Chemical and physical guidance of fish spermatozoa into the egg through the micropyle†,‡.

Eggs of teleost fish, unlike those of many other animals, allow sperm entry only at a single site, a narrow canal in the egg's chorion called the micropyle. In some fish (e.g., flounder, herring, and Alaska pollock), the micropyle is a narrow channel in the chorion, with or without a shallow depression around the outer opening of micropyle. In some other fish (e.g., salmon, pufferfish, cod, and medaka), the micropyle is like a funnel with a conical opening. Eggs of all the above fish have a glycoprotein tightly bound to the chorion surface around the micropyle. This glycoprotein directs spermatozoa into the micropylar canal in a Ca2+-dependent manner. This substance, called the micropylar sperm attractant or MISA, increases fertilization efficiency and is essential in herring. In flounder, salmon, and perhaps medaka, fertilization is possible without MISA, but its absence makes fertilization inefficient because most spermatozoa swim over the micropyle without entering it. The mechanism underlying sperm-MISA interactions is yet to be determined, but at least in herring the involvement of Ca2+ and K+ channel proteins, as well as CatSper and adenylyl cyclase, is very likely. In some other fish (e.g., zebrafish, loach, and goldfish), the chorion around the micropyle is deeply indented (e.g., zebrafish and loach) or it has radially or spirally arranged grooves around the outer opening of the micropyle (e.g., goldfish). MISA is absent from the eggs of these fish and sperm entry into micropylar canal seems to be purely physical.

Sperm attractant in the micropyle region of fish and insect eggs.

In some animals, such as fish, insects, and cephalopods, the thick egg coat has a narrow canal-a micropyle-through which spermatozoa enter the eggs. In fish, there is no indication that spermatozoa are attracted by eggs from a distance, but once spermatozoa come near the outer opening of the micropyle, they exhibit directed movement toward it, suggesting that a substance exists in this defined region to attract spermatozoa. Since Coomassie Blue (CB) binds preferentially to the micropyle region in flounder, herring, steelhead, and other fish, it probably stains this sperm guidance substance. This substance-a glycoprotein based on lectin staining-is bound tightly to the surface of the chorion, but can be removed readily by protease treatment. Although fertilization in fish (flounder) is possible after removal of this substance, its absence makes fertilization inefficient, as reflected by a drastic reduction in fertilization rate. The sperm "attraction" to the micropyle opening is species specific and is dependent on extracellular Ca(2+). Eggs of some insects, including Drosophila, have distinct micropyle caps with CB affinity, which also may prove to assist sperm entry. Our attempts to fertilize fly eggs in vitro were not successful.

Tubular injury in a rat model of type 2 diabetes is prevented by metformin: a possible role of HIF-1α expression and oxygen metabolism.

OBJECTIVE: Chronic hypoxia has been recognized as a key regulator in renal tubulointerstitial fibrosis, as seen in diabetic nephropathy, which is associated with the activation of hypoxia-inducible factor (HIF)-1α. We assess here the effects of the biguanide, metformin, on the expression of HIF-1α in diabetic nephropathy using renal proximal tubular cells and type 2 diabetic rats. RESEARCH DESIGN AND METHODS: We explored the effects of metformin on the expression of HIF-1α using human renal proximal tubular epithelial cells (HRPTECs). Male Zucker diabetic fatty (ZDF; Gmi-fa/fa) rats were treated from 9 to 39 weeks with metformin (250 mg ⋅ kg(-1) ⋅ day(-1)) or insulin. RESULTS: Metformin inhibited hypoxia-induced HIF-1α accumulation and the expression of HIF-1-targeted genes in HRPTECs. Although metformin activated the downstream pathways of AMP-activated protein kinase (AMPK), neither the AMPK activator, AICAR, nor the mTOR inhibitor, rapamycin, suppressed hypoxia-induced HIF-1α expression. In addition, knockdown of AMPK-α did not abolish the inhibitory effects of metformin on HIF-1α expression. The proteasome inhibitor, MG-132, completely eradicated the suppression of hypoxia-induced HIF-1α accumulation by metformin. The inhibitors of mitochondrial respiration similarly suppressed hypoxia-induced HIF-1α expression. Metformin significantly decreased ATP production and oxygen consumption rates, which subsequently led to increased cellular oxygen tension. Finally, metformin, but not insulin, attenuated tubular HIF-1α expression and pimonidazole staining and ameliorated tubular injury in ZDF rats. CONCLUSIONS: Our data suggest that hypoxia-induced HIF-1α accumulation in diabetic nephropathy could be suppressed by the antidiabetes drug, metformin, through the repression of oxygen consumption.

Measurement of melatonin in body fluids: standards, protocols and procedures.

BACKGROUND AND PURPOSE: The circadian rhythm of melatonin in saliva or plasma, or of the melatonin metabolite 6-sulfatoxymelatonin (a6MTs) in urine, is a defining feature of suprachiasmatic nucleus (SCN) function, the body's endogenous oscillatory pacemaker. The primary objective of this review is to ascertain the clinical benefits and limitations of current methodologies employed for detection and quantification of melatonin in biological fluids and tissues. DATA IDENTIFICATION: A search of the English-language literature (Medline) and a systematic review of published articles were carried out. STUDY SELECTION: Articles that specified both the methodology for quantifying melatonin and indicated the clinical purpose were chosen for inclusion in the review. DATA EXTRACTION: The authors critically evaluated the methodological issues associated with various tools and techniques (e.g. standards, protocols, and procedures). RESULTS OF DATA SYNTHESIS: Melatonin measurements are useful for evaluating problems related to the onset or offset of sleep and for assessing phase delays or advances of rhythms in entrained individuals. They have also become an important tool for psychiatric diagnosis, their use being recommended for phase typing in patients suffering from sleep and mood disorders. Additionally, there has been a continuous interest in the use of melatonin as a marker for neoplasms of the pineal region. Melatonin decreases such as found with aging are or post pinealectomy can cause alterations in the sleep/wake cycle. The development of sensitive and selective methods for the precise detection of melatonin in tissues and fluids has increasingly been shown to have direct relevance for clinical decision making. CONCLUSIONS: Due to melatonin's low concentration, as well as the coexistence of numerous other compounds in the blood, the routine determination of melatonin has been an analytical challenge. The available evidence indicates however that these challenges can be overcome and consequently that evaluation of melatonin's presence and activity can be an accessible and useful tool for clinical diagnosis.

The use of poly-L-lysine to facilitate examination of sperm entry into pelagic, non-adhesive fish eggs.

The fish egg is surrounded by a thick envelope called the chorion. The fertilizing spermatozoon enters the egg through a canal-like structure in the chorion, the micropyle. Examination of micropyle at fertilization is difficult if eggs are large and have no distinct landmarks surrounding the micropyle, or if they are positively buoyant in water. Eggs of many commercially important fishes (e.g., flounder, sea bream and eel) are buoyant in water or only slightly adhere to solid objects (e.g., sands, rock and water plants), which makes observation of spermatozoa at fertilization difficult. Here, we report that such eggs can be firmly attached to plastic and glass dishes that have been previously coated with poly-L-lysine. These adhering eggs can be fertilized and develop normally on the dishes. Observations of micropyles of three fish species, before and after sperm entry are presented.

Immunocytochemical localization of secretogranin III in the endocrine pancreas of male rats.

Secretogranin III (SgIII), a member of the granin protein family, is expressed specifically in neuronal and endocrine cells. To examine the precise localization of SgIII in the endocrine pancreas, pancreatic tissues of rats were analyzed immunocytochemically with a polyclonal anti-serum raised against rat SgIII. By light microscopy of semithin sections, the immunoreactivity for SgIII was readily detected in pancreatic A- and B-cells, faintly so in D-cells, and not at all in the exocrine pancreas. By immunoelectron microscopy, immunogold particles indicative of SgIII were observed in the peripheral regions of secretory granules, and universally in the pancreatic endocrine cells. Morphometrical analyses indicated that SgIII is most preferentially localized in the periphery of the secretory granule among granins. These findings suggest that SgIII is closely associated with the secretory granule membrane, serving to anchor the aggregates of other soluble constituents to the membrane.

Expression of membrane-bound and soluble guanylyl cyclase mRNAs in embryonic and adult retina of the medaka fish Oryzias latipes.

Localization of mRNAs for four membrane-bound guanylyl cyclases (membrane GCs; OlGC3, OlGC4, OlGC5, and OlGC-R2), three soluble guanylyl cyclase subunits (soluble GC; OlGCS-alpha(1), OlGCS-alpha(2), and OlGCS-beta(1)), neuronal nitric oxide synthase (nNOS), and cGMP-dependent protein kinase I (cGK I) was examined in the embryonic and adult retinas of the medaka fish Oryzias latipes by in situ hybridization. All of the membrane GC mRNAs were detected in the photoreceptor cells of the adult and embryonic retinas, but in different parts; the OlGC3 and OlGC5 mRNAs were expressed in the proximal part and the OlGC4 and OlGC-R2 mRNAs were expressed in the outer nuclear layer. The mRNA for nNOS was expressed in a scattered fashion on the inner side of the inner nuclear layer in the adult and embryonic retinas. The mRNAs (OlGCS-alpha(2) and OlGCS- beta(1)) of two soluble GC subunits (alpha(2) and beta(1)) were expressed mainly in the inner nuclear layer and the ganglion cell layer of the embryonic retina while the mRNAs of the soluble GC alpha(1) subunit and cGK I were not detected in either the adult or embryonic retina. These results suggest that NO itself and/or the cGMP generated by soluble GC (alpha(2)/beta(1) heterodimer) play a novel role in the neuronal signaling and neuronal development in the medaka fish embryonic retina in addition to the role played by phototransduction through membrane GCs in the adult and embryonic retinas.

Immunocytochemical localization of secretogranin III in the anterior lobe of male rat pituitary glands.

Secretogranin III (SgIII) is one of the acidic secretory proteins, designated as granins, which are specifically expressed in neuronal and endocrine cells. To clarify its precise distribution in the anterior lobe of the rat pituitary gland, we raised a polyclonal antiserum against rat SgIII for immunocytochemical analyses. By immunohistochemistry using semithin sections, positive signals for SgIII were detected intensely in mammotropes and thyrotropes, moderately in gonadotropes and corticotropes, but not in somatotropes. The distribution pattern of SgIII in the pituitary gland was similar to that of chromogranin B (CgB), also of the granin protein family, suggesting that the expressions of these two granins are regulated by common mechanisms. The localization of SgIII in endocrine cells was confirmed by immunoelectron microscopy. In particular, secretory granules of mammotropes and thyrotropes were densely and preferentially co-labeled for SgIII and CgB in their periphery. Moreover, positive signals for SgIII were occasionally found in cells containing both prolactin and TSH in secretory granules. These lines of evidence suggest that SgIII and CgB are closely associated with the secretory granule membrane and that this membrane association might contribute to gathering and anchoring of other soluble constituents to the secretory granule membrane.

Molecular cloning of a novel immunoglobulin superfamily gene preferentially expressed by brain and testis.

We have cloned and characterized a novel gene from both human and mouse that encodes a new member of the immunoglobulin superfamily. The gene is preferentially expressed in both brain and testis, and hence, termed BT-IgSF (brain- and testis-specific immunoglobulin superfamily). The predicted protein consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Human BT-IgSF protein (431 amino acids) is 88% identical to the mouse protein (428 amino acids) and both show significant homology to coxsackie and adenovirus receptor (CAR) and endothelial cell-selective adhesion molecule (ESAM). We examined the expression of BT-IgSF with various cultured cells and found that the gene was expressed in both neurons and glial cells in vitro. Furthermore, the expression was preferentially detected in pyramidal cell layers of the dentate gyrus and hippocampus and in commissure fibers of the corpus callosum, in brain tissue sections examined. These findings suggest that BT-IgSF plays a role in the development or function of the central nervous system.

Purification and Characterization of Sperm Creatine Kinase and Guanylate Cyclase of the Sea Urchin Hemicentrotus pulcherrimus.

Creatine kinase and guanylate cyclase were purified from Hemicentrotus pulcherrimus spermatozoa. The molecular weight of the purified sperm tail creatine kinase was estimated to be 137,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sperm tail guanylate cyclase was purified by chromatography on a WGA-Sepharose column connected to a Concanavalin A-Sepharose column, and a Superose 12 HR column. The molecular weight of the tail guanylate cyclase was estimated to be 128,000 by SDS-PAGE. The specific activity of the purified enzyme was 8.25 µmol of cGMP formed/min/mg protein. Sperm-activating peptide I (SAP-I) causes an electrophoretic mobility shift of H. pulcherrimus sperm guanylate cyclase from 131 kDa to 128 kDa. The 131 kDa form of guanylate cyclase was co-purified with a 76 kDa protein, whose molecular mass is similar to that of a SAP-I receptor. The purified 131 kDa form of guanylate cyclase had higher activity than the 128 kDa form. The 131 kDa and 128 kDa forms of guanylate cyclase contained 23.83 ± 0.65 and 4.16 ± 0.45 moles of phosphate per mol protein (mean ± S.D.; n = 3), respectively. The activities of guanylate cyclase and creatine kinase increased during the testis development. During spermatogenesis, sperm tail creatine kinase was detected immunohistochemically only in mature spermatozoa.

Differential Effects of the Egg Jelly Molecules FSG and SAP-I on Elevation of Intracellular Ca2+ and pH in Sea Urchin Spermatozoa: (FSG/SAP-I/[Ca2+ ]i /pHi ).

We examined the effects of two egg jelly components, a fucose sulfate glycoconjugate (FSG) and sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), on the intracellular pH (pHi ) and Ca2+ ([Ca2+ ]i ) of spermatozoa of the sea urchin Hemicentrotus pulcherrimus. FSG and/or SAP-I induced elevations of [Ca2+ ]; and pHi in the spermatozoa at pH 8.0. At pH 8.0, a second addition of FSG did not induced further elevation of the [Ca2+ ]i or pHi of spermatozoa treated with FSG, but addition or FSG after SAP-I or of SAP-I after FSG induced further increases of [Ca2+ ]i and pHi , At pH 6.6, FSG and/or SAP-I did not induce significant elevation of the [Ca2+ ]i , although SAP-I elevated the pHi , its half-maximal effective concentration being 10 to 100 pM. At pH 8.0, tetraethyl-ammonium, a voltage-sensitive K+ -channel blocker, inhibited induction of the acrosome reaction and elevations of [Ca2+ ]i and pHi by FSG, but did not affect those by SAP-I. These results suggest that FSG and SAP-I activate different Ca2+ and H+ transport systems.

Effects of Sperm-Activating Peptide I on Hemicentrotus pulcherrimus Spermatozoa in High Potassium Sea Water.

The effects of sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) on Hemicentrotus pulcherrimus spermatozoa in high [K+ ] sea water were examined. In high [K+ ] sea water, the respiration rates and motility of H. pulcherrimus spermatozoa were lower than those in normal sea water. SAP-I did not stimulate the lowered respiration rate or motility, although the peptide bound to the spermatozoa as it does in normal sea water. SAP-I elevated the sperm cGMP level in 100 mM K+ sea water (from 0.37 to 4.81 pmol/mg wet weight spermatozoa) more than those in normal sea water (from 0.21 to 0.93 pmol/mg wet weight). A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and SAP-I synergistically elevated the cGMP level from 0.35 to 33.08 pmol/mg wet weight in 100 mM K+ sea water. However, in high [K+ ] sea water, SAP-I did not increase the cAMP level even in the presence of IBMX. SAP-I caused rapid, transient elevation of the intracellular pH and Ca2+ concentration of spermatozoa in normal sea water but not in 100mM K+ sea water. SAP-I did not decrease the apparent molecular weight of sperm guanylate cyclase from 131,000 to 128,000 in high [K+ ] sea water. These results suggest that the SAP-I-induced elevation of the cGMP level in sea urchin spermatozoa occurs before or independently of membrane hyperpolarization induced by the opening of K+ channels.

Receptors for Sperm-activating Peptides, SAP-I and SAP-IIB, on Spermatozoa of Sea Urchins, Hemicentrotus pulcherrimus and Glyptocidaris crenularis: (Sea urchin spermatozoa/Sperm-activating peptides/Receptor/Cross-link).

Sperm-activating peptide analogues, GYGG-SAP-I (Gly-Tyr-Gly-Gly-Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and GYGG-SAP-IIB (Gly-Tyr-Gly-Gly-Lys-Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val) which exibit almost the same respiration-stimulating activity as the respective original peptide were chemically synthesized, radiolabeled with Na125 1, and used for experiments of binding and cross-linking to Hemicentrotus pulcherrimus or Glyptocidaris crenularis spermatozoa. In competitive binding, SAP-I caused 50% decrease in 125 l-GYGG-SAP-l binding to intact spermatozoa, sperm heads and sperm tails of H. pulcherrimus at the concentrations of 282 nM, 3 nM and 141 nM, respectively. The incubations of H. pulcherrimus sperm tails with 125 l-GYGG-SAP-I and the chemical cross-linking reagent, disuccinimidyl suberate, resulted in the appearance of two radiolabeled bands with apparent molecular masses of 71 kDa and 63 kDa (determined by sodium dodecyl sulfate-gel electrophoresis under reducing conditions). 125 l-GYGG-SAP-IIB bound almost equally to sperm heads and sperm tails of G. crenularis, and was cross-linked to 172 kDa, 62 kDa and 58 kDa proteins in sperm heads, and 157 kDa and 62 kDa proteins in sperm tails with disuccinimidyl suberate.