The human sperm basal body is a complex centrosome important for embryo preimplantation development

Abstract

The mechanism of conversion of the human sperm basal body to a centrosome after fertilization, and its role in supporting human early embryogenesis, has not been directly addressed so far. Using proteomics and immunofluorescence studies, we show here that the human zygote inherits a basal body enriched with centrosomal proteins from the sperm, establishing the first functional centrosome of the new organism. Injection of human sperm tails containing the basal body into human oocytes followed by parthenogenetic activation, showed that the centrosome contributes to the robustness of the early cell divisions, increasing the probability of parthenotes reaching the compaction stage. In the absence of the sperm-derived centrosome, pericentriolar material (PCM) components stored in the oocyte can form de novo structures after genome activation, suggesting a tight PCM expression control in zygotes. Our results reveal that the sperm basal body is a complex organelle which converts to a centrosome after fertilization, ensuring the early steps of embryogenesis and successful compaction. However, more experiments are needed to elucidate the exact molecular mechanisms of centrosome inheritance in humans.

Keywords: centriole; centrosome; compaction; embryo early development; fertilization; human; microtubule organizing centers; pericentriolar material.

Figure 1.

Sperm centrioles are stable structures. (A) Super-resolution imaging (STED) of human spermatozoa stained for centrin and β-tubulin. Scale bar: 1 μm. (B) Human sperm immunofluorescence (IF) of acetylated tubulin and β-tubulin in the human sperm centrioles visualized with STED. Scale bar: 1 μm. H, head; M, midpiece; T, tail. N = 2 different sperm samples.

Figure 2.

Sperm centrosome enrichment to identify centrosomal components. (A) IF images on human sperm to visualize Cep63, α-tubulin and DNA. Scale bar: 3 μm, inset: 1 μm. N = 4 different sperm samples. (B) Schematic representation of the sonication and enrichment protocol. Only normozoospermic samples with ≥50% of A + B motility were used. N = 3 different experiments. (C) Western blot analysis of cell lysates from the different sperm fractions shown in B to detect heads (protamine 1) and tails (γ-tubulin). N = 3 different experiments (see Supplementary Information for the uncropped blot). (D) Representative images of intact and sperm fractions stained for DNA (blue), centrin (green) and α-tubulin (red). Scale: 10 μm. N = 3 different experiments.

Figure 3.

Classification of the human sperm tail proteins. (A) Gene Ontology of the 3406 proteins based on their biological process. (B) Gene ontology of the same 3406 proteins based on their subcellular localization. (C) String network of the 251 identified centrosomal proteins. (D) Comparison of the pericentriolar material (PCM) proteins only identified in this work (19.1%—pink fraction) to all the previously published human sperm proteomic data (green fraction). (E–I) Antibodies against five of the centrosomal proteins identified in our proteomic analysis and co-stained with tubulin to visualize the centrioles. Nek9 (E), Poc1B (F) and WDR62 (G) co-localize with the proximal and distal centriole. Pontin (H) and Reptin (I) have a diffuse localization around the basal body. Scale: 3 μm, insets: 1 μm. N = 2 different sperm samples.

Figure 4.

The zygote centrosomal composition is biparentally inherited during fertilization. (A) Venn diagram showing overlap of 48 centrosomal proteins in the human oocyte and sperm. (B) Representative IF image of an in vitro matured metaphase II oocyte stained for pericentrin, tubulin and DNA. Scale: 10 μm. N = 10 oocytes. (C) IF images of abnormally fertilized human embryos at D + 2 stained for pericentrin, tubulin and DNA. Scale: 10 μm. N = 13 fertilized human embryos. (D) Pericentrin, tubulin and DNA staining of a D + 2 parthenogenetically activated human oocyte. Scale: 20 μm. N = 6 activated human oocytes.

Figure 5.

The sperm basal body localizes to the microsurgically separated tails. (A) Schematic representation of our functional assay. (B) IF for centrin, tubulin and DNA on intact sperm. Scale: 7.5 μm. (C) Representative IF images of a manually separated sperm tail stained for centrin, tubulin and DNA. Scale: 7.5 μm. (D) Graph showing the percentage of isolated tails with centrosomes. N = 2 different sperm samples.

Figure 6.

Sperm centrosome inheritance during fertilization ensures parthenotes compaction. (A) The graph on the left shows the percentage of parthenotes that form a blastocyst-like structure at D + 5 in controls and injected oocytes. The images on the right are representative pseudo-blastocysts obtained in control and injected oocytes. Scale: 20 μm. (B) Developmental progress of control versus injected parthenotes. The graph represents the number of parthenotes that achieved each cellular or embryonic stage. (C) Table with the number of control and injected parthenotes in each cellular and embryonic stage. (D) The graph on the left shows the rate of control and injected oocytes that arrested before or after compaction. On the right, representative images of non-compacted and compacted parthenotes. Scale: 20 μm. N = 10 control and N = 15 injected oocytes in 2 independent experiments.

Figure 7.

Microtubule organizing centers (MTOCs) can be formed de novo in control pseudo-blastocysts and after the activation of the embryonic genome. (A) Representative IF images of blastocyst and pseudo-blastocyst of abnormal fertilized oocytes, control and injected oocytes stained for pericentrin, tubulin and DNA. The lower panels are magnifications of the MTOCs for each condition. Scale: 20 μm. (B) Number of MTOCs per cell in abnormal fertilized oocytes, control and injected oocytes. (C) Table showing the percentage of scattered MTOCs per sample. N = 5 3PN embryos, N = 10 control and N = 15 injected oocytes.