Evolutionary conservation of a germ cell-specific lamin persisting through mammalian spermiogenesis.

We had identified earlier a germ cell-specific lamin of 60 kDa in rat which is related to somatic lamin B. This polypeptide was shown to be the only major component organizing the lamina structure of round spermatids. In the present study, we find that this 60-kDa polypeptide persists in the testicular and epididymal sperms of rat. We also show, by indirect immunofluorescence studies, that the 60-kDa protein is antigenically conserved in the germ cells of grasshopper, rooster, and frog and in plant meiocytes. The distribution of fluorescence among the various germ cell populations shows that the antigen is located around the nuclear cortex of pre- and postmeiotic germ cells, while it is distributed all over the pachytene nuclei. The anti-60-kDa polyclonal antibodies also reacted with a 60-kDa polypeptide in the Western blot analysis of nuclear matrix proteins of grasshopper germ cells. The similar fluorescent localization pattern of the antigen observed in various eukaryotic species strongly suggests that this germ cell-specific lamin may play a very crucial role during meiotic prophase, particularly during homologous chromosome pairing and recombination.     

Stage-dependent Dishevelled-1 expression during mouse spermatogenesis suggests a role in regulating spermatid morphological changes

Dishevelled (Dsh in Drosophila or DVL in mice) is a member of the highly conserved Wg/Wnt signaling pathway, which regulates important processes such as cell proliferation, polarity, and specification of cell fate. Three orthologous genes of Dishevelled (Dvl-1, Dvl-2, and Dvl-3) have been found in both humans and mice. They play pivotal roles in regulating cell morphology and a variety of changes in cell behaviors. In the present study, we show that the expression of Dvl-1 is stage-dependent during mouse spermatogenesis, although Dvl-2 and Dvl-3 show relative consistent expression. The expression of Dvl-1 mRNA first appears in pachytene spermatocytes, increases in round and elongating spermatids, and then turns to an undetectable level in mature sperm cells. Analyses of immunohistochemistry and immunofluorescence staining show that DVL-1 is present diffusely in the cytoplasm of pachytene spermatocytes and exhibits mainly a vesicular pattern and perinuclear distribution and a weak diffusely cytoplasmic signal in round and elongating spermatids. The vesicular pattern of DVL-1 has been observed by previous studies in somatic cells, and suggested to play roles in signal transduction. Immunoprecipitation experiments show that DVL-1 coimmunprecipitates with spermatogenic cells beta-actin rather than alpha-tubulin. These results indicate that DVL-1 may be involved in spermatid morphological changes during mouse spermiogenesis through mediating signal transduction and/or regulating actin cytoskeleton organization.     

Ultrastructural changes in the male germ cells of Bulinus truncatus during spermatogenesis

The structure of the spermatogonia, spermatocytes, spermatids and Sertoli cells of the hermaphroditic snail Bulinus truncatus was studied by electron microscopy. The spermatogonia are small, with relatively large nuclei. The acrosome develops from a small proacrosomal granule which is probably derived from the Golgi apparatus in the spermatocyte stage. Condensation and elongation of the nuclei were found in the spermatids. The shape and components of the Sertoli cells did not change during the spermatogonium and spermatocyte stages. Before spermiation the Sertoli cells have the morphological features of steroid-producing cells. The study showed that the Sertoli cells are involved in the nutrition and transportation of the spermatogenic cells, in spermiation and in hormone production.     

A novel germ cell-specific protein, SHIP1, forms a complex with chromatin remodeling activity during spermatogenesis

To determine the mechanisms of spermatogenesis, it is essential to identify and characterize germ cell-specific genes. Here we describe a protein encoded by a novel germ cell-specific gene, Mm.290718/ZFP541, identified from the mouse spermatocyte UniGene library. The protein contains specific motifs and domains potentially involved in DNA binding and chromatin reorganization. An antibody against Mm.290718/ZFP541 revealed the existence of the protein in testicular spermatogenic cells (159 kDa) but not testicular and mature sperm. Immunostaining analysis of cells at various stages of spermatogenesis consistently showed that the protein is present in spermatocytes and round spermatids only. Transfection assays and immunofluorescence studies indicate that the protein is localized specifically in the nucleus. Proteomic analyses performed to explore the functional characteristics of Mm.290718/ZFP541 showed that the protein forms a unique complex. Other major components of the complex included histone deacetylase 1 (HDAC1) and heat-shock protein A2. Disappearance of Mm.290718/ZFP541 was highly correlated with hyperacetylation in spermatids during spermatogenesis, and specific domains of the protein were involved in the regulation of interactions and nuclear localization of HDAC1. Furthermore, we found that premature hyperacetylation, induced by an HDAC inhibitor, is associated with an alteration in the integrity of Mm.290718/ZFP541 in spermatogenic cells. Our results collectively suggest that the Mm.290718/ZFP541 complex is implicated in chromatin remodeling during spermatogenesis, and we provide further information on the previously unknown molecular mechanism. Consequently, we re-designate Mm.290718/ZFP541 as "SHIP1" representing spermatogenic cell HDAC-interacting protein 1.     

The differential expression of lamin epitopes during mouse spermatogenesis

The presence of lamin proteins in mouse spermatogenic cells has been examined by using an anti-lamin AC and an anti-lamin B antisera which recognize somatic lamins A and C, and somatic lamin B, respectively. Anti-lamin B binds to the nuclear periphery of all cell types examined, including Sertoli cells, primitive type A spermatogonia, preleptotene, leptotene, zygotene and pachytene spermatocytes, and round spermatids. In sperm nuclei, the antigenic determinants are localized to a narrow domain of the nucleus. However, after removing the perinuclear theca, anti-lamin B localizes to the entire nuclear periphery in a punctate pattern, suggesting that it is binding to determinants previously covered by the theca constituents. On immunoblots anti-lamin B reacts with a ～ 68 kD polypeptide in all germ cells and, to a lesser extent, with four additional polypeptides present only in meiotic and post-meiotic nuclear matrices. Anti-lamin AC also reacts with the perinuclear region of the somatic cells in the testes, in particular, those of the interstitium and also the Sertoli cells of the seminiferous epithelium. In contrast to anti-lamin B, anti-lamin AC does not bind to the germ cells at any stage of spermatogenesis. In addition, nuclear matrix proteins from isolated spermatogenic cells do not bind anti-lamin AC on immunoblots, suggesting the lack of reactivity is not due to the masking of any antigenic sites. These data demonstrate that germ cells contain lamin B throughout spermatogenesis, even during meiosis and spermiogenesis when the nuclear periphery lacks a distinct fibrous lamina. © 1993 Wiley-Liss, Inc.     

Conservation of the gene structure and membrane-targeting signals of germ cell-specific lamin LIII in amphibians and fish

Targeting of nuclear lamins to the inner nuclear membrane requires CaaX motif-dependent posttranslational isoprenylation and carboxyl methylation. We previously have shown that two variants of lamin LIII (i.e., LIII and LIIIb) in amphibian oocytes are generated by alternative splicing and differ greatly in their membrane association. An extra cysteine residue (as a potential palmitoylation site) and a basic cluster in conjunction with the CaaX motif function as secondary targeting signals responsible for stable membrane association of lamin LIIIb. cDNA sequencing and genomic analysis of the zebrafish Danio rerio lamin LIII uncovers a remarkable conservation of the genomic organization and of the two secondary membrane-targeting signals in amphibians and fish. The expression pattern of lamin LIII genes is also conserved between amphibians and fish. Danio lamin LIII is expressed in diplotene oocytes. It is absent from male germ cells but is expressed in Sertoli cells of the testis. In addition, we provide sequence information of the entire coding sequence of zebrafish lamin A, which allows comparison of all major lamins from representatives of the four classes of vertebrates.     

Stage-dependent appearance of sulfhydryl oxidase during spermatogenesis in the testis of rat and hamster

Summary Sulfhydryl oxidase (SOx), an enzyme that catalyzes the oxidation of sulfhydryl compounds, appears in the spermatogenic cells of rat and hamster testes in a stage-dependent manner. It first appears in pachytene spermatocytes at stage I in both the animal species studied. SOx immunoreactivity is associated with mitochondria of these cells. The fate of such mitochondria is species-dependent. In rat, the immunoreactive mitochondria aggregate during maturation phase and are retained in the residual bodies. Spermatozoa free of SOx are released into the lumen. On the other hand, in hamster, the immunoreactive mitochondria arrange themselves around the midpiece of spermatozoa. In such a case, residual bodies lack SOx. The appearance of SOx coincides with the appearance of LDH-X in the spermatogenic cells. Like many other proteins such as LDH-X, RSA-1 and cytochrome c_t, SOx provides yet another example of differential gene activation associated with a developmental process of gametes.     

Immuno-electron microscopic localization of calmodulin and calmodulin-binding proteins in the mouse germ cells during spermatogenesis and maturation

When extracts of mouse testis were Western-blotted against a monoclonal antibody which reacts with calmodulin in the presence of Ca²⁺, all calmodulin was associated with the macromolecules of molecular weight above 50 kDa. Immuno-electron microscopy of testes using this antibody indicated that calmodulin is localized at higher density in the nucleus and cytoplasm of germ cells during the developmental phase between pachytene and round spermatid, showing the highest level just before meiotic divisions. There was no special association of calmodulin to any organelles in these cells. Extremely low levels of calmodulin occurred in spermatogonia and other testicular tissue cells. Calmodulin decreased dramatically as spermatids underwent metamorphosis, becoming detectable only at the perinuclear space of sperm heads. Further relocation to the postacrosomal region occurred during sperm transit to the cauda epididymis. Immunodetection after the calmodulin overlay on ultrathin sections revealed a sharp increase of calmodulin immunogold deposits in the nuclei of spermatids accompanying their condensation. The results indicate that some calmodulin-binding proteins, but not calmodulin itself, accumulate in the nuclei during the final steps of spermiogenesis.     

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation

Background

Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues.

Results

In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis.

Conclusions

Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.     

Identification of a short nuclear lamin protein selectively expressed during meiotic stages of rat spermatogenesis

The nuclear lamina is a karyoskeletal structure located at the nuclear periphery and intimately associated with the inner nuclear membrane. It is composed of a multigene family of proteins, the lamins, which show a conspicuous cell type-specific expression pattern. The functional role of lamins has not been definitively established but available information indicates that they are involved in the organization of nuclear envelope and interphase chromatin. Spermatogenesis is characterized, among other features, by stage-specific changes in chromatin organization and function. These changes are accompanied by modifications in the organization and composition of the nuclear lamina. In previous experiments we have determined that rat spermatogenic cells express a lamin closely related, if not identical, to lamin B1 of somatic cells; whereas rat somatic lamins A, C, D and E were not detected. Considering that chromatin reorganizations during spermatogenesis may be directly or indirectly related to changes of the nuclear lamina we have decided to further investigate lamin expression during this process. Here we report on the identification of a 52 kDa protein of the rat which, according to immunocytochemical and biochemical data, appears to be a novel nuclear lamin. Using meiotic stage-specific markers, we have also demonstrated that this short lamin is selectively expressed during meiotic stages of spermatogenesis.     

Native tesmin is a 60-kilodalton protein that undergoes dynamic changes in its localization during spermatogenesis in mice

Tesmin is a testis-specific protein. Four mouse tesmin cDNAs so far reported encode a testis-specific, metallothionein-like, 30-kDa protein (tesmin-30). An antibody against tesmin-30, however, detected a protein of 60 kDa (tesmin-60) from the mouse testis. To resolve the relationship between the two, the immunoprecipitated native tesmin-60 was sequenced. The result indicated that tesmin-30 is not full-length but is part of the C-terminal half of tesmin-60. The full-length cDNA (2.2 kilobases [kb]) encoding tesmin-60 (475 amino acid residues) and its genomic DNA (23 kb) were cloned and sequenced. A search of databases indicated that tesmin is a member of the CXC-hinge-CXC family. Immunohistochemistry indicated that tesmin exhibits dynamic subcellular localization changes during spermatogenesis. Before meiosis, it was localized in the cytoplasm of early to late spermatocytes and then translocated into the nucleus just before meiotic division. After meiosis, it appeared in spermatids, starting from the acrosomal vesicles, moving to the nuclear membrane and then to the caudal end as the spermatids elongated, and finally relocating into the cytoplasm. Oxidative stress by cobalt chloride, as well as by diethylmaleate, induced both premature translocation of tesmin from the cytoplasm to the nucleus and apoptotic signals in spermatocytes. The persistent existence of tesmin and its temporally and spatially dynamic localization suggest that tesmin is involved in multiple stages of spermatogenesis and spermiogenesis, possibly during sperm maturation and/or morphogenesis.     

Ultrastructural localization and distribution of Nardilysin in mammalian male germ cells

Background

NRD convertase, also termed Nardilysin, is a Zn⁺⁺ metalloendopeptidase that specifically cleaves the N-terminus of arginine and lysine residues into dibasic moieties. Although this enzyme was found located within the testis, its function in male reproduction is largely unknown. In addition, the precise distribution of this enzyme within germ cells remains to be determined.

Methods

To answer these questions, we developed an immuno-gold electron microscopy analysis to detect Nardilysin at ultrastructural level in mice. In addition, we performed a quantitative analysis of these gold particles to statistically estimate the distribution of Nardilysin in the different subcellular compartments of differentiating late spermatids/spermatozoa.

Results

Expression of Nardilysin in wild-type mice was restricted to germ cells and markedly increased during the last steps of spermiogenesis. In elongated spermatids, we found the enzyme mainly localized in the cytoplasm, more precisely associated with two microtubular structures, the manchette and the axoneme. No labelling was detected over the membranous organelles of the spermatids. To test whether this localization is dependent of the functional microtubules organization of the flagella, we analysed the localization into a specific mouse mutant ebo/ebo (ébouriffé) known to be sterile due to an impairment of the final organization of the flagellum. In the ebo/ebo, the enzyme was still localized over the microtubules of the axoneme and over the isolated cytoplasmic microtubules doublets. Quantification of gold particles in wild-type and mutant flagella revealed the specific association of the enzyme within the microtubular area of the axoneme.

Conclusions

The strong and specific accumulation of Nardilysin in the manchette and axoneme suggests that the enzyme probably contributes either to the establishment of these specific microtubular structures and/or to their functional properties.

     

Chromatin ultrastructure changes during spermatogenesis in armadillos

The chromatin fibres of the male gamete nucleus of Dasypus novemcinctus contain fibrils. Measurements indicate that the chromatin fibre diameter and the number of fibrils within the fibre increase, whereas the diameter of the fibrils decreases during spermiogenesis. It is suggested that the fibrils represent a DNA-protein complex. It is further suggested that the increased fibre diameter, and the increased number of fibrils correlates with the formation of larger fibres during the condensation of the spermatid nucleus, whereas the decrease in fibril diameter is correlated with the replacement of histones with new low molecular weight acid-soluble proteins.     

Stage-dependent appearance of sulfhydryl oxidase during spermatogenesis in the testis of rat and hamster. An immunohistochemical study

Sulfhydryl oxidase (SOx), an enzyme that catalyzes the oxidation of sulfhydryl compounds, appears in the spermatogenic cells of rat and hamster testes in a stage-dependent manner. It first appears in pachytene spermatocytes at stage I in both the animal species studied. SOx immunoreactivity is associated with mitochondria of these cells. The fate of such mitochondria is species-dependent. In rat, the immunoreactive mitochondria aggregate during maturation phase and are retained in the residual bodies. Spermatozoa free of SOx are released into the lumen. On the other hand, in hamster, the immunoreactive mitochondria arrange themselves around the midpiece of spermatozoa. In such a case, residual bodies lack SOx. The appearance of SOx coincides with the appearance of LDH-X in the spermatogenic cells. Like many other proteins such as LDH-X, RSA-1 and cytochrome ct, SOx provides yet another example of differential gene activation associated with a developmental process of gametes.