Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice
Jianqiang Bao, Carlos J. Perez, Jeesun Kim, Huan Zhang, Caitlin J. Murphy, Tewfik Hamidi, Jean Jaubert, Craig D. Platt, Janet Chou, Meichun Deng, Meng-Hua Zhou, Yuying Huang, Héctor Gaitán-Peñas, Jean-Louis Guénet, Kevin Lin, Yue Lu, Taiping Chen, Mark T. Bedford, Sharon Y.R. Dent, John H. Richburg, Raúl Estévez, Hui-Lin Pan, Raif S. Geha, Qinghua Shi, Fernando Benavides
Jianqiang Bao, Carlos J. Perez, Jeesun Kim, Huan Zhang, Caitlin J. Murphy, Tewfik Hamidi, Jean Jaubert, Craig D. Platt, Janet Chou, Meichun Deng, Meng-Hua Zhou, Yuying Huang, Héctor Gaitán-Peñas, Jean-Louis Guénet, Kevin Lin, Yue Lu, Taiping Chen, Mark T. Bedford, Sharon Y.R. Dent, John H. Richburg, Raúl Estévez, Hui-Lin Pan, Raif S. Geha, Qinghua Shi, Fernando Benavides
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Research Article Genetics Reproductive biology

Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice

Abstract

Ion channel-controlled cell volume regulation is of fundamental significance to the physiological function of sperm. In addition to volume regulation, LRRC8A-dependent volume-regulated anion channel (VRAC) activity is involved in cell cycle progression, insulin signaling, and cisplatin resistance. Nevertheless, the contribution of LRRC8A and its dependent VRAC activity in the germ cell lineage remain unknown. By utilizing a spontaneous Lrrc8a mouse mutation (c.1325delTG, p.F443*) and genetically engineered mouse models, we demonstrate that LRRC8A-dependent VRAC activity is essential for male germ cell development and fertility. Lrrc8a-null male germ cells undergo progressive degeneration independent of the apoptotic pathway during postnatal testicular development. Lrrc8a-deficient mouse sperm exhibit multiple morphological abnormalities of the flagella (MMAF), a feature commonly observed in the sperm of infertile human patients. Importantly, we identified a human patient with a rare LRRC8A hypomorphic mutation (c.1634G>A, p.Arg545His) possibly linked to Sertoli cell–only syndrome (SCOS), a male sterility disorder characterized by the loss of germ cells. Thus, LRRC8A is a critical factor required for germ cell development and volume regulation in the mouse, and it might serve as a novel diagnostic and therapeutic target for SCOS patients.

Authors

Jianqiang Bao, Carlos J. Perez, Jeesun Kim, Huan Zhang, Caitlin J. Murphy, Tewfik Hamidi, Jean Jaubert, Craig D. Platt, Janet Chou, Meichun Deng, Meng-Hua Zhou, Yuying Huang, Héctor Gaitán-Peñas, Jean-Louis Guénet, Kevin Lin, Yue Lu, Taiping Chen, Mark T. Bedford, Sharon Y.R. Dent, John H. Richburg, Raúl Estévez, Hui-Lin Pan, Raif S. Geha, Qinghua Shi, Fernando Benavides

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Figure 6

A potentially hypomorphic mutation (c.1634G>A, R545H) in the LRRC8A gene identified in a male sterile patient with Sertoli cell–only syndrome (SCOS).

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A potentially hypomorphic mutation (c.1634G>A, R545H) in the LRRC8A g...
(A) Whole exome sequencing (WES) identified a missense point mutation (c.1634G>A, p.Arg545His) in a highly conserved region of LRRC8A (top), resulting in the substitution of arginine (R) to histidine (H) (LRRC8AR545H) (bottom) in a male patient suffering from infertility. (B) Sanger resequencing validated the c.1634G>A mutation in this patient. A red rectangle highlights the mutation site. (C) H&E staining of testis biopsies from the patient (patient 90) and a fertile control. Spg, spermatogonia; Spc, spermatocyte; Spd, spermatid. Scale bar: 15 μm. (D) Three-dimensional homology modeling of protein structure for WT (top) and mutant LRRC8AR545H protein (bottom). Note that the R545H mutation altered not only the distribution of LRR domains, but also the topology of the transmembrane structure. The structural prediction was first performed via the Swiss-model server (https://swissmodel.expasy.org/) based on homology modeling data deposited in the protein data bank (PDB). Data were compiled and visualized using PyMOL (www.pymol.org). TM, transmembrane domain; LRRs, leucine-rich repeats. (E–G) In vitro current recordings in the Xenopus oocyte injection system. In vitro–transcribed cRNAs from fluorescently tagged LRRC8 family members as indicated were coinjected into Xenopus oocytes. Voltage-clamp traces of oocytes are shown for the coinjections of WT LRRC8A-VFP (8A-VFP) or LRRC8AR545H-VFP (8A[R545H]-VFP) with LRRC8C-mCherry (8C-mCh) or LRRC8D-mCherry (8D-mCh) as labeled (E) (n = 30). Comparison of the normalized slope conductance induced by coexpressing 8A-VFP and 8A(R545H)-VFP with 8C-mCherry or 8D-mCherry as indicated (F, left panel). Three batches of cRNAs were injected into 15 oocytes for each group in each assay. Representative Western blot performed using the same injected oocytes to demonstrate comparable expression levels of the injected cRNAs (F, right panel). Black dots indicate the expected size of fluorescently tagged WT or mutant LRRC8A. Comparison of the normalized slope conductance induced by coexpressing 8A HA-VFP and 8A(R545H) HA-VFP with 8C-mCherry or 8D-mCherry as indicated (G, left panel). ELISA-based luminescence assays utilizing the HA tag illustrated comparable expression of the injected, temdomly tagged cRNAs in the oocyte membranes (G, right panel). Three batches of cRNAs were injected into 15 oocytes for each group in each assay. PM, plasma membrane expression. Data represent mean ± SD of 3 independent experiments with 3 batches of cRNA preparations. *P < 0.05, **P < 0.01 (Student’s t test).