5-Ethynyl-2'-deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labeling to facilitate the analysis of the in vivo dynamics of DNA-centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo(-) and DNA polymerase beta. Incorporation of 5-ethynyl-2'-deoxycytidine (EdC) into DNA by these enzymes is, at most, modestly less efficient than native dC. EdC-containing DNA was visualized by using a click reaction with a fluorescent azide, following polymerase incorporation and T4 DNA ligase mediated ligation. Subsequent experiments in mouse male germ cells and zygotes demonstrated that EdC is a specific and reliable reporter of DNA replication, in vivo.

Over the past decade, PIWI-interacting RNAs (piRNAs) have emerged as the most intriguing class of small RNAs. Almost every aspect of piRNA biology defies established rules of the RNA interference world while the scope of piRNA functional potential spans from transcriptional gene silencing to genome defense to transgenerational epigenetic phenomena. This review will focus on the genomic origins, biogenesis, and function of piRNAs in the mouse testis - an exceptionally robust experimental system amenable to genetic, cell-biological, molecular, and biochemical studies. Aided and frequently guided by knowledge obtained in insect, worm, and fish germ cells, mouse spermatogenesis has emerged as the primary model in understanding the role of this conserved pathway in mammals.

Fetal oocyte attrition (FOA) is a conserved but poorly understood process of elimination of more than two-thirds of meiotic prophase I (MPI) oocytes before birth. We now implicate retrotransposons LINE-1 (L1), activated during epigenetic reprogramming of the embryonic germline, in FOA in mice. We show that wild-type fetal oocytes possess differential nuclear levels of L1ORF1p, an L1-encoded protein essential for L1 ribonucleoprotein particle (L1RNP) formation and L1 retrotransposition. We demonstrate that experimental elevation of L1 expression correlates with increased MPI defects, FOA, oocyte aneuploidy, and embryonic lethality. Conversely, reverse transcriptase (RT) inhibitor AZT has a profound effect on the FOA dynamics and meiotic recombination, and it implicates an RT-dependent trigger in oocyte elimination in early MPI. We propose that FOA serves to select oocytes with limited L1 activity that are therefore best suited for the next generation.

Pachytene piRNAs are a class of Piwi-interacting small RNAs abundant in spermatids of the adult mouse testis. They are processed from piRNA primary transcripts by a poorly understood mechanism and, unlike fetal transposon-derived piRNAs, lack complementary targets in the spermatid transcriptome. We report that immunopurified complexes of a conserved piRNA pathway protein Maelstrom (MAEL) are enriched in MIWI (Piwi partner of pachytene piRNAs), Tudor-domain proteins and processing intermediates of pachytene piRNA primary transcripts. We provide evidence of functional significance of these complexes in Mael(129) knockout mice that exhibit spermiogenic arrest with acrosome and flagellum malformation. Mael(129)-null mutant testes possess low levels of piRNAs derived from MAEL-associated piRNA precursors and exhibit reduced translation of numerous spermiogenic mRNAs including those encoding acrosome and flagellum proteins. These translation defects in haploid round spermatids are likely indirect, as neither MAEL nor piRNA precursors associate with polyribosomes, and they may arise from an imbalance between pachytene piRNAs and MIWI.

Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized. However, the protocols are often challenging to follow, partly due to difficulties related to sample preparation, instrument parameters, data display, and selection strategies. In addition, troubleshooting of flow cytometry experiments usually requires some fluency in technical principles and instrument specifications and settings. This unit describes setup and procedures for analysis and sorting of male meiotic prophase I (MPI) cells and other germ cells. Included are procedures that guide data acquisition, display, gating, and back-gating critical for optimal data visualization and cell sorting. Additionally, a flow cytometry analysis of spermatogenesis-defective testis is provided to illustrate the applicability of the technique to the characterization and purification of cells from mutant testis.

Piwi-interacting small RNAs (piRNAs) of fetal prospermatogonia of mice have been strongly implicated in transposon control. In contrast, little is known about biogenesis and function of abundant piRNAs from adult testes expressed in late spermatocytes and round spermatids. These so-called "pachytene" piRNAs are processed from long non-coding piRNA precursors and have no defined RNA targets in the transcriptome even though their binding partner Piwi, MIWI, is essential for spermiogenesis and fertility. Here we report that 129SvJae mice lacking Maelstrom (MAEL), a conserved piRNA pathway protein, exhibit spermiogenic arrest with defects in acrosome and flagellum formation. Further analysis revealed MAEL association with RNPs containing MIWI, TDRD6, and processed intermediates of pachytene piRNA precursors of various length. Loss of MAEL causes a 10-fold drop in pachytene piRNA levels but an increase in piRNAs from abundantly expressed mRNAs. These results suggest a MAEL-dependent mechanism for the selective processing of pachytene piRNA precursor into piRNAs. Strikingly, ribosome profiling of Mael-null testes revealed that reduced piRNA production is accompanied by reduced translation of over 800 spermiogenic mRNAs including those encoding acrosome and flagellum proteins. In light of recent reports of piRNA-independent protection of translationally repressed mRNPs by MIWI and piRNA-dependent turnover of MIWI, we propose that pachytene piRNAs function by controlling the availably of MIWI for the translational repression of spermiogenic mRNAs.

Depletion of oocytes from the embryonic ovary is a key feature of mammalian oogenesis; however, the rational and molecular bases for this phenomenon remain poorly understood. Presently in the field, the most systematic analysis used to understand the effect of a given molecular pathway on fetal oocyte attrition is to count the number of oocytes in ovaries at different stages of development. This analysis is commonly done using a sampling method based on sectioning of the ovary, a technique that includes many laborious steps culminating in an inaccurate estimate of oocyte number contained within that ovary. This inability to generate data that are directly comparable between labs hinders the field and raises questions about the timing and rate of oocyte depletion. Therefore, we set out to implement a robust method that can be easily used by most research laboratories to study the dynamics of oogenesis during fetal mouse ovary development in both normal and experimental conditions. Here we describe an approach to accurately count the total number of oocytes in embryonic ovaries. This method is based on whole-mount immunofluorescence, tissue clearing with sucrose and ScaleA2 reagent, and automatic detection and counting of germ cells in intact ovaries using confocal microscopy and three-dimensional software analyses. We demonstrate the power of the method by assessing variation of fetal oocyte numbers between left and right ovaries and between litters of mice. Finally, we anticipate that the method could be adopted to the analysis of substages of meiotic prophase I and ovarian somatic cells.

Stem cell differentiation involves a delicate balance of gene expression and transposon repression. In this issue of Developmental Cell, Shibata et al. (2016) show that a PIWI protein expressed in planarian stem cells is inherited by their differentiating descendants to ensure regenerative capacity of the flatworm via transposon silencing.

Meiotic prophase I (MPI), is an initial stage of meiosis characterized by intricate homologous chromosome interactions, synapsis, and DNA recombination. These processes depend on the complex, but poorly understood early MPI events of homologous chromosome search, alignment, and pairing. Detailed molecular investigation of these early events requires isolation of individual MPI substages. Enrichment for Pachytene (P) and Diplotene (D) substages of late MPI was previously accomplished using flow cytometry. However, separation of early MPI spermatocytes, specifically, of Leptotene (L) and Zygotene (Z) substages, has been a challenge due to these cells' similar characteristics. In this report, we describe an optimized Hoechst-33342 (Hoechst)-based flow cytometry approach for isolating individual MPI populations from adult mouse testis. We get significant enrichment for individual L and Z spermatocytes, previously inseparable from each other, and optimize the isolation of other MPI substages. Our flow cytometry approach is a combination of three optimized strategies. The first is optimization of testis dissociation protocol that yields more consistent and reproducible testicular single cell suspension. The second involves optimization of flow cytometric gating protocol where a critical addition to the standard protocol for cell discrimination based on Hoechst fluorescence, involves a back-gating technique based on light scattering parameters. This step specifies selection of individual MPI substages. The third, is an addition of DNA content restriction to the gating protocol to minimize contamination from non-meiotic cells. Finally, we confirm significant enrichment of high-purity Preleptotene (PreL), L, Z, P, and D MPI spermatocytes using stage-specific marker distribution. The technique will facilitate understanding of the molecular events underlying MPI. (c) 2014 International Society for Advancement of Cytometry

Germ cells bridge generations and are critical for the successful propagation of species. Early in development, a small group of embryonic cells embarks on a long but fascinating journey that will take them through most dramatic changes in their genomic architecture, epigenetic makeup and cellular morphology (Lesch and Page 2012). The germ cell lineage will multiply, protect and prepare a few original copies of the newly created embryonic genome for the ultimate purpose of fertilization. Failure to precisely fulfill these functions at any level of cellular organization leads to deleterious mutations, chromosomal numerical and structural abnormalities, subfertility or infertility of an organism.