Centriole Remodeling during Spermiogenesis in Drosophila

Abstract

The first cell of an animal (zygote) requires centrosomes that are assembled from paternally inherited centrioles and maternally inherited pericentriolar material (PCM) [1]. In some animals, sperm centrioles with typical ultrastructure are the origin of the first centrosomes in the zygote [2-4]. In other animals, however, sperm centrioles lose their proteins and are thought to be degenerated and non-functional during spermiogenesis [5, 6]. Here, we show that the two sperm centrioles (the giant centriole [GC] and the proximal centriole-like structure [PCL]) in Drosophila melanogaster are remodeled during spermiogenesis through protein enrichment and ultrastructure modification in parallel to previously described centrosomal reduction [7]. We found that the ultrastructure of the matured sperm (spermatozoa) centrioles is modified dramatically and that the PCL does not resemble a typical centriole. We also describe a new phenomenon of Poc1 enrichment of the atypical centrioles in the spermatozoa. Using various mutants, protein expression during spermiogenesis, and RNAi knockdown of paternal Poc1, we found that paternal Poc1 enrichment is essential for the formation of centrioles during spermiogenesis and for the formation of centrosomes after fertilization in the zygote. Altogether, these findings demonstrate that the sperm centrioles are remodeled both in their protein composition and in ultrastructure, yet they are functional and are essential for normal embryogenesis in Drosophila.

Keywords: PCL; Poc1; centriole; centrosome; centrosome reaction; embryo; fertilization; giant centriole; sperm; zygote.

Figure 1. Poc1 is enriched in the modified sperm centrioles

A) Poc1BGFP and Ana1tdTomato labels early spermatids (EE) but only Poc1BGFP labels spermatozoa (S) centrioles. B) Poc1BGFP is enriched during spermatogenesis while AslGFP and Ana1tdTomato are reduced. G, Spermatogonia; C, Spermatocyte: R, Round spermatid; A, almost needle spermatid: S, spermatozoa. C) In early-elongated spermatid with round nucleus (N), the PCL with a central tubule (white arrow) is found near the GC inside the CA. D) Correlative light (left panel) and electron microscopy (right panels) find that Ana1GFP labels the microtubule base GC and a novel PCL structures without microtubules in an intermediate elongated spermatid. E–F) A model (E) and TEM (F) of PCL in intermediate-elongated spermatid. G) TEM and a model of PCL in late spermatid. H) Correlative light (last panel) and electron microscopy find that Poc1BGFP (gPoc1ABGFP) labels a modified PCL structures in the spermatozoon. I) TEM and a model of PCL in spermatozoa. J) Summary of GC and PCL proteins during spermiogenesis. P, presence; R, reduced; “-”, undetectable. IN, initial spermatid; R, Round spermatid; EE, elongated spermatid; IE, EE, intermediate elongated spermatid; S, spermatozoa. Also see Figure S1, and Movies S1 and S2.

Figure 2. Poc1A and Poc1B have differential roles in centriole formation and sperm motility

A) Poc1 codes for two isoforms, Poc1A and B, that share seven N-terminal-WD domains coded by exon 1 and a middle segment coded by exon 2, but differ in their C-termini; coded by the end of exon 2 in Poc1A or exon 3 in Poc1B. Poc1 gene structure (black), the two Poc1 protein isoforms (green), the location of poc1 mutations (blue), and two anti-Poc1 antibodies (red). B) poc1^W87X and poc1^c06059 have the shortest GC labeling, poc1^k245 having mild shortening, and poc1^W45R have no effect (N=20). In these mutants, AslGFP and Ana2GFP labeled both the GC (square brackets) and PCL (line), but Ana1GFP/tdTomato selectively labeled the GC. C) The GC (square brackets) and PCL (line) of poc1^W45R sperm expressing gPoc1ABGFP have Ana1tdTomato-labeled PCL. ***, p<0.001; ** p<0.01. D) Maximum intensity projections and quantification of 3DSIM micrographs of sperm centrioles containing GFPAna1, Ana1GFP, and Poc1BGFP. P=0.0001. Poc1BGFP and GFPAna1 (N-terminal Ana1) are found in the PCL center, while Ana1GFP (C-terminal Ana1) is found in the PCL periphery. E) A TEM and model of PCL in intermediate-elongated spermatid of poc1^W87X that has disorganized electron dense wall and is missing the central tubule. Also see Figure S2.

Figure 3. Atypical centrioles are essential for normal fertility and embryogenesis

A) 0–3-minutes-old poc1^W45R_pat-embryos have abnormal PCL function at the decondensed male pronuclear (PN) stage. While Cnn and Asl labels both the GC and PCL of poc1^W45R_pat-embryos D-PLP labels only the GC. B) 0–3-minutes-old poc1_pat-embryos have abnormal centrosome function. Microtubules are not formed around the GC and PCL. C) 0–3-minutes-old poc1^W45R_pat-embryos have monopolar spindles, while control and poc1^W45R_pat-zygotes expressing gPoc1ABGFP have bipolar spindles. D) The pole of 0–3-minutes-old poc1^W45R_pat-zygotes has an Asl labeled GC and maternally expressed Sas-6 tdTomato labeled daughter centriole (DC). E) Unlike 1-hour-old control embryos (cont) or poc1^W45R_pat-embryos expressing gPoc1ABGFP, poc1_pat-embryos have delayed embryo development. F) 72-hour-old poc1^W45R_pat-embryos are arrested at cleavage stage with 2–4 nuclei and post-blastoderm stages (see representative figure). G) Poc1_pat-embryos have reduced larval hatching. A–G) In all these experiments the female had WT Poc1. Also see Figure S3.

Figure 4. Paternal Poc1 enrichment is essential for normal fertility and embryogenesis

A) gPoc1ABGFP, cPoc1AGFP, or the combination of cPoc1AGFP and cPoc1BGFP (cPoc1ABGFP), but not cPoc1BGFP, expressed in poc1^W87X restored sperm motility, indicating that Poc1A is essential for sperm axoneme architecture. B) In poc1^W87X mutants background, Poc1A and B labels specifically the base of the GC and PCL, respectively, in the of round spermatids; and Poc1B also labels the base of the GC in almost needle spermatids. C) Both Poc1A and B are essential for normal GC length. D) poc1^W87X_pat-embryos expressing various Poc1 construct have differential effects on embryo development and spindle formation. Paternal Poc1B is essential for embryo development. E) BamGAL4 expression of UASPoc1 RNAi affects the PCL but not GC length. F) Paternal BamGAL4 and UASPoc1 RNAi reduce early embryo development. G) Interpretive model: during spermatogenesis, the centrosome composition changes with the levels of some proteins decreasing (centrosome reduction) and others increasing (Poc1 enrichment). This remodeling eliminates the pericentriolar material (PCM) and produces centrioles with atypical structure, which after fertilization, form atypical centrosomes by recruiting PCM. The atypical centrosomes are essential for the formation of typical daughter centrioles, pronuclear migration, zygotic cell division, and embryo development. The typical daughter centrioles form normal centrosomes at later stage of embryogenesis. Also see Figure S4.