Reproductive characteristics and suitability of sterile dead end knockout nibe croaker as a recipient for intraperitoneal germ cell transplantation

Reproductive characteristics and suitability of sterile dead end knockout nibe croaker as a recipient for intraperitoneal germ cell transplantation | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Reproductive characteristics and suitability of sterile dead end knockout nibe croaker as a recipient for intraperitoneal germ cell transplantation Ryosuke Yazawa, Kyoichiro Saitoh, Akihiro Yamauchi, Onur Eyuboglu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3996577/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Apr, 2024 Read the published version in Marine Biotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract The use of sterile recipients is crucial for efficiently producing donor-derived offspring through surrogate broodstock technology for practical aquaculture applications. Although knockout (KO) of the dead end ( dnd ) gene has been used in previous studies as a sterilization method, it has not been reported in marine fish. In this study, nibe croaker was utilized as a model for marine teleosts that produce small pelagic eggs, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to produce dnd KO fish. The F1 generation, which carried a nonsense mutation in the dnd gene, was produced by mating founder individuals with wild-type counterparts. Subsequently, the F2 generation was produced by mating the resulting males and females. Among the F2 generations, 24.0% consisted of homozygous KO individuals. Histological analysis revealed that primordial germ cells (PGCs) were present in homozygous KO individuals at 10 days post hatching (dph), similar to wild-type individuals. However, by 20 dph, PGCs were absent in KO individuals. Furthermore, no germ cells were observed in the gonads of both sexes of homozygous KO individuals at 6 months old, which is the typical maturity age for wild-type individuals of both sexes. In addition, when cryopreserved donor nibe croaker testicular cells were transplanted, only donor-derived offspring were successfully obtained through the spontaneous mating of homozygous KO recipient parents. Results indicate that dnd KO nibe croaker lack germ cells and can serve as promising recipients, producing only donor-derived gametes as surrogate broodstock. Surrogate broodstock technology knock-out CRISPR/Cas9 sterile nibe croaker Nibea mitsukurii germ cell transplantation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Sciaenid fish, also known as croakers and drum fish, belong to the family Sciaenidae , which is a diverse group of marine fish. They are widely distributed and found in temperate estuaries and coastal environments. The aquaculture of Sciaenid fishes is being extensively practiced worldwide because they possess biological characteristics suitable for aquaculture, such as being eurythermal and euryhaline; adapt well to various farming conditions; and exhibit good growth performance in captive environments (FAO 2023; Cárdenas et al. 2018). Within the Sciaenidae family, large yellow croaker ( Larimichthys crocea ), red drum ( Sciaenops ocellatus ), meagre ( Argyrosomus regius ) are the main cultivated species worldwide, and shi drum ( Umbrina cirrosa ), Japanese meagre ( Argyrosomus japonicus ), and brown croaker ( Miichthys miiuy ) are consistently produced (FAO 2023). Moreover, as a new target species for aquaculture, other Sciaenid fishes are attracting global attention. Although most Sciaenidae species show high growth and survival rates under farming conditions, desirable strains, such as those with disease resistance and better feed conversion rates, must be established through genetic breeding. Surrogate broodstock technology can be a powerful tool to support genetic breeding, as the production of donor-derived gametes by transplantation of allogeneic or xenogeneic germ cells into recipient individuals is facilitated (Yoshizaki and Lee 2018 ; Yoshizaki & Yazawa, 2019 ; Takeuchi et al., 2020 ). This technology can enable the production of large, long-generation-time species using small surrogates with short generation time in small land-based tanks and can establish elite strains through genetic breeding in a short period of time, with low labor and cost. In addition, germ cells can be cryopreserved in liquid nitrogen semipermanently, and live fish derived from the cryopreserved germ cells could be produced at any time by transplantation of thawed germ cells into male and female recipients (Lee et al., 2013 , 2016 ; Yoshizaki & Lee, 2018 ). This process allows for the long-term preservation of elite strains without rearing live fish in a tank or net pen. We previously established a surrogate broodstock technology for Sciaenidae using nibe croaker, also known as blue drum ( Nibea mitsukurii ), as the recipient species (Takeuchi et al., 2009 ; Yoshikawa et al., 2017 ). Nibe croaker is distributed in central and southern Japan and the East China Sea (Kinoshita et al., 1988), and it is a promising recipient species for Sciaenids because it can mature in a short generation time (4 and 6 months in male and female, respectively), and at a small size (15 cm in total length) in land-based small tanks. In surrogate broodstock technology, sterilization of recipients is the key for the production of only donor-derived gametes from the transplanted recipients. Previously, in various fish species, sterile fish resulted from the gene knockdown (KD) or knockout (KO) of the dead end ( dnd ) gene (Baloch et al., 2021), which is necessary for the maintenance of primordial germ cells and their specification (Weidinger et al., 2003 ; Gross-Thebing et al., 2017 ), and they are used as recipients for germ cell transplantation (Saito et al., 2008; Yoshizaki et al., 2016 ; Li et al., 2017 ; Octavera & Yoshizaki, 2019 , 2020 ; Octavera et al. 2023 ; Marinović et al., 2019; Yoshikawa et al., 2020 ; Franěk et al., 2021 ). Recently, we have successfully produced F2-generation mutants in which the dnd gene was knocked out using the clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system in rainbow trout, indicating that these homozygous KO fish completely lack of endogenous germ cells and can be used as recipients for the surrogate broodstock technology (Fujihara et al, 2022 ). However, no reports have been found on this technique applied to marine teleost. As a first step in applying this technique to marine fish that produce small and fragile pelagic eggs, nibe croaker, a promising recipient species for Sciaenids, was utilized. In this study, we aimed to produce sterile nibe croaker without germ cells using the CRISPR/Cas9 system to knockout the dnd gene and evaluate their suitability as surrogate broodstock. Materials and methods Ethics All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals from Tokyo University of Marine Science and Technology, Japan. Fish Nibe croaker broodstock (approximate body weight (BW) of 300 g and total length of 250 mm) were maintained in a 0.5-m 3 circular fiber-reinforced plastic (FRP) tank at Tateyama Station (Banda), Field Science Center of Tokyo University of Marine Science and Technology (Chiba, Japan). Water temperature was maintained at 23°C–26°C using aquarium heaters, and the photoperiod was set at 16–18 h day length for the first 3 months, following the natural photoperiod. Fertilized eggs were collected by water flow into an egg collection net. Preparation of genome‑editing tools and their microinjection into fertilized eggs of nibe croaker A guide RNA (gRNA) targeting the dnd gene (GenBank Accession No. LC317114) of nibe croaker was designed on the basis of a report of dnd KO in Atlantic salmon (Wargelius et al., 2016 ). First, in the area of the nibe croaker dnd gene, “CHOP CHOP” ( http://chopchop.cbu.uib.no/index.php ) was used to search for candidate sequences. Then, microhomologies at both ends of the cleavage sites of the obtained target sequences were searched using the “search for the CRISPR target site with microhomology sequences” ( http://viewer.shigen.info/cgi-bin/crispr/crispr.cgi ) and candidate sequences that prevent in-frame mutations caused by microhomology within the sequence. Using the abovementioned process, a gRNA target site (5′-GCCCCACCGAGCTGAACAGGGGG-3′, Fig. 1 ) was designed. CRISPR RNA (crRNA) complementary to the target site sequence and trans-activating crRNA (tracrRNA) were chemically synthesized and then purified using high-performance liquid chromatography by Fasmac Co., Ltd. (Kanagawa, Japan). Fertilized eggs of nibe croaker were microinjected following the procedure of Kawamura et al. ( 2022 ) with a slight modification. The glass needles for microinjection were constructed using a puller (PC-10; Narishige Co., Ltd., Tokyo, Japan) and a microgrinder (EG-400; Narishige Co., Ltd.) to produce a needle with a 7-µm tip. Fertilized eggs were aligned in a groove of a 2% agar plate filled with sterilized seawater at 25℃. A total of 1 nL of CRISPR/Cas9 solution containing 40 ng/µL of crRNA, 40 ng/µL of tracrRNA, and 100 ng/µL of Alt-R S.p. Cas9 Nuclease V3 (Integrated DNA Technologies, Coralville, IA, USA) was injected into the cytoplasm of a one-cell-stage embryo using a micromanipulator (MP-2R; Narishige Co., Ltd.) and microinjector (IM-9B; Narishige Co., Ltd.) attached to a stereoscopic microscope (SZX-10; Nikon, Tokyo, Japan). The injected eggs were incubated in a 1-L glass beaker filled with sterilized seawater at 25°C until the embryo-formation stage (20 h postfertilization). The hatching rate of the injected eggs was calculated by dividing the number of successfully hatched larvae by the number of injected eggs and then multiplying the result by 100. Larval rearing of nibe croaker was performed as previously described (Takeuchi et al., 2009 ). In brief, injected eggs were transferred to a 100-L polycarbonate tank supplied with flow-through seawater and maintained at 23°C–26°C. The feeding of larvae began 3 dph. The rotifer Brachionus rotundiformis , fed with freshwater Chlorella (fresh Chlorella V12; Cholera Industry, Tokyo, Japan), was added to the tank two times a day. The densities of rotifers and Nannochloropsis sp. (Marine fresh; ISC, Fukuoka, Japan) in the tank were maintained at 30–40 individuals/mL and 5 × 10 5 cells/mL, respectively. Artemia nauplii were provided from 14 dph. The rotifers and Artemia nauplii were incubated with Hyper Gloss (Marine Tech Co., Ltd., Aichi, Japan) for 6 to 12 h before feeding to increase the n-3 fatty acid concentration in live food. Starting at 20 dph, the larvae were fed an artificial diet (Otohime; Marubeni Nisshin Feed, Tokyo, Japan). Founders were reared in 100-L tanks for approximately 60 days and then transferred into 0.5-m 3 tanks with flow-through seawater maintained under a natural photoperiod and water temperature. Production of homozygous KO mutants When the injected individuals reached 4 months of age, mature male specimens were confirmed by gently squeezing their abdomen to check spermiation. The sperm from these males were artificially inseminated with eggs derived from wild-type females to produce the F1 generation. The resulting F1-generation larvae were sampled and used for mutation detection by the T7 endonuclease I (T7EI) assay. This assay was performed following a procedure modified from Kawamura et al. ( 2022 ). In brief, genomic DNA was extracted from whole hatched larvae. A 244-bp fragment flanking the target site was amplified using the primers nc_dnd_Fw (5′-CCCACTGGATGCCTACGAG-3′) and nc_dnd_Rv (5′-TGGCAGGTCTTCGATACAGAG-3′). PCR was conducted in a 10-µL reaction volume containing 1× PCR Buffer II, 200 µM of dNTPs, 1.5 mM MgCl 2 , 1.25 U of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 10 ng of template DNA, and 1 µM of each primer. The thermal cycling conditions were as follows: 1 cycle of 95°C for 10 min, then 40 cycles of 95°C for 15 s, 52°C for 30 s, and 72°C for 30 s, followed by a final elongation step at 72°C for 5 min. The PCR products were purified using the ISOSPIN PCR Product (Nippon Gene, Tokyo, Japan). In addition, the purified PCR products were digested with T7E1 (New England Biolabs, Beverly, MA, USA) in accordance with the manufacturer’s instructions, and the digestion products were separated on a 2% agarose gel. The PCR products of F1 individuals, which have a mutation in the dnd gene, were sequenced using the dideoxy method (Sanger et al. 1977 ). F1 populations containing mutants were raised until males and females reached maturation at 1 year old. Then, they were subjected to the abovementioned T7EI assay to identify and select heterozygous dnd mutants. The resulting F1 heterozygous mutant females and males were mated to produce the F2 generation. Of the resulting F2 population, 50 individuals were sampled for genotyping: wild-type dnd (+/+), heterozygous KO dnd (+/−), and homozygous KO dnd (−/−), each with specific forward primers for the wild-type allele; nc_dnd_Fw_WTp (5′-AGGATCTGTTGATCCCCCTG-3′) and a specific forward primer for the mutant allele; nc_dnd_Fw_KOp (5′-TGGCAGGTCTTCGATACAGAG-3′) and PCR with common reverse primers; nc_dnd_Rv (5′-TGGCAGGTCTTCGATACAGAG-3′). PCR amplification with the primer sets was conducted using HiDi DNA polymerase (myPOLSBiotec, Konstanz, Germany) that efficiently amplifies from primers that are completely matched at the 3′-end and discriminates templated DNA containing single-nucleotide variations (Drum et al. 2014 ). In addition, PCR amplification was conducted in a 10-µL reaction volume tube containing 1× HiDi Buffer, 200 µM dNTPs, 1 U HiDi DNA polymerase, 25 ng of template DNA, and 0.4 µM of each primer. Then, the PCR mixture was cycled under the following conditions: 1 cycle of 95°C for 3 min, then 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min, followed by a final elongation step at 72°C for 3 min. Afterward, the PCR products were verified under UV light after electrophoresis on a 2.0% agarose gel. Histological analysis of the gonads of dnd KO fish In investigating the gonadal development of dnd KO nibe croaker in the F2 population, wild-type and homozygous dnd KO individuals were selected by PCR for genotyping, and histological and immunohistochemical analyses of the gonads obtained from 10-dph, 20-dph, and 6-months-old nibe croaker were performed. The gonads were paraffin embedded and sliced into 4-µm-thick sections in accordance with the conventional method. Immunohistochemistry was performed in accordance with the method of Yazawa et al. ( 2021 ), and germ cells were detected using an anti-nibe croaker Vasa polyclonal antibody (Yoshikawa et al., 2018 ). Primary antibodies, anti-nibe croaker Vasa polyclonal antibody, were diluted to 1:200 in Can Get Signal Immunostain (Toyobo). Secondary antibodies, goat anti-rabbit Alexa Fluor 488 (Invitrogen Life Technologies, San Diego, CA, USA), were used in accordance with the manufacturer’s instructions. The total number of PGCs was counted by observing the anti-Vasa staining on serial histological sections. Allogenic testicular cell transplantation into homozygous dnd KO recipients The testes from two male nibe croakers, individual “donor-A” (15.0 cm in standard length (SL), 59.3 g BW, and gonad somatic index (GSI) of 0.58%) and individual “donor-B” (14.5 cm in SL, 53.7 g BW, and GSI of 1.42%) caught using hook and line in Tateyama Bay, Chiba Prefecture, Japan, were isolated and cryopreserved following the procedure of Lee et al. ( 2013 ) with a slight modification. The isolated testes were minced and equilibrated in a cryotube containing 1 mL of cryomedium (1.3 M DMSO, 10% egg yolk, 0.1 M trehalose in Leibo-vitz’s L15 medium (Thermo Fischer Scientific, Waltham, MA)) on ice for 60 min and then frozen at − 1°C/min for 90 min using the Bicell freezing container (Nihon Freezer, Tokyo, Japan) in a deep freezer (− 80°C) before preservation in liquid nitrogen. After 6 months of cryopreservation, the cryotubes were thawed in a 25°C water bath for 1–2 min, and the cells were dehydrated in an L-15 medium (pH7.5) supplemented with 1% (vol/vol) fetal bovine serum and 25 mM Hepes. Subsequently, the thawed testes were enzymatically dissociated and used as donor testicular cells for intraperitoneal transplantation in accordance with the procedure reported by Takeuchi et al. ( 2009 ). Approximately 20,000 cells were transplanted into the peritoneal cavity of 12-dph F2 larvae resulting from mating between heterozygous dnd KO (300 recipients for each donor individual) using the method described by Takeuchi et al. ( 2009 ). Recipients were reared until 6 months, and homozygous dnd KO recipients were identified by genotyping PCR. The resulting homozygous dnd KO recipients were then used for the progeny test. In 12-month-old individuals, ten individuals among the homozygous dnd KO recipients identified from 50 transplanted recipients were subjected to histological observations of the gonads according to the aforementioned method. Progeny tests using dnd KO recipients Group mating experiments were conducted in each of the two strains using 6-month-old mature dnd KO recipients in a 0.5-m 3 FRP tank to confirm whether donor-derived offspring can be produced through the natural spawning of dnd KO recipients. In inducing spawning, the water temperature was increased from approximately 20°C to 26°C, and the photoperiod was fixed at 16 h. Floating eggs were collected using an egg collection net attached to the drain of the fish tank. The total number of spawned eggs, floating eggs, and hatched larvae obtained from each spawning event was recorded, and 1-dph larvae were sampled and used for DNA analysis. Microsatellite analysis was performed to confirm whether or not each resultant larva carried donor-derived nDNA. Two loci (501 and 1001) of microsatellite DNA markers designed on the basis of microsatellite regions on the genome of large yellow croaker (GenBank Accession No. GCA_900246015.1) were used for parentage assignment following a procedure described by Morishima et al. ( 2009 ) with a slight modification. The microsatellite marker loci were amplified using the primer set 501 (Fwd; 5′-GTGATGACAGACTGTGATAAGCT-3′, Rev; 5′-GTCACGACGTTGTAAGAGCATTTGATGTAGCAGTGT-3′) and 1001 (Fwd; 5′- ACTCAAACCCACACCTGACA-3′, Rev; 5′-GTCACGACGTTGTAAGGCCATTGGTGACTGATGA-3′). A 10-µL reaction volume comprising 1× ExTaq reaction buffer (TakaraBio, Tokyo, Japan), 200-µM dNTPs, 0.25 U of ExTaq DNA polymerase (TakaraBio), 20 ng of template DNA, 0.1 µM of forward primer, and a mixture of 0.01 µM of reverse primer and 0.1 µM of fluorescently labeled primer (Fam-GTCACGACGTTGTA for 501 and Vic-GTCACGACGTTGTA for 1001) was used to perform PCR amplification. The thermal cycling conditions were as follows: 95°C for 3 min, followed by 30 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 1 min, and a final elongation step at 72°C for 5 min. The resultant samples were electrophoresed on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems by Life Technology, USA), and the fluorescently labeled PCR products were analyzed using GeneScan (Applied Biosystems by Life Technology). Results Production of homozygous dnd KO nibe croaker The duration of the one-cell stage of nibe croaker embryos was approximately 50 min at a water temperature of 25°C. During this time, approximately 60 fertilized eggs were injected per individual. A total of 972 eggs were injected using fertilized eggs obtained from 15 spawning events to produce dnd mutant founders. The hatching rate of the injected group was 28.0% ± 9.0% (n = 15), whereas that of the noninjected control group was 70.0% ± 9.0% (n = 15). Of the injected individuals, 22 survived to 4 months old. T7EI analysis, performed using fin samples obtained from the 22 surviving individuals, revealed that the mutagenic efficiency was 68.2% (15 individuals out of 22 total individuals). Spermiation was confirmed in two male fish at 4 months of age, and milt from these two male fish was used to fertilize eggs stripped from a wild-type female nibe croaker. Subsequent T7EI analysis of 15 larvae from each offspring demonstrated a mutagenic efficiency of 0% in one line (Fig. 2 A) and 13.3% (two individuals out of 15 analyzed individuals, Fig. 2 B) in the other strain. Therefore, the F1 offspring in which mutant individuals were identified was reared until their maturity for the production of F2 generation. The mutation patterns of the dnd gene in F1 mutants were confirmed by sequencing, and all individuals (n = 8) were found to have identical mutations (Fig. 3 A). A three-base deletion and a one-base insertion were found to have occurred in the sequence encoding the 72nd amino acid in the coding region of the dnd protein. Given this mutation, a frameshift occurred, and the resulting stop codon was predicted to occur at position 104 in the coding region of the dnd protein (Fig. 3 B). These F1 heterozygous dnd mutants were reared to maturity. No difference in the fecundity of either male or female heterozygous dnd mutant individuals was observed compared with the wild type. Therefore, the F2 offspring was produced by crossing female and male heterozygous mutants. Genotyping of the resulting F2 larvae (a total of 50 larvae were analyzed) by using mutant-allele-specific PCR methods revealed that 20.0%, 56.0%, and 24.0% of the larvae were wild type, heterozygous dnd KO mutants, and homozygous dnd KO mutants, respectively (Fig. 4 ). Gonadal development in dnd KO nibe croaker The number of PGCs in 10-dph homozygous dnd KO mutants was compared with that in wild type through anti-Vasa staining in immunohistochemical analysis, and the result showed no difference in the number of PGCs between homozygous dnd KO mutants (12.4 ± 2.18 cells) and the wild type (13.6 ± 2.07, Fig. 5 ). At 20 dph, no evident PGCs were recognized by the anti-Vasa antibody in the genital ridges of homozygous dnd KO mutants, whereas the number of germ cells increased to 380 ± 44.7 in the genital ridges of the wild type (Fig. 6 ). Histological analysis of the gonads of homozygous dnd KO mutants at 6 months of age revealed that germ cells were completely absent in either the testes or ovaries in all investigated individuals, whereas numerous oocytes were observed in the ovaries, and spermatogenesis was in progress in the testis of the wild type (Fig. 7 ). Histological analysis further revealed that 17.6% of the larvae (three individuals out of 17 investigated) had ovary-like gonads with ovarian cavities and ovarian lamellae, whereas 82.4% (14/17) exhibited gonads resembling the testes of homozygous dnd KO mutants. However, among their siblings (wild-type and heterozygous dnd KO), 40.4% (13/33) and 59.6% (20/33) possessed an ovary and testes, respectively. Germ cell transplantation using dnd KO nibe croaker as recipients In assessing the suitability of dnd KO nibe croakers as recipients, germ cells (Fig. 8 A) obtained from two wild-caught male fish used as donors (referred to as donor-A and donor-B) were intraperitoneally transplanted into approximately 300 recipients, which were randomly selected from the F2 population, including homozygous dnd KO mutants. After transplantation, approximately 150 recipients from both donor groups survived to 6 months old. Mutant-allele-specific PCR was performed to identify homozygous dnd KO individuals out of 30 transplanted recipients from each donor group. Eight homozygous dnd KO individuals were identified from each of the donor groups and used for progeny tests. To observe the behavior of the donor germ cells in recipient gonads post-transplantation, histological observations were conducted on the gonads of 10 individuals of the homozygous dnd KO recipients, identified through screening among 50 transplanted recipients. The results revealed that in 12-month-old transplanted homozygous dnd KO recipients, 20.0% of females (two out of 10 observed fish, Fig. 8 B) and 40.0% of males (four out of 10 observed fish, Fig. 8 C) exhibited various stages of differentiated germ cells in their gonads compared with nontransplanted fish where both sexes lacked germ cells (Fig. 7 E-H). The rest of the observed recipients displayed a complete absence of germ cells, which is similar to the homozygous dnd KO in nontransplanted fish. Consequently, the gonads of 60% of the homozygous dnd KO recipients exhibited gametogenesis derived from transplanted germ cells. Production of donor-derived offspring from dnd KO surrogate parents Each of the eight (two female and six male) abovementioned homozygous dnd KO recipients was transferred to a 0.5-m 3 FRP tank, and spontaneous spawning was induced by elevating the water temperature. In donor-A transplanted recipients, 131,333 eggs were spawned; the ratio of floating eggs among the total spawned eggs was 10.0%, and the hatching rate among the floating eggs was 100%. In donor-B transplanted recipients, 125,333 eggs were spawned; the ratio of floating eggs among the total spawned eggs was 94.0%, and the hatching rate among the floating eggs was 95.6%. Microsatellite analysis was performed to determine whether or not each resultant larva carried donor-derived nDNA. Two loci (501 and 1001) of microsatellite DNA markers were used for parentage assignment in F1 larvae. Allele varieties at the 1001 locus were as follows: donor-A, 240/246-bp alleles and donor-B, 265/267-bp alleles. The recipient dnd KOs were as follows: 272 or 278-bp alleles. In addition, allele varieties at the 501 locus were as follows: donor-A, 292/316-bp alleles and donor-B, 329/331-bp alleles. The recipient dnd KOs were as follows: 304, 306, 360, or 364-bp alleles. The results of DNA analysis are summarized in Table 1 . The DNA analysis results of 17 and 18 F1 offspring in the donor-A group and donor-B group, respectively, demonstrated that 100% possessed donor-derived alleles in both microsatellite markers, and no recipient-derived alleles were detected (Table 1 ). These data indicate that the paternal and maternal genetic origins of the F1 offspring were donor wild-type nibe croaker. Subsequently, these donor-derived offspring were reared, and no evident abnormalities in the external morphology were observed (Fig. 9 ). Table 1 Summary of microsatellite analysis in F1 larvae obtained by a progeny test Donor-A Donor-B MS 501 1001 501 1001 Recipient 272, 278 304, 306, 360, 364 272, 278 304, 306, 360, 364 Donor 240/246 292/316 265/267 329/331 Genotype Frequency Genotype Frequency Genotype Frequency Genotype Frequency Offspring 240/240 23.5% 292/292 17.6% 265/265 5.6% 329/329 50.0% 240/246 52.9% 292/316 70.6% 265/267 55.6% 329/331 44.4% 246/246 23.5% 316/316 11.8% 267/267 38.9% 331/331 5.6% Discussion In this study, homozygous KOs of the dnd gene were successfully produced in nibe croaker by using the CRISPR/Cas9 system. The resulting dnd KO individuals showed the complete absence of germ cells in adult males and females. Furthermore, when germ cell transplantation was performed using the dnd KO mutants as recipients, they produced only donor-derived offspring through spontaneous spawning. Considering that F2 populations produced by the mating of heterozygous dnd KO F1 males and females were used as recipients in the study of germ cell transplantation, approximately 25% of the transplanted recipients were homozygous dnd KO fish, which is in accordance with Mendelian inheritance. Therefore, homozygous dnd KO recipients must be identified by genotyping PCR after transplantation. Given that the recipient larvae used for transplantation are too small and fragile at the developmental stage, genotyping each individual one by one was not feasible. Although the screening of the posttransplanted larvae was time consuming, the stable production of donor-derived offspring could be achieved after selecting the homozygous dnd KO recipients. In the present study, donor-derived gametes were produced in the gonads of 60% of the homozygous dnd KO recipients, which is high enough to be used for practical applications. When surrogate broodstock technology via germ cell transplantation is applied to practical aquaculture, recipients must produce only donor-derived offspring but not a mixture of donor- and recipient-derived offspring. Therefore, establishing sterilization methods in recipient species is of great importance. The methods commonly used for producing sterile recipients in previous studies, such as triploidization and gene knockdown (KD), require exposure of embryos to temperature shock or microinjection of morpholino oligonucleotides into fertilized eggs (Takeuchi et al. 2020 ). Although these treatments were relatively easy and reproducible in some other species such as salmonids, considerable marine fish show low survival and success rates following triploidization or microinjection into fertilized eggs. Therefore, the application of these sterilization methods to marine species producing small pelagic eggs may not be a reliable option. By contrast, the production of sterile recipients through genome editing demonstrated in this study was relatively stable and less labor intensive once heterozygous KO F1 strains are established. Another issue is sperm production from triploid males in some fish species (Piferrer et al. 2009 ). In the case of triploid nibe croaker, although triploids did not produce any functional gametes in both sexes, they produced small amounts of aneuploid (nonfunctional) gametes without developmental potency (Takeuchi et al, 2018 ). Several studies of dnd KD recipients have also reported the persistence of endogenous germ cells because of incomplete gene KD (Linhartová et al. 2015 ; Yoshizaki et al. 2016 ; Yoshikawa et al. 2020 ). On the contrary, in the case of KO, 100% sterile recipients can be obtained if appropriate screening is performed against the F2 generation produced by mating heterozygous F1 males and females, which could be an advantage in practical aquaculture applications. Interspecific hybridization represents another alternative strategy for producing sterile recipients for surrogate broodstock technology (Takeuchi et al. 2020 ). This approach is suitable for the mass production of recipients for fragile marine species that show high mortality during early developmental stages, as it can be achieved by simple artificial insemination without additional treatments. Recently, we showed that hybrids of female blue dram ( Nibea mitsukurii ) × male white croaker ( Pennahia argentata ) display germ cell–less sterile gonads, and these hybrid recipients effectively produced donor-derived offspring (Yoshikawa et al. 2018 ). However, hybrid viability and sterility vary depending on the combination of the two parental species, and these characteristics are completely unpredictable. Therefore, selecting a suitable sterilization technique that depends on the reproductive characteristics and early survival of each target species is important. PGCs were initially present in dnd KO nibe croaker at 10 dph, which is similar to wild-type individuals; however, their subsequent absence was observed in dnd KO individuals at 20 dph. Therefore, dnd KO nibe croaker still possessed their own germ cells when exogenous donor germ cells were transplanted into the recipients. Similar conditions were also observed in dnd KO rainbow trout, and the maternal supply of dnd mRNA might maintain their PGCs during early embryogenesis (Fujiwara et al. 2022). Therefore, there might be a competition between transplanted donor germ cells and endogenous PGCs until the latter disappears from the recipient’s genital ridges. Meanwhile, PGCs are absent during an earlier stage in dnd KD rainbow trout, and these phenotypic differences can be attributed to the fact that dnd KD simultaneously inhibits the translation of maternally supplied and zygotically expressed dnd mRNA (Yoshizaki et al. 2016 ). Therefore, the advantage of dnd KO fish as a recipient during germ cell migration and their incorporation stage was limited compared with that of dnd KD recipients. However, dnd KO recipients provide sufficient niches to support the proliferation and differentiation of donor-derived germ cells in their developing gonads. Furthermore, this advantage is superior to that of sterile triploids in which a large number of mitotic germ cells remained in the recipient gonads. Dnd KO nibe croaker has great application potential as sterile recipients in applying surrogate broodstock technology to gamete production in a variety of Sciaenid fishes. However, Sciaenid fishes, which are commercially cultivated worldwide, such as large yellow croaker ( Larimichthys ), red drum ( Sciaenops ), and meagre ( Argyrosomus ) belong to different genera of nibe croaker ( Nibea ). In general, the production of donor-derived gametes through transplantation among different genera is more difficult than that between two species belonging to the same genus (Takeuchi et al., 2020 ). To date, a few reports have been found on the successful donor-derived functional egg production in intergeneric transplants, such as slender bittering (donor: Tanakia lanceolata ) and Chinese rosy bitterlings (recipient: Rhodeus ocellatus ) (Octavera et al. 2023 ), as well as common carp (donor: Cyprinus carpio ) and goldfish (recipient: Carassius auratus ) (Franěk et al., 2021 ). Thus, confirming the compatibility with donor species by intragenus germ cell transplantation is necessary to advance the utilization of dnd KO nibe croaker recipients for gamete production of various Sciaenid fishes in aquaculture. Two challenges remain in the application of the dnd KO recipients developed in this study to actual seedling production. First, addressing the male-biased sex ratio in KO individuals is essential. Previous studies have reported that germ cell–deficient individuals such as medaka (Kurokawa et al. 2007 ), zebrafish (Weidinger et al. 2003 ; Slanchev et al. 2005 ; Li et al. 2017 ), nibe croaker (Yoshikawa et al. 2018 ), and chub mackerel (Kawamura et al. 2020 ) are either predominantly or entirely males. In the case of dnd KO nibe croaker, it exhibited a strong male-biased sex ratio similar to the abovementioned species. Meanwhile, a 1:1 sex ratio has been reported in germ cell–deficient fish produced by KD or KO of the dnd gene in several species, such as loach (Fujimoto et al. 2010 ), goldfish (Goto et al. 2012 ), salmon (Wargelius et al. 2016 ), grass puffer (Yoshikawa et al. 2020 ), and rainbow trout (Fujiwara et al, 2022). These reports indicate that the sex ratio of germ cell–deficient fish varies depending on species. For better reliability in increasing the proportion of female recipients, establishing a feminization technique by the administration of estrogen may be necessary, as reported in other species (Piferrer 2001 ). As discussed above, the second issue is the need to select homozygous KO individuals after the transplantation experiment. In achieving 100% production of homozygous dnd KO in the offspring, recent reports suggested the possibility of germ cell rescue by microinjecting wild-type dnd mRNA into fertilized eggs of Atlantic salmon, which are expected to be homozygous KO of the dnd gene (Güralp et al. 2020 ). If this method can be applied to other fish species, then it will be an effective technique for producing all-homozygous dnd KO populations. In the future, addressing these issues will improve this technology, which is suitable for the practical aquaculture industry. Declarations Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was partly supported by JSPS KAKENHI Grant Number 23H00344 and JST-Mirai Program Grant Number JPMJMI21C1. Author Contribution Kyoichiro Saitoh, Akihiro Yamauchi, Onur Eyüboğlu, Kana Ozawa, Wataru Kawamura, and Ryosuke Yazawa conducted experiments and analyzed the data. Ryosuke Yazawa, Tetsuro Morita, Yutaka Takeuchi, and Goro Yoshizaki designed the research. Ryosuke Yazawa and Goro Yoshizaki conceived the study and wrote the manuscript. All authors critically revised the report, commented on drafts of the manuscript, and approved the final report. Acknowledgement This work was partly supported by JSPS KAKENHI Grant Number 20H00430 and 20H00430 and JST-Mirai Program Grant Number JPMJMI21C1. References Cárdenas S (2012) Biología y acuicultura de corvinas en el mundo. AquaTIC 37:1–13 Chen S, Su Y, Hong W (2018) Aquaculture of the large yellow croaker. In: Klinger D, Guo Z (eds) Aquaculture in China: success stories and modern trends, 1st edn. Wiley-Blackwell, Hoboken, NJ, pp 297–308. https://doi.org/10.1002/9781119120759.ch3_10 . 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Supplementary Files supplementaryfile1.pdf Cite Share Download PDF Status: Published Journal Publication published 30 Apr, 2024 Read the published version in Marine Biotechnology → Version 1 posted Editorial decision: Revision requested 30 Mar, 2024 Reviews received at journal 28 Mar, 2024 Reviews received at journal 27 Mar, 2024 Reviewers agreed at journal 18 Mar, 2024 Reviewers agreed at journal 18 Mar, 2024 Reviewers invited by journal 18 Mar, 2024 Editor assigned by journal 15 Mar, 2024 Submission checks completed at journal 14 Mar, 2024 First submitted to journal 28 Feb, 2024 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-3996577","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280192889,"identity":"9332e05a-76b7-4fe0-a63c-fb38b4f2d99c","order_by":0,"name":"Ryosuke Yazawa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBADOQkGxgYGBjYGHjBXgggtxqRrSZwBptiIUGo+I/nxh59tdukz2w+3SXwos5NhYD/8gMFyB24tMjfSDAx725JzZ/MktknOOJfMw8CTZsAgeQa3FgmJBIME3m3MufMYEtukeduYgX7JYWCQbMOnJf3Dwb/b6tPl+B+CtNTzMPC/IaQlx7CZd9vhBGkJsC2HeRgkCNnC86aYWfbfccOZMx42W844d5yHTeKZwQG8fmFP3/zxzZlqeYnz6Q9vfCirtufnT374WBJPiDEIJMCZLOAIBEXNYckGPFr4D8CZzB9gLMaP+LSMglEwCkbBSAMAgFlKqdvDn+YAAAAASUVORK5CYII=","orcid":"","institution":"Tokyo University of Marine Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ryosuke","middleName":"","lastName":"Yazawa","suffix":""},{"id":280192890,"identity":"24adc2e6-1ca5-47c4-be49-865800e159aa","order_by":1,"name":"Kyoichiro Saitoh","email":"","orcid":"","institution":"Tokyo University of Marine Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kyoichiro","middleName":"","lastName":"Saitoh","suffix":""},{"id":280192891,"identity":"b69961c5-3345-418f-b803-67425d0b176e","order_by":2,"name":"Akihiro Yamauchi","email":"","orcid":"","institution":"Tokyo University of Marine Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akihiro","middleName":"","lastName":"Yamauchi","suffix":""},{"id":280192892,"identity":"bd811bca-3c24-433c-81ba-e7a83d977e14","order_by":3,"name":"Onur Eyuboglu","email":"","orcid":"","institution":"Tokyo University of Marine Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Onur","middleName":"","lastName":"Eyuboglu","suffix":""},{"id":280192893,"identity":"278db8a0-6135-4fd9-9322-6fc1e425fc30","order_by":4,"name":"Kana Ozawa","email":"","orcid":"","institution":"Tokyo University of Marine Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kana","middleName":"","lastName":"Ozawa","suffix":""},{"id":280192894,"identity":"6d0c481a-7c8e-4843-816c-f435e012f62d","order_by":5,"name":"Wataru Kawamura","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wataru","middleName":"","lastName":"Kawamura","suffix":""},{"id":280192895,"identity":"4ea0c49c-0c29-4605-8a4a-7a8f9cf9b1f2","order_by":6,"name":"Tetsuro Morita","email":"","orcid":"","institution":"Tokyo University of Marine Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tetsuro","middleName":"","lastName":"Morita","suffix":""},{"id":280192896,"identity":"03857f95-7e96-4424-bffc-9287ef39cd7b","order_by":7,"name":"Yutaka Takeuchi","email":"","orcid":"","institution":"Kanazawa University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yutaka","middleName":"","lastName":"Takeuchi","suffix":""},{"id":280192897,"identity":"5774540f-6a93-4453-8cd0-29ef0fa90e54","order_by":8,"name":"Goro Yoshizaki","email":"","orcid":"","institution":"Tokyo University of Marine Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Goro","middleName":"","lastName":"Yoshizaki","suffix":""}],"badges":[],"createdAt":"2024-02-28 12:05:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3996577/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3996577/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10126-024-10323-x","type":"published","date":"2024-05-01T00:47:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53008507,"identity":"d43fcf06-f02a-4ae7-a0a1-99426444a32b","added_by":"auto","created_at":"2024-03-19 15:19:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64030,"visible":true,"origin":"","legend":"\u003cp\u003eDesign location of the guide RNA targeting the nibe croker \u003cem\u003ednd\u003c/em\u003e gene for the CRISPR/Cas9 system. The nucleotide sequence represents the region where the gRNA was designed in the nibe croker \u003cem\u003ednd\u003c/em\u003e gene (GenBank Accession No. LC317114). The numbers indicated the position of nucleotide in the nibe croker \u003cem\u003ednd\u003c/em\u003e gene, LC317114. The underlined uppercase letters indicated the target site sequence, and the black box indicated the protospacer adjacent motif sequence\u003c/p\u003e","description":"","filename":"figfinal1.png","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/903925933093dc614a45e2c6.png"},{"id":53008511,"identity":"db2551d5-d2ea-4717-a26c-19ea710fe8fc","added_by":"auto","created_at":"2024-03-19 15:19:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":355186,"visible":true,"origin":"","legend":"\u003cp\u003eT7 endonuclease I assay of F1-generation larvae obtained from mosaic founders. Fifteen larvae from each of the two lines (A and B) were used for the assay. Amplicons from PCR targeting the \u003cem\u003ednd\u003c/em\u003e gene are shown as undigested (U, 244 bp) and digested (D, 208 bp) by T7 endonuclease I, which recognizes and cleaves mismatched DNA. No individuals showed mutations in one line (A), whereas two individuals (lanes 5 and 9) exhibited mutations in the other strain (B)\u003c/p\u003e","description":"","filename":"figfinal2.png","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/5bccc91fc01f789a8eba397a.png"},{"id":53008456,"identity":"eaf4e609-a643-4e1a-9b5c-0ce5abeece32","added_by":"auto","created_at":"2024-03-19 15:19:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":212669,"visible":true,"origin":"","legend":"\u003cp\u003eMutation patterns observed in the \u003cem\u003ednd\u003c/em\u003e gene in the strain produced by CRISPR/Cas9. (A) Nucleotide sequence encoding the 70–77th amino acid residues of the nibe croaker DND protein. The top sequence represents the wild type (WT) sequence, and the bottom represents the mutant (\u003cem\u003ednd\u003c/em\u003e mutant) nucleotide sequences. Letters in boxes indicate the estimated amino acid residues from each nucleotide sequence. (B) Estimated amino acid residues for the wild-type (top) and mutant (bottom) sequences were compared. Shaded letters represent the mutated amino acid sequences caused by two-nucleotide deletion. Asterisk indicates stop codon\u003c/p\u003e","description":"","filename":"figfinal3.png","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/248fa5e5c5ab3a332610dd78.png"},{"id":53008508,"identity":"80372490-f846-44f3-bfe6-e84aa118eebf","added_by":"auto","created_at":"2024-03-19 15:19:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":341973,"visible":true,"origin":"","legend":"\u003cp\u003eResults of genotyping by genomic PCR of the F2 generation obtained from the crosses of F1 heterozygous KO individuals. PCR results using wild-type allele-specific primers (WTp) and knockout-allele-specific primers (KOp) for the\u003cem\u003e dnd\u003c/em\u003egene in F2-generation offspring obtained by crossing F1 heterozygous individuals. Lanes 1–50: each larva, WT: wild type, Hetero: heterozygous KO, Homo: homozygous KO, NC: negative control without templated DNA\u003c/p\u003e","description":"","filename":"figfinal4.png","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/c12cf11e83412801faa40337.png"},{"id":53008502,"identity":"6dca2fb4-66d7-447a-8710-d3faa56adb5a","added_by":"auto","created_at":"2024-03-19 15:19:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":911880,"visible":true,"origin":"","legend":"\u003cp\u003eHistological observation of PGCs of wild type (WT) and homozygous \u003cem\u003ednd\u003c/em\u003eKO (KO) nibe croker at 10 dph. HE staining of the peritoneal cavity of WT and KO larvae by sagittal sections and immunohistochemistry (IHC) with the anti-nibe croaker Vasa polyclonal antibody in the same sections. Arrowheads indicate Vasa-positive PGCs. Bars = 20 µm\u003c/p\u003e","description":"","filename":"figfinal5.png","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/abb37007e3be49594e217a30.png"},{"id":53008518,"identity":"58746ec9-e90e-49e8-bef6-4416e843ef43","added_by":"auto","created_at":"2024-03-19 15:19:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1537351,"visible":true,"origin":"","legend":"\u003cp\u003eHistological observation of PGCs and genital ridges of wild type (WT) and homozygous \u003cem\u003ednd\u003c/em\u003e KO (KO) nibe croker at 20 dph. HE staining of the peritoneal cavity of WT (A) and KO (C) individuals by sagittal sections and immunohistochemistry (IHC) with the anti-nibe croaker Vasa polyclonal antibody in the same sections (B and F, respectively). The boxed areas in A, B, E, and F are shown at a higher magnification in C, D, G, and H. Bars = 20 µm (A, B, E, and F) and 10 µm (C, D, G, and H).\u003c/p\u003e","description":"","filename":"figfinal6.png","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/92e9f6d12708d07b0e6d169f.png"},{"id":53008444,"identity":"b75a13f3-b380-403a-afc1-4a612274506b","added_by":"auto","created_at":"2024-03-19 15:19:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2157848,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the ovary and testes of 6-month-old homozygous \u003cem\u003ednd \u003c/em\u003eKO (KO, right columns) nibe croaker with those of wild type (WT, left columns) by histological analysis. Ovary of WT (A and B) and homozygous KO (E and F). Testes of WT (C and D) and homozygous KO (G and H) at 6 months old. The boxed areas in A, C, E, and G are shown at a higher magnification in B, D, F, and H. Bars = 100 µm (A, C, E, and G) and 20 µm (B, D, F, and H)\u003c/p\u003e","description":"","filename":"figfinal7.png","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/fbf511cc671aac8ddfd8fd16.png"},{"id":53008503,"identity":"d4e9118a-2a26-4f55-b0cf-43db9278fb2a","added_by":"auto","created_at":"2024-03-19 15:19:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":706954,"visible":true,"origin":"","legend":"\u003cp\u003eTesticular cell suspension prepared from cryopreserved donor testes (A). Histological observations of the ovary (B) and testes (C) of the 12-month-old homozygous KO recipients that received transplanted germ cells. Bars = 10 µm (A) and 20 µm (B and C)\u003c/p\u003e","description":"","filename":"figfinal8.png","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/4d8760f1d6946279abed3391.png"},{"id":53008524,"identity":"da46ed13-f7d3-4ad5-b318-b447c4f96a67","added_by":"auto","created_at":"2024-03-19 15:19:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":782212,"visible":true,"origin":"","legend":"\u003cp\u003eExternal morphology of donor-derived nibe croaker offspring produced by mating between \u003cem\u003ednd\u003c/em\u003ehomozygous KO recipients, receiving donor germ cells\u003c/p\u003e","description":"","filename":"figfinal9.png","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/549d77fde46921f96452bb96.png"},{"id":55697699,"identity":"220a0170-41a0-4cff-ad8d-ec22c310d414","added_by":"auto","created_at":"2024-05-02 02:21:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5063007,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/12367503-be6f-4bf1-8878-5711c02608c1.pdf"},{"id":53008510,"identity":"365f8fab-2983-440d-87c8-54ba5d759478","added_by":"auto","created_at":"2024-03-19 15:19:22","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":438666,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3996577/v1/19a202a3acec62a89dcde17e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reproductive characteristics and suitability of sterile dead end knockout nibe croaker as a recipient for intraperitoneal germ cell transplantation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSciaenid fish, also known as croakers and drum fish, belong to the family \u003cem\u003eSciaenidae\u003c/em\u003e, which is a diverse group of marine fish. They are widely distributed and found in temperate estuaries and coastal environments. The aquaculture of Sciaenid fishes is being extensively practiced worldwide because they possess biological characteristics suitable for aquaculture, such as being eurythermal and euryhaline; adapt well to various farming conditions; and exhibit good growth performance in captive environments (FAO 2023; C\u0026aacute;rdenas et al. 2018). Within the \u003cem\u003eSciaenidae\u003c/em\u003e family, large yellow croaker (\u003cem\u003eLarimichthys crocea\u003c/em\u003e), red drum (\u003cem\u003eSciaenops ocellatus\u003c/em\u003e), meagre (\u003cem\u003eArgyrosomus regius\u003c/em\u003e) are the main cultivated species worldwide, and shi drum (\u003cem\u003eUmbrina cirrosa\u003c/em\u003e), Japanese meagre (\u003cem\u003eArgyrosomus japonicus\u003c/em\u003e), and brown croaker (\u003cem\u003eMiichthys miiuy\u003c/em\u003e) are consistently produced (FAO 2023). Moreover, as a new target species for aquaculture, other Sciaenid fishes are attracting global attention. Although most \u003cem\u003eSciaenidae\u003c/em\u003e species show high growth and survival rates under farming conditions, desirable strains, such as those with disease resistance and better feed conversion rates, must be established through genetic breeding.\u003c/p\u003e \u003cp\u003eSurrogate broodstock technology can be a powerful tool to support genetic breeding, as the production of donor-derived gametes by transplantation of allogeneic or xenogeneic germ cells into recipient individuals is facilitated (Yoshizaki and Lee \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yoshizaki \u0026amp; Yazawa, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Takeuchi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This technology can enable the production of large, long-generation-time species using small surrogates with short generation time in small land-based tanks and can establish elite strains through genetic breeding in a short period of time, with low labor and cost. In addition, germ cells can be cryopreserved in liquid nitrogen semipermanently, and live fish derived from the cryopreserved germ cells could be produced at any time by transplantation of thawed germ cells into male and female recipients (Lee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yoshizaki \u0026amp; Lee, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This process allows for the long-term preservation of elite strains without rearing live fish in a tank or net pen.\u003c/p\u003e \u003cp\u003eWe previously established a surrogate broodstock technology for \u003cem\u003eSciaenidae\u003c/em\u003e using nibe croaker, also known as blue drum (\u003cem\u003eNibea mitsukurii\u003c/em\u003e), as the recipient species (Takeuchi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Yoshikawa et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Nibe croaker is distributed in central and southern Japan and the East China Sea (Kinoshita et al., 1988), and it is a promising recipient species for Sciaenids because it can mature in a short generation time (4 and 6 months in male and female, respectively), and at a small size (15 cm in total length) in land-based small tanks.\u003c/p\u003e \u003cp\u003eIn surrogate broodstock technology, sterilization of recipients is the key for the production of only donor-derived gametes from the transplanted recipients. Previously, in various fish species, sterile fish resulted from the gene knockdown (KD) or knockout (KO) of the \u003cem\u003edead end\u003c/em\u003e (\u003cem\u003ednd\u003c/em\u003e) gene (Baloch et al., 2021), which is necessary for the maintenance of primordial germ cells and their specification (Weidinger et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Gross-Thebing et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and they are used as recipients for germ cell transplantation (Saito et al., 2008; Yoshizaki et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Octavera \u0026amp; Yoshizaki, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Octavera et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Marinović et al., 2019; Yoshikawa et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Franěk et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recently, we have successfully produced F2-generation mutants in which the \u003cem\u003ednd\u003c/em\u003e gene was knocked out using the clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system in rainbow trout, indicating that these homozygous KO fish completely lack of endogenous germ cells and can be used as recipients for the surrogate broodstock technology (Fujihara et al, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, no reports have been found on this technique applied to marine teleost.\u003c/p\u003e \u003cp\u003eAs a first step in applying this technique to marine fish that produce small and fragile pelagic eggs, nibe croaker, a promising recipient species for Sciaenids, was utilized. In this study, we aimed to produce sterile nibe croaker without germ cells using the CRISPR/Cas9 system to knockout the \u003cem\u003ednd\u003c/em\u003e gene and evaluate their suitability as surrogate broodstock.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals from Tokyo University of Marine Science and Technology, Japan.\u003c/p\u003e\n\u003ch3\u003eFish\u003c/h3\u003e\n\u003cp\u003eNibe croaker broodstock (approximate body weight (BW) of 300 g and total length of 250 mm) were maintained in a 0.5-m\u003csup\u003e3\u003c/sup\u003e circular fiber-reinforced plastic (FRP) tank at Tateyama Station (Banda), Field Science Center of Tokyo University of Marine Science and Technology (Chiba, Japan). Water temperature was maintained at 23\u0026deg;C\u0026ndash;26\u0026deg;C using aquarium heaters, and the photoperiod was set at 16\u0026ndash;18 h day length for the first 3 months, following the natural photoperiod. Fertilized eggs were collected by water flow into an egg collection net.\u003c/p\u003e\n\u003ch3\u003ePreparation of genome‑editing tools and their microinjection into fertilized eggs of nibe croaker\u003c/h3\u003e\n\u003cp\u003eA guide RNA (gRNA) targeting the \u003cem\u003ednd\u003c/em\u003e gene (GenBank Accession No. LC317114) of nibe croaker was designed on the basis of a report of \u003cem\u003ednd\u003c/em\u003e KO in Atlantic salmon (Wargelius et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). First, in the area of the nibe croaker \u003cem\u003ednd\u003c/em\u003e gene, \u0026ldquo;CHOP CHOP\u0026rdquo; (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://chopchop.cbu.uib.no/index.php\u003c/span\u003e\u003c/span\u003e) was used to search for candidate sequences. Then, microhomologies at both ends of the cleavage sites of the obtained target sequences were searched using the \u0026ldquo;search for the CRISPR target site with microhomology sequences\u0026rdquo; (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://viewer.shigen.info/cgi-bin/crispr/crispr.cgi\u003c/span\u003e\u003c/span\u003e) and candidate sequences that prevent in-frame mutations caused by microhomology within the sequence. Using the abovementioned process, a gRNA target site (5\u0026prime;-GCCCCACCGAGCTGAACAGGGGG-3\u0026prime;, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) was designed. CRISPR RNA (crRNA) complementary to the target site sequence and trans-activating crRNA (tracrRNA) were chemically synthesized and then purified using high-performance liquid chromatography by Fasmac Co., Ltd. (Kanagawa, Japan). Fertilized eggs of nibe croaker were microinjected following the procedure of Kawamura et al. (\u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e) with a slight modification. The glass needles for microinjection were constructed using a puller (PC-10; Narishige Co., Ltd., Tokyo, Japan) and a microgrinder (EG-400; Narishige Co., Ltd.) to produce a needle with a 7-\u0026micro;m tip. Fertilized eggs were aligned in a groove of a 2% agar plate filled with sterilized seawater at 25℃. A total of 1 nL of CRISPR/Cas9 solution containing 40 ng/\u0026micro;L of crRNA, 40 ng/\u0026micro;L of tracrRNA, and 100 ng/\u0026micro;L of Alt-R S.p. Cas9 Nuclease V3 (Integrated DNA Technologies, Coralville, IA, USA) was injected into the cytoplasm of a one-cell-stage embryo using a micromanipulator (MP-2R; Narishige Co., Ltd.) and microinjector (IM-9B; Narishige Co., Ltd.) attached to a stereoscopic microscope (SZX-10; Nikon, Tokyo, Japan). The injected eggs were incubated in a 1-L glass beaker filled with sterilized seawater at 25\u0026deg;C until the embryo-formation stage (20 h postfertilization). The hatching rate of the injected eggs was calculated by dividing the number of successfully hatched larvae by the number of injected eggs and then multiplying the result by 100. Larval rearing of nibe croaker was performed as previously described (Takeuchi et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). In brief, injected eggs were transferred to a 100-L polycarbonate tank supplied with flow-through seawater and maintained at 23\u0026deg;C\u0026ndash;26\u0026deg;C. The feeding of larvae began 3 dph. The rotifer \u003cem\u003eBrachionus rotundiformis\u003c/em\u003e, fed with freshwater Chlorella (fresh Chlorella V12; Cholera Industry, Tokyo, Japan), was added to the tank two times a day. The densities of rotifers and \u003cem\u003eNannochloropsis\u003c/em\u003e sp. (Marine fresh; ISC, Fukuoka, Japan) in the tank were maintained at 30\u0026ndash;40 individuals/mL and 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/mL, respectively. \u003cem\u003eArtemia\u003c/em\u003e nauplii were provided from 14 dph. The rotifers and \u003cem\u003eArtemia\u003c/em\u003e nauplii were incubated with Hyper Gloss (Marine Tech Co., Ltd., Aichi, Japan) for 6 to 12 h before feeding to increase the n-3 fatty acid concentration in live food. Starting at 20 dph, the larvae were fed an artificial diet (Otohime; Marubeni Nisshin Feed, Tokyo, Japan). Founders were reared in 100-L tanks for approximately 60 days and then transferred into 0.5-m\u003csup\u003e3\u003c/sup\u003e tanks with flow-through seawater maintained under a natural photoperiod and water temperature.\u003c/p\u003e\n\u003ch3\u003eProduction of homozygous KO mutants\u003c/h3\u003e\n\u003cp\u003eWhen the injected individuals reached 4 months of age, mature male specimens were confirmed by gently squeezing their abdomen to check spermiation. The sperm from these males were artificially inseminated with eggs derived from wild-type females to produce the F1 generation. The resulting F1-generation larvae were sampled and used for mutation detection by the T7 endonuclease I (T7EI) assay. This assay was performed following a procedure modified from Kawamura et al. (\u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). In brief, genomic DNA was extracted from whole hatched larvae. A 244-bp fragment flanking the target site was amplified using the primers nc_dnd_Fw (5\u0026prime;-CCCACTGGATGCCTACGAG-3\u0026prime;) and nc_dnd_Rv (5\u0026prime;-TGGCAGGTCTTCGATACAGAG-3\u0026prime;). PCR was conducted in a 10-\u0026micro;L reaction volume containing 1\u0026times; PCR Buffer II, 200 \u0026micro;M of dNTPs, 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1.25 U of AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 10 ng of template DNA, and 1 \u0026micro;M of each primer. The thermal cycling conditions were as follows: 1 cycle of 95\u0026deg;C for 10 min, then 40 cycles of 95\u0026deg;C for 15 s, 52\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s, followed by a final elongation step at 72\u0026deg;C for 5 min. The PCR products were purified using the ISOSPIN PCR Product (Nippon Gene, Tokyo, Japan). In addition, the purified PCR products were digested with T7E1 (New England Biolabs, Beverly, MA, USA) in accordance with the manufacturer\u0026rsquo;s instructions, and the digestion products were separated on a 2% agarose gel. The PCR products of F1 individuals, which have a mutation in the \u003cem\u003ednd\u003c/em\u003e gene, were sequenced using the dideoxy method (Sanger et al. \u003cspan class=\"CitationRef\"\u003e1977\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eF1 populations containing mutants were raised until males and females reached maturation at 1 year old. Then, they were subjected to the abovementioned T7EI assay to identify and select heterozygous \u003cem\u003ednd\u003c/em\u003e mutants. The resulting F1 heterozygous mutant females and males were mated to produce the F2 generation. Of the resulting F2 population, 50 individuals were sampled for genotyping: wild-type \u003cem\u003ednd\u003c/em\u003e (+/+), heterozygous KO \u003cem\u003ednd\u003c/em\u003e (+/\u0026minus;), and homozygous KO \u003cem\u003ednd\u003c/em\u003e (\u0026minus;/\u0026minus;), each with specific forward primers for the wild-type allele; nc_dnd_Fw_WTp (5\u0026prime;-AGGATCTGTTGATCCCCCTG-3\u0026prime;) and a specific forward primer for the mutant allele; nc_dnd_Fw_KOp (5\u0026prime;-TGGCAGGTCTTCGATACAGAG-3\u0026prime;) and PCR with common reverse primers; nc_dnd_Rv (5\u0026prime;-TGGCAGGTCTTCGATACAGAG-3\u0026prime;). PCR amplification with the primer sets was conducted using HiDi DNA polymerase (myPOLSBiotec, Konstanz, Germany) that efficiently amplifies from primers that are completely matched at the 3\u0026prime;-end and discriminates templated DNA containing single-nucleotide variations (Drum et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). In addition, PCR amplification was conducted in a 10-\u0026micro;L reaction volume tube containing 1\u0026times; HiDi Buffer, 200 \u0026micro;M dNTPs, 1 U HiDi DNA polymerase, 25 ng of template DNA, and 0.4 \u0026micro;M of each primer. Then, the PCR mixture was cycled under the following conditions: 1 cycle of 95\u0026deg;C for 3 min, then 30 cycles of 95\u0026deg;C for 30 s, 58\u0026deg;C for 30 s, and 72\u0026deg;C for 1 min, followed by a final elongation step at 72\u0026deg;C for 3 min. Afterward, the PCR products were verified under UV light after electrophoresis on a 2.0% agarose gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analysis of the gonads of\u003c/strong\u003e \u003cstrong\u003ednd\u003c/strong\u003e \u003cstrong\u003eKO fish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn investigating the gonadal development of \u003cem\u003ednd\u003c/em\u003e KO nibe croaker in the F2 population, wild-type and homozygous \u003cem\u003ednd\u003c/em\u003e KO individuals were selected by PCR for genotyping, and histological and immunohistochemical analyses of the gonads obtained from 10-dph, 20-dph, and 6-months-old nibe croaker were performed. The gonads were paraffin embedded and sliced into 4-\u0026micro;m-thick sections in accordance with the conventional method. Immunohistochemistry was performed in accordance with the method of Yazawa et al. (\u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e), and germ cells were detected using an anti-nibe croaker Vasa polyclonal antibody (Yoshikawa et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Primary antibodies, anti-nibe croaker Vasa polyclonal antibody, were diluted to 1:200 in Can Get Signal Immunostain (Toyobo). Secondary antibodies, goat anti-rabbit Alexa Fluor 488 (Invitrogen Life Technologies, San Diego, CA, USA), were used in accordance with the manufacturer\u0026rsquo;s instructions. The total number of PGCs was counted by observing the anti-Vasa staining on serial histological sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAllogenic testicular cell transplantation into homozygous\u003c/strong\u003e \u003cstrong\u003ednd\u003c/strong\u003e \u003cstrong\u003eKO recipients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe testes from two male nibe croakers, individual \u0026ldquo;donor-A\u0026rdquo; (15.0 cm in standard length (SL), 59.3 g BW, and gonad somatic index (GSI) of 0.58%) and individual \u0026ldquo;donor-B\u0026rdquo; (14.5 cm in SL, 53.7 g BW, and GSI of 1.42%) caught using hook and line in Tateyama Bay, Chiba Prefecture, Japan, were isolated and cryopreserved following the procedure of Lee et al. (\u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e) with a slight modification. The isolated testes were minced and equilibrated in a cryotube containing 1 mL of cryomedium (1.3 M DMSO, 10% egg yolk, 0.1 M trehalose in Leibo-vitz\u0026rsquo;s L15 medium (Thermo Fischer Scientific, Waltham, MA)) on ice for 60 min and then frozen at \u0026minus;\u0026thinsp;1\u0026deg;C/min for 90 min using the Bicell freezing container (Nihon Freezer, Tokyo, Japan) in a deep freezer (\u0026minus;\u0026thinsp;80\u0026deg;C) before preservation in liquid nitrogen. After 6 months of cryopreservation, the cryotubes were thawed in a 25\u0026deg;C water bath for 1\u0026ndash;2 min, and the cells were dehydrated in an L-15 medium (pH7.5) supplemented with 1% (vol/vol) fetal bovine serum and 25 mM Hepes. Subsequently, the thawed testes were enzymatically dissociated and used as donor testicular cells for intraperitoneal transplantation in accordance with the procedure reported by Takeuchi et al. (\u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). Approximately 20,000 cells were transplanted into the peritoneal cavity of 12-dph F2 larvae resulting from mating between heterozygous \u003cem\u003ednd\u003c/em\u003e KO (300 recipients for each donor individual) using the method described by Takeuchi et al. (\u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). Recipients were reared until 6 months, and homozygous \u003cem\u003ednd\u003c/em\u003e KO recipients were identified by genotyping PCR. The resulting homozygous \u003cem\u003ednd\u003c/em\u003e KO recipients were then used for the progeny test. In 12-month-old individuals, ten individuals among the homozygous \u003cem\u003ednd\u003c/em\u003e KO recipients identified from 50 transplanted recipients were subjected to histological observations of the gonads according to the aforementioned method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProgeny tests using\u003c/strong\u003e \u003cstrong\u003ednd\u003c/strong\u003e \u003cstrong\u003eKO recipients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGroup mating experiments were conducted in each of the two strains using 6-month-old mature \u003cem\u003ednd\u003c/em\u003e KO recipients in a 0.5-m\u003csup\u003e3\u003c/sup\u003e FRP tank to confirm whether donor-derived offspring can be produced through the natural spawning of \u003cem\u003ednd\u003c/em\u003e KO recipients. In inducing spawning, the water temperature was increased from approximately 20\u0026deg;C to 26\u0026deg;C, and the photoperiod was fixed at 16 h. Floating eggs were collected using an egg collection net attached to the drain of the fish tank. The total number of spawned eggs, floating eggs, and hatched larvae obtained from each spawning event was recorded, and 1-dph larvae were sampled and used for DNA analysis. Microsatellite analysis was performed to confirm whether or not each resultant larva carried donor-derived nDNA. Two loci (501 and 1001) of microsatellite DNA markers designed on the basis of microsatellite regions on the genome of large yellow croaker (GenBank Accession No. GCA_900246015.1) were used for parentage assignment following a procedure described by Morishima et al. (\u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e) with a slight modification. The microsatellite marker loci were amplified using the primer set 501 (Fwd; 5\u0026prime;-GTGATGACAGACTGTGATAAGCT-3\u0026prime;, Rev; 5\u0026prime;-GTCACGACGTTGTAAGAGCATTTGATGTAGCAGTGT-3\u0026prime;) and 1001 (Fwd; 5\u0026prime;- ACTCAAACCCACACCTGACA-3\u0026prime;, Rev; 5\u0026prime;-GTCACGACGTTGTAAGGCCATTGGTGACTGATGA-3\u0026prime;). A 10-\u0026micro;L reaction volume comprising 1\u0026times; \u003cem\u003eExTaq\u003c/em\u003e reaction buffer (TakaraBio, Tokyo, Japan), 200-\u0026micro;M dNTPs, 0.25 U of \u003cem\u003eExTaq\u003c/em\u003e DNA polymerase (TakaraBio), 20 ng of template DNA, 0.1 \u0026micro;M of forward primer, and a mixture of 0.01 \u0026micro;M of reverse primer and 0.1 \u0026micro;M of fluorescently labeled primer (Fam-GTCACGACGTTGTA for 501 and Vic-GTCACGACGTTGTA for 1001) was used to perform PCR amplification. The thermal cycling conditions were as follows: 95\u0026deg;C for 3 min, followed by 30 cycles of 94\u0026deg;C for 30 s, 62\u0026deg;C for 30 s, and 72\u0026deg;C for 1 min, and a final elongation step at 72\u0026deg;C for 5 min. The resultant samples were electrophoresed on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems by Life Technology, USA), and the fluorescently labeled PCR products were analyzed using GeneScan (Applied Biosystems by Life Technology).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eProduction of homozygous\u003c/b\u003e \u003cb\u003ednd\u003c/b\u003e \u003cb\u003eKO nibe croaker\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe duration of the one-cell stage of nibe croaker embryos was approximately 50 min at a water temperature of 25\u0026deg;C. During this time, approximately 60 fertilized eggs were injected per individual. A total of 972 eggs were injected using fertilized eggs obtained from 15 spawning events to produce \u003cem\u003ednd\u003c/em\u003e mutant founders. The hatching rate of the injected group was 28.0% \u0026plusmn; 9.0% (n\u0026thinsp;=\u0026thinsp;15), whereas that of the noninjected control group was 70.0% \u0026plusmn; 9.0% (n\u0026thinsp;=\u0026thinsp;15). Of the injected individuals, 22 survived to 4 months old.\u003c/p\u003e \u003cp\u003eT7EI analysis, performed using fin samples obtained from the 22 surviving individuals, revealed that the mutagenic efficiency was 68.2% (15 individuals out of 22 total individuals). Spermiation was confirmed in two male fish at 4 months of age, and milt from these two male fish was used to fertilize eggs stripped from a wild-type female nibe croaker. Subsequent T7EI analysis of 15 larvae from each offspring demonstrated a mutagenic efficiency of 0% in one line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and 13.3% (two individuals out of 15 analyzed individuals, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) in the other strain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTherefore, the F1 offspring in which mutant individuals were identified was reared until their maturity for the production of F2 generation. The mutation patterns of the \u003cem\u003ednd\u003c/em\u003e gene in F1 mutants were confirmed by sequencing, and all individuals (n\u0026thinsp;=\u0026thinsp;8) were found to have identical mutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A three-base deletion and a one-base insertion were found to have occurred in the sequence encoding the 72nd amino acid in the coding region of the \u003cem\u003ednd\u003c/em\u003e protein. Given this mutation, a frameshift occurred, and the resulting stop codon was predicted to occur at position 104 in the coding region of the \u003cem\u003ednd\u003c/em\u003e protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese F1 heterozygous \u003cem\u003ednd\u003c/em\u003e mutants were reared to maturity. No difference in the fecundity of either male or female heterozygous \u003cem\u003ednd\u003c/em\u003e mutant individuals was observed compared with the wild type. Therefore, the F2 offspring was produced by crossing female and male heterozygous mutants. Genotyping of the resulting F2 larvae (a total of 50 larvae were analyzed) by using mutant-allele-specific PCR methods revealed that 20.0%, 56.0%, and 24.0% of the larvae were wild type, heterozygous \u003cem\u003ednd\u003c/em\u003e KO mutants, and homozygous \u003cem\u003ednd\u003c/em\u003e KO mutants, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGonadal development in\u003c/b\u003e \u003cb\u003ednd\u003c/b\u003e \u003cb\u003eKO nibe croaker\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe number of PGCs in 10-dph homozygous \u003cem\u003ednd\u003c/em\u003e KO mutants was compared with that in wild type through anti-Vasa staining in immunohistochemical analysis, and the result showed no difference in the number of PGCs between homozygous \u003cem\u003ednd\u003c/em\u003e KO mutants (12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18 cells) and the wild type (13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). At 20 dph, no evident PGCs were recognized by the anti-Vasa antibody in the genital ridges of homozygous \u003cem\u003ednd\u003c/em\u003e KO mutants, whereas the number of germ cells increased to 380\u0026thinsp;\u0026plusmn;\u0026thinsp;44.7 in the genital ridges of the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Histological analysis of the gonads of homozygous \u003cem\u003ednd\u003c/em\u003e KO mutants at 6 months of age revealed that germ cells were completely absent in either the testes or ovaries in all investigated individuals, whereas numerous oocytes were observed in the ovaries, and spermatogenesis was in progress in the testis of the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Histological analysis further revealed that 17.6% of the larvae (three individuals out of 17 investigated) had ovary-like gonads with ovarian cavities and ovarian lamellae, whereas 82.4% (14/17) exhibited gonads resembling the testes of homozygous \u003cem\u003ednd\u003c/em\u003e KO mutants. However, among their siblings (wild-type and heterozygous \u003cem\u003ednd\u003c/em\u003e KO), 40.4% (13/33) and 59.6% (20/33) possessed an ovary and testes, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGerm cell transplantation using\u003c/b\u003e \u003cb\u003ednd\u003c/b\u003e \u003cb\u003eKO nibe croaker as recipients\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn assessing the suitability of \u003cem\u003ednd\u003c/em\u003e KO nibe croakers as recipients, germ cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) obtained from two wild-caught male fish used as donors (referred to as donor-A and donor-B) were intraperitoneally transplanted into approximately 300 recipients, which were randomly selected from the F2 population, including homozygous \u003cem\u003ednd\u003c/em\u003e KO mutants. After transplantation, approximately 150 recipients from both donor groups survived to 6 months old. Mutant-allele-specific PCR was performed to identify homozygous \u003cem\u003ednd\u003c/em\u003e KO individuals out of 30 transplanted recipients from each donor group. Eight homozygous \u003cem\u003ednd\u003c/em\u003e KO individuals were identified from each of the donor groups and used for progeny tests.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo observe the behavior of the donor germ cells in recipient gonads post-transplantation, histological observations were conducted on the gonads of 10 individuals of the homozygous \u003cem\u003ednd\u003c/em\u003e KO recipients, identified through screening among 50 transplanted recipients. The results revealed that in 12-month-old transplanted homozygous dnd KO recipients, 20.0% of females (two out of 10 observed fish, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) and 40.0% of males (four out of 10 observed fish, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) exhibited various stages of differentiated germ cells in their gonads compared with nontransplanted fish where both sexes lacked germ cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-H). The rest of the observed recipients displayed a complete absence of germ cells, which is similar to the homozygous \u003cem\u003ednd\u003c/em\u003e KO in nontransplanted fish. Consequently, the gonads of 60% of the homozygous \u003cem\u003ednd\u003c/em\u003e KO recipients exhibited gametogenesis derived from transplanted germ cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProduction of donor-derived offspring from\u003c/b\u003e \u003cb\u003ednd\u003c/b\u003e \u003cb\u003eKO surrogate parents\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEach of the eight (two female and six male) abovementioned homozygous \u003cem\u003ednd\u003c/em\u003e KO recipients was transferred to a 0.5-m\u003csup\u003e3\u003c/sup\u003e FRP tank, and spontaneous spawning was induced by elevating the water temperature. In donor-A transplanted recipients, 131,333 eggs were spawned; the ratio of floating eggs among the total spawned eggs was 10.0%, and the hatching rate among the floating eggs was 100%. In donor-B transplanted recipients, 125,333 eggs were spawned; the ratio of floating eggs among the total spawned eggs was 94.0%, and the hatching rate among the floating eggs was 95.6%.\u003c/p\u003e \u003cp\u003eMicrosatellite analysis was performed to determine whether or not each resultant larva carried donor-derived nDNA. Two loci (501 and 1001) of microsatellite DNA markers were used for parentage assignment in F1 larvae. Allele varieties at the 1001 locus were as follows: donor-A, 240/246-bp alleles and donor-B, 265/267-bp alleles. The recipient \u003cem\u003ednd\u003c/em\u003e KOs were as follows: 272 or 278-bp alleles. In addition, allele varieties at the 501 locus were as follows: donor-A, 292/316-bp alleles and donor-B, 329/331-bp alleles. The recipient \u003cem\u003ednd\u003c/em\u003e KOs were as follows: 304, 306, 360, or 364-bp alleles. The results of DNA analysis are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The DNA analysis results of 17 and 18 F1 offspring in the donor-A group and donor-B group, respectively, demonstrated that 100% possessed donor-derived alleles in both microsatellite markers, and no recipient-derived alleles were detected (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These data indicate that the paternal and maternal genetic origins of the F1 offspring were donor wild-type nibe croaker. Subsequently, these donor-derived offspring were reared, and no evident abnormalities in the external morphology were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\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\u003eSummary of microsatellite analysis in F1 larvae obtained by a progeny test\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" 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=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eDonor-A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003eDonor-B\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e1001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecipient\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e272, 278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e304, 306, 360, 364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e272, 278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e304, 306, 360, 364\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDonor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e240/246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e292/316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e265/267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003e329/331\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eOffspring\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e240/240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e292/292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e265/265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e329/329\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e240/246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e292/316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e70.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e265/267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e55.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e329/331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e44.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e246/246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e316/316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e267/267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e38.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e331/331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.6%\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\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, homozygous KOs of the \u003cem\u003ednd\u003c/em\u003e gene were successfully produced in nibe croaker by using the CRISPR/Cas9 system. The resulting \u003cem\u003ednd\u003c/em\u003e KO individuals showed the complete absence of germ cells in adult males and females. Furthermore, when germ cell transplantation was performed using the \u003cem\u003ednd\u003c/em\u003e KO mutants as recipients, they produced only donor-derived offspring through spontaneous spawning. Considering that F2 populations produced by the mating of heterozygous \u003cem\u003ednd\u003c/em\u003e KO F1 males and females were used as recipients in the study of germ cell transplantation, approximately 25% of the transplanted recipients were homozygous \u003cem\u003ednd\u003c/em\u003e KO fish, which is in accordance with Mendelian inheritance. Therefore, homozygous \u003cem\u003ednd\u003c/em\u003e KO recipients must be identified by genotyping PCR after transplantation. Given that the recipient larvae used for transplantation are too small and fragile at the developmental stage, genotyping each individual one by one was not feasible. Although the screening of the posttransplanted larvae was time consuming, the stable production of donor-derived offspring could be achieved after selecting the homozygous \u003cem\u003ednd\u003c/em\u003e KO recipients. In the present study, donor-derived gametes were produced in the gonads of 60% of the homozygous \u003cem\u003ednd\u003c/em\u003e KO recipients, which is high enough to be used for practical applications.\u003c/p\u003e \u003cp\u003eWhen surrogate broodstock technology via germ cell transplantation is applied to practical aquaculture, recipients must produce only donor-derived offspring but not a mixture of donor- and recipient-derived offspring. Therefore, establishing sterilization methods in recipient species is of great importance. The methods commonly used for producing sterile recipients in previous studies, such as triploidization and gene knockdown (KD), require exposure of embryos to temperature shock or microinjection of morpholino oligonucleotides into fertilized eggs (Takeuchi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although these treatments were relatively easy and reproducible in some other species such as salmonids, considerable marine fish show low survival and success rates following triploidization or microinjection into fertilized eggs. Therefore, the application of these sterilization methods to marine species producing small pelagic eggs may not be a reliable option. By contrast, the production of sterile recipients through genome editing demonstrated in this study was relatively stable and less labor intensive once heterozygous KO F1 strains are established.\u003c/p\u003e \u003cp\u003eAnother issue is sperm production from triploid males in some fish species (Piferrer et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In the case of triploid nibe croaker, although triploids did not produce any functional gametes in both sexes, they produced small amounts of aneuploid (nonfunctional) gametes without developmental potency (Takeuchi et al, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Several studies of \u003cem\u003ednd\u003c/em\u003e KD recipients have also reported the persistence of endogenous germ cells because of incomplete gene KD (Linhartov\u0026aacute; et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yoshizaki et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yoshikawa et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On the contrary, in the case of KO, 100% sterile recipients can be obtained if appropriate screening is performed against the F2 generation produced by mating heterozygous F1 males and females, which could be an advantage in practical aquaculture applications.\u003c/p\u003e \u003cp\u003eInterspecific hybridization represents another alternative strategy for producing sterile recipients for surrogate broodstock technology (Takeuchi et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This approach is suitable for the mass production of recipients for fragile marine species that show high mortality during early developmental stages, as it can be achieved by simple artificial insemination without additional treatments. Recently, we showed that hybrids of female blue dram (\u003cem\u003eNibea mitsukurii\u003c/em\u003e) \u0026times; male white croaker (\u003cem\u003ePennahia argentata\u003c/em\u003e) display germ cell\u0026ndash;less sterile gonads, and these hybrid recipients effectively produced donor-derived offspring (Yoshikawa et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, hybrid viability and sterility vary depending on the combination of the two parental species, and these characteristics are completely unpredictable. Therefore, selecting a suitable sterilization technique that depends on the reproductive characteristics and early survival of each target species is important.\u003c/p\u003e \u003cp\u003ePGCs were initially present in \u003cem\u003ednd\u003c/em\u003e KO nibe croaker at 10 dph, which is similar to wild-type individuals; however, their subsequent absence was observed in \u003cem\u003ednd\u003c/em\u003e KO individuals at 20 dph. Therefore, \u003cem\u003ednd\u003c/em\u003e KO nibe croaker still possessed their own germ cells when exogenous donor germ cells were transplanted into the recipients. Similar conditions were also observed in \u003cem\u003ednd\u003c/em\u003e KO rainbow trout, and the maternal supply of \u003cem\u003ednd\u003c/em\u003e mRNA might maintain their PGCs during early embryogenesis (Fujiwara et al. 2022). Therefore, there might be a competition between transplanted donor germ cells and endogenous PGCs until the latter disappears from the recipient\u0026rsquo;s genital ridges. Meanwhile, PGCs are absent during an earlier stage in \u003cem\u003ednd\u003c/em\u003e KD rainbow trout, and these phenotypic differences can be attributed to the fact that \u003cem\u003ednd\u003c/em\u003e KD simultaneously inhibits the translation of maternally supplied and zygotically expressed \u003cem\u003ednd\u003c/em\u003e mRNA (Yoshizaki et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, the advantage of \u003cem\u003ednd\u003c/em\u003e KO fish as a recipient during germ cell migration and their incorporation stage was limited compared with that of \u003cem\u003ednd\u003c/em\u003e KD recipients. However, \u003cem\u003ednd\u003c/em\u003e KO recipients provide sufficient niches to support the proliferation and differentiation of donor-derived germ cells in their developing gonads. Furthermore, this advantage is superior to that of sterile triploids in which a large number of mitotic germ cells remained in the recipient gonads.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDnd\u003c/em\u003e KO nibe croaker has great application potential as sterile recipients in applying surrogate broodstock technology to gamete production in a variety of Sciaenid fishes. However, Sciaenid fishes, which are commercially cultivated worldwide, such as large yellow croaker (\u003cem\u003eLarimichthys\u003c/em\u003e), red drum (\u003cem\u003eSciaenops\u003c/em\u003e), and meagre (\u003cem\u003eArgyrosomus\u003c/em\u003e) belong to different genera of nibe croaker (\u003cem\u003eNibea\u003c/em\u003e). In general, the production of donor-derived gametes through transplantation among different genera is more difficult than that between two species belonging to the same genus (Takeuchi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To date, a few reports have been found on the successful donor-derived functional egg production in intergeneric transplants, such as slender bittering (donor: \u003cem\u003eTanakia lanceolata\u003c/em\u003e) and Chinese rosy bitterlings (recipient: \u003cem\u003eRhodeus ocellatus\u003c/em\u003e) (Octavera et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), as well as common carp (donor: \u003cem\u003eCyprinus carpio\u003c/em\u003e) and goldfish (recipient: \u003cem\u003eCarassius auratus\u003c/em\u003e) (Franěk et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Thus, confirming the compatibility with donor species by intragenus germ cell transplantation is necessary to advance the utilization of \u003cem\u003ednd\u003c/em\u003e KO nibe croaker recipients for gamete production of various Sciaenid fishes in aquaculture.\u003c/p\u003e \u003cp\u003eTwo challenges remain in the application of the \u003cem\u003ednd\u003c/em\u003e KO recipients developed in this study to actual seedling production. First, addressing the male-biased sex ratio in KO individuals is essential. Previous studies have reported that germ cell\u0026ndash;deficient individuals such as medaka (Kurokawa et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), zebrafish (Weidinger et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Slanchev et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), nibe croaker (Yoshikawa et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and chub mackerel (Kawamura et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) are either predominantly or entirely males. In the case of \u003cem\u003ednd\u003c/em\u003e KO nibe croaker, it exhibited a strong male-biased sex ratio similar to the abovementioned species. Meanwhile, a 1:1 sex ratio has been reported in germ cell\u0026ndash;deficient fish produced by KD or KO of the \u003cem\u003ednd\u003c/em\u003e gene in several species, such as loach (Fujimoto et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), goldfish (Goto et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), salmon (Wargelius et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), grass puffer (Yoshikawa et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and rainbow trout (Fujiwara et al, 2022). These reports indicate that the sex ratio of germ cell\u0026ndash;deficient fish varies depending on species. For better reliability in increasing the proportion of female recipients, establishing a feminization technique by the administration of estrogen may be necessary, as reported in other species (Piferrer \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs discussed above, the second issue is the need to select homozygous KO individuals after the transplantation experiment. In achieving 100% production of homozygous \u003cem\u003ednd\u003c/em\u003e KO in the offspring, recent reports suggested the possibility of germ cell rescue by microinjecting wild-type \u003cem\u003ednd\u003c/em\u003e mRNA into fertilized eggs of Atlantic salmon, which are expected to be homozygous KO of the \u003cem\u003ednd\u003c/em\u003e gene (G\u0026uuml;ralp et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). If this method can be applied to other fish species, then it will be an effective technique for producing all-homozygous \u003cem\u003ednd\u003c/em\u003e KO populations. In the future, addressing these issues will improve this technology, which is suitable for the practical aquaculture industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was partly supported by JSPS KAKENHI Grant Number 23H00344 and JST-Mirai Program Grant Number JPMJMI21C1.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eKyoichiro Saitoh, Akihiro Yamauchi, Onur Ey\u0026uuml;boğlu, Kana Ozawa, Wataru Kawamura, and Ryosuke Yazawa conducted experiments and analyzed the data. Ryosuke Yazawa, Tetsuro Morita, Yutaka Takeuchi, and Goro Yoshizaki designed the research. Ryosuke Yazawa and Goro Yoshizaki conceived the study and wrote the manuscript. All authors critically revised the report, commented on drafts of the manuscript, and approved the final report.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis work was partly supported by JSPS KAKENHI Grant Number 20H00430 and 20H00430 and JST-Mirai Program Grant Number JPMJMI21C1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eC\u0026aacute;rdenas S (2012) Biolog\u0026iacute;a y acuicultura de corvinas en el mundo. AquaTIC 37:1\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Su Y, Hong W (2018) Aquaculture of the large yellow croaker. In: Klinger D, Guo Z (eds) Aquaculture in China: success stories and modern trends, 1st edn. 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Fish Sci 85:429\u0026ndash;437. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12562-019-01299-y\u003c/span\u003e\u003cspan address=\"10.1007/s12562-019-01299-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Surrogate broodstock technology, knock-out, CRISPR/Cas9, sterile, nibe croaker Nibea mitsukurii, germ cell transplantation","lastPublishedDoi":"10.21203/rs.3.rs-3996577/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3996577/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe use of sterile recipients is crucial for efficiently producing donor-derived offspring through surrogate broodstock technology for practical aquaculture applications. Although knockout (KO) of the \u003cem\u003edead end\u003c/em\u003e (\u003cem\u003ednd\u003c/em\u003e) gene has been used in previous studies as a sterilization method, it has not been reported in marine fish. In this study, nibe croaker was utilized as a model for marine teleosts that produce small pelagic eggs, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to produce \u003cem\u003ednd\u003c/em\u003e KO fish. The F1 generation, which carried a nonsense mutation in the \u003cem\u003ednd\u003c/em\u003e gene, was produced by mating founder individuals with wild-type counterparts. Subsequently, the F2 generation was produced by mating the resulting males and females. Among the F2 generations, 24.0% consisted of homozygous KO individuals. Histological analysis revealed that primordial germ cells (PGCs) were present in homozygous KO individuals at 10 days post hatching (dph), similar to wild-type individuals. However, by 20 dph, PGCs were absent in KO individuals. Furthermore, no germ cells were observed in the gonads of both sexes of homozygous KO individuals at 6 months old, which is the typical maturity age for wild-type individuals of both sexes. In addition, when cryopreserved donor nibe croaker testicular cells were transplanted, only donor-derived offspring were successfully obtained through the spontaneous mating of homozygous KO recipient parents. Results indicate that \u003cem\u003ednd\u003c/em\u003e KO nibe croaker lack germ cells and can serve as promising recipients, producing only donor-derived gametes as surrogate broodstock.\u003c/p\u003e","manuscriptTitle":"Reproductive characteristics and suitability of sterile dead end knockout nibe croaker as a recipient for intraperitoneal germ cell transplantation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-19 15:18:09","doi":"10.21203/rs.3.rs-3996577/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-30T08:31:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-28T08:13:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-28T03:38:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"383d42bf-0f96-4a3e-9c60-846adf40b55a","date":"2024-03-18T19:24:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8f51f3e2-c74e-4220-9b72-0521dead5c12","date":"2024-03-18T15:10:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-18T09:39:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-16T01:48:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-15T00:50:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biotechnology","date":"2024-02-28T11:57:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2ae06baa-55f0-4df2-962a-fd89b71f8f28","owner":[],"postedDate":"March 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-02T00:47:54+00:00","versionOfRecord":{"articleIdentity":"rs-3996577","link":"https://doi.org/10.1007/s10126-024-10323-x","journal":{"identity":"marine-biotechnology","isVorOnly":false,"title":"Marine Biotechnology"},"publishedOn":"2024-05-01 00:47:54","publishedOnDateReadable":"May 1st, 2024"},"versionCreatedAt":"2024-03-19 15:18:09","video":"","vorDoi":"10.1007/s10126-024-10323-x","vorDoiUrl":"https://doi.org/10.1007/s10126-024-10323-x","workflowStages":[]},"version":"v1","identity":"rs-3996577","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3996577","identity":"rs-3996577","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}