Fig 1.
Ultrastructure of early spermatocyte development in H. membranacea.
(A) TEM image of a spermatocyte in prophase of meiosis I. The nucleus (N) exhibits condensed chromatin (Chr) with synaptonemal complexes. Other organelles like the Golgi apparatus (G), the endoplasmic reticulum (ER) and mitochondria (M) are positioned in the vicinity of the nucleus. (B) Spermatocyte in prophase I. The nucleus (N) with a visible nuclear envelope (NE), a nucleolus (Nuc) and paired homologous chromosomes (Chr) are shown. The endoplasmic reticulum (ER) is positioned adjacent to the nucleus. (C) High-magnification view of a nucleus with condensed chromatin and a nucleolus. The central elements of synaptonemal complexes appear as single, double or triple complexes, a phenomenon known as polycomplexes (PC). The inset shows a synaptonemal complex at higher magnification. (DI) Tomographic slice of a 3D reconstruction of a nucleus in prophase I. In this semi-thick section, the chromatin is positioned around multiple synaptonemal complexes and polycomplexes. (DII) 3D model of the nuclear envelope (red), the chromatin (purple) and central elements of the polycomplexes (orange). Same cell as shown in (DI). (E) TEM image of a cell at prometaphase I. The chromosomes are condensed and the nuclear envelope is broken down. Mitochondria surround the chromosomes. The ER and electron-dense membrane-free aggregates (arrowheads) are visible in the cytoplasm. (F) TEM image of a spermatocyte presumably in meiosis II. The nuclear envelope appears open (arrow heads). (G) TEM of connective tissue (CT) separating the individual testicular follicles. The images are not shown according to the correct sequence of the developmental stages.
Fig 2.
Fine structure of differentiating sperm cells.
(A) Phase contrast image of a spermatid in an early stage of sperm maturation. The nucleus (N) and an irregular shaped nebenkern (NK) are shown. (B) Phase contrast image of a maturing spermatid. The nebenkern shows internal structures that cannot be resolved by phase contrast light microscopy. (C) Second example of a matured spermatocyte with an elongated, crescent-shaped nebenkern. Adjacent to the nucleus, a dark structure is visible that most likely represents a centriolar adjunct (CA) connecting the basal body to the nuclear envelope. (D) Spermatocyte with an axoneme (Ax) connected by the centriole adjunct material to the nucleus. The nebenkern appears elongated and spanning the whole volume of the cell. Scale bar for (A-D), 5 μm. (E) TEM image of a cell in late telophase II. The chromatin is still densely packed inside the nucleus, which is surrounded by endoplasmic reticulum (ER). Mitochondria (M) start to aggregate at a position close to the nucleus. (F-G) Sperm cell with clustered mitochondria, ER and Golgi (G). The nucleus (N) shows a fenestrated nuclear envelope (arrow heads). This fenestration is likely to be involved in a shrinkage and compaction of the nucleoplasm. (H) Nebenkern and nucleus with an attached basal body (BB) anchored through the centriolar adjunct structure (CA). The developing axoneme and the Golgi apparatus are visible in the vicinity of the nebenkern. The nuclear envelope appears fenestrated (arrowheads) at the opposite side of the nebenkern. (I) Maturing of the nebenkern into a complex multi-membrane structure. The cristae of the former mitochondria are still visible within the lumen of the nebenkern (arrowheads). Annulate lamellae (AL) are also visible presumably on an ER-derived structure next to it. (J) Nebenkern after maturation. In later sperm development, the nebenkern unfolds and assembles in the cytoplasm around the axoneme. In between this structure there are still cytoplasm and organelles like Golgi (G) visible.
Fig 3.
Partial 3D reconstruction of the nebenkern covering about 2.1 μm in the z-dimension.
(AI-AIV) Tomographic slices through a nebenkern. (BI-BIV) Segmentation of membranes (cyan) in the tomographic slices as shown in (A). (C-F) Side views of the 3D model of the nebenkern with two clipping planes (x-axis white, y-axis purple) perpendicular to each other, illustrating the interlocking of the internal membranes of the nebenkern. The membrane is shown in cyan and then membrane lumen of the nebenkern is shown in orange. (G) Front view of the 3D model of the nebenkern illustrating a zipper-like membrane structure. The arrow heads (differently colored) illustrate the direction of the membrane extrusions leading into the nebenkern from opposite positions. (H) Identical view showing microtubules (red) in close proximity of the membrane structure. (I-J) Back view on the nebenkern model with respective microtubule model. (K) Segmentation of the interconnected inner lumen of one out of two mitochondria-derived nebenkern constituents (shown in red). (L) Segmentation of the other mitochondria-derived nebenkern constituents (shown in yellow). (M) 3D model of both nebenkern constituents illustrating the interlocking of both membrane systems. (N) 3D model of the nebenkern with the membrane (cyan) and both segmented inner lumina of the constituents (red and yellow).
Fig 4.
Ultrastructure of spermiogenesis in H. membranacea.
(A) Thin-section-TEM image of a testes region. Multiple developing sperm cells are visible with axonemes (Ax) and nebenkern derivatives (NK). (B) Detailed view of a region with multiple Golgi complexes (G), axonemes (Ax) and nebenkern derivatives (NK) visible. (C) High-magnification view of a sperm cell illustrating the granular character of the centriolar adjunct (CA) next to the nucleus (N). An electron-dense structure is indicated (arrow heads). An outgrowing axoneme (Ax) is visible at an opposing position. (DI-DIII) Electron tomogram and segmentation of the nucleus with an attached basal body (BB). The centriolar adjunct surrounds the basal body with the outgrowing axonemal microtubules. A developing nebenkern is visible next to the axoneme. The nuclear envelope is shown in red, the nebenkern membrane in cyan, the centriole adjunct in yellow, the basal body in blue and outgrowing microtubules in green. (EI-EIII) Electron tomogram and segmentation of a region containing developing sperm tails. The Golgi complex and many axonemes are shown in cross- and longitudinal sections. In the 3D models, membranes are illustrated in cyan, axonemal microtubules in green and other microtubules in red.