Eugene Rivkin

The Acroplaxome Is the Docking Site of Golgi-Derived Myosin Va/Rab27a/b- Containing Proacrosomal Vesicles in Wild-Type and Hrb Mutant Mouse Spermatids

Abstract Acrosome biogenesis involves the transport and fusion of Golgi-derived proacrosomal vesicles along the acroplaxome, an F-actin/keratin 5-containing cytoskeletal plate anchored to the spermatid nucleus. A significant issue is whether the acroplaxome develops in acrosomeless mutant mice. Male mice with a Hrb null mutation are infertile and both spermatids and sperm are round-headed and lack an acrosome. Hrb, a protein that contains several NPF motifs (Asn-Pro-Phe) and interacts with proteins with Eps15 homology domains, is regarded as critical for the docking and/or fusion of Golgi-derived proacrosomal vesicles. Here we report that the lack of an acrosome in Hrb mutant spermatids does not prevent the development of the acroplaxome. Yet the acroplaxome in the mutant contains F-actin but is deficient in keratin 5. We also show that the actin-based motor protein myosin Va and its receptor, Rab27a/b, known to be involved in vesicle transport, are present in the Golgi and Golgi-derived proacrosomal vesicles in wild-type and Hrb mutant mouse spermatids. In the Hrb mutant, myosin-Va-bound proacrosome vesicles tether to the acroplaxome, where they flatten and form a flat sac, designated pseudoacrosome. As spermiogenesis advances, round-shaped spermatid nuclei of the mutant display several nuclear protrusions, designated nucleopodes. Nucleopodes are consistently found at the acroplaxome- pseudoacrosome site. Our findings support the interpretation that the acroplaxome provides a focal point for myosin-Va/ Rab27a/b-driven proacrosomal vesicles to accumulate, coalesce, and form an acrosome in wild-type spermatids and a pseudoacrosome in Hrb mutant spermatids. We suggest that nucleopodes develop at a site where a keratin 5-deficient acroplaxome may not withstand tension forces operating during spermatid nuclear shaping.

The actin-based motor myosin Va is a component of the acroplaxome, an acrosome-nuclear envelope junctional plate, and of manchette-associated vesicles

Protein and vesicle cargos can be mobilized during spermiogenesis by intramanchette transport utilizing microtubule-based protein motors (kinesins and dyneins). However, actin-based unconventional myosin motors may also play a significant role in targeting vesicle cargos to subcellular compartments during sperm development. Here we report that myosin Va, an actin-based motor protein, is a component of the acroplaxome of rodent spermatids. The acroplaxome is an F-actin/keratin-containing scaffold plate with a marginal ring fastening the caudal recess of the developing acrosome to the nuclear envelope during spermatid nuclear shaping. In contrast to the acroplaxome, fluorescently labeled phalloidin does not produce an obvious F-actin signal in the manchette. However, immunogold electron microscopy detects moderate but specific β-actin immunoreactivity along interconnected tube-like bundles of manchette microtubules. We also show that the membrane of vesicles co-fractionated with intact manchettes by sucrose gradient ultracentrifugation display immunogold-labeled myosin Va. Myosin Va vesicle localization is known to correlate with Rab proteins, monomeric GTPases of the Ras superfamily which recruit myosin Va/VIIa motor proteins through intermediate proteins. RT-PCR analysis demonstrates that transcripts for Rab27a and Rab27b and Slac2-c (a protein that links Rab27a/b to myosin Va/VIIa) are expressed in testis. These results indicate that two independent cytoskeletal tracks, F-actin in the acroplaxome and presumably in the manchette, and manchette microtubules, may facilitate short-range (from the Golgi to the acrosome) and long-range (from the manchette to the centrosome and axoneme) mobilization of appropriate cargos during spermiogenesis.

A protein associated with the manchette during rat spermiogenesis is encoded by a gene of the TBP‐1‐like subfamily with highly conserved ATPase and protease domains

We have used a rat pachytene spermatocyte cDNA expression library to clone TBP‐1 (for tat‐binding protein‐1; designated rat testis TBP‐1 [rtTBP‐1]), a new member of the family of putative ATPases associated with the 26S proteasome complex. The 1.63 kb rtTBP‐1 cDNA encodes a 49 kDa protein with 99% amino acid identity to human TBP‐1 protein. rtTBP‐1 protein contains a heptad repeat of six leucine‐type zipper fingers at the amino terminal end and highly conserved ATPase and DNA/RNA helicase motifs towards the carboxyl terminal region. Chromatofocusing fractionation of rat testis sucrose extracts demonstrates that the encoded product, recognized by an antiserum raised to the first 196 amino acids of human TBP‐1, consists of a protein triplet with a molecular mass range of 52‐48 kDa and acidic pI (5.0–5.9). An identical immunoreactive triplet was detected by immunoblotting in extracts of fractionated pachytene spermatocytes, round spermatids and epididymal sperm. In situ hybridization using digoxigenin‐labeled antisense RNA probes shows a predominant distribution of specific mRNA in the seminiferous epithelial region occupied by elongating spermatids and primary spermatocytes. Indirect immunofluorescence and immunogold electron microscopy studies show that rtTBP‐1 immunoreactive sites colocalize with α‐tubulin‐decorated manchettes of elongating spermatids. In addition, rtTBP‐1 immunoreactivity was detected in fibrillar and granular cytoplasmic bodies typically observed in spermatocytes and spermatids as well as in association with paraaxonemal mitochondria and outer dense fibers of the developing spermatid tail. Results of this study indicate that rtTBP‐1 is a member of the highly evolutionary conserved TBP‐1‐like subfamily of putative ATPases, sharing regions of identity—including ATP‐binding sites—with several subunits of the 26S proteasome, known to be involved in the ATP‐dependent degradation of ubiquitin‐conjugated proteins. Mol. Reprod. Dev. 48:77–89, 1997.

Cytoskeletal track selection during cargo transport in spermatids is relevant to male fertility

Spermatids generate diverse and unusual actin and microtubule populations during spermiogenesis to fulfill mechanical and cargo transport functions assisted by motor and non-motor proteins. Disruption of cargo transport may lead to teratozoospermia and consequent male infertility. How motor and non-motor proteins utilize the cytoskeleton to transport cargos during sperm development is not clear. Filamentous actin (F-actin) and the associated motor protein myosin Va participate in the transport of Golgi-derived proacrosomal vesicles to the acrosome and along the manchette. The acrosome is stabilized by the acroplaxome, a cytoskeletal plate anchored to the nuclear envelope. The acroplaxome plate harbors F-actin and actin-like proteins as well as several other proteins, including keratin 5/Sak57, Ran GTPase, Hook1, dynactin p150Glued, cenexin-derived ODF2, testis-expressed profilin-3 and profilin-4, testis-expressed Fer tyrosine kinase (FerT), members of the ubiquitin-proteasome system, and cortactin. Spermatids express transcripts encoding the non-spliced form of cortactin, a F-actin-regulatory protein. Tyrosine phosphorylated cortactin and FerT coexist in the acrosome-acroplaxome complex. Hook1 and p150Glued, known to participate in vesicle cargo transport, are sequentially seen from the acroplaxome to the manchette to the head-tail coupling apparatus (HTCA). The golgin Golgi-microtubule associated protein GMAP210 resides in the cis-Golgi whereas the intraflagellar protein IFT88 localizes in the trans-Golgi network. Like Hook1 and p150Glued, GMAP210 and IFT88 colocalize at the cytosolic side of proacrosomal vesicles and, following vesicle fusion, become part of the outer and inner acrosomal membranes before relocating to the acroplaxome, manchette, and HTCA. A hallmark of the manchette and axoneme is microtubule heterogeneity, determined by the abundance of acetylated, tysosinated, and glutamylated tubulin isoforms produced by post-translational modifications. We postulate that the construction of the male gamete requires microtubule and F-actin tracks and specific molecular motors and associated non-motor proteins for the directional positioning of vesicular and non-vesicular cargos at specific intracellular sites.

GMAP210 and IFT88 are present in the spermatid golgi apparatus and participate in the development of the acrosome–acroplaxome complex, head–tail coupling apparatus and tail

We describe the localization of the golgin GMAP210 and the intraflagellar protein IFT88 in the Golgi of spermatids and the participation of these two proteins in the development of the acrosome–acroplaxome complex, the head–tail coupling apparatus (HTCA) and the spermatid tail. Immunocytochemical experiments show that GMAP210 predominates in the cis‐Golgi, whereas IFT88 prevails in the trans‐Golgi network. Both proteins colocalize in proacrosomal vesicles, along acrosome membranes, the HTCA and the developing tail. IFT88 persists in the acrosome–acroplaxome region of the sperm head, whereas GMAP210 is no longer seen there. Spermatids of the Ift88 mouse mutant display abnormal head shaping and are tail‐less. GMAP210 is visualized in the Ift88 mutant during acrosome–acroplaxome biogenesis. However, GMAP210–stained vesicles, mitochondria and outer dense fiber material build up in the manchette region and fail to reach the abortive tail stump in the mutant. In vitro disruption of the spermatid Golgi and microtubules with Brefeldin‐A and nocodazole blocks the progression of GMAP210‐ and IFT88‐stained proacrosomal vesicles to the acrosome–acroplaxome complex but F‐actin distribution in the acroplaxome is not affected. We provide the first evidence that IFT88 is present in the Golgi of spermatids, that the microtubule‐associated golgin GMAP210 and IFT88 participate in acrosome, HTCA, and tail biogenesis, and that defective intramanchette transport of cargos disrupts spermatid tail development. Developmental Dynamics 240:723–736, 2011.

Rnf19a, a ubiquitin protein ligase, and Psmc3, a component of the 26S proteasome, Tether to the acrosome membranes and the head–tail coupling apparatus during rat spermatid development

We report the cDNA cloning of rat testis Rnf19a, a ubiquitin protein ligase, and show 98% and 93% protein sequence identity of testicular mouse and human Rnf19a, respectively. Rnf19a interacts with Psmc3, a protein component of the 19S regulatory cap of the 26S proteasome. During spermatid development, Rnf19a and Psmc3 are initially found in Golgi‐derived proacrosomal vesicles. Later on, Rnf19a, Psmc3, and ubiquitin are seen along the cytosolic side of the acrosomal membranes and the acroplaxome, a cytoskeletal plate linking the acrosome to the spermatid nuclear envelope. Rnf19a and Psmc3 accumulate at the acroplaxome marginal ring–manchette perinuclear ring region during spermatid head shaping and in the developing sperm head–tail coupling apparatus and tail. Rnf19a and Psmc3 may interact directly or indirectly with each other, presumably pointing to the participation of the ubiquitin–proteasome system in acrosome biogenesis, spermatid head shaping, and development of the head‐tail coupling apparatus and tail. Developmental Dynamics 238:1851–1861, 2009.

Rat hd Mutation Reveals an Essential Role of Centrobin in Spermatid Head Shaping and Assembly of the Head-Tail Coupling Apparatus

The hypodactylous (hd) locus impairs limb development and spermatogenesis, leading to male infertility in rats. We show that the hd mutation is caused by an insertion of an endogenous retrovirus into intron 10 of the Cntrob gene. The retroviral insertion in hd mutant rats disrupts the normal splicing of Cntrob transcripts and results in the expression of a truncated protein. During the final phase of spermiogenesis, centrobin localizes to the manchette, centrosome, and the marginal ring of the spermatid acroplaxome, where it interacts with keratin 5-containing intermediate filaments. Mutant spermatids show a defective acroplaxome marginal ring and separation of the centrosome from its normal attachment site of the nucleus. This separation correlates with a disruption of head-tail coupling apparatus, leading to spermatid decapitation during the final step of spermiogenesis and the absence of sperm in the epididymis. Cntrob may represent a novel candidate gene for presently unexplained hereditary forms of teratozoospermia and the “easily decapitated sperm syndrome” in humans.

Expression of Fer testis (FerT) tyrosine kinase transcript variants and distribution sites of FerT during the development of the acrosome‐acroplaxome‐manchette complex in rat spermatids

We report the association of testicular Fer, a non‐receptor tyrosine kinase, with acrosome development and remodeling of the acrosome‐associated acroplaxome plate during spermatid head shaping. A single gene expresses two forms of Fer tyrosine kinases in testis: a somatic form (FerS) and a truncated testis‐type form (FerT). FerT transcript variants are seen in spermatocytes and spermatids. FerS transcripts are not detected in round spermatids but are moderately transcribed in spermatocytes. FerT protein is associated with the spermatid medial/trans‐Golgi region, proacrosomal vesicles, the cytosolic side of the outer acrosome membrane and adjacent to the inner acrosome membrane facing the acroplaxome. FerT coexist in the acroplaxome with phosphorylated cortactin, a regulator of F‐actin dynamics. We propose that FerT participates in acrosome development and that phosphorylated cortactin may contribute to structural changes in F‐actin in the acroplaxome during spermatid head shaping. Developmental Dynamics 237:3882–3891, 2008.

Sak 57, an intermediate filament keratin present in intercellular bridges of rat primary spermatocytes

We have previously reported the purification of Sak 57 (for spermatogenic cell/sperm‐associated keratin of molecular mass 57 kDa) from outer dense fibers of rat sperm tails. Internal protein sequence analysis of Sak 57 revealed 70–100% homology to the 1A and 2A regions of the α‐helical rod domain of human, mouse, and rat keratins. A multiple antigen peptide was synthesized using the KQYEDIAQK sequence corresponding to the 2A region and a polyclonal antibody was produced in rabbit to detect Sak 57. During spermiogenesis, Sak 57 associates with the microtubular manchette before becoming a component of para‐axonemal keratin structures of the developing tail. We now report that during late meiotic prophase, intercellular bridges linking late pachytene‐diplotene spermatocytes display a distinct ribbon containing a Sak 57/β‐tubulin complex, separated by a nonimmunoreactive midzone. Indirect immunofluorescence demonstrates that the ribbon is the final stage of a three‐step developmental sequence: (1) a spindlelike arrangement radiating from equidistant spherical centers in early pachytene spermatocytes, (2) an ectoplasmic shell like framework in mid‐to‐late pachytene spermatocytes, and (3) a Sak 57/β‐tubulin‐containing ribbon found in intercellular bridges linking adjacent late pachytene‐diplotene spermatocytes. Shear forces causing a breakdown of one of the conjoined spermatocytes do not disrupt the cytoskeletal ribbon. Results of this work, together with previous observations during spermiogenesis, show that Sak 57 associates with cytoplasmic microtubules in a timely fashion. Upon completion of late meiotic prophase, the Sak 57/microtubule complex behaves as an intercellular ligament and contributes to both the strength of intercellular bridges and the cohesiveness of members of a spermatocyte lineage.

Purification, partial characterization, and localization of Sak57, an acidic intermediate filament keratin present in rat spermatocytes, spermatids, and sperm

We have purified a 57 kDa protein (designated Sak57, for spermatogenic cell/sperm‐associated keratin) from sodium dodecyl sulfate‐β‐mercaptoethanol(SDS‐βME)‐dissociated outer dense fibers isolated from rat sperm tails. Internal protein sequence analysis of Sak57 yielded two 15‐mer and 10‐mer fragments with 70–100% homology to human, rat, and mouse keratins and corresponding to the 1A and 2A regions of the α‐helical rod domain of keratins. A multiple antigenic peptide (MAP) was constructed using the 10‐mer amino acid sequence KAQYEDIAQK (corresponding to the 2A region) and used as antigen for the production of polyclonal antibodies in rabbit. Anti‐MAP sera were used for further analysis of the biochemical characteristics of Sak57 in testis and sperm tails using chromatofocusing, immunobloting, and [32P]orthophosphate‐labeling. We have found that rat testis displays two immunoreactive proteins: a soluble 83 kDa protein with pl range 5.9–6.3, regarded as a precursor, and both detergent‐insoluble and soluble 57 kDa protein with pl range 5.0–5.9, corresponding to the mature form Sak57. The testicular soluble form was phosphorylated. Rat sperm tail samples displayed only the Sak57 detergent‐insoluble form and its pl was more acidic (4.7–4.8). Whole‐mount electron microscopy of negatively stained preparations of sperm‐derived Sak57 resuspended in SDS‐βME revealed a rod‐shaped pattern. A decrease in the concentration of SDS‐βME resulted in the side‐by‐side aggregation of rod‐shaped Sak57 forming thick bundles. Indirect immunofluorescence was used to determine the localization of Sak57 in isolated outer dense fibers, epididymal sperm, spermatids, and pachytene spermatocytes. Confocal laser scanning microscopy was used to analyze the three‐dimensional arrangement of Sak57 in pachytene spermatocytes. Isolated outer dense fiber and sperm tails displayed an immunoreactive product in the form of linear clusters. In elongating spermatids (steps 10–11), Sak57 immunoreactivity was predominant in the head region whereas pachytene spermatocytes displayed a cortical cytoplasmic distribution. Results of this study demonstrate that Sak57 has the characteristics of a keratin intermediate filament and is present during meiotic and postmeiotic stages of spermatogenesis.