Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a marine teleost, Nibe croaker (Nibea mitsukurii)

AQUA-632139; No of Pages 13 Aquaculture xxx (2016) xxx–xxx

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Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a marine teleost, Nibe croaker (Nibea mitsukurii) Hiroyuki Yoshikawa a, Yutaka Takeuchi a,⁎, Yasuko Ino a, Junjie Wang a, Gaku Iwata b, Naoki Kabeya b, Ryosuke Yazawa b, Goro Yoshizaki b a b

Research Center for Advanced Science and Technology, Tokyo University of Marine Science and Technology, Banda 670, Tateyama, Chiba 294-0308, Japan Department of Marine Bioscience, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan

a r t i c l e

i n f o

Article history: Received 3 February 2016 Received in revised form 27 April 2016 Accepted 6 May 2016 Available online xxxx Keywords: Spermatogonial transplantation Perciformes Chromosome set manipulation Transgenic Fertilization

a b s t r a c t Intra-peritoneal spermatogonial transplantation could become important in the production of allogenic or xenogenic donor-derived offspring from surrogate parent fish in commercial aquaculture. However, the rates at which surrogates with chimeric germ-lines are produced and contribute donor-derived gametes and offspring to subsequent generations must be improved for this technology to become practical, especially for marine fishes. Here, we performed intra-peritoneal spermatogonial transplantation into triploid Nibe croaker Nibea mitsukurii to evaluate the suitability of functionally sterile triploids as surrogates. Donor testicular cells were collected from hemizygous pHSC-GFP transgenic (gfp/−) individuals and then transplanted into the peritoneal cavities of 12-day-old larvae (diploid or triploid). By 6 months after transplantation, 37% (male) and 29% (female) of triploid recipients had produced gfp-positive, donor-derived gametes; these rates were 7- and 4-fold higher, respectively, than the rates for diploid male and female recipients, respectively. Sperm and eggs from triploid recipients were artificially fertilized with eggs or sperm, respectively, from wild-type fish; approximately 50% of the resulting offspring were gfp-positive, suggesting that nearly all of offspring originated from donor-derived gametes that had descended from transplanted gfp/− germ cells. The fecundity and spawning frequency of surrogate triploid recipients were not significantly different to those of non-transplanted diploids, and the triploid recipients produced subsequent donor-derived offspring for at least two spawning seasons. Our results demonstrated that use of functionally sterile triploid recipients improved the percentage of recipients that produce donorderived gametes. Further, the use of triploid recipients resulted in exclusive production of donor-derived gametes in this marine teleost. Statement of relevance: Intra-peritoneal spermatogonial transplantation using allogenic triploid recipients was first demonstrated in marine teleosts. In addition to efficient and exclusive donor-derived gametogenesis, we found that use of functionally sterile triploid recipients improved the percentage of recipients that produce donor-derived gametes. Consequently, triploid surrogates were more suitable than diploid surrogates and that use of triploid surrogates may accelerate the application of surrogate broodstock technology in aquaculturetargeted, genetically valuable, or endangered marine teleosts. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Allogenic and xenogenic intraperitoneal germ-cell transplantation, which are referred to as the surrogate broodstock technologies, had been first established for a Salmonidae (Takeuchi et al. 2003, 2004). As an example, xenogenic transplantation of rainbow trout Oncorhynchus

⁎ Corresponding author. E-mail address: [email protected] (Y. Takeuchi).

mykiss germ cells into the peritoneal cavity of masu salmon Oncorhynchus masou hatchlings results in surrogate salmon parents that produce donor-derived trout gametes (Okutsu et al. 2007; Takeuchi et al. 2004). Interestingly, testis-derived donor germ cells developed into either spermatozoa within recipient testes or oocytes within recipient ovaries proving that transplanted donor spermatogonia differentiated into functional sperm or eggs based on the sex of the recipient (Lee et al. 2013; Okutsu et al. 2006, 2007, 2008; Yoshizaki et al. 2011, 2012), as was also shown with transplanted donor oogonia (Yoshizaki et al. 2010a, 2010b). In addition, cryopreservation method for producing

http://dx.doi.org/10.1016/j.aquaculture.2016.05.011 0044-8486/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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functional eggs and sperm from frozen whole testes has been demonstrated in a rainbow trout (Lee et al. 2013). Therefore, gametes carrying donor-derived traits can be produced in recipients by transplanting allogenic or xenogenic cryopreserved germ cells; this process could reduce the risk of losing valuable genetic resources and the costs associated with maintenance of broodstock for commercially desirable traits, endangered population and species, or with maintenance of expensive broodstock in marine teleosts (Kobayashi et al. 2006; Lee et al. 2013; Okutsu et al. 2007; Yoshizaki et al. 2011). Application of allogenic germ cell transplantation technique in commercially valuable marine fishes would also be expected to improve for producing 1) gametes with increased genetic diversity for use in stock enhancement by transplanting mixed germ cells isolated from gonads of donor individuals (Sato et al. 2014), 2) a large number of surrogate broodstock by transplanting germ cells isolated from a single donor fish with commercially desirable genetic traits (Morita et al. 2012), and 3) functional Y eggs derived from donor spermatogonia for use in obtaining YY “super-male” in species having an XY sex-determining system (Yoshizaki et al. 2010b). Methods for transplanting allogenic and xenogenic spermatogonia into the peritoneal cavity of larvae have been established for several marine fishes, such as Nibe croaker Nibea mitsukurii (Higuchi et al. 2011; Takeuchi et al. 2009), chub mackerel Scomber japonicus (Yazawa et al. 2010), yellowtail Seriola quinqueradiata (Morita et al. 2012), jack mackerel Trachurus japonicus (Morita et al. 2015), and yellowtail kingfish Seriola lalandi (Bar et al. 2015). Moreover, in order to identify spermatogonia in donor-cell preparations, several markers, such as vasa (Bar et al. 2015; Nagasawa et al. 2009), Ly75 (Nagasawa et al. 2012), and dead end (Bar et al. 2015; Yazawa et al. 2013) have been isolated; additionally, a cell-sorting technique (Kise et al. 2012) to enrich transplantable type Aspermatogonia is available for marine teleosts. However, successful production of donor-derived gametes and offspring has been only reported for yellowtail (Morita et al. 2012, 2015). To develop practical methods for surrogate broodstock technology in aquaculture, it is important to improve the efficiency of donorderived gamete production in recipient surrogates. Use of sterile recipients, specifically fish that have gonads but cannot produce their own gametes, is an important element of efficient production of donorderived gametes (Yoshizaki et al. 2012). In fact, only donor-derived trout gametes are produced from functionally sterile triploid masu salmon that undergo intra-peritoneal spermatogonial transplantation (Okutsu et al. 2007). As summarized in Felip et al. (2001b), functionally sterile triploid fish can be mass produced in several species of marine teleosts. Therefore, efficient production of donor-derived seeds from marine fish may result from combining surrogate broodstock technology with the use of triploid recipients. In previous studies, we established methods for generating triploid Nibe croaker N. mitsukurii (Sciaenidae, Perciformes) and documented that they are functionally sterile (Takeuchi et al., 2016). Nibe croaker produces small pelagic eggs that are similar to those of most commercially valuable marine fishes, such as tunas and yellowtails. A broodstock of this species can be maintained and induced to spawn in small fish tanks (500 l). Moreover, Nibe croaker has a short generation time; they reach sexual maturity by 6 months of age, and intraperitoneal spermatogonial transplantation methods are well established for this species (Higuchi et al. 2011; Takeuchi et al. 2009). We also established a transgenic lineage of Nibe croaker that carries an enhanced green fluorescent protein (gfp) gene driven by rainbow trout heat-shock-cognate 71 promoter/enhancer on pHSC-GFP (Yamamoto et al. 2011). Here, we combined these techniques and materials and demonstrated successful transplantation of donor spermatogonia obtained from pHSC-GFP transgenic Nibe croaker into the intraperitoneal cavity of triploid Nibe croaker recipients. In order to determine the potential of triploid recipients as surrogate broodstock for marine teleost, we assessed whether donor germ cells colonized the genital ridges of triploid recipients and whether gonads developed in these

triploid recipients that harbored donor-derived germ cells. Additionally, a gfp marker of the donor haplotype was used to examine the rate at which recipient surrogates produced donor-derived gametes and the rate at which donor-derived offspring appeared in subsequent generations. Additionally, the reproductive performance of triploid recipients and the quality of donor-derived gametes produced by triploid recipients were compared with those of non-transplanted diploid controls. Finally, we verified that donor-derived offspring could result from natural mating between male and female triploid recipients in an aquarium tank. 2. Materials and methods 2.1. Fish All Nibe croakers were maintained at the Tateyama Station (Banda), Field Science Center of Tokyo University of Marine Science and Technology in accordance with the Guide for the Care and Use of Laboratory Animals from Tokyo University of Marine Science and Technology. To maintain year-round spawning, the entire Nibe croaker broodstock was kept in 24–25 °C water in 1000-l circular fiber-reinforced plastic (FRP) tanks, and the photoperiod was maintained at 16–18 light. Sperm and ovulated eggs were collected by applying gentle pressure to the abdomen; no exogenous hormone was administered. To produce triploid fish, freshly ovulated eggs and milt were collected from wild-type Nibe croakers. After the artificial fertilization, eggs were treated with cold shock according to the method optimized by Takeuchi et al. (2016). Specifically, exactly 5 min after fertilization, fertilized eggs were placed in 10 °C seawater for 15 min. Then, treated eggs were transferred to a 100-l seed production tank containing 24–25 °C seawater. Hatchling larvae were reared as described previously (Takeuchi et al. 2009). 2.2. Testicular cell transplantation For each transplantation experiment, donor cells were prepared from testes taken from approximately 30 (range 20–53) individual gfp/− Nibe croaker males aged 3 months (mean total length 9.6 ± 1.1 cm, mean body weight 12.5 ± 4.3 g). The pooled testes were dissociated according to the following procedure. Dissociation and PKH26-labeling (Sigma-Aldrich, St. Louis, MO) of testicular cells were performed as described by Higuchi et al. (2011). A method described by Takeuchi et al. (2009) was used to transplant approximately 10,000 PKH26-labeled cells into the peritoneal cavity of each 4- to 5-mm larva (12 days post hatch (dph)) that had developed as a triploid from fertilized eggs; each triploid had been subjected to the aforementioned cold shock to induce triploidization. Transplantations were performed using 56–305 larvae in each experiment; eight independent experiments were conducted. 2.3. Observation of fluorescently labeled donor-derived germ cells in Nibe croaker recipients The rate at which transplanted testicular PKH26-labeled cells colonized recipient genital ridges was evaluated by assessing PKH26 signal in the genital ridges of 30-dph recipients under a fluorescence microscope (BX-51; Olympus, Tokyo, Japan) and a confocal microscope (FV1000, Olympus) as described previously (Takeuchi et al. 2009). Colonization rate (number of recipients with PKH26-labeled germ cell in the genital ridges/total number of observed recipients) and number of PKH26-labeled germ cells per genital ridge were determined. 2.4. Progeny tests with gametes from 6-month-old recipients To determine whether recipient males produced donor-derived sperm, genomic DNA was extracted from the milt of recipients and

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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analyzed via PCR with primers designed for the gfp gene (Yamamoto et al. 2011). Gentra Puregene cell kits (Qiagen GmbH, Dusseldorf, Germany) were used according to the manufacturer's instruction to extract sperm DNA. A 559-bp fragment from gfp was amplified with a primer set specific for gfp (5′-TAC GGC AAG CTG ACC CTG AAG TTC A3′ and 5′-TCA CGA ACT CCA GCA GGA CCA T-3′). Thermal cycling was carried out under the following conditions: initial denaturation for 5 min at 94 °C, 30 cycles of PCR (30 s at 94 °C, 30 s at 62 °C, and 30 s at 72 °C), and a final extension 5 min at 72 °C. Amplified DNA fragments were analyzed by electrophoresis through a 1% agarose gel. A β-actin (actb) primer set, described in Yamamoto et al. (2011), was used as a positive control for amplification of genomic DNA. To determine the concentration of sperm in milt samples, a milt subsample was diluted 1:1000 (v/v) in phosphate buffered saline (PBS), and sperm cells were counted on a Thoma hemocytometer; the original concentration of sperm (cells/ml) was then calculated. Each collected milt sample was added to seawater to observe sperm activity under a light microscope. The ratio of motile sperm relative to all sperm in one microscopic field of view was assessed visually, and the period of motility of sperm was also measured according to the procedure described by Yoshikawa et al. (2007). Briefly, 1 μl of milt from each fish was added to ambient seawater to observe motility under a light microscope (×150). The ratio of motile spermatozoa relative to all the spermatozoa in a microscopic view was assessed visually using six grades, i.e., N80%, about 60%, about 40%, about 20%, about 10%, and 0%. In addition, the amount of time motile spermatozoa spent moving was also measured. gfppositive milt samples obtained from diploid or triploid recipients were used to fertilize eggs collected from wild-type females, and at least 30 hatched, first-filial (F1) larvae from each male (one milt sample/male) were used for DNA analysis to evaluate the proportional contribution of donor-derived sperm to the population of F1 offspring. To assess production of donor-derived eggs, ovulated eggs from diploid or triploid female recipients were fertilized with milt from wildtype males. For each diploid recipient, 10–16 pooled DNA samples, each comprising DNA from 10 hatchlings from a cross between one diploid female recipient and a wild-type male, were investigated by PCR amplification of gfp. For each triploid female recipient, we first used genomic DNA PCR with gfp-specific primers to analyze independent pooled DNA samples that each comprised DNA from 10 unfertilized eggs obtained from an individual female. For those female triploid recipients that produced gfp-positive eggs, eggs were then harvested and fertilized with milt from a wild-type male. For each recipient female that produced gfp-positive eggs, at least 30 hatched larvae were analyzed by PCR to evaluate the proportional contribution of donor-derived eggs to the F1 offspring population. These progeny tests were repeated three to six times for male recipients and one to seven times for female recipients; for each test, a different clutch of eggs or sample of milt from a wild-type fish was used. For all experimental crosses, fertilized eggs were reared in a 100-l tank with 24–25 °C seawater. Approximately one hundred eggs (4- to 8-cell stage) were collected 1 h post fertilization; further cleavages were observed in a plastic petri dish under a stereo microscope to calculate fertilization rate. Then, these groups of fertilized eggs were each transferred to a 1000-ml glass beaker, reared for 30 h to the hatchling stage in a 25 °C incubator, and used to calculate the hatching rate. 2.5. Mating triploid female recipients with triploid male recipients To obtain donor-derived F1 offspring by natural spawning of male and female triploid-recipients, sexually mature triploid recipients of each sex were kept together a in 1000-l tank for 10 days (from Sep. 29 to Aug. 7, 2012). Spawned and potentially fertilized eggs were collected by water flow into an egg collector, and hatchlings were examined and used for DNA analysis. Developmental rates were determined at the somite-embryo stage (10 h post-fertilization), and hatching rates were evaluated as described before.

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Donor-derived F1 offspring of triploid-recipient parents were reared for 6 months to reach sexual maturity. Milt samples obtained from seven F1 males were used to fertilize eggs collected from wild-type females, and 32 hatched, second-filial (F2) larvae from each male were used for DNA analysis to determine whether gfp was present. In addition, sex ratios of a population of 6-month-old F1 fish were examined by dissecting the gonads. 2.6. Ploidy determination The ploidy of putative-triploid animals used for transplantation experiments was assessed. Whole larvae produced by cold-shock treatment, fin tissue from recipients dissected at 30 dph to assess colonization rates, and blood samples from mature 6-month-old recipients were used for flow cytometry to test the ploidy status of each individual recipient. The relative DNA content of each larval tissue, fin tissue, or blood sample was measured using Guava PCA-96 (Millipore, Billerica, MA). Cystain PI absolute T kits (Partec, Munster, Germany) were used according to the manufacturer's instructions to prepare larval samples and fin samples. Blood samples were collected from 6-monthold individuals with a heparinized syringe and then fixed in 70% ethanol for at least 30 min; approximately 1 × 106 of these fixed cells were resuspended in propidium iodide (PI) staining solution containing 60 μg/ml PI and 300 μg/ml RNase A in PBS. Larval, fin, or blood samples from normal diploids were used to represent the standard diploid DNA content value of the respective sample type. 2.7. Gonadal development and histological analysis of recipients To evaluate the gonadal development of recipients that had become 6 months old, testes or ovaries were collected, and the gonadosomatic index (GSI (%): total gonadal weight/total body weight × 100) was calculated. The testes and ovaries were also fixed with Bouin's fixative or Tissue-Tek Ufix (Sakura Finetech, Tokyo, Japan), respectively. Fixed tissues were then cut into 5 μm-thick sections using standard paraffinembedding methods and stained with hematoxylin–eosin (HE). Images of sections were obtained using a light microscope and a digital camera (DP-70; Olympus). 2.8. Statistical analysis Data are presented as means ± SEM. One-way ANOVA followed by the Tukey post-hoc test was used to determine significant differences among three group means. Differences between two group means were analyzed using Student's t-test. For all statistical tests, differences were considered statistically significant when the respective p-value was b 0.05. 3. Results 3.1. Colonization of recipient genital ridges by transplanted germ cells Donor cells were prepared from the testes of 3-month-old, hemizygous pHSC-GFP (gfp/−) donors; these testes consisted mainly of type-A spermatogonia (Fig. 1A). Dissociated testicular cells, labeled with PKH26 (Fig. 1B), were transplanted into the peritoneal cavities of a total of 1071 recipient12-dph larvae (total length: 4–5 mm) over the course of eight independent experiments. Each recipient had developed from cold-shock-treated wild-type eggs. Of the 1071 recipients, 434 survived transplantation to 30 dph (survival rate = 41.1 ± 3.4%). The triploidization rate among the 30-dph animals was 81.9 ± 6.5%; this rate was not significantly different from the rate at 3 dph (81.0 ± 7.4%) (p b 0.05), suggesting that triploids survived the transplantation procedure as well as diploids did. The other 18% of the transplanted 30-dph animals developed as diploids, not triploids, and were used as the transplanted diploid-recipient controls. Colonization of genital

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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ridges by PKH26-labeled donor germ cells with large nuclei was observed in recipients at 30 dph (Fig. 1C); such large nuclei are characteristic of type-A spermatogonia based on in situ hybridization of a germ cell marker, vasa, and 4′,6-diamidino-2-phenylindole (DAPI) staining (Takeuchi et al. 2009). Among 38 triploid recipients and 8 diploid recipients at 30 dph from eight independent transplantation experiments, 56.0 ± 9.8% (22 of 38 observed fish) and 63.3 ± 18.6% (5 of 8 observed fish) showed evidence of genital ridges colonized by PKH26-labeled germ cells, respectively. There was no significant difference in colonization rates between the two groups (p $_amp_$gt; 0.05). No significant difference was observed in numbers of PHK26-labeled germ cells observed in the genital ridges of triploid recipients (4.9 ± 1.4 cells, n = 11) when compared to those of diploid recipients (4.2 ± 1.2 cells, n = 5, p $_amp_$gt; 0.05). These 46 animals (38 triploids and eight diploids) that were used to assess colonization rates were sacrificed at 30 dph; the remaining 1025 cold-treated larvae subjected to transplantation were allow to develop past 30 dph. Of these 1025 recipients (diploid and triploid), 203 (19.8%) survived until 6 months old. Blood samples were used for ploidy analysis; 50.0% (38/76) of the surviving male recipients and 65.3% (83/127) of the surviving female recipients were triploid; in all, 121 (59.6%) of the 203 surviving recipients were triploid. The rest of the recipients (n = 82) were diploid. Both triploid and diploid recipients were used for the subsequent progeny tests to determine whether donor-derived germ cells produced functional gametes in the recipient gonads. 3.2. Production of donor-derived sperm by male recipients The production of donor-derived sperm by each surviving male recipient (n = 76, including 38 diploids and 38 triploids) was investigated. Milt was collected from each male recipient by abdominal pressure,

and 1-μl subsamples were individually dropped onto a glass slide to assess sperm production (Fig. 2A). Milts collected from non-transplanted triploid males (control), as shown for triploid, were transparent. Of the 38 male triploid recipients, 14 (36.8%) produced whitish milt that contained sperm as shown for 3N1M and 3N2M; no sperm was evident in any sample from the other 24 (63.1%) male triploid recipients. Notably, the color of the milt from the triploid male recipient, 3N1M, was similar to those of diploid males. Each of the 38 diploid recipients (100%) produced normal whitish sperm, as shown for 2N1M (Fig. 2A). Genomic DNA samples extracted from the milt of diploid or triploid recipients were analyzed by PCR amplification with gfp-specific primers designed to detect gfp and therefore the presence of donor-derived sperm (Fig. 2B). As summarized in Fig.2C, each of the 14 spermiating triploid recipients produced gfp-positive milt; therefore, 36.8% of triploid male recipients (14 out of 38) produced donor-derived sperm. In contrast, gfp signals were evident in the milt of only 2 (5.3%) of the 38 spermiating diploid-male recipients (Fig. 2C). 3.3. Production of donor-derived offspring from male recipients To assess sperm concentration and sperm activity, milt from two randomly selected male diploid recipients (2N1M and 2N2M) and three male triploid recipients with gfp-positive milt (3N1M, 3N2M, and 3N3M) were compared with milt from non-transplanted control diploids (Control 1–3) (Table 1). The concentrations of sperm in milt from both diploid recipients (6.3–6.5 × 109 cells/ml, 2N1M and 2N2M) and one of three triploid recipients (6.5 × 109 cells/ml, 3N1M) were not significantly different to those from control diploids (5.2– 6.1 × 109 cells/ml, Control 2 and 3, p N 0.05). Significantly lower sperm concentrations than the diploids (Control 1–3) were observed in milt from the other two triploid recipients (0.2–0.7 × 109 cells/ml,

Fig. 1. Testicular cell transplantation and colonization by PKH26-labeled donor spermatogonia. A) Sections of a donor testis stained with hematoxylin and eosin (HE). Arrowheads indicate type-A spermatogonia. B) Dissociated testicular cells labeled with PKH26. C) A genital ridge of a triploid recipient at 30 dph viewed via differential-interference contrast (DIC) and confocal microscopy. DIC (upper left), PKH26 image (upper right), DAPI image (bottom left), and merged (PKH26 + DAPI) images (bottom right) are each indicated. Arrows indicate a PKH26labeled, donor-derived germ cell with a large nuclei. Bar = 10 μm.

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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Fig. 2. Production of donor-derived sperm from diploid or triploid recipients. A) Photo of milt samples collected from non-transplanted diploid, non-transplanted triploid, diploid recipient (2N1M), or triploid recipients (3N1M and 3N2M). For each milt sample, a 1-μl subsample was dropped onto a glass slide. B) PCR amplification of gfp sequences from genomic DNA extracted from milt samples from individual male recipient. As an internal control, β-actin (actb) sequences were also amplified. C1, positive control (milt from pHSC-GFP fish); C2, negative control (milt from wild-type fish); 1–13, milt samples from diploid (1–10) and triploid (11–13) recipients. C) Percentage of individuals producing gfp-positive sperm, gfpnegative sperm, or no sperm among all diploid or triploid recipients.

3N2M and 3N3M, p b 0.05). The moving time of sperm from triploid recipients was not significantly different (3N1M and 3N2M, p N 0.05) or was significantly longer (3N3M, p b 0.05) to those of diploid recipients

(2N1M and 2N2M) and control diploids (Control 1–3) (Table 1). N 80% of sperm in the microscopic field were found to be motile in all of the fish examined (Table 1).

Table 1 Evaluation of sperm collected from the diploid and triploid recipients and non-transplanted controls. Individual #

Ploidy

Concentration (cells/ml × 109)

Motility

Moving time (s)

Recipient (2N1M) Recipient (2N2M) Recipient (3N1M) Recipient (3N2M) Recipient (3N3M) Control 1 Control 2 Control 3

Diploid Diploid Triploid Triploid Triploid Diploid Diploid Diploid

6.5 ± 0.1 a 6.3 ± 0.2 a 6.5 ± 0.2 a 0.2 ± 0.0 c 0.7 ± 0.1 c 5.2 ± 0.3 b 6.1 ± 0.3 a, b 5.7 ± 0.2 a, b

N80% N80% N80% N80% N80% N80% N80% N80%

321 ± 8 b, c 348 ± 16 c 363 ± 18 b, c 350 ± 15 b, c 452 ± 15 a 348 ± 19 b, c 388 ± 14 a, b, c 408 ± 9 a, b

Different letter designations (a, b, c) indicate statistically significant differences (p b 0.05).

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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Collected milt samples from each diploid recipient (2N1M and 2N2M) and from each triploid recipient (3N1M, 3N2M, and 3N3M) were used separately to fertilize eggs from wild-type females. For each of these five male recipients (with the exception of 2N2M who died after only three experimental crosses), experimental crosses were conducted six independent times during the period from March 19, 2012 to July 14, 2013. Fertilization and hatching rate of all of the crosses involving sperm from these five recipients (diploid or triploid) were summarized in Table 2. In only three of the six crosses (5/11/2012, 7/13/2012, and 5/10/2013) with a triploid male recipient, 3N2M, fertilization rates (2 to 4.5%) and hatching rates (0.4 to 0.8%) were very low. However, in all other crosses involving sperm from triploid male recipients, fertilization and hatching rates were not significantly different to those of crosses involving sperm of non-transplanted diploid males (control) and diploid male recipients (p N 0.05). PCR analysis of genomic DNA with gfp-specific primers indicated that approximately 50% of the F1 offspring from each triploid male recipient carried gfp (51.2 ± 1.2%, range = 43.8–59.4%). This result indicated that all F1 offspring originated from donor-derived sperm because the donor germ cells were diploid and had the gfp/− genotype. In contrast, 3.4 ± 1.9% (range = 0–9.4%) of the F1 hatchlings from 2N1M and 44.6 ± 0.8% (range = 43.1–45.3%) of those from 2N2M carried gfp, suggesting that recipient-derived sperm contributed a smaller proportion of individual in the F1 offspring of 2N1M but almost all proportion of individual in that of 2N2M. Additionally, approximately 50% of the offspring from the second spawning season (May 10 to July 14, 2013) of male triploid recipients (3N1M, 3N2M, and 3N3M) carried gfp; therefore, secondseason offspring were also probably all donor-derived (Table 2).

Notably, gfp-positive hatchlings were not obtained from the diploid recipient 2N1M in second spawning season. 3.4. Production of donor-derived eggs by female recipients The production of donor-derived eggs by the 127 surviving female recipients (83 triploids and 44 diploids) was investigated. Ovulation was confirmed for only 28.9% (24/83) of triploid recipients and for 100% (44/44) of diploid recipients. Pooled genomic DNA samples extracted from hatchlings or unfertilized eggs from diploid or triploid recipients, respectively, were analyzed via PCR amplification with gfp-specific primers to detect the production of donor-derived, gfp-positive eggs (Fig. 3A and B). As summarized in Fig. 3C, each of the 24 ovulating triploid recipients, which represented 28.9% of all triploid female recipients, produced gfp-positive eggs; in contrast, only 3 of the 44 ovulating diploid recipients (6.8%) produced gfp-positive, donor-derived eggs. The other 41 ovulating diploid recipients (93.2%) were found to produce only gfp-negative, recipientderived eggs based on PCR analyses of 100–160 eggs per recipient female. Notably, no triploid female recipient was observed to produce only gfp-negative eggs. Of the three diploid recipients produced gfp-positive hatchlings (2N1F, 2N2F, and 2N3F), the 2N3F-female spawned only once in the above-mentioned experiment; this fish could not be used for further progeny tests because it did not continue to spawn thereafter (Table 3). 3.5. Production of donor-derived offspring from female recipients Ovulated eggs from two of three diploid recipients (2N1F and 2N2F) and from six of 24 triploid recipients (3N1F thru 3N6F) were fertilized

Table 2 Progeny tests and the rates of gfp-positive offspring among F1 generation produced by male recipients. Male #

Ploidy

Female

Date of fertilization (month/day/yr)

No. of eggs

Fertilization (%)

Hatching (%)

Analyzed no. of F1 offspring

gfp + (%)

2N1M

Diploid

2N2Mb

Diploid

3N1M

Triploid

3N2M

Triploid

3N3M

Triploid

Wild-type1 Wild-type2 Wild-type3 Wild-type4 Wild-type7 Wild-type6 Wild-type1 Wild-type2 Wild-type3 Wild-type1 Wild-type2 Wild-type3 Wild-type4 Wild-type5 Wild-type6 Wild-type1 Wild-type2 Wild-type3 Wild-type4 Wild-type5 Wild-type6 Wild-type1 Wild-type2 Wild-type3 Wild-type4 Wild-type5 Wild-type6 Wild-type1 Wild-type2 Wild-type3 Wild-type4 Wild-type7 Wild-type5 Wild-type6

3/19/2012 5/11/2012 7/13/2012 5/10/2013 6/18/2013 7/14/2013 3/19/2012 5/11/2012 7/13/2012 3/19/2012 5/11/2012 7/13/2012 5/10/2013 6/25/2013 7/14/2013 3/19/2012 5/11/2012 7/13/2012 5/10/2013 6/25/2013 7/14/2013 3/19/2012 5/11/2012 7/13/2012 5/10/2013 6/25/2013 7/14/2013 3/19/2012 5/11/2012 7/13/2012 5/10/2013 6/18/2013 6/25/2013 7/14/2013

7000 1070 23,200 4500 25,000 37,800 8500 1330 15,200 5000 13,200 6400 4800 2700 52,400 3000 8600 9600 4200 2100 45,480 3000 6600 5600 2800 2400 39,200 3500 800 16,800 4800 10,000 2400 15,400

19.5 52.1 69.0 17.6 76.9 63.5 48.1 55.2 47.4 47.4 40.9 37.5 26.3 83.3 67.3 30.0 2.3 1.2 4.5 88.4 58.3 54.7 48.5 57.1 18.8 88.1 67.3 46.2 70.6 31.0 33.3 96.9 89.5 73.6

14.3 49.8 64.7 1.9 32.0 44.4 39.2 20.3 14.0 16.7 25.0 16.7 16.7 74.1 63.7 11.1 0.7 0.4 0.8 28.6 38.7 5.6 20.5 19.1 10.7 83.3 57.1 19.0 66.6 6.3 27.8 73.0 87.5 51.9

64 64 64 83 96 64 64 72 64 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 – – – – – – –

9.4 9.4 1.6 0.0a 0.0a 0.0a 45.3 43.1 45.3 43.8 43.8 43.8 53.1 50.0 59.4 50.0 50.0 50.0 53.1 53.1 50.0 46.9 50.0 59.4 53.1 53.1 59.4 – – – – – – –

Wild-type8 (control) Wild-type9 (control) Wild-type10 (control) Wild-type11 (control) Wild-type12 (control) Wild-type13 (control) Wild-type14 (control) a b

No gfp-positive embryos were obtained from the diploid male recipient (2N2M) in the second spawning season. Recipient number 2N2M died after third experimental cross.

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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with sperm from wild-type males (Table 3). Each experimental cross was conducted during the period from March 17, 2012 to June 8, 2013. In all the crosses involving eggs from triploid female recipients, fertilization rates were not significantly different to those of crosses involving eggs of non-transplanted diploids (wild-type1 to 6 in Table 2, p N 0.05). Eggs from triploid female recipients (22.4 ± 3.25%, range 1.2–62.5%, n = 25) showed significantly lower hatching rates than those from non-transplanted females (47.4 ± 11.5%, range 6.3–87.5%, n = 7, p b 0.05), although hatching rates were greatly variable inside each individual (Table 3). The fertilization and hatching rates obtained from diploid female recipients were not used for statistical analysis, because both of them spawned only once during this period. PCR analysis of genomic DNA with gfp-specific primers indicated that approximately 50% of the F1 offspring from female triploid recipients carried gfp (50.3 ± 0.9%, range = 43.8–56.7%). This result indicated that all F1 offspring originated from donor-derived eggs because the donor germ cells were diploid and had the gfp/− genotype. In contrast, in ovulating diploid recipients, 1.1% of the F1 hatchlings from 2N1F and 12.5% of those from 2N2F carried gfp, suggesting that recipient-derived eggs contributed a smaller proportion of individual in the F1 offspring of 2N1F and 2N2F. Additionally, for two ovulating triploid recipients, 3N1F and 3N2F, 50.0% and 56.3% of the offspring from the second spawning season (May 31 and June 8, 2013), respectively, carried gfp; therefore,

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second-season offspring from these females were also probably all donor-derived (Table 3). During the period from May 6 to June 8, 2012, which constituted the 34 days of their natural spawning season, serial experimental crosses were conducted to compare the reproductive performance of triploid female recipients (3N1F thru 3N6F, n = 6) with that of nontransplanted control diploids (n = 11). In 21 spawning events involving the six triploid female recipients, mean ovulation frequency (3.5 ± 0.5 times), mean number of ovulated eggs per female (56,500 ± 5600 eggs/spawning), mean fertilization rate (38.5 ± 4.6%), and mean hatching rate (18.9 ± 3.4%) were not significantly different to those for the 11 non-transplanted control diploids (ovulation frequency = 3.2 ± 0.3 times, number of ovulated eggs per female = 76.200 ± 5400 eggs/ spawning, fertilization rate = 26.1 ± 3.2%, and hatching rate = 5.7 ± 1.0% in total of 35 spawning events) (p N 0.05). 3.6. Production of donor-derived offspring by mating between triploid male and triploid female recipients To produce donor-derived offspring by natural spawning of triploid recipient surrogate parents, six female triploid recipients (3N1F–3N6F) and three triploid-recipient males (3N1M–3N3M), were maintained in a 1000-l tank for 10 days (from Sep. 29 to Aug. 7, 2012), and the resulting hatchlings were the analyzed for gfp (Table 4). During this 10-day period, five spawning events with an average of 141,000 ± 2200 eggs and 4629 ± 2446 hatchlings per event (hatching rate = 3.4 ± 1.7%, n = 5) were obtained. Across all five spawning events, the average percentage of gfp-positive F1 offspring was 74.2 ± 1.2% (range = 71.0–77.4%, n = 5). This result showed that all F1 offspring, which were obtained by natural mating of triploid male and female recipients, resulted from fertilization of donor-derived eggs with donorderived sperm because donor germ cells of all parents had the gfp/− genotype. Among the F1 offspring produced by natural spawning of triploid recipient parents, 33 individuals were reared to sexual maturity. This group of F1 offspring comprised 25 males (75.8%) and eight females (24.2%), suggesting that male to female sex ratio was 3:1. Furthermore, among the 25 F1 males, seven males that produced gfp-positive milt were crossed with wild-type females (Table 5), and three of these seven F1 males (F1M1, F1M3, and F1M7) produced only gfp-positive (100%) F2 offspring. Therefore, these three F1 males were homozygous pHSC-GFP transgenic fish (gfp/gfp) that originated from fertilization of donor-derived, gfp-positive eggs with donor-derived, gfp-positive sperm. 3.7. Morphological characteristics of the gonads from recipients that produced donor-derived gametes

Fig. 3. Production of donor-derived eggs from diploid or triploid recipients. A) PCR amplification of gfp from pooled DNA samples, which each comprised F1 offspring derived from one diploid recipient (n = 11). C1, negative-control DNA from wild-type fish; C2, positive-control DNA from pHSC-GFP fish; C3 and C4, positive-control DNA (C2) diluted 10-fold (C3) or 100-fold (C4) with fully concentrated wild-type DNA (C1); 1–11, pooled DNA samples from F1 offspring from 11 separate crosses each involving one diploid female recipient and a wild-type male. Each pooled sample comprised DNA from 10 hatchlings. For each of the 11 recipients, 10 to 16 pooled DNA samples were tested. B) PCR amplification of gfp from pooled DNA samples each comprising DNA from unfertilized eggs from one triploid recipient (n = 8). C1, positive control DNA from pHSC-GFP fish; C2, negative control DNA from wild-type fish; 1–8, pooled DNA samples of unfertilized eggs from triploid-recipients. Each pooled sample comprised DNA from 10 unfertilized eggs. Eight pooled DNA samples were tested for each of the eight triploid recipients. C) Percentage of individuals that produced gfp-positive eggs, only gfpnegative eggs, or no eggs (no ovulation) among diploid recipients or triploid-recipients.

To evaluate the gonadal morphology of male triploid recipients that produced donor-derived, gfp-positive sperm, testes were collected surgically (n = 5) from each of these males. Non-transplanted diploids (n = 10) and non-transplanted triploids (n = 10) were used as controls. External morphology and testes color of male triploid recipients (Fig. 4C) were undistinguishable from those of non-transplanted diploids and triploids (Fig. 4A and B). Although mean GSI of nontransplanted male triploids was significantly lower than that of nontransplanted male diploids (p b 0.05), mean GSI of male triploid recipients that produced gfp-positive sperm (1.9 ± 0.1%) was comparable with that of non-transplanted male diploids (2.5 ± 0.3%) (p N 0.05) (Fig. 4D). Based on histological observation, spermatogenesis with mass production of spermatozoa was observed in the testis of male triploid recipients (Fig. 5G) as well as non-transplanted male diploids (Fig. 5E). In contrast, non-transplanted male triploids produced very few spermatozoa, and cysts containing irregular shaped spermatids with heterogeneity in size were observed in these testes (Fig. 4F).

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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Table 3 Progeny tests and the rates of gfp-positive offspring among F1 generation produced by female recipients. Female #

Ploidy

Date of fertilization (month/day/yr)

No. of eggs

Fertilization (%)

Hatching (%)

Analyzed no. of F1 offspring

gfp + (%)

2N1F 2N2F 2N3F 3N1Fa

Diploid Diploid Diploid Triploid

3N2Fa

Triploid

3N3Fa

Triploid

3N4Fa

Triploid

3N5Fa

Triploid

a

Triploid

7/25/2012 7/27/2012 – 3/17/2012 5/06/2012 5/18/2012 5/22/2012 5/25/2012 6/04/2012 5/31/2013 3/23/2012 5/08/2012 5/23/2012 6/05/2012 6/08/2013 5/09/2012 5/18/2012 5/30/2012 6/04/2012 6/08/2012 5/08/2012 5/13/2012 5/22/2012 5/15/2012 6/03/2012 5/06/2012 5/23/2012 6/05/2012

76,000 70,000 – 52,400 53,600 7800 85,600 56,800 83,200 134,400 54,600 62,100 38,500 44,800 48,000 81,600 28,200 105,000 105,000 90,300 39,000 32,000 43,200 48,000 53,600 35,200 49,000 44,800

18.9 2.3 – 92.9 53.7 61.5 31.8 64.8 67.3 50.0 71.1 21.7 65.5 51.8 76.5 25.9 44.7 27.3 24.0 7.8 7.7 60.0 29.6 66.7 61.2 18.2 21.4 56.3

2.8 1.9 – 38.2 29.9 34.2 15.5 17.6 19.2 38.1 44.0 8.6 20.8 23.8 41.7 1.2 9.5 4.0 15.2 3.0 2.7 25.0 30.9 62.5 24.9 1.3 6.5 41.7

264 112 – 32 32 32 32 63 31 32 30 32 32 32 32 32 32 32 31 31 32 32 32 32 32 32 49 30

1.1 12.5 – 43.8 56.3 53.1 50.0 46.0 51.6 50.0b 53.3 56.3 46.9 53.1 56.3b 56.3 43.8 46.9 41.9 48.4 53.1 53.1 46.9 53.1 50.0 46.9 44.9 56.7

3N6F

Eggs from female recipients were artificially inseminated with milt from wild-type nontransplanted males. a Serial experimental crosses using all of the ovulated triploid recipients were conducted for the period from May 6 to June 8, 2012. b Percentage of donor-derived offspring obtained in the second spawning season.

Obvious unilateral hypertrophy of the ovary was observed in female triploid recipients that produced donor-derived, gfp-positive eggs (Fig. 5C); this overgrowth was evident when compared to that of nontransplanted diploids or non-transplanted triploids (Fig. 5A and B). The mean GSI of female triploid recipients (14.0 ± 1.7%, n = 5) was significantly higher than that of non-transplanted triploids (3.3 ± 0.5, n = 15, p b 0.05) but was not significantly different to that of nontransplanted diploids (11.2 ± 0.8%, n = 15, p N 0.05) (Fig. 5D). Interestingly, PCR analysis of genomic DNA from ovaries of female triploid recipients indicated that hypertrophic regions (region i Fig. 5C) were gfp-positive, and non-hypertrophic regions (region h Fig. 5C) were gfp-negative (Fig. 5E). Based on histological analysis, hypertrophic ovarian tissue from recipient triploids comprised mainly vitellogenic oocytes (Fig. 5I), as did most ovarian tissue of non-transplanted diploids (Fig. 5F); in contrast, very few vitellogenic oocytes were evident in non-hypertrophic ovarian tissue from recipient triploids (Fig. 5H) or in any ovarian tissue from non-transplanted triploids (Fig. 5G).

Table 4 Analysis of natural spawning in aquarium tank and rates of gfp-positive F1 offspring obtained by crossing of triploid-recipient parents.

Date of egg collection (month/day/yr)

No. of eggs

Somite embryo (%)

7/29/2012 7/31/2012 8/04/2012 8/06/2012 8/07/2012

94,670 111,000 205,000 110,400 184,600

1.9 4.5 10.5 9.8 8.8

Hatching (%)

Analyzed no. of F1 offspring

gfp + (%)

1.6 0.1 0.2 8.7 6.2

29 31 32 32 31

75.9 77.4 71.9 75.0 71.0

Spawning eggs were collected by water flow into an egg collector. Six female (3N1F– 3N6F) and three male (3N1M–3N3M) triploid recipients were reared in 1000-l tank for 10 days (Sep. 29 to Aug. 7, 2012).

4. Discussion In the present paper, production of only donor-derived gametes from triploid surrogate broodstock was demonstrated for the first time in germ-cell transplantation studies of marine fishes. Not only was the proportion of donor-derived offspring in the F1 generation improved, but the frequency of recipients that produced donor-derived gametes increased about 7-fold for male recipients and 4-fold for female recipients when triploids were used instead of diploids as recipients. Tolerance to the transplantation procedure was comparable between triploid and diploid recipient Nibe croaker larvae. Additionally, the quality and quantity of allogenic donor-derived eggs and sperm obtained from triploid Nibe croaker recipients and the frequency of ovulation in the triploid female recipients were as high as those for diploid recipients or non-transplanted diploids. These data demonstrated that triploid surrogates were more suitable than diploid surrogates and that use of triploid surrogates may accelerate the application of surrogate

Table 5 Progeny tests and the rates of gfp-positive offspring among F2 generation produced by F1 males.

Male

Female

No. of eggs

F1M1 F1M2 F1M3 F1M4 F1M5 F1M6 F1M7 wild-type17 wild-type18

Wild-type15 Wild-type15 Wild-type15 Wild-type16 Wild-type16 Wild-type16 Wild-type16 Wild-type15 Wild-type16

32,400 22,800 16,000 2400 2240 2320 1760 20,000 2240

Fertilization (%)

Hatching (%)

Analyzed no. of F2 offspring

gfp + (%)

95.4 93.0 87.5 23.3 50.0 44.8 36.4 90.0 42.9

49.4 43.9 46.9 66.7 59.4 60.3 30.1 55.0 35.7

32 32 32 32 32 32 32 – –

100.0 59.4 100.0 56.3 34.4 37.5 100.0 – –

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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Fig. 4. Testicular development of 6-month-old triploid recipients, non-transplanted diploids, or non-transplanted triploids. A)–C) Testes of a non-transplanted diploid (A), a nontransplanted triploid (B), and a triploid recipient (C). In each image, the posterior end of the testis is to the right. Bar = 1 cm. D) Gonadosomatic index of non-transplanted diploids, non-transplanted triploids, and triploid-recipients. Data are represented as mean ± SEM. Different letters indicate statistically significant differences (p b 0.05). E)–G) Histological section of testis from a non-transplanted diploid (E), a non-transplanted triploid (F), and a triploid recipient (G). SZ, spermatozoa; GB, type-B spermatogonia; SC, spermatocyte. Arrows indicate cysts containing irregular shaped spermatocytes found in the testis of non-transplanted triploids. Bar = 10 μm.

broodstock technology in aquaculture-targeted, genetically valuable, or endangered marine teleosts. Induced triploidy results in infertility for many teleost fishes, and it is generally believed that this sterility results from a failure in progress of meiosis (Pandian and Koteeswaran 1998). Failure of meiosis in triploids also leads reduced gonad size. However, triploid fish, especially male triploids, exhibited similar plasma levels of gonadal sex steroids (e.g. testosterone (T), 11-ketotestosterone) when compared with male diploids in most fish species examined to date (Felip et al. 2001a; Hamasaki et al. 2013; Mol et al. 1994; Nakamura et al. 1993; Tiwary et al. 2004). Although triploid females usually show lower levels of T and estradiol-17β (E2) than diploid females (Felip et al. 2001a; Tiwary et al. 2001), triploid females occasionally possess advanced stages of oocytes because of high plasma concentrations of T and E2 that are comparable to those of mature diploid females (Freund et al., 1995; Hamasaki et al. 2013). Moreover, in vitro culture experiments of ovarian follicles of brook trout Salvelinus fontinalis show that when controlling for follicular diameter the steroid biosynthetic capability of triploid ovarian follicles was comparable to that of diploid follicles (Schafhauser-Smith and Benfey 2003). Although, triploid Nibe croaker had very few sperm or vitellogenic oocytes in undersized testis and ovaries, respectively (Takeuchi et al., 2016), we showed here that triploid Nibe croaker recipient produced functional eggs and sperm that derived from transplanted allogenic diploid germ cells, but no functional gametes were produced from endogenous triploid germ cells. The gonadal sizes of recipients, which had successfully produced donor-derived gametes, were similar to those of sexually matured diploids. These observations indicated that 1) the gonadal endocrine system of triploid Nibe croaker could function properly if the recipient gonads harbored diploid germ cells, and 2) the gonadal microenvironment in triploid recipients was capable of producing functional gametes. Thus, we concluded that the use of Nibe croaker triploids as recipients for allogenic intra-peritoneal

spermatogonial transplantation could lead to efficient production of triploid surrogate broodstock that produced mostly or only donorderived gametes. In the triploid Nibe croaker recipients, the frequency of recipients that produced donor-derived gametes (i.e., surrogate parent rate) at 6 months old (28.9% in female, 36.8% in male) was not substantially lower than the rate of genital ridge colonization by donor-derived germ cells (i.e. colonization rate) observed at 30 dph (56.0%), which was equal to 18 days post transplantation (pt). However, in the diploid Nibe croaker recipients, the surrogate parent rate at 6 months old (6.8% in female, 5.3% in male) was markedly lower than the colonization rate at 18 days pt. (63.3%). Interestingly, this observation in diploid Nibe croaker recipients differed from our previous findings with allogenic germ-cell transplantations using diploid recipients in rainbow trout. In the trout, surrogate parent rate (40% in 2-year-old female, 50% in 1year-old male) with diploid recipients is comparable to the colonization rate at 20 days pt. (43%) (Okutsu et al. 2006). In yellowtail, both the colonization rate at 28 days pt. and the surrogate parent rate at 1.5 or 2.5 years old is nearly 100% (Morita et al. 2012). Moreover, in this study, PCR amplification of gfp gene from genomic DNA analysis demonstrated that no donor-derived germ cells were evident in the gonads of either diploid or triploid recipients that did not produce donorderived gametes (data not shown); these results indicated that some Nibe croaker donor germ cells that had colonized genital ridges had disappeared from the developing gonads of allogenic recipients, and the disappearance of the colonized donor cells during gametogenesis occurred more often in the diploid Nibe croaker recipient gonads than in those of triploids. Thus, we speculated that competition between donor-derived and endogenous germ cells during gametogenesis was more severe in the gonads of diploid Nibe croaker recipients than was observed with other fish species (Lee et al. 2013; Morita et al. 2012; Okutsu et al. 2006); we also speculated that this competition affected

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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Fig. 5. Ovarian development of 6-month-old triploid-recipients, non-transplanted diploids, or non-transplanted triploids. A)–C) Ovaries of a non-transplanted diploid (A), a nontransplanted triploid (B), and two individual triploid-recipients (C). In each image, the posterior end of the ovary is to the right, boxed regions indicated by h and i in 5C were also analyzed by PCR amplification and histology (see below). Bar = 10 mm. D) Gonadosomatic index of diploids, triploids and triploid-female recipients. Data are represented as mean ± SEM. Different letters indicate statistically significant differences (p b 0.05). E) PCR amplification of gfp from the DNA from ovary of a triploid recipient. Analyzed samples in the lanes designated h and i were collected from the boxed regions (h and i, respectively) indicated in C bottom. As an internal control, β-actin (actb) was also amplified. C1, positive control DNA from pHSC-GFP fish; C2, negative control DNA from wild-type fish. F)–I) Histological section of an ovary from a non-transplanted diploid (F), a non-transplanted triploid (G), or a triploid recipient (H and I). Histological section from the boxed regions (h and i, respectively) indicated in C are shown in H and I, respectively. VO indicates vitellogenic oocyte. Bar = 100 μm.

the survival of donor-derived germ cells. In conclusion, by comparing the surrogate parent rates between siblings, specifically triploid and diploid recipients, we clearly showed that efficient production of surrogate broodstock could be achieved by the use of functionally sterile triploid as recipients.

The rates of gfp-positive offspring obtained from diploid recipients were 0–45.3% in males and 1.1–12.5% in females, and these rates varied among diploid recipients. For example, 45.3% of the first set of F1 offspring from only one of the two diploid male recipients, 2N2M, were gfp-positive larvae. Because donor germ cells were hemizygous for the

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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gfp transgene, this data suggested that N 90% of this set of F1 offspring from this diploid male recipient had originated from donor-derived sperm. The predominance of donor-derived gametes over endogenous germ cell-derived gametes in diploid recipient gonads has been reported with allogenic and xenogenic spermatogonial transplantation in yellowtail (Morita et al. 2012) and salmonids (Okutsu et al. 2008), respectively. The preferential production of donor-derived cells might be explained if a high rate of proliferation of donor-derived spermatogonia following colonization of recipient genital ridges resulted in occupation of the germ cell niche among the supporting somatic cells by the donor-derived cells before the propagation of endogenous primordial germ cells (Yazawa et al. 2010). However, previous authors have also reported wide variation in germ-line transmission rates of donorderived haplotypes (e.g. 16.9–98.8% in yellowtail diploid male recipients, 0.2–91.3% in masu salmon diploid male recipients). In practical situations directed at production of donor-derived offspring via germ-cell transplantation techniques for aquaculture or conservation purposes, phenotypic markers (e.g., the albino mutant) and/or transgenes would not be used to mark the donor haplotype, thus massive analyses involving microsatellite PCR or PCR-RFLP would be required to distinguish donor-derived offspring from recipient-derived offspring. Varying the proportion of donor-derived offspring among F1 offspring of each diploid recipient would involve prohibitive technical difficulties for screening of donor-derived larvae, especially with marine fish that produce very large numbers of eggs. In contrast, as shown in this study of Nibe croaker and a previous study of salmonids (Okutsu et al. 2007, 2008), each mature triploid surrogate produced mostly or only donorderived gametes. Reportedly, recipients of donor spermatogonia produce large numbers of donor-derived sperm throughout their lifetimes; therefore, the progeny of the donor spermatogonia must undergo self-renewal and differentiation (Lee et al. 2013; Morita et al. 2012; Okutsu et al. 2007, 2008). In the current study, the densities of spermatozoa collected from three randomly selected triploid recipients that each produced only donor-derived sperm were 0.2–6.5 × 109 cells/ml. Notably, only 10 μl (approximately) of milt was collected from each recipient male per progeny test (6 times in total) in order to minimize the handling stress experienced by the broodstock fish; nevertheless, at least 0.1– 3.9 × 108 donor-derived spermatozoa were collected from each such triploid surrogate. Once fish spermatogonia are committed to differentiate, they lose their self-renewal capability and possess only a limited ability to proliferate (Schulz et al., 2010). In teleosts, differentiated spermatogonia are reported to undergo mitosis 6–16 times (Ando et al. 2000; Nóbrega et al. 2009; Schulz et al., 2010); these divisions are then followed by two consecutive cycles of meiosis; thus, one founder spermatogonium can produce between 256 and 262,144 spermatozoa. In the current study, approximately five donor germ cells were incorporated into the genital ridges of each triploid recipient. Thus, to produce 0.1–3.9 × 108 donor-derived spermatozoa from five differentiated spermatogonia, the donor-derived germ cells would need to have undergone mitosis 19–25 times, on average, before progeny spermatogonia committed to meiosis. This is far larger number of mitotic cycles than the predicted for proliferation of differentiated spermatogonia, if differentiated spermatogonia of Nibe croaker actually undergo similar numbers of divisions as other teleost to produce spermatozoa. In addition, subsequent production of donor-derived gametes for at least one year that included two consecutive spawning seasons was confirmed for triploid recipients of each sex. These data strongly suggested that the allogenic donor germ cells in Nibe croaker exhibited germ-line stem-cell activity in the triploid recipient gonads, and continually produce fully differentiated gametes. In this study, ovaries from triploid recipients consistently exhibited unilateral hypertrophy and produced the same amount of eggs as non-transplanted control diploid ovaries. The results of genomic DNAPCR analysis with gfp-specific primers showed that donor-derived gfppositive germ cells were present only on the hypertrophic side of the

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recipient ovaries. We previously showed that vitellogenic oocytes are rarely observed in a triploid ovary, although a large number of previtellogenic oocytes are usually evident (Takeuchi et al., 2016). Based on our current data and previous data, we proposed that unilateral hypertrophic ovaries observed in the recipient triploid females that produced donor-derived eggs resulted from colonization by and the progression of vitellogenesis for diploid donor-derived germ cells in triploid recipient ovaries. It is known that surgical removal of one ovary leads to compensatory hypertrophy (COH) in the remaining ovary in tetrapods (Billeter and Jorgensen 1976; Greenwald 1961; Hirshfield 1982; Staigmiller et al. 1972) and iteroparous fish, such as rainbow trout O. mykiss (Tyler et al. 1994, 1996), catfish Heteropneustes fossilis (Blosh) (Goswami and Sundararaj, 1968), Tilapia aurea and Oreochromis aureus (Dadzie and Hyder 1976); the remaining hypertrophic ovary is often equivalent in mass and fecundity to a pair of ovaries in a normal control animal. COH in the remaining ovary after unilateral ovariectomy could be explained by 1) production of larger eggs from the existing pool of vitellogenic follicles, 2) recruitment of a second population of primary oocytes into the vitellogenic pool, 3) maximization of the fecundity by reducing the number of atretic follicles, and/or 4) increases in the number of dividing oogonia with concomitant production of stem cells (Pandian 2012; Tyler et al. 1996). Because the average diameter of ovulated eggs produced by triploid female recipients did not differ from that for diploid females (data not shown), it seemed unlikely that an increase in egg size resulted in the unilateral hypertrophic ovaries in the triploid recipients. In order to understand the mechanism underlying unilateral hypertrophy of ovaries and the recovery of fecundity in the triploid recipient females, further study is needed to investigate the regulation of cell proliferation, apoptosis, and vitellogenesis for donor-derived diploid germ cells in the triploid recipient ovaries. Here, we transplanted type-A spermatogonia of Nibe croaker into sexually undifferentiated recipient larvae. Subsequently, mature triploid female recipients produced functional eggs derived from donor spermatogonia. This observation indicated that the colonizing spermatogonia of Nibe croaker have sexual plasticity, as has been shown with other teleost, such as rainbow trout (Lee et al. 2013; Okutsu et al. 2006, 2007, 2008) and yellowtail (Morita et al. 2012). Additionally, F1 offspring obtained from cross between triploid surrogate Nibe croaker parents exhibited an approximately 1:3 female to male sex ratio. Generally, for species in which male are heterogametic, crosses between a feminized XY male and regular XY male can yield zygote populations comprising XX, XY, and YY genotypes in 1:2:1 ratios, resulting in 1:3 female to male ratio in F1 offspring (Devlin and Nagahama 2002). In rainbow trout, the female recipients bearing transplanted spermatogonia were confirmed to produce Y-bearing eggs along with X-bearing eggs and to produce YY offspring (Okutsu et al. 2015; Yoshizaki et al. 2010b). Taken together, these observations strongly suggested that 1) male heterogametic sex determination in the Nibe croaker and 2) female Nibe croaker triploid recipients had produced Y-bearing eggs. Thus, YY super-males may have appeared among the F1 offspring. Our present findings demonstrated that spermatogonial transplantation technology could induce differentiation of Nibe croaker spermatogonia into functional eggs in a recipient ovary. This process could be extremely useful in fish species for which an all-male population is desired for aquaculture purpose but in which YY super-males have not been generated because of difficulties in inducing male-to-female sex change by conventional, sex steroid-based sex-reversal treatment. Here, we also propose that germ-cell transplantation might be useful for the purpose of stock enhancement or conservation of endangered marine teleosts. As discussed in our previous study of rainbow trout (Okutsu et al. 2015), the male-biased sex ratio caused by the contribution of YY super-males, which probably appeared in the F1 generation, to the spawning events would be temporary and would diminish after several generations. In addition, releasing offspring from the F3 generation that were obtained by mating of F2 individuals for restocking wild populations would overcome any potential risk of biasing the sex ratio

Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011

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H. Yoshikawa et al. / Aquaculture xxx (2016) xxx–xxx

in the natural population. Another alternative for preventing sexreversal of donor-germ cells in recipient gonads might be to use surrogate broodstock produced by the transplantation of ovarian germ cells prepared from sex-matched donors. Recently, in rainbow trout, donorderived eggs were obtained from female recipient fish that received transplanted ovarian germ cells, specifically oogonia, into the peritoneal cavity at the hatching stage (Yoshizaki et al. 2010a, 2010b). Thus, the development of ovarian germ transplantation in marine teleosts would be expected. Production of viable donor-derived eggs and sperm by transplanting rainbow trout spermatogonia into xenogenic masu salmon triploid recipients has been demonstrated (Okutsu et al. 2007, 2008). In the Nibe croaker, Higuchi et al. (2011) reported that donor spermatogonia prepared from yellowtail could colonize diploid Nibe croaker gonads, and these donor spermatogonia do not undergo gametogenesis and no donor-derived gametes are produced. In this case, the donor and recipient species belong to different families, and morphological and physiological differences of the gonads because of vast phylogenetic distance between the species could cause arrest of xenogenic donor germcell development in the recipient gonads. Another potential problem may be occurred by the immune rejection of xenogenic donor germ cells within the recipient gonads. Nevertheless, functional, donorderived sperm are produced in other cases of intra-family donorrecipient combinations (Morita et al. 2015; Saito et al. 2008). Thus, future study will focus on production of functional, donor-derived gametes by transplanting xenogenic germ cells of the Sciaenidae family, especially large-bodied commercially valuable species (e.g., mulloway Argyrosomus japonicus, shi drum Umbrina cirrosa, and red drum Sciaenops ocellatus), into triploid Nibe croaker recipients. Because of their small body size, short generation time, and potential triploid development, Nibe croaker would be a suitable recipient species for other sciaenids. Acknowledgments The Japan Society for the Promotion of Science (JSPS) supported this research through the “Funding Program for Next Generation WorldLeading Researchers (NEXT Program, GS010); this program was initiated by the Council for Science and Technology Policy (CSTP). This study was also supported, in part, by an Ocean Resource Use Promotion Technology Development Program grant sponsored by MEXT. References Ando, N., Miura, T., Nader, M.R., Miura, C., Yamauchi, K., 2000. A method for estimating the number of mitotic divisions in fish testis. Fish. Sci. 66, 299–303. Bar, I., Smith, A., Bubner, E., Yoshizaki, G., Takeuchi, Y., Yazawa, R., Chen, B.N., Cummins, S., Elizur, A., 2015. Assessment of yellowtail kingfish (Seriola lalandi) as a surrogate host for the production of southern bluefin tuna (Thunnus maccoyii) seed via spermatogonial germ cell transplantation. Reprod. Fertil. Dev. http://dx.doi.org/10.1071/ RD15136. Billeter, E., Jorgensen, C.B., 1976. Ovarian development in young toads, Bufo bufo bufo (L.): effects of unilateral ovariectomy, hypophysectomy, treatment with gonadotropin (hCG), growth hormone, and prolactin, and importance of body growth. Gen. Comp. Endocrinol. 29, 531–544. Dadzie, S., Hyder, M., 1976. Compensatory hypertrophy of the remaining ovary and the effects of methallibure in the unilaterally ovariectomized Tilapia aurea. Gen. Comp. Endocrinol. 29, 433–440. Devlin, R.H., Nagahama, Y., 2002. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208, 191–364. Felip, A., Piferrer, F., Carrillo, M., Zanuy, S., 2001a. Comparison of the gonadal development and plasma levels of sex steroid hormones in diploid and triploid sea bass, Dicentrarchus labrax L. J. Exp. Zool. 290, 384–395. Felip, A., Zanuy, S., Carrillo, M., Piferrer, F., 2001b. Induction of triploidy and gynogenesis in teleost fish with emphasis on marine species. Genetica 111, 175–195. Freund, F., Hoerstgen-Schwark, G., Holtz, W., Goetz, F.W., Thomas, P., 1995. Plasma Steroid Hormones in Adult Triploid Tilapia (Oreochromis niloticus). Proceedings of the Fifth International Symposium on the Reproductive Physiology of Fish, p. 116. Goswami, S.V., Sundararaj, B.I., 1968. Compensatory hypertrophy of the remaining ovary after unilateral ovariectomy at various phases of the reproductive cycle of catfish, Heteropneustes fossilis (Bloch). Gen. Comp. Endocrinol. 11, 401–413.

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Please cite this article as: Yoshikawa, H., et al., Efficient production of donor-derived gametes from triploid recipients following intra-peritoneal germ cell transplantation into a..., Aquaculture (2016), http://dx.doi.org/10.1016/j.aquaculture.2016.05.011