DAPL1 is a novel regulator of testosterone production in Leydig cells of mouse testis

1. Thibault, C., Levasseur, M. C. & Hunter, R. H. F. Reproduction in Mammals and Man (Ellipses, 1993).

2. Gann, P. H., Hennekens, C. H., Ma, J., Longcope, C. & Stampfer, M. J. Prospective study of sex hormone levels and risk of prostate

cancer. J. Natl. Cancer Inst. 88, 1118–1126 (1996).

3. Weiss, J. M. et al. Endogenous sex hormones and the risk of prostate cancer: A prospective study. Int. J. Cancer 122, 2345–2350

(2008).

4. Schenk, J. M. et al. Serum androgens and prostate cancer risk: Results from the placebo arm of the Prostate Cancer Prevention

Trial. Cancer Causes Control 27(175), 182 (2016).

5. Luo, S., Au Yeung, S. L., Zhao, J. V., Burgess, S. & Schooling, C. M. Association of genetically predicted testosterone with thromboembolism, heart failure, and myocardial infarction: Mendelian randomisation study in UK Biobank. BMJ 364, l476 (2019).

6. Dufau, M. L. Endocrine regulation and communicating functions of the Leydig cell. Annu. Rev. Physiol. 50, 483–508 (1988).

7. Manna, P. R. et al. Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP responseelement binding protein family. Mol. Endocrinol. 16, 184–199 (2002).

8. Zirkin, B. R. & Papadopoulos, V. Leydig cells: Formation, function, and regulation. Biol. Reprod. 99, 101–111 (2018).

9. Stocco, D. M. & Clark, B. J. Regulation of the acute production of steroids in steroidogenic cells. Endocr. Rev. 17, 221–244 (1996).

10. Hasegawa, T. et al. Developmental roles of the steroidogenic acute regulatory protein (StAR) as revealed by StAR knockout mice.

Mol. Endocrinol. 14, 1462–1471 (2000).

11. Stocco, D. M. & Clark, B. J. The role of the steroidogenic acute regulatory protein in steroidogenesis. Steroids 62, 29–36 (1997).

12. Stocco, D. M., Wang, X., Jo, Y. & Manna, P. R. Multiple signaling pathways regulating steroidogenesis and steroidogenic acute

regulatory protein expression: More complicated than we thought. Mol. Endocrinol. 19, 2647–2659 (2005).

13. Sun, L., Ryan, D. G., Zhou, M., Sun, T. T. & Lavker, R. M. EEDA: A protein associated with an early stage of stratified epithelial

differentiation. J. Cell Physiol. 206, 103–111 (2006).

14. Ma, X. et al. DAPL1, a susceptibility locus for age-related macular degeneration, acts as a novel suppressor of cell proliferation in

the retinal pigment epithelium. Hum. Mol. Genet. 26, 1612–1621 (2017).

15. Ma, X. et al. Regulation of cell proliferation in the retinal pigment epithelium: Differential regulation of the death-associated

protein like-1 DAPL1 by alternative MITF splice forms. Pigment Cell Melanoma Res. 31, 411–422 (2018).

16. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278

(2014).

17. Williams, W. P. 3rd., Jarjisian, S. G., Mikkelsen, J. D. & Kriegsfeld, L. J. Circadian control of kisspeptin and a gated GnRH response

mediate the preovulatory luteinizing hormone surge. Endocrinology 152, 595–606 (2011).

18. Turnham, R. E. & Scott, J. D. Protein kinase A catalytic subunit isoform PRKACA; History, function and physiology. Gene 577,

101–108 (2016).

19. Dyson, M. T. et al. Mitochondrial A-kinase anchoring protein 121 binds type II protein kinase A and enhances steroidogenic acute

regulatory protein-mediated steroidogenesis in MA-10 mouse leydig tumor cells. Biol. Reprod. 78, 267–277 (2008).

20. Matzkin, M. E., Yamashita, S. & Ascoli, M. The ERK1/2 pathway regulates testosterone synthesis by coordinately regulating the

expression of steroidogenic genes in Leydig cells. Mol. Cell Endocrinol. 370, 130–137 (2013).

21. Yamashita, S., Tai, P., Charron, J., Ko, C. & Ascoli, M. The Leydig cell MEK/ERK pathway is critical for maintaining a functional

population of adult Leydig cells and for fertility. Mol. Endocrinol. 25, 1211–1222 (2011).

22. Yao, B. et al. Gonadotropin-releasing hormone positively regulates steroidogenesis via extracellular signal-regulated kinase in rat

Leydig cells. Asian J. Androl. 13, 438–445 (2011).

23. Tremblay, J. J. Molecular regulation of steroidogenesis in endocrine Leydig cells. Steroids 103, 3–10 (2015).

24. Hattori, Y. et al. Attenuation of growth hormone production at the fetal stage is critical for dioxin-induced developmental disorder

in rat offspring. Biochem. Pharmacol. 186, 114495 (2021).

25. Zhang, H., Qu, X. & Han, L. Identification of death-associated protein-like 1 (DAPL1) as a novel prognostic biomarker of breast

cancer. https://​doi.​org/​10.​21203/​rs.2.​24569/​v1.

26. Rodrigues, T. C. et al. Upregulated genes at 2q24 gains as candidate oncogenes in hepatoblastomas. Future Oncol. 10, 2449–2457

(2014).

27. Smith, J. T. et al. Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. Endocrinology

146, 2976–2984 (2005).

28. Matthew, L.S. UCSC cell browser: Visualize your single-cell data. https://​doi.​org/​10.​1101/​2020.​10.​30.​361162.

29. Guo, J. et al. The adult human testis transcriptional cell atlas. Cell Res. 28, 1141–1157 (2018).

Scientific Reports |

(2021) 11:18532 |

https://doi.org/10.1038/s41598-021-97961-6

11

Vol.:(0123456789)

www.nature.com/scientificreports/

30. Yap, T. A. et al. AT13148 is a novel, oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity. Clin.

Cancer Res. 18, 3912–3923 (2012).

31. Shin, S.I., Studies on interstitial cells in tissue culture: steroid biosynthesis in monolayers of mouse testicular interstitial cells.

Endocrinology 81, 440-448 (1967).

32. Takeda, T., Taura, J., Hattori, Y., Ishii, Y. & Yamada, H. Dioxin-induced retardation of development through a reduction in the

expression of pituitary hormones and possible involvement of an aryl hydrocarbon receptor in this defect: A comparative study

using two strains of mice with different sensitivities to dioxin. Toxicol. Appl. Pharmacol. 278, 220–229 (2014).

33. Matsumoto, Y. et al. Maternal exposure to dioxin reduces hypothalamic but not pituitary metabolome in fetal rats: a possible

mechanism for a fetus-specific reduction in steroidogenesis. J. Toxicol. Sci. 35, 365-373 (2010). 34. Takeda T, et al. Maternal exposure to dioxin disrupts gonadotropin production in fetal rats and imprints defects in sexual behavior.

J. Pharmacol. Exp. Ther. 329, 1091-1099 (2009).

Acknowledgements

We appreciate the technical assistance from The Research Support Center, Research Center for Human Disease

Modeling, Kyushu University Graduate School of Medical Sciences. This research was supported in part by grants

from the Japan Society for the Promotion of Science (JSPS) [Scientific Research (A) 17H00788 and 21H04928,

Recipient YI], and the Ministry of Health, Labor and Welfare, Japan [Research on Food Safety (H30-Designated

Research-005 and R3-Designated Research 21KA2003, Recipient YI)]. Presented in part at meetings: Chen et al.,

Forum 2019: Pharmaceutical Health Science-Environmental Toxicology, the Pharmaceutical Society of Japan,

Kyoto, August, 2019; Chen et al., 47th Annual Meeting of the Japanese Society of Toxicology, Online, June-July,

2020; Chen et al., 24th North American ISSX meeting, September, 2021 (Virtual meeting).

Author contributions

H.-b.C.: data curation, formal analysis, investigation, methodology, project administration, resources, software,

validation, visualization, writing–original draft, writing-review, and editing. J.C.P.G.: investigation, writingreview, and editing. S.A.: methodology and resources. T.T., R.-s.L., Y.H., H.S., Y.M., Y.H.: methodology, writingreview, and editing. Y.T.: writing, review, and editing. Y.I.: conceptualization, funding acquisition, investigation,

project administration, supervision, writing–original draft, writing-review, and editing. All authors have read

and agreed to the published version of the manuscript.

Competing interests The authors declare no competing interests.

Additional information

Supplementary Information The online version contains supplementary material available at https://​doi.​org/​

10.​1038/​s41598-​021-​97961-6.

Correspondence and requests for materials should be addressed to Y.I.

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