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Keywords:

  • zebrafish;
  • oogenesis;
  • transgenics;
  • ovary;
  • oocyte;
  • stem cell;
  • ablation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We describe here a novel transgenic zebrafish, Tg(zpc:G4VP16/UAS:nfsB-mCherry) that effectively demonstrates the targeted oocyte ablation in the adult zebrafish ovary. This transgenic line expresses bacterial nitroreductase enzyme (nfsB) under the control of the oocyte-specific zona pellucida C (zpc) gene promoter. Adult transgenic females exposed to the prodrug metronidazole demonstrated near-complete ablation of growing oocytes, resulting in ovarian degeneration and complete cessation of reproductive function. Within 4 weeks of prodrug removal, treated fish demonstrated complete anatomical regeneration of the ovary and, within 7 weeks, ovarian function (fertility) was fully restored. Together, these results demonstrate functional renewal of the oocyte pool in the adult zebrafish ovary. Accordingly, this transgenic zebrafish model system provides a novel means to investigate ovarian growth dynamics in a genetically tractable vertebrate, and may be useful for evaluating signaling interactions that regulate gonadal development processes such as de novo oogenesis. Developmental Dynamics 240:1929–1937, 2011. © 2011 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The robust capacity of the zebrafish (Danio rerio) to regenerate fins, neural tissues (retinal cells, optic nerves, spinal cord), and cardiac muscle has led to the widespread use of this model species for tissue and organ regeneration studies (Chablais and Jazwinska,2010; Jopling et al.,2010; McCurley and Callard,2010; Cameron,2000; Poss et al.,2002,2003; Tawk et al.,2002; Lien et al.,2006; Sherpa et al.,2008). The innate regenerative capability of this species, combined with its relative ease of genetic manipulation, has consequently led to many novel insights into the molecular and cellular regulation of tissue regeneration (Curado et al.,2007,2008; Davison et al.,2007; Pisharath,2007; Pisharath et al.,2007; Moss et al.,2009; Pisharath and Parsons,2009; Zhao et al.,2009). To date however, regenerative studies have focused primarily on somatic tissues, whereas little is known about the regenerative capacity of reproductive (i.e., gonadal) tissues, which are of central importance for animal reproductive success.

Identifying factors that regulate homeostasis and repair of the reproductive organs (testis, ovary) is an important theme in reproductive biology. Previous studies have suggested that reproductive tissues in lower vertebrates such as fishes have a robust capacity for regeneration. For example, attempts to sterilize Chinese grass carp by surgical ovariectomy were largely unsuccessful, due to subsequent ovarian regeneration, possibly due to the presence of remaining ovarian tissue (Underwood et al.,1986). Similar experiments in rainbow trout have shown that ovarian regeneration following ovariectomy also occurs due to amplification of remaining gonadal tissue, as complete gonadectomy results in sterility (Kersten et al.,2001). Likewise, complete ovariectomy of Betta splendens resulted in gonadal regeneration and occasional female to male sex reversal, suggesting the existence of a bipotential germline stem cell (possibly extra-ovarian) underlying gonadal regeneration (Lowe and Larkin,1975). Putative oogonial stem cells have been histologically and functionally identified in a number of teleost species, including zebrafish (Abascal and Medina,2005; Draper et al.,2007; Grier,2000; Grier et al.,2007; Lo Nostro et al.,2003; Nakamura et al.,2010; Selman et al.,1993; Wallace and Selman,1990; Yoshizaki et al.,2010), but surprisingly little is known about the functional capacity or regulation of this cell population in any vertebrate species.

The data presented herein describe a novel bitransgenic zebrafish line, Tg(zpc:G4VP16/Tg(UAS:nfsB-mCherry), that uses a modified gene-directed enzyme prodrug therapy (GDEPT) approach to induce targeted ablation of differentiated germ cells (oocytes) from the adult ovary. Exposure to a prodrug leads to rapid and widespread ablation of oocytes from the ovary, which is followed by anatomical and functional regeneration after prodrug removal. This inducible model of targeted oocyte ablation provides a unique opportunity to evaluate ovarian dynamics under controlled experimental conditions, and thus contribute to a better understanding of oogenesis and organ regeneration.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Generation of Tg(zpc:G4VP16)/Tg(UAS: nfsB-mCherry) Zebrafish

Two Tg(zpc:G4VP16) germline founder males were generated using Tol2-mediated transgenesis (Kawakami,2004). Ovary-specific G4VP16 mRNA expression was confirmed by RT-PCR in adult (8–12 months old) female transgenic progeny, indicating appropriate control of heterologous transgene expression by the zpc promoter fragment (Onichtchouk et al.,2003; Fig. 1A). Confirmed transgenic adults were crossed with homozygous Tg(UAS:nfsB-mCherry) to generate the Tg(zpc:G4VP16/UAS:nfsB-mCherry) bitransgenic line; offspring with detectable cardiac CFP expression (zpc:GFVP16-positive) were further screened by genomic PCR to identify bitransgenic individuals [Tg(zpc: G4VP16/UAS:nfsB-mCherry); Fig. 1B].

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Figure 1. Generation of Tg(zpc:G4VP16/UAS:nfsB-mCherry) transgenic line. A: Ovary-specific G4VP16 mRNA expression was confirmed in adult Tg(zpc:G4VP16) females by RT-PCR. B: Genomic PCR of transgene inserts, zpc:G4VP16 and UAS:nfsB-mCherry, confirming bitransgenic genotype. C: Schematic diagram demonstrating cis-activation of nfsB-mCherry expression by zpc:G4VP16 in Tg(zpc:G4VP16/UAS:nfsB-mCherry) transgenic line. D: nfsB-mCherry expression in Tg(zpc:G4VP16/UAS:nfsB-mCherry) transgenics was confirmed by the detection of mCherry in ovarian tissues (oocytes), and by thepresence of mCherry protein in spawned eggs.

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zpc-driven G4VP16 expression in Tg(zpc:G4VP16/UAS:nfsB-mCherry) bitransgenics resulted in oocyte-specific expression and activity of an E. coli nitroreductase-mCherry fusion protein (nfsB-mCherry; Fig. 1C). G4VP16-driven cis-activation of nfsB-mCherry expression was confirmed by detection of mCherry fluorescence expression in mature ovarian tissues (Fig. 1D). Importantly, bitransgenic females were fully fertile, as evidenced by their production of healthy, mCherry-positive embryos (Fig. 1D) after out-crossing with wild-type males.

Mtz Treatment Induces Oocyte Ablation in Tg(zpc:G4VP16/UAS:nfsB-mCherry) Adult Females

Exposure of Tg(zpc:G4VP16/UAS: nfsB-mCherry) adult females 8–12 months of age to Mtz (5 mM) led to rapid and widespread oocyte ablation (Fig. 2). Initial signs of oocyte ablation were histologically apparent after only 4 days of Mtz treatment, affecting stage IV oocytes most prominently (Fig. 2B). Notably, the observed degenerative changes (zona radiata breakdown, follicle involution, and hypertrophy of granulosa cells; Fig. 2B) are consistent with the histological features of ovarian follicle atresia in teleosts, including zebrafish (Saidapur,1978; Wood and Van Der Kraak,2001). These observations suggest a relatively rapid onset of oocyte demise, but importantly, indicate continued normal function of ovarian somatic cells (e.g., follicular epithelium), suggesting an absence of bystander or off-target effects of prodrug treatment.

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Figure 2. Ovarian degeneration and regeneration following Mtz treatment. A: Representative micrograph demonstrating gross morphology (external, internal) and ovarian histology of untreated (control) fish; note distended abdomen, large ovarian lobes and abundant oocytes at multiple stages of development throughout ovary. B:Tg(zpc:G4VP16/UAS:nfsB-mCherry) female after 4 days exposure to Mtz (5 mM); external appearance and internal ovarian morphology are similar to that of untreated controls; histologically, analysis reveals evidence of degeneration in large oocytes (*). C:Tg(zpc:G4VP16/UAS:nfsB-mCherry) female after 14 days exposure to Mtz (5 mM); note severe ovarian atrophy, exhibited by loss of abdominal distention and recognizable oocytes. Histological analysis revealed extensive ovarian (oocyte) degeneration, and clusters of early-stage oocytes. D:Tg(zpc:G4VP16/UAS:nfsB-mCherry) female 14 days after Mtz removal (experiment d28); ovarian regeneration is observed. E:Tg(zpc:G4VP16/UAS:nfsB-mCherry) female 21 days after Mtz removal (experiment day 35); external appearance, ovarian morphology and histological features are indistinguishable from controls. Oocyte stages are as described in Selman et al. (1993). Degenerate oocytes are indicated by *; dashed line indicates abdominal morphology; ovarian lobes are outlined. Scale bar = 50 μm.

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Following 14 days exposure to Mtz, Tg(zpc:G4VP16/UAS:nfsB-mCherry) females showed extensive evidence of ovarian degeneration resulting from oocyte ablation (Fig. 2C). In comparison to control females, exposed females had lost the distended abdomen characteristic of healthy ovaries (Fig. 2A, C). Oocyte ablation was confirmed upon direct examination of the ovary after surgically exposing the peritoneal cavity (Fig. 2C). In contrast to control females, whose ovaries were populated by readily-visible oocytes, Mtz-treated bitransgenic females contained only small ovarian remnants, in which no growing oocytes were readily visible.

Histological examination of ovaries from Tg(zpc:G4VP16/UAS:nfsB-mCherry) females exposed to Mtz for 14 days confirmed widespread ablation of oocytes from treated fish as compared to controls (Fig. 2C). Interestingly, two histologically distinct zones could be observed within the ovarian remnant of treated animals (Fig. 2C). One zone was characterized by a complete absence of healthy oocytes; rather it was populated exclusively by collapsed, degenerating oocytes and residual ovarian stroma. The second region was comprised of a heterogeneous population of early-stage (previtellogenic) oocytes, most of which were Stage IB or younger, and the largest of which exhibited evidence of atretic degeneration (zona radiata breakdown). The zones containing these cohorts of young oocytes were distinctly compartmentalized, suggesting coordinated recruitment and growth of new oocyte cohorts.

Anatomical Ovarian Regeneration Following Mtz Removal

Fourteen days following Mtz removal (experiment d28), morphological evidence of ovarian regeneration (swollen abdomen) was observed in Mtz-treated females (Fig. 2J–L). Histological examination of ovaries at this time point revealed the presence of large numbers of young, healthy-looking oocytes, up to and including Stage III. By day 35 of experiment (21 days following Mtz removal), the ovaries of Mtz-treated females (Fig. 2M–O) were indistinguishable from those of controls (Fig. 2A–C), being populated with healthy-looking oocytes at all stages of development (Stage I–IV).

Immunohistochemical analyses provided evidence for mitotic proliferation of both germline and somatic cells following Mtz treatment, as indicated by BrdU incorporation in both Vasa-positive and Vasa-negative cells in treated ovaries (Fig. 3A). Colocalization of BrdU and Vasa protein in treated ovaries indicates the existence of a proliferating germline cell population that, importantly, appears unaffected by Mtz treatment (Fig. 3B).

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Figure 3. Immunohistochemical detection of BrdU incorporation and germline markers in ovarian tissue, indicating germline cell proliferation, following 14 days Mtz treatment (5 mM). A: Clusters of BrdU-positive cells were readily detectable at end of treatment period. B: Immunohistochemical identification of a candidate oogonial stem cell (arrowhead) positive for both BrdU (green) and the germline marker Vasa (Red); arrows indicate BrdU-positive somatic cells.

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Reproductive Function Following Mtz Treatment

As expected from the morphological and histological observations, weekly spawning trials revealed a precipitous decline in ovarian function (i.e., fertility) in Mtz-treated bitransgenic females (Fig. 4A,B) during the treatment period. Prior to Mtz treatment, the mean number of eggs spawned by Tg(zpc:G4VP16/UAS:nfsB-mCherry) females (204.2 ± 35.2, n = 5) did not differ significantly from the mean number of eggs spawned by control females (221.7 ± 59.8, n = 6, P = 0.82; Fig. 4A). After 7 days exposure to Mtz, the mean number of eggs spawned by Tg(zpc:G4VP16/UAS: nfsB-mCherry) females (43.8±18.9, n = 5) was significantly lower than controls (235.2±35; n = 6, P = 0.001). After 14 days exposure to Mtz, the mean number of eggs spawned by Tg(zpc:G4VP16/UAS:nfsB-mCherry) females (9.6 ± 6, n = 5) was again significantly lower than control females (181.3 ± 27, n = 6, P < 0.001). Seven days following prodrug removal (experiment d21), Mtz-treated Tg(zpc: G4VP16/UAS:nfsB-mCherry) females failed to spawn a single egg, whereas control females spawned an average of 209.5 ± 19.0 embryos (n = 6, P<0.001).

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Figure 4. Ovarian dynamics of Mtz-treated Tg(zpc:G4VP16/UAS:nfsB-mCherry) and control females. A: Mean total eggs produced by controls (dashed line) and Mtz-treated transgenics (solid line) over the 63-day study period. Mean ± SEM; control n = 6; treatment n = 5; *P<0.05. B: Eggs spawned by Tg(zpc:G4VP16/UAS:nfsB-mCherry) females were comprised of two distinct populations (not observed in the control group): viable (solid black line) and nonviable (solid grey line). Mean ± SEM; control, n = 6; treatment n = 5; *P<0.05 between control and viable; †P<0.05 between control and nonviable.

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Reproductive function (spawning) resumed in Mtz-treated Tg(zpc: G4VP16/UAS:nfsB-mCherry) females 14 days following prodrug removal (experiment d28). The mean number of eggs spawned by the Mtz-treated females (88.2 ± 25.2, n = 5) at this time point was not statistically different from the mean number of eggs produced by control females at this time point (183.8 ± 43; n = 6, P = 0.10). Importantly however, the eggs spawned by the Mtz-treated Tg(zpc: G4VP16/UAS:nfsB-mCherry) females at this time point were functionally compromised; 100% of the spawned eggs failed to undergo normal oocyte hydration (chorion expansion), and embryo development failed to proceed (i.e., blastula embryos failed to form).

The following week (d35; 21 days after prodrug removal), the mean total number of eggs spawned by Mtz-treated Tg(zpc:G4VP16/UAS:nfsB-mCherry) females (243.8 ± 77.3) exceeded that of control females (161.8 ± 23.9, n = 6), although the difference was not statistically significant (P = 0.30). However, the entire complement of embryos spawned by Mtz-treated females was again non-viable, with identical phenotypic defects observed in the previous week's cohort.

On experiment d42 (28 days after prodrug removal), the mean total number of eggs spawned by Mtz-treated Tg(zpc:G4VP16/UAS:nfsB-mCherry) females (227.2 ± 40.2, n = 5) was again similar to the mean total number of eggs spawned by control females (191.7 ± 17.4, n = 6, P = 0.41). However, the eggs spawned by Mtz-treated females at this time point were comprised of two distinct populations. Approximately half of the eggs spawned by Mtz-treated Tg(zpc: G4VP16/UAS:nfsB-mCherry) females in this week were nonviable (mean = 111.2 ± 29.7), with phenotypic defects identical to those observed in the previous 2 weeks. The remaining half (mean = 116.0 ± 27.3) were phenotypically normal, and developed into gastrula-stage embryos. When statistically compared as distinct groups (viable, nonviable), the mean numbers of eggs spawned in each group was significantly lower than the mean total number of eggs spawned by controls (viable, P = 0.04, nonviable, P = 0.04).

By experiment d56 (42 days after prodrug removal), the mean total number of eggs spawned by Mtz-treated Tg(zpc:G4VP16/UAS:nfsB-mCherry) females (157.2 ± 46.2, n = 5) again did not differ significantly from the mean number of eggs spawned by control females (151.7 ± 21.0, n = 6, P = 0.91). However the proportion of viable eggs among this total (mean = 121.6 ± 32.4) increased markedly from the previous week, and when analyzed as a distinct population, did not differ significantly from the mean number of eggs spawned by control females (P = 0.44). In reciprocal fashion, the proportion of nonviable eggs declined markedly (mean = 35.6 ± 18.4), which was significantly lower than the mean total number of eggs spawned by control females. On experiment d63 of the experiment (49 days after prodrug removal), the mean total number of eggs spawned by Mtz-treated Tg(zpc:G4VP16/UAS:nfsB-mCherry) females (166.0 ± 48.6) was similar to the mean total number of eggs spawned by control females (184.8 ± 28.3, n = 6, P = 0.73). The proportion of viable eggs spawned by Mtz-treated Tg(zpc:G4VP16/UAS:nfsB-mCherry) also increased further (mean = 160.8 ± 49.5), whereas the mean number of nonviable eggs (mean = 5.2 ± 2.2) approached negligible levels.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The data presented herein are the first to describe a novel transgenic zebrafish model that effectively demonstrates continual, sustained oogenesis in this model organism. The transgenic line is designed to express a bacterial nitroreductase enzyme, under the control of the oocyte-specific zpc gene promoter. This oocyte-specific expression leads to metabolic conversion of a prodrug (e.g., metronidazole) to a DNA alkylating agent specifically in differentiated oocytes, inducing DNA damage and thereby resulting in oocyte death and removal. The rapid onset of targeted oocyte ablation following prodrug treatment, observable as early as 4 days after the start of treatment, indicates that the transgenic model functions in the expected fashion. After 14 days exposure to Mtz, the ovary exhibited near complete ablation, being comprised mainly of degenerate ovarian tissue (degenerate oocytes), but interspersed with new cohorts of growing (early-stage) oocytes.

Prodrug removal resulted in markedly rapid anatomical and functional ovarian regeneration. Importantly, these observations suggest an absence of side-effects for both the prodrug and its metabolite on non-target cell populations (e.g., premeiotic germ cells, ovarian somatic cells; summarized in Fig. 5). While previous studies in zebrafish have demonstrated the efficacy of the GDEPT approach for studying tissue regeneration, most efforts have focused on embryonic or larval stages of development, and have targeted relatively restricted cell populations (e.g., pancreatic β cells; Moss et al.,2009). The results of this study are noteworthy in part because they demonstrate effective ablation of what is arguably the largest organ in the adult female zebrafish. The ovaries of reproductively mature females are comprised of thousands of follicle-enclosed oocytes, and can constitute upwards of 20% of total body weight. Despite the remarkable size of the adult ovary, near-complete ablation is achieved within 14 days of prodrug treatment.

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Figure 5. Summary model of structural and functional ovarian regeneration after targeted oocyte ablation in zebrafish. The rapid decline in both structure and function of the ovary is a consequence of the effects of Mtz on oocytes expressing nitroreductase. Prodrug removal leads to structural and functional regeneration, such that ovarian anatomy and function is fully restored 7 weeks following prodrug removal.

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These data also demonstrate the participation of germline stem cells in the sustained production of oocytes. Despite complete ablation of all growing mature oocytes, and a complete cessation of ovarian function (spawning), we observed rapid replenishment of the ovarian follicle pool following prodrug removal, and a complete rescue of ovarian function. These results differ significantly from what has been previously reported in a similar transgenic zebrafish, using the same promoter for oocyte ablation (Hu et al.,2010). However, in that study juvenile fish (28–42 days post-hatching) were exposed to prolonged 10-mM Mtz treatment. Under these conditions, oocyte production completely ceased, and fertility was not restored. Although these data would suggest that the effects of Mtz on oocyte production are non-reversible, the age of fish and treatment regime differ dramatically, and no data were given regarding the recovery time post-treatment. Furthermore, aged matched controls were not used to verify a lack of toxicity, which we observed during prolonged exposure at 10 mM, (data not shown). While the source of the new oocytes remains to be firmly established, recent evidence from studies in zebrafish and medaka strongly support the existence of a germline stem cell mechanism underlying de novo oogenesis in these species (Leu and Draper,2010; Wong et al.,2010; Nakamura et al.,2010). Consistent with this, we observed cell clusters in the ovarian regenerate that are anatomically consistent with previous descriptions of putative oogonia (Grier,2000; Draper et al.,2007), and that were positive for both BrdU incorporation and Vasa protein, a conserved germline-specific marker. While this study does not directly investigate or quantify the proliferative activity of these cells, the model system developed here could potentially be used to directly investigate the activity of this stem cell population.

While a return to full fertility (i.e., spawning healthy eggs) was observed 7 weeks after prodrug removal, most of the eggs that were spawned in the first few weeks following prodrug removal were non-viable. A likely explanation is that these eggs were derived from early-stage oocytes that had been damaged by prodrug treatment, but not severely enough to trigger oocyte atresia. Accordingly, successive cohorts spawned by these females contained increasing proportions of healthy eggs, such that by 7 weeks following pro-drug treatment, the vast majority of eggs spawned by Mtz-treated females were fully viable. These data suggest that there is continual sustainment of oogenesis in female zebrafish. This model system may, therefore, be useful to investigate the temporal dynamics of de novo oogenesis, including ovarian follicle formation, growth, and maturation.

Time course analyses of oocyte ablation suggested that sensitivity to prodrug treatment was positively associated with oocyte size. For example, after 4 days of Mtz treatment, signs of oocyte degeneration were most readily apparent in Stage-IV oocytes. We also noted the presence of what appeared to be healthy Stage IA oocytes, alongside atretic Stage IB oocytes, after long-term treatment with Mtz. This differential sensitivity may reflect the amount of nitroreductase that has accumulated in the oocytes of each size class. Despite reports showing that early-stage oocytes (e.g., Stage Ia/b) exhibit the highest levels of endogenous zpc expression (Del Giacco et al.,2000; Onichtchouk et al.,2003), the metabolic activity of nitroreductase likely corresponds with its intracellular concentration. It is therefore unsurprising that larger oocytes appear more susceptible to prodrug treatment, since they have likely accumulated greater concentrations of nitroreductase.

In conclusion, we have developed a novel transgenic zebrafish line that effectively demonstrates this model species' robust capacity for oocyte production, and is suggestive of the existence of a stem cell population capable of rescuing ovarian function following near complete ablation. This inducible and reversible model of ovarian function could, therefore, be of value to those seeking a better understanding of the ovarian kinetics and regulation of germline stem cells in a tractable vertebrate model.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Fish Maintenance

Zebrafish (Tubingen AB strain) were maintained at 28.5°C according to standard methods (Kimmel et al.,1995), and fed live food daily (Artemia, IVVE Group, Salt Lake City, UT). All experiments were conducted in accordance with guidelines established by the Subcommittee on Research Animal Care at Massachusetts General Hospital.

Chemicals and Reagents

Reagents for RT-PCR (SuperScript VILO, Platinum Taq DNA polymerase, oligonucleotide primers), were purchased from Invitrogen (Carlsbad, CA). FailSafe™ PCR Buffers were purchased from Epicenter Biotechnologies (Madison, WI), and commercial antibodies were purchased from Abcam (anti-BrdU; Boston, MA), Santa Cruz Biotechnology (goat anti-rat biotinylated; Santa Cruz, CA), and Invitrogen (Alexa Fluor 488 conjugated goat anti-rabbit IgG, Streptavidin Alexa Fluor 568). Antiserum against Zebrafish Vasa protein was a gift from Christianne Nüsslein-Volhard (Max Planck Institute for Developmental Biology, Tübingen, Germany), via Holger Knaut (Skirball Institute, New York, NY). All other reagents were from Sigma (St. Louis, MO) or ThermoFisher (Waltham, MA), unless otherwise stated. The Tol2-based cloning vector (pCHmcsG4VP16) was provided by Michael Nonet (Washington University, St. Louis, MO).

Generation of Tg(zpc:G4VP16) Transgenic Driver Line

Genomic DNA was purified from adult fin tissue using a commercial reagent (Direct PCR ViaGen Biotech, Inc., Los Angeles, CA), according to the supplier's instructions. A 452-bp fragment of the zona pellucida C (zpc) gene promoter (Onichtchouk et al.,2003) was PCR-amplified from the genomic DNA template using zpc-F1 and zpc-R1 primers (Table 1). These primers were engineered to contain BsiWI and ApaI restriction endonuclease sites, respectively. Amplification conditions were as follows: 94°C for 5 min; 30 cycles of 94°C (30 s), 58°C (30 s), and 72°C (30 s); 72°C (5 min). Agarose gel electrophoresis revealed a single reaction product of the expected size. The product was purified by gel extraction (QIAquick® Gel Extraction kit, Qiagen, Valencia, CA), subcloned into pCRII-TOPO® (Invitrogen, Inc.), sequenced to verify accuracy, and ligated into the multiple cloning site of pCHmcsG4VP16, using BsiWI and ApaI restriction sites. This generated a vector construct consisting of the zpc promoter fragment (446 bp) directly upstream of the modified yeast transcriptional activator GAL4-VP16 (G4VP16), flanked by Tol2 transposase recognition sites, to provide for efficient integration into genomic DNA (Kawakami,2004). To aid in screening for F0 founders, this bicistronic construct contains the cardiac myosin light chain 2 promoter (cmlc2) driving the fluorescent reporter Cyan Fluorescent Protein (CFP); putative F0 founders are readily identifiable by cardiac CFP expression as early as 24 hr post-fertilization (hpf).

Table 1. Oligonucletide Primer Sequencesa
Primer nameSequence
  • a

    Underlined regions indicate restriction endonuclease recognition sites.

zpc-F15′-GAATTCCCGTACGGATCCAAAATCCCC-3′
zpc-R15′-GGGCCCAGAAATGATATCCATGG-3′
nfsB-mCherry-F15′-GAAAGAGAAAGGCTACACCAG-3′
nfsB-mCherry-R15′-ACAGCTTCAAGTAGTCGGGG-3′
G4VP16-F15′-GCTACTGTCTTCTATCGAACAAGC-3′
G4VP16-R15′-GTCAAGGTCTTCTCGAGGAAAA-3′

One-cell-stage wild-type embryos were microinjected with ∼1 nl of injection solution, comprised of purified plasmid DNA (50 ng/μl) and synthetic mRNA encoding Tol2 transposase (20 ng/μl), suspended in Danieau buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES, pH 7.6). Cardiac CFP-positive embryos were identified by fluorescence microscopy at 24 hpf, raised until adulthood, and intercrossed to identify F0 individuals with germline transgene integration. Putative germline founders [Tg(zpc:G4VP16)] were outcrossed with wild-type adults to confirm germline integration, and CFP-positive progeny were raised to adulthood. PCR-based genotyping (described below) was performed to further confirm successful transgene integration.

Generation of Tg(zpc:G4VP16/UAS:nfsB-mCherry) Bitransgenic Line

Sexually mature Tg(zpc:G4VP16) individuals were crossed with homozygous Tg(UAS-nfsB:mCherry) adults (Davison et al.,2007) to generate the Tg(zpc:G4VP16/UAS:nfsB-mCherry) bitransgenic genotype. CFP-positive progeny from this cross were screened by genomic PCR to identify bitransgenic individuals.

To verify G4VP16-mediated activation of nfsB-mCherry transgene expression in Tg(zpc:G4VP16/UAS: nfsB-mCherry) adult females, ovarian tissues were dissected and examined for molecular (messenger RNA) expression of nfsB-mCherry by RT-PCR (nfsB-mCherry-F1, nfsB-mCherry-R1; Table 1), and mCherry protein expression by fluorescence microscopy. Freshly-spawned eggs from Tg(zpc: G4VP16/UAS:nfsB-mCherry) females were also examined for the presence of maternally-derived mCherry protein.

Genotyping

Genomic DNA was extracted from fin tissue as described above. Fragments of the UAS:nfsB-mCherry and zpc: G4VP16 transgene inserts were PCR-amplified in 25-μL reactions using primers nfsB-mCherry-F1, nfsB-mCherry-R1, G4VP16-F1, and G4VP16-R1 (Table 1) under the following conditions: 95°C (5 min); 35 cycles of 95°C (30 s), 58°C (30 s), 72°C (30 s); 72°C (5 min). PCR products were visualized by ethidium bromide staining after gel electrophoresis in 1% agarose.

Prodrug Treatment to Induce Oocyte Ablation

The prodrug selected for use in this study is metronidazole (Mtz, Sigma M1547), which has been validated as a suitable and effective prodrug in zebrafish GDEPT studies (e.g., Curado et al.,2007). Most published studies report using an Mtz concentration of 10 mM to induce cell ablation (e.g., Pisharath et al.,2007; Curado et al.,2008); however, these studies were conducted primarily in embryonic or larval stages, and employed short-term (e.g., 24-hr) exposure durations. Due to the considerable size of the zebrafish ovary (15–20% of body weight), and its propensity for compensatory regeneration (see Results section), we found in preliminary studies that a similar exposure regime was insufficient to achieve detectable oocyte ablation.

We, therefore, opted for a longer term exposure duration (14 days), but reduced the Mtz concentration (5 mM) to avoid any potential off-target effects. In preliminary optimization studies, no deleterious effects were observed after chronic exposure of adult wild-type females (8–12 months old) to 5 mM Mtz. Fish maintained a robust appetite, exhibited normal swimming behavior, and had histologically normal ovaries at the end of the treatment period. A concentration of 5 mM was, therefore, employed for all subsequent experiments.

Adult Tg(zpc:G4VP16/UAS:nfsB-mCherry) females were placed into 1.5-liter exposure chambers containing 5 mM Mtz dissolved directly (without vehicle) in holding water. The animals were maintained on a standard 14 hr:10 hr (light:dark photoperiod) throughout the exposure period, but the exposure chambers were shielded from direct illumination to prevent photodegradation of Mtz. Treatment waters were changed on a daily basis after feeding the animals to satiation. Control fish were maintained in identical rearing conditions, but without the addition of Mtz to holding water. A subset of fish were also exposed to 5 mM BrdU for the duration of the treatment period.

After the treatment period, a subset of animals from each group (n = 4) were euthanized by anesthetic overdose (Tricaine methanesulfonate; Sigma) and processed for histological analysis (described below). The remaining animals were transferred to vessels containing fresh water, and maintained in standard rearing conditions with daily water changes for the duration of the study period.

Histology, Immunohistochemistry, and Microscopy

After euthanization by anesthetic overdose, the abdominal cavity of each fish was surgically exposed to allow for fixation of the ovaries in situ. Briefly, whole fish were immersed in Bouin's fixative overnight at 4°C, after which the ovaries were removed and embedded in paraffin. The tissues were sectioned (8 μM), deparaffinized with xylene, and rehydrated in a graded ethanol series (100, 95, 80, and 50% ethanol, distilled water), followed by 2 × 5 min washes in PBS. Samples stained for BrdU incorporation were pretreated in 2N HCl at 37°C for 30 min to relax the chromatin, followed by neutralization in 0.1M sodium borate.

Ovarian sections were incubated for 10 min in 3% (v/v) hydrogen peroxide in methanol to block endogenous peroxidase activity, washed for 5 min in PBS, and blocked at room temperature for 1 hr using TNK buffer (0.1M Tris, 0.55M NaCl, 0.1mM KCL, 1% goat serum, 0.5% bovine serum albumin, and 0.1% Triton-X in PBS). Sections were then incubated in primary antibody solution (overnight, 4°C) in a humidified chamber, washed in PBS, and incubated in secondary antibody for 2 hr at RT. After washing with PBS, the sections were incubated with Vectastain ABC reagents (Lab Vision, Fremont, CA) for 30 min at RT, and diaminobenzidine (DAKO, Carpinteria, CA) was used for the color reaction. Sections were then counterstained with hematoxylin, re-hydrated through graded ethanol, and photographed with a Nikon TS2000 microscope equipped with a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI).

Breeding Trials to Assess Ovarian Function

Control and treatment animals were paired with untreated WT males every 7 days for the duration of the study period (including pre-treatment), and the number of eggs spawned was determined for each pair cross. Females (treatment, n = 5; control, n = 6) were housed individually for the duration of the breeding trials to prevent any unobserved spawning events.

Data Analysis

Data are presented as mean values ± standard error of the mean (SEM). The mean number of spawned embryos by treatment and control group animals was compared statistically using an unpaired Student's t-test. Differences among mean values were considered significant if P<0.05.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

D.C.W was supported by a Ruth L. Kirschstein National Research Service Award (F32-AG034809).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES