Abstract
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 testes-specific PFN3 is localized in the acroplaxome–manchette complex of developing spermatozoa. We demonstrate that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in vesicle transport for acrosome formation. Using CRISPR/Cas9 genome editing, we generated mice deficient for Pfn3. Pfn3–/– males are subfertile, 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, and mature sperm seems to suffer from a cytoplasm removal defect. An 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. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. TRIM27 is associated with PFN3 as determined by co-immunoprecipitation from testis extracts. Further, actin-related protein ARPM1 was absent in the nuclear fraction of Pfn3–/– testes and sperm. This suggests that lack of PFN3 leads to destabilization of the PFN3–ARPM1 complex, resulting in the degradation of ARPM1. Interestingly, in the Pfn3–/– testes, 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 implicate this protein as a candidate for male factor infertility in humans.
Highlights
Spermatogenesis is defined as the process of producing mature spermatozoa from spermatogonia
We examined the distribution of PFN3 at ultrastructural level during spermiogenesis with a PFN3 polyclonal antibody and a gold-conjugated secondary antibody using immunoelectron microscopy
We have shown that deletion of Pfn3 results in male subfertility hallmarked by reduced sperm count/vitality with sperm displaying type II globozoospermia
Summary
Spermatogenesis is defined as the process of producing mature spermatozoa from spermatogonia It comprises three phases, (i) spermatocytogenesis (mitotic), (ii) meiotic, and (iii) spermiogenesis. Haploid round spermatids are transformed into elongated spermatozoa (Oakberg, 1956; Agnew, 1997) This is achieved by a series of cellular reconstruction processes such as formation of the acrosome and tail, replacement of histones and protamineinduced DNA hypercondensation, removal of most of the cytoplasm, and rearrangement of mitochondria along the neck and tail region of the sperm. All mammalian spermatozoa are divided into two basic units based on their function—the head and the flagellum Both units are shaped and assembled during spermiogenesis, which is the cytomorphogenic phase of spermatogenesis (Khawar et al, 2019). In the final/maturation phase, the acrosome spreads over the anterior half in parallel to the elongation of the sperm head
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