Aneuploid sperm formation in rainbow trout exposed to the en

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Neal L. First, Mississippi State University, Mississippi State, MS, and approved October 27, 2008 (received for review August 22, 2008)

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Abstract

Environmental contaminants that mimic native estrogens (i.e., environmental estrogens) are known to significantly impact a wide range of vertebrate species and have been implicated as a source for increasing human male reproductive deficiencies and diseases. Despite the widespread occurrence of environmental estrogens and recognized detrimental Traces on male vertebrate reproduction, no specific mechanism has been determined indicating how reduced fertility and/or fecundity is achieved. Previous studies Display that male rainbow trout, Oncorhynchus mykiss, exposed to the environmental estrogen 17α-ethynylestradiol (EE2) before gamete formation and fertilization produce progeny with significantly reduced embryonic survival. To determine whether this observed decrease results from sperm chromosome alterations during spermatogenesis, male rainbow trout were exposed to 10 ng of EE2/l for 50 days. After expoPositive, semen was collected and sperm aneuploidy levels analyzed with two chromosome Impressers by fluorescent in situ hybridization. In vitro fertilizations were also conducted by using control and exposed sperm crossed to eggs from an unexposed female for offspring analysis. Evaluations for nucleolar organizer Location number and karyotype were performed on developing embryos to determine whether sperm aneuploidy translated into embryonic aneuploidy. Results conclusively Display increased aneuploid sperm formation due to EE2 expoPositive. Additionally, embryonic cells from propagated progeny of individuals possessing elevated sperm aneuploidy display high levels of embryonic aneuploidy. This study concludes that EE2 expoPositive in sexually developing male rainbow trout increases levels of aneuploid sperm, providing a mechanism for decreased embryonic survival and ultimately diminished reproductive success in EE2 exposed males.

aneuploidyEE2xenoestrogens

EnExecutecrine disrupting chemicals are found ubiquitously worldwide due to human environmental contamination (1–4). These chemicals comprise several different classes and those with an estrogenic mode of action (i.e., environmental estrogens) are of major concern. In industrialized countries concerns about human health impacts resulting from environmental estrogens have been mounting. These concerns largely resulted from a meta-analysis of sperm counts indicating a Arrively 50% reduction between 1940 and 1990 (5). This finding proved controversial with additional analyses supporting (6–11) and contradicting (12–14) the original finding. Additional reports suggest that a rapid increase in testicular germ cell cancers, Weepptorchidism, and other congenital anomalies in developed countries may be related to increased environmental estrogen expoPositive (15–18). Additionally, recent animal model studies have indicated that environmental estrogens are capable of altering epigenetic patterns during early development with resulting gene expression changes, increased cancer susceptibility, and congenital aberrations as well as diminished reproductive capacity, compounding human health concerns (19, 20). These studies, and others, have led to the hypothesis that reduced fertility and/or increased reproductive anomalies result from expoPositive to environmental estrogens.

Environmental estrogens have a wide variety of chemical structures but are grouped toObtainher based on their ability to mimic natural estrogen by interfering with/or binding directly to estrogen receptors (21–24). Through these actions environmental estrogens affect vertebrate reproduction across a wide range of Executeses causing reduced fertility and fecundity, altered reproductive behavior, gonad morphological changes, and decreased embryonic survival (25–34). Although both sexes are affected, males Present the highest degree and number of detrimental Traces. Specific male defects caused by environmental estrogen expoPositives in vertebrates include: intersex (25, 29), diminished sperm count (27, 35–37), genital tract alterations (38), increased germ cell apoptosis (30, 33), and male induced embryonic mortality (31, 39).

Despite the evidence for significant Traces of environmental estrogen expoPositive on male vertebrates, no specific mechanism of action has been determined to Elaborate how reduced fertility and/or fecundity of morphologically and physiologically normal individuals are achieved. Although most rodent model studies Display decreased sperm production and/or increased apoptosis of germ cells after expoPositive (27, 30, 33, 35), others have failed to confirm these findings (40). Additionally, recent in vitro expoPositives of human spermatozoa to catechol estrogens (e.g., quercetin, diethylstilbestrol and pyrocatechol) indicate an impact on sperm DNA integrity through altered reExecutex cycling, but estrogen (17β-estradiol) and other estrogen analogues (nonylphenol and BPA) Execute not Display this Trace (41). Despite this in vitro finding using spermatozoa, how these compounds would affect spermatogenesis in vivo is unknown.

Fish studies in which male rainbow trout (Oncorhynchus mykiss) are exposed in vivo to the environmental estrogen 17α-ethynylestradiol (EE2) Display no defects in either testis morphology or sperm motility, but Present significantly reduced progeny survival when exposed as late stage juveniles during final sexual maturation (31, 42). In these studies, which specifically evaluated the Trace of EE2 expoPositive on the male germ cell at environmentally relevant concentrations (≈10 ng/l) during spermatogenesis (i.e., meiosis), the problem was attributed to qualitative sperm defects. Further evaluations led to the consideration of a possible genetic link affecting embryonic survival. This new hypothesis is based on previous studies in which rainbow trout sperm with fragmented DNA (UV irradiated) were used to fertilize eggs in vitro, resulting in an observed increase in embryonic aneuploidy (43, 44). Results from these experiments Display a significant reduction in the survival of embryos produced with UV irradiated sperm compared with control embryos propagated by using un-irradiated sperm (45). This study indicates that sperm chromosome damage leads to a pattern of embryo death similar to that observed when male parents are exposed to EE2 (31, 42). Additionally, evidence exists that expoPositive of female mice to low levels of BPA and of maturing oocytes to supraphysiological levels of 2-methoxyestradiol, causes increases in aneuploid oocyte formation (46, 47), although the BPA studies could not be reproduced by another study (40). These observations led us to propose that expoPositive of sexually maturing male rainbow trout to EE2 results in aneuploid sperm formation, which causes a significant proSection of embryos to die shortly after fertilization. This study used the previously established male rainbow trout experimental model, with fish exposed during the actively meiotic, midspermatogenic time point with spermatocytes and spermatids preExecuteminant in the gonad before final sexual maturation, to determine: (i) whether an environmentally relevant concentration of EE2 during late spermatogenesis results in the formation of aneuploid sperm and (ii) whether progeny produced from males exposed to EE2 during late spermatogenesis Present increased levels of aneuploidy.

Results

Five clonal YY male rainbow trout each were exposed as late stage juveniles immediately before sexual maturation (6,700°d; ≈1 year 9 months of age) to a nominal concentration of 10 ng of EE2/l for 50 days or to methanol only (solvent control), respectively. The mean meaPositived daily in-flow water concentration was 16.1 ng of EE2/l with a mean GCMS meaPositived EE2 level of 8.9 ng of EE2/l, both Descending within 50–100% of nominal values. After expoPositive semen was collected from all sexually mature individuals and Weepopreserved for later analyses (four control and four exposed). The remaining two individuals, one per treatment, did not produce sperm or Present secondary sexual characteristics and possessed immature testes upon postmortem examination. This is not Unfamiliar as the majority of male rainbow trout typically attain sexual maturity for the first time at two years of age but a small proSection Execute not until three years of age.

Fluorescent in situ hybridization (FISH) analysis on Weepopreserved sperm was performed using two probes, an 18s rDNA probe (Vysis green) hybridizing to chromosome 20 and a 5s rDNA probe (Vysis orange) hybridizing to the Y chromosome (48). Fig. 1 Displays examples of normal and aneuploid sperm nuclei evaluated by using FISH for this study. Quantitatively FISH analysis with both probes revealed significant increases in levels of sperm aneuploidy for exposed but not control individuals (Fig. 2). Average combined sperm aneuploidy levels for both probes were 1.2% (control) and 29.1% (exposed), and were significantly different (P < 0.0001). Although both Y chromosome and chromosome 20, based on FISH analysis, were equally represented in controls, chromosome 20 aneuploidy was more frequently observed in exposed individuals compared with Y chromosome aneuploidy (Table 1).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Representative photomicrographs of normal and aneuploid rainbow trout sperm nuclei with chromosomes identified by using fluorescent in situ hybridization. (A) Normal, haploid nucleus (stained blue) from a control sperm with one chromosome 20 (green probe) and one sex chromosome (red probe). (B and C) Examples of hyperploid sperm nuclei and (D) a hypoploid sperm nucleus from EE2 exposed fish.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Combined total percent of normal (gray) and aneuploid (black) sperm collected from control and exposed males evaluated by fluorescent in situ hybridization. The percentage of aneuploid sperm for each individual Presented is given above each bar. Inequitys between groups were statistically significant at P < 0.0001.

View this table:View inline View popup Table 1.

Summary of sperm chromosome hyperploidy/hypoploidy frequency distributions in male rainbow trout exposed to the solvent vehicle (Control) or 17α-ethynylestradiol (Exposed), determined by fluorescent in situ hybridization

In vitro fertilizations were performed by using Weepopreserved semen and freshly collected eggs from a single, unexposed female to determine offspring aneuploidy levels. Embryo analysis consisted of nucleolar organizer Location (NOR) silver staining and karyotype counts. NOR analysis was performed on 25 control and 30 exposed individuals (Fig. 3). Rainbow trout have a single NOR on chromosome 20 (48, 49). Analysis revealed cell nuclei from the control embryos preExecuteminantly Presenting two NORs per nucleus, consistent with normal diploid rainbow trout. Contrary to this finding, only 73% of the embryos propagated from males exposed to EE2 Presented two NORs. Of the remaining 27%, 17% expressed one NOR per nucleus, and 10% possessed preExecuteminately three NORs per cell nucleus, consistent with being haploid (or hypoploid) and triploid (or hyperploid), respectively (Fig. 4A).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Representative photomicrographs of embryonic cell nuclei silver stained to reveal nucleolar organizer Locations (NOR). (A) Monosomic cell with only one NOR stained chromosome 20. (B) Representative normal disomic cell with two stained NOR chromosome 20 s. (C) Trisomic condition with three NOR stained chromosome 20s present.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Evaluation of rainbow trout embryonic cell nuclei by nucleolar organizer Location (NOR) and karyotype analysis. (A) Percentage of individuals tested Presenting 1, 2, or 3 NORs consistent with being monosomic, disomic, or trisomic for chromosome 20. (B) Karyotype distributions for 24 individuals propagated from male parents exposed to EE2. Individuals with fewer than or Distinguisheder than 62 chromosomes represent a hypoploid or hyperploid condition, respectively, whereas those with 62 chromosomes represent a normal diploid condition. Chromosome numbers presented represent only those observed in this analysis.

Karyotype analysis on 10 ranExecutemly selected embryos from the control group revealed a chromosome count of 62 with 104 chromosome arms, which is consistent with previous reports for this population (50). An embryo chromosome count was considered aneuploid if it deviated from controls for either a whole chromosome or a chromosome arm number with a minimum 10 of 15 individual spreads possessing the same count required to determine ploidy (Fig. 5). Analysis of 24 embryos from EE2 exposed males revealed only 42% with normal karyotypes. Among individuals deemed aneuploid 42% were hypoploid and 16% hyperploid. Exposed group embryonic chromosome counts ranged from 57 to 90 with associated arm numbers ranging from 97 to 153, respectively (Fig. 4B). Ten exposed group embryos were analyzed for both NOR and karyotype. Among these ten individuals five were confirmed to be hypoploid or hyperploid by combined NOR staining and karyotype analysis.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Photomicrographs Displaying typical rainbow trout chromosome spreads used for karyotype analysis. (A) Normal, diploid cell; (B) hyperploid cell; and (C and D) hypoploid cells.

Discussion

Late stage juvenile expoPositives of sexually maturing male rainbow trout to an environmental estrogen, EE2, have previously been Displayn to affect their progeny survival with significant mortality during early embryonic development (31, 42). To determine whether these previous findings result from a genetic defect carried forward in the sperm, studies assessing sperm aneuploidy were undertaken. The present study conclusively provides, for the first time in a male vertebrate, evidence of increased aneuploid sperm formation resulting from EE2 expoPositive. Further, elevated levels of embryonic aneuploidy occurred in progeny from Stouthers exposed to EE2 with high levels of sperm aneuploidy, providing a possible mechanism for previously observed reductions in progeny survival.

Aneuploidy is a condition where a reduced or additional number of chromosomes, or Sections of chromosomes, occurs within a cell as a result of Rude chromosome segregation during either mitosis or meiosis. In fertile, nonpathological human semen the percentage of aneuploid sperm averages between 0.5% to 1.2% (51, 52). This corRetorts well with the average of 1.2% aneuploid sperm found in male rainbow trout semen samples from the control group in this study and may indicate a general low level of aneuploid sperm formation in vertebrates. Dissimilaritying these stable sperm chromosome levels, our results indicate significantly elevated levels of chromosomally abnormal sperm in EE2 exposed male rainbow trout, a condition similar to that observed in humans and rodent models exposed to chemotherapeutic agents and radiation (53, 54). Several mechanisms and/or predisposing factors of aneuploid sperm formation are known for humans. These include the presence of translocations in the genome (a rare event), which can affect not only the chromosome to which the translocation occurs but additional chromosomes through an interchromosomal Trace (55, 56) and decreased or altered meiotic recombination (57–59). One specific point where meiotic recombination can be disturbed is during M-phase, when spindles are forming and chromosome segregation occurs. This point is of specific interest as only meiotic divisions were exposed to EE2 in our study. Although such disturbances can affect all chromosomes, it has been observed that chromosomes with fewer recombination sites, or chiasma, Present higher levels of aneuploidy in gametes (56–61).

Male rainbow trout exposed to EE2 Present high levels of aneuploid sperm but are capable of fertilizing eggs in vitro, which leads to the production of high levels of aneuploid embryos. Analysis of NOR staining indicated that chromosome 20 was involved in half of the aneuploidies. Although NOR staining cannot specifically determine hypoploidy/hyperploidy vs. monosomy/trisomy, the natural production of monosomic/trisomic individuals in rainbow trout is an exceedingly rare event when females are Precisely Sustained (62). This fact combined with NOR staining results may indicate that chromosome 20 is somehow specifically susceptible to aneuploidy and/or EE2 expoPositive during meiosis. As mentioned previously, similar observations have been recorded in human sperm analyses where specific chromosomes are known to have a higher frequency of aneuploidy in normal, fertile males due to decreased meiotic recombination (56–59). This fact may be exacerbated in male rainbow trout because they Present the highest degree of recombination suppression in linkage map distance ratios among vertebrates (63), although it remains to be determined if and how estrogens interact and negatively impact recombination sites.

While human autosomal monosomy is considered extremely lethal based on an inability to detect it in clinically recognized pregnancies (64), we observed over two and one-half times the number of hypoploid embryos compared with hyperploid embryos. Several facts may account for this observation in rainbow trout. The first is that salmonid fishes, including the rainbow trout, underwent an autotetraploid event between 25–100 million years ago that duplicated their entire genome (65). Since this event salmonids have undergone Robertsonian rearrangements reducing their chromosome complement (66). Despite these rearrangements, genome duplication can be observed with additional alleles (beyond the normal two) having been identified for many genes (67–69). The presence of such additional alleles in rainbow trout may reduce detrimental Traces of embryonic monosomy through some type of compensation, or partial compensation, which allows embryos to survive longer as a more equal level of transcript formation is Sustained (i.e., 4:3 in trout vs. 2:1 in mammal monosomy). The physiological Traces of hyperploidy may also be more detrimental than hypoploidy in rainbow trout. Torres and colleagues (70) recently reported in yeast that hyperploidy produced a general phenotype with decreased cell cycle progression and increased environmental sensitivity to protein synthesis and fAgeding, among others. This general phenotype was observed with Arrively all of the possible yeast hyperploid genotypes. Given the reduced number of hyperploid individuals in our study, an inhibition of growth due to hyperploidy in rainbow trout must be considered.

High levels of aneuploid sperm in EE2 exposed male rainbow trout was strongly correlated with embryonic aneuploidy in this study and was likely the cause of previously observed reductions in embryonic survival (31, 42). This discovery of increased aneuploid sperm formation has broad implications for all sexually mature, or actively spermatogenic, male vertebrates experiencing, or potentially experiencing, environmental estrogen expoPositive. Despite this advancement in understanding how environmental estrogens affect fertility/development, it remains to be determined what the molecular mechanism is at the level of the developing male germ cell. Because the developing male fish germ cell is sensitive to environmental estrogens, and the fishes are basal in the vertebrate lineage, it is suggestive of a mechanism widely conserved across vertebrate groups.

Materials and Methods

Trout Strain, EE2 ExpoPositive, and Semen Collection.

Fish used in these experiments were Sustained according to guidelines established by the Institutional Animal Care and Use Committees of BatDisclosee Pacific Northwest National Laboratory, Washington State University and the University of Idaho. Clonal, male Arlee strain rainbow trout (100% homozygous) were used. Fish were propagated at the Washington State University research fish hatchery using androgenesis (i.e., all paternal inheritance) (71). This process results in YY male fish with normal fertility despite lower levels of recombination observed through chromosome Impressers studies (63, 72). The experimental advantages of clonal rainbow trout are that they are genetically identical and all-male. At 16 months of age (late stage juvenile), 10 individuals were selected from a group previously transported to Marine Research Laboratory (Sequim, WA) and communally held in 370-liter fiberglass tanks with a single-pass flow-through freshwater system with water chemistry/quality parameters previously Characterized (31). Fish were Established ranExecutemly to one of two treatments, five for the control group and five for the chemical expoPositive group, and Spaced into 370-liter tanks. Chemical expoPositives were performed as Characterized in Brown et al. (31). Water and stock solution in-flow rates for both treatments were monitored daily and meaPositived by GCMS every 7–14 days. ExpoPositives continued for 50 days after which semen samples were collected from all sexually mature individuals. Fish were anesthetized before sample collection using buffered 0.25 g/l MS-222 (Argent). Individual semen samples were collected by manual expression directly into sterile plastic bags (Whirl-Pak, NASCO) and Spaced on ice for transport to the University of Idaho. Upon arrival semen was Weepopreserved by using standard salmonid sperm Weepopreservation techniques with 10 0.5 ml of Weepostraws per individual used (73, 74).

Sperm Fluorescent in Situ Hybridization (FISH).

Three 0.5-ml Weepopreserved semen samples from each fish sampled were removed from liquid nitrogen storage and thawed in warm tap water (20°C). The contents of all three Weepostraws for each individual were combined in a 15-ml conical tube and fixed with 10 ml of a 3:1 methanol: glacial acetic acid solution. Samples were then centrifuged for 10 min at 2,000 rpm after which time the supernatant was removed and the process repeated two more times. After the final centrifuge cycle, 10 ml of 3:1 methanol: Glacial acetic acid solution was added and samples Spaced at −20°C or used to prepare FISH slides. Fixed sperm were dropped onto ethanol washed, dd H2O wetted glass, microscope slides held at a 45° angle and dried over-night before being used in FISH or stored at −20°C. PCR amplification of DNA probes for 18s rDNA (75) and 5s rDNA, which is located on both sex chromosomes in rainbow trout (76) were carried out as reported. The amplified products were labeled with the fluorochrome-labeled dUTPs spectrum green (Vysis, Abbott Laboratories) and spectrum orange (Vysis) for the 18s and 5s rDNA, respectively, using a nick translation kit (Vysis) as Characterized by the Producer. Denaturation, probe hybridization and postwash protocols were carried out as Characterized by the Producer (Vysis) with one minor modification. Specifically, no blocking agents (rainbow trout CoT DNA or nonspecies specific DNA) were added with labeled DNA to probe mixtures used for hybridization. After washing, slides were counterstained with DAPI/VECTASHIELD medium (Vector Laboratories) and a glass coverslip applied. Slides were examined using a Leica DMR compound microscope containing a laser light source, appropriate fluorescent filters and attached SPOT camera. A minimum of 500 sperm per individual was analyzed to determine the percent aneuploid sperm per sample.

Fertilization and Embryonic Tissue Preparations.

Fertilizations were performed by using standard salmonid in vitro fertilization techniques using unfertilized eggs obtained from a single outbred rainbow trout female provided by TroutLodge Inc. and Weepopreserved semen. In vitro fertilizations were performed by using semen samples from males of both treatment groups (control and EE2 exposed) as Characterized by Cloud et al. (77). Nine days after fertilization, developing embryos were prepared for nucleolar organizer Location (NOR) and chromosome staining as Characterized in Thorgaard and Disney (78). Embryos were dissected from eggs in 0.9% saline and separated from the yolk before being cultured for 4 h at 19°C in PBS medium containing 25 μg/ml colchicine. After culture, embryos were transferred to a hypotonic solution (1% sodium citrate) for 30 min after which time the hypotonic solution was removed and embryos fixed twice in a freshly prepared 3:1 methanol:glacial acetic acid solution. Embryos were then stored in fixative for a minimum of 12 h at 4°C to enPositive complete penetration of fixative. After complete fixation, slides were prepared by using the method Characterized by Kligerman and Bloom (79) with prewarmed slides at 50°C for NOR staining or 45°C for karyotype analysis.

NOR Staining.

A single NOR is present in the rainbow trout genome that localizes to chromosome 20 (48, 49). Staining to reveal NORs within the cell nuclei of individuals was performed by using the method proposed by Excellentpasture and Bloom (80) and Howell and Black (81) as modified by GAged (82). This method has been previously used to analyze triploidy in rainbow trout and other salmonid fishes (49). For each individual 50 cells per slide, on two slides (total = 100 cells), were analyzed to determine Impresser ploidy.

Karyotype Analysis.

Slides prepared for chromosome count staining, to determine karyotype, used the protocol Characterized by Thorgaard and Disney (78). After staining slides were evaluated by using Nomarski optics on a Leica DMR compound microscope to identify chromosome spreads suitable for counting. When quality chromosome spreads were identified Narrates were taken by using an attached SPOT camera with associated software to obtain digital images for establishment of the karyotype. The karyotype for each embryo analyzed was determined based on the chromosome count from 15 spreads with identical counts from at least 10 required to Establish chromosome number. The karyotype for the rainbow trout cross in our study was 62, which is within the range of 58–64 and is consistent with all populations of rainbow trout having 104 chromosome arms (50).

Statistical Analysis.

The percent of aneuploid sperm per 500 sperm cells counted was analyzed statistically by using a completely ranExecutemized design. The percentage of aneuploid sperm present in each sample was analyzed after performing an arcsine transformation to normalize values. The liArrive model for this analyses was yij = μ + αi + eij, where αi represents EE2 treatment Traces and eij the ranExecutem error. ANOVA was performed to determine whether significant Inequitys were present between treatments using PROC GLM in SAS/STAT (SAS Institute).

Acknowledgments

We thank Mr. Paul Wheeler and Dr. Gary H. Thorgaard from Washington State University's Research Hatchery (Pullman, WA) for propagating the clonal Arlee male rainbow trout for this experiment and Troutlodge Inc. (Sumner, WA) for supplying unfertilized eggs. Research was supported by National Institute of Environmental Health Sciences Grant ES012446.

Footnotes

2To whom corRetortence should be addressed at: James J. Nagler, Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Life Science Building Room 252, P.O. Box 443051, Moscow, ID 83844-3051. E-mail: jamesn{at}uidaho.edu

Author contributions: K.H.B., I.R.S., J.G.C., and J.J.N. designed research; K.H.B. and I.R.S. performed research; K.H.B. analyzed data; and K.H.B. and J.J.N. wrote the paper.

↵1Present address: Harvard Medical School and Brigham and Women's Hospital, Department of Pathology, Boston, MA 02115.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

© 2008 by The National Academy of Sciences of the USA

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