The FSH-FSHR signaling pathway has traditionally been considered an essential regulator in reproductive development and fertility. But there has been emerging evidence of FSHR expression in extragonadal tissues/organs. This poses new questions and long-term debates regarding the physiological role of the FSH-FSHR pathway, and underscores the need for reliable, in vivo analysis of FSHR expression in animal models. However, conventional methods have proven insufficient for examining FSHR expression due to limitations, such as the scarcity of ‘reliable’ antibodies, rapid turnover/degradation of transcripts, and a lack of robust in vivo tools. To address this challenge, we developed Fshr-ZsGreen ‘knockin’ reporter mice under the control of the endogenous Fshr promoter using CRISPR/Cas9 genome-editing technology to append a P2A-ZsGreen targeting vector into a site between the last exon and the stop codon of the Fshr locus. With this novel genetic tool, we provide a reliable readout of Fshr expression at single-cell resolution level in vivo and in real time. Reporter animals were also subjected to additional analyses, including immunohistochemical staining, ddRT-PCR, and in situ hybridization, to define the accurate expression profile of FSHR in gonadal and extragonadal organs/tissues. Our compelling results not only demonstrated Fshr expression in gonadal tissues but also, strikingly, unveiled notably increased expression in Leydig cells, osteoblast lineage cells, endothelial cells in vascular structures, and epithelial cells in bronchi of the lung and renal tubes. The genetic decoding of the widespread distribution of Fshr expression highlights its physiological relevance beyond fertility and opens new avenues for therapeutic options for age-related disorders of the bones, lungs, kidneys, and hearts, among other tissues/organs. Exploiting the power of the Fshr knockin reporter animals, this report provides the first comprehensive genetic record of the spatial distribution of FSHR expression, correcting a long-term misconception about Fshr expression and offering prospects for extensive exploration of FSH-FSHR biology.

Atlas of Fshr Expression from Novel Reporter Mice