Background and PurposeThe 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 IdentificationA search of the English-language literature (Medline) and a systematic review of published articles were carried out.Study SelectionArticles that specified both the methodology for quantifying melatonin and indicated the clinical purpose were chosen for inclusion in the review.Data ExtractionThe authors critically evaluated the methodological issues associated with various tools and techniques (e.g. standards, protocols, and procedures).Results of Data SynthesisMelatonin 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.ConclusionsDue 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.

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.

OBJECTIVEChronic 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 METHODSWe 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.RESULTSMetformin 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.CONCLUSIONSOur 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.

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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.

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.

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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 Ca'+ concentration of spermatozoa in normal sea water but not in 100 mM 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.