Loss of Profilin3 impairs spermiogenesis by affecting acrosome biogenesis, autophagy, manchette development and mitochondrial organization

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis and metastasis of tumor cells. The testis specific PFN3 is localized in the acroplaxome-manchette complex of developing sperm. We demonstrate, that PFN3 further localizes during spermiogenesis in the Golgi complex and proacrosomal vesicles, suggesting a role in acrosome formation. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. Using CRISPR/Cas9 genome editing we generated mice deficient for Pfn3. Pfn3-/- males are sub-fertile displaying a type II-globozoospermia. We revealed, that Pfn3-/- sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3-/- sperm showed reduced sperm motility resulting from flagellum deformities. We show, that acrosome biogenesis is impaired starting from the Golgi phase. RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence mTOR was activated and AMPK was suppressed resulting in the inhibition of autophagy. This dysregulation of AMPK/ mTOR affected the autophagic flux which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. We conclude that this disruption leads to the observed malformation of the acrosome. Further, actin related protein ARPM1 was absent in the nuclear fraction of Pfn3-/- testis and sperm. This suggests, that lack of PFN3 leads to destabilization of the stable PFN3-ARPM1 complex resulting in the degradation of ARPM1. Interestingly, in the Pfn3-/- testis, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2) and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicates this protein as a candidate for male factor infertility in humans. Testicular mRNA of wildtype, Pfn3-deficient and Pfn3-heterozygous miceProfilins（前纤维蛋白，PFNs）是细胞内肌动蛋白聚合的关键调控蛋白，在小鼠和人类基因组中由4个Pfn基因编码。PFNs参与细胞运动性、细胞生长、神经发生以及肿瘤细胞转移等生理病理过程。睾丸特异性PFN3定位于发育中精子的顶体后环-微管领复合物（acroplaxome-manchette complex）。本研究证实，PFN3在精子形成过程中还可定位于高尔基体与前顶体囊泡，提示其参与顶体形成过程。 自噬参与前顶体囊泡的融合与转运过程，以促成顶体形成。本研究利用CRISPR/Cas9基因组编辑技术构建了Pfn3缺陷型小鼠模型。Pfn3敲除（Pfn3-/-）雄性小鼠生育力低下，表现为II型球形精子症。研究发现，Pfn3-/-小鼠的精子存在微管领发育异常，进而导致精子头部呈无定形形态；此外，Pfn3-/-小鼠精子因鞭毛畸形出现运动能力下降。 本研究显示，顶体发生从高尔基体阶段起即出现异常。RNA测序（RNA-seq）分析结果显示，Trim27表达上调，而Atg2a表达下调，进而激活雷帕霉素靶蛋白（mTOR）并抑制腺苷酸活化蛋白激酶（AMPK），最终抑制自噬过程。AMPK/mTOR通路的失调影响了自噬流，其特征表现为LC3B（微管相关蛋白1轻链3B）聚集以及SQSTM1蛋白水平升高。本研究认为，该紊乱正是顶体畸形的成因。 进一步研究发现，Pfn3-/-小鼠睾丸与精子的细胞核组分中缺乏肌动蛋白相关蛋白ARPM1，这提示PFN3缺失会破坏稳定的PFN3-ARPM1复合物，进而导致ARPM1被降解。值得注意的是，在Pfn3-/-小鼠睾丸中，我们检测到关键肌动蛋白调控蛋白丝切蛋白1（CFL1）、丝切蛋白2（CFL2）以及肌动蛋白解聚因子（ADF）的蛋白水平升高。 综上，本研究结果阐明了PFN3在雄性生育中的重要作用，并提示该蛋白可作为人类男性因素不育症的潜在候选靶点。野生型、Pfn3缺陷型及Pfn3杂合型小鼠的睾丸mRNA。