A CatSper-Uninvolved Mechanism to Induce Forward Sperm Motility in the Internal Fertilization.

Sperm-specific cation channel (CatSper), sperm-specific Na + /H + exchanger (sNHE), and soluble adenylyl cyclase (sAC) are necessary in the signaling pathways to control sperm motility in many animals, whereas some animals have lost some or all of them. In the present study, we examined CatSper-uninvolved signaling for vigorous undulation of the undulating membrane that is attached to the sperm tail and gives thrust for forward motility in the internally fertilizing newt Cynops pyrrhogaster. Reverse-transcription PCR failed to detect sNHE in the newt sperm. However, the pH of sperm cytoplasm was raised under a high extracellular pH equivalent to that of egg jelly, where sperm motility is initiated by sperm motility-initiating substance (SMIS). Carbonic anhydrase XII/ XVI and SLC4A4/8 were suggested to be present in the sperm, and transported bicarbonates raised the intracellular pH. In egg jelly extract that contained SMIS, the anion transporter inhibitor DIDS weakened the undulation of the undulating membrane, while bicarbonates enhanced it. The cyclic AMP concentration was found to increase in sperm cytoplasm in the egg-jelly extract. An inhibitor of sAC (KH7) weakened the undulation of the undulating membrane, and dibutyryl cyclic AMP blocked the inhibitory effect. Inhibitor of transmembrane AC (DDA) limitedly affected the undulation. The undulation was weakened by an inhibitor of protein kinase A (H89), and by an inhibitor of transient receptor potential (TRP) channels (RN1747). Our results support the conclusions that the high pH of the egg jelly triggers a signaling pathway through sAC, PKA, and TRP channels, and coacts with SMIS to induce forward sperm motility.

Differences of Extracellular Cues and Ca2+ Permeable Channels in the Signaling Pathways for Inducing Amphibian Sperm Motility.

Low osmolality of freshwater and/or sperm motility-initiating substance (SMIS) induce amphibian sperm motility through increases in intracellular Ca2+. In the internally fertilizing newt Cynops pyrrhogaster, the sperm motility-initiating substance engages T type voltage-dependent Ca2 + channels and N-methyl D-aspartate-type glutamate receptors to initiate sperm motility and L type voltage-dependent Ca2+ channels to enhance motility. In the present study, differences in the usages of SMIS and Ca2+ permeable channels for sperm motility regulation were examined in amphibians that undergo different reproductive modes. Proteins of 14-17 kDa were detected by antibody against the active site peptide of SMIS in the oviduct secretion of internal fertilizers (C. pyrrhogaster, Cynops ensicauda, and Ambystoma mexicanum) and arboreal fertilizers (Rhacophorus arboreus and Rhacophorus schlegelii), but not in Buergeria japonica, an external fertilizer in freshwater. In the pharmacological study, a blocker of some transient receptor potential channels (RN1734) additionally suppressed enhancement of sperm motility in C. pyrrhogaster. In R. schlegelii, blockers of four types of channels differently suppressed sperm motility induced by low osmolality with or without the active site peptide of SMIS. Notably, blockers of L type voltage-dependent Ca2+ channels (nifedipine) and N-methyl D-aspartate-type glutamate receptors (MK801) suppressed sperm motility in the presence and the absence of the peptide, respectively. Low osmolality-induced sperm motility was suppressed by RN1734 and MK801 in B. japonica, but not in Xenopus laevis. These results reveal complex differences in the signaling pathways for inducing sperm motility that may be partly related to reproductive modes in amphibians.

NMDA-type glutamate receptors mediate the acrosome reaction and motility initiation in newt sperm.

The N-methyl d-aspartate type glutamate receptor (NMDAR) is a ligand-gated cation channel that causes Ca2+ influx in nerve cells. An NMDAR agonist is effective to the sperm motility in fowls, although the actual role of NMDAR in sperm function is unknown. In the present study, RNA-seq of the spermatogenic testes suggested the presence of NMDAR in the sperm of the newt Cynops pyrrhogaster. Glutamate of at least 0.7 ± 0.5 mM was detected in the egg-jelly substances along with acrosome reaction-inducing substance (ARIS) and sperm motility-initiating substance (SMIS). In the egg-jelly extract (JE) that included the ARIS and SMIS, the acrosome reaction was inhibited by a NMDAR antagonists, memantine and MK801. MK801 also inhibited the spontaneous acrosome reaction in Steinberg's salt solution (ST). Furthermore, memantine and MK801 suppressed the progressive motility of the sperm in JE and spontaneous waving of the undulating membrane, which is the tail structure giving thrust for forward motility, in ST. The spontaneous waving of the undulating membrane was promoted when Mg2+ , which blocks Ca2+ influx through gated NMDARs, was removed from the ST. In addition, the ARIS-induced acrosome reaction was inhibited by a selective antagonist of the transient receptor potential vanilloid 4, whose activation might result in the membrane depolarization to release Mg2+ from the NMDAR. These results suggest that NMDAR acts together with other cation channels in the induction of the acrosome reaction and motility of the sperm during the fertilization process of C. pyrrhogaster.

Sperm motility initiating substance may be insufficient to induce forward motility of Cynops ensicauda sperm.

Sperm motility-initiating substance (SMIS) is a key protein for internal fertilization of the newt, Cynops pyrrhogaster, and commonly enhances forward sperm motility in some amphibian species, including external fertilizers. SMIS action varies among different species in correlation with a species-specific reproductive environment. In the present study, we identified the gene of C. ensicauda SMIS (CeSMIS) and examined the mechanism of SMIS action with reference to that of the closely related Cynops species. The CeSMIS was identified by a 176-amino acid sequence including seven amino acids critical for the initiation of sperm motility. The amino acid sequence showed 91% homology to the whole sequence of C. pyrrhogaster SMIS (CpSMIS). By immunostaining with an anti-CpSMIS antibody, CeSMIS was shown to be localized in the outer layer of the egg jelly. A peptide presenting the active site of SMIS was observed to bind to the axial rod of the midpiece in C. ensicauda sperm. The localization and binding patterns of CeSMIS were fundamentally similar to those of CpSMIS. However, the SMIS peptide did not induce forward motility of C. ensicauda sperm, although it induced a fast wave of the undulating membrane. Forward sperm motility was induced in the egg jelly extract containing CeSMIS. These results suggest that the mechanism of initiation of sperm motility is differentiated between C. ensicauda and C. pyrrhogaster.

Sperm storage influences the potential for spontaneous acrosome reaction of the sperm in the newt Cynops pyrrhogaster.

Sperm storage is supposed to influence sperm quality, although the details remain unclear. In the present study, we found that sperm stored in a sperm storage site, the vas deferens of Cynops pyrrhogaster, spontaneously undergo acrosome reaction following incubation in Steinberg's salt solution (ST). Percentages of acrosome-reacted sperm increased time-dependently to about 60% in 24 hr. The concentration of cyclic adenosine monophosphate (cAMP) was elevated after incubating sperm in ST, while dibutylyl cAMP induced an acrosome reaction. Chelating of extracellular Ca2+ suppressed the dibutylyl cAMP-induced acrosome reaction as well as spontaneous acrosome reaction in ST. These results suggest that cAMP elevation driven by Ca2+ influx can be a cue for spontaneous acrosome reaction. Relatively low Ca2+ concentration and pH in the vas deferens were sufficient to suppress spontaneous acrosome reaction within 1 hr. In addition, the cysteine rich secretory protein 2 gene was expressed in the vas deferens, indicating that it may be involved in the continuous suppression of spontaneous acrosome reaction. Sperm that underwent spontaneous acrosome reaction in ST was significantly increased when stored in the vas deferens for longer periods, or by males experiencing temperatures in excess of 12°C during hibernation conditions. Percentages of the spontaneously acrosome-reacted sperm were found to differ among males even though they were of identical genetic background. Taken together, C. pyrrhogaster sperm possess the potential for spontaneous acrosome reaction that does not become obvious in the vas deferens, unless promoted in correlation with sperm storage.

Evaluation of Fructo-, Inulin-, and Galacto-Oligosaccharides on the Maillard Reaction Products in Model Systems with Whey Protein.

The present study aimed to investigate the effects of fructo-, inulin-, and galacto-oligosaccharides (FOS, IOS, and GOS) on forming the Maillard reaction products such as browning, α-dicarbonyl compounds, and advanced glycation end products (AGEs). The model solutions at pH 6.8 containing each carbohydrate (mono-, di-, and oligosaccharides) and whey protein were incubated at 50 °C for 8 weeks. In the IOS model, sugars of DP3 or larger were significantly decreased at 4 weeks, whereas at 6 weeks in the FOS model. The residual amount of GOS after 8 weeks was higher than FOS and IOS; however, a large amount of 3-deoxyglucosone was formed compared to the other models. Nε-Carboxymethyllysine (CML) concentrations in oligosaccharide models were about half of those in monosaccharide and lactose models. The highest concentrations of glyoxal- and methylglyoxal-derived hydroimidazolones 3 (G-H3 and MG-H3) were observed in the IOS model, indicating the involvement of fructose units linked by ß-2 → 1 bonds. G-H3 and MG-H3 quantification could be a useful indicator to reflect the modification of an arginine residue by fructose if used acid-hydrolysis for AGE analysis. CML, G-H3, and MG-H3 were considerably formed even in the FOS model, which has no reducing terminal site, suggesting that degradation products of oligosaccharides probably participated in the formation of AGEs.

Immunohistochemistry for the differentiation of peritoneal disseminated carcinoma of unknown origin.

We report a woman with ascites, hydrothorax, pancreatic tumor, left cystic ovarian tumor, and an elevated serum cancer antigen 125 level. Exploratory laparotomy was performed to determine peritoneal disseminated carcinoma of unknown origin. Immunohistochemical analysis demonstrated positive staining for carcinoembryonic antigen, trypsin, and progesterone receptor and nonspecific or negative reaction for calretinin, estrogen receptor, amylase, lipase, Wilms tumor gene 1 protein, and inhibin or chromogranin A. These results together with the morphology of tubular structure suggested the pathological diagnosis of adenocarcinoma with pancreatic characteristics and contradicted ovarian cancer or mesothelioma. Immunohistochemistry is an adjunct tool to differentiate the primary site of carcinomatous peritonitis.

[Two cases of infectious mononucleosis-like syndrome associated with human herpes-virus 6].

We experienced two cases of infectious mononucleosis-like syndrome associated with human herpesvirus 6 (HHV-6). One of the patients had been under medication for depression and the other one for schizophrenia. Both of them were taking carbamazepine for more than a week along with the other drugs. The manifestation of the symptoms of those two were almost same, such as high fever, generalized eruption, liver dysfunction, lymph-adenopathy, and existence of atypical lymphocytes. Serological tests for EB virus, cytomegalovirus and herpes simplex virus showed no significant change while the tests for HHV-6 showed increased titers of IgG antibody during the courses. We also examined HHV-6 DNA by real time quantitative PCR tests for HHV-6, and they appeared significantly high in the peripheral blood samples.