Temperature response of enriched pre-pubertal caprine male germline stem cells in vitro.

Autor: Singh SP; Animal Physiology and Reproduction Division, ICAR-Central Institute for Research On Goats, Makhdoom, Farah, 281 122, Mathura, Uttar Pradesh, India. shiva.singh@icar.gov.in., Kharche SD; Animal Physiology and Reproduction Division, ICAR-Central Institute for Research On Goats, Makhdoom, Farah, 281 122, Mathura, Uttar Pradesh, India., Pathak M; Animal Physiology and Reproduction Division, ICAR-Central Institute for Research On Goats, Makhdoom, Farah, 281 122, Mathura, Uttar Pradesh, India., Soni YK; Animal Physiology and Reproduction Division, ICAR-Central Institute for Research On Goats, Makhdoom, Farah, 281 122, Mathura, Uttar Pradesh, India., Gururaj K; Animal Health Division, ICAR-Central Institute for Research On Goats, Makhdoom, Farah, 281 122, Mathura, Uttar Pradesh, India., Sharma AK; Animal Physiology and Reproduction Division, ICAR-Central Institute for Research On Goats, Makhdoom, Farah, 281 122, Mathura, Uttar Pradesh, India., Singh MK; Animal Genetics and Breeding Division, ICAR-Central Institute for Research On Goats, Makhdoom, Farah, 281 122, Mathura, Uttar Pradesh, India., Chauhan MS; ICAR-National Dairy Research Institute, Karnal, 132001, Haryana, India.
Jazyk: angličtina
Zdroj: Cell stress & chaperones [Cell Stress Chaperones] 2021 Nov; Vol. 26 (6), pp. 989-1000. Date of Electronic Publication: 2021 Sep 22.
DOI: 10.1007/s12192-021-01236-y
Abstrakt: The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4-5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90 + cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-β1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p < 0.05) higher number of viable cells and greater number of cell clusters, besides higher expression of pluripotency markers. The transcript expression of HSPs (more noticeably HSP72 than HSP73), anti-oxidative enzymes (GPx and CuZnSOD), and adhesion molecule (β1-integrin) was significantly (p < 0.05) downregulated when grown at 35.0, 38.5, or 40.0 °C compared with 37.0 °C. The expression of pluripotency-specific transcripts was significantly (p < 0.05) lower in cmGSCs grown at the culture temperature lower (35.5 °C) or higher (38.5 °C and 40.0 °C) than 37.0 °C. Overall, the culture temperature significantly affects the proliferation, growth characteristics, and expression of heat stress, pluripotency, and adhesion-specific markers in pre-pubertal cmGSCs. These results provide an insight to develop strategies for the improved cultivation and downstream applications of cmGSCs.
(© 2021. Cell Stress Society International.)
Databáze: MEDLINE
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