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Altered Biology of Testicular VSELs and SSCs by Neonatal Endocrine Disruption Results in Defective Spermatogenesis, Reduced Fertility and Tumor Initiation in Adult Mice

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Abstract

Reproductive health of men has declined in recent past with reduced sperm count and increased incidence of infertility and testicular cancers mainly attributed to endocrine disruption in early life. Present study aims to evaluate whether testicular stem cells including very small embryonic-like stem cells (VSELs) and spermatogonial stem cells (SSCs) get affected by endocrine disruption and result in pathologies in adult life. Effect of treatment on mice pups with estradiol (20 μg on days 5–7) and diethylstilbestrol (DES, 2 μg on days 1–5) was studied on VSELs, SSCs and spermatogonial cells in adult life. Treatment affected spermatogenesis, tubules in Stage VIII & sperm count were reduced along with reduction of meiotic (4n) cells and markers (Prohibitin, Scp3, Protamine). Enumeration of VSELs by flow cytometry (2–6 μm, 7AAD-, LIN-CD45-SCA-1+) and qRT-PCR using specific transcripts for VSELs (Oct-4a, Sox-2, Nanog, Stella, Fragilis), SSCs (tOct-4, Gfra-1, Gpr-125) and early germ cells (Mvh, Dazl) showed several-fold increase but transition from c-Kit negative to c-Kit positive spermatogonial cells was blocked on D100 after treatment. Transcripts specific for apoptosis (Bcl2, Bax) remained unaffected but tumor suppressor (p53) and epigenetic regulator (NP95) transcripts showed marked disruption. 9 of 10 mice exposed to DES showed tumor-like changes. To conclude, endocrine disruption resulted in a tilt towards excessive self-renewal of VSELs (leading to testicular cancer after DES treatment) and blocked differentiation (reduced numbers of c-Kit positive cells, meiosis, sperm count and fertility). Understanding the underlying basis for infertility and cancer initiation from endogenous stem cells through murine modelling will hopefully improve human therapies in future.

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Abbreviations

VSELS:

very small embryonic-like stem cells

SSCs:

spermatogonial stem cells

E2:

estradiol

DES:

diethylstilbestrol

TDS:

Testicular Dysgenesis Syndrome

EDCs:

endocrine-disrupting chemicals

FSH:

follicle stimulating hormone

E:

estrogen

T:

testosterone

LH:

luteinizing hormone

PBS:

phosphate buffer saline

FBS:

fetal bovine serum

CIS:

carcinoma in situ

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Acknowledgements

Authors are thankful to the Central Facilities at the Institute for their help. DB is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

NIRRH Accession Number

NIRRH/MS/RA/850/12–2019.

Funding

We are thankful to Indian Council of Medical Research for providing financial support and to UGC for fellowship (11-04-2016-329,600) to AK.

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  1. Stem Cell Biology Department, ICMR-National Institute for Research in Reproductive Health, Jehangir Merwanji Street, Parel, Mumbai, 400 012, India

    Ankita Kaushik, Sandhya Anand & Deepa Bhartiya

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  1. Ankita Kaushik
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  2. Sandhya Anand
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  3. Deepa Bhartiya
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DB planned and designed the study and arranged financial support. AK performed all the experiments. Data analysis, interpretation and manuscript preparation was done by DB, AK and SA. All authors have read and agreed to final version of the manuscript.

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Correspondence to Deepa Bhartiya.

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Kaushik, A., Anand, S. & Bhartiya, D. Altered Biology of Testicular VSELs and SSCs by Neonatal Endocrine Disruption Results in Defective Spermatogenesis, Reduced Fertility and Tumor Initiation in Adult Mice. Stem Cell Rev and Rep 16, 893–908 (2020). https://doi.org/10.1007/s12015-020-09996-3

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