Monica Brito

PRESENCE OF RNA IN THE SPERM NUCLEUS

The RNase-colloidal gold procedure for the ultrastructural localization of RNA was used for rat testis. Along with other structures, it was found that the testicular sperm nucleus was well stained. Similar labelling was observed in the nucleus of rat epididymal sperm and human sperm. The RNA was extracted from sperm and analyzed by electrophoresis on 10% polyacrylamide gel and 7 M urea. The electrophoretic profile revealed a complex set of bands ranging in size from tRNA to high molecular weight components. On the average, a content of about 0.1 pg of RNA per rat or human sperm was found.

Broad expression of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase provide evidence for gluconeogenesis in human tissues other than liver and kidney.

The importance of renal and hepatic gluconeogenesis in glucose homeostasis is well established, but the cellular localization of the key gluconeogenic enzymes liver fructose‐1,6‐bisphosphatase (FBPase) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in these organs and the potential contribution of other tissues in this process has not been investigated in detail. Therefore, we analyzed the human tissue localization and cellular distribution of FBPase and PEPCK immunohistochemically. The localization analysis demonstrated that FBPase was expressed in many tissues that had not been previously reported to contain FBPase activity (e.g., prostate, ovary, suprarenal cortex, stomach, and heart). In some multicellular tissues, this enzyme was detected in specialized areas such as epithelial cells of the small intestine and prostate or lung pneumocytes II. Interestingly, FBPase was also present in pancreas and cortex cells of the adrenal gland, organs that are involved in the control of carbohydrate and lipid metabolism. Although similar results were obtained for PEPCK localization, different expression of this enzyme was observed in pancreas, adrenal gland, and pneumocytes type I. These results show that co‐expression of FBPase and PEPCK occurs not only in kidney and liver, but also in a variety of organs such as the small intestine, stomach, adrenal gland, testis, and prostate which might also contribute to gluconeogenesis. Our results are consistent with published data on the expression of glucose‐6‐phosphatase in the human small intestine, providing evidence that this organ may play an important role in the human glucose homeostasis. J. Cell. Physiol. 197: 189–197, 2003© 2003 Wiley‐Liss, Inc.

Different involvement for aldolase isoenzymes in kidney glucose metabolism: Aldolase B but not aldolase A colocalizes and forms a complex with FBPase

The expression of aldolase A and B isoenzyme transcripts was confirmed by RT‐PCR in rat kidney and their cell distribution was compared with characteristic enzymes of the gluconeogenic and glycolytic metabolic pathway: fructose‐1,6‐bisphosphatase (FBPase), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK). We detected aldolase A isoenzyme in the thin limb and collecting ducts of the medulla and in the distal tubules and glomerula of the cortex. The same pattern of distribution was found for PK, but not for aldolase B, PEPCK, and FBPase. In addition, co‐localization studies confirmed that aldolase B, FBPase, and PEPCK are expressed in the same proximal cells. This segregated cell distribution of aldolase A and B with key glycolytic and gluconeogenic enzymes, respectively, suggests that these aldolase isoenzymes participate in different metabolic pathways. In order to test if FBPase interacts with aldolase B, FBPase was immobilized on agarose and subjected to binding experiments. The results show that only aldolase B is specifically bound to FBPase and that this interaction was specifically disrupted by 60 μM Fru‐1,6‐P2. These data indicate the presence of a modulated enzyme–enzyme interaction between FBPase and isoenzyme B. They affirm that in kidney, aldolase B specifically participates, along the gluconeogenic pathway and aldolase A in glycolysis.

Expression of GM‐CSF receptors in male germ cells and their role in signaling for increased glucose and vitamin C transport

We studied the expression and function of the granulocyte‐macrophage colony stimulating factor (GM‐CSF) receptor in male germ cells. RT‐PCR showed expression of mRNAs encoding the α‐ and β‐subunits of the GM‐CSF receptor in human testis, and the presence of the α‐ and β‐proteins was confirmed by immunoblotting with anti‐α and anti‐β‐antibodies. Immunolocalization studies showed the level of expression of GM‐CSF α‐ and β‐subunits in the germ line in the testis and in ejaculated spermatozoa. Receptor binding studies using radiolabeled GM‐CSF revealed that bull spermatozoa have about 105 high‐affinity sites with a Kd of 222 pM and ≈1100 low‐affinity sites with a Kd of 10 nM. GM‐CSF signaled, in a time‐ and dose‐dependent manner, for an increased uptake of glucose and vitamin C. J. Cell. Biochem. 80:625–634, 2001.

Purification of adhesive proteins from mussels.

The adhesive polyphenolic proteins from the mussels Mytilus chilensis and Choromytilus chorus have been purified based on their solubility in dilute perchloric acid and on differential precipitation with acetone containing about 0.3 N HCl. The specific activity of the proteins obtained was 0.16 mg of 3,4-dihydroxyphenylalanine per milligram of protein, or higher. The proteins have an apparent molecular weight of about 100,000 and they contain a high proportion of 3,4-dihydroxyphenylalanine, lysine, and proline.

Novel identification of peripheral dopaminergic D2 receptor in male germ cells

Dopamine is a recognized modulator in the central nervous system (CNS) and peripheral organ functions. The presence of peripheral dopamine receptors outside the CNS has suggested an intriguing interaction between the nervous system and other functional systems, such as the reproductive system. In the present study we analyzed the expression of D2R receptors in rat testis, rat spermatogenic cells and spermatozoa, in different mammals. The RT‐PCR analysis of rat testis mRNA showed specific bands corresponding to the two dopamine receptor D2R (L and S) isoforms previously described in the brain. Using Western blot analysis, we confirmed that the protein is present in rat testis, isolated spermatogenic cells and also in spermatozoa of a range of different mammals, such as rat, mouse, bull, and human. The immunohistochemistry analysis of rat adult testis showed that the receptor was expressed in all germ cells (pre‐ and post‐meiotic phase) of the tubule with staining predominant in spermatogonia. Confocal analysis by indirect immunofluorescence revealed that in non‐capacitated spermatozoa of rat, mouse, bull, and human, D2R is mainly localized in the flagellum, and is also observed in the acrosomal region of the sperm head (except in human spermatozoa). Our findings demonstrate that the two D2 receptor isoforms are expressed in rat testis and that the receptor protein is present in different mammalian spermatozoa. The presence of D2R receptors in male germ cells implies new and unsuspected roles for dopamine signaling in testicular and sperm physiology. J. Cell. Biochem. 100: 141–150, 2007.

Immunological studies of the polyphenolic proteins of mussels

Abstract 1. 1. The cross-reaction of the polyphenolic proteins purified from Mytilus chilensis, Aulacomya ater and Choromytilus chorus with an antiserum produced against the protein of the latter species, was studied. 2. 2. Contrary to the strong cross-reactivity found with the protein of M. chilensis , the immune serum exhibited a poor reaction with the protein of A. ater . 3. 3. This marked difference in reactivity may be related to the difference in the amino acid composition of these proteins.

Differential signalling for enhanced hexose uptake by interleukin (IL)-3 and IL-5 in male germ cells

We studied the expression and function of the IL (interleukin)-3 and IL-5 family of receptors in male germ cells. RT (reverse transcription)-PCR showed expression of mRNAs encoding the alpha and beta subunits of the IL-3 and IL-5 receptors in human testis, and the presence of IL-3 and IL-5 receptors alpha and beta proteins was confirmed by immunoblotting with anti-alpha and anti-beta antibodies. The immunolocalization studies showed expression of these receptors in the germ line in the human testis and in human and bovine ejaculated spermatozoa. Functional studies with bull spermatozoa indicated that IL-3 signalled for increased uptake of hexoses in these cells at picomolar concentrations compatible with expression of functional high-affinity IL-3 receptors in these cells. In contrast, IL-5 failed to induce increased hexose uptake in bull spermatozoa. Experiments using HL-60 eosinophils that express functional IL-3 and IL-5 receptors confirmed that IL-3, but not IL-5, signalled for increased hexose uptake. Our findings suggest that differential signalling for increased hexose uptake by heteromeric high-affinity IL-3 and IL-5 receptors in mammalian spermatozoa is a property that depends on the identity of the alpha-subunit forming part of the alphabeta-complex and is not a property specific to the germ cells.

Expression of granulocyte–macrophage colony stimulating factor (GM-CSF) in male germ cells: GM-CSF enhances sperm motility

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine capable of stimulating proliferation, maturation and function of hematopoietic cells. Receptors for this cytokine are composed of two subunits, alpha and beta, and are expressed on myeloid progenitors and mature mononuclear phagocytes, monocytes, eosinophils and neutrophils, as well as in other nonhematopietic cells. We have recently demonstrated that bull spermatozoa express functional GM-CSF receptors that signal for increased glucose and Vitamin C uptake. In this study, we analyzed the expression of GM-CSF in bovine and human germ cells and its influence in bovine sperm motility. Reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunoblotting analysis demonstrated that adult bovine and human testes expressed GM-CSF. In addition, immunolocalization studies confirmed the presence of GM-CSF in the germ cell line in bovine and human testes. Computer-assisted evaluation of patterns of sperm motility demonstrated that the addition of GM-CSF enhances several parameters of sperm motility in the presence of glucose or fructose substrates.

Isolation of the fibrous sheath of mammalian sperm flagella.

Publisher Summary Axoneme is found along the axis of the mammalian sperm tail. It has outer dense fibers, mitochondrial sheath, and fibrous sheath. Three segments of the sperm tail have been defined. The cross-sectional pattern of the middle segment contains nine outer dense fibers in close proximity with the central axoneme. On the outside, this complex is encircled by the mitochondrial sheath; the annulus marks the caudal end of this segment. In most species, the main segment, or key piece, is the longer flagellar segment. The axoneme is in contact with seven outer dense fibers, and on the outside, the complex is surrounded by the fibrous sheath. This structure, and the dense fibers, poses a challenging problem in reproductive biology with regard to both their function in sperm motility and the synthesis and assembly of their corresponding polypeptides during spermiogenesis. Incubation of sperm with a solution containing 6 M urea and 2-mercaptoethanol or dithiothreitol dissolves most of the structure, leaving the fibrous sheath and the sperm heads. The methods discussed in this chapter are sperm isolation, fibrous sheath isolation, and purification of the fibrous sheath polypeptides. Rat sperm are obtained from the caput and cauda epididymis of adult Holtzman male rats. The animals are sacrificed by cervical dislocation and the epididymides freed of fatty tissue, minced with scissors, and suspended in cold PBS. Treatment of rat sperm for 12 hours with solution B containing 6 M urea plus dithiothreitol induces the dissolution of most cell structures with the exception of the fibrous sheath and the sperm head. The sucrose gradient centrifugation step yields a pure fraction of fibrous sheaths at the interface between the 0.9 and 1.8 M layers. After centrifugation in the discontinuous sucrose gradient no opalescent layer at the interface between the 0.9 and 1.8 M sucrose layers was noticed.