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Figure 1 Ultrastructural landmarks of sporulation. A) An example of several premeiotic cells (T=0 hrs.), note the numerous lightly stained mitochondria (n = nucleus). B and C) Cells at the beginning of meiosis I (T=2 hrs.). In B note dense body (DB) and fiberous structures (*) located near the nuclear envelope (NE). In C note side-by-side, duplicated SPBs (arrows point to cytoplasmic side of SPBs) embedded in the nuclear envelope. D and F) Prospore membrane formation in meiosis II (T =7 hrs.). D shows meiosis II spindle with distinct MII SPB, note that the prospore membrane is associated with the enlarged, outer plaque and that it has an electron-dense bulb at its leading edge (long arrows). Short arrow denotes nuclear envelope in which SPB is embedded. D (insert) shows a prospore membranes’ leading edge, note that it is bulb shaped and studded with a distinct protein coat. E shows a cell completing prospore formation; note that a lobe of the nucleus (N) is being ‘pinched’ off and that mitochondria (M) are being sequestered into the forming spore.; arrows denote the bulb shaped leading edges of the prospore membrane. F, G, and H) Spore maturation. F (T = 7 hrs.) is an example of a prospore, note aggregation and accumulation of lipid droplets (L) around the double bilayer prospore membrane (arrow). G (T = 9 hrs.) is an example of a tetrad (partially shown) of immature spores; note that the spore walls (arrow) have begun to form and that lipid droplets are mostly absent. H (T = 11.5 hrs.) is an example of a mature spore within an ascus; note that the spore wall is thick and has a darkly staining outer layer (arrow). Vacuole (V). Bars: A = 1 mm; B, D and E = 0.25 mm; C = 0.125; F, G, and H = 0.5 mm. Enlargement in D (insert) is 135,864X.

CYTOLOGICAL CHANGES DURING SPORULATION

Previous cytological studies have described the morphological events of meiosis and spore formation. To relate these morphological events to the transcriptional data well as determine the synchrony of the sporulation culture, we examined cells taken from each time point by both light and electron microscopy.

T=0 hours: premeiosis

In the electron microscope, at T=0 hours SK1 cells appeared similar to growing mitotic cells (EM Fig. 1 A). Most cell sections contained one or two vacuoles. About half of all cell sections contained an average of 2 lipid droplets while the other half contained no lipid droplets. Approximately 40% of all cell sections observed contained a nucleus and in about 10% of these a single spindle and/or spindle pole body could be seen. In contrast to mitotic cells growing in rich medium, these pre-meiotic cells contained many mitochondria located all along the periphery of the cell. The observed "mitochondria" may well represent the network formed by a single large mitochondrion.

T = 2 hours: the beginning of meiosis I

One of the earliest observable events in meiosis is the duplication of the spindle pole body (SPB). Accordingly, the first landmark we looked for was the 'side-by-side' duplicated spindle pole body and the associated, short nuclear spindle. Only at time = 2 hours did we observe any side-by-side duplicated spindles (EM Fig. 1C); and they were seen in approximately 5% of the nuclear sections (n=50). About 25% of all sections through nuclei at this time contained a visible spindle and/or spindle pole body.

The early stages of meiosis are also marked by the appearance of a fibrous dense body (Byers and Goetsch, 1975) which is distinct from the nucleolus. The dense body or ‘polycomplex’ has been postulated as the location of the central elements of the incipient synaptonemal complex (Engles and Croes, 1968; Moens and Rapport, 1971; Zickler and Olson, 1975; Horesh et al., 1979). At T=2 hours we observed these bodies within many nuclei (EM Fig. 1B). We also observed fiber-like structures in about 20% of the nuclear sections observed at T=2 hours (EM Fig. 1B). These structures were often seen near the nuclear envelope and, like side-by-side SPBs, were not observed at any other time point. The functional significance of these structures is unknown.

Lastly we observed that most cell-sections (80%) now contained a single, large empty vacuole. Additionally, we observed numerous, cytoplasmic lipid droplets. More than 80% of the cell-sections observed contained lipid droplets and about half of these had 3 or more lipid droplets. Taken together all these ultrastructural data suggest that T=2 hours is the time of the beginning of meiosis I. Consistent with this conclusion, light microscopy observation of DAPI-stained nuclei revealed no binucleated or tetranucleated cells at this time point.

T = 5 hours: the end of meiosis I

At T=5 hours no morphological markers for meiosis I or II were observed. However, about 20% (n=50) of the nuclear sections contained a single spindle and/or spindle pole body. Interestingly, 80% of the cell sections no longer contained any identifiable vacuole and the remaining 20% contained a single large, empty vacuole. Additionally, 90% of the cell sections now contained lipid droplets and about 75% of these contained three or more lipid droplets. In the light microscope only 2% of the cells contained two DAPI-stained spots indicative of the completion of meiosis I. Taken together these results suggest that at T=5 hours the cells are just finishing meiosis I.

T = 7 hours: the beginning of meiosis II

The second meiotic division results in two pairs of SPBs positioned across from one another with each pair connected by an elongated spindle. In meiosis II the outer (cytoplasmic) plaque of each spindle pole body is enlarged, resulting in a structurally and functionally distinct meiosis II structure. From each of the four modified outer plaques a flattened membrane sac forms. This is the prospore membrane; it grows to surround and encapsulate each of the four lobes of the meiotic nucleus forming the tetrad of immature spores. At T=7 hours we observed this distinct modified outer plaque and/or its associated prospore membrane in approximately 10% of all cells observed (EM Fig. 1D). Most interestingly we could observe that the leading lip of prospore membrane was distinctly bulb-shaped and was studded with a distinct protein coat (EM Fig 1D, insert).

As at earlier time points, we saw an increase in the number of cytoplasmic lipid droplets. Almost 100% of the cells now contained lipid droplets and most cells now contained 6-8 lipid droplets. In cell-sections that contained immature spores, many of these droplets were observed aggregated around the prospore membrane (EM Fig. 1F). No vacuoles where observed within the meiotic cells at T=7.

Light microscopy observation of DAPI-stained nuclei showed that by T=7 hours 68% of the cells were mononucleate, 14% were binucleate and 28% were tetranucleate (representative of cells finishing meiosis II). These results suggest that at T=7 the mitotic culture had entered meiosis II and concomitant with that, spore wall formation had begun.

T = 9 and T = 11.5: from the end of meiosis to spore morphogenesis

Several distinct layers of wall material are laid down within the lumen of the double-bilayer prospore membrane during spore maturation. This deposition is thought to occur, at least in part, via the incorporation of the material in the lipid droplets that have been accumulating and aggregating around the prospore membrane (see EM Fig. 1F for an example). Mature spores are easily identified because the mature spore wall has a higher electron-density and the outer bilayer of the prospore membrane is lost (see EM Fig. 1H for an example). At T=9 hours we continued to observe immature spores (approximately 13% of the cells), but we also now observed mature spores in approximately 20% of the meiotic cells (EM Fig. 1F, G and H). By T=11.5 hours approximately 18% of the cells contained immature spores and another 18% contained mature spores. Consistent with the hypothesis that material from the lipid droplets are incorporated into the mature spore wall we observed a decrease in the total number of cytoplasmic lipid droplets (compare EM Fig. 1F to 1G).

In the light microscope, by t=9 the majority of cells undergoing nuclear division had progressed into and were now finishing meiosis II: 41% mononucleate, 7% binucleate and 42% tetranucleate. Similarly, at 11.5 hours we observed 29% mononucleate, 15% binucleate and 50% tetranucleate cells.

Mature spores observed at T=11.5 and T=20 (data not shown) contained identifiable mitochondria, a single round vacuole, several lipid droplets and nucleus (EM Fig. 1H). In other words, the mature spore resembled a small round mitotic haploid cell with a thick, darkly-stained wall. These mature spores were contained within an ascus that had few identifiable structures. The sporulation efficiency of the time course (after more than 24 hours in sporulation medium) was 67%, as assayed in the light microscope using DAPI-staining of nuclei and observation of tetrad containing asci.

Lastly, a previously undescribed mitochondrial structure was frequently seen (EM Fig. 2). About 45% of the cell sections at T = 9 and 38% of the sections at T = 11.5 had a drastically altered mitochondrial morphology, namely a series of filament-like (ca. 13.5 nm) structures that in cross section appeared as a double row of "teeth" and in longitudinal sections appeared as filaments that ran parallel to the inner mitochondrial membrane. The outer row of ‘teeth’ was invariably associated with the inner mitochondrial membrane and outer-most filament(s) was associated along its length with the inner mitochondrial membrane. These structures were predominately located on one side of the mitochondria (EM Fig. 2C and D) and were observed in mitochondria located within both the ascal and spore cytoplasm. It is not clear whether these structures are functional or simply a manifestation of mitochondrial decay.