Effects of progesterone on sperm motility in fathead minnow (Pimephales promelas)

Aquatic Toxicology 104 (2011) 121–125

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Effects of progesterone on sperm motility in fathead minnow (Pimephales promelas) Patrick J. Murack a , John Parrish b , Terence P. Barry b,∗ a b

University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706, USA University of Wisconsin-Madison, Department of Animal Sciences, 1675 Observatory Drive, Madison, WI 53706, USA

a r t i c l e

i n f o

Article history: Received 31 January 2011 Received in revised form 30 March 2011 Accepted 9 April 2011 Keywords: Progesterone Steroid Reproduction Fish Fathead minnow Sperm motility CASA CAFO Endocrine disruption

a b s t r a c t The steroid hormone progesterone (P4) is found at relatively high concentrations (∼300 ng/L) in association with concentrated animal feeding operations (CAFOs). In an effort to better understand the potential endocrine disrupting effects of P4 in male fish, computer assisted sperm analysis (CASA) was used to evaluate the effects of this steroid on sperm motility in the fathead minnow (Pimephales promelas). The rationale for focusing on sperm motility is that certain progestins have been shown to bind to surface membrane receptors on fish spermatozoa and increase sperm swimming velocity. It was hypothesized, therefore, that sperm swimming velocity might be a useful indicator of progestin exposure in fish. Adult male fathead minnows (ages 6–12 months) were exposed to environmentally relevant doses of P4, both longer-term (1 week, in vivo exposure) and short-term (minutes, in vitro exposure). Sperm were then video recorded and analyzed by CASA. When fathead minnows were continuously exposed for 1 week to low levels of progesterone in vivo there was a significant dose-dependent reduction in sperm motility. There was no effect of short-term P4 exposure on fathead minnow sperm swimming characteristics. Additional research is required to elucidate the mechanism by which progesterone alters sperm swimming in the fathead minnow. With further validation, the fathead minnow sperm motility assay may be a useful tool to rapidly screen for endocrine disrupting chemicals in the aquatic environment. © 2011 Elsevier B.V. All rights reserved.

1. Introduction More than 500 million tons of animal manure is produced annually in the U.S. from concentrated animal feeding operations (CAFOs). Both natural and synthetic steroids and their metabolites have been detected in runoff and soil samples from CAFOs (Durhan et al., 2005; Lange et al., 2002; Schiffer et al., 2001; Soto et al., 2004). In a recent survey of the steroid hormones associated with CAFOs in southern Wisconsin, researchers from the Wisconsin State Laboratory of Hygiene detected progesterone (P4) at concentrations as high as 350 ng/L in spring runoff (unpublished). This finding is compatible with reports that progestins can be excreted at high concentrations from hormone-implanted cattle, and that this class of steroid hormone can persist in the soil for several months (Schiffer et al., 2001).

Abbreviations: P4, progesterone; CASA, computer assisted sperm analysis; VSL, straight line velocity; VCL, curvilinear velocity; VAP, average path velocity; PMOT, percent motility; PLIN, percent linearity; CAFO, concentrated animal feeding operation. ∗ Corresponding author. Tel.: +1 608 262 6450; fax: +1 608 262 0454. E-mail address: [email protected] (T.P. Barry). 0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.04.006

Compared to androgens and estrogens, very little is known about the role of P4 as an endocrine disrupting chemical (EDC) in fish (Scott et al., 2010). Progestins mediate the process of final gamete maturation (spermiation) in male fish (Scott et al., 2010), and in some fish species progestins can bind to surface membrane receptors on mature spermatozoa and directly stimulate an increase in sperm motility (Thomas et al., 2004; Tubbs and Thomas, 2008, 2009). Thus, it was hypothesized that exposing fish to P4 from CAFO effluent could have a significant impact on the development, maturation and function of fish spermatozoa. Computer assisted sperm analysis (CASA) is an effective method to quantify changes in fish sperm swimming characteristics, and has been used in a wide variety of fish species for this purpose, including zebrafish, lake sturgeon, carp, bluegill, rainbow trout, and arctic char (Burness et al., 2004; Lahnsteiner et al., 2005; Linhart et al., 1995; Rudolfsen et al., 2006; Runnalls et al., 2007; Toth et al., 1995; Wilson-Leedy and Ingermann, 2007). To our knowledge, there have been no investigations on the impact of P4 on sperm swimming motility in fish. The present study was conducted to evaluate the effects of P4 on spermatozoa swimming characteristics in male fathead minnows – a fish species widely used in ecotoxicological investigations. The primary objectives were to (1) optimize a CASA protocol for use with fathead minnows based on a protocol originally developed

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for zebrafish (Wilson-Leedy and Ingermann, 2006), and (2) use this optimized fathead minnow CASA procedure to evaluate the dose-dependent effects of long-term in vivo (7 days) exposure to environmentally relevant concentrations of P4 on fathead minnow sperm motility.

2. Materials and methods 2.1. Chemicals and stock solutions The progesterone was purchased from Steraloids, Inc. (Newport, RI). All other chemicals were of the highest purity and purchased from Sigma Chemical Company (St. Louis, MO). Concentrated steroid stock solutions were prepared in 100% ethanol and diluted as required in distilled water. Ethanol concentrations in the final test solutions were less than or equal to 0.04%. Semen extender was prepared according to the method of Runnalls et al. (2007).

2.2. Fish Reproductively mature male fathead minnows (6–12 months of age) were housed in 25-gallon tanks with flow-through water at 24–26 ◦ C. The photoperiod was 16L:8D. Light intensity was ambient laboratory level (540–1080 lux). The fish were fed live and frozen artemia supplemented with formulated diet (flake feed and 45% protein salmon starter diet). Feeding frequencies and levels were adjusted as the fish grew (Denny, 1987; Parrott et al., 2006).

2.3. Semen preparation Males were sacrificed and their testes surgically removed. Each testis was placed into a 1.5 ml centrifuge tube containing 1 ml of semen extender. The tube was incubated in a water bath at 24 ± 2 ◦ C for 45 min after which time the testis was diced within the tube using a scalpel. The minced tissue preparation was vortexed for 2 s to release the mature spermatozoa. The tube was inverted several times to mix the semen and extender. Extended sperm preparations were used within 5 h of sacrificing the male.

2.4. Sperm activation and video recording Ten microliters of the sperm mixture was pipetted into a 12 mm × 75 mm test-tube. Forty microliters of water was then added to a test tube and vortexed for 2 s to activate the sperm. Some tubes received a 0.8 mM NaCl solution that prevents fathead minnow sperm from becoming motile (Wilson-Leedy et al., 2009). This saline treatment served as a positive control. Two microliters of the activated sperm mixture was pipetted onto a multiwell microscope slide, and covered with a glass cover slip. The sperm were observed using a Nikon (Melville, NY) Microphot-FX microscope (200× magnification) equipped with a Hamamatsu (Bridgewater, NJ) CCD video camera. There was initially considerable drift as the sperm mixture was drawn by capillary action under the cover slip. This interfered with the CASA analysis of sperm motility. Various methods were tried to reduce drifting including using lesser volume and slower pipetting rates, but these did not alleviate the problem. Therefore, the video recordings were initiated 80 s after sperm activation, a time when drift had completely subsided. Video images were recorded for 15 s (i.e., video was recorded between 80 and 95 s after sperm activation). The video recordings were saved onto a hard drive and later analyzed using CASA according to Kime et al. (2001).

Table 1 Input parameters for Image J adapted for fathead minnows. Input parameters

Input values

A. Minimum sperm size (pixels) B. Maximum sperm size (pixels) C. Minimum track length (frames) D. Maximum sperm velocity between frames (pixels) E. Minimum VSL for motile (␮m/s) F. Minimum VAP for motile (␮m/s) G. Minimum VCL for motile (␮m/s) H. Low VAP speed (␮m/s) I. Maximum percentage of path with zero VAP J. Maximum percentage of path with low VAP K. Low VAP speed 2 (␮m/s) L. Low VCL speed (␮m/s) M. High WOB (percent VAP/VCL) N. High LIN (percent VSL/VAP) O. High WOB two (percent VAP/VCL) P. High LIN two (percent VSL/VAP) Q. Frame rate (frames per second) R. Microns per 1000 pixels S. Print xy co-ordinates for all tracked sperm? T. Print motion characteristics for all motile sperm? U. Print median values for motion characteristics

5 50 16 20 3 5 15 5 1.0 25 10 15 80 80 200 200 16 2930 0 1 0

2.5. Analysis of sperm swimming parameters The sperm were analyzed using the software program “Computer Assisted Sperm Analysis” (CASA), which is a freeware plug-in of the program Image J (Wilson-Leedy and Ingermann, 2006). The program can be downloaded at http://rsb.info.nih.gov/ij/. The CASA program tracks sperm individually and quantifies specific swimming characteristics including: percent motility (PMOT), curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), and percent linearity (PLIN). These parameters are defined in Wilson-Leedy and Ingermann (2007). In previous investigations, all of these parameters have been shown to correlate with fish fertilization success (Rurangwa et al., 2001). The CASA input parameters used in the present investigation are shown in Table 1, and were based on those published for zebrafish spermatozoa by Wilson-Leedy and Ingermann (2006). These parameters can be used in all future studies using CASA to evaluate fathead minnow sperm motility, although specific parameters (A, B, D, Q and R) will require adjustment depending on what microscope and video recording equipment is used by different investigators. For example, in the current study, the video recording frame rate (Q) was 16 frames per second, and this value would need to be changed for video recordings made at a different frame rate. The input parameters A, B, D and R also need to be adjusted for different microscope magnification levels as these parameters are based on pixel number, not actual sperm size. All of the other CASA input values listed in Table 1 specify characteristics of fathead minnow sperm and do not need to be changed. CASA uses two selection criteria to identify motile sperm. First, the program identifies slow moving sperm by ensuring that they meet minimum VSL, VCL and VAP requirements. Second, all sperm that pass the first screen are further analyzed to determine if they are moving autonomously or due to bulk water flow or drift. This is accomplished by ensuring that the sperm meet at least one of the following conditions: (1) they are not moving in a perfectly straight line (values less than the set points for parameters M, N, J and I), (2) they are moving faster than drifting sperm (values more than set points for parameters L and K), or (3) they are moving with a high degree of path curvature (values less than set points for parameters O and P). See Wilson-Leedy and Ingermann (2006) for a complete description. Sperm motility and sperm curvilinear velocity both increased with the addition of 0.8 mM NaCl solution. These results were sim-

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ilar to those previously reported by Wilson-Leedy et al. (2009). In the salt trials, the video recordings were started 50 s after activation (rather than 80 s) so that there was still some drift to contend with for validation purposes, and also so that the data would be directly comparable with previously published literature recorded at the same time (Wilson-Leedy et al., 2009). In addition, the video recordings were made at 15 s intervals so that other sperm tracking software besides CASA could also use our protocol. In this regard, another widely used CASA program called the Hobson Sperm Tracker can only analyze 15 s of video. Recording lasted until the sperm were no longer moving (∼185 s). 2.6. Long-term exposure to P4 in vivo A flow-through exposure system similar to that described by Ankley et al. (1997) was used in this study. A 4-channel, model 07523-90 peristaltic pump (Cole Parmer, Vernon Hills, IL) delivered the control (ethanol vehicle) and two P4 stock solutions through size L/S 13 Masterflex® Norprene® tubing (Cole Parmer, Vernon Hills, IL) to three glass dilution chambers at a rate of 0.5 ml/min. Heated (25 ± 1◦ C), carbon-filtered City of Madison, WI water was added to the dilution chambers at a flow rate of 1.5 L/min which diluted the P4 stock solutions to the target P4 delivery concentrations of 0, 30, and 300 ng/L. The water then flowed from each dilution chamber to seven replicate 8-L exposure tanks (N = 7, 21 total tanks were used in the study). The target P4 concentrations were confirmed by measuring the P4 concentrations in three random exposure tanks from each treatment by HPLC–MS/MS (Wisconsin State Laboratory of Hygiene, ESS Organic Chemistry Method 1690). The P4 levels in nominal 0, 30, 300 ng/L tanks were 0 ± 0, 25.6 ± 7.1, 339.3 ± 23.3 (mean ± SEM, N = 3). Ethanol vehicle concentrations in the tanks were 0.00005% (v/v). The flow rate into each exposure tank was 250 ml/min (2.5 tank turnovers per hour). Each dilution chamber and all aquaria in the system were constantly aerated. The fish used in the in vivo exposure study were approximately 12 months in age. Male and female fathead minnows were “paired” (one male and two females) in the aquaria and allowed to acclimate for 1 week before exposure. The males were sacrificed at the end of the exposure with MS-222, and semen samples were prepared from each of the 21 males as described above. Twenty microliters of extended sperm was mixed in a test tube with 10 ␮l of distilled water, and 2 ␮l of the mixture was added to a slide and sperm motility data collected and analyzed as described above.

Fig. 1. The effect of 0.8 mM NaCl (NaCl) on the curlinear velocity (VCL) of fathead minnow sperm. Time after sperm activation is shown on the x-axis.

data between fish used in the two experiments are not directly comparable. 2.8. Statistical analysis The data were subjected to least squares mean analysis. Data points outside of three interquartile ranges were considered outliers (<5% of the data points). The data from the in vivo P4 exposure was analyzed by ANOVA and differences among treatment groups were compared using the least squares difference test at p = 0.05. A t-test was performed on the data from the in vitro study with the p-value set at 0.05. 3. Results 3.1. Effects of NaCl The sperm from male fathead minnows exposed in vitro to 0.8 mM NaCl had significantly higher VCL after 117.5 s than sperm from males activated with distilled water (Fig. 1). 3.2. In vivo P4 exposure

2.7. Short-term exposure to P4 in vitro The fish used in the short-term in vitro study were approximately 6 months in age. One male (58–72 mm) and one female were paired in a 2.5-gallon tank. The female was included to promote reproductive development and behavior in the male. After a one-day acclimation period with the female, the male was injected intraperitoneal with 100 IU of human chorionic gonadotropin (hCG) to induce spermiation, and returned to the tank with the female for 16–22 h. Ten microliters of sperm mixture was pipetted into a 12 mm × 75 mm test tube as described above. Forty microliters of the appropriate P4 stock solution (final P4 concentrations of 0, 1, 10 or 100 nM) was then added to a test tube and vortexed for 2 s to activate the sperm. The 80 s between the time the P4 was added to the sperm and the initiation of the video recording served as the P4 exposure duration (Thomas et al., 1997, 2004; Tubbs and Thomas, 2008). Because the fish used in the short-term exposure study were 6 months younger than the fish used in the long-term exposure study, the baseline spermatozoa swimming

The sperm from male fathead minnows exposed for 1 week to 300 nM of P4 had significantly lower VCL (curvlinear velocity), VAP (velocity average path), and VSL (straight line velocity) compared to sperm from control fish (Fig. 2). Exposure to 30 nM P4 had no effect on any of the measured sperm swimming parameters compared to controls (Fig. 2). 3.3. In vitro P4 exposure There were no significant differences in any of the measured swimming characteristics between control sperm and sperm exposed to P4 in vitro (Table 2). 4. Discussion A sperm motility assay using the freeware CASA program developed by Wilson-Leedy and Ingermann (2007) was modified for measuring sperm swimming parameters in the fathead minnow. As observed in previous studies with this and other small fish species

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Fig. 2. The effect of a one-week exposure to progesterone on fathead sperm motility examining PMOT (percent motility, %), PLIN (percent linearity, %), VCL (velocity curvlinear), VAP (velocity average path), and VSL (velocity straight line). Asterisks above bars designate a significant difference (p < 0.05) between the P4-exposed sperm and the control sperm. N = 4 for VCL, VAP, and VSL. Error bars represent standard error of mean (SEM). The fish used in this experiment were approximately 12 months of age.

Table 2 Effect of short-term in vitro exposure to P4 on the motility of fathead minnow sperm (N = 7 or 8).a Conc. (nM)

PMOT

0 1 10 100

57 53 54 53

± ± ± ±

6 7 7 7

VCL 66.9 78. 73.6 74.4

VAP ± ± ± ±

5.1 5.3 5.3 6.9

64.0 75.3 69.7 70.5

VSL ± ± ± ±

4.9 5.5 5.1 6.9

47.8 53.7 51.6 53.6

PLIN ± ± ± ±

4.7 4.3 4.6 5.2

74 71 74 77

± ± ± ±

3 2 3 3

a See text and legend of Fig. 2 for definitions of these five parameters. No significant differences due to progesterone were detected at p > 0.05. The fish used in this experiment were approximately 6 months of age.

(e.g., zebrafish), sperm swimming characteristics changed in time, and in response to elevated osmolality (Kime et al., 2001; Rurangwa et al., 2004; Wilson-Leedy et al., 2009). In contrast to our results, Hala et al. (2009) did not observe a change in VCL in fathead minnow spermatozoa with time. One explanation for this difference could be the age of the fish used in the respective studies. It has been shown that sperm from older fish have less robust swimming characteristics than sperm from fish in their reproductive prime (Kidd et al., 2001). Indeed, in the present investigation, the difference in sperm motility between the fish used in the in vitro and in vivo studies is probably best explained by fish age. The younger fish (in vivo ∼6 months) had more motile sperm than the older fish (in vitro ∼12 months). Optimal results were obtained when video recording was initiated 80 s after sperm activation when all sperm are still swimming robustly, but drift has completely subsided which permits detection of subtle changes in sperm swimming activity. This 80 s period was also likely a sufficient time for the P4 to initiate a rapid cellular response during the in vitro study based on evidence that the progestin 20␤-S binds to a surface membrane receptor on the sperm head of the Atlantic croaker and initiates an increase in sperm motility in less than 60 s (Thomas et al., 2004; Tubbs and Thomas, 2009). Two progestins, 17␣,20␤-P and 20␤-S, are important fish steroids that regulate the processes of final spermatozoa matu-

ration and spermiation (Scott et al., 2010). Although P4 itself is not typically considered a fish steroid, it could be a precursor in the biosynthesis of these other, more potent progestins. Thus, it was postulated that short-term exposure of the fathead minnow sperm to P4 might activate sperm swimming activity, either by binding directly to a sperm progestin receptor, or by being converted by enzymes present in the spermatozoa into a more active progestin. High levels of steroid-metabolizing enzymes, including hydroxysteroid dehydrogenases capable of such transformations, are present in mature fish spermatozoa (Asahina et al., 1990; Miura et al., 1991). The observation that there was no effect of short-term P4 exposure on sperm swimming suggests that (1) there is no progestin receptor on the fathead minnow sperm capable of binding P4, (2) there are no enzymes present in the spermatozoa that convert P4 into a more active steroid, (3) P4 is not a suitable steroid substrate for enzymes that might be present, or (4) there are no fast-acting steroid receptors present in fathead minnow sperm that mediate changes in sperm swimming ability (see Krietsch et al., 2006; Zhu et al., 2006). A key finding of the present investigation was that several sperm swimming characteristics were altered in fathead minnows exposed for 1 week to environmentally relevant concentrations of P4. Inasmuch as a reduction in sperm motility is one of the best predictors of decreased fertilization rates in fish (Rurangwa et al., 2001), these results suggest that P4 could be an EDC and have an important impact on reproduction in exposed feral fish populations. The mechanism by which P4 affected sperm swimming is not known, although it is possible that P4 itself, or a P4 conversion product such as 17,20-P, cortisol or estradiol-17␤ (E2 ), acted on one or more levels of the hypothalamic–pituitary–gonadal axis (HPG axis) to modify sperm development and/or maturation. Progesterone is not considered to be an active fish steroid, and therefore it is more likely that a P4 byproduct mediated the observed effects on sperm motility. The hypothesis that P4 was converted into cortisol or E2 is supported by the data showing a reduction in sperm motility, as both of these steroids have previously been shown to have inhibitory effects on sperm motility in fish (Pawlowski et al., 2004). The maturational steroids (e.g., 17,20-P) would be expected to have positive effects on sperm motility (Scott et al., 2010). Progesterone was likely absorbed across the gills of the exposed fish and could potentially bioaccumulate (Miguel-Queralt and Hammond, 2008). If bioaccumulation of P4 does occur, there is a possibility that even very low environmental concentrations of P4 could have an impact on sperm motility with longer exposure durations. Future studies with longer exposure times, and measurements of the circulating levels of various steroid hormones in the blood of P4-exposed males will help clarify the mechanism of action. In summary, a CASA protocol was developed to evaluate the effects of P4 exposure on spermatozoa motility in the fathead minnow. A one-week exposure of male fathead minnows to a nominal concentration of 300 ng/L P4 was found to reduce sperm motility. The data suggest that this steroid hormone that is found at these concentrations in association with CAFOs and sewage treatment plants may be an important EDC. The fathead minnow sperm motility assay is rapid and relatively simple to perform. Thus, with further development and validation, this assay could potentially become an important tool that can be used in conjunction with, or perhaps even replace, the time-consuming fish reproduction assays currently approved for screening potential EDCs in the aquatic environment. Acknowledgements The authors would like to thank members of the Laboratory of Fish Endocrinology and Aquaculture at the University of Wisconsin-

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