-expression during gonadal differentiation, we only observed a slight but not significant response of cyp19a1a to rzfGdf9. Knocking down the expression of gdf9 and cyp19a1a with vivo-morpholinos caused a male-skewed sex ratio. Our data suggested that Gdf9 is likely involved in promoting oocyte/ovary

C The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. 401 For permissions, please [email protected] 402 W. Chen et al., 2017, Vol. 96, No. 2 differentiation in the zebraﬁsh and it may act by suppressing amh expression, at least partly, in the somatic cells.

Summary Sentence Oocytes may inﬂuence gonadal differentiation by releasing growth differentiation factor 9.

Key words: Gdf9, Amh, aromatase, Cyp19a1a, gonadal differentiation, zebraﬁsh.

Introduction and ﬁsh [35,36], amhy in Patagonian pejerrey [3], and dmy in medaka [2]. Sex determination and differentiation are long-lasting questions in Although the important roles played by somatic cells in gonadal developmental and reproductive biology. So far, only a few sex-

differentiation are well accepted, lines of evidence from recent studies Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021 determining genes have been identified in vertebrates, e.g., SRY suggest that the germ cells also play an important role in determin- in mammals [1], DMY/dmrt1bY in Japanese medaka [2], amhy in ing gonadal differentiation. In mammals, germ line is required for Patagonian pejerrey [3] and tilapia [4], DMRT1 in chicken [5], and the initiation of follicle formation in females [37]. Without meiotic DMW in the African clawed frog [6]. In most vertebrate species, the germ cells, the ovarian pregranulosa cells transform into Sertoli-like mechanism of sex determination remains largely unknown, espe- testis cells [38]. Similarly, the germ cells have also been shown to be cially in teleost fish [7]. In contrast to sex determination, the genetic essential for female sex differentiation in the medaka [39,40], loach and physiological mechanisms that govern sex differentiation are [41], and zebrafish [42,43]. In the zebrafish, specific ablation of pri- better documented in fish [8]. mordial germ cells in the embryo by toxin or knocking down of germ In teleost fish, gonadal sex differentiation is highly plastic and cell-specific gene dnd by morpholino led to the formation of sterile can be influenced by both genetic and environmental factors such males [42,43]. Interestingly, low numbers of germ cells also led to as pH and temperature [9,10]. In the zebrafish, no sex chromo- male development in the zebrafish [44,45]. Recently, foxl3,agerm somes or sex-linked genes have been found [11,12], despite being cell-specific gene, was shown to promote germ cell differentiation to a popular vertebrate model for developmental biology and genetic oocytes by suppressing spermatogenesis in the medaka [46]. Further study. The zebrafish is considered a juvenile hermaphrodite; its go- evidence in the zebrafish shows that the germ cells are not only crit- nads first develop as ovary-like organs with oocyte-like germ cells ical for gonadal differentiation during the primary sex-determining (juvenile ovary) before further differentiation into true ovary and period, but also important in maintaining female status in adult stage testis [13–16]. During testis differentiation, the oocyte-like germ [47]. These results provide strong evidence that germ cells are crucial cells regress through apoptosis [17], which is accompanied by the for ovarian development and maintenance of gene expression in the appearance of spermatogonia and proliferation of the stromal so- ovarian somatic cells in fish including the zebrafish. However, the matic cells that gradually occupy the gonad (transforming gonad), mechanisms by which the germ cells influence the process remain leading to final differentiation into the testis [13]. Although gonadal entirely unknown. differentiation has been well documented in the zebrafish at morpho- Growth differentiation factor 9 (GDF9), a member of transform- logical and histological levels [13,14,17], the molecular mechanism ing growth factor beta (TGFβ) superfamily, is an oocyte-specific underlying the process remains largely unknown. growth factor in different vertebrates including mammals, chicken, It is widely accepted that P450 aromatase (cyp19a1), a key and zebrafish [48–50], and it is obligatory for early folliculogene- steroidogenic enzyme that converts androgen into estrogen, is ac- sis in mice [51]. Both the granulosa and theca cells are the targets tively involved in ovarian differentiation in fish [18]. There are of GDF9 [49], which regulates a wide range of activities including two different cyp19a1 loci in the zebrafish: cyp19ala is mainly ex- granulosa cell proliferation [52], secretion of steroid hormones [53], pressed in the gonad, while cyp19a1b mainly in the brain [19,20]. and biosynthesis of peptide growth factors [54]. The expression of Cyp19a1a showed an increased expression during ovarian differen- GDF9 reduced rapidly after fertilization in mice and was no longer tiation in many fish species such as the Japanese medaka [21], rain- detectable by day 1.5 postcoital (pc). The expression showed up bow trout [22], Nile tilapia [23,24], European seabass, and southern again in 4-day neonatal ovaries [55]. In sheep, GDF9 mRNA was flounder [25]. Similarly, cyp19a1a also increased expression during detectable in the germ cells before the formation of follicles [56]and ovarian differentiation in the zebrafish [26], but sharply decreased in the oocytes of newly formed primordial follicles around 78–85 when undergoing transformation into testis [15]. The importance days after birth in possums [57]. In the rainbow trout, gdf9 began to of aromatase in gonadal or sex differentiation is also supported express in early gametogenesis period (days 60 to 110 after complete by the evidence that treatment with aromatase inhibitor (AI) dur- yolk consumption) in the ovary [58]. These lines of evidence suggest ing the sex differentiation period led to the formation of testis in that GDF9 is expressed in early developing ovary in different species, the rainbow trout [27], Japanese flounder [28], Nile tilapia [27], but the exact time of its appearance varies depending on species, and Chinook salmon [29], common carp [30], and zebrafish [31,32]. its role in gonadal differentiation is largely unknown. Recently, it was also reported that AI treatment could induce To provide clues to its potential role in gonadal differentiation, sex reversal from female to male in adult zebrafish [33]. There- we undertook this study to analyze qualitatively and quantitatively fore, aromatase is being considered a key factor for ovarian for- the spatiotemporal expression profiles of gdf9 during gonadal differ- mation, development, and maintenance in fish. As aromatase is entiation in the zebrafish. In addition, we also analyzed the expres- expressed in the somatic follicle cells, its importance in sex dif- sion of a variety of genes that are potentially involved in gonadal ferentiation suggests a role for the somatic cells in determining or differentiation for comparison, especially cyp19a1a and amh, which influencing gonadal differentiation. This concept is also supported have been reported to be associated with female and male develop- by other genes such as Sry in mammals [34], amh in mammals ment, respectively [3,18,33]. To understand how Gdf9 acts during GDF9 in zebrafish gonadal differentiation, 2017, Vol. 96, No. 2 403 gonadal differentiation, we also generated a stable cell line that ex- fish, they were sampled from 27 to 40 dpf when gonads were dif- presses recombinant zebrafish Gdf9 (rzfGdf9). With recombinant ferentiating. The sampling times were determined according to our Gdf9, we demonstrated its signaling activity in cultured follicle cells previous work on the timeline of zebrafish gonadal development and its effects on the expression of cyp19a1a and amh. In addition, [59]. After quick cooling on ice to sacrifice the fish, each was halved we also examined the regulation of the activin–inhibin–follistatin by cross-sectioning around the communicating duct between the two family by Gdf9. air bladders (Figure 1). The posterior half of the body was kept in RNAlater at 4◦C overnight and transferred to –20◦C afterward. The anterior part was fixed in Bouin solution for histological examina- Material and methods tion to identify the stage of gonadal development. The staging of gonadal development was based on the report of Maack and Seg- Animals ner [13]. The presence of perinucleolar oocytes (PO) was used as The wild-type zebrafish (Danio rerio) were kept in flow-through the marker for the ovary, while the appearance of spermatogonia aquaria at 28±1◦C on 14L:10D lighting cycle. An albino strain, or spermatocytes marked the differentiation of testis. The gonads Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021 which has transparent body wall at juvenile stage, was maintained with both degenerating oocytes and developing spermatocytes were in the laboratory and used for microinjection of vivo-morpholinos considered transforming gonad or early testis. (VMO). The fish were fed twice a day with commercial fish food with supplement of live brine shrimp twice a week. All experiments were endorsed by the Research Ethics Committee of University of RNA isolation, reverse transcription and polymerase Macau and the Animal Experimentation Ethics Committee of The chain reaction Chinese University of Hong Kong. Total RNA was isolated from the gonads of larval or juvenile fish with Tri-Reagent (Molecular Research Center, Cincinnati, OH) according to the protocol of the manufacturer and our previous reports Culture of fish fry and sampling of gonads from larvae [50,60]. The RNA extracted from the gonads of each fish was reverse and juveniles transcribed into cDNA at 42◦C for 1 h in a total volume of 10 μl Mature females and males were kept separately for 4–5 days and reaction solution that contains total RNA, iScript reaction mix with placed together 1 day before spawning. The fertilized eggs were in- reverse transcriptase (Bio-Rad, Hercules, CA). cubated at 27±1◦C. After hatching, the larval fish were cultured Polymerase chain reaction (PCR) amplification was carried out under the same photoperiod (14L:10D) and fed with paramecium using gene-specific primers flanking an intron (Supplemental Table first followed by commercial baby fish diet (Tetra Baby, Melle, Ger- S1). The reaction was performed in a volume of 30 μl consisting of many) and Artemia salina nauplii. Samples were collected at different 10 μl reverse transcription (RT) reaction product (1:10 diluted with days to examine the expression of gdf9, amh, cyp19a1a, and other water), 1× PCR buffer, 0.2 mM of each dNTP, 2.5 mM MgCl2, related genes. The larvae from 13 to 20 dpf (day post fertilization) 0.2 μM of each primer, 0.6 U Taq polymerase with the profile of were cooled on ice and stored in RNAlater (Ambion, Austin, TX) at 30 s at 94◦C, 45 s at 56◦Cforgapdh,58◦Cforcyp19a1a, bactin, 4◦C overnight. They were then transferred to –20◦C for at least 1 dmrt1, 60◦Cforgdf9, star, sox9b,63◦Cforsox9a, and 65◦Cfor week before isolating the gonads for RNA extraction. For juvenile amh, and60sat72◦C for extension. The cycle numbers of PCR

Figure 1. Sampling of gonads from juvenile fish for histological examination and gene expression analysis. (A) The fish were severed in half with the anterior part being fixed for histology and the posterior part stored in the RNAlater solution for mRNA extraction. (B) The posterior part of the fish was dissected for gonads after fixation in the RNAlater solution. The gonads could be easily identified. (C) The isolated gonads ready for RNA extraction. 404 W. Chen et al., 2017, Vol. 96, No. 2 amplification were 30 for gdf9, cyp19a1a, and other genes and 27 detected by HRP-conjugated anti-fluorescein antibody (Roche) with for gapdh and bactin. TSA-fluorescein according to the manufacturer’s instruction. The sections were finally washed with PBS and mounted with Prolong Real-time qPCR quantification of gdf9, cyp19a1a, and Gold antifade reagent (Invitrogen, Carlsbad, CA); DAPI was added amh expression at this step. The slides were observed and the images recorded using Olympus Fluoview confocal microscope (Olympus, Tokyo, Japan). The expression levels of gdf9, cyp19a1a, amh, and ef1a in the gonads from each individual larval or juvenile fish at different developmental stages were determined by real-time qRCR. The standard for each gene was prepared by PCR amplification of cDNA fragments Recombinant production of zebrafish Gdf9 with specific primers (Supplemental Table S1). The amplicons were To produce recombinant zebrafish Gdf9, we used Flp-In CHO cells resolved by agarose gel electrophoresis, purified, and quantified by (Invitrogen) as the bioreactor [61]. Briefly, the open reading frag- electrophoresis together with the Mass Ruler DNA marker (MBI ment (ORF) of gdf9 was amplified from the ovarian cDNA with Fermentas, Hanover, MD). These amplified amplicons were used to pfu polymerase and a sense primer carrying the Kozak sequence Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021 construct standard curves in real-time qPCR assays. around the start codon ATG to enhance translation efficiency [62]. Real-time PCR was carried out on the iCycler iQ Real-Time PCR The ORF was cloned into the pcDNA5/FRT vector (Invitrogen) Detection System (Bio-Rad) in a volume of 30 μl that contained downstream of the CMV promoter to generate the expression con- 10 μl diluted RT reaction mix, 1× PCR buffer, 0.2 mM of each struct, pcDNA5/FRT/zfGDF9. The plasmid was co-transfected into dNTP, 2.5 mM MgCl2,0.2μM of each primer, 0.75 U Taq poly- the CHO cells with pOG44 encoding a recombinase by Lipofec- merase, 0.5× EvaGreen (Biotium, Hayward, CA), and 20 nM flu- tamine 2000 and selected by hygromycin B (500 μg/ml) (Invitrogen) orescein (Bio-Rad). The reaction profile consisted of 38 cycles of for 2 weeks. Individual clones were isolated by serial dilution in 96- 94◦C for 30 s, 60◦C for 30 s, 72◦C for 1 min, and 80◦C for 7 s for well plates, and a clone with integration of gdf9 ORF in the genome signal detection. A melt curve analysis was performed at the end of was chosen for production of recombinant Gdf9 according to the amplification to demonstrate reaction specificity, which was further protocol described by Schatz et al. and modified by our laboratory confirmed by agarose gel electrophoresis. [61,63]. The expression of gdf9 in the CHO cells was examined by northern blot hybridization [64], and the preparation of conditioned Fluorescent in situ hybridization medium containing recombinant Gdf9 was carried out according to our previous report [61]. The zebrafish gdf9, cyp19a1a, and amh cDNAs were amplified with specific primers (Supplemental Table S1), and the amplicons were cloned into pBluescript II KS (+). These plasmids were then used as the templates for probe synthesis. The sense and antisense probes Primary culture of zebrafish follicle cells and were labeled with DIG or fluorescein by RNA labeling kit (Roche Ap- incubation of ovarian fragments plied Science, Mannheim, Germany). The fish body with gonads was The primary culture of zebrafish ovarian follicle cells and incubation fixed in freshly prepared paraformaldehyde (4%) for 2–4 h at room of ovarian fragments were performed according to our previous re- temperature and stored in PBS buffer at 4◦C before processing for ports [65,66]. For follicle cell culture, the follicles of previtellogenic paraffin embedding and sectioning at 5 μm thickness. The sections (PV) to midvitellogenic (MV) stages from about 20 female zebrafish were mounted on slides coated with poly-(L)-lysine, and hybridized were isolated, washed with medium M199, and incubated in M199 ◦ with sense (control) or antisense cRNA probes of gdf9, cyp19a1a, supplemented with 10% FBS at 28 Cin5%CO2 for 6 days for and amh, respectively, according to the protocol we reported re- the follicle cells to proliferate. The medium was changed once on cently [60]. Briefly, the sections were deparaffinized, washed with the third day of incubation, and the follicle cells were harvested by PBS, and treated with proteinase K (4 μg/ml in PBS) for 10 min at trypsinization on the sixth day for subculturing in 48-well plates 37◦C. After rinsing with PBS, the sections were postfixed for 15 min (1 × 105/well). After 18 h of preincubation, the cells were treated with 4% paraformaldehyde in PBS before treatment with glycine with concentrated serum-free conditioned medium from the recom- (2 mg/ml) for 10 min, followed by 10 min treatment with 0.25% binant CHO cells for 20–180 min. After washing three times with acetic anhydride in 0.1 M triethanolamine buffer. After a 10-min M199, the follicle cells were subject to total RNA extraction with wash with 2 × SSC, the sections were prehybridized at room tem- Tri-Reagent for real-time qPCR analysis. For western blot analy- perature for 1 h in 66% formamide with 2 × SSC, and hybridized sis, proteins were extracted from the cultured cells, electrophoresed, at 58◦C overnight with DIG or fluorescein-labeled antisense or sense and blotted to nitrocellulose membrane. The immunoblot was in- probes in the hybridization buffer (60% deionized formamide, 7.5% cubated with p-Smad2 antibody (Cell Signaling, MA) (1:1000) fol- Dextran sulfate, 1 × Denhardt solution, 20 mM Tris-HCl, 2.5 mM lowed by incubation with the HRP-conjugated goat anti-rabbit an- EDTA, 350 mM NaCl, and 0.2 mg/ml tRNA). After a series of tibody (1:3000). Signals were detected using the Western Blotting washes with 5 × SSC, 50% formamide/50% 2 × SSC, 2 × SSC, and Luminol Reagent (Santa Cruz, CA) and the images were captured 0.2 × SSC at 58◦C, the sections were rinsed in maleic acid buffer on the Lumi-Imager F1 Workstation (Roche). (0.1 M maleic acid, 0.15 M NaCl, 0.01% Tween 20) and incubated For ovarian fragment incubation, the ovaries were dissected from with 100 μl 1% Blocking Reagent (Roche) at room temperature for six female zebrafish and placed in a petri dish containing 60% Lei- 60 min. The slides were then incubated with 100 μl diluted anti-DIG bovitz 15 medium (L-15, Gibco BRL, Grand Island, NY). The ovaries or anti-fluorescein horseradish peroxidase (HRP) (1:500 dilution in were carefully separated into small fragments with fine forceps and 1% Blocking Buffer) in a humidified chamber for 2 h at room tem- randomly distributed into 24-well tissue culture plate (Corning, NY). perature. After washing slides with maleic acid buffer three times, After treatment with recombinant conditioned medium for 12 h at the sections were treated with the TSA-Plus Fluorescein/cy5/TMR 28◦C, the ovarian fragments were collected for total RNA extraction (PerkinElmer, Waltham, MA) in dark for 10 min. The signal was followed by real-time qPCR analysis. GDF9 in zebrafish gonadal differentiation, 2017, Vol. 96, No. 2 405

Injection of vivo-morpholinos Results The antisense VMOs for gdf9 and cyp19a1a as well as stan- Expression of gdf9, cyp19a1a, and amh in dard control VMO were obtained from Gene Tools (Philomath, OR), and their sequences are as follows: 5-GGCGTAATAA differentiated gonads at 40 dpf TAAGCATACCTCTGT-3 (gdf9-MO), 5-GCCAGCTACAGAAC Juvenile fish around 40 dpf with similar size (1.3–1.5 cm in body ACAAACAGAAC-3(cyp19a1a-MO) and 5-CCTCTTACCTCAG length) were sampled, and the body was severed in half around the TTACAATTTATA-3(CTL-MO). The VMOs of gdf9 and cyp19a1a communicating duct between the anterior and posterior chambers of were designed to block mRNA splicing so as to knock down gdf9 the swim bladder. The anterior half was fixed for histological exam- and cyp19a1a expression. The sterile VMOs were prepared as 0.5 ination to identify gonad developmental stage, while the posterior mM (4.2 ng/nl) stocks in distilled water for gdf9-MO, cyp19a1a- half was stored in RNAlater for gonadal isolation and expression MO and CTL-MO. The larvae of 18 dpf with less than 10 mg body analysis (Figure 1A). The gonads could be easily identified and sepa- weight and undifferentiated gonads were anesthetized with MS222 rated under dissecting microscope after pretreatment with RNAlater

(0.25 mg/ml), weighted, and injected with VMOs at 12.5 ng/mg (Figure 1BandC). Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021 body weight. Vivo-morpholinos were injected into the gonad region Histological examination showed that the ovary contained tightly < μ around the posterior bladder with microinjector every 2 days and packed PO ( 100 m) (Figure 2A), and the testis was full of sper- seven injections were performed in total. The developmental state of matogonia, spermatocytes, and even spermatids (Figure 2B). By com- the gonads was determined by histological examination around 40 parison, the transforming gonads or early testis was small, contain- dpf when the gonads had almost completed differentiation. ing spermatogonia/spermatocytes, remnants of POs, and abundant stromal cells (Figure 2C). With the gonads categorized into the above three stages of Data analysis differentiation, we then examined the expression of a variety of The expression levels of gdf9, cyp19a1a, and amh were normalized genes that are potentially involved in gonadal differentiation with to that of internal control ef1a. All values were expressed as the gapdh and bactin being used as the housekeeping genes. The ex- mean ± SEM, and the data were analyzed by the Dunnett multiple pression of gapdh and bactin showed similar patterns in all three comparison test with Prism on Macintosh OS X (GraphPad Soft- types of gonads despite certain individual variation, indicating ware, San Diego, CA). The experiments were repeated at least three that our sampling method involving RNAlater was appropriate times. for analyzing gene expression (Figure 2D). Among the functional

Figure 2. Gene expression in three types of gonad at 40 dpf when sex differentiation was almost completed. (A) Differentiated ovary (outlined by dashed line) with well-developed PO at PG stage. (B) Differentiated testis with different stages of spermatogenic cells. (C) Transforming gonad (outlined by dashed line) with spermatogenic cells as well as undifferentiated or oocyte-like germ cells. (D) RT-PCR analysis for the expression of target genes in the three types of gonad. 406 W. Chen et al., 2017, Vol. 96, No. 2 genes analyzed, cyp19a1a, foxl2 (forkhead box protein L2) and positive signal could be more frequently detected in the testes sox9b (SRY-box containing gene 9b) are well known to be re- (Figure 2D). lated to ovarian differentiation [67], while dax1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1), amh (anti-Mullerian¨ hormone), dmrt1 (doublesex and Expression of gdf9, cyp19a1a,andamh in mab-3 related transcription factor 1), sox9a (SRY-box containing differentiating gonads at 27 dpf gene 9a), and star (steroidogenic acute regulatory protein) have To further illustrate the temporal expression of gdf9, cyp19a1a, and abundant expression in differentiated testis in ﬁsh [67]. Our re- amh during gonadal differentiation, we analyzed their expression sults in this experiment showed that gdf9 and cyp19a1a exhib- levels at an earlier stage (27 dpf, total body length 0.8–1.1 cm) when ited clear dimorphic expression patterns in the differentiating go- the differentiation was starting. Again, the developmental stage of nads. Their expression could be easily detected in the ovaries, but the gonads was identiﬁed by histological examination of the anterior not testes. In the transforming gonads, about half of individuals half of the body. Unlike the stage at 40 dpf when typical ovary and

showed detectable expression. Interestingly, although foxl2 is well testis were forming or had formed, the gonads at 27 dpf could also Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021 known to be important in controlling cyp19a1a expression, its ex- be categorized into three types, but of earlier developmental stages: pression could be detected ubiquitously in all gonads. The same the true ovary full of POs, juvenile ovary with small POs and/or pattern was observed for dax1. By comparison, although the ex- undifferentiated meiotic germ cells, and the gonad with undifferenti- pression of amh could also be detected in all three types of go- ated germ cells only (most likely destined to become testis). No testis nads, its expression level was obviously more abundant in the with typical spermatogenesis was observed at this stage (Figure 3A). testes than the ovaries. Similar pattern of expression was also Semiquantitative RT-PCR and real-time qPCR analyses showed observed for dmrt1 and sox9a. For sox9b and star, the expres- that the expression of gdf9 was dimorphic with strong expression sion could be detected in some individuals of all three types of in the differentiated ovary, weak expression in the juvenile ovary, gonads, but the expression pattern was not clear although the but very low expression in the undifferentiated gonad (Figure 3B

Figure 3. The expression and quantitative analysis of gdf9, cyp19a1a, and amh at 27 dpf during gonadal differentiation. (A) Histology of the three types of gonad: ovary (O), juvenile ovary (JO), and undifferentiated gonad (UG). (B) RT-PCR results show the expression of gdf9, cyp19a1a,andamh in O, JO, and UG. (C) Real-time qPCR analysis on the expression of gdf9, cyp19a1a,andamh in O, JO, and UG. Different letters indicate statistical signiﬁcance (P < 0.05). GDF9 in zebraﬁsh gonadal differentiation, 2017, Vol. 96, No. 2 407

was much weaker and sometimes fragmentary (Figure 5BandC). In comparison, the expression of cyp19a1a was weak in the ovary and could be detected sporadically in some follicle cells encompass- ing the oocytes (Figure 5D). On the contrary, amh was expressed in somatic cells in the juvenile ovary around 30 dpf, but the signal was weak (Figure 5E). In the transforming gonad, the expression of amh was the highest among the three stages and it was expressed in the somatic cells, presumably the Sertoli cells (Figure 5F).

Evidence for roles of gdf9 and cyp19a1a in ovarian differentiation Aromatase (cyp19a1a) is well known to be important in driving Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021 ovarian differentiation in ﬁsh [18]. However, as an oocyte-speciﬁc

Figure 4. Ontogenic expression of gdf9, cyp19a1a, and amh in undifferenti- growth factor, the role of gdf9 in this process remains entirely un- ated gonads from 13 to 20 dpf. The entire gonads were isolated from the known. In this experiment, we adopted a special morpholino, VMO, larval fish after fixing in the RNAlater and the total RNA extracted for qPCR to block the expression of gdf9 in vivo followed by sex ratio anal- analysis. ysis. The gdf9-VMO was injected into larval fish at 18 dpf before gonadal differentiation started. Since the effect of aromatase on ovarian development has been reported in the zebrafish [31,33], we also and C). Similarly, the expression of cyp19a1a could be detected in targeted cyp19a1a as the positive control with cyp19a1a-VMO that the ovary and juvenile ovary of some individuals, but not in the spans the boundary of exon 8 and intron 7 (Figure 6A). The VMOs undifferentiated gonad (Figure 3B and C). Similar to the observation for gdf9 and cyp19a1a were injected into the larvae of the same at 40 dpf, gdf9 and cyp19a1a were co-expressed during gonadal batches at the same time. The VMO for human actin was used as differentiation. As for amh, although its expression could be detected the negative control (CTL-VMO). About 70 larval fish in total were in all three forms of gonads by semiquantitative RT-PCR (Figure 3B), injected with each VMO. it showed a reciprocal expression pattern as compared with those of Injection of CTL-VMO did not change the sex ratio at 40 dpf gdf9 and cyp19a1a. Real-time qPCR showed that the expression when gonads had nearly finished sex differentiation. The numbers level of amh was very low in the newly formed ovary, increased in of differentiated females and males were similar with only a few the juvenile ovary, and reached the highest in the undifferentiated individuals still undergoing gonadal transformation from juvenile gonads destined to become the testis (Figure 3C). ovary to testis. In contrast, the injection of gdf9-VMO significantly decreased the number of female fish and increased that of males, and Expression of gdf9, cyp19a1a,andamh in there seemed to be more fish undergoing transformation. Similar undifferentiated gonads (13–20 dpf) results were obtained for cyp19a1a-VMO (30% ovary, 60% testis, Under our aquarium condition, the zebrafish gonads are either un- and 10% transforming gonad) (Figure 6B). differentiated or developed as juvenile ovary with small POs before 20 dpf. The differentiation into true ovary occurs between 25 and Effects of recombinant zebrafish Gdf9 on amh and 30 dpf, followed by testis formation around 40–45 days [59]. In this experiment, we examined the expression of gdf9, cyp19a1a, cyp19a1a expression in vitro and amh between 13 and 20 dpf before differentiation by real-time As shown above, cyp19a1a and amh were both expressed in the qPCR. Despite very low levels, the expression of all three genes (gdf9, somatic cells in the gonads and they exhibited reciprocal expres- cyp19a1a, and amh) could be detected in the gonads of 13-dpf fish sion patterns during gonadal differentiation. To demonstrate if (Figure 4). From 13 to 20 dpf, the expression of cyp19a1a and amh the oocyte-derived Gdf9 plays a role in regulating the expression remained low and stable despite a minor surge at 15 dpf. In con- of cyp19a1a and amh, we prepared recombinant zebrafish Gdf9 trast, there was an obvious increase in gdf9 expression starting from (rzfGdf9) with the CHO cells. The activity of rzfGdf9 was assessed 16 dpf, and the level remained high and stable to 20 dpf. Despite by its stimulation of Smad2 phosphorylation in cultured follicle cells. obvious trends, no statistical significance was detected due to large As shown in Figure 7A, the conditioned medium could significantly variations (Figure 4). increase the phosphorylation of Smad2, the protein involved in Gdf9 signaling [54,70], in a time-dependent manner. Treatment of the ovarian fragments did not have significant effect on cyp19a1a ex- Oocyte-specific expression of gdf9 during gonadal pression despite a slight increase; however, it significantly reduced differentiation the expression level of amh in a dose-dependent manner (Figure 7B). The expression of gdf9 was known to be oocyte-specific in adult ovaries of both mammals and fish [50,68,69]. However, little infor- mation is known about its location in early stage of development Effects of recombinant zebrafish Gdf9 on during gonadal differentiation. In this experiment, we examined the activin–inhibin–follistatin system in vitro distribution of gdf9 expression in differentiating gonads (27–30 dpf) In addition to cyp19a1a and amh, we also examined the effects of by fluorescent in situ hybridization. Strong signal of gdf9 expression rzfGdf9 on the expression of activin–inhibin–follistatin system in was detected in the ooplasm of all POs in the differentiated ovary cultured ovarian fragments. Our previous work has demonstrated (Figure 5A). In the juvenile ovary or transforming gonad, degener- that the subunits of activin and inhibin (inha, inhbaa, and inhbb) ating oocytes (around 30 μm in diameter) were sometimes present. are exclusively expressed in the somatic follicle cells [66,71,72] These oocytes still showed the expression of gdf9, but the signal and the expression of beta subunits inhbaa and inhbb increased 408 W. Chen et al., 2017, Vol. 96, No. 2 Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021

Figure 5. In situ hybridization for the localization of gdf9, cyp19a1a, and amh expression in the ovary, juvenile ovary, and transforming gonad. (A–C) Expression of gdf9 in the gonads. (D) Expression of cyp19a1a in the ovary. (E, F) The expression of amh in the juvenile ovary (E) and transforming gonad (F). significantly during follicle activation from primary growth (PG) to supporting the view that the gonadal somatic cells play an important PV stage, especially inhbaa, when gdf9 expression decreases [50,73]. or determining role in controlling sex differentiation. Interestingly, Interestingly, while rzfGdf9 reduced amh expression in cultured an increasing body of evidence in recent years also points to the im- ovarian fragments, it increased the expression of inhbaa and inhbb, portance of germ cells in the event [43,46]. The mechanisms underly- but had no effect on inhibin alpha subunit (inha) and follistatin (fst) ing germ cell signaling toward the somatic cells are unknown; how- (Figure 8). ever, this issue has attracted attention to the roles of oocyte-derived secreted factors. Among the best-characterized oocyte-derived factors is GDF9 (Gdf9/gdf9). Discussion As an oocyte-specific growth factor, GDF9 has been well docu- Adult zebrafish are gonochoristic with ovary and testis present in mented to play important roles in influencing the functions of the sur- different individuals; however, the juvenile zebrafish are considered rounding somatic follicle cells in mammals [80,81], supporting the hermaphroditic because they developed ovary-like gonad or juvenile notion that the oocyte as the germ cell functions as a signaling center ovary first followed by differentiation into true ovary and testis at in the follicle to orchestrate folliculogenesis. It increases the produc- later stage [13,17,74]. The juvenile ovary is characterized by meiotic tion of other growth factors and steroids by the surrounding follicle onset in all individuals and appearance of PO in most if not all in- cells [80,81], and maintains the expression of FSH receptor in the dividuals. The gonadal differentiation starts around 20–25 dpf and follicle so as to act as an anti-apoptotic factor suppressing granulosa completes around 30–45 dpf; however, large variation exists among cell apoptosis and follicular atresia [82]. In the zebrafish, apoptosis different individuals [13,16,17,59]. During sex differentiation, three is considered a major mechanism responsible for degeneration of the types of gonads could be observed, i.e., ovary, transforming gonad oocyte-like germ cells in juvenile ovary, therefore paving a way for (or early differentiated testis), and testis [16,17]. In this study, trans- testis development. This is supported by the evidence that apoptotic forming gonads started to appear around 25 dpf and the testis with signaling was stronger in the transforming gonads compared to that spermatogenic cells such as spermatocytes or even spermatids could in the developed ovary [17]. Whether the oocyte-secreted Gdf9 has be observed in some individuals after 30 dpf; however, large indi- protective effect to reduce apoptosis during gonadal differentiation vidual variation existed and the gonads of some individuals had not will be an interesting issue to explore in the zebrafish. completed their differentiation even at 40 dpf. In fish models, we previously reported that gdf9 was also specifi- Although no single primary sex-determining gene has been iden- cally expressed in the oocytes of the zebrafish, and its expression level tified in the zebrafish and its sex determination is believed to be poly- was the highest in early follicles. After fertilization, gdf9 transcripts genic [12], a variety of genes have been implicated in controlling its were still detectable in the embryos as maternal mRNA until gas- gonadal differentiation, including cyp19a1a, amh, dmrt1, sox9a/b, trulation stage [50]. In this study, we showed that the expression of ar, figla, and members of Wnt signaling pathway such as rspo1 gdf9 started in the gonads before sex differentiation at morphologi- [26,75–79]. Most of these genes are expressed in the somatic cells, cal level and its level increased substantially at 16 dpf. Interestingly, GDF9 in zebrafish gonadal differentiation, 2017, Vol. 96, No. 2 409 Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021

Figure 7. Effects of recombinant zebraﬁsh Gdf9 (rzfGdf9) on Smad2 phosphorylation in cultured follicle cells (A) and amh and cyp19a1a expression (B) in incubated ovarian fragments. The follicle cells were treated with concentrated conditioned medium from the recombinant CHO cells for 20 to 180 min followed by protein extraction for western blot analysis. For gene expression analysis with real-time qPCR, the test was carried out on cultured ovarian fragments. Different letters indicate statistical signiﬁcance (P < 0.05).

femaleness as suggested by Wang et al. [16] could contribute to the high data variation. During gonadal differentiation, the expression of gdf9 showed an obvious sexual dimorphic pattern with the level much higher in the differentiated ovary than testis, which was similar to that of Figure 6. Morpholino knockdown of gene expression in the juvenile zebrafish. cyp19a1a but reciprocal to amh expression. The expression of amh (A) Target sites of VMOs for gdf9 and cyp19a1a. (B) Sex ratios at 40 dpf in was much higher in the testis than that in the ovary. Our experiment in the fish treated with control, gdf9 and cyp19a1a VMOs. Different letters with rzfGdf9 showed that the oocyte-derived Gdf9 suppressed amh indicate statistical significance (P < 0.05). expression in vitro, and this effect could be part of the mechanism for the reciprocal patterns of gdf9 and amh expression during gonadal differentiation. The expression of amh in the gonads seems highly although it was obvious, the increase was not statistically signifi- diverse among different species. In the medaka, the expression of cant due to large data variation. Different from the analysis at later amh shows no sexual dimorphism with similar levels in males and stages, which involved categorization of samples based on histolog- females [83]. In the Japanese flounder, amh was not detectable in the ical characterization, the samples from 13 to 20 dpf were pooled ovary by RT-PCR or by northern blot analysis [84]. Although gdf9 without histological classification. The high data variation implies and cyp19a1a were co-expressed during gonadal differentiation, we that although gonadal differentiation was not visible at histologi- did not observe significant response of cyp19a1a to rzfGdf9 despite cal level from 15 to 20 dpf, the process might have already started a slight increase. The importance of Gdf9 in ovarian development at molecular level. The increase of gdf9 expression, which was not was evidenced by VMO-induced male-skewed sex ratio; however, it accompanied by cyp19a1a and amh, might be associated with the remains unclear whether the change in sex ratio was due to disrupted hermaphroditic phase of gonadal development (juvenile ovary) with signaling pathway towards ovary during gonadal differentiation or oocyte-like germ cells, and different levels of commitment toward sex reversal after differentiation. 410 W. Chen et al., 2017, Vol. 96, No. 2 Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021

Figure 8. Effects of rzfGdf9 on the expression of activin (inhbaa and inhbb), inhibin (inha), and follistatin (fst) in cultured ovarian fragments. The ovarian fragments were treated with concentrated conditioned medium from the recombinant CHO cells for 20 h followed by RNA extraction for real-time qPCR analysis. Different letters indicate statistical signiﬁcance (P < 0.05).

Interestingly, activin subunits (inhbaa and inhbb) whose expres- levels in the testis, suggesting that they likely participate in con- sion increased significantly during follicle activation, especially in- trolling gonadal differentiation quantitatively in a dose-dependent hbaa [73], responded positively to rzfGdf9 treatment. By compar- manner. ison, no significant responses of inha and fst were observed. This In summary, we demonstrated sexually dimorphic expression agrees well with the expression profile of inha during folliculogen- patterns of gdf9, amh, and cyp19a1a during gonadal differentia- esis, which increases significantly at late stages before oocyte matu- tion in larval and juvenile zebrafish. Gdf9 and cyp19a1a were co- ration and ovulation [66]. As for fst, it is abundantly expressed in expressed in the differentiating or differentiated gonads but in dif- the oocyte as compared to the somatic follicle cells [71,72], making ferent cells, and their expression patterns were closely correlated it less likely the target for Gdf9 in the ovarian fragments. This result with ovarian development, suggesting important roles for these two suggests that in addition to amh, the oocyte-derived Gdf9 may also molecules in ovarian differentiation and development. Knocking target other genes in the somatic follicle cells including the activin down the expression of gdf9 and cyp19a1a using VMOs both caused system, which represents a major paracrine signaling pathway from a reduction of female ratio. Future studies using genomic knockout the follicle cells toward the oocyte [85]. The stimulation of activin of gdf9 and cyp19a1a genes will provide further evidence for their subunits by Gdf9 appears puzzling because they exhibited recipro- roles and importance in gonadal development. cal expression patterns during follicle activation or PG-PV transition [50,73]. Our hypothesis for this regulatory relationship is that the de- Supplementary data creased mRNA level of gdf9 at this transition may likely represent an increased mRNA turnover and protein synthesis, which stimulates Supplementary data are available at BIOLRE online. activin output from the somatic follicle cells. The activin in turn Supplemental Table S1. Primers used in RT-PCR and real-time acts on the oocyte to stimulate its growth and follicle development qPCR∗ analysis. All the primers were synthesized by Integrated DNA [71,72]. Technologies, Inc. (Coralville, IA). In addition to gdf9, cyp19a1a, and amh, we also examined the expression of some other genes that are potentially involved in gonadal Disclosure differentiation. Interestingly, although these genes have been impli- The authors have nothing to disclose. cated in gonadal differentiation, favoring the development of either ovary (foxl2 and sox9b)ortestis(dmrt1, sox9a,anddax1), none of them exhibited a clear-cut dimorphic expression pattern as shown by References gdf9 and cyp19a1a. Despite this, some genes such as dmrt1, sox9a, 1. Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, and amh did show differential expression with higher expression Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow PN. A gene from GDF9 in zebrafish gonadal differentiation, 2017, Vol. 96, No. 2 411

the human sex-determining region encodes a protein with homology to a 23. D’Cotta H, Fostier A, Guiguen Y, Govoroun M, Baroiller JF. Aromatase conserved DNA-binding motif. Nature 1990; 346:240–244. plays a key role during normal and temperature-induced sex differen- 2. Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi tiation of tilapia Oreochromis niloticus. Mol Reprod Dev 2001; 59: T, Morrey CE, Shibata N, Asakawa S, Shimizu N, Hori H, Hamaguchi S 265–276. et al. DMY is a Y-specific DM-domain gene required for male development 24. Ijiri S, Kaneko H, Kobayashi T, Wang DS, Sakai F, Paul-Prasanth B, Naka- in the medaka fish. Nature 2002; 417:559–563. mura M, Nagahama Y. Sexual dimorphic expression of genes in gonads 3. Hattori RS, Murai Y, Oura M, Masuda S, Majhi SK, Sakamoto T, Fer- during early differentiation of a teleost fish, the Nile tilapia Oreochromis nandino JI, Somoza GM, Yokota M, Strussmann CA. A Y-linked anti- niloticus. Biol Reprod 2008; 78:333–341. Mullerian hormone duplication takes over a critical role in sex determi- 25. Luckenbach JA, Early LW, Rowe AH, Borski RJ, Daniels HV, Godwin nation. Proc Natl Acad Sci USA 2012; 109:2955–2959. J. Aromatase cytochrome P450: cloning, intron variation, and ontogeny 4. Li M, Sun Y, Zhao J, Shi H, Zeng S, Ye K, Jiang D, Zhou L, Sun L, Tao of gene expression in southern flounder (Paralichthys lethostigma). J Exp W, Nagahama Y, Kocher TD et al. A tandem duplicate of Anti-Mullerian Zool A Comp Exp Biol 2005; 303:643–656. hormone with a missense SNP on the Y chromosome is essential for male 26. Rodriguez-Mari A, Yan YL, Bremiller RA, Wilson C, Canestro C, Postleth- sex determination in Nile tilapia, Oreochromis niloticus. PLoS Genet wait JH. Characterization and expression pattern of zebrafish anti- Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021 2015; 11:e1005678. Mullerian hormone (amh) relative to sox9a, sox9b,andcyp19a1a, during 5. Smith CA, Roeszler KN, Ohnesorg T, Cummins DM, Farlie PG, Doran gonad development. Gene Expr Patterns 2005; 5:655–667. TJ, Sinclair AH. The avian Z-linked gene DMRT1 is required for male sex 27. Guiguen Y, Baroiller JF, Ricordel MJ, Iseki K, McMeel OM, Martin SAM, determination in the chicken. Nature 2009; 461:267–271. Fostier A. Involvement of estrogens in the process of sex differentiation in 6. Yoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida-Umehara two fish species: the rainbow trout (Oncorhynchus mykiss) and a tilapia C, Matsuda Y, Takamatsu N, Shiba T, Ito M. A W-linked DM-domain (Oreochromis niloticus). Mol Reprod Dev 1999; 54:154–162. gene, DM-W, participates in primary ovary development in Xenopus 28. Kitano T, Takamune K, Nagahama Y, Abe SI. Aromatase inhibitor and laevis. Proc Natl Acad Sci USA 2008; 105:2469–2474. 17α-methyltestosterone cause sex-reversal from genetical females to phe- 7. Penman DJ, Piferrer F. Fish gonadogenesis. Part I: Genetic and environ- notypic males and suppression of P450 aromatase gene expression in mental mechanisms of sex determination. Rev Fish Sci 2008; 16:16–34. Japanese flounder (Paralichthys olivaceus). Mol Reprod Dev 2000; 56: 8. Piferrer F, Guiguen Y. Fish gonadogenesis. Part II: Molecular biology and 1–5. genomics of sex differentiation. Rev Fish Sci 2008; 16:35–55. 29. Piferrer F, Zanuy S, Carrillo M, Solar II, Devlin RH, Donaldson EM. Brief 9. Devlin RH, Nagahama Y. Sex determination and sex differentiation in treatment with an aromatase inhibitor during sex-differentiation causes fish: an overview of genetic, physiological, and environmental influences. chromosomally female salmon to develop as normal, functional males. Aquaculture 2002; 208:191–364. J Exp Zool 1994; 270:255–262. 10. Strussmann CA, Nakamura M. Morphology, endocrinology, and envi- 30. Tzchori I, Zak T, Sachs O. Masculinization of genetic females of the com- ronmental modulation of gonadal sex differentiation in teleost fishes. Fish mon carp (Cyprinus carpio L.) by dietary administration of an aromatase Physiol Biochem 2002; 26:13–29. inhibitor. Isr J Aquacult-Bamid 2004; 56:239–246. 11. Liew WC, Orban L. Zebrafish sex: a complicated affair. Brief Funct 31. Uchida D, Yamashita M, Kitano T, Iguchi T. An aromatase inhibitor or Genomics 2014; 13:172–187. high water temperature induce oocyte apoptosis and depletion of P450 12. Liew WC, Bartfai R, Lim Z, Sreenivasan R, Siegfried KR, Orban L. Poly- aromatase activity in the gonads of genetic female zebrafish during sex- genic sex determination system in zebrafish. PLoS One 2012; 7:e34397. reversal. Comp Biochem Physiol A Mol Integr Physiol 2004; 137:11–20. 13. Maack G, Segner H. Morphological development of the gonads in 32. Shved N, Berishvili G, D’Cotta H, Baroiller JF, Eppler E, Segner H, Rei- zebrafish. J Fish Biol 2003; 62:895–906. necke M. A survey on the expression of IGF-I in the early developing bony 14. Takahashi H. Juvenile hermaphroditism in the zebrafish, Brachydanio fish with special emphasis on the tilapia, Oreochromis niloticus. Ann N Y rerio. Bull Fac Fish Hokkaido Univ 1977; 28:57–65. Acad Sci 2005; 1040:469–471. 15. Wang XG, Orban L. Anti-Mullerian hormone and 11 β-hydroxylase show 33. Takatsu K, Miyaoku K, Roy SR, Murono Y, Sago T, Itagaki H, Nakamura reciprocal expression to that of aromatase in the transforming gonad of M, Tokumoto T. Induction of female-to-male sex change in adult zebrafish zebrafish males. Dev Dyn 2007; 236:1329–1338. by aromatase inhibitor treatment. Sci Rep 2013; 3:3400. 16. Wang XG, Bartfai R, Sleptsova-Freidrich I, Orban L. The timing and 34. Polanco JC, Koopman P. Sry and the hesitant beginnings of male devel- extent of ‘juvenile ovary’ phase are highly variable during zebrafish testis opment. Dev Biol 2007; 302:13–24. differentiation. J Fish Biol 2007; 70:33–44. 35. Pala I, Kluver N, Thorsteinsdottir S, Schartl M, Coelho MM. Expression 17. Uchida D, Yamashita M, Kitano T, Iguchi T. Oocyte apoptosis during pattern of anti-Mullerian hormone (amh) in the hybrid fish complex of the transition from ovary-like tissue to testes during sex differentiation of Squalius alburnoides. Gene 2008; 410:249–258. juvenile zebrafish. J Exp Biol 2002; 205:711–718. 36. Munsterberg A, Lovellbadge R. Expression of the mouse Anti-mullerian 18. Guiguen Y, Fostier A, Piferrer F, Chang CF. Ovarian aromatase and es- hormone gene suggests a role in both male and female sexual- trogens: a pivotal role for gonadal sex differentiation and sex change in differentiation. Development 1991; 113:613–624. fish. Gen Comp Endocrinol 2010; 165:352–366. 37. Choi Y, Rajkovic A. Genetics of early mammalian folliculogenesis. Cell 19. Kishida M, Callard GV. Distinct cytochrome P450 aromatase isoforms Mol Life Sci 2006; 63:579–590. in zebrafish (Danio rerio) brain and ovary are differentially programmed 38. Guigon CJ, Magre S. Contribution of germ cells to the differentiation and and estrogen regulated during early development. Endocrinology 2001; maturation of the ovary: insights from models of germ cell depletion. Biol 142:740–750. Reprod 2006; 74:450–458. 20. Chiang EF, Yan YL, Guiguen Y, Postlethwait J, Chung B. Two Cyp19 39. Kurokawa H, Saito D, Nakamura S, Katoh-Fukui Y, Ohta K, Baba T, (P450 aromatase) genes on duplicated zebrafish chromosomes are ex- Morohashi K, Tanaka M. Germ cells are essential for sexual dimorphism pressed in ovary or brain. Mol Biol Evol 2001; 18:542–550. in the medaka gonad. Proc Natl Acad Sci USA 2007; 104:16958–16963. 21. Patil JG, Gunasekera RM. Tissue and sexually dimorphic expression of 40. Saito D, Morinaga C, Aoki Y, Nakamura S, Mitani H, Furutani-Seiki ovarian and brain aromatase mRNA in the Japanese medaka (Oryzias M, Kondoh H, Tanaka M. Proliferation of germ cells during gonadal sex latipes): implications for their preferential roles in ovarian and neural differentiation in medaka: Insights from germ cell-depleted mutant zenzai. differentiation and development. Gen Comp Endocrinol 2008; 158:131– Dev Biol 2007; 310:280–290. 137. 41. Fujimoto T, Nishimura T, Goto-Kazeto R, Kawakami Y, Yamaha E, 22. Vizziano D, Randuineau G, Baron D, Cauty C, Guiguen Y. Characteriza- Arai K. Sexual dimorphism of gonadal structure and gene expression in tion of early molecular sex differentiation in rainbow trout, Oncorhynchus germ cell-deficient loach, a teleost fish. Proc Natl Acad Sci USA 2010; mykiss. Dev Dyn 2007; 236:2198–2206. 107:17211–17216. 412 W. Chen et al., 2017, Vol. 96, No. 2

42. Slanchev K, Stebler J, de la Cueva-Mendez G, Raz E. Development without 63. Schatz SM, Kerschbaumer RJ, Gerstenbauer G, Kral M, Dorner F, Schei- germ cells: the role of the germ line in zebrafish sex differentiation. Proc flinger F. Higher expression of Fab antibody fragments in a CHO cell line Natl Acad Sci USA 2005; 102:4074–4079. at reduced temperature. Biotechnol Bioeng 2003; 84:433–438. 43. Siegfried KR, Nusslein-Volhard C. Germ line control of female sex deter- 64. Wang Y, Ge W. Cloning of zebrafish ovarian carbonyl reductase-like 20β- mination in zebrafish. Dev Biol 2008; 324:277–287. hydroxysteroid dehydrogenase and characterization of its spatial and tem- 44. Houwing S, Kamminga LM, Berezikov E, Cronembold D, Girard A, van poral expression. Gen Comp Endocrinol 2002; 127:209–216. den Elst H, Filippov DV, Blaser H, Raz E, Moens CB, Plasterk RHA, 65. Pang Y, Ge W. Gonadotropin regulation of activin βA and activin type Hannon GJ et al. A role for Piwi and piRNAs in germ cell maintenance IIA receptor expression in the ovarian follicle cells of the zebrafish, Danio and transposon silencing in zebrafish. Cell 2007; 129:69–82. rerio. Mol Cell Endocrinol 2002; 188:195–205. 45. Saito T, Goto-Kazeto R, Arai K, Yamaha E. Xenogenesis in teleost fish 66. Poon SK, So WK, Yu X, Liu L, Ge W. Characterization of inhibin α through generation of germ-line chimeras by single primordial germ cell subunit (inha) in the zebrafish - Evidence for a potential feedback loop transplantation. Biol Reprod 2008; 78:159–166. between the pituitary and ovary. Reproduction 2009; 138:709–719. 46. Nishimura T, Sato T, Yamamoto Y, Watakabe I, Ohkawa Y, Suyama M, 67. Sandra GE, Norma MM. Sexual determination and differentiation in Kobayashi S, Tanaka M. Foxl3 is a germ cell-intrinsic factor involved in teleost fish. Rev Fish Biol Fisher 2010; 20:101–121. Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021 sperm-egg fate decision in medaka. Science 2015; 349:328–331. 68. Halm S, Ibanez AJ, Tyler CR, Prat F. Molecular characterisation of 47. Dranow DB, Tucker RP, Draper BW. Germ cells are required to maintain growth differentiation factor 9 (gdf9) and bone morphogenetic protein a stable sexual phenotype in adult zebrafish. Dev Biol 2013; 376:43–50. 15 (bmp15) and their patterns of gene expression during the ovarian re- 48. McPherron AC, Lee SJ. GDF-3 and GDF-9: two new members of the productive cycle in the European sea bass. Mol Cell Endocrinol 2008; transforming growth factor-β superfamily containing a novel pattern of 291:95–103. cysteines. JBiolChem1993; 268:3444–3449. 69. Su YQ, Wu X, O’Brien MJ, Pendola FL, Denegre JN, Matzuk MM, Eppig 49. Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM. Paracrine JJ. Synergistic roles of BMP15 and GDF9 in the development and function actions of growth differentiation factor-9 in the mammalian ovary. Mol of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte- Endocrinol 1999; 13:1035–1048. granulosa cell regulatory loop. Dev Biol 2004; 276:64–73. 50. Liu L, Ge W. Growth differentiation factor 9 and its spatiotemporal ex- 70. Mazerbourg S, Klein C, Roh J, Kaivo-Oja N, Mottershead DG, Korchyn- pression and regulation in the zebrafish ovary. Biol Reprod 2007; 76:294– skyi O, Ritvos O, Hsueh AJ. Growth differentiation factor-9 signaling 302. is mediated by the type I receptor, activin receptor-like kinase 5. Mol 51. Dong JW, Albertini DF, Nishimori K, Kumar TR, Lu NF, Matzuk MM. Endocrinol 2004; 18:653–665. Growth differentiation factor-9 is required during early ovarian folliculo- 71. Tse AC, Ge W. Spatial localization of EGF family ligands and receptors in genesis. Nature 1996; 383:531–535. the zebrafish ovarian follicle and their expression profiles during follicu- 52. Vitt UA, Hayashi M, Klein C, Hsueh AJ. Growth differentiation factor-9 logenesis. Gen Comp Endocrinol 2010; 167:397–407. stimulates proliferation but suppresses the follicle-stimulating hormone- 72. Wang Y, Ge W. Spatial expression patterns of activin and its signaling induced differentiation of cultured granulosa cells from small antral and system in the zebrafish ovarian follicle: evidence for paracrine action of preovulatory rat follicles. Biol Reprod 2000; 62:370–377. activin on the oocytes. Biol Reprod 2003; 69:1998–2006. 53. Elvin JA, Yan C, Matzuk MM. Growth differentiation factor-9 stimu- 73. Wang Y, Ge W. Developmental profiles of activin βA, βB, and follistatin lates progesterone synthesis in granulosa cells via a prostaglandin E2/EP2 expression in the zebrafish ovary: evidence for their differential roles dur- receptor pathway. Proc Natl Acad Sci USA 2000; 97:10288–10293. ing sexual maturation and ovulatory cycle. Biol Reprod 2004; 71:2056– 54. Kaivo-Oja N, Bondestam J, Kamarainen M, Koskimies J, Vitt U, Cranfield 2064. M, Vuojolainen K, Kallio JP, Olkkonen VM, Hayashi M, Moustakas A, 74. Hsiao CD, Tsai HJ. Transgenic zebrafish with fluorescent germ cell: a use- Groome NP et al. Growth differentiation factor-9 induces Smad2 activa- ful tool to visualize germ cell proliferation and juvenile hermaphroditism tion and inhibin B production in cultured human granulosa-luteal cells. in vivo. Dev Biol 2003; 262:313–323. J Clin Endocrinol Metab 2003; 88:755–762. 75. Fenske M, Segner H. Aromatase modulation alters gonadal differentiation 55. McGrath SA, Esquela AF, Lee S-J. Oocyte-specific expression of in developing zebrafish (Danio rerio). Aquat Toxicol 2004; 67:105–126. growth/differentiation factor-9. Mol Endocrinol 1995; 9:131–136. 76. Schulz RW, Bogerd J, Male R, Ball J, Fenske M, Olsen LC, Tyler CR. 56. Mandon-Pepin B, Oustry-Vaiman A, Vigier B, Piumi F, Cribiu E, Cotinot Estrogen-induced alterations in amh and dmrt1 expression signal for dis- C. Expression profiles and chromosomal localization of genes controlling ruption in male sexual development in the zebrafish. Environ Sci Technol meiosis and follicular development in the sheep ovary. Biol Reprod 2003; 2007; 41:6305–6310. 68:985–995. 77. Jorgensen A, Morthorst JE, Andersen O, Rasmussen LJ, Bjerregaard P. Ex- 57. Eckery DC, Whale LJ, Lawrence SB, Wylde KA, McNatty KP, Juengel pression profiles for six zebrafish genes during gonadal sex differentiation. JL. Expression of mRNA encoding growth differentiation factor 9 and Reprod Biol Endocrinol 2008; 6:25. bone morphogenetic protein 15 during follicular formation and growth 78. Zhang Y, Li F, Sun D, Liu J, Liu N, Yu Q. Molecular analysis shows in a marsupial, the brushtail possum (Trichosurus vulpecula). Mol Cell differential expression of R-spondin1 in zebrafish (Danio rerio) gonads. Endocrinol 2002; 192:115–126. Mol Biol Rep 2011; 38:275–282. 58. Baron D, Houlgatte R, Fostier A, Guiguen Y. Large-scale temporal gene 79. Sreenivasan R, Jiang J, Wang X, Bartfai R, Kwan HY, Christoffels A, expression profiling during gonadal differentiation and early gametogen- Orban L. Gonad differentiation in zebrafish is regulated by the canonical esis in rainbow trout. Biol Reprod 2005; 73:959–966. wnt signaling pathway. Biol Reprod 2014; 90:45. 59. Chen W, Ge W. Gonad differentiation and puberty onset in the zebrafish: 80. Spicer LJ, Aad PY, Allen DT, Mazerbourg S, Payne AH, Hsueh AJ. Evidence for the dependence of puberty onset on body growth but not age Growth differentiation factor 9 (GDF9) stimulates proliferation and in- in females. Mol Reprod Dev 2013; 80:384–392. hibits steroidogenesis by bovine theca cells: Influence of follicle size on 60. Chen W, Ge W. Ontogenic expression profiles of gonadotropins (fshb and responses to GDF9. Biol Reprod 2008; 78:243–253. lhb) and growth hormone (gh) during sexual differentiation and puberty 81. Orisaka M, Jiang JY, Orisaka S, Kotsuji F, Tsang BK. Growth dif- onset in female zebrafish. Biol Reprod 2012; 86:73. ferentiation factor 9 promotes rat preantral follicle growth by up- 61. So WK, Kwok HF, Ge W. Zebrafish gonadotropins and their receptors: II. regulating follicular androgen biosynthesis. Endocrinology 2009; 150: Cloning and characterization of zebrafish follicle-stimulating hormone and 2740–2748. luteinizing hormone subunits - their spatial-temporal expression patterns 82. Orisaka M, Orisaka S, Jiang JY, Craig J, Wang YF, Kotsuji F, Tsang and receptor specificity. Biol Reprod 2005; 72:1382–1396. BK. Growth differentiation factor 9 is antiapoptotic during follicular de- 62. Kozak M. At least six nucleotides preceding the AUG initiator codon velopment from preantral to early antral stage. Mol Endocrinol 2006; enhance translation in mammalian cells. J Mol Biol 1987; 196:947–950. 20:2456–2468. GDF9 in zebrafish gonadal differentiation, 2017, Vol. 96, No. 2 413

83. Kluver N, Pfennig F, Pala I, Storch K, Schlieder M, Froschauer A, Gutzeit inhibiting substance during gonadal sex differentiation in Japanese ﬂoun- HO, Schartl M. Differential expression of anti-Mullerian hormone (amh) der, Paralichthys olivaceus. Biochem Biophys Res Commun 2004; 322: and anti-Mullerian hormone receptor type II (amhrII) in the teleost 508–513. medaka. Dev Dyn 2007; 236:271–281. 85. Ge W. Intrafollicular paracrine communication in the zebraﬁsh ovary: 84. Yoshinaga N, Shiraishi E, Yamamoto T, Iguchi T, Abe S, Kitano T. the state of the art of an emerging model for the study of vertebrate Sexually dimorphic expression of a teleost homologue of Mullerian folliculogenesis. Mol Cell Endocrinol 2005; 237:1–10. Downloaded from https://academic.oup.com/biolreprod/article/96/2/401/2968017 by guest on 27 September 2021