Super-fast generation of all-female grass carp via transplantation of female germline stem cell into zebrafish

Super-fast generation of all-female grass carp via transplantation of female germline stem cell into zebrafish | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Super-fast generation of all-female grass carp via transplantation of female germline stem cell into zebrafish Yonghua Sun, Ding Ye, Chaofan Wang, Junwen Zhu, Yongkang Hao, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5164959/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Surrogate reproduction has emerged as a powerful biotechnology in fish breeding, mainly aiming at shortening the maturation period of aquaculture species. Grass carp ( Ctenopharyngodon idellus ), possesses one of the largest body sizes and highest global production yields in freshwater aquaculture. However, the reproduction and genetic breeding of grass carp are significantly hindered, primarily due to its protracted sexual maturation period of nearly five years and the need for extensive cultivation space. In this study, we develop a super-fast strategy to breed all-female grass carp within half a year, leveraging surrogate production in a small-sized laboratory fish, zebrafish ( Danio rerio ). We characterized and purified female germline stem cells (GSCs) from grass carp juvenile ovary at three months post-fertilization, which is the first report of female GSCs in an aquaculture species. The grass carp female GSCs were transplanted into germ cell-depleted zebrafish larvae to generate surrogate zebrafish. The transplanted grass carp female GSCs underwent accelerated spermatogenesis in the zebrafish recipients. Three months after transplantation, the zebrafish recipients developed into males capable of producing all-X sperm derived from donor grass carp female GSCs. When these sperm were fertilized with wildtype grass carp eggs, a population of all-female grass carp was produced. Our study demonstrates that fish female GSCs with XX chromosomes can be differentiated into functional sperm in a short time under the control of zebrafish gonadal somatic niche, which opens a new avenue for precision breeding in aquaculture. Biological sciences/Developmental biology/Germline development Biological sciences/Biotechnology/Animal biotechnology zebrafish grass carp female germline stem cell sex control maturation period Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Aquaculture, currently accounting for more than half of the global fish production for human consumption, represents the fastest-growing food production sector 1 . To address the escalating demand of aquaculture production driven by human population growth, there is an urgent need for genetic improvement of aquaculture species through modern genetic breeding techniques. Surrogate reproductive technology, involving the production of gametes from a donor species in a recipient species, has emerged as a prominent approach for breeding aquaculture species 2 , 3 , 4 , 5 . This technology entails transplanting undifferentiated germ cells from a donor fish species into sterile larvae or juvenile fish of an easy-to-breed species. The outcome is the production of functional gametes from the donor fish in the recipient species, with the main goal of shortening the maturation period of donor gametes 6 , 7 . In fish surrogate reproduction, different donor cell types, such as primordial germ cells (PGCs) derived from early embryos and germline stem cells (GSCs) from juvenile or mature gonads were used as donor cells for transplanting into different host fish species 6 , 7 . Among them, spermatogonial stem cells, namely male GSCs have shown to be the most commonly used and the most successful type of donor cells, since male GSCs widely exist in the testes of different species, spanning from the juvenile stage to the adult stage 8 . In contrast, oogonial stem cells, namely female GSCs were only identified in two model fish, zebrafish and medaka 9 , 10 . Although a previous study showed that the transplanted rainbow trout ovarian germ cells differentiated toward sperm in a male host 11 , the presence and identification of female GSCs in aquaculture fish species remains elusive. The grass carp ( Ctenopharyngodon idellus ), a rapidly growing herbivorous freshwater fish species with large body size, has become a predominant species in both Chinese and global freshwater aquaculture 12 . The sex chromosome system of grass carp is characterized by XX/XY, with the identification of Y-specific sequences enabling the identification of genetic males 13 , 14 . However, the prolonged sexual maturation phase of grass carp, spanning approximately five years in the majority of cultivated areas, poses a significant obstacle to the implementation of conventional breeding methods, including hybridization, genetic selection, and sex control, for genetic improvement of grass carp 15 . Female grass carp exhibit considerably accelerated growth rates compared to their male counterparts, underscoring the significance of prioritizing the breeding of all-female grass carp 16 . Nevertheless, conventional techniques employed in breeding all-female populations, such as gynogenesis or sex reversal through steroid treatment 17 , 18 , require a substantial time and extensive effort, primarily attributable to the prolonged maturation period of grass carp. If female GSCs could be identified and isolated from grass carp juvenile ovaries, it might be possible to produce all-X sperm after transplanting female GSCs into recipient males with a short maturation period. Zebrafish ( Danio rerio ) is a small, laboratory-bred fish species that has been widely used as an animal model for studying vertebrate development, human diseases, and finfish aquaculture 1 , 19 , 20 . Previous studies have shown that laboratory-bred zebrafish strains lack a genetic sex determination system 21 , 22 . Instead, germ cells play an important role in sex differentiation of zebrafish, since a sufficient number of germ cells is required for female development of zebrafish 23 , and zebrafish lacking endogenous germ cells develop exclusively into males 24 . In our recent studies, when GSCs or 9 germplasm factors (9GM)-induced primordial germ cells (iPGCs) of Chinese rare minnow ( Gobiocypris rarus ) were transplanted into germ cell depleted zebrafish, all the zebrafish surrogates developed into males and produced rare minnow sperm 25 , 26 . This indicates that sex-chromosome lost and germ cell-depleted zebrafish could be used as an ideal surrogate host for producing donor-derived sperm. In contrast to the long maturation period of 5 years and a giant body size for grass carp, zebrafish have a sex maturation period of approximately three months 27 , with mature individuals typically weighing around 0.3g. Therefore, it is intriguing to test whether gametes from an aquaculture species with a prolonged maturation period and large body sizes, such as grass carp, could be produced by zebrafish with a significantly shorter maturation period and smaller body size (Fig. 1A), and to investigate how donor GSCs behave after transplanting into sterile zebrafish host. In this study, we characterized grass carp female GSCs from ovaries at three months post-fertilization (mpf) to 15 mpf, marking the first report of female GSCs of an aquaculture species. We further isolated female GSCs from 3 mpf grass carp and efficiently transplanted the purified female GSCs into sterile zebrafish larvae. We demonstrate that grass carp female GSCs with XX chromosomes could be differentiated into functional all-X sperm under the control of gonadal somatic niche of germ cell-depleted zebrafish within three months, which opens a new avenue for advancing precision breeding in aquaculture. Results Sexual dimorphism of gonads in grass carp at three months post-fertilization In zebrafish, it is well-known that they display sexual dimorphism of gonad sizes between juvenile testes and ovaries 23 , we wondered whether a similar phenomenon was observed in grass carp. Grass carp generally reach sex maturation at the age of 5 years, we therefore examined the gonadal development of grass carp at different ages. After dissection, the gonads were found to be located on both sides of the swim bladder (Fig. 1B, C). In the samples from 3 month mpf grass carp, 2 of the 10 gonads were wider and larger than others (Fig. 1D). We then detected the male-specific DNA fragments by PCR with those gonad samples, and found that big-sized gonads, No. 2 and 8, did not show any amplification of male-specific band, and the other eight gonad samples could give an amplification of male-specific bands, indicating that No. 2 and 8 grass carps were genetically females (Fig. 1E). These results suggest that the juvenile gonads of grass carp at 3 mpf exhibit a sexual dimorphism between genetic males and females, making it possible to distinguish juvenile ovaries from juvenile testes from the morphological view. Identification of female GSCs in grass carp It has been reported that GSCs are the only type of donor cells which could colonize, survive and differentiate into mature gametes in a host gonadal somatic niche 28 . Thus, an ideal donor gonad for GSC transplantation should be abundant in GSCs. To investigate the developmental status of germ cells, we analyzed grass carp gonads at different ages from 3 mpf to 5 years old (yr). At 3 mpf, although the germ cells were more abundant in the females than in the males, they were homogeneous in both types of gonads, (Fig. 2A, B), indicating that they were female and male GSCs. More importantly, the uniform germ cells displayed poorly condensed nucleus of large nucleolus, and expressed high level of Ddx4, a factor essential for germline development 29 , and Nanos2, a reliable GSC marker 10 , 30 , 31 (Fig. 2C-D1, Fig S1 ), just mimicking the characteristics of GSCs in zebrafish testes and ovaries 10 , 31 , 32 , further confirming that all the germ cells in juvenile ovaries or testes of grass carp at 3 mpf were GSCs. In 6 mpf grass carp of both sexes, likewise, there were only GSCs existing in the ovaries or testes 32 . All these indicate that female GSCs widely exist in the ovaries of female grass carp from 3 mpf to 6 mpf. In the ovaries at 15 mpf, plenty of stage I oocytes appeared (Fig. 2E, F), and the oogonia positive of Ddx4 and Pcna existed (Fig. 2G, Fig S2 A), and they were usually clustered with one or two obvious nucleolus (Fig. 2G1), suggesting that they were mitotic active female GSCs. In the testes at 15 mpf, all the germ cells showed poorly condensed nucleus of large nucleolus and expressed high level of Ddx4 and Pcna (Fig. 2H, H1, Fig S2 B), indicating that they were still undifferentiated and proliferative GSCs. We then checked the gonadal development and gametogenesis of grass carp from two to five years (Fig S3 A-D). The testes of two- and three-year-old grass carp were thin and light pink color, while the testes of four and five-year-old were thick and white (Fig S3 E-H). Histological staining and immunofluorescence further showed that the germ cells in the testes of the two-, three- and four-year old were mainly type A spermatogonia (Fig S3 E1-H3) 32 . The ovaries changed dramatically in morphology and color from two-year old to five-year old (Fig S3 I-L), indicating the differentiation progress of ovaries. Stage IB oocytes appeared in two-year old ovaries (Fig S3 I1), and the stage II oocytes appeared three-year old ovaries (Fig S3 J1). In the four- and five-year old ovaries, the yolk accumulated in the oocytes, indicating that stage III and IV oocytes appeared (Fig S3 K1-L1). Therefore, the ovaries from female grass carp at 3 mpf to 6 mpf and testes from male grass carps at 3 mpf to 4 years old could provide an ideal source of donor GSCs for surrogate reproduction. Purification and transplantation of female germline stem cells (GSCs) To test whether the female GSCs in grass carp juvenile ovaries could really contribute to germline development, we isolated female GSCs from 3 mpf ovaries and transplanted the female GSCs into germline-depleted zebrafish larvae (Fig. 3A). After percoll centrifuge of single-cell suspension from 3 mpf ovaries (Fig. 3B and C), the GSCs were highly enriched in percoll layers between 30% and 35%, and between 35% and 40%, with 40.5% and 46.9% GSCs among all the DAPI stained cells (Fig. 3B and C). The cells from these two layers were combined to serve as the donor female GSCs. The host zebrafish were germ cell-depleted by injection of antisense morpholino against dnd1 ( dnd1 _MO), which was verified by labeling of primordial germ cells (PGCs) by mCherry-UTRnanos3 mRNA injection 33 (Fig. 3D, E). The grass carp female GSCs cells, labeled by live cell tracker of green fluorescence, were transplanted into the genital ridge between the swim bladder and gut close to the primitive gonads of germ cell-depleted zebrafish larvae at 5 dpf (Fig. 3F), according to our recent study 25 . About 100 GSCs were transplanted into each larva. Five days post transplantation (dpt), large numbers of labeled grass carp GSCs were observed in the primitive gonad region of about 60% of host zebrafish (Fig. 3G), indicating that grass carp donor GSCs survived and colonized in zebrafish surrogates. Super-fast spermatogenesis of grass carp female GSCs in zebrafish host To determine whether donor grass carp GSCs could reconstitute gametogenesis in sterile zebrafish recipients within a relatively short maturation period, we examined the gonads of GSC transplanted (GSCT) zebrafish at 2 mpt and 3 mpt, and compared these with stage-matched zebrafish, grass carp, and dnd _MO-injected zebrafish (Table 1 ). At 2 mpt, a portion of the GSCT gonads were naive with undifferentiated grass carp germ cells (Fig. 4A, B). At 3 mpt, the gonads were dissected from GSCT fish, dnd1 _MO injected zebrafish and WT zebrafish for morphological analysis. The GSCT positive gonads appeared non-transparent, similar to the WT zebrafish testes, which were completely different from the germ cell-depleted testes (Fig. 4C-E), indicating that spermatogenesis occurred in the GSCT zebrafish. Table 1 Developmental statistics of GSCT zebrafish from 3 independent trials. Trial No No. with grass carp germ cells / No. observed at 5 dpt (%) No. colonized / No. observed at 2 mpt (%) No. colonized / No. observed at 3 mpt (%) No. producing grass carp sperm / No. checked at 3 mpt (%) Trial 1 85/189 (45.0%) 2/22 (9.1%) 1/10 (10%) 2/30 (6.7%) Trial 2 123/168 (73.2%) 6/21 (28.6%) 2/11 (18.2%) 6/47 (13.3%) Trial 3 158/256 (61.7%) 2/12 (16.7%) 2/24 (8.3%) 4/73 (5.3%) To further characterize the spermatogenesis progress of the transplanted grass carp female GSCs in host zebrafish, we conducted immunofluorescent staining of the testes using a series of spermatogenesis markers, including Ddx4, Sycp3, and Pcna, which indicate different types of spermatogenic cells 32 . In the developing testis, numerous cells were undergoing mitosis and meiosis in the GSCT positive testis (Fig. 4F-I), mimicking what was observed in the zebrafish adult testis at 3 mpf (Fig. 4J-M). Immunostaining of Ddx4 confirmed that the transplanted grass carp GSCs had successfully resumed spermatogenesis in the infertile zebrafish testis, and various germ cells including spermatogonia, spermatocytes, spermatids, and spermatozoa were present in the GSCT positive testis (Fig. 4G), consistent with observation in the WT zebrafish testis (Fig. 4K). In contrast, at 6 mpf, the grass carp testis only contained large numbers of spermatogonia, which were Ddx4 and Pcna-positive and Sycp3-negative, indicating they were in a proliferative rather than meiotic state (Fig. 4N-Q). RT-PCR amplification with grass carp and zebrafish ddx4- specific primers (Table S1 ) further validated that the germ cells in GSCT positive testes were derived from transplanted grass carp GSCs but not endogenous zebrafish GSCs (Fig. 4R). Additionally, we found that the cyst surfaces of spermatocytes in the GSCT testes were larger than those in the zebrafish adult testes, indicating that the spermatogonial generations in grass carp are more than in zebrafish (Fig. 4S). Taken together, these findings demonstrate that grass carp-originated female GSCs can efficiently proliferate and differentiate into mature sperm in zebrafish host testes, and that spermatogenesis cycle of grass carp-derived germ cells in GSCT zebrafish closely resembled that of zebrafish. Generation of grass carp sperm in GSCT zebrafish To investigate whether zebrafish recipients could produce functional grass carp sperm, we collected semen from all the surrogate zebrafish and examined the species-specific DNA fragment by PCR. PCR analysis of the genomic DNA confirmed that the semen produced by GSCT positive zebrafish males only contained grass carp DNA but not zebrafish DNA, as revealed by amplification of the species-specific genes (Fig. 5A). Although the GSCT positive zebrafish could produce matured grass carp sperm, we were interested in whether there was any difference between the sperm derived from the surrogate zebrafish and the grass carp males. By field emission scanning electron microscopy (FESEM) analysis, the morphology of GSCT sperm looked more similar to grass carp sperm than zebrafish sperm (Fig. 5B-D). The average tail length of GSCT sperm was nearly identical to that of grass carp sperm, but significantly longer than that of zebrafish sperm (Fig. 5E), and the average head diameter of GSCT sperm was significantly shorter than that of zebrafish sperm, and same to that of grass carp sperm (Fig. 5F). To further examine the genetic consistency among the sperm samples, the GSCT, grass carp and zebrafish sperm were subjected to RNA-seq analysis. The transcriptomes of the three samples were mapped to the reference genome of grass carp and zebrafish. By calculating the coverage of reads on each chromosome, the results revealed an average coverage of 99.7% for the Z-GC (zebrafish-produced grass carp sperms) group, whereas the Z (zebrafish sperms) group exhibited an average coverage of 17.8% (Fig. 5G), suggesting that the GSCT sperm are genetically identical to the grass carp sperm. Taken together, all the above data suggest that the zebrafish surrogate successfully produced grass carp-derived sperm. Generation of all-female offspring from female GSC transplanted zebrafish surrogate To identify whether the GSCT positive zebrafish males could produce functional grass carp sperm, we collected the GSCT sperm and performed in vitro fertilization assay. As controls, the progeny produced by GSCT sperm and zebrafish eggs, as well as those by grass carp sperm and zebrafish eggs were malformed and did not beyond 2 dpf (Fig S4). In contrast, the progeny generated by GSCT sperm and grass carp eggs developed normally, comparable to wildtype grass carp (Fig. 6B, C, E, F), and could be raised to later stages without any defects (Fig. 6G). PCR analysis of the genomic DNA confirmed that the offspring from GSCT sperm and grass carp eggs were indeed grass carp (Fig. 6G). Notably, no male-specific bands were detected in the GSCT sperm fertilized grass carps, whereas half of the wildtype grass carps exhibited the Y-fragment (Fig. 6H, I). Intriguingly, the grass carp sperm produced by the zebrafish surrogate could be artificially collected at regular intervals of 2–3 weeks, totaling up to 20 time per year. This is in stark contrast to grass carp, which produce sperm only once a year in the spring season. This finding suggests that the self-renewal and differentiation of female GSCs persist in the zebrafish testicular somatic cell microenvironment. In conclusion, these data indicate that zebrafish can repeatedly produce fully functional all-X grass carp sperm, which can be used to generate all-female grass carp populations within 3 months. Discussion Cyprinidae is the most diverse family of fish, with 12 subfamilies and more than 360 genera and more than 3000 species 34 . Cyprinids are the world's largest fish production of aquatic products, and grass carp production occupies the first place among cyprinids ( https://www.fao.org/fishery/en/collection/asfis/en ). Most of the fish species of the Cyprinidae family have a higher growth rate in females than males, highlighting the importance of all-female breeding of Cyprinidae fishes 16 . In this study, we found that the gonads of grass carp exhibit sexual dimorphism before differentiation, that is, the female gonad is larger than the male gonad and contains more germ cells. Therefore, with the help of the sexual dimorphism of the gonad morphology, the female GSCs were identified from the juvenile ovaries and transplanted into sterile zebrafish larvae, to achieve the super-fast production of all-X sperm of grass carp. This mono-sex breeding method for fish based on surrogate reproduction does not rely on molecular markers of sex linkage, so it is easy to be extended to other fish with sexual dimorphism and XX/XY genetic sex-determination type. Furthermore, this method can also be used to determine whether the genetic sex-determination type of a certain fish is XX/XY or ZW/ZZ type. GSCs are stem cells in the adult testes or ovaries that can self-renew and differentiate into sperm or oocytes. In mammals, since males produce sperm throughout their lives, it is presumed that there must be self-renewing GSCs in the testes, and the male GSCs have been identified and characterized since 1968 35 . However, in female mammals, it has long been believed that female germ cells enter meiosis before birth and subsequently arrest at meiosis I 36 . In 2004, the existence of female GSCs were discovered in mammalian ovaries for the first time 37 . Since then, the female GSCs have been identified in Drosophila 38 , pig 39 , human 40 , and two laboratory fish medaka and zebrafish 9 , 10 . However, the female GSCs have never been identified in aquaculture fish species. In our study, female GSCs were first identified in an aquaculture fish species, grass carp, based on both nuclear morphology and three molecular markers, Ddx4 labeling germ cells, Nanos2 labeling GSCs, and Pcna labeling mitotic cells 32 . More importantly, the grass carp female GSCs could differentiate into functional sperm after transplanting into sterile zebrafish host, demonstrating the differentiation potential of female GSCs into sperm in fishes. In future, the female GSC-based surrogate reproduction approach could be easily applied to all-female breeding of Cyprinidae family fishes, most of which possess an X/Y sex determination system. Generally, there are two main objectives of cross-species surrogate reproduction, to shorten the gamete maturation period of donor species and to reduce the cultivation space required for donor species. Consequently, overcoming the challenges posed by differences in maturation periods and body sizes between donor and host species is a critical issue in this field. Typically, smaller fish species reach sexual maturity earlier than larger species. It has been reported that the onset of puberty in fish is more closely linked to body size than to age 41 . Once a fish reaches a size sufficient for competing for resources, attracting mates, and providing parental care, the process of pubertal maturation begins 42 . Previous studies on surrogate reproduction have demonstrated the differences in body sizes and sexual maturation times between donor and recipient species to some extent 6 , 7 , 43 . For instance, in the case of Pacific bluefin tuna ( Thunnus orientalis ) as the donor and mackerel ( Scomber japonicus ) as the recipient, the size difference was as much as 6,000-fold 44 . The most dramatic difference in sexual maturation time was observed between loach ( Misgurnus anguillicaudatus ), which reaches sexual maturity in 12 months, and zebrafish, which matures in 3 months—a fourfold difference 45 , 46 . In the present study, the size disparity between zebrafish and grass carp was even more striking, with a weight difference of approximately 16,000-fold (~ 0.3g vs ~ 5kg). Additionally, the difference in their respective sexual maturation periods was 20-fold (3 months vs 5 years). These findings represent a substantial technical breakthrough in fish surrogate reproduction, particularly in addressing the challenges posed by size and maturation differences between donor and host species. In the future, this unique model system offers a fascinating opportunity to investigate how the female GSCs of grass carp can rapidly differentiate into functional sperm within such a short time. Methods Fishes The zebrafish of AB line, which were utilized as the experimental fish in this study, were obtained from the China Zebrafish Resource Center, National Aquatic Biological Resource Center (CZRC/NABRC) located in Wuhan, China. These zebrafish were maintained at a temperature of 28°C and subjected to a light and dark cycle of 14 hours of light followed by 10 hours of darkness. The embryos used for microinjection were collected through natural fertilization. The stages of embryonic development were determined based on the information provided in the referenced paper 47 . PCR analysis of genomic DNA The genomic DNA from grass carp tail was extracted by DNA extraction kit (Cwbio, China), and was used as the template for PCR. PCR was used to amplify a male-specific DNA fragment using the primers according to the previous study 13 (Table S1 ). The PCR reaction system was 20 µL in volume, and the PCR program was as follows: initial denaturation at 94 o C for 2 min, denaturation at 95 o C for 30 s, annealing at 55 o C for 15 s, and extension at 72 o C for 15 s for 35 cycles. To prepare the DNA template of sperm for PCR analysis, 10 µL of diluted sperm were used to extract genomic DNA using the Zebra Fish Direct PCR Kit (Foregene). PCR was performed with the primers listed in Table S1 . RNA isolation, cDNA synthesis, and PCR analysis The gonads were homogenized in the 500 µL RL1 buffer from a total-RNA isolation kit (Vazyme), added proteinase K to a final concentration of 100 µg/mL, and incubated in a 55 o C water bath for two hours. After centrifuge, the supernatant was applied to isolate RNA using the total-RNA isolation kit (Vazyme). RNA was reversed-transcribed into cDNA using HiScript III All-in-one RT SuperMix (Vazyme). PCR was performed with the primers listed in Table S1 . Histological analysis The gonads were dissected from the three-month, 15-month, two-year, three-year, four-year and five-year grass carps, cut into ~ 1cm*1cm*1cm pieces and fixed in Bouin’s solution followed by dehydration and infiltration. Samples were embedded and processed for paraffin sectioning using microtome (Thermo HM340E). Paraffin sections of 2 µm were mounted on slides and processed for hematoxylin and eosin (H&E) staining. The sections were captured with a digital camera under an upright microscope. Immunofluorescence analysis For frozen sections, the gonads were dissected from the three-month, 15-month, two-year, three-year four-year and five-year grass carps, cut into ~ 1cm*1cm*1cm pieces and fixed in 4% PFA at 4 o C overnight. The fixed samples were processed for frozen section according to the previous article 32 . The frozen section was cut into 12 µm and adhered to a PLL-coated cover glass. For suspended cells, 10 µL of the cells were pipetted onto a PLL-coated cover-glass, stay still for 30 mins at 4 o C, and fixed with 4% PFA for 20 minutes at room temperature. Samples on cover glasses were placed into a 24-well plate and applied to high through-put immunofluorescence according to the previous article 32 . The home-made antibodies against Ddx4, Pcna, Sycp3, and Nanos2 were used in this study 28 , 32 . The first antibody was used with a 1:500 dilution and the second antibody was used with a 1:1,000 dilution. For immunofluorescence co-staining, the antibodies of Sycp3, Pcna and Ddx4 were conjugated with Alexa Fluor 488, Cy3 and Alexa Fluor 680 and purified by a local company (Tanda Scientific, Wuhan, China). Fluorescence conjugated antibodies were used with a 1:200 dilution. After immunofluorescence, the sections and cells were counter-stained with 1 µg/mL DAPI for 10 minutes at room temperature. The morphological criteria used to distinguish different types of spermatogenic cells referred to a previous report 32 . The surface areas of spermatocytes were measured using Fiji 48 , and the data were plotted using Graphpad Prism software. Cell counting For counting cells, at least five images were taken from each sample under a fluorescence microscope using a 10x objective. Total cells and Ddx4-positive cells were counted according to the official manual using the particle analysis function of the Fiji software 48 . The percentage of Ddx4-positive cells was calculated using Excel. Preparation of female germline stem cells of grass carp Three-month-old grass carps were anesthetized with MS-222 (0.2 mg/mL) (Sigma). The gonads were dissected and placed in L-15 medium supplemented with 1x Penicillin and Streptomycin (Gibco). About 20 mg gonad was digested with 1 mL of compound digestive solution (0.25% trypsin (Beyotime), 300 U/mL collagenase (Aladdin), 0.05% DNase I (Roche) at 32 o C for three hours. The tissue was pipetted up and down every 30 minutes to facilitate digestion. The cell suspension was filtered through a 40 µm-cell strain, washed twice with D-PBS with 1% fetal bovine serum (FBS), and re-suspended in 1 mL of D-PBS with 1%FBS. The germ cells were enriched with percoll according to the previous article 25 . Cells from 30–40% Percoll fractions were collected and washed twice with D-PBS with 1% FBS and re-suspended with 1mL of D-PBS with 1% FBS. The cells were then stained with Cell Tracker DiO according to the manufacturer's guidance (Invitrogen), and finally suspended in 10 µL of L-15 medium with 1% FBS. Preparation of zebrafish recipient larvae Fertilized eggs were injected with 1nL of 100 µM of dead end 1 ( dnd1 ) antisense morpholino oligonucleotide (5′-GCTGGGCATCCATGTCTCCGACCAT-3′) to eliminate endogenous PGCs according to the previous study (Zhang et al., 2020). The injected embryos were raised in 0.3x Danieau’s buffer (1.5 mM HEPES buffer at pH 7.2, 17.4 mM NaCl, 0.21 mM KCl, 0.18 mM Ca(NO3)2, 0.12 mM MgSO4) until four to five day-post-fertilization (dpf), and served as the recipients. Germ cell transplantation Cell transplantation was performed under a stereomicroscope using a glass micropipette needle with an open end of ~ 50 µm diameter. Zebrafish recipient larvae were anesthetized with MS-222 (0.2 mg/mL) and placed on 2% agar bed. 30–50 donor cells were transplanted into the abdominal cavity under the swim bladder close to the primitive gonads using a micropipette as previously described 25 . The transplanted zebrafish larvae were recovered in 0.3x Danieau’s buffer at 28.5 o C, and transferred into the larvae tank containing water of fish facility the next day. At five day-post transplantation (5 dpt), the transplanted larvae fish were anesthetized with MS-222 (0.2 mg/mL) and directly examined under a fluorescence microscope. RNA sequencing and analysis Sperm samples were collected from five zebrafish, three GSCT zebrafish and five wild-type grass carps. 0.5 µL sperm from each fish was diluted into 20 µL Hank’s buffer. For one sample, 1µL of diluted sperm was used to extract RNA and synthesize cDNA in one reaction using the single cell full length mRNA-amplification kit (N712, Vazyme), and the cDNA was used to prepare DNA library for sequencing using the DNA Library Prep Kit for Illumina (TD504, Vazyme). Sequencing was performed on Illumina Nextseq 500 with read length of 150 bp paired-ended (PE). For each sample, about 6G clean data with Q30 > 90% were generated. The reference genome and annotation of grass carp (GCF_019924925.1) were downloaded from the NCBI database. Hisat2-build 49 was used to construct the reference genome index, and then the clean data was aligned to the reference genome. samtools 50 was used to convert sam files to bam files. The sliding window was taken as 1k, the genomic position was divided into segments, and the coverage of reads on each chromosome was calculated using bedtools coverage 51 . Sperm collection Three month-post transplantation (mpt) zebrafish were anesthetized with MS-222 (0.2 mg/mL), and the sperm of each fish was collected by a 10 µL-pipette, and put into 20 µL Hank’s buffer. The diluted sperm were either used for PCR analysis or scanning electron microscopy. FESEM analysis To prepare the sample for FESEM, 10 µL of sperm were mixed with 50 µL of 2% glutaraldehyde, and then loaded onto a PLL-coated cover-glass and stayed at 4 o C for overnight, dehydrated with 25%, 50%, 75%, 100% EtOH, and finally dried overnight in a hood. After coating with platinum, sperm were visualized using the FESEM (Hitachi S-4800). In vitro fertilization (IVF) The germ line chimera gonad from a zebrafish was dissected into 500uL Hank’s solution (0.137 mM NaCl, 5.4 mM KCl, 0.25 mM Na2 HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, and 4.2 mM NaHCO3) 52 . The gonads were cut into small pieces with ophthalmic micro scissors to release the sperm. 200 µL sperm were added to ~ 100 grass carp eggs, mixed well and flushed with the water from the fish facility. Fertilization rates were calculated using the embryos at six hours post fertilization, the number of developed embryos were counted, and the fertilization rate was calculated by the number of developed embryos divided by the total number. In all, five GSCT testis were used for IVF analysis. Statistical analysis The data was plotted using Graphpad Prism software. The values were presented as mean ± standard deviation (STD) and were analyzed using unpaired and two-tailed Student’s t-test. Statistical significance is represented by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001). Declarations All animal experiments were conducted according to the experimental protocols approved by the Animal Care and Use Committee of the Institute of Hydrobiology, Chinese Academy of Sciences. Data availability The original RNA-seq data has been deposited to the GEO record with the accession number GSE261835, and to the Science Data Bank (https://cstr.cn/31253.11.sciencedb.17281). Acknowledgement We thank Kuoyu Li from China Zebrafish Resource Center, National Aquatic Biological Resource Center for fish care. We thank Fang Zhou, Guangxin Wang, Zhixian Qiao, Xiaocui Chai, and Yuan Xiao from the Analysis and Testing Center of Institute of Hydrobiology, Chinese Academy of Sciences for the technical support of confocal microscopy, high-throughput sequencing, and scanning electron microscopy. This work was supported by the National Natural Science Foundation of China (32025037), Strategic Priority Research Program of the Chinese Academy of Sciences (CAS) (XDB0730300), National Key R&D Program of China (2023YFD2401603), Ministry of Agriculture and Rural Affairs (NK2022010207), Natural Science Foundation of Wuhan, Science and Technology Special Fund of Hainan Province (ZDYF2024XDNY256), and the Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture (CAS). Author contributions Y.S. conceived the idea and oversaw the project. D.Y. designed, performed the experiment and analyzed the data. C.W. participated in most of the experiments. J.Z. and Y.H. participated in transplantation experiments. L.L. performed zebrafish in vitro fertilization. Y.L. performed partial experiments on grass carp. Y.T. performed RNA-seq analysis. X.W. drew the schematic diagram of the experimental process. Z.R., H.W. and M.H. performed partial experiments on zebrafish. 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Nat Methods 12:357–360 Danecek P et al (2021) Twelve years of SAMtools and BCFtools. Gigascience 10 Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842 Westerfield M (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed Additional Declarations There is NO Competing Interest. Supplementary Files V1gcGSCTSupplTab.doc Supplementary table V1gcGSCTSuppl.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5164959","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":361699280,"identity":"fc3842eb-9f00-471f-af86-fb32f3e0f411","order_by":0,"name":"Yonghua 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1","display":"","copyAsset":false,"role":"figure","size":1108325,"visible":true,"origin":"","legend":"\u003cp\u003eThe differences on growth and gonadal size between the male and female of three-month old grass carp. (A) A three-year-old grass carp and a three-month old zebrafish. (B) A typical photograph of a three-month-old grass carp. (C) A typical anatomical picture showing the location of the gonad. (D) A typical photograph of the female and male gonads of a young three-month-old grass carp. (E) PCR detection of male-specific DNA fragments in grass carp (gc-Y) and a DNA fragment of grass carp mstn gene (gc_mstn).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5164959/v1/e8a1b680423cfe8edda8298d.png"},{"id":66041329,"identity":"ba0eb095-18ab-4c4e-a3c4-b9a124731502","added_by":"auto","created_at":"2024-10-07 05:59:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2589205,"visible":true,"origin":"","legend":"\u003cp\u003eHistological and immunofluorescence analysis on the gonads of threemonth- old and 15-month-old grass carp. (A, B) HE staining of gonads of female and male grass carp at three months of age. (C, D) Ddx4 IF staining of gonad sections from three-month-old female (C) and male grass carp (D). (C1,D1) A magnified view of the white box area in C and D. (E, F) HE staining of gonads of female and male grass carp at 15 months of age. (G, H) Ddx4 IF staining of gonad sections from 15-month-old female and male grass carp. (C1,D1) A magnified view of the white box area in G1 and H1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5164959/v1/0de58927d8b43ae9074b0bbb.png"},{"id":66039195,"identity":"db981503-58ba-4da4-8a57-e28c3cedc577","added_by":"auto","created_at":"2024-10-07 05:27:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":760713,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation of donor cells and transplantation. (A) Schematic diagram of the experimental process of producing all-X grass carp sperm with zebrafish. (B) A photo showing different cell layers after discontinuous density gradient centrifugation, and the IF staining of Ddx4 on the germ cells in different cell layers. (C) The percentages of germ cell in different cell layers. All data were collected from three independent experiments. All values are the mean±SEM. (D) A typical picture of wild-type zebrafish embryos with the endogenous PGCs labeled by mCherry. (E) A typical picture of dnd1_MO injected zebrafish embryos with the endogenous PGCs labeled by mCherry. (F) A photo showing that the cells were transplanted into the germinal ridge of a zebrafish recipient at five dpf. (G) A image showing a zebrafish recipient with green fluorescent donor cells at five dpt.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5164959/v1/2479bae3acac416d2ac1524a.png"},{"id":66039196,"identity":"f4e45773-8c2a-4558-abef-6ca32070f498","added_by":"auto","created_at":"2024-10-07 05:27:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2903564,"visible":true,"origin":"","legend":"\u003cp\u003eExamination of GSCT gonads. (A-B) The representative image showing the zebrafish gonad with or without the germ cells of grass carp at two mpt as showed by IF of Ddx4. (C-E) The representative images showing the chimeric gonad with grass carp germ cells (GSCT), the zebrafish gonad without grass carp germ cells (dnd1_MO) and a wildtype zebrafish testis at three mpt. (FQ) The representative images showing the co-staining of Ddx4 (magenta), Sycp3 (green), Pcna (yellow) and DAPI (blue) in the testis transplanted with female germline stem cell (GSCT) at 3 mpt (F-I), the zebrafish testis (zebrafish) at three mpf (J-M), grass carp gonad (grass carp) at six mpf (N-Q). (R) RT-PCR detection of ddx4 homolog in grass carp (gc_ddx4) and ddx4 homolog in zebrafish (zf_ddx4) in the gonads transplanted with female germline stem cell (dnd1_MO and GSCT positive), zf_testis and gc_gonad.(S) The surface area of spermatocytes in GSCT testes and WT zebrafish testes at 3 mpf.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5164959/v1/cc623a39ac6914bd11ba24e2.png"},{"id":66639753,"identity":"4eb5cf83-0728-4d68-8bd0-36360c72d4b7","added_by":"auto","created_at":"2024-10-15 06:01:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":385282,"visible":true,"origin":"","legend":"\u003cp\u003eExamination pf GSCT sperm. (A) PCR detection of a DNA fragment of grass carp mstn gene (gc_mstn), and a DNA fragment of zebrafish nr5a1a gene (zf_nr5a1a). The DNA template of the test samples were from the seminal fluid of GSCT fish. The genomic DNA from a male grass carp (gc_male), a female grass carp (gc_female) and zebrafish were used as controls. (B-D) The FESEM images of sperms derived from zebrafish, the GSCT positive zebrafish testis (GSCT) and grass carp. (E-F) The tail lengths (E) and the head diameters (F) of sperms derived from the zebrafish, GSCT gonad and the grass carp. (G) The coverage of reads on the chromosomes of grass carp. Z-GC, zebrafish-produced grass carp sperms;Z, zebrafish sperms\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5164959/v1/f321518c9342b69cc2319808.png"},{"id":66039200,"identity":"98e841ae-462f-4a27-8119-8fb51b1e2709","added_by":"auto","created_at":"2024-10-07 05:27:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":990577,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro fertilization assay of GSCT sperm. (A-F) In vitro fertilization assay using the GSCT sperm and wildtype grass carp eggs. The wildtype zebrafish (A, D) and the wildtype grass carp (B, E) at 2 dpf and 10 dpf were used as controls.(C, F) The fish fertilized from GSCT sperm and wildtype grass carp eggs. (G) A photo of all-female grass carp at 50 dpf. (H-I) PCR detection of male-specific DNA fragments in grass carp (gc-Y) and a DNA fragment of grass carp mstn gene (gc_mstn). The test samples used in (H) were from the 2 dpf-embryos fertilized by GSCT sperm and grass carp eggs. The DNA templates used in (I) were from the 2 dpf wildtype grass carp embryos. The genomic DNA from a male grass carp (gc_male), a female grass carp (gc_female) and zebrafish were used as the controls.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5164959/v1/76cb784ab3cabfffd8e5c4d0.png"},{"id":98626337,"identity":"2853dda9-4e3a-41b8-b36a-f7c0f98e4ae5","added_by":"auto","created_at":"2025-12-19 17:09:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9923504,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5164959/v1/0a00164d-6d94-470e-9772-ce6bb94abe87.pdf"},{"id":66039193,"identity":"34173af3-bd5a-4852-b5d4-c9cd69a9b62a","added_by":"auto","created_at":"2024-10-07 05:27:47","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":36352,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary table\u003c/p\u003e","description":"","filename":"V1gcGSCTSupplTab.doc","url":"https://assets-eu.researchsquare.com/files/rs-5164959/v1/66937e99e75deab57aaa604e.doc"},{"id":66039201,"identity":"bd8d7d8f-426c-4ea3-aef9-f791626492e6","added_by":"auto","created_at":"2024-10-07 05:27:48","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4622412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"V1gcGSCTSuppl.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5164959/v1/98406d26fb0aee13424c9248.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Super-fast generation of all-female grass carp via transplantation of female germline stem cell into zebrafish","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAquaculture, currently accounting for more than half of the global fish production for human consumption, represents the fastest-growing food production sector \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. To address the escalating demand of aquaculture production driven by human population growth, there is an urgent need for genetic improvement of aquaculture species through modern genetic breeding techniques. Surrogate reproductive technology, involving the production of gametes from a donor species in a recipient species, has emerged as a prominent approach for breeding aquaculture species \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This technology entails transplanting undifferentiated germ cells from a donor fish species into sterile larvae or juvenile fish of an easy-to-breed species. The outcome is the production of functional gametes from the donor fish in the recipient species, with the main goal of shortening the maturation period of donor gametes \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn fish surrogate reproduction, different donor cell types, such as primordial germ cells (PGCs) derived from early embryos and germline stem cells (GSCs) from juvenile or mature gonads were used as donor cells for transplanting into different host fish species \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Among them, spermatogonial stem cells, namely male GSCs have shown to be the most commonly used and the most successful type of donor cells, since male GSCs widely exist in the testes of different species, spanning from the juvenile stage to the adult stage \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In contrast, oogonial stem cells, namely female GSCs were only identified in two model fish, zebrafish and medaka \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Although a previous study showed that the transplanted rainbow trout ovarian germ cells differentiated toward sperm in a male host \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, the presence and identification of female GSCs in aquaculture fish species remains elusive.\u003c/p\u003e \u003cp\u003eThe grass carp (\u003cem\u003eCtenopharyngodon idellus\u003c/em\u003e), a rapidly growing herbivorous freshwater fish species with large body size, has become a predominant species in both Chinese and global freshwater aquaculture \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The sex chromosome system of grass carp is characterized by XX/XY, with the identification of Y-specific sequences enabling the identification of genetic males \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, the prolonged sexual maturation phase of grass carp, spanning approximately five years in the majority of cultivated areas, poses a significant obstacle to the implementation of conventional breeding methods, including hybridization, genetic selection, and sex control, for genetic improvement of grass carp \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Female grass carp exhibit considerably accelerated growth rates compared to their male counterparts, underscoring the significance of prioritizing the breeding of all-female grass carp \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Nevertheless, conventional techniques employed in breeding all-female populations, such as gynogenesis or sex reversal through steroid treatment \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, require a substantial time and extensive effort, primarily attributable to the prolonged maturation period of grass carp. If female GSCs could be identified and isolated from grass carp juvenile ovaries, it might be possible to produce all-X sperm after transplanting female GSCs into recipient males with a short maturation period.\u003c/p\u003e \u003cp\u003eZebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) is a small, laboratory-bred fish species that has been widely used as an animal model for studying vertebrate development, human diseases, and finfish aquaculture\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that laboratory-bred zebrafish strains lack a genetic sex determination system \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Instead, germ cells play an important role in sex differentiation of zebrafish, since a sufficient number of germ cells is required for female development of zebrafish \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and zebrafish lacking endogenous germ cells develop exclusively into males \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In our recent studies, when GSCs or 9 germplasm factors (9GM)-induced primordial germ cells (iPGCs) of Chinese rare minnow (\u003cem\u003eGobiocypris rarus\u003c/em\u003e) were transplanted into germ cell depleted zebrafish, all the zebrafish surrogates developed into males and produced rare minnow sperm \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This indicates that sex-chromosome lost and germ cell-depleted zebrafish could be used as an ideal surrogate host for producing donor-derived sperm. In contrast to the long maturation period of 5 years and a giant body size for grass carp, zebrafish have a sex maturation period of approximately three months \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, with mature individuals typically weighing around 0.3g. Therefore, it is intriguing to test whether gametes from an aquaculture species with a prolonged maturation period and large body sizes, such as grass carp, could be produced by zebrafish with a significantly shorter maturation period and smaller body size (Fig.\u0026nbsp;1A), and to investigate how donor GSCs behave after transplanting into sterile zebrafish host.\u003c/p\u003e \u003cp\u003eIn this study, we characterized grass carp female GSCs from ovaries at three months post-fertilization (mpf) to 15 mpf, marking the first report of female GSCs of an aquaculture species. We further isolated female GSCs from 3 mpf grass carp and efficiently transplanted the purified female GSCs into sterile zebrafish larvae. We demonstrate that grass carp female GSCs with XX chromosomes could be differentiated into functional all-X sperm under the control of gonadal somatic niche of germ cell-depleted zebrafish within three months, which opens a new avenue for advancing precision breeding in aquaculture.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSexual dimorphism of gonads in grass carp at three months post-fertilization\u003c/h2\u003e \u003cp\u003eIn zebrafish, it is well-known that they display sexual dimorphism of gonad sizes between juvenile testes and ovaries \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we wondered whether a similar phenomenon was observed in grass carp. Grass carp generally reach sex maturation at the age of 5 years, we therefore examined the gonadal development of grass carp at different ages. After dissection, the gonads were found to be located on both sides of the swim bladder (Fig.\u0026nbsp;1B, C). In the samples from 3 month mpf grass carp, 2 of the 10 gonads were wider and larger than others (Fig.\u0026nbsp;1D). We then detected the male-specific DNA fragments by PCR with those gonad samples, and found that big-sized gonads, No. 2 and 8, did not show any amplification of male-specific band, and the other eight gonad samples could give an amplification of male-specific bands, indicating that No. 2 and 8 grass carps were genetically females (Fig.\u0026nbsp;1E). These results suggest that the juvenile gonads of grass carp at 3 mpf exhibit a sexual dimorphism between genetic males and females, making it possible to distinguish juvenile ovaries from juvenile testes from the morphological view.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIdentification of female GSCs in grass carp\u003c/h3\u003e\n\u003cp\u003eIt has been reported that GSCs are the only type of donor cells which could colonize, survive and differentiate into mature gametes in a host gonadal somatic niche \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Thus, an ideal donor gonad for GSC transplantation should be abundant in GSCs. To investigate the developmental status of germ cells, we analyzed grass carp gonads at different ages from 3 mpf to 5 years old (yr). At 3 mpf, although the germ cells were more abundant in the females than in the males, they were homogeneous in both types of gonads, (Fig.\u0026nbsp;2A, B), indicating that they were female and male GSCs. More importantly, the uniform germ cells displayed poorly condensed nucleus of large nucleolus, and expressed high level of Ddx4, a factor essential for germline development \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and Nanos2, a reliable GSC marker \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;2C-D1, Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), just mimicking the characteristics of GSCs in zebrafish testes and ovaries \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, further confirming that all the germ cells in juvenile ovaries or testes of grass carp at 3 mpf were GSCs. In 6 mpf grass carp of both sexes, likewise, there were only GSCs existing in the ovaries or testes \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. All these indicate that female GSCs widely exist in the ovaries of female grass carp from 3 mpf to 6 mpf.\u003c/p\u003e \u003cp\u003eIn the ovaries at 15 mpf, plenty of stage I oocytes appeared (Fig.\u0026nbsp;2E, F), and the oogonia positive of Ddx4 and Pcna existed (Fig.\u0026nbsp;2G, Fig S2 A), and they were usually clustered with one or two obvious nucleolus (Fig.\u0026nbsp;2G1), suggesting that they were mitotic active female GSCs. In the testes at 15 mpf, all the germ cells showed poorly condensed nucleus of large nucleolus and expressed high level of Ddx4 and Pcna (Fig.\u0026nbsp;2H, H1, Fig S2 B), indicating that they were still undifferentiated and proliferative GSCs.\u003c/p\u003e \u003cp\u003eWe then checked the gonadal development and gametogenesis of grass carp from two to five years (Fig S3 A-D). The testes of two- and three-year-old grass carp were thin and light pink color, while the testes of four and five-year-old were thick and white (Fig S3 E-H). Histological staining and immunofluorescence further showed that the germ cells in the testes of the two-, three- and four-year old were mainly type A spermatogonia (Fig S3 E1-H3) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The ovaries changed dramatically in morphology and color from two-year old to five-year old (Fig S3 I-L), indicating the differentiation progress of ovaries. Stage IB oocytes appeared in two-year old ovaries (Fig S3 I1), and the stage II oocytes appeared three-year old ovaries (Fig S3 J1). In the four- and five-year old ovaries, the yolk accumulated in the oocytes, indicating that stage III and IV oocytes appeared (Fig S3 K1-L1). Therefore, the ovaries from female grass carp at 3 mpf to 6 mpf and testes from male grass carps at 3 mpf to 4 years old could provide an ideal source of donor GSCs for surrogate reproduction.\u003c/p\u003e\n\u003ch3\u003ePurification and transplantation of female germline stem cells (GSCs)\u003c/h3\u003e\n\u003cp\u003eTo test whether the female GSCs in grass carp juvenile ovaries could really contribute to germline development, we isolated female GSCs from 3 mpf ovaries and transplanted the female GSCs into germline-depleted zebrafish larvae (Fig.\u0026nbsp;3A). After percoll centrifuge of single-cell suspension from 3 mpf ovaries (Fig.\u0026nbsp;3B and C), the GSCs were highly enriched in percoll layers between 30% and 35%, and between 35% and 40%, with 40.5% and 46.9% GSCs among all the DAPI stained cells (Fig.\u0026nbsp;3B and C). The cells from these two layers were combined to serve as the donor female GSCs.\u003c/p\u003e \u003cp\u003eThe host zebrafish were germ cell-depleted by injection of antisense morpholino against \u003cem\u003ednd1\u003c/em\u003e (\u003cem\u003ednd1\u003c/em\u003e_MO), which was verified by labeling of primordial germ cells (PGCs) by \u003cem\u003emCherry-UTRnanos3\u003c/em\u003e mRNA injection \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;3D, E). The grass carp female GSCs cells, labeled by live cell tracker of green fluorescence, were transplanted into the genital ridge between the swim bladder and gut close to the primitive gonads of germ cell-depleted zebrafish larvae at 5 dpf (Fig.\u0026nbsp;3F), according to our recent study \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. About 100 GSCs were transplanted into each larva. Five days post transplantation (dpt), large numbers of labeled grass carp GSCs were observed in the primitive gonad region of about 60% of host zebrafish (Fig.\u0026nbsp;3G), indicating that grass carp donor GSCs survived and colonized in zebrafish surrogates.\u003c/p\u003e\n\u003ch3\u003eSuper-fast spermatogenesis of grass carp female GSCs in zebrafish host\u003c/h3\u003e\n\u003cp\u003eTo determine whether donor grass carp GSCs could reconstitute gametogenesis in sterile zebrafish recipients within a relatively short maturation period, we examined the gonads of GSC transplanted (GSCT) zebrafish at 2 mpt and 3 mpt, and compared these with stage-matched zebrafish, grass carp, and \u003cem\u003ednd\u003c/em\u003e_MO-injected zebrafish (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At 2 mpt, a portion of the GSCT gonads were naive with undifferentiated grass carp germ cells (Fig.\u0026nbsp;4A, B). At 3 mpt, the gonads were dissected from GSCT fish, \u003cem\u003ednd1\u003c/em\u003e_MO injected zebrafish and WT zebrafish for morphological analysis. The GSCT positive gonads appeared non-transparent, similar to the WT zebrafish testes, which were completely different from the germ cell-depleted testes (Fig.\u0026nbsp;4C-E), indicating that spermatogenesis occurred in the GSCT zebrafish.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDevelopmental statistics of GSCT zebrafish from 3 independent trials.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrial No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. with grass carp germ cells / No. observed at 5 dpt (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. colonized / No. observed at 2 mpt (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo. colonized / No. observed at 3 mpt (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo. producing grass carp sperm / No. checked at 3 mpt (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrial 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e85/189 (45.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2/22 (9.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/10 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2/30 (6.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrial 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e123/168 (73.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6/21 (28.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2/11 (18.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6/47 (13.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrial 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e158/256 (61.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2/12 (16.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2/24 (8.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4/73 (5.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo further characterize the spermatogenesis progress of the transplanted grass carp female GSCs in host zebrafish, we conducted immunofluorescent staining of the testes using a series of spermatogenesis markers, including Ddx4, Sycp3, and Pcna, which indicate different types of spermatogenic cells \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In the developing testis, numerous cells were undergoing mitosis and meiosis in the GSCT positive testis (Fig.\u0026nbsp;4F-I), mimicking what was observed in the zebrafish adult testis at 3 mpf (Fig.\u0026nbsp;4J-M). Immunostaining of Ddx4 confirmed that the transplanted grass carp GSCs had successfully resumed spermatogenesis in the infertile zebrafish testis, and various germ cells including spermatogonia, spermatocytes, spermatids, and spermatozoa were present in the GSCT positive testis (Fig.\u0026nbsp;4G), consistent with observation in the WT zebrafish testis (Fig.\u0026nbsp;4K). In contrast, at 6 mpf, the grass carp testis only contained large numbers of spermatogonia, which were Ddx4 and Pcna-positive and Sycp3-negative, indicating they were in a proliferative rather than meiotic state (Fig.\u0026nbsp;4N-Q). RT-PCR amplification with grass carp and zebrafish \u003cem\u003eddx4-\u003c/em\u003especific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) further validated that the germ cells in GSCT positive testes were derived from transplanted grass carp GSCs but not endogenous zebrafish GSCs (Fig.\u0026nbsp;4R). Additionally, we found that the cyst surfaces of spermatocytes in the GSCT testes were larger than those in the zebrafish adult testes, indicating that the spermatogonial generations in grass carp are more than in zebrafish (Fig.\u0026nbsp;4S). Taken together, these findings demonstrate that grass carp-originated female GSCs can efficiently proliferate and differentiate into mature sperm in zebrafish host testes, and that spermatogenesis cycle of grass carp-derived germ cells in GSCT zebrafish closely resembled that of zebrafish.\u003c/p\u003e\n\u003ch3\u003eGeneration of grass carp sperm in GSCT zebrafish\u003c/h3\u003e\n\u003cp\u003eTo investigate whether zebrafish recipients could produce functional grass carp sperm, we collected semen from all the surrogate zebrafish and examined the species-specific DNA fragment by PCR. PCR analysis of the genomic DNA confirmed that the semen produced by GSCT positive zebrafish males only contained grass carp DNA but not zebrafish DNA, as revealed by amplification of the species-specific genes (Fig.\u0026nbsp;5A).\u003c/p\u003e \u003cp\u003eAlthough the GSCT positive zebrafish could produce matured grass carp sperm, we were interested in whether there was any difference between the sperm derived from the surrogate zebrafish and the grass carp males. By field emission scanning electron microscopy (FESEM) analysis, the morphology of GSCT sperm looked more similar to grass carp sperm than zebrafish sperm (Fig.\u0026nbsp;5B-D). The average tail length of GSCT sperm was nearly identical to that of grass carp sperm, but significantly longer than that of zebrafish sperm (Fig.\u0026nbsp;5E), and the average head diameter of GSCT sperm was significantly shorter than that of zebrafish sperm, and same to that of grass carp sperm (Fig.\u0026nbsp;5F).\u003c/p\u003e \u003cp\u003eTo further examine the genetic consistency among the sperm samples, the GSCT, grass carp and zebrafish sperm were subjected to RNA-seq analysis. The transcriptomes of the three samples were mapped to the reference genome of grass carp and zebrafish. By calculating the coverage of reads on each chromosome, the results revealed an average coverage of 99.7% for the Z-GC (zebrafish-produced grass carp sperms) group, whereas the Z (zebrafish sperms) group exhibited an average coverage of 17.8% (Fig.\u0026nbsp;5G), suggesting that the GSCT sperm are genetically identical to the grass carp sperm. Taken together, all the above data suggest that the zebrafish surrogate successfully produced grass carp-derived sperm.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of all-female offspring from female GSC transplanted zebrafish surrogate\u003c/h2\u003e \u003cp\u003eTo identify whether the GSCT positive zebrafish males could produce functional grass carp sperm, we collected the GSCT sperm and performed in vitro fertilization assay. As controls, the progeny produced by GSCT sperm and zebrafish eggs, as well as those by grass carp sperm and zebrafish eggs were malformed and did not beyond 2 dpf (Fig S4). In contrast, the progeny generated by GSCT sperm and grass carp eggs developed normally, comparable to wildtype grass carp (Fig.\u0026nbsp;6B, C, E, F), and could be raised to later stages without any defects (Fig.\u0026nbsp;6G). PCR analysis of the genomic DNA confirmed that the offspring from GSCT sperm and grass carp eggs were indeed grass carp (Fig.\u0026nbsp;6G). Notably, no male-specific bands were detected in the GSCT sperm fertilized grass carps, whereas half of the wildtype grass carps exhibited the Y-fragment (Fig.\u0026nbsp;6H, I). Intriguingly, the grass carp sperm produced by the zebrafish surrogate could be artificially collected at regular intervals of 2\u0026ndash;3 weeks, totaling up to 20 time per year. This is in stark contrast to grass carp, which produce sperm only once a year in the spring season. This finding suggests that the self-renewal and differentiation of female GSCs persist in the zebrafish testicular somatic cell microenvironment. In conclusion, these data indicate that zebrafish can repeatedly produce fully functional all-X grass carp sperm, which can be used to generate all-female grass carp populations within 3 months.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCyprinidae is the most diverse family of fish, with 12 subfamilies and more than 360 genera and more than 3000 species \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Cyprinids are the world's largest fish production of aquatic products, and grass carp production occupies the first place among cyprinids (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fao.org/fishery/en/collection/asfis/en\u003c/span\u003e\u003cspan address=\"https://www.fao.org/fishery/en/collection/asfis/en\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Most of the fish species of the \u003cem\u003eCyprinidae\u003c/em\u003e family have a higher growth rate in females than males, highlighting the importance of all-female breeding of \u003cem\u003eCyprinidae\u003c/em\u003e fishes \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In this study, we found that the gonads of grass carp exhibit sexual dimorphism before differentiation, that is, the female gonad is larger than the male gonad and contains more germ cells. Therefore, with the help of the sexual dimorphism of the gonad morphology, the female GSCs were identified from the juvenile ovaries and transplanted into sterile zebrafish larvae, to achieve the super-fast production of all-X sperm of grass carp. This mono-sex breeding method for fish based on surrogate reproduction does not rely on molecular markers of sex linkage, so it is easy to be extended to other fish with sexual dimorphism and XX/XY genetic sex-determination type. Furthermore, this method can also be used to determine whether the genetic sex-determination type of a certain fish is XX/XY or ZW/ZZ type.\u003c/p\u003e \u003cp\u003eGSCs are stem cells in the adult testes or ovaries that can self-renew and differentiate into sperm or oocytes. In mammals, since males produce sperm throughout their lives, it is presumed that there must be self-renewing GSCs in the testes, and the male GSCs have been identified and characterized since 1968 \u003csup\u003e35\u003c/sup\u003e. However, in female mammals, it has long been believed that female germ cells enter meiosis before birth and subsequently arrest at meiosis I \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In 2004, the existence of female GSCs were discovered in mammalian ovaries for the first time \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Since then, the female GSCs have been identified in Drosophila \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, pig \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, human \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and two laboratory fish medaka and zebrafish \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, the female GSCs have never been identified in aquaculture fish species. In our study, female GSCs were first identified in an aquaculture fish species, grass carp, based on both nuclear morphology and three molecular markers, Ddx4 labeling germ cells, Nanos2 labeling GSCs, and Pcna labeling mitotic cells \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. More importantly, the grass carp female GSCs could differentiate into functional sperm after transplanting into sterile zebrafish host, demonstrating the differentiation potential of female GSCs into sperm in fishes. In future, the female GSC-based surrogate reproduction approach could be easily applied to all-female breeding of \u003cem\u003eCyprinidae\u003c/em\u003e family fishes, most of which possess an X/Y sex determination system.\u003c/p\u003e \u003cp\u003eGenerally, there are two main objectives of cross-species surrogate reproduction, to shorten the gamete maturation period of donor species and to reduce the cultivation space required for donor species. Consequently, overcoming the challenges posed by differences in maturation periods and body sizes between donor and host species is a critical issue in this field. Typically, smaller fish species reach sexual maturity earlier than larger species. It has been reported that the onset of puberty in fish is more closely linked to body size than to age \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Once a fish reaches a size sufficient for competing for resources, attracting mates, and providing parental care, the process of pubertal maturation begins \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Previous studies on surrogate reproduction have demonstrated the differences in body sizes and sexual maturation times between donor and recipient species to some extent \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. For instance, in the case of Pacific bluefin tuna (\u003cem\u003eThunnus orientalis\u003c/em\u003e) as the donor and mackerel (\u003cem\u003eScomber japonicus\u003c/em\u003e) as the recipient, the size difference was as much as 6,000-fold \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The most dramatic difference in sexual maturation time was observed between loach (\u003cem\u003eMisgurnus anguillicaudatus\u003c/em\u003e), which reaches sexual maturity in 12 months, and zebrafish, which matures in 3 months\u0026mdash;a fourfold difference \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In the present study, the size disparity between zebrafish and grass carp was even more striking, with a weight difference of approximately 16,000-fold (~\u0026thinsp;0.3g vs\u0026thinsp;~\u0026thinsp;5kg). Additionally, the difference in their respective sexual maturation periods was 20-fold (3 months vs 5 years). These findings represent a substantial technical breakthrough in fish surrogate reproduction, particularly in addressing the challenges posed by size and maturation differences between donor and host species. In the future, this unique model system offers a fascinating opportunity to investigate how the female GSCs of grass carp can rapidly differentiate into functional sperm within such a short time.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eFishes\u003c/h2\u003e\n \u003cp\u003eThe zebrafish of AB line, which were utilized as the experimental fish in this study, were obtained from the China Zebrafish Resource Center, National Aquatic Biological Resource Center (CZRC/NABRC) located in Wuhan, China. These zebrafish were maintained at a temperature of 28\u0026deg;C and subjected to a light and dark cycle of 14 hours of light followed by 10 hours of darkness. The embryos used for microinjection were collected through natural fertilization. The stages of embryonic development were determined based on the information provided in the referenced paper \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003ePCR analysis of genomic DNA\u003c/h2\u003e\n \u003cp\u003eThe genomic DNA from grass carp tail was extracted by DNA extraction kit (Cwbio, China), and was used as the template for PCR. PCR was used to amplify a male-specific DNA fragment using the primers according to the previous study \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). The PCR reaction system was 20 \u0026micro;L in volume, and the PCR program was as follows: initial denaturation at 94 \u003csup\u003eo\u003c/sup\u003eC for 2 min, denaturation at 95 \u003csup\u003eo\u003c/sup\u003eC for 30 s, annealing at 55 \u003csup\u003eo\u003c/sup\u003eC for 15 s, and extension at 72 \u003csup\u003eo\u003c/sup\u003eC for 15 s for 35 cycles. To prepare the DNA template of sperm for PCR analysis, 10 \u0026micro;L of diluted sperm were used to extract genomic DNA using the Zebra Fish Direct PCR Kit (Foregene). PCR was performed with the primers listed in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA isolation, cDNA synthesis, and PCR analysis\u003c/h2\u003e\n \u003cp\u003eThe gonads were homogenized in the 500 \u0026micro;L RL1 buffer from a total-RNA isolation kit (Vazyme), added proteinase K to a final concentration of 100 \u0026micro;g/mL, and incubated in a 55 \u003csup\u003eo\u003c/sup\u003eC water bath for two hours. After centrifuge, the supernatant was applied to isolate RNA using the total-RNA isolation kit (Vazyme). RNA was reversed-transcribed into cDNA using HiScript III All-in-one RT SuperMix (Vazyme). PCR was performed with the primers listed in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eHistological analysis\u003c/h2\u003e\n \u003cp\u003eThe gonads were dissected from the three-month, 15-month, two-year, three-year, four-year and five-year grass carps, cut into ~\u0026thinsp;1cm*1cm*1cm pieces and fixed in Bouin\u0026rsquo;s solution followed by dehydration and infiltration. Samples were embedded and processed for paraffin sectioning using microtome (Thermo HM340E). Paraffin sections of 2 \u0026micro;m were mounted on slides and processed for hematoxylin and eosin (H\u0026amp;E) staining. The sections were captured with a digital camera under an upright microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunofluorescence analysis\u003c/h2\u003e\n \u003cp\u003eFor frozen sections, the gonads were dissected from the three-month, 15-month, two-year, three-year four-year and five-year grass carps, cut into ~\u0026thinsp;1cm*1cm*1cm pieces and fixed in 4% PFA at 4 \u003csup\u003eo\u003c/sup\u003eC overnight. The fixed samples were processed for frozen section according to the previous article \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The frozen section was cut into 12 \u0026micro;m and adhered to a PLL-coated cover glass.\u003c/p\u003e\n \u003cp\u003eFor suspended cells, 10 \u0026micro;L of the cells were pipetted onto a PLL-coated cover-glass, stay still for 30 mins at 4 \u003csup\u003eo\u003c/sup\u003eC, and fixed with 4% PFA for 20 minutes at room temperature. Samples on cover glasses were placed into a 24-well plate and applied to high through-put immunofluorescence according to the previous article \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The home-made antibodies against Ddx4, Pcna, Sycp3, and Nanos2 were used in this study \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The first antibody was used with a 1:500 dilution and the second antibody was used with a 1:1,000 dilution. For immunofluorescence co-staining, the antibodies of Sycp3, Pcna and Ddx4 were conjugated with Alexa Fluor 488, Cy3 and Alexa Fluor 680 and purified by a local company (Tanda Scientific, Wuhan, China). Fluorescence conjugated antibodies were used with a 1:200 dilution. After immunofluorescence, the sections and cells were counter-stained with 1 \u0026micro;g/mL DAPI for 10 minutes at room temperature. The morphological criteria used to distinguish different types of spermatogenic cells referred to a previous report \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The surface areas of spermatocytes were measured using Fiji \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and the data were plotted using Graphpad Prism software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eCell counting\u003c/h2\u003e\n \u003cp\u003eFor counting cells, at least five images were taken from each sample under a fluorescence microscope using a 10x objective. Total cells and Ddx4-positive cells were counted according to the official manual using the particle analysis function of the Fiji software \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The percentage of Ddx4-positive cells was calculated using Excel.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003ePreparation of female germline stem cells of grass carp\u003c/h2\u003e\n \u003cp\u003eThree-month-old grass carps were anesthetized with MS-222 (0.2 mg/mL) (Sigma). The gonads were dissected and placed in L-15 medium supplemented with 1x Penicillin and Streptomycin (Gibco). About 20 mg gonad was digested with 1 mL of compound digestive solution (0.25% trypsin (Beyotime), 300 U/mL collagenase (Aladdin), 0.05% DNase I (Roche) at 32 \u003csup\u003eo\u003c/sup\u003eC for three hours. The tissue was pipetted up and down every 30 minutes to facilitate digestion. The cell suspension was filtered through a 40 \u0026micro;m-cell strain, washed twice with D-PBS with 1% fetal bovine serum (FBS), and re-suspended in 1 mL of D-PBS with 1%FBS. The germ cells were enriched with percoll according to the previous article \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Cells from 30\u0026ndash;40% Percoll fractions were collected and washed twice with D-PBS with 1% FBS and re-suspended with 1mL of D-PBS with 1% FBS. The cells were then stained with Cell Tracker DiO according to the manufacturer\u0026apos;s guidance (Invitrogen), and finally suspended in 10 \u0026micro;L of L-15 medium with 1% FBS.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003ePreparation of zebrafish recipient larvae\u003c/h2\u003e\n \u003cp\u003eFertilized eggs were injected with 1nL of 100 \u0026micro;M of \u003cem\u003edead end 1\u003c/em\u003e (\u003cem\u003ednd1\u003c/em\u003e) antisense morpholino oligonucleotide (5\u0026prime;-GCTGGGCATCCATGTCTCCGACCAT-3\u0026prime;) to eliminate endogenous PGCs according to the previous study (Zhang et al., 2020). The injected embryos were raised in 0.3x Danieau\u0026rsquo;s buffer (1.5 mM HEPES buffer at pH 7.2, 17.4 mM NaCl, 0.21 mM KCl, 0.18 mM Ca(NO3)2, 0.12 mM MgSO4) until four to five day-post-fertilization (dpf), and served as the recipients.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eGerm cell transplantation\u003c/h2\u003e\n \u003cp\u003eCell transplantation was performed under a stereomicroscope using a glass micropipette needle with an open end of ~\u0026thinsp;50 \u0026micro;m diameter. Zebrafish recipient larvae were anesthetized with MS-222 (0.2 mg/mL) and placed on 2% agar bed. 30\u0026ndash;50 donor cells were transplanted into the abdominal cavity under the swim bladder close to the primitive gonads using a micropipette as previously described \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The transplanted zebrafish larvae were recovered in 0.3x Danieau\u0026rsquo;s buffer at 28.5 \u003csup\u003eo\u003c/sup\u003eC, and transferred into the larvae tank containing water of fish facility the next day. At five day-post transplantation (5 dpt), the transplanted larvae fish were anesthetized with MS-222 (0.2 mg/mL) and directly examined under a fluorescence microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA sequencing and analysis\u003c/h2\u003e\n \u003cp\u003eSperm samples were collected from five zebrafish, three GSCT zebrafish and five wild-type grass carps. 0.5 \u0026micro;L sperm from each fish was diluted into 20 \u0026micro;L Hank\u0026rsquo;s buffer. For one sample, 1\u0026micro;L of diluted sperm was used to extract RNA and synthesize cDNA in one reaction using the single cell full length mRNA-amplification kit (N712, Vazyme), and the cDNA was used to prepare DNA library for sequencing using the DNA Library Prep Kit for Illumina (TD504, Vazyme). Sequencing was performed on Illumina Nextseq 500 with read length of 150 bp paired-ended (PE). For each sample, about 6G clean data with Q30\u0026thinsp;\u0026gt;\u0026thinsp;90% were generated. The reference genome and annotation of grass carp (GCF_019924925.1) were downloaded from the NCBI database. Hisat2-build \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e was used to construct the reference genome index, and then the clean data was aligned to the reference genome. samtools \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e was used to convert sam files to bam files. The sliding window was taken as 1k, the genomic position was divided into segments, and the coverage of reads on each chromosome was calculated using bedtools coverage \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eSperm collection\u003c/h2\u003e\n \u003cp\u003eThree month-post transplantation (mpt) zebrafish were anesthetized with MS-222 (0.2 mg/mL), and the sperm of each fish was collected by a 10 \u0026micro;L-pipette, and put into 20 \u0026micro;L Hank\u0026rsquo;s buffer. The diluted sperm were either used for PCR analysis or scanning electron microscopy.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eFESEM analysis\u003c/h2\u003e\n \u003cp\u003eTo prepare the sample for FESEM, 10 \u0026micro;L of sperm were mixed with 50 \u0026micro;L of 2% glutaraldehyde, and then loaded onto a PLL-coated cover-glass and stayed at 4\u003csup\u003eo\u003c/sup\u003eC for overnight, dehydrated with 25%, 50%, 75%, 100% EtOH, and finally dried overnight in a hood. After coating with platinum, sperm were visualized using the FESEM (Hitachi S-4800).\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eIn vitro fertilization (IVF)\u003c/h2\u003e\n \u003cp\u003eThe germ line chimera gonad from a zebrafish was dissected into 500uL Hank\u0026rsquo;s solution (0.137 mM NaCl, 5.4 mM KCl, 0.25 mM Na2 HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, and 4.2 mM NaHCO3)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The gonads were cut into small pieces with ophthalmic micro scissors to release the sperm. 200 \u0026micro;L sperm were added to ~\u0026thinsp;100 grass carp eggs, mixed well and flushed with the water from the fish facility. Fertilization rates were calculated using the embryos at six hours post fertilization, the number of developed embryos were counted, and the fertilization rate was calculated by the number of developed embryos divided by the total number. In all, five GSCT testis were used for IVF analysis.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eThe data was plotted using Graphpad Prism software. The values were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (STD) and were analyzed using unpaired and two-tailed Student\u0026rsquo;s t-test. Statistical significance is represented by asterisks (*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll animal experiments were conducted according to the experimental protocols approved by the Animal Care and Use Committee of the Institute of Hydrobiology, Chinese Academy of Sciences.\u003c/p\u003e\n\n\u003ch3\u003eData availability\u003c/h3\u003e\n\u003cp\u003eThe original RNA-seq data has been deposited to the GEO record with the accession number GSE261835, and to the Science Data Bank (https://cstr.cn/31253.11.sciencedb.17281).\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank Kuoyu Li from China Zebrafish Resource Center, National Aquatic Biological Resource Center for fish care. We thank Fang Zhou, Guangxin Wang, Zhixian Qiao, Xiaocui Chai, and Yuan Xiao from the Analysis and Testing Center of Institute of Hydrobiology, Chinese Academy of Sciences for the technical support of confocal microscopy, high-throughput sequencing, and scanning electron microscopy. This work was supported by the National Natural Science Foundation of China (32025037), Strategic Priority Research Program of the Chinese Academy of Sciences (CAS) (XDB0730300), National Key R\u0026amp;D Program of China (2023YFD2401603), Ministry of Agriculture and Rural Affairs (NK2022010207), Natural Science Foundation of Wuhan, Science and Technology Special Fund of Hainan Province (ZDYF2024XDNY256), and the Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture (CAS).\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eY.S. conceived the idea and oversaw the project. D.Y. designed, performed the experiment and analyzed the data. C.W. participated in most of the experiments. J.Z. and Y.H. participated in transplantation experiments. L.L. performed zebrafish in vitro fertilization. Y.L. performed partial experiments on grass carp. Y.T. performed RNA-seq analysis. X.W. drew the schematic diagram of the experimental process. Z.R., H.W. and M.H. performed partial experiments on zebrafish. D.Y. and Y.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSun Y, Zhu Z (2019) Designing future farmed fishes using genome editing. 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A guide for the laboratory use of zebrafish (Danio rerio). 4th ed\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"zebrafish, grass carp, female germline stem cell, sex control, maturation period","lastPublishedDoi":"10.21203/rs.3.rs-5164959/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5164959/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSurrogate reproduction has emerged as a powerful biotechnology in fish breeding, mainly aiming at shortening the maturation period of aquaculture species. Grass carp (\u003cem\u003eCtenopharyngodon idellus\u003c/em\u003e), possesses one of the largest body sizes and highest global production yields in freshwater aquaculture. However, the reproduction and genetic breeding of grass carp are significantly hindered, primarily due to its protracted sexual maturation period of nearly five years and the need for extensive cultivation space. In this study, we develop a super-fast strategy to breed all-female grass carp within half a year, leveraging surrogate production in a small-sized laboratory fish, zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e). We characterized and purified female germline stem cells (GSCs) from grass carp juvenile ovary at three months post-fertilization, which is the first report of female GSCs in an aquaculture species. The grass carp female GSCs were transplanted into germ cell-depleted zebrafish larvae to generate surrogate zebrafish. The transplanted grass carp female GSCs underwent accelerated spermatogenesis in the zebrafish recipients. Three months after transplantation, the zebrafish recipients developed into males capable of producing all-X sperm derived from donor grass carp female GSCs. When these sperm were fertilized with wildtype grass carp eggs, a population of all-female grass carp was produced. Our study demonstrates that fish female GSCs with XX chromosomes can be differentiated into functional sperm in a short time under the control of zebrafish gonadal somatic niche, which opens a new avenue for precision breeding in aquaculture.\u003c/p\u003e","manuscriptTitle":"Super-fast generation of all-female grass carp via transplantation of female germline stem cell into zebrafish","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-07 05:27:43","doi":"10.21203/rs.3.rs-5164959/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"18af06e9-a37b-47d2-adff-854e22b703b0","owner":[],"postedDate":"October 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38479588,"name":"Biological sciences/Developmental biology/Germline development"},{"id":38479589,"name":"Biological sciences/Biotechnology/Animal biotechnology"}],"tags":[],"updatedAt":"2025-12-19T02:50:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-07 05:27:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5164959","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5164959","identity":"rs-5164959","version":["v1"]},"buildId":"cTy_lsJlmDsVRNrSptgXS","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}