Background: Coexistence and transition of diverse sex determination strategies have been revealed in some ectothermic species, but the variation between males caused by different sex determination strategies and the underlying mechanism remain unclear. Here, we used the gynogenetic gibel carp (Carassius gibelio)withbothgenotypic sex determination (GSD) and temperature-dependent sex determination (TSD) strategies to illustrate this issue. Results: We foundout that malesofGSD andTSD in gibelcarphad similar morphology, testicular histology, sperm structure and sperm vitality. However, when maternal individuals were mated with males of GSD, sperm nucleus swelling and fusing with the female pronucleus were observed in the fertilized eggs. On the contrary, when maternal individuals were mated with males of TSD, sperm nucleus remained in the condensed status throughout the whole process. Subsequently, semen proteomics analysis unveiled that DNAreplication andgeneexpression-related pathways were inhibited in the sperm from males of TSD compared to males of GSD, and most differentially expressed proteins associated with DNA replication, transcription and translation were down-regulated. Moreover, via BrdU incorporation and immunofluorescence detection, male nucleusreplication wasrevealedtobepresent in the fertilized eggs by the sperm from males of GSD, but absent in the fertilized eggs by the sperm from males of TSD. Conclusions: These findings indicate that DNA replication and gene expression-related pathways are associated with the distinct sperm nucleus development behaviors in fertilized eggs in response to the sperm from males of GSD and TSD. And this study is the first attempt to screen the differences between males determined via GSD and TSD in gynogenetic species, which might give a hint for understanding evolutionary adaption of diverse sex determination mechanisms in unisexual vertebrates. Keywords: Genotypic sex determination, Temperature-dependent sex determination, Semen proteomics, Gynogenesis * Correspondence: email@example.com Yao-Jun Zhu and Xi-Yin Li contributed equally to this work. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Wuhan 430072, China Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhu et al. BMC Genomics (2018) 19:437 Page 2 of 15 Background were illustrated to be down-regulated in the sperm from Sex determination is amazingly plastic in vertebrates, and males of TSD. Moreover, male nucleus replication was only two kinds of contrasting strategies including genotypic sex revealed in the eggs fertilized by the sperm from males of determination (GSD) and environmental sex determination GSD, while no male nucleus replication was detected in the (ESD) have been extensively revealed [1–3]. The primary sex eggs fertilized by the sperm from males of TSD, via BrdU of individuals with GSD is determined at the moment of incorporation and immunofluorescence detection. These fertilization via genotypic elements with sex difference , results indicated that distinct sperm nucleus development and diverse systems of GSD have been identified such as behaviors between males of GSD and TSD were associated male heterogametic XX/XY system, female heterogametic with DNA replication and gene expression-related path- ZZ/ZW system and their numerous variants . While indi- ways in gibel carp. viduals with ESD do not have genetic difference between sexes, and their sex is determined during development  Results via environmental factors including temperature , photo- Similar morphological masculine characteristics between period , social factors , pH and dissolved oxygen (DO) males of GSD and TSD . These two seemingly distinct sex determination strat- Males of gibel carp are able to be determined via both egies are not mutually exclusive, and coexistence, interaction genotypic extra microchromosomes and larval rearing and transition of GSD and ESD have been observed in fishes temperature, respectively [9, 21]. So males of GSD with the [9, 10], amphibians  and reptiles . Along with genomic genotypic male-specific marker (MSM) [9, 21]could be anatomy and investigation of non-model organisms, diverse generated via sexual reproduction under normal rearing sex determination systems and evolutionary mechanisms of temperatureatabout20°C(Fig. 1a), while males of TSD transition among sex determination systems have been without MSM  could be produced through high rearing unveiled gradually [1, 2, 5], but the variation within a sex be- temperature treatment (32 °C) of gynogenetic larvae (Fig. tween different sex determination strategies and the under- 1b). Through comparative investigation, we found out that lying mechanism remain unclear. both males of GSD and males of TSD showed normal male Gibel carp (Carassius gibelio) with a wide geographic secondary sex characteristics during breeding season, such distribution in Eurasian continent and neighboring islands as slender body shape (Fig. 1c and i), pearl organs on gill [12–14], has two rounds of polyploidy origins including an cover (Fig. 1d and j) and prolate anus (Fig. 1e and k). More- ancient allopolyploidy and a recent autopolyploidy [13, 15]. over, mature testis (Fig. 1f and l) with spermatogenic cysts Compared with other unisexual vertebrates , rare but (Fig. 1g and m) and numerous sperms (Fig. 1h and n)were significant male incidences were observed in many natural also observed in both males of GSD and TSD. habitats [9, 17, 18] of gibel carp with unisexual gynogenetic ability [17, 19, 20]. Recently, both GSD and Similar sperm morphology, structure, vitality, and temperature-dependent sex determination (TSD) were found hatchability to coexist in gibel carp through analyses of natural popula- To reveal similarities and differences between the sperm tions and lab experimental progenies [9, 21]. Moreover, extra from males of GSD and TSD, we firstly examined the microchromosomes and high temperature were illustrated to sperm morphology under scanning electron microscope play genotypic and environmental male determination role (SEM) and both types of sperm had normal morphology respectively [9, 21], and a possible association between sex that a flagellum came from the basal body (Fig. 2a and d). determination mechanisms and reproduction modes were The sperm structure with nucleus (N), mid-piece (M) and revealed . Diverse sex determination mechanisms of GSD flagellum (F) were observed (Fig. 2b and e), and the flagel- and TSD [9, 21] and dual reproduction modes including uni- lum composed of 9 doublet microtubules and 2 central sexual gynogenesis and sexual reproduction [17, 19, 20] microtubules were also detected in both males of GSD make gibel carp an ideal system to investigate plastic sex de- and TSD (Fig. 2c and f) under transmission electron mi- termination strategies and their evolutionary consequences. croscopy (TEM). Moreover, sperm head width and sperm Here, we observed that there were no significant mascu- tail length were 2.34 ± 0.02 μm and 45.44 ± 0.55 μmin line characteristic differences between males of GSD and males of GSD as well as 2.29 ± 0.02 μm and 46.03 ± TSD in morphology and histology, and their sperms also 0.61 μm in males of TSD, respectively (T-test: P =0.10 had similar structure and vitality. However, distinct sperm and P = 0.48, respectively) (Fig. 2g). Subsequently, to un- nucleus development behaviors were revealed from the fer- veil sperm vitality, we analyzed average path velocity tilized eggs by two kind sperms of GSD males and TSD (VAP) (82.52 ± 2.39 μm/sec), straight-line velocity (VSL) males. Subsequently, we performed iTRAQ-based quantita- (73.84 ± 2.66 μm/sec) and curvilinear velocity (VCL) tive proteomics analyses of semen samples between GSD (88.26 ± 2.50 μm/sec) of sperm from males of GSD, which and TSD males. Compared to males of GSD, pathways were not significantly different from the VAP (77.23 ± assigned to DNA replication, transcription and translation 2.07 μm/sec), VSL (69.56 ± 1.91 μm/sec) and VCL (83.25 Zhu et al. BMC Genomics (2018) 19:437 Page 3 of 15 Fig. 1 Cultivation, morphology and testicular histology of males determined via GSD and TSD. a Cultivation of males determined via GSD. b Cultivation of males determined via TSD. c-n Morphological masculine characteristics and testicular histology between males of GSD and TSD. c, i Body shape. d, j Gill cover. e, k Anus. f, l Testes. g, m Haematoxylin–eosin staining of testes. h, n Immunofluorescence staining of testes via CgVasa antibody. ♀: female; ♂: male; (+): with male-specific marker (MSM); (−): without MSM ±1.98 μm/sec) of sperm from males of TSD (T-test: P = in all sampled males and the paternal individuals, whereas 0.10, P =0.19 and P = 0.12, respectively) (Fig. 2h). Besides, it was absent in all randomly picked females and the ma- there were also no significant differences on hatchability ternal individuals (Fig. 3a and Additional file 1: Table S1). between eggs fertilized by the sperm from males of GSD However, when maternal fish were mated with males of (59% ± 23%) and TSD (68% ± 17%) (T-test: P =0.76). TSD from strain A , no male offspring was generated and These results indicate that there are no significant differ- the MSM was absent in all randomly picked females and ences in sperm morphology, structure, vitality, and hatch- the parental individuals (Fig. 3b and Additional file 1: ability between males of GSD and TSD. Table S1), which were identical to the gynogenetic families (Fig. 3c and Additional file 1: Table S1). The similar results Different male incidence were also revealed previously in the wild population sam- Although there were many similarities between males of pled from Poyang Lake in China . GSD and TSD, male incidences in the offspring were re- vealed to be distinct between these two types of males. Distinct sperm nucleus events and development When the maternal fish from strain A were mated with behaviors males of GSD from strain A , 34.9% (±18.1%) males were Moreover, differential male nucleus events and develop- detected in the offspring, and the MSM  was present ment behaviors were also revealed from the fertilized eggs Zhu et al. BMC Genomics (2018) 19:437 Page 4 of 15 Fig. 2 Comparisons of sperm structure, size and vitality between males of GSD and TSD. SEM (a, d) and TEM (b, c, e, f) analysis of sperm from males of GSD and TSD. N: nucleus; M: mid-piece; F: flagellum. g Statistical data of sperm head width and sperm tail length. h Statistical data of sperm vitality. VAP: average path velocity, VSL: straight-line velocity, VCL: curvilinear velocity. Vertical bars: standard deviation in response to the sperm from males of GSD and TSD. Comparative semen proteomics and differentially When the eggs of maternal fish were fertilized by the sperm expressed protein identification from males of GSD, the fertilized egg encountered similar To reveal the underlying molecular mechanism of differen- sexual reproduction events and behaviors, including sperm tial sperm nucleus development behaviors in the fertilized nucleus swelling, male pronucleus fusing with female pro- eggs, iTRAQ-based quantitative semen proteomics were nucleus before 33 min, even though male pronucleus did performed on three semen samples from three males of not completely integrate into the first mitosis after 35 min GSD and three semen samples from three males of TSD (Fig. 4a). However, when the eggs of maternal fish respectively. A total of 13,722 unique peptides and 3948 were fertilized by the sperm from males of TSD, the proteins were identified from 389,273 spectra (Fig. 5a and entered sperm nucleus was always preserved in the con- Additional file 2: Table S2). Subsequently, 99.2% (3916 densed status throughout the whole first mitosis process proteins) of these proteins were annotated via any of the 6 (Fig. 4b), which was identical to a typical process of gyno- databases including non-redundant protein (NR), genesis stimulated by heterologous sperm from common Swiss-Prot, Translated EMBL Nucleotide Sequence Data carp (Fig. 4c). Thedataindicatethatthere existdistinct Library (TrEMBL), Clusters of Orthologous Groups of sperm nucleus events and development behaviors in the proteins (COG), Gene Ontology (GO) and Kyoto fertilized eggs between GSD and TSD males, in which the Encyclopedia of Genes and Genomes (KEGG) (Fig. 5b and sperm of GSD males undergoes sperm nucleus swelling Additional file 2: Table S2). In the KEGG classification, 3436 and pronucleus fusing similar to sexual reproduction, proteins were mapped to 44 terms belonged to 6 main cat- whereas the sperm of TSD males only triggers a typical egories (Fig. 5c). The pathway with most annotated proteins unisexual gynogenesis. was “global and overview maps” (739 proteins), followed by Zhu et al. BMC Genomics (2018) 19:437 Page 5 of 15 Fig. 3 Male incidence in the offspring. The maternal individuals from strain A were mated with males of GSD (a), males of TSD (b) and another species common carp (c). PCR detection analysis of MSM in the randomly-picked offspring and the parental individuals was shown at the bottom. ♀: female; ♂: male; (+): with MSM; (−): without MSM; P : male proportion; P : female proportion; Cc: Common carp; M: DL2000 marker. M F Detail data of replicates are given in additional file 1: Table S1 “signal transduction” (375 proteins), “folding, sorting and down-regulated pathways based on P<0.01 and the False degradation” (320 proteins), “transport and catabolism” (295 Discovery Rate (FDR) < 0.25 [22, 26]. And, all 3 proteins), and so on. Besides, 1972 proteins were classified sub-pathways assigned to the pathway of “DNA replication”, into 24 COG terms, such as “general function prediction such as “M/G1 transition”, “Synthesis of DNA” and “Regula- only”, “posttranslational modification, protein turnover, tion of DNA replication”, were detected as down-regulated chaperones”, “energy production and conversion”, “replica- sub-pathways (FDR = 0.12, 0.12 and 0.13, respectively). And, tion, recombination and repair” and so on (Fig. 5d). And two sub-pathways in the pathway of “Metabolism of RNA”, 2345 proteins were classified into 57 GO terms, and “cellu- such as “Processing of capped intron-containing pre-mRNA” lar process”, “cell” and “catalytic activity” were dominant in and “Regulation of mRNA stability by proteins that bind the category of “biological process”, “cellular component” AU-rich elements”, were illustrated to be down-regulated and “molecular function”, respectively (Fig. 5e). Compared (FDR = 0.003 and 0.13, respectively) (Fig. 6a). to semen proteomics from males of GSD, 753 differentially Subsequently, pathway enrichment analysis revealed expressed proteins(DEPs)were identified (fold change > 1.2, that these 753 DEPs were mapped to 275 pathways P < 0.05) in semen proteomics from males of TSD, including through KEGG classification, and the top 20 enriched 310 up-regulated DEPs (Additional file 3: Table S3) and 443 pathways were showed in Fig. 6b. Among the top 20 down-regulated DEPs (Additional file 4:Table S4). enriched pathways, the process of “Replication and repair” contained the most pathways, such as “Fanconi anemia Revelation of differentially regulated pathway pathway”, “Non-homologous end-joining”, “Base excision To identify the biological pathways that might be differen- repair” and “DNA replication” (Fig. 6b and Additional file 5: tially expressed in the semen between males of TSD and Table S5), and the process of “Translation” had three GSD, all the DEPs were used to performed overrepresenta- enriched pathways including “Ribosome biogenesis in eu- tion analysis via Reactome database [22–25], only “DNA karyotes”, “Ribosome” and “mRNA surveillance pathway”, replication” and “Metabolism of RNA” were revealed to be while the pathway of “Spliceosome” belonged to the Zhu et al. BMC Genomics (2018) 19:437 Page 6 of 15 Fig. 4 DAPI-stained differential male nucleus behaviors in fertilized eggs. The fertilized eggs of strain A inseminated by the sperms from males of GSD (a), males of TSD (b) and another species common carp (c). Thin arrows indicate sperm nucleus or male pronucleus. Thick arrows indicate female pronucleus. Arrowheads indicate second polar-body. Time showed on the right corner is the corresponding time after fertilization process of “Transcription” had the most DEPs (Fig. 6b and “Aminoacyl-tRNA biosynthesis” respectively, while only 3, Additional file 5: Table S5). 1, 1 and 2 up-regulated DEPs were detected in the pathway In the process of “Replication and repair”, a total of 27 of “RNA transport”, “Ribosome”, “mRNA surveillance DEPs were identified to include 24 (88.9%) down-regulated pathway” and “Aminoacyl-tRNA biosynthesis” respectively DEPs and 3 (11.1%) up-regulated DEPs. There were 10, 9, (Fig. 6d and Additional file 7: Table S7). And 35 DEPs were 8, 5, 5, 5 and 4 down-regulated DEPs in the pathway of identified in the process of “Transcription” to include 32 “Base excision repair”, “DNA replication”, “Fanconi anemia (91.4%) down-regulated DEPs and 3 (8.6%) up-regulated pathway”, “Nucleotide excision repair”, “Homologous DEPs. There were 30, 1 and 1 down-regulated DEPs in recombination”, “Non-homologous end-joining” and the pathway of “Spliceosome”, “RNA polymerase” and “Mismatch repair” respectively, while only 2, 1 and 1 “Basal transcription factors” respectively, while only 3 up-regulated DEPs were identified in the pathway of “DNA up-regulated DEPs were revealed in the pathway of replication”, “Fanconi anemia pathway” and “Nucleotide ex- “Spliceosome” (Fig. 6e and Additional file 8:Table S8). cision repair” respectively (Fig. 6c and Additional file 6: Thus, most DEPs associated with “Replication and Table S6). The process of “Translation” contains 41 (87.2%) repair”, “Translation” and “Transcription” were down-regulated DEPs and 6 (12.8%) up-regulated DEPs. down-regulated in the semen from males of TSD com- There were 18, 15, 9, 7 and 2 down-regulated DEPs in the pared with males of GSD, which was consistent with pathway of “RNA transport”, “Ribosome”, “mRNA surveil- the results of overrepresentation analysis via Reactome lance pathway”, “Ribosome biogenesis in eukaryotes” and database (Fig. 6a). Zhu et al. BMC Genomics (2018) 19:437 Page 7 of 15 Fig. 5 Summary information of semen proteomics. a Statistics of semen proteomic sequencing. b Overview of unigene/contig annotation in 6 databases including NR, Swiss-Prot, TrEMBL, COG, GO and KEGG. c Histogram of KEGG classification. d Histogram of COG classification. e Histogram of GO classification Zhu et al. BMC Genomics (2018) 19:437 Page 8 of 15 Fig. 6 Analysis of differentially regulated pathway. a Down-regulated biological pathways in the sperm from males of TSD via Reactome database overrepresentation analysis. b Top 20 enriched KEGG pathways of DEPs in males of TSD compared with males of GSD. The x-axis indicates the rich factor of each pathway and y-axis shows pathway. Detail data of the 20 pathways are given in the additional file 5: Table S5. c Histogram of DEPs assigned to the 7 pathways belonged to the process of “Replication and repair”. d Histogram of DEPs assigned to the 5 pathways belonged to the process of “Translation”. e Histogram of DEPs assigned to the 3 pathways belonged to the process of “Transcription”. Red and green columns indicate up-regulated and down-regulated DEPs respectively. Detail data of DEPs are given in the Additional file 6: Table S6, Additional file 7: Table S7 and Additional file 8: Table S8 respectively, related to Fig. 6c, d and e respectively Although many proteins have been identified from the to “Replication and repair”, “Translation” and “Transcription” seminal plasma in rainbow trout (Oncorhynchus mykiss) had intracellular distributions (Additional file 9:Table S9), andcommoncarp(Cyprinus carpio), the proteins which indicated that these DEPs in semen were identified involved in the DNA replication and gene expression were from sperm proteins. These findings suggest that most of the detected only in sperm proteins instead of the seminal DNA replication and gene expression-related proteins should plasma proteins . And all down-regulated DEPs assigned be disturbed in the sperm from males of TSD compared to Zhu et al. BMC Genomics (2018) 19:437 Page 9 of 15 males of GSD. Thereby, we propose a hypothesis that DNA the eggs of the same maternal fish were fertilized by the replication, translation and transcription-related pathways sperm from males of TSD, no DNA replication signal of might be associated with the distinct sperm nucleus behav- male nucleus was observed during the whole first mitosis, iors between males of GSD and TSD. and only female pronucleus underwent genome replication and completed the first cleavage (Fig. 7b), which was identi- Confirmation of sperm nucleus replication difference cal to the typical gynogenetic process (Fig. 7c). And the ab- To confirm the association between DNA replication and sence of male nucleus replication in the fertilized eggs by the sperm nucleus development behaviors in the fertilized eggs, males of TSD might be associated with down-regulation of we used BrdU incorporation and immunofluorescence de- DNA replication proteins in the semen from males of TSD tection to trace DNA replication status as described previ- compared to males of GSD. ously . When the eggs of maternal fish were fertilized by the sperm from males of GSD, both female and male nucleus Discussion experienced DNA replication as they migrated and contacted In this study, gibel carp males of genotypic and with each other. And the replicating pronuclei combined temperature-dependent sex determination were produced and formed zygote nucleus from 30 to 33 min, even though via sexual reproduction under normal rearing temperature the replicated male chromatin bubble was divorced from the and through high rearing temperature treatment of gynoge- maternal chromosomes at 35 min and failed to integrate into netic larvae, respectively. And males of GSD and TSD had the first zygotic mitosis at 42 min (Fig. 7a). However, when similar morphology, testis histology, and sperm structure Fig. 7 BrdU incorporation-marked distinct DNA replication of male pronucleus in fertilized eggs. The fertilized eggs of strain A inseminated by the sperms from males of GSD (a), males of TSD (b) and another species common carp (c). Thin arrows indicate the replicated male pronucleus and thick arrows indicate the replicated female pronucleus. Time showed on the right corner is the corresponding time after fertilization Zhu et al. BMC Genomics (2018) 19:437 Page 10 of 15 and sperm vitality. However, presence and absence of male sperm from gibel carp males of TSD (Fig. 4b and 7b)and nucleus swelling were observed in the fertilized eggs in re- the heterologous sperm from common carp (Fig. 4c and 7c), sponse to the sperm from males of GSD and TSD, respect- the potential mechanisms might be different from each ively. Subsequently, semen proteomics analysis revealed other. In many bisexual species, sperm nucleus swelling and that DNA replication and gene expression-related pathways replication were not species-specific, even between species were down-regulated in the semen from males of TSD, with far relationship [31–34]. Previous investigations re- compared to males of GSD. And the sperm from males of vealed that common carp sperm swelled and formed pro- TSD was illustrated to have lower expression of most DEPs nuclei obviously in the common carp egg extracts , and associated with DNA replication, transcription and transla- hybrids were easily produced between common carp males tion than the sperm from males of GSD. Besides, male nu- and other bisexual fish species [33, 34]. However, common cleus replication was only observed in the fertilized eggs by carp sperm were not able to swell and form pronuclei in the the sperm from males of GSD, which was not detected in gibel carp extracts , and a typical process of gynogenesis the eggs fertilized by the sperm from males of TSD. stimulated by the heterologoussperm wasobservedwhen When the sperm from males of GSD fertilized the eggs maternal gibel carp were mated with common carp . of maternal gibel carp, sperm nucleus swelling was corre- Thus, the condensation status of common carp sperm in the lated with sperm nucleus replication (Fig. 4a and 7a). eggs of gibel carp might be caused by the inhibition from Meanwhile, when the sperm from males of TSD fertilized gibel carp eggs instead of common carp sperm per se. the eggs of maternal gibel carp, sperm nucleus without Gynogenetic gibel carp has two rounds of polyploidy ori- swelling was associated with absence of sperm nucleus gins. The first polyploidy event might result in ancestral replication (Fig. 4b and 7b). These results indicated that tetraploid Carassius auratus [15, 36]and thelateround of male nucleus swelling is closely related with the DNA rep- multiple independent polyploidy events from sympatric lication of male nucleus. On the other hand, most proteins tetraploid C. auratus might lead to the extant hexaploid of the pathways assigned to DNA replication were re- gibel carp [13–15, 36]. The newly formed gibel carp with vealed to be down-regulated in the sperm from males of unisexual gynogynetic ability, might enter evolutionary tra- TSD, compared to the males of GSD (Fig. 6). In Caenor- jectory of diploidization, as diploidization process after habditis elegans, DNA replication initiation was also re- polyploidy was suggested to be the driving force of recur- ported to trigger rapid decondensation of chromatids rent polyploidy [13, 37, 38]. Perhaps, it is the two rounds of during the first embryonic mitosis after fertilization . polyploidy and diploidization as major evolutionary force Therefore, we deduce that down-regulation of DNA that leads to different sex determination mechanisms of replication-related proteins in the sperm from males of GSD and TSD, and acquires facultative reproduction strat- TSD might be associated with the absence of male nucleus egies of unisexual and bisexual reproduction modes in the replication and swelling in the fertilized eggs in response extant hexaploid gibel carp. to the sperm from males of TSD. TSD is the most common system in ESD and occurs in In overrepresentation analysis via Reactome database, the very many reptiles [5, 11], some fishes [39, 40]and amphib- pathway of “Metabolism of RNA” was also illustrated to be ians [11, 41]. In gibel carp, males of TSD trigger typical uni- down-regulated except the pathway of “DNA replication” sexual gynonenesis (Figs. 3, 4 and 7) which is able to (Fig. 6a). And in the top 20 enriched KEGG pathways, the achieve high fecundity , but TSD is commonly consid- pathways belonged to “Translation” and “Transcription” ered to be particularly vulnerable to climate change [43, were also down-regulated (Fig. 6b, d and e), which indicated 44]. Compared with TSD, males of GSD in gibel carp, that not only DNA replication-related pathway proteins which are able to stimulate reproduction mode similar to were down-regulated, but also gene expression-related path- bisexual reproduction (Figs. 3, 4 and 7), should have more way proteins were inhibited in the sperm form males of genetic contribution to the offspring than males of TSD TSD compared to males of GSD. In addition, most DEPs (Fig. 3). And the proportions of GSD is much higher than that are assigned to “Replication and repair”, “Transcription” that of TSD in sympatric population across mainland China and “Translation” are connected in the interaction network . Although some unisexual vertebrates have widespread analysis (Fig. 8). Thus, down-regulation of gene ecological distribution and long existence scale, unisexual expression-related proteins might be also associated with reproduction is suggested to be invariable failure as a absence of male nucleus replication and swelling in the long-term evolutionary strategy , that might be why fertilizedeggsbythe spermfrommales of TSD, though most species reproduce by obligate bisexual reproduction DNA replication was deduced to be critical to distinct male [16, 42]. Thus, the hexaploid gibel carp might be also under nucleus behaviors between males of TSD and GSD as the reproduction mode transition from unisexual gynogen- discussed above. esis to bisexual reproduction for a long evolutionary term Although sperm nucleus without replication and swelling [13, 19, 21], and males of GSD with the ability to stimulate were both observed in the fertilized eggs in response to the reproduction mode similar to bisexual reproduction in the Zhu et al. BMC Genomics (2018) 19:437 Page 11 of 15 Fig. 8 Network analysis for DEPs assigned to “Replication and repair”, “Translation” and “Transcription”. The fold changes of DEPs are presented with different size. Green color indicates down-regulated DEPs, while red color indicates up-regulated DEPs unisexual species might have more selective benefits than distinguished from males of TSD via the male-specific males of TSD with the ability to trigger unisexual marker (MSM) identified previously . gynogenesis. Artificial propagation and fish culture Conclusion In the breeding season of gibel carp, the selected maternal We have screened variations between males of GSD and fish were induced into spawning by intraperitoneal injec- TSD in gynogenetic gibel carp, and revealed that DNA tion as described previously . About 8–10 h after injec- replication and gene expression-related pathways are as- tion, the maternal fish started to ovulate, and the ovulated sociated with the distinct male nucleus development be- eggs were inseminated with sperms from gibel carp or red haviors in fertilized eggs in response to the sperm from common carp. The embryos were incubated in culture males of GSD and TSD, which might help us with un- dishes at 23 °C (±1 °C) during the periods of embryogenesis derstanding evolutionary adaption of diverse sex deter- and larval hatching, and then the hatched larvae were mination mechanisms in unisexual vertebrates. reared at 20 °C (±1 °C) in water boxes equipped with an in- flator pump. The larvae were fed with fairy shrimp for 35 d Methods since first feeding and then maintained in outdoor tanks Experimental animal source (5 m × 4 m × 1.5 m) with normal feed. All experimental fish including gibel carp (C. gibelio)and To produce males of TSD, the embryos of gynogenetic common carp (C. carpio) were collected from Wuhan family were firstly incubated in culture dishes at 23 °C Guanqiao Experimental Station, Institute of Hydrobiology, (±1 °C) until first feeding, and then the larval rearing Chinese Academy of Sciences. Strain A  of gibel carp temperature were gradually changed to 32 °C (±1 °C) as and red common carp were used for the analyses in this previously described . The larvae were reared in water study. The phenotypic sex of these fish were distinguished boxes equipped with inflator pump during the larval based on whether they ovulated and released eggs or pro- rearing period for 35 d. At last the fry were also main- duced sperm in the breeding season. Males of GSD were tained in outdoor tanks with normal feed. Zhu et al. BMC Genomics (2018) 19:437 Page 12 of 15 Haematoxylin–eosin and immunofluorescence staining C) during the periods of embryogenesis and larval The testes were dissected from males of GSD and males hatching. The hatchability was calculated as described of TSD, and fixed in 4% paraformaldehyde in PBS over- , that hatchability = (the number of hatched larvae / night at 4 °C. Then samples were dehydrated and em- the number of all fertilized eggs) × 100%. The unpaired bedded in paraffin, and were cut into 5 μm sections. HE T-test was used to estimate the significant difference of staining was performed as previously described  and sperm vitality and hatchability between males of GSD immunofluorescence staining using CgVasa antibody was and TSD via SPSS software v19.0.0. performed as described . DAPI staining in fertilized eggs Scanning electron microscope and transmission electron The ovulated eggs of the females from strain A in gibel microscopy carp were inseminated by sperms from males of GSD in + + The semen was fixed in PBS buffer (pH = 7.4) containing strain A , from males of TSD in strain A , and from an- 2.5% glutaraldehyde overnight at 4 °C, and the fixed sperm other species common carp. The fertilized eggs were specimens were dropped onto a tiny microslide. Then a digested by 0.25% trypsin to remove their shells and then stepwise ethanol dehydration (10, 30, 50, 70, 80, 90, 100, incubated at 23 °C for cytological observations. The fertil- 100%, 10 min each step) and a stepwise tert-butyl alcohol ized eggs of different developmental stage were fixed with dehydration (tert-butyl alcohol: ethanol = 1:3, tert-butyl al- 4% paraformaldehyde in PBS at 4 °C overnight. After cohol: ethanol = 1:1, tert-butyl alcohol: ethanol = 3:1, 100% washing with PBS three times, the nuclei were stained by tert-butyl alcohol, 10 min each step) were performed or- DAPI, and the images were acquired under confocal mi- derly. After electric conduction treatment, a S-4800 scan- croscopy (NOL-LSM 710 Carl Zeiss) as described . ning electron microscope (SEM) (Hitachi High-Tech) was used to examine the sperm. Head width and tail length of iTRAQ-based quantitative proteomics the sperm were measured by SEM and ImageJ software Total proteins of 6 semen samples from 3 males of GSD . In this study, 31 sperm from 3 males of GSD and 59 and 3 males of TSD in strain A were extracted respect- sperm from 3 males of TSD were measured for sperm ively as described before [52, 53], and the protein samples head width detection, and 39 sperm from 3 males of GSD were quantified by Bradford Assay and SDS-PAGE ana- and 37 sperm from 3 males of TSD were measured for lysis. The extracted proteins were digested using Trypsin sperm tail length detection. The unpaired T-test was used Gold (Promega) after being diluted by 100 mM triethyla- to estimate the significant difference between sperm from mine borane. Then peptides were labeled using iTRAQ males of GSD and TSD via SPSS software v19.0.0. Reagent8-plex Kit (AB SCIEX) according to the manufac- For transmission electron microscopy (TEM), the fixed turer’sprotocol . The labeled peptides with different sperm samples were washed in PBS and incubated with reagents were combined and desalted with a Strata X C18 PBS containing 1% OsO4. Then the samples were column (Phenomenex) and vacuum-dried. Subsequently, treated with 50, 70, 80, 90 and 95% ethanol orderly and the peptides were separated on a LC-20AB HPLC Pump processed with a mixed solution of acetone and epoxy system (Shimadzu), and the LC-MS/MS analysis was car- resin (1:1 for 1 h, and then 1:3 for 3 h). After infiltration ried out as described . with epoxy resin and ultrathin-section treatment, the The raw MS/MS data was converted into MGF format samples were stained with uranyl acetate and lead cit- by ProteoWizard tool msConvert, the exported MGF files rate. The specimens were observed with a HT7700 were searched in National Center for Biotechnology Infor- transmission electron microscope (Hitachi High-Tech). mation (NCBI) (https://www.ncbi.nlm.nih.gov/) and the Universal Protein Resource (UniProt) (http://www.unipro- Analysis of sperm vitality and hatchability t.org/) using MASCOT version 2.3.02 (Matrix Science) and The semen was diluted 500 times by Hank’s solution, then at least one unique peptide was necessary for the identified 10 μl diluted semen was dropped onto on a glass slide. protein. Gene Ontology (GO) database (http://www.ge- The prepared samples were observed and tested under a neontology.org/) , Clusters of Orthologous Groups of computer assisted sperm analyzer (CASA) system as soon proteins (COG) database (http://www.ncbi.nlm.nih.gov/ as the sperm was activated by dropping 1 μlwater. The COG/) and Kyoto Encyclopedia of Genes and Ge- parameters of sperm motility and kinematics were nomes (KEGG) database (http://www.genome.jp/kegg/ assessed by Animal Motility Software Manual Version 1.4. pathway.html) were used for analysis. And 61,163 sperms from 3 males of GSD and 83,516 sperms from 3 males of TSD were detected in total. Identification of differentially expressed proteins The ovulated eggs of maternal gibel carp were insemi- Automated software IQuant were used for protein quantifi- nated with sperm from males of GSD and TSD, and the cation as previously reported . The peptide-spectral embryos were incubated in culture dishes at 23 °C (±1 ° match (PSM) was pre-filtered at a PSM-level false discovery Zhu et al. BMC Genomics (2018) 19:437 Page 13 of 15 rate (FDR) of 1% for assessing the confidence of peptides. Additional file 3: Table S3. Up-regulated DEPs (males of TSD vs males In order to control the rate of false-positive at protein level, of GSD). (XLS 235 kb) an protein FDR at 1% was estimated after protein inference Additional file 4: Table S4. Down-regulated DEPs (males of TSD vs males of GSD). (XLS 243 kb) (protein-level FDR ≤ 0.01) . Proteins with 1.2 fold Additional file 5: Table S5. Detailed data of the top 20 enriched KEGG change and P-value less than 0.05 were determined as pathways, related to Fig. 6b (XLS 40 kb) differentially expressed proteins (DEPs). Additional file 6: Table S6. DEPs assigned to the process of “Replication and repair”, related to Fig. 6c. (XLS 28 kb) Reactome database overrepresentation and KEGG Additional file 7: Table S7. DEPs assigned to the process of pathway enrichment “Translation”, related to Fig. 6d. (XLS 29 kb) Overrepresentation analyses were performed in the Reac- Additional file 8: Table S8. DEPs assigned to the process of “Transcription”, related to Fig. 6e (XLS 27 kb) tome database (https://reactome.org/) using up-regulated Additional file 9: Table S9. Subcellular location of down-regulated DEPs anddown-regulatedDEPs. The P-value indicated the DEPs assigned to the process of “Replication and repair”, “Translation” statistical significance of each hit pathway, the false discov- and “Transcription”. (XLS 35 kb) ery rate (FDR) was calculated for estimating the false posi- tives via Benjamini-Hochberg approach in Reactome , Abbreviations FDR < 0.25 showed the confidence of “possible” or “hypoth- BrdU: 5-bromo-2-deoxy-uridine; CASA: computer assisted sperm analyzer; COG: Clusters of Orthologous Groups of proteins; DEPs: differentially esis”, FDR < 0.05 denoted statistical significance . All expressed proteins; DO: dissolved oxygen; ESD: environmental sex DEPs were used to perform KEGG pathway enrichment determination; F: flagellum; FDR: false discovery rate; GO: Gene Ontology; analysis using cluster profiler in R via Fisher’s exact GSD: genotypic sex determination; HE: haematoxylin–eosin; KEGG: Kyoto Encyclopedia of Genes and Genomes; M: mid-piece; MSM: male-specific test, P < 0.05 were considered as statistical significance. marker; N: nucleus; NCBI: National Center for Biotechnology Information; NR: non-redundant protein; PBST: PBS containing 0.1% Triton X-100; BrdU incorporation and immunofluorescence detection PSM: peptide-spectral match; SEM: scanning electron microscope; TEM: transmission electron microscopy; TrEMBL: Translated EMBL Nucleotide BrdU (5-bromo-2-deoxy-uridine) dissolved in PBS with Sequence Data Library; TSD: temperature-dependent sex determination; the concentration of 0.01 mg/ml was microinjected into Uniprot: Universal Protein Resource; VAP: average path velocity; eggs (1 nl each) within 10 min after fertilization. Then, the VCL: curvilinear velocity; VSL: straight-line velocity BrdU incorporation embryos were fixed in 4% formalde- Acknowledgements hyde, 0.25% glutaraldehyde, and 0.1% Triton X-100 in PBS We would like to thank You Duan for assistance in data analysis (Institute of at 4 °C overnight. After treating with 2 N HCl for 30 min, Hydrobiology, Chinese Academy of Sciences). We would like also to thank Fang Zhou for providing confocal services, and Zhen-Fei Xing and Yuan Xiao neutralization by 0.1 M sodium borate (pH = 8.5) and for providing SEM and TEM services (Analytical & Testing Center, Institute of washing in PBST (PBS containing 0.1% Triton X-100), the Hydrobiology, Chinese Academy of Sciences). embryos were subjected to immunofluorescence detection Funding according to standard protocols. Mouse α-BrdU antibody This work was supported by the Key Program of Frontier Sciences of the Chinese was used as primary antibody, and Rhodamine conjugated Academy of Sciences (QYZDY-SSW-SMC025), the National Natural Science goat anti-mouse IgG was used as secondary antibody. Foundation of China (31502148), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA08030201), the Young Elite Scientist Sponsorship Images were acquired under confocal microscopy Program by CAST (YESS20150156), the Earmarked Fund for Modern Agro-industry (NOL-LSM 710 Carl Zeiss) as described . Technology Research System (NYCYTX-49), the Autonomous Project of the State Key Laboratory of Freshwater Ecology and Biotechnology (2016FBZ01), the Autonomous Project of the Institute of Hydrobiology, Chinese Academy of Sciences (Y25A171) Analysis of protein interaction network and the Knowledge Innovation Program of the Chinese Academy of Science. The DEPs assigned to the process of “Replication and repair”, funding bodies had no role in the design of the study and collection, analysis, and “Translation” and “Transcription” were used to perform interpretation of data and in writing the manuscript. protein interaction network analysis via String (http:// Availability of data and materials string-db.org/). Amino acid sequences of DEPs were The proteomics raw data that support the findings of this study was uploaded to the String server, and database of Danio submitted to the Proteomics Identifications Database (PRIDE) under the project accession number PXD008919. rerio was selected. The threshold of minimum required interaction score was set to the highest confidence Authors’ contributions (score = 0.900), and the results were visualized by using JFG, XYL and YJZ conceived this project. YJZ, JZ, XYL, ZL and MD performed software Cytoscape3.5.1 . the experiments. YJZ, XYL, LZ and XJZ analyzed the data. JFG, XYL and YJZ wrote the manuscript. All authors read and approved the manuscript. Additional files Ethics approval and consent to participate Gibel carp (C. gibelio) and common carp (C. carpio) included in our experiment were collected from Wuhan Guanqiao Experimental Station, Additional file 1: Table S1. Detail data of male incidence, related to Institute of Hydrobiology, Chinese Academy of Sciences. All experiments in Fig. 3. (DOC 34 kb) the study were carried out according to the regulations of the Guide for Additional file 2: Table S2. All proteins identified by iTRAQ approach Care and Use of Laboratory Animals approved by the Animal Care and Use and their annotation information in 6 databases. (XLS 4444 kb) Committee of the Institute of Hydrobiology, Chinese Academy of Sciences. Zhu et al. BMC Genomics (2018) 19:437 Page 14 of 15 Competing interests 23. Croft D, O'Kelly G, Wu G, Haw R, Gillespie M, Matthews L, et al. 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Published: Jun 5, 2018