The CPEB Protein Orb2 Has Multiple Functions during Spermatogenesis in Drosophila melanogaster

Cytoplasmic Polyadenylation Element Binding (CPEB) proteins are translational regulators that can either activate or repress translation depending on the target mRNA and the specific biological context. There are two CPEB subfamilies and most animals have one or more genes from each. Drosophila has a single CPEB gene, orb and orb2, from each subfamily. orb expression is only detected at high levels in the germline and has critical functions in oogenesis but not spermatogenesis. By contrast, orb2 is broadly expressed in the soma; and previous studies have revealed important functions in asymmetric cell division, viability, motor function, learning, and memory. Here we show that orb2 is also expressed in the adult male germline and that it has essential functions in programming the progression of spermatogenesis from meiosis through differentiation. Like the translational regulators boule (bol) and off-schedule (ofs), orb2 is required for meiosis and orb2 mutant spermatocytes undergo a prolonged arrest during the meiotic G2-M transition. However, orb2 differs from boule and off-schedule in that this arrest occurs at a later step in meiotic progression after the synthesis of the meiotic regulator twine. orb2 is also required for the orderly differentiation of the spermatids after meiosis is complete. The differentiation defects in orb2 mutants include abnormal elongation of the spermatid flagellar axonemes, a failure in individualization and improper post-meiotic gene expression. Amongst the orb2 differentiation targets are orb and two other mRNAs, which are transcribed post-meiotically and localized to the tip of the flagellar axonemes. Additionally, analysis of a partial loss of function orb2 mutant suggests that the orb2 differentiation phenotypes are independent of the earlier arrest in meiosis

Orb2 expression during spermatogenesis.

<p>A) Drawing of the testis. Light blue: stem cell and spermatogonia region, green: spermatocytes, orange: spermatids, red: beginning of spermatids elongation, purple: elongated spermatids (dark blue: spermatid nuclear bundle). Terms used for describing directions of elongation are indicated here. B) Florescent <i>in situ</i> hybridization with <i>orb2</i> antisense probe of one testis. Image is stitched together from 4 frames with Fiji. B′) Zoom in view of the tip region. Arrow indicates position of the stem cell niche; square bracket outlines the spermatogonia region. C, C′) Testis stained with Orb2 antibody (red) and overlay of Orb2 and DNA (blue). C(I): Testis tip and spermatogonia region containing the 2,4,8-cell cysts. Arrow indicates position of the stem cell niche. C(II): Mature spermatocytes (arrow). C(III): Spermatocytes (arrow) and spermatids (arrowhead). C(IV): elongating spermatids. Arrow marks those with Orb2 expression, and arrowhead marks those without Orb2. D) Orb2 is concentrated around the growing spermatocyte nuclei. Arrowheads mark one nucleus. E) Orb2 localization near the distal (growing) tip of the elongating flagellar axonemes. Arrow, arrowhead and square bracket labels the leading edge of the axonemes, Orb2 concentrated region, and extended Orb2 expressing region respectively. F) Co-labeling of Orb2 in DJ-GFP testes. Orb2 is detected in cysts in which the 64 spermatid nuclei have coalesced into the nuclear bundle (*), but have not yet fully completed chromosome condensation (o). DJ-GFP is only expressed in spermatid cysts that have condensed their chromosomes. Some fully elongated spermatids with fully condensed nuclear bundles have neither Orb2 nor DJ-GFP (x). Insets are enlarged from the main panel. Scale bars are as shown in each panel.</p

Orb2 expression in the testes and fertility of different <i>orb2</i> alleles.

<p>A) <i>orb2</i> gene structure. <i>orb2</i> has three promoters (<i>orb2-1</i>, <i>2</i>, and <i>3</i>) and encodes multiple transcripts. <i>piggyBac</i> insertions <i>1556</i>, <i>1793</i>, <i>1925</i>, 6090, and <i>4965</i> are marked by green arrows. Only <i>1793</i>, <i>1925</i>, and <i>6090</i> are expected to disrupt <i>orb2</i> transcripts. Green box marks the poly-Q sequence that is deleted in <i>orb2^ΔQ</i> allele <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003079#pgen.1003079-Keleman1" target="_blank">[14]</a>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003079#pgen.1003079.s001" target="_blank">Figure S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003079#pgen.1003079-Hafer1" target="_blank">[19]</a> for further details. B) Orb2 expression in <i>piggyBac</i> alleles (6090, 1925, 1793, 1556, 4965), <i>orb2^ΔQ</i>, <i>piggyBac</i> insertion revertant (<i>6090⁻¹</i>), and <i>orb2³⁶</i>. (oe): over exposed. C) Fertility of <i>piggyBac</i> insertion alleles, revertant, <i>orb2^ΔQ</i>, <i>orb2³⁶ and orb2^Δ</i>. Fertility is consistent with Orb2 expression in the testes. <i>DF(3L)4416</i> (referred to as 4416) is a small deletion allele that removes part of the third chromosome that includes <i>orb2</i> gene region.</p

Orb expression in wild-type and <i>orb2³⁶</i> testes.

<p>A–D) Confocal images showing whole mount staining of testes with Orb (pink) and Orb2 (red) antibodies in DJ-GFP (green) expressing wild type testis. Arrow shows that Orb is expressed at the tip of those spermatids that express neither Orb2 nor DJ-GFP. Orb expression is sometimes seen in spermatids that have residual Orb2 (arrowhead). E, F) In <i>orb2³⁶</i> testes, Orb expression is observed in spermatids that are not fully elongated and its preferential localization near the tip of the elongating flagellar axonemes is lost. Orb: pink; DNA, yellow. Scale bar, 50 µm.</p

Spermatid nuclear bundles and Individualization Complex are not properly assembled in <i>orb2³⁶</i>.

<p>A, B) Formation and compaction of spermatid nuclear bundles in wild type (WT) and <i>orb2³⁶</i>. Arrowhead in A labels a condensed spermatid nuclear bundle. Arrowheads in B mark incompletely assembled spermatid nuclear bundles in <i>orb2³⁶</i>. Note that individual needle-shaped spermatid nuclei are larger than wild type, and most of the nuclei are scattered along the partially elongated spermatids. Double arrow indicates direction of spermatid elongation. C, D) Individualization complexes and DJ-GFP are missing from <i>orb2³⁶</i> testes. IC (Actin): red; DJ-GFP: green; DNA, blue. Inset shows complete IC (C) or scattered actin cones (D) in either wild type or <i>orb2³⁶</i> testes. E1, E2, F1, F2) MyosinVI (green) is a component of the Actin cones and is localized before the triangle shaped Actin signal in wild type IC (E1, E2, arrow). MyosinVI is also observed in the scattered actin cones in <i>orb2³⁶</i> testes (F1, F2, arrow). Scale bar in A and B: 20 µm.</p

<i>orb2³⁶</i> cysts arrest meiosis I at a step after the expression of the Twine phosphatase.

<p>A) Spermatocyte cysts in wild type typically have cytoplasmic CycA. B) <i>orb2³⁶</i> testes have multiple spermatocyte cysts with nuclear CycA, as observed in <i>bol </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003079#pgen.1003079-FranklinDumont1" target="_blank">[34]</a>. C) In wild type spermatocyte cysts, CycB is typically only found in the cytoplasm. D) CycB shows a similar nuclear enrichment as CycA in <i>orb2³⁶</i>. Arrow marks wild type and <i>orb2³⁶</i> cysts with cytoplasmic CycB, while arrowhead points to two <i>orb2³⁶</i> cysts with nuclear CycB. In contrast to <i>orb2</i>, cysts with cytoplasmic, but not nuclear CycB were seen in <i>bol</i> mutants (Xu, unpublished results). E, F) Twe-lacZ is over-expressed in <i>orb2³⁶</i>. Purple arrows: <i>orb2³⁶</i> cysts have much higher levels of Twe-lacZ than wild type. Orange arrow: <i>orb2³⁶</i> cysts containing immature spermatocytes prematurely express Twe-lacZ. Green arrow: Twe-lacZ persists in elongating <i>orb2³⁶</i> spermatids. G) Orb2 and Bol are found in the same complex in testes extracts. Testes extracts were immunoprecipitated with Bol or control IgG, in the presence or absence of RNase (RN) as indicated, and Westerns of the immunoprecipitated proteins were then probed with Orb2 antibodies. H) Testes extract from wild type (WT), <i>orb2</i> and <i>bol</i> were probed with antibodies against bulk Cdc2 (top) and phospho-Tyr15 Cdc2 (bottom). Note that <i>bol</i> and <i>orb2</i> have opposite effects on CDC2 Tyr15-P. The levels of phosphorylated Cdc2 in <i>bol</i> testes are higher than wild type, whereas they are lower than wild type in <i>orb2</i>. Ratio of phosphorylated Tyr15-P to unphosphorylated is as follows: WT = 1.5; <i>orb2</i> = 0.8; <i>bol</i> = 2.5. I) Semi-quantitative RT-PCR of <i>twine</i> and <i>gapdh</i> mRNA showing that <i>twine</i> mRNA levels in <i>orb2³⁶</i> are equivalent to wild type. Scale bar: 50 µm.</p

<i>orb2³⁶</i> spermatids have defects in flagellar axoneme elongation.

<p>A, B) Phase contrast images showing wild type (A) and <i>orb2³⁶</i> (B) elongated spermatid bundles. Wild type spermatid flagellar axoneme bundles (arrow) have a smooth morphology and extend in a nearly straight line. <i>orb2³⁶</i> bundles have rough and uneven morphology, are shorter, and zigzag back and forth. Individualized sperm (red arrowheads in A) are observed in wild type but not <i>orb2³⁶</i> testes. C1–C3) Wild type testes double stained with Orb2 (red) and Bol (green) antibodies showing co-localized Bol and Orb2 concentrated in a band near the tip of the elongating flagellar axonemes (arrowhead) and decreased expression level following this band. D1–D3) Bol localization at the tip is lost in <i>orb2³⁶</i> flagellar axonemes (arrow), while there is an uneven distribution along the flagellar axonemes extending behind the tip. E1–E3) Orb2 and Bol co-localization at the tip of the elongating flagellar axonemes in <i>orb2^ΔQ</i> is as in wild type. Scale bar: 20 µm.</p

Orb2 functions in meiosis and differentiation can be uncoupled in <i>orb2^ΔQ</i> allele.

<p>A) Percentage of scattered ICs is higher in <i>orb2^ΔQ</i> than wild type. A total of 34 wild type and 46 <i>orb2^ΔQ</i> testes were counted. B) Percentage of wild type or <i>orb2^ΔQ</i> testes having 0–3, 4–8, 9–13, 14–18, 19–23, 24–28, 29–33 or 34–38 ICs per testes. <i>orb2^ΔQ</i> testes have fewer ICs compared to wild type. C) <i>orb2^ΔQ</i> testes have normal spermatids. Green arrow: mature spermatocytes; yellow arrow: 64-cell spermatids cyst; arrowhead: spermatids at the beginning of elongation. D) Wild type testes stained with Orb2 (red), IC (Phalloidin, green) and DNA (blue). Yellow arrow points to where elongation usually stops in wild type testes. “d” marks the normal diameter of a testis at spermatogonia and early spermatocytes region. E–F) Flagellar axoneme bundles in <i>orb2^ΔQ</i> testes are over elongated. E) Overgrowth results in the swelling of the testis tip. Diameter of the <i>orb2^ΔQ</i> spermatogonia part of the testis is larger than that of the wild type (compare d in D and d′ in E), while the diameter of the nuclei side is relatively normal (compare d in D and d′, d″ in E). F) Another example of overly elongated flagellar axoneme bundles in <i>orb2^ΔQ</i> testis tip. The Orb2 positive axoneme bundle extended to the spermatogonial region and then changed its direction of elongation (arrows) to continue growing in the wrong direction. G, H) IC is not properly assembled in <i>orb2^ΔQ</i>. Phalloidin labeled Actin: green; DNA: blue. Arrow in G points to scattered actin cones of an <i>orb2^ΔQ</i> IC that remain relatively close together in one elongating spermatid cyst. Arrows in H are examples of widely scattered actin cones. In <i>orb2^ΔQ</i> testes with scattered IC, we can also observe what appear to be normal looking ICs, as indicated here by arrowhead in H. Scale bar: 50 µm.</p

<i>orb</i> is an Orb2 regulatory target.

<p>A) Western blots of extracts prepared from wild type, <i>orb2³⁶</i> and <i>orb2^Δ</i> testes were probed as indicated on the left. In this experiment Snf (Sans filles) was used as a loading control. Similar results were obtained using Tubulin as the loading control. B) <i>orb</i> mRNA can be immunoprecipitated with Orb2 antibodies from wild type testes extracts. <i>orb2³⁶</i> testis extract are used as a negative control for immunoprecipitation. After reverse transcription using oligo-dT primers, the <i>orb</i> cDNA was amplified using a primer set from the 3′ end of the male <i>orb</i> mRNA. L: 100 bp DNA ladder.</p