Continuation of mammalian species requires the formation and development of the sexually dimorphic germ cells. Cultured embryonic stem cells are generally considered pluripotent rather than totipotent because of the failure to detect germline cells under differentiating conditions. Here we show that mouse embryonic stem cells in culture can develop into oogonia that enter meiosis, recruit adjacent cells to form follicle-like structures, and later develop into blastocysts. Oogenesis in culture should contribute to various areas, including nuclear transfer and manipulation of the germ line, and advance studies on fertility treatment and germ and somatic cell interaction and differentiation.

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BackgroundCharacterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the “true” composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits.ResultsWe observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions.ConclusionsBias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0351-6) contains supplementary material, which is available to authorized users.

Gene targeting was used to create mice lacking sperm-associated antigen 6 (Spag6), the murine orthologue of Chlamydomonas PF16, an axonemal protein containing eight armadillo repeats predicted to be important for flagellar motility and stability of the axoneme central apparatus. Within 8 weeks of birth, approximately 50% of Spag6-deficient animals died with hydrocephalus. Spag6-deficient males surviving to maturity were infertile. Their sperm had marked motility defects and was morphologically abnormal with frequent loss of the sperm head and disorganization of flagellar structures, including loss of the central pair of microtubules and disorganization of the outer dense fibers and fibrous sheath. We conclude that Spag6 is essential for sperm flagellar motility and that it is important for the maintenance of the structural integrity of mature sperm. The occurrence of hydrocephalus in the mutant mice also implicates Spag6 in the motility of ependymal cilia.Fertilization is the process whereby sperm and eggs interact reciprocally to begin development. To initiate fertilization, mammalian sperm cells rely on the propulsive forces generated by their flagella to reach the site of fertilization in the oviduct and to penetrate the investments of the egg (8). All flagella contain an axoneme composed of structural elements and motor proteins that work in a coordinated and regulated fashion to produce wave forms that produce progressive movement (3,4,6,8,15,21). The axoneme consists of a central pair of microtubules (central apparatus) surrounded by nine doublets of microtubules with the associated force-generating dynein arms. The basic axonemal structure among cilia and flagella is conserved across species, and much of our understanding of the structure and function of the axoneme has been derived from the study of model organisms. Genetic studies on the green alga, Chlamydomonas, have revealed the importance of several genes for flagellar assembly, stability of specific axonemal structures, and motility (2-6, 15, 21). Inactivation of PF16, one of these Chlamydomonas genes, results in flagellar paralysis (2,20,21). Moreover, when the flagella from the pf16 mutant are demembranated to produce axonemes, the C1 microtubule is destabilized and C1-associated polypeptides are lost. We cloned the human and murine orthologues of PF16, named sperm-associated antigen 6 (Spag6), and found that the amino acid sequences of the mammalian and algal proteins were highly conserved, including the eight armadillo repeats required for the assembly of PF16 onto the C1 microtubule and for flagellar function (11,16,20,21). To determine if Spag6 plays a critical role in the function of the mammalian axoneme, we inactivated mouse Spag6. Males lacking Spag6 were infertile because their sperm had striking motility defects and were frequently decapitated and had disorganized flagellar structures. Approximately 50% of nullizygous males and females have enlarged heads and smaller bodies and die prematurely with hydrocephalus, presumably reflecting a...