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 beta-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.

Primordial germ cell-somatic cell partnership: a balancing cell signaling act.

Recent studies demonstrate that the normal progression of the germ cell lineage during gonadogenesis involves a delicate balance of primordial germ cell survival and death factors generated by surrounding somatic cells. This balance operates in a different fashion in females and males. The fine tuning primordial germ cell specification in the wall of the yolk sac, migration through the hindgut and dorsal mesentery, and colonization in the urogenital ridges involves the temporal and spatial activation of the following signaling pathways: Primordial germ cell specification involves bone morphogenetic proteins 2, 4 and 8b, and their migration is facilitated by the c-kit receptor-ligand duet. When colonization occurs: (1) neuregulin-beta ligand is expressed and binds to an ErbB2-ErbB3 receptor tyrosine kinase heterodimer on primordial germ cells; (2) Vasa, an ortholog of the Drosophila gene vasa, member of an ATP-dependent RNA helicase of the DEAD (Asp-Glu-Ala-Asp)-box family protein is also expressed by primordial germ cells; (3) Bcl-x (cell survival factor) and Bax (cell death factor) join forces to modulate the first burst of primordial germ cell apoptosis; (4) Cadherins, integrins, and disintegrins bring together primordial germ cells and somatic cells to organize testis and ovary. Information on other inducers of primordial cell survival, such as TER (teratoma) factor, is beginning to emerge.

Spermatid manchette: plugging proteins to zero into the sperm tail.

Spermiogenesis pursues three major objectives: (1) The safeguard of the male genome within the confines of a compact nucleus. (2) The accumulation of enzymes in the acrosome of be released at fertilization. (3) The development of a sperm propelling tail consisting of an axoneme surrounded by a scaffold of keratin-containing outer dense fibers and a fibrous sheath. Recent experimental data indicate that three keratins-Sak57, 0df1 and 0df2-and other proteins (the 26S proteasome and the 0df1-binding protein Spag4) are temporarily stored in the manchette before being sorted to the developing sperm tail. These findings support a general model for the manchette as an ephemeral structure timely developed and strategically positioned to provide a transient storage to both structural and signaling proteins. Some of the proteins are later sorted to the developing tail; others may participate in the reciprocal nuclear-cytoplasmic signaling pathways as the gene activity of the male genome gradually becomes silent. Mol. Reprod. Dev. 59: 347-349, 2001.

Decidualization and implantation: embryo-uterine bioinformatics at work.

The implantation of the blastocyst into a nurturing endometrium involves two overlapping steps: 1. The blastocyst-endometrial luminal epithelial attachment. 2. The decidualization of the endometrial stroma. An intriguing question is how does the blastocyst identify the uterine implantation site. Current research is focused on hypothetical soluble signaling molecules released by the blastocyst for conditioning a discrete uterine luminal epithelial domain for implantation. A still unresolved issue is the functional significance of receptor autophosphorylation following binding of uterine epithelial cell-derived heparin-binding epidermal growth factor-like growth factor to the epidermal growth factor receptor on trophoectodermic cell surfaces. With recent results hinting at the role of signaling proteins associated with the bone morphogenetic protein, fibroblast growth factor, WNT and hedgehog families to enable embryo implantation, the dynamics of uterine-embryo interaction becomes linked to fundamental cellular pathways of growth, differentiation and apoptosis.

Transition nuclear proteins during spermiogenesis: unrepaired DNA breaks not allowed.

The completion of spermiogenesis requires condensation of the haploid spermatid genome. This task is accomplished in a gradual and relentless manner by first erasing the nucleosomal organization of chromatin while the DNA is protected by transient nuclear proteins TP1 and TP2. Then, the more permanent protamines come into play to stabilize the spermatid genome until fertilization occurs. Mice lacking TPI manage to produce relatively structurally normal sperm, although fertility is reduced and chromatin condensation is abnormal despite the compensatory expression of TP2. TP1 and TP2 appear to have the house-keeping function of reestablishing continuity when chromatin breaks take place during the remodeling process. DNA single-strand breaks are frequently observed when spermiogenesis is half completed. There is a temporal relationship between TP1 and DNA breaks: TP1 nuclear levels increase and the frequency of DNA breaks become less prominent as spermiogenesis is reaching completion. TP1 seems to hold the broken ends together until an as-yet-unidentified ligase bridges the gap.

Offspring from normal mouse oocytes injected with sperm heads from the azh/azh mouse display more severe sperm tail abnormalities than the original mutant.

Sperm with abnormalities in the position and shape of the head were obtained from the azh/azh mutant and injected into the cytoplasm of mature mouse oocytes to determine whether sperm from the offspring display both head (club shape) and tail (looping, folding, and fusion) abnormalities observed in the mutant donor. Although quantitative differences were observed among the three examined offspring, we found that abnormalities in sperm head shape were less frequent than in the donor mutant, but that tail malformations predominated. In addition, we found that the frequency of tail abnormalities increased during sperm epididymal transit. A typical defect was the multiple folding of the sperm tail and eventual fusion of closely apposed plasma membranes. As a consequence, sperm forward motility and natural fertility were compromised. Results of this study indicate that the azh/azh mutant and offspring generated by intracytoplasmic sperm injection provide a valuable model for determining the role of the manchette and keratin-containing outer dense fibers and fibrous sheath during spermiogenesis. Furthermore, our findings stress the risk of enhancing a phenotypic abnormality caused by mutant male genotypes introduced through bypassing the biologic mechanisms of natural sperm selection during fertilization.

Apoptosis during spermatogenesis: the thrill of being alive.

Emerging clues about the apoptotic molecular mechanisms operating during spermatogenesis indicate that the activation mechanism of executioner caspases may diverge from the traditional signaling operating in the immune system. Two issues are now been debated: (1) Whether the massive apoptosis of spermatogenic cells observed during the first spermatogenic wave represents a mechanism for ensuring the steady state sperm output in a given segment of a seminiferous tubule. (2) Whether apoptosis just represents a local remodeling process commanded by the Sertoli cell constraints to satisfy the differentiation needs of a juxtaposed cellular domain of spermatogonial, spermatocyte, and spermatid cohorts in the adult testis.

Keratin 9 is a component of the perinuclear ring of the manchette of rat spermatids.

Previous work in our laboratory has shown that a 62- to 64-kDa protein was a major component of the perinuclear ring of manchettes fractionated from rat spermatids. Mass spectrometry analysis of this protein indicated the presence of a glycine-rich domain homologous to human keratin 9 (K9). Several antibodies to K9, raised against synthetic peptides of human K9, recognized the 64- to 62-kDa protein in the perinuclear ring of the manchette as well as in keratinocytes of the suprabasal layer of the rat and human footpad/sole epidermis in both immunoblotting and immunocytochemical experiments. Based on these data, human-derived K9 primers were used to clone rat K9 cDNA from epidermis by RT-PCR. Rat-specific K9 primers were then used to perform a two-step (nested) PCR to amplify the K9-specific rat testicular RNA and to obtain cDNA to demonstrate K9 gene expression in rat testis. The deduced amino acid sequence of rat K9 cDNA contains 618 amino acids with an estimated molecular mass of 63,020 Da, in agreement with that obtained by electrophoretic fractionation of rat manchette and epidermis footpad proteins. The deduced protein structure correlates with the recognizable pattern of keratins: a rod domain of 304 amino acids with well-conserved initiation and termination sequences (MQNLNSRLASY and EIETYRKLLEG, respectively), flanked by glycine/serine-rich head and tail domains of 141 and 173 amino acids, respectively. A high content of phenylalanine was detected in the head domain and a repetitive motif (SGGSYGGGS) in the tail domain. A comparison with human keratin 9 showed an overall nucleotide and amino acid similarity of 75%. An increased level of K9 transcripts was detected in a cDNA library prepared from fractionated round spermatids. Results of this study show that rat testis expresses K9 and that this protein is a component the perinuclear ring of the manchette of rat spermatids.

Fusion of membranes during the acrosome reaction: a tale of two SNAREs.

During spermiogenesis, hydrolytic enzymes are sorted from the Golgi apparatus to the acrosome, a supranuclear megavesicle. At fertilization, the enzymatic content of the acrosome is released by exocytosis when a portion of the plasma membrane enveloping the sperm head fuses with the outer membrane of the acrosome. Membrane fusion involves the interaction of a specific pair of proteins, called SNAREs (for soluble N-ethylmaleimide sensitive factor attachment protein receptor). v-SNARE is presumably associated with the membrane of the acrosomal vesicle. Target t-SNARE is associated with the plasma membrane. The interaction of v-SNARE and t-SNARE requires two additional proteins: Rab proteins, members of a family of small GTPases related to the Ras proteins, and a complex of two proteins, NSF-SNAP, recruited by the interacting v-SNARE-tSNARE pair. Syntaxin 2, a v-SNARE member, and Rab3A, a member of the Rab GTPases, have been localized in the acrosome of rodent sperm.

Nuclear transfer and cell transplantation: making more with less.

Pig cloning can be achieved by transfer of nuclei of differentiated somatic cells into enucleated oocytes. Then, developing embryos are placed into surrogate mothers for further development to full term. Although cloned pigs offer the possibility of unlimited organ supply for compatible xenotransplantation in humans, the yield of a predictable number of offspring is still at an experimental phase. Spermatogonial stem cells from a fertile donor can be transplanted to the testes of infertile recipients and generate sperm. At present, results from xenogeneic spermatogenesis by transplantation indicate that porcine, bovine and equine spermatogonia find the interior of the seminiferous tubular environment not favorable for further differentiation into meiotic prophase spermatocytes. Spermatogenic cell transplantation is a promising experimental alternative for understanding the conditions required by both the donor cells and the recipient testis to coexist and even cooperate towards the full development of fertilizing sperm.

The 26S proteasome: ubiquitin-mediated proteolysis in the tunnel.

The 26S proteasome is a self-compartmentalizing protease responsible for the degradation of intracellular proteins. This giant intracellular protease is formed by several subunits arranged into two 19S polar caps-where protein recognition and ATP-dependent unfolding occur-flanking a 20S central barrel-shaped structure with an inner proteolytic chamber. Proteins targeted to the 26S proteasome are conjugated with a polyubiquitin chain by an enzymatic cascade before delivery to the 26S proteasome for degradation into oligopeptides. As a self-compartmentalizing protease, the 26S proteasome circumvents proteins not destined for degradation and can be deployed to the cytoplasmic and nuclear compartments. The 26S proteasome is a representative of emerging group of giant proteases, including tricorn protease, multicorn protease, and TPPII (tripeptidyl peptidase II).

Structural features of the 26S proteasome complex isolated from rat testis and sperm tail.

We have previously cloned a cDNA encoding TBP-1, a protein present in the rat spermatid manchette and outer dense fibers of the developing sperm. TBP-1 contains a heptad repeat of six-leucine zipper fingers at the amino terminus and highly conserved ATPase and DNA/RNA helicase motifs toward the carboxyl terminus. TBP-1 is one of the 20 subunits forming the 19S regulatory complex of the 26S proteasome, an ATP-dependent multisubunit protease found in most eukaryotic cells. We now report the isolation of the 26S proteasome from rat testis and sperm tail and its visualization by whole-mount electron microscopy using negative staining. The 26S proteasome from rat testis was fractionated by Sephacryl S-400/Mono-Q chromatography using homogenates suspended in a 10% glycerol-supplemented buffer. Chromatographic fractions were analyzed by immunoblotting using a specific anti-TBP-1 serum. During the purification of Sak57, a keratin filament present in outer dense fibers from epididymal sperm, we detected a substantial amount of 26S proteasomes. Intact 26S proteasomes from rat testis display a rod-shaped particles about 45 nm in length and 11-17 nm in diameter. Each particle consists of a 20S barrel-shaped component formed by four rings (alphabetabetaalpha), capped by two polar 19S regulatory complexes, each identified by an element known as the "Chinese dragon head motif". TBP-1 is an ATPase-containing subunit of the 19S regulatory cap. Rat sperm preparations displayed both dissociated 26S proteasomes and Sak57 filaments. We hypothesize that 26S proteasomes in the perinuclear-arranged manchette are in a suitable location for recognition, sequestration, and degradation of accumulating ubiquitin-conjugated somatic and transient testis-specific histones during spermiogenesis. In the sperm tail, the 26S proteasome may have a role in the remodeling of the outer dense fibers and other tail components during epididymal transit.

Telomeres: more than chromosomal non-sticking ends.

Telomeres are specialized natural ends of eukaryotic chromosomes that, contrary to the ends of broken chromosomes, are stable and do not fuse with the ends of other chromosomes. In addition, telomeres protect chromosomal ends from degradation, facilitate completion of chromosomal DNA replication, and contribute to chromosome positioning within nuclei. Telomeric DNA consists of repetitive sequences and specific associated proteins, including the telomere repeat-binding factors TRF1 and TRF2. A lack of TRF2 enables end-to-end chromosome fusion. A structural disruption of telomeres not only causes chromosomal mechanical instability but also activates a programmed cell death cascade.

Molecular cloning of rat sperm galactosyl receptor, a C-type lectin with in vitro egg binding activity.

Rat sperm galactosyl receptor is a member of the C-type animal lectin family showing preferential binding to N-acetylgalactosamine compared to galactose. Binding is mediated by a Ca(2+)-dependent carbohydrate-recognition domain (CRD) identical to that of the minor variant of rat hepatic lectin receptor 2/3 (RHL-2/3). The molecular organization of the genomic DNA, cDNA, and derived amino acid sequence of rat testis galactosyl receptor have been determined and in vitro fertilization studies were conducted to ascertain its role. We have determined that the rat testis galactosyl receptor gene generates two mRNA species: one species, designated liver-type, is identical to RHL-2/3; the other, designated testis-type, contains one unspliced intron (86 nt) which alters the reading frame and changes the amino acid sequence of the carboxyl terminus. As a result, the CRD (glutamine-proline-aspartic acid/QPD) and flanked Ca(2+)-binding amino acid sequences were not present in the testis-type protein. Northern and Southern blots demonstrated presence of transcripts with unspliced intron in rat sperm but not liver. Similarly, antibody, raised against a synthetic 12-amino acid peptide (p12) encoded by the unspliced intron, recognized in immunoblots a 54 kDa receptor protein in protein extracts from testis but not from liver. Immunofluorescence and immunogold electron microscopy studies demonstrated that both protein species localized on the plasma membrane surface of the head and tail of rat sperm. Furthermore, capacitated rat sperm preincubated with polyclonal antisera to RHL-2/3 or to the CRD of the liver-type galactosyl receptor showed a statistically significant decrease in the in vitro fertilization rate. We conclude that rat sperm galactosyl receptor may play a role in egg binding and that an undetermined molecular mechanism operates to generate two proteins with identical intracellular amino terminal domain but only one of them displays a CRD and associated Ca(2+)-binding sites at the carboxyl terminal extracellular domain.

Galactosyl receptor, a cell surface C-type lectin of normal and tumoral prostate epithelial cells with binding affinity to endothelial cells.

BACKGROUND: The mechanism of bone metastasis of prostate cancer involves the interaction of cell surface receptor(s) on cancer cells with ligand(s) on bone marrow endothelial cell surfaces. The rat galactosyl receptor gene generates two mRNA species by differential splicing: one species encodes a protein identical to the minor form of hepatocyte asialoglycoprotein receptor and displays a galactose/N-acetyl-galactosamine-recognition domain; the other encodes a protein with identical intracellular and transmembrane domains but with a different extracellular domain lacking the carbohydrate-recognition domain (CRD). Both proteins appear to coexist as a heterooligomer on the surface of normal mouse, rat, and human prostate epithelial cells and human prostate cancer cells, including the PC-3 cell line. The CRD of galactosyl receptor mediates adhesion of normal and tumoral prostate cells to the surfaces of a human bone marrow endothelial cell line. The use of inhibitors targeting the CRD would be very valuable in hindering the binding of prostate cancer cells to endothelial cells, thus decreasing the incidence of hematogenous metastasis to bone. METHODS: Molecular biology, immunohistochemistry, flow cytometry, and a cell aggregation assay were used to determine the expression and role of the galactosyl receptor in cell adhesion. RESULTS: Immunoblotting experiments demonstrated that each component of the heterooligomer has a mass of 54 kDa, ascribed in part to associated carbohydrates. An oligonucleotide probe showed the presence of both galactosyl receptor forms in rat prostate and testis, but not in liver, kidney, and spleen. Antibodies to the CRD and a segment of the nonhomologous extracellular domain of the galactosyl receptor blocked cell adhesion to endothelial cell monolayers. CONCLUSIONS: The galactosyl receptor provides a valuable target for the development and use of synthetic ligands capable of disrupting endothelial cell-prostate cancer cell interaction, the first step in prostate cancer bone metastasis.

Cell death patterns of the rat spermatogonial cell progeny induced by sertoli cell geometric changes and Fas (CD95) agonist.

Spermatogonial-Sertoli cell co-cultures, prepared from sexually immature rats (7-10 days old) and maintained for experimental purposes for a maximum period of time of eight days, were used to determine whether Sertoli cell geometry can influence spermatogonial cell growth, viability and differentiation. We have found that when Sertoli cells are allowed to stretch, spermatogonial cell cohorts attached to Sertoli cell surfaces remain viable and exhibit typical cell oscillatory movements with a maximal oscillation radial length of 0.8 microm throughout the duration of the experiments. However, spermatogonial cell viability decreased when Sertoli cells were compelled to contract by preventing cell spreading onto a non-adhesive substrate. A video-microscopy analysis of spermatogonial cells progenies cocultured with contracted Sertoli cells revealed that conjoined members of the cohorts displayed a typical apoptotic sequence preceded by vigorous oscillatory cell movements (maximal oscillation radial length: 1.5 microm) followed by the release of apoptotic bodies and cessation of cell movements. This sequence of events occurred in a single cell. Upon completion of this sequence, another member of the cohort initiated the same cell death course until all members completed the cell death sequence. A similar apoptotic sequence was observed following addition of Fas (CD95/APO-1) antibody (ligand agonist) to the cocultures. Fragmentation of the actin-containing cytoskeleton was observed by indirect immunofluorescence in apoptotic spermatogonial cell cohorts, independent from the activating mechanism. We conclude that by forcing Sertoli cells to contract or by adding an apoptosis inducer to the cocultures, individual members of a spermatogonial cell cohort switch on a death (apoptosis) program in a coordinated fashion.

Structural and biochemical features of fractionated spermatid manchettes and sperm axonemes of the azh/azh mutant mouse.

The tubulin-containing axoneme and manchette develop consecutively during mammalian spermiogenesis. The nature of their molecular components and developmental sequence are not completely known. The azh/azh (for abnormal sperm headshape) mouse mutant is an ideal model for analyzing tubulin isotypes and microtubule-associated proteins of the manchette and axoneme in light of a potential role of the manchette in the shaping of the sperm head and formation of the tail. We have searched for possible differences in tubulin isotype variants in fractionated manchettes and axonemes of wildtype and azh/azh mutant mice using isotype-specific tubulin antibodies as immunoprobes. Manchettes from wild-type and azh/azh mutant mouse spermatids were fractionated from spermatogenic stage-specific seminiferous tubules and axonemes were isolated from epididymal sperm. We have found that: (1) Fractionated manchettes of azh/azh mutants are longer than in wild-type mice; (2) Manchette and sperm tail axonemes display a remarkable variety of posttranslationally modified tubulins (acetylated, glutamylated, tyrosinated, alpha-3/7 tubulins). Acetylated tubulin was more abundant in manchette than in axonemes; (3) An acidic 62 kDa protein was identified as the main component of the perinuclear ring of the manchette in wild-type and azh/azh mice; (4) Bending and looping of the mid piece of the tail of azh/azh sperm, accompanied by a dislocation of the connecting piece from head attachment sites, were visualized by phase-contrast, immunofluorescence and transmission electron microscopy in about 35% of spermatids/sperm; and (5) A lasso-like tail configuration was predominant in epididymal sperm of azh/azh mutants. We speculate that spermatid and sperm tail abnormalities in the azh/azh mutant could reflect structural and/or assembly deficiencies of peri-axonemal proteins responsible for maintaining a stiffened tail during spermiogenesis and sperm maturation.

Isolation of the rat spermatid manchette and its perinuclear ring.

The manchette is a transient structure that develops during spermiogenesis. It consists of three components: a perinuclear ring, a microtubule mantle inserted in the ring, and dense plaques attached at the distal end of the mantle. A procedure has been developed for the fractionation of intact manchettes from rat spermatids. Each fractionation step was monitored by indirect immunofluorescence using an antibody to unmodified alpha-tubulin. Indirect immunofluorescence and electron microscopy demonstrate that fractionated manchettes are relatively intact. A thermocleavage step was used to sever the microtubule mantle from the perinuclear ring. Microtubules of the mantle collected in a stabilizing buffer containing Taxol formed long bundles of side-by-side aligned microtubules. The perinuclear ring sample consisted of circular-shaped units of different diameter with truncated microtubules still attached to the ring, a property that enabled the initial recognition of the rings by alpha-tubulin antibody staining. Indirect immunofluorescence and immunoblotting experiments using isoform-specific antibodies to alpha-tubulins show that the manchette contains acetylated, tyrosinated, glutamylated alpha-tubulin and an alpha-3/7 tubulin isoform. The same alpha-tubulin isoforms were observed in the axoneme of the sperm tail. Two-dimensional polyacrylamide gel electrophoresis fractionation maps of silver-stained proteins of the intact manchette show four predominant proteins: alpha- and beta-tubulins, beta-actin, vimentin, and a 62-kDa protein. The latter persisted in thermocleaved perinuclear ring samples. Results of this study indicate that the newly developed procedure for the fractionation of manchettes will facilitate a direct characterization of posttranslationally modified tubulin variants, microtubule-associatedproteins, and the components of the perinuclear ring of this largely neglected structure of the spermiogenic process.

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 alpha-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.

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 alpha-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/beta-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/beta-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.