JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 306B:521–527 (2006)
High Efficiency of Meiotic Gynogenesis in Sea Lamprey Petromyzon marinus JACQUES RINCHARDy, KONRAD DABROWSKI, AND MARY-ANN GARCIA-ABIADO School of Environment and Natural Resources, The Ohio State University, Columbus, Ohio 43210
ABSTRACT
Induction of androgenesis and gynogenesis by applying a pressure (PS) or heat shock (HS) to double the haploid chromosomal set results in progenies possessing only chromosomes from a single parent. This has never been accomplished in representatives of Agnatha. The objective of this study was to induce gynogenesis and androgenesis in sea lamprey Petromyzon marinus. For gynogenesis experiments, ultraviolet (UV)-irradiated sperm was used to activate sea lamprey eggs and HS or PS were applied to inhibit the second meiotic division and consequently induce diploidy in the embryos. The UV irradiation of immobilized sperm was performed for 1 min at 1,719 J m2. HS of 35711C for 2 min and PS of 9,000 psi for 4 min were applied at different times after egg activation (8, 12, 20, and 24 min or 8, 16, and 24 min for HS or PS, respectively). Regardless of the induction time of the HS, survivals at pre-hatching stage were similar. In contrast, PS applied 8 min after activation appears to increase survival rate of pre-hatched embryos in comparison to 16 and 24 min after activation. In control groups, without shock treatment (no diploidization), there were no survivors. All deformed, gynogenetic embryos were confirmed to be haploids and died prior to burying themselves in the sand. We confirmed by flow cytometry that progenies produced using both shock methods surviving to the next stage, burying in the substrate, were diploid gynogenetic. For the androgenesis experiments, UV-irradiated eggs (1,719 J m2 for 1 min) were fertilized with non-treated sperm and HS was applied to restore diploidy of the eggs. Several attempts have been made to optimize the parameters used. HS of 35711C was applied 110, 140, 170, 200, and 230 min after activation for 2 min. Low yields of androgens were obtained and all animals died within a week after hatching. These techniques will allow to establish meiotic gynogenetic lines of sea lamprey for determining sex differentiation in this species and to analyze its hormonal and environmental regulation. J. Exp. Zool. (Mol. Dev. Evol.) 306B:521– 527, 2006. r 2006 Wiley-Liss, Inc.
With the opening of the St. Lawrence Seaway in 1932, the non-indigenous sea lamprey Petromyzon marinus gained access to the Great Lakes although this hypothesis was recently questioned based on some historical evidence and molecular biology fingerprinting (Daniels, 2001). The invasion of the sea lamprey in the Great Lakes had a devastating impact not only on the lake trout Salvelinus namaycush but also on all fish assemblages (Coble et al., ’90). Current attempts to control sea lamprey are mainly (1) treatment of streams with lampricides to kill larvae and (2) production and release of sterile males (sterilized with bisazir) to decrease spawning success. Total reliance on a few methods to control sea lamprey populations is considered unwise. Therefore, a search for alternative lamprey control measures is necessary to create an integrated program. r 2006 WILEY-LISS, INC.
Gynogenetic and androgenetic reproduction techniques have been used to identify the sex determination system in several fish species (Dabrowski et al., 2000; Bertotto et al., 2005). Gynogenesis involves the activation of the egg development by genetically incapable spermatozoa, and then the restoration of the diploidy of the egg by retention of the second polar body (heterozygous gynogenesis) or the suppression of the first Grant sponsor: Great Lakes Fishery Commission (GLFC). y Present address: School of Natural Resources and Environment, University of Michigan and US Geological Survey, Great Lakes Science Center, Ann Arbor, MI 48105. Correspondence to: Dr. K. Dabrowski, School of Environment and Natural Resources, The Ohio State University, 2021 Coffey Road, Columbus, OH 43210. E-mail:
[email protected] Received 13 January 2006; Accepted 23 February 2006 Published online 23 May 2006 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jez.b.21111.
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mitotic division (homozygous gynogenesis) of the eggs. If gynogenesis leads to all female offspring, the sex determination system of the species is XX/XY. In contrast, an equal number of females and males in the progenies indicate a ZZ/ZW sex determination system. In this case the superfemales (WW) and normal males (ZZ) are produced. Androgenesis involves the degradation of the egg DNA and the diploidization of the paternal genome. All male androgenetic progenies indicate a ZZ/ZW sex determination system, whereas the presence of males and females in equal proportion, a XX/XY sex determination system. In the latter case, males are super-males (YY) and their mating with normal females (XX) will produce 100% males. The objective of this study was to develop techniques to produce monosex population of sea lamprey which ultimately will be used to analyze hormonal regulation of sex differentiation and explain effects of environmental factors (temperature, pH; Hardisty, ’60; Lowartz and Beamish, 2000) on sex ration in wild populations. In a long term, these methods can be used to prevent successful reproduction of sea lamprey in the Great Lakes. Moreover, the production of homozygous sea lamprey, resulting from gynogenesis and androgenesis experiments, will be a useful tool in a variety of medical research areas, in which sea lamprey is intensely used as model animal (Zhang et al., 2005). MATERIALS AND METHODS
Animals Sea lampreys were obtained from the Hammond Bay Biological Station (Millersburg, MI). These animals were captured in the Cheboygan River (MI) on their anadromous migration after their parasitic phase in Lake Huron. They were transported by ground to the aquaculture facility at The Ohio State University, Columbus (OH). Sea lampreys were placed in aerated bags located in coolers with isolated ice bags to chill water to 4–81C for transport. At arrival, sea lampreys were acclimated to 141C. Males and females were assigned into separate circular tanks (400 L) in a semi-recirculated water system. Water temperature was maintained between 141C and 161C using a cooling system and photoperiod was kept under a 12 hr light and 12 hr dark regime. Fish sizes were as J. Exp. Zool. (Mol. Dev. Evol.) DOI 10.1002/jez.b
follows: females: 264.3748.9 g and 47.973.1 cm; males: 259.6750.3 g and 49.072.2 cm.
Induction of maturation and gamete collection Both genders were injected with luteinizing hormone releasing hormone analog (LHRHa, Sigma Chemical Co., St. Louis, MO), males with a single injection (200 mg kg1) and females with a priming (200 mg kg1) and a resolving (500 mg kg1) dose at 4-day interval. Gametes were collected by stripping and stored on ice until use. Prior to LHRHa injection and gamete collection, male and female sea lamprey were anesthetized with 0.01% tricaine (MS-222, Argent Chemical Laboratories, Redmond, WA) in 0.3% sodium bicarbonate (Langille and Hall, ’88).
Gynogenesis Induction of gynogens using heat shock (HS) was conducted with the eggs of five individual females (two on July 18, two on July 19, and one on August 2, 2003), whereas pressure shock (PS) experiments were conducted on August 4, 2003 using a pooled egg from three females.
Sperm irradiation Sea lamprey sperm (mixture from four males) was diluted (1:3; v:v) in modified immobilizing, Ringer solution (consisting of 137 mM NaCl, 12 mM KCl, 1 mM CaCl2, and 10 mM Hepes, pH 7.4) (Ciereszko et al., 2000) prior to ultraviolet (UV) irradiation and fertilization. Diluted sperm was exposed to UV irradiation using a DNA crosslinker (Stratalinker Model 2400, Stratagene, La Jolla, CA). The UV irradiation was performed for 1 min at 1,719 J m2 (Lin and Dabrowski, ’98). Throughout the irradiation, the sperm suspension was kept on ice and continuously stirred using a magnetic stirrer. After UV irradiation, the sperm suspension was placed inside a dark-colored Petri dish until fertilization to prevent photoreactivation (Ijiri and Egami, ’80).
Egg activation Eggs (3.75 g; 2,500 eggs) were activated with 2 ml of diluted sperm (0.5 ml sperm11.5 ml extender) exposed to UV. Then, eggs were subjected to HS or PSs. In each trial, the efficiency of the UV irradiation and the shock treatment was monitored by incubating egg samples not exposed to HS or PS as negative control (UVnoHS and UVnoPS, respectively). Eggs were also fertilized
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with non-UV-irradiated sperm as positive control. Water temperature during the fertilization for the gynogenesis experiments was 21711C.
Heat shock HS was used to inhibit the second meiotic division and consequently induce diploidy in the eggs activated with the UV-irradiated sperm. The HS was applied 8, 12, 16, 20, and 24 min after activation by submerging the eggs contained in the net in an intensely aerated conical bottom tank (50 l) at 35711C for 2 min.
Pressure shock PS was used to inhibit the second meiotic division and consequently induce diploidy in the eggs activated with the UV-irradiated sperm. Eggs were pressure shocked in a stainless steel hydrostatic pressure chamber (Aquacenter, Leland, MS) provided with a 0–103,421 kPa (0–15,000 psi) gauge, stainless steel bleed valve, and brass piston plunger with o-rings. The chamber was pressurized using a 12-ton hydraulic press (Grainger, Lake Forest, IL) with a 53.3 cm minimum working height. PS of 9,000 psi (62,053 kPa) was applied starting at 8, 16, and 24 min after activation for 4 min duration.
Androgenesis Androgenesis experiments were conducted with the eggs of a single female on August 15, 2003.
Egg irradiation Eggs were dispersed in a 5-cm Petri dish to form a single layer of eggs and then UV-irradiated for 1 min at 1,719 J m2 using a DNA crosslinker. Throughout the irradiation, the Petri dish mounted on a PowerPackTM (K-Nex, Inc., Hatfield, PA) was continuously rotating to ensure uniform egg irradiation.
Fertilization UV-irradiated eggs (3 g; 2,000 eggs) were fertilized with a pre-diluted 0.5 ml of a mixture of sperm from five males and then exposed to a HS. As in the gynogenesis experiments, the efficiency of the UV irradiation and HS was monitored by incubating egg samples that were not exposed to HS as a negative control (UVNoHS). Eggs not exposed to UV irradiation were also fertilized with sperm as positive control. Water temperature during the fertilization for androgenesis experiments was 22711C.
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Heat shock HS was applied 170, 200, and 230 min after activation at 35711C for 2 min as previously described.
Egg incubation, fertilization, and hatching rates Each batch of eggs was incubated in separate PVC jars with a screen bottom placed in California hatching trays (Flex-a-Lite Consolidated, Inc., Tacoma, WA) with recirculating, UV sterilized water at 22711C. Starting on day 2 of incubation until prior to hatching, formaldehyde treatments were applied daily at a concentration of 50 ml l1 to prevent development of fungi. Fertilization rate was assessed at two-cell embryos (5 hr post fertilization) and prior to hatching (Ciereszko et al., 2000). At hatching, sea lamprey embryos from each trial were transferred into separate troughs (13 l) on a flow-through system supplied with dechlorinated city water. One week after hatching, embryos were transferred to identical troughs with the bottom covered by a 1.270.2 thick layer of beach sand. Additional aeration was provided to each trough to maintain dissolved oxygen level near saturation.
Assessment of ploidy Embryonic stages, pre-hatched, and larvae of sea lamprey from all groups were sampled for flow cytometry analyses. Embryonic and pre-hatched sea lampreys were frozen in 5% dimethylsulfoxide (DMSO), whereas larvae were processed immediately without fixation. Larvae were killed and the head and tail regions were removed from the rest of the body. The body was cut into 3–5 pieces and washed three times in saline (0.7% sodium chloride solution). The tissue portions or the embryos were transferred to 12 75-mm2 sterile plastic tubes with snap cap (Fisher Scientific, Pittsburgh, PA) containing 800 ml propidium iodide stain (Garcia-Abiado et al., 2001). Approximately 20 ml of rainbow trout red blood cells were placed into each of 5–10 vials to serve as internal standard. The propidium iodide stain was prepared by dissolving 50 mg propidium iodide (Sigma Chemical Co.) and 10 mg Ribonuclease A (Sigma Chemical Co.) in 1 l of ISOTON II. The tissues were incubated overnight at 41C, gently syringed (21 and 26-G needles, Becton-Dickinson and Company, Franklin Lakes, NJ) and filtered using 60-mm Nitex netting (Argent Chemical Laboratories). Flow cytometry analysis was perJ. Exp. Zool. (Mol. Dev. Evol.) DOI 10.1002/jez.b
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RESULTS
Gynogenesis experiments In the first experiment, when the HS was used to inhibit the second meiotic division and consequently induce diploidy in the eggs activated with the UV-irradiated sperm, survival at the 2-cells and at the pre-hatching stages of control (eggs fertilized with non-UV-irradiated sperm) significantly differed among females from 48.1% to 97.8% and from 38% to 98.8%, respectively (Fig. 1). These differences were also reflected in the different treatments (no UVHS and UVHS at different times after fertilization). Flow cytometry analyses revealed that all control embryos were diploids (100%, n 5 15) (Fig. 2B) without deformities (Fig. 3A). A few embryos were also observed when eggs were activated with UV-irradiated sperm but not exposed to HS (Fig. 1). Flow cytometry analyses revealed that all these embryos were haploids (n 5 2) (Fig. 2D). Haploid embryos were morphologically deformed based on embryo size and curved body (Fig. 3D) and all of them died within a week after hatching. J. Exp. Zool. (Mol. Dev. Evol.) DOI 10.1002/jez.b
2-cells
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formed on a Coulter EPICS Elite flow cytometer (Coulter Corporation, Miami, FL) equipped with a 488 nm, 15 mW air-cooled Argon laser. A minimum of 10,000 gated cells were collected at a rate of 500 events per second. Propidium iodide signal was measured using a 610-nm long pass transmission filter and represented in linear mode. Single parameter statistics on sample and internal standard peak positions were generated using a Standard Elite Workstation Software. The peak positions between diploid sea lamprey (near 500) and rainbow trout red blood cells internal standard (near 600) are very close to each other in the histogram. This made the identification of sample and standard peaks cumbersome during the preliminary flow cytometry analyses. For each batch of samples analyzed, separate incubation of the standard rainbow trout blood and sea lamprey samples in propidium iodide stain were made. The ratio of sample peaks vs. the mean peak of standards was determined. Flow cytometry analyses of embryonic and prehatched sea lamprey samples frozen in DMSO were problematic. The minimum requirement of 10,000 gated cells was not met in some of the samples analyzed. The increase of incubation period of samples in propidium iodide stain from 24 to 48 hr resulted in visualization of peaks in 40–60% of the samples analyzed.
Female 1 Female 2 Female 3 Female 4 Female 5
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0 Control UVnoHS UVHS 8UVHS 12 UVHS 16UVHS 20 UVHS 24 Treatment
Fig. 1. Survival (%, mean7SD) of sea lamprey embryos at 2-cells and at pre-hatching stages of eggs subjected to various treatments. Control: eggs fertilized with untreated sperm; UVnoHS: eggs fertilized with UV-irradiated sperm and not exposed to heat shock; UVHS8, 12, 16, 20, 24: eggs fertilized with UV-irradiated sperm and exposed to heat shock 8, 12, 16, 20, or 24 min after fertilization.
Survival of gynogenetic sea lamprey was low (o50%) regardless of the treatments except when eggs from females 3 were used (Fig. 1). At hatching, some gynogenetic sea lamprey larvae were deformed and died within a week (Fig. 3B and C). Gynogenetic sea lamprey were either diploid (70.5%) or haploid (29.5%) (n 5 61) (Fig. 2C). In the second experiment, when PS was used to inhibit the second meiotic division and consequently induce diploidy with the UV-irradiated sperm, a pool of eggs from three females was used. Survival to the 2-cells and pre-hatching stages of sea lamprey embryos in the control groups were 8676% and 7979%, respectively (Fig. 4). Flow cytometry analyses revealed that all control embryos were diploid (100%, n 5 27). All eggs fertilized with UV-irradiated sperm but not
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Fig. 2. Representative distribution of fluorescence of cells prepared from sea lamprey ammocoetes measured by flow cytometry. (A) Rainbow trout blood cells, (B) sea lamprey larvae control (eggs fertilized with untreated sperm) diploid, (C) sea lamprey larvae UVHS (eggs fertilized with UV-irradiated sperm and exposed to heat shock) diploid, and (D) UVnoHS (eggs fertilized with UV-irradiated sperm and not exposed to heat shock) haploid.
exposed to PS produced deformed larvae which died within a week. A few embryos (24, 7, and 10, respectively) were observed at hatching when eggs were fertilized with UV-irradiated sperm and exposed to PS 8, 16, and 24 min after fertilization (Fig. 4), but they died prior to burying themselves in the sand.
Androgenesis experiments Androgenesis experiments were conducted with the eggs of a single female and survival rates of control were 98.470.7% and 71.773.0% at 2-cells and pre-hatching stages, respectively (Fig. 5). Regardless of the treatment, survival of J. Exp. Zool. (Mol. Dev. Evol.) DOI 10.1002/jez.b
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Fig. 3. Sea lamprey larvae at 10 days post hatching. (A) Control (eggs fertilized with untreated sperm), (B) and (C) UVHS normal larvae (eggs fertilized with UV-irradiated sperm and exposed to heat shock), (D) UVnoHS deformed larvae (eggs fertilized with UV-irradiated sperm and not exposed to heat shock). Deformed larvae were identified morphologically based on larvae size (50% length in comparison to control) and curved body.
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Fig. 4. Survival (%, mean7SD) of sea lamprey embryos at 2-cells and at pre-hatching stages from eggs subjected to various treatments. Control: eggs fertilized with untreated sperm; UVnoPS: eggs fertilized with UV-irradiated sperm and not exposed to pressure shock; UVPS8, 16, 24: eggs fertilized with UV-irradiated sperm and exposed to pressure shock 8, 16, or 24 min after fertilization.
androgenetic sea lamprey prior to hatching was low (o1%) and all animals died within a week after hatching (Fig. 5). DISCUSSION For the first time, we report quantitative parameters to induce gynogenetic and androgeJ. Exp. Zool. (Mol. Dev. Evol.) DOI 10.1002/jez.b
UVnoHS UVHS170 UVHS200 UVHS230 Treatment
Fig. 5. Survival (%, mean7SD) of sea lamprey embryos at 2-cells and at pre-hatching stages from eggs subjected to various treatments. Control: eggs fertilized with untreated sperm; UVnoHS: UV-irradiated eggs fertilized with sperm and not exposed to heat shock; UVnoHS170, 200, 230: UV-irradiated eggs fertilized with sperm and exposed to heat shock 170, 200, or 230 min after fertilization.
netic sea lamprey. The parameters used in the present study were based on those reported in Chondrostei fish, shovelnose sturgeon and paddlefish (Mims et al., ’97; Mims and Shelton, ’98), and teleost fish, rainbow trout (Diter et al., ’93), and muskellunge (Lin and Dabrowski, ’96, ’98; Dabrowski et al., 2000; Garcia-Abiado et al., 2001; Rinchard et al., 2002). For gynogenesis experiments, UV-irradiated sperm was used to activate sea lamprey eggs and HS or PS was applied to inhibit the second meiotic division and consequently induce diploidy in the embryos. The UV irradiation was performed for 1 min at 1,719 J m2. We used sea lamprey sperm (homologous sperm) in these experiments. The negative control was used as an indirect reference for sperm DNA destruction and spontaneous diploidization was not observed. HS of 35711C for 2 min and PS of 9,000 psi for 4 min were applied at different times after egg activation (8, 12, 20, and 24 min or 8, 16, and 24 min for HS or PS, respectively). Regardless of the application time of the HS, survivals at pre-hatching stage were similar. In contrast, PS applied 8 min after activation appears to increase survival rate of prehatched embryos in comparison to 16 and 24 min after activation. All deformed, gynogenetic embryos were haploid and died prior to burying themselves in the sand. Therefore, we concluded that all progenies produced using both methods were diploid gynogens.
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For the androgenesis experiments, UV-irradiated eggs were fertilized with non-treated sperm and HSs were applied to restore diploidy of the eggs. Several attempts have been made to optimize the parameters used. Although we were able to produce viable embryos, all died within a week after hatching. Lin and Dabrowski (’96) reported that high dose of UV irradiation may lead to chromosomal fragmentation in gynogenetic teleost fish, damaged molecules in the eggs, and ultimately to the appearance of deformed embryos. However, if gynogenetic animals will survive to reach gonad differentiation, this should not be a limitation to describe sex determination system (XX/XY or WZ/ZZ) in sea lamprey. In both types of shocks to induce gynogenesis, yields of embryos produced with initially highfertility eggs (see controls in females 3, 4, and 5; Fig. 1) were exceptionally high (40–80% with 70% diploids) in comparison to teleost fish (Dabrowski et al., 2000; Bertotto et al., 2005). Physical shocks (thermal or pressure shocks) used to restore egg diploidy and resulting inbreeding deficiency are the major factors responsible for the low survival in teleosts (Chourrout, ’87; Dunham, ’90; Ihssen et al., ’90; Yamazaki and Goodier, ’93); however, this was not the same in the sea lamprey. Although it is imperative to have a good quality of eggs for induction of gynogenesis, there is also evidence of some exceptional differences in response to the strenuous diploidization attempts between Agnatha and teleost fishes. ACKNOWLEDGMENTS We are grateful to the Great Lakes Fishery Commission (GLFC) for financial support. We would like to acknowledge the staff at the Hammond Bay Biological Station for the supply of adult sea lampreys, as well as Dr. Andrzej Ciereszko, Kyle Ware, Tobie Wolfe for their assistance. LITERATURE CITED Bertotto D, Cepollaro F, Libertini A, Barbaro A, Francescon A, Belvedere P, Barbaro J, Colombo L. 2005. Production of clonal founders in the European sea bass, Dicentrarchus labrax L., by mitotic gynogenesis. Aquaculture 246:115–124. Chourrout D. 1987. Genetics manipulations in fish: review of methods. In: Tiews K, editor. Selection, hybridization and genetic engineering in aquaculture, Vol. II. Berlin: Heenemann. p 111–125.
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J. Exp. Zool. (Mol. Dev. Evol.) DOI 10.1002/jez.b
