Abstract
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 Fshr endogenous promoter using CRISPR/Cas9 genome-editing technology to append a P2A-ZsGreen targeting vector into a locus between the last exon and the stop codon of Fshr. 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 intragonadal 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 pattern of Fshr expression highlights its physiological relevance beyond reproduction and 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Here is a summary of the revision: 1.New data on Fshr expression are input to the revised Manuscript: (1)Fshr expression in the testis and adipose tissues (WAT and BAT) of B6 mice; (2)Fshr expression in the testis of B6 by RNA-smFISH; (3)Comparison of Fshr expression in the testis and ovary between Fshr-ZsGreen and B8 mice by ddRT-PCR to prove Fshr expression without interruptions by insertion of P2A-ZsGreen vector; (4)Reduction of Fshr expression in osteocytes within the femoral sections from DMP1-CreERT2:Fshrfl/fl mice; (5)Fshr expression in an established Leydig cell line-TM3 by immunofluorescence and ddRT-PCR, also show Fshr located in the nuclei of TM3 cells; (6)Fshr expression at scRNA-seq level from 5 public single cell portals as Supplementary Data 3 to support our findings of the widespread expression pattern of Fshr, particularly in Leydig cells. 2.Re-organization of Figure 2 with a new legend. 3.A new paragraph is added to the Discussion Section of the revised MS to explain the function of P2A peptide in generation of GFP reporter mice and why Fshr express is not interrupted by the P2A-ZsGreen insertion in Fshr-ZsGreen reporter. 4.Deletion of Figure 1-D-c, as it is not necessary. 5.Replace of Figure 8-A (the left panel) with a reduced exposure time image.
