Fishman EL , Jo K , Nguyen QPH , Kong D , Royfman R , Cekic AR , Khanal S , Miller AL , Simerly C , Schatten G , Loncarek J , Mennella V , Avidor-Reiss T .

R01 GM098394 NIGMS NIH HHS , R21 HD092700 NICHD NIH HHS

	Fig. 1. Centriolar proteins localize to the DC (group v). a The current dogma is that mature sperm have one centriole. The spermatid neck has a PC and DC, and the ejaculated spermatozoon has a PC and an empty space, the vault, which marks the location of the degenerated DC (Manandhar et al.6). b Graphical summary of the groups of the localization patterns of proteins in the sperm. Group i is centriolar proteins that were localized to the PC area only and is consistent with the dogma. Group ii is PCM proteins that were absent from the sperm, as is consistent with the dogma. Group iii are PCM proteins that were unexpectedly localized to the striated columns or capitulum. Group iv are centriolar proteins that were unexpectedly localized to the striated columns or capitulum, or were unexpectedly absent altogether. Group v are proteins that were unexpectedly present in the DC and the PC, which are inconsistent with the dogma. Group v is shown in c–f. c Antibodies against the centriole tip proteins CETN1/2 and POC5 labeled the PC and DC. d Antibodies against CEP63 and CPAP labeled near the CETN1/2 or POC5-labeled PC and DC. e Antibodies against CEP290 labeled adjacent to the CETN1/2-labeled DC, presumably marking the junction of the DC with the axoneme. f Three distinct antibodies against POC1B labeled both the PC and DC. Unlike other centriolar proteins, POC1B was enriched in the DC relative to the PC (POC1B-P: 2.1 ± 0.5, POC1B-537: 2.74 ± 1.64, POC1B-5G5A7: 1.92 ± 0.41, N > 6). POC1B-P, Rabbit polyclonal antibody from the Pearson lab; POC1B-537, Rabbit polyclonal antibody from the Avidor-Reiss lab; POC1B-5G5A7 Rat monoclonal Ab from the Avidor-Reiss lab. N nucleus, ne sperm neck, t tail; scale bars 1 μm
	Fig. 2. POC1B associates with splayed microtubules at the axoneme base. a The PC and the DC were labeled with POC1B using Correlative Light and Electron Microscopy. These structures were found to be associated with the splayed microtubules (arrowheads, between the axoneme and electron light vault). Scale bars 200 nm. b Four distinct antibodies against tubulins labeled the DC and the PC with greater intensity than in the axoneme. Scale bars 1 μm. c, d Two examples of serial longitudinal sections showed splayed microtubules around an electron light vault using high-pressure freezing-freeze substitution of the sperm neck (c). Serial cross-sections showed splayed microtubules (d, arrowheads). Scale bars 200 nm. e–h Models with side and top views of the human spermatozoon DC (e) and centriole (f), the fly spermatid's PCL (g), and beetle spermatid's PCL (h). Ax axoneme, M mitochondria, mp tail midpiece, N nucleus, ne neck, O outer dance fibers, pp tail principal piece, S striated columns, V vault
	Fig. 3. The precise location of DC proteins. a Diagrams of the six types of DC rods morphologies. b 3D-SIM showed “V”-shaped rods of centriolar proteins POC5 and CETN1/2. c Graph depicting the abundance of the six types of DC rods morphologies. The most common type for both CETN1/2 and POC1B is “V” shape (N~140). POC5 shows both a high rate of “V” shape (~25%), and a higher rate of “reduced” shape. d A diagram depicting the dimensions of the rods (height and width) and their distances from each other (from center to center of the rods) at their tips and bases (N ≥ 20). e 3D-SIM showed “V”-shaped rods of centriolar protein POC1B flanking the splayed microtubules shown with DM1A, α-tubulin (red). f Quantification of the relationship between POC1B shape and tubulin shape in 3D-SIM. Most cells that have “V”-shaped tubulin also have “V”-shaped POC1B, suggesting that their morphology correlates. p = 0.016 by Chi-squared. g Model of the DC in the ejaculated spermatozoon based on electron microscopy, confocal microscopy, and 3D-SIM. Sc striated columns, Ax axoneme, V vault. Scale bars 1 μm
	Fig. 4. POC5 and CETN 1/2 are enriched and redistributed, while CEP152 is reduced. a STORM with POC5 antibodies recognized the PC and DC (left panel). Zoom in on the DC (two middle panels) identifies two major rods (marked as “1” and “2”) and one minor rod (marked as “3”). A diagram (second row, right panel) depicting the dimensions of the rods and their distances from each other (from center to center) at their tips and bases (N ≥ 7). Scale bar 100 nm. b A section of a seminiferous tubule (left panel) depicting the changes in CEP152 (pink) and CETN1/2 (green) in various stages (cells are numbered 1–9). During spermatogenesis, CEP152 and CENT1/2 localizes to 2–4 foci in spermatogonia (cells 1–3). In spermatocytes, CEP152 is maintained, often in large foci surrounding much smaller CENT1/2 lines (cell 4). In round spermatids, CEP152 is localized to the ends of elongated CENT1/2 foci and reduces into two small foci straddling the CENT1/2 line (cells 5–6). Finally, in the elongated spermatids (cells 7–9), CEP152 is localized to the tips of the CENT1/2 “V” shape, before it is finally dramatically reduced. Scale bar 10 μm in low-magnification images (left), and 1 μm in centriole high-magnification images (right). c During spermatogenesis, CEP152 is dramatically reduced (blue), CETN1/2 is slightly reduced (yellow), and POC5 is enriched (red) (N ≥ 3). p-values determined by ANOVA with Fisher’s LSD post hoc. d A section of a seminiferous tubule (left panel) depicting the changes in the distribution of POC5 (pink) and CETN1/2 (green) in various stages (cells 1–4). Both POC5 and CENT1/2 localize to dot-shaped centrioles in spermatocytes (cell 1). In round spermatids, both POC5 and CENT1/2 elongate into a DC that is longer than the PC (cell 2). Finally, in the elongated spermatids (cells 3–4) both CETN1/2 and POC5 appear as “V” shapes. Scale bar 10 μm in low-magnification images (left), and 1 μm in centriole high-magnification images (right). e Quantification of the DC shape by POC5 immunolabeling during spermatogenesis. Most spermatogonia are dot shaped; most round spermatids are elongated; and most elongated spermatids are bifurcated (N ≥ 3, p ≤ 0.00001). p by Chi-squared
	Fig. 5. The remodeled DC is functional in vitro. a–c The human remodeled DC recruits γ-tubulin from Xenopus egg extract. Demembranated human ejaculated spermatozoa did not have γ-tubulin, but after exposure to the extract, γ-tubulin was present near the DC (a). Demembranated spermatozoa with a DC inactivated by methanol pretreatment did not recruit γ-tubulin (b). Quantification (N ≥ 3) of the percent of γ-tubulin-positive sperm pretreated with either buffer or methanol (MeOH) and then exposed to extract (+) or buffer (−) (c). p < 0.001 by t-test. Scale bar 1 μm. Error bars represent +1 standard deviation
	Fig. 6. The remodeled DC is functional in bovine zygotes. a, b The axoneme with a CEP152-labeled DC was observed near the male pronucleus (a) and at the spindle poles (b). a Adjacent pronuclei (red), and the axoneme (arrows) at low magnification on the left. A pair of CEP152-labeled centrioles (arrowhead, right side high magnification) are found at the axoneme base. The bifurcated axoneme (arrows) is observable using DIC (lower right high magnification). b A spindle (red) with an attached axoneme (red, Ax, arrows) and a pair of CEP152-labeled centrioles (green, arrowheads, pole 2) at one pole and CEP152-labeled centrioles at the other pole (green, arrowhead, pole 1). Centrioles are within an aster (red). Scale bar 10 μm (left), and 1 μm (right). c–f Zygote with closely apposed pronuclei with centrioles retained at axoneme base has separated centrioles co-labeled with CEP152 (green) and SAS-6 (red) within a single aster (blue) (f). The upper inset in c shows the centrioles relative to the pronuclei (blue). The lower inset in c shows a DIC image of the axoneme (arrows) overlaid with CEP152-labeled centrioles (box, green). The CEP152 (green, arrowheads, d) and SAS-6-labeled centrioles are within an aster (blue, arrowheads, e). The composite image (f) shows the centrioles (arrowheads) at the bifurcated axoneme base (arrows). Scale bar 10 μm (left), and 1 μm (right). g–m A zygote with two separated centriole pairs (box “1” and “2” in g that each reside in an aster; blue). Upper inset in g, shows DNA (blue), and the labeled centrioles (boxes) relative to the pronucleus (blue). Lower inset in g shows a DIC image of the bifurcated axoneme (arrows) and CEP152-labeled centriole pair 1 (box). h–j Enlarged images of centriole pair 1 showing details of CEP152 (green, arrowheads, h) and SAS-6 (red, arrowheads, j) within the aster (blue). Image j shows centriole pair 1 (arrowheads) at the bifurcated axoneme base (arrows). k–m Enlarged images of centriole pair 2 showing CEP152 (green, arrowhead, k) and SAS-6 (red, arrowheads, l) within the aster (blue). Image (m) shows centriole pair 2 (arrowheads). Scale bar 10 μm (left), and 1 μm (right)

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