Key Phases of Cellular Differentiation During Spermatogenesis

The testis sheath is a closed tube made up of muscle and pigment cells, separated from the lumen by a basement membrane. At the apical tip (the closed end of the tube), the basement membrane is thickened adjacent to a group of approx 20 somatic cells called the hub (see Fig. 2A). About eight germ-line stem cells are found around the hub, each associated with two cyst progenitor somatic stem cells. Division of the germ-line stem cell, accompanied by division of the two cyst progenitor cells, results in a spermatogonium encapsulated by two cyst cells. These two cyst cells will never divide again, but they will grow and remain intimately associated with the germ-line cells. Division of the sper-matogonium generates two spermatogonia, and three subsequent spermatogo-nial mitotic divisions (see Fig. 3A) followed by premeiotic S-phase results in a cyst of 16 primary spermatocytes still surrounded by two cyst cells. Cells within each cyst remain interconnected by cytoplasmic bridges called ring canals, derived from the cleavage furrow after incomplete cytokinesis. A specialized membrane-rich region of cytoplasm, the fusome, extends through these bridges to connect all of the cells in the cyst (21).

The primary spermatocyte period is primarily one of cell growth (approx 25-fold increase in cell volume ref. 2), with certain morphological changes indicating the early, middle, and mature primary spermatocyte. Notable is the transition to the polar spermatocyte stage (see Fig. 1B), where the phase dark mitochondria are aggregated to one side of the cell and the phase light nucleus

Fig. 2. FITC Phalloidin shows the hub, meiotic cleavage furrows and investment cones. Formaldehyde-fixed testis squash preparations stained with FITC phalloidin (A,C,E), which labels F-actin, and counterstained with propidium iodide to reveal the DNA (B,D). (A,B) The apical tip of a testis. The somatic hub structure appears as a small rosette where there is a more extensive array of F-actin. The large cells around this rosette are the germ-line stem cells. (C,D) Several cells at telophase of meiosis I. The F-actin is concentrated in the contractile rings of the cleavage furrows separating sister cells. (E) Investment cones are formed around the nuclei of elongated sperma-tids; they are then displaced from the nuclei as they progress along the cyst, investing each spermatid with its own plasma membrane and extruding the minor mitochondrial derivative and excess cytoplasm into a cytoplasmic waste bag. This set of investment cones has progressed part way along the spermatid tails.

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Fig. 3. Phosphorylation of histone H3 identifies mitotic and meiotic chromosomes. Methanol/acetone fixed testes stained with an antibody against phosphorylated histone H3 (B,E green [yellow] in C and F) and counterstained with propidium iodide (A,D, red in C and F). (A-C) A cyst of four spermatogonia (arrow) near the apical tip of the testis undergoing mitosis. Note the bright staining of spermatogonial cell DNA and the relatively weak staining of the DNA of the maturing primary spermatocytes to the left of the figure. (D-F) A cyst of cells completing meiosis I (large arrow) have phosphorylated histone H3. Early in elongation (small arrow), the nuclei are all clustered to one side of the cyst and histone H3 is no longer phosphorylated. (See color plate 1 in the insert following p. 242.)

Fig. 3. Phosphorylation of histone H3 identifies mitotic and meiotic chromosomes. Methanol/acetone fixed testes stained with an antibody against phosphorylated histone H3 (B,E green [yellow] in C and F) and counterstained with propidium iodide (A,D, red in C and F). (A-C) A cyst of four spermatogonia (arrow) near the apical tip of the testis undergoing mitosis. Note the bright staining of spermatogonial cell DNA and the relatively weak staining of the DNA of the maturing primary spermatocytes to the left of the figure. (D-F) A cyst of cells completing meiosis I (large arrow) have phosphorylated histone H3. Early in elongation (small arrow), the nuclei are all clustered to one side of the cyst and histone H3 is no longer phosphorylated. (See color plate 1 in the insert following p. 242.)

resides in the other side. Later, the asymmetry is lost as the cells become apolar spermatocytes. Throughout the primary spermatocyte stage, the decondensed chromosomes are in three nuclear domains, corresponding to the two major autosome and the sex chromosome bivalents (see Fig. 1I). Primary spermatocytes have a very prominent phase dark nucleolus, which is associated with the sex chromosome bivalent. Bulk transcription shuts down as primary spermatocytes mature (22,23), so transcripts for genes required late in spermatogenesis need to have accumulated in the cells by this stage. They are then stored in RNP particles in the cytoplasm until translation (24) (see Fig. 4B).

As mature primary spermatocytes (see Fig. 1C) enter the meiotic divisions the nucleus becomes rounder and the chromosomes condense and move away from the nuclear envelope (see Fig. 1J). Mitochondria aligned on the meiotic spindle make this structure readily visible in phase-contrast preparations (see

Fig. 4. In situ to wild type with cyclin B, Mst87F. (A) In situ hybridization to cyclin B transcript reveals a two-phase expression pattern. cyclin B message is expressed in the mitotic cells at the apical tip of the testis, but is absent from cells undergoing premeiotic S-phase. cyclin B message then reappears in early primary spermatocytes, persists until meiosis, and is degraded before the cysts progress to onion stage. (B) Mst87F encodes a protein important for the structure of the sperm heads. The transcript first accumulates in primary spermatocytes and persists through to very late stage of spermatogenesis, when it is translated.

Fig. 4. In situ to wild type with cyclin B, Mst87F. (A) In situ hybridization to cyclin B transcript reveals a two-phase expression pattern. cyclin B message is expressed in the mitotic cells at the apical tip of the testis, but is absent from cells undergoing premeiotic S-phase. cyclin B message then reappears in early primary spermatocytes, persists until meiosis, and is degraded before the cysts progress to onion stage. (B) Mst87F encodes a protein important for the structure of the sperm heads. The transcript first accumulates in primary spermatocytes and persists through to very late stage of spermatogenesis, when it is translated.

Figs. 1D and 5). An aster can be seen on one side of the nucleus, and this separates as the centrosomes migrate to opposite poles to set up the bipolar spindle. During meiotic telophase the cleavage furrow separating the sisters pinches the spindle to generate a "bow tie" effect (see Fig. 1F, [telophase II], Fig. 5 [bottom left panel], and 2C) and, finally, give a secondary spermatocyte cyst consisting of 32 cells connected by ring canals, encapsulated by 2 cyst cells. Meiosis II follows after a very short interphase (see Fig. 1E). Morphologically, meiosis II is very similar to meiosis I, although there is clearly half as much DNA, only 1 centriole per centrosome, the cells are smaller, and the final product is a cyst of 64 interconnected spermatids, still surrounded by the 2 cyst cells.

After the second meiotic division, all of the mitochondria aggregate and fuse to form two giant mitochondria. These are interleaved by the onion stage to make a Nebenkern, which, by transmission electron microscopy, resembles an onion slice (25) and, by phase contrast, is a dark sphere adjacent to the phase light nucleus (see Figs. 1G,J). The centriole inserts into the nuclear membrane and axoneme elongation initiates (2). A phase dark dot, the pseudonu-cleolus or protein body, appears inside the otherwise featureless phase light nucleus. During elongation, the mitochondria unfurl from each other and two distinct phase dark mitochondrial derivatives can be seen elongating alongside

the flagellar axoneme at the comet stage (see Fig. 1H). All of the spermatids in a cyst develop in synchrony; during elongation, their heads become more closely aligned. This is already visible at the comet stage where the nuclei are found at one side of the cyst, and the tails extending toward the other side (see Fig. 3D). Intercellular bridges remain at the distal end of the elongating cysts (26). During elongation, the two cyst cells behave somewhat differently, each encompassing one end of the cyst. The head end cyst cell contacts the terminal epithelium near the base of the testis, so that cysts elongate with their heads anchored and the tails pushing up the length of the testis. During elongation, the nuclei become invisible with phase contrast as they compact and are transformed from a sphere into a needle shape (see Fig. 1L).

Finally, the fully elongated spermatids individualize and coil. Individualization initiates with an actin-based structure, the investment cone, at the head end of each spermatid (27). This progresses along the length of each tail (see Fig. 2E), stripping off all excess cytoplasm and the minor mitochondrial derivative into a cytoplasmic waste bag (see Fig. 6C) that is eventually shed into the lumen of the testis. Individual sperm are coiled into the seminal vesicle ready for transfer to the female during copulation.

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