Developmental Biology

In mammals, the semen is ejaculated into the female reproductive tract, and the sperm travel to the oviduct to fertilize the egg. A comprehensive understanding of the pre- and post-ejaculatory intrauterine environment is one of the key points for overcoming infertility; however, the dynamics of the intrauterine environment and its physiological role in the uterus, namely in the internal fertilization process, remain unclear. Conventional methods for collecting uterine fluids from the uterus post-ejaculation of mice show challenges regarding the ambiguous ejaculation timing. Here, we established a method for a mating environment with exact ejaculation timing. We also created a simple method for collecting pre- and post-ejaculatory uterine fluid without using forceps. Our methods achieved time-dependent biochemical and histological analyses of uterine fluids to provide fundamental information regarding protein composition and uterine structure changes during pre- and post-ejaculation. This protocol is suitable for analyzing temporal changes in reproductive phenomena, thereby contributing to elucidating the physiological role of the uterus in the process of intrauterine fertilization.

Infertility has emerged as a global health concern, impacting around 8%–12% of couples during their reproductive years. Due to limitations in obtaining human biological samples, mouse models have been widely used for investigating gene functions. Fertility assessment in mouse models is a critical component in reproductive biology for studying gene function and elucidating mechanisms of reproductive disorders. However, natural mating observation of mice may yield inconsistent results, especially in the absence of standard guidelines, prolonged experimental cycles, and operational complexity. This protocol establishes a comprehensive breeding strategy for evaluating murine fertility through systematic vaginal plug monitoring and litter size quantification within defined timeframes. Key steps include (1) standardized male–female pairing protocols, (2) daily vaginal plug inspection, and (3) longitudinal tracking of pregnancy outcomes. This protocol presents a straightforward and easily implementable protocol for mouse mating cage setup and statistical analysis, enabling reliable fertility assessment under natural breeding conditions.

Immunofluorescence staining is a technique that permits the visualization of components of various cell preparations. Manchette, a transient structure that is only present in elongating spermatids, is involved in intra-manchette transport (IMT) for sperm flagella formation. Sperm flagella are assembled by intra-flagellar transport (IFT). Due to the big complexes formed by IMT and IFT components, it has been challenging to visualize these components in tissue sections. This is because the proteins that make up these complexes overlap with each other. Testicular tissue is digested by a combination of DNase I and Collagenase IV enzymes and fixed by paraformaldehyde and sucrose. After permeabilization with Triton X-100, testicular cells are incubated with specific antibodies to detect the components in the manchette and developing sperm tails. This method allows for cell type–specific resolution without interference from surrounding cells like Sertoli, Leydig, or peritubular myoid cells. Additionally, isolated cells produce cleaner immunofluorescence signals compared to other methods like tissue section/whole mount, making this method the best fit for visualizing protein localization in germ cells when spatial context is not being considered. Hence, this protocol provides the detailed methodology for isolating male mice germ cells for antibody-targeted immunofluorescence assay for confocal/fluorescence microscopy.

Super-resolution imaging of RNA–protein (RNP) condensates has shown that most are composed of different immiscible phases reflected by a heterogenous distribution of their main components. Linking RNA–protein condensate’s inner organization with their different functions in mRNA regulation remains a challenge, particularly in multicellular organisms. Drosophila germ granules are a model of RNA–protein condensates known for their role in mRNA storage and localized protein production in the early embryo. Present at the posterior pole of the embryo within a specialized cytoplasm called germplasm, they are composed of maternal mRNAs as well as four main proteins that play a key role in germ granule formation, maintenance, and function. Germ granules are necessary and sufficient to drive germ cell formation through translational regulation of maternal mRNAs such as nanos. Due to their localization at the posterior tip of the ovoid embryo and small size, the classical imaging setup does not provide enough resolution to reach their inner organization. Here, we present a specific mounting design that reduces the distance between the germ granule and the objectives. This method provides optimal resolution for the imaging of germ granules by super-resolution microscopy, allowing us to demonstrate their biphasic organization characterized by the enrichment of the four main proteins in the outermost part of the granule. Furthermore, combined with the direct visualization of nanos mRNA translation using the Suntag approach, this method enables the localization of translation events within the germ granule’s inner organization and thus reveals the spatial organization of its functions. This approach reveals how germ granules serve simultaneously as mRNA storage hubs and sites of translation activation during development. This work also highlights the importance of considering condensates’ inner organization when investigating their functions.

During the first meiotic prophase in mouse, repair of SPO11-induced DNA double-strand breaks (DSBs), facilitating homologous chromosome synapsis, is essential to successfully complete the first meiotic cell division. Recombinases RAD51 and DMC1 play an important role in homology search, but their mechanistic contribution to this process is not fully understood. Super-resolution, single-molecule imaging of RAD51 and DMC1 provides detailed information on recombinase accumulation on DSBs during meiotic prophase. Here, we present a detailed protocol of recombination foci analysis of three-color direct stochastic optical reconstruction microscopy (dSTORM) imaging of SYCP3, RAD51, and DMC1, fluorescently labeled by antibody staining in mouse spermatocytes. This protocol consists of sample preparation, data acquisition, pre-processing, and data analysis. The sample preparation procedure includes an updated version of the nuclear spreading of mouse testicular cells, followed by immunocytochemistry and the preparation steps for dSTORM imaging. Data acquisition consists of three-color dSTORM imaging, which is extensively described. The pre-processing that converts fluorescent signals to localization data also includes channel alignment and image reconstruction, after which regions of interest (ROIs) are identified based on RAD51 and/or DMC1 localization patterns. The data analysis steps then require processing of the fluorescent signal localization within these ROIs into discrete nanofoci, which can be further analyzed. This multistep approach enables the systematic investigation of spatial distributions of proteins associated with individual DSB sites and can be easily adapted for analyses of other foci-forming proteins. All computational scripts and software are freely accessible, making them available to a broad audience.

Key features

• Preparation of spread nuclei, resulting in a flattened preparation with easy antibody-accessible chromatin-associated proteins on dSTORM-compatible coverslips.

• dSTORM analysis of immunofluorescent repair foci in meiotic prophase nuclei.

• Detailed descriptions of data acquisition, (pre-)processing, and nanofoci feature analysis applicable to all proteins that assemble in immunodetection as discrete foci.

Graphical overview