Spermatogenesis-Specific Features of the Meiotic Program in Caenorhabditis elegans

In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex

Spermatogenesis-Specific Features of the Meiotic Program in Caenorhabditis elegans

In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex

Changes in cell and microtubule morphologies distinguish spermatocytes that are entering and proceeding through the meiotic divisions.

<p>Left column (DIC/DNA) are unfixed and Hoechst-stained spermatocytes from flattened male gonad preparations viewed simultaneously under DIC and UV epifluorescence. Right columns are immunofluorescence analysis of methanol fixed spermatocytes doubled labeled with antibodies to α-tubulin (green) and pHisH3-ser10 (red) and stained with DAPI (blue in 2× enlarged merged image, white in enlarged DNA image). (A) karyosome, (B) late diakinesis, (C) metaphase I, (D) anaphase I, (E) metaphase II, (F) anaphase II, (G) budding figure. Primary spermatocytes are 12 microns in diameter.</p

A summary of the progression of nuclear events during late sperm formation.

<p>A summary of the progression of nuclear events during late sperm formation.</p

Inner and outer kinetochore components are differentially enriched and localized in sperm meiosis compared with oocyte meiosis.

<p>Immunolocalization of kinetochore components (green) and DAPI-stained DNA (red) during spermatogenesis and in metaphase oocytes. Yellow coloration in images and the schematics indicates extensive co-localization. (A) Barely detectable levels of the inner kinetochore component HCP-3^CENP-A encase spermatocyte chromosomes, but is at high levels distribute evenly all over oocyte chromosomes. (B) High levels of the inner kinetochore component HCP-4^CENP-C surround spermatocyte chromosomes starting in diakinesis and persist after meiosis is complete. HCP-4^CENP-C distributes evenly all over oocyte chromosomes. (C) The outer kinetochore components HIM-10 and (D) HCP-2^CENP-F surround spermatocyte and oocyte chromosomes at high levels. (E) The outer kinetochore component HCP-1^CENP-F is not detectable in sperm meiosis but surrounds chromosomes during oocyte meiosis. Scale bars represent 2 µm. Panels in each column are sized the same as (A) except as marked for oocyte meiotic chromosomes.</p

Immunolocalization of chromosome segregation markers during spermatocyte meiotic divisions.

<p>Proteins involved in chromosome segregation are green and DNA is red. (A) AIR-2 localizes to the short arms of bivalents during metaphase I and between sister chromatids during metaphase II. (B) p-HisH3-ser10 concentrates on regions bound by AIR-2. (C) The cohesin protein REC-8 is detectable along the equatorial plane on metaphase I and II plates. (D) PLK-1 protein is present on centrosomes throughout the meiotic divisions, chromosomes during metaphase, and between chromosomes during anaphase. Scale bars in (A) represent 2 µm. Panels in each column are sized the same as (A).</p

Karyosome formation during late spermatogenesis in <i>C. elegans</i>.

<p>Numbered nuclei highlighted by circles in (A) and (B) or arrows in (D) and (E) correspond to meiotic stages shown in (C). Pachytene (green), the condensation zone (teal), meiotic divisions (blue), post-meiotic region (purple). (A,B) DAPI-visualized nuclei in isolated and fixed male gonads (C) Enlarged images of DAPI-visualized nuclei in dissected and fixed male gonads. (D,E) A dissected and flattened non-fixed male gonad stained with the DNA dye Hoechst 33258 (D) and visualized by DIC optics (E). Scale bars represent 10 µm.</p

Activation of signaling pathway components during the G2/M transition in spermatogenesis.

<p>(A) α-tubulin costaining with (B) PLK-1. PLK-1 shifts from a ring around the nuclear envelope in diplotene and karyosome nuclei and then localizes to centrosomes (arrowheads) during the transition to diakinesis. In prometaphase, PLK-1 is detected both on centrosomes and on chromosomes. (C) Cyclin B levels increase during diakinesis and stays localized around chromosomes in prometaphase. (D) 2× enlarged images of DNA from cyclin B immunostained imaged in (C). (E) Sequence of karyosome to budding figure spermatocytes from flattened and fixed male gonad preparations immunostained with α-tubulin (green) and PLK-1 (red). DNA is shown in blue.</p

The progression of germ cell nuclei during gamete formation in <i>C. elegans</i>.

<p>DAPI-visualized nuclei in isolated and fixed gonads. (A) One arm of a bilaterally symmetric, two-armed, wild-type hermaphrodite gonad undergoing oogenesis and (B) a wild-type male gonad undergoing spermatogenesis. Regions of the gonad are labeled: mitotic (yellow), transition zone and pachytene (green), condensation zone of hermaphrodites (red) and males (teal), meiotic division zone (blue), and haploid spermatids (purple). The meiotic divisions of oocytes, which occur after fertilization, are not shown. Scale bars represent 50 µm.</p

Immunolocalization of subcellular marker proteins defines events in the male condensation zone.

<p>Marker proteins, listed in parenthesis below, are shown in green. DNA is shown in red. (A) The nuclear envelope (lamin) disassembles during prometaphase (B) The nucleolus (fibrillarin) disappears before karyosome formation. (C) pHisH3-ser10 initiates in late diplotene, increasing in karyosomes, and shifts between homologous chromosomes during prometaphase. A spermatid chromatin mass is present in the bottom right of the prometaphase image. (D) High levels of active RNA polymerase levels (phosphorylated on the C-terminal domain on serine 2) decrease dramatically upon karyosome formation. (E) Centrosomes (SPD-2) separate and enlarge during the transition to diakinesis. Scale bars represent 2 µm. Panels in each row are sized the same as in (A).</p