Search

The calcium-binding protein spermatid-associated 1 (CABS1) localizes to the principal piece of mature sperm flagella. Deletion of CABS1 results in subfertility in male mice, possibly due to an impaired annulus in the sperm flagella. However, it is unknown whether there are other mechanisms by which CABS1 affects male fertility. Our current investigation has uncovered that CABS1 is located in the midsection of the flagellum in testicular sperm and the principal piece in epididymal sperm. Moreover, male mice lacking CABS1 exhibit a defect in the progressive motility of sperm. Furthermore, the regulation of calcium levels, which has been reported to have a significant impact on sperm motility, capacitation, and the acrosome reaction, is also affected when sperm are exposed to alkalized high-salt buffer (pH 8.0) and progesterone (100 μM) in Cabs1-null spermatozoa. This alteration in calcium response may contribute to changes in the phosphorylation of PKA substrates and subsequent phosphorylation of tyrosine residues. Additionally, the absence of CABS1 leads to a defective fibrous sheath and abnormal configuration of doublet microtubules in post-testicular sperm. These findings indicate that the absence of CABS1 also disrupts the structural integrity of the fibrous sheath, resulting in male subfertility. The highly conserved nature of CABS1 in humans suggests that it could potentially be a contributing factor to asthenozoospermia in men., (© 2024 Wiley Periodicals LLC.)

SEPT12 is a testis-specific gene involved in the terminal differentiation of male germ cells. SEPT12 protein is required for sperm head-tail formation and acts as a fundamental constituent of sperm tail annulus. In this study, we screened genetic variations in exons 5, 6, 7 of the SEPT12 and assessed the annulus status in teratozoospermic, globozoospermic, and patients with immotile short tail sperm. DNA sequencing was performed for 90 teratozoospermic and 30 normozoospermic individuals. Immunocytochemistry, transmission electron microscopy and western blotting were conducted to evaluate annulus status and the expression level of SEPT12 in patients with a distinct exonic variation (c.474G>A), respectively. Five polymorphisms identified within the desired regions of the SEPT12, among them c.474G>A had the potential to induce aberrant splicing results in the expression of a truncated protein. The annulus was detected in most of the spermatozoa from teratozoospermic and normozoospermic men with c.474G>A. In contrast, in the patient with short tail sperm defect carrying c.474G>A, 99% of spermatozoa were devoid of the annulus. Based on our findings there would be no association between exons 5, 6, 7 polymorphisms of the SEPT12 gene and the occurrence of mentioned disease but c.474G>A would be a predisposing factor in male infertility., (© 2019 Wiley Periodicals, Inc.)

The molecular and cellular characteristics of male germ cell development remain largely unknown in corals. This study focused on the expression pattern of acetylated α-tubulin (Ac-α-Tu), which is involved in male germ cell development in various animals across taxa, to gain a better understanding of male germ cell development in the stony coral Euphyllia ancora. Immunohistochemical analysis of the different stages of male germ cells showed the presence of filamentous Ac-α-Tu in the early to late stages of male germ cells-such as spermatogonia, spermatocytes, and spermatids-as well as in the flagella of mature sperm. Immunocytochemical and transmission electron microscope analyses demonstrated that early-stage male germ cells possess long flagella containing Ac-α-Tu. The presence of filamentous Ac-α-Tu was also immunohistochemically demonstrated in the male germ cells from 14 other coral species, implying that possession of flagella with Ac-α-Tu is a common characteristic of male germ cells in stony corals. Therefore, as a distinctive cellular characteristic of male germ cells, Ac-α-Tu could be used as a male germ cell marker in stony corals; indeed, immunolabeling for Ac-α-Tu may be a useful method to aid in the identification and morphological observation of male germ cells in various corals in basic and applied biology (e.g., aquaculture) as well as in ecological studies., (© 2017 Wiley Periodicals, Inc.)

The gene 1700040L02Rik (GenBank accession number NM_028491, NP_082767.1) was selected by in silico screening as candidate that encodes a calcium-binding protein in sperm from a database of predicted mouse cilia-related genes. The predicted amino acid sequence revealed the presence of coiled-coil domain at the C-terminus and a CLAMP motif containing a leucine zipper domain in the middle of the protein. Assessment of a recombinant version of this protein by Stains-all and ruthenium red staining and by direct measurement of terbium binding revealed its calcium-binding activities. We therefore named this protein CABCOCO1 for calcium-binding coiled-coil protein-1. Immunohistochemical analyses showed its localization in spermatogenic cells of mouse testis. CABCOCO1 was first observed in the cytoplasm of murine spermatocytes, concentrated around centrioles of spermatids and co-localized with the centrosomal protein pericentrin. During the stage when centrosome number is reduced, CABCOCO1 relocalized to the murine sperm flagellum. On the other hand, in porcine sperm, whose proximal centriole remains intact while the distal centriole degenerates during spermiogenesis, CABCOCO1 localized both in the basal body and the flagellum. These results suggested that CABCOCO1 is involved in the control of sperm flagellar movement. Mol. Reprod. Dev. 83: 912-926, 2016 © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Triosephosphate isomerase 1 (TPI1) is a member of the glycolytic pathway, which is a critical source of energy for motility in mouse sperm. By immunoblotting, we detected two male, germ line-specific TPI1 bands (Mr 33,400 and 30,800) as well as the somatic-type band (Mr 27,700). Although all three bands were observed in spermatogenic cells, somatic-type TPI1 disappeared from sperm during epididymal maturation. In vitro dephosphorylation analysis suggested that the two male, germ line-specific TPI1 bands were not the result of phosphorylation of the 27,700 Mr TPI1 band. The Mr 33,400; 30,800; and 27,700 TPI1 bands corresponded to the respective sizes of the proteins predicted to use the first, second, and third possible initiation codons of the Tpi1 cDNA. We performed immunofluorescence on epididymal sperm and determined that TPI1 specifically localized in the principal piece. The antibody staining was stronger in cauda epididymal sperm than in caput epididymal sperm, a finding consistent with the identification of TPI1 as a cauda epididymal sperm-enriched protein. Immunofluorescence with sodium dodecyl sulfate (SDS)-insoluble flagellar accessory structures showed a strong TPI1 signal only in the principal piece, indicating that TPI1 is a component of the fibrous sheath. Northern blot hybridization detected longer Tpi1 transcripts (1.56 kb) in mouse testis, whereas somatic tissues had shorter transcripts (1.32 kb). As there is only one triosephosphate isomerase gene in the mouse genome, we conclude that the three variants we see in sperm result from the use of alternative translation start codons in spermatogenic cells., (© 2013 The Authors. Molecular Reproduction and Development Published by Wiley Periodicals, Inc.)

EPPIN (epididymal protease inhibitor; SPINLW1), an antimicrobial cysteine-rich protein containing both Kunitz and whey acidic protein (WAP)-type four disulfide core protease inhibitor consensus sequences, is a target for male contraception because of its critical role in sperm motility. Here, we characterized EPPIN's expression and cellular distribution in rat tissues and its in vivo regulation by androgens in the epididymis. EPPIN (mRNA and protein) was abundantly expressed in the rat testis and epididymis; we also found that the vas deferens, seminal vesicles, and brain were novel sites of EPPIN expression. PCR studies demonstrated that in addition to Sertoli cells, spermatogenic cells expressed Eppin mRNA. EPPIN was immunolocalized in Sertoli cells and spermatogenic cells (pachytene spermatocytes and round and elongated spermatids) and in epithelial cells and spermatozoa from efferent ductules and epididymis. EPPIN staining was observed on the middle and principal pieces of the flagellum of testicular spermatozoa. Epididymal spermatozoa had more intense EPPIN staining on the flagellum, and the EPPIN staining became apparent on the head and neck regions. This suggested that the EPPIN found on maturing spermatozoa was secreted primarily by the epithelial cells of the epididymis. Surgical castration down-regulated EPPIN expression levels (mRNA and protein) in the caput and cauda epididymis, an effect reversed by testosterone replacement. Altogether, our data suggested that EPPIN expression in rats is more widespread than in humans and mice, and is androgen-dependent in the epididymis. This species could be used as an experimental model to further study EPPIN's role in male fertility., (Copyright © 2012 Wiley Periodicals, Inc.)

Sperm structure has evolved to be very compact and compartmentalized to enable the motor (the flagellum) to transport the nuclear cargo (the head) to the egg. Furthermore, sperm do not exhibit progressive motility and are not capable of undergoing acrosomal exocytosis immediately following their release into the lumen of the seminiferous tubules, the site of spermatogenesis in the testis. These cells require maturation in the epididymis and female reproductive tract before they become competent for fertilization. Here we review aspects of the structural and molecular mechanisms that promote forward motility, hyperactivated motility, and acrosomal exocytosis. As a result, we favor a model articulated by others that the flagellum senses external signals and communicates with the head by second messengers to affect sperm functions such as acrosomal exocytosis. We hope this conceptual framework will serve to stimulate thinking and experimental investigations concerning the various steps of activating a sperm from a quiescent state to a gamete that is fully competent and committed to fertilization. The three themes of compartmentalization, competence, and commitment are key to an understanding of the molecular mechanisms of sperm activation. Comprehending these processes will have a considerable impact on the management of fertility problems, the development of contraceptive methods, and, potentially, elucidation of analogous processes in other cell systems., (Copyright © 2011 Wiley Periodicals, Inc.)

In this study, we performed extensive proteomic analysis of sperm from the ascidian Ciona intestinalis. Sperm were fractionated into heads and flagella, followed by further separation into Triton X-100-soluble and -insoluble fractions. Proteins from each fraction and whole sperm were separated by isoelectric focusing using two different pH ranges, followed by SDS-PAGE at two different polyacrylamide concentrations. In total, 1,294 protein spots representing 304 non-redundant proteins were identified by mass spectrometry (MALDI-TOF). On comparison of the proteins in each fraction, we were able to identify the proteins specific to different sperm compartments. Further comparison with the testis proteome allowed the pairing of proteins with sperm-specific functions. Together with information on gene expression in developing embryos and adult tissues, these results provide insight into novel cellular and functional aspects of sperm proteins, such as distinct localization of actin isoforms, novel Ca(2+)-binding proteins in axonemes, localization of testis-specific serine/threonine kinase, and the presence of G-protein coupled signaling and ubiquitin pathway in sperm flagella., (Copyright © 2011 Wiley-Liss, Inc.)

Spetex-1, which has been isolated by differential display as a haploid spermatid-specific gene, encodes a protein with two coiled-coil motifs located in the middle piece of flagella in rodent spermatozoa. The middle piece of flagella is composed of axoneme and peri-axonemal elements including outer dense fibers (ODFs) and satellite fibrils. Pre-embedding immunoelectron microscopy clearly demonstrated that Spetex-1 is located at satellite fibrils associated with ODFs in the middle piece of flagella of rat spermatozoa. Extraction of Spetex-1 from spermatozoa by SDS or urea required dithiothreitol, suggesting crosslinking by disulfide bond is involved in the assembly of satellite fibrils containing Spetex-1. We identified putative Spetex-1 orthologs in many animal species, and both cysteine residues and coiled-coil motifs were well conserved in mammalian orthologs of Spetex-1. When Spetex-1 was co-transfected into COS-7 cells with myc-tagged Tektin4, another filamentous protein associated with ODFs, the two molecules were co-localized in various sizes of aggregates in the cells. These data suggested that Spetex-1, a new component of satellite fibrils, might be involved in the structural stability of the sperm flagellar middle piece and functions in co-operation with Tektin4., (Copyright 2010 Wiley-Liss, Inc.)

Incubation of boar spermatozoa in Krebs-Ringer-Henseleit medium with either 10 mM lactate or 10 mM citrate induced a fast and robust increase in the intracellular levels of ATP in both cases, which reached a peak after 30 sec of incubation. Utilization of both citrate and lactate resulted in the export of CO(2) to the extracellular medium, indicating that both substrates were metabolized through the Krebs cycle. Incubation with citrate resulted in the generation of extracellular lactate, which was inhibited in the presence of phenylacetic acid. This indicates that lactate is produced through the pyruvate carboxylase step. In addition, there was also a significant increase in tyrosine phosphorylation induced by both citrate and lactate. Boar sperm has a sperm-specific isoform of lactate dehydrogenase (LDH), mainly located in the principal piece of the tail. Kinetic studies showed that boar sperm has at least two distinct LDH activities. The major activity (with an estimated Km of 0.51 mM) was located in the supernatants of sperm extracts. The minor LDH activity (with an estimated Km of 5.9 mM) was associated with the nonsoluble fraction of sperm extracts. Our results indicate that boar sperm efficiently metabolizes citrate and lactate through a metabolic pathway regulated by LDH., ((c) 2005 Wiley-Liss, Inc.)

The leucine zipper motif has been identified as an important and specific interaction motif used by various sperm tail proteins that localize to the outer dense fibers. We had found that rat Odf1, a major integral ODF protein, utilizes its leucine zipper to associate with Odf2, another major ODF protein, Spag4 which localizes to the interface between ODF and axonemal microtubule doublets, and Spag5. The rat Spag5 sequence indicated a close relationship with human Astrin, a microtubule-binding spindle protein suggesting that Spag5, like Spag4, may associate with the sperm tail axoneme. RT PCR assays indicated expression of Spag5 in various tissues and in somatic cells Spag5 localizes to endoplasmic reticulum and microtubules, as expected for an Astrin orthologue. MT binding was confirmed both in vivo and in in vitro MT-binding assays: somatic cells contain a 58 kDa MT-associated Spag5 protein. Western blotting assays of rat somatic cells and male germ cells at different stages of development using anti-Spag5 antibodies demonstrated that the protein expression pattern changes during spermatogenesis and that sperm tails contain a 58 kDa Spag5 protein. Use of affinity-purified anti-Spag5 antibodies in immuno electron microscopy shows that in rat elongated spermatids and epididymal sperm the Spag5 protein associates with ODF, but not with the axonemal MTs. This observation is in contrast to that for the other Odf1-binding, MT-binding protein Spag4, which is present between ODF and axoneme. Our data demonstrate that Spag5 has different localization in somatic versus male germ cells suggesting the possibility of different function., (Copyright 2005 Wiley-Liss, Inc)

Keratin 9 (K9) is one of the components of the perinuclear ring of the manchette found in developing spermatids but is predominantly expressed in the epidermis of the footpad (palm and sole in human epidermis). As an initial step to determine the function of K9 protein in sperm development, we have generated a mutant mouse by homologous recombination of the targeting vector containing the disrupted K9 gene in which the neo(r) gene was inserted into the intron 6. This insertion resulted in the expression of two K9 mRNAs: a wild-type K9 mRNA, in which intron 6 with the neo(r) gene was completely spliced out, and a mutated form in which only a portion of the intron 6 between neo(r) gene and exon 7 was spliced out. While both heterozygous (K9(+/neo)) and homozygous (K9(neo/neo)) mutant mice expressed the wild-type form of K9 protein, the expression profile of the wild-type K9 in K9(neo/neo) mutants was modified. In addition, the open reading frame of the aberrant mRNA terminated at the exon 6/intron 6 splice site, resulting in a truncated K9 protein. Both K9(+neo) and K9(neo/neo) male mice displayed spermatids with ectopic manchette. Coiled tails were seen in maturing spermatids and epididymal sperm of mutant mice and sperm with deformed tails displayed forward motility. A predominant sperm anomaly was residual cytoplasm at the end of the mitochondria-containing middle piece tail segment. The residual cytoplasm displayed vesicles with random in situ motion, suggesting a transport impediment toward the distal end of the sperm tail. All mutant mice were fertile. Surprisingly, in oocyte nuclear injection experiments using K9(neo/neo) sperm donor, 76% of the resulting animals displayed a deletion of the neo(r) gene from the intron 6 of the mutated K9 allele. Results of this study support the view that intron 6 influences the transcriptional efficiency of the K9 gene by decreasing production of wild-type K9 and changing the expression of K9 proteins., ((c) 2005 Wiley-Liss, Inc.)

Tektins are composed of a family of filament-forming proteins associated with ciliary and flagellar microtubules. A new member of the TEKTIN gene family, which was designated as rat Tektin4, was obtained by PCR technique combined with yeast two-hybrid screening. Rat Tektin4 cDNA consists of 1,341 bp encoding a 52 kDa protein of 447 amino acids. Tektin4 protein contains a Tektin domain including a nonapeptide signature sequence (RPNVELCRD), which is a prominent feature of Tektins. Its amino acid sequence showed 29% approximately 58% identities to that of other Tektin family proteins registered in the public databases. Tektin4 gene, which was mapped to rat chromosome 10q12, is composed of six exons and spanning 5 kb. Reverse-transcriptional-PCR (RT-PCR) analysis indicated that Tektin4 was predominantly expressed in testis and its expression was upregulated during testis development. In situ hybridization analysis showed that Tektin4 mRNA was localized in round spermatids in the seminiferous tubules of the rat testis. Tektin4 protein was predominantly localized in the flagella of spermatozoa, suggesting that it might works as a flagellar component requisite for flagellar stability or sperm motility.

Based on cDNA and amino acid sequence, we demonstrate that hamster contraception associated protein 1 (CAP1) protein (an homolog of DJ-1 in mouse, CAP1/SP22/RS in rat and DJ-1/RS in human) is conserved during evolution. Through solubilization studies, it was demonstrated that hamster CAP1 has a peripheral membrane localization. SDS-PAGE analysis revealed that the migration pattern for hamster CAP1 compared to the other rodent counterparts, rat and mouse was different; indicating species-specific differences in the protein (possibly due to post-translational modifications). This protein also shows a ubiquitous presence in both somatic and germ tissues, and has been localized to the sperm tail. It was noticed that hamster CAP1 was lost from the mid piece of spermatozoa during capacitation. Interestingly, following in vitro treatment with ornidazole, CAP1 was lost from the spermatozoa and immunofluorescence studies showed that the major loss was from the mid piece of the spermatozoa. Another interesting feature highlighted about hamster CAP1 is its tendency to exist in two pI isoforms. Summarily, hamster CAP1 appears to exhibit species-specific differences compared to its rodent counterparts with respect to its unique peripheral localization, its size, two pI isoforms, and fate during capacitation, which may have implications in its functions., (Copyright 2004 Wiley-Liss, Inc.)

Experimental evidence indicates that the membrane-associated proteins polycystin-1 and polycystin-2 operate as a receptor-calcium channel complex that regulates signaling pathways essential for modulation of renal tubulogenesis. Polycystic kidney disease is characterized by defective renal tubular structure and results from mutations in either PKD1 or PKD2 genes. Recent data suggest that polycystin-1 and polycystin-2 might localize to primary cilium in principal cells of renal collecting tubules and are thought to act as mechanosensors of fluid flow and contents. Ciliary bending by fluid flow or mechanical stimulation induce Ca(2+) release from intracellular stores, presumably to modulate ion influx in response to tubular fluid flow. Polycystins are also emerging as playing a significant role in sperm development and function. Drosophila polycystin-2 is associated with the head and tail of mature sperm. Targeted disruption of the PKD2 homolog results in nearly complete male sterility without disrupting spermatogenesis. Mutant sperm are motile but are unable to reach the female storage organs (seminal receptacles and spermathecae). The sea urchin polycystin-1-equivalent suPC2 colocalizes with the polycystin-1 homolog REJ3 to the plasma membrane over the acrosomal vesicle. This localization site suggests that the suPC2-REJ3 complex may function as a cation channel mediating acrosome reaction when sperm contact the jelly layer surrounding the egg at fertilization. Future studies leading to the identification of specific ligands for polycystins, including the signaling pathways, might define the puzzling relationship between renal tubular morphogenesis and sperm development and function., (Copyright 2004 Wiley-Liss, Inc.)

Intramanchette transport (IMT) and intraflagellar transport (IFT) share similar molecular components: a raft protein complex transporting cargo proteins mobilized along microtubules by molecular motors. IFT, initially discovered in flagella of Chlamydomonas, has been also observed in cilia of the worm Caenorhabditis elegans and in mouse ciliated and flagellated cells. IFT has been defined as the mechanism by which protein raft components (also called IFT particles) are displaced between the flagellum and the plasma membrane in the anterograde direction by kinesin-II and in the retrograde direction by cytoplasmic dynein 1b. Mutation of the gene Tg737, encoding one of the components of the raft protein complex, designated Polaris in the mouse and IFT88 in both Chlamydomonas and mouse, results in defective ciliogenesis and flagellar development as well as asymmetry in left-right axis determination. Polaris/IFT88 is detected in the manchette of mouse and rat spermatids. Indications of an IMT mechanism originated from the finding that two proteins associated with the manchette (Sak57/K5 and TBP-1, the latter a component of the 26S proteasome) repositioned to the centrosome and sperm tail once the manchette disassembled. IMT has the features of the IFT machinery but, in addition, facilitates nucleocytoplasmic exchange activities during spermiogenesis. An example is Ran, a small GTPase present in the nucleus and cytoplasm of round spermatids and in the manchette of elongating spermatids. Upon disassembly of the manchette, Ran GTPase is found in the centrosome region of elongating spermatids. Because defective molecular motors and raft proteins result in defective flagella, cilia, and cilia-containing photoreceptor cells in the retina, IMT and IFT are emerging as essential mechanisms for managing critical aspects of sperm development. Details of specific role of Ran GTPase in nucleocytoplasmic transport and its relocation from the manchette to the centrosome to the sperm tail await elucidation., (Copyright 2002 Wiley-Liss, Inc.)

The outer dense fibers (ODF) are the main cytoskeletal structures of the sperm tail found in animals with internal fecundation. They consist of at least 14 polypeptides from which only a few are identified due to difficulties in isolation of the protein components. Here we report the isolation and molecular characterization of Odf3, encoding a novel protein of rat sperm ODF. Odf3 is transcribed in testes and more specifically in spermatids but it is also expressed in epididymides and brain suggesting a possible involvement in building of the cellular cytoskeleton. Odf3 encodes a putative protein of approximately 110 kDa. Secondary structure predictions indicated that ODF3 is a coiled-coil protein. The identification of coiled-coil proteins as constituents of outer dense fibers reveals a model for ODF formation., (Copyright 2002 Wiley-Liss, Inc.)

During mammalian spermatogenesis, many specific molecules show the dynamics of expression and elimination, corresponding with the morphological differentiation of germ cells. We have isolated a novel cDNA designated F77 from mouse testis by cDNA subtractive hybridization between normal and sterile mice, using the C57BL/6 congenic strain for the hybrid sterilityhyphen;3 lpar;Hsthyphen;3rpar; allele from Mus spretus. The full-length F77 mRNA was 3.4 kb and showed significant nonmatching with entries in the databases. F77 was mapped at a proximal position between D8Mit212 and D8Mit138 on mouse chromosome 8, in which no corresponding genes related to its nucleotide sequence were found. F77 mRNA was not detected in any other organs except the testis of adult fertile mice. F77 protein was only seen in normal adult testis and epididymis. In contrast to normal C57BL/6 mice, F77 mRNA and protein were not seen in germ cell-deficient Kit(W)/Kit(Wv) mice. By in situ hybridization, F77 mRNA was detected mainly at round spermatids in the sexually mature testis, and immunohistochemical analysis revealed that F77 protein was located at the tail of elongated spermatids. We are proposing the name, sperm-tail-associated protein (Stap), for the gene encoding F77 cDNA. Mol. Reprod. Dev. 59: 350-358, 2001., (Copyright 2001 Wiley-Liss, Inc.)

Outer dense fibers (ODF) and the fibrous sheath (FS) are major cytoskeletal structures in the mammalian sperm tail. The molecular mechanisms underlying their morphogenesis along the axoneme or their function are poorly understood. Recently, we reported the cloning and characterization of Odf2, a major ODF protein, and Spag4, an axoneme-binding protein, by virtue of their strong interaction with Odf1, the 27 kDa major ODF protein. We proposed a crucial role for leucine zippers in molecular interactions during sperm tail morphogenesis. Here we report the cloning and characterization of a novel gene, Spag5, which encodes a 200 kDa testicular protein that interacts strongly with Odf1. Spag5 is transcribed and translated in pachytene spermatocytes and spermatids. It bears 73% similarity with the mitotic spindle protein Deepest of unknown function. We identified two putative leucine zippers in the C-terminal part of the Spag5 protein, the downstream one of which is involved in interaction with Odf1. Interestingly, these motifs are present in Deepest. These results highlight the importance of the leucine zipper in sperm tail protein interactions. Mol. Reprod. Dev. 59: 410-416, 2001., (Copyright 2001 Wiley-Liss, Inc.)

Previously we reported the cloning of a member of the cysteine-rich secretory protein family, tpx-1, from a testis expression library using an outer dense fiber (ODF)-specific antiserum. Using immunohistochemical and immunoelectron microscopic techniques and Western blotting of purified sperm tail components, we have determined that tpx-1 exists as 25 and 27 kDa proteins in two components of rat spermatid: the ODFs and the acrosome. Tpx-1 mRNA is first expressed in the late pachytene spermatocytes, but the production of these tpx-1 proteins is translationally delayed for 4-5 days before being incorporated into the developing sperm acrosome, surrounding the elongating and condensing spermatid nucleus. Concurrent with sperm head formation, tpx-1 protein was incorporated into the developing sperm tail, and specifically the ODFs. The tpx-1 protein was seen within structures resembling granulated bodies in the cytoplasmic lobe of elongating spermatids and was incorporated subsequently into the growing tail in a manner consistent with ODF development. In addition, tpx-1 protein was localized at the ultrastructural level of the connecting piece of the neck and longitudinal columns of the fibrous sheath, suggesting common protein components in these cytoskeletal structures. As such, tpx-1 may have functional significance in the processes of sperm head development and tail function.

RS is 20-kDa microtubule-associated protein found in several human tissues. Sequence analysis showed that the polypeptide is highly related to a rat protein whose level has been previously reported to be correlated with sperm fertility. The present study examines the intracellular distribution of RS in spermatozoa from both humans and rats employing a specific antibody to the polypeptide and immunofluorescence microscopy. We demonstrate that in humans, RS is mainly a flagellum protein, but in rats, it is also abundant in the sperm head. In the sperm tail, RS was found to be co-localized with beta-tubulin, a major component of the axoneme, suggesting that RS is also associated with the flagellum axoneme. Contrary to a previous report, incubation of isolated spermatozoa from both humans and rats in the presence of ornidazole, a reported male contraceptive drug in rats, did not result in modulation in the level of RS, suggesting that the drug does not act directly on sperm RS. Mol. Reprod. Dev. 55:189-196, 2000., (Copyright 2000 Wiley-Liss, Inc.)

Cyclic AMP has been shown essential for activation of sperm motility. When immotile hamster caudal epididymal spermatozoa were suspended in a Ca2+-deficient solution, they showed a sluggish motility. Spermatozoa were demembranated and transferred to an ATP-containing reactivation solution. Demembranated spermatozoa did not exhibit reactivated flagellar movement unless cAMP was added. Conversely, when the immotile epididymal spermatozoa were suspended in a Ca2+-containing solution, they were immediately activated to display a vigorous motility; demembranated spermatozoa also exhibited reactivated flagellar movement in the reactivation solution without cAMP. Further investigation of microtubule sliding properties revealed that the effects of Ca2+ on live spermatozoa were identical with the effects of cAMP on demembranated spermatozoa both in microtubule sliding velocity and sliding disintegration pattern. Moreover, a 36-kDa flagellar protein was found to be phosphorylated in a cAMP-dependent manner and coupled to the motility activation. A polyclonal antibody against this protein was developed and showed specific immunolocalization and significant inhibitory effects on microtubule sliding disintegration. These results indicate that extracellular Ca2+ owes its effect to triggering intracellular cAMP production, and cAMP-dependent phosphorylation of a 36-kDa phosphoprotein activates hamster sperm motility through regulation of microtubule sliding properties.

The outer dense fibers (ODF) of the mammalian sperm tail comprise a unique, specialized, and very prominent structure, consisting of nine fibers surrounding the axoneme. The ODF may play an important but as yet undefined role in sperm morphology, integrity and function. Study of the ODF is hampered by insufficient knowledge of their protein composition and the genetic regulation of their synthesis. We report here on the characterization of cDNAs encoding the Odf2 proteins of outer dense fibers of mouse sperm. We isolated two cDNA clones with variable 5' regions. Variability in sequence is restricted to specific regions in the N-terminal part of the encoded proteins, whereas the C-terminal part is highly conserved in Odf2 proteins both between species and within a species. This variability is confirmed at the protein level. The outer dense fibers could be detected immunologically in total sperm tails allowing a direct comparison of their length in relation to the length of the sperm tail. Odf2 transcripts could be demonstrated in testicular RNA and are restricted to germ cells. The start of transcription is in step 5 spermatids of tubular stage V and the RNA could be detected in the cytoplasm of differentiating spermatids in all subsequent tubular stages.

We have previously reported that Sak57 (for Spermatogenic cell/Sperm-associated keratin of molecular mass 57 kDa) is an acidic keratin found in rat spermatocytes, spermatids, and sperm. Sak57 displays conserved amino acid sequences found in the 1A and 2A regions of the alpha-helical rod domain of keratins in human, rat, and mouse. We now report indirect immunofluorescence, confocal laser scanning microscopy and immunogold electron microscopy data showing that Sak57 is associated with the microtubular mantie of the manchette, a transient microtubular structure largely regarded as formed by tubulin and microtubule-associated proteins. The immunocytochemical localization of Sak57 was detected with a polyclonal antiserum to a multiple antigenic peptide (MAP) containing an amino acid sequence known to be present in the 2A region of the alpha-helical rod domain. During spermiogenic steps 8-12, Sak57 immunoreactive sites were restricted to microtubular mantie of the manchette which encircles the spermatid nucleus during shaping and chromatin condensation. At later stages (spermiogenic steps 12-14), Sak57 immunoreactive sites in the spermatid head region disappeared gradually as specific immunoreactivity appeared along the already assembled axoneme of the developing spermatid tail. Immunogold electron microscopy confirmed the presence of Sak57 immunoreactivity among microtubules of the manchette and on outer dense fibers and the longitudinal columns linking the ribs of the fibrous sheath. Mature spermatids (spermiogenic step 19) displayed tails with an immunofluorescent banding pattern contrasting with the lack of Sak57 immunoreactivity in the head region. Results from this study suggest that, during early spermiogenesis, a microtubular-Sak57 scaffolding is associated with the spermatid nucleus during shaping and chromatin condensation. During late spermiogenesis, the dispersion of the manchette coincides with the progressive visualization of Sak57 in the paraaxonemal outer dense fibers and longitudinal columns of the fibrous sheath in the developing spermatid tail.

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein alpha-subunits, and A 86, which detects all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein alpha-subunits failed to detect any specific antigens in enriched tail membranes. AS 36, recognizing the beta 2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive alpha-subunits. However, membrane preparations of nonpurified human spermatozoa contained alpha i2 subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein alpha-subunits; the putative beta-subunit was identified as a beta 2-subunit.

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin I lectin column. Epididymal fluid antigens have apparent M(rs) of 38-26 kD, whereas the membrane-associated form of the molecule has an M(r) of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secreted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm.

Outer dense fibers (ODFs) are located on the outside of the axoneme in the midpiece and principal piece of the mammalian sperm tail and may help to maintain the passive elastic structures and elastic recoil of the sperm tail. We have identified and describe here a human gene that is homologous to the Mst(3)CGP gene family of Drosophila melanogaster and encodes an ODF protein of 241 amino acids. The transcribed region has a size of approximately 1 kb and contains two exons of 416 bp and 406 bp, respectively, not including the 3' untranslated region. The gene is expressed in testis but not in human spleen, kidney, or brain and resembles in this respect the expression of the Drosophila Mst(3)CGP gene family in the male germline. An antiserum raised against a synthetic peptide derived from the N-terminus of the encoded sequence identified a protein of approximately 32 kDa in an extract of human sperm flagella. By Southern-blot analyses and in situ hybridization, the ODF gene was localized to band q22 of chromosome 8. The isolation of a human gene encoding a sperm tail protein may provide the ability to identify and investigate, on the molecular level, possible reasons for human male infertility that are dependent on flagellar disturbances.

We identified a rat sperm flagellar surface antigen using an IgG1 monoclonal antibody (MC31) against rat epididymal sperm. Avidin-biotin-peroxidase immunohistochemistry demonstrated that the antigen was first expressed in the cytoplasm of early primary spermatocytes, then gradually became restricted to the principal piece of the sperm flagellum during spermatogenesis. However, when the sperm reached the corpus epididymidis, the antigen was expressed on the surface of both the principal piece and the midpiece of the flagellum. The epithelial cells of the epididymis were not stained with MC31. Immunogold electron microscopy showed that the antigen was present on the surface of the sperm flagellar plasma membrane. Immunoblotting of Triton X-100 extracts of epididymal sperm after one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions demonstrated that MC31 detected a major antigen of 26,000-28,000 daltons (26-28K). Two-dimensional isoelectric focusing and SDS-PAGE indicated that the 26-28K antigen had an isoelectric focusing point (pl) of 5.8-5.3; minor antigens were also detected from 26K (pl 5.8) to 35K (pl 5.0). These results indicate that the antigen recognized by MC31 is an acidic 26-35K protein that originates in the testis, is integrated into the sperm flagellar plasma membrane of the principal piece during spermatogenesis, and then is expressed on the entire flagellar surface during epididymal transit.

In previous work a specific membrane protein with an estimated Mr of 20.1 kDa was purified from rabbit sperm tails and designated as rSMP-B protein. Antibodies were raised against rSMP-B protein and used to isolate and identify the cDNA coding the rSMP-B protein from a rat testis lambda gt11 expression library. The nucleotide sequence of the cDNA was determined in a previous study. Single-stranded 35S-labeled RNA probes were prepared. With the techniques of in situ hybridization, rSMP-B mRNA was detected in spermatids of rat and rabbit testis. The present results support our previous observation that immunization of male rabbits with the rSMP-B protein results in the arrest of spermatogenesis at the spermatid stage. Overall, rSMP-B protein appears to be involved in spermiogenesis, and the synthesis of the mRNA encoding the protein occurs in germ cells during the postmeiotic haploid phase of spermatogenesis.