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Reproductive stage- and season-dependent culture characteristics of enriched caprine male germline stem cells

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Abstract

The present study aims to evaluate season- and reproductive-stage dependent variation in culture characteristics and expression of pluripotency and adhesion markers in caprine-male germline stem cells (cmGSCs). For this, testes from pre-pubertal (4–6 months) and adult (~ 2 years) bucks during non-breeding (July–August; n = 4 each) and breeding (October–November; n = 4 each) seasons were used to isolated testicular cells by two-step enzymatic digestion. After cmGSCs enrichment by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), cell viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer up to 3rd-passage (P-3). The culture characteristics of cmGSCs were compared during primary culture (P-0) and P-3 with different assays [BrdU-assay (proliferation), MTT-assay (senescence), and Cluster-forming activity-assay] and transcript expression analyses by qRT-PCR. Moreover, the co-localization of UCHL-1, CD90, and DBA was examined by a double-immunofluorescence method. In adult bucks, significantly (p < 0.05) higher cell numbers with the ability to proliferate faster and form a greater number of cell clusters, besides up-regulation of pluripotency and adhesion markers expression were observed during the breeding season than the non-breeding season. In contrast, such season-dependent variation was lacking in pre-pubertal bucks. The expression of transcripts during non-breeding seasons was significantly (p < 0.05) higher in pre-pubertal cmGSCs than in adult cells (UCHL-1 = 2.38-folds; CD-90 = 6.66-folds; PLZF = 20.87-folds; ID-4 = 4.75-folds; E-cadherin = 3.89-folds and β1-integrin = 5.70-folds). Overall, the reproductive stage and season affect the population, culture characteristics, and expression of pluripotency and adhesion specific markers in buck testis. These results provide an insight to develop an efficient system for successful cell culture processes targeting cmGSCs.

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The datasets that pertain to the current study can be made available from the corresponding author on reasonable request.

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Acknowledgements

The authors are thankful for the support extended by the Director of the Institute for providing the necessary facilities to carry out this study. The technical assistance of Mr. Atul Kumar Sharma and the help of Mr. Raj Kumar are duly acknowledged.

Funding

The study was supported by a grant from the Department of Biotechnology (DBT), Government of India, New Delhi (Grant No. BT/PR27544/AAQ/1/715/2018).

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Authors and Affiliations

  1. Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh, 281122, India

    Shiva Pratap Singh, Suresh Dinkar Kharche, Manisha Pathak, Yogesh Kumar Soni & Ravi Ranjan

  2. Animal Genetics and Breeding Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh, 281122, India

    Manoj Kumar Singh

  3. ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India

    Manmohan Singh Chauhan

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  1. Shiva Pratap Singh
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  2. Suresh Dinkar Kharche
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  4. Yogesh Kumar Soni
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  5. Ravi Ranjan
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  6. Manoj Kumar Singh
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  7. Manmohan Singh Chauhan
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Contributions

Conceptualization: SPS, SDK; Methodology: SPS, MP; Formal analysis and investigation: SPS, RR, YKS; Writing—original draft preparation: SDK; MKS; Writing—review and editing: SPS, SDK, MSC; Funding acquisition: SPS; Supervision: MSC.

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Correspondence to Shiva Pratap Singh.

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Singh, S.P., Kharche, S.D., Pathak, M. et al. Reproductive stage- and season-dependent culture characteristics of enriched caprine male germline stem cells. Cytotechnology 74, 123–140 (2022). https://doi.org/10.1007/s10616-021-00515-x

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