Heterotrimeric G protein subunit Gγ13 is critical to olfaction.

The activation of G-protein-coupled olfactory receptors on the olfactory sensory neurons (OSNs) triggers a signaling cascade, which is mediated by a heterotrimeric G-protein consisting of α, ß, and γ subunits. Although its α subunit, Gαolf, has been identified and well characterized, the identities of its ß and γ subunits and their function in olfactory signal transduction, however, have not been well established yet. We, and others, have found the expression of Gγ13 in the olfactory epithelium, particularly in the cilia of the OSNs. In this study, we generated a conditional gene knock-out mouse line to specifically nullify Gγ13 expression in the olfactory marker protein-expressing OSNs. Immunohistochemical and Western blot results showed that Gγ13 subunit was indeed eliminated in the mutant mice's olfactory epithelium. Intriguingly, Gαolf, ß1 subunits, Ric-8B and CEP290 proteins, were also absent in the epithelium whereas the presence of the effector enzyme adenylyl cyclase III remained largely unaltered. Electro-olfactogram studies showed that the mutant animals had greatly reduced responses to a battery of odorants including three presumable pheromones. Behavioral tests indicated that the mutant mice had a remarkably reduced ability to perform an odor-guided search task although their motivation and agility seemed normal. Our results indicate that Gαolf exclusively forms a functional heterotrimeric G-protein with Gß1 and Gγ13 in OSNs, mediating olfactory signal transduction. The identification of the olfactory G-protein's ßγ moiety has provided a novel approach to understanding the feedback regulation of olfactory signal transduction pathways as well as the control of subcellular structures of OSNs.

Oxalate upregulates expression of IL-2Rß and activates IL-2R signaling in HK-2 cells, a line of human renal epithelial cells.

The role of inflammation in oxalate-induced nephrolithiasis is debated. Our gene expression study indicated an increase in interleukin-2 receptor ß (IL-2Rß) mRNA in response to oxalate (Koul S, Khandrika L, Meacham RB, Koul HK. PLoS ONE 7: e43886, 2012). Herein, we evaluated IL-2Rß expression and its downstream signaling pathway in HK-2 cells in an effort to understand the mechanisms of oxalate nephrotoxicity. HK-2 cells were exposed to oxalate for various time points in the presence or absence of SB203580, a specific p38 MAPK inhibitor. Gene expression data were analyzed by Ingenuity Pathway Analysis software. mRNA expression was quantitated via real-time PCR, and changes in protein expression/kinase activation were analyzed by Western blotting. Exposure of HK-2 cells to oxalate resulted in increased transcription of IL-2Rß mRNA and increased protein levels. Oxalate treatment also activated the IL-2Rß signaling pathway (JAK1/STAT5 phosphorylation). Moreover, the increase in IL-2Rß protein was dependent upon p38 MAPK activity. These results suggest that oxalate-induced activation of the IL-2Rß pathway may lead to a plethora of cellular changes, the most common of which is the induction of inflammation. These results suggest a central role for the p38 MAPK pathway in mediating the effects of oxalate in renal cells, and additional studies may provide the key to unlocking novel biochemical targets in stone disease.

Partial bladder outlet obstruction in mice may cause E-cadherin repression through hypoxia induced pathway.

PURPOSE: Posterior urethral valves are the most common cause of partial bladder outlet obstruction in the pediatric population. Posterior urethral valves is a devastating clinical problem that ultimately results in urinary incontinence, neurogenic bladder and renal impairment. Despite improvements in medical and surgical management at least a third of patients with this condition progress to end stage renal disease and half will have problems with urinary incontinence. To achieve better understanding of the mechanism associated with clinical events we generated partial bladder outlet obstruction in male mice. In this model we investigated pathological consequences and underlying molecular mechanisms secondary to partial bladder outlet obstruction. MATERIALS AND METHODS: Five to 8-week-old male C57BL/6 mice were divided into a surgical obstruction group and a sham operated group that served as controls. Bladders and kidneys were harvested from each group 1, 2, 3, 5 and 7 days postoperatively, respectively. We examined histological and biochemical alterations, and further investigated our hypothesis that partial bladder outlet obstruction induces hypoxia activated profibrotic signaling and changes in gene expression in the bladder. RESULTS: Mice with partial bladder outlet obstruction demonstrated significant increases in bladder mass and urinary retention compared to sham operated mice. Obstruction caused fibrosis in the bladder and induced up-regulation of profibrotic genes, hypoxia-inducible factors and epithelial-mesenchymal transition-inducing transcription factors, resulting in E-cadherin down-regulation. CONCLUSIONS: Obstruction induced significant histological and molecular alterations, including activation of the hypoxia-inducible factors pathway in the mouse bladder. Activation of epithelial-mesenchymal transition-inducing transcription factors by hypoxia-inducible factors might have an important role in the pathogenesis of partial bladder outlet obstruction.

Functional characterization of bitter-taste receptors expressed in mammalian testis.

Mammalian spermatogenesis and sperm maturation are susceptible to the effects of internal and external factors. However, how male germ cells interact with and respond to these elements including those potentially toxic substances is poorly understood. Here, we show that many bitter-taste receptors (T2rs), which are believed to function as gatekeepers in the oral cavity to detect and innately prevent the ingestion of poisonous bitter-tasting compounds, are expressed in mouse seminiferous tubules. Our in situ hybridization results indicate that Tas2r transcripts are expressed postmeiotically. Functional analysis showed that mouse spermatids and spermatozoa responded to both naturally occurring and synthetic bitter-tasting compounds by increasing intracellular free calcium concentrations, and individual male germ cells exhibited different ligand-activation profiles, indicating that each cell may express a unique subset of T2r receptors. These calcium responses could be suppressed by a specific bitter-tastant blocker or abolished by the knockout of the gene for the G protein subunit α-gustducin. Taken together, our data strongly suggest that male germ cells, like taste bud cells in the oral cavity and solitary chemosensory cells in the airway, utilize T2r receptors to sense chemicals in the milieu that may affect sperm behavior and fertilization.

Olefin metathesis in carotenoid synthesis.

Olefin metathesis is a powerful and widely applicable synthetic method for carbon-carbon double bond formation. However, its application to the synthesis of conjugating polyene chains has been very limited because of possible undesired side reactions. We attempted to apply this method to the synthesis of symmetrical carotenoids. In this paper, the syntheses of violaxanthin and mimulaxanthin are described using the olefin metathesis protocol.

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.

HANP1/H1T2, a novel histone H1-like protein involved in nuclear formation and sperm fertility.

We cloned a testis-specific cDNA from mice that encodes a histone H1-like, haploid germ cell-specific nuclear protein designated HANP1/H1T2. The HANP1/H1T2 protein was specifically localized to the nuclei of murine spermatids during differentiation steps 5 to 13 but not to the nuclei of mature sperm. HANP1/H1T2 contains an arginine-serine-rich domain and an ATP/GTP binding site, and it binds to DNA, ATP, and protamine. To investigate the physiological role of HANP1/H1T2, we generated Hanp1/H1T2-disrupted mutant mice. Homozygous Hanp1/H1T2 mutant males were infertile, but females were fertile. Although a substantial number of sperm were recovered from the epididymides, their shape and function were abnormal. During sperm morphogenesis, the formation of nuclei was disturbed and protamine-1 and -2 were only weakly detectable in the nuclei. The chromatin packaging was aberrant, as demonstrated by electron microscopy and biochemical analysis. The mutant sperm exhibited deficient motility and were not competent to fertilize eggs under in vitro fertilization conditions; however, they were capable of fertilizing eggs via intracytoplasmic sperm injection that resulted in the birth of healthy progeny. Thus, we found that HANP1/H1T2 is essential for nuclear formation in functional spermatozoa and is specifically involved in the replacement of histones with protamines during spermiogenesis. At the time of submission of the manuscript, we found an independent publication by Martianov et al. (I. Martianov, S. Brancorsini, R. Catena, A. Gansmuller, N. Kotaja, M. Parvinen, P. Sassone-Corsi, and I. Davidson, Proc. Natl. Acad. Sci. USA 102:2808-2813, 2005) that reported similar results.

Noncoding RNAs of the mammalian testis: the meiotic transcripts Nct1 and Nct2 encode piRNAs.

In eukaryotic cells, the vast majority of transcribed sequences are extragenic with no known functions. Translin is a DNA/RNA-binding protein involved in mRNA transport and translation in postmeiotic male germ cells. In an effort to identify meiotic target RNAs of Translin, reversible RNA protein cross-linking and immunoprecipitations with an affinity purified antibody to Translin were performed. Four new meiotically expressed mRNAs and one noncoding RNA with Translin binding sites were identified. Following sequencing, the noncoding RNA, Nct1, was 100% identical to a site on mouse chromosome 2. A second partially homologous sequence, Nct2, was detected nearby. Nct 1 and 2 contained sequences identical to piRNAs. Nct1 and 2 appear to be male germ cell-specific transcripts and are predominantly detected in pachytene spermatocytes. Focusing on the abundant single-copy PIWI-interacting RNA (piRNA), germline small RNA (gsRNA10) (the gsRNA10 sequence is identical to 29 nt in Nct1), we find that gsRNA10 increases greatly as spermatogenesis proceeds with concomitant decreases in Nct1 and 2. The piRNA gsRNA10 binds to the germ cell-specific Y-box protein, MSY2, but not to Translin. Although the size of the primary transcript(s) encoding the piRNAs in the locus on chromosome 2 is not known, we propose that Nct1 and 2 are part of a piRNA precursor.

Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function.

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.

Transient expression analysis of the mouse ornithine decarboxylase antizyme haploid-specific promoter using in vivo electroporation.

The testicular isoform of the ornithine decarboxylase antizyme (OAZt) gene is expressed exclusively in the haploid spermatids of mice. The 357-bp region, which includes a TATA-less promoter and an untranslated region, is sufficient for OAZt gene expression in the spermatids of transgenic mice. In this study, in vivo transient transfection to living mouse testes was used to define the transcriptional regulatory elements of the OAZt gene promoter. We found that the 10-bp element that contains an initiator (Inr) plays a central role as the core promoter, in combination with a downstream element, while two cyclic adenosine monophosphate-responsive element (CRE)-like sites in the upstream region also contribute to promoter activity. The electrophoretic mobility shift assay showed binding of the testis-specific factors to these elements. Our results show that the in vivo DNA transfer technique enables detailed analysis of haploid germ cell-specific gene regulation in mice.

Sarco/Endoplasmic reticulum Ca2+-ATPases (SERCA) contribute to GPCR-mediated taste perception.

The sense of taste is important for providing animals with valuable information about the qualities of food, such as nutritional or harmful nature. Mammals, including humans, can recognize at least five primary taste qualities: sweet, umami (savory), bitter, sour, and salty. Recent studies have identified molecules and mechanisms underlying the initial steps of tastant-triggered molecular events in taste bud cells, particularly the requirement of increased cytosolic free Ca(2+) concentration ([Ca(2+)](c)) for normal taste signal transduction and transmission. Little, however, is known about the mechanisms controlling the removal of elevated [Ca(2+)](c) from the cytosol of taste receptor cells (TRCs) and how the disruption of these mechanisms affects taste perception. To investigate the molecular mechanism of Ca(2+) clearance in TRCs, we sought the molecules involved in [Ca(2+)](c) regulation using a single-taste-cell transcriptome approach. We found that Serca3, a member of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) family that sequesters cytosolic Ca(2+) into endoplasmic reticulum, is exclusively expressed in sweet/umami/bitter TRCs, which rely on intracellular Ca(2+) release for signaling. Serca3-knockout (KO) mice displayed significantly increased aversive behavioral responses and greater gustatory nerve responses to bitter taste substances but not to sweet or umami taste substances. Further studies showed that Serca2 was mainly expressed in the T1R3-expressing sweet and umami TRCs, suggesting that the loss of function of Serca3 was possibly compensated by Serca2 in these TRCs in the mutant mice. Our data demonstrate that the SERCA family members play an important role in the Ca(2+) clearance in TRCs and that mutation of these proteins may alter bitter and perhaps sweet and umami taste perception.

Expression of the voltage-gated potassium channel KCNQ1 in mammalian taste bud cells and the effect of its null-mutation on taste preferences.

Vertebrate taste buds undergo continual cell turnover. To understand how the gustatory progenitor cells in the stratified lingual epithelium migrate and differentiate into different types of mature taste cells, we sought to identify genes that were selectively expressed in taste cells at different maturation stages. Here we report the expression of the voltage-gated potassium channel KCNQ1 in mammalian taste buds of mouse, rat, and human. Immunohistochemistry and nuclear staining showed that nearly all rodent and human taste cells express this channel. Double immunostaining with antibodies against type II and III taste cell markers validated the presence of KCNQ1 in these two types of cells. Co-localization studies with cytokeratin 14 indicated that KCNQ1 is also expressed in type IV basal precursor cells. Null mutation of the kcnq1 gene in mouse, however, did not alter the gross structure of taste buds or the expression of taste signaling molecules. Behavioral assays showed that the mutant mice display reduced preference to some umami substances, but not to any other taste compounds tested. Gustatory nerve recordings, however, were unable to detect any significant change in the integrated nerve responses of the mutant mice to umami stimuli. These results suggest that although it is expressed in nearly all taste bud cells, the function of KCNQ1 is not required for gross taste bud development or peripheral taste transduction pathways, and the reduced preference of kcnq1-null mice in the behavioral assays may be attributable to the deficiency in the central nervous system or other organs.

Expression profiling reveals meiotic male germ cell mRNAs that are translationally up- and down-regulated.

Gametes rely heavily on posttranscriptional control mechanisms to regulate their differentiation. In eggs, maternal mRNAs are stored and selectively activated during development. In the male, transcription ceases during spermiogenesis, necessitating the posttranscriptional regulation of many paternal mRNAs required for spermatozoan assembly and function. To date, most of the testicular mRNAs known to be translationally regulated are initially transcribed in postmeiotic cells. Because protein synthesis occurs on polysomes and translationally inactive mRNAs are sequestered as ribonucleoproteins (RNPs), movement of mRNAs between these fractions is indicative of translational up- and down-regulation. Here, we use microarrays to analyze mRNAs in RNPs and polysomes from testis extracts of prepuberal and adult mice to characterize the translation state of individual mRNAs as spermatogenesis proceeds. Consistent with published reports, many of the translationally delayed postmeiotic mRNAs shift from the RNPs into the polysomes, establishing the validity of this approach. In addition, we detect another 742 mouse testicular transcripts that show dramatic shifts between RNPs and polysomes. One subgroup of 35 genes containing the known, translationally delayed phosphoglycerate kinase 2 (Pgk2) is initially transcribed during meiosis and is translated in later-stage cells. Another subgroup of 82 meiotically expressed genes is translationally down-regulated late in spermatogenesis. This high-throughput approach defines the changing translation patterns of populations of genes as male germ cells differentiate and identifies groups of meiotic transcripts that are translationally up- and down-regulated.

Meiotic messenger RNA and noncoding RNA targets of the RNA-binding protein Translin (TSN) in mouse testis.

In postmeiotic male germ cells, TSN, formerly known as testis brain-RNA binding protein, is found in the cytoplasm and functions as a posttranscriptional regulator of a group of genes transcribed by the transcription factor CREM-tau. In contrast, in pachytene spermatocytes, TSN is found predominantly in nuclei. Tsn-null males show a reduced sperm count and high levels of apoptosis in meiotic cells, suggesting a critical function for TSN during meiosis. To identify meiotic target RNAs that associate in vivo with TSN, we reversibly cross-linked TSN to RNA in testis extracts from 17-day-old and adult mice and immunoprecipitated the complexes with an affinity-purified TSN antibody. Extracts from Tsn-null mice were used as controls. Cloning and sequencing the immunoprecipitated RNAs, we identified four new TSN target mRNAs, encoding diazepam-binding inhibitor-like 5, arylsulfatase A, a tetratricopeptide repeat structure-containing protein, and ring finger protein 139. In contrast to the population of postmeiotic translationally delayed mRNAs that bind TSN, these four mRNAs are initially expressed in pachytene spermatocytes. In addition, anti-TSN also precipitated a nonprotein-coding RNA (ncRNA), which is abundant in nuclei of pachytene spermatocytes and has a putative polyadenylation signal, but no open reading frame. A second similar ncRNA is adjacent to a GGA repeat, a motif frequently associated with recombination hot spots. RNA gel-shift assays confirm that the four new target mRNAs and the ncRNA specifically bind to TSN in testis extracts. These studies have, for the first time, identified both mRNAs and a ncRNA as TSN targets expressed during meiosis.

Isolation and characterization of a novel cDNA encoding a DNA-binding protein (Hils1) specifically expressed in testicular haploid germ cells.

A cDNA encoding a protein homologous with histone H1 has been cloned from a haploid germ cell specific cDNA library. Deduced amino acid sequence (170 amino acids) showed 40% identity with histone H1 globular domain. Messenger RNA of the gene was observed exclusively in the testis, and was accumulated after post-natal day 23. Western blotting analysis showed that the protein encoded by this gene is about 19 kDa in molecular weight, and it was exclusively recovered from the nuclei of testicular germ cells. Immunohistochemical analysis showed that the protein was localized to the nuclei of round and elongating spermatids, consistent with the results of immunoblot analysis. Thus, the gene product was named Hils1 (histone H1 like protein in spermatids 1). In vitro DNA-binding experiments using DNA-cellulose mini-columns showed that Hils1 was able to bind to both double and single stranded-DNAs in a non-sequence-specific manner. These findings suggest that Hils1 may play an important role in the structural changes of spermatid nuclei, such as nuclear condensation, and gene regulation of haploid germ cell differentiation.

Cloning and characterization of the human tektin-t gene.

Tektin-t is a part of the tektin protein family, the members of which form filamentous polymers in the walls of ciliary and flagellar microtubules. In mice, tektin-t protein has been localized to the tail of mature sperm, suggesting that it has a role in the formation of sperm flagella and/or sperm motility. In the present study, we have cloned a human orthologue of mouse haploid germ cell-specific tektin-t. The cloned human cDNA and the predicted amino acid sequence showed 82 and 83% identity with mouse tektin-t respectively. Included were a sequence conserved in the tektin B1 family, the TEKTIN2 motif, and the consensus sequence in the tektin protein family composed of nona-peptide. Northern blot analysis of mRNA isolated from various human organs showed that the transcript was approximately 1.7 kb in length and strongly expressed in the testis. Human (h-)tektin-t protein, having a molecular weight of 54 kDa, was exclusively expressed in the testis, whereas two additional stronger bands of 46 and 56 kDa existed in sperm. The h-tektin-t localized specifically to the principal piece of flagella and to the post-acrosomal region. The h-tektin-t gene was assigned to chromosome 1 by a radiation hybrid mapping technique.

Control of mouse hils1 gene expression during spermatogenesis: identification of regulatory element by transgenic mouse.

Histone H1-like protein in spermatids 1 (Hils1) is a testis- specific histone H1-like protein exclusively expressed in haploid spermatids and should be involved in chromatin remodeling during mouse spermatogenesis. Spatial and temporal regulation of the hils1 gene expression would be critical for the formation of functional sperm, controlled at both transcriptional and translational levels. Previously, we reported that transcripts of the hils1 gene are exclusively expressed in mouse testis from 23 days of age whereas the Hils1 protein is not detected until 28 days of age, suggesting that hils1 is a member of a class of translationally regulated genes. By analyzing transgenic mice, we could demonstrate that 318-base pair (bp) 5'-proximal region corresponding to the first 70-bp proximal TATA-less promoter, and 248 bp of 5'-untranslated region is sufficient to confer testis- and spermatid-specific transcription as well as posttranscriptional control of the mouse hils1 gene in vivo.

Characterization of a novel postacrosomal perinuclear theca-specific protein, CYPT1.

The perinuclear theca (PT) is a unique cytoskeletal structure that surrounds the nucleus of the sperm. The posterior acrosome segment of the PT (postacrosomal PT) is thought to play roles in shaping the nucleus during differentiation of the spermatid and in activating the oocyte during fertilization. We isolated a cDNA clone that encoded a novel haploid germ cell-specific cysteine-rich perinuclear theca protein, CYPT1. The transcripts were expressed exclusively in testicular germ cells after meiotic division. Sequence analysis revealed that CYPT1 comprised 168 amino acids and that the N-terminal was rich in basic amino acids, including cysteine clusters. Immunohistochemical and biochemical analyses localized CYPT1 to the postacrosomal PT of elongated spermatids and mature sperm. The cypt1 had three paralogs that were expressed in adult testis. A comparison of genomic structure suggested that two of the three cypt1 paralogs were generated by gene triplication on the X chromosome, while one paralog was retrotransposed to an autosome. Interestingly, the 5'-flanking regions of these genes were highly homologous with the promoter region of the spermatid-specific gene Zfy-2. CYPT1 and the proteins of the paralogous genes constitute a novel, basic cysteine-rich sperm protein family that may contribute to the function of the postacrosomal PT during nuclear shaping.

Novel actin-like proteins T-ACTIN 1 and T-ACTIN 2 are differentially expressed in the cytoplasm and nucleus of mouse haploid germ cells.

We isolated cDNA clones for the novel actin-like proteins T-ACTIN 1 and T-ACTIN 2, which are expressed specifically in the mouse testis. These clones were from a subtracted cDNA library that was enriched for haploid germ cell-specific cDNAs. The mRNA sizes and deduced molecular masses of t-actin 1/mACTl7b and t-actin 2/mACTl7a were 2.2 kilobases (kb) and 1.8 kb, and Mr 43.1 x 10(3) and Mr 47.2 x 10(3), respectively. The two deduced amino acid sequences had 60% homology, and they had approximately 40% homology with other actins. The T-ACTINs contained some of the conserved regions seen in other actins. Although the cellular locations of these two proteins are quite different (T-ACTIN-1 was found in the cytoplasm and T-ACTIN-2 was located in the nucleus), the expression of their proteins and mRNAs is controlled during development and limited during spermiogenesis. In contrast, only T-ACTIN-2 was present in sperm heads and tails. These results suggest that T-ACTINs play important roles in sperm function and in the specific morphogenesis of spermatozoa during spermiogenesis.

Molecular cloning and characterization of the human orthologue of the oppo 1 gene encoding a sperm tail protein.

We report here the molecular cloning and characterization of a human orthologue of oppo 1, a mouse gene encoding a male germ-cell-specific sperm tail protein, and the organization of its genomic structure. The mRNA of the human oppo 1 gene (h-oppo 1) was expressed exclusively in the testis, and the 30 kDa protein encoded by the mRNA was detected in human testis and sperm. Immunohistochemical analyses showed that human OPPO 1 protein was localized in the flagellae of ejaculated sperm. A human genomic DNA database search indicated that the h-oppo 1 gene mapped to chromosome 17. The genomic structure of h-oppo 1 showed differences in exon/intron usage, the sequence of the 5'-flanking region, and the first intron was rich in Alu repeats as compared with the mouse oppo 1 gene. Comparison of the two genomic sequences indicated that human oppo 1 has evolved independently, resulting in substantial differences in the genomic structure after the human-mouse split, whereas the sequence of the basic functional unit of the oppo 1 gene seems to have been relatively well conserved.