Impaired sperm maturation in RNASE9 knockout mice.

Ribonuclease, RNase A family, 9 (RNASE9) is a ribonuclease A superfamily member that is expressed only in the epididymis. It is a small, secreted polypeptide, it lacks ribonuclease activity, and its function(s) is unknown. However, epididymis-specific expression suggests a role in sperm maturation. We generated Rnase9(-/-) mice to study RNASE9 function in vivo. We confirm that RNASE9 expression is restricted to the epididymis. Within the epididymis, RNASE9 is first detected in midcaput, persists through the distal caput and corpus, and wanes in the cauda. Rnase9(-/-) mice are born at the expected Mendelian ratio, have normal postnatal growth and development, and have no outwardly apparent phenotype. Spermatogenesis is normal, and Rnase9-null sperm are morphologically normal. Rnase9(-/-) males have normal fertility in unrestricted mating trials, and fertilization rates in in vitro fertilization assays are indistinguishable from wild-type mice. Visual observations coupled with analyses of sperm velocities shortly after swim out from the corpus shows that motility of Rnase9-null sperm is significantly impaired. However, no differences between wild-type and Rnase9-null sperm are detected by computer-assisted sperm analysis 10-90 min after sperm isolation from the corpus or cauda. Assessment of capacitation-dependent signaling pathways in Rnase9-null sperm showed that, while levels of tyrosine phosphorylation of sperm proteins were normal, there was decreased phosphorylation of protein kinase A substrates upon capacitation compared to wild-type mice. In conclusion, RNASE9 is dispensable for fertility, but the absence of RNASE9 during epididymal transit results in impaired sperm maturation.

WITHDRAWN: Fibulin 2, a tyrosine o-sulfated protein, is up-regulated following retinal detachment.

The manuscript has been withdrawn by the author.

SPACA7 is a novel male germ cell-specific protein localized to the sperm acrosome that is involved in fertilization in mice.

Sperm acrosome associated 7 (SPACA7) is a novel protein of unknown function with no homology to any known protein. Spaca7 transcripts are detected only in testis and predict a 158-residue mature polypeptide with one potential N-glycosylation site and no cysteines. Orthologs are present in various species, including mice and humans. We developed a polyclonal antibody to mouse SPACA7 to study its expression and function. Western blotting and immunofluorescence microscopy detected SPACA7 only in testis, and it was detected in testis starting at Postnatal Day 21 and into adulthood. Immunofluorescence staining of testicular germ cells detected weak SPACA7 expression as early as zygotene spermatocytes. Higher expression was observed in round spermatids, where SPACA7 was localized to a perinuclear spot adjacent to the Golgi and to the acrosome of elongating spermatids and spermatozoa. Immunogold electron microscopy demonstrated that SPACA7 is localized within the proacrosomal granule of round spermatids and the acrosome of spermatozoa. Finally, we showed that SPACA7 was retained within the acrosome of epididymal sperm and was released upon the acrosome reaction. To assess if SPACA7 was involved in fertilization, in vitro fertilization assays in the presence of anti-SPACA7 IgG were performed. Anti-SPACA7 inhibited fertilization of cumulus-intact eggs and prominently delayed cumulus dispersal. However, anti-SPACA7 did not inhibit fertilization of cumulus-free eggs. Our findings indicate that release of SPACA7 from the acrosome accelerates cumulus dispersal and facilitates fertilization via unknown mechanisms. This study is the first to document the expression of endogenous SPACA7 and a function for this novel acrosomal protein.

Salivary gland hypofunction in tyrosylprotein sulfotransferase-2 knockout mice is due to primary hypothyroidism.

BACKGROUND: Protein-tyrosine sulfation is a post-translational modification of an unknown number of secreted and membrane proteins mediated by two known Golgi tyrosylprotein sulfotransferases (TPST-1 and TPST-2). We reported that Tpst2-/- mice have mild-moderate primary hypothyroidism, whereas Tpst1-/- mice are euthyroid. While using magnetic resonance imaging (MRI) to look at the thyroid gland we noticed that the salivary glands in Tpst2-/- mice appeared smaller than in wild type mice. This prompted a detailed analysis to compare salivary gland structure and function in wild type, Tpst1-/-, and Tpst2 -/- mice. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative MRI imaging documented that salivary glands in Tpst2-/- females were (≈) 30% smaller than wild type or Tpst1-/- mice and that the granular convoluted tubules in Tpst2-/- submandibular glands were less prominent and were almost completely devoid of exocrine secretory granules compared to glands from wild type or Tpst1-/- mice. In addition, pilocarpine-induced salivary flow and salivary α-amylase activity in Tpst2-/- mice of both sexes was substantially lower than in wild type and Tpst1-/- mice. Anti-sulfotyrosine Western blots of salivary gland extracts and saliva showed no differences between wild type, Tpst1-/-, and Tpst2-/- mice, suggesting that the salivary gland hypofunction is due to factor(s) extrinsic to the salivary glands. Finally, we found that all indicators of hypothyroidism (serum T4, body weight) and salivary gland hypofunction (salivary flow, salivary α-amylase activity, histological changes) were restored to normal or near normal by thyroid hormone supplementation. CONCLUSIONS/SIGNIFICANCE: Our findings conclusively demonstrate that low body weight and salivary gland hypofunction in Tpst2-/- mice is due solely to primary hypothyroidism.

Tyrosine sulfation of native mouse Psgl-1 is required for optimal leukocyte rolling on P-selectin in vivo.

BACKGROUND: We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity. METHODOLOGY/PRINCIPAL FINDINGS: Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT → B6) or Tpst1;Tpst2 double knockout mice (Tpst DKO → B6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1. CONCLUSIONS/SIGNIFICANCE: These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis.

T cell signal regulation by the actin cytoskeleton.

T cells form an immunological synapse (IS) that sustains and regulates signals for cell stimulation. Enriched in the IS is the Src family kinase Lck. Conversely, the membrane phosphatase CD45, which activates Src family kinases, is excluded, and this is necessary to avoid quenching of T cell receptor phosphosignals. Data suggest that this arrangement occurs by an enrichment of cholesterol-dependent rafts in the IS. However, the role of rafts in structuring the IS remains unclear. To address this question, we used fluorescence resonance energy transfer (FRET) to interrogate the nanoscopic structure of the IS. The FRET probes consisted of membrane-anchored fluorescent proteins with distinct affinities for rafts. Both the raft and nonraft probes exhibited clustering in the IS. However, co-clustering of raft donor-acceptor pairs was 10-fold greater than co-clustering of raft-nonraft pairs. We measured the effect of disrupting rafts in the IS on CD45 localization and Lck regulation by treating stimulated T cells with filipin. The filipin specifically disrupted co-clustering of the raft FRET pairs in the IS and allowed targeting of CD45 to the IS and dephosphorylation of the regulatory tyrosine of Lck. Clustering of the raft probes was also sensitive to latrunctulin B, which disrupts actin filaments. Strikingly, enriching the cortical cytoskeleton using jasplakinolide maintained raft probe co-clustering, CD45 exclusion, and Lck regulation in the IS following the addition of filipin. These data show the actin cytoskeleton maintains a membrane raft environment in the IS that promotes Lck regulation by excluding CD45.

Lack of tyrosylprotein sulfotransferase activity in hematopoietic cells drastically attenuates atherosclerosis in Ldlr-/- mice.

OBJECTIVE: Leukocyte recruitment is a major contributor in the development of atherosclerosis and requires a variety of proteins such as adhesion molecules, chemokines, and chemokine receptors. Several key molecular players implicated in this process are expressed on monocytes and require protein-tyrosine sulfation for optimal function in vitro, including human CCR2, CCR5, CX3CR1, and PSGL-1. We therefore hypothesized that protein-tyrosine sulfation in hematopoietic cells plays an important role in the development of atherosclerosis. METHODS AND RESULTS: Lethally-irradiated Ldlr(-/-) mice were rescued with hematopoietic progenitors lacking tyrosylprotein sulfotransferase (TPST) activity attributable to deletion of the Tpst1 and Tpst2 genes. TPST deficient progenitors efficiently reconstituted hematopoiesis in Ldlr(-/-) recipients and transplantation had no effect on plasma lipids on a standard or atherogenic diet. However, we observed a substantial reduction in the size of atherosclerotic lesions and the number of macrophages in lesions from hyperlipidemic Ldlr(-/-) recipients transplanted with TPST deficient progenitors compared to wild-type progenitors. We also document for the first time that murine Psgl-1 and Cx3cr1 are tyrosine-sulfated. CONCLUSIONS: These data demonstrate that protein-tyrosine sulfation is an important contributor to monocytes/macrophage recruitment and/or retention in a mouse model of atherosclerosis.

Early postnatal pulmonary failure and primary hypothyroidism in mice with combined TPST-1 and TPST-2 deficiency.

Protein-tyrosine sulfation is a post-translational modification of an unknown number of secreted and membrane proteins mediated by two known Golgi tyrosylprotein sulfotransferases (TPST-1 and TPST-2). Tpst double knockouts were generated to investigate the importance of tyrosine sulfation in vivo. Double knockouts were born alive at the expected frequency, were normal in size, and their tissues do not synthesize sulfotyrosine. However, most pups die in the early postnatal period with signs of cardiopulmonary insufficiency. A combination of clinical, magnetic resonance imaging, and histological data indicated that lungs of Tpst double knockouts fail to expand at birth resulting in acute pulmonary hypertension, right-to-left shunting, and death by asphyxia in the early postnatal period. Some double knockouts survive the postnatal period, but fail to thrive and display delayed growth that is due in part to hypothyroidism. In addition, we find that Tpst2-/- mice have primary hypothyroidism, but that Tpst1-/- mice are euthyroid. This suggests that a protein(s) required for thyroid hormone production is sulfated and cannot be sulfated in the absence of TPST-2. Thus, Tpst1 and Tpst2 are the only Tpst genes in mice, tyrosine sulfation is required for normal pulmonary function at birth, and TPST-2 is required for normal thyroid gland function.

Targeted disruption of tyrosylprotein sulfotransferase-2, an enzyme that catalyzes post-translational protein tyrosine O-sulfation, causes male infertility.

Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo and gain insight into other potential TPST substrates, TPST-2-deficient mice were generated by targeted disruption of the Tpst2 gene. Tpst2(+/-) mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. Tpst2(-/-) mice have moderately delayed growth but appear healthy and attain normal body weight by 10 weeks of age. In contrast to Tpst1(-/-) males that have normal fertility, Tpst2(-/-) males are infertile. Tpst2(-/-) sperm are normal in number, morphology, and motility in normal media and appear to capacitate and undergo acrosomal exocytosis normally. However, they are severely defective in their motility in viscous media and in their ability to fertilize zona pellucida-intact eggs. Adhesion of Tpst2(-/-) sperm to the egg plasma membrane is reduced compared with wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility.

Tissue factor-dependent coagulation is preferentially up-regulated within arterial branching areas in a baboon model of Escherichia coli sepsis.

Endothelium plays a critical role in the pathobiology of sepsis by integrating systemic host responses and local rheological stimuli. We studied the differential expression and activation of tissue factor (TF)-dependent coagulation on linear versus branched arterial segments in a baboon sepsis model. Animals were injected intravenously with lethal doses of Escherichia coli or saline and sacrificed after 2 to 8 hours. Whole-mount arterial segments were stained for TF, TF-pathway inhibitor (TFPI), factor VII (FVII), and markers for endothelial cells (ECs), leukocytes, and platelets, followed by confocal microscopy and image analysis. In septic animals, TF localized preferentially at branches, EC surface, leukocytes, and platelet aggregates and accumulated in large amounts in the subendothelial space. FVII strongly co-localized with TF on ECs and leukocytes but less so with subendothelial TF. TFPI co-localized with TF and FVII on endothelium and leukocytes but not in the subendothelial space. Focal TF increases correlated with fibrin deposition and increased endothelial permeability to plasma proteins. Biochemical analysis confirmed that aortas of septic baboons expressed more TF mRNA and protein than controls. Branched segments contained higher TF protein levels and coagulant activity than equivalent linear areas. These data suggest that site-dependent endothelial heterogeneity and rheological factors contribute to focal procoagulant responses to E. coli.