Spermatid-specific Protein Research Articles

Cell-Cell Interactions in the Testis that Support Male Fertility

The testis has two important functions: the production of sperm for fertility and the synthesis and release of androgens, such as testosterone, that maintain sperm production, sexual function, health, and wellbeing. Sperm production takes place within the seminiferous tubules, supported by the somatic Sertoli cells, and is stimulated by gonadotrophins produced by the pituitary and androgens produced by the Leydig cells in the interstitium. Spermatogenesis is a highly complex and co-ordinated process regulated via cell-cell interactions between multiple cell types. Sertoli cells produce many factors, in a cyclic manner, that are required for germ cell development and, in turn, they receive signals from developing germ cells to maintain their function. Regulation of the spermatogenic cycle involves different cell types co-operating to produce a “pulse” of retinoic acid that stimulates immature germ cells to become committed to differentiation. Research in the past decade has shown that Sertoli cells do much more than regulate germ cell development; they maintain the development and function of peritubular myoid cells, the testicular vasculature and the Leydig cells. Sertoli cells send signals to interstitial immune cells to maintain an immunosuppressive environment and deposit thousands of proteins into the interstitium, including spermatid-specific proteins that likely help to promote peripheral immune tolerance. Leydig cells produce testosterone that acts on receptors in Sertoli cells to initiate and maintain spermatogenesis, and in peritubular myoid cells to support germ cell development. Leydig cells exist in a niche with specialised macrophages that can regulate Leydig cell function and steroidogenesis, highlighting that cross-talk between testis cells and the immune system is important for testis function. Defining the specific signals that operate between the different cell types that regulate key testis functions will help to uncover new therapies to support male fertility and testicular testosterone production over the life course.

Nuclear elongation during spermiogenesis depends on physical linkage of nuclear pore complexes to bundled microtubules by Drosophila Mst27D.

Spermatozoa in animal species are usually highly elongated cells with a long motile tail attached to a head that contains the haploid genome in a compact and often elongated nucleus. In Drosophila melanogaster, the nucleus is compacted two hundred-fold in volume during spermiogenesis and re-modeled into a needle that is thirty-fold longer than its diameter. Nuclear elongation is preceded by a striking relocalization of nuclear pore complexes (NPCs). While NPCs are initially located throughout the nuclear envelope (NE) around the spherical nucleus of early round spermatids, they are later confined to one hemisphere. In the cytoplasm adjacent to this NPC-containing NE, the so-called dense complex with a strong bundle of microtubules is assembled. While this conspicuous proximity argued for functional significance of NPC-NE and microtubule bundle, experimental confirmation of their contributions to nuclear elongation has not yet been reported. Our functional characterization of the spermatid specific Mst27D protein now resolves this deficit. We demonstrate that Mst27D establishes physical linkage between NPC-NE and dense complex. The C-terminal region of Mst27D binds to the nuclear pore protein Nup358. The N-terminal CH domain of Mst27D, which is similar to that of EB1 family proteins, binds to microtubules. At high expression levels, Mst27D promotes bundling of microtubules in cultured cells. Microscopic analyses indicated co-localization of Mst27D with Nup358 and with the microtubule bundles of the dense complex. Time-lapse imaging revealed that nuclear elongation is accompanied by a progressive bundling of microtubules into a single elongated bundle. In Mst27D null mutants, this bundling process does not occur and nuclear elongation is abnormal. Thus, we propose that Mst27D permits normal nuclear elongation by promoting the attachment of the NPC-NE to the microtubules of the dense complex, as well as the progressive bundling of these microtubules.

Cabs1 Maintains Structural Integrity of Mouse Sperm Flagella during Epididymal Transit of Sperm.

The calcium-binding protein spermatid-associated 1 (Cabs1) is a novel spermatid-specific protein. However, its function remains largely unknown. In this study, we found that a long noncoding RNA (lncRNA) transcripted from the Cabs1 gene antisense, AntiCabs1, was also exclusively expressed in spermatids. Cabs1 and AntiCabs1 knockout mice were generated separately (using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 methods) to investigate their functions in spermatogenesis. The genetic loss of Cabs1 did not affect testicular and epididymal development; however, male mice exhibited significantly impaired sperm tail structure and subfertility. Ultrastructural analysis revealed defects in sperm flagellar differentiation leading to an abnormal annulus and disorganization of the midpiece–principal piece junction, which may explain the high proportion of sperm with a bent tail. Interestingly, the proportion of sperm with a bent tail increased during transit in the epididymis. Furthermore, Western blot and immunofluorescence analyses showed that a genetic loss of Cabs1 decreased Septin 4 and Krt1 and increased cyclin Y-like 1 (Ccnyl1) levels compared with the wild type, suggesting that Cabs1 deficiency disturbed the expression of cytoskeleton-related proteins. By contrast, AntiCabs1−/− mice were indistinguishable from the wild type regarding testicular and epididymal development, sperm morphology, concentration and motility, and male fertility. This study demonstrates that Cabs1 is an important component of the sperm annulus essential for proper sperm tail assembly and motility.

Relationship Between Spermatid Specific Thioredoxin-3 Protein and Antisperm Antibody in Patients with Varicocele Compared with a Control Group

The spermatogenesis process are very complex and involves many process to lead to give normal sperm, these sperms a very important to fertility in the men). Protein in the male fertility that is very importantwhich give the significant job in fertilization. The infertile varicocele patient grade 1,2 and 3 (n= 25 , 25and 25 respectively) compare with control patient (n=15). The present study Shown spermatid – specificthioredoxin 3 level a significant increase in varicocele patient grade 1,2 and 3 compare with control group(mean± Std. Error 17.97± 0.8 & 22.92±0.75 Respectively) .Also in Antisperm antibody a significant increasein varicocele patient grade 1,2 and 3 compare with control group (mean± Std. Error 73.85±2.71 & 45.6±1.44Respectively ). Consecutive (r= -0. 840, r = -0. 745 and r = -0. 475). Also theirs a negative correlationbetween Antisperm antibody level and the sperms concentration , Sperm Progressive motility percent andsperm normal morphology present in varicocele patient grade 1,2 and 3 Consecutive (r= -0.443 , r = -0.370and r = -0.384 ) .The result show a positive correlation was found between Spermatid specific thioredoxin-3 protein leveland antisperm antibody level in varicocele patients .(The present study conclude that a negative function ofSpermatid specific thioredoxin-3 protein and antisperm antibody which that appears on sperm function andsperm parameters , The study showed that spermatid specific thioredoxin-3 protein and antisperm antibodyhas a harmful effect with the time) .

The control of male fertility by spermatid-specific factors: searching for contraceptive targets from spermatozoon's head to tail.

Male infertility due to abnormal spermatozoa has been reported in both animals and humans, but its pathogenic causes, including genetic abnormalities, remain largely unknown. On the other hand, contraceptive options for men are limited, and a specific, reversible and safe method of male contraception has been a long-standing quest in medicine. Some progress has recently been made in exploring the effects of spermatid-specifical genetic factors in controlling male fertility. A comprehensive search of PubMed for articles and reviews published in English before July 2016 was carried out using the search terms ‘spermiogenesis failure', ‘globozoospermia', ‘spermatid-specific', ‘acrosome', ‘infertile', ‘manchette', ‘sperm connecting piece', ‘sperm annulus', ‘sperm ADAMs', ‘flagellar abnormalities', ‘sperm motility loss', ‘sperm ion exchanger' and ‘contraceptive targets'. Importantly, we have opted to focus on articles regarding spermatid-specific factors. Genetic studies to define the structure and physiology of sperm have shown that spermatozoa appear to be one of the most promising contraceptive targets. Here we summarize how these spermatid-specific factors regulate spermiogenesis and categorize them according to their localization and function from spermatid head to tail (e.g., acrosome, manchette, head-tail conjunction, annulus, principal piece of tail). In addition, we emphatically introduce small-molecule contraceptives, such as BRDT and PPP3CC/PPP3R2, which are currently being developed to target spermatogenic-specific proteins. We suggest that blocking the differentiation of haploid germ cells, which rarely affects early spermatogenic cell types and the testicular microenvironment, is a better choice than spermatogenic-specific proteins. The studies described here provide valuable information regarding the genetic and molecular defects causing male mouse infertility to improve our understanding of the importance of spermatid-specific factors in controlling fertility. Although a male contraceptive ‘pill' is still many years away, research into the production of new small-molecule contraceptives targeting spermatid-specific proteins is the right avenue.

Cryopreservation of adult primate testes

For primates that may unexpectedly die, spermatozoa recovered from the epididymis can be used for assisted reproductive technologies (ART). However, in cases of unsuccessful epididymal sperm retrieval, adult testes are the only source of sperm. This study reports cryopreservation of testicular tissue of three adult primates (mandrill, chimpanzee, and marmoset). For successful cryopreservation, optimization of concentrations of commonly used cryoprotectant such as dimethyl sulfoxide (DMSO) and combinations of foetal bovine serum (FBS) and DMSO is essential. Testicular tissues were cryopreserved in 10 % DMSO (D10) with 80 % FBS (D10S80) or 20 % DMSO (D20) with 20 % FBS (D20S20). The cryopreserved testes were evaluated for efficacy of cryopreservation by assessing the viability and acrosome status of retrieved sperm, expression of spermatozoa- and spermatid-specific proteins (PRM2 and TNP1) by immunohistochemistry, and expression of pro- and anti-apoptosis- (BAX/BCL2 ratio), early apoptosis- (Annexin V), and cell proliferation-specific (PCNA) proteins by western blot analysis. The results suggest that mandrill testis can be effectively cryopreserved in D10S80, marmoset testis in D10S80 and D20S20, whereas chimpanzee testis in D20.

A mouse protein that localizes to acrosome and sperm tail is regulated by Y-chromosome

BackgroundAcrosomal proteins play crucial roles in the physiology of fertilization. Identification of proteins localizing to the acrosome is fundamental to the understanding of its contribution to fertilization. Novel proteins are still being reported from acrosome. In order to capture yet unreported proteins localizing to acrosome in particular and sperm in general, 2D-PAGE and mass spectrometry analysis of mouse sperm proteins was done.ResultsOne of the protein spots identified in the above study was reported in the NCBI database as a hypothetical protein from Riken cDNA 1700026L06 that localizes to chromosome number 2. Immunofluorescence studies using the antibody raised in rabbit against the recombinant protein showed that it localized to mouse acrosome and sperm tail. Based on the localization of this protein, it has been named mouse acrosome and sperm tail protein (MAST, [Q7TPM5 (http://www.ncbi.nlm.nih.gov/protein/Q7TPM5)]). This protein shows 96% identity to the rat spermatid specific protein RSB66. Western blotting showed that MAST is expressed testis-specifically. Co-immunoprecipitation studies using the MAST antibody identified two calcium-binding proteins, caldendrin and calreticulin as interacting partners of MAST. Caldendrin and calreticulin genes localize to mouse chromosomes 5 and 8 respectively. In a Yq-deletion mutant mouse, that is subfertile and has a deletion of 2/3rd of the long arm of the Y chromosome, MAST failed to localize to the acrosome. Western blot analysis however, revealed equal expression of MAST in the testes of wild type and mutant mice. The acrosomal calcium-binding proteins present in the MAST IP-complex were upregulated in sperms of Yq-del mice.ConclusionsWe have identified a mouse acrosomal protein, MAST, that is expressed testis specifically. MAST does not contain any known motifs for protein interactions; yet it complexes with calcium-binding proteins localizing to the acrosome. The misexpression of all the proteins identified in a complex in the Yq-del mice invokes the hypothesis of a putative pathway regulated by the Y chromosome. The role of Y chromosome in the regulation of this complex is however not clear from the current study.

An in vitro, short‐term culture method for mammalian haploid round spermatids amenable for molecular manipulation

Extensive chromatin remodeling is a characteristic feature of mammalian spermiogenesis. To date, methods for the molecular manipulation of haploid spermatids are not available as there is a lack of a well-established culture system. Biochemical experiments and knockout studies reveal only the final outcome; studying the incremental details of the intricate mechanisms involved is still a challenge. We have established an in vitro culture system for pure haploid round spermatids isolated from rat testes that can be maintained with good viability for up to 72 hr. Changes in cell morphology and flagellar growth were also studied in the cultured spermatids. Further, we have demonstrated that upon treatment of cells with specific histone deacetylase inhibitors, sodium butyrate and trichostatin A, there is an increase in the hyperacetylation status of histone H4, mimicking an important event characteristic of histone replacement process that occurs during later stages of spermiogenesis. We have also tried various methods for introducing DNA and protein into these round spermatids in culture, and report that while DNA transfection is still a challenging task, protein transfection could be achieved using Chariot™ peptide as a transfection reagent. Thus, the method described here sets a stage to study the molecular roles of spermatid-specific proteins and chromatin remodelers in the cellular context.

Parp2 is required for the differentiation of post-meiotic germ cells: Identification of a spermatid-specific complex containing Parp1, Parp2, TP2 and HSPA2

Spermiogenesis is a complex male germ cell post-meiotic differentiation process characterized by dramatic changes in chromatin structure and function, including chromatin condensation, transcriptional inhibition and the sequential replacement of histones by transition proteins and protamines. Recent advances, in mammalian cells, suggest a possible role of poly(ADP-ribosyl)ation catalyzed by Parp1 and/or Parp2 in this process. We have recently reported severely compromised spermiogenesis in Parp2-deficient mice characterized by a marked delay in nuclear elongation whose molecular mechanisms remain however unknown. Here, using in vitro protein–protein interaction assays, we show that Parp2 interacts significantly with both the transition protein TP2 and the transition chaperone HSPA2, whereas Parp1 binds weakly to HSPA2. Parp2–TP2 interaction is partly mediated by poly(ADP-ribosyl)ation. Only Parp1 poly(ADP-ribosyl)ates HSPA2. In addition, a detailed analysis of spermatid maturation in Parp2-deficient mice, combining immunohistochemistry and electron microscopic approaches, reveals a loss of spermatids expressing TP2, a defect in chromatin condensation and abnormal formation of the manchette microtubules that, together, contribute to spermatid-specific cell death. In conclusion, we propose both Parps as new participants of a spermatid-specific protein complex involved in genome reorganization throughout spermiogenesis.

First characterization of CLPH, a spermatid-specific protein

Researchers analyze a novel phosphoprotein that likely functions in mammalian spermiogenesis.

Characterization and Linkage Mapping of an ENU-Induced Mutant Mouse with Defective Spermatogenesis

repro23 is an autosomal recessive mutation of the mouse generated by the N-ethyl-N-nitrosourea (ENU)-induced mutagenesis program at The Jackson Laboratory. The repro23/repro23 homozygous mouse shows male-specific infertility caused by defective spermatogenesis. In the present study, we investigated the testicular pathology of the affected mouse and performed linkage analysis to determine the chromosomal localization of the repro23 locus. Histological examination of the affected testis showed that the seminiferous epithelium of the repro23/repro23 mice contained spermatogonia and early stage spermatocytes, but no spermatids or spermatozoa. Immunohistochemical staining for Hsc70t, a spermatid specific protein, confirmed the absence of elongating spermatids. These findings indicated interruption of the spermatogenesis during meiosis in the repro23/repro23 mouse. By linkage analysis using 137 affected mice of F(2) progeny obtained from crosses between repro23/repro23 female and JF1/Ms (+/+) male mice, the repro23 locus was mapped to 2.2-Mb region of mouse chromosome 7. Although this region contains several potential candidate genes for the repro23 mutation, no gene already identified as a cause of defective spermatogenesis was in this region. Therefore, the gene responsible for the repro23 mutation is suggested to be a novel gene which plays an essential role in mammalian spermatogenesis.

High Throughput, Parallel Imaging and Biomarker Quantification of Human Spermatozoa by ImageStream Flow Cytometry

Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.

A New ENU-Induced Mutant Mouse with Defective Spermatogenesis Caused by a Nonsense Mutation of the Syntaxin 2/Epimorphin (Stx2/Epim) Gene

Repro34 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing male-specific infertility caused by defective spermatogenesis. In the present study, we investigated pathogenesis and molecular lesions in relation to spermatogenesis in the repro34/repro34 homozygous mouse. Histological examination of the testis showed that the seminiferous epithelium of the repro34/repro34 mouse contained spermatogonia and spermatocytes but no round and elongating spermatids. Instead of these haploid cells, multinucleated giant cells occupied the niche of the seminiferous tubules. Immunohistochemical staining for Hsc70t, an elongating spermatid specific protein, confirmed the absence of elongating spermatids. Furthermore, RT-PCR showed that there were significantly reduced expressions of the marker genes specifically expressed in the spermatid and that there was no difference in the expressions of the spermatocyte specific marker genes. These findings indicated interruption of the spermatogenesis during transition from the spermatocyte to spermatid in the repro34/repro34 mouse. The repro34 locus has been mapped on a 7.0-Mb region of mouse chromosome 5 containing the Syntaxin 2/Epimorphin (Stx2/Epim) gene, and targeted disruption of this gene has been reported to cause defective spermatogenesis. We therefore sequenced the entire coding region of the Stx2/Epim gene and found a nucleotide substitution that results in a nonsense mutation of this gene. The expression pattern of the Stx2/Epim gene during the first wave of spermatogenesis, increased expression at later stages of spermatogenesis, was in agreement with the affected phase of spermatogenesis in the adult repro34/repro34 testis. We therefore concluded that the male infertility of the repro34/repro34 mouse is caused by the interruption of spermatogenesis during transition from the spermatocyte to spermatid and that the nonsense mutation of the Stx2/Epim gene is responsible for the interruption of spermatogenesis.

SPAN-XB Core Promoter Sequence Is Regulated in Myeloma Cells by Specific CpG Dinucleotides Associated with MeCP2 Protein.

SPAN-Xb is a spermatid-specific protein encoded by the SPAN-XB gene on chromosome Xq27.1. It is a novel Cancer-Testis antigen in multiple myeloma. We recently demonstrated that SPAN-Xb expression in myeloma cells is regulated through promoter methylation and could be upregulated by IL-7 and GM-CSF. In this present study, we set out to investigate the mechanism of SPAN-XB expression and the promoter association with the methyl-CpG binding protein, MeCP2. Elucidation of these interactions is likely to shed light on potential therapeutic strategies to upregulate antigen levels that could be used to improve the outcome of SPAN-Xb-based tumor vaccines.We previously showed that the putative SPAN-XB promoter resides within exon 1 and contains 433 bp, starting from position −546 to position −104 upstream of the translational start site. Using a panel of truncated promoter constructs generated from the 3′ and 5′ ends of the promoter gene, we localized the core sequence of SPAN-XB promoter to the 73 base pairs at the 3-end of the promoter, a region that lacks CpG dinucleotides within the full length promoter. There are 11 CpG dinucleotides within the putative SPAN-XB promoter. We previously found that DNA methylation provides the primary regulatory mechanism for SPAN-XB gene expression.We also identified that hypomethylation at positions −310, −307, −299 and −221 strongly predicted for SPAN-XB expression, suggesting the involvement of these four CpG dinucleotides in the regulation of SPAN-XB gene expression through DNA methylation. In the present study, using reporter gene expression assays, we found that the core promoter function is significantly modulated by these adjacent CpG sequences so that mutation of the CpG dinucleotides outside the core sequence resulted in changes in the promoter function. We also previously demonstrated by bisulfite conversion and sequence analysis the association between methylation at specific CpG dinucleotides with repression of SPAN-XB gene. Here, we extended our study to determine whether or not the methylated cytosine binding protein, MeCP2, interacts with the SPAN-XB promoter gene in myeloma cells. Chromatin immunoprecipitation assays revealed a specific association of the MeCP2 with SPAN-XB promoter, and MeCP2 binding strongly correlated with repression of the SPAN-XB gene in myeloma cell lines and CD138-enriched fresh myeloma cells. Reactivation of the SPAN-XB gene by 5-azacytidine treatment resulted in the loss of MeCP2 from this site. We, therefore, conclude that SPAN-XB promoter consists of a core sequence and a regulatory element; the core element resides within the 73 base pairs at the 3′ end of the full length promoter. SPAN-XB gene expression by the core sequence is regulated in myeloma cells by specific CpG nucleotides and associated with MeCP2 binding to the promoter sequence.

SPAN-Xb Expression in Myeloma Cells Can Be Upregulated by Pharmacologics.

SPAN-Xb is a spermatid-specific protein. We recently identified SPAN-Xb as a novel Cancer-Testis (CT) antigen in multiple myeloma (MM) (Blood 2003; 101: 955–960) and that it frequently elicited immune responses in vivo in these patients, suggesting that SPAN-Xb may be a suitable target for immunotherapy of MM. SPAN-Xb, however, was only detected in the tumor cells from 20% of MM patients. To increase the applicability of a SPAN-Xb tumor vaccine for MM, we have set out to determine if SPAN-Xb expression could be upregulated by pharmacologics in 8 MM cell lines.Since most CT antigen expression occurs due to promoter demethylation, we first determined the ability of a hypomethylating agent, 5-azacytidine, to upregulate SPAN-Xb expression. Prior to exposure to 5-azacytidine, SPAN-Xb transcripts were only detected by RT-PCR in ARP-1 and RPMI 8226 cells. Following incubation in 5-azacytidine (2 uM) for 96 hours, the levels of SPAN-Xb transcripts were upregulated, as determined by RT-PCR and real time PCR, in all MM cell lines (ARK-B, ARP-1, H929, IM9, MM1-R, RPMI 8226 and U266) except KMS-11 (Table 1). This was associated with a corresponding increase in the level of SPAN-Xb protein expression, as shown by immunocytochemistry using a murine monoclonal antibody directed at SPAN-Xb.We next determined if SPAN-Xb gene expression could be increased by cytokine pre-incubation of these cells. IL-2 (100 IU/ml) and IL-4 (1000 IU/ml) did not affect SPAN-Xb gene expression. On their own, IL-7 (0.1 ng/ml) and GM-CSF (10 U/ml) did not affect SPAN-Xb gene expression significantly. However, when combined, especially with 5-azacytidine, a summative effect of these pharmacologics on SPAN-Xb gene expression, as determined by real time PCR, was observed in IM9 cells (Table 2). Our results indicate the potential to upregulate SPAN-Xb expression in MM for therapeutic purpose using 5-azacytidine, GM-CSF and IL-7 and provide insight into the possible mechanisms of regulation of SPAN-Xb gene expression.Table 1Cellspre-5-azacytidinepost-5-azacytidineARK-B0456ARP-12601376H9290116IM90532KMS-1100MM1-R0428RPMI 82262721348U2660984SPAN-Xb mRNA copy numbers before and after 5-azacytidine exposureTable 2PharmacologicsSPAN-Xb mRNA5-azacytidine532IL-74GM-CSF9IL-7 + GM-CSF365-azacytidine + IL-710885-azacytidine + GM-CSF9525-azacytidine + IL-7 + GM-CSF1632medium only0SPAN-Xb mRNA copy numbers after exposure to combination pharmacologics

Crystallization and preliminary crystallographic analysis of RSB-66, a novel round spermatid-specific protein.

Crystals of the RSB-66 protein have been grown at 291 K using NaCl as precipitant. In the refinement of the crystallization this protein, the crystallographic PCR method was used and was found to help in obtaining the best crystals more quickly and easily. The diffraction pattern of the crystal extends to 2.7 A resolution in-house. A full set of X-ray diffraction data were collected to 2.7 A from a single crystal. The crystals belong to space group P4212, with unit-cell parameters a = 90.4, b = 90.4, c = 122.2 A, alpha = beta = gamma = 90 degrees. The presence of two or three molecules per asymmetric unit gives a crystal volume per protein mass (V(M)) of 3.22 or 2.14 A(3) Da(-1), respectively.

Cloning, expression and characterization of mouse spermatid specific thioredoxin-1 gene and protein.

Thioredoxins are proteins that participate in different cellular processes via redox-mediated reactions. For humans, we have recently described two novel members of this family that display a male germ cell specific expression pattern, named spermatid specific thioredoxin (Sptrx-1 and Sptrx-2 respectively). We report here the cloning and characterization of the mouse Sptrx-1 gene and protein, which are similar to those described for the human orthologue. The mouse Sptrx-1 open reading frame encodes for a protein of 462 aa composed of an N-terminal repetitive domain of a 15 residue motif followed by a C-terminal domain typical of thioredoxins. The mouse Sptrx-1 gene sequence is interrupted by only one intron of 525 bp located in the 5'-UTR, and using fluorescence in-situ hybridization we have mapped its chromosomal location to 17E1.2-1.3. Northern blot analysis identified the testis as the only tissue expressing mouse Sptrx-1 mRNA, and by in-situ hybridization we found a strong labelling in the testicular seminiferous tubules, mostly in the round spermatids. Affinity purified antibodies against human Sptrx-1 crossreacted well with the mouse protein confirming its expression in seminiferous tubules at the later stages of spermiogenesis. Recombinant mouse Sptrx-1 displayed protein disulphide reducing activity in an enzymatic assay coupled to NADPH and thioredoxin reductase. The availability of the mouse Sptrx-1 gene sequence is the first step towards the generation of knock-out mice, whose characterization will provide significant information regarding the in-vivo function of Sptrx-1 and its possible implication in several sperm anomalies.

Squid spermiogenesis: molecular characterization of testis-specific pro-protamines.

Cuttlefish spermiogenesis is characterized by a two-step nuclear protein transition: histones-->spermatid-specific protein (protein T)-->sperm protamine (protein Sp). A similar situation can be observed in another Cephalopod species, the squid Loligo pealeii. The protein T from Loligo consists of two structural variants, T1 and T2 (molecular masses: 10788 and 10791 Da respectively), phosphorylated to different degrees (2-6 phosphate groups). The primary structures of these two variants and of the protamine variant Sp2 were established from sequence analysis and mass spectrometric data of the proteins and their fragments. T1 and T2 are closely related proteins of 79 residues. The complete structural identity of the C-terminal domain (residues 22-79) of protein T2 with the sperm protamine Sp2 (molecular mass 8562 Da, 58 residues) strongly suggests that the testis-specific protein T2 is indeed the precursor of the protamine. The transition between the precursor protein T and protein Sp results from a hydrolytic cleavage similar to that found in many proteins that are synthesized as precursors. The processing mechanism involves the specific cleavage of a Gly-Arg bond in the sequence Met/Leu18-Lys-Gly-Gly-Arg-Arg23. Furthermore, the study provides molecular evidence on the taxonomic relationship between Loligo and Sepia.

Cuttlefish spermatid-specific protein T. Molecular characterization of two variants T1 and T2, putative precursors of sperm protamine variants Sp1 and Sp2.

In cuttlefish, as in selachians and mammals, spermiogenesis is characterized by the double nuclear protein transition histones----intermediate protein (protein T)----protamine (protein Sp). The cuttlefish protein T, which consists of two structural variants phosphorylated at different degrees, is the first invertebrate spermatid-specific protein to be fully characterized and sequenced. The primary structures of these two variants were established from sequence analysis and mass spectrometric data of the proteins and their fragments. T1 and T2 are two highly related proteins of 78 and 77 residues, respectively, which differ only by four conservative substitutions, two inversions Ser in equilibrium with Arg, and the deletion of 1 residue of arginine in variant T2. The asymmetrical distribution of the hydrophobic and basic residues determines two well defined domains: an amino-terminal domain (residues 1-21) devoid of arginine and aromatic residues and containing all the aliphatic hydrophobic residues and a highly basic carboxyl-terminal domain (residues 22-77 or 78) that contains 77% of arginine, all the tyrosine residues, and most of the phosphorylated serine residues present in the protein. The complete structural identity of the basic carboxyl-terminal domain of spermatidal proteins T1 and T2 with the protamine variants Sp1 and Sp2 isolated from cuttlefish spermatozoa strongly suggests that T1 and T2 could be precursors of Sp1 and Sp2, respectively.

Mammalian spermatid specific protein, TP2, is a zinc metalloprotein with two finger motifs

An analysis of the recently reported cDNA derived amino acid sequences of mouse (Kleene and Flynn, J. Biol. Chem. 262, 17272-17277, 1987) and rat (Luersson et al., Nucl. Acids Res. 17, 3585, 1989). TP2 has revealed the presence of two potential zinc finger motifs involving cysteine and histidine residues. TP2, as purified from rat elongating spermatids, is shown here to contain 0.2 atoms of zinc bound per molecule of the protein by atomic absorption spectroscopy. On incubation with 10 microM ZnCl2, in vitro, and subsequent exhaustive dialysis, TP2 had 2 atoms of zinc bound per molecule. The involvement of cysteine residues of TP2 in coordination with zinc was also suggested by the observation that TP2 could be labeled, in situ, with iodoacetamidofluorescein only after preincubation of spermatid nuclei with EDTA. The zinc finger domains of TP2 may play an important role in initiation of chromatin condensation and/or cessation of transcriptional activity during mammalian spermiogenesis.