- Email: [email protected]
Differential expression of two Piwil orthologs during embryonic and gonadal development in pufferfish, Takifugu fasciatus
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb
Differential expression of two Piwil orthologs during embryonic and gonadal development in pufferfish, Takifugu fasciatus
T
Xin Wena,b, Dan Wanga,b, Xinru Lia,b, Cheng Zhaoa,b, Tao Wanga,b, Xiaoming Qianc, ⁎ Shaowu Yina,b, a
Key Laboratory of Biodiversity and Biotechnology of Jiangsu Province, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China Co-Innovation Center for Marine Bio-Industry Technology of Jiangsu Province, Lianyungang, Jiangsu 222005, China c Jiangsu Zhongyang Group Company Limited, Haian, Jiangsu 226600, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Takifugu fasciatus Piwil Gametogenesis Gonadal development Embryonic development
Piwil was an important regulator gene in germ cell division during gonadal development. Two Piwi-like genes, Piwil1 and Piwil2, were first cloned from T. fasciatus. The full-length cDNAs of Piwil1 and Piwil2 were of 2933 and 3394 bp, respectively. Piwil1 and Piwil2 possessed an open reading frame (ORF) of 2565 and 3138 bp, encoding 854 and 1045 amino acids, respectively. The tissue distribution analysis demonstrated that Piwil1 and Piwil2 were expressed at higher levels in gonad compared to other tissues (brain, liver, gill, etc.). The timecourse dynamic expressions of Piwils during embryonic indicated that Piwil1 and Piwil2 were mainly enriched in the early embryonic development. In testis, the expression of Piwil1 and Piwil2 increased at first but then decreased at mRNA and protein levels. However, the expression of Piwil1 and Piwil2 in the ovary showed a downward trend from the beginning. In addition, the expression levels of Piwil1 and Piwil2 were weak in mature testes or ovaries. The immunohistochemistry analysis revealed that Piwil1 and Piwil2 were abundantly expressed in cytoplasm of spermatogonia, spermatocytes, oocyte I and oocytes II, which were mainly presented in the early stages of gonadal development. Our results suggested that Piwil was related to the differentiation of germ cells, and might play an important role in embryonic development. Therefore, our findings provided valuable information of Piwils in the reproductive cycle of T. fasciatus.
1. Introduction Gametogenesis is central to sexual reproduction (Wang and Croll, 2004; Lim et al., 2013), which including spermatogenesis and oogenesis (Kowalczykiewicz et al., 2012). In invertebrates, gametogenesis is a process regulated by internal and external factors, but mainly influenced by internal factors (Zhang et al., 2014; Mueller et al., 2015), such as azoospermia-like (DAZL) (Gill et al., 2011), heat shock proteins (Hsp) (Wolgemuth and Gruppi, 1991), vasa (Feng et al., 2011; Hartung et al., 2014) and Piwi-like (Klein et al., 2016). Recently, it has been demonstrated that Piwi-like (Piwil) plays a crucial role in regulation during gametogenesis (Sugio et al., 2008), and contains the highly homologous PAZ and Piwi domain (Cox et al., 1999; Unajak et al., 2006), while Piwil is involved in a novel Piwi-interacting RNAs (piRNAs) pathway for gametogenesis and therefore less understood. To date, a series of studies have demonstrated that Piwils (Piwil1 and Piwil2) are essential in gene silencing and transposon regulation during germ cell differentiation and gonadal development in animals
⁎
(Klattenhoff and Theurkauf, 2008; Grentzinger et al., 2012; Kawaoka et al., 2012). Piwil is first identified and isolated in Drosophila, which functions as a crucial factor in maintaining self-renewal and division of germline stem cell (Cox et al., 1999). Mutations of Piwil proteins, as shown in mammals are limited to the male germline (Girard et al., 2006). In humans (Homo sapiens), four homologs of Piwils have been identified, including Hiwi, Hiwi2, Hiwi3 and Hili (Qiao et al., 2002), which are specifically expressed in spermatocytes and round spermatids. The Mus musculus Piwil (Miwi1, Miwi2, and Mili) (KuramochiMiyagawa et al., 2001; Carmell et al., 2007) encode proteins that are specifically expressed in the cytoplasm of spermatocytes and spermatids. Research reported that spermatogenesis procedure stopped at the initial stage of spermatids after knockout of the Miwi in mouse (Carmell et al., 2007). Expression of the three Piwil (Piwil1, Piwil2 and Piwil4) mRNAs in porcine was proved to be tissue specific and restricted exclusively to the gonads (Kowalczykiewicz et al., 2012). Generally, Piwil has been studied extensively in mammals, but we are acquainting scarcely with its function in teleost. The research on the function of
Corresponding author at: Key Laboratory of Biodiversity and Biotechnology of Jiangsu Province, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210023, China. E-mail address: [email protected] (S. Yin).
https://doi.org/10.1016/j.cbpb.2018.03.005 Received 27 October 2017; Received in revised form 23 February 2018; Accepted 19 March 2018 Available online 24 March 2018 1096-4959/ © 2018 Elsevier Inc. All rights reserved.
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
quantitative analysis. All of the samples were stored at −80 °C prior to further analysis. Total RNA was extracted using High Purity RNA Fast Extract Reagent (BioTeke, Beijing, China) according to the manufacturer's instructions. Purified RNA was reversely transcribed into cDNA by HiScript™ QRT SuperMix (Vazyme, New Jersey, USA) and then immediately stored at −20 °C for subsequent quantitative real-time PCR (qRT-PCR) analyses.
Piwil mainly concentrated in model organism of teleost (Bak et al., 2011), such as zebrafish (Tan et al., 2002) and medaka (Zhao et al., 2012). Zebrafish Piwil has two members; Ziwi (Piwil1) and Zili (Piwil2), both of them are expressed in female and male gonads. In addition, Ziwi mutation can lead to apoptosis of zebrafish germ cells (Houwing, 2009). For another, Zili is an important factor in the differentiation and meiosis of zebrafish reproductive cells (Ings and Gj, 2006). Moreover, lack of Opiwi can significantly reduce the number of PGCs in vivo and in vitro, and affect the distribution of PGCs in developing embryos of medaka (Li et al., 2012). Furthermore, sex steroid can regulate the expression of Piwil during embryonic development of Xenopus (Zhang et al., 2010). These researches indicating that Piwil are not only related to the differentiation of germ cells, but also plays an important role in embryonic development. However, the mechanism of Piwil in embryonic and gonadal development is not well deciphered. As a widely distributed species in the South China Sea, the East China Sea, inland waters in China and Korean Peninsula (Akira et al., 2005), Takifugu fasciatus (T. fasciatus) is an important farmed fish with high commercial value (Liu et al., 2013). T. fasciatus was found to spawn once a year between Aprils and May (Hua and Chen, 1996). However, the culture efficiency of T. fasciatus has been plagued by developmental asynochronization, low gamete viability and slow maturation procedure, which seriously restricts the development of T. fasciatus industrialization. Therefore, to solve these problems, it is very necessary to carry out further study on the Piwil1 and Piwil2 in the breeding process of T. fasciatus. In the present study, Piwil1 and Piwil2 of T. fasciatus were identified. Moreover, we aimed (1) to characterize the identified Piwil1 and Piwil2 at molecular level; (2) to elucidate their expressions at transcript level in both embryonic development and mature individuals; (3) to analyze their temporal expression profiles at mRNA and protein levels during different stages of gonadal development; and (4) to locate the expression distribution of Piwil1 and Piwil2. Overall, studies on the mechanism of Piwils in embryonic and gonadal development can provide a theoretical basis for gametogenesis and improving breeding efficiency of T. fasciatus.
2.3. Histological sections of the gonads According to the method of reference (He et al., 2001), the gonad tissue was fixed in 4% paraformaldehyde for at least 24 h. Subsequently, dehydration was carried out at different concentrations of alcohol (75%, 85%, 90%, 95% and 99.5%). The dehydrated tissues were embedded in paraffin and then stored at −20 °C for chill-down. The thickness of the slice was 6–7 μm, and these samples were then used for HE (hematoxylin-eosin) staining and immunohistochemical analysis, respectively. 2.4. Piwil cloning, and phylogenetic analysis To obtain the full-length cDNAs of Piwil1 and Piwil2 in T. fasciatus, total RNA from testis was used as template. Briefly, 5′-rapid amplification of cDNA ends (RACE) and 3′-RACE were performed using the 5′ RACE System (Version 2.0, Invitrogen, US) and SMARTer™ RACE cDNA Amplification Kit (Clontech, US), respectively. Primers (Table S1) were designed based on NCBI databases derived from conserved regions of approximate species. The anchor primers were supplied along with the kit, and touchdown PCR was programmed according to the recommended conditions using 5′ and 3′ gene specific primers. The resulting PCR products were cloned into the pMD18-T simple vector and then subjected to sequence analysis. All sequences were assembled to obtain the full-length cDNAs of Piwils. The software of Simple Modular Architecture Research Tool (SMART) was used to predict protein domain structure. Phylogenetic tree was constructed by the neighbor-joining (NJ) method of Molecular Evolution Genetic Analysis (MEGA 5.0) software. The number at each node indicated the percentage of bootstrapping after 1000 repetitions. The Ensembl and GenBank databases were used to identify related genes in other vertebrates to initially derive the syntenic relationships between species.
2. Materials and methods 2.1. Experimental animals and fertilized eggs All the experimental fish and fertilized eggs, supplied from Zhongyang Group Co., Ltd. of T. fasciatus (Jiangsu Province, China). Classification of gonad maturation state in this study refers to the previous research (Hua et al., 1999). According to the difference of gonad tissue slice (Fig. S3) and gonadosomatic index (GSI, Fig. 2) of experimental fish, gonad development is divided the process into five phases (I, II, III, IV and V). Fertilized eggs were derived from gametes produced by mature individuals and then cultured in hatchery barrels after artificial insemination. All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in China. This study was also approved by the Ethics Committee of Experimental Animals at Nanjing Normal University (grant No. SYXK 2015-0028, Jiangsu).
2.5. Real-time RT-PCR The tissue distribution and temporal expression profiles of Piwil1 and Piwil2 in the different tissues of adult individual, different stages of gonadal and embryonic development were assessed by qRT-PCR. Table S1 listed all the primers for Piwil1, Piwil2 and β-actin of T. fasciatus. The experiments were carried out in triplicate with a total volume of 20 μL in ABI stepone™ plus (Applied Biosystems, USA), consisting of 4 μL of diluted cDNA template, 10 μL of Faststart Universal SYBR Green Master (Roche, Basel, Switzerland), 1 μL of each primer (6 mmol/μL) and 4 μL ddH2O. Briefly, after a denaturation step at 95 °C for 10 min, the reaction was carried out with 40 cycles at a melting temperature of 94 °C for 10 s, and an annealing temperature of 55 °C for 30 s. β-actin of T. fasciatus was employed as the housekeeping gene, and the relative expression level of Piwils in T. fasciatus was determined by the 2−ΔΔCt method. In the tissue-specific expression analysis, the calculated relative expression level of Piwils in each tissue of T. fasciatus was compared with its respective level in liver. In the different stages of gonadal development, the fold-change of I, II, III and IV were determined by comparing the expression of V phases. Similarly, the I phase of embryonic development was used as a reference to measure differences in other periods of expression.
2.2. Tissue collection, total RNA extraction and cDNA synthesis Briefly, experimental T. fasciatus (n = 6 for mature individuals) in the late preparatory phase were anaesthetized with an MS-222 solution (0.05%, Sigma, USA), and tissues (brain, gill, heart, intestine, muscle, spleen, liver, kidney, ovary and testis) were collected under the normal physiological conditions. In order to evaluate the role of Piwils in T. fasciatus, tissues of ovary and testis at the different stages of gonadal development were used for western blot and immunohistochemistry analysis. Samples of ten periods during embryogenesis were identified by microscopic examination (Fig. 4) and collected immediately for 45
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
Fig. 1. Tissue distribution analysis of Piwil1 and Piwil2 transcripts of T. fasciatus by qRT-PCR. The relative expression of Piwils at the mRNA level in each tissue was calculated by the 2−ΔΔCt method using T. fasciatus β-actin as an internal reference gene and liver as a calibrator. Vertical bars represented the S.D. (n = 3).
the gonads at the different stages using 3, 3-diaminobenzidine tetrahydrochloride (DAB). Tissues sectioned of 6–7 μm were prepared. After removing paraffin with xylene, the sections were rehydrated in a series of ethanol (95%, 85% and 75%) and water solutions. Antigen retrievals were performed by using a citric acid buffer (0.1 M citric acid and sodium citrate, pH 6.0). The sections were washed in 3% H2O2 solution and blocked with 10% normal rabbit serum to block nonspecific binding, and the sections were incubated at 4 °C overnight with Piwils antibody (Piwil1, 1:100, D221136, Sangon, and Piwil2, 1:100, D160710, Sangon). The samples were then incubated with a secondary antibody which was labeled as peroxidase (anti-mouse/rabbit, DAKO K5007). The signals were stained by DAB (3, 3′-diaminobenzidine). As the negative control, the tissue sections were prepared by using Trisbuffered saline instead of the primary antibody.
2.6. Western blot analysis Polyclonal antibodies of anti-Piwil1 (D221136) and anti-Piwil2 (D160710) were purchased from Sangon Biotech (Shanghai) Co., Ltd. Western blot analysis was performed to detect Piwil1 and Piwil2 proteins in the gonads by using anti- Piwil1 and anti- Piwil2 antibodies, respectively. Briefly, about 50 mg of tissue homogenate was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 12%) and then electro-transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Non-specific bindings were blocked by incubating membranes with TBS containing 0.05% Tween-20 and 5% albumin bovine V (Solarbio, Beijing, China). Subsequently, the PVDF membranes were incubated with primary antibodies against Piwil1 (rabbit, anti-Piwil1; 1:500), Piwil2 (rabbit, anti-Piwil2; 1:300) or β-actin (mouse, 1: 1000; Sigma, St. Louis, MO, USA), and then the membranes were washed and incubated with secondary antibody (goat anti-rabbit IgG or goat antimouse IgG, SAB, Baltimore Ave, MD, USA). Immunoreactive bands were visualized using the Renaissance chemiluminescence reagent (Perkin-Elmer Life Science, USA). Densitometry analysis was performed by using ImageJ (1.48) software.
2.8. Statistical analysis All data were expressed as mean ± standard deviation (SD) of triplicate experiments, and the results were subjected to one-way analysis of variance (one-way ANOVA) with SPSS v22.0. Differences were considered to be significant at P < 0.05.
2.7. Immunohistochemistry Immunohistochemistry was performed to localize Piwil proteins in 46
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
3. Results
During embryonic development (Fig. 4), the expression of Piwil1 gradually increased and reached the peak at IV phase and then began to decline. However, Piwil2 expression showed a downward trend during the embryonic development.
3.1. Characterization of the full-length cDNAs of Piwil1 and Piwil2 in T. fasciatus The determined full-length cDNA of Piwil1 (MF374799) was 2933 bp, which contained a 5´-UTR of 47 bp, a 3′-UTR of 321 bp and an open reading frame (ORF) of 2565 bp, encoding a polypeptide of 854 aa residues displayed a calculated Mw of 96.64 kDa. Fig. S1A illustrated that the predicted sequence was composed of Piwi domain (residues 393–837) and PAZ domain (residues 270–408). The determined full-length cDNA of Piwil2 (MF374800) was 3394 bp, which contained a 5′-UTR of 66 bp, a 3′-UTR of 190 bp and an ORF of 3138 bp, encoding a protein of 1045 aa residues displayed a calculated Mw of 116.44 kDa. Similarly, the deduced Piwil2 (Fig. S1B) sequence exhibited a typical Piwi domain architecture (residues 583–1028), and including a PAZ domain (residues 458–598).
3.5. Western blot analysis
3.2. Homology comparison and evolutionary analysis Multiple sequence alignment showed the sequence similarity of Piwil1 and Piwil2 among different species (Table S2). Piwil1 of T. fasciatus showed sequence identity from 73% to 99%. Moreover, Piwil2 of T. fasciatus showed sequence identity from 61% to 99%. Phylogenetic analysis indicated that Piwil1 and Piwil2 (Fig. S2) were in the same branch to T. rubripes, but in different branches with other fish.
In the different development stages of gonads, the positive signals of Piwils were detected by immunohistochemistry (Fig. 6A and B). The histological sections illustrated those significant differences among different stages of gonadal development (Fig. S3). Moreover, Piwil1 and Piwil2 were detected in the cytoplasm of oocytes at stages I and II. Both Piwil1 and Piwil2 were predominantly localized in spermatogonia and spermatocytes, and weak signals were also detected in spermatids. In contrast, no expression of Piwil1 was occurred in mature spermatozoa.
3.3. Tissue distribution of Piwil1 and Piwil2 in T. fasciatus
4. Discussion
As shown in Fig. 1, it reveals that Piwil1 and Piwil2 of T. fasciatus were predominantly distributed in the testes and ovaries. The expression level of Piwil1 in the gonads was 72–3760 times higher than that of other tissues. Similarly, the difference in expression of Piwil2 between the gonads and other tissues reached a maximum of 243 times and 4 times at least. In addition, the signal of Piwil1 and Piwil2 were stronger in the testes tissue than that in the ovaries tissue.
In the present study, we obtained the full-length cDNAs of Piwil1 and Piwil2 in T. fasciatus. Subsequently, the qRT-PCR analysis showed that Piwil1 (Fig. 1) was highly abundant in the gonad, and negligibly expressed in the other tissues (brain, liver, gill, etc.), which were similar to those which found in Oncorhynchus mykiss (Rolland et al., 2009). Different from T. fasciatus, Piwil1 transcribed in zebrafish (Tan et al., 2002) and common carp (Zhou et al., 2012) were detected only in testes and ovaries. However, Olpiwi1 was not only enriched in the gonads of medaka (Zhao et al., 2012), but also highly expressed in the intestine, suggesting that it might have other functions. Therefore, the differences in Piwil1 expression might be caused by species variation and genetic background. The highest expression of Piwil2 was detected in the gonad of T. fasciatus, followed by the gill and brain. Similarly, both medaka (Li et al., 2012) and tongue sole (Zhao et al., 2012), the expression of Piwil2 was highly abundant in gonad, and was weakly visible in other tissues. However, some studies reported Piwil2 was only expressed in the gonad of zebrafish (Hartung et al., 2014), common carp (Zhou et al., 2014) and rainbow trout (Rolland et al., 2009). Taken together, Piwil was not only associated with reproduction of fish, but might also have
Western blot analysis revealed that the molecular weight of Piwil1, Piwil2 and β-actin of T. fasciatus was approximately 97, 116 and 42 kDa, respectively. During the development of testis and ovary (Fig. 5), the protein expression of Piwil1 and Piwil2 showed a trend of rising first and then decreasing, and a weak expression in V phase. In testis, the abundance of Piwil1 and Piwil2 reached the peak at III and II phase, respectively. In ovary, both Piwil1 and Piwil2 expressions reached the peak at II phase. 3.6. Protein localization of Piwils during gonad development
3.4. Temporal expression profiles of Piwil1 and Piwil2 in T. fasciatus The time-dependent expression profiles of Piwils were considerably altered during embryonic and gonadal development. Gonadosomatic index (GSI, Fig. 2) analysis revealed significant differences among different stages of gonadal development. In testis (Fig. 3A), the Piwil1 and Piwil2 expressions were significantly up-regulated before III phase, then, decreased and reached the lowest level at the V phase. In Ovary (Fig. 3B), the expression of Piwil1 and Piwil2 reached the peak level at I phase, subsequently, its expression showed a downward trend at other periods and dropped to the lowest level at the V phase.
Fig. 2. Gonadosomatic index (GSI) of testes (A) and ovaries (B) in the T. fasciatus. Gonadosomatic index (GSI) was calculated as GSI (%) = [gonad weight (g)/total body weight (g)]*100%. Error bars represented standard deviation of three replicates. Significant differences (P < 0.05) among five developmental phases (I, II, III, IV and V) in testes and ovaries were indicated by different lowercase letters (a, b, c, d and e). 47
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
Fig. 3. Temporal expressional analysis of T. fasciatus Piwil transcripts in the gonad using qRT-PCR. A shown the expressions of Piwils in the testis, while B were in the ovary. The relative expressions of Piwils at the mRNA level in each stage were calculated by the 2−ΔΔCt method using T. fasciatus β-actin as an internal reference gene and stage V as a calibrator. Vertical bars represented the S.D. (n = 3). Significant differences (P < 0.05) among five developmental phases (I, II, III, IV and V) were indicated by different lowercase letters (a, b, c, d and e).
Fig. 4. Temporal expressional analysis of T. fasciatus Piwil transcripts during embryonic development by qRT-PCR. The relative expressions of Piwils at the mRNA level in each stage were calculated by the 2−ΔΔCt method using T. fasciatus β-actin as an internal reference gene and stage I as a calibrator. Vertical bars represented the S.D. (n = 3). I: zygote; II: 8-cell; III: blastula; IV: gastrula; V: neurulation; VI: optic stage; VII: tail-bud stage; VIII: muscular contraction; IX: heart pulsation stage; X: new-hatching larvae.
48
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
Fig. 5. Western blot analysis of T. fasciatus in gonad at the different stages. Densitometry analysis was performed using ImageJ software. All experiments were repeated three times (n = 3). Significant differences (P < 0.05) among five developmental phases (I, II, III, IV and V) were indicated by different letters.
expression levels presented a trend of rising first then decreasing, and reached the lowest level in the V stage during testes development of T. fasciatus. Similarly, in C. semilaevis (Zhang et al., 2014), at early developmental stages of gonads revealed that the expression of Piwil2 was up-regulated at 95–150 days after hatching. When the gonads mature, the expression of Piwil2 gene was lower than that in 150 day larvae. The cause of this phenomenon might be the germ cells began to divide rapidly after finishing sex differentiation. Analysis by western blot (Fig. 5) found that the expression of Piwil1 and Piwil2 in T. fasciatus at the protein level also presented a corresponding trend with that in mRNA level. However, the mRNA level of Piwil2 began to decrease after reaching the highest point at the II stage. But its performance was slightly delayed at the protein level and began to decline at the III stage, suggesting that the relation between mRNA and protein was not strictly linear, and the amount of the two molecules was mainly determined by translation and protein degradation (Abreu et al., 2009). When it came to ovarian development, our results (Fig. 4C and D) were in agreement with carp ovaries (Zhou et al., 2012), the Piwil1 and Piwil2 transcription were abundant in the early stage of ovarian development and decreased in pre-ovulation. In T. fasciatus, similar to testis, the highest Piwil1 and Piwil2 proteins abundance were slightly delayed in the ovary compared with that in the mRNA levels. Moreover, we found that
other functions that have not been revealed yet. So further studies are needed to conduct. Previous studies revealed that Piwils had an impact on early embryonic development in humans (Fernández et al., 2014), bovine (Russell et al., 2006), and drosophila (Gu and Elgin, 2013). Furthermore, Li found that Opiwi also played an important role in early embryonic development of fish (such as medaka) (Li et al., 2012). In this regard, our data were in agreement with medaka, as we found Piwil1 and Piwil2 exhibit highly expression at early developmental stages of T. fasciatus. In addition, the expression of Piwil1 and Piwil2 decreased after V stage (neurulation), which were similar to those found in turbot Scophthalmus maximus (Wang et al., 2017) and Xenopus (Zhang et al., 2010). Tan (Tan et al., 2002) and Kleppe (Kleppe et al., 2015) reported Piwil1 expressed in all stages in embryonic development of zebrafish and Atlantic salmon, which were roughly consistent with our results. Taken together, these results suggested that Piwils were actively involved in the process of embryonic development, especially in the early stages. Gonadal development involved in the process of both germline and somatic cells differentiation (Vermeirssen et al., 2004). In this process, germline cells specifically differentiate into sperm (spermatogenesis) or eggs (oogenesis) (Hayes, 1998). In this study, Piwil1 and Piwil2 mRNA 49
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
Fig. 6. A. Distribution of Piwils protein in the different stages of testes by immunohistochemistry. Testis was sampled at stages I–V. A, B, C, D and E are the positions of Piwil1 at the different stages of testis, corresponding to I, II, III, IV and V stage, respectively. Similarly, F, G, H, I and J were related to Piwil2 localization. K, L, M, N and O were negative controls. SC, spermatocytes; SG, spermatogonia; ST, spermatids; SZ, spermatozoa. Scale bar was 0.4 mm. B. Distribution of Piwils protein in the different stages of ovaries by immunohistochemistry. Ovary was sampled at stages I–V. A, B, C, D and E were the positions of Piwil1 at the different stages of ovary, corresponding to I, II, III, IV and V stage, respectively. Similarly, F, G, H, I and J were related to Piwil2 localization. K, L, M, N and O were negative controls. I, II, III, IV, oocytes at stage I, II, III, IV, respectively. Scale bar was 0.2 mm.
the expression of Piwil1 and Piwil2 of T. fasciatus in the V phase at both mRNA and protein level were negligible, which was similar to Xiwi1 in Xenopus. Intriguingly, another genes of the piwil family in Xenopus, Xili were abundant in mature oocyte (Wilczynska et al., 2009), which suggested that the functions of Xiwi1 (piwil1) and Xili (piwil2) were different. However, different from the testis, the expression of Piwil1 and Piwil2 in the ovary of T. fasciatus showed a downward trend from the beginning (I phase), suggesting that the role of Piwils varies between oogenesis and spermatogenesis. Therefore, the different trends of Piwil1 and Piwil2 expression between testis and ovary illustrated in T. fasciatus that the mechanism of spermatogenesis and oogenesis was different. It was intriguing to note the similarities and differences which possibly reflecting the reason of developmental asynochronization in T. fasciatus. Many studies demonstrated that Piwil1 was expressed in the cytoplasm of oocytes, and Piwil2 had a dynamic nucleocytoplasmic distribution in oocytes (Houwing et al., 2007; Houwing et al., 2008). In our study, the immunohistochemistry analysis (Fig. 6) revealed that Piwil1 and Piwil2 were abundantly expressed in cytoplasm of spermatogonia and spermatocytes, which was similar to Ziwi and Zili in the process of spermatogenesis (Houwing et al., 2007; Houwing et al., 2008). Being consistent with zebrafish and turbot Scophthalmus maximus (Wang et al., 2017), Piwil1 and Piwil2 were detected in all stages of oogenesis, and abundantly expressed in oocyte I and oocytes II. Similarly, a strong signal of medaka Piwils was detected in oogonia of cytoplasm while a faint expression was observed in mature oocytes (Zhao et al., 2012). Furthermore, early stages of these germ cells were mainly presented in the early stages of gonadal development. Altogether, these results could explain why Piwil1 and Piwil2 were highly expressed in prespawning of T. fasciatus. Taken together, we revealed that the expression of Piwil1 and Piwil2 during embryonic and gonadal development of T. fasciatus in the present study. In summary, our data not only explored the underlying mechanism of Piwils in reproductive cycle, but also laid the foundation for improving the breeding efficiency of T. fasciatus. Acknowledgements This work was supported by the National Spark Program of China (2015GA690040), the National Finance Projects of Agro-technical Popularization (TG15-003), NSFC for Talents Training in Basic Science (J1103507), and Project Foundation of the Academic Program Development of Jiangsu Higher Education Institution (PAPD). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpb.2018.03.005. References Abreu, R.D.S., Penalva, L.O., Marcotte, E.M., Vogel, C., 2009. Global signatures of protein and mRNA expression levels. Mol. BioSyst. 5, 1512.
50
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
migration. Curr. Mol. Med. 12, 1040–1049. Lim, S.L., Tsend-Ayush, E., Kortschak, R.D., Jacob, R., Ricciardelli, C., Oehler, M.K., Grutzner, F., 2013. Conservation and expression of PIWI-interacting RNA pathway genes in male and female adult gonad of amniotes. Biol. Reprod. 89. Liu, M., Ai, H., Xiao, W., Shen, Y., Cui, X., Zhang, S., 2013. Identification of interferon-γinducible-lysosomal thiol reductase (GILT) gene from Mefugu (Takifugu obscures) and its immune response to LPS challenge. Dev. Comp. Immunol. 41, 120. Mueller, C.A., Eme, J., Manzon, R.G., Somers, C.M., Boreham, D.R., Wilson, J.Y., 2015. Embryonic critical windows: changes in incubation temperature alter survival, hatchling phenotype, and cost of development in lake whitefish (Coregonus clupeaformis). J. Comp. Physiol. B. 185, 315–331. Qiao, D., Zeeman, A.M., Deng, W., Looijenga, L.H., Lin, H., 2002. Molecular characterization of Hiwi, a human member of the piwi gene family whose overexpression is correlated to seminomas. Oncogene 21, 3988–3999. Rolland, A.D., Lareyre, J.J., Goupil, A.S., Montfort, J., Ricordel, M.J., Esquerré, D., Hugot, K., Houlgatte, R., Chalmel, F., Le, G.F., 2009. Expression profiling of rainbow trout testis development identifies evolutionary conserved genes involved in spermatogenesis. BMC Genomics 10, 546. Russell, D., Baqir, S., Bordignon, J., Betts, D.H., 2006. The impact of oocyte maturation media on early bovine embryonic development. Mol. Reprod. Dev. 73, 1255–1270. Sugio, M., Takeuchi, K., Kutsuna, J., Tadokoro, R., Takahashi, Y., Yoshida-Noro, C., Tochinai, S., 2008. Exploration of embryonic origins of germline stem cells and neoblasts in Enchytraeus japonensis (Oligochaeta, Annelida). Gene Expr. Patterns 8, 227–236. Tan, C.H., Lee, T.C., Weeraratne, S.D., Korzh, V., Lim, T.M., Gong, Z., 2002. Ziwi, the zebrafish homologue of the Drosophila piwi: co-localization with vasa at the embryonic genital ridge and gonad-specific expression in the adults. Mech. Dev. 119, S221–S224. Unajak, S., Boonsaeng, V., Jitrapakdee, S., 2006. Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 145, 179. Vermeirssen, E.L.M., de Quero, C.M., Shields, R.J., Norberg, B., Kime, D.E., Scott, A.P., 2004. Fertility and motility of sperm from Atlantic halibut (Hippoglossus hippoglossus) in relation to dose and timing of gonadotrophin-releasing hormone agonist implant. Aquaculture 230, 547–567. Wang, C., Croll, R.P., 2004. Effects of sex steroids on gonadal development and gender determination in the sea scallop, Placopecten magellanicus. Aquaculture 238, 483–498. Wang, H., Wang, B., Liu, X., Liu, Y., Du, X., Zhang, Q., Wang, X., 2017. Identification and expression of piwil2 in turbot Scophthalmus maximus, with implications of the involvement in embryonic and gonadal development. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 208. Wilczynska, A., Minshall, N., Armisen, J., Miska, E.A., Standart, N., 2009. Two Piwi proteins, Xiwi and Xili, are expressed in the Xenopus female germline. RNA 15, 337–345. Wolgemuth, D.J., Gruppi, C.M., 1991. Heat shock gene expression during mammalian gametogenesis and early embryogenesis. Results Probl. Cell Differ. 17, 138. Zhang, D., Duarteguterman, P., Langlois, V.S., Trudeau, V.L., 2010. Temporal expression and steroidal regulation of piRNA pathway genes (mael, piwi, vasa) during Silurana (Xenopus) tropicalis embryogenesis and early larval development. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 152, 202–206. Zhang, L., Liu, W., Shao, C., Zhang, N., Li, H., Liu, K., Dong, Z., Qi, Q., Zhao, W., Chen, S., 2014. Cloning, expression and methylation analysis of piwil2 in half-smooth tongue sole (Cynoglossus semilaevis). Mar. Genomics 18 (Pt A), 45. Zhao, H., Duan, J., Cheng, N., Nagahama, Y., 2012. Specific expression of Olpiwi1 and Olpiwi2 in medaka (Oryzias latipes) germ cells. Biochem. Biophys. Res. Commun. 418, 592–597. Zhou, Y., Wang, F., Liu, S., Zhong, H., Liu, Z., Tao, M., Zhang, C., Liu, Y., 2012. Human chorionic gonadotropin suppresses expression of Piwis in common carp (Cyprinus carpio) ovaries. Gen. Comp. Endocrinol. 176, 126–131. Zhou, Y., Zhong, H., Liu, S., Yu, F., Hu, J., Zhang, C., Tao, M., Liu, Y., 2014. Elevated expression of Piwi and piRNAs in ovaries of triploid crucian carp. Mol. Cell. Endocrinol. 383, 1–9.
Akira, K., Hiroyuki, D., Tsutomu, N., Harumi, S., Shigehisa, H., 2005. Takifugu obscurusis a euryhaline fugu species very close to Takifugu rubripes and suitable for studying osmoregulation. BMC Physiol. 5, 18. Bak, C.W., Yoon, T.K., Choi, Y., 2011. Functions of PIWI proteins in spermatogenesis. Clin. Exp. Reprod. Med. 38, 61–67. Carmell, M.A., Girard, A., Kant, H.J.G.V.D., Bourc'His, D., Bestor, T.H., Rooij, D.G.D., Hannon, G.J., 2007. MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline. Dev. Cell 12, 503. Cox, D.N., Chao, A., Baker, J., Chang, L., Qiao, D., Lin, H.F., Cox, D.N., Chao, A., Baker, J., Chang, L., Qiao, D., Lin, H., 1999. A novel class of evolutionary conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev. 12, 3715–3727. Feng, Z.F., Zhang, Z.F., Shao, M.Y., Wei, Z., 2011. Developmental expression pattern of the Fc-vasa-like gene, gonadogenesis and development of germ cell in Chinese shrimp, Fenneropenaeus chinensis. Aquaculture 314, 202–209. Fernández, A.F., Toraño, E.G., Urdinguio, R.G., Lana, A.G., Fernández, I.A., Fraga, M.F., 2014. The Epigenetic Basis of Adaptation and Responses to Environmental Change: Perspective on Human Reproduction. Springer New York. Gill, M.E., Hu, Y.C., Lin, Y., Page, D.C., 2011. Licensing of gametogenesis, dependent on RNA binding protein DAZL, as a gateway to sexual differentiation of fetal germ cells. Proc. Natl. Acad. Sci. U. S. A. 108, 7443–7448. Girard, A., Sachidanandam, R., Hannon, G.J., Carmell, M.A., 2006. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442, 199. Grentzinger, T., Armenise, C., Brun, C., Mugat, B., Serrano, V., Pelisson, A., Chambeyron, S., 2012. RNA-mediated transgenerational inheritance of an acquired trait. Genome Res. 22, 1877–1888. Gu, T., Elgin, S.C., 2013. Maternal depletion of Piwi, a component of the RNAi system, impacts heterochromatin formation in Drosophila. PLoS Genet. 9, e1003780. Hartung, O., Forbes, M.M., Marlow, F.L., 2014. Zebrafish vasa is required for germ-cell differentiation and maintenance. Mol. Reprod. Dev. 81, 946. Hayes, T.B., 1998. Hayes TB. Sex determination and primary sex differentiation in amphibians: genetic and developmental mechanisms. J. Exp. Zool. 281, 373–399. He, D., Chen, Y., Cai, B., 2001. Histological studies on the gonadal development of an endemic Tibet fish gymnocypris namensis. Acta Hydrobiologica Sinica 1–13. Houwing, S., 2009. Piwi-piRNA Complexes in the Zebrafish Germline. Utrecht University. Houwing, S., Kamminga, L.M., Berezikov, E., Cronembold, D., Girard, A., Van, d.E.H., Filippov, D.V., Blaser, H., Raz, E., Moens, C.B., 2007. A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in zebrafish. Cell 129, 69. Houwing, S., Berezikov, E., Ketting, R.F., 2008. Zili is required for germ cell differentiation and meiosis in zebrafish. EMBO J. 27, 2702–2711. Hua, Y., Chen, Y., 1996. A study on the sexual identification of Takifugu obscurus in reproduction period. J. Fish. China 20, 207–213. Hua, Y., Yang, Z., Chen, Y., Qian, L., Wang, L., Gu, M., 1999. Characteristics of sexual gonad development and artificial propagation of Takifugu obscurus. Freshw. Fish. 29, 000003. Ings, J.S., Gj, V.D.K., 2006. Characterization of the mRNA expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles. Mol. Reprod. Dev. 73, 943–954. Kawaoka, S., Mitsutake, H., Kiuchi, T., Kobayashi, M., Yoshikawa, M., Suzuki, Y., Sugano, S., Shimada, T., Kobayashi, J., Tomari, Y., 2012. A role for transcription from a piRNA cluster in de novo piRNA production. RNA 18, 265. Klattenhoff, C., Theurkauf, W., 2008. Biogenesis and germline functions of piRNAs. Development 135, 3–9. Klein, J.D., Qu, C.X., Yang, X.Y., Fan, Y.P., Tang, C.L., Peng, J.C., 2016. c-Fos repression by Piwi regulates Drosophila ovarian germline formation and tissue morphogenesis. PLoS Genet. 12. Kleppe, L., Wargelius, A., Johnsen, H., Andersson, E., Edvardsen, R.B., 2015. Gonad specific genes in Atlantic salmon (Salmon salar L.): characterization of tdrd7-2, dazl2, piwil1 and tdrd1 genes. Gene 560, 217–225. Kowalczykiewicz, D., Pawlak, P., Lechniak, D., Wrzesinski, J., 2012. Altered expression of porcine Piwi genes and piRNA during development. PLoS One 7, e43816. Kuramochi-Miyagawa, S., Kimura, T., Yomogida, K., Kuroiwa, A., Tadokoro, Y., Fujita, Y., Sato, M., Matsuda, Y., Nakano, T., 2001. Two mouse piwi-related genes: miwi and mili☆. Mech. Dev. 108, 121–133. Li, M., Hong, N., Gui, J., Hong, Y., 2012. Medaka piwi is essential for primordial germ cell
51
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb
Differential expression of two Piwil orthologs during embryonic and gonadal development in pufferfish, Takifugu fasciatus
T
Xin Wena,b, Dan Wanga,b, Xinru Lia,b, Cheng Zhaoa,b, Tao Wanga,b, Xiaoming Qianc, ⁎ Shaowu Yina,b, a
Key Laboratory of Biodiversity and Biotechnology of Jiangsu Province, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China Co-Innovation Center for Marine Bio-Industry Technology of Jiangsu Province, Lianyungang, Jiangsu 222005, China c Jiangsu Zhongyang Group Company Limited, Haian, Jiangsu 226600, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Takifugu fasciatus Piwil Gametogenesis Gonadal development Embryonic development
Piwil was an important regulator gene in germ cell division during gonadal development. Two Piwi-like genes, Piwil1 and Piwil2, were first cloned from T. fasciatus. The full-length cDNAs of Piwil1 and Piwil2 were of 2933 and 3394 bp, respectively. Piwil1 and Piwil2 possessed an open reading frame (ORF) of 2565 and 3138 bp, encoding 854 and 1045 amino acids, respectively. The tissue distribution analysis demonstrated that Piwil1 and Piwil2 were expressed at higher levels in gonad compared to other tissues (brain, liver, gill, etc.). The timecourse dynamic expressions of Piwils during embryonic indicated that Piwil1 and Piwil2 were mainly enriched in the early embryonic development. In testis, the expression of Piwil1 and Piwil2 increased at first but then decreased at mRNA and protein levels. However, the expression of Piwil1 and Piwil2 in the ovary showed a downward trend from the beginning. In addition, the expression levels of Piwil1 and Piwil2 were weak in mature testes or ovaries. The immunohistochemistry analysis revealed that Piwil1 and Piwil2 were abundantly expressed in cytoplasm of spermatogonia, spermatocytes, oocyte I and oocytes II, which were mainly presented in the early stages of gonadal development. Our results suggested that Piwil was related to the differentiation of germ cells, and might play an important role in embryonic development. Therefore, our findings provided valuable information of Piwils in the reproductive cycle of T. fasciatus.
1. Introduction Gametogenesis is central to sexual reproduction (Wang and Croll, 2004; Lim et al., 2013), which including spermatogenesis and oogenesis (Kowalczykiewicz et al., 2012). In invertebrates, gametogenesis is a process regulated by internal and external factors, but mainly influenced by internal factors (Zhang et al., 2014; Mueller et al., 2015), such as azoospermia-like (DAZL) (Gill et al., 2011), heat shock proteins (Hsp) (Wolgemuth and Gruppi, 1991), vasa (Feng et al., 2011; Hartung et al., 2014) and Piwi-like (Klein et al., 2016). Recently, it has been demonstrated that Piwi-like (Piwil) plays a crucial role in regulation during gametogenesis (Sugio et al., 2008), and contains the highly homologous PAZ and Piwi domain (Cox et al., 1999; Unajak et al., 2006), while Piwil is involved in a novel Piwi-interacting RNAs (piRNAs) pathway for gametogenesis and therefore less understood. To date, a series of studies have demonstrated that Piwils (Piwil1 and Piwil2) are essential in gene silencing and transposon regulation during germ cell differentiation and gonadal development in animals
⁎
(Klattenhoff and Theurkauf, 2008; Grentzinger et al., 2012; Kawaoka et al., 2012). Piwil is first identified and isolated in Drosophila, which functions as a crucial factor in maintaining self-renewal and division of germline stem cell (Cox et al., 1999). Mutations of Piwil proteins, as shown in mammals are limited to the male germline (Girard et al., 2006). In humans (Homo sapiens), four homologs of Piwils have been identified, including Hiwi, Hiwi2, Hiwi3 and Hili (Qiao et al., 2002), which are specifically expressed in spermatocytes and round spermatids. The Mus musculus Piwil (Miwi1, Miwi2, and Mili) (KuramochiMiyagawa et al., 2001; Carmell et al., 2007) encode proteins that are specifically expressed in the cytoplasm of spermatocytes and spermatids. Research reported that spermatogenesis procedure stopped at the initial stage of spermatids after knockout of the Miwi in mouse (Carmell et al., 2007). Expression of the three Piwil (Piwil1, Piwil2 and Piwil4) mRNAs in porcine was proved to be tissue specific and restricted exclusively to the gonads (Kowalczykiewicz et al., 2012). Generally, Piwil has been studied extensively in mammals, but we are acquainting scarcely with its function in teleost. The research on the function of
Corresponding author at: Key Laboratory of Biodiversity and Biotechnology of Jiangsu Province, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210023, China. E-mail address: [email protected] (S. Yin).
https://doi.org/10.1016/j.cbpb.2018.03.005 Received 27 October 2017; Received in revised form 23 February 2018; Accepted 19 March 2018 Available online 24 March 2018 1096-4959/ © 2018 Elsevier Inc. All rights reserved.
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
quantitative analysis. All of the samples were stored at −80 °C prior to further analysis. Total RNA was extracted using High Purity RNA Fast Extract Reagent (BioTeke, Beijing, China) according to the manufacturer's instructions. Purified RNA was reversely transcribed into cDNA by HiScript™ QRT SuperMix (Vazyme, New Jersey, USA) and then immediately stored at −20 °C for subsequent quantitative real-time PCR (qRT-PCR) analyses.
Piwil mainly concentrated in model organism of teleost (Bak et al., 2011), such as zebrafish (Tan et al., 2002) and medaka (Zhao et al., 2012). Zebrafish Piwil has two members; Ziwi (Piwil1) and Zili (Piwil2), both of them are expressed in female and male gonads. In addition, Ziwi mutation can lead to apoptosis of zebrafish germ cells (Houwing, 2009). For another, Zili is an important factor in the differentiation and meiosis of zebrafish reproductive cells (Ings and Gj, 2006). Moreover, lack of Opiwi can significantly reduce the number of PGCs in vivo and in vitro, and affect the distribution of PGCs in developing embryos of medaka (Li et al., 2012). Furthermore, sex steroid can regulate the expression of Piwil during embryonic development of Xenopus (Zhang et al., 2010). These researches indicating that Piwil are not only related to the differentiation of germ cells, but also plays an important role in embryonic development. However, the mechanism of Piwil in embryonic and gonadal development is not well deciphered. As a widely distributed species in the South China Sea, the East China Sea, inland waters in China and Korean Peninsula (Akira et al., 2005), Takifugu fasciatus (T. fasciatus) is an important farmed fish with high commercial value (Liu et al., 2013). T. fasciatus was found to spawn once a year between Aprils and May (Hua and Chen, 1996). However, the culture efficiency of T. fasciatus has been plagued by developmental asynochronization, low gamete viability and slow maturation procedure, which seriously restricts the development of T. fasciatus industrialization. Therefore, to solve these problems, it is very necessary to carry out further study on the Piwil1 and Piwil2 in the breeding process of T. fasciatus. In the present study, Piwil1 and Piwil2 of T. fasciatus were identified. Moreover, we aimed (1) to characterize the identified Piwil1 and Piwil2 at molecular level; (2) to elucidate their expressions at transcript level in both embryonic development and mature individuals; (3) to analyze their temporal expression profiles at mRNA and protein levels during different stages of gonadal development; and (4) to locate the expression distribution of Piwil1 and Piwil2. Overall, studies on the mechanism of Piwils in embryonic and gonadal development can provide a theoretical basis for gametogenesis and improving breeding efficiency of T. fasciatus.
2.3. Histological sections of the gonads According to the method of reference (He et al., 2001), the gonad tissue was fixed in 4% paraformaldehyde for at least 24 h. Subsequently, dehydration was carried out at different concentrations of alcohol (75%, 85%, 90%, 95% and 99.5%). The dehydrated tissues were embedded in paraffin and then stored at −20 °C for chill-down. The thickness of the slice was 6–7 μm, and these samples were then used for HE (hematoxylin-eosin) staining and immunohistochemical analysis, respectively. 2.4. Piwil cloning, and phylogenetic analysis To obtain the full-length cDNAs of Piwil1 and Piwil2 in T. fasciatus, total RNA from testis was used as template. Briefly, 5′-rapid amplification of cDNA ends (RACE) and 3′-RACE were performed using the 5′ RACE System (Version 2.0, Invitrogen, US) and SMARTer™ RACE cDNA Amplification Kit (Clontech, US), respectively. Primers (Table S1) were designed based on NCBI databases derived from conserved regions of approximate species. The anchor primers were supplied along with the kit, and touchdown PCR was programmed according to the recommended conditions using 5′ and 3′ gene specific primers. The resulting PCR products were cloned into the pMD18-T simple vector and then subjected to sequence analysis. All sequences were assembled to obtain the full-length cDNAs of Piwils. The software of Simple Modular Architecture Research Tool (SMART) was used to predict protein domain structure. Phylogenetic tree was constructed by the neighbor-joining (NJ) method of Molecular Evolution Genetic Analysis (MEGA 5.0) software. The number at each node indicated the percentage of bootstrapping after 1000 repetitions. The Ensembl and GenBank databases were used to identify related genes in other vertebrates to initially derive the syntenic relationships between species.
2. Materials and methods 2.1. Experimental animals and fertilized eggs All the experimental fish and fertilized eggs, supplied from Zhongyang Group Co., Ltd. of T. fasciatus (Jiangsu Province, China). Classification of gonad maturation state in this study refers to the previous research (Hua et al., 1999). According to the difference of gonad tissue slice (Fig. S3) and gonadosomatic index (GSI, Fig. 2) of experimental fish, gonad development is divided the process into five phases (I, II, III, IV and V). Fertilized eggs were derived from gametes produced by mature individuals and then cultured in hatchery barrels after artificial insemination. All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in China. This study was also approved by the Ethics Committee of Experimental Animals at Nanjing Normal University (grant No. SYXK 2015-0028, Jiangsu).
2.5. Real-time RT-PCR The tissue distribution and temporal expression profiles of Piwil1 and Piwil2 in the different tissues of adult individual, different stages of gonadal and embryonic development were assessed by qRT-PCR. Table S1 listed all the primers for Piwil1, Piwil2 and β-actin of T. fasciatus. The experiments were carried out in triplicate with a total volume of 20 μL in ABI stepone™ plus (Applied Biosystems, USA), consisting of 4 μL of diluted cDNA template, 10 μL of Faststart Universal SYBR Green Master (Roche, Basel, Switzerland), 1 μL of each primer (6 mmol/μL) and 4 μL ddH2O. Briefly, after a denaturation step at 95 °C for 10 min, the reaction was carried out with 40 cycles at a melting temperature of 94 °C for 10 s, and an annealing temperature of 55 °C for 30 s. β-actin of T. fasciatus was employed as the housekeeping gene, and the relative expression level of Piwils in T. fasciatus was determined by the 2−ΔΔCt method. In the tissue-specific expression analysis, the calculated relative expression level of Piwils in each tissue of T. fasciatus was compared with its respective level in liver. In the different stages of gonadal development, the fold-change of I, II, III and IV were determined by comparing the expression of V phases. Similarly, the I phase of embryonic development was used as a reference to measure differences in other periods of expression.
2.2. Tissue collection, total RNA extraction and cDNA synthesis Briefly, experimental T. fasciatus (n = 6 for mature individuals) in the late preparatory phase were anaesthetized with an MS-222 solution (0.05%, Sigma, USA), and tissues (brain, gill, heart, intestine, muscle, spleen, liver, kidney, ovary and testis) were collected under the normal physiological conditions. In order to evaluate the role of Piwils in T. fasciatus, tissues of ovary and testis at the different stages of gonadal development were used for western blot and immunohistochemistry analysis. Samples of ten periods during embryogenesis were identified by microscopic examination (Fig. 4) and collected immediately for 45
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
Fig. 1. Tissue distribution analysis of Piwil1 and Piwil2 transcripts of T. fasciatus by qRT-PCR. The relative expression of Piwils at the mRNA level in each tissue was calculated by the 2−ΔΔCt method using T. fasciatus β-actin as an internal reference gene and liver as a calibrator. Vertical bars represented the S.D. (n = 3).
the gonads at the different stages using 3, 3-diaminobenzidine tetrahydrochloride (DAB). Tissues sectioned of 6–7 μm were prepared. After removing paraffin with xylene, the sections were rehydrated in a series of ethanol (95%, 85% and 75%) and water solutions. Antigen retrievals were performed by using a citric acid buffer (0.1 M citric acid and sodium citrate, pH 6.0). The sections were washed in 3% H2O2 solution and blocked with 10% normal rabbit serum to block nonspecific binding, and the sections were incubated at 4 °C overnight with Piwils antibody (Piwil1, 1:100, D221136, Sangon, and Piwil2, 1:100, D160710, Sangon). The samples were then incubated with a secondary antibody which was labeled as peroxidase (anti-mouse/rabbit, DAKO K5007). The signals were stained by DAB (3, 3′-diaminobenzidine). As the negative control, the tissue sections were prepared by using Trisbuffered saline instead of the primary antibody.
2.6. Western blot analysis Polyclonal antibodies of anti-Piwil1 (D221136) and anti-Piwil2 (D160710) were purchased from Sangon Biotech (Shanghai) Co., Ltd. Western blot analysis was performed to detect Piwil1 and Piwil2 proteins in the gonads by using anti- Piwil1 and anti- Piwil2 antibodies, respectively. Briefly, about 50 mg of tissue homogenate was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 12%) and then electro-transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Non-specific bindings were blocked by incubating membranes with TBS containing 0.05% Tween-20 and 5% albumin bovine V (Solarbio, Beijing, China). Subsequently, the PVDF membranes were incubated with primary antibodies against Piwil1 (rabbit, anti-Piwil1; 1:500), Piwil2 (rabbit, anti-Piwil2; 1:300) or β-actin (mouse, 1: 1000; Sigma, St. Louis, MO, USA), and then the membranes were washed and incubated with secondary antibody (goat anti-rabbit IgG or goat antimouse IgG, SAB, Baltimore Ave, MD, USA). Immunoreactive bands were visualized using the Renaissance chemiluminescence reagent (Perkin-Elmer Life Science, USA). Densitometry analysis was performed by using ImageJ (1.48) software.
2.8. Statistical analysis All data were expressed as mean ± standard deviation (SD) of triplicate experiments, and the results were subjected to one-way analysis of variance (one-way ANOVA) with SPSS v22.0. Differences were considered to be significant at P < 0.05.
2.7. Immunohistochemistry Immunohistochemistry was performed to localize Piwil proteins in 46
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
3. Results
During embryonic development (Fig. 4), the expression of Piwil1 gradually increased and reached the peak at IV phase and then began to decline. However, Piwil2 expression showed a downward trend during the embryonic development.
3.1. Characterization of the full-length cDNAs of Piwil1 and Piwil2 in T. fasciatus The determined full-length cDNA of Piwil1 (MF374799) was 2933 bp, which contained a 5´-UTR of 47 bp, a 3′-UTR of 321 bp and an open reading frame (ORF) of 2565 bp, encoding a polypeptide of 854 aa residues displayed a calculated Mw of 96.64 kDa. Fig. S1A illustrated that the predicted sequence was composed of Piwi domain (residues 393–837) and PAZ domain (residues 270–408). The determined full-length cDNA of Piwil2 (MF374800) was 3394 bp, which contained a 5′-UTR of 66 bp, a 3′-UTR of 190 bp and an ORF of 3138 bp, encoding a protein of 1045 aa residues displayed a calculated Mw of 116.44 kDa. Similarly, the deduced Piwil2 (Fig. S1B) sequence exhibited a typical Piwi domain architecture (residues 583–1028), and including a PAZ domain (residues 458–598).
3.5. Western blot analysis
3.2. Homology comparison and evolutionary analysis Multiple sequence alignment showed the sequence similarity of Piwil1 and Piwil2 among different species (Table S2). Piwil1 of T. fasciatus showed sequence identity from 73% to 99%. Moreover, Piwil2 of T. fasciatus showed sequence identity from 61% to 99%. Phylogenetic analysis indicated that Piwil1 and Piwil2 (Fig. S2) were in the same branch to T. rubripes, but in different branches with other fish.
In the different development stages of gonads, the positive signals of Piwils were detected by immunohistochemistry (Fig. 6A and B). The histological sections illustrated those significant differences among different stages of gonadal development (Fig. S3). Moreover, Piwil1 and Piwil2 were detected in the cytoplasm of oocytes at stages I and II. Both Piwil1 and Piwil2 were predominantly localized in spermatogonia and spermatocytes, and weak signals were also detected in spermatids. In contrast, no expression of Piwil1 was occurred in mature spermatozoa.
3.3. Tissue distribution of Piwil1 and Piwil2 in T. fasciatus
4. Discussion
As shown in Fig. 1, it reveals that Piwil1 and Piwil2 of T. fasciatus were predominantly distributed in the testes and ovaries. The expression level of Piwil1 in the gonads was 72–3760 times higher than that of other tissues. Similarly, the difference in expression of Piwil2 between the gonads and other tissues reached a maximum of 243 times and 4 times at least. In addition, the signal of Piwil1 and Piwil2 were stronger in the testes tissue than that in the ovaries tissue.
In the present study, we obtained the full-length cDNAs of Piwil1 and Piwil2 in T. fasciatus. Subsequently, the qRT-PCR analysis showed that Piwil1 (Fig. 1) was highly abundant in the gonad, and negligibly expressed in the other tissues (brain, liver, gill, etc.), which were similar to those which found in Oncorhynchus mykiss (Rolland et al., 2009). Different from T. fasciatus, Piwil1 transcribed in zebrafish (Tan et al., 2002) and common carp (Zhou et al., 2012) were detected only in testes and ovaries. However, Olpiwi1 was not only enriched in the gonads of medaka (Zhao et al., 2012), but also highly expressed in the intestine, suggesting that it might have other functions. Therefore, the differences in Piwil1 expression might be caused by species variation and genetic background. The highest expression of Piwil2 was detected in the gonad of T. fasciatus, followed by the gill and brain. Similarly, both medaka (Li et al., 2012) and tongue sole (Zhao et al., 2012), the expression of Piwil2 was highly abundant in gonad, and was weakly visible in other tissues. However, some studies reported Piwil2 was only expressed in the gonad of zebrafish (Hartung et al., 2014), common carp (Zhou et al., 2014) and rainbow trout (Rolland et al., 2009). Taken together, Piwil was not only associated with reproduction of fish, but might also have
Western blot analysis revealed that the molecular weight of Piwil1, Piwil2 and β-actin of T. fasciatus was approximately 97, 116 and 42 kDa, respectively. During the development of testis and ovary (Fig. 5), the protein expression of Piwil1 and Piwil2 showed a trend of rising first and then decreasing, and a weak expression in V phase. In testis, the abundance of Piwil1 and Piwil2 reached the peak at III and II phase, respectively. In ovary, both Piwil1 and Piwil2 expressions reached the peak at II phase. 3.6. Protein localization of Piwils during gonad development
3.4. Temporal expression profiles of Piwil1 and Piwil2 in T. fasciatus The time-dependent expression profiles of Piwils were considerably altered during embryonic and gonadal development. Gonadosomatic index (GSI, Fig. 2) analysis revealed significant differences among different stages of gonadal development. In testis (Fig. 3A), the Piwil1 and Piwil2 expressions were significantly up-regulated before III phase, then, decreased and reached the lowest level at the V phase. In Ovary (Fig. 3B), the expression of Piwil1 and Piwil2 reached the peak level at I phase, subsequently, its expression showed a downward trend at other periods and dropped to the lowest level at the V phase.
Fig. 2. Gonadosomatic index (GSI) of testes (A) and ovaries (B) in the T. fasciatus. Gonadosomatic index (GSI) was calculated as GSI (%) = [gonad weight (g)/total body weight (g)]*100%. Error bars represented standard deviation of three replicates. Significant differences (P < 0.05) among five developmental phases (I, II, III, IV and V) in testes and ovaries were indicated by different lowercase letters (a, b, c, d and e). 47
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
Fig. 3. Temporal expressional analysis of T. fasciatus Piwil transcripts in the gonad using qRT-PCR. A shown the expressions of Piwils in the testis, while B were in the ovary. The relative expressions of Piwils at the mRNA level in each stage were calculated by the 2−ΔΔCt method using T. fasciatus β-actin as an internal reference gene and stage V as a calibrator. Vertical bars represented the S.D. (n = 3). Significant differences (P < 0.05) among five developmental phases (I, II, III, IV and V) were indicated by different lowercase letters (a, b, c, d and e).
Fig. 4. Temporal expressional analysis of T. fasciatus Piwil transcripts during embryonic development by qRT-PCR. The relative expressions of Piwils at the mRNA level in each stage were calculated by the 2−ΔΔCt method using T. fasciatus β-actin as an internal reference gene and stage I as a calibrator. Vertical bars represented the S.D. (n = 3). I: zygote; II: 8-cell; III: blastula; IV: gastrula; V: neurulation; VI: optic stage; VII: tail-bud stage; VIII: muscular contraction; IX: heart pulsation stage; X: new-hatching larvae.
48
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
Fig. 5. Western blot analysis of T. fasciatus in gonad at the different stages. Densitometry analysis was performed using ImageJ software. All experiments were repeated three times (n = 3). Significant differences (P < 0.05) among five developmental phases (I, II, III, IV and V) were indicated by different letters.
expression levels presented a trend of rising first then decreasing, and reached the lowest level in the V stage during testes development of T. fasciatus. Similarly, in C. semilaevis (Zhang et al., 2014), at early developmental stages of gonads revealed that the expression of Piwil2 was up-regulated at 95–150 days after hatching. When the gonads mature, the expression of Piwil2 gene was lower than that in 150 day larvae. The cause of this phenomenon might be the germ cells began to divide rapidly after finishing sex differentiation. Analysis by western blot (Fig. 5) found that the expression of Piwil1 and Piwil2 in T. fasciatus at the protein level also presented a corresponding trend with that in mRNA level. However, the mRNA level of Piwil2 began to decrease after reaching the highest point at the II stage. But its performance was slightly delayed at the protein level and began to decline at the III stage, suggesting that the relation between mRNA and protein was not strictly linear, and the amount of the two molecules was mainly determined by translation and protein degradation (Abreu et al., 2009). When it came to ovarian development, our results (Fig. 4C and D) were in agreement with carp ovaries (Zhou et al., 2012), the Piwil1 and Piwil2 transcription were abundant in the early stage of ovarian development and decreased in pre-ovulation. In T. fasciatus, similar to testis, the highest Piwil1 and Piwil2 proteins abundance were slightly delayed in the ovary compared with that in the mRNA levels. Moreover, we found that
other functions that have not been revealed yet. So further studies are needed to conduct. Previous studies revealed that Piwils had an impact on early embryonic development in humans (Fernández et al., 2014), bovine (Russell et al., 2006), and drosophila (Gu and Elgin, 2013). Furthermore, Li found that Opiwi also played an important role in early embryonic development of fish (such as medaka) (Li et al., 2012). In this regard, our data were in agreement with medaka, as we found Piwil1 and Piwil2 exhibit highly expression at early developmental stages of T. fasciatus. In addition, the expression of Piwil1 and Piwil2 decreased after V stage (neurulation), which were similar to those found in turbot Scophthalmus maximus (Wang et al., 2017) and Xenopus (Zhang et al., 2010). Tan (Tan et al., 2002) and Kleppe (Kleppe et al., 2015) reported Piwil1 expressed in all stages in embryonic development of zebrafish and Atlantic salmon, which were roughly consistent with our results. Taken together, these results suggested that Piwils were actively involved in the process of embryonic development, especially in the early stages. Gonadal development involved in the process of both germline and somatic cells differentiation (Vermeirssen et al., 2004). In this process, germline cells specifically differentiate into sperm (spermatogenesis) or eggs (oogenesis) (Hayes, 1998). In this study, Piwil1 and Piwil2 mRNA 49
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
Fig. 6. A. Distribution of Piwils protein in the different stages of testes by immunohistochemistry. Testis was sampled at stages I–V. A, B, C, D and E are the positions of Piwil1 at the different stages of testis, corresponding to I, II, III, IV and V stage, respectively. Similarly, F, G, H, I and J were related to Piwil2 localization. K, L, M, N and O were negative controls. SC, spermatocytes; SG, spermatogonia; ST, spermatids; SZ, spermatozoa. Scale bar was 0.4 mm. B. Distribution of Piwils protein in the different stages of ovaries by immunohistochemistry. Ovary was sampled at stages I–V. A, B, C, D and E were the positions of Piwil1 at the different stages of ovary, corresponding to I, II, III, IV and V stage, respectively. Similarly, F, G, H, I and J were related to Piwil2 localization. K, L, M, N and O were negative controls. I, II, III, IV, oocytes at stage I, II, III, IV, respectively. Scale bar was 0.2 mm.
the expression of Piwil1 and Piwil2 of T. fasciatus in the V phase at both mRNA and protein level were negligible, which was similar to Xiwi1 in Xenopus. Intriguingly, another genes of the piwil family in Xenopus, Xili were abundant in mature oocyte (Wilczynska et al., 2009), which suggested that the functions of Xiwi1 (piwil1) and Xili (piwil2) were different. However, different from the testis, the expression of Piwil1 and Piwil2 in the ovary of T. fasciatus showed a downward trend from the beginning (I phase), suggesting that the role of Piwils varies between oogenesis and spermatogenesis. Therefore, the different trends of Piwil1 and Piwil2 expression between testis and ovary illustrated in T. fasciatus that the mechanism of spermatogenesis and oogenesis was different. It was intriguing to note the similarities and differences which possibly reflecting the reason of developmental asynochronization in T. fasciatus. Many studies demonstrated that Piwil1 was expressed in the cytoplasm of oocytes, and Piwil2 had a dynamic nucleocytoplasmic distribution in oocytes (Houwing et al., 2007; Houwing et al., 2008). In our study, the immunohistochemistry analysis (Fig. 6) revealed that Piwil1 and Piwil2 were abundantly expressed in cytoplasm of spermatogonia and spermatocytes, which was similar to Ziwi and Zili in the process of spermatogenesis (Houwing et al., 2007; Houwing et al., 2008). Being consistent with zebrafish and turbot Scophthalmus maximus (Wang et al., 2017), Piwil1 and Piwil2 were detected in all stages of oogenesis, and abundantly expressed in oocyte I and oocytes II. Similarly, a strong signal of medaka Piwils was detected in oogonia of cytoplasm while a faint expression was observed in mature oocytes (Zhao et al., 2012). Furthermore, early stages of these germ cells were mainly presented in the early stages of gonadal development. Altogether, these results could explain why Piwil1 and Piwil2 were highly expressed in prespawning of T. fasciatus. Taken together, we revealed that the expression of Piwil1 and Piwil2 during embryonic and gonadal development of T. fasciatus in the present study. In summary, our data not only explored the underlying mechanism of Piwils in reproductive cycle, but also laid the foundation for improving the breeding efficiency of T. fasciatus. Acknowledgements This work was supported by the National Spark Program of China (2015GA690040), the National Finance Projects of Agro-technical Popularization (TG15-003), NSFC for Talents Training in Basic Science (J1103507), and Project Foundation of the Academic Program Development of Jiangsu Higher Education Institution (PAPD). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbpb.2018.03.005. References Abreu, R.D.S., Penalva, L.O., Marcotte, E.M., Vogel, C., 2009. Global signatures of protein and mRNA expression levels. Mol. BioSyst. 5, 1512.
50
Comparative Biochemistry and Physiology, Part B 219–220 (2018) 44–51
X. Wen et al.
migration. Curr. Mol. Med. 12, 1040–1049. Lim, S.L., Tsend-Ayush, E., Kortschak, R.D., Jacob, R., Ricciardelli, C., Oehler, M.K., Grutzner, F., 2013. Conservation and expression of PIWI-interacting RNA pathway genes in male and female adult gonad of amniotes. Biol. Reprod. 89. Liu, M., Ai, H., Xiao, W., Shen, Y., Cui, X., Zhang, S., 2013. Identification of interferon-γinducible-lysosomal thiol reductase (GILT) gene from Mefugu (Takifugu obscures) and its immune response to LPS challenge. Dev. Comp. Immunol. 41, 120. Mueller, C.A., Eme, J., Manzon, R.G., Somers, C.M., Boreham, D.R., Wilson, J.Y., 2015. Embryonic critical windows: changes in incubation temperature alter survival, hatchling phenotype, and cost of development in lake whitefish (Coregonus clupeaformis). J. Comp. Physiol. B. 185, 315–331. Qiao, D., Zeeman, A.M., Deng, W., Looijenga, L.H., Lin, H., 2002. Molecular characterization of Hiwi, a human member of the piwi gene family whose overexpression is correlated to seminomas. Oncogene 21, 3988–3999. Rolland, A.D., Lareyre, J.J., Goupil, A.S., Montfort, J., Ricordel, M.J., Esquerré, D., Hugot, K., Houlgatte, R., Chalmel, F., Le, G.F., 2009. Expression profiling of rainbow trout testis development identifies evolutionary conserved genes involved in spermatogenesis. BMC Genomics 10, 546. Russell, D., Baqir, S., Bordignon, J., Betts, D.H., 2006. The impact of oocyte maturation media on early bovine embryonic development. Mol. Reprod. Dev. 73, 1255–1270. Sugio, M., Takeuchi, K., Kutsuna, J., Tadokoro, R., Takahashi, Y., Yoshida-Noro, C., Tochinai, S., 2008. Exploration of embryonic origins of germline stem cells and neoblasts in Enchytraeus japonensis (Oligochaeta, Annelida). Gene Expr. Patterns 8, 227–236. Tan, C.H., Lee, T.C., Weeraratne, S.D., Korzh, V., Lim, T.M., Gong, Z., 2002. Ziwi, the zebrafish homologue of the Drosophila piwi: co-localization with vasa at the embryonic genital ridge and gonad-specific expression in the adults. Mech. Dev. 119, S221–S224. Unajak, S., Boonsaeng, V., Jitrapakdee, S., 2006. Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 145, 179. Vermeirssen, E.L.M., de Quero, C.M., Shields, R.J., Norberg, B., Kime, D.E., Scott, A.P., 2004. Fertility and motility of sperm from Atlantic halibut (Hippoglossus hippoglossus) in relation to dose and timing of gonadotrophin-releasing hormone agonist implant. Aquaculture 230, 547–567. Wang, C., Croll, R.P., 2004. Effects of sex steroids on gonadal development and gender determination in the sea scallop, Placopecten magellanicus. Aquaculture 238, 483–498. Wang, H., Wang, B., Liu, X., Liu, Y., Du, X., Zhang, Q., Wang, X., 2017. Identification and expression of piwil2 in turbot Scophthalmus maximus, with implications of the involvement in embryonic and gonadal development. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 208. Wilczynska, A., Minshall, N., Armisen, J., Miska, E.A., Standart, N., 2009. Two Piwi proteins, Xiwi and Xili, are expressed in the Xenopus female germline. RNA 15, 337–345. Wolgemuth, D.J., Gruppi, C.M., 1991. Heat shock gene expression during mammalian gametogenesis and early embryogenesis. Results Probl. Cell Differ. 17, 138. Zhang, D., Duarteguterman, P., Langlois, V.S., Trudeau, V.L., 2010. Temporal expression and steroidal regulation of piRNA pathway genes (mael, piwi, vasa) during Silurana (Xenopus) tropicalis embryogenesis and early larval development. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 152, 202–206. Zhang, L., Liu, W., Shao, C., Zhang, N., Li, H., Liu, K., Dong, Z., Qi, Q., Zhao, W., Chen, S., 2014. Cloning, expression and methylation analysis of piwil2 in half-smooth tongue sole (Cynoglossus semilaevis). Mar. Genomics 18 (Pt A), 45. Zhao, H., Duan, J., Cheng, N., Nagahama, Y., 2012. Specific expression of Olpiwi1 and Olpiwi2 in medaka (Oryzias latipes) germ cells. Biochem. Biophys. Res. Commun. 418, 592–597. Zhou, Y., Wang, F., Liu, S., Zhong, H., Liu, Z., Tao, M., Zhang, C., Liu, Y., 2012. Human chorionic gonadotropin suppresses expression of Piwis in common carp (Cyprinus carpio) ovaries. Gen. Comp. Endocrinol. 176, 126–131. Zhou, Y., Zhong, H., Liu, S., Yu, F., Hu, J., Zhang, C., Tao, M., Liu, Y., 2014. Elevated expression of Piwi and piRNAs in ovaries of triploid crucian carp. Mol. Cell. Endocrinol. 383, 1–9.
Akira, K., Hiroyuki, D., Tsutomu, N., Harumi, S., Shigehisa, H., 2005. Takifugu obscurusis a euryhaline fugu species very close to Takifugu rubripes and suitable for studying osmoregulation. BMC Physiol. 5, 18. Bak, C.W., Yoon, T.K., Choi, Y., 2011. Functions of PIWI proteins in spermatogenesis. Clin. Exp. Reprod. Med. 38, 61–67. Carmell, M.A., Girard, A., Kant, H.J.G.V.D., Bourc'His, D., Bestor, T.H., Rooij, D.G.D., Hannon, G.J., 2007. MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline. Dev. Cell 12, 503. Cox, D.N., Chao, A., Baker, J., Chang, L., Qiao, D., Lin, H.F., Cox, D.N., Chao, A., Baker, J., Chang, L., Qiao, D., Lin, H., 1999. A novel class of evolutionary conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev. 12, 3715–3727. Feng, Z.F., Zhang, Z.F., Shao, M.Y., Wei, Z., 2011. Developmental expression pattern of the Fc-vasa-like gene, gonadogenesis and development of germ cell in Chinese shrimp, Fenneropenaeus chinensis. Aquaculture 314, 202–209. Fernández, A.F., Toraño, E.G., Urdinguio, R.G., Lana, A.G., Fernández, I.A., Fraga, M.F., 2014. The Epigenetic Basis of Adaptation and Responses to Environmental Change: Perspective on Human Reproduction. Springer New York. Gill, M.E., Hu, Y.C., Lin, Y., Page, D.C., 2011. Licensing of gametogenesis, dependent on RNA binding protein DAZL, as a gateway to sexual differentiation of fetal germ cells. Proc. Natl. Acad. Sci. U. S. A. 108, 7443–7448. Girard, A., Sachidanandam, R., Hannon, G.J., Carmell, M.A., 2006. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442, 199. Grentzinger, T., Armenise, C., Brun, C., Mugat, B., Serrano, V., Pelisson, A., Chambeyron, S., 2012. RNA-mediated transgenerational inheritance of an acquired trait. Genome Res. 22, 1877–1888. Gu, T., Elgin, S.C., 2013. Maternal depletion of Piwi, a component of the RNAi system, impacts heterochromatin formation in Drosophila. PLoS Genet. 9, e1003780. Hartung, O., Forbes, M.M., Marlow, F.L., 2014. Zebrafish vasa is required for germ-cell differentiation and maintenance. Mol. Reprod. Dev. 81, 946. Hayes, T.B., 1998. Hayes TB. Sex determination and primary sex differentiation in amphibians: genetic and developmental mechanisms. J. Exp. Zool. 281, 373–399. He, D., Chen, Y., Cai, B., 2001. Histological studies on the gonadal development of an endemic Tibet fish gymnocypris namensis. Acta Hydrobiologica Sinica 1–13. Houwing, S., 2009. Piwi-piRNA Complexes in the Zebrafish Germline. Utrecht University. Houwing, S., Kamminga, L.M., Berezikov, E., Cronembold, D., Girard, A., Van, d.E.H., Filippov, D.V., Blaser, H., Raz, E., Moens, C.B., 2007. A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in zebrafish. Cell 129, 69. Houwing, S., Berezikov, E., Ketting, R.F., 2008. Zili is required for germ cell differentiation and meiosis in zebrafish. EMBO J. 27, 2702–2711. Hua, Y., Chen, Y., 1996. A study on the sexual identification of Takifugu obscurus in reproduction period. J. Fish. China 20, 207–213. Hua, Y., Yang, Z., Chen, Y., Qian, L., Wang, L., Gu, M., 1999. Characteristics of sexual gonad development and artificial propagation of Takifugu obscurus. Freshw. Fish. 29, 000003. Ings, J.S., Gj, V.D.K., 2006. Characterization of the mRNA expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles. Mol. Reprod. Dev. 73, 943–954. Kawaoka, S., Mitsutake, H., Kiuchi, T., Kobayashi, M., Yoshikawa, M., Suzuki, Y., Sugano, S., Shimada, T., Kobayashi, J., Tomari, Y., 2012. A role for transcription from a piRNA cluster in de novo piRNA production. RNA 18, 265. Klattenhoff, C., Theurkauf, W., 2008. Biogenesis and germline functions of piRNAs. Development 135, 3–9. Klein, J.D., Qu, C.X., Yang, X.Y., Fan, Y.P., Tang, C.L., Peng, J.C., 2016. c-Fos repression by Piwi regulates Drosophila ovarian germline formation and tissue morphogenesis. PLoS Genet. 12. Kleppe, L., Wargelius, A., Johnsen, H., Andersson, E., Edvardsen, R.B., 2015. Gonad specific genes in Atlantic salmon (Salmon salar L.): characterization of tdrd7-2, dazl2, piwil1 and tdrd1 genes. Gene 560, 217–225. Kowalczykiewicz, D., Pawlak, P., Lechniak, D., Wrzesinski, J., 2012. Altered expression of porcine Piwi genes and piRNA during development. PLoS One 7, e43816. Kuramochi-Miyagawa, S., Kimura, T., Yomogida, K., Kuroiwa, A., Tadokoro, Y., Fujita, Y., Sato, M., Matsuda, Y., Nakano, T., 2001. Two mouse piwi-related genes: miwi and mili☆. Mech. Dev. 108, 121–133. Li, M., Hong, N., Gui, J., Hong, Y., 2012. Medaka piwi is essential for primordial germ cell
51