Relationship: 2130: Antagonism, Androgen receptor leads to Decrease, AR activation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

This KER is applicable to mammals as AR expression and activity is highly conserved (Davey & Grossmann, 2016). AR activity is important for sexual development and reproduction in both males and females (Prizant et al., 2014; Walters et al., 2010). AR function is required during development, puberty, and adulthood. It is, however, acknowledged that this KER most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.

Key Event Relationship Description

The androgen receptor (AR) is a ligand-activated steroid hormone nuclear receptor (Davey & Grossmann, 2016). In its inactive state, the AR locates to the cytoplasm (Roy et al., 2001). When activated, the AR translocates to the nucleus, dimerizes, and, together with co-regulators, binds to specific DNA regulatory sequences to regulate gene transcription (Davey & Grossmann, 2016) (Lamont and Tindall, 2010). This is considered the canonical AR signaling pathway. The AR can also activate non-genomic signalling (Jin et al., 2013). However, this KER focuses on the canonical pathway.

The two main AR ligands are the androgens testosterone (T) and the more potent dihydrotestosterone (DHT). Androgens bind to the AR to mediate downstream androgenic responses, such as male development and masculinization (Rey, 2021; Walters et al., 2010). Antagonism of the AR would decrease AR activation and therefore the downstream AR-mediated effects.  

Evidence Supporting this KER

The biological plausibility for this KER is considered high.

The AR belongs to the steroid hormone nuclear receptor family. The AR has 3 main domains essential for its activity, the N-terminal domain, the ligand binding domain, and the DNA binding domain (Roy et al., 2001). Ligands, such as T and DHT, must bind to the ligand binding domain to activate AR allowing it to fulfill its role as a transcription factor. The binding of the ligand induces a change in AR conformation allowing it to translocate to the nucleus and congregate into a subnuclear compartment (Marcelli et al., 2006; Roy et al., 2001) homodimerize and bind to the DNA target sequences and regulate transcription of target genes. Regulation of AR target genes is greatly facilitated by numerous co-factors. Active AR signaling is essential for male reproduction and sexual development and is also crucial in several other tissues and organs such as ovaries, the immune system, bones, and muscles (Ogino et al., 2011; Prizant et al., 2014; Rey, 2021; William H. Walker, 2021).

AR antagonists can compete with or prevent in different ways  AR ligand binding, thereby preventing AR activation. Antagonism of the AR can prevent translocation to the nucleus, compartmentalization, dimerization and DNA binding. Consequently, AR cannot regulate transcription of target genes and androgen signalling is disrupted. This can be observed using different AR activation assays such as AR dimerization, translocation, DNA binding or transcriptional activity assays (Brown et al., 2023; OECD, 2020).

The empirical evidence for this KER is considered high

The effects of AR antagonism have been shown in many studies in vivo and in vitro.

Several stressors can act as antagonists of the AR and lead to decreased AR activation. Some of these are detailed in an AOP key event relationship report by (Pedersen et al., 2022) and shown below, exhibiting evidence of dose-concordance:

Stressors

• Cyproterone acetate: Using the AR-CALUX reporter assay in antagonism mode, cyproterone acetate showed an IC50 of 7.1 nM (Sonneveld, 2005)
• Epoxiconazole: Using transiently AR-transfected CHO cells, epoxiconazole showed a LOEC of 1.6 µM and an IC50 of 10 µM (Kjærstad et al., 2010).
• Flutamide: Using the AR-CALUX reporter assay in antagonism mode, flutamide showed an IC50 of 1.3 µM (Sonneveld, 2005).
• Flusilazole: Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.8 µM and an IC50 of 2.8 (±0.1) µM (Draskau et al., 2019).
• Prochloraz: Using transiently AR-transfected CHO cells, prochloraz showed a LOEC of 6.3 µM and an IC50 of 13 µM (Kjærstad et al., 2010).
• Propiconazole: Using transiently AR-transfected CHO cells, propiconazole showed a LOEC of 12.5 µM and an IC50 of 18 µM (Kjærstad et al., 2010).
• Tebuconazole: Using transiently AR-transfected CHO cells, tebuconazole showed a LOEC of 3.1 µM and an IC50 of 8.1 µM (Kjærstad et al., 2010).
• Triticonazole: Using hAR-EcoScreen Assay, triticonazole showed a LOEC for antagonisms of 0.2 µM and an IC50 of 0.3 (±0.01) µM (Draskau et al., 2019).
• Vinclozolin: Using the AR-CALUX reporter assay in antagonism mode, vinclozolin showed an IC50of 1.0 µM(Sonneveld, 2005).”(Pedersen et al., 2022)

Other evidence:

Known AR antagonists are used for treatment of AR-sensitive cancers such as flutamide for prostate cancer (Mahler et al., 1998).

Quantitative Understanding of the Linkage

 The quantitative relationship between AR antagonism and AR activation will depend on the type of antagonist.

Nuclear translocation in HeLa cells transfected with AR-GFP show a response within 2 hours after ligand exposure (Marcelli et al., 2006; Szafran et al., 2008). Another assay focusing on AR binding to promoters in LNCaP cells has shown that after ligand binding, AR is able to translocate and bind to the DNA sequences within 15min showing the speed of AR activation (Kang et al., 2002).

AR antagonism can lead to increased AR transcript stability and levels as a compensatory mechanism in prostate cancer cells (Dart et al., 2020). In turn, in presence of increased AR levels, AR antagonists can exhibit agonistic activity (Chen et al., 2003).

References

Brown, E. C., Hallinger, D. R., Simmons, S. O., Puig-Castellví, F., Eilebrecht, E., Arnold, L., & Bioscience, P. A. (2023). High-throughput AR dimerization assay identifies androgen disrupting chemicals and metabolites. Front. Toxicol, 5, 1134783. https://doi.org/10.3389/ftox.2023.1134783

Chen, C. D., Welsbie, D. S., Tran, C., Baek, S. H., Chen, R., Vessella, R., Rosenfeld, M. G., & Sawyers, C. L. (2003). A R T I C L E S Molecular determinants of resistance to antiandrogen therapy. NATURE MEDICINE, 10(1). https://doi.org/10.1038/nm972

Dart, D. A., Ashelford, K., & Jiang, W. G. (2020). AR mRNA stability is increased with AR-antagonist resistance via 3′UTR variants. https://doi.org/10.1530/EC-19-0340

Davey, R. A., & Grossmann, M. (2016). Androgen Receptor Structure, Function and Biology: From Bench to Bedside. In Androgen Receptor Biology Clin Biochem Rev (Vol. 37, Issue 1).

Draskau, M. K., Boberg, J., Taxvig, C., Pedersen, M., Frandsen, H. L., Christiansen, S., & Svingen, T. (2019). In vitro and in vivo endocrine disrupting effects of the azole fungicides triticonazole and flusilazole. Environmental Pollution, 255, 113309. https://doi.org/10.1016/j.envpol.2019.113309

Jin, H. J., Kim, J., & Yu, J. (2013). Androgen receptor genomic regulation. In Translational Andrology and Urology (Vol. 2, Issue 3, pp. 158–177). AME Publishing Company. https://doi.org/10.3978/j.issn.2223-4683.2013.09.01

Kang, Z., Pirskanen, A., Jänne, O. A., & Palvimo, J. J. (2002). Involvement of Proteasome in the Dynamic Assembly of the Androgen Receptor Transcription Complex. Journal of Biological Chemistry, 277(50), 48366–48371. https://doi.org/10.1074/jbc.M209074200

Kjærstad, M. B., Taxvig, C., Nellemann, C., Vinggaard, A. M., & Andersen, H. R. (2010). Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals. Reproductive Toxicology, 30(4), 573–582. https://doi.org/10.1016/j.reprotox.2010.07.009

Lamont, K. R., and Tindall, D. J. (2010). Androgen Regulation of Gene Expression. Adv. Cancer Res. 107, 137–162. doi:10.1016/S0065-230X(10)07005-3.

Mahler, C., Verhelst, J., and Denis, L. (1998). Clinical pharmacokinetics of the antiandrogens and their efficacy in prostate cancer. Clin. Pharmacokinet. 34, 405–417. doi:10.2165/00003088-199834050-00005/METRICS.

Marcelli, M., Stenoien, D. L., Szafran, A. T., Simeoni, S., Agoulnik, I. U., Weigel, N. L., Moran, T., Mikic, I., Price, J. H., & Mancini, M. A. (2006). Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility. Journal of Cellular Biochemistry, 98(4), 770–788. https://doi.org/10.1002/jcb.20593

OECD (2020). Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. OECD Guide. Paris: OECD Publishing doi:10.1787/9789264264366-en.

Ogino, Y., Miyagawa, S., Katoh, H., Prins, G. S., Iguchi, T., & Yamada, G. (2011). Essential functions of androgen signaling emerged through the developmental analysis of vertebrate sex characteristics. Evolution & Development, 13(3), 315–325. https://doi.org/10.1111/j.1525-142X.2011.00482.x

Pedersen, E. B., Christiansen, S., & Svingen, T. (2022). AOP key event relationship report: Linking androgen receptor antagonism with nipple retention. Current Research in Toxicology, 3, 100085. https://doi.org/10.1016/j.crtox.2022.100085

Prizant, H., Gleicher, N., & Sen, A. (2014). Androgen actions in the ovary: balance is key. Journal of Endocrinology, 222(3), R141–R151. https://doi.org/10.1530/JOE-14-0296

Rey, R. A. (2021). The Role of Androgen Signaling in Male Sexual Development at Puberty. Endocrinology, 162(2). https://doi.org/10.1210/endocr/bqaa215

Roy, A. K., Tyagi, R. K., Song, C. S., Lavrovsky, Y., Ahn, S. C., Oh, T. S., & Chatterjee, B. (2001). Androgen receptor: Structural domains and functional dynamics after ligand-receptor interaction. Annals of the New York Academy of Sciences, 949, 44–57. https://doi.org/10.1111/j.1749-6632.2001.tb04001.x

Sonneveld, E. (2005). Development of Androgen- and Estrogen-Responsive Bioassays, Members of a Panel of Human Cell Line-Based Highly Selective Steroid-Responsive Bioassays. Toxicological Sciences, 83(1), 136–148. https://doi.org/10.1093/toxsci/kfi005

Szafran, A. T., Szwarc, M., Marcelli, M., & Mancini, M. A. (2008). Androgen Receptor Functional Analyses by High Throughput Imaging: Determination of Ligand, Cell Cycle, and Mutation-Specific Effects. PLoS ONE, 3(11), e3605. https://doi.org/10.1371/journal.pone.0003605

Walters, K. A., Simanainen, U., & Handelsman, D. J. (2010). Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. In Human Reproduction Update (Vol. 16, Issue 5, pp. 543–558). Hum Reprod Update. https://doi.org/10.1093/humupd/dmq003

William H. Walker. (2021). Androgen Actions in the Testis and the Regulation of Spermatogenesis. In Advances in Experimental Medicine and Biology: Vol. volume 1381 (pp. 175–203).

Relationship: 2124: Decrease, AR activation leads to Altered, Transcription of genes by the AR

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

This KER is applicable for both sexes, across developmental stages into adulthood, in numerous cells and tissues and across mammalian taxa. It is, however, acknowledged that this KER most likely has a much broader domain of applicability extending to non-mammalian vertebrates. AOP developers are encouraged to add additional relevant knowledge to expand on the applicability to also include other vertebrates.

Key Event Relationship Description

The androgen receptor (AR) is a ligand-dependent nuclear transcription factor that upon activation translocates to the nucleus, dimerizes, and binds androgen response elements (AREs) to modulate transcription of target genes (Lamont and Tindall, 2010, Roy et al. 2001). Decreased activation of the AR affects its transcription factor activity, therefore leading to altered AR-target gene expression. This KER refers to decreased AR activation and altered gene expression occurring in complex systems, such as in vivo and the specific effect on transcription of AR target genes will depend on species, life stage, tissue, cell type etc.

Evidence Supporting this KER

The biological plausibility for this KER is considered high

The AR is a ligand-activated transcription factor part of the steroid hormone nuclear receptor family. Non-activated AR is found in the cytoplasm as a multiprotein complex with heat-shock proteins, immunophilins and, other chaperones (Roy et al. 2001). Upon activation through ligand binding, the AR dissociates from the protein complex, translocates to the nucleus and homodimerizes. Facilitated by co-regulators, AR can bind to DNA regions containing AREs and initiate transcription of target genes, that thus will be different in e.g. different tissues, life-stages, species etc.

Through mapping of AREs and ChIP sequencing studies, several AR target genes have been identified, mainly studied in prostate cells (Jin, Kim, and Yu 2013). Different co-regulators and ligands lead to altered expression of different sets of genes (Jin et al. 2013; Kanno et al. 2022). Alternative splicing of the AR can lead to different AR variants that also affects which genes are transcribed (Jin et al. 2013).

Apart from this canonical signaling pathway, the AR can suppress gene expression, indirectly regulate miRNA transcription, and have non-genomic effects by rapid activation of second messenger pathways in either presence or absence of a ligand (Jin et al. 2013).

The empirical evidence for this KER is considered high

In humans, altered gene expression profiling in individuals with androgen insensitivity syndrome (AIS) can provide supporting empirical evidence (Holterhus et al. 2003; Peng et al. 2021). In rodent AR knockout (KO) models, gene expression profiling studies and gene-targeted approaches have provided information on differentially expressed genes in several organ systems including male and female reproductive, endocrine, muscular, cardiovascular and nervous systems (Denolet et al. 2006; Fan et al. 2005; Holterhus et al. 2003; Ikeda et al. 2005; Karlsson et al. 2016; MacLean et al. 2008; Rana et al. 2011; Russell et al. 2012; Shiina et al. 2006; Wang et al. 2006; Welsh et al. 2012; Willems et al. 2010; Yu et al. 2008, 2012; Zhang et al. 2006; Zhou et al. 2011).

Exposure to known antiandrogens has been shown to alter transcriptional profiles, for example of neonatal pig ovaries (Knapczyk-Stwora et al. 2019).

Dose concordance has also been observed for instance in zebrafish embryos; a dose of 50 µg/L of the AR antagonist flutamide resulted in 674 differentially expressed genes at 96 h post fertilization whereas 500 µg/L flutamide resulted in 2871 differentially expressed genes (Ayobahan et al., 2023).

AR action has been reported to occur also without ligand binding. However, not much is known about the extent and biological implications of such non-canonical, ligand-independent AR activation (Bennesch and Picard 2015).

Quantitative Understanding of the Linkage

There is not enough data to define a quantitative relationship between AR activation and alteration of AR target gene transcription, and such a relationship will differ between biological systems (species, tissue, cell type, life stage etc).

AR and promoter interactions occur within 15 minutes of ligand binding, RNA polymerase II and coactivator recruitment are proposed to occur transiently with cycles of approximately 90 minutes in LNCaP cells (Kang et al. 2002). RNA polymerase II elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb/min (Maiuri et al. 2011). Therefore, depending on the cell type and the half-life of the AR target gene transcripts, changes are to be expected within hours.

AR has been hypothesized to auto-regulate its mRNA and protein levels (Mora and Mahesh 1999).

References

Ayobahan, S. U., Alvincz, J., Reinwald, H., Strompen, J., Salinas, G., Schäfers, C., et al. (2023). Comprehensive identification of gene expression fingerprints and biomarkers of sexual endocrine disruption in zebrafish embryo. Ecotoxicol. Environ. Saf. 250, 114514. doi:10.1016/J.ECOENV.2023.114514.

Bennesch, Marcela A., and Didier Picard. 2015. “Minireview: Tipping the Balance: Ligand-Independent Activation of Steroid Receptors.” Molecular Endocrinology 29(3):349–63.

Chamberlain, Nancy L., Erika D. Driverand, and Roger L. Miesfeldi. 1994. The Length and Location of CAG Trinucleotide Repeats in the Androgen Receptor N-Terminal Domain Affect Transactivation Function. Vol. 22.

Denolet, Evi, Karel De Gendt, Joke Allemeersch, Kristof Engelen, Kathleen Marchal, Paul Van Hummelen, Karen A. L. Tan, Richard M. Sharpe, Philippa T. K. Saunders, Johannes V. Swinnen, and Guido Verhoeven. 2006. “The Effect of a Sertoli Cell-Selective Knockout of the Androgen Receptor on Testicular Gene Expression in Prepubertal Mice.” Molecular Endocrinology 20(2):321–34. doi: 10.1210/me.2005-0113.

Fan, Wuqiang, Toshihiko Yanase, Masatoshi Nomura, Taijiro Okabe, Kiminobu Goto, Takashi Sato, Hirotaka Kawano, Shigeaki Kato, and Hajime Nawata. 2005. Androgen Receptor Null Male Mice Develop Late-Onset Obesity Caused by Decreased Energy Expenditure and Lipolytic Activity but Show Normal Insulin Sensitivity With High Adiponectin Secretion. Vol. 54.

Holterhus, Paul-Martin, Olaf Hiort, Janos Demeter, Patrick O. Brown, and James D. Brooks. 2003. Differential Gene-Expression Patterns in Genital Fibroblasts of Normal Males and 46,XY Females with Androgen Insensitivity Syndrome: Evidence for Early Programming Involving the Androgen Receptor. Vol. 4.

Ikeda, Yasumasa, Ken Ichi Aihara, Takashi Sato, Masashi Akaike, Masanori Yoshizumi, Yuki Suzaki, Yuki Izawa, Mitsunori Fujimura, Shunji Hashizume, Midori Kato, Shusuke Yagi, Toshiaki Tamaki, Hirotaka Kawano, Takahiro Matsumoto, Hiroyuki Azuma, Shigeaki Kato, and Toshio Matsumoto. 2005. “Androgen Receptor Gene Knockout Male Mice Exhibit Impaired Cardiac Growth and Exacerbation of Angiotensin II-Induced Cardiac Fibrosis.” Journal of Biological Chemistry 280(33):29661–66. doi: 10.1074/jbc.M411694200.

Jin, Hong Jian, Jung Kim, and Jindan Yu. 2013. “Androgen Receptor Genomic Regulation.” Translational Andrology and Urology 2(3):158–77.

Kang, Zhigang, Asta Pirskanen, Olli A. Jänne, and Jorma J. Palvimo. 2002. “Involvement of Proteasome in the Dynamic Assembly of the Androgen Receptor Transcription Complex.” Journal of Biological Chemistry 277(50):48366–71. doi: 10.1074/jbc.M209074200.

Kanno, Yuichiro, Nao Saito, Ryota Saito, Tomohiro Kosuge, Ryota Shizu, Tomofumi Yatsu, Takuomi Hosaka, Kiyomitsu Nemoto, Keisuke Kato, and Kouichi Yoshinari. 2022. “Differential DNA-Binding and Cofactor Recruitment Are Possible Determinants of the Synthetic Steroid YK11-Dependent Gene Expression by Androgen Receptor in Breast Cancer MDA-MB 453 Cells.” Experimental Cell Research 419(2). doi: 10.1016/j.yexcr.2022.113333.

Karlsson, Sara A., Erik Studer, Petronella Kettunen, and Lars Westberg. 2016. “Neural Androgen Receptors Modulate Gene Expression and Social Recognition but Not Social Investigation.” Frontiers in Behavioral Neuroscience 10(MAR). doi: 10.3389/fnbeh.2016.00041.

Knapczyk-Stwora, Katarzyna, Anna Nynca, Renata E. Ciereszko, Lukasz Paukszto, Jan P. Jastrzebski, Elzbieta Czaja, Patrycja Witek, Marek Koziorowski, and Maria Slomczynska. 2019. “Flutamide-Induced Alterations in Transcriptional Profiling of Neonatal Porcine Ovaries.” Journal of Animal Science and Biotechnology 10(1):1–15. doi: 10.1186/s40104-019-0340-y.

Lamont, K. R., and Tindall, D. J. (2010). Androgen Regulation of Gene Expression. Adv. Cancer Res. 107, 137–162. doi:10.1016/S0065-230X(10)07005-3.

MacLean, Helen E., W. S. Maria Chiu, Amanda J. Notini, Anna-Maree Axell, Rachel A. Davey, Julie F. McManus, Cathy Ma, David R. Plant, Gordon S. Lynch, and Jeffrey D. Zajac. 2008. “ Impaired Skeletal Muscle Development and Function in Male, but Not Female, Genomic Androgen Receptor Knockout Mice .” The FASEB Journal 22(8):2676–89. doi: 10.1096/fj.08-105726.

Maiuri, Paolo, Anna Knezevich, Alex De Marco, Davide Mazza, Anna Kula, Jim G. McNally, and Alessandro Marcello. 2011. “Fast Transcription Rates of RNA Polymerase II in Human Cells.” EMBO Reports 12(12):1280–85. doi: 10.1038/embor.2011.196.

Mora, Gloria R., and Virendra B. Mahesh. 1999. Autoregulation of the Androgen Receptor at the Translational Level: Testosterone Induces Accumulation of Androgen Receptor MRNA in the Rat Ventral Prostate Polyribosomes.

Peng, Yajie, Hui Zhu, Bing Han, Yue Xu, Xuemeng Liu, Huaidong Song, and Jie Qiao. 2021. “Identification of Potential Genes in Pathogenesis and Diagnostic Value Analysis of Partial Androgen Insensitivity Syndrome Using Bioinformatics Analysis.” Frontiers in Endocrinology 12. doi: 10.3389/fendo.2021.731107.

Rana, Kesha, Barbara C. Fam, Michele V Clarke, Tammy P. S. Pang, Jeffrey D. Zajac, and Helen E. Maclean. 2011. “Increased Adiposity in DNA Binding-Dependent Androgen Receptor Knockout Male Mice Associated with Decreased Voluntary Activity and Not Insulin Resistance.” Am J Physiol Endocrinol Me-Tab 301:767–78. doi: 10.1152/ajpendo.00584.2010.-In.

Roy, Arun K., Rakesh K. Tyagi, Chung S. Song, Yan Lavrovsky, Soon C. Ahn, Tae Sung Oh, and Bandana Chatterjee. 2001. “Androgen Receptor: Structural Domains and Functional Dynamics after Ligand-Receptor Interaction.” Pp. 44–57 in Annals of the New York Academy of Sciences. Vol. 949. New York Academy of Sciences.

Russell, Patricia K., Michele V. Clarke, Jarrod P. Skinner, Tammy P. S. Pang, Jeffrey D. Zajac, and Rachel A. Davey. 2012. “Identification of Gene Pathways Altered by Deletion of the Androgen Receptor Specifically in Mineralizing Osteoblasts and Osteocytes in Mice.” Journal of Molecular Endocrinology 49(1):1–10. doi: 10.1530/JME-12-0014.

Shiina, Hiroko, Takahiro Matsumoto, Takashi Sato, Katsuhide Igarashi, Junko Miyamoto, Sayuri Takemasa, Matomo Sakari, Ichiro Takada, Takashi Nakamura, Daniel Metzger, Pierre Chambon, Jun Kanno, Hiroyuki Yoshikawa, and Shigeaki Kato. 2006. Premature Ovarian Failure in Androgen Receptor-Deficient Mice. Vol. 103.

Supakar, P. C., C. S. Song, M. H. Jung, M. A. Slomczynska, J. M. Kim, R. L. Vellanoweth, B. Chatterjee, and A. K. Roy. 1993. “A Novel Regulatory Element Associated with Age-Dependent Expression of the Rat Androgen Receptor Gene.” Journal of Biological Chemistry 268(35):26400–408. doi: 10.1016/s0021-9258(19)74328-2.

Tut, Thein G., Farid J. Ghadessy, M. A. Trifiro, L. Pinsky, and E. L. Yong. 1997. Long Polyglutamine Tracts in the Androgen Receptor Are Associated with Reduced Trans-Activation, Impaired Sperm Production, and Male Infertility*. Vol. 82.

Wang, Ruey Sheng, Shuyuan Yeh, Lu Min Chen, Hung Yun Lin, Caixia Zhang, Jing Ni, Cheng Chia Wu, P. Anthony Di Sant’Agnese, Karen L. DeMesy-Bentley, Chii Ruey Tzeng, and Chawnshang Chang. 2006. “Androgen Receptor in Sertoli Cell Is Essential for Germ Cell Nursery and Junctional Complex Formation in Mouse Testes.” Endocrinology 147(12):5624–33. doi: 10.1210/en.2006-0138.

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Yu, I. Chen, Hung Yun Lin, Ning Chun Liu, Ruey Shen Wang, Janet D. Sparks, Shuyuan Yeh, and Chawnshang Chang. 2008. “Hyperleptinemia without Obesity in Male Mice Lacking Androgen Receptor in Adipose Tissue.” Endocrinology 149(5):2361–68. doi: 10.1210/en.2007-0516.

Yu, Shengqiang, Chiuan Ren Yeh, Yuanjie Niu, Hong Chiang Chang, Yu Chieh Tsai, Harold L. Moses, Chih Rong Shyr, Chawnshang Chang, and Shuyuan Yeh. 2012. “Altered Prostate Epithelial Development in Mice Lacking the Androgen Receptor in Stromal Fibroblasts.” Prostate 72(4):437–49. doi: 10.1002/pros.21445.

Zhang, Caixia, Shuyuan Yeh, Yen-Ta Chen, Cheng-Chia Wu, Kuang-Hsiang Chuang, Hung-Yun Lin, Ruey-Sheng Wang, Yu-Jia Chang, Chamindrani Mendis-Handagama, Liquan Hu, Henry Lardy, Chawnshang Chang, and † † George. 2006. Oligozoospermia with Normal Fertility in Male Mice Lacking the Androgen Receptor in Testis Peritubular Myoid Cells.

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Relationship: 2273: Altered, Transcription of genes by the AR leads to Reduced granulosa cell proliferation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Key Event Relationship Description

Decreased transcription of genes that are downstream of AR activation leads to reduced granulosa cell proliferation of the early-stage gonadotropin-independent ovarian follicles. Therefore, the follicle growth to the antral stage is decreased.

Evidence Supporting this KER

AR is a ligand-activated nuclear transcription factor expressed in the ovaries across mammalian species, including humans(Gervásio et al., 2014). During the gonadotropin independent follicular stage, AR activation is hypothesized to promote follicle growth, whereas in later stages it has been shown to inhibit growth and induce apoptosis(Franks and Hardy, 2018; Harlow et al., 1988). In humans, both mRNA and protein of AR are present in the oocyte, stroma cells, theca cells, but most prominently in granulosa cells of small antral follicles(Gervásio et al., 2014; Jeppesen et al., 2012).

In the mouse ovary, AR mRNA and protein are present in the oocyte, theca, and granulosa cells(Gill et al., 2004; Hirai et al., 1994; Szoltys and Slomczynska, 2000; Tetsuka and Hillier, 1996; Tetsuka et al., 1995). In the cow and sheep ovary, AR mRNA is present in granulosa and theca cells, and most prominently in granulosa of antral and early antral follicles(Hampton et al., 2004; Juengel et al., 2006). In the pig ovary, AR mRNA is mainly expressed in the granulosa cells until the antral stage(Cárdenas and Pope, 2002; Slomczynska et al., 2001). In the monkey ovary, AR mRNA and protein are present in theca, but mainly granulosa cells of antral and early antral follicles(Hillier et al., 1997; Weil et al., 1998).

In human follicles, the expression of the AR transcript is observed after the primordial stage and is most pronounced during the small antral stage(Rice et al., 2007). Throughout early folliculogenesis, AR expression controls transcription of genes involved in promoting growth and differentiation of granulosa cells and formation of antrum(Gervásio et al., 2014). Genes under the control of AR that are involved in these processes include Kit ligand (KITL), Bone morphogenetic protein 15 (BMP15), and Hepatocyte growth factor (HGF)(Astapova et al., 2019; Prizant et al., 2014).

In the monkey ovary, high levels of AR mRNA correlates with high levels of granulosa cell proliferation(Vendola et al., 1998; Weil et al., 1998) Increased AR activation is associated with increased follicle growth and increased granulosa cell proliferation in small antral rat follicles, supporting the important role for AR during this developmental stage(Lim et al., 2017a).

AR may mediate early follicle growth through FSHR, supported by studies correlating mRNA levels of AR and FSHR in granulosa cells of small antral follicles(Nielsen et al., 2011,^,Weil et al., 1999). In mice, FSH-mediated in vitro follicle growth is increased by androgens, suggesting that androgens through AR may act synergistically with FSHR, which in turn increases follicle growth to antral follicles(Sen et al., 2014).

It is also hypothesized that FSHR activation through AR leads to increased AMH expression in granulosa cells of primary to small antral follicles(Lin et al., 2021). In turn, elevated levels of AMH lead to inhibition of FSH-induced aromatase activity, resulting in higher androgen levels that inhibit further follicular growth.

AR activation has been associated with Insulin-like Growth Factor 1 (IGF1) and Insulin-like Growth Factor 1 Receptor (IGFR1) and other key factors of the IGF signaling pathway, which is essential for granulosa growth and differentiation(Baumgarten et al., 2014)^,(Vendola et al., 1999). In human granulosa cells of primordial and primary follicles, AR and IGF-related factors are highly enriched at the transcriptional level(Steffensen et al., 2018).

AR activation affects the level of connexins, proteins that form gap junctions between granulosa cells and the oocyte and hence regulate intracellular communication; a prerequisite for folliculogenesis(Kamal et al., 2020).

In humans, the importance of AR in follicular growth becomes evident with the beneficial effects of androgens in assisted reproductive technology outcomes(Bosdou et al., 2012; Casson et al., 2000; Fábregues et al., 2009; Kim et al., 2011, 2014; Nagels et al., 2015; Noventa et al., 2019; Petya Andreeva, Ivelina Oprova, Luboslava Valkova, Petya Chaveeva, Ivanka Dimova, 2020). Although the mechanism remains elusive, it has been suggested that androgen priming of women seeking fertility treatment promotes follicle growth resulting in an increase in the FSH-sensitive follicle pool(Hu et al., 2017). Gene expression studies in human small antral follicles reveal significant association of AR and FSHR levels, suggesting that the increase in follicle growth could be mediated through regulating AR transcription in granulosa cells(Hu et al., 2017; Nielsen et al., 2011). Epidemiological studies have shown that upon androgen pretreatment, increase in the number of antral follicles and mean follicular diameter were observed(Balasch et al., 2006; Kim et al., 2011). This increase supports the hypothesis that androgen receptor signaling is important for early follicle growth. Studies observing no effects upon androgen pre-treatment claim that dose and duration of the selected androgen might lead to contradicting results(Yeung et al., 2014).

Hypoandrogenism provides further evidence for an important role for androgen actions in human follicle development. Lower levels of DHEA or testosterone have been associated with women that have diminished ovarian reserve or premature ovarian aging(Gleicher et al., 2013). In the case of untreated primary adrenal insufficiency, the androgen deficient patient exhibit significantly reduced fertility(Erichsen et al., 2010).

Conclusions on the androgen significance can also be drawn from clinical evidence where women are exposed to an androgen excess.  Hyperandrogenism in the case of congenital adrenal hyperplasia and exogenous androgen treatments in trans males lead to polycystic ovaries(Walters and Handelsman, 2018). This indicated that the androgens stimulate early follicle growth and inhibit further maturation(Walters and Handelsman, 2018). In polycystic ovarian syndrome, a syndrome characterized by accumulation of small antral follicles in the ovarian cortex, a plausible cause for this morphology is hyperandrogenaemia(Balen et al., 2003; Lebbe and Woodruff, 2013).

Androgen Receptor Knock Out (ARKO) mouse model

In vitro/Ex vivo

In vivo

Quality assessment of the studies was performed and can be found at: QA of Empirical Evidence

Genomic and non-genomic effects are not distinguished in the studies included in the KER analysis. Hence, it cannot be concluded that all observations are solely due to directly perturbed transcription. However, since AR transcribes genes necessary for early folliculogenesis (KITLG, BMP15, HGF), it is reasonable to assume that genomic mechanisms are involved.

Other uncertainties to consider: different anti-androgenic compounds have different effects on the AR (e.g. different IC₅₀, C_max); compounds that are anti-androgenic may also affect other mechanisms/modalities; downstream effects of perturbed AR transcriptional function might depend on the duration of exposure as well as the developmental stage of the follicles. In humans, effects can be inconclusive since a part of the population can have androgen-related conditions such as polycystic ovary syndrome (PCOS)(Gleicher et al., 2011).

Quantitative Understanding of the Linkage

The nature of the response-response relationship between decreased AR activation and reduced granulosa cell proliferation in the early stage of follicular development is not clear. Some of the aforementioned studies claim the effects were dose-dependent; however, the limited number of concentrations tested prevents a solid conclusion(Murray et al., 1998; Wang et al., 2001; Yang and Fortune, 2006). Therefore, at present, the quantitative understanding of this KER is rated ‘low’.

Studies included in establishing this KER exhibit observed changes in vitro at 24h in pig granulosa cells, and in vivo studies after 48h in rats(Hickey et al., 2004, 2005; Kumari et al., 1978). The conclusion that can presently be drawn is that the approximate timescale of the changes in KEdownstream relative to changes in KEupstream is less than 48h. However, the species differences between the time scales of folliculogenesis need to be taken into consideration in order for human extrapolations to be made.

The E3 ubiquitin ligase protein Ring Finger Protein 6 (RNF6) regulates AR levels in granulosa cells through polyubiquitination and AR transcriptional activity for KITLG expression in small antral follicles(Lim et al., 2017b, 2017a).

Epidermal growth factor receptor (EGFR) may mediate the androgen-induced granulosa cell proliferation(Franks and Hardy, 2018).

Sixteen different mutations of the AR gene (Xq11.2-q12) that cause androgen insensitivity syndrome have been identified(Jiang et al., 2020).

The number of CAG repeats on the N-terminal domain of the AR has been associated with effects on fertility and ovarian reserve(Hickey et al., 2002; Lledo et al., 2014).

Activated AR can transcriptionally regulate its own expression through a negative feedback loop(Gelmann, 2002). However, in granulosa cells of monkey ovaries, AR was shown to have the opposite effect, thus creating an autocrine positive feedback(Weil et al., 1998). More studies are needed to understand when AR regulation of its own expression is positive or negative.

During the early stages of folliculogenesis, mainly from the secondary to the small antral stage, activated AR can induce FSH activities in granulosa cells and promote granulosa cell differentiation and follicle maturation, even though follicles are still not gonadotropin-dependent(Gleicher et al., 2011). These activities include changes in androgen metabolism due to altered expression of steroidogenic enzymes. Therefore, it has been suggested that androgens can bind to the AR to establish a loop between activated AR and FSH action(Gleicher et al., 2011; Lenie and Smitz, 2009).

A positive feedback loop connecting activated AR and AMH through FSHR is also hypothesized. Activated AR could lead to increased expression of AMH through FSHR activation, resulting in inhibition of FSH-induced aromatase, ultimately increasing levels of androgens and AR activation(Dewailly et al., 2016). Typically, elevated levels of androgens, for instance in transgender males and PCOS patients, correlate with increased levels of AMH, however, contradictory results exist connecting elevated androgen or FSH levels to reduced AMH(Caanen et al., 2015; Li et al., 2011).

There is also evidence of an intra-follicular feedback loop that regulates steroidogenesis during the secondary follicle stage, causing downregulation of androgen synthesis upon exogenous androgen exposure and upregulation upon androgen receptor antagonism(Lebbe et al., 2017).

As mentioned, genes involved in early folliculogenesis like KITLG and HGF are under the control of AR. Those two genes have been shown to create a positive feedback loop in mice, by increasing the expression levels of each other(Guglielmo et al., 2011).

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Weil, S.J., Vendola, K., Zhou, J., Adesanya, O.O., Wang, J., Okafor, J., and Bondy, C.A. (1998). Androgen receptor gene expression in the primate ovary: cellular localization, regulation, and functional correlations. Journal of Clinical Endocrinology & Metabolism 83, 2479–2485. .

Xue, K., Liu, J.Y., Murphy, B.D., and Tsang, B.K. (2012). Orphan nuclear receptor NR4A1 is a negative regulator of DHT-induced rat preantral follicular growth. Molecular Endocrinology 26, 2004–2015. https://doi.org/10.1210/me.2012-1200.

Xue, K., Kim, J.Y., Liu, J.Y., and Tsang, B.K. (2014). Insulin-like 3-induced rat preantral follicular growth is mediated by growth differentiation factor 9. Endocrinology 155, 156–167. https://doi.org/10.1210/en.2013-1491.

Yang, M.Y., and Fortune, J.E. (2006). Testosterone stimulates the primary to secondary follicle transition in bovine follicles in vitro. Biology of Reproduction 75, 924–932. https://doi.org/10.1095/biolreprod.106.051813.

Yeung, T.W.Y., Chai, J., Li, R.H.W., Lee, V.C.Y., Ho, P.C., and Ng, E.H.Y. (2014). A randomized, controlled, pilot trial on the effect of dehydroepiandrosterone on ovarian response markers, ovarian response, and in vitro fertilization outcomes in poor responders. Fertility and Sterility 102, 108-115.e1. https://doi.org/10.1016/j.fertnstert.2014.03.044.

Relationship: 3142: Reduced granulosa cell proliferation leads to disrupted, ovarian cycle

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

The supporting empirical evidence includes rodent studies and biological plausibility additionally includes humans. However, the taxonomic applicability can be expanded to other mammals as follicle growth is the basic principle driving the ovarian cycle in all mammalian species.

Key Event Relationship Description

This KER connects reduced granulosa cell proliferation of early-stage follicles (gonadotropin-independent, up to antral stage) to ovarian cycle irregularities, which include disturbances in the ovarian cycle (e.g. longer cycle) and/or ovulation problems (deferred ovulation or anovulation). Reduced granulosa cell proliferation manifests as reduced follicle growth, which can be observed by follicle counting or staging.

Evidence Supporting this KER

Ovarian follicles are composed of a centrally located immature oocyte surrounded by supporting somatic cells where granulosa cells make up the inner layer most closely associated with the oocyte. In humans, it is important to emphasize that all follicles are formed during fetal life and this pool of primordial follicles makes up the ovarian reserve. Once primordial follicles are activated, they can grow into primary follicles as granulosa cells proliferate and change in morphology. This transition is driven by factors produced by either the granulosa cells or the oocytes, notably Kit ligand (KITL). As granulosa cells continue to proliferate, the follicles become secondary, a process regulated by factors including Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15). As more layers of granulosa cells are established, the follicle forms an antral cavity. Up to this stage, the growth and survival of the follicles is not dependent on gonadotropins, and thus do not involve the hypothalamus-pituitary-gonadal signaling axis, which is activated during puberty.

For later stages of follicular maturation and ovulation, the gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are essential. They are secreted from the anterior pituitary gland upon stimulation from the gonadotropin-releasing hormone (GnRH) from the hypothalamus. LH stimulates androgen production in theca cells of late-stage ovarian follicles, and is used by the neighbor granulosa cells to produce estrogens upon FSH stimulation. The hormones produced by these late-stage follicles and the corpora lutea formed by ovulated follicles, control the release of hormones from hypothalamus and anterior pituitary. The ovarian cycle results from the cyclic changes that occur in the female reproductive tract and are initiated and regulated by the hypothalamic-pituitary-ovarian (HPO) axis.

All stages of follicles can be found in the ovaries of reproductively active adult mammals, with the majority of the follicles being primordial. Only a minority of the primordial follicles ever complete folliculogenesis, with the majority dying by atresia. The granulosa cells are key determinants of follicle fate. Therefore, disruption of granulosa cell proliferation can halt follicular growth. By reducing the number of available earlier-stage gonadotropin-independent follicles, this will also affect the pool of more mature follicles. Consequently, fewer steroid hormones that regulate the ovarian cycle will be produced, leading to disturbances such as longer cycles. Additionally, less mature follicles will be available for ovulation, leading to e.g. deferred ovulation or anovulation. Biological evidence in humans can be provided by the irregular menstrual cycles observed in perimenopausal individuals (O’Connor et al., 2001).

Selected studies demonstrating an effect on early follicles by reducing their population and leading to effects on estrus cyclicity were selected as empirical evidence for the KER. An effect was observed by histologically identifying, staging, and counting the follicles in all of the included studies. Studies that observed a decrease in primordial follicle counts were excluded, as this indicates an effect on the follicular reserve, rather than on follicle growth.

Table 1. In vivo rodent exposure studies demonstrating reduced granulosa cell proliferation leading to ovarian cycle irregularities.

Table 2. In vivo rodent model demonstrating reduced granulosa cell proliferation leading to ovarian cycle irregularities.

The studies included as empirical evidence report decreased numbers of growing follicles of early stages. However, some of the studies also report increased atresia, which could imply that the observations of altered follicle numbers are a result of stage-specific atresia rather than decreased follicular growth.

During our evidence collection, we identified studies that observed changes in follicle numbers with no effects on the ovarian cycle (Guerra et al., 2011; Ulker et al., 2020). An additional uncertainty is that estrus cyclicity is an endpoint potentially affected by different experimental set-ups, for example, group size, study length and statistical analyses (Goldman et al., 2007). Lastly, ovarian cycle irregularities indicate disturbances in any parts of the Hypothalamic-Pituitary-Ovarian (HPO) axis, which regulates reproductive processes. Therefore, direct effects on hypothalamus and pituitary can lead to uncertainties.

References

Binder, A. K., Peecher, D. L., Qvigstad, A. J., Gutierrez, S. D., Magaña, J., Banks, D. B., & Korach, K. S. (2023). Differential Strain-dependent Ovarian and Metabolic Responses in a Mouse Model of PCOS. Endocrinology (United States), 164(4). https://doi.org/10.1210/endocr/bqad024

Brehm, E., Rattan, S., Gao, L., & Flaws, J. A. (2018). Prenatal exposure to Di(2-ethylhexyl) phthalate causes long-term transgenerational effects on female reproduction in mice. Endocrinology, 159(2), 795–809. https://doi.org/10.1210/en.2017-03004

Brehm, E., Zhou, C., Gao, L., & Flaws, J. A. (2020). Prenatal exposure to an environmentally relevant phthalate mixture accelerates biomarkers of reproductive aging in a multiple and transgenerational manner in female mice. Reproductive Toxicology, 98, 260–268. https://doi.org/10.1016/j.reprotox.2020.10.009

Brown, C., Larocca, J., Pietruska, J., Ota, M., Anderson, L., Smith, S. D., Weston, P., Rasoulpour, T., & Hixon, M. L. (2010). Subfertility Caused by Altered Follicular Development and Oocyte Growth in Female Mice Lacking PKBalpha/Akt1 1. BIOLOGY OF REPRODUCTION, 82, 246–256. https://doi.org/10.1095/biolreprod.109.077925

Du, G., Hu, J., Huang, Z., Yu, M., Lu, C., Wang, X., & Wu, D. (2019). Neonatal and juvenile exposure to perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS): Advance puberty onset and kisspeptin system disturbance in female rats. Ecotoxicology and Environmental Safety, 167, 412–421. https://doi.org/10.1016/j.ecoenv.2018.10.025

Feng, X., Wang, X., Cao, X., Xia, Y., Zhou, R., & Chen, L. (2015). Chronic exposure of female mice to an environmental level of perfluorooctane sulfonate suppresses estrogen synthesis through reduced histone h3k14 acetylation of the StAR promoter leading to deficits in follicular development and ovulation. Toxicological Sciences, 148(2), 368–379. https://doi.org/10.1093/toxsci/kfv197

Goldman, J. M., Murr, A. S., & Cooper, R. L. (2007). The rodent estrous cycle: Characterization of vaginal cytology and its utility in toxicological studies. In Birth Defects Research Part B - Developmental and Reproductive Toxicology (Vol. 80, Issue 2, pp. 84–97). https://doi.org/10.1002/bdrb.20106

Guerra, M. T., de Toledo, F. C., & Kempinas, W. D. G. (2011). In utero and lactational exposure to fenvalerate disrupts reproductive function in female rats. Reproductive Toxicology, 32(3), 298–303. https://doi.org/10.1016/j.reprotox.2011.08.002

Jaroenporn, S., Malaivijitnond, S., Wattanasirmkit, K., Watanabe, G., Taya, K., & Cherdshewasart, W. (2007). Assessment of Fertility and Reproductive Toxicity in Adult Female Mice after Long-Term Exposure to Pueraria mirifica Herb. In Journal of Reproduction and Development (Vol. 53, Issue 5).

Lin, D., Chen, Y., Liang, L., Huang, Z., Guo, Y., Cai, P., & Wang, W. (2023). Effects of exposure to the explosive and environmental pollutant 2,4,6-trinitrotoluene on ovarian follicle development in rats. Environmental Science and Pollution Research, 30(42), 96412–96423. https://doi.org/10.1007/s11356-023-29161-w

Mandour, D. A., Aidaros, A. A. M., & Mohamed, S. (2021). Potential long-term developmental toxicity of in utero and lactational exposure to Triclocarban (TCC) in hampering ovarian folliculogenesis in rat offspring. Acta Histochemica, 123(6). https://doi.org/10.1016/j.acthis.2021.151772

Muto, T., Imano, N., Nakaaki, K., Takahashi, H., Hano, H., Wakui, S., & Furusato, M. (2003). Estrous cyclicity and ovarian follicles in female rats after prenatal exposure to 3,3′,4,4′,5-pentachlorobiphenyl. Toxicology Letters, 143(3), 271–277. https://doi.org/10.1016/S0378-4274(03)00175-9

O’Connor, K. A., Holman, D. J., & Wood, J. W. (2001). Menstrual cycle variability and the perimenopause. American Journal of Human Biology, 13(4), 465–478. https://doi.org/10.1002/ajhb.1078

Sen, A., & Hammes, S. R. (2010). Granulosa cell-specific androgen receptors are critical regulators of ovarian development and function. Molecular Endocrinology, 24(7), 1393–1403. https://doi.org/10.1210/me.2010-0006

Ulker, N., Yardimci, A., Kaya Tektemur, N., Bulmus, O., Kaya, S. O., Bulmus, F. G., Turk, G., & Canpolat, S. (2020). Irisin may have a role in pubertal development and regulation of reproductive function in rats. https://doi.org/10.1530/REP

Walters, K. A., Middleton, L. J., Joseph, S. R., Hazra, R., Jimenez, M., Simanainen, U., Allan, C. M., & Handelsman, D. J. (2012). Targeted loss of androgen receptor signaling in murine granulosa cells of preantral and antral follicles causes female subfertility. Biology of Reproduction, 87(6). https://doi.org/10.1095/biolreprod.112.102012

Xu, C., Chen, J. A., Qiu, Z., Zhao, Q., Luo, J., Yang, L., Zeng, H., Huang, Y., Zhang, L., Cao, J., & Shu, W. (2010). Ovotoxicity and PPAR-mediated aromatase downregulation in female Sprague-Dawley rats following combined oral exposure to benzo[a]pyrene and di-(2-ethylhexyl) phthalate. Toxicology Letters, 199(3), 323–332. https://doi.org/10.1016/j.toxlet.2010.09.015

Yamada, T., Ichihara, G., Wang, H., Yu, X., Maeda, K.-I., Tsukamura, H., Kamijima, M., Nakajima, T., & Takeuchi, Y. (2003). Exposure to 1-Bromopropane Causes Ovarian Dysfunction in Rats. https://academic.oup.com/toxsci/article/71/1/96/1639572

Yin, X., Di, T., Cao, X., Liu, Z., Xie, J., & Zhang, S. (2021). Chronic exposure to perfluorohexane sulfonate leads to a reproduction deficit by suppressing hypothalamic kisspeptin expression in mice. Journal of Ovarian Research, 14(1). https://doi.org/10.1186/s13048-021-00903-z

Zhou, Z., Lin, Q., Xu, X., Illahi, G. S., Dong, C., & Wu, X. (2019). Maternal high-fat diet impairs follicular development of offspring through intraovarian kisspeptin/GPR54 system. Reproductive Biology and Endocrinology, 17(1). https://doi.org/10.1186/s12958-019-0457-z

Relationship: 394: disrupted, ovarian cycle leads to decreased, Fertility

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

In many instances, human female reproductive toxicity of an agent is suspected based on studies performed in experimental animals. The neuroendocrinology, steroid biochemistry, and other physiologic events in the females of most small experimental species often used (mouse, rat, hamster) are similar in their susceptibility to disruption by toxicants (Massaro, 1997).

Although the assessment of the human ovarian cycle may have a variety of biomarkers distinct from those in rats, many of the underlying endocrine mechanisms associated with successful follicular development, ovulation, pregnancy, and parturition are homologous between the two (for review see (Bretveld et al., 2006). For this reason, a toxicant-induced perturbation of ovarian cycles in female rats suggest that a compound may function as a reproductive toxicant in human females.

Mice

• environmental air pollution (Mohallem et al., 2005)
• phthalates (DEHP)
• abortion rate of 100% in F0 dams in the 500-mg/kg/day was observed, in F1 females found that the total number of F2 embryos (exposed to DEHP only as germ cells) was not impaired. However, in the 0.05- and 5-mg DEHP groups, 28% and 29%, respectively, of the blastocysts were degenerated, compared with 8% of controls (Schmidt et al., 2012).
• Lamb et al. studied fertility effects of DEHP in mice (both sexes) and found that DEHP caused dose-dependent decreases in fertility. DBP exposure resulted in a reduction in the numbers of litters per pair and of live pups per litter and in the proportion of pups born alive at the 1.0% amount, but not at lower dose levels. A crossover mating trial demonstrated that female mice, but not males, were affected by DBP, as shown by significant decreases in the percentage of fertile pairs, the number of live pups per litter, the proportion of pups born alive, and live pup weight. DHP in the diet resulted in dose-related adverse effects on the numbers of litters per pair and of live pups per litter and proportion of pups born alive at 0.3, 0.6, and 1.2% DHP in the diet. A crossover mating study demonstrated that both sexes were affected. DEHP (at 0.1 and 0.3%) caused dose-dependent decreases in fertility and in the number and the proportion of pups born alive. A crossover mating trial showed that both sexes were affected by exposure to DEHP. These data demonstrate the ability of the continuous breeding protocol to discriminate the qualitative and quantitative reproductive effects of the more and less active congeners as well as the large differences in reproductive toxicity attributable to subtle changes in the alkyl substitution of phthalate esters (Lamb et al., 1987).

Rat phthalates (DEHP)

• female rats exposed to a high dose of DEHP (3,000 mg/kg/day) had irregular estrous cycles and a slight decline in pregnancy rate (Takai et al., 2009). At 1,000 mg/kg bw/day over a period of 4 weeks did not disturb female fertility or early embryo development.

• There was significant evidence that 5, 15, 50, and 400 mg /kg/day females differed from the control females in the relative amount of time spent in oestrous stages, however no changes were revealed in the number of females with regular cycles, cycle length, number of cycles, and in number of cycling females across the dose groups as compared to the control females The litter size (number of live pups) produced by the P0 generation was significantly reduced in the 400 mg/kg/day dose group (Blystone et al., 2010).

Human

Studies showing a correlation between decreased fertility and;

• professional activity (Olsen, 1994)
• phthalates (DEHP) In occupationally exposed women to high concentration of phthalates exhibit hypoestrogenic anovulary cycles and was associated with decreased pregnancy rate and higher miscarriage rates (Aldyreva,M.V.,Klimove,T.S.,Iziumova,A.S.,Timofeevskaia,L.A., 1975).
• smoking (Hull, North, Taylor, Farrow, & Ford, 2000)
• the use of certain drugs or radiation exposure (Dobson & Felton, 1983)

For the taxonomic applicability see also the Table 1.

Key Event Relationship Description

The ovarian cycle irregularities impact on reproductive capacity of the females that may result in impaired fertility:

1. Irregular cycles may reflect impaired ovulation. Extended vaginal estrus usually indicates that the female cannot spontaneously achieve the ovulatory surge of LH (Huang and Meites, 1975). The persistence of regular vaginal cycles after treatment does not necessarily indicate that ovulation occurred, because luteal tissue may form in follicles that have not ruptured. However, that effect should be reflected in reduced fertility. Conversely, subtle alterations of cyclicity can occur at doses below those that alter fertility (Gray et al., 1989).

2. Persistent or constant vaginal cornification (or vaginal estrus) may result from one or several effects. Typically, in the adult, if the vaginal epithelium becomes cornified and remains so in response to toxicant exposure, it is the result of the agent’s estrogenic properties (i.e., DES or methoxychlor), or the ability of the agent to block ovulation. In the latter case, the follicle persists and endogenous estrogen levels bring about the persistent vaginal cornification. Histologically, the ovaries in persistent estrus will be atrophied following exposure to estrogenic substances. In contrast, the ovaries of females in which ovulation has been blocked because of altered gonadotropin secretion will contain several large follicles and no corpora lutea. Females in constant estrus may be sexually receptive regardless of the mechanism responsible for this altered ovarian condition. However, if ovulation has been blocked by the treatment, an LH surge may be induced by mating (Brown-Grant et al., 1973; Smith, E.R. and Davidson, 1974) and a pregnancy or pseudopregnancy may ensue. The fertility of such matings is reduced (Cooper et al., 1994).

3. Significant delays in ovulation can result in increased embryonic abnormalities and pregnancy loss (Fugo and Butcher, 1966; Cooper et al., 1994).

4. Persistent diestrus indicates temporary or permanent cessation of follicular development and ovulation, and thus at least temporary infertility.

5. Prolonged vaginal diestrus, or anestrus, may be indicative of agents (e.g., polyaromatic hydrocarbons) that interfere with follicular development or deplete the pool of primordial follicles (Mattison and Nightingale, 1980) or agents such as atrazine that interrupt gonadotropin support of the ovary (Cooper et al., 1996). Pseudopregnancy is another altered endocrine state reflected by persistent diestrus. The ovaries of anestrous females are atrophic, with few primary follicles and an unstimulated uterus (Huang and Meites, 1975). Serum estradiol and progesterone are abnormally low.

6. Lengthening of the cycle may be a result of increased duration of either estrus or diestrus.

Evidence Supporting this KER

In females, normal reproductive function involves the appropriate interaction of central nervous system, anterior pituitary, oviducts, uterus, cervix and ovaries. During the reproductive years the ovary is the central organ in this axis. The functional unit within the ovary is the follicle which is composed of theca; granulosa cells and the oocyte. The somatic compartment synthesizes and secrets hormones (steroids and growth factors) necessary for the orchestration of the inter-relationship between the other parts of the reproductive tract and the central nervous system. Oestrus cycle is under strict hormonal control, therefore perturbations of hormonal balance lead to perturbations of normal cyclicity (change in number of cycles or duration of each phase) and/or ovulation problems leading to impaired female reproductive function. However, there are other mechanisms that might result in impaired fertility (e.g cellular maturation in ovary).

Many chemicals are found to interfere with reproductive function in the female. This interference is commonly expressed as a change in normal morphology of the reproductive tract or in ovarian cycle irregularities (disturbance in the duration of particular phases of the estrous cycle and/or ovulation problems). Monitoring estrous cyclicity provides a means to identify alterations in reproductive functions which are mediated through nonestrogenic as well as estrogenic mechanisms (Blasberg, Langan, & Clark, 1997), (Clark, Blasberg, & Brandling-Bennett, 1998). Adverse alteration in the nonpregnant female reproductive system have been observed at dose levels below those that result in reduced fertility or produce other overt effects on pregnancy or pregnancy outcomes. A disruption of cycling caused by xenobiotic treatment can induce a persistent estrus, a persistent diestrus, an irregular pattern with cycles of extended duration and ovulation problems. Common classes of chemicals have been shown to cause cycle irregularities in rats, humans, and non-human primates. Examples include the polychlorinated biphenyls (PCBs) and dioxins, which are associated with such irregularities in rats and humans (e.g (Li, Johnson, & Rozman, 1995) (Meerts et al., 2004), (Chao, Wang, Lin, Lee, & Päpke, 2007) and various agricultural pesticides, including herbicides, fungicides, and fumigants for review see (Bhattacharya & Keating, 2012),(Bretveld, Thomas, Scheepers, Zielhuis, & Roeleveld, 2006).

Table 1 Summary the empirical evidence supporting the KER.

It is known that exposure to 17-β-estradiol can disrupt the normal 4- to 5-day estrous cycle in adult female rats by inducing an extended period of diestrus consistent with pseudopregnancy within 5–7 days after the exposure (Gilmore & McDonald, 1969). This is due to the estrogen-dependent increase in prolactin that rescues ovarian corpora lutea and the subsequent synthesis and release of progesterone (Cooper, R. L., and Goldman, 1999). Significant evidence that the estrous cycle (or menstrual cycle in primates) has been disrupted should be considered an adverse effect (OECD, 2008).

Chemicals may be found to interfere with reproductive function in the female. This interference is commonly expressed as a change in normal morphology of the reproductive tract or a disturbance in the duration of particular phases of the estrous cycle. However, menstrual cyclicity is affected by many parameters such as age, nutritional status, stress, exercise level, certain drugs, and the use of contraceptive measures that alter endocrine feedback. In nonpregnant females, repetitive occurrence of the four stages of the estrous cycle at regular, normal intervals suggests that neuroendocrine control of the cycle and ovarian responses to that control are normal. Even normal, control animals can show irregular cycles. However, a significant alteration compared with controls in the interval between occurrence of estrus for a treatment group is cause for concern. Generally, the cycle will be lengthened or the animals will become acyclic. Therefore changes in cyclicity should be interpreted with caution and not judged adverse without a comprehensive consideration of additional relevant endpoints in a weight-of-evidence approach.

Inconsistencies

Two generation studies by Tyl et al with Butyl benzyl phthalate (BBP) did not observe effects in F0 females on any parameters of estrous cycling, mating, or gestation. However, F1 females carrying F2 litters at and reduced number of total and live pups/litter at birth, with no effects on pre- or postnatal survival (Tyl et al., 2004).

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