Mammalian Spermiogenesis Research Articles

Condensation of DNA by Spermatid Basic Nuclear Proteins

Two transition proteins, TP1 and TP2, participate in the repackaging of the spermatid genome early in mammalian spermiogenesis, coincident with the first detectable changes in chromatin condensation. Using an optical trap and a two-channel flow cell to move single DNA molecules into buffer containing protein, we have measured the rates of DNA condensation and decondensation induced by the binding of Syrian hamster transition proteins TP1 and TP2 and protamines P1 and P2. The results show that both transition proteins condense free DNA, with rates similar to those of protamine 1 and 2. DNA molecules condensed with TP1 were significantly less stable than DNA condensed by protamine or by TP2. Experiments conducted with a peptide corresponding to the C-terminal 25 residues of TP2 showed that this domain is responsible for condensing DNA. Experiments conducted with two fragments of TP1 containing arginine and lysine residues demonstrated that DNA binding by TP1 must involve more than these basic sequences. Zinc facilitated the condensation of DNA by P2 but not by TP2. The dissociation rates of TP2 and P2 from DNA were not affected by the addition of zinc.

Previously uncharacterized histone acetyltransferases implicated in mammalian spermatogenesis.

During spermiogenesis (the maturation of spermatids into spermatozoa) in many vertebrate species, protamines replace histones to become the primary DNA-packaging protein. It has long been thought that this process is facilitated by the hyperacetylation of histone H4. However, the responsible histone acetyltransferase enzymes are yet to be identified. CDY is a human Y-chromosomal gene family expressed exclusively in the testis and implicated in male infertility. Its mouse homolog Cdyl, which is autosomal, is expressed abundantly in the testis. Proteins encoded by CDY and its homologs bear the "chromodomain," a motif implicated in chromatin binding. Here, we show that (i) human CDY and mouse CDYL proteins exhibit histone acetyltransferase activity in vitro, with a strong preference for histone H4; (ii) expression of human CDY and mouse Cdyl genes during spermatogenesis correlates with the occurrence of H4 hyperacetylation; and (iii) CDY and CDYL proteins are localized to the nuclei of maturing spermatids where H4 hyperacetylation takes place. Taken together, these data link human CDY and mouse CDYL to the histone-to-protamine transition in mammalian spermiogenesis. This link offers a plausible mechanism to account for spermatogenic failure in patients bearing deletions of the CDY genes.

Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair

An important chromatin remodeling process is taking place during spermiogenesis in mammals and DNA strand breaks must be produced to allow the accompanying change in DNA topology. Endogenous DNA strand breaks are indeed detected at mid-spermiogenesis steps but are no longer present in mature sperm. Both in vitro and in vivo evidence suggests that the DNA-binding and condensing activities of a set of basic nuclear ‘transition proteins’ may be crucial to the integrity of the chromatin remodeling process. We propose that these proteins are necessary for the repair of the strand breaks so that DNA fragmentation is minimized in the mature sperm.

Keratins: unraveling the coordinated construction of scaffolds in spermatogenic cells.

Recent work shows that two groups of keratins are expressed during mammalian spermatogenesis. One group, belonging to the classic epidermis-type keratins, is present in spermatogonia, spermatocytes, and spermatids. A member of this group, Sak57, a keratin 5 homologue, has been shown to co-align with microtubules and provide a scaffolding shell while also strengthening intercellular cytoplasmic bridges conjoining members of spermatogonial and spermatocyte cohorts. The other, keratin 9, is a component of the perinuclear ring of the manchette, a microtubular structure developed during the elongation and condensation of the spermatid nucleus. The second group, the outer dense fiber (Odf) proteins, is expressed preferentially during mammalian spermiogenesis. The family of Odf proteins-Odf1, Odf2, and Odf3-includes an expanding group of proteins co-assembled along the axoneme during the development of the sperm tail. Investigations on the assembly of epidermis-type and Odf sperm tail-targeted keratins are now focused on a group of chaperone-like Odf-binding molecules, designated Spags. Spags appear to drive Odfs to a precise destination. A daunting task is to determine how members of the family of keratins get the signal to produce linear scaffolds in specific spermatogenic cell populations and transport keratins to microtubule-containing structures such as the manchette and axoneme.

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice.

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.

Characterization of Sptrx, a Novel Member of the Thioredoxin Family Specifically Expressed in Human Spermatozoa

Thioredoxins (Trx) are small ubiquitous proteins that participate in different cellular processes via redox-mediated reactions. We report here the identification and characterization of a novel member of the thioredoxin family in humans, named Sptrx (sperm-specific trx), the first with a tissue-specific distribution, located exclusively in spermatozoa. Sptrx open reading frame encodes for a protein of 486 amino acids composed of two clear domains: an N-terminal domain consisting of 23 highly conserved repetitions of a 15-residue motif and a C-terminal domain typical of thioredoxins. Northern analysis and in situ hybridization shows that Sptrx mRNA is only expressed in human testis, specifically in round and elongating spermatids. Immunostaining of human testis sections identified Sptrx protein in spermatids, while immunofluorescence and immunogold electron microscopy analysis demonstrated Sptrx localization in the cytoplasmic droplet of ejaculated sperm. Sptrx appears to have a multimeric structure in native conditions and is able to reduce insulin disulfide bonds in the presence of NADPH and thioredoxin reductase. During mammalian spermiogenesis in testis seminiferous tubules and later maturation in epididymis, extensive reorganization of disulfide bonds is required to stabilize cytoskeletal sperm structures. However, the molecular mechanisms that control these processes are not known. The identification of Sptrx with an expression pattern restricted to the postmeiotic phase of spermatogenesis, when the sperm tail is organized, suggests that Sptrx might be an important factor in regulating critical steps of human spermiogenesis.

Lack of sharing of Spam1 (Ph-20) among mouse spermatids and transmission ratio distortion.

Gametic equality is thought to exist, despite haploid gene action in mammalian spermiogenesis, because of product sharing via the intercellular bridges of conjoined spermatids. However, mice carrying different t-alleles have been known to produce functionally different sperm, leading to transmission ratio distortion (TRD), whose mechanism is unknown. The reduced Spam1 mRNA levels, previously shown to be associated with TRD and reduced fertility in mice carrying the Rb(6.16) or the Rb(6.15) Robertsonian translocation, are reflected in the levels of its encoded membrane protein (Spam1) and its accompanying insoluble hyaluronidase activity. Studies of the temporal expression pattern of Spam1 reveal that it is haploid expressed, with both the RNA and protein first appearing on Day 21.5. RNA fluorescence in situ hybridization and immunocytochemistry show both the mRNA and the protein to be compartmentalized. Compartmentalization of the mRNA along with its immediate translation and insertion of the protein in the plasma membrane suggests the nonsharing of Spam1 transcripts among spermatids, resulting in functionally different sperm in males with different Spam1 alleles. Evidence for biochemically different sperm in these heterozygous males was revealed by flow cytometry and confocal microscopy. Our findings support the notion that the Spam1 antigen is not shared, and we may have uncovered a mechanism for TRD.

EXAFS analysis of the zinc-binding domain of boar spermatidal transition protein 2.

Boar Spermatidal Transition Protein 2 (TP2; 137 amino acid residues) is supposed to play an important role in initiation of chromatin condensation and cessation of transcriptional activity during mammalian spermniogenesis. Boar TP2 has three potential zinc finger motifs and binds three atoms of zinc per molecule. However the structure of the zinc-binding domain of boar TP2 has not been completely determined. To elucidate the local structure around the zinc atoms of boar TP2, we performed an X-ray absorption fine structure (XAFS) measurement on the zinc-binding domain of TP2(TP2Z)(residues 1-103) in the fluorescence mode. By EXAFS analyses we have demonstrated that each of the three zinc atoms is coordinated by approximately two sulfur and two nitrogen atoms on average. The average Zn-S and Zn-N distances were found to be 2.36 and 2.01 A, respectively. The sulfur and nitrogen atoms are attributed to cysteine and histidine residues, respectively, from comparison of the EXAFS spectra with model compounds ZnS and ZnTPP zinc(II) tetraphenylporphyrin).

Targeting and fusion proteins during mammalian spermiogenesis.

Regulated exocytosis is controlled by internal and external signals. The molecular machinery controlling the sorting from the newly synthesized vesicles from the Golgi apparatus to the plasma membrane play a key role in the regulation of both the number and spatial location of the vesicles. In this context the mammalian acrosome is a unique vesicle since it is the only secretory vesicle attached to the nucleus. In this work we have studied the membrane trafficking between the Golgi apparatus and the acrosome during mammalian spermiogenesis. During bovine spermiogenesis, Golgi antigens (mannosidase II) were detected in the acrosome until the late cap-phase spermatids, but are not found in testicular spermatozoa (maturation-phase spermatids). This suggests that Golgiacrosome flow may be relatively unselective, with Golgi residents retrieved before spermination is complete. Surprisingly, rab7, a protein involved in lysosome/endosome trafficking was also found associated with the acrosomal vesicle during mouse spermiogenesis. Our results suggest that the acrosome biogenesis is associated with membrane flow from both the Golgi apparatus and the endosome/lysosome system in mammalian spermatids.

Stimulation of DNA repair by the spermatidal TP1 protein.

The chromatin remodeling process that takes place during spermiogenesis in mammals is characterized by a transient increase in DNA single-strand breaks (SSB). The mammalian transition proteins (TPs) are expressed at a high level at mid-spermiogenesis steps coincident with chromatin remodeling and could be involved in the repair of these lesions since SSB are no longer detected in terminally differentiated spermatids. We report that TP1 can stimulate the repair of SSB in vitro and demonstrate that in vivo repair of UV-induced DNA lesions is enhanced in mammalian cells stably expressing TP1. These results suggest that, aside from its role in DNA compaction, this major transition protein may contribute to the yet unidentified enzymatic activity responsible for the repair of SSB at mid-spermiogenesis steps. These results also suggest that the TP1 proteins have the potential to participate in the repair process following genotoxic insults and therefore may play an active role in the maintenance of the integrity of the male haploid genome during spermiogenesis.

Identification of Two Novel Zinc Finger Modules and Nuclear Localization Signal in Rat Spermatidal Protein TP2 by Site-directed Mutagenesis

Spermatidal protein TP2, which appears transiently during stages 12-16 of mammalian spermiogenesis, is a DNA condensing zinc metalloprotein with a preference to GC-rich DNA. We have carried out a detailed site-directed mutagenesis analysis of rat spermatidal protein TP2 to delineate the amino acid residues involved in coordination with two atoms of zinc. Two zinc fingers modules have been identified involving 4 histidine and 4 cysteine residues, respectively. The modular structure of the two zinc fingers identified in TP2 define a new class of zinc finger proteins that do not fall into any of the known classes of zinc fingers. Transfection experiments with COS-7 cells using wild type and the two zinc finger pocket mutants have shown that TP2 preferentially localizes to nucleolus. The nuclear localization signal in TP2 was identified to be (87)GKVSKRKAV(95) present in the C-terminal third of TP2 as a part of an extended NoLS sequence.

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.

CpG islands in chromatin organization and gene expression.

CpG islands are stretches of DNA sequence that are enriched in the (CpG)n repeat and are present in close association with all housekeeping genes as well as some tissue-specific genes in the mammalian genome. Methylation of CpG islands strongly influences both structural organization and function of chromatin. The presence of a CpG island in a given chromosomal domain can, by itself, give rise to relatively open and active chromatin. Recently, several histone acetyltransferases, histone deacetylases, and chromatin remodeling factors have been found to be part of the transcription machinery. It is becoming increasingly clear that CpG islands and their methylation status may influence the function or recruitment of these newly discovered chromatin remodeling factors, especially the histone deacetylases. In addition, CpG islands may also play a significant role in the reorganization of chromatin during mammalian spermiogenesis.

Centrosome reduction during mammalian spermiogenesis

Spermiogenesis is the terminal differentiation of postmeiotic germ cells into spermatozoa. The cellular and molecular pathways of centrosome reduction during mouse, rhesus, and human spermiogenesis are described. This chapter discusses the fundamental events and stages of centrosome reduction during mammalian spermiogenesis. The developmental stages of isolated mouse spermatids show remarkable events of centrosomal changes: round spermatid stage, elongating spermatid stage, and late elongating stage. The process of centrosome reduction extends throughout the entire testicular and epididymal stages of spermiogenesis and comprises four stages: (a) loss of microtubule nucleating function, (b) formation of transitional microtubule organizing centers (MTOCs), (c) loss of centrosomal proteins, and (d) centriole degeneration. The observation of a systematic degeneration of distal centriole and γ-tubulin in nonrodent mammalian sperm implies that centrosome reduction could be a ubiquitous phenomenon taking place in all mammalian sperm to varying degrees, the mouse sperm representing the highest state and exhibiting complete loss of both centrioles and centrosomal proteins. The complete absence of centrosomes in mouse sperm consolidates the hypothesis that murine oocytes can compensate for the lack of paternal centrosomal contribution. The lack of a standard distal centriole and γ-tubulin in rhesus and human sperm does not interfere with the formation of the zygotic centrosome during fertilization and bipolar spindles during cleavages.

Expression of the spermatid-specific Hsp70 antigen is conserved in mammals including marsupials.

The anatomical location of testes in mammals ranges from a location close to that observed in the embryo to a lower position usually involving a pendant scrotum. In scrotal mammals, the abdominal position of the cryptorchid testis, which elevates its temperature, is detrimental to spermatogenesis and causes infertility. Spermatocytes are sensitive but late spermatids are relatively resistant to thermal stress suggesting that the latter might be protected in some way. In general, most organisms express Hsp70 proteins, which play a crucial role in the protection of cells against thermal stress. We have found previously that the Hsc70t protein, a member of the Hsp70 family of proteins, is constitutively expressed in the late spermatids of mice. Here, we have utilized immunohistochemistry with anti-mouse Hsc70t antiserum to examine the expression of the spermatid-specific Hsp70 antigen in the testes of several mammalian species with different degrees of testes migration. Our data indicate that the antigen is conserved in the mammals including marsupials. We also examined whether antigens of Hsp70-related proteins were expressed in non-mammalian vertebrates including not only homoiothermal but also poikilothermal animals. The spermatid-specific Hsp70 antigens were not detectable in the testes of the animals examined. From results of immunohistochemistry with BRM22 monoclonal antibody which reacts broadly with Hsp70 family proteins, however, we revealed constitutive expression of antigens of Hsp70-related proteins in spermatogenic cells of the vertebrates. These results suggest that the expression of spermatid-specific Hsp70 protein may be involved in the developmental pathway during spermiogenesis in mammals rather than in thermotolerance.

Generation of Multiple Site-Specific Mutations in a Single Polymerase Chain Reaction Product

Site-directed mutagenesis has been very valuable in studying the structure–function relationship of proteins (1). PCR-mediated mutagenesis has been the method of choice in recent years to change the desired amino acid from one to another. Several procedures have been described to achieve the desired mutation using PCR technology (2–13). Although in most of the cases the desired change is only with respect to one amino acid, sometimes one needs to generate several changes in the amino acid sequence to understand the biological function of a given protein. Such a necessity is apparent when one is dealing with a zinc finger class of proteins wherein multiple residues of cysteine and histidine are involved in the coordination of zinc. We have been working over the past several years on the structure–function relationship of a spermatidal specific protein TP2 which appears transiently during mammalian spermiogenesis.

Architectural DNA-Binding Properties of the Spermatidal Transition Proteins 1 and 2

Mammalian spermiogenesis is characterized by replacement of somatic histones by a set of basic nuclear transition proteins thought to be actively involved in the chromatin remodeling process. The two major transition proteins of the elongating spermatids, namely TP1 and TP2, were expressed and purified using a bacterial expression system. Both topoisomerase and ligase-mediated supercoiling assays demonstrated that TP1, as well as TP2, did not produce detectable changes in the twist and/or writhe of DNA molecules upon binding. Ligase-mediated circularization assay further demonstrated that neither of the transition proteins under study produced bends in linear DNA but that they both have the capacity to stimulate oligomerization of linear DNA fragments. We further established that the transition proteins arein vitrosubstrates for the Ca+2-phospholipid-dependent protein kinase (PKC) as well as the cAMP-dependent protein kinase (PKA). PKC phosphorylation was found to strongly weaken the DNA-condensing ability of TP2. These results suggest that the major transition proteins represent architectural factors able to stabilize DNA in a nonsupercoiled state, thereby promoting DNA condensation.

Haploid transcripts persist in mature human spermatozoa.

Mammalian spermiogenesis is marked by the morphological and functional differentiation of round haploid spermatids into mature spermatozoa. A molecular restructuring of the chromatin accompanies this process facilitated by the transition proteins and protamines which compact and condense the genetic material within the developing spermatid. Previous studies from this laboratory have demonstrated that human protamines PRM1, PRM2 and transition protein TNP2 transcripts are associated with round and elongating spermatids. Extending this investigation, we examined the occurrence of these transcripts in mature spermatozoa by in-situ hybridization analysis using [35S]-labelled cRNA probes. These results demonstrate that PRM1, PRM2 and TNP2 haploid-specific transcripts are present in mature spermatozoa. Quantitative analysis of the localized signal also indicates that the PRM1, PRM2 and TNP2 transcripts persist at a similar ratio to that previously described for these transcripts in human testes, i.e. PRM2 > PRM1 approximately equal to TNP2. The persistence of these transcripts in mature spermatozoa warrants further investigation.

Increased activity associated with the MAST205 protein kinase complex during mammalian spermiogenesis.

The morphological and biochemical changes that occur in the haploid male germ cell during spermiogenesis facilitate the natural delivery of the paternally imprinted chromosomes into oocytes. Despite the obvious morphological changes, little is known about the molecular events underlying spermiogenesis. We recently cloned a novel 205-kDa manchette microtubule-associated serine/threonine protein kinase (MAST205) from mouse testis. The objective of this study was to further delineate the role of MAST205 in mammalian spermiogenesis. While MAST205 RNA levels were similar in pachytene spermatocytes, round spermatids, and residual bodies, MAST205 protein could be detected only in round spermatids and residual bodies. Kinase activity associated with MAST205 immunoprecipitates was low in pachytene spermatocytes, high in round spermatids, and maximal in residual bodies, indicating that MAST205-associated kinase activity is modified during spermatid maturation. Furthermore, MAST205 protein and the associated kinase activity were not detected in epididymal spermatozoa, indicating that MAST205 protein is either excluded from, or degraded in, the latter cell type. Multiple heterologous protein species were seen in immunoprecipitates from 35S-labeled mouse seminiferous tubules using an affinity-purified MAST205 antiserum. Consistent with this observation, MAST205 eluted as part of a 1-2 x 10(6) dalton protein complex when extracts of mouse testis were fractionated by Superose 6 column chromatography. MAST205 mRNA was detected in human testis indicative of conservation in other mammalian species. Taken together, these results indicate that the MAST205 complex functions in spermatid maturation in mammals.

The Testis-Specific High-Mobility-Group Protein, a Phosphorylation-Dependent DNA-Packaging Factor of Elongating and Condensing Spermatids

Mammalian spermiogenesis is characterized by a striking restructuring of the spermatid chromatin caused by the replacement of nucleohistones with transition proteins and their subsequent replacement with nucleoprotamines. The onset of nuclear elongation and chromatin condensation in spermatids is accompanied by a general decrease in the transcriptional activity of the DNA. A recently identified testis-specific high-mobility-group (tsHMG) protein, similar to the human mitochondrial transcription factor I and to the linker-associated protein delta of Tetrahymena thermophila micronuclei, is thought to play a structural role in this process. We confirm by immunoblot analysis of fractionated germ cells that the presence of tsHMG is restricted to transcriptionally quiescent elongating and condensing spermatids. Purified recombinant tsHMG protein displays preferential binding to supercoiled plasmid DNA, which reversibly protects the DNA against the DNA-relaxing activity of eukaryotic topoisomerase I and also impairs the transcriptional activity of this template when assayed in vitro. The tsHMG protein can also introduce negative supercoils into a relaxed plasmid substrate in a topoisomerase I-dependent manner. We also show that the tsHMG protein is the substrate of a Ca2+-phospholipid-dependent protein kinase (protein kinase C) present in testis extracts of adult mice and demonstrate that phosphorylation by protein kinase C is required for both the DNA-binding and the topoisomerase I-dependent supercoiling activities of tsHMG. Our results support the hypothesis that the spermatid tsHMG protein is a topological factor (transition protein) that can modulate the activity of topoisomerase I. This activity could contribute to the important transition in chromatin structure which leads to the decrease in DNA metabolism observed at the early stages of spermatid elongation.