Human cyritestin genes (CYRN1 and CYRN2) are non-functional.

The mouse cyritestin gene is a member of the ADAM (a disintegrin and metalloprotease) gene family and codes for a membrane-anchored sperm protein. Recently, it was shown that cyritestin is critical for male fertility in the mouse. Spermatozoa of cyritestin-deficient mice are not able to bind to the zona pellucida of the oocyte and therefore unable to fertilize the egg. However, zona-free oocytes can be fertilized and the resulting embryos show normal development. In contrast to the mouse, where only one gene for cyritestin (Cyrn) is reported, two cyritestin genes (CYRN1 and CYRN2) are known in humans. The human CYRN1 and CYRN2 genes are located on chromosomes 8 and 16, respectively. We report that 27% of fertile men are deficient for the CYRN1 gene but that all have a CYRN2 gene, suggesting that the CYRN2 gene is the orthologous mouse cyritestin gene in humans and might be involved in sperm-egg interactions. However, the characterization of CYRN2 transcripts from testicular RNA of CYRN1-deficient men demonstrated many termination codons in the synthesized cyritestin cDNA. Furthermore, Western-blot analysis with human testicular protein extracts using an anti-cyritestin antibody failed to detect any cyritestin protein. These results demonstrate clearly that both cyritestin genes are non-functional in humans.

Male mice deficient for germ-cell cyritestin are infertile.

Cyritestin is a membrane-anchored sperm protein belonging to the ADAM (f1.gif" BORDER="0"> f2.gif" BORDER="0">isintegrin and f1.gif" BORDER="0"> f3.gif" BORDER="0">etalloprotease) family of proteins, which are proposed to be involved in cell-cell adhesion through binding to integrin receptors. Several lines of evidence support a role of cyritestin and other members of this protein family in the fusion of sperm and the egg plasma membrane. In an effort to elucidate the physiological function of cyritestin, we have disrupted its locus by homologous recombination. Male homozygous null mutants are infertile, even though spermatogenesis, mating, and migration of sperm from the uterus into the oviduct are normal. In vitro experiments showed that infertility is due to the inability of the cyritestin-deficient sperm to bind to the zona pellucida. However, after removal of the zona pellucida, sperm-egg membrane fusion monitored by the presence of pronuclei and generation of 2- and 4-cell embryos did not reveal any differences from the wild-type situation. These results demonstrate that cyritestin is crucial in the fertilization process at the level of the sperm-zona pellucida interaction.

Proteins with tandemly arranged repeats of a highly charged 16-amino-acid motif encoded by the Dhmst101 gene family are structural components of the outer sheath of the extremely elongated sperm tails of Drosophila hydei.

Fruit fly species of the genus Drosophila show a remarkable variation in sperm length. Some of them produce gigantic sperm several times the total male body length. Sperm of Drosophila hydei, for example, are more than 20 mm long. Little is known about the advantage of such elongated sperm or about the proteins that stabilize their thin flagellar tails. Recently, two members of a novel gene family Dhmst101(1) and Dhmst101(2), whose gene products are associated with the sperm tail, were isolated and characterized. Here a third member of this gene family, Dhmst101(3), is described. It was previously demonstrated that all three genes are located in a single small cluster on chromosome 5 of D. hydei. They are located within 15 kb of genomic DNA, oriented in the same direction and transcribed testis-specifically. The encoded sperm tail-specific proteins are mainly composed of tandemly arranged repeats of a highly charged, cysteine-containing motif of 16 amino acids with the consensus sequence KKKCA/EEAAKKEKEAAE. Experiments with synthetic repeat monomers and dimers have demonstrated a tendency for alpha-helical rod formation, which increased strongly with an increase in repeat number. Therefore, Dhmst101 proteins with 7-60 repeats with regularly spaced cystein-residues are thus expected to form long alpha-helical rods cross-linked by numerous Cys-Cys bridges. Here we apply immunoelectron microscopy and monospecific antibodies, alpha-mst101, raised against the KKKCAEAAKKEKEAAE-motif to investigate the distribution of Dhmst101 proteins within the sperm tail of D. hydei. We show that Dhmst101 proteins are part of the outer sheath of the sperm tail where they presumably help to provide a tight but elastic envelope for the extremely extended spermatozoa of D. hydei.

PACSIN, a brain protein that is upregulated upon differentiation into neuronal cells.

To identify genes that are differentially expressed during self-repair processes in mouse brain, we screened a subtracted cDNA library enriched for brain-specific clones. One of these clones, H74, detected a 4.4-kb mRNA predominantly expressed in brain and dorsal root ganglia neurons. Expression increased continuously during the lifespan and the state of differentiation, but decreased after entorhinal-cortex lesion. A full-length cDNA clone was isolated from a cerebellum cDNA library and characterized. Sequence analysis and database search revealed high sequence similarity to FAP52, a protein expressed in focal-adhesion contacts, and uncharacterized Echinococcus and Caenorhabditis elegans gene products. Furthermore, peptide sequences derived from human cDNA fragments showed up to 65% sequence identity at the amino acid level. The presence of a C-terminal src homology 3 (SH3) domain and its phosphorylation by casein kinase 2 (CK2) and protein kinase C (PKC) imply a role in signaling. Here we demonstrate that the gene encodes a phosphoprotein, referred to as PACSIN, with a restricted spatial and temporal expression pattern.

Dead box for the living.

Members of a large family of proteins, called the DEAD box family, are ribonucleic acid binding proteins with ATPase activity. Recent investigations into the developmentally and cell type-specific expression patterns of one family member, p68 RNA helicase, suggest that this protein might play a role in organ differentiation and/or maturation, and that its expression is subject to complex regulation.

Intratesticular distribution of cyritestin, a protein involved in gamete interaction.

Cyritestin, a member of the ADAM family of proteins, has been shown to be involved in the interaction between sperm and egg during fertilization. The protein is a transmembrane protein associated with the sperm acrosome. In the present study, electron microscopy was used to trace the distribution of the cyritestin molecule in intratesticular germ cells, particularly in haploid round spermatids where the acrosomal structure is differentiating. Our results indicate that cyritestin is transported to the forming acrosomal vesicle through the Golgi apparatus to become part of the acrosomal membrane. Differential staining with antibodies recognizing either the metalloprotease-like domain or the cytoplasmic domain of cyritestin indicates that processing of the molecule leading to the loss of the pro- and metalloproteinase-like domains begins during germ cell stage 6 and is completed before stage 15.

Molecular cloning, chromosomal localization, and expression analysis of CYRN1 and CYRN2, two human genes coding for cyritestin, a sperm protein involved in gamete interaction.

Germ cell cyritestin is a membrane-anchored protein belonging to the ADAM family of proteins. Sequencing of eight human cyritestin cDNA clones revealed that they are identical at their 5' and 3' ends but differ from each other in the length of an internal deletion, suggesting that the human cyritestin mRNA is alternatively spliced. Internal deletions that are present in some cDNA isoforms do not cause a frameshift in the C-terminal coding region. Analysis of the predicted amino acid sequences demonstrated that the human cyritestin is a polymorphic protein that could include membrane-anchored and soluble forms. Southern blot analysis and characterization of human cyritestin genomic fragments revealed that the human genome contains two copies of the cyritestin gene instead of one as in the mouse. The human CYRN1 and CYRN2 genes were assigned to the region p12-21 of chromosome 8 and q12 of chromosome 16, respectively. Northern blot and RT-PCR analyses revealed that both human genes are expressed in the testis. Amino acid sequence comparisons between cyritestin and other members of the metalloprotease-disintegrin family of proteins suggested that human and mouse cyritestin and monkey tMDCI are homologous molecules.

Expression pattern and cellular distribution of the murine homologue of AF10.

We have cloned Af10, the murine homologue of the MLL partner gene AF10. The predicted open reading frame of Af10 contains 1069 aa which are 90% identical to those of AF10. Af10 contains an N-terminal cysteine-rich region with a LAP/PHD finger, a leucine zipper domain and a glutamine-rich region at the C-terminus, features also found in the human proteins AF10 and AF17. A single 5. 5-kb transcript was detected in murine tissues with the highest level of expression in the testes. A polyclonal antibody raised to the cysteine-rich region of AF10 was able to identify a double band of 140 kDa on Western analysis in mouse testicular extracts. After subcellular separation Af10 was identified in both the nuclear and cytoplasmic extracts, again as a double band of 140 kDa in size. In situ hybridisation studies were performed with sense and antisense digoxigenin-labelled oligonucleotides. High levels of expression were noted in postmeiotic germ cells, especially in spermatids from around stage VI to stage VIII. High levels of expression were also seen in the white matter of the cerebellum, extending into the granular layer. The expression in differentiated rather than in proliferating cells suggests that the role of Af10 may lie in the suppression of proliferation rather than in differentiation. Since the LAP/PHD finger domains are lost in the MLL-AF10 fusion, arguably such a function could be carried out by this domain.

Decreased in vitro fertilization efficiencies in the presence of specific cyritestin peptides.

Synthetic peptides corresponding to specific regions of the mouse acrosomal transmembrane protein cyritestin have been used as competitors in in vitro fertilization experiments. One peptide representing a putative egg-receptor binding site within the disintegrin domain of cyritestin lowered the fertilization rate to 30% of the normal value.

The C5 Unit: a semi-automatic cell culture device suitable for experiments under microgravity.

This paper presents data on a novel, semi-automatic cell culturing device called 'C5 Unit' (connectable circuit cell culture chamber) which was developed and adapted to the quality and size criteria set by the characteristics of the ESA Biorack. The suitability of the hardware for culturing cells under microgravity conditions was demonstrated by successful culture of primary mouse cells from neonatal cerebellum and testis aboard the Space Shuttle during the IML-2 mission.

Delayed translation and posttranslational processing of cyritestin, an integral transmembrane protein of the mouse acrosome.

This paper presents data on the cellular localization of the testis-expressed mouse Cyrn gene product, cyritestin. This cysteine-rich protein is a member of a family including various rodent and primate proteins and snake venom proteins of the metalloproteinase and disintegrin types. By using antibodies raised against recombinant proteins generated in bacteria and against synthetic peptides we show that (i) Cyrn mRNA is present in germ cells 4 days prior to translation; (ii) cyritestin protein is localized in the acrosomal region of spermatids and spermatozoa; and (iii) cyritestin has an apparent molecular weight of 110,000 Daltons, but is subject to processing during epididymal sperm transport, resulting in a shorter molecule lacking approximately 55 kDa from the N-terminal half. We conclude that cyritestin becomes exposed on the sperm surface after successful acrosome reaction and thus may play a role in sperm function rather than in testicular germ cell maturation.

Developmentally regulated mouse gene NK10 encodes a zinc finger repressor protein with differential DNA-binding domains.

Using oligonucleotides complementary to the conserved inter-finger region of a variety of previously described zinc finger-encoding genes, a novel mouse gene was cloned and characterized. The gene is localized on chromosome 8 and comprises five exons. Its corresponding mRNA is developmentally regulated in various tissues and includes an open reading frame encoding a protein of 72,422 daltons. It shares amino-terminal homologies with human KRAB (or FPB) boxes, and contains 13 zinc fingers of the C2-H2 type. The NK10 KRAB domains exhibit repressing activity when tested in GAL4 fusion protein assays. Cloning of putative target sequences revealed that the individual domains differentially contribute to zinc-dependent target DNA binding.

Male germ cell extracts contain proteins binding to the conserved 3'-end of mouse p68 RNA helicase mRNA.

The 3'-untranslated regions of human and mouse p68 RNA helicase mRNA are highly conserved, suggesting a functional role of the nucleic acid sequence itself in regulation of p68 RNA helicase expression. Secondary structure evaluations revealed no indications for a predominant folding pattern within the 3'-UTR. To test the potential of the 3'-sequence to serve as a target for specific binding proteins, gel shift assays were performed. In vitro-synthesized RNA was incubated with cytoplasmic as well as nuclear extracts from mouse male germ cells. Evidence was obtained that such specific proteins exist in germ cell extracts. Photo-crosslinking experiments suggested that a 30 kDa protein was involved in these binding events.

Assignment to chromosome 11 of mouse p68 RNA helicase gene (Hlr1) and pseudogene (Hlr1-ps1).

The gene encoding murine p68 RNA helicase (Hlr1) was mapped to the distal portion of mouse chromosome 11 by linkage analysis of DNA restriction length polymorphisms using an interspecific genetic backcross between (C57BL/6J x SPRET/Ei) F1 hybrids and SPRET/Ei mice. A closely related gene (Hlr1-ps1) was identified, isolated, and mapped to the proximal part of the same chromosome. Sequence analysis and PCR results suggest that Hlr1-ps1 is a pseudogene, flanked by DNA stretches similar to mouse insertion element IE118.

Recombinant polypeptides transplanted from pUMA vector-derived cRNAs are translocated through microsomal membranes and exported out of frog oocytes.

pSP64 derivatives were constructed to obtain cloning vectors suitable for in vitro transcription and subsequent in vitro synthesis of recombinant proteins equipped with N-terminal signal sequences. The amino acid sequence of the signal peptide was adapted and slightly modified from the one occurring in the neural cell adhesion molecule, NCAM. Its ability to direct recombinant proteins into secretory pathways was tested by in vitro translation in microsomal membrane-containing reticulocyte lysates and by injection of the pUMA-derived cRNAs into Xenopus laevis oocytes.

Ectopic expression of NCAM in mouse fibroblasts stimulates self-aggregation, and promotes integration into primary cerebellum cell aggregates.

We have undertaken aggregation experiments using mouse LMTK(-)-fibroblasts transfected with various isotypes of the neural cell adhesion molecule, NCAM. We found that self-aggregation of NCAM-positive fibroblasts is enhanced compared to control-transfected cells. The aggregation properties are partly dependent on the expressed NCAM isotype. Fibroblasts expressing a NCAM 140 isotype with exons a3 and pi were further tested in primary cerebellum cell re-aggregation experiments. While control-transfected fibroblasts could not be found in forming aggregates, fibroblasts ectopically expressing NCAM were integrated into neural cell aggregates. Time-lapse photography indicated that the nascent primary cell aggregates actively participated in the integration process by migration and attachment to nearby NCAM-positive fibroblasts.

Chromosomal assignment of three novel mouse genes expressed in testicular cells.

The chromosomal positions of three genes that are selectively expressed in mouse testis cells have been identified. These genes include (i) TAZ83, which codes for an early- to mid-pachytene germ cell stage-expressed, cysteine-rich transmembrane protein (cyritestin) with homologies to various snake toxins and guinea pig sperm-egg fusion proteins; (ii) TNZ1, which is expressed in neonatal Leydig cells; and (iii) TAZ4, a testis-specific gene isolated by immunoscreening with antiserum raised against Sertoli cell membranes. Our experimental data, derived from chromosomal in situ hybridizations and RFLP studies of genetic backcrosses, indicate that (i) the TAZ83 (cyritestin) gene maps to chromosome 8, band A2, near the Plat locus; (ii) TNZ1 is located in the proximal region of chromosome 11; and (iii) TAZ4 is located at band D in the distal portion of chromosome 11, near the Hlr1 locus, with a related sequence, TAZ4-rs1, in the proximal part of chromosome 1.

The Drosophila hydei gene Dhmst101(1) encodes a testis-specific, repetitive, axoneme-associated protein with differential abundance in Y chromosomal deletion mutant flies.

To understand the effect of the megabase-sized, Y chromosomal fertility genes on different stages of spermatogenesis in Drosophila hydei, an immunoscreening was performed to search for testis-specific protein-encoding cDNAs. The array of isolated clones contained cDNA sequences derived from a gene on chromosome 5 at 101BC. The gene, Dhmst101(1), is a member of a small gene family and is specifically expressed in adult testis tissue. The mRNA encodes a protein of 344 amino acids with a deduced apparent molecular weight of 37,793 Da. The main portion of the protein sequence comprises repetitive, highly charged amino acid units and shows repeat number variations among several D. hydei laboratory stocks. Immunocytochemistry with antibodies raised against synthetic peptides localized the protein product in elongated spermatids. This pattern of expression and the evaluation of biophysical considerations on the protein sequence data suggest that the Dhmst101(1) gene product may have some importance for the structural integrity of the sperm tail. Moreover, Y chromosomal deletions affecting correct spermiogenesis lead to degradation of the Dhmst101(1) gene product.

High-level expression in male germ cells of murine P68 RNA helicase mRNA.

The complete cDNA coding for mouse P68 RNA helicase was cloned and its nucleotide sequence was determined. The sequence is about 95% identical to the human equivalent. Whereas the 5'-untranslated region is less conserved (71%), the 3'-ends of mouse and human mRNAs are nearly identical. Between stop codon and poly(A)-tail both sequences are 97% conserved. At the level of amino acid sequence, the similarity of both, mouse and human, DEAD box family proteins is as high as 98%. In situ hybridizations using cDNA subfragments as probes revealed a testis-selective expression of P68 RNA helicase mRNA. The signal was restricted to late pachytene spermatocytes and haploid spermatids. Northern blot analyses corroborated these results but suggested that expression of related mRNA species occurs in a variety of other tissues.