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Phospholipase D Family Member 6 Is a Surface Marker for Enrichment of Undifferentiated Spermatogonia in Prepubertal Boars.

ABSTRACT: Spermatogonial stem cells (SSCs) have a functional ability to maintain self-renewal and sustain production of spermatozoa throughout the reproductive lifespan of a male. Studies on SSCs can thus better the understandings of spermatogenesis and unravel the mechanisms for self-renewal and differentiation of male germline stem cells. However, the rarity of SSCs in the testis and the lack of reliable surface markers obstruct the related study and further application of SSCs. This is especially the case in livestock animals. In this study, we identified that phospholipase D family member 6 (PLD6) is a surface marker for undifferentiated spermatogonia in boar testes. By magnetic-activated cell sorting, PLD6+ cell fraction comprises 84.45%?±?0.35% of undifferentiated spermatogonia (marked by PLZF). Xenotransplantation of PLD6+ cells into the recipient mouse testis revealed a ninefold increase of donor cell-derived colony formation compared with that in the unselected cell group, indicating the significant enrichment of SSCs. Furthermore, based on the sorted PLD6+ cells with a high SSC content, we established a feeder-free culture system that could maintain porcine undifferentiated spermatogonia for 4 weeks in vitro with the expression of typical markers throughout the culture period. In conclusion, this study demonstrates that PLD6 is a surface marker of undifferentiated spermatogonia in testes of prepubertal boars and could be utilized to unprecedentedly enrich porcine undifferentiated spermatogonia. These data provide the basis for future studies on the refinement of germ cell culture and manipulation of porcine undifferentiated spermatogonia.

SUBMITTER: Zhang P

PROVIDER: S-EPMC5756458 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Phospholipase D Family Member 6 Is a Surface Marker for Enrichment of Undifferentiated Spermatogonia in Prepubertal Boars.

Zhang Pengfei P Qin Yuwei Y Zheng Yi Y Zeng Wenxian W

Stem cells and development 20171206 1

Spermatogonial stem cells (SSCs) have a functional ability to maintain self-renewal and sustain production of spermatozoa throughout the reproductive lifespan of a male. Studies on SSCs can thus better the understandings of spermatogenesis and unravel the mechanisms for self-renewal and differentiation of male germline stem cells. However, the rarity of SSCs in the testis and the lack of reliable surface markers obstruct the related study and further application of SSCs. This is especially the c ...[more]

PMID: 29113556

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Project description:BACKGROUND:Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)(4)-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. METHODOLOGY/PRINCIPAL FINDINGS:PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. CONCLUSIONS/SIGNIFICANCE:Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.

Dataset Information

Phospholipase D Family Member 6 Is a Surface Marker for Enrichment of Undifferentiated Spermatogonia in Prepubertal Boars.

Publications

Phospholipase D Family Member 6 Is a Surface Marker for Enrichment of Undifferentiated Spermatogonia in Prepubertal Boars.

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