Impacts of Genetic and Environmental Factors on Reproductive Organ Development

National Institute Of Environmental Health Sciences

Investigators

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Abstract

1. Identify gene-regulatory networks underlying sex determination of murine primordial germ cells Accurate specification of female and male germ cells during embryonic development is critical for sexual reproduction. Primordial germ cells (PGCs) are the bipotential precursors of mature gametes that commit to an oogenic or spermatogenic fate in response to sex-determining cues from the fetal gonad. The critical processes required for PGCs to integrate and respond to signals from the somatic environment in gonads are not understood. In this study, we developed the first single-nucleus multiomics map of chromatin accessibility and gene expression during murine PGC development in both XX and XY embryos. Profiling of cell-type specific transcriptomes and regions of open chromatin from the same cell captured the molecular signatures and gene networks underlying PGC sex determination. Joint RNA and ATAC data for single PGCs resolved previously unreported PGC subpopulations and cataloged a multimodal reference atlas of differentiating PGC clusters. We discovered that regulatory element accessibility precedes gene expression during PGC development, suggesting that changes in chromatin accessibility may prime PGC lineage commitment prior to differentiation. Similarly, we found that sexual dimorphism in chromatin accessibility and gene expression increased temporally in PGCs. Combining single-nucleus sequencing data, we computationally mapped the cohort of transcription factors that regulate the expression of sexually dimorphic genes in PGCs. Finally, we determined the temporal expression patterns of various signaling pathways during PGC sex determination, and our discovery analyses identified potentially new cell communication pathways between supporting cells and PGCs. Our results illustrate the diversity of factors involved in programming PGCs towards a sex-specific fate. 2. Understand how embryonic testes form We discovered a protein called NR2F2 that plays a crucial role in regulating testicular function by controlling the development of fetal Leydig cells, the androgen producing cells in the testes. Testes include various cell types, such as fetal Leydig cells, which produce and secrete hormones that are important for male reproductive development. Impairments in the development of these cells can result in ambiguous genitalia, improper location of the urethral opening, undescended testicles, and infertility. Yet relatively little is known about the origin and development of fetal Leydig cells. To fill this knowledge gap, the researchers analyzed cells in the embryonic mouse testis and used single-nucleus multiomics. They identified cell populations that give rise to mature fetal Leydig cells and pinpointed the molecular mechanisms underlying this process of cell differentiation. In particular, cells containing a nuclear receptor called Nr2f2 serve as progenitors for fetal Leydig cells and regulate the appearance of this unique cell type. Deletion of Nr2f2 in mouse testes led to insufficient testosterone production, ambiguous genitalia, improper location of the urethral opening, and undescended testicles – features found in humans with Nr2f2 mutations. According to the authors, the study reveals molecular targets for diagnosing and treating Nr2f2-associated conditions affecting male reproduction. 3. Decipher the process of male external genitalia formation We have identified a novel cell population that migrates from the hindgut into the external genitalia in the male mouse embryo. This cell population is positive for the orphan nuclear receptor Nr5a1, which serve as a lineage marker for this population. The Nr5a1+ cell population facilitates urethra closure by modulating the function and differentiation of peri-urethra mesenchymal cells via the EGF/Neuregulin pathway. Without the Nr5a1+ cell population, urethral closure fails to occur normally, consequently leading to hypospadias, one of the most common birth defects. We will use this knowledge and the models that we established to interrogate the potential impacts of maternal heat stress on the incidence of hypospadias in male offspring. As a result of climate change, pregnant women are at risk of being exposed to high temperatures during gestation. Such heat stress during pregnancy could lead to an increase in the incidence of hypospadias in male offspring. Males born with hypospadias are susceptible to other disorders later in life (infertility, kidney diseases, etc.), which affects reproductive outcomes and quality of life. Our work will establish a much needed animal model to better understand the impacts of maternal heat stress on hypospadias. The research outcomes will lay the foundation for future research and provide critical knowledge to the field. Our ultimate goal is to develop ways, based on the results of this project, to mitigate the systemic impacts of high heat on the health of mothers and their offspring.