Project description:Spermatogenesis is an intricate developmental process occurring in testes by which spermatogonial stem cells (SSCs) self-renew and differentiate into mature sperm. The molecular mechanisms for SSC self-renewal and differentiation, while have been well studied in mice, may differ between mice and domestic animals including pigs. To gain knowledge about the molecular mechanisms for porcine SSC self-renewal and differentiation that have to date been poorly understood, here we isolated and enriched primitive spermatogonia from neonatal porcine testes, and exposed the cells to retinoic acid, a direct inducer for spermatogonial differentiation. We then identified that retinoic acid could induce porcine primitive spermatogonial differentiation into leptotene spermatocyte-like cells, which was accompanied by a clear transcriptomic alteration, as revealed by the RNA-sequencing analysis. We also compared retinoic acid-induced in vitro porcine spermatogonial differentiation with the in vivo process, and compared retinoic acid-induced in vitro spermatogonial differentiation between pigs and mice. Furthermore, we analyzed retinoic acid-induced differentially expressed long non-coding RNAs (lncRNAs), and demonstrated that a pig-specific lncRNA, lncRNA-106504875, positively regulated porcine spermatogonial proliferation by targeting the core transcription factor ZBTB16. Taken together, these results would help to elucidate the roles of retinoic acid in porcine spermatogonial differentiation, thereby contributing to further knowledge about the molecular mechanisms underlying porcine SSC development and, in the long run, to optimization of both long-term culture and induced differentiation systems for porcine SSCs.
Project description:A bioenergetic balance between glycolysis and mitochondrial respiration is particularly important for stem cell fate specification. It however remains to be determined whether undifferentiated spermatogonia switch their preference of bioenergy production during differentiation. In this study, we found that ATP generation in spermatogonia was gradually increased upon retinoic acid-induced differentiation. To accommodate this elevated energy demand, retinoic acid signaling concomitantly switched ATP production in spermatogonia from glycolysis to mitochondrial respiration, accompanied by increased levels of reactive oxygen species. In addition, inhibition of glucose conversion to glucose-6-phosphate or pentose phosphate pathway blocked the formation of c-Kit+ differentiating germ cells, suggesting that metabolites produced from glycolysis are required for spermatogonial differentiation. We further demonstrated that the expression levels of several metabolic regulators and enzymes were significantly altered upon retinoic acid-induced differentiation by both RNA-seq analyses and quantitative proteomics. Taken together, our data unveil a critically regulated bioenergetic balance between glycolysis and mitochondrial respiration which is required for spermatogonial proliferation and differentiation.
Project description:Retinoic acid triggers differentiation of spermatogonial stem cells by activating the expression of differentiating genes. Long noncoding RNAs (lncRNA) are transcripts longer than 200 nucleotides that do not code for proteins, providing a new perspective of RNA species in gene regulation. However, the function of lncRNAs in determining SSC differentiation has not been investigated. Here, we used high-throughput sequencing to profile all lncRNAs in SSCs, and report the dataset of lncRNAs during SSC differentiation.
Project description:Mouse spermatogonial stem cells (SSCs) continuously self-renew on the feeder layers in serum-free culture medium supplemented with glial cell line-derived neurotrophic factor and fibroblast growth factor 2. To identify novel nuclear proteins involved in SSC maintenance, comparative proteomic profiling of nuclear proteins was performed between self-renewing and differentiation-initiated SSCs in culture. The self-renewing SSC cultures were established from C57BL/6 mouse testes. Nuclear fractions from self-renewing SSC cultures treated with ethanol as a vehicle control (spermatogonial stem cells) and differentiation-initiated SSC cultures treated with 0.3 μM retinoic acid for 24 h (spermatogonial progenitor cells) were isolated for proteomic analysis.
Project description:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.