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Developmental tracing of oocyte development in gonadal soma-derived factor deficiency medaka (Oryzias latipes) using a transgenic approach
Accepted Manuscript Developmental tracing of oocyte development in gonadal somaderived factor deficiency medaka (Oryzias latipes) using a transgenic approach
Guijun Guan, Kaiqing Sun, Xi Zhang, Xiaomiao Zha, Mingyou Li, Yan Yan, Yunzhi Wang, Jianbin Chen, Meisheng Yi, Yunhan Hong PII: DOI: Reference:
S0925-4773(16)30132-0 doi: 10.1016/j.mod.2016.12.006 MOD 3436
To appear in:
Mechanisms of Development
Received date: Revised date: Accepted date:
29 September 2016 25 December 2016 26 December 2016
Please cite this article as: Guijun Guan, Kaiqing Sun, Xi Zhang, Xiaomiao Zha, Mingyou Li, Yan Yan, Yunzhi Wang, Jianbin Chen, Meisheng Yi, Yunhan Hong , Developmental tracing of oocyte development in gonadal soma-derived factor deficiency medaka (Oryzias latipes) using a transgenic approach. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mod(2017), doi: 10.1016/j.mod.2016.12.006
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ACCEPTED MANUSCRIPT Developmental
tracing
of
oocyte
development
in
gonadal
soma-derived factor deficiency medaka (Oryzias latipes) using a transgenic approach
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Guijun Guan1*#, Kaiqing Sun1#, Xi Zhang2, Xiaomiao Zha3, Mingyou Li1,Yan Yan2, Yunzhi Wang2, Jianbin Chen2, Meisheng Yi4 and Yunhan Hong2 1
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Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China; 2Reproductive Endocrinology & Infertility, Sun Yat-sen Memorial Hospital, Sun Yat-sen University Yanjiang Road 107, Guangdong 510120, China; 3Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore; 3Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Zhuhai Key Laboratory of Marine Bioresources and Environment, School of Marine Sciences, Sun Yat-sen University, Guangzhou, China
Running title: Gsdf essential for folliculogenesis in medaka
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Key words: meiosis; gonadal soma-derived factor (Gsdf); folliculogenesis; TGF-beta
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superfamily
To whom correspondence should be addressed:
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Dr. Guijun Guan
[email protected] College of Fisheries and Life Sciences Shanghai Ocean University HuchengHuan Road 999, Shanghai 201306, China Tel/Fax: +86 21 61900499 Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Gonadal soma-derived factor (gsdf) is reported to be a male initiator in medaka based on loss- and gain- of function via targeted disruption, or transgenic over-expression. However, little is known about how gsdf promotes undifferentiated gonad entry into male pathways or prevents entry into the female pathway. We
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utilized a visible folliculogenesis system with a reporter cassette of dual-color fluorescence expression to identify difference between oocyte development from
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wildtype and gsdf deficiency medaka. A red fluorescent protein (RFP) is driven by a major component of the synaptonemal complex (SYCP3) promoter which enables
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RFP expression solely in oocytes after the onset of meiosis, and a histone 2b-EGFP fused protein (H2BEGFP) under the control of an elongation factor (EF1α) promoter,
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wildly used as a mitotic reporter of cell cycle. This mitosis-meiosis visible switch revealed that early meiotic oocytes present in gsdf deficiency were more than those in
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wildtype ovaries, corresponding to the decrease of inhibin expression detected by real-time qPCR analysis, suggesting gsdf is tightly involved in the process of medaka
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oocyte development at early stage.
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ACCEPTED MANUSCRIPT Introduction In spite of the enormous diversity of sex-determining genes which triggers undifferentiated gonads to form ovaries or testes [1], the cascade of cellular differentiation and gonadal organization are well-conserved among vertebrates including fish [1, 2]. For instance, doublesex- and mab-3 domain related
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transcriptional factor 1 (dmrt1) and SRY-related high mobility group-box gene 9 (sox9) genes are essential for vertebrate testicular differentiation from fish to mammals [3, 4].
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Gonadal soma-derived factor (gsdf) restricts expression in gonadal somatic cells in teleosts [5], and targeted disruption of gsdf in medaka feminizes XY fish in the
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presence of the master sex determining gene, the DM domain gene on chromosome Y (dmy/dmrt1bY) [6, 7]. This was reported by two groups independently using a
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customized zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALENs) approach [8, 9]. Thus, bi-potential gonads enter female pathways
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for ovarian development without gsdf irrespective of the presence or absence of dmy. In contrast, addition of gsdf is sufficient for masculinization in the absence of dmy in
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Oryzias latipes [8]. gsdf is the sex determining gene instead of dmy in the closely
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related O. luzonensis [10]. Therefore, gsdf is essential for the initiation of testicular differentiation via gain- and loss of function. Silencing gsdf but not dmrt1 was reported to cause morphological changes in early embryonic medaka gonads, and
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germ cells were increased to an ovarian level [8, 11], indicating that gsdf suppresses primordial germ cell (PGC) proliferation and the commencement of the male pathway
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prior to dmrt1 functionality in medaka [8]. Teleost gsdf was first identified as an orthologue of mammalian bone morphogenetic protein-7 (BMP7) from a complementary DNA (cDNA) library subtraction using cDNAs derived from somatic cells at the genital ridge versus those without a genital ridge in rainbow trout embryos [12]. Trout gsdf suppresses PGC proliferation via antisense gripNA oligonucleotides, which translationally inhibit Gsdf protein products. These data were supported by evidence from in vitro experiments that type-A spermatogonia proliferation relied on recombinant Gsdf in a dose-dependent manner and spermatogonia proliferation was blocked by antiserum 3
ACCEPTED MANUSCRIPT against Gsdf [12]. Germ cell development appeared sexual dimorphism at early stage when male germ cell commits mitotic proliferation, whilst female germ cell enters meiosis [13]. However, how germ cells arise from PGCs in early undifferentiated gonads to create a female germline is still unclear. In general among vertebrate species, female germline is the acquisition of
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femaleness, and follicles are functional ovarian units comprising of oocytes surrounded by one or more layers of somatic cells [14-16]. Folliculogenesis is a
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progressive and extensive series of developmental stages, initiated from primordial follicles with an oocyte surrounded by a single layer of flattened pre-granulosa cells.
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In mammals, most follicles degenerate in pre- or post-natal life, and few surviving primordial follicles are recruited to the growing follicle population, and then fewer
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will ultimately ovulate [17, 18]. Follicle development requires tight communication between germline and somatic cells including granulosa and theca cells, which
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nourish germ cells in an autocrine/paracrine manner to coordinate and control oocyte fate in vertebrates [14, 19, 20]. Transforming growth factor-β (TGF-β) superfamily
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members are required for mammalian oocyte development [21]. BMP-4 and BMP-7
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are members of TGF- family expressed by ovarian stromal cells and/or theca cells, and they have been implicated as positive regulators of the primordial-to-primary follicle transition during diminished ovulation in sheep and cows but this process is
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species-dependent and different in poly-ovulatory species such as rat [22]. Other family members, activin and inhibin also have been demonstrated to play roles with
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BMPs in follicle development in the zebrafish [23]. To explore roles of gsdf in sexual differentiation in medaka, we compared ovarian differentiation at molecular and cellular levels between wildtype XX females with intact gsdf, XX and XY females with truncated Gsdf expression. Developmental oocytes were monitored by a transgenic approach using a dual-color reporter cassette expression of green and red fluorescent proteins under the promoter regulation of a mitosis-meiosis switcher, and relevant gene expression was quantified with real-time PCR. 4
ACCEPTED MANUSCRIPT Materials and Methods Fish Medaka fish were housed in re-circulating systems at 26-28 °C, and a light-dark cycle of 14-h dayliggsdf(+/-) and 10-h darkness. Fish handling was performed in strict compliance with the guidance of the Committee for Laboratory Animal Research at
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Shanghai Ocean University and National University of Singapore. Oocyte developmental stages were monitored with histological classification according to the
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literature [24]. Phenotypic sex was assessed using secondary sex characteristics (dorsal and anal fin shape) and confirmed with gonad biopsy of 6-month adult fish.
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Genotyping was performed with PCR and primers of PG17.5-17.6 to amplify a dmy-specific fragment and a fragment from dmrt1 of medaka [7]. Genotyping of the
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gsdf allele was performed with primers as previously described [8]. Generation of intact and mutant Gsdf lines with green and red signals in ovary
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A pGRY reporter cassette was constructed in accordance with published methods [25]. pGRY contains a synaptonemal complex component (SYCP3) promoter (2.1kb
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in length), which was PCR amplified and flanked with SacI and EcoRI restriction
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sites. This was inserted in front of RFP (pDsRed-N1, Invitrogen, Carlsbad, CA, USA) to drive RFP expression for cytoplasmic labeling of cells in meiosis-prophase. A 2.5 kb EFH2BGFP fragment containing a medaka elongation factor 1α (EF1α) promoter
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[26], driven by human histone H2B and GFP fusion product (pBOS-H2BGSDF, BD Biosciences, San Jose, USA) for nuclear labeling was used because this approach
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allows ubiquitous expression of the fusion protein between human histone H2B and GFP under EF1α promoter regulation. In addition, medaka protamine fused to yellow fluoresent protein (mprmYFP) driven by protamine promoter (2 kb of 5’ UTR and 812 bp of 3’ UTR fragments in length) was ahead of SYCP4RFP and designed for both monitoring of a mitosis-meiosis switch in ovary, and an histone-protamine switch in spermatogenesis via green-red-yellow color transition in testis [27]. Primers used for vector construction was listed in Table S1. This multi-color construct was transfected into haploid embryonic stem cells, and nuclearly transferred into unfertilized eggs to generate a transgenic GRY line according to published reports 5
ACCEPTED MANUSCRIPT [25]. A homozygous line was identified from transgenic fish crossed with wildtype (gsdf +/+) fish to produce 100% GFP-positive offspring. A stable homozygous line was obtained by crossing identified homozygote fish. Male and female gsdf heterozygotic fish were mated with GRY transgenic fish to obtain heterozygotic (gsdf +/-) fish with green and red (GR) signals expression in gonads. Crossing these
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siblings ultimately generated homozygous gsdf -/- GRY XX and XY female fish. Fertility test
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Three pairs of adult males and females from Tg lines were fertility tested as a control group in pair-wise mating for two weeks. These Tg males were subsequently used to
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breed with gsdf KO females in low fertility and sterile group (n=3) in pair-wise mating for two weeks as testing groups. Cumulative production of ovulated and
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fertilized eggs were counted and listed in Table 1 and Table S3. Infertile females were ultimately validated by gonad biopsy with no mature oocytes formation.
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Histological analysis
Gonads were fixed with Bouin’s solution, dehydrated in a graded series of ethanol and
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subsequently embedded into paraffin blocks (Sigma, St. Louis, MO), or 4%
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paraformaldehyde fixed in phosphate buffer (4% PFA; PBS pH 7.4), embedded directly into tissue freezing medium (Leica, Germany) and 5-μm cross sections were cut (Leica RM2265 microtome, Germany) and stained with hematoxylin and eosin
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(H&E) for histological analysis (Jiancheng Biotech. China), or subjected to cryosectioning (Leica CM1950, Germany) for fluorescent observation. All
fish.
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morphological data were verified by similar observations from at least 3 individual
Fluorescent photography Fluorescent signals of GFP and RFP were captured with a Nikon Ds-Ri2 system under a stereomicroscope (Nikon, Japan), or photographed with confocal microscopy (Zeiss LSM 710, Carl Zeiss Germany). Quantitative real-time PCR (qPCR) Total RNA was extracted from gonads and reverse transcribed into first-strand cDNA with AMV reverse transcriptase (Takara, Dailian, China) and oligo-dT(18) primers 6
ACCEPTED MANUSCRIPT according to manufacturer instructions. Primers used for qPCR have been used previously [28] and appeared in Table S2. qPCR was carried out using cDNA as template and SYBR Fast qPCR mix (Takara, Dailian, China) and a 7500 real-time PCR system (Applied Biosystems, Foster City, CA) to measure fluorescence. Thermal cycling conditions were 1 cycle of 2 min at 95 °C followed by 40 cycles of 15 sec denaturation at 95 °C and a 34 sec extension at 64 °C. Relative abundance of each -actin was used to
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sample gene was evaluated using the formula: R=2- △△ Ct,
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normalize expression values. Statistical comparisons were evaluated by ANOVA followed by a pair-wise Student’s t-test (p<0.05 was considered to statistically
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significant unless specified). Data are means of three to five independent experiments.
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ACCEPTED MANUSCRIPT Results Abnormal gonad development in Gsdf deficient fish Normal XX females and XY males have typical secondary sexual characteristics with respect to the shape of anal fins which are under the regulation of sex determining gene dmy/dmrt1bY [6, 7] and sex hormones regulation through the
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hypothalamic-pituitary-gonadal (HPG) axis. In gsdf knockout (gsdf KO) fish, anal fin shapes resembled normal XX females (Figure 1a), irrespective of dmy or an XX or
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XY genotype. Moreover, all gsdf KO fish (100%) have larger abdominal ovaries than normal adult females, containing a giant ovary greater than one in normal females
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(Figure 1b) [8].
Normal XX female has an asynchronous-type ovary with developmental oocytes
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in various growth phases from small primordial to larger follicles in medaka (Figure 1c-c’) [29], whereas normal XY gonad develops into a testis (Figure 1b, 1d-d’) under
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the control of dmy/dmrt1bY [6, 7]. In gsdf KO XY mutants, the gonad develops into an ovary, in spite of the presence of dmy [8] (Figure 1e, e’). Folliculogenesis was
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damaged as numerous small and previtellogenic oocytes (Pvo), but few vitellogenic
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oocytes (Vo) present in gsdf KO fish, therefore resulted in low fertility with rare ovulation or absolute infertile (Figure 1e). Normal females usually give 20-50 eggs per day and ovulate daily for several months to more than one year. Oocyte
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maturation was largely prohibited in gsdf KO fish. The fertility of Gsdf KO fish (3 to 4-month mature adults) was tested by pair-wise mating. Only five from 38 tested Gsdf
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KO fish gave eggs in a reproductive period shorter than normal females. Numbers of ovulated eggs from gsdf KO fish were about two third fold lower than the normal females, with the fertilization rate much lower to one half of the normal females, implying the lack of Gsdf caused the lower fecundity and fertilization directly and/or indirectly (Table 1).
Monitoring oocyte development through a transgenic strategy H2BGFP was first designed to label the chromatin architecture with GFP signal which has been demonstrated to be wildly useful as a mitotic imaging of cell cycle [30, 31]. 8
ACCEPTED MANUSCRIPT Medaka translation elongation factor 1α (EF-1α) promoter can drive GFP expression in almost all embryonic tissues from the early gastrula stage [26]. EF1αH2BGFP was designed with EF1α controlling H2BGFP to ensure ubiquitous nuclear GFP signal expression with the assistance of nuclear localization signal (NLS) [25]. It enables a visualization of cytological changes in vivo according to fluorescent reporter
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expression. We modified the reporter by adding a SYCP3 promoter driving RFP and protamine promoter driving YFP with a map of vector illustrated in Figure 2a-b.
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Oocytes entering meiosis were monitored under a fluorescent microscope by the mitosis-meiosis switch of H2BGFP-SYCP3RFP based on green-red transition (Figure
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2-3). Light or dark orange was obtained by a merged imaging of green-red channels. Although the light orange resembles yellow color which is difficult to be
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distinguished from YFP signals within pGRY report-cassett. Since mPRM-YFP was ultilized for monitoring germ cells in post meiotic phase which was only detected in
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testis based on enhancer activity of 5’- and 3’-cis-element conserved from mammals to medaka protamine genes [27, 32]. Only gsdf roles in oocyte development was
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addressed with assistance of this transgenic line, hence we focused on green-red color
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transition which reflected mitosis-meiosis exchange bona fide in oocyte development. As expected, H2BGFP signals were detected throughout early embryogenesis of transgenic fish revealed in Figure 2c. In 10 days after hatching (10 dah) fry, most
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germ cells were green within the gonad of XY males (Figure 2d-d’) and few were red or light yellow (see arrows) in gonads of XX females, confirming that these oocytes
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entered meiosis in XX fry (Figure 2e-e’). In normal Tg adult ovaries, oogonia consists a large nucleus with weak GFP signals in gonia hardly being detected before meiosis phase (Figure 3b’-b” asterisks). After the onset of meiosis, oogonia turned to be primitive and primary oocytes in red with the expression of RFP under SYCP3 regulation (Po in Figure 3b”), then a light orange color observation due to the combination of GFP-RFP expression in these germ cells (Oc in Figure 3b”). The red color was progressively fainter and disappeared in the cytoplasm of growing oocytes during successively developmental stages, and only green GFP were observed in the nuclei of previtellogenic oocytes onwards vitellognic and fully mature stages (Pvo and 9
ACCEPTED MANUSCRIPT Vo in Figure 3a”). Therefore this fluorescence reporter cassette provides a good tool to monitor the whole process of oocyte development from the onset of meiosis onwards full maturation in medaka.
Abnormal folliculogenesis displayed by gsdf KO Tg fish line
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In order to define any difference between Gsdf present and absent female ovaries during folliculogenesis, we crossed the transgenic line of GRY with gsdf KO fish. The
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process of oocyte development was monitored precisely by these transgenic fluorescence signals through transgenic expression of the reporter cassette in normal
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females with intact Gsdf or in abnormal females with Gsdf null mutants. According to the criteria of oocyte stage classification described by Iwamatsu’s group [29], all
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developmental stages from primordial to mature oocytes present in normal XX female ovaries (Figure 4a). However, few mature oocytes were obtained in gsdf KO females
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with lower fertility (Figure 4b). No mature oocytes were observed in infertile gsdf KO fish (Figure 4c), indicating a severe prohibition of oocyte growth in gsdf deficiency
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females. In contrast of large follicle (LF) and primordial follicles (PF, in yellow
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resulted from RFP and GFP signal combination) present in normal ovaries (Figure 4a’-a”), numerous meiotic oocytes in dark orange and some oocytes in light orange color were observed in both gsdf KO with lower fertility and absolutely infertile
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female ovaries (Figure 4b’-b”; 4c’-c” arrowheads). These results implied that oogonia were able to enter meiosis, but oocyte growth was largely restrained after the onset of
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meiosis in gsdf KO mutants. Expression profile of key genes displaying in wildtype and mutant fish To investigate molecular events underlying gsdf-deficient XY feminization, expression of key genes essential for ovary or testis development were measured with qPCR assay using total RNAs as templates from gonads of 3 to 4 month-old adults. Genes of cytochrome P450 19a1 gene (cyp19a1) and 17-hydroxylase (cytochrome P450 17A, cyp17a) are molecular markers that are chiefly expressed in ovary or testis [33]. Sample RNAs were extracted and mixed from five adults of each group to avoid individual variation. qPCR data confirm predominant expression of cyp19a1 and 10
ACCEPTED MANUSCRIPT cyp17a in ovary or testis as expected (Figure 5a-b). Expression patterns in heterozygote males and females are similar to wildtype fish, in consistent with a similar fertility and a similar histological morphology compared to wildtype testes and ovaries (Figure S1). High cyp19a1 but low cyp17a expression was detected in gonads of gsdf null XX-XY females (Figure 5a-b), resembling to the complete ovary
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developed in gsdf KO fish. Expression of inhibin A (inha) had a similar pattern to that of cyp17a, higher in males but lower in females, indicating that these genes are
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involved in male testicular differentiation in medaka (Figure 5b-d). In contrast to the severe inhibition of inha in female ovary, there were no statistically significant
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differences between testis and ovarian expression, suggesting that expression of activin A (actBA) may not be subjected to gsdf regulation (Figure 5c). It has been
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indicated that smaller follicles produce more activin, whilst developed follicles secrete more inhibin [21]. Decrease of inhibin expression in gsdf-/- medaka was consistent
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heterozygous ovaries (Figure 5d).
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with rare developed follicles present in gsdf-/- mutants in contrast to wildtype and
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ACCEPTED MANUSCRIPT Discussion Requirement of gsdf in normal folliculogenesis Gsdf is conserved in teleost but absent in tetrapods [5] and expression of a gsdf ortholog is correlated to testicular differentiation in protogynous fish [34, 35], supporting an essential role for testis development. XY feminization and XX
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masculinization were obtained via loss- and gain-of function approaches in medaka and tilapia [8-10, 36, 37], suggesting a functionally conserved role of gsdf in testicular
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differentiation across species. Nevertheless, Gsdf is also essential for ovary development. We found that targeted disruption of gsdf resulted in high sterility
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(33/38 of gsdf KO mutants, 86.8%), which was caused by abnormal folliculogenesis with restrained oocyte growth and maturation in medaka. It has been reported that the
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TGF-β superfamily is critical for regulation of oocyte development, including recruitment, selection and growth of follicles from the primordial stage to ovulation in
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mammals. TGF-β is secreted by somatic (granulosa and thecal cells) and/or oocytes [21]. Granulosa and thecal cells in fish are functionally homologous to those of
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mammals [38], which support and nurture oocytes growth in an autocrine/paracrine
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manner to the microenvironment. Granulosa cells promote oocyte development and survival via thecal cells that supply granulosa cells with steroids such as the estrogenic precursors androstenedione. Medaka oogenic stages include formation of
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the primordial follicle, recruitment of selectable follicles from the growing pool to form primary, secondary, and tertiary follicles, and ovulation and subsequent
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formation of a corpus luteum (CL) [14]. Although regulators of initial stages in which germ cell clusters or cysts are broken up during follicle development have not been identified, the interaction between oocytes and somatic cells is apparently important for the formation of primordial follicles. It was presumed that absence of gsdf expression in somatic cells caused granulosa cells abnormal differentiation and/or development, therefore providing a poor nourishment for oocytes growth and rendering a numerous small follicles scatting in ovaries, with rare oocytes developed and reached to the mature stage (Figure 1 and 4). It was interesting that medaka anti-Mullerian hormone (amh) inhibited type A spermatogonia proliferation in the 12
ACCEPTED MANUSCRIPT testis but not oogonia proliferation in the ovary [39]. Hyperproliferation of self-renewing type A germ cells and XY feminization was also noted in loss-of-function mutants of its receptor (Amhr2/hotei) [39]. An alternative or redundancy function is considerable existing between amh and gsdf, as they both belong to TGF-β superfamily members and structurally share the conserved TGF-
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domain. The redundancy of amhr2 and TGF-β receptors may explain this although receptor specifically binding to Gsdf has yet to be identified in teleosts.
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Early meiotic arrest associated with gsdf deficiency
Cellular and molecular processes that accompany female germ cells have
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primarily been characterized in Drosophila and the mouse [40, 41]. Nevertheless, acquisition of femaleness in vertebrate female germlines have been investigated using
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this model species [14], after the successful identification of germline stem cells in medaka [42]. Similar to birds and mammals, after PGC differentiation, oogonia
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undergo mitotic proliferation to leave daughter cells connected by intercellular bridges, forming clusters or nests surrounded by somatic pre-follicular or supporting
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cells. Female germ cells can arrange the tubular structure of supporting cells and
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maintain feminization [15]. Also, in medaka and zebrafish, the maintenance of female-specific estrogen-producing cells depends on the presence of germ cells [16, 43]. Therefore, the effect of abnormal oocytes on surrounding follicular cells
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differentiation should not be excluded. Folliculogenesis is also regulated by release of pituitary hormone, FSH and LH
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[44] which are well conserved in vertebrates. Fish folliculogenesis is a unique process of vitellogenesis with oocyte expansion, instead of activation and formation of fluid-filled antra as seen in mammals. The relationship between pituitary activity and oocyte maturation has been depicted in medaka during oogenesis in ultrastructural detail, revealing the Balbiani Body in late oocyte stages after the appearance of synaptonemal complexes as visualized under a electron microscope [45]. We observed many primitive oocytes (Po) at early meiotic stage (dark orange) in gsdf KO ovaries (Figure 4) before the Balbiani Body formation. In contrast to normal ovaries where all stages of pre-vitellogenic, vitellogenic, and post-vitellogenic oocytes 13
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numerous Po (dark orange) but few oocytes at late-stage meiosis (light
orange) displayed in gsdf mutants, indicating oocyte development and vitellogenesis were severely restrained in the gsdf KO ovary. It is possible that the poor nutrition from abnormal granulosa cells in gsdf KO mutants could retard oocyte development. Another explanation could not be excluded that the loss of Gsdf attenuated
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suppression of germ cell proliferation, thus increased the number of germ cells entering meiosis, which subsequently caused the shortage of essential factor for
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normal folliculogenesis provided by granulosa cells. Phenomenon of previtellogenic oocytes accumulation in gsdf KO medaka corresponds to report that the mammalian
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TGF-β superfamily effects oocyte growth-arrest and promotion of germ cell proliferation [21].
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gsdf deficiency associated with vitellogenesis prohibition and failure of oocyte maturation in medaka
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In mature females, vitellogenesis is under the control of estradiol, a major endogenous estrogen in all vertebrates. Increasing estradiol stimulates hepatic
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production of vitellogenin (VTG), which is transported in the circulation to the ovary
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[46]. In gsdf KO ovaries, most oocytes are previtellogenic (Figure 1e-e’), suggesting that vitellogenesis and yolk intake are largely prohibited. Less activation of hepatic vitellogenesis in response to less serum estrogen could explain rare vitellogenic
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oocytes in gsdf KO fish. Fecundity, fertility (percent fertilized) and hatching success (percent hatched) were much lower in gsdf KO mutants, decreased to 2/3~1/2 fold of
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those in normal females (Table 1). In spite of an equivalent level of cyp19a1 expression in gsdf KO and wildtype ovaries (Figure 5a), serum estrogen was lower in gsdf null mutants than normal females (data unpublished), suggesting that gsdf influenced estrogen synthesis/conversion irrelevant to gonadal cyp19a1 expression. TGF-β signaling participates in the maintenance of the primordial follicle pool in medaka, zebrafish and mouse ovary [15, 16, 47]. Activin, one member of TGF-β subfamily, acts as an ovarian mediator of pituitary gonadotropin (s) to stimulate oocyte maturation in zebrafish [48, 49]. Although activin A was constantly expressed in gsdf deficient gonads, its counterpart inhibin expression was prohibited in gsdf KO 14
ACCEPTED MANUSCRIPT gonads, suggesting the gsdf effects on inhibin-mediated folliculogenesis in medaka, which is the fundermental pathway evolutionally conserved across species. Assessment of germ cells entering meiosis relies on morphological criteria and mechanisms underlying morphological change are unclear. The fluorescent reporter system enables the mitotic-meiotic process visible on a cellular level. Molecular and
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cytological evidence of abnormalities in gsdf KO medaka suggest that gsdf is involved in proliferation and differentiation of early primordial follicle formation. Defective
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Gsdf signaling may also affect the pituitary-gonad axis to regulate FSH-LH secretion via gonadal activin/inhibin expression feedback. Nearly all TGF-β isoform-specific
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knock-out mice die perinatally and the role of TGF-β remains unclear. It is expected
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addition of zebrafish or rodent models.
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that this gsdf KO medaka would assist us understand early follicular development in
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ACCEPTED MANUSCRIPT ACKNOWLEDGMENTS This work was supported by Shanghai University First-class Disciplines Project of Fisheries and the International Research Group Program; a grant to GG from the Natural Science Foundation of Shanghai (16ZR1415200); the National Science Foundation of China to ML (31372520); and grants to YH from the National Research Singapore
(NRF-CRP7-2010-03).
We
also
thank
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Foundation
LetPub
(www.letpub.com) for the linguistic assistance during the preparation of this
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manuscript.
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ACCEPTED MANUSCRIPT Competing interests The authors declare that they have no competing interests. Additional files and information. Additional files: Table S1. Primer sets for pGRY construction; Table S2. Primer sets for qPCR analysis; Table S3. Fertility of heterozygotes; Figure S1. Morphology
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analysis of heterozygote (gsdf+/- XX and XY) gonads.
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ACCEPTED MANUSCRIPT Figures legends Figure 1. Morphology analysis of wildtype (XX and XY) gonads and gsdf deficient mutant (XY gsdf -/-) gonad. a). Secondary sex character of gsdf -/- females resemble normal XX females in the shape of anal fins. b). An ovary was developed in XY gsdf -/- instead of testis in XY gsdf +/+ male, which is larger than ovary from XX
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gsdf +/+ female. c-e). represent HE staining gonadal sections of wildtype females, males and gsdf deficiency mutants, with low magnification present in c’-e’). Pvo,
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pre-vitellogenic oocytes ; Vo: vitellogenic oocytes.
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Figure 2. Visualization of oocyte development via a transgenic line. a). Map of pGRY vector containing tri-cistronic cassette; b). Schematic illustration of pGRY
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designed for monitoring switch of mitosis-meiosis and histone-protamine transition reflected by YFP-RFP-GFP fluorescent proteins. c). Strong GFP signals were
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observed in whole body during embryogenesis. 1, dark-eye Tg+; 2, albino i1Tg+; 3, albino i3Tg-. d-e) (high magnification in d’-e’). Germ cells labeled with GFP signals
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(arrows) were observed in 10-daf XY fry, both GFP and RFP (arrowheads) were
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detected in 10-daf XX fry. n: numbers of individuals subjected to fluorescence observation.
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Figure 3. Oocyte development monitored by a visible fluorescence Tg fish line. Cryosection images of oocytes at various stages present in transgenic fish ovary at
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low magnification (a, RFP; a’, GFP; a”, merged) and high magnification (white dotted frame) (b-b’-b”). Fluorescence in germ cells exchanged from light green to orange (merged of green and red) before and after the onset of meiosis during oocyte growth. c. An illustration of oocyte development from mitotic gonia to meiotic oocytes. GFP signals were first observed in nuclei of oogonia like germ cells (Og). After the oneset of meiosis, red signals from SYCP3 promoter driving RFP turn on in primitive oocytes (Po), become strong and then lost progressively, left nuclear GFP signals solely in nuclei of previtellogenic oocytes (Pvo) and vitellogenic oocytes (Vo). Oc: oocyte. Enlarged views framed in yellow dotted line are under illustrations. 18
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Figure 4. External and histological views of normal XX females and Gsdf KO mutants of low fertility or infertility. Ovary of wildtype XX with normal fertility present in (a), Gsdf KO mutants with low fertility in (b); and infertility in (c). Arrows stand for matured oocytes. Histological analysis of ovaries in low magnification (a’,
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b’, c’), and in high magnification (a”, b”, c”). nuclei were DAPI counter staining.
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Figure 5. Figure 5. Quantitative analyses of gene expression in wild-type (+/+) and gsdf mutant (-/-) medaka. Relative mRNA expression was normalized with the
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value of -actin in gonads, Vertical bar, Mean ± SEM (n=5). The asterisks indicate
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significant differences compared to the gsdf (+/+) O group (* P ＜ 0.05; **, P<0.01;
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***, P<0.001).
Figure S1. Morphology analysis of heterozygote (gsdf+/- XX and XY) gonads. Oogenesis and spermatogenesis of gsdf+/- XX (a) and gsdf+/- XY (b) resemble with
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normal females and males in Figure 1.
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Kwok, H.F., et al., Zebrafish gonadotropins and their receptors: I. Cloning and characterization
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Table 1. Cumulative production of eggs by transgenic (Tg-GRY) and gsdf KO-GRY Control group Strain
Low fertility group
Sterile group (*)
TgA♂x TgB♂x TgC♂x TgA♂x TgA♂x TgB♂x TgB♂x TgC♂x TgC♂x Tg D♀ Tg D♀ Tg D♀ Hm1♀
Hm2♀
Hm3♀
Hm4♀
Hm5♀
Hm6♀
249/14d 379/14d 245/14d 127/14d 246/14d 140/14d 0
0
0
Ovulated eggs/day (Average)
17.8/d
Fertilized eggs/2 weeks
249/14d 379/14d 242/14d 108/14d 76/14d
Fertilized eggs/day ( Average)
17.8/d
27.1/d
17.3/d
3 daf embryos/ fertilized eggs
17.8/d
27.1/d
242/245 108/127 53/76
100%
100%
98.8%
17.8/d
27.1/d
242/245 108/127 49/76
62/140
100%
100%
98.8%
44.2%
9.1/d
85%
85%
17.5/d
5.4/d
69.7%
10/d
0
0
0
71/14d
0
0
0
5.1/d
0
0
0
71/140
0
0
0
0
0
0
50.7%
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64.4%
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Hatched fry/fertilized eggs
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27.1/d
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Ovulated eggs/14 days
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(*) Sterility was confirmed by gonadal biopsy that no mature oocytes were observed in each ovary from three individuals listed here and all tested in sterile group.
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ACCEPTED MANUSCRIPT Highlights of paper
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1. We established a transgenic fish line which is able to distinguish oocytes from various stages of development with a multi-color fluorescent combination. 2. Targeted disruption of gsdf resulted in a severe problem in female folliculogenesis and fertility, whereas the abnormal folliculogenesis was visualized under fluorescent microscope. 3. According to a TGF- domain sharing between gsdf and inhibin, and inhibin severe suppression in gsdf KO ovary, gsdf deficient medaka provides a good model for insights of TGF- signaling pathway conserved in folliculogenesis from fish to humans.
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Guijun Guan, Kaiqing Sun, Xi Zhang, Xiaomiao Zha, Mingyou Li, Yan Yan, Yunzhi Wang, Jianbin Chen, Meisheng Yi, Yunhan Hong PII: DOI: Reference:
S0925-4773(16)30132-0 doi: 10.1016/j.mod.2016.12.006 MOD 3436
To appear in:
Mechanisms of Development
Received date: Revised date: Accepted date:
29 September 2016 25 December 2016 26 December 2016
Please cite this article as: Guijun Guan, Kaiqing Sun, Xi Zhang, Xiaomiao Zha, Mingyou Li, Yan Yan, Yunzhi Wang, Jianbin Chen, Meisheng Yi, Yunhan Hong , Developmental tracing of oocyte development in gonadal soma-derived factor deficiency medaka (Oryzias latipes) using a transgenic approach. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Mod(2017), doi: 10.1016/j.mod.2016.12.006
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ACCEPTED MANUSCRIPT Developmental
tracing
of
oocyte
development
in
gonadal
soma-derived factor deficiency medaka (Oryzias latipes) using a transgenic approach
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Guijun Guan1*#, Kaiqing Sun1#, Xi Zhang2, Xiaomiao Zha3, Mingyou Li1,Yan Yan2, Yunzhi Wang2, Jianbin Chen2, Meisheng Yi4 and Yunhan Hong2 1
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Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China; 2Reproductive Endocrinology & Infertility, Sun Yat-sen Memorial Hospital, Sun Yat-sen University Yanjiang Road 107, Guangdong 510120, China; 3Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore; 3Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Zhuhai Key Laboratory of Marine Bioresources and Environment, School of Marine Sciences, Sun Yat-sen University, Guangzhou, China
Running title: Gsdf essential for folliculogenesis in medaka
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Key words: meiosis; gonadal soma-derived factor (Gsdf); folliculogenesis; TGF-beta
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superfamily
To whom correspondence should be addressed:
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Dr. Guijun Guan
[email protected] College of Fisheries and Life Sciences Shanghai Ocean University HuchengHuan Road 999, Shanghai 201306, China Tel/Fax: +86 21 61900499 Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Gonadal soma-derived factor (gsdf) is reported to be a male initiator in medaka based on loss- and gain- of function via targeted disruption, or transgenic over-expression. However, little is known about how gsdf promotes undifferentiated gonad entry into male pathways or prevents entry into the female pathway. We
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utilized a visible folliculogenesis system with a reporter cassette of dual-color fluorescence expression to identify difference between oocyte development from
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wildtype and gsdf deficiency medaka. A red fluorescent protein (RFP) is driven by a major component of the synaptonemal complex (SYCP3) promoter which enables
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RFP expression solely in oocytes after the onset of meiosis, and a histone 2b-EGFP fused protein (H2BEGFP) under the control of an elongation factor (EF1α) promoter,
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wildly used as a mitotic reporter of cell cycle. This mitosis-meiosis visible switch revealed that early meiotic oocytes present in gsdf deficiency were more than those in
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wildtype ovaries, corresponding to the decrease of inhibin expression detected by real-time qPCR analysis, suggesting gsdf is tightly involved in the process of medaka
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oocyte development at early stage.
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ACCEPTED MANUSCRIPT Introduction In spite of the enormous diversity of sex-determining genes which triggers undifferentiated gonads to form ovaries or testes [1], the cascade of cellular differentiation and gonadal organization are well-conserved among vertebrates including fish [1, 2]. For instance, doublesex- and mab-3 domain related
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transcriptional factor 1 (dmrt1) and SRY-related high mobility group-box gene 9 (sox9) genes are essential for vertebrate testicular differentiation from fish to mammals [3, 4].
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Gonadal soma-derived factor (gsdf) restricts expression in gonadal somatic cells in teleosts [5], and targeted disruption of gsdf in medaka feminizes XY fish in the
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presence of the master sex determining gene, the DM domain gene on chromosome Y (dmy/dmrt1bY) [6, 7]. This was reported by two groups independently using a
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customized zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALENs) approach [8, 9]. Thus, bi-potential gonads enter female pathways
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for ovarian development without gsdf irrespective of the presence or absence of dmy. In contrast, addition of gsdf is sufficient for masculinization in the absence of dmy in
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Oryzias latipes [8]. gsdf is the sex determining gene instead of dmy in the closely
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related O. luzonensis [10]. Therefore, gsdf is essential for the initiation of testicular differentiation via gain- and loss of function. Silencing gsdf but not dmrt1 was reported to cause morphological changes in early embryonic medaka gonads, and
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germ cells were increased to an ovarian level [8, 11], indicating that gsdf suppresses primordial germ cell (PGC) proliferation and the commencement of the male pathway
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prior to dmrt1 functionality in medaka [8]. Teleost gsdf was first identified as an orthologue of mammalian bone morphogenetic protein-7 (BMP7) from a complementary DNA (cDNA) library subtraction using cDNAs derived from somatic cells at the genital ridge versus those without a genital ridge in rainbow trout embryos [12]. Trout gsdf suppresses PGC proliferation via antisense gripNA oligonucleotides, which translationally inhibit Gsdf protein products. These data were supported by evidence from in vitro experiments that type-A spermatogonia proliferation relied on recombinant Gsdf in a dose-dependent manner and spermatogonia proliferation was blocked by antiserum 3
ACCEPTED MANUSCRIPT against Gsdf [12]. Germ cell development appeared sexual dimorphism at early stage when male germ cell commits mitotic proliferation, whilst female germ cell enters meiosis [13]. However, how germ cells arise from PGCs in early undifferentiated gonads to create a female germline is still unclear. In general among vertebrate species, female germline is the acquisition of
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femaleness, and follicles are functional ovarian units comprising of oocytes surrounded by one or more layers of somatic cells [14-16]. Folliculogenesis is a
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progressive and extensive series of developmental stages, initiated from primordial follicles with an oocyte surrounded by a single layer of flattened pre-granulosa cells.
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In mammals, most follicles degenerate in pre- or post-natal life, and few surviving primordial follicles are recruited to the growing follicle population, and then fewer
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will ultimately ovulate [17, 18]. Follicle development requires tight communication between germline and somatic cells including granulosa and theca cells, which
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nourish germ cells in an autocrine/paracrine manner to coordinate and control oocyte fate in vertebrates [14, 19, 20]. Transforming growth factor-β (TGF-β) superfamily
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members are required for mammalian oocyte development [21]. BMP-4 and BMP-7
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are members of TGF- family expressed by ovarian stromal cells and/or theca cells, and they have been implicated as positive regulators of the primordial-to-primary follicle transition during diminished ovulation in sheep and cows but this process is
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species-dependent and different in poly-ovulatory species such as rat [22]. Other family members, activin and inhibin also have been demonstrated to play roles with
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BMPs in follicle development in the zebrafish [23]. To explore roles of gsdf in sexual differentiation in medaka, we compared ovarian differentiation at molecular and cellular levels between wildtype XX females with intact gsdf, XX and XY females with truncated Gsdf expression. Developmental oocytes were monitored by a transgenic approach using a dual-color reporter cassette expression of green and red fluorescent proteins under the promoter regulation of a mitosis-meiosis switcher, and relevant gene expression was quantified with real-time PCR. 4
ACCEPTED MANUSCRIPT Materials and Methods Fish Medaka fish were housed in re-circulating systems at 26-28 °C, and a light-dark cycle of 14-h dayliggsdf(+/-) and 10-h darkness. Fish handling was performed in strict compliance with the guidance of the Committee for Laboratory Animal Research at
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Shanghai Ocean University and National University of Singapore. Oocyte developmental stages were monitored with histological classification according to the
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literature [24]. Phenotypic sex was assessed using secondary sex characteristics (dorsal and anal fin shape) and confirmed with gonad biopsy of 6-month adult fish.
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Genotyping was performed with PCR and primers of PG17.5-17.6 to amplify a dmy-specific fragment and a fragment from dmrt1 of medaka [7]. Genotyping of the
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gsdf allele was performed with primers as previously described [8]. Generation of intact and mutant Gsdf lines with green and red signals in ovary
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A pGRY reporter cassette was constructed in accordance with published methods [25]. pGRY contains a synaptonemal complex component (SYCP3) promoter (2.1kb
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in length), which was PCR amplified and flanked with SacI and EcoRI restriction
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sites. This was inserted in front of RFP (pDsRed-N1, Invitrogen, Carlsbad, CA, USA) to drive RFP expression for cytoplasmic labeling of cells in meiosis-prophase. A 2.5 kb EFH2BGFP fragment containing a medaka elongation factor 1α (EF1α) promoter
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[26], driven by human histone H2B and GFP fusion product (pBOS-H2BGSDF, BD Biosciences, San Jose, USA) for nuclear labeling was used because this approach
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allows ubiquitous expression of the fusion protein between human histone H2B and GFP under EF1α promoter regulation. In addition, medaka protamine fused to yellow fluoresent protein (mprmYFP) driven by protamine promoter (2 kb of 5’ UTR and 812 bp of 3’ UTR fragments in length) was ahead of SYCP4RFP and designed for both monitoring of a mitosis-meiosis switch in ovary, and an histone-protamine switch in spermatogenesis via green-red-yellow color transition in testis [27]. Primers used for vector construction was listed in Table S1. This multi-color construct was transfected into haploid embryonic stem cells, and nuclearly transferred into unfertilized eggs to generate a transgenic GRY line according to published reports 5
ACCEPTED MANUSCRIPT [25]. A homozygous line was identified from transgenic fish crossed with wildtype (gsdf +/+) fish to produce 100% GFP-positive offspring. A stable homozygous line was obtained by crossing identified homozygote fish. Male and female gsdf heterozygotic fish were mated with GRY transgenic fish to obtain heterozygotic (gsdf +/-) fish with green and red (GR) signals expression in gonads. Crossing these
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siblings ultimately generated homozygous gsdf -/- GRY XX and XY female fish. Fertility test
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Three pairs of adult males and females from Tg lines were fertility tested as a control group in pair-wise mating for two weeks. These Tg males were subsequently used to
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breed with gsdf KO females in low fertility and sterile group (n=3) in pair-wise mating for two weeks as testing groups. Cumulative production of ovulated and
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fertilized eggs were counted and listed in Table 1 and Table S3. Infertile females were ultimately validated by gonad biopsy with no mature oocytes formation.
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Histological analysis
Gonads were fixed with Bouin’s solution, dehydrated in a graded series of ethanol and
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subsequently embedded into paraffin blocks (Sigma, St. Louis, MO), or 4%
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paraformaldehyde fixed in phosphate buffer (4% PFA; PBS pH 7.4), embedded directly into tissue freezing medium (Leica, Germany) and 5-μm cross sections were cut (Leica RM2265 microtome, Germany) and stained with hematoxylin and eosin
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(H&E) for histological analysis (Jiancheng Biotech. China), or subjected to cryosectioning (Leica CM1950, Germany) for fluorescent observation. All
fish.
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morphological data were verified by similar observations from at least 3 individual
Fluorescent photography Fluorescent signals of GFP and RFP were captured with a Nikon Ds-Ri2 system under a stereomicroscope (Nikon, Japan), or photographed with confocal microscopy (Zeiss LSM 710, Carl Zeiss Germany). Quantitative real-time PCR (qPCR) Total RNA was extracted from gonads and reverse transcribed into first-strand cDNA with AMV reverse transcriptase (Takara, Dailian, China) and oligo-dT(18) primers 6
ACCEPTED MANUSCRIPT according to manufacturer instructions. Primers used for qPCR have been used previously [28] and appeared in Table S2. qPCR was carried out using cDNA as template and SYBR Fast qPCR mix (Takara, Dailian, China) and a 7500 real-time PCR system (Applied Biosystems, Foster City, CA) to measure fluorescence. Thermal cycling conditions were 1 cycle of 2 min at 95 °C followed by 40 cycles of 15 sec denaturation at 95 °C and a 34 sec extension at 64 °C. Relative abundance of each -actin was used to
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sample gene was evaluated using the formula: R=2- △△ Ct,
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normalize expression values. Statistical comparisons were evaluated by ANOVA followed by a pair-wise Student’s t-test (p<0.05 was considered to statistically
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significant unless specified). Data are means of three to five independent experiments.
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ACCEPTED MANUSCRIPT Results Abnormal gonad development in Gsdf deficient fish Normal XX females and XY males have typical secondary sexual characteristics with respect to the shape of anal fins which are under the regulation of sex determining gene dmy/dmrt1bY [6, 7] and sex hormones regulation through the
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hypothalamic-pituitary-gonadal (HPG) axis. In gsdf knockout (gsdf KO) fish, anal fin shapes resembled normal XX females (Figure 1a), irrespective of dmy or an XX or
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XY genotype. Moreover, all gsdf KO fish (100%) have larger abdominal ovaries than normal adult females, containing a giant ovary greater than one in normal females
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(Figure 1b) [8].
Normal XX female has an asynchronous-type ovary with developmental oocytes
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in various growth phases from small primordial to larger follicles in medaka (Figure 1c-c’) [29], whereas normal XY gonad develops into a testis (Figure 1b, 1d-d’) under
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the control of dmy/dmrt1bY [6, 7]. In gsdf KO XY mutants, the gonad develops into an ovary, in spite of the presence of dmy [8] (Figure 1e, e’). Folliculogenesis was
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damaged as numerous small and previtellogenic oocytes (Pvo), but few vitellogenic
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oocytes (Vo) present in gsdf KO fish, therefore resulted in low fertility with rare ovulation or absolute infertile (Figure 1e). Normal females usually give 20-50 eggs per day and ovulate daily for several months to more than one year. Oocyte
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maturation was largely prohibited in gsdf KO fish. The fertility of Gsdf KO fish (3 to 4-month mature adults) was tested by pair-wise mating. Only five from 38 tested Gsdf
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KO fish gave eggs in a reproductive period shorter than normal females. Numbers of ovulated eggs from gsdf KO fish were about two third fold lower than the normal females, with the fertilization rate much lower to one half of the normal females, implying the lack of Gsdf caused the lower fecundity and fertilization directly and/or indirectly (Table 1).
Monitoring oocyte development through a transgenic strategy H2BGFP was first designed to label the chromatin architecture with GFP signal which has been demonstrated to be wildly useful as a mitotic imaging of cell cycle [30, 31]. 8
ACCEPTED MANUSCRIPT Medaka translation elongation factor 1α (EF-1α) promoter can drive GFP expression in almost all embryonic tissues from the early gastrula stage [26]. EF1αH2BGFP was designed with EF1α controlling H2BGFP to ensure ubiquitous nuclear GFP signal expression with the assistance of nuclear localization signal (NLS) [25]. It enables a visualization of cytological changes in vivo according to fluorescent reporter
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expression. We modified the reporter by adding a SYCP3 promoter driving RFP and protamine promoter driving YFP with a map of vector illustrated in Figure 2a-b.
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Oocytes entering meiosis were monitored under a fluorescent microscope by the mitosis-meiosis switch of H2BGFP-SYCP3RFP based on green-red transition (Figure
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2-3). Light or dark orange was obtained by a merged imaging of green-red channels. Although the light orange resembles yellow color which is difficult to be
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distinguished from YFP signals within pGRY report-cassett. Since mPRM-YFP was ultilized for monitoring germ cells in post meiotic phase which was only detected in
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testis based on enhancer activity of 5’- and 3’-cis-element conserved from mammals to medaka protamine genes [27, 32]. Only gsdf roles in oocyte development was
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addressed with assistance of this transgenic line, hence we focused on green-red color
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transition which reflected mitosis-meiosis exchange bona fide in oocyte development. As expected, H2BGFP signals were detected throughout early embryogenesis of transgenic fish revealed in Figure 2c. In 10 days after hatching (10 dah) fry, most
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germ cells were green within the gonad of XY males (Figure 2d-d’) and few were red or light yellow (see arrows) in gonads of XX females, confirming that these oocytes
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entered meiosis in XX fry (Figure 2e-e’). In normal Tg adult ovaries, oogonia consists a large nucleus with weak GFP signals in gonia hardly being detected before meiosis phase (Figure 3b’-b” asterisks). After the onset of meiosis, oogonia turned to be primitive and primary oocytes in red with the expression of RFP under SYCP3 regulation (Po in Figure 3b”), then a light orange color observation due to the combination of GFP-RFP expression in these germ cells (Oc in Figure 3b”). The red color was progressively fainter and disappeared in the cytoplasm of growing oocytes during successively developmental stages, and only green GFP were observed in the nuclei of previtellogenic oocytes onwards vitellognic and fully mature stages (Pvo and 9
ACCEPTED MANUSCRIPT Vo in Figure 3a”). Therefore this fluorescence reporter cassette provides a good tool to monitor the whole process of oocyte development from the onset of meiosis onwards full maturation in medaka.
Abnormal folliculogenesis displayed by gsdf KO Tg fish line
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In order to define any difference between Gsdf present and absent female ovaries during folliculogenesis, we crossed the transgenic line of GRY with gsdf KO fish. The
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process of oocyte development was monitored precisely by these transgenic fluorescence signals through transgenic expression of the reporter cassette in normal
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females with intact Gsdf or in abnormal females with Gsdf null mutants. According to the criteria of oocyte stage classification described by Iwamatsu’s group [29], all
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developmental stages from primordial to mature oocytes present in normal XX female ovaries (Figure 4a). However, few mature oocytes were obtained in gsdf KO females
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with lower fertility (Figure 4b). No mature oocytes were observed in infertile gsdf KO fish (Figure 4c), indicating a severe prohibition of oocyte growth in gsdf deficiency
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females. In contrast of large follicle (LF) and primordial follicles (PF, in yellow
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resulted from RFP and GFP signal combination) present in normal ovaries (Figure 4a’-a”), numerous meiotic oocytes in dark orange and some oocytes in light orange color were observed in both gsdf KO with lower fertility and absolutely infertile
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female ovaries (Figure 4b’-b”; 4c’-c” arrowheads). These results implied that oogonia were able to enter meiosis, but oocyte growth was largely restrained after the onset of
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meiosis in gsdf KO mutants. Expression profile of key genes displaying in wildtype and mutant fish To investigate molecular events underlying gsdf-deficient XY feminization, expression of key genes essential for ovary or testis development were measured with qPCR assay using total RNAs as templates from gonads of 3 to 4 month-old adults. Genes of cytochrome P450 19a1 gene (cyp19a1) and 17-hydroxylase (cytochrome P450 17A, cyp17a) are molecular markers that are chiefly expressed in ovary or testis [33]. Sample RNAs were extracted and mixed from five adults of each group to avoid individual variation. qPCR data confirm predominant expression of cyp19a1 and 10
ACCEPTED MANUSCRIPT cyp17a in ovary or testis as expected (Figure 5a-b). Expression patterns in heterozygote males and females are similar to wildtype fish, in consistent with a similar fertility and a similar histological morphology compared to wildtype testes and ovaries (Figure S1). High cyp19a1 but low cyp17a expression was detected in gonads of gsdf null XX-XY females (Figure 5a-b), resembling to the complete ovary
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developed in gsdf KO fish. Expression of inhibin A (inha) had a similar pattern to that of cyp17a, higher in males but lower in females, indicating that these genes are
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involved in male testicular differentiation in medaka (Figure 5b-d). In contrast to the severe inhibition of inha in female ovary, there were no statistically significant
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differences between testis and ovarian expression, suggesting that expression of activin A (actBA) may not be subjected to gsdf regulation (Figure 5c). It has been
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indicated that smaller follicles produce more activin, whilst developed follicles secrete more inhibin [21]. Decrease of inhibin expression in gsdf-/- medaka was consistent
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heterozygous ovaries (Figure 5d).
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with rare developed follicles present in gsdf-/- mutants in contrast to wildtype and
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ACCEPTED MANUSCRIPT Discussion Requirement of gsdf in normal folliculogenesis Gsdf is conserved in teleost but absent in tetrapods [5] and expression of a gsdf ortholog is correlated to testicular differentiation in protogynous fish [34, 35], supporting an essential role for testis development. XY feminization and XX
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masculinization were obtained via loss- and gain-of function approaches in medaka and tilapia [8-10, 36, 37], suggesting a functionally conserved role of gsdf in testicular
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differentiation across species. Nevertheless, Gsdf is also essential for ovary development. We found that targeted disruption of gsdf resulted in high sterility
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(33/38 of gsdf KO mutants, 86.8%), which was caused by abnormal folliculogenesis with restrained oocyte growth and maturation in medaka. It has been reported that the
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TGF-β superfamily is critical for regulation of oocyte development, including recruitment, selection and growth of follicles from the primordial stage to ovulation in
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mammals. TGF-β is secreted by somatic (granulosa and thecal cells) and/or oocytes [21]. Granulosa and thecal cells in fish are functionally homologous to those of
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mammals [38], which support and nurture oocytes growth in an autocrine/paracrine
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manner to the microenvironment. Granulosa cells promote oocyte development and survival via thecal cells that supply granulosa cells with steroids such as the estrogenic precursors androstenedione. Medaka oogenic stages include formation of
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the primordial follicle, recruitment of selectable follicles from the growing pool to form primary, secondary, and tertiary follicles, and ovulation and subsequent
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formation of a corpus luteum (CL) [14]. Although regulators of initial stages in which germ cell clusters or cysts are broken up during follicle development have not been identified, the interaction between oocytes and somatic cells is apparently important for the formation of primordial follicles. It was presumed that absence of gsdf expression in somatic cells caused granulosa cells abnormal differentiation and/or development, therefore providing a poor nourishment for oocytes growth and rendering a numerous small follicles scatting in ovaries, with rare oocytes developed and reached to the mature stage (Figure 1 and 4). It was interesting that medaka anti-Mullerian hormone (amh) inhibited type A spermatogonia proliferation in the 12
ACCEPTED MANUSCRIPT testis but not oogonia proliferation in the ovary [39]. Hyperproliferation of self-renewing type A germ cells and XY feminization was also noted in loss-of-function mutants of its receptor (Amhr2/hotei) [39]. An alternative or redundancy function is considerable existing between amh and gsdf, as they both belong to TGF-β superfamily members and structurally share the conserved TGF-
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domain. The redundancy of amhr2 and TGF-β receptors may explain this although receptor specifically binding to Gsdf has yet to be identified in teleosts.
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Early meiotic arrest associated with gsdf deficiency
Cellular and molecular processes that accompany female germ cells have
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primarily been characterized in Drosophila and the mouse [40, 41]. Nevertheless, acquisition of femaleness in vertebrate female germlines have been investigated using
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this model species [14], after the successful identification of germline stem cells in medaka [42]. Similar to birds and mammals, after PGC differentiation, oogonia
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undergo mitotic proliferation to leave daughter cells connected by intercellular bridges, forming clusters or nests surrounded by somatic pre-follicular or supporting
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cells. Female germ cells can arrange the tubular structure of supporting cells and
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maintain feminization [15]. Also, in medaka and zebrafish, the maintenance of female-specific estrogen-producing cells depends on the presence of germ cells [16, 43]. Therefore, the effect of abnormal oocytes on surrounding follicular cells
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differentiation should not be excluded. Folliculogenesis is also regulated by release of pituitary hormone, FSH and LH
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[44] which are well conserved in vertebrates. Fish folliculogenesis is a unique process of vitellogenesis with oocyte expansion, instead of activation and formation of fluid-filled antra as seen in mammals. The relationship between pituitary activity and oocyte maturation has been depicted in medaka during oogenesis in ultrastructural detail, revealing the Balbiani Body in late oocyte stages after the appearance of synaptonemal complexes as visualized under a electron microscope [45]. We observed many primitive oocytes (Po) at early meiotic stage (dark orange) in gsdf KO ovaries (Figure 4) before the Balbiani Body formation. In contrast to normal ovaries where all stages of pre-vitellogenic, vitellogenic, and post-vitellogenic oocytes 13
ACCEPTED MANUSCRIPT present,
numerous Po (dark orange) but few oocytes at late-stage meiosis (light
orange) displayed in gsdf mutants, indicating oocyte development and vitellogenesis were severely restrained in the gsdf KO ovary. It is possible that the poor nutrition from abnormal granulosa cells in gsdf KO mutants could retard oocyte development. Another explanation could not be excluded that the loss of Gsdf attenuated
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suppression of germ cell proliferation, thus increased the number of germ cells entering meiosis, which subsequently caused the shortage of essential factor for
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normal folliculogenesis provided by granulosa cells. Phenomenon of previtellogenic oocytes accumulation in gsdf KO medaka corresponds to report that the mammalian
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TGF-β superfamily effects oocyte growth-arrest and promotion of germ cell proliferation [21].
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gsdf deficiency associated with vitellogenesis prohibition and failure of oocyte maturation in medaka
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In mature females, vitellogenesis is under the control of estradiol, a major endogenous estrogen in all vertebrates. Increasing estradiol stimulates hepatic
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production of vitellogenin (VTG), which is transported in the circulation to the ovary
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[46]. In gsdf KO ovaries, most oocytes are previtellogenic (Figure 1e-e’), suggesting that vitellogenesis and yolk intake are largely prohibited. Less activation of hepatic vitellogenesis in response to less serum estrogen could explain rare vitellogenic
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oocytes in gsdf KO fish. Fecundity, fertility (percent fertilized) and hatching success (percent hatched) were much lower in gsdf KO mutants, decreased to 2/3~1/2 fold of
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those in normal females (Table 1). In spite of an equivalent level of cyp19a1 expression in gsdf KO and wildtype ovaries (Figure 5a), serum estrogen was lower in gsdf null mutants than normal females (data unpublished), suggesting that gsdf influenced estrogen synthesis/conversion irrelevant to gonadal cyp19a1 expression. TGF-β signaling participates in the maintenance of the primordial follicle pool in medaka, zebrafish and mouse ovary [15, 16, 47]. Activin, one member of TGF-β subfamily, acts as an ovarian mediator of pituitary gonadotropin (s) to stimulate oocyte maturation in zebrafish [48, 49]. Although activin A was constantly expressed in gsdf deficient gonads, its counterpart inhibin expression was prohibited in gsdf KO 14
ACCEPTED MANUSCRIPT gonads, suggesting the gsdf effects on inhibin-mediated folliculogenesis in medaka, which is the fundermental pathway evolutionally conserved across species. Assessment of germ cells entering meiosis relies on morphological criteria and mechanisms underlying morphological change are unclear. The fluorescent reporter system enables the mitotic-meiotic process visible on a cellular level. Molecular and
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cytological evidence of abnormalities in gsdf KO medaka suggest that gsdf is involved in proliferation and differentiation of early primordial follicle formation. Defective
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Gsdf signaling may also affect the pituitary-gonad axis to regulate FSH-LH secretion via gonadal activin/inhibin expression feedback. Nearly all TGF-β isoform-specific
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knock-out mice die perinatally and the role of TGF-β remains unclear. It is expected
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addition of zebrafish or rodent models.
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that this gsdf KO medaka would assist us understand early follicular development in
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ACCEPTED MANUSCRIPT ACKNOWLEDGMENTS This work was supported by Shanghai University First-class Disciplines Project of Fisheries and the International Research Group Program; a grant to GG from the Natural Science Foundation of Shanghai (16ZR1415200); the National Science Foundation of China to ML (31372520); and grants to YH from the National Research Singapore
(NRF-CRP7-2010-03).
We
also
thank
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Foundation
LetPub
(www.letpub.com) for the linguistic assistance during the preparation of this
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manuscript.
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ACCEPTED MANUSCRIPT Competing interests The authors declare that they have no competing interests. Additional files and information. Additional files: Table S1. Primer sets for pGRY construction; Table S2. Primer sets for qPCR analysis; Table S3. Fertility of heterozygotes; Figure S1. Morphology
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analysis of heterozygote (gsdf+/- XX and XY) gonads.
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ACCEPTED MANUSCRIPT Figures legends Figure 1. Morphology analysis of wildtype (XX and XY) gonads and gsdf deficient mutant (XY gsdf -/-) gonad. a). Secondary sex character of gsdf -/- females resemble normal XX females in the shape of anal fins. b). An ovary was developed in XY gsdf -/- instead of testis in XY gsdf +/+ male, which is larger than ovary from XX
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gsdf +/+ female. c-e). represent HE staining gonadal sections of wildtype females, males and gsdf deficiency mutants, with low magnification present in c’-e’). Pvo,
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pre-vitellogenic oocytes ; Vo: vitellogenic oocytes.
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Figure 2. Visualization of oocyte development via a transgenic line. a). Map of pGRY vector containing tri-cistronic cassette; b). Schematic illustration of pGRY
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designed for monitoring switch of mitosis-meiosis and histone-protamine transition reflected by YFP-RFP-GFP fluorescent proteins. c). Strong GFP signals were
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observed in whole body during embryogenesis. 1, dark-eye Tg+; 2, albino i1Tg+; 3, albino i3Tg-. d-e) (high magnification in d’-e’). Germ cells labeled with GFP signals
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(arrows) were observed in 10-daf XY fry, both GFP and RFP (arrowheads) were
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detected in 10-daf XX fry. n: numbers of individuals subjected to fluorescence observation.
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Figure 3. Oocyte development monitored by a visible fluorescence Tg fish line. Cryosection images of oocytes at various stages present in transgenic fish ovary at
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low magnification (a, RFP; a’, GFP; a”, merged) and high magnification (white dotted frame) (b-b’-b”). Fluorescence in germ cells exchanged from light green to orange (merged of green and red) before and after the onset of meiosis during oocyte growth. c. An illustration of oocyte development from mitotic gonia to meiotic oocytes. GFP signals were first observed in nuclei of oogonia like germ cells (Og). After the oneset of meiosis, red signals from SYCP3 promoter driving RFP turn on in primitive oocytes (Po), become strong and then lost progressively, left nuclear GFP signals solely in nuclei of previtellogenic oocytes (Pvo) and vitellogenic oocytes (Vo). Oc: oocyte. Enlarged views framed in yellow dotted line are under illustrations. 18
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Figure 4. External and histological views of normal XX females and Gsdf KO mutants of low fertility or infertility. Ovary of wildtype XX with normal fertility present in (a), Gsdf KO mutants with low fertility in (b); and infertility in (c). Arrows stand for matured oocytes. Histological analysis of ovaries in low magnification (a’,
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b’, c’), and in high magnification (a”, b”, c”). nuclei were DAPI counter staining.
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Figure 5. Figure 5. Quantitative analyses of gene expression in wild-type (+/+) and gsdf mutant (-/-) medaka. Relative mRNA expression was normalized with the
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value of -actin in gonads, Vertical bar, Mean ± SEM (n=5). The asterisks indicate
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significant differences compared to the gsdf (+/+) O group (* P ＜ 0.05; **, P<0.01;
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***, P<0.001).
Figure S1. Morphology analysis of heterozygote (gsdf+/- XX and XY) gonads. Oogenesis and spermatogenesis of gsdf+/- XX (a) and gsdf+/- XY (b) resemble with
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Table 1. Cumulative production of eggs by transgenic (Tg-GRY) and gsdf KO-GRY Control group Strain
Low fertility group
Sterile group (*)
TgA♂x TgB♂x TgC♂x TgA♂x TgA♂x TgB♂x TgB♂x TgC♂x TgC♂x Tg D♀ Tg D♀ Tg D♀ Hm1♀
Hm2♀
Hm3♀
Hm4♀
Hm5♀
Hm6♀
249/14d 379/14d 245/14d 127/14d 246/14d 140/14d 0
0
0
Ovulated eggs/day (Average)
17.8/d
Fertilized eggs/2 weeks
249/14d 379/14d 242/14d 108/14d 76/14d
Fertilized eggs/day ( Average)
17.8/d
27.1/d
17.3/d
3 daf embryos/ fertilized eggs
17.8/d
27.1/d
242/245 108/127 53/76
100%
100%
98.8%
17.8/d
27.1/d
242/245 108/127 49/76
62/140
100%
100%
98.8%
44.2%
9.1/d
85%
85%
17.5/d
5.4/d
69.7%
10/d
0
0
0
71/14d
0
0
0
5.1/d
0
0
0
71/140
0
0
0
0
0
0
50.7%
RI
9.1/d
64.4%
D
MA
NU
Hatched fry/fertilized eggs
17.5/d
SC
(%)
27.1/d
PT
Ovulated eggs/14 days
AC
CE
PT E
(*) Sterility was confirmed by gonadal biopsy that no mature oocytes were observed in each ovary from three individuals listed here and all tested in sterile group.
28
ACCEPTED MANUSCRIPT Highlights of paper
AC
CE
PT E
D
MA
NU
SC
RI
PT
1. We established a transgenic fish line which is able to distinguish oocytes from various stages of development with a multi-color fluorescent combination. 2. Targeted disruption of gsdf resulted in a severe problem in female folliculogenesis and fertility, whereas the abnormal folliculogenesis was visualized under fluorescent microscope. 3. According to a TGF- domain sharing between gsdf and inhibin, and inhibin severe suppression in gsdf KO ovary, gsdf deficient medaka provides a good model for insights of TGF- signaling pathway conserved in folliculogenesis from fish to humans.
29