A Protamine Knockdown Mimics the Function of Sd in Drosophila melanogaster

Segregation Distorter (SD) is an autosomal meiotic drive system found worldwide in natural populations of Drosophila melanogaster. This gene complex induces the preferential and nearly exclusive transmission of the SD chromosome in SD/SD+ males. This selfish propagation occurs through the interplay of the Sd locus, its enhancers and the Rsps locus during spermatid development. The key distorter locus, Sd, encodes a truncated but enzymatically active RanGAP (RanGTPase-activating protein), a key nuclear transport factor in the Ran signaling pathway. When encoded by Sd, RanGAP is mislocalized to the nucleus interior, which then traps Ran inside the nucleus and disrupts nuclear import. As a result of this aberrant nuclear transport, a process known as the histone-to-protamine transition that is required for proper spermatid condensation fails to occur in SD/SD+ males. In this process, sperm-specific protamine proteins enter the spermatid nucleus and replace the formerly chromatin-complexed histones. Previously, we have shown that mutations affecting nuclear import and export can enhance distortion in an SD background, thus verifying that a defect in nuclear transport is responsible for the unequal transmission of chromosomes. Herein, we show that specifically reducing protamines induces distortion in an SD background, verifying that protamines are transported via the RanGAP/GEF pathway and indicating that E(SD) plays a significant and unique role in the process of distortion.

LEM-domain proteins are lost during human spermiogenesis but BAF and BAF-L persist

During spermiogenesis the spermatid nucleus is elongated, and dramatically reduced in size with protamines replacing histones to produce a highly compacted chromatin. After fertilisation, this process is reversed in the oocyte to form the male pronucleus. Emerging evidence, including the coordinated loss of the nuclear lamina (NL) and the histones, supports the involvement of the NL in spermatid nuclear remodelling, but how the NL links to the chromatin is not known. In somatic cells, interactions between the NL and the chromatin have been demonstrated: LEM-domain proteins and LBR interact with the NL and respectively, the chromatin proteins BAF and HP1. We therefore sought to characterise the lamina-chromatin interface during spermiogenesis, by investigating the localisation of six LEM-domain proteins, two BAF proteins and LBR, in human spermatids and spermatozoa. Using RT-PCR, IF and western blotting, we show that six of the proteins tested are present in spermatids: LEMD1, LEMD2 (a short isoform), ANKLE2, LAP2β, BAF and BAF-L, and three absent: Emerin, LBR and LEMD3. The full-length LEMD2 isoform, required for nuclear integrity in somatic cells, is absent. In spermatids, no protein localised to the nuclear periphery, but five were nucleoplasmic, receding towards the posterior nuclear pole as spermatids matured. Our study therefore establishes that the lamina-chromatin interface in human spermatids is radically distinct from that defined in somatic cells. In ejaculated spermatozoa, we detected only BAF and BAF-L, suggesting that they might contribute to the shaping of the spermatozoon nucleus and, after fertilisation, its transition to the male pronucleus.

Ultrastructural, autoradiographic and electrophoretic examinations of Chara tomentosa spermiogenesis

Ultrastructure of a spermatid nucleus changes many times during spermiogenesis. Condensed chromatin forms irregular clusters during phases I-II, a continuous ring adjacent to a nuclear envelope during phases III-V and a network occupying the whole nucleus during phase VI. In advanced spermiogenesis dense chromatin disappears and short randomly positioned fibrils arise, then long parallel ones are found (phase VIII) which during phase IX form a lamellar structure. In mature spermatozoids (phase X) chromatin becomes extremely condensed. ³H-arginine and ³H-lysine incorporation into spermatids during 2-min incubation is intensive during phases IN, decreases during phases VI, VII and becomes very low during phases VIII-IX. Capillary electrophoresis has shown that during <em>Chara tomentosa</em> spermiogenesis replacement of histones with basic proteins whose mobility is comparable to that of salmon protamines takes place. At the beginning of spermiogenesis core and linker histones are found in spermatids. During early spermiogenesis protamine-like proteins appear and their amount increases in late spermiogenesis when core histones are still present. In mature spermatozoids only protamine-like proteins represented by 3 fractions: 9.1 kDa, 9.6 kDa, 11.2 kDa are found. Disappearance of linker histones following their modification precedes disappearance of core histones. The results indicate that dynamic rearrangement of chromatin ultrastructure and aminoacid incorporation rate during spermiogenesis are reflected in basic nuclear protein changes.

Involvement of Importin-4 in the Transport of Transition Protein 2 into the Spermatid Nucleus

ABSTRACT Mammalian spermiogenesis is characterized by a unique chromatin-remodeling process in which histones are replaced by transition protein 1 (TP1), TP2, and TP4, which are further replaced by protamines. We showed previously that the import of TP2 into the haploid spermatid nucleus requires the components of cytosol and ATP. We have now carried out a detailed analysis to characterize the molecular components underlying the nuclear translocation of TP2. Real-time PCR analysis of the expression of different importins in testicular germ cells revealed that importin-4 and importin-β3 are significantly up-regulated in tetraploid and haploid germ cells. We carried out physical interaction studies as well as an in vitro nuclear transport assay using recombinant TP2 and the nuclear localization signal of TP2 (TP2NLS) fused to glutathione S-transferase in digitonin-permeabilized, haploid, round spermatids and identified importin-4 to be involved in the import of TP2. A three-dimensional model of the importin-4 protein was generated using the crystal structure of importin-β1 as the template. Molecular docking simulations of TP2NLS with the importin-4 structure led to the identification of a TP2NLS binding pocket spanning the three helices (helices 21 to 23) of importin-4, which was experimentally confirmed by in vitro interaction and import studies with different deletion mutants of importin-4. In contrast to TP2, TP1 import was accomplished through a passive diffusion process.

A novel method for isolating spermatid nuclei from cytoplasm prior to ROSI in the mouse

In the current widely used round spermatid injection (ROSI) protocol for the mouse, the spermatid nucleus is separated from most of the cytoplasm before ROSI by drawing a spermatid in and out of a pipette. This results in the highest rate of normal fertilization. However, this separation method is not always consistent and can be time-consuming. An alternative separation method that cuts away the cytoplasm using the tip of an injection pipette was developed. After removing the cytoplasm, ROSI was performed following both post- and pre-activation protocols and development in vitro and in vivo were examined. The new method consistently removed the bulk of the cytoplasm, as shown by quantifying mitochondria. ROSI without the cytoplasm resulted in significantly higher rates of fertilization than ROSI with the cytoplasm into either post- or pre-activated oocytes. Furthermore, the offspring production rates of ROSI without the cytoplasm were also high (50% and 49% for the post- and pre-activation protocols, respectively). This new method for separating the cytoplasm is an alternative way of producing offspring using ROSI.