In vitro oogenesis from murine premeiotic germ cells using a new three-dimensional culture system.

A faithful reconstitution of the complete process of oogenesis in vitro is helpful for understanding the molecular mechanisms, genetics, and epigenetic changes related to gametogenesis; it can also be useful for clinical drug screening, disease research, and regenerative medicine. To this end, given the consensus that murine female germ cells initiate meiosis at E13.5, substantial works have reported the successful generation of fertile oocytes using E12.5 female gonads as starting materials. Nevertheless, our data demonstrated that murine germ cells at E12.5 have heterogeneously initiated a meiotic transcriptional program based on a measurement of pre-mRNAs (unspliced) and mature mRNAs (spliced) at a single-cell level. Therefore, to establish a platform that faithfully recapitulates the entire process in vitro (from premeiotic murine germ cells to fully developed oocytes), we here report a novel three-dimensional organoid culture (3-DOC) system, which successfully induced fully developed oocytes from E11.5 premeiotic female germ cells (oogonia). Compared with 2D culture and other 3D culture methods, this new culture system is more cost-effective and can create high-quality oocytes similar to in vivo oocytes. In summary, our new culture platform provides an experimental model for future research in regenerative medicine and reproductive biology.

Single-cell transcriptomics allows novel insights into the endocrine-disrupting chemicals induced mammalian reproductive disorder.

Increasing environmental pollution has led to the spread of many endocrine-disrupting chemicals (EDCs) around the world, which are toxic substances in the form of compounds that pose a great threat to the reproductive health of mammals and become a potential cause of many reproductive function-related diseases. In the past decade, the rapid development of single-cell RNA sequencing (scRNA-seq) technology has greatly promoted the study of the toxic mechanisms of EDCs in the mammalian reproductive system, including DEHP, ZEN, BPA, and BDE47. These studies aim to resolve the interference of EDCs in critical stages of reproductive development, including prepubertal and pubertal in males, meiosis I and early follicle formation in females. This paper introduces the sequencing process and analysis methods of current mainstream scRNA-seq technology, systematically reviews the outstanding contributions and specific research ideas of this technology in the study of reproductive system toxicity, lists representative cases of using this technology to explore reproductive damage caused by EDCs, and summarizes in detail the connection between environmental pollution and reproductive development disorders. It provides an important theoretical basis and direction for further exploring the mechanism of damage to the physiological functions of toxic substances on the reproductive system and the prevention and treatment of reproductive diseases.

Murine skin-derived multipotent papillary dermal fibroblast progenitors show germline potential in vitro.

BACKGROUND: Many laboratories have described the in vitro isolation of multipotent cells with stem cell properties from the skin of various species termed skin-derived stem cells (SDSCs). However, the cellular origin of these cells and their capability to give rise, among various cell types, to male germ cells, remain largely unexplored. METHODS: SDSCs were isolated from newborn mice skin, and then differentiated into primordial germ cell-like cells (PGCLCs) in vitro. Single-cell RNA sequencing (scRNA-seq) was then applied to dissect the cellular origin of SDSCs using cells isolated from newborn mouse skin and SDSC colonies. Based on an optimized culture strategy, we successfully generated spermatogonial stem cell-like cells (SSCLCs) in vitro. RESULTS: Here, using scRNA-seq and analyzing the profile of 7543 single-cell transcriptomes from newborn mouse skin and SDSCs, we discovered that they mainly consist of multipotent papillary dermal fibroblast progenitors (pDFPs) residing in the dermal layer. Moreover, we found that epidermal growth factor (EGF) signaling is pivotal for the capability of these progenitors to proliferate and form large colonies in vitro. Finally, we optimized the protocol to efficiently generate PGCLCs from SDSCs. Furthermore, PGCLCs were induced into SSCLCs and these SSCLCs showed meiotic potential when cultured with testicular organoids. CONCLUSIONS: Our findings here identify pDFPs as SDSCs derived from newborn skin and show for the first time that such precursors can be induced to generate cells of the male germline.

Comparative transcriptomic analysis reveals that TPX2 and AURXA are involved in porcine PCV2 infection.

Porcine circovirus type 2 (PCV2) has been a notorious killer for the pig industry, causing substantial economic losses worldwide. However, its pathogenesis is still poorly understood. Comparative transcriptomic analysis and weighted gene co-expression network analysis (WGCNA) were performed in different porcine tissues after PCV2 infection. Our comparative transcriptomic analysis obtained 40 key differentially expressed genes (DEGs), and our WGCNA identified 458 hub genes. Significantly, both TPX2 microtubule nucleation factor (TPX2) and Aurora kinase A (AURKA) are included in these key DEGs and hubs genes. Our gene ontology (GO) analysis indicated that the key DEGs and hub genes participated in cell cycle regulation and immune response. The expressive levels of TPX2 and AURKA went down in the spleen but up in the kidneys after infection with PCV2. We conclude that TPX2 and AURKA played an essential role in PCV2 infection.