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PubMed2004

Functional expression of TRESK-2, a new member of the tandem-pore K+ channel family.

A new member of the tandem-pore K+ (K(2P)) channel family has been isolated from mouse testis complementary DNA. The new K(2P) channel was named TRESK-2, as its amino acid sequence shares 65% identity with that of TRESK-1. Mouse TRESK-2 is a 394-amino acid protein and possesses four putative transmembrane segments and two pore-forming domains. TRESK-2 has a long cytoplasmic domain joining the second and third transmembrane segments and a short carboxyl terminus. In the rat, TRESK-2 mRNA transcripts were expressed abundantly in the thymus and spleen and at low levels in many other tissues, including heart, small intestine, skeletal muscle, uterus, testis, and placenta, as judged by Northern blot analysis. TRESK-2 mRNA was also expressed in mouse and human tissues. In COS-7 cells transfected with TRESK-2 DNA, a time-independent and noninactivating K+-selective current was recorded. TRESK-2 was insensitive to 1 mm tetraethylammonium, 100 nm apamin, 1 mm 4-aminopyridine, and 10 microm glybenclamide. TRESK-2 was inhibited by 10 microm quinidine, 20 microm arachidonate and acid (pH 6.3) at 49, 43, and 23%, respectively. Single channel openings of TRESK-2 showed marked open channel noise. In symmetrical 150 mm KCl, the current-voltage relationship of TRESK-2 was slightly inwardly rectifying, with the single channel conductance 13 picosiemens (pS) at +60 mV and 16 pS at -60 mV. In inside-out patches, TRESK-2 was unaffected by the intracellular application of 10 microm guanosine 5'-O-(thiotriphosphate). These results show that TRESK-2 is a functional member of the K(2P) channel family and contributes to the background K+ conductance in many types of cells.

4-Aminopyridine↗
PubMed1994

Characterization of a novel murine testis-specific serine/threonine kinase.

Using degenerate oligos corresponding to two highly conserved motifs within the protein kinase catalytic domain and a PCR-based cloning strategy, we have isolated a cDNA fragment encoding a new member of the Ser/Thr (serine/threonine) family of protein kinases. Expression analysis revealed that the fragment recognized two transcripts (1.6 and 1.4 kb) exclusively in testis. Using this fragment as a probe, we have cloned a full-length cDNA from a mouse testis cDNA library. The sequence has a 1092-bp open reading frame encoding a protein of 364 amino acids. The N-terminally localized kinase catalytic domain has all the conserved motifs found in other Ser/Thr kinases. Northern blot analysis using the full-length sequence as a probe revealed that the cloned gene corresponds to the 1.6-kb transcript, suggesting the existence of at least two testis-specific novel Ser/Thr kinases. We propose the name testis-specific kinase-1 (TSK-1) for the gene described here. A GenEMBL databank search revealed highest homology to the human gene encoding rac protein kinase-beta and the group of yeast Ser/Thr kinases encoded by SNF-1, nim-1, KIN-1 and KIN-2.

3T3 Cells↗
PubMed2004

A generational study of glyphosate-tolerant soybeans on mouse fetal, postnatal, pubertal and adult testicular development.

The health safety of transgenic soybeans (glyphosate-tolerant or Roundup Ready) was studied using the mammalian testis (mouse model) as a sensitive biomonitor of potential toxic effects. Pregnant mice were fed a transgenic soybean or a non-transgenic (conventional) diet through gestation and lactation. After weaning, the young male mice were maintained on the respective diets. At 8, 16, 26, 32, 63 and 87 days after birth, three male mice and an adult reference mouse were killed, the testes surgically removed, and the cell populations measured by flow cytometry. Multi-generational studies were conducted in the same manner. The results showed that the transgenic foodstuffs had no effect on macromolecular synthesis or cell growth and differentiation as evidenced by no differences in the percentages of testicular cell populations (haploid, diploid, and tetraploid) between the transgenic soybean-fed mice and those fed the conventional diet. Additionally, there were no differences in litter sizes and body weights of the two groups. It was concluded that the transgenic soybean diet had no negative effect on fetal, postnatal, pubertal or adult testicular development.

Animals↗
PubMed1999

Spatiotemporal patterns of expression of neurotrophins and neurotrophin receptors in mice suggest functional roles in testicular and epididymal morphogenesis.

Several reports have established that the action of neurotrophins is not restricted to the nervous system but can affect a broad range of non-neuronal cells. Nerve growth factor (NGF) is present in adult testis and has been suggested as a potential regulator of meiosis in rat seminiferous epithelium. Here we present an extensive immunohistochemical study on neurotrophins and their receptors (p75 and trk) in the developing mouse testis and epididymis, and in fetal human testis. During the early steps of testicular and epididymal organization in the mouse, strong p75 immunoreactivity is detectable in the gonadal ridge in the mesenchyme that is excluded from the evolving testicular cords, and in the mesenchymal cells of the mesonephros. Later in organogenesis, most of the p75-positive interstitial cells of the testis coexpress neurotrophin-3 (NT-3) and the truncated trk B receptor in a developmentally regulated pattern. Our Western blot data confirm the expression of these molecules. These findings suggest that neurotrophin receptors play a role in early inductive events during critical periods of testicular and epididymal development. During fetal and postnatal histogenesis, an increasing number of NT-3- and p75-positive mesenchymal cells start to express alpha-smooth muscle isoactin, suggesting a role for the so-called neurotrophic system in the differentiation of testicular myoid cells and epididymal smooth muscle cells. In the testis of an 18-wk gestational-age human fetus, immunohistochemical analysis has shown intense immunoreactivity of mesenchymal cells to antibodies for neurotrophin receptors p75, trk A, and trk C, and their ligands NGF and NT-3. In addition, we found that in the human fetal testis, the interstitial cells that are differentiating into peritubular myoid cells are associated with a dense network of nerve fibers. Our data suggest that neurotrophins and their receptors are involved in a multifunctional system that regulates cell differentiation and innervation in the developing testis and epididymis.

Animals↗
PubMed2003

A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets.

Understanding mammalian spermatozoan development and the events surrounding fertilization has grown slowly, in part because of uncertainty about the number and identity of the cellular components involved. Determination of those transcripts expressed specifically by germ cells should provide an inclusive list of probable critical proteins. Here, total mouse testis transcript profiles were trimmed of transcripts found in cultures enriched in Sertoli or interstitial cells to yield a germ cell-enriched transcript profile. Monitoring of changes of this profile in the developing testis identified 1,652 genes whose transcript abundance increased markedly coincident with the onset of meiosis. Remarkably, 351 of these genes (approximately equal to 20%) appear to be expressed only in the male germline. Germ cell-specific transcripts are much less common earlier in testis development. Further analysis of the UniGene EST database coupled with quantitative PCR indicates that approximately 4% of the mouse genome is dedicated to expression in postmeiotic male germ cells. Most or many of the protein products of these transcripts are probably retained in mature spermatozoa. Targeted disruption of 19 of these genes has indicated that a majority have roles critical for normal fertility. Thus, we find an astonishing number of genes expressed specifically by male germ cells late in development. This extensive group provides a plethora of potential targets for germ cell-directed contraception and a staggering number of candidate proteins that could be critical for fertilization.

Animals↗
PubMed1981

Conservatism of the H-Y/H-W receptor.

Soluble H-Y antigen is taken up by cells of the homogametic gonad of cattle, dog, chicken and South African clawed frog. After in vitro exposure to mouse testis supernatant or male fetal calf serum, XX ovary cells or ZZ testis cells, which are normally H-Y-, acquire the H-Y+ (H-W+) phenotype and absorb mouse H-Y antibody in standard serological assays. In addition, H-Y antigens of the different species can compete for attachment to target cells of a single species. In a new competitive binding radioassay, uptake of tritiated human H-Y is blocked in XX bovine fetal ovarian cells exposed to non-labeled H-Y of mouse or fetal bull. Because of H-Y antigens of the different species are cross-reactive serologically, positive reaction of H-Y from one species with gonadal cells of another signifies structural conservatism of the H-Y/H-W gonadal "receptor". It follows that establishment of the H-Y/H-W-receptor complex is a common and critical early event in primary sex differentiation of the vertebrates, directing the initially indifferent embryonic gonad towards the heterogametic mode, which may be testicular or ovarian, depending on the species.

Animals↗
PubMed2001

LH induces orphan nuclear receptor Nur77 gene expression in testicular Leydig cells.

The orphan nuclear receptor Nur77 (NR4A1) is a member of the nuclear receptor superfamily and plays an important role in the regulation of genes involved in steroidogenesis and cell death. Northern blot analysis revealed that the expression of Nur77 mRNA was increased after puberty in mouse testis, and hCG treatment of peripubertal animals induced this gene expression in the testis. Moreover, LH treatment induced a transient increase in Nur77 mRNA, and this induction was LH dose dependent in mouse Leydig tumor cell line, K28. Western blot analysis showed that LH transiently induced Nur77 protein. The protein kinase inhibitor H-89, bisindolymaleimide I, and wortmannin strongly inhibited this inductive effect of LH on Nur77 gene expression. Transient transfection assay demonstrated that LH significantly increased the Nur77 promoter-driven luciferase reporter activity in a dose-dependent manner, and LH also increased the activity of a luciferase reporter gene driven by a promoter containing multi copies of a Nur77-responsive element. Moreover, EMSA showed that Nur77 DNA-binding activity was increased in response to LH. Finally, overexpression of dominant negative Nur77 reduced LH-mediated progesterone biosynthesis. Taken together, these results demonstrate that LH induces Nur77 gene expression, and Nur77 may play an important role in the LH-mediated steroidogenesis in Leydig cells.

Animals↗
PubMed2004

Developmental stage-specific expression of Rbm suggests its involvement in early phases of spermatogenesis.

Rbm is a male infertility gene located on the Y chromosome that is expressed in the testis. To investigate the specific events of spermatogenesis in which Rbm plays a role, the precise pattern of expression of Rbm in the mouse testis was determined. An antibody was generated against the Rbm protein and used to detect a single specific band of 43 kDa in size in mouse testicular lysates. In situ hybridization, immunoblot and immunohistochemistry analyses together indicated that Rbm was expressed in spermatogonia, preleptotene spermatocytes, late leptotene to early pachytene spermatocytes but not in mid-pachytene spermatocytes or subsequent stages of differentiation, including haploid germ cells. These observations suggest that Rbm functions in early but not later stages of male germ cell development.

Animals↗
PubMed2004

Expression of human hormone-sensitive lipase (HSL) in postmeiotic germ cells confers normal fertility to HSL-deficient mice.

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) is a multifunctional fatty acyl esterase that is essential for male fertility and spermatogenesis and that also plays important roles in the function of adipocytes, pancreatic beta-cells, and adrenal cortical cells. Gene-targeted HSL-deficient (HSL-/-) male mice are infertile, have a 2-fold reduction in testicular mass, a 2-fold elevation of the ratio of esterified to free cholesterol in testis, and unique morphological abnormalities in round and elongating spermatids. Postmeiotic germ cells in the testis express a specific HSL isoform. We created transgenic mice expressing a normal human testicular HSL cDNA from the mouse protamine-1 promoter, which mediates expression specifically in postmeiotic germ cells. Testicular cholesteryl esterase activity was undetectable in HSL-/- mice, but in HSL-/- males expressing the testicular transgene, activity was 2-fold greater than normal. HSL transgene mRNA became detectable in testes between 19 and 25 days of age, coinciding with the first wave of postmeiotic transcription in round spermatids. In contrast to nontransgenic HSL-/- mice, HSL-/- males expressing the testicular transgene were normal with respect to fertility, testicular mass, testicular esterified/free cholesterol ratio, and testicular histology. Their cauda epididymides contained abundant, normal-appearing spermatozoa. We conclude that human testicular HSL is functional in mouse testis and that the mechanism of infertility in HSL-deficient males is cell autonomous and resides in postmeiotic germ cells, because HSL expression in these cells is in itself sufficient to restore normal fertility.

Animals↗
PubMed2000

Sperm antigen 6 is the murine homologue of the Chlamydomonas reinhardtii central apparatus protein encoded by the PF16 locus.

A cDNA encoding sperm antigen 6 (Spag6), the murine homologue of the Chlamydomonas reinhardtii PF16 protein-a component of the flagella central apparatus-was isolated from a mouse testis cDNA library. The cDNA sequence predicted a 55.3-kDa polypeptide containing 8 contiguous armadillo repeats with 65% amino acid sequence identity and 81% similarity to the Chlamydomonas PF1 protein. An antipeptide antibody generated against a C-terminal sequence recognized a 55-kDa protein in sperm extracts and localized Spag6 to the principal piece of permeabilized mouse sperm tails. When expressed in COS-1 cells, Spag6 colocalized with microtubules. The Spag6 gene was found to be highly expressed in testis and was mapped using the T31 radiation hybrid panel to mouse chromosome 16. Mutations in the Chlamydomonas PF16 gene cause flagellar paralysis. The presence of a highly conserved mammalian PF16 homologue (Spag6) raises the possibility that Spag6 plays an important role in sperm flagellar function.

Algal Proteins↗
PubMed1998

Tex261, a novel gene presumably related but distinct from steroidogenic acute regulatory (StAR) gene, is regulated during the development of germ cells.

Tex261 is a new gene cloned from a subtractive cDNA library from 10-day postnatal mouse testis. Tex261 transcribes three mRNAs of 3.5, 1.6 and 1.4 kb. The 3.5 kb and 1.4 kb transcripts are expressed in different gonadal and somatic tissues analyzed. However, the 1.6 Kb transcript is only detected in testis and differentially regulated during development. This 1.6 kb mRNA is highly expressed in adult testis, with detection beginning at 15 days of postnatal life, which coincides with the presence of pachytene cells in prepuberal mouse. This expression was confirmed in pachytene cells by run-off transcription assay and by in situ hybridization. A region of 86 amino acids from the predicted Tex261 was recently reported as a part of the steroidogenic acute regulatory protein StAR gene by its sequence identity to a rat StAR cloned cDNA. We demonstrate her that, in the mouse, StAR and Tex261 are two different genes with different expected functions, yet, a high identity (43%) at amino acid level is detected in a region of 153 amino acids corresponding to a transmembrane protein.

Amino Acid Sequence↗
PubMed2002

The glucose transport facilitator GLUT8 is predominantly associated with the acrosomal region of mature spermatozoa.

The glucose transporter 8 (GLUT8) is a recently identified member of the family of sugar transport facilitators. In human tissues GLUT8 is predominantly expressed in testis in a gonadotropin-dependent manner. It is shown here that the onset of mRNA synthesis of GLUT8 during the maturation of mouse testis coincides with the appearance of mature spermatozoa. Furthermore, immunohistochemistry with antiserum against the C-terminus of GLUT8 indicated that the protein was associated with spermatozoa within the seminiferous and the epididymal tubules. The GLUT8 immunoreactivity was detected within the head of mouse and human spermatozoa in the acrosomal region, and appeared to be located at the plasma membrane as well as within the cells. This specific expression and localization of GLUT8 suggests that the transport facilitator plays a major role in the fuel supply of mature spermatozoa, and that it is a potential target for inhibition of sperm cell function.

Acrosome↗
PubMed2001

Isolation and characterization of three genes paralogous to mouse Ring3.

Syntenic chromosomal areas share paralogous genes which are believed to have been generated by repeated duplication of an ancestral gene. The human RING3 gene is known to have paralogous relationships with the ORFX, BRDT, and HUNK1 genes. In addition to the mouse Ring3 cDNA clones previously reported, we isolated mouse Orfx, Brdt, and Hunk1 cDNA clones using mouse testis RNA. Among these four paralogous genes, structure and expression profiles were compared. The proteins encoded by these genes exhibited similar amino acid sequences including two conserved bromodomains. While the Ring3, Orfx, and Hunk1 genes were ubiquitously expressed in various tissues of adult mouse, the Ring3, Orfx, and Brdt genes produced testis-specific transcripts and the Hunk1 gene produced a striated muscle-specific transcript. The diversification of expression patterns of Ring3-related genes during evolution may reflect nucleotide variations in regulatory elements associated with ubiquitous or tissue-specific gene expression.

Animals↗
PubMed2004

Isolation and characterization of two serpentine membrane proteins containing glycerophosphodiester phosphodiesterase, GDE2 and GDE6.

Serpentine membrane protein with a glycerophosphodiester phosphodiesterase (GP-PDE) motif, GDE3, is involved in morphological change of cells and accelerates the program of osteoblast differentiation, suggesting that mammalian GP-PDEs play an important role in the regulation of cytoskeletal modification. Here, we isolated two cDNAs encoding serpentine membrane proteins, GDE2 and GDE6, containing GP-PDE motif from mouse cDNA libraries. The deduced sequence of GDE2 contains 607 amino acids with seven putative transmembrane regions. GDE6 was composed of 633 amino acids also with seven putative transmembrane regions. In amino acid sequences, GDE2 and GDE6 are 43.7% and 34.3% identical to GDE3, respectively. Although GDE3 mRNA is highly expressed in bone tissue and spleen, GDE2 mRNA was expressed in a variety of mouse tissues including lung and heart, while the GDE6 transcript was particularly abundant in spermatocytes of mouse testis. Immunohistochemical analyses using anti-GDE2 antibody showed that GDE2 protein is expressed in the epithelial cell layer of mouse lung. These results suggest that GP-PDEs are differentially expressed in mouse tissues, and might have distinct roles.

Amino Acid Sequence↗
PubMed1999

Characterization of a potent sperm-agglutinating monoclonal antibody and its cognate antigens.

OBJECTIVE: To identify sperm antigens that are capable of eliciting infertility-related sperm-agglutinating antibodies. DESIGN: In vitro laboratory experiments. SETTING: University research laboratory. PATIENT(S): Fertile semen donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm agglutination, immunofluorescence localization, and flow cytometric analysis of surface expression of A36 antigens. Antigen analysis by Western immunoblotting. RESULT(S): Monoclonal antibody A36 induced intensive head-to-head, tail-to-tail, and head-to-tail agglutination of motile human spermatozoa. Antigens recognized by A36 were localized on the acrosomal cap and in the principal tail regions of motile, noncapacitated human sperm. Changes in subcellular levels and localization of the A36-recognized epitope occurred after capacitation and acrosomal loss. A36 reacted with a polymorphic series of proteins in Western blots of sperm extracts from humans and various other animal species, including mouse testis extracts. A common 53-kd antigen was recognized by the antibody in the different antigenic preparations. CONCLUSION(S): A mouse antibody to human sperm, monoclonal antibody A36, caused intensive agglutination of noncapacitated human spermatozoa and reacted with antigens on the acrosomal cap and in the principal tail regions. Of the multiple polypeptides that were reactive with the monoclonal antibody in sperm extracts from humans and other animal species, a common 53-kd antigen was recognized.

Agglutinins↗
PubMed1997

CD106 (VCAM-1) in testicular immunoregulation.

The expression of CD18, CD49d/CD29, CD44, CD54 and CD106 was studied in the testis of normal mice at various ages, in the cryptorchid testis, in the testis of estrogen-treated mice and in the testis of non-obese diabetic (NOD) mice, using immunocytochemistry to see which of these lymphocyte and endothelial adhesion proteins may be involved in lymphocyte regulation in the testis. CD18-, CD49d/CD29-, CD44- and CD54-expressing cells were not found in the normal > 10-week-old BALB/c mouse testis. Leydig cells expressed CD106 strongly at this age. In contrast to the > 10-week-old testis, only very few interstitial cells of the 2-week-old normal mice expressed CD106. The expression of CD106 increased gradually with age so that at 6 weeks of age the expression of CD106 was moderate in the interstitial tissue. In the experimentally abdominal testis, CD106 was expressed in the interstitial tissue as strongly as in the contralateral scrotal testis. CD44- and CD18-expressing cells were occasionally present in the interstitial tissue of the abdominal testis, but not in the contralateral scrotal testis. CD54 was present in the epithelium of the ductuli efferentes. In the testis of the estrogen-treated mice, CD106 was expressed in the interstitial tissue as strongly as in the normal mice. Occasional CD44- and CD18-expressing cells were found in the testicular capsule. In the testis of adult NOD mice, CD106 was present in the interstitial tissue, but none of the other studied proteins. Immunoblotting of CD106 from the adult testis under reducing conditions demonstrated a single broad band with a M(r) of 51-65 kDa. This is a novel isoform of CD106. In a modified Stamper-Woodruff assay, lymphocytes bound to the testicular interstitial tissue. In co-incubations of native Leydig cells and lymphocytes, anti-CD106 antibodies prevented formation of Leydig cell-lymphocyte rosettes more than isotype-matched irrelevant control antibodies, suggesting that Leydig cell lymphocyte binding occurs through CD106-CD49d interactions. In lymphocyte cultures in the presence of anti-CD3, anti-CD28, the M(r) > 5 K fraction of testis extract (containing CD106 as shown by immunoblotting) and anti-CD106 or control antibody, anti-CD106 did not consistently affect T cell 3H-TdR incorporation. The present results suggest that CD106 expressed by the Leydig cells may act as an adhesion-promoting molecule or a co-stimulatory factor for T cells migrating to the testis.

Animals↗
PubMed1997

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain.

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333-372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.

A Kinase Anchor Proteins↗
PubMed1995

Characterization of Fsc1 cDNA for a mouse sperm fibrous sheath component.

The fibrous sheath is a major cytoskeletal structure in the principal piece of the mammalian sperm flagellum. We have cloned a cDNA and used it to characterize the expression of mRNA for a mouse sperm fibrous sheath protein. Peptides from a tryptic digest of fibrous sheath proteins were separated by HPLC and a 31 amino acid sequence was obtained from one of the peptides. Through the use of degenerate oligonucleotide polymerase chain reaction (PCR) primers predicted from this sequence, an 80-bp product was amplified from mouse testis first-strand cDNA. This was utilized as a probe to isolate a 2.9-kb cDNA clone from a mouse round spermatid cDNA library. Sequence analysis of the cDNA clone showed that it encodes a protein with an open reading frame of 849 amino acids and includes the original peptide sequence. The predicted protein has a molecular weight of 93,795 and contains 32 cysteine residues and 32 potential phosphorylation sites. It has no significant homology with other known cytoskeletal proteins. Northern blot analysis detected an mRNA of approximately 3 kb that was abundant in round spermatids of the mouse and in testes from six other mammalian species, but not in twelve somatic tissues from the mouse. In situ hybridization analysis indicated that the mRNA is first detected in step 1-6 spermatids, is most abundant in step 8-12 spermatids, and decreases in amount in step 13-15 spermatids, suggesting that expression of the mRNA occurs in the postmeiotic phase of spermatogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗