Dysregulation of Immature Sertoli Cell Functions by Exposure to Acetaminophen and Genistein in Rodent Cell Models
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. TM4 Mouse Sertoli Cell Line Culture
2.3. Primary Immature Rat Sertoli Cell Isolation
2.4. Sertoli Cell Treatments
2.5. Cell Viability/Proliferation with MTT Assay
2.6. Cell Proliferation with EdU Assay
2.7. Gene Expression Measured by qRT-PCR
2.8. PGD2 & PGE2 ELISA
2.9. Immunofluorescent (IF) Staining
2.10. Whole Transcriptome Sequencing (Total RNA-Seq)
2.11. Statistical Analysis
3. Results
3.1. Sertoli Cell Viability and Proliferation Are Dysregulated by Exposure to APAP and GEN
3.2. Eicosanoid Pathway Is Dysregulated in Immature Sertoli Cells Exposed to APAP, GEN, and Their Mixtures
3.3. Estrogen Receptors Dysregulation in Immature Sertoli Cells by APAP and GEN
3.4. Dysregulation of Immature Sertoli Cell Differentiation by APAP and GEN
3.5. APAP and GEN Disrupt TM4 Cells and PND8 Sertoli Cell Transcriptomes
4. Discussion
4.1. Dysregulation of Sertoli Cell Development by APAP and GEN
4.2. Similarities and Differences between TM4 Cells and PND8 Immature Sertoli Cells
4.3. Is There a Link between APAP and GEN Effects on Sox9 and Eicosanoid Pathway?
4.4. Mechanisms Dysregulated in Immortalized and Primary Immature Sertoli Cells
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Forward Primer | Reverse Primer | Product Size (bp) |
---|---|---|---|
Rat | |||
Gapdh | CCATTCTTCCACCTTTGATGCT | TGTTGCTGTAGCCATATTCATTGT | 98 |
Sox9 | TGCTGAACGAGAGCGAGAAG | ATGTGAGTCTGTTCGGTGGC | 160 |
Amh | GTGGGTGGCAGCAGCACTAGG | CGGGCTGTTTGGCTCTGATTCCCG | 69 |
Ar | CGGTCGAGTTGACATTAGTGAAGGACC | ATTCCTGGATGGGACTGATGGT | 66 |
Cox1 | AGGTGTACCCACCTTCCGTA | GGTTTCCCCTATAAGGATGAGGC | 242 |
Cox2 | ACGTGTTGACGTCCAGATCA | CTTGGGGATCCGGGATGAAC | 234 |
Ptgds | ATTCAAGCTGGTTCCGGGAG | CAGGAACGCGTACTCATCGT | 242 |
Esr1 | GCCACTCGATCATTCGAGCA | CCTGCTGGTTCAAAAGCGTC | 107 |
Ppar-α | TGCGGACTACCAGTACTTAGGG | GCTGGAGAGAGGGTGTCTGT | 72 |
Mouse | |||
Rps29 | TGAAGGCAAGATGGGTCAC | GCACATGTTCAGCCCGTATT | 127 |
Cox1 | CCTCTTTCCAGGAGCTCACA | TCGATGTCACCGTACAGCTC | 70 |
Cox2 | CAGGACTCTGCTCACGAAGG | ATCCAGTCCGGGTACAGTCA | 231 |
Ptgds | GGCTCCTGGACACTACACCT | CTGGGTTCTGCTGTAGAGGGT | 160 |
Ptges | GGCTCCTCCAAAGACGGAAA | TGGCACAGCATGGGTCTTAG | 226 |
Ptgdr2 | CACGTGTCGGTGCTGTTG | GATGAGTCCGTTTTCCACCA | 63 |
Amh | GGGGAGACTGGAGAACAGC | AGAGCTCGGGCTCCCATA | 67 |
Ar | ACCAGATGGCGGTCATTCAG | TGTGCATGCGGTACTCATTG | 135 |
Sox9 | TCGGACACGGAGAACACC | GCACACGGGGAACTTATCTT | 96 |
Esr1 | TCTCCTCAAACACATCCCGTG | GGCGAGTTACAGACTGGCTC | 96 |
Gper | CCTGGACGAGCAGTATTACGATATC | TGCTGTACATGTTGATCTG | 77 |
Err-α | CGCTGTCAGCTGGAGGAA | ACCTTGGGCTCCGGCA | 199 |
Err-β | TCTTCCCAGCTCCCACAGTA | CCCCATGCAAGCTTCGTAGT | 106 |
TM4 APAP (50 µM) | |||||
---|---|---|---|---|---|
Downregulated | Upregulated | ||||
Gene | p-value | Fold change | Gene | p-value | Fold change |
Ereg | 2.36 × 10−11 | −4.90 | Dag1 | 1.13 × 10−4 | 2.09 |
Hbegf | 8.14 × 10−10 | −3.37 | Rpa1 | 1.29 × 10−4 | 2.11 |
Lif | 1.39 × 10−7 | −2.96 | Ubc | 2.08 × 10−7 | 2.20 |
Cth | 1.14 × 10−11 | −2.84 | Mki67 | 1.08 × 10−14 | 2.29 |
H1f1 | 4.41 × 10−18 | −2.55 | Kif20a | 4.78 × 10−6 | 2.32 |
H1f0 | 3.34 × 10−21 | −2.54 | Ckap2l | 2.18 × 10−6 | 2.43 |
Il1rl1 | 5.63 × 10−8 | −2.51 | Acat2 | 1.76 × 10−4 | 3.28 |
Mtmr10 | 4.93 × 10−5 | −2.51 | Mettl26 | 1.93 × 10−5 | 8.46 |
H4f16 | 4.61 × 10−10 | −2.39 | |||
Prkg2 | 2.33 × 10−10 | −2.35 | |||
PND8 rat Sertoli APAP (50 µM) | |||||
Downregulated | Upregulated | ||||
Gene | p-value | Fold change | Gene | p-value | Fold change |
Hoxb1 | 4.11 × 10−2 | −13.14 | Ppp1r3d | 3.85 × 10−2 | 12.17 |
Senp18 | 3.59 × 10−2 | −11.41 | |||
Hjv | 3.63 × 10−2 | −10.06 | |||
Sypl2 | 4.26 × 10−2 | −7.85 | |||
Hs3st2 | 3.37 × 10−2 | −7.70 | |||
Mal | 4.63 × 10−2 | −7.57 | |||
Nmur2 | 4.35 × 10−2 | −7.48 | |||
Esrrg | 4.85 × 10−2 | −5.88 | |||
Synb | 4.50 × 10−2 | −5.66 | |||
Akain1 | 4.52 × 10−2 | −5.23 | |||
TM4 GEN (50 µM) | |||||
Downregulated | Upregulated | ||||
Gene | p-value | Fold change | Gene | p-value | Fold change |
Fosl1 | 5.86 × 10−28 | −15.38 | Akr1c14 | 1.29 × 10−18 | 3.57 |
Ereg | 6.71 × 10−35 | −14.81 | Mboat2 | 8.25 × 10−8 | 3.85 |
Tgtp1 | 1.06 × 10−17 | −13.57 | Gja1 | 3.97 × 10−38 | 3.89 |
Gbp3 | 8.73 × 10−60 | −13.20 | Dhcr24 | 4.89 × 10−5 | 4.01 |
Tgtp2 | 1.37 × 10−19 | −13.16 | Nbl1 | 1.07 × 10−5 | 4.17 |
Hbegf | 1.64 × 10−42 | −12.08 | Scd2 | 1.10 × 10−72 | 4.88 |
Ccl20 | 3.98 × 10−5 | −10.50 | Lars2 | 9.28 × 10−5 | 5.29 |
Ifit3 | 6.09 × 10−85 | −9.99 | Acat2 | 3.36 × 10−9 | 5.35 |
Iigp1 | 2.80 × 10−36 | −8.57 | Mettl26 | 3.78 × 10−6 | 7.98 |
Usp18 | 1.67 × 10−54 | −8.45 | Mettl7a1 | 2.43 × 10−11 | 8.48 |
PND8 rat Sertoli GEN (50 µM) | |||||
Downregulated | Upregulated | ||||
Gene | p-value | Fold change | Gene | p-value | Fold change |
Depdc1b | 7.54 × 10−4 | −10.00 | Ptx3 | 1.17 × 10−43 | 2.62 |
H2ac10 | 1.39 × 10−37 | −7.87 | Mdm2 | 5.71 × 10−67 | 2.93 |
E2f8 | 4.22 × 10−8 | −5.69 | Bbc3 | 2.14 × 10−21 | 3.02 |
Sgo2 | 2.73 × 10−6 | −4.99 | Csf2rb | 6.13 × 10−5 | 3.17 |
Cenpq | 1.56 × 10−3 | −4.96 | Cd80 | 4.64 × 10−9 | 3.36 |
Pbk | 1.09 × 10−4 | −4.94 | Abcb1b | 2.99 × 10−101 | 3.66 |
Cdca3 | 8.23 × 10−8 | −4.71 | Star | 2.53 × 10−188 | 4.36 |
Depdc1 | 1.17 × 10−4 | −4.71 | Cdkn1a | 1.35 × 10−155 | 4.86 |
Hist1h2bc | 7.92 × 10−5 | −4.67 | Il6 | 3.29 × 10−8 | 5.18 |
Uhrf1 | 1.10 × 10−21 | −4.44 | Gdf15 | 2.02 × 10−22 | 9.17 |
TM4 APAP + GEN (50 µM) | |||||
Downregulated | Upregulated | ||||
Gene | p-value | Fold change | Gene | p-value | Fold change |
Fosl1 | 1.19 × 10−27 | −22.36 | Greb1 | 4.85 × 10−12 | 3.54 |
Ereg | 2.19 × 10−31 | −17.81 | Thra | 3.82 × 10−6 | 3.60 |
Mx1 | 3.19 × 10−8 | −15.73 | Nbl1 | 3.52 × 10−4 | 3.63 |
Il1rl1 | 1.39 × 10−53 | −15.63 | Ulk1 | 1.74 × 10−6 | 3.75 |
Tgtp2 | 8.87 × 10−17 | −14.41 | Mreg | 3.26 × 10−6 | 3.76 |
Gbp3 | 3.49 × 10−49 | −13.68 | Scd2 | 1.59 × 10−43 | 3.88 |
Ccl20 | 7.76 × 10−5 | −12.90 | Dzip1 | 1.14 × 10−6 | 4.05 |
Dusp5 | 1.04 × 10−7 | −12.49 | Acacb | 6.33 × 10−4 | 4.18 |
Hbegf | 3.01 × 10−34 | −12.06 | B4gat1 | 1.30 × 10−3 | 4.64 |
Ifit3 | 6.59 × 10−75 | −11.33 | Acat2 | 1.39 × 10−7 | 5.23 |
PND8 rat Sertoli APAP + GEN (50 µM) | |||||
Downregulated | Upregulated | ||||
Gene | p-value | Fold change | Gene | p-value | Fold change |
H2ac10 | 3.82 × 10−39 | −8.28 | Pde4d | 2.74 × 10−12 | 2.49 |
Pimreg | 9.09 × 10−4 | −6.10 | Nqo1 | 9.70 × 10−23 | 2.54 |
E2f8 | 9.53 × 10−8 | −5.40 | Csf2rb | 1.12 × 10−3 | 2.56 |
Uhrf1 | 6.39 × 10−26 | −5.24 | Il6 | 8.36 × 10−4 | 2.73 |
Cdca3 | 1.40 × 10−8 | −5.23 | Eda2r | 5.03 × 10−26 | 2.82 |
Pbk | 2.67 × 10−4 | −4.45 | Cd80 | 3.24 × 10−9 | 3.40 |
Hist1h2bg | 5.98 × 10−7 | −4.40 | Cdkn1a | 8.58 × 10−94 | 3.40 |
Ckap2l | 5.00 × 10−10 | −4.20 | Abcb1b | 3.69 × 10−113 | 3.94 |
Hist1h2bc | 2.33 × 10−4 | −4.17 | Star | 3.91 × 10−179 | 4.21 |
Cdca5 | 5.13 × 10−6 | −4.14 | Gdf15 | 8.82 × 10−15 | 5.88 |
TM4 Cells | PND8 Rat Sertoli Cells | |||||
---|---|---|---|---|---|---|
Pathway Description | Enrichment Score | p-Value | Genes | Enrichment Score | p-Value | Genes |
Necroptosis | 18.7 | 7.39 × 10−9 | 22 | 3.4 | 3.50 × 10−2 | 4 |
Viral carcinogenesis | 10.1 | 4.01 × 10−5 | 20 | 9.9 | 5.17 × 10−5 | 9 |
Transcriptional misregulation in cancer | 13.5 | 1.36 × 10−6 | 21 | 7.0 | 9.35 × 10−4 | 7 |
MicroRNAs in cancer | 6.8 | 1.10 × 10−3 | 14 | 4.0 | 1.80 × 10−2 | 5 |
p53 signaling pathway | 5.9 | 2.61 × 10−3 | 8 | 12.5 | 3.73 × 10−6 | 7 |
Cellular senescence | 6.0 | 2.47 × 10−3 | 14 | 10.8 | 1.95 × 10−5 | 9 |
TNF signaling pathway | 4.5 | 1.16 × 10−2 | 9 | 3.9 | 2.10 × 10−2 | 4 |
Viral protein interaction with cytokine and cytokine receptor | 5.2 | 5.72 × 10−3 | 8 | 3.1 | 4.32 × 10−2 | 3 |
TM4 Cells | |||
---|---|---|---|
Pathway Description | Enrichment Score | p-Value | Genes |
Ribosome biogenesis in eukaryotes | 15.8 | 1.40 × 10−7 | 15 |
NOD-like receptor signaling pathway | 8.2 | 0.000270 | 16 |
AMPK signaling pathway | 4.6 | 0.0100 | 10 |
NF-kappa B signaling pathway | 4.0 | 0.0185 | 8 |
IL-17 signaling pathway | 3.6 | 0.0280 | 7 |
Steroid biosynthesis | 3.5 | 0.0291 | 3 |
TGF-beta signaling pathway | 3.4 | 0.0343 | 7 |
Cytosolic DNA-sensing pathway | 3.0 | 0.0500 | 5 |
PND8 Rat Sertoli Cells | |||
---|---|---|---|
Pathway Description | Enrichment Score | p-Value | Genes |
Foxo signaling pathway | 8.6 | 0.000180 | 7 |
Melanoma | 3.7 | 0.0252 | 3 |
Glioma | 3.6 | 0.0283 | 3 |
PI3K-Akt signaling pathway | 3.5 | 0.0298 | 7 |
Pancreatic cancer | 3.5 | 0.0316 | 3 |
Rheumatoid arthritis | 3.5 | 0.0316 | 3 |
Hematopoietic cell lineage | 3.4 | 0.0351 | 3 |
JAK-STAT signaling pathway | 3.3 | 0.0368 | 4 |
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Corpuz-Hilsabeck, M.; Mohajer, N.; Culty, M. Dysregulation of Immature Sertoli Cell Functions by Exposure to Acetaminophen and Genistein in Rodent Cell Models. Cells 2023, 12, 1804. https://doi.org/10.3390/cells12131804
Corpuz-Hilsabeck M, Mohajer N, Culty M. Dysregulation of Immature Sertoli Cell Functions by Exposure to Acetaminophen and Genistein in Rodent Cell Models. Cells. 2023; 12(13):1804. https://doi.org/10.3390/cells12131804
Chicago/Turabian StyleCorpuz-Hilsabeck, Maia, Nicole Mohajer, and Martine Culty. 2023. "Dysregulation of Immature Sertoli Cell Functions by Exposure to Acetaminophen and Genistein in Rodent Cell Models" Cells 12, no. 13: 1804. https://doi.org/10.3390/cells12131804