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3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling combined with image segmentation, centroid mapping and nearest-neighbor analyses in open-source environments, 3D maps of nuclear structures are analyzed in individual cells and normalized to fluorescence standards on the nanometer scale. This protocol fills an unmet need for the application of 3D-SIM to the technically challenging nuclear environment, and subsequent quantitative analysis of 3D nuclear structures and epigenetic modifications. In addition, it establishes practical guidelines and open-source solutions using ImageJ/Fiji and the TANGO plugin for high-quality and routinely comparable data generation in immunostaining experiments that apply across model systems. From sample preparation through image analysis, the protocol can be executed within one week.

X-chromosome dosage compensation in female placental mammals is achieved by X-chromosome inactivation (XCI). An exception are human pre-implantation embryos, where dosage compensation occurs by X-chromosome dampening (XCD). Here, we examined whether XCD extends to human prenatal germ cells given their similarities with naïve pluripotent cells. We found that female human primordial germ cells (hPGCs) display reduced X-linked gene expression before entering meiosis. Moreover, in hPGCs, both X-chromosome are active and express the long non-coding RNAs XACT and XIST , the master regulator of XCI, which are silenced upon entry into meiosis. These findings uncover XACT as hPGC-marker, describe XCD associated with XIST -expression in hPGCs, and suggest that XCD evolved in humans to regulate X-linked genes in pre-implantation embryos and PGCs. Additionally, we found a unique X-chromosome regulation in human primordial oocytes. Therefore, future studies of human germline development must consider the sexually dimorphic X-chromosome dosage compensation mechanisms in the prenatal germline.