The ovary is perhaps the most dynamic organ in the human body, only rivaled by the uterus. The molecular mechanisms that regulate follicular growth and regression, ensuring ovarian tissue homeostasis, remain elusive. We have performed single-cell RNA-sequencing using human adult ovaries to provide a map of the molecular signature of growing and regressing follicular populations. We have identified different types of granulosa and theca cells and detected local production of components of the complement system by (atretic) theca cells and stromal cells. We also have detected a mixture of adaptive and innate immune cells, as well as several types of endothelial and smooth muscle cells to aid the remodeling process. Our results highlight the relevance of mapping whole adult organs at the single-cell level and reflect ongoing efforts to map the human body. The association between complement system and follicular remodeling may provide key insights in reproductive biology and (in)fertility.

cPseudomonas aeruginosa is an opportunistic pathogen that causes considerable morbidity and mortality, specifically during intensive care. Antibiotic-resistant variants of this organism are more difficult to treat and cause substantial extra costs compared to susceptible strains. In the laboratory, P. aeruginosa rapidly developed resistance to five medically relevant antibiotics upon exposure to stepwise increasing concentrations. At several time points during the acquisition of resistance, samples were taken for whole-genome sequencing. The increase in the MIC of ciprofloxacin was linked to specific mutations in gyrA, parC, and gyrB, appearing sequentially. In the case of tobramycin, mutations in fusA, HP02880, rplB, and capD were induced. The MICs of the beta-lactam compounds meropenem and ceftazidime and the combination of piperacillin and tazobactam correlated linearly with beta-lactamase activity but not always with individual mutations. The genes that were mutated during the development of beta-lactam resistance differed for each antibiotic. A quantitative relationship between the frequency of mutations and the increase in resistance could not be established for any of the antibiotics. When the adapted strains are grown in the absence of the antibiotic, some mutations remained and others were reversed, but this reversal did not necessarily lower the MIC. The increased MIC came at the cost of moderately reduced cellular functions or a somewhat lower growth rate. In all cases except ciprofloxacin, the increase in resistance seems to be the result of complex interactions among several cellular systems rather than individual mutations.T he medical consequences of antibiotic resistance, such as fewer options for and increased costs of treating infectious diseases, are well recognized. The pathway to resistance consists of sequential mutations or acquisition of resistance genes driven by the selective pressure caused by antibiotic exposure (1). Once resistance has been acquired, the cell rarely reverses to become sensitive again, compensating for the metabolic costs instead (2, 3). The increased level of resistance caused by an antibiotic treatment typically prescribed by primary care physicians is very noticeable when subsequent further treatment is necessary (4). Hence, in order to limit the development of resistance when antibiotics have to be used, treatment protocols need to be devised to prevent this side effect. Rational design of such protocols requires knowledge of the molecular mechanisms that cause resistance. One of the central questions is whether similar mechanisms are operational for all drugs or whether resistance to each drug is induced in a distinct manner. Other basic questions center on evolutionary pathways to clinically significant resistance and the persistence of molecular changes after treatment.Molecular changes that cause the development and persistence of drug resistance can be identified by combining experimental evolution and whole-genome sequencing (WGS), provided that the proper c...

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In contrast to mouse, human female germ cells develop asynchronously. Germ cells transition to meiosis, erase genomic imprints, and reactivate the X chromosome. It is unknown if these events all appear asynchronously, and how they relate to each other. Here we combine exome sequencing of human fetal and maternal tissues with single-cell RNA-sequencing of five donors. We reconstruct full parental haplotypes and quantify changes in parental allele-specific expression, genome-wide. First we distinguish primordial germ cells (PGC), pre-meiotic, and meiotic transcriptional stages. Next we demonstrate that germ cells from various stages monoallelically express imprinted genes and confirm this by methylation patterns. Finally, we show that roughly 30% of the PGCs are still reactivating their inactive X chromosome and that this is related to transcriptional stage rather than fetal age. Altogether, we uncover the complexity and cell-to-cell heterogeneity of transcriptional and epigenetic remodeling in female human germ cells.

Human ovarian folliculogenesis is a highly regulated and complex process. Characterization of follicular cell signatures during this dynamic process is important to understand follicle fate (to grow, become dominant, or undergo atresia). The transcriptional signature of human oocytes and granulosa cells (GCs) in early-growing and ovulatory follicles have been previously described; however, that of oocytes with surrounding GCs in small antral follicles have not been studied yet. Here, we have generated a unique dataset of single-cell transcriptomics (SmartSeq2) consisting of the oocyte with surrounding GCs from several individual (non-dominant) small antral follicles isolated from adult human ovaries. We have identified two main types of (healthy) follicles, with a distinct oocyte and GC signature. Using the CellphoneDB algorithm, we then investigated the bi-directional ligand–receptor interactions regarding the transforming growth factor-β (TGFβ)/bone morphogenetic protein (BMP), wingless-type (MMTV)-integration site (WNT), NOTCH, and receptor tyrosine kinases (RTK) signaling pathways between oocyte and GCs within each antral follicle type. Our work not only revealed the diversity of small antral follicles, but also contributes to fill the gap in mapping the molecular landscape of human folliculogenesis and oogenesis.