Development of the acrosome and alignment,elongation and entrenchment of spermatids in procarbazine-treated rats

Intraperitoneally administered procarbazine caused, among other features previously reported (Russell et al., 1983), specific defects in the acrosome of cap phase spermatids of the rat seminiferous epithelium. The effect of procarbazine was to fragment and eventually cause resorption of the acrosomes of a small number of steps 5–9 spermatids. Although the acrosome was lost, dose union of the leaflets of the nuclear envelope underlying the acrosomal sac was maintained as was the marginal fossa and acrosomal zonule. Spermatids at steps 8 and 9 of development, which had lost their acrosomes, showed nuclei which were eccentric within the cell—a feature which normally occurs at these steps of spermiogenesis in acrosome intact cells. Even without an acrosomal sac, the plasma membrane of these cells (in stage VIII) became orientated to the region of the nuclear membrane which would have underlaid the acrosome. Although abundant, Sertoli ectoplasmic specialization did not become aligned with the spermatid head. The spermatid failed to become orientated within the seminiferous epithelium and failed to enter the crypts within the Sertoli cell as usually occurs during the elongation process. Thus, the presence of an acrosome is not likely related to the formation of an eccentric nucleus or the alignment of the surface of the nucleus which would normally underlay the acrosome with the cell's plasma membrane (internal alignment). The presence of an acrosome may be related to the alignment of the spermatid head with the ectoplasmic specialization, which in turn may influence the orientation and positioning of the late spermatids within the seminiferous epithelium (external alignment) and their position within recesses of the Sertoli cell. This study also suggests a role for the manchette in the process of elongation of the spermatid.     

A membrane protein,TMCO5A,has a close relationship with manchette microtubules in rat spermatids during spermiogenesis

We isolated the transmembrane and coiled‐coil domains 5A (Tmco5A) gene using polymerase chain reaction‐based subtraction technique and showed that Tmco5A was predominantly expressed in rat testes starting at 4 weeks of postnatal development. When expressed in COS7 cells, TMCO5A was found to be distributed in the endoplasmic reticulum‐nuclear membrane (ER‐NM) of cells as a membrane‐associated protein, while TMCO5AΔC lacking the transmembrane region (TM) mislocalized and diffused throughout the cytoplasm. The result suggested that TM is responsible for the retention of TMCO5A at the ER‐NM. Immunocytochemical and immunoblotting analyses indicated that TMCO5A was localized along the posterior part of the nuclei in both round and elongated rat spermatids but disappeared from epididymal spermatozoa. Double immunolabeling of isolated spermatids with the anti‐TMCO5A and the anti‐β tubulin antibodies showed that TMCO5A was always found to be closely associated with developing manchette microtubules but did not completely colocalize with them. On the other hand, we found that almost all TMCO5A colocalized with SUN4, a linker of nucleoskeleton and cytoskeleton complex protein present at the posterior part of spermatid nuclei. These data suggested that TMCO5A is located closer to the nuclei than the manchette microtubules. It is likely that TMCO5A, in association with manchette microtubules, is involved in the process of spermiogenesis.     

Isolation and characterization of the spermatid-specific Smrp1 gene encoding a novel manchette protein

The manchette, which is the structure that appears around the nuclei of elongated spermatids, is assumed to be involved in nuclear shaping during spermiogenesis and the transport of various proteins between the nucleus and sperm tail. In this report, we describe the molecular cloning and characterization of a mouse spermatid-specific manchette-related protein 1 (Smrp1) from a spermatid-specific subtracted mouse testis cDNA library. The isolated Smrp1 cDNA clones could be divided into three variants based on sequence analysis. Computer-assisted analysis showed that these variants were splice variants from a single locus of the mouse genome. The three putative proteins consisted of 296, 260, and 175 amino acids, respectively. Although 155 amino acids of the N terminus were common to the three proteins, they were distinguished by their C-terminal regions. Western blot analyses using specific antisera showed that SMRP1 expression was specific to the testes and that only the 261-amino-acid form was translated into protein. Immunohistochemistry revealed that SMRP1 was localized to the cytoplasm of step 9-12 elongated spermatids. The protein appeared in a cap formation that covered the caudal sides of the elongated nuclei. This localization pattern coincided with that of the manchette. SMRP1 may play an important role as a functional protein that co-operates with manchette proteins.     

Spergen-1 might be an adhesive molecule associated with mitochondria in the middle piece of spermatozoa

Spergen-1, a recently identified molecule specifically expressed in haploid spermatids in testis, is a small protein of 154 amino acids with a mitochondria-targeting signal at the N terminus. To examine the localization of spergen-1 protein in germ cells, we performed immunocytochemistry with the anti-spergen-1 antibody on frozen sections of rat testis and purified spermatozoa. Immunolabeling for spergen-1 was detected in mitochondria of elongating spermatids and of the middle pieces of matured spermatozoa. Immunoelectron microscopy revealed that spergen-1 was localized to the surface of mitochondria in the middle piece of spermatozoa. To investigate the properties of spergen-1, COS-7 cells were transfected with vectors encoding various spergen-1 mutants. The transfection experiments showed that spergen-1 expressed in the cells tended to agglutinate mitochondria and assemble them into aggregations and that the C-terminal region of spergen-1 as well as the N-terminal mitochondrial targeting signal was requisite for induction of mitochondrial aggregation. These results suggest that spergen-1, a mitochondria-associated molecule in spermatozoa, has a property to induce mitochondrial aggregation at least in cultured cells. We hypothesize that spergen-1 might function as an adhesive molecule to assemble mitochondria into the mitochondrial sheath around the outer dense fibers during spermiogenesis.     

Phosphorylation state of protamines 1 and 2 in human spermatids and spermatozoa

The basic nuclear proteins of a fraction of elongating spermatids from human tests and of a fraction of motile spermatozoa from the ejaculate, separated by ion-exchange chromatography, were compared. Analysis by acetic acid-urea polyacrylamide gel electrophoresis (PAGE) showed that, in both fractions, four proteins of lower mobility were coeluted with protamine 1 by 23% guanidinium chloride (GuCI) while protamine 2 alone was eluted by 50% GuCI. Treatment with alkaline phosphatase identified those four proteins as phosphorylated protamines, and cyanogen bromide (CNBr) treatment of the dephosphorylated protamines distinguished them as variants of protamine 2 and not of protamine 1. Thus far, phosphorylated forms of protamine 1 have not been detected in either spermatids or spermatozoa. Those observations indicate that protamine 2 functions in the cycle of phosphorylation-dephosphorylation, which is essential to the process of sperm chromatin condensation, while the role of protamine 1 in human spermiogenesis is not yet defined. The presence of phosphorylated protamine in motile, presumably mature spermatozoa appears to be characteristic of human sperm but not of the sperm of other mammals and is probably the basis for the heterogeneity of chromatin condensation frequently observed in human spermatozoa.     

Testis fascin (FSCN3): a novel paralog of the actin-bundling protein fascin expressed specifically in the elongate spermatid head

During spermiogenesis, significant morphological changes occur as round spermatids are remodeled into the fusiform shape of mature spermatozoa. These changes are correlated with a reorganization of microfilaments and microtubules in the head and tail regions of elongating spermatids. There is also altered expression of specialized actin- and tubulin-associated proteins. We report the characterization of a novel, spermatid-specific murine paralog of the actin-bundling protein fascin (FSCN1); this paralog is designated testis fascin or FSCN3. Testis fascin is distantly related to fascins but retains its primary sequence organization. cDNA clones of mouse testis fascin predict a 498 amino acid protein of molecular mass 56 kD that shares 29% identity with mouse fascin. Mapping of murine and human FSCN3 genes shows localization to the 7q31.3 chromosome. Northern analysis indicates that FSCN3 expression is highly specific to testis and that in situ hybridization further restricts expression to elongating spermatids. Antibodies raised against recombinant FSCN3 protein identify a band at 56 kD in testis, epididymis, and epididymal spermatozoa, suggesting that testis fascin persists in mature spermatozoa. In accord with the in situ hybridization results, immunofluorescent microscopy localizes testis fascin protein to areas of the anterior spermatid head that match known distributions of F-actin in the dorsal and ventral subacrosomal spaces. It is possible that testis fascin may function in the terminal elongation of the spermatid head and in microfilament rearrangements that accompany fertilization.     

Differential expression of lysosomal associated membrane protein (LAMP-1) during mammalian spermiogenesis

The mammalian acrosome is a secretory vesicle of mature sperms that plays an important role in fertilization. Recent evidence had pointed out that some components found at endosomes in somatic cells are associated with the developing acrosome during the early steps of spermiogenesis. Moreover, the mammalian acrosome contains many enzymes found within lysosomes in somatic cells. In this work, we studied the dynamics of some components of the endosome/lysosome system, as a way to understand the complex membrane trafficking circuit established during spermatogenesis. We show that the cation independent-mannose-6-phosphate receptor (CI-MPR) is transiently expressed in the cytoplasm of mid-stage spermatids (steps 5-11). On the other hand, gamma-adaptin, an adaptor molecule of a complex involved in trafficking from the Golgi to lysosomes, was expressed in cytoplasmic vesicles only in pachytene and Cap-phase spermatids (steps 1-5). Our major finding is that the lysosomal protein LAMP-1 is differentially expressed during spermiogenesis. LAMP-1 appears late in spermatogenesis (Acrosome-phase) contrasting with LAMP-2, which is present throughout the complete process. Both proteins appear to be associated with cytoplasmic vesicles and not with the developing acrosome. None of the studied proteins is present in epididymal spermatozoa. Our results suggest that the CI-MPR could be involved in membrane trafficking and/or acrosomal shaping during spermiogenesis.     

Localization and quantitative expression of mRNAs encoding the testis-specific histone TH2B,the phosphoprotein p19, the transition proteins 1 and 2 during pubertal development and throughout the spermatogenic cycle of the rat

Expression of the testis-specific histone TH2B, the phosphoprotein p19, and the transition proteins TP1 and TP2, was localized in the rat testis and quantified, using in situ hybridization of their mRNAs with radiolabeled probes and image analysis. In a first study, expression was assessed during testicular development between day 2 and day 65 postpartum. TH2B mRNAs appeared first in preleptotene spermatocytes (PL) on day 12 and in pachytene spermatocytes (PS) on day 18; p19 mRNAs were present in PS from day 18 onward, and TP1 and TP2 mRNAs were detected in round spermatids (RS) from day 32 onward. In the second trial, the expression of these four genes was studied throughout the cycle of spermatogenic epithelium in mature animals. TH2B mRNAs were localized in B spermatogonia at stage V, and in PL at stages VII and VIII but no longer in leptotene and zygotene spermatocytes. Thereafter, TH2B mRNAs were observed in PS from stages III–IV to XIII. The steady-state mRNA level per cell was high in PS with a maximum at stages IX–X. p19 mRNAs were present in PS from stages III–IV onward and in RS up to stages 1–2 of spermiogenesis. The maximum mRNA level per cell was observed in PS between stages VII and XIII. The presence of TP1 mRNAs was restricted to spermatids from steps 6 to 15–16 of spermiogenesis while TP2 mRNAs were detected in spermatids only between step 7 and step 13. The highest steady-state amounts of mRNAs were observed between step 7 and step 14 for TP1 and between step 10 and step 12 for TP2. Mol. Reprod. Dev. 51:22–35, 1998. © 1998 Wiley-Liss, Inc.     

Complementary DNA cloning and characterization of rat spergen-2, a spermatogenic cell-specific gene 2 encoding a 56-kilodalton nuclear protein bearing ankyrin repeat motifs

Differential display in combination with a cDNA cloning approach were used to isolate a novel gene, spergen-2, which has an open reading frame of 1500 nucleotides and encodes a protein of 500 amino acids that contains ankyrin repeat motifs and a putative nuclear localization signal. Expression of spergen-2 is developmentally upregulated in testis. In situ hybridization revealed that spergen-2 mRNA is expressed in spermatocytes and round spermatids (steps 1-6). Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spergen-2 protein is predominantly expressed in nuclei of late spermatocytes (stages IX-XIV) and spermatids (steps 1-11), indicating the restricted expression of spergen-2 during spermatogenesis. In nucleoplasm of spermatogenic cell nuclei, spergen-2 tends to localize in the interchromosome space with relatively low DNA density. These findings indicate a potential role of spergen-2 in spermatogenesis, especially in cell differentiation from late pachytene spermatocytes to spermatids or in early spermatid differentiation.     

Molecular cloning and characterization of SRG-L, a novel mouse gene developmentally expressed in spermatogenic cells

Full-length cDNA of a novel mouse gene upregulated in late stages of spermatogenic cells was cloned from mouse testis using overlapping RT-PCR and RACE. The mRNA of the gene was expressed mainly in diplotene/pachytene spermatocytes, round and elongating spermatids. We named this gene as SRG-L (Spermatogenesis Related Gene expressed in late stages of spermatogenic cells, GenBank Accession No. AY352586). The tissue-specific analysis showed a higher expression level in testis and spleen. The gene is mapped on chromosome 8q33.1 and contains 18 exons. The full-length of cDNA is 2,843 bp with an open reading frame (ORF) of 2,625 bp that encodes a 104 kDa protein (874 amino acids) with a putative transmembrane region. The bioinformatics analysis revealed that the SRG-L has two conserved regions, transglutaminase-like homologues domain and D-serine dehydratase domain, rich phosphorylation sites and methylation sites. The SRG-L protein was detected in diplotene/pachytene spermatocytes and spermatids by immunohistochemical staining and Western blot. The results suggest that SRG-L may play definite roles regulating differentiation of germ cells during spermatogenesis, particularly during meiosis and spermiogenesis.     

Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes,Tact1 and Tact2

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.     

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

We have previously reported the purification of Sak 57 (for spermatogenic cell/sperm-associated keratin of molecular mass 57 kDa) from outer dense fibers of rat sperm tails. Internal protein sequence analysis of Sak 57 revealed 70–100% homology to the 1A and 2A regions of the α-helical rod domain of human, mouse, and rat keratins. A multiple antigen peptide was synthesized using the KQYEDIAQK sequence corresponding to the 2A region and a polyclonal antibody was produced in rabbit to detect Sak 57. During spermiogenesis, Sak 57 associates with the microtubular manchette before becoming a component of para-axonemal keratin structures of the developing tail. We now report that during late meiotic prophase, intercellular bridges linking late pachytene-diplotene spermatocytes display a distinct ribbon containing a Sak 57/β-tubulin complex, separated by a nonimmunoreactive midzone. Indirect immunofluorescence demonstrates that the ribbon is the final stage of a three-step developmental sequence: (1) a spindlelike arrangement radiating from equidistant spherical centers in early pachytene spermatocytes, (2) an ectoplasmic shell like framework in mid-to-late pachytene spermatocytes, and (3) a Sak 57/β-tubulin-containing ribbon found in intercellular bridges linking adjacent late pachytene-diplotene spermatocytes. Shear forces causing a breakdown of one of the conjoined spermatocytes do not disrupt the cytoskeletal ribbon. Results of this work, together with previous observations during spermiogenesis, show that Sak 57 associates with cytoplasmic microtubules in a timely fashion. Upon completion of late meiotic prophase, the Sak 57/microtubule complex behaves as an intercellular ligament and contributes to both the strength of intercellular bridges and the cohesiveness of members of a spermatocyte lineage. © 1996 Wiley-Liss, Inc.     

Molecular cloning and characterization of a gene encoding a 13.1 kDa antigenic protein of Naegleria fowleri

An antigen-related gene was cloned from a cDNA expression library of Naegleria fowleri by immunoscreening with sera obtained from mice that were either immunized with an amoebic lysate or infected with trophozoites. The coding nucleotide sequence of the cloned gene consisted of 357 bases that were translated into 119 amino acids. This gene was designated as nfa1. The predicted amino acid sequence of Nfa1 protein has two potential glycosylation and three potential phosphorylation sites, and its predicted secondary structure consists of four helices and three corners. The deduced amino acid sequence of Nfa1 protein shares 43% identity with the myohemerythrin (myoHr) protein from a marine annelid, Nereis diversicolor, including 100% identity in conserved regions and iron-binding residues. A phylogenetic tree constructed from amino acid sequences placed the N. fowleri Nfa1 protein outside of a cluster of myoHr proteins from eight invertebrates. A purified recombinant protein that migrated as a 13.1 kDa species in SDS-PAGE was produced. This recombinant protein exhibited a strong immunoreactivity with infected, immune, and anti-Nfal sera. In addition, an anti-Nfa1 serum reacted with an amoeba lysate in immunoblotting analysis. The present nfal gene encoding the myoHr-like protein is the first myoHr gene cloned from protozoa, and the Nfal antigen may be useful in diagnostic studies