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

Shiva Pratap Singh; Suresh Dinkar Kharche; Manisha Pathak; Yogesh Kumar Soni; Ravi Ranjan; Manoj Kumar Singh; Manmohan Singh Chauhan

doi:10.1007/s10616-021-00515-x

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

Cytotechnology. 2022 Feb;74(1):123-140. doi: 10.1007/s10616-021-00515-x. Epub 2022 Jan 20.

Authors

Shiva Pratap Singh¹, Suresh Dinkar Kharche¹, Manisha Pathak¹, Yogesh Kumar Soni¹, Ravi Ranjan¹, Manoj Kumar Singh², Manmohan Singh Chauhan³

Affiliations

¹ Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281122 India.
² Animal Genetics and Breeding Division, ICAR-Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281122 India.
³ ICAR-National Dairy Research Institute, Karnal, Haryana 132001 India.

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 3^rd-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.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-021-00515-x.

Keywords: Culture characteristics; Enriched germline stem cells; Goats; Reproductive stage; Season.