Chronic Hypoxia Impairs Gamete Maturation in Atlantic Croaker

Apr 29, 2009 - 750 Channel View Drive, Port Aransas, Texas 78373. Received January 9, 2009. Revised manuscript received. March 23, 2009. Accepted Apri...
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Environ. Sci. Technol. 2009, 43, 4175–4180

Chronic Hypoxia Impairs Gamete Maturation in Atlantic Croaker Induced by Progestins through Nongenomic Mechanisms Resulting in Reduced Reproductive Success PETER THOMAS* AND MD. SAYDUR RAHMAN University of Texas at Austin, Marine Science Institute, 750 Channel View Drive, Port Aransas, Texas 78373

Received January 9, 2009. Revised manuscript received March 23, 2009. Accepted April 14, 2009.

Recent studies have shown that chronic hypoxia exposure impairs reproduction in fish by interfering with endocrine function, although the mechanisms of endocrine disruption remain unclear. The effects of chronic exposure (4 or 10 weeks) to hypoxia (dissolved oxygen, DO: 1.7 mg L-1) on gamete maturation and its endocrine control, as well as the consequences for reproductive success, were investigated in Atlantic croaker (Micropogonias undulatus). Circulating levels of the progestin hormone that induces gamete maturation, 17,20β,21-trihydroxy4-pregnen-3-one (20β-S), were significantly decreased in croaker of both sexes chronically exposed to hypoxia and were associated with impairment of oocyte meiotic maturation and sperm motility. Interestingly, expression of the novel membrane receptor mediating these nongenomic 20β-S actions, membrane progestin receptor alpha (mPRR), was significantly decreased on oocyte and sperm plasma membranes of hypoxiaexposed fish. Hypoxia-induced impairment of gamete maturation was accompanied with a dramatic decline in the percent fertilized eggs in a spawning trial. Moreover, the fertilized eggs from hypoxia-exposed donors displayed decreased hatching success and larval survival. The results suggest that nongenomic progestin signaling controlling the final stages of the reproductive cycle in fish is impaired under hypoxic conditions.

Introduction Successful completion of the reproductive cycle in fishes involves the initiation and precise coordination of numerous reproductive processes in the brain, gonads, and other tissues, beginning with gonadal differentiation and culminating with the release and fertilization of mature gametes. The timing and coordination of these reproductive processes are under complex endocrine control by hormones secreted by the hypothalamus-pituitary-gonad (HPG) axis (1). In view of their complexity, it is not surprising that fish reproduction and its endocrine control are susceptible to interference by exposure to a variety of environmental stressors, including xenobiotic chemicals, confinement in captivity, and physical stressors such as hypoxia (1-5). Impairment of reproduction in fish environmentally exposed to stressors is a concern because * Corresponding author phone: 1-361-749-6788; fax: 1-361-7496777; e-mail: [email protected]. 10.1021/es9000399 CCC: $40.75

Published on Web 04/29/2009

 2009 American Chemical Society

reduced reproductive success could eventually lead to a decrease in population abundance (6). A major mechanism of endocrine disruption and reproductive impairment in vertebrates by xenobiotic chemicals involves interference with classical, genomic steroid hormone actions mediated through activation of nuclear steroid receptors (7, 8). However, there is now extensive evidence that steroids also exert important rapid, cell surface-initiated, nongenomic actions (i.e., nonclassical) by activating steroid membrane receptors (9-12). For example, a novel seventransmembrane receptor recently discovered in fish and other vertebrates (13), progestin membrane receptor alpha (mPRR), has been shown to mediate nongenomic progestin actions at the end of the reproductive cycle to induce maturation of fish gametes, enabling fertilization to occur (12, 13). The progestin hormone, 20β-S, binds to mPRR on Atlantic croaker gametes and induces the resumption of oocyte meiotic maturation and sperm hypermotility through activation of G proteins and their associated second messengers (12, 14). Interestingly, recent studies suggest that a variety of xenobiotic chemicals that disrupt nuclear steroid receptor signaling can also interfere with nongenomic progestin actions on fish gametes mediated by mPRR (15, 16). Rapid progress has been made in elucidating the signaling pathways and the physiological functions of mPRs in the few years since their discovery (12). However, the molecular structure of the ligand binding pocket remains unknown and on the basis of their negative results Brosens and coworkers have challenged the conclusions of other researchers that mPR functions as a membrane progestin receptor in mammals (12, 17, 18). This controversy has recently abated with the confirmation by another research group that recombinant mPRs produced in a third expression system, yeast, have the binding characteristics of progestin receptors (19, 20). No such controversy currently exists over the functions of mPRs in teleosts, where extensive evidence has been obtained in a variety of species that mPRs mediate progestin regulation of gamete maturation via a nongenomic mechanism (20). There is mounting concern over the long-term ecological effects of the recent increase in the incidence and extent of seasonal hypoxia in many coastal regions throughout the world as a result of increased anthropogenic inputs of nutrients (21, 22). Marked impairment of gametogenesis and reproductive endocrine function was observed in Atlantic croaker collected from hypoxic sites in two Gulf of Mexico estuaries (23, 24), and in croaker, Gulf killifish (Fundulus grandis), and common carp (Cyprinus carpio), continuously exposed to low DO concentrations for several weeks in controlled laboratory studies (23, 25-27). Hypoxia-induced disruption of this phase of the reproductive cycle in croaker was associated with declines in circulating levels of androgens in males and estrogens in females which in turn was related to impaired luteinizing hormone (LH) secretion and hypothalamic neuroendocrine function (23). Hypoxia exposure also affects the final stage of the reproductive cycle in Gulf killifish and common carp resulting in reductions in sperm motility, retarded oocyte maturation, decreased egg production, and reproductive success (25-27). However, the mechanism of endocrine disturbance is unknown because the progestin hormone control of gamete maturation has not been investigated under hypoxic conditions. In the present study the effects of chronic hypoxia exposure on oocyte meiotic maturation and sperm motility in Atlantic croaker, their regulation via nongenomic progestin signaling pathways, and reproductive success were investigated. The results VOL. 43, NO. 11, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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provide the first evidence that hypoxia can interfere with reproduction in a vertebrate species by impairing nongenomic steroid signaling.

Materials and Methods Chemicals. 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) was purchased from Steraloids, Inc. (Wilton, NH). Luteinizing-releasing hormone analog (LHRHa, des-Gly10,[D-Ala6]LHRH (1-9) ethylamide) was obtained from Bachem (Torrance, CA). All other chemicals were purchased from SigmaAldrich (St. Louis, MO) unless noted otherwise. Experimental Fish. Adult young-of-the year Atlantic croaker (14-15 cm length, 30-38 g body weight), obtained from local fisherman, were acclimated to laboratory conditions at the University of Texas Marine Science Institute, Port Aransas in large indoor tanks (4725 L) with a recirculating seawater system for 1 month prior to experimentation. Fish were maintained under fall conditions (temperature: 22-25 °C, photoperiod: 11 L:13D) and fed commercial pellets daily (Rangen, Angleton, TX, 5% BW day-1) to promote gonadal development. Effects of Chronic Hypoxia Exposure on Gamete Maturation. Fish at a mid phase of gonadal growth (assessed by sampling several individuals from each tank) were exposed to hypoxic (1.7 ( 0.01 mg L-1 DO) or normoxic conditions (6.5 ( 0.02 mg L-1) for 4 weeks in 2000 L recirculating saltwater (salinity 32‰) tanks (25 fish of mixed sex tank-1, two tanks treatment-1) under fall temperature/photoperiod conditions and fed pellets (3% BW) daily as described previously (23). The dissolved oxygen levels in the tanks were lowered by reducing both the aeration and water recirculation, and oxygen levels were monitored twice daily with an oxygen meter (YSI 556 Multiprobe System, YSI Incorporated, Yellow Springs, OH) as described in Supporting Information (SI) Figure S1. Approximately 20% of the water in each exposure tank was exchanged every week and other water quality parameters did not change significantly during the experimental period (pH 7.7-7.9, ammonia 0.1-0.2 mg L-1, and nitrite 0.01-0.02 mg L-1, SI Figure S2). At the end of the 4-week exposure period in both control and hypoxia-exposed male and female fish the gametes had reached an advanced stage of development based on the presence of sperm and morphometric characteristics of oocytes (milt could be expressed from males and females had large oocytes, 425-450 µm in diameter). Fish were rapidly captured, anesthetized with MS-222 (20 mg L-1), and humanely sacrificed according to guidelines approved by the University of Texas at Austin Animal Resource Center. Blood and gonadal tissues were collected for subsequent measurement of progestin levels and bioassays of gamete maturation, respectively. In Vitro Oocyte Maturation Bioassay. The in vitro croaker oocyte maturation assay was conducted as described previously (28). Ovarian tissues were weighed and transferred to DMEM culture media containing 120 mM NaCl, 4.2 mM KCl, 3 mM MgCl2, and 1 mM CaCl2 supplemented with 100 mg L-1 streptomycin, 60 mg L-1 penicillin G, and 14.3 mM Na2HCO3 (pH 7.4, 299 mOsm kg-1 H2O). Ovarian fragments (∼100 large oocytes) were transferred to 24-well tissue culture plates containing 1 mL culture media and incubated for 18 h with hCG (10 IU) or 20β-S (20 nM) with hCG. Oocyte maturation was assessed at the end of incubation period by counting the oocytes under a binocular microscope that had completed germinal vesicle breakdown (GVBD). The media was stored at -80 °C until assayed for steroid content by radioimmunoassay. Radioimmunoassay of 20β-S Content. The concentration of 20β-S in plasma and in the culture media collected from the ovarian follicle incubations was measured by a radioimmunoassay validated for croaker plasma and media (29). 4176

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In Vitro Sperm Motility Bioassay. Milt was collected with a syringe after applying gentle pressure to the abdomen to express the milt; care was taken to avoid contamination of the milt with urine or seawater. The motility of fresh sperm samples was determined by an in vitro bioassay protocol developed for croaker sperm and recorded with a video camera as described previously (15). Preparation of Sperm and Ovarian Membranes. Sperm and ovarian membranes were isolated as described previously (13, 14) with minor modifications. Milt and ovarian fragments were suspended in HAED buffer (25 mM HEPES, 10 mM NaCl, 10 mM MgCl2, 1 mM dithioerythritol, 1 mM EDTA, pH 7.6) with 1.5 mg mL-1 HALT protease inhibitor cocktail (Pierce, Rockford, IL) and homogenized thoroughly with a glass homogenizer. The homogenate was centrifuged at 500g for 30 min to remove the nuclear fraction. The supernatant was centrifuged at 17 000g for 45 min to obtain the plasma membrane pellets. Final membrane pellets were resuspended in HAED buffer and stored at -80 °C for subsequent mPRR protein analysis. Western Blot Analysis for mPRr Protein. Plasma membrane proteins were solubilized by boiling for 10 min in SDS loading buffer (0.5 M Tris-HCl, 0.5% Bromophenol Blue, 10% glycerol), and cooled on ice for 5 min. The solubilized protein (10 µg total protein) was resolved on a 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane and blocked with 5% milk in phosphate buffered saline (PBS; 136 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 10 mM KH2PO4, 0.1% Tween20) for 1 h at 24 °C. Membranes were probed with a primary antibody (dilution 1:2000) directed against a synthetic 15 amino acid peptide sequence in the N-terminal region of spotted seatrout mPRR (13) which is 100% identical to the corresponding region in Atlantic croaker mPRR (GenBank accession no. EU095257). Membranes were then washed with PBS, incubated for 1 h with a goat polyclonal to rabbit IgG (HRP) secondary antibody (1: 10 000; Novus Biologicals, Littleton, CO). The protein was visualized by the addition of WestPico chemiluminescent substrate (Pierce, Rockford, IL) and photographed on Hyperfilm (Amersham Biosciences) in dark condition. Effects of Chronic Hypoxia Exposure on Spawning Success. Fish at mid to late phases of gonadal crudescence were exposed to hypoxic (1.7 and 2.7 mg L-1 DO) or normoxic (control) conditions as described previously (23) for 10 weeks in 2000 L recirculating seawater tanks (water quality data shown in SI Figure S3) maintained under ambient fall environmental conditions during the period of gonadal crudescence. At the end of the exposure period, 7-10 females from each experimental group were injected with 50 ng LHRHa g-1 BW (Bachem, Torrance, CA) to induce gamete maturation and spawning. Fish were then placed in spawning tanks with males (1:1) from the same experimental group and allowed to spawn naturally overnight. Eggs were collected from the outflow pipe in fine mesh egg bags and transferred to a measuring cylinder. Percent fertilization success was calculated from the proportion of the eggs that formed a perivitelline space and remained buoyant in the cylinder (accuracy >95%, confirmed by observing cell division under a binocular microscope). For each experimental group, 10 fertilized live eggs were transferred to each of ten bowls with 100 mL of oxygenated filtered seawater and incubated in an incubator for 48 h. During the 48 h incubation period, eggs and larvae were observed for development and counted in 12 h increments. During each observation, approximately 30% of the water was replaced with filtered oxygenated water. Spawning success was calculated as the percent of live, hatched fingerlings compared to the total number of fertilized eggs spawned. Statistical Analysis. Results were analyzed by one-way ANOVA and Fisher’s protected least-significant difference

FIGURE 2. Effects of four weeks hypoxia exposure on membrane progestin receptor alpha (mPRr) protein levels on croaker oocyte membranes (gel loading 10 µg) determined by Western blot analysis (A) and densitometry (B). Representative Western blot shown for samples from individual fish. Each bar represents mean ( SEM (N ) 12). Asterisk indicates significant difference from normoxic control (Student’s t-test, p < 0.05). M, marker.

FIGURE 1. Effects of four weeks hypoxia exposure on gonadal growth and the progestin hormone status of female croaker and maturation of their oocytes in vitro compared to controls (exposed to normoxic conditions). (A) ovarian growth expressed as gonadosomatic index (GSI %), (mean ( SEM, N ) 12). (B) plasma 20β-S levels (mean ( SEM, N ) 11-12). (C) Oocyte maturation is expressed as percent germinal vesicle breakdown (GVBD) in an in vitro bioassay conducted as described previously (28). Oocytes were incubated in response to hCG (10 IU) and 20β-S (20 nM) plus hCG for 18 h. All treatments were tested in triplicate (mean ( SEM, N ) 12). (D) in vitro production of 20β-S by intact ovarian follicle in response to hCG (mean ( SEM, N ) 12). Asterisk indicates significant difference from normoxic control (Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001). (PLSD) test for multiple comparisons and Student’s t test for paired comparisons using Systat (Systat, San Jose, CA) and GraphPad Prism (GraphPad, San Diego, CA) computer software. A probability of p < 0.05 was considered statistically significant.

Results Progestin Status in Females and Oocyte Maturation In Vitro. Ovarian growth was significantly impaired in croaker exposed to hypoxia (1.7 mg L-1 DO) for four weeks compared to normoxic controls (Figure 1A) which was associated with a slight reduction in the size of fully grown oocytes (mean oocyte diameter: normoxia 455 ( 20, hypoxia 434 ( 16, mean ( SEM, N ) 10-12, p < 0.05). Plasma levels of the croaker maturation-inducing steroid, 20β-S, were also significantly reduced after chronic hypoxia exposure (Figure 1B). Ovarian

follicles from control fish were responsive to gonadotropin treatment in the oocyte maturation bioassay, more than 50% of the large oocytes had reached the germinal vesicle breakdown (GVBD) stage of oocyte maturation after 18 h incubation (Figure 1C) which was associated with the in vitro production of substantial amounts of 20β-S (Figure 1D). In contrast, fewer than 20% of the large follicle-enclosed oocytes from the hypoxia-exposed donor females had undergone oocyte maturation after this period of gonadotropin treatment (Figure 1C) and 20β-S production was only 25% that of the controls (Figure 1D). Treatment with 20 nM 20β-S did not further augment the oocyte maturation response to gonadotropin in the hypoxia-treatment group (Figure 1C), suggesting that the impairment of oocyte maturation was not solely due to decreased in vitro production of 20β-S. The amount of the mPRR protein on the plasma membranes of large oocytes from the low DO-exposed fish was significantly reduced compared to controls, suggesting 20β-S signaling is impaired after hypoxia exposure (Figure 2). A second study demonstrated that oocyte maturation in response to gonadotropin treatment in vitro was also decreased after 10 weeks hypoxia exposure (SI Figure S4). Progestin Status in Males and Sperm Motility In Vitro. Testicular growth was significantly impaired after 4 weeks exposure to 1.7 mg L-1 DO and was half that of the controls, although milt containing mature sperm could be collected from both treatment groups (Figure 3A). Circulating levels of 20β-S were also significantly decreased in hypoxia-exposed males compared to controls (Figure 3B). A similar decrease in plasma 20β-S levels was observed after 10 weeks exposure to 2.7 mg L-1 DO (SI Figure S6A). A high percentage of the sperm collected from normoxia-exposed fish were motile and treatment with 20β-S further increased the number of hypermotile sperm (Figure 3C). A significantly lower percentage of the sperm from hypoxia-exposed fish were motile (Figure 3C). Treatment with 20β-S increased the % motile sperm of both control and hypoxia-treated fish. Similar to the effects observed on oocytes, mPRR protein expression was significantly reduced on sperm membranes from hypoxia-exposed fish (Figure 4). Reductions in sperm motility were also observed after 10 weeks exposure to 1.7 and 2.7 mg L-1 DO (SI Figures S5, S6B). Fertilization, Hatching Success and Larval Survival. Induction of gamete maturation, ovulation and spawning VOL. 43, NO. 11, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Effects of four weeks hypoxia exposure on membrane progestin receptor alpha (mPRr) protein levels on croaker sperm membranes (gel loading 10 µg) determined by Western blot analysis (A) and densitometry (B). Representative Western blot shown for samples from individual fish. Each bar represents mean ( SEM (N ) 13). Asterisk indicates significant difference from normoxic control (Student’s t-test, p < 0.05). M, marker.

FIGURE 3. Effects of four weeks hypoxia exposure on gonadal growth and the progestin hormone status of male croaker and sperm motility assessed in an in vitro bioassay compared to controls. (A) testicular development expressed as gonadosomatic index (GSI %), (mean ( SEM, N ) 13). (B) plasma 20β-S levels (mean ( SEM, N ) 10-12). (C) percent sperm motility in vitro. Sperm were pretreated with the predilutant buffer, alone and 20β-S (20 nM) for 10 min prior to the addition of activating solution as described previously (15). Percent motile sperm was assessed 10 s after activation. All treatments were tested in triplicate. Each bar represents mean ( SEM (N ) 6-8). Asterisk indicates significant difference from normoxic control (Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001). CTL, control. with an injection of LHRHa resulted in a high percentage of eggs that were fertilized for all control fish exposed to normoxic conditions (mean % fertilization: 82.8 ( 11.5, N ) 7, Figure 5A). In contrast fertilization success was markedly reduced in eggs from donors that had been chronically exposed to hypoxia (mean % fertilization: 2.7 mg L-1 DO, 10.7 ( 2.6; 1.7 mg L-1 DO, 10.4 ( 6.0, N ) 7-10, p < 0.05). Moreover, the hatching rate of the fertilized eggs from hypoxia-exposed fish was only 15-22% that of the controls (Figure 5B), so that only 1.4-2.4% of the spawned eggs from these treatment groups hatched compared to 59% of the eggs from the controls. Survival of larvae from the hypoxiaexposed fish continued to decline after hatching so that by 48 h post hatch survival was 10% or less that of the controls (Figure 5C).

Discussion The present results provide the first demonstration that exposure to hypoxia can decrease nongenomic steroid signaling leading to impaired reproductive function in vertebrates. Impaired maturation of gametes from both male and female hypoxia-exposed croaker was associated with declines in nongenomic progestin signaling. This final stage of the reproductive cycle is induced by elevated circulating 4178

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FIGURE 5. Effects of 10 weeks hypoxia exposure on (A) fertilization rate (mean ( SEM, N ) 7-10), (B) hatching rate, and (C) larval survival rate in croaker. Significant differences identified with a multiple range test, Fisher’s PLSD, are indicated with different letters (p < 0.05). DO, dissolved oxygen. levels of progestin hormones, which in turn are regulated by a surge in luteinizing hormone secretion (1, 30). Progestins regulate meiotic maturation of oocytes and sperm hypermotility by nongenomic mechanisms through activation of the novel seven-transmembrane progestin receptor, mPRR (13, 14). The results show that plasma levels of the progestin hormone in croaker, 20β-S, and gamete expression of the mPRR protein were decreased in both sexes after hypoxia exposure. Recent studies indicate that expression of mPRR

on fish oocyte and sperm membranes needs to be above critical threshold levels in order for them to respond to progestin hormones and undergo maturation. For example, the development of oocyte maturational competence (i.e., the ability of oocytes to respond to progestins and complete oocyte maturation) is associated with a several-fold increase in mPRR concentrations during the initial phase of oocyte maturation (12, 13). In sperm, a clear relationship has been established between the abundance of mPRR and sperm motility for fish as well as in humans (14, 20). In addition, there is evidence that both of these sperm parameters are positively correlated with fertilization success in croaker (14). Thus it is not surprising that fertilization and overall reproductive success were significantly impaired in the present study. Taken together, these results indicate that gamete maturation and fertilization in croaker are particularly susceptible to interference by hypoxia exposure. Hypoxia appears to interfere with nongenomic progestin signaling in fish oocytes and sperm through different mechanisms to those reported previously for a variety of xenobiotic chemicals. Evidence has been obtained for direct competition by several environmental estrogens with progestin binding to the receptor, resulting in antagonism of progestin induction of maturation in vitro of oocytes and sperm from croaker and a closely related species, spotted seatrout (15, 16, 31). However, a major effect of hypoxia on progestin signaling in the current study was to cause decreases in the concentrations of 20β-S in the blood and mPRR in the gametes, both of which are regulated by LH. We have previously shown hypoxia causes a reduction in plasma LH levels in this species through inhibition of the hypothalamic serotonergic neuroendocrine stimulatory pathway controlling LH secretion (23). Impairment of this neuroendocrine system, in addition to attenuating progestin signaling, causes a decrease in estrogen signaling and slows oocyte growth (23). The smaller size of the follicle-enclosed oocytes from some of the hypoxia-exposed fish most likely also partially accounts for their reduced responsiveness to hormonal stimulation and the low rate of fertilization success. No information is currently available on the effects of hypoxia exposure on progestin signaling during gamete maturation in any other teleost species. However, decreased progestin signaling and a reduction in gonadotropin secretion have been observed in mice exposed to hypoxia at the time of implantation and in hypoxia-exposed term trophoblasts (32, 33). Also the hypoxia-induced decrease in LH secretion in croaker has been confirmed in a second fish species, common carp, around the time of spawning (26). Reductions in oocyte maturation, sperm motility and egg production have been reported in several species of fish chronically exposed to hypoxia (25-27). We propose, therefore, that the widespread impairment of gamete maturation in hypoxiaexposed fish is due, at least partly, to decreases in LH secretion and gonadal progestin production and signaling. In support of this, there is clear evidence that the adverse effects of hypoxia earlier in the reproductive cycle on gametogenesis in fish are associated with declines in LH secretion, and gonadal sex steroid production and signaling (23-27). Thus it is likely that impairment of sex steroid signaling during both early gametogenesis and gamete maturation is partly mediated by hypoxia-induced inhibition of the same neuroendocrine pathway. Progestin signaling after hypoxia exposure is being investigated in other teleost species and neuroendocrine function is being evaluated during gamete maturation to test this hypothesis. Longer term exposure of carp to more severe hypoxia (6-12 weeks exposure to 1 mg L-1 DO) caused greater impairment of sperm motility than that observed in croaker (27), whereas oocyte maturation in vivo was inhibited to the same extent as that seen with croaker oocytes in vitro (26).

The effects of hypoxia on oocyte maturation in vivo and natural spawning of croaker may be more severe than that observed in the present study in which impairment of the neuroendocrine reproductive system was completely or partially bypassed by the treatments with gonadotropin and LHRHa, respectively. Moreover both the in vitro oocyte maturation bioassay and the spawning trials were conducted under normoxic conditions. The finding that no natural ovulation or spawning occurred in another teleost species, carp, continuously exposed to hypoxia (26) is consistent with this suggestion. The long-term impacts on fishery resources of the recent marked increase in coastal hypoxia throughout the world are unknown. Atlantic croaker, like many other coastal fishes, remain in the estuaries or inshore coastal regions until gonadal growth and gametogenesis have been completed, and subsequently migrate further offshore to spawn. Clear evidence was obtained from our previous studies that this initial hormone-dependent phase of gonadal and gamete development in croaker is susceptible to interference by hypoxia. Marked decreases in endocrine function and gametogenesis were observed in croaker environmentally exposed to hypoxia in several northeastern Gulf of Mexico estuaries and under controlled hypoxia conditions in the laboratory (23, 24). Preliminary results show similar impairment of endocrine function and gametogenesis in croaker collected from hypoxic sites off the Louisiana coast in the “dead zone”, an extensive hypoxic region which covers 9000-16 000 km2 during summer months in the northern Gulf of Mexico (34). Several modeling approaches are being used to predict the long-term effects of this reproductive impairment on egg production (fecundity) and croaker population abundance (35, 36). Physiological modeling of endocrine function during gametogenesis in female croaker has shown a close correlation between the effects of environmental exposure to hypoxia on vitellogenesis (yolk production) and the production of fully grown oocytes (fecundity) capable of undergoing maturation and fertilization (35). In addition, matrix projection models are being used to simulate and predict the long-term effects on population abundance of persistent reductions in croaker fecundity as a result of exposure to hypoxia and other environmental stressors (36). The results of the present laboratory study demonstrate that hypoxia exposure also disrupts the final phase of the croaker reproductive cycle resulting in reduced reproductive success and larval survival. If a similar disruption of gamete maturation and fertilization occurs in croaker environmentally exposed to hypoxia, this could lead to a decrease in the reproductive success of croaker on their spawning sites with potential serious long-term impacts on recruitment and population abundance. Accurate predictions of the population responses of croaker and other marine fishes to coastal hypoxia in the northern Gulf of Mexico dead zone will be required for effective coastal fisheries management in this region. Therefore, future field studies on the reproductive effects of hypoxia on croaker in the northern Gulf of Mexico as well as population modeling should also consider its possible impacts on gamete maturation, fertilization and hatching success.

Acknowledgments This research was supported by grants from the Environmental Protection Agency’s Science to Achieve Results (STAR) Estuaries and Great Lakes (EaGLe) program through the Consortium for Ecosystem Research for the Gulf of Mexico (CEER-GOM) grant no. R-82945801 (to P.T.) and from The National Oceanic and Atmospheric Administration Coastal Ocean Program Gulf of Mexico GOMEX grant no. NA06NOS4780131 (to P.T). The assistance of James Kummer with the hormone analyses and VOL. 43, NO. 11, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Susan Lawson with the fish care and maintenance of the DO conditions in the experimental tanks are greatly appreciated.

Supporting Information Available Photographs and schematic diagram of experimental setup (Figure S1), average weekly physio-chemical parameters of four weeks (Figure S2) and ten weeks (Figure S3) experimental periods, percent oocyte GVBD, sperm motility, and plasma 20β-S levels after 10 weeks exposure (Figures S4-S6). This material is available free of charge via the Internet at http:// pubs.acs.org.

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