Generation of all-male-like sterile zebrafish by eliminating primordial germ cells at early development
Generation of all-male-like sterile zebrafish by eliminating primordial germ cells at early development
Li Zhou 0
Yongyong Feng 0
Fang Wang 0
Xiaohua Dong 1
Lan Jiang 0
Chun Liu 0
Qinshun Zhao 1
Kaibin Li 0
0 Pearl River Fishery Research Institute, Chinese Academy of Fishery Sciences , No. 1 Xingyu Road, Xilang, Liwan District, Guangzhou, Guangdong, 510380 , China
1 MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University , 12 Xuefu Road , Pukou High-tech Development Zone , Nanjing, Jiangsu
OPEN Production of all-male and sterile fish may not only substantially improve yield but also be crucial for the application of genome modified species in aquaculture. Previously, it was reported that the fish lacking primordial germ cells (PGCs) becomes infertile, and nitroreductase, an enzyme converting non-toxic metronidazole (MTZ) into toxic metabolites, induces targeted toxicity to kill the cells expressing it. In this study, we generated a transgenic zebrafish line of Tg(nanos3:nfsB-mCherry-nanos3 3?UTR) in which the NfsB nitroreductase is solely expressed in PGCs. Treating the embryos derived from the female transgenic zebrafish with MTZ from 0 through 2 dpf (days post fertilization), we found that the germ cells were completely eliminated in the ones older than 2.5 dpf. At 20 dpf, the MTZ-treated juvenile had no germ cells in their gonads. At 100 dpf, the MTZ-treated adult exhibited male-like morphology and showed normal mating behaviors although they had no germ cells but only supporting cells in their gonads. Taken together, our results demonstrated that conditional elimination of PGCs during early development make the zebrafish male-like and infertile. It may provide an alternative strategy to make sterile and all-male farmed fish that is good for increasing aquaculture yield and preventing the genome modified species from potential ecological risks.
Published; xx xx xxxx
Infertility treatment of farmed fish is a promising strategy for increasing aquaculture production as well as
mitigating the potential ecological risks from biological invasion by escaped farmed species1. In addition, inhibiting
the development of sex organs can improve fish growth, enhance the quality of aquaculture products, increase the
utilization rate of feed, and reduce production costs2. Species-specific infertile techniques are therefore desirable
for efficient large-scale farming1.
Fish are sterilized by eliminating germ cells or interfering with reproductive function. The polyploid induction
technique is frequently utilized in aquaculture, especially for rainbow trout (Oncorhynchus mykiss)3 and crucian
carp (Carassius carassius)4. The sterile individuals obtained by this technique are the offspring of multiple parental
hybridizations with different ploidies. The germ cells may become infertile due to chromosome synapsis disorder
during meiosis5, which can be maintained by ?fertile parent and infertile filial generation?1. Surgery, drug
treatment, and morpholino targeting reproductive cells have also been utilized to make fish infertile2. However, these
strategies have poor specificity, low efficiency, and are thus unsuitable for large-scale aquaculture production.
The germ cells of fish are derived from primordial germ cells (PGCs), so loss of PGCs impedes gonadal
development6. Transgenic techniques causing stagnation of PGC development offer an alternative sterilization strategy.
The Maternal Sterility Technology (MST) employs maternal expression of a pro-apoptotic protein to eliminate
PGCs and obtain infertile individuals7, but the efficacy of this pro-apoptotic protein on early development has
not been assessed in practice. It is also challenging to maintain infertile parant8. Disrupting PGC migration
during the early stage of development is another promising infertility technique for industrialized application. For
instance, Wong et al. overexpressed stromal cell-derived factor 1a (Sdf1a), a chemokine vital for PGC homing, in
zebrafish by heat induction, resulting in mis-migration of the PGCs and the development of sterile fish9.
Nitroreductase (NTR), an enzyme derived from Escherichia coli, can convert non-toxic metronidazole (MTZ)
into toxic metabolites, thereby inducing targeted toxicity in fish cells expressing NTR10. Indeed, this NTR/MTZ
system is frequently employed for targeted ablation in studies of organ regeneration and function. For instance, it
has been applied to conditionally eliminate ventricular cardiomyocytes11, hepatocytes12, and pancreatic ? cells13
in zebrafish (Danio rerio) to investigate the mechanism underlying organ repair following injury. In addition,
it can be applied for the targeted elimination of gonadal cells. Hsu et al. utilized the promoters of asp, odf, and
sam to specifically drive the expression of the NTR coding gene nfsB in zebrafish gonadal tissues and found that
MTZ treatment could induce male infertility14. Dai et al. employed dnd promoter to construct NTR transgenic
fish and demonstrated that MTZ treatment could induce male transformation and severely impair reproduction
A series of marker genes, including the maternal genes vasa16, piwi17, and dazl18, have been identified that
allow for study of fish PGC origin, migration, and differentiation. The maternal gene nanos3 encoding the
RNA-binding protein is also specifically expressed in PGCs. In this study, we used the zebrafish nanos3 regulatory
sequences driving the expression of NfsB-mCherry specifically in PGCs and examined the effect NTR/MTZ
system on zebrafish gonad development. Our results showed that conditionally eliminating PGCs during early
development completely ablate the development of germ cells in gonad, resulting in sterile and male-like adult
Generation of Tg(nanos3:nfsB-mCherry-nanos3 3?UTR) zebrafish. To generate the transgenic
zebrafish of Tg(Tol2-nanos3-nfsB-mCherry-nanos3 3?UTR), we microinjected the transgenic construct (Fig.?1A)
together with transponase mRNA into zebrafish fertilized eggs. Totally, we obtained 80 F0 zebrafish. To obtain the
germline transmission transgenic zebrafish, we screened the F1 embryos using the strategy described in Fig.?1B.
Briefly, 20 sexual mature females of the F0 were mated with wild-type males to produce F1 embryos. When
reaching 24 hpf, the F1 embryos were examined whether they had the mCherry expression under fluorescent
microscopy. Among the founders, 18 did not produce any fluorescent embryos. Only Founder #9 and Founder #13
produced 54 of 228 and 24 of 159 embryos carrying the fluorescent signals respectively. Among the embryos, the
ones derived from Founder #9 had stronger fluorescent intensity than the ones from Founder #13. We therefore
raised the fluorescent F1 embryos produced from Founder #9 for setting up the germline transmission transgenic
At 2 months after fertilization, the caudal fins of F1 zebrafish derived from Founder #9 were clipped to prepare
the genomic DNA template for genotyping with PCR amplification using nfsb gene-targeted specific primers.
Results from agarose gel electrophoresis of the PCR products revealed that 19 of 24 F1 zebrafish were positive for
the expected 602 bp product (Fig.?1C) and the transgene identities were further confirmed by Sanger sequencing.
The transgenic zebrafish were named Tg(nanos3:nfsB-mCherry-nanos3 3?UTR) or prfri001. That the other 5
F1 did not carry the transgene but exibit fluorescent at early developemnt is due to the expression of marternal
To examine the expression of the transgene, the female F1 carrying transgene nfsB-mCherry was selected to
mate with male wild type for producing F2 embryos. As shown in Fig.?1D?F, the localized mCherry fluorescence
was found in the genital ridge in the F2 embryos at 24 hpf. Fluorescent cells were arranged and aggregated in two
We next performed quantitative PCR to determine how many copies of transgene were recombined into the
transgenic zebrafish genome using the method as described previously19,20. Briefly, we first set up the
standard curve comparison between the copies of housing keeping actb DNA and the transgene Tg(Tol2-nanos
3-nfsB-mCherry-nanos3 3?UTR) verse their PCR cycles (Fig.?1H). We then performed the quantitative PCR
using the template isolated from the F2 zebrafish carrying the transgene. The experiments were repeated three
times and resulted in an average Ct with 21.773 for the transgenic nsfb. Calculated with the standard curve, we
found the copy number of the transgenic nsfb is about 0.55. Therefore, we concluded that only one copy of
transgene were inserted into the genome of transgenic zebrafish prfri001.
The transgenic zebrafish embryos treated with MTZ exhibit ablation of PGCs. After treating the
embryos derived from the female transgenic zebrafish of Tg(nanos3:nfsB-mCherry-nanos3 3?UTR) with 10 mM
MTZ for 48 h from 0 hpf through 48 hpf (referring as MTZ-treated embryos hereafter), we examined the number
and distribution of PGCs under fluorescence microscopy. When observed at 24 hpf, the control embryos without
MTZ treatment (referring as control embryos hereafter) displayed most labeled PGCs colonizing the genital
ridge (Fig.?2A) whereas the MTZ-treated embryos had obviously fewer PGCs colonizing in the genital ridge
and some labeled cells were incomplete migration towards the genital ridge (Fig.?2B). Throughout development,
the most of labeled cells were arranged around the genital ridge in the control embryos (Fig.?2C) whereas fewer
cells were seen in the genital ridge and the labeled cells were incomplete migration towards the genital ridge
of the MTZ-treated embryos at 36 hpf (Fig.?2D). When observed at 48 hpf, almost no fluorescent cells can be
seen in the MTZ-treated embryos (Fig.?2F) while the PGCs labeled by mCherry fluorescent were rich around
the gential ridge of the control transgenic embryos (Fig.?2E). By 60 hpf, the fluorescent intensity of PGCs was
slightly reduced and dispersed in the control embryos (Fig.?2G), and this became more evident at 72 hpf (Fig.?2I).
However, no fluorescent cells or PGCs were found in the MTZ-treated embryos at neither 60 hpf (Fig.?2H) nor
72 hpf (Fig.?2J). Taken together, the results suggest that MTZ treatment not only affected early migration but also
targeted cells for elimination of the PGCs.
No germ cells are present in the gonads of 20-dpf juvenile developed from the MTZ-treated
transgenic zebrafish embryos. The differentiation and development of gonad in zebrafish are relatively
distinctive during early stage. Both male and female zebrafish undergo a juvenile ovary stage and subsequently
proceed into the gonadal differentiation stage21. Histological examination of H&E-stained sections showed that
the gonadal tissues of 20-dpf juvenile derived from control embryos mainly localized in the upper posterior cavity,
specifically nearby the posterior chamber of the swim bladder, the hepatopancreas, and intestinal tract (Fig.?3A),
where numerous perinucleolar oocytes in the gonad indicated the juvenile ovary stage (Fig.?3C). However, the
20-dpf juvenile derived from the MTZ-treated embryos displayed a gonad-like structure beneath the swim
bladder (Fig.?3B), primarily consisting of supporting cells with no detectable perinucleolar early oocytes or other
germ cells (Fig.?3D). Thus, MTZ treatment appears to completely eliminate the germ cells in the juvenile zebrafish
developed from the female transgenic zebrafish Tg(nanos3:nfsB-mCherry-nanos3 3?UTR), while somatic cells
adjacent to the gonadal tissues normally differentiates into supporting structures in the absence of germ cells.
To confirm the above observation, we performed in situ hybridization on the sections of gonad with the
antisense RNA probe of vasa. The results showed that no vasa positive cells were found in the gonad tisssues of the
20-dpf juvenile developed from MTZ-treated embryos (Fig.?3F) while the vasa expression was obviously present
in the gonad of the juvenile derived from the control embryos (Fig.?3E). Morever, results from RT-PCR analysis
revealed that the 20-dpf juvenile derived from MTZ-treated embryos exibited no expressions of vasa and ziwi
that were normally expressed in the juvenile developed from control embryos (Fig.?3G). And the 20-dpf juvenile
neither from MTZ-treated embryos nor from control embryos had the expressions of sox9a or foxl2 (Fig.?3G).
The results suggest that no germ cells were present in the 20-dpf juvenile developed from MTZ-treated embryos.
Adult transgenic zebrafish derived from MTZ-treated embryos exhibit male-like morphology.
The appearances of male and female adult zebrafish differ substantially. Females are plumper than males due to
expansion of the abdomen with ovarian development. In contrast, males are longer and slender with larger fins,
especially the anal fin, than females. Sexually mature males are bright yellow-brown (the normal nuptial color),
especially in the anal region and caudal fin. The abdominal regions adjacent to each fin are also brightly colored
(Fig.?4A), especially at daybreak. Mature males are observed chasing females counterparts, a normal mating
behavior. Interstingly, the adult zebrafish derived from MTZ-treated embryos exhibited no observable disparities
in feeding, behavior, and growth. The majority of them looked long and slender at 60 dpf, and displayed obvious
male characteristics (Data not shown). Morever, no evident abdominal expansion was observed in 124 of the
adult zebrafish derived from the MTZ-treated embryos. They all displayed the normal nuptial color (Fig.?4B,D).
In contrast, about 39% (52 of 133) of the adult zebrafish derived from the control embryos appeared to be females
and the other 61% (81 of 133) exhibited male morphology (Fig.?4A,C) when reaching 100 dpf.
Effect of MTZ treatment on reproductive capability. The adult zebrafish derived from the
MTZ-treated embryo showed normal mating behavior (chasing), and stimulated females to lay eggs. However, at
approximately 4 h after spawning, the eggs, prusumbly fertilized by 10 of the different adult zebrafish with male
apperances, all gradually became white and died. In the control group, the eggs fertilized by the adult zebrafish
derived from control embryos developed normally, and the hatching rate all exceeded 90%. The results
demonstrate that the eggs were not able to be fertilized by the adult zebrafish derived from the MTZ-treated embryos
though they exhibited male-like morphology and normal reproductive behaviors.
Effect of MTZ on the gonad structure of mature zebrafish. The testes of the sexually mature male
zebrafish locate bilaterally at the dorsal side of the digestive tract, closely adhered to the abdominal wall and the
ventral side of the swim bladder, occasionally surrounded by adipose tissues. Both cylinder-shaped testes extend
from the middle abdominal cavity to the anal fin, converging to form a V shape at the end of the posterior
abdominal cavity with separation at the gonopore. Bilateral gonads were fully developed in the adult zebrafish derived
from the control embryos, and were cream white in color and plump in shape (Fig.?4E). The gonadal position of
the adult zebrafish derived from MTZ-treated embryos was similar to that of control males, and their gonadal
tissues still formed a bilateral structure. Additionally, their general shape of the testis was preserved, and bilateral
gonadal tissues also converged to the gonopore. However, after removal of the adipose tissues, the gonadal tissues
appeared completely withered and shrunken (Fig.?4F) compared to testes of the adult zebrafish developed from
the control embryos (Fig.?4E).
The fine structural features of mature zebrafish testes were then examined in thin sections throughout the
transverse axis (Table?1). In the adult zebrafish developed from the control embryos, the testis was likely divided
into two functional parts including bilateral tetses and intersection point. Bilateral testes are responsible for sperm
production. They were surrounded by membrane structures and both consisted of a large quantity of irregularly
shaped seminiferous tubules, which were interleaved and intimately arranged. Cross sections of seminal vesicles,
the central structure of the seminiferous tubule, were generally circular or oval. The seminal vesicles consisting
of germ cells were enveloped by Sertoli cells. Sperm were formed within the seminal vesicles, and the germ cells
within it were derived from the same spermatogonia with nearly synchronous development. However, the germ
cells between adjacent seminal vesicles differed in developmental phases. Spermatogonia, primary spermatocytes,
Sc I, secondary speratocytes, Sc II, spermatids, and spermatozoa could be observed within a single cross section
(Fig.?5A,C,D). In contrast, the adult zebrafish developed from MTZ-treated embryos exhibited neither germ cells
at any developmental phases nor seminiferous tubule-like structures. Rather, the gonadal structure was composed
of grid-shaped supporting tissues consisting of Sertoli cells and connective tissues (Fig.?5B,E,F). Sertoli cells were
seen in multi-porous structures due to the lack of filling by functional sperm cells, consistent with the gross
withering and shrinkage of the gonad.
In the adult zebrafish developed from control embryos; however, the testes intersection point stored the sperm.
It consisted mainly of seminal vesicle-like structures distinct from those of bilateral testes. No spermatogonia or
spermatids were seen; rather, the structure consisted of a large quantity of honeycomb-shaped tissues composed
of multiple irregular cavities forming ridge structures. These cavities were full of sperm, similar to functional
seminal vesicles (Fig.?5G,H). We proposed that the sperm were generated in the seminiferous tubules and
transferred from the seminal duct for temporary storage. Cross-sections close to the intersection point also revealed
long strips of tissue in a dumbbell-shape. There was no significant change in the structures adjacent to the cloacel
opening, whereas the cross-sections gradually became round, consistent with the overall appearance of the
testes at this position (Fig.?5G,H). The gonadal structures of the adult zebrafish developed from the MTZ-treated
embryos were similar to those of controls, but no sperm were detected within the seminal vesicles. Cavities
consisting of Sertoli cells were seen, which subsequently formed network structures (Fig.?5I,J). In the adult zebrafish
developed from control embryos, the seminal vesicles were full of semen to nourish the sperm and maintain
ionic equilibrium (Fig.?5G,H). In the adult zebrafish developed from MTZ-treated embryos, however, the testis
lacked germ cells, failed to produce and store the sperm, and were withered, all manifested as collapsed network
structures under the microscope (Fig.?5I,J).
To confirm the histological observation, we performed in situ hybridization on the sections of gonads with the
antisense RNA probe of vasa. The results revealed that no vasa positive cells were found in the gonad tisssues of
adult zebrafish (100-dpf) derived from MTZ-treated embryos (Fig.?5M) while the vasa expressions were
obviously present in the primary spermatocytes (Sc I) and secondary speratocytes (Sc II) of the testis in the adult male
zebrafish derived from control embryos (Fig.?5K), and in the primary oocyte (Po) of the ovary in the adult female
zebrafish derived from control embryos (Fig.?5L). Morever, results from RT-PCR analysis showed that the gonads of
100-dpf adult zebrafish developed from MTZ-treated embryos had no expressions of vasa and ziwi that are normally
expressed in the ovary or tetsis in the adult zebrafish derived from control embryos (Fig.?5N). Consistently, foxl222,23,
expressed in ovary but not in testis of the adult zebrafish developed from the control embryos, was not found to
express in the gonads of adult zebrafish developed from the MTZ-treated embryos. In contrast, sox9a, strongly
expressed in testis22 but weakly in ovary of the adult zebrafish developed from the control embryos, was also found to
express weakly in the gonads of adult zebrafish developed from the MTZ-treated embryos (Fig.?5N). Taken together,
the adult zebrafish developed from the MTZ-treated embryos had no germ cells at all.
Fish gonads are derived from two types of cells: PGCs and somatic cells adjacent to the genital ridge. PGCs are
critical determinants of zebrafish sex as they can differentiate into either ovogonium or spermatogonia. During
early embryonic development, PGCs aggregate at a specific position, undergo oriented migration to the gonadal
primordium, and develop with surrounding somatic-derived cells to form a functional gonad where PGCs
differentiate into two types of germ cells namely sperm and oocyte, and somatic cells adjacent to germinal primordium
develop into the structures supporting the gonadal tissues24.
The NTR/MTZ system can specifically eliminate cells or tissues, and currently serves as an effective approach
for investigating the mechanism of organ function and regeneration11,25. Previously, Hsu et al. generated
transgenic zebrafish Tg(asp:nsfB), Tg(odf:nsfB) and Tg(sam:nsfB) in which NsfB was expressed specifically in testis
under the promoter of asp (A-kinase anchoring protein-associated protein), odf (outer dense fibers) and sam
(sperm acrosomal membrane-associated protein)14. After treated with MTZ, 68%, 59%, 54% of the transgenic
zebrafish displayed male sterile, respectively. Because asp, odf and sam were all expressed in spermatid or sperm,
the transgenic zebrafish treated with MTZ still had spermatogonia in their testis although the spermatid was
ablated by MTZ treatment. Therefore, it is reasonable to hypothesize that the normal sperm can be developed
and the transgenic zebrafish are fertile again once MTZ is removed because the spermatogonia are not affected by
MTZ at all. In contrast, two other groups25,26 generated transgenic zebrafish Tg(zp:nsfB) in which the expression
of nsfB is specifically driven by the promoter of ovarian specific expression gene of zp (zona pellucida). When
treated with MTZ, the transgenic zebrafish exhibited female sterile because the disrupted formation of zona
pellucida blocked the oogenesis although oogonium were not affected at all. Once MTZ was removed, oogenesis was
recovered and normal oocyte was developed from the unaffected oogonium, resulting in the female transgenic
Unlike sperm and oocyte that are formed in testis and ovary respectively, PGCs are the germ stem cells that
are specified in the embryos at early development and are fate to differentiate into gametes. Therefore, the fish
would be sterile if their PGCs are eliminated in the embryos at early development. Actulaly, in the absence of
PGCs, the somatic cells of zebrafish and medaka (Oryzias latipes) gonads tend to spontaneously
differentiate into male characteristics, while gonadal differentiation with female characteristics requires PGCs15,27?31,
although the underlying mechanisms remain to be elucidated. Consistenly, Wong and Collodi reported that
overexpressing stromal-derived factor 1a (Sdf1a), providing the directional cue that guides the migration of
PGCs to the gonadal tissue in a gradient, disrupted the normal PGC migration pattern, resulting that the
embryos developed into sterile males9. Additionally, microinjection of dnd morpholino into zebrafish fertilized
eggs interfered with the migration and formation of PGCs, resulting in zebrafish with male characteristics22.
In this study, we set up a transgenic zebrafish line Tg(nanos3:nfsB-mCherry-nanos3 3?UTR). Driven by nanos3
promoter, the transgene was expressed during oogenesis but not spermatogenesis, and the mRNA message
was deposited in oocyte. After fertilization, the message in the oocyte was alloted into PGCs but not somatic
cells because of the 3?UTR of nanos3 that allows the mRNA be stable in PGCs but be degradated in somatic
cells32. In other words, only the PGC cells in the embryos derived from female transgenic zebrafish can express
the fusion protein of NfsB-mCherry. Therfore, once the embryos are incubated with MTZ, the PGC cells in
the embryos derived from the female transgenic zebrafish will be killed no matter the embryos themselves are
transgenic or not. Conforming to the above hypothesis, our results demonstrated that early MTZ treatment
completely eliminated PGCs in the embryos derived from the female transgenic zebrafish. With development,
no germ cells were detected in the gonads of the zebrafish developed from the MTZ-treated embryos (Fig.?3),
and only cavity-shaped structures derived from somatic cells were observed (Fig.?4), resulting in all male-like
sterile adults without reproductive function (Figs?4 and 5).
As an anti-anaerobe and protozoacide, prolonged treatment with high-dose MTZ may induce adverse effects33.
Therefore, the duration and dose of MTZ treatment are the key parameters for targeted conditional elimination.
Previously, Curado et al. utilized 10 mmol/L MTZ to treat transgenic NTR juvenile zebrafish under the control of
different promoters and found that cell ablation occurred in the heart, pancreas, and liver at 24 h, accompanied by
functional defects34. White et al. adopted 5 mmol/L MTZ to treat transgenic animals and conditionally removed
the ovary26. Hsu et al. treated transgenic zebrafish using 5 mmol/L MTZ for 14 d and demonstrated that the testis
was completely or partially destroyed14. In this study, we demonstrated that a dose of 10 mmol/L Mtz for 48 h
from 1-cell stage exerted no significant adverse effects on zebrafishembryos and all the embryos treated with
Mtz developed into male-like sterile adult. The results revelaed that short-term MTZ intervention with 10mM is
sufficient to effectively eliminate germ celles at early development. However, 5mM is not optimal amount to kill
all the germ cells in zebrafish embryos because Dai et al. administrated 5 mM MTZ to treat transgenic zebrafish
Tg(dnd:NTR-EGFP + 3?UTR) for 20 days from 18 dpf and found that all MTZ-treated transgenic fish exclusively
developed into males with subfertilities15.
In aquaculture, males of multiple species, such as tilapia (Oreochromis niloticus) and yellow catfish (Tachysurus
Fulvidraco), outperform females in terms of breeding and growth. For example, if male and female tilapia are
raised together, they reach sexually mature at the age of 3?5 months and they then mate ad libitum to produce
offspring. The adults derived from the offspring are in small body size, far less than the market size. Therefore,
production of all-male fish may substantially improve aquaculture yield of the defined fish species35,36. In this
study, we found no germ cells were detected in the gonads of MTZ-treated zebrafish at 20 dpf. In addition, no
PGCs-derived germ cells except somatic cell-derived supporting structures were observed in the gonads of
MTZ-treated transgenic zebrafish. Interestingly, the MTZ-treated adult zebrafish retained the morphology and
behavioral characteristics of males, but failed to produce sperm despite chasing and attempting to mate with
females for spawning. The results demonstrate that zebrafish with conditional elimination of PGCs at early
development grow up as sterile male-like fish. Therefore, the NTR/MTZ system established to conditionally eliminate
PGCs in this study provides an alternative novel strategy for producing all-male fish, which is probably a practical
direction for fishery industrialization. Provided that the female transgenic fish is a heterzygote for the transgene,
only half of their offspring will be transgenic and the other half will be non-trangenic but their germ cells were all
killed when they are incubated with MTZ. Therefore, one can obtian non-trangenic all male-like fish in
aquaculture industry using this developed techanique system.
Transplantation of fish germ cells offers a novel strategy for rapid breeding and seed conservation37,38, but this
strategy is unfeasible for industrialized application due to low transplantation efficiency (which may be enhanced
depending on the donor fish cells and the quality of the recipient fish). Currently, multiple recipient fish
species lack a sterile line39,40. Conventionally physical and chemical approaches have been employed, but they have
either failed to completely eliminate the germ cells or resulted in substantial variability of outcome, which greatly
increases the workload for subsequent screening. Furthermore, these non-targeted methods can alter other
tissues2. Thus, the experimental endpoint cannot be controlled and reproducibility is poor. In this study, the germ
cells of adult zebrafish developed from the MTZ-treated embryos were completely eliminated, which can avert
the germ cell chimeras between the donor and recipient fish and facilitate subsequent screening. Alternatively,
the somatic cell-derived supporting structures remain in intact, thereby creating a favorable environment for the
development and differentiation of donor cells and enhancing transplantation efficiency. Thus, induced
elimination of germ cells as demonstrated in this study may facilitate the systematic development of appropriate recipient
fish for the transplantation of allogenic germ cells.
Ethics statement. Experimental protocols using zebrafish as a research subject in this study were approved
by the Aquatic Animal Research Committee at Pearl River Fishery Research Institute, China. All methods were
performed in accordance with the relevant guidelines and regulations.
Generation of transgenic zebrafish. A Tol2-nanos3-nfsB-mCherry-nano3-3?UTR vector (Fig.?1A)
was constructed using traditional molecular recombination. In the expression construct of Tol241, the CDS of
nfsB-mCherry, encoding a fusion protein of NfsB nitroreductase fused with mCherry reporter, was inserted
between the promoter of zebrafish nano3 and its 3?UTR40 in order for the transcription of the fused gene to be
controlled by zebrafish nano3 promoter and the transcript to be only stably present in PGCs42. A transgenic
line was established by microinjecting the transgenic construct plus transponase mRNA into zebrafish fertilized
eggs as we described previously43. F0 zebrafish were bred to sexual maturity, and the females selected and mated
with wild-type males to produce F1 progeny. The transgenic F1 was determined by the strong occurrence of
mCherry fluorescence in PGCs at 24 hpf under fluorescence microscopy. The number of fluorescence-labeled
embryos derived from per female was recorded and embryos without fluorescence were abandoned. At 2 months
of age, the caudal fin of fluorescence-labeled zebrafish (F1) was clipped for the template preparation of genomic
DNA using a commercial kit (YSY, China). PCR amplification was performed using nfsB gene-specific primers
(CATTCCACTAAGGCATTTGA and GTTTTGCGGCAGACGAGATT) and the PCR products were
characterized by agarose gel (1%) electrophoresis and Sanger sequencing. F1 Zebrafish carrying the nfsB gene were used
for strain breeding and subsequent analysis.
Transgene copy number determination. Quanntative PCR was performed using the equipment of
ABI 7500. To determine the copy numbe of transgene in the genome of trangenic zebrafish, two standarad curves
(log of copy number vs amplification cycle threshold)19,20, one for endogenous actb and the other for trangene
nfsb, were first set up by the real time PCR using the pair of primers of ACGAACGACCAACCTAAACTCT
and TTAGACAACTACCTCCCTTTGC (actb), and CATCCAGCACCAACTCCCA and CCAGCTTCAGCC
AGACATCGT (nsfB), respectively. The standard template of actb DNA sample was the genome DNA isolated from the
caudal fin of wild type zebrafish using the tradiational genomic DNA isolation mthod. In contrast, the standard
template of nsfB DNA sample was the cloning vector of the transgene. The copy number of target template was calcualted
using the equations of DNA concentration (ng/?L)?10?6 ?1?L?10?3 ?6.02?1023/(6593?650) (for transgene nsfb)
or DNA concentration (ng/?L) ? 10?6 ? 1 ?L ? 10?3 ? 6.02 ? 1023/(1.7 ? 109 ? 650) (for actb). The PCR reaction was
performed in a 20 ?L volume including 10 ?L of SYBR Premix Ex Taq II (2?), 0.8?L of 10?M forward primer, 0.8 ?L of
10 ?M reverse primer, 0.4 ?L of ROX Refernce Dye II (50?), 6 ?L of ddH2O, and 2 ?L of standard DNA template. The
PCR was run in 95?C 30 s, followed by 40 cycles of 95 ?C 5 s, 60 ?C 34 s, and 72 ?C 15 s. Each amplification was repeated
3 times. The cuve was plotted with X axis representing log of copy number and Y denoting the average of Ct.
To determine the copy nummber of the transgene in the transgenic zebrafish genome, genomic DNA was
isolated from the caudal fin of the transgenic zebrafish as descibed above and then used as the template of the
quantative PCR amplifying the trangene and the endogenous actb gene. The copy number of trangene was
finnally calcualated from the standard curve by comparing it with actb that are two copies in the zebrafish genome.
MTZ treatment. Female zebrafish with positive phenotype (mCherry fluorescence) and genotype (nfsb)
were mated with wild type male and the fertilized eggs collected. Metronidazole (MTZ) was dissolved in DMSO
and adjusted to 10 mM with water. Sixty mCherry-positive fertilized eggs were obtained and treated with 10 mM
MTZ in a 500 mL beaker at 28 ? 1 ?C under darkness. After 24 h, the medium was exchanged with fresh MTZ.
After another 24 h, the solution was exchanged for egg water and incubation was continued. Growth and
development status were monitored regularly hereafter. All treatments were conducted in triplicate.
Effect of MTZ treatment on PGC morphology. After 24, 36, 48, 60 and 72 h of MTZ treatment, four
embryos per group were randomly selected and PGC morphology and distribution assessed under fluorescence
Effect of MTZ treatment on gross appearance of zebrafish. Zebrafish body and color changes were
observed at each time point and compared between the MTZ treatment and control groups.
Effect of MTZ treatment on gonadal tissues of 20-dpf zebrafish. Ten zebrafish were randomly
selected after 20 dpf (days post fertilization) of MTZ treatment and fixed in 4% paraformaldehyde solution
overnight at 4 ?C. The juvenile were then dehydrated in gradient ethanol, made translucent by xylene treatment,
embedded by paraffin, sectioned at 3 ?m longitudinally along the body axis or transversely, stained with
hematoxylin and eosin (H&E), and finally mounted. The histology of gonadal tissues was compared between MTZ-treated
and control groups under light microscopy.
Assessment and validation of reproductive capability. Ten of 100-dpf zebrafish developed from the
MTZ-treated embryos were randomly selected and mated with wild-type female zebrafish, and another 10
transgenic male zebrafish were chosen as normal controls. Spawning and reproduction status were compared between
the two groups.
Gross observation of gonad tissues of adult zebrafish. Five of 100-dpf zebrafish developed from the
MTZ-treated embryos or control males were randomly chosen and sacrificed under anesthesia using 0.2 mg/
ml Tricaine. The size and morphology of the zebrafish gonad were compared between MTZ-treated and control
transgenic males. Differences in morphology and positions of liver, intestine, kidney, and other organs were also
examined to evaluate extraneous effects of MTZ.
Comparison of gonadal histology in adult zebrafish. Ten of 100-dpf zebrafish developed from the
MTZ-treated embryos and control males were randomly chosen, fixed, decalcified for 2 weeks, transverse
sectioned, and stained with H&E. The gonadal structure and germ cellular morphology were compared.
RT-PCR. Gonads were isolated from the zebrafish developed from MTZ-treated embryos or control embryos
without MTZ treatment at 20 dpf and 100 dpf under the stereoscope. Samples were homogenized with a mortar
and pestle in liquid nitrogen. Total RNA was then extracted with TRIzol Reagent (Invitrogen) according to the
manufacturer?s instructions. The quality of the extracted RNA was determined by agarose gel electrophoresis.
cDNA was reverse transcribed using the TAKARA PrimeScript? RT reagent Kit with gDNA Eraser (TAKARA,
Japan). cDNA was stored at ?20 ?C for gene expression analysis.
PCR reactions were performed in 25 ?l volume containing 9.5 ?l ddH2O, 1 ?l cDNA template, 1 ?l (10 ?M) of
each primer, 12.5 ?l 2 ? Taq PCR MasterMix (TianGen, China). The seqeucnes of the forward and reverse primers
used to detect gene expressions were CCCAATATGGATGACTGGGAG and GTCATTTTCCATGAGCTACC
(for vasa), CTCAGATGGTGGTGGTGATCT and ACGGTCACACTGTTCCTTCAG (for ziwi), AGTCCACACG
TTTCCTGATTG and ATCCTGTGGAATTCTGTGACG (for sox9a), CCCAGCATGGTGAACTCTTAC
and C G TG ATC C C A ATATG AG C AG T ( for fox l 2 ) , G AG A AG ATC TG G C ATC AC AC C T TC and
GGTCTCGTGGATACCGCAAGATTC (for actb). The PCR was run in 95?C 5 min, followed by 35 cylces of
95 ?C 30 s, 60 ?C 30 s and 72 ?C 30 s, and finally extended for 5 min at 72 ?C. The PCR products was seperated by
1.0% native agarose gel electrophoresis.
In situ hybridization. Gonads were isolated from the zebrafish developed from MTZ-treated embryos or
control embryos without MTZ treatment at 20 dpf and 100 dpf under the stereoscope. They were fixed in 4%
paraformaldehyde solution overnight at 4 ?C, dehydrated and embedded as described above, then sectioned at 7 ?m
transversely. Some sections stained with H&E and others for hybridizations. A 1223 bp vasa cDNA fragment was
inserted into a pGEM-T vector for the synthesis of antisense RNA probes under the drive of T7 promoter by using
the digoxigenin RNA Labeling Kit (Roche). The RNA probes were treated with RNase-free TURBO DNase and
purified with SigmaSpinTM Sequencing Reaction Clean-Up (Sigma). The sections were digested with proteinase
K (10 ?g/ml) for 10 min and hybridized with the probes at 65 ?C for 14 hours. Chemical in situ hybridization was
conducted by developing the signals with BCIP/NBT substrates on sections and post fixed in 50% glycerin. The
results was photopgraphed under microsope uisng digital CCD camera.
This work was supported by National Key Technology R&D Program of the Ministry of Science and Technology
of China (No. 2015BAI09B05).
K.L., Q.Z. and L.J. conceived and designed the experiments; L.Z., Y.F., F.W., C.L., and X.D. did experiments; K.L.,
Q.Z., L.Z., and Y.F. analyzed the data and wrote the paper.
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-20039-3.
Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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