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Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus)
MARINE ENVIRONMENTAL RESEARCH Marine Environmental Research 58 (2004) 547–551 www.elsevier.com/locate/marenvrev
Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus) Iris Knoebl a, Michael J. Hemmer b, Nancy D. Denslow a
a,c,*
Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610, USA b US Environmental Protection Agency, Gulf Ecology Division, Gulf Breeze, FL 32561, USA c Department of Biochemistry and Molecular Biology, University of Florida, P.O. Box 100156, Gainesville, FL 32610, USA
Abstract Several genes normally induced by estradiol (E2 ) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E2 and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E2 and NP elicited a doseresponse increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Vitellogenin; Zona radiata protein; Estradiol; Nonylphenol; Real time PCR
1. Introduction In adult female fish, the native estrogen, 17b-estradiol (E2 ) regulates gene expression of mRNAs in the liver that encode vitellogenin (VTG), the egg yolk protein precursor, and several egg membrane proteins (zona radiata, ZRP). Male fish do not normally express these genes to a significant level, however when exposed to some
*
Corresponding author. Tel.: +1-352-392-9665; fax: +1-352-392-4441. E-mail address: [email protected]fl.edu (N.D. Denslow).
0141-1136/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2004.03.043
548
I. Knoebl et al. / Marine Environmental Research 58 (2004) 547–551
estrogenic chemicals, plasma VTG levels can exceed levels measured in females (Folmar et al., 2000). It is now clear that fish have multiple VTGs (LaFleur, Byrne, Haux, Greenberg, & Wallace, 1995) and ZRPs (Arukwe & Goksøyr, 2003) that are differentially controlled by E2 . The purpose of this study was to evaluate the comparative induction of these genes at the mRNA level using real time PCR, in fish exposed to several aqueous concentrations of E2 and NP for several days.
2. Materials and methods Adult sheepshead minnow (Cyprinodon variegatus) (SHM) males (between 50–65 cm and 2–5 g) were treated by aquatic exposure as described previously (Hemmer et al., 2001) to either E2 or NP. Measured concentrations of E2 (58, 93, 182 and 527 ng/ l) and NP (0.64, 5, 12, 23 and 43 lg/l) were tested. The fish (N ¼ 4) exposed to E2 were sampled after 2, 5 and 8 days, and fish exposed to NP after 2 and 13 days. Quantitative real-time PCR was used to measure mRNA levels of VTG1, VTG2, ZRP2 and ZRP3. Total RNA was isolated from livers using RNA-STAT 60 Total RNA/mRNA isolation reagent (Tel-Test, Inc., Friendswood, TX) following the manufacturer’s protocol and genomic DNA was removed using a kit (DNA-Free, Ambion, Inc., Austin, TX) following the manufacturer’s protocol. cDNA was prepared from 5 lg total RNA in a 50 ll reaction volume using Stratascript (Stratagene, Inc., La Jolla, CA) reverse transcriptase following the manufacturer’s protocol. The gene sequences were analyzed by Primer Express software (PE Applied Biosystems, Foster City, CA) to select forward and reverse primers and fluorogenic probes. The forward (F) and reverse (R) primers and probes (P) are as follows (50 – 30 ): VTG1 (Accession # AF239720) F ¼ ATGTCACTGTGAAGGTCAACGAA, R ¼ ACCTGTTGGGTGGCGGTAA, P ¼ TGGAAATTCCCCACAGCAACCTGC (amplicon length ¼ 68), VTG2 (Accession # AF239721) F ¼ TGGACTGGATGAGAGGCCAAA, R ¼ GTGCTGTACTCCTGCCTGACTTC, P ¼ TGTGGACT CTGTGGAAAGGCTGACGG (amplicon length ¼ 72), ZRP2 (Accession # AY598615) F ¼ TTTTCCACAGTGGGACTTACTTATTG, R ¼ CTACAGGGAG GAGTCGTGTTCTG, P ¼ TGGTTGCCCATATGTTGATGACCGC (amplicon length ¼ 76), ZRP3 (Accession # AY598616) F ¼ TTCATTAATAACTTTGGGTGTTTCGTT, R ¼ ACTTTCAGAGTTTTGGAACCTGAAG, P ¼ CTGACTCAAAGTTCCTGGCTCGCACTG (amplicon length ¼ 128). The fluorogenic probes were labeled at the 50 end with the reporter dye FAM (6-carboxyl-fluorescein) and at the 30 end with the quencher dye TAMRA (6-carboxytetramethyl-rhodamine). The selected primers and probe were purchased from ABI Prism (PE Applied Biosystems, Foster City, CA). Amplification of each gene product was performed in duplicate using a kit (Brilliant core reagents, Stratagene, Inc.) according to the manufacturer’s protocol, in the ABI PRISM 5700 (Applied Biosystems, Inc.). Each 25 ll reaction contained 0.1 lg cDNA. A standard curve of known molar quantities of each plasmid was used to calibrate the Q-PCR data and the values interpolated from the curve were converted to pg mRNA/lg total RNA for each sample. All samples were normalized to 18S ribosomal RNA. The 18S ribosomal primers and a labeled probe
I. Knoebl et al. / Marine Environmental Research 58 (2004) 547–551
549
were purchased as a kit (PE Applied Biosystems, Foster City, CA). The 18S values for all samples was highly reproducible with mean Ct values of 16.83 (0.73 SD).
3. Results and discussion Exposure of male SHM to E2 and NP induced the expression of both ZRPs and both VTGs, genes normally induced only in female fish. These results indicate that all four genes are biomarkers of exposure to estrogens in SHM and are in agreement with studies in other fish species (Arukwe & Goksøyr, 2003). No clear differences in induction sensitivity were seen among the VTGs and the ZRPs by exposure to either E2 or NP, but differences in magnitude of induction were observed. Exposure to increasing doses of E2 induced expression of both VTGs and both ZRPs (Fig. 1). VTG1 was the most highly induced, reaching a concentration of 1001 pg mRNA/lg total RNA after eight days at 182 ng/l E2 . The other genes increased to lower levels, reaching maximum concentrations of 43 pg mRNA/lg total RNA for VTG2, 71 pg mRNA/lg total RNA for ZRP2 and 362 pg mRNA/ug total RNA for ZRP3. The increase in ZRP3 mRNA from 182 to 527 ng/l between days 5 and 8 is not statistically significant. These data suggest that by five days of constant exposure, the ER mediated pathway becomes saturated with an exposure to at least 182 ng/l E2 . Exposure to NP also stimulates induction of all four genes tested (Fig. 2). As with the E2 exposures, VTG1 was the most highly expressed gene, increasing to 2088 pg VTG2
VTG1
pg mRNA/ ug Total RNA
1000
500
75
pg mRNA/ ug Total RNA
Day 2 Day 5 Day 8
1500
Day 2 Day 5 Day 8
50
25
0
0
0
100
200
300
400
500
0
600
100
200
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600
ZRP3
ZRP2 Day 2 Day 5 Day 8
80 60 40 20
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pg mRNA/ ug Total RNA
100
pg mRNA/ ug Total RNA
300
E2 ng/L
E2 ng/L
Day 2 Day 5 Day 8
400 300 200 100
0
0
0
100
200
300
E2 ng/L
400
500
600
0
100
200
300
400
500
600
E2 ng/L
Fig. 1. Dose-response increase in VTG and ZRP mRNAs, as measured by real time PCR, in male sheepshead minnows exposed to four concentrations of estradiol (E2 ). Bars represent standard errors.
550
I. Knoebl et al. / Marine Environmental Research 58 (2004) 547–551 VTG1
VTG2 100
Day 2 Day 13
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pg mRNA/ ug Total RNA
pg mRNA/ ug Total RNA
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ug/L NP
ZRP2
ZRP3 Day 2 Day 13
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0
0
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ug/L NP
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50
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Fig. 2. Dose-response increase in VTG and ZRP mRNAs, as measured by real time PCR, in male sheepshead minnows exposed to four concentrations of para-nonylphenol (NP). Bars represent standard errors.
mRNA/lg total RNA at the highest concentration and time. As in the E2 exposures, ZRP3 (637 pg mRNA/lg total RNA) was induced more than ZRP2 or VTG2 (54 and 62 pg mRNA/lg total RNA, respectively). These data suggest that a constant exposure to a concentration of 12 lg/l NP for two days or 5 lg/l for 13 days is sufficient to induce the ER-mediated pathway. This is an important environmental consideration as there are locations where measured levels of NP are higher than 15 lg/l (Petrovic, Sole, Lopez, & Barcelo, 2002). Research by others has shown mixed results. For example Yadetie, Arukwe, Goksøyr, and Male (1999), found dose-response increases for both VTG and ZRP mRNA, but no differences between VTG and ZRP mRNA levels in Atlantic salmon exposed to NP. In work with rainbow trout injected with E2 , Celius, Matthews, Giesy, and Zacharewski (2000), reported that ZRP mRNA was more responsive to low levels of E2 than VTG mRNA. While our results do not indicate a difference in sensitivity at the concentrations tested between the ZRPs and VTGs, there was a clear difference in the magnitude of induction between VTG1 and VTG2 as well as between ZRP2 and ZRP3 after exposure to both E2 and NP. The SHM VTG1 and VTG2 sequences are similar to the killifish (Fundulus heteroclitus) VTG1 and VTG2 sequences, which are also expressed in a 10 to 1 ratio, respectively (LaFleur et al., 1995). The lack of difference in sensitivity between the VTGs and the ZRPs in SHM, as was observed in rainbow trout, may be due to the fact that SHM are serialspawners. As such, eggs are in constant development, whereas in seasonal spawners such as rainbow trout, they are produced only during the reproductive season.
I. Knoebl et al. / Marine Environmental Research 58 (2004) 547–551
551
Acknowledgements This study was funded by the US Environmental Protection Agency Cooperative Agreement #CR826357-10, and the Biotechnology Program, University of Florida.
References Arukwe, A., & Goksøyr, A. (2003). Comparative Hepatology, 2, 4–24. Celius, T., Matthews, J. B., Giesy, J. P., & Zacharewski, T. R. (2000). Journal of Steroid Biochemistry and Molecular Biology, 75, 109–119. Folmar, L. C., Hemmer, M., Hemmer, R., Bowman, C., Kroll, K., & Denslow, N. D. (2000). Aquatic Toxicology, 49, 77–88. Hemmer, M. J., Hemmer, B. L., Bowman, C. J., Kroll, K. J., Folmar, L. C., Marcovich, D., Hoglund, M. D., & Denslow, N. D. (2001). Environmental Toxicology and Chemistry, 20, 336–343. LaFleur, G. J., Jr, Byrne, B. M., Haux, C., Greenberg, R. M., & Wallace, R. A. (1995). International symposium on reproduction physiology of fish, Fish Symposium 95 (pp. 336–338). Austin, TX: University of Texas. Petrovic, M., Sole, M., Lopez, de Alda M. J., & Barcelo, D. (2002). Environmental Toxicology and Chemistry, 21, 2146–2156. Yadetie, F., Arukwe, A., Goksøyr, A., & Male, R. (1999). The Science of the Total Environment, 233, 301– 310.
Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus) Iris Knoebl a, Michael J. Hemmer b, Nancy D. Denslow a
a,c,*
Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610, USA b US Environmental Protection Agency, Gulf Ecology Division, Gulf Breeze, FL 32561, USA c Department of Biochemistry and Molecular Biology, University of Florida, P.O. Box 100156, Gainesville, FL 32610, USA
Abstract Several genes normally induced by estradiol (E2 ) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E2 and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E2 and NP elicited a doseresponse increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Vitellogenin; Zona radiata protein; Estradiol; Nonylphenol; Real time PCR
1. Introduction In adult female fish, the native estrogen, 17b-estradiol (E2 ) regulates gene expression of mRNAs in the liver that encode vitellogenin (VTG), the egg yolk protein precursor, and several egg membrane proteins (zona radiata, ZRP). Male fish do not normally express these genes to a significant level, however when exposed to some
*
Corresponding author. Tel.: +1-352-392-9665; fax: +1-352-392-4441. E-mail address: [email protected]fl.edu (N.D. Denslow).
0141-1136/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2004.03.043
548
I. Knoebl et al. / Marine Environmental Research 58 (2004) 547–551
estrogenic chemicals, plasma VTG levels can exceed levels measured in females (Folmar et al., 2000). It is now clear that fish have multiple VTGs (LaFleur, Byrne, Haux, Greenberg, & Wallace, 1995) and ZRPs (Arukwe & Goksøyr, 2003) that are differentially controlled by E2 . The purpose of this study was to evaluate the comparative induction of these genes at the mRNA level using real time PCR, in fish exposed to several aqueous concentrations of E2 and NP for several days.
2. Materials and methods Adult sheepshead minnow (Cyprinodon variegatus) (SHM) males (between 50–65 cm and 2–5 g) were treated by aquatic exposure as described previously (Hemmer et al., 2001) to either E2 or NP. Measured concentrations of E2 (58, 93, 182 and 527 ng/ l) and NP (0.64, 5, 12, 23 and 43 lg/l) were tested. The fish (N ¼ 4) exposed to E2 were sampled after 2, 5 and 8 days, and fish exposed to NP after 2 and 13 days. Quantitative real-time PCR was used to measure mRNA levels of VTG1, VTG2, ZRP2 and ZRP3. Total RNA was isolated from livers using RNA-STAT 60 Total RNA/mRNA isolation reagent (Tel-Test, Inc., Friendswood, TX) following the manufacturer’s protocol and genomic DNA was removed using a kit (DNA-Free, Ambion, Inc., Austin, TX) following the manufacturer’s protocol. cDNA was prepared from 5 lg total RNA in a 50 ll reaction volume using Stratascript (Stratagene, Inc., La Jolla, CA) reverse transcriptase following the manufacturer’s protocol. The gene sequences were analyzed by Primer Express software (PE Applied Biosystems, Foster City, CA) to select forward and reverse primers and fluorogenic probes. The forward (F) and reverse (R) primers and probes (P) are as follows (50 – 30 ): VTG1 (Accession # AF239720) F ¼ ATGTCACTGTGAAGGTCAACGAA, R ¼ ACCTGTTGGGTGGCGGTAA, P ¼ TGGAAATTCCCCACAGCAACCTGC (amplicon length ¼ 68), VTG2 (Accession # AF239721) F ¼ TGGACTGGATGAGAGGCCAAA, R ¼ GTGCTGTACTCCTGCCTGACTTC, P ¼ TGTGGACT CTGTGGAAAGGCTGACGG (amplicon length ¼ 72), ZRP2 (Accession # AY598615) F ¼ TTTTCCACAGTGGGACTTACTTATTG, R ¼ CTACAGGGAG GAGTCGTGTTCTG, P ¼ TGGTTGCCCATATGTTGATGACCGC (amplicon length ¼ 76), ZRP3 (Accession # AY598616) F ¼ TTCATTAATAACTTTGGGTGTTTCGTT, R ¼ ACTTTCAGAGTTTTGGAACCTGAAG, P ¼ CTGACTCAAAGTTCCTGGCTCGCACTG (amplicon length ¼ 128). The fluorogenic probes were labeled at the 50 end with the reporter dye FAM (6-carboxyl-fluorescein) and at the 30 end with the quencher dye TAMRA (6-carboxytetramethyl-rhodamine). The selected primers and probe were purchased from ABI Prism (PE Applied Biosystems, Foster City, CA). Amplification of each gene product was performed in duplicate using a kit (Brilliant core reagents, Stratagene, Inc.) according to the manufacturer’s protocol, in the ABI PRISM 5700 (Applied Biosystems, Inc.). Each 25 ll reaction contained 0.1 lg cDNA. A standard curve of known molar quantities of each plasmid was used to calibrate the Q-PCR data and the values interpolated from the curve were converted to pg mRNA/lg total RNA for each sample. All samples were normalized to 18S ribosomal RNA. The 18S ribosomal primers and a labeled probe
I. Knoebl et al. / Marine Environmental Research 58 (2004) 547–551
549
were purchased as a kit (PE Applied Biosystems, Foster City, CA). The 18S values for all samples was highly reproducible with mean Ct values of 16.83 (0.73 SD).
3. Results and discussion Exposure of male SHM to E2 and NP induced the expression of both ZRPs and both VTGs, genes normally induced only in female fish. These results indicate that all four genes are biomarkers of exposure to estrogens in SHM and are in agreement with studies in other fish species (Arukwe & Goksøyr, 2003). No clear differences in induction sensitivity were seen among the VTGs and the ZRPs by exposure to either E2 or NP, but differences in magnitude of induction were observed. Exposure to increasing doses of E2 induced expression of both VTGs and both ZRPs (Fig. 1). VTG1 was the most highly induced, reaching a concentration of 1001 pg mRNA/lg total RNA after eight days at 182 ng/l E2 . The other genes increased to lower levels, reaching maximum concentrations of 43 pg mRNA/lg total RNA for VTG2, 71 pg mRNA/lg total RNA for ZRP2 and 362 pg mRNA/ug total RNA for ZRP3. The increase in ZRP3 mRNA from 182 to 527 ng/l between days 5 and 8 is not statistically significant. These data suggest that by five days of constant exposure, the ER mediated pathway becomes saturated with an exposure to at least 182 ng/l E2 . Exposure to NP also stimulates induction of all four genes tested (Fig. 2). As with the E2 exposures, VTG1 was the most highly expressed gene, increasing to 2088 pg VTG2
VTG1
pg mRNA/ ug Total RNA
1000
500
75
pg mRNA/ ug Total RNA
Day 2 Day 5 Day 8
1500
Day 2 Day 5 Day 8
50
25
0
0
0
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200
300
400
500
0
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100
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ZRP3
ZRP2 Day 2 Day 5 Day 8
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pg mRNA/ ug Total RNA
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E2 ng/L
E2 ng/L
Day 2 Day 5 Day 8
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0
0
0
100
200
300
E2 ng/L
400
500
600
0
100
200
300
400
500
600
E2 ng/L
Fig. 1. Dose-response increase in VTG and ZRP mRNAs, as measured by real time PCR, in male sheepshead minnows exposed to four concentrations of estradiol (E2 ). Bars represent standard errors.
550
I. Knoebl et al. / Marine Environmental Research 58 (2004) 547–551 VTG1
VTG2 100
Day 2 Day 13
2000
pg mRNA/ ug Total RNA
pg mRNA/ ug Total RNA
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1500 1000
Day 2 Day 13
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ZRP3 Day 2 Day 13
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Day 2 Day 13
750
500
250
0
0
0
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30
ug/L NP
40
50
0
10
20
30
40
50
ug/L NP
Fig. 2. Dose-response increase in VTG and ZRP mRNAs, as measured by real time PCR, in male sheepshead minnows exposed to four concentrations of para-nonylphenol (NP). Bars represent standard errors.
mRNA/lg total RNA at the highest concentration and time. As in the E2 exposures, ZRP3 (637 pg mRNA/lg total RNA) was induced more than ZRP2 or VTG2 (54 and 62 pg mRNA/lg total RNA, respectively). These data suggest that a constant exposure to a concentration of 12 lg/l NP for two days or 5 lg/l for 13 days is sufficient to induce the ER-mediated pathway. This is an important environmental consideration as there are locations where measured levels of NP are higher than 15 lg/l (Petrovic, Sole, Lopez, & Barcelo, 2002). Research by others has shown mixed results. For example Yadetie, Arukwe, Goksøyr, and Male (1999), found dose-response increases for both VTG and ZRP mRNA, but no differences between VTG and ZRP mRNA levels in Atlantic salmon exposed to NP. In work with rainbow trout injected with E2 , Celius, Matthews, Giesy, and Zacharewski (2000), reported that ZRP mRNA was more responsive to low levels of E2 than VTG mRNA. While our results do not indicate a difference in sensitivity at the concentrations tested between the ZRPs and VTGs, there was a clear difference in the magnitude of induction between VTG1 and VTG2 as well as between ZRP2 and ZRP3 after exposure to both E2 and NP. The SHM VTG1 and VTG2 sequences are similar to the killifish (Fundulus heteroclitus) VTG1 and VTG2 sequences, which are also expressed in a 10 to 1 ratio, respectively (LaFleur et al., 1995). The lack of difference in sensitivity between the VTGs and the ZRPs in SHM, as was observed in rainbow trout, may be due to the fact that SHM are serialspawners. As such, eggs are in constant development, whereas in seasonal spawners such as rainbow trout, they are produced only during the reproductive season.
I. Knoebl et al. / Marine Environmental Research 58 (2004) 547–551
551
Acknowledgements This study was funded by the US Environmental Protection Agency Cooperative Agreement #CR826357-10, and the Biotechnology Program, University of Florida.
References Arukwe, A., & Goksøyr, A. (2003). Comparative Hepatology, 2, 4–24. Celius, T., Matthews, J. B., Giesy, J. P., & Zacharewski, T. R. (2000). Journal of Steroid Biochemistry and Molecular Biology, 75, 109–119. Folmar, L. C., Hemmer, M., Hemmer, R., Bowman, C., Kroll, K., & Denslow, N. D. (2000). Aquatic Toxicology, 49, 77–88. Hemmer, M. J., Hemmer, B. L., Bowman, C. J., Kroll, K. J., Folmar, L. C., Marcovich, D., Hoglund, M. D., & Denslow, N. D. (2001). Environmental Toxicology and Chemistry, 20, 336–343. LaFleur, G. J., Jr, Byrne, B. M., Haux, C., Greenberg, R. M., & Wallace, R. A. (1995). International symposium on reproduction physiology of fish, Fish Symposium 95 (pp. 336–338). Austin, TX: University of Texas. Petrovic, M., Sole, M., Lopez, de Alda M. J., & Barcelo, D. (2002). Environmental Toxicology and Chemistry, 21, 2146–2156. Yadetie, F., Arukwe, A., Goksøyr, A., & Male, R. (1999). The Science of the Total Environment, 233, 301– 310.