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In Vitro Meiosis of Male Germline Stem Cells.

ABSTRACT: In vitro spermatogenesis has been achieved by culturing mouse embryonic stem cells (ESCs) together with a cell suspension of male juvenile gonad. However, for human fertility treatment or preservation, patient-specific ESCs or juvenile gonad is not available. We therefore aim to achieve in vitro spermatogenesis using male germline stem cells (GSCs) without the use of juvenile gonad. GSCs, when cultured on immortalized Sertoli cells, were able to enter meiosis, reach the meiotic metaphase stages, and sporadically form spermatid-like cells. However, the in vitro-formed pachytene-like spermatocytes did not display full chromosome synapsis and did not form meiotic crossovers. Despite this, the meiotic checkpoints that usually eliminate such cells to prevent genomic instabilities from being transmitted to the offspring were not activated, allowing the cells to proceed to the meiotic metaphase stages. In vitro-generated spermatid-like cells should thus be thoroughly investigated before being considered for clinical use.

SUBMITTER: Lei Q

PROVIDER: S-EPMC7664054 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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In Vitro Meiosis of Male Germline Stem Cells.

Lei Qijing Q Lai Xin X Eliveld Jitske J Chuva de Sousa Lopes Susana M SM van Pelt Ans M M AMM Hamer Geert G

Stem cell reports 20201101 5

In vitro spermatogenesis has been achieved by culturing mouse embryonic stem cells (ESCs) together with a cell suspension of male juvenile gonad. However, for human fertility treatment or preservation, patient-specific ESCs or juvenile gonad is not available. We therefore aim to achieve in vitro spermatogenesis using male germline stem cells (GSCs) without the use of juvenile gonad. GSCs, when cultured on immortalized Sertoli cells, were able to enter meiosis, reach the meiotic metaphase stages, ...[more]

PMID: 33176123

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Project description: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.

Project description:BackgroundMale germline stem cells (mGSCs) offer great promise in regenerative medicine and animal breeding due to their capacity to maintain self-renewal and to transmit genetic information to the next generation following spermatogenesis. Human testis-derived embryonic stem cell-like cells have been shown to possess potential of mesenchymal progenitors, but there remains confusion about the characteristics and origin of porcine testis-derived stem cells.MethodsPorcine testis-derived stem cells were obtained from primary testicular cultures of 5-day old piglets, and selectively expanded using culture conditions for long-term culture and induction differentiation. The stem cell properties of porcine testis-derived stem cells were subsequently assessed by determining the expression of pluripotency-associated markers, alkaline phosphatase (AP) activity, and capacity for sperm and multilineage differentiation in vitro. The gene expression profile was compared via microarray analysis.ResultsWe identified two different types of testis-derived stem cells (termed as C1 and C2 here) during porcine testicular cell culture. The gene expression microarray analysis showed that the transcriptome profile of C1 and C2 differed significantly from each other. The C1 appeared to be morphologically similar to the previously described mouse mGSCs, expressed pluripotency- and germ cell-associated markers, maintained the paternal imprinted pattern of H19, displayed alkaline phosphatase activity, and could differentiate into sperm. Together, these data suggest that C1 represent the porcine mGSC population. Conversely, the C2 appeared similar to the previously described porcine mGSCs with three-dimensional morphology, abundantly expressed Leydig cell lineage and mesenchymal cell-specific markers, and could differentiate into testosterone-producing Leydig cells, suggesting that they are progenitor Leydig cells (PLCs).ConclusionCollectively, we have established the expected characteristics and markers of authentic porcine mGSCs (C1). We found for the first time that, the C2, equivalent to previously claimed porcine mGSCs, are actually progenitor Leydig cells (PLCs). These findings provide new insights into the discrepancies among previous reports and future identification and analyses of testis-derived stem cells.

Dataset Information

In Vitro Meiosis of Male Germline Stem Cells.

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In Vitro Meiosis of Male Germline Stem Cells.

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