Albumin is required for the guinea pig sperm capacitation but is not essential for acrosome reaction and fusion with eggs.

The importance of serum albumin in supporting guinea pig sperm capacitation, acrosome reaction, and fusion with eggs in vitro was studied by incubating the spermatozoa in albumin-free medium containing different synthetic polymers. Serum albumin was found to be an obligatory component in the incubating medium for the capacitation of guinea pig spermatozoa. Albumin in the medium is not essential for the acrosome reaction and fusion with the eggs, but these phenomena take place most efficiently in presence of albumin.     

An epididymis-specific carboxyl esterase CES5A is required for sperm capacitation and male fertility in the rat

Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo. Previously, our group cloned and characterized a carboxyl esterase gene Ces5a in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CES5A in sperm maturation. By local injection of Lentivirus-mediated siRNA in the CES5A-expressing region of the rat epididymis, Ces5a-knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CES5A plays an important role in sperm maturation and male fertility.     

A fragment of prosaposin (SGP-1) from rooster sperm promotes sperm-egg binding and improves fertility in chickens

A protein isolated from the supernatant of cryopreserved rooster sperm was found to increase the capability of cryopreserved rooster sperm to bind in vitro to the perivitelline membrane of a chicken egg and substantially raise fertility after artificial insemination (AI). That activity was partially purified and termed universal primary sperm-egg binding protein (UPSEBP). Insufficient protein remained from 6 x 10(11) sperm, despite retention of bioactivity, to allow sequencing. We deduced that the protein may be related to prosaposin (also termed SGP-1, for sulfated glycoprotein-1), and we used published amino acid sequences of prosaposin as a guide for synthesis of peptides. Certain peptides were found to increase in vitro sperm-egg binding and increase fertility of frozen-thawed or fresh rooster sperm, in a manner similar to semipurified UPSEBP. Active epitopes were in a 60 amino acid sequence, reflecting the intervening sequence between saposins A and B, plus short extensions into saposins A and B. Highest activity was found when this synthetic peptide was oxidized to form a disulfide bond between terminal cysteines. Antibody against a synthetic peptide consisting of 58 of these 60 amino acids bound to a 7-9 kilodalton protein in UPSEBP. Collectively, the data support the conclusion that UPSEBP is a fragment of prosaposin. Because prosaposin is in semen in humans and animals, these observations have broad implications for possible cause and therapy of one type of subfertility.     

Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity

Apoptosis, necrosis, and inflammation are hallmarks of cisplatin nephrotoxicity; however, the role and mechanisms of necrosis and inflammation remains undefined. As poly(ADP-ribose) polymerase 1 (PARP1) inhibition or its gene deletion is renoprotective in several renal disease models, we tested whether its activation may be involved in cisplatin nephrotoxicity. Parp1 deficiency was found to reduce cisplatin-induced kidney dysfunction, oxidative stress, and tubular necrosis, but not apoptosis. Moreover, neutrophil infiltration, activation of nuclear factor-κB, c-Jun N-terminal kinases, p38 mitogen-activated protein kinase, and upregulation of proinflammatory genes were all abrogated by Parp1 deficiency. Using proximal tubule epithelial cells isolated from Parp1-deficient and wild-type mice and pharmacological inhibitors, we found evidence for a PARP1/Toll-like receptor 4/p38/tumor necrosis factor-α axis following cisplatin injury. Furthermore, pharmacological inhibition of PARP1 protected against cisplatin-induced kidney structural/functional damage and inflammation. Thus, our findings suggest that PARP1 activation is a primary signal and its inhibition/loss protects against cisplatin-induced nephrotoxicity. Targeting PARP1 may offer a potential therapeutic strategy for cisplatin nephrotoxicity.     

Mice lacking Raf kinase inhibitor protein-1 (RKIP-1) have altered sperm capacitation and reduced reproduction rates with a normal response to testicular injury

Raf kinase inhibitor protein-1 (RKIP-1) belongs to the phosphatidyl ethanolamine-binding family of proteins (PEBP), which are highly conserved throughout evolution and widely expressed in tissues of mammalian organisms. RKIP-1 is a modulator of extracellular signal-regulated kinase (ERK), nuclear factor-kappa B (NF-kappaB), and G protein coupled receptor (GPCR) signaling cascades and is implicated as a factor in numerous physiological processes and disease states including metastasis. Testicular germ cells also express high levels of RKIP mRNA during spermatogenesis, particularly from late pachytene spermatocytes through step 15 elongate spermatids. Therefore, the sensitivity of spermatogenesis to injury was compared in wild-type and RKIP-1(-/-) mice. Unlike what has been described with tumor suppressors such as p53, RKIP-1(-/-) and wild-type mice were equally sensitive to germ cell toxicity by x-irradiation as assessed by terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) positivity 9 hours after a 5 Gy exposure and testicular spermatid head counts 15.5 days after 0.5 Gy exposure. Recent findings also indicate that RKIP is a decapacitation factor receptor on sperm. The present study demonstrates that sperm from RKIP-deficient mice are precociously capacitated compared with their wild-type counterparts. Data from mating experiments indicate decreased reproduction rates between crosses of RKIP-1(-/-) male mice and either heterozygous or RKIP-1(-/-) females. Furthermore, RKIP immunolocalization of epididymal sperm supports transfer of the protein from germ cell cytoplasm to the sperm via the cytoplasmic droplet during epididymal transport. Overall, these studies indicate an important role for RKIP in reproduction as a modulator of capacitation but not in the regulation of testicular injury.     

Evidence for a role of cyclooxygenase (prostaglandin synthetase) and prostaglandins in the sperm acrosome reaction and fertilization

Three cyclooxygenase (prostaglandin synthetase) inhibitors, indomethacin, phenylbutazone, and oxyphenbutazone, decreased fertilization in vitro when mixed with capacitated mouse spermatozoa before addition of the treated gametes to oocytes. Fertilization was inhibited whether the oocytes were intact, follicle cell-free, or both follicle cell-free and zona-free. At various concentrations of inhibitor, no effect was observed on the motility or forward progression of the spermatozoa. These cyclooxygenase inhibitors also decreased the guinea pig acrosome reaction. Inhibition of the acrosome reaction did not occur when a mixture of the prostaglandins (PGE2 or PGF2 alpha) and one of the inhibitors was added to the spermatozoa. Alone, these prostaglandins tended to enhance the rate at which the acrosome reaction took place. Lowered calcium levels reduced the occurrence of the acrosome reaction, an effect that could be reversed at least partially by the addition of PGE2. Even in the nominal absence of calcium, some acrosome reaction took place when PGE2 was present in the medium. These results support an essential role for cyclooxygenase and arachidonic acid metabolites, including prostaglandins, in the events leading to the acrosome reaction and fertilization.     

Ncx (Enx, Hox11L.1) is required for neuronal cell death in enteric ganglia of mice

Background/Purpose

Ncx (Enx, Hox11L.1)-deficient (Ncx-/-) mice develop mega-ileo-ceco-colon with a larger number of neuronal cells in the enteric ganglia. We investigated mechanisms related to this abnormality and directed our attention to the effects on gastrointestinal tract functions.

Methods

The number of NADPH diaphorase or cuprolinic blue-positive neuronal cells in the enteric ganglia was examined during growth of the mice. Neuronal cell death of enteric ganglia was assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling. Function of the gastrointestinal tract was determined by measuring excretion time of the barium chloride given into the stomach.

Results

The number of neuronal cells decreased in control mice older than 2 weeks, and neuronal cell death was evident in the ganglia. However, the number of neuronal cells did not decrease in Ncx-/- mice, and cell death was rare. Excretion time of barium chloride was prolonged in all Ncx-/- mice examined and was improved by the administration of an inhibitor of nitric oxide synthase.

Conclusions

Ncx participates in cell death of enteric neurons. Motor abnormality of the gastrointestinal tract in Ncx-/- mice may be attributed to the large number of neuronal cells.     

【原创论文】组蛋白甲基转移化酶基因SETDB1促进前列腺癌细胞的增殖、迁徙和侵袭

SETDB1已在部分人类肿瘤中被确立为癌基因。本研究的目的是检测艇珏坫J基因及蛋白在人前列腺癌组织、细胞系、前列腺增生组织、前列腺上皮细胞中的表达。并研究SETDB1基因对前列腺癌细胞在体外的增殖、迁徙、侵袭的作用。方法：应用实时定量聚合酶链反应（Real-timeqPCR）免疫组化检测SETDB1基因及蛋白在人前列腺癌组织、细胞系、前列腺增生组织、前列腺上皮细胞中的表达。借助siRNA下调SETDB1在前列腺癌细胞系的表达,分别应用细胞计数实验、细胞克隆形成实验、流式细胞技术;细胞划痕实验、细胞小室侵袭实验对SETDB1下调后的细胞进行检测。结果：SETDB1在人前列腺组织、细胞中高表达,下调SETDB1在前列腺癌细胞系的表达后,前列癌细胞的增殖、迁徙、侵袭能力降低。结论：SETDB1可以作为前列腺癌的癌基因进一步研究。     

Mechanistic target of rapamycin kinase(Mtor)is required for spermatogonial proliferation and differentiation in mice

Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility.However,the exact mechanisms underlying the behavior of spermatogonia,including spermatogonial stem cell(SSC)self-renewal and spermatogonial proliferation and differentiation,are not fully understood.Recent studies demonstrated that the mTOR complex 1(mTORC1)signaling pathway plays a crucial role in spermatogonial development,but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined.In this study,we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia.The Mtor knockout(KO)mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age.These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre.Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice,suggesting that spermatogonial differentiation was inhibited.Spermatogonial proliferation was also impaired in Mtor KO mice,leading to a diminished spermatogonial pool and total germ cell population.Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.     

Characterization of cDNA clones for porcine α(l,3)galactosyl transferase: The enzyme generating the Galα(l,3)Gal epitope

Abstract: Galoα(l,3)Gal is a terminal carbohydrate found on many glycosylated cell surface molecules of species other than humans and Old World monkeys, and is produced by the α(l,3)galactosyl transferase enzyme's adding galactose to a substrate. We have previously shown, by the transfection of COS cells with the cloned mouse α(l,3)galactosyl transferase, that most human anti-pig antibodies react with Galα(l,3)Gal. Using cross-species hybridization with the mouse α(l,3)galactosyl transferase cDNA, bacteriophage λ, gt11 and λgt10 pig cDNA libraries were screened and overlapping clones isolated which encode the pig α(l,3)galactosyl transferase. Sequencing of the clones demonstrated a single open reading frame coding for a protein with high homology to murine (75% identity) and bovine (82% identity) α(l,3)galactosyl transferases. Southern blot analysis shows the porcine α(l,3)galactosyl transferase gene to be a single copy gene, and northern analysis demonstrated an mRNA of 3.9 kb. After splicing the clones to produce a single full length clone, transfection of Galα(l,3)Gal^- COS cells led to strong reactivity with human serum and with the IB4 lectin (which reacts only with Galα(l,3)Gal), indicating that the expression of the transferase led to the expression of Galα(l,3)Gal. The cloning of the cDNA gene for the pig α(l,3)galactosyl transferase is the first step in the production of a transgenic pig lacking the transferase and therefore the Galα(l,3)Gal epitope; such animals could serve as donors for human transplantation.     

Osteopontin is required for unloading-induced osteoclast recruitment and modulation of RANKL expression during tooth drift-associated bone remodeling,but not for super-eruption

Unloading of teeth results in extensive alveolar bone remodeling, causing teeth to move in both vertical (“super-eruption”) and horizontal direction (“drift”). In order to decipher the molecular mechanisms of unloading-induced bone remodeling during tooth movement, we focused on the role of osteopontin (OPN) in the un-opposed molar model, comparing wild-type (WT) and OPN-null mice. Our data indicated that OPN was not required for the continuous eruption of un-opposed teeth while OPN was necessary for the drift of teeth. OPN expression and osteoclast counts were greatly increased on alveolar bone surfaces facing the direction of the drift in WT mice, while osteoclast counts were diminished in OPN?/? mice. RANKL expression in the distal periodontal ligament of WT molars increased significantly by day 6 following unloading, while overall levels of RANKL expression were decreased in both WT and OPN-null mice. In vitro treatment of MC3T3 cells, WT BMCs and OPN?/? BMCs with recombinant OPN resulted in significantly increased RANKL expression in all three cell types. The PI3K and MEK/ERK pathway inhibitors Ly294002 and U0126 reduced RANKL expression levels in vitro. Treatment of BMCs and MC3T3 with OPN also resulted in increased ERK phosphorylation and reduced OPG levels. Together, our studies suggest that increased OPN expression during unloading-induced drifting of teeth enhances localized RANKL expression and osteoclast activity on drift-direction alveolar bone surfaces via extracellular matrix signaling pathways.     

V-ATPase subunit ATP6AP1 (Ac45) regulates osteoclast differentiation, extracellular acidification, lysosomal trafficking, and protease exocytosis in osteoclast-mediated bone resorption

Lysosomal trafficking and protease exocytosis in osteoclasts are essential for ruffled border formation and bone resorption. Yet the mechanism underlying lysosomal trafficking and the related process of exocytosis remains largely unknown. We found ATP6ap1 (Ac45), an accessory subunit of vacuolar-type H(+)-ATPases (V-ATPases), to be highly induced by receptor activator for nuclear factor kappa B ligand (RANKL) in osteoclast differentiation. Ac45 knockdown osteoclasts formed normal actin rings, but had severely impaired extracellular acidification and bone resorption. Ac45 knockdown significantly reduced osteoclast formation. The decrease in the number of osteoclasts does not result from abnormal apoptosis; rather, it results from decreased osteoclast precursor cell proliferation and fusion, which may be partially due to the downregulation of extracellular signal-regulated kinase (ERK) phosphorylation and FBJ osteosarcoma oncogene (c-fos), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and "transmembrane 7 superfamily member 4" (Tm7sf4) expression. Notably, Ac45 knockdown osteoclasts exhibited impaired lysosomal trafficking and exocytosis, as indicated by the absence of lysosomal trafficking to the ruffled border and a lack of cathepsin K exocytosis into the resorption lacuna. Our data revealed that the impaired exocytosis is specifically due to Ac45 deficiency, and not the general consequence of a defective V-ATPase. Together, our results demonstrate the essential role of Ac45 in osteoclast-mediated extracellular acidification and protease exocytosis, as well as the ability of Ac45 to guide lysosomal intracellular trafficking to the ruffled border, potentially through its interaction with the small guanosine-5'-triphosphatase (GTPase) Rab7. Our work indicates that Ac45 may be a novel therapeutic target for osteolytic disease.     

Tslc1 (nectin-like molecule-2) is essential for spermatozoa motility and male fertility

The nectin-like molecule-2 (TSLC1) is a cell-cell adhesion molecule expressed in testicular germ cells. To directly examine the role of Tslc1 in male fertility, we generated Tslc1+/- mice that have greater than 90% reduction in Tslc1 expression. Tslc1+/- males exhibited reduced fertility and rarely transmitted the Tslc1 mutant allele, whereas Tslc1+/- females were consistently able to transmit the mutant allele. Histologic and electron microscopic analyses of the testes in Tslc1+/- mice demonstrated disruption of the junctional scaffold between germ cells and Sertoli cells. Reduced Tslc1 expression had no effect on germ cell proliferation or apoptosis. While evidence of normal spermatozoal maturation was supported by Fluorescence Activated Cell Sorting (FACS) analysis, spermatozoa from Tslc1+/- mice demonstrated markedly reduced motility without compromised viability. Collectively, these results establish an essential role for Tslc1 in spermatozoal maturation and motility, distinct from other members of the nectin family.