Atlas of FSHR expression from novel reporter mice

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.

传统观点认为，促卵泡激素-促卵泡激素受体（FSH-FSHR）信号通路是生殖发育与生育能力的关键调控因子。然而，越来越多的证据表明促卵泡激素受体（FSHR）在性腺外组织/器官中存在表达。这引发了关于FSH-FSHR通路生理功能的新问题与长期争论，也凸显了在动物模型中对FSHR表达进行可靠体内分析的必要性。然而，传统方法因存在诸多局限（如可靠抗体稀缺、转录本快速周转/降解，以及缺乏稳定可靠的体内研究工具），已被证实不足以用于检测FSHR的表达。为应对这一挑战，我们利用CRISPR/Cas9基因组编辑技术，将P2A-ZsGreen靶向载体插入Fshr基因最后一个外显子与终止密码子之间的位点，构建了受Fshr内源性启动子调控的Fshr-ZsGreen基因敲入（knockin）报告小鼠。借助这一新型遗传工具，我们实现了对体内Fshr表达的实时、单细胞分辨率水平的可靠检测。我们还对报告小鼠进行了免疫组织化学染色、数字微滴逆转录聚合酶链式反应（ddRT-PCR）及原位杂交等补充分析，以明确FSHR在性腺及性腺外组织/器官中的准确表达谱。我们的研究结果极具说服力，不仅证实了Fshr在性腺内组织中的表达，更令人惊讶的是，还揭示了其在间质细胞（Leydig cells）、成骨细胞谱系细胞、血管结构中的内皮细胞，以及肺支气管和肾小管上皮细胞中的表达显著增加。对Fshr广泛表达模式的遗传解码，凸显了其在生殖与生育之外的生理相关性，并为骨骼、肺、肾、心脏等组织/器官的年龄相关疾病开辟了新的治疗途径。利用Fshr敲入报告小鼠的优势，本研究首次提供了FSHR表达空间分布的全面遗传记录，纠正了关于Fshr表达的长期误解，并为深入探索FSH-FSHR生物学提供了前景。