Different Effects of Sugars and Methods to Preserve Post-Thaw Functional Properties of Cryopreserved Caprine Spermatogonial Stem Cells.

The present study aimed to identify the effects of sugar and methods (slow freezing [SF] vs. fast freezing [FF]) on post-thaw in vitro functional characteristics of cryopreserved caprine spermatogonial stem cells (cSSCs) and the cells obtained from cryopreserved testis tissue of prepubertal Barbari bucks. For this, in experiment 1, cSSCs were isolated and cryopreserved by either SF or FF method with different non-permeable (sugars; trehalose [140 mm; 140T or 400 mm; 400T] and sucrose [140 mm; 140S or 400 mm; 400S]) or/and permeable (5% ethylene glycol [EG] and dimethyl sulfoxide) cryoprotectants. After 1 week of cryopreservation, the cSSCs were thawed and cultured for evaluation of their characteristics. Further, in experiment 2, the effectiveness of sugars (trehalose [140 mm] or sucrose [140 mm]) for cryopreservation of testicular tissues of prepubertal Barbari bucks using the SF or FF method was evaluated. After 1 week of cryopreservation, the tissues were thawed and cSSCs were isolated and cultured for 3 weeks. In both experiments, cSSCs were evaluated for recovery rate, proliferation, metabolic viability, senescence, and stemness markers' expression. The recovery rate was 1.3-, 1.3-, and 1.1-fold higher in the 140T group compared with EG, 140S, and 400S groups, respectively. Similarly, the expression of stemness markers (protein gene product 9.5 and octamer-binding transcription factor-4) was relatively higher in 140T group compared with the other groups. In experiment 2, the recovery rate of cells per unit tissue weight was significantly (p < 0.05) higher when cryopreserved using 140 mm trehalose compared with other groups. The results of immunocytochemical analyses imply the expression of pluripotent stem cell markers in cSSCs following cryopreservation. Overall, the outcome of the study demonstrates different effects of sugars and methods on post-thaw functional properties of cSSCs with superiority of 140 mm trehalose using SF method over other treatment groups. These results are important for ex vivo expansion and differentiation of cSSCs for fertility preservation and their other downstream applications.

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

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 ß1-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.

Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells.

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O2) environment, whereas, their in-vitro expansion is typically performed under normoxia (20-21% O2). The comparative information about the effects of low and normal O2 levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (ß-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O2) and normoxia (21% O2). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p < 0.05) improved, thus, yielding a significantly (p < 0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O2 conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and ß-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.